关键词: 露地菊, 组织培养, 二氢黄酮醇4–还原酶基因, 遗传转化, 分子检测

Abstract: In our study，with the leaves of the aseptic seedlings of Chrysanthemum morifolium‘Shenyun’，the effect of plant growth regulator combination on regeneration buds induced from callus was analyzed，and the regeneration system of‘Shenyun’was established. The study results indicated that the optimum condition for regeneration buds inducing on MS medium is NAA 1.0 mg · L^-1 + BA 1.0 mg · L^-1 for Chrysanthemum morifolium‘Shenyun’. Using the BrDFR gene of turnip‘Tsuda’，the expression vector of non-antibiotic screening was constructed. Leaf explants of the chrysanthemumaseptic seedlings were infected with Agrobacterium tumefaciens EHA105 pCAMBIA1301 and the BrDFR gene of turnip‘Tsuda’was introduced into‘Shenyun’. The selection system was based on the use of Escherichia coli phosphomannose isomerase（PMI）gene as a selectable marker gene and mannose as a selective nutrient. The explants were selected on medium supplemented with a combination of mannose and sucrose as a carbon source at different concentrations. Among the leaf explants which were infected，there were 7 shoots surviving after selection by the medium containing 10 g · L^-1 mannose. PCR analysis and southern blot analysis confirmed that T4 and T7 independent transformed lines were positive for PMI and DFR genes.

Key words: Chrysanthemum morifolium, tissue culture, dihydroflavonol 4-reductase gene, genetic transformation, molecular detection