双亲灭活原生质体融合技术在草菇耐低温菌株选育上的应用

Application of Inactivated Parental Strain Protoplasts Fusion Technology in Selection of Volvariella volvacea with Higher Antifreeze Capacity

为了选育耐低温草菇菌株,以草菇V23、V3552为亲本,采用酶解法制备原生质体,用紫外诱变、化学诱变两种方法对草菇菌株进行诱变,筛选到耐低温的突变株;然后利用紫外灭活(20 W,30 cm,110 s)和热灭活(50℃3 min)的双亲灭活标记法对突变株进行化学融合,结果表明在400 g/L的PEG6000、pH 8.0、融合时间30 min和融合温度32℃的条件下融合率最高,达到0.517%,共获得200个融合子。经过0℃低温筛选,最终获得15株草菇耐低温菌株,菌丝在0℃的耐冻能力提高了4.5倍。经出菇实验证明其子实体与出发菌株相比具有明显的耐低温性,液化现象明显推迟,说明该方法筛选出的菌株具有进一步应用开发的价值。

The protoplasts of VolvarieUa volvacea V23 and V3552 were prepared by enzymatic method, and then were taken mutagenesis by UV and chemical 2 methods. A fusant displaying higher antifreeze capacity, as compared to that of its parents, was obtained by PEG-induced fusion between UV-inactivated protoplasts of V3552 EMS and heat-inactivated protoplasts of V23 UV through low temperature （ 0℃ ） selection. The best fusion condition is gained at 400g/LPEG6000, pH 8.0, for 30min fusion time, at 32℃ fusion temperature. The fusion rate could be as high as 0. 517%. And 200 fusants were obtained. Through screening with 0℃ low temperature, 15 VolvarieUa volvacea cold tolerant bacterial strains were obtained. The cold tolerant ability of hypha at 0℃ was improved by 4. 5 times. The results proved that the fruit displayed higher antifreeze capacity, as compared with bacterial strains; and the liquefaction phenomenon was obviously postponed. This indicated that strains screened by this method were of further development value.