Abstract:In order to optimize the preparation and regeneration of the protoplasts of Pleurotus eryngii GIM5.344 strain and to establish a stable and effective genetic transformation system，lytic enzymes，enzymatic hydrolysis temperature，enzymatic hydrolysis time，regeneration medium stabilizer in protoplast preparation conditions，as well as concentrations of hygromycin B and the polyethylene glycol（PEG）-mediated transformation method were optimized.The effects of different protoplast preparation and regeneration conditions on protoplast yield and regeneration rate were investigated.The results showed that the use of lysing enzyme 1 for enzymatic hydrolysis was the most effective.When enzymatic hydrolysis was conducted at 30 ℃for 2 h using regeneration medium containing mannitol，the protoplast yield reached 7 × 108 CFU/mL and the regeneration rate reached 0.40%.The transformation method that yielded the highest number of transformants was the solid pre-culture followed by a double -layer culture method.This method involved pre -culturing the protoplasts at 28 ℃on non -selective regeneration medium for 48 h after PEG -mediated transformation.Subsequently，a layer of culture medium containing 30 μg/mL hygromycin was added for selection of putative transformants.The green fluorescent protein gene in the obtained transformants exhibited stable and effective expression.