| Groundcover chrysanthemum(Chrysanthemum morifolium) plays a vital role in landscape greening for its various flowers,rich color and high ornamental value. In this study, turned to 'Song Jiang Baihe' as experimental material, a petal symmetry of cloned genes. The gene was CYC gene family by structural analysis and homology comparison. Chrysanthemum, leaf regeneration system were optimized, and LjCYC2 gene into the chrysanthemum, the establishment of a genetic transformation system, and obtained transgenic plants.1. A flower asymmetry cDNA clone was obtained with RT-PCR from groundcover chrysanthemum 'Song Jiangbai He'. Its cDNA length was 898 bp. Analysis of its amino acid sequence showed it the CYC family gene, and has high identity to asymmetry gene of many plants.2. Study of the efficient regeneration system. The regeneration frequency of'Donglin 9' was 90%, in medium of MS+0.5mg/L BA +1.5mg/L NAA. The bud differentiation rate in upper young leaves of'Donglin 9'was as high as 96%, with the differentiation rate being for leaves at middle and lower parts which near the roots were 62% and 37%. After a 2-4d preculture in medium of MS+0.5 mg/L BA +1.5 mg/L NAA, the regeneration frequency of 'Donglin 9'was enhanced. Gradient of BA concentration on callus induction was no significant difference. However, within a certain range, high concentrations of BA will promote callus by adventitious bud differentiation. When the BA concentration above 0.5mg/L, the contrary, it will inhibit the callus formation. In this study, BA concentration of 0.5mg/L in the differentiation medium when Ml grew 30d, explant differentiation of adventitious buds up, the differentiation rate of 92%.3. Study of the efficient transformation system. Hygromycin reduced bud differentiation of leaf explants.'Donglin 9'lost ability to differentiation in the culture medium containing 10 mg/L hygromycin and once-differentiated buds died when soaked in solution at 30mg/L hygromycin. The efficient transformation system was as follows:the suitable concentration of Cef was 300 mg/L, the infection time was 10min, the co-culture time was 2d, the delay selection time was 5d, and the addition of Acetosyringone could improve the efficiency of transformation.4. Establish LjCYC2 gene into chrysanthemum genetic transformation system, by PCR detection of a positive lines. |
