| One , two or more genes of four cellulose synthase genes (acsA> acsE^ acsC and acsD) from Acetobacter xylinum were respectively transformed into a brown cotton line G-007, a restorer line of cotton cytoplasmic male sterility, by Agrobacterium-mediated transformation and the pollen tube pathway transformation, in order to improve cotton fiber quality. Four transgenic cotton plants with better fiber properties were selected from the transformant progeny through the detection of hyg resistant, GUS activity, PCR and Southern hybridization. Genetic and agronomic analysis of transgenic plants was also conducted. A system of cotton transformation for the foreign genes was primarily established by comparing the transformation methods, the main results was listed as follows:1. The tissue culture system of cotton transformation recipientIn order to regenerate plants of brown cotton by the technique of somatic embryogenesis, explants (cotyledon, radicle and hypocotyl )were placed on the MS medium containing O.lmg/L KT and 0.2mg/L2, 4-D to induce callus, then the callus were subcultured on the MS medium containing 0.5mg/L ZT for 2-5 times to develop somatic embryos which were differentiated on the MSB medium containing 0.5mg/L ZT and 0.2mg/L NAA. Among the explants, the regeneration percentage of the cotyledon was 16.38%, the radicle was 10.2%, the hypocotyl was only 1.81%. the result shown that additional of 5 umol Ni"1^ and Zn++ in the above mediums could increase to 16.5% the regeneration percent of green planets from hypocotyl.In the regeneration of shoot apex, 85% tiny shoot apexes from fivedays-old seedings could survive when cultured on the MSB medium containing 0.3mg/L NAAN KT and 30g/L glucose. If these developing shoot apexes were subcultured on the medium containing 2g/L active carbon, the surviving percent of explants could be increased to 92%, and the root percent also increased from 70% to 87%.Regenerated planets with 2-3cm height obtained from the above two methods could be transferred to 1/2MS Medium consisting of 0.5mg/L NAA to let roots and shoots elongate. The plants could be transplanted to the field until the shoots elongated to 5cm.2. The cellulose synthase gene transformation based on tissue culture.A clone of Agrobacterium GV3101 was inoculated in LB to activate for two times, then they were diluted by liquid MS medium. Cotyledons were infected by the diluted Agrobacterium for 15-20 minutes and co-cultivated for 60 hours at 25℃, then transferred to selection medium consisting of 50mg/L hygromycin and 500-700mg/L carbenicillin to induce and subculture the resistant callus. The resistant calluses were placed on the medium consisting of 30mg/L hygomycin or non-hygromycin to differentiate planets. The GUS activity detection should be followed at every step of the explant culture and planet development. At last, 3 resistant plantlets rooted on the medium consisting of 20mg/L hygromycin were obtained. After the analysis of the resistant callous by PCR and Southern blot, it was found that the transformation frequency of single target gene was 61.2%, two linkage target genes about 50%, two pairs of linkage target genes 21.3%.Cotton shoot apexes from 3-5 days-old seedlings was used as recipient of Agrobacterium-mediated transformation. After 72 hours co-cultivation of shoot apexes with Agrobacterium tumefaciens, they would be cultivated on the medium without hygromycin for 5-7 days and then be transferred to the medium, in which hygromycin concentration increased gradually, to give rise to planets. In the 1026 shoot apexes, we can only got 1 transgenic plant.3. The situ transformation of cellulose synthase genesThe situ transformation in this study include two approaches, the direct DNA uptake of egg via the pollen tube pathway and the infection of pollen grain by Agrobacterium tumefaciens. The egg could be transformed by ovary injection and pollen uptake of foreign DNA in the first approach. Increase of the transformation frequency to 3.2% was obtained by extirpating the w...
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