Establishment of an Agrobacterium-mediated transformation system for Periploca sepium Bunge

DOI Web Site 参考文献30件
  • Chen Ren
    Department of Forest and Forest Products Sciences, Faculty of Agriculture, Kyushu University
  • Gyokusen Mayumi
    Department of Forest and Forest Products Sciences, Faculty of Agriculture, Kyushu University
  • Nakazawa Yoshihisa
    Department of Biotechnology, Graduate School of Engineering, Osaka University
  • Su Yinquan
    Faculty of Forestry, Northwest Sci-Tech University of Agriculture and Forestry
  • Gyokusen Koichiro
    Department of Forest and Forest Products Sciences, Faculty of Agriculture, Kyushu University

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Agrobacterium-mediated transformation of Periploca sepium Bunge using proliferated clonal shoots was investigated to identify important factors affecting the transformation efficiency. Agrobacterium tumefaciens strains EHA105 and LBA4404 were used, both of which harbored a pKAFCR21 binary vector, which contained two reporter genes (GUS and sGFP, encoding β-glucuronidase and the synthetic green-fluorescent protein with S65T mutation) and two marker genes (encoding neomycin phosphotransferase II and hygromycin phosphotransferase). The factors evaluated were Agrobacterium strain, co-cultivation treatment, and antibiotic selection regime. The results revealed that the transformation efficiency could be synergistically increased to as high as 50–60% by infecting explants with Agrobacterium strain EHA105/pKAFCR21 and co-cultivating in the presence of 150 mg l−1 dithiothreitol, followed by selection at 100 mg l−1 kanamycin. Genomic DNA PCR, Southern hybridization, and quantitative real-time reverse transcription PCR analyses confirmed that the transgenes (GUS or sGFP) had presented, integrated, and expressed in all the tested transformant plants. The optimized protocol provides a basis for further genetic alteration of P. sepium for medicinal compounds and cis-polyisoprene production.

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