ISOLATION OF CELLS AND PROTOPLASTS FROM LEAVES OF IN-VITRO PROPAGATED PEACH (PRUNUS-PERSICA) PLANTS

Yields of 10(6)-10(8) peach mesophyll cells and protoplasts . gfw-1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4-10 mm in length, were a1 superior source for protoplast isolation than medium or big expanded leaves, 22-30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml-1. Purification of protoplasts on a1 sucrose gradient yielded about 10(7) protoplasts . gfw-1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.