Preventing transgene escape with RNAi

By Graeme O'Neill
Friday, 13 June, 2008

There are no genetically modified triffids stalking suburbia, and no unkillable "superweeds" choking the farm. But for nervous consumers in Australia and around the world, the spectre of transgene escape from GM crops looms larger than any real-world threat.

More than a decade of anti-GM scaremongering has persuaded many consumers that transgenic crops pose unacceptable risks to their long-term health, and to the environment, scientific and epidemiological evidence to the contrary.

In a study published in Science in 2002, Dr Mary Rieger of the Australian Weeds Cooperative Research Centre in Adelaide, showed the risks of transgene introgression into weedy crop relatives creating "superweeds" were very low.

Rieger found that hybridisation between GM herbicide tolerant canola and the wild radish (Raphanus raphanistrum) in the field is very rare - among 53 million seedlings raised from Roundup-Ready canola, she found only two herbicide-tolerant hybrids.

The public's concerns are twofold: that pollen from transgenic crops will contaminate nearby conventional and organic crops, and that GM crops will hybridise with weedy relatives, creating intractable superweeds.

GM crops have now been grown for more than a decade, with no adverse episodes for human health, no greater impact on the environment than their non-GM counterparts, and, in most cases, real environmental benefits.

But the anti-GM movement's concerns have eroded consumer confidence in GM crops and foods, and there are good commercial reasons to devise an effective way of preventing transgene escape through wind-blown or bee-borne pollen.

Physical containment of transgenic crops and their pollinators, or the use of extended buffer zones to isolate GM crops from conventional and organic crops, are costly and impractical. Gene technology itself proffers a low-cost genetic solution.

At CSIRO Plant Industry's Merbein research laboratories in north-western Victoria, Professor Steve Swain, Dr Davinder Singh and Dr Angelica Jermakow have devised an ingenious, broadly applicable strategy to prevent transgene escape from GM crops.

It exploits a combination of post-transcriptional gene silencing - RNA interference - and "gene imprinting": natural, methylation-induced suppression of particular genes, according to whether they are inherited from the female or male parent.

The CSIRO researchers have demonstrated their system in an Arabidopsis model. A basic difference from the Technology Protection System developed in the US in the late 1990s is that the transgenic plants still produce viable seed - provided similarly-engineered cultivars cross-pollinate with each other.

If GM pollen is transferred to a non-GM crop of the same species, or a weedy relative, fertilisation will fail and no viable seed will result.

The system would prevent contamination of non-GM crops, prevent transgenic crops hybridising with weedy relatives, yet still allow farmers to save GM seed for replanting the following season.

In the case of crops like maize and sunflowers, farmers have not saved seed since the 1930s because F1 hybrid seed does not "come true" to type. After initial resistance to the new-fangled hybrids, farmers gave up saving seed in return for the superior yield, disease resistance and profitability of F1 hybrids.

The Merbein research team's new system addresses two of the anti-GM movement's major objections to GM crops: there would be no "contamination" of non-GM crops, and no superweeds.

But because the solution to these objections depends on the very technology to which anti-GM activists object it seems unlikely that the anti-GM movement would endorse its use.

There would also be an issue if companies developing GM crops would have to find a way to levy fees for re-use of their proprietary technology if farmers saved GM seed from season to season.

---PB--- Pollen and ovule

The approach involves introducing two transgene constructs into the selected crop or cultivar, attached to promoter sequences from early-acting developmental genes expressed in the endosperm of the developing seed, with an essential role in seed development.

The two genes are from the MEDEA (or MEA) polycomb gene group in Arabidopsis, which are also known as Fertilisation Independent Seed 1 (FIS1), and Fertilisation Independent Seed 2 (FIS2).

One transgene construct comprises the protein-coding sequence of a seed-lethal gene - yet to be selected - under the control of the MEA promoter. For demonstration purposes, Swain's team used the GUS reporter gene as a proxy.

The second transgene uses the FIS2 promoter to drive expression of a so-called hairpin gene, designed to silence expression of the seed-lethal gene via RNA interference. The construct codes for a double-stranded hairpin RNA molecule that programs the plant's cells to destroy the messenger RNA of the seed-lethal gene, blocking synthesis of the encoded protein.

The ingenuity of the approach lies in the fact that, in the MEA:GUS construct, the MEA promoter begins to drive expression of GUS 48 hours after pollination when it is inherited from the male (pollen) parent.

If it is inherited via the female (ovule parent), it is expressed before fertilisation occurs, and again after pollination.

In contrast, the FIS2:GUS construct, designed to silence GUS, is expressed throughout seed development when maternally inherited, but is repressed by imprinting when inherited from the pollen parent.

On self-pollinated plants or transgenic plants fertilised by pollen from non-transgenic plants, the seeds develop normally because the FIS2:hairpin gene prevents the seed-lethal gene being expressed throughout seed development.

But if transgenic pollen finds its way onto the flowers a non-transgenic crop, or a weedy relative, seed development aborts because the non-GM seed parent has no inbuilt RNAi defence against the MEA:seed lethal gene carried by GM pollen.

One of the intriguing aspects of the system is that the system would not prevent fertilisation if the GM crop were fertilised by stray pollen from a non-GM crop, or by weedy relatives. In the first instance, this could make non-GM farmers, including organic farmers, liable for "contaminating" GM crops and compromising farmers' ability to save and re-sow GM seed for which they have paid a premium.

This would turn a key strategy of the anti-GM movement on its head: the threat of legal action if pollen from GM crops "contaminates" conventional and organic crops, compromising their GM-free status, which according to activists, attracts a premium in international markets.

Swain says the CSIRO system could be adapted to prevent non-GM pollen fertilising GM crops.

But in practice, GM farmers would be unlikely to require this type of protection against non-GM pollination, because the Rieger study has shown that cross-pollination between weeds and GM crops is extremely rare, and would involve no risk to human health.

And in a field crowded with self-pollinating GM plants, the likelihood that pollen from any weedy relative, or even a nearby non-GM crop, will pollinate a single GM flower, is very small, and would poses no credible threat to human health.

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