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采用单一变量法研究了黑木耳神农A8与秦单4#原生质体的制备、再生及融合的最佳条件。培养7 d的黑木耳菌丝体用0.5 mol/L甘露醇做稳渗剂,2.0 mol/L的溶壁酶酶解,30℃下水浴3.5 h时,黑木耳神农A8和秦单4#原生质体释放量分别达到0.94×107、1.44×107个/mL;选择RM5再生培养基,神农A8和秦单4#原生质体再生率最高,分别为1.91%、1.53%;原生质体融合的最佳条件为:使用30%聚乙二醇(PEG4000)和0.05 mol/L CaCl2溶液(pH 10)为助融剂,35℃融合30 min。通过荧光镜检和撷抗反应得到融合子,为黑木耳的遗传育种奠定理论基础。
The optimal conditions for preparation, regeneration and fusion of black fungus Shennong A8 and Qindan 4 # protoplasts were studied by single variable method. After cultured for 7 days, the mycelia of Auricularia Auricula were treated with 0.5 mol / L mannitol as stabilizing agent and lysozyme of 2.0 mol / L for enzymolysis. When the water bath at 30 ℃ was 3.5 h, the black fungus Shennong A8 and Qindan 4 # The body regeneration rate reached 0.94 × 107 and 1.44 × 107 cells / mL respectively. The regeneration rate of Shennong A8 and Qindan 4 # protoplasts were the highest (1.91% and 1.53%, respectively) As follows: 30% polyethylene glycol (PEG4000) and 0.05 mol / L CaCl2 solution (pH 10) as a flux, 35 ℃ fusion 30 min. Fusions were obtained by fluorescence microscopy and pick-up reaction, laying a theoretical foundation for the genetic breeding of black fungus.