Plant Cell, Tissue and Organ Culture (2006) 86:111–115
DOI 10.1007/s11240-005-9062-2
Ó Springer 2006
In vitro plant regeneration of Arachis correntina (Leguminosae)
through somatic embryogenesis and organogenesis
Marı́a Laura Vidoz*, Pablo Klusacek, Hebe Yolanda Rey & Luis Amado Mroginski
Facultad de Ciencias Agrarias (UNNE), Instituto de Botánica del Nordeste (IBONE), Sargento Cabral
2131, 3400, Corrientes, Argentina (*requests for offprints: Fax: +54-3783-427131;
E-mail: mlvidoz@agr.unne.edu.ar)
Received 26 May 2005; accepted in revised form 21 November 2005
Key words: Arachis correntina, explant type, organogenesis, plant regeneration, somatic embryogenesis
Abstract
In vitro protocols for plant regeneration of Arachis correntina through both somatic embryogenesis and
organogenesis were developed using immature leaves as explants. Morphologically normal somatic
embryos were obtained on culture media composed of 20.70 or 41.41 lM picloram (PIC) with the addition
of 0.044 lM 6-benzylaminopurine (BA), resulting in a 33 and 24% of conversion into plants, respectively.
The source of explants and the developmental stage of the leaves had a marked effect on somatic
embryogenesis. The second folded immature leaves from in vitro growing plants were the most responsive
producing up to 30% embryogenesis in MS+41.41 lM PIC. Embryos converted into plants after transfer
to MS medium devoid of growth regulators and these plants were successfully acclimatised. Adventitious
shoots were obtained on culture media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or
naphthaleneacetic acid (NAA) with or without 0.044 lM BA, achieving plant regeneration in the induction
media. The highest percentage of bud formation was obtained on culture medium composed
of MS+10.74 lM NAA+0.044 lM BA (12.5%). Roots were formed on all culture media tested.
Regenerated plants were transferred to pots and grew well under greenhouse conditions.
Abbreviations: BA – 6-benzylaminopurine; 2,4-D – 2,4-dichlorophenoxyacetic acid; NAA – naphthaleneacetic acid; PIC – picloram-4-amino-3,5,6-trichloropicolinic acid
The groundnut, Arachis hypogaea, is the most
widely spread of the 69 species of the genus
Arachis, all of which are native to South America
(Krapovickas and Gregory, 1994). However, several other species, such as A. pintoi Krapov. and
W.C. Gregory, A. glabrata Bentham and A. repens
Handro, are also useful as forage and ornamental
or ground cover species (Dos Santos et al., 2003).
The growing concern over the collection, rescue,
conservation, multiplication and characterisation
of wild species of Arachis germplasm lays in the
fact that they contain useful genes for the genetic
improvement of peanut (Gagliardi et al., 2000).
Cultivated and many wild peanuts are stored
as seeds and, even with optimum storage
practices, seed germinability and germplasm
losses are inevitable (Dunbar et al., 1993).
Consequently, in vitro germplasm conservation
constitutes a viable option for their preservation
providing that efficient in vitro protocols for
plant regeneration are developed (Gagliardi
et al., 2000; Vijaya Laxmi and Giri, 2003).
Additionally, these protocols are required for
the utilisation of genetic engineering in the
improvement of Arachis species (Ozias-Akins
and Gill, 2001).
Diploid wild peanuts of the genus Arachis
section Arachis are close relatives of the cultivated
peanut and present potential usefulness as tropical forages. Among these, Arachis correntina
112
(2n=2x=20 chromosomes) occupies a prominent
position for its resistance to rust, peanut mottle
virus, tomato spotted wilt virus, aphids, mites,
thrips and jassids (Kameswara Rao et al., 2003).
A. correntina (Burkart) Krapov. and W.C. Gregory, is a perennial species that grows in the North
West of the province of Corrientes (Argentina) as
a component of natural pastures (Krapovickas
and Gregory, 1994). In vitro plant regeneration
from leaf explants via organogenesis has been
recently achieved in A. correntina using thidiazuron (Mroginski et al., 2004) whereas somatic
embryogenesis has never been reported in this
species previously.
In this paper, we report two simple in vitro
regeneration systems for Arachis correntina
through either somatic embryogenesis or organogenesis from immature leaf explants.
Seeds of Arachis correntina, a predominantly
autogamous species, were collected from different
plants growing in the same area in Pirayui,
Corrientes, Argentina by Luis Mroginski (herbarium specimen Krapovickas and others 11905
deposited in CTES) and cultivated in the garden
of the Instituto de Botánica del Nordeste
(IBONE). Explants were obtained from one-yearold plants growing: (a) in the garden of the
IBONE; (b) in an acclimatised room with
27±2 °C, a photoperiod of 14 h and
336 lmol m)2 s)1; and (c) in vitro with 27±2 °C,
a photoperiod of 14 h and 116 lmol m)2 s)1,
obtained by in vitro culture of shoot tips from
the plants growing in the garden according to Rey
and Mroginski (2003). Explant tissues, randomly
collected within the three groups of plants mentioned before, consisted of: (I) shoot tips (1–2 mm
long), (II and III) first and second immature folded
leaves beyond the shoot tip respectively, (IV)
portions of the third emerging folded leaflets,
and (V) portions of unfolded leaflets of the fourth
leaf.
