茶树菇原生质体再生及单核体菌株的特性

Protoplast regeneration and monokaryotation characteristics of Cyclocybe aegerita

【背景】茶树菇遗传育种工作是茶树菇产业持续发展的保障和关键,原生质体的制备及单核体菌株的获得可为茶树菇遗传育种工作的开展提供技术支持。【目的】获得茶树菇原生质体的再生特性、单核化特性及其交配型,为开展茶树菇的杂交育种、融合育种、诱变育种、遗传转化和功能基因挖掘等奠定基础。【方法】以茶树菇保藏菌种Aa11的菌丝为材料,采用甘露醇溶液和溶壁酶溶液直接处理平板菌丝制备茶树菇原生质体,而后对原生质体进行分离和再生培养。通过原生质体单核菌丝体两两单单对峙培养,观察对峙培养过程中的菌落形态变化。【结果】当接种块数量为7、酶解温度为33-34℃、酶解时间为60-80 min时,原生质体数量为107个/mL。茶树菇原生质体在涂布平板7 d后肉眼才可见明显的再生菌落形成,在再生培养基上再生率为0.71%,单核化率为41.1%;再生异核体和再生单核体在形成再生菌落时有时间差,从第7天开始往后连续3 d的再生菌落均为异核体菌株,往后第4天开始陆续出现单核体菌落,之后时间内的菌落均为单核体菌株。试验共得到290个原生质体单核体,分为A1B1和A2B2两种亲本交配型,A1B1和A2B2二者的比例为138:152;两种交配型的单核体在菌落形态上都表现为气生菌丝旺盛、颜色浓白,A1B1单核体的生长速度要快于A2B2单核体。双亲菌落接触部位存在一条明显比周围菌丝体稀疏的分界线,双亲菌落接触区域的分界线均向菌落扩大速度慢的亲本菌落弯曲。【结论】两种交配型单核体菌株及其对峙培养后的菌落形态变化特征可为茶树菇杂交育种的亲本选择提供依据。

[Background] Genetic breeding is the key guaranteeing the sustainable development of the Cyclocybe aegerita industry. The preparation of protoplasts and monokaryons can provide technical support for the genetic breeding of C. aegerita. [Objective] To obtain the regeneration characteristics,monokaryotation characteristics, and mating types of the protoplasts for the cross breeding, fusion breeding, mutagenesis breeding, genetic transformation, and functional gene mining of C. aegerita.[Methods] The mycelia of C. aegerita Aa11 were directly treated with mannitol solution and lysozyme solution to prepare the protoplasts which were then isolated and regenerated. The changes of colony morphology were observed during the hybridization of protoplasts derived from mononuclear mycelia.[Results] When 7 blocks were inoculated for enzymatic hydrolysis at 33–34 ℃ for 60–80 min, the suspension reached a concentration of 107 protoplasts/mL. The protoplasts of C. aegerita could not form regenerative colonies until 7 days after the coating and showed a regeneration rate of 0.71% and a monokaryotation rate of 41.1% on the regeneration medium. The regenerated dikaryon and monokaryon strains presented a time difference in the formation of colonies. From day 7, all the regenerated colonies in 3 consecutive days were heterokaryonic strains, and mononuclear colonies began to appear successively since day 4. Almost all of the colonies in the following days were mononuclear strains. A total of 290 monokaryons were obtained in this study and classified into two parental mating types A1B1 and A2B2(A1B1:A2B2 ratio of 138:152). The monokaryons of both mating types showed vigorous white aerial hyphae, and the monokaryon growth of A1B1 was faster than that of A2B2. A dividing line, bending towards the parent colony with a slower expansion rate, appeared in the contact area of the parent colonies, with the mycelia significantly sparser than the surrounding mycelia.[Conclusion] The morphological characteristics of the two monokaryon strains and their hybridization provide a basis for the selection of parents in the cross breeding of C. aegerita.