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Somatic Embryogenesis and Plant Regeneration from Cultured Zygotic Embryos of Soybean (Glycine max L. Merr.)
Somatic Embryogenesis and Plant Regeneration from Cultured Zygotic Embryos of Soybean (Glycine max L. Merr.) N.
HAMMATT
and M. R.
DAVEY
Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG72RD England Received May 27, 1986 . Accepted August 12, 1986
Summary A culture sequence has been established for the induction of somatic embryogenesis from cultured zygotic embryos of soybean. Globular, heart stage and cotyledonary embryos produced embryoids on agarose-solidified medium consisting of B5 salts and vitamins, 2 % w/v sucrose, 0.1 mg ·1- 1 IBA and 10% v / v coconut milk, the heart stage embryos being most responsive. Embryoids were obtained in each of four genotypes evaluated and such mature embryoids produced plants. Germination of mature embryoids to plants was enhanced by desiccation. The application of this methodology is discussed in relation to soybean breeding.
Key words: Glycine max, soybean, embryoids, plant regeneration, somatic embryogenesis, tissue culture, zygotic embryos.
Introduction
Plant regeneration from tissue culture is useful in clonally propagating novel genotypes and generating somaclonal variation (Evans et aI., 1984; Karp and Bright, 1985). Furthermore, it is an essential step in producing whole plants from genetically transformed cells and protoplasts, and somatic hybrids and cybrids from heterokaryons resulting from protoplast fusion (Bravo and Evans, 1985). Interest in exploiting somatic cell techniques for improvement of soybean [Glycine max (L.) Merr.] has resulted in increasing efforts to regenerate plants from cultured tissues of the commercial crop and wild Glycine species, but reports of plant regeneration from soybean cultures remain limited. Kimball and Bingham (1973) first regenerated plants from hypocotyl explants through shoot formation. Subsequently, attention focussed on somatic embryogenesis as the principle mode of regeneration. Christianson et aL (1983) observing embryoids in a soybean suspension culture derived from a morph~genetic callus of embryo origin. These workers succeeded in germinating the embryoids to produce plants. Medium containing 5 mg .1- 1 2,4-D was used to initiate morphogenetic tissue. Other workers have also used high levels of auxins to induce somatic embryogenesis in tissues of G. max. Ranch et aL (1985) and Lazzeri et aL (1985) produced embryogenic callus using the same concentration of Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid, ABA, abscisic acid, IBA, indolebutyric acid, NAA, a-naphthaleneacetic acid.
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2,4-D (5mg·l- 1 2,4-D) while Lazzeri et ai. (1985) also obtained a similar response with a medium containing 10 mg .1- 1 NAA. In each case, plants were obtained following maturation and germination of embryoids. Lippmann and Lippmann (1984) stimulated embryogenesis from embryo cotyledons using a range of 2,4-D levels (0.2 - 8.0 mg .1- 1), but were unable to obtain whole plants. In the case of wild Glycine species, plants have been regenerated from hypocotyl explants (Kameya and Widholm, 1981), and callus derived from embryo cotyledons (Grant, 1984) and seedling hypocotyls (Widholm and Rick, 1983), cotyledons, leaves and petioles (Hammatt et al., 1987 b) of G. canescens. Hammatt et al. (1986 a, 1987) extended these studies to seedling tissues of G. clandestina, G. /alcata and G. latrobeana. Regeneration in G. tomentella has been reported from seedling hypocotyls (Kameya and Widholm, 1981) and leaf callus (Hammatt et aI., 1987b). Attempts have also been made to regenerate plants from protoplasts. However, despite observations of early stages of embryogenesis in protoplast-derived cell suspensions of G. soja and G. tabacina (Gamborg et al., 1983), the only report, to date, of plant recovery in the genus has been from hypocotyl protoplasts of G. canescens (Newell and Luu, 1985). Because of the difficulty of recovering plants from somatic tissues of Glycine species, additional information is essential to advance knowledge of the factors controlling regeneration in this genus. This paper describes a method for recovering plants from zygotic embryos of G. max via secondary embryogenesis.
