金耳原生质体制备与再生研究

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3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3 d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0.6 mol/L蔗糖为渗稳剂,36℃酶解4 h,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5 d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3 h,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。

Research on Protoplast Preparation and Regeneration Conditions of Tremella aurantialba

Objective] This study aimed to explore the protoplast preparation and regeneration 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 protoplasts: 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 solution 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 × 107 protoplasts/ ml.The optimal regeneration condition was using mycelium cultured in liquid medium for 5 d,for enzymolysis in enzyme solution containing 1.5% of lywallzyme at 33 ℃ for 3 h,with 0.6 mol / L sucrose as osmotic stabilizer,and the regeneration rate had reached 0.22%.Conclusion] This study provided valuable support for protoplast fusion,ultraviolet mutation breeding and genetic engineering research.