PJB-2009-161
AGROBACTERIUM-MEDIATED TRANSFORMATION OF POTATO USING PLRV-REP. AND PVY CP GENES AND ASSESSMENT OF REPLICASE MEDIATED RESISTANCE AGAINST NATURAL INFECTION OF PLRV
M. ARIF1, P. E. THOMAS2, J. M CROSSLIN2 AND C. R. BROWN3
Abstract
Replicase-and coat protein gene-mediated resistances against potato leafroll virus (PLRV) and potato virus Y (PVY), respectively, demonstrated to be an effective way of protecting potato against two major virus problems (PLRV & PVY) world-wide. Potato cultivar Desiree was transformed using Agrobacterium tumefaciens with LBA4404pBinplusPLRV-replicase construct. A total of 25 lines were generated from kanamycin-resistant calli. Shoots were excised and placed onto shoot medium containing 250mg/L cefotaxime and 50mg/L kanamycin sulfate in tissue culture tubes. Genomic DNA was extracted from shoot samples and polymerase chain reaction (PCR) analysis was done using specific primers. A total of 116 plants of 25 lines were tested and most of the plants were positive showing a band of 449bp specific to PLRV-replicase gene insert. The plants showing maximum PCR reaction were selected. Potato cultivar Desiree and Norkotah Russet were also transformed using two constructs containing a coat protein gene of PVY (RC4pBinPAubi3P and RC435S). The efficiency of transformation of Desiree with RC435S was high but only a few lines of Norkotah Russet were generated with RC4pBinPAubi3P construct. A total of 13 lines of Desiree were generated from kanamycin resistant calli using RC435S PVY CP construct. On the basis of PCR analysis of 42 putative transformants
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