Phenotyping For Plant Breeding 2013

Siva Kumar
Panguluri · Are Ashok Kumar

Editors
Phenotyping
for Plant
Breeding
Applications of Phenotyping Methods
for Crop Improvement
Phenotyping for Plant Breeding
Siva Kumar Panguluri • Are Ashok Kumar
Editors
Phenotyping for Plant

Breeding
Applications of Phenotyping Methods
for Crop Improvement
Editors
Siva Kumar Panguluri Are Ashok Kumar
Pharmaceutical Sciences Sorghum Breeding
College of Pharmacy ICRISAT
University of South Florida Patancheru, India
Tampa, FL, USA
ISBN 978-1-4614-8319-9 ISBN 978-1-4614-8320-5 (eBook)

DOI 10.1007/978-1-4614-8320-5
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013946933
© Springer Science+Business Media New York 2013

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Preface
Plant breeding is defined as the art and science of changing genetic architecture of
plants for the benefit of mankind and it has been in practice for thousands of years,
since the beginning of agriculture. However, it is being practiced more scientifically
ever since the rediscovery of Mendel’s laws in 1900 and has become increasingly
precise by the use of new molecular tools. In addition to simple selection methods,
crop improvement involves sexual hybridization of desirable parents followed by
selections in the segregating populations so as to select desirable combinations and
eliminate the undesirable ones. Even today, this is the predominant practice in crop
improvement, although various tools like mutation induction, wide hybridization,
exploitation of somaclonal variation, genomic tools, and genetic transformation are
also employed. Thus plant breeders have been largely engaged with creation of
variation and selection to improve the crop plants over decades.
Plant breeder’s task is to select the plants that most likely meet the breeding
objectives. Selecting a desirable combination and rejecting the undesirable one
remains a challenging task given the fact that selections have to be exercised on a
large number of plants/progenies with due consideration to a large number of traits,
both qualitative and quantitative. The greatest apprehension haunting a breeder is
the loss of superior plant/progeny during selections. As a consequence, the number
of selections increased, sometimes by selecting the undesirable combinations,
which burdens both time and resources required to handle them. Thus, success of a
breeding program largely depends on use of an appropriate phenotyping method
enabling a breeder to make judicious selections. Plant breeders have been using new
tools like trait selection and use of markers to select gene(s) (marker-assisted selec-
tion) and/or genomes (genome-wide selection) to enhance the speed, accuracy, and
scope of selection process. These techniques complement the selection process in
breeding but cannot replace phenotyping for two reasons: first the design of these
tools itself needs high-throughput phenotyping and second the need for the geno-
typed selections be confirmed by phenotypic data. Thus even the application of new
tools essentially requires appropriate phenotyping systems.
v
vi Preface
A phenotype is any measurable characteristic or trait of a plant and is a result

of combination of genes expressing in the plant (referred to as genotype), environ-
mental influence, and their interactions. Phenotyping to a plant breeder means char-
acterizing the performance of the plants for desired trait(s). Phenotyping is central
to plant breeding to carry out selections; in addition, it is also done to study genetics
of the traits, to associate markers with traits, to understand trait diversity, etc.
Although routinely used, it still remains a factor of paramount importance for the
success of breeding programs and to derive valid conclusions from genetic studies.
In fact generating reliable phenotype data is now considered as a major limiting
factor in breeding programs. Even in this era of genomics where state-of-the-art
genotyping techniques and bioinformatics tool are available, the progress and valid-
ity of the results are largely constrained by the generation of reliable and reproduc-
ible phenotype data.
The objectives of a crop-breeding program in general are to develop improved
varieties/hybrid parents with specific adaptation, high yield potential possessing
pest and disease resistance, abiotic stress tolerance, enhanced nutritional content,
high quality, market preferred traits, etc. These additional traits are as important as
increasing yield and are often of critical significance as they offer protection from
yield losses, improve quality, and thus enhance the economic returns. Plant breed-
ing is often a painstakingly slow process; therefore a breeder often has to look many
years ahead of the requirements of farmers and consumers to prioritize crop-
breeding objectives. These objectives are location specific and depend on the eco-
nomic importance of the trait.
We have therefore in this book discussed the phenotyping techniques for priori-
tized traits in some of the agriculturally important crops. This book broadly dis-
cusses various established methods of phenotyping for important biotic and abiotic
constraints and other traits of interest. Thus it serves the requirements of a practical
plant breeder who is often perplexed with the selection process requiring a good
phenotypic method. A large number of reviews and books are now available on the
use of molecular and genetics tools in plant breeding, although not many breeders
have access to use them in their breeding programs. On contrary, we don’t find
comprehensive information on phenotyping of plants which indeed can be routinely
used in breeding programs, and a large number of breeders even in developing
counties can use such phenotyping techniques. A crop breeder has to pull informa-
tion from many different publications before she/he chooses an appropriate screen-
ing method. This book is also important in the context of dwindling numbers of
plant breeders who can guide students and younger generations on practical issues
of selections, and a majority of students now consider plant breeding an old-
fashioned science where modern tools are not applied. While the fact remains that
plant breeding has played an important role in increasing the crop production
through improved cultivars and will continue to play a key role in future in meeting
future food, fodder, fiber, and fuel demands.
Preface vii
This book is intended to serve as a useful guide to practicing plant breeders to

use appropriate phenotyping methods for improving the major traits in selective
crops. This also helps the teachers and students in plant breeding to better understand
the phenotyping and its importance in plant breeding.
Tampa, FL, USA Siva Kumar Panguluri

Patancheru, India Are Ashok Kumar
Contents
1 Phenotyping Rice for Molecular Plant Breeding ................................... 1

M.S. Madhav, G.S. Laha, A.P. Padmakumari, N. Somasekhar,
S.K. Mangrauthia, and B.C. Viraktamath
2 Phenotyping in Wheat Breeding .............................................................. 41
Govindan Velu and Ravi Prakash Singh
3 Phenotyping in Sorghum [Sorghum bicolor (L.) Moench] .................... 73
Are Ashok Kumar, Hari C. Sharma, Rajan Sharma,
Michael Blummel, P. Sanjana Reddy, and Belum V.S. Reddy
4 Chickpea Phenotyping .............................................................................. 111
A. Saeed and Siva Kumar Panguluri
5 Phenotyping for Groundnut (Arachis hypogaea L.)
Improvement. ............................................................................................ 129
Janila Pasupuleti and S.N. Nigam
6 Phenotyping of Tomatoes.......................................................................... 169
Amolkumar U. Solanke and P. Ananda Kumar
Index ................................................................................................................. 205
ix
List of Reviewers
Arun Balasubramaniam Department of Genetics and Plant Breeding, Institute of

Agricultural Sciences, Banaras Hindu University, Varanasi, U.P., India
Liao Boshou Oil Crops Research Institute, Chinese Academy of Agricultural
Sciences, Wuhan, Hubei, China
Majid R. Foolad Department of Horticulture, The Pennsylvania State University,
University Park, PA, USA
Rebecca Ford Department of Agriculture and Food Systems, Melbourne School
of Land & Environment, The University of Melbourne, Melbourne, VIC, Australia
Anju Mahendru Singh Division of Genetics, Indian Agricultural Research
Institute, New Delhi, India
Esten Mason University of Arkansas, Fayetteville, AR, USA
Fred J. Muehlbauer USDA-ARS, Washington State University, Pullman, WA, USA
Suchismita Mondal Wheat Breeder at CIMMYT, Ciudad Nezahualcóyotl Area,
Mexico
M.V. Rajam Department of Genetics, University of Delhi, New Delhi, India
S. Ramesh Department of Genetics and Plant Breeding, College of Agriculture,
University of Agricultural Sciences, Bangalore, Karnataka, India
A. Luke Rathnakumar Directorate of Groundnut Research (DGR), National
Research Centre for Groundnut (NRCG), Junagadh, Gujarat, India
Khela Ram Soren IIPR, Kanpur, India
A.V. Umakanth Directorate of Sorghum Research, Hyderabad, AP, India
xi
Chapter 1
Phenotyping Rice for Molecular Plant
Breeding
M.S. Madhav, G.S. Laha, A.P. Padmakumari, N. Somasekhar,

S.K. Mangrauthia, and B.C. Viraktamath
Abstract Rice is an important food crop, has the plasticity in growing in different
ecologies in many countries around the world, which makes this crop to expose to
many diseases and pests. The recent development in the genomics has led to the
intensive efforts in molecular breeding for improvements of some of the qualitative
traits. To make the successful molecular breeding programme, accurate phenotyp-
ing techniques need to be coupled with high-throughput genotyping. The chapter
discusses the various phenotypic methods available for different diseases, pests and
abiotic stress like drought.
Keywords Rice • Diseases • Pests • Phenotype
Molecular breeding programmes in most of the crops including rice is on increase day
by day and lot of public and private partners are joining hand in this programme to
develop varieties through relatively faster technology than the classical plant breeding
programme. In recent years there are tremendous improvements in development of
markers and genotyping techniques in rice enabling the researchers to genotype rap-
idly and accurately. But for any successful molecular breeding programme, the pre-
cise phenotyping technique needs to be accurate and the standard uniform techniques
need to be followed across the environments, since the phenotype is dependent on
environment. In this context, we focused on the phenotyping techniques for major
diseases, insect pests, nematodes and abiotic stress like drought.
M.S. Madhav (*) • S.K. Mangrauthia • B.C. Viraktamath

Crop Improvement Section, Directorate of Rice Research (DRR),
Indian Council of Agricultural Research, Rajendranagar,
Hyderabad 500030, Andhra Pradesh, India
e-mail: sheshu24@gmail.com; skmdrr@gmail.com; viraktamath123@rediffmail.com
G.S. Laha • A.P. Padmakumari • N. Somasekhar
Crop Protection Section, Directorate of Rice Research (DRR), Indian Council of Agricultural
Research, Rajendranagar, Hyderabad 500030, Andhra Pradesh, India
e-mail: lahags@yahoo.co.in; padmaapk@lycos.com; nssekhar@hotmail.com
S.K. Panguluri and A.A. Kumar (eds.), Phenotyping for Plant Breeding: 1
Applications of Phenotyping Methods for Crop Improvement,
DOI 10.1007/978-1-4614-8320-5_1, © Springer Science+Business Media New York 2013
2 M.S. Madhav et al.
1.1 Phenotyping of Rice Diseases
Plant pests including diseases are the important production constraints in rice.
Rice crop threatened by a number of pests and diseases. Among these, stem borers,
brown plant hopper (BPH) and diseases of fungal and bacterial origin were ranked
the most dangerous followed by others (Geddes and Iles 1991). Due to change in
cultivation practices which are heavily dependent on chemical fertilizers and the
apparent changes in the climate, the intensity and scenario of rice pests and diseases
has changed over the years. Many diseases which were earlier considered as minor,
have assumed the proportion of major ones. For example, false smut of rice, which
was earlier considered as a sign of bumper harvest, is appearing in threatening
intensity in many rice growing areas in India and other Asian and south Asian
countries (Ladhalakshmi 2007; Muthuraman et al. 2007). Many diseases which
were earlier restricted to certain parts of the country, have now spread to newer
areas. Rice diseases which can cause major economic losses are blast, bacterial
blight, sheath blight, rice tungro virus disease and brown spot. The most economic
and environmentally safe strategy to manage these diseases is deployment of resis-
tant varieties. Therefore, phenotyping for resistance in different germplasm is an
important criterion in disease management. The most ideal method of evaluating
resistance against different rice diseases is to grow the germplasm in the fields
(in hot spots) and exposing them to natural infection. However, this is labor oriented
and results may fluctuate due to inconsistent and uneven degree of natural infection.
To obtain certain and uniform occurrence of the disease, artificial inoculation of the
plants is required. The following sections describe the methods adopted for artificial
inoculation and screening for accurate phenotyping of rice diseases.
1.1.1 Major Rice Diseases
1.1.1.1 Bacterial Blight of Rice
Pathogen and Its Isolation
Bacterial blight of rice is caused by Xanthomonas oryzae pv. oryzae (Ishiyama)

Swings et al., which is gram negative, non-spore forming and rod shaped bacterium.
Bacterial blight is a typical vascular disease and has two distinct phases i.e. leaf
blight phase and kresek (wilt phase), among these leaf blight phase is most com-
mon. One of the most important criteria in artificial inoculation of the pathogen is
purity and its multiplication. Before isolation, the infected leaf samples are checked
for bacterial ooze under microscope. The positive samples are then surface steril-
ized with 0.1 % mercuric chloride or 95 % ethanol for 30 s followed by 2–3 times
rinsing with sterile distilled water. The infected leaf (preferably the portion with
advancing lesion) is then cut into small sections (2–3 mm) and put in a drop of
1 Phenotyping Rice for Molecular Plant Breeding 3
sterile distilled water on a sterilized glass slide or in a small vial containing sterile
water. After about 4–5 min, when the bacterial ooze comes out from the cut ends of
infected leaf bits into water, a loop full of water can be streaked on to a suitable
medium. The bacterium can be isolated on a number of culture media viz., potato
semi-synthetic medium, peptone sucrose agar (PSA) or modified Wakimoto’s
medium. After 4–5 days of incubation at 28±2 °C, pinhead sized colonies of the
bacterium can be observed in culture plates which can be further purified by sub-
culturing. The identity of the bacterium can be confirmed through pathogenicity test
by inoculating on to susceptible rice varieties like TN1.
Mass Culturing and Artificial Inoculation
The bacterium can be multiplied by streaking on culture plates using any of the
above mentioned media. Multiplication in broth culture is not preferred as it is dif-
ficult to detect any contamination during culturing. Using 3–4 days old culture, a
bacterial suspension (108–109 cfu/ml) is made with distilled water. This suspension
is then used for artificial inoculation. A number of methods have been used for arti-
ficial inoculation of bacterial blight pathogen. The methods can be broadly divided
into two groups.
Methods for creating leaf blight phase. Reitsma and Schure (1950) used spraying
method (spraying the plants with the bacterial suspension) and needle prick or pin
prick method (pricking the leaves with a needle dipped in bacterial suspension or
putting a drop of bacterial suspension on the leaf and then pricking the leaf with a
pin through the bacterial suspension droplet). Needle prick inoculation method is
suitable for accurate evaluation of resistance but it is laborious and time-consuming
and is not suitable for large scale screening. Several modifications were made to pin
prick inoculation. Mukoo and Yoshida (1951) and Yoshida and Muko (1961) devel-
oped multi-needle prick inoculation method, which was more convenient and prac-
ticed by a number of research workers. Though, the number of needles may vary
from 1–100, usually 4–6 needles are sufficient for successful inoculation. Usually,
the needles are mounted on a rubber pad and a cotton pad soaked in bacterial sus-
pension provides the inoculum so that in one operation, leaves are punctured and
gets inoculated with the bacteria (Ou 1985). Goto et al. (1953) used injection inocu-
lation method where disease was created by injecting the bacterial suspension in the
leaf veins. In spraying method, the disease development is generally slower when
compared with the pin prick method. This method was slightly modified by Rao and
Srivastava (1970) where leaf tips of the seedlings were clipped before spray inocu-
lation to create more disease pressure. The cut-and-spray inoculation method of
Ezuka and Horino (1976) was a similar modification in which the leaves of rice
plants at maximum tillering stage were clipped with pruning shears followed by
immediate spraying with bacterial suspension.
A leaf clipping method was developed at AICRIP (All India Coordinated Rice
Improvement Project) wherein the leaves (45–50 days old plants) are cut with
scissor dipped in bacterial suspension. This method is very efficient and very
Fig. 1.1 Diagram key for

assessment for bacterial leaf
blight in field
1=1-5% 3=6-12% 5=13-25% 7=26-50% 9=51-100%
convenient for inoculation of large number of plants in practical breeding work in

the field and glass house (Kauffman et al. 1973). Presently, this method of artificial
inoculation is being used by most of the research workers around the world.
Method for creating Kresek phase. Reitsma and Schure (1950) used immersion
inoculation (immersing the seedlings in a bacterial suspension) to reproduce kresek
phase of the disease. Root dip-inoculation method was developed for mass screen-
ing of breeding materials (Yoshimura and Iwata 1965; Yoshimura and Yamamoto
1966). In this method, the rice seedlings are pulled off from the nursery and their
roots and crown parts are dipped in the bacterial suspension for 24–48 h before
transplanting in the main field. This method is very efficient in creating kresek
symptoms. Crown inoculation method for creating kresek symptoms was developed
by Durgapal et al. (1979) in which the seedlings were pricked at the crown region
and dipped in bacterial suspension for 10 min and then transplanted in pots or fields.
They also reported that pricking the crown at 5-leaf stage did not induce any injury
and provided most reproducible results.
Observations
Observations are recorded 15 days after inoculation. For assessing resistance, the
Standard Evaluation System for Rice (SES) developed at International Rice
Research Institute (IRRI), Philippines (Anonymous 1996) is usually followed
(Fig. 1.1). Generally, the scores from several plants are averaged and categorized as
resistant (mean score below 4), moderately resistant (mean score 4–5) and suscep-
tible (mean score more than 5). Many researchers prefer absolute lesion length as
criteria for characterizing host reaction though the length of the lesion for categoriz-
ing resistance/susceptibility varied among the research workers. Lee et al. (1999)
categorized the plant reaction according to lesion length as resistant (<3 cm), mod-
erately resistant (3.1–5.0 cm), moderately susceptible (5.1–7.0 cm), and susceptible
(>7.1 cm). Chen et al. (2000) classified a plant as resistant if the average lesion
length was shorter than 3 cm, moderately resistant if the lesion length was 3–6 cm,
moderately susceptible if the lesion length was 6–9 cm and susceptible when lesion
length was >9 cm. Shanti et al. (2001) followed lesion length up to 4 cm as resistant
and lesion length greater than 4 cm as susceptible while Sanchez et al. (2000) and
Chen et al. (2002) recorded plants with lesion length less than 6 cm as resistant and
those with lesion length greater than 6 cm as susceptible.
1.1.1.2 Blast
Blast caused by the fungus Pyricularia grisea (Cook) Sacc. [teleomorph:

Magnaporthe oryzae (Hebert) Barr] is the most widespread and destructive rice
disease causing substantial loss in yield both in upland and irrigated rice production
system. The fungus affects the leaves, nodes and panicles and produces characteris-
tic symptoms viz. leaf blast, node blast and panicle or neck blast, respectively.
Commonly used media for culturing rice blast fungus are oat meal agar, rice leaf
extract agar, rice polish agar etc. The panicles and leaves showing typical blast
symptoms are surface sterilized with 70 % ethyl alcohol for 10 s and then washed
repeatedly 3–4 times in sterile distilled water. The portions of infected tissue are
then excised with a sterile blade or scalpel and put in a sterile Petri plate lined with
filter paper moistened with sterile water. The plates are then incubated for 24–48 h
at 25–27 °C temperature to induce sporulation. When the lesions become grey
(sporulating lesions), they are held over a plate containing thin layer of water agar
and gently tapped to dislodge the spores. The plates are then observed under a dis-
section microscope and the portions of the agar having single conidia are marked.
The portion of agar is then cut with a sterile scalpel and transferred into a culture
medium plate by putting the agar bit upside down. The mono-conidial culture can
then be sub-cultured in fresh agar plates or tubes. Alternatively, the tissues with
active sporulation can be tapped directly onto a culture medium (preferably supple-
mented with some antibacterial agents). The typical single colony (growing from a
single blast spore) can then be further purified by transferring into fresh culture
medium. The fungus can be maintained for long term in sterile filter paper discs at
−20 °C (Valent et al. 1986).
The fungus can be mass multiplied on a number of natural media. The fungus can
be easily cultured on autoclaved sorghum seeds soaked with 0.2 % yeast extract
powder and then incubating them at 28 °C for 7 days. Mass production of conidia of
blast pathogen can also be done by growing the fungus on autoclaved barley grains
(barley grains: water, 1:1.2 w/w) (Chen et al. 2001). After incubation when the
grains are covered with white and grey hyphae, the grains are washed with sterile
distilled water to remove the hyphae from the surface of the grains and the washed
grains are then put in a sterile Petri plate lines with moistened sterile filter papers
and incubated at 28 °C for 48 h under fluorescent light to allow sporulation. The
fungus can also be mass multiplied by growing them on rice polish agar or oat meal
agar medium and incubating at 25 °C for 7 days in dark after which the plates are
placed under continuous fluorescent light at 25 °C for 4 days to induce sporulation
(Mekwatanakarn et al. 2000). Sporulation of the fungi can be obtained by scraping
the mycelia growth with a sterile rubber spatula and then exposing the plates to fluo-
rescent light at 25–28 °C (Bonman et al. 1987). Conidial suspensions are then made
by washing the grains or scrapping the culture plates with sterile distilled water and
then filtering the solution through cheese cloth. Tween 20 can be added to the conid-
ial suspension at 0.05 % (v/v). The concentration of the suspension should be adjusted
to approximately 105 conidia/ml using a hemocytometer before inoculation.
Screening using uniform blast nursery (UBN). Varietal resistance is usually done at
the seedling stage. A dry upland nursery bed is more favorable than a flooded field
for evaluation of blast resistance. A heavy application of N fertilizer (120–160 kg
N/ha) and high humidity (>95 %) should be maintained in the microclimate of the
nursery. Temperature for infection and disease development is 24–26 °C. Considering
all these parameters, a uniform blast nursery (UBN) method of evaluation of blast
resistance was developed at IRRI (Ou 1965). This method can accommodate a large
number of entries, requires small quantity of seed, and ensures uniform infection.
Briefly in this method, the seedlings will be raised in upland nursery. Test entries are
sown in 50–100 cm long rows with a row to row distance of 10 cm. After every 20
test entries, seeds of a highly susceptible variety are sown. The entire nursery should
be surrounded on all sides by two rows of susceptible variety to act as spreader/
infector rows to ensure heavy disease pressure. Initial inoculum can be introduced
by transplanting infected plants or spreading plant parts such as pieces of infected
leaves, nodes, or panicles in the spreader rows. Spore suspension of specific isolates
can be applied, if necessary. For proper development and spread of the disease, care
need to be taken for dense planting, high N fertilizer application, and maintenance
of prolonged dew period by covering the plots with plastic film at night and supple-
mental overhead sprinkling of water 3–4 times a day depending on the weather
conditions. Proper check varieties should be kept for comparison of the results.
Plants at 15 day old stage are inoculated and observations are taken after 10–15 days
of inoculation.
Screening in trays/pots. To determine the phenotypic reaction of rice seedlings to
specific isolates of the pathogen, artificial inoculation under controlled condition is
essential. Seeds of the test cultivars should be sown in rows in a plastic tray in glass
house. Seedlings of 18–20 days age will be the right stage for spraying freshly
prepared conidial suspension. Inoculated trays are then incubated at 25 °C tempera-
ture and >95 % relative humidity for 7 days in greenhouse (Bonman et al. 1987;
Table 1.1 Descriptive key (SES) for recording leaf blast disease severity (Anonymous 2002)
Score Description of symptoms
0 No lesions observed
1 Small brown specks of pin-head size or long brown specks without sporulating centre
2 Small roundish to slightly elongated, necrotic grey spots, about 1–2 mm in diameter
with a distinct brown margin
3 Lesion type is the same as in scale 2, but significant number of lesions are on the
upper leaves
4 Typical susceptible blast lesions, 3 mm or longer, infecting less than 4 % of the leaf area
5 Typical blast lesions infecting 4–10 % of the leaf area
8 Typical blast lesions infecting 51–75 % of the leaf area and many leaves are dead
9 More than 75 % leaf area affected
Chen et al. 2001). Long et al. (2001) described a method of creating blast disease by
growing the fungus on autoclaved rice seeds and then applying the colonized rice
grains on the soil in between the rows 10 days after sowing either in fields or in
nursery beds. They reported that the disease incidence was high when 25–30
infested grains were applied in an area of 0.1 m2. Kuribayashi and Terazawa (1953)
artificially induced the disease by injecting the spore suspension into the leaf sheaths
of rice seedlings. In this method, the lesions appear on the young leaves which
unfold in a few days.
Artificial inoculation for neck blast. Inoculations can be done through injection of 1 ml
of spore suspension with a syringe into the leaf sheaths of emerging panicles (about
half way emerged). This method develops 100 % infection (Ou and Nuque 1963).
In another most commonly used method, the neck region (5–6 cm long) can be cut
placed in Petriplate having moistened filter paper soaked with benzimidazole solution
(Chai and Jin 1995).The necks are then smeared with aqueous solution of conidia
containing 2 % carboxymethyl cellulose. The Petri plates are then covered and incu-
bated under light at 28 °C and observations are taken after 10 days of inoculation.
Observations
Disease scoring will be carried out in 10–15 days after inoculation when the disease
severity in susceptible control plants has reached to the maximum. For all practical
purposes, the observations are recorded following the SES (Anonymous 2002). This
scale is mainly used for recording blast reaction in the nursery stage. In general, the
average score 3 or below is taken as resistant, 4–5 as moderately resistant and score
greater than 5 is taken as susceptible. Sometimes, based on these scores, disease
severity index or disease index is calculated (Table 1.1).
Mackill and Bonman (1992) recorded blast reactions after 7 days of inoculation
following a 0–5 scale, where 0 = no evidence of infection; 1 = brown specks smaller
Table 1.2 SES scale based on symptoms for measuring neck blast
SCALE (based on symptoms)
0 No visible lesion observed or lesions on only a few pedicels
1 Lesions on several pedicels or secondary branches
3 Lesions on a few primary branches or the middle part of panicle axis
5 Lesions partially around the base (node) or the uppermost internode or the lower part
of panicle axis near the base
7 Lesions completely around panicle base or uppermost internode or panicle axis near
base with more than 30 % of filled grains
9 Lesions completely around panicle base or uppermost internode or the panicle axis
near the base with less than 30 % of filled grains.
Table 1.3 SES Scale for Score Description

neck blast based on incidence
0 No incidence
of severely neck infected
panicles 1 Less than 5 %
3 5–10 %
5 11–25 %
7 26–50 %
9 More than 50 %
than 0.5 mm in diameter; 2 = brown specks about 0.5–1 mm in diameter; 3 = round-

ish to elliptical lesions about 1–3 mm in diameter with gray centers and brown
margins; 4 = typical spindle shaped blast lesions, 3 mm or longer with little or no
coalescence of lesions and 5 = same as 4 but half of one or more leaves killed by
coalescence of lesions. Plants with score 0–3 are considered resistant and those with
scores of 4–5 are considered as susceptible.
Padmanabhan and Ganguly (1959) described a scale for recording blast reaction
of germplasm. The scale describes as A = reddish flecks only; B = minute reddish
spots showing no differentiation into distinct zones; C = Circular spots about 2–3
mm in diameter with a central ashy zone and a purple brown margin; D = broadly
spindle shaped spots, only slightly longer than breadth, 3–5 mm in diameter and
E = large, distinct spindle shaped spots with a central ashy zone and marginal zones
3–5 mm broad and up to several cm in length. The cultivars were with class A and
B were classified as resistant, those with C as moderately resistant and with D and
E as susceptible.
Scale for measuring neck blast is based on the percentage of panicles infected.
In addition, girdling of the neck (partial or complete) and site of infection (on the
main or the smaller branches) may also be considered (Table 1.2).
However, for mass evaluation of germplasm against panicle blast, the number of
severely infected panicles is considered (Anonymous 2002) as follows in Table 1.3.
1.1.1.3 Sheath Blight
Sheath blight of rice caused by the fungus Rhizoctonia solani Kuhn [teleomorph:
Thanetophorus cucumeris (Frank) Donk] is second most important fungal disease
next to blast. The teleomorph belongs to Basidiomycetes. It belongs to anastomosis
group 1 IA (AG-1-IA). In addition to R. solani, two other species of Rhizoctonia
viz., R. oryzae causing rice sheath spot and R. oryzae-sativae causing aggregate
sheath spot have been found to be associated with this disease. All the three patho-
gens may occur concurrently and sometimes referred to as rice sheath blight disease
complex. The fungus can be readily isolated into culture medium. The fungus pro-
duces abundant sclerotia (dark compact mass of hyphae capable of surviving under
unfavorable environment) in culture media and also on infected plants. The infected
sheath/leaf samples are first washed in tap water, cut into small pieces (2–5 mm),
washed 2–3 times in sterile distilled water and then dried using sterile blotting
papers. These sheath/leaf pieces are then placed on 2 % water agar (WA) plates and
incubated at 28 °C for 24–48 h. The sclerotia collected from the infected plant parts
can also be used for isolating the fungus following the above method. The emerging
hyphal tip of a single mycelium is then transferred to potato dextrose agar medium
(PDA) to obtain pure culture of the fungus. The fungus can be maintained in PDA
slants at 4 °C.
Several methods for artificially inducing the disease have been used by various
workers. Yoshimura and Nishizawa (1954) found that placing sterile straw bits inoc-
ulated with the fungus among the tillers in each hill and wrapping them for 1 week
was most efficient in inducing the disease. They also found that maximum tillering
stage of the plant is most suitable for varietal screening. Amin (1975) described an
improved method ‘stem-tape-inoculation’ for sheath blight disease by placing
R. solani colonized stem bits on to the non-injured sheath of 6-week old rice plants
using a cellotape at about 6–10 cm above the water line. The disease development
is faster in this method. Freshly developed sclerotia of the fungus can also be used
as inoculum source in this method. However, this method is time consuming and
impractical for screening large number of germplasm under field conditions.
Bhaktavatsalam et al. (1978) developed a simple, rapid and mass inoculation
technique to induce sheath blight disease in rice and to evaluate germplasm and
breeding lines in fields and glass house. The pathogen is multiplied on autoclaved
stem pieces (2–3 inches in length) of water sedge (Typha angustata) soaked with
1 % peptone solution for 8–10 days. Four to five stem bits colonized with fungal
mycelia (and sclerotia) are then placed in between the tillers in the central region of
the hill, 5–10 cm above the water line and then tied with a rubber band to maintain
high humidity in the micro-climate. In glass house tests, the inoculated plants are
kept in a humid chamber for 4–5 days for rapid pathogen establishment after which
the plants are transferred into glass house benches. This method is very easy, less
time consuming and highly reproducible. In case of non-availability of Typha
plants, the fungus can be multiplied in cut stem pieces of rice plants or very young
sorghum plants.
Toothpick method of inoculation of R. solani was described by Zou et al. (2000)
and Rodrigues et al. (2001). Wooden toothpicks (1 cm in length) are placed in
Erlenmeyer flasks containing a shallow layer of potato dextrose broth and auto-
claved. Ten to fifteen autoclaved toothpicks are then placed in a PDA plate keeping
small gaps in between the toothpicks. The plates are then inoculated with 4–5 myce-
lial plugs from actively growing culture and incubated for 5–6 days so that the
fungus colonizes the toothpicks. Plants at the maximum tillering stage are then
inoculated by placing one R. solani colonized toothpick into the lowest inner sheath
of the main tiller. The plants are then kept in a moist chamber for varying period of
time for the development of the disease. This method is highly reproducible and has
been used by many workers for artificially inducing the disease.
Singh et al. (2002) described a method of artificially inducing rice sheath blight
disease by carefully placing a freshly harvested sclerotium inside the leaf sheath.
Adding few drops of water is required to maintain high humidity inside leaf sheath.
The plants are then kept in a humid chamber for rapid disease development. This
method has also limitations in screening large number of germplasm accessions.
A micro-chamber screening method was described by Jia et al. (2007) to evalu-
ate sheath blight disease resistance under glass house conditions, wherein the rice
seedlings are inoculated at 3–4 leaf stage with PDA agar plugs containing mycelium
and then covered with a 2- or 3-litre transparent plastic bottle for maintaining high
humidity after inoculation. This method can be used to accurately quantify resis-
tance to sheath blight pathogen under controlled greenhouse conditions but has
limited application in screening large number of germplasm accessions and in field
evaluation. Recently, Ram Singh et al. (2010) standardized inoculation method for
evaluating mass screening of for sheath blight resistance in nursery beds. In this
method, 30–40 days old seedlings were inoculated by broadcasting R. solani
inoculum raised on barley grains and Typha pieces (1:1 v/v). A positive correlation
(r = 0.931) between disease score (0–9) in nursery and field screening tests was
obtained. Though nursery screening was not found an absolute indication of resis-
tance in the field but it could be utilized in shortlisting of rice genotypes for screen-
ing against sheath blight in the field. Park et al. (2008) described a method of
inducing rice sheath blight disease. The fungal mycelium grown in liquid culture is
harvested and cut into small balls (approximately 0.5 cm in diameter) with forceps.
Rice plants at late tillering stage are then inoculated with R. solani by placing a
mycelial ball beneath leaf sheath and immediately covering with aluminium foil.
The plants are then kept in a humid chamber for rapid disease development. This method
has also limited application in screening large number of germplasm accessions.
A detached cut-leaf inoculation technique was developed by Dath (1987) for
assessing reaction of a large number of varieties in the laboratory to sheath blight.
Briefly, this technique involves placing leaf blades cut to 6–8 cm long over a thin
layer of water or moist filter paper contained in Petri dishes and inoculating the cut
leaf blades by pacing 10-day-old sclerotia over them. The Petri plates are then incu-
bated at 26–28 °C. Water-soaked lesions appear within 24 h of incubation and clear
cut lesions are formed between 48–72 h. The disease can be scored 96 or 120 h after
inoculation. The laboratory screening compares well with the field screening. When
floated on tap water, the leaf pieces remained green and fresh even for 4–5 days.
Since the spread of the infection was very fast, the leaf pieces could be safely floated
on tap water and no kinetin solution was required. The detached leaf bioassay
method has also been described by Jia et al. (2007).
At IRRI, several methods of artificial inoculation were tested and inoculation of
rice plants at the booting stage by placing a specified quantity (about 5 g) of rice
grain and rice hull mixture (1:5 w/w) colonized with fungus was found best and
highly reproducible (Sharma et al. 1990).
Observations
Observations are taken 14 days after inoculation. Yoshimura and Nishizawa (1954)
proposed a formula for a sheath blight disease index, considering the number of
tillers having flag leaf and the sheath infection
3n1 + 2 n 2 + 1n3 + 0 n 4
Disease index ( D.I.) = × 100
N
where, N = total number of tillers in a plant/hill; n1 = Number of tillers having
infection on the upper four leaf sheaths and leaves including the flag leaf and its
sheath; n2 = Number of tillers having infection on three leaf sheaths below flag leaf;
n3 = Number of tillers having infection on the 2nd and 3rd leaf sheath below the flag
leaf; n4 = Number of healthy tillers.
SES developed by International Network for Genetic Evaluation of Rice
(INGER) adopted a 0–9 scale based on the relative lesion height (Fig. 1.2). The rela-
tive lesion height is the average vertical height of the uppermost lesion on leaf or
sheath expressed as a percentage of the average plant height. This is the most widely
followed procedure for assessing rice sheath blight disease intensity (Table 1.4).
The observations can then be summarized as percentage disease intensity (sever-
ity index) as follows:
Sum of all disease ratings ×100

DI (%) or SI (%) =
Total number of ratings × Maximum disease grade
Tang et al. (2007) calculated sheath blight index by separating all the tillers from
10 hills into 6 disease ratings as follows: 5 – lesion on the panicle; 4 – lesion on the
flag leaf; 3 – lesion on the second topmost leaf; 2 – lesion on the third topmost leaf;
1 – lesion on any leaf except the top three leaves; and 0 – no lesion.
Fig. 1.2 Diagram key for 100%

assessment for sheath blight
in field
80%
60%
40%
20%
1=<20% 3=20-30% 5=31-45% 7=46-65% 9=>65%

Lesion limited to plant height(%)
Table 1.4 SES (Anonymous 2002) for sheath blight of rice

Disease grade Description
0 No infection
1 Vertical spread of the lesions up to 20 % of plant height
3 Vertical spread of the lesions up to 21–30 % of plant height
The number of tillers in each category was counted to calculate ShBI as follows:
100 × ( 5n1 + 4 n 2 + 3n3 + 2 n 4 + 1n 5)

SHBI (%) =
5 × ( n1 + n 2 + n3 + n 4 + n 5 + n6 )

where n1 to n6 are the tiller numbers of each category rated 5 to 0, respectively.
Rush et al. (1976) proposed a 0–9 disease grade scale for recording sheath blight
symptoms where 0 indicates no infection and 9 indicates plants dead or collapsed.
Based on the grades, disease index (DI) was calculated by the formula:
DI (%) = (100 × total grade points) / (Number of sheath counted × maximum grade)
In case of detached leaf bioassay, disease observations were recorded after

5 days of inoculation (Vidhyasekaran et al. 1997). The intensity of the symptoms was
graded into four categories based on the leaf area affected: 1 = 1–10 %, 2 = 11–25 %;
3 = 26–50 %, and 4 = more than 50 % affected leaf sheath area. Taheri and Tarighi
(2010) adopted a modified procedure for recording the sheath blight severity in
detached leaf bioassay. The intensity of the symptoms was graded into five classes
based on the leaf area infected: 0 = no infection, 1 = 1–25 %, 2 = 26–50 %; 3 = 51–75 %,
and 4 = 76–100 % infected leaf area. Disease index (DI) was calculated by the for-
mula DI = [(0n0 + 1n1 + 2n2 + 3n3 + 4n4)/4N] × 100,where, n0 is the number of
plants with score 0, n1 the number of plants with score 1, n2 the number of plants
with score 2, n3 the number of plants with score 3, n4 the number of plants with score
4, and N the total number of plants used in the experiment.
1.1.1.4 Rice Tungro Virus
Rice tungro disease is caused by two unrelated virus viz., rice tungro bacilliform
virus (RTBV) and rice tungro spherical virus (RTSV). RTSV is a plant picornavirus
with a single stranded positive sense RNA genome while RTBV is a pararetrovirus,
with double stranded and circular DNA genome. RTBV resembles the members of
the Badnavirus (Bacilliform DNA virus) group. The plants infected by RTSV alone
do not show any definite symptoms. The plants infected by RTBV alone show mod-
erate stunting and discolouration. However, rice plants infected with both RTBV
and RTSV produce severe tungro symptoms (Krishnaveni et al. 2009). Rice tungro
virus is transmitted from infected plants to healthy plants only by means of insect
vectors called green leaf hoppers. The virus is mainly transmitted by two species of
green leaf hopper, Nephotettix virescens and N. nigropictus. The zigzag leafhopper,
Recilia dorsalis has also been reported to transmit the virus but it is much less
important. Initially, the virus is identified and purified by transmitting the virus from
the infected sample to healthy plants by green leaf hoppers (GLH). The virus is then
multiplied on susceptible varieties like Taichung Native 1 and these tungro infected
plants are maintained in separate glass house chamber. Simultaneously, virus free
GLH is maintained on TN 1 plants in insect cages.
Method of Inoculation
When GLH (nymphs and adults) feed on diseased plants (source plants), the virus
particles get attached to mouth parts (stylets) of the insects. Though, the insects can
pick up the virus in 30 min time, an acquisition feeding period of 1–2 days is pre-
ferred so that insects acquire maximum number of virus particles. A period (30–60
min) of pre-acquisition fasting can improve the efficiency of transmission. Once,
the insects acquire the virus, they become viruliferous. When these viruliferous
insects feed on healthy plants, they transmit the virus. The shortest inoculation feed-
ing period is reported to be 7 min. However, longer feeding period results in higher
transmission rates. It has been reported that the insect can remain viruliferous for
2–6 days and thereafter they become non-infective unless they acquire the virus
again. Generally, tungro virus disease symptoms appear 10–14 days after introduc-
tion of virus in plants.
Table 1.5 SES Scale (1996) for rice tungro disease

Score Description
1 No symptoms
3 1–10 % plant height reduction with no distinct leaf discoloration
5 11–30 % plant height reduction with no leaf discoloration
7 31–50 % plant height reduction and yellow to orange leaf discoloration
9 More than 50 % plant height reduction and yellow to orange leaf discoloration
For artificial screening of rice germplasm, the entries are line sown in plastic
trays. Twenty days old plants are then inoculated by releasing 2–3 viruliferous
insects per plant. The trays are then kept in a cage for effective transmission.
Observations are taken 14 days after inoculation following SES (Anonymous 2002)
(Table 1.5).
1.1.1.5 Brown Spot
Brown spot caused by fungus Bipolaris oryzae (Breda de Haan) Shoemaker

[Synonyms: Helminthosporium oryzae, Drechslera oryzae; teleomorph:
Cochliobolus miyabeanus (Ito and Kuribayashi) Drechsler ex Dastur] is a major
problem in upland rice and rice grown in hill ecosystem. High intensity of the dis-
ease has also been reported from low land fields. The disease tends to be severe on
rice grown on poor soils of low pH, deficient in essential and trace elements and
especially when the available potash is low. The disease was a major contributing
factor of great ‘Bengal famine’ during 1942. The fungus can be readily cultured in
a number of artificial media. Infected seeds or leaf segments having typical brown
spot symptoms are incubated on moist blotter. Profuse mycelial growth with abun-
dant conidia is formed on the infected samples. The fungus then can be isolated in
a similar way like that of blast. Alternatively, the affected parts (seeds or segments
of leaves showing brown spots) are surface sterilized with 0.1 % mercuric chloride
for 60 s followed by washing with sterile distilled water 3–4 times, blotted dry and
then plated on to freshly prepared PDA plates (preferably supplemented with some
antibacterial agents). The fungus is then sub-cultured from mycelia growing from
the infected parts (after examining the spores under microscope). The fungus can be
multiplied on potato dextrose agar media, potato sucrose agar or rice polish agar for
4–5 days in dark. Heavy production of conidia of this fungus can be achieved by
growing the fungus on sucrose praline agar medium (Shoemaker 1962). The spores
are harvested by scrapping the plates, filtered through muslin cloth and concentra-
tion is adjusted to 1 × 105 spores/ml. The spore suspension is then used for spraying
18–20 days old seedlings grown in a tray or pots or in a nursery bed until run off.
Observations can be recorded 2 weeks post inoculation following SES (Anonymous
2002) as follows in Table 1.6.
Table 1.6 SES for brown Score Description (affected leaf area)
spot of rice
0 No incidence
1 Less than 1 %
2 1–3 %
3 4–5 %
4 6–10 %
5 11–15 %
6 16–25 %
7 26–50 %
8 51–75 %
9 76–100 %
1.1.2 Emerging Rice Diseases
1.1.2.1 Sheath Rot
Sheath rot caused by the fungus Sarocladium oryzae is especially important on

those rice crops which are affected by other biotic or abiotic stresses particularly
stem borer and rice tungro virus. The disease is of common occurrence in cytoplas-
mic male sterile lines (A lines) in hybrid rice seed production. The fungus can be
isolated and maintained in PDA medium. Several inoculation techniques have been
tried by different researchers to artificially induce the disease. Several workers
reported that insertion of rice or sorghum or pearl millet grains colonized with the
fungus between the flag leaf sheath and culm was most dependable and consistently
produced severe infection (Raju and Singh 1978; Estrada et al. 1979; Mukherjee
and Singh 1980). Estrada and Crill (1980) successfully induced typical sheath rot
symptoms with S. oryzae spore suspension prepared in 25 % beef extract peptone
solution. Kang and Rattan (1983) induced sheath rot disease by injecting spore sus-
pension or inserting mycelial pieces from PDA culture. Reddy and Subbayya (1989)
reported successful induction of the disease by inserting single leaf bit inoculum in
between culm and leaf sheath.
Among various measurement scales proposed by different workers, disease inci-
dence based 0–9 scale of SES (Anonymous 2002) is commonly followed for scor-
ing sheath rot of rice, which is given below in Table 1.7.
However, this scale is not adequate to identify resistant donors. Satyanarayana
and Reddy (1979) suggested a 1–9 scale for sheath rot disease based on the cover-
age of the lesions on the leaf sheath and damage to the panicles. Raychoudhuri and
Purakayastha (1980) suggested a method of quantifying disease severity based on
the size of the necrotic spot. Mukherjee et al. (1981) suggested a 0–5 scale scoring
system and classified the entries based on the lesion area which was calculated by
multiplying length and breadth of the lesions. Narayanasamy and Viswanathan
(1990) developed a new qualitative and quantitative 1–9 scoring system as follows
in Table 1.8.
Table 1.7 SES for sheath Score Incidence of severely affected tillers
rot of rice
0 No incidence
1 Less than 1 %
3 1–5 %
5 6–25 %
7 26–50 %
9 51–100 %
Table 1.8 New scoring system for sheath rot of rice

Score Description
1 Small brown lesion on boot leaf sheath and panicle emergence normal
3 Lesions enlarge, coalesce and cover about 5 % of the leaf sheath and panicle
emergence normal
5 Lesions cover 6–15 % leaf sheath area and 75 % of the panicle is emerged
7 Lesions cover 16–50 % of leaf sheath area and 50 % of the panicle is emerged
9 Lesions cover >50 % leaf sheath area and about 25 % of the panicle is emerged
The disease index was then calculated by the following formula:

DI = (1 × A) + (3 × B) + (5 × C) + (7 × D) + (9 × E,) where A, B, C, D and E are per-
centage of tillers in grade 1, 3, 5, 7 and 9, respectively. The varietal reaction was then
categorized based on DI as HR (DI = 0–99), R (DI = 100–199), MR (DI = 200–299),
MS (DI = 300–499), S (DI = 500–699) and HS (DI = 700–899).
1.1.2.2 False Smut
False smut of rice caused by the fungus Ustilaginoidea virens (Cke.) Tak [teleo-
morph: Villosiclava virens] has assumed the status of a serious disease in recent
years possibly due to high input rice cultivation, increase in area under hybrid rice
cultivation and apparent changes in the climate. Though the fungus is very slow
grower, it has been isolated and grown in different culture media. Lu et al. (2009)
isolated the fungus on XBZ agar medium. The potato sucrose broth can be used for
mass production of conidia.
Spore suspension (5 × 104 conidia/ml) is prepared either by growing the fungus
in potato sucrose broth or by collecting from the smut balls. Lu et al. (2009) and
Ashizawa et al. (2010) artificially created the disease by injecting 2 ml spore sus-
pension into each panicle at the booting stage (boot still inside leaf sheath) between
4–6 P.M. The method is highly dependable, but applicable to only small number of
samples. Kulkarni and Moniz (1975) successfully induced the disease by applying
chlamydospore suspension into fertilized and unfertilized ovaries with camel hair
brush. Chhottaray (1991) reported that spraying of spore suspension at the flower-
ing period produced maximum infection.
Table 1.9 SES for false smut of rice

Score Percentage of infected florets Category
0 No incidence Highly resistant
1 Less than 1 % Resistant
3 1–5 % Moderately resistant
5 6–25 % Moderately susceptible
7 26–50 % Susceptible
9 51–100 % Highly susceptible
Table 1.10 Rating scale for Class Description

recording severity of false
0 No smut balls
smut of rice
1 1 smut ball or infected grain per panicle
2 2 smut balls or infected grains per panicle
3 3–5 smut balls or infected grains per panicle
4 6–9 smut balls or infected grains per panicle
5 >10 smut balls or infected grains per panicle
The SES for measuring false smut disease developed at IRRI, Philippines
(Anonymous 2002) is based on percentage of infected florets as follows in Table 1.9.
Lu et al. (2009) followed a 0–5 scale for recording false smut intensity as follows
in Table 1.10.
The disease index (%) was then calculated as
DI (%) =
∑ (N × V ) × 100
i i
∑N ×Vi max

where, Ni is the number of panicles in different classes; Vi is the class value and Vmax
is the maximum class value.
1.1.3 Stem Rot
Stem rot of rice caused by Sclerotium oryzae Catt. (anamorph: Helminthosporium

sigmoideum Cav.; teleomorph: Magnaporthe salvanii (Catt.) Krause and Wesbster)
can cause severe yield loss in certain production system due to increased lodging,
smaller panicles, production of light and chalky grains and poor milling quality. For
isolation of the fungus, the infected stem or withered stubble can be cut into 1 cm
length pieces, disinfected with 1 % sodium hypochlorite solution for 1–2 min fol-
lowed by washing in sterile distilled water (3–4 times), blotted dry and then put into
2 % water agar plates (preferably containing some antibacterial agents). After 2–3
days of incubation at 28 °C, the hyphal tip growing from the infected plant parts are
transferred onto a fresh PDA or potato sucrose agar plates. Alternatively, the fungus
can be isolated from the sclerotia collected from the infected parts. The collected
scleortia are first washed in 70 % ethanol for 1 min, washed in sterile distilled water,
blotted dry and then put onto potato dextrose agar medium. The fungus then can be
further purified by sub-culturing.
Incorporation of diseased rice stubbles or floating the sclerotia on the field plot
water surface had been suggested for large scale field inoculation (Kawamura
1941; Luthra and Satter 1936). Using this method, Ferreira and Webster (1975)
inoculated 7 weeks plants grown in pots by placing 150 mg of sclerotia of the
fungus on the surface of the water of each pot. Oster (1990) mass produced scle-
rotia in rough rice-rice hull mixture and used the sclerotia to inoculate 45 days old
plants by spreading the sclerotia on the water surface. A stem tape and rice grain
culture inoculation method was proposed to evaluate stem rot resistance in rice
genotypes in fields (Amin 1976). The disease can also be artificially created by cut
stem wound inoculation method (Hseih 1966). Mixing of sclerotia in pots (80 mg
sclerotia per 3 kg soil) was also advocated by Sharma and Mehrotra (1988).
Sprinkling a sclerotial inoculum on the soil surface around seedlings immediately
prior to flooding was also advocated for artificially creating the disease (Cother
and Nicol 1999). Miah et al. (1977) used two methods to artificially induce the
disease viz. placing the mycelial mass grown on PDA agar among the injured
culms of the hill and second by placing the inoculum from the autoclaved rice
grains in the hills and then tying the hill with a rubber band or a thread as in sheath
blight disease.
Several scales have been followed by different research workers to measure the
disease. Cralley (1936) categorized the plants into four different groups’ viz. (i)
plants with diseased sheaths only, (ii) plants with mildly diseased culms, (iii) plants
with moderately diseased culms and (iv) plants with severely diseased culms. The
cultivars were then classified as resistant, moderately resistant and susceptible based
on the disease index. Krause and Webster (1973) developed a 1–5 scale based on
severity of the disease as 1 = healthy, no visible symptoms (H); 2 = light, symptoms
and sclerotia on the outer leaf sheath only (L); 3 = mild, symptoms and sclerotia in
the inner leaf sheaths but culm green and healthy (M); 4 = moderate, mild discolor-
ation of the culm but interior of the culm healthy (M*) and 5 = severe, culms infected
internally, may be collapsed (S). Based on these scores, the disease index (DI) was
then calculated as follows
DI = [1(n1 × H) + 2(n 2 × L) + 3(n3 × M) + 4(n 4 × M* ) + 5(n 5 × S)] /

Total number of tillers examined
Hseih (1966) developed an efficient 0–9 rating scale to categorize resistance in
laboratory based on lesion length. To ensure uniform adoption of a rating scale, a
0–9 scale was developed (Anonymous 1996). This scale is being followed by most
of the rice researchers for recording severity of stem rot, which is described below
in Table 1.11.
Table 1.11 SES scale for % Age of stems with

recording observations on Score lesions and sclerotia
stem rot severity
0 No incidence
1 Less than 1 %
3 1–5 %
5 6–25 %
7 26–50 %
9 51–100 %
1.2 Phenotyping for Important Insect Pests
Though various options are available for pest management, host plant resistance is
the major component which is deployed to mitigate pest damage. It is inherent in the
plant, eco-friendly, cheaper and compatible with most of the available options for
pest management. But identification of the promising donors/resistant sources from
the germplasm and pre-breeding lines for pest resistance is an onerous task. The
successful selection of lines depends on the accurate phenotyping. Based on the pest
behaviour and the breeding objective various methods have been in vogue (Heinrichs
et al. 1985; Anonymous 2002). Here we briefly discuss the methodology for screening
of the two major pests viz., planthoppers and yellow stem borer.
1.2.1 Planthoppers
Planthoppers include brown planthopper (BPH), Nilaparvata lugens Stal and white
backed planthopper (WBPH), Sogatella furcifera Horvath.
1.2.1.1 Damage Symptoms
Planthoppers are homopteran insects which suck the sap from the base of the plant
resulting in yellowing and drying of the affected plant. Both adults and nymphs
cause damage to the plant. The drying in the field progresses in the form of concen-
tric circles and the symptom is known as ‘hopper burn’. Crop loss is usually consid-
erable and complete destruction of the crop occurs in severe cases. WBPH
preferentially feeds at the base of upper leaves, while BPH prefers base of the plant.
Severely attacked seedlings do not grow, remain stunted, wilt and eventually die.
If the infestation is at the panicle initiation stage, the number of grains and the
panicle length decrease. But when attacked later, during the maturation period, gain
filling is affected. When hoppers are present in large numbers late in crop growth
stage they are seen moving on the foliage and panicles. Oviposition punctures and
Table 1.12 Scoring of plants for planthoppers under field conditions

