Studies On Pleurotus Geesteranus And Tricholoma Giganteum Protoplast Fusion Breeding

Pleurotus geesteranus is a popular edible mushroom with delicious and smooth taste,and also has other excellent characteristics of the goods.Meanwhile,it is rich in protein,vitamins,carbohydrates,unsaturated fatty acids,folic acid and trace elements,etc.,which has high nutritional value.Furthermore,it has anti-tumor,anti-oxidation,lowering cholesterol and blood lipids,as well as other functions.The mycelium of P.geesteranus has strong saprophytic ability,strong adaptability to environment and biological efficiency.Tricholoma giganteum is a kind of high temperature edible fungi,which is able to produce mushrooms at high temperature of 22³5?.At the same time,based on simple and extensive cultivation techniques,T.giganteum possesses high biological efficiency and high nutritional value,having a very good development prospects in the market in recent years.However,its long growth cycle restricts the development of its industrialization.In the study,P.geesteranus and T.giganteum were used as parent stains to conduct protoplast fusion breeding.A systematic research was performed on the protoplasts preparation and regeneration of both fungi,as well as the fusion condition optimization and other properties,so as to select new type of medium-and high-temperature modification fungi with high nutritional properties,combining unique flavor of T.giganteum,and having characteristics of long storage time,not easy to be browning,rapid mycelial grow th and without casing soil when fruiting.Meanwhile,the morphology,antagonistic test,enzyme activity and molecular identification of the fusion strain were analyzed in the paper,to provide certain method and reference for the genetic breeding of edible mushroom and breed improvement in the future.The protoplast preparation conditions of two kinds of mushroom were optimized with the application of P.geesteranus and T.giganteum as parent stains.Two parental strains were marked to compare the heat inactivation conditions by the inactivation labeling technique and the protoplast fusion conditions of PEG were optimized by using Uniform Design Method in the study.After obtaining fusant,the morphology,antagonistic experiment and molecular biology were sc reened and identified,and the obtained fusants were identified to have parental gene.In the following experiments,the biological characteristics and cultivation techniques of the fusants were studied.The research contents and results are as follows:1.The screening of parent stains:In this study,the mycelial growth rate and protoplast yield were taken as the index to determine the X01 strain of P.geesteranus and J01 strain of T.giganteum as the parent strains of protoplast fusion.2.Design and optimization of protoplast fusion technique of P.geesteranus and T.giganteum:The protoplast fusion technique of P.geesteranus and T.giganteum was designed and optimized based on Uniform Design Method.The results showed that protoplast thermal inactivation condition was water bath for 25 min at 50?;PEG concentration was 25.00%,fusion time was 14 min,and fusion temperature was 34?,respectively;Tris was 0.014 M and CaC l₂ was 0.025 M.The fusion rate of P.geesteranus and T.giganteum reached the maximum when the p H value was 7.40.3.The screening of the fusion strain:The fusion strains were selected and identified by morphological and molecular methods.Two fusion strains were screened by comparison with morphological characteristics and antagonistic experiments of their parent strains,R7-1 and R7-2,which showing rapid mycelial growth and being new strains by applying ISSR technique.4.Optimization of mother medium:The mother culture med ium of R7-1?R7-2 were optimized.The optimum mother culture medium of R7-1 was:glucose 1%,peptone 1%,yeast extract 0.1%,V_B1 20mg/L,light calcium carbonate 0.10%,magnesium sulfate 0.30%,potassium dihydrogen phosphate 0.10%;the mycelium of R7-1 grew fastest?3.70 mm/d?in this medium at its suitable p H 9.35.The optimum mother culture medium of R7-2 was:glucose 2.80%,peptone 1.00%,yeast extract 0.1%,V_B1 5.05mg/L,light calcium carbonate0.10%,magnesium sulfate 0.26%,potassium dihydrogen phosphate 0.10%,the mycelium of R7-2 grew fastest?3.81 mm/d?in this medium at its suitable p H 7.09.5.The stock culture medium of R7-1?R7-2 were compared.In the wheat medium,R7-1 and R7-2 both growed fastest and had the shortest germination time and the shortest time of the mycelium continued to grow full of the bottles and the difference was significant,followed by corn granule medium.The mixed medium was the slowest growth.6.The cultivation medium of R7-1?R7-2 were optimized.The optimum cultivation medium of R7-1 was:Sawdust 18.17%,cottonseed hull 15.42%,corncob 28.66%,straw powder 13.86%,bagasse 4.67%,potassium dihydrogen phosphate 0.04%,bran 17.97%,lime 1.20%.The optimum cultivation medium of R7-2 was:Sawdust 8.84%,cotton seed hull 19.59%,corncob 33.14%,straw powder 5.72%,bagasse 11.45%,potassium dihydrogen phosphate 0.04%,bran 19.89%,lime 1.33%.