Selection Of Pleurotus Eryngii Mutant Strains By Protoplast Regeneration

This study used fruiting of Pleurotus eryngii mycelium as experimental material. The Pleurotus eryngii was supplied by Fire Kirin development co., LTD. The study isolated mycelium as starting strain and got asexual mutant strain through the protoplast preparation and regeneration.To optimize the conditions of protoplast preparation, the study designed the single variable experiments based on the enzyme system constitution, enzyme concentration, pH, enzymatic time, enzymatic temperature, osmotic pressure, stabilizer concentration and fungus age. Study got the result that higher yield protoplasm can be obtained by the protoplast preparation conditon with5d culture time,0.6mol/mL mannitol,2%lywallzyme, pH5.5,30℃,50r/min, digesting for2.5h, higher yield protoplasm can be obtained. After counting many times by using microscope, the average concentration of protoplast reach to6.10x106/mL.Protoplast regeneration was the key for this experiment, so study optimized the conditions of protoplast regeneration based on the kinds of osmotic stabilizer and constitution of culture medium and temperature of regeneration and culture. Study got the result that the better regeneration condition was useing0.6mol/mL Sucros as osmotic stabilizer, Maltose comprehensive culture medium (composed by the Maltose10g, Soluble starch10g, Glucose4g, Yeast extract4g, H2PO4lg, MgSO4-7H2O1g, VB11g,0.6mol Sucros,1000mL H2O and pH5.5), and regeneration culture temperature is28℃.Asexual mutant strains by protoplast regeneration were selected, finally8new strains were selected and numbered R’l-R’8. Then we make antagonism experiment, averaged daily mycelium growth value measurement and culture with Trichoderma viride, Penicillium and Aspergillus nige on these8new strains and the original strain. Finally got the result that R’1, R’2and R’8have strong aerial mycelia; After several passage, R’5and R’7have similar colony morphology; R’5, R’7and R’6have irregular colonies; R’3, R’4and original strain form fruiting bodies on plate culture medium; all strains can’t resist Trichoderma viride, but can resist Penicillium well, and the resistence to Aspergillus nige depends on strain kinds.