茶树菇与鸡腿菇原生质体融合及再生

Protoplast Fusion and Regeneration of Agrocybe aegerita and Copyinds comatus

研究茶树菇与鸡腿菇原生质体制备、融合及再生。以菌龄4d的菌丝体为原料,采用1%果胶酶和1%蜗牛酶混合酶液(体积比1:1),酶解4h,酶解温度为30℃,pH6.0的0.6mol/L甘露醇溶液为渗透压稳定剂,制备得到茶树菇与鸡腿菇原生质体。两种原生质体融合的最佳条件为:使用35%聚乙二醇(PEG4000)和0.2mol/L CaCl2溶液(pH10)为助融剂,30℃融合30min。利用灭活原生质体作为标记筛选出融合子,融合的细胞在培养基中实现再生,通过菌落表型的比较和拮抗实验验证再生出的为融合子。

In this study,the preparation,fusion and regeneration of protoplasts from Agrocybe aegerita and Copyinds comatus were explored.Protoplasts were prepared from the four-day-old mycelia through hydrolysis for 4 h with a mixture of 1% pectase and 1% snailase both in 0.6 mol/L mannitol stabilizing the osmotic pressure（1：1,V/V） at 30 ℃.The optimal protoplast infusion conditions were determined to be：fusion at 30 ℃ for 30 min in the presence of 35% PEG（PEG4000） and 0.2 mol/L CaCl2（pH 10） as the fusogenic agents.Inactivated protoplasts were used as the marks to pick fusants out,which were then regenerated on PDA plate medium.After the regeneration of fusants,a comparison of colonial phynotype and antagonistic experiments were made to validate them.