This study was conducted to optimize the enzyme concentrations and incubation time to isolate high quantity and high viable protoplasts from in vitro grown leaves of Gleditschia caspica Desf. 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. Comparative tests between different concentrations of enzymes and incubation times were made and results showed that an enzyme combination of 1.0% Cellulase and 0.5% Macerozyme for a digestion period of 16 h resulted in the best yield of protoplasts (3.35×105 protoplast per gram of fresh weigh). By this time of incubation viability of isolated protoplastswas85%87%. The protoplasts were cultured in a modification of the Kao and Michayluk culture medium, using two plating densities (2×105 and 3×105 protoplasts ml1).
