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

• Published in: Plant cell reports Vol. 19; no. 12; pp. 1203 - 1211
• Main Authors: YANG, Z. N, INGELBRECHT, I. L, LOUZADA, E, SKARIA, M, MIRKOV, T. E
• Format: Journal Article
• Language: English
• Published: Berlin Springer 01.12.2000; Springer Nature B.V
• Subjects: agglutinins; Agrobacterium; Biological and medical sciences; Biotechnology; Citrus paradisi; Citrus tristeza virus; Cultivars; Fruits; Fundamental and applied biological sciences. Psychology; Gene expression; Genetic engineering; Genetic technics; gna gene; Methods. Procedures. Technologies; Plant tissues; Proteins; Seedlings; Shoots; Transgenic animals and transgenic plants; Transgenic plants; uidA gene; uncp gene; Monocotyledones; Insecticide; Plant pathogen; Citrus fruit; Biological control; Rutaceae; Citrus paradisi; Agrobacterium tumefaciens; Tissue culture; Microorganism resistance; Gene; Dicotyledones; Angiospermae; Bacteria; Transgenic plant; Coat protein; Closterovirus; Citrus tristeza virus; Vegetals; Genetic transformation; Pesticides; Galanthus nivalis; Gene expression; Virus; Closteroviridae; Agglutinin; Pest resistance; Spermatophyta; Rhizobiaceae; Amaryllidaceae; Cultivar; 4-MUG: 4-Methylumbelliferyl-d-glucuronic acid; NOS: Nopaline synthase; Keywords Grapefruit; GUS: β-Glucuronidase; LB: Luria broth; GNA: Galanthusnivalis agglutinin; nptII: Neomycin phosphotransferase II gene; uncp: Untranslatable coat protein gene; DBA3: Regeneration medium; gna: Galanthus nivalis agglutinin gene; MS: Murashige and Skoog; uidA: β-Glucuronidase gene; CaMV: Cauliflower mosaic virus; FMV: Figwort mosaic virus; Galanthus nivalis agglutinin; 34S promoter; Abbreviations BG: Shoot elongation medium; CTV: Citrus tristeza virus
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s002990000257

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.