An efficient regeneration system of black gram ( Vigna mungo L.) through organogenesis

Plant Science 134 (1998) 199–206

An efficient regeneration system of black gram (Vigna mungoL.) through organogenesis

Dilip K. Das, N. Shiva Prakash, Neera Bhalla-Sarin *

School of Life Sciences, JNU, New Delhi 110 067, India

Received 5 January 1998; received in revised form 24 February 1998; accepted 24 February 1998

Abstract

Regeneration has been achieved in Vigna mungo L. through organogenesis using explants from axillary shootsoriginating from the nodes of seedlings germinated in cytokinin containing medium. Seeds germinated in thidiazuron(TDZ) at 0.5 mg l−1 supplemented MS medium produced :11 axillary shoots/cotyledonary node. Stem and petioleexplants derived from these axillary-shoots produced callus along with shoot-buds after 2 weeks of culture on halfstrength MS supplemented with 0.1 mg l−1 a-napthaleneacetic acid. Shoot-buds were also produced from varioussites of injury caused by incisions on the stem explants. Full strength MS salts inhibited bud formation. Histologicalstudies indicated the differentiation of shoot-buds from the cortical cells. The pH of the regeneration medium had asignificant effect on regeneration efficiency. The shoot-buds elongated and rooted on one third strength MS medium.The plantlets were transferred to soil after 3 weeks and 90–95% of the plantlets thus obtained could survive transferto soil. The regeneration protocol described is highly reproducible and equally effective for all the four genotypestested, viz. T-9, Pusa-1, Pusa-2 and PS-1 yielding about 5 shoot-buds/stem explant (with apex). The regenerationsystem is efficient as it results in the recovery of 4–5 plants within a period of 8 weeks, starting from an explant.© 1998 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Black gram; Vigna mungo L.; Regeneration; Organogenesis

Abbre6iations: BAP, 6-benzylaminopurine; IAA, indole-3-acetic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; MSmedium, Murashige and Skoog (1962) medium; MS1/2, halfstrength MS medium; MS1/3, one third strength MS; MS1/

2N0.1, half strength MS medium supplemented with NAA at0.1 mg l−1; MSN1/2, MS medium with NH4NO3 and KNO3

reduced to half; NAA, a-naphthaleneacetic acid; TDZ, thidi-azuron.

* Corresponding author.

1. Introduction

Genetic engineering is predicted to make signifi-cant contributions towards crop improvement inthe coming years. Availability of a regenerationprotocol is one of the prerequisites towards thisend. The present study was undertaken to estab-lish an efficient and reproducible regeneration sys-

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D.K. Das et al. / Plant Science 134 (1998) 199–206200

tem for black gram (Vigna mungo L.), an impor-tant tropical grain legume rich in phosphoric acid.The seeds have 60% carbohydrate, 24% proteinand 1.3% fat on dry weight basis. Besides itsutility for human consumption, it also serves as anutritive fodder for milch cattle. The crop is usedas green manure and its deep root system bindssoil particles preventing erosion of the soil [1].Previously, axillary shoot proliferation of V.mungo L. has been reported when cotyledons withintact cotyledonary nodes were cultured on cy-tokinin containing medium [2]. Somatic embryo-genesis from immature cotyledon explants of V.mungo L. has been reported which however, failedto form well developed plantlets [3]. In this com-munication, we report a very efficient and repro-ducible organogenetic mode of regeneration fromstem and petiole explants of black gram (V.mungo L.). The method involved two steps: first,production of multiple-shoots from the cotyle-donary node of V. mungo L. which resembles themethod of regeneration reported previously [2]and second, culture of stem and petiole explantsderived from the multiple-shoots for induction oforganogenesis. Various factors involved in theregeneration response such as strength of MSmajor salts, supplementation of different hor-mones, pH of the medium and genotype havebeen studied. Histological studies revealed thatregeneration occurs from the cortical cells of theexplants. About 90–95% of the plantlets obtainedcould survive the transfer to soil. No phenotypicvariations were observed in the regenerated plantsas well as in the R2 generation plants.

