| Using Corynebacterium crenatum AS1.542 as material, the optimum conditions of the protoplast preparation were obtained by studying the factors of protoplast preparation. The preparation and regeneration technology of Corynebacterium protoplast was established and optimized by seeking after the regeneration conditions of protoplast. Using N~+ ion beam implanting AS1.542 and its protoplast, the method of implantation was explored and improved , and its factors and mutagenic effects were discussed elementarily. It was expected that it would offer further help for the basic theory of the breeding and production practice.The research showed that the strain was inoculated to complete medium and cultured 11 hours under the conditions of 30℃ , 200rpm before the preparation of the protoplast. 4% of inoculum was adopted to culture 2.5 hours, and added penicillin in order to attain 0.8u/ml in the culture and continued culturing 2 hours. 5ml culture substance was extracted and centrifugated, and washed by hypertonic solution to prepare for the protoplast. After exploring the influential factors of lysozyme treatment, the optimum condition of lysozyme treatment was decided. It was treated 15 hours under the conditions of 1g/L lysozyme, 37℃ and 150rpm.The preparation rate of the protoplast was over 95%,and its regeneration was 6.2%.To study the effect and mechanism of low-energy ion beam on Corynebacterium protoplast, the protoplast of C. crenatum and its complete cells were implanted by low-energy nitrogen ion beam of 30KeV. Their survival curves were compared and analyzed. Their curves were down in the first, then up and down at last with the increase of the implanting doses. At the same time, there are many obvious difference in the curve. First of all, The mutation rates were different, the total and positive mutation rates of the former were higher than the later. Since the former was more sensitive than the later when they were implanted by ion beam, more mutants were... |
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