Studies On The Protoplast Fusion Between Tuber Indicum And Ganoderma Lucidum Karst

Tuber indicum,a precious mycorrhizal mushroom,had delicious in taste, high nutritional value, a variety of active substances, higher health care efficacy and commercial value.Grown under special condition, long growth cycle and low yield, However, artificial cultivation was hard to realize,so the market demand. G.lucidum.karst,a characteristically traditional Chinese medical herb,had many clinical efficacies, large scale artificial cultivation has been realized yet. Studied on the ProtoPlast PreParation, regeneration and fusion,in addition,seleeted the fusant and made the identifieation,the research was to get the fusan aim at promoting strain production, artificial cultivation of Tuber indicum and lay the foundation for breeding research of fusion strains.Diseussed about the mycelia liquid medium screening, protoplast preparation and regeneration system between Tuber indicum and G.lucidum.karst used by orthogonal design method. The results are as follows:The best liquid medium for Tuber indicum is Glucose2.00%, Maltose1.50%, Peptone0.15%,Yeast Extract Powder0.25%, KH2PO4 0.30%、MgSO4·7H2O 0.15%,VB10.0008%,pH nature. The best liquid medium for G.lucidum.karst is: Glucose2.00%, Maltose 1.50%, Sucrose 0.30%,Yeast Powder0.20%, KH2PO40.30%, MgSO4·7H2O 0.15%,VB10.0004%,pH nature.The optional protoplast preparation system of Tuber indicum was that 2d mycelia of liquid static cultivation were digested in to 2% Lywallzyme with 0.6mol/L KCl,at 36℃,for 4h,the preparation rate was 4.9×107/ml; For G.lucidum.karst the optional protoplast preparation system was that 8d mycelia of liquid static cultivation were digested in to 1% Lywallzyme, 1%Snail Enzyme, 1%Cellulase,with 0.6mol/L mannitol,at 30℃,for 3h, the preparation rate was 1.05×108/ml.The protoplast regeneration system for Tuber indicum was that 2d mycelia of liquid static cultivation were digested in to 2% Lywallzyme with 0.6mol/L KCl,at 30℃,for 3h,the regeneration rate was 87.4×10-2; For G.lucidum.karst the protoplast regeneration system was that 8d mycelia of liquid static cultivation were digested in to 2% Lywallzyme with 0.6mol/L sucrose,at 36℃,for 3.5h,the regeneration rate was 67.9×10-2.Utilized two inactivation mark were to make the protoplast fusion between Tuber indicum and G.lucidum.karst.the results showed that heat inactivation on Tuber indicum at 55℃ for 10min,while UV inactivation on G.lucidum.karst below 30W UV-lamp,30cm away vertically irradiated for 100s,both the lethal ratio were 100%.mingled with 30%PEG-6000 and 40mmol/LCaCL2 (pH 8.0) promoted at 30℃ for 25min,the fusion rate was 8.43×10-7.Through observing colony phenotype, spawn shap,growth rapidity antagonistic experiment and combined with RAPD,ERIC molecular marker,the results showed that the two fusants were fusion products of the parents.clearly,fusant Ra had closed relationship with Tuber indicum,fusant Rb had closed relationship with G. lucidum. karst.