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Alfalfa (Medicago sativa) is an important forage crop belonging to the Fabaceae family. It is cultivated across the world for fodder and originated in Asia. Alfalfa cultivar Regen-SY was used in this study which is a hybrid of first-generation self-parents from Regen-S (M. sativa) and Regen-Y (Medicago falcata) research cultivars. The main objective of the study was to optimize conditions for the isolation and liquid culture of alfalfa Regen-SY protoplasts. Several factors like enzyme combination, incubation time, plant age, centrifugation speed and shaker speed affecting protoplast isolation and culture were optimized in the study. The yield and viability of the protoplasts was determined by using hemocytometer and Fluorescein diacetate (FDA) staining respectively. Results showed that factors like enzyme combination, incubation time, plant age, centrifugation speed and Mannitol concentration significantly (p ≤ 0.05) affect protoplast yield and viability whereas shaker speed didn't result in any significant difference in the yield and viability of protoplasts. Using optimum conditions protoplasts were cultured in the liquid medium and microcalli formation was achieved after five weeks of the culture. The protocol established in this study will assist researchers in the isolation and culture of protoplasts in alfalfa and will accelerate the research processes like protoplast fusion and genetic engineering.

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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.

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