Selection Of Volvariella Volvacea With Low Temperature Tolerance By Genome Shuffling

The straw mushroom, Volvariella volvacea, is one of edible mushrooms cultivated in large areas, but it has lower antifreeze capacity. Low temperature (4℃) induced Volvariella volvacea mycelium autolyzed and fruiting body liquefied, which brings great difficulty to the preservation of strains and storage of fruiting body.Our study was the first to screen low-temperature-resistant strains of Volvariella volvacea using genome shuffling technology. Three low-temperature-resistant strains were obtained by parents inactivated mark and low-temperature screening condition.In this study, the conditions of preparation and regeneration of protoplast were optimizated and the mutant library was constructed. Main results were as follows:1. The optimum conditions of protoplast preparation of Volvariella volvacea were mycelium cultured for60h,0.6mol/L mannitol as osmotic stabilizer,1.5%lywallzyme enzymolysis for3h at32℃. The optimum conditions of protoplast regeneration were mycelium cultured for84h,0.6mol/L mannitol as osmotic stabilizer,1.5%lywallzyme enzymolysis for2h at32℃, then cultured in constant temperature incubator at32℃.2. The protoplasts of16strains were mutagenized with ultraviolet, chemical, electron beam and60Co-γ.194mutant strains were obtained through low temperature (0℃) selection and stability testing. These mutant strains could survive about24h in0℃. Their antifreeze capacity of0℃increased1.4times compare to original strains. The construction of mutant libraries laid a good foundation for genome shuffling.3. Mutants were chemical (PEG) fusion by ultraviolet-inactivated (20W,30cm,110s) and heat inactivated (50℃,3min) parents inactivated notation. The results showed that the highest fusion rate was0.517%at the condition (40%PEG6000, pH8.0, fusion for30min at32℃). 4. The15strains (F4)which had higher antifreeze capacity(0℃,48h) were obtained by4rounds of genome shuffling. Their antifreeze capacity of0℃increased3.8times compare to original strains. There were9strains genetically stable in the15F4, and3strains were capable of fruiting normally. The storage experiment at10℃showed that, the storage life of the3strains were25、32、48h respectively, which were1.56、2.00、3.00times that of the original.The experiment results showed that it was feasible to improve antifreeze capacity of Volvariella volvacea through genome shuffling technology. Genome shuffling was firstly reported to be applied in edible fungus breeding in our paper. It provided a new method for edible fungi breeding.