GM Crop Database

Database Product Description

G94-1, G94-19, G168 (DD-Ø26ØØ5-3)
Host Organism
Glycine max (Soybean)
Trait
Modified seed fatty acid content, specifically high oleic acid expression.
Trait Introduction
Microparticle bombardment of plant cells or tissue
Proposed Use

Production for human consumption.

Product Developer
DuPont Canada Agricultural Products

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2000
Canada 2000 2000 2000
Japan 2001 2000 1999
United States 1997 1997 1997

Introduction Expand

Three lines of a new variety of soybean (G94-1, G94-19 and G168), high in the monounsaturated fatty acid oleic acid, were generated by the transfer of a second copy of a soybean fatty acid desaturase gene (GmFad2-1) to a high yielding commercial variety of soybean (line A2396). The fatty acid desaturase is responsible for the synthesis of linoleic acid, which is the major polyunsaturated fatty acid present in soybean oil. The presence of a second copy of the fatty acid desaturase gene causes a phenomenon known as "gene silencing" which results in both copies of the fatty acid desaturase gene being "switched off", thus preventing linoleic acid from being synthesised and leading to the accumulation of oleic acid in the developing soybean seed.

The genetic modification affects only the seed, and endogenous GmFad2-1 gene is still expressed in other plant parts such as the leaves. These changes result in a superior, more heat stable soybean oil with improved nutritional and functional properties. High oleic soybean oil contains oleic acid levels higher than those found in olive oil and rapeseed oil.

Other genes transferred along with the GmFad2-1 gene were the uidA gene and the bla gene. The uidA gene is a colourimetric marker used for selection of transformed plant lines during the soybean transformation procedure. It codes for the enzyme beta-glucuronidase and is derived from the bacterium Escherichia coli. The bla gene is a marker used to select transformed bacteria from non-transformed bacteria during the DNA cloning and recombination steps undertaken in the laboratory prior to transformation of the plant cells. It codes for the enzyme beta-lactamase and confers resistance to some beta-lactam antibiotics, such as penicillin and ampicillin.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
gus beta-D-glucuronidase SM CaMV 35S A. tumefaciens nopaline synthase (nos) 3'-untranslated region Silenced in the lines submitted for approval.
bla beta lactamase SM bacterial promoter Not expressed in plant tissues
gm-fad2-1 delta(12)-fatty acid dehydrogenase FA

seed specific promoter from the soybean beta-conglycinin gene

3' poly(A) signal from phaseolin gene from Phaseolus vulgaris

1 locus

Native; coordinate suppression

Characteristics of Glycine max (Soybean) Expand

Center of Origin Reproduction Toxins Allergenicity

Southeast Asia; wild soybean species endemic in China, Korea, Japan, Taiwan.

Self-polinated; rarely displays any dormancy characteristics; does not compete well with other cultivated plants.

Raw soybeans contain trypsin inhibitors, which are toxin when eaten.

Soy allergies are common, and eating soy products can cause rashes and swelling of the skin in sensitive individuals.

Modification Method Expand

The soybean sublines G94-1, G94-19 and G168 were produced via biolistic transformation of the parent soybean line (Asgrow A2396) with a mixture of two plasmids pBS43 and pML102. These sublines were advanced using traditional breeding techniques to produce high oleic soybean varieties.

Plasmid pBS43 contained the GmFad2-1 gene of soybean (Glycine max) and the beta-glucuronidase (GUS; uidA) gene from Escherichia coli. The GmFad2-1 gene was inserted in the sense orientation and expression of the GmFad2-1 was regulated by a seed specific promoter from the soybean beta-conglycinin gene and a transcription terminator from the phaseolin gene from Phaseolus vulgaris. The uidA gene was used during the transformation process as a reporter gene to select plants cells which carried the introduced genes. Expression of the GUS encoding gene was controlled by the 35S promoter from cauliflower mosaic virus (CaMV) and a nopaline synthase (nos3') terminator.

Plasmid pML102 contained the dapA gene from Corynebacterium glutamicum which encodes the enzyme dihydrodipicolinic acid synthase (DHDPS). Corynebacterium DHDPS catalyzes a step in the biosynthesis of the amino acid lysine but is insensitive to feedback inhibition by lysine. Expression of this enzyme in soybean seeds results in accumulation of the essential amino acid lysine. The dapA gene is controlled by the seed specific promoter Kunitz trypsin inhibitor (Kti3) from soybean that allows high levels of expression. The dapA gene was fused to a chloroplast transit peptide sequence to direct the DHDPS protein into the chloroplast where lysine biosynthesis is carried out.

