A highly efficient protocol for transforming Cuscuta reflexa based on artificially induced infection sites

The parasitic plant genus Cuscuta is notoriously difficult to transform and to propagate or regenerate in vitro. With it being a substantial threat to many agroecosystems, techniques allowing functional analysis of gene products involved in host interaction and infection mechanisms are, however, in high demand. We set out to explore whether Agrobacterium-mediated transformation of different plant parts can provide efficient alternatives to the currently scarce and inefficient protocols for transgene expression in Cuscuta. We used fluorescent protein genes on the T-DNA as markers for transformation efficiency and transformation stability. As a result, we present a novel highly efficient transformation protocol for Cuscuta reflexa cells that exploits the propensity of the infection organ to take up and express transgenes with the T-DNA. Both, Agrobacterium rhizogenes and Agrobacterium tumefaciens carrying binary transformation vectors with reporter fluorochromes yielded high numbers of transformation events. An overwhelming majority of transformed cells were observed in the cell layer below the adhesive disk's epidermis, suggesting that these cells are particularly susceptible to infection. Cotransformation of these cells happens frequently when Agrobacterium strains carrying different constructs are applied together. Explants containing transformed tissue expressed the fluorescent markers in in vitro culture for several weeks, offering a future possibility for development of transformed cells into callus. These results are discussed with respect to the future potential of this technique and with respect to the special characteristics of the infection organ that may explain its competence to take up the foreign DNA.