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Decision Document 2001-34:
Determination of the Safety of Optimum Quality Grains L.L.C.'s High Oleic Soybean
(Glycine max) lines G94-1, G94-19, and G-168
Derived from Transformation Event Line 260-05

This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir94-08: Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits and its companion document T1-10-96 The Biology of Glycine max (Soybean), and the guidelines Dir95-03: Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office (PBO) of the Plant Health and Production Division, and the Feed Section of the Animal Health and Production Division, has evaluated information submitted by Optimum Quality Grains, L.L.C. regarding soybean sublines G94-1, G94-19 and G-168 derived from transformation event line 260-05 These plants have been modified to produce oil with elevated levels of oleic acid. The CFIA has determined that these plants with novel traits (PNT's) should pose no concerns with respect to environmental safety, the safety to livestock consuming feed derived from the PNT, and is considered substantially equivalent to soybean products currently approved in Canada as livestock feed.

Unconfined release into the environment and use as livestock feed of Optimum Quality Grains L.L.C.'s high oleic soybean sublines G94-1, G94-19 and G-168 are therefore authorized as of February 10, 2000. Also, any soybean, Glycine max lines derived from G94-1, G94-19 and G-168 are also authorized for release provided that: no interspecific crosses are performed; the intended use of the plants is the same; and it is known, following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybeans, in terms of their potential environmental impact and livestock feed safety.

Table of Contents

1. Brief Identification of the Plant with Novel Traits (PNT)
2. Background Information
3. Description of the Novel Traits
   1. High Oleic Acid
   2. Beta-glucuronidase
   3. Ampicillin Resistance
   4. High Lysine Content
   5. Development Method
   6. Stable Integration into the Plant's Genome
4. Assessment Criteria for Environmental Safety
   1. Potential of the PNT to Become a Weed of Agriculture or be Invasive of Natural Habitats
   2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
   3. Altered Plant Pest Potential
   4. Potential Impact on Non-Target Organisms
   5. Potential Impact on Biodiversity
5. Nutritional Assessment Criteria for Use as Livestock Feed
6. Regulatory Decision

I. Brief Identification of the Plant with Novel Traits (PNT's)

II. Background Information

Optimum Quality Grains L.L.C. has developed the soybean sublines G94-1, G94-19 and G-168 with elevated levels of oleic acid in their oil, (hereafter referred to as high oleic soybean sublines) from transformation event line 260-05. The high oleic trait is intended to alter the heat stability of soybean oil for food applications such as frying. The development of 260-05 used recombinant DNA technology to introduce an additional copy of the endogenous soybean GmFad2-1 gene into the elite soybean line A2396. This gene codes for a seed specific delta-12 desaturase involved in the synthesis of polyunsaturated fatty acids in the developing seeds. The insertion of a second copy of the GmFad2-1 gene causes a silencing of the endogenous GmFad2-1 gene in the seed resulting in lowered levels of the delta-12 desaturase, which in turn results in elevated levels of oleic acid in the seed. A second gene, bla, (beta-lactamase), conferring resistance to the antibiotic ampicillin, was inserted into the high oleic soybean lines. However this gene lacks the necessary DNA sequences for expression in plants. The bla gene was present on the transforming plasmid and used as a marker during the cloning process of the plasmid in bacterial cells. A third gene, uidA, encoding beta-glucuronidase (GUS) was inserted to allow selection of transformed plant cells by using a simple visual assay to detect tissue which expressed the gene.

Along with the high oleic acid trait, the soybean transformation line 260-05 was intended to express elevated lysine levels in the seed meal. To increase seed lysine content, the elite soybean line A2396 was co-transformed with a second plasmid composed of two gene expression cassettes, one encoding an ampicillin resistance marker gene (bla) and a seconden coding dihydrodipicolinic acid synthase (dapA ). Dihydrodipicolinic acid synthase is insensitive to feedback inhibition by lysine, thus expression of this enzyme in soybeans would result in the accumulation of free lysine in the seeds. The high oleic soybean sublines, G94-1, G94-19 and G-168 do not have an intact copy of the dapA gene, thus the high lysine phenotype is not expressed.

