Somatic embryogenesis and in vitro plant regeneration in moth bean [Vigna aconitifolia (Jacq.)...

ORIGINAL ARTICLE

Somatic embryogenesis and in vitro plant regenerationin moth bean [Vigna aconitifolia (Jacq.) Marechal]:a recalcitrant grain legume

Kailash Choudhary Æ M. Singh Æ M. S. Rathore ÆN. S. Shekhawat

Received: 3 July 2008 / Accepted: 27 April 2009 / Published online: 24 May 2009

� Korean Society for Plant Biotechnology and Springer 2009

Abstract An efficient in vitro regeneration protocol for

moth bean [Vigna aconitifolia (Jacq.) Marechal] via

somatic embryogenesis has been developed. Embryogenic

callus cultures were established from the cotyledonary

node as explant on semi-solid Murashige and Skoog (MS)

medium supplemented with 0.75 mg l-1 2,4-dichlorophe-

noxyacetic acid (2,4-D) and 1.5 mg l-1 6-benzylamino-

purine (BA) and with various additives (50 mg l-1

ascorbic acid and 25 mg l-1 each of adenine sulphate,

citric acid and L-arginine). Numerous somatic embryos

differentiated on MS basal nutrient medium supplemented

with 0.25 mg l-1 2,4-D and 0.5 mg l-1 of kinetin (Kin).

Sustained cell division resulted in the formation of cell

aggregates, which progressed to the globular- and heart-

shaped somatic embryos and then, if they differentiated

properly, to the torpedo shape and cotyledonary stages. The

transfer of embryos onto fresh MS basal medium

containing 0.2 mg l-1 BA and 2.0 mg l-1 gibberellic acid

enabled the embryos to achieve complete maturation and

germination. More than 80% of somatic embryos were

converted into true-to-type fertile plants. In vitro-regener-

ated plantlets with well-developed roots were successfully

hardened in a greenhouse and established in soil.

Keywords Embryogenic callus � Germination �Heart and torpedo shape � True-to-type

Introduction

Moth bean [Vigna aconotifolia (Jacq.) Marechal] is an

important food grain legume crop that is cultivated over the

entire world. It is regarded as a quality pulse crop in India

and Pakistan for its excellent protein quality, high digest-

ibility and freedom from flatulent effects associated with

other pulses, such as chickpea and lentil. The seeds of

Vigna aconitifolia are a rich source of protein and minerals,

including calcium, magnesium, iron, zinc and manganese

(Siddhuraju et al. 1994), and exhibit fairly high levels of

crude lipid. The demand for this crop has been steadily

increasing in the Indian subcontinent. However, it is

characterized by a low yield potential, which is thought to

be due to a number of biotic and abiotic factors as well as

low genetic variability. In some growing seasons, losses

have been reported to exceed 50% due to the incidence of

many pests and diseases (Poehlman 1991).

There is a need to increase the productivity of this crop

as well as enhance its nutritional value and other essential

agronomic qualities. One of the first steps in any yield

improvement program is the creation of a broadly based

gene pool. In the last several years, numerous attempts

have been made to develop disease-resistant as well as

high-yielding varieties of moth bean through interspecific

hybridization. However, due to interspecific cross-

K. Choudhary (&)

Department of Biotechnology, Lachoo Memorial College

of Science and Technology, J.N.V. University,

Jodhpur 342001, Rajasthan, India

e-mail: [email protected]

M. Singh

Biotechnology Laboratory, FASCL, Mody Institute

of Technology and Science, Lakshmangarh,

Sikar 332311, Rajasthan, India


M. S. Rathore � N. S. Shekhawat

Department of Botany, Biotechnology Center,

J.N.V. University, Jodhpur 342001, Rajasthan, India


123

Plant Biotechnol Rep (2009) 3:205–211

DOI 10.1007/s11816-009-0093-8

incompatibility and hybrid sterility, it has proven impos-

sible to develop such improved moth bean varieties. Thus,

the low genetic variability of moth bean caused by a high

degree of self-pollination has imposed limitations on its

improvement using conventional methods of breeding. One

alternative approach is genetic engineering, which has in

recent years been effectively used to develop desirable

breeding lines of many important crop plants (Fisk and

Dandekar 1993; Wambugu 1999; James 2004). To date, a

reproducible and reliable transformation system that would

enable genes of interest to be inserted into moth bean lines

is not available in existing genotypes. Consequently,

genetic transformation combined with traditional breeding

methods may prove helpful in improving both the quality

and yield of moth bean.

