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Plant Cell, Tissue and Organ Culture 11,233 240 (1987) ©Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands Short Communication Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek) 233 HELENA MATHEWS Plant Biotechnology Section, Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Bombay-400085, India Key words: Vigna radiata, shoot regeneration, callus, organogenesis Abstract. The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differen- ces in hormonal requirements, the various explants showed preferential growth in different basal media. Introduction Successful plant regeneration from tissue cultures of grain legumes is rather limited [15]. However, programmes on genetic upgrading of legumes based on cell and tissue culture technology necessitates a clear understanding of the system in vitro. Mung bean is one of the most important pulse crops of tropics. A method of producing multiple seedlings from single seeds of mung bean by in vitro culture of excised cotyledons was reported earlier [12]. This communication presents the organogenesis responses of various seedling explants in culture. Materials and methods Seeds of mung bean Vigna radiata L. Wilczek cv ML-5 (source: Directorate of Pulses Research ICAR, Kalyanpur, Kanpur 208 024, U.P. India) were surface sterilised and cultured on Sucrose 1%, Na2Fe EDTA and agar 0.6%. After 48 hours of incubation in the dark, the sprouted seeds were used for excising cotyledons and cotyledons with deapexed embryo explants (i.e. one cotyledon removed and the other cotyledon retained with the embryo axis from which the primordial leaves and the terminal shoot apex cut off). Cotyledonary node, primordial leaves, hypocotyls and root explants were

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

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Page 1: Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

Plant Cell, Tissue and Organ Culture 11,233 240 (1987) ©Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands

Short Communication

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

233

HELENA MATHEWS Plant Biotechnology Section, Bio-Organic Division, Bhabha Atomic Research Centre, Trombay, Bombay-400085, India

Key words: Vigna radiata, shoot regeneration, callus, organogenesis

Abstract. The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differen- ces in hormonal requirements, the various explants showed preferential growth in different basal media.

Introduction

Successful plant regeneration from tissue cultures of grain legumes is rather limited [15]. However, programmes on genetic upgrading of legumes based on cell and tissue culture technology necessitates a clear understanding of the system in vitro. Mung bean is one of the most important pulse crops of tropics. A method of producing multiple seedlings from single seeds of mung bean by in vitro culture of excised cotyledons was reported earlier [12]. This communication presents the organogenesis responses of various seedling explants in culture.

Materials and methods

Seeds of mung bean Vigna radiata L. Wilczek cv ML-5 (source: Directorate of Pulses Research ICAR, Kalyanpur, Kanpur 208 024, U.P. India) were surface sterilised and cultured on Sucrose 1%, Na2Fe EDTA and agar 0.6%. After 48 hours of incubation in the dark, the sprouted seeds were used for excising cotyledons and cotyledons with deapexed embryo explants (i.e. one cotyledon removed and the other cotyledon retained with the embryo axis from which the primordial leaves and the terminal shoot apex cut off). Cotyledonary node, primordial leaves, hypocotyls and root explants were

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taken from 4 day old seedlings while 7 day old seedlings provided the source for shoot tip explants (0.5-0.7 mm). In order to study the effect of precon- ditioning on explants, the seeds were germinated on Murashige and Skoog (MS) [ 16] supplemented with different concentrations of Benzylaminopurine - BA (5 #M, 25 #M and 50 pM). Influences of auxins, a-naphthalene acetic acid (NAA), indole-3-butyric acid (IBA), 2,4-Dichlorophenoxy acetic acid (2,4-D) and cytokinin BA were studied in different combinations at 0.5-20#M levels. The basal media used were Miller [13], MS [16], Nitsch [17], Phillips and Collins (PCL2) [18], modified Blaydes medium [3] and modified Gamberg's medium [5]. Ascorbic and citric acid (100mg/1 each) were added in the medium to prevent browning of the tissue during subcul- tures. The pH of the medium was adjusted to 5.8 before solidifying with agar 0.7%. Medium was sterilised by autoclaving at 121 °C, 15 psi for 15 minutes. All cultures were kept in continuous light of 8.2 watts/m 2 at 25 + 2 °C with a relative humidity of 60%. Number of replicates in each experiment was 24 and observations were made after 30-35 days. The in vitro plants were initially transferred to sterilised soil in paper cups. These were kept in the same environment as that of the cultures for about 8-10 days and watered on alternate days. Later these were transplanted to field.