Optimum Quality Grains, L.L.C. has submitted information and data to the CFIA on the identity of transformation event line 260-05 and the high oleic soybean sublines derived from it, the molecular characterization, a detailed description of the modification method, data and information on the stability of the gene insertion, and the role of the inserted genes and regulatory sequences in donor organisms.

The soybean lines were field tested in Iowa, Michigan, Wisconsin, Maryland, Minnesota, and Indiana, USA, under confined conditions. Field trials were not conducted in Canada, however the data provided included information generated in regions of the United States with environments similar to those were soybeans are cultivated in Canada. The high oleic soybean sublines were approved for commercialization in the USA in 1997.

Agronomic characteristics such as maturity, yield, plant height, oil and protein content, and disease susceptibility were compared to those of its unmodified G. max counterpart. Other than the intentional change in oil composition, no significant differences were apparent. The plant morphology of the high oleic soybean sublines and their unmodified counterpart was also compared and no differences were detected. Allergenicity studies using human sera showed that there were no significant differences in the allergen content between the high oleic soybean sublines and their unmodified counterpart.

The Plant Biosafety Office of the Plant Health and Production Division, CFIA has reviewed the above information, in light of the assessment criteria for determining environmental safety of plants with novel traits, as described in the regulatory directive Dir94-08. The review included the following:

• potential of the PNT to become a weed of agriculture or be invasive of natural habitats
• potential for gene-flow to wild relatives whose hybrid offspring may become more weedy or invasive
• potential for the PNT to become a plant pest
• potential impact of the PNT or its gene products on non-target species, including humans
• potential impact on biodiversity

The Feed Section of the CFIA's Animal Health and Production Division, has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed as described in Dir95-03. The evaluation included:

• potential impact to livestock
• potential impact on livestock nutrition.

III. Description of the Novel Traits

1. High Oleic Acid

• The high oleic trait results from the suppression of expression of the soybean's endogenous seed-specific delta-12 desaturase encoded by GmFad2-1, by the insertion and expression of an additional copy of the gene. Silencing of the GmFad2-1 gene results in a block in the conversion of oleic acid to linoleic acid, resulting in levels of oleic acid in the seed of approximately 80% as compared to approximately 20% in the seed of conventional elite soybean lines. Trace amounts (0.5%) of a linoleic acid isomer were also detected. This isomer is not normally present in non-hydrogenated soy oil but is present in butterfat at similar levels and in partially hydrogenated vegetable oils (1-3%).

2. Beta-glucuronidase Expression

• The uidA gene expresses beta-glucuronidase (GUS). This gene was derived from the bacteria Escherichia coli. Expression of the encoded enzyme allows detection in the laboratory of transformed plant tissue by using a simple colour change assay. The expression of the gene was below the limits of detection when the high oleic soybean sublines were assayed.

3. Ampicillin Resistance

• An ampicillin resistance gene ( bla) derived from E. coli was used during the development of the genetic sequence used to produce soybean transformation line 260-05 and as a result is also present in the high oleic soybean sublines. This gene was not intended to have any agronomic purpose.
• The bla gene is not functional as it does not have the necessary regulatory sequences for expression in plants. Analysis of the high oleic soybean sublines demonstrated that, as expected, the gene was not expressed.

4. High Lysine Content

• A gene encoding dihydrodipicolinic acid sythase (dapA) was also introduced into elite soybean line A2396. The dapA gene was derived from Corynebacterium glutanicum, a common bacterium which is used in the fermentation industry for amino acid production. The C. glutanicum dihydrodipicolinic acid synthase is insensitive to feedback inhibition by lysine, thus expression of this enzyme in soybeans would result in the accumulation of free lysine in the seeds
• The high oleic soybean sublines G94-1, G94-19 and G-168 derived from transformation event line 260-05 have only a non-functional copy of the dapA gene that lacks the complete genetic elements necessary for protein expression in the plant. Data was provided which demonstrated that the enzyme was not expressed in the high oleic soybean sublines and that lysine levels were within the normal range for soybean cultivars.

