Plant Cell Reports
Plant Cell Reports (1996) 15:531-535
9 Springer-Verlag1996
Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.] Rina Ahmed, S. Dutta Gupta, and Deepesh N. De Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur 721302, India Received 23 February 1995/Revised version received 5 September 1995 - Communicated by G. C. Phillips
Abstract. Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/1 BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/1 BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 rag/1 IBA. The regenerated plants were successfully transferred to glasshouse conditions. Abbreviations: MS: Murashige and Skoog (1962) medium; 2,4-D: 2,4-dichlorophenoxyaceticacid; NAA: 1-naphthaleneacetic acid; IAA: Indole-3-acetic acid; IBA: Indole-3-butyric acid; BAP: 6- benzylaminopurine; KN: Kinetin Introduction Regeneration of whole plants from cultured tissues or cells is a prerequisite for successful application of in vitro techniques to gene transfer, mass propagation and somaclonal variation studies. Adventitious regeneration via somatic embryogenesis is highly desirable as the process affords high rates of multiplication and often results in true-to-type plants (Parrott et al. 1991; Senaratna 1992). Considerable advancements have been made recently towards the development of embryogenic cultures for a number of grain legumes including Glycine (Lazzeri et al. 1987; Komatsuda and Ohyama 1988), Arachis (Sellars et al. 1990; Baker and Wetzstein 1994), Pisum (Kysley and Jacobsen 1990 ), Vicia
Correspondence to: S. D. Gupta
(Pickardt et al. 1989), Phaseolus (Malik and Saxena 1992), Cicer (Barna and Wakhlu 1993) and Vigna (Kumar et al. 1988). In most of the reported examples, the primary explants used were immature zygotic cotyledons and embryos. The first report of plantlet regeneration from callus tissues derived from the leaf of a grain legume came from the winged bean, Psophocarpus tetragonolobus (L.) DC (Gregory et al. 1980), although, the regeneration of plants was via shoot bud formation. The regeneration of plants in winged bean was also achieved from epicotyl segments (Mehta and Mohan Ram 1981; Tran Thanh Van etal. 1986; Venketeswaran et al. 1992). However, no information is available on plant regeneration via somatic embryogenesis. The winged bean, an underexploited legume, has been acclaimed as one of the crops with most potential for easing the problem of protein malnutrition throughout the humid tropics (N.A.S. Report 1979). Its protein content and oil quality are comparable to soybean. This interest has prompted us to initiate studies on tissue culture of the winged bean. In the present report somatic embryogenesis leading to the formation of complete plants from leaf callus of winged bean is described.
Materials and methods Seeds of winged beangenotypeEC-38825-2(obtainedfromNBPGR, Akola), were surfacesterilizedwith 70% ethanolfor 1 min, followed by treatmentwith 0.1% mercuricchloridefor 10-12 min. Theywere then rinsedthoroughlyin steriledistilledwaterand germinatedon MS basal media (Murashige and Skoog 1962) containing 0.8% agar (Hi-media, Bombay)under a 16 h photoperiod(35 # mol m-2s-1) at 25+1 o C. All media were adjusted to pH 5.8 before autoclavingat 121o C (106 kpa) for 15 min. Seed germinatedwithin 7 d and alter another 3 d the leaves were excised from the seedlings, cut transverselyinto 1-2 ram long sectionsand used as explants.
532 Somatic embryogenesis. For somatic embryogenesis, excised leaf segments were oriented with abaxial side in contact with the primary culture medium and incubated for 42 d. The primary culture medium used for somatic embryo induction consisted of MS medium containing 3% (w/v) sucrose, 0.8% agar, and supplemented with 2,4-D, NAA or IAA at vari0us concentrations (0.0, 0.1, 0.5, 1.0, 2.0 and 4.0 mg/l) either singly or in combination with BAP or KN at 0.0, 0.5, 1.0 and 2.0 mg/1 concentrations. Each induction medium treatment had 25 replications (culture tubes, 25 m m x 100 mm), each replication consisting of two explants, arranged in a completely randomised design. After 6 weeks, compact calli were produced on primary culture media either with 2,4-D (1-4 mg/l), 2,4-D (1-4 mg/l) plus BAP (0.5-2 mg/l), or NAA (0~5-4 mg/1) plus BAP (I-2 rag/l). Other treatments resulted in either browning of explants, ealli with roots or a little friable callus. Since no somatic embryos were visible on any of the cultures, compact calli obtained with 1 mg/1 BAP in combination with either NAA (0.5-4 mg/1) or 2,4-D (i-4 mg/l), and 2,4-D (1-4 mg/1) alone, were transferred separately to secondary culture media and observed for somatic embryogenesis. The secondary culture medium consisted of MS containing 3% sucrose with or without growth regulators. In search for a growth regulator treatment that would stimulate somatic embryo development, the auxins 2,4-D, NAA and IAA were used in the secondary culture medium either singly or in combination with BAP. The concentrations used for auxins were 0.I, 0.2, 0.5 and 2.0 mg/l and for cytokinin were 0.5, 1.0, 2.0 and 5.0 mg/1. There were 15 replications (culture tubes) of 2 calli each for every treatment. Cultures were incubated at 25+ 1~ C under white light (15/zmol m 2 s-1) with a 16 h photoperiod.
