Plant Biotechnology and
Molecular Markers
Plant Biotechnology and
Molecular Markers
Edited by
P.S. Srivastava
Alka Narula
Centre for Biotechnology, Faculty of Science
Jamia Hamdard, New Delhi, India
Sheela Srivastava
Department of Genetics
University of Delhi South Campus
New Delhi, India
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Professor Sant Saran Bhojwani
Sant Saran Bhojwani was born to Mrs. Nam Adhari and Mr. Parmanand on 20th November, 1940
in the serene and tranquil environment of Dayalbagh, about 3 km from the hustle-bustle of the
Agra city. He had his early education in Dayalbagh and graduated and postgraduated from Agra
University. Soon after finishing M.Sc. (Botany), Dr Bhojwani served his alma-mater (R.E.I.
Dayalbagh, Agra) as lecturer for one year before joining the University of Delhi as a doctoral
student. His supervisor, late Professor B.M. Johri assigned him a challenging research problem,
with the warning that his Ph.D. degree would depend on his demonstrating the cellular totipotency
of endosperm, a completely unorganized, short-lived, triploid tissue. Earlier, many students of
Professor Johri and scientists elsewhere in the world could establish tissue cultures of endosperm
but failed to induce the organogenic differentiation. It was remarkable that within six months of
his joining Delhi University, Dr Bhojwani achieved differentiation of normal shoot buds from
the endosperm of Exocarpus cupressiformis; a parasitic flowering plant (Nature, 1965). At this
stage a very renowned American plant physiologist, Prof. F.C. Steward, visited the University of
Delhi who even after observing the cultures could not believe that the endosperm tissue could
form shoots and remarked, “Young man, take a bet with me. All the shoots in the cultures are
diploid. If that is the case remember me or else forget me”. However, when the shoots of
endosperm origin were cytologically analysed, all of them were found to be triploid, which is of
considerable practical importance in plant genetics and improvement. Subsequently, Dr Bhojwani
established the cellular totipotency of endosperm cells by reporting regeneration of triploid
shoots and/or plants in Scurrula pulverulenta, Acacia nilotica (Garg et al., 1996), Morus alba
vi
PROFESSOR SANT SARAN BHOJWANI
(Thoma et al., 2000) and Azadirachta indica (Chaturvedi et al., 2003). In the meantime, many
other scientists confirmed the observations of Bhojwani.
Dr. Bhojwani and his students worked on a range of basic and applied aspects of in vitro plant
morphogenesis. During 1971-1972 he worked with Dr Norman Sunderland at the John Innes
Institute, Norwich, U.K. under the British Council Fellowship Programme and reported quantitative
changes in nucleic acid and protein contents of microspores during the induction of androgenesis
in tobacco using histochemistry and cytophotometry (J. Exp. Bot. 1973). In 1972, he spent three
months in the laboratory of Professor Edward C. Cocking, FRS, at the University of Nottingham,
U.K. and reported for the first time isolation of microspore protoplasts using helicase enzyme.
The report appeared in Nature, New Biology (1972).
Dr Bhojwani had another opportunity to work in the U.K. for a year during 1975-1976 under
the Royal Society Commonwealth Bursary. This time he spent the whole year with Professor
Cocking and worked on wheat tissue culture (Z. Planzenphysiol., 1977) and protoplast isolation
and culture in cotton (Plant Sci. Lett. 1977). At this point of time there was considerable interest
in the application of biotechnological techniques to crop improvement. However, a major limitation
in achieving this goal was the recalcitrance of legumes, cereals and other major crop plants for
plant regeneration from cultured cells, an essential step in genetic engineering and somatic
hybridization. This prompted Dr Bhojwani to critically review the literature on tissue culture of
crop plants which was presented as an invited lecture in a meeting organised by the Agricultural
Research Council, London and later published in Euphytica (1977). The review, discussing the
progress and problems of tissue culture of major crop plants and emphasizing the need for
extensive further research in the area, was a highly cited publication which paved the way for a
fresh spurt of research to achieve high frequency regeneration in tissue cultures of these plants.
In 1978 Dr Bhojwani was awarded the prestigious Senior Fellowship of the National Research
Advisory Council of New Zealand, and the family moved to Palmerston North to join the Plant
Physiology Division of the D.S.I.R., New Zealand. Before the expiry of the term of the Fellowship,
the D.S.I.R. offered Dr Bhojwani a position of Senior Scientist (Scientist 105) and the Government of
New Zealand granted Permanent Residence to him and his family. In 1980 he was confirmed in
the job.
The stay of Dr Bhojwani in New Zealand was very productive. He published numerous
papers on the micropropagation of Willow (N.Z.J. Bot., 1980), Garlic (Sci. Hortic., 1980),
Clover (Physiol. Plant. 1981), Japanese Pear (Sci. Hortic, 1984), and Feijoa (Acta Hortic. 1987).
He also worked on Trifolium spp and reported, for the first time, regeneration of full plants from
mesophyll protoplasts of white clover (Plant Sci. 1982, Euphytica, 1984). Virus-free garlic
plants of a Japanese variety imported into New Zealand were produced by shoot tip culture to
facilitate its release through quarantine (Sci. Hortic 1982/83). Impressed by the work and publications
of Dr Bhojwani the D.S.I.R. decided to promote him to Scientist 106, an opportunity which was
pre-empted by his decision to return to India in 1981. However, his post in the D.S.I.R. was
not filled for at least two years expecting that Dr Bhojwani might decide to return to New
Zealand. He did return to New Zealand in 1983 but only as a Visiting Scientist for three months
to finish some experiments which remained incomplete in 1981 and process the data for
publication.
After his sojourn in New Zealand, Dr Bhojwani made a modest beginning as a Research
Associate at the University of Delhi and started guiding Ph.D. students in 1981. Fortunately, a
PROFESSOR SANT SARAN BHOJWANI
vii
major research project on “Micropropagation of Important Horticultural and Silvicultural species
of India” was sanctioned to him by the UGC, under which he and his students developed an
efficient protocol for clonal propagation of the leguminous tree species, Leucaena leucocephala,
and in vitro nodulation of micropropagated plants by Rhizobium to enhance their field survival.
He also demonstrated that sugar cubes, produced by Daurala Sugar Mills, was a fair substitute
of ‘Analar’ Grade Sugar used in Plant tissue culture media. The sugar cubes were more than 10
times cheaper than the ‘Analar’ Grade sucrose. In 1985 the Department of Environment and
Forests, Government of India, awarded another major research project to Dr Bhojwani to work
on “In Vitro Conservation of Endangered Plants”. It led to the development of protocols for
micropropagation and cold storage of Himalayan Species of three medicinally important plants,
viz., Picrorhiza kurroa, Podophyllum hexandrum and Saussurea lappa. In collaboration with the
scientists at the Biochemical Engineering and Biotechnology Department of IIT Delhi, Dr
Bhojwani studied the kinetics of cell growth in suspension cultures of Podophyllum hexandrum
and in vitro production of Podophyllotoxin, an anticancerous drug (Biotechnol. Lett. 2001, J.
Biosci. Bioengg., 2002).
Dr Bhojwani guided six Ph.D.’s on plant regeneration alone from somatic and gametic cells
of Brassica spp and published several papers (Plant Cell Tissue Organ Cult. 1985, 1991; Biol.
Plant., 1989; Plant Sci. 1990a,b; Euphytica 1993). A detailed investigation on direct shoot
regeneration from excised cotyledons of B. juncea proved a viable system for genetic transformation
of this important oleiferous crop of India. His group also achieved high frequency androgenesis
and selection of agronomically useful androclones in B. juncea. This work was supported by
funds from MOMBUSHO, Japan and European Commission, Brussels.
Dr Bhojwani undertook two major projects on mulberry biotechnology and investigated
micropropagation of some elite clones and production of gynogenic haploids (Euphytica, 1999)
and endosperm derived triploids (Pant Cell Rep. 2000) of this invaluable tree for silk industry,
the sole source of feed for silkworms. Recently, he has reported the production of gynogenic
haploids (Plant Cell Rep. 2003) and triploids (J. Plant Physiol., 2003) of Neem.
Dr Bhojwani has published 75 research papers in journals of international repute, 10 critical
reviews and 19 invited chapters in books published from India and abroad. In addition, he has
authored and edited several books. His first book “The Embryology of Angiosperms” (Vikas
Publishers, New Delhi) has been a popular text book for graduate and post-graduate students in
India and many other countries. Running into its 5th edition, the book has been translated into
Japanese (1995) and Korean (2001). In 1983, Dr Bhojwani brought out another book titled
“Plant Tissue Culture : Theory and Practice”, published by Elsevier, The Netherlands. This has
been regarded as the first standard text book on the subject and became so popular worldwide
that the publishers brought out its paperback edition in 1986. It was translated into Korean in
1986. Under a project funded by the Department of Biotechnology, Dr Bhojwani completed a
mammoth task of compiling ‘A Classified Bibliography of Plant Tissue Culture’, covering the
entire literature on the subject up to 1984. He spent two weeks in the U.K. under the INSA-Royal
Society Exchange Programme to complete the volume. It soon became a popular reference book.
A supplement to this volume, covering the literature of the next five years, was brought out in
1989. Both the volumes were published by Elsevier, The Netherlands. Dr Bhojwani has edited
four volumes, viz., “Plant Tissue Culture : Applications and Limitations” (1990; Elsevier),
“Morphogenesis in Plant Tissue Cultures” (1999; Kluwer Academic Publishers, The Netherlands),
viii
PROFESSOR SANT SARAN BHOJWANI
“Current Trends in the Embryology of Angiosperms” (2002; Kluwer Academic Publishers) and
“Agrobiotechnology and Plant Tissue Culture” (2003; Science Publishers, U.S.A.).
Dr Bhojwani has been in great demand by the organisers of conferences, seminars, workshops,
training courses and refresher courses because of his contributions and in-depth knowledge in
the subject of Plant Tissue Culture. He is a voracious speaker and scientists and students look
forward to his informative and thought-provoking lectures. In one of the meetings of the Indian
Association of Plant Tissue Culture held at NBRI, Lucknow, in 1976-77, the late Professor P.N.
Mehra, Padamshri, who chaired the lecture of Dr Bhojwani, was so impressed by his lecture that
he asked the audience to give standing ovation to the young scientist. Dr Bhojwani has participated
in several National and International Conferences in India and overseas. He was invited to
deliver a lecture at the conference on “Problems Related to Mass Propagation of Horticultural
Species”, Belgium (1985). The organisers of the Conference on Tissue Culture of Tropical
Plants in Bagota, Colombia invited him to deliver a Plenary Lecture and Chair a session. Dr
Bhojwani was a member of the International Advisory Committee of the VIII Conference of the
International Association of Plant Tissue Culture held in Florence, Italy, where he organised a
workshop. He was also a member of the International Advisory Committees of the 1st, 2nd and
3rd Asia-Pacific Conferences in Taejon, South Korea (1993), Shanghai, China (1997) and Singapore
(2000). He delivered plenary lectures in the Conferences held in South Korea and Singapore. In
1987 an International Symposium on Gene Manipulation for Plant Improvement was organised
in Kuala Lumpur, Malaysia and Dr Bhojwani was invited to deliver a plenary lecture. Dr Bhojwani
also attended the VI Conference of the International Association of Plant Tissue Culture held in
Minnesota, U.S.A. (1986) and the International Botanical Congress in Yokohama, Japan (1993).
He also delivered an invited talk in the latter. He was the only Indian invited as a Resource
Person to a workshop on “Production and Utilization of Double-Haploid Lines in Rice Breeding”
organised by the International Agency for Atomic Energy in Suwon, South Korea, in 1999. Dr
Bhojwani delivered plenary lectures and chaired sessions in International Conferences in Dhaka,
Bangladesh. Recently, he was invited to participate in the 15th Biennial Conference of the New
Zealand Chapter of International Association of Plant Tissue Culture and Biotechnology” at
Leigh, New Zealand and presented a paper on “Pollen Embryogenesis in Brassica ssp.
Dr Bhojwani has been a recipient of many honours. He was elected Full Member of the New
Zealand Institute of Agricultural Sciences (1981). In 1990 he became Invited Member of the
Technology Transfer Association of Japan. He was awarded the Nawashina Memorial Medal. Dr
Bhojwani was elected as a Fellow of the National Academy, Allahabad in 1994 and has been
awarded many National and International Fellowships to visit laboratories in other countries.
Besides the British Council Fellowship and Royal Society Bursary to work in U.K., Dr Bhojwani
was awarded the Senior Fellowship of the NRAC, New Zealand; Fellowship of the Japanese
Society for the Promotion of Science; Biotechnology Overseas Associateship, Government of
India; CIDA/NSERC Research Associateship, Canada; INSA-KOSEF Fellowship of South Korea,
and Fellowship of the Kernforschungsanlage, Germany.
Dr Bhojwani has been on the Editorial Board of many journals. To mention a few, Scientia
Horticulture, Holland; Journal of Biochemistry and Biotechnology, New Delhi; Phytomorphology,
Delhi, Plant Tissue Culture, Dhaka and Chromosome, Calcutta.
Dr Bhojwani has been a member of the Academic Council’s of the TERI School of Advanced
Study and C.C. Singh University, Meerut. He is the Chairman of the Research Advisory Committee
PROFESSOR SANT SARAN BHOJWANI ix
of the Central Tassar Research & Training Institute, Ranchi. He was a Visiting Senior Fellow of
the Tata Energy Research Institute, New Delhi and made a major contribution to the designing
and production and planning of the DBT-Sponsored Plant Tissue Culture Pilot Plant. He was
also a consultant to the Commercial Plant Tissue Culture Laboratories such as A.V. Thomas,
Cochin and Aranaya Micropropagation, New Delhi.
After serving the University of Delhi for 35 years, Professor Bhojwani took voluntary retirement
to serve the Dayalbagh Educational Institute (Deemed University), Agra as its Honorary Director.
Married to Shaku, Dr Bhojwani discharged his family obligations well and timely with both the
children married and settled happily with their families. His daughter, Anjli Sarup, married to Mr
Gursewak Maneesh, is living in Allahabad whereas his son Nova, with his dentist wife, Kokila
has recently moved to the U.S.A. as a Software Engineer. His wife Shaku in the true Indian
tradition extended her full support to the husband and deserves appreciation for her forbearance
and active interest throughout his career, especially during the long periods when Professor
Bhojwani was away completing academic assignments.
EDITORS
Preface
The genesis of the volume, Plant Biotechnology and Molecular Markers, has been the occasion
of the retirement of Professor Sant Saran Bhojwani from the Department of Botany, University
of Delhi. For Professor Bhojwani, retirement only means relinquishing the chair as being a
researcher and a teacher which has always been a way of life to him. Professor Bhojwani has
been an ardent practitioner of modern plant biology and areas like Plant Biotechnology and
Molecular Breeding have been close to his heart. The book contains original as well as review
articles contributed by his admirers and associates who are experts in their area of research.
While planning this contributory book our endeavour has been to incorporate articles that
cover the entire gamut of Plant Biotechnology, and also applications of Molecular Markers.
Besides articles on in vitro fertilization and micropropagation, there are articles on forest tree
improvement through genetic engineering. Considering the importance of conservation of our
precious natural wealth, one article deals with cryopreservation of plant material. Chapter on
molecular marker considers DNA indexing as markers of clonal fidelity of in vitro regenerated
plants and prevention against bio-piracy. A couple of write-ups also cover stage-specific gene
markers, DNA polymorphism and genetic engineering, including raising of stress tolerant plants
to sustain productivity and help in reclamation of degraded land.
The readiness with which the colleagues acceded to our request and the quality of articles
reflect the esteem in which they hold Professor Bhojwani. It is hoped that in honouring Professor
Bhojwani, this volume will further the frontiers of knowledge in Biotechnology as a whole and
Plant Biotechnology in particular.
While finalizing this volume we have received unsolicited support from all our colleagues
and friends which we acknowledge gratefully. Mr M.S. Sejwal and Mr Manish Sejwal, Anamaya
Publishers, New Delhi, have been forthcoming with suggestions and have shown utmost patience
while preparing the proof and final publication. They deserve the appreciation of all contributors
as well as of the editors.
EDITORS
Contents
Professor Sant Saran Bhojwani
Preface
1. In Vitro Androgenesis: Events Preceding Its Cytological Manifestation
Shashi B. Babbar, Nishi Kumari and Jitendera K. Mishra
2. Doubled Haploids: A Powerful Biotechnological Tool for Genetic
Enhancement in Oilseed Brassicas
Deepak Prem, Kadambari Gupta and Abha Agnihotri
3. Double Fertilisation in vitro and Transgene Technology
Erhard Kranz, Yoichiro Hoshino, Takashi Okamoto and Stefan Scholten
4. Polymorphism of Sexual and Somatic Embryos as Manifestation of Their
Developmental Parallelism Under Natural Conditions and in Tissue Culture
Tatyana B. Batygina
v
xi
1
18
31
43
5. Molecular Biology and Genetic Engineering of Polyamines in Plants
M.V. Rajam, R. Kumria and S. Singh
60
6. Biotechnological Approaches Towards Improvement of Medicinal Plants
Alka Narula, Sanjeev Kumar, K.C. Bansal and P.S. Srivastava
78
7. Production of Phytochemicals in Plant Cell Bioreactors
Saurabh Chattopadhyay, A.K. Srivastava and V.S. Bisaria
8. Development of Biotechnology for Commiphora wightii: A Potent Source
of Natural Hypolipidemic and Hypocholesterolemic Drug
Sandeep Kumar, S.S. Suri, K.C. Sonie and K.G. Ramawat
9. Biotechnology in Quality Improvement of Oilseed Brassicas
Abha Agnihotri, Deepak Prem and Kadambari Gupta
10. Role of Biotechnology for Incorporating White Rust Resistance in
Brassica Species
Kadambari Gupta, Deepak Prem and Abha Agnihotri
117
129
144
156
11. Current Trends in Forest Tree Biotechnology
E.M. Muralidharan and Jose Kallarackal
169
12. Cloning Forestry Species
Vibha Dhawan and Sanjay Saxena
183
xiv CONTENTS
13. Micropropagation of Woody Plants
J.S. Rathore, Vinod Rathore, N.S. Shekhawat, R.P. Singh,
G. Liler, Mahendra Phulwaria and H.R. Dagla
195
14. Biotechnology in Mulberry (Morus spp.) Crop Improvement: Research
Directions and Priorities
S.B. Dandin and V. Girish Naik
206
15. Development of High Efficiency Micropropagation Protocol of an
Adult Tree—Wrightia tomentosa
S.D. Purohit, P. Joshi, K. Tak and R. Nagori
217
16. In Vitro Regeneration and Improvement in Tropical Fruit Trees: An
Assessment
Madhulika Singh, Uma Jaiswal and V.S. Jaiswal
228
17. Tissue Culture of Cashewnut
Sumita Jha and Sudripta Das
244
18. Changing Scenarios in Indian Horticulture
Sanjay Saxena and Vibha Dhawan
261
19. Cryopreservation: A Potential Tool for Long-term Conservation of
Medicinal Plants
Sonali Dixit, Sangeeta Ahuja, Alka Narula and P.S. Srivastava
278
20. Molecular Mapping and Marker Assisted Selection of Traits for Crop
Improvement
Anushri Varshney, T. Mohapatra and R.P. Sharma
289
21. Studies on Male Meiosis in Cultivated and Wild Vigna Species
S. Rama Rao and S.N. Raina
331
22. Transgenic Crops for Abiotic Stress Tolerance
Deepti Tayal, P.S. Srivastava and K.C. Bansal
346
23. Cell Differentiation in Shoot Meristem: A Molecular Perspective
Jitendra P. Khurana, Lokeshpati Tripathi, Dibyendu Kumar, Jitendra
K. Thakur and Meghna R. Malik
366
INDEX
387
Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
1. In Vitro Androgenesis: Events Preceding
Its Cytological Manifestation
Shashi B. Babbar, Nishi Kumari and Jitendera K. Mishra
Department of Botany, University of Delhi, Delhi 110 007, India
Abstract: In vitro sporophytic development from the microspores of angiosperms through a process
referred to as androgenesis is an important field of research because of its fundamental as well as
applied importance. Besides providing possibility of developing haploid plants in large numbers
having application in plant breeding, the phenomenon offers an experimental system for studying
the events associated with transition from gametophytic to sporophytic phase. Of particular interest
are the changes occurring in the pollen prior to the onset of androgenic divisions, as these are likely
to throw light on the very process of androgenic induction. The present article reviews the ultrastructural,
biochemical and molecular changes that take place prior to cytological manifestation of androgenesis.
1.
Introduction
In angiosperms, the gametophytic phase is short lived and is completely dependent on the
sporophyte. Male gametophytes in these plants are referred to as pollen grains or microspores.
Pollen grains being haploid possess each gene in a single copy. Though destined to function as
male gametophytes, under suitable conditions, pollen grains are capable of developing into
sporophytes through a process called androgenesis. A new field of in vitro androgenesis was
initiated due to the landmark discovery of Guha and Maheshwari [1, 2], who reported development
of embryos from microspores in cultured anthers of Datura innoxia. Since this path-breaking
discovery, investigations in this field have aimed to: (i) extend the technique to more and more
taxa, (ii) identify the intrinsic and extrinsic requirements for successful development of plants
from microspores and (iii) understand the mechanism of induction. It is the last aspect, which
has remained enigmatic. The most baffling aspect of in vitro androgenesis is the transition from
gametophytic to sporophytic development.
The exact mechanism underlying this event, conditioning the male gametophyte to embark
upon an altogether different mode of development, is yet unknown. Studies using cytochemical,
biochemical, electron microscopic and molecular techniques have indicated that some structural
and biochemical changes do take place in microspores when they switch over to sporophytic
pathway. However, the critical turning point, at which the entity pre-ordained to become a
gametophyte switches over to an altogether different development pathway is yet not identified.
Therefore, the changes that take place at ultrastructural, biochemical and molecular levels,
preceding initiation of divisions leading to sporophytic organization of microspore, may either
be the cause or result of onset of androgenesis, depending on whether they are taking place
before or after this unidentified transition point [3]. Nevertheless, a brief review of such events
which precede the expression of androgenesis is necessary, to reflect upon the induction mechanism
initiating sporophytic development of a microspore.
2
2.
BABBAR ET AL
Trigger for Androgenic Induction
As early as 1975, Vasil and Nitsch [4] stated that angiosperm pollen is a versatile entity and its
normal course of development is precisely controlled by certain factors present within the
anther. Further, they opined that severing the contact with the plant and culturing of anthers on
a nutrient medium may be causing an imbalance in this precisely controlled influence, resulting
in the sporophytic development in pollen. However, the information gathered subsequently
indicates that developments preceding sporophytic divisions in microspore under in vitro conditions
can take place even in the absence of culture medium [5]. Thus, it seems that it is not the
prerogative of culture medium to suppress gametophytic development of pollen as the intrinsic
control can also be broken down by other factors.
2.1 Stress as a Major Trigger
It is known for a long time that stress treatment or sub-optimal conditions can alter development
programs. Stress is an important component of androgenic induction. The role of stress in
androgenic induction is explained by the following two hypotheses: (i) stress may cause
developmental defects by increasing the expression or stabilization of a critical target gene and
(ii) repression of critical genes below a threshold level [6].
In case of Brassica napus, the type of stress treatment given to cultured microspores for the
induction of androgenesis can vary from heat to gamma irradiation and colchicine [7–10]. Heat
treatment is the most commonly used pretreatment to initiate androgenesis in B. napus [11]. The
temperature must be around 32°C for 8 h in order to induce androgenesis sufficiently. This is
time and dose dependent and any interruption in between, leads to the failure of androgenesis
[12, 13]. This temperature of 32°C is near the temperature above which most microspores and
pollen grains of B. napus die [14]. It appears that stress alone, rather than in combination with
tissue culture conditions, is needed to initiate androgenesis. Thus, 32°C temperature treatment
itself can initiate the redirection process in situ before initiating in vitro culture [13]. Gamma
irradiation especially in combination with the temperature treatment has a stimulatory effect on
the induction of androgenesis [8].
In tobacco, androgenic process is usually induced in bi-cellular pollen grains, which are
initially cultured under glutamine and sugar starvation conditions before being transferred to a
high glucose medium. This is a very efficient method for androgenic induction in a large
percentage of pollen grains [15–18].
The elicitation of a general stress response in young microspores is associated with the
appearance of small heat-shock protein (smHSP) transcripts that precedes induction of androgenesis
[14, 19]. Generally, larger the temperature difference between the donor plant growth conditions
and in vitro culture conditions, stronger is smHSP signal. Similar results have been obtained
when colchicine and gamma irradiation are used as stress stimuli [8, 10]. No smHSP was,
however, produced below 25°C, a temperature too low to elicit a stress response. Thus, appearance
of smHSPs may be used as a molecular marker to indicate whether or not the pollen grains have
responded to the stress elicitors and therefore, are capable of initiating androgenesis [13, 14].
The changes in the pollen differentiation process leading to androgenesis can be initiated only
in microspores of specific developmental stages. Therefore, stress response has to be considered
together with the stage of microspore development [13].
In Vitro Androgenesis: Events Preceding Its Cytological Manifestation 3
3.
Impact of Microspore Developmental Stage
For most of the species, a suitable stage for the induction of androgenesis lies between just
before or just after first pollen mitosis. During this phase of development, the microspores are
non-committal in their developmental potential, as most of the sporophyte-specific gene products
are eliminated from the cytoplasm before meiosis [20] and the gametophyte-specific genes are
generally transcribed only after first pollen mitosis [21]. After the first mitosis, the cytoplasm
gets populated with gametophytic information and it gradually becomes irreversibly programmed
to form the male gametophyte [22]. A variety of external stimuli are applied during the microspore
development/culture in order to mask the gametophytic program and induce the expression of
sporophyte-specific genes, thereby making them to switch over to sporophytic mode of development.
Based on two model systems, i.e. Brassica napus and Nicotiana tabacum and also on the
findings on wheat (Triticum aestivum), Touraev et al. [23] suggested that microspores are competent
to change this developmental program within a relatively wide developmental window. In cereals,
the uninucleate stage up to first pollen mitosis, with species-specific variations, has frequently
been recommended [24]. A stage around first pollen mitosis has been considered optimal for the
induction of androgenesis in Secale cereale [25, 26].
It is found that the developmental stage of microspore affects the induction in a major way.
In callus/embryoid induction, a positive curvilinear trend was observed in each case and the
highest induction was obtained when B. napus microspores had undergone mitosis [10].
4.
Pathways of Androgenesis
Based on the studies in different plants, the five routes of androgenesis that have been identified
are: (i) by repeated divisions of the vegetative cell, (ii) by repeated divisions of the generative
cell, (iii) by repeated divisions of both, (iv) through symmetrical divisions in uninucleate microspore
giving rise to two identical cells rather than unequal generative and vegetative cells (B-pathway)
and (v) origin from fusion product of generative and vegetative. A species can exhibit predominance
of one or the other pathway [27].
Thus, division of otherwise quiescent vegetative cell, more than one division in generative
cell or formation of two-celled unit with identical cells from a uninucleate microspore can be
taken as the first sign of deviation from gametophytic development.
Of the abovementioned pathways, the fourth pathway is the most widely studied and is
considered to be the major pathway of androgenesis. This pathway involves symmetrical division
of microspore. Stress serves as a major signal for this symmetrical division. Since colchicine
treatment has been shown to increase the number of symmetrically dividing embryogenic pollen
grains in cultures of Brassica napus [9, 10, 28], it has been suggested that symmetry during
cytokinesis is an important factor in deflecting the gametophytic program of the pollen grain
towards the androgenic one.
The significance of microspore division symmetry for vegetative cell-specific transcription
and generative cell differentiation has been addressed in microspores of transgenic tobacco
plants transformed with promotor of vegetative cell-specific tomato lat52 gene fused to reporter
gus gene [29]. In vitro maturation, in the presence of high concentrations of colchicine, blocks
the first pollen mitosis effectively, resulting in the formation of uninucleate pollen grains expressing
both the abovementioned genes which are capable of germination and a pollen tube growth,
despite the absence of a generative cell. Lower amounts of colchicine induced symmetric division
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BABBAR ET AL
producing two similar daughter cells, both expressing the gus gene. These results demonstrate
that division asymmetry, at the first pollen mitosis, is essential for the correct generative cell
differentiation. Moreover, the activation of vegetative cell-specific transcription and functional
maturation may be uncoupled from cytokinesis [29].
Touraev et al. [17] on the other hand, have shown that cultivation of pollen grains containing
even two equal sized cells under the maturation conditions, lead to the development of mature
pollen grains. This indicates that rather than the symmetry of first pollen mitosis irreversible
commitment to embryogenesis is essential. In an interesting study, Zonia and Tupy [30] have
shown that lithium disrupts the partitioning of membrane-associated calcium, blocks polar nuclear
migration and subsequently, induces a symmetrical mitosis in microspores of tobacco.
Eady et al. [29] proposed two models to explain the significance of first pollen mitosis for
pollen determination and differentiation. According to the first model of passive repression, low
levels of gametophytic expressed factors (which are the result of asymmetric division), are
present in the generative cell. On the contrary, the symmetrically dividing cells, or in the case of
colchicine-blocked uninucleate microspores, no such repression of the vegetative cell-specific
genes occurs. According to the second model, there exists an active repressor, blocking transcription
in the generative cell, which upon asymmetric division, is again selectively retained in the
generative cell.
5.
Sub-Cellular Changes Associated with Androgenic Induction
As the microspore switches from its normal gametophytic to sporophytic pathway, numerous
structural, biochemical and molecular changes take place at the cell level.
5.1 Structural Changes
The first ever report, mentioning any cytological change, preceding the sporophytic cell division,
was that of Sunderland and Wicks [31]. They observed that in tobacco anthers, cultured either
at uninucleate microspore stage or during first haploid mitosis, the grains undergo either normal
mitosis resulting in typical vegetative and generative cells or rarely modified first mitosis resulting
into two identical cells. Subsequently, after a lag phase of some days, two types of grains can be
distinguished. These types are characterized by their differential staining; one stains lighter than
the other. The former develops into an embryoid, whereas in the latter, starch is deposited and
some of these may even germinate [31, 32]. These observations imply that before cell divisions,
a dedifferentiation process takes place in such grains, which are destined to develop into embryoids.
This dedifferentiation process mainly involves degradation of gametophytic information in
embryogenic grains. This is substantiated to some extent by electron microscopic investigations
conducted on cultured anthers of Nicotiana tabacum [33]. During the first two days of culture,
microspores underwent normal gametophytic differentiation at the same rate
as under in vivo conditions. In anthers cultured for 8-12 days, two types of grains could be
distinguished. In the first type, presumed to be embryogenic, vegetative cell was occupied by
multi-vesiculate structures resembling lysosomes. In these grains, cytoplasm was scarce in
organelles and by twelfth day these grains were virtually devoid of organelles, except plastids.
In contrast, vegetative cell of other grains was with full complements of cytoplasmic organelles,
however, lysigenous cavities, encountered in embryogenic type, were conspicuously absent
[34]. With the first division of vegetative cell, the embryogenic microspores were re-populated
In Vitro Androgenesis: Events Preceding Its Cytological Manifestation 5
with various cell organelles, thus, once again becoming rich in cytoplasm [35]. Based on these
observations, authors suggested that prior to the first division of vegetative cell, leading to
sporophytic development, there is a controlled degradation of organelles in embryogenic
microspores. In contrast, similar ultrastructural studies conducted on Datura innoxia showed
that there was no degradation and re-synthesis of cytoplasm in its vegetative cell prior to
androgenic induction [36]. The incongruity in observations was ascribed to the difference in
post-mitotic development in these taxa. The post first haploid mitotic cytoplasm synthesis in N.
tabacum is fast and microspores pass on to the stage 6 [37] from 5 quickly. Whereas, stage 5 in
D. innoxia is extended. Dunwell and Sunderland [36] believed that in N. tabacum degradation
of gametophytic cytoplasm was necessitated because the signal for sporophytic development is
perceived only after its synthesis is over. On the other hand, slow development of D. innoxia
microspores results in triggering of androgenesis before gametophytic information is at all or
completely synthesized.
The microspores of maize after the culture possess cytoplasm scarce in organelles and low
density of ribosomes. The first structural changes occurring at the sub-cellular level, include
occurrence of nuclear chromatin at de-condensed stage, nucleolus comprising exclusively of the
fibrillar component, cytoplasm with scarce organelles, a low ribosomal density and 2-3 fold
increase in the number of nuclear pores, before the onset of the first pollen mitosis [38].
In Brassica napus, the first division of cultured microspores destined to become embryogenic
is generally symmetrical. The first stage of differentiation in culture is the dispersion of the
central vacuole. The centre of the cell then becomes occupied with a highly pleomorphic single
nucleus. The large central nucleolus is gradually lost, being replaced by 3-6 smaller nucleoli.
The plastids, which were previously dispersed throughout the cytoplasm subsequently become
aggregated around the nucleus and lay down large quantities of starch granules. Another
cytoplasmic feature is the appearance of large numbers of aggregated globules, which are not
bound by membrane but may at times have small aggregates of ribosomes on their surface
[39].
The central nucleus of the microspore undergoes a symmetrical mitotic division to form two
cells within the exine. A normal middle lamella, followed by fibrillar wall, is then laid down in
each of these cells. The cytoplasm of each of these cells contains two principal domains. The
vacuolar domain contains dispersed or aggregated material while the second domain contains
evenly staining globules. Both the vacuoles and the globules are distributed evenly in the
cytoplasm. Over the course of this first division, the ER cisternae increase in number and the
plastids continue to accumulate starch and become more irregular in their outline [39].
In Hyoscyamus niger, which exhibits sporophytic development predominantly through the
generative cell, potentially embryogenic uninucleate microspore could be identified within 6 h
of culture. Such microspores had an increased ratio of volume densities of the nucleolar granular
zone to the fibrillar zone and dispersed to condensed chromatin [40]. The de-condensation of
chromatin is associated with the early and rapid synthesis of DNA during development. The
dispersed distribution of chromatin in potentially embryogenic grains may thus reflect increased
DNA synthesis early in culture. These investigations have shown that continued DNA synthesis
in generative cell in cultured microspores of H. niger, followed by mitosis and cytokinesis, result
in the development of pollen embryos [41]. After first pollen mitosis, the generative cell maintains
its large granular nucleolus. The volume fraction of the cytoplasm occupied by mitochondria and
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BABBAR ET AL
plastids and the area fraction occupied by RER and golgi cisternae, differ in the generative cells
of potentially embryogenic and non-embryogenic pollen [42].
5.1.1 Cause of Symmetrical Division
Symmetrical division, rarely found in normal gametophytic development, is essential for the
sporophytic embryogenesis in some taxa [28, 43, 44]. Simmonds [45] reported that during heat
treatment a pre-prophase band (PPB) of microtubules develops in Brassica microspores, which
was thought to determine the division symmetry.
During gametophytic development of microspore, the first pollen mitosis lacks a PPB
[46, 47] and results in a non-consolidated cell plate. Such a cell plate is considered to be an
important feature in this division because the generative cell is destined to be mobile within the
vegetative cell. Normally microtubules are involved in anchoring the nucleus at the cell edge.
Electron microscopic studies have revealed the presence of microtubules connecting the nuclear
and plasma membranes [48]. Heat or colchicine treatments induce de-polymerization of microtubules
[10, 45, 49], which results in displacement of the nucleus from its peripheral position, indicating
thereby microtubular disruption in the nuclear-cortical zone [49].
Pre-prophase band (PPB) of microtubules is a good indicator of the embryogenic potential in
microspores of Brassica. PPB is cortical in location and constitute the attachment site of the
future cell plate. Moreover, wall maturation does not occur if the cell plate is attached at a site
not previously occupied by PPBs [49]. The appearance of the PPB in heat-treated microspores
of Brassica napus predicts a cytokinesis leading to a stable cell wall, a critical event in the
initiation of multicellular organism comprising stationary cells separated by stable cell wall. Due
to cell wall consolidation, deviation from the normal pollen mitosis occurs and symmetric
division takes place [28, 39, 44, 50, 51, 52]. These observations suggest that symmetric division
blocks the normal microspore development, which in turn, results in a default developmental
pathway leading to androgenic induction [28, 39, 44, 45, 52]. Thus, at least in B. napus, the PPB
is both a marker of embryogenic development and its integrity is critical to the development
of first consolidated wall, which marks the beginning of a multicellular structure leading to
embryogenesis.
During normal microspore ontogeny, there is a polar distribution of cytoplasmic organelles
away from the microspore nucleus, prior to the first haploid mitosis, resulting in a generative cell
that is deficient in organelles following division [46]. Ultrastructural examination of the early
stages of androgenesis have revealed that potentially embryogenic microspores could be identified
by the loss of polar distribution of organelles, resulting in generative cells possessing a full
complement of organelles required for the continuous growth and division of embryos [42].
Since components of the cytoskeleton are also known to mediate the movement and position of
cytoplasmic organelles, these results could be interpreted as an indication that the early processes
of androgenic induction involve alteration in the structure and function of the cytoskeleton even
in those microspores in which the first division is asymmetric [28].
5.2 Biochemical Changes
Biochemical changes mainly include changes in protein synthesis [19, 53-56], phosphorylation
[57–59], and changes in secondary metabolite metabolism [38, 60, 61].
In Vitro Androgenesis: Events Preceding Its Cytological Manifestation 7
5.2.1 Change in Protein Synthesis
The induction of microspore embryogenesis must be accompanied by the activation of specific
transcriptional factors which result in altered patterns of gene expression [55]. Change in the
protein expression patterns during induction of microspore embryogenesis has been investigated
by 2-D gel electrophoresis in Brassica and tobacco [19, 62].
Since culture of Brassica microspores at 32°C for 8 h leads to irreversible commitment to the
sporophytic pathway [19], changes in protein synthesis during this period was examined by
using in situ [35S] methionine labeling followed by 2-D gel electrophoretic analysis [55]. The
qualitative and quantitative analysis of 2-D [35S] methionine protein patterns revealed that six
polypeptides are specifically labeled under embryogenic culture conditions. Eighteen polypeptides
incorporated [35S] methionine at higher rate under embryogenic culture conditions (32°C) than
in controls (18°C). These results indicated that only a limited number of proteins detectable in
the 2-D gels of microspore extracts were associated with the induction of androgenesis [55].
The microspores of Brassica are irreversibly induced towards androgenic pathway during
4-8 h of the 8-h high temperature pretreatment. Analysis of in vitro translated total mRNA
indicates that proteins of molecular weight 84, 67 and 66 kDa and to some extent, 27 kDa are
synthesized during this period. Interestingly, these proteins were absent or present in low amounts
in freshly isolated (0 h), potentially embryogenic microspores. Microspores, unable to undergo
embryogenesis, contained very low amount of these proteins [19].
In a hybrid cultivar of Zea mays, during the induction of androgenesis, a 32 kDa protein
(MAR 32) is induced which accumulates in the anthers during the cold pre-treatment. Different
responsive and non-responsive genotypes have been evaluated and accumulation of MAR 32like proteins observed only in certain responsive genotypes [54].
5.2.1.1 Heat-shock and Protein Synthesis
Heat-shock induces a program of gene expression in which synthesis of a family of proteins socalled heat-shock proteins (HSPs) takes place [63, 64]. In microspore culture, elevation of
temperature to 32°C for 8 h is accompanied by de novo synthesis of a number of heat-shock
proteins of 70 kDa class [56]. The HSPs act as molecular chaperones in the folding, refolding,
assembly and transport of cellular proteins and are as such essential for cell survival [64].
Detailed analysis has shown that out of eight isoforms of HSP68, only one shows a three-fold
increase. An immuno-cytochemistry study has revealed a co-distribution of HSP68 with DNAcontaining organelles, presumably mitochondria. Of the six HSP70 isoforms detected, one increased
to six-fold in the embryogenic culture condition. During normal pollen development, HSP70 is
localized in the nucleoplasm during the S-phase of cell cycle and later in the cytoplasm. In early
bi-cellular pollen of Brassica, the nucleus of the vegetative cell, which normally does not divide
and never expresses HSP70, shows intense labeling of nucleoplasm with anti-HSP70 after 8 h
of culture under embryogenic condition [56]. Such studies demonstrate a strong correlation
between the phase of cell cycle and the nuclear localization of HSP70 with the induction of
embryogenesis. On the basis of these studies, it is speculated that HSPs might be involved in the
altered pattern of cell division, which leads to the induction of androgenesis [56].
In Brassica microspore culture, six proteins were identified, which were exclusively synthesized
under embryogenic (32°C) conditions [55]. Of these, four that were specifically synthesized
during the first 8 h at 32°C were not synthesized further after two days of culture. In a study on
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BABBAR ET AL
tobacco, a dramatic increase in the level of a low molecular weight HSP transcript has been
detected in embryogenic microspores, following the inductive starvation treatment [65]. Based
on these studies, it is speculated that these proteins represent ideal markers for the induction
phase of microspore embryogenesis [66].
5.2.1.2 Phosphorylation of Proteins
Phosphorylation of proteins plays an important role in the reception of signals from exogenous
factors by cells and in the expression of cellular functions [67, 68]. The embryogenic microspores
of Nicotiana rustica as well as N. tabacum exhibited a specific pattern of protein phosphorylation.
The characteristic pattern of phosphorylation was neither observed in microspores following
gametophytic development nor in non-embryogenic microspores [57, 58]. Kyo and Harada [58]
speculated that these phosphoproteins might be the essential factors for the onset of microspore
embryogenesis. Moreover, as these were not detected after the beginning of sporophytic cell
divisions, their function in the process of pollen embryogenesis appears to be transient [58].
Using density gradient centrifugation, it has been shown that these phosphoproteins were localized
in the plasma membrane [59], thus, indicating their role in signal perception.
Comparison of the 2-D patterns of phosphorylated proteins, in 2-day-old embryogenic and
non-embryogenic microspore cultures of Brassica napus, revealed a much higher phosphorylation
state of HSP70 under embryogenic conditions [55]. It was reported that in cultured microspores
of Brassica napus, HSP70 immuno-reacted with the monoclonal antibody MPM-2, which recognizes
a mitosis-specific phosphorylated epitope, in embryogenic microspores [69]. In another study,
Cordewener et al. [56] showed the difference in the rate of synthesis and the intracellular
translocation of HSP70 in the embryogenic and non-embryogenic cultures. In a recent study,
Cordewener et al. [66] have reported that change in synthesis of HSP70, translocation and
protein phosphorylation are associated with the switch in the developmental pathway of Brassica
microspores from gametophytic to sporophytic development.
5.2.1.3 Ubiquitin-mediated Degradation Pathway
A recent study based on immuno-cytochemistry using polyclonal antibody to ubiquitin revealed
a developmentally regulated loss of free ubiquitin and ubiquitinated proteins in embryogenic
microspores of maize. After immuno-localization experiments, a steady low level of UBQ/
UBQ-Ps was revealed in most cell types of anthers excluding degenerated and non-induced
microspores [38]. The localization of ubiquitinated compounds correlated particularly with those
MCMs (multi-cellular microspores) which were considered potentially androgenic on the basis
of their ultrastructural characteristics, than with cells displaying symptoms of elevated proteolytic
activity and degradation [70]. These results confirmed that the ubiquitin-mediated pathway is
involved in gene expression and regulation of cellular processes [71]. Similarly, Callis and
Bedinger [72] had also reported a positive correlation between loss of free ubiquitin and ubiquitinated
proteins, and pollen development and maturation. The results obtained by Alche et al. [38]
confirmed that the return to the sporophytic pathway is once again accompanied by an increase
in the levels of UBQ and UBQ-Ps species. The increase in the level of these proteins was opined
to represent not only a consequence of the deviation of the microspore to the sporophytic
pathway, but even a direct factor responsible for the androgenic induction [38].
In Vitro Androgenesis: Events Preceding Its Cytological Manifestation 9
5.2.2 Development of Phenolic Compounds
Delalonde et al. [60] reported that prior to the induction of androgenesis in maize, cold pretreatment
is required and this leads to the accumulation of phenolic compounds. A possible role of phenolic
compounds has been shown in the modulation of IAA-oxidase activity [73, 74]. IAA protection
or degradation effects may be partially linked to the individual phenolic capacity existing in
different varieties. Some diphenols could protect auxin by inhibiting IAA-oxidase [74] and some
monophenols on the contrary, could increase this activity, thus increasing the degradation of IAA
[75]. This accumulation of phenolic compounds can be attributed to the protection of IAA in
vitro from IAA-oxidase. Consequently, the genotype with maximum in vitro protection for IAA
is regarded as the best genotype for androgenesis [60]. This is interesting as one of the reasons
for observed enhancement due to cold pretreatment was earlier considered to be the delayed
browning of anthers. In fact, in Datura metel androgenic response was considerably enhanced
if anthers were cultured on medium incorporated with cysteine (an anti-oxidant, presumably
inhibiting the activity of phenol oxidases) and polyvinylpyrrolidone (an adsorbent of phenols).
However, as the polyphenols are known to inhibit IAA oxidase activity, the observed enhancement
due to decreased levels of IAA because of the increased activity of IAA oxidase was not ruled
out [76].
5.3 Molecular Changes
The molecular basis of developmental switch from pollen maturation to embryogenesis is still
not well understood. A large amount of data is available on gene expression during pollen
development in vivo [22, 77]. Microspores isolated from tobacco anthers at different stages of
development show an increase in the transcriptional and translational activities, accompanying
a new program of gene expression in the period immediately following the first pollen mitosis
[77-79]. In maize, this change in gene expression has been correlated with the cytological stage
at which the microspores become incompetent for embryogenesis [80]. However, in comparison,
information available on the molecular events during the developmental switch to embryogenesis
is fragmentary.
Bhojwani et al. [53], for the first time, reported that in an embryogenic microspore, there is
a change in the nucleic acid and protein content prior to embryogenesis. They reported a decrease
in the RNA content of vegetative cell of the embryogenic pollen grains of Nicotiana tabacum
prior to division, suggesting thereby that suppression of the gametophytic program already
acquired, is the first step in the induction process. Later, Garrido et al. [62] reported a decrease
in the overall synthesis of RNA and protein in tobacco pollen during 7-day starvation treatment.
In another study, Kyo and Harada [15] reported an increase in the rate of protein synthesis in
tobacco pollen cultured under maturation conditions. Kyo and Harada [15] proposed that the
degradation of proteins and/or the suppression of synthesis of proteins were necessary to switch
from normal pollen development to embryogenesis.
In contrast, studies conducted on Hyoscyamus niger highlighted the need of de novo RNA
synthesis in embryogenic grains. The metabolic changes in embryogenic grains become discernible
within 1-2 h of culture [40]. This study revealed that embryogenic grains are the only one to be
labeled during 24 h of culture of anther segments on 5-3H uridine incorporated medium for
1-2 h, thus indicating de novo RNA synthesis in embryogenic grains. The extended exposure
time to the labeled uridine for 6 h increased the number of labeled grains, suggesting that in all
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BABBAR ET AL
the embryogenic grains, RNA synthesis is not started simultaneously. Further experiments utilizing
actinomycin D, incorporated to the basal medium, revealed that even one hour of culture followed
by transfer to inhibitor incorporated medium is sufficient for 15 per cent of the embryogenic
grains to escape inhibition. The segments cultured on the basal medium for 24 h before being
transferred to inhibitor adjuvated medium, developed embryos at the same frequency as observed
in fragments continuously cultured on the basal medium. This observation implied that RNA
required for initiation of androgenesis is synthesized during first 24 h of culture, whereas for
subsequent development (up to at least heart-shaped stage of embryos) either RNA synthesis is
not required or its synthesis remains unaffected by actinomycin D. Raghavan [81] further reported
that within one hour of culture, in some of the microspores, poly (A) containing RNA (presumably
mRNA triggering embryogenic development) content, monitored by (3H) polyuridylic acid binding,
is increased. Contrary to Raghavan’s observations, Sopory [82] did not find any effect on the
frequency of embryo production, if anthers of dihaploid Solanum tuberosum were cultured on
actinomycin D incorporated medium for initial four days. However, continuous culture of anthers
in the presence of this transcriptional inhibitor reduced the response, thus, indicating that fresh
RNA synthesis is required only after four days of culture. In contrast, puromycin (a translational
inhibitor) totally inhibited the response. Based on these observations it was proposed that for
induction of androgenesis in Solanum tuberosum, conserved messenger exists and for initiation
of sporophytic development only translation is required [82].
The studies on Hyoscyamus niger also revealed that in this plant the transcription in the
generative nucleus is a more important prerequisite for the subsequent embryogenic pathway, in
comparison to that in the vegetative one. Thus, embryogenic divisions are initiated only in those
microspores in which the generative nucleus alone or along with the vegetative nucleus synthesizes
RNA and pollen grains in which RNA synthesis occurs almost exclusively in the vegetative
nucleus become starch filled and non-embryogenic [40, 81]. These observations are consistent
with data obtained from previous studies by Raghavan [41, 83], according to which in H. niger
organogenetic part of embryoid is formed by repeated divisions of generative nuclei.
In tobacco microspores, starvation treatment as an induction stimulus, results in the
dedifferentiation of male gametophyte, followed by a redifferentiation process including the
acquisition of embryogenic competence and de-repression of the cell division. During normal
pollen development, the generative nucleus passes through S phase to G2 phase soon after first
pollen mitosis, while the vegetative nucleus remains arrested in the G1-phase [84]. During
starvation, a large fraction of the pollen shows DNA replication in the vegetative cell. However,
inhibition of DNA replication in the vegetative cell, caused by the addition of hydroxy-urea to
the starvation medium, did not affect the formation of embryos after transfer to a hydroxy-ureafree medium with sucrose [84]. Thus, Zarsky et al. [84] concluded that DNA replication during
starvation was not essential for embryogenic induction, but emphasized that an event preceding
S-phase is important. In addition, the induction of changes in development is characterized by
the activation of specific transcription factors, which in turn, cause altered pattern of gene
expression. RNA and protein synthesis cultured in tobacco pollen showed a gradual decrease
during the starvation treatment [15, 62, 65].
Two major approaches have been utilized to identify the molecular markers for pollen
embryogenesis [85]. In one, gene products expressed during zygotic embryogenesis have been
used as probes for differentiating pollen embryos. This method led to the characterization of one
In Vitro Androgenesis: Events Preceding Its Cytological Manifestation 11
of the first markers, the 12S storage glycoprotein, found in microspore-derived embryos of
Brassica napus [86]. It is a useful marker since it demonstrates that non-zygotic embryos do
accumulate proteins characteristic of zygotic embryos. Boutilier et al. [87], applying the above
approach prepared a cDNA library of Brassica microspore embryoids and found that the expression
of the napin seed storage proteins coincides with the induction of microspore embryogenesis and
could therefore be used as a molecular marker for earliest stage of induction. Napin genes were
highly expressed in the embryogenic microspores, but not in the microspores undergoing pollen
development or in the somatic tissues. Three members of the distinct Bnm NAP sub-family of
napin seed storage protein genes (Bnm NAP2, Bnm NAP3 and Bnm NAP4) were responsible for
the majority of napin gene expression in embryogenic microspores. The elevated temperatures
induced expression of napin gene only in embryos and microspores that were competent for
embryogenesis [87].
The second approach for the identification of developmental markers is based on a comparison
of gene expression during micro-gametogenesis and induced embryogenesis in microspores.
Reynolds and Kitto [88] prepared a cDNA library of young pollen embryoids of wheat and
screened it with cDNA probes prepared from pollen at different stages of development [88]. Two
clones, pEMB4 and 94 were expressed very early during culture, suggesting that these genes are
associated with morphogenesis and are not simply expressed as a consequence of differentiation.
The accumulation patterns of clones may indicate the activation of specific genes associated
with the major morphological and physiological activities connected with the formation and
differentiation of pollen embryoids in vitro. These genes are spatially and temporally specific,
and were not expressed in microspore culture. pEMB4 may be an example of a “transition” gene,
which is normally expressed only at the time of first haploid mitosis. However, as a consequence
of embryogenic induction, this gene remains turned on in the developing embryos [88].
A cysteine-labelled metallothionein (EcMt) gene, isolated from a wheat pollen embryoid, was
transcribed only in embryogenic microspores, pollen embryoids and developing zygote embryos
of wheat. Increase in the transcript was directly correlated to the synthesis of abscisic acid [89].
Treatment of the cultures with fluridone, the inhibitor of ABA biosynthesis, suppressed not only
ABA accumulation, but also the EcMt gene transcripts and the ability of microspores to become
embryogenic [89]. To demonstrate the direct involvement of ABA in this process, exogenous
ABA was added to fluridone treated cultures. The inhibitory effect on ABA on both gene
expression and androgenesis was negated. Kawashima et al. [90] speculated that based on
sequence similarities, the wheat EcMt is a functional analogue of the animal Mt and that it may
play a role in zinc homeostasis during androgenesis in which zinc Mt sequester or disperse zinc
dependant DNA and RNA polymerases as well as in transacting zinc fingers proteins during
differentiation. Since the EcMt gene transcript is not expressed during normal pollen development,
but only in the microspore-derived embryoids or developing zygotic embryos, this may be an
example of a new expressed sporophytic gene [89]. Also, since the EcMt transcripts appear only
in the embryogenic microspores after 6 h in vitro, this may serve as a marker for the early events
of microspore embryogenesis, as the first structural change associated with embryogenic induction
is observed within 12 h of culture [88–90].
Zarsky et al. [65] identified a cDNA clone for a low molecular weight heat shock protein from
a library prepared from RNA isolated from Nicotiana tabacum binucleate pollen grains. This
study revealed that although the gene was expressed normally during the later stages of pollen
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BABBAR ET AL
ontogeny, it was transcriptionally activated by starvation-induced pollen embryogenesis and is
therefore, an example of a developmentally regulated gene associated with androgenic induction.
In another study, Vrinten et al. [91] prepared cDNA clones corresponding to genes differentially
expressed during the early stages in barley microspore culture. These genes were isolated and
characterized. Three cDNAs representing genes, not previously identified in barley, were isolated.
The first gene ECA1 (early culture abundant) was expressed only during the early stages of
culture and with a reduced expression in low-density culture. This lacked significant homology
with any other known gene or protein. The second one, ECGST (early culture glutathione Stransferase), having homology with members of group glutathione S-transferase genes was
thought to be important in protecting cells from oxidative stress during culture process. The third
one ECLTP (early culture lipid transfer proteins) had homology with lipid transfer proteins
(LTP’s) and an expression pattern similar to that of an LTP known to be a marker of early stages
of embryogenesis in the carrot somatic embryogenesis system.
Significant genotypic effects suggest that genetic factors are also important in determining
the androgenic potential of microspores. There has been considerable work to isolate genes that
control these events. In maize, the products of a number of crosses between highly embryogenic
and non-embryogenic genotypes were analyzed using RFLP markers [92, 93]. Cowen et al. [92]
used 98s families to map genes which were associated with the anther culture response. These
families were derived from the cross of a highly embryogenic line (139/39-05) and a nonresponsive line (B73). The analysis showed that the anther culture response is associated with
two major recessive genes on chromosome 3 and 9, which are epistatic, two minor genes on
chromosomes 1 and 10. Thus, tightly linked RFLP markers may serve as starting points for the
characterization of genes conferring high androgenic capacity. Since only a small percentage of
the microspores within an anther form embryos, it has been suggested that the anther culture
response is limited to those microspores bearing certain favorable genetic factors.
6.
Conclusions and Prospects
Despite many years of efforts by a number of groups, the mechanism of induction of in vitro
androgenesis is still poorly understood. Though, the information on cellular, biochemical and
molecular changes preceding its cytological expression is meager and limited only to a few taxa,
certain generalizations can be made that may provide the framework for future investigations.
Induction of androgenesis is developmentally regulated. Once the pollen maturation gene
products begin to accumulate after first pollen mitosis, the developmental pathway is determined
and androgenesis cannot take place. Thus, only microspores at certain stages of development can
be redirected to undergo androgenesis. The optimal developmental stage for induction of
androgenesis is around the first pollen mitosis. In most of the cases, if cultured at bi-celled stage
it is the vegetative nucleus that contributes to the embryo formation. However, if cultured at
uninucleate stage, besides development through this pathway, B-pathway also becomes operative.
The androgenic induction requires some external stress to competent microspores. The stress
may be in form of heat, cold, chemicals, starvation or water stress. The stress response during
induction involves some disruption and organization of the cytoskeleton, altering the polarity of
the cytoplasmic components of competent microspores. Imposition of stress is correlated with
the appearance of smHSP transcripts in the affected cells. Without elicitation of the stress
response, as indicated by the appearance of smHSP transcripts, androgenesis cannot proceed.
In Vitro Androgenesis: Events Preceding Its Cytological Manifestation 13
Induction results in an altered pattern of synthesis and accumulation of RNA and proteins in
potentially embryogenic microspores, leading to the first sporophytic divisions. Although the
identity of most of these genes is unknown, in several cases, they are stress-related or are
associated with zygotic embryogenesis. The stress-related genes may be concerned with a general
reprogramming of the cell or provide some type of protection from that stress. Perhaps stress is
the single most important factor determining whether androgenesis will be initiated or not.
Some of the potential areas for future research in the field of androgenic induction could be:
(a) The emphasis so far has been on gene products, which are up regulated during the
androgenic induction period. Future studies should concentrate on gene products, which
are down regulated.
(b) Up-regulation of many genes may also be due to certain other factors and this might not
be a potential molecular marker for induction. This can be verified by using molecular
techniques such as antisense technology against gene in question so as to determine
whether or not the same is really involved in the induction process.
(c) There is no information as to how division in the vegetative cell becomes self-sustaining.
Studies in this area could be rewarding.
(d) It has not been unequivocally demonstrated whether there are any cytological and
histochemical differences between the two similar looking cells or nuclei, after the equal
division of microspore. This requires further investigation.
(e) Information about the signal transduction pathway operating during androgenesis is
practically lacking. The physiological, biochemical and molecular changes that occur
within the competent microspores and/or pollen in response to this information are poorly
understood.
The investigation, on the abovementioned aspects of androgenesis, will require an integrated
approach using a combination of the physiological, biochemical, molecular and genetic dissections
of the regulatory relationship between gene expression and androgenic induction.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
2. Doubled Haploids: A Powerful Biotechnological Tool
for Genetic Enhancement in Oilseed Brassicas
Deepak Prem2, Kadambari Gupta2 and Abha Agnihotri1
1
Bioresources and Biotechnology Division, TERI, Habitat Place, Lodhi Road, New Delhi 110003, India
2
Centre for Bioresources and Biotechnology, TERI-School of Advanced Studies, Habitat Place,
Lodhi Road, New Delhi 110 003, India
Abstract: The review presents the detailed advantages of using doubled haploids for crop improvement
programs. It also elaborates the use of the doubled haploid technique in terms of its conjugation
with other biotechnological approaches. The hall mark of doubled haploid technique lies in versatile
potential of its use in various breeding programs and its ability to compress the time taken for
breeding a desired genotype in comparison to conventional breeding methods. The factors affecting
the production of haploids/doubled haploids have been discussed in the light of relevant research
undertaken towards genetic enhancement for value addition in oilseed brassicas.
1.
Introduction
The oils of plant origin have important edible and non-edible uses in human life. Various oil
yielding crop species have been domesticated to produce high oil yielding seeds for edible or
industrial purposes. The oilseed bearing crops include perennial trees like coconut and palm and
annuals like groundnut, soybean, sunflower and oleiferous brassicas. The oilseed brassicas include
B. carinata, B. nigra and rapeseed-mustard (collective term for B. napus, B. juncea and B.
campestris). Among various oilseed crops mentioned above oilseed brassicas command a substantial
market proportion. Rapeseed mustard with an average world production of 3.6 × 107 million ton
and an acreage of 24.7 million Ha, ranks third at global level preceded by soybean and cotton
seed (FAO on line statistics, http://www.fao.org/).
A considerable proportion of the world production of oilseed brassicas has been contributed
by developing countries, in particular China and India (FAO year book 1990-1999, http://
www.fao.org/). In India, among the nine annual oilseed crops grown in the country, oilseed
brassicas collectively rank second in terms of production and acreage next only to groundnut.
Among the various Brassica species, B. juncea occupies the maximum acreage followed by B.
campestris especially in the north gangetic plain [1]. B. napus is a relatively new introduction to
India but is gaining rapid popularity in Punjab and Himachal Pradesh [2]. Thus, in India too, the
oilseed brassicas are a major contributor to vegetable oil production and a considerable amount
of research effort is directed towards their genetic enhancement.
On account of its economic importance, oilseed brassica attracted the early plant breeders and
geneticists, and pioneering research was undertaken for elucidating its cytogenetic composition
[3-6]. The cytogenetic relationship between various oilseed brassicas has been represented by U
[7] and is popularly referred to as the U’s triangle. Brassicas comprise three diploid species
namely B. nigra, B. oleracea and B. campestris and three allotetraploids namely B. carinata, B.
Doubled Haploids: A Powerful Biotechnological Tool for Genetic Enhancement 19
juncea and B. napus which have arisen out of interspecific hybridization between the diploid
species followed by spontaneous chromosomes doubling. Evidence in support of this relationship
has been accumulated from cytological, biochemcial and molecular investigations [8, 9].
The oilseed brassicas show variable pollination behaviour in terms of existence of selfcompatible and incompatible forms. B. campestris is cultivated in the form of three ecotypes
namely brown sarson, yellow sarson and toria. These ecotypes comprise both—self-compatible
and incompatible plant types. B. napus, B. carinata and B. juncea are predominantly selfpollinated whereas B. nigra and B. oleracea are often cross-pollinated species [10, 11]. Therefore,
a variety of breeding methodologies ranging from inbred development through pure line selection
to hybrid cultivar development have been utilized for genetic enhancement of oilseed brassicas.
The choice of the breeding methodology, irrespective of the breeding goal, largely depends upon
the predominance of self- or cross-pollination, and the availability of naturally occurring genetic
variations [2]. Nevertheless, the recognition of breeding goals are essential before a strategy to
attain the same is spelt out.
2.
Need for Genetic Enhancement in Oilseed Brassicas
The major breeding objectives for genetic enhancement of oilseed brassica are aimed at enhancing
the commercial value of the end product either quantitatively or qualitatively. On one hand
breeding objectives like development of high yielding varieties and generation of varieties
having resistance to abiotic stress (salt, drought and frost) and biotic stress (insects and diseases)
target production in terms of quantitative enhancement. On the other hand, improvement in oil
content, and quality of oil and meal target the qualitative improvement of the produce. In recent
years greater emphasis has been laid upon nutritional quality enhancement of oilseed brassicas
with the aim of providing better nutrition and value addition [12].
Various methods are being routinely used for generation of genetic variability and genetic
enhancement. These range from conventional tools, such as selection from available germplasm
and hybridization for transfer of desired genes (either in vivo or in vitro through embryo rescue
and somatic hybridization) to the development of transgenics and use of molecular markers for
selection. Among the biotechnological tools used for genetic improvement doubled haploids
have emerged as an exciting tool for brassica breeders since this technology has a versatile
ability to blend with and expedite the existing approaches [13].
3.
Doubled Haploids: The Concept and Its Utility
Doubled haploids are plants produced by spontaneous or artificial doubling of the chromosomes
of haploid plants. Such a plant is valuable because the chromosomes that are created by artificial/
spontaneous doubling are exact copies of the chromosomes that were present in the haploid
plant-justifying the term doubled haploid. Doubled haploids offer a major advantage by attainment
of homozygosity in a single step thus significantly reducing the breeding cycle along with its use
in conjugation with other biotechnological methods for expediting the crop improvement programs
[14]. The major advantages of the doubled haploids are briefly summarized as follows.
3.1 Attainment of Homozygosity
According to conventional breeding approaches, homozygosity may be achieved by repeated
selfing and rigorous selection for several generations [15]. Normally this exercise requires about
20
PREM, GUPTA AND AGNIHOTRI
10 to 12 years for varietal development programs, and is most efficient in self-pollinated crops
that do not show inbreeding depression. Doubled haploids greatly reduce the time required for
obtaining homozygous plants if an efficient haploid generation protocol is available. Moreover,
attaining homozygosity for recessive and quantitatively controlled traits is an even greater mammoth
task because of the involvement of many loci and masking of recessive allels in heterozygous
state [16]. Doubled haploids may be generated in a single step thus fixing the genotypic combinations
in a single generation [17, 18]. This technique also considerably reduces the time required for
homozygous line development in cross-pollinated crops and may be especially useful in parent
development for hybrid production [19]. Thus doubled haploids essentially compress the breeding
cycle by accelerating the development of homozygous lines [14].
3.2 Utilization of Gametic Gene Combinations
Doubled haploids offer the unique advantage of utilization of the haploid phase for selection.
This fact assumes greater importance for selection in case of induced mutations, which are
generally recessive in nature or other quantitative traits, controlled by recessive allels [20]. Since
haploids would express recessive genes, transgressive segregants for recessive traits can efficiently
be recovered through diploidization of chromosomes. This concept also offers the advantage
of significantly smaller population size required to find the least likely recombinant in case of
quantitative traits, since in a doubled haploid population selection is actually effective on gametic
gene combinations [17, 18]. Therefore, doubled haploid populations have been used to study the
inheritance of important quantitative traits [21–23].
3.3 Versatile Compatibility with Other Approaches
In addition to the above stated advantages of the doubled haploids, they can also be profitably
utilized for mutation breeding, disease resistance, biotechnological gene transfer etc. Some of
the applications of doubled haploids in conjugation with other breeding approaches are reviewed
below:
• Mutation breeding: Mutations are immediately expressed in haploid and doubled haploid
plants, hence these are very lucrative targets for mutation research. In B. napus imidazoline
herbicide resistance has been introduced using doubled haploid technique in conjugation with
chemical mutagenesis. These resistant lines have been evaluated in field trials [24, 25].
Besides this, chemical as well as physical mutagens have been utilized to develop resistance
to Phoma lingam [26] and Alternaria brassicola [27] in B. napus by treating cultured microspores.
In vitro mutagenesis at haploid level also led to the development of high oleic acid, thinner
seed coat, high oil and protein, and low fibre content in B. napus lines [22, 28] and modified
erucic acid content in B. carinata [29].
• Disease resistance: Microspore cultures are one of the most excellent targets for in vitro
selection for disease resistance, provided that the disease defence system is active at such an
early stage of plant development [13]. Gametoclonal variation exhibited by haploids generated
through anther/microspore culture, along with host specific and non-host specific toxins as
medium supplements, could be used for in vitro selection of resistant genotypes.
• Biotechnological gene transfer: Microspores or anther form a good explant source for gene
transfer systems such as PEG, electroporation, microinjection and biolistic methods. Microspore
Doubled Haploids: A Powerful Biotechnological Tool for Genetic Enhancement 21
derived embyros have been used as recipient cell system for Agrobacterium-mediated gene
transfer in B. napus [30–34].
• Molecular breeding: DNA based procedures such as RFLP/AFLP analysis are being increasingly
employed in plant breeding due to their enormous range of application. DNA markers provide
unprecedented refinement in genetic analysis through the construction of nearly saturated
genetic maps. This provides the breeder with a highly efficient marker aided selection tool.
Double haploids being truly homozygous for all loci are now being routinely used for genetic
mapping of brassicas since they reduce the time required for making RFLP maps and for
generating polymorphic mapping populations [35–38]. Doubled haploid production have
been utilized to study gene linkages and interactions [39].
• Breeding for desired oil profile: Doubled haploid technique has also proved useful in the
development of mustard cultivars expressing specific oil profile, such as modified erucic acid,
reduced linolenic acid, increased linoleic, palmitic and oleic acid content for specific purposes
[28, 40, 41]. Hence double haploids provide a powerful breeding tool for obtaining designer
mustard varieties having specific fatty acid profile.
4.
Development of Haploids and Doubled Haploids
Availability of haploids is a prerequisite for doubled haploid production. Spontaneously occurring
haploid plants having half the normal number of chromosomes were discovered in 1920s, but
utilization of haploid plants was not a practical technique until methods for the controlled
production of haploid plants were developed. Apart from spontaneous occurrence, which is rare
and is confined to a few species [42], haploid plants may be produced by interspecific/intergeneric
crossing followed by selective chromosome elimination, for example, as in barley and wheat ×
maize. Developments in tissue culture techniques in the early 1960’s opened the doorway for
haploid plant production by in vitro culturing of unfertilized ovules (gynogenesis) or from the
mature/immature pollen grains (androgenesis). Although doubled haploids have been obtained
via gynogenesis [43–45], the majority of published successes have resulted from anther and
microspore culture. A breakthrough in this direction was achieved when Guha and Maheshwari
[46] for the first time demonstrated that anthers of Datura innoxia cultured in vitro produce
embryos that originate from immature pollen grains or microspores. However, the difficulties
associated with anther culture are low frequencies of haploids, difficulty in distinguishing
spontaneous doubled haploids from diploids which regenerate from somatic tissue, and the
considerable time and labour which may be needed to generate the desired doubled haploid
population required for successful utilization in breeding program [47].
Developments in isolated microspore culture for several crop species attracted the interest of
brassica breeders for generation of doubled haploids. Oilseed brassicas being responsive to cell
and tissue culture techniques have been extensively researched upon for both anther and isolated
microspore culture [13, 48–50].
5.
Anther Culture in Oilseed Brassicas
Successful anther culture in brassicas was first reported by Canadian scientists in early 1970s.
Keller and Armstrong [51] reported development of embryoids from cultured anthers of B. napus.
Following this several reports on B. napus anther culture were published [49, 52–58]. Similar to
these, anther culture has also been reported in B. nigra [59], B. campestris [60–62]
22
PREM, GUPTA AND AGNIHOTRI
and in B. juncea [63–67]. These reports on anther culture of various oilseed brassicas primarily
elucidate the possibility of doubled haploid production. However, the major bottleneck of this
technique lies in low frequency of embryogenesis resulting in the realization of a very few
embryos. Several factors may be responsible for this low embryo yield. It has been proposed that
anther wall generated toxins may inhibit microspore embryo development inside cultured anthers
[68, 69] and the congested conditions inside the anther may limit the nutrient supply to growing
microspores [52]. Much of these drawbacks of anther culture have been effectively overcome by
the development of isolated microspore culture technique.
6.
Microspore Culture in Oilseed Brassicas
As in the case of anther culture, the first report of successful isolated pollen culture was also
reported in B. napus [49]. This, in fact, was the first report of isolated pollen grain or microspore
embryogenesis in plant species other than those belonging to the family Solanacae. Following
this many reports have elucidated the potential of isolated microspore culture in oilseed brassicas
for developing haploid embryos.
Among the various oilseed brassicas, B. napus is primarily the most researched upon species
for doubled haploid production using isolated microspore culture. Many laboratories have developed
specialized protocols for isolated microspore culture of B. napus [56, 70–74]. Several reports
on isolated microspore culture have also been published in B. carinata [18, 70, 75], B. nigra
[76, 77], B. campestris [62, 76, 78–83] and B. juncea [23, 77, 83–87]. However, apart from B.
napus, limited success has been achieved in other species towards cultivar development, due to
the lack of efficient microspore embryogenesis.
7.
Factors Influencing Microspore Embryogenesis
The ability to induce totipotency in anther cultures/isolated microspore cultures is greatly influenced
by several factors. These include genetic and exogenic factors that may have profound implications
on microspore development in vitro. Various factors that influence microspore embryogenesis in
oilseed brassicas are:
7.1 Genotype
The genotype of the donor plants have been reported to have a profound effect on the microspore
embryogenic response. Genotypic variations in haploid embryo development have been observed
in several brassica species [88]. Genotypic variability for microspore embryogenesis response in
isolated microspore culture has been reported in B. campestris [18, 79, 80, 82], B. juncea [23,
77, 85] and in most reports sited for B. napus above. Recently microspore embryogenic ability
has been studied as a stably inherited trait using the diallele mating system in B. napus by Zhang
and Takahata [89]. The study elucidates that both additive and dominant effects are significant
for microspore embryogenesis as a genetically controlled trait. Similar dominant gene action has
been reported by Cloutier et al. [39] reiterating the strong genotype influence on microspore
embryogenic ability. Ajisaka et al. [90] have further reported two putative chromomosome
regions associated with microspore embryogenic ability in B. campestris.
7.2 Donor Plant’s Growth Condition
The donor plant’s growth condition has a marked effect on the physiological processes of the
Doubled Haploids: A Powerful Biotechnological Tool for Genetic Enhancement 23
plant thus it invariably influences the microspore embryogenic ability. Proper light, temperature,
humidity and nutrients are all necessary to develop healthy plants. Varying growth conditions of
donor plants have been tested for their influence on microspore embryogenesis, ranging from
plants grown under field conditions to plants grown under artificial or growth room conditions.
In B. campestris, plants grown under cold temperature conditions, 10/5°C day/night cycle showed
enhanced microspore embryogenic capability [80]. However, plants grown under relatively higher
temperature regime 28/15°C day/night cycle upto bolting and then transferred to low temperature
conditions as mentioned above, have also been reported to show enhanced microspore embryogenic
response [82]. Low temperature regime for donor plants has been reported to show a positive
correlation with increased microspore embryogenesis in B. napus [72, 74, 76, 91–93]. However,
in this species too, donor plants grown till bolting at higher temperature regime (25–28°C day/
12–15°C night) and then shifted to a low temperature (10/5°C day/night cycle) have been
reported to show higher microspore embryogenic ability [74]. Limited reports are available on
the influence of donor plant’s growth condition on microspore embryogenesis in B. juncea. A
relatively higher temperature regime (20–21°C day/15–18°C night) has been reported to be
congenial for microspore embryogenic response in isolated microspore cultures of B. juncea
[23, 77, 85]. In view of this it seems likely that the plants may be grown under normal in vivo
temperature conditions for healthy and vigorous vegetative growth, however, a shift to lower
temperature regime is essential for enhancing microspore embryogenic response.
7.3 Microspore Development Stage
The microspore development stage is a prime important factor that influences the microspore’s
ability to turn totipotent. This is primarily due to the fact that microspores would only respond
to embryo formation at a developmental stage when they are not committed to develop into
pollen grains [58, 64, 92]. Moreover, in oilseed brassicas the microspore development is
asynchronous and microspores of different developmental stages may be observed in a developing
anther. Therefore, selection of buds that have maximum proportion of embryogenic microspores
is essential for efficient microspore embryo yield. The microspore development may be divided
into three basic stages viz. the tetrad stage (when the microspore mother cell splits into four
haploid cells), the uninucleate stage (when the uninucleate microspore prepares for the nuclear
division to form the vegetative and generative nuclei) and the binucleate stage (when the microspore
contains a generative and a vegetative nucleus). Each of the abovementioned microspore
development stage has been extensively researched upon in B. napus to determine the exact
stage at which the microspore is not under a differentiation pressure or is not committed towards
pollen development.
It has been established that there is an optimum development stage (embryogenic window)
that corresponds to the late uninucleate to early binucleate stage of development, during which
large number of microspores could undergo embryogenesis [14, 94]. Further to this it has been
proposed that non-embryogenic microspores produce inhibitory substances that suppress embryo
development in the embryogenic microspores [68, 69]. This may be because of the rupturing of
non-embryogenic binucleate microspores [71] thus reducing the embryogenic frequency and
altering the morphology of embryos [69, 92]. Replacement of culture media after microspore
isolation helps in reducing the autotoxins thus allowing normal embryo development. Similar
influence of microspore developmental stage on microspore embryogenesis has been reported in
24
PREM, GUPTA AND AGNIHOTRI
B. campestris [80, 82], B. carinata [75], and B. juncea [66, 77, 83, 85]. These studies have
established that the late uninucleate stage is most responsive to embryogenesis and that selection
of buds with majority of late uninucleate microspores increases the frequency of embryo formation
in isolated microspore culture in oilseed brassicas.
7.4
Microspore Density
The density of isolated microspores in the culture media is another essential factor responsible
for normal development of embryos. Microspore culture density ranging from 1 to 10,000 cells/
ml has been reported for different species. Varying density of microspores have been studied
by various scientists to get the optimum embryo yield such as 1 × 104 microspores/ml [83],
2 × 104/ml [92], 3–4 × 104/ml [95–98], 8 × 104/ml [74] and 10 × 104/ml [99]. However, the
most favourable density has been found to range between 5 and 8 × 104 cells/ml for B. napus and
1 and 4 × 104 cells/ml for B. juncea.
7.5
Media Composition
The basic media composition for microspore culture protocols in various oilseed brassicas have
mostly been the same over the years. Initial reports of anther culture in oilseed brassicas emphasized
the role of increased sucrose concentration in culture media [51, 60]. Subsequent to this, it was
established that high sucrose concentration (10 to 13%) is essential for microspore embryogenesis
in anther as well as isolated microspore culture in B. napus [49, 52, 56]. Keller et al. [60]
demonstrated that L-serine was an important constituent of the anther culture media in B. napus.
Later, Lichter [52] demonstrated that basal Nitsch and Nitsch [100] medium supplemented with
glutamine, glutathione and L-serine was most congenial for anther culture in the same species.
Off late Nitsch and Nitsch medium [100] modified by Lichter [52], commonly known as NLN
medium, has become the most frequently used media for isolated microspore culture in oilseed
brassicas.
7.6
Growth Additives
Activated charcoal has been reported to be beneficial for embryo growth and normal development
in both anther and microspore culture of B. napus [55, 72]. However, its role in triggering
microspore embryogenesis has not been reported, rather it has been reported to be beneficial for
normal growth of induced embryos [72, 83, 101]. Growth regulators have been used in both
isolated microspore as well as anther culture of oilseed brassicas. Initial reports on microspore
culture emphasised the role of 6-benzyl amino purine and 1-naphthalene acetic acid for induction
of microspore embryogenesis [49, 52, 102]. However, off late it has been reported that growth
regulators are not essential for inducing microspore embryogenesis [71, 77, 94]. Several scientists
have also propounded the role of colchicine as an embryogenesis inducing agent [57, 91, 103105]. However, owing to high toxicity, potential hazard and great care required in handling
colchicine, this concept has not been utilized extensively. Till date the major role of colchicine
has been limited for doubling chromosome number of haploid plants.
Apart from the abovementioned factors, post culture incubation conditions are also reported
to have profound effect on induction and development of microspore derived embryos. For most
reports cited above an initial heat shock of 30–32°C for 3–10 days has been reported to be
essential for microspore embryogenesis. Further to induction of microspore embryos, determination
Doubled Haploids: A Powerful Biotechnological Tool for Genetic Enhancement 25
of ploidy of the produced embryos and colchiploidy for chromosome doubling are essential
part of any successful doubled haploids production protocol. Determination of ploidy is usually
done either through root cytology or using flow cytometry. Colchiploidy may be carried out by
axillary bud treatment or by dipping the plantlet’s roots in colchicine [74]. Having overcome the
successful induction of the embryogenesis, the doubling of the chromosomes and regeneration
of doubled haploid plants is not a severe bottleneck for utilizing this technique for various crop
improvement programs.
8.
Conclusions
The oleiferous brassica species have been extensively utilized as an important source of edible
oil. Oilseed brassicas stand third in world’s oilseeds production and acreage, whereas in India,
it ranks second next to groundnut. A considerable amount of work has been undertaken for
genetic enhancement of oilseed brassicas to increase its commercial value through various
conventional as well as modern approaches. Among the biotechnological techniques used, double
haploids have emerged as a promising tool. In addition to compressing the breeding cycle by
accelerating the development of homozygous plants, it has versatile compatibility with other
approaches like mutation breeding, transgenics, molecular breeding, etc. However, for successful
utilization of doubled haploids in crop improvement programs, generation of substantial doubled
haploid population is essential, which in turn is possible with the availability of an efficient
haploid production protocol. Haploids have been produced by selective chromosome elimination,
gynogenesis or androgenesis. Among the three, androgenesis, i.e. anther/microspore culture has
been the most successfully utilized approach. Both anther and microspore culture have been
researched upon in oilseed brassicas, however, extensive work has been done elucidating the
potential of the isolated microspore culture technique for developing haploid embryos. A number
of genetic and exogenic factors influence the efficiency of microspore embryogenesis. This
article has presented some of the important factors affecting microspore development and their
implications on establishing an efficient microspore culture protocol. The doubled haploids are
already being extensively utilized for the quality and agronomic improvement in B. napus but its
practical utilization in other economically important oilseed brassicas is yet to be realized. The
work in this direction is in progress at several institutions opening many a new vistas for
utilization of this versatile technique for genetic enhancement and value addition in the oilseed
brassicas.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
3. Double Fertilisation in vitro and Transgene Technology
Erhard Kranz,1 Yoichiro Hoshino2, Takashi Okamoto3 and Stefan Scholten1
1
2
Centre for Applied Plant Molecular Biology, AMP II, Institute for General Botany,
University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany
Field Science Centre for Northern Biosphere, Hokkaido University, Kita 11, Nishi 10,
Kita-Ku, Sapporo 060-0811, Japan
3
Department of Biological Science, Tokyo Metropolitan University, Minami-Osawa 1-1,
Hachioji, Tokyo 192–0397, Japan
Abstract: The procedure of in vitro fertilisation with single isolated maize gametes is the well
characterised model system to study fertilisation and early zygotic embryogenesis of higher plants.
It allows individual development of zygotes and primary endosperm cells. Both in vitro produced
zygotes and primary endosperm cells are able to develop into embryos, fertile plants and endosperm
in culture. These zygotes and primary endosperm cells are able to self-organise independently from
maternal tissue. Many developmental steps of both the in vitro-produced embryo and endosperm
are comparable to the situation in planta. Application of molecular techniques to the in vitro
fertilisation system can dissect specific expression patterns of known genes, for example, cell cycle
regulators and to isolate unknown genes and their products. Expression of foreign genes is possible
in gametes and zygotes. This allows to unravel the roles of genes during fertilisation and early
development. The ability of gametes and zygotes to express transgenes enable us to follow the
expression of GFP based reporter genes for the visualisation of subcellular components in these
living cells.
1.
Introduction
Two fertilisation events occur in angiosperm species [1, 2]. During these processes one sperm
fuses with the egg and the resulting zygote subsequently develops into an embryo. The other
sperm fuses with the secondary nucleus in the central cell forming a primary endosperm cell
which develops into endosperm. Now these two fertilisation events can be accomplished in
vitro. As has been possible for a long time with animal and lower plant gametes, in vitro
fertilisation (IVF) can be performed with single higher plant gametes. It has been performed
mainly with maize (for reviews, see for example [3-7]). The application of single cell culture
techniques allows single zygotes to develop into embryos and fertile plants, as well as single in
vitro fertilised central cells to develop into endosperm. In culture, the zygote without an endosperm
and the primary endosperm cell without an embryo are able to develop in a manner similar to
that in vivo. They are able to self-organise without mother tissue. Thus, an in vitro model system
for investigations of zygotic embryogenesis and endosperm development is now available to
dissect more precisely the early processes which are developmentally important.
To date comprehensive cytological and ultrastructural in vivo data on double fertilisation in
maize are available [8-13], but there is a lack of molecular information on gamete interactions
and only few data exist on fertilisation-induced molecular events after zygote formation. Therefore,
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KRANZ ET AL
cDNA libraries of egg cells and zygotes were generated to explore gene expression after fertilisation
[14, 15]. Analyses of these libraries showed that expression of several genes is up- or downregulated
after in vitro gamete fusion [16]. Expression of some cell cycle genes was investigated in single
gametes and zygotes of maize to follow the re-entering of the gametes into the cell cycle
between in vitro fertilisation and first cell division [17]. This is possible, because the gene
expression status of single cells can be investigated by the use of reverse transcriptase polymerase
chain reaction (RT-PCR) methods [17, 18].
This article is focused on advances in zygote and primary endosperm cell development in
vitro and describes the application of transgene technology to study early developmental processes.
2.
In vitro Fertilisation
Whereas animal and lower plant IVF-systems can easily make use of naturally free-living
gametes, sperm, egg and central cells of angiosperms presuppose their isolation, because the
embryo sac is generally deeply embedded in the ovule, and the sperm cells are enclosed in pollen
grains or tubes. Micromanipulation techniques and skills are prerequisites for the isolation,
fusion and culture of single cells. These methods were developed originally for experiments with
somatic cells [19-22]. They were adapted and improved for investigations with gametic cells
[23-25]. By use of these methods, experimental access to single gametes, fertilisation and postfertilisation events under continuous microscopic observation with defined conditions are possible,
for example, isolation, selection and fusion of pairs of gametic protoplasts. Also, this allows to
design detailed experiments to follow precisely timed early events of zygote, embryo and endosperm
formation after gamete fusion.
Isolated gametes (Fig. 1 a and b) are protoplasts and therefore can be fused by techniques that
have proved to be successful in the fusion of somatic protoplasts. These are electrofusion and the
fusion methods using polyethylene glycol or calcium to induce cell fusion. Sperm and egg cell
fusion are electrically induced in maize [17, 23, 25-27] and in wheat [28]. Using maize, the same
method was applied to central cell fertilisation [29, 30]. Calcium mediated cell fusion of sperm
and egg cells [31-33] and of sperm and central cells [29] were also performed in maize. Possibly
attributed to the large differences between the cell sizes isolated sperm cells fuse fast, generally
in less than one second with egg and central cells [25].
a
b
c
Fig. 1. Isolated female gametic cells and two-celled embryo derived from in vitro fertilised egg
cell from maize. (a) egg cell. Bar = 33 µ m. (b) central cell. Bar = 105 µ m. (c) two-celled
embryo, 43 h after in vitro fertilisation. Bar = 38 µ m.
Double Fertilisation in vitro and Transgene Technology 33
3.
Embryo and Endosperm Development
Development of a single isolated egg cell to the zygote, embryo and finally to a fertile hybrid
plant or from an isolated central cell to endosperm after IVF have exclusively been reported in
maize [27, 29].
Embryo and endosperm development occurs in culture independently from each other and
without female tissue. Sustained growth of in vitro fertilised egg cells or isolated zygotes has
been achieved by co-cultivation of zygotes and feeder cells. The feeder effect depends on the
medium composition which must fulfil the demands of both, the zygote and the feeder cells for
optimal growth. In vitro development of maize zygotes turned out to be genotype-independent.
The generally high developmental capacity may be attributed to the natural predestination of the
zygote to form an embryo. Originating from a single in vitro zygote, transition stage embryos,
consisting of a meristematic region and a suspensor form a scutellum-like compact white tissue,
and subsequently, a coleoptile and a plantlet. Clearly, the plant formation occurs without a
maturation period, as seed formation is circumvented. Seeds which are obtained from regenerated
plants are of the F2 generation [27].
The maize in vitro zygote is metabolically highly active. Newly formed cell wall material can
be detected as early as 30 sec after in vitro gamete fusion [26]. After IVF, karyogamy was
observed as early as 35 min [27] to 45 min [34] in egg cells and 1 h in central cells [29]. It is
completed both in the egg and in the central cell within 2 h after fertilisation in vitro (HAF). The
time course of karyogamy was determined by using isolated, DAPI-stained nuclei of fertilised
egg and central cells [29]. Two types of karyogamy were observed in in vitro fertilised central
cells. The sperm nucleus fuses either with one of the two polar nuclei or with the secondary
nucleus which can be formed prior to pollination and fertilisation. This was also found in maize
[35]. In vitro produced maize zygotes [17, 23, 26, 27, 32, 33] and in vitro produced wheat
zygotes [28] divide in culture. Depending on culture conditions, in maize it occurs as early as 29
HAF (E. Kranz, unpublished data), but generally 42-46 HAF (Fig. 1c) [26, 27]. Maize zygotes
divide in plants about 16 h after karyogamy [35].
Zygotic polarity is mostly of maternal origin. The distinct polarity [23, 26, 27, 36] of in vitro
and in vivo maize zygotes may mainly derive from the uneven distribution of cytoplasm within
the egg. Comparable to the situation in the embryo sac in plants, where the cell wall generally
surrounds the egg only at the micropylar region, the isolated and cultured egg restores its
polarity by forming new cell wall material in a polar fashion [26]. Maize eggs fused either with
barley, Coix or Sorghum sperm cells divide asymmetrically, just as the maize egg divides after
homologous fusion [26]. The maize egg also divides asymmetrically after fusion with a wheat
sperm. However, when a wheat egg is fused with a maize sperm, the plane of the dividing zygote
is rather symmetrical and characteristic to the situation in the wheat egg fused previously with
a wheat sperm [6]. Thus, the underlying processes performing the plane of the first cell division
asymmetrically are also of maternal origin.
In higher plants, the function of cell cycle regulatory genes during the first zygotic cell cycle
remains to be investigated. In maize, cyclin genes are differentially expressed during the first
embryonic cell division cycle which is regulated zygotically rather than maternally as in many
animal zygotes. Maize sperm cells express the cell division cycle-specific genes cdc2ZmA/B and
the mitotic cyclin Zeama; CycA1; 1. However, the other mitotic cyclins Zeama; CycB1; 2 and
Zeama; CycB2; 1 are not expressed in the male gametes [17]. What is generally the contribution
34
KRANZ ET AL
of the sperm cell in egg cell division? Isolated egg cells of maize and fusion products of two
maize egg cells do not divide [23, 26]. However, as in somatic cell culture [37], a short treatment
of high amounts of 2,4-D can trigger cell division in cultured isolated egg and central cells [26,
38]. Also, in mutants of Arabidopsis, unfertilised central cells can develop into endosperm [39,
40]. In animal and lower plant systems, egg activation and fertilisation-induced signalling events
have been widely studied. Investigations like these are now also feasible in angiosperms by using
single gametes. In maize egg cells and in vitro zygotes, membrane Ca2+ and the calcium receptor
protein calmodulin are mainly localised in the vicinity of their nuclei [27]. It is well known that
calcium ions play a central role in the regulation of metabolic processes and signal transduction
[41]. A localised elevation to micromolar Ca2+ levels from the increased Ca2+ influx across the
plasma membrane is needed for early fertilisation events, for example, the generation of the
fertilisation potential and cell wall secretion in the brown alga Fucus serratus [42]. In maize, a
transient elevation of free cytosolic Ca2+ in egg cells after fertilisation was reported [33]. An
influx of extracellular Ca2+ induced by gamete fusion was measured by the use of an extracellular
Ca2+ selective vibrating probe. The Ca2+ influx spread subsequently through the whole egg cell
plasma membrane as a wave front, starting in the vicinity of the sperm cell fusion side [43, 44].
In maize, central cell fertilisation can also be performed [29]. The isolated maize central cell
does not divide without fertilisation, as the egg cell generally does not divide in culture. However,
single fertilised central cells develop into a characteristic tissue, comparable to the in vivo
situation. The transition from the syncytium to the stage of cellularisation of in vitro endosperm
occurs within 3-5 days after fertilisation. As found in plants, cell divisions are highly frequent
and synchronised after cellularisation. In maize endosperm develops initially more rapidly in the
micropylar than in the antipodal area of the fertilised embryo sac [45]. It is characterised by
densely cytoplasmic cells predominately located at the base of the suspensor and larger vacuolated
cells in other regions near the embryo [46]. In vitro produced endosperm consists of one globular
part containing small cells with dense cytoplasm and one oblong part with more large cells.
Compared to the oblong part, the globular part develops more rapidly in culture. The similarity
in morphological polarisation both of the embryo and the endosperm might indicate underlying
similar developmental processes and might have a common origin. The central cell might be
regarded as a modified egg cell and early endosperm, evolved from a second embryo, develops
as a special kind of embryogenesis [5, 47, 48, 49].
In plants, endosperm development is terminated. Plant regeneration from in vitro produced
endosperm has not been observed. However, shoot bud development from isolated and cultured
endosperm of several species was reported [50]. Also, plant regeneration was achieved from
callus cultures which originated from excised endosperm (for review see [51]). In maize, plant
regeneration from excised immature endosperm derived callus and suspension cultures [52-54],
has not been reported.
4.
Transgene Expression in Gametes and Early Development
Transgenic technology provides a way to gain insights of gene function by altering the expression
level of a given gene, for example, by overexpression or expression of antisense RNA. A lot of
studies have shown that these techniques are well suited to unravel the role of genes important
for development, as for example transcription factors [55, 56]. Moreover, the novel marker,
green fluorescent protein (GFP), isolated from Aequorea victoria extends the possibilities of
Double Fertilisation in vitro and Transgene Technology 35
transgenic technology. Due to its non-toxic nature and the non-invasive visualisation by fluorescence
microscopy, GFP permits real-time observations of dynamic changes in living cells. GFP fusion
proteins can be used to study subcellular localisation, movements of proteins and organelles in
vivo [57, 58]. Fusions of GFP with entire proteins of known or unknown function have shown
where these proteins are located and whether they move from one compartment to another [59].
The GFP based cameleon calcium indicator, developed by Miyawaki et al. [60] may be used to
characterise the spatial and temporal distribution of calcium ions during fertilisation and early
embryonic development in vivo. Recently the function of this indicator was shown in guard cells
of Arabidopsis [61]. Other GFP based approaches, being of special interest for the application
to the in vitro fertilisation system, enable visualisation of cytoskeleton components. A microtubule
reporter gene (gfp-mbd) was constructed by fusing a GFP gene to the microtubule binding
domain of the mammalian microtubule-associated protein 4 (MAP4) gene. GFP-MBD labels
cortical microtubules after transient expression of the reporter gene in living epidermal cells of
faba bean [62]. Granger and Cyr [63] showed that constitutive expression of the microtubule
reporter gene in stable transformed tobacco BY-2 cells allows spatial and temporal resolution of
microtubule arrays as they reorganise throughout the cell cycle. Labelling of microtubular structures
in intact Arabidopsis plants was recently shown by Camilleri et al. [64]. By using GFP fusion
proteins, which bind to actin [65] the visualisation of dynamic changes of this component of the
cytoskeleton might be achieved.
In addition to the cytoskeleton, GFP that possesses specific intracellular sorting signals for
defined cell compartments can be used to tag, for example, endoplasmic reticulum, golgi apparatus
and vacuoles. Dynamic changes or reorganisation of these cellular components during zygote
and endosperm development can be observed by using transgenic gametes for in vitro fertilisation.
These examples show that expression of transgenes in isolated gametes and in vitro produced
zygotes will become a valuable tool for cytological and functional analyses of these developmental
stages. So far mainly two strategies are followed to study expression of foreign genes in gametes
and zygotes: direct delivery of DNA into these untransformed cells via microinjection and the
use of transgenic gametes and zygotes derived from stable transformed plant lines.
4.1 Microinjection
Transient expression of transgenes after microinjection of plasmid DNA in zygotes was reported
by Leduc et al. [36]. In this study the gus gene under control of the maize histone H3C4
promoter followed by an actin intron and two anthocyanin regulatory genes under control of the
35S promoter were used as reporter genes. They were injected in zygotes of maize and isolated
24 h after pollination. Transient expression, with a frequency of 3.5% on an average was
reported in zygotes 4 days after injection. Pónya et al. [66] demonstrated transient expression of
reporter genes after microinjection of plasmid DNA into egg cells and isolated zygotes of wheat.
A gfp gene under control of the ubiquitin promoter was injected into egg cells, whereas the gus
gene driven by the 35S promoter was injected into zygotes. Transient expression frequencies of
46 and 52% on an average for egg cells and zygotes, respectively were reported. High-frequency
AC fields, applied to immobilise the cells on an electrode were suggested by the authors to be
a possible reason for such high expression frequencies. However, this remains to be determined.
In general, immobilisation of cells for microinjection is performed with a holding capillary or
by embedding them in low melting point agarose. After injection of embedded isolated maize
36
KRANZ ET AL
zygotes we obtained transient expression frequencies up to 30% (E. Kranz, unpublished results).
In these experiments the GFP gene under control of an enhanced 35S promoter followed by the
first intron of the hsp70 gene [67] was used. GFP fluorescence was monitored about 18 h after
injection and culture. The described studies focus on the transient expression of transgenes after
microinjection of plasmids into egg cells or zygotes. The advantage of this method is that results
can be obtained immediately after injection of DNA into a cell of interest. It might be a suitable
method for evaluation of promoter activities in the target cells.
Holm et al. obtained stable transformed plants via microinjection of DNA into isolated zygotes
[68]. Basis of these experiments was an efficient regeneration system for isolated barley zygotes.
This co-culture system with barley microspores undergoing embryogenesis allows isolated zygotes
to develop into embryo-like structures with a frequency of 75%. Fertile plants were regenerated
from approximately 50% of these embryo-like structures [69]. After microinjection of the gus
gene under control of the rice actin promoter into isolated barley zygotes, presence of the
construct was confirmed by PCR with a mean frequency of 21% of the derived structures. GUS
expression was found in few cases. Two lines of green plants were shown to be transgenic, one
of them for an intact copy of the expression cassette beside fragments of the construct. However,
the gus gene was not expressed. Degradation of the introduced DNA was discussed to be a
possible reason for the rarely found expression of the transgene after microinjection [68]. After
circumvention of these problems stable transformation via microinjection of zygotes would be
of great advantage for applied purposes, since the use of selectable marker genes is not required.
The regenerants can be screened directly for the presence of the transgene. Efficient regeneration
systems for isolated zygotes which are the basis for this transformation method, were established
for wheat [70, 71] and maize [36]. In vitro produced maize zygotes can also be efficiently
regenerated into plants [27]. In vitro fertilisation provides the possibility to inject DNA into egg
cells before fertilisation. This option might have an impact on the integration event.
4.2
Transgenic Plant Lines
For investigation of stable integrated transgene expression in maize, gametes and zygotes transgenic
plant lines can be generated by microprojectile bombardment of immature embryos. An advantage
of stable transformation over transient expression assays is the fact that transgenic lines can be
used for various experiments without the need for new time-consuming microinjection experiments.
Nevertheless, due to the long generation time of maize, the time needed to establish and characterise
transgenic maize lines has to be considered.
There is little information on transcription and translation activity in maize gametes and
zygotes. The competence of maize gametes and zygotes to express stable integrated transgenes
was shown in our laboratory with plants transgenic for the gfp gene. The same gfp vector (35S:
gfp, [67]), as used for microinjection experiments, was introduced. It was optimised for high
expression levels of GFP in monocotyledonous plants and codes for a plant codon usage optimised
S65T version of the gfp gene. In female gametophytes the 35S: gfp construct was expressed.
Egg cells, synergids, and central cells showed GFP fluorescence, whereas no fluorescence was
detected in transgenic male gametes [72]. After fertilisation of non-transgenic egg cells and
central cells with transgenic sperm cells expression of the transgene was induced early after
fertilisation, and GFP was detected in zygotes and early endosperm (S. Scholten, unpublished
results). The 35S promoter construct was active in egg cells, central cells, zygotes, embryos and
Double Fertilisation in vitro and Transgene Technology 37
early endosperm. This opens the possibility to design new experiments and to express various
transgenes under control of this promoter construct during fertilisation and early development.
Therefore, we constructed expression vectors adapted to the requirements of maize to label
microtubules and actin filaments in living cells according to the constructs described by Marc et
al. [62] and Kost et al. [65]. Double labelling experiments with spectral GFP variants [73] or the
recently isolated red fluorescent protein [74] might enable analyses of dynamic changes and the
interactions of actin filaments and microtubules in vivo. We chose GFP to label microtubules and
the red fluorescent protein to label actin filaments. Transient expression analyses of these two
constructs revealed that double labelling of both cytoskeletal components is possible in living
cells (Fig. 2). The next step will be the generation and characterisation of transgenic plant lines
expressing both constructs in gametes and zygotes. Once established, these lines could be used
to study dynamics and interactions of the main cytoskeletal components during early development
in vivo.
a
b
c
Fig. 2. Transient expression of microtubule and actin tagged fluorescent proteins. Scutellar tissue of
immature maize embryos were bombarded with constructs (see text for details) to tag both major
cytoskeletal components with fluorescent proteins. (a-c) Cell with tagged microtubules and actin
filaments. (a) Microtubules tagged with green fluorescent protein. (b) Actin filaments tagged with
red fluorescent protein. (c) Overlay of (a) and (b) showing both major cytoskeletal components
tagged with fluorescent proteins within the same cell.
