草菇原生质体制备、再生及转化研究

Studies on Preparation,Regeneration and Transformation of the Protoplasts of Volvariella volvacea

用酶解法对草菇原生质体制备及再生条件进行了研究.结果表明:液体静置培养4 d的菌丝,以0.6 mol/L的甘露醇作渗透压稳定剂,溶壁酶质量浓度15 mg/mL,34℃下酶解3 h,原生质体制备率最高;草菇原生质体预培养16 h后涂布在再生培养基上,再生率最高.同时,采用PEG法将潮霉素抗性基因转入草菇的原生质体中,经过抗性筛选,得到了具有潮霉素抗性的草菇转化子.

The conditions for Volvariella volvacea protoplast preparation and regeneration were studied by lywallzyme digestion. The results showed that the highest yield of V. volvacea protoplast was obtained with the mycelia of static cultivation for 4 d by using 15 mg/mL lywallzyme digestion for 3 h at 34 ℃ and 0.6 mol/L mannitol as osmotic pressure stabilizer. The highest regeneration rate was gained on PDA plates after the pre-incubation in liquid regeneration media for 16 h. The transformants of V. volvacea resistant to hygromycin were obtained after screening on the selection medium by using PEG-mediated protoplast trans- formation with the hpt gene.