Studies on Coat Protein Mediated Resistance Against Chilli Veinal Mottle Virus Associated with Murda Complex Disease in Chilli Capsicum annuum L

Abstract

Molecular identification, characterization, development of recombinant gene construct newlineand transformation work was carried out in Departments of Plant Pathology and newlineBiotechnology, University of Agricultural Sciences, Dharwad. Chilli plants showing typical newlinesymptoms of murda complex were collected and total RNA was isolated from diseased and newlinehealthy samples of chilli. Subsequently cDNA was synthesized from the RNA using oligo dT newlineprimer and reverse transcriptase enzyme. The cDNA was used as a template for amplification newlineof ChiVMV, GBNV, TMV and CMV using gene specific primers. Similarly, total DNA from newlinevirus infected and healthy samples of chilli plants was isolated and used as template for newlineamplification of ToLCV using gene specific primers. None of chilli murda complex disease newlinesamples were amplified for ToLCV, GBNV, TMV and CMV except ChiVMV with amplicon newlineof ~531 bp was amplified. Thus, the investigation focused on molecular identification, newlineconvincingly revealed ChiVMV association with the chilli murda complex. newlineThe ChiVMV coat protein gene (~531 bp) was cloned into pTZ57R/T cloning vector. newlineGene sequence and BLAST analyses clearly revealed that ChiVMV coat protein gene had 95 newlineper cent homology with the reported nucleotide sequences. Recombinant gene construct was newlinedeveloped by sub-cloning the ChiVMV-CP gene into plant transformation pHS100 vector. newlineRecombinant gene construct was mobilized into Agrobacterium tumefaciens LBA4404 strain. newlineAgrobacterium mediated flower dip method of transformation was adopted to develop newlinetransgenic chilli plants expressing ChiVMV-CP gene. newlineAmong 55 T1 plants, 18 putative transformants showed positive results for PCR and newlineGUS analysis. Post challenge inoculation studies identified twelve T1 transgenic plants viz., 1- newline1, 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 newlineand six T1 transgenic plants viz., 3-1, 5-4, 6-2, 7-1, 7-3 and 11-3 T1 showed delayed newlinesymptoms. Resistant T1 transgenic plants can be used to mitigate ChiVMV with further newlinevalidation.