5.
Prospects
IVF with single gametes can now be used for wide hybridisation approaches to create new
hybrid and cybrid plants. Possibly due to zygotic and postzygotic incompatibility mechanisms,
resulting hybrid plants might be restricted to hybridisation between more closely related species.
This has been demonstrated in egg activation studies: Cell divisions were triggered in isolated
maize eggs by sperm cells of several cereal species. However, zygotic incompatibility was
observed after in vitro fusion of maize eggs with Brassica sperm cells [26].
Also, IVF techniques are valuable experimental tools for the elucidation of various processes
of double fertilisation and early development of embryo and endosperm under defined conditions.
Clearly, important progress towards a better understanding of these processes will continue to
come from analyses of mutants. However, experimental access to single higher plant gametes
and zygotes will facilitate studies on fertilisation and early developmental processes which are
difficult to investigate in plants. These studies together with gene cloning, protein isolation and
38
KRANZ ET AL
characterisation will certainly allow a comparison with fertilisation-induced processes occurring
in lower plants and animals [75, 76].
Fertilisation-induced signal transduction events, changes in the endoplasmic reticulum,
cytoskeleton, and nuclear movement are now possible to be studied under defined conditions,
e.g., an exact time point after gamete fusion. Such studies can be performed both in the zygote
and in the primary endosperm cell allowing comparative studies. Thus, they will provide a more
precise picture of co-ordinated processes during early developmental stages of the embryo and
the endosperm [5, 77, 78].
Molecular analyses are possible with few cells. In maize, cDNA libraries from egg cells and
in vitro zygotes were constructed by using RT/PCR techniques to isolate and to study the
function of the cloned egg and fertilisation induced genes [6, 14, 15]. PCR protocols were
adapted for expression studies of known genes by use of single cells [18], for example, to follow
gene expression of cell cycle regulatory genes in a time course during zygote development [17].
Also, fertilised central cells and primary endosperm cells are promising target cells for the
isolation of unknown genes and expression studies by using especially endosperm specific genes
[77, 79-81]. For functional analyses of gene products, existing protocols such as immunocytochemical techniques for protein detection and methods for protein isolation are being currently
adapted to single cells and to small cell aggregates in our laboratory. These tools will provide a
valuable contribution to the elucidation of common features and differences in zygotic and
somatic developmental processes.
Many processes involved in early endosperm development might well be studied during
development of in vitro produced or isolated primary endosperm cells by using defined culture
conditions. These are, for example, the suppression of phragmoplast formation between nuclei,
the mitotic hiatus, the synchronised re-initiation of mitosis, the periclinal phragmoplast formation,
the initiation of cellularisation via formation of nucleocytoplasmic domains (NCD) of a radial
microtubular array, alveolation, the programming of nuclear location and division planes during
cell wall formation in the syncytium [82].
In this respect the expression of GFP based marker genes might be a valuable tool. Stable
transgenic lines showed that central cells and early endosperm as well as egg cells and early
embryos are competent to express transgenes. In vitro fertilisation and culture systems enable
direct observation and monitoring of the development of individual cells. Combination of this
option with the expression of GFP based marker genes for subcellular structures will facilitate
new strategies to analyse cytological characteristics during fertilisation, early zygotic and endosperm
development. The use of fluorescent protein based markers for cytoskeletal components is of
high interest, since the plant cytoskeleton has crucial functions in cellular processes that are
essential for cell morphogenesis and development [83]. Once established through transgenic
maize plants, cytological markers might be of value to correlate expression data with specific
developmental stages, e.g., cell cycle phases through visualisation of microtubular structures.
Also, the GFP based cameleon calcium indicator [60] might be a possibility to characterise the
spatial and temporal distribution of calcium ions during fertilisation and early embryonic
development in vivo. The function of this indicator was demonstrated in guard cells of Arabidopsis
[61].
Additionally, transgenic approaches provide the opportunity for functional analyses during
fertilisation and very early zygotic and endosperm development. Transcription factors being
Double Fertilisation in vitro and Transgene Technology 39
expressed in egg cells, and cell cycle regulators, both might have a critical role during fertilisation,
further development or morphogenesis and thus are interesting candidates for antisense and
overexpression studies. A more comprehensive view on the fertilisation processes and early
development will certainly be the result of linking in vitro fertilisation with transgenic technology.
Acknowledgements
This article is dedicated to Sant S. Bhojwani, an outstanding scientist and a person we highly
regard, on the occasion and in honour of his 62nd birthday.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
4. Polymorphism of Sexual and Somatic Embryos as
Manifestation of Their Developmental Parallelism
Under Natural Conditions and in Tissue Culture
Tatyana B. Batygina
Department of Embryology and Reproductive Biology, Komarov Botanical Institute of the
Russian Academy of Science, Prof. Popov str., 2, 197376 St. Petersburg, Russia
Abstract: The new approach and strategy of investigation have permitted to reveal the existence
of somatic embryo not only in vitro, but also in natural conditions.
Plant organism is able to form somatic embryos at all stages of its development and on different
organs (vegetative and generative) along with sexual reproduction. This ability enlarges the plasticity
and tolerance of reproductive system. The system approach also permitted to introduce the new
notion—“embryoidogeny”, as a special category of asexual reproduction (vegetative) in situ, in
vivo and in vitro and consider its role in the reproductive system of flowering plants [1, 2]. The
embryoidogeny includes the following forms: ovular (nucellar and integumental), embryonic
(monozygotic-cleavage) and homophasic vivipary (foliar, cauligenic and rhizogenic). It is somatic
embryo that is the elementary structural unit of all these reproductive forms.
The development of sexual and somatic embryos proceeds in parallels manifesting their great
polymorphism. The only difference between them is the origin: zygotic embryo of heterophasic
reproduction, embryoid of homophasic one. Various transitional forms from embryo, embryoid to
bud can be revealed.
There are sexual embryos, embryoids and buds that appear as three elementary structural units
of reproduction and propagation.
The correlation of different modes of reproduction in plants, i.e. the reproductive strategy of
species [3] is predominantly determined by the process of adaptive evolution. This prolonged
process has led to the emergence of a great diversity of reproductive structures. These are zygotic
embryos, adventive buds, propagules, somatic embryos, etc. All the diversity of embryo-like
structures appear in plant tissue culture in vitro and on plants in situ and in vivo that was
generalised by the term “embryoid” [4]. Later the following definition was created to expand the
term: embryoid (somatic embryo)—the originative structure arising asexually in situ, in vivo and
in vitro. Embryoid is bipolar at all stages of development as also the sexual embryo. Unlike these
two structures, a bud develops as a pole itself and can give rise to an individual only after
regeneration of roots [3, 5].
Similarity of sexual and somatic embryo development is considered to be one of the main
points under the discussion of terms of the morphogenetic pathways such as embryogenesis,
embryoidogenesis and gemmorhizogenesis. The point of discussion is: to what extent the
morphogenesis of somatic embryo corresponds to that of sexual embryo, i.e. to what extent it
obeys Errera’s, Sachs’s and Hertwig’s laws of cell division and the laws of embryogeny [6, 7].
44 BATYGINA
The attention paid to the role of embryoidogeny in general system of plant reproduction is
insufficient. Terminology connected with embryo-like structures and their classification also
remains debatable.
Only separate aspects of somatic embryo morphogenesis in vitro have been studied in terms
of comparative embryology. The comparison of somatic embryos under natural conditions (cleavage,
nucellar, integumentary, cauligenous, rhizogenous, foliar) with sexual embryos and with somatic
embryos from in vitro culture is practically absent.
Well known Indian embryologists Swamy and Krishnamurthy [8] came to a conclusion that
the development of somatic embryo in vitro would never stand comparison with that of zygotic
one. They have stated that embryoid is devoid of the main features of sexual embryo and from
the morphological standpoint more closely related to a shoot bud (Fig. 1). Their arguments are
the following: embryoid is devoid of the internal differentiation because its initial cell lacks
polarisation; embryoid lacks an orderly pattern of cell divisions; the laws of cell divisions of
Errera and Sachs are not obeyed; suspensor does not form; the protoderm formation is belated
and also incomplete; embryoid lacks typical centres of polar organization—the hypophysis and
epiphysis; it often lacks the initials of hypocotyls as well; the organization of main root is
suppressed, plerome and periblem do not develop, and so on.
The latest data on the development of somatic embryo in situ and in vitro give evidence with
regard to the abovementioned signs proposed by Swamy and Krishnamurthy to distinguish
sexual embryos from somatic embryos, as optional. They can not be used in all cases of definition
of morphological status of sexual embryo, as well as of embryoid.
The system approach [9, 10] allowed to compare the modes of sexual and somatic embryo
formation on the model objects in situ, in vivo and in vitro for the first time. It has been revealed
that the developmet of a somatic embryo in situ and in vitro recapitulates a sexual embryo with
the origin being the only difference (sexual embryo results from heterophasic reproduction,
somatic embryo—homophasic reproduction). A new type of asexual reproduction in flowering
plants—embryoidogeny—has been established. Somatic embryo occurs to be an elementary
structural unit of embryoidogeny [4, 11-13].
In connection with this it seems necessary to carry out a comparative analysis of sexual and
somatic embryo development (in situ, in vivo and in vitro). The following structures have been
chosen for comparison: (1) the initial cells of sexual and somatic embryos [14]; (2) sexual
embryos of plants with different types of embryogenesis, and of seedling development with
respect to their reproductive biology and ecology; (3) somatic embryos developing on different
parts of plant (seed, leaf, stem, root); (4) somatic embryos, obtained from different explants
(plants from diverse taxa) with various modes of initiation and of development in in vitro
culture.
The present article examines sexual and somatic embryos in terms of certain characteristics,
for example: polarity, symmetry, the formation of epiphysis and hypophysis, development of
main organs, shoot and root apices, etc.
1. Sexual Embryos Under Natural Conditions
A high totipotency peculiar to the mature zygote, preserved in ca and cb cells has been recognized
(Fig. 2). Souéges took the degree of participation of these cells in embryo formation as a basis
for the classification of embryogenic types. He established six basic types (megarchetypes) [15].
Polymorphism of Sexual and Somatic Embryos
First
Zygote division
Filamentous Formation of
stage
protoderm
Formation of
polar centres
Formation of
cotyledons
45
Mature embryo
A
Free cells
B
C
Nests of cells
from interior
Surface cells
Callus
D
E
Fig. 1. Diagrammatic representation of the development of a sexually produced typical dicotyledon
embryo (A), of embryoids (B, C and D) and of an adventitious shoot bud (E) [8].
12
Cellules
4
Cellules Tétrade
llu
le
8
Cellules
4
Cellules Tétrade
11e
2e
3e
4e
5e
Génération (proembryon bicelluaire)
1
16
Cellules
Ba
Ce
le
le
llu
Ce
llu
Ce
8
Cellules
ica
12
Cellules
Ap
16
Cellules
Ap
ica
Bas le
ale
16
Cellules
le
46 BATYGINA
Ce
llu
sal
e
le
sal
e
2e
3e
4e
5e
11e
Génération (proembryon bicelluaire)
2
4
Cellules Tétrade
le
llu
i
Ap
Ce
8
Cellules
Ba
12
Cellules
Ce
llu
ca
le
le
11e
2e
3e
4e
5e
Génération (proembryon bicelluaire)
3
Fig. 2. Diagrams show the comparison of division rate of ca and cb cells in the proembryos of Polygonum
persicaria (1), Oenothera biennis (2) and Erodium cicutarium (3). In different taxa ca and cb cells
make different contribution to the formation of embryo body [15].
I ca = pco
cb = pvt + phy + icc + icc + CO + S
II ca = pco + pvt
cb = phy + icc + iec + CO + S
III ca = pco + pvt + 1.2 phy
cb = 1/2 phy + icc + icc + CO ± S
IV ca = pco + pvt + phy + ice
cb = icc + Co + S
V ca = pco + pvt + phy + pee + iec
cb = CO + S
VI ca = pco + pvt + phy + icc + iec + Co
cb = S
Subsequently two more embryogenic types have been established (Paeonad- and Graminadtypes), that account for the total number being eight [16] (Fig. 3). Totipotency gradually reduces
during the subsequent embryo development in the course of histogenic differentiation and
specialization. Only separate cell loci remain totipotent in the embryo, seedling and plant. High
totipotency at the first stages of development may determine the great polymorphism of embryos.
Souéges [17] has revealed the initials and loci of hypophysis and epiphysis in the embryos of
different angiosperm species. Their derivatives give rise to the shoot and root apices (Fig. 4).
The formation of hypophysis and epiphysis initials and loci takes place at the early stages of
embryogenesis, but the point of their differentiation is taxon-specific. Ontophylogenetic approach
allows to reveal, at least, following five groups of zygotic embryos (Fig. 5):
Polymorphism of Sexual and Somatic Embryos
Cruciferae-type
(= Onagrad =
Onagraceae =
Onagraceen)
Chenopodiaceae-type
(= Chenopodiad =
Chenopodiaceen)
Asteraceae-type
(= Asterad =
Asteraceen)
Piperad-type
Caryophyllaceae-type
(= Caryophyllad =
Caryophyllaceen)
Paeoniad-type
(= Paeoniaceae)
Solanaceae-type
(= Solanad =
Solanaceen)
Graminad-type
(= Gramineae =
Poaceae)
Fig. 3.
cb
The main types of embryogenesis in angiosperms [16].
a
a
m
ca b
b
m
2
q
ci
ci
1
3
e
e q
e
m
m
n
n
n′
n′
4
e
q
q e
q e
m
n′
e
e
o
p
7
p
8
q
r
pl
pl
m
t
Fig. 4.
n
o
m
p
9
m
h
n
q
m
pe h
Pl
t
o
q
m
n
6
5
q
de
r
47
o
h
p
pe
iec
h
10
11
iec
12
13
Early and middle stages of Geum urbanum L. embryo development [18].
1. The embryo exhibits typical initials and loci of hypophysis and epiphysis (for example,
Geum urbanum—Asterad-type of embryogenesis [18]);
2. The embryo exhibits only a typical initial of hypophysis and its locus (for example,
48 BATYGINA
ca
cb
m
ci
e
e
e
h
h
h
e
e
h
1. Geum urbanum (Asterad-type)
(after Soueges, 1948)
e
e
ca
e
iec
h
MR
e
e
cb
2. Polemonium caeruleum (Chenopodiad-type)
(after Soueges, 1948, Kapil et al., 1968)
MR
ca
p
cb
h
h
iec
h
MR
3. Arabidopsis thaliana (Onagrad-type)
(after Yakovlev, Alimova, 1976)
ca
cb
iec
4. Morina kokanica (Asterad-type)
(after Kamelina, 1987)
MR
ca
cb
5. Nelumbo nucifera (Asterad-type)
(after Titova, Batygina, 1987)
AR
ca
m
cb
ca
m
ci
q
q
n
n′
n
n′
MR
q
m
n′
o
p
6. Triticum aestivum (Graminad-type)
(after Batygina, 1969)
AR
ca
cb
ca
ci
ca
n
ca
m
n
1
l′
m
h
7. Platanthera bifolia (Onagrad-type)
(after Veyret, 1965)
1
l′
h′
1
1
l′
l′
m
h′
h′
h′
Fig. 5. Polymorphism of sexual embryos. AR: adventive root, MR: main root
[18, 19, 21-23, 39, 50].
Polymorphism of Sexual and Somatic Embryos
49
Arabidopsis thaliana and Capsella bursa-pastoris—Onagrad-type of embryogenesis
[19, 20]);
3. The embryo exhibits only a typical epiphysis initial and its locus (for example, Polemonium
caeruleumn—Chenopodiad-type of embryogenesis [17, 21]);
4. The embryo lacks typical initials and loci of hypophysis and epiphysis (for example,
Triticum aestivum—Graminad-type of embryogenesis [22, 11, 5]);
5. The initials of hypophysis and (or) epiphysis arise in the embryo, but they do not develop
the typical loci of hypophysis and (or) epiphysis (for example, Platantera bifolia and
Gymnadenia conopsea—Onagrad-type of embryogenesis [23, 24].
Polarity and morphological axis are normally established in the embryos of first four groups,
and shoot and root apices develop orderly in the first three groups—the apex of the main root,
and in the fourth—the apices of the adventive roots. The mature embryo of the fifth group
exhibits morphological polarity, but shoot and root apices are not distinguished exomorphically.
The embryos of all groups can be classified as normal types, not irregular, though they lack traits
of the classical embryo according to Swamy and Krishnamurthy.
The development of epicotyl varies greatly from taxon to taxon (for example, from epicotyl
locus to a well developed plumule). The same situation applies to the root (a developed main
root, or just its initials, or the adventive roots, or the full absence of roots can be observed in the
embryo).
Comparative analysis of structure and genesis of the mature embryo in different angiosperm
taxa gives the evidence that almost all signs proposed by Swamy and Krishnamurthy (see
before) and many other scientists to characterise sexual embryo vary greatly.
Somatic embryos under natural conditions are shown in Fig. 6. ‘Asexual (homophasic)
reproduction in flowering plants is accomplished by two elementary structural units of different
morphological essence: a somatic embryo (individual as the whole) and an adventive bud (only
a part of an organism). The majority of botanists when speak about vegetative reproduction bear
in mind only gemmorhizogenesis. Both structures seem to develop in parallel during evolution,
and realised side-by-side with sexual embryo (heterophasic reproduction) to a diverse extent in
different taxa. Embryoids in natural conditions could be provisionally divided into two groups:
embryoids developing in the flower and those developed on vegetative organs. Morphogenesis
of somatic embryo, and subsequently of a seedling in situ occurs in the whole system of parental
organism under its influence. The situation is similar to sexual embryo. This peculiarity of the
development probably is the reason for the orderly embryogenesis and a high per cent of seedlings.
On the contrary, the development of somatic embryos in vitro never produces high quantity of
normal plant-regenerants.
Unique form of embryoidogeny (monozygotic-cleavage) can be observed in Paeonia (Fig. 7)
[25, 26]. The zygotic embryo phase in the seed of all Paeonia species finishes at the stage of
protoderm formation in coenocyte-cellular structure (this being the heterophasic reproduction).
Then sexual embryo is cloned, when its epidermal cells give rise to somatic embryos. The
somatic embryo does not differ from sexual one of a typical dicot (Asterad-type of embryogenesis)
in morphogenesis. The establishment of polar axis takes place since the stage of initial cell. The
orderly development of root and shoot apices subsequently occurs. The genesis of Paeonia
somatic embryo and its structure seems to be determined by the conditions within embryo sac
50 BATYGINA
MR
1. Paeonia anomala (Asterad-type) (after Brukhin, Batygina, 1984)
2. Euonimus macroptera (Type-?) (after Naumova, 1987)
MR
MR
3. Ranunculus sceleratus (Onagrad-type) (after Konar et al., 1972a, changed)
AR
4. Bryophyllum calycinum (Type-?) (after Batygina et al., 1996, changed)
Fig. 6. Polymorphism of somatic embryos in natural conditions. AR: adventive root, MR: main
root [27, 28, 35, 51].
and ovule, where it takes place. Probably this also guides the development of nucellar and
integumentary embryoids. However, unlike the Paeonia embryo, these develop during cloning
of a parental sporophyte. The first divisions in them are irregular, the initials and loci of hypophysis
and epiphysis are not observed, but the development of protoderm, shoot and root apices and the
differentiation of the main root with all its elements correspond to those of sexual embryos [27].
Somatic embryos
and degenerating sexual embryo
Sexual embryo
2n
n + n 2n
2n
Heterophasic reproduction
2n
Homophasic reproduction
Fig. 7. Switching over the programme from heterophasic to homophasic reproduction in
Paeonia seed [26].
Polymorphism of Sexual and Somatic Embryos
51
The external shape and internal structure of Paeonia somatic embryo and of nucellar embryos
(ovular embryoidogeny) are similar to that of zygotic embryo.
The development of somatic embryo on the stem of Ranunculus sceleratus in situ takes place
in similar way [28]. It occurs according to onagrad-type. The authors have mentioned the
absence of suspensor differentiation, unlike sexual embryos of this species. The formation of
somatic embryos from epidermal leaf cell derivatives was observed in Crassula multicava [29].
The inner structure in these embryos, particularly of shoot and root apices may be compared
with that of the majority of zygotic embryos in spite of absence of typical initials and loci of
hypophysis and epiphysis. The development of sexual and somatic embryos of the same species
in natural conditions occurs according to the same type of embryogenesis.
Thus, the conducted analysis of somatic embryos in different taxa, developing in natural
conditions shows a large variety. The structure and genesis of somatic embryos in situ are taxonspecific and to a considerable extent determined by the place of their formation, and also by the
environment. For example, somatic embryos arisen in the seed more often develop the main
root. Those formed on vegetative organs usually develop adventive roots.
2. Somatic Embryos in in vitro Culture
As is known the in vitro culture provides to the researchers two different model systems (callus
and suspension) to obtain somatic embryos. These systems are distinguished by many parameters
that result in different structure and behaviour of initiating cells and determine the whole genesis
of somatic embryos. The development of sexual and somatic embryos of the same species in
natural conditions occurs according to the same type of embryogenesis. The development in vitro
of somatic embryos may be traced by the example of several species, contrasted by a number of
features [30].
In the callus culture of Triticum aestivum the endogenous initiation of meristematic zone has
been revealed (Fig. 8). This zone occurs to be a special tissue which consists of cell rows strictly
oriented to the callus surface [31, 32, 33]. Later, the cells of this zone become the initials of
somatic embryos. It is noticeable that in wheat microspore culture the embryoids arise exogenously
(from callus epidermal cells). At the early stages of development the sequence and place of
divisions in sexual embryo and embryoids are relatively similar (Graminad-type) [22, 5], though
the embryoids have some variability, preserved at the subsequent stages of their development.
The initials are differently oriented inside the cluster, the polar axes of young embryoids are
situated in different planes in reference to the callus surface. The delay of divisions of ca
derivatives has also been observed as compared with cb derivatives. Subsequently, it causes the
disturbance in their division sequence and of vacuolisation character and as a result, the appearance
of embryoids with “linear” structure of the apical pole and with abnormal histogenesis. In
zygotic embryogenesis the apical pole gives rise to the most of scutellum (cotyledon) and to the
plumule, while in the abnormal embryoids apical cells are destroyed. Finally the majority of
abnormal embryoids degenerate. In certain cases the normal plumule formation has been observed,
though the development of adventive roots disturbed (these embryoids may be possibly considered
as a transitional forms). Usually in the studied cultivars of Triticum the regeneration of plants by
means of embryoidogenesis does not occur.
The comprative analysis of the development of somatic embryo in callus culture and of sexual
embryo in situ in Aconitum heterophyllum (Ranunculaceae) has shown relative similarity.
52 BATYGINA
1
2
3
4
5
6
7
8
13
9
10
11
12
14
15
16
21
17
18
19
20
Fig. 8. Early stages of wheat zygotic embryo development in natural conditions (1–6), and somatic
embryos in tissue culture at different stages of development (1–12 drawings and 13–21 LM [49]).
Single initial cells of callus give rise to embryoids which subsequently pass all main stages of
normal embryogenesis—tetrads (T-shaped), quadrants, octants, globule, heart- and torpedoshaped. Embryoidogenesis in A. heterophyllum occurs as a whole according to Onagrad-type of
embryogenesis, which is peculiar for Ranunculaceae [35]. It is noticeable that embryoids exhibit
well differentiated suspensors. In the course of the experiment different abnormalities have been
observed in the embryoid development, the most frequent has been the precocious differentiation
of xylem in the plerome area at the heart-shaped stage. This is evidently connected with the
disbalance of carbohydrates and hormones in the culture medium and callus tissue. As a result
the number of regenerants essentially decreases.
The comparative analysis of data on structure and development of embryoids in Daucus
carota (produced in callus and suspension culture) has revealed its relative similarity to that of
zygotic embryo [35, 36].
Thus a considerable resemblance of sexual and somatic embryo development has been revealed
in the main morphogenetic regularities: polarity (bipolarity), symmetry (radial, bilateral,
dorsoventral), the pattern of cell divisions and histogen differentiation (formation of morphogenetic
Polymorphism of Sexual and Somatic Embryos
53
fields), morphogenetical and morphophysiological correlations and allometry. Same critical
stages are also exhibited in the development of sexual and somatic embryos: the laying down of
the first cell wall, protoderm formation, differentiation of organs, autonomy, etc.
The embryoidogenesis does not recapitulate zygotic embryogenesis only in the cases of
abnormal development (first divisions are irregular in the embryo, and the laws of embryogeny
are disobeyed, the protoderm formation is abnormal, the formation of root and shoot apices is
distorted, and so on).
3. Transitional Forms from Embryo to Bud (Fig. 9)
The analysis of literature and the original data on the development of sexual and somatic
embryos in natural conditions and in vitro has led us to conclude, that there are structures which
differ from typical sexual and somatic embryos and from bud by their morphology. In connection
with this we introduce now a term “transitional form” (in the terms of evolution) which means
a structure exhibiting traits of an embryo (for example, globular, heart- and torpedo-shaped
stages of development) and of a bud (formation of adventitious roots during regeneration) [5, 12,
37]. The embryos of Nelumbonaceae (Nelumbo nucifera), Ceratophyllaceae (Ceratophyllum
demersum), Poaceae, Orchidaceae and Orobanchaceae illustrate the possible ways of such transition.
A similar phenomenon can be observed among somatic embryos.
The embryos of Nelumbo (hydrophyte) and Ceratophyllum (hydatophyte) lack epiphysis and
hypophysis. In the embryo of Nelumbo the main root is substituted by the adventive roots during
germination. The latter originate at the base of plumule leaves at the later stages of embryogenesis.
However, the embryo of Nelumbo is bipolar from the very beginning of its development [38, 39].
The embryo of Ceratophyllum seems to be bipolar at the first stages of its genesis, as it
exhibits a group of cells which can be taken for initials of a main root. However, in the mature
embryo only a well developed plumule can be found, and no main root. As for the adventive
roots, they do not arise neither in the seed, nor in the seedling. The seedling of Ceratophyllum,
thus, lacks typical bipolarity [40, 41].
In the majority of Poaceae (xerophytes) the mature embryo exhibits well formed plumule and
developed adventive roots (their number depends on species, where and when the parental plant
grows). The main root had been transformed into coleorhiza in the course of evolution [42–45].
However, a number of investigators are of the opinion that the main root exists in the grasses
embryo. The embryo is bipolar and dorsoventral from the first stages of development, although
the question remains, whether the grasses embryo preserve the primary polarity or it is replaced
by a secondary polarity in the course of embryogenesis.
In Orchidaceae the shoot and root apices are not exhibited morphologically in the mature
embryo. Later during protocorm formation the bud and the adventive root arise and the secondary
polarity establishes [9, 46, 47].
The embryos of parasitic plants can serve as best model for investigation on reduction of
typical initials of epiphysis and hypophysis of shoot and root apices (Fig. 10). A considerable
peculiarity of its genesis is great variability of the first developmental stages. It is displayed in
the diverse contribution of ca and cb derivatives into the formation of embryo body. For example,
in Aeginetia indica even the first few divisions are irregular, so that the type of embryogenesis
can not be elucidated.
54 BATYGINA
MR
1. Capsella bursa-pastoris (Onagrad-type), mesophyte
(after Maheshwari, 1950)
2. Nelumbo nucifera (Asterad-type), hydrophyte
(after Titova, Batygina, 1996)
AR
AR
MR
3. Ceratophyllum demersum (Asterad-type),
hydatophyte (after Shamrov, Batygina, 1984, 1988)
AR
AR
4. Triticum aestivum (Graminad-type), xerophyte
(after Batygina, 1987)
AR
5. Dactylorhiza maculata (Onagrad-type) mycorrhizal plant
(after Batygina, Vasilyeva, 1983)
6. Aeginetia indica (Onagrad-type), parasitic plant
(after Teryokhin, Nikiticheva, 1981)
7. Agropyron repens (after Esau, 1969)
AR
Fig. 9. Parallellism of the first stages of morphogenesis in sexual and vegetative reproduction.
AR: adventive root, MR: main root [9, 11, 20, 39-41, 46, 52].
Xenoparasitism
Scr
oph
ula
ria
cea
sc
Cu
Fig. 10.
ro
Py
e
u
ea
tac
e
Orc
hid
ace
lac
eae
55
Alleloparasitism
Polymorphism of Sexual and Somatic Embryos
ae
Four main types of embryo reduction corresponding to four forms of parasitism in different
angiosperm families [48].
However, the structure of mature embryo provides evidence that embryogenesis in A. indica
as well as in other Orobanchaceae corresponds to onagrad-type. The differentiation of hypocotyl
and radicle initials is distorted to different extents in Orobanchaceae. Besides that, in holoparasitic
plants the protoderm formation is irregular in some parts of the embryo [46–48].
The analysis of sexual embryos in parasitic plants with diverse degree of reduction proves
that bipolarity can be observed from the very first stages of embryogenesis. But it vanishes in the
course of development and secondary polarity is established only during sprouting (in contact
with host-plant).
The comparison of morphology and embryology of the abovementioned sexual embryos
provides evidence to their transitional forms (because they exhibit morphological features of
sexual embryo and of bud). The whole complex of enumerated peculiarities can be interpreted
as an evidence of evolutionary tendencies towards a transition from normal sexual embryo to a
bud (or vice versa).
Such reproductive structures as propagules that arise on vegetative organs of plants (homophasic
reproduction) bear similarity to the abovementioned transitional forms (heterophasic reproduction).
In Bryophyllum, bipolar propagules arise on the leaf. They lack the initials and loci of hypophysis
and epiphysis. Globular-, heart- and torpedo-shaped stages are observed in their development
(and this brings them closer to somatic embryos). However, the absence of main root and the
development of adventive roots show that propagules are similar to the adventive bud. Morphogenesis
of these structures may be compared with that of sexual embryo in Poaceae [5] which also may
be considered as a transitional form.
The investigation of somatic embryogenesis of Triticum in vitro has revealed that some of the
obtained embryoids are the transitional forms. They have a well developed plumule, but the
morphogenesis of their adventive roots is distorted, if compared with sexual embryos [32].
Polymorphism of sexual and somatic embryos (in situ, in vitro) shows that morphogenetic
reorganisations may occur at the level of cell, organ and organism and they can affect different
stages of embryo and seedling development. At the most early stages of embryoidogenesis these
56 BATYGINA
reorganisations may be displayed as irregular divisions, with contribution of ca and cb derivatives
into embryo formation; at the middle stages, hypophysis and epiphysis may vanish and at the
later stages the embryo and even the seedling can lack the main or adventive roots. The embryo
differentiation as a whole can thus be reduced from the beginning of its development. In sexual
embryo the point of primary polarity establishment can shift as a result of adaptive evolution, or
the primary polarity can be substituted by secondary one during the seedling formation [37].
The development of sexual and somatic embryos (in situ and in vitro) obeys the laws of cell
division (Errera’s, Sachs’ and Hertwig’s) and the laws of embryogeny (Souèges’, Johansen’s).
However, with the variability of the first zygote division and of the subsequent stages of
embryogenesis (eight types of embryogenesis and more than 50 variations [7]), the existence of
transitional forms provide evidence that these laws could not be taken as absolute.
In every intitial cell, zygote, embryo, regardless of embryogenesis type and plant species, all
the genetic information is available that is responsible for cell division pattern in the proembryo
and for subsequent morphogenetic events. In the course of evolution a certain type of embryogenesis
had been determined for each taxon, but the initial morphogenetic potential of both sexual and
somatic embryos is usually realised under stress conditions (hybridisation, mutations, tissue
culture, etc.).
4. Conclusions
1. A sexual embryo, an embryoid and a bud are three elementary structural units of seed and
vegetative reproduction. A plant is able to form somatic embryos at all stages of its development
and on different organs (vegetative and generative), side by side with the sexual reproduction.
This ability enlarges the plasticity and tolerance of the reproductive system.
A high heterogeneity of seeds increases the adaptivity of a plant and of the whole population.
2. Sexual and somatic embryos formed in situ and in vitro reveal the great polymorphism.
However, the obvious uniformity of morphogenesis and of the main regularities of shoot and
root apex development appear in sexual and somatic embryos. During its development a plant
individual exhibits also formation of shoot and root apices. polarity, symmetry and so on,
whether it is the development of sexual embryo, embryoid, or regeneration of a bud.
3. The development of sexual and somatic embryos exhibits certain parallels, manifested in
the great polymorphism of these structures, which depends on their high adaptive abilities. The
only difference between them concerns the origin: zygotic embryo as a result of heterophasic
reproduction and embryoid of homophasic one. Transitional forms from one structure to another
can be revealed.
Somatic embryo does not stand comparison with zygotic embryo only in the case of
developmental abnormalities.
Sexual and somatic reproduction appear not to be strictly divided.
Acknowledgements
I would like to express my gratitude to Dr. valentina E. Vasilyaeva for valuable advises and to
Miss Elena Bragina for the help in illustrating and technical help in manuscript preparation.
The research was carried out with the support of Russian Foundation for Fundamental Research,
grants number 02–04–49807.
Polymorphism of Sexual and Somatic Embryos
57
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
5. Molecular Biology and Genetic Engineering of
Polyamines in Plants
M.V. Rajam, R. Kumria and S. Singh
Plant Polyamine and Transgenic Research Laboratory, Department of Genetics,
University of Delhi-South Campus, Benito Juarez Road, New Delhi 110021, India
Abstract: The involvement of polyamines in various cellular and metabolic processes has been
well established, but their mechanism of action and extent of involvement and regulation in various
responses is not clearly understood. The use of specific biosynthetic inhibitors as well as mutants
has been employed to study the intricacies of their regulation but many queries still remain unanswered.
The cloning of the genes of polyamine metabolism allowed for the generation of transgenic plants
with over-expression or down-regulation of a particular gene. These transgenic plants could be used
to study the effects on plant development, metabolic shifts as well as stress responses. Similarly, the
up- or down-regulation of the entire polymine metabolism is also possible by introduction of two
or more genes into plants which would provide greater insight into the mechanisms of polyamine
functions.
1.
Introduction
Polyamines (PAs) are naturally occurring polycationic aliphatic amines, which due to their
ubiquity and versatility are involved in the regulation of various cellular and molecular processes.
They are positively charged compounds with their charge distributed along the molecule. The
common PAs, spermidine (SPD) and spermine (SPM) and their diamine precursor putrescine
(PUT) play a critical role in the normal functioning of all cells [1]. They are involved in the
cellular functioning both at the molecular and physiological levels due to their association with
various macromolecules (DNA, RNA and proteins) and membranes as well as their high
concentration in the cytosol thus behaving as osmolytes [2]. The role of PAs is much better
studied in animal systems than plants, though they have been suggested to have a role as new
plant growth regulators either by mediating the plant hormone effects or independently signalling
other responses [3–5].
PAs exist in three forms in the cell, viz. as free cations, covalently bound to low molecular
weight phenolic compounds like hydroxycinnamic acids (conjugated form of PAs) and bound to
marcomolecules or membranes (bound form of PAs). Though the major form is the free cationic
form of PAs, there are instances when the amounts of conjugated form exceed the free form and
these are known to be critical in certain physiological processes including seed germination,
flower development, defence responses and stress reactions [2, 6–8]. Besides PUT, SPD and
SPM, there are certain unusual PAs found in nature, e.g. thermo-SPM which have been detected
in bacteria residing in hot springs and they seem to be important in protecting the enzymes from
heat denaturation [9–10] and aminobutylhomo-SPD found in fast growing cells of root nodule
bacteria Rhizobium [11]. NorSPD and norSPM are found in thermophilic red algae, brown algae,
Molecular Biology and Genetic Engineering of Polyamines in Plants 61
and Chlamydomonas, Nitella and Chlorella [12, 13]. Similarly, some unusual PAs have been
reported in plants, homo-SPD was first detected in sandalwood [14] and also in mosses and ferns
[12]. In leguminous plants, other unusual PAs like canavalmine, homoagmatine,
aminopropylcanavalmine and aminobutylcanavalmine have been detected [15, 16]. NorSPD and
NorSPM have been detected in alfalfa grown under drought conditions and have been postulated
to play a protective role under stress conditions [17]. As a matter of fact it has been suggested
that PA distribution, especially of SPM, may serve as a phylogenetic marker [12].
The study of plant PAs has come a long way since the first report by Bagni [18] regarding the
stimulatory effect of a PA (PUT) on growth of Helianthus tuberosus explants. Since then, PAs
have been demonstrated to be associated with regulation of somatic embryogenesis [19–22], root
and shoot formation [23–25], flower and fruit development [26], stress responses [2, 4, 5, 27]
and senescence [28–29]. In fact, PAs may serve as ‘biomarkers’ for in vitro morphogenetic
potential including plant regeneration via somatic embryogenesis [5, 29]. The multifaceted
functions of PAs as well as the variations in their levels in response to changes in the physiological
state, point towards their role as possible second messengers, though their high titres do not
support the view. Various studies have been conducted to investigate the involvement of PAs in
cell functioning, using mutants of PA biosynthetic genes and specific substrate-based inhibitors
of PAs. Though much information could be generated regarding the involvement and possible
mechanisms of action, no clear picture of their functioning emerged. Hence, transgenic plants
expressing PA biosynthetic genes in constitutive and regulated manner were generated, with an
aim to answer some of the queries regarding the functioning and role of PAs.
This article deals with the molecular biology of PAs in plants, with special reference to
transgenic plants expressing PA biosynthesis genes.
2.
Polyamine Metabolism
The diamine PUT is universally derived from ornithine by the rate limiting enzyme ornithine
decarboxylase (ODC). Plants, bacteria and some fungi have an alternate pathway for PUT
synthesis from arginine by arginine decarboxylase (ADC). In plants, ADC forms the major
pathway for PA biosynthesis. The PUT, hence derived from either of the two pathways is
converted into higher PAs, the triamine SPD and the tetraamine SPM by the addition of aminopropyl
groups obtained from decarboxylated S-adenosylmethionine (dcSAM). The dcSAM is formed
by the decarboxylation of SAM by SAMDC, SAM is in turn synthesized from methionine by
SAM synthase [2]. SAM is a major methyl donor in the cellular metabolism and also forms a
part of ethylene biosynthesis. SAM is converted to 1-aminocyclopropane-1-carboxylic acid
(ACC) by ACC synthase, which is converted to ethylene by ACC oxidase (Fig. 1).
The catabolism of PAs involves the enzymes diamine oxidase (DAO) and polyamine oxidase
(PAO). DAO preferentially acts on diamines (PUT, cadaverine) to form pyrroline, ammonia
(NH3) and hydrogen peroxide (H2O2), though it can break down SPD to aminopropyl pyrroline,
NH3 and H2O2 [3]. PAO, on the other hand, breaks down SPD and SPM to pyrroline and
aminopyrroline respectively, and diamine propane (DAP) and H2O2 [3]. Pyrroline formed due to
the activity of DAO and PAO is further converted to γ-aminobutyric acid (GABA) by a nicotinic
acid diamine (NAD) dependent dehydrogenase [30]. Besides oxidases there might exist alternate
pathways for PA catabolism as there are many plants in which oxidases have not been detected
[31]. Alternative diversion of the PAs into other metabolic pathways also plays a role in the
regulation and the dynamics of the PA metabolism.
62
RAJAM, KUMRIA AND SINGH
ADC
ARG
MET
Agm
NCP
ODC
SAM Synthase
ORN
PUT
SAMDC
SPD Synthase
APG
ACC
Synthase
dcSAM
SPD
SPM Synthase
SAM
ACC
ACC Oxidase
APG
Ethylene
SPM
Fruit Ripening
Senescence
Fig. 1. Metabolic and functional inter-relationships between polyamine and ethylene metabolism. Dotted
lines show inhibitory effecs of respective metabolism on the other, whereas dashed lines depict
stimulatory effects. ARG, arginine; ORN, Ornithine; MET, Methionine; PUT, Putrescine; SPD,
Spermidine; SPM, Sperimine; ODC, Ornithine decarboxylase; ADC, Arginine decarboxylase;
SAM, S-Adenosyl methionine; SAMDC, SAM decarboxylase; APG, Aminopropyl group, ACC,
Aminocyclopropylcarboxylic acid; Agm, Agmatine; NCP, N-Carbomyl putrescine.
PUT forms the precursor for pyrrolidine ring of the nicotine and tropane alkaloids. PUT is
converted to N-methyl PUT by PUT methyl transferase (PMT) which then forms the pyrrolidine
ring of nicotine and other tropane alkaloids [32].
3.
Role of Polyamines in Biological Processes
Most of the PA functions can be attributed to their polycationic nature and the distribution of the
charge along the molecule, which allows them to bind to a variety of molecules, including
nucleic acids, protein and cell membranes in the cell and regulate their functions. A very crucial
binding of PAs is with membrane phospholipids thus stabilizing them and reducing chlorophyll
loss when bound to the thylakoid membrane [29, 33], as well as preventing the lowering of
membrane potential and the Ca2+, PO 4–3 fluxes of mitochondrial membrane under saline stress
[34, 35]. Similarly, PAs have been found associated with cell wall components like lignin and
pectins [36] and have been implicated in maintaining cell wall characteristics by strengthening
the links between cell wall components [37]. PAs also play a role in cell wall expansion and are
part of modulators involved in host-pathogen interactions [38, 39]. The binding of SPD to the
plasma membrane proteins in zucchini hypocotyls has been characterized. SPD was found to
have a specific binding to a 44 and a 66 kDa protein [40].
The binding of PAs with DNA is also known to be responsible for playing some part in the
regulation of synthesis and function of DNA, including gene expression. It had been proposed
that PAs affect growth by interacting with DNA [41-43] and SPM plays a part in B to Z DNA
Molecular Biology and Genetic Engineering of Polyamines in Plants 63
transitions [43, 44]. SPM has been reported to stabilize the triplex DNA formation and aggregation
[45]. PAs have also been reported to stimulate DNA, RNA and protein synthesis [46]. They are
involved in joining of okazaki fragments and the depletion of PAs leads to accumulation of short
DNA pieces [47]. As a matter of fact odc has been suggested to be a proto-oncogene and its over
expression leads to cell transformation [46].
PAs are also known to bind with RNA molecules, and protect them from RNases [48]. SPD
and SPM are known to stimulate the reading of amber mutations and play an active role in the
expression of specific genes [49–50]. The antisenescence effects of PAs are in part due to the
inhibition of ribonuclease synthesis and activity by PAs [2, 51]. PAs also inhibit the protease
activity, thereby delaying the degradative processes initiated during stress or senescence [52].
PAs regulate their own biosynthesis by inducing a ribosomal frame shifting in the translation
of ODC antizyme [53]. Besides, being involved at the DNA and RNA levels, PAs are also known
to regulate protein synthesis and activity. SPM has been reported to have a specific role in
activity of cyclic AMP-independent caesin kinase [54, 55]. A branched quarternary PA, tetrakis
(3-aminopropyl) ammonium along with SPM has been reported to support protein synthesis at
high temperature in a thermophilic bacteria [56]. SPD stimulates protein synthesis in chloroplast
especially in light [57]. PUT, SPD, SPM and cadaverine enhanced the phosphorylation of several
plasma membrane proteins in tobacco, cucumber and Arabidopsis [58]. PUT has been reported
to increase the phosphorylation of many soluble proteins, unlike SPD and SPM, which decreased
the phosphorylation of soluble proteins [58]. PAs are known to regulate cell division and also
prolong the cell division phase by inhibiting the synthesis of phenylpropanoids. The conjugation
of PAs with phenolics regulated the free PA levels and therefore led to the cessation of cell
division [59].
The critical role played by PAs in growth and development along with their role in protein
phosphorylation/dephosphorylation and the binding of PAs (SPD) to specific protein in the thinlayer of tobacco together point towards the possibility of these being considered as signal
transduction molecules [60, 61]. Though the high PA titres in the cell are quite unlike secondary
messengers, which increase rapidly, and transiently in response to stimuli. Therefore, this aspect
of PAs warrants more attention to be able to pinpoint their role and mechanism of involvement,
if any, in signal transduction [58, 61, 62].
The study of physiology, biochemistry and genetics of the PA metabolism was initiated with
the generation of mutants of PA metabolism in E. coli, yeast and plants [63]. The E. coli mutants
defective in their PUT synthesis were the first to be isolated, these preferentially used the ODC
pathway in the absence of any PA supplements and the ADC pathway in the presence of arginine
[64, 65]. The substitution of PUT by its analogs which could not be converted into the higher
amines could not restore the growth of the mutants, whereas the inclusion of SPD analogs was
helpful, these results demonstrated the critical requirement of SPD for growth [66]. The E. coli
mutants defective in the SAMDC function were also isolated, but their growth rate was almost
unaffected. Further, an E. coli strain deficient in ADC, ODC and SAMDC function was isolated
which was able to grow at a reduced growth rate [67]. This strain was utilized in elucidating the
role of PAs in ribosomal complex formation and hence protein synthesis [68]. Mutant studies in
Saccharomyces cerevisiae revealed the presence of a single biosynthetic pathway (ODC) for the
synthesis of PUT as well as the absolute requirement of SPD and SPM for growth and sporulation
in yeast [69].
64
RAJAM, KUMRIA AND SINGH
PA mutants have also been raised in the model plants, Arabidopsis and tobacco [63]. The
Arabidopsis mutants with lower levels of ADC and ODC activities had abnormal root, shoot and
floral morphology [63]. Malmberg and McIndoo [70] have isolated tobacco mutants with abnormal
floral morphologies, including flowers with large non-functional stigma, anthers with nonviable pollen and ovary with most ovules turned into anthers. These mutants were deficient in
the function of SAMDC and brought out the role of SPD and SPM in flower development. The
ODC mutants failed to flower, demonstrating the involvement of ODC in floral initiation [71].
Tobacco mutants have been raised by activation T-DNA tagging, such that the regions adjacent
to the insertional position over-expressed the gene, and these were selected on selective concentration
of MGBG [72]. The mutated plants showed altered phenotypes, abnormal floral morphology,
male sterility and parthenocarpy, with higher SAMDC activity and SPD levels.
The study of PAs pertains much to the availability of specific, irreversible, substrate- or
product-based inhibitors of its biosynthetic enzymes [73]. The substrate analogue of ODC,
α-difluoromethylornithine (DFMO) was the first inhibitor to be synthesized [74]. Similarly αdifluoromethylarginine (DFMA) has been used as a potent inhibitor of ADC [75]. Another set of
similar substrate-based analogous for ODC and ADC were monofluoromethylornithine (MFMO)
and monofluoromethylarginine (MFMA), respectively. These have been reported to be much
more potent than DFMO and DFMA [73]. The substrate-based inhibitor for lysine decarboxylase,
α-difluoromethyllysine (DFML) is also available [73, 76]. A very potent inhibitor of the enzyme
SAMDC is methylgloxyl bis (guanylhydrazone) (MGBG), though it has been put to limited use
due to its non-specific effects on the respiratory enzymes [77]. The inhibitors have provided
insight into the dynamics of inter-conversion and regulation of the levels of PAs in the cell. Yet
no clear picture of their mechanism of action or extent, period or stage of involvement emerges.
The polycationic nature of PAs allows them to interact with various molecules whose functioning
is modulated and regulated by them. PAs also have a role in free radical scavenging due to their
polycationic nature [78]. Although there are many other suggested possible mechanisms regarding
the functions of PAs, the exact role, the extent of involvement and the mechanism of action of
PAs is as yet not very clearly understood.
However, the development of transgenics expressing PA biosynthetic genes driven by constitutive
promoters offered a good opportunity for the study of the PA functions. Since PAs are fundamental
to the process of morphogenesis, the generation of such transgenics presented a problem. Also
the transgenics recovered had abnormal phenotypes [61, 79–81]. Inducible promoters were
applied to overcome these problems but ambiguous effect of the inducer on the PA metabolism
and the problem of sustained induction remained as hurdles. The transgenics raised have been
used to study the role of PAs in plant development [79–80, 82] and also the dynamics of their
metabolism [83], but detailed studies were not conducted on the plant development and stress
responses of the transgenics over-expressing PA biosynthesis genes as well as their response
during in vitro morphogenesis. Therefore, much work is needed on the genetic manipulation of
PAs in plants to clearly demonstrate the role of PAs in a variety of cellular and molecular
processes.
4.
Cloning of Polyamine Metabolic Genes
The PA biosynthetic genes were first isolated from animal systems, yeast and bacteria. The
odc gene has been cloned from human, rat, mouse, holstein, Trypanosoma, Leishmania, yeast,
Molecular Biology and Genetic Engineering of Polyamines in Plants 65
Neurospora and E. coli [84–88]. Recently PA biosynthetic genes have been cloned from plants
too. The odc gene has been cloned from Datura, tobacco and tomato [89–91].
The adc gene has been cloned from oat, tomato, pea, Arabidopsis and soybean [92–96]. The
samdc gene has been cloned from Arabidopsis, Datura, potato [97], spinach [98], Catharanthus
roseus [99], Tritordeum [100], Pharbitis nil [101], tomato, tobacco [63], rice [102] and also from
human genome [103]. The spd syn gene has been cloned from N. sylvestris, Hyocyamus niger
and Arabidopsis [104]. Recently spm syn gene has been cloned from human genome [105].
The genes coding for the enzymes involved in formation of conjugated PAs have also been
cloned. The gene for the enzyme homo-SPD synthase has been cloned from bacteria (Acetobacter)
[106], Senecio vernalis [107] and Eupatorium cannavulgaris [108]. Besides, the biosynthetic
enzyme, the gene for the catabolic enzyme PAO has been cloned from maize [109] and DAO
from lentil [110] and pea [111]. The cloned PA metabolic genes are summarized in Table 1.
Table 1.
The polyamine metabolic genes cloned from different organisms
PA metabolic gene
Source
odc
E. coli, Trypanosoma, Leishmania, Datura, tomato, mouse, yeast and human
adc
Oat, pea, soybean and tomato
samdc
Potato, Arabidopsis, Spinach, Catharanthus, Tritordeum and Pharbitis nil
spd syn
Nicotiana sylvestris, Hyoscyamus niger and Arabidopsis
spm syn
Human
homo-spd syn
Acetobacter, Senecia vernalis and Eupatorium cannavulgaris
dao
Lentil and pea
pao
Maize
The plant decarboxylases (both the ADC and ODC) belong to the group IV of decarboxylases
and have the 23 amino acids which have been shown to be critical for the activity, conserved in
them [90]. There exists a strong homology between the oat, tomato and soybean adc genes,
further their catalytic motifs are more than 75% identical [93, 96]. The genes for related pathways
like, homo-SPD synthase and PUT-N-methyltransferase seem to be evolved from the genes of
the basic PA metabolism [108]. There are evidences for the presence of more than one copy of
adc or odc gene in the plant [90, 96].
5.
Transgenic Plants Expressing Polyamine Metabolic Genes
Transgenic plants expressing PA biosynthetic genes were generated to gain better understanding
of the PA metabolism, reconfirm the effects of the modulation of PA titres caused by the
inhibitors at the molecular level and also to overcome the limitations of the inhibitor-based
experiments. The transgenic approach was highly specific to the target gene and moreover it
provided a tool for manipulating the metabolic flux with the persistant shift in the PA metabolism.
Even though some of the plant PA biosynthesis genes have been isolated and characterised, most
transgenics have been raised using genes from heterologous source as these were the first to be
isolated. In most of the transgenics generated, CaMV35S promoter has been used to drive the
transgene, though tetracycline (tet)-inducible promoter has also been used in cases where extreme
66
RAJAM, KUMRIA AND SINGH
deleterious effects of the transgene were expected [2, 5]. Some of the transgenic plants expressing
PA biosynthesis genes are listed in Table 2.
Table 2.
Gene
odc
odc
adc
samdc
samdc-odc
dao
Transgenic plants expressing polyamine metabolic genes
Gene source
Yeast
Mouse
Mouse
Mouse
Mouse (antisense)
Oat
Oat
Human
Potato (sense)
Potato (antisense)
Human-Mouse
Pea (sense)
Pea (antisense)
Transgenic plant
Tobacco root cultures
Tobacco
Carrot
Rice
Rice*, Tobacco*
Tobacco
Rice
Tobacco
Potato
Potato
Tobacco*
Pea
Pea
*Unpublished data from our laboratory.
The first report of the introduction of yeast odc gene was in root cultures of tobacco using
Agrobacterium rhizogenes [112]. The study was aimed to increase the nicotine content of the
culture as PUT is a precursor for nicotine. Hence, over-production of PUT was attempted by
using a double enhancer sequence containing promoter but only a 3-fold increase in ODC
activity and a 2-fold increase in nicotine was observed. This was suggested to have been the
result of a tight regulation of nicotine biosynthesis or the activity of the enzyme PUT methyl
transferase becoming limiting. PUT might be incorporated into many other secondary metabolic
pathways, like alkaloids, which are present in significant amounts in Solanaceous plants and
also conjugation of PUT could have been another pathway for the PUT synthesised due to the
over-expression of the odc gene. No significant increase in SPD and SPM levels was observed
as their biogenesis is also precisely regulated. No plants were regenerated from the transformed
root cultures [112]. The tobacco transgenic plants expressing the mouse odc gene were the first
PA transgenic plants raised by DeScenzo and Minocha [79]. Two constructs were used for the
transformation of tobacco, one having complete coding sequence of odc and the other in which
350 bp of 3′ end were removed. The truncated gene produced a functional peptide 37 amino
acids less at the C terminus and an increased half-life. The enzyme activity when checked at the
pH optimum for mouse ODC was much higher in transgenics compared to endogenous plant
ODC activity in the controls, whereas at the pH optimum for the plant ODC, no significant
change was observed. PUT was found to be 2–3 fold higher in leaves and 4–12 fold higher in
callus, though no significant increase was observed in SPD and SPM content as the amounts of
SAM were suggested to be limiting. The transgenics having high PUT titres were stunted with
wrinkled leaves and reduced stamens.
Carrot cell lines are known to have no detectable ODC activity, only the ADC pathway is
functional in them. Carrot cell lines were transformed with mouse odc gene driven by CaMV35S
Molecular Biology and Genetic Engineering of Polyamines in Plants 67
promoter [83] and the effect of the high PUT titres on somatic embryogenesis was studied. The
transformed cell lines showed improved somatic embryogenesis which could be correlated to
higher PUT amounts. The somatic embryos were formed even in the presence of DFMA which
inhibited the carrot ADC, therefore all the PA requirements of the embryos were fulfilled by the
introduced mouse odc gene. Exogenous addition of PUT was not found to be helpful thus
suggesting that a fast turnover of PUT is also essential besides the high concentration for
somatic embryogenesis. These transformed carrot cell lines were used to study the shift in
metabolic flux as compared to the control cell lines [113]. 14C labelled arginine, ornithine,
methionine or PUT was fed to the cell cultures and amount of label incorporated in different PAs
and their fractions was analyzed. 14C labelled PUT was much higher in trasgenic cell lines when
14
C ornithine was given as substrate and there was no difference in labelled PAs when 14C
arginine was fed to the cultures. In correlation the conversion of 14C-methionine to ethylene was
much lower in transgenics due to a shift in the dynamics towards PA metabolism as more of PUT
was available to be converted to SPD and SPM [113].
Alterations in the PA levels during Agrobacterium-mediated genetic transformation with a
reporter gene (gus) and mouse odc gene were found to affect the regeneration potential of indica
rice [114]. It has been suggested that the modulation of PA metabolism may be used to improve
the regeneration from transformed calli in rice and other crops [114].
In a recent study, it was demonstrated that over-expression of human odc gene in transgenic
rice plants alters PA pools in a tissue-specific manner [115]. It was suggested that ODC rather
than ADC is responsible for the regulation of PUT synthesis in plants. In these transgenics,
significant changes in the levels of all three major PAs were observed in seeds and also in
vegetative tissues (leaves and roots) as compared to oat odc transgenics, wherein PUT and SPM
levels were higher in seeds only.
Transgenic tobacco plants over-expressing human samdc gene driven by CaMV35S promoter
were generated by Noh and Minocha [80]. These plants were found to have 2–6 fold higher SPD
than the untransformed controls, there was an increase in SPM too though PUT decreased. Since
high amounts of SPD is cytotoxic, the regenerants obtained might have been moderate accumulators
of SPD as the increase in SPD and SPM was not comparable to the dramatic decrease in PUT.
The cytotoxicity of the high amounts of SPD did not allow the regeneration of any plants overexpressing potato samdc in potato, hence a tet-inducible promoter was used to drive samdc. The
sense samdc plants showed 7-fold increase in SPD, 3-fold in SPM and a decrease in PUT on tetinduction. Similarly potato plants expressing antisense samdc gene driven by both 35S promoter
and tet-inducible promoter were raised [81]. The antisense samdc plants were stunted, branched,
necrotic with few small tubers; this was attributed to increase in ethylene levels caused by the
channelling of SAM for the formation of ethylene due to down-regulation of SAMDC. A decrease
in PUT levels was observed due to down-regulation of ODC and ADC by the elevated levels of
ethylene. Phenotypic abnormality, i.e. delay in flowering was also observed in case of tobacco
plants transformed with Agrobacterium rhizogenes and was attributed to a delay in the appearance
of conjugated PAs due to the decrease in ODC and ADC activities [116].
Bhatnagar et al. [117] studied the genetic manipulation of PA metabolism in poplar cells by
introducing mouse odc gene. It was observed that over-expression of the heterologous gene
resulted in high levels of PUT and increase in PUT degradation in the transgenic cells. In
continuation of the above study [118], they showed that there was an increased turnover of PUT
68
RAJAM, KUMRIA AND SINGH
as well as its conversion to SPD as compared to the untransformed cells, the increase being
proportionate to the cellular content of this diamine. Furthermore, the increase in PUT catabolism
in the transgenic cells did not result in any major changes in the activity of DAO or the half-life
of PUT [118].
The fact that elevated levels of PAs were detrimental for plant regeneration and were cytotoxic,
led to the use of tet-inducible promoter for driving oat adc gene introduced into tobacco [119].
The PUT levels were increased by 16-46% on tet-induction and more significant increase was
seen in the conjugated and bound fractions of PAs. A prolonged induction of the transgene at an
early stage of development led to plant growth inhibition, necrotic, wrinkled leaves, but no such
effects were seen in case of older plants on tet-induction of the transgene. These results clearly
brought forward the differential role of PAs at various developmental stages [119].
Rice transgenics over-expressing the oat adc gene have also been raised [82]. These transgenics
showed a 4-7 fold increase in the activity of the ADC enzyme, along with a 4-fold increase in
the PUT titres. The high PUT titres were found to be inhibitory to plant regeneration from the
transformed calli. The effect of the strength of the promotor driving the adc gene on the PA
metabolism as well as the morphogenic capacity of the transformed calli has also been analyzed
[120]. In this study, oat adc gene under the strong maize ubiquitin promoter 1 (ubi-1) was
introduced into rice but even then no significant change in PA levels was observed in seeds or
in the vegetative tissues. However, Noury et al. [121] reported that only one specific transgenic
line showed a significant increase in PUT and SPM levels in vegetative tissues and seeds. R1
generation rice transgenics expressing the oat adc gene driven by ABA responsive promoter
were tested for their response to various environmental stresses [122].
Since PAs are known to play a role in stress responses, particularly the activity of the enzyme
ADC is known to increase under stress along with increase in PUT levels, rice transgenics with
adc gene were tested for their tolerance to abiotic stress (drought) and it was reported that no
chlorophyll loss was observed after 8-days of drought as compared to the untransformed control
plants [82].
Tobacco transgenics over-expressing mouse odc gene affects cellular PAs and in vitro
morphogenesis, and confers salt stress tolerance [123]. Further, it was seen that favourable
changes in PAs titres and the optimum PUT: SPD ratio in transgenic lines showed better regeneration,
and previously similar results were reported in indica rice genotypes [124, 125].
Transgenic pea plants with PA catabolic gene dao in sense and antisense orientation have also
been generated in order to study the role of DAO in nodulation. The sense plants showed an
increased DAO activity and reduced PUT levels. It was observed that DAO activity was not
involved in nodule formation, but may have a role in regulation of PA levels in host cells [126].
Though the SPD synthase and the catabolic enzyme PAO genes have been isolated, no
transgenic plants have been raised with them, neither are there any transgenics reported expressing
the adc and odc genes in antisense orientation for studying the effects of long-term downregulation of these genes on plant development.
Transgenic plants of tobacco and/or rice were also generated in our laboratory with oat adc,
human samdc and spd syn, and the transgenics have been analysed for the effects on cellular PA
concentrations, PA biosynthetic enzyme activities, plant development and abiotic stress responses
[unpublished data]. The PA titres in adc and samdc tobacco transgenics were also analysed. In
case of adc transgenics a significant increase in the PUT and SPD levels with no apparent
Molecular Biology and Genetic Engineering of Polyamines in Plants 69
changes in SPM was observed. The increase in the PA levels was comparable to the concurrent
increase in the activity of ADC as well as SAMDC. The ODC activity was found to be decreased
in these transgenics. The activity of the PUT catabolic enzyme DAO was measured, and a higher
activity was found in all the transgenic lines tested, suggesting that the increase in DAO activity
may be important to maintain optimum PA levels in the cell [127]. In case of SAMDC transgenics,
in addition to an increase in SPD and SPM levels, there was a significant increase in PUT levels,
which might be due to the re-conversion of SPD to PUT via acetyl-SPD or γ-amino butyraldehyde.
These transgenics have exhibited very high SAMDC activity, which was accompanied by higher
DAO activity. They also showed marginal increase in ODC activity. Tobacco transgenics with
adc and samdc genes were also tested for stress responses. They showed increased tolerance to
salinity (250 mM NaCl) and PEG (10%) mediated drought. Interestingly, these transgenics also
showed enhanced resistance against fungal (caused by Verticillium dahliae and Fusarium oxysporum)
and bacterial (caused by Ralstonia solanecearum) wilts [127].
Some of the above single PA transgenics (e.g. odc transgenics) exhibited morphological
aberrations like stunted plants with wrinkled leaves, which might be due to altered PA levels and
PUT: SPD ratios. This problem may be overcome by up-gradation of the entire PA pathway in
transgenic plants by the simultaneous introduction of the PUT synthesis gene (i.e. odc) and SPD
synthesis gene (i.e. samdc or spd syn) by co-transformation. Indeed, such an attempt has been
made in our laboratory, and double transgenic tobacco plants were produced with mouse odc and
human samdc genes. It was observed that the double transgenic tobacco plants were normal and
did not show any morphological abnormalities. Further, regeneration was also better from the
transformed leaf explants with both odc and samdc genes as compared to explants from single
transformants. These results further substantiate the role of PUT : SPD ratio in in vitro plant
regeneration [124, 125]. Further, stress assays with double tobacco transgenics revealed increased
tolerance to salinity and bacterial wilt [127].
Since most of the phytopathogenic fungi have only the ODC pathway for the synthesis of
PAs, a novel method for the control of fungal plant infections by the selective inhibition of the
fungal ODC by using its specific inhibitor (DFMO) has been reported [1, 2, 128]. The selective
inhibition of the fungal ODC might also be achieved at the molecular level by the use of the
antisense RNA technology [2, 128]. The endoparasitic fungi which infect the plant might take up
the antisense odc transcripts produced in the transgenic plants along with the nutrients from the
plant cells, thereby leading to the inhibition of the fungal ODC and growth [2, 128]. The above
hypothesis was tested for the antisense odc transformed tobacco plants for the control of fungal
wilt caused by Verticillium dahliae. The transgenic lines tested showed increased resistance to
fungal infection as compared to the untransformed control plant. However, some more studies
would be needed to prove this hypothesis [129].
There seemed to be distinctive roles played by either of the PA biosynthetic enzymes, with the
ODC being critical in the early development of roots as the root development was affected
during the regeneration of the antisense odc transformed rice and tobacco plants. The high ODC
activity or perhaps the high PUT titres were on the other hand inhibitory for the early growth and
development probably because they might lead to an increase in DAO activity as PUT forms the
substrate for the enzyme, which results in the production of H2O2, higher concentrations of
which are cytotoxic. Even though there existed distinctive roles for either of the two enzymes,
it was observed in case of sense odc plants that the high titres of PUT could compensate for the
70
RAJAM, KUMRIA AND SINGH
decrease in the activity of the enzyme ADC, as was observed in the response of sense odc plants
to abiotic stress.
6.
Conclusions and Future Prospects
The PA transgenics have provided a lot of information regarding the long-term effects of the
shifts in the metabolism on plant growth and development, but they can be further used for
gathering more information regarding the effects of the variations in the PA metabolism on other
fundamental metabolic pathways in the cell. Such studies may provide an insight into the extent
of involvement of each metabolic pathway in a specific response or phenotype. Also, there
seems to be distinctive roles played by either of the PA biosynthetic enzymes, as can be deduced
from the variable phenotypes observed in the various transgenics; this suggests a possible role
for the improvement of modulation of the specific responses.
The transgenics also have the advantage of being distinctive from each other due to the
random integration of the transgene, which influences the expression of the transgene and hence
the shifts in the physiology in each plant were different. Therefore, an array of different transgenic
lines each showing variability in the expression of transgene that were generated, could provide
information regarding the physiological effects of variable gene expression. A clearer picture
would emerge for these transgenics with the analysis of the fate of the PAs accumulated in these
plants, as the PA catabolic enzymes as well as their channelling into the other pathways like
ethylene and tropane alkaloids etc. also play a role in the metabolic flux of the cell. Besides the
PA metabolism, the variations in the dynamics of other related metabolic pathways and their
cumulative effect on the phenotype and physiological responses of the transgenics would give
a better view of the cross-talk amongst pathways and its role in the functioning of the cell.
Also, the PA transgenics raised need to be tested for their tolerance to various stresses to
elucidate the role played by the transgenics in stress responses and also the degree of tolerance
imparted by them. These transgenics may also be used for studying various other biological
processes, including senescence and fruit ripening. The use of the PA biosynthesis genes to
generate stress tolerant plants has a major hurdle of the plants having an abnormal phenotype.
Therefore a better approach to the problem would be to up-grade the entire metabolism (by the
introduction of the odc/adc gene in conjunction with SPD and SPM synthesis gene samdc/spd
syn) instead of a singular step, so that the plants would possess a normal phenotype and yet be
tolerant to stresses. In fact, this approach was examined in our laboratory and proved to be
correct. Further, it has been observed that the optimum PUT : SPD ratio is very important for
normal plant growth and development [124, 125]. Therefore, the maintenance of a balance
between PUT and SPD ratio appears to be very important for obtaining normal transgenics. This
may be achieved by transforming the plants with the genes for both PUT and SPD synthesis. We
were able to produce a large number of normal tobacco transgenic plants with mouse odc and
human samdc genes by co-transformation as well as step-wise transformation. Such an approach
would be quite useful in producing normal transgenic plants with PA biosynthesis genes and they
would also impart abiotic stress tolerance.
Acknowledgements
The research work in our laboratory has been generously supported by the grants from the
Department of Biotechnology (Grant No. BT/R & D/08/40/96), Department of Science and
Molecular Biology and Genetic Engineering of Polyamines in Plants 71
Technology (Grant No. SP/SO/AO6/96), and the Indian Council of Agricultural Research [Grant
No. F-1 (21)/96-FFC], New Delhi to MVR. Award of Senior Research Fellowship from the
University Grants Commission, New Delhi to RK and Junior Research Fellowship from Council
of Scientific and Industrial Research, New Delhi to SS is gratefully acknowledged. We are also
thankful to Mr. Amit Arora for his excellent secretarial assistance.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
6. Biotechnological Approaches Towards Improvement of
Medicinal Plants
Alka Narula, Sanjeev Kumar*, K.C. Bansal** and P.S. Srivastava
Centre for Biotechnology, Faculty of Science, Hamdard University, New Delhi 110 062, India
*Amity Institute of Biotechnology, Amity IT University Campus, Sector 125, Noida 201 303, India
**NRC on Plant Biotechnology, IARI, New Delhi 110012, India
Abstract: Herbs are now in great demand in both developed and developing countries because of
their proven efficacy and little or no reported side effects. Secondary metabolites, the active
principles expensive to produce and accumulate are usually biosynthesized in smaller quantities.
This has resulted in ruthless exploitation of medicinally important plants creating imbalance in
supply and demand. An alternative technology could be the application of in vitro culture of
desirable medicinal plants to increase the plantation propagules and enhance the yield of specific
drug components. Successful micropropagation protocols for various medicinal plants have been
developed and their conservation has also become feasible through synthetic seeds and cryopreservation
technologies. Besides other techniques, genetic engineering of medicinal plants using Agrobacteriummediated transformation has many advantages that include fast growth and high level of stable
production of secondary metabolites making them commercially and economically feasible. Genetic
fidelity of tissue culture raised plants can be ascertained by using molecular markers.
1.
Introduction
Medicinal plants have been the subject of man’s curiosity and purpose since time immemorial.
The importance of medicinal plants in the treatment of chronic diseases needs no elaboration. In
fact, even with the tremendous advancement in the field of synthetic chemistry, almost 50% of
the commercial drugs available in the market remain of plant origin. The herbal system was,
however, pushed to the background with the advent of allopathic system. It is now back with a
venegence and the age-old system of herbal medicine is being revived due to its long lasting
curative effect, easy availability, natural way of healing and rare or no reported side effects. Due
to growing world population, increased anthropogenic activities, rapidly eroding natural ecosystem
etc., the natural habitat for a great number of plants are dwindling and many of them are facing
extinction [1]. The inevitable ruthless exploitation of herbs leading to their rapid depletion from
the wild is a cause for concern. In fact, the pace of depletion has outpaced the pace of conservation.
New strategies are being therefore formulated for rapid multiplication and conservation of
medicinal plants. Besides the conventional methods, biotechnology has proved useful in the
improvement of herbs that yield drugs. In this resurgent era of herbal drugs it is very difficult to
make an accurate assessment of the volume and value of herbal trade in India. Consequently, it
varies widely [2]. According to estimates by the Ayurvedic Drug Manufacturers Association
(ADMA), the current value of trade in Indian System of Medicine (chiefly Ayurveda, Siddha and
Unani) and Homeopathy is around Rs. 4205 crores, roughly close to US$ 1 billion [see 3].
Therefore, the value of medicinal plants is also reflected in the economics of global market
Biotechnological Approaches Towards Improvement of Medicinal Plants 79
which was estimated to be 60 billion US dollars in 2000 (Fig. 1). The Asian region rich in
biological wealth and genetic diversity also has a substantial share in herb trade (Fig. 2).
100 (Thailand)
4 (India)
220 (China)
Fig. 1. The market value of natural health products in 2000 was already worth US $ 500 billion of
which 220 was projected for China, 100 for Thailand and only 4 billion for India.
6 (Other
countries)
3 (Germany)
1.6 (France)
0.6 (Italy)
1 (India)
10 (Rest of Europe)
4 (USA)
Fig. 2. In 2000, country-wise share of herbal medicine was US $ 60 billions with India’s share of
only 1 billion. The predicted annual growth rate was 7% for this sector.
It is surmountable that medicinal plant biotechnology has grown from cell technology, specifically
plant tissue culture. Regeneration of plants has been achieved with cells and tissues excised from
various medicinal herbs. The powerful techniques of plant cell and tissue culture, and recombinant
DNA and bioprocessing technologies etc., coupled with sophisticated analytical tools such as
NMR, HPLC, GC-MS, LC-MS etc., have offered mankind a great opportunity to exploit the
totipotent, biosynthetic and biotransformation capabilities of plant cells under in vitro conditions.
The scope for in vitro germplasm preservation and large-scale production of plant secondary
metabolites has brightened.
Advantages of extracting secondary metabolites using plant tissue culture are:
1. The source of these metabolites, i.e., most of higher plants have specific agroclimatic
80
ALKA NARULA ET AL
requirements. Hence specific metabolites can be produced in cultures all through the year
even in places where these crops are not grown.
2. The already limited supply of these raw materials can not be exhausted considering the
future needs.
3. If not in all, at least in remarkable number of cases cells under culture tend to produce
greater amounts of these metabolites than that is accumulated in nature.
In addition, in vitro technology also facilitates: (i) conservation of genetic diversity and germplasm
of medicinal plants through cryopreservation, and (ii) gene transfer through recombinant DNA
technology and the molecular markers in the form of AFLP and RAPD.
In this article, emphasis has been laid on the fact that protection and preservation of germplasm
of medicinal plants is indispensible, without which the knowledge of herbal medicines will
remain futile. Also, various ways of enhancing the yield of active components are reviewed.
2.
Materials and Methods
2.1
Micropropagation
Juvenile Explants: Seeds of the desired plant species are washed with 0.5–2.0% cetrimide
followed by treatment with 0.1% mercuric chloride and dipped in 70% alcohol, thereafter
washed with sterile distilled water. Such sterilized seeds are implanted on basal medium for
germination. Various seedling explants such as hypocotyl, epicotyl, cotyledon and radicle are
incoulated on suitable media with growth regulators.
Mature Explants: Explants such as stem segment, shoot apex, axillary buds, leaf, root, anther,
etc., from field grown plants are surface sterilized with 1–2% cetrimide followed by treatment
with streptomycin sulphate and bavistin solution. They may then be treated with 0.1% mercuric
chloride, 70% alcohol and finally washed with sterile distilled water. After sequential sterilization,
explants are implanted on media with auxin and cytokinin in appropriate combination and
concentration.
Most of the cultures are maintained in a culture room at 25 ± 2°C with 55 ± 5% relative
humidity and 10–14 hr light/dark period with irradiance of 60–100 µmol m–2s–1 provided by
white cool flourescent tubes. The cultures are monitored at regular intervals and the regenerants
are maintained on the best suited medium. The rooted plantlets are hardened and then transplanted
to pots and finally transferred to field.
2.2 Secondary Metabolite Analysis
Cultures harvested during different stages of growth and differentiation are analysed for the
presence of secondary metabolites (alkaloids, steroids, flavonoids, glycosides, furanocoumarins,
etc.). Quantification of isolated compounds is made either through spectrophotometry, High
Performance Liquid Chromatography (HPLC) or Gas Liquid Chromatography (GLC). Stage
showing highest yield of the active principle is selected as the harvesting stage for that particular
culture.
As they are present in low amounts in plants, attempts can be made to enhance the yield by
Biotechnological Approaches Towards Improvement of Medicinal Plants 81
supplementing the medium with elicitors, precursors or manipulating the hormonal combination
of the medium or subjecting biotic/abiotic stress to the cultures.
2.3 Cryopreservation of Cultures
Various explants have been used for cryopreservation of medicinally important plants. The
general protocol involves treating the cultures with appropriate cryoprotectant such as DMSO,
glycerol, sucrose or proline, etc. and then plunging in liquid nitrogen (–196°C). After freezestorage, the cultures are thawed at 35–40°C, washed and recultured. Complete plantlets can be
regenerated from such frozen cultures.
2.4 Synthetic Seeds
This technology involves the encapsulation of propagules (somatic embryos/axillary buds/shoot
apices, etc.) which functionally mimic seeds and can develop into plantlets under suitable
conditions. For encapsulation, the propagules may be embedded in a matrix that serves as
endosperm, containing carbon source, nutrients, growth regulators and antimicrobial agents.
Sodium alginate is commonly used. However, there are several other agents including guargum,
calcium alginate, gelrite, sodium alginate with gelatin, potassium alginate, sodium pectate, etc.
In addition, polyethylene oxide homopolymers, synthetic sodium-magnesium-lithium silicate,
sodium crylate, etc. have been tried as coating agents. After mixing in the encapsulation matrix,
the propagules are picked up by pipette and then dropped into a solution of calcium chloride.
Thereafter, they are kept undisturbed for surface complexing to obtain encapsulated beads. The
beads are kept in a solution of 2.5% CaCl2 for 40 min on a shaker. After the completion of
incubation period, the beads are recovered by decanting the CaCl2 solution and washed 3–4
times with basal medium. Such encapsulated propagules cultured on nutrient medium or different
substrates like filter paper, soilrite, etc. can develop into plants.
2.5 Transformation
In recent years, Agrobacterium-mediated transformation has emerged as an efficient method for
genetic manipulation of plants. Although direct DNA transfer methods, particularly particle
bombardment, are also being employed, other gene transfer methods include electroporation and
electrophoresis, laser microbeam technique, microinjection, liposome fusion and injection.
Agrobacterium-mediated transformation, however, has major advantages over these systems.
After establishing a reliable protocol for micropropagation the explants can be incubated with
Agrobacterium suspension, blotted dry on whatman filter paper and transferred to regeneration
medium for co-cultivation. The co-cultivated explants are then transferred to the selection medium.
After selection, the explants are allowed to grow on regeneration medium + Cefotaxime. The
putative transgenics can be rooted and after hardening transferred to field. The transgenic nature
of regenerated plants can be confirmed by polymerase chain reaction (PCR) and southern blot
analysis.
2.6 Molecular Markers for Ascertaining Clonal Fidelity
DNA-based markers provide an efficient tool for screening tissue culture raised plants because
these markers are not affected by environmental factors and present more reliable results. PCR-
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ALKA NARULA ET AL
based markers such as RAPD have been used for detecting off-types from micropropagated
plants. AFLP, and SNPs are now preferred as it combines the reliability of RFLP with RAPD.
For AFLP analysis, total genomic DNA can be isolated from desired plant parts by using a
suitable method. It can then be restricted with restriction enzymes followed by ligation with
specific adapters. Pre-amplification of the adapter-ligated DNA can be done by using selective
nucleotides. The samples are electrophoresed on acrylamide gel and autoradiographed. AFLP
amplification products are scored for their presence and absence across the individuals tested.
Genetic similarity between pairs is estimated by Jaccard’s coefficient. The phenetic dendrogram
can be constructed by UPGMA (unweighed pair group method of arithmetic averages) in order
to group individuals into discrete clusters.
3.
Results and Discussion
3.1 Micropropagation of Medicinal Plants and Yield of Secondary Metabolites
In vitro cultured cells and tissues can be induced to differentiate into plants through organogenesis
[4-8] (Figs. 3 to 8) or somatic embryogenesis [9]. The response of any tissue in vitro is attributed
to the composition of the medium besides other factors including a balance between growth
regulators [10]. MS medium originally developed for rapid growth of tobacco tissue culture is
the most frequently used for majority of the species. Already there are credible reports of in vitro
propagation of medicinal plants by using various explants, such as leaf [11], stem [12,
13], shoot buds [14, 15] anthers [16], roots [17], shoot tips, nodal segments [18, 19], and seedlings
[4, 20, 21] (Table 1).
The earliest detailed reference of plant cell cultures as an industrial route to natural product
synthesis dates back to 1956. Despite the success and the related surge in information, the
expected progress during the following decades remained slow. After 1973, a turning point in
cell culture technology demonstrated reasonable yield of desired secondary metabolites [16, 22].
Earlier it was believed that enhancement in the yield of secondary metabolites was dependent on
prolonged tissue cultures or organogenesis [23]. Subsequently, it has been revealed that the
ability of product biosynthesis continues throughout during the culture regime and can be detected
at various stages of growth and differentiation [24].
Yield of secondary metabolites can be enhanced by modifying the chemical milieu and
culture conditions (Table 2). Zenk et al. [25] observed that the composition of culture medium
not only affects growth and production of metabolites but also plays a critical role in initiation
of morphogenic events in the culture. Consequently, almost all the major components of the
growth medium have been tested for their varied effects on different types of differentiated and
undifferentiated cultures [26].
The most commonly used carbon source for tissue culture media is sucrose. The other carbon
sources tested for supporting growth include glucose, galactose and also complex carbohydrates
such as milkwhey and molasses. Increased sugar concentration favoured synthesis of shikonin
in cell cultures of Lithospermum erythrorhizon, diosgenin production in Dioscorea, and
anthraquinone in cell cultures of Gallium mollugo. On the contrary, lesser amounts of sucrose
favoured the production of ubiquinone 10 in Coleus blumei [see 27]. Saccharose as sugar source
has shown strongest effect for secondary metabolite content increase in cultures of Catharanthus,
Biotechnological Approaches Towards Improvement of Medicinal Plants 83
B
A
C
D
Fig. 3. Artemisia annua. Stem segments cultured on MS + (in mg l–1) NAA (0.1) + BAP (3.0) + GA3 (0.1)
+ Asp (50) + Arg (50) + Glu (100) + Cyst hyd (5.0). (A) Multiple shoots in 10-week-old culture;
(B) Further growth of A, after 15 weeks; (C) Closer view of B and (D) Plants at preflowering
stage.
84
ALKA NARULA ET AL
A
B
C
D
Fig. 4. Bacopa monniera. Nodal segments cultured on MS + (in mg l–1) NAA (0.1) + BAP (0.5) + CH (500).
(A) Multiple shoots after 6-weeks; (B) Plantlets, 8-week-old; (C) Closer view of B and (D)
10-week-old plantlets.
Biotechnological Approaches Towards Improvement of Medicinal Plants 85
A
B
Fig. 5. Crocus sativus. (A) Callus and direct root differentiation from bulb scale and
(B) emergence of multiple shoot buds from cultured scales.
Nicotiana, Chenopodium, Thalictrum, Dioscorea, and Rhamnus in tandem with the concentration
supplied, and with a parallel increase in dry weight [28].
Higher concentrations of phosphate results in an increase in the production of indole alkaloids
in Catharanthus roseus, whereas in callus cultures of Peganum, low phosphate levels stimulate
the secondary metabolism [see 29]. Transfer of suspension cultures of Thuja occidentalis from
86
ALKA NARULA ET AL
A
B
C
Fig. 6. Dioscorea bulbifera. Culture of nodal segments on MS + (in mg l–1) IAA (0.1) +
Kn (5.0) + CH (500). (A) 24-week-old regenerants; (B) Aerial bulbils on regenerants
after 16 weeks and (C) In vitro formed tubers.
Biotechnological Approaches Towards Improvement of Medicinal Plants 87
A
B
C
Fig. 7. Pluchea lanceolata. Culture of leaf explant on WB (2%) + Kn (5.0 mg l–1). (A) Multiple shoots
after 12 weeks; (B) Close up of a shoot and (C) Transplanted plantlets in soil: soilrite (1:1).
A
B
C
Fig. 8. Thevetia neriifolia. (A) Regenerating callus from juvenile leaves cultured on MS + (in mg l–1) IAA
(0.5) + BAP (1.0); (B) Regeneration of shoot from callus on MS + (in mg l–1) IBA (0.1) + BAP (2.0)
and (C) Growth of isolated shoot with callus at the base on MS + (in mg l–1) IBA (0.1) + BAP (0.5).
+
MS to B5 medium induced the synthesis of terpenoids. Both, different NH 4 content and the
stress due to transfer to specific media, seemed responsible for accelerated shikonin production
in suspension cultures of Lithospermum erythrorhizon [30]. The type and amount of N source
seems to affect the yield of secondary products. The ratio of nitrate and ammonia in the culture
Therapeutic use
Abortifacient
Leucoderma
Antimalarial,
anti-HIV
Tuberculosis, nerve
defects, cold, cough
Anti-inflammatory
Antispasmodic,
narcotic, analgesic,
antiasthamatic
Memory vitalizer
Antileukaemic
Plant
Abrus precatorius
Ammi majus
Artemisia annua
Allium wallichii
Arnica montana
Atropa acuminata
Bacopa monniera
Catharanthus roseus
Table 1.
Juvenile explants
and mature stem
segments
Nodal segments
MS + NAA (2.0) + BAP (5.0) +
Asp (100) + CH (1000)
MS + NAA (0.2) + BAP (5.0)
+ CH (500)
MS + NAA (0.1) + BAP (0.5)
+ CH (500)
RT + IAA (1.0)
RT + IBA (1.0)
Isolated shoots
Callus
Callus, multiple
shoots
Plantlets
Elongated shoots
Plantlets
Shoot buds
Plantlets
MS + (in µM) NAA (5.3) + 2iP
(5.0) + Phloroglucinol
(0.6 mM) + Ad (0.2 mM)
MS + BAP (1.0) + IBA (1.0)
Multiple shoots
Multiple shoots
Multiple shoots
In vitro flowering
and fruiting
Plantlets
Multiple shoots
Plantlets
Plantlets
Response
MS + Zt (20 µM)
MS + (in µM) NAA (0.5) + BAP
(13.0) + GA3 (0.3) + Glu(700) +
Asp (300) + Arg (300) + Cyst HCl (30)
MS + (in µM) NAA (1.0)
+ BAP (13) + CM (2 %)
MS + (in µM) NAA (0.5)
+ BAP (13.0)
MS + IAA (0.5) + Kn (2.0)
+ CH (1000)
MS + IAA (0.5) + Kn (5.0)
+ CH (500) + Ad (40)
MS + IBA (0.2) + Glu (100)
MS + NAA (0.1) + BAP (0.5)
MS + NAA (0.1) + Kn (0.5)
Medium*
Shoot tips, nodal
segments
Nodal segments
Seedlings (without
the root portion)
Cotyledonary
leaves
Stem segments
Hypocotyl
segments
Immature
inflorescence
segments
Isolated shoots
Nodal segments
Epicotyl segments
Cultured explant
Examples of micropropagation of some medicinal plants
[13]
[6]
[139]
[138]
[137]
[7]
[136]
[20]
Reference
88
ALKA NARULA ET AL
Substitute for
quinine
Anti-inflammatory
Anticholinergic
Antifertility
Dysentry, colic pain
Source of cardiac
glycosides
Antitumor
Abortifacient
Antidiabetic and
used against
hepatitis B virus
Laxative
Clerodendrum
inerme
Coleus forskohlii
Datura innoxia
Dioscorea bulbifera
Holarrhena
antidysentrica
Isoplexis canariensis
Nothapodytes
foetida
Peganum harmala
Phyllanthus
caroliniensis
Plantago ovata
Multiple shoots
Plantlets
Multiple shoots
MS + BAP (15 µM)
MS + IBA (35 µM)
MS + BAP (5.0 µM)
MS + (in µM) BAP (2.25)
+ IAA (0.17)
MS + (in µM) BAP (0.22)
+ IAA (0.17)
Plantlets
Multiple shoots
Plantlets
Multiple shoots
Plantlets
Callus
Somatic
embryogenesis
MS (1/2)) + (in µM) BAP (2.22)
+ IBA (0.49)
MS + (in µM) NAA (0.1) + BAP (5.0)
MS + IBA (8.0 µM)
MS + (in µM) BAP (5.0)
+ Kn (1.25-5.0)
MS
MS + (in µM) 2,4-D (4.5) + Kn (2.3)
MS + (in µM) BAP (4.4) + NAA (2.7)
+ CM (10 %)
Nodal segments
Isolated shoots
Shoot buds
Callus
Cotyledonary node
with shoot tip
Leaf segments
Multiple shoots
Plantlets
Plantlets
Plantlets
MS + IAA (0.1) + Kn (5.0 ) + CH
(500) + Charcoal (2000)
WB + NAA (1.0) + BAP (2.0)
+ CH (500)
WB + IBA (0.1)
Multiple shoots
Plantlets
MS + (in µM) IAA (0.57) + Kn (0.46)
MS + IAA (2.0)
Callus, multiple
shoots
Plantlets
Multiple shoots
MS + BAP (2.0) + NAA (0.5)
MS + NAA (0.1) + BAP (5.0) +
Zt (1.0) + Asp (100) + Glu (100)
MS +TDZ (1.36 µM)
Isolated shoots
Seedling explants
Node with axillary
buds
Nodal segments
Anthers
Shoot tips
Leaf segments
Callus
(Contd)
[147]
[146]
[145]
[144]
[21]
[143]
[142]
[16]
[141]
[140]
Biotechnological Approaches Towards Improvement of Medicinal Plants 89
Therapeutic use
Anticancerous
Leprosy, skin
diseases
Psoriasis
Cardiovascular
diseases, sedative
or tranquillizer
Abortifacient,
diuretic
Antidiabetic
Antihepatotoxic
Steroidal drugs
Asthma, bronchitis
anti-tumorous
Anticancerous
Tranquillizer
Plant
Plumbago rosea
P. zeylanica
Psoralea corylifolia
Rauwolfia
serpentina
Sterculia foetida
Stevia rebaudiana
Silybum marianum
Solanum khasianum
Tylophora indica
Typhonium
flagelliforme
Valeriana jatamansi
Shoots buds
Shoot buds
(from rhizome)
Leaf segments
Callus
Somatic embryos
Leaf segments
Callus
Shoots
Nodal segments
Nodal segments
[14]
MS + BAP (4.44 µM)
MS + (in µM) NAA (4.03)
+ BAP (4.44)
Multiple shoots
Plantlets
[8]
MS + (in µM) IBA (2.46) + BAP (1.33) Plantlets
[155]
[156]
Callus
Multiple shoots
Plantlets
[154]
[153]
[152]
[151]
[150]
[149]
[148]
Reference
MS + (in µM) 2,4–D (9.04) + Kn (0.05) Callus
MS + 2iP (9.84 µM)
Somatic embryos
MS
Plantlets
MS + 2,4- D (3.0) + Kn (1.0)
MS + BAP (3.0)
MS + NAA (2.0)
Multiple shoots
Plantlets
Plantlets
Multiple shoots
MS + (in µM) BAP (8.87)
+ IAA (5.71)
MS (1/2) + IBA (4.90 µM)
MS + IAA (0.1) + Kn (5.0)
MS + NAA (0.1) + Zt (0.5)
Multiple shoots
Plantlets
Multiple shoots
Plantlets
Multiple shoots
MS + BAP (4.0)
MS + IAA (2.0)
MS + BAP (1.0) + NAA (0.1)
MS + NAA (2.0) + BAP (1.5)
Nodal segments
Cotyledonary nodes
MS + BAP (2.0)
Multiple shoots
Plantlets
Multiple shoots
Plantlets
MS + (in µM) Ad (27.2) + IBA (2.46)
MS + IBA (4.92 µM)
MS + BAP (0.5)
MS + IAA (5.7)
Plantlets
Multiple shoots
Response
MS + IAA or IBA (0.1) + BAP (1.5)
+ Ad (50)
MS (1/2) + IBA (0.25)
Medium*
(Contd)
Shoot apices
Nodal segments
Nodal segments
Nodal segments
Cultured explant
Table 1.
90
ALKA NARULA ET AL
Antistress,
antitumor,
anti-inflammatory
Withania somnifera
Multiple shoots
Plantlets
MS + IBA (10.0)
Multiple shoots and
flowering
Flower maturation,
plantlets
MS + BAP (1.0)
MS + NAA (0.5) + BAP (0.1)
Shoots with
immature flowers
Leaf segments
MS + NAA (0.1) + BAP (2.0)
Nodal segments
[158]
[157]
MS = Murashige and Skoog medium, WB = Wood and Braun medium, RT = Revised Tobacco medium (Khanna and Staba, 1968)
Ad = Adenine, Arg = Arginine, Asp = Asparagine, BAP = Benzylamino purine, CH = Casein hydrolysate, CM = Coconut milk,
2,4-D = 2,4-dichlorophenoxyacetic acid, Glu = Glutamine, IAA = Indole-3-acetic acid, IBA = Indole-3-butyric acid, 2iP = 2 iso-pentenyladenine,
Kn = Kinetin, NAA = α−naphthaleneacetic acid, TDZ = Thidiazuron, Zt = Zeatin.
*Concentration of growth hormones are in mgl–1 unless mentioned otherwise.
Anticancerous
Vitex negundo
Biotechnological Approaches Towards Improvement of Medicinal Plants 91
92
ALKA NARULA ET AL
Table 2.
Effect of stage of culture and conditions on the yield of some secondary metabolites
Plant
Active constituent
Culture conditions and yield of secondary metabolite Reference
Agaveamaniensis
Sapogenin steroid
Absence of calcium ions in media increased the
sapogenin steriod content, while relatively high
concentration of Mg, Co and Cu showed inhibitory
effects
[35]
Ammi majus
Xanthotoxin
Xanthotoxin content monitored during different stage
of growth and differentiation revealed highest content
at plantlet differentiation stage bearing immature green
fruits (in vitro)
[136]
Artemisia annua
Artemisinin
Enhanced artemisinin content was found in vitro
[7]
Beta vulgaris
Betalains
B5 medium supplemented with Co (5 µM) increased
the betalains production
[36]
Catharanthus
roseus
Catharanthine
and vindoline
Multiple shoot cultures raised directly from sterile
seedlings inoculated on MS medium containing BA
(4 µM) produced mainly catharanthine and vindoline
in amounts higher than in the parent plant
Zt or BA were more active than Kn in increasing the
content of alkaloids. But all the three cytokinins
enhanced the production of indole alkaloids
High degree of differentiation was correlative to the
increased vinblastine production
[159]
Indole alkaloids
(ajmalicine,
serpentine)
Vinblastine
2+
[160]
[13]
Cinchona
ledgeriana
Quinine
Shoot cultures contained much higher levels of quinine
and related alkaloids than the cell suspensions
[161]
Datura
stramonium
Hyoscyamine and
scopolamine
Maximum contents were found in the stem and leaves
of young plants; hyoscyamine being always the
predominant component
[162]
Daucus carota
Anthocyanins
Increase of 63.41% in production of anthocyanins by
addition of 1.0 nM Co2+
[163]
Digitalis lanata
Digitoxin
Addition of Mn2+ (10 mM) at day zero of culture
increased the digitoxin content
[164]
Dioscorea
deltoidea
Diosgenin
Hypocotyl callus on RT+2,4-D (0.1)+ Cholesterol
(10–100) +YE (0.5%) yielded higher diosgenin content
[see 142]
Ephedra andina
E. distachya
E. equisitina
E. fragilis
E. gerardiana
E. intermedia
E. major
E. minima
E. saxatilis
Alkaloids
(1-ephedrine and
d-pseudoephedrine)
Trace quantities of alkaloids were present in cultures.
The ability to produce alkaloids diminished to zero
with successive subcultures
[165]
Lepidine content was much higher in 8-month-old
regenerants grown on ZnSO4 (900 µM) or CuSO4
(100 µM)
[37]
Lepidium sativum
Lepidine
Biotechnological Approaches Towards Improvement of Medicinal Plants 93
Papaver
bracteatum
de-sanguinarine
Increase in concentration of Cu alone brought an
increase in the content
[166]
Rauwolfia
sellowii
Alkaloid
Increased alkaloid content in leaf callus
[167]
R. serpentina
Alkaloid
Total alkaloid content in the plantlets was higher as
compared to field grown plants
A group of new alkaloids, the raumaclines and some
related alkaloids were isolated from cell suspensions
fed with high level of ajmaline
[168]
[169]
Silybum
marianum
Silybin
Higher silybin content in regenerants grown on ZnSO4
(200 µM) or CuSO4 (75 µM)
[170]
Solanum
aviculare
Solasodine
Addition of cholesterol to the medium improved the
yield
[171]
S. laciniatum
Solasodine
Decreasing the sucrose concentration increased the
solasodine content significantly in shoot cultures
[172]
S. nigrum, and
S. nigrum var.
judaicum
Glycoalkaloids
Total glycoalkaloids were higher in plantlets
[173]
Stizolobium
hassjoo
L-DOPA
Supplementation with 2.5 µM Co2+ stimulated 25 times
the synthesis of L–DOPA
[174]
Withania
somnifera
Withanolides
Maximum accumulation of withaferin A was noted
in shoot tips proliferating on B5 medium; the
withanolide D content was low. In MS medium
withaferin A accumulation was low than in B5
medium, while withanolide D accumulation was
higher Among the BA and Kn, BA favoured both
shoot multiplication and withanolide synthesis. In
the absence of any carbon source withanolide
accumulation was very low in shoot tips (0.002%)
Withaferin A accumulated maximum in the presence
of 10% sucrose. The maximum accumulation of
withanolide D was at 4% sucrose
[175]
media also influences growth and secondary metabolite production. Fujita et al. [30] report
increase in the yield of shikonin with increase in the concentration of sole nitrogen source, nitrate
till 6.7 mM, but the production decreased with above 10 mM nitrate level. Decreased levels of
N are reported to stimulate the production of secondary metabolites such as, certain polyphenols,
anthocyanins [13], etc.
Manipulation of concentrations of microelements in the nutrient media offers a strategy to
increase the production of secondary metabolites in plant cell cultures [32]. Trace elements have
indeed been considered as abiotic elictors or as inducing factors [33] that trigger the biosynthesis
of secondary metabolites. There are results showing the effect of divalent ions; Co2+ and Cu2+
seem to have received more attention because of their positive effects on the production of
secondary metabolites [30, 34, 35]. Increase in Co2+ from 1 to 5 µM resulted in the enhanced
production of betalains in Beta vulgaris [36]. Enhanced shikonin production in the cultures of
94
ALKA NARULA ET AL
Lithospermum erythrorhizon have been attributed to the increased concentration of both copper
and sulphate [30].
Phytoxicity by heavy metals due to industrial pollution has caused degradation of cultivable
land. Plants allowed to grow on such soil receiving sludge high in heavy metals show reduction
in the quality and productivity. Efforts thus are required to raise metal tolerant plants. Tissue
culture techniques have helped not only in raising metal tolerant plants but have also demonstrated
that subjecting the cultures of medicinal plants to abiotic stress can be crucial in increasing the
yield of secondary metabolites [37]. Several investigations have indeed demonstrated the possibility
to raise metal tolerant plants in vitro [see 38]. Heavy metals have different role in metabolic
functions. Some of them including Cu and Zn are required as micronutrients in biological
systems to act as cofactor and/or as part of prosthetic groups of enzymes in a wide variety of
developmental pathways [39], Cu a constituent of the medium is an essential microelement for
plant growth [40, 41]. It is required for several biochemical and physiological pathways. Cu at
higher concentrations exhibits strong toxicity and hamper plant growth as do some other heavy
metals, such as Cd, Pb or Hg which have no function in plant metabolism. Copper is released as
particulates in stack effluents primarily from Cu smelters. Greater concern of Cu comes from
prolonged applications in fungicidal treatments [42]. Addition of Cu in the medium is reported
to promote somatic embryogenesis as well as its subsequent development in Citrus [43]. Cu
stimulated regeneration in wheat, Nicotiana tabacum and Bacopa monniera [44]. This was also
so at lower concentrations with Dioscorea bulbifera (Narula, unpublished). Cu has proved to be
more effective than Zn in enhancing the yield of xanthotoxin in Ammi majus and lepidine in in
vitro cultures of Lepidium sativum [4, 5, 37]. Heavy metals and others have also induced a
positive effect on alkaloid production in Catharanthus roseus [45]. Fe2+ [46] and Cu2+ [30]
induced positive effects on the synthesis of shikonin. Endress [47] and Obrenovic [48] have
already demonstrated profitable role of Cu2+ on the accumulation of betacyanins in callus cultures
of Portulacca grandiflora and Amaranthus caudatus seedlings. Higher concentrations of Cu2+ in
the media supported increased accumulation of sapogenin steroid in the in vitro cultures of
Agave amaniensis [49, 50].
