双孢蘑菇同核原生质体育种技术研究 Ⅰ.同核原生质体的分离与鉴定

Study on Homokaryotic Protoplast Breeding Technique in Agaricus bisporus I.Isolation and Identification of Homokaryotic Protoplast in Agaricus bisporus

通过对溶壁酶组成及浓度、渗透压稳定剂、菌丝培养方法等研究，建立了有效的双孢蘑菇原生质体制与再生系统，采用１．５％Ｌｙｗａｌｌｚｙｍｅ、０．６Ｍ ＫＣｌ为渗透压稳定剂，２５℃酶解４小时，原生质体的产量可达到１０＾７个／ｍｌ，纯化后的原生质体在０．６Ｍ蔗糖为渗透压稳定剂的ＰＤＭＡ培养基中，２５℃恒温培养５￣７天，再生率达１％以上，根据原生质体再生菌落出现时间的先后顺序，并以菌落形态特征，菌丝生长速度、

Based on testing of different combinations and concentration of lysozymes, osmotic stabilizers and different methods of mycelium incubation, an efficient system of protoplast preparation and regeneration in Agaricus bisporus was established in present paper. It was proved that after the mycelium being digested with 1.5% lywallzyme in 0.6M KCl for 4h at 25℃, the protoplast yield reached 107/ml, and that after the purified protoplasts being incubated in PDMA medium with 0.6M sucrose as osmotic stabilizer for 5-7 days at 25℃, the regeneration rate was over 1%. Based on different regeneration time, using the difference of colonial morphology, activity of Cx enzyme and fruiting ability as main markers for identification of homokaryotic protoplast, the homokaryotic protoplasts were isolated from protoplasts of 12 strains with a rate of 2% to 15%, averaging out at 11.75%.