Study On The Protoplast Fusion Between Cordyceps Sinensis And Cordyceps Militaris

Cordyceps sinensis, a characteristically traditional Chinese medical herb, had many clinical efficacies. Grown under the special condition, however, artificial cultivation was hard to realize. Cordyceps militaris, resembled as Cordyceps sinensis, distributed widely and was planted mannually at moderate or low altitude. Studied on the protoplast preparation, regeneration and fusion, in addition, selected the fusant and made the identification, the research was to get the fusant replaced of Cordyceps sinensis in the future, which owned 2 parents’ merits.Discussed about the protoplast preparation and regeneration system between Cordyceps sinensis and Cordyceps militaris used by orthogonal analysis, the optimal composition was that 3d mycelia of liquid static cultivation were digested into 2.0% lywallzyme and 1.0% snailase, 2.0% lywallzyme and 1.0% cellulose, respectively, at 32℃and 30℃for 2h, with osmotic pressure stabilizer of 0.6mol/L KCl as protoplast preparation and 0.6mol/L mannitol added to the optimal liquid media as protoplast regeneration. The preparation rate was 2.98×10⁸/mL and 2.083×10⁷/mL, while the regeneration rate was 42.09×10^-1% and 35.43×10^-1%, respectively.Utilized 2 inactivation mark was to make the protoplast fusion between Cordyceps sinensis and Cordyceps militaris. The results showed that heat inactivation on Cordyceps sinensis at 60℃for 10min, while UV inactivation on Cordyceps militaris below the 30W UV-lamp, 10cm away vertically irradiated for 13min, both the lethal ratio was 100%. Mingled with 40% PEG6000 and 80mmol/L CaCl₂（pH7） promoted at 35℃for 20min, the fusion rate was 2.74×10^-5.Observed colony phenotype, growth rapidity and antagonistic phenomenon, combined with RAPD molecular mark, the fusant was verified to the product of the 2 parent strains above.