Studies On Flammulina Velutipes And Tricholoma Giganteum Massee Protoplast Fusion Breeding

Flammulina velutipes is one of the famous varieties of edible fungi industrial production. However, enterprise economic benefits reduce due to the large energy consumption of the low temperature controlling. Tricholoma giganteum, one of a rare edible and medicinal fungus in the world with tremendous market potential, is restricted on the development of being industrialized and large-scale by its slow growth of mycelium and long-time-fruiting. Protoplast fusion technology is an important breeding techniques in the edible research area. It save time and manpower, and is good at overcoming the incompatibility of distant hybridization. The present study was designed to take advantage of protoplast fusion technique to screen a new strain of Flammulina velutipes which can grow fruiting in high temperature environments and a Tricholoma giganteum new strain which can be fruited during shorter time. In this study, we screened one of Flammulina velutipes and one of Tricholoma giganteum with excellent traits as a parent strain. After being inactivated, we optimized the protoplast fusion condition with the two parent strains,treated by PEG. Partial zygotes were identified by ISSR analysis. The physiological,biochemical and biological characteristics of these zygotes were studied. The main results were as follow:1 ? Parental strains were screened. In this study, a comprehensive analysis after, we determined JD03 and KD04 as parental strains for the protoplast fusion, according to the growth rate of mycelium, the extent of the monocytes protoplast preparation and production of protoplasts number.2?The condition of protoplast fusion of Tricholoma giganteum and Flammulina velutipes was optimized by uniform design method. The results showed that the optimum protoplast fusion condition was: PEG 25.91%, fusion time 24.52 min, fusion temperature 25.6?, Tris0.016mol/L, Ca Cl2 0.014mol/L, p H 8.34. The highest fusion rate was 1.68×10-3 at thiscondition.3?Fusion strains were screened and identified according to the morphological analysis and molecular biology. Four zygotes having good character were obtained. They grew fast and their mycelium lock-like joint structure is clearly visible. Obvious antagonism happened between the strains and parents. Three of them(R13?R14?R15) were identified by using ISSR. The colony character,morphological state of these three zygotes were different from their parents. Genetic correlation coefficient and cluster analysis were analyzed. The result showed that the genetic correlation coefficients between all zygotes and Tricholoma giganteum were 0.49 and between all zygotes and Flammulina velutipes were 0.45; The fusion exhibited new traits different from the parent, which show a preliminary successful fusion between Tricholoma giganteum and Agaricus bisporus.4?The mycelia growth optimum medium of R13?R14?R15 were optimized. The mycelia growth optimum medium of R13 was: glucose 1%, peptone 1%, yeast extract 0.1%, VB120mg/L, light calcium carbonate 0.55%, magnesium sulfate 0.17%, potassium dihydrogen phosphate 0.1%; the mycelium of R13 grew fastest(3.744 mm/d) in this medium at its suitable p H9.5. The mycelia growth optimum medium of R14 was: glucose 1%, peptone0.6%, yeast extract 0.1%, VB1 2.1mg/L, light calcium carbonate 0.55%, magnesium sulfate0.12%, potassium dihydrogen phosphate 0.55%, the mycelium of R14 grew fastest(3.805mm/d) in this medium at its suitable p H7.6. The mycelia growth optimum medium of R15was: glucose 2.8%, peptone 0.1%, yeast extract 0.1%, VB1 3.3mg/L, light calcium carbonate 0.55%, magnesium sulfate 0.14%, potassium dihydrogen phosphate 0.55%, the mycelium of R15 grew fastest(2.916 mm/d) in this medium at its suitable p H10.5.5?The cultivation properties of the fusion strains R13?R14?R15 were studied. It indicated that the sparse mycelium of R13?R14?R15 grew fastest in wheat grain medium, making shortest germination time, which is significantly different from others. Following is corn grain medium in which the mycelium of R13?R14?R15 grew bushy. Mycelial growth in the mixed medium was the slowest.