Transferring Of Rol Genes To The Cotton (Gossypium Hirsutum L.) Genome And Researching Into The Effects Of The Genes On The Plant Development

Upland Cotton (Gossypium hirsutum L.) is one of the most important economic crops. Because of its poor root regeneration ability and the difficulty in keeping the plants lively after transplantation, we often adopt the nourishment pot transplanting and the soil protecting membrine techniques to resolve the problem and make the growth and developmental period be earlier during the cotton growing season in the central area of China. Even so, the recovery period of the transplanted young plants still at least lasts 15 days, hence not only affecting the earlier ripening of cotton, also affecting the ratio of the three kinds of bolls (bolls set before hot summer days, bolls set during hot summer days, and the bolls set during Autumn). Therefore breeding new cotton varieties with the strong rooting ability is an important way to overcome the barrier that we meet at the early stage of cotton production.Transgene technology could break through species' reproductive isolation and make the fully use of natural gene sauces. Plant genetic engineering provides a modern and effective method for plant breeding. In this study, the artificially reconstructed rooting genes (rol genes) were used to transform some varieties of upland cotton to strengthen the rooting ability, improve the growth situation of roots, and supply excellent germplasm for cotton breeding programme.In this study, the system of cotton shoot apex transformation has been established. Upland cotton genome was transformed with rol genes by Agrobacertium mediated transformation. We had obtained transgenic plants and their progenies with strong rooting ability.The main results in this experiment were as follows:1. The establishment of regeneration system of cotton shoot apex: Cotton shoot apex were cultured on MS basic medium containing different concentration and combinations of 2, 4-D and kinetin to directly generate the adventitious buds. The results showed that the MS basic medium could induce adventitious bud formation and the basic MS medium supplemented with 0. Img/L kinetin has an advantage to the differentiation of adventitious buds.2. The kanamycin resistance assay showed that 50mg/L kanamycin could completely inhibit the shoot formation. Most of the untransformed regenerative shoot gradually became white and finally died. 200mg/L cefotaxime sufficiently inhibits Agrobacterium growth and didn't affect the explants differentiation.3. Agrobacertium-medialed transformation of cotton shoot apex: Adopted explants were drenched in 0.6-0.8 OD concentrations Agrobacertium about 15 min, then co-cultivated 2-3d at 28℃. The explants were transferred onto the shoot inducing mediumonly supplied with cefotaxime of 200 mg/ L, and cultured 3 d to suppress the bacterium growth, then transferred to the selective medium supplemented with 50 mg/ L kanamycin and 200mg/L cefotaxime to isolate kanamycin-resistance plants.4. The regenerants were grafted to the cotton seedling with 1 to 2 flatted true leaves. The survival rate of plantlets reached 30%-40%. In this way, we obtained 21 transgenic plants.5. By PCR analysis and Southern blotting, the results showed that the foreign genes had integrated into the genome of TO transgenic plants successfully; the efficiency of transformation retained 100%. Also by PCR analysis on the TI generation of transgenic plants, 20%-80% of which were transgenic. Therefore it is concluded that most of the TO transgenic plants were chimera.6. Great difference in growth characters existed, while compared TO and TI generations with the control, for examples: the transgenic plants usually have smaller bolls, shortened internodes; boll-bearing branch changed to foliage branch, and stunted growth etc.7. The rooting ability assay of TI transgenic plants showed that the length of taproot, fresh and dry weights significantly gained an advantage over the control.