PJB-2016-88
A STUDY ON THE ISOLATION OF PROTOPLASTS FROM MESOPHYLL CELLS OF DENDROBIUM QUEEN PINK
RAMSHA AQEEL1,2, MARIUM ZEHRA1,2, SYEDA KAHKASHAN KAZMI2, SAIFULLAH KHAN2,3*, HAMMAD AFZAL KAYANI2,4 AND AMEER AHMED MIRBAHAR2
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.
To Cite this article:
Download
