金耳原生质体制备与再生研究

摘要

[Objective] This study aimed to explore the protoplast preparation and re- generation conditions of Tremella aurantialba. [Method] Optimal combination of five factors affecting the protoplast preparation and regeneration of Tremella aurantialba was selected by using orthogonal experiment, including enzyme system, culture age, enzymolysis temperature, enzymolysis duration and osmotic stabilizer. [Result] The results showed that there were three release modes of Tremella aurantialba proto- plasts： release from top in the early period of enzymolysis, release from the side and release in situ. The optimal protoplast preparation condition was selecting mycelium cultured in liquid medium for 3 d, for enzymolysis in mixed enzyme solu- tion containing 1% of cellulase ＋1% of snailase ＋1% of lywallzyme at 36 ℃; for 4 h, with 0.6 mol/L sucrose as osmotic stabilizer, and the obtained preparation rate had reached 1.76 ×10^7 protoplasts/ml. The optimal regeneration condition was using mycelium cultured in liquid medium for 5 d, for enzymolysis in enzyme solution con- taining 1.5% of lywallzyme at 33 ℃ for 3 h, with 0.6 mol/L sucrose as osmotic sta- bilizer, and the regeneration rate had reached 0.22%. [Conclusion] This study provid- ed valuable support for protoplast fusion, ultraviolet mutation breeding and genetic engineering research.%【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0．6m01／L蔗糖为渗稳剂，36℃酶解4h，得到原生质体制备率达1．76×10^7个／ml；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。