GM Crop Database

Database Product Description

T45 (HCN28) (ACS-BNØØ8-2)
Host Organism
Brassica napus (Argentine Canola)
Trait
Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Bayer CropScience (Aventis CropScience(AgrEvo))

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2002 2002 2003
Canada 1997 1996 1996
China 2004 2004
European Union 2009 2009 View
Japan 1997 1997 1997
Korea 2005 2005
Mexico 2001 2001
United States 1998 1998 1998

Introduction Expand

Canola (Brassica napus) line T45 (synonym HCN28) was developed using recombinant DNA techniques to allow the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides such as Basta®, Rely®, Finale®, and Liberty®.

Prior to the novel food safety assessment of T45, Health Canada had approved the glufosinate tolerant canola lines HCN92 (Innovator) and HCN10, both derived form the transformation event Topas 19/2. The primary difference between T45 and Topas 19/2 derived transformants is that the former does not contain an antibiotic resistance marker gene. The phosphinothricin-N-acetyltransferase (pat) gene introduced into T45, which conferred the novel trait of glufosinate herbicide tolerance, was also used as a selectable marker for screening transgenic plants, thereby eliminating the need for an antibiotic resistance marker in the construct.

Glufosinate is a short name for the ammonium salt, glufosinate-ammonium. It is a broad-spectrum contact herbicide and is used to control a wide range of weeds after the crop emerges or for total vegetation control on land not used for cultivation. Glufosinate herbicides are also used to desiccate (dry off) crops before harvest.

Glufosinate is a natural compound isolated from two species of Streptomyces fungi. It inhibits the activity of an enzyme, glutamine synthetase, which is necessary for the production of glutamine and for ammonia detoxification. The application of glufosinate leads to reduced glutamine and increased ammonia levels in the plant tissues. This causes photosynthesis to stop and the plant dies within a few days. Glufosinate also inhibits the same enzyme in animals. Glufosinate ammonium is currently registered in Canada as a non-selective herbicide for both non-crop and crop uses. It is highly biodegradable, has no residual activity, and very low toxicity for humans and wild fauna.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
pat phosphinothricin N-acetyltransferase HT CaMV 35S 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
Streptomyces viridochromogenes pat

S. viridochromogenes is ubiquitous in the soil. It exhibits very slight antimicrobial activity, is inhibited by streptomycin, and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

Canola line HCN28 was produced by Agrobacterium-mediated transformation of B. napus cultivar 'AC Excel'. 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 gene coding for glufosinate ammonium tolerance. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome.

The pat gene used in transformation event T45 is a synthetic version of the gene isolated from Streptomyces viridochromogenes, strain Tu 494. The nucleotide sequence was modified to provide codons preferred by plants without changing the amino acid sequence of the enzyme. Expression of the pat gene was regulated by including promoter and terminator sequences from the 35S transcript of cauliflower mosaic virus (CaMV).

The original transformant (T45) was backcrossed twice with B. napus line AC Excel. HCN28 was developed from the resultant BC2 using the single seed descent method.

Characteristics of the Modification Expand

The Introduced DNA

In order to demonstrate the stable integration of the pat gene in T45 and its progeny, the genomic DNA from three different generations of event T45 (F5, F7 and R2) was analyzed by Southern blot. These analyses indicated that the original transformant (T45) contained a single copy of the pat gene integrated at a single site. Additional analyses to indentify the possible incorporation of plasmid DNA sequences outside of the T-DNA region were negative, indicating that the T-DNA was the only new DNA inserted into the host genome.

Genetic Stability of the Introduced Trait

HCN28 was at least four generations removed from the original transformant (T45) during which time the presence and expression of the pat gene remained stable.

Expressed Material

The only newly expressed material in T45 was the PAT protein. The identity and levels of expression of PAT enzyme were determined using a double antibody sandwich (DAS)-ELISA performed on total protein extracts obtained from seeds of transgenic T45 plants. Concentrations of PAT protein in canola seeds ranged from 95 to 246 ng/g of seed tissue.

The levels of PAT protein detected by DAS-ELISA were highest in the untoasted canola meal, up to 38 µg/g protein. The presence of the PAT protein was expected in the meal due to the use of a constitutive promoter (CaMV 35S), which drives PAT expression throughout the plant, but only minimally in pollen. The untoasted canola meal showed detectable enzymatic activity. After the meal was toasted, all activity was destroyed indicating that the enzyme was denatured during the first stages of processing. Levels of PAT protein detected in toasted canola meal were much lower than those in untoasted meal, and ranged from 2-5 µg/g of canola meal. This reduction is likely due to a decrease in antibody reactivity as a result of PAT protein denaturation due to the meal toasting process where temperatures in excess of 90°C are encountered.