Materials and Methods Seeds and Plant Growth
Seeds of Glycine max (L.) Merr. cultivars Essex and Fiskeby V. and the breeding lines AP 120 and ACCO 101. were obtained from Dr. W. Ellingson, Nickersons American Plant Breeders, Ames, Iowa, USA. Plants were grown in 7 cm diameter plastic plant pots in Levington Universal compost (Fisons Horticultural Division, Ipswich, U.K.), and maintained under a 12 hour photoperiod 60cm below mercury vapour lamps (5000 lux, night temperature 16 °c, mean day temperature 22 °C). Culture media
Five media were used: i) B5GE which contained B5 salts and vitamins (Gamborg et aI., 1968), 2 % w/v sucrose, 0.1 mg .1- 1 lEA and 10 % v/v coconut milk. A double strength solution was prepared, filter sterilised (0.2 JLm pore size) and solidified by addition of an equal volume of 1.2 % w/v Sigma Type VII agarose which had been autoclaved (121 oC, 20 min) and allowed to cool to 40°C. ii) HMSO consisting of half strength MS salts and vitamins (Murashige and Skoog, 1962) and 1.5 % w/v sucrose. iii) SR6 containing MS salts and vitamins, 3 % sucrose and 5 mg .1- 1 2,4-D as employed by Christianson et ai. (1983), Ranch et ai. (1985) and Lazzeri et ai. (1986) to induce embryogenic tissues from soybean embryo explants. iv) B5E containing B5 salts and vitamins, 2 % w/v sucrose and 0.1 mg .1- 1 lEA, as used for plant recovery from cultured embryos of soybean (Tilton and Russell, 1984). v) HB50 containing B5 salts and vitamins at half strength and 1.5 % w/v sucrose. SR6, B5E, HB50 and HMSO were solidified with 0.6 % w/v Sigma agar prior to autoclaving (121 °c, 20 min).
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Embryo isolation and culture Pods containing immature seeds (1.5-2.3mm in size) were surface sterilised in 20% v/v «Domestos» bleach solution (Lever Bros., London) for 30 min, followed by thorough rinsing in four changes of sterile tap water. The pods were opened by slitting along both sutres and separating the two halves of the pericarp. Each immature seed was removed by severing the funicle. Immature embryos were removed from the seeds by dissection (Tilton and Russell, 1984) this being carried out in a bathing solution (MSO) consisting of MS salts and vitamins and 3 % v/v sucrose to prevent embryo desiccation. Each embryo was collected in a drop of MSO medium using a Pasteur pipette and transferred to the surface of B5GE medium. The MSO solution was removed, leaving the embryo on the surface of the agarose medium. Embryos were cultured in Sterilin Scm diameter, 2cm deep Petri dishes, each containing 15ml of agarose medium. A modification of this procedure was used to culture a small proportion of globular embryos from the cultivar Essex. A Pasteur pipette containing an embryo was inserted into the medium almost to the bottom of the dish, and individual embryos transferred to the interface between the medium and the base of the culture vessel. Cultures were maintained in the dark at 27 ± 2 °C for two weeks, before transfer to glass shelves 30 cm below Thorn Pluslux cool lighting tubes (continuous illumination, 1.6 W m - z, 27 ±2 0C). Embryoids were germinated in the Petri dishes under the same light and culture conditions. Regenerated plantlets were maintained in 175 ml Powder round jars (Beatson Clark, Rotherham, U.K.), each containing 30 ml of agar solidified HMSO medium.
Desiccation of embryoids Cotyledons of Mitchell and AP 120, (3.0 mm long) were excised from embryos obtained from immature seeds 6.0 mm in length and cultured on SR6 medium for 4 weeks. The resulting embryoids were matured following a further 4 week culture period on B5E medium. For desiccation, the population of mature embryoids produced (some with and some without viable, apical meristems), were transferred to empty Scm diameter, 2cm deep Sterilin Petri dishes where they were kept under continuous illumination (1.6 Wm -z,27±2 0C) until they had shrivelled to about 40- 50 % of their original volume (1-4 weeks). Germination rates were scored after such desiccated embryoids were returned to HB50 medium for a further 2 weeks.
