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

Abstract

ABSTRACTA current bottleneck in the functional analysis of the emerging parasitic model plant Cuscuta and the exploitation of its recently sequenced genomes is the lack of efficient transformation tools. Here, we describe the development of a novel highly efficient Agrobacterium-mediated transformation protocol for Cuscuta reflexa based on the parasitic structure referred to as adhesive disk. 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. Co-transformation 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 possibility for development of transformed cells into callus.ONE SENTENCE SUMMARYA protocol that yields high numbers of transformed cells in the adhesive disks of Cuscuta reflexa upon exposure to agrobacteria brings closer the vision of generating genetically modified Cuscuta.