genetically modified soybeans to produce high oleic acid

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Soybean (Glycine max) is a leguminous plant originally native from East Asia, and their seeds contain approximately 20% of oil and 40% of protein. The major producers (90%) of soybean worldwide are: the US, Argentina, Brazil and China. The major uses of soybean include: human oil consumption, source of protein for livestock feed and as a biodiesel source.

Soybean seeds contain five different types of fatty acids: palmitic acid (16:0 - 12% of total fatty acid content), stearic acid (18:0 - 4%), oleic acid (18:1 - 23%), linoleic acid (18:2 - 53%), and linolenic acid (18:3 - 8%). The high percentage of linoleic and linolenic acid fatty acids results in an oxidative instability of the oil, which leads to rancidity, off-flavours and decreased shelf-life of food products (Clemente and Cahoon, 2009; Kinney, 1994). To avoid oxidative rancidity, the soybean oil industry partially hydrogenates the oil, reducing the polyunsaturated fatty acid content below 18%. Oil hydrogenation involves the addition of hydrogen atoms to the double bonds of the unsaturated fatty acids. A major limitation is that partial hydrogenation generates trans-fatty acids predominantly, which have been linked to cardiovascular diseases (Danaei et al, 2009).

One solution to this problem is to change the fatty acid composition of soybeans using biotechnology. Pioneer Hi-Bred International, Inc., a DuPont Company, generated genetically modified soybeans which produce an increased level of oleic acid in the seeds, with a concomitant reduction in the levels of linolenic and linoleic acid. The advantage of this manipulation is that the soybean oil produced is more stable and therefore, it does not require partial hydrogenation. On September 2008, Food Standards Australia and New Zealand (FSANZ) received an application from Pioneer Hi-Bred requesting the permission to sale and use of food derived from high oleic acid soybean line DP-305423-1.

Synthesis of fatty acids.

The synthesis of fatty acids in plants occurs in plastids, and it involves the repeated elongation, by two carbon units, of malonyl to sixteen (palmitic acid) and eighteen carbon units (stearic acid) (Durrent et al, 2008). The first desaturation step also occurs in the plastid, catalysed by stearoyl-CoA desaturase and forms oleic acid.

These fatty acids are esterified to acyl carrier proteins (ACP), and then released by thioesterases so they can be transported to the endoplasmic reticulum for more desaturation. The conversion of linoleic acid from oleic acid, is catalysed by a microsomal ω-6-desaturase, encoded by the GmFad 2-1 gene, which is constitutively and specifically expressed in the seeds (Heppard et al 1996). The introduction of a third double bond, conversion of linolenic to linoleic acid, is catalysed by ω-3 desaturase, which is encoded by the GmFad 3 gene.

Development of genetically modified high oleic soybean.

Soybean 305423were transformed by the insertion of two linear DNA fragments. The first fragment, PHP19340A (2,924 bp), was excised from plasmid PHP 19340, using the Asc I restriction enzyme and purified using agarose gel electrophoresis (Fig. 2). The PHP19340A fragment contains the Gm-fad 2-1 gene, and the regulatory components necessary for its expression derived from the Kunitz trypsin inhibitor gene 3 (KTi3), also from soybean: a KTi3 promoter and KTi3 terminator sequence (Jofuku and Goldberg, 1989).

The gm-fad 2-1 fragment has a sequence that is identical to a portion of the coding region of the endogenous soybean fad2-1 gene, which codes for the ω-6-desaturase, and to the fad 3 gene, which codes for the ω-3-desaturase enzyme (Heppard et al., 1996). Upstream initiation of transcription of the gm-fad2-1 gene by the promoter KTi3 causes a silencing effect on expression of endogenous fad2-1 gene in 305423 soybean seed. This results in the inhibition of conversion of oleic acid to linoleic acid, and increased levels of monounsaturated (oleic) acid in soybeans seeds.

This regulatory strategy to the expression of the endogenous fad2-1 gene is known as "gene silencing", which is mediated by co-suppression. In this case, the introduced fragment leads to an overproduction of sense mRNA, which in turns leads to production of dsRNA. This results in the degradation of RNA from both the gm-fad2-1 gene partial sequence and the endogenous fad2-1 gene.

The second fragment PHP 17752A (4,512 bp), was obtained from plasmid PHP17752, excised using the AscI restriction enzyme, and purified by gel electrophoresis (Fig. 4). It contains the gm-hra gene (1971 bp), which is an optimized form of the endogenous als gene from soybean, with transcription regulated by the S-adenosyl-L-methionine synthetase (SAMS) constitutive promoter, and terminated by the endogenous als gene terminator. The insertion of the gm-hra gene produces a modified form of the acetolactate synthase (ALS) enzyme, which is tolerant to a wide range of ALS-inhibiting herbicides. This gene was generated by mutagenesis of the endogenous soybean als I gene, resulting in the substitution of two amino acids in the gm-hra protein sequence (Falco and Li, 2003). This tolerance to ALS-inhibiting herbicides makes it possible to use it as a selectable marker for the transformation in 305423-1 soybean.

The soybean cells were transformed using the biolistic method ( Klein et al, 1987). This means that both fragments, PHP19340A and PHP17752A, were adsorbed to the surface of microscopic gold particles and bombarded into soybean somatic embryogenic cells using a particle gun. In this manners, the fragments were incorporated into the soybean chromosomal DNA. The bombarded soybean tissue is then transferred to a culture maintenance medium for recovery, and after a few weeks the tissue was transferred to a culture maintenance medium containing chlorsulfuron to select presumed transformants.