2. Materials and methods

2.1. Plant material

Seeds of black gram genotypes, T-9, PS-1,Pusa-1 and Pusa-2 were obtained from the PulseDivision (IARI, New Delhi). All the media usedin the investigation were supplemented with 3%sucrose, adjusted to pH 5.8 (unless otherwise spe-cified) before autoclaving at 1.4 kg cm−2 (121°C)for 15 min.

The seeds were surface sterilized with 0.2%HgCl2 for 5 min, rinsed 3× in sterile doubledistilled water and germinated aseptically on MSmedium [4], supplemented with either BAP at 2mg l−1 (MSB2) or thidiazuron (TDZ) at 0.5 mgl−1 (MST0.5). Two seeds were implanted in eachculture tube (25×150 mm) containing 15 mlmedium and illuminated by two Phillips coolwhite fluorescent lamps (40 W) at photon fluencedensity of 50 mmol m−2 s−1 for 16 h per day. Thetemperature was maintained at 2592°C. Threedays after germination, the shoots were removedand cut at the region below cotyledonary attach-ment. The apical portion was placed on the samemedium as used for germination (MSB2 orMST0.5, respectively) such that the cotyledonary

Fig. 1. Schematic diagram of the regeneration protocol of V.mungo L. (a) Surface sterilised seeds were germinated inMSB2. (b) The hypocotyl along with root was excised and therest of the seedlings were cultured on MSB2. (c) Multiple-shoots were excised, cut into two to three pieces and culturedon MS1/2N0.1. (d) Regenerated shoots were separated fromthe callus and cultured on MS1/3 for rooting. (e) Plantlets weretransferred to vermiculite. Dotted lines represent the regions ofexcision.

D.K. Das et al. / Plant Science 134 (1998) 199–206 201

nodes were in direct contact with the medium(Fig. 1) for 14 days.

Multiple-shoots originating from the cotyle-donary nodes were removed from the nodal por-tion and used as the source of explants. Portionsof stem (with or without shoot-apex), leaf andpetiole explants were tested for their efficiency ofregeneration by culturing them on half strengthMS (MS1/2) supplemented with different auxins(NAA, 2,4-D, IAA added before autoclaving) at0.1 and 0.5 mg l−1, respectively (Fig. 1). Some ofthe stem explants (with intact shoot-apex) werecultured on MS1/2N0.1 after inflicting three tofour incisions half way down the stem and spaced:2–3 mm from each other. Regenerated shoot-buds elongated on MS1/3. Shoots, 2–3 cm long,were excised from the callus and rooted on MS1/3.Plantlets were transferred to autoclaved vermi-culite, and covered with a transparent plastic bagto maintain high humidity and kept at low illumi-nation (1 Phillips cool white fluorescent lamp) for1 week and watered daily with Hoagland’s nutri-ent solution [5]. After 14 days the plastic bagswere opened and gradually removed over a periodof 3–4 days, before the plantlets were transferredto soil.

To study the effect of pH on regeneration, thestem explants with intact apices derived from themultiple-shoots obtained from the seedlings (cv.Pusa-2) as in above, were transferred, respectively,to MS1/2N0.1 adjusted to different pHs (4.5, 5.5,7.0 and 8 with 0.1 N HCl or 0.1 N NaOH). Thenumber of shoot-buds were counted after 3 weeksof culture.

2.2. Histological studies

After 3, 6, 9, 12 and 15 days of culture onMS1/2N0.1, stem explants with intact apices werefixed in FAA, dehydrated in ethanol series,infiltrated with wax and sectioned as described [6].Five explants from each stage were observed.

2.3. Statistical analysis

Data presented in the tables were subjected toF-test to detect differences between treatmentsand Tukey’s multiple comparison test was used toseparate treatment means.

Table 1Comparative effect of TDZ and BAP supplementation (mgl−1) in the growth medium, on the number of axillary shootformation from the cotyledonary node of V. mungo L. (cv.Pusa-2) after 14 days of excision of hypocotyl and root

MS medium Mean no. ofshoots/explant9S.D.supplemented with:

TDZ 0.1 4.490.89a

TDZ 0.25 4.690.89a

10.691.51bTDZ 0.50BAP 2.0 7.692.60ab

BAP 3.0 6.691.52ab

Values are means of three replicates with ten explants in eachreplication).Means with the same letter do not differ significantly (P=0.05) as indicated by one-way ANOVA followed by Tukey’smultiple comparison test.

