Title: Agrobacterium-mediated transformation of potato using PLRV-rep and PVY CP genes and assessment of replicase mediated resistance against natural infection of PLRV Authors
Submitted to: Pakistan Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: October 1, 2008
Publication Date: June 1, 2009
Citation: Arif, M., Thomas, P.E., Crosslin, J., Brown, C.R. 2009. Agrobacterium-mediated transformation of potato using PLRV-rep and PVY CP genes and assessment of replicase mediated resistance against natural infection of PLRV. Pakistan Journal of Botany. 41:1477-1488. Interpretive Summary: Potato leafroll virus (PLRV) and Potato virus Y (PVY) are the two most economically important viruses of cultivated potato worldwide. This paper reports the production of genetically engineered potatoes of cultivar 'Desiree' with some level of resistance to PLRV under field conditions. Cultivar Desiree is particularly high-yielding in many regions of the world and virus resistance in this cultivar is of particular interest in some developing nations because of the inability to chemically aphids, which transmit both PLRV and PVY.
Technical 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, 28 plants were selected for further propagation and evaluation of resistance against respective virus isolates. All of the transgenic clones containing PLRV-replicase gene constructs showed lesser rates of infection by PLRV from field exposure than the cultivars used as control.