GM Crop Database

Database Product Description

GT200 (MON89249-2)
Host Organism
Brassica napus (Argentine Canola)
Trade Name
Roundup Ready®
Trait
Glyphosate herbicide tolerance.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

This experimental line will not be commercialized.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1997 1997 1996
Japan 2001 2001 2006
United States 2002 2002 2003

Introduction Expand

Canola (Brassica napus) line GT200 was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option in canola crops. Two novel genes were introduced into canola line GT200, which in combination provide field level tolerance to glyphosate. The novel plants express a glyphosate tolerant version of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) isolated from Agrobacterium tumefaciens strain CP4 (CP4 EPSPS). Glyphosate containing herbicides act by binding to and inactivating the wild type EPSPS enzyme, which is involved in the synthesis of aromatic amino acids, tyrosine, phenylalanine and tryptophan (shikimate biochemical pathway).

The EPSPS enzyme is present in all plants, bacteria and fungi, but not in animals, which do not synthesize their own aromatic amino acids. The modified enzyme (CP4 EPSPS) has a reduced binding affinity for glyphosate and allows the plant to function normally in the presence of the herbicide.

The second gene, isolated from Ochrobactrum anthropi strain LBAA, encodes the enzyme glyphosate oxidase, which accelerates the normal degradation of the herbicide glyphosate into aminomethylphosphonic acid (AMPA) and glyoxylate. AMPA is the principal metabolite of glyphosate and is degraded by several microorganisms while glyoxylate is commonly found in plant cells and is broken down by the glyoxylic pathway for lipid metabolism.

These two enzymes, CP4 EPSPS and glyphosate oxidase (GOX), allow GT200 canola to be protected against herbicide damage when glyphosate is used for weed control in the cultivation of canola.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase HT chloroplast transit peptide from A. thaliana 1 Native
goxv247 glyphosate oxidoreductase HT chloroplast transit peptide from A. thaliana 1 Native

Characteristics of Brassica napus (Argentine Canola) Expand

Center of Origin Reproduction Toxins Allergenicity

The species is native to India.

Canola flowers can self-pollinate, and they can also be cross-pollinated by insects and by wind.­

Brassica species can contain erucic acid and various glucosinolates, which can be toxic. However, commercial canola varieties have been bred to reduce the levels of these substances. Canola may contain elevated levels of tannins, which reduce the digestibility of seed protein, and sinapine, which is a bitter substance that can reduce the palatability of feeds made from canola meal.

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Occupational exposure to pollen and seed flour have been associated with allergic reactions in humans. There are no known allergic reactions to canola oil.

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Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Agrobacterium tumefaciens strain CP4 CP4 epsps

Agrobacterium tumefaciens is a common soil bacterium that is responsible for causing crown gall disease in susceptible plants. There have been no reports of adverse effects on humans or animals.

Modification Method Expand

Canola line GT200 was produced by Agrobacterium-mediated transformation of the B. napus cultivar 'Westar'. The T-DNA region of the Ti plasmid was "disarmed" by removal of virA genes, normally associated with the pathogenicity and disease-causing properties of A. tumefaciens, and replaced with the genes coding for glyphosate tolerance. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome.

GT200 was produced using a plasmid that contained the CP4 EPSPS gene and the gox gene. Both genes were controlled by the same constitutive promoter and fused to a chloroplast transit peptide sequence to allow targeting of the newly translated CP4 EPSPS and GOX enzymes into the chloroplast, the site of aromatic amino acid biosynthesis and endogenous EPSPS and GOX activity.

There was no incorporation of translatable plasmid DNA sequences outside of the T-DNA region and no antibiotic resistance marker genes were introduced into GT200.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from GT200 indicated the presence of a single site of insertion that contained one complete copy of the CP4 EPSPS gene and a complete copy of the gox gene and their respective regulatory sequences. The GOX protein introduced into canola line GT200 was shown to have a 99% similarity to the modified GOXv247 in transgenic line GT73. Both proteins are 46.1 KDa (there is a 17 Da difference) and differ by three amino acid substitutions.

Genetic Stability of the Introduced Trait

Segregation analysis and Southern blots were consistent with a single site of insertion of the CP4 EPSPS and gox genes into the GT200 genome. GT200 was several generations removed from the original transformant and a comparative analysis of third-generation material, using Southern blots and PCR analysis, determined that the introduced DNA was stably inserted and stably inherited of several generations.

