|CEVIK, BAYRAM - Suleyman Demirel University|
|NIBLETT, C - University Of Florida|
Submitted to: Turkish Journal of Agriculture and Forestry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2012
Publication Date: 3/15/2012
Citation: Cevik, B., Lee, R.F., Niblett, C.L. 2012. Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus. Turkish Journal of Agriculture and Forestry. 36:195-206.
Interpretive Summary: This manuscript reports the transformation of grapefruit with the wild-type and mutants of the RNA-dependent RNA polymerase (RdRp) of Citrus tristeza virus (CTV) using Agrobacterium-mediated transformation. From a total of 4540 putative transformed etiolated epicotyle segments of grapefruit, 1402 were kanamycin resistant. A total of 146 plants were selected for rooting then which were expressing the green fluorescent protein and ß-glucuronidase (GUS) markers. A total of 70 transgenic plants which survived were screened for presence of the GUS and CTV-RdRp transgenes by polymerase chain reaction; 51 contained both the GUS and the CTV-RdRp construct. Additional screening of the transgenic plants and evaluation of the plants for resistance to CTV will be done in the future.
Technical Abstract: Citrus paradisi Macf. cv. 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 CTV genome and used to generate the wild-type and two 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-'GDD). Etiolated epicotyl segments of 'Duncan' grapefruit (Citrus paradisi Macf. cv. Duncan) were transformed with each of these constructs using 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 ß-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). Fifty one GUS and CTV transgene positive transgenic plants (15 with RdRp, 21 with RdRp-mGDD and 15 with RdRp-'GDD) were identified.