Investigations on mild strains and transformation of PRSV-CP gene in papaya
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Date
2009
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UAS Dharwad
Abstract
Management of PRSV using pathogen derived resistance and cross protection was
attempted. Roving survey was conducted in entire Karnataka and parts of Maharashtra to
know the incidence of PRSV and for collection of mild strains under natural conditions.
Northern Karnataka witnessed high range of PRSV incidence compared to southern. The
average of all the districts ranged between 1.42 to 92 per cent. Other than Kodagu incidence
was recorded in all the districts. In Maharashtra incidence ranged between 35.50 to 86.17 per
cent.
During survey 16 mild strains were collected and evaluated for cross protection ability
but, none of them proved as mild. Mutation of local PRSV-UASD strain using UV
irradiation, sodium salts, sodium azide and EMS was also unsuccessful. Coat protein of
PRSV-UASD CP gene (864bp) was cloned in Agrobacteruim tumefaciens for plant
transformation. An in vitro regeneration in papaya was attempted by direct regeneration and
somatic regeneration. Neither the explants nor the growth regulators in any treatments were
successful.
Embryo culture was attempted using embryos excised from immature seeds formed
after 95 to 115 days of anthesis developed highest callus induction. Shoots regenerated from
callus clump but, did not survive further.
The PRSV-CP gene was cloned in Agrobacterium and cocultivated with papaya
callus. It regenerated in to putataive transformants on kanamycine selective medium but, did
not elongate further. Construct of PRSV-UASD-CP was finally transformed in to tobacco.
In PCR and one step RT-PCR, amplicon of 864bp was evident of expression of
transgene at transcriptional level. The PCR positive tobacco plants were challenged with
PRSV-UASD strain. They remained healthy for 70 days without any viral symptoms,
indicating successful expression of PRSV-UASD-CP gene conferring resistance to PRSVUASD
inoculation.