Damage score Description
0 No damage
1 Slight yellowing of a few plants
3 Leaves partially yellow in case of BPH but no hopper burn
5 Leaves with pronounced yellowing and some stunting or wilting and 10–25 %
of plants with hopper burn, remaining plants severely stunted
7 More than half of the plants wilting or with hopper burn, remaining plants
severely stunted
9 All plants dead
Table 1.13 Scoring of plants Damage score Number/hill

for BPH incidence based on
0 0
insect population
1 Less than 5 insects
3 5.1–10
5 10.1–20
7 20.1–40
9 >40
honey dew excretion predispose the damaged plant to fungal infections and sooty
mould growth. Though the damage caused by both the species are similar the
resistance mechanisms in rice plant do differ. To identify new sources of resistance
and study the mechanisms and genetics of resistance, the genetic resources are
screened for each of the pests individually.
1.2.1.2 Screening Methodology
Phenotyping Under Field Conditions
Evaluation under field conditions is carried out in a hot spot by adjusting the plant-
ing dates so as to coincide with the maximum pest incidence. After every 10 rows
of test material a row of susceptible check (variety TN1) should be planted alternat-
ing with a resistant check i.e. PTB 33 for BPH and MO 1 for WBPH. All around the
test entries, infestor rows of tall, susceptible, long duration variety are planted.
Nursery should be well protected before transplanting and a high dose of nitroge-
nous fertilizer may be given at later stages of crop growth to increase susceptibility
to pest. When hopper burn symptoms start appearing either in the susceptible checks
or in any of the test entries, varieties may be scored on the 0–9 scale (Anonymous
2002) as mentioned in Table 1.12. However, there may be few tolerant lines which
are healthy, despite infested by high populations. In such conditions the lines may
be scored on the number of hoppers per hill (Table 1.13). The planthopper popula-
tion should be counted on 10 plants of each genotype at 10 days interval commenc-
ing from 60 days onwards till 10 days before harvest. In recent years, WBPH is
Fig. 1.3 Seed box lay out for

greenhouse screening against
BPH/WBPH (adapted from
Bentur et al. 2011)
gaining prominence and in most of the locations in India it is found along with BPH
(DRR 2000–2010). If the field population comprises of BPH and WBPH then the
ratio of the two pests in time and space is of paramount importance. If hopper burn
is not observed despite high pest population, per cent tiller mortality can be recorded
in all the test varieties. The disadvantages with this methodology are that only field
tolerance can be identified if the pest pressure is high. The exact reaction of the
varieties to mixed populations cannot be quantified. Many of the times the pest
infestation levels are not very high to categorize the germplasm. Jena and Kim
(2010) opined that field evaluation is unreliable due to seasonality, unpredictable
and uneven distribution of BPH.
Phenotyping Under Greenhouse
A pure colony of BPH and WBPH maintained on a susceptible variety is the pre-
requisite for an efficient screening program. The Standard Seedbox Screening
Technique (SSST) (Heinrichs et al. 1985; Velusamy et al. 1986 and Bentur et al.
2011) is the most basic technique based on which most of the theories on
planthopper-rice interactions have been studied. In the SSST, sprouted seeds of the
test cultivars are sown in a single row in a seed box of about 60 × 40 × 10 cm.
Suitable susceptible and resistant checks are sown in similar rows in the same box
(Fig. 1.3). Seven days after sowing, seedlings are thinned to about 20 plants per row.
At three leaf stage, trays are infested with 10 s instar nymphs per plant from the
greenhouse colony. When TN1 plants on one side show severe damage, rotate the
tray by 180O for even reaction. When plants of susceptible check TN 1 show 90 %
mortality or damage, the test entries should be scored on individual plant basis on a
0–9 scale for BPH and WBPH (Table 1.14).
As the infestation in the field occurs in the tillering stage and beyond, the
modified seedbox screening test (MSST) was designed to overcome certain
Table 1.14 Scoring of plants in SSST under greenhouse conditions

Damage Brown planthopper White backed planthopper
score (DS) (Bentur et al. 2011) (Heinrichs et al. 1985)
0 No damage No damage
1 Very slight damage Very slight damage
3 Lower leaf wilted with two green First and second leaves with orange
upper leaves tips; slight stunting
5 Two lower leaves wilted with one More than half the leaves with
green upper leaf yellow orange tips; pronounced
stunting
7 All three leaves wilted but stem More than half the plants dead;
still green remaining plants severely stunted
and wilted
9 Plants dead Plants dead
inconsistencies in the SSST. In the MSST, seeds are sown and thinned as in the
SSST, but infested at 20 days after sowing with four second-instar nymphs per
plant. Plants are evaluated at the time when susceptible checks are killed using the
same scale as in the SSST. With the MSST, usually mortality of the susceptible
check is caused by F1 BPH, that is, the original nymphs mature and reproduce in the
seedbox, and their offspring kill the plants (Velusamy et al. 1986).
Apart from these there are some special tests viz. functional plant loss index,
days to wilt test and insect growth parameters like nymphal survival, honeydew
excretion, days to adult, and hatchability of eggs which can be quantified (Heinrichs
et al. 1985; Bentur et al. 2011) to elucidate the mechanisms of resistance in the dif-
ferent test varieties.
1.2.2 Stem Borers
Of all the stem borers prevailing in India, yellow stemborer (YSB), Scirpophaga
incertulas, is the predominant pest and causes damage at all the stages of crop
growth. Stem borer is a chewing pest and only the larva causes damage by boring
into the plant.
1.2.2.1 Damage Symptoms
Neonate larvae emerging from an egg mass generally nibble the leaf. Consequently,
small shot holes appear on the plant with slight yellowing or halo. The larvae dis-
perse through slings and bore into the plant. Due to larval feeding within the stem
the central leaf turns brown, fails to open and dries. The symptom is known as dead
heart (DH). When this dead portion is pulled, it comes out easily. The dead heart
symptom is visible from nursery to pre-booting stage. If the damage occurs in rice
nursery then the establishment of plants in the main field would be difficult due to
mortality of the tillers. Presence of entrance or tiny exit holes on the stem and tillers,
disintegrated tissues, frass or broken stems are subsequent signs of feeding. At later
stages this symptom is visible in non-productive side tillers which are economically
unimportant but harbour larval populations. Dead heart damage can be compen-
sated and sometimes the compensated tillers produce small panicles with shriveled
grains. The extent of dead heart formation varies with the age of the crop, stage,
variety and point of larval entry into the plant.
In the reproductive phase, stem borer larvae may feed within the stem without
severing the growing plant parts at the base. Larval damage at early booting stage
can cause empty panicles, which remain enclosed in the sheath. Most commonly
when infestation occurs at booting stage the panicles are fully emerged but are
completely empty. Hence called as white heads (WH). At times the panicles are
partially emerged or remaining enclosed in the leaf sheath (Islam Zahirul and Karim
1997). Older plants often break where the stem is hollowed out causing lodging.
The damage to rice grains by stem borer attack is usually represented by the inci-
dence of chaffiness.
Catling and Islam (1999) summarized the yield loss caused by stem borers as
(i) a loss of panicle bearing stems due to the production of ‘dead hearts’ or from
damaged but symptomless stems attacked in the vegetative stage; (ii) smaller
panicles borne by compensatory nodal tillers; (iii) ‘white heads’ produced in the
reproductive phase; and (iv) a decrease in filled grains and lowering of panicle
weight from late damage.
1.2.2.2 Phenotyping Methods
There is no artificial diet available till date where YSB had completed its life cycle
successfully. Hence during the screening season when adult moths are available in
the field, they are collected every day and brought to the glasshouse and released on
to 30 day old potted rice plants to lay the egg masses. Eggs are laid both on leaf
blade and stem. These egg masses free from egg parasitoids at the black head
stage or the neonate larvae emerging from these eggs are used for screening (Bentur
et al. 2011).
1.2.2.3 Phenotyping Under Field Condition
Four rows of susceptible variety (TN 1 or Pusa Basmati 1) are raised as infestor
rows around the material to be screened to attract stem borers. But Pusa Basmati 1
is more susceptible to stem borer and increases the infestation (Padmakumari and
Pasalu 2003) and can be used as infestor rows. W 1263 can be used as a resistant
check. If any special material bred for stem borer tolerance is available for evalua-
tion, then the following methodology involving artificial infestation may be adopted.
Since two different types of symptoms are evident and the mechanisms operating
them may be different, it is advisable to always screen the plants at both the phases
Table 1.15 SES for Vegetative phase Reproductive phase

recording observations
Damage score DH (%) WE (%)
on stem borer severity
0 No damage No damage
1 1–10 % 1–5 %
3 11–20 % 6–10 %
5 21–30 % 11–15 %
7 31–60 % 16–25 %
9 61 % and above 25 % and above
Fig. 1.4 Field screening by pinning yellow stem borer egg mass
of crop growth viz. vegetative (maximum tillering) and reproductive (booting)

stages. For a valid screening test, the damage in the susceptible check should be
more than 20 % dead hearts and 10 % white ears, respectively following Anonymous
2002 as indicated in Table 1.15.
1.2.2.4 Test Insects
Egg Mass as Source of Inoculum
Egg mass at black head stage are pinned @ one/sq m on to the boot leaf of a plant
near the auricle and the area is covered with a polythene sheet (Fig. 1.4). This would
minimize the labour requirement when compared to release of larvae. The main
disadvantage is that we need a lot of egg mass for release and we are not sure how
many larvae would have entered the plant as the invasive mortality is high. The
damage is visible after 3 days but it is apt to take observations on seventh and
twenty-first day after infestation or when the susceptible check records 20 % DH
or 10 % WE.
Neonate Larvae as Source of Inoculum
The method was slightly modified from Heinrichs et al. (1985). Neonate larvae are
released on to the auricle of the tiller @2 larvae/tiller. This level of infestation was
arrived at, so as to effectively screen all the entries because 10 larvae per hill or one
larvae/tiller may not be sufficient to cause enough damage as tiller number varies
with variety. It was observed that under open field conditions the mortality of the
larvae is very high during invasion and dispersion. Once the larvae are released,
observations are taken at 7 day interval from 7 days after infestation (DAI) till 28
DAI. At boot leaf stage, the larvae are released into the boot by slightly opening the
top leaf sheath under greenhouse conditions. Under field conditions they are released
into the whorl from where the flag leaf emerges.
Observations to be Recorded
At vegetative phase, total tillers per hill and dead hearts are recorded. In reproduc-
tive phase, number of panicle bearing tillers per hill and the number of white ears
caused are counted. The damage in different genotypes are scored following SES
(Anonymous 2002). At times the white ear damage is not evident in all the test
entries but grain filling is affected. Hence grain yield is the major criteria for selec-
tion of the plants after infestation (Padmakumari et al. 2009). The actual impact of
infestation in the test entries can be calculated as (Grain yield in test entry − Grain
yield in control) × 100 / Grain yield in control under infested conditions. The geno-
types showing lesser difference in yield over uninfested control should be selected
as field tolerant genotypes.
Test entries may be susceptible at vegetative phase and tolerant at reproductive
phase and vice versa. Entries with damage score of one at either or both phases of
crop growth are promising and considered as tolerant for stem borer damage. A test
entry with a damage score “0” must be carefully evaluated. This may be true
reaction or it could be an escape due to insufficient pest pressure. Hence a careful
correlation with the pest infestation and the stage of the crop is very much essential.
But they need to be confirmed under either greenhouse or field conditions with
sufficient pest pressure.
1.2.2.5 Limitations of the Field Evaluation
Stem borer is a regular pest all over India. However, under field evaluation we do
not observe pest pressure to be uniform through out the crop period. If there is only
one planting time (due to paucity of seed material), then the material would not be
screened at both the stages of crop growth. So we rarely get entries having low
damage at both the phases of crop growth period under sufficient pest pressure.
Under such conditions most of the time the escapes are scored as nil damage. Escape
could be due to earliness of a variety or due to delayed flowering in which case the
material will not be screened under sufficient pest pressure.
1.3 P
henotyping for Root-Knot Nematode (Meloidogyne
spp.) Resistance
Root-knot nematodes (Meloidogyne spp.) particularly, Meloidogyne graminicola

are serious pests of rice (Bridge et al. 2005). These nematodes are obligate seden-
tary endoparasites (Abad et al. 2003). When infected by root-knot nematode, the
plants become stunted and chlorotic with reduced growth due to disturbance in
nutrient and water uptake leading to the crop failure. The nematode infected roots
show typical knots or galls at the tips or in the middle of roots (Fig. 1.5). Root-knot
nematodes occur in almost all rice growing ecosystems including deep water
(Bridge and Page 1982) and high altitude hill ecosystems (Srivastava et al. 2008).
Generally, root-knot nematode damage is usually more pronounced in rainfed
upland systems. However, changing climate, cultivation practices and introduction
of new water saving rice production technologies like SRI, AWD, aerobic rice, etc
are aggravating this problem in irrigated rice in recent years (Prasad et al. 2010).
Due to limitations of current control measures, incorporation of nematode resis-
tance in appropriate rice cultivars, combined with valuable agronomic traits, would
be of enormous value to the management and preventative control of nematodes in
Fig. 1.5 Nematode
infested root
Fig. 1.6 Giant cell formed

by nematode
rice (Atkinson et al. 2003). Nematode resistant cultivars represent a highly practical
means of nematode control in smallholder, subsistence agriculture prevalent in
many rice growing areas. In this context, breeding for nematode resistance with the
aid of biotechnological tools like marker assisted selection, transgenic approach,
etc., are receiving increased attention. Employing appropriate phenotyping methods
is crucial for the success of any resistance breeding program and more so in molecu-
lar breeding approach. The Biology and detailed methodology for phenotyping for
resistance to root-knot nematodes is described below.
1.3.1 Biology
The second stage juveniles of root-knot nematodes upon entry into the roots estab-
lish at a point in the stele and start deriving their nutrition from the giant cells
induced by the nematode secretions (Fig. 1.6). The nematode induces formation of
galls due to hypertrophy and hyperplasia of infected tissue (Williamson and
Gleason 2003). The second stage juvenile undergoes successive moults to become
an adult. The males are vermiform with weak stylet. The females are saccate and
pear shaped with a bent neck which remains inserted in the stelar tissues. Each
root-knot may contain one or more females. The eggs are laid in a gelatinous matrix
and each egg mass contains 150–300 eggs. The females of M. graminicola remain
embedded in the root cortex and eggs are laid inside the roots, unlike other root-
knot nematodes where the females protrude out of the cortical tissues of the root
(Jena and Rao 1976). The eggs get dispersed in the soil due to root decay and the
juveniles hatch from eggs to invade the fresh plant roots if available or wait for the
following season.
1.3.2 Identification of Nematode Species/Isolate
Accurate identification of nematode species and isolate is an essential prerequisite

for screening for resistance. For identification of Meloidogyne species perineal pat-
terns of adult females, the North Carolina differentials host tests (Harman and
Sasser 1985) and isozyme phenotype techniques (Esbenshade and Triantaphyllou
1990) are most widely used. With the advent of PCR based molecular diagnostic
tests, accurate identification of species or isolate can be done based on analyses of
ribosomal DNA of even single nematode (Powers and Harris 1993). Generally,
most aggressive nematode isolate is used for phenotyping of breeding lines which
facilitates selection of genotypes possessing highest level of resistance. Another
approach is to use of a mixture of isolates so that genotypes with broad spectrum
resistance against a wide range of isolates are selected (Hussey and Boerma 1981).
1.3.2.1 Maintenance of Pure Cultures
Maintenance of pure cultures of nematode species or isolate is crucial for providing

inoculum needed for phenotyping large number of breeding lines or mapping popula-
tions. Pure cultures of nematode species or isolates are maintained on susceptible host
plants in glasshouse in plastic or earthen pots containing sterile soil mix. Second stage
infective juveniles collected from a single egg mass are used as starting inoculum.
Care should be taken to avoid chances of contamination in culture pots in the glass-
house. Purity should be checked periodically by using techniques mentioned above.
1.3.2.2 Nematode Inoculum
The egg masses, egg suspension, or second stage infective juveniles (J2) of root-
knot nematode can be used as inoculums. Most commonly J2 are used as inoculum
because of ease of collection and handling. Among inoculums types, the order of
highest control of inoculums quantity and quality is J2 > Eggs > Egg masses. For
collecting J2, roots are washed free of water, egg masses were removed directly
from roots using forceps (for Meloidogyne incognita) or galled roots are teased with
needles (for M. graminicola) under stereo-zoom microscope and placed on a modi-
fied Baermann funnel setup (Hooper 1986). After 24–48 h, J2 hatched into tap water
are collected in a glass beaker. Concentration of nematodes is determined by count-
ing number of J2 in 3 one ml aliquots under stereo-zoom microscope and averaging
it. Finally, the volume is made up with water to obtain a concentration of 100 J2/ml.
1.3.3 Inoculation of Plants
Nematodes are inoculated by making holes in moist soil around the plants.
Inoculation can be done manually by using pipettes or bottle top dispensers.
However, for large scale inoculations involving large mapping populations a digital
dispensing pump can be used (Hussey and Janssen 2002). Care should be taken to
shake the nematode suspension intermittently to avoid settling of nematodes while
inoculation of large number of plants. Excess watering is avoided especially during
first 2 days after inoculation.
1.3.4 Phenotyping Protocol
Phenotyping of large plant populations is done in glasshouse conditions. Pre-

germinated rice seeds are sown in small plastic pots or seedling trays containing
sterile soil mix. Two weeks after sowing, each plant is inoculated with 200 second
stage juveniles, by making small holes in soil around the plants. Plants are watered
daily and fertilized once a week or as required. At least ten replications are main-
tained for each genotype. One susceptible cultivar is included as a standard check
along with test genotypes for comparison in each test. Scoring for nematode resis-
tance is done 45–60 days after inoculation to ensure that at least 2 generations of
nematode is completed (Jena and Rao 1976; Prasad et al. 2000).
1.3.4.1 Scoring for Root-Knot Nematode Resistance
Scoring for root-knot nematode resistance is done using indices based on galls
(degree of root galling or number of galls) and eggs (egg mass number or total num-
ber of eggs per root system) developed on each plant. Among scoring parameters,
the order of greatest sensitivity of scoring is total number of eggs > egg mass num-
ber > number of galls > degree of galling. In the initial stages of evaluation where
population size is usually large gall index is used for scoring, as it is relatively easy.
For advanced breeding lines scoring based egg mass number or total number of eggs
per root system is performed to get more reliable results (Luzzi et al. 1987). When
galling or egg mass number used for evaluation, an index (0–10) is developed based
local susceptible cultivar included in each test is used for scoring the genotypes
(Jena and Rao 1976; Prasad et al. 2000).
1.3.4.2 Degree of Galling
In this method, a root gall index of 0–10 is developed based on the actual percent-
age of roots galled (0 = no galls; 1 = 10 % roots with galls; 2 = 30 % roots with
galls; 3 = 30% roots with galls; 4 = 40% roots with galls; 5 = 50% roots with galls;
6 = 60% roots with galls; 7 = 70% roots with galls; 8 = 80% roots with galls; 9 =
90% roots with galls; 10 = all roots are galled). Plants are scored using this index
(Bridge et al. 2005).
Fig. 1.7 Stereomicroscopic
picture of nematode egg mass
inside root
1.3.4.3 Number of Galls
Forty-five days after inoculation, the plants were uprooted carefully, roots were
cleaned and number of galls were counted. The genotypes are assigned a gall index
value based on number of galls per plant (0 = no galls; 1 = 1–5 galls; 3 = 6–15 galls;
5 = 16–30 galls; 7 = 31–50 galls and 9 = >50 galls per plant). The genotypes are
scored based on their gall index: 0 = Highly Resistant; 1.0 = Resistant; 3.0 =
Moderately Resistant; 5 = Moderately Susceptible; 7 = Susceptible; 9 = highly sus-
ceptible (IRTP 1984; Prasad et al. 2000).
1.3.4.4 Egg Mass Number
For enumerating egg mass number, roots are fixed in 4 % formalin, stained with
lacto phenol-cotton blue or acid fuchsine, cleared in pure lacto phenol and the num-
ber of egg masses was recorded. Well developed egg masses can be easily viewed in
stained roots when observed under stereo-zoom microscope (Fig. 1.7). For each
entry, the egg mass index was computed using the formula: (Number of egg masses
in test genotype × 4)/Number of egg masses in susceptible check cultivar. The geno-
types are rated based on the egg mass index: 0 – 1.0 (Resistant); 1.1 – 2.0 –
(Moderately resistant); 2.1 – 3.0 (Susceptible); 3.1 and above (Highly susceptible)
(Jena and Rao 1976; Prasad et al. 2000).
1.3.4.5 Number of Eggs
For collecting eggs, cleaned roots are vigorously shaken or macerated in 1.05 %
NaOcl solution and the solution passed through the 500u sieve. The eggs retained
on the sieve are rinsed with water and collected in a beaker. Egg number can be
expressed on per plant or per gram root basis. The ratio of total number of eggs per
plant to the number of eggs in susceptible check cultivar is used for comparing and
scoring the genotypes.
1.4 Phenotyping for Drought Tolerance
Drought is a major threat to agricultural production and drought tolerance is a prime

target for molecular breeding and transgenic approaches to crop improvement. To
achieve meaningful results, these approaches must be linked with suitable pheno-
typing protocols at all stages (Salekdeh et al. 2009). In most upland rice environ-
ment, drought is unpredictable and will not occur every year. Plant breeders must
therefore select varieties capable of producing well in both favorable and unfavor-
able years. Therefore, a comprehensive and careful field evaluation of mapping
populations and transgenic plants is urgently needed in order to provide reliable
information on the effectiveness of QTLs, candidate genes and transgenes.
Discovery of genes involved in drought stress will provide new targets for breeding
and genetic engineering of rice and other crops for better tolerance (Mangrauthia
et al. 2008).
Capacity for precise phenotyping under reliable conditions probably represents
the most limiting factor for the progress of genomic studies on drought tolerance.
Often field experiments designed to evaluate genetic differences in drought toler-
ance are faced with contrasting requirements. There is a need for a high precision
because the differences may be small and subtle, and detailed physiological
measurements (i.e. evaluation of the photosynthetic activity) are difficult when a
large numbers of genotypes are involved. To achieve an accurate phenotyping, it is
also important to control stress levels and timing. The intensity of drought stress is
often different from year to year and within fields because of variations in soil
composition which determine the capability of the soil to retain water. To reduce the
signal-to-noise ratio in field based experiments there is a need to select research
plots with low spatial variability in soil properties. Application of nutrients and the
control of weeds/pests should be carried out precisely and uniformly; experimental
design should control within-replica variability. Variations in rainfall amount and
distribution strongly influence the level and timing of the stress; the use of rain
shelters and supplementary irrigation can help to control the stress conditions and
improve the quality of the phenotyping work (Cattivelli et al. 2008).
In the last decade many genetically engineered plants have been proposed and
tested for improved performance under drought. Nevertheless, in many reports
desiccation and salt stresses applied are ‘shock’ treatments, while for most crops
drought tends to develop slowly as the soil dries. The evaluation of drought tolerance
of transgenic plants has often been based on survival capacity, with very limited
analyses of the transgene effects on yield potential. Furthermore, the assessment of
plant water status with different methods, often with a visual score, makes the com-
parison among different reports difficult.
1.4.1 Selection Criteria
The connection between information derived from large scale molecular analysis
and the way a plant responds in an agronomic environment can be partially improved
by following a set of rigorous criteria for phenotyping in both controlled and field
situations. This starts with defining the drought target environment. Although rain-
fall and climate are fundamental, the impact of micro-environmental and agronomic
effects must also be considered, as well as the potential for all these factors to inter-
act with genetic background.
For efficient screening, Blum (2002) suggested two points for assessing the
utility of traits. The first is that important drought resistance traits are normally
constitutive and not stress adaptive. Constitutive traits, such as flowering time, the
stay-green trait (delayed onset of leaf senescence), and root depth, can be routinely
screened (without a drought challenge); generally, their role towards drought resis-
tance may be considered greater than stress responsive traits. Stress adaptive traits
(responsive, induced) are those that are expressed only under drought. Such traits
include active cellular accumulation of compatible solutes (osmo protectants),
antioxidant agents, heat shock proteins, and molecular chaperones, as well as
osmotic adjustment and membrane stability. Blum’s second point is that plant
water status, rather than plant function, controls crop performance under drought.
Therefore, those genotypes that can maintain higher leaf water potential (LWP)
and relative water content (RWC) are drought resistant simply because of their
superior internal water status. For screening, traits, includes constitutive and
induced traits, which need to be highly heritable and easily measured (Kamoshita
et al. 2008).
Selection criteria to be used in order to obtain high yields under both stress and
non-stress environments have been debated by breeders for decades. Screening for
increased yield potential is generally performed under ideal conditions. Such selec-
tion environments will serve to improve yield under drought if yield under drought
and yield under well watered conditions are positively correlated. Selection for
yield potential is therefore an important element in developing varieties that pro-
duce acceptable yields under moderate levels of stress. Selecting genotypes that
complete their flowering before the onset of water stress is possible if the timing of
drought is predictable and terminal. However, in many areas where upland rice is
grown, brief periods of drought stress, particularly around flowering, occur unpre-
dictably during the middle of the monsoon. Drought resistant varieties are essential
in these areas. In short, selection for drought resistance can be performed by
measuring yield under stress conditions and/or measuring a secondary character

correlated with yield under stress conditions. A secondary trait is only useful in
breeding if it is inexpensive to measure, highly correlated to yield under drought
and if it shows higher heritability than that of yield itself under stress.
1.4.1.1 Selection for Yield Under Drought
Direct selection for yield under favorable or water-limited conditions is the selec-
tion strategy that has been the most commonly used by cereal breeders to improve
yield in water-limited environments. This method has not been widely used in rice
until recently, but is now increasingly so, as drought resistance is being recognized as
an important trait to improve in rice. Kumar et al. (2007) reported higher broad-sense
heritability of grain yield under severe terminal drought stress in 2 years in the CT
9993/IR 62266 populations than for secondary and integrative drought-resistance
traits such as harvest index, spikelet sterility, flowering delay, relative water content,
root pulling force, or root dry weight, all under field conditions. In Brazil, direct
selection for yield using a managed drought environment that matches the environ-
ment in the breeding domain has resulted in the identification of tolerant genotypes
and development of higher yielding cultivars for upland rice. This approach has also
been used for rainfed lowland rice in Thailand and Cambodia. Despite its increasing
use, direct selection for yield under drought conditions poses problems; establishing
drought stress in the field is complicated to manage and rainfall can always occur at
undesirable moments, even during the dry season. In upland rice, drought stress
usually enables clear differentiation between resistant and susceptible lines on the
basis of yield only if it reduces yields by at least 70–80 % of unstressed yields.
Direct selection for yield under managed stress, when combined with concurrent
selection for yield potential, is an effective and underutilized approach to develop-
ing stress-tolerant upland rice varieties (Bernier et al. 2008).
1.4.1.2 Selection for Secondary Traits Under Drought
Integrative traits or secondary traits are likely to be more closely associated with
grain yield under various drought conditions than are primary traits, and they may
have wider applicability in breeding provided they add efficiency to selecting for
yield per se. Heritability of secondary traits (such as plant water status, leaf death
score and leaf rolling) and integrated traits (such as spikelet sterility) are often com-
parable to or higher than the heritability of yield under stress. Combining selection
based on yield with secondary traits into selection indices can improve selective
response, if the physiological processes contributing to grain yield in the target
environment are well understood and if the secondary traits can be repeatable and
inexpensively measured. The number of days to flower is normally measured under
both stress and non-stress conditions. Selection may be performed for lines with
ideal maturity dates in well-watered conditions and no large flowering delay under
drought. Selection for lines that maintain high spikelet fertility under drought stress
and/or a low rate of leaf drying under drought stress is also common. Flowering delay
and spikelet fertility have both been reported to have a moderate heritability and a
high correlation with grain yield under stress at flowering stage (Bernier et al. 2008).
Jearakongman (2005) showed higher broad-sense heritability for pre-dawn LWP, leaf
death and leaf rolling scores, panicle exertion rate, plant height, total number of
grains per panicle, and number of unfilled grains per panicle than broad sense herita-
bility of yield under terminal stress among fifty five IR 64 near-isogenic lines.
Similarly, in the CT 9993/IR 62266 population, Babu et al. (2003) measured
secondary and integrated traits in drought-affected fields and reported high
broad-sense heritability for relative water content, canopy temperature, leaf rolling,
leaf drying, harvest index and spikelet fertility compared with the broad-sense heri-
tability of yield under stress. Leaf water potential is strongly correlated with spike-
let sterility under drought stress and is less influenced by timing of stress than spikelet
sterility. Leaf water potential has been reported to have a relatively high correlation
with grain yield when drought stress was applied around flowering in an upland rice
experiment. A major limitation of LWP is that it is tedious to measure, with the result
that only a small number of lines can be tested at a time. Further, the variation in
potential yield and phenology can be corrected for by calculating a drought response
index (DRI) (Ouk et al. 2006) and drought-tolerant genotypes may be selected by
using DRI.
1.4.1.3 Selection for Other Secondary Traits Under Drought
Several authors have suggested that carbon isotope discrimination (CID), an indi-
rect measure of stomatal conductance, could be an effective selection criterion for
grain yield under drought. In wheat, grain yield was found to be positively corre-
lated to stem CID under the conditions of South Australia and to grain CID in the
conditions of Syria, South of France and Spain. There are few circumstances where
negative correlations between grain yield and CID have been observed, despite the
fact that it is generally assumed that lower CID indicates higher transpiration effi-
ciency. This may be due to the fact that lower CID leads to slower crop growth
under the absence of water deficit and that a higher transpiration rate does not
necessarily imply faster rate of soil water depletion (Bernier et al. 2008).
Leaf or grain ash (mineral) content was proposed as an alternative selection cri-
terion for yield under drought, thereby avoiding the high cost of CID analysis. As
most minerals are mainly transported passively in the xylem and accumulated in
transpiring plant tissues, greater transpiration consequently increases the amount of
passively transported minerals into the leaves. Correlation was found between CID,
leaf ash content and grain yield in different C3 species. Other studies failed to iden-
tify a significant relationship with grain ash content, but found a significant relation-
ship with leaf ash content under flowering and grain filling drought stress. Despite
encouraging results concerning the feasibility of using ash content for selection, this
trait has not been used to produce drought-resistant varieties in any crop up to now,
possibly due to the irregularity of relationships between grain yield and ash content
(Bernier et al. 2008).
1.4.1.4 Selection for Primary Traits Under Drought
Primary drought-resistance traits are associated with fewer genes and are under
simpler genetic control than yield under stress. Hence, the heritability of primary
traits should be higher than for secondary and integrative traits and yield, assuming
that trait expression is accurately measured. Primary traits such as deep and thick
roots, osmotic adjustment are comparable to, or higher than, that of yield under
severe stress. However, measurements of root traits under drought conditions in the
field usually have large errors, and the broad-sense heritability of root traits mea-
sured in the field are in general lower than those measured under hydroponic sys-
tems and pot systems. Root characteristics are not widely used traits by breeders
because the root system is difficult to study. Root architecture is highly dependant
on the environment; plants change their resource partitioning according to the grow-
ing conditions and this process of phenotypic plasticity is poorly understood.
When breeding for drought tolerance at the grain-filling stage, it has been sug-
gested that breeders select for plants with a high capacity of remobilizing non-
structural carbohydrate stored in stems. This is routinely done in some wheat
breeding programs; this selection criterion is not used by rice breeders. This may be
due to the possible negative association between stem reserve storage and yield
potential or lodging resistance, or to the impossibility of combining this trait with
resistance to leaf drying (stay-green). It has been shown that, for most crop species,
osmotic adjustment (OA) does not have a negative impact on yield potential and
does have a positive effect under some drought-stress conditions. There is a large
genetic variation in osmoregulation potential between different rice varieties, so
breeding for this trait may be feasible. It remains to be seen whether or not signifi-
cant improvements in drought resistance can be achieved through an increase in OA
capacity of rice. There is large genetic variation in cuticle thickness and composition
within the rice germplasm and this trait exhibits high heritability; therefore it appears
possible to breed rice with a thicker epicuticular layer. However, measuring wax
layer thickness and/or composition on a large number of lines is slow and expensive
and therefore impossible to use by breeders (Bernier et al. 2008). Measurement of
traits such as osmotic adjustment or cell membrane stability could involve greater
errors than measurement of constitutive traits because of differences in the degree of
water stress if experimental conditions are not precisely controlled.
1.5 Conclusion
The selection of suitable technique for accurate scoring for the phenotyping is the
key in selection of appropriate progeny in breeding programmes. The phenotyping
methods for seedling under artificial inoculation conditions and natural infestation
in some cases vary and the scale to measure also vary. To get good artificial inocula-
tion for the disease and pest, the biology of the pest and the conditions that favor the
pest is need to understand critically.
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Chapter 2
Phenotyping in Wheat Breeding
Govindan Velu and Ravi Prakash Singh
Abstract Approximately 25 % of global agricultural land is utilized for wheat

cultivation, making wheat the largest food crop worldwide in terms of area. Wheat
is the second most produced cereal crop after Maize with more than 650 million
tons produced every year. Wheat productivity is increasing at less than 1 percent
annually, while the annual productivity must increase at 2 % annually to meet the
global demand. The potential of increasing arable land is limited; hence future
increases in wheat production must be achieved by enhancing the productivity per
unit area. Increasing grain yield, yield stability, resistance/tolerance to biotic and
abiotic stresses, and end-use quality characteristics are among the most important
wheat breeding goals.
The Green Revolution wheat varieties performed well in terms of responsiveness
to fertilizer application and water-use efficiency. But now there is not a lot more
water to spare, and fertilizer usage in some places has already passed saturation
point, so a new Green Revolution will have to make even more efficient use of exist-
ing resources. Efficient phenotyping techniques are essential to develop new wheat
varieties with higher yield potential, tolerate high temperatures and improved water-
use efficiency or drought tolerance due to climate change and the dwindling supply
of irrigation water. This book chapter describes various phenotyping techniques
being used in national and international wheat breeding programs.
Keywords Wheat • Phenotyping • Grain yield potential • Biotic and abiotic stress
tolerance • End-use quality • Biofortification
G. Velu (*) • R.P. Singh

International Maize and Wheat Improvement Center (CIMMYT),
Apdo. Postal 6-641, 06600 Mexico DF, Mexico
e-mail: velu@cgiar.org
42 G. Velu and R.P. Singh
2.1 Introduction
Global food demand is expected to be doubled by 2050, while production

environments and natural resources are continuously shrinking and deteriorating
(Barrett 2010). Sustainable food security in a world with a growing population and
changing diets is a major challenge under climate change. Although estimates of
food insecurity vary, the number of undernourished people already exceeds 1 billion
and feeding this many people will require more than incremental changes (Fedoroff
et al. 2010). Food production may need to increase by as much as 70 % by 2050
when the global population may likely reach 9 billion people (World Bank 2008).
Food security depends not only on gross production of staples, but also on ability to
provide income for farmers in developing countries, a diverse and balanced food
basket, and the socio-economic factors that determine whether poor people, particu-
larly women, are able to purchase, store, prepare and consume sufficient food.
The next 40 years will also have to deal with the potentially profound damage to
farming from climate change, which in some parts of the world could reduce yields
by one third. And disturbingly, for the first time since the Green Revolution, crop
yields are growing more slowly than population. Between 1961 and 1990 wheat
yields were rising at nearly 3 % a year (FAO 2008). During that period the world’s
population was growing by an average of 1.8 % per year. Between 1990 and 2007
population growth slowed down to 1.4 %, but the rise in annual wheat yields slack-
ened to 0.5 % (Fig. 2.1). To be more precise, growth in population and demand for
food have both slowed down, but crop yields have slowed at a higher rate.
Looking into the future, global wheat requirements are expected to increase from
the current 685 million tons from 225 million hectares in 2009 with an average
productivity of 3.04 t ha−1 (FAO 2011) to about 900 million tons in the year 2020
with an average productivity of 4 t ha−1 to meet demand (Ortiz et al. 2007). Thus,
wheat not only has a key role to play in current food security, but also in future
global food security and poverty reduction. Challenges for wheat breeders include
increasing productivity, especially for agro-climatically marginal areas, and defend-
ing past yield gains against diseases and pests, of which rust may well be the most
Fig. 2.1 Global annual yield

growth of major staples
(adapted from FAO, 2008)
2 Phenotyping in Wheat Breeding 43
important. In addition, water and nutrient use efficiency could increase productivity
and profitability of resource poor farmers.
Phenotypic plant breeding will always be an important practice. This belief is
due to: (1) it is progressively less expensive than marker assisted selection and
marker assisted breeding, (2) visual selection for some traits remains a remarkably
effective way of handling massive populations and experimental lines that are gen-
erated in a breeding program, (3) our understanding of the genome, while constantly
increasing, may not fully explain the complexity of the phenotype. Complete QTL
interactions will be difficult to identify, and hence more difficult to breed for suc-
cessfully (Dudley 2008). Most importantly, all of the marker data, however obtained
or used, must be associated with carefully measured phenotypes to establish the
value of the marker(s), and (4) the environment in which we grow plants may
change quite often, especially for biotic stresses. The first indication of these changes
is the phenotype, hence plant breeders must always pay attention to the phenotype
(e.g. the rise of Ug99 [TTKS] stem rust [Puccinia graminis f. sp. tritici]. Phenotyping
wheat lines for performance in diverse environments and cropping systems, disease
and insect resistance, and end-use quality is most important strategy for the devel-
opment of widely adaptable, stable, and disease/pest resistant germplasm.
2.2 Shuttle Breeding Program for Higher Yield Potential

and Resistance to Biotic Stresses
Development of broadly adapted, durable disease resistant, high yielding and stable
wheat germplasm is the primary objective of any wheat breeding program across
the world. To breed for wide adaptation and high yield potential, the International
Maize and Wheat Improvement Center’s (CIMMYT) wheat breeding program shut-
tles segregating materials between alternative sites within Mexico. The shuttle
breeding methodology is unique to CIMMYT; it was proposed and implemented by
Dr. Norman E. Borlaug (Borlaug 1968), initially accompanied by much criticism,
but finally widely acclaimed. This methodology has been responsible for the pro-
duction of photoperiod insensitive and widely adapted improved germplasm. In par-
ticular, the shuttle breeding process involving contrasting locations in regard to
latitude, altitude and rainfall has proven a most efficient way to introduce and select
genes for photoperiod insensitivity. Shuttle breeding in Mexico consists of growing
segregating populations in two distinct environments, i.e., Ciudad Obregon (Sonora)
and Toluca (State of Mexico).
The Ciudad Obregon location is irrigated, similar to the Nile Delta in Egypt and
the Punjab in India, whereas Toluca is a high rainfall location (1,000 mm during the
wheat growing season) situated 2,600 meters above sea level. Planting in Obregon
occurs in November, and the maturation of plants coincides with increasing
high temperatures in April and May. The harvested materials, after selection, are
then transferred to Toluca where planting is undertaken in May and June and har-
vesting is completed in October and November, when temperatures are declining.
This shuttle methodology allows the harvest of two generations of segregating

populations per year and cuts the length of the normal breeding cycle in half.
The procedure has inherently allowed only photoperiod insensitive plants to flower
at both locations and complete the maturity period within this very tight time limit.
Ciudad Obregon is located at 27.5°N, 40 masl, in the state of Sonora. It is a dry,
irrigated, low-altitude site, located in a desert climate. Mean rainfall during the
wheat crop cycle is about 50 mm. Irrigated yields in the region are high, in the order
of 8–11 tons ha−1 in experimental plots and 6–8 tons ha−1 in farmers’ fields. With a
reduction in the number of irrigations, various types of drought stress can be cre-
ated. This is one of the two most important breeding and screening sites for the
CIMMYT wheat program. Inoculation of stem rust (Puccinia graminis f. sp. tritici)
and leaf rust (P. triticina) by spray applications of susceptible border-mixtures
ensures adequate infection of the entire targeted field. Rust inoculation is carried out
in the latter part of January, with the spring wheat grown from November until May.
The alternate site for the CIMMYT shuttle breeding program is Toluca, which is
located at 19°N, 2640 masl, and west of Mexico City in the State of Mexico. This
temperate, high-rainfall, high-altitude site is the most important CIMMYT summer
cycle location. This environment is conducive for disease expression, especially of
stripe rust (P. striiformis), Septoria tritici and Fusarium head blight (FHB). Spray
applications to susceptible border-mixtures provide stripe rust infection. Dispersal
of infected straw at the tillering growth stage initiates epidemics of S. tritici and
FHB. Spring wheat is grown from May until October. When planted in November,
winter wheats are exposed to vernalizing temperatures during the winter that are
low enough to initiate flowering.
The concept of photoperiod insensitivity was not known to science when the
CIMMYT shuttle breeding program was initiated. It only became apparent when
Dr. Borlaug began sending the breeding material to the Indian subcontinent and the
Middle East in the early 1960s. The photoperiod insensitive genes, Ppd1 and Ppd2,
abound in CIMMYT’s spring wheats, and along with the dwarfing genes, Rht1 and
Rht2, resulted in a new plant type, which was not only lodging tolerant, but dra-
matically higher yielding with high biomass due to pleiotropic effects or close
linkage.
2.3 Historical Analysis of Yield Gains in the Cimmyt

Wheat Breeding Program
There has been a continuous involvement of CIMMYT researchers in the evolution

of plant types for different agro-ecological conditions. Since the mid-1950s, there
has been a continuous rise in wheat yields in Mexico and elsewhere. The modern
cultivars evolved from changes in plant type and structure (dwarfing genes), physi-
ological aspects (photoperiod insensitivity genes), durable disease resistance (incor-
poration of rust resistance), robustness, delayed leaf senescence, and other changes
in spike number, morphology, grain number, and size.
The yield potential of semi-dwarf wheat cultivars, regardless of their origin, has
continued to increase at the rate of 1 % annually (Sayre et al. 1997). Yield potential
of bread wheat in Mexico increased from 4.5 t ha−1 for the tall cultivar “Yaqui 50’ to
6.5 t ha−1 for the first semi-dwarf mega-cultivars ‘Siete Cerros 66’ and ‘Sonalika’,
which were widely grown Green Revolution cultivars. Increased yields in these
cultivars were due to incorporation of dwarfing genes Rht1 and Rht2 that provided
lodging tolerance, and responsiveness to fertilization and irrigation. Dwarfing genes
were successfully transferred from short statured Japanese cultivar ‘Norin 10’.
A better understanding of various genetic and non-genetic factors contributed to
further yield increases and the development of superior cultivars like ‘Baviocora 92’
with a yield potential of about 9.0 t ha−1, which represented a narrow genetic base or
specific adaptation (Rajaram et al. 2002). However, ‘Veery’, a derivative of a
Russian cultivar (winter wheat) and an Indian line of CIMMYT origin (spring
wheat), and a CIMMYT advanced line ‘Attila’, represent a wider genetic base or
wider adaptation and are known to be widely adapted and a stable performer in
different parts of the world. This supports the hypothesis of Kronstad where he
proposes the use of a wider genetic base in the breeding program (Kronstad 1996).
Although thousands of crosses were made between winter and spring wheats,
‘Veery’ and ‘Attila’ were the successful spring types with the winter wheat parents
‘Kavkaz’ and ‘Nord Desperez’, respectively, and led to the development of mega-
cultivars that were subsequently grown on millions of hectares around the world.
These yield increases were often associated with the presence of the alien
translocation commonly known as 1B.1R, where the short arm of chromosome 1B
is replaced by the short arm of chromosome 1R of rye (Secale cereale) (Rajaram
et al. 1995). Some of the new wheat genotypes developed in recent years have
shown further increases in yield potential of 10–15 % over ‘Attila’ (Singh et al.
2007). Historical analysis of Elite Spring Wheat Yield Trial (ESWYT), tested over
the past 15 years (1995–2009) across locations in many countries, showed mean
yields of the five highest yielding entries with an annual gain of 0.66 % compared
to ‘Attila’ (Sharma et al. 2012) and an another study using 30 years international
data revealed about 0.7 % yield gain over the years in Mexico (Lopes et al. 2012)
(Fig. 2.2). Results from this study demonstrate continuous genetic yield gains in the
elite spring bread wheat lines developed and distributed by CIMMYT for the global
irrigated and rainfed environments.
2.4 Grain Yield
The most significant objective of any wheat breeding program is to enhance the
grain yield potential. Grain yield is a complexly inherited trait of low to moderate
heritability and is strongly influenced by the environmental conditions. Higher grain
yields are usually associated with delayed maturity, increased plant height and
lower protein content (Heisey 2002). Yield enhancement is often achieved by not
only selecting for greater yield potential but also by selecting for resistance to biotic
Fig. 2.2 Genetic yield gain over 30 years in a historic set of CIMMYT germplasm (adapted from
Lopes et al. 2012)
and abiotic stresses that may limit the expression of cultivar’s maximum yield
potential. Breeding for enhanced yield depends on: (i) the genotypic variation for
yield potential of parents used in the crossing program, (ii) selection intensity, and
(iii) the degree to which genotypic differences in yield potential are expressed in the
selection nursery. To achieve higher yield potential, plant breeders employ a range
of crossing and selection methods. The role of wheat breeders includes: (1) intro-
duce genetic variation, (2) inbreed and select among the variants, and (3) evaluate
the selected lines in the diverse and varied environments where the lines may even-
tually be grown as a released cultivar (Baenziger and DePauw 2009).
The introduction of variation has historically been done by sexual crosses to
make hybrids (usually single, three-way, double, or back crosses). Wheat breeding
at CIMMYT until the early 1980s relied on simple, three-way (top) or four-way
(double) crosses followed by the pedigree method of selection. Breeders realized
that best advanced breeding lines were rarely derived from double crosses, with the
possible reason being that the genetic variation generated by such crosses was large
and the chances of recovering plants with desirable combinations of genes was low
due to insufficient population sizes. With the globalization of CIMMYT’s Bread
Wheat Breeding Program in the 1980s, CIMMYT breeders relied on simple and
three-way crosses and occasionally single backcrosses (Wang et al. 2003).
2.5 Crossing Block
Historically, many wheat breeding programs made relatively few crosses, often 60
or less. However, most wheat breeders would consider 250 crosses as the minimum
and many programs make 1,000 or more crosses. The total number of crosses made
will depend upon the number of parent lines available, the percentage used for cul-
tivar development versus those made for parent development, as well as, how the
resultant populations will be used.
The CIMMYT crossing block (CB) consists of collections of elite breeding

source material from the ongoing breeding program and elite germplasm from dif-
ferent parts of the world. In order to facilitate crossing operations, each CB is sown
on four different dates, about 7 days apart. The production of high yielding, widely
adapted, stable and durable resistant spring wheat germplasm with acceptable qual-
ity is the primary consideration of the CIMMYT wheat program. For this reason,
parental lines are being grouped and sub-grouped based on their country of origin
or specific character expression (e.g. disease resistance, abiotic stress tolerance,
industrial quality). Also, CB entries include: (1) major varieties released in different
target countries, (2) elite CIMMYT and other germplasm identified from interna-
tional and national testing, and (3) advanced lines exhibiting desirable expression of
one specific trait or group of traits, including those made available by the pathology,
wide cross, physiology and other sections within the wheat program.
Not all entries in a CB are involved in crossing. Before making crosses the male
master (MM) and female master (FM) lists are first determined. The male master list
represents the best entries in the CB based on field observations (agronomic type in
the field from the past cycles and the current cycle) and relevant databases (yield,
adaptation, disease resistance, end-user type, and protein content etc.,). These entries
serve as male parents for simple crosses, but simple crosses are also made among
these parents. The FM list includes some entries that still require specific improve-
ments but carry certain traits of great interest, like high yield potential. These entries
will only be used as female parents for crossing to the entries from the MM list.
F1: about 1,000–1,200 crosses are made per cycle. Crosses are always directed
toward targeted breeding, taking into account the relevant requirements such as,
higher grain yield, Ug99 resistance, and heat-tolerance. The total number of F1 seed
from a cross will depend upon the cross type and the size of the population needed
in the next generation to adequately represent the genotypic array. Mostly introduced
materials are used as females in their original crosses, in order to possibly expand the
genetic base of CIMMYT wheat cytoplasm, but also for practical/logistical reasons.
As a rule, 3 spikes are emasculated and pollinated for simple crosses.
F1Top or backcross: A majority of F1’s are top crossed to one or more third parents,
or a single (limited) backcross is made back to the adapted parent. About 15–20
spikes are used for top or backcrosses in order to get 400–500 seeds to capture
maximum variation from the simple cross F1’s. Backcrosses are carried out to stabi-
lize variability, as the genetic distance between parents becomes greater, and are
proving very effective in expanding adaptation and performance while introgressing
new genetic diversity. The single backcross approach was initially aimed at incor-
porating resistance to rust diseases based on multiple, additive, minor genes (Singh
and Huerta-Espino 2004). However, it soon became apparent that the single back-
cross approach also favored selection of genotypes with increased yield potential.
The underlying reason was that single backcrosses shift the progeny mean towards
the higher side of the curve and favors the retention of most of the desired additive
genes from the recurrent parent while simultaneously allowing the incorporation
and selection of additional useful small-effect genes from the donor parent.
Per cycle, about 500–750 top and/or backcrosses are made. An epidemic is
created of the prevalent diseases, either in Ciudad Obregon or Toluca. In the first
segregating generation of such crosses, the best plant types with good agronomy
and resistance to diseases will be selected. Individual spikes from selected plants are
then bulk threshed and advanced to F2 stage.
2.6 F1 and Segregating Populations
Once the variation has been introduced, the wheat breeder must decide how best to
select and inbreed. In every selection protocol there is a hierarchy in which the
breeder must choose in what order the traits will be selected. Simple selection tech-
niques can be used to eliminate undesirable phenotypes from the F2 population.
Some examples of simple selection techniques include inoculating segregating pop-
ulations with a particular disease so resistant types can be selected (Pozniak and
Hucl 2004), and planting populations in a given environment so the winter or spring
growth habit segregants will be winterkilled or not vernalized (Dowell et al. 2006).
These selection protocols can quickly eliminate obvious undesirable types at rela-
tively low cost and with high efficiency. The resulting population is smaller but
contains valuable traits at a higher frequency than an unselected population.
Therefore, the population size becomes more manageable.
Once a cross has been made and classified, its segregating progenies are selected
in a shuttle breeding fashion between Toluca and Ciudad Obregon. All the elite
CIMMYT advanced materials go through this shuttle breeding program, and it
allows the breeders to get improved germplasm with wider adaptability, disease
resistance and higher yield potential.
The CIMMYT wheat breeding program was using the modified-bulk selection
scheme, where individual plants were harvested in the F2 generation to grow as an
F3 generation, bulk selection was then practiced in the F3–F5 generations. Individual
plants or spikes were once again harvested in the F5 or F6 generation (Rajaram et al.
2002). Following the study by Singh and coworkers which showed that selection
schemes had little or no effect on the performance of progeny lines, but it was the
choice of parents that determined the progeny response, a ‘selected-bulk breeding
scheme’ was introduced in the bread wheat improvement program (Singh et al.
1998). Under this scheme, in all segregating generations until F5 or F6, one spike
from each of the selected plants is harvested as a bulk and a sub-sample of seed used
to grow the next generation. Individual plants or spikes are then harvested in the F5
or F6 generation. This scheme allows the retention of a larger sample of selected
plants without increasing the cost and was found to be highly efficient in terms of
operational costs. Moreover, retaining a large sample of plants in segregating popu-
lations increases the frequency of rare segregants carrying the most desirable genes.
Below, detailed information on advancement of each of the segregating and
advanced generation materials under the selected-bulk breeding scheme for the
bread wheat improvement program is included.
F2: Each of the F2 populations consists of about 1,500–2,000 plants per cross, which
are space-planted at 10–15 cm between plants. This includes simple, top and
(limited) backcrossed F2 populations. An epidemic is created of the prevalent
diseases. The poorest F2 populations are completely discarded. Within the better F2
populations, the best plants are selected by the breeders based on good agronomic
type, appropriate height, synchronous tillering, desired spike type, large spike, good
fertility, durable disease resistance, and desired maturity. Individual spikes from
selected plants are bulk threshed and advanced to the F3 generation.
F3 to F5: The bulk seed (20 g) from selected F2 plants is planted in two beds of 8–10
m in length to achieve about 400 plants/population. An epidemic is created of the
prevalent diseases. The best plants are selected by the breeders based on agronomic
type, fertility, lodging tolerance, durable disease resistance, and expected yielding
ability, plus somewhat for phenotypic uniformity. Subsequently, in the F5 generation
selected plants are harvested and threshed on an individual basis and the seed is
visually observed for grain filling characteristics, boldness, lack of diseases, yellow
berry, other markings, and color. About 30–50 % of the plants are thus discarded.
F6: Selected individual plants or spikes from the F5 generation are planted in small
plots of 0.7 to 1.0 m in length in a paired row. This system of planting allows thou-
sands of entries to be planted in a small area. Again, an epidemic is created of the
prevalent diseases. The best and most uniform lines are visually selected and har-
vested in bulk. The plump and bold grain lines retained after visual seed selection.
Best entries with desirable agronomic features and good grain characteristics are
promoted to yield trials (YT).
YT: Selected best entries from the F6 generation are promoted to the first-year YTs.
YTs are planted in an Alpha-Lattice-Latinized design with 2–3 replications, 2
checks and 28 entries in each trial. Checks are normally high-yielding commercial
cultivars or the highest yielding cultivar identified from the breeding program. YTs
are conducted in Ciudad Obregon (Fig. 2.3). Trial entries are planted in 2 beds of 3
rows each of 2 m length. Plots are harvested after physiological maturity using plot
combine harvesters, and then weighed manually for grain yield. High-yielding
entries are selected on relative yield over checks, agronomic type, disease resis-
tance, and additional industrial quality tests, including alveograms (using Ciudad
Obregon seed), and the best lines are promoted to the Elite Yield Trial (EYT) or
second year yield testing.
PCs:(Parcela chica- Spanish acronym for small plots) are planted separately at the
same time as the yield trials, with exactly the same entries as in the YTs, in an area
where relevant diseases are artificially inoculated. The PCs provide disease resis-
tance data. In addition, the PCs form small seed multiplication plots, where rouging
can be carried out to provide clean, pure seed for subsequent cycles.
EYT: EYTs are conducted using the best yielding entries from the YTs. The EYTs
are grown under representative and relevant environmental conditions. Latinized
alpha-lattice design with 2–3 replications is used. Again the EYTs are conducted in
Fig. 2.3 Standard yield trial plots at Cuidad Obregon, Mexico
Ciudad Obregon, under five different environmental conditions, where the targeted
conditions can be simulated: for example, Mega-environment 1 (ME1) by applying
5–6 irrigations; ME4 by supplying limited water under drip-irrigation, and by
restricting the number of irrigations (one irrigation during pre-sowing and booting
stages), thus creating very stringent drought conditions; ME5 by planting late (in
February) resulting in considerable heat stress at the time of flowering and grain
filling. Thus each target environment (e.g. ME1: irrigated, ME4: drought, ME5:
heat) is somewhat represented or simulated during the yield trial phase. Each entry
is planted in 2 beds, each 80 cm in width, 3–4 m long with 3 rows per bed. Also,
another environment created for a planting method called melgas (melga means
“irrigation basin” in Spanish) with 30 plots/melga basin. In melgas every entry is
sown as an 8-row plot, 3.8 m long.
EPCs: EPCs (Elite Parcela Chica) are planted separately at the same time and with
exactly the same entries as in the EYTs, in an area where relevant diseases are arti-
ficially inoculated. The EPCs provide disease resistance data. In addition the EPCs
provide clean, pure seed for subsequent cycles.
2.7 IBWSN: (International Bread Wheat Screening Nursery)
After considering grain yield, stress (biotic/abiotic) tolerance and end-use industrial
quality characteristics, and data from across the five environments, the best lines are
selected as candidates for the International Bread Wheat Screening Nursery (IBWSN)
for distribution to collaborators across the world. The lines that enter into the IBWSN
are top yielders in the EYTs, but also showed good resistance and performance in the
EPCs which are always planted at the same time under disease stress, where artificial
inoculation with virulent races (rust) takes place. These advanced lines are expected
to be good for diseases since they have been screened for resistance since the F1
generation onwards. Industrial quality is also taken into account, but a very high-
yielding line that has low industrial quality may still be included in the IBWSN, since
many countries still value quantity over quality. The lines also should not have lodged
excessively in the EYTs, and must be very uniform.
2.8 ESWYT (Elite Spring Wheat Yield Trial)
The best entries from the IBWSN enter into the Elite Spring Wheat Yield Trial
(ESWYT). This trial consists of 50 entries of 2 replications arranged in an alpha-
lattice design. Each ESWYT has 45 new bread wheat lines, four CIMMYT checks
(not necessarily the same each year) and one local check that presumably is the best
locally adapted commercial variety at individual sites. To maintain genetic diversity,
representative entries from diverse genetic backgrounds are included in the trial.
Individual experimental plots grown by the cooperators usually vary in size and are
adapted to the local yield trial planting practices used by the cooperators. A differ-
ent randomization is used for each site. The trial management practices are based on
standard crop husbandry practices for specific sites. A field book with instructions
for trial management and data recording are provided to each collaborator along
with seed shipment. Seed packages are prepared and dispatched to collaborators in
different countries by CIMMYT’s Seed Information and Distribution Unit in
Mexico. The CIMMYT wheat program distributes several yield trials annually that
are targeted to specific wheat growing environments and management conditions in
many developing and developed countries through its collaborative international
wheat improvement network. The ESWYT is targeted at irrigated environments
with higher production; this trial is often grown in other environments also, as mate-
rials adapted to other environments are also identified by the cooperators. There are
more than 100 sites worldwide where CIMMYT’s yield trials are grown annually;
however, data recovery is about 50 %. The sites are representative of different mega-
environments based on their classification by CIMMYT (Rajaram et al. 1995).
The diverse global wheat testing locations where ESWYT trials are grown encom-
pass a great deal of contrasting environmental conditions that might be expected in
the future due to climate change.
2.8.1 Variety Release
The final stage before cultivar release is the extensive evaluation phase. At this
stage, there is often little that one can do, other than extensively test, to build a data-
base that ensures that accurate information has been obtained to make the right
decision. This must be done over time and locations and should target environments
where the cultivar will most likely be recommended to be grown, but also surround-
ing environments that will test its robustness. However, these trials still need to be
undertaken in the most efficient manner (generally considered to be using incom-
plete block designs or nearest neighbor analyses (Stroup et al. 1994), grown in the
most representative locations, and correctly interpreted (Roozeboom et al. 2008).
As the lines continue to be advanced, the complexity and expense of the selections
assays will also increase. While considerable information is available on how to
analyze genotype × environment interaction, there are two aspects that need to be
considered in detail. The first is ensuring that the locations are representative of
critical regions within the target environments. The second aspect is that given the
correct testing locations, it is important to learn from those locations how to inter-
pret the data. In this case, every testing site in every year tells a story. The successful
breeders will be able to understand why one line did well or poorly at a site based
upon the line’s and site’s history.
2.9 Biotic Stresses
2.9.1 Resistance to Diseases
The major fungal diseases of wheat caused by biotrophs, include the three rusts
(stem, leaf and yellow), powdery mildew, bunts and smuts; whereas, those caused
by hemibiotrophs include Septoria tritici blotch, Septoria nodorum blotch, spot
blotch, tan spot and Fusarium head blight (FHB). The biotrophs are highly special-
ized and significant variation exists in the pathogen population for virulence to spe-
cific resistance genes. Evolution of new virulence through migration, mutation,
recombination of existing virulence genes and their selection is more frequent in
rust fungi. Therefore, breeding for resistance to these diseases needs a strategic
approach to enhance the durability of resistance.
The three rust diseases, stem (or black), leaf (or brown) and stripe (or yellow),
caused by fungi Puccinia graminis f. sp. tritici, P. triticina and P. striiformis f. sp.
tritici, respectively, continue to cause losses, often major, in various parts of the
world and hence receive high attention in breeding. The phenomenon of the erosion
of race-specific resistance genes, or their combinations, has led scientists to look for
alternative approaches to resistance management. Van der Plank was the first epide-
miologist to clearly define the theoretical basis of the concepts of resistance (Van
der plank 1963). This approach was widely recommended for breeding leaf rust
resistance by Caldwell (1968), stem rust resistance by Borlaug (1972), and yellow
rust resistance by Johnson (1988). The wide application of such a concept in breed-
ing for leaf rust resistance, commonly known as slow rusting, has dominated in
CIMMYT’s bread wheat improvement program for almost 40 years with major
impacts (Marasas et al. 2003). Recently, we have begun to understand better the
genetic basis of race-nonspecific or durable resistance to rust diseases and this