Results
Embryos removed from immature seeds 1.5 - 2.3 mm in length were between the late globular and early cotyledonary stages of development. A trend could be identified in the response of embryos of different stages to B5GE medium, although milk from different batches of coconuts gave variable responses in callus production and frequency of embryogenesis. The most common response of cotyledonary stage embryos was the production of white, friable, wet callus which became necrotic within 4 - 6 weeks of culturing embryos on B5GE medium. Only one cotyledonary stage embryo, from AP 120, produced a single embryoid of 32 tested. In the case of ACCO 101, cotyledonary stage embryos, that did not produce callus, often became torpedo shaped and germinated into whole plants following transfer to HB50 medium. Heart stage embryos gave the best response to B5GE medium, with the highest frequency of embryoid induction and largest number of embryoids .per responding embryo. Of the four genotypes studied for the response of heart shaped embryos to B5GE medium, Fiskeby was most responsive with 38 % (42 of 112) of embryos producing embryoids, while 18 % (25 of 140) of embryos from AP 120 and 9 % (10 of
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HAMMATI
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DAVEY
Fig.!: Globular embryoids (e) developing from a zygotic embryo of soybean; c, hand s, cotyledon, hypocotyl and suspensor respectively of explanted zygotic embryo. x 37.
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110) from ACCO 101 also responded. Responding embryos each produced, on average, two embryoids, although up to six were produced in some cases. Only heart shaped embryos of Essex failed to respond. Embryoids appeared on the surface of embryos after 6 - 8 weeks of culture and, in their early development, could be identified as small cream coloured protruberances (Fig. 1). Such structures progressed through the globular, heart and cotyledonary stages (Fig.2). Development to the heart stage followed a similar pattern to the process of zygotic embryogenesis. However, development to the cotyledonary stage always involved enlargement and maturation of only one of the cotyledons (Fig. 3), even when the orientation of the embryoids, relative to the medium surface, was altered to ensure contact of both cotyledonary primordia with the medium. Embryogenesis was not restricted to any specific part of the zygotic embryos. Globular stage embryos usually died within 2-3 days when cultured on the surface of B5GE medium, although a single incidence of callus formation and the production of an embryoid was observed in AP 120. However, when globular embryos from the cultivar Essex were cultured between B5GE medium and the bottom of the dish, embryos produced friable, white callus after ten days in the dark, and, at this stage, were subcultured to the surface of fresh B5GE medium. The white, peripheral tissues died following this transfer, but revealed a core of green, nodular callus from which embryoids subsequently developed, each with a single cotyledon. Following cotyledon maturation, each embryoid was detached from the parent embryo by severing its suspensor, and transferred to HMSO medium. Although germination was erratic, embryoids subcultured regularly to fresh HMSO medium at 2-3 week intervals germinated after 2-3 months to plantlets (Fig.4). When desiccation was investigated as a factor capable of promoting embryoid germination, desiccated embryoids rapidly imbibed water following transfer to HB50 medium and, within 12 h, had resumed their original size. Within two weeks, 35 % (26 of 77) of Mitchell and 72 % (54 of 75) of AP 120 embryoids had developed a root; shoot development also ensued in embryoids with viable apical meristems. In contrast, less than 5 % (14 of 300) of embryoids transferred directly from B5E medium to HB50 medium, without desiccation, showed root and shoot development. In some cases, roots from both desiccated and unstressed embryoids became brown and necrotic. However, excision of the necrotic organs and subculture of the young plantlets to fresh HMSO medium stimulated production of a more prolific root system and vigorous plantlet growth.
Fig. 2: Early cotyledonary stage embryoids (e) still attached to the zygotic embryo. One cotyledon (c) of the cultured zygotic embryo has enlarged. x 22.5. Fig. 3: Embryoid at an early stage of germination showing maturation of one cotyledon (c) and early development of the shoot (s) and root (r). X 9.5. Fig. 4: Embryoid-derived plantlet of soybean. x 4.5.
