Genetic engineering and evaluation of pathogen-derived resistance to potato leafroll virus
Potato leafroll virus (PLRV) is the most serious virus affecting the potato crop worldwide, causing significant economic losses both in yield and quality. Genetic engineering was successfully applied to develop PLRV resistance in North American potato genotypes. The molecular mechanism, and effectiveness and stability of the engineered resistance under field conditions were investigated. In vitro regeneration response was studied in tuber disc culture of tetraploid and diploid potato genotypes in order to establish an efficient regeneration system for potato transformation. Most of the culture development characteristics were affected by the ploidy level. The highest frequency of regeneration was obtained from diploid lines where explants grew at a moderate rate, producing green calli at multiple sites on discs with moderate level of senescence. Considerable morphological variation and high frequencies of chromosome doubling were observed in diploid-derived plants. Some culture characteristics such as callus size, form of culture growth and frequency of regeneration were correlated significantly with the magnitude of some morphological changes. Agrobacterium-mediated leaf disc and stem segment transformation systems were used to transfer the coat protein (CP) gene of PLRV into tetraploid and diploid potato genotypes. Despite the high response of tetraploid cvs to both transformation systems, no response to stem segment transformation was observed in diploid lines. More than 400 putative transformants were regenerated from two chipping cultivars Snowden and FL1607, and diploid line, 7506-01, and were tested for integrative transformation. Varying levels of resistance ranging from 0-100% were obtained from 21 transgenic lines after inoculating twice under field conditions. Protein product of the CP gene was not detected in any of the transgenic lines, while a positive relationship was detected between the resistance and RNA transcript level, suggesting that genetically engineered resistance to PLRV is likely RNA mediated. No PLRV was detected in highly resistant lines throughout the growing season, across two environments and two clonal generations, while virus titre increased significantly in susceptible transgenic lines and nontransgenic controls in the second clonal generation after infection. This study suggests that pathogen-derived resistance to PLRV is agronomically viable and can effectively control PLRV disease in potato.