Somatic embryo germination and plant regeneration.To determine germination rate, somatic embryos having a distinct cotyledonary notch were plated on MS medium supplemented with various concentrations of IBA or IAA (0.0, 0.02, 0.05, 0.1 and 0.5 rag/l) alone and in combination with 0.2 mg/1 BAP. Five replications of 15 embryos per dish (15 x 100 nun) were maintained at 250 C with 16 h light provided by cool white fluorescent tubes (50/zmol m 2 s-l). The numbers of germinated somatic embryos (root-shoot emergence) in each dish were determined after 2 weeks incubation. Plantlet formation was evaluated after another I week incubation under the same cultural conditions. Establishment of plants in soil. Once roots and shoots developed without any callusing, plantlets were transferred to culture tubes (40 x 190 mm) containing half-strength MS medium devoid of growth hormones, gelled with 0.8 % agar. In vitro raised plants were removed from the culture tubes and, after washing their roots in running tap water, were transferred to earthen pots containing a mixture of soil, vermiculite and farmyard manure (1:1:1) and covered with plastic bags and transferred to glasshouse conditions.
Analyses of data. Percentage data for somatic embryogenesis were subjected to arcsin transformation for proportions before analysis, and were converted back to percentages in the data table. Significant differences between treatment means were determined using Duncan's Multiple Range Test.
Results and Discussion
Somatic embryogenesis The protocol used to obtain somatic embryogenesis consisted of two distinct stages, induction and development. Calli induced On 2,4-D and 2,4-D plus
BAP upon transfer to secondary media resulted in no embryo induction. Somatic embryos were induced from calli initiated on primary culture medium with 0.5 mg/1 NAA in combination with 1.0 mg/l BAP. On this treatment, 86 % (43 of 50) of leaf explants responded to form calli. Higher concentrations of NAA from 1-4 mg/l failed to induce somatic embryos. In our studies with direct somatic embryogenesis of winged bean,a relatively narrow range of low concentrations of NAA from 0.1-0.5 mg/1 in combination with 1-2 mg/1 BAP was found capable of inducing somatic embryos (data not presented). No somatic embryos were detected while calli were on primary culture medium, which was also seen in alfalfa (Meijer and Brown 1987). Somatic embryos appeared as early as 14 d after transfer to secondary media containing IAA (0.1-2.0 mg/1) along with BAP (0.5-5.0 mg/1). The frequency of somatic embryogenesis and the number of somatic embryos varied in accordance with the tested combinations (Table 1). Maximum embryogenesis of 60% was obtained with 0.5 mg/1 NAA and 1.0 mg/l BAP supplemented primary medium followed by transfer to a secondary medium with 0.1 mg/1 IAA and 2.0 mg/1 BAP. However, secondary medium with 0.2 mg/1 IAA and 1.0-2.0 mg/1 BAP was statistically comparable to the best treatment. The present findings on NAA/BAP stimulated embryo induction are in contrast to previous results in winged bean (Venketsewaran et al. 1992) and also differ from previous experience with legumes in general. Venketsewaran et al. (1992) obtained somatic embryos or embryo-like structures with 2,4-D and NAA alone or in combination. However, pertinent details are lacking in the report. 2,4-D was found to be effective for the induction of somatic embryos in soybean (Lazzeri et al. 1987; Liu et al. 1992), peanut (Eapen and George 1993; Baker and Wetzstein 1992, 1994), pea (Kysely and Jacobsen 1990) and alfalfa (Parrot 1991; Finstad et al. 1993), while NAA in the induction medium was less effective. Conflicting results have been reported for the role of cytokinins in legume somatic embryogenesis. In white clover, somatic embryos were obtained with BAP without the presence of an auxin in the medium (Maheswaren and Williams 1986). Cytokinin-stimulated embryo induction from non-embryonic tissue has also been documented in Phaseolus (Malik and Saxena 1992). Finstad et al. (1993) in their studies with alfalfa found that the inclusion of kinetin in the induction medium with 2,4-D greatly enhanced the embryogenic response. Conversely, the addition of BAP, zeatin or kinetin to the auxin supplemented medium strongly inhibited somatic embryogenesis in soybean (Lazzeri et al. 1987), pea (Kysely and Jacobsen 1990) and peanut
533
Fig. 1 a-d. la. lb. lc. ld.