3. Results and discussion

Multiple-shoot formation was observed fromthe cotyledonary nodes of V. mungo L. seedlingsgerminated in BAP (2 or 3 mg l−1) or TDZ (0.1,0.25 or 0.5 mg l−1) containing MS medium (fullstrength). The formation of multiple-shoots wasobserved only when the main shoots were cut atthe region below cotyledonary attachment as de-scribed in Section 2 (Fig. 1). The multiple-shootsranged from 1–3 cm in length. Among the differ-ent media tested, the highest number of multiple-shoots (11 shoots/stem explant with intact apex)was observed on MS medium supplemented withTDZ at 0.5 mg l−1, after 2 weeks of culture(Table 1). Stem and petiole explants were derivedfrom these multiple-shoots and cultured on MS1/2

supplemented with different auxins at variousconcentrations (see Section 2). The cut surfaces ofall the explants which were in contact with themedium swelled on the second day of culture andcallus was initiated from the swollen portion oneighth day. Formation of shoot-buds and exten-sive root initiation were observed from these ex-plants in the second week of culture. The rootswere separated from the shoot-buds by an inter-vening callus (Fig. 2a). Among the different aux-ins tested, NAA at 0.1 mg l−1 was found to bethe best for obtaining the maximum number ofshoot-buds (5.9/explant) (Table 2). Shoot-buds

D.K. Das et al. / Plant Science 134 (1998) 199–206202

Fig. 2. Organogenesis of V. mungo L. (a) Regenerating explants after 3 weeks on MS1/2N0.1. (a1–a3) Close up of explants markedin (a). Arrow-heads point to the shoot-buds. Note the root separated from shoot by intervening callus (ca).

were obtained from the various sites of injury onthe stem explants (with intact apex) which werecultured on MS1/2N0.1 after multiple incisions(Fig. 3).

Histological studies revealed that the meristem-atic regions were not formed until after 12 days ofculture of the stem explants (with intact apex) onMS1/2N0.1. Formation of meristematic cells was

restricted to the cortical region of the stem ex-plant (Fig. 4).

Inclusion of complete MS salts in the mediumresulted in vitrification of callus within 10 days ofculture and hence the number of shoot-initialswas low (Table 3). Vitrification could not bereduced by supplementation of AgNO3 (10–50mM) in complete MS salts. However, the extent of

D.K. Das et al. / Plant Science 134 (1998) 199–206 203

Table 2Comparative effect of various auxin supplementations at dif-ferent concentrations (mg l−1) on shoot-bud formation in V.mungo L. (cv. Pusa-2) from stem (with apex) explants, after 3weeks of culture

MS1/2 medium % Explants re- No. of shoot-buds/supplemented sponding explant9S.D.with:

NAA 0.1 83.3 5.9090.17a

4.8390.23b83.3NAA 0.591.72,4-D 0.1 4.5090.66b

3.7790.51c2,4-D 0.5 75.03.1690.14c91.7IAA 0.1

83.3IAA 0.5 2.0290.63d

Values are means of three replicates with four explants in eachreplication.Means with the same letter do not differ significantly (P=0.05) as indicated by one-way ANOVA followed by Tukey’smultiple comparison test.

Fig. 4. Cross section of a stem explant cultured on MS1/2N0.1for 12 days. Note the meristem regions (m) within the cortex(×10).

vitrification was drastically reduced when halfstrength MS (MS1/2) was employed for regenera-tion. Reduction of nitrogen (NH4NO3 and KNO3)alone among all the salts of MS major, to half(MSN1/2), also resulted in improved regenerationwhich was not statistically different from the re-sponse on MS1/2 (Table 3). The effect of concen-tration of nitrogen in the nutrient medium onorganogenesis in legumes is not well studied andfull strength MS is the most commonly employed

medium. However, MS and Nitsch’s basalmedium has been found to be unfavorable fororganogenesis from the shoot-tips of V. radiata[7]. In contrast, in Arachis hypogaea, a higherefficiency of organogenesis from embryo axes wasobserved on full strength MS medium when com-pared with Gamborg’s B5 or Potato extractmedium [8]. Full strength MS medium was foundto be superior to Schenk and Hildebrandt orHeller’s medium for organogenesis from cotyle-dons and stem node explants of V. radiata [9].