Plant growth regulators (auxins and cytokinins) are also effective triggers of secondary
metabolites. An optimum concentration of 2,4-D (25 mgl–1) favoured the production of L-Dopa
in cell cultures of Mucuna. Low concentration of 2, 4-D (0.1 ppm) proved favourable for
alkaloid production in cell cultures of Cinchona ledgeriana. In addition to concentration, the
type of auxin used also exerts a strong influence on secondary product formation. 2,4-D in
general proved less suitable for protein synthesis than IAA [51]. Zenk et al. [52] have reported
that in Morinda citrifolia presence of 2,4-D reduced the production of anthraquinones but NAA
enhanced the accumulation of anthraquinones. The alkaloid synthesis and biomass accumulation
increased on nutrient media containing NAA as compared to 2,4-D in Nothapodytes foetida [53].
Like auxins, cytokinins also influence secondary metabolite production. BAP enhanced shikonin
production in L. erythrorhizon and kinetin promoted L-Dopa synthesis in callus cultures of
Stizolobium hassjoo. Likewise, in the presence of BAP maximum accumulation of withanolide
occurred. There was a decline in withanolide in the cultures of Withania somnifera with an
increase (2.0–5.0 mg l–1) in the concentration of BAP [54].
In experiments conducted by Decendit et al. [55] Zt or BAP proved more effective than
Kinetin in Catharanthus cell cultures. At 1 µM Zt or BAP production of alkaloids was doubled.
Biotechnological Approaches Towards Improvement of Medicinal Plants 95
Higher concentrations resulted in the decrease of alkaloids. According to Bhatt et al. [56] besides
growth regulators, a combination of IAA and sucrose in the medium can also stimulate the
production of solasodine in the tissue cultures of Solanum nigrum.
Higher concentrations of NAA + Kn or IAA + Kn promoted the yield of diosgenin in D.
bulbifera. Among the two auxins tried, NAA + Kn induced much higher content (Narula,
unpublished). Corroborative results were obtained in D. deltoidea tissue cultures grown in the
presence of 2,4-D, IBA, BA and GA singly and in combinations. The medium with 2,4-D
favoured diosgenin production most consistently. GA and high BAP concentrations proved toxic
[57]. GA or kinetin are otherwise reported to increase the steroid content in Phaseolus aureus
and Corylus avellana and doubled production of diosgenin in Solanum xanthocarpum tissue
cultures [57]. Zhao et al. [58] observed that an increase in jaceosidin production was accomplished
by increased concentration of NAA. This concurs with the results of Matsumoto et al. [59] who
used cell suspension cultures of Populus.
Addition of precursors of desired compounds to the culture medium also enhances the yield
of secondary products. Ajmalicine production in Catharanthus roseus could be stimulated to
approximately 10-fold by supplying secologanin [60]. Quinine in Cinchona cultures, rosemarinic
acid in Coleus blumei and capsaicin production by cell cultures of Capsicum frutescens [61], and
addition of loganin (precursor of secologanin) into the medium for enhanced yield of secologanin
[62] are some examples where precursor addition caused an increase in the yield of related
metabolites. Addition of various precursors (L-ornithine, L-arginine, L-phenylalanine, DL-βphenyllactic acid and tropinone) alone was ineffective in stimulating hyoscyamine production in
Datura innoxia. But, a combination of these precursors alongwith DL-β-phenyllactic acid and
Tween 20 increased the yield [63].
The recognition that certain specific secondary metabolite products, such as phytoalexins are
produced by plants which are active against microorganisms has led to the concept of using such
stimulators for in vitro cultures also. These compounds have been described as ‘elicitors’ by
Keen et al. [64]. Elicitors can be of biotic or abiotic origin [65]. Biotic elicitors are prepared
from fungal, yeast or bacterial cultures, fungal mycelial extracts, culture filtrates, and fractions
or compounds obtained from microbial cell walls. Autoclaved fungal mycelia induced the
accumulation of diosgenin in Dioscorea deltoidea cultures [66]. The production of berberine and
shikonin enhanced in the cultured cells treated with fungal extracts [67, 68]. A beta-glucan
elicitor prepared by ethanol precipitation of yeast, Saccharomyces cerevisiae elicited alkaloid
production in cultured cells of Eschscholtzia. In Tabernaemontana divaricata cultures, reserpine
accumulation increased by treating the cells with an elicitor prepared from Candida albicans
[69]. Purified fractions from bacteria also elicited diosgenin and capsaicin production [66].
Elicitation of capsaicin in Capsicum frutescens cultures could be achieved by supplementing the
culture medium with chitosan, curdlan and xanthan.
The abiotic elicitors include physical and chemical stresses such as UV radiation, exposure to
heat or cold, ethylene, fungicides, antibiotics, salts of heavy metals, salinity, etc. [70]. It has also
been recorded that the synthesis of alkaloids can be similarly elicited with jasmonic acid and its
esters playing a key role in regulating the response [71]. In fact, it is reported that fungal cell
wall elicitors and methyl jasmonate (MeJa) can activate inducible secondary metabolism in
soybean cell cultures by different mechanisms. Treatments with exogenous MeJa can elicit the
accumulation of several classes of alkaloids in a wide range of plant species [72]. Hairy root
96
ALKA NARULA ET AL
cultures of Datura stramonium showed maximum alkaloid in the presence of MeJa followed by
fungal elicitors and oligogalacturonide [73]. Jasmonate can elicit natural product formation not
only in plants but also in cell cultures [74, 75]. Methyl jasmonate therefore could be an useful
tool for the enhancement of lignan production in biotechnological processes. Feeding experiments
with the precursor coniferyl alcohol resulted in fast increase in the pinoresinol content [76].
Some of the examples where elicitors caused enhancement in the yield of medicinal compounds
are cited in Table 3.
Table 3. Some examples of in vitro production of medicinal compounds when elicitors were used in cell
suspensions
Plant
Elicitor Used
Active Principle
Reference
Catharanthus roseus
Botrytis species homogenate
Fungal homogenate
Catharanthene
Terpenoid, indole alkaloid
[176, 177]
[178]
Eschscholtzia californica
Yeast
Sanguinarine
[179]
Hyoscyamus albus
Phytophthora cinnamomi
Lubimin
[180]
Lithospermum erythrorhizon
Oligogalacturonides
Dihydroechinofurane
[181]
Morinda citrifolia
Polysaccharides
Anthraquinones
[182]
Papaver bracteatum
Fungal
Verticillum
Sanguinarine
Sanguinarine
[183]
[184]
P. somniferum
Fungal homogenate
Botrytis species homogenate,
Pythium aphanidermatum
Sanguinarine
Sanguinarine
[185]
[186]
Sanguinaria canadensis
Verticillum
Sanguinarine
[184]
Thalictrum rugosum
Yeast carbohydrate
Berberine
[67]
Tripterygium wilfordii
Botrytis species
Trichoderma virideae
Rhodotorula rubra
Sclerotinia sclerotiorum
Oleanane triterpenes
[187]
It is not only the chemical milieu but also the physical factors which play a significant role
in secondary metabolite production. Light as physical source, for example, has an effect on
growth and development of plants as well as in stimulation of secondary metabolite production
[77, 78]. In fact, quality, intensity and duration of light play a decisive role in the accumulation
of secondary compounds [79, 58]. Production of diosgenin and related compounds seem to be
controlled by different media ingredients as well as by light [80].
In some cases, direct effect of hydrogen ion concentration on secondary compound production
has been demonstrated. For example, alkaloid synthesis in Lupinus polyphyllus cultures rose
with a decrease in pH from 5.5 to more acidic, 3.5. Even physical conditions of the medium have
proved crucial for the production of secondary metabolites in cultures. Cell suspension cultures
have been favourites for the production of valuable secondary metabolites in cultures. These
cultures initiated by transfer of most friable sector of an established callus tissue into an agitated
liquid medium received more homogenous stimuli. A close correlation between the growth of
Biotechnological Approaches Towards Improvement of Medicinal Plants 97
cultures and yield of products has been envisaged. Since the product accumulates through the
growth cycle, the product and biomass show a close correlation.
The first commercial production of a natural plant product by cell suspension cultures was
developed in Japan for the production of naphthoquinone, shikonin. In suspension cultures of
Rauwolfia sellowii alkaloid content was maximum at the end of the exponential growth phase.
It has been argued that for industrial scale production of plant secondary metabolites, the cells
should be suspended in liquid so that the entire operation of harvesting, inoculation and other
treatments could be accomplished by pumping the suspended cells. Immobilized plant cells used
in the same way as immobilized enzymes have also played an important role in the secondary
product formation [81]. Although the enzymatic activity of immobilized cells is about half that
of suspending cells, these have the advantage of being reusable as a biocatalyst over a considerable
period [82].
3.2 Synthetic Seed
The concept of ‘synthetic seed’ was first introduced by Toshio Murashige in 1977 and later the use
of synthetic seeds or artificial seeds was realized by Redenbaugh and coworkers [83] and others.
Synthetic seeds help in reducing the cost of transport and in maintaining the uniformity. Besides,
of much importance is the ability to provide large-scale delivery of elite genotypes selected from
hand pollinated hybrids or genetically engineered plants. The first successful examples of synthetic
seed technology have been in alfalfa [84] and celery [85]. Various vegetative propagules like
axillary buds, shoot tips, bulbs, protocorms have been used [86]. The production of ‘Syn’ seeds has
been reported in several medicinal plants like, Atropa belladonna, Hyoscyamus muticus, Mentha
arvensis, Picrorhiza kurroa [87], Dioscorea alata, D. floribunda, [88], Clitoria ternatea [89] and
Guazuma crinita [90].
3.3 Cryopreservation
Cryopreservation offers long-term conservation of germplasms. In addition to germplasm
conservation, it also ensures genetic stability and retention of biosynthetic potential [91].
Cryopreservation has been achieved by using various explants (Table 4). The period over which
the cultures retain viability vary widely with the species and a maximum of 3 years has been
recorded in Digitalis [92].
Meristems have been preferred over cell and callus cultures because they are genetically more
stable. Shoot tips of medicinal plants such as Cichorium sp. [93], Dioscorea deltoidea, D.
floribunda [91], Holostemma annulare [94] and Mentha sp. [95] have been cryopreserved
successfully. Genetic erosion due to periodic subculture and storage can be overcome by freeze
preservation of callus and cell suspensions in liquid nitrogen. Cell suspensions of medicinal
plants, e.g., Atropa belladonna, Datura innoxia, Nicotiana tabacum, Panax ginseng, etc. retain
their biosynthetic potential after freezing. Cryopreservation of somatic embryos helps in storage
at appropriate stage that can be used whenever required. The somatic embryos of carrot, orange
and asparagus frozen in liguid nitrogen yielded high viability and regenerated complete plants.
The potential of zygotic embryos is manifold in plants with recalcitrant seed, in fruit and timber
trees and plantation crops. In wide hybridization programs, especially dealing with intergeneric
crosses which are incompatible due to degeneration of embryos, can be possibly dissected out
at immature stages and cryopreserved. Zygotic embryos of rice, wheat, barley, mustard and
98
ALKA NARULA ET AL
Table 4.
Plant
Examples of some cryopreserved medicinal plants
Explant/Culture
Method used
Reference
Anisodus acutangulus
Cell suspensions
Liquid nitrogen (–196°C)
[188]
Atropa belladonna
Pollen embryos
Protoplasts
Liquid nitrogen (–196°C)
[163]
Catharanthus roseus
Cell suspensions
Low temperature (0-30°C), –196°C
[189]
Datura innoxia
Protoplasts
Exposed to vapors, immersed in liquid nitrogen
[163]
Dioscorea alata
Shoot tips
Encapsulation
Dehydration
[88]
[190]
D. balanica
Callus
Direct immersion in liquid nitrogen
[191]
D. bulbifera
Shoot tips
Encapsulation
Dehydration
[88]
[190]
D. floribunda
Shoot tips
Encapsulation
Dehydration
[88]
Eucalyptus sp.
Shoot tips
Encapsulation
Dehydration
[192]
Ipomea batatas
Shoot tips
Vitrification
[193]
Medicago sativa
Somatic embryos
Encapsulation
[83, 194]
Mentha sp.
Shoot tips
Encapsulation
Vitrification
[195]
Nicotiana tabacum
Protoplasts
Liquid nitrogen (–196°C)
[163]
Panax ginseng
Cell suspensions
Hardening, –30, –70, then –196°C
[196]
Trifolium repens
Shoot tips
Vitrification
[197]
coconut cryopreserved by quick freezing, followed by thawing at 35-40°C produced viable
plants but viability varied considerably [96].
The storage of pollen has been of prime interest to plant breeders. Cryopreservation of pollen
enables en masse production of haploid plants, maintenance of stability of haploids and conservation
of genetic resources. Segments of anthers and pollen embryos of Atropa belladonna, Brassica
campestris, Nicotiana tabacum and Primula obconica have been successfully frozen and entire
plants have been regenerated after one year of storage [97]. Freshly isolated protoplasts of
Atropa belladonna, and Nicotiana tabacum, and Datura innoxia [98], and Glycine max [99]
subjected to freezing in liquid nitrogen for various time periods have survived and retained their
morphogenetic potential.
3.4 Molecular Markers
As micropropagation developed from a laboratory curiosity to commercial industry, different
considerations became important concerning the feasibility of approaches for long-term economic
benefits. The foremost concern has been the maintenance of the genetic integrity of micropropagated
plants with regard to the explant source so that the advantages (high yield, uniform quality,
shorter rotation period, etc.) in the use of elite genotypes over natural seedlings is maintained
Biotechnological Approaches Towards Improvement of Medicinal Plants 99
[100,101]. Rani and Raina [101] have emphasized that micropropagation cannot be rewarding
unless complete genetic fidelity is maintained. Thus for obtaining true-to-type plants, axillary
branching or somatic embryogenesis have mostly been adopted. These two methods have generally
been considered to be immune to genetic changes that may arise during cell division or differentiation
under in vitro conditions [102]. Rani and Raina [101] showed that the field-transferred enhanced
axillary branching derived plants of Eucalyptus camaldulensis were genetically stable in terms
of genome size, RFLPs of nuclear and organellar genomes and RAPD and oligonucleotide
fingerprinting patterns. The concept of uniformity among micropropagated plants, however,
received a jolt when somaclonal variations were reported. Somaclonal variations can pose a
threat to the genomic integrity of regenerated plants. Several strategies were therefore adopted
to detect variants based on morphological traits, cytogenetical analysis for the determination of
numerical and structural variation in the chromosomes and isozymes. But, these met with severe
limitations. Molecular markers have thus been used to study genetic diversity, phylogeny and
fingerprinting as well as to construct physical genetic maps in medicinal plants. The range of
marker system includes RAPD, AFLP, microsatellites and RFLP.
RFLP was introduced as hybridization based marker for single copy loci. Since RFLP is
capable of detecting multiple alleles, it reveals greater level of heterozygosity and has a higher
information content. The major drawback of RFLP is that it screens very few loci per assay. It
is expensive, labour intensive and technically complex as it involves the use of radioactive
probes. It also requires larger amounts of genomic DNA making its application impractical for
efficiently cataloguing of genetic resources [103]. RAPD technique is quite simple, inexpensive
but less reliabile. AFLP has many advantages that make it applicable in assessment of genetic
diversity, genetic mapping and tagging studies. AFLP has now become a preferred technique as
it combines RFLP and RAPD [104]. AFLP markers offer best method for detecting mutations by
randomly surveying the genome. This technique does not require prior sequence information.
Besides, it has wide genome coverage as compared to other DNA-based markers [105] which
makes it an ideal tool for detecting genetic variation. This technique has been used for analyzing
genetic variation in somatic embryoids of pecan [106]. Singh et al. [107] reported application of
AFLP markers for ascertaining clonal fidelity in tissue culture raised progenies of a medicinally
important plant, Azadirachta indica. AFLP markers are now being routinely employed for assessment
of genetic variation in economically important plant species including chichory [108], Withania
sp. [109], etc. (Table 5).
3.5 Genetic Engineering in Medicinal Plants Through Agrobacterium
The stable introduction of foreign genes into plants represents one of the most significant
developments in plant biotechnology. Attempts have been made to manipulate pharmaceutically
important medicinal plants for their secondary metabolic pathways by using transgenic technique.
Since secondary products are often biosynthesized in mulit-step enzymatic reactions in specifically
differentiated cells, manipulations of such pathways to alter metabolic production is complex,
complicated and unpredictable [110].
Transformation has many advantages over conventional cell culture systems that may include
fast growth and stable high level production of secondary metabolites making them favourable
for biotechnological exploitation. Agrobacterium tumefaciens and A. rhizogenes have proved
efficient and have provided highly versatile vehicles for introduction of genes into the desired
100
ALKA NARULA ET AL
Table 5.
Application of molecular markers in some medicinal plants
Plant
Marker
Application
Reference
Achillea ospenifolia
Oligonucleotide
Stability of micropropagated plants
finger printing, RAPD
[198]
Allium sativum
RAPD
Genetic diversity in plants regenerated by
somatic embryogenesis from long-term-callus
[199]
Artemisia annua
OPGMA-RAPD
Artemisinin and chemotypic variants
[200]
Azadirachta indica
AFLP
Genetic diversity
[201]
A. indica
AFLP
Clonal fidelity in tissue culture raised plants
[202]
Cichorium sp.
AFLP
Diagnostic marker for endive and chicory
group
[203]
Codonopsis pilosula
RAPD
Geographic variation
[204]
Datura sp.
AFLP
Genetic diversity
[205]
Digitalis obscura
RAPD
Genetic variation
[206]
Dioscorea bulbifera
RFLP
RAPD
Linkage (physical) map
Genetic variability and relationship
within the species
[207]
[208]
D. rotundata and
D. cayenensis
AFLP
Genetic diversity
[209]
Duboisia
RFLP
Hybrid origin identification
[210]
Moringa oliefera
AFLP
Genetic variation
[211]
Panax ginseng
RAPD
Genetic stability in micropropagated plants
[212]
P. ginseng and
P. quniquefolium
RFLP
Ginseng drug
[213]
Plantago major
RAPD
Identifying subspecies
[214]
Rehmannia sp.
RAPD
Homogenity
[215]
Tylophora indica
RAPD
Genetic variation
[216]
plant genome. As a consequence of transfer and integration of genes through plasmids into the
plant DNA, the transformed tissues and hairy roots have provided encouraging results. These
transformed tissues have thus become potential sources for stable production of plant metabolites
(Table 6).
Hairy root cultures of Trigonella-foenum-graecum L. produced twice the amounts of diosgenin
than the non-transformed roots [111]. Several studies have indicated that Agrobacterium rhizogenes
affects the levels of polyamine in transformed plants [112] that may influence growth and the
production of secondary metabolites. Atropa baetica hairy roots synthesized and accumulated a
conspicuously high amount of tropane alkaloids [113].
In Hyoscyamus albus, hyoscyamine content was more in the transformed roots followed by
stem and leaves [114]. Doerk-Schmitz et al. [114], however, reported low proportion of scopolamine
in hairy roots of Hyoscyamus albus but there was high content of hyoscyamine even after several
Biotechnological Approaches Towards Improvement of Medicinal Plants 101
Table 6.
Some examples of Agrobacterium-mediated transformation in medicinal plants
Plant
Strain
Result
Reference
Ammi majus
Agrobacterium rhizogenes
A4 (20233)
Hairy roots produced higher
content of visnagin
[217]
Artemisia annua
A. rhizogenes
LBA 9402
1-month-old
transgenic
produced more artemisnic
acid and arteannuin B
[218]
A. annua
A. tumefaciens
C58 , N2 73
Artemisinin content was slightly
higher in shoots
[219]
Atropa belladonna
A. rhizogenes
A4 , TR 105
Higher atropine levels
[220]
A . belladonna
A. tumefaciens
LBA 4404
Higher scopolamine
[221]
A . belladonna
A. rhizogenes
15834
Increased scopolamine content
[222]
A . belladonna
A. rhizogenes 15834 and
A. tumefaciens rol
ABC genes
Higher alkaloid
[223]
Catharanthus roseus
A. rhizogenes
At reduced pH more alkaloid released
[224]
Cinchona ledgeriana
A. tumefaciens A6
Five times more alkaloids
(cinchonine and cinchonidine)
[225]
C. ledgeriana
A. rhizogenes
LBA 9402
Quinine, cinchonidine and quinidine
reached a maxima after 45 days
[226]
Datura candida hybrid
(D.candida ×
D.candida)
A. rhizogenes
Scopolamine and hyoscyamine
showed increase
[227]
D.
D.
D.
D.
A. rhizogenes LBA 9402
Maximum hyoscyamine content in
D. stramonium and scopolamine in
D. innoxia
[24]
D. innoxia
A. rhizogenes A4,
15834 and A4–24
A4 and 15834 strains more
effective
Higher hyoscyamine content
[228]
D. innoxia
A. rhizogenes LBA9402,
A41027, R1601
R1601 gave best response
Hyoscyamine and scopolamine
content was higher and among the
two alkaloids hyoscyamine content
was much higher
[229]
D. innoxia
A. rhizogenes
Permeabilization with Tween 20 for
30 hr period increased the alkaloid
concentration in the medium
[230]
D. stramonium
A. rhizogenes TR-105
Heat shock given to the cultures
resulted in higher hyoscyamine
release in the medium
[231]
innoxia
stramonium
ferox
wrightii
(Contd)
102
ALKA NARULA ET AL
Table 6.
Plant
Strain
(Contd)
Result
Reference
D. stramonium
A. rhizogenes TR-105,
Hyoscyamine and scopolamine
ATCC 15834, A4, 1855,
bioproductivity was higher in
A41027, ATCC 13333
hairy root culture
Among these strains TR-105
proved most effective
[232]
D. stramonium
A. rhizogenes
Release of alkaloids increased at
low pH (3.5)
[224]
D. stramonium
A. rhizogenes LBA 9402
Live fungal pellets caused enhanced
hyoscyamine production
[233]
D. stramonium
A. rhizogenes A4 rol
ABC and tms gene
Higher hyoscyamine production
[234]
D. stramonium
A. rhizogenes TR-105
An inverse relation between alkaloid
accumulation and growth, hyoscyamine
content showed an increase
[235]
D. stramonium
A. rhizogenes ATCC 15834
Highest hyoscyamine yield with
culture medium in which SO42–
and K+ was dominant
[236]
D. stramonium
A. rhizogenes A4
Lower calcium concentrations
reduced the hyoscyamine synthesis
[237]
D. quercifolia
A. rhizogenes LBA 9402
5% sucrose in Gamborg B5 medium
proved best for growth and higher
hyoscyamine accumulation
[238]
Hyoscyamus albus,
H. desertorum,
H. muticus
A. rhizogenes LBA 9402
Hyoscyamine and scopolamine
content was highest in H. albus
[24]
H. albus
A. rhizogenes MAFF 0301724
Higher yield of hyoscyamine
[239]
H. muticus
A. rhizogenes LBA 9402,
C58CI, pRTGUS 104
High hyoscyamine content at 3%
sucrose in two of the clones, high
nitrogen content had negative effect
on hyoscyamine production and
growth. Copper (11 µM) stimulated
hyoscyamine production
[240]
Hyoscyamus × gyorffyi
(H. niger × H. albus)
A. rhizogenes LBA 9402,
In 14 clones of H. gyorffyi
A41027, R 1601
hyoscyamine percentage being much
Among these strains R 1601 higher than scopolamine
gave the best response
[241]
Panax ginseng
A. rhizogenes
Produced saponin, and ginsenosides
more effectively
[242]
P. ginseng
A. rhizogenes A4, 15834
A4 proved more effective
Higher content of glycosides
[243]
P. ginseng
A. rhizogenes A4
Faster growth of callus and higher
yield of ginsenosides
[244]
Biotechnological Approaches Towards Improvement of Medicinal Plants 103
Pgq (Panax hybrid)
(P. ginseng ×
P. quinquefolium)
A. rhizogenes ATCC 15834
Ginsenoside content was higher
[245]
Rauwolfia serpentina
A. rhizogenes 15834
Increased levels of ajmaline and
serpentine
[246]
Solanum eleagnifolium
A. tumefaciens T 37
Transgenic shoots showed higher
solasodine content
[247]
Scopolia lurida and
S. stramonifolia
A. rhizogenes LBA 9402
Produced little alkaloids
[24]
Withania somnifera
A. rhizogenes LBA 9402
Productivity of withanolide D was
higher
[248]
subcultures and the transgenic plants could be regenerated directly from such roots via organogenesis
[115]. Strains of Agrobacterium are reported to affect the growth behaviour and production of
secondary metabolites. Influence of A. rhizogenes strains on biomass and alkaloid prductivity in
hairy root lines of Hyoscyamus muticus and H. albus was also studied by Zehra et al. [116]. A4
induced hairy root lines of H. albus and H. muticus were faster growing than those induced by
strain LBA 9402. The atropine yield of A4 induced lines of H. albus was significantly higher
(3.5-fold) than the LBA 9402 induced lines [116].
Cu2+ enhanced both, the growth and the alkaloid yield in Hyoscyamus albus hairy roots.
Similar results have been obtained in the production of shikonin derivative by cell suspension
cultures of Lithospermum erythrorhizon [117]. Copper concentration up to 11 µM stimulated
hyoscyamine production but had no influence on growth of hairy root cultures of Hyoscyamus.
Two-year-old transformed root cultures of Catharanthus roseus accumulated higher ajmalicine
and catharanthine than the non-transformed cultures. Addition of MeJa increased the yield of
both the alkaloids [118]. A positive correlation between STR activity and alkaloid accumulation
has also been found in tissues of Cinchona ledgeriana and C. roseus seedlings [72, 119]. TDC
(trytophane decarboxylase) activity in developing Cinchona seedlings increased after a large
pool of tryptophan was formed. It fell to undetectable levels once the tryptophan was converted
into tryptamine [119].
Serotonin content enhanced if the hairy root cultures of Peganum harmala were fed with
tryptamine. But the alkaloid content was not affected [120]. In Panax ginseng roots were transformed
with A. rhizogenes. Inomata et al. [121, 122] found that periodic changes of medium maintained
the high growth rate and the ginsenoside production varied during different stages of growth.
Mallol et al. [122] also observed that the capacity to produce and accumulate ginsenoside is
associated with biomass. The results concur with other investigations as well as our results with
Datura. Transformation stimulated increased biomass and tropane alkaloid production in axenic
root cultures of Calystegia sepium and Atropa belladonna [123]. Compared to transformed
plants, non-transformed plants contained low amounts of tropane alkaloids, especially 6 βhydroxy hyoscyamine and scopolamine in the roots [124].
In transgenic lines of Nicotiana tabacum feeding of lysine to root cultures with low LDC
(lysine decarboxylase) activity enhanced cadaverine and anabasine levels [125]. Several hairy
root cultures of N. tabacum having lDC gene increased cadaverine levels and this was used for
the formation of anabasine to obtain a 3-fold increase of this alkaloid. Transformation has indeed
104
ALKA NARULA ET AL
helped in the enhancement of secondary metabolites in a number of cases though not to commercial
levels. In Artemisia, use of arnesyl diphosphate synthase gene promoted artemisinin 3-4 times
higher in hairy roots [126]. In Catharanthus roseus where str (strictocidine synthase) is highly
desirable for increased terpenoid indole alkaloid (TIA) production, high STR activity positively
influenced the flow of metabolites through the indole pathway [127].
Subroto and Pauline [128] reported the production of steroidal alkaloids in Solanum aviculare
that was growth associated. It has been shown by Schaller et al. [129] that the introduction of
extra copies of a chimeric hmgr gene (obtained from Hevea brasiliensis) increased the accumulation
of sterols by 6-fold in tobacco plants. HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase)
plays a major role in the regulation of sterol biosynthesis in plants. In Solanum aviculare,
Cavalcante Argôlo [130] obtained transgenic hairy root clones that grew faster and accumulated
up to 4.2 times more solasodine when grown under dark. Upregulation of the hmgr gene in
tobacco has also been shown to give rise to highly significant increase in sterol accumulation
[131].
odc and adc genes play important role in the biosynthetic pathway of alkaloids. A stable
transformation system has been developed by us for Datura innoxia using androgenic callus that
was transfected with Agrobacterium tumefaciens strain LBA4404 carrying odc and adc genes.
Transformed cultures showed higher amounts of hyoscyamine and early regeneration.
Tiburcio and Galston [132] reported that in Nicotiana tabacum ODC pathway is important for
cell division and growth, the effects of inhibitors on alkaloid biosynthesis show that ADC is
more important in synthesis of pyrrolidine alkaloids.
Imanishi et al. [133] in tobacco and Robins et al. [134] in Datura stramonium also found that
arginine decarboxylase activity is more important for hyoscyamine formation. However, expression
of yeast odc gene in Nicotiana rustica roots induced an increased accumulation of nicotine [135]
that shows that plant secondary products can be elevated by means of genetic manipulation.
4.
Epilogue
Biotechnology of medicinal plants has not received the attention it deserves. While other group
of plants have been the area of concern for yield improvement including disease and pest
resistance, no organised technology has been adopted for improving the yield of medicinal
plants. The methodology should include credible selection, micropropagation and studies on
abiotic stress related yield as stress has been implicated in the biosynthesis of secondary metabolites.
Recombinant DNA technology, molecular biology and metabolic engineering need to be integrated
to make a combined concerted effort that will help in improving the productivity of drug
components.
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B.D. Benjamin, G. Roja, M.R. Heble, Agrobacterium rhizogenes mediated transformation of Rauwolfia
serpentina: regeneration and alkaloid synthesis, Plant Cell Tiss. Org. Cult. 35 (1993) 253–257.
M.A. Alvarez, J.R. Talou, N.B. Paneiego, A.M. Giulietti, Solasodine production in transformed organ
cultures (roots and shoots) of Solanum eleagnifolium Car. Biotech. Lett. 16 (1994) 393–396.
S. Ray, B. Ghosh, S. Sen, S. Jha, Withanolide production by root cultures of Withania somnifera
transformed with A. rhizogenes, Planta Med. 62 (1996) 571–573.
Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
7. Production of Phytochemicals in Plant Cell
Bioreactors
Saurabh Chattopadhyay, A.K. Srivastava and V.S. Bisaria
Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, Delhi,
New Delhi 110016, India
Abstract: Plant cell culture provides a viable alternative over whole plant cultivation for the
production of useful phytochemicals. In order to successfully cultivate the plant cells at large scale,
some engineering parameters such as cell aggregation, mixing, aeration and shear sensitivity are
taken into account for selection of a suitable bioreactor. Increased productivity in a bioreactor can
be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stage, etc.), feeding
of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous
analysis of these parameters would pave the way towards the successful commercialization of plant
cell bioprocesses.
1.
Introduction
Higher plants are inexhaustible sources of a wide range of biochemicals such as flavors, fragrances,
natural pigments, pesticides and pharmaceuticals. Currently many of these compounds are isolated
by solvent extraction from the naturally grown whole plants. This continued destruction of
plants has posed a major threat to the plant species getting extinct over the years. Clearly, the
development of alternative methods to whole plant extraction for the production of these compounds,
especially of medicinal value, is an issue of considerable socio-economic importance. These
factors have generated considerable interest in the use of plant cell culture technologies for the
production of phytochemicals [1]. In plant cell culture, the isolated cells from the whole plant (or
parts derived thereof) are cultivated under appropriate physiological conditions and the desired
product is extracted from the cultured cells. The recent developments in plant tissue culture
techniques and their processing have shown promising results to improve the productivity by
many folds.
2.
Cell Suspension Cultures
The first step in plant tissue culture is to develop a callus culture from the whole plant. A callus
can be obtained from any portion of the whole plant containing dividing cells. To maximize the
formation of a particular compound, it is desirable to initiate the callus from the plant part that
is known to be a high producer. However, from an engineering perspective, cell suspension
cultures have more immediate potential for industrial application than plant tissue and organ
cultures, due to extensive expertise which has been amassed for submerged microbial cultures.
While tissue and root cultures offer genetic stability as well as, in some instances, superior
metabolic performances over suspension cultures of the cell lines, the development of appropriate
118
CHATTOPADHYAY ET AL
bioreactors and operating techniques for these systems involve high investment and laborious
experimentation [2]. Accordingly, most of the research efforts have been directed towards
commercialization of plant cell suspension cultures. A suspension culture is developed by
transferring the relatively friable portion of a callus into liquid medium and is maintained under
suitable conditions of aeration, agitation, light, temperature and other physical parameters. However,
various strategies may have to be adopted to obtain a fairly homogeneous suspension culture.
Mitsui Petrochemical Industry, Japan was the first to produce shikonin (a dyestuff) on commercial
scale. While the large-scale cultivation of plant cell suspension cultures is desirable for industrial
production of plant-derived biochemicals, the production technology comparable to that used for
microbial systems needs to be further developed. Although the basic equipment- and processrelated requirements for suspension cultures of plant cells are similar to those of submerged
microbial cultures, some of the features used for microbial cultures are not suitable for plant cell
cultures because of striking differences in the nature and growth pattern of the two types of cells.
The implications of these differences on culturing of the plant cells are summarized in Table 1
[3]. Certain engineering considerations are normally addressed before embarking on the mass
scale propagation of plant cells.
3.
Engineering Considerations
Plant cell suspensions can now be successfully cultivated in bioreactors of various configurations.
However, many of the unique properties of plant cells in culture such as sensitivity to shear, slow
growth rates, and low oxygen requirements are manifested in complex ways at large scale
cultivation. As the scale of operation increases, mixing inside the bioreactor vessel becomes
difficult, resulting in non-uniform concentration of the nutrients and limited oxygen transfer to
respiring cells. Changes in the rheological nature of the fluid, wall growth, and clumping of cells
resulting in sedimentation, lead to suboptimal utilization of the bioreactor. These problems
Table 1. Differences between the characteristic features of plant and microbial cells and their implications
for bioreactor design
Characteristic features of a
typical plant cell
Implications for reactor design
Lower respiration rate
Lower oxygen transfer rates required
More shear sensitive
May require operation under low-shear conditions by, for example,
employing low-shear impellers and bubble-free aeration
Growth as aggregates
May have mass transfer limitations that limit the availability of nutrients
to cells within the aggregates
Aggregation important for
secondary metabolism
An optimal aggregate size may be required for product synthesis by
manipulation of media constituents and environmental conditions
Volatile compounds (e.g. CO2 or
ethylene) may be important
for cell metabolism
May need to sparge gas mixtures containing them
Product synthesis may be
non-growth-associated
May require a two-step cultivation system for maximal product
synthesis
Adapted from [3].
Production of Phytochemicals in Plant Cell Bioreactors 119
necessitate a more rigorous analysis of bioreactors to be used for the large scale cultivation of
plant cells for metabolite production [4–7].
3.1 Aggregation
Plant cells are significantly larger and slower growing cells than most microbial organisms.
Aggregation is common, largely due to failure of the cells to separate after division, although the
secretion of extracellular polysaccharides, particularly in the later stages of growth, may contribute
to increased adhesion. This tendency of the plant cells to grow in clumps results in sedimentation,
insufficient mixing and diffusion-limited biochemical reaction. This so-called cell-cell contact is
desirable for the biosynthesis of many secondary metabolites by the plant cells. Hence controlled
aggregation of plant cells is of interest from process engineering point of view.
3.2 Mixing
Mixing promotes better growth by enhancing the transfer of nutrients from liquid and gaseous
phases to cells and the dispersion of air bubbles for effective oxygenation. Although plant cells
have higher tensile strength in comparison to microbial cells, their shear sensitivity to hydrodynamic
stresses restricts the use of high agitation for efficient mixing. Plant cells are, therefore, often
grown in stirred tank bioreactors at very low agitation speeds. Mixing of plant cells grown on a
large scale is also hampered by the rheological characteristics of the culture broth [6]. Plant cell
suspensions are viscous at high concentrations and behave like non-Newtonian fluids. NonNewtonian behavior of the culture broth also restricts effective mass and heat transfer inside the
bioreactor, leading to non-uniform nutrient concentration and temperature, and the development
of dead zones inside the culture vessel. Excretion of polysaccharides at the later stages of cell
growth, the extent and nature of which depend on the nature of the plant cells and the carbohydrate
source used for growth, also results in a rapid increase in viscosity. Inadequate mixing may lead
to clumping of cells, thereby complicating the nature of the reacting system; also the inner
cells of the clumps become nutrient deficient, which may have either an adverse or a positive
effect on the cell growth and product formation [4]. Adequate mixing can be achieved by proper
design of the impeller; helical-ribbon impeller has been reported to enhance mixing at the high
density of plant cell suspension cultures [8].
3.3 Oxygen and Aeration Effects
Oxygen requirements of plant cells are comparatively lower than that of microbial cells due to
their low growth rates. In some cases, high oxygen concentration is even toxic to the cells’
metabolic activities and may strip nutrients such as carbon dioxide from the culture broth.
Hence, effective oxygen transfer in plant cell cultures must be carefully analyzed when a bioreactor
system is being selected. The intensity of culture broth mixing, the degree of air bubble dispersion,
the culture medium’s capacity for oxygen, and the hydrodynamic stress inside the culture vessel
affect proper aeration of the culture. Effects of aeration on plant cell suspension cultures have
focused largely on the influence of kLa, the mass transfer coefficient, in which the aeration and
agitation are linked. The kLa value gives a direct measure of effective oxygenation of culture
fluid and helps one choose a suitable bioreactor to cultivate plant cells. Increased viscosity of the
culture broth decreases kLa and signals the need for intensive agitation of the culture for better
mixing and oxygen transfer. A balanced analysis of mixing and oxygen transfer as reflected in
120
CHATTOPADHYAY ET AL
kLa value is, therefore, required to achieve reasonable cell yield and product formation. The
effect of initial kLa on growth and alkaloid production by suspension cultures of Catharanthus
roseus was studied in 12.5 liter stirred tank bioreactor using either a cross sparger or a sinter
sparger, and a 6-bladed Rushton impellor for agitation [9]. It has been observed that, at higher
kLa values, serpentine was produced when the cells were in the log phase, whereas production
of serpentine and ajmalicine was maximum at kLa values of 16 h–1 and 4.5 h–1, respectively [9].
High aeration may lead to severe foaming, which has considerable influence on the cell
growth and secondary metabolite production [10]. A number of antifoams such as polypropylene
glycol 1025 and 2025, Pluronic PE 6100, and Antifoam-C have often been employed to
control foaming; however, in some cases this resulted in reduction in cell growth and product
formation [11].
3.4 Shear Sensitivity
The sensitivity of plant cells to hydrodynamic stress associated with aeration and agitation can
be attributed to the physical characteristics of the suspended cells, viz. their size, the presence
of thick cellulose based cell wall, and existence of large vacuoles. Mechanically agitated vessels
lead to damaging and breaking the cells through the hydrodynamic stress generated by aeration,
agitation, and other operations. The air-lift bioreactor has also been used to achieve better
oxygen transfer and good growth. Bubble-free aeration of the culture fluid through a moving
membrane provided another suitable alternative for transferring gas without inducing cell damage
through shear stress.
The immediate consequence of the shear effect on plant cells is cell damage, which has been
quantitatively measured by using a number of system responses such as reduction in cell viability
[12], release of intracellular compounds [13], changes in morphology and/or aggregate patterns
[14], and changes in metabolism [15]. The effects of hydrodynamic and interfacial stress on
plant cell suspension cultures with various modes of quantitative analysis of system response at
shake flask as well as bioreactor levels have recently been reviewed by Kieran and co-workers
[16].
4.
Plant Cell Bioreactors
A suitable bioreactor can be designed for a specific plant cell system from the following
considerations [4–7]:
• optimum aeration-agitation with respect to capacity of oxygen supply and intensity of
hydrodynamic stress effects on the plant cells.
• intensity of culture broth mixing and air-bubble dispersion.
• control of temperature, pH and nutrient concentration inside the bioreactor.
• control of aggregate size (which may be important to enhance secondary metabolite
production).
• maintenance of aseptic conditions for relatively longer cultivation period.
A number of different types of bioreactors (Fig. 1) have been used for mass cultivation of
plant cells taking the above considerations into account. Stirred tank bioreactors have been most
extensively applied in order to achieve the optimum process parameters. In spite of the fact that
stirred tank reactors exert more hydrodynamic stress on plant cells, they have great potential
Production of Phytochemicals in Plant Cell Bioreactors 121
when used with low agitation speed and modified impeller. The first commercial application of
large scale cultivation of plant cells was carried out in stirred tank reactors of 200 and 750 liter
capacities to produce shikonin by cell cultures of Lithospermum erythrorhizon [3]. Cells of
Catharanthus roseus [17], Digitalis lanata [18], Panax notoginseng [19], Taxus baccata [20]
and Podophyllum hexandrum [21] have been cultured in stirred tank bioreactors with suitable
modifications for production of phytochemicals. Another type of reactor known as bubble column
reactor has also been used for large scale cultivation of plant cells. The major advantages of this
reactor are the absence of moving parts and ease of maintaining sterile environment, as no
sealing parts are required. Cudrania tricuspidata, being highly shear sensitive, was cultivated in
bubble column reactor [22]. A modification of bubble column reactor ‘balloon type bubble
bioreactor’ has been recently adopted for the production of taxol by Taxus cuspidata [23]. The
major disadvantage of this reactor is insufficient mixing. A reactor having more uniform flow
pattern with slight modification of stirred tank reactor (a draught tube is inserted instead of the
impeller) is air-lift bioreactor. The cells of Catharanthus roseus [24], Digitalis lanata [25],
Cudrania tricuspidata [22, 26], Lithospermum erythrorhizon [26] and Taxus chinensis [27] have
been successfully cultivated in air-lift bioreactors for production of secondary metabolites. The
major disadvantages of this reactor are the development of dead zones inside the bioreactor,
insufficient mixing at high cell densities and rupture of cells due to collision between air bubbles
Stirred tank
reactor
Bubble column
reactor
Rotating drum reactor
Fig. 1.
Air-lift reactor
(Draught tube)
Air-lift reactor
(Outer loop)
Membrane reactor
Configurations of different bioreactors used for plant cell cultivation.
122
CHATTOPADHYAY ET AL
and the cells. Another type of reactor used in plant cell cultivation is rotating drum reactor,
which has higher oxygen transfer ability and relatively lower hydrodynamic stress. This consists
of a horizontally rotating drum on rollers connected to a motor. Rotary drum reactor has been
shown to be superior over other reactors for the cultivation of Vinca rosea [28] and Lithospermum
erythrorhizon [26]. The performance of the different bioreactors has been summarized by Panda
and co-workers [4].
5.
Process Strategies
5.1 Selection of Cultivation Techniques
Various modes for culturing plant cells have been employed in suspension culture in order to
maximize product formation. The fed-batch mode is used in cases where the addition of a high
concentration of substrate affects the growth. The technique of repeated batch cultivation (semicontinuous mode) provides an appropriate approach towards the continuous cultivation of plant
cells when the rate of product synthesis (or biotransformation of added precursors) parallels the
rate of growth. For non-growth-associated products, the use of a two-stage culture, where cells
are propagated in a growth medium and then transferred to a production medium, would be the
ideal choice for maximizing product synthesis. Obviously, it would be important to recognize
the best physiological state of the cell for maximal product accumulation. Once the type of
bioreactor is selected for a specific plant cell process, the mode of operation will depend on the
dynamics of the specific culture [29].
Batch cultivations are characterized by constantly changing environmental conditions, and
are capable of producing metabolites associated with any kinetic pattern. Therefore, a number of
plant cell systems have been cultivated under batch mode to scale-up the process. Although
batch cultivation strategy has been widely adopted for scale-up of plant cell bioprocesses, it has
not always been successful in improving the production of desired metabolites; in many cases
the production of secondary metabolites has been reported to be decreased in scale-up process.
A variety of plant cells cultivated under batch mode for production of secondary metabolites are
summarized in Table 2. Stirred-tank bioreactors with modified impellers that impart improved
mixing under low shear have been advocated for cultivation of fragile plant cells in large scale
suspension cultures. Panax ginseng has been successfully cultivated at a large scale in 2000 liter
and 20000 liter stirred tank bioreactors to produce 500–700 mg/l of ginseng saponins [30].
Panax ginseng cell lines have been cultivated in both stirred tank and air-lift bioreactors for the
production of gingenoside. Different types of impellers (flat-blade, angled-blade disc turbine,
anchor impeller) at various impeller speeds have been used for the cell growth of Panax ginseng
and it has been observed that angle-blade disc impeller at 100–150 rpm resulted in highest cell
growth, indicating the shear sensitivity of the cells [31]. Another cell line of P. ginseng cultivated
in a 2 liter stirred tank bioreactor with a marine propeller grew fairly well up to an unusually
high agitation speed of 1000 rpm [31], indicating shear-resistant nature of the cell line.
The conditions derived from the batch cultivation can be used to design suitable fed-batch or
continuous operation to overcome the inhibitions by controlled addition of a limiting nutrient.
Fed-batch cultivation has been able to improve the productivity of ginseng by Panax ginseng
[32], and taxane by Taxus chinensis [33]. Fed-batch cultivation of Coptis japonica had a significant
effect on production of berberine at high cell density, as batch cultivation in stirred tank bioreactor
Production of Phytochemicals in Plant Cell Bioreactors 123
Table 2. Production of secondary metabolites by plant cell suspension cultures under different modes of
cultivation
Plant cell
Product
Bioreactor type, capacity
and mode of cultivation
Product
(mg/l)
Reference
Anchusa officinalis
Rosmarinic
acid
Stirred tank bioreactor,
2.5 liter, batch
3500
[36]
Aralia cordata
Anthocyanin
Jar culture vessel, 500 liter,
continuous
1090
[37]
Catharanthus roseus
Ajmalicine
Catharanthine
Serpentine
Tryptamine
Air-lift bioreactor,
20 liter, batch
6.4
3
1.6
16.1
[38]
[38]
[38]
[38]
Coptis japoinca
Berberine
Stirred tank bioreactor, 2.5 liter
batch
Fed-batch
Continuous
800
2320
3500
[34]
[34]
[34]
Holarrhena
antidysenterica
Conessine
Stirred tank bioreactor,
6 liter, batch
106
[39]
Lithospermum
erythrorhizon
Shikonin
Stirred tank bioreactor,
200 and 750 liter,
two-stage culture
4000
[40]
Nicotiana tabacum
Cinnamoyl
putrescines
Stirred tank bioreactor
batch
Fed-batch
160
400
[35]
[35]
Ginseng saponin
Centrifugal impeller
bioreactor, 2.5 liter, batch
Turbine bioreactor,
2.5 liter, batch
Air-lift bioreactor,
1 liter, batch
Erlenmeyer flask,
0.25 liter, batch
800
[41]
490
[41]
3120
[42]
1570
[43]
5800
[44]
13.8
Authors’
work
Authors’
work
Authors’
work
Panax notoginseng
Perilla frutescens
Podophyllum hexandrum
Anthocyanin
Podophyllotoxin
Erlenmeyer flask, 0.5 liter
Stirred tank bioreactor, 3 liter
batch
Fed-batch (intermittent
feeding)
Continuous with cell
retention
43.2
48.8
Taxus chinensis
Taxane
Erlenmeyer flask, 0.25 liter
274.4
[33]
Taxus cuspidata
Taxol
Wilson type bioreactor
22
[45]
124
CHATTOPADHYAY ET AL
damaged the cells due to high osmotic pressure of the culture medium. Further, the biomass
concentration was reduced due to accumulation of inhibitory products during cell growth. This
problem was resolved by suitable fed-batch cultivation, which enhanced both cell growth and
berberine production [34]. An increased production of cinnamoyl putrescines has also been
observed by fed-batch cultivation of Nicotiana tabacum in stirred tank bioreactor [35]. Production
of podophyllotoxin has been enhanced to 43.2 mg/l by fed-batch cultivation of Podophyllum
hexandrum in stirred tank bioreactor as compared to 13.8 mg/l in batch cultivation (Table 2).
Steady state continuous flow or chemostat operation, with a constant withdrawal of culture
medium and cells is commonly used for the production of growth-associated products, typically
primary metabolites and biomass. It also provides a system to eliminate product inhibition, if
any. The continuous culture technique has also been adopted for the cultivation of several plant
cells such as, Coptis japonica [34], Catharanthus roseus [46], and Nicotiana tabacum [47]. A
high cell density of Coptis japonica produced 3500 mg/l berberine when cultivated in continuous
mode in 2.5 l stirred tank bioreactor [34]. However, the cellular content of berberine in continuous
culture decreased to less than 50% of that observed in batch cultivation because the production
of berberine in C. japonica was a part of the non-growth-associated kinetics (Table 2).
Cell retention systems have been occasionally employed for the enhancement of growth and
product yield in various microbial systems for their ability to achieve high cell density in
continuous cultivation. In situ cell retention systems have been particularly successful in improving
the productivity of product inhibited cultivations, mainly because the bioreactor could be operated
at high dilution rates to flush out inhibitory products and at the same time the cells could be
retained by the filtration device [48]. Spin filter device has been applied for the somatic
embryogenesis of plant cell suspension cultures and for industrial plant propagation [49].
Podophyllum hexandrum has been cultivated in stirred tank bioreactor in continuous mode using
cell retention device and this further enhanced the production of podophyllotoxin to 48.8 mg/l
(authors’ work) (Table 2).
5.2 Precursor Feeding
Precursors of biosynthetic pathways have been used in various plant cell suspension cultures to
improve the production of secondary metabolites. Factors such as the concentration and the time
of addition of the precursor are to be considered when applying the precursor to the cell culture
medium. The addition of loganin, tryptophan and tryptamine enhanced the production of secologanin
[50], and indole alkaloids [51] by Catharanthus roseus suspension cultures. Paclitaxel yields in
the cell culture of Taxus cuspidata were improved up to six times by feeding phenylalanine and
other potential paclitaxel side-chain precursors (e.g. benzoic acid, N-benzoylglycine and serine)
[52]. Cholesterol, a precursor of alkaloid biosynthesis, was found to have a strong effect on the
production of conessine by Holarrhena antidysenterica cell suspension culture [39]. The time of
addition of cholesterol as well as its concentration had a significant effect on alkaloid synthesis.
A step feeding strategy, in which 50 mg/l cholesterol was added in 4 installments during different
phases of growth, enhanced the production of the alkaloid from 63 mg/l to 106 mg/l in 6 liter
stirred tank bioreactor; this study highlighted the importance of the physiological state of the
culture for effective transformation of the precursor to alkaloid [39].
Production of Phytochemicals in Plant Cell Bioreactors 125
5.3
Permeabilization of Plant Cells
Plant secondary metabolites are normally produced intracellularly which adds up to the cost of
downstream processing of a specific product. It is, therefore, desirable to extract the products
into the culture medium. Removal of secondary metabolites from the vacuoles of the cells would
also reduce the product inhibition and increase the productivity. Many attempts have been made
to permeabilize the plant cell membranes in a reversible manner with organic solvents.
Dimethylsulfoxide (DMSO) has been used in many cases, because it is known to extract sterols
from the membranes of the eukaryotic cells. Of various cells tested, only Catharanthus roseus
survived the treatment of DMSO [53]. Taxol has recently been extracted by various organic
solvents such as hexadecane, decanol and dibutylphthalate, in the range of 5–20% (v/v), in the
culture medium of Taxus chinensis [54]. Selection of a specific solvent system with due consideration
to its effect on cell growth may lead to substantial release and increase in the production of
secondary metabolites.
6.
Future Prospects
Plant cell cultivation is a suitable alternative to whole plant cultivation for the production of
desired compounds. However, due attention must be given to the relevant engineering parameters
influencing cell growth and secondary metabolite production. The inherent difficulties associated
with in vitro plant cell cultivation, e.g., genetic variation of plant cell lines, sensitivity to shear
stress, complex regulatory mechanism etc. are to be properly addressed for a specific cell line.
Design of a suitable bioreactor with low-shear impeller, and selection of an appropriate mode of
cultivation is required for increased metabolite production. Selection of suitable metabolic
precursors, extraction of intracellular metabolites by organic solvents can also lead to significant
enhancement in productivity of secondary metabolites.
The third author Prof. V.S. Bisaria along with Prof. Saroj Mishra and Dr. A.K. Panda of
National Institute of Immunology, New Delhi, initiated a collaborative project on production of
alkaloids by cell cultures of Holarrhena antidysenterica, a plant growing in the lawns of Department
of Botany, University of Delhi, Delhi, with the cooperation of Prof. S.S. Bhojwani about 15
years ago. Since then the activity at IIT Delhi has increased many folds with substantial inputs
of modeling and simulation expertise of the second author Dr. A.K. Srivastava, into the cultivation
strategies of plant cells in bioreactors. During the period of association, the authors were greatly
benefited by the vast expertise of Prof. Bhojwani. The authors, therefore, feel honored to dedicate
this article to Prof. Bhojwani at the time of his superannuation from a very fulfilling and
academically rewarding experience.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
8. Development of Biotechnology for Commiphora wightii:
A Potent Source of Natural Hypolipidemic and
Hypocholesterolemic Drug
Sandeep Kumar, S.S. Suri, K.C. Sonie and K.G. Ramawat
Laboratory of Bio-Molecular Technology, Department of Botany, M.L. Sukhadia University,
Udaipur 313001, India
Abstract: Commiphora wightii has become an endangered species due to its overexploitation for
its gum-resin. Guggulsterones present in gum-resin are potent lipid and cholesterol lowering natural
agents. Drugs based on these are currently used clinically in India and Europe. The plant is endemic
to Indian subcontinent, therefore major contributions on its biology, chemistry, pharmacology and
biotechnology have been made by Indian scientists. Biotechnological approaches made for
guggulsterone production by cell cultures and for its micropropagation are reviewed.
1.
Introduction
Commiphora wightii (Arnott.) Bhandari, commonly known as ‘Indian bdellium’, or ‘guggul’, is
an important medicinal plant of the herbal heritage of India. For centuries, guggul has been used
extensively by Ayurvedic physicians to treat a variety of afflictions, including arthritis, inflammation,
bone-fractures, obesity and disorders of lipid metabolism. It provides ‘guggul’, an oleogumresin whose medicinal and curative properties are mentioned in the classic Ayurvedic medical
text, the Sushruta Samhita 3000 years ago. The plant has become endangered because of over
exploitation for its gum-resin, associated with slow growth of the plant, poor seed set and
excessive tapping for gum-resin, which causes mortality of the plant. Gum-resin yields
guggulsterones effective against high blood cholesterol and lipids.
2.
Distribution
Commiphora is widely distributed in tropical regions of Africa, Madagascar and Asia. It is
generally distributed in arid regions and is particularly widespread on the Indian side of Thar Desert.
In the Indian subcontinent Commiphora species occur in Pakistan, Baluchistan and India. Of
the total 185 species, only three (C. wightii, C. stocksii and C. berryi) have been found in India.
C. wightii occurs in Rajasthan, Gujarat and Maharashtra [1].
3.
Biology
Commiphora wightii (Arnott.) Bhandari (syn. C. mukul, C. roxburghii, Balsamodendron mukul)
belongs to the family Burseraceae. A characteristic feature of the family is the presence of resinducts in the parenchymatous bark.
The plant is a shrub reaching 3 m in height with crooked, knotty branches ending in sharp
spines. The papery bark peels in flakes from the older parts of the stem, whereas younger parts
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is pubescent and grandular leaves are trifoliate. The flowers are sessile and single or in groups
of 2-3. The fruit, 6-8 mm in diameter is a drupe, which becomes red on ripening. Fruit yield and
seed set is low (about 16% in Aravalli ranges). In drier parts it is even lower. The chromosome
number of C. wightii is 2n = 26 [1]. Recently, Gupta et al. [2, 3] reported apomictic seed
development associated with polyembryony in guggul. Female plants set seeds irrespective of
the presence or absence of pollen. Hand pollination experiments and embryological studies have
confirmed the occurrence of non-pseudogamous apomixis, nucellar polyembryony and autonomous
endosperm formation. It was inferred that apomixis may have a significant role in the speciation
of tropical trees. Apomixis may be favoured by natural selection if the population densities are
low and distance between individual trees is greater than the permissible cross-pollination range.
Multiple sapling formation by the germination of polyembryonic seeds of C. wightii has also
been observed [4] thereby confirming the multiple embryo formation in seeds. In another study,
Gupta et al. [3] described the cause of low seed set in C. wightii on the basis of pollen-stigma
interaction in the non-pseudogamous apomictic plants. They observed that although pollen grain
germinated on stigma, pistil did not support pollen tube growth perhaps due to changed orientation
of the cells of transmitting tissue and absence of proteins in the intercellular matrix. This results
in poor seed set.
C. wightii is an excellent fuel wood and burns even wet due to the presence of resin in the
stem. The plant is cut mercilessly by villagers for cooking the food and used with other wet
woods to facilitate burning [5]. Due to abovementioned inherent biological and social problems
the plant has become an endangered species. An interesting biological property of ecological
significance of resin has also been reported. Essential oil constituents of resin have been shown
to enhance sexual maturation of immature adults of the desert locust [6].
4.
Chemistry of Gum-Resin
The presence of guggulsterones differentiates C. wightii from 184 other Commiphora species.
Gum-resin obtained from Boswellia serrata, another tree from the family Burseraceae and
common in the same regions, is also known locally as salai guggul or white guggul, but B. serrata
does not contain guggulsterones. It is used as an anti-inflammatory drug.
Phytochemical investigation of guggul gum-resin has been carried out by the group of Dev
[7, 8]. Guggul (oleogum-resin) of C. wightii is a mixture of 38.5% resins, 32.3 % gum, 1.45%
volatile oil, 19.5% minerals, 3.2% organic foreign matter and 3.6% other impurities. During the
separation of various products from the complex mixture, the neutral fraction was reported to
contain ketonic compounds (5.13%). It is this ketonic fraction that contains biologically important
active principles of C21 or C27 steroid, viz. Z-guggulsterol (0.01%), guggulsterol–VI (0.02%),
Z-guggulsterone (1.6%), E-guggulsterone (0.4%), guggulsterol-III (0.03%), guggulsterol-I (0.8%),
guggulsterol-IV, guggulsterol-V (Fig. 1) and some defence related secretory ketones [9–12].
Oleogum-resin is a complex mixture and needs stepwise separation [7]. Purification involves
separating soluble (45%) and insoluble (55%) components with the aid of ethylacetate, alcohol
or petroleum-ether. Insoluble fraction is associated with toxic effects while the soluble fraction
contains the guggulsterones and other constituents that are thought to impart the hypolipidemic
and anti-inflammatory effects. A method of high performance liquid chromatographic (HPLC)
separation was proposed. Recently a HPLC method for quantitative determination of E- and Zguggulsterones in C. mukul resin [13], serum [14, 15] and diet supplement [16] has been developed.
Development of Biotechnology for Commiphora wightii 131
18
H
H
17
O
16
O
3
O
O
4
Z
E
Structure of E- and Z-guggulsterones
H
OH
OH
H
CH3
OH
OH
3
O
O
4
Z-Guggulsterol
Guggulsterol-1
OH
OH
OH
OH
3
HO
Fig. 1.
4
Guggulsterol-II
O
Guggulsterol-III
Structure of guggulsterones and guggulsterols isolated from C. wightii.
In addition to these steroids, the gum-resin of C. wightii contains diterpenoids (combrene-A
and mukulol), steroids derived from pregnane and cholestane, and various carbohydrate derivatives
[17]. Upon steam distillation, the gum-resin furnishes an aromatic essential oil. The oil contains
the monoterpenes—myrcene, camphorene, polymyrcene and caryophyllene [9]. The aerial parts
of C. wightii contain β-sitosterol, myricyl alcohol and amino acids [18]. The flowers are rich in
flavonoids, most notably quercetin [19].
From the resin of C. tenuis growing in Ethiopia, 37 mono- and sesqui-terpenes were detected
and identified by GLC and GLC-MS [20]. The main components of the monoterpenoid fraction
were α-pinene (60.8%), β-pinene (8.8%), sabinene (6.3%), α-thujene (8.9%), limonene (5.5%),
3-carene (3.7%), β-myrcene (1.8%) and β-elemene (1.1%) constituting 97% of the oil. Identified
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KUMAR ET AL
sesquiterpenoid components constituted approximately 1.6% of the oil (Fig. 2). Oleanolic acid
acetate and three other triterpenes were also identified in this oil obtained by wounding the plant.
Limonene
Fig. 2.
5.
α -Pinene
β -Pinene
Myrcene
Structure of various monoterpenes isolated from C. wightii.
Medicinal Properties and Pharmacology
Many Indian medicinal plants have come under scientific scrutiny since the middle of the
nineteenth century [8]. Commiphora wightii is one such plant from which a modern medicine for
hyperlipidemia has been prepared based on ancient information. In ancient times, guggul was
used primarily as treatment for inflammatory conditions, including arthritis. The development of
gum guggul as a potent hypolipidemic agent was first reported by Satyavati working at Banaras
Hindu University, Varanasi, leading to the discovery of new anti-cholesterol drug from a plant
source [21, 22]. This work was initiated on the basis of information gathered from ancient concept
of the pathogenesis of atherosclerosis and obesity described in Sushruta Samhita.
In Ayurveda, guggul is highly valued for the treatment of several ailments including rheumatoid
arthritis, lipid disorder and obesity. Currently several formulations of Ayurveda for arthritis, joint
pain, sciatica and other ailments contain guggul. Several reports conclusively established
scientifically with modern pharmacological tests the hypolipidemic and hypocholesterolemic
properties of C. wightii extract [22–28].
The effect of guggul was very promising in experimental animal systems [24, 29, 30 and
references therein]. Immediately after trials guggul and its purified extract was established effective
hypolipidemic agent in patients with ischemic heart disease, hyper-cholesterolemia, obesity and
hyperlipidemia. In different trials with patients, a reduction of serum cholesterol (24 to 59%)
and triglycerides (22 to 30%) was recorded [31-33]. Hypolipidemic and antioxidant effects of
guggulipid, a drug prepared from guggul, were demonstrated in patients with hypercholesterolemia
[21, 28, 29, 34, 35]. Guggulipid used as adjunct to dietary therapy decreased the total cholesterol
levels by 11.7%, low density lipoprotein (LDL) cholesterol by 12.5%, triglycerides by 12% and
the total cholesterol/high density lipoprotein (HDL) cholesterol ratio by 11% [28]. ‘Guggulipid’,
a purified ketonic fraction, is presently used in India and Europe for hyperlipidemia and
hypercholesterolemia.
In addition to its lipid lowering activity, guggul may also promote cardiovascular health
through its ability to act as an antioxidant and to inhibit platelet aggregation. The guggulsterones
inhibited the oxidative modifications of lipid and protein components of LDL induced by copper
(Cu) in vitro in a concentration dependent manner. Furthermore, guggulsterones also inhibited
the formation of hydroxyl (OH–) free radicals created in a non-enzymatic system in a concentration
dependent manner [36]. Myocardial necrosis is associated with increased levels of lipid peroxides,
xanthine oxidase activity and a lowering of superoxide dismutase, which may lead to increased
Development of Biotechnology for Commiphora wightii 133
formation of free radicals with subsequent cardiac cell damage. Guggulsterones, in a manner
similar to two other cardioprotective drugs (propranolol and nifedipine), reversed this elevation
of lipid peroxides and xanthine oxidase and the decrease in superoxide dismutase activity [37].
The extract of C. wightii along with that of Terminalia arjuna, Inula racemosa showed protection
against isoproterenol induced myocardial necrosis in rats [38] or that with Allium sativum and
A. cepa showed protection against increased cholesterol and blood serum triglycerides, thereby,
confering protection against atherosclerosis and myocardial infraction [27]. Mester et al. [39]
reported total inhibition of platelet aggregation in vitro induced by adenosine diphosphate,
serotonin and adrenaline by isolated E- and Z-guggulsterones.
Guggulsterone-Z and guggulsterone-E are responsible for lipid lowering properties in human
blood and at least four mechanisms have been proposed to explain their activity. First, guggulsterones
might interfere with the formation of lipoproteins by inhibiting the biosynthesis of cholesterol
in the liver [40]. Second, guggulsterones have been shown to enhance the uptake of LDL by the
liver through stimulation of the LDL receptor binding activity in the membranes of hepatic cells
[41]. Third, guggulsterones increase the fecal excretion of bile acids and cholesterol resulting in
a low rate of absorption of fat and cholesterol in the intestine [40]. Finally, guggulsterones
directly stimulate the thyroid gland [42, 43]. Guggul induced triiodothyronine production with
possible involvement of lipid peroxidation, demonstrated thyroid stimulatory effect of guggul
administration in the experimental mice [44]. Because serum lipids, including cholesterol, are
reduced in response to increased levels of circulating thyroid hormones, the effect of guggulsterones
on the thyroid gland might explain the hypolipidemic activity and weight loss property of guggul.
The anti-inflammatory effect of guggul from C. wightii on osteoarthritis [21, 45–50] has also
been established. Anti-inflammatory effect is common in other plants of the family Burseraceae,
viz., Boswellia dalzielli, B. carteri, B. serrata and C. incisa [48, 51] and references therein).
The extract of C. molmol, another species from middle east, possesses anti-thrombosis activity
[52], anti-ulcer and cyto-protective property [53], anti-inflammatory effect [54] and cytotoxic
and anti carcinogenic effect [55, 56]. On the basis of non-mutagenic, antioxidative and cytotoxic
potential of C. molmol extract, its use in cancer therapy was recommended [55]. Similarly,
sesquiterpenes responsible for hypoglycemic activity were isolated from C. myrrha [57].
Dev [7] reported that the steroid profile of C. wightii parallels the catabolism of cholesterol
to C21 steroids. Thus, there is sufficient evidence that both in mammalian tissues and in plants,
the catabolism of cholesterol to pregnane derivatives proceeds by either of the two major pathways
as shown in Fig. 3. It is because of this property and increased demand for the natural product
for hyperlipidemia, the plant has attained great importance in recent years [8].
6.
Gum-Resin Production
In C. wightii the balsam (oleogum-resin) is present in ‘balsam canals’ in the phloem of larger
veins of the leaf and in the soft base of the stem. The development and widening of gum-resin
canal in young stem occurs schizogenously. The lumen of canal is surrounded by an epithelial
layer of parenchyma containing dense cytoplasm and shows the presence of gum and resin
droplets [58]. The walls of epithelial cells facing the lumen are thin and of fibrillar mesh. The
resinous material is synthesized within the epithelial cells and is presumably transported into the
canal lumen through the relatively porous wall [59]. Among various plant growth regulators
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KUMAR ET AL
applied on stem with lanolin paste, only kinetin increased the lumen size, while auxin and
morphactin had adverse effect causing increase in number of epithelial cells [60].
H
OH
H
Cholesterol
H
OH
OH
OH
OH
H
OH
OH
OH
O
3
OH
Fig. 3.
4
Pregnenolone
Metabolism of cholesterol to pregnenolone.
Gum is tapped in the winter season. Plants over 5 years old with a basal diameter more than
7.5 cm are suitable. Circular incisions of 1.5 cm deep are made on the main branches and stem
at a uniform distance of 30 cm apart and at an angle of 60° with the stem. The yellow, fragrant
latex oozes out through the incisions and slowly solidifies into vermicular or stalactitic pieces
which are collected manually. Subsequent collections of gum-resin are made at an intervals of
10–15 days. About 200–500 g dry guggul is usually obtained from a plant in one season.
Application of ethephon on the cuts enhances guggul production 22 times over that obtained in
control. This technique developed by Bhatt et al. [61] is inexpensive, safe and requires no skills,
and hence can be used by the tribals very easily. They established that guggul production is
maximum with the onset of summer (a stress induced secondary product formation) as supported
Development of Biotechnology for Commiphora wightii 135
by observations with bright field and fluorescence microscopy. But in the long term, excessive
production through ethephon application exhausts the plant and resultantly, kills the plant.
7.
Vegetative Propagation
Fruit set and yield of fruits per plant are very low in natural conditions. Poor seed set, poor seed
viability and harsh arid conditions are responsible for complete failure of plant establishment in
nature from seed. Plants bear fruits in April to May and August to October. About 27% fruits
contained single embryo and 7% fruits contained 2 embryos while 66% fruits were without
embryo [62]. Therefore, attempts were made to propagate the material by conventional methods
of stem cuttings and attempts are being made to propagate the plants through non-conventional
biotechnological methods.
7.1 Conventional Methods
Rooting of stem cuttings has its own drawbacks in the arid environment like termites attack,
desiccation and heat adversely affecting rooting. Rooting response of stem cuttings was shown
to be improved by application of plant growth regulators [63, 64], by selecting cuttings of
suitable length and diameter [65] and treating them with potassium salts [66]. However, such
methods are not suitable for large-scale multiplication as stock material with sufficient biomass
is not available as well as % response of the cutting is variable and affected by seasons [1].
7.2 Biotechnological Methods
Biotechnological research on C. wightii has been supported by central funding agencies since
1979 but nothing concrete has come out of these programmes which shows the difficult nature
of the material. In nature, the plant is a very slow growing woody shrub. Explants obtained from
the mature plants (stem, leaf or petiole) produce fast growing, white and amorphous callus on
MS medium containing kinetin and 2,4-dichlorophenoxy acetic acid (2,4-D). Resin exudation
from the explants makes the process of sterilization difficult. It is equally difficult to find tender
stem explants for in vitro growth. Due to these reasons detailed investigations using explant as
source material are hampered [67].
7.2.1 Clonal Propagation
Clonal propagation as a biotechnological approach is commonly applied for vegetative propagation
of selected materials. Barve and Mehta [68] described a method for clonal propagation of C.
wightii using stem explants grown on Murashige and Skoog medium [69] containing benzyladenine
(BA, 4.0 mg1–1), kinetin (4.0 mg1–1), glutamine 100 mg1–1, thiamine HCl 10 mg1–1 and activated
charcoal 0.3%. Shoots obtained from explants were incubated to elongate on medium containing
lower concentration of BA (0.40 mg1–1) and kinetin (0.4 mg1–1). These elongated shoots were
rooted by treating them with IAA and IBA for 24 h in dark and then transferred onto low salt
basal medium with activated charcoal. Six-week-old plants (5–6 cm in height) from half strength
White’s modified medium were used in transplantation. At hardening stage 60% of the transferred
plants survived. Micropropagated C. wightii plants once established in soil showed vigorous and
uniform growth with no morphological abnormalities. The lack of selected high yielding plants
and limited number of plants produced by this method are limiting factors for use of this
technique for large scale multiplication.
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7.2.2 Somatic Embryogenesis
Somatic embryogenesis in callus cultures of C. wightii has been achieved. Somatic embryo
formation was first observed in immature zygotic explants or intact ovules transferred on B5
medium [62]. Though the frequency of explants producing embryonic culture was low, immature
zygotic embryos were the only suitable explants to produce embryonic callus after reciprocal
transfers between B5 medium [70] containing 0.1 mg1–1 2,4,5-trichlorophenoxy acetic acid and
0.1 mg1–1 kinetin and that devoid of it. All other media failed to produce embryonic callus.
Further, somatic embryogenesis in callus obtained from immature zygotic embryos was possible
because of selection of embryonic cells. Embryonic cells were small, densely filled with cytoplasm
and isodiametric (Fig. 4) as compared to non-embryonic cells, which were large, elongated and
vacuolated [67]. Maximum growth of embryonic callus was recorded on MS-2 medium
supplemented with 0.25 mg1–1 BA and 0.1 mg1–1 IBA. MS-2 salts supported higher growth of
callus as compared to tissues grown on B5 medium containing same concentrations of plant
growth regulators.
Fig. 4.
Embryonic cells of C. wightii.
Embryonic callus transferred on MS-2 medium containing various combinations of ΙΑΑ and
BA produced globular, torpedo and a few early cytoledonary stage embryos (Fig. 5). Maximum
number of somatic embryos was observed on the medium containing 0.1 mg1–1 ΙΑΑ and 0.25
mg1–1 BA. Torpedo and cotyledonary stage embryos obtained from experiments for development
of somatic embryos of previous experiments were used. Activated charcoal, ABA, and agar-agar
were incorporated in MS-2 medium to generate stress and enhance maturation in somatic embryos.
Maximum number of cotyledonary stage embryos were produced on the medium containing
0.5 g1–1 activated charcoal and 10 g1–1 sucrose. In this experiment embryos were placed on filter
Development of Biotechnology for Commiphora wightii 137
Fig. 5.
Somatic embryos formed from embryonic callus of C. wightii.
paper-bridge using liquid medium. Cotyledonary stage somatic embryos kept on various maturation
media were transferred onto MS-2HF medium. Somatic embryos grown on media containing
plant growth regulators, irrespective of their concentration and combination, produced callus. A
high percentage of embryos remained ungerminated, while about 10–25% produced secondary
somatic embryos. Therefore, proper selection of mature embryos was required for high percentage
of germination. Somatic embryos showed precocious germination and callusing except those
grown on MS-2HF medium, which could be maintained for several months. By using static
medium or liquid medium with filter paper bridge, about 25% torpedo staged embryos matured
into cotyledonary stage embryos and out of these about 25% were converted into plantlets.
Thickening of hypocotyl was observed in such plantlets without elongation of internodes (Fig.
6). MS-2 medium containing 20 µg1–1 gibberellic acid was most effective for shoot elongation
in such plantlets. About 200 plantlets were successfully established in garden soil (Fig. 7) to
verify the survivability of regenerants. Survivability was 95% for the plantlets [71–75].
The embryo formation from zygotic embryo and ovule explants may be a case of induced
polyembryony as already reported in this plant [2, 4]. This achievement opened new avenues of
research on C. wightii like cell culture and embryogenesis in bioreactor, formation of resin
canals in vitro and production of guggulsterones in these organized cultures.
138
KUMAR ET AL
Fig. 6.
Young planlets formed after germination of somatic embryos.
7.3 Guggulsterone Production
Unavailability of sufficient guggul from natural sources and destruction of plants from most of
the localities, initiated the search for alternative methods of guggulsterone production. Cell
culture is an excellent alternative to produce secondary metabolites. Cell suspension cultures of
C. wightii were derived from leaf callus in MS medium containing 0.15 mg1–1 each of 2,4-D and
kinetin. Cells were immobilized in calcium alginate beads. The immobilized cells in stationary
phase suspension cultures were less viable but they were active in the synthesis of guggulsterols.
Guggulsterol production was 1.97% in stem explants, 0.22% in callus culture (2-month-old) and
0.32% in cell suspension culture (25-day-old) [76]. Guggulsterone is produced in resin canals
and hence unorganized cultures proved unproductive for this purpose. However, use of organized
cultures, in vitro produced embryos and hypocotyls may prove better sources and are being
evaluated.
8.
Prospects and Research Need
Commiphora wightii has become an endangered plant species because of high demand for its
gum-resin. This plant has become a torchbearer of efficacy of plants used in the Indian system
of medicine for treatment of complex human syndrome. Therefore, many more plants are being
reinvestigated for their properties mentioned in the ancient system of medicine. The complexity
of molecules in mixture warrants their production by natural sources using biotechnological
methods.
Development of somatic cell cultures in static and liquid medium opened new avenues of
research on this material because large quantities of aseptic material in organized form can be
Development of Biotechnology for Commiphora wightii 139
Fig. 7.
Potted plantlets of C. wightii, about 4 months old.
obtained. It is known that the organized material produces several-folds higher amount of active
principle as compared to unorganized cultures. This can effectively be used for immobilization
of embryos, germinated seedlings, seedling parts, and so on. All these are available in aseptic
condition in a material which is otherwise difficult to sterilize in large quantities due to presence
of resin and hence sticky nature of the explants. Large amount of aseptic material is required for
bioreactor culture and failure of the system due to contamination adds a lot to the cost factor of
running the system. Besides, the advantages of mass propagation and development of artificial
seeds are evident from the results and need not to be emphasized again (Fig. 8). The other new
approach is the development of hairy root culture system using Agrobacterium rhizogenes for the
production of active principle again on the lines as described above for the organized cultures.
Somatic
embryo
Embryogenic
callus
Plantlets
Direct somatic
embryogenesis
Plant
Clonal
propagation
Immobilization
of organs
Guggulsterone
production
Fig. 8.
Indirect somatic
embryogenesis
Growth in
bioreactor
Scale-up technology
for mass propagation
Schematic presentation of approaches for guggulsterones production and mass
propagation of plants
140
KUMAR ET AL
Acknowledgement
This work was supported by grants from Department of Biotechnology, Government of India,
New Delhi (grant No.BT/R&D/08/23/95) to K.G. Ramawat.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
9. Biotechnology in Quality Improvement of Oilseed Brassicas
Abha Agnihotri1, Deepak Prem2 and Kadambari Gupta2
1
Bioresources and Biotechnology Division, TERI, Habitat Place, Lodhi Road, New Delhi 110 003, India
2
Centre for Bioresources and Biotechnology, TERI-School of Advanced Studies, Habitat Place,
Lodhi Road, New Delhi 110 003, India
Abstract: The review presents a comprehensive description of the advances made towards desired
quality improvements in rapeseed mustard. The two nutritionally undesired elements, namely erucic
acid in the seed oil and glucosinolates in the deoiled meal, are discussed in reference to their
nutritional implications and inheritance. The genetic manipulation of fatty acid biosynthetic pathway
for diversified uses is also presented. It elaborates the use of biotechnological methods in terms of
their conjugation with various other conventional approaches for genetic enhancement and value
addition by developing the desired superior genotypes of brassicas for nutritional and industrial
purposes.
1.
Introduction
Production of crop varieties with increased sustainable production is the most challenging task
facing the plant breeders in the current century. Considering the limited resources such as
diminishing and deteriorating cultivable land, water supply, fertilizers etc., improvement in
terms of yield and quality within a limited time frame, is the demand of the present and future
generations. Since the increased yield alone may not sustain the needs of human nutrition,
improvement of nutritional quality and value addition for diversified uses are of prime importance.
The oilseeds form the second largest agricultural commodity in India. Among the nine annual
oilseed crops grown in the country, oilseed brassica rank second in importance contributing
about 30% to the total oilseed produced. It is one of the best edible oils available, having lowest
amount of saturated fats as compared to other vegetable oils, provides both essential fatty acids
and also the animal feed through oil free meal rich in protein having well balanced aminogram.
The oleiferous Brassicas being the provider of edible oil to a major proportion of our population
are prime targets for quality improvement. The presence of high amounts of two nutritionally
undesired elements in Indian varieties (40–50% erucic acid in the seed oil and upto 300 µm/g
glucosinolates in the deoiled meal) pose a huge challenge to plant breeders working on improving
oil and deoiled meal quality in Brassicas. The canola quality exotic rapeseed cultivars, commonly
known as double low or ‘oo’, having less than 2% erucic acid in the seed oil and less than
30 µm glucosinolate/g oil free meal [1] were not found suitable for cultivation under Indian
agroclimatic conditions. Since, among the oilseed Brassicas, B. juncea acquires the maximum
share of cultivated area in our country, the improvement of nutritional quality in B. juncea is
most desired to suit our needs. The brassica fatty acid profile is also amenable to alterations for
developing designer crops for specific food or non-food industrial applications thus having
prospects of diversified uses.
Biotechnology in Quality Improvement of Oilseed Brassicas 145
2.
Seed Oil Quality
The nutritional quality of vegetable oils is considered significant in modern living. Oil quality
is described in terms of saturated, monounsaturated and polyunsaturated fatty acids. Mustard oil
contains the lowest amounts of saturated fatty acids as compared to other vegetable oils and also
has a very good proportion of n3 and n6 polyunsaturated fatty acids, thus considered beneficial
for food consumption. In most vegetable oils, the unsaturated fatty acids consists mainly of oleic
and linoleic acid. However, mustard oil is an exception since in addition to oleic acid (8–15%),
linoleic acid (13–20%) and linolenic acid (6–14%), it also contains erucic acid (41–50%), and
palmitic and stearic acids in trace amounts [2, 3]. Erucic acid contributes approximately 50% of
the total fatty acids in mustard oil. However, it is nutritionally undesirable and the high erucic
acid B. napus oil is reported to be less metabolisable [4]. High erucic acid content is also known
to cause impaired myocardial conductance, increased blood cholesterol and cardiac lipidosis
with accumulation of erucic acid in mammalian system [5, 6].
High concentration of oleic acid is preferred for cooking purposes since it is thermostable.
Both linoleic and linolenic acids are essential fatty acids that need to be supplied in diet from
external sources. However, high linolenic acid in the oil being prone to peroxydation causes
flavor revision and oil deterioration [7] and therefore 3 to 5% is preferred to meet the dietary
requirement. Linoleic and linolenic acid are both produced by a common biosynthetic desaturation
pathway [8]. Therefore, selection for high linoleic acid has tended to increase the level of
linolenic acid, while selection for low linolenic acid tends to decrease the level of linoleic acid
also. For this reason selection within the same germplasm may not be able to meet the breeding
objectives. Therefore, the fatty acid scenario in mustard oil implies that efforts should be made
towards development of cultivars having low levels of erucic and linolenic acids, high levels of
oleic and moderate linoleic acids.
The success of developing B. juncea with low erucic acid suitable for Indian agroclimatic
condition has been limited due to search for appropriate gene pool. Stefansson et al. [9] and
Downey [10] identified genotypes with a genetic block in the biosynthesis of eicosenoic and
erucic acid in summer rape (B. napus) and summer turnip rape (B. campestris), respectively.
Studies for inheritance of erucic acid content have shown that it is controlled by multiple genes
and the seed erucic acid level is controlled by the embryo genotype in B. napus [11, 12]. Kirk
and Hurlstone [13] have reported two genes showing dominance and acting in an additive
manner for erucic acid biosynthesis in B. juncea. Kirk and Oram [14] identified zero erucic
genotypes of B. juncea and following this low erucic acid genetic stock among Indian accessions
of B. juncea was also identified [15]. Recently the development of early maturing, low erucic
acid strains of B. juncea and B. napus, suitable to grow under Indian agroclimatic conditions,
have been reported and are under the advance stages of testing [16, 17]. In B. juncea, the
predominantly grown oilseed brassica in India, the main emphasis has been on successful reduction
of erucic acid and the work in the direction of developing cultivars with variable fatty acid
profile for edible or industrial purposes is just beginning.
3.
Deoiled Meal Quality
The defatted Brassica meal contains about 40% protein with a well balanced aminogram and is
used as animal feed [18]. Brassica oil meal is particularly rich in lysine and methionine, which
are essential amino acids not found in cereal grains. For this reason, Brassica oil meal has been
146
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used for animal feed. However, the feeding value of rapeseed-mustard meal has been limited
because of the presence of sulfur containing compounds called glucosinolates present in the
vegetative tissues and seeds of cruciferous plants [19]. The various kinds of glucosinolates
present in B. juncea, in decreasing order of their abundance are gluconapin, sinigrin, progoitrin,
napoleiferin and glucobrassicanapin. At cellular level, glucosinolates are stored in the vacuole
[20, 21] and myrosinase, a glycoprotein enzyme responsible for hydrolysis of glucosinolates is
stored in a tonoplast-like membrane bound organelle called the idioblast [22]. On mechanical
injury, myrosinase catalyzed hydrolysis of glucosinolates occur to form thiocyanates, isothiocyanates
and nitriles. Although glucosinolates as such do not cause much harm, their breakdown products
are undesirable in animal feeds. These compounds impart a characteristic flavor and odor to
Brassica vegetables and condiments but may reduce palatability and adversely affect iodine
uptake by the thyroid glands in non-ruminant animals such as swine and poultry. Thus they
reduce the feed efficiency in terms of development and weight gain [19, 23].
To avoid glucosinolate hydrolysis products to accumulate in Brassica oil meal, the myrosinase
enzyme is heat inactivated as one of the first steps in oil extraction process. However, this in turn
also causes the breakdown of other proteins, which may adversely affect the nutritional value of
the oil meal quality. In India heat treatment of seed before oil extraction is usually not done,
therefore the extracted edible oil has relatively large proportion of glucosinolates breakdown
products imparting the characteristic pungency in the oil. In view of these facts, a prime breeding
objective for B. juncea quality breeders is to develop low glucosinolate varieties.
Work in the direction of glucosinolate inheritance in Brassicas was started in 1970’s and was
revolutionised by the discovery of a low glucosinolate B. napus cultivar ‘Bronowski’ from
Poland [24]. Since then, this genotype has provided the source of low glucosinolate gene for
practically all cultivated Brassicas. Kondra and Stefansson [25] had proposed that in B. napus
the maternal genotype rather than the embryo genotype controlled seed glucosinolates. They
have proposed low glucosinolate level to be controlled by as many as 11 recessive alleles that
do not show independent segregation. Rather than linkage to be operative, a simultaneous action
of genes for a common biosynthetic pathway for synthesis of all glucosinolates has been suggested.
The formation of individual glucosinolates is thought to occur through a chain break at the end
of the biosynthetic pathway [26]. Further, Lein [27] has also determined an additional influence
of cytoplasm on glucosinolate synthesis.
Till date no germplasm source for low glucosinolate genes has been reported in B. juncea.
However, Love et al. [28] developed the low glucosinolate B. juncea line BJ-1058 using interspecific
hybridization between Indian mustard and a low glucosinolate strain of B. campestris having the
Bronowski gene block. Glucosinolates also show tissue level variation within the same plant
[29-31] and the leaf glucosinolate quantity and profile can be correlated to the seed glucosinolate
level only in small seedlings suggesting that the glucosinolate content in the leaves and seeds
may be under different genetic control [32, 33]. The glucosinolate profile at the seedling level
may serve as a tentative tool to predict glucosinolate profile of seeds, but its authenticity is
doubtful since differentiation is not clearly understood and may produce unpredictable and
drastic changes [34]. There have been some reports indicating that the genes for glucosinolate
contents in vegetative tissue are pleiotropic or linked with the grain filling stage [35, 36], and
that glucosinolates may contribute towards resistance to insect pests and pathogens [32, 33].
Biotechnology in Quality Improvement of Oilseed Brassicas 147
4.
Quality Status in B. juncea
Most of the work related to quality improvement has been globally concentrated on B. napus
and the work on genetic enhancement of B. juncea quality, the predominant species of Asian
subcontinent, is somewhat limited. The facts discussed above suggest that an ideal genotype of
mustard from the point of view of nutritional quality would be one having low erucic acid in the
seed oil, low levels of glucosinolate in the seed or reproductive tissue and high glucosinolate
content in the vegetative tissue. This may seem to be a mammoth task but it has already been
accomplished in B. napus [33] and is being extensively researched on for B. juncea.
Several double low strains/cultivars of B. napus are available globally, however, the progress
of work to develop double low B. juncea has not been very successful as yet, primarily due to
the lack of suitable donor germplasm. Following the successful introgression of low glucosinolate
genes, the double low B. juncea strains have been developed in Canada through cross breeding
of BJ-1058 and LDZ (a zero erucic acid, high oil content B. juncea strain). The progeny of this
cross was backcrossed to the B. juncea var. Cutlass, in order to incorporate white rust resistance
genes. This material has shown promising results in field trials, and is being improved for its
fatty acid profile [37]. In India also, attempts have been made to introgress the double low
characteristics in various Indian mustard cultivars [38, 39] but the desired success is yet to be
achieved and these low erucic/low glucosinolate/double low strains are being improved for
agronomic characteristics.
5. Conventional Approaches for Development of Double Low Cultivars
The conventional breeding techniques for quality improvement vary greatly and have evolved
from simple mass selection to hybrid cultivar development. The breeding strategies depend on
the objective and practical scientific considerations such as inheritance pattern of genes responsible
for a particular trait. The backcrossing approach has been successfully used to transfer simply
inherited traits such as low erucic acid. The erucic acid content of the seed is controlled by the
genotype of the embryo, that is, the individual F2 seeds borne on F1 plants have different erucic
acid level. This fact led to the development of the half seed technique [40]. This approach has
been used by Kirk and Hurlstone [13] to develop low erucic B. juncea lines. The use of backcross
technique for development of low glucosinolate B. juncea is limited due to the non-availability
of any natural low glucosinolate source and polygenic inheritance, but it has been successfully
used for development of low glucosinolate B. napus [41].
Both B. napus and B. juncea are predominantly self-pollinated species [42] and thus the
commonly used breeding tool of pedigree selection can be employed for cultivar development.
A clearly defined breeding objective and identified suitable parent is a pre-requisite to start a
pedigree selection program [40]. Backcrossing in conjugation with pedigree selection has been
used successfully for the development of early maturing canola quality B. napus cultivars in
India [17, 43]. Further, modifications in the methods can be done as per available germplasm
resources or breeding objectives. However, due to the involvement of multiple recessive genes,
development of double low B. juncea by conventional methods alone is proving to be a lengthy
process, thus necessitating the need for incorporation of suitable biotechnological tools such as
doubled haploids production, mutagenesis and molecular approaches to facilitate the quality
improvement in a targeted manner.
148
6.
AGNIHOTRI, PREM AND GUPTA
Quality Improvement Using Doubled Haploids
The efficient production of doubled haploid plants from anther or microspore cultures has
become an important new tool for Brassica breeders [44]. After initiation of work for this school
of thought in late 1970’s, efficient protocols to induce embryogenesis in isolated microspore
cultures of B. napus have been reported by several workers [45-48]. A promising double low
variety of B. napus (cyclone) developed through doubled haploid technique using isolated
microspore culture, is being commercially cultivated in Canada.
Doubled haploids provide several advantages over conventional breeding approaches. The
selection of desired genotypes at F1 haploid level followed by diploidization fixes the desired
genes i.e., leads to the production of pure lines that do not segregate further. Hence homozygosity
can be achieved in one step equivalent to repeated in-breeding for several generations (8–10
years). Doubled haploids being homozygous, also considerably reduce the time required for
parental identification for hybridization programmes [49].
This technique also offers the advantage of significantly smaller population size required to
find the least likely recombinants particularly when several genes are involved [50]. Since both
glucosinolates and erucic acid are multiple recessive gene governed traits, doubled haploids
offer a powerful tool to reduce the perfect population size to approximately 70 to 80 fold than
what would be needed to be handled via conventional methods. An integrated approach involving
application of doubled haploid technique with early selection for high erucic acid in the cotyledons
of microspore derived embryos obtained from the F1 hybrids of winter oilseed rape established
a positive correlation in the erucic acid content of embryos and the seeds derived from them,
thus elucidating the efficiency of selection at microspore embryo stage [51].
In addition to the complicated inheritance of glucosinolates, additional effects of maternal
inheritance [25] and cytoplasmic influence [27] on seed glucosinolates have been reported.
Since glucosinolates in seed are governed by maternal genotype rather than the seed embryo’s
genotype [52], the F1 seeds would show glucosinolate content as per maternal parent genotype.
This means that effective selection can only be done in F3 seeds produced by the F2 population
and in order to follow this procedure under field conditions at least 3 years would be required to
reach initial screening. However, the pollen/microspore is not the target site for glucosinolate
storage [53] and has minimal cytoplasm, therefore seeds produced by the doubled haploids
would reflect the genotype of the doubled haploid plant rather than the parent. Moreover, since
haploids would express recessive genes, transgressive segregants for recessive traits can efficiently
be recovered through diploidization of recessive haploids.
7.
Quality Improvement Through Mutagenesis
Mutagens are also being effectively employed to generate considerable variability in fatty acid
composition [54-57]. Chemical mutagenesis has been used to produce B. napus lines with
reduced linolenic and increased linoleic acid contents [58]. The low linolenic acid lines have
been used as genetic base for the development of B. napus with less than 3% linolenic acid and
more than 22% linoleic acid [1]. Doubled haploid lines of B. carinata with modified erucic acid
content have also been identified through chemical mutagenesis by EMS treatment of isolated
microspores [55, 59]. Mutagens have not only been used to produce variable fatty acid composition
but also to substantially increase the oil content. Kumar et al. [56] have reported an increase in
oil content upto 4.55% using gamma irradiation in B. juncea. An increase in oleic acid content
Biotechnology in Quality Improvement of Oilseed Brassicas 149
coupled with decrease in erucic acid was also observed in the treated varieties. Wong and
Swanson [60] and Auld et al. [61] recovered high oleic acid producing doubled haploids through
chemically induced mutagenesis in microspore cultures of B. napus.
The use of induced mutations for altering fatty acid profile allows for the selection of variants
with either complete or incomplete sets of functionally altered genes responsible for fatty acid
synthesis [62]. However, a major limitation of mutagenesis is that apart from the genes controlling
the target trait, it may cause several changes in the genetic background thus affecting the
non-target traits. For example, mutants for the high oleic acid content have been shown to be
associated with undesirable agronomic characteristics [63]. It is considered likely that several
genes code for ∆12 desaturase enzymes (that are responsible for conversion of oleic to linoleic)
in B. napus seed and that some of these genes also regulate production of ∆12 desaturase in the
vegetative tissue. Mutation exposure would lead to non-tissue specific changes in both the seed
and vegetative tissue ∆12 desaturase genes, which could have detrimental effects on the vegetative
tissue where the correct fatty acid composition is required for normal membrane structure and
function [63].
As discussed above, mutagenesis aims at altering the existing fatty acid profile and does not
have the ability to add genes for new biosynthetic pathways for production of novel fatty acids.
Although mutagenesis has been fairly successful for producing genotypes with altered fatty acid
compositions, its non-tissue specific action restricts its use for cultivar development. Nevertheless,
the lines derived from mutation breeding programs serve as important donor material in forthcoming
crop improvement programmes, and the abovementioned bottlenecks can be quite satisfactorily
overcome by using the transgenic approach.
8.
Quality Improvement Through Genetic Engineering
The potential use of genetic engineering to modify plant seed oil composition has been recognized
for a number of years. The oilseed crops have the potential to produce high quality edible oils
as well as speciality oils having commercial applications. For instance jojoba is a rich source of
wax esters, coconut is rich in capric, lauric and myristic fatty acid, palm has a high proportion
of palmitic, oleic and stearic acid, whereas linseed is a rich source of linoleic acid [64]. These
fatty acids are used in a wide range of products ranging from the production of soaps, detergents,
cosmetics, surfactants, lubricants, plastics, varnishes and pharmaceuticals. Due to the nondomestication of most of the potential sources and their restricted availability, at present the fatty
acids for industrial applications are mostly derived from petrochemicals. However, in the near
future, with the biased use of global reserves of fossil derived hydrocarbons alternative sources
of industrial fatty acids from the environment friendly oil crops are sought after [65]. This can
be achieved either by altering the existing fatty acids profile or by adding new genes for synthesis
of novel fatty acids.
In most of the oil bearing crops, the biosynthetic pathway of fatty acid synthesis is similar
[66] and their differential accumulation in the seed is genetically controlled depending upon the
species. During the seed development process, photosynthetically fixed carbon is imported into
the seed in the form of sucrose, and is converted into the storage products with the help of
enzymes present in the seed. The seed contains all the enzymes that are required for the conversion
of sucrose into any of the storage products. However, it is the rate of sucrose uptake by the
various biosynthetic pathways that lead to the differential accumulation of a particular storage
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product in the seed [67]. Thus the genetic manipulation of any of the biosynthetic pathway can
lead to a specific ratio of seed storage product, according to the end use of the seed. This can be
done either by modifying the length of the existing hydrocarbons in fatty acid chain (modifying
the chain elongation enzymes) or by changing the position of double bonds (modifying desaturase
enzymes).
The seed specific or tissue specific genetic modifications may be used for creating changes in
endogenous fatty acid biosynthesis pathway or addition of new biosynthetic pathways. The use
of seed specific antisense technology has allowed for the selective modulation of key enzyme
activities in the developing seed, while keeping the rest of the genetic background of the plant
absolutely constant [68]. Co-suppression based on post-transcriptional gene silencing of endogenous
desaturase gene has shown promising results in developing high oleic acid genotypes of rapeseedmustard [69]. The recently derived RNAi approach has also shown a great potential for endogenous
desaturase silencing. Using this concept total silencing of the ∆12 desaturase gene in B. napus
has been acheived, resulting in the production of genotypes accumulating 89% oleic acid in the
seed oil [70].
The rapeseed oil normally contains low levels of lauric acid (C12) and stearic acid (C18) at
a concentration of 1–2% and 0.1–0.2%, respectively. High lauric rapeseed can be used as a
substitute in detergent markets, leading to displacement of conventional lauric oils derived from
coconut or palm kernel, whereas high stearic rapeseed is a useful substitute in margarine markets
and replaces conventional hydrogenated rapeseed oil. The two most notable achievements in oil
modification to-date are the 40% stearic and 40% lauric rapeseed varieties (laurical) first produced
and entered in field trials by Calgene in 1993–94 [71]. Thus laurical was the first genetically
manipulated rapeseed variety given permission for commercial cultivation in 1995 in US. The
∆9 stearoyl ACP desaturase gene which normally converts stearic to oleic acid was partially
inactivated in rapeseed using antisense technology, resulting in the accumulation of a seed oil
containing up to 40% stearic acid [68]. This high stearic variety contains an antisense copy of
a Brassica stearate desaturase gene which inhibits the function of the normal rapeseed stearic
desaturase gene, resulting in an accumulation of stearic acid, rather than their saturation to
oleate. The resulting high stearic oil has many advantages over the normal rapeseed oil for the
production of certain solid fats, such as margarines.
With the advent of transgenic technology, the genes coding for enzymes that synthesize
industrially important fatty acids can be transferred from non-traditional crops into more important
oil crops. The canola oil having low erucic acid has food applications in margarine, salad and
salad dressings while the high erucic rapeseed has industrial application. The canola quality
rapeseed has also been genetically modified for containing high levels of β-carotene. This high
carotenoid canola oil may prove very beneficial to combat the vitamin A deficiency in developing
world [73]. Various species of Brassicaceae have been transformed with mutated Sn-2 acyltransferase
gene from yeast and have been reported to show increase in seed oil content, seed weight and
erucic acid content [72].
Lauric oils are mainly used in soaps and detergents although their use in confectionary fats
and milk formulas is also being investigated. Lauric acid which is present at insignificant levels
in rapeseed is found at high levels in the seed oil of the California Bay plant, Umbelluria
californica, due to the presence in the latter species of a lauryo-ACP thioesterase. This gene has
been cloned from the Bay plant and inserted into rapeseed causing premature chain-termination,
Biotechnology in Quality Improvement of Oilseed Brassicas 151
resulting in a novel variety with a seed oil containing almost 25% lauric acid [74]. Following this
an Sn-2 acyl transferase gene (LPAAT) from coconut has been introduced in lauric rapeseed to
increase the accumulation of lauric acid in the seed triacyl glycerol molecules [75].
Similar to the development of lauric acid producing rapeseed, several novel genes coding for
altered fatty acid synthesis have been used for altering seed fatty acid profile. Some worthy
examples are Caprilic and Capric acid (from Cuphea spp.), myristic acid (from Myristica fragrans),
Crepenylic acid (from Crepis alpira), Richinolic acid (from Castor), Vernolic acid (from Crepis
palaestina) and petroselenic acid (from Coriandrum sativum) [64]. Thus, in future, plant derived
oils may be an important source of industrial oil derived chemical or oleo chemicals.
8.
Conclusion
Genetic enhancement for improvement in the quality of rapeseed-mustard is a prime breeding
target for Brassica breeders all over the world. In addition to being the second most important
edible oilseed crop in India, the rapeseed-mustard oil also finds its use in industrial applications
[76, 77]. The advent of biotechnology has provided the plant breeders with new and more
accurate tools that have the ability to compress the time taken in directed evolution of crop
species. Efforts towards developing improved quality rapeseed varieties have been consolidated
and expedited at global level through the use of biotechnological techniques such as doubled
haploid and mutagenesis in conjugation with the conventional methods. The advances in molecular
approaches, the genetic engineering and transformation, has made it possible to develop many
designer rapeseed varieties with specific fatty acids profile for edible and industrial purposes.
