小麦光腥黑粉菌原生质体的制备与再生

Protoplast preparation and regeneration of Tilletia foetida

以优化原生质体的制备方法,建立小麦光腥黑粉菌的遗传转化体系为目的,研究了小麦光腥黑粉菌菌株培养时间、细胞壁裂解酶、酶解时间和渗透压稳定剂等对原生质体制备的影响。结果表明,以水琼脂培养基上培养7d的菌丝为初始材料,配置1.5%崩溃酶+1.5%溶壁酶+1.5%蜗牛酶的复合酶液,28℃酶解2 h后原生质体的获得率最高;以1.2 mol/L的氯化钾为渗透压稳定剂,得到的原生质体数量最多,且释放的原生质体能够均匀散乱分布,不聚集成堆;较适宜原生质体再生的培养基为TB3培养基,可产生较多的单菌落。

To establish protoplast-mediated genetic transformation system of Tilletia foetida, conditions for protoplast isolation and regeneration of T. foetida were optimized. The results showed that mycelia incubated for 7 days were the most suitable for protoplast release. Mixture solution of 1.5% driselase, 1.5% lyase and 1.5% snailase were the most favorable for protoplast preparation. The most suitable incubation time with enzyme for maximum release of protoplasts was 2 h at 28 ℃. The most effective osmotic stabilizer for the protoplast release was 1.2 mol/L KCl, and protoplast also scattered distribution. For regeneration of the protoplast prepared freshly, TB3 medium was better than PDA medium, which could produce more single colonies.