Expressed Material

The expression of both CP4 EPSPS and GOX was quantified in the leaves and seeds of canola line GT200. Enzyme linked immunosorbent assay (ELISA) was used to determined average levels of CP4 EPSPS and GOX as follows: in seeds 31 µg/g CP4 EPSPS (f.w.) and 105 µg/g GOX (f.w); and in leaves 31 µg/g CP4 EPSPS and 69 µg/g GOX (f.w.)

Environmental Safety Considerations Expand

Field Testing

The canola line GT200 was field tested in Canada (1992-1994). Agronomic characteristics such as vegetative vigor, overwintering capacity, time to maturity, seed production and yield of the transformed line GT200 were compared to unmodified canola counterparts and determined to be within the normal range of expression found in commercial canola cultivars. Stress adaptation was evaluated, including resistance to major B. napus pests such as the fungal pathogen Leptosphaeria maculans (blackleg) and the susceptibility of GT200 was within the ranges currently displayed by commercial varieties. The only significant difference between GT200 canola and the parental non-transformed variety was tolerance to glyphosate. Overall the field data reports demonstrated that canola GT200 had no potential to pose a plant pest risk.

Outcrossing

Transgenic canola line GT200 displayed normal reproductive characteristics. B. napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that gene flow is most likely to occur with B. rapa.

The genes coding for glyphosate tolerance, CP4 EPSPS and gox are not expected to confer an ecological advantage to potential hybrid offspring. If glyphosate tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless these populations were routinely subject to herbicide treatments. This may occur in managed ecosystems where glyphosate herbicide Roundup® was applied for broad spectrum weed control, or when used to control weeds in plant varieties developed for tolerance to Roundup®. In the event that a glyphosate tolerant plant survived, the herbicide-tolerant individual would be easily controlled using mechanical and other available chemical means. It was concluded that gene flow from the transgenic line GT200 to canola relatives was possible, but would not result in increased weediness or invasiveness of these relatives.

Weediness Potential

No competitive advantage was conferred to GT200 canola, other than that conferred by resistance to glyphosate herbicides. Resistance to glyphosate did not confer any pest resistance, alter reproductive biology or change any physiology related to survival. Volunteer glyphosate tolerant plants would be easily managed using mechanical or alternative herbicides with different modes of action. It was concluded that GT200 canola had no altered weed or invasiveness potential compared to currently commercialized B. napus varieties.

Secondary and Non-Target Adverse Effects

It was determined that genetically modified canola line GT200 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and were not expected to impact on threatened or endangered species. The novel proteins CP4 EPSPS and GOX expressed in GT200 did not result in altered toxicity or allergenicity properties as determined from studies using simulated digestive fluids, nutritional composition and amino acid sequence homology studies.

Impact on Biodiversity

The transgenic canola line GT200 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of canola/rapeseed production. Since outcross species are only found in agricultural ecosystems, transfer of novel traits would not have an impact on unmanaged environments. It was determined that the relative impact on biodiversity of GT200 was equivalent to that of currently commercialized canola lines.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The human consumption of canola products is limited to the refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortening, salad oil and cooking oils. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, hence no amounts of CP4 EPSPS or GOX proteins were detected in the refined oil of GT200 canola. The genetic modification of GT200 canola will not result in any change in the consumption pattern for this product. As the introduced gene products are not detectable in the refined oil produced from transgenic canola, there will be no human exposure to these proteins.

Nutritional Data

The analysis of nutrients from transgenic GT200 canola and non-transgenic canola did not reveal any significant differences in the levels of crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. Raw seeds of GT200 were shown to have the same levels of glucosinolates and erucic acid as commercial canola varieties. Seed protein profiles, amino acid and fatty acid compositions were all within the range of the unmodified counterparts. The consumption of refined oil from GT200 was judged to have no significant impact on the nutritional quality of the food supply.

Several animal feeding studies were conducted to demonstrate the wholesomeness of glyphosate tolerant canola meal. Canola meal is used only for animal feed and is not considered a human food fraction. The feeding studies were conducted to establish the nutritional adequacy of canola meal for animal feeds and represented a worse case scenario in terms of canola consumption by humans. These studies included a four-week rat study on processed and unprocessed meal, a five-day quail (Northern Bobwhite) study on unprocessed meal, and a ten-week trout study on processed meal. The studies all supported the safety of meal from glyphosate-tolerant canola as a component in animal feed.

The rat feeding study examined six week-old Sprague-Dawley rats (10/sex/group) fed either 0, 5 or 15% w/w ground (unprocessed) and processed (toasted and defatted) glyphosate-tolerant canola (GT200 and GT73) and 'Westar' canola meal and a diet control (commercial rodent chow with no added canola meal). The study determined that rats fed on diets with unprocessed glyphosate tolerant canola seed had relative liver and kidney weights 5 - 20% greater compared to the diet controls. However, there were no differences in absolute or relative organ weights between the glyphosate-tolerant canola and parental line groups.

