A STUDY ON THE ISOLATION OF PROTOPLASTS FROM MESOPHYLL CELLS OF DENDROBIUM QUEEN PINK

摘要

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 Mmannitol, 10 mM CaCl2.2H2O and 10 mM 2 (N-morpholino)-ethanesulfonic acid (MES) at pH 5.8. Protoplast highest yield with15.7x104 protoplasts per 1.5 gm freshly chopped leaves were obtained when digested in enzyme solution for 4 hrs on a rotaryshaker with an agitation speed of 45 rpm in dark conditions. Protoplasts were filtered with 45μm nylon sieve and washed with0.3 M mannitol solution supplemented with 10 mM CaCl2.2H2O and 10 mM MES, and purified with 0.3 M sucrose solutiongradient. Purification of protoplasts on a sucrose mannitol gradient yielded clean protoplasts that were free from debris.