Regeneration of fertile plants from protoplasts of soybean (Glycine max L. Merr.): genotypic differences in culture response

Fourteen soybean (Glycine max L.) genotypes were evaluated for their regenerability from protoplasts using a procedure previously descibed for the cultivar Clark 63. Protoplasts were isolated from immature cotyledon tissue and were cultured in liquid or agarose gelled KP8, MS or B5 medium with different sugars. Significant differences were observed in plating efficiency, which was as high as 63% in Jack and A-2396, and as low as 38% in X-3337. Upon regular dilution with K8 medium, 1–2 mm diameter colonies were formed in 5–6 weeks with all the genotypes tested. These colonies were then transferred onto MSB (MS salts; Murashige & Skoog, 1962 + B5 organics; Gamborg et al., 1968) medium with 0.5 mg l^–1 each of 2,4-D, BA and KN and 500 mg l^–1 CH for further growth. Once the colonies had become green, compact and nodular, and were 8–10 mm in size, they were transferred to regeneration medium. Upon regular subculturing, calli of six genotypes; A-2396, Chamberlain, Heilong-26, Jack, Resnick and XP-3015 developed shoots, with the regeneration frequency being highest 27% in Jack (52 calli out of 192 produced 8–12 shoots). The regenerated shoots from different genotypes were elongated and rooted. So far, sixty three complete plants have been obtained, including twelve A-2396, nineteen Chamberlain, fifteen Jack, nine Resnick and eight XP-3015. A total of thirty five plants have been transplanted into pots in the greenhouse. Sixteen have set seeds and others are producing flowers and pods.Abbreviations BA 6-benzyladenine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - GA₃ gibberellic acid - IBA indolebutyric acid - KN kinetin - MES 2N-morpholino] ethane sulfonic acid - NAA naphthaleneacetic acid - TH thidiazuron - ZT zeatin