Abstract
The hairy root culture system has several desirable features as an experimental model for functional analyses of the genes involved in root metabolism and/or physiology. We developed a binary vector set for efficient target gene overexpression and RNA interference (RNAi) in transformed (hairy) roots. The vectors, pHR-OX and pHR-RNAi, contain a cluster of rol (rooting locus) genes, together with the single GATEWAYTM conversion cassette (in pHR-OX vectors) or inverted repeats of the GATEWAYTM conversion cassette separated by an intron sequence (in pHR-RNAi vectors), flanked by a dual CaMV 35S promoter and nopaline synthase polyadenylation signal, on the same T-DNA. Transformation experiments with pHR-OX vectors using Arabidopsis, potato, and tobacco as model plants revealed that by inoculating Agrobacterium tumefaciens harboring these vectors, a large number of independently transformed roots could be induced from explants in a short period of time, and subsequent establishment of root culture lines was possible. A model experiment that focused on the sterol biosynthetic pathway in Arabidopsis validated the utility of pHR-RNAi vectors for gene functional analysis in cultured roots. Use of our vector system may facilitate identification of regulatory or biosynthetic genes for the production of valuable secondary metabolites in plant roots.