The work is in progress towards such quality improvement in Indian mustard, B. juncea also.
Such targeted value addition in the quality of oilseed Brassicas will not only provide for an
improved source of human nutrition but also for non-food, fuel/non-fuel industrial products that
could reduce the load on ever depleting natural oil and gas resources.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
10. Role of Biotechnology for Incorporating White Rust
Resistance in Brassica Species
Kadambari Gupta2, Deepak Prem2 and Abha Agnihotri1
1
Bioresources and Biotechnology Division, TERI, Habitat Place, Lodhi Road, New Delhi 110 003, India.
2
Centre for Bioresources and Biotechnology, TERI–School of Advanced Studies, Habitat Place,
Lodhi Road, New Delhi 110003, India
Abstract: The productivity of the oilseed brassica species, the second most important edible
oilseed crop in India, is adversely affected by several fungal diseases, white rust being one of the
them. White rust caused by Albugo candida may cause 17–34% yield losses which may enhance
upto 60% under environmental conditions favourable to disease infestation. While most of the
cultivated species of brassicas are susceptible to this fungal disease, some sources of white rust
resistance have been reported in widely related species. Apart from conventional methods of selection
and hybridization, several in vitro techniques have been used to utilize these genes for incorporating
resistance/tolerance in the cultivated varieties. The article presents a comprehensive status of
Indian cultivated brassicas vis-a-vis white rust resistance, and the use of biotechnological tools
such as embryo rescue, somatic hybridization, somaclonal variation and molecular techniques for
incorporation of disease resistance.
1.
Introduction
Oleiferous Brassicas are the important cash crops of India and stands second only to groundnut
among the nine annual oilseeds being cultivated. It contributes 27 and 25.3% to the total oilseed
production and hectarage, respectively, and is cultivated in about 6.81 million ha with 6.96 m
tons production of oilseed [1]. The productivity level of 1022 kg ha–1 is far below that of the
developed countries (2500–3000 kg/ha) and the world average of 1500 kg/ha (Economic Survey,
2000–2001, GOI). This is mainly due to certain abiotic and biotic factors that adversely influence
the average yield of cultivated varieities.
Amongst the abiotic factors, drought, frost and salinity are a major cause of concern since they
may cause yield losses to the magnitude of 20–70% [2]. The important biotic factors are weeds
and insects-pests causing 17–41% and upto 60% loss, respectively, whereas fungal diseases
alone can cause major damage to the crop contributing to a yield loss of upto 70% under favourable
conditions for the disease infestation.
The fungal diseases that attack Brassica species in India throughout the cultivated areas
include Alternaria blight caused by Alternaria brassicae and Alternaria brassicicola, white rust
caused by Albugo candida and Downy mildew caused by Peronospora parasitica. The other
diseases that attack Brassicas, but are distributed more commonly in the temperate regions
include sclerotinia stem rot caused by Sclerotinia sclerotiorum, blackleg by Leptosphaeria maculans,
club root by Plasmodiophora brassicae and powdery mildew caused by Erysiphe cruciferarum.
Role of Biotechnology for Incorporating White Rust Resistance in Brassica Species 157
Among the abovementioned fungal diseases white rust has emerged as a major limiting factor
in production of Brassicas causing a loss of 17–34% [3, 4] which may reach upto 60% depending
upon the severity of infection and environmental conditions [3, 5-9]. In addition, Downy mildew
that alone does not cause much of damage, when combined with white rust causes synergistic
damage resulting upto 35% yield loss [10].
B. juncea, the most predominantly grown Brassica species in India, is highly susceptible to
white rust disease. Although B. nigra, B. oleracea, B. napus, B. carinata and some species of B.
campestris have been reported to be comparatively tolerant to this disease, adequate amount of
resistance is not available in cultivated Brassica species.
This article discusses the white rust disease in terms of its symptoms, effect on plant system,
its physiology, inheritance, available sources of resistance along with the biotechniques utilized
to achieve the adequate amount of resistance in Brassicas.
2.
White Rust
2.1 Symptoms
White rust caused by fungal pathogen Albugo candida (Pers.) Kunzee belonging to family
Albuginacae appears in almost all rapeseed mustard growing states of India. A. candida can
infect all above ground parts of the plant, producing characteristic white blisters known as sori
[10, http: // www. extento. hawaii. edu/kbase/crop/Type/a_candi.htm]. The fungal pathogen
attacks the plant at both vegetative and reproductive phase. In the vegetative phase the fungal
pathogen infects leaves and cotyledons causing local infection resulting in the appearance of
white to creamy yellow pustules on the abaxial (lower) surface corresponding to tan yellow
pustules on the adaxial (upper) surface of the leaves such that disease can be easily recognized
from the upper surface of the affected leaves. The pustules rupture after maturity and release
white coloured dust of spores known as sporangia. With the increase in duration of disease,
tissues around the pustules become necrotic and lead to senescence of leaves.
At the flowering stage the fungus causes systemic infection, leading to extensive distortion,
hypertrophy, hyperplasia and sterility resulting in severe inflorescence malformation known as
staghead [10, 11]. This leads to early foliar infection and abnormalities in reproductive organs
leading to complete sterility. This systemic staghead infection of the inflorescence is often in
association with Peronospora parasitica [4, 12, 13]. However in a recent report it has been
elucidated that the inflorescence malformation in B. juncea is due to A. candida and not because
of P. parasitica [14].
2.2 Effect on Plant
White rust has a significant impact on the yield and quality of seeds. It also has a profound effect
on important end products such as total oil content, fatty acid composition and seed protein
content. The fungal infection tends to decrease dry matter, and increase erucic acid [15]. High
proportion of erucic acid is reported to cause impaired myocardial conductance and increased
blood cholesterol and is thus nutritionally undesirable [16]. A positive correlation exists between
the amount of chlorophyll, sugars, flavonoids, waxy deposition on leaves, total phenols and the
extent of infection by Albugo candida. The moderately resistant cultivars contain higher amount
of the abovementioned biochemicals than the susceptible cultivars at all stages of growth
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GUPTA, PREM AND AGNIHOTRI
[17-21]. Phenols, in particular, have been reported to impart resistance whereas more proteins
led to higher disease severity [22, 23]. Thus, white rust infection not only damages the plant
morphology but also disrupts its physiological metabolism.
2.3 Biology of the Fungus
The biggest challenge in breeding white rust resistant brassicas is in the fact that as many as 13
pathotypes of Albugo parasitize different cruciferous plant species [24 -28]. Besides infecting
cruciferous plant species, Albugo also finds its host in several wild species of different families
such as Portulacaceae, Chenopodiaceae, Amaranthaceae, Convolvulaceae, Boraginaceae to
name a few. The white rust races are classified based upon their ability to infest different host
species. The different races that infect Brassica species are given in Table 1. However, these races
at times, may not retain their species specificity and can also attack the related species, i.e. host
specificity in Albugo candida is not an absolute adaptation to a particular species especially when
the races are from hosts sharing a common genome.
Table 1.
Various pathotypes of white rust and their specific host species
White Rust Pathotypes
Race
Race
Race
Race
Race
Race
Race
Race
Race
Race
Race
Race
Race
1
2
3
4
5
6
7
8
9
10
11
12
13
Host Species
Raphanus sativus
Brassica juncea
Armoracia rusticana
Capsella bursa pastoris
Sisymbium officinale
Rorrippa islandica
Brassica campestris
Brassica nigra
Brassica oleracea
Sinapis alba
Brassica carinata
Brassica juncea (Indian isolates)
Brassica campestris var. toria (Indian isolates)
Source: Singh et al. [28].
2.4 Inheritance of Resistance
The information on the genetics of host parasite interaction for white rust has centered on the
level of specificity between the races of pathogen and genotypes of related host species [29].
Genetic analysis of available white rust resistance through biometrical techniques has elucidated
a digenic mode of inheritance with duplicate gene action in B. napus [9, 30] and monogenic
dominant resistance in B. juncea [31-38]. Cheung et al [39] and Prabhu et al. [40] have confirmed
this through gene mapping. The white rust resistance in the three Brassica species, B. campestris,
B. nigra and B. carinata, is reported to be under the control of a single dominant gene [27,
41-44]. It is suggested that a few major genes in Brassica are responsible to initiate the disease
resistance whereas other minor genes may be involved in the control of the intensity of sporulation
of the fungus in the plant [45]. However, additive genetic variations were also found to be
predominant for the intensity of white rust resistance [35, 46-50] and thus differential expressions
have been obtained.
Role of Biotechnology for Incorporating White Rust Resistance in Brassica Species 159
2.5 Sources of Resistance
The traits for resistance to white rust are found to be present in some species of Brassica as well
as in related weedy and wild speices. Among the various Brassica species grown in India, B. napus
and most cultivars of B. oleracea [41, 51, 52], some species of B. campestris [53-57] and B.
carinata [58] have been reported to exhibit moderate resistance and thus utilized as a source
of resistance to white rust. Recently moderate resistance has also been found in B. tournefortii
[59, 60] and in certain species of related genus Diplotaxis [61] and Sinapis alba [58]. Eruca
sativa has been identified as a potential source of white rust resistance and all the accessions of this
genera are reported to be resistant to race 2 that attacks B. juncea [62].
3.
Disease Control Strategies
The different strategies adopted to control plant diseases include non-chemical and chemical
control. The non-chemical control includes hot water treatment and biological control [63, 64].
The chemical control, though found to be effective, results in development of resistance in the
pathogens and residual toxicity [10], thus having detrimental effect on non-target species. Besides
this, the fungicide sprays affect crop physiology independent of disease occurrence. These may
decrease the triacylglycerol fraction of the oil and increase the diacylglycerol fraction [15], thus
affecting the oil quality. Owing to the abovementioned problems associated with chemical control,
focus has been to develop new biotechnological techniques for crop protection and production.
An upcoming alternative approach to crop protection is the use of externally applied biotic
and abiotic stress inducers that activate plant’s natural defence mechanism. They create a
hypersensitive response in the plant thus leading to systemic acquired resistance. For instance,
actigard, an isonicotinic acid derivative identified by Syngenta has been recently commercially
utilized to prevent downy mildew on spinach [65]. Under such biological stresses, plant synthesize
a variety of compounds that include phytoalexins and pathogenesis-related proteins [66-68].
However, work in this direction is still in infancy and commercial products are yet to be realized
on a large scale.
Among the biological approaches, exploitation of genetic resistance, present in the existing
plant species for its incorporation in the cultivated varieties, is seen as the eco-friendly and
environmentally safe approach [10, 69]. This includes sexual hybridization, wide hybridization,
somatic hybridization, somaclonal variations and genetic engineering. The possibility of using
these biotechniques to enhance the scope and efficiency of transfer of desired traits with special
emphasis on white rust for improvement of crop Brassicas are briefly discussed.
3.1 Selection and Hybridization
Several germplasm lines and popular cultivars have been screened for white rust resistance and
varying degree of response to A. candida has been reported in B. juncea [58, 70–75]. However, it
was found that the selected lines are not stable for the trait, and breakdown of resistance occurs in
successive years [74]. The breakdown of resistance may be due to mutations in the existing
pathotypes leading to new pathotypes. This indicates that there is a continuous need to expand the
genetic base for white rust resistance. Intervarietal transfer of disease resistance has been attempted
and resistant F2 generation plants have been selected by Chauhan et al. [76]. However, response
of the plants to pathogen is yet to be studied under field conditions.
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GUPTA, PREM AND AGNIHOTRI
B. juncea cultivars have also been hybridized with other species of Brassica such as B. napus
[52, 77, 78] and advanced progenies of the cross were identified to possess similar degree of
response to white rust as the resistant donor. B. carinata has also been utilized for transfer
of disease resistance in B. juncea and moderate disease tolerance was observed in the hybrids
[51, 79]. However, B. juncea accessions having resistance to white rust are under different stages
of development.
3.2 Wide Hybridization
Genes conferring resistance to biotic and abiotic stress are frequently scattered in weedy and
widely related species that can be used for the incorporation of resistance in cultivated varieties
[80]. However, majority of these belong to secondary and tertiary gene pools. Their exploitation
is problematic because of the difficulties in obtaining hybrids and subsequent gene transfer in
desirable genetic background due to pre- and post-fertilization barriers.
Pre-fertilization considerations include spatial separation, asynchrony of flowering, pollination
system, floral characteristics and competitiveness of pollen whereas post-fertilization considerations
include genetic/sexual compatibility, hybrid viability, fertility of progeny and successful
introgression. For successful gene introgression all pre- and post-fertilization requirements must
be met, failure of any one requirement will lead to non-introgression of the gene, and thus would
not produce the desirable results [81, 82]. A large number of interspecific and intergeneric sexual
hybrids have been produced using in vitro techniques to study the compatability barriers that
exists between the species [83]. These techniques have been scantily utilized in Brassicas for
transfer of disease resistance traits, however, there are a few reports for transfer of Alternaria
blight resistance. Chevre et al [84] generated hybrids between B. napus and S. alba through
ovary culture to transfer resistant traits for Alternaria blight. Similar study was made utilizing
B. campestris and B. spinenscens through sequential embryo rescue technique by Agnihotri et al.
[85]. However, Alternaria blight resistant genotypes are yet to be realized and only a preliminary
report by Gupta and Agnihotri [86] is available for transfer of resistance to white rust in Brassica
species utilizing this technique.
3.3 Somatic Hybridization
Somatic hybridization involves enzymatic removal of the cell wall and the resulting spherical
protoplasts are fused together. Fusion of protoplasts at the level of plasma membrane is nonspecific, and there is no barrier to interspecific, intergeneric or even intertribal fusion of cells.
The resulting hybrid cells are cultured and subsequently regenerated to give rise to somatic
hybrids. The use of this technique can bypass both pre- and post-fertilization barriers [87, 88].
Among the cultivated Brassica species, the main focus so far has been on B. napus and B.
oleracea and this technique has not been utilized effectively in other Brassica species. Attempts
have been made to transfer resistance traits to some fungal diseases in Brassicas through somatic
hybridization such as Leptosphaeria maculans (Black leg) in B. napus [89-96] and B. olearacea
[97]; Plasmodiophora brassicae in B. napus [98] and B. oleracea [99]; Alternaria blight in B.
napus [98, 100] and B. oleracea [101-105]. Therefore, as apparent several studies have been
undertaken to transfer black leg, downy mildew and Alternaria blight disease resistance, mainly
in B. napus and B. oleracea, and only a few reports are available for transfer of white rust
resistance in B. oleracea [106, 107] and B. juncea [108]. However, in both the cases the somatic
Role of Biotechnology for Incorporating White Rust Resistance in Brassica Species 161
hybrids obtained were sterile and could not be utilized further. Hence, production of hybrids that
are either sterile or do not survive, mainly due to meiotic irregularities, is the major drawback
of this technique.
3.4 Somaclonal Variation
Somaclonal variation, regarded as the spontaneous epigenetic variations that occur in vitro, have
been a source of genetic variation suitable for crop improvement. Somaclonal variations have
been utilized for many abiotic stress resistance traits, however, it has been scantily used for
disease resistance. Somaclones have been selected for salt tolerance [109], high yield [110] and
for transfer of Alternaria blight disease resistance in B. juncea [111]. So far only one study has
been published for resistance to white rust in B. juncea through generation of somaclones [111],
reporting a stable and heritable resistance till R2 generation in the field.
3.5 Molecular Techniques
Recent developments in DNA marker technology has led to a better understanding of the complex
genome of various crop plants. Molecular markers that are tightly linked to the trait of interest,
besides helping in identifying the desired species at any growth stage of the plant [112], also
helps to select for the trait under strict quarantine laws [113-115]. However, even then the crop
plants have to be tested for virulence against the pathogen to confirm the effectiveness of the
marker associated with the resistant gene [116]. Marker assisted selection, or MAS as it is
commonly known, has been successfully utilized in identifying oil quality in B. napus [117-119]
and B. campestris [120, 121], seed coat colour in B. napus [122] and B. juncea [123], and for
fungal disease Leptosphaeria maculans in B. napus [44, 124].
Work is in progress to identify the genes responsible for resistance to white rust for use in
molecular assisted selection. In B. juncea, resistance to white rust race 2 was observed to be
controlled by a single dominant allele. With the help of restriction fragment length polymorphism
(RFLP) a locus Acr [39] and Ac21 [40] have been identified in B. juncea. Recently, flanking
markers have been identified for a white rust resistant locus, AcAl in B. napus [34] and in B.
campestris [45], and Ac2t in a Polish B. juncea accession [125]. Similarly, 3 genes namely Ac71,
Ac72 and Ac73 have been identified for resistance to white rust race 7 in B. campestris [43].
However, the use of these markers in molecular assisted selection has not been successful as yet
[39, 40, 43] mainly because of their specificity to their respective host species.
Work has been undertaken to identify the molecular markers that could be used in precise and
efficient screening. Two markers, WR2 and WR3 [40] and OPNOl1000 and OPBO61000 [125]
linked to white rust resistance have been identified which flank the resistant locus. Prabhu et al.
[40] have reported that although these markers were effective in identifying the presence or
absence of the resistance gene in the population of the cultivars, these are specific to the Russian
source of white rust resistance. Work is in progress to study the mechanism of resistance response
and mapping of the loci responsible for resistance to white rust [43, 39]. Although, a few markers
linked to white rust resistance locus have been identified in some species of Brassica, work has
to be consolidated to employ these markers in routine marker-assisted selection for efficient
utilization. Furthermore, the focus has now been shifted from identifying trait linked markers to
the mapping of the genes to utilize them more efficiently in developing new cultivars [126].
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During the past decade, different strategies have been used to produce transgenic plants that
are less susceptible to disease caused by phytopathogenic fungi and bacteria [127]. For achieving
transgene derived resistance, genes from organisms other than plants and endogenous plant
genes have been utilized. The basic concept of utilizing these genes revolves around induction
of systematically acquired resistance utilizing transgene mediated production of pathogenesis
related (PR) proteins [127]. Almost 14 distinct PR-protein groups have been identified from
different plants [68, 128] and have been utilized for transgene mediated production of PRproteins. This technique has been successfully utilized in cereals [68, 129, 130, 131]. However,
transgene mediated resistance for white rust has not been exploited in crop Brassicas because of
poor understanding of pathogenesis related proteins and their role in trigerring systematically
acquired resistance towards Albugo candida.
4.
Conclusion
Oilseed Brassicas contribute to about 30% of the edible oilseeds being produced in India.
However, in spite of the horizontal increase in the area and production, vertical increase in
productivity per unit area has remained far below the yield potential of presently cultivated
varieties. This is mainly due to various abiotic and biotic stresses, fungal diseases being one of
them. The major fungal diseases affecting oilseed Brassicas are Alternaria blight, white rust and
downy mildew, which together cause severe yield losses under environmental conditions favouring
disease infestation. White rust caused by Albugo candida is reported to be under the control of
digenic inheritance with duplicate gene action in B. napus and monogenic dominant resistance
in B. juncea. Upto 13 races of Albugo candida affecting different Brassica species have been
reported that are host specific. Strategies for controlling white rust have mainly focussed on
development of disease resistant cultivars, exploiting the resistance available within crop species
and also utilizing the modern biotechnological tools to tap resistance from secondary or tertiary
gene pool. Utilization of resistance genes from wide species has been a promising proposition
since some of the widely related species of crop Brassicas such as Eruca sativa, species of
Diplotaxis and B. tournefortii have been reported to have resistance/tolerance to white rust.
These sources of resistance have been utilized by various scientists for introgression of resistance
genes in the cultivated varieties. Some work has also been undertaken to identify and clone the
resistance genes and develop molecular markers for precise selection. Systematic characterization
of the Indian gene pool of Brassicas and its related species for identification of white rust
resistant genes is important and work in this direction is being pursued at various national and
international institutions. Utilization of these genes through the use of biotechnological tools
will help in expediting the development of varieties having resistance to white rust.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
11. Current Trends in Forest Tree Biotechnology
E.M. Muralidharan and Jose Kallarackal
Division of Genetics and Division of Plant Physiology, Kerala Forest Research Institute,
Peechi 680653, Kerala, India
Abstract: Modern tools of biotechnology offer a variety of options through which it is possible to
match the strides made in crop improvement in agriculture and horticulture. Current trends in forest
tree biotechnology indicate that this is indeed happening and that some of the hurdles of conventional
forest tree improvement are no longer a serious bottleneck. The progress made in in vitro culture
of forest trees and the current status of application of the technology is discussed. The trends in use
of molecular tools particularly the wide variety of DNA markers available and the identification of
novel genes controlling traits of interest are examined. The current status of the technology in
genetic transformation of forest trees is also reviewed. The bio-safety issues in forest biotechnology
especially those relating to transgenic trees are presented without bias to either side of the ongoing
debate.
1.
Introduction
With growing realization of the ecological role of forests in sustaining life on earth and the
consequences of indiscriminate exploitation being felt in several parts of the world, afforestation
and reforestation have in recent decades been high on the agenda of most nations. While agriculture
and horticulture advanced by leaps and bounds in bringing about a quick domestication of a
large number of plant species that supply the world most of its food, fodder and fibre, another
primary need, namely, shelter, was based on wood biomass that continued to be taken out from
the natural forests. Productive lands being always chosen for growing food crops, the trees have
been relegated to the fringes except where agroforestry has been the tradition. Until the latter
part of the last century, much of the wood based produce had been taken out of the natural
forests, which was considered inexhaustible. It has become clear that this activity is no longer
sustainable or environmentally sensible. Unbridled clearing of tropical forests has been particularly
severe in several of the poorer nations that happen to be the richest in biodiversity. The loss of
biodiversity is often inestimable in some of the biodiversity hot spots.
In consonance with the traditional emphasis given to agriculture, in plant biotechnology too,
the accent has always been on crop plants. The relatively short history of domestication, the long
lifecycles and the large size of most trees and inaccessibility in the wild have always been a
disadvantage to researchers.
However, intensive forestry has become the order of the day when the availability of land and
other factors are making traditional forestry practices increasingly unsustainable. Trees with
shorter rotations, and genetically improved for disease and pest resistance, superior form etc.,
have been deployed in plantations in many of the developed nations where genetic improvement
programmes has been initiated. The advent of biotechnology in the past two decades, however,
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broadened the scope of genetic improvement of trees, mainly by removing the hurdles encountered
in conventional breeding programmes. Partly due to the environmental concerns and also due to
the increasing realization of the advantages of intensively managed plantations of fast growing
tree crops, the interest in application of biotechnology to forest crops has been kindled. Mixing
of trees with other crop plants (agroforestry) is also gaining more popularity in several countries.
Biotechnological research has a major role to play in developing the right kind of trees for interplanting among annual crops [1]. While the role of traditional knowledge and conventional
technology in conservation and utilization of plant resources cannot be underplayed, it is clear
that to keep pace with the growth and development of human civilization, strategic changes in
our approach will be necessitated.
This article takes a perspective view of the area of forest tree biotechnology and evaluates the
progress made to date and examines the current trends in four areas of research and application,
namely, in vitro culture techniques and micropropagation, application of molecular biological
tools to forestry, genetic transformation of trees and biosafety vis-à-vis forestry.
2.
In vitro Culture of Forest Trees
More than half a century of developments in in vitro culture of trees has not significantly
changed the empirical methods used to induce morphogenesis in cultures. The latter half of the
last century saw the development of protocols for in vitro culture and plantlet regeneration in
several of the important tree crops. But in spite of the volume of work done, it is still rare to find
protocols that are dependable in terms of efficiency and repeatability. Additions have been made
to the list of plant growth regulators found to be useful for culture, with TDZ and ancymidol,
being used with success in several plant species including trees.
The factors underlying the maturation of trees, which is a great hurdle in the propagation of
important species, are still imperfectly understood. Two approaches to circumvent this problem
are (i) maintenance of juvenility over long periods through cryopreservation or long term in vitro
storage, while field trials are on with clones of juvenile origin, so that promising clones can be
mass multiplied easily at the end of the testing period, and (ii) the induction and use of juvenile
material from mature trees. The problem of physiological aging which has been noticed in
micropropagated plantlets of juvenile origin and which eventually results in losses, e.g. in volume,
is an issue of great concern. Rejuvenation of 20-year-old Radiata Pine through somatic embryogenesis induced from vegetative apical meristems [2] does indicate a possible solution to this
problem. Aderkas and Bonga [3] have reviewed the factors influencing rejuvenation in trees
using methods of enhancing micropropagation through manipulations that involve application of
osmotic, temperature or hormonal stress. There is a need to re-evaluate the morphogenetic
competence of tissues from different levels of the tree. No significant differences were found in
bud break, shoot multiplication or callus derived from cambium taken from top branches and
epicormic shoots of Robinia pseudoacacia [4]. Epicormic shoots are considered more juvenile
compared to the top branches and generally believed to be better explants for in vitro morphogenesis.
In tree species of the humid tropics the presence of endophytes within the tissues pose a
serious problem in establishment of sterile cultures. Although this fact is rarely highlighted in
literature, it is without doubt a vexing problem that needs to be tackled with a combination of
techniques such as proper selection of explant type, prophylactic treatments of plants, use of
antimicrobial agents for pre-treatment of explants and for inclusion in the culture media.
Current Trends in Forest Tree Biotechnology 171
Browning of explants, contaminations and the effect of seasons still remain major obstacles
for establishment of cultures from mature trees. In mature female trees of Ceratonia siliqua [5],
shoot culture initiation was greatly influenced by season, with the highest survival percentage
observed in spring.
Some definite advantages have been shown for micropropagated forest trees. A comparison
was made between the growth of trees produced by micropropagation from nodal stem sections
or callus tissue of a 20-year-old silver birch (Betula pendula) tree with that of seedlings [6].
Micropropagated trees were more uniform in height and trunk girth than seedling trees, and
more than 80% of the trees flowered within three years of field planting, whereas only 39% of
seedling trees flowered within the same period. Besides, micropropagated trees had lesser bark
fissuring (a desirable character) than seedling trees. In Loblolly pine [7] the early reduced
growth and mature morphology observed in plantlets derived from cotyledons does not occur in
micropropagation from fascicular and axillary shoots or epicotyls. Increased biomass production
was observed in micropropagated plants when compared to seedling progenies of the plus trees
of two species of eucalyptus [8].
The recent advancements made in plant regeneration through somatic embryogenesis in several
hardwood and conifer trees [9, 10] will greatly facilitate efficient mass propagation, conservation
and genetic transformation. Eucalyptus are perhaps the most popular among the plantation tree
crops around the world. In vitro regeneration systems in the different Eucalyptus species have
been developed particularly using somatic embryogenesis [11-14]. The development of transgenics
is thus facilitated. Shoot induction as well as somatic embryogenesis were induced on zygotic
embryos in several genotypes. Among the other important tree species where improvement
continues to be made in technology is the American chestnut (Castanea dentata) a timber and
nut-yielding tree that has been on the decline due to the chestnut blight, where use of tissue
culture and genetic engineering for restoration is approaching reality. Plantlet regeneration
through germinated somatic embryos and microshoots derived from somatic embryos were
obtained from developing ovules [15].
Among broad-leaved trees, micropropagation using shoot cultures appears to be the predominant
method for cloning. Several important genera of forest trees can now be successfully
micropropagated either through multiple shoot induction or somatic embryogenesis e.g. Acacia,
Albizzia, Casuarina, Dalbergia, Prosopis, Eucalyptus, Populus, Ficus, Bambusa, Dendrocalamus,
Phyllostachys besides important tree species like Azadirachta, Gmelina, Tectona, and species of
Salix, Shorea, Cassia etc. [16-18]. India, with its rich biodiversity of tree species and with a long
list of successful reports of in vitro culture [16], still has very few trees micropropagated on a
large scale. However, the Micropropagation Technology Parks set up by the Department of
Biotechnology (DBT) have been successful in scaling-up of protocols for forestry species such
as eucalyptus, bamboo, poplars and teak used in energy plantations and reforestation [19].
Among the forest trees of the world, teak (Tectona grandis) is an important species in which
work in standardizing micropropagation, cryopreservation and transformation continues to be
carried out in several countries [20, 21]. The absence of an in vitro regeneration and transformation
method in teak has been a major bottleneck for development of transgenics in this important
timber species. Improved cryopreservation of in vitro shoot tips were obtained in teak [22].
Somatic embryogenesis has been reported from hypocotyls, endosperm, stem segments and
protoplasts of sandalwood. Improvements in the techniques are being made [23-25]. The use of
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extracts of a cyanobacterium, Plectonema boryanum for somatic embryogenesis, in the absence
of hormones, and the successful encapsulation in a composite gel of 50% silica gel and 4%
sodium alginate and their germination were also reported in sandal [26]. Somatic embryogenesis
and proliferation through repetitive embryogenesis were achieved in Dalbergia sissoo, an important
timber tree of the sub-tropics, from callus derived from immature embryos [27]. Direct organogenesis
and plantlet regeneration were also obtained from semi-mature and mature cotyledons of D.
sissoo [28].
Bamboo and rattan (canes) are forest plants of great importance to the tropical countries
especially of South East Asia where a significant proportion of the population utilise them as a
source for a wide range of products of both traditional household uses and for food and industrial
uses (paper and pulp). Interest in scientific management of bamboo and rattan resources has
however been relatively recent. Application of biotechnology to the genetic improvement and
conservation of these plants assumes importance since the potential for improvement in productivity
is tremendous and resource management through conservation and establishment of plantations
is gaining importance in many parts of the world.
In bamboo, tissue culture and micropropagation have been very successful and the technology
has been commercialized for several years now. Regeneration has been obtained from a variety
of explants [16, 29, 30], but rapid and high rate of multiplication are achieved through sprouting
of nodal explants taken from in vitro raised seedlings. The formation of shoot cultures from
secondary branches of culms of mature culms is much more difficult and reports are still few
[31, 32]. Cultures derived from seeds and nodes of in vitro raised shoot cultures or mature culms
have also produced embryogenic callus and regenerated plantlets [29]. Artificial seeds of
Dendrocalamus strictus were produced [33] by encapsulation of somatic embryos. Minimal
growth and storage up to 8 months was achieved in Bambusa arundinacea and Thyrsostachys
siamensis cultures stored at 5°C or 10°C or on media containing different concentrations of 2chloroethyltrimethyl ammonium chloride (CCC) or butanedioic acid mono (2, 2-diemthyl-hydrazide)
diaminozide. Such in vitro methods can therefore be expected to increase the availability of
planting material for much longer periods than is possible through seeds.
The results of small-scale field trials using tissue culture plants derived from mature culms
and seedlings of bamboo were reported by Mascarenhas et al. [34]. They observed early culms
formation and improvement in several other growth parameters in tissue culture raised plants as
compared to seed-raised plants.
Although the induction of suspension cultures and the isolation of protoplasts from different
species of bamboo have been reported [35, 36], further progress has apparently not been obtained
in utilizing the cultures. Virus-free plantlets and salt resistant plantlets have been regenerated
through in vitro culture [35]. The flowering cycle in bamboo is unique and gregarious flowering
resulting in the death of the entire population takes place in cycles of 12–120 years depending
on the species. This has been a hurdle in the propagation and breeding of bamboo. In vitro
flowering is a first step in bringing about a control on the phenomenon so that studies could be
carried out. This phenomenon has been reported in several bamboo species [37, 38]. It however
appears that an understanding of the factors responsible for flowering both in vitro as well as in
nature, is still eluding us and the benefits of the procedure cannot yet be realized. The in vitro
strategies available for genetic improvement of bamboo have been discussed [39].
Rattans are climbing palms, which are the source of rattan or cane, which is an important raw
Current Trends in Forest Tree Biotechnology 173
material for wickerwork in several countries of the South East Asia. Overexploitation of the wild
resources and lack of sufficient plantations have resulted in several species being endangered.
Tissue culture procedures have been standardized for different species in India [40, 41], Thailand
[42], Philippines [43] and Malaysia [44]. Maziah [45] found a growth dependency of in vitro
micropropagated Calamus manan on vesicular arbuscular mycorrhiza (VAM) prior to transplanting
to field. Multiple shoots and in vitro flowering were reported in C. thwaitesii [46] in embryo
cultures. Regeneration from mature plant tissues is however not very successful in rattans.
Evidence of somatic embryogenesis from root tip explants of mature plants of C. manan has
been reported [47]. Mass multiplication of superior genotypes will depend on perfection of this
technique. Until then tissue culture will perhaps be useful for rare species which do not produce
enough seeds for meeting the demand for planting stock. Genetic transformation of rattan will
also be facilitated if an efficient regeneration system based on somatic embryogenesis is available.
A large volume of literature is available on in vitro culture of conifers [48-51]. Somatic
embryogenesis and high quality plantlet regeneration have been achieved and several patents
attest to the commercial interest in this technology [52-54]. Somatic embryogenesis has been
achieved in about 30 species [51] and methods involving immature embryos have been used
wherein cleavage polyembryony is induced as in the natural case or the formation of an embryonal
suspensor mass (ESM) from the different parts of the embryo is obtained. The different pathways
to embryogenic cultures and the importance of osmolarity regulation for normal development
and conversion of somatic embryos are now understood [51]. Organogenesis from embryonic
cotyledons of Radiata pine is a well-established procedure and field trials have been conducted
in New Zealand [55]. Rejuvenation, as evidenced by complete restoration of rooting competence
of Sequoia sempervirens was achieved [56] through in vitro grafting of adult shoot tips onto
juvenile rootstocks in vitro repeatedly for five times. They also found that rejuvenation was
correlated with a disappearance of adult-associated esterase and peroxidase isozymes and an
appearance of isoesterases and isoperoxidases that were characteristic of juvenile-phase shoots
and hence these isozymes could serve as markers to assist phase-change investigations. The
development of efficient in vitro regeneration systems in conifers through somatic embryogenesis
has facilitated the genetic transformation of a number of conifers. The potential for automated
systems for plantlets regeneration and delivery to soil have also been discussed by Gupta et al.
[57].
3.
Application of Molecular Biological Tools to Forestry
A wide variety of DNA-based markers have been developed and procedures are getting simpler
and inexpensive. Nuclear and chloroplast based Single Sequence Repeat (SSR), interSSR, Random
Amplification of Polymorphic DNA (RAPD), SSCP, Amplified Fragment Length Polymorphism
(AFLP), microsatellite DNA, Expressed Sequence Tags (EST) and Sequence Tagged Sites (STS)
are some of the commonly used markers used for genome and QTL mapping for conservation
and understanding of the evolutionary genetics and sequences controlling traits of economic
interest of forest trees.
Genetic linkage maps and mapping of QTLs have been prepared for a wide range of trees
using RAPD and AFLP markers [58, 59]. Molecular markers linked to specific traits can predict
inheritance and is one of the most important applications of biotechnology in tree improvement.
Wilcox et al. [60] used genome mapping in loblolly pine and identified a locus behaving as a
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single dominant gene imparting resistance to fusiform rust disease. Years of conventional genetic
analysis had failed to detect any such resistance factor. The characterization of the phytoplasma
causing the spike disease of sandalwood and its detection has been aided by the use of PCR [61].
The power and versatility of neutral DNA-based marker technologies allow for flexible highresolution investigation of genetic variation at different levels of the population [62]. Both
nuclear and organelle DNA can be studied with advantage since the rate of evolution of the two
are different. Grattapaglia et al. [62], studied several tropical tree species with different DNA
technologies to devise strategies for in situ and ex situ conservation. Markers can be powerful
tools for tree improvement at both the early and advanced stages of the breeding programme.
Marker assisted selection (MAS) will be particularly useful in studying the inheritance of quality
traits in later stages of tree breeding. RAPDs with their dominant inheritance only detect one
allele per locus and hence the information content is low, but high quality genetic maps of trees
can be generated using simple reagents, if some care is taken to ensure repeatability [63-65].
AFLP markers too are versatile and use simple reagents, but a single assay generates much
higher information [66, 67]. Faster and cheaper DNA marker technologies are becoming available.
DNA chips that permit simultaneous analysis of thousands of loci are just around the corner and
increased accessibility will make their routine use possible for forestry conservation programmes.
Genes associated with control of flowering are of particular interest in trees because of their
potential to promote or inhibit flowering or in determination of gender. Ecological effects of
transgenic trees could in a large measure be under control through manipulation of flowering.
Our understanding of the genetic pathways controlling phase change, flower initiation and
flower development has improved in recent years. Homology between the Arabidopsis flowering
genes, LEAFY and flowering organ identity gene AGAMOUS and genes involved in flowering
in conifers have been identified [67, 68]. Expressed Sequence Tag (EST) studies have been used
to identify genes important in the regulation of flowering. The range of options that are available
for control of flowering in transgenic trees [69] is encouraging.
Much research has been done to develop new clones of trees that are resistant or tolerant to
different kinds of environmental stress such as salinity, drought, flooding etc. Abiotic stress
affecting productivity of tree crops such as drought and heat-shock stress also has attracted the
attention of forest biotechnologists. Mayne et al. [70] identified a S-Adenosyl Methionine Synthetase
(SAM-S) cDNA by differential screening of a cDNA library constructed from root mRNA from
jack pine (Pinus banksiana) seedlings exposed to two cycles of drought conditioning. The
increase in the rate of SAM-S enzyme activity after drought conditioning was also correlated
with increase in rates of ethylene and betaine synthesis. Investigators [71] have cloned and
characterised three cDNAs (PgEMB22, 27 and 29) predicted to encode low-molecular-weight
(LMW) heat-shock proteins (HSPs) from white spruce (Picea glauca) somatic embryos by
differentially screening a zygotic embryo cDNA library. They were developmentally regulated
during somatic embryo development and germination and also showed strong response to heatshock stress. Abscisic acid and polyethylene glycol, stimulators for spruce embryo maturation,
could also induce the HSP genes. A cDNA clone (pLP6) of a gene, which is repressed under
water deficit and by wounding, was isolated from a loblolly pine (Pinus taeda) cDNA library and
characterized [72]. The predicted polypeptide for pLP6 bears strong resemblance to a number of
Class I chitinases although some of the diagnostic domains are absent.
Tolerance to oxidative stress can help the plants to survive in many adverse conditions. To
Current Trends in Forest Tree Biotechnology 175
achieve this, stem explants of a poplar hybrid (Populus tremula × P. alba [P. canescens]) clone
were co-cultivated with Agrobacterium tumefaciens strain C58pMP90 having binary vectors
with constructs with bacterial genes for either glutathione reductase (GR) (gor) or glutathione
synthetase (GS) (gshII) [73]. When gor was targeted to the chloroplasts, leaf GR activities were
up to 1000 times greater than in all other lines. These results suggest that overexpression of GR
in the chloroplasts increased the antioxidant capacity of the leaves and that this improved the
capacity to withstand oxidative stress. The high chloroplastic GR expressors showed increased
resistance to photoinhibition. The herbicide methyl viologen inhibited CO2 assimilation in all
lines, but the increased leaf levels of glutathione and ascorbate in the high chloroplastic GR
expressor persisted despite this treatment.
Use of markers has been of indirect application in improving the productivity of forest trees.
For the reclamation or reforestation of poor soils the use of nitrogen fixing trees are of great
potential. Improved survival and productivity of such trees are obtained if specific strains of
nitrogen-fixing bacteria are used for inoculation of the seedlings. The identification of such
strains through conventional microbiological means is extremely slow and unreliable and therefore,
studies on suitability and nodulation behaviour and persistence in soil will be facilitated by the
use of genetic markers. In the identification of Frankia strains that are symbionts of Casuarina
the use of amplified nifH and rDNA segments has been found useful [74, 75].
Lignin, the complex polymer constituent of the secondary cell walls of xylem tracheids and
fibres of trees is of great interest to breeders, wood and paper/pulp industry and biotechnologists
alike. While playing an essential role in the plant structure and function, lignin causes severe
problems in the efficient utilization of biomass for pulp production as well as for food. Modification
of the lignin composition and content through suppression of the key enzymes involved in the
biosynthesis is the target of several genetic transformation studies around the world. Some of the
genes of interest in the lignin biosynthesis pathway are caffeic acid/5-hydroxy ferulic acid
o-methyltransferase, Phenylalanine ammonialyase (PAL), p-Coumaric acid: CoA ligase genes
[76-79]. Transgenics with alteration of wood colour phenotypes [79], reduced lignin, repression
of lignin biosynthesis, high-cellulose, accelerated growth [80, 81] have been obtained. Sequencing
of cDNAs isolated from specialized tissues of wood has been used as a tool to identify genes
involved in wood formation [82]. Such studies will help to increase our knowledge of the
environmental influence on wood properties.
4.
Genetic Transformation of Trees
A significant number of crops such as corn, soybean, tomato and cotton in many developed
nations and also in China consist of genetically engineered plants. However, transgenic trees are
yet to be released commercially. Progress is being made rapidly and the constraint appears to be
the time taken to complete the field trials. Among the important tree crops where transgenics
have been reported are eucalyptus [83], quaking aspen [84], sweet gum—Liquidambar styraciflua
[85], larches, spruces and pines [86]. The particle bombardment and Agrobacterium mediated
gene transfer systems have both been used successfully.
Advancements in molecular tools have made possible the development of flexible and adaptable
expression vector systems for plant transformation utilizing Agrobacterium or biolistics. They
aim at a cassette system that allows quick and easy replacement of promoter, terminator, markers
or the gene of interest. Besides the genes of interest, the promoters are a factor of importance.
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Wound induced promoters [87] hold great potential for use with insect resistance genes to avoid
constitutive expression, which is potentially a threat to non-target organisms including plant
friendly ones.
Genetic engineering for resistance to pests, diseases and environmental stress is a major
objective in forestry. As of today the Bacillus thuringiensis (Bt ) and the Cry family of genes
against insect pests are the most widely studied. Although the strategy appears not without
drawbacks—effective concentrations of the toxin are difficult to achieve and resistance may
develop over time. Further research to modify the genes involved and to engineer multiple genes
into trees may find acceptance. Proteinase inhibitors are another choice. Here too, high levels of
protein are required for killing the target organisms and there is a need to target expression to
specific organs of the tree. Controlling the cytokinin producing genes [88] forms an alternative
strategy that is closer to the natural defense mechanisms of plants and therefore safer and more
acceptable than Bt genes. As discussed above options are becoming available for preventing any
gene flow from transgenics to wild plants like induction of sterility [69].
Agrobacterium-mediated gene transfer is the method of choice for many plant biotechnology
laboratories because of the high percentage of single-copy and single-locus insertion events
compared to biolistics. In Norway spruce [89], increased transformation efficiencies of 1000fold from initial experiments were obtained, where little or no transient expression was detected
by varying the strain of Agrobacterium, source material and co-cultivation conditions. In loblolly
pine, transient expression increased 10-fold utilizing modified Agrobacterium strains. Both
A. tumefaciens and A. rhizogenes have been used for stable transformation in conifers. The
reporter or selectable marker genes most commonly transferred to conifers are the uidA
and nptII genes and among the useful genes, the Bt and genes controlling lignin synthesis
[81, 85, 86], herbicide resistance and control of flowering [68].
5.
Biosafety vis-à-vis Forestry
The advocates of a ban on plant genetic engineering cry hoarse over the lack of sufficient testing
of the modified organisms. They, in particular, point to the risks involved in escape of genes to
wild relatives of the crop plants, resulting in the modified plants turning into weeds or unproductive
strains, and the danger of toxic gene products on animal and human systems on ingesting them.
The pro-genetic engineering lobby, with scientists and the corporate sector with a stake in the
spoils, dismiss the arguments. Admittedly, all the possible risks have not been assessed and the
debate is far from over. It would, therefore, be prudent for all concerned in these early years of
GMO’s to bring in an extra measure of caution lest a Thalidomide or Minimata be repeated.
Regulations have been primarily designed for annual plant species with which agriculturists
are familiar. Genetically modified trees have to be treated as a separate category because unlike
annuals, a much larger time frame is involved in growing and testing the perennial species, and
several aspects of the biology of most tree species are relatively less studied and understood. The
possibility of trees modified for fast growth and resistance to stress, turning to weeds or smothering
other vegetation in a low intensity management regime is not to be ignored. The interaction of
the introduced gene in the genome over a long period of time needs careful monitoring. Danger
of silent genes getting activated (atavism) during the different growth phases of the tree is
another possibility, given our inadequate understanding of developmental biology. Faster growing
trees, transgenic or otherwise, carry a price tag—greater water and nutrient demand and reduced
Current Trends in Forest Tree Biotechnology 177
opportunity for nutrient cycling leading to site deterioration over a few rotations. This leads to
the use of fertilizers, or in tropical areas, the increased probability of abandoning the site. In
biotechnological applications for pests and disease resistance, Bt genes are known not to discriminate
between pests and friendly insects and can also affect the biodiversity of the plantation, which
is important although not comparable with that of a natural ecosystem. However, it is possible
to restrict the range of insects affected through modification of the genes involved.
Transparency in the regulatory and supervisory processes in testing of GMO’s is yet to be
evident. The issues relating to transgenic forest plantations has been debated in IUFRO, which
represents the professional forest scientists all over the world, and a position statement released
to promote informed public debate. The statement calls for a scientific appraisal of the transgenic
technology and points out that advantages of the technology also lies in the significant environmental
benefits accrued out of increased productivity leading to decreased dependence on natural forests,
diminished use of pesticides and pulping chemicals and that options exist for mitigating the risks
posed by gene flow to wild relatives [90]. FAO has also addressed the issue by organising a
debate on the risks of gene flow from genetically modified organisms including transgenic
trees [91].
Biosafety regulations are either non-existent or inadequate in many developing countries and
many may not have the capability or desire to implement them. In the absence of an international
system of regulations, unscrupulous entrepreneurs can be tempted to take advantage and introduce
GMO’s commercially or for testing without adequate safeguards. The complacency that characterises
testing of GMO’s has more to do with the confidence that the scientists have in their understanding
of the way genes behave rather than abundant caution regarding the broader environmental
consequences. A comprehensive internationally coordinated programme involving governments,
non-governmental organisations and biotechnology companies to assess, study and monitor
GMO trials and lend confidence to researchers and public is required to remove the stalemate.
6.
Conclusion
Technologies in molecular biology and tissue culture could play an increasing role in the choice
of genotypes for successful establishment of plantations and agroforestry practices. Research
areas such as micropropagation, somatic embryogenesis, genetic engineering, marker-aided
selection and molecular diagnostics are merging with traditional forestry to help identify and
produce better-suited trees for plantations and agroforestry. A combination of classical and
molecular biological research could be used to improve pest and stress resistance of selected
genotypes, modify structure and function, and monitor pests of trees. This merger of approaches,
as well as continued technological development, could accelerate the production and selection
of suitable tree genotypes for forestry.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
12. Cloning Forestry Species
Vibha Dhawan and Sanjay Saxena
Bioresources and Biotechnology Division, Tata Energy Research Institute, Habitat Place,
Lodhi Road, New Delhi 110 003, India
Abstract: Over-exploitation of Indian forests has led to progressive decline in the forest cover and
its productivity. Since a large number of people depend on forest resources for their livelihoods and
many more for meeting their energy needs it is imperative to enhance the productivity levels of our
forests for a sustainable harvesting. Way back in 1999, the Department of Biotechnology, Government
of India took the initiative of setting-up Tissue Culture Pilot Plants for micropropagation of various
plant species. One of these facilities was established at TERI and so far about 12 million plants
have been despatched out of which 3.7 million are of forest species alone. The field trials have
clearly established clonal uniformity of tissue cultured plants and substantial increase in productivity
levels.
1.
Introduction
Forest play a major role in maintaining climatic stability, conserving water and soil, housing
biological diversity and serve as a valuable source of various timber and non-timber products.
India with its wide geographical distribution is endowed with rich forest resource and has two
of the eighteen hotspots in biodiversity in the world. Traditionally, communities have lived in
close harmony with the forest and their dependence on this important resource has taught them
to be caring for sustenance. In the recent past, however, this situation has changed tremendously
and the forests are being heavily over-exploited leading to reduction in forest cover, lowering of
plant density, and eroding of floral and faunal diversity. On account of intense population
pressures (both human and cattle) and heavy reliance of inhabitants on forests to cater their daily
needs, the forests are under severe strain. This problem is quite complicated and has no easy
solutions given by the fact that forests belong to the State Governments and thus conservation
is viewed as the Government’s responsibility while harvesting in many of these areas is the
privilege of the local communities. There are many industries, which are dependent on forest
resource, and at the time of independence many concessions were given to them, which includes
making timber available at subsidized rate. Many such actions have caused heavy damage to our
forests. Much of this loss is yet to be made up. This problem is further aggravated by the fact that
there is no incentive for industries to get themselves involved into growing of raw material
required by them. Further, due to fewer job options in the rural parts, cattle rearing and collection
of non-timber forest produce remain the favourite revenue-earning activities. Since cattle are left
in open for grazing, natural regeneration of the forests has become very difficult. This has
resulted in denudation of several areas and spread of wastelands in the country. To repair the
damage already caused to our forests and to restrict their future abuse, the Government of India
has formulated several plans and policies directed towards large-scale afforestation and using
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improved planting material for higher yields. Due to increasing human needs, it is not possible
to divert agricultural land for forestry purposes and the only option therefore, is to improve the
productivity of existing forests. This involves selection of suitable species (matching species
with sites), using superior quality planting material, managing the plantations properly, and
developing a programme for sustainable harvesting.
While the importance of conventional breeding in improving the productivity cannot be
undermined, breeding of trees is technically difficult and time consuming. Since the generation
time in most tree species is very long, in many cases one may not even see the results of the
experiments in lifetime. It may take several decades to release an improved variety/clone. Fortunately,
most trees species are cross-pollinated and consequently lot of genetic variability exists in nature
today. For immediate gains, it would be worthwhile to exploit this genetic variability existing in
nature by selecting and cloning superior genotypes/individuals from their natural population.
2.
Cloning of Tree Species by Conventional Methods
Compared to horticultural plants, research in tree species has lagged behind. There has been very
little interest shown by the private sector in this field and that too mainly in short rotation crops
and softwoods. General interest in cloning of hardwood species started with eucalyptus. Aracruz
(Brazil) took the lead in raising commercial plantations of eucalyptus through rooting of cuttings.
The concept of raising plantations by pulp and paper companies is not new and many countries
including United States, Australia and New Zealand are involved in similar kind of activities but
largely with pines. Commendable work has also been done in Thailand and Indonesia on hardwood
species such as acacia, casuarina, eucalyptus, teak etc. and increase in productivity has been
reported.
Of late, in India too, foresters who are the custodians of our forests have realized the importance
of planting clonal material. Massive projects have been launched by several state forest departments
for raising clonal nurseries. Some initiatives have also been taken by private companies such as
ITC Bhadrachalam, JK industries, West Coast Paper Mills and Andhra Pradesh Forest Cooperation.
However, the total plantlet production through conventional techniques is far from adequate.
This is largely because till date methods largely of rootings of cuttings have been developed only
for a few species and that too are effective only when the mother tree is in a juvenile phase.
3.
Major Constraints in Vegetative Propagation
• Because of large size of the propagules only few functional cuttings can be derived from
the desired clone/genotype.
• In many tree species the cuttings lose their ability to root by the time a particular clone
is evaluated for its useful traits.
• The cutting-raised-plants tend to form adventitious roots which unlike the tap root of
seedlings do not penetrate very deep inside the ground, thereby, making the plant highly
prone to felling by strong winds.
• Propagation through cuttings also poses a potential risk for spread of various systemic
diseases.
• Propagation through cuttings is extremely slow and season specific
• Depending on the species and the efficiency of asexual methods of propagation, the
production cost of cutting raised plants is marginally to significantly higher in comparison
Cloning Forestry Species 185
to seed-raised plants. This is quite a crucial factor for the foresters as they are often given
large target of afforestation and have limited funds at their disposal.
4.
Micropropagation
Tissue culture perhaps is the most commercially exploited field of plant biotechnology. It overcomes
many of the constraints that the conventional methods of propagation are inflicted with. Cloning
of plants under aseptic conditions, commonly called micropropagation results in mirror images
of selected mother plants on a large-scale within a short period of time. Micropropagation
assumes greater significance in those species which cannot be regenerated or are difficult to
regenerate by conventional methods such as seeds and vegetative propagation, where conventional
methods are inadequate to meet the demand of planting material, and vast variability exists in
seed-raised progenies.
5.
Advantages of Micropropagation
Rapid multiplication: By using an efficient protocol one may produce over a million plants
starting from a single bud.
Saving of space: Unlike conventional cuttings, that measure 8–12 inches in length and occupy
lot of space, large number of cultures can be accumulated within a small area.
Production of disease free plants: The plants produced by tissue culture are free of almost all
bacterial and fungal diseases. In those species where virus infestation is known to affect the
quality of the plant as well as the productivity, virus elimination can be achieved by tissue
culture. Since virus elimination is a time consuming and expensive process, it can only be
applied if one produces a large number of plants from a single explant, which is free of known
viruses.
Clonal uniformity: Unlike seedlings which represent only the half siblings, the tissue culture
raised plants are true-to-the-mother type and there is no segregation of genes or change in genetic
character/traits in the progeny during the regeneration process.
Independent of seasonal constraint: Since plants are produced in controlled conditions of
light, temperature and humidity, there is no effect of the outside environment on the regeneration
process. While the plants can be produced independent of season inside the lab, the transplantation
process remains dependent on the season.
6.
Tissue Culture of Trees
Tissue culture of woody species was first reported by Gautheret way back in 1933. However, the
progress made with trees has been rather slow as compared to herbaceous species. This is largely
because tree species have a distinct juvenile and an adult phase and trees, especially in their
adulthood, are more recalcitrant to tissue culture technology. Further, they have a long gestation
period and thus, field evaluation and commercial exploitation takes much longer vis-a-vis agricultural
and horticultural species.
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DHAWAN AND SAXENA
Although micropropagation process has been in use in the developed world for several decades,
in India the commercial importance of this technique was realized only in late 1980s and early
1990s when several tissue culture companies were set-up. However, most of these companies
were export-oriented units with focus on ornamentals. By and large these companies had a
general reluctance to venture into tissue culture of forest trees on account of following reasons
:
• Policy matter
• Technical problems
• Commercial factors
: Being export-oriented units with a buy-back arrangement with their
collaborators, these companies have largely been catering to the
demand of their collaborators.
: There are several technical problems associated with tissue culture
of forest species and there are very few groups in the country who
have the technical capability to deal with them.
: As compared to ornamentals (most of which are seasonal and demand
frequent replacement), the market of forest species is relatively small
on account of their perennial nature and selective users. The paucity
of funds with most of the State Forest Departments who are expected
to be the main users also deterred these companies to take up tissue
culture of forest species.
6.1 Tissue Culture Pilot Plant
To meet this challenge, the Department of Biotechnology (DBT), Government of India in 1989
decided to set-up a Tissue Culture Pilot Plant (TCPP) at TERI for mass propagation of forest
species using tissue culture technique to augment the biomass production in the country. Located
within TERI’s 36-hectare campus at Gual Pahari, Gurgaon, Haryana, here all the infrastructural
facilities ranging from modern laboratories and greenhouses to nurseries required for mass
production of tissue cultured plants, are available. In 1997, this Tissue Culture Pilot Plant was
upgraded into a Micropropagation Technology Park (MTP) to provide an effective platform for
transfer of the proven tissue culture related technologies to the entrepreneurs. Thus MTP has an
annual capacity of over two million plants.
6.2
Objectives of Micropropagation Technology Park
• Propagate superior clones of various plant species on a large-scale using tissue culture
technology
• Multiply those species on a mass-scale which are difficult to regenerate by conventional
methods of propagation
• Multiply species in vitro where conventional methods of propagation are inadequate to
meet the demand of planting material
• Enhance further the productivity of in vitro raised plants using symbionts such as rhizobia
and mycorrhizae
• Impart training in the field of plant tissue culture
• Technology transfer to new entrepreneurs or industry for commercialization
• Function as a think-tank for the tissue culture industry
Cloning Forestry Species 187
6.3
Achievements of TCPP/MTP
• Established a modern, indigenously designed tissue culture laboratory with an annual
production capacity of two million plants at Gurgaon (Haryana).
• Developed micropropagation protocols for a large number of species and refined procedures
for several others so as to make them suitable for large-scale propagation.
• Supplied over 12 million plants of various species to different state forest departments,
non-governmental organization, agro-based companies, private growers etc.
• Successfully demonstrated the application of tissue culture technology at the farmers’ field.
• Established high survival, plant uniformity, and better growth rates of tissue culture plants
as compared to conventional propagules.
• Successful technology transfer to industry.
• Contractual research/plant production for clients.
• Created awareness about MTP and the tissue culture technology through lectures,
demonstrations, seminars/workshops and exhibitions.
• Conducted several training programmes.
6.3.1 Criteria for the Selection of Hardwood Species
For the species that can be conventionally propagated through seeds and exhibit wide variability,
tissue culture is of immense value if the plus trees are mass multiplied. To achieve this, the
selection of the mother tree must be done very carefully. Also it is necessary to select newer and
newer clones to avoid monoculture and degeneration of clone. Some of the criteria followed for
selections are:
General Criteria
• A tree can be evaluated earliest at half its rotation age. For eucalyptus and populus this
age could vary from 4 to 5 years while for species like teak this may be as long as 30
years.
• Superior growth in height and diameter of the bole is judged in relation to neighboring
trees of the same or similar age. Isolated trees cannot be marked as plus trees.
• The marked tree should be free of all diseases.
Criteria for Stem Form
• Straight bole.
• Leading shoot must be showing active growth.
• No spiral grain.
Criteria for Crown and Branching Habit
• It should provide dense mass of healthy foliage.
• Good natural pruning and well-healed knot scars.
• Branches should be small in relation to the stem at the point of origin.
6.4 Multiplication of Tree Species at TERI
Following tree species have been/are being multiplied at TERI’s production facility:
• Anogeissus spp. (A. pendula and A. latifolia)
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DHAWAN AND SAXENA
•
•
•
•
•
Bamboos (Bambusa tulda, Bambusa arundinacea and Dendrocalamus strictus)
Eucalyptus (E. tereticornis, E. camaldulensis and E. citriodora)
Paulownia fortunei
Poplars (P. deltoides and P. euphratica)
Leucaena hybrids
Till March 31, 2002 nearly 3.7 million plants of various forest species alone had been dispatched
to various State Forest Departments, NGOs and private growers for field trials and routine
plantations. These plants would cover an area of over 4000 hectares (Tables 1 and 2).
Table 1.
Area covered under tissue cultured plants of various forest species till March 31, 2002
S.
No.
Species
No. of plants
dispatched
1.
2.
3.
4.
5.
6.
7.
Anogeissus pendula
Anogeissus latifolia
Eucalyptus spp.
Populus deltoides
Dendrocalamus strictus
Paulownia fortunei
Miscellaneous
447144
127532
1600481
1235410
95385
220981
11094
Total
3738027
3
3
3
3
5
4
3
Spacing
Approximate
area covered (ha)
×
×
×
×
×
×
×
402.46
114.79
960.67
1854.96
238.46
441.96
16.65
m
m
m
m
m
m
m
3
3
2
5
5
5
5
m
m
m
m
m
m
m
4029.95
Table 2. Number of tissue cultured plants of various forest species
dispatched to different states for field trials and routine plantations
till March 31, 2002
State
Total number of plants dispatched
Assam
Bihar
Delhi
Gujarat
Haryana
Himachal Pradesh
Jammu & Kashmir
Madhya Pradesh
Maharashtra
Orissa
Punjab
Rajasthan
Uttar Pradesh
Tamil Nadu
West Bengal
Kerala
Karnataka
Miscellaneous
17163
20517
85113
20905
1704507
1400
29700
45106
2735
4321
30075
616580
1034635
8404
10779
1300
4835
99952
Total
3738027
Cloning Forestry Species 189
6.5 Field Trials
Although TERI has been into production of forest species by tissue culture since 1991, the
evaluation of the tissue cultured plants started much later because it is recommended that the
performance of a forest species should be evaluated only after it had completed half its rotation
age. The field trial data of various species available thus suggest the following:
• High survival rate of tissue cultured plants in the field at times even when the soil and
other growth conditions are not favourable and life saving irrigation facilities are lacking.
• The plants showed high degree of clonal uniformity.
• Most of clones (CPTs) selected and multiplied at TERI outperformed the local clones or
seedling raised plants in biomass production. Depending on the nature of the clone and
its suitability at a particular location, the gains varied from marginal to significant (upto
200%).
• In the initial trials of Populus deltoides some problem of formation of ‘kinks’ was observed
which was later rectified by modifying the regeneration procedures and management
practices.
6.5.1
Anogeissus spp.
Anogeissus pendula
A. pendula is a very slow growing tree that grows 9 to 15 m in height and 1 m in girth. The tree
is essentially an inhabitant of dry and hot regions of Haryana, Madhya Pradesh, Rajasthan and
southern Uttar Pradesh, where the annual rainfall ranges between 400 and 800 mm. It can also
withstand a temperature regime of 3°C to 47°C. While the leaves are used as fodder, the timber
is valued for its strength and working qualities. It is used extensively for making various items
of domestic and agricultural use. The wood is also consumed for making charcoal of high
calorific value. The utility of this species makes it highly vulnerable to felling by rural communities
and grazing by their livestock. Regeneration through seeds is extremely difficult and is not much
in practice as the viability of seeds is very low (0.2–0.4%). Methods of vegetative propagation
by cuttings are not yet available.
In the absence of identified plus trees and recalcitrant nature of adult tissues, cultures were
established from seeds. For tissue culture work, the mother trees were carefully selected on the
basis of their phenotype and the seeds were collected only from tall and healthy looking trees.
The shoots obtained from aseptically raised seedlings served as the explant. The shoots were
multiplied by axillary branching method and rooted individually on a suitable rooting media. After
4 weeks of hardening inside the greenhouse and polyhouse, the plants were hardened further in
the nursery for at least 3 months before transfer to the field.
Till March 31, 2002, over 4.4 lakh plants of A. pendula had been lifted from TERI’s facilities
by various forest departments and other agencies for field trials and routine plantations. Most of
these plants were lifted by Haryana Forest Department and planted in Aravalli Hills. The feedback
received from the concerned forest departments suggests a transplantation success of over 85%.
Since A. pendula is a very slow growing species other growth parameters such as height, girth,
etc. do not hold much relevance in early years of plantation. Hence, the emphasis has been
accorded only to the survival success. In the absence of conventional seedlings no controls were
possible. Generally, the tissue cultured plants of a particular genotype exhibited similar growth
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pattern. However, as expected, there was some variation in the performance of the plants of
different genotypes.
In a 36-month-old trial conducted at National Research Centre for Agro-forestry, Jhansi,
AP-28 has proved to be the most promising genotype. The tallest plant of this particular genotype
attained a height of 365 cm. Since these plants were grown under routine plantations, performance
of individual genotypes could not be monitored. Furthermore, in the absence of conventional
propagules, the comparison of tissue cultured plants with seedlings was not possible.
The available data clearly suggest that by and large tissue cultured plants of Anogeissus
pendula have survived well in the field. This is despite the fact that most of these plants were
planted in extremely hostile conditions (poor soil and no irrigation) prevailing in the barren hills
of Aravalli. Low survival in few trials was mainly due to unsuitability of the site or biotic
interference. The plants are constantly being monitored for their growth performance and in
coming years more data would be available on the subject. Production of over 4.4 lakh plants and
high survival of tissue cultured plants in the field fully testify the utility of in vitro technology
in mass propagation of a forest species which was almost impossible regenerate by conventional
methods.
Anogeissus latifolia
Commonly referred to as ‘dhaura’, A. latifolia is a large tree that grows upto 33 m in height and
2.4 m in girth. It is commonly found in the forests of the sub-Himalayan tract and Shivalik hills.
The tree is found at its best in Madurai, Coimbatore, and Salem districts of Tamil Nadu and some
parts of Maharashtra, Karnataka and UP. Like A. pendula, A. latifolia is also a good fodder and
timber tree. The timber is fairly durable and is deployed for making furniture, cart-axles, shafts,
poles, tool handles etc. The regeneration problems in conventional methods are similar to that
described for A. pendula and, therefore, justify the need of in vitro techniques for plant propagation.
To initiate cultures, seeds were collected from healthy looking trees growing in Udaipur (Rajasthan).
After removing the seed coat, the seeds were put for germination and the shoots derived from
3-week-old seedlings were used for further multiplication.
Till date, nearly 1.2 lakh tissue cultured plants have been lifted from TCPP/MTP. Almost 80%
of these have been planted in Haryana. Since a majority of plants were transferred to the field
only during the last couple of years, it is premature to comment upon the specifics of various
growth parameters. However, initial feedback received on the performance of tissue cultured
plants suggest high survival rate and vigorous growth. A large percentage of plants were grown
as routine plantation for which the forest departments do not maintain any record. In some
cases the trials were laid initially but were abandoned later due to heavy biotic pressure or
various administrative reasons. Nevertheless, repeated requests for the plants made by the endusers suggest that in vitro plants are doing well in the field.
6.5.2 Bamboos
Bamboos are one of the fastest growing perennial grasses belonging to the family Poaceae. On
account of their versatility and immense utility, bamboos have been used for a variety of purposes
since times immemorial. Being straight, light, hard and strong, bamboos are extensively used for
construction of houses, scaffoldings, ladders, bridges, fences, furniture, sticks, tool handles,
pipes, basket mats and a large number of items of domestic and agricultural use. Bamboo leaves
Cloning Forestry Species 191
are used for thatching and are also valued as fodder. However, the most important use of bamboo
is in the paper and pulp industry to which it serves as the basic raw material.
Over-exploitation of bamboo resources by paper and pulp industry, bad management practices
and interference by biotic factors such as grazing and forest fires are some of the major factors
that have resulted in scarcity of bamboos. Although propagation of bamboos take place both by
seeds as well as vegetatively, however, both the methods of propagation are beset with many
problems that restrict their large-scale use. In view of the constant increase in demand, the
scarcity of planting material and the problems associated with conventional methods of propagation,
development of effective in vitro methods of propagation are highly desirable.
TERI scientists have developed in vitro regeneration protocol for four bamboo species viz.,
Bambusa tulda, Bambusa arundinacea, Dendrocalamus longispathus and D. strictus. However,
keeping in view the demand of various species and area of distribution, the emphasis was laid
only on mass propagation of D. strictus.
Commonly known as ‘lathi bamboo’, D. strictus is a densely tufted bamboo with strong
culms that grow 20 to 50 ft in height and 1 to 3 inches in diameter. It is the most widely grown
bamboo species in India. D. strictus is found in almost all parts of the country except northern
parts of West Bengal, Assam and other very moist areas. It grows well on dry, properly drained
soil up to a height of 2,000 m. Unlike most other bamboos, culms of D. strictus are either solid
or have a very narrow lumen. Because of this property, D. strictus is relatively harder and
stronger than other bamboo species.
For initiation of cultures, seeds were dehusked and after surface sterilization cultured on 2,4D containing medium for induction of callus and somatic embryos. The somatic embryos were
multiplied for several passages on a suitable multiplication medium. On being transferred to a
germination medium the somatic embryos formed plantlets. Till March 31, 2002 over 95,000
plants had been dispatched to various states.
6.5.3 Eucalyptus spp.
Commonly known as ‘safeda’, eucalyptus is a versatile tree that grows in almost all parts of the
country, from coastal areas to an elevation of 200 m. It can attain a height of 40–50 m and a girth
of 1 to 1.4 m. The tree is valued for its fast growth, high adaptability to grow in different kind of
soil and climatic conditions, and multiple uses. The wood is heavy, hard and mostly straight
grained. In India, the plantation-grown wood is mainly used for scaffolding, construction of houses,
making rayon-grade pulp and paper pulp, agricultural implements, furniture, boxes, carts, etc.
Eucalyptus is one of the fastest growing tree species producing large amount of biomass.
Because of its rapid and straight growth that casts very little shadow, eucalyptus is extremely
popular as an agro-forestry species. It is easy to cultivate and can even be grown in nutritionally
deficient soils. One of the major advantages of growing eucalyptus is that the animals do not
browse it and therefore, its protection does not pose any problem. Also, after planting once, one
can have three harvests without going for re-planting.
Conventionally, eucalyptus is propagated through seeds. However, due to segregation of
genes, the seed-raised population is highly heterogeneous. Clonal propagation of eucalyptus by
rooting of cuttings has met with limited success. Not only it is difficult to obtain large number
of plants of a particular clone by conventional vegetative methods, but also there is a potential
risk of spread of various diseases along with the propagules. In contrast, using tissue culture
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DHAWAN AND SAXENA
technology, a large number of healthy and disease-free plants of selected clones can be produced
within a short span of time.
At TERI, we have been successful in multiplying three species of eucalyptus i.e., E. tereticornis,
E. camaldulensis and E. citriodora. However, most of the production has been of E. tereticornis
only. Disease-free trees in possession of various desirable traits, such as faster growth, higher
biomass, straight bole etc. were selected from the natural population or field plantations of
different state forest departments. Referred to as ‘Candidate Plus Trees (CPTs)’, such superior
clones served as the source material for tissue culture work. The CPTs were coppiced in a
particular season to obtain juvenile shoots. Single node segments (explants) derived from such
coppiced shoots were then used to initiate cultures. Under the influence of the media, the axillary
bud present at the node sprouted and formed shoot(s). In vitro shoots were then multiplied and
rooted separately on well defined media. The tissue-cultured plants were hardened inside the
greenhouse before their transfer to the field.
Till March 31, 2002, 1.6 million tissue-cultured plants of eucalyptus had been supplied to
various state forest departments, NGOs and private growers for field trials and routine plantations.
Field data confirms high transplantation success (more than 90%), uniform and faster growth,
higher yields and better timber qualities. In some of the clones selected and multiplied at TERI,
the yield is more than twice as compared to conventional (seed raised) plants. Whereas seedraised plantations have yielded a maximum mean annual increment (MAI) of 20 m3/ha/year,
TERI clones have shown growth with MAI up to 40 m3/ha/year. Under natural (non-irrigated)
conditions, the average yield of TERI clones after 6 years of planting is estimated to be around
120 tons/ha as against only 80 tons/ha in case of seed-raised plants. Higher yields can be
expected if the plantations are raised under irrigated conditions.
6.5.4 Paulownia fortunei
Paulownia is receiving increasing attention as a short rotation woody species. A species of
Chinese origin, it is characterized by fast growth, attractive growth habit and flowers, and
biomass production. Besides timber, Paulownia leaves are used for fodder and flowers for honey
production. The value of Paulownia for afforestation, mine site reclamation and inter-cropping
systems has also been demonstrated. Despite all its potential uses, the species could not be
evaluated at the commercial level in India because of lack of planting material.
Although conventionally Paulownia can be propagated through seeds as well as vegetatively
from root or shoot cuttings, yet these methods are not adequate to meet the demand of planting
material that is required for carrying out extensive field trials. At TERI, success has been
achieved in developing a complete micropropagation protocol of P. fortunei using adult tissue.
Till March 31, 2002, over 2.2 lakh tissue-cultured plants had been dispatched from TERI’s MTP.
Since P. fortunei is an exotic species and its planting material is not so easily available, the ideal
edapho-climatic conditions required for its growth in India are not yet known. With the availability
of planting material it will now be possible to carry out extensive field trials in different geographical
conditions. Based on the performance suitable Paulownia growing areas may be identified and
industrial plantations be raised.
Cloning Forestry Species 193
6.5.5
Populus spp.
Populus deltoides
P. deltoides, which was first introduced in eastern UP, has now become a common tree in Tarai
region and states of Punjab and Haryana. It thrives well in tropical and sub-tropical regions of
India. It is an excellent source of biomass, and as a raw material its wood accounts for 50–60%
for plywood and nearly 90% for match stick industry. The wood being light and of low density
is an excellent source of packaging material.
To maintain its clonal nature, the species is always propagated through vegetative means and
the seeds are mainly used for breeding purposes. For vegetative propagation, stem cuttings derived
from superior trees are used.
It is recommended that only the leader shoot are used to derive cuttings because cuttings
obtained from the side branches are not successful and the plant tends to die within 2–3 years of
raising. However, the number of cuttings that can be obtained from the leader shoot of a particular
tree is rather small. Therefore, in order to meet the ever-increasing demand of industry, it would
be useful to carry out micropropagation of P. deltoides.
Mass propagation of several superior clones of P. deltoides such as G-3, G-48, D-121, L-34
and S7C15 using tissue culture technology has been undertaken at TERI and over 1.2 million
plants have been dispatched. In the initial lots of tissue cultured plants that were transferred to
the field, many of the plants showed kinks/bends in the stem. Sometimes the percentage of such
plants was as high as 40%. However, cuttings derived from such kink-bearing plants in the
following year produced almost normal plants. More than 95% of the plants were straight and
the remaining plants showed decreased degree of bends. Disappearance of bends confirms the
fact that this problem was not due to any change in the genetic make-up of the plants during the
course of in vitro process. The clonal fidelity of the tissue cultured plants was further confirmed
by DNA fingerprinting. In order to overcome the problem of kink formation in tissue cultured
plants, the regeneration protocol as well as the management practices adopted in the field were
modified. Following the same, the frequency of shoots bearing kinks became negligible.
Populus euphratica
P. euphratica is a unique species that can tolerate drought as well as water logging. Besides P.
deltoides, TERI has also worked out efficient protocol for in vitro regeneration of P. euphratica.
However, due to restricted geographical distribution and limited demand, the emphasis continues
to be on the mass propagation of P. deltoides.
Conclusions
TERI is one of those few organizations not only in India but in the world that are involved in
large-scale production of superior quality planting material of various tree species using tissue
culture technology. The dispatch of over 3.7 million tissue cultured plants of tree species clearly
demonstrates the technical feasibility of using tissue culture technology for large-scale production
of forest species. The clonal uniformity, and thereby increase in productivity, signifies enormous
potential the technology has to offer for increasing land productivity and thus face the challenge
of meeting biomass needs of the country.
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References
1.
Saxena, S., Dhawan, V. (1999). Regeneration and large-scale propagation of bamboo (Dendrocalamus
strictus Nees) through somatic embryogenesis. Plant Cell Reports. 18: 438–443.
2. Saxena, S., Dhawan, V. (2001). Large-scale production of Anogeissus pendula and A. latifolia by
micropropagation. In Vitro Cell. Devp. Biol.-Plant. 37: 586–591.
Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
13. Micropropagation of Woody Plants
J.S. Rathore, Vinod Rathore, N.S. Shekhawat, R.P. Singh*, G. Liler,
Mahendra Phulwaria and H.R. Dagla
Biotechnology Unit, Department of Botany, JNV University, Jodhpur 342 001, India
E-mail: biotechunit@satyam.net.in
*Department of Botany, Shri B.R.M. Govt. College, Nagaur, Rajasthan, India
Abstract: Micropropagation protocols for cloning of mature trees of Balanites aegyptiaca, the
Hingota (Balanitaceae); Citrus limon, the Nimbu (Rutaceae) and Syzygium cuminii, the Jamun
(Myrtaceae) have been developed. In order to harvest responsive nodal explants the mother tree(s)
were pruned during the winter. Fresh shoot sprouts derived from the trees were used as explants.
The nodal explants produced multiple shoots in vitro by activation of axillary meristems on MS
medium + 0.45 µ M BAP. Shoots were further multiplied in culture by (i) repeated transfer of the
mother explants and (ii) the subculturing of the nodal segments of in vitro differentiated shoots.
Shoots multiplication in Citrus limon could be achieved by amendment of the nutrient medium. The
in vitro cloned shoots of the three species were rooted in vitro and ex vitro. Ex vitro root induction
was followed to produce plants. Micropropagated plants were hardened in the green house. The
hardened and acclimatized plants were transferred to pots and subsequently to field. The cloned
plants are growing normal. The protocols defined are reproducible. These can be used for mass
multiplication of selected clones and genetic improvement of these species.
1.
Introduction
In vitro technologies are continuously expanding in the field of biology. Plant tissue culture has
become a general title for a very broad subject. While in the beginning it was possible to culture
plant cells either as established organs, such as roots or as disorganized masses, it is now possible
to culture plant cell in a variety of ways, individually (as single cells in microculture systems),
collectively (as calluses or suspensions, on petri-dishes, in Erlenmeyer flasks or in large-scale
fermentors), or as organized units as shoots, roots, ovules, flowers, fruits etc. [1]. In case of
Arabidopsis, which has been the subject of the most intensive research effort into technology
development [2], it is even possible to culture complete plants for generations from seed
germination to seed set without having to revert to an in vivo phase [3]. In its most general
definition plant cell culture covers all aspects of the cultivation and maintenance of plant material
in vitro. The cultures produced are being put to an ever-increasing variety of uses. At the early
stages, in vitro cultured systems were developed as experimental tools for basic research and
studies on plant cell division, growth, differentiation, physiology and biochemistry [4]. Such
systems were seen as ways to reduce the degree of complexity associated with whole plants,
providing additional exogenous control over endogenous processes, to enable more reliable
conclusions to be made through simpler experimental designs [5]. However, in the recent past
tissue culture technology has been increasingly used in highly applied contexts. Successes in a
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number of areas have been achieved. There has been major change in both the number of people
making use of these techniques and also in an enhancement of the degree of sophistication
associated with in vitro technology. Techniques of micropropagation and production of diseasefree plant stocks have been defined and refined to such an extent that they have become standard
practice for a range of (usually vegetatively propagating) crop plants. Thus creating what is now
a multi-million plant/multi-million dollar industry [3, 6, 7]. Moreover, the discipline within this
technology in which advances have been most rapid and will eventually have the greatest impact
on both fundamental and applied plant sciences is that of genetic modification of plant cell.
Micropropagation deals with the propagation of plants, in vitro, has many advantages over
conventional vegetative propagation. Its application in horticulture, agriculture and forestry is
currently expanding world-wide. The goal of micropropagation is to mass-produce genetically
identical, physiologically uniform, developmentally normal and pathogen-free plantlets, which
can be acclimatized in a reduced time period and at a lower cost. Development of both automated
environmental control systems and improved in vitro culture systems are essential for a significant
reduction in production cost [8]. However, commercial use of micropropagation is still limited,
because of its relatively high production cost resulting mainly from high labor costs, low growth
rate in vitro, and poor survival rate of the plantlets during acclimatization [9]. Altman and
Loberant [10] have elegantly reviewed principles and practices of micropropagation.
Micropropagation of woody/tree/forest plant is feasible [11-13]. However, with some exceptions
traditional in vitro methods are not as yet practical or commercially viable for most forest trees.
Therefore, improvement in current procedures and their scaling-up is required. Although it has
been argued that from the environmental perspective, the genetic diversity of forest should be
maintained/conserved, hence the traditional use of mixed population of seedling for forestation
be applied and clonal forestry may not be appropriate. The case of cuttage propagation and
micropropagation for all types of woody perennials is strongly affected by ontogenetic age [14].
Cloning in vitro and in vivo of adult and/or mature plants is adversely affected by characteristics
accompanying maturation such as reduced growth rate, reduced or total lack of rooting ability
or sometimes the unpleasant phenomenon of plagiotropy [15]. Maturation, a complex phenomenon,
is the major problem preventing a wider application of tissue culture technology among woody
plant species. Micropropagation of woody plants of stressed environments which experience
types of (annually recurring) abiotic-stresses, become more difficult as the seasonal and
environmental factor influence the behavior of explant(s) in culture to a great extent.
In simple terms, plant tissue culture can be considered to involve three phases. First, isolation
of the plant (tissue) from its usual environment. Second, the use of aseptic techniques to obtain
clean material free of bacterial, fungal, viral and even algal contaminations. Third, the culture
and maintenance in vitro in a strictly controlled physical and chemical environment [9, 16, 17].
The components of this environment are then in the hands of the researcher who gains a considerable
degree of external control over the subsequent rate of the plant material concerned. Hall [3]
suggested an extra fourth phase where recovery of whole plants for rooting and transferring to
soil is the ultimate goal. The success of this technology is to a great extent dependent upon
abiding by a number of fundamental rules and following a number of basic protocols.
During last four decades a number of plant tissue culture technologies have been developed
for a number of plant species in India [18-20]. Govil and Gupta [21] have reviewed
commercialization of plant tissue culture in India. It has been suggested that plant tissue culture
Micropropagation of Woody Plants 197
would play a very important role in conservation, propagation and genetic improvement of
plants of our country and also in restoration ecology and restoration of degraded habitats [22].
Since 1980, we have been working on development of tissue culture protocols for application
in propagation and genetic improvements of woody plants of arid and semi-arid regions (namely
the Indian Thar Desert and the Aravallis). Some of the woody plant species (as important
biomass producer) are keystone species of these regions of the country. We developed tissue
culture processes for cloning and mass propagation, using nodal shoot explants of mature and
selected woody plants namely Aegle marmelos [23], Capparis decidua [24], Celastrus paniculatus
[25], Maytenus emarginata [26], Zizyphus spp. [27]. We also cloned shoots of Prosopis cineraria
[28] and Tecomella undulata [29]. Micropropagated shoots were rooted by pulse treatment with
root-inducing auxins. Several species of Anogeissus (Combretaceae) were first micropropagated
in our laboratory using cotyledonary nodes [30–35]. Later, Saxena and Dhawan [36] of Tata
Energy Research Institute (TERI), New Delhi reported the micropropagation of Anogeissus
latifolia and A. pendula, also using juvenile explants.
Now we describe the development of micropropagation protocols for cloning of Balanites
aegyptiaca (Hingota), Citrus limon (Nimbu) and Syzygium cuminii (Jamun). These woody species
are economically and ecologically important as they yield valued products. Balanites aegyptiaca
(Balanitaceae) is a tree of arid regions. This has multiple uses particularly for the aboriginals and
rural people. The stem-bark is used as a fish-poison, and the pulp of fruit as detergent/soap for
washing cloths/hair. Hard and durable timber is utilized for making agricultural appliances and
household articles. The powder of mature fruits is taken orally by the women to prevent unwanted
pregnancy. The roots and fruits of B. aegyptiaca yield ‘Diosgenin’—a sapogenin widely used for
production of pharmaceutical steroid and oral contraceptives [37]. Citrus (Rutaceae) is considered
as number one fruit of the world for its nutritional values, the magnitude of fruit production and
an array of commercial products which are derived from it. Citrus limon is an important horticultural
species. Similarly, the Black-plum S. cuminii (Myrtaceae) is a tropical fruit tree which has
multiple uses [38]. Also this tree has very high water use efficiency and thus is effective biomass
producers. We developed cloning processes using nodal segments of rejuvenated (fresh shoot
sprouts) shoots of selected mature trees.
2.
Materials and Methods
2.1 Source Plants
Selected mature tree(s) of Balanites aegyptiaca, Citrus limon and Syzygium cuminii were pruned
during December-January. Shoot sprouts were harvested during the months of February-MarchApril. Fresh shoot sprouts collected during the months of March/April were used as explants.
The nodal explants were dressed and treated with 0.1–0.2% Bavistin and 0.1% Tetracylin for
10–15 min. These were surface sterilized with 0.1% HgCl2 (5–6 min), then with 90% ethanol
(60 sec) and were thoroughly washed with sterile water. These were finally treated with chilled
sterile antioxidant solution (0.1% ascorbic acid; 0.05% citric acid and 0.1% PVP) for 15–20 min.
The explants were inoculated on MS [39] medium supplemented with different concentrations
of BAP or kinetin.
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2.2 Culture Establishment and Axillary Bud Breaking
The nodal explants of the three species were inoculated in culture tubes on agar-gelled MS medium
supplemented with different concentrations of benzylaminopurine (BAP) or kinetin. The cultures
were incubated at 28 ± 2°C in a culture room with 10 h per day photoperiod. The responses of
the explants were recorded regularly.
2.3 Amplification of Shoots in Culture
Shoot of B. aegyptiaca were multiplied by subculturing of nodal shoot segments of in vitro
generated shoot on MS medium + 0.2 µ M BAP. Shoot amplification in Citrus limon was
achieved when the mother explants were repeatedly transferred or nodal explants subcultured on
amended MS (50% of NH4NO3, KNO3) with 0.25 µ M BAP. Multiplication of shoots of Syzygium
cuminii was achieved by (a) repeated transfer of mother explants and (b) subculturing of in vitro
produced shoots on above mentioned amended medium with K2SO4 (100 mgl–1), KCl (70 mgl–1)
and ammonium citrate (50 mgl–1). Subculturing was done after 20–25 days. The cultures were
amplified in 250 ml flaks or bottles. These were kept under the controlled conditions of temperature
(28 ± 2°C), light (40–50 µ mol m–2 s–1 SFP for 12 h/d photoperiod) and 60% RH.
2.4 Rooting of Cloned Shoots
Experiments were conducted to induce the roots in vitro and ex vitro from the micropropagated
shoots. For in vitro rooting 4 to 5 cm long shoots were excised and cultured on agar-gelled full,
half, one-third and one-fourth strengths of MS medium containing 0.1% of activated charcoal
and different concentrations (1.23 to 16.1 µ M) of IBA or NAA or NOA. These shoots were
cultured at 30°C, under different regimes of light (8–10 h photoperiod per day). For ex vitro
rooting, the individual shoots were pulse-treated with sub-lethal concentrations of root-inducing
auxins and cultured on autoclaved soilrite in glass bottles ( jam bottles). These bottles were kept
in the green house at 30 ± 2°C.
2.5 Acclimatization of Micropropagated Plants
In vitro rooted plantlets were washed with sterile water to remove adhered nutrient agar and
transferred to sterile soilrite in the culture bottles. In case of ex vitro rooted plantlets after roots
were visible, the culture bottles were shifted from low temperature/high relative humidity (RH)
regime of green house to the region which experienced relatively high temperatures and low RH.
Also the rooted plantlets were exposed gradually to external environment by loosening/removing
the caps of the culture bottles. Micropropagated and hardened plantlets were transferred to
polybags containing mixture of organic manure, garden soil and sandy soil. These plants were
watered regularly. The green-house-hardened plants were kept in nursery covered with agronet.
3.
Results
3.1 Selection of Explants
Nodal shoot segments harvested from pruned and non-pruned tree(s) were used as explants for
establishment of cultures of three species. Explants prepared from fresh (rejuvenated) shoots
regenerated from pruned plants during the months of March-April proved to be the most suitable
for culture establishment. The explants harvested from non-pruned tree(s) proved to be difficult
Micropropagation of Woody Plants 199
to surface sterilize as these carried recalcitrant microbial contaminations; these rarely showed
bud breaking, caused excessive browning of the culture medium and exhibited browning/darkening
of cut ends/explants. Thus management and pruning of mother tree was found to be essential for
harvesting shoots to be used as responsive explants.
3.2 Establishment of Shoot Cultures
The surface sterilized nodal explants could be cultured on MS media containing 0.45 µM BAP
or higher concentrations of BAP or kinetin. The axillary meristems were activated and bud
breaking was observed after 10-15 days of inoculation in 85-90% of the explants of three species.
Maximum number of shoots differentiated on MS medium supplemented with 0.45 µ M BAP.
Shoot differentiated from each node were 2-3 in Citrus limon, 1-2 in B. aegyptiaca and 3-4 in
S. cuminii, respectively. Kinetin proved to be less effective as compared to BAP in the activation
of axillary buds. More than 0.45 µ M of either of BAP or kinetin caused callusing from the explants
and proved to be inhibitory.
3.3 Amplification of Shoots in vitro
After the activation of meristems, bud breaking and axillary bud differentiation, the shoots were
further multiplied on suitable culture media. Shoots of B. aegyptiaca were multiplied by subculturing of segments of in vitro produced shoots (Fig. 1). Shoot amplification occurred on
MS + 0.22 µ M BAP. Shoots of C. limon were multiplied by repeated transfer of mother explants
on amended MS medium + 0.22 µ M of BAP. About 12-15 shoots differentiated from each
mother explant (Fig. 2). Three-fold rate of shoot multiplication was achieved by repeated transfer
of the mother explants.
By repeated transfer of mother explants on amended MS medium, shoots of S. cuminii
multiplied. Two- to three-fold rate of shoot multiplication was achieved (Fig. 3). The cultures
were transferred on to fresh media after 20-25 days. The cultures were maintained at high light
intensity (50–60 µ mol m–2s–1) at 28–30°C. The shoot cultures of all the three species are being
multiplied and maintained for the last 3 years.
3.4
Rooting of Cloned Shoots
3.4.1 In Vitro Rooting
Isolated shoots of all the three species rooted on half-strength MS medium with 0.1% activated
charcoal. Ninetyfive to 100% of the shoots of C. limon rooted on half-strength MS medium + 27.0
µ M of NAA. From each shoot six to eight roots regenerated. Rooting was poor on media
supplemented with IBA or NOA. Of the shoots of B. aegyptiaca, 80-90% rooted in vitro on halfstrength MS medium + 0.2 µ M IBA + 0.1% activated charcoal (Fig. 4). Ninety percent of the shoots
of S. cuminii rooted on half-strength MS medium + 0.1% activated charcoal + 9.8 µ M of IBA.
3.4.2 Ex vitro Rooting
About 90-95% of the in vitro amplified shoots of C. limon rooted ex vitro (Fig. 5) if pulsed with
0.98-2.46 µ M IBA. The rooting percentage was 85-90% if the shoots were treated with equimolar
NOA. The shoots treated with 1.07-2.68 µ M NAA showed maximum rooting. The ex vitro roots
were visible after 10-12 days of pulse treatment.
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RATHORE ET AL
4
1
2
3
7
8
5
6
9
Fig. 1. Multiplication of shoots of Balanites aegyptiaca in vitro by subculturing; Fig. 2. Amplification of
shoots of Citrus limon; Fig. 3. Amplification of shoots of Syzygium cuminii by repeated transfer of mother
explant; Fig. 4. Rooting of shoots of Balanites aegyptiaca in vitro on half-strength MS medium + 0.2 µ M
IBA + 0.1% activated charcoal; Fig. 5. Ex vitro rooted plantlets of Citrus limon being hardened in the
green house; Fig. 6. Ex vitro rooting of shoots of Syzygium cuminii; Figs. 7, 8 and 9. Cloned plants of
Balanites aegyptiaca, Citrus limon and Syzygium cuminii, respectively.
The shoots of S. cuminii could also be rooted ex vitro. A pulse treatment with 2.46 µ M of IBA
for 10–15 min was found to be sufficient to induce ex vitro roots from the shoots. Cent-per-cent
of the shoots rooted on soilrite in the green house within 20–25 days. If the shoots were pulsed
with NAA, 65% of these rooted (Fig. 6) after 30–35 days.
Micropropagation of Woody Plants 201
About 70% of the shoots of B. aegyptiaca rooted on soilrite after 12–15 days if treated with
1.0–2.5 mM IBA for 2–5 min.
3.5 Hardening of Micropropagated Treelets
In vitro rooted plants were hardened by transfer to soilrite containing bottles in the green house.
These were kept near pad section for 8–10 days and gradually shifted towards fan section. After
10 days the caps of culture bottles were loosened and gradually removed. Plantlets rooted ex
vitro were acclimatized in the green house. After formation of roots the plants were exposed to
low RH and high temperatures. Ex vitro rooted plantlets were found to be easy to harden and
acclimatize than those rooted in vitro. Hardened and acclimatized plants were transferred to
black bags containing garden soil; sandy soil and organic manure (Figs. 7, 8 and 9). Several
plants have been transferred to the field. These are growing normal. Flowering of these is yet to
be recorded.
4.
Discussion
The research work presented in this article demonstrates that the mature woody plants can be
cloned using appropriate in vitro methods. We have described the development protocols for
cloning of Balanites aegyptiaca, Citrus limon and Syzygium cuminii. These are valuable woody
species that yield products of economic value. Selection of the individual plant with desired
(superior) characters is possible only after certain age, when reproductive maturity is reached.
Such selected and mature plants give high yield of quality product. Once the selection is done
it is necessary to maintain genetic fidelity of the clone. This is done by vegetative propagation
in vivo or in vitro (micropropagation). Cloning of mature woody plants in vitro and in vivo is
adversely affected by characteristics accompanying maturation such as reduced growth rate,
reduced or total lack of rooting ability or sometimes the unpleasant phenomenon of plagiotropy
[14, 15, 40]. Maturation, a complex phenomenon, is the major problem preventing a wide
application of tissue culture technology among woody species. Nevertheless, a number of woody
species/trees have been micropropagated. Success with several species have been achieved
mainly by the use of special starting (explanting) material, by special pre-treatment to mother/
source plant(s) in vivo or by in vitro culture [11, 26, 28]. All of these tricks, which improve
clonal propagation are often described by the general term rejuvenation. It is clear that rejuvenation
is a pre-requisite for possible cloning of adult trees and that the success in practice mainly
depend on the ability to rejuvenate them. We found that in all the three species under investigation,
pre-treatment (pruning during winter) of mother plant was desirable otherwise the explant did
not respond in culture. The shoot sprouts (flushes) from plants pruned during winter proved to
be the only useful (suitable explants) for culture initiation. Rejuvenation (also known as phase
reversal or return to the juvenile form) includes the complete reversal of maturation as a result
of sexual reproduction or vegetative propagation via shoot formation (through activation of preexisting axillary- or apical-meristems) or somatic embryogenesis. Re-invigoration is defined as
the reversal of ageing (which leads to reduced vigor and rooting ability). Reinvigoration can be
used when rooting-ability and vigor are increased as a result of, for example, pruning, hedging,
repeated culturing, BAP-treatment and grafting [41]. The nodal explants of B. aegyptiaca, C.
limon and S. cuminii derived from fresh shoot sprouts, responded in culture and produced
multiple shoots on BAP (0.45 µ M) supplemented medium. The shoots could be further amplified
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in vitro by (i) repeated transfer of explants and (ii) subculturing, but on medium with comparatively
lower concentration of BAP. In quite a number of plant species repeated subculture/transfer of
adult shoots (mother explants) were reported to induce invigoration and complete rejuvenation,
by which shoot multiplication and rooting ability are strongly improved. It is suggested that once
the cultures/explants were established these become conditioned and they required low cytokinin
for further multiplication. In case of Citrus limon the cultures could be multiplied by lowering
the concentrations of certain salts (NH4NO3 and KNO3). Chaturvedi [42] et al. critically reviewed
tissue culture employing vegetative explants in Citrus spp. It is stated that maximum tissue
culture research has been done in Citrus during the last four decades however the results of
practical value are meager. We have successfully established procedure for large-scale shoot
multiplication of Citrus limon. This is important contribution in Citrus tissue culture with
practical utility.
The micropropagated shoots were rooted in vitro on half-strength MS medium + 0.1% activated
charcoal supplemented with IBA (B. aegyptiaca and S. cuminii) and NAA (Citrus limon). Bonga
and Von Aderkas [43] suggested that roots from rejuvenated shoots of woody plants, is induced
in vitro by IBA or NAA. Probably the nature of auxin required and the concentration for in vitro
root regeneration are species specific.
In the present case the micropropagated shoots of all the three species could be rooted ex
vitro. The main advantage of ex vitro over in vitro rooting is that root damage during transfer to
soil is less likely. Furthermore, rooting rates are often higher and root quality is better when the
rooting takes place ex vitro [43]. McClelland et al. [44] studied the effect of in vitro and ex vitro
root initiation on subsequent microcutting root quality in three woody plants. They suggested
greater resistance of ex vitro rooted plants to stress. Arya et al. [25] found that the ex vitro rooted
plantlets of a woody climber, Celastrus paniculatus were easy to harden. The duration of time
and cost of plant production are also reduced by switching to ex vitro root generation. IBA
proved to be more effective auxin for pulsing of the shoots for ex vitro root induction. The auxin,
most commonly used for root formation is IBA. It is generally assumed that the greater ability
of IBA as compared with other auxins to promote rooting is due to its relatively higher stability
[45, 46]. It has been possible to induce ex vitro induction in number of woody species [35] of
stressed environments.
The rooted plantlets of all the species could be hardened in the green house and pot transferred
with ease. The survival rates have been satisfactory. Development of protocol for micropropagation
of B. aegyptiaca is important contribution as this could be applied for cloning of plants selected
for higher yield of diosgenin. Selected and tested plants of Citrus limon bearing desired attributes
of horticultural importance can also be cloned using our protocol. Yadav et al. [38], and Jain and
Babbar [47] reported in vitro micropropagation of Syzygium cuminii. They used explants from
young seedlings. This method of cloning is not preferred for fruit trees. Multiple shoot induction
from 1- to 2-year-old seedlings of S. travancoricum was recorded by Anand et al. [48]. Mathew
and Hariharan [49] reported in vitro shoot multiplication in S. aromaticum. Shah Valli Khan et
al. [50] reported in vitro micropropagation of mature S. alternifolium. In this article we have
described a process for cloning of mature tree of Black-plum (S. cuminii). This is the most
desired level of cloning. The micropropagated plantlets of all the three woody species could be
hardened and pot transferred. The processes defined are highly reproducible and efficient and
these can be utilized for cloning of selected trees of these species.
Micropropagation of Woody Plants 203
Acknowledgements
N.S. Shekhawat is grateful to the Department of Biotechnology (DBT), Government of India for
financial assistance for the establishment of Micropropagation Unit and Green House (Grant No.
BT/R&D/08/03/93), University Grants Commission (UGC), New Delhi for providing support
under UGC-SAP-DSA Programme (1997–2001) to the Department of Botany and the Department
of Science and Technology, Government of India for providing support under DST-FIST Programme.
We are grateful to the Head, Department of Botany for providing facilities for this work. We also
appreciate the technical assistance provided by Shri M.S. Panwar.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
14. Biotechnology in Mulberry (Morus spp.) Crop
Improvement: Research Directions and Priorities
S.B. Dandin and V. Girish Naik
Central Sericultural Research and Training Institute, Srirampura, Mysore 570 008, India
Abstract: Mulberry (Morus spp.) is a crop plant of economic importance in sericulture. Mulberry
improvement through conventional breeding has substantially contributed to the success of sericulture
industry. However, the application of biotechnology in mulberry crop improvement holds a great
promise especially in those areas where conventional research has not achieved the desired success.
The biotechnological research in genome characterization with isozyme and DNA markers,
micropropagation, regeneration from callus, somatic hybridization, in vitro conservation technologies
like slow-growth storage and cryopreservation, genetic transformation etc., have contributed to the
success in mulberry improvement. Besides discussing the progress achieved so far in mulberry
biotechnology, the article also emphasizes the future priorities in this direction both in terms of
supportive and strategic research.
1.
Introduction
Application of biotechnological methods for crop improvement has significantly contributed to
the success of modern day agriculture. Enhancement of yield potential, improvement of quality,
resistance to pests and diseases, tolerance to abiotic stresses and resistance to herbicides are the
main focus of crop improvement in many agricultural crops through biotechnological approach.
Mulberry (Morus spp.) is a crop of economic importance in the sericulture industry. Its foliage
forms the sole source of food for the domesticated silkworm, Bombyx mori L. Mulberry is a
dioecious, heterozygous and perennial tree. In spite of the problems associated with tree crop
improvement, considerable progress has been achieved in mulberry breeding through conventional
approaches. However, biotechnology application holds a great promise in further improvement
of mulberry crop especially in those areas where conventional research has not achieved the
desired success. Already considerable progress has been made in this direction. The article
attempts to consolidate the important outcome of the biotechnological applications in mulberry
and also discusses the need for future research priorities in mulberry improvement, utilization
and conservation.
2.
