- Turkish Journal of Agriculture and Forestry
- Vol: 30 Issue: 3
- Genetic Transformation of Citrus paradisi with Antisense and Untranslatable RNA-dependent RNA Polyme...
Genetic Transformation of Citrus paradisi with Antisense and Untranslatable RNA-dependent RNA Polymerase Genes of Citrus tristeza closterovirus
Authors : Bayram Çevik, Richard F. Lee, Charles L. Niblett
Pages : 173-182
View : 7 | Download : 2
Publication Date : 9999-12-31
Article Type : Makaleler
Abstract :Protein and RNA-mediated forms of pathogen-derived resistance (PDR) have been developed against many viruses in different plants. However, no resistance has been reported against Citrus tristeza virus (CTV), a closterovirus, in Citrus species transformed with coat protein genes or other sequences of CTV. The successful use of replication-associated genes in RNA-mediated resistance in other crops prompted the use of the RNA-dependent RNA polymerase (RdRp) gene of CTV for the development of RNA-mediated PDR in Citrus. The RdRP gene was amplified from CTV isolate DPI3800 from Florida and used to generate antisense (RdRp-AS) and untranslatable (RdRp-UT) constructs with point mutation consecutive stop codons in the 5’ end of the RdRp gene for use in plant transformation. A total of 3120 etiolated epicotyl segments of Duncan grapefruit (Citrus paradisi Macf. cv. Duncan) were transformed with these constructs using Agrobacterium tumefaciens-mediated transformation. From these segments 1040 kanamycin-resistant shoots were regenerated, and a total of 131 putative transgenic shoots were identified by fluorescent microscopy and histochemical b-glucuronidase (GUS) assays. One hundred GUS positive plants were rooted and 66 plants survived and were established on soil. A total of 41 plants were tested by polymerase chain reaction (PCR) for the presence of the GUS gene and for the transgenes. Eighteen GUS-positive and transgene-positive plants (8 with RdRp-AS, and 10 with RdRp-UT) were identified.Keywords : Citrus tristeza virus, replicase and RNA-mediated resistance, plant transformation, RNA-dependent RNA polymerase