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, cilt.36, sa.2, ss.195-206, 2012 (SCI İndekslerine Giren Dergi) identifier identifier

  • Cilt numarası: 36 Konu: 2
  • Basım Tarihi: 2012
  • Doi Numarası: 10.3906/tar-1008-1192
  • Dergi Adı: TURKISH JOURNAL OF AGRICULTURE AND FORESTRY
  • Sayfa Sayıları: ss.195-206

Özet

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.