Transgenic Studies On Resisting To Premature Senescence In Cotton

The plant bivalent expression vector pBG121 containing PCSAG12-ipt genewas transformed into different cotton cultivars CARI 10, CARI24 andCARI36 by using pollen-tube pathway method. Through orthogonalexperiment, we discussed the effects of genotype, introduction (afterpollination) time, DNA concentration and EMS concentration on the rate oftransformation in cotton. The optimal condition, which the rate oftransformation was promoted, was obtained. Then using the optimal condition,we introduced PCSAG12-ipt gene into CARI10. Transgenic plants were obtained.Trough ELISA of CTK, we observed the exogenous gene expressed normallyin senescencing leaves. High content CTK elevated the activity and content ofsome substance that resistant to senescence. Finally the genetic segregationlaw of T2 was analysed .The main results showed as follows:1. When introducing exogenous DNA into cotton via pollen tube pathway, there were many factors affecting transformation rate. The importance of these factors by the transformation rate was: EMS concentration > acceptor genotype > DNA concentration > introducing time. Correlated with transformation rate, the acceptor genotype and the EMS concentra- tion had significant differences between different treatments; the DNA concentration and the introducing time didn't have significant differences between different treatments. So an optimal condition, which the rate of transformation was promoted, was obtained: the genotype was CARI24, the DNA concentration was 0.01μg/μl, the EMS concentration was 0.01 μg/μl,the introducing time was 36 hours afer pollination.2. In order to verify the function of the senescence-inhibition chimeric gene - 3 -棉花抗早衰的转基因研究 and the optimal transformation condition that was screened, we selected CARI10 as the acceptor genotype, introduced 0.01μg/μl DNA and 0.01 μg/μl EMS at 36 hours after pollination. Another treatment (CK) was the same as above except EMS. CK had buffer of the same volume as EMS in optimal condition. T1 seedlings were screened by kanamycin selection, PCR analysis and GUS histochemical staining. The applied result showed that: compared with CK, the transformation rate of optimal condition enhances two times.3. In the late period of CARI10 development, chlorophyll content, SOD activity, POD activity, CAT activity, MDA content, soluble protein content, cytokinin content were determined. The result showed that the transgenic cotton had higher activity of SOD, POD, CAT and more content of chlorophyll and soluble protein at the same stage. The MDA content of transgenic cotton was a little lower than untransgenic plants. Through CTK analysis and morphological observation, transgenic plants showed signific- ant senescence delaying characteristics.4. The genetic segregation law of transgene: Transformed T2 seedlings were screened by kanamycin selection. mostly transgenic cottons could inherit steadily and their genetic segregation pattern showed 3:1 Mendel segregation. Some lines didn't segregate normally. According to this paper, the use of PCSAG12-ipt gene to improve cotton varieties that had the defect of premature senescence was feasible. In this way, the potential yield of that cotton could be developed greatly and the new lines for cotton breeding could be produced.