GM surgery to detoxify feed crop

Molecular geneticists at Melbourne University's Institute of Land and Food Resources plan to use RNA interference gene silencing to remove a potent neurotoxin from vetch (Vicia sativa).

Hardy, and an efficient nitrogen fixer, vetch is grown in Australia as a green manure crop. Its seeds contain up to 32 per cent protein, one of the highest figures for any legume, and it is a nutritious fodder for ruminant livestock.

Ruminants digest it without harm, but the raw seeds can paralyse or even kill monogastric species like chickens and pigs -- and humans, if the seeds are not leached or boiled to remove the toxin.

Two PhD students in BioMarka, a division of the Institute of Land and Food Resources, Indonesian-born Andi Maddeppungeng and Philippines-born Annabelle Novera, are working on complementary research projects to ultimately silence the gene that causes vetch seeds to produce the toxin -- the beta-cyanoalanine synthase gene.

Their supervisor, Dr Rebecca Ford, said Maddeppungeng was developing a regeneration and transformation system for the Australian vetch cultivar 'Blanche fleur'. Novero is characterising the family of synthase genes in vetch to which the offending enzyme belongs.

"We need to develop a reliable regeneration system for future high throughput of transformants, because Vicia species are renowned for their recalcitrance in tissue culture," said Ford.

Maddeppungeng recently managed to regenerate non-transgenic plantlets through somatic embryogenesis from leaf and embryo axis tissues, and is now developing a transformation system using green fluorescent protein as a marker.

Subsequently, the plan is to transform vetch with the most appropriate part of the gene sequence, in a self-complimentary, intron-containing 'hairpin' RNA (ihpRNA) loop construct, silencing production of the neurotoxin.

But Ford said the project was complicated by the fact that beta-cyanoalanine synthase is involved in the metabolism of cysteine, an essential constituent of high-sulphur proteins required for balanced nutrition of livestock. Silencing the vetch beta-cyanoalanine synthase gene could lead to elevated levels of cysteine and cyanide in plant tissues. Also, due to their biochemical closeness, 'backup' enzymes for cysteine synthase in vetch may also be capable of synthesizing beta-cyanoalanine.

Novero has so far pulled out four members of the cyanoalanine and closely related cysteine synthase gene families from vetch using degenerate primers designed from other species such as Arabidopsis, spinach and potato.

These will be biochemically and molecularly characterised for their ability to produce the neurotoxin. DNA sequences will then be identified that could be selectively silenced by RNA interference.

In related legumes, beta-cyanoalanine synthase is normally degraded by asparaginase, but Ford says this process is blocked in vetch. Activating the asparaginase pathway might be an alternative approach to remove the neurotoxin.

If the BioMarka group succeeds in developing a non-toxic vetch, it would give wheat farmers a potentially profitable grain legume crop that would enrich fallow ground with nitrogen, could be grazed by ruminants, and the seed used for high-protein feed formulations for pigs and chickens.

Ford said similar technology could be applied to remove a different toxin, BOAA, from chickling vetch, or grass pea, (Lathyrus sativa), a grain legume widely grown in Asia and Africa, and occasionally consumed by humans as a survival food during drought or after flooding.

Chickling vetch has been responsible for several epidemics of a paralysing disorder called lathyrism in countries like Ethiopia, India and Bangladesh over the past three decades. In the early 1970s, a lathyrism epidemic during an Ethiopian famine left an estimated 2.5 per cent of the population irreversibly paralysed.

Genetic surgery to detoxify Lathyrus sativa would remove a health risk to millions of people in Africa and Asian nations.