An efficient in vitro plant regeneration system is

required for successful crop improvement through genetic

engineering. In the case of grain legumes, crop

improvement is mostly hampered by the recalcitrant nat-

ure of leguminous tissues under in vitro conditions.

Several attempts have been made to establish in vitro

regeneration protocols for moth bean (Shekhawat and

Galston 1983; Kaur-Sawhney et al. 1985; Prem Anand

et al. 2000; Chandra and Pental 2003; Kaviraj et al.

2008), and considerable research has been done on cow-

pea (Vigna unguiculata) and mung bean (Vigna radiata)

in the USA and Europe during the last century. In con-

trast, limited attention has been paid to other Vigna spe-

cies. Ignacimuthu et al. (1997) and Das et al. (1998)

found that cotyledonary nodes of Vigna are the most

responsive explant material for the induction of multiples

shoots via organogenesis. The induction of somatic

embryogenesis via suspension culture in Vigna spp. was

reported by Prem Anand et al. (2000). The Agrobacte-

rium-mediated transformation of cotyledonary node

explants was reported in Vigna species by Ignacimuthu

(2000), and shoot regeneration from various Vigna species

was studied by Avenido et al. (2001). Chandra and Pental

(2003) reviewed the regeneration and genetic transfor-

mation of grain legumes, including Vigna species. The

development of somatic embryos (SEs) induced in cell

suspension cultures of Vigna unguiculata has been

reported by many researchers (Kumar et al. 1988a; Prem

Anand et al. 2000; Ramakrishnan et al. 2005). Saini and

Jaiwal (2005) reported the transformation of a recalcitrant

grain legume, Vigna mungo, using Agrobacterium tum-

efaciens in shoot apical meristem cultures. However, the

in vitro regeneration protocols developed by these

researchers did not produce the desired results using moth

varieties from India. Given this background, the aim of

our study was to develop and establish a reproducible in

vitro plant regeneration system in moth bean varieties of

India.

Materials and methods

Two varieties of moth bean [Vigna aconitifolia (Jacq.)

Marechal], Jwala and Jaria, the most commonly grown

varieties in a stressed ecosystem, were used in our inves-

tigation. Both varieties were provided by the Seed Cor-

poration, Government of Rajasthan, Jodhpur, India. The

seeds were washed thoroughly under tap water to remove

microbes and other adherents, pretreated with a 0.1%

aqueous solution of Bavistin (a systemic fungicide) and a

1% solution of streptomycin (an antibiotic), respectively,

each for 15 min, and then washed several times with sterile

water. The pretreated seeds were then surface sterilized

with a 0.1% mercuric chloride (HgCl2) solution for 5 min,

followed by several washes with sterile water. The surface-

sterilized seeds were then dipped in 90% ethanol for 30–

60 s and allowed to air dry under a laminar air flow bench.

The sterilized seeds were inoculated on plant growth reg-

ulator (PGR)-free Murashige and Skoog (MS; Murashige

and Skoog 1962) basal medium for activation of the

embryo and subsequent germination. Different types of

explants, namely epicotyls, cotyledonary nodes, cotyle-

dons, hypocotyls and leaflets, from in vitro-grown seed-

lings were used to raise callus cultures. Cotyledonary nodes

and other explants were collected from 10- to 15-day-old

aseptically grown seedlings.

