PLANT-REGENERATION FROM PROTOPLASTS ISOLATED FROM CALLUS OF GENTIANA-CRASSICAULIS

Fast growing calli induced from hypocotyl segments of Gentiana crassic aulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellul ase, 1% pectinase, and 0.5% Hemicellulase. Protoplasts were cultured i n KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LY 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture met hod. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically d iluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm i n size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA(3), 1 mg/l NAA and 6% sucrose to obtain embryogeni c calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.