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Discussion This investigation has identified conditions for the induction of somatic embryogenesis from zygotic embryos of soybean and subsequent plant regeneration. The medium (B5GE) is distinct from those reported by Christianson et al. (1983), Ranch et al. (1985) and Lazzeri et al. (1986) as high levels of 2,4-D were not included in the formulation, suggesting that high 2,4-D levels previously reported are important, but not essential, for somatic embryogenesis. The concentration of auxin in the coconut milk used in the present experiments was unlikely to increase the total auxin concentration in the medium to levels comparable to or exceeding those used by other workers. The reports, to date, of plant regeneration through somatic embryogenesis in soybean have all identified embryonic tissues as the best source, although each group of workers has used embryos at different stages of development. These techniques should be useful in generating somaclonal variation in soybean following evaluation on a wider range of cultivars and breeding lines. Desiccation has been shown to lead to precocious germination of zygotic embryos in soybean. Adams and Rinne (1981) allowed seeds to dry out in detached pods as early as three weeks post anthesis, to stimulate precoccious germination in these embryos. Subsequently, Ackerson (1984) desiccated excised soybean embryos and induced germination in vitro, which was correlated with lowered ABA levels in the desiccated tissues. Prevost and Le Page-Degivry (1985) have also shown that there is an inverse correlation between ABA levels and in vitro germination of zygotic embryos of the grain legume Phaseolus vulgaris. On the evidence in these reports, desiccation of embryoids in the present work may also be related to reduction in the endogenous ABA level. Germination of embryoids was also stimulated in embryoids produced on B5GE by regular subculture to HMSO medium and this transfer routine may also result in a gradual depletion of the endogenous ABA. The precocious germination of soybean embryoids on HB50 medium, following desiccation, may be useful in stimulating this response in embryoids of other legumes where germination has previously proved to be difficult. The wild relatives of soybean are a potential source of novel germplasm for soybean improvement (BroU(~ et al., 1982). Although G. max can be hybridised sexually with its wild progenitor G. soja (Hadley and Hymowitz, 1973), attempts to hybridise soybean with other wild Glycine species have only been successful with G. tomentella by culturing ovules (BroU(~ et aI., 1982; Newell and Hymowitz, 1982) and immature seeds (Singh and Hymowitz, 1985) to circumvent pod abortion following fertilization. Another technique with potential for recovering hybrids is the isolation of embryos and their culture to whole plants (Tilton and Russell, 1984), but these workers reported difficulty in inducing a response in cultured globular and early heart stage embryos of soybean. The method reported here shows that embryogenesis with plant regeneration can be achieved from very early stage embryos and this procedure should be applicable to the recovery and multiplication of sexual hybrids between soybean and wild Glycine species.
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Acknowledgements N. Hammatt was supported by a SERC CASE studentship. The authors thank Prof. E. C. C-ocking and Dr. R. S. Nelson (Shell Research Ltd.) for constructive discussions. The assistance of Mr. B. V. Case (photography), and Mr. P. Anthony and Mr. I. Gilder (growth of plant material) is acknowledged.
References ACKERSON, R. c.: Abscisic acid and precocious germination in soybeans. }. Exp. Bot. 152, 414-421 (1984). ADAMS, C. A. and R. W. RINNE: Seed maturation in soybeans [Glycine max (L.) Merr.] is independent of seed mass and of the parent plant, yet is necessary for production of viable seeds. }. Exp. Bot. 32, 615-620 (1981). BRAVO, J. E. and D. A. EVANS: Protoplast fusion for crop improvement. Plant Breed. Rev. 3, 193-218 (1985). BROUE, P., J. DOUGLASS, J. P. GRACE, and D. R. MARSHALL: Interspecific hybridisation of soybeans and perennial Glycine species indigenous to Australia via embryo culture. Euphytica 31,715-724 (1982). CHRISTIANSON, M. L., D. A. WARNICK, and P. S. CARLSON: A morphogenetically competent soybean suspension culture. Science 222,632-634 (1983). EVANS, D. A., W. R. SHARP, and H. P. MEDINA-FILHO: Somaclonal and gametoclonal variation. Am.}. Bot. 71, 759-774 (1984). GAMBORG, O. L., R. A. MILLER, and K. OlIMA: Nutrient requirements of suspension cultures of soybean roots. Exp. Cell Res. 50, 151-158 (1968). GAMBORG, O. L., B. P. DAVIS, and R. W. STAHLHUT: Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean. Plant Cell Rep. 2, 213-215 (1983). GRANT,}. E.: Plant regeneration from cotyledonary tissue of Glycine canescens, a perennial wild relative of soybean. Plant Cell Tiss. Org. Cult. 3, 169-173 (1984). HADLEY, H. H. and T. HYMOWITZ: Speciation and Cytogenetics. - In: B. E. CALDWELL (ed.): Soybeans: Improvement, Production and Uses. 97-116. Amer. Soc. Agron. Monogr. 