绿菇子实体菌株的原生质体分离及再生菌株的获得

收集30℃下，暗培养36～48hr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

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

Protoplasts of Russula virescens(Schaef)Fr.were i solated fromthe spores produced by the mycelia,which collected from the culture s 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 s nailase(1－20mg／ml),Mes(0.1M)and various osmotica(0.6 osmole).The yield of th e protoplast was in the range of 0.1×106 to 1.75×107.The purified pro toplasts being incubated at 30℃ formed germinating tubes and regenerated myceli a in 2-7 hours using liquid thin layer culture.The regenerated colonies were vi sible after 48 hours.The regeneration rates were in the range of 3% to 67.7% an d dependent on the conditions for isolation of the protoplasts.The regenerated c olonies appeared with one day delay if the protoplasts cultured at 15℃.Formatio n and regeneration of the protoplasts were influenced by the time for culture of the mycelia,concentration of the digesting enzyme,time and temperature for diges t ion,identity of the osmotic stabilizers and shaking in digestion.If the protopla st 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 wit h other osmotic stabilizers,even digested for 8-24 hours.