Technical Research Of Protoplast Fusion Between Pleurotus Geesterani And Pleurotus Citrinopileatus Sing

Protoplast fusion between Pleurotus geesterani Singer and Pleurotus citrinopileatus Sing is studied in this paper,the aim is to pick out new strains of Pleurotus with excellent advantages from two parents,enrich the source of Pleurotus.The main research contents and results are as follows:Comparing the growth conditions of Pleurotus geesterani Singer and Pleurotus citrinopileatus Sing in different culture medium,It is proposed to select Pleurotus geesterani Singer and Pleurotus citrinopileatus Sing as the parent materials in test of protoplast fusion.The optimum conditions of protoplast preparation,regeneration and fusion were selected by the single factor test and orthogonal test,which contained these study:fungus age,enzyme concentration,enzymolysis time,enzymolysis temperature,types of steady seepage and its concentration,kinds of regeneration medium,concentration of fusion agent,ways of protoplast regeneration,types of protoplast regeneration culture medium,etc.The results showed:The optimum conditions for the final system of x188-1 are determined:the yield of protoplast and regeneration rate of protoplast reached 3.4×107 CFU/mL and 1.5%respectively,under the conditions of 8 days mycelium in the enzyme solution of 1.6%lywallzyme digested for 160 min at temperature of 29 ? with 0.6 mol/L mannitol +10 mmol/L Tris-HCI as osmotic pressure stabilizer.The optimal conditions for the protoplasmic system of py-1:the yield of protoplast and regeneration rate of protoplast reached 4.7 ×107 CFU/mL and 1.7%respectively,under the conditions of 8 days mycelium in the enzyme solution of 1.6%lywallzyme digested for 160 min at temperature of 29 ?with 0.6 mol/L mannitol +10 mmol/L Tris-HCI as osmotic pressure stabilizer.In different acidity condition of parents mycelium growth rate were determined,the results show that:under the condition of pH3,there is a huge difference in the growth of two parent strains,pH3 is selected as one of the natural genetic markers for the breeding of protoplast fusion.The single-nucleated strains of x188-1 were fused with the single-nucleated strains of py-1 after inactivating the x188-1 mononuclear strain,and then the fusants were selected under the condition of pH3.The regeneration results of protoplast under different inactivation treatment were observed,and the optimum temperature and time were selected.The results show that 65? water bath 25 min as a condition of inactivated can achieve completely inactivated,with protoplast damaged slightly,stable heat inactivated effect,strong repeatability.Observing protoplast fusion and protoplast survival situation,selecting the best concentration and the degree of polymerization of PEG fusion agents,the results show that under the condition of 35%PEG4000,It has the highest rate of protoplast fusion,and less damage of protoplast.The feasibility of protoplast fusion is proved by the observation of protoplast fusion.The high temperature inactivation and pH screening were selected as the initial screening markers in this experiment,and then a fusant was obtained through the microscopy of clamp connection,and a stable fusant R1 was obtained after purification.The antagonistic experiment showed that there was a certain genetic difference between the fusant and the parents,and the ISSR molecular marker technique proved that the fusant contained genetic material from two parents.