In Vitro Cell. Dev. Biol. 29P:102-108. July1993 © 1993Tissue Culture Association 1054-5476/93 $01.50+0.00 GENOTYPE EFFECTS ON PROLIFERATIVE EMBRYOGENESIS AND PLANT REGENERATION OF SOYBEAN M. A. BA1LEY, H. R. BOERMA, ~D W. A. PARROT? Department of Crop and Soil Sciences, The University of Georgia, Athens, Georgia 30602 (Received 10 November 1992; accepted 10 February 1993; editor J. J. Finer) SUMMARY Proliferative somatic embryogenesis is a regeneration system suitable for mass propagation and genetic transformation of soybean [Glycine max (L.) Merr.]. The objective of this study was to examine genotypic effects on induction and maintenance of prohferative embryogenic cultures, and on yield, germination, and conversion of mature somatic embryos. Somatic embryos were induced from eight genotypes by explanting 100 immature cotyledons per genotype on induction medium. Differences in frequency of induction were observed among genotypes. However, this step was not limiting for plant regeneration because induction frequency in the least responding genotype was sufficient to initiate and maintain proliferative embryogenic cultures. Six genotypes selected for further study were used to initiate embryogenic cultures in liquid medium. Cultures were evaluated for propagation of globular-stage tissue in liquid medium, yield of cotyledon-stage somatic embryos on differentiation medium, and plant recovery of cotyledon-stage embryos. Genotypes also differed for weight and volume increase of embryogenic tissue in hquid cultures, for yield of cotyledon-stage embryos on differentia- tion medium, and for plant recovery from cotyledon-stage embryos. Rigorous selection for a proliferative culture pheno- type consisting of nodular, compact, green spheres increased embryo yield over that of unselected cultures, but did not affect the relative ranking of genotypes. In summary, the genotypes used in this study differed at each stage of plant regeneration from proliferative embryogenic cultures, but genotypic effects were partially overcome by protocol modifi- cations. Key words: somatic embryogenesis; conversion; desiccation; Glycine max. INTRODUCTION Proliferative embryogenic cultures of soybean were developed by Finer and Nagasawa (1988) and consisted of dense, globular-stage clumps of embryogenic tissue which were rapidly propagated and maintained by routine subculture in hquid medium at low inoculum densities. Recently, these cultures have proven amenable to trans- formation (Finer and McMullen, 1991), although plant regenera- tion and transformation from this system have been reported for only one genotype (cv. Fayette). Genotype has considerable influ- ence on the efficiency of plant regeneration via somatic embryogen- esis of soybean (Komatsuda and Ohyama, 1988; Komatsuda et al., 1992) and thus may influence the apphcation of proliferative em- bryogenic cultures. As is the case with zygotic embryogenesis (Kermode, 1990; Hughes and Galau, 1989), somatic embryogenesis has several dis- tinct developmental stages. Inasmuch as plant recovery is poten- tially limited at any stage of regeneration, it is essential to quantify all stages when examining the effect of experimental variables such as genotype. Large differences in embryogenic potential among ge- notypes, for example, are of httle relevance if the embryos derived from prolific genotypes are incapable of conversion to plants. The objective of this study was to evaluate the extent of genotypic effects on induction, growth, and differentiation of prohferative embryo- genie cultures, and on recovery of fertile plants from mature so- matic embryos. The importance of selecting high-quality, embryo- genic tissue for overcoming genotype limitations was also investi- gated. MATERIALS ANDMETHODS Plant material. Eight genotypes (Century, Centennial, Davis, Hutche- son, Lee, Peking, Williams 82, and Masshokutou kou 502 [ = P1417138]) were selected to represent a diversity of germplasms and maturity groups. Ten soybean plants from each genotype were planted in a greenhouse, with staggered planting dates to synchronize anthesis between the different matu- rity groups. In addition, flowers were removed from early flowering geno- types until all genotypos were in bloom. Induction of somatic embryos. Immature pods containing embryos 3 to 5 mm in length were harvested over a l-too, period. Pods were surface-ster- ilized for 30 s in 70% 2-propanol, followed by 10 min in 1.05% sodium hypochlorite and three rinses with sterile water. Cotyledon explants were excised by removing the embryonic axis and pushing the cotyledons out of the seed coat. Cotyledons 2 to 3 mm in length were cultured with the abaxial surface in contact with induction medium which consisted of MS salts (Mu- rashige and Skoog, 1962), B5 vitamins (Gamberg et al., 1968), 6% su- crose, 40 mg/liter 2,4-dichlorophenoxyacetic acid (2,4-D), and pH 7.0 (MSD40 medium). Twenty explants were cultured on 30 ml of medium dispensed in a 100 X 20-mm plastic disposable petri dish. Five dishes (replicates) per genotype were prepared, sealed with Nescofilm (Karlan Research Products, Corp., Santa Rosa, CA), and incubated at 28 ° C with a 23-h photoperiod. Illumination was provided by cool white fluorescent fix- tures providing 5 to 10 #E • m-2 • s -1. The proportion of explants respond- ing was evaluated after 6 wk in culture. Responses scored were the propor- 102