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Researchers Report Data on Use of RNAi to Alter Linseed Oil Content

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NEW YORK (GenomeWeb) – Amid increasing use of RNAi as a crop-modification technology, a research team led by scientists at Australia's Commonwealth Scientific and Industrial Research Organization has published a report detailing the use of the gene-silencing technology to boost levels of a preferred oil in flax seeds.

The work, which appeared in Plant Cell Reports, demonstrates that RNAi can effectively inhibit target gene expression in flax in a tissue-specific manner, with the effects strongly and stably passed on to subsequent generations. It has also laid the groundwork for similar efforts in other crops, leading to a newly developed high-oleic safflower oil.

Flax is a commercially important crop with its fiber used to manufacture items such as linen, rope, and canvas, and its oil has long been used in industrial products such as paints and varnishes.

Traditionally, efforts to use linseed oil for food applications have been hampered by its high levels of alpha-linolenic acid (ALA) — about 50 percent — which is susceptible to oxidation and quickly turns rancid.

To address this, in the early 1990s researchers from CSIRO and grain handling firm Viterra bred a form of flax called solin, trademarked as Linola, that is high in linoleic acid but with very low ALA content — about 2 percent — allowing it to remain edible after storage and opening up the use of flaxseed oil in foods including margarine and salad dressing.

But in recent years, there has been a shift in consumer demand away from polyunsaturated fatty acids, such as linoleic acid toward healthier monounsaturated fatty acids (MUFA) such as oleic acid, CSIRO researcher Allan Green told GenomeWeb in an email. Due to this trend, Linola is no longer commercialized.

"Additionally, high oleic oils also have important industrial applications due to the higher oxidative stability," Green and his collaborators from Viterra wrote in Plant Cell Reports, while purified oleic acid is also a valuable industrial chemical feed stock. Further, a high oleic oil crop could serve as a platform for engineering other valuable fatty acids.

Various groups have made attempts to boost oleic acid while cutting linolenic acid in different oilseed crops by targeting its two delta-12 fatty acid desaturase 2 (FAD2) genes, which encode a fatty acid biosynthesis enzyme that converts oleic acid into linoleic acid.

"It is present in all plants and is expressed throughout the plant to enable synthesis of polyunsaturated fatty acids needed for cellular membrane structure and function," Green noted in his email. "In seeds that accumulate oils as their energy storage reserve [including flax], the Fad2 gene is highly expressed. Silencing this gene prevents the conversion of oleic to linoleic, leading to the shift in oil composition to high-oleic type."

In 2010, researchers from the University of Missouri demonstrated this approach in soybeans, showing that oleic acid content in the plant could be raised to 80 percent — a benchmark for commercial applications — by combining FAD2 mutations to reduce the expression of the normal FAD2 enzyme.

This, however, required the generation and mapping of mutant alleles in each gene, as well as the combination of multiple mutations to achieve the best results — an especially tedious process since oleic acid levels are controlled by multiple FAD2 gene family members in soybeans, according to the Plant Cell Reports paper.  

Having successfully used RNAi to develop high-oleic cottonseed oil, Green and his team set out to use the technology to silence FAD2 genes in solin in hopes of revitalizing its use with an improved MUFA profile.

To do so, the researchers created hairpin RNAs against both flax FAD2 genes, which were delivered to the flax plant using a seed-specific expression cassette. As Green noted, FAD2 expression is required for normal plant function, and so it is critical that hairpin expression be limited to seeds.

Delta-12 desaturases were suppressed in the developing seeds of the transgenic plants, resulting in oleic acid levels above 80 percent, along with a corresponding reduction in linolenic acid. Notably, these plants appeared phenotypically normal and seeds from them had normal germination and seedling development. This effect was also passed along to subsequent generations without any evidence of diminution of phenotype.

Despite the promising outcome, Green said that commercialization of this newly modified solin is on hold given the current resistance to genetically modified oils — a particular issue when it comes to foods.

Still, the work suggests that RNAi-modified flax could serve as a platform for the production of a variety of "unusual fatty acids, such as ricinoleic acid [and] dihydrosterculic acid, for other applications," the study's authors concluded in their paper.

Meanwhile, Green and collaborators have been applying their FAD2-silencing approach to crops other than flax, and recently developed safflower oil with oleic content hitting 95 percent, he told GenomeWeb.

"This resulted in an oil with the highest known purity of a single fatty acid, and we are entering commercial development of this product at the moment," with a particular focus on the industrial chemical and lubricant markets, he added.

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