Exploration on Preparation and Regeneration Conditions for Protoplast of Trichoderma viride

The effects of culture time of mycelium,enzyme combinations,enzyme concn.,enzymolysis time,hydrolysis temperature,osmotic stabilizer types,regeneration methods etc on preparation and regeneration of protoplast of Trichoderma viride were studied.The results showed that the optimum condition for preparation of protoplast of T.viride was culture time of mycelium of 60 h,enzyme combination of 1.5% lysozyme +0.5% snail enzyme,hydrolysis temperature of 30-35 ℃ and enzymolysis time of 4 h with pH value of 6.0-6.5 and osmotic stabilizer of 0.7 mol/L mannitol.Under this condition the yield of protoplast could reach 1.68×107/ml.The optimum condition for regeneration of protoplast of T.viride was PDA as the regeneration medium with osmotic stabilizer of 0.5 mol/L sucrose and regeneration method of double-layer solid culture.Under this condition the regeneration rate of the protoplast was 6.05%-6.12%.