4种地被菊再生及遗传转化条件的比较

Comparison on the Regeneration System and Genetic Transformation Condition in 4 Ground Cover Chrysanthemum Cultivars

为建立地被菊‘神韵’、‘繁星粉’、‘都市丽人’和‘粉玫瑰’的离体再生体系,明确其遗传转化条件,为通过转基因技术培育地被菊新品种奠定基础,以叶片为外植体,研究了激素配比对4种地被菊再生芽诱导的影响;通过农杆菌介导比较了4种地被菊的遗传转化条件。研究结果表明,‘神韵’、‘繁星粉’、‘都市丽人’和‘粉玫瑰’可分别在含有NAA 1.0 mg/L+BA 1.0 mg/L、NAA 0.3 mg/L+BA 1.0 mg/L、NAA1.5 mg/L+BA 0.5 mg/L、NAA 0.5 mg/L+BA 1.5 mg/L的MS培养基上获得最佳分化率;其叶片外植体的遗传转化条件为24 h预培养、OD600分别为0.4、0.5、0.3和0.4的农杆菌侵染10 min、共培养48 h、延迟培养2天、甘露糖筛选浓度为10 g/L、头孢霉素抑菌浓度为300 mg/L。部分抗性植株经PCR检测获得了目的条带,初步证明目的基因已经整合到4种地被菊的基因组中。

In order to establish the regeneration system and definite the transformation condition of the four ground cover Chrysanthemum cultivars,‘Shenyun＇,‘Fanxingfen＇,‘Dushiliren＇and‘Fenmeigui＇, the leaves of the aseptic seedlings were used as explants to analyze the effect of plant growth regulator hormone combination on regeneration buds induced from callus, and then the genetic transformation condition of these four Chrysanthemum morifolium was compared through the mediation of Agrobacterium tumefaciens. This study will lay the research foundation for cultivating new Chrysanthemum morifolium using the transgenic means. The result indicated that the optimum condition for regeneration buds inducing on MS medium was NAA 1.0 mg/L＋ BA 1.0 mg/L, NAA 0.3 mg/L＋ BA 1.0 mg/L, NAA 1.5 mg/L＋ BA 0.5 mg/L and NAA 0.5 mg/L ＋ BA 1.5 mg/L for‘Shenyun＇,‘Fanxingfen＇,‘Dushiliren＇and‘Fenmeigui＇respectively. The genetic transformation conditions of the aseptic seedling leaves were pre-cultured for 24 hours, infected with A. tumefaciens for 10 minutes which OD600 was 0.4, 0.5, 0.3 and 0.4 respectively, co-cultivated for 48 hours, later-cultivated for 2 days, the mannose concentration was 10 g/L and the cefsulodin concentration was 300 mg/L. The target bands of genes could be obtained in some resistant plants of these four Chrysanthemum morifolium by PCR detection, which indicated that the target gene was integrated into their genomes.