Isolation and Fusion of Protoplasts from Mesophyll Cells of Dendrobium Pompadour

Abstract

Protoplasts were isolated from Dendrobium Pompadour leaves grown in vitro. Yields of 3x105-2.5x106 mesophyll protoplasts per gram fresh weight were obtained depending on factors such as digesting enzyme, osmoticum concentration, and leaf size. Small leaves (2.5-3 cm in length and 0.3 g in weight) were digested with 5 ml of enzyme solution containing 1% Cellulase Onozuka R-10, 1% Macerozyme R-10, 0.5% Driselase, and 0.4 M mannitol. The mixture of leaves and enzyme solution was incubated at 30 C for 3 h on a gyr otary shaker with an agitation speed of 80 rpm. Purification of protoplasts on a sucrose gradient yielded very clean protoplasts that are free of raphide crystals. The fusion of protoplasts was carried out using 40% polyethylene glycol. Video microscopic studies revealed that fusion occurred when osmotic gradient was lowered.