Murashige and Skoog (1962) (MS) basal medium with 3% sucrose was used. For culture establishment, PIC (20.70, 41.41, 62.11 and 82.82 lM),
2,4-D (9.05, 22.62, 45.25 and 67.87 lM) and NAA
(10.74, 26.85, 53.71 and 80.56 lM) were tested alone
or in combination with 0.044 lM BA. To achieve
somatic embryo maturation and conversion, MS
devoid of plant growth regulators was utilised.
Media pH was adjusted to 5.8 with KOH or HCl
before the addition of 0.7% agar (Sigma A-1296).
Media were sterilised by autoclaving for 20 min
(0.101 MPa). Explants were placed with the abaxial side down on 3 ml of culture medium in 11 ml
glass tubes. Tubes were sealed with Resinite AF
50Ò (Casco SACIF Buenos Aires, Argentina) and
incubated in a growth room at 27±2 °C with 14 h
photoperiod (116 lmol s)1 m)2 provided by coolwhite fluorescent tubes – Philips TLD 84).
Rooted shoots and germinated somatic embryos were washed under running tap water and
transferred to pots containing a mixture of soil,
sand and perlite (1:1:1). Plantlets were acclimatised
in a growth chamber at 27±2 °C with 14 h
photoperiod (336 lmol s)1 m)2 provided by
cool-white fluorescent tubes – Philips TLD 84).
Ten explants were cultured per treatment, and
experiments were repeated three times. Results are
presented as the means of the replications with the
standard error (mean±SEM). Analysis of variance (ANOVA) was performed followed by
Duncan’s multiple comparison test (p<0.05).
In order to study the influence of PIC on
somatic embryogenesis, 2–4 mm long immature
leaves (explant types II and III) collected from
in vitro plants were cultured using the PIC concentrations mentioned above, with and without
the addition of 0.044 lM BA. Within 7 days of
culture, nodular calli were observed in most
cultures, which gave rise to somatic embryos after
20 days. Direct somatic embryogenesis was also
observed (Figure 1a).
The highest percentages of somatic embryogenesis were obtained in the media composed of
MS+41.41 or 62.11 lM PIC (45 and 56% respectively), the last one being significantly better than
the rest. These media also produced more embryos
per explant, especially the first one (43 embryos per
explant). Globular, heart, torpedo and cotyledonary shaped normal embryos were observed, with
numerous variations of abnormal embryos (two or
multiple fused, horn-shaped embryos) similar to
those described by Wetzstein and Baker (1993).
Embryo quality was better in media composed of
MS+20.70 or 41.41 lM PIC+0.044 lM BA, with
up to 35% of normal embryos in the latter,
conducing to a higher rate of ex vitro-established
plants (33 and 24%, respectively).
To evaluate the types of explant, the best media
in the previous experiment (MS+41.41 lM PIC
and MS+41.41 lM PIC+0.044 lM BA) were
used. The stage of development, the size of the
113
Figure 1. Somatic embryogenesis from immature leaves of A. correntina. (a) Somatic embryos emerging from the basal part of an
immature leaf after 30 days of culture, bar: 5 mm. (b) Somatic embryos before being transferred to MS medium devoid of PIC,
after 60 days of culture, bar: 5 mm. (c) Somatic embryo germinating on MS medium 10 days after transfer, bar: 10 mm. (d) Welldeveloped plantlet 30 days after being transferred to MS medium, bar: 20 mm. (e) Acclimatised plant growing under greenhouse
conditions, after 60 days of transfer to soil, bar: 40 mm.
leaves and the source of explants considerably
affected somatic embryo induction of A. correntina. Overall, the multiple comparisons test showed
that the in vitro origin is superior in both percentage of somatic embryogenesis and number of
embryos per explant. Regarding the type of
explants, type III (second immature folded leaf)
was superior (Table 1).
The percentage of somatic embryogenesis
obtained in the culture medium experiments was
higher than that of the explant type experiments
(56% versus 30%). Such results could be ascribed
to the fact that the first experiment was performed
with plants that were established in vitro for
40 days while for the second experiment, the
plants had been growing in vitro for 120 days.
Somatic embryos were isolated and transferred
to MS without growth regulators (Figure 1b),
where embryo maturation and conversion into
plants was achieved 90 days after immature leaves
were cultured. These plants were transferred to
pots (Figure 1c–e), with an ex vitro survival rate of
80%, where they grew normally and produced
seeds.
