Elementary Studies On Transferring F3'5'H Gene Into Chrysanthemum Mediated By Agrobacterium Tumefaciens

Flower colors are mean the colors of the entire genitalia of phanerogam which meaning petalines' colors in narrow sense ,but generalized mean the colors of flower apparatus including calyces,stamen even the petaline from the bud .Flower pigment mostly being composed of three kinds substances—carotenoids, flavonoids and alkaloids. Flavonoids can produce blue color. F3'5'H gene which coded key enzyme decides the location and degree of achromaticity DFR; it can make biosynthesis of anthocyanin tending to produce blue flower. Flower color is a one of the important qualitative characters and economic parameters for the ornamental plants. Because of the colors for many focal flowers are limited, for example, the roses, carnations, and tulips and so on, which flowers are lack of the color for blue and violet in nature. And the reforms for the ornamental plants colors are provided with the advantages such as the visual arts, and the large economic potential. Gene engineering technology is superior to the Cross breeding and Directive breeding technology with its short cycle , low cost and high benefit. Though traditional breeding technology has been used for a long time.So the F3'5'H gene is transferred into Chrysanthemum to breeding new variety Chrysanthemum.Making sure the F3'5'H gene which one of the most important blue flower genes express in different species can be benefit of breeding precious flowers else.Chrysanthemum is from human cultivation and natural hybridization for many years, the abundance sources of chrysanthemum are high hybridization, multiple and species variation mechanism. Chrysanthemum is the famous flower in China; pass for four flowers man of hornour with plum, orchid, bamboo. It is not only cherished and canonize by Chinese, but also attentioned and favored by all the countries around the world. Conventional breeding methods have obtainted great achievement in chrysanthemum breeding for many years already; however it has localization which couldn't utilize gene resources from other species. Along with establishing and improving chrysanthemum genetic transformation system, expressing exogenous gene in cell, improving of transformation systems ect, directional decorating certain targeting properties and keeping it inhere propertyes, changing and developing in design and color, florescence, model, resistance. The different standards in classifing genetic transformation systems of chrysanthemum reported on broad are not disagreed with some traditional varieties in home. So it has been significant in researching genetic transformation system which adapt to chrysanthemum cultivars in China.The main experiment and results of this paper are such as follows:1,optimizte of efficient regeneration system of four chrysanthemum varietiesFour varieties of chrysanthemum researched in this paper are 'Dong ju', 'Jiu mi rong', 'shan cheng zhi guang', 'Huang guan'. The result describes an efficient regeneration system of four chrysanthemum varieties with differentiation medium, several factors such as different genotypes, combinations of hormone, pre-culture, explant affected callus, shoot inducement of chrysanthemum. A rapid and efficient regeneration system of chrysanthemum has been established. The results shown that the optimal differentiation medium are: ' Dong ju' MS +6-BA 2.0 mg/L+IAA 2.0 mg/L; ' Jiu mi rong' MS+6-BA 1.0mg/L+IAA2.0mg/L; 'Shan cheng zhi guang' MS+6-BA2.0mg/L +IAA 5.0 mg/L; 'Huang guan' MS+6-BA 1.0 mg/L+IAA 3.0 mg/L. Compared to regeneration of four chrysanthemum varieties that the highest regeneration frequence of Shan cheng zhi guang can reach 100.0%. Dong ju , Jiu mi rong, Huang guan are 86.7%,85.3% and 81.2%.Therefore we have choosed the system of explant of Shan cheng zhi guang leaves been transformation in this research subsequently.2,Construction of plant expressional vector pC2301-F3'5'H and transffere into LBA4404.pT-F3'5'H and pC2301 both digested with Kpn I and BamH I were constructed for plant sense expressional vector pC2301- F3'5'H of F3'5'H gene. Then we have transffered the plant expressional vector pC2301-F3'5'H into agrobaterium LBA4404.3,Introduction of F3'5'H gene into chrysanthemum by Agrobacterium tumefaciens.Young leaves were excised from asepsis chrysanthemum Shan cheng zhi guang, cut into6×6mm² , dipped into Agrobaterium (OD₆₀₀ adjusted to 0.5) for 20min. After patted dry redundant agrobaterium on the sterile filter paper, been transferred into MSG: MS+6-BA 2.0 mg/L+IAA 5.0 mg/L medium and co-cultured for 3 days in darkness. Subsequently, the explants were transferred to MSY:MS+6-BA 3.0 mg/L+IAA 5.0 mg/L+Cef 200 mg/L for 4 days to delay selection culture before formal selection (MSS1:MS+6-BA 2.0 mg/L+IAA 0.5 mg/L+ Km 20 mg/L +Cef 100 mg/L). When the resistant shoots were cut down until 1cm and then been rooted on MSS:1/2MS+ Km 10 mg/L. The transferred the plantlets with normal roots were used for further identify analysis.4,The accidence identification analyse of transformed plants by PCR.35s promoter and F3'5'H gene were amplified respectively by common PCR to identify transformed plants. The PCR results suggested that transgenic has 15 positive plantlets named Y^F3'5'H while untransgenic named y⁰. Integration of F3'5'H gene into chrysanthemum was confirmed by using PCR amplification.The transgenetic were obtained the same strip with the marker. We principle identify that the target gene has been translated into genome of Chrysanthemum varieties Shan chenng zhi guang successfully.