The quail feeding study examined thirty northern bobwhite (Colinus virginianus) chicks fed diets for five days and observed for a further three days. Treatment groups were fed a control diet, glyphosate-tolerant canola (both lines GT200 and GT73) or the parental line 'Westar', where canola was incorporated at a rate of 20% of the total weight of the diet. The study did not find any significant effects on body weight or feed consumption between birds in the control or treatment groups. There were no mortalities or overt signs of toxicity in either treatment or control groups.
Similarly a trout feeding study examined triplicate groups of 15 fish rainbow trout (Oncorhynchus mykiss) fed glyphosate tolerant canola meal (both lines GT200 and GT73) at 0, 5, 10, 15 or 20% weight of the dry diet for 10 weeks (i.e., 45 fish/treatment). There was statistical overlap in weight gain of fish fed each dietary treatment and no differences were detected between glyphosate-tolerant canola diets and controls. Fish fed the glyphosate-tolerant canola did not exhibit any adverse effects.

Toxicity and Allergenicity

It was determined that there were no toxicity or allergenicity concerns with GT200, since refined canola oil is the only product for human consumption and does not contain any detectable amount of protein. The absence of toxicity was further demonstrated by examining the amino aid sequence homology and the characteristics of the novel proteins CP4 EPSPS and GOX.

The amino acid sequences of both the CP4 EPSPS and GOX proteins were compared to the amino acid sequences of known protein toxins. No significant similarities were found other than would be expected given that certain functional domains are generally conserved between proteins.

The novel enzyme CP4 EPSPS is one of many EPSPS proteins that occur in plants, fungi and bacteria. The EPSPS proteins are naturally present in foods derived from plants and microbes and have no history of being toxic or allergenic. Similarly, the gox gene was sourced from a common soil bacterium, which has no history of pathogenicity. The GOX enzyme degrades glyphosate and experiments demonstrated that the enzyme displayed a narrow substrate specificity, and appeared not to have any secondary effects on other plant specific pathways.

The digestibility of both proteins in model digestion systems was done in vitro using simulated gastric fluid and simulated intestinal fluid) as mammalian digestion models. Unlike typical protein toxins and allergens, both the CP4 EPSPS and GOX proteins were rapidly digested.

Abstract Collapse

Argentine or oilseed rape (Brassica napus) was grown as a commercial crop in over 50 countries, with a combined harvest of 48.9 million metric tonnes in 2006. The major producers of rapeseed are China, Canada, India, Germany, France, the United Kingdom and Australia. Canola is a genetic variation of B. napus that was developed through conventional breeding to contain low levels of the natural rapeseed toxins, glucosinolate and erucic acid. Canola is grown for its seed, which represents a major source of edible vegetable oil and is also used in livestock feeds.

The only food use of canola is as a refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortenings, cooking and salad oils. Canola meal, a byproduct of the oil production process, is added to livestock feed rations. An increasing amount of oil is being used for biodiesel production, especially in Europe.

The removal of weeds early in the growing season is extremely important in canola production. Young canola seedlings are not very competitive and early weed pressure has detrimental effects on final yield. Precautions such as pre-plant tillage or herbicide application are common approaches for reducing weed competition. Once established, canola forms a dense canopy and is very competitive, making weed control less of a concern.

At present, it is expensive, but possible, to control annual grassy weeds (wild oats, volunteer cereals), perennial weeds (Canada thistle, perennial sow thistle, quackgrass), and certain annual broadleaf weeds in canola. The broadleaves are the most difficult to control because there are few herbicide options available. Many producers will use as many as four herbicides per year in an effort to control weeds.

Traditionally, it has been difficult to manage weeds in canola rotations because of the need to use Group 1 herbicides (Assure®, Fusion®, Poast®, Select®, Venture DG®) that are also commonly used to control weeds in flax, wheat and other cereals. Growers favour these herbicides because they are easy to work with, are extremely effective, and may be applied over a period of time. However, the constant use of Group 1 herbicides has placed large acreages of cultivated land at high risk of developing Group 1 weed resistance. The establishment of several herbicide-tolerant weed populations demonstrates this problem.

The herbicide-tolerant canola line GT200 was developed to allow for the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option. In order to obtain field tolerance to glyphosate herbicide, two novel genes, CP4 EPSPS and goxv247, were introduced into B. napus cv. Westar by Agrobacterium-mediated transformation.
The CP4 EPSPS gene codes for the enzyme 5-enolpyruvylshikimate-3-phosphate synthase that is present in all plants, bacteria and fungi. The EPSPS gene put into GT200 was isolated from strain CP4 of the common soil bacterium Agrobacterium tumefaciens and is a glyphosate tolerant form of EPSPS. The EPSPS enzyme is part of an important biochemical pathway in plants called the shikimate pathway that is involved in the production of aromatic amino acids and other aromatic compounds. When conventional canola plants are treated with glyphosate, the plants cannot produce the aromatic amino acids needed to grow and survive. EPSPS is not present in mammals, birds or aquatic life forms, which do not synthesize their own aromatic amino acids. For this reason, glyphosate has little toxicity to these organisms. The EPSPS enzyme is naturally present in foods derived from plant and microbial sources.

The canola line GT200 contains a second gene that codes for a modified version of glyphosate oxidase (GOX) enzyme that is ubiquitous in nature. The goxv247 gene inserted into GT200 was isolated from strain LBAA of the bacterium Ochrobactrum anthropi. Glyphosate oxidase (GOX) enzyme accelerates the normal breakdown of the herbicide glyphosate into two non-toxic compounds, aminomethylphosphonic acid (AMPA) and glyoxylate. AMPA is the principal breakdown product of glyphosate and is degraded by several microorganisms, while glyoxylate is commonly found in plant cells and is broken down by the glyoxylic pathway for lipid metabolism.

The canola line GT200 was field tested in Canada from 1992 to 1994. Agronomic characteristics such as vegetative vigor, overwintering capacity, time to maturity, seed production and yield of the transformed line GT200 were compared to unmodified canola counterparts and determined to be within the normal range of expression found in commercial canola cultivars. The only significant difference between GT200 canola and the parental non-transformed variety was tolerance to glyphosate. It was demonstrated that the transformed canola line did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Canola line GT200 was not expected to impact on threatened or endangered species.

Brassica napus may outcross (up to 30% of the time) with plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that cross hybridization is most likely to occur with B. rapa. Because of the ability of canola to outcross with related plants, the formation of glyphosate tolerant hybrids is possible. However, the glyphosate-tolerance trait is not expected to provide a competitive advantage to hybrid plants unless grown in managed environments routinely subjected to glyphosate applications. In the event that a glyphosate tolerant hybrid survived, the herbicide-tolerant individual could be easily controlled using mechanical and/or other available chemical means.

The human consumption of canola products is limited to the purified oil. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, hence no amounts of CP4 EPSPS or GOX proteins were detected in the refined oil of GT200 canola. As the introduced gene products are not detectable in the refined oil produced from transgenic canola, there will be no human exposure to these proteins and the consumption of refined oil from GT200 was judged to have no significant impact on the nutritional quality of the food supply.

The livestock feed safety of GT200 canola was established based on several standard criteria. The analysis of nutrients from transgenic GT200 canola and non-transgenic canola did not reveal any significant differences in the levels of crude protein, crude fat, crude fibre, ash and gross energy in either the whole seed or processed meal. Raw seeds of GT200 were shown to have the same levels of glucosinolates and erucic acid as commercial canola varieties. Seed protein profiles, amino acid and fatty acid compositions were all within the range of the unmodified counterparts.

Several animal feeding studies were conducted to demonstrate the wholesomeness of glyphosate-tolerant canola meal. Canola meal is used only for animal feed and is not considered a human food fraction. These studies included a four-week rat study on processed and unprocessed meal, a five-day quail (Northern Bobwhite) study on unprocessed meal, and a ten-week trout study on processed meal. The studies all supported the safety of the inclusion of GT200 canola in animal feed.

It was determined that there were no toxicity and allergenicity concerns with GT200. There is no dietary exposure to CP4 EPSPS and GOX since these proteins are not present in the refined canola oil consumed by humans. Furthermore, the structure and function of CP4 EPSPS protein was determined to be similar to the same enzyme naturally present in foods. Neither CP4 EPSPS nor GOX were found to have any amino acid sequence similarities with known toxins or allergens, and there are no known reports of toxicity or allergenicity of EPSPS or GOX proteins derived from plants, bacteria and fungi. Direct laboratory studies of the CP4 EPSPS and GOX proteins revealed the ready digestibility of both proteins under conditions simulating mammalian digestion, providing further evidence supporting the lack of toxicity or allergenicity concerns with GT200.

Links to Further Information Expand


This record was last modified on Friday, March 26, 2010