Protoplast Isolation and Culture of Aromatic Rice (Oryza ativa L.) Variety KDML 105

Abstract

Protoplasts were isolated from 3 sources of cells; leaves of seedlings, embryo-derived calli and cell suspension by incubating them in enzyme solution containing various kinds and concentrations of enzymes for 1, 3 and 5 hrs, repecitively. The results showed that leaves from 5-day-old seedlings was the most suitable source of cells for protoplast isolation which gave the highest yield of protoplasts 9.14 x 10 per 1 g fresh weight of leaves. The kinds and concentrations of enzymes optimum for protoplast isolation were different depended on the sources of cells used for isolation. The enzyme solution containing 2% cellulase Onozuka RS, 1% macerozyme R 10 and 0.1% pectolyase was suitable for isolation of protoplast from the leaves whereas calli and cell suspension gave high yielded protoplasts when incubated in enzyme solution containing 4% cellulase Onozuka RS, 1% macerozyme R 10 and 0.2% pectolyase. The incubation period of 3 hrs was optimum for protoplast isolation from leaves, calli and cell suspension. However, protoplasts isolated from cell suspension possessed hgiher percentage of viability (80-90%) than protoplasts isolated from leaves and calli (50-60%). The isolated protoplast could not divide to form colony or callus when cultured on N6 or K8P medium