Somatic embryogenesis and plant regeneration in leaf derived callus of winged bean A general surface view of embryo masses A globular (right) and a heart-shaped (left) embryo with well developed suspensor Regeneration of a plantlet developed from a somatic embryo Somatic embryo derived plant after transfer to glasshouse conditions, 130 d after initiation of leaf explant culture
(Eapen and George 1993). Thus, generalities for auxin/cytokinin requirements for somatic embryogenesis in legumes are difficult to formulate. The effectiveness of growth regulators appear to be very species and tissue specific (Baker and Wetzstein 1994). Somatic embryo development occurred on secondary media supplemented with IAA and BAP.
There was a general trend for the percent embryogenesis and mean number of somatic embryos per responding callus to decrease as the level of IAA increased from 0.2 to 2.0mg/l (Table 1). The apparent inhibition of somatic embryo development with IAA in winged bean is analogous to alfalfa and peanut, where somatic embryos develop upon removal of the auxin from the medium (Denchev et al. 19911 Baker and
534 Table 1. Secondary media effects on percent embryogenesis and mean n u m b e r of somatic embryos for winged bean. Somatic embryos were only induced from calli initiated on p r i m a r y medium with 0.5 mg/l NAA and 1.0 mg/l BAP. The percent embryogenesis and mean number of somatic embryos were recorded 21 d after transfer to secondary medium containing the in~cated I A A and BAP combinations. Growth regulator (mg/1) IAA
0.1
0.2
0.5
2.0
%Embryogenic cultures~
Total no.of somatic embryos
Mean no. of somatic embryos/ responding callus 4- standard erroP
BAP 0.5 1.0 2.0 5.0
3.3 ab 36.6 a~ 60.0 f 20.0 boa
12 205 327 84
18.6+0.3 a 18.1+0.2 ~b 14.0+0.44
0.5 1.0 2.0 5.0
@ 43.3 ef 53.3 ef 23.34
0 123 282 116
0~ 16.3+0.3 a~ 17.6+0.2 ~b 16.5+0.4 a~
0.5 1.0 2.0 5.0
@ 3.3 ~b 23.34 16.6 at~
0 10 106 67
0~
0.5 1.0 2.0 5.0
0~ 0~ 0~ 6.6 ~
0 0 0 25
0~ 0~ 0~ 12.5+0.7 a
15. i +0.3 ~a 13.4+0.44
Means within columns followed by the same letter are not significantly different at P = 0.05 according to Duncan Multiple Range zest. Indicates insufficient number of responding caUi to determine mean.
Wetzstein 1992). However, unlike alfalfa and peanut, somaticembryo development in winged bean cultures could not be seen in hormone-free secondary medium (data not shown). Exposure to low levels of IAA along with high levels of BAP in the secondary medium was required for somatic embryo development. Combinations of IAA and BAP were effective in shoot bud initiation from leaf explants (Gregory et al. 1980) and thin cell layer explants (Tran Thanh Van et al. 1986) of winged bean: Cultivar differences may account for tlae different observations reported. Genotype is generally considered to be a critical factor in determining regeneration potential in legumes (Komatsuda and Ohyama 1988; Sellers et al. 1990). Embryogenesis from winged bean leaf callus through the two:step culture method isillustratedin Fig. 1a-b, showing cultures on secondary medium containing 0.1 mg/l IAA and 2.0 rag/1 BAP. A general surface morphology of a cluster of compact embryogenic callus
undergoing differentiation is shown in Fig. la. Sequential somatic embryo differentiation takes place through the normal developmental stages. Fig. lb shows typical heart-shaped and globular-shaped embryos emerging from compact embryogenic callus. The developmental pattern of somatic embryos was comparable to that of the well established system of carrot and alfalfa (Dudits et al. 1991).
