绿菇子实体菌株的原生质体分离及再生菌株的获得  

Isolation of the protoplasts from the strain isolated from fruit body of Russula virescens and regeneration of the protoplasts

作　　者：黄萍[1] 沈孝善[1] 

机构地区：[1]贵州省农业科学院生物技术研究所,贵州贵阳550006

出　　处：《西南农业学报》2001年第1期91-95,共5页Southwest China Journal of Agricultural Sciences

摘　　要：收集 30℃下 ,暗培养 36～ 4 8hr的菌体 ,放入酶液中并把菌丝分散 ,经过 3～ 8hr的酶解 ,菌体产生的分生孢子 95%以上成为原生质体。经过滤、离心纯化 ,原生质体的产量在 0 1×10 6～ 1 75× 10 7个 /g (鲜重 )。以液体浅层培养 ,在 30℃下培养离心纯化的原生质体 2～ 7hr ,原生质体形成萌发管 ,4 8hr形成肉眼可见的菌落。再生率随分离原生质体的条件变化很大 ,在3 0 %～ 6 7 7%。在 15℃下培养也能形成再生菌落。培养菌体的时间、酶浓度、酶解时间、酶解温度、震荡与否影响原生质体的形成和再生。以CaCl2 高渗缓冲液酶解 7～ 2 4hr ,原生质体的再生率为 4 5 7%～ 6 7 6 % ,是使用其他高渗缓冲液酶解 3～ 5hr的 2～Protoplasts of Russula virescens(Schaef)Fr.were isolated from the spores produced by the mycelia,which collected from the cultures initiated from actively growing cultures(incubated at 30℃ less than 72 hours)and cultured in the dark at 30℃ for 36-62 hours,in solutions containing snailase(1-20mg/ml),Mes(0 1M)and various osmotica(0 6 osmole).The yield of the protoplast was in the range of 0 1×10 6 to 1 75×10 7.The purified protoplasts being incubated at 30℃ formed germinating tubes and regenerated mycelia in 2-7 hours using liquid thin layer culture.The regenerated colonies were visible after 48 hours.The regeneration rates were in the range of 3% to 67 7% and dependent on the conditions for isolation of the protoplasts.The regenerated colonies appeared with one day delay if the protoplasts cultured at 15℃.Formation and regeneration of the protoplasts were influenced by the time for culture of the mycelia,concentration of the digesting enzyme,time and temperature for digestion,identity of the osmotic stabilizers and shaking in digestion.If the protoplast were isolated with calcium dichloride as the osmoticum,the regeneration rate was as high as 45 7%-67 7%,2-6 folds of that of the protoplasts isolated with other osmotic stabilizers,even digested for 8-24 hours. -

关 键 词：绿菇 分生孢子 原生质体 再生 分离 渗透压稳定剂 二氯化钙 食用菌 

分 类 号：S646.19[农业科学—蔬菜学]