GM Crop Database

Database Product Description

HCN92 (ACS-BNØØ7-1)
Host Organism
Brassica napus (Argentine Canola)
Trade Name
Liberty-Link™ Innovator
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 2001 2003
Canada 1995 1995 1995
China 2004 2004
European Union View
Japan 1996 1996 1996
Korea 2005 2008
Mexico 1999 1999
South Africa 2001 2001
United States 1995 2002

Introduction Expand

Canola (Brassica napus) line HCN92 (synonyms: Topas19/2, Innovator) was developed through a specific genetic modification to allow the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides such as Liberty®. The pat gene, which encodes the enzyme phosphinothricin-N-acetyltransferase (PAT) and confers tolerance to glufosinate ammonium, was isolated from the common aerobic soil actinomycete, Streptomyces viridochromogenes strain Tü 494 and introduced into the parent canola line.

The PAT enzyme was used as a selectable marker enabling identification of transformed plant cells as well as a source of field tolerance to phosphinothricin containing herbicides. Glufosinate ammounium acts by inhibiting the plant enzyme glutamine synthetase, a key enzyme that detoxifies ammonia by incorporating it into glutamine. Inhibition of this enzyme leads to an accumulation of ammonia in the plant tissues, which kills the plant within hours of application. PAT catalyses the acetylation of phosphinothricin detoxifying it into an inactive compound. The modified canola permits farmers to use phosphinothricin-containing herbicides for weed control in the cultivation of canola.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
pat phosphinothricin N-acetyltransferase HT CaMV 35S CaMV 35S poly(A) signal 1 Native
nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens octopine synthase 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 HCN92 was produced by Agrobacterium-mediated transformation of microspores from B. napus cultivar 'Topas'. 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 glufosinate ammonium tolerance and aminoglycoside resistance. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome.

The original transformant (Topas 19/2) was produced using plasmid pOCA/Ac and contained a synthetic pat gene coding for phosphinothricin acetyltransferase (PAT) and the aminoglycoside resistance marker neo coding for neomycin phosphotransferase II. Expression of the pat gene was regulated by the cauliflower mosaic virus (CaMV) 35S promoter and terminator sequences. The neo gene was driven by the nopaline synthase promoter and an octopine synthase terminator sequence.

HCN92 was derived from traditional breeding crosses between non-genetically modified canola and a line resulting from transformation event (Topas 19/2). The transformed line was initially crossed with line ACSN3, followed by a second cross with cultivar 'AC Excel'. The seed from the final cross was advanced and single seed from F3 plants were bulked to form HCN92.

Characteristics of the Modification Expand

The Introduced DNA

The synthetic pat gene was modified to provide codons preferred by plants without changing the amino acid sequence of the PAT enzyme. The PAT protein under study was found to have a similar molecular weight to the bacterial PAT protein.

Southern blot analyses indicated that the DNA was integrated in the original transformant at one insertion site and the insertion contained 2 linked copies of the pat gene. Additional Southern blot analyses were performed to demonstrate that plasmid backbone sequences outside of the T-DNA region were not incorporated into the host genome.

Genetic Stability of the Introduced Trait

HCN92 was several generations removed from the original transformant and comparisons between the original transgenic plant (Topas19/2) and HCN92 showed no differences in the presence and expression of the pat and neo genes, nor in the insertion site.

Expressed Material

Constitutive expression of the PAT protein was detected in roots, leaves, buds and seeds. Seed expression levels ranged from 150 to 223 ng/g of seed tissue. PAT protein was not detected in stem tissue, protein extracts from the pollen, or unprocessed honey. Maximum expression was 0.001% of total plant protein.

The neo gene was linked to a weak constitutive promoter, and expression was consistently stronger in root tissue, but was also observed in buds, leaves, and crude seed samples. The enzyme was not detected in unprocessed honey or pollen samples and was inactivated during processing of canola seed into feed ingredients.

Environmental Safety Considerations Expand

Field Testing

The canola line HCN92 was field tested in Canada from 1990 to 1994. Agronomic and adaptation characteristics such as vegetative vigour, seed production and dormancy were within the normal range of expression of characteristics in unmodified counterparts. Seed morphology and average seed weight did not change, indicating that seed dispersal potential was not altered. Stress adaptation was evaluated, including resistance to major B. napus pests and pathogens (e.g., blackleg, sclerotinia, flea beetles) and determined to fall within the ranges currently displayed by commercial varieties. The only significant difference between HCN92 canola and the parental non-transformed variety was its resistance to glufosinate ammonium. Overall the field data reports demonstrated that canola HCN92 has no potential to pose a plant pest risk.

