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DEVELOPMENT OF MOLECULAR RESISTANCE IN POTATO AGAINST POTATO LEAF ROLL VIRUS AND POTATO VIRUS Y THROUGH AGROBACTERIUM-MEDIATED DOUBLE TRANSGENESIS

M. Arif^1*, P.E. Thomas², J.M. Crosslin² AND C.R. Brown³

Abstract: Potato leafroll virus (PLRV) and potato virus Y (PVY) are the two major viral problems for the potato production all over the world.  Transgenic approaches involving the expression of viral genes are being developed to provide protection for plants against viral diseases. The purpose of this study was to develop double transgenic plants of potato using PLRV replicase and PVY coat protein genes tandemly placed in a single T-DNA transformant through Agrobacterium-mediated transformation. A total of 17 lines of putative transformants of potato cv. Desiree were generated from kanamycine resistant calli originated from co-inoculation of separate Agrobacterium cultures containing PVY CP and PLRV replicase genes.  Shoots were excised and cultured onto shoot medium containing 250mg/L cefotaxime and 50mg/L kanamycin sulfate in test tubes. Polymerase chain reaction (PCR) analysis was conducted of 39 plants of 16 transformed lines using primers each of PVY CP and PLRV-replicase genes; 10 plants of 8 lines and 7 plants of 6 lines showed presence of of PVY CP and PLRV-replicase genes, respectively. However, 22 plants of 14 lines harbored both PVY-CP (508bp) and PLRV-replicase (449bp) genes. Sixteen plants of 11 double transgenic lines that showed high level of expression of both PLRV-replicase and PVY-CP genes. Transformants and control standards were exposed to field virus infection augmented by placement of aphids on Datura leaves infected with PLRV. Two clones (Des (CP+LR) 9.1 and Des (CP + LR) 9.2) showed incidence of infection statistically lower than the lowest infection of one of the standards (Ranger).  The Monsanto clone (21-350) showed no infection. The two resistant clones may be identical as they were derived from the same callus.

¹Fulbright Research Fellow, ²Research Plant Pathologist, and ³Research Geneticist, United States Department of Agriculture, Agricultural Research Service (USDA-ARS), Pacific West Area, Irrigated Agriculture Research and Extension Center (IAREC), Vegetable and Forage Crop Production Research Unit, 24106-North Bunn Road, Prosser, WA 99350-9687, USA.