16, Madison (1973). HAMMATT, N., M. R. DAVEY, and R. S. NELSON: Plant regeneration from seedling cotyledons, leaves and petioles of Glycine clandestina WendI. PhysioI. Plant. 68, 125-128 (1986). HAMMATT, N., R. S. NELSON, and M. R. DAVEY: Regeneration studies in perennial Glycine species (Plant Cell Tiss. Org. Cult, submitted, 1987). KAMEYA, T. and J. WIDHOLM: Regeneration from hypocotyl sections of Glycine species. Plant Sci. Lett. 21, 289-294 (1981). KARP, A. and S. W. }. BRIGHT: On the causes and origins of somaclonal variation. Oxf. Surv. Plant Mol. Cell BioI. 2, 199-234 (1985). KIMBALL, S. L. and E. T. BINGHAM: Adventitious bud development of soybean hypocotyl sections in culture. Crop Sci. 13, 758-760 (1973). LAZZERI, P. A., D. F. HILDEBRAND, and G. B. COLLINS: A procedure for plant regeneration from immature cotyledon tissue of soybean. Plant Molec. BioI. Rep. 3, 160-168 (1985). LIPPMANN, B. and G. LIPPMANN: Induction of somatic embryos in cotyledonary tissue of soybean Glycine max (L.) Merr. Plant Cell Rep. 3,215-218 (1984). MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473-497 (1962). NEWELL, C. A. and T. HYMOWITZ: Successful wide hybridisation between the soybean and a wild perennial relative, G. tomentella Hayata. Crop Sci. 22, 1062-1065 (1982). NEWELL, C. A. and H. T. Luu: Protoplast culture and plant regeneration in Glycine canescens F. J. Herm. Plant Cell Tiss. Org. Cult. 4, 145 -149 (1985).
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PREVOST, 1. and M. TH. LE PAGE-DEGIVRY: Inverse correlation between ABA content and germinability throughout the maturation and the in vitro culture of the embryo of Phaseolus vul· garis. J. Exp. Bot. 170, 1457-1464 (1985). RANCH, J. P., L. OGLESBY, and A. C. ZIELINSKI: Plant regeneration from embryo-derived tissue cultures of soybeans. In vitro 21,653-658 (1985). SINGH, R. J. and T. HYMOWITZ: An intersubgeneric hybrid between Glycine tomentella Hayata and the soybean, G. max (L.) Merr. Euphytica 34, 187 -192 (1985). TILTON, V. R. and S. H. RUSSELL: In vitro culture of immature soybean embryos. J. Plant Physiol. 115, 191-200 (1984). WIDHOLM, J. M. and S. RICK: Shoot regeneration from Glycine canescens tissue cultures. Plant Cell Rep. 2, 19-20 (1983).
J. Plant. Physiol.
Vol. 128. pp. 219-226 {1987}
HAMMATT
and M. R.
DAVEY
Plant Genetic Manipulation Group, Department of Botany, University of Nottingham, University Park, Nottingham NG72RD England Received May 27, 1986 . Accepted August 12, 1986
Summary A culture sequence has been established for the induction of somatic embryogenesis from cultured zygotic embryos of soybean. Globular, heart stage and cotyledonary embryos produced embryoids on agarose-solidified medium consisting of B5 salts and vitamins, 2 % w/v sucrose, 0.1 mg ·1- 1 IBA and 10% v / v coconut milk, the heart stage embryos being most responsive. Embryoids were obtained in each of four genotypes evaluated and such mature embryoids produced plants. Germination of mature embryoids to plants was enhanced by desiccation. The application of this methodology is discussed in relation to soybean breeding.
Key words: Glycine max, soybean, embryoids, plant regeneration, somatic embryogenesis, tissue culture, zygotic embryos.
Introduction
Plant regeneration from tissue culture is useful in clonally propagating novel genotypes and generating somaclonal variation (Evans et aI., 1984; Karp and Bright, 1985). Furthermore, it is an essential step in producing whole plants from genetically transformed cells and protoplasts, and somatic hybrids and cybrids from heterokaryons resulting from protoplast fusion (Bravo and Evans, 1985). Interest in exploiting somatic cell techniques for improvement of soybean [Glycine max (L.) Merr.] has resulted in increasing efforts to regenerate plants from cultured tissues of the commercial crop and wild Glycine species, but reports of plant regeneration from soybean cultures remain limited. Kimball and Bingham (1973) first regenerated plants from hypocotyl explants through shoot formation. Subsequently, attention focussed on somatic embryogenesis as the principle mode of regeneration. Christianson et aL (1983) observing embryoids in a soybean suspension culture derived from a morph~genetic callus of embryo origin. These workers succeeded in germinating the embryoids to produce plants. Medium containing 5 mg .1- 1 2,4-D was used to initiate morphogenetic tissue. Other workers have also used high levels of auxins to induce somatic embryogenesis in tissues of G. max. Ranch et aL (1985) and Lazzeri et aL (1985) produced embryogenic callus using the same concentration of Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid, ABA, abscisic acid, IBA, indolebutyric acid, NAA, a-naphthaleneacetic acid.
