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园艺学报 ›› 2013, Vol. 40 ›› Issue (8): 1517-1526.

• 观赏植物 • 上一篇    下一篇

露地菊离体再生体系建立及BrDFR基因遗传转化

许志茹,陈智华,姜艳东,侯 杰,佟 玲,李玉花*   

  1. (东北林业大学生命科学学院,林木遗传育种国家重点实验室,哈尔滨 150040)
  • 收稿日期:2013-01-16 出版日期:2013-08-25 发布日期:2013-08-25

Establishment of the Regeneration System and Genetic Transformation of BrDFR Gene in Chrysanthemum Cultivar

XU Zhi-ru,CHEN Zhi-hua,JIANG Yan-dong,HOU Jie,TONG Ling,and LI Yu-hua*   

  1. (College of Life Sciences, Northeast Forestry University,State Key Laboratory of Forest Tree Genetic Improvement,Harbin 150040,China)
  • Received:2013-01-16 Online:2013-08-25 Published:2013-08-25

摘要: 以露地菊(Chrysanthemum morifolium)品种‘神韵’无菌苗叶片为外植体材料,研究植物生长调节剂配比对其愈伤组织诱导再生芽的影响,以建立离体再生体系。研究结果表明,露地菊‘神韵’在含NAA 1.0 mg · L-1 + BA 1.0 mg · L-1的MS培养基上获得了最高的再生芽分化率。利用已经克隆的‘津田’芜菁BrDFR基因构建非抗生素筛选的表达载体。以露地菊‘神韵’的无菌苗叶片为受体,通过农杆菌介导进行BrDFR基因的遗传转化。以载体的PMI基因为筛选标记,通过甘露糖筛选获得了‘神韵’的遗传转化再生植株。经过PCR验证和Southern杂交检测证实BrDFR基因已经整合到‘神韵’基因组中。

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