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A clean pair of genes

Agrobacterium tumefaciens is an unusual creature. A cancer of trees, its talent lies in exploiting wounded tree trunks, infiltrating their cells, and inserting part of its DNA into the plant DNA. The infected cells start producing new transgenic substances, and these form a tumour, or gall, in the bark, where Agrobacterium lives and prospers. The bacteria, and its associated disease, crown gall, are extremely common. Similar bacteria cause the unsightly marring of grapes that plagues vine-growers across the world. But Agrobacterium's ability to incorporate its own DNA in that of another cell has also made it a useful tool of the scientist. Replacing the cancer-forming parts of the bacterium with genes that offer desirable traits, and then allowing the bacteria to incorporate these in the DNA of the crop plant, is the basis of much genetic modification.

Indelible markers

When scientists attempt to transform a cell, their chances of success are relatively small. For example, if a petri dish containing an arbitrary one million cells is exposed to a modification technique, just one cell may be successfully transformed. And how to know which one? The current method is to ensure that any cell that is successfully transformed contains not just the new desired gene (which is impossible to detect unless all the one million cells could be grown to plants and tested), but also a second, marker gene which can be detected more easily. If that marker gene gives the cell resistance to antibiotics, for example, it is easy to identify the transformed cell by simply exposing the whole dish to an antibiotic. All the untransformed cells die, and only the one or two transformed cells live on and grow.

The catch is that the transformed cells, and the plants that grow from them, not only contain the desired gene but also the antibiotic resistance gene, or whatever marker was used. Fine for the laboratory, but not for the field. The fact that many GM crops thus far developed contain unwanted genetic material, such as antibiotic resistance, is one of the key issues raised by regulatory bodies. Finding, and refining ways of producing plants that only have the desired genes, and not the unwanted markers, is the work of numerous biotechnology companies and publicly-funded research programmes.

Rooting out nematodes...

Artificially coloured nematode (red) in coloured rice root (blue) (JIC)
Artificially coloured nematode (red) in coloured rice root (blue)
JIC

The John Innes Centre (JIC) on the outskirts of Norwich in the UK is participating in one such programme, the Plant Sciences Research Programme of the UK Department for International Development (DFID). One project is focussing on making upland rice plants resistant to nematodes, the microscopic worms that bore into the roots of plants in order to feed, grow and lay eggs. Paddy rice tends to suffer less from nematodes, as regular flooding drowns the worms, but upland rice, and most other major crops, are vulnerable and suffer poor health and vigour as a result. Nematodes feed on a wide range of plant species, so crop rotations do little to reduce their prevalence. Chemical treatments are possible, but nematicides rank amongst the most toxic of pesticides, undesirable for both users and the environment, and too expensive for most farmers in the developing world.

...with clean gene technology

Nematode-resistant rice plants (JIC)
Nematode-resistant rice plants
JIC

When Agrobacterium incorporates its DNA into a plant cell, one of two things happens. Either all the DNA transferred from the bacteria is put into the same place in the plant cell, or different bits of the bacteria's DNA are put in different places within the plant cell. If they are put in different places, which happens roughly half of the time, a new set of possibilities opens up. Similar to the case of whether two brown-eyed parents will give birth to a blue-eyed baby, there is a chance that the offspring of a plant transformed with two genes, such as a nematode resistance and a marker gene, will contain only one of them. In other words the next generation of a transgenic plant can be clean of any genetic material except the desired 'gene of interest'. This happens with about two progeny plants in every hundred.

In the case of nematode resistance, the 'genes of interest' are actually genes from the rice plant itself. Rice grains, like most seeds, contain a substance called protease inhibitor. This acts as a natural protection for the plant, as it inhibits digestion in a number of pest species, although, fortunately, not in humans. Unfortunately rice roots, food source and breeding ground for the nematode, do not naturally produce this protease inhibitor. But with help from Agrobacterium a second copy of the protease inhibitor gene programmed to express in roots has been successfully introduced into rice using the clean gene method. Trials at the University of Leeds of plants bred at the JIC, have shown an 80 per cent reduction in nematode egg production, with most nematodes failing to grow and mature to the egg laying stage. Trials at WARDA in Cote D'Ivoire are presently halted by the instability there, but the JIC plants are due to be trialled at the Rice Research Institute of Hefei, near Shanghai, China. If it proves as successful in the field as the research station, nematode-resistant rice seed could be distributed to developing countries before the end of the decade.

Date published: March 2005

 

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