knowledge is being routinely applied in breeding. The development and deploy-
ment of wheat cultivars with such resistance will provide a long-term genetic solu-
tion to rust control.
2.9.2 Breeding for Resistance to Rusts
In the late 1960s and 1970s, there was a revival of the concept of general (race-
nonspecific) resistance and its application in crop improvement (Caldwell 1968).
In the early 1990s, once the genetic basis and diversity of slow rusting resistance
became more clear, high yielding lines that combined four or five additive, minor
genes for both leaf and yellow rusts and with near-immune levels of resistance were
developed through 3- and 4-way crosses involving lines carrying different minor
genes (Singh et al. 2000).
Recently, simple and three-way crosses have been commonly used in the
CIMMYT breeding program, with one or more parents carrying adult plant resis-
tance (APR), and these are being used to breed new high yielding, near-immune
wheat materials resistant to all three rusts. To transfer minor gene based resistance
into a susceptible adapted cultivar or any other selected genotype, a ‘single backcross
selected bulk’ scheme is being used in CIMMYT wheat breeding program. In this
scheme high yielding lines are crossed with a resistance donor parent; 20 spikes
from F1 plants from each cross are then back-crossed to the improved lines to obtain
400–500 BC1 seeds. Rust resistant and agronomically desirable plants are selected
from large segregating populations grown under artificially created rust epidemics,
where the pathogen race that had virulence for race-specific resistance genes pres-
ent in the populations are used to create the epidemics. Selection is practiced from
the BC1 generation onward for resistance and other agronomic features under high
rust pressure. Because additive genes are partially dominant, BC1 plants carrying
most of the genes show intermediate resistance and can be selected visually. About
1,500–2,000 plants are space sown in the F2, whereas about 600–800 plants are
maintained in the F3–F5 populations. Plants with desirable agronomic features, low
to moderate terminal disease severity in early generations (BC1, F2 and F3) and with
low terminal severity in later generations (F4 and F5) are retained. Under the selected
bulk scheme, one spike from each selected plant is harvested as a bulk until the F4
generation, and plants are harvested individually in the F5. Bulking of selected
plants poses no restriction on the number of plants that can be selected in each gen-
eration as the harvesting and threshing are quick and inexpensive, and the next
generation is derived from a sample of the bulked seed. Because high resistance
levels require the presence of four or five additive genes, the level of homozygosity
from the F4 generation onward is usually sufficient to identify plants that combine
adequate resistance with good agronomic features. Moreover, selecting plants with
low terminal disease severity under high disease pressure means that more additive
genes may be present in those plants. Selection for seed characteristics is carried out
on seeds obtained from individually harvested F5 plants. Small plots of the F6 lines
are then evaluated for agronomic features, homozygosity of resistance etc., before
conducting yield trials.
2.9.3 Targeted Breeding for Resistance to UG99 Group

of Races of Stem Rust Pathogen
Characterization of existing spring wheat breeding materials for resistance to Ug99

and its derivative races of stem rust pathogen in field trials in Kenya and as seedlings
in greenhouses at USDA-ARS Cereal Disease Laboratory (St. Paul, MN, USA) dur-
ing 2005–2009 resulted in the identification of several wheat lines with varying
levels of adult plant resistance (APR). Information on the resistance was made
available on www.globalrust.org and also summarized by Njau et al. (2010). Wheat
lines ‘Kingbird’, ‘Kiritati’, ‘Pavon 76’, ‘Muu’, ‘Parula’ and a few others were iden-
tified as carrying a high level of APR.
In the absence of molecular markers for APR genes and the absence of the Ug99
race in Mexico, a shuttle breeding scheme between Mexican field sites (Ciudad
Obregon in northwestern Mexico during winter, and Toluca or El Batán in the high-
lands near Mexico City during summer) and Njoro, Kenya, was initiated in 2006 to
screen and select breeding materials resistant to the Ug99 race of stem rust fungus
in Njoro, near Nairobi, Kenya. The Ug99 race of stem rust was recognized as a
major threat to wheat production and food security as more than 80 % of the world
wheat’s is susceptible to this race.
The ‘single-backcross, selected-bulk’ breeding approach (Singh and Trethowan
2007) is being applied for transferring multiple minor genes to adapted back-
grounds. The BC1 plants are selected for desired agronomic features and resistance
to leaf and stripe rusts, and harvested as bulk in Mexico. F2 plants derived from the
BC1, simple, and top crosses with desirable agronomic features and resistance to
leaf and stripe rusts are selected for agronomic traits and resistance to other diseases
at Ciudad Obregon or Toluca and harvested as bulks (Table 2.1). If the F2 popula-
tions were grown at Ciudad Obregon, where the quarantine disease Karnal bunt may
occur, the F3 populations are grown at Toluca for another round of selection. About
1,000 seeds of each F3 and F4 population obtained from the Toluca harvest are
grown at Njoro, Kenya for selection under high stem rust pressure during the off-
season. Populations not carrying sufficiently resistant plants are discarded. Selection
of plants with high to adequate resistance is carried out, selected plants are bulk-
harvested and plump grains are selected for establishing F4 and F5 populations of
about 1,000 plants during the main season at Njoro under high stem rust pressure.
Because stem rust affects grain filling, we expect plants with insufficient resistance
to have shriveled grains. Selection in main season is carried out in the same manner
as off-season and about 400 plump seeds harvested from the selected plants are
returned to Mexico and grown at Ciudad Obregon under high leaf rust pressure for
Table 2.1 Flow of breeding materials in the Mexico–Kenya shuttle breeding scheme
Year Locations Activities
1 Cd. Obregon New crosses made
El Batán F1 grown, BC1 and F1-Top made on selected F1
2 Cd. Obregon BC1 and F1-Top (350 plants), F2 (1,000 plants from simple
crosses) grown and selected for agronomic traits and leaf
rust resistance. Spikes from selected plants are harvested
as a bulk and plump grains are retained
Toluca F2 (1,000 plants from BC1 and F1-Top) and F3 (350 plants
from F2 simple) grown and selected for agronomic traits,
resistance to stripe rust, and Septoria tritici. Spikes from
selected plants are harvested as a bulk and plump grain are
retained
3 Njoro F3 and F4 (800 plants) grown under stem and stripe rust
pressures. Plants with high to adequate resistance are
tagged and harvested as a bulk. Plump grains are retained
Njoro F4 and F5 (800 plants) grown, spikes from short plants
resistant to stem and stripe rust are selected and harvested
as a bulk. Plump grains are retained
4 Cd. Obregon F5 and F6 (350 plants) grown and selected for agronomic traits
and resistance to leaf rust. Plants are harvested individu-
ally and those with plump grains are retained
El Batán and Toluca Advanced lines grown as small plots, selected for agronomic
traits and resistance to stripe rust and Septoria tritici
blotch at Toluca and leaf rust at El Batán. Best lines are
harvested in El Batán and those with plump grains are
promoted to yield trials
5 Cd. Obregon, Njoro Advanced lines grown as replicated yield trials at Cd.
and Santa Catalina Obregon and as small plots at all three sites. They are
phenotyped for leaf rust, stem rust and stripe rust at Cd.
Obregon, Njoro and Santa Catalina, respectively, and the
best lines are retained
El Batán, Toluca, Seed of International Nurseries Candidates multiplied at El
and Njoro Batán. Lines are also grown at all sites and phenotyped for
leaf rust, stripe rust, stem rust, Septoria tritici blotch,
Fusarium head blight, etc. Quality analysis is conducted
using Cd. Obregon grain
6 Cd. Obregon, Mexicali 2nd year yield trials conducted in 5 environments at Obregon,
and Njoro seed multiplication for international distribution at
Mexicali and phenotyped for stem rust resistance at Njoro
El Batán International Yield Trials and Screening Nurseries prepared
and distributed
7 International Countries with wheat seasons between April–December
8 International Countries with wheat seasons between October–June
Source: Singh et al. (2006, 2010)
final selection as individual plants in the F5 and F6 generations. Small plots of

advanced lines obtained by selecting individual plants in Ciudad Obregon are grown
at the El Batán and Toluca field sites to select for agronomic characteristics and
resistance to leaf and stripe rusts (Singh et al. 2006; 2010).
2.9.4 Rust Screening Methodology and Selection Environment
The probability of identifying resistant parents and resistant progenies is increased

by the availability of a reliable screening methodology and a favorable environment
for disease development. Depending on the disease and choice of the type of resis-
tance, the methodology may require simple tests in the greenhouse on seedlings or
adult plants and replicated field tests. Protocols for screening for resistance to most
diseases are well established and can be employed in breeding for resistance.
Inclusion of check cultivars for resistance and susceptibility is important to assess
the disease pressure and degree of resistance. Choice of field sites with reliable
environmental conditions is crucial for progress when selection is to be carried out
in field conditions.
To study the genetics of wheat rusts, and to breed for resistance, it is essential to
have efficient and reproducible methods of producing infection under controlled
and field conditions. When breeding for resistance to rusts, it is usually sufficient to
produce infection on all of the plants in a test. The test must be adequate to accu-
rately determine the infection types on host genotypes.
The CIMMYT wheat breeding program handles a large amount of breeding
material, so rust testing is usually done under field conditions. Exposing the breed-
ing materials under field conditions has several advantages. In most cases, except in
a limited number of locations, where natural epidemics occurred for rusts, artificial
inoculation is required. Good conditions should be provided for plant growth and
the spread of the rust. To obtain a uniform rust pressure the spreaders are sown as
borders around the entire experimental field and as small hills at one end of each
plot. Most nurseries include rust susceptible spreaders at intervals of every 25 to 50
entries. The spreaders are used for inoculation and insure that there is ample spore
production and spread of the rust. The more frequent the spreader rows, the more
uniform the epidemic is expected to be. The spreader can be planted as a single
susceptible cultivar but it is often useful to mix two or three susceptible cultivars of
different maturities in order to extend the epidemic over a longer period. Check
lines with known rust reactions are commonly planted at regular intervals to provide
a check for rust reaction and for agronomic features. Segregating generations such
as F2–F5 are usually planted in long rows, but the advanced generations are planted
in short rows. Under both of these conditions, spreader rows are good enough to
supply ample spores for disease epidemic.
Periodically, the disease pressure should be monitored in the spreader rows to
ensure that there is adequate disease development for the supply of ample spores.
Sometimes there may be problems with an uneven rust distribution. In this case the
rust infection will be heavier in areas nearest to the spreaders, particularly early in
the development of the epidemic. Uneven spore loads can be a problem when select-
ing for some types of resistance such as slow rusting. The resistance can also be
overwhelmed by heavy spore loads and the differences between plants or lines
become obscured. In special cases, it may not be desirable to have spreader rows but
instead it may be beneficial to inoculate the whole nursery uniformly.
Use of one or a mixture of races depends on the purpose of the test. One race
should be used if the purpose is to test for the presence of a specific gene for resis-
tance or to test for resistance to a particularly important race, such as a highly viru-
lent race like Ug99. A mixture should be used if the purpose is to simulate a natural
field epidemic or to select for resistance against all of the races that are normally
present in the field.
2.9.5 Field Inoculation Techniques
Several techniques have been used to inoculate field nurseries with urediospores,
including injection, dusting, spraying and transplanting infected plants. In the
CIMMYT rust nurseries, spraying of urediniospores suspended in a light mineral oil
(Soltrol 170) is practiced. This oil has proven to be a satisfactory carrier of the spores,
it has low phytotoxicity and the spores are readily wetted and suspended in oil, which
is its major advantage over water. A low concentration of spores (0.5 mg per ml) is
sufficient to give good results. Sprayers that produce a very fine mist are used and for
larger areas, several types of hand or power-operated low volume sprayers are avail-
able. Rust infection is initiated approximately 6–8 weeks after planting.
2.9.6 Measuring Rust Severity
Breeders need to decide the degree of resistance that is acceptable and select only
plants with at least that level of resistance. A common procedure is to record the
percentage of the leaf or stem that is covered with uredia. The leaves or stems are
compared with diagrams on which various percentages of the area have been cov-
ered with spots of various sizes and represent pustules. Based on the size of pustules
and the associated necrosis or chlorosis, infection responses are classified into four
discrete categories: R= resistant, MR= moderately resistant, MS= moderately sus-
ceptible and S= susceptible (Roelfs et al. 1992). Infection responses overlapping
between any particular two categories are denoted using a dash. For instance, ‘MR-
MS’ denotes an infection response class that overlaps the MR and MS categories.
Rust severity can also be assessed using 0–100 % following the modified Cobb
Scale (Peterson et al. 1948). Entries are evaluated for rust severity two to three times
between heading and plant maturity. The area under the disease progress curve
(AUDPC) can be determined following the formula described in Roelfs et al. (1992).
2.9.7 Greenhouse or Growth Chamber Tests
Greenhouse or growth chamber tests can be used for limited amounts of material
when it is particularly important to test with a specific race or to speed up the
breeding process. All greenhouse studies in CIMMYT are conducted at El Batán, in

Mexico, where a collection of rust races is preserved. In the greenhouse, plants are
inoculated with a specific race by spraying with urediniospores suspended in Soltrol
oil using an atomizer. After inoculation, plants are transferred to a dew-chamber
overnight to ensure germination and infection of the pathogen. Greenhouse evalua-
tion of rust infection type responses follow the 0–4 Scale described in Roelfs et al.
(1992). The slow rusting components; latent period, receptivity, and uredinium size
etc., are scored on flag leaves in repeated greenhouse experiments according to the
method described by Das et al. (1993), Lee and Shaner (1985), and Singh and
Huerta-Espino (2003).
Host response at seedling stage is normally scored as susceptible or resistant
depending on the infection type produced against a particular race of the pathogen.
Testing for major or specific resistance is normally done on the primary leaf under
controlled conditions. Screening involves use of a single isolate in each test and
includes susceptible check lines and differential lines that possess designated genes
for resistance. Inoculation is performed 7 days after planting and disease observa-
tions are taken based on infection type reaction 10–14 days after inoculation.
2.9.8 Resistance to Pests
More than 100 insect species have been identified as pests in wheat. A limited num-
ber of insect pests are systemically important to wheat worldwide, including the
greenbug, hessian fly, Russian wheat aphid and stem sawfly. The insects and mites
that have a negative impact on wheat production have complex biology, varied
reproductive behaviors, diverse food and survival habits, and powers of dispersal.
This makes screening for pest resistance very challenging.
2.10 Tolerance to Abiotic Stresses
Climate change-induced temperature increases (heat stress) and drought are esti-
mated to reduce wheat production in developing countries by 20–30 % (Lobell et al.
2008; Rosegrant and Agcaoili 2010). Heat and drought tolerance of crops varies
greatly and wheat is among the most sensitive of the major staples. Wheat breeders
have successfully developed cultivars better adapted to heat and moisture stress
conditions resulting in a 0.5 to 1.3 % grain yield increase per annum in many drier
wheat producing areas (Byerlee and Traxler 1995; Manes et al. 2012). Considerable
success has been achieved in breeding for lodging resistance by developing semi-
dwarf cultivars, resulting in the Green Revolution. The improved lodging resistance
conferred by reducing culm length and increasing harvest index has further allowed
exploitation of yield promoting factors like response to irrigation and fertilization.
In recent years major emphasis has been given to tolerance to heat and drought as
these stresses limit productivity in many parts of the world.
2.10.1 Canopy Temperature (CT)
Under drought, selection for cooler CT permits genetic gains for grain yield and
genotypes with cooler canopies have been shown to extract more water from deeper
soil profiles (Reynolds et al. 2007). Canopy temperature depression (CTD) is usu-
ally expressed as canopy temperature (Tc) minus air temperature (Ta), and it is posi-
tive when the canopy is cooler than the air. It has been used as a selection criterion
in wheat breeding in terms of heat and drought stress tolerance (Balota et al. 2007).
According to Munjal and Rana (2003), a cooler canopy and high stomatal conduc-
tance during the grain filling period is assumed to be the basic morpho-physiological
criteria for higher grain yield under heat stressed conditions. Furthermore, Balota
et al. (2008) reported that wheat cultivars with a high CTD showed a trend of higher
yield under heat and drought stress.
Canopy temperature depression can be measured almost instantaneously using
an infrared (IR) thermometer in a breeding plot. Since the measurement integrates
the temperature of several plants at once, the error normally associated with traits
measured on individual plants is reduced. Investigations into this methodology in
warm environments (Amani et al. 1996) have shown that CTD was best associated
with performance when measured at higher vapor pressure deficit (i.e., warm, sunny
conditions and during grain-filling). Irrigation status was not a confounding factor
within the normal frequencies of water application. Under drought, studies found
that optimum time for CTD measurements are in the morning and afternoon between
full ground cover and late booting, and during grain filling. Line performance seems
to be better predicted when CTD is measured in the morning during grain filling or
prior to heading.
2.10.2 Rapid Screening in Breeding Populations
CTD is routinely used by CIMMYT’s wheat breeding program for rain-fed environ-
ments to enrich for alleles associated with dehydration resistance. All F3 and F4
bulks (1,000 per cycle) are screened for CT under drought; a larger number of
plants, which are expressing favorable agronomic traits but with warmer CT (com-
pared to checks) are discarded.
When CTD was compared with other potential selection traits (grain number,
biomass, phenological data, and yield) measured in the selection environment, none
of the other traits showed a greater association with performance in the target envi-
ronment than CTD. In addition to yield, breeding objectives must take into account
multiple factors, such as disease tolerance and phenology. Therefore, it would be
logical when incorporating CTD into a selection protocol, to select for relatively
genetically simple traits such as agronomic type and disease resistance in the early
generations (e.g., F2–F3). Selection for complex traits, such as CTD, could be
employed in subsequent generations, when more loci are homozygous, perhaps in
preliminary yield trials.
CTD readings are normally measured using the infrared thermometer (Model
IRTS-P, Apogee Instrument, Inc., Logan, UT, USA) with a 4° field of view, which
is equipped with an extendible thermistor to read air temperature. The data for each
plot are the mean of 4–5 readings taken from the same side of each plot, at an angle
of approximately 30° to the horizontal, in a range of directions that cover different
regions of the plot and integrate many leaves. Measurements are normally made in
mid-afternoon because of low wind, as a high wind velocity may disturb the tem-
perature in and around the canopy.
Canopy temperature is largely a function of stomatal conductance (Amani et al.
1996) that can also be measured rapidly using a viscous flow porometer (Condon
et al. 2008). An evaluation of the effectiveness of integrating CT into other criteria
used by breeders showed that selecting for cooler plots, in addition to visual selec-
tion for plant type, improved the ability to identify the very highest yielding lines
(van Ginkel et al. 2008). Economic analysis supported the idea that incorporating
selection for Stomatal Aperture Traits (SAT) into a breeding program is likely to
result in increased efficiency associated with the ability to cull more lines, thereby,
reducing the size of yield trials (Brennan et al. 2007).
The development of relatively easy to use spectral radiometers offers another
high throughput screening approach for comparing spectral reflectance indices
(SRIs) of genotypes. The composition of light reflected by canopies is a function of
many physiological factors including light interception, hydration status of tissues
and pigment content and composition of photosynthetic tissue (Araus et al. 2001).
A number of SRIs have been shown to be correlated with the yield of genotypes
(Montes et al. 2007).
2.11 Grain Quality
Enhancing wheat quality improves processing efficiencies, makes more desirable

and more diverse consumer products and ensures the competitiveness of farmers,
grain merchandisers, millers and end processors. Wheat quality criteria vary drasti-
cally depending on the end-use. Similarly, wheat cultivars may show large differ-
ences in their processing and end-use quality attributes. Therefore, while setting
breeding priorities and strategies, one must determine: the cultivar’s intended end-
uses and/or the demands of the targeted market, specific quality traits to breed for,
and genotype × environment × management interactions that may influence the
quality of the resulting cultivar.
Once the parental lines are characterized and the crossing plan defined, the
probability of selecting desirable lines depends on the intensity and effectiveness of
the quality-selection pressure applied; the best results are obtained by breeding
for the targeted environment (warm, dry, wet and erratic) and screening F3–F5 lines
for desirable genes and allelic variations controlling grain-compositional traits
(Arbelbide and Bernardo 2006), complemented by rapid, high-throughput
conventional small scale tests such as flour sedimentation and Near Infrared
Reflectance Spectroscopy (NIRS), which are related to end-use processing quality
(Peña et al. 2002). Conventional small scale quality tests explain end-use quality
only partially; in advanced breeding stages (F6–F8), quality screening should be
based on more specific food processing (dough viscoelasticity and mixing proper-
ties, starch pasting properties, baking performance) and end-product quality attri-
butes (Peña et al. 2002). Finally, multi-location yield trials exposing advanced elite
lines to environmental variation and farmer’s crop management practices are nec-
essary to identify the few genotypes combining stable yield and quality attributes
across locations and years.
2.11.1 Grain Hardness
Grain hardness is a grain quality trait associated with the milling properties of wheat
(Miller et al. 1982) and with the baking quality of the resulting milling products.
Milling times, milling energy requirements and the level of starch damage produced
in the milled flour are all influenced by grain hardness. Hard wheats require longer
milling times and more milling energy, and produce a larger amount of damaged
starch. Rapid small-scale methods (based on grinding time, grinding volume, or
particle size distribution) used to determine grain hardness make it relatively easy to
screen for hardness as early as the F3 generation. Near infrared reflectance and
transmittance (NIR, NIT) analysis of the particle size distribution of whole grain
flour or analysis of the intact grain samples are particularly fast and useful in early
generation screening.
2.11.2 Starch
Native starch, which is the main component of the wheat grain (70–75 % dry
weight), shows little influence on the functional properties of wheat flours used in
bread, cookie and cake making. However, damaged starch (mechanically damaged
during flour milling), by exposing its components (amylose and amylopectin) to
interact with other constituents of the baking formula, influences importantly the
water absorption and fermentation time requirements of bread-making dough, as
well as the staling and crumb textural properties of bread. Some small amount of
damaged starch is desirable in bread-making flours but highly undesirable in cookie-
and cake-making flours, as it may reduce considerably the expansion capacity of the
cookie dough (Miller and Hoseney 1997). This is the reason that the cookie and
cake industries use soft wheat flour, which has a minimum amount, if any, of
mechanically damaged starch and, consequently, low flour water absorption.
The Amylograph/Viscograph and more recently the Rapid Visco Analyser (RVA)
are used to obtain a complete profile of starch pasting properties. While the first
requires a large sample size and a considerably long testing time, the RVA requires
a 3 to 4 g sample and only a few minutes to reveal the pasting profile of the tested
material. Therefore, the RVA is now considered a rapid test suitable for the early
selection of wheat lines possessing desirable starch pasting viscosity for noodle
making (Bhattacharya and Corke 1996; Panozzo and McCormick 1993).
2.11.3 Proteins
Protein content is a key specification for wheat since it is related to many processing
properties, such as water absorption and gluten strength. Protein content also can
be related to finished-product attributes, such as texture and appearance. Bakers
use protein content results to anticipate water absorption and dough development
time for processes and products, because higher protein content usually requires
more water and a longer mixing time to achieve optimum dough consistency. Grain
protein content (GPC) in wheat varies between 8 % and 17 %, depending on genetic
make-up and on external factors associated with the crop. A unique property of
wheat flour is that when in contact with water its insoluble protein forms; a visco-
elastic protein mass known as gluten. Gluten, comprising roughly 78 to 85 % of
total wheat endosperm protein, is a very large complex composed mainly of poly-
meric (multiple polypeptide chains linked by disulphide bonds) and monomeric
(single chain polypeptides) proteins known as glutenins and gliadins, respectively
(MacRitchie 1994). Glutenins confer elasticity, while gliadins confer mainly vis-
cous flow and extensibility to the gluten complex. Thus, gluten is responsible for
most of the viscoelastic properties of wheat flour doughs and is the main factor
dictating the use of a wheat variety in bread and pasta making. Gluten viscoelastic-
ity, for end-use purposes, is commonly known as flour or dough strength.
The sodium dodecyl sulphate (SDS) sedimentation tests (Axford et al. 1979) can
be used to obtain a semi-quantitative estimation of the amount of glutenin (or indi-
rectly, of general gluten strength). These tests, which are based on the expansion of
mainly glutenins (also known as gel proteins) in lactic acid or SDS/lactic acid solu-
tion, are currently the most rapid and reliable single small-scale tests (Weegels et al.
1996). These tests are widely used to screen early generation wheat lines in relation
to their general gluten strength type (strong to weak).
SDS-PAGE of whole protein extracts can be used in breeding programs as an
early generation technique to select lines possessing desirable high molecular
weight glutenin (HMWG) subunit composition and in advanced stages to define
desirable HMWG combinations in progeny of new crosses. Low molecular weight
glutenin (LMWG) subunits are also important in determining gluten viscoelasticity
(Weegels et al. 1996).
Some of the key quality tests conducted in CIMMYT wheat breeding programs
are elucidated below.
2.11.4 Falling Number
The level of enzyme activity can be measured by the falling number test. Yeast in
bread dough, for example, requires sugars to develop properly and therefore needs
some level of enzyme activity in the dough. Too much enzyme activity, however,
means that too much sugar and too little starch are present. Since starch provides the
supporting structure of bread, too much activity results in sticky dough during pro-
cessing and poor texture in the finished product. If the falling number is too high,
enzymes can be added to the flour in various ways to compensate. If the falling
number is too low, enzymes cannot be removed, which results in a serious problem
that makes the flour unusable.
The falling number instrument analyzes viscosity by measuring the resistance of
a flour-and-water paste to a falling stirrer. Falling number results are recorded as an
index of enzyme activity in a flour sample and the results are expressed in time as
seconds. A high falling number (for example, above 300 s) indicates minimal
enzyme activity and sound quality wheat flour. A low falling number (for example,
below 250 s) indicates substantial enzyme activity and sprout damaged wheat flour.
2.11.5 Sedimentation Test
The sedimentation test provides information on the protein quantity and the quality
of flour samples. Positive correlations were observed between sedimentation vol-
ume and gluten strength or loaf volume attributes. The sedimentation test is used as
a screening tool in wheat breeding as well as in milling applications. The sedimenta-
tion test is conducted by holding the flour sample in an acid solution. During the
sedimentation test gluten proteins swell and precipitate as a sediment. Sedimentation
values can be in the range of 20 or less for low-protein wheat with weak gluten to
as high as 70 or more for high-protein wheat with strong gluten.
2.11.6 Farinograph
The farinograph results are used as parameters in formulation to estimate the amount
of water required to make dough, to evaluate the effects of ingredients on mixing
properties, to evaluate flour blending requirements, and to check flour uniformity.
The results are also used to predict processing effects, including mixing require-
ments for dough development, tolerance to over-mixing, and dough consistency
during production. Farinograph results are also useful for predicting finished prod-
uct texture characteristics. For example, strong dough mixing properties are related
to firm product texture. The farinograph determines dough and gluten properties of
a flour sample by measuring the resistance of dough against the mixing action of
paddles (blades). Farinograph results include absorption, arrival time, stability time,
peak time, departure time, and mixing tolerance index.
2.11.7 Alveograph
The alveograph determines the gluten strength of dough by measuring the force
required to blow and break a bubble of dough. The results include P Value, L Value,
and W Value. Stronger dough requires more force to blow and break the bubble (a
higher P value). A bigger bubble means the dough can stretch to a very thin mem-
brane before breaking and indicates the dough has higher extensibility; that is, its
ability to stretch before breaking (L value). A bigger bubble requires more force and
will have a greater area under the curve (W value).
The alveograph test provides results that are common specifications used by
flour millers and processors to ensure a more consistent process and product. The
alveograph is well suited for measuring the dough characteristics of weak gluten
wheats. Weak gluten flour with low P value (strength of gluten) and long L value
(extensibility) is preferred for cakes and other confectionery products. Strong gluten
flour will have high P values and is preferred for breads.
2.11.8 Mixograph
The mixograph test quickly analyzes small quantities of flour for dough gluten
strength. Wheat breeders use mixograph results to screen early generation lines for
dough gluten strength. Flour water absorption measured by the mixograph often
serves as bake absorption in bread baking tests. The mixograph determines dough
and gluten properties by measuring the resistance of dough against the mixing
action of pins. Mixograph results include water absorption, peak time, and mixing
tolerance. The mixograph curve indicates gluten strength, optimum dough develop-
ment time, mixing tolerance (tolerance to over-mixing), and other dough character-
istics. The amount of water added (absorption) affects the position of the curve on
the graph paper. Less water increases dough consistency and moves the curve
upward. The mixograph test measures and records the resistance of dough to mixing
with pins. Peak Time is the dough development time, beginning the moment the
mixer and the recorder are started and continuing until the dough reaches maximum
consistency. This indicates optimum mixing time and is expressed in minutes.
Mixing tolerance is the resistance of the dough to breakdown during continued mix-
ing and affects the shape of the curve. This indicates tolerance to over mixing and is
expressed as a numerical score based on comparison to a control. Weak gluten flour
has a shorter peak time and less mixing tolerance than strong gluten flour.
2.11.9 Quality Criteria Used in the CIMMYT

Wheat Breeding Program
Wheat quality improvement is an important component in the CIMMYT wheat

breeding program. Therefore, wheat progenitors and lines derived from the crosses
are assessed for diverse quality attributes. Besides relevant quality data, wheat
breeders receive an end-use quality classification, which is intended to aid the
breeder in identifying the distribution of wheat end-use quality types (Peña 2009).
Table 2.2 shows wheat quality classes related to grain quality attributes. Based on
wheat quality attributes, an end-use quality type is classified.
2.11.10 Breeding for Improved Human Nutrition
Micronutrient malnutrition arising from dietary deficiency of bio-available minerals

and vitamins affects more than half of the world’s population, especially women
and preschool children. In particular, zinc (Zn) and iron (Fe) deficiencies are a
growing public health concern, especially in the developing world. A new public
health approach to alleviate deficiencies of these mineral nutrients in developing
countries is through biofortification of staple food crops. Biofortification involves
development of micronutrient-dense staple crops using the best traditional plant
breeding approach. The HarvestPlus (www.harvestplus.org) initiative of the
Consultative Group on International Agricultural Research (CGIAR) has aimed to
develop and distribute biofortified varieties of major staple crops, including bread
wheat, that have high concentrations of these essential micronutrients. CIMMYT is
leading the efforts in development and dissemination of high-yielding, disease-
resistant wheat varieties with significantly increased Zn and Fe concentrations.
Genotypic variation for grain Zn and Fe concentration in wheat has been demon-
strated (Graham et al. 1999). A genetic study of a bread wheat mapping population
showed continuous variation for both Zn and Fe suggesting that it is a quantitative
trait controlled by several genes (Shi et al. 2008). As our understanding of the
underlying genetic control of Zn concentration is poor, breeding has focused on
crossing materials of unrelated parentage and intermediate micronutrient status
with the aim of identifying transgressive segregants. Provided sufficiently large F2
and F3 population sizes are maintained and genetic drift minimized, the F4 and later
generations are screened for Zn concentration once a higher level of homozygosity
has been achieved. The most promising sources for grain Zn concentration are wild
relatives, primitive wheats, and landraces. Current breeding efforts at CIMMYT
have focused on transferring genes governing increased Zn from T. spelta and
T. dicoccon based synthetics, landraces, and other reported high Zn sources to high
yielding elite wheat backgrounds.
Table 2.2 Bread wheat quality classification

Hardness class and Abbreviated
grain color Gluten type descriptiona End-use typeb
Acceptable Quality Typesc
Hard Wheat
Hard-white (HW) Strong (S) HW-S, HR-S 1a, 1b (Pan type breads, mechanized
and hard-red industry)
(HR)
HW, HR Medium strong HW-MS, 2a (Leavened breads in general,
(MS) HR-MS baguette, etc., semi-mechanized
industry)
2a (Flat breads: two-layer breads,
baladi, etc.)
2a (Dry and fresh noodles: alkaline,
white, instant)
2b (Steamed bread, North-China
style)
HW MS HW-MS 2b (Flat breads: single-layer, chapati,
etc.)
HW, HR Weak (W) HW-W, HR-W 3a, 3b (Dense breads: Moroccan,
etc., flour tortilla)
Soft Wheat
Soft-white (SW) S (and MS) SW-S(MS), 4a (Steamed bread, South China
and soft-red SR-S(MS) style)
(SR)
SW MS SW-MS 4a (White-salted noodles, China,
Japan, and Korea)
SW, SR W SW-W, SR-W 4b (Biscuits, cakes, and steamed
bread of SE Asia)
Quality types unacceptable for mechanized and semi-mechanized bread makingd. These can be
considered as feed (or utility) wheat
H Tenacious (T) HW-T, HR-T 5 (Feed or utility wheat)
H Weak-Tenacious HW-WT, 5 (Feed or utility wheat)
(WT) HR-WT
S T SW-T, SR-T 5 (Feed or utility wheat)
S WT SW-WT, SR-WT 5 (Feed or utility wheat)
Source: Peña (2009)
a
Criteria to determine grain hardness class-gluten type abbreviated code: Grain hardness (Hard, H
or Soft, S), followed by grain color (White, W or Red, R), followed by a hyphen, and then by
gluten type (Strong, S; Medium-strong, MS; Tenacious, T; Weak, W; and Weak-tenacious, WT).
Example: HW-S, hard and white with strong gluten; SR-T, soft and red with tenacious (short)
gluten
b
End-use type number followed by letter “a” has higher protein content than the same followed by
the letter “b”: Type 1a should have grain protein above 12.5 % (12.5 % M. B.); Types 2a and 3a
should have grain protein above 11.5 % (12.5 % M. B.); Type 4a should have grain protein above
11.0 % (12.5 % M. B.); Type 5 has no differentiation regarding protein content
c
All wheat lines/varieties falling within the quality type 1a to 3b must possess moderate to high
gluten extensibility
d
Quality types 4a, 4b and 5 are characterized for having slightly- to non-extensible gluten charac-
ter, which is generally undesirable for making bread or flat bread.
New primary hexaploid synthetic wheats and landraces with significantly higher
Zn concentrations are used as donor parents. Limited backcross populations of
between 400 to 800 plants with elite materials and subsequent F2 (1,200–2,400
plants) and F3–F4 (400–800 plants) are grown, and plants with desired agronomic
features selected. BC1-F4s and BC2-F3s are grown in small plots for selection of
agronomic traits and leaf and yellow rust resistance. Agronomically superior, rust
resistant F4/F5 lines are then measured for Zn concentration. Selected advanced
lines with higher Zn and desirable agronomic traits are tested for grain yield poten-
tial and grain Zn concentration in replicated yield trials (Velu et al. 2010). Best leads
with high yield potential along with considerable Zn concentration (above the target
Zn concentration of 33 mg/kg) are then deployed to the national partners as a
HarvestPlus Yield Trial (HPYT). The first set of advanced lines derived from crosses
of high yielding wheats with genetic resources possessing high Zn and Fe such as
Triticum spelta, landraces and synthetic wheat based on T. dicoccon were tested at
nine locations in South Asia and Mexico for Zn and Fe concentration, grain yield
and other traits. Although G × E interaction was significant, high heritabilities were
observed for Zn and Fe concentrations at individual sites and across environments,
reflecting non-crossover type of interaction (Velu et al. 2012). This trend was con-
firmed by the high genetic correlations between locations that showed similar rank-
ing of entries across locations, indicating that it is possible to select the best adapted
entries with high Zn concentration. Pooled data across locations showed increments
of 28 % and 25 % over the checks for Zn and Fe. A considerable number of entries
exceeded intermediate to full breeding target Zn concentrations, indicating that it is
possible to develop Zn-biofortified varieties with competitive yields and other
farmer preferred agronomic traits.
This breeding method relies on the development of very large populations and
significant investment in Inductively Coupled Plasma (ICP) analysis for micronu-
trient status. A major shortage is that selection pressure for Zn cannot be applied in
early generations as the evaluation of single plants does not give an accurate mea-
sure of micronutrient status. Soil analyses as well as grain analysis of systematic
checks at CIMMYT’s research station in Ciudad Obregon showed that soil Zn
concentration may have been much more heterogeneous than soil Fe concentra-
tion. Large variation in soil Zn can confound or mask genetic differences among
lines, thereby preventing the identification of lines with genetically superior con-
centrations of grain Zn. One strategy to reduce this problem is to use a systematic
check, alpha lattice designs, and spatial analyses of segregating and advanced
populations. Another potential strategy that needs further study is the use of
Zn-containing fertilizer (foliar or soil applied) to homogenize soil Zn concentra-
tion (Oury et al. 2006). The development of cheaper and more rapid screening
assays for Zn, such as X-ray florescence (XRF) screening techniques, allows wheat
breeders to apply greater selection pressure in early generations, thereby minimiz-
ing the effect of “misclassified” lines on eventual outcomes.
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Chapter 3
Phenotyping in Sorghum [Sorghum bicolor
(L.) Moench]
Are Ashok Kumar, Hari C. Sharma, Rajan Sharma, Michael Blummel,

P. Sanjana Reddy, and Belum V.S. Reddy
Abstract Sorghum is one of the most important cereal crops grown in the semi-arid
tropics (SAT) of Asia, Africa and Americas for its food, feed, fodder and fuel value.
Sorghum production is constrained by several biotic and abiotic stresses. Genetic
enhancement of sorghum for grain and stover yield, nutritional quality and plant
defense traits (abiotic and biotic) which stabilize the crop performance requires
thorough knowledge on crop genetic and crop breeding principles. Rapid progress
in biotechnology provided powerful and cost-effective molecular/genomic tools to
develop desired products in sorghum. However, development of robust and efficient
phenotyping methods for traits of interest is critical to make use of these new tools.
There is no publication with efficient phenotyping protocols for sorghum research
compiled at one place for use by sorghum workers. This book chapter is an attempt
to fill that gap and we hope various phenotyping methods discussed hereunder will
be useful to sorghum researchers in developing improved products by using them in
combination with appropriate breeding/genomic tools.
Keywords Sorghum • Yield and quality • Biotic and abiotic stresses • Breeding
• Phenotyping • Genotyping • Genomics
A.A. Kumar (*) • H.C. Sharma • R. Sharma • B.V.S. Reddy

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),
Patancheru 502324, Andhra Pradesh, India
e-mail: a.ashokkumar@cgiar.org
M. Blummel
International Livestock Research Institute (ILRI), ICRISAT, Patancheru
502324, Andhra Pradesh, India
P.S. Reddy
Directorate of Sorghum Research, Rajendranagar 500030,
Andhra Pradesh, India
74 A.A. Kumar et al.
3.1 Introduction
Sorghum is an often cross-pollinating (6 % cross-pollination on an average) diploid

(2n = 2x = 20) belonging to Gramineae family with a genome (730 Mb), about 25 %
the size of maize or sugarcane. It is a C4 plant with higher photosynthetic efficiency
and higher abiotic stress tolerance (Nagy et al. 1995; Reddy et al. 2009) Its small
genome makes sorghum an attractive model for functional genomics of C4 grasses.
Drought tolerance makes sorghum especially important in dry regions such as
northeast Africa (its center of diversity), India and the southern plains of the United
States (Paterson et al. 2009). Genetic variation in the partitioning of carbon into
sugar stores versus cell wall mass, and in perenniality and associated features such
as tillering and stalk reserve retention, make sorghum an attractive system for the
study of traits important in perennial cellulosic biomass crops (Paterson et al. 1995).
Its high level of inbreeding makes it an attractive association genetics system.
Sorghum is one among the climate resilient crops that can better adapt to climate
change conditions (Cooper et al. 2009; Reddy et al. 2011). This chapter deals with
the biology and classification of sorghum, major sorghum improvement methods,
traits of global importance in sorghum improvement research and various phenotyp-
ing methods used for improving sorghum for these traits. We hope it serves as a
practical tool for the sorghum workers across the world.
3.1.1 Global Importance
Sorghum [Sorghum bicolor (L.) Moench] is the fifth most important cereal crop
globally and is the dietary staple of more than 500 million people in over 30 coun-
tries, primarily in the developing world. It is grown on 40 m ha in more than 90
countries in Africa, Asia, Oceania, and the Americas. Among those, USA, Nigeria,
India, Mexico, Sudan, China, and Argentina are the major sorghum producers glob-
ally. Sorghum accounts for 6 % of the global coarse cereals production in the world
and is particularly well suited to hot and dry agro-ecologies in the world. Global
sorghum productivity is low (1.4 t ha−1) with wide variation in different parts of the
world (Reddy et al. 2011).
Sorghum grain is mostly used directly for food (55 %), and is consumed in the
form of porridges (thick or thin) and flat breads. However, sorghum is also an
important feed grain (33 %), especially in Australia and the Americas. Stover (crop
residue after grain harvest) is an important feed source to livestock in mixed crop-
livestock systems prevalent in semi-arid tropics. Of late, sweet sorghum with sugar-
rich juicy stalks is emerging as an important biofuel crop (Reddy et al. 2008).
Sorghum grain is a rich source of micronutrients, particularly Fe and Zn (Kumar
et al. 2011a) and is also a rich and cheap source of starch. Thus, sorghum is a unique
crop with multiple uses as food, feed, fodder, fuel and fiber. It is generally grown in
rainy season (spring) but in India and in some parts of Africa it is grown in both
rainy and postrainy seasons (Reddy et al. 2009).
3 Phenotyping in Sorghum [Sorghum bicolor (L.) Moench] 75
Table 3.1 Five basic and ten hybrid races

Basic races Intermediate/hybrid races
1. Race bicolor (B) 6. Race guinea-bicolor (GB)
2. Race guinea (G) 7. Race caudatum-bicolor (CB)
3. Race caudatum (C) 8. Race kafir-bicolor (KB)
4. Race kafir (K) 9. Race durra-bicolor (DB)
5. Race durra(D) 10. Race guinea-caudatum (GC)
11. Race guinea-kafir (GK)
12. Race guinea-durra(GD)
13. Race kafir-caudatum (KC)
14. Race durra-caudatum (DC)
15. Race kafir-durra (KD)
3.1.2 Taxonomy and Classification
Sorghum was first described by Linnaeus in 1753 under the name Holcus. In 1974,
Moench distinguished the genus Sorghum from genus Holcus (Celarier 1959;
Clayton 1961). Subsequently, several authors have discussed the systematics, origin
and evolution of sorghum since Linnaeus (de Wet and Huckabay 1967; de Wet and
Harlan 1971; Doggett 1988; Dahlberg 2000). Sorghum is classified under the family
Poaceae, tribe Andropogoneae, subtribe Sorghinae, genus Sorghum Moench
(Clayton and Renvoize 1986). Some authors further divided the genera into five
subgenera: sorghum, chaetosorghum, heterosorghum, parasorghum and stiposor-
ghum (Garber 1950; Celarier 1959). Variation within these five subgenera except
the subgenera sorghum has been described (Celarier 1959). Sorghum bicolor sub
spp. bicolor contains all of the cultivated sorghums. Doggett (1988; Dubey 1994)
described them as annual plants, with stout culms up to 5 m tall, often branched, and
frequently tillering.
Harlan and de Wet (1972) have developed a simplified classification of cultivated
sorghum which proved to be of real practical utility for sorghum researchers. They
classified Sorghum bicolor (L.) Moench, subspp. bicolor into five basic and ten
hybrid races as depicted below (Table 3.1).
The 15 races of cultivated sorghum can be identified by mature spikelets alone,
although head type is sometimes helpful. The classification is based on five funda-
mental spikelet types (Harlan and de Wet 1972). However, some of the commercial
grain sorghum types are utilized in improvement programs, the characteristics of
which are given in Table 3.2.
The Biodiversity International [formerly International Plant Genetic Resources
Institute (IPGRI)] Advisory Committee on Sorghum and Millets Germplasm has
accepted and recommended this (Harlan and de Wet 1972) classification to be used
in describing sorghum germplasm (IBPGR/ICRISAT 1980). Large genetic diversity
reported in sorghum and sorghum gene bank at ICRISAT holds ~38,000 global
collections of sorghum germplasm which represents 80 % of the variability in sor-
ghum (Kumar et al. 2011a).
Table 3.2 Characteristics of commercial grain sorghum types

