Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat

Abstract

Immature embryos (IEs) of barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) were co-cultivated with Agrobacterium tumefaciens and the transfer of T-DNA to the cereals was assessed by analysis of the expression of auidA reporter gene. This approach revealed that T-DNA transfer is preferentially targeted to distinct anatomical regions of the cereal embryos that have a low capacity for regenerative growth. This preference may limit the efficiency of generating genetically transformed plants. With the aims of maximizing the frequency of T-DNA delivery and altering the preference of delivery in favour of regeneration-competent areas of IEs, various parameters of the inoculation and co-cultivation procedure were evaluated. It was found that controlling the density of the Agrobacteriuminoculum and/ or the concentration of the Murashige and Skoog (MS) basal salts in the medium produced changes both in the total number and relative distribution of T-DNA transfer events, and these changes may have positive implications for the production of stable transformants. The use of pre-cultured embryos were also important for T-DNA targeting.