After several weeks in the selective medium, chlorsulfuron resistant tissue was obtained, and samples were taken for molecular analysis to confirm the presence of the gm-fad2-1 and gm-hra transgenes by Southern analysis. Transformed tissues were selected and used for plant regeneration and seed production.

The transformed plants that incorporated the introduced gene fragments and had a good agronomic performance were selected for a breeding program. During the breeding programme the plant was selfed or crossed with a number of elite Pioneer cultivars to ensure that the 305423 germoplasm was incorporated into a wide range of soybean cultivars.

Using the Southern blot technique, the number of insert copies and their integrity was evaluated. The probes used were homologous to the promoter and terminator regions for each of the inserted fragments, PHP 19340A and PHP17552A. One intact copy of the PHP19349A fragment, containing the complete KTi3 promoter, gm-fad2-1 partial gene sequence and KTi3 terminator was found to be inserted into the genome of the 305423 soybean. Same results were obtained for the PHP17752 fragment.

The generational stability of the fragment was also evaluated and concluded that the introduced DNA fragments were stable across multiple generations (REF).

Comparison between non gm-soybean and gm-soybean.

Rigourous safety tests has been required before the use of soybean 305423-1 is allowed to use or sale for human or animal consumption. In Australia, food containing high oleic acid soybean 304523-1 has been evaluated according to the Safety Assessment of Genetically Modified Foods guidelines developed by FSANZ. First, a compositional analysis was required. For this, seeds from non-gm soybean and 305423 soybean were analysed for protein, fat, ash, acid detergent fiber, neutral detergent fiber, fatty acids, amino acids, isoflavones and antinutrients. The major difference between the two types of soybean was in the fatty acid content. As expected, the 305423 soybean had a higher level of oleic acid (76.5%) compared to the non-gm soybean (21%). In addition, the mean level of linoleic acid in the non-gm was 52.5 % whereas in the 305423 the meal level was 3.6%. There were no significant differences in the levels of protein, ash, fiber, amino acid content, isoflavones and antinutrients between the two types of soybean tested.

The nutritional quality of the 305423 soybean was assessed by comparing diets containing the gm-soybean and non-gm soybean in a poultry feeding study, which lasted 42 days (REF). Results from this study revealed that there were no significant differences in the mortality, body weight gain, feed efficiency, organ yield, carcass yield, breast, thigh, wing and leg yield and abdominal fat of the chickens fed with 305423 soybean compared to chickens fed non-GM control soybean diets.

The potential toxicity of the gm-hra protein was assessed by comparing the amino acid sequence similarity between the gm- and non-gm protein, using the National Center for Biotechnology Information (NCBI) Protein dataset. No significant similarities between the gm-hra protein and toxic proteins were found. Similar results were obtained when the gm-hra amino acid sequence was compared with known allergenic proteins, using the Food Allergy Research and Resource Program (version 6.0) database.

Based on the comparisons between the gm and non-gm soybeans on the compositional analysis, nutritional studies, potential toxicity and allergenicity of 304523 soybean, it was concluded that the 304523 soybean was safe for both, human and animal consumption.

Approval of Soybean line DP-305423-1 in Australia and other countries.

Once the safety assessment indicated that there were no public health nor safety concerns asscociated with the use of genetically modified high oleic acid soybean line 305423, the FSANZ directory decided on December 2009, to approve the variation to Standard 1.5.2 - Food produced using Gene Technology, to include food derived from high oleic acid soybean line DP-305423-1 in the Table to clause 2.

Soybean line DP-305423-1 has also been approved in: the United States by the Food and Drug Administration, Department of Agriculture (2009), Canada by the Health Canada and the Candian Food Inspection Agency (2009), Japan by the Ministry of Health, Labour and Welfare, Ministry of Agriculture, Forestry and Fisheries (2009), and Mexico (Ministerio de Agricultura) (2008). Currently there are submissions (NL-2007-45) for food import approvals of soybean 305423 line in the European Union

Consumer and producer opinions to the specific GM food

Currently, most of the food has been genetically modified to have resistance towards pesticides or certain diseases, or to increase tolerance towards the cold, drought or high salinity conditions. Therefore, genetically modified food offers great advantages to the producer, but little advantages to the consumer. In addition, consumers in general, have a negative perception of genetically modified foods. The major concerns relate to the unintended harm to other species, gene transfer to other species, development of resistance to pesticides, and the unknown effects to human health. However, the development of high oleic acid soybean line 305423 is one of the first genetically modified crops to offer nutritional advantages to the consumer, and this might change the public perception of genetically modified foods in the future.

From a nutritional point of view, the consumption of high oleic acid has been shown to have positive effects on total cholesterol levels when compared to equal intakes of hydrogenated oils, therefore their use is recommended for the prevention of cardiovascular diseases (Lichtenstein et al. 2006). Consumption of high oleic fatty acid has also been shown to decrease systolic blood pressure (Bondia-Pons et al., 2007). Another advantage is that partial hydrogenated oils have a high level of trans fatty acids. Metabolic and epidemiologic studies indicate that the consumption of trans fatty acids adversely affects blood lipid levels, and are considered a risk for cardiovascular disease (Ascherio et. al., 1999).

Positive and negative effects that may be associated with producing, processing or consuming the specific GM food.

Currently, there are no plans for cultivation of soybean line 305423 in either Australia or New Zealand. The Application only refers to the use of high oleic acid oil in the food industry, to be used in frying foods such as french fries, fried chicken, potato chips, tortilla chips and salad oil.

The use of high oleic acid oil derived from soybean line 305423 offers several advantages to the food industry. The major one is that it is resistant to oxidation, and therefore, it is suitable for frying applications without the production of off flavours or off odours during the cooking process (REF).