Creation And Phenotyping Of Mutants In Tobacco YAN Guanghao

Mutants play an important role in cloning of functional genes in crops and crop breeding. In this research, we selected tobacco as the experimental materials, which including two varieties of Honghuadajingyuan and Xanthi, and conducted creation and phenotypic identification of tobacco mutants through two approaches, agrobacterium-mediated T-DNA insertion and irradiation mutagenesis.1. Optimization of transformation system:The M8 gene that interferes the expression of RNA was carried into the explants of tobacco by binary vector pCMI8 which contains hromycin resistance gene via agrobacterium-mediated transformation. After stringent selection of the infected explants on the medium with hygromycin and following assays, the transgenic tobacco plants were generated from the hygromycin resistant explants. The transgenic plants were confirmed by the examination of PCR, and it is very useful for us to explore that whether the M8 gene that interferes the expression of RNA has been transformed and expressed or not in tobacco. The result showed that the gene has been integrated into the genome of tobacco. Analyzing the transformation effects of recipient material, The high effective regeneration systems were obtained by the comparison of different regeneration system. The system used for leaf discs culture:MS base medium complemented with1mg/L 6-BA+0.1mg/L NAA (for callus induction) and with 0.2mg/L NAA (for shoots generation). Effects of the explant growth state, Hpt sensibility, the period of preculture, bacterial infection and co-cultivation on the transgenic efficiency were investigated. As a result, the best explant was leaves which growing on MS medium. During the period of shaking of bacterial, the best concentration of kanamycin was 50mg/L for selecting positive bacterial and rifamycin was 50mg/L for killing other bacterials. For the stage of callus induction and the formation of shoot and root, the best concentration of hygromycin was 25mg/L for selecting positive bacterial and carbenicillin was 250mg/L for killing bacterials.The optimized period for preculture was 2 days in the dark at 25℃, for bacterial infection was 8-10 minutes and OD600 0.4-0.6, and for co- cultivation was 3 days to acquire best growth effectiveness for resistant callus induction. 2. Mutants of T-DNA insertion:We obtained at least 9 transgenic plants, phenotypes of which are significant difference compared wild tobacco. For example, the content of mesophyll cell is high, and the leaf is thick and deep green. The positive plants grow out two or three terminal buds before florescence and many axillary buds even though no topping.3. Mutants of irradiation mutagenesis:The seeds of tobacco Honghuadajingyuan were irradiated by 60Coγray of 400 Gy. The results indicated that a large number of mutants were collected from M0 inducing germination percentage of Mo tobacco seed, plant leaf trait mutants (leaf color, shape and margin variation), stem trait mutants (plant height, axillary bud number variation), growth period mutants and flower mutants. The mutant populations are very useful to genetic analysis of gene, gene mapping and cloning, and further the research on functional genomics. These mutants will be useful to serve the tobacco improvement.