Studies On The Method Of Genetic Transformation And Utilization Of Salt-tolerant And Herbicide-resistant Genes In Cotton

Cotton, Gossypium spp., is an economically important crop. Genetic engineering plays a major role in further improving the yield, stress-tolerance and quality of fiber. There are mainly three genetic transformation methods to incorporate valuable alien genes into the cotton genome, namely, vector mediated transformation, DNA direct transformation and germ line transformation. The germ line transformation methods avoid tissue culture steps and direct transformation of genes into in-vivo plants. In the research, we took the advantages of Agrobacterium-mediated transformation and pollen tube pathway to develop a new method of direct transformation mediated via the pollen tube pathway. At the same time, the four different transformation methods, that is, ovarian injection of plasmid DNA, pollination by using pollen co-cultivated with Agrobacterium, Agrobacterium drop/dip and apical meristem explants methods were standardized and compared. Transformation efficiency of the four methods and genetic characteristics of transformed plants were evaluated. In addition, homozygous transgenic bar cotton plants were obtained and used as paternal parent to have hybrid production. Heterosis utilization and hybrid purity controlling in virtue of the anti-herbicide transgenic parent plant were simultaneously studied. Thus a new seed production technique was set up to control the purity of hybrid cotton in field. Furthermore, the transformation of BADH and AtNHX1 into cotton by the method of pollen tube pathway was conducted to enhance the salt tolerance in transgenic cotton. The main experimental results are presented as follows:1. Development of Agrobacterium transfected pollen via pollen tube pathway method for genetic transformation of Gossypium hirsutum L.A new method for the genetic transformation of cotton pollen by means of Agrobacterium co-cultivation and pollen tube pathway mediated transformation was reported. The bar gene, which is involved in herbicide tolerance, was transferred into pollen grains of upland cotton. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by Agrobacterium strain LBA4404 harboring a vector pCB4, which contains the gene of interest of herbicide resistance, bar gene, constructed with the CaMV 35S promoter and NOS (nopaline synthase) terminator sequences. The integration and expression of bar in the genome of transgenic plants were analyzed with basta bioassay tests, PCR and Southern blot hybridization. At the same time, the heredity, agronomic characters and economic traits of transgenic plants were analyzed and studied. The results indicated that the transformation efficiency was up to 5.5%, which was obviously higher than that of pollen tube pathway. The exogenous gene was confirmed to integrate into the cotton with single-copy. Progeny analysis and molecule testing showed a classical Mendelian pattern of inheritance in transgenic cottons. The integration of exogenous DNA did not show adverse effect on agronomic characters and economic traits of transgenic plants. The method of pollen tube pathway via ovarian injection proved to be a valuable transformation method.2. Comparison of transformation methods for genetic transformation in Gossypium hirsutum L.Protocols for consistent production of transgenic herbicide tolerant upland cotton were established and compared the different transformation methods. Agrobacterium strain LBA4404 harboring a vector pCB4 carrying the bar gene was utilized to transform into upland cotton cultivar 'Coker312'. The transformation efficiency and genetic characters of different transformation methods were evaluated and studied. We obtained transgenic plants in all four methods, that is, ovarian injection of plasmid DNA (method-I), pollination by using pollen co-cultivated with Agrobacterium (method-11), Agrobacterium drop/dip (method-Ill) and apical meristem explants (method-IV) method. The herbicide basta leaf sensitivity test, polymerase chain reaction (PCR), and southern hybridization confirmed that the bar gene was integrated with the plant genome. But they were differed with respect to transformation efficiency, time consume, reproducibility and simplicity of methods. The method-II found to be economically and technically viable compared with other three methods. The time consume by this method-II was on par with method-1 and reproducibility was on par with method-IV. In addition, method-11 used minimum facilities than tissue culture base method-IV. The transformation efficiency was higher in method-IV, but the percentage established transgenic plants were less than that of method-II. Furthermore, the growth and development of transgenic plants were influenced by the transformation of method-lV. Method-Ill was the least choice-able method for cotton transformation due to the inferior performance.3. Study on transformation of salt resistant genes, BADH and AtNHX1, in cottonThe transformation of BADH gene from Atrplex hortensis and AtNHX1 gene from Arabidopsis thalians into upland cotton cultivar-ZMS35 by the method of pollen tube pathway was conducted to enhance the salt tolerance in transgenic cotton. The results of PCR and Southern-blot indicated that the target genes BADH and AtNHX1 had been integrated into the cotton genome. Five transgenic BADH plants and nine transgenic AtNHX1 plants were ultimately obtained. The identification on salt-resistance of T₂ transgenic seeds in artificial experiment plot with 0.8% NaCl suggested that the germination of transgenic BADH and AtNHX1 cotton was 63.4% and 82.4% respectively, which was significantly higher than that of non-transgenic control cottons. However, the identification of salt tolerance during the middle and later stages of transgenic cotton growth period was not done for further investigation because of lack of time. Preliminary results showed that transgenic plants exhibited good salt tolerance during seedling stage and possessed high utilization value.4. Heterosis utilization and hybrid purity controlling in virtue of the herbicideresistant transgenic parent plantThe herbicide resistant transgenic bar gene cotton germplasm, BR001, which was developed in the research, was used as paternal parent to cross with different transgenic Bt insect resistant cultivars. 7 hybrid combinations with pest and herbicide resistant characteristics were obtained. Agronomic characters, fiber qualities and yield traits of 7 hybrid combinations were investigated to analyze heterosis of transgenic cotton, while using the extending transgenic Bt hybrid cotton cultivar, ZMS29, as control (CK). The results suggested that all hybrid combinations showed significant higher-parental heterosis and 6 combinations showed positive competitive heterosis in lint yield. Chemical herbicides thus could be applied in the farm and to reduce the cotton plant cost, owing to the hybrid combinations carrying anti-herbicide characteristics. Clearly, the inscet and herbicide resistant transgenic cotton with good fiber quality and agronomic traits possessed fine prospects for productive application. Hybrid F₁ displayed herbicide resistant characters because the herbicide resistant character of paternal parent was controlled by one dominant gene. When F₁ hybrid seedlings were sprayed with herbicide to prevent and control the field weeds, false hybrids could be killed and excluded from population hybrid seeds at the same time to guarantee the purity of hybrid cotton. The study demonstrated the practicability of the purity test techniques by utilizing marker characters (pigment gland). The results indicated that the purity control techniques possessed the advantageous of accuracy, rapidity, economic and high-efficiency. Undoubtedly, it exhibited important utilization value to hybrid cotton production. In addition, the research made an objective evaluation on current market situation of seed purity of hybrid cotton. And the influence on yield and fiber quality by different purity of hybrid cotton was also studied. The results suggested that yield and fiber quality was evidently decreased when the purity of hybrid cotton below 90%. Therefore, purity of hybrid seed improvement could significantly increased yield and fiber quality. The results indicated the significant importance of the purity control techniques established in the study on yield increase and fiber quality improvement of hybrid cotton.