A Preliminary Study On Protoplast Fusion Between Edible Mushroom Strains Hypsizigus Marmorens And Flammulina Velutipes

The capacity and rule of laccase production of six different types of edible fungus were analyzed and compared with two media. The results showed:Laccase activities of Pleurotus sajor-caju、Pleurotus geesteranus and coprinus comatus reached peak at6th day in liquid medium. The laccase activity of Flammulina velutipes increased continuously, which reached the peak at10th day. Laccase activities of Pleurotus sajor-caju、Pleurotus geesteranus and coprinus comatus reached the peak when the mycelium growed to the half part of big test tube in cultivation material. The laccase activity of Flammulina velutipes increased continuously, which reached the peak in the period of anlage formed. Under two culture conditions, Pleurotus sajorcaju、 Pleurotus geesteranus、Flammulina velutipes and coprinus comatus had high laccase activity with short growth cycle, while Hypsizigus marmoreus and pholiota namek had little laccase with long growth cycle. The rule of laccase production is same under two culture conditions, the strains with high production capacity of laccase growed fast and produce laccase at early stage of growth cycle,the strains with weak production capacity of laccase growed slow.Flammulina velutipes fl98with high laccase activity and Hypsizigus marmoreus HM03with no activity of laccase were selected as origin strains. New strains were breeded with protoplast fusion technology. Conditions optimized results of protoplast preparation showed that:protoplast production of Hypsizigus marmoreus reached to5.1×106per milliliter under the conditions of2%lywallzyme3h and0.6mol/L mannitol as osmotic stabilizer. Protoplast production of Flammulina velutipes reached to6.7×107per milliliter under the condition of1.5%enzyme2h and0.6mol/L KC1as osmotic stabilizer. Rgeneration rate of protoplast is2.8%and4.7%separately.Under the condition of25%PEG(4000),50mmol/L CaCl2·H2O,0.6mol/L mannitol, and the osmotic stabilizer0.6mol/L mannitol, protoplast of Hypsizigus marmoreusHM03and heat-treated protoplast of Flammulina velutipes fl98were fused. Four fusion strains II B、 ⅡC、 ⅢA and V6with higher laccase activity were screened by the RB-PDA plate coloration from the regeneration of fusion protoplasts.Fusion strain ⅡC discoloured steady and have short growth cycle with RB-PDA plate screening. There was obvious antagonistic lines between fusion strain ⅡC and the parents with morphological identification. Fusion strain ⅡC and the parents were identified with RAPD and ISSR molecular markers. One hundred and thirty one bands were amplified by11RAPD primers. The similarity index of fusion strain ⅡC was71%with Hypsizigus marmoreusHM03and was40%with Flammulina velutipes fl98. Sixty one bands were amplified by5ISSR primers. The similarity index fusion of strain ⅡC was57%with Hypsizigus marmoreusHM03and37.5%with Flammulina velutipes fl98. The results showed fusion strain ⅡC is a new strain and the genome of the fusion strain ⅡC is similar with Hypsizigus marmoreus. The result of fruit body production showed the stipe length and diameter of fusion strain ⅡC is longer than Hypsizigus marmoreus HM03and the pileus color is whiter than that of Hypsizigus marmoreus HM03.