Agrobacterium tumefaciens-Mediated Genetic Transformation of the Cardenolide-Producing PlantDigitalis minorL.

Abstract

A repeatable transformation system has been established for Digitalis minor using Agrobacterium tumefaciens. Leaf explants from 30-day-old seedlings were inoculated with either EHA105 (carrying the nptII and gusA genes) or AGL1 (with the bar and gusA genes) strains. Among the tested factors influencing T-DNA transfer to plants, the EHA105 strain and the addition of acetosyringone to the co-culture medium increased transformation. The highest transformation efficiency (8.4 %) was obtained when freshly isolated explants, soaked in a bacterial suspension with an OD₅₅₀ of 0.9, were subcultured on selection medium after a 4-day co-culture with the bacteria. Evidence of stable transgene integration was obtained by PCR, growth on media selective for nptII or bar genes, and expression of the gusA gene. Southern hybridisation, performed in six plants, provided information about the number of inserts. More than 200 transgenic plants were recovered from 65 independent explants. Thirty of these plants were successfully established in soil. This is the first report on transgenic Digitalis spp plants using an A. tumefaciens-mediated leaf disc transformation procedure. AS:acetosyringone BA:benzyladenine CEF:cefotaxime GEN:geneticin IAA:indoleacetic acid KAN:kanamycin MS:Murashige & Skoog medium (1) PPT:phosphinothricin