Shoot-buds were successfully obtained fromboth stem and petiole explants but the leaf blade

Fig. 3. Shoot-buds arising from an explant with multipleincisions after 3 weeks of culture. The stem explant (withintact apex) was cultured on MS1/2N0.1 after making fourconsecutive incisions (half-way deep through the explant) at adistance of :2–3 mm from each other. Buds (black arrows)can be seen to arise far away from the bottom of the explantand are associated with calli produced from the incisions.White arrow-heads point to the sites of incisions. be, bottomof the explant.

Table 3Comparative effect of different strengths of MS supplementedwith NAA (0.1 mg l−1) on the efficiency of shoot-bud forma-tion from Vigna mungo L (cv. Pusa-2) stem (with apex)explants

Medium Mean no. of shoot-buds/explant

5.8390.52aMS1/2

4.8390.29bMS1/3

MSN1/2 5.3790.33ab

MS 2.2590.35c

Data were recorded after 3 weeks of culture.Means with the same letter do not differ significantly (P=0.05) as indicated by one-way ANOVA followed by Tukey’smultiple comparison test.

D.K. Das et al. / Plant Science 134 (1998) 199–206204

Table 4Comparative response of different explants of V. mungo L. (cv.Pusa-2) derived from the axillary shoots of plants germinatedin MSB2 on the regenerating medium after 3 weeks

% Explants re-Explant No. of shoot-buds/explant9S.D.sponding

Stem with apex 83.3 5.9090.17a

100 3.6690.33bStem (withoutapex)

0cLeaf blade 02.8190.69d80Petiole

Values are means of four replicates with ten explants in eachreplication.Means with the same letter do not differ significantly (P=0.05) as indicated by one-way ANOVA followed by Tukey’smultiple comparison test.

medium. Explants derived from the main-shoot ofplants germinated either on MSB2 or MS basalmedium failed to respond on MS1/2N0.1. It is wellknown that the physiological and the hormonalstatus of the explants can exert a critical influenceon regeneration. Previously, pre-conditioning ofhypocotyl explants of Pisum sati6um on IAA con-taining medium for 10 days was reported to beessential for obtaining shoots on zeatin containingmedium [14]. In the present investigation, seedgermination in the presence of a cytokinin mayhave a pre-conditioning effect on the physiologi-cal status of the axillary shoots which in turnyield stem and petiole explants amenable for re-generation. The short duration of two weeks forthe formation of shoot-buds in V. mungo L. iscomparable to the model regeneration system oftobacco.

The shoot-initials obtained on MS1/2N0.1, elon-gated rapidly and attained a height of 7–8 cmwithin 2 weeks of transfer to one third strengthMS medium. The roots were also initiated onMS1/2N0.1 resulting in the formation of sturdyplantlets (Fig. 5a). For root initiation, MS1/3

proved to be better than MS1/2 or complete MS.Although the mean number of roots initiatingdirectly from the plantlets was higher on MS1/2,secondary root production was better on MS1/3,which is reflected in the higher fresh-weight ofroots on MS1/3 (Table 6).

Rooted plantlets were initially transferred tovermiculite (Fig. 5b) and then to soil (Fig. 5c).Forty one out of 45 and 35 out of 38 plantstransferred to soil in two separate experiments,

explants produced only calli on MS1/2N0.1 whichcontinued to proliferate without exhibiting shoot-bud formation. The stem explants proved superiorto the petiole explants as the former yieldedgreater number of shoot-buds/explant (Table 4).The stem explants with intact shoot-apex gave riseto significantly higher number of shoot-buds ascompared to the explants without the shoot-apex.The nature of the explant is known to influenceregeneration of legumes. In peanut, embryonatedcotyledons produced greater number of shootscompared to de-embryonated cotyledon explants[10]. However, somatic embryogenesis fromcotyledons was found to be suppressed by thepresence of embryonic axis in A. hypogaea [11].