R e s u l t s

a) Cotyledon explants

Response of cotyledon explants (after the complete removal of embryo axes) on different basal media was reported in the earlier communication, [12]. Addition of BA 5 pM to MS basal medium induced slight callus formation at the proximal end of the cotyledon followed by shoot differentiation (Fig. 1A and B) in 80% of the cultures. Further development and elongation of the shoots took place on transfer to MS + BA 2.5 pM (Fig. 1C). These shoots when isolated and cultured on MS basal medium, formed roots and developed into complete plants. When MS medium was supplemented with auxin NAA (1-20 pM) - the cotyledons produced a tuft of roots from the proximal region. Out of the various auxin cytokinin combinations tested IBA and BA were found to be the best for callus induction and differentia- tion. Although the explants responded to a range of combinations of IBA (5-20#M) and BA (5-20#M) the maximum response (80%) of the coty- ledons was in MS + IBA 20pM ÷ 5/~M (Fig. 1D). However when the callus alone was isolated and cultured on same medium, callus grew profuse- ly without any shoot bud differentiation.

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Fig. 1 A-F. Culture of cotyledons and deapexed embryos of mung bean: A. Proliferation of the proximal end of the cotyledon on MS + BA 5 #M, 10 days after culture. B. The same after 30 days of culture, showing shoot regeneration. C. Development of cotyledonary shoots on MS + BA 2.5/~M. D. Shoot regeneration of cotyledonary explant on MS + IBA 20#M + BA 5#M. E. Deapexed embryo producing multiple shoots on MS + BA 2.5/tM. F. Deapexed embryo producing multiple shoots on MS + BA 10/~M.

b) Cotyledons with deapexed embryo axes

The cotyledon with deapexed embryo axis on MS basal medium produced

fresh shoot pr imordium at the cut end and developed into complete plant

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with shoot and root system. When the explant was placed on MS + BA (2.5-10#M) the deapexed region of the embryo produced large number of shoots while the basal end of the embryo axis produced callus, without developing into the root system (Fig. 1E and F). At lower level of cytokinin (BA 2.5-5.0 pM), 2-3 thick shoots and at higher level of BA (10/~M) 8--10 slender shoots were formed from the deapexed region. In the latter case, they had to be transferred to MS + BA 2.5pM for better growth of the shoots. The well developed shoots (2-3 cm in size) could be isolated and cultured on MS basal medium for rooting.

c) Shoot tips explants

Isolated shoot tips of about 0.5--0.7 mm in size (with only apical meristem) developed into complete plants on the basal medium in 30-35 days (Table 1). PCL 2 medium gave plantlets in 58% of the total explants, while MS and Nitsch's media proved altogether unfavourable for shoot tip explants, where they necrosed.

Modified Gamborg's medium favoured callus development from shoot tip explants. Callus was profuse when this medium was supplemented v~ith BA 2.5/~M and 2,4-D 5 pM or NAA (10 pM). Callus growth was enhanced further when explants were used from preconditioned seedlings (MS + BA 50 pM). Within 15 days the shoot tip explant of size 0.7 mm proliferated to a friable callus of 1 cc while the same from non-conditioned seedling took about 30-35 days to attain similar size. Addition of ascorbic and citric acid (100mg/1 each) was essential to prevent browning of the callus during subcultures. However the callus did not differentiate under any hormonal/ nutrient combinations.

Table 1. Culture of shoot tip on various basal media

Media % cultures with shoot and roots (complete plants)

Miller [13] 8 Murashige & Skoog [16] nil Nitsch [17] nil Phillips & Collins [18] 58 Modi. Blayde's [3] 25 Mod. Gamborg [5] 41

No. of replicates 24 Culture period 30 days.

