Abstract:
Protoplasts were successfully isolated from one month old In vitro grown plantlets of Dendrobium cultivar Queen pink. The enzyme solution used was composed of 1% Cellulase Onozuka R-10, 0.5% Macerozyme R-10, 0.1% Pectinase, 0.3 M mannitol, 10 mM CaCl2 .2H2O and 10 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH 5.8. Protoplast highest yield with 15.7x104 protoplasts per 1.5 gm freshly chopped leaves were obtained when digested in enzyme solution for 4 hrs on a rotary shaker with an agitation speed of 45 rpm in dark conditions. Protoplasts were filtered with 45µm nylon sieve and washed with 0.3 M mannitol solution supplemented with 10 mM CaCl2 .2H2O and 10 mM MES, and purified with 0.3 M sucrose solution gradient. Purification of protoplasts on a sucrose mannitol gradient yielded clean protoplasts that were free from debris.
