The Expression Of DFL Gene In Tobacco And The Transformation Of Chrysanthemum

LEAFY(LFY) gene is a floral meristem identity gene. It has important function in plants' inflorescence progress. Research found that it controlling the switch from vegetative to reproductive development. Its overexpression can induce the plant flower early. DFL gene is LFY homologous gene in the Dendranthema lavandulifolium. The structure and expression pattern of DFL gene is similar to other plants' LFY gene. This research make molecular identification to Nicotiana tabacum transformed DFL gene. The identification of transgenic tobacco with DFL gene provides reference for further research on genetic transformation of ornamentals' florescence. The success of the transformation establishes technical foundation for the transformation of chrysanthemum.Chrysanthemum is our traditional flower. People like it very much. Chrysanthemum is used for the construction of garden. In order to make Chrysanthemum blossom in every time and everywhere, now the research of florescence control gradually becomes an important aspect in the breeding research. DFL gene is separated from D. lavandulifolium. It can reduce the diversity between family and genus for the Chrysanthemum's transformation. It has practical significance for the breeding Chrysanthemum that has our intellectual property rights.1. This research make molecular identification to eight Nicotiana tabacum plants transformed DFL gene. Six tobacco plants were identified by PCR and three transgenic plants were identified using Southern-blotting. The florescence and morphological characters of the transgenic plants were observed. Through the contrast analysis with untransformed tobacco plant, we found that the florescence was changed. Three of them flowered 15 to 23 days earlier. Several morphological characters of the transgenic tobacco plants were changed. For example, some leaves crimpled or the color became yellow. The seeds of transformed plants are selected under antibiotic. The seeds could grow under 50mg/L Hyg.2. Regeneration experiment used leaves as explants. Through the regenerationexperiment of 'helanbai', SA and SW with leaves we found that 'helanbai' and SA were regenerated with low frequency while the frequency of SW was 75%. So SW was chosen as suitable material for the transformation experiment.3. Established steady and efficient regeneration system. SW was used for regenerated experiment. The regenerated experiment with young leaves and shoots as explants showed that the frequency of shoots is lower than leaves. The prime medium for regeneration of leaves was: MS +6-BA 1.0 mg/L +NAA0.2mg/L( pH6.3 ). The frequency was 75%. The optimum medium for rooting was 1/2MS+ NAA0.2mg/L. The survival rate was more than 90% through the rooting and transplant experiment.4. Susceptibility to antibiotics of SW had been studied. SW was susceptible to Hyg. The critical concentration for leaves regeneration and reducing root of SW was 5mg/L Hyg. 200-500mg/L carbenicillin was used in transformation experiment of SW.5. Transformation system of SW was established. Agrobacteriumt umefaciens was used for the transformation experiment The leaves of SW were pre-cultured for 0—4d on media. The suitable concentration of Agrobacteriwn tumefaciens was OD₆₀₀0.4-0.6. The leaves were immersed in the Agrobacteriumt umefaciens for 10min then were co-cultivated for 2d on media (MS) at 20-25℃ in darkness and delayed cultivated (MS + 6-BA 1.0 mg/L +NAA0.2mg/L + Carb500mg/L) for 2d at 25℃. Then the leaves were transferred to selection media (MS + 6-BA 1.0 mg/L +NAA0.2mg/L + Carb500 mg/L +Hyg5 mg/L) at 25 ℃. Secondary culture every 20d. Put the transgenic regeneration shoot into rooting media with 3—5 mg/L Hyg.