5. Development Method

• The soybean elite line A2396 (Asgrow Seed Co.) was transformed by particle bombardment, using two modified E. coli plasmid vectors, one containing the GmFad2-1, bla and uidA genes and the other containing the dapA and bla genes.
• Transformants were visualized by GUS activity and the presence of the GmFad2-1 gene was confirmed by polymerase chain reaction. Several small sample chips were taken from seeds of transformant 260-05 and screened for fatty acid composition. The chips expressing the high oleic trait were selected and the high oleic soybean sublines were developed from the seeds.
• There are no novel gene products expressed. The novelty of the soybean sublines is the high oleic acid content achieved via silencing of the endogenous GmFad2-1 gene. Although the high oleic soybean sublines contain transgenes for GUS, and ampicillin resistance, and an incomplete copy of the dapA gene, the expression of these genes cannot be detected.

6. Stable Integration into the Plant's Genome

• The high oleic soybean sublines G94-1, G94-19 and G-168 have genes inserted at two loci in the genome, designated as locus A and locus C. The original transformation event line 260-05 also had genes inserted at a third locus, designated as locus B, however this locus was lost during the selective breeding process to produce the high oleic sublines. Locus A is the GmFad2-1 silencing locus, it contains two intact copies of the GmFad2-1 gene along with associated regulatory sequences, one intact copy of the uidA (GUS) gene and associated regulatory sequences, one truncated copy of the uidA gene, and two intact copies of the ampicillin resistance (bla) gene with bacterial regulatory sequences. Locus C contains a single truncated copy of the dapA gene. The structural part of the gene is intact, but the promoter carries a deletion, so as a result no protein is expressed.
• The high oleic acid phenotype was shown to be stable over 8 generations. Data was also presented which demonstrated the environmental stability of the trait over a number of locations.
• The data presented for the high oleic soybean sublines was from plant material 4 to 8 generations removed from the original transformation event line 260-05.

IV. Assessment Criteria for Environmental Safety

1. Potential of the PNT to be a Weed of Agriculture or be Invasive of Natural Habitats

The CFIA has evaluated data submitted by Optimum Quality Grains L.L.C. on the reproductive and survival biology of the high oleic soybean sublines and determined that the morphological traits, plant maturity, seed yield, disease and insect resistance, and quality traits, except for the intentional alteration to the oil profile, were within the normal range of expression of characteristics in unmodified G. max counterparts. No competitive advantage in the environment was conferred by the insertion of the novel genes.

Based on the submitted data, the CFIA has determined that the high oleic soybean sublines derived from transformation event line 260-05 did not show any stress adaptation. The sublines were tested at several sites and no significant differences in agronomic performance was observed when compared to unmodified counterparts under the same conditions.

The biology of G. max, described in T1-10-96, shows that unmodified plants of this species are not weedy, nor invasive of unmanaged habitats in Canada. According to the information provided by Optimum Quality Grains L.L.C. the high oleic soybean sublines were determined to not be significantly different from their traditionally developed counterparts in this respect in the U.S. field trial environments similar to the normal soybean cultivation areas in Canada. No competitive advantage was conferred to the high oleic soybean plants, nor has the altered oil trait rendered them weedy. Volunteer plants can easily be managed by mechanical means or by the use of available herbicides.

The above considerations, together with the fact that the novel trait has no intended effect on weediness or invasiveness, led the CFIA to conclude that the high oleic soybean sublines have no altered weediness or invasiveness potential compared to currently commercialized soybean varieties.

2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive

The biology of soybean, as described in T1-10-96 shows that soybeans exhibit a high percentage of self-fertilization and cross pollination is usually less than one percent, suggesting that any pollen flow from cultivated soybeans to related species is minimal. Natural hybridization between cultivated soybean and the wild annual species Glycine soja can occur. G. soja is not naturalized in North America, and although this species could occasionally be grown in research plots, there are no reports of its escape from such plots to unmanaged habitats.

The CFIA has therefore concluded that the potential for transfer of the high oleic trait from the high oleic soybean sublines through gene flow to other soybean lines is negligible in managed agricultural ecosystems, and that there is no potential for gene transfer to wild species.