PAT protein was not detected in crude oil from the non-transgenic control, but was detected at a low level in crude oil samples from transgenic samples. Amounts detected ranged from 0.296 to 0.460 ng/g, well below the validated limit of quantitation of 250 ng/g. The PAT protein was not detected by DAS-ELISA in any of the refined oils obtained from transgenic or non-transgenic sources, and there were no detectable levels of PPT-acetyltransferase activity. Refined canola oil generally contains no protein of any kind.

Environmental Safety Considerations Expand

Field Testing

The canola line HCN28 (derived from T45) was field tested in Canada (1993 - 1995), in the United States (1996, 1997) and in Chile, Japan, the United Kingdom, and Australia. Agronomic characteristics such as cotyledon width, pod and leaf length, flowering period, time to maturity, plant height, lodging score, seed yield and thousand seed weight were compared to unmodified canola. Stress adaptation was evaluated, including resistance to major B. napus pests such as white rust (Albugo candida) and blackleg (Leptosphaeria maculens) and determined to fall within the ranges currently displayed by commercial varieties. The only significant difference between HCN28 canola and the parental non-transformed variety was its resistance to glufosinate ammonium. Overall the field data reports demonstrated that canola HCN28 has no potential to pose a plant pest risk.

Outcrossing

Brassica napus plants, including HCN28, are known to outcross up to 30% with other plants of the same species. Potential gene movement may occur at low levels with plants of related species such as B. rapa and B. juncea and at extremely low levels with B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies showed that introgression of the herbicide tolerance gene was most likely to occur with B. rapa.
Incorporation of the pat gene was not expected to confer an ecological advantage to potential hybrid offspring. If glufosinate ammonium-tolerant individuals arose through interspecific or intergeneric hybridization, the novel traits would confer no competitive advantage to these plants unless challenged by glufosinate ammonium. This may occur in managed ecosystems where glufosinate ammonium was applied for broad spectrum weed control, or when used to control weeds in plant varieties developed for tolerance to Liberty®. In the event that a glufosinate ammonium tolerant B. napus survived, these herbicide-tolerant individuals would be easily controlled using mechanical and other available chemical means. The use of good crop management practices should prevent the occurrence of persistent resistant hybrids. It was concluded that gene flow from the transgenic line HCN28 to canola relatives was possible, but would not result in increased weediness or invasiveness of these relatives.

Secondary and Non-Target Adverse Effects

It was determined that genetically modified canola line HCN28 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and was not expected to impact on threatened or endangered species. The PAT enzyme responsible for glufosinate ammonium tolerance is ubiquitous in nature and there have been no adverse effects, toxic or allergenic, ever reported.

Impact on Biodiversity

HCN28 has no novel phenotypic characteristics which would extend its use beyond the current geographic range of canola production. Since outcross species are only found in disturbed habitats, transfer of novel traits would not impact unmanaged environments. It was concluded that the potential impact on biodiversity of HCN28 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 PAT were detected in the refined oil of HCN28 canola. Furthermore, processing would destroy the enzymatic activity of the PAT protein. As such, there will be no human exposure to the introduced PAT protein as result of the consumption of refined oil from line HCN28.

Nutritional Data

Compositional analyses of processed canola oil from HCN28 and current commercial canola cultivars were compared for fatty acids, glucosinolates, chlorophyll and phytosterol content. These comparisons indicated no statistically significant differences for the components analyzed. The use of refined oil from HCN28 would therefore have no significant impact on the nutritional quality of the food supply.

Because canola meal is used as a livestock feed, additional compositional studies were conducted that demonstrated there were no significant differences in the moisture, oil, protein, crude fibre, ash, phytosterol and gross energy of HCN28, Innovator, and non-transgenic lines Excel, Legend and Cyclone. The pat gene did not result in any secondary effects in the seed composition or gross energy levels between glufosinate tolerant and non-transformed B. napus varieties.

Toxicity and Allergenicity

It was determined that there were no toxicity or allergenicity concerns with HCN28, 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 PAT protein.
The potential toxicity of the PAT protein was evaluated in a 14-day repeated dose feeding study with rats. Subject groups consisting of male and female Wistar rats were fed a diet amended with either 5000 or 50,000 ppm purified PAT protein and monitored for dietary intake, weight gain, and observable adverse effects. There were no clinical signs noted and food consumption and body weights were unaffected by treatment. No treatment-related changes were seen in hematology or urinalysis parameters. Organ weight data, macroscopical and microscopical findings also did not distinguish treated groups from controls. Based on these results, there was no evidence of acute toxicity for the PAT protein.