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2,4-D (5mg·l- 1 2,4-D) while Lazzeri et ai. (1985) also obtained a similar response with a medium containing 10 mg .1- 1 NAA. In each case, plants were obtained following maturation and germination of embryoids. Lippmann and Lippmann (1984) stimulated embryogenesis from embryo cotyledons using a range of 2,4-D levels (0.2 - 8.0 mg .1- 1), but were unable to obtain whole plants. In the case of wild Glycine species, plants have been regenerated from hypocotyl explants (Kameya and Widholm, 1981), and callus derived from embryo cotyledons (Grant, 1984) and seedling hypocotyls (Widholm and Rick, 1983), cotyledons, leaves and petioles (Hammatt et al., 1987 b) of G. canescens. Hammatt et al. (1986 a, 1987) extended these studies to seedling tissues of G. clandestina, G. /alcata and G. latrobeana. Regeneration in G. tomentella has been reported from seedling hypocotyls (Kameya and Widholm, 1981) and leaf callus (Hammatt et aI., 1987b). Attempts have also been made to regenerate plants from protoplasts. However, despite observations of early stages of embryogenesis in protoplast-derived cell suspensions of G. soja and G. tabacina (Gamborg et al., 1983), the only report, to date, of plant recovery in the genus has been from hypocotyl protoplasts of G. canescens (Newell and Luu, 1985). Because of the difficulty of recovering plants from somatic tissues of Glycine species, additional information is essential to advance knowledge of the factors controlling regeneration in this genus. This paper describes a method for recovering plants from zygotic embryos of G. max via secondary embryogenesis.
Materials and Methods Seeds and Plant Growth
Seeds of Glycine max (L.) Merr. cultivars Essex and Fiskeby V. and the breeding lines AP 120 and ACCO 101. were obtained from Dr. W. Ellingson, Nickersons American Plant Breeders, Ames, Iowa, USA. Plants were grown in 7 cm diameter plastic plant pots in Levington Universal compost (Fisons Horticultural Division, Ipswich, U.K.), and maintained under a 12 hour photoperiod 60cm below mercury vapour lamps (5000 lux, night temperature 16 °c, mean day temperature 22 °C). Culture media
Five media were used: i) B5GE which contained B5 salts and vitamins (Gamborg et aI., 1968), 2 % w/v sucrose, 0.1 mg .1- 1 lEA and 10 % v/v coconut milk. A double strength solution was prepared, filter sterilised (0.2 JLm pore size) and solidified by addition of an equal volume of 1.2 % w/v Sigma Type VII agarose which had been autoclaved (121 oC, 20 min) and allowed to cool to 40°C. ii) HMSO consisting of half strength MS salts and vitamins (Murashige and Skoog, 1962) and 1.5 % w/v sucrose. iii) SR6 containing MS salts and vitamins, 3 % sucrose and 5 mg .1- 1 2,4-D as employed by Christianson et ai. (1983), Ranch et ai. (1985) and Lazzeri et ai. (1986) to induce embryogenic tissues from soybean embryo explants. iv) B5E containing B5 salts and vitamins, 2 % w/v sucrose and 0.1 mg .1- 1 lEA, as used for plant recovery from cultured embryos of soybean (Tilton and Russell, 1984). v) HB50 containing B5 salts and vitamins at half strength and 1.5 % w/v sucrose. SR6, B5E, HB50 and HMSO were solidified with 0.6 % w/v Sigma agar prior to autoclaving (121 °c, 20 min).