Somatic embryo germination and conversion Embryo germination and plantlet development was achieved on MS medium supplemented with 0.2 mg/1 BAP plus 0.05 to 0.5 mg/1 IBA (Table 2). Optimal germination (47.9%) was obtained on a medium with 0.2 mg/l BAP and 0.1 mg/1 IBA. The conversion percentage varied from 5.3 to 35.9 %. Somatic embryos which did not undergo extension of root-shoot axis increased in size and formed callus. The germination and conversion rates may be improved by maturation and desiccation of somatic embryos, as have been demonstrated in soybean (Parrot et al. 1988; Buchheim et al. 1989; Komatsuda et al. 1992). Table 2. Effect of IBA in combination with 0.2 mg/1 BAP on the frequency of embryo germination and plantiet formation in leafcaUus cultures of winged bean. Each treatment induded 15 somatic embryos and there were 5 replicates. Growth regulator (mg/l) IBA
BAP
No. of somatic embryos germinated (Mean4-SE)
0.05 0.1 0.5
0.2 0.2 0.2
1.4+1.1 7.2+1.3 4.2+0.8
% Germination
% Plantlet formation
(Mean+SE)
(Mean+SE)
9.3+1.5 47.9+1.7 27.9+1.1
5.3+1.1 35.9+1.5 7.9+1.1
Establishment of plants in soil Somatic embryo derived plantlets were transferred to culture tubes containing half-strength MS medium lacking growth hormones. After 30 d incubation, nearly all of the plantlets (94.5%, 35 of 37) had initiated further root development and shoots with many healthy leaves (Fig. lc). Gradually plants were potted in soil: vermiculite: farmyard manure (1:1:1) mixture and moved to glasshouse conditions. Of these plants, 40 % (14 of 35) survived after transfer to glasshouse conditions. Plants derived from somatic embryos appeareO morphologically normal (Fig. ld). To our knowledge this is the first report of plant regeneration via somatic embryogenesis from leaf callus of winged bean. With 60 % embryogenesis, the system
535
demonstrates the feasibility for further development of high frequency plant regeneration in winged bean. Subsequent studies to optimize the development of somatic embryos to plantlets and into plants may make the system amenable for transformation and other biotechnological studies. Acknowledgements. Financial support from the CSIR, New Delhi to RA is gratefully acknowledged. RA is also indebted to Dr. Manoranjan Singh, IICB for initial supervision.
References Baker CM, Wetzstein HY (1992) Plant Cell Reports 11:71-75 Baker CM, Wetzstein HY (1994) Plant Cell Tiss Org Cult 36:361-368 Bama KS, Wakhlu AK (1993) Plant Cell Reports 12:521-524 Buchheim JA, Colburn SM, Ranch JP (1989) Plant Physiol 89: 768-775 Denchev P, Velcheva M, Atanassov A (1991) Plant Cell Reports 10:338-341 Dudits D, Borge L, Gyorgyey J (1991) J. Cell Sci 99:475-484 Eapen S, George L (1993) Plant Cell Tiss Org Cult 35:151-156 Finstad K, Brown DCB, Joy K (1993) Plant Cell Tiss Org Cult 34: 125-132 Gregory HM, Haq N, Evans PK (1980) Plant Sci Lett 18:395-400 Komatsuda T, Ohyama K (1988) Theor Appl Genet 75:695-700 Komatsuda T, Wenbin L, Seibi O (1992) Plant Cell Tiss Org Cult 28:103-113 Kumar AS, Gamborg OL, Nabors MW (1988) Plant Cell Reports 7:138-141 Kysely W, Jacobsen HJ (1990) Plant Cell Tiss Org Cult 20:7-14 Lazzeri PA, Hildebrand DF, Collins GB (1987) Plant Cell Tiss Org Cult 10:197-208 Liu W, Moore PJ, Collins GB (1992) In Vitro Cell Dev Biol 28 P: 153-160 Maheswaren G, Williams EG (1986) Ann Bot 57:109-117 Malik KA, Saxena PK (1992) Plant Cell Reports 11:163-168 Mehta U, Mohan Ram HY (1981) Ann Bot 47:163-166 Meijer EG, Brown DC (1987) Physiol Plant 69:591-596 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 N.A.S. Report (1979) The Winged bean. A high protein crop for the tropics, National Academy of Sciences, Washington, D.C. Parrot WA, Dryden G, Vogt S, Hildebrand DF, Collins GB, Williams EG (1988) In Vitro Cell Dev Biol 24:817-820 Parrot WA (1991) Plant Cell Reports 10:17-21 Parrot WA, Merkle SA, Williams EG (1991) In: Murray DR (ed) Advanced methods in plant breeding and biotechnology, CAB International, UK, pp 158-200 Pickardt T, Huancaruna PE, Schieder O (1989) Protoplasma 149: 5-10 Sellers RM, Southward GM, Philips GC (1990) Crop Sci 30: 408-414 Senaratna T (1992) Biotech Adv 10:379-392 Tran Thanh Van K, Lie-Schricke H, Marcotte JL, Trinh TH (1986) In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 2. Springer-Verlag, Berlin Heidelberg, New York,pp 556-567 Venketeswaran S, Dias MADL, Weyers UV (1992) Acta Hort 280: 202-205
