| The inducing of callus, formation of embryo and plant regeneration, and some factors which affected the tissue culture such as the type of media, the concentration of inorganic salt, the concentration and ratio of plant hormones, the type and concentration of carbon source, the genotype and the explant type were studied systematically in this paper, using three cultivars and germplasm of Upland cotton, G. hirsutum, in order to optimize the culture technique of cotton tissue culture for callus induce, somatic embryo formation and plant regeneration, and to establish the system of cotton tissue culture, which will lie a good basis on the field of researching in somatic cell culture and transgenic research work in cotton. On the other hand, the tissue culture of transgenic cotton and its genetic background as well as three pairs of pigment gland near-isogenic lines of G. hirsutum were carried out to study the effects of extraneous genes, pigment gland and gossypol on cotton tissue culture, and the main results were as follows:1. Optimization in system of the tissue culture in upland cottonExplants from five-day asepsis seedlings were inoculated on the MSB medium containing 0.1mg/L KT to induce embryonic callus directly, or inoculated on the MSB medium with 0. 1mg/L KT and 0.01-0. 1mg/L 2, 4-D to induce callus, then transfer to the medium containing 0.1mg/L KT and 2g/L active coal to induce embryonic callus. The embryonic callus was golden-yellow and grain type, which was related to the induction of adventitious roots, and all the explants that could induce embryonic callus can produce adventitious roots. In addition, the types of the explants were also related to the induction efficiency of embryonic callus. The differentiation ratio of embryonic callus from cotyledon was higher than that of the hypocotyls. The regeneration of cotton from embryoid to plant did not need hormone, but the active coal and KNO3 could improve the frequency of the normal regeneration seedling. After the regeneration, plantlets should be planted on the rhizogenesis medium and growth medium for a few weeks, and then transplanted into the nutritive pots. By the method of keeping wet and transplanting step by step, a high survival rate for tube seedlings could be transplanted.2. The effect of foreign genes on cotton tissue cultureThe comparing tissue culture for Bt-transgenic cotton, R-CAZ, and its genetic background, CAZ, were carried out, and the fresh weight of callus that induced fromhypocotyl and cotyledon were analyzed by T- test. The results shown that the t-value of fresh weight for the hypocotyls callus was 47.667, >t0.01, P<0.01; and that of cotyledon was 86.711, >t0.01, P<0.01, which showed that the Bt gene had a very significant effects on the induction of the callus in cotton tissue culture. The root numbers that induced from stem tip culture were also analyzed by T- test, and the t-value was 128.187, >t0.01, P<0.01, it also showed the Bt gene had a very markedly effects on the ability of the adventitious root inducing in stem tip culture. The extraneous genes made the Bt-transgenic cotton into a easy material for tissue culture. American Bt-transgenic cultivars, 32B and 33B, Chinese Bt-transgenic cultivars, GK22 and HD109, and the double gene (CPTI and Bt) cotton cultivars, CK321, were also studied in this experiment, the inducing ratio for callus were all over 95%, and the callus quality and its growth potential were similar to that of Coker 312, the CK. Comparing with their genetic background, all the materials used in the experiment prove that extraneous Bt genes could change transgenic varieties to be the tissue culture easy cultivars. However, there were not significant effects on cotton tissue culture of Bar gene transgenic materials obtained through the pollen tube pathway.3. The effect of pigment gland and gossypol on cotton tissue cultureThe pigment gland and gossypol could induce the adventitious root in cotton stem tip culture dramatically. The ability of the stem tip culture with the g... |
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