Study On Novel Germplasm Of Flowering Controllable Chrysanthemum

The chrysanthemum(Dendranthema grandiflorum (Ramat) Kitamura), one of the traditional famous flowers and major cut flowers worldwide, have a long history and occupy the important economic status in many countries and regions. But the majority of chrysanthemum always blossom in late autumn, their commercial and ornamental value is strictly limited by seasons. In recent years, several flowering time genes have been used in chrysanthemum genetic engineering to improve the florescence properties. SOC1 is a key gene in the pathway regulating Flowering time in Arabidopsis, but it hasn't been researched in the chrysanthemum genetic engineering. This study used chrysanthemum named 'Huang-jinhu' as material and set up a high efficient adventitious bud regeneration system and a stable genetic transformation system. Also, we transformed the vector earring flowering time gene SOC1 driven by inducible promoter, rd29A, into chrysanthemum and obtained 13 transgenic plants. Then we researched the transgenic plants under stress treatments, expecting to get a novel germplasm of flowering controllable chrysanthemum. The main results are as followings.1. The experiment established 'Huang Jin Hu' chrysanthemum leaves adventitious buds regeneration system firstly, the regeneration rate on MS medium which added to 3 mg/16-BA and 1.7 mg/l NAA could got 96.7%.2. The plant expression vector PV003 was constructed, in which a cassette of flowering gene SOC1 driven by inducible promoter rd29A was inserted. The vector was transformed into Agrobacterium tumefacien LBA4404.3. The optimal antibiotics concentration was screened in this study. Both adventitious buds and roots regeneration mediums added to 10 mg/l kanamycin for selecting resistant seedlings. To restrain the growth of Agrobacterium, we used 75 mg/l cefotaxime as bacteriostasis antibiotic.4. Several key factors contained preincubation time, Agrobacterium concentration and infecting time which affect successful transformation were researched. Transgenic protocol for'Huang Jin Hu'was developed. Leaf explants were not precultured was best for transformation. The suitable concentration of Agrobacterium and the time for transforming was OD600 0.8 and 12 min. Cocultivated 2 days at 25℃in dark after infection. Then transferred the explants to the selection medium which added to kanamycin.5. The resistant seedlings were detected by PCR and the results indicated that 13 out of 71 seedlings which resisted to Knamycine were positive. The transformation rate was 6.34%.6. After the transgenic plants being treated with 250 mM NaCl respectively, we analysed the level of SOC1 gene expression by RT-PCR. It was showed that the SOC1 expression was elevated to different extent in various seedlings. Expression of the SOC1 gene analyzed by RT-PCR under drought stress conditions in Y4 was also up-regulated. All the results suggested that exogenous gene could express in the transgenic plants induced by salt and drought stress.