Both plasmids contained the antibiotic resistance marker, beta-lactamase (bla) gene. The bla gene confers resistance to the antibiotic ampicillin and permits the selection of transformed E. coli cells during laboratory recombinant DNA steps. The bla gene contains its own E. coli regulatory sequences and was therefore not expressed in the transformed plants.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from the original transformant (event 260-05) identified inserts at three loci which were designated as locus A, B and C. At one locus (locus A), the GmFad2-1 construct was causing silencing of the endogenous GmFad2-1 gene, resulting in seeds like G168 with a high oleic acid content only. Locus A was characterized using Southern blotting and shown to contain two copies of the GmFad2-1 expression cassette as indicated by two hybridizing bands on the Southern blot. The second locus (locus B) contained a copy of GmFad2-1 that was over-expressing, thus decreasing the oleic acid levels to around 4% (G175). This locus also contained a functioning dapA gene as evidenced by an increase in the seed lysine levels. Locus B contained only a single copy of the GmFad2-1 expression cassette as indicated by a single hybridizing band on the Southern blot. In seeds with both locus A and locus B (G94), lysine levels were increased and oleic acid levels were increased but not as high as in those with locus A alone.

Lines G94 and G168 were selected for further characterization as they contained the silencing locus A with the high oleic acid phenotype. As G94 plants contained both locus A and locus B, an additional round of selection was used on the segregating R2 plants to isolate plants containing locus A and not locus B. Southern blot analysis on R2 leaf tissue grown from G94 R2 seed identified two sub lines, G94-1 and G94-19, that contained locus A without locus B which had been removed through segregation. Locus B was not further characterized for the purposes of this application.

The three sub lines, G94-1, G94-19 and G168, identified as containing the GmFad2-1 silencing locus A, were selected as the high oleic acid soybeans for subsequent analyses. G94-1, G94-19 and G168 containing only locus A and locus C and were advanced to produce high oleic soybeans. The presence of the high oleic phenotype was the primary screen for the advancement of sublines since G94-1, G94-19 and G168 were GUS negative. These sublines were all homozygous for the GmFad2-1 encoding sequences arranged in the sense orientation.

Genetic Stability of the Introduced Trait

All three sublines G94-1, G94-19 and G168 were kept separate for six generations and all maintained an identical Southern pattern indicating the stability of the introduced trait for high levels of oleic acid. Multiple years of field testing at several locations determined that the fatty acid phenotype for these plants was stable. 

Expressed Material

There were no new proteins expressed in the sublines G94-1, G94-19 and G168. Biochemical and molecular tests confirmed that the beta-lactamase gene was not active in plants, and thus no protein (enzyme) was produced by this gene. The GUS gene was also silent and beta-glucuronidase was not expressed. Similarly no protein affecting the lysine levels was produced in any of the sublines.

Suppression of the GmFad2-1 genes in soybean sublines G94-1, G94-19 and G168 resulted in the expression of levels of oleic acid in soybean oil exceeding 80%, compared to 23% found in a typical conventional soybean oil.

Environmental Safety Considerations Expand

Field Testing

High oleic soybean lines derived from sublines G94-1, G94-19 and G168 were field tested in the United States (1995-1996), Canada, Puerto Rico and Chile. Field test data concerning yields, visual observations of agronomic properties including susceptibility to diseases and insects indicated high oleic soybeans were within the range normally displayed by non-modified varieties.

Outcrossing

Gene introgression from transformed soybean sublines G94-1, G94-19 and G168 is extremely unlikely as there are no relatives of cultivated soybean in the continental United States and Canada, and soybean plants are almost completely self-pollinated. Furthermore, the reproductive characteristics such as pollen production and viability were unchanged by the genetic modification resulting in G94-1, G94-19 and G168.

Cultivated soybean, Glycine max, naturally hybridizes with the wild annual species G. soja. Although G. soja is endemic to China, Korea, Japan, Taiwan and the former USSR it is not naturalized in North America, although it may possibly be grown in research plots. It was concluded that the potential for transfer of the high oleic acid from the transgenic sublines to soybean relatives through gene flow was negligible in managed ecosystems, and that there was no potential for transfer to wild species in Canada and continental United States.

Weediness Potential

No competitive advantage was conferred to sublines G94-1, G94-19 and G168, other than that conferred by high levels of oleic acid in the seed. Non-modified soybean varieties do not show any special weediness characteristics and modifying the fatty acid composition only in the seeds would not be expected to have any effect on weediness, nor in any other way be harmful to the environment. It was concluded that soybean lines G94-1, G94-19 and G168 had no altered weed or invasiveness potential compared to commercial soybean varieties.

Secondary and Non-Target Adverse Effects

Field observations of soybean sublines G94-1, G94-19 and G168 revealed no negative effects on nontarget organisms, suggesting that the relatively higher levels of the oleic acid in the tissues of these sublines are not toxic to organisms. Indirect metabolic alterations caused by the genetic modification were assessed and determined to have no impact on nontarget organisms. Furthermore, the lack of known toxicity of oleic acid suggests no potential for deleterious effects on beneficial organisms. It was determined that genetically modified soybean lines G94-1, G94-19 and G168 did not have a significant adverse impact on organisms beneficial to plants or agriculture, or on nontarget organisms, and were not expected to impact threatened or endangered species.

