Salt of the earth

With global climate models projecting a temperature rise of one to four degrees by mid-century, evaporation rates will rise, and rainfall is likely to decline by a further 20 per cent across Australia's drought-ridden south-east.

These climatic trends will intersect and ramp up salt concentrations in wheatbelt soils that, in many regions, already have a toxic layer of ancient leached salt just below the root zone. Moisture and nutrients below this layer are inaccessible to the crop.

For the fortunate few blessed with low-salinity soils, high-protein durum wheat may require a little extra nitrogen to achieve the high protein levels prized by pasta manufacturers, but profits usually match or exceed those from bread wheat.

But durum varieties, which are all tetraploids, are less tolerant of salt than hexaploid bread wheats, or barley, and the sodium ions in sodium chloride cause yield depression, eroding any price advantage over bread wheat.

At CSIRO Plant Industry in Canberra, Dr Rana Munns' plant physiology research group has discovered a sodium-tolerance gene that promises to provide durum growers with genetic insurance against salinity, and which could see durum wheat plantings expand into mildly salinised regions of southern NSW, Victoria, SA and WA.

The CSIRO team owes its original discovery to veteran NSW Primary Industries durum wheat expert Dr Ray Hare, at Tamworth, home of the Australian National Durum Wheat Improvement Program.

Rana Munns and Ray Hare have been friends since they were teenagers growing up in the same suburb in Sydney.

In 1998, Munns and Hare were discussing the problem of durum's lack of salt tolerance. Hare envisaged a day when durum wheats with improved salt tolerance would be grown in moderately salinised soils across southern Australia.

Munns asked Hare to provide her with selections from the durum wheat seed bank in Tamworth. Hare took 64 of them to Canberra for screening for salt tolerance, with no foreknowledge that they would find any significant variation.

They were surprised when their tests identified several lines that actively excluded sodium ions from their roots. They had very low sodium levels in their leaf tissues.

In the early 1970s, Hare and Dante The undertook their PhD research projects at the Plant Breeding Institute at the University of Sydney. Dante The was attempting to improve the resistance to stem rust of hexaploid wheats by transferring a resistance gene from a land race of the primitive diploid wheat, einkorn (Triticum monococcum).This gene transfer was achieved via a series of crosses, firstly monococcum to tetraploid durum wheat. Then the resistant durum progeny were crossed to bread wheat.

The intermediary resistant durum lines inadvertently carried the sodium exclusion genes from the monococcum and it is these lines that have been used by CSIRO.

Einkorn wheat

Archaeologists have found 9000-year old carbonised grains of einkorn wheat in the Karacadag Mountains of Anatolia, in Turkey, confirming that Neolithic farmers domesticated einkorn wheat from a large-seeded wild grass, Triticum boeoticum, not long after the last glacial period.

Dante The successfully crossed the einkorn wheat with two rust-susceptible durum varieties, Marrocos and Glossy Hugenot.

He lodged some of his project seed lines with the durum seed bank in Tamworth, where, more than three decades later, Hare remembered them and included several among the 64 varieties to be screened by Munns' group.

Einkorn was known to be resistant to stem rust, because it was one of a dozen wheat types that pioneering US plant pathologist Professor E. C. Stakman selected for his celebrated "macroarray" of standard, rust-resistant wheat lines 75 years ago.

Stakman's "Holy 12" carry unique combinations of resistance genes. By dusting them with rust spores, breeders can assess the virulence of emergent rust strains, and whether their breeding lines will possess resistance. Einkorn was one of three primitive, diploid progenitors of today's hexaploid bread wheats.

An improbable genetic collision in a Neolithic farmer's field somewhere in the Fertile Crescent at least 6000 years ago combined the entire genomes of jointed goat grass (Triticum tauschii), a diploid, and a tetraploid spelt-like wheat (probably Triticum dicoccum), itself the product of an earlier merger between einkorn wheat and an as-yet unidentified diploid wild grass.

While the A, B and D genomes share a nucleus, and collaborate functionally, they retain their separate chromosomal identities.

When a tetraploid durum wheat is used as the female parent, and its inflorescence is dusted with pollen from diploid einkorn, their chromosome counts mismatch, leaving half of the durum chromosomes unpaired. Yet the hybrids produce viable seed - this was how The developed his einkorn/durum hybrids.

---PB---

Goat grass' gift to civilization was a suite of glutenin and gliadin alleles that give rise - literally - to the remarkable extensibility of bread wheat dough, and modern bread's soft, airy texture.

Munns says the tauschii genome also endowed bread wheat with a gene controlling sodium transport in root cells that selectively admits potassium (K) ions while excluding sodium (Na) ions.

Kna1 is the source of bread wheat's superior salt tolerance, while its absence explains durum wheat's vulnerability to sodium toxicity.

Dante The wasn't looking for salt-tolerance in his "durum" lines, because he was selecting for rust resistance. He could not have known there was a sodium-exclusion gene on the same chromosome segment - nor that there was a second one on a neighbouring chromosome.