Plant Cell Reports (1996) 15:531-535
9 Springer-Verlag1996
Somatic embryogenesis and plant regeneration from leaf derived callus of winged bean [Psophocarpus tetragonolobus (L.) DC.] Rina Ahmed, S. Dutta Gupta, and Deepesh N. De Department of Agricultural and Food Engineering, Indian Institute of Technology, Kharagpur 721302, India Received 23 February 1995/Revised version received 5 September 1995 - Communicated by G. C. Phillips
Abstract. Somatic embryos were obtained from callus cultures derived from leaf explants of the winged bean, Psophocarpus tetragonolobus (L.) DC. Initiation and development of the somatic embryos occurred with a two-step culture method. Callus cultures initiated on MS medium with NAA and BAP, upon transfer to a new medium with IAA and BAP, produced somatic embryos. Maximum embryogenesis of 60% was obtained on induction medium with 0.5 mg/l NAA plus 1.0 mg/1 BAP followed by transfer to a secondary medium with 0.1 mg/l IAA and 2.0 mg/1 BAP. Optimal embryo germination and plantlet development was achieved on MS medium with 0.2 mg/l BAP plus 0.1 rag/1 IBA. The regenerated plants were successfully transferred to glasshouse conditions. Abbreviations: MS: Murashige and Skoog (1962) medium; 2,4-D: 2,4-dichlorophenoxyaceticacid; NAA: 1-naphthaleneacetic acid; IAA: Indole-3-acetic acid; IBA: Indole-3-butyric acid; BAP: 6- benzylaminopurine; KN: Kinetin Introduction Regeneration of whole plants from cultured tissues or cells is a prerequisite for successful application of in vitro techniques to gene transfer, mass propagation and somaclonal variation studies. Adventitious regeneration via somatic embryogenesis is highly desirable as the process affords high rates of multiplication and often results in true-to-type plants (Parrott et al. 1991; Senaratna 1992). Considerable advancements have been made recently towards the development of embryogenic cultures for a number of grain legumes including Glycine (Lazzeri et al. 1987; Komatsuda and Ohyama 1988), Arachis (Sellars et al. 1990; Baker and Wetzstein 1994), Pisum (Kysley and Jacobsen 1990 ), Vicia
Correspondence to: S. D. Gupta
(Pickardt et al. 1989), Phaseolus (Malik and Saxena 1992), Cicer (Barna and Wakhlu 1993) and Vigna (Kumar et al. 1988). In most of the reported examples, the primary explants used were immature zygotic cotyledons and embryos. The first report of plantlet regeneration from callus tissues derived from the leaf of a grain legume came from the winged bean, Psophocarpus tetragonolobus (L.) DC (Gregory et al. 1980), although, the regeneration of plants was via shoot bud formation. The regeneration of plants in winged bean was also achieved from epicotyl segments (Mehta and Mohan Ram 1981; Tran Thanh Van etal. 1986; Venketeswaran et al. 1992). However, no information is available on plant regeneration via somatic embryogenesis. The winged bean, an underexploited legume, has been acclaimed as one of the crops with most potential for easing the problem of protein malnutrition throughout the humid tropics (N.A.S. Report 1979). Its protein content and oil quality are comparable to soybean. This interest has prompted us to initiate studies on tissue culture of the winged bean. In the present report somatic embryogenesis leading to the formation of complete plants from leaf callus of winged bean is described.
