Transgenic plants of grapefruit cv. Rio Red (Citrus paradisi Macf.) have been obtained by Agrobacterium tumefaciens-mediated gene transfer using seedling-derived epicotyl segments as explants and kanamycin as the selective agent. The transformation procedure includes a shoot elongation phase with a liquid medium overlay, which provides additional selection against non-transgenic shoots. Transformed shoots are invigorated and multiplied on a non-selective medium prior to grafting, thus assuring that plants can be recovered from transgenic shoots. We have constructed a binary vector, pBin34SGUS, with an intron-containing β-glucuronidase gene (uidA) under the control of the Figwort mosaic virus 34S promoter. The 34S promoter efficiently drives uidA gene expression both in transient assays and in transgenic Rio Red leaf tissue, although at levels five- to sevenfold lower than the Cauliflower mosaic virus 35S promoter. An untranslatable coat protein gene (uncp) of the Citrus tristeza virus strain SY568 and the Galanthus nivalis agglutinin gene (gna) were inserted into pBin34SGUS and transgenic plants have been obtained. Stable integration of the uncp and gna genes was confirmed by Southern hybridization and gna gene expression was confirmed by Western blot analysis.
Keywords: 34S promoter; 4-MUG: 4-Methylumbelliferyl-d-glucuronic acid; Abbreviations BG: Shoot elongation medium; Agrobacterium tumefaciens; CTV: Citrus tristeza virus; CaMV: Cauliflower mosaic virus; DBA3: Regeneration medium; FMV: Figwort mosaic virus; GNA: Galanthusnivalis agglutinin; GUS: β-Glucuronidase; Galanthus nivalis agglutinin; Genetic transformation; Keywords Grapefruit; LB: Luria broth; MS: Murashige and Skoog; NOS: Nopaline synthase; gna: Galanthus nivalis agglutinin gene; nptII: Neomycin phosphotransferase II gene; uidA: β-Glucuronidase gene; uncp: Untranslatable coat protein gene.