The Research Of Protoplast Isolation, Regeneration And Fusion Of Gliocladium Virens And Beauveria Vuillenun

Gliocladium virens is the important plant fungus pathogeny inhibitor, Beauveriavuillenun is the green environmental protection biopesticidal epiphyte. Their protopla-st fusion belongs to different genus protoplast fusion. Along with the biologicaltechnology and the genetic engineering is developing, different genus protoplast fusio-n frequentlyobtains success, it is provided the feasibility for Gliocladium virens andB. vuillenun protoplast fusion.This article will take Gliocladium virens F051 and B. vuillenun as original strainsand attempt to use the PEG6000 to help protoplast fusion, associating with fungicidetolerance and heat trement to one original stain in order toselect biocontrol engineer-ing strains on the selective regeneration culture medium. The biocontrol engineerigstrains will have pesticide and sterilization dualeffection. The concrete researchmeth-od and the result as follows:1 Weighting the dry hypha of F051 and B. Vuillenun mensurate their hypha growthcurve. The result shows, F051 cultures 20h to enter the logarithmic phase of growth,up to 28h; but B. vuillenun cultures 70h to enter logarithmic phase of growth, up to80h.2 Discussing 9 kinds of factors which affect the protoplast formation rate and rege-neration rate of F051 and B.vuillenun by means of one way experiment, the resultas follows:(1) F051: the optimum condition of F051 protoplast isolation and the regenerationoptimum condition is: conidiophores cultured 24h using culture medium A, 0.6 mol/L MgSO₄ (regeneration used 0.6 mol/L sucrose)as osmotic stabilizer, the sodium hy-drogen phosphate citric acid as the buffer system, the cellulase: Snailase: Lysozyme=4mg/mL: 2 mg/mL: 2 mg/mL, in 32℃water baths dige-sting 2h.(2) B. vuillenun: the optimum condition of B. vuillenun protoplast isolation and the r-egeneration is: conidiophores cultured 78h using L-broth culture medium, 0.8 mol/LKCl (regeneration used 0.6 mol/L sucrose)as osmotic stabilizer, the citric acid sodiu-m citrate as the buffer system, the cellulase: Snailase: Lysozyme=5mg/mL: 2.5mg/mL: 2.5mg/mL, in 32℃water baths digesting 3h.3 F051 and B.vuillenun's ability to resist to 7 kind of agricultural chemicals is me-nsurated, because two original strains have different ability of resist to Carbendazi-m.Finally we select Carbendazim as the medicine to identify the resistense and sele-ct 120μg/mL as mark concentration. 4 Choosing the heat trement to destroy the protoplast of B. vuillenun, temperatureand time two factors which affect protoplast death rate are mensurated, the result sh-ows: protoplast of B. vuillenun will all dead and not regenerate any colony treat 90min with water baths above 55℃and 60min with water baths above 65℃. Final-ly we choose treating 90min in 55℃waterbaths as heatinactivated condition.5 The influence of protoplast mutation of mark concentration to F051and heatinacti-vated condition to B.vuillenun is mensurated. The mutant strain disturbing the fusantstrain is eliminated.6 The culture medium for fusant strain regeneration is selected and finally improve-d YEPD culture medium is choosed for fusant strain regeneration.7 Protoplasts then fuse between F051 and B.vuillenun by 30% PEG 6000+0.01mol/LCaCl₂ in 32℃water baths. The process of protoplast fusion is watched under themicroscope. But recombinant progenies couldn't obtain on the improved YEPD rege-neration culture medium which has mark concentration of Carbendazim.The reasionmay be:(1) The molecular weight of PEG is too big, and concentration is too high.It is poison to fusant strains; (2)The fusant strains are not instability; (3)The regene-ration rate of fusant strains is low.