Genome Characterization
Understanding of genetic structure of the plant is very important for crop improvement, utilization
and conservation. Mulberry being a perennial, heterozygous tree, traditional methods of analysis
have not provided sufficient insight into the genetic architecture. Compared to the phenotypic
characters, molecular markers are highly heritable, consistent, fast and easy to measure and
evaluate. Among the molecular markers, isozyme and DNA markers are widely employed for
genome characterization and analysis of plants and animals.
Biotechnology in Mulberry (Morus spp.) Crop Improvement 207
2.1 Isozyme Markers
Hunter and Markert [1] were first to introduce isozymes as genetic markers in plants. Hirano [2]
used peroxidase isozyme technique to evaluate the affinities in mulberry and its relatives and
showed that the results supported the conventional view. The study [3] of inheritance of peroxidase
isozyme of mulberry was initiated in Japan and established that particular isozyme banding type
was significantly correlated with leaf stalk length. Hirano [4] also used isozyme technique to
analyze 284 mulberry varieties. He used seven enzyme systems and a sap protein to characterize
these varieties. Based on the electrophoretic pattern he categorized 131 varieties into seven
groups and established the gentic relationship among them. The study also demonstrated the
correlation between amino acid content and peroxidase enzyme in the leaf. Katagiri and coworkers [5] successfully utilized peroxidase isozyme technique to differentiate hexaploid mulberry
strains collected from Mexico. In India, peroxidase isozyme studies were reported on introduced
species from Indonesia [6], triploids [7] and aneuploids [8] of mulberry. Even though isozyme
analysis is comparatively easy, less costly and the markers are codominant in expression, they
are less attractive compared to the DNA markers because of lack of sufficient polymorphism.
2.2 DNA Markers
Studies on mulberry genome was first initiated in Japan. Katagiri and coworkers [9] successfully
isolated chloroplast DNA from mulberry. Later Machii [10] reported the isolation of total DNA
by ultracentrifugation method. Chengfu and coworkers [11] detected DNA marker variation
using RAPD technique in 12 mulberry varieties with 24 primers. Relationships among the
operational taxonomical units (12 species and 2 varieties) of Morus were examined with 20
random decamer primers, generating 238 polymorphic markers [12]. Phylogenetic analysis of
RAPD data indicated that grouping so obtained is in conformity with morphological classification.
Polymorphism in genomic DNA of five parents and their four resulting hybrids were analyzed
by RAPD technique [13]. Of the F1 patterns, most of the markers appeared were same as their
respective parents, however, few were unique not found in their parents. Sharma and coworkers
[14] assessed the genetic diversity in Morus germplasm collections using fluorescence-based
AFLP markers. The wide range in the genetic similarity (0.58–0.99) indicated that the mulberry
germplasm collection represents a genetically diverse population. However, the study also concluded
that the genetic base of cultivated mulberry is narrow. A recent study [15] showed that as many
as five RAPD and one DAMD primers generated profiles can together differentiate all the nine
mulberry varieties in terms of unique bands. Central Sericultural Germplasm Resources Centre,
Hosur in collaboration with Seribiotech Research Laboratory, Bangalore has characterized number
of mulberry germplasm using DNA fingerprinting techniques [16–19]. RAPD analysis of 15
mulberry species revealed few species diagnostic markers indicating the usefulness of the technique
in identification. Phylogenetic analysis of RAPD and ISSR markers showed the separation of
wild and cultivated mulberry species into a different cluster. Study of 44 cultivated mulberry
varieties and 27 M. laevigata collections with RAPD marker data has resulted in generation of
useful information on genetic diversity and identity. A research project on “Genome analysis of
mulberry” [20] is currently underway at Central Sericultural Research and Training Institute at
Mysore. The results indicate that RAPD can be effectively used to DNA fingerprint mulberry
cultivars and also can be successfully employed to study the inheritance pattern and for the
development of molecular linkage map.
208
3.
DANDIN AND NAIK
Micropropagation by Tissue and Organ Culture
Most of the initial studies on mulberry tissue culture concentrated on the regeneration of complete
plantlets from various explants like shoot tip, axillary bud, winter bud, leaf, cotyledon, hypocotyls
etc. Ohayama [21] for the first time successfully obtained complete plant from axillary bud of
M. alba on MS medium supplemented with growth regulators. Since then, shoot proliferation
was observed in many mulberry genotypes using different explants and supplementing the media
with cytokinins like BAP [22–43]. However, BAP had a negative response at a higher concentration
on shoot proliferation of mulberry genotypes [44–45]. Modification of basal media with macroand micro-salt were tested on different genotypes for shoot proliferation [41]. Micropropagation
of shoots of M. indica was tested with MS salts and B5 vitamins [46]. The pH level of various
media tested ranged from 4 to 5.6 for shoot multiplication in different mulberry genotypes
[47–48]. However, the optimum pH level appears to be in the range of 5.6–5.8 for many genotypes.
Best shooting response was obtained at 0.8% of agar concentration [41, 46, 48].
Auxin rich media induced rooting within 10–14 days of culture in M. laevigata [44]. In several
mulberry species, rooting was enhanced by treatment with NAA and IAA [21, 23, 41]. Combination
of IBA, IAA and IPA helped root proliferation in M. australis [45], M. lhou, M. cathayana and
M. serrata [49]. In M. alba shoots produced roots in auxin-free media [48].
Hardening of regenerated plantlets is an essential perquisite for successful establishment in
the field. Various kinds of potting mixtures like steam sterilized peat and agroperlite (2:1),
autoclaved soil, soilrite mixture and vermi-compost have been used for establishment of regenerated
plants [39, 49]. Half-strength Hogland’s nutrient solution and water was used to irrigate the
plantlets [39, 45, 49].
4.
Callus Formation and Differentiation
Induction of callus of mulberry genotypes from different explants sources like stem segments
[50], young leaf [51, 52] and hypocotyls segments [53, 54] were successfully attempted on MS
media supplemented with 2-4 D. Addition of Kn, IAA and NAA in the media resulted in the
better proliferation of calli. Calli can be prolonged in the culture medium up to eight weeks in
good conditions by adding ABA and PABA [55]. Calli of M. bombycis, M. alba and M. multicaulis
were regenerated in medium supplemented with auxins and cytokinins [56, 57]. Shoot regeneration
from callus of M. alba was obtained [26, 58] on MS medium supplemented with BAP. In M.
bombycis shoot bud induction was reported in the callus on LS medium supplemented with BAP
[42]. Addition of GA3 and DTT to the culture medium enabled to break the pseudo-dormancy
and obtained regeneration in stored calli [59].
Rhizhogenesis of calli was frequently reported from the cultures on media containing auxins
[60–61]. Rooting was also obtained from the cell suspension of the callus from hypocotyl tissue
[62]. From the callus of internodal segment and leaf explants of M. laevigata, rhizogenesis was
observed on MS medium supplemented with NAA [63]. Recently, few workers have reported the
complete regeneration of mulberry plants from callus culture using TDZ [64, 65].
5.
Development of Haploids
As already discussed, mulberry is a dioecious, outbreeding and heterozygous tree species.
Development of homozygous lines through conventional method has not been successful.
Homozygous lines are extremely important in genetical studies and exploitation of hybrid vigor
Biotechnology in Mulberry (Morus spp.) Crop Improvement 209
in crop improvement programme. In this background, constant efforts have been made in mulberry
to develop homozygous lines through the production of haploids. Lin and coworkers [66]
successfully reported the regeneration of haploid plants from uninucleate anthers in a Chinese
mulberry variety. Venkateshwaralu and Katagiri [67] reported globular and heart-shaped embryoids
on B5 medium in a Japanese variety. Similar results were also obtained by Sethi and coworkers
[68] in an Indian mulberry variety on MS medium. Katagiri [69] reported the colony formation
and induction of callus from pollen culture studies. Katagiri and Venkateswaralu [70–71] observed
embryo like structures on culturing the pollen in B5 medium. Addition of fructose to B5 medium
[72] resulted in profuse division and obtained a compact calli. On MS media supplemented with
glutamine, coconut water and 2–4 D, Tewary and coworkers [73] obtained globular embryoids
from pollens isolated from anthers starved at 10–12°C for 72 h in S-1 variety. Lakshmi Sita and
Ravindran [74] for the first time reported the gynogenic haploids plants from the ovary culture
of mulberry. Dennis Thomas and coworkers [75] developed a reproducible protocol for the
production of gynogenic haploids of a female clone of mulberry (M. alba L. Cv. K-2) from
unpollinated ovary culture.
6.
Protoplast Isolation, Culture and Somatic Hybridization
Genetic barrier in hybridization due to sexual incompatibility and other associated problems can
be successfully overcome by somatic hybridization of protoplast cells. Methodology for isolation
of protoplast, its culture and fusion of cells play a critical role in successful regeneration of
plants. Protoplast isolation in mulberry was attempted from callus [76] and mesophyll cells
[77–78]. Ohnishi and Kiyama [79] showed that primary callus culture gave a better protoplast
yield than secondary callus cultures of mulberry. Tewary and Lakshmi Sita [78] reported the
optimized concentration of cellulase (2%), macerozyme (1%) and macerase (0.5%) for better
protoplast yield in mulberry. Katagiri [80] observed the colony formation in cultures of mulberry
mesophyll protoplasts. Differences in division of mesophyll protoplasts cultured on different
media and under different light intensities were studied in few mulberry species. Ming and coworkers
[81] demonstrated the regeneration of complete plant from the callus derived from mesophyll
protoplast of mulberry through organogenesis and somatic embryogenesis on MS medium. Protoplast
fusion in mulberry was successful using chemical fusogen [76] and electro-fusion [82].
7.
In Vitro Methods for Conservation of Genetic Resources
Conventional approach to germplasm conservation of reclalcitrant seed species as well as
vegetatively propagated crops did not overcome the inherent limitation in storage technique. In
contrast, in vitro conservation methods offered suitable alternative to seed and field gene bank.
In vitro conservation refers to maintenance of germplasm in a relatively stable form under more
or less defined nutrient conditions in artificial environment. Potential advantages of conserving
mulberry genetic resources by in vitro methods are:
(i)
(ii)
(iii)
(iv)
(v)
(vi)
can be utilized for germplasm collection in the field
rapid multiplication of germaplsm genotypes
pathogen-free germplasm can be maintained
require very small storage space
loss due to diseases, pests and natural calamities avoided
germplasm exchange is easier as quarantine requirement is effectively met
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DANDIN AND NAIK
Some of the methods which are attempted/employed for conservation of mulberry genetic
resources are discussed below:
7.1 Synthetic Seeds
Synthetic seeds, also called artificial seeds, are prepared by encapsulating the apical/axillary
buds with 3–5% sodium alginate and 100 mM calcium chloride solution as a complexing agent.
Sodium alginate solution is mixed to liquid medium supplemented with all macro- and micronutrients and growth regulators necessary for the development of mulberry plant [83]. About
50 ml of sodium alginate and 120 ml of calcium chloride solution making a total of 170 ml is
sufficient to make 200–220 encapsulated beads (artificial seeds). The artificial seeds can be
germinated either in vitro or in vivo. However, success of germination in vivo is comparatively
less. For in vitro germination of artificial seeds of mulberry, MS medium was found suitable. A
cytokinin supplement in MS medium enhances the germination but inhibits root formation. Even
though, encapsulation of apical buds are of limited value in germplasm conservation, these
artificial seeds retain viability upto 45 days at +4°C and for a long period under cryopreservation.
These artificial seeds are also useful in germplasm exchange.
7.2 Slow-Growth Storage
Slow-growth condition in in vitro provide a secondary storage method for field gene bank, a
storage mode for experimental, or a rescue of germplasm for plant distribution [84]. Slowgrowth storage may provide short- or medium-term conservation strategy for germplasm materials
depending on the period of storage achieved. This is done by maintaining the cultures under
growth limiting condition, which reduces the requirements of sub-culturing and associated risks.
Even though, in vitro slow-growth storage appears to be good choice for conservation of
vegetatively propagated species, the information on germplasm conservation is limited to few
genera. In vitro technique has been utilized to conserve wide range of species including temperate
woody plants, fruit trees, horticultural species, and numerous tropical species. A recent FAO
survey indicates that only 37,600 accessions are conserved in vitro worldwide. Slow-growth
storage is routinely used in the conservation of only few species like banana, potato and cassava.
In mulberry, single shoots of M. nigra L. stored on multiplication medium at 4°C for 16-hour
photoperiod survived for only six months. Survival was enhanced to 42% at nine months by
storing them at 25°C with activated charcoal as supplement [85]. High viability (80%) for six
months was observed in 15 genotypes of M. alba stored at 4°C and in dark on shoot proliferation
medium [86]. Rooting was observed in all the shoots and shoots retained their multiplication
potential.
In vitro techniques are becoming increasingly popular in storing and distributing germplasm
throughout the world. Certification programme insist on in vitro cultures for providing virus-free
plants from stock collections. However, additional research is needed to be carried out in the
field of genetic stability of in vitro grown plants. Field experimentation and molecular analysis
are needed to confirm the genetic stability. Additional research to develop standard method
along with regular evaluation of culture materials will provide safe storage for in vitro cultures.
7.3 Cryopreservation
Cryopreservation (storage in liquid nitrogen at –196°C) is considered an ideal method for long-
Biotechnology in Mulberry (Morus spp.) Crop Improvement 211
term germplasm storage. At cryogenic temperature cell divisions and all metabolic activities are
stopped, minimizing the possibility of any genetic change. Cryopreservation can be applied to
different plant parts/structures including seed, apical or axillary buds, embryos, pollen and in
vitro cultures. Sakai [87] was first to report the survival of plant tissue exposed to ultra-low
temperature, when he demonstrated that very hardy mulberry twigs could withstand freezing in
liquid nitrogen (LN) after dehydration mediated by extra-organ freezing. Generally, the technique
of preserving at low temperature improvised with chemical cryoprotectant, slow dehydration,
cooling followed by rapid immersion in LN, storage in LN, rapid thawing, washing and recovery.
As mentioned earlier; cryopreservation technique possibility was first demonstrated using
mulberry. Since then, considerable work on cryopreservation of mulberry has been undertaken
especially in Japan. Shoot tips of pre-frozen winter buds of M. bombycis Koidz. cv Kenmochi
were able to withstand storage in LN [88], however, grafts and cuttings could not on immersing
in LN. With modification of this method Wang and coworkers [89] were able to regenerate plants
of M. multicaulis Loud cv. Lusang through tip culture of frozen winter buds. Shoot segments
were prefrozen at –3°C for 10 days, –5°C for 3 days, –10°C for 1 day and –20°C for 1 day before
immersion in LN. Buds were cultured on MS medium after thawing in air at 0 to 20°C. Observed
survival rate was 55 to 90%. Excised shoot tips from winter buds of M. bombycis cv. Kenmochi
prefrozen to –20°C at 5°C/day were able to produce more shoots compared to the buds prefrozen
at 10°C/day [90]. Prior to prefreezing at –20°C, partial dehydration to 38.5% improved the
recovery rates. The survival rates of the winter buds stored in LN from one month to 3.5 years
did not change [91]. Direct dehydration with silica gel at 25°C of excised shoot tips (2 mm long)
from winter bud could be done before immersion in LN. With decreasing water content shoot
formation increased and at about 19% water content, a maximum of 80% survival rate was
observed. Encapsulation by alginate coating of winter hardened shoot tips of many Morus
species had 81% of shoot formation with 22–25% water content [90, 92]. In vitro grown shoot
tips of thirteen cultivars of mulberry were tested for cryopreservation. Slow freezing (0.5°C/min
to – 42°C), vitrification (PVS2, 90 min) and air drying (24% water content) or encapsulationdehydration (33% water content) was tested for survival, which ranged from 40 to 81.3%. Niino
and coworkers [93] also reported long-term storage of mulberry winter buds by cryopreservation.
Winter buds from M. bombycis with about 10 mm vascular tissue were kept at 0°C for 1 day
before freezing. Buds were cooled to –10°C steps at daily intervals from 0 to –30°C. They were
kept for one day at –30°C prior to immersion in LN or before transfering to –135°C. After storage,
buds were rapidly thawed at 37°C in a water bath and then cultured on MS medium supplemented
with 2% fructose and 1 mg/l 6-BAP. Rate of shoot formation did not vary much in buds stored
in LN or deep freezer at –135°C after a storage period of 3.5 years.
8.
Genetic Transformation
Genetic transformation has been successfully attempted in many agricultural crops. According
to an estimate about 50 million ha of transgenic crops were cultivated worldwide in 2001. These
estimates do not include those cultivated in China. In spite of the resistance to the genetically
modified plants (GMPs) from some quarter, the popularity is gaining among the cultivators.
“Golden rice” is a remarkable achievement and a major leap. This genetically modified rice is
nutritionally enriched with Vitamin A and iron content, which can effectively prevent malnutrition
among the population, especially in Asian countries, where rice is a staple food.
212
DANDIN AND NAIK
Even though, genetically transformed mulberry is yet to be released for cultivation, preliminary
work in this direction has been initiated. Machii [94] used Agrobacterium tumefaciens LBA
4404 as vector to incorporate a foreign gene into mulberry. He transferred kanamycin resistance
gene and β-glucouronidase (GUS) gene through Ti plasmid PB1121 to mulberry leaf discs and
showed their expression in transformed plantlets. Oka and Tewary [95] induced hairy roots in in
vitro grown mulberry (M. indica L.) hypocotyls using Japanese wild Agrobacterium rhizogenes
strains. Specific amplification of DNA fragment by PCR showed that portions of the rol genes
in the T-DNA core region of the Ri plasmid were integrated into the hairy roots. A genomic
clone, Mahmg 1, was isolated from M. alba and its expression characterized in mulberry and
transgenic tobacco [96].
9.
Future Priorities
Biotechnological tools are of immense value in generating genetical information in crops especially
in problematic plants like trees. It holds a great promise in mulberry improvement, utilization
and conservation.
India has large resources of mulberry, which needs to be characterized unambiguously with
DNA marker technology and the total diversity required to be assessed as a supportive research
work for breeding. Developing DNA fingerprints of indigenous mulberry cultivars and important
genotypes for their individualization will be of immense value to the breeders as well as for the
curators of gene banks. DNA fingerprints of mulberry can be successfully used as ‘molecular
I.D. cards’ in context of IPR/patent protection and also protection of Plant Breeders’ Rights.
Based on the molecular data, a core collection is required to be developed for efficient utilization
of mulberry germplasm for crop improvement. There is an urgent need to identify DNA markers
for important agronomic traits, resistances to biotic stresses and tolerance to adverse edaphic and
climatic conditions, which can be utilized to hasten the mulberry breeding programme and
thereby saving considerable physical and financial resources. The major values of molecular
markers lie in the long-term strategic research. An important aspect in this direction is the study
of quantitative trait loci (QTLs) of mulberry. Absence of any linkage map based on morphological/
agronomic traits, necessitates the immediate development of molecular framework linkage of
mulberry. The map can be used to tag genes of agronomic importance and to perform map based
cloning of target genes. Genetic transformation techniques needs to be further fine tuned for
stable expression of cloned genes. The silkworm is completely dependent on mulberry leaves for
their entire nutritional requirement. Hence, the development of transgenic mulberry with qualitatively
superior protein and carbohydrate content of the other known system, which may have significant
impact on silk production, is also an important area.
In the in vitro culture front, further refinement is required to obtain a consistent regeneration
of plants from callus culture. This will help in a long way in successful regeneration of transformed
cells. Further, in vitro protocols already developed needs to be practically utilized for medium
and long-term conservation of mulberry genetic resources.
Biotechnology in Mulberry (Morus spp.) Crop Improvement 213
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
15. Development of High Efficiency Micropropagation
Protocol of an Adult Tree—Wrightia tomentosa
S.D. Purohit*, P. Joshi, K. Tak and R. Nagori
Post Box No. 100, Plant Biotechnology Laboratory, Department of Botany,
M.L. Sukhadia University, Udaipur 313 001, Rajasthan, India
*e-mail: sdp_56@hotmail.com
Abstract: Highly efficient and reproducible micropropagation protocol for Wrightia tomentosa
using sexually adult material has been developed. Multiple shoots were induced from nodal shoot
segments through forced axillary branching in vitro. Nature and management of the donor tree,
season of collecting explant and their orientation on the medium strongly influenced the initial
establishment of cultures. Explants collected in April-June period and placed vertically on the MS
medium containing 2 mgl–1 BAP produced shoots from axillary nodes in vitro. Management of
donor tree by serial harvesting of explants every fortnight was necessary to obtain vigorous growth
of shoots in vitro. Explants of the fifth flush (F5) were found most suitable to obtain more than 7
shoots per node on the above medium. The rate of multiplication in subsequent subcultures was a
little more than two and half-fold. Incorporation of phloroglucinol (100 mgl–1) into the multiplication
medium containing BAP (2 mgl–1) accelerated the rate of multiplication to 3-fold per subculture.
Similar response could be obtained by using 10 mM thidiazuron (TDZ) alone in the multiplication
medium. Nodal segments from in vitro raised shoots were also used to initiate a new culture cycle.
The shoots could be multiplied for at least 24 months without loss of vigor. More than sixty per cent
shoots obtained after sixth subculture developed roots when treated with pre-autoclaved indole-3butyric acid solution (100 mgl–1) for 10 min and implanted on modified MS medium (major salts
reduced to 1/4 strength and 400 mgl–1 activated charcoal). Successfully rooted plants were hardened
in vitro in glass bottles containing SoilriteTM irrigated with 1/4 strength MS salt solution (pH 5.0).
More than 5,000 plantlets were successfully hardened in vitro and transferred to greenhouse for
acclimatization. The survival rate of the plants during hardening was more than 95 per cent.
1.
Introduction
Wrightia tomentosa (Roxb.) Roem et Schult (Apocynaceae), once a common tree species of
Aravallis in Rajasthan (India), has traditionally been exploited for its ivory-white wood in making
toys and as fuel. High rate of seedling mortality, lack of suitable method for natural regeneration
and overexploitation has reduced its population drastically and the plant has been listed as an
endangered species [1]. There is, therefore, a strong need for an alternative method to produce
large number of plants of superior types for conservation and regeneration. Micropropagation
methods have been widely applied for clonal propagation of tree species for afforestation, woody
biomass production and conservation of elite and rare germplasm [2, 3]. These methods have been
successfully integrated with modern forest tree management programs for rapid restoration of the
degraded lands [4]. A lab-scale protocol for micropropagation of W. tomentosa using adult material
was reported by Purohit et al. [5]. Only a limited number of plants could be produced by that
218
PUROHIT ET AL
method with major constraint being in hardening of in vitro developed plants. The present article
describes a highly efficient and reproducible micropropagation protocol that is being taken up for
scaling-up production by this laboratory for large-scale plantation by the foresters.
2.
Materials and Methods
Trees of W. tomentosa selected and marked for quality of wood (age of tree more than 30 years)
were used as a source of explants. Shoots were harvested from these plants round the year,
divided into four distinct periods viz. April-June, July-September, October-December and JanuaryMarch. Management of donor tree was done by lopping one major fork and juvenile shoots
produced near cut ends were collected for explantation. Such newly flushed shoots were serially
harvested fortnightly and successive flushes were termed as first (F1), second (F2), third (F3),
fourth (F4) and fifth (F5). Nodal shoot segments (1.5, 3.0 and 4.5 cm long and 0.2–0.5 cm in
diameter) were prepared as explants. Two different orientations of explant on medium was
tested. Horizontal placement was done in two ways: (i) explant lying horizontally on medium
(H1) and (ii) one side of node, having axillary bud, inserted in medium while other side exposed
to air (H2). Vertical placement of explant was done in three different ways: (a) node completely
immersed in medium (V1), (b) node on the surface of medium (V2) and (c) node one centimeter
above the medium (V3).
Explants were washed thoroughly with sterilized distilled water containing few drops of
Tween-20 and then surface-sterilized with 0.1% (w/v) mercuric chloride for 5 min followed by
thorough washing with sterile distilled water. Surface sterilized explants were inoculated on
standard multiplication medium containing MS salts [6] with 2 mg l–1 BAP. Explants were also
inoculated on MS medium containing different concentrations of Kn (0.5–5.0 mg l–1), TDZ
(50–10,000 nM) and GA3 (1.0–2.0 mg l–1). Proliferated shoots from nodes of F5 flush were further
subcultured on MS medium with various concentrations of TDZ (0.1, 1.0 and 10.0 µM) or BAP
(2 mgl–1). Phloroglucinol (50, 100 and 250 mg l–1) was also added to standard shoot multiplication
medium. Cultures in conical flasks (100, 150 ml) covered with non-absorbent cotton plugs were
kept under controlled conditions of temperature (28 ± 2°C), light (45 µmol m–2 s–1 for 16 h/day
provided by fluorescent tubes) and 60–70% relative humidity. Once culture conditions for optimum
shoot induction from explants were established, the shoots produced in vitro were subcultured
on fresh medium every 3 weeks.
Shoots having passed through three, six and nine passages in multiplication medium were
used for rooting. Shoots (2.0–3.0 cm) were excised and their cut ends were dipped in different
concentrations of IBA solution (50, 100, 200 and 500 mg l–1) for different duration (5–15 min)
followed by their implantation on standard rooting medium containing quarter strength MS salts,
sucrose (1%) and agar (0.6%). Activated charcoal (50, 100, 200 and 400 mg l–1) was also tested
in standard rooting medium. Initially the culture vessels were kept wrapped with black paper or
in darkness for 5–7 days at 30 ± 2°C temperature at 60–70% relative humidity.
Rooted shoots from 3-week-old cultures were hardened prior to ex vitro exposure. Hardening
was attempted by three different methods. In first method, individual plantlets were planted
carefully in Soilrite™ (Karnataka Explosives, Bangalore, India) filled netted pots (3 cm high)
and placed in horizontally kept pickle bottles (30 cm long) which accommodated 25 such pots
(W1). In second method, individual plantlets, transferred to netted pots were placed in glass
troughs (30 cm diameter) covered with polythene sheets (W2). Thirdly, autoclaved 400 ml screw
Development of High Efficiency Micropropagation Protocol for an Adult Tree 219
cap glass bottles one-fourth filled with soilrite™ irrigated with 40 ml inorganic salt solution
(major salts of MS medium reduced to 1/4 strength, pH 5.0) were used (W3). Each bottle containing
4 plantlets were kept in culture room for 30 days.
After 30 days, plantlets hardened by methods described as W1 and W2 were shifted individually
to plastic pots (10 cm high) and covered with polythene bags. Gradually, humidity was lowered
by perforating polythene cover, then opening it for 1 h/day and finally completely removing it.
Plantlets hardened in vitro (W3) were kept in closed bottles till they touched the caps of bottles
(nearly after 30 days). The caps were loosened and finally opened in misthouse (with 70–85%
RH). After one month, plants were transferred to pots and kept under greenhouse conditions where
a gradient of humidity (80–40%) was maintained by a Fan-Pad evaporative cooling system.
2.1 Statistical Analyses
Standard analysis procedures [7, 8] were followed for CRD analysis. Abnormality, non-additivity
and heterogeneity of variance in raw data of different experiments were minimized using square
root ( χ and χ + 0.5) transformation [9]. In ANOVA, test for significance (F test), standard
error of mean and critical difference at 5 and 1 per cent probability was calculated on transformed
data which were tabulated along with retransformed values in each experiment. In case of
discrimination amongst two treatments ‘t’ test was used [8].
3.
Results
3.1 Initiation of Shoot Cultures
Bud break frequency was strongly influenced by the nature and management of donor tree, season
of explant collection and their orientation on the medium. April-June was found to be the best
period for collection of explant to obtain maximum (98%) bud break response with minimum
(5.0%) loss due to contamination (Table 1). The explants collected during the months of JulySeptember developed fungal growth associated with shoot bud proliferation. Least bud break
response was observed in the winter months of October-December. Explants prepared from oneyear-old mature branches responded poorly in cultures as compared to explants taken from freshly
flushed branches (Table 2).
Table 1. Effect of season of harvest on shoot initiation from mature node explants of W. tomentosa on
standard multiplication medium
Period
April–June
July–September
October–December
January–March
Per cent
contamination*
5
90
81
72
±
±
±
±
1.08
8.78
7.02
8.60
Per cent
response*
Callus
intensity
98 ± 13.85
78 ± 12.68
20 ± 4.12
45 ± 8.02
++
++++
+++++
+++
*Mean ± SE.
Explants of different size showed varied response in terms of bud break and amount of
associated callus during shoot initiation. Maximum bud break was found in explant measuring
220
PUROHIT ET AL
Table 2.
Effect of source of explant on shoot initiation in W. tomentosa on standard multiplication (SM)
medium
Source of
explant
Per cent explant
sprouted
Mean number of
shoots per node
Mean shoot
length (cm)
52
98
2.17
3.83*
0.85
2.92*
Mature branches
Juvenile branches
*Significant at 1% level using t test.
3.0 and 4.5 cm in length (Table 3). However, the size of explant did not make significant difference in shoot bud proliferation both in terms of their number and length. In very small explants
(1.5 cm) the basal callus developed upto node, posing difficulty in further subculturing.
Table 3.
Effect of length of explant on shoot initiation in W. tomentosa on standard multiplication
medium
Size of
explant
Per cent explant
sprouted
1.5 cm
3.0 cm
4.5 cm
82
98
97
SEm±
CD.05
0.07
*Figures in parentheses are
Mean number of
shoots per node
Mean shoot
length (cm)
4.32 (2.08)
4.64 (2.15)
4.47 (2.11)
1.92
2.33
2.00
0.18
NS
NS′
χ transformed values.
Orientation of explant on medium was a significant factor in shoot proliferation from nodal
segments. Between horizontal and vertical orientation of explants on medium, the latter was
found significantly superior (Table 4). Maximum number of shoots were produced in explants
oriented in vertical position V3 followed by V2 and horizontal position H2, both statistically at
par in terms of number and length of shoots. When positioned vertically, callusing was associated
with lower internodal region of explant only while it extended to whole surface in horizontally
placed explants. Nodal explants placed 1.0 cm above the medium provided callus-free shoot
proliferation.
Management of donor tree from which the explants were collected was a very important step
in accelerating the number of shoots per node during initial phases of cultures establishment.
Explants obtained from serially lopped branches producing different flushes of juvenile shoots
exhibited graded increase in shoot bud proliferation. An increase in per cent bud break response
and number of shoots per explant from first flush (F1) to fifth flush (F5) was noted with a
concomitant decrease in per cent contamination (Table 5). Explants from F5 flush exhibited
initiation of ca 7.33 axillary shoots per node as compared to 3.98 shoots induced in explants
from F1 flush. However, effect of flushes on length of proliferated shoots was non-significant.
Explants from F5 flushes responded differently as compared to that of F1 flush (Table 6).
About 90 per cent bud break response was observed when explants from F5 flush were inoculated
on the MS medium containing any of the four growth regulators tested. In explants from F1
Development of High Efficiency Micropropagation Protocol for an Adult Tree 221
Table 4.
Effect of node orientation on shoot initiation in W. tomentosa on standard multiplication medium
Orientation of nodal
explant
Horizontal placement
Both sides of node on the medium (H1)
One side inserted into the medium (H2)
Mean
Vertical Placement
Node immersed in the medium (V1)
Node on the surface of the medium (V2)
Node nearly 1 cm above the medium (V3)
Per cent explant
sprouted
Mean number of
shoots per node*
Mean shoot
length (cm)
98
94
2.45 (1.57) c
3.29 (1.81) b
2.85 (1.69)
1.42 c
2.50 b
1.96
10
95
98
1.30 (1.14) d
3.98 (1.99) b
5.31 (2.30) a
1.00 d
3.83 b
3.33 a
3.28 (1.81)*
0.09
0.25
0.35
2.38*
0.18
0.52
0.71
Mean
SEm±
CD.05
CD.01
*Figures in parentheses are χ transformed values.
Means followed by different letters in the same column differ significantly.
*Placement of explant (horizontal or vertical) differ significantly.
Table 5. Effect of serial harvesting on in vitro response by MN of W. tomentosa on standard multiplication
medium
Flush
number
I flush (F1)
II flush (F2)
III flush (F3)
IV flush (F4)
V flush (F5)
SEm±
CD.05
CD.01
Per cent
contamination
Per cent explant
sprouted
10
10
8
5
1
88
90
91
95
98
Mean number of
shoots per node*
3.98
5.14
5.49
6.51
7.33
(1.99)
(2.27)
(2.34)
(2.48)
(2.71)
0.06
0.17
0.23
d
c
bc
b
a
Mean shoot
length (cm)
2.54
2.18
2.43
2.17
2.31
0.17
NS
NS
*Figure in parentheses are χ transformed values.
Means followed by different letters in the same column differ significantly.
flush, mean number of axillary shoots initiated were statistically insignificant on tested
concentrations of any of the four growth regulators except GA3 with least shoot formation.
However, influence of PGRs on shoot initiation was marked in explants from F5 flush, maximum
being on BAP (2 mg l–1). None of the other three growth regulators (Kn, TDZ and GA3) at any
of the concentrations showed better response in terms of number of axillary shoots induced per
node. Numerically, 2 mg l–1 BAP produced maximum number of shoots, followed by 250 nM
TDZ.
222
PUROHIT ET AL
Table 6.
Effect of different PGRs on shoot initiation in W. tomentosa
F1 flush
MS + PGR
BAP
Kn
TDZ
GA3
2 mg l–1
0.5 mg l–1
2.0 mg l–1
2.5 mg l–1
5.0 mg l–1
50 nM
250 nM
500 nM
1,000 nM
10,000 nM
mg l–1
2 mg l–1
SEm±
CD.05
CD.01
F5 flush
Per cent
explant
sprouted
Mean number
of shoots/node*
Per cent
explant
sprouted
Mean number
of shoots/node
84
48
58
52
65
61
60
52
61
53
35
24
3.74
2.91
2.69
3.19
2.44
2.44
3.44
2.69
2.91
2.69
1.72
1.45
97
89
90
88
95
98
92
98
97
92
95
92
6.52 (2.55)a
3.73 (1.93)b
3.19 (1.79)bc
3.11 (1.79)bcd
2.91 (1.71)bcd
2.91 (1.71)bc
3.48 (1.87)bc
2.91 (1.71)bcd
3.48 (1.87)bc
3.19 (1.79)bc
2.23 (1.49)cd
1.93 (1.39)d
(1.93)a
(1.71)ab
(1.64)ab
(1.79)ab
(1.56)abc
(1.56)abc
(1.85)a
(1.64)ab
(1.76)ab
(1.64)ab
(1.31)bc
(1.21)c
0.14
0.40
NS
0.13
0.38
0.52
*Figure in parentheses are χ + 0.5 transformed values.
Means followed by different letters in the same column differ significantly.
3.2 Shoot Multiplication
Shoots after their initial proliferation from F5 explants on medium containing 2.0 mgl–1 BAP
along with the mother explant were further subcultured onto standard multiplication medium after
every 3 weeks. Substitution of BAP with TDZ in subcultures increased shoot multiplication rate,
highest being on medium containing 10 µ M TDZ (2.92 fold) which was significantly superior
to the rate obtained on standard multiplication medium containing 2.0 mg l–1 BAP (Table 7).
Incorporation of phloroglucinol (PG) in standard multiplication medium increased the rate of
shoot multiplication above 3-fold. PG also induced healthy cultures with dark green and lustrous
leaves (Table 8).
Table 7. Effect of different concentrations of TDZ on shoot multiplication in
W. tomentosa cultures
MS + TDZ (µM)
Multiplication fold*
Callus intensity
Control (SM medium)
0.1
1.0
10.0
2.42b
1.92c
2.67ab
2.92a
+
+
+++
+++
SEm±
CD.05
CD.01
0.14
0.42
0.58
*Means followed by different alphabets in the same column differ significantly.
Development of High Efficiency Micropropagation Protocol for an Adult Tree 223
Table 8.
Effect of different concentrations of phloroglucinol on shoot
multiplication in W. tomentosa
MS + BAP (2 mgl–1)
+PG (mgl–1)
Multiplication fold*
Control
50
100
250
SEm±
CD.05
CD.01
2.34
1.08
3.33
1.92
0.20
0.61
0.83
b
c
a
b
Callus intensity
+++
+++
+++
++++
*Means followed by different letters differ significantly.
3.3 Rooting in Shoots
Those shoots having passed through six multiplication cycles, responded to rooting treatments
(Table 9). With the increasing number of shoot multiplication cycles, the rooting response was
more favorable, showing early root initiation and reduced callusing.
Table 9.
Effect of number of subcultures on rooting in IBA pulse treated shoots of W. tomentosa on
standard rooting medium
Shoots harvested
after subculture
III subculture
VI subculture
IX subculture
Per cent rooting
response
Mean number of
days to rooting
Callus
intensity
00.0
15.4
40.3
00.0
35.6
18.1
+++
+++
+
* IBA pulse treatment (100 mgl–1 for 10 min).
Concentration of IBA and duration of treatment affected the root induction process considerably.
Among various IBA concentrations tested for pulse treatment, rooting response was maximum
in shoots treated with 100 mg l–1 IBA solution for 10 min (Table 10). Such shoots exhibited
ca 2.92 roots with 2 cm mean root length. Higher or lower concentrations of IBA did not improve
rooting response. Rooting percentage was found to be positively related with concen-tration of
activated charcoal (AC) added to rooting medium (Table 11). Maximum rooting response (69.7%)
was observed when the IBA-treated shoots were placed in medium containing 400 mgl–1 AC.
Addition of AC helped in early root initiation, increased root number and reduced callusing at
the root-shoot junction.
3.4
Hardening and Acclimatization
Plantlets raised in vitro initially posed problems in hardening and acclimatization. Rooted plants
when transferred directly to pots without prior hardening started wilting, no sooner they were
transferred, and desiccated completely within 24 h. Seedlings were also used in experimentation
to understand their requirements for hardening. Even the seedlings were prone to transplantation
shock similar to in vitro developed plantlets.
224
PUROHIT ET AL
Table 10.
Effect of IBA pulse treatment on rooting in shoots of W. tomentosa on standard rooting medium
Strength of
IBA solution
(mgl–1)
Duration
(min)
50
5
10
15
5
10
15
5
10
15
5
10
15
100
200
500
SEm±
CD.05
CD.01
Per cent
rooting
response
Mean number of
roots*
11
15
18
40
59
48
18
21
19
0
0
0
1.30
1.49
1.84
2.00
2.92
1.90
2.37
1.96
1.79
–
–
–
(1.14)
(1.22)
(1.36)
(1.41)
(1.71)
(1.38)
(1.54)
(1.40)
(1.34)
d
cd
bcd
bc
a
bc
ab
bc
bcd
0.08
0.23
0.31
Mean root
length (cm)
0.78
1.25
1.50
1.50
2.00
1.85
1.85
1.00
1.00
–
–
–
d
bc
b
b
a
a
a
cd
cd
Callus
intensity
–
+
+
++
++
+++
+++
+++
++++
0.11
0.31
0.42
*Means followed by different alphabet in same column differ significantly.
Table 11. Effect of activated charcoal (AC) on rooting in pulse treated shoots of W. tomentosa on standard
rooting medium
Medium +
AC (mgl–1)
Control
AC
SEm±
CD.05
CD.01
50
100
200
400
Per cent
rooting
response
59
45
50
65
69.7
Mean number
of roots*
2.89
2.93
2.76
3.06
3.78
0.07
0.21
0.30
(1.70)
(1.71)
(1.66)
(1.75)
(1.94)
b
b
b
b
a
Mean root
length (cm)
1.87
1.92
2.11
2.41
2.06
0.11
0.34
0.46
b
b
ab
a
b
Mean shoot
length (cm)
Callusing
intensity
3.74
3.67
3.56
3.89
3.60
+
–
–
–
–
0.18
NS
NS
*Values in parentheses are χ transformed values.
Means followed by different letters in the same column differ significantly. IBA pulse treatment (100 mgl–1 for
10 min).
Owing to fast desiccation of rooted plants on direct pot transfer, other methods of hardening
were employed. Rooted plants with nearly 4 cm long shoot, 1 to 2 cm long root and 4 to 6 leaves
in number were hardened by three different methods as described in materials and methods.
Apical growth was visible in more than 95 per cent of plantlets reared through any of the three
methods. After 30 days, plantlets attained an average height of 5.4 cm with 2.1 cm long root
(root shoot ratio being 0.37) and 6-8 broad leaves. Such plantlets were transferred to individual
Development of High Efficiency Micropropagation Protocol for an Adult Tree 225
plastic pots and covered with polythene bags in case of W1 and W2 while the caps of glass bottles
were loosened in W3 plantlets. Plantlets exhibited wide variation in growth during this period.
Most of the plantlets in all the three methods exhibited good shoot growth while root growth
was better only in case of plantlets hardened through W3 method. Generally, the plants were ca
9.08 cm long with 3.67 cm long roots and 8 to 10 leaves. These plants on an average accumulated
20.22 mg dry matter in shoots (without leaves), 7.95 mg in roots and 13.54 mg in each leaf.
During gradual exposure to ex vitro conditions in greenhouse, all plants remained green and
healthy for initial 15 days. Plants hardened through W1 and W2 modes exhibited yellowing of
leaves and leaf fall in next 15 days. The rate of survival was 26.8% after 30 days of transfer to
pots which declined to 2.4% after 60 days. Plantlets hardened through W3 mode grew vigorously
having rigid and thick stem, highly branched root system and green, lustrous, healthy and broad
leaves. Survival rate of such plants was more than 95 per cent. More than 5,000 plantlets have
been successfully hardened and acclimatized and are ready for transplantation into field (Fig. 1).
Fig. 1.
4.
Hardened plants of W. tomentosa kept in a nursery.
Discussion
An adult superior tree can be micropropagated for desired attributes by enhanced axillary
proliferation. The buds residing in the axil of twigs are induced to proliferate and generate
multiple shoot buds in vitro. Proliferation of these axillary buds may be difficult due to microorganism contamination [10], phenolic oxidation [11] and tissue maturity [12]. Maturity of
tissue is accompanied with reduced growth rate, reduced/lack of rooting ability and sometimes
plagiotrophy [13]. By reverting a part of tree to complete /partial juvenility by in vivo and in
vitro methods problems associated with maturity can be minimized. In W. tomentosa the explants
collected from previously lopped trees showed better proliferation when shoots were harvested
serially. Severe pruning has been found to be an efficient method for rejuvenation [14]. In
Quercus robur forced flushing method, related to severe pruning, was adopted [15].
The season of explant collection greatly influenced establishment of W. tomentosa cultures in
vitro. Effect of season on bud sprouting was also noted in many tree species viz. Tectona grandis
226
PUROHIT ET AL
[16], guava [17], Tecomella undulata [18], Prosopis cineraria [19] and W. tinctoria [20]. Vertical
orientation of W. tomentosa explants was found better than horizontal orientation in terms of
number of proliferated shoots. On the contrary, horizontal orientation of explants was found
better in Fraxinus angustifolia [21] and Quercus robur [15].
Cytokinins promote cell division in plant tissues under specific conditions and are found
obligatory for shoot differentiation [22]. Hu and Wang [23] reported superiority of BAP among
cytokinins in differentiation of shoots from explants of trees. Buising et al. [24] suggested that
transient exposure of soybean embryonic axes to BAP interrupted chromosomal DNA replication
and reprogrammed the developmental fate of a large number of cells in shoot apex. In our case,
maximum number of shoots were obtained in medium containing BAP in comparison to other
PGRs. Recently, TDZ has been found to be one of the most active cytokinin-like substances used
for woody plant tissue culture [25]. In present study, TDZ did not supercede the response
obtained with BAP in shoot induction, but it did enhance multiplication of shoots in subcultures.
Incorporation of phloroglucinol in the multiplication medium improved shoot multiplication
rate. Similar results have been reported in apple root stock M.7 by Jones [26]. The effect could
be related to hastening of rejuvenation process in vitro by phloroglucinol.
Rooting by dip treatment of auxin has been recommended by Harry and Thorpe [27]. It is
supposed to eliminate the inhibitory effect on root growth when IBA is incorporated in the media
[28]. Purohit et al. [5] have recommended IBA pulse treatment for rooting in W. tomentosa shoots.
This method of root induction has been successfully employed in the present studies also.
Hardening is most critical factor for achieving success in pot transfer of regenerated plantlets.
We have observed that a gradual shifting of plants from medium to culture bottles containing low
salt concentration without sucrose allowed stress, compelling plants to become partially autotrophic.
This step proved useful in achieving more success in hardening.
The results have demonstrated the feasibility of application of this protocol for raising large
number of W. tomentosa which would greatly help in afforestation programmes in Aravallis in
Rajasthan (India). Large numbers of plants are ready for field transfer that can be used for field
evaluation studies.
Acknowledgements
Authors thank Dr. N.S. Shekhawat, Incharge, Plant Biotechnology Laboratory, J.N. Vyas University,
Jodhpur, India for providing hardening facilities. Thanks are also due to the Department of
Biotechnology, Govt. of India, New Delhi, for financial support.
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Copyright © 2004 Anamaya Publishers, New Delhi, India
16. In Vitro Regeneration and Improvement in Tropical
Fruit Trees: An Assessment
Madhulika Singh, Uma Jaiswal and V.S. Jaiswal
Laboratory of Morphogenesis, Department of Botany, Banaras Hindu University, Varanasi 221 005, India
Abstract: In vitro regeneration protocol has been developed for many tropical fruit trees by using
juvenile as well as mature explants. Regeneration via somatic embryogenesis have been obtained
in a number of cases e.g., while in citrus, sugar apple and papaya, etc. induction of androgenic
haploids are successful, in guava and feijoa only callus results in anther cultures. Somaclones have
helped in the selection of seedless Musa. Synthetic seed technology has aided in raising plantlets
from encapsulated embryos of guava, mango, papaya, etc. Gene transfer techniques can further
prove to be useful in the improvement of varieties.
1.
Introduction
Commercial cultivation of fruits is still in infancy and even at present the yield of fruits in most
cases remains low and are not within the means of working classes of the developing world.
Many tropical fruits remain under utilized due to the complex circumstances that encompass
their production and marketing. Moreover, the green revolution could make a relatively little
impact on fruit cultivar development. Increase in fruit production can be achieved by advances
in horticultural practices, post harvest handling and disease and pest control. Conventional
propagation methods, i.e. grafting, air layering and removal of suckers, for improving the tropical
fruit crop trees already exist for many important tropical fruits but the long juvenile period has
made these techniques time consuming and cumbersome. Clonal propagation and selection of
fruit crops using tissue and organ culture techniques have considerable potential for the improvement
of economically important fruit trees that have been under cultivation for many generations.
Improvement of plant quality and yield by cell manipulation through the sophisticated methods
of genetic engineering has to rely on tissue culture for the final product. Generation of new
variability through somaclonal variant selection, production of androgenic and gynogenic haploids
to achieve homozygosity, rapid fixation of specific traits in hybrids, freeing plants from disease
causing organisms by shoot tip culture and production of industrial compounds by cell culture
are some well-known applications of plant tissue culture. Tissue culture technique and other
biotechnological intervention have proved fairly successful and could be commercialised for
some temperate fruit crops [1]. However, due to difficulty in controlling normal somatic embryo
development and achieving high rates of their germination the progress in the application of
tissue culture for clonal multiplication of tropical fruit trees and biotechnological tools has been
rather slow. The purpose of this review is to present the current status of in vitro regeneration and
improvement of tropical fruit trees.
In Vitro Regeneration and Improvement in Tropical Fruit Trees 229
2.
In vitro Regeneration of Tropical Fruit Trees
One of the earliest attempts to regenerate tropical fruit trees through in vitro culture technique
was made by Maheshwari and Rangaswamy [2]. Subsequently, several species of tropical fruits
have been regenerated through the process of organogenesis as well as somatic embryogenesis.
Organogenesis involves adventitious and axillary shoot production. The adventitious shoot
production comprises de novo shoot meristem formation from callus tissue or directly from
organized tissues such as epidermal or subepidermal cells. The axillary shoot production involves
shoot formation from axillary buds, shoot tips and meristems. The regenerated shoots are excised
and used to produce additional shoots. The axillary shoot production is a direct method involving
multiplication of preformed buds, usually without any callus formation and produces in general,
genetically stable cultures. It produces the smallest number of plants, since the number of shoots
produced is limited by the number of axillary buds placed in culture. Although the initial
multiplication rate is low, it increases during the first few subcultures and eventually reaches a
steady state, which may be maintained through numerous subcultures. Somatic embryogenesis
is the process in which structures are formed containing a shoot and root connected by a closed
vascular system (directly analogous to zygotic embryos).
2.1 Regeneration via Organogenesis
The main factors that influence the mode and rate of in vitro regeneration are the nature of
explant, composition of the medium and the physical conditions in which the cultures grow.
Organogenesis has been induced in vitro both from seedlings and mature tree explants (Table 1).
Adventitious shoots have arisen directly from internode segments without a callus phase in
Citrus [3]. Direct shoot organogenesis and plant regeneration have also been reported from
seedling leaf explants of Annona squamosa [4] and Garcinia mangostana {5, 6] and from
hypocotyl and seedling petioles of A. cherimola [7]. Adventitious shoots have differentiated
following callus initiation and proliferation in Citrus [8, 9]. New vegetative growth that occurs
from the base of the main stem during the period of vigorous vegetative growth in guava [10]
serves as a reliable source of shoot tip and nodal explants. Papayas have been decapitated in
order to stimulate lateral branching and to increase the number of explants from stock plants
[11]. Shoot tip culture is the basic technique for Musa propagation [12–15]. It has been successfully
applied to the rapid propagation of AA and AAA bananas, cooking ABB bananas and to a limited
extent to AAB plantains and ‘Silk’ and ‘Pome’ AAB dessert bananas.
Organogenesis of tropical fruit species have generally been based on MS medium [16]. In a
few cases (mangosteen, Musa) other media have been used for optimum morphogenesis (Table
1). In most studies callus initiation and shoot induction have been reported on the same medium
which contains cytokinin, BA (Table 1) or a cytokinin together with an auxin. A high cytokinin
to auxin ratio favours caulogenesis. Occasionally, shoot formation can occur following subculture
of callus initiated on a medium with either a high auxin to cytokinin ratio, or high cytokinin to
auxin ratio, or with cytokinin alone [17]. Usually, the auxin, NAA has been preferred for its
synergistic effect on shoot induction. Some tropical fruit trees which have been regenerated via
organogenesis have been listed in Table 1.
2.2 Regeneration via Somatic Embryogenesis
Somatic embryogenesis has several distinct advantages over organogenesis [17–19]. In woody
230
MADHULIKA SINGH ET AL
Table 1.
Species
Annona cherimola
Annona squamosa
Annona squamosa
Artocarpus heterophyllus
Carica papaya
Carica papaya
Carica papaya
Carica papaya
Citrus acida
In vitro regeneration of tropical fruit trees: Organogenesis
Explant
Mature/
Juvenile
Medium
Growth
Regulator
Reference
H, P
L
H
ST
S
C
N
L, P, S, R
Ep
J
J
J
M
J
J
J
M
J
MS
MS
WPM
MS
MS
MS
MS
MS
MS
[7]
[4]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
J
J, M
J
J
J
J
J
J, M
J
–
MS
J,M
M
M
J, M
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
NAA, BA
BA
BA, NAA, IBA
BA, KIN, NAA
KIN, IAA
BA, NAA
BA, KIN, IBA, NAA
BA, IBA
BA, NAA, IAA,
2, 4–D, GA3
BA
NAA, KIN
NAA, BA
BA, TDZ, NAA, GA3
BA, NAA
BA, KIN, NAA
NAA, KIN
NAA, BA
BA, KIN, NAA,
BA, NAA
BA, NAA
NAA, KIN
BA, KIN NAA, IBA
BA, NAA
BA, IBA
J, M
J, M
J, M
M
J, M
–
J
J
M
M
M
M
M
J
J
J
M
–
J
MS
MS
MS
WPM
*
MS
MS, WPM
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS
MS, Knop’s
MS
Citrus aurantifolia
S, R
C. aurantium
S
C. grandis
S, L
C. grandis
S, T
C. halimii
H
C. jambhiri
S, R
C. Iimetoides
S
Citrus limon
S
Citrus limon
S, R
C. madurensis
S
C. paradisi
S, L
C. reticulata
S
C. reticulata
ST
C. sinensis
S, L
C. sinensis
S
Citrus sinensis × Poncirus
trifoliata
S
C. sinensis × P. trifoliata
R
Garcinia mangostana
L, C
Garcinia mangostana
L
Garcinia mangostana
L
Garcinia mangostana
Seed
Garcinia mangostana
L
Litchi chinensis
Seed C
Mangifera indica
L
Poncirus trifoliata
ST
Psidium guajava
ST
Psidium guajava
N
Psidium guajava
N
Psidium guajava
N
Psidium guajava
ST
Psidium guajava
Seedling
Psidium guajava
S
Musa
ST
Syzygium cumini
ST, N
BA
BA, 2, 4-D
BA
BA, IBA
BA, auxin
BA, NAA
BA, TDZ, NAA
BA, IBA
KIN, IAA, IBA
BA, IBA
BA, NAA, IBA
BA, NAA, IBA
BA, NAA, IBA
BA
BA, NAA, IBA
BA, NAA, IBA
BA, NA
BA, BAA, IBA
[68]
[69]
[8]
[70]
[71]
[72]
[9]
[69]
[72]
[73]
[74]
[69]
[75]
[8]
[69]
[3]
[76]
[5]
[77]
[78]
[6]
[79]
[80]
[81]
[82]
[83]
[10]
[84]
[85]
[86]
[87]
[88]
[89]
[90]
C = cotyledon, Ep = epicotyl, H = hypocotyl, L = leaf, N = node, P = petiole, R = root, S = stem, ST = shoot
tip, M = mature, J = juvenile, MS = Murashige and Skoog, WPM = Woody plant medium [91], Knop’s = Knop’s
medium [92], * = specific formulation, BA = 6-benzylamino purine, KIN = kinetin, TDZ = thidiazuron,
NAA = α-naphthalene acetic acid, IAA = Indole-3-acetic acid, IBA = indole-3-butyric acid, Zea = zeatin, 2ip =
2-isopentenyl adenine, GA3 = gibberellic acid.
In Vitro Regeneration and Improvement in Tropical Fruit Trees 231
species somatic embryogenesis is achieved less frequently than other methods of regeneration.
However, most of the tropical fruit trees have been regenerated via somatic embryogenesis.
Among the tropical fruit trees in vitro somatic embryogenesis was first reported in Citrus. The
initial attempt on induction of somatic embryogenesis in Citrus was made by Stevenson [20].
Later on, Maheshwari and Rangaswamy [2] reported the induction of somatic embryogenesis in
Citrus by showing the formation of subcuticular globular proembryos from nucellus explants.
Since then, the list of species has been extended and numerous publications have appeared on
the initiation of somatic embryogenesis (both direct and indirect) using diverse explants
(Table 2).
Among the different explants used to induce somatic embryogenesis in tropical fruit trees,
nucellus has been the most appropriate. Somatic embryogenesis has been induced directly in
cultured nucelli of Citrus [21] and indirectly in mango [22–24] and papaya [25]. Immature
zygotic embryo has also proved to be regenerable tissue for many species (Table 2). The culture
of zygotic embryo is a relatively easy in vitro procedure. Embryo culture has been used to
multiply Litchi which is one of the most recalcitrant tropical fruit species.
Somatic embryogenesis is reported to follow two different patterns [26]. In the first,
embryogenesis proceeds from the cells that are embryogenic in origin [27, 28] and in the second,
embryogenesis is induced in highly differentiated tissues such as leaf, stem, nucellus and
inflorescence [29, 30]. Embryogenesis from proembryogenic determined cells (PEDC) requires
only an in vitro environment to follow the requisite pattern of cell division [19]. Since mature
tissues are highly differentiated than those of proembryos, embryogenesis from the former
tissues proceeds via the other route described by Sharp et al. [26], i.e. through induced embryogenic
determined cells (IEDC) [31]. These highly differentiated tissues must undergo major epigenetic
changes to initiate somatic embryogenesis. Therefore, IEDC requires an in vitro environment
initially to dedifferentiate and then to redifferentiate quiescent cells to an embryogenic state.
Direct and indirect embryogenesis, are two additional terms used to describe PEDC and IEDC
embryogenesis respectively [17].
A number of media have been used for the induction of embryogenic cultures. However, most
of the successful reports are based on Murashige and Skoog’s (MS) medium (Table 2). The effect
of medium composition and strength on induction of somatic embryogenesis has been demonstrated
in some species, e.g. Citrus [30], papaya [32] and mango [22, 23, 33]. Generally, embryogenic
callus has been obtained following explanting onto the medium containing 2,4-D or other synthetic
auxins, like dicamba, NAA, etc. (Table 2). The requirement of exogenous auxin for the induction
of somatic embryogenesis depends on the nature of the explant used. Although cytokinins have
sometimes been incorporated into the induction medium, they are probably not critical for
induction. But, in a few cases, e.g. in longan [34], induction of embryogenic callus has been shown
to be cytokinin-dependent. Nitrogen in the form of glutamine has been shown to be essential for
somatic embryogenesis in mango [22–24, 33]. Addition of polyamines to the culture media
promoted somatic embryogenesis in coconut and papaya [35]. The complex organic nutrients
such as coconut water, casein hydrolysate, malt extract, etc., have also been used in the induction
medium for some species [36, 37]. Sucrose is the commonly used carbon source and a
relatively high concentration of sucrose (5–6%) is optimum for somatic embryogenesis in guava
[38], Citrus [39], mango [22–24, 33] and longan [34]. In addition to culture medium and explants,
different genotypes of a species influence the ability of somatic embryogenesis [23, 37, 40].
232
MADHULIKA SINGH ET AL
Table 2.
Species
Carica papaya
Carica papaya
Carica papaya
Carica papaya
Carica papaya
Carica papaya
C. papaya × C. cauliflora
Citrus aurantifolia
Citrus aurantium
Citrus clementina
Citrus grandis
Citrus jambhiri
Citrus limon
Citrus limon
Citrus limon
Citrus limon
Citrus microcarpa
Citrus nobilis
Citrus paradisi
Citrus reticulata
Citrus reticulata
Citrus reticulata
Citrus sinensis
Citrus sinensis
C. unshiu
Cocos nucifera
Cocos nucifera
Eugenia spp.
Euphoria longan
Eriobotrya japonica
Feijoa sellowiana
Mangifera indica
Mangifera indica
Mangifera indica
Mangifera indica
Mangifera indica
Mangifera indica
Musa (AAA, ABB)
Myrciaria cauliflora
In vitro regeneration of tropical fruit trees: Somatic embryogenesis
Explant
S
ZE
Protoplasts
isolated somatic
embryos
P
H
H
ZE
Nu
Nu
Nu
Nu
Nu
Nu
In (style)
ST
Nu
Nu
Nu
Nu
Nu
ST
L, E, C, R
Nu
Nu
Juice vesicle
Inf
Inf
ZE
L
Nu
ZE
Nu, ZE
Nu
Nu
Nu, ZE
Nu
Protoplasts isolated
from pro embryogenic masses
Rh, Basal
Sheath
Nu
Mature/
Juvenile
Medium
Growth
Regulator
Reference
J
J
J
MS
MS
KIN, IAA
2, 4–D, KIN
[63]
[32]
[93]
J
J
J
J
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
M
J
M, J
M
M
M, J
M
J
MS
MS
MS
MS
MS
MT
MS
MS
MT
MS
MS
MS
MS
W
MT
MT
W
MS
MS
MS
MS
MS
MS
2, 4-D, BA
2, 4-D, IBA
2, 4-D, ABA
BA, ABA
IAA, KIN, GA3
MS
B5
MS
MS
MS
MS,
MS,
MS,
MS,
MS,
[94]
[95]
[25]
[96]
[97]
[98]
[99]
[21]
[98]
[21]
[29]
[75]
[100]
[2]
[101]
[102]
[103]
[75]
[30]
[21]
[100]
[104]
[105]
[106]
[114]
[34]
[107]
[108]
[109]
[22]
[110]
[23]
[24]
[111]
M
SH, MS
Dicamba, Zea
[112]
M
MS
2,4-D
[113]
BA, NAA
BA, KIN, NAA, IBA
–
–
–
–
–
BA, KIN, NAA, IBA
KIN, NAA
–
–
KIN, GA, NAA
2, 4-D, BA, zip
B5
B5
B5
B5
B5
2,4-D
2, 4-D, KIN
2, 4-D, BA
2, 4-D, KIN
2, 4-D
2,4-D, KIN
BAP, 2, 4–D GA3
2, 4-D, GA3
2, 4-D, GA3
2, 4-D, NAA, KIN
GA3
C = Cotyledon, Ep = epicotyl, H = hypocotyl, Inf = inflorescence, L = leaf, Nu = nucellus, P = petiole, R = root,
Rh = rhizome, S = stem, ST = shoot tip, ZE = zygotic embryo, M = mature, J = juvenile, MS = Murashige and Skoog,
B5 = Gamborg et al. [115], MT = Murashige and Tucker [39]; W = White’s [116], * = specific formulation, 2,4-D = 2,
4 dichlorophenoxy acetic acid, BA = 6-benzylamine purine, KIN=kinetin, TDZ = thidiazuron, NAA = α-naphthalene
acetic acid, IAA = Indole-3-acetic acid, IBA = Indole-3-butyric acid, Zea = zeatin, GA3 = gibberellic acid.
In Vitro Regeneration and Improvement in Tropical Fruit Trees 233
Among the tropical fruit trees, regeneration of viable plantlets from somatic embryos is a
more frequently encountered problem than the production of somatic embryos from somatic
embryogenesis. The problem may occur at any stage of development like maturation, germination,
shoot apex elongation or acclimatization. Although somatic embryogenesis has been reported
for several tropical fruit tree species (Table 2), the quality of somatic embryos with regard to
their germinability or conversion into plants has been very poor This is because the apparently
normal looking somatic embryos are actually incomplete in their development. Unlike seed
embryos, the somatic embryos normally do not go through the final phase of embryogenesis
called ‘embryo maturation’ which is characterised by the accumulation of embryo specific
reserve food materials and proteins which impart desiccation tolerance to the embryos [41].
Abscisic acid (ABA) which prevents precocious germination and promotes normal development
of embryos by suppression of secondary embryogenesis and pluricotyledonary [18] is reported
to promote embryo maturation in several species. A number of other factors such as temperature
shock, osmotic stress, nutrient deprivation and high density inoculation can substitute for ABA,
presumably by inducing the embryos to synthesize the hormone. ABA is known to trigger the
expression which is normally expressed during down phase of seeds [41]. Cytokinin can be
important for somatic embryo maturation and has been demonstrated to influence development
of cotyledon and shoot apex [17].
2.3 Anther Culture
In vitro androgenesis has been described as a process of deviation of development from normal
gametophytic to a sporophytic pathway. This deviation generally leads to callus production or
embryo formation. The plants can subsequently be obtained either via organogenesis or
embryogenesis from the androgenic callus or via direct germination of androgenic embryos. In
the tropical fruit trees androgenesis and plantlet regeneration have been reported in Citrus [42],
sugar apple [43], papaya [44], longan [45] and Litchi [46]. The androgenic callus formation from
in vitro culture of anthers has been reported in guava [47] and Feijoa [48]. This meagre progress
with anther culture particularly in woody tropical fruit species that are difficult to culture suggests
that the technique could be a reproducible method in regeneration of tropical fruit trees, but an
extensive research in this area is still needed. Anther culture following pollen storage has
potential for conservation [49]. Cryogenic storage of pollen would be space efficient and economical.
3.
Somaclonal Variation
The term ‘somaclonal variation’ refers to the phenotypic and genotypic variation observed in
plants regenerated from any form of cell culture [50]. The degree of variation has been shown
to depend on tissue being cultured [51] and also on the length of time that the cells or tissues
have been maintained in vitro [51]. Somaclonal variation may be a viable approach for obtaining
horticulturally useful traits in tropical fruit trees. In addition, a long generation time for most
fruit species like, seedlessness in Musa, etc. make this approach even more appealing for many
species. Progress has been made with a few fruit species to use this technique to obtain disease
resistance [117, 120–122], salt tolerance [119], thornlessness [99] and toxin resistance [118].
Additional research still needs to be conducted to assess the phenotypic and genotypic stability
of these traits.
234
4.
MADHULIKA SINGH ET AL
Synthetic Seed
The ‘synthetic’ or ‘artificial’ seed technology is an exciting and rapidly growing area of research
in plant cell and tissue culture. Production of artificial seeds has unravelled new vistas in plant
propagation. It is an excellent technique for propagation of rare hybrids, elite germplasm and
genetically engineered plants. Germplasm can be stored effectively in the form of synthetic
seeds. They serve as the most efficient delivery system. Synthetic seeds have been produced
using either of the two methods: a hydrated system [52] or a desiccated one [53]. In the tropical
fruit trees, the artificial seed technology is progressing well. Encapsulation of somatic embryos
and plantlet regeneration have been reported in guava [38], mango [54] and papaya [55]. Plants
were also regenerated from encapsulated shoot tips of banana [56, 57].
5.
Transgenic Plants: Achievements in Tropical Fruit Trees
The development of recombinant DNA technology and efficient systems of controlling
morphogenesis from the culture of cells and tissue have opened the opportunity for genetic
manipulation of plants at the cellular level. The goal of gene transfer techniques is to produce
improved varieties through the incorporation of horticulturally important genes (such as pest and
disease resistance, drought and cold tolerance, herbicide resistance, improved fruit quality,
reduced juvenility, dwarfism, etc.) into existing cultivars. Methods available for plant transformation
are arranged in three main groups: (i) those using biological vectors (virus- or Agrobacteriummediated transformation), (ii) direct DNA transfer techniques (chemical-, electrical-, or microlaserinduced permeability of protoplasts or cells), and (iii) non-biological vector system (microprojectiles,
microinjection or liposome fusion). A comprehensive review on transformation methods has
been compiled by Potrykus [58]. Agrobacterium based transformation shows an advantage over
other methods since it targets transgenes to the nucleus and integrates them into the host DNA.
Several trasformations have been reported based on Agrobacterium-mediated transformation
of cells or explants, e.g. in Citrus [125], papaya [134] and mango [131]. The recovery of
transgenic plant is mainly dependent on the frequency of gene introduction and the ability of the
transformed cells to differentiate into plants, i.e. an efficient in vitro regeneration protocol is a
pre-requisite. Pang and Sanford [133] were the first to demonstrate transformation of papaya by
co-cultivating leaf discs, stems and petioles with A. tumefaciens. Although transformation was
confirmed by nopaline assays, they were not able to regenerate the callus into plant. Fitch et al.
[129] first demonstrated papaya with the neomycin phosphotransferase II (NPTII) and βglucuronidase (GUS) genes using immature embryo explants via microprojectile bombardment.
Fitch et al. [130] regenerated papaya plants resistant to papaya ring spot virus by incorporating
PRV cp gene. The frequency of transformation in both cases was very low. In Citrus, successful
transformation is reported using different methods (Table 3), but transformation frequencies
were much lower. Transformation has been reported in a few tropical fruit trees, some of which
are listed in Table 3.
Although elegant protocols have been worked out using the biological vector Agrobacterium
tumefaciens as well as direct gene transfer in basic and applied science, there are still many
problems which have to be solved in terms of a reproducible method, but these problems are
more related to the biological or genetical phenomena than to the delivery of DNA into plant
cells. The different methods could deliver DNA into the cells, but the events in the cell and the
genetic compartments, organelles and nucleus are not controlled and the genetic integration of
Electroporation
Embryogenic
callus subcultured in
liquid medium
Protoplast of
nucellar callus
Cell suspension
culture derived
from embryo
callus
Internodal stem
segments of
seedlings
Internodal stem
segments of 5
week old
seedlings
Epicotyl
segment
Zygotic and
somatic embryos
and hypocotyl
Immature zygotic
embryos
Citrus jambhiri
(rough lemon)
Citrus sinensis
CVS ‘Trovita’
‘Washington
navel’
Citrus sinensis
CV ‘Pineapple’
Citrus sinensis ×
Poncirus
trifoliata
(root stock)
Poncirus
trifoliata
(root stock)
Carica papaya
Carica papaya
Explant
Particle bombardment
Microprojectile
bombardment
Agrobacterium
Agrobacterium
Agrobacterium
Agrobacterium
Direct DNA
transfer with 20%
PEG6000
Direct DNA
transfer by
Electroporation
Transformation
method
coat
protein of
papaya ring
gus, npt II and
the coat protein
of papaya ring
spot virus
gus, npt II
gus with
intron and
npt II
gus with
intron and
npt II
npt II and
hpt
cat and
npt II
gus
Foreign gene
Transgenic papaya
plants having increased
resistance to PRV
Transformed somatic
embryos and leafy
shoots. Resistance
to Kan
Transformed plants
(Resistance to Kan)
Transformed shoots
grafted in vitro onto
seedling rootstocks
Transformed shoots
grafted in vitro onto
seedling rootstocks
Transgenic plants,
embryoids resisting
Kan
Selection of microcolonies with paramomycin (20–40 µg/ml).
Transgenic plants
Reduced colony
formation
Result
Tropical fruit trees in which stable transformed plants have been obtained
Citrus reticulata
CVS ‘Onta’
(Ponkan) ‘Kara’
(mandarin)
Species
Table 3.
(Contd)
[130]
[129]
[128]
[127]
[126]
[125]
[124]
[123]
Reference
In Vitro Regeneration and Improvement in Tropical Fruit Trees 235
Somatic
proembryos
Embryogenic
cell suspension
initiated using
immature male
flower
Musa (AAA group)
Explant
Mangifera indica
Species
Microprojectile
bombardment
Agrobacterium
Transformation
method
Table 3.
Foreign gene
npt II, Vid A
or BBTV
gus, npt II
spot virus
gus, npt II
(Contd)
Resistance to
Kanamycin
Proembryos resistant
to Kanamycin
Result
[132]
[131]
Reference
236
MADHULIKA SINGH ET AL
In Vitro Regeneration and Improvement in Tropical Fruit Trees 237
foreign DNA is random. Targeted transformation is still at its infancy [59]. Gene silencing and
interactions between different transgenes result in unexpected expression patterns of foreign
genes [59, 60]. Several independent transformants with a specific gene construct are still necessary
to find one transgenic plant with the proposed expression pattern [60]. Transgene-mediated
suppression of a gene by antisense constructs can be achieved. Up to now, plant biotechnology
has mainly focused on a single gene strategy. It is still cumbersome to change physiological
traits which are determined by multiple genes and/or quantitatively inherited.
6.
Conclusions
Considerable progress has been made in the recent past on in vitro plant regeneration via
organogenesis and somatic embryogenesis in tropical fruit trees by manipulation of growth
media and culture conditions as well as testing a variety of explant sources. To improve the
propagation system and to overcome the main bottlenecks, in particular, maturation and low
germination frequency, the knowledge of developmental physiology need to be enhanced.
Refinements in protocols are also necessary to get good quality embryos to facilitate storage,
germination and encapsulation of these embryos. Numerous characteristics in tropical fruit trees
which cannot be improved by conventional breeding need biotechnological intervention.
Besides the fundamental aspects, a wide array of practical problems need to be solved such
as mechanical handling and automated planting. In addition, it would be necessary to reduce the
production cost for commercial application.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
17. Tissue Culture of Cashewnut
Sumita Jha and Sudripta Das
Centre of Advanced Study, Department of Botany, University of Calcutta,
35, B.C. Road, Kolkata 700 019, India
Abstract: Cashewnut, Anacardium occidentale L. (Anacardiaceae), is one of the most recalcitrant
species in tissue culture. Even after eighteen years of research since the first report on cashew tissue
culture, limited success has been achieved in obtaining a reproducible protocol for induction,
development and conversion of somatic embryos. Some success has been achieved in micropropagation
using cotyledonary nodal explants but there are no reports on propagation using explants from
mature trees. Embryo culture has been successful in raising whole plants from immature embryos
to overcome the problem of embryo abortion in cashew. Micrografting has also been employed by
a modified side-grafting procedure by which shoot tips from glass-house raised seedlings and field
plants were grafted on in vitro raised seedling rootstock. Induction of somatic embryos on different
explants excised from immature zygotic embryos like excised cotyledons and excised hypocotyl
with radicle, as well as on intact zygotic embryos as small as 1–2 mm has been obtained. The
establishment of viable aseptic culture of immature zygotic embryos in cashewnut was restricted
due to the exudation of phenolics in the media and the oxidation and browning of explants. Somatic
embryos were induced directly on intact zygotic embryo explants of A1–A3 size in two out of the
five genotypes that were studied, after 4 weeks of culture initially in M1 media and 4 weeks in the
M2 media. Induction of somatic embryos was related to the size of intact zygotic embryo explant,
and to the presence or absence of callusing in explants. Embryos were also induced on excised
cotyledons and excised hypocotyls with radicle, but the best results of number of somatic embryos
induced per explant (23 embryos) and frequencies of maturation (59.5%) and germination (23.2%)
were obtained in intact zygotic embryos of A2 size, belonging to the variety KV-26. A preconditioning
or post-maturation period was necessary for germination of somatic embryos and germination was
achieved after 4–5 weeks of preconditioning on MS media containing BA (1.0 mg/l) (M3), however,
only in 8.05–23.2% of the cases somatic embryos were induced on different explants. Plants
regenerated from somatic embryos were transferred to MS basal medium for proliferation of
shoots, which was found to be very slow. Attempts to transfer somatic embryo derived plants to
potted soil were not successful. The results obtained have important implications for the further use
of in vitro culture techniques for this recalcitrant species for the recovery of somaclones and transgenics.
1.
Introduction
Plantation crops are high value commercial crops, of great economic importance and play a vital
role in the country’s export trade. There is an urgent need today to concentrate more on the
research aspects of plantation crops, particularly cashew nut, for rapid propagation and qualitative
and quantitative improvement of the yield. Conventional breeding techniques have contributed
much to the improvement of perennial tree species. However, the resources of useful genetic
variation are nearing exhaustion. Reforms in breeding techniques are therefore imperative. In
conventional plant breeding, long periods are necessary to develop new varieties of woody
species and also to replace varieties. Particularly with cross-pollinated species propagated with
Tissue Culture of Cashewnut 245
seeds, it is difficult to maintain the superior characteristics of a new variety. The application of
tissue culture techniques for propagation and improvement of woody plant species, particularly
tree species, holds great promise. By using this approach, a large number of individual plantlets
with improved characteristics may be propagated in a short time. From a genetic point of view,
rapid propagation in tree species has three advantages, production of population with uniformly
superior genotypes; maintenance of characteristics and combinations that cannot be maintained
by sexual propagation, rapid multiplication and storage of a superior variety or hybrid is possible
through artificial seed technology. If traditional breeding techniques can be combined with
biotechnology, new and remarkable progress will be achieved in improvement of tree species. As
compared with herbaceous plants, perennial crops present some difficulties for using biotechnologies
for their improvement. Despite three decades of research, the generation of woody plant species
by cell and tissue culture techniques has been elusive [1, 2]. Even though some tree species can
be micropropagated from mature trees, many others can presently be propagated only from
tissues of juvenile specimens i.e., embryos or young seedlings [3].
Cashewnut, Anacardium occidentale L., belonging to family Anacardiaceae, is an evergreen,
tall, tropical fruit tree, upto 12 m in height, which forms a thin peripheral canopy, studded with
protruding inflorescences. The kernel of the seed, which remains after removal of testa, is the
cashewnut of commerce.
Cashewnut is cultivated in many tropical countries, the main producers of the nut being
Brazil, India, Mozambique and Tanzania. Although cashew was introduced in India in the 16th
century by the Portuguese, the gene pool that was available to breeders was very low. However,
some of the research centres in India, namely Bapatla, Vengurla and Ullal (in South India) were
instrumental in assembling the germplasms and evaluating them for yield, quality and other
agronomic characters. There are a few named cultivars but efforts were made to select superior
high yielding types and propagate them by asexual methods [4]. Though there has been considerable
improvement in the crop through conventional breeding, progress has been slow because the tree
is heterozygous and takes 10–12 years to reach full cropping. It is propagated mainly by seeds
often resulting in high degree of variability.
In cashewnut, area has increased from 0.176 million hectares in 1961 to 0.659 million hectares
in 1996–97. The production in cashew has gone up from 0.079 million tonnes to 0.430 million
tonnes in 1996–97. India exported cashew kernels worth Rs 13,000 million (US $ 362 million
during 1996–97). Export of cashew is rising @ 27% per annum. These export earnings are
exceeded only by coffee and rice among agricultural exports. To overcome the problem of low
production in cashew in India, areas under high yielding varieties with clonal saplings is being
increased and orchards are being replaced by new high yielding varieties [5]. However, conventional
methods of propagation are not efficient enough to provide high yielding planting materials
[5, 6]. Reforms in breeding techniques are therefore imperative to meet the increasing demand
of cashew nut in the international market. Techniques like micropropagation via multiple axillary
branching and in vitro organogenesis or embryogenesis offer prospects of faster multiplication
of elite genotypes. The application of plant biotechnological methodologies for the improvement
of cashewnut is mainly limited by the difficulty of regenerating plants in a reproducible and
efficient fashion. Cashew, like other members of Anacardiaceae, is strongly recalcitrant in in
vitro culture and only limited successes have been achieved to date in this cash crop.
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JHA AND DAS
1.2 Organogenesis
In India, cashew tissue culture work was initiated for the first time at Calicut University by
Philip and Unni [7] and later Philip [8] reported direct shoot and root organogenesis from
proximal ends of cotyledon explants. Leva and Falcone [9] used microcuttings, young leaves and
cotyledons from mature seed as explants to evaluate micropropagation using buds, and regeneration
from callus cultures. Morphogenesis was achieved from globular calli on leaves and from nodular
structures on cotyledons on SH medium with a high concentration of NAA and 6-BAP in
combination. Sue et al. [10] screened morphogenetic capacities of different explants. Callus
formation was obtained from leaf and petiole explants when different levels of NAA/BAP or
2-4, D/BAP were combined on MS modified medium. Adventitious and secondary roots induction
occurred only when NAA was present at high levels (6 mg/1) along with BAP (1 mg/1). Hegde
et al. [11] reported direct regeneration of plantlets from cotyledonary segments cultured on
LS medium supplemented with kinetin and NAA. There are no reports/publications dealing
with successful regeneration of plantlets through shoot organogenesis in vitro from any type of
explants to date.
1.3 Apical and Axillary Node Culture
Progress with application of micropropagation has been achieved using microshoots and
cotyledonary nodes using in vitro germinated seedling explants. However seedling explants are
normally extremely heterozygous due to outbreeding. Shoot tip/node culture from identified
elite trees would ensure genetic fidelity of in vitro raised plants. However, many problems persist
with explant viability when shoot tips/nodes are used from mature trees. Cashewnut was found
difficult to propagate in vitro from mature plant tissues (nodal segments or shoot apices) due to
recalcitrant nature, microbial contaminations and high phenolic exudation. Lievens et al. [12]
cultured nodal cuttings from 6- to 15-month old seedlings and showed axillary growth and shoot
bud proliferation. Leva and Falcone [9] cultured shoots on MS and SH media and obtained shoot
growth and rooting. They reported that the presence of GA3 in combination with zeatin riboside
improved node formation. Our own attempts to raise aseptic cultures from explants taken from
mature trees have failed due to browning and non-viability of explants. Boggetti et al. [13] used
glass-house raised plants (1 month,1 year and 5 years old) to develop methods for multiplication
of nodal explants. They reported that sprouting of buds decreased strongly with increase in age
of mother plants. Shoots developed in presence of cytokinins were short and produced axillary
branches while Gibberellins supported bud sprouting but suppressed rooting. Cytokinins never
induced the multiple bud formation and only one bud developed at each axil. However, in
presence of cytokinins, the number of side branches per microshoots increased and were excised
to give new lateral shoots. Microshoots rooted in vitro at a frequency of 42% when cultured for
five days with 100 µm IBA.
In cashew, attempts were also made to induce multiple shoot formation using seedling
explants. Although highly heterozygous, seedling explants can be used to propagate individual
seedling genotypes and provides a potential method for propagation of cashew altered by genetic
transformation.
D’Silva and D’Souza [14] reported multiple shoot induction from cotyledonary nodes. Sucrose
concentration was reported to affect the number of buds developing from cotyledonary nodes. In
vitro studies on rapid propagation of five cultivars of cashew were undertaken in our laboratory
Tissue Culture of Cashewnut 247
[15]. Shoot tip, leaf axil and cotyledonary nodes from seedlings could be induced to multiply on
MS medium containing BA, kinetin and zeatin in combination (Fig. 1a-d). Factors affecting
multiplication rates, included age of explant source, explant type, medium composition, light
requirements, and transfer frequencies. Cotyledonary nodes produced more buds than other type
of explants. Nodes had a 90% viability when transferred daily to fresh medium containing
activated charcoal for 7 days while exposed to continuous dark. Microshoots from the different
varieties could be rooted by the use of IBA. In one variety, high frequency rooting could be
obtained by treating shoots with Agrobacterium rhizogenes. Genotype was found to affect in
vitro response. The rates of multiplication in three varieties VTH-174 (Andhra Pradesh), Ullal
(Karnataka) and VRI-I(M10/4) (Tamil Nadu) was low as compared to KV-26 (West Bengal).
b
a
c
d
Fig. 1. (a) Swelling and bulging in cotyledonary node explants; (b) and (c) Proliferation and growth of
shoots obtained from multiplication of cotyledonary node and (d) Acclimatized plants in pots
before being transferred to the field.
248
JHA AND DAS
Genotypes also differed in the ability of microshoots to root. Boggetti et al. [13] reported
difference in response of three genotypes studied (two Brazilian and one Tanzanian elite selection)
and axillary branching from explants was achieved only with one genotype (Tanzanian).
1.4 Embryo Culture
One of the problems in cashew breeding is of embryo abortion. Low percentage of fruit set
(3-4%) have been reported in cashewnuts [6]. In our study, immature zygotic embryos from five
varieties and of various sizes could be cultured to stimulate normal embryological development
[16].
1.5 Micrografting
Different grafting techniques and cuttings have been experimented and the best season and
climatic conditions determined according to technical informations obtained in Brazil [EC-STD
1999]. In vitro micrografting was performed onto cashew and other Anacardiaceae seedlings,
mainly, Rhus typhina [17]. Terminal apices from cultured shoots of cashew (1–3 mm) were
micrografted onto stems and rooted understocks. For cashew/cashew micrografting, mature
cashewnuts from the cashew germplasm were scarified, surface sterilized and cultured onto MS
medium in agar to germinate. Once the seedlings reached 5–8 cm in height, they were decapitated
just below the cotyledons and then grafted with a short apex. Shoot tips from glass house raised
seedlings and field plants micrografted by a modified side-grafting procedure on in vitro raised
seedling rootstocks (cashew or other Anacardiaceae) gave a successful rate of 40–80% [17].
Terminal apices from cultured shoots of cashew were micrografted onto stems and rooted
microcuttings of Rhus typhina. On cashew/cashew micrografting at different rootstock position,
significant differences were reported in the elongation growth rates and hypocotyl grafts grew
stronger than epicotyl ones [18]. Rooting of micrografted shoots of mature tree origin was poor
(13%) because the shoots were poorly rejuvenated.
1.6 Somatic Embryogenesis
In vitro somatic embryogenesis potentially offers alternative forms of large scale propagation of
plants. Somatic embryos can be used for biotechnological applications such as genetic modification
of trees to select desired stress tolerance traits and gene transfer. Jha [19] reported morphogenesis
in callus cultures derived from zygotic embryos and occurrence of globular protuberances which
developed into embryo-like structures. Hegde et al. [20] observed embryogenesis in cotyledonary
segments. However, the obtained embryos could not be germinated. Cardoza and D’Souza [21]
reported induction of direct somatic embryos from radicular end of zygotic embryos. Secondary
embryos developed from the primary embryos. However, conversion of embryos to whole plant
was not achieved. Recently, Ananthakrishnan et al. [22] and Cardoza and D’Souza [21] used
nucellus tissues from developing seeds for induction of somatic embryogenesis. Ananthakrishnan
et al. [22] reported induction of calli from nucellar explants excised from 1-month old developing
fruits of cashew on Murashige and Skoog’s medium containing 6.78 µM 2,4-D. Differentiation
of somatic embryos from calli was noticed when they were transferred to MS liquid medium
supplemented with 4.52 µM 2,4–D. Different stages of somatic embryo development were traced
but there was no further development of the torpedo stage in the liquid medium containing
2,4-D. Conversion of somatic embryos to whole plants was not obtained. Cardoza and D’Souza
Tissue Culture of Cashewnut 249
[21] have reported development of globular somatic embryos from nucellar callus in presence of
picloram. Somatic embryos maturated in presence of picloram and putrescine and germination
was obtained in MS basal medium. In our laboratory we initiated tissue culture studies in cashew
nut for induction of somatic embryos from different explants from juvenile and mature trees but
failed to raise embryogenic cultures from such explants. We then initiated tissue cultures using
immature zygotic embryos to study the induction, development, maturation and conversion of
somatic embryos to whole plants.
2.
Materials and Methods
2.1 Plant Materials
Plant materials were collected from the Arabari Forest Range, West Bengal and NRCC, Puttur.
Immature green nuts of cashew of different improved varieties (viz.VTH-174, M-10/4, Ullal, M44/3 and KV-26) were collected from mature trees.
2.2 Sterilization of Explants
The immature green nuts were surface disinfected using a sequence which included rinsing for
5 min with 70% alcohol followed by a 0.1% HgCl2 treatment for 20 min. The nuts were washed
thoroughly with sterile distilled water, opened aseptically and the intact immature embryos,
ranging from 1 to 10 mm, were excised out. The isolated zygotic embryos were inoculated either
intact or explants like cotyledons, hypocotyls with radicle or epicotyl with plumule, were excised
from the zygotic embryos and inoculated.
2.3 Induction of Somatic Embryos
Different basal media namely, Murashige and Skoog [23], Gamborg [24] and Lloyd and McCown’s
Woody Plant Medium [25], supplemented with various auxins like NAA, 2,4-D, IAA, IBA and
cytokinins like BA, kinetin, either singly or in combinations were used. Growth adjuvants such
as yeast extract, casein hydrolysate and proline were supplemented to the media in different
experiments. PVP (0.5%) and activated charcoal (0.3%) were added to the media to prevent
browning of explants.
2.4 Maturation and Germination
Globular somatic embryos, induced after 6–8 weeks, developed upto cotyledonary stages in MS
basal media with various growth regulators, but lacking PVP, charcoal and yeast extract. Maturation
of somatic embryos was obtained in this media, after incubation for 6 weeks. However, the
cotyledonary embryos did not germinate in the same media. For germination, mature somatic
embryos were subjected to different treatments, under dark conditions. Basal salts such as MS,
WPM, in combination with different growth regulators such as BA (1-5 mg/l), abscisic acid
(ABA, 0.1 mg/l), mannitol (1.0 g/l) and sucrose (30-100 g/l) were used, singly or in combination.
Germination of somatic embryos was obtained after 4-5 weeks.
2.5 Cytological Study
For the study of mitotic chromosomes, shoot tips as well as root tips of regenerated plants were
pretreated with 0.002 M 8-hydroxyquinoline for 4 hours, fixed in Carnoy’s mixture (alcohol :
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JHA AND DAS
chloroform : acetic acid (6 : 3 : 1) and stained with 2% aceto-orcein : 1(N) HCl (9 : 1).
Photomicrographs were taken with Wild-Leitz MPS 52 microscope.
3.
Results
3.1 Problem of Browning and Establishment of Viable Cultures from Zygotic Embryos
In our laboratory, immature intact zygotic embryos of various sizes (1-10 mm, A1–A4), excised
cotyledons, excised hypocotyls with radicle and epicotyl with plumule were used as primary
explants for induction of somatic embryos. The establishment of viable aseptic cultures of
cashewnut was restricted due to the exudation of phenolics in the media and the oxidation and
browning of explants. Considering intact embryos as explants, it was found that the oxidation
and browning of explants, as well as the exudation of phenolics was more pronounced in
embryos 5-10 mm (size A3–A4) in length than in 1-2 mm (size A1–A2) embryos, suggesting that
the size of the explant was in some way directly related to the extent of browning of explants and
exudation of phenolics (Table 1). Oxidation was maximum in A4 embryo explants (80%) where
exudation of phenolics preceded browning of explants and within 2 weeks of culture initiation,
most of the explants turned brown. When excised explants were taken into consideration, maximum
exudation of phenolics was observed in excised cotyledons, exudation taking place mostly from
the cut-end (Table 3). In A2 zygotic embryos, the frequency of oxidation was lower in intact
embryos, than in excised cotyledons or excised hypocotyl with radicle, as exudation of phenolics
takes place profusely from cut-end of explants. The exudation of phenolics and the subsequent
browning of explants was reduced to some extent by frequently subculturing the explants (initially
after every 2nd day for the 1st week, followed by weekly subcultures during the induction
phase), use of activated charcoal (0.3%) and PVP (0.5%) in the culture media, and dark incubation.
The best response, so far as viability and establishment of aseptic viable cultures is concerned,
was obtained in MS basal media containing 2 mg/l NAA, 2 mg/1 Kn, 500 mg/1 YE, 0.3%
activated charcoal and 0.5% PVP (M1). Although it is believed that auxins enhance phenolic
oxidation, but NAA was used at an optimum concentration because, in the presence of NAA,
the normal germination of zygotic embryos and development of shoot and root was restricted.
Cultures were maintained in this media for 4 weeks, under continuous dark conditions.
Some intact zygotic embryos showed a tendency to initiate callus after 3 weeks of culture on
induction media (M1), under dark conditions. Callus induction from immature embryo explants
was variable, depending on the length of incubation, concentration of growth regulators used and
size of zygotic embryos. Callus induced, continued to proliferate if maintained on induction
media and similar cultural conditions. Callus was induced both with 2,4-D (2-4 mg/l) and NAA
(2 mg/1) but not in the presence of other auxins. Callus induced with 2,4–D was brown in colour
and leached exudates in media, turning the media completely brown within 10-12 days of culture.
Such calli necrosed after 6 months. Callus induced with 2 mg/l (NAA) was creamish-brown in
colour, did not show much leaching or exudation, and could be subcultured and maintained
after induction. Callus proliferated from all stages of embryos sampled except for A2 size.
Results of preliminary experiments indicated that the maximum frequency of callusing was from
embryos of A4 size when cultures were incubated for 4 weeks (Table 1, Fig. 3a, b). Most of
this callus developed at the epicotyl region of the embryo, along the plumule and at the site of
explant contact with the media. But these calli were not embryogenic, there was no differentiation
Frequency
of explant
browning
(%)
2.0
10.6
50.5
80.0
Size***
A1
A2
A3
A4
4.0
0
17.5
55.4
Frequency
of explants
forming
callus (%)
80
90
38
0
No. of explants
forming
embryos
(N = 100)
12.2 ± 0.8
23.06 ± 0.04
4.02 ± 0.23
–
No. of embryos/
explants ± S.E.
39.5
59.5
38.6
–
Frequency
of embryo
maturalion
(%)*
15.2
23.2
11.6
–
Frequency
of embryo
germination
(%)**
18.0
22.5
36.0
55.2
10.6
VTH-174
M-44/3
Ullal
M-10/4
KV-26
4.0
17.2
26.5
21.0
–
Frequency
of explants
forming
callus (%)
10
0
0
0
90
No. of explants
forming
embryos
(N = 100)
8.0 ± 0.4
–
–
–
23.06 ± 0.04
No. of embryos/
explants± S.E.
*Somatic embryo maturation: transformation from globular to cotyledonary/torpedo stage.
**Somatic embryo germination: development of root and shoot—a complete plantlet.
Media: For Induction—MS+NAA(2 mg/l)+Kn(2 mg/l)+YE(500mg/l)+PVP(0.5%)+act. charcoal (0.3%) (M1);
MS + NAA(0.1 mg/l) + Kn(l mg/l)(M2), For Maturation—M2; For germination—MS + BA (l mg/1) (M3).
Frequency
of explant
browning
(%)
Genotype
20.95
–
–
–
59.5
Frequency
of embryo
maturation
(%)*
10.6
–
–
–
23.2
Frequency
of embryo
germination
(%)**
Difference in response of explants from cashew genotypes during induction, maturation and germination of somatic embryos
Intact zygotic
embryos of
A2(3-4 mm)
size
Explant
type
Table 2.
Intact immature
zygotic embryos
(var. KV-26)
Explant
type
Table 1. Effect of immature zygotic embryo size on frequency of induction of somatic embryos and their maturation and germination in
cashewnut
Tissue Culture of Cashewnut 251
Type
55
0
20.5
8.4
–
8.0 ± 0.5
23.06 ± 0.4
10.0 ± 0.0
No. of embryos/
explants± S.E.
Excised hypocotyls
with radicle
Excised cotyledons
↓
M3 for 4-5weeks →
Germination
MS basal media → ↓
Conversion to whole plants
↓
M2 for 6 weeks →
Maturation
M1 for 4 weeks → ↓
M2 for 4 weeks → ↓
Induction of globular somatic embryos
Intact
zygotic embryo
Zygotic embryo explants (3–4 mm)
↓
Scheme for direct somatic embryogenesis in cashewnut
–
58.6
59.5
20.2
Frequency of
embryo maturation
(%)*
*Somatic embryo maturation: transformation from globular to cotyledonary/torpedo stage.
**Somatic embryo germination: development of root and shoot—a complete plantlet
Media : For Induction—MS + NAA(2 mg/l) + Kn(2 mg/l) + YE(500 mg/l) + PVP(0.5%) + act. charcoal (0.3%) (M );
1
MS +NAA(0.1 mg/l) + Kn (l mg/l)(M2). For Maturation—M2; For germination—MS + BA(l mg/1) (M ).
3
90
35
No. of explants
forming embryos
(N = 100)
10.6
26.3
Frequency of
explant browning
(%)
–
20.1
23.2
8.05
Frequency of
embryo germination
(%)**
Effect of explant type on frequency of induction of somatic embryos and their maturation and germination in cashewnut
Intact
Intact zygotic
Excised
embryos of
cotyledons
A2 (3-4 mm)
size—(var-KV-26) Excised hypocotyl
with radicle
Excised epicotyl
with plumule
Explant source
Table 3.
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JHA AND DAS
Tissue Culture of Cashewnut 253
a
b
c
d
e
f
Fig. 2. (a–f) Somatic embryogenesis leading to whole plant regeneration in cashewnut, Anacardium
occidentale L.; (a–c) Cluster of somatic embryos induced on excised cotyledonary node explants.
(d–f) Somatic embryos at different stages of development induced on excised radicle explants.
of somatic embryos from these calli cultured in presence of different combinations and
concentrations of NAA and Kn or in auxin unsupplemented media.
254
JHA AND DAS
a
b
c
d
e
f
Fig. 3. (a, b) Embryogenic calli induced on intact zygotic embryo explants; (c–f) Different
developmental stages of somatic embryos leading to maturation.
Tissue Culture of Cashewnut 255
3.2 Induction of Somatic Embryos
No zygotic embryo germination or growth of zygotic embryonal axis was observed in intact
zygotic embryo explants in the presence of NAA (M1). Cultures were transferred, after 4 weeks
in M1, to media consisting of MS inorganics, Kn (1 mg/1) and NAA (0.1 mg/1), and lacking PVP,
charcoal and yeast extract (M2), and kept for 4 weeks. The suppression of growth of cashew
zygotic embryonal axis in M media was followed by the appearance of white protrusions. These
white protrusions subsequently developed into globular somatic embryos. Somatic embryos
were thus induced directly on intact immature embryo explants of A1–A3 size, after 4 weeks of
culture in M1 media and another 4 weeks in M2 media. Embryos were observed in culture where
there was no callusing, particularly in A2 explants, and the highest number of embryos induced
(23 embryos) per explant were obtained (Table 1). It is interesting that A3 explants produced few
somatic embryos, yet yielded adventitious roots (10-12 roots/explant), in the same media. However,
rooting ability was not observed in A1, A2 or A4 explants suggesting cellular competency to
differentiate somatic embryos or adventitious roots or calli (since A2 explants failed to produce
any calli) change during cashew ontogeny. No fully developed somatic embryos were observed
in A4 cultures and hardly any (4-5 embryos/explant) in A3 cultures, indicating that the stage of
development of zygotic embryos affects somatic embryo development. Occasionally, we observed
secondary embryogenesis, on primary somatic embryos, while still attached to the mother tissue.
3.3
Development of Somatic Embryos
Maturation
Maturation of globular somatic embryos was obtained on the same culture media M2 after
incubation for another 6 weeks. The frequency of somatic embryos maturing i.e. globular embryos
ultimately forming torpedo or cotyledonary stages, was maximum in the case of embryos induced
on intact zygotic embryos of A2 size (59.5%), followed by embryos induced on A1 and A3 size
zygotic embryos (39.5% and 38.6 %, respectively) (Table l, Fig. 2a-f, 3c-f). But there was no
germination of the obtained embryos into complete plantlets in the same media.
Post-Maturation and Germination
It was observed that a preconditioning period or post-maturation treatment of these developing
mature embryos was necessary for their germination. Of the various combinations tried out, MS
basal media supplemented with BA (1.0 mg/1) (M3) was found to be the most effective for
germination of mature somatic embryos.
Germination was obtained after 4-5 weeks of preconditioning in the abovementioned media
M3, however, in only 11.6-23.2% of the somatic embryos induced on the different intact zygotic
embryo explants (A1–A3).
Plants regenerated from the somatic embryos were transferred to MS basal media, for proliferation
of shoots. However, proliferation of shoots was found to be very slow in the somatic embryo
derived plants. Attempts to transfer somatic embryo derived plants to potted soil was not successful.
Trials are being carried out to suitably harden the regenerated plants for successful survival in
the soil.
3.4 Difference in Response of Different Genotypes During Induction of Somatic Embryos
In a set of experiments, the varying responses of intact zygotic embryo explants of A2 size of
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JHA AND DAS
different genotypes were evaluated during somatic embryo induction and further development
(Table 2). Globular somatic embryos were successfully differentiated in only two of the five
genotypes studied, in var. VTH-174 and KV-26. The number of globular embryos induced per
explant was quite low, but the frequency of differentiation of somatic embryos on intact zygotic
embryo explant of the 2 genotypes was contrasting, 10.3% explants forming somatic embryos in
VTH-174 as against 90.0% in KV-26. The frequencies of maturation of somatic embryos was
low to high in the two genotypes (20.95–59.5%) and germination was obtained in only 10.6%
and 23.2% of somatic embryos in VTH-174 and KV-26, respectively. Callus was induced in A2
size zygotic explants of all the genotypes except KV-26, although this callus did not lead to
development of embryogenic calli.
3.5
Induction of Somatic Embryos from Explants Excised from
Germinated Zygotic Embryos
In a separate set of experiments, explants like excised cotyledons, hypocotyl with radicle and
epicotyl with plumule (all excised from A2 zygotic embryos of variety KV-26) were compared
with intact zygotic embryos (A2) of var. KV-26, in order to ascertain the exact part of the
embryonal axis where somatic embryos were induced and the differences in frequencies of
induction, maturation and germination of somatic embryos (Table 3). The best results were
obtained from intact zygotic embryo explants so far as the number of globular somatic embryos
induced per explant and the frequencies of maturation and germination of somatic embryos.