For the initiation of callus cultures, explants harvested

from in vitro-grown seedlings were inoculated on various

types of media, namely MS, modified MS (MMS; nitrate

level reduced to half strength; Shekhawat et al. 1998) and

White’s medium (White 1963), supplemented with addi-

tives (50 mg l-1 ascorbic acid, 25 mg l-1 each of adenine

sulphate, citric acid and L-arginine). Experiments were

carried out to evaluate the effects of various types, con-

centrations and combinations of auxins [2,4-dichlorophe-

noxyacetic acid (2,4-D) and a-naphthalene acetic acid

(NAA)] and cytokinins [benzylaminopurine (BA) and ki-

netin (Kin)] for callus initiation and multiplication. The

cell cultures were transferred onto the various media (MS,

MMS and White’s) containing different types, concentra-

tions and combinations of cytokinins and auxins for further

multiplication. These cultures were incubated in a growth

room at 20–25 lmol m-2 s-1 spectral flux photon (SFP),

60–70% relative humidity (RH) and 28 ± 2�C under a 12/

12-h light/dark photoperiod. Callus initiated from various

types of explants were harvested carefully and transferred

to media supplemented with various concentrations and

combinations of 2,4-D and Kin for the induction of somatic

embryogenesis. These cultures were incubated in a growth

room at 25–30 lmol m-2 s-1 SFP, 60–70% RH and

28 ± 2�C under a 12/12-h light/dark photoperiod. After

20–25 days of culture, the entire organogenic/embryogenic

callus with SEs were transferred onto fresh medium with

206 Plant Biotechnol Rep (2009) 3:205–211

123

the same composition. In the second round of subculturing,

the SEs were removed and transferred onto the multipli-

cation media. Experiments were conducted with the aim of

obtaining germination and the development of SEs into

complete plantlets. For conversion of SEs into plantlets, the

embryogenic cultures/SEs were transferred onto MS med-

ium containing BA (0.0–0.5 mg l-1), combinations of

NAA and BA, gibberellic acid (GA3; 1.0–3.0 mg l-1) and

combinations of GA3 and BA. These cultures were incu-

bated in a growth room at 35–40 lmol m-2 s-1 SFP, 60–

70% RH and 28 ± 2�C under a 12/12-h light/dark

photoperiod.

The plants were carefully removed from the culture

vessels, washed with water to remove any adhering nutrient

agar, transferred to glass bottles containing microbe-free

soilrite and moistened with a one-fourth-strength MS

macro-salt solution. The polycarbonate-capped bottles

containing the plants produced through SE germination

were placed in the greenhouse near a cellulose pad to

maintain 70% RH and 25–27�C through evaporative

cooling. After 10–12 days, the polycarbonate caps of the

bottles were loosened to allow free gaseous exchange with

the external environment and to expose the plantlets to ex

vitro conditions. The bottles were slowly shifted from the

pad section to the fan section (30% RH and 32 ± 2�C) in

the greenhouse. The hardened plantlets were transferred to

soil in black polybags for complete acclimatization. These

were kept in a nursery covered with an agro-net. The plants

were then transferred to the field under the environmental

conditions of Rajasthan.

Experiments were set up in a completely randomized

block design and repeated three times. Each treatment had

a minimum of ten replicates. Observations on the induction

process, rate of multiplication and differentiation of SEs

were scored after a regular interval. The data were sub-

jected to statistical analysis (Gomez and Gomez 1984).

Results and discussion

The application of tissue culture in the improvement of

moth bean has received little attention from researchers.

Plant regeneration among Vigna species is limited relative

to that among other grain legumes, with the callus phase

and its duration negatively correlated with the regeneration

ability of explants. Moreover, somaclonal variations can

influence the phenotype of regenerated plants during

indirect organogenesis. The explant is considered to be a

critical factor that determines the success—or not—of

most tissue culture experiments, particularly in somatic

embryogenesis (Kantharajah and Golegaonkar 2004;

Varisai Mohamed et al. 2004). The results of our study

demonstrate that it is possible to induce plants from SEs in

moth bean.

The successful induction of callus was achieved in all

types of explants used in our moth bean experiments.

However, among all of the explant material tested in our

study, the callus obtained from leaflets and cotyledonary

nodes of 10- to 15-day-old seedlings of V. aconitifolia

responded the quickest and had the highest rate of prolif-

eration and the best texture (Fig. 1a). Consequently, these

explants were preferred over the others for raising the

primary culture. The callus produced from hypocotyls was

soft and slow growing, eventually turning brown/black, and

that produced from epicotyls was yellowish puffy, fast and

non-regenerative.