Grain
sorghum type Brief morphological description Geographical location
Durra Hairy rachis, flattened kernels and dry stalks Mediterranean, Near East,
Middle East
Shallu Partly pubescent involute glumes, cone-shaped India, tropical Africa
lax panicles, corneous kernels, dry and
non-sweet stalks
Guineense Involute and nearly glabrous glumes and compact Central and Western Africa
panicles
Kafir Awnless, compact cylindrical panicles and juicy South Africa
non-sweet stalks
Kaoliang Stiff stalks, thick hard rind, stiff spreading Eastern Asia
and few panicle branches, and dry and
no-sweet stalks
Milo Yellow midrib, transverse wrinkle of the glumes, East Africa
compact, awned panicles, large round kernels
Feterita Large kernels, brown testa, and dry and Sudan
non-sweet stalks
Hegari Rounded kernels, brown testa mid-compact Sudan
ellipsoid and branched panicles, and white
kernels with a bluish-white appearance
3.2 Floral Biology and Crop Improvement Methods
Sorghum is a short day plant, and blooming is hastened by short days and long
nights. However, varieties differ in their photoperiod sensitivity (Quinby and Karper
1947). In traditional varieties, reproductive stage is initiated when day lengths return
to 12 h. Floral initiation takes place 30–40 days after germination. Usually, the floral
initial is 15–30 cm above the ground when the plants are about 50–75 cm tall (House
1980). Floral initiation marks the end of the vegetative phase. The time required for
transformation from the vegetative primordial to reproductive primordial is largely
influenced by the genotype and the environment. The grand growth period in sor-
ghum follows the formation of a floral bud and consists largely of cell enlargement.
Hybrids take less time to reach panicle initiation, more days to expand the panicle
and a longer grain filling period than their corresponding parents (Maiti 1996).
3.2.1 Mode of Reproduction and Artificial Hybridization
Sorghum is an often cross-pollinating crop and natural cross pollination varies from
0.6 to 30 % depending on the genotype, panicle type, wind direction and velocity
(House 1980). Inflorescence is a raceme, consisting of one to several spikelets. The
spikelets usually occur in pairs, one being sessile and the second borne on a short
pedicel, except the terminal sessile spikelet, which is accompanied by two pedicelled
spikelets. In sorghum anthesis starts with the exertion of complete panicle from
the boot leaf. Flowers begin to open 2 days after complete emergence of the panicle.
The sorghum head begins to flower at its tip and anthesis proceeds successively
downward. Anthesis takes place first in the sessile spikelets. It takes about 6 days for
completion of anthesis in the panicle with maximum flowering at 3 or 4 days after
anthesis begins. Anthesis takes place during the morning hours, and frequently
occurs just before or just after sunrise, but may be delayed on cloudy damp morn-
ings. Maximum flowering is observed between 0600 and 0900 h. Because all heads
in a field do not flower at the same time, pollen is usually available for a period of
10–15 days. At the time of flowering (anthesis), the glumes open and all the three
anthers fall free, while the two stigmas protrude, each on a stiff style. The anthers
dehisce when they are dry and pollen is blown into the air. Pollen in the anthers
remains viable several hours after pollen shedding. Flowers remain open for
30–90 min. Dehiscence of the anthers for pollen diffusion takes place through the
apical pore. The pollen drifts to the stigma, where it germinates; the pollen tube, with
two nuclei, grows down the style, to fertilize the egg and form a 2n nucleus (Aruna
and Audilakshmi 2008). Stigmas get exposed before the anthers dehisce subjecting
to cross pollination. Pollination for crossing purposes should start soon after normal
pollen shedding is completed during morning hours.
Sorghum is amenable for crossing and selfing quite easily. For selfing, after
panicle exertion, bagging should be done by snipping off the flowered florets at the
tip. Crossing is done by emasculation of selected panicles and dusting of pollen
from identified plants. Hand emasculation is the most commonly practiced in
sorghum. Because of this ease in crossing, hybridization is most commonly
followed in sorghum for trait improvement.
3.2.2 Crop Improvement Methods
The crop improvement methods depend on the pollination control mechanisms and
cultivar options. Considering that sorghum is predominantly a self-pollinated crop,
breeding methods that are being followed in sorghum are those that are designed for
self-pollinated crops. The hybrids are superior to pure lines. The discovery of
cytoplasmic-nuclear male sterility helped to produce hybrids seed on mass scale
using three-line system (A, B and R) for commercial cultivation of hybrids. Also,
sorghum can be handled as cross pollinated crop for breeding purposes; the recurrent
population methods can be deployed using genetic male sterility genes.
3.2.2.1 Pure Line Selection
Pure line selection is practiced in two situations (a) when there is a need to develop
a variety from a land race population, and (b) while developing a variety from a
segregating population. For e.g. in sorghum, for postrainy season adaptation, the
local landraces from Maharashtra were collected and single plant selections were
made for a couple of generations and the performance for grain and stover yields of
the lines were compared. The line showing better performance than the check vari-
ety for yield traits is released for commercial cultivation (Audilakshmi and Aruna
2008). In case of segregating populations, the individual plants are heterozygous in
the beginning as they are the products of crossing between two homozygotes and
attain homozygosity in successive generations upon self-pollination. Individual
plant selections have to be carried out for at least 5–6 generations to achieve the
desired level of homozygosity of a pure line. Higher number of plants (3,000–
10,000) of segregating population is evaluated and selection is practiced to obtain
desired plants.
3.2.2.2 Mass Selection
Mass selection differs from pure line selection, wherein a number of desirable
plants (instead of only one), are selected and compositing is done on the
harvested seed to produce the next generation (Allard 1960). This method has a
few drawbacks, such as, it is not known whether the plants being grouped are
homogenous and some of them if heterogeneous would segregate further in fol-
lowing generations, and repeated selection would be required (Sharma 1988).
Mass selection is generally practiced to purify a variety. A large number of single
plants are selected from impure variety population, each line progeny tested and
similar type progenies bulked to form the pure seed lot. The success of the method
depends upon high heritability, that is, the presence of additive gene action and
minimal influence of genotype × environment interaction on the expression of the
selected trait.
3.2.2.3 Hybridization-Based Methods
The term hybridization refers to crossing of two genetically different individuals as

it combines the traits of two varieties and provides an opportunity to select plants
with desirable features of both parents through recombination in the segregating
progenies. As the natural variability for most traits is limited or already exploited,
there is a need to create new variability by making artificial hybrids to make any
further dent in developing improved varieties through selection in the segregating
populations. As most of the traits of interest in sorghum are quantitatively inher-
ited, sorghum breeders generally use pedigree method of selection in segregating
populations. In pedigree method, the records of the ancestry or pedigree of each
progeny is maintained and it is easy to trace back the parentage and selection. With
the pedigree system, the F2 generation represents the first opportunity for selection.
Selection for superiority is based on the vigor and other agronomic features of
progenies (families). In F2, selection is limited to individuals. In F3 and subsequent
generations, until a reasonable level of genetic homozygosity is reached, selection

is practiced both within and between families. Of the >700 sorghum female parents
(A-/B-pairs) developed by ICRISAT for various traits of global importance, more
than 600 parents are used in crossing to develop them using pedigree method
(Reddy et al. 2007).
Bulk population breeding is an economic method of obtaining homozygous lines
in self-fertilized crops. However it is not widely used in sorghum. Back cross
method is widely used in sorghum improvement particularly for resistance genes,
transferring male sterility to the identified maintainer lines by test crossing. Similarly
it is the most sought after method for transferring QTLs for shoot fly resistance and
stay-green trait (Kumar et al. 2011a).
The choice of parents for hybridization programs is critical for its success and
requires careful and critical evaluation of potential parents for various attributes
such as yielding ability, disease resistance, adaptation, quality of the produce and
morphological features relevant to crop management practices. Since new strains
are intended to have superior yield potential than the existing varieties, one of the
parents is invariably the adapted variety of the area. The other parent is primarily
chosen for complimenting the specific weakness of the variety, which needs to be
replaced. The general combining ability of a parent is likely to be reflected ade-
quately in the parental performance of the trait. Besides selection of the parents on
the yield performance and general and specific combining abilities in the partial
diallel crosses or line × tester crosses, it is desirable to analyze the potential parents
for important traits such as panicle length, number of primary/secondary branches,
grain per primary branch, and grain size (Audilakshmi and Aruna 2008).
A single genetic male sterility recessive gene in homozygous condition confers
male sterility. Population improvement methods can also be deployed in sorghum
by making use of this system which provides long-term breeding strategy to derive
diverse and broad genetic-based superior varieties/hybrid parents (Reddy and Ashok
Kumar 2008). More than 50 sorghum hybrid parents (A-/B-pairs) at ICRISAT were
developed using population improvement methods.
3.2.3 Marker Technologies and Genetic Transformation
Traditional methods of plant breeding have made significant contributions to sor-

ghum improvement as indicated by the progress in productivity in different parts of
the world (global average productivity 1.4 t ha−1in 2007 compared to 1.1 t in 1970).
However, the traditional methods have been slow in improving complex traits like
grain yield, grain quality, drought tolerance, resistance to grain mold, shoot fly,
midge, and Striga. For efficient genetic management of such traits, biotechnology
offers new and potentially powerful tools to plant breeders. Of the several biotech-
nological tools, DNA marker technology and genetic transformation have wide
application in sorghum improvement programs across the globe.
3.2.3.1 DNA Marker Technology in Sorghum
DNA markers have the potential to enhance the operation of a plant breeding
program through a number of ways ranging from finger printing of elite genetic
stocks, assessment of genetic diversity, addressing genome evolution, phylogeny
relevant to germplasm management, increasing the efficiency of selection for diffi-
cult traits through their tight linkages with DNA markers, to make environment-
neutral selection for map based cloning (Ejeta et al. 2000; Subudhi and Nguyen
2000). The long-term utility of marker-assisted selection in sorghum improvement
is likely to be jointly determined by the identification and mapping of phenotypes
with a direct impact on productivity and quality but which are difficult to study and
manipulate by classical means (Paterson 1994).
Construction of linkage map is the most fundamental step required for a detailed
genetic study and to follow marker-assisted breeding approach in any crop (Tanksley
et al. 1989). The use of DNA markers in marker-assisted breeding is based on the
tight linkages found between these markers and genes of interest. Such linkage infers
the presence of a desirable gene by assaying for the DNA marker. For example, while
transferring disease resistance gene to susceptible cultivars traditionally, progenies
are screened for the presence of disease resistance genes by inoculation with the
pathogen. With DNA-marker technology screening the plants with several different
pathogens simultaneously is possible without the need to inoculate the pathogens
(Lu 1994). However, expression of such resistance genes under variable field envi-
ronments needs to be tested. Sorghum genome mapping based on DNA markers
began in early 1990s and since then several maps of sorghum have been constructed
(Subudhi and Nguyen 2000). Several qualitative traits and QTLs of agronomic
importance have been mapped with the help of different classes of DNA markers.
Some of them include QTLs for yield components like kernels weight panicle–1,
threshing (%), dehulling yield (%) (Rami et al. 1998; Deu et al. 2000; Hart et al.
2002), panicle length (Pereira et al. 1995; Rami et al. 1998; Deu et al. 2000), tiller
number (Paterson et al. 1995; Hart et al. 2002), flowering or maturity (Crasta et al.
1999), number of seed branches panicle−1 (Pereira et al. 1995), 100/1,000 seed weight
(Pereira et al. 1995; Rami et al. 1998; Deu et al. 2000), number of seeds panicle−1
(Rami et al. 1998; Paterson et al. 1998; Deu et al. 2000) and seed size (Paterson et al.
1998). Apart from grain yield components, fodder quality traits like stay-green
(Tuinstra et al. 1996; Tuinstra et al. 1997; Crasta et al. 1999; Xu et al. 2000; Subudhi
et al. 2000a, b; Tao et al. 2000; Haussmann et al. 2002) and juicy midrib (Xu et al.
2000) have been investigated and mapped. Depending on their relative effects and
position, many QTLs could be used as targets for marker-assisted selection and pro-
vide opportunity for accelerating breeding programs (Subudhi and Nguyen 2000).
The QTL studies (Tuinstra et al. 1996, 1997; Crasta et al. 1999; Xu et al. 2000;
Ejeta et al. 2000; Kebede et al. 2001) identified several genomic regions of sorghum
associated with pre- and post-flowering drought tolerance. The molecular genetic
analysis of QTLs influencing stay-green trait, an important post-flowering drought
resistance (Xu et al. 2000; Tao et al. 2000; Haussmann et al. 2002) resulted in the
identification of up to four QTLs. Subudhi and Nguyen (2000) confirmed all the
four QTLs (Stg-1, -2, -3, -4) that were identified earlier by Xu et al. (2000) by evalu-
ating Recombinant Inbred Line (RIL) populations derived from B 35 and BTx 700
in two locations for 2 years. By generating a dense linkage map using RFLP mark-
ers, Ejeta et al. (2000) mapped the locus for one of the better characterized mecha-
nisms of resistance to Striga.
For disease resistance in sorghum, Rami et al. (1998) for the first time detected
three QTLs explaining 33.8 % of phenotypic variations in grain mold incidence.
Later, Rooney and Klein (2000) identified five QTLs on linkage groups D, E, F, G
and I with each QTL accounting for 10–24 % of the phenotypic variation for grain
mold. Rodriguez-Herrera et al. (1999) found that eight grain mold resistant RILs
from Sureno × TX 430 had consistently higher levels of anti-fungal proteins than
those in susceptible lines. Klein et al. (2001) also identified five QTLs for grain
mold each accounting between 10 and 23 % of phenotypic variation whose expres-
sion varied with location and the year tested.
For insect resistance, Sajjanar (2002) identified eight QTLs for shoot fly resis-
tance components. One major QTL for glossiness was detected on linkage group
J with phenotypic expression ranging from 34.3 to 46.5 % in the three screening
environments with highest expression in postrainy season. The largest consistent
effect for glossiness due to this QTL on linkage group “J” co-mapped with genomic
regions associated with dead hearts (%) under high shoot fly pressure. This QTL
may be a useful target for MAS for shoot fly resistance in sorghum.
At ICRISAT–Patancheru, India, QTL mapped for shoot fly resistance using RILs
populations derived from BTx 623 × IS 18551 and 296B × IS 18551. A linkage map
with reasonable genome coverage has been constructed and six QTLs have been
identified in at least two screening environments. The phenotypic variance explained
by each of these QTL ranged from 62.9 % for glossiness to 4.5 % for seedling vigor
(Ramesh et al. 2005). Satish et al. (2009) identified 29 QTLs for five component
traits of shoot fly resistance using the RIL populations of the cross 296 B × IS 18551.
Interestingly, some more additional QTL regions where resistance alleles were con-
tributed by the susceptible parent (296B) are also identified. All these can be used in
MAS for shoot fly resistance improvement in sorghum.
3.2.3.2 Genetic Transformation Technology
Recent advances in transgenic technology have enabled the transfer of agronomi-

cally desirable traits into crop species from diverse sources across reproductive
barriers. Entire process of crop improvement through transgenic technology can be
divided into (a) production of transgenic plants, (b) transgenic breeding program,
(d) release of products. Sorghum is recalcitrant to tissue culture and thereby to
genetic transformation compared to other cereals (Seetharama et al. 2003). Model
genotypes that can be readily transformed with far greater efficiency and reproduc-
ibility are not available in sorghum, and thus the genotypes of interest are directly
used (Visarada 2008). In order to overcome the difficulties encountered in in vitro
protocols, in planta methods and direct transformation of developing tissues with
Table 3.3 Transformation of sorghum with agronomically important traits at research level
Trait Transgenes Method Organization References
Resistance to Bt cry1Ac Bombardment ICRISAT, (Girijashankar et al.
stem borer India 2005)
Resistance to Bt cry1Aa & Bombardment DSR (ICAR), (Visarada et al.
stem borer cry1B India 2004)
Resistance to Rice chitinase Agrobacterium and Kansas Univ., (Zhu et al. 1998;
stalk rot bombardment USA Krishnaveni et al.
2001)
Drought HVA1 Bombardment Michigan (Devi et al. 2004)
resistance Univ.,
USA
Drought mtlD, Agrobacterium and CRIDA (Maheswari et al.
resistance p5CSf129A bombardment (ICAR), 2006)
and codA India
Anthracnose Chitinase Particle KIRDI, Kenya (Moses et al. 2011)
tolerance (harchit) and bombardment
chitosanase
(harcho)
gene guns are employed, though the transformation efficiency is far lower than the
methods described above. After production, the transgenic plants are evaluated for
the levels of expression of transgene trait and the stable inheritance of the transgene
in subsequent generations. Development of transgenic sorghum plants for agro-
nomically important traits at research level is presented in Table 3.3.
3.3 Crop Improvement Objectives and Phenotyping

for Major Traits of Interest
Sorghum improvement deals with production of new crop cultivars which are supe-
rior to existing cultivars for traits of interest. Availability of genetic variability for
these traits, knowledge about their heritability and inheritance, availability of effec-
tive phenotyping methodologies are fundamental for success of any crop improve-
ment program. In fact, the efficiency of phenotyping and its robustness decides the
success of the crop improvement program in terms of producing a tangible product
or technology. In sorghum, a large collection of germplasm is available at ICRISAT
(~38,000 accessions) and other places with characterization information available
for various morphological, agronomic and adaptive traits. Inheritance of major
traits is well studied and phenotyping techniques developed for efficient selection/
screening for major traits of interest. There is continuous exchange of material and
information across research groups. As a result, a large number of sorghum culti-
vars were developed and commercialized across the world for traits of interest. For
e.g. during the period 1976–2010, a total of 242 sorghum cultivars were released in
44 countries using the ICRISAT-bred sorghum material by the private and public
sector organizations (Kumar et al. 2011a). The list is quite exhaustive if we consider
cultivars developed by other centers in all sorghum growing countries. Focused
sorghum improvement programs backed by the germplasm sources, information on
heritability and gene action for traits of interest, phenotyping tools, established
selection procedures and massive adaptive trials in partners’ locations and above
all, collaborative research contributed for the large scale development and commer-
cialization of improved cultivars, though conventional methods were used. The phe-
notyping tools employed in sorghum improvement program for various traits of
global importance are discussed here under.
3.3.1 Adaptation
Sorghum is produced in rainy (hot) season in most parts of the world for various
uses- food, feed, fodder and industrial starch etc. where as in India it is grown in
both rainy and postrainy (cold) seasons. Limited sorghum area (mostly forages) is
there under summer season but it is small compared to the global area of 40 m ha.
3.3.1.1 Rainy Season
This is the most important adaptation globally spanning from May/June to August/
September with more than 30 m ha sorghum area across various continents falling
under this category. A variety of sorghums belonging to different races (direct or
hybrid), different cultivar types (mostly hybrids and varieties) and different grain
color (red, brown, white etc.,) types are grown for a variety of end-uses in more than
90 sorghum growing countries. For a plant breeder, the target materials and criterion
for selection depends upon the prevailing seed systems and the utilization pattern of
the crop and the consumer preference. For e.g., medium tall dual-purpose hybrids
with bold white seeds are preferred in India for both food and feed use whereas
grain types with red pericarp are preferred in Africa for food and brewing purposes
in East Africa while tall, long duration guinea sorghums are preferred in West Africa
for food. Similarly, medium tall/short hybrids are preferred in USA, South America
and Australia for mechanical harvesting for use as animal feed. In sorghum, plant
height, pigmentation, time to flowering, crop duration, panicle exertion, panicle
size, glume coverage, grain number, grain size and color and grain threshability are
major selection criteria in addition to the grain yield. In dual purpose types, apart
from grain yield, stover yield and quality are also important selection criteria.
A plant breeder needs to select appropriate germplasm and breeding methods keep-
ing the end product in mind; the maturity duration of the cultivar should correspond
with the length of growing period in the target area with the grain development stage
coinciding with dry period to get the best quality grain. The important biotic con-
straints in rainy season sorghum include shoot fly, stem borer, midge, grain mold,
striga and among abiotic constraints, drought predominates (Reddy et al. 2010a).
3.3.1.2 Postrainy Season
It is a unique adaptation to India (approximately 4.5 m ha) where the crop is grown
from September/October to January/February with residual and receding moisture
in black soils. The postrainy sorghum grain is preferred for food use in India owing
to its bold globular lustrous nature. However, no differences were observed between
the flat breads made from rainy (but matured under rain-free condition) and post-
rainy sorghums (ST Borikar, personal communication). The stover from postrainy
crop is the most important animal feed particularly in the dry periods. In addition to
the traits mentioned under rainy season adaptation, photoperiod sensitivity, tem-
perature insensitivity and grain luster are the major selection criterion. Varieties are
the cultivar choice but there is good scope for hybrid development using the white
grained rainy season adapted lines as female parents and land race restorers as pol-
linators. While terminal drought is the major production constraint, shoot fly, aphids
and charcoal rot play havoc with postrainy season production (Kumar et al. 2011a).
3.3.2 Yield and Yield Attributes
Grain yield is the most important trait in sorghum breeding as in other crops; how-
ever stover yield is equally important in sorghum particularly in countries like India.
Breeding for grain yield improvement is carried out by selecting genotypes directly
for grain yield and for component traits. For higher yield, genotypes with a plant
height of around 1.5 m are desirable which are amenable for mechanical harvesting
with medium maturity duration (100–120 days). Longer duration types give higher
yields but the length of growing period (LGP) in most sorghum growing areas does
not allow for breeding long duration types, with the exception of West Africa. If we
reduce the crop duration, it is likely that the yield goes down. Therefore the breeder
has to first fix the plant height and maturity duration for a given environment.
However, in the context of climate change, longer duration types need to be main-
tained in the breeding program considering the fact that when temperatures increases
by 2 °C, the longer duration types behave as medium duration types and produce
higher yields than other types (Cooper et al. 2009). Another important consideration
is photoperiod sensitivity. It is the ability of a genotype to mature at a given period
in the calendar year irrespective of its planting date. It is feasible to identify the
photoperiod-sensitive genotypes by planting them in different dates (at 15 or 30
days interval) and recording the days for 50 % blooming in the genotypes. The
genotypes that take less time for flowering when planted late can be considered
photoperiod-sensitive. In sorghum improvement in West Africa and postrainy sor-
ghum in India, photoperiod sensitivity is a key trait. Among the component traits,
long panicles, bold grains, number of grains per panicle, 100-seed weight contribute
for grain yield and most of these traits have high heritability enabling the plant
breeder to improve for these traits through simple selection. The gap between flag
leaf sheath and panicle base should be minimum to have good grain filling and the
glume coverage on grains is to be less for higher threshability. Grain size can be
visually judged and grain color can be selected as per the consumer /market prefer-
ence in the given adaptation (Reddy et al. 2009; House 1980).
3.3.2.1 Grain and Stover Yield
In areas where sorghum stover is important as animal feed, breeding dual-purpose

types is the best choice. Heterosis for grain and stover yield is high in sorghum and
therefore hybrids development should be targeted. A heterosis of 30–40 % for grain
yield is reported compared to the best varieties (Kumar et al. 2011a). Hybrid par-
ents’ development is critical for exploiting heterosis and therefore genetic and cyto-
plasmic diversification of hybrid parents is a major breeding objective. Population
improvement is also being followed for improving the grain and stover yields.
Quality of grain and stover is as important as grain yield. This is more so in the
postrainy season sorghum where consumers prefer bold, lustrous white grain types,
which is generally available only in landrace varieties (Reddy et al. 2009). The grain
luster is visually scored on a scale 1–3 where 1 = lustrous and 3 = dull among the white
grained types. The genetic base of these landraces is narrow and therefore it is more
challenging to improve for postrainy season adaptation. Similarly heterosis is low when
both parents are derived from landraces. A more practical method for developing post-
rainy season hybrids is by using rainy season adapted lines (mostly caudatum types) as
females and landrace varieties as pollinators. While improving the stover yield, one has
to keep in the mind the stover digestibility, protein content in addition to the stover
yields. The stover yields have to be recorded on oven dried samples after harvesting the
grains and for stover quality, indirect selection using NIRS is the most practical method.
3.3.2.2 Height and Maturity
Plant height is a major consideration in sorghum improvement and in fact it is one

the criteria for classifying sorghums as grain sorghums, dual-purpose sorghums,
fodder sorghums, sweet sorghums and forage sorghums. In sorghum, four loci are
known to be involved in the control of plant height. These genes are assigned the
symbols Dw1, Dw2, Dw3, and Dw4. Tallness is partially dominant to dwarfness.
The zero dwarf type (dominant [DW-] at all loci) may reach a height of 4 m. The
change from four to three dominant genes may result in a height change of 50 cm or
more. If genes at one or more of the loci are recessive, the difference in height
resulting from the recessive condition at an additional locus may have a smaller
effect in reducing plant height. The difference between a 3-dwarf (recessive genes
[dw dw] at three loci) and a 4-dwarf type may be only 10 or 15 cm (House 1980).
Breeders have to keep in mind these facts while selecting genotypes with appropri-
ate height. The plant height is always recorded from base of the plant to tip of the
panicle. Plant height and days to flowering data gives an idea about the genotype in
terms of suitability for various uses.
Quinby (1967) identified factors at four loci that influence maturity, Ma1, Ma2,
Ma3, and Ma4. Generally tropical types are dominant (Ma-) at all four of these loci,
and a recessive condition (mama) at any one of them will result in more temperate
zone adaptation which takes more time for maturity. Most sorghum improvement
programs target medium maturity types (crop duration less than 120 days) as they
yield high, however the targeted maturity is to be decided based on the length of
growing period (LGP) of the target area. In general, sorghum takes 35–40 days from
flowering to maturity. The grain is to be harvested at physiological maturity stage.
The hilum turns dark at physiological maturity and this is an important criterion for
harvesting (House 1980).
3.3.3 Resistance Breeding
Sorghum is affected by various biotic and abiotic factors leading to severe reduction
in productivity and production. A combination of genetic and management methods
are more effective in overcoming these constraints.
3.3.3.1 Phenotyping for Host Plant Resistance to Insect Pests
Nearly 150 insect species have been reported as pests on sorghum (Sharma 1993),
of which sorghum shoot fly (Atherigona soccata), stem borers (Chilo partellus,
and Busseolafusca), aphid (Melanaphis sacchari), sorghum midge (Stenodiplosis
sorghicola), and mirid head bugs (Calocorisangustatus and Eurystylusoldi) are
the major pests worldwide. They cause an estimated loss of $1,089 million in the
semi-arid tropics (International Crops Research Institute for the Semi-Arid
Tropics (ICRISAT) 1992). Early planting, use of pest-resistant cultivars, inter/
mixed cropping, and need based application are the major components of pest
control in sorghum (Sharma 1985). Host-plant resistance is one of the most effec-
tive and economic means of pest management in sorghum. It is compatible with
other methods of pest control and there is no cost involvement for the farmers
(Sharma 1993). Screening for resistance to insects under natural infestation is
unreliable, and takes a long time. Therefore, several field, cage, and screen house
techniques have been standardized for evaluating sorghum germplasm, breeding
lines, mapping populations, and transgenic plants for resistance to different insect
pests (Sharma et al. 1992a, 2003).
Sorghum Shoot Fly, Atherigona soccata. Sorghum shoot fly, A. soccata is a key
pest of sorghum in Asia, Africa, and the Mediterranean Europe. The larva cuts the
growing point, resulting in wilting and drying of the central leaf, known as a dead-
heart. The damaged plants produce side tillers, which may also be attacked. The
shoot fly population begins to increase in July, peaks in August–September, and
declines thereafter. Infestations are high when sorghum plantings are staggered due
to erratic rainfall.
Interlard-Fishmeal Technique (Multi-choice Field-Screening). Adequate shoot fly

density for resistance screening can be achieved by manipulating the sowing date,
using infester rows, and spreading fishmeal (which attracts the shoot flies) in the field
(Sharma et al. 1992a). Shoot fly population can be monitored through fishmeal-baited
traps to determine the periods of peak abundance of the shoot fly (Taneja and Leuschner
1985a). This information can be used for planting the test material so that the suscep-
tible stage of the crop coincides with the optimum shoot fly pressure. Late-sown crops
are subjected to high shoot fly infestation. At ICRISAT-Patancheru, sowing test mate-
rial in mid-July in the rainy season, and during October in the postrainy season is
effective to screen for resistance to shoot fly. The interlard-fishmeal technique, which
is useful for increasing shoot fly abundance under field conditions, involves planting
four rows of a susceptible cultivar (such as CSH 1, or Swarna) 20 days before the sow-
ing of test material. Moistened fishmeal is spread uniformly 1 week after seedling
emergence or kept in plastic bags in the interlards to attract shoot flies from the sur-
rounding areas. Four infester rows should be planted for every 20 rows of the test
material. One generation of the shoot fly is completed on interlards, and the emerging
flies infest the test material (Taneja and Leuschner 1985a; Sharma et al. 1992a).
No Choice Cage-Screening Technique. To confirm resistance to shoot fly observed
under field conditions, and to study the resistance mechanisms, the cage-screening
technique developed by Soto (Soto 1972) has been modified to simulate field condi-
tions. The cage-screening technique can be used for multiple- or no-choice tests.
For a multiple-choice test, the test genotypes are sown in the field in 3.4 × 2 m beds,
with a row spacing of 15 cm. Ten days after seedling emergence, the plants are cov-
ered with a 3.4 × 2 × 1 m screened cage, and the shoot flies are introduced into the
cage. The shoot flies are collected from fishmeal-baited traps in the field (Sharma
et al. 1992a). Eggs and deadhearts are recorded after 1 week. For a no-choice test,
only one genotype is sown in 1 × 1 m beds. Six beds can be covered with a
2 × 3 × 0.5 m cage having six compartments. Twenty shoot flies are released into
each compartment, and observations are recorded as described above.
Damage Evaluation for Resistance Screening. Data on number of eggs and the plants
with eggs, plants with deadhearts should be recorded when there are maximum dif-
ferences between the susceptible (>80 % deadhearts in Swarna) and resistant (<40 %
deadhearts in IS 18551) checks, or record data twice at 14 and 21 days after seedling
emergence. Also record the number of tillers, and tillers with panicles at maturity as
a measure of genotype's recovery resistance. Grain yield under protected and unpro-
tected conditions can also be used as a measure of resistance to sorghum shoot fly.
Resistance can also be measured in terms of leaf glossiness (1 = highly glossy, and
5 = nonglossy) and trichome density on the undersurface of leaves (Sharma and
Nwanze 1997). These traits are associated with resistance to shoot fly.
Spotted Stem Borer, Chilo partellus. Spotted stem borer, Chilo partellus is
common in Asia and east and southern Africa. The first indication of stem borer
infestation is the appearance of small-elongated windows (pin holes) in whorl
leaves. The third-instar larvae migrate to the base of the plant, bore into the shoot,
and damage the growing point resulting in the production of a deadheart. Normally,
two leaves dry up as a result of stem borer damage. Larvae continue to feed inside
the stem throughout the crop growth. Extensive tunneling of the stem and pedun-
cle leads to drying up of the panicle, production of a partially chaffy panicle or
peduncle breakage. Stem borer infestation starts about 20 days after seedling
emergence, and the deadhearts appear on 30–40 day old-crop. In northern India,
moth catch in light traps begins to increase during the last week of July and peaks
during August to September, while in southern India, the peak in moth catches has
been recorded during January to February. Screening for resistance to spotted
stem borer can be carried out under natural and artificial infestation (Jotwani 1978;
Taneja and Leuschner 1985b; Sharma et al. 1992a).
Use of Hot-Spots. Screening for stem borer resistance can be carried out at hot-spot
locations, where the pest populations are known to occur naturally and regularly at
levels that often result in severe damage. Hot-spot locations for C. partellus are
Hisar in Haryana and Warangal in Andhra Pradesh, India; Agfoi and Baidoa in
Somalia; Panmure and Mezarbani in Zimbabwe; Kiboko in Kenya; and Golden
Valley in Zambia.
Sowing Date. To screen for resistance under natural infestation, especially at the hot-
spot locations, adjust the sowing date of the crop such that the crop is at a susceptible
stage when the stem borer abundance is at its peak. Determine the periods of maximum
borer abundance through pheromone traps, light traps, or by monitoring borer infesta-
tion in the crop planted at regular intervals. In northern India,
C. partellus is most abundant in August to September, and the crop sown between the
1st and 3rd week of July suffers maximum stem borer damage. At ICRISAT-Patancheru,
maximum number of moths in the light traps have been recorded during September,
followed by smaller peaks during November and February (Sharma et al. 1992a).
Mass Rearing and Artificial Infestation. Artificial infestation with laboratory-reared
insects has been successfully used for screening test material for resistance to C.
partellus (Taneja and Leuschner 1985b; Dang et al. 1970; Reddy and Davies 1979).
For field infestation, the Bazooka applicator, developed at the International Maize
and Wheat Improvement Center (Trigo) 1977), has been modified to suit the require-
ments for infesting sorghum. Infest 15–20-day-old plants in the field with 5–7 larvae
per plant. Deadheart formation decreases progressively as the infestation is delayed.
Shoot fly infestation interferes with screening for resistance to stem borer. Spray
fenvalerate or endosulfan to suppress shoot fly infestation one week before artificial
infestation with stem borer. Screening for resistance to stem borer, C. partellus can
also be carried out using diet incorporation assay (Kumar et al. 2005).
Damage Evaluation. Stem borer attack in sorghum causes leaf damage, deadheart for-
mation, stem and peduncle tunneling, and production of chaffy panicles. Record the
extent of leaf feeding 2 weeks after artificial infestation, and 4–5 weeks after crop emer-
gence under natural infestation. Record the total number of plants, the number of plants
showing the leaf-feeding symptoms, and the leaf-feeding score on a 1–9 scale (1 = <10 %
leaf area damaged, and 9 > 80 % leaf area damaged). Data on deadhearts is recorded 3
weeks after artificial infestation, and 4–6 weeks after crop emergence under natural
infestation. Record the total number of plants, plants showing borer deadhearts, and the
visual score (1–9 scale) (1 = <10 % plants with deadhearts, and 9 = >80 % plants with
deadhearts). At crop maturity, record observations on the number of partial and com-
pletely chaffy panicles, the number of broken panicles. Recovery resistance could be
recorded in terms of number of plants with tillers and the number of tillers with produc-
tive panicles on a 1–9 scale (1 = >80 % plants with 2–3 uniform and productive tillers,
and 9 = <20 % plants with one or no productive tillers). Data on stem tunneling may be
recorded by measuring plant height and the peduncle length in five plants at random in
each plot. Measure the stem and peduncle tunneling separately and express it as a per-
centage of stem/peduncle length. The use and importance of various criteria to select for
stem borer resistance have been discussed by Singh et al. (2010).
Sugarcane Aphid, Melanaphis sacchari. Sugarcane aphid, M. sacchari is a serious
pest of sorghum in Asia and Africa. It feeds on the under surface of leaves and
secretes honeydew. The infested leaves begin to die, first turning yellow-brown at
the edges. The infestation starts from lower leaves and proceeds upwards. Under
severe infestation, the plants become pale yellow, with soot molds, wither and dry
up. Infestation becomes severe by panicle initiation stage.
Screening for Resistance. Screening for resistance to aphids can be carried out
under natural infestation in the field or infesting the test material under greenhouse
conditions using uniform number of insects per plant at the flag leaf stage. Crops
planted between 20 September and 15 October are heavily infested by the aphids.
Screening Under Greenhouse or Net-House Conditions. The plants can be infested
artificially by stapling a 10 cm aphid infested leaf cutting to the 5th leaf of each plant
under screen house or under nylon net in the field at the flag leaf stage. The nylon net
excludes the natural enemies and results in fast build-up of the aphid population. The
test lines can also be tested for aphid resistance by using clip cages. Fifth leaf at the
boot leaf stage can be infested with ten mature aphids inside a 5 cm diameter leaf cage.
The cages are placed in the mid-portion of each leaf. Rate of multiplication of the
aphids inside the clip cages can be recorded after 10 days (Sharma HC, unpublished).
Damage Evaluation. Aphid damage can be evaluated at the hard–dough stage on a
1-9 scale (1 = plants with a few aphid colonies with no apparent feeding symptoms,
9 = 5-6 leaves with severe aphid damage, and completely covered with aphid colo-
nies). Under no-choice screen house and clip cage methods, data can also be
recorded on numbers of aphids, and this also provides information on antibiosis
mechanisms of resistance to the aphids.
Sorghum Midge, Stenodiplosis sorghicola. Sorghum midge, S. sorghicola larvae feed
on the developing ovary resulting in production of empty spikelets. The damaged
panicles present a blasted appearance. Midge damaged spikelets have a pupal case
attached to the glumes or have a small exit hole of the midge parasite on the upper
glume. The major difficulty in identifying source material with stable resistance
against sorghum midge is the variation in the flowering of sorghum cultivars and
day-to-day variation in midge populations. Because of these problems, genotypes
rated as resistant under natural infestation often turn out to be susceptible in the
following seasons or at other locations. Techniques to screen for midge resistance have
been described by Jotwani (1978), Page (1979), and Sharma et al. (1988a, 1992a).
Hot-Spots. Hot-spot locations are useful to screen for resistance to sorghum midge.
Hot-spot locations for sorghum midge are Dharwad, Bhavanisagar, and Pantnagar
in India, Sotuba in Mali, FarakoBâ in Burkina Faso, Alupe in Kenya, and Kano in
Nigeria. Midge infestations are also high at several locations in Australia, the USA,
and Latin America.
Sowing Date. To screen test the material for resistance to sorghum midge under
natural conditions, it is necessary to determine the appropriate time for sowing at
different locations. Determine the periods of maximum midge density through fort-
nightly sowings of a susceptible cultivar. Adjust sowing dates so that the flowering
of the test material coincides with greatest insect density. At ICRISAT-Patancheru,
maximum midge damage has been observed in the crop planted during the 3rd week
of July. The peak in midge density occurs during October, and a second but smaller
peak has been observed during March in the postrainy season, for which planting is
carried out during mid-December (Sharma et al. 1992a).
Infester Row Technique. Midge abundance can be increased through infester rows
and spreading sorghum panicles containing diapausing midge larvae in the infester
rows (Sharma et al. 1988a). Four infester rows of a susceptible cultivar such as
CSH 1 should be planted 20 days before the test material after every 20 rows of the
test material. Alternatively, early-flowering (40–45 days) lines (IS 802, IS 13249,
and IS 24439) can be sown along with the test material. Midge-infested chaffy
panicles containing diapausing midge larvae, collected during the previous season
should be moistened 10–15 days to stimulate the termination of larval diapause
and spread in the infester rows at initiation of flowering in the infester rows. Midge
population multiplies for 1–2 generations on the infester rows before infesting the
test material, and increases midge damage by three to five times. High relative
humidity is important for adult emergence, oviposition, and subsequent damage.
Use overhead sprinkler irrigation to increase relative humidity in midge-screening
trials during the postrainy season or periods of low relative humidity. Group the
test material according to maturity (early, medium, and late) and height (dwarf,
medium, and tall) for proper comparisons. The test material can also be planted
twice at 15-day intervals to minimize the chances of escape from midge damage.
No Choice Head Cage Technique. Caging midge flies with sorghum panicles inside
a head cage to screen for midge resistance under uniform insect pressure (Sharma
et al. 1988b). Collect 20 adult female midges in a plastic bottle (a 200 ml aspirator)
between 0800 and 1100 from flowering sorghum panicles and release 40 midges
into each cage, and repeat the operation the next day. Infest 5–10 panicles in each
genotype, depending upon the stage of material and the resources available. Midge
damage decreases as the time of collection and release advances from 0830 to
1230 h. Examine the cages 5–7 days after infestation and remove any other insects
such as head bugs, panicle-feeding caterpillars, and predatory spiders from inside
the cage. Remove the cages 15 days after infestation and evaluate the midge
damage. The head cage technique is quite simple, easy to operate, and can be used
on a fairly large scale to confirm the field resistance of selected genotypes.
Damage Evaluation. Feeding by the midge larva inside the glumes leads to sterile or
chaffy spikelets. However, the symptoms (chaffiness) of natural sterility and exten-
sive grain damage by sucking insects are superficially similar to the damage caused
by sorghum midge. The midge-infested panicles have either small white pupal cases
attached to the tip of damaged spikelets or have small parasite exit holes in the
glumes. Genotypes flowering on different dates should be tagged with different-
colored labels or tapes or marked with paint along with panicles of resistant and
susceptible checks for proper comparison. Selection for resistance should be based
in relation to reaction of resistant and susceptible checks flowering on the same day.
Percentage chaffy spikelets is the most appropriate criterion by which to evaluate
sorghum lines for midge resistance. Record midge damage in 250 spikelets col-
lected from five panicles at random at 15 days after flowering or at maturity. In
samples collected at the milk stage, squeeze the chaffy spikelets between the thumb
and first finger or with forceps, and record the numbers of spikelets producing a red
ooze (this indicates midge damage). Express the data as a percentage of chaffy or
midge-damaged spikelets. The midge infested panicles can also be evaluated at crop
maturity visually on a 1–9 scale (1 = <10 %, and 9 = >80 % midge-damaged
spikelets). The test material can be maintained under infested and non-infested
conditions by using a cloth bags or sprayed with insecticides at flowering to control
the sorghum midge. Harvest all panicles from the middle row(s) at the time of matu-
rity and record grain yield. Express the loss in grain yield in the infested plots or
panicles as a percentage of the grain yield in non-infested plots or panicles. Glume
size and tightness, which are associated with resistance to sorghum midge, can also
be evaluated on a 1–5 scale (1 = glume short, shining and tight, and 5 = glumes long,
nonglossy, and soft upon touch) (Sharma and Nwanze 1997).
Head Bug, Calocoris angustatus. Head bugs, C. angustatus is a serious pests of
grain sorghum in India, while E.oldi is important in West Africa. The nymphs and
adults suck the sap from the developing grain resulting in tanning and shriveling of
the grain. Head bug damage leads to both qualitative and quantitative losses in grain
yield (Sharma and Lopez 1990). Head bug damage spoils the grain quality, and
renders the food unfit for human consumption. Such grain also shows poor seed
germination. Head bug damage also increases the severity of grain molds. Techniques
to screen for resistance to head bugs have been discussed by Sharma and Lopez
(1992) and Sharma et al. (1992a, b, 2003).
Hot-Spots. In India, ICRISAT-Patancheru, Bhavanisagar, Kovilpatti, Coimbatore,
and Dharwad are the hot-spot locations to screen for resistance to head bugs. At
ICRISAT- Patancheru, head bug density is very high during September to October.
Sowing Date. Adjust sowing dates such that flowering of the test material coincides
with maximum head bug density. Determine the periods of maximum head bug
abundance through fortnightly sowings. Maximum bug numbers at ICRISAT-
Patancheru have been recorded during September and a second but smaller peak has
been recorded during March. Crops sown during the 2nd week of July suffer the
maximum head bug damage.
Infester-Row Technique. Sow infester rows of mixed-maturity cultivars 20 days ear-
lier than the test material. Alternatively, sow early-flowering (40–45 days) sorghums
(IS 802, IS 13249, and IS 24439) along with the test material as infester rows along
with the test material. Sow four rows of a susceptible cultivar after every 20 rows of
the test material. Collect head bugs from other fields and spread them in the infester
rows at the panicle emergence to augment the bug abundance. Sow the test material
in two sets, at an interval of 10–15 days to reduce the chances of escape in the early-
and late-flowering lines. For better results, group the test material according to
maturity and height. The sowing date of each maturity group can also be suitably
adjusted so that flowering occurs during peak activity period of the head bugs.
No Choice Head Cage Technique. To overcome the problem of variation in flower-
ing among the test cultivars, and fluctuations in insect abundance, the head cage
technique developed for midge resistance screening has been found to be useful to
screen for resistance to head bugs as well (Sharma et al. 1992b). Collect ten head
bug pairs in a 200-ml plastic bottle aspirator and release them inside the cage.
Examine the infested panicles after 1 week and remove panicle-feeding caterpillars
or predatory spiders if any. Remove the muslin cloth bag along with the bugs 20
days after infestation, kill the bugs with ethyl acetate or benzene (2 ml bag−1), or
keep the bags in deep-freeze for 30 min. Count the total number of bugs in each
cage. Evaluate the panicles for head bug damage at maturity as described under
damage evaluation.
Damage Evaluation for Resistance Screening. Sorghum head bugs suck the sap
from developing ovary and result in shriveling and tanning of the grains. Head bug
damage can be evaluated by tagging five panicles at random in each genotype at the
half-anthesis stage. Sample the panicles for head bugs at 20 days after flowering or
infestation in a polyethylene or muslin cloth bag containing a cotton swab soaked in
2 ml of ethyl acetate or benzene. Count the total number of adults and nymphs.
Evaluate head bug damage at maturity on a 1–9 scale (1 = all grains fully developed
with a few feeding punctures, and 9 = most of the grains highly shriveled and almost
invisible outside the glumes).
Harvest all panicles from the middle row(s) of each plot or genotype at maturity
and record panicle and grain weight. Plots or panicles of lines being tested can also
be maintained under infested and un-infested conditions by using cloth bags to
exclude the head bugs or chemical control. Express the loss in grain yield of infested
plots or panicles as a percentage of the grain yield in non-infested plots or panicles.
Grain weight and percentage floaters in sodium nitrate solution can also be used as
selection criteria (Sharma and Lopez 1992). Take a sample of 1,000 grains at random
from each replication or panicle. Equilibrate the moisture content (24 h at 37 °C),
and record the grain weight. Prepare a sodium nitrate solution of a specific density of
1.31 in a beaker. Place the 1,000 grain sample in the beaker containing sodium nitrate
solution, and count the number of grains floating on the surface, and express them as
a percentage of the total number of grains. Glume covering of the grain and grain
hardness that are associated with resistance to head bugs can also be used as an indi-
rect criterion to select for resistance to head bugs (Sharma et al. 1992b).
Grain Mold. Grain mold is a major production constraint in Asia and parts of Africa.
The white grain medium duration genotypes are more prone to grain mold attack as
their grain development coincides with heavy rainfall. A complex of pathogenic and
saprophytic fungi causes grain mold, and the major fungi associated with early
infection events are Fusarium spp., Curvularia lunata, Alternaria alternata and
Phoma sorghina (Thakur et al. 2003, 2006). Damage resulting from early infection
includes reduced kernel development, discoloration of grains, colonization and deg-
radation of endosperm, and decreased grain density, germination and seedling vigor
(Thakur et al. 2006). Several species of Fusarium associated with grain mold com-
plex have been shown to produce mycotoxins, such as fumonisins and trichothe-
cenes that are harmful to human and animal health (Thakur et al. 2006; Sharma
et al. 2011). Phenotyping for grain mold reaction is done under field conditions
during rainy season (June–September). No artificial inoculation is required since
sufficient natural inocula of mold fungi are present during the rainy season over
sorghum fields in India for natural field epiphytotic conditions (Bandyopadhyay
et al. 1988; Thakur et al. 2007). The test lines are sown in the first half of June so
that grain maturing stages coincided with periods of frequent rainfall in August–
September. To enhance mold development, high humidity (>90 % RH) is provided
through sprinkler irrigation of test plots twice a day for 30 min each between 10 and
12 noon, and between 4 and 6 PM on rain-free days from flowering to physiological
maturity (when most grains in the middle of the panicle develop a black layer at the
hilum). The visual panicle grain mold rating (PGMR) is taken at the prescribed
physiological maturity (Thakur et al. 2006) using a progressive 1–9 scale, where
1 = no mold infection, 2 = 1–5 %, 3 = 6–10 %, 4 = 11–20 %, 5 = 21–30 %, 6 = 31–40 %,
7 = 41–50 %, 8 = 51–75 % and 9 = 76–100 % molded grains on a panicle to catego-
rize the test entries into resistant (1–3 score), moderately resistant (3.1–5.0 score),
susceptible (5.1–7.0 score) and highly susceptible (>7.0 score) reaction types. The
resistant and susceptible checks are invariably included for comparison. More
recently, a greenhouse screening method has been developed at ICRISAT Patancheru
that facilitates screening sorghum lines against individual mold pathogen under
controlled conditions (Thakur et al. 2007).
Resistance to grain mold is a polygenic trait and both additive and non-additive
gene action in conditioning resistance has been reported. To develop grain mold resis-
tant hybrids, at least one parent should possess grain mold resistance (Kumar et al.
2011b). Hard grain and colored glumes contribute to grain mold resistance in white
grain types and red grain types possess better grain mold resistance than white grain
types. Several resistant accessions (IS 2815, IS 21599, IS 10288, IS 3436, IS 10646,
IS 10475 and IS 23585) have been used in breeding to develop restorer lines, varieties
and hybrid parents. White/chalky white-grained mold resistant accessions such as IS
20956, -21512, -21645 IS 2379 and -17941 have been selected from the sorghum
mini-core collection (Sharma et al. 2010). In a trait-specific breeding program, a num-
ber of grain mold resistant lines with maintainer reaction have been converted into
male-sterile lines. Fifty-eight seed parents with A1 cytoplasm with white grain, red
grain and brown grain have been developed. Also, the grain mold resistant accession
IS 9470 with A1 (milo), A2, A3, and A4 (maldandi), and IS 15119 with A3 and A4 (mal-
dandi) cytoplasms have been converted into male-sterile lines and these have been
characterized. More recently, some test hybrids developed using mold resistant
advanced hybrid parents (A- and R-lines) have shown promising results for mold
resistance and grain yield at ICRISAT (Kumar et al. 2011a; Thakur et al. 2007).
Anthracnose and Leaf Blight. Sorghum anthracnose caused by Colletotrichum sub-
lineolum Hann. Kabátet Bub. (syn. C. graminicola (Ces.) G.W. Wils.), is one of the
most important foliar disease of sorghum (Marley et al. 2001; Valério et al. 2005).
Estimated grain losses caused by anthracnose are about 50 % on susceptible culti-
vars (Thakur et al. 2007). Severe infection and disease development occur during
prolonged periods of cloudy, warm, humid and wet weather. Sorghum plants are
more vulnerable to infection from flowering through the grain development phase.
The pathogen causes seedling blight, leaf blight, stalk rot, head blight and grain
molding, and thus limits both forage and grain production. Among these, foliar
anthracnose is the most pronounced and devastating on forage and grain sorghum,
especially on sweet sorghum cultivars.
Leaf blight caused by Exserohilum turcicum (Pass) Leonard and Suggs, is
another widely distributed, and most damaging foliar disease of sorghum, causing
significant grain losses due to the reduction of the photosynthetic leaf area (Bergquist
2000). In case of early infection in susceptible cultivars, up to 50 % grain yield
losses may occur. However, in case of late infection, disease development is slow
and yield losses are minimal. The disease is considered more important on dual-
purpose grain sorghum, but is especially severe on sweet sorghum (Hennessy et al.
1990; Thakur et al. 2007).
Screening techniques for phenotyping of both the diseases are same. Both green-
house and field screening for these diseases have been standardized. For field
screening, the test lines are evaluated along with susceptible check H 112 in the
anthracnose/leaf blight screening nurseries. Anthracnose screening is carried out
during rainy season and leaf blight nursery is planted in the late rainy season (2nd
week of September) at ICRISAT, Patancheru, India. The inoculum of both the
pathogens (C. sublineolum and E. turcicum) is multiplied by inoculating autoclaved
sorghum grains with an actively growing pure culture of a local isolate and
incubating at 28 ± 1 °C for 10 days under a 12-h photoperiod. The accessions in the
screening nursery are whorl-inoculated with infested sorghum grains (colonized by
C. sublineolum or E. turcicum) @ 3–4 grains/plant at 30 days after seedling emer-
gence. High humidity is maintained with overhead sprinklers twice a day on rain-
free days until the soft dough stage. Disease severity is recorded on 10 uniformly
flowering plants at the soft-dough stage using a progressive 1–9 scale, where 1 = no
disease and 9 = 76–100 % leaf area covered with lesions (Thakur et al. 2007). Based
on the disease score, the test lines are categorized as resistant (1.0–3.0 score), mod-
erately resistant (3.1–5.0 score), susceptible (5.0–7.0 score) and highly susceptible
(>7.0 score). Greenhouse screening involves spray-inoculation of 21-day-old plants
with the inoculum of C. sublineolum or E. turcicum (1 × 105 conidia ml−1) using a
handheld atomizer. Inoculated plants are incubated in a humidity chamber (25 °C,
RH >95 %) for 24 h, and then transferred to greenhouse benches under mist to

maintain high humidity. Disease severity is recorded on a 1–9 scale as described
above, 14 days after inoculation.
Several sorghum lines have been identified as moderately to highly resistant to
both anthracnose and leaf blight. Some of the lines with stable anthracnose resis-
tance are: IS 3547, IS 6958, IS 6928, IS 8283, IS 9146, IS 9249, IS 18758, M 35610,
A 2267–2, SPV 386 and ICSV 247. Four accessions IS 473, IS 23521, IS 23644 and
IS 23684 have been found to have stable resistance to both leaf blight and anthrac-
nose. At ICRISAT Patancheru, in a trait-specific breeding program, some of these
lines with white-grain have been used to develop resistant lines and hybrid parents.
Some anthracnose tolerant hybrid seed parents, such as ICSA/B 260 to ICSA/B 295
are available at ICRISAT. Similarly some leaf blight tolerant hybrid seed parents,
such as ICSA/B 296 to ICSA/B 328 were developed during 1989–1998 and are
available at ICRISAT, Patancheru (Thakur et al. 2007).
Charcoal Rot. Charcoal/stalk rot of sorghum is caused by the soil-borne fungus
Macrophomina phaseolina (Tassi) Goid. It is a major disease in dry regions of
Asia, Africa, Americas and Australia. The disease is relatively more severe and
destructive on high yielding sorghum cultivars when grain filling coincides with
low soil moisture in hot dry weather (Mughogho and Pande 1984). In India, the
postrainy (Rabi) sorghums that are generally grown on residual soil moisture often
get exposed to soil moisture stress during the grain filling stage if there are no rains.
Dry weather conditions during this time may further increase the moisture loss
from the soil. Under such a situation, plants are severely stressed due to increased
senescence in root and stem cells that adversely affects the production and translo-
cation of carbohydrates in the plant parts. These conditions predispose plants to
infection by the charcoal rot fungus. Affected stalks become soft at the base and
often lodge even due to moderate wind or by bending the plants. Thus pre-mature
lodging is the most apparent symptom of charcoal rot. When infected stalk is split
open, the pith is found disintegrated across several nodes. The cortical tissues are
disintegrated and vascular bundles get separated from one another. Numerous min-
ute, dark, charcoal-colored sclerotia of the pathogen are formed on these vascular
tubes. The disease reduces grain yield and stover quality. Loss in grain yield is
mainly due to lodging of the crop and loss in stover quality (and yield) is due to
rotting and decaying of the stalk.
Phenotyping for charcoal rot involves artificial inoculation of the test lines with
tooth pick infested with inoculum of M. phaseolina. The tooth picks are inoculated
with actively growing pure culture of the virulent local isolate of M. phaseolina and
incubated at 25 ± 1 °C for 10 days. The test lines are grown in field in the post rainy
season and are artificially inoculated by inserting toothpick infested with inoculum
of M. phaseolina into the second internode of the stalk at 10 days after 50 % flower-
ing. Irrigation is withheld in the experimental plots at 50 % flowering to ensure
adequate soil moisture stress to facilitate disease development. The inoculated
plants in test lines are scored for charcoal rot severity at the physiological maturity
(25–35 days after inoculation) using a 1–5 scale, where: 1 = one internode invaded,
but rot does not pass through any nodal area; 2 = two internodes; 3 = three
internodes; 4 = more than three internodes; and 5 = most internodes extensively

invaded, shredding of stalk and death of plant (Thakur et al. 2007). Data are also
recorded for per cent soft rot, and length of infection. Charcoal rot rating of test
lines is compared with that of the known resistant and susceptible checks to identify
resistant lines.
Sorghum genotypes that show stay-green trait (e.g., E36-1 and B35) are gener-
ally tolerant to charcoal rot. Some other lines, such as SLB 7, SLB 8, SLR 17, and
SLR 35 are also reported to be tolerant to charcoal rot. Drought tolerant, lodging
resistant and non-senescent sorghum genotypes are supposed to have good tolerance
to charcoal rot. However, finding such genotypes with high grain yield under desir-
able agronomic background are often not easy. Involving the stay-green trait sources
in crosses with other high yielding lines, several improved hybrid parents have been
developed. Among the hybrid seed parents, ICSA/B 307, -351, -371, -373, -375,
-376, -405, -589, -675, -678 and 702, and among male parents/varieties ICSV 21001
through 21025 are quite promising for stay-green trait. Based on number of nodes
infected, infection length and per cent soft, two hybrids (ICSA 675 × SPV 1411 and
ICSA 675 × ICSV 700) have been found tolerant to charcoal rot.
Striga. The witch weed (Striga spp.), a serious parasitic angiosperm of cereal crops,
is the most limiting biotic factor in the production of sorghum in sub-Saharan Africa
(Ejeta 2007). The weed survives by extracting water and nutrients from the host
plant and produces phytotoxins which are harmful to the host crop. It causes a char-
acteristic “witch” appearance of the host crop manifested by stunting and withering.
The yield losses range from 20 to 80 % and even total crop failure in severe infesta-
tion. Up to 5 and 95 % yield losses have been recorded for resistant and susceptible
sorghum hybrids, respectively (Obilana 1980). Striga seeds remain dormant and
viable in the soil for up to 20 years. With every planting, some of the dormant seeds,
stimulated by crop exudates, germinate and infest the host crop while reproducing
and increasing the Striga seeds in the soil thus escalating the problem. Several host
resistance mechanisms have also been suggested in the literature including low ger-
mination stimulant production, low production of the haustorial initiation factor,
avoidance mechanisms, presence of physical barriers, hypersensitive response (HR)
and antibiosis (Ejeta et al. 2000). Low germination stimulant production is the only
mechanism that has been studied and exploited for breeding purposes (Hess et al.
1992; Ejeta et al. 2000). Haustoria formation and attachment occur on the hosts and
non-host roots in a similar manner, but parasitic penetration in the non-host is
arrested only at the epidermis of the root with clear necrosis. An in vitro culture is an
important tool in identification of Striga resistance genes and characterization of
their mechanisms of expression. With the development of the agar gel assays (Hess
et al. 1992), important sources of resistance were identified and, reliable genetic
information generated (Ejeta et al. 1992). An extended agar gel assay was developed
by Mohamed et al. (2010) for screening for resistance to striga.
Abiotic Stresses. As sorghum is grown in a range of environments across tropical
and temperate regions, it is subjected to various abiotic stresses in different growing
countries. The inherent climatic variability and the projected changes in climate
profoundly influence the sorghum production in these regions. Most important
abiotic stresses affecting sorghum include drought, heat, salinity, acid soils etc.
Efforts are underway to address these issues in various sorghum improvement
programs.
Drought Tolerance. Drought is the most important abiotic constraint and the crop
may get exposed to drought during any stage of the growth. The response of sorghum
plant varies with the growth stage at which the drought occurs and therefore one
needs to breed for different droughts. Four growth stages in sorghum are considered
vulnerable to drought: germination and seedling emergence, post-emergence or
early seedling stage, midseason or pre-flowering, and terminal or post-flowering.
Terminal drought is the most limiting factor for sorghum production worldwide. In
sub-Saharan Africa, drought at both seedling establishment and terminal stages is
very common. In India, the rainy season sorghum most often faces mid-season or
end of season drought but end of season drought is a common phenomenon in
postrainy sorghum. The variable moisture availability at both pre-flowering and
post-flowering stages during the rainy season can have severe impact on grain and
biomass yield. Similarly the terminal drought severely affects the grain and stover
yields in postrainy season. The extent of grain yield losses due to drought stress
depends on the stage of the crop and the timing, duration, and severity of drought
stress (Reddy et al. 2009).
Sorghum responses to moisture stress at all four growth stages have been well
characterized. Variation in these responses has been observed and found to be heri-
table. Since the phenotypic responses of genotypes differing in drought tolerance
can be masked if drought occurs at more than one stage, screening techniques have
been developed to identify drought-tolerant genotypes at each of the growth stages,
separately. Of the several mechanisms to circumvent drought stress in sorghum,
drought escape (related to shorter maturity durations), drought avoidance (mainte-
nance of higher leaf water potential, LWP), and drought tolerance (related to greater
osmotic adjustment, OA) are important and have been well characterized. However,
LWP and OA did not correlate well enough with grain yield in field conditions to
merit selection based on them; in addition, screening techniques developed based
on LWP and OA were not cost effective in sorghum breeding. Empirical screening
based on imposing drought at various growth stages and measuring plant morpho-
logical and yield responses is the most effective approach. Long mesocotyl in seed-
ling establishment and recovery from mid-season stress after release by rains are
important traits that can be easily deployed in lines. The stay-green trait has been
well exploited to enhance post-flowering drought tolerance in sorghum.
At ICRISAT, growth-stage-specific breeding for drought tolerance, which
involves alternate seasons of screening in specific drought and well-watered envi-
ronments, has been used to breed sorghum that can yield well in both high-yield-
potential environments as well as in drought-prone environments (Reddy et al.
2009). Since hybrids exhibits relatively better performance than open pollinated
(OP) cultivars for grain yield under water-limited environments, hybrid cultivar
development (including their parents) should be given strategic importance for
enhancing sorghum production in water-scarce environments (Celarier 1959). The
progress in enhancing drought tolerance in sorghum through conventional
approaches is limited by the quantitative inheritance of drought tolerance and yield

