The Study On Pest And Herbicide Resistance Transgenic Tobacco And Cotton

With the development of modern agriculture, the new agriculture which the green, the environmental protection, time-saving receives general farmer's welcome more and more, therefore the compound character's transgenic crops also arise at the historic moment. The important factor that serious influence our country agricultural development is insect pest, often causes massive reduce output of crop; at the same time in recent years, the weed competed for water and nutrients, multiplying plant disease and the insect pest, affected the crops quality and the output. So the study on transgenic crops that insect resistance, herbicide-resistant is important for our country agricultural development.In this work, insect-resistant gene that fusion CPTI and GFM Cry1A expression box driven by CaMV35S promoter, and herbicide-resistant gene EPSPS expression box driven by RUBP promoter, which constructed into plant expression vector pGBI121 together, obtain insect-resistant and herbicide-resistant plant expression vector pGBIF4ABCRUBPEPSPS. Then Plant expression vector pGBIF4ABCRUBPEPSPS were transferred into tobacco by Agrobacterium mediated transformation.45 regeneration tobacco plants were obtained in the process of transplanting survived 29. Genome from 10 regeneration tobacco plants leaves which randomly selected from29, after PCR analysis, 8 transgenic tobacco insect-resistant and herbicide-resistant positive, the efficiency of transformation is 80 percent.The results of southern blot show most of Agrobacterium mediated transformation tobacco are single-copy, only 3rd tobacco is double. Further research found that transgenic tobacco leaves can grow callus in the medium contains 1 m mol glyphosate, can grow regeneration shoots in the MSB medium contains 0.03 m mol glyphosate, and can regenerated in the MSB medium contains 0.05 m mol glyphosate. We transferred the vector pGBIF4ABCRUBPEPSPS into upland cotton Y18 by pollen tube pathway transformation method. We injected 5700 flowers of cotton, after kna screening, we obtain 10 kana positive cotton plants, then PCR and direct sequencing and blast analysis we found 7 transgenic cotton plants insect-resistant and herbicide-resistant positive. Meanwhile, in order to expand genotypes of cotton for tissue culture, and to provide more excellent germplasm materials for cotton tissue culture, we exploration agrobacterium mediated system for the regeneration of upland cotton variety Y18 , the results as follows:1) The best method for asepsis is peeled cotton seeds and then sterilized by 0.1%Hgcl2 for 5min.It is not only effectively prevent contamination, but also will not affect the germination rate and growth of asepsis seedling.2) The time when the explants can be used in genetic transformation, peeled cotton seeds had been grown on MS1 culture medium for 7 days. Then they were easily infected by agrobacterium and induced callus, and will not cause material waste.3) Ascertain of the screening pressure of antibiotics .Through concentration grading experiment, 110mg/L kana was regarded as the screening pressure of resistance-callus.4) The optimal concentration and infection time when Agrobacterium infect explants.When the Agrobacterium that had entered log-growth period(OD0.3-0.6) was used to infect hypocotyl for 5min have the best effect .5) Optimum of culture medium. Callus-inducing medium: MSB+IBA0.8 mg/L,KT 0.4 mg/L,pH=6.0;Embryo-callus inducing medium: MSB*+ IBA0.4 mg/L,KT 0.2 mg/L,pH=6.5;Embryogenic bud and regenerated seeding inducing medium: MSB*+ IBA0.4 mg/L,KT 0.2 mg/L,Asp,Gln each 1g/L,active carbon1.5g/L,pH=6.2 .Preliminary Exploration of the Application of antibacterial peptide in cotton,tobacco tissue culture :The treatment of peeled cotton seeds by 0.1% mercuric chloride for 5 min, washed with sterile water3-5 times, and then immersed in 0.12mg/ml antibacterial peptide liquid, was effective in preventing most common fungal contamination of the cotton tissue culture, and did not effect the development of cotton roots. The using of antibacterial peptide in callus induction stage showed no impact on cotton callus; the using of antibacterial peptide in the tobacco tissue culture showed no impact on callus,shoots and shoots regeneration. Individual treatment by antibacterial peptide for the bacteria, the bactericidal effect was less than antibiotics, but the joint treatment of antibacterial peptide and antibiotics prevented to bacterial contamination effectively. In this preliminary study antibacterial peptide applied in cotton, tobacco tissue culture, and presented good performances in fungal and bacterium contamination control.