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[目的]对金耳原生质体制备与再生体系进行研究。[方法]采用正交试验法筛选金耳原生质体制备和再生体系中酶体系、菌龄、温度、时间、渗稳剂的最佳用法。[结果]金耳原生质体的释放方式有三种类型:酶解初期的顶端释放、侧位释放和后期的原位释放。其最佳制备条件为:液体静置培养3d的菌丝体,在1%纤维素酶+1%蜗牛酶+1%溶壁酶的混合酶液中,以0.6mol/L蔗糖为渗稳剂,36℃酶解4h,得到原生质体制备率达1.76×107个/ml;最佳再生条件为:液体静置培养5d的菌丝体,在1.5%溶壁酶的酶液中,以0.6mol/L蔗糖为渗稳剂,33℃酶解3h,得到原生质体再生率达0.22%。[结论]为金耳的原生质体融合、紫外线诱变育种、基因工程研究等提供了有益支持。
[Objective] The research aimed to study the preparation and regeneration system of protoplast of golden ear. [Method] The orthogonal test was used to screen the optimal usage of enzyme system, bacterial age, temperature, time and stabilizing agent in the process of preparation and regeneration of golden ear protoplast. [Result] There were three types of release ways of protoplast of golden ear: apical release, lateral release and in-situ release in the early stage of enzymolysis. The optimum conditions for the preparation of mycelia were as follows: the liquid was cultured on the mycelium for 3 days, and in the mixed enzyme solution of 1% cellulase + 1% snail enzyme + 1% lysozyme, 0.6mol / L sucrose as the stabilizing agent , 36 ℃ digestion 4h, the protoplast preparation rate of 1.76 × 107 个 / ml; optimum regeneration conditions: liquid stationary culture 5d mycelium, 1.5% lytic enzyme solution to 0.6mol / L sucrose as a stabilizing agent, digested at 33 ℃ for 3h, the protoplast regeneration rate was 0.22%. [Conclusion] The study provided the beneficial support for protoplast fusion of golden ear, UV mutagenesis and genetic engineering.