The potential of the PAT protein expressed in T45 transgenic canola to elicit allergic reactions was evaluated by comparing the nucleotide sequence of the pat gene from S. viridochromogenes with available nucleotide sequences contained within the GENBank database. Significant similarity was judged to be >60% identity for >100 base pairs. Using this criterion, the only significant similarity at the nucleotide sequence level was between the pat gene and a previously characterized PPT acetyltransferase from S. viridochromogenes (70.7% identity in 546 nucleotide overlap) and the analogous bar gene from S. hygroscopicus (65.8% identity in 546 nucleotide overlap). When comparing the deduced amino acid sequence of the PAT protein with other proteins in the database, the only noted significant similarity was with an rRNA methyltransferase also isolated from a Streptomyces species (68.2% identity in 132 amino acid overlap). The results of the GENBank database search failed to identify any homologies with known toxins or allergens that have been sequenced.

Also, unlike known protein toxins and allergens, the PAT protein was extremely susceptible to proteolytic degradation. Crude PAT protein containing extracts from transgenic canola plants were compared with purified PPT-acetyltransferase with respect to acid and/or enzymatic hydrolysis under conditions simulating the mammalian stomach. Samples were incubated at 37°C in stomach fluid from beagle dogs for up to 15 minutes, following which aliquots were removed, adjusted to pH 8.0, and tested for acetyltransferase activity. The enzymatic activity of purified PPT-acetyltransferase was completely lost within 1 minute of exposure to stomach fluid (pH 1.1), and within 10 minutes of exposure to fluid adjusted to pH 4.0. While the acetyltransferase activity of crude extracts from transgenic plants was lost equally quickly at pH 1.1, the rate of inactivation was slower at higher pH values.

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 canola line T45 was genetically engineered to express tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®). Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Essentially, glufosinate acts enough like glutamate, the molecule used by glutamine synthetase to make glutamine, that it blocks the enzyme's usual activity. Glutamine synthetase is also involved in ammonia detoxification. The action of glufosinate results in reduced glutamine levels and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant withering and death.

Glufosinate tolerance in T45 is the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, Streptomyces viridochromogenes, the same organism from which glufosinate was originally isolated. The PAT enzyme catalyzes the acetylation of phosphinothricin, detoxifying it into an inactive compound. The PAT enzyme is not known to have any toxic properties.
Line HCN28, which was derived from T45, was field tested in Canada (1993 - 1995), in the United States (1996, 1997) and in Chile, Japan, the United Kingdom, and Australia. Agronomic characteristics such as cotyledon width, pod and leaf length, flowering period, time to maturity, plant height, lodging score, seed yield and thousand seed weight were compared to those of unmodified canola. The only significant difference between HCN28 canola and the parental non-transformed variety was its tolerance of glufosinate ammonium. It was demonstrated that the transformed canola line did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Canola line HCN28 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 glufosinate-tolerant hybrids is possible. However, the glufosinate-tolerance trait is not expected to provide a competitive advantage to hybrid plants unless grown in managed environments routinely subjected to glufosinate applications. In the event that a glufosinate-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 refined oil. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, and hence no PAT protein was detected in the refined oil of HCN28 canola. As such, there will be no human exposure to this novel protein as a result of the consumption of refined oil from line HCN28. Compositional analyses of processed canola oil from HCN28 and current commercial canola cultivars were compared for fatty acids, glucosinolates, chlorophyll and phytosterol content. These comparisons indicated no statistically significant differences for the components analyzed. The use of refined oil from HCN28 would therefore have no significant impact on the nutritional quality of the food supply.

Canola meal from line HCN28 was also assessed to determine its safety for use in animal feed. Additional compositional studies were conducted, revealing no significant differences in the moisture, oil, protein, crude fibre, ash, phytosterol and gross energy content between HCN28 and other commercially available canola lines, thus supporting the safety of the inclusion of HCN28 canola in animal feed.

It was determined that there were no toxicity and allergenicity concerns with HCN28. There is no dietary exposure to PAT since this protein is not present in the refined canola oil consumed by humans. PAT was not found to have any amino acid sequence similarities with known toxins or allergens. The PAT enzyme does not possess the proteolytic or heat stability characteristic of toxic compounds, and was readily digested under conditions simulating mammalian digestion. An acute oral toxicity study revealed that no negative effects were experienced by rats fed high doses of the PAT protein (up to 50,000 ppm for a period of 14 days), providing further evidence supporting the lack of toxicity or allergenicity concerns related to the expression of PAT protein in HCN28 canola.

Links to Further Information Expand

Australia New Zealand Food Authority Canadian Food Inspection Agency Canadian Food Inspection Agency, Plant Biotechnology Office European Commission: Community Register of GM Food and Feed European Food Safety Authority Food Standards Australia New Zealand Japanese Biosafety Clearing House, Ministry of Environment Office of Food Biotechnology, Health Canada Office of the Gene Technology Regulator U.S.Department of Agriculture, Animal and Plant Health Inspection Service US Food and Drug Administration USDA-APHIS Environmental Assessment

This record was last modified on Tuesday, June 16, 2015