Abstract: 【Objective】 To provide reference for the studies on cell mutagenesis,fusion and gene editing of Hypsizygus marmoreus.【Methods】 The single factor test and response surface method were used to screen and optimize the main factors affecting the preparation and regeneration conditions of the protoplasts of Hypsizygus marmoreus—bacterial age,osmotic pressure stabilizer types,lysozyme concentration and enzymolysis time.【Results】 The single factor test results showed that the optimal preparation condition of protoplasts were 7 days of bacterial age,with the concentration of 0.6 mol/L mannitol as osmotic pressure stabilizer,the lysozyme concentration of 2%,enzymolysis for 5 h. The optimal regeneration conditions of protoplasts were 6 days of bacterial age,with the concentration of 0.6 mol/L mannitol as osmotic pressure stabilizer,the lysozyme concentration of 2%,enzymolysis for 3 h. The response surface method showed that when 0.6 mol/L mannitol was used as the osmotic pressure stabilizer,the best preparation and regeneration conditions were 7 days of bacterial age,2% of lysozyme concentration,and 4 h of enzymolysis,which had good fitting with the predicted value of the model.【Conclusion】 Based on the optimal preparation conditions determined by response surface method,higher protoplast yield and regeneration rate could be obtained.

Key words: Hypsizygus marmoreus, Protoplast, Yield, Regeneration rate, Single factor test, Response surface method