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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备与再生系统。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。羧甲基纤维素酶(Cx酶)活性及子实体形成能力上的差异为鉴定同核体的主要标记,从两种形态类型,不同来源的12个双孢蘑菇菌株中均分离到同核原生质体,同核率为2％～15%,平均为11.75％。
Through the research on the composition and concentration of lywallzyme, osmotic pressure stabilizing agent and mycelium culture method, the effective preparation and regeneration system of Agaricus bisporus protoplast was established. Using 1.5% Lywallzyme and 0.6M KCl as osmotic stabilizer, the protoplast production reached 10 ~ 7 / ml after enzymatic hydrolysis at 25 ℃ for 4 hours. The purified protoplasts were incubated in 0.6M sucrose as osmotic pressure stabilizer for PDMA The medium was incubated at 25 ° C for 5 to 7 days at a regeneration rate of 1% or more. According to the order in which the protoplast regenerated colonies appeared, the colony morphology and the mycelium growth rate were used. The difference in the activity of carboxymethyl cellulose (Cx enzyme) and the formation ability of fruiting body was the main marker for identification of the same nucleus. From the 12 species of Agaricus bisporus with two morphological types and different origins, the same nuclear protoplast Body, with the nuclear rate of 2% to 15%, an average of 11.75%.