Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus

Authors: BAYRAM ÇEVİK, RICHARD F. LEE, CHARLES L. NIBLETT

Abstract: Citrus paradisi Macfad. ‘Duncan’ was transformed with constructs coding for the wild-type and mutant RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) for exploring replicase-mediated pathogen-derived resistance (RM-PDR). The RdRp gene was amplified from a CTV genome and used to generate the wild-type and 2 mutant RdRp constructs for plant transformation. One mutant had the key amino acids GDD changed to AAA (RdRp-mGDD), and the second mutant had a deletion encompassing the GDD domain (RdRp-\DeltaGDD). Etiolated epicotyl segments of Duncan grapefruit (Citrus paradisi Macfad. 'Duncan') were transformed with each of these constructs using the Agrobacterium-mediated transformation method. From 4540 transformed epicotyl segments, 1402 kanamycin-resistant shoots were regenerated. After testing for expression of green fluorescent protein (GFP) and \Beta-glucuronidase (GUS) reporter genes by fluorescence microscopy and histochemical staining, respectively, 146 GUS-positive plants were rooted and 97 surviving plants were established in soil in pots. A total of 70 plants were tested for the presence of the GUS gene and CTV RdRp transgenes by polymerase chain reaction (PCR). A total of 51 GUS and CTV transgene-positive transgenic plants (15 with RdRp, 21 with RdRp-mGDD, and 15 with RdRp-\DeltaGDD) were identified.

Keywords: Citrus tristeza virus, plant transformation, replicase-mediated resistance, RNA-dependent RNA polymerase

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