Materials and methods Seeds of winged beangenotypeEC-38825-2(obtainedfromNBPGR, Akola), were surfacesterilizedwith 70% ethanolfor 1 min, followed by treatmentwith 0.1% mercuricchloridefor 10-12 min. Theywere then rinsedthoroughlyin steriledistilledwaterand germinatedon MS basal media (Murashige and Skoog 1962) containing 0.8% agar (Hi-media, Bombay)under a 16 h photoperiod(35 # mol m-2s-1) at 25+1 o C. All media were adjusted to pH 5.8 before autoclavingat 121o C (106 kpa) for 15 min. Seed germinatedwithin 7 d and alter another 3 d the leaves were excised from the seedlings, cut transverselyinto 1-2 ram long sectionsand used as explants.
532 Somatic embryogenesis. For somatic embryogenesis, excised leaf segments were oriented with abaxial side in contact with the primary culture medium and incubated for 42 d. The primary culture medium used for somatic embryo induction consisted of MS medium containing 3% (w/v) sucrose, 0.8% agar, and supplemented with 2,4-D, NAA or IAA at vari0us concentrations (0.0, 0.1, 0.5, 1.0, 2.0 and 4.0 mg/l) either singly or in combination with BAP or KN at 0.0, 0.5, 1.0 and 2.0 mg/1 concentrations. Each induction medium treatment had 25 replications (culture tubes, 25 m m x 100 mm), each replication consisting of two explants, arranged in a completely randomised design. After 6 weeks, compact calli were produced on primary culture media either with 2,4-D (1-4 mg/l), 2,4-D (1-4 mg/l) plus BAP (0.5-2 mg/l), or NAA (0~5-4 mg/1) plus BAP (I-2 rag/l). Other treatments resulted in either browning of explants, ealli with roots or a little friable callus. Since no somatic embryos were visible on any of the cultures, compact calli obtained with 1 mg/1 BAP in combination with either NAA (0.5-4 mg/1) or 2,4-D (i-4 mg/l), and 2,4-D (1-4 mg/1) alone, were transferred separately to secondary culture media and observed for somatic embryogenesis. The secondary culture medium consisted of MS containing 3% sucrose with or without growth regulators. In search for a growth regulator treatment that would stimulate somatic embryo development, the auxins 2,4-D, NAA and IAA were used in the secondary culture medium either singly or in combination with BAP. The concentrations used for auxins were 0.I, 0.2, 0.5 and 2.0 mg/l and for cytokinin were 0.5, 1.0, 2.0 and 5.0 mg/1. There were 15 replications (culture tubes) of 2 calli each for every treatment. Cultures were incubated at 25+ 1~ C under white light (15/zmol m 2 s-1) with a 16 h photoperiod.
Somatic embryo germination and plant regeneration.To determine germination rate, somatic embryos having a distinct cotyledonary notch were plated on MS medium supplemented with various concentrations of IBA or IAA (0.0, 0.02, 0.05, 0.1 and 0.5 rag/l) alone and in combination with 0.2 mg/1 BAP. Five replications of 15 embryos per dish (15 x 100 nun) were maintained at 250 C with 16 h light provided by cool white fluorescent tubes (50/zmol m 2 s-l). The numbers of germinated somatic embryos (root-shoot emergence) in each dish were determined after 2 weeks incubation. Plantlet formation was evaluated after another I week incubation under the same cultural conditions. Establishment of plants in soil. Once roots and shoots developed without any callusing, plantlets were transferred to culture tubes (40 x 190 mm) containing half-strength MS medium devoid of growth hormones, gelled with 0.8 % agar. In vitro raised plants were removed from the culture tubes and, after washing their roots in running tap water, were transferred to earthen pots containing a mixture of soil, vermiculite and farmyard manure (1:1:1) and covered with plastic bags and transferred to glasshouse conditions.
Analyses of data. Percentage data for somatic embryogenesis were subjected to arcsin transformation for proportions before analysis, and were converted back to percentages in the data table. Significant differences between treatment means were determined using Duncan's Multiple Range Test.