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Fig. 2 A E. Primordial leaves and cotyledonary node explants in culture and the in vitro transplants: A. Primordial leaf explant producing callus at base. B. Primordial leaf showing callus from base as well as tip. C. Shoot formation ofcotyledonary node explant on MS + BA 6.1 pM. D. Multiple shoots initiated on MS + BA 3.4/~M from cotyledonary node explant on transfer to MS + IBA 6.1/~M. E. Cotyledon derived plants at pod bearing stage, in the field.

d) Primordial leaves

Primordial leaves f rom 4 day old seedlings (incubated in dark) were subcul- tured on different basal media. Murashige and Skoog as well as Millers was found favourable for callus induction. Basal medium (either MS or Miller's fortified with BA 5-25 pM and IBA 25 #M produced callus f rom leaf base and leaf tip (Fig. 2A and B). Precondit ioning of seedlings for 48 hours with BA 25 or 50 #M was effective in enhancing callus development, but did not influence callus differentiation.

e) Cotyledonary node explants

Cotyledonary node segments produced shoots f rom the nodal region on MS + BA 3 .4#M as well as on MS + IBA 6 .1pM (Fig. 2C). On MS + BA (3.4#M), the number o f shoots (6-8) were more than that in

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MS + IBA (2-4 shoots) - but the shoots in the latter were bigger and well developed compared to small and underdeveloped shoots in the former. In subsequent experiments, the cotyledonary segment was cultured initially on MS + BA 3.4#M for a period of 20 days and then transferred to MS + IBA (6.1#M) where most of the shoots induced in cytokinin medium, grew into well developed shoots (Fig. 2D). Shoots about 2-3 cm could be isolated and rooted on MS basal medium for complete plant development.

Cotyledonary nodes cultured on MS + NAA (11.2#M) or MS + IBA 6.1/~M + BA (3.4-6.6#M) produced callus which did not undergo dif- ferentiation under any change in media combinations. Rhizogenesis was occasionally observed.

f) Root segments

Root segments in culture produced brownish callus on MS with IBA and BA at a wide range of combinations 2.5-20/~M. Addition of ascorbic and/or citric acid did not help to reduce darkening of the root derived callus. Callus did not show any form of organogenesis.

g) Transplantation

Rooted in vitro plants f rom shoot tip, deapexed embryo, cotyledon and cotyledonary node explants could be successfully transplanted to soil. After a period of 8-10 days these were transferred to pots in the field where they grew into normal plants and set seeds. Fig. 2E shows cotyledon derived plants at maturity.

Discussion

In legume tissue cultures, alterations of basal media composition have been attempted by various workers [1]. The preference of mung bean tissues to specific basal medium indicate the different nutritional requirements of different explants. Plantlet development from shoot tip explants of mung bean varied from 0-58% depending on the basal medium used. Goel et al 1983 [8] reported plantlet development from shoot tip explants of Vigna mungo and Vigna radiata while Haque et al 1984 [10] obtained multiple shoots in shoot tip cultures of Vigna mungo. In mung bean addition of auxin and/or cytokinin resulted in dedifferentiation of shoot apex meristems but no regeneration was observed. The recalcitrant callus of grain legumes has

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always been a subject of discussion in literature [4, 14, 15]. Except for the limited success in pea callus [11] and the recent report in soybean [2] the differentiation in grain legumes is often restricted to primary callus and not in established callus cultures [6, 9, 19]. However judicial selection of explants and medium components would help to raise in vitro plants in large numbers. The formation of multiple shoots in mung bean from deapexed embryonal axis could be of practical application in raising hybrid seedlings of difficult crosses. Cotyledonary node and cotyledon explants also give rise to multiple shoots either directly or via primary callus production. In addition to rapid cloning of valuable genotypes, these findings would be of use in solving or reducing the problem of chimaerism and diplontic selection in mutation experiments [7, 20].

Acknowledgements

I thank Dr. P.S. Rao, Bio-Organic Division, Bhabha Atomic Research Centre, for his interest in the work and Dr. C.R. Bhatia, Nuclear and Agriculture Division for all the guidance in growing in vitro plants in the field.

References

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