Peach (Prunus persica) is one of the important fruits in moderate regions. In deciduous fruit trees, such as peach, owing to their heterozygosity in genotype and long juvenile period classical methods of breeding is difficult and time-consuming. Therfore, parasexual breeding methods by using their protoplasts will be very beneficial. In this study, protoplasts were isolated from four different size of leaf (4-10 mm, 10-16mm, 16-22, and 22-30 mm in lenght) of three cultivars (Elberta, Red Haven, and Mashhadi) of peach under 5 different digestion time (10, 14, 18, 22, and 26 h). The results showed that effect of cultivar on viability and quantity of isolated protoplasts was not significant. The yields of 106 – 109 peach protoplasts/ gfw-1 were obtained depending on factors such as digesting time, and leaf size. For protoplasts isolation, leaf segments were incubated on enzyme solution that consists of cellulase onozuka R-10 (2%) and macerozyme (1%). In respect of leaf size, small leaves, 4-10 mm in lenght, were a best source for protoplast isolation than medium or big expanded leaves, 22-30 mm in lenght. Furthermore, the high yields of protoplasts were obtained when the leaf segments were incubated on digestion enzymes for 18-22 h. By this time of incubation, viability of isolated protoplasts was 85%-90% for all cultivars.
