Plantlet regeneration from immature cotyledon protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of Glycine max L. Merr. cv. Clark 63 and cultured in liquid or in agarose-gelled modified KP8 medium. Plating efficiencies of 45-50% were obtained in liquid medium and 55-60% in 1.2% (w/v) agarose beads. Upon regular dilution with K8 medium rapidly growing green microcalli (1-2 mm in size) were obtained in 5-6 weeks, which upon transfer to MSB medium with 0.5 mg 1(-1) each of 2,4-D, BA, Kn and 500 mg 1(-1) CH produced compact green calli in 4-6 weeks. After 3-4 regular subcultures of 14 days each on MSB medium containing 0.5 mg 1(-1) each of BA, Kn, ZT, 0.1 mg 1(-1) NAA and 500 mg 1(-1) CH, about 21% of the compact calli formed multiple shoots. Addition of glutamine, asparagine and GA3 enhanced shoot regeneration up to 30%. Shoots of 0.5-1.0 cm length were transferred to 1/2 MS medium with 0.01 mg 1(-1) TH and 0.5 mg 1(-1) GA3 for elongation. In 2 to 3 weeks, approximately 60% of the shoots were 2-3 cm in length. These shoots were rooted on 1/2 MS with 1% sucrose and 0.2 mg 1(-1) IBA or 0.5 mg 1(-1) NAA. So far, twenty six plants have been transferred to the greenhouse, where they all have set seed.