Impact on Biodiversity

Sublines G94-1, G94-19 and G168 have no novel phenotypic characteristics that would extend their use beyond the current geographic range of soybean production. Since there are no wild relatives of soybean in Canada and the continental United States and since soybean is not an invasive species, the novel trait will not be transferred to plant species in unmanaged environments.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The transgenic soybean G94-1, G94-19 and G168 sublines are the same as non-modified soybeans with the exception of the fatty acid profile which is quite different from that of traditional soybean oil and more similar to that of other high oleic oils such as olive or canola oil. The genetic modifications resulted in a superior, more heat and oxidatively stable soybean oil due to the reduced levels of the oxidatively unstable polyunsaturated fatty acids. The high oleic soybean oil has improved nutritional and functional properties compared to conventional soybean oil or partially hydrogenated soybean oil and will fit into food applications where a highly stable oil is required, such as in fat frying operations. Studies in the United Kingdom determined that the use of high oleic soybean oil may lower the intake of dietary linoleic acid but concluded that the small decrease in saturated fatty acid was likely to be beneficial, by reducing the risk factor for coronary heart disease. It was further concluded that these results should generally be applicable to the European Community and most other countries.

Nutritional Data

The analysis of nutritional properties of high oleic soybean sublines G94-1, G94-19 and G168 for total protein, oil, carbohydrate, crude fiber, ash, individual amino acid, phytic acid, trypsin inhibitor, stachyose, raffinose and isoflavone contents, determined that there were no differences in the levels of these components compared to non-transgenic soybeans. High Oleic soybean oil contains approximately 10% saturated fats, greater than 80% oleic acid, and low levels of polyunsaturated fatty acids: approximately 2% linoleic acid and 3.5% linolenic acid. Trace amounts (0.5%) of a linoleic acid 9,15 isomer were also detected, which while absent from non-hydrogenated soybean oil, is present at similar levels in butterfat, and is often found at considerably higher levels (typically 1-3%) in partially hydrogenated vegetable oils. The levels of accumulation of two important seed storage proteins, glycinin and beta-conglycinin, were changed. Glycinin levels increased while beta-conglycinin levels decreased. These differences were not expected to have a negative impact as variation in the levels of the various seed storage proteins were also noted in some commercial soybean lines. Antinutritional factors including trypsin inhibitors, phytic acid and the oligosaccharides raffinose and stachyose normally present in non-modified soybeans were similar to those found in the transgenic sublines G94-1, G94-19 and G168. Glycitein and lectin levels were somewhat elevated in the transgenic soybean sublines versus A2396, but were within the middle range reported in the literature. It was determined that the consumption of refined oil from soybean sublines G94-1, G94-19 and G168 would have no significant impact and may improve the nutritional quality of the food supply in the United States and Canada.

Feeding studies carried out with pigs and chickens demonstrated that processed soybean meal derived from high oleic soybeans was nutritionally equivalent to processed soybean meal derived from the conventional soybeans. It was determined that high oleic soybeans may be used as any other non-modified soybean meal and would be treated as any other commodity soybean meal.

Toxicity

A complete compositional analysis was performed on the transgenic soybean lines. High oleic soybeans did not differ in composition from the parent line or commodity soybean lines (literature ranges) regarding total fat, protein, fiber and ash, amino acids, minerals and vitamins, anti-nutritional factors (phytic acid, trypsin inhibitor, lectins, raffinose, stachyose) and isoflavones. These results demonstrated that there were no significant quantitative or qualitative differences between transgenic soybean sublines G94-1, G94-19 and G168 and elite soybean lines with regard to all of these components. No new proteins were expressed in these novel soybeans, and the variation in seed protein was considered to be within the natural range of variation.  It was concluded that there was no need to carry out a complete toxicological assessment since the high oleic acid soybean lines were, with the exception of high oleic acid content, equivalent to non-modified soybeans.

Allergenicity

Allergenicity studies demonstrated that there were no significant quantitative or qualitative differences between the high oleic soybean and non-modified elite soybean. The allergenicity potential was the same for transgenic soybean sublines G94-1, G94-19 and G168 and elite soybean with regard to their allergen content.

Abstract Collapse

Soybean (Glycine max) is grown primarily for its seed, which has many uses in the food and industrial sectors, and represents one of the major sources of edible vegetable oil and of proteins for livestock feed use. The major producers of soybeans were the United States, Brazil, Argentina, China, India, Paraguay and Canada.

A major food use of soybean in North America and Europe is as purified oil, used in margarines, shortenings, and cooking and salad oils. It is also a major ingredient in food products such as tofu, tempeh, soya sauce, simulated milk and meat products, and is a minor ingredient in many processed foods. Soybean meal is used as a supplement in feed rations for livestock.