coupled with the complexity of the timing, severity and duration of drought.
Biotechnology appears to offer promising tools, such as marker-assisted selection,
for genetic enhancement of drought tolerance in sorghum. Four stable and major
QTLs were identified for the stay-green trait and are being introgressed through
MAS into elite genetic backgrounds at ICRISAT, QDPI, Purdue University, and
Texas A&M University (Nagy et al. 1995). However drought phenotyping assumes
critical importance for using any of these methods.
High Temperature Tolerance. Sorghum grows well in a temperature range of
15–40 °C but temperatures below and above this may have a bearing on crop ger-
mination, establishment, flowering and seed setting. It was reported that sorghum
flowers and set seed under high temperatures (up to 43 °C) provided soil moisture
is available (House 1985). In many regions of the world, sorghum production
encounters heat and drought stress concurrently but heat and drought tolerances
are unique and independent traits (Jordan and Sullivan 1982). Despite the level of
adaptation of sorghum in the semi-arid tropics, seedling establishment is still a
major problem. Failure of seedling establishment due to heat stress is one of the
key factors that limits yields and affect stability of production (Peacock 1982).
Thomas and Miller (1979) reported that sorghum seedlings respond differently
when exposed to varying temperatures, and genetic variation for thermal tolerance
in sorghum has been shown to exist in certain lines that are capable of emerging at
soil temperature of about 55 °C. Peacock et al. (1993) and Howarth (1989) have
discussed the need for greater diversity in sorghum seedling tolerance to heat in
superior genotypes, as this will improve the crop establishment in the semi-arid
tropics. Genetic variability for heat tolerance among the genotypes at seedling
stage was demonstrated by Wilson et al. (1982). Using screening techniques such
as leaf disc method (Jordan and Sullivan 1982) and leaf firing ratings by ICRISAT
breeders, genetic variability past the seedling stage was demonstrated and positive
correlation found between grain yield and heat tolerance thus making breeding for
heat tolerance a viable option. Genetic variability for heat tolerance in sorghum
was also reported by other researchers (Sullivan and Blum 1970; Seetharama et al.
1982; Jordan and Sullivan 1982). Understanding the genetic control of heat
tolerance in sorghum is a prerequisite for formulating an appropriate breeding
program. Khizzah et al. (1993) studied four sorghum parental lines RTx430,
BTx3197, RTx7000, and B35 and their F1 and reciprocals, and F2 progenies during
their reproductive phase to assess the genetic basis of heat tolerance in sorghum.
They reported that inbreds were more heat tolerant compared to their F1 progenies.
Also, cultivars which had good late season-field drought tolerance appeared to be
heat tolerant, suggesting a possible relationship between drought and heat
responses. They also reported cytoplasmic effects for heat tolerance. Using the F2
frequency distribution of the crosses with B 35, Khizzah et al. (1993) made the
following assumptions, (a) two loci were responsible for expression of heat toler-
ance, and (b) complete dominance at both gene pairs, but one gene when dominant
is epistatic to the other. Reported low to high heritability of heat tolerance in sor-
ghum suggests the feasibility of genetic enhancement. B35 and BTx3197 could be
used as sources for heat tolerance in sorghum improvement programs (Khizzah

et al. 1993). The importance of additive gene effects over dominance effects for
heat tolerance index was reported by Setimela et al. (2007). However, selection for
heat tolerance has limited success as (a) laboratory techniques to screen for heat
tolerance have not been effective in improving heat tolerance in field studies; (b)
field screening for heat tolerance is difficult to manage and is often confounded
with drought tolerance (Rooney 2004). Due to the confounding effects, though the
heat and drought tolerance are independent traits, the selection for drought toler-
ance traditionally has been assumed to improve heat tolerance.
Salinity and Acid Soils. Of all the soil mineral stresses or chemical toxicities, acid-
ity, and associated Al3+ toxicity and salinity are probably the most important con-
straints to sorghum productivity in tropical environments. Saline and sodic soils
cause mineral stresses on approximately 0.9 billion hectares of land (Gourley et al.
1997). Salinity causes reduction in germination (Igartua et al. 1994), growth (Maiti
et al. 1994), yields (Macharia et al. 1994) and modifies the physiological and bio-
chemical processes of the plant (Dubey and Singh 1999) in sorghum. Salinity causes
more serious damage in the seedling emergence stage than in any other stage in
sorghum (Macharia et al. 1994). Though sorghum is known to be relatively more
tolerant to soil salinity than maize (Igartua et al. 1994; Krishnamurthy et al. 2007),
genetic enhancement of sorghum for salinity tolerance would further increase
sorghum productivity in such soils. High soil acidity (80 % Al3+ saturation) signifi-
cantly reduce early vigor and green leaf area at maturity, and induce the lines to
flower early, besides reducing the head and grain weights quite substantially in
sorghum (Reddy et al. 2000).
An evaluation of a number of germplasm lines, breeding lines and a few popular
cultivars indicated existence of significant genetic variability in sorghum for grain
yield and other agronomic traits under saline soil conditions (Ramesh et al. 2005).
Similarly, Krishnamurthy et al. (2003) have identified some elite sorghum varieties
and improved lines promising for agronomic traits and also exhibited better salinity
tolerance under induced salinity (at 250 μM NaCl solution; EC: 23.4 dS m−1) in a
series of pot-culture experiments. The best way to screen large number of genotypes
is planting them in fields with high salinity (with an average ECe of 10 dS m−1 or
more) and select for desirable agronomic traits and salinity tolerance (Reddy et al.
2010b). In highly saline areas in West Asia and Caucasus, sorghum along with pearl
millet is a good option for forage production. Sweet sorghums with high juice brix
(%) are likely to be tolerant to salinity.
Haug (1984) considered the capacity of the cellular membrane for binding Al3+
as a possible mechanism of Al3+ tolerance, whereby Al3+ is prevented from entering
and accumulating in the cell. In situ selection in a given problem soil in the field is
a reasonable and reliable approach. While some argue that selection in a given prob-
lem soil in natural field conditions is less desirable as it does not allow one to address
resistance to Al3+ toxicity per se, as most acid soils produce both Al3+ and Mn toxici-
ties, and both elements interact differentially with other elements such as Ca, P and
Mg, others regard such an approach as a more practical approach, since such a work
may result in selection of genotypes resistant to the different toxicity problems and
their interactions in such soils (Blum 1988). The open-panicled Guinea race and the
hybrid Guinea bicolor lines had a higher overall percentage of acid tolerant sor-
ghum entries than those of other races and hybrids evaluated (Gourley 1988).
Variation in soil acidity stress factors with location, soil depth, rainfall, temperature,
effective cation exchange capacity (ECEC), natural content of essential elements,
level of toxic ions, p-fixation capacity and amount and quality of organic matter
(OM) (Gourley et al. 1997), has made breeding for soil acidity tolerance, a complex
and slow process. Nevertheless, much progress has been made since EMBRAPA
sorghum breeding program for tolerance to acid soils (Schaffert et al. 1975),
International Sorghum and Millets (INTSORMIL) sorghum acid-soil breeding proj-
ect and Inter-American Development Bank funded ICRISAT project on “Research
Network for developing sorghum for acid soils in Latin America” were imple-
mented in Brazil and Columbia in 1981. Many good sources of Al toxicity tolerance
have been identified (Gourley et al. 1997). In ICRISAT—Latin American Partnership
Sorghum Program, diverse sets of 378 pairs of A/B-grain sorghum lines, 784 grain
sorghum restorers/varieties and 94 forage sorghum lines were screened during
1996–1998 at Cali, Quilichao, La Libertad, Carimagua and Matazul and selections
were made based on early vigor (scale 1–5, where 1 = most vigorous, 5 = least vigor-
ous), plant height (m) at maturity, stay green at maturity (scale 1–5, where 1 = most
green, 5 = least green), grain yield (t ha−1) and biomass (t ha−1) in grain sorghum
lines. Fresh forage weight, recovery score on 1–5 scale (1 = high recovery, 5 = less
recovery) after the first cut, and tiller number were also used as an additional criteria
for advancing the forage sorghum lines (Reddy et al. 2000).
3.3.4 Nutritional Quality Traits
Sorghum being one of the major food crops in the world and has predominant
role in meeting the dietary energy and micronutrient requirements in the low
income group populations, improving sorghum nutrition quality is of paramount
importance.
3.3.4.1 Protein and β-Carotene Contents
Protein content is relatively more studied in sorghum where in high genetic vari-
ability reported. Gains in protein content were reported by various authors
(Virupaksha and Sastry 1968; Ramesh and Hudda 1994; De Mesa-Stonestreet et al.
2010). The best method for phenotyping for protein content is through using
Microkjeldahl method or Technicon autoanalyser (TAA) method (Johnson and
Craney 1971; Jambunathan et al. 1983). A study on limited number of germplasm
lines, hybrid parents in sorghum did not show appreciable variability for β-carotene
content in sorghum (Reddy et al. 2005). Similar is the case with yellow endosperm
lines where in the β-carotene did not exceed 1.1 ppm. For phenotyping for this trait,
spectrophotometry can be followed but estimation using High-Performance Liquid
Chromatography (HPLC) gives more accurate information.
3.3.4.2 Grain Fe and Zn Concentration
Large scale screening of sorghum core germplasm accessions, hybrid parents and
commercial hybrids showed high genetic variability for grain Fe and Zn concentra-
tions and most of this variation is heritable (Reddy et al. 2005; Kumar et al. 2012).
Significant positive association exists between grain Fe and Zn concentrations
(r2 = 0.6–0.8) and it is possible to simultaneously improve both the traits (Kumar
et al. 2009; Reddy et al. 2010b). Additive gene action plays significant role in con-
ditioning the grain Zn concentration while both non-additive and additive gene
actions condition the grain Fe concentration (Kumar et al. 2013). The Fe and Zn
concentrations can be estimated using Inductively Coupled Plasma Spectrometry
(Houk 1986). This is a precise but destructive and laborious method. Most rapid and
low cost method for assessing grain Fe and Zn concentrations is by using X-ray
fluorescence spectrometry (XRF) method which is non-destructive and can be used
routinely to screen the breeding materials. There is high correspondence between
the values obtained by both the methods indicating that XRF can be used for assess-
ing grain Fe and Zn concentrations particularly for discarding the poor lines in the
breeding material.
3.3.5 Fodder Quality Traits
Sorghum is an important source of fodder particularly in Asia and Africa. While it

is mostly the stover used as animal feed in these areas, forage sorghums are quite
popular in Americas, Europe and Australia.
3.3.5.1 Forage/Fodder Yield and Animal Feed Quality
Conventional laboratory analysis cannot cope with phenotyping the large set of
sample entries from multidimensional sorghum improvement programs. Near
Infrared Spectroscopy (NIRS) is a non-evasive technique that can be employed for
phenotyping for relevant sorghum stover/forage/ fodder traits after calibration and
validation with laboratory fodder traits obtained with conventional chemical and
biological laboratory analysis. We summarize here the good-of-fitness NIRS equa-
tions used for the prediction of nitrogen, cell wall (NDF) cellulose (ADF), lignin
(ADL), in vitro digestibility (IVOMD), in vitro metabolizable energy (ME) and in
vitro fermentation kinetics using a modified exponent model that included a lag
phase (Table 3.4).
Blummel et al. (Kumar et al. 2010) investigated 24 sorghum stovers with sheep
for organic matter digestibility (OMD) and intake (OMI), and for digestible
organic matter intake (DOMI) and for relations between above laboratory fodder
quality traits and these in vivo measurements (Table 3.3). For each of the in vivo
measurements, chemical (NDF, ADF, ADL) and in vitro (IVOMD, ME) traits
were identified which accounted for at least 50 % of the variation in the respective
Table 3.4 Blind prediction of chemical and in vitro fodder quality nutritional traits of
sorghum stover by near infrared spectroscopy
Trait Range Mean SD SEP R2
N 0.1 2.5 0.7 0.4 0.1 0.94
NDF 34.2 84.3 66.8 6.5 2.7 0.83
ADF 27.5 60.1 42.6 6.5 2.0 0.91
ADL 1.7 12.1 4.6 1.9 0.6 0.82
ME 4.7 12.0 7.2 1.0 0.3 0.91
IVOMD 30.0 77.8 49.0 6.6 1.9 0.90
A 33.3 75.3 53.6 7.0 3.5 0.76
C 0.02 0.07 0.04 0.01 0.004 0.57
Lag 10.2 3.6 −1.9 3.11 0.8 0.92
T50 6.3 33.00 16.0 4.4 1.8 0.82
animal performance traits. Using multiple regression procedures and stringent

cross-validation (“blind-predictions”) procedures OMD, OMI and DOMI could
be predicted with R2 for comparing observed and predicted values of 0.36, 0.65
and 0.75, respectively.
3.3.5.2 Stalk Sugar Traits
Sweet sorghum is a multi-purpose crop that yields food, fodder and fuel. It is being
used for syrup and ethanol production in USA (http://nssppa.org/Sweet_Sorghum_
FAQs.html verified on 5th December 2012) EU (http://esse-community.eu/verified
on 5th December), China, Philippines, Mali, India and other countries (Reddy et al.
2008, 2010b; Wortmann et al. 2010). Phenotyping for stalk sugar and related traits
in sweet sorghum is done by recording observations on the fresh stalk yield (t ha−1),
stem girth (cm), soluble solids concentration (°Bx), juice yield (t ha−1) and/or juice
volume (l t−1), juice extraction (%) and sugar yield is extrapolated using the equation
sugar yield (t ha−1) = Juice yield × Brix (%) × 0.75 (Blümmel et al. 2010).
Sorghum improvement has come a long way from using simple classical meth-
ods like mass selection to advanced level of selection using molecular markers for
trait improvement. Efforts are underway to use new genomic tools for sorghum
improvement facilitated by the availability of aligned genome sequence. While the
genotyping tools are increasingly available and more affordable now, the phenotyp-
ing is not receiving equal attention. One should keep in mind that without good
quality phenotyping data, the genotyping data is of no use, no matter how it was
generated. Therefore the progress in sorghum improvement in the years to come
depends upon the quality of the phenotyping data that we generate for traits of inter-
est and most appropriate use of genomic tools available.
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Chapter 4
Chickpea Phenotyping
A. Saeed and Siva Kumar Panguluri
Abstract The phenotype is known as a reflection of dynamic interaction between

the genome, environmental conditions and the multiple plant responses that
determines the plant performance. The phenotype is the final goal for plant
breeders. Phenotyping is the basis of plant selection and has been going on for
millennia. Breeders have done phenotyping since the beginning of the art.
Nowadays phenotyping is used as a powerful approach in several plants improve-
ment programs particularly for stress resistance/tolerace breeding. Recent
advances in phenomics, genomics and bioinformatics provide the possibility of
undertaking large scale screening and sequencing of germplasm accessions so
that modern breeding approaches can be realized in near future for chickpea
improvement. This article reviews some basic concepts of phenotyping, types of
the employed methods and bioassays referring to important biotic and abiotic
stresses, and continues with answering the question “how phenotypic data can be
integrated with genotypic data?” It concluded with a discussion about gains
which have been made with molecular assisted breeding (MAB) in chickpea and
opportunities for MAB in chickpea in the immediate future are also briefly
discussed.
Keywords Phenotyping • Chickpeas • Molecular assisted breeding
A. Saeed
Department of Seed and Plant Improvement, West Azerbaijan Agricultural & Natural
Resources Research Center, Urmia, Iran
e-mail: sdalisaeid@yahoo.com
S.K. Panguluri (*)
Pharmaceutical Sciences, College of Pharmacy, University of South Florida,
Tampa, FL 33612, USA
e-mail: spanguluri@gmail.com
112 A. Saeed and S.K. Panguluri
4.1 General Information
Chickpea (Cicer arietinum L.) is the only cultivated species belonging to the Cicer
genus, and is believed to have originated in south-eastern Turkey and the adjoining
parts of Syria and north-western of Iran. Chickpea is known to be a nutraceutical (or
health benefiting food) because of its high nutritional value and near absence of
anti-nutritive components (Millan et al. 2006). Chickpea fixes substantial amounts
of atmospheric nitrogen that is available to subsequent crops and adds much needed
organic matter that improves soil health, long-term fertility and sustainability of the
ecosystems (Ahmad et al. 2005) and plays a vital role in farming systems as an
alternative to continuous cereal production or fallow.
The cultivated chickpea as the third most important food legume is grown on
11 million ha with 9.77 million tons production (FAO [Homepage on the Internet]
2010). It is grown in over 57 countries of the world in South and West Asia, North and
East Africa, southern Europe, Australia, South America, North America and Central
America (FAO [Homepage on the Internet] 2010). Despite the economic value of
chickpea and relatively long period of traditional breeding, the productivity of the crop
has not greatly improved in recent years (Taylor and Ford 2007). The world average
yield (0.88 t ha−1) is far below potential seed yield of about 5 t ha−1 (Ahmad et al. 2005;
Berger and Turner 2007) due to lack of well adapted cultivars for autumn or winter
sowing, poor farm management and susceptibility to several biotic and abiotic stresses.
In recent years, major emphasis in breeding has been to stabilize chickpea yield.
The main goal of chickpea breeding is to improve productivity by upgrading the
genetic potential of cultivars or by eliminating or reducing the effects of biotic and
abiotic stresses (Singh 1997). Since the limited diversity in the crop (Udupa et al.
1993), hybridization (genotyping based method) programs were emphasized in
many national or international research centers; however phenotyping based meth-
ods also was being adopted in many countries. Unfortunately, global climate change
is likely to increase the occurrence and severity of biotic and especially abiotic
stresses and consequently, food security in this century rely increasingly on the
release of cultivars with improved resistance/tolerance to these stresses and with
high yield stability (Collins et al. 2008). It is clear that, although in the past we have
not endeavored to adapt crop needs to climate changes and environmental stresses,
newly released cultivars should be genetically provided to improve their ability to
withstand drought, cold and other environmental constraints (Tuberosa 2010). In
this challenging status, phenotyping as a new bottleneck in plant science, along with
molecular assisted breeding, offer unprecedented opportunities for breeders.
4.2 The Importance of Phenotyping
Phenomics using high throughput phenotyping tools has played an important role
on major challenges such as sustainability of agriculture, production stability and
climate changes which have led to biotic and abiotic stresses increasing in plants
(Tadele et al. 2010). It has improved breeding efficiency by better use of genomics
4 Chickpea Phenotyping 113
data or applicability of them. Phenotyping is by nature a process which requires

choices in variables, sampled organs and times of sampling, whose relevance
depends on a breeding program (Tardieu and Schurr 2009). Variables involved in
plant phenotyping are multi-dimensional, with time constants ranging from seconds
(gas exchange) to several days (crop architecture, cell cycle).
Phenotyping has been taken into consideration for several concepts. Ecologically
it is necessary to collect information about the plant species existing into several
areas and identify their microhabitat as well as their exact location (Tsaftaris and
Noutsos 2009). By comparing the phenotypes of a plant in controlled and outdoor
conditions it is possible to confirm any differences that were identified in the field
sites, and could be gained not only directly in different environmental conditions
but also in possible plant architecture (Tsaftaris and Noutsos 2009).
The landraces of chickpea and its wild relatives hold a wealth of genes (Upadhyaya
et al. 2011), which, if use in breeding programs can be effectively increase stress resis-
tance level of agronomically superior cultivars. This emphasized the need for preser-
vation of germplasm which led to assembling and maintaining a very large number of
germplasm accessions (over 97,400) by many countries and conserving them in their
genebanks (WIEWS-FAO [Internet] 2009). However, despite the genomic studies for
major part of these materials, the lack of phenomics is completely evident.
Phenotyping technology is basis for reducing the genotype–phenotype gap,
especially for quantitative traits, which are the major determinants of many stress
resistances (Furbank and Tester 2011). A notable case that clearly emphasises the
importance of phenotyping is provided by QTL cloning (Tuberosa 2010). The status
will be ideal if the alternative QTL alleles can be clearly scored phenotypically and
the trait itself is mapped as one of the markers (Salvi and Tuberosa 2007).
Plant phenomics has assisted breeders for more than a decade, by offering cutting
edge molecular breeding technology (Morris et al. 2006) and trait-specific breeding
methods (Yin et al. 2003) for their own crop development. It offers them to accelerate
relation of complex traits to the genetic diversity by applying new technologies such
as greenhouse automation and plant imaging systems with modern digital approaches
and computing (Varaprasad and Sivaraj 2009). Advances in phenotyping are therefore
a key factor for success in modern breeding as well as for basic plant researches.
Modern phenotyping is not conceivable without the development of new
methods involving novel sensor technologies, bioinformatics as well as plant
environment modelling, able to relevantly analyse the large amount of informa-
tion originating from genotypes, transcriptomics, proteomics, metabolomics,
fluxomics (Zhang et al. 2010) and phenotypic analyses.
4.3 The Role of Phenotyping in Chickpea Breeding
For several years the phenotyping methods—mostly without using modern tech-
nologies—such as screening for economically important traits (e.g. flower and seed
color and size, growth duration, yield, and biomass, disease resistance, drought,
cold and salinity tolerance, quality traits such as cooking time, amino acid content,
flatulence and digestibility which high level of diversity has been documented
(Muehlbauer and Tullu 2012) and improved cultivars have been developed and
released. On the other hand, the amount of molecular information has increased
enormously for chickpea genomics (Muehlbauer and Rajesh 2008). As a conse-
quence, more effective and reliable phenotyping data has become a new opportu-
nity for modern genetic chickpea improvement and chickpea phenomics has
become a major field of research in its breeding. Since chickpea production has
recently been challenged by climate changes, biotic and abiotic stresses have
become important in breeding programs. As regards the applied phenotyping
methods, we have represented a number of high lighted investigations for some
important stresses in chickpea.
4.4 Phenotyping for Biotic Stresses in Chickpea
Diseases, insects, weeds and birds are the major biotic stresses affecting chickpea’s
productivity. Fungal diseases such as ascochyta blight, botrytis grey mould, alter-
naria blight and stemphylium blight, whilst soil-borne fungal diseases include fusar-
ium wilt, verticillium wilt, dry root rot, collar rot and wet root rot constrain crop
productivity (Buhariwalla et al. 2005). Singh et al. (2008) have been reported impor-
tant chickpea diseases and pests. The main diseases affecting chickpea are ascochyta
blight and fusarium wilt caused by Ascochyta rabiei and Fusarium oxysporum,
respectively. Under favourable environmental conditions, where relative humidity is
>60 % and temperature 10–20 °C, ascochyta blight has devastating destructive power
and develops in epiphytotic proportions (Nasir et al. 2000). Thus improving resis-
tance to this disease is a major aim of chickpea breeders around the world (Millan
et al. 2006). Siddique et al. (2012) were screened more than 2,000 breeding lines for
ascochyta blight resistance and 335 superior lines were introduced to Australia.
Fusarium wilt also is capable of causing total crop loss and is difficult to effec-
tively manage via crop rotation or foliage fumigation since the pathogen is soil
borne (Sharma et al. 2004). Seven distinct physiological races of Fusarium oxyspo-
rum have been identified so far, of which 1, 2, 3 and 4 are prevalent in India (Haware
and Nene 1982) and the remaining races (0, 5 and 6) are reported from Spain
(Jimenez-Diaz et al. 1989). Breeding programmes at national and international cen-
tres have resulted in the release of several chickpea cultivars resistant to fusarium
wilt but they do not show resistance across locations (Infantino et al. 1996) due to
the prevalence of location specific races of the wilt pathogen (Singh et al. 2008).
The genotypes of chickpea differ for their time to develop initial symptoms of
wilt that indicates different degrees of resistance. Nene and Haware (1980) pheno-
typically evaluated 102 accessions representing six annual wild Cicer species for
resistance to fusarium wilt. Some accessions of C. bijugum, C. judaicum and
C. reticulatum were completely free from wilt damage, and are therefore potential
source of genes for fusarium wilt resistance that could be transferred to the culti-
vated species.
Cyst nematode (Heterodera ciceri) is another major biotic stress to chickpea.

Di Vito et al. (1996) phenotyped 7,258 lines of C. arietinum and 102 lines of
wild Cicer species for resistance to cyst nematode by screening on 0–5 scale.
Only 1 % of the lines of cultivated species showed resistance with a rating of 2,
which clearly indicates lack of adequate resistance to cyst nematode in the
cultivated species.
Leaf miner (Liriomyza cicerina) causes considerable damage to chickpea. Seven
thousand germplasm accessions of cultivated species and 200 accessions of 8 wild
annual species have been evaluated phenotypically by Singh and Weigand (1994).
The germplasm of cultivated species lack adequate resistance and only ten lines
with moderate levels of resistance have been identified. In case of wild species, two
accessions of C. cuneatum and ten of C. judaicum have exhibited resistance with a
rating of 2 (highly resistant) and some accessions of C. judaicum, C. pinnatifidum
and C. reticulatum were identified with a rating of 3 (resistant).
Bruchids (Callosobruchus chinensis) are the important insect pests of chickpea
in the Mediterranean region. Singh et al. (1998) evaluated germplasm accessions
of cultivated and annual wild species for resistance to leaf miner and bruchids.
All the accessions of C. chorassanicum, C. cuneatum and some accessions from
C. judaicum, C. bijugum, C. pinnatifidum and C. echinospermum showed resis-
tance to leaf miner.
Pod borer (Helicoverpa armigera) is the most destructive and attacks chickpea
plants, pods and seeds (Indian Institute of Pulses Research [Homepage on the
Internet] 2010). An integrated breeding approach has been adopted for pod borer
control in chickpea involving both non-preference/antibiosis and avoidance (Singh
et al. 2008). Chickpea cultivars and wild Cicer species have been found to differ
significantly in their ability to inhibit Helicoverpa armigera gut proteinases
(Patankar et al. 1999). Gene technology is now the excellent path for the genetic
improvement of chickpeas (Chopra and Kamma 2011) especially for pod borer
resistance. There are at least four closely related transformation protocols from
which to choose and all indications are that the systems are robust and reproducible.
It is possible to predict that genes for protection against major insect pests, such as
pod borer, will soon be installed in chickpea germplasm for use in varieties adapted
to local conditions around the world (Popelka and Higgins 2007).
Most breeding programs for ascochyta blight resistance rely on field screening to
identify the most resistant selections. These are then evaluated for other agronomic
and quality traits and the resistance to blight reevaluated and confirmed. In these
circumstances phenotype will depend upon the presence or absence of resistance
gene (Abdel-Monaim 2011), the pathogene, a uniform level of the disease and care-
ful measurement. Therefore in the following cases we will get less than satisfactory
results: insufficient disease pressure to distinguish resistant and susceptible lines
(Scott et al. 2012); non-uniform disease inoculation (Baranger et al. 2006); non-
uniform environment (Baranger et al. 2006) for disease development (e.g. humidity)
and errors in labeling, data recording, analysis, etc. All of these factors will result in
the non-genotype factors. The factors affecting the phenotype having a larger role in
the results than do genotypic factors (Allard 1999) with the consequence that it will
be difficult to detect resistance QTL, even though the resistance gene is present in
the population. However to overcome on weaknesses of these type of phenotyping,
use of modern approaches of digital phenotyping is too appropriate.
4.5 Phenotyping for Abiotic Stresses
The important challenges facing modern agriculture are the complex field environ-
ment with its heterogeneous conditions led to abiotic stresses (Mittler and Blumwald
2010) and global climatic changes. A combination of approaches will be needed
(genotyping and phenotyping) to accurately identify tolerant plants for resistance/
tolerance to abiotic stresses such as drought, cold and freezing. Currently environ-
ment simulation facilities that constrain firmly controlled spatially and temporally
(either stable or fluctuating) environmental conditions (Walter et al. 2009) are cen-
tral for phenotyping designs with predictive value for field performance. Such simu-
lation systems are also necessary to design experiments (Faller et al. 2003)
addressing the capacity of crops to acquire and utilise resources such as nutrients,
water or light that are by nature not homogeneously distributed or only available in
temporally and distinct patterns (Birch et al. 2003).
Although chickpea is sensitive to salinity at both the vegetative and reproductive
phase (Samineni et al. 2011) drought and cold are the major constraints to its pro-
ductivity worldwide (Croser et al. 2003; Stoddard et al. 2006). Some production
losses are due to intermittent drought during the vegetative phase while others are
due to terminal drought during reproductive development (Serraj et al. 2004). The
main strategies and related traits to cope with drought are: (a) drought escape (com-
plete growth cycle before drought is too severe) and related traits are early maturity;
reduced period after flowering; (b) drought avoidance (get more water or use less)
and related traits are deep root and transpiration efficiency; (c) drought tolerance
(maintains functioning under drought) and related trait is osmotic adjustment
(Bhatnagar-Mathur et al. 2010).
It is difficult to develop phenotypic screens for intermittent drought tolerance
since the timing and intensity of this type of drought are fairly unpredictable,
whereas screening for terminal drought has been successful in many crops
(Upadhyaya et al. 2010). Nevertheless, it should be noted that drought hardly ever
occurs in the absence of other stress factors. An example of this is provided by the
conditions of terminal drought stress frequently experienced during grain filling
(Cattivelli et al. 2008) wheat, barley and chickpea grown in West Mediterranean
environments as a result of high temperatures. A partial solution to this problem, at
least for traits other than grain yield and its components, which are best evaluated
under field testing, is to collect phenotypic data (Witt et al. 2012) from plants grown
in controlled conditions (greenhouse, growth chamber etc.). This will allow for an
accurate control of the main environmental parameters—temperature, air humidity,
light, etc.—governing water flow in the space, and hence the water balance. It is
particularly important for omics-profiling studies (Collins et al. 2008) where even
small fluctuations in environmental conditions can considerably change gene

expression. On a broader scale, environmental characterisation can be improved
through the use of geographic information systems (GIS) for crop monitoring
(Sivakumar et al. 2004), for water balance models (Padilla et al. 2011) and for their
combination.
Terminal drought is a usual feature in semi-arid tropics where chickpea is grown
in the post-rainy season on progressively receding soil moisture conditions (Siddique
et al. 2000). Zaman-Allah et al. (2011) tested whether plant water use at the
vegetative stage and under non-limited water conditions could relate to the degree
of sensitivity of chickpea to terminal drought. Transpiration response to a range of
vapour pressure deficiencies under controlled and field conditions was measured
with canopy conductance using gravimetric measurements and thermal imagery in
eight chickpea genotypes with comparable phenology and contrasting seed yield
under terminal drought.
Lichitenzveiz et al. (2006) used an intraspecific cross of kabuli and desi cultivars
and mapped two quantitative trait loci (QTLs) responsible for days to flowering.
This work may help in identifying genes responsible for early flowering (Kamoshita
et al. 2008) which may affect developing varieties to escape terminal drought. QTL
studies need large number of individuals is genotyped and phenotyped for the quan-
titative trait of interest (Sen et al. 2009). Since this can be a costly attempt, breeders
employ cost-saving methods such as selective genotyping, in which a selected por-
tion of the phenotyped individuals are genotyped (Sen et al. 2009).
Phenotyping for every stress needs a standardized and repeatable protocol (Beebe
et al. 2011) that lays out the following objectives: identification of the phenotype/
trait and measuring type and tools; definition of the experimental design and logis-
tics (sampling dates; replication; material to be used; variable and co-variables) and
growth conditions (soil and air temperature, T°C–photoperiod, etc., treatment to be
applied and maintenance conditions). In environments where escape is the main
cause of drought tolerance (Hussain 2006), the presence of large differences in
number of days to 50 % flowering in chickpea genotypes will bias the interpretation
of the influence of drought-adaptive traits on yield under stress conditions. In a
similar manner, the existence of large differences in plant height and/or root mass
among the progeny of a mapping population or accessions, may lead to an over
estimate of the effects of relevant QTLs owing to competition between neighbour-
ing plots, especially when their surface area is small (Tuberosa 2012). Such QTL
effects will most likely reduce once phenotypic evaluation has been carried out with
more phenologically homogeneous factors (Tuberosa 2012).
In the Mediterranean climates (in parts of Western and South Australia, sections
of Central Asia) and West Asia, chickpea is usually sown in spring. Studies on cold
tolerance in chickpea were initiated when it was realized that yields can be consid-
erably improved by shifting sowing time from traditional spring to winter in the
Mediterranean environments (Millan et al. 2006). Cicer species and cultivars vary
in their genetic and phenotypical response to cold stress (Croser et al. 2003). This is
probably due to close association of plant response to low temperatures, phenologi-
cal stage of the plant effect, the preceding temperature regimes, and prevailing
environmental conditions (Croser et al. 2003). Chickpea seedlings are protected by

the buffering role of soil during germination (Wery 1990). Increasing in frost
resistance after seedling emergence could be explained by the hardening process-
lowering of cells’ freezing point by accumulation of organic (mainly saccharose and
glucose) and mineral compounds (Toker et al. 2007). Chickpea suffers from cold
stress (<10 °C) especially during reproductive phase resulting in flower and pod the
abortion, poor pod set (Croser et al. 2003), and consequently reduced seed yield and
seed quality (Mittler and Blumwald 2010). It is important in the development of
cold-tolerant lines to identify genetic variation for cold tolerance through simple
available screening methods (Saxena 1993). As a first step Singh and Singh (1989)
screened 3,158 germplasm accessions of kabuli chickpea at a high elevation plateau
site in Turkey and identified cold tolerant genotypes. According to Singh et al.
(1995) more than 10,000 germplasm and breeding lines were phenotyped and addi-
tional sources of tolerance identified. Saeed et al. (2010) screening a mini core
germplasm collection comprising 225 chickpea lines under controlled conditions
and field experiments, identified 13 cold tolerant lines.
In recent decades crossing programs have been carried out based on phenotyping
results in most research centers, however all the progenies usually were not success-
ful (tolerance to stresses especially for cold). That was due to lack information
about genetic distance between lines used in crosses. It is clear that more the genetic
distance more diversity and high variation advantages are available (Berilli et al.
2011). Information about current genetic diversity permits the classification of
available germplasm into heterotic groups, which is particularly important to hybrid
breeding programs in chickpea. It is well documented that crosses between unre-
lated, and consequently genetically distant parents, show greater hybrid vigor than
crosses between closely related parents (Stuber 1994).
Wild relatives of Cicer species are a promising source of genes for stresses toler-
ance. For example, in cold highland areas cultivars suitable for winter or autumn
sowing could be achieved by the use of genes for cold tolerance from the C. reticu-
latum (Toker 2005). Complementary studies using molecular markers with assis-
tance new phenotyping tools should be undertaken to identify genes controlling
frost tolerance of good donor parent as well as wild spp. accessions in marker
assisted selection (Saeed et al. 2010).
Collecting a reliable record of meteorological parameters (rainfall, tempera-
ture, wind, evapotranspiration, light intensity, etc.) allows for more meaningful
interpretation of the results and identification of the environmental factors limit-
ing yield (Ozkan and Akcaoz 2002). It has been revealed that because of inter-
twining of genetic and environmental factors, biased observations and complex
interactions, pervious classic screening trials for different stresses have not been
(always) straightforward. It has faced with some logistical and statistical prob-
lems, which have caused difficulties in breeding workflows and hence low reli-
ability. Keeping in mind these weaknesses of phenotyping methods, the importance
of phenomics as modern phenotyping by using novel approaches to get reliable
results is characterized.
4.6 Management of Phenotyping Data
The phenotyping technology is a reliable tool for collection of accurate data

(Vankadavath et al. 2009) to minimise the experimental errors introduced by uncon-
trolled, multivariate environmental and experimental variability. Investigating the
effects of stresses on crops when difficulties arise in standardising, controlling and
monitoring the environmental conditions under which plants are grown and the data
are collected, especially in the field phenotyping is found as a critical approach.
A major challenge for the crop and field phenotyping systems is the vast array of data
types collected from a variety of sensors, both imaging and radiometric and geno-
typic information (Furbank et al. 2009). Statistically the phenotype (what we mea-
sure) depends upon: (a) the genotype of the plant (G); (b) the effect of environment
in which the plant is grown (E); (c) possible interactions between the genotype and
the environment (G × E); and (d) errors on our measurements (δ). A successful phe-
notyping needs to maximize the ratio of the genotypic to phenotypic variance because
the differences among phenotypes reflect the differences in their genotypes.
The gap between genes and phenotypes is particularly large in analyses of plant
environment interaction (Allard and Bradshaw 2005), urgently needed for research
on chickpea production in the context of climate change. Novel genetic methods in
chickpea have mostly been applied on relatively simple phenotypic traits such as
flowering time, which do not require complex phenotypic approaches (Tardieu and
Schurr 2009). The use of the same genetic methods in analyses of tolerance to biotic
and abiotic stresses requires the analysis of defined genetic material in multiple well
characterized environmental scenarios.
The success of field trials, their management and interpretation of phenotypic
data can be increased greatly through the application of proper experimental designs
to allow for better control of within-replicate variability (Bänziger et al. 2000) and
to reduce or eliminate spatial trends. Equally important are statistical methods for
analysing the data, especially when the effects of G × E interactions and epistasis are
considered (Ceccarelli 1996).
Encountering with the provisional variability of drought/cold-adaptive features can
be associated with through in-depth analysis of QTL × E interactions (Chen et al. 2010).
This is also possible by recognizing inherent traits of each genotype relating to its inter-
action with particular environmental conditions. All these need the development of
models able to identify such variables and to simulate the behavior of genotypes in any
environmental conditions (Collins et al. 2008). Models with Predictive ability have
been created for phenotypic variation in multiple traits (Sun et al. 2010) which consider
a range of environmental conditions, complex traits and developmental time. They use
molecular marker techniques (DNA polymorphisms), environmental data, genomic
information (gene expression, metabolites, proteins …) and image analysis by fluores-
cence measurements as input data. The philosophy of these modeling is analyzing of
complex traits (yield) with context dependent variation into component traits that do
not show strong context dependencies which can be integrated with environmental
information over the growing season to produce yield (Peccoud et al. 2004).
Key plant functions, like cell division, expansive growth, gas exchange, water
and mineral status/transport and the role of plant architecture at the above- and
below-ground need to be quantified during the dynamic responses of plants to
environmental conditions throughout their development (Tardieu and Schurr
2009). Innovative technologies and novel approaches—often based on non- or
minimal-invasive techniques—become increasingly available. These allow
quantitative analysis of the spatially and temporally developing processes and
structures at a mechanistic level, providing the basis for understanding the
dynamic interactions between genetic and biochemical processes with physio-
logical phenotypes. They also potentially allow genetic analyses of structural
and functional traits at high throughput, not achievable until recently (Tardieu
and Schurr 2009).
Nowadays, breeders use genotype-to-phenotype models (Messina et al. 2011)
which have relatively simple genetic basis and few additive genes/QTLs. These
models predict the phenotypic performance of the plant on the basis of genetic,
genotypic, genomic and environmental information and help in defining strategies
for improvement of crop performance (Peccoud et al. 2004). In chickpea breeding
programs by using digital imaging tools externally provided phenotype data should
be combined with in-house generated genotype data to maximise phenotyping
potential. It is notable that a more powerful genetic analysis will be achieved just by
phenotyping at the right moment.
4.7 Powerful Breeding with Phenotyping–Genotyping
Molecular breeding has revolutionized conventional breeding techniques in all

areas within the last 20 years (Carmen de Vicente 2004). Molecular breeding has
played significant role in chickpea breeding programs by way of integrating appro-
priate marker assisted selection (MAS) technique with empirical selection
(Chaturvedi and Nadarajan 2010). The availability of molecular data, based on
pedigrees and phenotypic evaluation, now makes breeding analysis a reliable real-
ity. The development of molecular breeding in chickpea offers substantial opportu-
nities for enhancing the effectiveness of classical breeding programmes. With this
molecular breeding (also called molecular assisted breeding, MAB), the transfer of
multiple genes of interest and genomic regions (quantitative trait loci, QTL)
involved in polygenic traits, has been successfully used in many crops in the
developed countries, but more rarely in developing countries (Stafford 2009). These
techniques have shortened the duration of breeding programs. It has also improved
the precision of crosses in chickpea and allowed breeders to produce strains with
combined traits that were impossible before the application of DNA technology
(Stuber et al. 1999).
In recent years in chickpea breeding use of biochemical and molecular markers
has been usual in breeding programs (Kumar et al. 2011). However, morphological
markers (Khan et al. 2011) are used in many experiments. Biochemical markers
are superior to morphological markers in that they are generally independent of

environmental growth conditions. Isozymes also have been tested in chickpea
(Labdi et al. 1996). There was a low level of polymorphism detected in cultivated
chickpea using isozyme/allozyme markers (Gaur and Slinkard 1990). The main
problem of isozymes is the lack of polymorphism required for the marker assisted
selection (MAS), especially among closely related cultivars. This may identify
variation in the form of allozymes at a particular isozyme locus. That variation can
then be used if it can be shown to be linked to a gene of interest. The fact that there
is minimal polymorphism at most isozyme loci makes isozymes not particularly
useful for breeding.
For many benefits molecular marker systems have been used in chickpea
including restriction fragment length polymorphism (RFLP), random amplified
polymorphic DNA markers (RAPDs), amplified fragment length polymorphisms
(AFLPs), sequence characterized amplified regions (SCARs), inter-simple
sequence repeat (ISSRs), simple sequence repeat (STMS or SSR), resistance
gene analogs (RGAs), DNA amplification fingerprinting (DAF), and expressed
sequence tags (ESTs). Molecular methods such as RAPDs, AFLPs, SSRs and
microsatellites used for detecting DNA sequence variation are being used as
complementary strategies to traditional approaches for assessment of genetic
diversity (Karp 2002).
Molecular markers are not influenced by developmental or environmental fac-
tors, they are relatively easy to detect, often abundant throughout the genome even
in highly bred cultivars, and can be detected at virtually any stage of plant develop-
ment. It is fortunate that the wild species relatives of chickpea are available for
breeding purposes. Techniques of gene transfer using molecular markers should
allow breeders to use this genetic resource with the expectation of obtaining useful
recombinants within a reasonable period of time (Muehlbauer et al. 2004). Molecular
marker systems, by their very nature, offer direct examination of the introgressed
genotype at any given locus. Loci under examination with these systems may or
may not have an overt phenotypic effect, but may offer a subtle reversion of plant
type toward the wild phenotype due to the infusion of wild species DNA (Muehlbauer
et al. 2004). Molecular markers are most useful in the context of a well developed
genetic linkage map. Such maps have been developed for chickpea (Pfaff and Kahl
2003; Rajesh et al. 2008) particularly for drought tolerance (Jain and Chattopadhyay
2010), double podding and other morphological traits (Upadhyaya et al. 2011) and
tolerance to fusarium wilt (Sharma et al. 2005).
All the molecular tools can be integrated into breeding programmes in order to
be able to efficiently analyse high numbers of crosses at the early seedling stage
(Kulwal et al. 2012). Through this approach, both the phenotype and the genotype
of new varieties can be analysed and the performance of new specific introgressed
traits can be predicted. This is known as genotype-to-phenotype mapping (Messina
et al. 2011). The goals of this integration of technologies in classical breeding are to
create genotype-to-phenotype trait knowledge for breeding objectives and to use
this knowledge in product development (http://www.orgsyngentafoundation./index.
cfm?pageID=652). Saeed et al. (2011) in a study confirmed the importance of
molecular studies beside of phenotypical ones in detecting genetic variation among

genotypes that will provide suitable diverse parents for carrying out a new crossing
programme. They found relatively acceptable genetic diversity within available
C. arietinum accessions by using SSR markers.
The effectiveness of marker-based approaches intimately depends on how well
and how accurately the target trait has been assessed phenotypically in mapping
populations. In fact, low heritability impairs the probability of detecting the
presence of QTLs (Bernardo 2004), thereby increasing Type II errors (i.e., false
negatives). An accurate and relevant phenotyping is of even greater importance
when applying genomewide selection, an approach that disregards QTL identifi-
cation and relies on the molecular profiling and accurate phenotyping of each
progeny (Bernardo 2008; Heffner et al. 2010). Ultimately the accuracy and preci-
sion of phenotyping determines how realistic the QTL mapping results are
(Semagn et al. 2010).
New phenotyping technologies can be developed and accomplished into
phenomics methods. These technologies often provide images (e.g. multi-spectral,
infrared, etc.) allowing the analysis of spatial heterogeneity (Eberhardt and Teal
2009) and thus addressing the inherent local and modular organisation of crops.
Further developments include tomographic methods (e.g. X-ray computer tomogra-
phy (CT), magnetic resonance imaging (MRI) or positron emission tomography
(PET) that literally provide insight into plant organs and non-invasive analysis of
below-ground structures and functions and thus the basis for in-depth phenotyping
(Tardieu and Schurr 2009). The development of environmental sensors with good
spatial definition and accuracy (Kotamäki et al. 2009) allow monitoring of the rel-
evant environmental conditions. High throughput phenotyping platforms which are
used in controlled conditions, able to accept several hundred up to several thousand
of plants in a single run in controlled or tightly monitored environmental conditions
and allowing high precision phenotypic measurements. These platforms are most
often dedicated to a limited number of applications, compatible with their technical
choices, namely phenotypic target (e.g. plant architecture, photosynthesis, vegeta-
tive growth), species (plant size/architecture, monocots vs. dicots) and type of envi-
ronmental cues (biotic, water, nutrients) (Tardieu and Schurr 2009).
Furthermore field phenotyping platforms also able to follow growth, gas
exchange and status of canopies with large number of plants and genotypes, by
using proxy detection (imaging or vegetation indices) with sensors placed on trac-
tors, robots or flying platforms (Montes et al. 2007). These are combined with
accurate environmental monitoring and can be linked to devices able to manipu-
late environmental conditions (e.g. rain out shelters, free-air carbon dioxide
enrichment (FACE), heating devices, etc.). However the identification of the most
essential objectives in chickpea phenotyping in terms of biological questions,
sensors and methods, the development and implementation of innovative pheno-
typing concepts that address the identified objectives and the development of new
methods are needed.
4.8 Opportunities for Marker Assisted Breeding (MAB)

in Chickpea in the Immediate Future
Marker assisted breeding in chickpea can be developed from phenotyping technol-

ogy in the immediate future as the following:
– Relieve phenotyping bottleneck with robotics, noninvasive imaging and analysis
using powerful computing.
– Possibility of providing “whole of life cycle”, quantitative measurements of
plant performance from the growth cabinet to the field.
– Delivering genomic advances to phenomics for modeling objectives—e.g. model
systems.
– Accelerate time from gene discovery to trait discovery and release of innovative
new varieties.
Therefore breeders should assist modern phenotyping for functional analysis of
specific genes, forward and reverse genetic analysis and production of new varieties
with beneficial characteristics. By this they will be able to manage trials in different
growth conditions with using many different lines in mutant populations, mapping
populations, breeding populations and germplasm collections. It is notable that new
improved chickpea phenotypes can be created through genetic improvement
(QTLs). Which the detection of these QTLs requires high quality molecular mark-
ers and high throughput phenotyping. Nevertheless high throughput and accurate
phenotyping, modeling and collaboration with plant breeders remain important
challenge particularly when defining ideal characteristics for chickpea and target
environments.
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Chapter 5
Phenotyping for Groundnut (Arachis
hypogaea L.) Improvement
Janila Pasupuleti and S.N. Nigam
Abstract Groundnut (Arachis hypogaea L.) is grown world over for oil and food
uses. It is a self-pollinated crop with low genetic diversity. The origin of the crop
from single hybridization event followed by chromosome doubling as well as cross-
ing barriers of cultivated species with wild species due to ploidy differences ren-
dered the crop with narrow genetic variability. Developing new varieties with
increased yield potential and resistance to biotic and abiotic stresses that meet the
needs of the growers, processors and consumers is the primary objective of ground-
nut breeding. In this chapter, we discuss about phenotyping tools used in groundnut
improvement programs for various targeted traits. Both field and laboratory tools
are described to screen for resistance to diseases caused by fungi, bacteria, virus and
nematodes. Phenotyping based on Cumulative Thermal Time (CTT) is used to
select for early maturity. Phenotyping for complex traits can be challenging. Either
empirical approach that involves measuring the yield under imposed drought stress
or salinity conditions or trait based approach using surrogates or a combination of
both are used for phenotyping abiotic stresses. Phenotyping for Aspergillus con-
tamination needs improvement to derive reliable and reproducible results. Estimation
of quality and nutritional parameters generally involves use of destructive and labo-
rious chemical or physical procedures. Near infrared reflectance spectroscopy
(NIRS), a robust and non-destructive method is gaining popularity for estimation of
oil, protein, carbohydrate and fatty acid contents. Methods for estimating oil, pro-
tein, sugar and micronutrient concentrations and fatty acid composition of seeds and
haulm quality traits are described.
Keywords Groundnut • Phenotyping • Screening • Yield • Resistance • Disease •

Drought • Quality
J. Pasupuleti (*) • S.N. Nigam

International Crops Research Institute for the Semi-Arid Tropics,
Patancheru 502324, Andhra Pradesh, India
e-mail: p.janila@cgiar.org
130 J. Pasupuleti and S.N. Nigam
5.1 Introduction
5.1.1 Global Importance of Groundnuts
Groundnut, also known as peanut, was grown on nearly 24.07 million ha worldwide
with a total production of 37.64 million tons and an average dry pod yield of
1,564 kg ha−1 in 2010 (FAOSTAT 2012). China, India, Nigeria, USA, Senegal,
Myanmar, Indonesia, Sudan (undivided), Argentina, Ghana and Vietnam are the
major groundnut growing countries in descending order and totally account for
84 % of the world groundnut production. Countries in Asia, Africa and South
America account for over 97 % of the world groundnut area and about 95 % of the
world groundnut production. Production is concentrated in Asia (50 % of the global
groundnut area and 64 % of the global groundnut production) and Africa (46 % of
the global groundnut area and 28 % of the global groundnut production), where the
crop is grown mostly by smallholder farmers under rainfed conditions with limited
or no inputs. In the last decade (2000–2010), the global groundnut production
increased marginally. The annual increase in production was 0.4 % which was due
to both, an annual increase in yield (0.1 %) and area (0.3 %) (Fig. 5.1). The increase
in global production of groundnut, contributed by increase in yield is a result of
adoption of improved varieties and/or better crop management practices. In India, it
is reported that improved varieties alone contributed to 30 % increase in pod yield
over two decades (Reddy and Basu 1989).
European Union is the major importer of groundnut oil and groundnuts with and
without shells; the value of imports in 2009 was about 254 million USD (FAOSTAT
2012). During the same year, Argentina stood first in groundnut oil exporting
Fig. 5.1 Three-year moving center average for groundnut yield, production and area harvested in
world
5 Phenotyping for Groundnut (Arachis hypogaea L.) Improvement 131
countries with a value of 84 million USD, while India was the top exporter of shelled
groundnut (of value 28 million USD) and China of groundnut with shells (of value
53 million USD) (FAOSTAT 2012).
Groundnut seed can be consumed raw (non-heated), boiled, and roasted or
crushed for edible oil. Its haulms are used as animal feed and shells that constitute
about 25 % of the total pod mass are used as fuel, as filler in the feed and fertilizer
industries and in manufacture of particle boards etc. Globally, over 50 % of the
groundnut produced is crushed for extraction of oil for human consumption and
industrial uses and slightly less than 40 % is used directly as food, raw or processed
as snack (Birthal et al. 2010). The cake obtained after extraction of oil is used in
animal feed industry, in making enriched easily digestible food for children and
aged persons and as soil amendment. In USA about 75 % of the production is used
as food, while in Asia only 35 % is used for food purposes. Groundnut oil is an
excellent cooking medium because of its high smoking point (Singh and Diwakar
1993). In USA, Canada, and Australia, groundnut is grown to make peanut butter
rather than to extract oil. Groundnut is also used to make confectioneries and its
flour to make baked products.
Groundnuts seeds are rich in energy due to high oil (48–50 %) and protein con-
tent (25–28 %); they provide 564 K calories of energy from 100 g of kernels
(Jambunathan 1991). The seeds contain many health enhancing nutrients, minerals,
antioxidants and vitamins and are rich in mono-unsaturated fatty acids. They con-
tain antioxidants like p-coumaric acid and resveratrol and are excellent source of
vitamin E and many important B-complex groups of thiamin, pantothenic acid, vita-
min B-6, folates and niacin. Groundnut is a dietary source of biologically active
polyphenols such as the stilbene trans-resveratrol (Sobolev and Cole 1999), flavo-
noids (Wang et al. 2008) and isoflavones. Groundnut haulms constitute nutritious
fodder for livestock. They contain protein (8–15 %), lipids (1–3 %), minerals
(9–17 %), and carbohydrate (38–45 %) at levels higher than cereal fodder. The
digestibility of nutrients in groundnut haulm is around 53 % and that of crude pro-
tein is 88 % in animals. Haulms release energy up to 2,337 cal kg−1 of dry matter.
5.1.2 Taxonomy and Classification
Groundnut (Arachis hypogaea L.) is an annual herb belonging to the family