There was high frequency of browning of cotyledon explants and exudation of phenolics, we
noted an abundance of mucilage in these cultures. No somatic embryos were obtained on epicotyl
explants but somatic embryos were induced on the radicle end of hypocotyl explants. The
number of somatic embryos obtained from hypocotyl explants was low (8 embryos/explant), as
compared to the cotyledon explants (10 embryos/explant), but the maturation and germination
frequencies were higher in somatic embryos from hypocotyl explants (58.6% and 20.1%,
respectively).
3.6 Cytological Observations
The chromosome number in root tip of zygotic embryo derived plants was noted to be 2n = 40,
in all the 40 regenerated plants derived from somatic embryos that were analysed, with no
irregularities in chromosome behaviour. Plants regenerated by somatic embryogenesis were thus
euploid, and were free of any noticeable phenotypic variability.
4.
Discussion
The developmental stage of the explant and the explant type determined the type of response
obtained in vitro in cashew immature zygotic embryo cultures. Although primary cultures from
immature embryos followed a pattern of growth somewhat similar to that observed for cultured
mature embryos, several aspects were unique to immature embryo explants, particularly of size.
The results indicate changes in cellular competency to differentiate somatic embryos or callus or
adventitious roots during cashew zygotic embryo ontogeny. Callus initiation was observed from
all stages of development of embryos tested, but for A2 embryos. However, embryos from early
stages of development (A1, A3) showed a tendency to produce less primary callus than older
Tissue Culture of Cashewnut 257
embryos (A4). Also, no somatic embryos were observed in explants where callus was induced
and vice-versa, suggesting a negative correlation.
Growth and development of zygotic embryo axis in cashewnut was restricted in the presence
of high levels of auxin (NAA), irrespective of the size of the embryo. This observation supports
earlier findings. Maheswaran and Williams [26], in studies on Trifolium repens, proposed that
growth suppression of the main embryonal axis is associated with the breakdown in the integrity
of the cells as a single embryogenic group, and escape of individual or smaller group of cells to
function autonomously. In Anacardium occidentale, suppression of growth in the embryonal
axis in high auxin media (NAA at 2 mg/l) is followed by appearance of white protrusions in low
auxin media (NAA at 0.1 mg/l), which subsequently develop into somatic embryos.While a high
level of auxin (NAA at 2 mg/l) was essential for the explants to gain embryogenic competence,
prolonged exposure (for more than 4 weeks) in such high auxin supplemented media did not
favour somatic embryo differentiation. Hence, it may be assumed that the embryogenic competence
of the explant cells gained during 4 weeks of exposure in high auxin supplemented media, leads
to the development of globular embryos, when such explant were subcultured onto low auxin
and high cytokinin media (NAA 0.1 mg/l and Kn 1.0 mg/l) after 4 weeks. Another noteworthy
feature in cashew somatic embryogenesis was while globular embryos developed in the presence
of NAA (0.1 mg/l) and Kn (1.0 mg/1), they did not germinate to rooted plants if cultured on the
same media. The mature somatic embryos had to be cultured on MS basal media containing BA
(1.0 mg/1) for further germination and regeneration of whole plants. However, conversion of
mature somatic embryos to whole plants as obtained in the present study, does not occur at a
desirable high frequency. Thus, somatic embryogenesis in cashew is not a one step process. The
pattern of embryogenesis is somewhat similar to the direct embryogenic pathway in coffee, as
reviewed by Sharp et al. [27].
Somatic embryos were induced directly on immature intact zygotic embryo explants in only
two out of the five genotypes studied for induction of somatic embryogenesis, at a varying
frequency of induction and the number of somatic embryos differentiated per explant. The
inability to induce somatic embryos in certain genotypes, as was observed in cashew, is well
documented. The ability of closely related plants to produce somatic embryos directly on explants
is also under genetic control and differences between varieties are often found [28]. Stamp and
Meredith [29] obtained somatic embryos on the zygotic embryos of four cultivars of Vitis
vinifera, but could not induce them to form on the cultivar ‘Pinot noir’. The direct formation of
somatic embryos on apple leaf segments was genotype dependent [30]. The frequencies of callus
induction in different genotypes varies in cashew. Many reports (e.g. Espinasse et al. [31]), in
Helianthus) illustrate how the capacity of explanted tissues to form callus and the subsequent
growth rate of callus cultures, can both be variety dependent. There was variation in the frequencies
of explant browning among genotypes in cashew. The extent of blackening or browning and
growth inhibition which occurs in cultures is reported to be genotype dependent in species, that
naturally contain high levels of tannins or other hydroxyphenols as in cashew, Juglans, Quercus
and Rhododendron [28]. Differences were also found between species of the same genus and
cultivars within a species. Cultivars of Sorghum bicolor released such large quantities of pigmented
phenolics, that the medium darkened and cultures readily became necrotic [32].
Plant age and the degree of differentiation of tissues are often interrelated and produce interactive
effects in vitro. Both the size and degree of development of certain organs like cotyledons, hypocotyls
258
JHA AND DAS
and epicotyls depend on age [28]. During seed ontogeny, the physiological changes and their
accompanying hormonal control may play a role in the differential responses of explants of varying
maturity, as was found in cashew. Zygotic embryos, at the A3–A4 size, undergo rapid enlargement
within the hardening shell in situ. During this period, sugar and protein content may be decreasing.
These changes may somehow relate to changes in the morphogenic potential of cashew embryo
explants. Competence of zygotic embryos of Picea for somatic embryogenesis has been shown to
be limited to a specific stage of development prior to the accumulation of proteins [33]. Such
relationships between changes in seed physiology, nutrient content and cellular competency to
form adventitious structures, remains to be understood.
In A. occidentale, regenerated plants from somatic embryos were cytologically stable and
normal. Since these somatic embryos can be repetitively embryogenic, they may be used as target
tissues for transformation studies in cashewnut. The results obtained in this study have important
implications in the further use of in vitro culture techniques for this species.
Although diploid normal rooted plants derived from somatic embryos have been successfully
regenerated in the present study, procedure for successful survival of such plants on transfer to
potted soil have yet to be standardized. The plants obtained following multiplication and proliferation
of microshoots from cotyledonary nodes in Anacardium occidentale have survived on transfer to
potted soil, but somatic embryo derived plants transferred (23 plants) under identical conditions
have failed to survive. Further studies are needed for success of propagation through somatic
embryogenesis of cashew.
There are two major limitations to the application of somatic embryogenesis for propagation
and genetic manipulation—first of these is the low multiplication rates, that is, the low numbers
of field plantable clonal plantlets produced per embryogenic culture, and second is the inability
to initiate embryogenic cultures from mature trees.
The limitations of low multiplication rates can be further subdivided into problems such as low
frequency somatic embryo production, production of malformed embryos, incomplete maturation,
low germination and low conversion of germinants to plantlets capable of surviving transfer to
ex vitro conditions.
Somatic embryogenic cultures in many species fail to demonstrate continued embryo production.
Generally, there are also reports of direct embryogenesis, where, for example, individual somatic
embryos arise from explanted zygotic embryo cotyledon tissues and in such cases only a single
population of embryos is produced, some of which may mature and convert to plantlets. In some
cases however, these primary embryos fail to mature and give rise to successive cycles of new
embryos. This successive generation of new embryos is known as repetitive, recurrent or secondary
embryogenesis. It is this phenomenon that gives somatic embryogenesis its great potential for
mass propagation and gene transfer, since a single culture undergoing repetitive embryogenesis
is theoretically capable of regenerating an unlimited number of somatic embryos.
A more frequent problem than low embryo production is that of regenerating viable plantlets
from somatic embryos. The bottleneck may occur at any stage, including maturation, germination,
shoot apex elongation or acclimatization. The standard use of simple two-step media sequences
to promote the induction and developmental stages of embryogenesis is proving inadequate to
accommodate the multiple and distinct phases that somatic embryos undergo in the course of
their ontogeny and subsequent development. Therefore, the more closely the pattern of somatic
embryo gene expression matches that of zygotic embryos, the greater are the chance of obtaining
Tissue Culture of Cashewnut 259
highly efficient regeneration systems. Such normalization of gene expression patterns will be
achieved through the optimization of media and culture protocols for each individual stage of
embryo development.
Proliferation of embryogenic cells takes a number of forms and is apparently influenced by
a variety of factors, some of which can be controlled during the culture process, and some of
which are yet undefined. Factors such as the effects of plant growth regulators, reduced nitrogen,
plant species and genotype of the cultured material have been investigated for induction and
proliferation of embryos. By recognizing the critical factors involved at each stage, and those
that exert their influence throughout the process, the protocols at each stage can be tailored to
more closely simulate the conditions in planta.
Besides factors such as explant type and growth regulator regimes, a substantial number of
other factors can affect the induction of the embryogenic state. Perhaps one of the most important
is plant genotype as suggested from our earlier studies in cashew. Inasmuch as the induction of
somatic embryogenesis plausibly involves activation of the same genetic pathways as zygotic
embryogenesis, somatic embryogenesis should be a universal phenomenon for all seed-bearing
plants. Nevertheless, individual genotypes, within a given species, such as cashew, vary greatly
in embryogenic capacity. Such genotypic differences in embryogenic capacity might reflect
current differences in the ability to activate key elements in the embryogenic pathway. In addition,
individual genotypes may have unique requirements for optimal regeneration capacity, and it
would also be expected to have impact on culture proliferation rates, another problem in cashew.
With informed manipulation of these factors, not only will proliferative embryogenic cultures
realize their potential for virtually unlimited propagule production, but the somatic embryos
produced will come to have the vigor and germination associated with their zygotic counterparts.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
18. Changing Scenarios in Indian Horticulture
Sanjay Saxena and Vibha Dhawan
Bioresources and Biotechnology Division, Tata Energy Research Institute, Habitat Place,
Lodhi Road, New Delhi 110 003, India
Abstract: India holds enormous potential for production of horticultural crops. However, despite
ranking high in terms of overall production, in most species the yields are far below the world
average. Enhancement in productivity levels is not only necessary because land is a finite resource
but also to remain cost competitive in the global market. Along with productivity, it is also imperative
to improve the quality of the produce. This will not only enable the Indian farmers to compete with
the imported products more effectively but also gear them to sell their produce in the international
market. This communication highlights some of the strategies that could possibly be adopted to
improve production, productivity and the quality of horticulture produce in the country.
1.
Introduction
Horticulture covers a wide spectrum of crops such as fruits, vegetables, roots and tubers, medicinal
and aromatic plants, plantation crops, ornamentals and spices. It provides nutritional security,
offers a remunerative means for diversification of land use for improving productivity and
returns, and increases employment opportunities. Horticultural products especially spices are
also valuable foreign exchange earners for the country. However, since independence till the
beginning of the Eighth Five Year Plan the government policies were focussed towards developing
agriculture in the country with virtually no attention been paid to horticulture. Subsequently, the
government realized the value of horticulture in Indian economy and gave a major thrust to the
same by raising the budgetary allocation from a meager amount of Rs. 24 crores in the Seventh
Five Year Plan to Rs. 1000 crores in the Eighth Five Year Plan. Besides this 40 times hike in the
allocation, a large number of concessions, subsidies and incentives were given to the growers.
One important trend observed in the last few years is that horticulture development has gradually
moved out of its rural confines into urban areas and from traditional agricultural enterprise to
the corporate sector. The adoption of improved technology, greater commercialization and
professionalism in the management of production and marketing has brought about a perceptible
change in the concept of horticulture development in the country.
Today India holds a major share in the world trade of spices. It also ranks second in the world
both in production of fruits as well as vegetables. However, this increase in production is largely
on account of increase in area under cultivation rather than increase in productivity levels.
Barring few crops such as grapes, litchi and citrus where the yields have improved during the last
decade, in most other important fruit crops the yields have either declined or have shown only
a marginal increase (Table 1). It is only recently that there has been an awareness to increase our
land productivity and quality of product, and government is taking serious measures for adoption
of modern technologies such as use of hybrid seeds, tissue culture for producing clonal plants,
molecular techniques, biofertilizers, high-tech agro-techniques etc.
262
SAXENA AND DHAWAN
Table 1.
Status of some important fruit crops in India
Fruit
Year
Area (000 ha)
Production (000 tonnes)
Yield (kg/ha)
Apple
1991–92
1999–2000
1991–92
1999–2000
1991–92
1999–2000
1991–92
1999–2000
1991–92
1999–2000
1991–92
1999–2000
194.6
238.3
1077.6
1486.9
94.0
150.9
57.1
75.5
27.2
64.4
32.4
44.3
1147.7
1047.4
8751.6
10503.5
1095.1
1710.5
768.5
1025.3
396.3
800.3
668.2
1137.8
5900
4395
8120
7064
11650
11335
13470
13572
14540
12427
20650
25684
Mango
Guava
Pineapple
Sapota
Grapes
Source: CMIE, 2001.
Except for grapes wherein India has the highest productivity in the world, in most other fruit
crops the productivity levels are much lower (Table 2).
Table 2.
Fruit
Apple
Apricot
Banana
Citrus
Grapes
Papaya
Pineapple
Yield (kg/ha) of various fruit crops in the world in 2001
World
average
10767.4
6281.7
16338.5
13709.2
8480.1
15983.0
17932.3
Leading country
India
6493.5
4125.0
32653.1
19195.9
26760.6
11298.2
13750.0
Name
Yield
Belgium-Luxembourg
Switzerland
Nicaragua
USA
India
Costa Rica
Panama
52631.6
21515.2
55398.2
33692.5
26760.6
46933.3
52681.7
Source: FAO, 2002.
India’s position with respect to productivity of vegetables is no better either (Table 3). In most
vegetables the yields are marginally to significantly lower than the world average. In fact, the
difference between our yields and that of the leading country in the respective crop is so large
that it may not be even worthwhile to draw a comparison. It is true that one cannot expect India
to be the world leader in each and every crop as climatic conditions that strongly influence the
productivity level may not be ideal for all the crops in India. Also, the practices followed by
small and marginal farmers in our country are very different from those followed in large
mechanized farms in USA and European countries. However, these figures clearly bring out the
potential that exists before us in improving the yields of our crops.
It is really hard to believe that a country ranking second in the world in terms of production
of fruits and vegetables having below world average per capita consumption of these commodities
(Table 4). This is despite the fact that most of our population is vegetarian.
The floriculture sector in India has shown a steady growth in exports over the years, but in
Changing Scenarios in Indian Horticulture
Table 3.
Fruit
Green beans
Cabbage
Carrot
Cauliflower
Okra
Potatoes
Onion (dry)
Pumpkins, squash
and gourds
Tomatoes
263
Yield (kg/ha) of various vegetable crops in the world in 2001
World
average
India
6901.7
20778.9
21204.7
17755.0
6896.3
15967.8
17070.2
12496.0
26769.9
Leading country
Name
Yield
2800.0
17916.7
14583.3
16250.0
10967.7
18642.8
9800.0
Kuwait
Korea
Austria
Kuwait
Cyprus
New Zealand
Austria
23130.4
61591.6
57067.7
45284.0
16438.4
50000.0
59777.8
9750.0
17000.0
Netherlands
Netherlands
55000.0
433333.4
Source: FAO, 2002.
Table 4.
Per capita supply of vegetables and fruits in the world in 2000
Country
India
Asia
European Union (EU)
USA
World average
Vegetables (kg/year)
Fruits (kg/year)
62.5
116.2
112.5
125.8
101.9
41.5
46.2
83.0
124.8
59.8
Source: FAO, 2002.
terms of value we stand 25th in the world trade which is an indication of the long road that lies
ahead of us.
2.
Constraints
Some of the constraints in developing horticulture sector in the country include: (a) inadequacy of
good quality seeds and other planting material, (b) low productivity, (c) poor quality of the product,
(d) inadequate efforts for product diversification and consumption, (e) lack of awareness, (f ) slow
pace in adoption of improved technology, (g) lack of infrastructure for post-harvest management
and marketing, (h) inadequacy of trained manpower and human resources in horticulture, (i) lack
of proper database on demand projection, price realization etc. and ( j) poor transportation system,
credit facilities and price support.
3.
Strategies
To meet the growing demand of horticultural products on account of increase in population and
average household income, and to improve per capita consumption there is an urgent need to
increase the production of fruits and vegetables in the country. However, it must be emphasized
that this increase in production is to be achieved largely through increase in productivity rather
than increase in area, as has been the case so far. This is because land being a finite resource will
become a limiting factor at some point of time. Improvement in productivity levels is also
264
SAXENA AND DHAWAN
necessary to remain cost competitive in the global market. This is more so because recently,
under WTO agreement India has withdrawn several restrictions on import of agricultural/horticultural
items resulting in easy availability of superior quality food items at a reasonable price. This will
pose a major challenge to the Indian farming community. To counteract this threat it is absolutely
essential for the Indian farmer to enhance productivity and cut down the cost of production
without compromising on the quality. High quality standards would not only put them on an
even platform with the imported products but will also enhance their prospects of exploiting the
foreign market to export their produce for better price realization. Under this changing scenario
of Indian horticulture some of the suggested strategies for improving production, productivity
and quality of horticultural products in the country are as follows.
3.1 Use of Disease-Free Planting Material
The level of technological and extension support with regard to planting material that is provided
by the government in agricultural sector is not available to farmers practicing horticulture.
Consequently, even after more than 55 years of independence a majority of the farmers in the
country use uncertified seeds or other forms of planting material to grow horticultural crops.
This is on account of unawareness, lack of financial resources and even unavailability of authentic
planting material in several crops. The problem of disease-infested planting material is even
more pronounced in those crops where the propagules are regenerated vegetatively. In many
crops such as potato (tubers), sugarcane (ratoons), strawberry (runners), banana (suckers) etc.,
the propagules derived from the previous crop are used to raise the new crop. Such propagules
accumulate diseases on account of perpetual exposure to the field conditions leading to decline
in yields. They also contribute towards spread of diseases in virgin areas.
Tissue culture offers rapid and reliable means of large-scale production of disease-free planting
material. All the plants raised through tissue culture are free from most of bacterial and fungal
diseases. One can also produce virus-free plants by meristem culture followed by micropropagation.
Therefore, the use of tissue-cultured material in horticulture should be encouraged.
3.2 Use of High Yielding Superior Quality Planting Material
Ever since the beginning of human civilization, mankind has been selecting superior individuals
and improving them further through crossing of different parents with desirable traits.
Simultaneously, the technique of vegetative cloning of plants was also perfected. With the
advancements in biotechnology, the pace of obtaining plants with desirable traits has gained
further momentum because it is now possible to create new and unique genetic combinations
that involve distinctly or even totally unrelated parents.
Cloning of elites can be done conventionally through cuttings, grafting or using other vegetative
propagules such as roots, suckers, rhizomes etc. However, vegetative propagules are generally
available in small numbers. They are often bulky and difficult to transport over long distances.
In many plant species particularly in trees, large-scale propagation through cuttings is not possible
because by the time the plant is evaluated for its productivity, the cuttings had already lost their
capacity to root. Moreover, the vegetative propagules if infested with a disease would result in
the spread of the disease even in the virgin areas. In contrast, through micropropagation, one can
not only obtain disease-free plants, but all the tissue-cultured plants are clonaly uniform i.e., they
are genetically alike and behave just like the mother plant. This way all the desirable traits of the
Changing Scenarios in Indian Horticulture
265
mother plant can be passed on to the progenies unaltered. A desirable genotype that could not be
multiplied because of virus infestation could now be freed of known viruses and then mass
multiplied. Since the technology has the potential of producing millions of plants starting from
a single shoot-tip, it becomes economically and technologically viable to free otherwise elite
genotypes of horticultural species of the known viruses and then mass multiply them.
Considerable progress has been made within the country with regard to tissue culture of
horticultural species and micropropagation protocols have been developed for several species.
These can broadly be classified as follows.
3.2.1
Fruit Crops
Banana
This is an important fruit crop of India grown widely in almost all parts of the country, especially
in Southern parts. The plant is propagated through suckers drawn from the previous crop.
Consequently, with every passing generation, there is an accumulation of diseases leading to
decline in vigour and yields. Also, there remains a major risk of the spread of the disease along
with the propagule. Tissue cultured plants of banana have become extremely popular because of
higher and consistent yield as compared to conventional propagules. The success met with tissue
cultured plants of banana at the field level has generated lot of interest towards the technology
among the farmers and they have become more receptive to the idea of trying other plants. There
are several varieties of banana for which micropropagation protocols are now available, and
depending upon the regional priorities and the end-use that the crop would be put to, suitable
varieties are selected for cultivation. This includes Robusta, Dwarf Cavendish, Grandinaine,
Williams, Elakki, Basari, Madukar etc. Today, several private companies in India like Khoday
Biotek, A.V. Thomas, Cochin; Décor Plant Culture, Mumbai; Godrej Plant Biotech, Mumbai;
Growmore Bio-tech (P) Ltd., Hosur; Harrison Malyalam Limited, Hosur etc. together are producing
more than a million tissue cultured plants of banana for distribution among the local farmers.
Although tissue culture of banana is a success story, there are reports to suggest that after certain
passages (usually around 10) the cultures become more prone to somaclonal variations and
therefore, as much as possible, subculturing beyond 10 passages should be avoided.
Strawberry
Conventionally, strawberry is propagated through runners. Plants raised through runners give
proper yield only up to two generations. Beyond two generations the plant may look healthy but
the yields are significantly lower. Moreover, continuous use of runners of the previous crop for
the new one results in accumulation of various pathogens resulting in lower yields. Also, there
is a potential risk of spread of diseases along with the propagules. In contrast, the plants raised
through tissue culture are free of diseases. During the last few years several day-neutral varieties
have been developed in USA. Importing material of such varieties on a large-scale will not only
be expensive for the grower but would involve outflow of valuable foreign exchange. Also, it
would be difficult for a small farmer to procure planting material from abroad. Using tissue culture
one can bulk-up the planting material of these new varieties within the country and make the plant
available to the growers at a much lower price. Considerable success has been made in this regard
at TERI’s tissue culture facilities and more than 3 lakh tissue cultured plants have so far been
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dispatched to various growers. It is rather interesting that a species of hill is now being cultivated
in plains. Overseas, the strawberry mother plants are produced through tissue culture and their
subsequent multiplication is done through runners. However, it is important to note that unlike
in India, these runners are produced under very hygienic conditions and therefore, give normal
yields. Perhaps, it would be worthwhile to adopt similar kind of approach in India as well.
Banana and strawberries are the two fruit crops for which the tissue culture technology has
been fully commercialized. In addition, there are few other fruit crops such as apple, pineapple,
Prunus, raspberry, pomegranate and Zizyphus that are produced by various tissue culture companies
operating in India on a small-scale. The demand for these species is restricted mainly on account
of narrow geographical distribution. Success has also been achieved in regenerating plants of
citrus, guava etc. by tissue culture inside the lab. However, these protocols either suffer from
certain deficiencies because of which they could not be applied for commercial propagation or
there are other technical problems that restrict their usage. For example, those species, which are
multiplied through grafting, the self-rooted scions produced through tissue culture may not
perform well in the field. In such cases grafting becomes inevitable and there is a possibility that
one may not be able to multiply both the scion and the stock by tissue culture. Even where
production of both scion and stock could be achieved, the grafts may not be very successful. It
is only a matter of time, when such technical problems would be overcome and these species
could be multiplied on a commercial scale. There are still few species such as mango and Litchi
in which there is an urgent need to develop tissue culture procedures but no significant progress
has been made so far.
3.2.2 Cash Crops
Potato and sugarcane are two major cash crops in which tissue culture technology has been
applied quite extensively.
Potato
Potato is highest consumed single vegetable of the world and accounts for the largest area under
cultivation. It serves as an important component of the Indian cuisine and also finds its way in
processed food industry as chips and French fries. Conventionally, potato is propagated through
tubers that tend to accumulate diseases with repeated cycles of propagation. This accumulation
of diseases eventually brings about significant reduction in yields. Through tissue almost diseasefree planting material (plantlets, microtubers and minitubers) could be produced on a large-scale
as being done by several labs in the country. The secondary farmers, who are planting minitubers,
are benefited through higher yields and better price realization for their produce. Potato chip is
a major agri-industry, which is highly dependent on the quality of the potato tuber used in
processing. Most of the potato varieties grown in India do not yield an even slice resulting in
heavy wastage. Also they contain fairly high percentage of sugars that get oxidized and impart
brown colour to the chips during baking process. This results in serious losses and low cost
realization. Using tissue culture new and exotic varieties of potatoes that are primarily meant for
making of potato chips have been successfully multiplied on a large scale within a short period
of time. At present only 8% of the plantation is by certified seeds. Multiplication of new varieties
(both suitable for processing and table varieties) will not only ensure better price realization to
the farmers, but will also benefit the food processing industry.
Changing Scenarios in Indian Horticulture
267
Sugarcane
Sugarcane is an important cash crop and a major source of raw material for the sugar industry
in India. Conventionally, sugarcane is propagated through ratoons and as in the case of potato,
tends to accumulate lot of diseases over a period of time adversely affecting the production and
the productivity. One of the commonest diseases in sugarcane is ‘Red rot’. Till date there is no
variety available which is totally resistant to ‘Red rot’. Consequently, any variety of sugarcane
has a very short life and has to be replaced with a new variety periodically. Since the conventional
methods of cuttings are very slow, it takes several years before a newly released variety of
sugarcane is available to the farmers on a large-scale. The plants raised through tissue culture are
not only free of most bacterial and fungal diseases but also through this method of propagation,
the newly released varieties could be made available to the growers within a short period of time.
Presently, several varieties of sugarcane obtained from different sources are being multiplied on
a large-scale at TERI’s production facilities. The plants have survived very well in the field and
their performance is very encouraging. The tissue-cultured plants produced more number of
tillers as compared to their conventional counter parts. It is desirable to use the first generation
tissue-cultured plants as seed stock rather than sending them to mills for recovery of sugar. The
ratoon crop from tissue cultured plants give higher yields (11/2 times higher than the conventional)
and in few varieties up to 10% increase in sugar recovery (depending upon climate and management
practices) has been observed. We may have surplus sugar even without the use of tissue cultured
plants but in today’s era of globalization, it is important to produce food including sugarcane at
a competitive price. Also, the current emphasis on ethanol production from molasses for subsequent
use in mixing with the petrol has further renewed interest in this crop.
3.2.3 Spices and Aromatic Plants
Micropropagation protocols are now available for several spices and aromatic plants that are
found in India (Table 5). However, of all the spices, commercialization is largely confined to
cardamom and black pepper and among aromatic plants it is mainly Vanilla and Patchouli. With
increased awareness and development of efficient micropropagation protocols demand for other
spices and aromatic plants is also catching up.
3.2.4 Medicinal Plants
During the recent past there has been a lot of interest developed towards the tissue culture of
medicinal plants. This is mainly due to the fact that there has been a substantial increase in the
demand of medicinal plants in the world market that provides ample opportunities to the Indian
growers for good economic returns. However, Indian exports have suffered badly on account of
inferior and inconsistent quality, and uncertain supplies. Tissue culture can provide solutions to
many of these problems as superior quality plants containing high active principle can be produced
in very large numbers. In anticipation of a surge in the demand from the growers, several tissue
culture companies such as Nandan Agro Farms, Hyderabad; Labland Biotech, Mysore; Unicorn
Natural Products (P) Ltd., Hyderabad; PCD Enterprises, Nainital; Growmore Biotech (P) Ltd.,
Hosur; Whitefield Agrotech, Bangalore; Cipla, Mumbai; Greenearth Biotechnologies Ltd.,
Bangalore etc. have undertaken mass multiplication of medicinal plants. This includes species
such as Chlorophytum borolivilianum, Withania somnifera, Phyllanthus, Aloe vera, Commiphora
mukul, Gymnema sylvestris, Catharanthus roseus etc. Since tissue culture of medicinal plants
has been discussed in great detail in this volume, the same is not being repeated here.
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Table 5.
S.
No.
Status of tissue culture work in various spices and aromatic plants in India
Species
Explant
Mode of
propagation
Remarks
1. Piper nigrum
(Black pepper)
Shoot tip and
nodal segments
Axillary
Tissue cultured plants were successfully
transferred to the field; reports suggest
early bearing in some tissue cultured
plants
2. Piper longum
(Indian long
pepper)
Shoot tip and
nodal segments
Axillary
Tissue cultured plants transferred to the
field flowered early and had more number
of axillary shoots
3. Piper chaba
(Java long
pepper)
Shoot tip and
nodal segments
Axillary
Two year old tissue cultured plants transferred
to the field flowered early and had more
number of axillary shoots
4. Piper betle
(Betelvine)
Shoot tip and
nodal segments
Axillary
Transplantation success was 80%
5. Piper
colubrinum
Shoot tip and
nodal segments
Axillary
Field survival was 85%; TC plants flowered
earlier than the control
6. Piper barberi
Shoot tip and
nodal segments
Axillary
Micropropagation protocol can be used for
rapid multiplication of this endangered
species
7. Elettaria
cardamomum
(Cardamom)
Rhizome bits
with vegetative
buds
Axillary
Technology for micropropagation has been
fully commercialized. Up to 40% increase in
yield has been reported in the TC plants over
the control
8. Amomum
subulatum
(Large
cardamom)
Rhizome bits with
vegetative buds
Axillary
The rooted plantlets could be separated and
transferred to the soil with 80% success
9. Zingiber
officinale
(Ginger)
Vegetative buds,
immature
inflorescences and
rhizome bits
Axillary
Field evaluation suggest that it takes three
crop seasons for the micropropagated plants
to develop rhizomes of normal size, hence
they can not be used directly for commercial
planting
Vegetative buds,
rhizome explants
with buds
Axillary
It takes three crop seasons for the micropropagated plants to develop rhizomes of
normal size, hence they can not be used
directly for commercial planting
11. Curcuma longa
(Turmeric)
Vegetative buds
and rhizomes
Axillary
Being small, the micropropagated plants can
not be used directly for commercial planting
12.
Vegetative buds
and rhizome bits
Axillary
Transplantation success was over 80%; the
rhizomes produced by micropropagated plants
are small and not suited for commercial
planting
10.
Curcuma amada
(Mango ginger)
Curcuma
aromatica
(Kasturi
turmeric)
Changing Scenarios in Indian Horticulture
269
13.
Kaempferia
galanga
(Galangal)
Vegetative buds
and rhizome bits
Axillary
As above
14.
Kaempferia
rotunda
Vegetative buds
and rhizome bits
Axillary
As above
15.
Vanilla fragrans
(Vanilla)
Seeds, shoot tip
and nodal segments
Axillary
Process of micropropagation of vanilla has
been fully commercialized and there are
several companies that are engaged in mass
multiplication of this species
16.
Cinnamomum
zeylanicum
Shoot tip and
nodal segments
Axillary
TC plants established in the field with over
90% success. Plants grew to a height of 1-2 ft
within one year of planting
17.
Cinnamomum
camphora
(Camphor)
Shoot tip
Axillary
TC plants grew up to a height of 8 ft within
one year of field planting; there was lack of
expansion of leaves in some cases
18.
Cinnamomum
cassia
(Chinese cassia)
Shoot tip and
nodal segments
Axillary
In vitro plants hardened up to the nursery
stage; field transfer not reported
19.
Thymus vulgaris
(Thyme)
Seedlings and
shoot tips
Axillary
Transplantation success inside the greenhouse
was only 60%
20.
Mentha piperita
(Peppermint)
Shoot tips and
nodal segments
Axillary
Hardening survival at the greenhouse stage
was 60%
21.
Mentha spicata
(Spearmint)
Seedlings and
shoot tips
Axillary
As above
22.
Marjorana
hortensis
(Marjoram)
Seedlings and
shoot segments
Axillary
Field transfer of in vitro plants not reported
23.
Origanum vulgare Seeds and
(Oregano)
shoot segments
Axillary
As above
24.
Salvia officinalis
(Sage)
Seedlings and
shoot tips
Axillary
As above
25.
Lavendula
angustifolia
(Lavender)
Seedlings and
shoot tips
Axillary
As above
26.
Ocimum sanctum Seedlings and
(Sacred basil)
nodal explants
Axillary
As above
27.
Petroselinum
crispum
(Parsley)
Seedlings and
nodal explants
Axillary
Greenhouse survival was only 40%
28.
Apium
graveolens
(Celery)
Seedlings and
stem cuttings
Axillary
Greenhouse survival was 50%
(Contd)
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Table 5.
S.
No.
Species
Explant
(Contd)
Mode of
propagation
Remarks
29.
Pimpinella
anisum
(Anise)
Seedlings and
stem cuttings
Axillary
Greenhouse survival was only 60%
30.
Anethum
graveolens
(Dill)
Seedlings and
stem cuttings
Adventitious
shoots
60% plants survived at the greenhouse
stage; field transfer not reported
31.
Foeniculum
vulgare
(Fennel)
Seedlings, shoots
and nodal segments
Adventitious
shoots
60% plants survived at the greenhouse
stage; field transfer not reported
32.
Bunium persicum Petiolar segments
(Kala zira)
Somatic
embryogenesis
Few plants were transferred to the pots;
plants obtained by germinating small
tubers do not establish in soil
33.
Crocus sativus
(Saffron)
Shoot meristem
with a pair of leaf
primordium
Somatic
embryogenesis
50% of the in vitro formed corms
germinated upon transfer to the field
34.
Syzygium
aromaticum
(Clove)
Shoot-tips
and axillary buds
Axillary
Field transplantation not achieved
Sources : IISR, Calicut, 1997 and DBT, 2000.
It is rather sad that it is only those growers who are export oriented are going for tissue
cultured plants while those who cater to the domestic demand continue to rely on harvesting
from natural forest or adopting conventional means of propagation. However, the growers are
not entirely to be blamed for this situation because all said and done, the farmers are looking for
higher economic returns and the cost of tissue cultured plants is certainly higher than the
conventional propagules. While in exports, the impact of higher cost of the tissue-cultured plants
is more than neutralized through higher returns, the domestic market for the medicinal plants
continues to be highly disorganized with growers not getting any significantly higher returns on
account of the superior quality of their produce. Unless and until the domestic market matures
and the farmer is paid not only for the quantity but also for the quality, the present trend of
harvesting from natural forest will continue. It is heartening that over the last few years there has
been some positive change in this direction and many pharmaceutical companies involved in
plants and plant-based products are willing to pay a higher price for a better quality raw material.
As a result even the domestic suppliers are now exploring the possibilities of cultivating tissuecultured plants. However, it is only a beginning and much more is desired. This includes:
(a) development of efficient micropropagation protocols so that the cost of the plantlet is really
low, because we must realize that when the supplier is simply collecting the plant from the
natural forest he is not incurring any cost towards the planting material. Even if he undertakes
cultivation, the conventional propagules are much cheaper than the tissue cultured plants, (b) in
several cases, although the plants have been raised through tissue culture and are therefore, free
Changing Scenarios in Indian Horticulture
271
of most diseases, no proper studies have been made to estimate the actual active principle in the
tissue cultured plants. In such a situation it becomes difficult for the grower to convince his
buyer about the superiority of the crop. Therefore, while propagating plants through tissue
culture, active principle estimation is must, (c) it is known that the amount of active principle
produced by a plant is affected not only by the genotype but also the environment in which it has
been grown. Therefore, it is imperative that besides emphasizing on a good genotype, equal
attention is paid to work out the most suitable climatic conditions for growing that clone so that
the end product is of really high quality, and (d) initiatives are required from the government to
organize the market of medicinal plants as much as possible so that the growers are not left to
the mercy of the unscrupulous traders who do not pay the growers their due. The multiplication
of elite planting material and growing them under properly managed fields will ensure uniform
quality of the extract. This will help the pharmaceutical industry in India as well as contribute
in capturing international market.
3.2.5 Ornamentals
So far as tissue culture of ornamentals is concerned lot of work has been done in the country and
almost every tissue culture company is working on some or the other ornamental species. Some
of the major species that are being produced commercially include Ficus spp., Syngonium,
Spathiphyllum, Dieffenbachia, Philodendron, Cordyline, Calathea, Orchids, Gerbera, Zantedeshia,
Anthuriums, etc. There are many reviews appearing in recent past that provide a detailed account
of the mode of propagation and the success met with various ornamentals. Therefore, this aspect
of tissue culture of ornamentals is being deliberately omitted here. On the whole, ornamentals
are the easiest species to deal with in tissue culture. Most of those species, which in general, are
not propagated through tissue culture is not on account of non-availability of micropropagation
protocols but because there is either no requirement or the tissue-cultured plants are significantly
more expensive than the conventional propagules. Somehow the growth of tissue cultured
ornamentals has been much slower as compared to the international market. This is because of
several reasons as follows:
Grower’s Account
• Lack or incomplete scientific knowledge amongst growers is one of the major factors in
the popularity of tissue cultured plants including ornamentals.
• Reluctance on part of growers to accept change and adopt new technology.
• Many a times, the traders for business reasons or out of ignorance tend to oversell tissue
culture technology raising very high expectations among the minds of the growers. If
these expectations fall short, then it adversely affects the demand of the tissue cultured
plants.
Seller’s Account
• Tissue culture plants are sold through retailers who are more concerned about their profits
rather than the quality they are selling. Hence, if a trader makes more profit by selling
conventional plants then he will promote only those rather than tissue-cultured plants.
• Although tissue cultured plants can be produced inside the lab round the year, however,
they can be taken to the open nursery only in a particular season (this problem is more
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pronounced in those regions where there is extremity of both summer and winter, e.g. most
of north India). This way the tissue-cultured plants are available to the client only in a
specific season. In contrast, the traders are able to bring conventionally raised ready-toplant plants from other locations at low rates and sell them at a premium during off-season
thereby affecting the demand of the tissue-cultured plants in the proper season.
Buyer’s Account
• In domestic market, except for few metropolitan towns where the per capita income is
high and people are more quality conscious, in other areas the demand is influenced more
by the price rather than the quality; since conventionally raised plants are cheaper, they
sell more.
• Purchase of ornamental plants is usually not done on the basis of the regeneration process
but mainly on looks (a customer prefers to buy which is aesthetically appealing without
caring whether the plant has been raised conventionally or through tissue culture).
Technical/Other Reasons
• Field failures: There are certain species such as Dahlia, Chrysanthemum etc. in which the
tissue cultured plants are very weak, lanky and produce tiny flowers (much smaller than
the normal ones). In contrast the cutting-raised plants are healthy and produce normal
sized flowers. It is, therefore, necessary to combine both the micropropagation and
macropropagation techniques wherein few disease-free mother plants are produced by
tissue culture. They are transferred to the field and subsequently, cuttings are derived
from them.
• Imbalances of capacities in production and consumption areas: To keep the hardening
cost as low as possible, most of the tissue culture companies are concentrated in those areas
where the climate is relatively moderate such as Bangalore, Pune etc. If the plants produced
in this part of the country are to be sold in north India or far off places then the transportation
cost becomes prohibitory thereby affecting the market of tissue cultured plants.
• There are certain plants for which there is a genuine demand for tissue cultured material
but the micropropagation protocols are not available.
As against the domestic market, tissue culture raised ornamentals find a better international
market. In fact, most of the tissue-cultured plants that are exported from India are ornamentals.
However, India has failed to make any dent in the international market and its share in the world
trade of tissue cultured plants is insignificant. Some of the factors responsible for dismal performance
in foreign markets are:
• Non-adherence of delivery schedules by the producer.
• Supply of plants of inferior/inconsistent quality; also, there in no effective mechanism
from the government to prevent such dispatch from the country. In fact this factor has
contributed most in tarnishing the image of the country in foreign market.
• Inability of the Indian companies to produce plants as per the specifications of the client
• International competition.
• Lack of cooperation and coordination among the Indian companies resulting in undercutting of prices and thereby lower price realization.
Changing Scenarios in Indian Horticulture
273
High yielding planting material can also be obtained through breeding and genetic engineering.
So far several new varieties have been developed through breeding that are being grown
commercially. The only drawback with hybrid seeds is that they are relatively expensive and can
be used for raising only a single crop, that is, for every crop new seeds have to be purchased.
Recently, genetic engineering technology has gained lot of grounds and several improved varieties
carrying several agronomically important traits such as higher yield, disease and insect resistance,
drought tolerance, herbicide resistance, enhanced nutritional status etc. have been developed.
The first and the only transgenic crop for which the Government of India has granted permission
for commercial cultivation is Bt cotton. Although in India research is underway to develop
transgenic plants of several other economically important plant species, it will take some more
time before these genetically modified plants become available to the Indian farmers for commercial
use. Even after the availability of transgenic plants, it will take some more time to popularize
them among the masses. This will be more so in food crops for human consumption.
3.3 Use of High-Tech Agro-Techniques
Inadequate technological upgradation by the farmers has been one of the major reasons of low
productivity. During the past few decades several technological advancements have taken place
in the production technology which requires greater commercialization and professionalism for
production of fruits. Some of these include high density planting, drip irrigation, protected cultivation,
biofertilizers, use of new and high-yielding varieties etc.
Introduction of high density planting is one of the major advances in the field of fruit production.
High-density plantation is a worldwide phenomenon that has been successfully adopted in apple,
banana, peach, plum, pear, pineapple and papaya. In India too, substantial increase in yield on
account of high density planting have been reported in banana, mango, papaya and guava. In
pineapple, a plant density of 63,758 plants per hectare coupled with improved management
practices increased the yields from 15-20 tonnes per hectare to 70-80 tonnes per hectare. However,
high density planting in India is largely restricted to few demonstration plots only with most of
the orchards being still under the traditional low-density system. Much needs to be done in this
regard.
Use of drip irrigation not only results in higher productivity (10–50% over conventional
methods) but also saves 50–70% water. In addition, using drip, fertilizers, pesticides and other
soluble chemicals can be applied along with irrigation water leading to their efficient use,
reduced incidence of diseases, less weed growth, better quality products, and low labour and
operational costs. Today, 260,000 ha of area in the country is under drip irrigation using which
higher yields have been obtained in grapes, banana, mango, guava, pomegranate, sapota, cabbage,
coconut, arecanut, roses etc. Although drip irrigation is gaining popularity in the country there
is still a long way to go. The major constraints in popularization of this system are high cost of
the equipment and maintenance as the equipment is highly sensitive to clogging with the existing
water quality and finally, the water-soluble fertilizers are far too expensive and available only at
selected outlets. Besides bringing about technological improvement, government initiatives in
terms of subsidies are also desired.
The advent of protected cultivation in microclimate regulated/modified greenhouses result in
production of high quality vegetables, flowers and other ornamentals. As compared to western
world, the concept of protected cultivation in India is rather recent and is largely confined to
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cultivation of ornamentals. In spite of its late entry, Indian industry has made rapid strides and
nearly 250–300 ha of land is now under protected cultivation of floricultural crops. Nearly
ninety percent of the area is under roses and the remaining is shared by other ornamentals. High
initial and operational costs have deterred the Indian farming community to adopt this system.
Many soil microorganisms enhance nutrient uptake in plants. Those which have a direct
beneficial effect on the plants, may have considerable potential as biofertilizers. Two main groups
of plant-beneficial micro-organisms are: (i) nitrogen fixing micro-organisms such as blue-green
algae (cyanobacteria), soil bacteria of the genera Azotobacter, Klebsiella, Bradyrhizobium, Rhizobium
and Actinomycetes and (ii) mycorrhizal fungi. Mycorrhiza is the form of a symbiotic relationship
between certain fungi, particularly vesicular-arbuscular (VA) mycorrhizae and the roots of vascular
plants. Unlike rhizobial associations, VA mycorrhizal fungi are non-specific and can affect a wide
range of host plants. In certain circumstances, mycorrhizal infection can significantly increase
the rate of uptake of nutrients, particularly phosphorus and nitrogen from deficient soils. In
addition, they can mobilize other trace elements such as copper, zinc and iron. Besides enhancing
the productivity, mycorrhizae are also very effective in reclamation of wastelands.
Biofertilizers can be an effective substitute for chemical fertilizers. They are not only
environmentally benign but also the crops grown without the use of chemical fertilizers command
a better price realization especially in the international market. Use of biofertilizers in horticultural
crops has not gained much popularity in India as yet. This is largely due to lack of awareness,
non-availability and technical constraints associated in the use of biofertilizers. Relatively low
cost of chemical fertilizers on account of government subsidies has also deterred the farmers to
switch over from chemical fertilizers to biofertilizers on a large-scale.
3.4 Application of Frontier Technologies
As described earlier, breeding, micropropagation, biofertilizers, genetic engineering and other
molecular techniques, either singly, or in combination can play a significant role in augmenting
horticultural production in the country. In addition to quantitative gains these frontier technologies
can also bring about marked improvement in the quality of the horticultural products. Except for
genetic engineering where there are still some perceived technological problems that needed to
be resolved before the GM crops are cleared for mass consumption in India, all other technologies
are well proven and should therefore be promoted for adoption by various end users. More than
the cost, it is the lack of awareness that is hindering the wider use of these technologies.
3.5 Post-harvest Management
Although there is no consensus with regard to exact quantum of post-harvest losses of fruits and
vegetables in India, however, taking into account the estimates made by various agencies this
figure could be anywhere between 25 and 30%. In terms of value, the estimated loss could be
over Rs. 23,000 crores per annum. These post-harvest losses in supply chain of horticulture
produce are attributed to: (a) mishandling of produce, (b) improper and inadequate facilities for
storage, and (c) improper packaging and transportation. If these losses can be minimized, the
additional horticultural produce available will help in achieving per capita increase in consumption
of fruits and vegetables. Surpluses, if any, may be diverted to the food processing industry in the
country. Post-harvest losses is one important aspect of food production that has not received the
attention it deserves and still much is desired to be done.
Changing Scenarios in Indian Horticulture
275
The areas that need to be targeted to reduce post-harvest losses can be broadly classified into
following three categories:
• Storage losses: The first step towards cutting down on the storage losses is to minimize
the damage to the produce during harvest. After harvesting, the fruits and the vegetables
should be washed and treated to eliminate or minimize bacterial and fungal infection and
attack by insects and other pests. Then only the properly packed produce should be
sent for refrigeration or cold storage. The present cold storage capacity in India is about
87 lakh tonnes out of which nearly 80-90% is utilized for storage of potato and potato
seed. It is imperative on part of the Central and the State Governments to develop
adequate infrastructure for proper storing of fruits, vegetables and flowers at different
temperatures depending upon the requirement of the species.
• Transportation: Transportation is the link between the farmer and the consumer. The
transportation of perishable horticultural produce to the consumer requires the selection
of the fastest and most efficient mode of transport to deliver the consignment in the best
possible condition at the lowest possible cost. In this regard, infrastructure for both rail
and road transport, the mainstay of Indian transport system will have to be strengthened.
Packaging, loading and unloading, and containerization are some of the other related
aspects of transportation that are as important as the transportation process per se but are
often neglected. Uniformity in the size of the containers/crates used for different purposes,
will not only lead to effective utilization of space during transportation, but will also reduce
the time spent in loading and unloading operations.
• Processing: Although not an integral part of post-harvest management, facilities for food
processing will help in value addition of the product resulting in better cost realization.
At present, we do not even cultivate varieties that are better suited for processing purposes.
Also, it is usually the substandard produce or surplus, which is diverted for processing.
3.6 Horticulture Informatics
To develop plans and strategies for any developmental activity, a comprehensive database is a
must. In India, as compared to agriculture, horticulture sector is much less organized. Consequently,
with regard to statistics on area, production and productivity, information on very few horticultural
crops is available. In floriculture crops, the situation is even worse as statistics on productionrelated aspects is either missing or there is huge variation in the data provided by various sources.
It is, therefore, desirable to create authentic databases on various aspects of horticulture including
area, varieties, total production, yield, post-harvest losses, export, processing, price realization
etc. for all the horticultural crops. Some projections on demand of various crops in future should
also be made available to the grower so as to enable them to plan their strategy for better price
realization. Other databases providing information on soil type, climate, rainfall, pest attack and
marketing will also be very useful.
3.7 Marketing
Marketing is an integral component of any production process. However, so far as horticultural
crops are concerned very little effort has been made by the government in this regard. Unlike for
agricultural crops there are no governmental agencies such as FCI that makes direct purchase of
horticultural crops from the farmers. Also, there is no minimum support price offered by the
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government for the horticultural crops. Apart from local ‘Mandis’ (wholesale markets) there are
no other places where the farmer could go to sell his produce. Very often the poor farmers do not
get the real value of their produce and suffer heavily on account of middlemen who control all
the business transactions in these ‘Mandis’. There are many exotic vegetables such as asparagus,
broccoli, celery, Chinese cabbage, kale, leek lettuce, parsley, etc. that can fetch very good price
to the farmers. However, the farmers hesitate to grow such crops as their market is highly
restricted and not so easily accessible to them. Similarly, on the export front, there are very few
international airports in India where facilities for cold storage are available. In addition, there is
hardly any information/statistics available on the world markets, price trends, marketing agencies,
etc. Much needs to be done for easy and organized marketing of horticultural crops in India.
3.8 Credit Facilities
Credit availability at reasonable interest rate is absolutely essential for the small and marginal
farmers to enable them to adopt modern technology and improved horticultural practices. Access
to credit facilities is also very critical for sustaining competitiveness. Agricultural/horticultural
credit to the needy growers has increased over the years but how far the credit that is made
available to the grower is put to use is a point to ponder. There is a serious problem of overdues
that has been inhibiting credit expansion and economic viability of the lending institutions. Loan
waivers by State Governments for making political gains have caused severe problems of recovery.
3.9 Research and Extension
With the objective of increasing production, new high-yielding varieties with other desirable
traits such as disease resistance, better flavour etc. must be developed on a continuous basis.
Wherever possible, the conventional breeding must be linked or supplemented with latest molecular
techniques to accrue higher gains. GM technology holds lot of potential that must be tapped.
There are several exotic vegetable and fruit crops that could be sold at a premium both in the
domestic as well as international markets. In spite of the fact that many of these crops can
possibly be grown in India, their cultivation has not been undertaken on the desired scale
because either the agroclimatic conditions suitable for their growth are not known or the cultural
practices required for raising crop have not been established. In many cases it is mere lack of
awareness about the new introductions in the country that has deprived the Indian farmer from
growing them. It is, therefore, important to strengthen our extension network in horticulture as
we have done in agriculture.
4.
Conclusions
India has made significant achievements in the field of horticulture in terms of overall production,
however, there is still a lot that remains to be done with regard to productivity and quality. There
are several impediments in our way to progress and accordingly a multi-pronged approach is
desired to deal with the problem. This is just not a requirement but also a necessity because with
the opening of Indian market there would be a large-scale invasion of foreign horticultural
products in the country that could seriously effect the very existence of the Indian farmer.
However, instead of a threat the integration of the Indian market with the world markets should
be taken as an opportunity that every Indian farmer must try to seize effectively. This would be
possible only through concerted and integrated efforts on part of the policy makers, researchers,
administrators and the growers.
Changing Scenarios in Indian Horticulture
277
References
1.
2.
CMIE (2001) Agriculture. Centre for Monitoring Indian Economy Pvt. Ltd., Mumbai, India (322 p).
DBT (2000) Plant Tissue Culture from Research to Commercialization—A Decade of Support. Department
of Biotechnology, Ministry of Science & Technology, Govt. of India (224 p).
3. FAO (2002) http://apps.fao.org/.
4. IISR (1997) Protocols for micropropagation of spices and aromatic crops. Nirmal Babu K., Ravindran
P.N. and Peter K.V. (Eds). Indian Institute of Spices Research, Calicut, Kerala (35p).
Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
19. Cryopreservation: A Potential Tool for Long-term
Conservation of Medicinal Plants
Sonali Dixit, Sangeeta Ahuja1, Alka Narula2 and P.S. Srivastava2
Amity Institute of Biotechnology, Amity Campus, Sector 44, Noida 201303, India
1
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville,
Virginia-22903, USA
2
Centre for Biotechnology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
Abstract: Medicinal plants are one of the most important groups of plant genetic resources. Their
use in biotechnology has assumed considerable significance because of overexploitation of these
plants to meet the increasing demand. As cells cultured in vitro are prone to spontaneous changes,
continuous culture of plant cells is often undesirable. Cryopreservation is a safe and cost-effective
technique for preservation of germplasm and management of in vitro produced materials for
biotechnological applications. The present article is a brief account of cryopreservation techniques
and their application for medicinal plant conservation.
1.
Introduction
Traditional medicinal systems are part of a time-honoured and time-tested culture, that still
intrigues people today. A culture that has successfully used plants to treat primary and complex
ailments for over 3,000 years obviously has a contemporary relevance. In an age when toxic
drugs are increasingly unwelcome and when people are using viable alternatives, this heritage
of medicinal plants must be documented and conserved for effective use in future.
During the past decade, a dramatic increase in exports of medicinal plants attests to worldwide
interest in these products. Nevertheless, most of these plants being taken from the wild, hundreds
of species are now threatened with extinction because of overharvesting, destructive collection
techniques, and conversion of habitats to crop-based agriculture.
Preservation of these genetic resources is currently at the forefront of conservation activities
and biotechnology has played an important role in international conservation programs [1].
Traditionally, plant genetic resource management involves conserving germplasm as seeds at
low temperature, or as field plantings (field genebanks) for vegetatively propagated plant species.
These approaches are now complemented by in vitro conservation methods that can be used in
combination with traditional practices and offer added security for field genebank conservation
[2]. The ideal genetic resource conservation program consists of active collections that are
available for distribution or characterization and base collections held for the sole purpose of
long-term preservation. Base collections of vegetatively propagated plants are more difficult to
achieve and recently, cryopreservation has been identified as the best option for long-term
conservation of germplasm of these species [3, 4, 5]. Cryopreservation, i.e., non-lethal storage
of plant tissues at ultra-low temperature usually that of liquid nitrogen (–196°C) is the only
Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants 279
available method for the long-term conservation of germplasm of these problem species.
Cryopreservation has manifold applications in conservation and biotechnology. A number of
medicinal plant species have been subjected to cryopreservation (Table 1). The major advantage
of storage of biological material at such a low temperatue is that both metabolic processes and
biological deterioration are considerably slowed or even halted [6, 7]. Additionally, continued
maintenance of plants in tissue culture can lead to loss of morphogenic, genetic and biosynthetic
capacity, which may confound successful exploitation [8]. It scores advantages over other
conservation strategies as it minimizes the risk of contamination, cost of maintenance and cost
of labor [9]. Through cryopreservation, it may be possible to establish a reserve of freshly
initiated competent cultures which after thawing and recovery can be reintroduced into culture.
2. International and National Programmes for Cryopreservation of
Medicinal Plants
Interests and concern of the international scientific community in this area has lead to formulations
of several national and international level programmes, which are devoted to cryopreservation of
medicinal plants. For example, G-15 Genebanks for medicinal and aromatic plants were initiated
from the Summit Level Meeting of Group on South-South Consulting and Cooperation of the G15 countries held in Kuala Lumpur (January 1990). Malaysia together with Indonesia and India
represent the Asian region where India is a Regional Coordinator. India has also been given the
overall responsibility for coordinating the activities of the G-15 nations for the establishment of
gene banks for medicinal and aromatic plants. Under the aegis of this programme, Department
of Biotechnology, Government of India, has constituted a network of three national gene banks
at Tropical Botanical Garden and Research Institute (TBGRI), Thiruvananthapuram; Central
Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow; and National Bureau of Plant
Genetic Resources (NBPGR), New Delhi. One of the important mandates of this group is to
develop cryopreservation protocols for long-term conservation of medicinal plants.
3.
Cryopreservation Techniques
Some plant organs such as orthodox seeds and frost-hardy dormant buds contain very low
amounts of water and can thus be cryopreserved directly, without any pretreatment. However,
most of the experimental systems employed in cryopreservation (cell suspensions, calli, shoot
tips, embryos) contain high amounts of cellular water and are thus extremely sensitive to freezing
injury since most of them are not inherently freezing-tolerant. Cells have thus to be dehydrated
artificially to protect them from the damages caused by the crystallization of intracellular water
into ice. The techniques employed and the physical mechanisms upon which they are based are
different in classical and new cryopreservation techniques [10]. Classical techniques involve
freeze-induced dehydration, whereas new techniques are based on vitrification, i.e. the transition
of water directly from the liquid phase into an amorphous phase or glass, whilst avoiding the
formation of crystalline ice.
Classical cryopreservation techniques involve slow cooling down to a defined prefreezing
temperature followed by rapid immersion in liquid nitrogen. They are generally operationally
complex since they require the use of sophisticated and expensive programmable freezers. In
some cases, their use can be avoided by performing the freezing step with a domestic or laboratory
freezer [11].
280
DIXIT, AHUJA AND SRIVASTAVA
Table 1.
Technique
Summary of different techniques used for cryopreservation
Explants
Protocol
Reference
1. Vitrification
Shoot tips/embryogenic
tissues/cell cultures
Explant is treated with LS (1 M glycerol)
for 20 min at 25°C followed by dehydration
with PVS2 (30% glycerol, 15% EG, 15%
DMSO) at 0°C for 90 min, rapid freezing in
LN, rapid thawing at 40°C for 1–2 min, UL
(1.2 M sucrose) and culture for recovery
growth
[27]
2. Encapsulation
dehydration
Shoot tips/embryogenic
tissues
Explant is encapsulated in calcium alginate
and precultured in high sucrose solution
(0.5–0.75 M), followed by dehydration in
laminar airflow for 4–5 h, rapid freezing in
liquid nitrogen, rapid thawing at 40°C for
1–2 min and culture for recovery growth
[28]
3. Encapsulationvitrification
Shoot tips
Excised encapsulated meristems containing
2 M glycerol +0.4 M sucrose were
dehydrated with PVS2 for 2h at 0°C and
subsequently plunged in LN
[29]
4. Pregrowth
Zygotic and somatic
embryos
Pre-growth technique consists of cultivating
samples in the presence of cryoprotectants,
then freezing them rapidly by direct
immersion in liquid nitrogen
[30]
5. Pregrowth
desiccation
Stem segments
Pre-growth desiccation refers to the preculture of the explant on a medium with high
concentration of sucrose or ABA or Proline
and desiccation/drying followed by freezing
in liquid nitrogen
[31]
6. Desiccation
Large number of recalcitant and intermediate
seeds
Desiccation is usually performed in the
air current of a laminar flow cabinet, but
more precise and reproducible dehydration
conditions are achieved by using a flow
of sterile compressed air or silica gel
[10]
7. Droplet
freezing
Shoot tips
Apices are pretreated with liquid
cryoprotectant in medium then placed on
aluminum foil in minute droplets of
cryoprotectant and frozen directly by rapid
immersion in LN
[32]
Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants 281
In the new vitrification-based procedures, cell dehydration is performed prior to freezing by
exposure of samples to concentrated cryoprotective media and/or air desiccation. This is followed
by rapid cooling. As a result, all factors, which affect intracellular ice formation, are avoided.
Glass transitions (changes in the structural conformation of the glass) during cooling and rewarming
have been recorded with various materials using thermal analysis. Vitrification-based procedures
offer practical advantages in comparison to classical freezing techniques. Like ultrarapid freezing
(above), they are more appropriate for complex organs (shoot tips, embryos) which contain a
variety of cell types, each with unique requirements under conditions of freeze-induced dehydration.
By precluding ice formation in the system, vitrification-based procedures are operationally less
complex than classical ones (e.g., they do not require the use of controlled freezers) and have
greater potential for broad applicability, requiring only minor modifications for different cell types
[10]. A common feature to all these new protocols is that the critical step to achieve survival
is the dehydration step, and not the freezing step, as in classical protocols. Seven different
vitrification-based procedures can be identified: (1) encapsulation-dehydration; (2) a procedure
actually termed vitrification; (3) encapsulation-vitrification; (4) desiccation; (5) pregrowth;
(6) pregrowth-desiccation; and (7) droplet freezing (Tables 2 and 3).
Table 2.
Advantages and disadvantages of commonly used cryopreservation techniques
Technique
Advantages
Slow freezing
Stability from relatively nontoxic
cryoprotectants
Requires expensive equipment, slow recovery, low
applicability to tropical species
Vitrification
No special equipment needed, fast
procedure, fast recovery
Vitrification solutions are toxic to many plants, cracking is possible, requires careful timing of solution changes
Encapsulationdehydration
No special equipment needed, non
toxic cryoprotectants, simple
thawing procedures
Requires handling each bead several times, some plants
do not tolerate high sucrose concentrations
Dormant bud
desiccation
Easy, useful for many temperate
tree species
Requires freezing equipment, larger storage space,
recovery requires grafting or budding, works best in
cold temperate regions
4.
Disadvantages
Some Important Case Studies
4.1 Cryopreservation of Shoot Tips of Dioscorea spp.
In vitro grown shoot tips of two medicinally important species of Dioscorea, D. floribunda and
D. deltoidea, were successfully cryopreserved using vitrification and encapsulation-dehydration
techniques. For vitrification the excised shoot tips were precultured for 16h on MS medium
containing 0.3M sucrose followed by loading for 20 min at 25°C (2 M glycerol + 0.4 M sucrose),
dehydration with plant vitrification solution (PVS2) (30% glycerol, 15% Ethylene Glycol (EG),
15% DMSO and 0.4 M sucrose) for 90 minutes at 0°C prior to plunging in liquid nitrogen (LN).
After storage in LN for atleast 1 h the shoot tips were unloaded for 20 min at 25°C (1.2 M
sucrose) and transferred to medium for recovery growth. During recovery growth, apices of D.
floribunda and D. deltoidea regenerated directly with a frequency of 30 and 75%, respectively
(Fig. 1 A, C, D).
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DIXIT, AHUJA AND SRIVASTAVA
Table 3.
Summary of cryopreservation studies on some important medicinal plant species
Plant
Explant
Atropa belladonna
Anisodus acuntangulus
Catharanthus roseus
Coleus blumei
Chicory
Cinchona ledgeriana
Datura innoxia
D. stramonium
Dioscorea caucasia
D. balanica
D. bulbifera
D. floribunda
D. deltoidea
Protoplasts, cells
Suspension cultures, cells
Cells
Cells
Shoot tips
Protoplasts
Protoplasts
Cell suspension
Organogenic callus
Organogenic callus
Somatic embryos
Shoot tips
Cell cultures
Shoot tips
Cell cultures
Cell cultures
Leaf
Axillary buds
Shoot tips
Suspension cultures
Suspension cultures
Suspension Cultures
Shoot tips
Hairy roots
Cells
Cell cultures
Transformed Cells
Suspension cells
Shoot tips
Digitalis lanata
D. thapsi
Eucalyptus
Gentiana scabra
Holostemma annulare
Nicotiana tabacum
N. sylvestris
N. plumbaginifolia
Olea europe
Panax ginseng
P. quinquefolium
Papaver somniferum
Polygonum avuculare
Trifolium repens
Reference
[33]
[34]
[25]
[25]
[35]
[36]
[37, 38]
[38]
[39]
[39]
[13]
[40, 41]
[42]
[13]
[43]
[17]
[44]
[45]
[46]
[47]
[48]
[48]
[49]
[15]
[50]
[51]
[52]
[53]
For encapsulation-dehydration the shoot tips pregrown in 0.3 M sucrose were encapsulated in
calcium alginate followed by preculture in 0.75 M sucrose, dehydration for 51/2 and 5 h respectively,
rapid freezing and rapid thawing. The encapsulated shoot tips were recovered with high frequency
direct regeneration in both the species (Fig. 1B) [12, 13]. Interestingly, the diosgenin content in
the plants recovered after cryopreservation was found to be stable using HPLC analysis. Molecular
studies using RAPD analysis proved that the plants were genetically stable [14].
4.2 Cryopreservation of Somatic Embryos of Dioscorea bulbifera
Somatic embryos/embryogenic tissues of Dioscorea bulbifera were cryopreserved using
encapsulation-dehydration technique. The embryogenic tissues of about 1–2 mm in diameter,
with a group of embryoids were encapsulated into calcium alginate beads. These were then
precultured in 0.5 M sucrose for 7 d followed by dehydration under laminar airflow for 4 h. High
frequency (75%) of embryogenic survival was recorded after storage in LN (Fig. 2 A-D). The
plants hence produced and transferred to field have been found to be morphologically similar to
the non-treated controls. The diosgenin content in the plants recovered after cryopreservation
Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants 283
A
B
C
Fig. 1.
D
Recovery growth of shoot tips of D. deltoidea after cryopreservation. (A) Close-up cryopreserved
shoot tip showing growth without any intermediary callus phase; (B) High frequency direct
regeneration from cryopreserved shoot tips; (C) Close-up of shoot tip showing recovery growth
cryopreserved using encapsulation-dehydration and (D) Well developed shoots.
284
DIXIT, AHUJA AND SRIVASTAVA
C
A
B
D
Fig. 2. Recovery growth of encapsulated embryogenic tissue of D. bulbifera using encapsulation-dehydration
technique. (A) Development of somatic embryos directly emerging out of an alginate bead;
(B) Maturation of somatic embryos. Note the numerous cotyledonary stage embryos on the cryopreserved tissue; (C) Single somatic embryo growing to give rise to a complete plantlet after
freezing; (D) Plants established in small pots transferred from in vitro cultures.
was analyzed using HPLC and the content was found to be stable. Molecular studies using
RAPD analysis proved that the plants were genetically stable [13].
4.3 Cryopreservation of Hairy Roots of Panax ginseng
The protocol for cryopreservation of hairy roots of Panax ginseng was developed by Yoshimatsu
et al. [15]. Hairy root segments including root tips were placed on to phytohormone-free halfstrength Murashige and Skoog solid medium and stored at 4°C in the dark for 4 months. The root
segments resumed elongation when the temperature was raised to 25°C in the dark. For
cryopreservation, the root tips were precultured with 0.1 mg l–1 2,4-D for 3 d and dehydrated with
PVS2 for 8 min before immersion in liquid nitrogen. Sixty percent survival could be obtained.
The hairy roots regenerated from cryopreserved root tips grew well and showed the same ginsenoside
productivity and patterns as those of the control hairy roots cultured continuously at 25°C. The
conservation of T-DNAs in the regenerated hairy roots was proved by PCR analysis.
4.4 Cryopreservation of Transformed Calli of Papaver somniferum
The transformed P. somniferum cells maintained on MS solid medium at 22°C in the dark were
Cryopreservation: A Potential Tool for Long-term Conservation of Medicinal Plants 285
precultured in 50% loading solution (1 M glycerol + 0.2 M sucrose) at 20°C in the dark for 1 d,
dehydrated with PVS2 at 25°C for 35 min without loading, and then cryopreserved in liquid
nitrogen. After rapid thawing and washing, the cells were precultured on MS solid medium at
22°C in the dark. All the four clones used for cryopreservation regenerated successfully showing
the same morphological characteristics as the untreated cultures. To confirm the conservation of
T-DNA derived from Agrobacterium rhizogenes, the existence of T-DNA in the regenerants was
examined by PCR analysis. Amplification of T-DNA bands was clearly observed in the regenerated
cells as well as in the untreated ones. Preliminary evaluation of genetic stability using RAPD
analysis was performed and no significant difference was observed between the untreated and
cryopreserved cells [16].
4.5 Cryopreservation of Cell Cultures of Digitalis thapsi
Cell cultures of Digitalis thapsi were treated for 3 d with 0.15 M mannitol followed by treatment
with a cryoprotectant solution composed of 0.5 M DMSO, 0.5 M glycerol and 1 M sucrose, slow
cooled for 30 min at –20°C and freezed by rapid immersion in LN, rapid thawing and transfer
of cells without washing to a standard semi-solid medium. High viability (60%) was recorded
and the cultures originating from cryopreserved cells retained their capacity to accumulate
carotenoids [17].
5. Monitoring Genetic Stability of Regenerants from
Cryopreserved Germplasm
It is important to consider that the plants regenerated from cryopreserved germplasm have been
exposed to a range of different experimental conditions including tissue culture, pre-growth,
cryoprotection, freezing-thawing, recovery (re-growth) and manipulations to enhance regeneration.
All these stages have the potential to influence genetic stability [18]. Successful post-thaw
storage recovery must not only be assessed in terms of survival (viability) of plant tissues, but
also the ability to regenerate and produce complete plants. This necessitates a tissue culture
regeneration system. It is therefore essential to consider the effects that in vitro regeneration will
have on the genetic stability of the surviving plants as these may show somaclonal variation
[19]. For instance, the time taken to regenerate plants and the quality of germplasm recovered
after cryopreservation are likely to be important features in maintenance of genetic stability and
operation of a functional gene bank [20-22]. It is thus imperative to assess the genetic stability
of plant material regenerated from cryopreserved germplasm and to determine if it is genetically
identical to the mother stock (Germplasm prior to storage in LN).
Studies on ginsenoside production from cryopreserved cells of Panax ginseng revealed that
total amount of ginsenosides together with product remain unchanged [15]. Furthermore, it was
demonstrated that alternative conservation method such as preservation under mineral oil for six
months as well as continuous subculturing for 14 months failed to preserve biosynthetic capacity
of the cells [23]. Benson and Hamil [24] reported stability in the biosynthetic capacity in
transformed roots of Beta vulgaris after cryopreservation. All recovered cultures of Coleus
blumei showed the same growth and production characteristics of rosmaric acid as controls [25].
Stability was also demonstrated for recovery after different storage periods in liquid nitrogen
(from 1 day to 15 months). These works clearly show that cultures were stable even after
286
DIXIT, AHUJA AND SRIVASTAVA
successive cryopreservation cycles. Similar conclusions of successful application of cryopreservation
can be made from freezing experiments with biotin producing callus cultures [26].
6.
Conclusions
Cryopreservation of medicinal plants has multifacet advantages. The technology of cryopreservation
has been refined and it enables the storage of in vitro cultures for the long-term conservation of
medicinal plants. The retention of biosynthetic potential of the retrieved cultures amply demonstrates
the use of this technology for the storage of rare, high alkaloid/secondary metabolites/medicines
producing cell cultures for pharmaceutical purposes. Medicinal plants are potential candidates
for transformation as well and cryopreservation of transgenic lines is an important line of
research. The successful cryopreservation of cell cultures of tobacco, Datura, Panax, Dioscorea,
Catharanthus, Anisoidus, Atropa, etc., without any evidence of deterioration coupled with potential
expected benefits warrant the extension of this technique to other species. As far as genetic
stability is concerned the chances of an undesired cell selection after cryogenic storage seem to
be less important than expected. It is even more remarkable that in most of the cases the
important characters of the preserved cell lines did not change. Finally, it may be concluded that
routine cryopreservation procedures developed and applied on a large scale in medicinal plants
will help in conservation of this important group of genetic resource.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
20. Molecular Mapping and Marker Assisted Selection of
Traits for Crop Improvement
Anushri Varshney, T. Mohapatra and R.P. Sharma
National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute,
New Delhi-110 012, India
Abstract: Genetic markers, the heritable entities that are associated with economically important
traits can be used by plant breeders as selection tools. By using molecular markers, breeders can bypass traditional phenotype-based selection methods, which involve growing plants to maturity and
closely observing their physical characteristics in order to infer underlying genetic make up. The
purpose of this chapter is to describe the available genetic marker types and provide the status of
gene mapping and marker-assisted selection in important crop species. This review highlights how
genetic markers have been used in mapping genes both for qualitative and quantitative traits and
defines the potential use of genetic maps for marker-assisted selection.
1.
Introduction
Crop plants have evolved initially by incidental consequences of human gatherers, and more
recently through sophisticated plant breeding programmes. While changes in cultural practices
and mechanization have had significant impact on agricultural productivity, yield gains in most
crops have been due to genetic improvement. Although the gains have already achieved, further
improvement of agricultural productivity and quality are demanded continuously mainly due to
population growth, the increasing cost of inputs such as water, fertilizer and energy, concerns
about the effects of agrochemicals on the ecosystem, and rapidly changing consumer preferences.
Plant breeding, a process being used for centuries is largely depending on selection for desirable
traits. These selections often take many cycles of breeding in order to place desirable agronomic
and quality characteristics from different parents into a single genotype. Recent advances in
biotechnology have led to the development of a number of novel tools that offer the promise of
making plant breeding more precise and faster. Among the most promising are molecular markers,
which are segments of plant DNA that breeders use to detect the presence or absence in experimental
plants of specific alleles of interest and thus use them as selection tools [1, 2]. Such a selection
of desirable plants based on linked markers is termed as Marker-Assisted Selection (MAS). By
using molecular markers, breeders can by-pass traditional phenotype-based selection methods,
which involve growing plants to maturity and closely observing their physical characteristics in
order to infer underlying genetic make up. Several molecular marker systems have been developed
and put to use. The more frequently used ones are discussed as follows.
290
2.
VARSHNEY, MOHAPATRA AND SHARMA
Molecular Markers
2.1 RFLP
Restriction fragment length polymorphisms, the first molecular marker developed by Botstein et
al. [3] are detected by the use of restriction enzymes that cut genomic DNA molecules at specific
nucleotide sequences (restriction sites), thereby yielding variable size DNA fragments. Identification
of genomic DNA fragments is done by Southern blotting, a procedure whereby DNA fragments,
separated by electrophoresis, are transferred to nitrocellulose or nylon filter [4]. In this, filterimmobilized DNA is allowed to hybridize to radioactively labeled probe DNA. RFLP is a codominant marker in which the probes are usually small (500 to 3000 bp), cloned DNA fragments
(e.g. genomic or cDNA). The filter is placed against photographic film, where radioisotope
disintegration from the probe results in visible bands.
2.2 RAPD
Random amplified polymorphic DNA is a dominant marker based on polymerase chain reaction
(PCR). It employs a single decamer primer of arbitrary sequence, which is annealed to the
template DNA typically at 37°C [5]. The variation in RAPD profile is in the form of presence
or absence of a band resulting from variation in primer binding sites. A major limitation of this
marker system is non-reproducibility due to low annealing temperature. However, utility of a
desired RAPD marker can be increased by sequencing its termini and designing longer primers
(e.g. 24 nucleotides) for specific amplification of markers [6]. Such sequenced characterized
amplified regions (SCARs) are similar to sequence-tagged-sites (STS, [7]) in construction and
application.
2.3 CAPS
Cleaved amplified polymorphic sequences are based on the restriction enzyme site variation in
the DNA fragments generated by PCR [8]. The source of the sequence information for the primers
can come from a gene bank, genomic or cDNA clones, or cloned RAPD bands. This marker is
a co-dominant marker.
2.4 SSRs
Simple sequence repeats or microsatellites are ubiquitous in eukaryotes. SSR polymorphism
reflects variation in the number of repeat units in a defined region of the genome. The frequency
of repeats longer than 20 bp has been estimated to occur every 33 kb in plants. Nucleotide
sequence flanking the repeat is used to design primers to amplify different number of repeat units
in different varieties. These primers are very useful for rapid and accurate detection of polymorphic
loci and the information could be used for developing a physical map based on these sequence
tags. This type of polymorphism is highly reproducible.
2.5 AFLP
The amplified fragment length polymorphism markers are generated by selective amplification of
DNA fragments obtained by restriction enzyme digestion [9]. High molecular weight DNA is
digested by two restriction enzymes: one hexacutter (e.g. EcoRI) and one tetracutter (e.g. Mse I).
Adapter molecules are ligated to the ends of DNA fragments. Two primers possessing sequence
Molecular Mapping and Marker Assisted Selection of Traits 291
complementarity to the adapter as well as few extra random nucleotides at their 3′ ends are used
for selective amplification of fragments employing PCR. The amplified products are separated
on sequencing gels or even ordinary PAGE and visualized by silver staining. Alternatively, the
primers are labeled either by radioisotope or fluorescent dye so that the AFLP profile can be
obtained by autoradiography or by using image analysis. The highest number of amplified
products (50-100) is produced in AFLP among all the DNA profiling systems. This increases the
probability of detecting polymorphism many folds. The technique is, at present, lengthier and
costlier than other PCR based techniques. It requires good quality DNA for ensuring complete
digestion by enzymes. Partial digestion of DNA results in non-reproducible variation in DNA
profiles.
2.6 SNP
Molecular markers are polymorphic when there is DNA sequence variation between the individuals
under study. Molecular markers are, therefore, simply an indicator of sequence polymorphism.
Sequence polymorphism between individuals can take many forms, for instance, it can be due
to the insertion or deletion of multiple bases, or it can be due to single nucleotide polymorphisms
(SNPs; [10]). Insertions, deletions and SNPs are important in determining sequence variation
between individuals. SNPs are abundant in plant genomes. They are being used for genotyping
human populations for certain genetic diseases. The cost of developing SNPs is very high, since
for each locus DNA has to be sequenced and suitable PCR primers designed. The primers must
then be used to amplify the corresponding fragment from all other possible genotypes. These
fragments must then be sequenced and the sequences compared with one another to determine
the SNPs for each haplotype [11]. The term ‘haplotype’ is used in the context of SNPs instead
of the term ‘allele’. There are number of methods for identifying SNPs within a genetic locus
namely direct sequencing, single-strand conformation polymorphism (SSCP), chemical cleavage
of mismatches (CCM) and enzyme mismatch cleavage (EMC).
3.
Molecular Mapping of Genes of Agricultural Importance
Earlier, construction of genetic linkage maps using morphological markers could not be initiated
in most crop plants due to lack of sufficient number of molecular markers. Map construction was
highly laborious, took many years and required several mapping populations since all the
morphological markers could not be obtained in a single cross. These maps contained limited
number of markers and, therefore, could not be used for efficient mapping of target genes. With
the availability of a large number of molecular markers, like RFLP, RAPD, AFLP, microsatellites,
etc. saturation mapping of plant genomes has become a reality. Molecular genome maps have
been constructed in almost all important crop plants. The number of markers employed to
construct these maps and marker density varies greatly. Most of these maps are based on RFLP
markers. The recent mapping efforts have included mostly the PCR based markers such as
AFLP, STMS, RAPD, CAPS, SCAR and STS. Among the crop plants, the rice genome map is
considered most saturated. The map reported by Harushima et al. [12] contained the maximum
number of markers (2275). Significantly, this map was made using a single F2 population.
Recently, this map has been further saturated by combining additional STS and STMS markers
[13]. In most of the crop plants F2 population has been used since it could be generated in the
shortest possible time with the least effort. However, for mapping genes, particularly those for
292
VARSHNEY, MOHAPATRA AND SHARMA
Table 1.
Crop
Molecular mapping of agriculturally important genes in crop plants
Pathogen/Trait
1. Disease resistance
Rice
Pyricularia oryzae
Pyricularia grisea
Xanthomonas oryzae pv.
oryzae (Bacterial blight)
Rice yellow mottle virus
Rice stripe
Tungro
Puccinia striiformis f.
sp. tritici
Rhizoctonia solani Kuhn
Wheat
Erysiphe graminis
p.v. tritici
Gene
Marker(s)
Reference
Pi-2,4
Pi11
Pi-5(t), Pi–7(t)
Pi-Z–6
Pi-10
Pi-12(t)
Pi-18(t)
Pib
Pikm
Pita-2, Pita
Pi-5 (t)
Pi20
Pi44
Pb1
Pi-1(t)
QTL (1)
Xa-1, Xa-3, Xa-4
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
AFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
[18]
[139]
[140]
[141]
[142]
[143]
[144]
[145]
[146]
[147]
[148]
[149]
[150]
[151]
[152]
[153]
[20]
Xa-5
Xa-13
Xa-21
Xa-1
Xa3, Xa4, Xa5, Xa10
Xa13
Xa22(t)
Xa-1
Xa 23 (t)
RYMV (QTL)
RYMV
Stv-bi
RTSV
Yr5
RFLP
RFLP
RFLP
RAPD
RFLP
RFLP
RFLP
RAPD
SSR
RFLP
RFLP & STS
RFLP
RFLP
RGA
[19]
[154]
[155]
[156]
[156]
[157]
[158]
[159]
[160]
[161]
[162]
[163]
[164]
[165]
Rsb1
RFLP, RAPD,
AFLP, SSR
[166]
Pm1, Pm2, Pm3b,
Pm4a
Pm1, Pm2
Pm2
Pm3b, Pm4a
Pm2
Pm1
Pm12
Pm21
Pm
Pm4b
RFLP
[167]
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RAPD
RFLP
AFLP
[168]
[169]
[170]
[171]
[172]
[173]
[174]
[175]
[176]
Molecular Mapping and Marker Assisted Selection of Traits 293
Crop
Pathogen/Trait
Gene
Pm4a & Pm4b, Pm6
Pm13
Adult plant resistance to
powdery mildew
Common bunt
Karnal bunt
Durable stem rust
Puccinia recondite
Stripe rust
Loose smut
Septoria nodorum
Septoria tritici
Fusarium head blight
Yellow rust
MlG
APR
Bt-10
Bt-11
KB
Sr2
Sr2
Sr2
Sr22
Lr9
Lr18
Lr1
Lr9
Lr 19
Lr24
Lr24
Lr 29
Lr32
Lr34
Lr24
Lr10
Lr10
Lr23
Lr27
Lr31
Lr34
Lr34
Lr13
Lr35
Lr28
Lr3
Lr3
Yr15
Yr15
YrH52
T19
T10
–
–
–
–
YrMoro
Marker(s)
STS, RFLP
RFLP, RAPD,
STS, DDRT-PCR
SSR
STMS, RFLP
RAPD
RAPD
RFLP
RFLP
STS
RFLP
RFLP
RFLP, RAPD
N-band
RFLP
RFLP
RFLP
RFLP
RAPD
RAPD
RFLP
RFLP
RAPD, SCAR
STS
RFLP
RFLP
RFLP
RFLP
RFLP, RAPD
RFLP
RFLP, STMS
PCR
RAPD, STS
RFLP
mRNA fingerprinting,
cDNA cloning
RFLP
RAPD, STMS
STMS, RFLP
Monoclonal
antibody
RAPD, RFLP
RAPD
AFLP
AFLP, RFLP
AFLP
AFLP, STS
Reference
[177]
[178]
[179]
[180]
[181]
[182]
[183]
[169]
[184]
[185]
[186]
[187]
[188]
[189]
[190]
[190]
[190]
[191]
[192]
[190]
[169]
[193]
[194]
[195]
[195]
[195]
[195]
[195]
[196]
[197]
[197]
[37]
[198]
[199]
[200]
[201]
[40]
[202]
[203]
[204]
[205]
[206]
[207]
[208]
(Contd)
294
VARSHNEY, MOHAPATRA AND SHARMA
Table 1. (Contd)
Crop
Pathogen/Trait
Gene
Marker(s)
Reference
Tilletia indica
Wheat streak mosaic virus
QTL (1)
Wsm1
SSR, AFLP
STS, RAPD
[209]
[210]
Maize
Heliminthosporium turcicum
Maize dwarf mosaic virus
Cercospora zeamaydis
Maize streak virus
Maize mosaic virus
Maize stripe virus
Sugarcane mosaic virus
Ht1
mdm1
QTL (>10)
QTL (1)
QTL (1)
QTL (1)
Scm1
Scmv1, Scmv2
Scmv1, Scmv2
RFLP
RFLP
–
RFLP
RFLP
RFLP
RFLP, SSR
RGA-CAPs
AFLP, SSR
[211]
[212]
[213]
[214]
[215]
[216]
[217]
[218]
[219]
Barley
Erysiphe graminis
QTL (2)
Rar1
Rrs 13
Rph Q
QTL-Rphq (6)
Rph7.g
QTL (3)
RFLP
AFLP
RFLP
RAPD
AFLP
RFLP
–
[220]
[221]
[222]
[223]
[224]
[225]
[226]
QTL (1)
–
[227]
Yd2
AFLP
[228]
rym5
CAPs, SSR
[229]
Vhv1
QTL (2)
AFLP
–
[230]
[231]
Rynchosporium secalis
Puccinia hordei
Xanthomonas campestris
pv. hordei
Puccinia striiformis
f.sp. hordei
Barley yellow dwarf
Luteovirus
Barley yellow mosaic
virus
Cochliobolus sativus
Pyrenophora graminea
Sorghum
Sporisorium reilianum
Shs
RFLP/RAPD
[232]
Tomato
Stemphylium vesicarum
Cladosporium fulvum
Fusarium oxysporum
Pseudomonas syringae
Leveillula tourica
Verticillium dahliae
Sm
cfa
I2
Pto
Lv
Ve
Ve
Mi
QTL (3)
Ol-1
QTL (1)
RFLP
RFLP
RFLP
RFLP
RAPD/RFLP
RAPD
RFLP
RFLP
RFLP, RAPD, SCAR
RFLP, RGA
[233]
[234]
[235]
[236]
[237]
[238]
[239]
[240]
[241]
[242]
[89]
Rx1, Rx2
Nb
QTL (11)
R2
Nxphu
Ryadg
RFLP
AFLP
RFLP
AFLP
RFLP
RFLP
[243]
[244]
[97]
[99]
[245]
[246]
Medoidogyne sp.
Psuedomonas solanacearum
Oidium lycopersicum
Alternaria solani
Potato
Potato virus X
Phytophthora infestans
Potato X potexvirus
Potato Y potyvirus
Molecular Mapping and Marker Assisted Selection of Traits 295
Crop
Pathogen/Trait
Gene
Marker(s)
Reference
Soybean
Phytophthora sojai
Soybean mosaic virus
Pseudomonas syringae
pv. glycinea
Rps 1
Rsv
Rpg 1
RFLP
RFLP/SSR
RFLP
[247]
[248]
[249]
Common
bean
Uromyces appendiculatus
PI 181996
Ur-9, Fin
I
QTL (7)
Co-42, Co-7
RAPD
RAPD
RAPD
RFLP
RAPD, SCAR
[250]
[251]
[252]
[253]
[254]
Potyvirus
Xanthomonas campestris
Colletotrichum lindemut
hianum
Pea
Pea seed borne mosaic virus
Ascochyta pisi
sbm-1
QTL (3)
RFLP
RFLP
[255]
[256]
Tobacco
Chalara elegans
Brr
RAPD
[257]
Apple
Venturia inaequalis
Vf
Vf
RAPD
AFLP & SCAR
[258]
[259]
Melon
Fusarium sp.
Form 2
RAPD
[260]
Mungbean
Erysiphe polygoni
QTL (3)
RFLP
[261]
Cocoa
Phytophthora palmivora
QTL (5)
AFLP
[262]
Oil palm
Fusarium sp.
QTL (1)
SSR & AFLP
[263]
Rubber
Microcyclus ulei
Phyllochora herberi
QTL (8)
Phr
–
Isozyme
[264]
[265]
Sugarcane
Puccinia melanocephala
–
RFLP
[266]
Brassica
Leptosphaeria maculans
(Desm.) Ces.et de Not
Plasmodiophora brassicae
Sclerotinia sclerotiorum
QTL (10)
–
[267]
Pb-Bn1, QTL (2)
QTL (1)
–
RFLP, AFLP,
SSR, RAPD
[268]
[269]
Chick pea
Fusarium sp.
Race 4
ISSR (Inter-Simple
Sequence repeat)
[270]
Pearl millet
Puccinia substriata var. indica Rr1
RAPD,RFLP
[271]
Rose
Diplocarpon
Rdr1
RAPD, AFLP
[272]
Grape
Powdery mildew
Run1
AFLP
[273]
Pepper
Potato virus Y
Pvr4
RAPD, SCAR
[274]
Cassava
Cassava Mosaic Virus
CMD2
SSR, RFLP
[275]
Rye
Rust
Lr26
Sr31
Yr9
SrR
AFLP, RGA,
STS
[276]
Banana
Banana Streak Virus
–
AFLP
[277]
Tobacco
Ralstonia solanacearum
QTL (1)
AFLP
[278]
Lentil
Colletotrichum truncatum
LCt-2
RAPD, AFLP
[279]
(Contd)
296
VARSHNEY, MOHAPATRA AND SHARMA
Table 1. (Contd)
Crop
Pathogen/Trait
2. Nematode and insect resistance
Potato
Globodera rostochiensis
Globodera rostochiensis,
G. pallida
G. pallida
Gene
Marker(s)
Reference
QTL (2)
Grp1
–
[280]
AFLP, CAPs & RFLP [281]
QTL (1)
QTL (1)
AFLP, SSR
AFLP
[282]
[283]
Tomato
Globodera rostochiensis
Meloidogyne spp.
Hero
Mi-1
SSR
RFLP
[284]
[285]
Sorghum
Head bug
Schizaphids graminum
B2/b2
QTL (1)
RFLP, SSR
RAPD, SSR
[286]
[287]
Soybean
Helicoverpa zea Boddie
Heterodera glycines Ichinohe
QTL (1)
QTL (1)
RFLP
RFLP
[77]
[288]
Wheat
Diuraphis noxia Mordvilko
Dn2
RAPD, SCAR
Dn2, Dn4
RFLP
Dn4
SSR
Dn6
H23, H24
RFLP
H3, H5, H6, H9- H17 RAPD
H21
RAPD
H6
RAPD, STS
Rlnn1
AFLP, RFLP
Cre1
RFLP
[289]
[290]
[291]
Cre1
Ccn-D1
RFLP
RAPD, RFLP
[127]
[299]
Hessian fly
Pratylenchus neglectus
Cereal cyst nematode
resistance
[292]
[293, 294]
[295]
[296]
[297]
[298]
Maize
Ostrinia nubilalis
QTL (1)
RFLP, SSR
[300]
Rice
Orseolia oryzae (Gall midge)
Gm2
Gm4(t)
Gm7
Bph1
Bph10
Bph(t)
GLH
Grlp3
Grlp11
Grh1
WBPH
WBPH
RFLP
RFLP
AFLP, SCAR
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
[14]
[118]
[301]
[302]
[303]
[23]
[17]
[304]
[304]
[305]
[306]
[307]
Sd-1
AFLP, SSR, RFLP
[308]
Sub1
Salt
OSA3
RFLP
RFLP
RFLP
[24]
[25]
[26]
Brown planthopper
Green leafhopper
Whitebacked planthopper
Apple
Dysaphis devecta Wlk.
3. Abiotic stresses
Rice
Submergence tolerance
Salt tolerance
Molecular Mapping and Marker Assisted Selection of Traits 297
Crop
Pathogen/Trait
Gene
Marker(s)
Reference
Phosphorus uptake
Aluminium tolerance
QTL (1) (Pup1)
QTL (1)
RFLP
–
[27]
[28]
Thermosensitive earliness
per se
Eps-Am1 (QTL)
RFLP
[309]
Aluminium tolerance
Alt2
AltBH
RFLP
RFLP
[310]
[311]
Tolerance to salt stress
Kna1
Protein poly
morphism
[312]
Aluminium tolerance
Alt (QTL)
AFLP, SSR
[313]
4. Male sterility, wide compatibility and fertility restoration
Petunia
Restorer of fertility
Rf
RAPD, AFLP
[314]
Rice
tgms1.2
RFLP
[29]
Hybrid breakdown
Wide compatibility
tms2
tms3
tgms
tgms-vn1 (tms4)
pms1
pms2
pms3
ms-h(t)
Rf-1
Rf?
Rf2
Rf3
Rf5
Rfu
Rf?
Hwd1, hwd2
S5
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
[30]
[31]
[32]
[33]
[315]
[315]
[316]
[317]
[318]
[319]
[320]
[321]
[322]
[323]
[114]
[324]
[325]
Wheat
Fertility restoration
Rf4, Rf3
RFLP
[326]
Rye
CMS
Self-fertility
Rfg1
S1Z1S5
RFLP, RAPD
Isozyme, RFLP
[227]
[228]
Brassica
CMS restorer
Rfp1
RFLP, RAPD
[229]
Sorghum
Fertility restorer
rf4 (QTL)
AFLP
[330]
Sunflower
Fertility restoration
Rf1
RAPD, AFLP, SCAR
[331]
Cotton
CMS fertility restoration
Rf1
RAPD, SSR
[332]
Coffee
Pollen viability restoration
QTL (3)
AFLP
[333]
5. Grain quality
Sorghum
Grain quality and yield
components
QTL (6)
RFLP, AFLP & SSR
[334]
Rice
Fgr
RFLP
[34]
Wheat
Barley
Male sterility and fertility
restoration
Grain aroma
(Contd)
298
VARSHNEY, MOHAPATRA AND SHARMA
Table 1.
Crop
Wheat
Pathogen/Trait
Gene
Marker(s)
Reference
Cooked-kernel elongation
Amylose
KNE
Wx
RFLP
RFLP
[35]
[335]
Flour colour
Grain yield
Red grain colour
High molecular weight
glutanin
Grain protein content
QTL (1)
QTL (1)
R3, R1
Glu-D1
RFLP, AFLP
RFLP
RFLP
PCR-based
[336]
[337]
[169]
[338]
Bread making quality
Amylose content
QTL (1)
QTL (1)
ha
ha
Glu-D1(1Dx5)
Wx-B1
STMS, RFLP
SSR
RFLP
RFLP
PCR
RFLP
[44]
[339]
[169]
[340]
[341]
[342]
Grain oil content
QTL(2)
AFLP, SSR
[343]
QTL (1)
RAPD
[110]
EgHypar and
EgTub A1
SSCP (Single Strand
Confirnation
Polymorphism)
[344]
[263]
Kernel hardness
Sunflower
(Contd)
6. Yield, its components and other traits
Eucalyptus
Wood density, stem growth
and stem form
Lignification genes
Oil palm
Fruit morphology and fertility Sh
AFLP
Carnation
Flower type
QTL (1)
RAPD, SCAR & RFLP [345]
Soybean
Specific leaf weight and
leaf size
Stearic acid content
QTL (1)
RFLP
[346]
Fas
SSR
[80]
Plant height
Rht-B1
Rht-D1
Rht12
Rht8
Rht-B1, Rht-D1
Rht-B1, Rht-D1
QTL (1)
QTL (1)
Rht-B1b (Rht-1)–
RFLP
STMS, RFLP
SSR
RFLP
RFLP
AFLP
AFLP
PCR-based
[347]
[43]
[41]
[348]
[347]
[349]
[349]
[350]
QTL (1)
RFLP
[351]
QTL (1)
RFLP
[352]
Major gene
Vrn1
Vrn1
Vrn1
Vrn-Am1, Vrn-Am2
Vrn-D1
STMS, STS
RFLP
RFLP
RFLP
RFLP
STMS
[45]
[353]
[169]
[43]
[354]
[355]
Wheat
Dwarfing genes
Haploid formation
Green plant formation
Semi-dwarfing genes
Rht-D1b (Rht2)
Ear emergence time,
plant height
Preharvest sprouting
tolerance
Vernalization response
Molecular Mapping and Marker Assisted Selection of Traits 299
Crop
Pathogen/Trait
Gene
Marker(s)
Reference
Cadmium uptake
ABA production and response
Coleoptile pigmentation
Milling yield
Eyespot
Tan spot
Na+/K+ discrimination
Cdu1
–
Rc1
–
Pch2
–
–
RAPD
RFLP
RFLP
RFLP, STMS
RFLP
RFLP
RFLP
[356]
[357]
[169]
[358]
[359, 360]
[361]
[362]
Apple
Growth and development
in juvenile apple trees
QTL (1)
RAPD
[363]
Potato
For foliar glycoalkaloid
and aglycones
QTL (1)
RFLP
[364]
Peach
Fruit quality
QTL (1)
Isozymes, RAPD,
RFLP, AFLP
[365]
Rose
Recurrent blooming, double
corolla, thorn density of the
shoots
QTL (1)
AFLP
[366]
Grape
Seedlessness, berry weight
QTL (1)
AFLP, SSR, isozyme,
RAPD, SCAR
[367]
Barley
Intermedium spike-C and
non-brittle rachis1
int-c
btr-1 (QTL)
AFLP
[368]
Pea
Rhizobium nodulation
sym9, sym 10
AFLP, RFLP
[369]
Sugarbeet
Sucrose content, yield and
quality
QTL (1)
RFLP, AFLP
[370]
Rice
Photoperiod sensitivity
Semidwarf gene
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
RFLP
[371]
[122]
[372]
[122]
[373]
[374]
[375]
[376]
Yield
Root morphology
Se1
sd1
Sdg
Sh2
Sh4
Sht
QTL (5)
QTL-Hd-1,
Hd-2 & Hd-3
QTL (1)
QTL (1)
SSR, STS
–
[36]
[377]
Early height growth
QTL (1)
RAPD
[105]
Maize
Popping explosion volume
QTL (4)
SSR
[378]
Cotton
Fibre strength
QTL (2)
SSR, RAPD
[379]
Sunflower
Agronomic traits [grain
weight by plant (GWP),
100-grain weight (TGW),
percentage of oil in grain
(POG), sowing to flowering
date (STF)]
QTL (1) for TWP,
QTL (6) for POG,
QTL (2) for STF
AFLP, SSR
[341]
Shattering-resistance gene
Seed dormancy, heading date
Heading date
Pinus
palustris
Mill. × P.
elliottii Engl.
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VARSHNEY, MOHAPATRA AND SHARMA
quantitative traits, permanent mapping populations such as recombinant inbred lines (RILs) and
doubled haploids are preferred since they can be maintained over years by selfing and replicated
over locations and seasons.
Availability of molecular markers and saturated linkage maps has enabled mapping of genes
for qualitative as well as for quantitative traits. The qualitative traits that are controlled by one
gene show simple Mendelian pattern of monogenic inheritance such as genes controlling biotic
stresses, fruit flesh colour in peach, kernel colour in corn, flower colour in Petunia, etc. The
mapping of such genes with different molecular markers is listed in Table1. Quantitative traits
such as yield, drought and cold tolerance, wood density etc. that show continuous variation are
controlled by many genes. The individual genes controlling the expression of quantitative traits
are now called Quantitative Trait Loci (QTL). QTL with relatively strong effects are good targets
for marker-assisted selection especially if the trait is difficult to measure. Many QTL have
relatively small effects. These QTL are difficult to accurately map, especially with population
sizes typical of most mapping studies. So, now the population size for QTL mapping has
increased to about 300-500 individuals. However, in order to have an understanding of what the
gene does and how it interacts with other genes, it is useful to know where the genes are within
the genome and in relation to other genes of interest. The number of QTL controlling a trait
varies from 1 to more than 10 for different crop plants (Table 1). The progress in mapping of
genes of agricultural importance in some major crops is described here.
3.1 Rice
A large number of genes for qualitative and quantitative traits including disease resistance,
insect resistance, cooking quality, drought and flooding tolerance etc. have been mapped using
DNA markers as listed in Table1. Mapping of disease resistance genes are of major concern for
imparting stability to rice production. Few examples of mapping such genes using molecular
markers is described here. For instance, RFLP markers have been used to map gall midge
resistance gene Gm2 using recombinant inbred lines derived from a cross between ‘Phalguna’
(resistant variety) and ‘ARC6650’ (a susceptible land race) [14]. Another gall midge resistance
gene, Gm4t, which is non-allelic to Gm2 and is known to confer resistance against insect
biotypes 1, 2, 3 and 4 has also been tagged using RAPD in combination with bulk segregant
analysis of a F3 population [15]. Several of the putative resistance gene analogues (RGAs) have
been cloned, sequenced and found to be tightly linked to known disease resistance genes [16].
Genetic mapping of resistance to rice tungro spherical virus (RTSV) and green leaf hopper
(GLH) in ARC11554 was achieved using RAPD and RFLP markers [17]. Yu et al. [18] mapped
a major locus Pi-2(t) for resistance to blast caused by the fungus Magnaportha grisea using
RFLP markers. Several of the major genes to the bacterial leaf blight (BLB) pathogen, Xanthomonas
oryzae pv. oryzae, have been tagged with RFLP or RAPD markers [19–21]. Two microsatellite
markers tightly linked to BLB were located at approximately 2 and 18 cM from the xa5 locus
[22]. RFLP tagging of a gene for resistance to brown plant hopper (BPH) was reported by Mei
et al. [23].