The induction and maintenance of callus was carried out

on several media. The formulation of MS medium proved

to be the best amongst the media tested, hence full-strength

MS was used routinely. Semi-solid MS medium supple-

mented with 0.75 mg l-1 2,4-D and 1.5 mg l-1 BA was

clearly the best for achieving callus culture initiation from

all types of explants (Table 1). At increased concentrations

of 2,4-D (to a maximum of 5 mg l-1), the callus initially

grew as a white mass and then turned brown; at lower

concentrations (\0.5 mg l-1), very little soft callus was

produced, and the response of explants was too delayed.

On MS medium supplemented with 1.0–2.0 mgl-1 of

NAA, very little brown/black callus was produced, which

differentiated into roots. Similarly, on medium supple-

mented with 0.1–1.0 mgl-1 of indole-3-acetic acid (IAA),

slow-growing rhyzogenic callus was produced. Regenera-

tive callus was separated from non-regenerative callus on

the basis of morphology and texture. The callus initiated

was sub-cultured regularly at 25- to 28-day intervals.

Following the induction of callus cultures, the regener-

ative/embryogenic cultures were separated and subcultured

on media with different compositions supplemented with

various concentrations and combinations of PGRs. The

growth of callus was optimum on MS medium supple-

mented with 1.0 mg l-1 2,4-D and 1.0 mg l-1 Kin

(Fig. 1b). The cultures obtained on this specific medium

were granular and highly regenerative/embryogenic. At

higher concentrations of 2,4-D ([3.0 mg l-1), the callus

could not be multiplied, becoming brown, compact and

finally drying out; at a lower concentration (1.0 mg l-1),

slow-growing callus was produced. Both BA (1.0 mg l-1)

and 2-iso-pentenyladenine (2-iP; 1.0 mg l-1) in combina-

tion with 2,4-D (1.0 mg l-1) were found to be less effec-

tive in terms of the multiplication and maintenance of

embryogenic cultures. Ultimately, the combination of

1.0 mg l-1 2,4-D and 1.0 mg l-1 Kin was determined to be

the most suitable PGR combination for the multiplication

of callus.

Plant Biotechnol Rep (2009) 3:205–211 207

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Granular and regenerative callus was obtained from

cotyledonary nodes when inoculated on MS medium

containing 0.25 mg l-1 2,4-D and 0.5 mg l-1 Kin, and

numerous dark-green, globular structures with enriched

cytoplasm and visible nucleus appeared within 7 days of

culture (Fig. 1c; Table 2). These structures ultimately

developed into heart-shaped SEs (Fig. 1d).

A low response was observed on hormone-free medium

and on medium containing various concentrations (0.1–

1.0 mg l-1) of 2,4-D, but without Kin, with about 15–20

somatic embryos per culture vessel. The regenerative callus

was repeatedly transferred to fresh medium to produce

fresh crops of SEs. The embryogenic nature of the cultures

could be maintained by regular subculturing (Fig. 1e), with

the optimal subculture interval being 25–28 days. Any

delay in subculturing led to the drying out and dediffer-

entiation of the SEs into non-regenerative callus. A high

rate of SE production (15–20) was obtained with repeated

subculturing of 100 mg fresh weight of inoculum per

culture vessel (Fig. 1d). The culture conditions of

25–30 lmol m-2 s-1 SFP, 60–70% RH and 28 ± 2�C

under a 12/12-h light/dark photoperiod were found effec-

tive for inducing somatic embryogenesis.

Somatic embryos germinated following their transfer to

auxin-free medium, with some showing normal germina-

tion and others producing primary roots. Of the SEs

transferred to hormone-free MS medium, 20% germinated;

in comparison, 60% of the SEs transferred to MS medium

containing 0.2 mg l-1 BA germinated. Somatic embryos

transferred to MS medium containing 0.1 mg l-1 BA and

0.1 mg l-1 NAA did not germinate at all but produced

callus. The addition of various concentrations of GA3 (0.2–

4.0 mg l-1) to MS medium positively affected the germi-

nation of SEs. About 50% of the SEs germinated on

medium containing 2.0 mg l-1 of GA3. The MS medium

with 0.2 mg l-1 BA and 2.0 mg l-1 GA3 was found to be

the most suitable for inducing the germination of SEs

(Fig. 1g): 80% of SEs germinated on this medium

(Table 3). Further increase in the concentration of BA and

GA3 caused abnormal germination and the plantlets formed

Fig. 1 Somatic embryogenesis

and plant regeneration in moth

bean. a Callus induction from

leaflet explants on Murashige

and Skoog (MS)

medium ? 0.75 mg l-1 2,4-

dichlorophenoxyacetic acid

(2,4-D) and 1.50 mg l-1

benzylaminopurine (BA).