Fabaceae (Leguminosae). It is an allotetraploid (2n = 2x = 40) with “A” and “B”
genomes. All species, except the cultivated species (A. hypogaea) and A. monticola
in Section Arachis, and certain species in Section Rhizomatosae, are diploid
(2n = 2x = 20). The diploid progentors, A. duranensis and A. ipaensis contributed
“A” and “B” genomes, respectively, to the cultivated groundnut (Kochert et al.
1996). A single hybridization event between the diploid progenitors followed by
chromosome doubling about 3,500 years ago lead to origin of cultivated groundnut.
The “A” genome set of chromosomes are relatively smaller than the “B” genome.
Southern Bolivia and Northern Argentina are thought to be center of origin of this
crop (Gregory et al. 1980; Kochert et al. 1996). The center of diversity of the genus
includes Western Brazil, Bolivia, Paraguay and Northern Argentina (Gregory et al.
1980). A. duranensis occurs throughout the region, while A. ipaensis has only been
found in Southern Bolivia. The genetic diversity of the genus is classified into four
gene pools (Singh and Simpson 1994): primary genepool consisting of A. hypogaea
and A. monticola, secondary consisting of diploid species from Section Arachis that
are cross compatible with A. hypogaea, tertiary consisting of species of the Section
Procumbentes that are weakly cross-compatible with A. hypogaea and fourth gene-
pool consisting of the remaining wild Arachis species classified into seven other
sections.
The cultivated groundnuts are classified into two subspecies, subsp. fastigiata
Waldron and subsp. hypogaea Krap. et Rig. The subsp. fastigiata contains four
botanical varieties, var. vulgaris, var. fastigiata, var. peruviana and var. aequatori-
ana. The subsp. hypogaea contains two varieties, var. hypogaea and var. hirsuta.
Each of these botanical types has different plant, pod and seed characteristics
(Krapovickas and Gregory 1994). The A. hypogaea subsp. hypogaea has alternate
pairs of vegetative and reproductive axes on branches (alternate branching) and
does not bear flowers on the main axis, inflorescence is simple, generally has two
seeds per pod, with moderate seed dormancy, seed coat is generally tan in colour
and medium to late maturing. In var. hypogaea, cultivars with medium seed size are
runner market type and those with large seeds are Virginia market type. In contrast,
A. hypogaea subsp. fastigiata var. vulgaris (Spanish market type) has floral axes on
main stem, irregular pattern of vegetative and reproductive branches with reproduc-
tive branches predominating on branches (sequential branching), inflorescence
compound, mostly two seeds per pod and with little or no dormancy. The A. hypo-
gaea subsp. fastigiata var. fastigaita (Valencia market type) has floral axes on main
stem, sequential branching, inflorescence usually simple, two or four seeds per pod
and little or no seed dormancy.
As a consequence of crosses made between different botanical types in the
course of breeding new improved groundnut varieties, several intermediate types
having the specific traits of more than one botanical type are now under cultivation
across the world. Hybridization between two botanical types can break linkages
between the traits, which have otherwise co-segregated for over centuries to form
distinct botanical types.
5.2 Breeding Methodologies
5.2.1 Mode of Reproduction and Artificial Hybridization
Groundnut is a self-pollinated crop, but natural hybridization can occur where

bee activity is high (Nigam et al. 1983). Flowering begins 17–35 days after seed-
ling emergence depending on the cultivar and environmental conditions. In var.
hypogaea and var. fastigiata, the inflorescence is simple and that of var. vulgaris
is compound. Flowers are born in the axils of the leaves and never at the same
node as vegetative branch. One or more flowers may be present at a node. The
flower consists of five petals, ten monadelphous stamens, two of which are not
fully developed while the other eight are fertile, and a pistil. Among the eight
fertile anthers, four are globose and the other four are oblong type. The pistil
consists of ovary, style and stigma. The ovary contains a single sessile carpel and
one to six ovules. The style is glabrous throughout its length and covered with
bristles near the club-shaped stigma. The stigma becomes receptive to pollen
about 24-h before anthesis and remains so for about 12 h after anthesis, and the
dehiscence of anthers takes place 2–3 h prior to opening of the flower in the
morning. Fertilization occurs about 6 h after pollination. In a week after fertiliza-
tion, the peg or gynophore carrying the ovary and fertilized ovule grows and
enters the soil where the pods develop. The tip orients itself horizontally away
from tap root (diageotropic).
Emasculation of groundnut can be accomplished on warm bright days between
afternoon and evening. A well-developed bud is selected for emasculation, and all
the other buds at the node are removed mechanically. The selected bud is carefully
opened and the anthers are fully removed. A small coloured thread is tied on the
node of the emasculated flower for identification at the time of pollination next
morning. A healthy flower from the male parent (pollen source) is plucked and the
pollen is gently squeezed on to the stigma of the emasculated flower or, alternately,
the pollen is squeezed on to forceps, and then transferred to the stigma of emascu-
lated flower. The maximum physiological development of pollen is in the early
hours of the day. It was observed that pollen remained viable up to 8 days when
stored in a sealed desiccator with calcium chloride in a refrigerator at 6°C.
Hybridizing groundnuts in the greenhouse may result in over 70 % success, higher
than that obtained in field. The procedure for hybridization in groundnut has been
described in detail by Nigam et al. (1983).
5.2.2 Breeding Methodologies
Several reviews describing breeding methodologies in groundnut have been pub-

lished (Wynne and Gregory 1981; Isleib et al. 1994; Knauft and Wynne 1995) and
a large number of cultivars following these methodologies have been released across
the world that not only have high yield potential but possess resistance/tolerance to
biotic and abiotic stresses and have improved quality traits (for oil and food uses).
ICRISAT has contributed to the release of 138 groundnut cultivars between 1986
and 2010 through its partners in National Agricultural Research System (NARS) in
36 countries of Asia and Africa. The breeding methods used for self-pollinated
crops are applied in groundnut breeding. They include mass selection, pedigree,
bulk, single seed descent and back-cross methods.
5.2.2.1 Introduction and Mass Selection
Like in any other crop, at initial stages of a breeding program, introductions played
an important role in groundnut also as they are either directly used as cultivars or
Mass-selection is practiced in introduced genotypes to develop a new cultivar. JL
24, a popular short-duration groundnut cultivar in India, is a classic example for
selection made in the material, EC 94943, introduced from Taiwan. The selection,
made at the Oilseeds Research Station, Jalgaon, Maharashtra, was released as JL 24
(Phule Pragati) in 1979 for cultivation in India (Patil et al. 1980). Subsequently, it
was introduced into Africa and was released in several countries there.
5.2.2.2 Hybridization and Handling of Segregating Populations
To combine traits from different parents in improved groundnut cultivars, the par-
ents, selected for desirable traits are hybridized followed by selections for desirable
trait combinations in segregating populations. Single crosses, three-way crosses,
and double crosses are also used to derive segregating populations. Multiple cross-
ing systems, such as the convergent cross, to create adequate genotypic variability
before selection (Wynne and Gregory 1981) were also used. In groundnut, pedigree
and bulk-pedigree methods of breeding are most frequently used to handle segregat-
ing populations derived from hybridization. Confirming the hybridity of F1 plants
based on the morphological characteristics and pod and kernel features is important
in groundnut and it is done by growing male and female parents along with F1’s.
Since seed multiplication rate is low in groundnut (1:10), it is advisable to make
large number of pollinations to get sufficient number of hybrid seeds to generate
large enough F2 population. In pedigree method individual plants are selected in F2
population and F3 progeny rows are grown in the next season. Selection of single
plants is continued in F3 and F4 progeny rows. More than one individual plant is
selected and bulked from best progenies in F5 generation and repeated in F6 genera-
tion. The F7 generation is advanced to either preliminary yield trials or seed increase,
if sufficient seeds are not available for including in trials. Bulk-pedigree method,
aimed at improving traits with low heritability, is a modified method of bulk method
in which individual plants of F2 are harvested in bulk up to F4 generation and then
single plant selections are made and subsequent generations are handled as in pedi-
gree method (Wynne and Gregory 1981).
Another modified bulk method of selection is using single-seed descent method.
The main advantage of this procedure is that the characters with low heritability can
be improved as the genetic variance for these traits is maintained. Single seed decent
method is becoming popular as this has the advantage to save space and resources
(Isleib et al. 1994). In single-seed decent method, one or two seed from each plant
of F2 and F3 are advanced and in F4 generation single plant selection is done and
raised as individual plant progenies in F5 generation. The handling of material from
here is similar to that of pedigree method. In the recent times, with the advent of
molecular markers linked to the traits of interest and QTL identification and
mapping, backcrossing is used frequently in breeding programs. In fact, Marker

Assisted Backcrossing (MABC) is the most frequently used method of breeding in
groundnut to transfer a desired allele or QTL into the target genotype (recurrent
parent) (Chu et al. 2011).
5.2.2.3 Sources of Variability
The cultivated accessions of Arachis in gene banks across the world and the
advanced breeding lines available with the breeder are often used as parents in
breeding programs and hence serve as important sources of variability. Induced
mutants and interspecific derivatives are other important sources of variability. The
gene banks are also the repositories of wild Arachis species and interspecific deriva-
tives. The gene bank at ICRISAT, India has the largest collection of groundnut
genetic resources that include 14,310 accessions of Arachis hypogaea and 413
accessions of wild Arachis species (Upadhyaya et al. 2001). The other large collec-
tions are available at United States Department of Agriculture (Holbrook 2001),
Texas A&M, North Carolina State University, National Center of Genetic Resources
(CENARGEN) in Brazil (Holbrook and Stalker 2002), National Bureau of Plant
Genetic Resources (NBPGR) in India, and Chinese Academy of Agricultural
Sciences (Boshou and Holbrook 2007).
5.2.2.4 Population Improvement
Population improvement procedures are not commonly used in groundnut as it is a

highly self-pollinated crop with cleistogamous flowers. Nevertheless, diallel selec-
tive matings and modified recurrent selection schemes were applied to a limited
extent in groundnut improvement (Wynne 1976) that led to development of some
higher yielding groundnut cultivars with a broad genetic base (Monteverde-Penso
et al. 1987). The difficulties involved in making large number of pollinations have
limited the use of recurrent selection schemes although its potential was identified
to improve several traits in groundnut.
5.2.2.5 Mutation Breeding
The above described methods of breeding enable reshuffling of the existing vari-
ability and fixing the desirable combinations, while through induced mutations
new variability is created. Mutation breeding has also been extensively used in
groundnut breeding. Mutation breeding is often used to improve a superior breeding
line or a popular cultivar for a single-specific trait such as, bold kernel size, disease
resistance etc. Both physical and chemical mutagens have been used in groundnut
to induce mutations. Under its joint FAO-IAEA program, about 72 groundnut vari-
eties were developed through mutation breeding and released for cultivation
worldwide over the last five decades (http://mvgs.iaea.org). By 1996 in China,

14.7 % of new groundnut varieties were bred from induced mutants and they
accounted for 19.5 % of the cumulative cultivated area in China (Qui et al. 1998).
In India, the Nuclear Agriculture and Biotechnology Division of the Bhabha Atomic
Research Centre, Mumbai has developed cultivars using either mutation breeding or
a combination of mutation and hybridization (Kale et al. 1999, 2000).
5.2.2.6 Wide Hybridization
Unlike cultivated groundnut, wild Arachis species are reported to possess high lev-
els of resistance to rust, leaf spots, nematodes, peanut bud necrosis virus (PBNV),
tobacco streak virus (TSV), tomato spotted wilt virus (TSWV), groundnut rosette
virus (GRV), groundnut rosette assistor virus (GRAV), leaf miner, Spodoptera, jas-
sids, thrips, aphids and abiotic stresses (Dwivedi et al. 2003; Rao et al. 2003;
Nautiyal et al. 2008; Kalyani et al. 2007; Dwivedi et al. 2008). Wide hybridization
has been used to expand the available variation using wild species and several inter-
specific derivatives have been developed for use as donors of desirable traits or
released as cultivars. Synthetic amphidipoids of Arachis sp. can be useful to develop
wild introgression lines in cultivated background (Fonceka et al. 2009) and can
facilitate better utilization of wild species in breeding programs as use of synthetic
amphidiploid circumvents the crossing barrier between wild and cultivated species
of Arachis (Fonceka et al. 2009; Mallikarjuna et al. 2010). Synthetic amphidiploids
have been bred at ICRISAT using different combinations of wild Arachis including
the progenitors of cultivated groundnut, A. duranensis and A. ipanensis (Mallikarjuna
et al. 2010).
5.2.3 Marker Technologies and Genetic Transformation
Although low genetic diversity in cultivated groundnut gene pool was a serious
bottleneck until recently in developing the genetic maps based on mapping popula-
tions of cultivated groundnut lines, availability of large number of simples sequence
repeat (SSR) markers facilitated the development of the first SSR-based genetic
map based on recombinant inbred line (RIL) population derived from TAG
24 × ICGV 86031 (Varshney et al. 2009a). With an objective of estimating the
marker order for a maximum number of marker loci based on a single map, a com-
posite map comprising of 175 marker loci has been developed by Hong et al. (2010).
Although several genetic maps have become available for cultivated groundnut,
single nucleotide polymorphic (SNP) markers have not yet been integrated into
these maps. Linked markers for nematode resistance (Burrow et al. 1996; Garcia
et al. 1996), aphid vector of groundnut rosette disease (Herselman et al. 2004), yield
and yield parameters (Selvaraj et al. 2009), drought tolerance related traits (Varshney
et al. 2009b; Ravi et al. 2011), resistance to foliar disease (Khedikar et al. 2010) and
nutritional quality traits (Sarvamangala et al. 2011) were identified. Following

marker assisted breeding, “Tifguard High O/L” cultivar was developed through
three rounds of accelerated backcrossing to pyramid nematode resistance and the
trait for high oleic:linoleic acid (high O:L) ratio in seeds (Chu et al. 2011).
At ICRISAT, marker assisted backcrossing is underway for transfer of QTLs
conferring resistance to foliar fungal diseases. Pandey et al. (2012) reviewed the
development of genomic resources such as development of molecular markers,
genetic and physical maps, generation of expressed sequenced tags (ESTs), devel-
opment of mutant resources, and functional genomics platforms that facilitate the
identification of QTLs and discovery of genes associated with tolerance/resistance
to abiotic and biotic stresses and agronomic traits.
Reports of the transformation and development of groundnut transgenics using
a variety of genes such as the bar gene for herbicide tolerance (Brar et al. 1994),
cry1A (Singsit et al. 1997), a chimeric Cry1X gene (Entoori et al. 2008), a gene
encoding the nucleocapsid protein of the tomato spotted wilt virus (Yang et al.
1998), a gene that confers resistance to Indian peanut clump virus, gus
(Venkatachalam et al. 2000; Rohini and Rao 2000), chitinase (Rohini and Rao
2001), DREB1A for drought tolerance (Bhatnagar Mathur et al. 2007), non-heme
chloro-peroxidase gene (cpo-p) from Pseudomonas pyrrocinia having antifungal
activity (Niu et al. 2009), and AtNHX1, a gene driven by 35S promoter for salt
and drought tolerance (Asif et al. 2011) have been reported. Recent developments
in the area of transgenic research through modification of aflatoxin biosynthesis
pathway or use of genes with antifungal and anti-aflatoxin properties also appear
to be encouraging. A post-transcriptional gene silencing (PTGS) model to knock
out the production of allergenic protein, Ara h 2 in groundnut by specific degrada-
tion of the endogenous target messenger RNA (mRNA) was demonstrated (Dodo
et al. 2005). Groundnut allergy is an IgE-mediated hypersensitivity reaction.
ICRISAT has developed a pipeline of genetically engineered groundnuts for sev-
eral traits that are in different stages of product development including pathogen-
derived resistance to viruses, anti-fungal genes for resistance to fungal pathogens
and aflatoxin contamination, nutritional enhancement by the over-production of
β-carotenes and for tolerance to drought stresses.
5.3 Breeding Objectives and Phenotyping
Developing new varieties with increased yield potential and resistance to biotic and
biotic stresses that meet the needs of the growers, processors and consumers is the
primary objective of groundnut breeding. With the constraints that limit the yield
potential and emerging market and consumer preferences, groundnut breeders
always have a challenging task to breed new genotypes to meet these requirements.
Once the objectives are defined, the traits that meet the objectives will be identified.
Phenotyping the target trait is an important aspect in any breeding program.
Appropriate trait phenotyping enables the breeder to make desirable selections in
segregating generations and advance the breeding lines in yield trials. In this
section, we discuss various phenotyping tools to qualify/quantify traits that meet
various objectives of breeding groundnut. Phenotyping may not be possible for all
the traits that are targeted in a breeding program due to their complex nature, in such
cases, simple surrogates, if known, could be used.
5.3.1 Maturity Duration
Regions of groundnut cultivation have varying lengths of growing period (LGP) (90
to over 150 days); based on the LGP, three maturity groups are identified, early or
short (90–110 days), medium (110–130 days) and late (over 130 days). Temperature
plays a critical role in determining duration of maturity; higher temperature reduces
the crop duration, while reverse is true at lower temperatures. Early maturing variet-
ies are important in various agroecological regions like (1) rainfed semi-tropics
where growing season is short (100–110 days), and/or end of season drought is
frequent, a typical scenario in South India, (2) irrigated with short cropping window
in multiple cropping systems, a typical situation in South East Asia (<100 days),
and (3) rice fallows, where crop is grown on residual moisture (<100 days). Further,
breeding for short-duration in groundnut has become more relevant with the pre-
dicted decline of LGP by 5 % or more across the tropics by the climate change,
agriculture and food security (CCAFS) research.
As the “calendar days” concept of determining maturity duration is location and
season specific, it cannot be applied in breeding for short-duration varieties for
other locations or for the same location over years. As growth and development in
groundnut is largely driven by temperature (Ong 1986), the concept of cumulative
thermal time or degree days (CTT or °Cd), which is both, location and season neu-
tral, was developed at ICRISAT to breed short-duration varieties with stable matu-
rity duration across locations (Vasudeva Rao et al. 1992). Taking 10 °C as base
temperature, CTT or °Cd (cumulative thermal time or cumulative degree days) for
each day is calculated from maximum and minimum temperatures. The daily ther-
mal time is accumulated each day from planting to harvest to arrive at the CTT or
°Cd. At ICRISAT location, the standard CTTs for the 75- and 90-day crop growing
periods is 1,240 and 1,470 °Cd, respectively. These standard CTTs are used to pre-
dict the harvest dates for early-maturing groundnut variety trials and breeding mate-
rials in each season. The CTT or °C d for other locations can also be worked out
using the formula: H∑P{[(Tmax * Tmin)/2]-Tbase}, where Tmax is daily maximum
temperature, Tmin is daily minimum temperature, Tbase is mean base temperature
for groundnut, P is planting date and H is harvest date. In breeding for short-
duration, both, segregating progenies and preliminary and advanced yield trails are
harvested at 1,470 °Cd and selection is practiced based on number of mature pods
per plant and yield. The selection is more intense in elite trial that is staggered har-
vested twice, first when the crop is exposed to 1,240 °Cd and next at 1,470 °Cd. The
selection for early maturity is based on the increase in pod yield, shelling outturn
and 100-seed weight from 1,240 and 1,470 °Cd, and the genotypes recording the
least increase are selected. For breeding medium and late duration varieties, both,
the segregating generations and the entries in yield trials are harvested when the
plants show the signs of physiological maturity and selections are carried out based
on yield and yield attributes including percentage of sound mature kernels. As the
breeding for multiple traits is becoming common practice, resistance to biotic and
abiotic stress, quality and other consumer and market preferred traits are targeted
along with the duration.
5.3.2 Yield and Yield Attributes
Selection for yield has been the basis for improving groundnut productivity in the
world (Nigam et al. 1991), but gains from such selection are slow due to large envi-
ronmental effects. Yield and yield attributes are quantified for appropriate selection
and advancement of both, segregating progenies and test entries (advanced breeding
lines). In segregating generations, selection and advancement for yield and yield
attributes is, in general, qualitative, but in yield trials, conducted to test the perfor-
mance of advanced breeding lines, yield attributes are measured (quantified) in rep-
licated trials following appropriate field designs and statistical procedures. In
segregating generations, for both individual plant or bulk selections, visual observa-
tions on growth habit, branching pattern, yield and yield attributes, pod and seed
characteristics, proportion of pod yield to the total biomass, in-situ germination
(due to lack of dormancy), peg strength etc. are taken into account for generation
advance. But in yield evaluation trials, the specific observations are recorded on:
days to 50 or 75 % emergence in a plot, initial and final plant population in a plot,
days to flowering (initiation, 50 or 75 % plants in a plot flowering), growth habit,
branching habit, reaction to diseases and insect pests, individual plant observations
(number of primary and secondary branches, height of main axis and length of pri-
mary branches, number of pods, pod yield and seed yield per plant), days to matu-
rity, plot pod and haulm yields, shelling outturn, 100-seed weight, pod characteristics
(size, number of seeds per pod, reticulation, beak and constriction), seed size, shape
and color and fresh seed dormancy etc. Depending upon the objectives of the trial,
appropriate observations on yield/yield attributes are selected for recording data.
Replicated yield trials are conducted to determine the performance of advanced
breeding lines. Each breeding program may have its own protocol of evaluation of
advanced breeding lines for yield and other agronomic traits. At ICRISAT, the
performance of advanced breeding lines is evaluated following a three-tier system
of evaluation that includes trait-specific preliminary, advanced and elite trials
organized based on growth habit. Test entries (advanced breeding lines) are first
compared with appropriate controls in smaller plots in preliminary trials orga-
nized based on growth habit (Spanish or Virginia bunch) and conducted in both
rainy and postrainy season. Based on performance in two seasons, the promising
entries are promoted to the next level of trials—advanced trials. The entries at this
level are evaluated in a larger plot size in both rainy and postrainy season. The best
performing entries from advanced trials are promoted the elite trials which are
again evaluated for two seasons in bigger plots. Based on the performance over six
seasons (3 years), the selected entries are identified for inclusion in international
trials which are made available to National Agricultural Research System (NARS)
partners on request. The promising entries from international trials and from their
own breeding programs are evaluated in multilocation trials at the state or national
level. Multilocation yield trials allow breeders to identify location specific and/or
widely adapted stable varieties for release, assess the stability of resistance/
tolerance to biotic and abiotic stresses and estimate G × E for different traits of
economic importance. To avoid confounding effects of the phenology of the crop,
the comparison of test entries with controls are done within a taxonomic group.
Maintaining optimum plant population in all the test entries and controls is an
important aspect in the conduct of yield trials, as plant population is directly cor-
related with pod yield and there is little compensation mechanism for the low plant
stand/missing plants in groundnut.
5.3.3 Resistance Breeding
The potential and realized yields are represented in Fig. 5.2 (adapted from Johansen
and Nageswara Rao 1996), where yield potential is defined as the maximum yield
obtainable by the best genotypes in a specified agroclimatic environment when the
known biotic and abiotic constraints are overcome. From Fig. 5.2, it is apparent that
the yield gap, the difference between yields realized by farmers and potential yield,
is large for major producing countries or regions. It implies that there is consider-
able scope for increasing yield by identifying genotypes resistant to biotic and abi-
otic stresses and/or by addressing these constraints through management options.
The phenotyping tools useful in breeding varieties for tolerance/resistance to abiotic
and biotic factors are discussed in the following sections.
5.3.3.1 Abiotic Stress
Drought and high temperature are the most important abiotic stresses that are wide-
spread in groundnut growing areas. The others include salinity and acid soils.
Drought is a major abiotic constraint in the semi-arid tropics affecting yield and
quality in groundnut. Yield losses due to drought depend on its timing, intensity and
duration. Depending on the time of occurrence, drought can be characterized as
early season, mid-season and end-of-season drought. An annual estimated loss in
groundnut production equivalent to US$520 million (at the market prices of 1994)
is caused by drought. Almost half of it (US$208 million) can be recovered through
genetic enhancement for drought tolerance with a benefit: cost ratio of 5.2 (Johansen
and Nigam 1994). Further, drought predisposes pre-harvest Aspergillus infection in
Fig. 5.2 Representation of realized and potential yields and their relationships (source: Johansen
and Nageswara Rao 1996)
the field that affects quality of produce. Linked closely with drought is high tem-
perature stress. The CGIAR’s climate change for agriculture and food security
(CCAFS) research has shown that high temperature stress (above 30 °C) will be
widespread in East and Southern Africa, India, South East Asia and Northern Latin
America, which are important groundnut growing areas.
5.3.3.2 Phenotyping for Drought Tolerance
The breeding approaches include developing short-duration varieties to escape end-

of-season drought and drought tolerant varieties that can withstand moisture stress
through various mechanisms. Both, empirical (also called conventional) that involve
selection for superior yield performance under drought conditions and trait-based
approaches are used in breeding. Studies have shown that both these approaches led
to same gains in breeding for drought tolerance (Rao and Nigam 2003). In empirical
approach, genotypes tolerant/resistant to drought are identified by assessing their
total and pod dry matter productivity under drought stress (Rao and Nigam 2003).
The evaluations are done in both well watered and imposed water stress conditions
and genotype with superior pod and biomass yield in well watered conditions along
with least reduction in pod and biomass yield under water stress are selected. For
such evaluations in rainy season, supplemental irrigation is provided during dry
spell for well watered treatment, while for stress treatment no irrigation is provided.
However, the differences get nullified when the rainfall in the rainy season is well
distributed; hence postrainy (or dry) season results are more reliable where stress is
imposed by withholding of scheduled alternate irrigations from 60 DAS up to har-
vest and there is less interference of rainfall. The empirical approach is both labour
and resource intensive; nevertheless it is most extensively used. At ICRISAT and
elsewhere in national programs of Asia, Africa and the USA (Branch and Kvien
1992), breeders have been successful in developing drought tolerant groundnut
genotypes using empirical approach. ICGV 91114, a drought tolerant groundnut
variety developed at ICRISAT and released in India in 2006, is slowly replacing old
and less productive varieties in highly drought prone district of Anantapur (Birthal
et al. 2011) in Andhra Pradesh state. When a larger number of genotypes have to be
screened, line source sprinkler technique can be used that evaluated genotypes
under varying intensities of drought and empirical approach is used for evaluating
the performance of the genotypes. However, strong winds and rains influence this
technique and it requires complex statistical analysis (Singh et al. 1991).
Trait-based approach involves phenotyping for the traits like transpiration, tran-
spiration efficiency (TE), water use efficiency (WUE) and harvest index and it is
expected that genotypes selected for these traits will have stable yields across erratic
rainfall. Measurement of WUE and TE that requires special growing facilities such
as rainout shelter and lysimeters to grow plants under controlled water regimes are
not often used in breeding programs. WUE is determined by gravimetric approach
that involves determination of total water transcribed by a plant over a specific
period of crop growth and the total biomass the plant accumulated over the same
period. For this the plant are grown in suitable containers that are weighed once or
twice daily and the difference on subsequent days is corrected by adding an extra
amount of water. As a whole plant, WUE can be determined during the period
between 25 and 65 days after sowing. TE is also assessed gravimetrically by grow-
ing the genotypes in lysimeters under well-watered or drought conditions.
Transpiration is measured by regularly weighing the lysimeters, in which the soil
surface is mulched with a 2-cm layer of polythene beads to avoid water evaporation
from the soil (Ratnakumar et al. 2009).
As it is cumbersome to measure WUE and TE in an applied breeding program,
instead its surrogates such as specific leaf area (SLA) and SPAD chlorophyll meter
readings (SCMR) are used. “Carbon isotope discrimination” is an important surro-
gate to WUE/TE but determining is cumbersome, expensive and sometimes not
correlated to increased yield and hence not often used in breeding programs
(Sheshashayee et al. 2003). SLA is obtained by dividing the area of a fresh leaf by
its oven-dry mass, expressed in m2 kg−1. Leaf area meter is used to determine the
leaf area and the samples are dried in an oven for 2 days at 70 °C to obtain the oven-
dry mass. A direct close relationship of TE with SCMRs was reported in groundnut
(Rao et al. 2001) and SCMR also has a direct linear relationship with extracted leaf
chlorophyll and leaf nitrogen concentration. The advantages such as easy and rapid
measurement, nondestructive method and light weight made SPAD chlorophyll
meters the best choice for use in the trait-based groundnut breeding programs
(Serraj et al. 2004). The SCMRs are recorded on the second or third leaf from the
top that is completely expanded and two readings covering on either side of midrib
are taken on each leaflet and the average SCMR is computed. Care is taken to cover
the SPAD meter sensor with leaf lamina and interface with midrib and veins are
avoided to improve the accuracy of the readings. At ICRISAT both empirical and
trait based approaches are used; while selection in segregating generations is based
on total dry matter and pod yield under stress, in yield trials, in addition to these,
SCMR and SLA are also used as selection criteria.
5.3.3.3 Screening for High Temperature Tolerance
A common method of selecting plants for heat-stress tolerance is to grow breeding

lines in a hot target production environment and identify individuals that do not
compromise on pod yield and quality at elevated temperatures (Ehlers and Hall
1998). Heat tolerance screening in glasshouses may be a more effective method as
screening can be carried out throughout the plant life cycle, from seedling to repro-
ductive stages. Better control of temperatures and other experimental parameters in
glasshouse are additional advantages. Breeding for heat tolerance in groundnut is at
infancy and the best screening method and selection criteria are yet to be identified.
Two key stages, flowering including microsporogenesis (3–6 days before flower-
ing), and fruit-set were measured to assess tolerance to high air temperature in
groundnut (Craufurd et al. 2002, 2003). Vara Prasad et al. (1999) showed that 34 °C
is threshold temperature for pollen production in groundnut. Membrane thermosta-
bility is also used to evaluate the genetic variability for heat stress in groundnuts by
using the heat killing temperature and heat killing time as the selection index
(Talwar et al. 2002).
5.3.3.4 Screening for Tolerance to Salinity
Screening for salinity tolerance can either be done under controlled conditions (pot
culture method) or in the field. However, each of the two methods has inherent limi-
tations; while screening under controlled conditions raises the question of its appli-
cability in the field, the experimental error is huge under field conditions. Since a
combination of both the methods was suggested for efficient screening, a screening
protocol was standardized using 100–125 mM of NaCl water under a facility that
allows both a rigorous control on salt treatment and yield evaluation (Vadez et al.
2005). Such a facility, located outdoors, is available at ICRISAT and is equipped
with moveable rainout shelters and uses large pots filled with natural soil. Salt appli-
cation is made on a per unit soil basis dissolved in irrigation water to ensure uniform
distribution. In addition to measuring reduction in yield under salt stress, the other
key indicators under salinity stress are: large stem proportion that may serve as
sodium sink to confer tolerance, maintaining the leaf size and relatively less
reduction in nodulation. Singh et al. (2008) screened groundnut materials in two
consecutive seasons, first by imposing the salinity treatment as irrigation with
saline water (6–7 dS m−1) during summer season and then on residual salinity in
next season. The salinity was build up to a range of 4.0–8.0 EC measured regularly
during cropping season.
5.3.3.5 Screening for Tolerance to Aluminum Toxicity
At high soil acidity, it is not usually the hydrogen ion activity that limits plant
growth but rather the toxicity and deficiencies of elements. Aluminum toxicity is
the single most important factor that effects plant growth and yield under acid soils.
Pot experiments were conducted by adding AlCl3 (40 mg Al l−1, pH 4) to the soil in
a pot to create Al toxic conditions before sowing (Boshou et al. 2000) and reduction
in yield under Al toxicity is measured to determine tolerance to acid soils. The pri-
mary response to aluminum stress occurs in the roots and it was shown that root
dry weight per plant, root volume per plant and shoot dry weight per plant were the
key indicators for evaluating Al tolerance. The concept of “average Al tolerance
coefficient” for the evaluation of Al tolerance in groundnut genotypes was also put
forth (Boshou et al. 2000). Solution culture using AlCl3.6H2O @ 5–15 mg/l (Yang
and Jing 2000) or 40 ppm aluminum solution prepared using Al2(SO4)3·6H2O
(Pratap et al. 2002) and soil block culture and field experiments (Yang et al. 1998)
were also used to study varietal responses to Al toxicity. Field evaluations following
duplicate tests, one on natural un-reclaimed acid soil and other lime-amended plot
is desirable.
5.3.3.6 Fungal Diseases
Groundnut is attacked by several diseases caused by fungi of which foliar fungal

diseases, Aspergillus infection and pod and stem rot are widespread and important.
Late leaf spot (LLS) caused by Phaeoisariopsis personata (Berk. & Curt.) Van Arx,
early leaf spot (ELS) caused by Cercospora arachidicola Hori and rust caused by
Puccinia arachidis Spegazzini are among the major foliar fungal diseases. Both leaf
spots are commonly present in all groundnut growing areas but the severity of each
disease varies between locations and seasons. An estimated global yield loss of
US$600 million due to LLS was reported (Dwivedi et al. 2003). At ICRISAT, breed-
ing for foliar fungal disease has resulted in development of several genotypes with
high level resistance to rust and moderate resistance to LLS (Singh et al. 2003).
There is scope to further enhance the level of resistance to these diseases. Aflatoxins
are potent carcinogen produced by Aspergillus infection forcing several countries to
have strict regimes in place on permissible levels of aflatoxins in their imports of
groundnuts. Aspergillus flavus is predominant in Asia and Africa, while Aspergillus
parasiticus is the predominant species in America. Lamb and Sternitzke (2001)
estimated that aflatoxin contamination costs over $20 million in losses to the south-
east U.S. groundnut industry. Stem and pod rot, caused by Sclerotium rolfsii is a
potential threat to groundnut production in many warm, humid areas, especially
where irrigated groundnut cultivation is expanding.
5.3.3.7 Screening for Foliar Fungal Diseases
High yield potential and high degree of resistance do not generally go together
(Nigam et al. 1991). In most breeding programs a balance is struck between these two
traits—combining high yield potential with moderate levels of resistance to avoid
penalty in yield potential. Advanced breeding lines and segregating generations are
screened in a disease screening nursery under infector row system during rainy sea-
son and selections are done for both, resistance to disease and superior yield under
disease pressure (Tallury et al. 2009). Both, field and controlled conditions screening
can be used, although field screening is widely used in breeding programs.
Field Screening for Leaf Spots and Rust. Different infector row arrangements are
practiced for advanced breeding lines and segregating populations. Segregating
population are generally grown in ridge and furrow system. After every 5–10 rows
of segregating populations, an infector row (a mixture of short- and medium-
duration, highly susceptible to foliar diseases varieties) is planted. The frequency of
occurrence of infector rows depends on the location and season of foliar diseases
screening. The screening block is surrounded by rows/plots of infector rows on all
sides. To verify uniform disease spread, plots of a foliar diseases susceptible variety
are also interspersed along with segregating populations. Screening of advanced
breeding lines is done in replicated plots along with susceptible controls in broad
bed and furrow system. After every fourth bed of test material, a bed of mixture of
susceptible varieties forming infector row is also sown. Infector rows/beds are inoc-
ulated with a conidial (for leaf spot)/urediniospore (for rust) suspension at flowering
stage. If needed, the inoculation can be further repeated. In addition, the artificially
inoculated potted “spreader” plants are also placed throughout the field to serve as
an additional source of inoculum. After inoculation, perfo-irrigation is provided
daily for 15 min in the evening hours for 30 days to favour building up of humidity
required for disease development. A 9-point scale, as given by Subrahmanyam et al.
(1995), is followed for scoring for leaf spots and rust reaction in the field (Table 5.1).
The genotypes recording a score of 1–4 are considered to be resistant. Disease scor-
ing is done two to three times at intervals depending up on the requirement. Yield
and yield contributing traits are also recorded in yield trials for making selections
based on both disease reaction and yield under disease pressure.
Detached Leaf Technique for Leaf Spots and Rust. Detached leaf method is a rapid
technique for screening resistance to leaf spots (Foster et al. 1980) and rust (Mayee
and Munde 1979) in groundnut. Detached, healthy groundnut leaves rooted in ster-
ile sand in trays are inoculated with a concentration of 30,000 spores ml−1 for LLS
and 105 urediniospores ml−1 for rust, followed by incubation in the growth chamber
Table 5.1 A 1–9 scale for recording reaction of foliar diseases in groundnut in the field
(Subrahmanyam et al. 1995)
Disease Disease
score Description severity (%)
(A) Late and early leaf spot diseases
1 No disease 0
2 Lesions present largely on lower leaves; no defoliation 1–5
3 Lesions present largely on lower leaves, very few on middle leaves; 6–10
defoliation of some leaflets, evident on lower leaves
4 Lesions on lower and middle leaves, but severe on lower leaves; 11–20
defoliation of some leaflets, evident on lower leaves
5 Lesions present on all lower and middle leaves; over 50 % defoliation 21–30
of lower leaves
6 Severe lesions on lower and middle leaves; lesions present but less 31–40
severe on top leaves; extensive defoliation of lower leaves;
defoliation of some leaflets, evident on middle leaves
7 Lesions on all leaves but less severe on top leaves; defoliation of all 41–60
lower and some middle leaves
8 Defoliation of all lower and middle leaves; severe lesions on top leaves; 61–80
some defoliation of top leaves evident
9 Almost all leaves defoliate, leaving bare stems; some leaflets may 81–100
remain, but show severe leaf spots
(B) Rust disease
1 No disease 0
2 Pustules sparsely distributed, largely on lower leaves 1–5
3 Many pustules on lower leaves, necrosis evident; very few pustules on 6–10
middle leaves
4 Numerous pustules on lower and middle leaves; severe necrosis on 11–20
lower leaves
5 Severe necrosis of lower and middle leaves; pustules may be present on 21–30
top leaves, but less severe
6 Extensive damage to lower leaves; middle leaves necrotic, with dense 31–40
distribution of pustules; pustules on top leaves
7 Severe damage to lower and middle leaves; pustules densely distributed 41–60
on top leaves
8 100 % damage to lower and middle leaves; pustules densely distributed 61–80
on top leaves
9 Almost all leaves withered; bare stems seen 81–100
at 24 °C temperature and 85 % relative humidity and a 12 h light/12 h dark regime.

LLS disease development is determined every 2 days from 5 to 37 days after inocu-
lation and observations are recorded on incubation period (days), latent period
(days), lesion number, lesion diameter (mm) and leaf area damage (%) (Janila et al.
2013). Rust pustules appear some 10 days after inoculation on susceptible geno-
types. Disease severity is scored on a 1–9 scale, where 1 = no disease, and
9 = 81–100 % foliage destroyed (Subrahmanyam et al. (1983). The other compo-
nents that the recorded include incubation period (days), infection frequency
(lesions per cm2), pustule diameter (mm), pustule ruptured (%), spores per mm2 of
pustule area and Urediniospore germination (%). Incubation period is days from
inoculation to appearance of first lesion/pustule, and latent period is days from inoc-
ulation to the appearance of first sporulating lesion/ruptured pustule. Lesion/pustule
diameter is measured using vernier caliper under a magnifying glass. Leaf area
damage as percent is assessed by comparing the leaves with diagrams depicting
leaves with known percentage of their areas affected (Hassan and Beute 1977).
Urediniospores per unit area and germination are measured under a microscope.
Screening for Aspergillus Infection and Aflatoxin Contamination. The infection of
Aspergillus can occur before harvest in the field, during post-harvest drying and
curing and in storage. Infection can result in aflatoxin contamination in groundnut
kernels. Resistance to Aspergillus in groundnut operates at three independent sites,
pods, seed coat and cotyledons (Utomo et al. 1990). A three-step evaluation is
adopted at ICRISAT for screening: pre-harvest infection (Mehan 1989), in vitro
seed colonization and aflatoxin production (Mehan and McDonald 1980). Holbrook
et al. (1994) developed a large scale field system for screening groundnut germ-
plasm for resistance to aflatoxin contamination at Yuma, Arizona as a screening site
because it consistently has hot and dry conditions.
Field screening for pre-harvest infection involves growing the genotypes in rep-
licated trials in an Aspergillus flavus sick plot and imposing drought late in the
season to promote the infection (Mehan 1989) as drought stress predisposes
Aspergillus infection. Postrainy/dry season allows creation of moisture stress condi-
tions in the field without interference from rains by withholding irrigations from 60
to 70 days after sowing up to harvest. To ensure sufficient inoculum load at the pod
zone, soil inoculation is repeated three to four times from 25 days after flowering.
At ICRISAT, a highly toxigenic A. flavus isolate AF 11-4 is multiplied on auto-
claved sorghum seed in the conical flasks. After 5 days, the inoculum is removed
from the flasks and mixed with autoclaved sorghum seed (1:5 ratio) before the field
application. For inoculum application, the soil near the groundnut plants is opened
about 3–5 cm deep on either side of the plants in a row. The mean soil temperature
of 29–31 °C in the podding zone is preferred. The pods are harvested at maturity
and carefully shelled. The shelled kernels are tested in laboratory for pre-harvest
seed infection by incubating them in petri plates at 99 % relative humidity.
Screening of groundnut genotypes can also be done by investigating in-vitro seed
invasion and colonization and aflatoxin contamination (IVSCAF), an indicative of
resistance at the sites of seed coat and cotyledon, respectively. To study IVSCAF, the
seeds are inoculated with A. flavus and incubated for seed invasion and colonization
is recorded as incidence percentage. The seed sample is prepared either by scarifica-
tion or removal of testa. Then they are placed on the surface sterile filter paper moist-
ened with 5 ml of sterile water in a 10-cm plastic petri dish and inoculated with 25 μl
of a suspension containing approximately 1 × 106 conidia per ml of A. flavus and
incubated at 28 °C. After 8 days, samples were removed from the incubator and rated
separately for mycelial growth, green color, and development of “fluffy” colonies on
a proportional scale of 0 (no growth, green color, or fluffy colonies) to 10 (dense
mycelium on all quarters, dark green color, or all fluffy colonies) in one-point
increments (Xue et al. 2003). Since it is the aflatoxin contamination and not the
Aspergillus infection itself which is important, estimation of aflatoxin concentration

seems to be the best technique to screen genotypes for tolerance. Hence aflatoxin
contamination of genotypes is estimated for both, pre-harvest and in vitro infected
seed samples. The cotyledons are tested for aflatoxin contamination using enzyme-
linked immunosorbent assay (ELISA). Commercial kits are also available in the
market such as, Aflatest, Agriscreen, Aflacup 10, Aflacup 20 and EZ-Screen with
1–20 ppb detection power (www.oxnet.ksu.edu/grsiest). For selection of genotypes,
it is plot-wise data and not mean over the replications that is taken into consideration.
For instance, the genotypes with low infection/contamination in all the replication
are selected, while those with low value in one replication and high in other are
rejected. In China, several new groundnut cultivars with improved productivity and
resistance to aflatoxin contamination are extensively used in production. In addition,
integrated management approaches have been recommended to farmers based on
agro-ecological characteristics in different regions for aflatoxin management
(Boshou et al. 2009). To increase breeding efficiency, studies on mechanisms of
resistance to preharvest aflatoxin contamination were conducted and the most prom-
ising mechanisms identified were resistance to drought and root-knot nematode
(Holbrook et al. 2009).
Screening for Stem and Pod Rot. Breeding lines along with known susceptible
controls are screened in sick plots in replicated trials. Field screening under
uniform, high disease pressure is a useful way to identify resistant genotypes for
stem rot disease (Shokes et al. 1996; Pande et al. 1994), but this method has limita-
tions like presence of natural antagonists and aggregation of inoculum resulting in
disease escape. Low pathogen populations can also be a cause of concern in field
screening. To overcome the limitations, it is desirable to use the same field each
year to encourage build-up of inoculum in the soil. To facilitate pod rot develop-
ment, it is important to increase the interval between irrigations during pod devel-
opment stage. Shew et al. (1987) used oat seed inoculation method to increase the
pathogen population and ensure uniform distribution in the sick plot. Shokes et al.
(1998) followed a method of inoculating individual plants to improve the precision
of screening. It was described as “agar disc technique” which is effective over field
screening and oat inoculations. Breeding lines and cultivars were evaluated by
inoculating 55–65 days old plants with aggressive isolates of S. rolfsii that were
grown on grain-based (oats, corn) medium in the laboratory and the cultivars with
maximum yield under disease pressure were selected and some of them were
released in 2002 and 2003 (Gorbet et al. 2004).
Greenhouse screening, a simple and commonly used screening technique (Shew
et al. 1987; Pande et al. 1994), for S. rolfsii, is described below:
1. Isolate S. rolfsii by hyphal-tip culture on potato dextrose agar
2. Prepare mycelial or sclerotial inoculum from young cultures of highly virulent
isolates grown on dehydrated autoclaved groundnut shells or sorghum grains
3. Grow groundnut seedlings in 15-cm diameter pots containing a 1:2 mixture of
sand and greenhouse potting mix
4. Add ten mature, well-dried sclerotia along with mycelial growth to the pots 8
weeks after sowing or just at pegging
5. Cover the surface with cloth and keep it wet to ensure soil surface and crown are
kept humid
6. Incubate the inoculated plants in a green-house at 28–30 °C and relative humid-
ity >85 %,with 12-h light and dark periods
7. Harvest the plants 30 days after inoculation and count the lesions on stems.
Calculate average length of the three longest lesions on each stem
5.3.3.8 Virus Diseases
Groundnut is host to several virus diseases, but only a few of them are economically
important - groundnut rosette disease (GRD) in Africa, peanut bud necrosis disease
(PBND) in India, tomato spotted wilt virus (TSWV) in USA, peanut stripe potyvi-
rus (PStV) in East and South East Asia, peanut stem necrosis disease (PSND) in
pockets in Southern India and peanut clump virus disease (PCVD) in West Africa.
In 1995, GRD epidemic affected approximately 43,000 ha of groundnut in Eastern
Zambia with an estimated loss of US$4.89 million. In the following year, groundnut
production in Malawi was reduced by 23 % due to GRD epidemic (Waliyar et al.
2007). The loss in pod yields vary with the strain type of peanut stripe poty virus
(PStV) and it can reach as high as 55 % in China (Kunrong et al. 1999). PSND came
to notice in India in 2000, when it caused an epidemic in Anantapur district in
Andhra Pradesh affecting 225,000 ha and causing an economic loss of US$65 mil-
lion (Reddy et al. 2002). Effective laboratory and field screening techniques have
been developed to screen for resistance to these viruses. Sources of resistance were
identified for GRD and PBND and used in breeding programs. Some wild diploid
species have been identified as resistant to PStV.
Groundnut Rosette Disease (GRD). GRD is transmitted by Aphis craccivora and
three agents are involved in causing the symptoms. They are groundnut rosette virus
(GRV), groundnut rosette assistor virus (GRAV) and satellite RNA. An effective field
screening method for GRV resistance is in operation in breeding programs in Africa
(Nigam and Bock 1990). This method involves planting of infector rows of a suscep-
tible variety after two rows of test genotypes, followed by transplanting infected
plants that are heavily aphid infested at every 1.5 m among the infector rows after
seedling emergence. Further, the infection is supplemented by releasing glasshouse-
raised viruliferous aphids in the screening field. In order to identify and eliminate
escapes from the apparently healthy plants in the field, the apparently healthy plants
are individually harvested and their progenies are screened for GRV resistance in the
glasshouse following mechanical sap inoculation. The test plants in glass house are
inoculated with viruliferous aphids fed on GRAV infected plants or by grafting scion
from GRAV-infected plants (Olorunju et al. 1992; Naidu and Kimmins 2007).
Olorunju et al. (1991) devised a method of estimating disease severity index
(DSI) that was modified by Subrahmanyam et al. (1998) by reducing the individual
plant disease scoring scale to a 1–3 scale, where 1 = plants with no visible disease
symptoms on foliage and no stunting, 2 = plants with obvious rosette leaf symptoms
stunted to about 50 % of the size of symptom less plants and 3 = plants with severe
rosette leaf symptoms and stunting greater than 50 %. Disease severity index (DSI)
is calculated based on the score. Resistance to GRD was discovered in the late
1950s in local landraces of Burkina Faso. By utilizing them, cultivars resistant to
GRD, such as K 11149A, K1124D, 69–101, RMP-91 and RG 1 were bred and
released in Africa. These cultivars are now used as sources of resistance as the land
races were semi-erect and late maturing (Bockelee-Morvan 1983; Mayeux et al.
2003) and resistant varieties with 19–92 % higher yield than susceptible were
released in Malawi and Nigeria (Ntare et al. 2002).
Peanut Bud Necrosis Disease (PBND). The disease is caused by peanut bud necrosis
virus (PBNV) and is transmitted by thrips, T. palmi. Screening for resistance to this
disease is done in endemic areas with infector rows of susceptible plants (ex. Cowpea)
sown to ensure sufficient inoculum load. In Thailand, Pensuk et al. (2002) found field
disease incidence at 50 or 60 DAS as most appropriate parameter to identify resis-
tance to PBNV in groundnut genotypes. Ten plants in each plot were randomly
selected and disease score on a 1–5 scale for PBNV on each plant were recorded
where 1 = healthy plant, 2 = spots on some leaves but no systemic symptoms, 3 = sys-
temic symptoms without stunting, 4 = systemic symptoms with stunting and 5 = severe
necrosis or die as described by Pensuk et al. (2002). The genotypes are then rated
based on percentage of infected plants (Buiel 1995), the scoring of the infected plants
is done every 2–3 weeks. Testing for PBNV resistance by mechanical inoculation
under controlled Greenhouse conditions can also be used (Dwivedi et al. 1995).
Tomato Spotted Wilt Virus (TSWV). TSWV is transmitted by thrips in a persistent
manner but it is not seed or pollen borne (German et al. 1992; Peters 2003). TSWV
and related viruses have a wide host range and are reported to infect over 650 spe-
cies of plants among both monocots and dicots (Culbreath et al. 2003). Field screen-
ing, similar to that used for PBND, can be adopted for TSWV screening. Culbreath
et al. (1997) described a new intensity rating method based on percent of row length
severely affected by TSWV, which takes much less time and effort than determining
disease incidence based on individual plants and this is a practical alternative to
individual plant assessment for characterization of genotype responses to TSWV.
For stable resistance across locations, a multilocation field screening of genotypes
is required due to potential strain variation in TSWV (Culbreath et al. 2000).
A glasshouse screening method involving mechanical transmission protocol is also
described for confirmation of field observations (Mandal et al. 2001).
Peanut Stripe Virus Disease (PStVD). Peanut stripe potyvirus (PStV) is transmitted
by aphids, A. craccivora, A. gossypii and Myzus persicae. It is also seed-transmitted.
Wongkaew and Dollet (1990) grouped isolates of PStV, obtained from different
countries, into eight strains. Field screening for PStV under infector rows of a sus-
ceptible variety at regular interval is followed. Wakman and Ansar (1989) trans-
planted PStV infected plants in infector rows and also released aphids onto infected
plants. Planting of the screening nursery at a time when natural aphid activity is
more (dry season) will ensure better spread of the virus in the field. Scoring for
PStV reaction is done based on percentage disease incidence, types of symptoms
observed, and yield estimation (Middleton et al. 1988). To improve the efficiency,
screening has to be done in locations with high incidence of PStV.
Peanut Mottle Virus Disease (PMVD). Mottle disease, caused by peanut mottle
potyvirus (PMV) is transmitted in a non-persistent manner by several aphid species
including Aphis craccivora and infected groundnut seeds. Screening for resistance
to PMV has been done under greenhouse conditions following mechanical sap inoc-
ulation and aphid transmission. The disease reaction is determined by symptoms.
Peanut Stem Necrosis Disease (PSND). It is caused by tobacco streak ilavirus (TSV)
and transmitted by adults of thrips species, F. schultzei, S. dorsalis and Megalurothrips
usitatu. A screening method, where Parthenium was grown one month before sow-
ing the test genotypes around the field in which PSND screening would be carried
out, gave encouraging results. An artificial inoculation method involving infected
sap dilution at 1:10 and inoculation twice at 12 and 15 days after sowing was found
to be very good in screening groundnut germplasm and to identify stable resistance
(Nigam et al. 2012). Screening for TSV/PSND resistance should be carried out
when temperature conditions are favorable (28–32 °C) for virus multiplication and
symptom expression.
Peanut Clump Virus Disease (PCVD). It is caused by a peanut clump furovirus (PCV)
and is transmitted by soil inhabiting fungus Polymyxa graminis. Hot spot locations have
been used for screening for resistance to peanut clump disease. A convenient and reli-
able glasshouse screening method was suggested by Reddy et al. (2005) using mechani-
cal sap inoculation, where French bean is used as source of inoculum.
5.3.3.9 Bacterial Diseases
Bacterial wilt is most predominant among bacterial diseases of groundnut. It is

caused by Ralstonia solanacearum. It was first reported from Indonesia (1905) and
later in Georgia, USA (1931). Presently, the disease is one of the major biotic con-
straint in China, Indonesia and Vietnam. Yield losses range from 10 to 30 %. In
China, annual losses in groundnut pod yield due to bacterial wilt are estimated over
50,000 t (Mehan et al. 1994). Evaluation of breeding lines for wilt resistance is
largely based on field screening in wilt-sick plots under uniform high disease pres-
sure. Screening in hot-spot locations of China, Vietnam and Indonesia is common.
Greenhouse screening using pure culture, controlled soil temperature and moisture
and inoculum concentration and placement can give more precise information.
Several sources of resistance originating from Indonesia and China are used in
breeding programs in groundnut growing countries in East and South East Asia. For
field screening, test genotypes are sown in replicated plots along with susceptible
checks, arranged systematically throughout the wilt-sick filed (Sharma and Soekarno
1992) and percentage of wilted plants in each genotype are recorded based on visual
observations. Lines showing up to 10 % wilt incidence are considered highly resis-
tant and those with 10–20 % incidence are resistant. Lines with less than 30 % sur-
vival are highly susceptible (Mehan et al. 1994). Following extensive screening of
about 5,000 breeding lines and germplasm accessions in wilt-sick plots in China
and Indonesia, many lines with varying levels of resistance have been reported
(Duan et al. 1993; Sharma and Soekarno 1992; Mehan et al. 1994). Several glass-
house screening techniques resulting in successful inoculation with pure cultures of
bacterium have been developed using plants at seedling stage. These include stem
inoculation (stem puncture), hypodermic injection and root inoculation (Kelman
1953). Of which, root inoculation technique appears to be the best way to evaluate
the plants for resistance, while stem inoculation may eliminate certain lines which
might have field resistance (Mehan et al. 1994). Soaking seeds in bacterial suspen-
sion (6 × 108 cfu ml−1) for 30 min is another useful inoculation technique (Li and Tan
1984). Infested soil placed in the pots or other containers can also be used as a
source of inoculum for screening under controlled conditions.
5.3.3.10 Nematodes
Globally, nematodes cause 11.8 % of pod yield losses in groundnut. The root-knot
nematodes, Meloidogyne spp. and the lesion nematodes, Pratylenchus spp. are
important in groundnut (Sharma and McDonald 1990). The root-knot nematode
causes substantial yield losses in severely infested fields, resulting primarily from
stunted plant growth and premature plant death. The parasitic species, M. areneria,
M. javanica and M. hapla have worldwide distribution, while M. incognita was not
found to be parasitic so far on groundnut (Sharma and McDonald 1990). Only race
1 of M. areneria and M. hapla is parasitic in USA, India and China, while M. javan-
ica, common in Egypt and India is not parasitic in USA. Kalahasti malady, a nema-
tode disease caused by Tylenchorhynchus brevilineatus causes brownish-black
discoloration on pod surface and reduced pod size was first observed in 1975–1976
in Chitoor district of Andhra Pradesh, India. Since then the disease has been wide-
spread and serious.
Screening for Nematode Resistance. Resistance of plant-parasitic nematodes is
commonly defined as a reduction or inhibition of nematode reproduction.
Phenotyping can be done following the screening procedure described by Holbrook
et al. (1983) for resistance to M. arenaria. In this method, plants were inoculated
with 3,500 eggs of nematode prepared using the NaOCl method (Hussey and Barker
1973) and applied 10 days after planting. Approximately 70 days after inoculation,
the roots were placed in 1,000 ml cups containing 300 ml of 0.05 % (v/v) phloxin B
solution for 3–5 min. Each plant was indexed for root galls and egg masses based on
a scale of 0–5 (0 = no galls or no egg masses, 1 = 1–2, 2 = 3–10, 3 = 11–30, 4 = 31–100,
and 5 = more than 100 galls or egg masses per root system). To identify resistant
source for Tylenchorhynchus brevilineatus (Kalahasti malady disease), screening in
farmer’s field in Chittoor district of Andhra Pradesh, a hot spot location was fol-
lowed. The nematode density was estimated using a modified Baermann funnel
technique (Southey 1970). The disease scoring was done on a 1–5 scale in which
1 = no disease symptoms evident; 2 = a few small dark brown to black lesions to
cover 1–25 % on some pods, pods of normal size; 3 = many small lesions coalescing
to cover 25–50 % of pod surfaces, all pods affected, pods of normal size; 4 = many
lesions coalescing to cover 50–75 % of pod surfaces, all pods affected, pods of
smaller than normal sixe; and 5 = many lesions coalescing to cover 75 % of pod
surfaces, all pods affected, pods of much smaller than normal size (Mehan et al.
1993). The screening methods were useful to identify resistant source and breed
cultivars with resistance to root knot nematodes (Simpson et al. 2003). A tolerant
cultivar to Kalahasti malady, Tirupati 3 has been released for cultivation in endemic
areas in India (Mehan et al. 1993).
5.3.3.11 Insect Pests
Aphids (Aphis craccivora Koch), three different species of thrips (Frankliniella

