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

Cevik B., Lee R. F., Niblett C. L.

TURKISH JOURNAL OF AGRICULTURE AND FORESTRY, vol.36, no.2, pp.195-206, 2012 (SCI-Expanded) identifier identifier

  • Publication Type: Article / Article
  • Volume: 36 Issue: 2
  • Publication Date: 2012
  • Doi Number: 10.3906/tar-1008-1192
  • Journal Indexes: Science Citation Index Expanded (SCI-EXPANDED), Scopus, TR DİZİN (ULAKBİM)
  • Page Numbers: pp.195-206
  • Süleyman Demirel University Affiliated: Yes


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-Delta GDD). 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-Delta GDD) were identified.