b Regenerative callus on

MS ? 1.00 mg l-1 2,4-D and

1.00 mg l-1 of kinetin (Kin).

c Globular somatic embryos on

MS ? 0.25 mg l-1 2,4-D and

0.5 mg l-1 Kin. d High

frequency of heart-shaped

somatic embryo. e Excised

somatic embryos during

subculture. f Abnormal

germination of somatic

embryos. g Germination of

somatic embryos on

MS ? 0.2 mg l-1 BA and

2.0 mg l-1 gibberellic acid

(GA3). h Cloned plantlets of

moth bean raised from

germination of somatic embryos


123

were not normal (Fig. 1f). Germinated plants had healthy

roots, and the plants were successfully hardened by trans-

ferring them from the cellulose pad section (70% RH and

25–27�C) to the fan section (30% RH and 32 ± 2�C) of the

greenhouse. These plants were transferred to polybags

containing soil, sand and vermicompost (1:1:1; w/w/w) and

were successfully hardened in the greenhouse (Fig. 1h),

following which they were transferred to the field.

During our study we were able to successfully regenerate

normal moth bean plants from somatic embryos. However,

we identified two major limitations to the application of

somatic embryogenesis for the propagation and genetic

manipulation of moth bean plants: (1) low multiplication

rates—that is, low numbers of field-plantable clonal

plantlets were produced per embryogenic culture; (2)

inability to initiate embryogenic cultures from mature

plants. The limitation of low multiplication rates can be

further broken down into such problems as a low frequency

of somatic embryo production, production of malformed

embryos, incomplete maturation, low germination and low

conversion of germinated embryos to plantlets capable of

surviving the transfer to ex vitro conditions.

Our investigation revealed that the induction of

embryogenic callus from cultured explants mainly depen-

ded on the exogenous application of 2,4-D and BA. Kumar

et al. (1988a, b), however, used NAA and Kin to initiate the

suspension cultures of Vigna aconitifolia and Phaseolus

acutifolius. In our study, embryogenic tissues swelled and

proliferated from cotyledonary nodes or leaflets as explants

on MS semi-solid medium fortified with 0.75 mg l-1 2,4-D

and 1.5 mg l-1 BA. Somatic embryogenesis in cultures

from immature cotyledons of soybean at higher doses of

2,4-D with high concentrations of sucrose were reported by

Finer and Nagasawa (1988), whereas we found that

embryogenesis could be initiated at a lower concentration

of 2,4-D (0.75 mg l-1) and BA (1.5 mg l-1). Girija et al.

(2000) reported the induction of embryogenic callus from

immature cotyledon explants of Vigna radiata on MS

medium supplemented with 5.0 mg l-1 NAA. Somatic

embryogenesis and plant regeneration from cotyledonary

explants of V. radiata was reported by Kaviraj et al. (2008).

Devi et al. (2004) reported plant regeneration via somatic

embryogenesis in two Indian cultivars of mung bean using

different explants, such as mature cotyledons, hypocotyls,

nodal segments, and leaf explants on MS medium supple-

mented with several different combinations of growth

regulators. The development of SEs from leaf explants

Table 1 Effect of various types, concentrations and combinations of

plant growth regulators on callus induction from cotyledonary seg-

ments of Vigna aconitifolia on Murashige and Skoog (1962) medium

with additives

PGR treatments (mg l-1) Days taken for

callus initiation

% Response

2,4-D 0.5 15 30

2,4-D 1.0 15 50

2,4-D 2.0 12 70

2,4-D 5.0 – –

NAA 1.0 18 35

NAA 2.0 18 30

IAA 0.5 15 25

IAA 1.0 – –

2,4-D 0.5 ? BA 0.5 20 55

2, 4-D 0.75 ? BA 1.0 25 75

2,4-D 0.75 ? BA 1.5 25 95

2,4-D 1.0 ? BA 1.75 25 85

2,4-D 2.0 ? BA 2.0 25 75

PGR plant growth regulator, 2,4-D 2,4-dichlorophenoxyacetic acid,

NAA a-naphthalene acetic acid, BA benzylaminopurine, IAA indole-3-

acetic acid

Table 2 Treatment of different PGR combinations on the induction

of somatic embryogenesis from cotyledonary segments of V.aconitifolia

PGR treatments (mg l-1) Somatic embryos per

culture vessel (n)

