Plant Cell Reports

, Volume 19, Issue 12, pp 1203–1211

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

  • Z. N. Yang
  • I. L. Ingelbrecht
  • E. Louzada
  • M. Skaria
  • T. E. Mirkov
GENETIC TRANSFORMATION AND HYBRIDIZATION

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 Galanthusnivalis 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 

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Copyright information

© Springer-Verlag Berlin Heidelberg 2000

Authors and Affiliations

  • Z. N. Yang
    • 1
  • I. L. Ingelbrecht
    • 1
  • E. Louzada
    • 2
  • M. Skaria
    • 2
  • T. E. Mirkov
    • 1
  1. 1.Department 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: +1-956-9680641US
  2. 2.Citrus Center, The Texas A&M University – Kingsville, 312 N. Intl. Blvd., Weslaco, TX 78596, USAUS

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