The pH of the medium was found to influencethe efficacy of the regeneration system. The maxi-mum number of shoot-buds (5.3 buds/explant)was obtained from stem explants on MS1/2N0.1with pH adjusted to 5.5 (Table 5). Studies on theeffect of pH on regeneration in legumes arescanty. pH was found to have no consistent effecton the efficiency of embryogenesis or rootingfrom mature embryos of soybean [12]. On thecontrary, in chickpea, pH 6.5 proved to be theoptimum for embryo maturation which was ad-versely affected by pH above 7.0 and below 4.0[13].

Regeneration was observed only when the ex-plants were derived from the axillary shoots ofplants germinated in cytokinin containing

Table 5Effect of pH of the regenerating medium (MS1/2N0.1) on thenumber of shoot-buds formed in V. mungo L. (cv. Pusa-2)from stem (with apex) explants after 3 weeks of culture

pH Mean no. of shoot-buds/explant

4.5 4.7990.18a

5.5 5.2690.64b

7.0 2.7290.54c

8.0 1.2790.25d

Means with the same letter do not differ significantly (P=0.05) as indicated by one-way ANOVA followed by Tukey’smultiple comparison test.

D.K. Das et al. / Plant Science 134 (1998) 199–206 205

Fig. 5. (a) Rooted plantlets after 2 weeks in MS1/3; (b) Plantlets 1 week and (c) 3 weeks after transfer to vermiculite.

survived and set seeds. The R2 generation plantsappeared phenotypically normal. The absence ofsomaclonal variation could be because of thecomparative short duration of (:2–3 weeks)of undifferentiated callus phase during regenera-tion.

The method of regeneration worked with equalefficacy for the four released varieties of V.mungo L. which were utilized (Table 7). How-ever, additional genotypes need to be tested forconfirming whether the regeneration protocol isgenotype independent. The entire process of re-generation starting from an explant up to thestage of establishment of regenerants in soil took

:8 weeks and 4–5 plants could be recoveredfrom each cultured stem explant (with apex).Agrobacterium mediated transformation has beenused for obtaining kanamycin resistant calli in V.mungo L. [15]. The standardization of tissue cul-ture conditions for efficient regeneration mighthelp in the generation of transgenic V. mungo L.plants.

Acknowledgements

The authors are thankful to The Pulse Divi-sion, IARI, New Delhi for providing black gram

Table 6Effect of different strengths of MS medium on rooting efficiency of V. mungo L.(cv. Pusa-2) regenerated shoots

MSO MS1/2MS1/3

0.0 3.691.67 5.8392.71No. of roots initiating from the shoot9S.D.42.62932.20 23.48913.72Fresh wt. of roots (mg) 0.0

4.9092.934.3092.22Length of the longest root9S.D. (cm) 0.0

Values are means of three replicates with ten explants in each replication.

D.K. Das et al. / Plant Science 134 (1998) 199–206206

Table 7Comparative response of different genotypes of V. mungo L.for regeneration from stem explants (with apex) after 3 weeksof culture

No. of shoot-buds/explant9S.D.Genotype

T-9 5.4090.05a

5.1692.16aPusa-15.3591.55aPusa-25.4091.50aPS-1

Values are means of three replicates with ten explants in eachreplication. Explants were derived from multiple-shoots ofseedlings as described.in Section 2.Means with the same letter do not differ significantly (P=0.05) as indicated by one-way ANOVA followed by Tukey’smultiple comparison test.

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seeds; to Professor R.N.K. Bamezai, SLS, JNU,for providing the microphotographic facility; toDr V.K. Tewari of NIHFW, New Delhi for thehelp in statistical analysis; to Professor S.K. So-pory, Dr Nirmala and Professor S.C. Maheshwariof ICGEB for going through the manuscript andtheir valuable comments. Biotechnology NationalAssociateship awarded to DKD by DBT, Govern-ment of India, is gratefully acknowledged.

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