Quercus brantii is one of the important species of west forests of Iran. Oaks are playing important role in ecosystem stability of ese forests. In forest trees, such as Oak, owing to their heterozygosity in genotype and long juvenile period, classical methods of breeding is difficult and timeconsuming.Therefore, parasexual breeding methods by using their protoplasts will be very beneficial. In this study, vital protoplasts were isolated from in vitro grown leaves of Quercus brantii. Different concentrations of hydrolytic enzymes were tested for protoplast isolation (0.5%, 1% and 1.5% of Cellulase Onozuka R10 in combination with 0.1%, 0.3%, 0.5% and or 0.7% Macerozyme). Several incubation times were used (6, 10, 14, 18, and 22 h) for digestion. The highest yields were obtained when 1.5% Cellulase and 0.1% Macerozyme were used as digestion enzyme solution (2.90×105 protoplast per gram of fresh weigh). Comparative tests between different incubation times showed that digestion period of 18 h resulted in the best yield of protoplasts, in which, viability of isolated protoplasts was 75%78%. The protoplasts were cultured in a modification of the Kao and Michayluk culture medium, using two plating densities (1×105 and 2×105 protoplasts ml1).
