Isolation, culture and whole plant regeneration from protoplasts in elite indica riccultivars-Pusa Basmati 1 and Jaya

摘要

Callus was induced from mature seed and immature embryos in two indica varieties viz. Pusa Basmati 1, and Jaya. The media composition, MS + 2, 4-D (2.0 L~(-1)) +Kin (0.5 mg L~(-1)) was the optimum combination for callus induction. At constant hormonelevel [2,4-D (1.5 L~(-1)) + Kin (0.5 L~(-1))], N_6 media was found to be as good as MS medium. The cell suspension was developed from the callus by regular sub-culturing in R2 liquid medium. Attempts were made to isolate protoplasts from callus (primaryand secondary), epicotyls (3-4 days, 7 days old) and actively growing cell suspensions. Among the cell wall degrading enzymes viz. cellulase (1-4%), macerozyme (0.5-2.0%) and pectolyase (0.1%) used in different combinations, Cellulase (1%) and pectolyase(0.1%) was found most suitable to isolate protoplasts. Cell wall formation and division of protoplast derived from cell suspension was achieved within 72 hours when cultured on KPR medium containing glucose as osmoticum. On the other hand, the growth and division of callus-derived protoplasts was very slow and there were sporadic divisions after 7 days. The protoplasts which were cultured on membrane filters using Lolium multiflorum as the feeder, gave rise to callus within 30 days.