Detailed Description and Protocols

TransBacter™ focuses on the use of bacteria outside the genus Agrobacterium for gene transfer to plants.

CLEANED UP Tobacco.Sme3R.plant12.seedlings

^{Segregating population of progeny from Nicotiana
transformed with

Sinorhizobium meliloti containing
pCAMBIA1105.1R.
Photo taken by Dr Brian Weir, CAMBIA.}

Since the discovery in the 1970's that Agrobacterium tumefaciens is capable of transferring genes to plants, it has become the most important tool in plant biotechnology. It is also widely considered to be the only bacterial genus capable of transferring genes to plants. We have shown that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to diverse plants. Bacteria from two families, and three genera, Rhizobium sp. NGR234, Sinorhizobium meliloti and Mesorhizobium loti, were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a binary vector (info). Stable transformation of three plant species, tobacco, rice and Arabidopsis was achieved with these non-Agrobacterium species.

Transformation was performed using minor modifications of published protocols, using leaf disk (info), scutellum-derived callus (info) or floral dip (info) methods, respectively. The resulting plants expressed hygromycin resistance and GUS activity, contained 1-3 copies of the T-DNA by Southern blot analysis, and showed the normal range of T-DNA insertions into host genomes by 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 (info), selective plating (info), and use of a tagged binary vector (info) 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-transmissable, 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 timescales.

This alternative to Agrobacterium-mediated technology may provide two distinct advantages:

It may lead to better use of natural bacteria-plant interactions, for instance with benign epiphytic bacteria, to achieve more efficient and optimal plant transformation for species that have proven challenging.
The cumbersome patent thicket that surrounds Agrobacterium gene transfer technology has rendered this tool largely unusable for most companies, institutions and individuals for commercial purposes. The patents in the thicket explicitly refer to and claim Agrobacterium. Our observations lead to a comprehensive 'work-around' to these patents, as the species now capable of gene transfer are very distinct from Agrobacterium.

This new gene transfer technology is freely available here through this novel open-source inspired BIOS license that will encourage ethical, shared and transparent development and use of the technology (Nature 431: 494, 2004). It is envisaged that this will help to break the dominance of plant transformation by a few multinational companies and will forge a highly cooperative and public-spirited technology development process. This step towards independence from corporate control of enabling technology may foster diverse applications of biotechnology to emerge with a focus on public good and low-margin priorities typical of the needs of developing world agriculture, production environments and economies.