Soybean oil is rich in polyunsaturated fatty acids and is considered a “healthy” oil due to its effects on decreasing blood cholesterol levels. However, polyunsaturated fatty acids will oxidize and are unstable at high temperatures, which makes them unsuitable for high temperature cooking. For many food applications, soybean oil requires additional processing, such as hydrogenation, before it can be used in margarines, shortening, and deep fat frying products. The hydrogenation process changes the fatty acid profile of soybean oil by converting the polyunsaturates into monounsaturated fatty acids or saturated fats depending on the use of the final product. Chemical hydrogenation also generates trans-isomers of oleic acid and other trans-fatty acids reported to have negative health effects by raising blood cholesterol levels.

Soybean lines G94-1, G94-19 and G168 were developed through a specific genetic modification to produce a soybean oil that contains high levels of oleic acid, a monounsaturated fatty acid. These high oleic soybeans contain a second copy of fatty acid desaturase gene (fad2), which is naturally present in soybeans. The fad2 gene codes for the enzyme, delta-12 desaturase, which is involved in fatty acid synthesis. Unlike conventional soybeans, the presence of a second copy of the fad2 gene in the high oleic soybeans G94-1, G94-19 and G168 causes a phenomenon known as "gene silencing" which results in both copies of the fatty acid desaturase gene being "switched off". This blocks the fatty acid biosynthetic pathway and results in the accumulation of oleic acid. As a consequence, polyunsaturated fatty acids (linoleic acid and linolenic acid) are only produced in very small amounts.

The transgenic fad2 gene in G94-1, G94-19 and G168 was isolated from G. max and introduced into a commercial soybean variety using particle acceleration (biolistic) transformation. The genetic modification affects only the seed, allowing fatty acid biosynthesis to function normally in other plant parts such as the leaves.

High oleic soybean oil contains levels of oleic acid exceeding 80%, higher than the levels found in olive oil and rapeseed oil. This oil is lower in saturated fat, contains no trans-fatty acids, and remains in a user-friendly liquid form. The high levels of oleic acid make the oil more heat-stable for cooking and edible spray applications.

G94-1, G94-19 and G168 were tested in field trials in the United States and Canada. Data collected from these trials demonstrated that G94-1, G94-19 and G168 did not differ from conventional soybeans in agronomic characteristics including seed production and susceptibility to diseases and insects. These tests also demonstrated that the transformed lines did not exhibit weedy characteristics, or negatively affect beneficial or nontarget organisms, and were not expected to impact threatened or endangered species.

Soybean does not have any weedy relatives with which it can crossbreed in the continental United States and Canada. Cultivated soybean can naturally cross with the wild annual species G. soja, however G. soja, which occurs naturally in China, Korea, Japan, Taiwan and the former USSR, is not naturalized in North America. Additionally, soybean plants are almost completely self-pollinated, and reproductive and growth characteristics were unchanged by the genetic modification resulting in G94-1, G94-19 and G168. It was therefore concluded that the potential for transfer of the trait for high oleic acid from the transgenic line to soybean relatives through gene flow (outcrossing) was negligible in managed ecosystems, and that there was no potential for transfer to wild species in Canada and the continental United States.

The food and livestock feed safety of high oleic soybean lines G94-1, G94-19 and G168 was established based largely on the fact that there were no new proteins present in these soybean lines. It was determined that the allergenic potential of the high oleic soybeans was the same as for conventional soybeans. The nutritional equivalence of soybeans compared to conventional (non-GM) soybeans was demonstrated by the analyses of key nutrients, including proximates (e.g., protein, fat, fibre, ash, and carbohydrates), amino acid and fatty acid composition, as well as anti-nutrients. High oleic soybean oil contains approximately 10% saturated fats, greater than 80% oleic acid, and low levels of polyunsaturated fatty acids (approximately 2% linoleic acid and 3.5% linolenic acid). Trace amounts (0.5%) of a linoleic acid 9,15 isomer were also detected, which while absent from non-hydrogenated soybean oil, is present at similar levels in butterfat, and is often found at considerably higher levels (typically 1-3%) in partially hydrogenated vegetable oils. The equivalence of high oleic soybeans to conventional soybeans was confirmed in feeding studies with pigs and broiler chickens. Soybean meal from high oleic soybeans was nutritionally equivalent to processed soybean meal derived from the conventional soybeans.

High oleic soybeans are a value-added commodity. These premium soybeans are grown under contract to preserve the identity of high oleic soybeans from the point of planting through to delivery of seed to the processing plant. Other than this, management and production practices for growing high oleic soybeans are much the same as growing any regular variety of soybean.

Links to Further Information Expand


This record was last modified on Tuesday, September 15, 2015