Transformation of potato (cv. Late Harvest) with the potato leafroll virus coat protein gene, and molecular analysis of transgenic lines

Potato leafroll virus (PLRV) is one of the most destructive potato viruses in South Africa. In order to establish resistance against PLRV in the potato cultivar Late Harvest, the coat protein (CP) gene of a South African isolate of the virus was isolated, cloned into the plant transformation vector pBII21 and inserted into potatoes using Agrobacterium tumefaciens-mediated transformation. Six plantlets, which appeared to be phenotypically normal, were regenerated from leaf disks under kanamycin selection. These lines were analysed for stable transgene insertion and expression. The presence of the PLRV CP, uidA (GUS) and nptII (kanamycin resistance) genes were shown using PCR: Southern blot analysis verified that the PLRV CP gene had been inserted into the genome of the transgenic potato lines. Coat protein could not be detected, but RNA dot blots demonstrated PLRV CP gene expression at the mRNA level. Expression of the uidA gene was investigated using a fluorometric assay, and it was observed that lines containing the PLRV CP gene in the antisense orientation exhibited GUS activity.