Studies On The Application Of Interspecific Protoplast Fusion Techniques To The Breeding Of Bac.subtilis With High Cellulase Activity

In this paper, He—Ne laser, PEG and inactivated protoplast technique were adopted in the interspecific hybridization of parents- Bac.subtilis(abbreviated as BS) and Tri.viride (abbreviated as TV) in order to cultivate hybridized strains of BS with high cellulase activity. The results showed that:The best zymolysis conditions for BS and TV protoplasts release and regeneration through orthogonal experiment were as follows: 1mg·mL^-1 lysozyme treated BS protoplasts for 1.5 hours at 37℃ and 4 mg·mL^-1 lywallzyme treated TV protoplasts for 2 hours at 30℃. In these conditions, the number of protoplasts release was respectively 8.87×10⁸ units/mL and 8.62×10⁷units/mL and the regeneration rate was respectively 54.1% and 29.3%.The BS and TV was respectively irradiated by laser for 4 and 6 minutes, they could reach the highest positive mutation rate, 57.8% and 45.6%.Additionally, the proteinase activity of mutant BS18# and the cellulase activity of mutant TV7# which were cultivated could reach 736.8U/mL and 759.4U/mL. Furthermore, the hereditary stability of mutations was well.In the interspecific inactivated protoplast fusion of BS and TV induced by PEG which was conducted under such experimental condition, the optimal condition was 40% PEG(6000) which induced protoplast for 6 minutes at 50℃,the fusion rate was the highest which is 7.02×10^-6;While by laser, the optimal irradiating time was 6 minutes, the fusion rate was the highest which is 8.99×10^-6;While by PEG associated with laser, after adding 1mL 40% PEG(6000) pre-heated at 50℃ to mixed protoplasts ,they were irradiated by laser for 4 minutes, the fusion rate is the highest which was 1.23×10^-5. It was concluded that the higher fusion rate could be gotten by laser associated with PEG, so this is the best means to fuse the protoplasts.The BS and TV were selected as a couple of parental strains ,and the mutants BS18# and TV7# induced by laser which had good features were selected as another couple of parental strains, which respectively induced by PEG and laser together, the fusion of conditions is that after adding lmL 40% PEG(6000) pre-heated at 50℃ tomixed protoplasts ,they were irradiated by laser for 2—4 minutes, The fusants with fine characteristics could be selected and cultivated through initial and repeated sieving. Fusants were tested by the esterase isoenzyme patterns, enzyme-producing abilities and hereditary stability. The results showed that the fusants inherited the fine features from their parental strains, which indicated that they were new strains produced by the fusion of the parental strains. By comparison the enzyme-producing abilitiy of the mutants' fusants was higher than the parental strains and got three hybridized strains of BS with high cellulase activity, in which the 13# hybridized strain had highest cellulase activity 119.3U/mL.