3. Altered Plant Pest Potential

Soybean is not a plant pest in Canada (T1-10-96), and the intended effect of the novel trait is not related to plant pest potential. In addition, morphological and agronomic characteristics of the high oleic soybean sublines were shown to be within the range of values displayed by currently commercialized G. max varieties, thus leading to the conclusion that plant pest potential was not inadvertently altered. The CFIA has therefore determined that the high oleic soybean sublines do not display any altered pest potential.

4. Potential Impact on Non-Target Organisms

The high oleic trait is intended only to alter the heat stability of soybean oil in food applications. No novel toxins or allergens are produced by the high oleic soybean sublines when compared to unmodified soybean lines. No effect would be expected on non-target organisms as no genes have been inserted into the plant which confer pest resistance and the high oleic soybean sublines do not produce a novel protein.

Based on the above, the CFIA has determined that the unconfined release of the high oleic soybean sublines will not result in altered impacts on interacting organisms, including humans, when compared with currently commercialized counterparts.

5. Potential Impact on Biodiversity

The high oleic soybean sublines have no novel phenotypic characteristics that are likely to extend their use beyond the current geographic range of soybean production in Canada. The overall relative impact on plant biodiversity is expected to be neutral. Impact on animal and microbial biodiversity is also expected to be neutral since the introduced trait would not be expected to alter the plant's metabolism such that novel compounds would be produced.

The CFIA has therefore concluded that the potential impact on biodiversity of the high oleic soybean sublines is equivalent to that of currently commercialized soybean lines.

V. Nutritional Assessment Criteria for Use as Livestock Feed

Soybean meal derived from high oleic soybeans was demonstrated to be nutritionally equivalent to traditional soybean meal. Amino acid profile, and concentration of fibre, minerals, and trypsin inhibitor were not different in high oleic soybean meal from the parental line soybean meal. Slight but statistically significant differences in protein and fat concentration were observed between the high oleic soybean meal and that derived from the parental line, but both of these nutrients were well within the normal range for soybean meal. Soybean oil, and full fat soybean seeds, while not intended for the livestock feed market, could enter the feed market. The fatty acid composition of seeds and oil is intentionally different from traditional soybean seeds and oil. This difference in fatty acid profile in the seeds and oil would have little impact on the soybean meal (oil extracted) and little impact on the total diet in normal feeding practices.

In a 17-day feeding trial with poultry fed high oleic vs control soybean meal, there was no difference in performance between the two treatments. Trials with dairy cattle and swine, fed full fat high oleic acid soybeans also showed no differences in performance. Milk from cows fed full fat soybeans did not differ in fat content, but the fatty acid profile is altered (intentionally), compared with milk from traditional soybeans.

VI. Regulatory Decision

Based on the review of data and information submitted by Optimum Quality Grains L.L.C. and through comparison of the high oleic soybean sublines G94-1, G94-19 and G-168 derived from transformation event line 260-05 with their unmodified soybean G. max counterpart, the Plant Biosafety Office, Plant Health and Production Division, CFIA has concluded that the novel trait is not expected to confer any intended or unintended ecological advantage to the high oleic soybean sublines following unconfined release. The potential for transfer of the novel trait to other soybean plants is insignificant, due to the biology of the species. There is no potential for transfer to wild relatives in Canada.

Based on the review of data and information submitted by Optimum Quality Grains L.L.C., the Feed Section, Animal Health and Production Division, CFIA, has concluded that the novel trait does not in itself raise any concerns regarding the safety or nutritional composition of the high oleic soybean lines. Soybean and several of its byproducts are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As whole seeds and plants have been assessed and found to be substantially equivalent to traditional soybean varieties, the high oleic soybean sublines and their byproducts are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada

Unconfined release into the environment and use as livestock feed of Optimum Quality Grains L.L.C.'s high oleic soybean sublines G94-1, G94-19 and G-168 are therefore authorized. Also, any soybean, Glycine max lines derived from G94-1, G94-19 and G-168 are also authorized for release provided that: no interspecific crosses are performed; the intended use of the plants is the same; and it is known, following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown soybeans, in terms of their potential environmental impact and livestock feed safety.

This bulletin is published by the Plant Health and Production Division. For further information, please contact the Plant Biosafety Office or the Feed Section at: