GENETIC TRANSFORMATION AND HYBRIDIZATION

Plant Cell Reports

, Volume 19, Issue 12, pp 1203-1211

First online:

Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.)

  • Z. N. YangAffiliated withDepartment of Plant Pathology and Microbiology, The Texas A&M University Agricultural Experiment Station, 2415 E. Hwy 83, Weslaco, TX 78596, USA e-mail: e-mirkov@tamu.edu Fax: +1-956-9680641
  • , I. L. IngelbrechtAffiliated withDepartment of Plant Pathology and Microbiology, The Texas A&M University Agricultural Experiment Station, 2415 E. Hwy 83, Weslaco, TX 78596, USA e-mail: e-mirkov@tamu.edu Fax: +1-956-9680641
  • , E. LouzadaAffiliated withCitrus Center, The Texas A&M University – Kingsville, 312 N. Intl. Blvd., Weslaco, TX 78596, USA
  • , M. SkariaAffiliated withCitrus Center, The Texas A&M University – Kingsville, 312 N. Intl. Blvd., Weslaco, TX 78596, USA
  • , T. E. MirkovAffiliated withDepartment of Plant Pathology and Microbiology, The Texas A&M University Agricultural Experiment Station, 2415 E. Hwy 83, Weslaco, TX 78596, USA e-mail: e-mirkov@tamu.edu Fax: +1-956-9680641

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Abstract

 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 Grapefruit Agrobacterium tumefaciens Genetic transformation 34S promoter Galanthus nivalis agglutinin
Abbreviations BG: Shoot elongation medium CaMV: Cauliflower mosaic virus CTV: Citrus tristeza virus DBA3: Regeneration medium FMV: Figwort mosaic virus GNA: Galanthusnivalis agglutinin gna: Galanthus nivalis agglutinin gene GUS: β-Glucuronidase LB: Luria broth MS: Murashige and Skoog 4-MUG: 4-Methylumbelliferyl-d-glucuronic acid NOS: Nopaline synthase nptII: Neomycin phosphotransferase II gene uidA: β-Glucuronidase gene uncp: Untranslatable coat protein gene