Proteinase inhibitor II gene in transgenic poplar: Chemical and biological assays

Abstract

Transgenic poplar lines were developed to investigate the role of a proteinase inhibitor in pest resistance of woody plants. Using an Agrobacterium binary vector system, the clone ‘Hansen’ ( Populus alba L. × P. grandidentata Michx.) was transformed with chimeric genes containing the coding region of potato proteinase inhibitor II ( PIN2) linked to either a bacterial nopaline synthase ( nos) or a cauliflower mosaic virus (35 S) promoter. All transferred DNA also contained a selectable marker in the form of a nos promoter linked to a neomycin phosphotransferase II ( NPT II) structural gene. The presence of the transferred PIN2 and NPT II sequences in poplar was confirmed for nine transgenic lines using polymerase chain reaction (PCR). Expression of PIN2 in leaves of transgenic poplar was demonstrated by enzyme-linked immunosorbent assays (ELISAs) and western blots. Two unique polypeptides from transgenic poplar, of ca 8 kDa and ca 12 kDa, indicate that PIN2 was translated appropriately. Resistance to the imported willow leaf beetle was tested in laboratory bioassays. The untransformed clone ‘Hansen’ and 11 transgenic lines were submitted to freshly hatched larvae to determine effects on pupal weight, larval development time and leaf area consumed. A significant difference from the untransformed clone in leaf area consumed was detected in one transgenic line, Tr665. Trends were indicated for several other transgenic lines for the other parameters.