Genetic Transformation of Citrus paradisi with Antisense and Untranslatable RNA-dependent RNA Polymerase Genes of Citrus tristeza closterovirus

Authors : Bayram ÇEVİK, Richard F. LEE, Charles L. NIBLETT

Pages : 173-182

View : 13 | Download : 5

Publication Date : 2006-05-01

Article Type : Research Paper

Abstract :Protein and RNA-mediated forms of pathogen-derived resistance insert ignore into journalissuearticles values(PDR); have been developed against many viruses in different plants. However, no resistance has been reported against Citrus tristeza virus insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(RdRp-AS); and untranslatable insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(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 insert ignore into journalissuearticles values(PCR); for the presence of the GUS gene and for the transgenes. Eighteen GUS-positive and transgene-positive plants insert ignore into journalissuearticles values(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