Results and Discussion
Somatic embryogenesis The protocol used to obtain somatic embryogenesis consisted of two distinct stages, induction and development. Calli induced On 2,4-D and 2,4-D plus
BAP upon transfer to secondary media resulted in no embryo induction. Somatic embryos were induced from calli initiated on primary culture medium with 0.5 mg/1 NAA in combination with 1.0 mg/l BAP. On this treatment, 86 % (43 of 50) of leaf explants responded to form calli. Higher concentrations of NAA from 1-4 mg/l failed to induce somatic embryos. In our studies with direct somatic embryogenesis of winged bean,a relatively narrow range of low concentrations of NAA from 0.1-0.5 mg/1 in combination with 1-2 mg/1 BAP was found capable of inducing somatic embryos (data not presented). No somatic embryos were detected while calli were on primary culture medium, which was also seen in alfalfa (Meijer and Brown 1987). Somatic embryos appeared as early as 14 d after transfer to secondary media containing IAA (0.1-2.0 mg/1) along with BAP (0.5-5.0 mg/1). The frequency of somatic embryogenesis and the number of somatic embryos varied in accordance with the tested combinations (Table 1). Maximum embryogenesis of 60% was obtained with 0.5 mg/1 NAA and 1.0 mg/l BAP supplemented primary medium followed by transfer to a secondary medium with 0.1 mg/1 IAA and 2.0 mg/1 BAP. However, secondary medium with 0.2 mg/1 IAA and 1.0-2.0 mg/1 BAP was statistically comparable to the best treatment. The present findings on NAA/BAP stimulated embryo induction are in contrast to previous results in winged bean (Venketsewaran et al. 1992) and also differ from previous experience with legumes in general. Venketsewaran et al. (1992) obtained somatic embryos or embryo-like structures with 2,4-D and NAA alone or in combination. However, pertinent details are lacking in the report. 2,4-D was found to be effective for the induction of somatic embryos in soybean (Lazzeri et al. 1987; Liu et al. 1992), peanut (Eapen and George 1993; Baker and Wetzstein 1992, 1994), pea (Kysely and Jacobsen 1990) and alfalfa (Parrot 1991; Finstad et al. 1993), while NAA in the induction medium was less effective. Conflicting results have been reported for the role of cytokinins in legume somatic embryogenesis. In white clover, somatic embryos were obtained with BAP without the presence of an auxin in the medium (Maheswaren and Williams 1986). Cytokinin-stimulated embryo induction from non-embryonic tissue has also been documented in Phaseolus (Malik and Saxena 1992). Finstad et al. (1993) in their studies with alfalfa found that the inclusion of kinetin in the induction medium with 2,4-D greatly enhanced the embryogenic response. Conversely, the addition of BAP, zeatin or kinetin to the auxin supplemented medium strongly inhibited somatic embryogenesis in soybean (Lazzeri et al. 1987), pea (Kysely and Jacobsen 1990) and peanut
533
Fig. 1 a-d. la. lb. lc. ld.
Somatic embryogenesis and plant regeneration in leaf derived callus of winged bean A general surface view of embryo masses A globular (right) and a heart-shaped (left) embryo with well developed suspensor Regeneration of a plantlet developed from a somatic embryo Somatic embryo derived plant after transfer to glasshouse conditions, 130 d after initiation of leaf explant culture
(Eapen and George 1993). Thus, generalities for auxin/cytokinin requirements for somatic embryogenesis in legumes are difficult to formulate. The effectiveness of growth regulators appear to be very species and tissue specific (Baker and Wetzstein 1994). Somatic embryo development occurred on secondary media supplemented with IAA and BAP.
There was a general trend for the percent embryogenesis and mean number of somatic embryos per responding callus to decrease as the level of IAA increased from 0.2 to 2.0mg/l (Table 1). The apparent inhibition of somatic embryo development with IAA in winged bean is analogous to alfalfa and peanut, where somatic embryos develop upon removal of the auxin from the medium (Denchev et al. 19911 Baker and
534 Table 1. Secondary media effects on percent embryogenesis and mean n u m b e r of somatic embryos for winged bean. Somatic embryos were only induced from calli initiated on p r i m a r y medium with 0.5 mg/l NAA and 1.0 mg/l BAP. The percent embryogenesis and mean number of somatic embryos were recorded 21 d after transfer to secondary medium containing the in~cated I A A and BAP combinations. Growth regulator (mg/1) IAA
0.1
0.2
0.5
2.0
%Embryogenic cultures~
Total no.of somatic embryos
Mean no. of somatic embryos/ responding callus 4- standard erroP
BAP 0.5 1.0 2.0 5.0
3.3 ab 36.6 a~ 60.0 f 20.0 boa
12 205 327 84
18.6+0.3 a 18.1+0.2 ~b 14.0+0.44
0.5 1.0 2.0 5.0
@ 43.3 ef 53.3 ef 23.34
0 123 282 116
0~ 16.3+0.3 a~ 17.6+0.2 ~b 16.5+0.4 a~
0.5 1.0 2.0 5.0
@ 3.3 ~b 23.34 16.6 at~
0 10 106 67
0~
0.5 1.0 2.0 5.0
0~ 0~ 0~ 6.6 ~
0 0 0 25
0~ 0~ 0~ 12.5+0.7 a
15. i +0.3 ~a 13.4+0.44
Means within columns followed by the same letter are not significantly different at P = 0.05 according to Duncan Multiple Range zest. Indicates insufficient number of responding caUi to determine mean.