Table 1. Effect of explant type on the percentage of somatic embryogenesis (SE) and mean number of embryos per explant (N°) of
A. correntina, after 40 days of culture
Type of explants
MS+41.41 lM PIC
MS+41.41 lM PIC+0.044 lM BA
Explant origin
Explant origin
Garden
I
II
III
IV
V
Acclimatised
room
In vitro
Garden
Acclimatised
room
In vitro
% SE
N°
% SE
N°
% SE
N°
% SE
N°
% SE
N°
% SE
N°
0
13.3
6.7
0
0
0
4.3
4.7
0
0
10
10
13.3
6.7
0
2
8
8.7
2.7
0
6.7
10
30*
10
0
4
4.7
19*
6.3
0
6.7
3.3
10
10
0
3.7
4
7
5
0
3.3
3.3
10
0
0
3.3
4
2.7
0
0
16.7
16.7
13.3
13.3
3.3
7.3
7.7
14.7
5
2.3
(I) Shoot tips (1–2 mm long), (II and III) first and second immature folded leaves beyond the shoot tip respectively, (IV) portions of the
third emerging folded leaflets, and (V) portions of unfolded leaflets of the fourth leaf.
*Represents significant difference according to Duncan’s multiple comparison test (p £ 0.05).
114
obtained when the culture medium was composed
of MS+10.74 lM NAA+0.044 lM BA. Shoot
elongation and shoot rooting occurred in the
induction media, and plant regeneration was
achieved in only one step in 90% of the cases.
In our studies, 2,4-D induced direct and
indirect adventitious shoot and root formation.
Similar results were reported for A. pintoi, (Rey
et al., 2000), but not for A. hypogaea, in which
somatic embryogenesis occurred in media containing 2,4-D (Lakshmanan and Taji, 2000; Little
et al., 2000).
Bud and shoot formation was observed in
almost all culture media containing NAA, regardless BA addition to the media. Similarly, shoot
organogenesis was obtained in a number of
Arachis species using different combinations of
NAA and BA (Bajaj et al., 1981; McKently et al.,
1991; Cheng et al., 1992; Mansur et al., 1993; Rey
et al., 2000). Additionally, in our experiments
roots were produced with all NAA concentrations
tested. Our results differ from a previous report in
A. correntina (Mroginski et al., 2004) in which
explants produced only callus when PIC, 2,4-D or
NAA were added to the induction medium. This
difference in responses could be attributed to
the type of explant used: fully expanded leaves in
the former while immature leaves were used in the
present study.
Our findings provide a fast, simple procedure
for regenerating plants via somatic embryogenesis
or organogenesis, with little or no callus formation.
The minimization of the callus stage is a desirable
Our results coincide with those that reported the
effectiveness of PIC on the induction of somatic
embryos of A. hypogaea (Sellars et al., 1990; Eapen
and George, 1993; Little et al., 2000), A. pintoi (Rey
et al., 2000), A. glabrata (Vidoz et al., 2004) and
A. paraguariensis (Sellars et al., 1990).
In A. hypogaea, the addition of cytokinins
decreased the number of somatic embryos per
explant (Eapen and George, 1993) or was ineffective in enhancing somatic embryogenesis (Chengalrayan et al., 1994). In our studies, although the
highest percentage of somatic embryogenesis was
obtained in a culture medium devoid of cytokinins,
a better morphology and greater number of established plants were obtained when embryos developed on media containing 0.044 lM BA. This is in
agreement with a previous report of the positive
influence of BA addition on somatic embryogenesis
of A. pintoi (Rey et al., 2000). The positive influence
of BA on A. correntina somatic embryo conversion
is probably due to its effect on embryo maturation,
since it was successfully used in A. hypogaea for this
purpose (Venkatachalam et al., 1999).
After 50 days of culturing folded leaves (explant
types II and III) in media containing 2,4-D or
NAA, several responses were obtained: expanded
leaves, friable calli, roots with or without callus,
and shoot buds. Only the lowest concentration of
2,4-D (9.05 lM) originated buds, regardless the
addition of 0.044 lM BA (Figure 2). However, bud
formation was achieved employing several concentrations of NAA with or without the addition of
0.044 lM BA. The highest percentage (12.5%) was
80
70
% Responses
60
50
40
30
20
*
10
*
0
MS +
9.05
22.62
45.25
67.87
9.05
22.62
45.25
67.87
10.74
26.85
2.4-D
53.71
80.56
10.74
26.85
53.71
80.56 µM
NAA
+ BA 0,044µM
% Buds
+ BA 0,044µM
% Roots
Figure 2. Effect of 2,4-D, NAA and BA supplemented to MS on adventitious bud and root formation from immature leaves of
A. correntina, after 50 days of culture. *Represents significant difference according to Duncan’s multiple comparison test (p £ 0.05).
115
feature for in vitro protocols because callus cells are
more genetically unstable than differentiated ones
(Pittman et al., 1983). Moreover, an abbreviated
tissue culture phase is preferred in order to reduce
somaclonal variation and detrimental effects on the
regeneration of transformed plants (Ozias-Akins
and Gill, 2001). Consequently, in vitro culture of
immature leaves can be used for an effective system
of plant regeneration through either somatic
embryogenesis or organogenesis. To induce
somatic embryogenesis, two steps and the use of
PIC were necessary whereas organogenesis was
achieved in only one step employing NAA or
2,4-D. Plantlets obtained were normal and could be
acclimatised successfully.
Acknowledgements
HY Rey and LA Mroginski are members of
the National Research Council (CONICET) of
Argentina. We gratefully acknowledge the financial support received from UNNE, CABBIO,
CONICET, and USDA.
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Hebe Rey