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Embryo isolation and culture Pods containing immature seeds (1.5-2.3mm in size) were surface sterilised in 20% v/v «Domestos» bleach solution (Lever Bros., London) for 30 min, followed by thorough rinsing in four changes of sterile tap water. The pods were opened by slitting along both sutres and separating the two halves of the pericarp. Each immature seed was removed by severing the funicle. Immature embryos were removed from the seeds by dissection (Tilton and Russell, 1984) this being carried out in a bathing solution (MSO) consisting of MS salts and vitamins and 3 % v/v sucrose to prevent embryo desiccation. Each embryo was collected in a drop of MSO medium using a Pasteur pipette and transferred to the surface of B5GE medium. The MSO solution was removed, leaving the embryo on the surface of the agarose medium. Embryos were cultured in Sterilin Scm diameter, 2cm deep Petri dishes, each containing 15ml of agarose medium. A modification of this procedure was used to culture a small proportion of globular embryos from the cultivar Essex. A Pasteur pipette containing an embryo was inserted into the medium almost to the bottom of the dish, and individual embryos transferred to the interface between the medium and the base of the culture vessel. Cultures were maintained in the dark at 27 ± 2 °C for two weeks, before transfer to glass shelves 30 cm below Thorn Pluslux cool lighting tubes (continuous illumination, 1.6 W m - z, 27 ±2 0C). Embryoids were germinated in the Petri dishes under the same light and culture conditions. Regenerated plantlets were maintained in 175 ml Powder round jars (Beatson Clark, Rotherham, U.K.), each containing 30 ml of agar solidified HMSO medium.
Desiccation of embryoids Cotyledons of Mitchell and AP 120, (3.0 mm long) were excised from embryos obtained from immature seeds 6.0 mm in length and cultured on SR6 medium for 4 weeks. The resulting embryoids were matured following a further 4 week culture period on B5E medium. For desiccation, the population of mature embryoids produced (some with and some without viable, apical meristems), were transferred to empty Scm diameter, 2cm deep Sterilin Petri dishes where they were kept under continuous illumination (1.6 Wm -z,27±2 0C) until they had shrivelled to about 40- 50 % of their original volume (1-4 weeks). Germination rates were scored after such desiccated embryoids were returned to HB50 medium for a further 2 weeks.
Results
Embryos removed from immature seeds 1.5 - 2.3 mm in length were between the late globular and early cotyledonary stages of development. A trend could be identified in the response of embryos of different stages to B5GE medium, although milk from different batches of coconuts gave variable responses in callus production and frequency of embryogenesis. The most common response of cotyledonary stage embryos was the production of white, friable, wet callus which became necrotic within 4 - 6 weeks of culturing embryos on B5GE medium. Only one cotyledonary stage embryo, from AP 120, produced a single embryoid of 32 tested. In the case of ACCO 101, cotyledonary stage embryos, that did not produce callus, often became torpedo shaped and germinated into whole plants following transfer to HB50 medium. Heart stage embryos gave the best response to B5GE medium, with the highest frequency of embryoid induction and largest number of embryoids .per responding embryo. Of the four genotypes studied for the response of heart shaped embryos to B5GE medium, Fiskeby was most responsive with 38 % (42 of 112) of embryos producing embryoids, while 18 % (25 of 140) of embryos from AP 120 and 9 % (10 of
J. Plant. Physiol.
Vol. 128. pp. 219-226 {1987}
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HAMMATI
and M. R.
DAVEY
Fig.!: Globular embryoids (e) developing from a zygotic embryo of soybean; c, hand s, cotyledon, hypocotyl and suspensor respectively of explanted zygotic embryo. x 37.
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110) from ACCO 101 also responded. Responding embryos each produced, on average, two embryoids, although up to six were produced in some cases. Only heart shaped embryos of Essex failed to respond. Embryoids appeared on the surface of embryos after 6 - 8 weeks of culture and, in their early development, could be identified as small cream coloured protruberances (Fig. 1). Such structures progressed through the globular, heart and cotyledonary stages (Fig.2). Development to the heart stage followed a similar pattern to the process of zygotic embryogenesis. However, development to the cotyledonary stage always involved enlargement and maturation of only one of the cotyledons (Fig. 3), even when the orientation of the embryoids, relative to the medium surface, was altered to ensure contact of both cotyledonary primordia with the medium. Embryogenesis was not restricted to any specific part of the zygotic embryos. Globular stage embryos usually died within 2-3 days when cultured on the surface of B5GE medium, although a single incidence of callus formation and the production of an embryoid was observed in AP 120. However, when globular embryos from the cultivar Essex were cultured between B5GE medium and the bottom of the dish, embryos produced friable, white callus after ten days in the dark, and, at this stage, were subcultured to the surface of fresh B5GE medium. The white, peripheral tissues died following this transfer, but revealed a core of green, nodular callus from which embryoids subsequently developed, each with a single cotyledon. Following cotyledon maturation, each embryoid was detached from the parent embryo by severing its suspensor, and transferred to HMSO medium. Although germination was erratic, embryoids subcultured regularly to fresh HMSO medium at 2-3 week intervals germinated after 2-3 months to plantlets (Fig.4). When desiccation was investigated as a factor capable of promoting embryoid germination, desiccated embryoids rapidly imbibed water following transfer to HB50 medium and, within 12 h, had resumed their original size. Within two weeks, 35 % (26 of 77) of Mitchell and 72 % (54 of 75) of AP 120 embryoids had developed a root; shoot development also ensued in embryoids with viable apical meristems. In contrast, less than 5 % (14 of 300) of embryoids transferred directly from B5E medium to HB50 medium, without desiccation, showed root and shoot development. In some cases, roots from both desiccated and unstressed embryoids became brown and necrotic. However, excision of the necrotic organs and subculture of the young plantlets to fresh HMSO medium stimulated production of a more prolific root system and vigorous plantlet growth.
