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

Yields of 10(6)-10(8) peach mesophyll cells and protoplasts . gfw-1 we re obtained depending on factors such as digesting enzymes, and leaf s ize. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found b est for protoplast isolation and mediocre for cell isolation among sev eral enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our inve stigation. Small leaves, 4-10 mm in length, were a1 superior source fo r protoplast isolation than medium or big expanded leaves, 22-30 mm in length. The high yields of protoplasts could be obtained only when ke eping 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 di fferent growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.