Abstract:Protoplasts were prepared from the five-day-old mycelia through hydrolysis for 3 h with a mixture of 1.5%lywallzyme and 1.5%snailase both in 0.6 mol/L KCl stabilizing the osmotic pressure at 30 ℃.After two protoplast of Flammulina velutipes and Pleurotus ostreatus infusion and static culture 2 days at 25 ℃，it was subsequently coated on the regenerative medium.The culture temperature of 34 ℃was used as the marks to pick fusants out，which were then regenerated on potato dextrose agar（PDA）plate medium.After the regeneration of fusants，a comparison of mycelium morphology，cytology observation，antagonistic experiments，and mushroom experiments were made to validate them.In this study，a fusion strain which could fruiting at 34 ℃ was selected from 18 regenerated strains.The fusion strain was named F1P8 because of its strong fruiting body，more primordium formation and good mushroom shape.