Fig. 2: Early cotyledonary stage embryoids (e) still attached to the zygotic embryo. One cotyledon (c) of the cultured zygotic embryo has enlarged. x 22.5. Fig. 3: Embryoid at an early stage of germination showing maturation of one cotyledon (c) and early development of the shoot (s) and root (r). X 9.5. Fig. 4: Embryoid-derived plantlet of soybean. x 4.5.
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Discussion This investigation has identified conditions for the induction of somatic embryogenesis from zygotic embryos of soybean and subsequent plant regeneration. The medium (B5GE) is distinct from those reported by Christianson et al. (1983), Ranch et al. (1985) and Lazzeri et al. (1986) as high levels of 2,4-D were not included in the formulation, suggesting that high 2,4-D levels previously reported are important, but not essential, for somatic embryogenesis. The concentration of auxin in the coconut milk used in the present experiments was unlikely to increase the total auxin concentration in the medium to levels comparable to or exceeding those used by other workers. The reports, to date, of plant regeneration through somatic embryogenesis in soybean have all identified embryonic tissues as the best source, although each group of workers has used embryos at different stages of development. These techniques should be useful in generating somaclonal variation in soybean following evaluation on a wider range of cultivars and breeding lines. Desiccation has been shown to lead to precocious germination of zygotic embryos in soybean. Adams and Rinne (1981) allowed seeds to dry out in detached pods as early as three weeks post anthesis, to stimulate precoccious germination in these embryos. Subsequently, Ackerson (1984) desiccated excised soybean embryos and induced germination in vitro, which was correlated with lowered ABA levels in the desiccated tissues. Prevost and Le Page-Degivry (1985) have also shown that there is an inverse correlation between ABA levels and in vitro germination of zygotic embryos of the grain legume Phaseolus vulgaris. On the evidence in these reports, desiccation of embryoids in the present work may also be related to reduction in the endogenous ABA level. Germination of embryoids was also stimulated in embryoids produced on B5GE by regular subculture to HMSO medium and this transfer routine may also result in a gradual depletion of the endogenous ABA. The precocious germination of soybean embryoids on HB50 medium, following desiccation, may be useful in stimulating this response in embryoids of other legumes where germination has previously proved to be difficult. The wild relatives of soybean are a potential source of novel germplasm for soybean improvement (BroU(~ et al., 1982). Although G. max can be hybridised sexually with its wild progenitor G. soja (Hadley and Hymowitz, 1973), attempts to hybridise soybean with other wild Glycine species have only been successful with G. tomentella by culturing ovules (BroU(~ et aI., 1982; Newell and Hymowitz, 1982) and immature seeds (Singh and Hymowitz, 1985) to circumvent pod abortion following fertilization. Another technique with potential for recovering hybrids is the isolation of embryos and their culture to whole plants (Tilton and Russell, 1984), but these workers reported difficulty in inducing a response in cultured globular and early heart stage embryos of soybean. The method reported here shows that embryogenesis with plant regeneration can be achieved from very early stage embryos and this procedure should be applicable to the recovery and multiplication of sexual hybrids between soybean and wild Glycine species.
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Acknowledgements N. Hammatt was supported by a SERC CASE studentship. The authors thank Prof. E. C. C-ocking and Dr. R. S. Nelson (Shell Research Ltd.) for constructive discussions. The assistance of Mr. B. V. Case (photography), and Mr. P. Anthony and Mr. I. Gilder (growth of plant material) is acknowledged.
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