| The major objective of this study was to breed tobacco varieties were selected as experimental materials. Their cotyledons were used as explants so that an experiment could be conducted on the induction and differentiation, the growth, and the rooting of adventitious buds. As different hormones with various mass-concentrations were added into the media used for each stage of tissue culture, the growth of explants in each medium was investigated, and the optimal hormone combination was sifted out through the investigation. Thus a system for in high efficient regeneration of tobacco was established. Based on the above system, the Kanamycin with different mass-concentrations was respectively added into the media, which were used for inducing the callus of cotyledons and for differentiating adventitious buds during each stage. After that, the effects of Kanamycin on the growth of explants were observed so as to determine its minimum lethal mass-concentration, which was to be used for studying genetic transformation of tobacco. Subsequently, parameters that affecting the efficiency of Agrobaterium tumefaciens-mediate transformation of tobacco were comprehensively studied. By comparing research, all the factors were found out that influence Agrobaterium tumefaciens transformation efficiency, which are chosen right the number of pre-culturing days, bacterial concentration, suitable infection time, the number of co-culturing days and concentration of antibiotics. And a transformation protocol was established. On the basis of the established transformation system, rolB genes were transferred to tobacco by PCR.The results of this study were introduced separately as following:1. Establishment of plant regeneration system of tobaccoThe optimum sterilized measure to be immerged in 75% ethanol for 1 minutes and then in 0.1% HgCl2 for 7 minutes in succession. The optimal adventitious bud induction medium was MS+6BA0.5mg/L+IBA0.1mg/L. And the most optimal rooting medium was 1/2MS+NAA1.0mg/L. And the appropriate substance for plantlets transplantation is soil and vermiculite and perlite in 2:1:1.2. Establishment of genetic transformation system of tobaccoThe results showed that precultured explants for 3 days, Agrobacteritunm in the phrase of loizarithlm Growth was deluted to 1/30 of original concentration, immerged for 5min. The co-cultured of the explants in baterium is for 3 days. Resistant calli or buds are obtained about 15 days on MS media supplemented with 500mg/L Cb and 80mg/L Kan. The resistant buds go on culturing on 1/2 MS midium supplemented. Through this process better transformation rates can be got.The factors influencing Agrobacterium-mediated tabacco transformation were studied in systematism, in order to establish a universal high efficient and stability tabacco gene transferring system.3. Molecular detection of transgenic plantletsPCR assay was the common way to validate the transgenic plants. The experiment was conducted by total DNA of transgenic plants as template, by using non-transgenic plantlets as negative control group, plasmid DNA as positive control group, then carried through PCR amplification.And PCR results showed:Extended strip in transgenic plantlet with target strip in plasmid while no strip extended in non-transgenic plantlets,66.7 percent of kanamycin-resistant plants were masculine plants. This demonstrated that rolB gene had been transformed and integrated into genome of transgenic plants. |
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