Investigations on mild strains and transformation of PRSV CP gene in papaya

Abstract

Management of PRSV using pathogen derived resistance and cross protection was newlineattempted. Roving survey was conducted in entire Karnataka and parts of Maharashtra to newlineknow the incidence of PRSV and for collection of mild strains under natural conditions. newlineNorthern Karnataka witnessed high range of PRSV incidence compared to southern. The newlineaverage of all the districts ranged between 1.42 to 92 per cent. Other than Kodagu incidence newlinewas recorded in all the districts. In Maharashtra incidence ranged between 35.50 to 86.17 per newlinecent. newlineDuring survey 16 mild strains were collected and evaluated for cross protection ability but, newlinenone of them proved as mild. Mutation of local PRSV-UASD strain using UV irradiation, newlinesodium salts, sodium azide and EMS was also unsuccessful. Coat protein of PRSV-UASD CP newlinegene (864bp) was cloned in Agrobacteruim tumefaciens for plant transformation. An in vitro newlineregeneration in papaya was attempted by direct regeneration and somatic regeneration. newlineNeither the explants nor the growth regulators in any treatments were successful. newlineEmbryo culture was attempted using embryos excised from immature seeds formed after newline95 to 115 days of anthesis developed highest callus induction. Shoots regenerated from newlinecallus clump but, did not survive further. newlineThe PRSV-CP gene was cloned in Agrobacterium and cocultivated with papaya callus. It newlineregenerated in to putataive transformants on kanamycine selective medium but, did not newlineelongate further. Construct of PRSV-UASD-CP was finally transformed in to tobacco. newlineIn PCR and one step RT-PCR, amplicon of 864bp was evident of expression of newlinetransgene at transcriptional level. The PCR positive tobacco plants were challenged with newlinePRSV-UASD strain. They remained healthy for 70 days without any viral symptoms, newlineindicating successful expression of PRSV-UASD-CP gene conferring resistance to PRSVUASD newlineinoculation.