Genes have also been mapped using RFLP markers for submergence tolerance [24], salt
tolerance [25 and 26], phosphorus uptake [27] and Al tolerance [28]. As far as male sterility and
fertility restoration is concerned, several reports have been available on mapping genes using
RFLP markers [29, 30–33]. Similarly, for traits like grain aroma, cooked kernel elongation,
Molecular Mapping and Marker Assisted Selection of Traits 301
genes have been mapped using RFLP markers [34, 35]. Recently, quantitative trait loci for yield
have been mapped using SSR and STS markers [36].
3.2 Wheat
Several reports on mapping of genes for various disease and insect resistance, abiotic stresses,
grain quality and other traits are listed in Table 1. Wheat rust disease is a major concern and
many successful results have been reported on gene mapping. A sequence-tagged-site (STS)
marker linked to Lr28, a wheat leaf rust resistance gene has been identified by Randomly
Amplified Polymorphic DNA (RAPD) analysis of near isogenic lines (NILs) of Lr28 in eight
varietal backgrounds. Of the 80 primers tested, one RAPD marker distinguished the NILs and
the donor parent from susceptible recurrent parent [37]. Comparisons between near isogenic
lines (NILs) and their recurrent parents have been useful for identifying molecular markers
linked to host genes showing resistance to pathogens. Inter-simple sequence repeat (ISSR primers)
markers for stem rust (SR39) and leaf rust (Lr 35) resistance genes have been developed by Gold
et al. [38], which would facilitate the transfer of these genes to elite wheat lines. Microsatellite
markers have been used for detecting DNA polymorphism in yellow rust-resistance accessions
of Triticum dicoccoides [39]. Nine microsatellite markers were identified to be linked to striperust resistance gene YrH52 [40]. Microsatellite markers have also been used to tag several genes
or QTL, including the genes Rht8 [41, 42], Rht12 and Vrn1 [43], and QGpc.ccsu.2D.1, a QTL
for grain protein content [44]. The problem of pre-harvest sprouting, particularly in amber
kernels, is quite common in major wheat growing regions of the world, including India. The
improvement in grain protein content and its composition in bread wheat is also a difficult task
and remains a major concern to plant breeders. The QTL for pre-harvest sprouting tolerance [45]
and grain protein content [44, 46] have been tagged using sequence tagged multiple sites (STMS)
and sequence tagged sites (STS) markers.
3.3 Brassica
Brassica juncea (Indian mustard), B. rapa (turnip rape) and B. napus (rapseed) are the major
oilseed Brassicas. In this group of crops, molecular markers have been employed for mapping
of genes primarily for disease resistance and oil and meal quality. Several efforts have been
made to identify markers for resistance to white rust caused by the fungus Albugo candida
(Pers.) Kuntze, which is a widespread and destructive disease in these crops with yield reductions
of 30-60% in severely infested fields [47]. A locus (ACA1) controlling resistance to A. candida
has been mapped in B. napus using RFLP markers [48]. A single locus controlling resistance to
AC2 in B. rapa was mapped using RFLP markers and a segregating population from Per (resistant
to both AC2 and AC7) × ‘R500’ (susceptible) [49]. A co-segregating RFLP marker (X140a) and
two closely linked RFLP markers (X42 and X83) were identified which were useful for MAS
and map based cloning of a single gene (Acr) responsible for conferring resistance to A. candida
in B. juncea [50]. Prabhu et al. [51] mapped a resistance gene (Ac2t) in B. juncea from a Russian
source imparting resistance to a predominant Canadian isolate of A. candida. B. juncea accession
BEC-144 from Poland shows resistance to the Indian isolates of the white rust pathogen.
Identification of two markers linked in coupling and repulsion phases flanking the gene controlling
resistance to A. candida in BEC-144 was reported by Mukherjee et al. [52]. This work has been
further extended to develop AFLP and CAPS markers for this gene. Moreover the CAPS marker
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VARSHNEY, MOHAPATRA AND SHARMA
has been validated in different populations revealing thereby its utility in marker assisted selection.
Kole et al. [53] mapped genes for resistance to white rust in B. rapa using a recombination
inbred population and a genetic linkage map consisting of 144 RFLP markers and 3 phenotypic
markers. Molecular markers have been generated for the genes conferring resistance to
Leptosphaeria maculans in B. napus by various workers. The resistance locus LmFr1 was linked
to markers cDNA 011 and cDNA 110 [54], and localized onto the linkage group 6 (LG6); [48].
Loci pb-3 and pb-4 conferring resistance to Plasmodiophora brassicae in B. oleracea were
identified and linked to RFLP and AFLP markers [55]. Similarly, Figdore et al. [56] also identified
markers 14a on LG1, marker 48 on LG4 and 177b on LG9 linked to clubroot resistance (resistance
to Plasmodiophora brassicae wor. Race 7) in B. oleracea.
A number of studies have been undertaken to generate markers for fatty acids such as linolenic
acid, linoleic acid, oleic acid, palmitic acid and erucic acid. Two RAPD markers, K-011100 and
25a were generated and linked to the linolenic acid concentration [57, 58]. RAPD markers
linked to oleic, linolenic and linoleic acids were identified in B. napus [59]. RAPD marker linked
to the linolenic acid content was converted to a co-dominant SCAR marker [59]. Markers linked
to genomic regions controlling linolenic acid concentration in B. napus corresponding to fad3
(omega-3-desaturase) gene in A. thaliana [60] were also identified [61-63]. In another study,
a single QTL containing 6 markers associated with oleic, palmitic and linoleic acid content was
detected in B. rapa [64]. Sharma et al. [65] recently mapped two major QTLs influencing oleic
acid level in B. juncea using both single factor analysis of variance and interval mapping. Erucic
acid loci have been linked to molecular markers by [66-68] using BSA or RFLP analysis in B.
napus. In each of the studies, two QTL were detected. These QTL have been positioned on LG 6
and LG12 [66] or on LG7 and LG15 [63]. In an independent study, two QTL associated with the
erucic acid level in B. napus were detected [67] and mapped onto two different loci termed as
E1 and E2. QTL E1 and E2 correspond to the two alleles of the β-ketoacyl-synthase (KCS)
derived from B. campestris and B. oleracea, the two parental species of B. napus and encode the
Fatty acid elongation 1 (Fae1) protein [69]. In B. rapa (Syn campestris) erucic acid loci were
linked to RFLP markers [70].
The seed coat colour gene has been tagged to various RFLP and RAPD markers. The RFLP
markers linked to seed coat colour in B. napus were identified using the Bulked Segregant
Analysis (BSA) approach [71]. Seed coat colour trait in B. campestris was tagged with RAPD
markers using B. campestris-oleracea additional lines [72]. A 3:1 ratio of segregation of
brown : yellow seed in B. rapa indicated a monogenic control of this trait and was mapped to
LG5 [70]. Upadhyay et al. [73] studied segregation of the trait in an F2 population of B. juncea
and reported duplicate dominant gene action giving a phenotypic ratio 15:1. Two RFLP markers
flanking one of the interacting loci were identified. In a recent report, the seed coat colour trait
was tagged using a combined approach of BSA and AFLP in B. juncea [74].
3.4 Soybean
In soybean, emphasis is laid on genetic mapping of pest and disease resistance genes. Apart from
that quantitative traits such as oil quality, plant height, sprout yield etc. have been characterized
using molecular maps. The soybean cyst nematode (SCN) (Heterodera glycines Inchinoe) is the
most economically significant soybean pest. Two SSR markers BARC-Satt 309 and BARC-Satt
168 have been reported that segregate and map 0.4 cM from rhg1 [75]. When these markers were
Molecular Mapping and Marker Assisted Selection of Traits 303
used to assay lines from SCN-susceptible × SCN - resistant crosses, they proved to be highly
effective in identifying lines carrying rhg1 resistance from those carrying the allele for SCN
susceptibility at the rhg1 locus. In another study, field resistance to SCN race 3 in soybean cv.
Forrest was found conditioned by two QTLs. The underlying genes are presumed to include rhg1
on linkage group G and rgh4 on linkage group A2. A high density map for the intervals carrying
rhg1 and rhg4 have been developed using AFLP markers. A12-way analysis of variance showed
two loci controlling SCN resistance in Essex × Forrest RILs [76]. Using 139 RFLPs QTLs
associated with resistance to corn earworm (Helicoverpa zea Boddie) were identified [77]. With
the help of AFLP, four markers closely linked to soybean mosaic virus resistance gene, Rsv1 was
mapped, thus demonstrating the utility of genetic mapping for generating markers tightly linked
to important plant disease resistance genes [78]. Soybean death syndrome (SDS) caused by
Fusarium solani f. sp. glycines results severe yield losses. Two QTLs for resistance to SDS were
mapped in cv. Pyramid using SSR markers namely, BARC-Satt 163 and BARC-Satt 080. Similarly,
a QTL was identified from cv. Douglas using SSR marker BARC-Satt 307. Njiti et al. [79]
suggested that gene pyramiding would be an effective method for developing cultivars with
stable resistance to SDS.
Increasing the stearic acid content to improve soybean oil quality is a desirable breeding
objective for food processing applications. Three SSR markers, Satt 070, Satt 474 and Satt 556
were identified to be associated with stearic acid content by Spencer et al. [80]. Identification of
these markers may be useful in molecular marker-assisted breeding programmes targeting
modifications in soybean fatty acids. RFLP markers have also been used to identify QTLs
associated with plant height, lodging and maturity. The major locus associated with plant height
was identified as Dt1 on LG L. Dt1 was also associated with lodging. In addition, with the help
of RFLP markers, two QTLs for plant height (K007 on LG H and A516b on LG N) and one QTL
for lodging (cr517 on LG J) were identified. For maturity, independent QTLs were identified in
intervals between R051 and N100, and between B032 and CpTI on LG K [81]. RFLP markers
were also used for identifying QTLs associated with soybean sprout-related traits. Four QTLs
were associated with sprout-yield in the combined analysis done for two years by Lee et al. [82].
They also found that the QTLs conditioning sprout yield were in the same genomic locations as
the QTLs for seed weight. These data demonstrates MAS may be feasible for enhancing sproutyield in soybean.
3.5
Pea
Aphanomyces root rot, caused by Aphanomyces euteiches Drechs, is the most important disease
of pea worldwide. No efficient chemicals are available to control the pathogen. Thus, to facilitate
breeding for Aphanomyces root rot resistance and to better understand the inheritance of partial
resistance [83], identified QTLs associated with the disease using DNA markers AFLPs, RFLPs,
SSRs, ISSRs and STS. The resulting genetic map consisted of 324 linked markers distributed
over 13 linkage groups covering 1,094 cM. A total of seven genomic regions were associated
with Aphanomyces root rot resistance. The first one was named as Aph1, which was considered
a major QTL. Two other specific QTLs, namely Aph2 and Aph3 were identified, which were
mapped near the r (wrinkled/round seeds) and af (normal afila leaves) genes. Four other minor
QTLs were identified. The resistant alleles of Aph3 and the two minor QTLs were derived from
the susceptible parent. RAPD and SACR markers linked to genes affecting plant architecture of
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VARSHNEY, MOHAPATRA AND SHARMA
pea, namely, three ramosus genes (rms2, rms3 and rms4) and two genes conferring flowering
response to photoperiod (sn and dne) have been reported by Rameau et al. [84]. In another study,
QTLs affecting seed weight in pea were mapped using RFLP markers. Four QTLs were identified
in marker intervals on three different linkage groups [85].
3.6 Chickpea
Ascochyta blight is an economically important disease of chickpea caused by the fungus Ascochyta
rabiei. Udupa and Baum [86] identified and mapped a major locus (ar1) using SSRs, which
confers resistance to pathotype I, and two independent recessive major loci (ar2a), with
complementary gene action conferring resistance to pathotype II. In another study, integration of
co-dominant STMS markers improved the mapping of ascochyta resistance in chickpea [87].
Resistance gene analogs (RGAs) of Cicer were isolated by different PCR approaches and mapped
in an inter-specific cross, segregating for Fusarium wilt by RFLP and CAPS markers. A total of
13 different RGAs were isolated and classified into nine distinct classes. This study by Huettel
et al. [88] provides a starting point for the characterization and genetic mapping of candidate
resistance genes in Cicer that is useful for MAS and as a pool for resistance genes of Cicer.
3.7 Tomato
Tomato is an important vegetable crop. Extensive work has been done on genetic mapping of
agriculturally important genes in this crop. Early blight (EB) caused by a devastating fungus,
Alternaria solani Sorauer causes plant defoliation, reduces yield and fruit quality, and contributes
to significant crop loss. QTL mapping using 14 RFLP markers and 23 RGAs identified 10
significant QTLs for EB by Foolad et al. [89]. Potato virus Y (PVY) is also an important disease
causing organism which affects the yield of tomato. Resistance against PVY was identified in
the wild tomato relative Lycopersicon hirsutum PI247087. The locus pot-1 was mapped using
AFLP markers to the short arm of tomato chromosome 3, in the vicinity of the recessive py-1
locus for resistance to corky root rot [90]. Another important pathogen, cucumber mosaic virus
(CMV) gene, Cmr was mapped using RFLP and isozyme markers in L. chilense and was located
on chromosome 12. The chromosome 12 markers were found to be significantly associated with
CMV resistance in both qualitative and quantitative models of inheritance. This knowledge of
the map location of Cmr should accelerate introgression by marker-assisted selection [91].
Identification of tightly linked markers for the genes of importance has facilitated isolation of
genes from tomato. For instance, map based cloning strategy was designed to isolate the rootknot nematode resistance gene Mi in tomato using PCR-based flanking markers. Fine structure
mapping of recombinants with newly developed AFLP and RFLP markers from physically
mapped cosmid subclones localized Mi to a genomic region of about 550 kb [92]. Two recessive
mutations have been discovered in tomato that completely suppress the formation of flower and
fruit pedicel abscission zones, i.e. jointless ( j) and jointless-2 ( j-2). Both the genes were
tentatively localized to chromosome 11 about 30 cM apart. However, RFLP and RAPD markers
helped in correctly identifying and mapping the j-2 locus on chromosome 12 instead of chromosome
11 [93] that enabled map-based cloning of this gene.
Improving organoleptic quality is an important but complex goal for fresh market tomato
breeders. A total of 26 traits involved in organoleptic quality variation were evaluated. Physical
traits included fruit weight, diameter, colour, firmness and elasticity. Chemical traits were dry
Molecular Mapping and Marker Assisted Selection of Traits 305
matter weight, titratable acidity, pH, and the contents of soluble solids, sugars, lycopene, carotene
and 12 aroma volatiles. A total of 81 significant QTLs were detected for the 26 traits using DNA
markers [94]. RFLP mapping of 32 independent tomato loci corresponding to genes known to
influence fruit ripening and/or ethylene response was reported by Giovannoni et al. [95]. The
placement of ripening and ethylene-response loci on the tomato RFLP map would facilitate both
the identification of candidate gene sequences corresponding to identified single gene and QTL
contributing to fruit development and ethylene response.
3.8 Potato
In vegetable crops like potato, genetic mapping has been done primarily on disease resistance.
Phytophthora infestans is a very devastating fungus causing late blight of potato. Eleven resistance
alleles (R1-R11) are known which confer race-specific resistance to this fungus. In two of the
reports, R6 and R7 alleles were mapped by RFLP markers on chromosome XI similar to R3
allele [96] and R2 allele was mapped using AFLP marker [97]. A study on mapping of the
resistance gene of root knot nematode (Meloidogyne chitwoodi) derived from Solanum
bulbocastanum in a BC2 population using RFLP markers have been reported by Brown et al.
[98]. RFLP mapping has also been carried out for the potato virus X controlled by a single gene,
Nxphu. Four RFLP markers CT220, TG328, CT112 and TG424 from the long arm of chromosome
IX that were linked to the hypersensitive phenotype have been reported by Tommiska et al. [99].
Mapping of QTL for resistance to potato cyst nematode (Globodera rostochiensis) has been
reported by several researchers. In one of the studies, the nematode resistance locus Gpa2 was
mapped on chromosome 12 of potato using 733 AFLP markers. This study also showed that
Gpa2 is linked to the Rx1 locus conferring resistance to potato virus X [100]. Linkage maps
using AFLP and RFLP markers were constructed and used to identify three QTLs on chromosomes
V, VI and XII, respectively, for resistance against the potato cyst nematode [101]. In a recent
study by Baker at al. [102], nine resistance gene homologues (RGHs) were identified in two
diploid clones of potato with a specific primer pair based on conserved motifs in the LRR
domain of the potato cyst nematode resistance gene Gpa2 and the potato virus X resistance gene
Rx1. AFLP marker was used to facilitate the genetic mapping of the RGHs in the four haplotypes
under investigation.
3.9 Sugarcane
Sugarcane is an important cash crop and in order to analyse the inheritance of quantitative traits,
extensive study on Quantitative Trait Allele (QTA) mapping was done. The first extensive QTL
mapping study performed in cultivated sugarcane was reported by Hoarau et al. [103] based on
a population of 295 progenies derived from the selfing of cultivar R570, using about 1,000 AFLP
markers. The population was evaluated in a replicated trail for four basic yield components,
plant height, stalk number, stalk diameter and brix, in two successive crop-cycles. Forty putative
QTAs were found for the four traits of which five appeared in both years. In another study,
mapping of QTLs for sugar yield and related tarits, namely pol, stalk weight, stalk number, fiber
content and ash content were done using 735 DNA markers. Fifty of the 61 mapped QTLs were
clustered in 12 genomic regions of seven sugarcane homologous groups [104].
3.10 Forest Trees
Molecular markers have been successfully applied in tree species also which may be incorporated
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into existing improvement programmes in an efficient and cost efficient manner. In loblolly
pine, marker-trait associations for components of radial wood density profiles had been found
and verification populations have been established to confirm these associations. RAPD markers
were employed to map the genome and quantitative trait loci controlling the early growth of a
pine hybrid F1 tree (Pinus palustris Mill. × P. elliottii Engl.) and a recurrent slash pine tree (P.
elliottii Engl.) in a (long leaf pine × slash pine) × slash pine BC1 family consisting of 258
progeny [105]. With the help of RFLP markers 13 different height increment and eight different
diameter-increment QTLs were detected in loblolly pine by Kaya et al. [106]. Similarly, chemical
wood property traits were analysed for the presence of QTLs in a three-generation outbred
pedigree of loblolly pine (Pinus taeda L.) using DNA markers [107]. Kumar et al. [108] reported
multiple-marker mapping of wood density loci in an outbred pedigree of radiata pine using DNA
markers. The effect of locations of QTL was found to be significantly associated with the
expression of wood density at different ages. These results are encouraging for the application
of marker information to early selection in order to increase juvenile wood density.
The single dominant gene (R) that confers resistance to the white pine blister rust fungus
(Cronartium ribicola Fisch.) in Pinus lambertiana Dougl. has been mapped using RAPD markers.
Thirteen RAPD loci were identified by Harkins et al. [109] that were linked to R. This would
help in subsequent high-resolution mapping experiments to identify very tightly linked markers
to facilitate the eventual cloning of R.
RAPD markers have been used to determine the genetic location and effects of genomic
regions controlling wood density, stem growth and stem formation in Eucalyptus [110]. A total
of 86 and 92 markers distributed among 11 linkage groups covered 1295 cM and 1312 cM for
E. urophylla and E. grandis, respectively. This application of marker information will help in
early selection of hybrid trees to be vegetatively propagated for the production of clonal varieties.
4.
Marker-Assisted Selection (MAS)
Molecular marker-assisted selection involves scoring for the presence or absence of a desired
plant phenotype indirectly based on DNA banding pattern of linked markers on a gel or on
autoradiogram depending on the marker system. The rationale is that the banding pattern revealing
parental origin of the bands in segregants at a given marker locus indicates presence or absence
of a specific chromosomal segment which carries the desired allele. This increases the screening
efficiency in breeding programmes in a number of ways such that:
(a) the segregants can be scored at the seedling stage for traits that are expressed late in plant
development. This includes traits such as grain quality, male sterility and photoperiod
sensitivity.
(b) it is possible to screen for traits that are extremely difficult, expensive or time consuming
to score and measure such as tolerance to drought, salt, mineral deficiencies and toxicity,
root morphology, resistance to nematodes or to specific races or biotypes of diseases or
insects.
(c) selection can be practiced for several traits simultaneously, which is difficult or even
impossible by conventional means.
(d) heterozygotes are easily identified and distinguished from either homozygotes without
resorting to progeny testing. This saves time and effort.
Molecular Mapping and Marker Assisted Selection of Traits 307
MAS is an attractive option for improvement of certain traits of interest for which phenotypic
evaluation is often expensive or unreliable. MAS provides a potential for increasing selection
efficiency by allowing earlier selection and reducing plant population size during selection.
Breeders can rapidly determine inheritance patterns at the genomic level by directly examining
the genetic make up of experimental plants when they are still seedlings. This is especially
useful for traits that cannot be identified until the plant is mature such as fruit characteristics and
for traits that are difficult to test such as disease resistance. Resistant plants are selected based
on DNA markers that are linked to the gene(s) controlling the trait, instead of actually evaluating
the disease resistance of the plants. Incorporating natural resistance genes into varieties is the
most effective, economic and environmentally safe means of controlling the disease. This is the
response to the demand for cost-effective, “green” solutions since it eliminates the need for
expensive chemicals to control diseases. It is a uniform method of scoring, tells percentage of
genome from each parent, and tells which parts of each chromosome come from each parent. In
addition to that as the precision in selection is increased, less unwanted side effects appear in the
following generation of plants. MAS can also be used to pyramid two or more desirable genes
in a new plant variety.
4.1 Some MAS Advantages in Backcrossing Breeding
There are cases where many conventional backcross programmes fail. For example, despite
carefully made backcrosses to the recurrent parent, progeny derived exclusively from selfpollination due to failure of crossing, have been found in backcross programmes. Hence, in
conventional breeding, breeders are often not working on the genetic material that they assume
they are, because crossing fails more often than expected. A reason why backcrossing fails is the
misclassification of a plant for the presence of the donor gene (disease escape instead of disease
resistance) and is used as a parent in further backcrossing. All these problems and others like
need to make time and resource consuming selfed generations to identify a recessively controlled
character are avoided in marker assisted selection.
4.2 MAS Status in Different Crops
Enormous work is being carried out on marker-assisted selection in India and abroad. The
progress made in some major crops is presented here.
Rice
The ongoing significant efforts on marker-assisted breeding and gene pyramiding in rice include
resistance to blast, blight, gall midge, dwarfing and also drought. Bacterial blight (BB) caused
by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most destructive diseases of rice throughout
the world and in some areas of Asia it can reduce crop yield by upto 50%. The most effective
approach to combat BB is the use of resistant varieties [111]. So far, 19 resistant genes have been
identified [112] and some of these have been incorporated into modern rice varieties. However,
the large-scale and long-term cultivation of varieties carrying one of the most important resistant
gene Xa-4 has resulted in significant shifts in the rice frequency of Xoo [113]. In many areas of
Indonesia, India, China and Philippines, rice varieties with only Xa-4 for defense against Xoo
have become susceptible to the pathogen. Thus, DNA marker- assisted selection was used to
pyramid four bacterial resistance genes, Xa-4, xa-5, xa-13 and Xa-21. Breeding lines with two,
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three and four resistance genes were developed and tested for resistance to bacterial blight
pathogen. The pyramid lines showed a wider spectrum and a higher level of resistance than lines
with only a single gene. To speed up the gene pyramiding process and to facilitate future markeraided selection, Huang et al. [114] developed PCR markers for two recessive genes xa-5 and xa13, and used these to survey a range of rice germplasm. The results of the germplasm survey will
be useful for the selection of parents in breeding programmes aimed at transferring these bacterial
blight resistance genes from one varietal background to another. In India, at Punjab Agricultural
University (PAU), Ludhiana, three BB resistance genes xa-5, xa-13 and Xa-21 were pyramided
in PR106 and Pusa 44 background. After multi-location and replicated testing, two PR106
pyramid lines were identified and these have been included in All India Coordinated Testing
during 2002. This is the first ever marker- assisted product reaching testing at national level.
Pusa 44 pyramid lines were tested in multi-location replicated trials during 2002 [115].
Blast caused by the fungus Magnaportha grisea is another devastating disease of rice. The
most economical and effective approach to reduce the yield loss is to breed varieties that are
resistant to the disease. However, the resistance often breaks down within a few years of cultivar
release. Many genes for qualitative blast resistance have been mapped using molecular markers
(Table 1) and some of those markers have also been tried in MAS for blast resistance. For
example, RG64, a RFLP marker on chromosome 6 is tightly linked (2.8 cM) to Pi-2(t), a major
gene for blast resistance [18]. The RG64 rice genome clone was sequenced and primers based
on the DNA sequences were found useful in producing polymorphism between the susceptible
and resistant varieties after the monomorphic PCR product was digested with restriction enzymes
[116]. The CAPS marker was then used to identify rice plants carrying Pi-2(t) from an F2
population derived from the cross between CO39 and CO10151. The effectiveness of the selection
for resistant plants based on linked DNA markers was then compared with phenotyping for blast
resistance through progeny testing in the F3 families by blast inoculation. Results indicated that
identification of plants carrying Pi-2(t) in a large segregating population is possible using one
linked marker as well as flanking markers. The accuracy of identification of homozygous resistant
genotypes was 96% when RG64 marker was used. The accuracy of selection increased to 100%
when two markers flanking the Pi-2(t) were scored simultaneously. These results illustrate that
marker-assisted identification of linked target gene in a segregating population is efficient in
identifying resistant genotypes [117]. This work has been extended futher to pyramid three
major genes for blast resistance.
Another objective of marker-assisted breeding in rice is to transfer resistance against gall
midge (Orseolia oryzae), a major insect pest of rice. The resistance to gall midge biotypes is
governed by single dominant genes. At national level, effort has been made to screen rice
germplasm for new sources for resistance genes effective against one or more biotypes of the
pest [118]. PCR based markers have been designed and currently are being used in markerassisted selection at different research institutions in the country.
The semi-dwarf gene (sd-1) in rice is one of the most important single genes in the history of
rice improvement. This single, recessive gene causes reduced culm length and has been widely
used to confer lodging resistance, high harvest index, responsiveness to nitrogen fertilizer, and
favourable plant type, in the breeding of high-yielding rice varieties [119]. In rice, molecular
mapping of sd-1 has been reported by several workers [120–122]. Chao et al. [123] used 20
mapped clones as probes, based on an existing rice RFLP map [124], and conducted experiments
Molecular Mapping and Marker Assisted Selection of Traits 309
to establish the location of sd-1 gene. They evaluated the efficacy of marker-assisted selection
in F2 and F6 plants derived from the cross Milyang 23/Gihobyeo. The application of MAS for
sd-1 gene has potential to greatly improve the efficiency of the Australian rice-breeding programme.
In their breeding programme, the semi-dwarf character when detected before maturity even as
a heterozygote eliminates the need for progeny testing in a backcrossing programme [125].
Wheat
The wheat stem and leaf rusts are two major pathogens, which can potentially devastate wheat
crops. Resistant cultivars have long been depended upon to control disease epidemics. Most of
the genes for resistance have been mapped using molecular markers and currently being used in
MAS at different national as well as international centres. In India, RAPD markers linked to
Lr19 (leaf rust) gene has been converted into SCAR markers. Lr 28 gene was also tagged by two
flanking RAPD marker S464700 and S326350. These linked molecular markers are further in use
in pyramiding of rust resistance genes, which is difficult, and time consuming by conventional
breeding procedures [126]. A population of 220 BC1F2 plants segregating for two genes Cre1
and Cre3 was evaluated with three molecular markers Xglk 605, Xcdo 588 and Cd 2.2 and the
markers were found to provide a reliable means of gene pyramiding and selecting plants carrying
the genes in wheat breeding programmes [127].
With overall goal of transferring new developments in genomics to wheat breeding and
production, investigators at 12 public wheat-breeding and research programmes across the US
including University of California, Colorado State University, Cornell University, Kansas State
University, Montana State University, University of Idaho, University of Minnesota, Purdue
University, University of Nebraska, USDA and Washington State University have constituted a
national wheat Marker Assisted Selection (MAS) consortium that aims to use molecular markers
as chromosome landmarks in MAS programme to facilitate introgression of small chromosome
segments carrying the genes of interest. Available molecular markers will be used to transfer
genes for resistance to fungi, viruses and insects as well as gene variants related to improved
bread, pasta and noodle quality. These genes will be incorporated into a minimum of 240
adapted cultivars or breeding lines belonging to all major market classes of US wheat, and since
they are transferred by normal recombination, the resulting lines will not be classified as transgenics.
These improved cultivars will transfer the value of genomic research to the wheat growers’
fields [128].
4.3 Other Crops
Use of molecular markers in marker-assisted selection has been carried out for improvement of
several other crops such as sunflower, tomato, sugar beet, barley, soybean, apple etc. throughout
the world. These are briefly described crop-wise as follows.
Sunflower
MAS for two rust resistance genes in sunflower was reported using RAPD markers Ox20600 and
OO04950 linked to the gene RAdv responsible for rust resistance in the proprietary inbred line P2.
This gene confers resistance to most of the pathotypes of Puccinia helianthi identified in Australia.
These RAPD markers were converted into SCAR markers and the robustness of these markers
were demonstrated through the amplification in a diverse range of sunflower germplasm. This
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will be useful in further attempts for molecular-assisted breeding to produce durable resistance
in sunflower to P. helianthi [129].
Tomato
MAS has been carried out for several traits including fruit characteristics in tomato. More
recently, MAS was used to transfer the ability to accumulate acylsugars to cultivated tomato.
RFLP and PCR-based markers were used through three backcross generations to select plants
containing five target regions associated with acylsugar accumulation [130]. In another example,
MAS has been demonstrated for QTL influencing blackmold resistance. Blackmold, caused by
the fungus Alternaria alternata, is a major ripe fruit disease of processing tomatoes. Five QTLs
were selected for introgression from Lycopersicon cheesmanii into cultivated tomato using
marker-assisted selection. RFLP and PCR-based markers flanking and within the chromosomal
regions containing QTLs were used for MAS during backcross and selfing generations [131].
Barley
The effectiveness of molecular marker-assisted selection for malting quality trait in barley was
reported by Han et al. [132]. In this study, the flanking markers, Brz and Amy2, and WG622 and
BCD402B, for two major QTL regions present on chromosomes 1 and 4 were used for MAS.
The MAS for QTL1 was more effective than phenotypic selection. It could substantially eliminate
undesirable genotypes by early genotyping and keeping only desirable genotypes for later phenotypic
selection. The MAS was also used for verification of yield QTL in a barley cross. The objectives
of this study were to verify the value of four QTLs for selection and to compare the efficiency
of alternative MAS strategies using these QTL vs. conventional phenotypic selection for grain
yield. It was shown that MAS was as good as phenotypic selection [133].
Soybean
MAS offers the potential to reduce linkage drag and to pyramid genes with similar phenotypic
effects into elite genotypes. One such example was seen in soybean breeding programme where
a QTL conditioning corn earworm resistance in the accession PI229358 and a synthetic Bacillus
thuringiensis cry1Ac transgene from the recurrent parent ‘Jack-Bt’ were pyramided into BC2F3
plants by marker-assisted selection. Segregating individuals were genotyped at SSR markers
linked to an antibiosis/antixenosis QTL on linkage group M, and were tested for the presence of
cryAc1. MAS was used during and after the two backcrosses to develop a series of BC2F3 plants
with or without cryAc1 transgene and the QTL conditioning for resistance in BC2F3 plants that
were homozygous for parental alleles at markers. This work by Walker et al. [134] demonstrated
the usefulness of SSR for MAS in soybean, and showed that combining transgene and QTLmediated resistance to lepidopteran pests might be a viable strategy for insect control.
Apple
MAS is also a promising method to select resistant individuals in horticultural crops like apple.
Molecular tools have the potential to give very early information on the genetics of apple
seedlings. The aims of apple breeding such as high fruit quality, consistently high yields and
durable disease and pest resistance can be achieved more efficiently. Progress in MAS for apple
breeding is being achieved mainly in the area of disease resistance especially in the durable
Molecular Mapping and Marker Assisted Selection of Traits 311
incorporation of scab and mildew resistance. The AL07-SCAR and M18-CAPS molecular analysis
in progenies, in which both parents are heterozygous for the resistance gene, made it possible to
identify clearly the homozygous plants for the Vf gene (resistance for apple scab) and these
plants showed higher level of resistance than the heterozygous one. Progenies were developed
from crosses with parents that carry different resistance genes such as Vf, Vm, Vb, Pl1 and QTLs
in different combinations [135–137].
Kentucky Bluegrass
The MAS has wide range of utility in this grass species. It was reported by Albertini et al. [138]
that MAS has helped in avoiding costly and time-consuming phenotypic progeny tests in Poa
pratensis to study mode of reproduction. Genotypic apomixis in Kentucky bluegrass involves
the pathenogenetic development of unreduced eggs from aposporic embryo sacs. Two SCAR
primer pairs were tested and identified the apomictic and sexual genotypes among progenies of
sexual × apomictic crosses with low bias. Furthermore, when tested on a wide range of Italian
and exotic P. pratensis germplasm, they were able to unequivocally distinguish sexual from
apomictic genotypes. This system should, therefore, allow new selection models to be set up in
this species.
5.
Future Prospects of MAS
The above review of the available literature reveals that during the last 17 years since the
publication of the first paper on the use of RFLP markers for construction of linkage maps in
tomato and maize in 1986, molecular markers have been extensively used for mapping and
tagging of hundreds of different agriculturally important genes/QTL in various crop species.
With the availability of linked markers, the first requirement for successful MAS has been
fulfilled. Besides, the feasibility of MAS based on these linked markers has been demonstrated
in several crops both for qualitative as well as quantitative traits as evident from the above
description. However, MAS is yet to be used routinely in plant breeding programmes.
Utility of the MAS in crop plants is currently limited by factors such as recombination
between the marker and the target gene, low level of polymorphism between parents with
contrasting traits and lower resolution of QTLs due to interaction with the environment. With the
recent developments in both structural and functional genomics, it would not be difficult to find
solutions to these problems. Availablity of high-density genetic and physical maps will enable
finding markers physically closer to the target gene that would not allow failure of MAS due to
genetic recombination. Moreover, cloning and characterization of the target genes, which are
possible based of their position on the linkage map, would allow development of allele-specific
markers. Use of such markers would completely eliminate the possibility of breakdown of the
marker-trait linkage. Besides, markers based on the sequences of the genes would facilitate
allele mining in the germplasm resources, thereby leading to identification and utilization of
newer alleles in crop improvement. Different alleles of a gene would differ for a number of
nucleotides at different positions in their sequence that would be the basis of developing highly
polymorphic single nucleotide polymorphism (SNP) markers. The problem of low level of
polymorphism in narrow crosses can thus be circumvented. Use of MAS for QTL, particularly
those having little effect on trait expression and highly interacting with environment would
require greater amount of research effort and newer experimental strategies.
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Complete integration of MAS with the conventional plant breeding programmes demands
consideration of two important factors: a) size of population and b) cost. Plant breeding experiment
requires screening of large segregating populations routinely over generations. Genotyping of
large number of samples manually is an extremely difficult task. MAS to be practicable should
be amenable to automation that would allow handling of large number of samples. Development
and use of PCR based markers such as STS and SCAR will be a key to success of MAS in crop
improvement. As the technology develops and gets modified to analyze large number of samples,
the cost will automatically go down. The investment in gene tagging, and selection based on
molecular markers should be weighted against the overall cost and time involved in traditional
breeding program. Even though the cost of MAS is higher at present level of estimation, its
integration with traditional plant breeding is desirable because of immense possibilities it offers.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
21. Studies on Male Meiosis in Cultivated and
Wild Vigna Species
S. Rama Rao and S.N. Raina*
Cytogenetics Laboratory, Department of Botany, J.N. Vyas University, Jodhpur 342 005, Rajasthan, India
*Cellular and Molecular Cytogenetics Laboratory, Delhi University, Delhi-110 007, India
Abstract: Male meiosis was studied in 11 species and one sub-species which include both cultivated
and wild ones to understand the cytogenetic mechanism underlying the speciation and evolution in
the genus Vigna. The present observations together with earlier published data indicate that the most
common gametic number is n = 11, 2n = 22 and in all probability this gametic number (n = 11) is
the basic number of the genus. Majority of the species studied presently showed normal eleven
bivalents at diakinesis/metaphase I, and 0–2 univalents found in four taxa have been ascribed to early
separation of precocious separation of rod bivalents. Complete bivalent formation in the teraploid
species V. galbrescens indicates its allopolyploid origin. Apparently, non-random distribution of
chiasmata in majority of the species investigated, seems to be an important cytogenetic phenomenon
in the genus. B-chromosomes [1–2] were recorded in two species. The data collected on anphase
I/II distribution of bivalents/chromosomes show that inversion heterozygosity in the genus is not
occasional but might be at floating stage in the population(s). Further, the study of meiosis in these
wild and cultivated species of Vigna clearly confirms that gene mutations and chromosomal repatterning
played a significant role in speciation and evolution of the genus.
1.
Introduction
The genus Vigna comprises about 120 species distributed widely in tropical and subtropical regions
of both hemispheres. It is one of the most important genus of Fabaceae and some thirty species
including V. radiata (mung bean), V. mungo (urd bean), V. aconitifolia (moth bean), V. umbellata
(rice bean), V. trilobata, V. vexillata, V. angularis (adzuki bean), V. lanceolata, V. mariana, V.
ambacensis, V. fisheri, V. unguiculata (cowpea), V. reticulata and V. capensis are cultivated
extensively for their pulse (protein content 17–24%) crop, vegetable, fodder crop, cover crop,
green manure and soil erosion control value [1–5]. The close morphological resemblance between
Vigna and two closely related genera (Phaseolus and Dolichos) has made it difficult for the
taxonomists to clearly delimit the species of the genus from that of Phaseolus and Dolichos, and
it is the phylogenetic classifications [6–8] which has set at rest most of such confusion. The
morphological features that distinguish Vigna from Phaseolus and Dolichos are curved, rather a
coiled or twisted keel and lateral rather than a terminal stigma [6], respectively, and Verdcourt
[7] has included yellow flowered species of Phaseolus in section certotropis piper in the subgenus
Certotropis of the genus Vigna. Several species of Phaseolus including P. aureus, P. radiatus, P.
angularis, P. mungo, P. trilobatus, P. pubescens, P. calcaratus have now been transferred to large
and heterogenous genus Vigna. Much of our present cytological understanding of Vigna is
restricted to mere chromosome numbers, a few inter-specific hybrids, nuclear DNA amounts
332
RAMA RAO AND RAINA
and often conflicting accounts about the chromosome complements and associations both
within and between the species [9–43]. The detailed studies on mitotic complements, male
meiosis, colchitetraploidy, interspecific hybrids and various other cytogenetical parameters, an
important prerequisite for providing evidence of past evolutionary events of theoretical and
practical importance and for logical manipulations to the advantage of economically important
taxa as in cereals, is very much limited in Vigna. The reasons could be factors like inherent
difficulty in obtaining good analyzable cytological preparations, small chromosome size, overall
stability of chromosome morphology and symmetry, and no success in raising cytogenetic
stocks like translocation testers and/or aneuploids. Such factors have also proved an impediment
in ascertaining precisely the genome relationships between species in a few successful interspecific
hybrids. The information available from interspecific crosses between V. radiata, V. angularis,
V. mungo, V. umbellata, V. minima and V. trilobata has confirmed that there exists a certain
degree of homology between different genomes [21, 34–41, 44]. The failure of crosses between
other species, especially those with wild species could not, however, be taken up due to lack
of genomic homology between them. There is, for example, complete bivalent pairing in the
F1 hybrids between V. umbellata and V. angularis, raised by embryo rescue culture techniques
[45].
Besides this, there is an overall stability in chromosome morphology and symmetry between
the species of the genus Vigna. The species differentiation cannot be, in most cases, correlated
with chromosome differentiation. In such case, meiosis could be yet another parameter for
understanding cytogenetic system in the genus. The comprehensive study about chromosome
associations, chiasma distribution and its frequency, and chromosome distribution during anaphase
would also throw some light on the nature of cytogenetic mechanisms underlying evolution in
the genus. In spite of several inherent disadvantages in the material a concerted attempt has been
made to bring out details of male meiosis in eleven taxa comprising ten species and one subspecies
as detailed below.
2.
Material and Methods
The seeds of various species and subspecies of Vigna were kindly supplied by the United States
Department of Agriculture (USDA), Maryland, USA and the National Bureau of Plant Genetic
Resources, New Delhi, India. For meiotic analysis flower buds of appropriate size were collected
from field grown plants and anthers were squashed in 1% aceto-carmine. On an average 25 cells
were analyzed at diplotene/diakinesis and metaphase I for recording chromosome associations
and recombinational frequencies through chiasma analysis. 15–20 cells were also analysed at
AI/AII for distributional pattern of chromosomes. For percentage pollen stainability, the pollen
grains were stained in 1 :1 (glycerin : acetocarmine) mixture and on average 10 slides were
scored for stainable pollen. Photomicrographs from temporary preparations were taken using
Agfa-Copex Pan photonegative film (ASA–20).
3.
Results
The meiotic data has been summarized in Tables 1 to 3.
V. aconitifolia
V. aureus
V. luteola
V. mungo
V. radiata
V. repens
V. umbellata
V. unguiculata
V. unguiculata
ssp. sesquipedaceae
V. sps. Tvnu-72
V. glabrescens
Species
Table 1.
20
15
14
22
22
44
2.83
2.67
2.76
2.84
3.03
4.95
No. of cells
analysed
26
20
20
25
25
25
25
26
2n
22
22
22
22
22
22
22
22
DNA amount
(×10–12 g)
18-24
18-24
15-21
18-25
17-26
15-24
13-22
18-21
13-22
13-22
32-42
19.90 ± 2.86
19.93 ± 2.37
39.57 ± 2.50
±
±
±
±
±
±
±
±
1.29
1.79
1.76
1.77
1.63
1.80
2.27
0.98
20.92
20.85
18.80
21.84
20.60
18.52
18.00
19.80
Chiasmata
Mean
Range
±
±
±
±
±
±
±
±
1.77
2.05
2.26
1.29
1.94
1.66
1.96
1.62
18.80 ± 2.70
16.40 ± 1.50
39.57 ± 2.50
18.77
15.80
16.10
17.56
17.12
14.24
14.96
16.07
Chiasmata
terminalized
1.10
3.53
2.15
4.95
2.70
4.28
3.48
4.28
3.04
3.73
Unterminalized
0.94
0.82
1.0
0.89
0.76
0.85
0.80
0.83
0.76
0.83
0.81
Terminalization
coefficient
Average number, range of chiasmata, terminalization coefficient and pollen stainability in Vigna species
80.34
89.92
69.09
87.09
90.00
88.39
89.80
94.00
93.16
82.30
99.10
Percentage
pollen
stainability
Studies on Male Meiosis in Cultivated and Wild Vigna Species 333
26
20
20
25
25
25
25
26
20
15
14
22
22
22
22
22
22
22
22
22
22
44
aconitifolia
aureus
luteola
mungo
radiata
repens
umbellata
unguiculata
unguiculata
ssp. sesquipedaceae
V. sps. Tvnu-72
V. glabrescens
V.
V.
V.
V.
V.
V.
V.
V.
V.
No. of
cells
analysed
2n
Species
18
13
9
26
20
20
25
25
24
23
26
No.
90.0
86.7
64.29
100.0
100.0
100.0
100.0
100.0
96.0
92.0
100.0
Percentage
31
33
39
52
44
72
50
54
98
108
80
No.
0–4
1–4
2–7
0–4
1–4
2–6
2–8
2–5
0–3
1–4
0–5
1.50 ± 0.94
2.20 ± 0.94
2.79 ± 1.63
±
±
±
±
±
±
±
±
183
130
258
234
174
148
225
221
176
164
206
Range No.
0.94
1.00
1.66
1.00
0.94
1.11
1.51
0.74
2.04
2.25
3.60
2.00
2.16
3.92
4.32
3.07
Mean
Rod bivalents
±
±
±
±
±
±
±
±
1.09
1.08
1.66
1.00
1.20
1.17
1.58
0.74
6–11
7–10
4–9
7–11
6–10
5–9
3-9
6-9
286
218
220
275
275
274
272
286
Range No.
10.65 10–11
10.86 8–11
21.21 16–22
0.08 ± 0.40
0.24 ± 1.38
Mean
Univalents
4 0.60 ± 2.3
4 1.57 ± 3.25
22 1.57 ± 3.25
2
6
Range No.
11.0
9–11
11.0
9–11
11.0
11.0
11.0
10.96 10–11
10.88 9–11
11.0
Mean
Total bivalents
9.15 ± 1.50 4–11 214
8.66 ± 1.40 5–10 163
18.42 ± 1.34 16–21 297
8.96
8.76
7.40
9.00
8.84
7.04
6.56
7.92
Mean
Ring bivalents
Average number, range of associations at diakinesis/metaphase I
Cells with 11/22 II
Table 2.
0–2
0–12
0–12
0–2
0–2
Range
334
RAMA RAO AND RAINA
Studies on Male Meiosis in Cultivated and Wild Vigna Species 335
Table 3.
Anaphase I distribution (U = Univalents, B = Bivalents)
Species
2n
No. of cells
analysed
Chromosome
distribution
No. of
cells
Percentage
V. aconitifolia
V. aureus
V. luteola
V. mungo
V. radiata
V. repens
22
22
22
22
22
22
15
20
20
20
20
20
V. umbellata
22
15
V. unguiculata
V.unguiculata ssp.
sesquipidaceae
V. sps. Tvnu-72
V. glabrescens
22
22
20
15
11:11
11:11
11:11
11:11
11:11
11:11
10:1U:11
11:11
10:1U:11
11:11
11:11
15
20
20
20
20
16
4
14
1
20
15
100.0
100.0
100.0
100.0
100.0
80.0
20.0
93.3
6.7
100.0
100.0
22
44
15
25
11:11
22:22
23:21
21:1B:21
22:2U:20
15
18
3
3
1
100.0
72.0
12.0
12.0
4.0
3.1
Associations
Diploids
V. aconitifolia (n = 11): All the cells analyzed had eleven bivalents (Figs. 1 to 3). On the average
there were 8.92 ring and 2.0 rod bivalents. Same gametic number has been reported by Purseglove
[15], Bhatnagar et al. [17], Sarbhoy [25] and Tschechow and Karataschowa [46].
V. aureus (n = 11): Eleven bivalents were observed in all the cells analyzed. The bivalents on
the average resolved into 8.70 ring and 2.20 rod bivalents.
V. luteola (n = 11): Eleven bivalents, observed in all the cells, on the average resolved into
7.40 ring and 3.60 rod bivalents. The nucleolus in a few cells at diplotene/diakinesis was seen
associated with as many as 2-4 bivalents. Same gametic number has been reported by Sen and
Bhowal [48].
V. mungo (n = 11): Eleven bivalents were encountered in all the twentyfive cells analysed. On
the average there were 9.0 ring and 2.0 rod bivalents. In this species also the nucleolus was
observed to be associated with more than one bivalent. The same gametic number has been
reported by several workers [9, 13, 14, 18, 19, 21, 22, 25, 28, 30, 34, 49, 50].
V. radiata (n = 11): All the cells analyzed had eleven bivalents at diakinesis/metaphase I. The
average number of ring and rod bivalents was 8.80 and 2.16, respectively. As in V. luteola and
V. mungo few cells in this species had 1–3 bivalents associated with the nucleolus. The same
gametic number has been reported by various researchers [9–12, 14, 17, 19, 20–22, 26, 28, 30,
34, 49, 50]. Univalents ranging from 2 to 4 were observed in a few cells by Sarbhoy [25]. A
variety of the species (V. radiata var. glabra), which was given the rank of separate species by
later authors, had 22 II (2n = 44) instead of normal 11 II [16] prevalent in the species.
V. repens (n = 11): In 24 out of 25 cells eleven bivalents were observed (Figs. 5 and 6). In the
336
RAMA RAO AND RAINA
1
2
5
4
8
3
6
9
7
10
11
Figs. 1 to 11. Male meiosis in Vigna. Fig. 1. V. aconitifolia. Diakinesis, 11 II. Figs. 2 and 3. V. aconitifolia.
Metaphase I, 11 II. Fig. 4. V. aconitifolia Anaphase I, 11: 11. Figs. 5 and 6. V. repens.
Metaphase I, 11 II. Fig. 7. V. repens. Anaphase I 11:11 Fig. 8. V. umbellata. Diakinesis, 11
II. Figs. 9 and 10. V. umbellata. Metaphase I, 11 II. Fig. 11. V. umbellata. Anaphase I, 11: 11.
remaining one cell there were ten bivalents and two univalents. The average number of associations
per cell was 10.96 II + 0.08 I. On the average 7.04 were ring and 3.92 rod bivalents.
V. umbellata (n = 11): Majority (92.0%) of the cells analyzed had eleven bivalents (Figs. 8 to
10). In the remaining cells besides bivalents, univalents ranging from 0 to 2 were also observed.
The average number of associations per cell was 10.88 II + 0.24 I. On the average there were
6.56 ring and 4.32 rod bivalents. The same gametic number has been reported by a number of
workers [21, 25, 26, 34, 51, 52].
Studies on Male Meiosis in Cultivated and Wild Vigna Species 337
V. unguiculata (n = 11): All the cells analyzed had eleven bivalents (Figs. 12 to 14). On the
average there were 7.92 ring and 3.07 rod bivalents per cell. Same gametic number has been
reported by Karpechenko [9], Mukherjee [54], while Kawakami [10], Rao [55], Floresca et al.
[56], Miege [57], Faris [6], Frahm-Leliveld [12] and Yarnell [58] have all reported 12 as the
gametic number.
V. unguiculata ssp. sesquipedaceae (n = 11): Majority (90%) of the cells analyzed had eleven
bivalents (Figs. 16 to 18). In the remaining 10% cells univalents ranging from 0 to 2 were
observed. The average number of associations per cell therefore was 10.65 II + 0.60 I. Ring
bivalents (9.15) outnumbered rod bivalents (1.50).
V. sps. Tvnu 72 (n = 11): This species obtained from Dr. Barnhart, USA had eleven bivalents
in 86.6% cells while in the remaining (13.3%) cells univalents ranging from 0 to 2 were observed.
The average number of associations per cell therefore was 10.88 II + 0.26 I. Ring bivalents
(8.66) were predominant over rod (2.20) bivalents.
Tetraploid
V. glabrescens (n = 22): The species, previously designated as V. radiata var. glabra, was the
only one in the present investigation which had 2n = 44. Majority (64.3%) of the cells had
normal 22 bivalents (Figs. 20 and 21). The other 35.7% cells had a mixture of bivalents and
univalents ranging from 16 to 21 and 2 to 12, respectively. Multivalents were not observed in
any of the cells analyzed. The average number of associations per cell was 21.21 II + 1.57 I. The
mean frequency per cell of ring and rod bivalents was 18.42 and 2.79, respectively.
3.2 Chiasma Frequency
The average number of chiasmata per cell ranged from 18 to 24, 18 to 24, 15 to 21, 18 to 25, 17
to 26, 15 to 24, 13 to 22, 18 to 21, 13 to 22, 13 to 22 mean number being 20.92, 20.85, 18.80,
21.84, 20.60, 18.52, 18.00, 19.80, 19.90, 19.93 in V. aconitifolia, V. aureus, V. luteola, V. mungo,
V. radiata, V. repens, V. umbellata, V. unguiculata, V. unguiculata ssp. sesquipedaceae, V. sps.
Tvnu-72, respectively, out of which on the average 18.77, 15.90, 16.10, 17.56, 17.12, 14.24,
14.96, 16.07, 18.80, 16.40 were terminalized giving terminalization coefficient of 0.89, 0.76,
0.85, 0.80, 0.83, 0.76, 0.83, 0.81, 0.94 and 0.82, respectively. The corresponding values for V.
glabrescens were 32–42, 39.57, 39.57 and 1.0, respectively. The maximum number of chiasmata
observed for any bivalent was four. The most common observation was 1 chiasma in rod and 2–
3 in ring bivalents. The average number of chiasmata per cell among the species with n = 11 was
highest in V. mungo (21.84) and lowest in V. umbellata (18.0).
3.3 B-chromosomes
A single Feulgen positive B-chromosome was observed in two (V. unguiculata, V. mungo) out of
the eleven species investigated presently. A large number of PMCs in these two species were,
however, apparently without B-chromosome. They did not have any perceptible effect on the
morphology, meiotic behaviour and pollen fertility.
3.4 Anaphase I, II
In seven out of the present eleven taxa anaphase I had equal distribution of chromosomes at the poles
(Figs. 4, 7, 11, 15, 19 and 22; Table 3). Three species (V. glabrescens, V. repens and V. umbellata)
338
RAMA RAO AND RAINA
13
12
17
16
20
14
18
21
15
19
22
Figs. 12 to 22. Male meiosis in Vigna. Figs. 12 to 14. V. unguiculata. Metaphase I, 11 II. Note B-chromosome
in Fig. 12. Fig. 15. V. unguiculata. Anaphase I, 11: 11. Figs. 16 to 18. V. unguiculata ssp.
sesquipedaceae. Metaphase I, 11 II. Fig. 19. V. unguiculata ssp. sesquipedaceae. Anaphase
I, 11: 11. Figs. 20 and 21. V. glabrescens. Metaphase I. 22 II. Fig. 22. V. glabrescens. Early
anaphase I.
Studies on Male Meiosis in Cultivated and Wild Vigna Species 339
had also equal distribution of chromosomes in as many as 64.0, 80.0 and 93.3% cells, respectively.
The remaining cells had unequal distribution and/or lagging univalents/bivalents (Table 3). In the
same very species few cells analyzed at anaphase I and II had 1-2 bridge fragment configurations.
3.5 Pollen Stainability
All but one V. glabrescens had 82–99% stainable pollen. The pollen stainability in V. glabrescens
was only 69.08%.
4. Discussion
Barring V. glabrescens, all the ten taxa investigated here had same gametic number (n = 11).
Even in V. glabrescens the gametic number (n = 22) was multiple of n = 11. The previous data
on male meiosis includes gametic numbers of 31 more species not presently investigated and it
is evident from the combined data that as many as 36 species out of the total 51 have n = 11
followed by n = 10, n = 9, n = 12, n = 22 in 6, 4, 1, 1 species, respectively. The remaining three
species are reported to have two gametic numbers (n = 10, 11; n = 11, 12) within the species.
In ascertaining the true basic number of the genus it is essential to have information regarding
gametic and/or zygotic numbers of as many species as possible. From the review of literature,
it is however clear that altogether 51 species out of total 150 species have only been evaluated
for above aspects. Taking mitotic data also into consideration it becomes clear that there exists
four basic numbers (x = 9, 10, 11, 12) in the genus and the most common among these is x = 11,
met in about 78% of the species investigated so far. Two different patterns of origin of more than
one basic number in a genus could be recognized. One that the genus might be polybasic in
origin and second possibility would be that the genus had only one basic number and during the
course of evolution one or more basic numbers originated from a relatively primitive basic
number.
According to Frahm-Leliveld [12] and Goswami [27] the basic numbers of 10 and 12 in the
genus are derivatives of n = 11. The reason for change in gametic number given are structural
alterations including centric fusion. Similarly, Froni-Martinus [59] believes that n = 9 observed
in V. candida might have arisen from n = 11 by structural alterations. Since all the species
investigated here had either n = 11 or n = 22, the present author could not evaluate the comparative
karyomorphology of the species with n = 11 to that of species with other basic numbers for
making out alterations in morphology as a result of the change in basic number. The present
author, however, supports the view of the other cytogeneticists referred above [12, 25, 27, 59],
regarding 11 as the true basic number of the genus Vigna and all other basic numbers (x = 9, 10,
12) as derivative of x = 11 occurred during the course of evolution. The reasons for its validity
are that this number is not only found in majority (78%) of the species analyzed cytologically so
far, but is found in taxa that are not only morphologically distinct but are also widely distributed
in tropics and subtropics of the world. Furthermore, the only polyploid species reported so far
is built on this number. The occurrence of the two basic numbers within the species in V.
unguiculata (x = 11, 12) and V. vexillata (x = 10, 11) is very interesting and hybridization
between plants differing in basic numbers within species will be of importance in ascertaining
the relationship of the two basic numbers within the species.
The plants representing V. aconitifolia, V. aureus, V. luteola, V. mungo, V. radiata and V.
unguiculata were characterized by the presence of perfectly normal eleven bivalents at diakinesis/
340
RAMA RAO AND RAINA
metaphase I (Table 2). In comparison, a few cells in V. repens, V. umbellata, V. unguiculata ssp.
sesquipedaceae and V. sps. Tvnu-72 had univalents, ranging from 2 in V. repens and V. unguiculata
sp. sesquipedaceae to 6 in V. sps. Tvnu-72, in hardly 4.0, 2.0, 10.0 and 14.3% cells, respectively
(Table 2). They in most cells behaved normally at anaphase I leading to organized and/or equal
distribution to the respective poles. The occurrence of stray univalents might be attributed to
early separation of synapsed homo/homeologues with or without formation of chiasmata or
precocious separation of rod bivalents. Such behaviour will convert bivalents to pair of univalents
which generally move to respective poles. The reason(s) for the prevalence of relatively high
frequency (1.57) per cell of univalents (Table 2) in 35.21 per cent cells of the tetraploid (V.
glabrescens) species is unknown.
The highest chiasma frequency in the diploid species was recorded in V. mungo (21.84) and
lowest in V. umbellata (18.0) (Table 1). All other species had values between these two extremes.
Majority of the species had in general 1 or 2 chiasmata in the bivalents. Due to very small size
of bivalents, the exact location of chiasma even in early diplotene could not be made out clearly.
They however seemed to be located at distal ends, and most of them got terminalized even at
pro-metaphase I stage. Same situation existed in the lone tetraploid species (V. glabrescens)
where average frequency of chiasma per cell was 39.57 and all of them were observed at the
terminal region even at diplotene thus giving terminalization coefficient of one (Table 2).
The seemingly nonrandom distribution of chiasmata is corroborated by the fact that the
frequency per cell in a particular species was not dependent on the size of chromosomes. In V.
umbellata and V. mungo, for example, although there is a difference of 4.0 in chiasma frequency
per cell, the 2C nuclear DNA amounts [42] are exactly the same. The occurrence of localization
of chiasmata, which prevents some chromosome segments from recombining and thus keeping
special gene combinations intact, apparently seems to be a strong feature at least in the species
of Vigna investigated here. The presence of localized chiasma, proximal or distal, found in large
number of plants [60, 61] has been attributed to the course of chromosome pairing, interference
pattern or availability of only short segments for pairing of the chromosomes.
In eight out of eleven taxa presently investigated the distribution of chromosomes at anaphase
I and presumably at anaphase II was normal and no abnormalities due to structural alteration
and/or unequal distribution of chromosomes were observed (Table 3). However, in the remaining
three species (V. glabrescens, n = 22, V. repens, n = 11, V. umbellata, n = 11) unequal distribution
of chromosomes and lagging univalents/bivalents were encountered in a few cells. Similarly,
bridge fragment configurations were also observed in these very same species, though only in
stray cells. Occurrence of bridge fragment configuration has also been reported in some accessions
of V. aconitifolia and V. mungo [23, 24, 28]. A dicentric bridge and an acentric fragment at
anaphase I could result from a single crossover within the inverted region involving two nonsister
chromatids or because of diagonal three strand double crossovers involving three chromatids
within the inverted region. Dicentric bridge and an acentric fragment might also arise following
chromosome breakage and reunion or by inverted crossing over during meiosis [62, 63]. The
presence of two dicentric bridges and two acentric fragments at anaphase II observed in a single
cell of V. glabrescens is the outcome of complementary four strand double crossovers involving
all the chromatids within the inverted region and one crossover between centromere and the
region outside the inverted loop. The low frequency of bridge fragment configuration in the cells
investigated might be attributed to the failure of crossing over and/or nonpairing within and
Studies on Male Meiosis in Cultivated and Wild Vigna Species 341
between the small inverted segments, respectively. From the present investigations together with
earlier reports [23, 25, 28, 34], it is amply clear that inversion heterozygosity in the genus is not
occasional as reported in large number of plants. They might be at a stage of floating inversions
in the genus as in Campanula [64]. Inversion heterozygosity has been instrumental in establishing
species relationships in Drosophila, Lilium and Paeonia [61, 65, 66]. How far they have got
established in the genus Vigna could only be determined after a detailed study is conducted in
as many species as possible.
The incidence of polyploidy in Vigna seems almost non-existent. So far, polyploidy has been
reported in only one species (V. glabrescens) based on the basic number 11. The detailed meiosis
of V. glabrescens conducted by Swindel et al. [16] and the present authors reveal that all the
associations observed were in the form of bivalents. Other associations like quadrivalents were
altogether absent, and therefore on the basis of chromosome associations it is evident that V.
glabrescens having 2C DNA content of 4.95 pg is allotetraploid in nature. V. glabrescens has
unmistakable morphological similarity with V. radiata and the fact remains that it was for a long
time considered to be a variety of V. radiata (V. radiata var. glabra). One of the putative parent
involved in its synthesis is therefore considered to be V. radiata [7] which has 2.67 pg of 2C
nuclear DNA amount. The other species involved in its synthesis should have DNA content of
the order 2.3 pg, an amount less than that of V. ambacensis (2.43), V. oblongifolia (2.55), V.
trilobata (2.60), V. angularis (2.70), V. repens (2.76), V. caracalla (2.82), V. mungo (2.83),
V. umbellata (2.84), V. vexillata (2.89), V. parvifolia (2.94), V. unguiculata (3.03) and more than
V. lancifolia (2.13) for which the DNA amounts have been analysed so far. To determine the
other species involved in the synthesis of V. glabrescens, therefore, one should find out the
species having DNA amounts of the order of 2.13 pg and if they are more than one in number,
the species should be involved in the synthesis of amphidiploids, V. radiata being common in all
crosses, for the precise identification of other species involved in the synthesis of V. glabrescens.
The perusal of previous as well as present data on male meiosis and mitotic complements brings
out clearly that due to inherent difficulty in obtaining good analyzable cytological preparations,
very small size of chromosomes and overall stability of chromosome morphology and symmetry,
the understanding of innate cytogenetic mechanisms underlying evolution, an important prerequisite
for providing evidence of past evolutionary history of theoretical and practical importance
and for logical manipulations to practical advantage of economically important taxa and wild
relatives as in cereals is not forthcoming in the genus Vigna. The little information one could gather
from the above studies so far is that (apparently) nonrandom distribution of chiasmata, cryptic
structural hybridity and paracentric inversion might have played role in the evolutionary process
of the genus. There has been no success in raising cytogenetic stocks like translocation testers
and/or aneuploids. The limitations in conventional chromosome research has also provided an
impediment in ascertaining precisely the genome relationships between species in a few successful
interspecific hybrids including the species belonging Asiatic group. The little information one
could gather from the crosses between V. radiata, V. angularis, V. mungo, V. umbellata, V. minima
and V. trilobata is that there exists certain degree of homology between the genomes involved
[13, 21, 32–41, 67, 68].
In an important genus like Vigna where cellular cytogenetics is of little consequence in
determining phylogenetic relationships and genome architecture between species, the evidence
of such differentiation could be obtained by molecular cytogenetics, such as relative quality and
342
RAMA RAO AND RAINA
quantity of DNA change, longitudinal differentiation of chromosomes, overall sequence architecture
of chromosomes, DNA/DNA hybridization, organelle DNA variation in situ hybridization of
chromosomes such as FISH and McFISH etc. An attempt in this regard has already been made
in the present laboratory and elsewhere. Microdensitometry measurements in 13 diploid species
showed that the divergence and evolution of the species was accompanied by small but significant
quantitative DNA variation ranging from 2.13 pg in V. lancifolia to 3.03 pg in V. unguiculata
[42]. There was continuity in the distribution of DNA changes between the diploid complements
of various species. The lone tetraploid species (V. glabrescens) had 4.95 pg of DNA. Significant
differences between the species in areas of chromatin were also observed and compaction of
DNA per unit area in interphase nuclei increases with increasing DNA amounts. Like Vicia,
Lathyrus, Festuca and Lolium [69–71] the difference in DNA amount between Vigna species is
equally distributed among all chromosomes within the complement irrespective of their size
differences [42]. Molecular composition of DNA is available in only V. radiata and it has been
reported that it consists of about 35% repetitive sequences [72, 73].
Acknowledgements
The authors are thankful to the United States Department of Agriculture (USDA), Maryland,
USA and the National Bureau of Plant Genetic Resources, New Delhi for providing seed samples.
SRR wish to thank Dr. Arun Kumar for his help in preparing the manuscript and illustrations.
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
22. Transgenic Crops for Abiotic Stress Tolerance
Deepti Tayal1,2, P.S. Srivastava2 and K.C. Bansal1*
1
National Research Centre on Plant Biotechnology, Indian Agricultural Research Institute,
New Delhi 110 012, India *e-mail: kailashbansal@hotmail.com
2
Centre for Biotechnology, Faculty of Science, Jamia Hamdard, New Delhi 110 062, India
Abstract: Crop cultivars that are high yielding and also possess tolerance to abiotic stresses mainly
drought and salinity are always on the shopping list of Indian farmers. However, in the past it had
been technically difficult to produce such improved cultivars through conventional breeding due to
the complex nature of the traits involved. Currently, the modern transgenic approach is being
utilized to develop cultivars that are tolerant to abotic stresses. Efforts have been made and are
currently in progress worldwide to understand the genetic and molecular basis of this complex trait.
Substantial progress has been made in the identification of genes involved in abiotic stress tolerance,
and their transfer to model plant species as well as crop species of economic importance for
increased stress tolerance. In this chapter, we have reviewed results of the economically important
transgenic crop plants that have been developed with altered expression of the genes implicated in
stress tolerance. Most of the transgenic crops have shown enhanced tolerance to abiotic stress
factors in pot culture experiments. However, data on their field performance under real stress
situation are lacking. Nevertheless, analysis of the transgenic plants has proved useful in providing
basic understanding of the function of the stress-induced genes in stress tolerance. Further progress
is anticipated by the rapid discovery of more novel genes on a genome wide basis followed by
determination of their precise physiological role in stress tolerance through functional genomics,
and subsequent generation of transgenic crops with enhanced tolerance to multiple abiotic stresses
and improved yields.
1.
Introduction
Abiotic stresses represent the most limiting environmental factors affecting agricultural productivity.
To overcome these limitations and to improve production, to feed the ever-increasing population,
it is imperative to develop crop cultivars that are stress tolerant. When crop plants are subjected
to environmental stress conditions, they fail to express their full genetic potential for production.
The effect of stress depends on the developmental stage, genotype of plant species as well as
duration and intensity of the stress. Generally, plants respond to these stresses under low or
moderate levels, but when the stress levels exceed a certain critical level (which varies from crop
to crop), the physiological mechanisms imparting tolerance to plants start breaking down causing
ultimately plant death. Consequently, the abiotic stress factors cause a massive loss to the
productivity of crop plants. According to the ICRISAT Report [1], biotic and abiotic stress
factors lead to a loss of US$ 15.74 billion in five most important crops of semi arid tropics—
sorghum, pear millet, pigeonpea, chick pea and groundnut. These crops are the main food source
for poor people of the developing countries. Amongst the various stresses affecting crop plants,
loss due to abiotic stresses is much more significant as compared to the losses that occur due to
insect/pests, weeds and diseases (Fig. 1).
Transgenic Crops for Abiotic Stress Tolerance 347
3
Abiotic factors
2.5
Insects
Diseases
Weeds
Pigeonpea
Groundnut
US $
2
1.5
1
0.5
0
Fig. 1
Sorghum
Pearlmillet
Chickpea
Loss due to abiotic factors, insects, diseases and weeds
(Source: ICRISAT1)
Classical plant breeding methods involving inter-specific or inter-generic hybridization and
in vitro induced variation have been applied to improve the abiotic stress tolerance of various
crop plants but without much success. The conventional breeding strategies are limited by the
complexity of stress tolerance traits, low genetic variance of yield components under stress
condition and lack of efficient selection criteria. It is important, therefore, to look for alternative
strategies to develop stress tolerant crops. Recently, marker assisted selection of specific traits
that are linked to yield, e.g. osmotic adjustment, membrane stability or physiological tolerance
indices, has been recommended. However, QTL that are linked to tolerance at one stage in plant
development can differ from those linked to tolerance at other stages. Furthermore, desirable
QTLs can require extensive breeding to restore suitable traits along with the introgressed tolerance
trait. The best alternative, therefore, is the direct introduction of genes by genetic engineering to
incorporate tolerance traits in target crops.
Research over the past two decades has provided a better understanding of the molecular
biology of stress responses in plants. Many genes and gene products have been identified which
get induced upon exposure of plants to various abiotic stresses—drought, salinity, low and high
temperature stress, etc. Consequently, biotechnological tools have been applied to transfer some
of these useful genes implicated in stress tolerance to plants. In addition to these stress-induced
proteins, genes encoding enzymes of the biosynthetic pathways of different osmolytes such as
proline, glycine betaine, trehalosc, sorbitol, pinitol, etc. have been cloned and exploited in
improving abiotic stress-tolerance in plants through genetic engineering.
In this chapter we have made an attempt to summarize the progress made towards understanding
the role of different genes implicated in stress tolerance and the genetic engineering efforts
towards developing stress tolerant transgenics in crop plants of economic importance.
2.
Genes involved in Abiotic Stress Tolerance
Under different abiotic stress conditions, a large number of genes show elevated transcript levels
in plants (Table 1). Up-regulation of these genes does not always confirm their role in stress
tolerance. Changes in gene expression may be due to disruption of physiological and metabolic
processes of the cell. However, precise physiological function of any such gene can be studied
by its altered expression (overexpression or suppression) in transgenic plants. Indeed, transgenic
348
TAYAL, SRIVASTAVA AND BANSAL
approach has emerged as a valuable tool in determining or confirming the precise function of the
stress-induced genes and to develop stress-tolerant transgenic crop plants. Normally, genes
isolated under stress conditions are first tested in model species such as tobacco and Arabidopsis
for their role in stress tolerance before transferring them to economically important crop species.
Stress-induced genes and gene products that accumulate under abiotic stresses have been reviewed
by Shinozaki et al. [2], Grover et al. [3] and Abdin et al. [4]. There are four categories of stress
induced genes/proteins with known function which have been exploited for generating stress
tolerant transgenic plants (Fig. 2). These genes and their role in model plant species have been
described below.
Drought
Salinity
Abiotic stresses
Low
High
temperature
temperature
High light/UV-B
Signal Perception and Transduction
Protein kinases
Transcription factors
Gene Expression
Factors for
protection of
cellular machinery
Enzymes for
osmolyte
Reactive O2
scavenger
Membrane
proteins
Genetic engineering for regulated expression
in transgenic crop plants
Stress tolerant transgenics with improved crop
productivity under stress
Fig. 2
Schematic representation of stress perception and transduction, stress-induced gene expression,
and genetic engineering utilizing the candidate genes for developing stress tolerant transgenic
crop plants.
2.1 Genes Involved in Osmolyte Biosynthesis
Osmolytes are highly soluble compatible solutes that are neutral at physiological pH. Being
neutral at physiological pH, increased concentration of compatible osmolytes does not interfere
with macromolecular conformation. Enhanced accumulation of osmolytes under stress conditions
lead to the lowering of osmotic potential of cells which inturn results in uptake of water and
maintenance of cell turgor. Various kinds of osmolytes are known to accumulate in plants under
stress such as proline, mannitol, glycine betaine, trehalose, etc. [5].
Proline, an important osmolyte is synthesized from glutamate by the catalytic action of
enzyme ∆1-pyrroline 5 carboxylate synthetase (P5CS). Overexpression of this enzyme in transgenic
tobacco showed enhanced biomass production, better plant growth and flower development
Transgenic Crops for Abiotic Stress Tolerance 349
Table 1.
Some examples of osmotic stress induced genes and gene products
Category
Proteins
Genes
Reference
Genes
involved in
osmolyte
biosynthesis
Enzymes for synthesis of:
Proline
Polyols-mannitol, ononitol
Fructans, Trehalose
Polyamine-putrescine
Quarternaryamine-glycine betaine
Osmotin-induced proline
P5CS
MtlD, IMT
SacB, TPSI
ADC, ODC
codA, CDH, CMO
Osmotin
[6]
[8,9]
[10,11]
[12,13]
[15-17]
[7]
Genes
encoding
factors for
protection of
cellular
machinery
Antifreeze proteins
LEA proteins
LEA-like proteins
Osmotin
AFP1, AFP2
LEAI, Dehydrins, HVA1, LEAIV
COR14, COR15
Osmotin
[18, 19]
[20-23]
[24, 25]
[7]
Genes
encoding
membrane
proteins
Water channel proteins
Aquaporins
γ-TIP, PM28A, AthH2
[26-28]
Transport proteins
H+ ATPase
Ca2+ ATPase
K+ transporters
K+ channels
Na+/H+ antiporter
Superoxidase dismutase
Ascorbate peroxidase
Glutathione synthetase
Glutathione reductase
AHA3, PMA2
LCA1
HKt1, Hak1
AKt1, AKt2
AtNHX1
Cu/Zn SOD, MnSOD, FeSOD
Apx
GS
GR
[29,30]
[31]
[32,33]
[34,35]
[36]
[37-39]
[40]
[41]
[42]
Genes for
reactive
oxygen
scavenger
proteins
Genes
encoding
transcription
factors
Genes
encoding
protein
kinases
Catalase
Glyoxalase
Ethylene responsive element
binding factors (ERF)
Basic domain Leucine Zipper (BZIP)
Myb and Myc like protein
Catalase
Glyl, Glyll
CBF1, Tsi, DREBF1
[43]
[44, 45]
[46-48]
ABF3, ABF4
Atmyb2, rd22BPI
[50]
[51]
Mitogen activated protein kinases
MAPK
MAPKK
MAPKKK
Ca2+ dependent protein kinases
At MPK 3/6, At MPK4
At MPKK 4/5, At MKK 1/2
At MEKK1, At ANP1
AtCDPK1, AtCDPK2
[52]
[53]
under drought and salinity stress conditions [6]. We recently reported accumulation of free
proline in transgenic tobacco plants over-expressing osmotin gene [7]. The transgenic plant
showed tolerance to osmotic stress caused by drought and salinity; however, the precise role of
osmotin in imparting tolerance could not be ascertained.
350
TAYAL, SRIVASTAVA AND BANSAL
Among polyols, mannitol overproduction through E. coli mannitol 1-phosphate dehydrogenase
(MtlD) gene expression in transgenic tobacco provided enhanced tolerance against salinity stress
[8]. Overaccumulation of D-mannitol upto a remarkable limit of 600 mM in cytosol provided
osmotic tolerance to transgenic tobacco [9]. Similarly, fructan synthase (Sac B) gene responsible
for fructan biosynthesis, showed tolerance to freezing and PEG-mediated water stress in transgenic
tobacco [10]. Engineering of trehalose metabolism by transferring trehalose 6-phosphate synthase
(TPS1) gene in tobacco plants showed improved drought tolerance. However, the transgenic
plants exhibited stunted growth, reduced sucrose content and lancet-shaped leaves [11]. Experiments
are in progress to circumvent these growth related problems.
Polyamines are also known to have positive effects on plants exposed to abiotic stresses.
Spermine and spermidine are two major polyamines synthesized from putrescine. Ornithine
decarboxylase (ODC) and arginine decarboxylase (ADC) are the key enzymes involved in
putrescine biosynthesis. Although the role of putrescine in stress tolerance remains to be elucidated,
the biosythesis is stimulated in the presence of osmotic stress [12-14].
A quaternary amine, glycine betaine is another important osmolyte whose enhanced accumulation
was observed in halophytes and bacterium under drought and salinity stress. Choline oxidase
(COD) from Arthrobacter globiformis, or choline dehydrogenase (CDH) and choline monoxygenase
(CMO) in plants are the key enzymes involved in glycine betaine biosynthesis. Transgenic
tobacco and Arabidopsis plants producing COD, CDH and CMO have shown enhanced tolerance
against salinity stress [15, 16]. However, it has been reported that availability of choline is a
limiting factor in glycine betaine producing transgenic plants. This problem can be overcome to
some extent by exogenous choline supply [17]. Engineering osmolyte biosynthesis is emerging
as a viable approach in producing transgenics for enhanced tolerance to osmotic stresses in
plants. However, the major focus is on enhanced biosynthesis of trehalose and glycine betaine
through genetic engineering.
2.2 Genes Encoding Factors for Protection of Cellular Machinery
Protection factors such as antifreeze proteins (AFPs) bring about lowering of freezing point by
inhibiting binding of additional water molecules to ice crystals. Larger ice crystals have more
harmful effects on tissues as compared to small crystals. When a synthetic fusion protein (based
on type I AFP) was expressed in yeast [18], inhibition of recrystallization was observed as a
result of AFP expression. Transformed yeast cells also showed a two-fold increase in survival
after rapid freezing. In another study, no effect on freezing tolerance in transgenic tobacco plants
expressing the type II antifreeze protein was observed [19]. Further studies are required for
defining the exact role of AFPs in abiotic stress tolerance.
Another kind of protection factors are late embryogenesis (LEA) proteins that are highly
hydrophilic. They accumulate in seeds during desiccation. Group II LEA proteins, also known
as dehydrins help in maintaining folded form of proteins and thereby function as chaperons [20].
These proteins are generally induced during cold acclimatization and dehydration. Overexpression
of group I LEA proteins from wheat in yeast cells showed attenuation of growth inhibition in
high osmolarity media. However, their role in freezing tolerance remains unknown [21]. Group
III Lea proteins have been suggested to function against desiccation tolerance by sequestration
of ions [22]. But there is no report available on transfer of HVA1 gene encoding LEA III proteins
in model species. Overexpression of group IV LEA protein in yeast has shown tolerance against
low temperature and salinity stress [23].
Transgenic Crops for Abiotic Stress Tolerance 351
Another group of LEA-like proteins are hydrophilic COR proteins having repeated amino
acid sequence motifs forming amphipathic α-helix. Based on this property, these proteins have
been suggested to increase freezing and dehydration tolerance by stabilizing proteins and
membranes. Constitutive expression of cold regulated COR15a gene in transgenic Arabidopsis
plants showed an increase in both chloroplast and protoplast freezing tolerance [24]. However,
effect of low temperature was not measured in transgenic Arabidopsis plants over-expressing the
barley gene Cor14b [25].
2.3 Genes Encoding Membrane Proteins
The membrane proteins involved in osmotic stress tolerance include water channel and transport
proteins. Water channel proteins control cellular water transport in response to drought and salt
stress. The recently identified aquaporins are complex family of water channel proteins having
control over water flux in and out of the cell. Aquaporins also maintain proton gradient for
osmotic balance by preventing the ion flow through water channel. Phosphorylation of aquaporins
through membrane bound protein kinase has been suggested as an essential factor for regulation
of activity of water channel proteins [26,27). Transgenic Arabidopsis plants with antisense
construct of plasma membrane aquaporin have revealed the role of aquaporins in maintaining
cytosolic osmoregulation [28].
In high saline environments, plants take up excessive amounts of Na+ and Cl– at the cost of
+
K and Ca2+. K+ is required as a cofactor for many enzymes and Ca2+ is essential in signal
transduction. A number of transport proteins play an important role in maintaining ion homeostasis
under stress condition. In salt tolerant plants, H+ ATPase maintains H+ ion flux across the plasma
membrane [29, 30]. Ca+ homeostasis for reducing toxic effects of NaCl is maintained by Ca+
ATPase [31]. K+ transporters and K+ channels maintain K+ and Na+ uptake for mediating ion
homeostasis [32–35]. Na+/H+ antiporters use electrochemical proton gradient for transporting
Na + into vacuole. This gradient is provided by vacuolar H + translocating enzymes.
Compartmentation of Na+ into vacuole helps in accumulating water into the cell and thus in
maintaining osmotic balance. Consequently, overexpression of the gene encoding vacuolar
Na+/H+ antiporter showed tolerance to 200 mM NaCl in Arabidopsis thaliana [36].
2.4 Genes for Reactive Oxygen Species Scavenger Proteins
Under stress conditions, plants produce various active oxygen species (AOS) such as superoxide
.
O⋅2 , hydrogen peroxide H2O2, and hydroxyradical OH . Plants generally respond to these active
oxygen species by inducing antioxidant system involving superoxide dismutase (SOD), ascorbate
peroxidase (APx), glutathione synthetase (GS), glutathione reductase (GR) and catalase enzymes.
For detailed analysis of contribution of these antioxidant enzymes to stress tolerance, a large
number of experiments have been conducted with transgenic model plants overproducing the
antioxidant enzymes [37–43]. Most of these transgenics provided tolerance against oxidative
stress, and photooxidative and ozone damage.
Glyoxalate system is known for being involved in protection against cytotoxicity. The first
evidence investigating the role of glyoxalase I enzyme in imparting tolerance to plants under
salinity stress came through the studies of Veena et al. [44]. The same group has now overexpressed
glyoxalase II in tobacco either independently or in concert with gly I . Transgenic plants inheriting
both the genes showed many fold increase in salinity tolerance over the single gene transgenics
depicting a synergistic effect of the gly I and gly II genes [45].
352
TAYAL, SRIVASTAVA AND BANSAL
2.5 Genes Encoding Transcription Factors
Transcription factors play an important role in controlling the expression of stress-responsive
genes. Few important families of transcription factors are:
2.5.1 Ethylene Responsive Element Binding Factors (ERF)
All ERFs are suggested to have a conserved 58-59 amino acid domain that can bind to C-repeat/
dehydration responsive element (DRE). DRE motifs are involved in regulation of ABA independent
gene expression under drought, salinity and cold stress. Therefore, overexpression of single ERF
gene may help in improving tolerance to a range of abiotic stresses. The role of ERF gene in
freezing tolerance was confirmed, through overexpression of CRT/DRE binding factor CBF1 in
Arabidopsis thaliana [46]. The ERF gene imparted tolerance to multiple stress factors such as
drought, salinity and cold stresses imposed together [47]. Overexpression of tobacco stress
induced gene (Tsi) in transgenic tobacco further confirmed the role of ERF gene in conferring
tolerance to osmotic stress [48].
2.5.2 bZIP Transcription Factor
bZIPs belong to a large family of transcription factor genes and possess a basic domain adjacent
to leucine-zipper motif. A number of bZIP proteins are found to be involved in stress signaling
[49]. The first genetic evidence of importance of bZIP proteins in stress tolerance was provided
by overexpressed ABRE binding factor/ABA responsive element binding protein of bZIP family
in transgenic Arabidopsis thaliana [50].
2.5.3 Myb and Myc Binding Proteins
Myb-like proteins contain helix turn helix related motif and Myc-like proteins have basic helix
loop helix domain for DNA binding. Expression of this class of transcription factors is induced
by ABA. In Arabidopsis, application of exogenous ABA induces a dehydration responsive gene
rd22. Expression of this gene requires protein synthesis as revealed by the use of cycloheximide,
an inhibitor of protein synthesis. The promoter of rd22 contains a 67bp DNA sequence, which
is sufficient for the expression of the gene. Abe et al. [51], identified the presence of MYB and
MYC recognition sites in the 67bp region by transforming tobacco plants with this region.
cDNA encoding MYB related DNA binding protein was termed as At MYB2 and gene encoding
MYC related protein was given the name rd22 BP1.
2.6
Protein Kinases
2.6.1 Mitogen Activated Protein Kinases (MAPKs)
MAPKs are serine/threonine protein kinases which phosphorylate a number of substrates involved
in various cellular responses including gene expression. They play essential role in plant signal
transduction pathways. MAPK cascade is regulated by MAPK kinases (MAPKK) and MAPKK
kinases (MAPKKK). In this cascade, signal is sensed by MAPKKK first that phosphorylates the
MAPKK, which in turn phosphorylates the MAPK. A number of abiotic stress factors such as
wounding, low temperature, high osmolarity, high salinity and reactive oxygen species act as a
signal in activating MAPK cascade. To our knowledge there seems to be no report as yet on
transgenic with overexpression of MAPK cascade genes. Studies are underway on cloning these
Transgenic Crops for Abiotic Stress Tolerance 353
genes on the basis of sequence homology and specific antibody recognition. A detailed analysis of
mitogen activated protein kinase signaling cascade has been presented by Guillaume et al. [52].
2.6.2 Calcium Dependent Protein Kinases
A number of abiotic stress factors such as cold, salt and drought elevate Ca2+ levels in cells for
achieving control over various cellular mechanisms. Ca2+ influx mediates this control by
phosphorylation/dephosphorylation of various proteins through Ca2+ dependent protein kinases
(CDPKs). These kinases contain a calmodulin like regulatory domain and a Ca+ binding site at
C terminal. Around 40 different CDPKs have been investigated in Arabidopsis thaliana. Sheen
[53] introduced eight CDPK isoforms of Arabidopsis into maize protoplasts, and found that only
two isoforms, AtCDPK1 and AtCDPK2 induced the expression of specific stress genes thereby
suggesting the presence of specific CDPK isoforms for different stress signaling pathways.
3.
Development of Stress-Tolerant Transgenic Crops
3.1 Wheat
Wheat is an important cereal crop.There are only few reports on transgenics for abiotic stress
tolerance in this economically important crop. For instance, improved biomass productivity and
water use efficiency was observed when wheat cultivar Hi-Line was transformed with HVA1
gene encoding LEAIII protein. These transgenic lines were shown to have higher dry mass, root
fresh and dry weight and shoot dry weight as compared to control plants [54]. For elucidating
the role of HKT1, transformed wheat with sense and antisense construct of HKT1 were raised
[55]. The transgenic plants exhibited better growth and reduced Na+/K+ ratios as compared to
control plants under saline conditions.
The role of mannitol accumulation in imparting stress tolerance is known in model transgenic
plants. Based on this fact, transgenic plants were generated with mtlD gene of E.coli in sense and
antisense orientation [56]. Wheat plants do not synthesize mannitol by their own metabolism.
The transgenic wheat plants with mtlD gene accumulated very low level of mannitol which was
not sufficient for osmotic adjustment. However, the transgenic plants showed improved growth
under water stress and salinity conditions probably due to protein stabilizing effect of osmolytes
under stress [56].
3.2 Rice
Rice is a highly drought and salt sensitive crop. A number of studies have been performed for its
improvement through genetic engineering. In 1998, Sakamoto et al. [57] developed transgenic
rice by introducing codA gene from Arthrobacter globiformis for glycinebetaine synthesis.
These transgenic plants could not show tolerance against salinity stress but their stress recovery
rate was high. They showed that transgenic plants with CodA enzyme targeted to chloroplasts
were more efficient in protecting photosynthetic machinery against stress than transgenic plants
with codA expression in cytosol. Further, the role of codA gene in improving salt stress tolerance
was confirmed by Mohanty et al. [58], in transgenic lines of Indica rice. In a recovery period
after exposure to 0.15 M NaCl for one week, the transgenic plants survived well whereas control
plants failed to recover and died. For elucidating the role of photorespiration in protection
against salt stress, transgenic rice plants over expressing chloroplast glutamine synthetase (GS2)
HVA1
HKT1
MtlD
CodA
CodA
GS2
P5CS
OSCDPK7
HVA1
HVA1
PMA80 & PMA1959
ADC
SAMDC
Catalase
OtsA +OtsB
TPS + TPP
Glutathione synthetase
Glutathione reductase
CodA
AtNHX1
Osmotin
Antisense P5CR
MnSOD
MnSOD
MnSOD
FeSOD
MitMnSOD + ChlMnSOD
Alfin
Rice
Mustard
Soybean
Alfalfa
Gene introduced
Wheat
Transgenic
crop
Table 2.
A. thaliana
Tobacco
Tobacco
Tobacco
A. thaliana
Tobacco
Alfalfa
E.coli
E.coli
A. globiformis
A. thaliana
Tobacco
A. globiformis
A. globiformis
Rice
Mothbean
Rice
Barley
Barley
Wheat
Oat
Tritordeum
Wheat
E.coli
E.coli
Barley
Wheat
E. coli
Source of
the gene
Tolerance to drought stress as compared to control ones
Enhanced freezing stress tolerance
Enhanced water deficit tolerance
Enhanced winter survival
Increased winter survival, no change in oxidative stress tolerance
Improved biomass, stress tolerance not detected
Improvement against salinity tolerance
Enhanced cadmium accumulation and tolerance
Targeted expression in chloroplast showed cadmium tolerance
Tolerance against salt and water stress
Enhanced salt tolerance
Enhanced drought and salt tolerance
Early recovery from salt induced damage
Tolerance against salt stress
Enhanced tolerance to salt stress and cold stress
Increased biomass under salt and water stress
Improved tolerance against cold and salt/ drought
Significantly increased tolerance to water deficit and salt stress
Improvement in drought and salt tolerance
Enhanced dehydration and salt stress tolerance
Enhanced tolerance to drought and salinity
Enhanced NaCl stress tolerance
Improved tolerance against low temperature stress
High tolerance against drought, salinity and low temperature stress
Enhanced tolerance to drought, salinity and cold stress
Improved biomass under water deficit conditions
Enhanced growth under salinity
Improved growth under water stress and salinity
Performance of transgenics under stress
Some examples of transgenic crop plants tolerant to abiotic stresses
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[70]
[71]
[72, 73]
[74]
[75]
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
[65]
[66]
[67]
[68]
[69]
[54]
[55]
[56]
Reference
354
TAYAL, SRIVASTAVA AND BANSAL
ODC
Carrot
Mouse
Barley
Tobacco
CBF3
HVA1
Yeast
A. thaliana
A. thaliana
HAL1
ATNHX1
CBF1
Oat
Tomato
S. cerevisiae
Oyster
mushroom
Potato
Tobacco
Synthesized
based on Winter
flounder
E.coli
Osmotin
AFP (synthetic
antifreeze protein)
OtsA
OtsB
TPS1
Glyceraldehyde -3
phosphate dehydrogenase
CDSP32
Tomato
Potato
Cu, ZnSOD
Osmotin like protein
Potato
–
–
Pea
A. thaliana
MnSOD
MnSOD
Apx
GR
Cotton
Response against abiotic stress not studied
Higher osmotic tolerance
[100]
[99]
[98]
[95]
[96]
[97]
[94]
Enhanced tolerance to oxidative damage
Improved salt tolerance
Improved fruit yield and K+/Na+ selectivity
Tolerance against 200mM NaCl stress, enhanced tolerance to
water deficit stress, catalase activity increased and H2O2 decreased
Elevated tolerance to chilling and oxidative stresses,
catalase activity induced
[92]
[93]
[91]
[89]
[90]
[86]
[88]
[83]
[84]
[85]
Improved drought tolerance
Improved salt tolerance
No trehalose accumulation,abiotic stress tolerance not determined
Enhanced tolerance to oxidative stress
No appreciable role in freezing tolerance but showed increased
tolerance to late-blight
Enhanced tolerance to drought and salt stress
Enhanced tolerance to freezing stress
Enhanced tolerance to photooxidative and low temperature effect
No tolerance conferred to low temperature and high light
Protection to photosynthesis against moderate chilling and high
photon flux density
Transgenic Crops for Abiotic Stress Tolerance 355
356
TAYAL, SRIVASTAVA AND BANSAL
were generated [59]. One of the transgenic lines retained more than 90% PSII activity whereas
control plants lost it completely after two weeks of stress. The same transgenic line also exhibited
resistance to cold stress as observed in a preliminary experiment. Zhu et al. (60) overexpressed
full length cDNA of ∆1-pyrroline 5 carboxylate synthetase (P5CS) in rice under ABA-inducible
promoter complex (AIPC) [60]. The transgenic plants showed better fresh root weight as compared
to controls under salt stress (100 mM NaCl). The transgenics showed higher growth rate as
compared to the control plants under water stress as well.
A full-length cDNA encoding CDPK was cloned from rice and overexpressed in rice under
the control of CaMV 35S promoter for detecting its physiological function [61]. The transgenic
plants showed tolerance against cold and salt/drought stresses. Overexpression of the OsCDPK
in rice induced the expression of many other genes such as rab16A, SalT and Wsi18 under salt/
drought but not under cold stress. This suggested the presence of two distinct ABA-induced
pathways using a single CDPK, one that is induced by salt/drought stress, whereas, the other
induced by cold stress.
For engineering Lea group of genes in rice, suspension culture of rice, Oryza sativa L. (cv.
Nipponbare) were transformed with HVA1 gene encoding the LEA III group of proteins [62].
Later, the HVA1 gene was overexpressed for the improvement of abiotic stress tolerance in
Basmati rice [63]. The transgenic plants maintained growth rates higher than control plants
under water deficit and salt stress conditions. However, the use of stress-inducible promoter
gave better results in term of stress tolerance than the constitutive promoter under stress conditions.
Further, transgenic plants harboring PMA80 gene (encoding LEA II group protein) and PMA
1959 gene (encoding LEA group I protein) were developed separately [64] and the role of these
proteins against dehydration and salt tolerance was studied. The tolerance level of transgenic
plants with PMA80 gene was higher than the plants with PMA 1959 gene.
In an attempt to decipher the role of polyamines in stress tolerance, Malabika Roy and Ray
Wu [65, 66] generated transgenic plants with adc gene and samdc gene, respectively. Both the
types of transgenic plants which accumulated polyamines to a significant level exhibited increased
tolerance to environmental stress to almost an equal extent.
The evidence confirming tolerance against low temperature stress in rice came through
overexpression of catalase gene. The transgenic rice plants overexpressing the catalase gene
displayed less damage as compared to control plants against a treatment of 5°C for 8 days. This
enhanced tolerance to cold stress in transgenics was attributed to higher detoxification of H2O2
by enhanced catalase activity [67].
Tolerance to multiple abiotic stresses was introduced by the overexpression of trehalose
biosynthetic genes (OtsA and OtsB) as fusion gene in rice plants [68]. The transgenic plants
showed better growth, less photo-oxidative damage and more favourable mineral balance than
that of the non-transgenic controls under drought, salinity and low temperature stresses. More
recently, Jhang et al. [69] reported tolerance against drought, salinity and low temperature by
introducing gene encoding a bifunctional fusion protein trehalose 6-phosphate synthase and
trehalose 6-phosphate phoshatase, in transgenic rice plants. High level of trehalose accumulation
resulted in multiple stress tolerance that was attributed to enzymatic activities of both the enzymes.
3.3 Mustard
Mustard is one of the important oilseed crops grown all over the world. Brassica juncea, the
Transgenic Crops for Abiotic Stress Tolerance 357
Indian mustard is the second most important oilseed crop in India. A number of transgenics have
been developed in Brassica species with improved abiotic stress tolerance. Among them,
overexpression of glutathione synthetase showed enhanced accumulation and tolerance to cadmium
[70]. In another study, overexpression of glulathione reducatase (GR) targeted to cytoplasm did
not show any cadmium tolerance, whereas targeted expression in chloroplasts did show higher
cadmium tolerance [71].
Transgenic mustard showing tolerance to salinity stress have been developed [72]. Glycine
betaine biosynthesis pathway gene codA encoding choline oxidase was introduced into B. juncea.
The transgenic plants showed significantly improved performance as compared to control plants
in terms of chlorophyll loss, photosystem II activity and shoot growth under stress conditions
[73]. A very interesting example of salt tolerance came through overexpression of AtNHX1 in
transgenic B. napus. These plants grew well in the presence of 200 mM NaCl, flowered and set
seeds. An increase in proline content was observed attributing to osmotic adjustment [74].
Our recent studies have shown that overexpression of osmotin gene in transgenic Indian
mustard enabled plants to tolerate drought and salinity stresses. The transgenic plants exhibited
increased level of water retention by excised leaves at the laboratory bench as compared to the
wild type plants of cultivar Pusa Jaikisan. In addition, loss of chlorophyll in the presence of salt
stress (100-200 mM NaCl) was retarded in transgenic leaf discs [75].
3.4 Soybean
Soybean is an important source of nutrition to human beings. Antisense soybean transgenic
plants with L-∆1-pyrroline 5 carboxylate reductase (P5CR) gene under the control of an inducible
heat shock promoter (IHSP) confirmed the potential role of proline in stress tolerance. Investigation
of antisense plants under stress conditions provides a means to understand plant metabolic
pathways. The IHSP was fully activated at 32 and 42°C along with mannitol stress. The antisense
expression of P5CR gene resulted in significant decrease in proline accumulation in transgenic
plants. In contrast, control plants showed higher proline content, thus exhibiting better growth
under similar stress conditions [76]. This clearly suggests that overexpression of genes encoding
enzymes required for proline biosynthesis will lead to increased stress tolerance in transgenic
crops including soybean.
3.5 Alfalfa
Medicago sativa is an important perennial forage legume all over the world. First transgenic
alfalfa plant with improved tolerance to abiotic stress was developed by McKersie et al. [77] by
overexpressing MnSOD cDNA under CaMV35S promoter. The transgenic plants showed more
rapid growth recovery after exposure to freezing stress than that of control plants. After 3 years
of field trials in 1996, data suggested that these plants also showed improved survival to water
deficit stress, as determined by chlorophyll fluorescence and electrolyte leakage. During these
experiments only few transgenic alfalfa plants were obtained [78]. Moreover, variety RA3 used
for transgenic development was popular at the time of experiment but showed poor agronomic
performances. Consequently, two different clones of alfalfa, N4 and S4 were transformed with
MitSOD and ChlSOD genes [79]. Results confirmed the hypothesis that MnSOD overexpression
improves survival of transgenic alfalfa against abiotic stresses. Further McKersie et al. [80]
overexpressed FeSOD in transgenic alfalfa for investigating its role in stress tolerance as compared
358
TAYAL, SRIVASTAVA AND BANSAL
to the MnSOD. The transgenics showed increased FeSOD activity along with increased winter
survival. However, this improvement in winter survival was not due to improvement in oxidative
stress tolerance associated with photosynthesis.
For testing the synergy between SOD transgenes and stress tolerance, gene-pyramiding studies
were conducted. Samisk et al. [81], crossed a hemizygous Mit-MnSOD plant and hemizygous
Chl-MnSOD transgenic plants. F1 progeny containing joint expression of the two genes (MitMnSOD + Chl-MnSOD) had lower shoot and storage organ biomass compared to either of the
parent, whereas, the progeny containing either of the transgene had significantly higher shoot
and storage organ biomass.
In another experiment, overexpression of transcription factor Alfin1 in transgenic alfalfa
improved growth properties of plants exposed to 128 mM NaCl stress for 17 days [82]. Alfin1,
cDNA encodes zinc finger family of transcription factor which binds to promoter fragment of
root-specific MsPRP2 gene. The MsPRP2 gene is also induced by NaCl stress. The transgenic
plants with Alfin1 overexpression showed MsPRP2 accumulation, thereby confirming the role of
Alfin1 in imparting enhanced NaCl stress tolerance to alfalfa plants.
3.6 Cotton
As photosynthesis in cotton is highly sensitive to low temperature and high light stress, focus
has, therefore, been on developing transgenic to protect the photosynthetic machinery under
abiotic stresses. Overproduction of MnSOD in chloroplasts of cotton conferred a substantially
enhanced tolerance to photo-oxidative stress (high light) and low temperature [83]. However,
Payton et al. [84] failed to achieve photosynthetic stability against low temperature stress in
transgenic cotton overexpressing chloroplast MnSOD [84]. Attempts were also made to improve
cotton for tolerance against abiotic stresses by overproducing chloroplast targeted glutathione
reducatase (GR) and ascorbate peroxidase (APx) [85]. Elevated levels of GR or Apx activity
improved photosynthetic capacity after chilling treatment at 10°C and high photon flux exposure.
