Increasing expression of an anti-picloram single-chain variable fragment (ScFv) antibody and resistance to picloram in transgenic tobacco (Nicotiana tabacum)

Systematic research involving four chimeric gene constructions designed to express the same antipidoram single-chain variable fragment (scFv) antibody is described. Agrobacterium-mediated transformation produced at least 25 transgenic tobacco plants with each of these, and the number of T-DNA loci in each plant was determined using kanamycin-resistance segregation assays. The relative amounts of active and total scFv in each plant were evaluated using quantitative enzyme-linked immunosorbent assay and immunoblot technologies, respectively. No significant differences in scFv activity were found among the four groups of single-locus plants, although the 35S/M construct was found to produce significantly more total anti-picloram scFv than the other three constructs. A dose-response bioassay involving T-1 seedlings from several of the highest expressers of active scFv demonstrated resistance to a constant exposure of picloram at 5 x 10(-8) M. Other approaches for increasing antibody-based herbicide resistance are discussed, as further improvements are needed before practical application of this technology.