Breeders identified the Kna1 locus in the early 1990s, but the gene remained elusive, because it is present in all bread wheat cultivars, and no deletion mutants were available for comparative DNA analysis.

The's salt tolerant einkorn/durum hybrids provided a crucial clue that allowed Munns and her colleagues to pinpoint and clone the gene from bread wheat, by first cloning its homoeologue from the synthetic 'durums'.

The first gene from the synthetic 'durums' came to light four years ago, as a quantitative trait locus (QTL) contributing to salt tolerance. The gene was called Nax1, for Na exclusion. A second gene was discovered on a neighbouring chromosome, and dubbed Nax2.

"We knew that Nax2 from the 'durum' and Kna1 from bread wheat were physiologically equivalent," Munns says. "We got hold of some chromosome deletion lines, and a gene very similar to Nax2 was found on the same chromosome as the gene known as Kna1. It was even on the same segment of chromosome, so Nax2 is the einkorn homoeologue of Kna1 from the D genome in bread wheat.

"We published it eight months ago in Plant Physiology. We now have a set of three genes - two in the durum wheat, and an extra one in bread wheat. "These are three major salt-tolerance genes in wheat, and physiologically, that's the pathway to improving bread wheat's salt tolerance as well."

Roots to shoots

The CSIRO researchers have already inserted extra copies of Nax1 and Nax2 into bread wheat, using advanced conventional breeding techniques, and will conduct field trials this year to determine if there is an additive effect on salt tolerance.

Nax2 and Kna1 are 94 per cent similar at the DNA sequence level, and Munns' team was not expecting that Nax2 would provide any additive benefit in salt tolerance, because the combined effect would merely be equivalent to over-expressing Kna1.

So they were surprised - and delighted - to find that the plants had 25 per cent less sodium in their leaves than normal bread wheat, equivalent to adding a second, independently acting gene to the three sodium-tolerance genes already present in bread wheat.

So what does Nax2 do differently to Kna1? Using a sodium isotope to track the movement of sodium ions from roots to shoots, Munns' team found that both sodium-transporter molecules were selectively removing sodium ions from nutrients absorbed by the roots before the solutions were sent to the shoots.

"Kna1 and its sister Nax2 works in the older tissues of the roots, pulling sodium out of the water moving up the xylem vessel," she says. "Nax1 also operates in the roots, but it works at the level of the shoots as well. Nax1 removes any residual sodium that makes it to the base of the leaf, so virtually no sodium enters the shoot."

Some of the sequence differences between the genes lie in the promoter region, which may explain why Nax1 is expressed both in roots and shoots.

Munns' team is attempting to over-express the genes in experimental, transgenic barley lines to see if they improve the plants' salt tolerance.

"Barley is already highly salt tolerant, but through a different mechanism," Munns says. "It's not a sodium excluder, and has a high rate of sodium transport to the shoots, so it sequesters sodium instead of excluding it.

"We thought it would be interesting to see if a complementary, exclusion mechanism would improve barley's salt tolerance."

Four years ago the discovery of a QTL for salt tolerance allowed Munns to mark the anonymous gene, so breeders could track it through a conventional hybridisation program - a much quicker and more reliable alternative to measuring sodium concentrations in hybrids.

Collaborating breeders have introduced the gene into a number of durum lines, which are being trialled at a number of sites in NSW, under Ray Hare's supervision. Hare says few wheatbelt soils are free of salt. He cites the case of farmer who planted a sorghum crop on the deep black soils of northern NSW, which are at least four metres deep.

The farmer was astonished when the apparently healthy crop ran out of water and died. Even though there was water deeper in the soil profile, it was inaccessible to the crop because of the presence of a saline layer about a metre beneath the surface.

Similar saline layers occur, usually at much shallower depth, in wheatbelt soils across southern Australia - both durum and bread wheats would benefit from increased salt tolerance.

"Even if you could give growers a five per cent yield increase on these soils, by enhancing their ability to access saline groundwater, it would help," Hare says.

"Rana's glasshouse work shows this is quite achievable. It would be a partial solution to the salinity problem. What we really need is enough rain to wash the salt further down in the soil profile."

Hare believes the transfer of the einkorn Nax1 gene to bread wheat, via The's 'durum' hybrids, exemplifies the enormous potential to improve bread wheat through hybridisation with tetraploid varieties.

He is most interested in east Asian tetraploids, from countries like Korea, China and Japan, which have been isolated from bread wheat's centre of origin in Asia Minor for millennia.

"East Asian tetraploids seem to share a mutation that is absent from western varieties, that facilitates gene flow when they are hybridized with bread wheat," he says. "Chinese spring wheats are widely used in cytogenetic research, because they're so easy to cross."

Salt tolerance is one thing, drought tolerance another. Munns says early single-gene experiments with transgenic wheats have shown that drought hardiness is genetically complex, and will be much more difficult to achieve.