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Author: Abdul Kareem M. × End date: 2018 × Type: Thesis ×
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    ThesisItemOpen Access
    Studies on Coat Protein Mediated Resistance Against Chilli Veinal Mottle Virus Associated with Murda Complex Disease in Chilli (Capsicum annuum L.)
    (University of Agricultural Science, Dharwad, 2017-06) Abdul Kareem M.; Byadgi, A.S.
    Molecular identification, characterization, development of recombinant gene construct and transformation work was carried out in Departments of Plant Pathology and Biotechnology, University of Agricultural Sciences, Dharwad. Chilli plants showing typical symptoms of murda complex were collected and total RNA was isolated from diseased and healthy samples of chilli. Subsequently cDNA was synthesized from the RNA using oligo dT primer and reverse transcriptase enzyme. The cDNA was used as a template for amplification of ChiVMV, GBNV, TMV and CMV using gene specific primers. Similarly, total DNA from virus infected and healthy samples of chilli plants was isolated and used as template for amplification of ToLCV using gene specific primers. None of chilli murda complex disease samples were amplified for ToLCV, GBNV, TMV and CMV except ChiVMV with amplicon of ~531 bp was amplified. Thus, the investigation focused on molecular identification, convincingly revealed ChiVMV association with the chilli murda complex. The ChiVMV coat protein gene (~531 bp) was cloned into pTZ57R/T cloning vector. Gene sequence and BLAST analyses clearly revealed that ChiVMV coat protein gene had 95 per cent homology with the reported nucleotide sequences. Recombinant gene construct was developed by sub-cloning the ChiVMV-CP gene into plant transformation pHS100 vector. Recombinant gene construct was mobilized into Agrobacterium tumefaciens LBA4404 strain. Agrobacterium mediated flower dip method of transformation was adopted to develop transgenic chilli plants expressing ChiVMV-CP gene. Among 55 T1 plants, 18 putative transformants showed positive results for PCR and GUS analysis. Post challenge inoculation studies identified twelve T1 transgenic plants viz., 1-1, 1-2, 2-2, 2-5, 3-3, 3-4, 4-2, 6-4, 7-2, 7-5, 8-1 and 10-5 with complete resistance to ChiVMV and six T1 transgenic plants viz., 3-1, 5-4, 6-2, 7-1, 7-3 and 11-3 T1 showed delayed symptoms. Resistant T1 transgenic plants can be used to mitigate ChiVMV with further validation.