Application Of Inactivated Protoplast Fusion To The Breeding Of Cordyceps Militaris

Cordyceps militaris (L. Fr) Link, is a type of famous traditional Chinese medicine with high economic value, which belongs to Ascomycota, Pyrenomycete, Sphaeriales, Clavicipitaceae and Cordyceps group.Nine strains of C. militaris were collected from all over the country and numbered 1-9. Agriculture properties of nine strains were observed by bottle cultivation indoors. And cordycepin, adenosine, ureter and sugar contents of mycelium were studied by DNS and HPLC. The results showed that No.4 strain which forming an amount of primordium earliest was suitable for the weather in WuHan, No.8 strain was the best in all tested strains for its identical strong fruit body and high contents of cordycepin and sugar. No.4 strain and No.8 strain were selected to breed filial generation with fine agricultural characters and high contents of bioactive metabolite.By the research on protoplast regeneration and shortest inactivation time, it shows the highest regeneration percentage was obtained when mycelia had been hydrolysised for 5 hours by lytic enzymes, the shortest UV-kill time and heat-kill time was 8 min, 14 min respectively. After mixed equally by fusion agent PEG, the fusion products regenerated because of inactivated sites complementarity while inactivated parents didn't.A "semisolid-solid" double layer method was used to cultivate the protoplasts in 25℃, 228 regeneration strains were selected randomly after 4 days.53 regeneration strains were screened by analysis on esterase isozyme after cultured in liquid medium for 20 days. Determination of cordycepin shows, 36 strains in 53 preliminary screened strains content of cordycepin resemble or much higher than that of No.8. The PCR product of 53 regeneration strains by ISSR primer showed, 26 strains had both specific bands of parents that prove those 26 strains were fusion products on molecular biology level.Seventeen strains in 36 high yield strains formed young fruit body in bottle cultivation, but the primordium amounts were much less than the parents. Concluded from the results of cordycepin content and bottle cultivation, 16 strains were selected to test genetic stability. Sixteen regeneration strains were subcultured 4 times, determinated cordycepin content in each generation to test genetic stability. The result showed, cordycepin contents in 14 strains were stable. In those stable strains, 11 strains possessed molecular evidence including 5 strains that cordycepin yield increased conspicuously. The content of cordycepin in these high yield fusion products No.59, No.61, No.69, No. 136, No. 186 increase 1.32, 1.17,1.49, 1.73, 1.33 times than that of No.8, respectively.This study showed, basic stable fusion products could be obtained by inactivated protoplast fusion to the C. militaris breeding. Cordycepin content increased in most of fusion products although no fusion product detected show better agricultural characters than the parents. The high yield strain could be applied in fermentation for cordycepin product after further genetic stability test.