Preparation And Mutagenesis Of Protoplast Of Cordyceps Militaris

Cordyceps sinensis which borned in Qinghai is a famous Chinese traditional medicine. Cordyceps sinensis has many clinical efficacies. Because of its natural source is very extremely limited, the application is limited too. The effects to the preparation of Cordyceps militaris protoplast, including age of the Cordyceps militaris, stabilizing agent of osmotic pressure, constitution of enzymes and time of action, were investigated by the orthogonal design. By the research, we found the optimum condition was that to mix the lysozyme and cellulase of the concentration of 1% as the enzyme solution, use the stabilizing agent of mannitol and act on the 4 days' strains 3 hours. Under the optimization of protoplast preparation, the protoplasts were mutanted by UV and NTG. ,We selected five times—10s, 15s, 20s, 25s, 30s with UV. The results indicated 25s is the best time. Then experiments were done with different concentrations of NTG and time of reaction to act on the protoplast. The mannitol of different mutants was extracted. According to the colorimetry to measure the content of mannitol as the standard, the high yield strains, using 0.2mg/ml NTG acting 15min, were screened out, among which the highest one had the concentration of 33.9% of mannitol. After several generations, the concentration of mannitol is stabile.By the standard of nature Cordyceps sinensis, some active components of strains of UV and NTG mutation such as cordycepin, mannite, polysaccharose and aminoacid were studied. The result indicated:The cordycepin content of nature Cordyceps sinensis was 0.038%. The cordycepin content of UV mutation was 0.053%. The cordycepin content of NTG mutation was 0.068%.The mannite content of nature Cordyceps sinensis was 6.35%. The mannite content of UV mutation was 5.97%. The mannite content of NTG mutation was 6.81%.The polysaccharose content of nature Cordyceps sinensis was 8.92％. The polysaccharose content of UV mutation was 8.41%. The polysaccha-rose content of NTG mutation was 9.08%.The general aminoacid content for nature Cordyceps sinensis was 259.87mg/g. The general aminoacid content for UV mutation was 232.96 mg/g. The general aminoacid content for NTG mutation was 257.67mg/g.From the results, the active components of strains of UV and NTG mutation are higher than nature Cordyceps sinensis. Moreover the active components of strains of NTG mutation is highest among them. So the latter is applied to officinal field instead of the nature Cordyceps sinensis. Therefore, it has great research significance to explore and optimize the culture conditions on Cordyceps militaris of NTG mutation in order to make for its industrialized mass production in future.