By Shelley Jambresic, Checkbiotech

Agrobacterium tumefaciens is a naturally-occurring soil bacterium that causes tumours, or galls on plants. For a long time it was widely considered that Agrobacterium is the only bacterial genus capable of transferring genes to plants. The discovery that this gall formation is due to the integration of bacterial DNA into the plant genome laid the foundations of plant biotechnology.

This ability to transfer genes from bacteria to plants has been widely exploited by researchers for genetic engineering and plant improvement.

Transgenic plants, like genetically modified maize, cotton or soy, are plants that have been genetically engineered by adding one or more genes to the plant’s genome. The majority of transgenic plants cultivated for commercial use have had genes added which confer resistance to insects and/or herbicides.

Most of the technologies involved, however, are ‘protected’ by hundreds of patents worldwide, many of which are held by multinational corporations. The complexity of the patent landscape has formed very serious ‘real and perceived’ obstacles to the effective use of these technologies for agricultural improvements, both by small-medium enterprise in the industrialized world, and by the developing countries. The unfortunate industry consolidation this leads to has caused a lack of public trust and transparency, which is a root of the public disquiet about GMOs.

Most people think patent licenses are readily obtained by purchasing a license. “The costs are not just simple financials,” says Dr Richard A. Jefferson from CAMBIA, a private international non-profit institute in Australia.

“Transaction costs – the time, money, energy and emotion expended to even get to the negotiating table – can defeat a project before it starts. Further, many patents are simply not being licensed, and only one patent right withheld can block progress.”

To address this problem, CAMBIA is creating new tools and technologies to support innovation and collaboration in the life sciences. One of CAMBIA's major activities is the BIOS Initiative (Biological Innovation for Open Society), which has parallels with the open source software movement, but which is focussing on patented technologies in the life sciences. BioForge is the associated internet-based platform for collaborative research and development.

“A major goal of BIOS and the BioForge is to reduce transaction costs, and free technology and patent blockages so that the focus of innovators can be on innovation, not simply on ‘getting to the starting gate’,” Jefferson told Checkbiotech.

Jefferson and his team recently published the observation that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer (Broothaerts et al, Nature 433:629-633).

“Our technology is patented – just as Linux is copyrighted – and requires a special license for use, as does ‘free or open source software’. However, these licenses are free as long as users agree to share improvements with other users (Licensees), share all biosafety data, agree that licensees may not assert rights over improvements on other licensees.” This unique patent license, called a BiOS license, serves as a useful template for the CAMBIA-led open source movement.

Rather than using the pathogen Agrobacterium, the CAMBIA researchers used the benign plant-associated bacteria responsible for the symbiotic relationship between plants and bacteria – the symbiosis between roots and Rhizobia. Rhizobia are soil bacteria that ‘fix’ nitrogen from its inert molecular form (N2), and convert it into nitrogenous compounds useful for the plant. Jefferson and his research team modified these plant-associated symbiotic bacteria, and made them competent for the gene transfer by using ‘classical’ bacterial genetic techniques.

The DNA transferred into the plant’s cell by Agrobacterium tumefaciens is called transferred DNA (T-DNA). The T-DNA, while in the bacterium, is located on the large and complex bacterial tumour-inducing (Ti) plasmid. If the Ti-plasmid is removed from the bacteria, the tumour growth, which normally results from pathogenic infection by Agrobacterium, does not occur.

To use the Ti plasmid as a vector for inducing new genes into plants, it is necessary to disarm the plasmid so that it does not cause tumours. This task is accomplished by deleting the genes on the Ti-plasmid, which encode the enzymes controlling the synthesis of the plant growth hormones auxin and cytokinin. A cloned gene encoding a new function can then be inserted into the T-DNA region of the disarmed Ti plasmid. A more typical and modern process puts the T-DNA on a separate, readily manipulated plasmid called a binary vector.

The researchers further modified the Ti plasmid to allow it to be more easily mobilized into a diverse group of Rhizobial genera, including Rhizobum, Sinorhizobium and Mezorhizobium, and tested the gene transfer on different plant species.

“With Arabidopsis, Heidi Mitchell at CAMBIA has already achieved an efficiency about half that of the method with Agrobacterium and we expect this will readily rise with some work by others and us,” said Dr. Jefferson. “With tobacco it generates more lines than we can sensibly monitor - so that's more than enough.”

Experiments with rice were successful too, but showed lower efficiency. However Dr. Jefferson is confident, “We're a very small team. Putting our protocols and material in the BioForge, and having the community explore new bacterial strains and new conditions will together generate substantial increases in efficiency in the very near future.” Dr. Jefferson also told Checkbiotech that the team at CAMBIA has already increased the frequency above that published in the Nature article in February.

A significant advantage of the new method over the one with Agrobacterium is that Rhizobial species are not pathogens, allowing the plants to grow without the stresses associated with Agrobacterium infection. This may also allow for the transfer of genes into new target cells and tissues. Very good results were obtained using the combination of Sinorhizobium with tobacco.

“Brian Weir, in our team, discovered that young leaves are more susceptible to gene transfer by Sinorhizobium than older leaves, whereas with Agrobacterium this makes little difference,” explained Dr. Jefferson. This wide range of interactions may be an advantage in transferring genes into other, previously intractable, plant species or cell types. “With some work we anticipate the number of gene transfer competent species to greatly increase, using many benign plant-microbe interactions as a basis for such gene transfer.”

However, for Dr. Jefferson and his team the important issue is not only the efficiency or the used species, but also the whole concept of rapid and unfettered use and improvement of the technology by the larger community. “The power of ‘open source’ is in the highly-parallel, efficient and transparent development and optimisation of conditions. There is now a ready mechanism for this improvement to happen, and excellent motivation to do so.”

About his team’s future plans, Dr. Jefferson further explained, “Besides the optimization of these technologies, we’re looking at new ways of using plants as living ‘instruments’ to allow better management of nutrients and other scarce resources. We’re also going to develop programs on the BioForge to build ‘open source’ toolkits for such critical technologies as precise ‘homologous’ recombination.”

“Part of our effort over the next years will be in advocacy and communications: spelling out how technology choice, development and dissemination can have a major impact on contributions of science and technology to social equity. It also has to be made clear that such choices need not conflict with scientific curiosity, career development or productive and fair business strategies.”

Gene transfer to plants by diverse species of bacteria
Nature. 433, 629 - 633
10 February 2005