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Many strains were degenerated during cultivation,because of automatically mutation and environmental factors.Purification and rejuvenation is essential to conserve the original state of cultivated strains.An urgent need in the production is scientific detection methods of strain degeneration degree.The strain As2796 was used as object;many separators are isolated by protoplast regeneration,tissue isolation and hyphal tip isolation.By testing the growth speed on PDPY media,wheat spawn media,composted substrate,27 primary screened strains which perform better than the As2796 were chose.All the 27 primary screened strains were cultivated in substrate to compare agronomic traits(yield,diameter of pileus, thickness of pileus,diameter of stipe,stipe length,single fruit weight).Using grey correlation analysis to evaluate the characteristics of all these separators.According to the correlation coefficient,P2、P28、Tip8、TI12 have been selected as the rejuvenated isolates out of all the 27 primary screened strains.The strain As2796 has slim stipe,and its pileus is very thin,with yield of 7.406 Kg/m~2,but the correlation coefficient of As2796 is 0.6230.The rejuvenated isolate P28 showed the highest correlation coefficient of 0.8195.What is more,P28 has the highest yield,8.444 Kg/m~2,14%higher than As2796.Though the yield of P2 is a little less than As2796,its marketable fruit body percentage is higher.The correlation coefficient of P2 is the second largest one among all the cultivated strains.TI12 has the largest single fruit weight,its shape is round and even,holds the third largest correlation coefficient,and its yield is located in the fifteenth position among all the cultivated strains.Tip8 correlates with ideal strain at highest correlation coefficient among all the tip isolates and performs best of all tip isolates,but its yield is not very high,only situated in eighteenth location.There is some relationship between strain degeneration and cell apoptosis.8 strains, obtained from different ways of conservation,were used to be the research objects to detect the extent of apoptosis in different ways of conservation.By testing DNA Ladder and the accumulated level of reactive oxygen species(ROS),different strain manifestes different apoptosis degree.High temperature would make terrible lesion to fungal hypha, make it slim,frizzy,and slow growth.The rejuvenated strains P28,Tip5、TI12 were also detected.The apoptosis degree of TI12 is less than that of P28 and Tip5.The apoptosis degree of strains conserved in 4℃is higher than that of strains which is conserved in room temperature. According to cultivation test and apoptosis detection,protoplast regeneration、tissue isolation、tip isolation are effective for rejuvenation.Strain conservation in natural composted substrate can reduce the speed of degeneration.
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