Wetzstein 1992). However, unlike alfalfa and peanut, somaticembryo development in winged bean cultures could not be seen in hormone-free secondary medium (data not shown). Exposure to low levels of IAA along with high levels of BAP in the secondary medium was required for somatic embryo development. Combinations of IAA and BAP were effective in shoot bud initiation from leaf explants (Gregory et al. 1980) and thin cell layer explants (Tran Thanh Van et al. 1986) of winged bean: Cultivar differences may account for tlae different observations reported. Genotype is generally considered to be a critical factor in determining regeneration potential in legumes (Komatsuda and Ohyama 1988; Sellers et al. 1990). Embryogenesis from winged bean leaf callus through the two:step culture method isillustratedin Fig. 1a-b, showing cultures on secondary medium containing 0.1 mg/l IAA and 2.0 rag/1 BAP. A general surface morphology of a cluster of compact embryogenic callus
undergoing differentiation is shown in Fig. la. Sequential somatic embryo differentiation takes place through the normal developmental stages. Fig. lb shows typical heart-shaped and globular-shaped embryos emerging from compact embryogenic callus. The developmental pattern of somatic embryos was comparable to that of the well established system of carrot and alfalfa (Dudits et al. 1991).
Somatic embryo germination and conversion Embryo germination and plantlet development was achieved on MS medium supplemented with 0.2 mg/1 BAP plus 0.05 to 0.5 mg/1 IBA (Table 2). Optimal germination (47.9%) was obtained on a medium with 0.2 mg/l BAP and 0.1 mg/1 IBA. The conversion percentage varied from 5.3 to 35.9 %. Somatic embryos which did not undergo extension of root-shoot axis increased in size and formed callus. The germination and conversion rates may be improved by maturation and desiccation of somatic embryos, as have been demonstrated in soybean (Parrot et al. 1988; Buchheim et al. 1989; Komatsuda et al. 1992). Table 2. Effect of IBA in combination with 0.2 mg/1 BAP on the frequency of embryo germination and plantiet formation in leafcaUus cultures of winged bean. Each treatment induded 15 somatic embryos and there were 5 replicates. Growth regulator (mg/l) IBA
BAP
No. of somatic embryos germinated (Mean4-SE)
0.05 0.1 0.5
0.2 0.2 0.2
1.4+1.1 7.2+1.3 4.2+0.8
% Germination
% Plantlet formation
(Mean+SE)
(Mean+SE)
9.3+1.5 47.9+1.7 27.9+1.1
5.3+1.1 35.9+1.5 7.9+1.1
Establishment of plants in soil Somatic embryo derived plantlets were transferred to culture tubes containing half-strength MS medium lacking growth hormones. After 30 d incubation, nearly all of the plantlets (94.5%, 35 of 37) had initiated further root development and shoots with many healthy leaves (Fig. lc). Gradually plants were potted in soil: vermiculite: farmyard manure (1:1:1) mixture and moved to glasshouse conditions. Of these plants, 40 % (14 of 35) survived after transfer to glasshouse conditions. Plants derived from somatic embryos appeareO morphologically normal (Fig. ld). To our knowledge this is the first report of plant regeneration via somatic embryogenesis from leaf callus of winged bean. With 60 % embryogenesis, the system
535
demonstrates the feasibility for further development of high frequency plant regeneration in winged bean. Subsequent studies to optimize the development of somatic embryos to plantlets and into plants may make the system amenable for transformation and other biotechnological studies. Acknowledgements. Financial support from the CSIR, New Delhi to RA is gratefully acknowledged. RA is also indebted to Dr. Manoranjan Singh, IICB for initial supervision.
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