2,4-D 0.10 3.5 ± 1.08

2,4-D 0.25 8.2 ± 1.03

2,4-D 0.50 3.2 ± 0.78

2,4-D 0.25 ? Kin 0.50 17.1 ± 1.9

2,4-D 0.25 ? Kin 1.0 12.7 ± 1.33

Values are given as the average ± SD

Kin Kinetin

Table 3 Effect of various concentrations of PGRs in MS medium on

the germination of somatic embryos of V. aconitifolia

PGR treatments (mg l-1) Germination percentage

of somatic embryos

Hormone free 20 ± 2.15

BA 0.1 30 ± 3.15

BA 0.2 60 ± 2.20

BA 0.5 40 ± 1.50

GA3 1.0 10 ± 2.00

GA3 2.0 50 ± 3.15

GA3 4.0 40 ± 2.10

BA 0.2 ? GA3 1.0 70 ± 0.75

BA 0.2 ? GA3 2.0 80 ± 0.90

BA 0.2 ? GA3 3.0 30 ± 1.75

BA 0.3 ? GA3 2.0 40 ± 1.50

Values are given as the average ± SD

GA3, Gibberellic acid


123

cultured in MS liquid medium has been reported in Caj-

anus cajan (Anbazhagan and Ganapathi 1999), cowpea

(Prem Anand et al. 2000) and horsegram (Varisai Mo-

hamed et al. 2004). Girija et al. (2000) reported that

1.5 mg l-1 2,4-D and 50 mg l-1L-proline can stimulate

embryogenic cells and subsequent embryo development in

Vigna radiata, whereas we induced somatic embryogenesis

from embryogenic callus on MS medium containing

0.25 mg l-1 2,4-D and 0.50 mg l-1 Kin.

The proliferation of embryogenic cells takes a number

of forms and is apparently influenced by a variety of fac-

tors, some of which can be controlled during the culture

process, and some of which are as yet undefined. Attempts

have been made to develop the early ontogeny stages of SE

differentiation from cultures of legumes, i.e. Vigna species

(Eapen and George 1990; Kulothungan et al. 1995; Girija

et al. 2000; Prem Anand et al. 2000). Our approach was

similar to previously reported procedures in which SE

development occurred through a division of spherical or

elongated cells.

Factors such as the effects of PGRs, reduced nitrogen,

plant species and genotype of the cultured material have an

effect on the induction and proliferation of embryos (El

Abidine Triqui et al. 2008). Even radiation can play a key

role in somatic embryogenesis (Begum et al. 2008). By

recognizing the critical factors involved at each stage as

well as those that exert their influence throughout the

process, the protocols at each stage can be tailored to more

closely simulate conditions in planta (Salaj et al. 2008).

Kysely et al. (1987) and Kysely and Jacobsen (1990)

found that cytokinin drastically reduced the frequency of

SE development in pea (Pisum sativum). However, we

found that the Kin in combination with 2,4-D promoted

somatic embryogenesis in callus, whereas in media with

only 2,4-D as PGR, the cell growth and embryo differen-

tiation processes were not regulated, and there was an

inhibition of maturation and further development. Loiseau

et al. (1995) reported that, in pea, the addition of cytokinins

(BA, zeatin and Kin) to auxin-containing medium reduced

embryo conversion. This is in contradiction to our results in

which maturation and germination occurred in full-strength

MS medium with a lower level of BA (0.2 mg l-1) and

GA3 (2.0 mg l-1).

In conclusion, we have developed a reliable in vitro

regeneration protocol for moth bean. Using this protocol, it

may be possible in future studies to successfully transfer

useful candidate genes conferring disease, insect and pest

resistance to moth bean varieties.

Acknowledgments The authors are grateful to CSIR, New Delhi,

India for providing financial support for the investigation reported

here and to the authorities of the Seed Corporation of India for pro-

viding seed materials used in the investigation.

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