MODGIL, MANJUNEHA KUMARI2021-09-142021-09-142021-09https://krishikosh.egranth.ac.in/handle/1/5810175718ABSTRACT Apple ‘Oregon Spur’ is a commercial cultivar recommended in India, but it is susceptible to Marssonina blotch, a fungal disease which causes premature leaf fall thus affects the fruit production. In order to avoid the excessive use of fungicides, a novel innovation Host Induced Gene Silencing (HIGS) approach, which is RNAi based gene silencing has been used to impart resistance to ‘Oregon Spur’ in the present studies. Before transformation trials, an efficient system for in vitro regeneration of shoots from leaf explants has been established by examining the type and concentration of plant growth regulators, dark-light incubation. Upto 95% of leaves could be induced to form direct and healthy adventitious shoots on MS medium supplemented with 4mg/l BA and 0.2mg/l NAA. Though the frequency of shoot regeneration increased to 100% on 2mg/l TDZ and 0.5mg/l NAA. All concentrations of TDZ produced abnormal and vitrified shoots. The effect of different concentration of antibiotics was also observed on shoot regeneration from shoot explants. Kanamycin at higher concentrations above 5mg/l inhibited shoot induction, whereas cefotaxime at 100-300mg/l promoted organogenesis. To eliminate bacterial overgrowth after co-cultivation, 500mg/l cefotaxime was found optimal. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pRI101-AN hpRNA:CaM containing targeted region of calmodulin (CaM) gene of M. coronaria in sense and antisense orientation under CaMV 35S promoter and nptII gene under NOS promoter was used for transformation experiment. Putative RNAi transformants were selected on shoot regeneration medium supplemented with 5mg/l kan and 500 mg/l cef after a pause to kan for 2 wks. 10min infection and 2 d co-cultivation periods were found effective. A total of 15 putative RNAi shoots/lines from 18 events were obtained which were multiplied and rooted. Eleven lines out of 15 putatively created RNAi lines successfully integrated CaM sense-antisense region and nptII genes, yielding 381 and 725 bp respectively with specific primers. Gene integration was further confirmed by reverse transcriptase PCR analysis. Semi quantitative RT-PCR revealed low to medium level of expression of CaM off-targeted region in eleven transgenic RNAi lines which confirmed that gene is expressing in each line. Four lines E10a, E1b and E1a showed higher relative expression in qRT-PCR analysis and in these lines fungus was not able to colonize the leaf explants during bioassay experiment. In vitro detached leaf assay revealed lesion development and disease progression in wild type after 20 dpi which were not visible in eleven lines. Microscopic examination showed fully developed, septate mycelium and necrosis of whole tissue in wild type while not found in transformants which is associated with down regulation of CaM gene expression. Thus, it is concluded that deciphering the role of CaM gene using hpRNAi construct through HIGS approach has provided resistance to Marssonina blotch in RNAi transgenic lines of ‘Oregon Spur’. Signature of Major Advisor Signature of the Student CountersignedEnglishThesis