3.7 Potato
Potato is highly sensitive to abiotic stresses. Perl et al. [86] developed transgenic plants by
transforming potato tubers with Cu/Zn superoxide dismutase. These transgenic lines showed
elevated tolerance to superoxide generating herbicide paraquat (methyl viologen). Induction of
osmotin-like protein by low temperature stress in potato was shown by Zhu et al. [87]. However,
transgenic potato expressing sense and antisense genes for osmotin-like proteins showed no
statistical difference among sense/antisense transgenics and control plants against low temperature
stress measured as electrolyte leakage. This ruled out the possibility of role of osmotin-like
proteins as a major freezing tolerance determinant [88]. Our unpublished results with osmotin
overexpressed transgenic potato have confirmed the role of osmotin protein in imparting tolerance
to osmotic stresses caused by drought and salinity [89].
The first evidence of potato transgenics tolerating freezing stress came through the expression
of a synthetic AFP-PHA (antifreeze protein gene fused to phytohemagglutinin) gene construct.
Phytohemagglutinin acted as signal peptide directing the antifreeze protein molecule to extra
cytoplasmic space where ice crystallization occurs. Transgenic plants showing maximum level
of AFP expression showed the highest degree of tolerance against freezing stress [90] as evidenced
by significantly reduced electrolyte leakage in transgenics as compared to the wild type plants.
Transgenic Crops for Abiotic Stress Tolerance 359
Goddijin [91] tried to develop stress tolerant transgenic potato by engineering trehalose biosynthesis.
However, surprisingly no trehalose accumulation was observed in transgenics, which was attributed
to trehalase activity. Later, the role of trehalose as an osmoprotectant was confirmed by expressing
TPS1 (Trehalose 6-phosphate synthase) gene in potato plants [92]. Although, the transgenic
potato plants showed abnormal morphological characteristics, such as dwarfism, yellowish lancet
shaped leaves and aberrant root development, drought resistance capacity of these plants was
significantly increased.
Overexpression of glyceraldehyde 3-phosphate dehydrogenase in transgenic potato showed
increased tolerance to salt stress [93]. Overexpression of chloroplastic drought-induced stress
protein (CDSP32) conferred protection to transgenic potato against photooxidative stress induced
by incubation with either methyl viologen or t-butyl hydroperoxide or by exposure to low
temperature. On the contrary, plants without CDSP32 expression showed enhanced damage to
photosynthetic membrane [94].
3.8 Tomato
Tomato is a widely grown vegetable crop. Higher ability to withstand salt tolerance was observed
in transgenic lines of tomato expressing HAL gene [95]. Further elucidation of these transgenics
for long term salinity effects showed many improved characteristics as compared to control. On
exposure to 35 mM NaCl concentration, 58% reduction in fruit yield was observed in control
plants whereas in transgenic plants expressing AtNHX1 the loss was 30%. Similarly, loss of leaf
water content was higher in controls than the transgenics under 100 mM NaCl. These plants also
maintained higher K+/Na+ selectivity values [96]. A remarkable example of tolerance to 200mM
NaCl and preserving fruit quality at such a higher concentration was shown in transgenic tomato
plants overexpressing Na+/H+ antiporter gene [97]. Accumulation of salts was observed in leaves
without affecting the fruit quality.
Overexpression of a transcription factor gene encoding CBF1 in transgenic tomato conferred
enhanced tolerance to water deficit stress. Lack of water for 4 weeks showed 80% survival in
transgenic plants as compared to less than 6% survival of control plants. Water content of
transgenics was relatively high during stress treatment. However, these plants showed retardation
in growth resulting into reduction in number and fresh weight of fruits [98].
3.9 Oat
Oat, a cereal crop serves as an important component of human and animal diets. This crop
requires sufficient water for growth and grain production. Overexpression of HVA1 in transgenic
plants showed higher osmotic tolerance than non-transgenics. Under NaCl and mannitol mediated
stresses, there were significant differences in wilting, death of old leaves and necrosis of young
leaves between the transgenic and non-transgenic plants [99].
3.10 Carrot
To our knowledge, there seems be no report on transgenic carrot development tolerant to drought,
salinity or low temperature stress. Efforts have been made in this direction to increase polyamine
levels by expressing ornithine decarboxylase (ODC) in transgenic cell lines of carrot. Detailed
metabolic studies of transgenic lines revealed higher rate of putrescine anabolism as well as
catabolism producing spermine and spermidine as compared to non-transgenic cell lines [100].
However, the effect of abiotic stress was not tested on these transgenic cell lines.
360
4
TAYAL, SRIVASTAVA AND BANSAL
Conclusions and Future Perspectives
Development of crop cultivars tolerant to abiotic stresses is an important goal of national and
international institutions engaged in plant research. Both traditional plant breeding methods and
transgenic technology are being employed to achieve the above objective. Since conventional
breeding approaches were not found sufficient, scientists are now trying to explore the advantages
of the transgenic technology to develop transgenic crops tolerant to abiotic stresses viz. drought,
salinity, cold and high temperature, etc. Although numerous studies have demonstrated the
feasibility of developing such transgenics in an array of crop species, substantial data are lacking
on the response of these transgenics subjected to field stress conditions.
Abiotic stress tolerance is a complex trait that is controlled by multiple genes. Studies in early
1990s demonstrated that a battery of genes get up-regulated in plants that are exposed to drought
or salinity stress. However, function of majority of these stress-induced genes/gene products
remained largely unknown. With the advent of high throughput sequencing of genes (genomics)
and proteomics, more and more ESTs/cDNA/genes or proteins are being added to the list by the
global effort with little information on elucidation of their function or the mechanism of stress
tolerance in plants. Genome wide approaches coupled with reverse genetics approach will surely
allow deciphering the role of specific gene / gene combinations in stress tolerance. Undoubtedly,
studies on stress signal perception and transduction have identified genes that play a significant
role in controlling the expression of stress-induced genes. As a result, transgenic development
with genes encoding transcription factors and/or protein kinases have provided tolerance to
multiple stresses to significantly high levels, and has increased the hope of generating transgenic
crops cultivars with improved stress tolerance.
Although there are numerous examples of transgenics over-expressing genes encoding enzymes
for increased osmolyte biosynthesis, stress-induced proteins, ROS scavengers and membrane
proteins, their field performance is awaited. To make the transgenic route more effective with
respect to tolerance against abiotic stresses at the field level, it will be important that crop
species-specific research programmes are undertaken for developing transgenics considering the
crop phenology, water requirement, type of stress experienced by the crop, growth stages sensitive
to stress, and the existing response of the crop to a given stress. For each crop, it is necessary to
understand the basis of effective engineering strategies leading to greater stress tolerance.
Information on genetic regulation and complex interaction of genes with environment is scanty
and further studies in this direction will help understanding the molecular mechanism of stress
tolerance in plants. Difference in molecular response of plants subjected to drought and salinity
stress also need to be addressed, as salt-specific effects are different than drought [101]. For
making the transgenic technology more effective, it is required that transgenes are expressed in
a specific tissue in a developmental or stress-inducible manner. Moreover, integration of cellular
and whole plant response is required for combining increased stress tolerance with high yields.
Acknowledgement
DT thanks Council of Scientific and Industrial Research (CSIR), New Delhi for financial assistance
in the form of SRF.
Transgenic Crops for Abiotic Stress Tolerance 361
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Plant Biotechnology and Molecular Markers
P.S. Srivastava, Alka Narula and Sheela Srivastava (Editors)
Copyright © 2004 Anamaya Publishers, New Delhi, India
23. Cell Differentiation in Shoot Meristem:
A Molecular Perspective
Jitendra P. Khurana*, Lokeshpati Tripathi, Dibyendu Kumar,
Jitendra K. Thakur and Meghna R. Malik
Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi 110021, India
Abstract: The basic body plan of higher plants is laid down during embryogenesis, however, the
entire adult plant develops post-embryonically through the activity of two meristems (shoot and
root apical meristems) established originally at the opposite ends of the embryo. This article
focuses on the shoot apical meristem (SAM), which is primarily responsible for the formation of
leaves and stems in the vegetative phase and converts into reproductive meristem at a specific stage
of development. The SAM comprises a central zone harboring a reservoir of pluripotent stem cells
and a peripheral zone, which gives rise to primordia for organs such as leaves and flowers. Studies
in the past decade have unravelled some of the molecular pathways that determine stem cell fate in
the central portion of the SAM as well as regulate organ formation from peripheral zone of SAM.
These studies are providing insight into the information flow between various zones and cell layers
of the SAM that helps in stabilizing the size of the stem cell population, so vital for cellular
proliferation and regulation of plant growth and development.
1.
Introduction
A vascular plant begins its life cycle as a simple unicellular zygote. The zygote develops into the
embryo and eventually into the mature sporophyte. These developmental events involve cell
division, enlargement and differentiation, and organization of cells into tissues or organs. After
a certain period of vegetative growth, the plant enters the reproductive stage with the development
of specialized structures. The most striking feature of plant development is that plants continue
to develop new organs after embryogenesis. Thus, portions of embryonic tissue persist in the
adult and juvenile tissues in plants throughout their life cycle. These tissues are a specialized
group of stem cells, called meristems, primarily concerned with the formation of new cells. Stem
cell populations act as self-renewing populations of cells that give rise to one or more differentiated
cell types. Angiosperm shoot and root apical meristems consist of such a population of cells
located at their tips. The primary growth initiated in the apical meristems expands the plant
body, increases its surface, leading eventually to reproductive development. In addition, many
plants possess additional extensive meristems, the cambia, which aid in increasing the volume
of the conducting system and forming supporting and protective cells.
During vegetative growth, shoot apical meristems (SAMs) give rise to repeating units of
shoot called phytomers. Depending on their location within the shoot, the successive phytomers
differ in internode length, leaf size and shape, axillary bud etc. A typical phytomer consists of
a node to which leaf is attached, a subtending internode and an axillary bud at the base of the leaf
(Fig. 1). The axillary bud has a meristem similar to SAM and can give rise to indeterminate
Cell Differentiation in Shoot Meristem: A Molecular Perspective 367
Shoot apical meristem
within apical bud
Leaf
Node
Phytomer
Axillary bud
Internode
Fig. 1
Schematic representation of the plant shoot apex (adapted from Langdale, 1994)
structures. SAMs are formed at distinct times and locations in the embryo, in the axils of the
leaves and in modified form as floral meristems. The plant architecture is essentially determined
by the pattern of meristem formation.
SAMs perform two main functions—maintaining a self-renewing population of initial cells
and producing the fundamental parts of shoot system—leaves and stem. These two functions
must be spatially coordinated. Leaves develop on the flanks of SAM in a defined arrangement,
whereas initial cells are present in the center of the SAM. Unravelling the communication
pathways that guide the SAM cells to continuously coordinate these two processes is critical for
understanding and ultimately manipulating the plant form. Mutants defective in SAM formation
and/or function have been identified in Arabidopsis and some other plant species. These studies
have not only helped in understanding the functional domains of SAM but also indicated that the
localized signaling between different cells and regions of SAM is an important component of
SAM functioning.
2.
Structure and Organization of the Shoot Apical Meristem
Vegetative shoot apices vary in size, shape, cytological zonation and meristematic activity. The
shape and size of the apex change during the development of a plant from embryo to reproduction,
and also in response to seasonal variations [1]. The average diameter of SAM is about 100 to 200
microns, although SAMs as small as 50 microns and as large as 900 microns have been reported
from diverse species. The size and shape of SAM also vary within a plant during plastochron
(time interval between production of successive leaf primordia). Toward the end of the plastochron,
just before the emergence of a distinct leaf primordium, the SAM is largest, whereas at the
beginning of the plastochron, just after the leaf primordia has been produced, the SAM is at its
smallest [2]. The changes in the morphology of the shoot apex occurring during one plastochron
may be referred to as plastochronic changes.
In dicotyledonous plants like Arabidopsis, the SAM, covered and protected by leaves in a
bud, consists of a small dome of cells, organized into regions with different functions and fates.
The outermost region of meristem consists of one or more sheets of cells that divide anticlinally
and together comprise the tunica. The tunica lies above corpus, which comprises of a mass of
cells that divide obliquely and periclinally.
368
KHURANA ET AL
Cell layers within SAM are shown as L1, L2, L3 etc., where L1 refers to the outermost layer,
L2 to the next subjacent layer and so on (Fig. 2). One to five layers of tunica have been reported
for dicots, with two layers present in most species. In Arabidopsis, SAM consists of three layers
— L1 and L2 comprise the tunica while L3 is the corpus. Monocots have one to four layers with
one and two predominating. In maize, both tunica (L1) and corpus (L2) are single layered [1, 3].
Clonal layers
Shoot apical meristem
L1
Floral meristem
L2
L3
Histological zones
CZ
PZ
PZ
RZ
Fig. 2
Diagrammatic sketch of Arabidopsis SAM showing the clonal layers (L1-L3) and the histological
zonation (adapted from Fletcher and Meyerowitz, 2000). PZ, peripheral zone; CZ, central zone;
RZ, rib meristem zone.
The outermost layer L1 gives rise to a cell layer that covers all organs while the cells in L2
layer give rise to sub-epidermal tissue, the procambium and the part of the ground meristem. The
cells in the L3 layer give rise to the rest of the ground meristem and pith. However, studies using
genetic mosaics have shown that the position of the cell and not its clonal origin determines its
fate [4]. All three layers contribute to organ formation and the growth of the stem, indicating that
coordination of cell proliferation and cell fate specification is required during development [5].
One of the mechanisms responsible for communication of developmental signals could involve
the transfer of signalling molecules, proteins and RNA, through plasmodesmata that establish a
cyclic continuity between neighboring cells [6].
SAMs are also radially organized into zones. This pattern is termed cytohistological zonation,
which is reflective of different rates of mitotic activity of cells in different regions of the
meristem. The SAM consists of two different zones: (i) a central zone made up of stem cells (or
central mother cells) that are large and exhibit low mitotic index and (ii) a peripheral zone of
apical initial cells, which are undifferentiated and exhibit higher mitotic index, which surrounds
the central zone and is the site of leaf formation. Both the central and peripheral zones overlap
the tunica and corpus. At the flanks of the meristem is the morphogenic zone where organ
primordia are formed [7]. Initials in the peripheral zone lying below the central zone constitute
Cell Differentiation in Shoot Meristem: A Molecular Perspective 369
the rib meristem, which gives rise to the pith of shoot axis and is responsible for shoot elongation.
In addition to their cell division rates, these zones differ in terms of cytology, expression pattern
of marker genes and membrane potentials [5].
The capacity of the SAM for self-perpetuation and cell-type specification implies that SAM
harbors an extensive communication system. In apical meristems, cells are interconnected via
plasmodesmata, which are probably used to exchange molecules. The shoot meristem is
compartmentalized into a central and peripheral symplastic field, as revealed by use of tracer
dyes, that could restrict the diffusion of molecules to the cells within their boundaries [8].
Microinjection techniques have revealed that cells in the central zone are cytoplasmically coupled
in two symplasmic fields: one within the tunica and other within the corpus, which harbor
distinct signal networks. In addition, cells in tunica peripheral zone appear to be coupled into
symplasmic ring, surrounding the central field. These observations substantiate the tunica-corpus
concept as well as the apical zonation model, and indicate that tunica-corpus concept is
physiologically relevant!
3.
Origin of Shoot Meristem During Embryogenesis
In dicots, the SAM develops between the two cotyledon primordia, in the central portion of the
embryo. In contrast, SAM develops laterally on the embryo in monocots, at the base of the single
scutellum (Fig. 3). In maize, the first five leaves are produced during embryogenesis whereas in
Arabidopsis, two small leaf primordia are detectable at the end of embryogenesis [2,3]. The
origin and development of SAM during embryogenesis has been equivocal with respect to
whether cotyledons are formed from the SAM or if the SAM and cotyledons arise independently.
According to one school of thought, the cotyledons and SAM respond to positional signals and
establish their respective cell fates independently in the apical region of the globular embryo [2].
Alternatively, specification of either one may require prior specification of the other.
The vegetative meristem with characteristic tunica-corpus structure is not evident until the
torpedo stage of embryogenesis in Arabidopsis [9]. After cotyledons and provasculature are
clearly distinguishable, the apical histological zonation is visible and this is considered to be an
indicator for the activity of SAM [10]. It has thus been presumed that either the apical portion
of the globular embryo forms the SAM or the SAM is formed only after the tunica-corpus
structure is evident at the early torpedo stage of embryogenesis.
It has been shown by histological studies and clonal analysis in different species, such as
Arabidopsis [9] and cotton [11], that the cotyledons and SAM develop from distinct regions of
the globular embryo. The existence of mutations that adversely affect SAM formation but not
cotyledons (e.g. STM mutations described below) suggest that cotyledons are specified independent
of SAM specification. At the same time, mutations leading to loss of both the cotyledons and the
SAM exist. Such mutations could prevent the specification of cotyledons and thereby SAM
formation or affect the gene products involved in signal transduction to both SAM and cotyledon
primordia [2,9]. Alternatively, there are evidences to support the view that cotyledons are
homologous to leaves and that they are the first products of embryonic SAM. Thus, in bipolar
embryos, the SAM originates in the globular stage before the cotyledons arise, at the same time
when root apical meristem is also defined [10].
Recent molecular genetic analysis of mutants defective in embryonic shoot meristem development
has improved our understanding greatly. These mutants were identified during systematic screens
370
KHURANA ET AL
A.
100 microns
sc
B.
sc
c
c
c
sm
sm
sm
If
su
rm
100 microns
Fig. 3
su
SAM formation during embryogenesis. A. Arabidopsis: Shaded cells indicate pattern of STM
expression. The diagrams (from left to right) represent different stages of embryo development
from globular to mature stage. B. Maize: Shaded areas show pattern of kn1 expression. The maize
SAM develops on the side of the embryo; sm, shoot apical meristem; c, coleoptile; sc, scutellum;
If, first leaf; su, suspensor; rm, root apical meristem (adapted from Evans and Barton, 1997).
for mutants affected in SAM formation or embryo development. Some of these are described
below in some detail.
The lesions in the GURKE gene cause characteristic apical defects and the cotyledons are
either reduced, absent or reduced to knob-like structures [12-13]; roots are also short and hypocotyls
malformed. It could be ascribed to abnormal cell divisions occurring within the apical region
leading to establishment of no or only rudimentary cotyledonary primordia during early heartstage of embryogenesis. Post-embryonically also, gurke seedlings give rise to abnormal leaves
and stem-like structures, suggesting that GURKE gene, although involved primarily in the
organization of the apical region in the embryo, may play a role in post-embryonic development
[13]. The gk mutations are allelic with emb22 mutations, which do not have apical deletion
phenotype but are generally defective in morphogenesis and cellular differentiation and form
abnormal, thick tube-shaped leaves in culture. This suggests gk alleles to be weak alleles of
emb22 [2,12].
Cell Differentiation in Shoot Meristem: A Molecular Perspective 371
Mutations in MONOPTEROS gene affect both root and shoot apical meristem formation.
Mutant alleles of the mp gene eliminate both the hypocotyls and the root and many mutant
seedlings have two fused cotyledons or single cotyledon and also lack a shoot meristem [12]. MP
gene encodes a protein with features of transcriptional regulator harboring a DNA binding
domain that possibly binds upstream regulatory elements of auxin-inducible promoters and thus
modulate gene activities in response to auxin signals [14]. However, the role of MONOPTEROS
in embryonic SAM formation is still unclear.
The Arabidopsis mutants defective in TOPLESS (TPL) gene fail to form cotyledons and SAM
during embryogenesis. Severely affected tpl mutants consist of only root and hypocotyls with no
SAM or cotyledons, while the less severely affected seedlings may have both cotyledons and
SAM depending on the severity of phenotype. The TPL gene may be required for specifying
shoot fate in a general way or some aspect of embryonic pattern formation, but not for postembryonic SAM function [2].
The mutant analysis has also shown WUSCHEL gene to be necessary for maintaining structural
and functional integrity of the shoot meristem. The wus mutant apices have aberrant organization
and form a flat structure with abnormal cells in comparison to wild-type shoot meristem. The
SAM stem cells are miss-specified and undergo differentiation without becoming incorporated
into organ primordia, terminating into flat apices. Subsequently, new shoot meristems are initiated,
which again terminate after making a few leaves. The wus plants also initiate numerous adventitious
meristems, which form only a few organs and terminate prematurely. This stop/start phenotype
suggests separate regulatory mechanisms for shoot meristem initiation and maintenance. In wus
embryos, the shoot meristem appears to be initiated but displays defective organization, suggesting
WUS gene product is required for central zone function [15]. WUS encodes a novel sub-type of
the homeodomain protein family [16]. Homeodomain proteins regulate transcription in many
diverse species and are generally involved in developmental or cell type specification. The
characteristic homeodomain consists of about 60 amino acids that bind DNA in a sequencespecific manner. The helix-loop-helix-turn-helix structure and twelve highly conserved amino
acid residues are also essential features [17]. The WUS homeodomain conforms to these features
and is about 30% identical and 45%-50% similar to homeodomain sequences from diverse
organisms. WUS expression is initiated in the four sub-epidermal cells of the apical region of
embryo at the 16-cell stage and becomes gradually confined to sub-epidermal cells in the center
of the embryonic SAM through several asymmetric divisions. These WUS-expressing cells
specify the overlying cells to maintain their specification as stem cells [16].
The maize homeobox gene KNOTTED 1 (KN1) is a useful molecular marker for the SAM
[18-19]. The onset of KN1 expression during embryogenesis coincides with the first histological
recognization of SAM in maize embryos [19]. KN1 is not expressed in determinate products of
meristems such as leaves and floral organs and, even within the vegetative meristem, the KN1
mRNA declines tremendously in cells destined to form the next leaf [20]. KN1 was first defined
by dominant gain-of-function mutations affecting leaf development [21]. In both maize and
Arabidopsis, KN1-like genes are represented as a multigene family. The KN1 homeobox (KNOX)
genes characterized in maize share a high degree of similarity within and outside the homeodomain
and show overlapping expression patterns [22-23]. KN1 mutant analysis indicates that maize
KN1 gene product serves to maintain the cells in a meristematic or undifferentiated state and
thus KN1 may be involved in maintenance of the morphogenetic zone of the SAM [20–21].
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In Arabidopsis, SHOOTMERISTEMLESS (STM) displays a pattern of expression very similar
to KN1 [24]. STM expression is first detected in a few cells in the center of the apical half of the
mid-globular stage embryos, when the embryo consists of about 32-64 cells. In late-globular
stage embryos, STM mRNA appears in a stripe across the top half of the globular embryo and
later becomes restricted to the notch between the cotyledons in the heart, torpedo and mature
stage embryos [24]. Histological analysis indicates that STM mRNA accumulates in the cells
predicted to give rise to SAM [9,24]. The expression of STM mRNA persists in seedling and
adult plants and is detectable in vegetative, axillary, inflorescence and floral meristem. However,
STM mRNA is absent from leaves and leaf primordia, and even within the meristem, STM
expression is down-regulated at the site of primordia formation. The analysis of Arabidopsis
seedlings carrying mutations at the STM locus has indicated that STM gene affects embryonic
SAM initiation and spacing of the cotyledons [9,25]. The stm mutant shoot meristems, however,
initiate primordia in aberrant phyllotaxis with phenotypic defects and the cells in the center
appear to be incorporated into the ectopic primordia and undergo differentiation, suggesting
STM is required to maintain undifferentiated population of cells within the SAM. Alternatively,
it is proposed that STM is required for the initiation of the shoot meristem after appearance of
cotyledons, as in stm mutants cells in presumptive SAM fail to organize into a tunica-corpus
structure [9]. On the basis of these observations, it has been proposed that in the peripheral
region, STM is required to inhibit organ outgrowth and differentiation, while in the central
region, STM is required to inhibit differentiation and to inhibit SAM-specific program of
development [26].
The STM encodes a KN1-type of homeodomain protein of 382 amino acid residues and is,
therefore, likely to act as transcriptional regulator [24]. A possible target for STM is UNUSUAL
FLORAL ORGANS (UFO) gene, as UFO accumulates in early heart-shaped embryo in STMdependent manner, although ufo mutants are unaffected in SAM development. However, UFO
negatively regulates growth of inflorescence meristem and floral meristems, but promotes the
expression of floral organ identity gene APETALA3 [27].
Using transposon-mediated activation tagging, a mutant of Arabidopsis, drn-D (Dornroschen),
has been identified where shoot meristem activity is arrested prematurely, with the formation of
radicalized lateral organs [28]. The expression of the homeobox gene STM is downregulated
during development of drn-D mutant. Strikingly, the expression of CLV3 and WUS, which act
antagonistically to regulate stem cell fate in meristems is increased in drn-D mutants. The
cloning of DRN gene, revealed that it encodes an AP2/ERF-type transcription factor that is
probably involved in regulation of gene expression patterns, and eventually the cell fate in
developing meristems. Mutations in CUC1 and CUC2 (CUP-SHAPED COTYLEDON) cause
defects in separation of cotyledons, sepals and stamens as well as in the formation of SAM in
Arabidopsis. The CUC1 and CUC2 genes are functionally redundant and thus the defects described
above are most apparent in cuc1cuc2 double mutants. These two genes encode members of NAC
family of proteins that share a highly conserved N-terminal domain termed the NAC domain
[29–30]. Petunia NO APICAL MERISTEM (NAM) gene is another member of this family [31].
Petunia embryos in nam mutation background fail to develop a SAM and cotyledons are partially
fused. The NAM transcripts are first detected at late heart stage of embryogenesis and accumulate
in cells at the boundaries of meristems and primordia, indicating that NAM helps in determining
positions of meristem and primordia [31]. However, cuc1cuc2 and nam mutants give rise to
Cell Differentiation in Shoot Meristem: A Molecular Perspective 373
adventitious shoots showing normal vegetative and reproductive development, suggesting that
CUC1, CUC2 and NAM genes are not essential for SAM maintenance during later development
[31–32]. Another gene encoding NAC-domain protein, CUC3, with high similarity to CUC1 and
CUC2 has been identified [33]. The CUC3 mutant analysis indicates that CUC3 functions in
establishing the boundary between the cotyledons and in SAM formation. Expression analysis
in the overexpressor lines and in loss-of-function mutants suggests that CUC1, CUC2 and CUC3
act upstream of STM and are redundantly required for STM expression through yet other unidentified
factors [30, 32].
In the developing SAM, the ZWILLE/PINHEAD (ZLL/PNH) gene is also required for maintaining
stem cells in an undifferentiated state. In zll/pnh mutants, the cells in the SAM primordium do
not maintain STM expression and differentiate, and the defective SAMs that are formed terminate
shortly after germination. The adventitious meristems developing on these mutants resemble the
wild-type, although occasionally zll/pnh mutants do not initiate meristems in the axils of the
cauline leaves. However, the defect is limited to secondary inflorescences and may be secondary
to fasciation. Thus, ZLL/PNH seems to be required specifically for SAM formation in the
embryo [34]. According to another study, there is no correlation between fasciation and failure
to form axillary meristems and that ZLL/PNH is required for the efficient formation of axillary
meristems during post-embryonic development. This is consistent with the persistent expression
of ZLL/PNH mRNA in the meristem and the adaxial leaf domain [35]. The expression of ZLL/
PNH is first detected at four-celled stage. As the development proceeds, the expression becomes
progressively confined to presumptive SAM region and the provascular tissue.
The ZLL/PNH gene encodes a member of novel family of proteins found in many eukaryotes
and that includes the product of ARGONAUTE1 (AGO1) gene involved in leaf development and
meristem cell maintenance [36]. The rabbit translation initiation factor eIF2C is another family
member, suggesting a role for ZLL/PNH and AGO1 in translational control of development [37].
As mutations in ZLL/PNH result in specific defects, the gene could be involved in tissue- and/
or stage-specific translational control. ZLL/PNH function is necessary for regulating spatial STM
expression at later stages of embryogenesis and, thus, it is proposed that ZLL/PNH relays
positional information required to maintain stem cells of developing shoot meristem in an
undifferentiated state and is required for partitioning of the embryo apex by regulating spatial
STM expression [34–35]. Embryos doubly mutant for ZLL/PNH and AGO1 fail to progress to
bilateral symmetry and do not accumulate STM transcripts, suggesting these genes could encode
partially redundant functions in regulating growth and gene expression patterns during
embryogenesis [35]. Recent work on Drosophila homologue of ZLL/PNH and AGO1, called
PIWI, suggests that these genes have an ancestral function in stem-cell maintenance [38]. Thus,
signaling from differentiated cells to stem cells may represent a basic mechanism for stem cell
maintenance among diverse eukaryotes [37].
3.1 Axillary Shoot Meristem Formation
Shoot apical meristems form throughout the lifecycle of most higher plants. Three types of
SAMs form post-embryonically: vegetative SAMs in lateral positions along the main shoot axis,
inflorescence lateral SAMs, and floral meristems. Molecular genetic analysis indicates that
axillary SAMs share many common genetic determinants with the primary SAM formed in the
embryo [39]. However, the formation of the axillary shoot is inhibited by the shoot apical
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meristem and depends on the subtending leaf primordium. Axillary meristem formation in
Arabidopsis occurs in two waves: an acropetal wave forms during vegetative development, and
a basipetal wave forms during reproductive development [40]. Axillary meristems are believed
to originate from the SAM as detached meristematic cells, in the axils of leaf primordia [40].
However, in some cases as Heracleum, axillary meristems appear to arise from leaf primordium
rather than from separate meristematic cells [7, 41].
Numerous mutants affected in axillary meristem initiation have been identified, for example
auxin resistant mutants in maize and decreased apical dominance mutants in petunia [42]. In
tomato, lateral suppressor (ls) mutant prevents the initiation of the vegetative lateral SAMs but
not of floral or sympodial mersitems. In this mutant, the SAM was shown to be smaller than
normal, which may restrict the initiation of axillary bud primordia. The ls mutant has no effect
when present in L1 layer, whereas chimeras with the mutation in L2 and L3 have ls phenotype,
suggesting that internal cell layers are important for regulating lateral SAM formation. Another
mutant in tomato, torosa-2 (to-2), reduces the number of vegetative axillary buds. Both ls and
to-2 mutants have reduced cytokinin levels suggesting a possible cause of the mutant phenotype.
However, increasing cytokinin levels in the mutants failed to increase lateral SAM formation
although dormancy of existing buds was eliminated in to-2 [2, 42].
In Arabidopsis, two mutants identified with defects in axillary bud formation are in fact
defective in primary SAM. For example, pinhead mutations affect embryonic SAM where a leaf
or pin-like organ terminates the growth of the meristem [43]. In addition, the pinhead mutations
reduce the inflorescence SAMs in the axils of cauline and rosette leaves and are defective in
floral meristem formation. In comparison, the rev (revoluta) mutants unusually develop large
leaves, stems and floral organs but reduced numbers of vegetative and floral axillary shoots.
These mutants are defective in apical meristem activity resulting in premature termination of
shoot apex and in formation of abnormal or incomplete structures in place of axillary shoots,
indicating REVOLUTA plays a role in meristem maintenance [41]. The REV gene has been
identified and found to encode a predicted homeodomain leucine zipper transcription factor that
also contains START sterol-lipid binding domain [39]. REV gene is expressed at the earliest
stages of lateral shoot meristem and floral meristem formation. Within the inflorescence shoot
meristem, REV expression appears to mark the next 3–5 flower primordia forming on the flanks
of the shoot meristem, and its expression matches that of WUS and STM at stage 1 and stage 2,
respectively. These observations suggest that REV acts to establish meristem identity or activates
other meristem regulators. Within the organs, REV is expressed largely on the adaxial portion of
the organ and may be involved in establishing adaxial fate in cotyledons, leaves and floral
organs. REV is also expressed in developing vasculature and may be required for proper
differentiation of vascular elements within the SAM. The different activities of REV appear to
be functionally distinct [39]. Recently, another gene involved in axillary meristem formation has
been identified by the analysis of an Arabidopsis mutant, las (lateral suppressor), which are
unable to form lateral shoots during vegetative development [44]. This study also examined the
transcript accumulation of LAS and STM and showed that LAS gene works upstream to other
regulators of shoot branching like REV. Members of the MADS box gene family are highly
conserved transcription factors that play diverse roles in regulating plant development. Potato
MADS box 1 (POTM1) expresses most abundantly in vegetative meristems of potato (more
specifically in tunica and corpus layers), the procambium, the lamina of newly formed leaves
Cell Differentiation in Shoot Meristem: A Molecular Perspective 375
and developing axillary meristems [45]. The transgenic suppression lines of POTM1 exhibited
decreased apical dominance and enhanced axillary bud growth, coupled with 2-3 fold increase
in cytokine levels. This implies that POTM1 regulates cell growth in vegetative meristems and
indirectly regulates axillary bud development.
3.2 Adventitious Shoot Meristem Formation
Meristems also develop at locations other than those formed during embryogenesis, in the axils
of leaves and during reproductive development. These meristems form normally on many different
organs of adventitious shoots in a variety of plant species, such as root bearing shoots of
Convolvulus arvensis, and shoots initiating from cambial tissue of tree stumps. Epiphylly is
another example wherein organs or shoots develop upon a leaf as observed in plants such as
Bryophyllum, in which plantlets form along the margin of the blade. An epiphyllous shoot may
represent fusion or displacement of a normal axillary meristem or may be a true adventitious
shoot. Displacement of normal axillary buds into organ surfaces results in meristem formation
followed by change in meristem position relative to other plant parts due to differential growth,
e.g., Coryphantha [42]. However, in Bryophyllum, vegetative adventitious shoots are rather
initiated by remeristemization of differentiated tissues of mature leaves. In Bryophyllum, epiphyllous
shoots arise from cells of the leaf margin that stop dividing and remain blocked in G1 phase of
the cell cycle. In mature leaf, these cells become reactivated to form an undifferentiated meristem
that acquires zonation and forms a small shoot [42].
Adventitious SAMs have also been produced in transgenic plants, overexpressing KN-like
genes and cytokinin biosynthetic pathway genes. Tobacco and Arabidopsis transgenic plants
overexpressing maize KN1 gene show lack of apical dominance and are severely dwarfed [23,
46]. Leaves are thickened and lobed and, in severe cases, small shoots originate from these
diminutive leaves [46–47]. However, the ectopic expression of KN-like genes does not lead to
adventitious shoot production in species such as tomato. The analysis of transgenics overexpressing
a related gene KNAT1 in Arabidopsis showed that simple leaves are transformed into lobed
leaves with stipules in the sinus, the region at the base of the two lobes. Ectopic meristems also
arise in the sinus region close to the veins. The shoot-like characteristics of these leaves suggest
that KN1-related genes may have an important role in the regulation of leaf diversity [47].
The overexpression of isopentenyltransferase (ipt), bacterial gene involved in cytokinin
production, in transgenic tobacco plants leads to adventitious shoot meristem formation at the
site of Agrobacterium infection as well as the shooty phenotype of the transformed cells in
culture. The phenotype of the tobacco plants expressing either ipt or KN1-like genes are quite
similar. It has been observed that ipt transgenics have higher steady-state mRNA levels of
KNAT1 and STM, similar to cytokinin overproducing shoot meristem mutant amp1 [48], suggesting
that cytokinins possibly act upstream of KNAT1 and STM in the same pathway. This provides a
link between the hormone and developmental genes and indicates for a probable role for cytokinins
in the SAM formation [48–49].
Another study has reported that ectopic expression of TBP-2, the TATA box binding protein,
induces apical shoot proliferation in Arabidopsis [50]. The ectopic meristem-like structures
arose essentially from highly undifferentiated leaf primordium or from young ectopic shoots
during more advanced vegetative growth phases. The expression of some shoot meristem regulatory
genes such as STM, KNAT1, and CLV1 is altered in Arabidopsis apical shoots. This suggests that
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TBP-2 protein might be needed for apical meristem function and that high TBP-2 levels prevent
cell differentiation in undifferentiated cells, possibly by increasing transcription initiation and,
thus, play a role in controlling shoot production [50].
4.
Coordination of SAM Proliferation
The SAMs remain relatively constant in size throughout the life cycle of most higher plants, but
still continue to produce lateral organs. This indicates that cells within the SAM somehow
continually assess their relative positions and subsequently decide to divide, differentiate, maintain
status quo or restrict cell proliferation in the SAM. Several mutants accumulating rather too
many cells in the SAM have been identified in Arabidopsis. The first class is represented by the
recessive clavata mutants (clv1, clv2, clv3). clavata is derived from the latin word clava and
means ‘club’. The clavata mutants harbor excess cells in the central zone of the SAM and floral
meristems, resulting in fasciation of the shoot and generation of flowers with extra floral organs
and distortion of siliques, giving them a club-shaped appearance. Although the central zones of
both the shoot and floral clv-meristems appear enlarged, the organs initiating from the peripheral
zone grow normally [51–53]. The comparative analysis of cell division patterns suggests that
increase in meristem size in clv SAMs is due to reduction in drafting of these cells into the
peripheral zone, rather than increase in the cell division rates in the central zone [54]. The
second class of mutants include the mgoun mutants (mgo1 and mgo2), which display reduction
in the number of leaves and floral organs, larger meristems and fasciation of the inflorescence
stem. The molecular genetic analysis of these mutants indicates that MGO genes play a role in
organ primordia initiation and determining their number [55].
The epistasis analysis revealed that CLV1 and CLV3 act in the same pathway to control
meristem-cell proliferation [52]. The shoot and floral meristems of clv2, third mutant in this
group, are phenotypically similar to those of weak clv1 and clv3 mutants, and clv1/clv3 are
epistatic to clv2 with regard to size of shoot meristem and the number of flowers initiated by the
floral meristem. Thus CLV2, like CLV1 and CLV3, helps in preventing the accumulation of
undifferentiated cells at the shoot and flower meristems [53]. However, clv2 mutants display
other organ defects (e.g. reduced anthers/stamens) as well, suggesting that while CLV2 is required
to regulate meristem development, it also functions independent of CLV1 and CLV3 to regulate
organ development [53].
The cloning and molecular analysis of CLV genes has provided new insights into the mechanism
by which they regulate meristem cell proliferation. Both CLV1 and CLV2 genes encode
transmembrane leucine-rich repeat (LRR) transmembrane proteins [56–57]. The CLV2 receptorlike protein (RLP) carries only a short C-terminal domain, whereas the CLV1 protein harbors a
C-terminal serine/threonine kinase domain. There is evidence to suggest that CLV1 and CLV2
most likely form a heterodimeric receptor molecule localized in the plasma membrane [57].
LRRs are a common motif of protein-binding domains both in plants and animals suggesting
that the CLV receptor interacts with an extracellular protein ligand. LRR-receptor kinases have
been implicated in signal transduction cascades and more than 50 genes have already been
identified in diverse plant species in many cases. For example, the Arabidopsis ERECTA (ER)
gene encodes an LRR-receptor kinase. It is expressed in SAMs and flowers and is thought to
mediate cell-cell communication to accelerate cell division and elongation [58–59]. The
BRASSINOSTEROID INSENSITIVE 1 (BRI1) gene also encodes an LRR-receptor kinase that
Cell Differentiation in Shoot Meristem: A Molecular Perspective 377
most likely acts as a receptor for plant steroid hormone brassinolide [60–61]. Mutations in BRI1
gene cause dwarfism and plants grown in dark display light-grown phenotype. However, analysis
of CLV1/BRI1 chimeric receptors in clv1 mutant background provides evidence that CLV1 and
BRI1 kinase domains are not interchangeable [62]. The results of this study also indicate that
CLV1 can act outside the meristem to regulate the pedicel length in erecta mutant background.
In addition, several plant disease resistance genes encode LRR-receptors or receptor kinases that
enable plants to sense and respond appropriately to specific bacterial and fungal pathogens [63].
Interestingly, CLV3 encodes a secretary protein of 96 a.a. and carries an 18 a.a. N-terminal
signal peptide, suggesting CLV3 protein may be extracellular ligand, which may interact with
the extracellular domains of CLV1 and CLV2 receptors [37, 64]. CLV3 mRNA is detected
mainly in L1 and L2 layer of the central zone and probably demarcates the stem cells in these
layers; it is however not detected in the flanks of the meristem. In contrast, CLV1 is expressed
mostly in an underlying domain in the L3 layer and is not detected in the L1 layer. The CLV2
mRNA is detected in all the shoot tissues of the plant. This suggests that CLV3 may signal in a
non-cell autonomous manner from overlying to the underlying regions of Arabidopsis SAM
[56–57, 64]. However, until recently the experimental evidence that CLV3 acts as an extracellular
signaling molecule was lacking. Employing genetic and immunological assays, CLV3 has been
shown to localize to the apoplast [66]. Apoplastic localization permits CLV3 to signal from the
stem cell population to the organizing centre in the underlying cells, activating the CLV1/CLV2
receptor complex. Essentially a similar conclusion was drawn in a parallel study [65] whereby
it was shown that CLV3 functions as a mobile intercellular signal but its spread is regulated by
its receptor CLV1. This enables the shoot meristem to permit the peripheral cell differentiation
and yet maintains a stable niche for the stem cells in the middle. Biochemical and genetic
analysis shows that CLV1 function depends on the presence of functional CLV2 and CLV3, and
that CLV3 acts as a ligand for CLV1 as a part of multimeric complex [57, 67]. In vivo CLV1
forms an inactive complex of approximately 185 kDa, which is thought to consist of a CLV1
disulfide linked to CLV2, and an active complex of approximately 450 kDa, containing 185 kDa
complex, and a type-2C kinase-associated protein-phosphatase (KAPP), which has been shown
to act as a negative regulator of CLV1 signal transduction pathway [68–69].
The role of WUS gene product in CLV signal transduction pathway has been implicated (Fig.
4). As mentioned earlier, the WUS gene promotes stem cell fate and encodes a homeodomain
transcription factor that is expressed in the L3 layer of the shoot and flower meristems throughout
development. Mutations in WUS or the CLV genes have opposite phenotypes, indicating that
these genes promote and restrict stem cell formation in the central zone, respectively. wus
mutants are largely epistatic to clv mutants, indicating WUS functions downstream of and could
be a target gene for repression by CLV genes [15–16]. Recent studies have shown that WUS
mRNA is not confined to its normal expression domain in clv mutants, but expands both apically
and laterally, indicating CLV signaling restricts the boundary of WUS expression [70–71]. The
enlarged size of the shoot meristem in clv mutants may be a consequence of deregulation of
WUS activity, as a result of which more stem cells would be specified, causing expansion of the
central zone and eventual fasciation of the meristem. Indeed, constitutive expression of CLV3 in
transgenic Arabidopsis plants caused severe reduction in the levels of the WUS transcripts and
phenotypically these plants resembled wus mutants [70]. Transgenic plants overexpressing WUS
under the control of CLV promoter resembled clv mutants with large and fasciated meristems
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KHURANA ET AL
A.
CLV3
CLV1
WUS
B.
CLV3
CLV1
WUS
C.
CLV3
CLV1
WUS
Fig. 4
Control of stem cell maintenance. A. Expression of CLV3, CLV1 and WUS share common regions.
Expression of WUS is restricted to very thin area by CLV3 signaling involving CLV1 receptor
complex. B. Expression of WUS is decreased due to constitutive CLV3 signaling. C. When CLV
pathway is broken, restriction over limited area of WUS expression is relieved, resulting into
excessive stem cell accumulation and expansion of the meristem. The arrow indicates positive
regulation and blunt-ended line indicates negative regulation (adapted from Fletcher, 2002).
[71]. The expression of WUS under the control of ANT promoter that confers expression in organ
primordia and developing organs, leads to the termination of leaf formation and a large bulge of
cells similar to meristem cells is formed. In addition, these cells express the stem cell marker
CLV3, suggesting that WUS is sufficient to induce CLV3 expression at the correct position and
thus specify stem cell identity [71]. In a recent study, it has been found that expression of CLV3
depends on WUS function only in the embryonic shoot meristem. At later stages of development,
WUS stimulates CLV3 expression together with STM gene [70].
It is possible that there may be additional factors that function together with WUS and are
regulated by CLV to promote stem cell pathway. Mutations in the POLTERGEIST (POL) gene
have been identified as partial suppressors of meristem defects in clv mutants. When CLV genes
are functional, pol mutants are nearly indistinguishable from wild type plants. Like WUS, POL
may either encode for a transcription factor or a protein that regulates one [72]. It is proposed
that WUS together with other genes like POL acts to promote stem cell fate and CLV3 expression
in the overlying cells in a non-cell-autonomous manner. CLV3 signaling would act to repress the
activity of these regulatory factors. This mutual regulation, involving positive and negative
interactions, provides a feedback system maintaining the meristem size [70].
Cell Differentiation in Shoot Meristem: A Molecular Perspective 379
The role of CLV and STM genes in regulating SAM cell proliferation is rather antagonistic.
Unlike wus and clv mutants, clvstm double mutants display an additive phenotype, suggesting
that they act in separate pathways [73]. SAM development is defective in both stm and wus
mutants as both stm and wus mutants lack stem cells, however, terminal phenotypes are different
in two cases. Genetic analysis indicates that STM acts upstream of WUS and, although WUS and
STM expressions are initiated independently of each other in different meristem domains, expression
of one cannot be maintained in absence of the other. Therefore, while WUS (and possibly POL)
may be required to specify stem cells, STM activity allows their progeny to proliferate before
incorporation into lateral organ primordia [16, 25]. The stem cell promoting activity of STM and
WUS genes is restricted by CLV genes. It has been observed that mRNAs of various components
of the signaling network accumulate in non-overlapping domains of the meristem, suggesting
the involvement of all the cell layers in regulating meristem structure. This indicates the existence
of an intercellular communication network through symplasmic domains and exchange of
cytoplasmic components [74–75].
5.
Regulation of SAM by the Lateral Organ Primordia
The STM gene is required for SAM development in Arabidopsis and is expressed throughout the
meristem but is absent in organ primordia, indicating that STM expression is repressed in cells
that give rise to organ primordia [24]. In maize, expression of KNOX genes is regulated by
ROUGH SHEATH 2 (RS2), a MYB protein found only in lateral organ primordia and their
initials [76–77]. In rs2 mutants, KNOX genes are ectopically expressed in developing leaves
indicating that RS2 represses KNOX gene expression in lateral organs. PHANTASTICA (PHAN),
an RS-2 related gene in Antirrhinum, is expressed in organ founder cells and required to inhibit
expression of an STM orthologue AmSTM1 in lateral organs [78]. asymmetric leaves 1 (as1) is
a mutation in Arabidopsis that disrupts development of cotyledons, leaves and floral organs.
Loss of AS1 activity leads to misexpression of KNOX genes, KNAT1 and KNAT2, closely related
to STM, while STM expression itself is unchanged, and as1stm double mutants have as1 phenotype
except failure to form flowers, suggesting that STM is a negative regulator of AS1 [79]. In stem
cells, STM negatively regulates AS1, while in the organ founder cells STM is downregulated
permitting AS1 expression, which in turn downregulates KNAT1 and KNAT2 genes. Thus, stm
mutants fail to develop a meristem due to misexpression of AS1 in stem cells causing them to
differentiate. Lack of AS1 in as1stm double mutant allows meristem function possibly by
derepression of KNAT1 and KNAT2. The clv1, clv3 and wus mutants show additive interactions
with as1, suggesting AS1 along with STM acts independently of these genes in stem cell specification
pathway [79]. Recently, another gene ASYMMETRIC LEAVES2 has been identified, which along
with AS1 is involved in establishing the entire vein system and repression of class I knox genes
in the leaves [80].
6.
Meristem Patterning and Floral Determination
Floral meristems arise post-embryonically from the shoot apical meristem. Floral meristems are
modified shoot meristems since they have a similar structure and function. Since the two meristems
are functionally very similar, it has long been speculated that same genes may be involved in
regulating their function. In Arabidopsis, LEAFY and APETALA genes are involved in floral
meristem specification and mutations in these genes convert floral meristems into shoot meristems
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[81–82]. Conversely, mutations in TERMINAL FLOWER gene convert shoot meristems into
floral meristems [83]. An important difference between the two is that floral meristems are
determinate structures, producing flowers with fixed number of parts namely stamens, petals,
sepals and carpels, while shoot meristems produce indefinite number of leaves.
Two groups have independently shown that AGAMOUS (AG) gene, which has a role in
meristem termination and floral organ patterning, interacts with WUS [84–85]. AG is a MADS
box gene expressed in third and fourth whorls of developing flower and specifies stamens and
carpels. The ag mutants develop indeterminate flowers containing only sepals and petals [86].
In contrast, wus mutants display premature termination of SAM and floral meristems after
formation of a few organs, and the flowers formed lack carpels and most stamens. Thus, it is
possible that the two genes interact to control floral development.
In ag mutants, WUS expression persists in flowers whereas in wild type it is switched off
when carpel primordia are established. Also, in plants with reduced WUS expression in floral
meristems, AG expression domains are smaller, indicating WUS is required for AG activation.
Moreover, over expression of WUS using LFY and APETALA 3 (AP 3) promoters can cause
ectopic formation of stamens and carpels. It has been observed that wus mutants are epistatic
over ag mutants, suggesting that AG functions as a negative regulator of WUS [84-85]. In vitro,
WUS protein binds to consensus homeodomain target sites within the AG regulatory region and
these sites are necessary for expression of AG reporter gene in planta [85]. However, activation
of AG expression by WUS is restricted to floral meristems, suggesting requirement of additional
flower specific factors. It has been shown that one of these factors could be floral meristem
identity gene LEAFY [84-85]. Endogenous WUS is unable to activate AG expression in lfy
mutants [84] however, overexpression of WUS can activate AG promoter in absence of LFY
suggesting LFY requirement is not absolute [85]. LFY protein directly binds to sites in the AG
regulatory region and binding of both WUS and LFY is essential for the expression of AG since
activation of AG reporter gene in yeast occurs only when both LFY and WUS are coexpressed.
However, the two proteins bind their recognition sites independently and binding is not cooperative
[85].
These data suggest that stem cell termination in floral meristems requires a autoregulatory
mechanism involving WUS and AG. WUS is responsible for activation of AG expression in the
center of the floral meristem. AG once established, represses WUS either alone or in combination
with other factors, which results in the termination of the stem cell maintenance. This mechanism
is restricted to floral meristem since AG activation by WUS requires LFY [84–85]. This loop is
analogous to negative feed back loop between WUS and CLV3, which regulates stem cell
population in shoot apical meristem.
7.
Conclusions and Perspectives
The development of higher plants depends on the activity of the shoot meristem, a dynamic
structure consisting of self-renewing stem cells. The shoot meristem arises early during
embryogenesis and subsequently forms the basic subunits of the shoot, leaf and stem, in repeated
patterns. SAM maintenance depends on two antagonistic processes: stem cell self-renewal and
organ initiation. In order to achieve this, the cells need to be placed in proper fields of positional
information. Recent molecular and genetic studies have identified many components of the
intercellular pathways that play important roles in regulating meristem function. As a result of
Cell Differentiation in Shoot Meristem: A Molecular Perspective 381
these studies, the view that emerges is that SAM is a dynamic structure in which cell fate is not
predetermined; rather cell fate is in accordance with their positions relative to each other. These
studies also bring into limelight the significance of interactions between the SAM and the
developing leaf being necessary for axis specification. Thus, cells in the SAM continuously
modify their gene expression patterns in accordance with their environment. In this regard, a
number of receptors, protein ligands and putative transcription factors have been identified,
which are essential for meristem regulation. Sequencing of the Arabidopsis genome has helped
identify many possible components of the meristem signaling machinery such as target genes
and receptor molecules, however, their potential roles in SAM development need to be identified.
The signaling pathways known are just the tip of the iceberg and other pathways involving plant
hormones and other morphogens may also exist. The emphasis therefore must lie on understanding
the coordination between the events occurring during development and the intercellular
communication between the cells in the meristem. These areas offer a great deal of challenges
and promises for the future.
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Index
Abiotic stress, 70, 94, 159-61, 174, 196, 206, 296,
346, 347, 348, 350, 352, 353, 355-60
Abortifacient, 88-90
Abrus precatorius, 88
Acacia, 171
ACC synthase, 61
Acentric fragment, 340
Acetobacter, 65
Achillea ospenifolia, 100
Aconitum heterophyllum, 51, 52
Acropetal wave, 374
Actigard, 159
Actin, 35-37
Actinomycetes, 274
Actinomycin D, 10
Adapter molecules, 290
Adaptive evolution, 43
Additive phenotype, 379
Adenosine diphosphate, 133
Adrenaline, 133
Aeginetia indica, 53-55
Aegle marmelos, 197
Aequorea victoria, 34
AFLP, 80, 82, 99,100, 173, 174, 207, 290-92, 294,
296, 298, 299, 301-04
AFP-PHA, 358
ag mutants, 380
AGAMOUS (AG) gene, 174, 380
Agave amaniensis, 92, 94
Aglycones, 299
Agmatine, 62
AGO1, 373
Agrobacterium, 21, 67, 78, 81, 99, 101, 103, 175,
176, 234, 235, 375
Agrobacterium rhizogenes, 66, 67, 99-102, 139, 176,
212, 247, 285
Agrobacterium tumefaciens, 99, 101, 103, 104, 175,
176, 212, 234
Agronet, 198
Agroperlite, 208
Agropyron repens, 54
Ajmalicine, 92, 95, 120, 123
Ajmaline, 103
Albizzia, 171
Albuginacae, 157
Albugo candida, 156-59, 162, 301
Alfalfa, 61, 97, 354, 357, 358
Alfin, 358
Alginate, 81, 138, 172, 210, 211, 280, 282
Allele mining, 311
Allium cepa (onion), 133, 263
Allium sativum, 100, 133
Allium wallichii, 88
Allometry, 53
Allopolyploid, 18, 331, 341
Aloe vera, 267
Alternaria alternata, 310
Alternaria blight, 156
Alternaria brassicae, 156
Alternaria brassicola, 20, 156
Alternaria solani, 294, 304
Amaranthaceae, 158
Amaranthus caudatus, 94
Amber mutations, 63
American chestnut, 171
γ -aminobutyric acid (GABA), 61
Aminobutylcanavalmine, 61
Aminobutylhomo-SPD, 60
Aminocyclopropylcarboxylic acid, 62
Aminopropyl pyrroline, 61
Aminopropylcanavalmine, 61
Ammi majus, 88, 92, 94, 101
Amomum subulatum, 268
Amp1, 375
Amphipathic, 350
Amylose, 298
Anabasine, 103
Anacardiaceae, 244, 245, 248
Anacardium occidentale, 244, 245, 253, 257, 258
Anchusa officinalis, 123
Ancymidol, 170
Androgenesis, 1-3, 5-7, 10-13, 21, 25, 233
Androgenic, 2, 3, 5-9, 12, 13, 104, 228, 233
Anethum graveolens, 270
Anise, 270
Anisodus acutangulus, 98, 282, 286
Annona, 229, 230
Anogeissus, 187-90, 197
ANOVA, 219
ANT promoter, 378
388
INDEX
Anthraquinone, 82, 94, 96
Anthuriums, 271
Antiasthamatic, 88
Anticarcinogenic, 90, 91, 133
Anticholesterol, 132
Anticholinergic, 89
Antidiabetic, 89, 90
Antifertility, 89
Antifreeze proteins (AFPs), 349
Antihepatotoxic, 90
Antiinflammatory, 88, 89, 91, 133
Antileukaemic, 88
Antimalarial, anti-HIV, 88
Antioxidant, 132, 133, 197, 351
Antirrhinum, 379
Antisenescence, 63
Antisense, 34, 39, 66-69, 237
Antispasmodic, 88
Antistress, 91
Antithrombosis, 133
Antitumor, 89, 91
Antiulcer, 133
Antixenosis, 310
APETALA genes, 372, 379, 380
Aphanomyces euteiches, 303
Aphanomyces root rot, 303
Apium graveolens, 269
Apocynaceae, 217
Apomixis, apomictic, 130
Apoplast, 377
Apple, 226, 262, 266, 295, 296, 299, 309, 310
Apple scab, 311
Apricot, 262
Aquaporins, 349, 351
Arabidopsis, 34, 35, 38, 48, 49 63-65,174, 195, 302,
347, 348, 350-55, 367-72, 374-77, 379, 381
Aralia cordata, 123
Arginine decarboxylase (ADC), 61-70, 104, 348,
356
ARGONAUTE1 (AGO1) gene, 373
Armoracia rusticana, 158
Arnesyl diphosphate synthase, 104
Arnica montana, 88
Aromatic plants, 261, 267
Arteannuin B, 101
Artemisia, 83, 88, 92, 100, 101,104
Artemisinin, 92, 101, 104
Artemisnic acid, 101
Arthrobacter globiformis, 348, 353, 354
Arthritis, 129, 132
Artocarpus heterophyllus, 230
Ascochyta, 295, 304
Ascochyta blight, 304
Ascorbate peroxidase (Apx), 175, 349, 351, 358
Asparagus, 97, 276
Asterad-, Asteraceae-type, 47, 49, 50, 54
‘Asymmetric leaves’ 1, 2 (as1, 2), 379
Atherosclerosis, 132, 133
Atropa, 286
Atropa acuminata, 88
Atropa baetica, 100
Atropa belladonna, 97, 98, 101, 103, 282
Atropine, 101, 103
Azadirachta indica, 99, 100, 171
Azotobacter, 274
Bacillus thuringiensis (Bt), 176, 177, 273, 310
Bacopa monniera, 84, 88, 94
Bacterial wilt, 69
Balanites aegyptiaca, 195, 197, 200, 201
Balsamodendron mukul, 129, 133
Bambusa arundinacea (bamboo), 171, 172, 188,
190, 191
Banana, 210, 229, 234, 262, 264-66, 273, 295
Barley, 12, 21, 33, 36, 97, 294, 299, 309, 310, 354,
355
Barley gene Cor, 14b, 351
Barley yellow dwarf, 294
Barley yellow mosaic, 294
ß-carotene, 150
B-chromosome, 331, 337
ß-element, 131
Berberine, 95, 96, 123, 124
Beta vulgaris, 92, 93, 285
Betacyanins, 94
Beta-glucan, 95
Betaine synthesis, 174
Betalains, 92
Betelvine, 268
Betula pendula, 171
Bioprocesses, 79, 117
Bioreactor, 117-125, 137, 139
Bio-safety, 169, 170, 177
Biotic stress, 19, 95, 156, 159,160,162, 191, 212,
346
Black pepper, 268
Blackleg, 156
Blackmold resistance, 310
Black-plum, 197, 202
Bombyx mori, 206
Boraginaceae, 158
Boswellia, 130, 133
Index 389
Botrytis, 96
Bradyrhizobium, 274
Brassica, 7, 8, 11, 18, 21, 37, 144-146, 148, 151,
156-62, 295, 297, 356
Brassica aegyptiaca, 198-202
Brassica campestris, 18, 19, 21-24, 98,145, 146,
157-161, 302
Brassica campestris-oleracea, 302
Brassica carinata, 18-20, 22, 24, 157-160
Brassica carteri, 133
Brassica juncea, 18, 19, 22-24, 144-48, 151, 157-62,
301, 302, 356, 357
Brassica napus (rape seed), 2, 3, 5, 6, 8, 11, 18-25,
145-150, 157-162, 301, 302, 357
Brassica nigra, 18, 19, 21, 157, 158
Brassica oleracea, 18, 19, 157-160, 302
Brassica rapa (turnip rape), 301, 302
Brassica serrata, 133
Brassica spinenscens, 160
Brassica tournefortti, 162
Brassinolide, 377
BRASSINOSTEROID INSENSITIVE 1 (BRI1) gene,
376, 377
BRI1 kinase, 377
Bridge fragment, 339, 340
Broccoli, 276
Bronowski gene, 146
Bryophyllum, 50, 55, 375
Bunium persicum, 270
Burseraceae, 129, 130, 133
Butanedioic acid mono (2, 2-diemthyl-hydrazide)
diaminozide, 172
t-butyl hydroperoxide, 359
3-carene, 131
Citrus sinensis × Poncirus trifoliata, 230
Cabbage, 263
Cadaverine, 63, 103
Caesin kinase, 63
Caffeic acid, 175
Calamus, 173
Calathea, 271
California bay plant, 150
Calmodulin, 34, 353
Calystegia sepium, 103
Cameleon, calcium indicator, 35, 38
Campanula, 341
Camphor, 269
Camphorene, 131
CaMV 35S promoter, 65-67, 356, 357
Canavalmine, 61
Candida albicans, 95
Canola, 144, 147, 150
Capparis decidua, 197
Capric acid, 149,151
CAPS, 290, 291, 296, 301, 304, 308, 311
Capsaicin, 95
Capsella bursa-pastoris, 49, 54, 158
Capsicum frutescens, 95
N-carbomyl putrescine, 62
Cardamom, 268
Cardiac glycosides, 89
Cardiac lipidosis, 145
Cardioprotective, 133
Cardiovascular, 90
Carica papaya, 230, 232, 235
Carica papaya × C. cauliflora, 232
Carnation, 298
Carotenoids, 150, 285, 306
Carrot, 12, 66, 97, 263, 359
Caryophyllaceae-type, 47
Caryophyllene, 131
Cashew nut, 244-46, 248, 250-53, 255-59
Cassava, 210, 295
Castanea dentata, 171
Casuarina, 171, 175
Catalase, 349, 355, 356
Catharanthene, 92, 96, 123
Catharanthus, 65, 82, 85, 88, 92, 94-96, 101, 103,
104, 120-25, 267, 282, 286
Cauliflower, 263
Cauligenic/caulogenesis, 43, 229
Cauline, 374
CDPK, CDPK isoforms, 353, 356
Celastrus paniculatus, 197, 202
Celery, 97, 269, 276
Central cell, 31-34, 36, 38
Ceratonia siliqua, 171
Ceratophyllaceae, 53
Ceratophyllum demersum, 53, 54
Cercospora zeamaydis, 294
Cereal cyst nematode, 296
Chalara elegans, 295
Chaperon, 350
Chenopodiaceae, 158
Chenopodiate/Chenopodiaceae-type, 47, 49
Chenopodium, 85
Chinese cabbage, 276
Chinese cassia, 269
Chitinases, 174
Chitosan, 95
Chlamydomonas, 61
390
INDEX
Chlorella, 61
2-chloroethyltrimethyl ammonium chloride (CCC),
172
Chlorophytum borolivilianum, 267
Cholestane, 131
Cholesterol, 92, 124, 125, 129, 132-34, 157
Choline, 348
Choline dehydrogenase (CDH), 348
Choline monoxygenase (CMO), 348
Choline oxidase (COD), 348, 357
Chrysanthemum, 272
Cicer (chick pea), 295, 346, 304
Cichorium/chicory, 97, 99, 100, 282
Cinchona, 92, 94, 95, 101, 103, 282
Cinchonidine/cinchonine, 101
Cinnamomum, 269
Cinnamoyl, 123
Citrus, 94, 97, 202, 228, 229, 231, 233, 234, 261,
262, 266
Citrus acida, 230
Citrus aurantifolia, 230, 232
Citrus aurantium, 230, 232
Citrus berryi, 129
Citrus clementina, 232
Citrus grandis, 230, 232
Citrus halimii, 230
Citrus incisa, 133
Citrus jambhiri, 230, 232, 235
Citrus ledgeriana, 101
Citrus limetoides, 230
Citrus limon, 195, 197-202, 230, 232
Citrus madurensis, 230
Citrus microcarpa, 232
Citrus nobilis, 232
Citrus paradisi, 230, 232
Citrus reticulata, 230, 232, 235
Citrus sinensis, 230, 232, 235
Citrus unshiu, 232
Cladosporium fulvum, 294
Clavata mutants, 376
Clerodendrum, 89
Clitoria ternatea, 97
Clove, 270
Club root, 156
CoA ligase, 175
Cochliobolus sativus, 294
Cocoa, 295
Coconut/ coconut water, 18, 149-151, 209, 231,
232
CodA, 353
Codonopsis pilosula, 100
Coffee, 245, 297
Coix, 33
Colchicine, 2-4, 6, 24, 25, 332
Cold stress/tolerance, 234, 300, 353
Coleus, 89, 95, 282, 285
Colletotrichum, 295
Combretaceae, 197
Commiphora, 129-33, 135-139, 142, 267
Conessine, 123, 125
Coniferyl alcohol, 96
Constitutive CLV3 signalling, 378
Convolvulaceae, 158
Convolvulus arvensis, 375
Coptis japoinca, 123, 124
Copulus, 187
Cordyline, 271
Coriandrum sativum, 151
Corky root rot, 304
Corn earworm, 303, 310
Corpus, 367-69, 374
Corylus avellana, 95
Coryphantha, 375
Cosmid, 304
Cotton, 175, 297, 299, 358
p-Coumaric acid, 175
Crassula multicava, 51
CRD analysis, 219
Crepenylic acid, 151
Crepis, 151
Crocus sativus, 85, 270
Cronartium ribicola, 306
Cruciferae-type, 47
cry, 176
Cryopreservation, 78-81, 97, 98, 170, 171, 206, 210,
211, 233, 278-286
Cucumber mosaic virus, 304
Cudrania tricuspidata, 121, 122
Culms, 172
Cuphea, 151
Curcuma, 268
Curdlan, 95
Cyanobacterium, 172
Cyclin genes, 33
Cycloheximide, 352
Cysteine, 9, 11
Cytokine, 375
Dactylorhiza maculata, 54
Dahlia, 272
Dalbergia, 171, 172
DAMD primers, 207
Index 391
Datura, 65, 92, 100, 103, 286
Datura candida hybrid, 101
Datura ferox, 101
Datura innoxia, 1, 5, 21, 89, 95, 97, 98, 101, 104,
282
Datura metel, 9
Datura quercifolia, 102
Datura stramonium, 96, 101, 102, 104, 282
Datura wrightii, 101
Daucus carota, 52, 92
DDRT-PCR, 293
Decanol, 125
Decarboxylases, 65
Dehydrins, 350
Dehydrogenase, 61
Dendrocalamus, 171, 172, 188, 191
Dephosphorylation, 63, 353
de-sanguinarine, 93
Desaturase, 149, 150
Diamineoxidase (DAO), 61, 65, 66, 68, 69
Dibutylphthalate, 125
Dicamba, 231, 232
Dieffenbachia, 271
α-difluoromethylarginine (DFMA), 64
α-difluoromethyllysine (DFML), 64
α-difluoromethylornithine (DFMO), 64, 69
Digenic, 158, 162
Digitalis, 97
Digitalis lanata, 92, 121, 122, 282
Digitalis obscura, 100
Digitalis thapsi, 282, 285
Digitoxin, 92
Dimethylsulfoxide (DMSO), 81, 125, 280, 281,
285
Dioscorea, 82, 85, 281, 286
Dioscorea alata, 97, 98
Dioscorea balanica, 98, 282
Dioscorea bulbifera, 86, 89, 94, 95, 98, 100, 282,
284
Dioscorea caucasia, 282
Dioscorea cayenensis, 100
Dioscorea deltoidea, 92, 95, 97, 281-83
Dioscorea floribunda, 97, 98, 281, 282
Dioscorea rotundata, 100
Diosgenin, 82, 92, 95, 96, 100, 197, 202, 282
Diplocarpon, 295
Diplotaxis, 159, 162
Diterpenoids, 131
Diuraphis noxia, 296
Diuretic, 90
DL-β-phenyllactic, 95
DNA chips, 174
DNA finger printing/ marker, 169, 193, 207, 212,
301, 306, 307
Dolichos, 331
Dominant marker, 290
Double haploids, 18-22, 25, 147-149, 300
Double low, 144, 147
Downy mildew, 157, 159, 162
d-pseudoephedrine, 92
drn-D (Dornroschen) gene, 372
Drosophila, 341, 373
DTT, 208
Duboisia, 100
Duplicate gene, 158, 162
Durable stem rust, 293
Dysaphis devecta, 296
Ectopic, ectopic primordia, 372, 375, 379
Eicosenoic, 145
EIF2c, 373
Electrofusion, 32, 209
Electroporation, 81, 235
Elettaria cardamomum, 268
Elicitors, Elicitation, 93, 95, 96, 281
Elite genotypes, 98
Embryogenesis, 4, 6, 9-13, 22-25, 31, 34, 36, 43, 44,
46, 49, 51-53, 55, 56, 148, 258, 366, 369-71,
373, 375, 380
Embryogenic, 4-13, 22, 23
Embryoid, 3, 4, 11, 21, 43, 44, 49, 50-52, 55, 56
Embryoidogeny, embryoidogenesis, 43, 44, 49, 5153, 55
Embryonal suspensor mass (ESM), 148, 173
Encapsulated, Encapsulation, 81, 98, 210, 211, 228,
234, 237, 280-84
Endoparasitic, 69
Endophytes, 170
Endosperm, 31, 33, 34, 36-38
1-ephedrine, 92
Ephedra, 92
Epicotyl, 80, 171
Epigenetic, 161, 231
Epiphylly/epiphyllous, 375
Epiphysis, 44, 46, 49-51, 53, 55, 56
Epistasis, 376, 377, 380
ERECTA (ER) gene, 376, 377
Eriobotrya japonica, 232
Erodium cicutarium, 46
Errera’s law, 44
Eruca sativa, 159, 162
Erucic acid, 20, 21, 144, 145, 147-50, 157, 302
392
INDEX
Erysiphe cruciferarum, 156
Erysiphe graminis, 292, 294
Erysiphe polygoni, 295
Escherichia coli, 63, 65, 353-55
Eschscholtzia, 95, 96
Ethephon, 134, 135
Ethylene, 67, 174, 306
Ethylene glycol, 281
Eucalyptus, 98, 99, 171, 175, 184, 188, 191, 192,
282, 298, 306
Eucalyptus camaldulensis, 188, 192
Eucalyptus citriodora, 188, 192
Eucalyptus grandis, 306
Eucalyptus tereticornis, 188, 192
Eucalyptus urophylla, 306
Eugenia spp., 232
Euonimus macroptera, 50
Eupatorium cannavulgaris, 65
Euphoria longan, 232
Faba bean, 35
Fabaceae, 331
Fatty acids, 144, 145, 157
Feeder cells, 33
Feijoa, 228, 232, 233
Fertility restoration, 297
Festuca, 342
Ficus, 171, 271
Flavonoids, 80, 131, 157
Flooding tolerance, 300
Floral meristems, 379
Fluridone, 11
Foeniculum vulgare, 270
Forage legume, 357
Frankia, 175
Fraxinus angustifolia, 226
Freeze preservation, 97, 281, 352, 355, 357
Frost-hardy, 156, 279
Fructan synthase (Sac B), 348
Fructans, 349
Fucus serratus, 34
Furanocoumarins, 80
Fusarium, 69, 294, 295, 303, 304
Fusarium head blight, 293
Fusiform rust disease, 174
Fusogen, 209
Galangal, 269
Gall midge biotypes, 296, 300, 308
Gallium mollugo, 82
Garcinia mangostana, 229, 230
Gelatin, 81
Gelrite, 81
Gemmorhizogenesis, 43, 49
KNOTTED gene, 371
Gene bank, 210, 212, 290
Gene pyramiding, 289, 303, 307, 358
Gene silencing, 237
Gene tagging, 312
Generative cell, 3-6, 23
Genetic diseases, 291
Genetic fidelity, 78, 99, 201
Genetic markers, 207, 289
Genetic mosaics, 368
Genomics, 311, 360
Genotypic apomixis, 311
Gentiana scabra, 282
Gerbera, 271
Geum urbanum, 47, 48
Gingenoside, 122
Ginger, 268
Ginseng saponin, 122, 123
Ginsenoside, 102, 103, 285
Globodera, 296
Glucobrassicanapin, 146
Gluconapin, 146
Glucosinolates, 144, 146-48
Glutamine, 2, 135, 209, 231
Glutamine synthetase, 353
Glutathione reductase (GR) (gor), 175, 349, 351,
354, 356, 358
Glutathione synthetase (GS) (gshII), 349, 351, 354,
356
gly I, 351
Glyceraldehyde 3-phosphate dehydrogenase, 359
Glycine betaine, 347-349, 353, 357
Glycine max, 98
Glycoalkaloids, 93, 299
Glycoprotein, 11, 146
Glycosides, 80, 102
Glyoxalase/ glyoxalate, 349, 351
Gmelina, 171
Gourds, 263
Graminad, 46, 47, 49, 51, 54
Grapes, 261, 262, 295, 299
Green beans, 263
Green fluorescent protein (GFP), 34
Groundnut, 18, 25, 156, 346
Guanylhydrazone, 64
Guargum, 81
Guava, 226, 228, 229, 233, 234, 262, 266, 273
Guazuma crinita, 97
Index 393
Guggul, 129, 130, 132-34
Guggulipid, 132
Guggulster, 138
Guggulsterol-I, II, III, IV, V, VI, 130, 131, 138
Guggulsterone, 129-33, 137, 139
Gum-resin, 129-134, 138
GURKE gene, 370
Gus, GUS, 4, 35, 36, 67, 212, 234-36
Gymnadenia conopsea, 49
Gymnema sylvestris, 267
Gynogenesis, gynogenic, 21, 209, 228
5-hydroxyferulic acid o-methyltransferase, 175
6-ß-hydroxy hyoscyamine, 103
8-hydroxyquinoline, 249
H2O2, 351, 356
Hairy roots, 95, 100, 101, 103, 212, 284, 304
Haploids, 1, 5, 6, 11, 19, 21, 209, 228
Heat-shock proteins (HSPs), 7, 174
Helianthus, 61, 257
Helicoverpa zea, 296, 303
Heliminthosporium turcicum, 294
Hepatitis B, 89
Heracleum, 374
Herbicide resistance, 176, 206, 234
Heterodera glycines, 296, 302
Heterophasic, 43, 44, 49, 50, 55, 56,
Hevea brasiliensis, 104
Hingota, 195, 197
Holarrhena, 89, 117, 123, 125
Holoparasitic, 55
Holostemma annulare, 97, 282
Homeodomain/protein, 371, 372, 377, 380
Homophasic, 43, 44, 49, 50, 55, 56
Homo-SPD synthase, 65
hpt, 235
Hydrophilic COR proteins, 350
Hydroxycinnamic acids, 60
Hydroxyphenols, 257
Hyoscyamine, 92, 95, 100-02, 104
Hyoscyamus albus, 96, 100, 102, 103
Hyoscyamus desertorum, 102
Hyoscyamus muticus, 97, 102, 103
Hyoscyamus niger, 5, 9, 10, 65
Hyoscyamus × gyorffyi, 102
Hyper-cholesterolemia, 132
Hyperlipidemia, 132, 133
Hyperplasia/Hypertrophy, 157
Hypocholesterolemic, 132
Hypoglycemic, 133
Hypolipidemic, 130-33
Hypophysis, 44, 46, 47, 49-51, 53, 55, 56
IAA-oxidase, 9
Idioblast, 146
IHSP, 357
Imidazoline, 20
Immobilization, 97, 138, 139
Indian bdellium, 129
Indian long pepper, 268
Indole alkaloids, 85, 92, 124
Introgression/introgressed, 160, 162, 304, 310,
347
Inula racemosa, 133
inversion heterozygosity, 331, 341
Ipomea batatas, 98
ipt, 375
Ischemic, 132
Isonicotinic acid, 159
Isopentenyltransferase (ipt), 375
Isoplexis canariensis, 89
Isoproterenol, 133
Isothiocyanates, 146
Isozymes, 99, 206, 207, 304
ISSR-ISSR primers, 207, 295, 301, 303
Jaccard’s coefficient, 82
Jaceosidin, 95
Jack pine, 174
Jamun, 195, 197
Jasmonate/Jasmonic acid, 95, 96
Java long pepper, 268
Jojoba, 149
Juglans, 257
Kaempferia, 269
Kala zira, 270
Kale, 276
Kasturi turmeric, 268
Kentucky bluegrass, 311
Kinase-associated protein-phosphatase (KAPP),
377
Kinases, 349, 360
Kinks, 189, 193
Klebsiella, 274
KN1 gene, 370-72, 375
KNAT1, 2, 375, 379
KNOX genes, 371, 379
Larch, 175
Large cardamom, 268
las (lateral suppressor), 374
394
INDEX
Late blight of potato, 306
Late embryogenesis (LEA) proteins, 349, 350,
356
Lathyrus, 342
Lauric acid, 149-51
Lauryo-ACP thioesterase, 150
Lavendula angustifolia, 269
Laxative, 89
LDC (lysine decarboxylase), 103
L-DOPA, 93, 94
Leek lettuce, 276
Leishmania, 64, 65
Lentil, 65, 295
Lepidine, 92, 94
Lepidium, 92, 94
Leprosy, 90
Leptosphaeria maculans, 156, 160, 161, 295, 302
Leucaena hybrids, 188
Leucine zipper, 349
Leucine-zipper motif, 352
Leucoderma, 88
Leveillula tourica, 294
Lignan, 96
Lignin, 62, 175, 176
Lilium, 341
Limonene, 131, 132
Linoleic/linolenic acid, 21, 145, 148, 149, 302
Linseed, 149
Lipid peroxides, 132, 133
Lipoproteins, 132, 133
Liposome, 81, 234
Liquidambar styraciflua, 175
Litchi, 230, 231, 261, 266
Lithospermum erythrorhizon, 82, 87, 94, 96, 103,
121-23
Loblolly pine, 171, 174, 176, 306
Loganin, 95, 124
Lolium, 342
Loose smut, 293
LRR-receptor/kinases, 376, 377
Lubimin, 96
Lupinus polyphyllus, 96
Luteovirus, 294
Lycopene, 305
Lycopersicon, 304, 310
Lysine, 103, 145
Lysine decarboxylase, 64
MADS box gene, 374, 380
Magnaportha grisea, 300, 308
Maize viruses, 294
Maize (Zea mays), 7, 12, 21, 31-38, 65, 68, 294,
296, 299, 311, 353, 368, 370, 371, 374, 379
Mangifera indica (mango), 228, 230-32, 234, 236,
262, 273
Mangosteen, 229
Mannitol 1-phosphate dehydrogenase (MtlD), 348
Map-based cloning, 304
MAPK kinases, 352
Marjoram, 269
Marjorana hortensis, 269
Marker assisted selection/breeding, 289, 300, 302,
303, 307-09
Maytenus emarginata, 197
McFISH, 342
Medicago sativa, 98, 357
Meloidogyne, 294, 296, 305
Melon, 295
Memory vitalizer, 88
Mentha, 97, 98, 269
Metallothionein, 11
Methionine, 61, 62, 67, 145
Methyl jasmonate (MeJa), 95, 96, 103
Methyl viologen, 175, 359
mgo genes, 376
Microcyclus ulei, 295
Micrografting, 244, 248
Microinjection, 35, 36, 81, 234, 369
Microprojectile, 36, 234-36
Microsatellite markers, 290, 300, 301
Microshoots, 171, 246-48, 258
Microspore embryogenesis, 7, 8
Microtubers, 266
Mildew, 311
Milkwhey, 82
Minimata disease, 176
Misexpression, 379
Mitotic complements, 332
Molecular markers, 78, 80, 81, 98-100, 161, 206,
289, 291, 300-03, 308-12
Monoclonal antibody, 8
Monofluoromethylarginine (MFMA), 64
Monofluoromethylornithine (MFMO), 64
Monogenic, 158, 162, 300, 302
Monoterpenes, 131, 132
Monozygotic, 43, 49
Morina kokanica, 48
Morinda citrifolia, 94, 96
Moringa oliefera, 100
Morphactin, 134
Morus (mulberry), 206-12
Morus alba, 208-10, 212
Index 395
Morus australis, 208
Morus bombycis, 208, 211
Morus cathayana, 208
Morus indica, 208, 212
Morus laevigata, 207, 208
Morus lhou, 208
Morus multicaulis, 208, 211
Morus nigra, 210
Morus serrata, 208
mtlD, 353
Mucuna, 94
Mukulol, 131
Multigene family, 371
Multimeric complex, 377
Musa, 228-30, 232, 233, 236
Mushroom, 355
Mustard, 97, 147, 356, 357
Mutagenesis, 147-49, 151, 159
Myb and Myc like protein, 349, 352, 379
Mycorrhizae, 186, 274
Myocardial, 132, 133, 145, 157
Myrcene, 131, 132
Myrciaria cauliflora, 232
Myricyl alcohol, 131
Myristic acid, 149, 151
Myristica fragrans, 151
Myrosinase, 146
Myrtaceae, 195, 197
Naphthoquinone, 97
Napin, 11
Napoleiferin, 146
Nelumbo, 48, 53, 54
Nelumbonaceae, 53
Neomycin phosphotransferase (npt II), 175, 234-36
Neurospora, 65
Nicotiana, 82
Nicotiana plumbaginifolia, 282
Nicotiana rustica, 8, 104
Nicotiana sylvestris, 65, 282
Nicotiana tabacum, 3-5, 8, 9, 11, 94, 97, 98, 103,
104, 123, 124, 282
Nicotine, 62, 66, 104
Nifedipine, 133
Nitella, 61
Nitriles, 146
Nitrogen-fixing bacteria, 175
NO APICAL MERISTEM (NAM) gene, 372
nopaline, 234
NorSPD/NorSPM, 60, 61
Norway spruce, 176
Nothapodytes foetida, 89, 94
Nucellar/embryos, 43, 44, 50, 51, 231, 248, 249
Obesity, 129, 132
Oenothera biennis, 46
Oidium lycopersicum, 294
Oil palm, 295, 298
Oilseed, 145, 149, 151, 156, 301, 356
Okazaki fragments, 63
Okra, 263
Olea europe, 282
Oleanane triterpenes, 96
Oleanolic acid, 132
Oleic, 148, 149
Oleic acid, 20, 21, 145, 148-50, 302
Oleiferous, 25, 144, 156
Oleogum-resin, 129, 130, 133
Oligogalacturonide, 96
Oligonucleotide fingerprinting, 99, 100
Omega-3-desaturase, 302
Onagrad, 49-52, 54, 55
Ononitol, 349
Orange, 97
Orchidaceae, 53
Orchids, 271
Oregano, 269
Organoleptic, 304
Origanum vulgare, 269
Ornithine, 61, 62, 67
Ornithine decarboxylase (odc), 61-70, 104, 348, 359
Orobanchaceae, 53, 55
Orseolia oryzae, 296, 308
Orthologue, 379
Oryza sativa (Rice), 36, 65-69, 97, 211, 245, 291,
296, 297, 299, 300, 307, 308, 353, 354, 356
Osmolytes, 60, 347, 348, 353, 360
Osmoprotectant, 359
Osmoregulation, 351
Osmotic stress/osmotic tolerance, 233, 348, 351,
352, 355
Osmotin, 348, 349, 357, 358
Osteoarthritis, 133
Ostrinia nubilalis, 296
Oxidation, 244, 350
Oxidative stress, 174, 175, 355, 357
Oxygenation, 119, 120
Ozone, 351
Paclitaxel, 124
Paeonad, 46, 47
Paeonia, 49-51, 341
396
INDEX
Palm, 18, 149, 150
Palmitic, 21, 149
Panax ginseng, 97, 98, 100, 102, 103, 122, 124, 282,
284-86
Panax notoginseng, 121, 123
Panax quniquefolium, 100, 282
Papaver, 93, 96, 282, 284
Papaya, 228, 229, 231, 133, 234, 262, 273
Papaya ring spot virus, 235
Parsley, 269, 276
Particle bombardment, 81, 175, 235
Patchouli, 267
Paulownia fortunei, 188, 192
PCR/markers, 81, 284, 285, 290, 291, 304, 308, 310,
312
Pea, 65, 66, 68, 295, 299, 303, 304, 355
Peach, 273, 299, 300
Pear, 273
Pearl millet, 295, 346
Pecan, 99
Pedigree selection, 147
Peganum harmala, 85, 89, 103
Pepper, 295
Peppermint, 269
Perilla frutescens, 123
Peronospora parasitica, 156, 157
Peroxidase, 173, 207
Petroselenic acid, 151
Petroselinum crispum, 269
Petunia, 297, 300, 372, 374
PgEMB22, 27 and 29, 174
Pgq (Panax hybrid), 103
PHANTASTICA (PHAN), 379
Pharbitis nil, 65
Phaseolus, 95, 331
Phenetic dendrogram, 82
Phenolics, 9, 93, 157, 158, 225, 244, 246, 250, 256,
257
Phenylalanine ammonialyase (PAL), 175
Phenylpropanoids, 63
Philodendron, 271
Phloroglucinol, 217, 218, 222, 226
Phoma lingam, 20
Phosphorylation, 6, 8, 63, 351, 353,
Photooxidative, 351, 355, 356, 359
Phyllanthus, 89, 267
Phyllochora herberi, 295
Phyllostachys, 171
Phytoalexins, 95, 159
Phytohemagglutinin, 358
Phytomers, 366
Phytophthora, 96, 294, 295, 306
Picea, 174, 258
Picrorhiza kurroa, 97
Pigeonpea, 346
Pimpinella anisum, 270
Pine blister rust, 306
Pineapple, 262, 266, 273
ß-pinene, 131, 132
PINHEAD (ZLL/PNH), 373
Pinitol, 347
Pinoresinol, 96
Pinus, 299
Pinus banksiana, 174
Pinus elliottii, 306
Pinus lambertiana, 306
Pinus palustris, 306
Pinus taeda, 174, 306
Piper barberi, 268
Piper, 268
Piperad-type, 47
Plagiotrophy, 196, 201, 225
Plantago major, 100
Plantago ovata, 89
Plasmodiophora brassicae, 156, 160, 295, 302
Plastochronic, 367
Platantera bifolia, 49, 48
Plectonema boryanum, 172
Pluchea lanceolata, 87
Plum, 273
Plumbago, 90
Pluronic PE 6100, 120
Poa pratensis, 311
Poaceae, 53, 55, 190
Podophyllotoxin, 123, 124
Podophyllum hexandrum, 121, 123, 124
Polemonium, 48, 49
POLTERGEIST (POL) gene, 378
Polyamine oxidase (PAO), 61
Polyamines, PAs, 60-70, 100, 231, 259, 348, 356
Polyethylene glycol (PEG), 32, 174, 235, 348
Polygonum, 46, 282
Polypropylene glycol 1025 and 2025, 120
Polyvinylpyrrolidone (PVP), 9, 249, 250, 255
Pome, 229
Pomegranate, 266
Poncirus trifoliata, 230, 235
Populus/poplar, 95, 171, 175, 188, 191,193
Populus alba, 175
Populus canescens, 175
Populus deltoides, 188, 189, 193
Populus euphratica, 188, 193
Index 397
Populus tremula, 175
Portulacca grandiflora, 94
Portulaceae, 158
Potato, 66, 67, 210, 263, 264, 266, 267, 275, 294,
296, 299, 355, 358, 359, 374
Potato virus, 294, 304
Potexvirus, 294
Potyvirus, 294
Powdery mildew, 156, 293
Pratylenchus neglectus, 296
Precursor, 81, 95, 122, 124, 125
Pregnane, 131, 133
Pregnenolone, 134
Primula obconica, 98
Progoitrin, 146
Proline, 81, 249, 280, 347-49, 357
Prosopis cineraria, 171, 197, 226
Protein kinase, 351, 352
Protocorm, 53, 97
Protoderm, 44, 49, 50, 53, 55
Prunus, 266
PRV cp gene, 234
Pseudomonas, 294, 295
Psidium guajava, 230
Psoralea corylifolia, 90
Psoriasis, 90
Puccinia helianthi, 309, 310
Puccinia hordei, 294
Puccinia melanocephala, 295
Puccinia recondite, 293
Puccinia striiformis, 292, 294
Puccinia substriata, 295
Pulse treatment, 200
Pumpkins, 263
Puromycin, 10
Purseglove, 335
PUT-methyl transferase (PMT), 62
PUT-N-methyltransferase., 65
Putrescine, PUT, 60-63, 66-70, 123, 249, 349, 359
PVS2, 280, 281, 284, 285
Pyramid genes, 307-10
Pyrenophora graminea, 294
Pyricularia grisea, 292
Pyrrolidine alkaloids, 104
D1-pyrroline-5-carboxylate synthetase (P5CS),
348
Pyrroline/pyrrolidine ring, 61, 62
QTA, 306
QTL, 173, 212, 289, 300-03, 306, 310, 311, 347
Quaking aspen, 175
Quercetin, 131
Quercus, 225, 226, 257
Quinidine, 101
Quinine, 89, 92, 95, 101
Rab16A, 356
Radiata pine, 170, 173
Ralstonia solanacearum, 69, 295
Ramosus genes, 304
Ranunculaceae, 51, 52
Ranunculus sceleratus, 50, 51
RAPD, 80, 82, 99, 100, 173, 174, 207, 282, 284,
285, 290-94, 296-304, 306, 309
Rapeseed, 18, 144, 146, 150, 151, 157
Raphanus sativus, 158
Raspberry, 266
Rauwolfia, 90, 93, 97, 103
Red fluorescent protein, 37
Rehmannia sp., 100
Remeristemization, 375
Reporter gene, 31, 35
Reserpine, 95
Resin/ducts, 129, 130, 133, 137
rev (revoluta) gene, 374
RFLP, 12, 82, 100, 161, 290-92, 294, 296-303, 306,
308, 310, 311
RGA-CAPs, 294, 300
Rhamnus, 85
Rheumatoid, 132
Rhizobium, 60, 274, 186, 299
Rhizoctonia solani, 292
Rhododendron, 257
Rhodotorula rubra, 96
Rhus typhina, 248
Rib meristem, 369
Rice stripe, 292
Rice tungro, 300
Rice yellow mottle virus, 292
Richinolic acid, 151
Ring spot virus, 234
Robinia pseudoacacia, 170
Rorrippa islandica, 158
ROS scavengers, 360
Rose, 274, 295, 299
Rosmaric acid, 285
Rosmarinic acid, 95, 123
Rough lemon, 235
Rubber, 295
Rutaceae, 195, 197
Rynchosporium secalis, 294
‘Syn’ seeds (artificial seeds), 78, 81, 97, 139, 172,
210, 228, 234, 245
398
INDEX
NaCl/salt-stress/tolerance, 172, 233, 296, 297, 353,
355-59
Sabinene, 131
Saccharomyces cerevisiae, 63, 95, 355
Sacred basil, 269
Saffron, 270
Sage, 269
Salai guggul, 130
Salvia officinalis, 269
SAM, 367, 369, 371-75, 379, 380
SAM decarboxylase, 62-69, 356
SAMdc (S-adenosylmethionine), 61
Samdc/spd syn, 70
Samdc-odc, 66
SAM-S (S-Adenosyl Methionine Synthetase),
174
Sandalwood, 61, 171, 172, 174
Sanguinaria canadensis, 96
Sanguinarine, 96
Sap protein, 207
Sapogenin, 92, 94, 197
Saponin, 102
Saturated linkage maps, 291, 300
SCAR marker, 290, 291, 293, 294, 296-99, 302,
309, 311, 312
Scavenger proteins, 351
Schizaphids graminum, 296
Sclerotinia sclerotiorum, 96, 156, 295
Scopolamine, 92, 100-03
Scopolia, 103
Secale cereale (rye), 32, 95, 297
Secologanin, 95, 124
Secondary embryogenesis, 255
Semidwarf gene, 298, 299, 308
Senecio vernalis, 65
Septoria, 293
Sequoia sempervirens, 173
Serotonin, 103, 133
Serpentine, 92, 103, 120, 123
Sesquipedaceae, 333-35
Sesquiterpenes, 131-133
Shattering-resistance, 299
Shikonin, 82, 87, 93-95, 97, 103, 118, 121, 123
Shoot apical meristem (SAM), 366
SHOOTMERSITEMLESS (STM), 372
Shorter rotation, 98
Signal transduction, 34, 38, 63, 351, 352, 376, 377
Silver birch, 171
Silver staining, 291
Silybin, 93
Silybum marianum, 90, 93
Sinapis alba, 158-60
Sinigrin, 146
Sisymbrium officinale, 158
ß-sitosterol, 131
Small heat-shock protein (smHSP), 2
Sn-2 acyl transferase gene, 151
SNP, 82, 291, 311
Solanaceae, 47
Solanaceous, 66
Solanum, 93
Solanum aviculare, 93, 104
Solanum bulbocastanum, 306
Solanum eleagnifolium, 103
Solanum khasianum, 90
Solanum nigrum, 95
Solanum tuberosum, 10
Solanum xanthocarpum, 95
Solasodine, 93, 95, 103
Somaclonal, 99, 159, 161, 228, 233, 244, 265, 285
Somatic embryogenesis, 55, 61, 67, 82, 94, 99, 124,
136, 170-73, 201, 209, 228, 229, 231-33, 237,
248, 252, 253, 257-59
Somatic hybridization, 159, 160, 206, 209
Sorghum, 33, 257, 294, 296, 297
Soybean, 18, 65, 95, 175, 226, 295, 296, 298, 302,
303, 309, 310, 357
Soybean death syndrome (SDS), 303
Spathiphyllum, 271
Spearmint, 269
Spermidine (SPD), 60, 62, 359
Spermine (SPM), 60, 62-70, 348, 359
Spike disease, 174
Spinach, 65, 159
Sporisorium reilianum, 294
Spruce, 174, 175
Squash, 263
SSR markers, 173, 290, 292, 294, 296, 298, 299,
301-03, 310
Stearic acid, 149, 150, 303
Stearoyl ACP desaturase gene, 150
Stemphylium vesicarum, 294
Sterculia foetida, 90
Steroid/steroidal drugs/alkaloids/sterols, 80, 90, 95,
104, 130, 131, 133, 197
Stevia rebaudiana, 90
Stizolobium, 93, 94
Strawberry, 264-66
Strictocidine synthase, 104
Stripe rust, 293, 301
Suckers, 164, 228, 265
Sugar apple, 228, 233
Index 399
Sugar beet, 299, 309
Sugarcane, 264, 267, 295, 306
Sugarcane mosaic virus, 294
Summer turnip rape, 145
Sunflower, 18, 297-99, 309
Superoxidase dismutase (SOD), 132, 133, 349, 351,
358
Sweet gum, 175
Syngonium, 271
Syzygium, 195, 197-02, 230, 270
Tabernaemontana divaricata, 95
TATA box, 375
Taxane, 123
Taxol, 123
Taxus, 121-25
TBP-2 protein, 376
Teak, 171
Tecomella undulata, 197, 226
Tectona grandis, 171, 225
TERMINAL FLOWER, 380
Terminalia arjuna, 133
Terminalization coefficient, 337, 340
Terpenoid indole alkaloid (TIA), 104
Tet-inducible, 67, 68
Tetraamine SPM, 61
Thalictrum rugosum, 85, 96
Thalidomide, 176
Thermophilic red algae, 60
Thermosensitive, 297
Thermo-SPM, 60
Thevetia neriifolia, 87
Thiocyanates, 146
Thuja occidentalis, 85
α-thujene, 131
Thymus vulgaris, 269
Thyroid, 133
Thyrsostachys siamensis, 172
Tilletia indica, 294
Tobacco (see also Nicotiana), 7, 9, 10, 35, 63-70,
104, 175, 212, 286, 294-96, 304, 306, 309-11,
348, 352, 354, 355, 359, 374, 375,
Tomatoes, 263
TOPLESS (TPL) gene, 371
Tracer dyes, 369
Tranquillizer, 90
Transferase, 66
Transformation, 81, 99, 103, 104, 169-71, 173, 175,
219, 234, 237, 258, 286
Transmembrane leucine-rich repeat (LRR), 376
Transporters, 351
Trehalase, 348, 349, 356, 358, 359
Trehalose 6-phosphate phoshatase, 356
Trehalose 6-phosphate synthase, 348, 356, 359
Triacyl glycerol, 151, 159
Triamine SPD, 61
2,4,5-trichlorophenoxy acetic acid, 136
Trichoderma virideae, 96
Trifolium repens, 98, 257, 282
Triglycerides, 132, 133
Triiodothyronine, 133
Tripterygium wilfordii, 96
Triterpenes, 132
Triticum, 51, 55
Triticum aestivum, 3, 48, 49, 51, 54
Triticum dicoccoides, 301
Tritordeum, 65, 354
Tropane alkaloids, 62, 100
Tropinone, 95
Trypanosoma, 64, 65
Tryptamine, 103, 123, 124
Trytophane decarboxylase, 103
Tuberculosis, 88
Tungro, 292
Tunica/tunica-carpus, 367-69, 372, 374
Turmeric, 268
Tylophora indica, 90, 100
Typhonium, 90
Ubiquinone, 10, 82
Ubiquitin, 8
Ubiquitin promoter, 35, 68
uidA, 176
Umbelluria californica, 150
UNUSUAL FLORAL ORGANS (UFO), 372
UPGMA, 82
Uromyces appendiculatus, 295
Valeriana jatamansi, 90
Vanilla fragrans (vanilla), 267, 269
Venturia inaequalis, 295
Vernolic acid, 151
Verticillium dahliae, 69, 96, 294
Vesicular arbuscular mycorrhiza (VAM), 173, 274
vid A, 236
Vigna, 331-33, 336, 341
Vigna aconitifolia (moth bean), 331, 333-37, 339,
340, 354
Vigna ambacensis, 331, 341
Vigna angularis (adzuki bean), 331, 332, 341
Vigna aureus, 333-35, 337, 339
Vigna candida, 339
400
INDEX
Vigna capensis, 331
Vigna caracalla, 341
Vigna radiata (mung bean), 295, 331-35, 337, 339,
342
Vigna fisheri, 331
Vigna galbrescens, 331, 333-35, 337-42
Vigna lanceolata, 331
Vigna lancifolia, 342
Vigna luteola, 333-35, 337, 339
Vigna mariana, 331
Vigna minima, 332, 341
Vigna mungo (urd bean), 331-35, 337, 339-41
Vigna oblongifolia, 341
Vigna parvifolia, 341
Vigna repens, 333-37, 340, 341
Vigna reticulata, 331
Vigna sps, Tvnu-72, 333, 337, 340
Vigna trilobata, 331, 332, 341
Vigna umbellata (rice bean), 331-337, 340, 341
Vigna unguiculata (cowpea), 331, 333-35, 337-42
Vigna vexillata, 331, 339, 341
V inblastine, 92
Vinca rosea, 122
V indoline, 92
Visnagin, 101
V itamin A, 150, 211
Vitex negundo, 91
Vitis, 257
Vitrification, 98, 211, 279-81
Water stress, 356
Wax esters, 149
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Wheat, 11, 33, 35, 36, 51, 97, 292, 296- 98, 301,
309, 353, 354
Wheat streak mosaic virus, 294
White blisters, 157
White guggul, 130
White rust, 147, 156-62
White spruce, 174
Withaferin A, 93
Withania, 91, 93, 94, 99, 103, 267
Withanolide D, 93, 94, 103
Wound induced promoters, 176
Wrightia tinctoria, 226
Wrightia tomentosa, 217-26
Wsi18, 356
wus mutant, 371, 372, 374, 377-80
WUSCHEL gene, 371
Xanthan, 95
Xanthine oxidase, 132, 133
Xanthomonas, 292, 294, 295, 300, 307
Xanthotoxin, 92, 94
Yeast odc, 104
Yellow rust, 293
Zantedeshia, 271
Zinc finger family, 358
Zingiber officinale, 268
zip, 232
Zizyphus, 197, 266
Zucchini, 62