schultzei, Thrips palmi and F. fusca), leaf miner (Aproaerema modicella), jassids
(Empoasca kerri and E. fabae) and Spodoptera are the major pests in groundnut,
among which aphids, thrips and Spodoptera have worldwide distribution and cause
serious damage (Whitman and Amin 1988). In addition, termites, white grubs and
storage pests also cause damage to the groundnuts. Detection of host–plant resis-
tance to insect pests is a lengthy procedure and has to be carried out with maximum
care. Plant resistance to major insect pest in cultivated and wild species of Arachis
has been confirmed to the following species: thrips, aphids, leafhoppers, Helicoverpa
sp., Spodoptera sp., and leaf miner (Lynch 1990). However, tapping of resistance to
insect pests from wild species into cultivated species has not been successful so far
(Sharma et al. 2003). Screening procedures for resistance to common insect pests
are described in detail by Ranga Rao and Wightman (1996). The technique employed
for screening differs with insect involved and sometimes location itself.
Screening for Resistance to Aphids. Resistance to aphids is important as they trans-
mit major virus diseases. Field screening of breeding lines is done along with known
susceptible checks under heavy infestation [>100 aphids per plant at 30 days after
emergence (DAE)] under natural conditions, where ten plants are selected and total
number of aphids on them are recorded (Padagham et al. 1990). This procedure is
tedious, thus for mass screening the lines with ≤50 % infestation than the suscep-
tible control are selected and further evaluated for confirmation by screening in
glasshouse. Glasshouse screening is done by transferring two adult aphids on each
plant (15–20 DAE) using a fine tipped camel hair brush and scoring is done based
on number of aphids developed on each plant. At least ten single plants should be
taken for each genotype for the glasshouse screening and lines with 50 % less infes-
tation than the susceptible control are selected (Zeyong et al. 1995).
Screening for Resistance to Thrips. Resistance to thrips is important as they also
transmit major virus diseases. Field screening is done by sowing the test genotypes
along with highly susceptible lines or infector rows of a susceptible crop such as
cowpea to coincide with peak periods of thrips infestation/migration followed by
scoring for the thrips damage (Amin et al. 1985). The screening should coincide
with period of peak infestation/migration, which varies with location and season at
a given location. Ekvised et al. (2006) suggested that plant damage parameters are
more useful than thrips number in identifying differences among groundnut culti-
var as these parameters are more consistent across evaluation dates and years. A
rating scale of 1–9 for scoring thrips injury is used, where 1 = 0–10 % damage,
2–3 = 11–30 % damage, 4–5 = 31–50 % damage, 6–7 = 51–70 % damage and
8–9 = 71–100 % damage (that is also read as 1 = highly resistant, 2–3 = resistant,
4–5 = moderately resistant, 6–7 = susceptible, and 8–9 = highly susceptible (Ranga
Rao and Wightman 1996; Dwivedi et al. 1995).
Screening for Resistance to Spodoptera. Since this pest is highly sporadic on farm,
a simple effective artificial filed screening technique was developed at ICRISAT
(Ranga Rao and Wightman 1996). In this method, test genotypes are sown along
with known susceptible checks in replicated design and the area is surrounded by
15 cm aluminum barrier to arrest the escape of the larvae from the experimental
area. Another set of test material should be planted outside the barrier to have a pest-
free comparison. The artificially reared fourth instar larvae from the insectary are
released in test rows planted in the field. Ten random plants from the central rows
are selected and leaf area is measured and relative performance of lines is assessed
based on loss of leaf area. The genotypes with less than 20 % damage were identi-
fied as resistant (Ranga Rao and Wightman 1996).
Screening for Resistance to Leaf Miner. Field screening is done by growing test
entries along with known susceptible controls in replicated design. However, sporadic
nature of the pest makes field screening non-reliable over the years of testing. At
ICRISAT, an artificial screening method under laboratory conditions was developed
(Ranga Rao and Wightman 1996). This involves maintaining leaf miner cultures in
small cages under glasshouse and after obtaining moths from the insectary, 30 pairs
of moths per cage are released on test entries. Resistance to leaf miner is assessed by
following 1–9 scale in 20 leaves collected at random (Ranga Rao and Wightman
1996). The lines having less than 20 % damage are classified as resistant.
Screening for Tolerance to Jassids. Screening for resistance to jassids is done under
field conditions by growing test entries along with known susceptible controls in
replicated design. The screening nursery is preferably grown to coincide the natural
peak infestation of jassids (Ranga Rao and Wightman 1996). At ICRISAT center the
peak infestation is seen during August-September and February–March. During
peak population periods scoring should be done for jassid injury on a scale of 1–9.
The scoring has to be done at least twice with 15 days interval. Resistance can also
be estimated by counting the percentage of yellowed foliage by visual rating at time
of peak infestation from 10 leaves randomly collected from 3, 4 or 5 leaf positions
on the main stem, in a plot of 12.5 m2 (Dwivedi et al. 1986).
Storage Pests. Groundnut borer or weevil or bruchids (Caryedon serratus) and rust-
red flour beetle (Tribolium castaneum) are major storage pests in groundnut. Others
include merchant grain beetle (Oryzaephilus Mercator), Khapra beetle (Trogoderma
granarium), Elasmolomus sordidus, and rice moth (Corcyra cepahlonica).
Groundnut borer, found in Asia and Africa, is the only species that can penetrate
intact pods to infest the kernels. Rust-red flour beetle is distributed throughout the
tropics and is major pest on shelled groundnuts. Breeding for resistance to storage
pests has not been an objective in groundnut breeding programs, nevertheless, eval-
uation of advanced breeding lines for plant resistance to post-harvest infection of
storage pests is important, as the new high-yielding varieties have frequently proved
to be more susceptible to insect attack during storage than the indigenous genotypes
(Dick 1987). The screening should be performed under controlled temperature and
humidity as they influence the length of the insects’ development period. Known
susceptible and resistant genotypes should be included in the screening and the
duration of the storage in the experiment should be designed as per the requirement.
The parameters such as loss of pod/kernel mass, length of development period of
the pest, mortality of juvenile stages, amount of food consumed and oviposition rate
of the storage pest can be assessed to indicate the cultivar resistance to storage pest
infections (Dick 1987). The differences in these parameters obtained in screening
trials reflect differences between genotypes when both the kernel and insects used
in the experiment are uniform. The insect cultures to supply insects for screening
should be relatively constant in density and the kernel should be preconditioned to
the experimental temperature and humidity for a period of at least 2 weeks.
5.3.4 Biological Nitrogen Fixation
Biological nitrogen fixation (BNF) in groundnut can be improved through both, culti-
var selection and Rhizobium strain improvement (Nambiar et al. 1982). In breeding
programs the genotypes with high BNF can be selected based on various parameters
such as, nodule number, nodule mass, top weight and total nitrogen (Wynne et al.
1980; Nambiar et al. 1982) and nitrogenase activity (Nigam et al. 1985). Of these
parameters, nodule number and nodule mass and top weight is simple to measure and
most commonly used. Nitrogenase activity is measured using acetylene reduction
(Ar) assay (Herdina and Silsbury 1990), carried out in a closed vessel containing 10 %
acetylene using detached nodules, de-topped roots, or whole plants. Gas chromato-
graph (GC) is used to determine the amount of ethylene formed and expressed as
nano-moles or micromoles of ethylene produced per hour per plant or per weight unit
of nodules. The acetylene reduction assay provides a measure of nitrogenase activity
under the experimental conditions and it can vary on field based on seasonal condi-
tions and moreover it does not measure atmospheric nitrogen that is fixed by the plants
hence not frequently used. Total leaf nitrogen is another parameter to indicate BNF of
genotypes and it is determined by Kjeldhal method, discussed in detail under seed
protein content estimation. It can also be estimated by robust methods such as,
Technicon Autoanalyser (Pulse Instrumentation Ltd, Saskatoon, SK) (Singh and
Jambunathan 1980) or near infrared reflectance spectroscopy (NIRS) (Misra et al.
2000). At ICRISAT (ICRISAT Annual reports, 1981) and North Carolina State
University, Raleigh, USA (Wynne et al. 1980, 1983) high performing germplasm
lines were identified based on evaluation of above parameters.
5.3.5 Confectionary and Nutritional Traits
Traits for confectionary purposes are important for both, food uses and export
markets. For confectionary uses, groundnuts are bred possessing all/some of these
traits: greater proportion of sound mature kernels (SMK), flavor, 100 seed weight
exceeding 55 g, >11 % of sugar content, >24 % of protein content, blanchability
(>60 %) and low oil content (<45 %) (Ramanathan 2004) and variability for these
traits is already known (Dwivedi and Nigam 2005). Seed coat colour and seed shape
are the other important confectionary attributes. There are several nutritional attri-
butes, for which groundnut improvement is targeted, of which protein and oil con-
tent and fatty acids composition are important. While low oil content is preferred for
confectionary uses, it is high oil content that is important for oil extraction as high
oil content in groundnut is translated into economic benefits to both farmer and
millers (Narasimham et al. 1985). It is known that fatty acid composition deter-
mines oil quality; oleic and linoleic acids account for 80 % of the fatty acids found
in groundnut oil. Groundnuts are bred for high oleic to linoleic ratio. Gorbet and
Knauft (1997) registered the first high oleic line, SunOleic 95R, and more cultivars
were developed since then (Chu et al. 2011).
Phenotyping for both, confectionary and nutritional traits involves analysis of
groundnut kernels, therefore they are discussed together under physical and chemi-
cal traits.
5.3.5.1 Physical Traits
Sound Mature Kernels (SMK) and Seed Size, Shape and Color. Higher proportion of
sound mature kernels (SMK) is an important attribute as it indicates proportion of
fully mature kernels. SMK % is the ratio of weight of SMK to weight of total kernels
(that includes immature/shriveled kernels). Depending upon the end use seed size also
become an important consideration in confectionery groundnuts. Seed size is mea-
sured in counts (number of seeds per ounce in trade) or as seed length (mm) and seed
width (mm), and 100-seed weight (g) Dwivedi and Nigam (1995). The US peanut
kernel grades based seed count are as follows: Virginia Extra Large—28/32 counts/
oz, Virginia Medium—38–42 counts/oz, Virginia # 1—45/55 counts/oz, Runner
Jumbo—38/42 counts/oz, Runner Medium—40/50 counts/oz, Runner # 1—60/70
counts/oz, Spanish Jumbo—60/70 counts/oz, Spanish # 1—70/80 counts/oz. Kernels
are also graded using grading sieves with holes of prescribed dimensions (NPCA
1988). The seed measurements also reflect the shape of the seed. When used as
roasted-in-shell, pod traits–pod size, pod shape, pod appearance and cleanliness etc
become important. Groundnut testa color varies from light brown to deep red and 20
different testa colors are known, of which the preferred colours are tan, rose tan and
red (Dwivedi and Nigam 2005). Red testa colour is a preferred trait in snack industry.
Seed coat colour is scored based on visual observations taking care to avoid recording
observations on stored seed as seed coat upon storage turns darker.
Blanchability. Blanchability is removal of testa or seed coat (skin) from raw or

roasted groundnuts and this attribute is of economic importance in processed
groundnut food products, which include peanut butter, salted groundnuts, candies,
and bakery products and groundnut flour. To determine the blanchability of geno-
types a laboratory type blancher, based on the model developed by Wright and
Mozingo (1975), was fabricated at ICRISAT Center (Singh et al. 1996). A pre-
heating temperature of 110 °C for 35 min, with 200 g sample for blanching time of
2 min (120 s) and blanching pressure of 17.6 psi was standardized for blanching at
ICRISAT. The following blanchability parameters are taken into account when
breeding lines are selected for this trait—total blanchability (TB, includes fully
blanched intact kernels and fully blanched splits), whole blanched (WB, fully
blanched intact kernels), whole unblanched (UB, unblanched intact kernels), par-
tially blanched (PB, partially blanched intact kernels), blanched splits (SB, fully
blanched splits) and unblanched splits (UBS).
5.3.5.2 Chemical Traits
Chemical analyses can be done on random samples or samples consisting of only

SMKs. If needed the samples may be divided into sub-samples and the mean of the
sub-sample readings can be taken to improve the accuracy of estimation.
Flavour and Sugars. The sensory attributes that make up roasted peanut and flavor
quality are important traits to evaluate in development of new cultivars. Roasted
groundnuts are evaluated by organoleptic test by a carefully selected “taste panel”
for flavor attributes. The desirable flavors include almond, coffee, fresh, nutty,
popcorn, smoky and sweet and not off-flavour (Fletcher 1987). Firm and crispy
texture of the roasted groundnuts is preferred and soft and mushy roasted ground-
nuts lack consumer preference. Free sugars and amino acids have been found to be
the major flavor precursors in roasted groundnut (Newell et al. 1967). Seed sugars
provide a source of carbon for the production of flavour compounds and also
impart desirable taste. Soluble sugars of raw groundnuts are
estimated by extracting sugars from defatted flour from freeze dried and cold
stored groundnuts samples using 80 % methanol and fractured by high perfor-
mance liquid chromatography (HPLC) (Basha 1992). A simpler colorimetric
method for sugar determination uses phenol–sulfuric acid (Dubois et al. 1956).
Sugar can also be estimated by Anthrone reagent method in which carbohydrates
are first hydrolysed into simple sugars using dilute hydrochloric acid. In hot acidic
medium glucose is dehydrated to hydroxymethyl furfural. This compound forms
with anthrone a green coloured product with an absorption maximum at 630 nm.
Total sugars are expressed as per cent of total seed weight.
Seed Oil Content and Fatty Acids. Oil content is estimated by Soxhlet method, a
gravimetric approach that involves estimation of solvent extracted oil from a given
quantity of ground sample. More robust methods like nuclear magnetic resonance
(NMR) (Jambunathan et al. 1985) and near infrared reflectance spectroscopy (NIRS)
(Misra et al. 2000) are also used. A high correlation (r = 0.97) between the estimates
of Soxhlet and NMR methods was reported by Jambunathan et al. (1985). For NMR
oven dried samples are used to determine oil content. NIRS facilitates non-destructive
method of estimation and single-intact kernel (Fox and Cruickshank 2005) or pod
(Sundaram et al. 2010) can be used for estimating oil content and fatty acids. It can
also be done using grounded meal sample. Single-seed based oil content determina-
tion enables screening of segregating populations and reject the low oil content seeds
at early generations thus optimize both, time and resources. The oil content deter-
mined by Soxhlet method is used to both, calibrate and validate NIRS. The fatty acid
analysis of breeding lines is carried out on a gas chromatograph (GC) by estimating
fatty acid methyl esters (Phillips and Singleton 1981). NIRS can be used for robust
estimation of fatty acid, but prior calibration and validation with readings of GC is
required. Oil and fatty acids are expressed as per cent of seed weight.
Seed Protein Content. The wet chemistry method, Kjeldhal procedure is used for
estimation of nitrogen content which can then be converted to protein content.
Quantifying nitrogen content by Kjeldhal method involves digesting the sample in
strong acid such as, sulphuric acid to produce ammonium sulphate, followed by
liberation of ammonia by adding strong alkali (sodium hydroxide). The ammonia is
then captured by boric acid and the exact amount of nitrogen is determined by titrat-
ing the excess acid with sodium carbonate. The nitrogen content thus estimated is
expressed as protein content after conversion, the conversion factor for groundnut is
5.46. Although Kjeldhal method is fairly accurate, it is quite cumbersome and time-
consuming and hence robust methods of determining protein content such as,
Technicon Autoanalyser (Pulse Instrumentation Ltd, Saskatoon, SK) (Singh and
Jambunathan 1980) or NIRS (Misra et al. 2000) can also be used. Protein content of
seed is expressed as per cent of seed weight.
Estimation of Iron and Zinc Content and Other Nutritional Factors. Triacid method was
used for digesting the groundnut seed samples and then Fe and Zn contents are mea-
sured by atomic absorption spectrometer (AAS). For which one gram ground sample is
digested with 10 ml triacid mixture consisting of nitric acid, sulfuric acid and perchloric
acid in the ratio of 10:0.5:2 (v/v). Digestion is done overnight (for cold digestion) in
digestion chamber. The sample is digested initially at 120 °C for 1 h followed by diges-
tion at 230 °C for about 2 h to get clear and colorless solution. The digestion tubes were
allowed to cool down and the contents were dissolved in water and diluted to 75 ml with
distilled water. This aliquot is taken for the estimation of Fe and Zn concentration. The
concentrations are measured by AAS, Varian Spectra AA 20 and results were expressed
in mg kg−1 (Sahrawat et al. 2002). Same procedure is used for estimation of other micro-
nutrients like calcium, potassium, magnesium, manganese and copper. X-ray diffraction
spectroscopy (XRF), a non-destructive method that does not require digestion of the
samples can be more useful when large number of breeding populations and genotypes
are to be studied. The other nutritional factors such as, niacin (Whitley et al. 2011),
tocopherols, folic acid (Dean et al. 2009), proanthocyanidins (flavonoid), and quercetin
(flavonols) (Choo and Siong 1996; Wang et al. 2008) were quantified in groundnuts
using high performance liquid chromatography (HPLC).
5.3.6 Haulm Yield and Quality
Groundnut haulms fodder is used for livestock and it is this important dual purpose
usage of groundnut that prompted groundnut breeders and livestock nutritionists to
collaboratively explore the feasibility of genetic enhancement of not only pod traits
but also haulm yield and haulm quality. No inverse relationships exist between
haulm fodder quality traits and pod and haulm yield, which is important to improve
the haulm fodder quality without jeopardizing the pod yield (Nigam and Blummel
2010). Harvested groundnuts are air dried in the field, after which the pods were
stripped and biomass weighed to determine haulm yield (kg ha−1). Although haulm
yield has been an important parameter in selecting genotypes, determining haulm
quality of the breeding lines is not used often. Haulm nitrogen content, in vitro
organic matter digestibility (OMD) (%) and metabolisable energy (ME) (MJ kg−1)
are important parameters for which breeding line are evaluated to determine haulm
quality. Haulm quality is analyzed for haulm nitrogen content done by Kjeldhal
method as described above. Estimation of OMD and ME are described by Menke
and Steingass (1988). About 200 mg samples were placed in polyester/polyethylene
bags (size 5 cm × 3 cm; pore size 25 μm), incubated at 39 °C with 35 ml rumen
liquor-buffer mixture in 100 ml glass syringes and measured after 0, 3, 6, 12, 24, 48,
72 and 96 h incubation. After finishing the in vitro digestion trials, bags were gently
rinsed with cold tap water and dried at 65 °C for 48 h to determine OMD. The resi-
dues were analyzed for Organic Matter (OM) and Organic Matter Digestibility
(OMD). Each measurement was performed in triplicate. Gross Energy (GE) content
was determined by PARR6300 (ARC 1965) and ME is determined by equation,
ME = GE × OMD × 0.815. Near infrared reflectance spectroscopy (NIRS) can be
used to determine haulm quality parameters. The NIRS is calibrated for haulm
nitrogen content, OMD and ME based on wet chemistry readings and then used for
determining haulm nitrogen content, OMD and ME of haulm (Nigam and Blummel
2010). After recording weight the haulms were ground to pass through a 1 mm sieve
particle mesh and such a fine powder is used for haulm quality analysis. The ground
samples are scanned on NIRS to determine haulm quality traits.
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Chapter 6
Phenotyping of Tomatoes
Amolkumar U. Solanke and P. Ananda Kumar
Abstract Tomato is the most important vegetable crop after potato consumed
worldwide. It is serving as model plant for fruit development and ripening biology.
It is also serving as reference plant for genomics of other solanaceaous crops.
Recently, tomato genome has been completely sequenced. As large amount of
genetic and genomics resources are available in tomato. In-depth phenotyping of
existing and generated variation in tomato may serve a valuable tool to correlate this
huge data with agronomically important traits. For detailed phenotyping, IPGRI has
developed tomato plant characteristics descriptors. By selecting few important
descriptors, compact phenotypic catalog has been developed in tomato. Many
mutant populations have been characterized using this catalog to store massive phe-
notyping data. Many wild species are naturally crossable with cultivated tomato;
which are sources of many fruit quality traits along with biotic and abiotic stress
tolerance alleles. Many researchers already used these species to introgressed agro-
nomically important alleles into cultivated tomato. In future appropriate phenotyp-
ing of these lines along with mutant populations using new platforms like Tomato
Analyzer software and new imaging technologies can help to intensify the pheno-
typing of tomato and thus availability of high throughput phenotyping platforms in
future will hasten the speed of tomato breeding program and also help to correlate
complex phenotypic data with genome.
Keywords Tomato • Mutants • Introgression lines • Breeding • Tomato descriptors

• Phenotypic catalog • High throughput phenotyping
A.U. Solanke • P.A. Kumar (*)

National Research Center on Plant Biotechnology, Indian Agricultural Research Institute,
New Delhi 110012, India
e-mail: kumarpa@nrcpb.org
170 A.U. Solanke and P.A. Kumar
6.1 Introduction
Tomato is one of the most important vegetable crops grown worldwide. Fruit is the
edible part of tomato. According to UN Food and Agriculture Organization (FAO),
it is the second most consumed vegetable crop after potato, and its world production
is 153 million tonnes (FAOSTAT 2009; http://faostat.fao.org). China is the largest
producer of tomato with production of 45 million tonnes, followed by United States
(14 million tonnes), India (11 million tonnes) and Turkey (10 million tonnes).
Tomato is the integral part of balanced diet throughout the world because of its
nutritional value and sugar content (Willcox et al. 2003). Tomato fruit is a rich
source of vitamin A, vitamin C, minerals and phenolic antioxidants. The carotenoid
present in tomato is the preventive agent against diseases like prostate cancer, age-
related muscular degeneration and photoprotection from ultraviolet rays (Stahl et al.
2006; Basu and Imrhan 2007; Foolad 2007). Among nine tomato species, Solanum
lycopersicum is the only domesticated species. There are several other wild species
of this genus. Some other important wild species are S. pennellii, S. habrochaites,
S. peruvianum and S. pimpinellifolium. These species have served as buffer stock of
alleles for disease resistance, stress tolerance and improvement of fruit quality
throughout its breeding history.
The last two decades have witnessed tremendous progress in the genetics and
genomics of tomato, leading to the development of improved tomato, tolerant to
different biotic and abiotic stresses (Otoni et al. 2003; Cuartero et al. 2006) and with
better fruit quality as well as nutritive value (Sevenier et al. 2002; Dixon 2005).
Tomato transgenics have also been raised to define functions of various genes
involved in developmental processes. Advanced cytological, genetic and physical
maps are also available for tomato. Tomato genome has many important features
which make it so special. It has the smallest haploid genome size of 953 Mb amongst
the Solanaceae members (Arumuganathan and Earle 1991) with nearly 77 % and
23 % of its genome concentrated in the pericentromeric heterochromatin and distal
euchromatin, respectively (Peterson et al. 1996). Thus, more than 75 % of genome
is generally devoid of genes and majority of genes are present in the long contigu-
ous stretches of gene-dense euchromatic region located on the distal portions of
each chromosome arms which is approximately 220 Mb. A large number of ESTs
are also available for tomato on Tomato Gene Index, NCBI and SGN websites.
In addition to this large number of studies are also available for macro and micro
level synteny of tomato with other species of Solanaceae. With such saturated
molecular information, tomato is ultimate candidate for a meaningful analysis of
gene-to-phenotype relationship (Gupta et al. 2009).
Desirable phenotype is the ultimate goal of crop improvement. A phenotype is
the ensemble of observable characteristics or traits of an organism such as its mor-
phology, development, biochemical or physiological properties, behavior, and prod-
ucts of behavior. Phenotypes are always results of the expression of genes under the
influence of environmental factors. Genetic improvement through all possible way
is the only way to create new phenotypes. Now a day’s QTL (quantitative trait loci)
6 Phenotyping of Tomatoes 171
mapping is the way to measure new phenotypes. But for QTLs, there is need of high
quality molecular markers and high quality phenotyping. Process of phenotyping is
not easy, as it is the complex interaction of genetic and environmental factors. As
tomato is emerged as model crop for genomics of fruit biology, high throughput
phenotyping of tomato plant and fruit is very important. In case of tomato, some
important phenotypic characters are shape, size, colour and position of fruit; num-
ber, position and shape of leaves or architecture of plant.
In this chapter, the importance of tomato phenotyping for crop improvement has
been discussed. Along with increase in yield, fruit quality and ripening are other
most important aspects of tomato breeding; therefore these are also covered here in
detail. Role of phenotyping in biotic and abiotic stress tolerance is also covered in
this chapter. To generate desirable phenotypes, genetic diversity or wide gene pool
plays important role. Therefore importance of natural and manmade genetic varia-
tion, their high throughput phenotyping also covered. Special emphasis was given
on mutant populations, which is a source of new phenotypes with special insight
into genotypes of specific phenotype through TILLING. Finally chapter was sum-
marized with few important examples of phenotyping for gene characterization in
tomato through QTLs, MAS and map based cloning.
6.2 Origin and History
There are nine species of tomato, among which Solanum lycopersicum (previously
known as Lycopersicon esculentum) is the only cultivated species. In some regions
small fruited S. pimpinellifolium is also grown for cultivation. The plant of tomato
was initially known as pomi d’oro, mala aurea (golden apple), poma amoris (love
apple), and garden apple. Its name “tomato” was derived from Spanish word,
“tomate” which again derived from Mexican Nahuatl “tomatl” (Foolad 2007).
Andean region particularly Chile, Peru, Ecuador, Bolivia and Colombia is consid-
ered as center of diversity for tomato. The wild S. lycopersicum var. cerasiforme
(cherry tomato) is considered as ancestor of modern tomato which is indigenous in
tropical and subtropical America. In pre-Colombian times wild ancestor spread to
Mexico where its domestication occurs. That’s why Mexico is considered as centre
of domestication of tomato. From Mexico, it moved to Europe by Spanish in six-
teenth century. Tomatoes were initially grown only for ornamental purpose because
of its bright red fruits. The fruits of tomato were considered as poisonous because of
its close relation with deadly nightshade (Solanum dulcamara). But slowly in the mid
of sixteenth century tomato consumption was started in the southern Europe even
though its acceptance was slow. Europeans in seventeenth century spread it to China,
South and South-east Asia and further it extend to Japan and USA in eighteenth
century. It is interesting that very little commercial production is undertaking in
Ecuador-Peru which is considered to be native of tomato and it is very important crop
of North America where it entered recently. Commercial production of tomato started
in USA and now it is cultivated throughout world as major vegetable crop.
6.3 Botanical Description
Tomato belongs to the family Solanaceae, which is also called nightshade family.
In Solanaceae tomato is in division Magnoliophyta, class Magnoliopsida, subclass
Asteridae, order Solanales and suborder Solanineae. This family consists of ~3,000
species belonging to 90 genera; of which ~1,500 species belong to genus Solanum
(van der Knapp et al. 2004). This family is the most variable in terms of agricultural
utility, the 3rd most economically important crop family, after the grasses and
legumes. This is the most important family in relation to vegetable crops and it is
third most economically important plant taxon including potato, tomato, pepper,
eggplant, petunia, and tobacco. Some species of solanaceae also have medicinal
properties. In terms of habitats and habits, members of Solanaceae are extremely
diverse in nature. Some species are found in deserts whereas other found in wettest
tropical rain forests. Some species are very small herbs whereas others are tall trees.
The cultivated tomato and its almost all wild relatives have a diploid genome
with 12 chromosomes. The cultivated tomato was originally named by Linnaeus as
Solanum lycopersicum. Further tomato species were separated from other Solanum
plants and designated the genus Lycopersicon with the species esculentum and
named it as Lycopersicon esculentum. Lycopersicon plants have anthers that dehisce
laterally with mostly pinnate leaves whereas other Solanum species have anthers
that dehisce from the terminal ends with simple leaves. But more recently on the
basis of phylogenetic relationships between Solanum and Lycopersicon, and also
based on much molecular and morphological information, new taxonomists classi-
fied cultivated tomato as S. lycopersicum. The other species of Lycopersicon have
also been reassigned as Solanum.
Tomato is weak stemmed, trailing, much branched, short lived perennial plants.
But because of commercial production they are cultivated as annuals. There are two
growth habits in tomato i.e. determinate and indeterminate. In case of determinate
plants lateral axis terminates in a blossom giving self-topping. Determinates have
about five flower per clusters and each cluster developing about five to seven fruits.
There are usually two leaves between clusters. In case of indeterminate habit, con-
tinuous extension of the main shoot by side shoots occurs and plant growth is con-
tinuous. The terminal buds do not set fruit in indeterminates but produces more
leaves. The growing tip inflorescences are formed after every three leaves. Normally
monopodial branching occurs at the base of the plant, which becomes sympodial at
higher level. Stem, leaves, petiole and peduncle are covered with small glossy
reddish-yellow glandular hairs and long sharp non-glandular trichomes. The leaves
are alternate, spirally arranged, unevenly pinnate, compound with variously intended
or lobed margins. Normal size of leaf is 15–30 cm by 10–25 cm. Inflorescence is the
extra axillary cymes with dichotomous polycotomous branching. The flowers are
always appearing in the cluster on main axis and on the lateral branches. The flow-
ers are bright yellow in colour. These are pentamerous, bisexual, regular, complete,
ebracteate and hypogynous. The pistil has two or more carpels. The anthers connote
appearing in the throat of the corolla and they dehisce through the poricidal dehiscence.
Fig. 6.1 Various shapes of

tomato. A: sphere, B:
ponderosa, C: san marzand,
D: lemon, E: plum, F: oblate,
G: beefheart, H: currant, I:
cherry, J: calabash and K:
pear. Photograph was taken
by author. Source of tomato
fruits: Dr. Raj Kumar and Dr.
Harshawardhan Choudhary,
Division of Vegetable
Science, Indian Agricultural
Research Institute, New
Delhi, India
The style is usually shorter than anther because of which tomato is highly self-pol-
linated. The fruits are soft fleshy berries with small hairs during immature stage.
There are various shapes and sizes of tomato fruits viz. sphere, ponderosa, san mar-
zand, lemon, oblate, beefheart, cyrrant, chery, plum, calabash and pear (Fig. 6.1).
The transverse section of fruit show five main parts i.e. outer and inner walls, skin,
locular tissue, pulp and seeds. The pericarp constitutes the wall of the fruit and to a
considerable extent the quality of the fruit depends on the pericarp. The layer of the
mesocarp increases the size of the cells which results in the growth of the pericarp.
The locule number varies from variety to variety. The locules are divided by the
radial walls of the pericarp known as columella. Each locule has a lot of seeds. The
seeds are enclosed in a jelly-like substance which contains parenchymatous cells
that develop round the ovule. Fruit ripening is most important feature in tomato.
There are different stages of fruit development in tomato like immature green,
mature green, breaker and red-ripe with some intermediate stages. These are men-
tioned in Table 6.1. The fruit colour of ripened tomato also varies from dark red to
yellow, which is shown in Fig. 6.2.
Table 6.1 Description of tomato fruit developmental stages

No. Stage Description
1. Immature green Pale green colour, no full expansion of fruit
2. Mature green Dark green colour, fully expanded fruit
MG1: firm locular tissue, knife cut seeds
MG2: softened locular tissue, seeds not cut with knife
MG3: some get in the locule, no red colour
MG4: locular tissue predominantly gel, some red colour in columella
3. Breaker Break of colour from green to tannish-yellow
4. Turning 10–30 % change in colour from green to tannish yellow or red
5. Pink 30–60 % change in colour from green to pinkish red
6. Light-red Light red colour throughout surface, firm fruit
7. Red-ripe Dark red colour with softness
Fig. 6.2 Various colour of

the fresh market tomato.
Photograph was taken by
author. Source of tomato
fruits: Dr. Raj Kumar and Dr.
Harshawardhan Choudhary,
Division of Vegetable
Science, Indian Agricultural
Research Institute, New
Delhi, India
6.4 Tomato as a Model Plant
Tomato is emerged as a model system for a large number of significant studies, both
in basic and applied levels. Tomato is also playing an important role as model plant
for the study of fruit development (Tanksley 2004; Giovannoni 2007; Paran and van
der Knaap 2007). There are many reasons for tomato as model plant. Some of them
includes, short life cycle, photoperiod insensitivity, high self fertility and homozy-
gosity, high reproduction capacity, ability of controlled pollination and hybridiza-
tion. It also includes the smaller genome size with lack of gene duplication. High
transformation efficiency for generation of large number of transgenic plants also
made it as a model plant. In addition to this, wide arrays of mutants are available
with tomato (Menda et al. 2004; Giovannoni 2007). The high molecular weight
insert genomic libraries in YAC and BAC are also available in tomato for map-based
cloning and gene mapping (Bonnema et al. 1996; Hamilton et al. 1999; Budiman
et al. 2000). Because of all these advantages of tomato, the first map-based cloning
of disease resistance gene Pto was carried out in tomato which provided the under-
standing of the molecular basis of Pto-mediated resistance to bacterial speck dis-
ease (Pedley and Martin 2003). The first transgenic product that entered in the
market was FLAVR SAVR variety of tomato (Kramer and Redenbaugh 1994).
Many molecular investigations have been carried out in tomato plant. Some of these
are the molecular interaction between tomato and leaf mold Cladosporium fulvum
(Rivas and Thomas 2005), the wound-induced gene expression by the peptide sys-
temin and the lipid-derived jasmonic acid (Wasternack et al. 2006). Since tomato
has unique developmental aspect of formation of fleshy fruit which is not found in
Arabidopsis and rice, the genetic, developmental, and molecular basis of fruit size
and shape variation (Tanksley 2004) and fruit ripening (Giovannoni 2007) have
been widely studied using tomato as a model plant.
6.5 Purpose of Tomato Cultivation
Global tomato production is divided into two sharp divisions, fresh market tomato
production for table purpose and for processing industry. In USA 95 % of their
produce is used for processing purpose, while in European countries like Italy,
Spain, Brazil and Turkey, it varies from 25 to 70 % (Heuvelink 2005). In Asian
countries like China and India, tomatoes are mainly produced for table purpose and
processing tomato production is not more than 10 %. Thus USA, Italy, Spain and
Turkey dominate the world processing tomato industry. The Netherlands is the
global leader of fresh market tomato with production of tomatoes in greenhouse.
Varieties of fresh market and processing have different growth habits. The specific
characteristics of fresh market and processing tomatoes are given bellow.
6.5.1 Fresh Market Tomato
Tomatoes sold in the local markets for eating as raw or in salad or after cooking are
roughly categorized as fresh market tomatoes. These tomatoes are mainly grown in
kitchen garden or at commercial level in green houses. The varieties are generally
indeterminate and required trellising. But normally varieties used for commercial
cultivation are determinate. Plants are supported by staking, which keep fruits away
from the soil. Pruning method is used for increasing fruit size. Harvesting of fresh
market fruits is always done by hands. Preferences for fresh market tomato vary
among different localities and among different countries. Japanese consumers pre-
fer pink-coloured tomatoes, Indians like dark red juicy tomatoes whereas Taiwanese
like green-shouldered tomatoes with slight reddening on the blossom-end.
Preferences for size and shape of the fruit are also vary among various countries.
A large number of varieties are available for fresh market tomato production, which
range from small, sweet cherry tomatoes to large beefsteak tomatoes varies in
colour, shelf-life and flavours (Dorais et al. 2001). These tomatoes are mainly,
classic round, cherry and cocktail, plum and baby plum, beefsteak, and vine or truss
tomatoes as per their size, shape and use. Specific traits of interest in fresh market
cultivars include large, round fruit with adequate firmness and shelf-life, uniform
fruit size, shape and colour, appearance, freedom from external blemishes or abnor-
malities, texture, taste and flavor (Foolad 2007).
6.5.2 Processing Tomato
Processing tomatoes are used to produce paste, sauce, whole-peeled tomato, catsup
or other products. The choice for this purpose is the deep red colour of fruit. pH
should not be more than 4.5. The solid content should be high with maximum vis-
cosity. Plantation of these tomatoes are done on commercial basis, harvesting is
always done by machinery and therefore varieties for this purpose are usually deter-
minate growth habit with short vines. The fruit set is concentrated with uniform fruit
set and ripening. These tomatoes are grown in open field directly by drill method or
some time by transplanting, and do not required trellising or staking. Thus the char-
acters of processing cultivars include compact, determinate plant habit and concen-
trated flowering and fruit set suitable for once-over machine harvest, ease of fruit
separation from the vine, and specific fruit quality characteristics such as colour,
pH, total acidity, soluble solids, total solids, and viscosity (Foolad 2007).
6.6 Descriptors of Tomato
Rick et al. (1990) formulated the S. lycopersicum and related Solanum species
keys and published in the Tomato Genetics Cooperative Report, 40:31. That was
further modified and now International Plant Genetic Resources Institute
(IPGRI) Rome, Italy generated various descriptors of tomato for proper pheno-
typing of tomato genetic resources. They divided these descriptors into passport,
management, environment and site, characterization and evaluation categories.
Passport descriptor provides the basic information used for the
general management along with parameters that should be observed when the
accession is originally collected. Management descriptors supply the basis for
the management of accessions during multiplication and regeneration.
Environment and site descriptors explain the environmental and site-specific
parameters that are important when characterization and evaluation trials are
held. Evaluation descriptors are vulnerable to environmental differences but are
generally useful in crop improvement which includes yield, agronomic perfor-
mance, stress susceptibilities and biochemical and cytological traits.
Characterization descriptors are the significant among all because these enable
an easy and quick discrimination between phenotypes. The characters covered in
these descriptors are generally highly heritable. These are the phenotypes which
can be easily seen by the naked eye and are equally expressed in all environments.
In these parameters, other additional traits thought to be desirable by curator can
also add along with photographs. Here we are giving details of characterization
descriptors as these are the most important for proper and thorough phenotyping
(Table 6.2).
6.7 Tomato Phenotypic Catalog
As these tomato descriptors are very comprehensive, to characterize the huge mutant
populations, there was need of constructing a compact phenotypic catalog. This cata-
log should cover almost all aspect of plant phenotypes from architecture to its behav-
ior under different environmental conditions. Here we are providing tomato
phenotypic catalog, which have been used by different researchers to characterized
core collection and mutant populations (Menda et al. 2004; Vankadavath et al. 2009;
Minoia et al. 2010). Although this phenotypic catalog does not include root altera-
tion or behavior of plants under different environment, it can provide solution to the
need of high throughput scoring. In future, there is need to modify the phenotypic
catalog so that it can be suitable for new bioinformatics tools used in high throughput
phenomics. The major categories of tomato phenotypic catalog are described below:
1. Seed: Germination per cent, duration of germination and incase of mutant pop-
ulation, seedling lethality can be measures in this category. Further seed size
can also be includes under seed.
2. Plant size: Overall plant architecture is considered in this category. Plants can
be grouped as extremely small, average size or very large.
3. Plant habit: Diverse plant size due to shorter or longer internode distance, num-
ber of branches or arrested growth after initial stages in case of mutants can be
grouped in this category. Plants with different behavior other than mentioned
habits can group separately as “other plant habit”.
4. Leaf morphology: Leaf morphology of tomato can be divided on the basis of its
size like large or small; width like narrower or wider; and texture like smooth
or rough. In case of mutant population many other morphological sub-categories
are possible like rolled leaf, thick leaf or no leaf. These can be grouped into
“other leaf morphology”.
5. Leaf colour: Colour of the leaf from dark green to pale yellow and from white
to purple can be categorized here.
6. Flowering time: This is very important category in which flowering can be
grouped as early of late. In case of indeterminate and determinate accessions,
this can also be grouped as continuous or one time flowering.
7. Inflorescence structure: Nature of inflorescence can grouped in this category as
less or very high inflorescence, structure of inflorescence etc.
Table 6.2 The descriptor list characterizing morphological traits (modified after Descriptors of
Tomato by IPGRI 1996)
IPGRI
descriptor
Sl. No. number Descriptor name Descriptor state
Seedling Records at the seedling level when primary leaves are fully opened and the terminal
bud is around 5 mm in size
1 7.1.1.1 Hypocotyl colour 1 Green
2 1/4 purple from the base
3 1/2 purple from the base
4 Purple
2 7.1.1.2 Hypocotyl colour intensity 3 Low
5 Intermediate
7 High
3 7.1.1.3 Hypocotyl pubescence 0 Absent
1 Present
4 7.1.1.4 Primary leaf length (mm) Average of ten cotyledonous leaves
5 7.1.1.5 Primary leaf width (mm) Average of ten cotyledonous leaves
Plant Records should be taken when the fruits of the 2nd and 3rd truss are ripened
6 7.1.2.1 Plant growth type Observed on the whole plot, after
admixtures have been removed
1 Dwarf
2 Determinate
3 Semi-determinate
4 Indeterminate
7 7.1.2.2 Plant size Visual estimation of the whole plot
3 Small
5 Intermediate
7 Large
8 7.1.2.3 Vine length (cm) Measured on ten randomly selected
plants from the soil level to the tip
of the longest stem of a plant
9 7.1.2.4 Stem pubescence density 3 Sparse
5 Intermediate
7 Dense
10 7.1.2.5 Stem internode length 3 Short
5 Intermediate
7 Long
11 7.1.2.6 Foliage density 3 Sparse
5 Intermediate
7 Dense
12 7.1.2.7 Number of leaves under 3 Few
1st inflorescence 7 Many
13 7.1.2.8 Leaf attitude 3 Semi-erect
5 Horizontal
7 Drooping
(continued)
Table 6.2 (continued)

IPGRI
descriptor
14 7.1.2.9 Leaf type 1 Dwarf
2 Potato leaf type
3 Standard
4 Peruvianum
5 Pimpinellifolium
6 Hirsutum
7 Other (specify in descriptor 7.4 notes)
15 7.1.2.10 Degree of leaf dissection 3 Low
5 Intermediate
7 High
16 7.1.2.11 Anthocyanin colouration Indicate the environmental conditions of
of leaf veins the trial (e.g. temperature and
luminous intensity)
1 Obscure vein
2 Normal (clear)
Inflorescence
17 7.2.1.1 Inflorescence type Observe the 2nd and 3rd truss of at least
ten plants
1 Generally uniparous
2 Both (partly uniparous, partly
multiparous)
3 Generally multiparous
18 7.2.1.2 Corolla colour 1 White
2 Yellow
3 Orange
19 7.2.1.3 Corolla blossom type 1 Closed
2 Open
20 7.2.1.4 Flower sterility type 1 Stemless
2 Functional
3 Pollen
21 7.2.1.5 Petal length (mm) Average of ten petals from different
flowers of different plants
22 7.2.1.6 Sepal length (mm) Average of ten sepals from different
23 7.2.1.7 Style position The relative position of the style
compared with the stamens. Average
of ten styles from different flowers of
different plants
1 Inserted
2 Same level as stamen
3 Slightly exserted
4 Highly exserted
24 7.2.1.8 Style shape 1 Simple
2 Fasciated
3 Divided
(continued)

IPGRI
descriptor
25 7.2.1.9 Style hairiness 0 Absent
1 Present
26 7.2.1.10 Stamen length (mm) Average of ten stamens from different
27 7.2.1.11 Dehiscence 1 Poricidal
2 Longitudinal
Fruit All observations on the fruit should be taken, when possible, on the 3rd fruit of the
2nd and/or 3rd truss at the full maturity stage, provided normal fertilization has
occurred. Record the average of 10 fruits from different plants
28 7.2.2.1 Exterior colour of Recorded before maturity
immature fruit 1 Greenish-white
3 Light green
5 Green
7 Dark green
9 Very dark green
29 7.2.2.2 Presence of green 0 Absent (uniform ripening)
(shoulder) trips on the 1 Present (fruit shoulders—upper part of
fruit the fruit, around calyx—are green
while pistilar area of the fruit is red)
30 7.2.2.3 Intensity of greenback 3 Slight
(green shoulder) 5 Intermediate
7 Strong
31 7.2.2.4 Fruit pubescence 3 Sparse
5 Intermediate
7 Dense
32 7.2.2.5 Predominant fruit shape Recorded after the fruits turn colour
1 Flattened (oblate)
2 Slightly flattened
3 Rounded
4 High rounded
5 Heart-shaped
6 Cylindrical (long oblong)
7 Pyriform
8 Ellipsoid (plum-shaped)
33 7.2.2.6 Fruit size At maturity
1 Very small (<3 cm)
2 Small (3–5 cm)
3 Intermediate (5.1–8 cm)
4 Large (8.1–10 cm)
5 Very large (>10 cm)
34 7.2.2.7 Fruit size homogeneity (Within a plant)
3 Low
5 Intermediate
7 High
35 7.2.2.8 Fruit weight (g)
(continued)

IPGRI
descriptor
36 7.2.2.9 Fruit length (mm) Recorded from stem end to blossom end,
to one decimal place, at maturity
37 7.2.2.10 Fruit width (mm) Recorded at the largest diameter of
cross-sectioned fruits to one decimal
place, at maturity
38 7.2.2.11 Exterior colour of mature Recorded at maturity
fruit 1 Green
2 Yellow
3 Orange
4 Pink
5 Red
39 7.2.2.12 Intensity of exterior colour 3 Light
5 Intermediate
7 Dark
40 7.2.2.13 Secondary fruit shape Recorded on fruits of the second and
third truss, after the fruits turn
colour
1 Flattened (oblate)
2 Slightly flattened
3 Rounded
4 High rounded
5 Heart-shaped
6 Cylindrical (long oblong)
7 Pyriform
8 Ellipsoid (plum-shaped)
41 7.2.2.14 Ribbing at calyx end 1 Very weak
3 Weak
5 Intermediate
7 Strong
42 7.2.2.15 Easiness of fruit to detach Recorded during harvesting
from the pedicel 3 Easy
5 Intermediate
7 Difficult
43 7.2.2.16 Fruit shoulder shape 1 Flat
3 Slightly depressed
5 Moderately depressed
7 Strongly depressed
44 7.2.2.17 Pedicel length (cm) Measured from peduncle to calyx
45 7.2.2.18 Pedicel length from Recorded from abscission layer to calyx.
abscission layer (cm) Average of ten pedicels from different
plants
46 7.2.2.19 Presence/absence of 0 Absent
jointless pedicel 1 Present
(continued)

IPGRI
descriptor
47 7.2.2.20 Width of pedicel scar Recorded at the widest part on ten
(mm) randomly selected fruits from
different plants
3 Narrow (covered by the calyx)
5 Medium (slightly apparent around the
calyx)
7 Wide (very apparent around the calyx)
48 7.2.2.21 Size of corky area around Recorded at the widest part on ten
pedicel scar (mm) randomly selected fruits
3 Small
5 Intermediate
7 Large
49 7.2.2.22 Easiness of fruit wall 3 Easy
(skin) to be peeled 5 Intermediate
7 Difficult
50 7.2.2.23 Skin colour of ripe fruit Observe the peeled fruit skin
1 Colourless
2 Yellow
51 7.2.2.24 Thickness of fruit wall Measured with a dial caliper
(skin) (mm)
52 7.2.2.25 Thickness of pericarp Measured from an equatorial section of
(mm) the fruits
53 7.2.2.26 Flesh colour of pericarp 1 Green
(interior) 2 Yellow
3 Orange
4 Pink
5 Red
54 7.2.2.27 Flesh colour intensity 3 Light
5 Intermediate
7 Dark
55 7.2.2.28 Colour (intensity) of core 1 Green
2 White
3 Light
5 Intermediate
7 Dark
56 7.2.2.29 Fruit cross-sectional shape 1 Round
2 Angular
3 Irregular
57 7.2.2.30 Size of core (cm) Measured on 10 cross-sectional
randomly selected fruits at the widest
part of the core
58 7.2.2.31 Number of locules Counted on at least ten fruits
59 7.2.2.32 Shape of pistil scar 1 Dot
2 Stellate
3 Linear
4 Irregular
(continued)

