The Study On Transformation Of LjCYC2 Gene Into Helianthus Annuus

In this study, LjCYC2 gene of which function was clarified in Lotus japonicus was used as target gene. A high efficient Agrobacterium-mediated transformation system for ornamental sunflower was established. And the transgenic plants were used for studying the function of LjCYC2 gene in ornamental sunflower. The results were as follows:1. The seeds of sunflower "Fuyang"(golden yellow) were used as material. An efficient system of multiplication, in vitro flowering and rooting of ornamental sunflower was established through adding several kinds of plant growth regulators into MS medium. The system can provide the basis for the establishment of genetic transformation system and identification of flower type, color, and traits of ornamental sunflower. The results showed that low concentration of BA, especially MS containing 0.05mg/L BA, had a better effect on multiplication; Rooting was influenced by the levels of nutrition, and the optimum rooting medium was 1/2MS; GA3 and PP333 can inhibit the formation of flower bud, however, low concentration of BA can promote the blooming of sunflower in vitro and the optimum medium for the flowering was MS supplemented with 0.05mg/L BA.2. The shoot tip of sunflower "Fuyang"(golden yellow) were used as explants and a high efficiency Agrobacterium-mediated transformation system was established. The study showed that the optimal condition for Agrobacterium culture was 28℃,150 rpm and 15-18h in dark; The suitable hygromycin concentration for selection is 10mg·L-1; The suitable cefotaxime concentration for inhibiting Agrobacterium growth is 500 mg·L-1; Proper use of sonication could enhance the efficiency of transformation, and it could reach 7 percentage when 25kHz sonication was used for 2 min before Agrobacterium infection for 5min. However, vacuum infiltration could interfere the process of selection.3. Using LjCYC2 F 5'-GGACTAGTATGTTCCCTTTCAGCTCAAG-3'and LjCYC2 R 5'-GGACTAGTTGGACGTGGATTTGTGCAC-3' as primers, the PCR results of 19 plants that resist-hygromycin showed the bands, suggesting that the gene had been inserted into the genome of ornamental sunflower. However, the RT-PCR results of number 16-19 plants didn't show the bands, suggesting that the silence of the genes maybe occur. In the plants of number 10 and 14, the disk florets were degenerative while the ray florets became wider and there was a gap in the top of the floret. The character of the shape of transgenic ornamental sunflower was identical with the function of LjCYC2 gene in Lotus japonicus.