Gene transfer to plants by diverse species of bacteria: an open-source platform for plant biotechnology

Since the discovery in the 1970s that Agrobacterium tumefaciens is capable of transferring genes to plants, it has become the most important tool in plant biotechnology. It has also been widely considered that Agrobacterium is the only bacterial genus with this capacity. Here we show that several other genera of bacteria can be modified to mediate gene transfer to diverse plant species. Three bacterial species from three genera and two families, Rhizobium sp. NGR234, Sinorhizobium meliloti and Mesorhizobium loti, were made competent for gene transfer by acquiring both a modified, disarmed Ti plasmid and a binary vector. Stable transformation of three plant species—tobacco, rice and Arabidopsis—was achieved using these non-Agrobacterium species. Transformation was performed with minor modifications of published protocols, using the leaf disk method for tobacco, scutellum-derived callus for rice and floral dip for Arabidopsis. The resulting plants expressed hygromycin resistance and glucoronidase (GUS) activity, contained 1–3 copies of the T-DNA as indicated by southern blot analysis, and showed the normal range of T-DNA insertions into host genomes determined by the polymerase chain reaction (PCR)–mediated sequencing of integration sites. To ensure that the gene transfer did not result from contamination with Agrobacterium cells, controls including species-specific PCR, selective plating, and use of a tagged binary vector were implemented. Thus, diverse plant-associated bacteria, when harbouring a disarmed Ti plasmid and binary vector (or presumably a co-integrate or whole Ti plasmid), are readily able to transfer T-DNA to plants. The Ti plasmid is self-transmissible, perhaps indicating the existence of a ubiquitous natural mechanism effecting horizontal gene transfer from bacteria to plants. If so, then much of the plant genomic DNA sequence thus far determined, which seems bacterial in origin, may have derived from such transfer over recent evolutionary time scales.