IPGRI
descriptor
60 7.2.2.33 Fruit blossom end shape 1 Indented
2 Flat
3 Pointed
61 7.2.2.34 Blossom end scar 1 Open
condition 2 Closed
3 Both
62 7.2.2.35 Fruit firmness (after Recorded by pressing together in the
storage) palm on the side of a fruit at its
widest girth, i.e. sideways, 10 days
after harvesting in full ripeness
3 Soft
5 Intermediate
7 Firm
Seed
63 7.3.1 Seed shape 1 Globular
2 Ovate
3 Triangular with pointed base
63 7.3.2 1,000-seed weight (g)
65 7.3.3 Seed colour 1 Light yellow
2 Dark yellow
3 Grey
4 Brown
5 Dark brown
Notes 7.4 Any additional information, especially in the category of “other”
under various descriptors
above, may be specified here
Photographs
8. Flower morphology: Morphology of individual flower can categorize here. In

case of mutant population flower homeotic mutants, flower organ size and
flower organ width can mention in this category.
9. Flower colour: This is not very important category, but can help to distinguish
different accessions. Here flowers can grouped as per their colour like white,
pale yellow or strong yellow flower.
10. Fruit size: As fruit is the economically important part of tomato, this is very
important category. On the basis of size of fruit, plant population can be grouped
as small or large fruit. Since large amount of variation is possible for this char-
acter, it can be further sub-categorized.
11. Fruit morphology: This is also important character as people from different
countries prefer different shape and size of tomatoes. Fruits can be grouped as
sphere, ponderosa, san marzand, lemon, oblate, beefheart, cyrrant, chery, plum,
calabash and pear shape tomatoes.
12. Fruit colour: Colour of fruit from green, red, purple, yellow, pink or stripped
can be categorized here.
13. Fruit ripening: Early or late ripening of fruits can place in this category. Some
never ripening mutants or wild accessions can also sub-categorize.
14. Sterility: Partial, full or no sterility of accession can grouped in this category.
15. Disease and stress response: Although this is agronomically very important
category, its analysis is possible when population is subjected to different
environmental stresses or infected with disease causing organisms. But pre-
liminary responses like necrosis, wilting, or other disease responses can be
categorized here.
6.8 Sources of Variation for Tomato Phenotyping
The domestication of the tomato through centuries of breeding and selection

resulted in great reduction of genetic variability and therefore cultivated tomato
contains a small fraction of genetic variation (Bai and Lindhout 2007). Still some
variation in size, shape and colour is available in cultivated tomato (Figs. 6.1 and
6.2). Genetic diversity present in the form of wild relatives and landraces is also
enormous and can be used for tomato crop improvement. Tomato genetic material
collected at various genebanks and core collections are the buffer stock for breeding
program. To characterize agronomically important traits, new mutant populations
have been developed; these are also source of variation in tomato. Some important
sources for tomato variation are described below.
6.8.1 Natural Variation
Wild species of tomato are the main source utilized in maximum breeding programs
to improve the cultivated tomato. There are nine wild species along with cultivated
tomato. These are S. pimpinellifolium (currant tomato), S. lycopersicoides, S. chees-
manii, S. chmielewskii, S. chilense, S. neorickii, S. peruvianum, S. habrochaites,
and S. pennellii. Several wild accessions have been identified with desirable horti-
cultural characteristics such as high fruit quality, disease resistance and tolerance to
abiotic stresses. Dr. C.M. Rick had spent his life on collection of tomato species and
research on tomato genetics. He grouped these species in two complexes as per their
crossing ability with cultivated tomatoS. lycopersicum. These complexes are the
L. esculentum complex and the L. peruvianum complex. Only for this section we are
referring old nomenclature. Six other species included in the L. esculentum complex
are relatively easy to cross with the cultivated tomato. These species are L. esculen-
tum (var. cerasiforme; var. grandifolium; var. pyriforme and var. validium), L. pimp-
inellifolium, L. cheesmanii (f. minor), L. parviflorum, L. chmielewskii, L. hirsutum
(f. typicum and f. glabratum), L. pennellii. All species from this group are diploid in
nature (2n = 2x = 24). Except L. hirsutum f. typicum and some L. pennellii species
other are self-compatible. Flower morphology of these species is similar. They have
yellow flowers and the stamens are joined to produce an anther cone. Fruit colour of
these species varies depending on each species. Several members of this complex
have provided sources of pest resistance in the cultivated tomato. The other complex
is L. peruvianum which have two extremely diverse species. These can hybridize
only with great difficulty. Both are diploid (2n = 2x = 24) and naturally habitat in
very adverse environmental conditions. These two species are valuable and poten-
tial resource of alleles for cultivated tomato. Their fruits are green in colour. These
species are mostly self-incompatible. Use of these species to cultivated tomato is
limited due to various barriers present in sexual hybridization through conventional
breeding. But in total all related wild tomato species are a rich source of desirable
genes for crop improvement.
6.8.2 Core Collection
It is estimated that around 70,000 accessions of the cultivated and wild species of
tomato are preserved in genebanks around the world. The centers where these
accessions have been stocked are the Asian Vegetable Research and Development
Center (AVRDC) at Tainan, Taiwan; the United States Department of Agriculture
(USDA); Plant Genetic Resources Unit at Geneva (PGRU), NY, USA; and the CM
Rick Tomato Genetics Resource Center (TGRC), University of California, Davis,
USA. Good collections of tomato germplasm are also maintained in The Netherlands,
Russia, India, Japan, Peru, and Cuba.
To preserve genetic and phenotypic diversities core collection of around 7,000
tomato varieties have been developed by Dani Zamir and Zach Lippman. They have
collected it from different parts of the world, from Europe to United States and also
from Asia and planted it in the Hebrew University at Jerusalem. This core set con-
tains several wild species of tomato, domesticated lines along with landraces. These
land races have been collected from farmers who have domesticated them at differ-
ent environmental conditions and also at different local farm management practices.
Scientist working on this core collection have selected accessions on the basis of
phenotypic characters. They selected 400 accessions for colour ranging from red,
purple, green, yellow, orange and many more; 50 accessions are selected for colour
of fruit skin ranging from solid red to semi transparent and from also with zebra pat-
tern; 100 accessions are selected for fruit shape which varies from round, oval, pear
to fragmented one; and 100 accessions are selected for fruit firmness, size. Accessions
are also selected for plant and flower architecture. The content of soluble sugar (brix)
is also analyzing for this core collection. The phenotypic data of this core collection
is available on EU-SOL website (EU-SOL Newsletter, 2009). This phenotypic data
can be further used by scientist from the world to integrate their information on
aspects like metabolites and genetic markers to enrich tomato genomics. In 2008,
Dani Zamir organized a tomato phenotyping workshop to educate researchers about
tomato traits. The core collection is the part of a large genebank, so it is available to
all for crop improvement.
6.8.3 Breeding Techniques
Plant breeding is the art as well as science of genetic improvement of crop plants by
crossing genotypically different but crossable relatives to produce new varieties
with increased productivity and quality. As discussed earlier, because of limited
variability present within the cultivated species of tomato, maximum crosses for
crop improvement are interspecific, between the cultivated tomato and related wild
species. Almost all wild species of tomato have been used for incorporation of
genes/QTLs into cultivated tomato. Many crosses have been generated using red
fruited S. pimpinellifolium for mapping experiments mainly because of its close
genetic relationship with the cultivated tomato. It is easy to develop these crosses
and handling of segregating populations. Because of the presence of a self-
compatible accession (LA716) of S. pennellii, a green fruited and distantly related
tomato was able to cross with cultivated one. This cross was further used to develop
the first molecular linkage map and the high-density molecular map of tomato
(Tanksley et al. 1992). S. pennellii, S. pimpinellifolium, and S. lycopersicoides have
been used extensively, whereas wild species S. chilense and S. peruvianum have
been used occasionally because of their lack of natural crossability with cultivated
tomato. Other breeding populations used time to time are S. peruvianum intra-
specific population (van Ooijen et al. 1994), inter-specific BC1 of S. lycopersi-
cum × S. pimppinellifolium (Grandillo and Tanksley 1996); S. lycopersicum ×
S. habrochaites (Bernacchi and Tanksley 1997) and BC1 S. lycopersicum × S. lyco-
persicoides (Chetelat et al. 2000). These species are used mainly for the incorpora-
tion of few major disease resistance genes into cultivated tomato.
6.8.4 ILS, RILS, NIL and BILS
Wild natural relatives available for any crop species cannot be used directly for crop
improvement as wild species often carry many agronomically undesirable charac-
ters. But wild ancestors of crop plants can be used as source of genetic variation for
disease resistance and abiotic stress tolerance, the characters that loose during
domestication process. For proper and firm phenotyping for gene mapping and QTL
development, some more stable segregating populations have been developed in
tomato. Such lines are recombinant inbred lines (RILs), backcross inbred lines
(BILs), inbred backcross lines or IBC, e.g., BC2S3, BC3S2), advanced backcross
populations (AB; e.g., BC2 and BC3), and introgression lines (ILs).
Development of introgression lines harboring a single homozygous donor
segment introgressed into a uniform, cultivated elite background is such a source
which can help plant breeder to exploit wild species. The mapping power of ILs is
always greater than traditional QTL mapping populations and as a result often larger
numbers of QTLs. Introgression lines are the homozygous immortal lines with
homogenous genetic backgrounds constant phenotype and therefore used for
mapping of many traits. QTLs can be dissected into separate monogenic components,
which increase the reliability of measuring phenotypic traits. IL libraries provide
optimal starting material for the fine-mapping of the mapped loci and for generating
improved breeding lines. Eshed and Zamir (1994) developed a library of 50 intro-
gression lines of S. lycopersicum in M82 cultivar. Each line contains a single
segment of chromosomes from S. pennellii (LA 716) a green fruit bearing species.
After some years they have added additional 26 introgression lines in it. Now total
number of ILs is 76. These 76 ILs cover the entire S. pennellii genome in S. lycop-
ersicum background. Thus using these lines it is possible to divide S. lycopersicum
genome into 107 bins or fragments (Pan et al. 2000). These ILs have been used to
dissect many quantitative traits such as fruit weight, soluble solid content, pH, yield
(Eshed and Zamir 1995) or carotenoid content in relation to fruit colour (Liu et al.
2003). ILs have also been used to clone genes corresponding to several colour muta-
tions and determinate growth phenotype (Pnueli et al. 1998; Ronen et al. 1999),
QTLs controlling sugar content in fruit (Fridman et al. 2000) and fruit weight (Frary
et al. 2000). Genetic locations of putative candidate genes involved in fruit size and
composition were also mapped using this population (Causse et al. 2004). These lines
were used for mapping SSR and CAPS markers as well (Frary et al. 2005). These
tomato ILs have been found to be valuable resources to associate genetic and molecu-
lar information to physical position on chromosomes. These ILs are useful for physi-
cal mapping in tomato genome sequencing project along with FISH mapping. This
permanent source generated from diverse selection of accessions can be screened for
multiple phenotypes to identify alleles of economic importance (Zamir 2001).
Other mapping population in tomato is a total of 99 NILs (near isogenic lines)
and BILs (backcross inbred lines) developed from a cross between processing
tomato cultivar E6203 of S. lycopersicum and a single plant of S. habrochaites
accession LA1777 (Monforte and Tanksley 2000). These lines have several chro-
mosomal segments from the wild donor parent in an S. lycopersicum genetic back-
ground. But in most of the lines, a single-defined introgression from the
S. habrochaites parent in the S. lycopersicum genetic background is present.
Therefore these are almost similar to introgression lines. The complete set of 99
lines is providing availability of more than 85 % of the genome of the LA1777 plant
in cultivated tomato background. One more set of an IL lines have been developed
by introgressions from S. lycopersicoides chromosome fragments in the background
of S. lycopersicum (Canady et al. 2005).
These ILs are also used for high-resolution mapping by developing F2 populations
of crosses between targeted ILs and the recurrent parent. Using this strategy, ILs have
been used to develop NILs for particular chromosomal region for fine-mapping and
map-based cloning of several genes and QTLs controlling various traits. The IL popu-
lations can also be used for MAS pyramiding of important QTLs, as it has been done
in case of tomato yield and soluble solids QTLs (Gur and Zamir 2004). These ILs also
help to generate NILs for particular genes or QTLs, which can be used for validation
of individual effects of QTLs in uniform S. lycopersicum background, marker-assisted
transferring of individual or combination of QTLs to different genetic backgrounds,
identification of presence of association and understanding its nature (linkage or
pleiotropy) between different traits, estimation of QTL × QTL, QTL × genetic back-
ground and QTL × environment interactions, and fine-mapping, possible cloning and
characterization of underlying genes or QTLs (Foolad 2007). Many QTLs controlling
important traits in tomato, including various disease resistance and fruit quality
characters have been identified using such IL led NILs.
6.8.5 Mutant Populations
Mutations are the modifications in the structure of gene. Sometimes these altera-
tions can phenotypically observable. In case of tomato, these are changes in fruit
colour or shape, or an alteration in some physiological process (such as the sucrose
accumulator) which is not visibly detectable. Spontaneous mutations arise usually
in large populations and these are rare events. But tomato breeders find out these
natural mutants and are using as sources of variation to create or enhance existing
genetic resources. M.C. Rick Tomato Genetic Resource Center, Davis phenotypi-
cally characterized and cataloged thousands of tomato monogenic stocks and
mutants in addition to the wild and cultivated accessions. But first effort for devel-
opment of saturated mutants population was done by Emmanuel and Levy 2002
using T-DNA tagging, transposon tagging and enhancer traps. Further, new
activation-tagged MicroTom population was generated and used as potential
resource for gene identification (Mathews et al. 2003; Matsukura et al. 2008). In
2004, Menda et al. developed a comprehensive mutant population in inbred variety
M82 using EMS (ethyl methane sulfonate) and fast-neutron as mutagens. They col-
lected and characterized 13,000 M2 families of tomato (a total of 6,000 EMS and
7,000 fast-neutron families). Mutagen 0.5 % EMS was used to get C/G to T/A tran-
sitions at intensity of LD15. Fast-neutrons treatment at 12 and 15 Gy was used to
crate deletions and rearrangements of chromosomal DNA. Out of these 3,417 muta-
tions have been cataloged which represent phenotypes similar to monogenic mutant
collection at M.C. Rick Tomato Genetic Resource Center, Davis and thousands are
new mutants. The images and description of the mutants are available on Solanaceae
Genome Network site under the title “The Genes That Make Tomatoes”. Other
mutant population is developed by Minoia et al. (2010) in Red Setter variety of S.
lycopersicum. This is processing variety with a vegetative cycle of about 110 days.
The resource was named as LycoTILL because of its use for TILLING. They used
EMS as mutagen at the concentration of 0.7 and 1 %. The population consist of
6,677 M2 and 5,872 M3 families of tomato mutants. The phenotypic data of popula-
tion is available on online database LycoTILL. This resource of Red Setter TILLING
platform is open to all researchers and available via web for requesting mutation
screening services. One more mutant population and database was developed in
Japan by Saito et al. (2011) in the background of MicroTom variety. This variety is
model organism for molecular genomics studies because of its small size of
20–30 cm, short generation time of 3–4 months, easy in proliferation and high effi-
ciency of genetic transformation. 12,000 M2 mutants were developed in MicroTom
variety, 6,000 using EMS and remaining 6,000 using gamma-radiation as mutagens.
Among these mutants, a total of 9,183 independent M2 families comprising
91,830 M2 plants were analyzed for phenotypic alteration and 1,048 individual
mutants were identified. The whole data was integrated into in silico database
TOMATOMA, a relational system interfacing modules between mutant line names
and phenotypic categories. TOMATOMA is also freely accessible database. Similar
kind of work has been initiated by Dr. Rameshwar Sharma’s group in India. They
developed EMS mutagenized TILLING population of 10,000 plants in Arka Vikas
variety (Vankadavath et al. 2009; Sreelakshmi et al. 2010). To characterize this large
population, they also developed software called PHEMONE. Further this group is
also setting up TILLING platform to isolate mutants for desired tomato genes.
6.9 High Throughput Phenotyping
Increase in the genomics information now increases pressure on the breeders for
providing ample and accurate phenotypic data. Precise and efficient phenotypic
characterization is one of the important parameter for the plant breeder for develop-
ing new variety or hybrid superior to existing one. All breeding techniques like,
marker assisted selection, QTL mapping, association mapping, analysis of mutants
population, sampling of several individuals in one or more ecological places requires
proper phenotypic analysis. Manual collection of massive phenotypic data is time
consuming and requires huge man power. Even after collection of massive data, its
management becomes difficult. Thus, the biggest challenge for massive phenotyp-
ing is to design the tools that can allow researchers to collect, access, organize,
integrate, analyze and manage phenotypic database across the population. New
imaging technologies and bioinformatics software are now available, that in combi-
nation with computational tools are helping for high throughput phenotyping. Here
we are discussing about some facilities which can be used for high throughput
phenotyping.
6.9.1 Tomato Analyzer for Fruit Analysis
Tomato analyzer is software to analyze morphological traits like size, shape and
colour of tomato fruit (Gonzalo et al. 2009; Rodríguez et al. 2010). This software
can collect data from scanned digital images and measures 37 attributes related to
two-dimensional shapes in a semi-automated and reproducible manner. To analyze,
fruit should be properly clean, dry and cut with a sharp serrated knife or razor blade
to avoid change in shape. Depending on the type of analysis and the attributes to be
evaluated, fruit should be cut longitudinally or transversely through center and
scanned with a black or very dark background to prevent shadblow. Regarding
shape and size of tomato fruit, it provides information about ten attributes. These
are Basic measurements, Fruit shape index, Blockines, Homogeneity, Distal fruit
end shape, proximal fruit end shape, Asymmetry, Internal eccentricity, Latitudinal
section, and Morphometrics. This software is designed to recognize objects of par-
ticular size and image resolution on the basis of pixels or dots per inch (dpi). It
determines boundaries of the fruit from scanned image and these boundaries are
used for all fruit shape measurements (Brewer et al. 2006). The colour test module
of Tomato analyzer is designed to quantify the colour parameters inside the bound-
aries recognized by the software (Darrigues et al. 2008). RGB (red, green and blue)
is the base for colour measurement. The average RGB values of each pixel is calcu-
lated by colour test module and further translated it to the CIELAB colour space
which uses L*, a*, b* to describe colour. This software is very useful to analyze
large number of tomato fruits at a time. The data may be exported to an excel file in
batch mode. This software is a valuable and effective tool for identifying and con-
firming tomato fruit shape QTLs. This can also objectively classify tomato fruits
into various shape size and colour categories.
6.9.2 Computer Aided Data Acquisition Tool Phenome
To generate and store high throughput phenotypic data of around 10,000 M2

mutant and 1,500 mapping population, Vankadavath et al. (2009) developed
software called PHENOME with mobile computing technology. This software
was developed to meet the needs of cataloging divergence in morphological
traits for a large tomato mutant population raised for TILLING experiment.
This software allow researcher to accumulate, categorize and manage large
volume of phenotypic data. Using this software a large number of individual
tomato plants can be phenotyped with the help of a Palm personal Digital
Assistant (PDA) and build-in barcode scanner. This software works in combi-
nation with a set of data entry forms for phenotype data collection. First of all
“data recording system form” should be filled in PDA. Here, tomato plant
descriptors have been feed in the PDA using CASL (Compact application solu-
tion language). The barcode system attached to the PDA, so that data should be
accurately categorize. The availability of touch-screen for data entry allows
easy navigation to user. The PDA was used to collect phenotypic data from
heterogenous tomato plants.
6.9.3 Image Based High-Content Phenotyping
With the advancement in imaging technologies, image based high content pheno-
typing is emerged as a tool for high throughput phenotyping. This technology is a
comprehensive culture room facility with advance plant imaging cameras from all
sides of the plants, with conveyer facilities to move the pots and bar-coding
facilities to improve accuracy of data storage with advance computational system.

The plants are images several times during their different growth phases to quantify
growth and development of plants. LemnaTec GmbH, Germany is one such com-
pany providing this facility under the name of LemnaTec Scanalyzer (http://www.
lemnatec.com). Normally, they provide one top and two side cameras, but in case
of tomato phenotyping, four side cameras are using along with top view as tomato
does not have main or principal symmetry axis. The morphological parameters gen-
erated for tomato plants through imaging are Moment ratio, Total plant height,
Height over pot, Height hanging down, Plant width, Projected plant area, Rotational
moment, Roundness, and Compactness. This facility can phenotype whole plant,
leaves, branches along with fruits. The data analyzed by computers is presented in
the form of absolute values and in terms of percent change. Data storage, analysis
and mining facilities are available with a speedily accessible array of hard drives on
a server. These comprehensive high-content phenotyping thus provide analysis of
the visual results of regularly grown plants. Whole phenotypic data can be quanti-
fied in the time series throughout the life span for each character by generating
dynamic phenotypic data for each plant. This data can provide growth rate, stay-
green period, flowering period, ripening dynamics and behavior of plants under
stress or nutrient deficient conditions. Along with this, it also provide dynamic
information like closing and opening of stomata using IR-imaging, behavior of
plants against disease or pastes, and reaction of plants under different stress condi-
tions like salt, heat or drought. The valuable data obtained from such phenotyping
may act as powerful tool to identify relation between genes and any type of devel-
opmental or phenotypic trait for QTLs.
6.10 Aspects of Tomato Breeding
Use of plant breeding techniques to develop new improved tomato cultivars was
started about 200 years ago in Europe, mainly in Italy; whereas in the USA, it was
started only a century ago. Early domestication and breeding programs on tomato
has mainly focused on productivity and resistance to biotic stresses as tomato breed-
ers have mainly selected for yield, uniformity and disease resistance (Zamir 2001).
The initial breeding objectives of tomato breeding were development of multipur-
pose cultivars to meet several needs of fresh market as well as processing industries.
Later, these objectives were changed as per requirements of fresh market tomatoes
and processing tomatoes, separately, due to different quality requirements. As a
result of which today’s fresh market and processing cultivars are quite distinct. Even
after that few characters like disease resistance, broad adaptability, early maturity,
ability to set fruit at adverse environmental conditions, resistance to rain-induced
cracking, tolerance to major ripe-fruit rots, adequate vine cover, fruit firmness, and
several other fruit quality are still common in both type of tomatoes (Foolad 2007).
Some of the major tomato breeding aspects are briefly discussed below where phe-
notyping plays an important role.
6.10.1 Yield
The universal goal of breeding in any crop is to raise production of an economically

important part of the plant. Similar way, for both fresh market and processing
tomato breeding programs, increase in fruit yield per unit area is the major objec-
tive. Most of the time, basis for release of new cultivar is higher yield over existing
varieties, along with few quality, disease resistance, tolerance to abiotic stresses,
earliness, and improved fruit sugar contents. The production of tomato increases
many fold globally in this last century and its main reason is an improvement in the
yield along with increase in the cultivated area and improved cultivation practices.
It is estimated that, on an average about half of the increase in crop productivity has
been due to cultivar improvement through plant breeding. As yield is quantitative
character and large number of genes are associated with it, high throughput pheno-
typing for QTL analysis along with molecular marker data can play important role
for increase in yield of tomato.
6.10.2 Fruit Quality
Fruit quality is the second most important objective after yield. Characters for which
both fresh market and processing tomato breeding have been concentrated are fruit
size, shape, colour, firmness, total solids, ripening, nutritional quality and flavor.
Total soluble solid of the fruit is important for the processing industry and therefore
received more interest of breeders than any other fruit trait. 4–7.5 % total solid of its
fresh weight is present in the cultivated tomato and the soluble (SS) and insoluble
solids (ISS) account for about 75 and 25 %, respectively. Quality of the processing
products like tomato juice, catsup, sauce, soup, and paste are depends on the viscos-
ity of the fruit pulp. There are some wild species of tomato, like S. pimpinellifolium,
S. chmielewskii and S. cheesmanii, where concentration of SS is higher than culti-
vated tomato. Even after genetic diversity is available, breeders achieved very little
success in combining high SS with high yield. This may be due to complex nature
of this characteristic.
Colour of fruit is other quality aspect which is important in tomato. Red colour
of tomato is popular in both processing and table purpose tomato, but variable fruit
colours are also desirable in fresh market tomato. In processing industry, colour of
tomato influences the grades and standards of the processed products. In fresh mar-
ket tomato, fruit colour has significant effect on its marketability. The attention to
fruit colour has recently been increased due to the increasing knowledge of the
health benefits of different carotenoids present in fruits and vegetables. Tomato is a
good source of lycopene which is an effective natural antioxidant. Thus, an impor-
tant goal of many tomato breeding programs is to develop cultivars with enhanced
fruit lycopene content. Fruit firmness, pH, acidity, and vitamin contents are other
important tomato quality characteristics. Acidity influences the storage capacity of
processed tomato. Tomatoes are good source of vitamins A and C. Plant carotenoids,
in particular β-carotene, a major carotenoid in orange–yellow tomatoes, are the pri-
mary sources of vitamin A in tomato. Taste and flavor are other important quality
aspects which breeders are considering for breeding program. Numerous aromatic
volatile compounds play a main role in tomato fruit flavor. These are complex traits
which rely on many components that are needed in appropriate proportion. Major
components of the taste are sugars and acids and their ratio within the fruit. High
levels of SS are directly correlated with tomato flavor, and previous research
approved that tomato flavor can be improved by breeding for high SS and high acid-
ity. Fructose and citric acid are more important for sweetness and sourness
in tomato. pH of the fruit is a better objective measure of sharp taste. A single
incomplete-dominant gene (Fgr) has been identified in S. habrochaites that increases
the proportion of fructose over glucose which contributes for fruit sweetness (Levin
et al. 2000). It is difficult to phenotype for quality characters but high throughput
phenotyping along with metabolite profiling can help to improve the quality aspects
(Gur et al. 2004).
6.10.3 Fruit Ripening
Table purpose tomatoes are harvested at early stage before ripening and transported
to the markets for sale. The reason for this is to avoid post-harvest damage to fruits
caused by various physical and biotic or abiotic factors, as tomatoes are firm at
breaker than fully ripe stage. During transportation or in storage these tomatoes are
then allowed to ripe. But under storage ripening is not natural and therefore affects
quality attributes of fresh tomato. Thus improvement of self life without affecting
quality attributes of fruit is important objective to tomato breeding. Many studies on
tomato ripening process has discovered important genes and pathways involved in
the fruit ripening and softening process of tomato. The role of ethylene in initiation
of ripening is now well established (Alba et al. 2005). An enzyme polygalacturo-
nase (PG) in fruit softening has also been well characterized (Giovannoni 2004).
Identification and characterization of several ripening mutants such as never ripe
(Nr), non ripening (nor), and ripening inhibitor (rin), genes help to understand rip-
ening process. The activity of PG is almost absent in these mutants during ripening.
Another ripening mutant of tomato fruit which show signs of prolonged keeping
quality is controlled by a single gene alc (Kinzer et al. 1990). Traditional breeding
utilized Nr, nor, and rin genes and developed lines and cultivars with delayed ripen-
ing. Hybrids developed using these mutants in heterozygous conditions have been
successful in providing for delayed ripening, longer shelf life, and increased firm-
ness. Many commercial cultivars of tomato are developed using these mutants. In
future good understanding of the ripening process and the contributing genetic and
physiological factors is necessary to improve self life by understanding fruit ripen-
ing process. Proper phenotyping using software like Tomato analyzer can help in
breeding program for these characters.
6.10.4 Mechanical Harvesting
There is necessity for one time harvest in processing tomatoes. This characteristic can
allow the mechanization of fruit harvesting by machinery. For mechanical harvesting
of processing tomato cultivars should have determinate growth habit, small vine size,
concentrated flowering and fruit set, slow fruit maturing and softening, and high har-
vest index. Currently, whole processing tomato production in the USA is mechanized,
and almost all commercial cultivars are compact and highly determinate which are
suitable for once-over machine harvest. Similarly, most of the fresh market tomato
cultivars for field production are determinate but with larger vine than processing
types. The determinate growth habit in tomato is due to self-pruning (sp) gene. The sp
gene was fine mapped, cloned, and physically characterized in tomato (Pnueli et al.
1998). The introduction of the sp allele into processing tomato cultivars changed the
architecture of tomato plant with a major modification. But somehow fruits of deter-
minate type plants in all cultivar backgrounds have less sugar content than indetermi-
nate types. The fruit yield and quality of determinate plants are often inferior to those
of indeterminate plants (Georgelis et al. 2004). High throughput phenotyping with
larger population can help to find out proper recombinants to solve this problem.
6.10.5 Insect Resistance
Due to breeding for yield and quality aspects in the early stage of improvement program
the traits like insect resistance was side lined. Therefore the cultivated tomato cultivars
are attacked by numerous insects like, mites, whiteflies, aphids, Lepidoptera (e.g.,
tomato fruit worm, beet armyworm, cotton bollworm, southern armyworm, soybean
podworm, and Egyptian cottonworm), Coleoptera (e.g., Colorado potato beetle and
tobacco flea beetle), Diptera (e.g., leafminers and fruit fly), thrips, sinkbugs, and
cutworms. These insects can cause overwhelming losses. Earlier, as compare to disease
resistance, insect resistance in tomato has received considerably less attention. But
recently few commercial cultivars have been developed with specific insect resistance.
Many wild relatives like S. habrochaites and S. pennellii are good source of insect resis-
tance for cultivated tomato (Farrar et al. 1994). Resistance to at least nine insect species
has been reported in S. pennellii, including greenhouse whitefly, carmine and two-spotted
spider mites, and the potato aphid. Some insect resistance genes have also been reported
in S. lycopersicum var. cerasiforme, S. pimpinellifolium, S. cheesmanii and
S. chmielewskii, S. peruvianum and S. chilense (Farrar et al. 1994). Many of these
resources have not been utilized for insect resistance breeding till date, due to incorpora-
tion of some undesirable characters along with resistance characters. Breeding for insect
resistance in tomato has generally encountered more difficulties than breeding for dis-
ease resistance because of linkage drag (Foolad 2007). It is expected that identification
of markers associated with insect resistance and their proper phenotyping will help ease
some of the difficulties in developing insect resistant cultivars.
6.10.6 Disease Resistance
Diseases are major culprit for the economic losses as crop damage occur throughout
the world in processing and fresh market tomato industries (http://faostat.fao.org).
Tomato is susceptible to more than 200 diseases which are caused by pathogenic
fungi, bacteria, viruses, or nematodes. The phenotypic descriptors are also available
for diseases of tomato plant for more than 30 of the major tomato diseases natural
wild source of resistance has been identified. These mainly include diseases like
fusarium wilt, verticillium wilt, root-knot nematode, alternaria stem canker, gray
leaf spot, and some bacterial and viral diseases. Wild tomato like S. pimpinellifo-
lium, S. peruvianum, and S. habrochaites species have been utilized as the source of
resistance for almost all tomato diseases. Both vertical and horizontal resistances
are available for some diseases like late blight and powdery mildew. Proper pheno-
typing is very important in case of disease resistance breeding as all original char-
acterization, disease evaluation, and incorporation of resistance genes required
thorough phenotypic selection. Still today many of the disease resistance breeding
in tomato is using similar protocols. During the past two decades, the use of molec-
ular markers and MAS techniques have facilitated identification, mapping, and
transferring of many disease resistance genes and quantitative trait loci (QTLs) in
tomato with the help of classical phenotyping. The use of marker technology and
disease phenotyping for disease resistance breeding in tomato is a routine proce-
dure. As breeding for disease resistance remains a major goal of most public and
private tomato breeding programs, imaging technology for disease resistance breed-
ing can open the doors for new resistant varieties.
6.10.7 Abiotic Stress Tolerance
Cultivated tomato is more sensitive to different environmental stresses, including

salinity, drought, excessive moisture, extreme temperatures, mineral toxicity and
deficiency, and environmental pollution due to nonstop selection procedure at favor-
able conditions. As a result of which limited genetic variation for abiotic stress tol-
erance remain within the cultivated tomato and most of the commercial cultivars are
considered sensitive to different abiotic stresses. But almost all wild relatives of
cultivated tomato viz. S. chilense, S. peruvianum, S. pennellii, S. pimpinellifolium,
S. habrochaites, S. cheesmanii, S. chmielewskii, S. parviflorum S. lycopersicoides,
S. rickii, S. juglandifolium and S. ochranthum are tolerant to one or another stress
(Foolad 2007). Several tomato wild species have been utilized for genetic and phys-
iological characterization of abiotic stress tolerance and for breeding programs by
Foolad and Lin (1998; Foolad et al. 2001, 2003). But as this is complex character,
there is very few report of stress-tolerance via traditional breeding. For successful
tomato production under environmental stress, tolerance may be needed at all major
stages of plant development, including seed germination, the vegetative stage, and
flowering and fruit production. Each developmental stage may require a different
screening procedure and simultaneous or sequential screening may be impractical
or impossible with manual efforts. Phenotypic selection under field conditions is
difficult because uncontrollable environmental factors adversely affect the accuracy
and repeatability of the experiments. Selection for stress tolerance using phenotypic
measurements also requires specialized personnel and extensive investments in
field nurseries or greenhouse facilities. High throughput phenotyping using culture
rooms well equipped with imaging facilities and conveyers may help to in-depth
characterization of these phenotypes to correctly correlate it with QTLS and finally
with stress tolerance genes.
6.11 Use of Tomato Phenotyping for Breeding
It is very important to understand the use of phenotyping under breeding process.

Map-based or positional cloning is one such a method in which identifying or clon-
ing of a gene responsible for a particular phenotype can be identified by looking for
the linkage phenotype to the markers, whose physical location in the genome is
known. Thus here particular phenotype in the breeding population is correlated with
the markers. Once the phenotypically defined trait has been closely linked to one or
more DNA markers, it becomes possible to clone the gene. The benefit of this tech-
nique is that the investigator can start even without prior information of the gene or
its product. For characters which are governed by many genes, quantitative trait loci
(QTLs) are also identified using same method. As mentioned before for mapping
phenotypic traits to genetic map, near isogenic lines (NILs) are most suitable popu-
lation, but bulk segregation analysis (BSA) in F2 generation on the basis of con-
trasting alleles also helps in high resolution mapping (Churchill et al. 1993). Further
chromosome walking or chromosome jumping are used to isolate any gene related
to a desired trait but recently the flanking marker approach (chromosome landing),
which helps to land directly on that part of chromosome where target gene is present
has found more popularity (Tanksley et al. 1995).
Tomato is the first plant in which disease resistance gene for Pseudomonas syrin-
gae pv. tomato have been identified using phenotypic correlation with marker data.
Further, many genes have been tagged in similar way, including genes for disease
resistance, biochemical metabolism, as well as regulators of developmental pro-
cesses and fruit characteristics. A comprehensive list of genes and QTLs isolated
from tomato using map based cloning is presented in Table 6.3. Many single gene
mutants related to fruit ripening are present in tomato. The genes responsible for
some mutants are cloned and other have been mapped on different chromosomes
viz; nor (Giovannoni et al. 1995), pat (Beraldi et al. 2004), joinjtless-2 (Budiman
et al. 2004) and sun (van der Knaap et al. 2004). Some developmental mutants like
Lazy-2 (Behringer and Lomax 1999), poc (Madishetty et al. 2006), rmc (Larkan
et al. 2007) as well as some genes responsible for disease resistance like Cf-ECP2
Table 6.3 Genes/QTLs cloned by positional mapping from tomato

Gene Phenotype Chromosome Reference
Si Self incompatibility 1 Bernatzky (1993)
Pto Pseudomonas syringae pv. 5 Martin et al. (1993)
tomato resistance
Fen Fenthion sensitivity 5 Martin et al. (1994)
jointless Control flower abscission 11 Zhang et al. (1994)
zone development
sucr Sucrose accumulator 3 Chetelat et al. (1995)
Hero Nematode resistance 4 Ganal et al. (1995)
rin Ripening inhibitor 5 Giovannoni et al. (1995)
nor Non-ripening 10 Giovannoni et al. (1995)
ls Lateral suppressor 7 Schumacher et al. (1995)
fw2.2 Major fruit weight locus 2 Alpert and Tanksley (1996)
Cf-2 Cladosporium fulvum 6 Dixon et al. (1996)
resistance
Ms14 Pollen abortion and male 11 Gorman et al. (1996)
sterility
Tm-2a Tobacco Mosaic Virus (TMV) 9 Pillen et al. (1996)
resistance
Sw-5 Tomato spotted wilt virus 9 Brommonschenkel and
(tospovirus) resistance Tanksley (1997)
l2 Fusarium oxysporum f. sp. 11 Ori et al. (1997)
lycopersici resistance
hp High pigment 2 Yen et al. (1997)
LeMT 1-4 Metallothioneins-like 4, 6 and 9 Giritch et al. (1998)
gene family
Mi Root-knot nematode resistance 6 Kaloshian et al. (1998)
Cf-4 Cladosporium fulvum resistance 1 Thomas et al. (1998)
Sp Self pruning 6 Pnueli et al. (1998)
Lazy-2 Shoot gravitropism 5 Behringer and Lomax (1999)
Cf-ECP2 Cladosporium fulvum resistance 1 Haanstra et al. (1999)
through factor ECP2
ovate Pear shaped fruit 2 Ku et al. (1999)
Asc Alternaria alternata 3 Mesbah et al. (1999)
(stem canker) resistance
Fgr Fructose to glucose ratio 4 Levin et al. (2000)
jointless-2 Control flower abscission zone 12 Zhang et al. (2000)
development
Bs4 Xanthomonas campestris pv. 5 Ballvora et al. (2001a, b)
Vesicatoria resistance
sun Early fruit development 7 Van der Knaap and Tanksley
(2001)
fer Iron-uptake response in roots 6 Ling et al. (2002)
Mi-9 Root-knot nematode resistance 6 Ammiraju et al. (2003)
Spr2 Suppressor of prosystemin- 6 Li et al. (2003)
mediated response 2
(continued)

Gene Phenotype Chromosome Reference
OI-4 Powdery mildew (Oidium 6 Bai et al. (2004)
neolycopersici) resistance
pat Parthenocarpic fruit 3 Beraldi et al. (2004)
jointless-2 Control flower abscission zone 12 Budiman et al. (2004)
development
sun Early fruit development 7 Van der Knaap and Tanksley
(2004)
Brix9-2-5 Increase sugar yield of tomato 9 Fridman et al. (2004)
LeEix1 Fungal defense response 7 Ron and Avni (2004)
LeEix2
ACX1A Impaired JA biosynthesis 8 Li et al. (2005)
Mi-3 Root-knot nematode resistance 12 Yaghoobi et al. (2005)
Dgt Slow gravitropic response 1 Oh et al. (2002)
(Haanstra et al. 1999), Mi-9 (Ammiraju et al. 2003), Mi-3 (Yaghoobi et al. 2005),
Ol-4 (Bai et al. 2004) and QTLs like sucr (Chetelat et al. 1995) and Fgr (Levin et al.
2000) have also been positionally mapped by phenotypically screening populations
along with marker data.
6.12 Conclusions
In the era of genomics, to correlate huge genotypic data with the agronomically
important traits, thorough phenotyping is most important. In recent years, world-
wide efforts on tomato genomics have generated massive data pertaining to differ-
ent aspects of tomato fruit biology. High throughput phenotyping facilities in
combination with molecular markers are increasing the speed of breeding procedure
to identify new QTLs for complex characters like yield, fruit quality and abiotic
stress tolerance. Many mutant populations have also been generated to correlate
change is phenotypes to change in gene structure using TILLING method. Analysis
of large mutant population related to quality aspects is possible only with the help
of proper phenotyping platforms and software like Tomato Analyzer. Thus integra-
tion of genomics with phenomics is the future of tomato crop improvement and
breeding program.
6.13 Databases for Tomato Information
EU-SOL BreeDB database: https://www.eu-sol.wur.nl/

Genes that Make Tomatoes: http://zamir.sgn.cornell.edu/mutants/index.html
Kazusa Full-length Tomato cDNA Database: http://www.pgb.kazusa.or.jp/kaftom/
LycoTILL Tomato Mutant DB: http://www.agrobios.it/tilling/index.html

MiBASE Micro-Tom Database: http://www.pgb.kazusa.or.jp/mibase/
PlantGDB Solanum lycopersicum: http://www.plantgdb.org/SlGDB/
SOL genomics network: http://solgenomics.net/
Solanaceae source: http://www.nhm.ac.uk/research-curation/research/projects/
solanaceaesource/
Tomato functional genomics database: http://ted.bti.cornell.edu/
Tomato National BioResource Project: http://tomato.nbrp.jp/indexEn.html
TOMATOMA Tomato mutant archive: http://tomatoma.nbrp.jp/index.jsp
Tomato SNP: http://www-plb.ucdavis.edu/labs/maloof/tomatosnp/index.asp
Tomato metabolites: http://webs2.kazusa.or.jp/komics/
Tomato Genetics Resource Center, University of California, Davis, USA: http://
tgrc.ucdavis.edu/
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Index
A B
Abiotic stress Bacterial blight
in chickpeas artificial inoculation
cold tolerance, 125–126 kresek phase creation method, 12
salinity, 124 leaf blight phase creation method,
terminal drought, 124–125 11–12
in groundnut mass culturing, 11
aluminum toxicity, 152 pathogen and isolation, 10–11
drought tolerance, 148–151 SES scores, 12–13
high temperature tolerance, 151 Biological nitrogen fixation (BNF), 163
salinity tolerance, 151–152 Biotic stress
in sorghum, 104 in chickpeas
drought tolerance, 105–106 ascochyta blight, 122, 123
high temperature tolerance, bruchids, 123
106–107 cyst nematode, 123
salinity and acid soils, 107–108 fusarium wilt, 122
in tomatoes, 203–204 leaf miner, 123
in wheat pod borer, 123
canopy temperature, 67 in wheat
heat and drought tolerance, 66, 67 pest resistance, 66
Aluminum toxicity rust disease resistance, 60–62, 64–66
in groundnut, 152 stem rust pathogen, UG99 resistance
in sorghum, 107–108 of, 62–63
Alveograph test, 72 Bipolaris oryzae. See Brown spot
Anthracnose Blast
accessions resistance, 103 mass culturing and artificial inoculation,
field screening, 102 13–16
greenhouse screening, 102–103 pathogen and isolation, 13
Arachis hypogaea L. See Groundnut Breeding techniques
Artificial inoculation in groundnut, 141
bacterial blight mass selection, 142
kresek phase creation method, 12 mutation breeding, 143–144
leaf blight phase creation method, objectives, 145
11–12 pedigree and bulk-pedigree methods, 142
for neck blast, 15 population improvement, 143
Atherigona soccata. See Sorghum shoot fly single-seed descent method, 142–143
DOI 10.1007/978-1-4614-8320-5, © Springer Science+Business Media New York 2013
206 Index
Breeding techniques (cont.) quality tests

sources of variability, 143 alveograph, 72
wide hybridization, 144 falling number test, 71
in tomatoes, 194 farinograph, 71–72
abiotic stress tolerance, 203–204 mixograph, 72
crop yield, 200 sedimentation test, 71
disease resistance, 203 shuttle breeding program, 51–52
fruit quality, 200–201 in Toluca sites, 51, 52
fruit ripening, 201 Colletotrichum sublineolum. See Anthracnose
insect resistance, 202 Colour test module, of Tomato analyzer, 198
mechanical harvesting, 202 Crossing block (CB), 54
never ripe genes, 201 elite breeding source material, 55
non ripening genes, 201 entries, 55
objectives, 199 F1 seed
ripening inhibitor genes, 201 and segregating populations, 56–58
Brown planthopper (BPH). See Planthoppers top/back cross, 55–56
Brown spot, 22, 23 male/female master list, 55
CTD. See Canopy temperature depression
(CTD)
C
Callosobruchus chinensis, 123
Calocoris angustatus. See Head Bug D
Canopy temperature depression (CTD), 67–68 Disease index (DI)
Carbon isotope discrimination (CID), 42, 150 false smut, 25
Charcoal rot, 103–104 sheath blight, 19
Chickpeas sheath rot, 24
atmospheric nitrogen fixation, 120 stem rot, 26
biochemical markers, 128–129 DNA marker technology, in sorghum, 88–89
economic value, 120 Drought response index (DRI), 42
genotype-to-phenotype mapping, 129 Drought tolerance
high throughput phenotyping, 130 genetic difference, 39
marker assisted breeding, 131 in groundnut
molecular markers, 128, 129 annual production loss, 148
phenotyping empirical breeding approach, 150
abiotic stress, 124–126 harvest index, 150
biotic stress, 122–124 moisture stress, 149
data management, 127–128 SPAD chlorophyll meters, 150–151
importance of, 120–121 trait-based approach, 150
role of, 121–122 transpiration efficiency, 150
Chilo partellus. See Spotted stem borer water use efficiency, 150
Cicer arietinum L. See Chickpeas selection criteria
CIMMYT wheat breeding program carbon isotope discrimination, 42
bread wheat quality classification, 73, 74 constitutive features, 40
in Ciudad Obregon sites, 51, 52 integrative/secondary features, 41–42
crossing block, 54 primary features, 43
elite breeding source material, 55 for yield, 41
entries, 55 in sorghum, 105–106
F1 seed, 55–58 stress intensity, 39
male/female master list, 55 in wheat, 66, 67
CTD, 67
genetic yield gains, historical analysis of,
52–53 E
grain yield potential, 53–54 Elite Spring Wheat Yield Trial (ESWYT)
iron and zinc concentrations, 73, 75 genetic diversity, 59
Index 207
management practice and instructions, 59 leaf spot (see Leaf spots, in groundnut)
variety release, 59–60 rust disease, 152–155
Exserohilum turcicum. See Leaf blight stem and pod rot, 156–157
genetic transformation, 144–145
groundnut borer, 162–163
F importance of, 138–139
Falling number test, 71 insect pests, resistance to
False smut aphids, 161
disease index, 25 jassids, 162
SES score, 25 leaf miner, 162
spore suspension, 24 thrips, 161–162
Farinograph test, 71–72 marker technologies, 144–145
Fresh market tomatoes, 183–184 maturity duration, 146–147
nematodes, 160–161
reproduction mode, 140–141
G rust-red flour beetle, 163
Genetic transformation simples sequence repeat markers, 144
in groundnut, 144–145 storage pests, 162–163
in sorghum, 89–90 taxonomy, 139–140
Grain mold, in sorghum, 101–102 virus diseases, 157–159
Groundnut yield and yield attributes, 147–148
abiotic stress Groundnut rosette disease (GRD), 144, 157–158
aluminum toxicity, 152
drought tolerance, 148–151
high temperature tolerance, 151 H
salinity tolerance, 151–152 Head bug
allergy, 145 damage evaluation, 100–101
artificial hybridization, 140–141 hot-spot locations, 99
bacterial wilt, 159–160 infester-row technique, 100
biological nitrogen fixation, 163 no choice head cage technique, 100
breeding methods, 141 sowing dates, 99–100
mass selection, 142 Helicoverpa armigera, 123
mutation breeding, 143–144 Heterodera ciceri, 123
objectives, 145 High throughput phenotyping
pedigree and bulk-pedigree methods, chickpeas, 130
142 tomatoes
population improvement, 143 image based high-content phenotyping,
single-seed descent method, 142–143 198–199
sources of variability, 143 PHENOME, 198
wide hybridization, 144 Tomato analyzer, 197–198
classification, 139–140 Hopper burn symptoms, 27, 28
confectionary and nutritional traits
blanchability, 165
flavour and sugars, 165 I
haulm yield and quality, 167 IBWSN. See International Bread Wheat
iron and zinc content, 166 Screening Nursery (IBWSN)
oil content and fatty acids, 165–166 Image based high-content phenotyping,
protein content, 166 198–199
seed colour, 164 Infester row technique
seed size and shape, 164 head bug, 100
sound mature kernels, 164 sorghum midge, 98
fungal diseases Interlard-fishmeal technique, 95
aflatoxin contamination, 155–156 International Bread Wheat Screening Nursery
Aspergillus infection, 155–156 (IBWSN), 58–59
208 Index
K O
Kjeldhal method, 166 Organic matter digestibility (OMD)
Kresek phase creation method, 12 haulm quality, 167
sorghum, 109, 110
L
Leaf blight P
phase creation method, 11–12 Palm personal Digital Assistant (PDA), 198
in sorghum, 102–103 Panicle grain mold rating (PGMR), 101
Leaf spots, in groundnut Peanut. See Groundnut
detached leaf method, 153–155 Peanut bud necrosis disease (PBND), 158
early and late, 152 Peanut clump virus disease (PCVD), 159
field screening, 153 Peanut mottle virus disease (PMVD), 159
9-point scale, 153, 154 Peanut stem necrosis disease (PSND), 159
Leaf water potential (LWP), 40, 42, 105 Peanut stripe virus disease (PStVD), 158–159
Liriomyza cicerina, 123 Pedigree method
LycoTILL database, 196 groundnut, 142
sorghum, 86–87
PHENOME, 198
M Planthoppers
Macrophomina phaseolina. See Charcoal rot damage symptoms, 27–28
Marker assisted backcrossing (MABC) phenotyping
method, for groundnut, 143 under field conditions, 28–29
Marker assisted breeding (MAB), in under greenhouse conditions, 29–30
chickpeas, 131 scoring of plants, 28
Marker assisted selection (MAS) technique Pod rot, in groundnut, 156–157
chickpeas, 128, 129 Positional cloning method, genes/QTLs
shoot fly resistance, in sorghum, 89 isolation, 204–206
Mass selection Processing tomatoes, 184
in groundnut, 142 Pyricularia grisea. See Blast
in sorghum, 86
Melanaphis sacchari. See Sugarcane aphid
Meloidogyne spp. See Root-knot nematodes R
Metabolisable energy (ME), for haulm quality, Rapid visco analyser (RVA), 70
167 Relative water content (RWC), 41, 42
Mexico–Kenya shuttle breeding scheme, Rhizoctonia solani. See Sheath blight
62, 63 Rice
MicroTom variety, 196–197 diseases
Mixograph test, 72 bacterial blight, 10–13
Modified seedbox screening test (MSST), blast, 13–16
29–30 brown spot, 22, 23
Mutation breeding, for groundnut, false smut, 24–25
143–144 rice tungro disease, 21–22
sheath blight, 17–21
sheath rot, 23–24
N stem rot, 25–27
Neck blast drought tolerance, 39–43
artificial inoculation for, 15 molecular breeding programmes, 9
SES scale, 16 pests
No choice cage-screening technique, 95 planthoppers, 27–30
No choice head cage technique root-knot nematode, 34–39
head bug, 100 stem borers, 30–33
sorghum midge, 98–99 Rice tungro bacilliform virus (RTBV), 21
Index 209
Rice tungro spherical virus (RTSV), 21 artificial hybridization, 84–85

Root-knot nematodes basic and hybrid races, 83
biology, 35 classification, 83
giant cell formation, 35 commercial grain characteristics, 83, 84
inoculation of plants, 36–37 crop improvement methods
inoculum types, 36 bulk population breeding, 87
occurrence, 34 cultivar options, 85
phenotyping protocol hybridization-based methods, 86–87
egg mass number, 38 mass selection, 86
glasshouse conditions, 37 pedigree method, 86–87
number of eggs, 39 pollination control mechanisms, 85
number of galls, 38 population improvement methods, 87
root gall index, 37 pure line selection, 85–86
scoring parameters, 37 DNA marker technology, 88–89
pure cultures, maintenance of, 36 floral initiation, 84
second stage juveniles, 35 forage/fodder yield, 109–110
species/isolate identification, 36 genetic transformation technology, 89–90
Rust disease host-plant resistance
in groundnut anthracnose and leaf blight, 102–103
detached leaf method, 153–155 charcoal rot, 103–104
early and late, 152 grain mold, 101–102
field screening, 153 head bug, 99–101
9-point scale, 153, 154 shoot fly, 94–95
in wheat sorghum midge, 97–99
disease resistance, 60–62 spotted stem borer, 95–97
field inoculation techniques, 65 sugarcane aphid, 97
greenhouse/growth chamber tests, witch weed, 104
65–66 importance of, 82
reliable environmental conditions, 64 nutrition quality
screening methodology, 64–65 β-carotene contents, 108
severity assessment, 65 grain Fe and Zn concentrations, 109
protein content, 108
reproduction mode, 84–85
S stalk sugar traits, 110
Sarocladium oryzae. See Sheath rot taxonomy, 83
Scirpophaga incertulas, 30–33 yield and yield attributes
Sclerotium oryzae Catt. See Stem rot grain and stover yield, 93
SES. See Standard Evaluation System for Rice height, 93
(SES) maturity, 94
Sheath blight, 17–21 photoperiod sensitivity, 92
Sheath rot, 23–24 Sorghum bicolor (L.) Moench. See Sorghum
Sodium dodecyl sulphate (SDS) sedimentation Sorghum midge, 97
tests, 70 damage evaluation, 99
Sorghum hot-spot locations, 98
abiotic stress, 104 infester row technique, 98
drought tolerance, 105–106 no choice head cage technique, 98–99
high temperature tolerance, 106–107 sowing date, 98
salinity and acid soils, 107–108 Sorghum shoot fly, 94–95
adaptation Soxhlet method, 165–166
postrainy season, 92 Spotted stem borer
rainy season, 91 artificial infestation, 96
agronomically important traits, 90 damage evaluation, 96–97
animal feed quality, 109–110 hot-spot locations, 96
210 Index
Spotted stem borer (cont.) carotenoid, 178

indication of infestation, 95–96 characterization descriptors, 184–191
mass rearing, 96 colours of, 181, 182
sowing date, 96 core collection, 193
Standard Evaluation System for Rice (SES) developmental stages, 181, 182
bacterial blight, 12 environment and site descriptors, 184
brown spot, 22, 23 evaluation descriptors, 184
false smut disease, 25 fresh market tomato, 183–184
neck blast, 15, 16 genes/QTLs isolation, map-based/
rice tungro disease, 22 positional cloning method,
sheath blight, 19, 20 204–206
sheath rot, 23, 24 genomes, 178
stem borer severity, 32 high throughput phenotyping,
stem rot severity, 26, 27 197–199
Standard seedbox screening technique (SSST), history, 179
29, 30 introgression lines, 194–195
Stem borers management descriptors, 184
dead heart damage, 30–31 mutant populations, 196–197
larval damage, 31 natural variation, 192–193
limitations, of field evaluation, 33 near isogenic lines, 195
test insects nutritional value and sugar content, 178
egg mass, 32 origin, 179
grain yield, 33 passport descriptor, 184
neonate larvae, 33 phenotypic catalog
vegetative phase, 33 disease and stress response, 192
white heads, 31 flowering time, 185
Stem rot flower morphology and colour, 191
of groundnut, 152, 156–157 fruit colour, 191
of rice fruit ripening, 192
disease index, 26 fruit size and morphology, 191
isolation of fungus, 25–26 inflorescence structure, 185
SES scale, 26, 27 leaf morphology and colour, 185
yield loss, 25 plant size and habit, 185
Stenodiplosis sorghicola. See Sorghum midge seed, 185
Striga spp. See Witch weed sterility, 192
Sugarcane aphid, 97 processing tomatoes, 184
Pto-mediated resistance, 183
quantitative trait loci, 195–196
T recombinant inbred lines, 194
Tomatoes shapes and sizes, 181
advantages, 182 TOMATOMA database, 197
backcross inbred lines, 195 Tomato spotted wilt virus (TSWV), 158
botanical description, 180–182
breeding techniques, 194
abiotic stress tolerance, 203–204 U
crop yield, 200 Uniform blast nursery (UBN) method, 14
disease resistance, 203 Ustilaginoidea virens. See False smut
fruit quality, 200–201
fruit ripening, 201
insect resistance, 202 W
mechanical harvesting, 202 Wheat breeding
never ripe genes, 201 abiotic stress
non ripening genes, 201 canopy temperature, 67
objectives, 199 heat and drought tolerance, 66, 67
ripening inhibitor genes, 201 biofortification, 73
Index 211
biotic stress IBWSN, 58–59

pest resistance, 66 objective, 51
rust disease resistance, 60–62, 64–66 spectral radiometers, 68
stem rust pathogen, UG99 resistance stomatal aperture traits, 68
of, 62–63 White backed planthopper (WBPH).
challenges, 50–51 See Planthoppers
CIMMYT (see CIMMYT wheat breeding Witch weed, 104
program)
ESWYT
genetic diversity, 59 X
management practice and instructions, 59 Xanthomonas oryzae pv. oryzae. See Bacterial
variety release, 59–60 blight
grain quality
breeding priorities/strategies, 68
hardness, 69 Y
protein content, 70 Yellow stemborer (YSB).
starch, 69–70 See Stem borers

Phenotyping For Plant Breeding 2013

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Phenotyping For Plant Breeding 2013

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Siva Kumar

Panguluri · Are Ashok Kumar

Phenotyping for Plant

ISBN 978-1-4614-8319-9 ISBN 978-1-4614-8320-5 (eBook)

Library of Congress Control Number: 2013946933

© Springer Science+Business Media New York 2013

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

A phenotype is any measurable characteristic or trait of a plant and is a result

This book is intended to serve as a useful guide to practicing plant breeders to

Tampa, FL, USA Siva Kumar Panguluri

1 Phenotyping Rice for Molecular Plant Breeding ................................... 1

Index ................................................................................................................. 205

Arun Balasubramaniam Department of Genetics and Plant Breeding, Institute of

M.S. Madhav, G.S. Laha, A.P. Padmakumari, N. Somasekhar,

Keywords Rice • Diseases • Pests • Phenotype

M.S. Madhav (*) • S.K. Mangrauthia • B.C. Viraktamath

1.1 Phenotyping of Rice Diseases

1.1.1 Major Rice Diseases

1.1.1.1 Bacterial Blight of Rice

Pathogen and Its Isolation

Bacterial blight of rice is caused by Xanthomonas oryzae pv. oryzae (Ishiyama)

Mass Culturing and Artificial Inoculation

Fig. 1.1 Diagram key for

1=1-5% 3=6-12% 5=13-25% 7=26-50% 9=51-100%

convenient for inoculation of large number of plants in practical breeding work in

Pathogen and Its Isolation

Blast caused by the fungus Pyricularia grisea (Cook) Sacc. [teleomorph:

Mass Culturing and Artificial Inoculation

Table 1.3 SES Scale for Score Description

than 0.5 mm in diameter; 2 = brown specks about 0.5–1 mm in diameter; 3 = round-

1.1.1.3 Sheath Blight

Pathogen and Its Isolation

Mass Culturing and Artificial Inoculation

Sum of all disease ratings ×100

Fig. 1.2 Diagram key for 100%

1=<20% 3=20-30% 5=31-45% 7=46-65% 9=>65%

Table 1.4 SES (Anonymous 2002) for sheath blight of rice

100 × ( 5n1 + 4 n 2 + 3n3 + 2 n 4 + 1n 5)

In case of detached leaf bioassay, disease observations were recorded after

1.1.1.4 Rice Tungro Virus

Table 1.5 SES Scale (1996) for rice tungro disease

1.1.1.5 Brown Spot

Brown spot caused by fungus Bipolaris oryzae (Breda de Haan) Shoemaker

1.1.2 Emerging Rice Diseases

1.1.2.1 Sheath Rot

Sheath rot caused by the fungus Sarocladium oryzae is especially important on

Table 1.8 New scoring system for sheath rot of rice

The disease index was then calculated by the following formula:

1.1.2.2 False Smut

Table 1.9 SES for false smut of rice

Table 1.10 Rating scale for Class Description

1.1.3 Stem Rot

Stem rot of rice caused by Sclerotium oryzae Catt. (anamorph: Helminthosporium

DI = [1(n1 × H) + 2(n 2 × L) + 3(n3 × M) + 4(n 4 × M* ) + 5(n 5 × S)] /

Table 1.11 SES scale for % Age of stems with

1.2 Phenotyping for Important Insect Pests

1.2.1.1 Damage Symptoms

Table 1.12 Scoring of plants for planthoppers under field conditions

Table 1.13 Scoring of plants Damage score Number/hill

1.2.1.2 Screening Methodology

Phenotyping Under Field Conditions

Fig. 1.3 Seed box lay out for

1.1 Phenotyping of Rice Diseases

1.1.1 Major Rice Diseases

1.1.1.1 Bacterial Blight of Rice

1.1.1.3 Sheath Blight

1.1.1.4 Rice Tungro Virus

1.1.1.5 Brown Spot

1.1.2 Emerging Rice Diseases

1.1.2.1 Sheath Rot

1.1.2.2 False Smut

1.1.3 Stem Rot

1.2 Phenotyping for Important Insect Pests

1.2.1.1 Damage Symptoms

1.2.1.2 Screening Methodology

1.2.2 Stem Borers

1.2.2.1 Damage Symptoms

1.3.2 Identification of Nematode Species/Isolate

1.3.3 Inoculation of Plants

1.3.4 Phenotyping Protocol

Phenotyping of large plant populations is done in glasshouse conditions. Pre-

1.4 Phenotyping for Drought Tolerance

1.4.1 Selection Criteria