Outcrossing

B. napus plants, including HCN92, 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 glufosinate ammonium and aminoglycoside resistance were 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 HCN92 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 HCN92 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. For example, studies on honey bees foraging in HCN92 and observations on brood development determined that there were no negative impacts on bees. Plant residue studies were conducted to determine whether successive crops were affected by residues from transgenic HCN92. Results indicated that there were no differences between HCN92 and unmodified canola, indirectly demonstrating that soil bacteria were not negatively affected by plant residues from HCN92. Overall, the PAT enzyme responsible for glufosinate ammonium tolerance has very specific enzymatic activity, does not possess proteolytic or heat stability typical of toxic compounds, and does not affect the metabolism of the plant. Both the PAT enzyme and NPTII proteins are ubiquitous in nature and no adverse effects have been reported to be associated with either enzyme.

Impact on Biodiversity

HCN92 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. Studies demonstrated that HCN92 was not invasive of natural habitats, and that it was no more competitive than its unmodified counterparts, both in natural and managed ecosystems. It was concluded that the potential impact on biodiversity of HCN92 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 and NPTII proteins were detected in the refined oil of HCN92 canola. Furthermore processing would destroy the enzymatic activity of the PAT and NPTII protein. As such, there will be no human exposure to these proteins as a result of the consumption of refined oil from line HCN92.

Nutritional Data

The analysis of nutrients from transgenic HCN92 canola and non-transgenic canola did not reveal any significant differences in the levels crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. Compositional analysis of processed canola oil indicated that no statistically significant differences between HCN92 and current commercial canola cultivars for the components analysed including fatty acids, chlorophyll and phytosterol content. Furthermore, the content of glucosinolates and erucic acid in glufosinate tolerant canola HCN92 was identical with that of seed from non-transgenic canola. These results collectively demonstrated that the novel herbicide tolerant genes introduced into HCN92 had no observable secondary effects impacting on composition or nutritional quality. The use of refined oil from line HCN92 was judged to be equivalent to traditional canola varieties and would therefore have no significant impact on the nutritional quality of the U.S. and Canadian food supply.

Canola meal was assessed to determine its safety for use in animal feed. In vivo tests performed on broiler chickens fed canola in their diets confirmed the safety previously established with purified PAT protein. It was also determined that PAT protein represented only 0.005% of protein in finished canola meal and enzyme activity was always destroyed in toasted canola meal. The overall results lead to the conclusion that there were no significant risks to livestock following ingestion of transgenic canola meal from HCN92.

Toxicity and Allergenicity

It was determined that there were no toxicity or allergenicity concerns with HCN92, 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 PAT and NPTII, and acute toxicity study in mice.

The amino acid sequences of both the PAT and NPTII proteins were compared to the amino acid sequences of known protein toxins using the GENBank sequence database. No significant similarities were found. Furthermore, the acute oral toxicity of PAT protein was studied in rats and showed no negative effects.

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 HCN92 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 HCN92 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.
The canola line HCN92 was field tested in Canada from 1990 to 1994. Agronomic and adaptation characteristics such as vegetative vigour, seed production and dormancy 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 HCN92 canola and the parental non-transformed variety was its resistance to 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 HCN92 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 HCN92 canola. As such, there will be no human exposure to this novel protein as a result of the consumption of refined oil from line HCN92.

A comparative analysis of nutrients from transgenic HCN92 canola and non-transgenic canola did not reveal any significant differences in the levels crude protein, crude fat, crude fibre, ash and gross energy in either the whole seed or processed meal. Compositional analysis of processed canola oil indicated that no statistically significant differences existed between HCN92 and current commercial canola cultivars in fatty acid, chlorophyll, phytosterol, glucosinolate and erucic acid content. These results collectively demonstrated that the novel herbicide-tolerant genes introduced into HCN92 had no observable secondary effects on nutritional composition or quality. The use of refined oil from line HCN92 was judged to be equivalent to that of traditional canola varieties and would therefore have no significant impact on the nutritional quality of the food supply.

Canola meal from HCN92 canola was also assessed to determine its safety for use in animal feed. In vivo tests performed on broiler chickens fed diets containing transgenic canola confirmed the safety previously established with purified PAT protein. It was also determined that PAT protein represented only 0.005% of protein in finished canola meal and enzyme activity was always destroyed in toasted canola meal. The overall results lead to the conclusion that there were no significant risks to livestock consuming transgenic HCN92 canola meal.

It was determined that there were no toxicity and allergenicity concerns with HCN92. 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. An acute oral toxicity study revealed that no negative effects were experienced by mice fed high doses of the PAT protein, providing further evidence supporting the lack of toxicity or allergenicity concerns related to the expression of PAT protein in HCN92 canola.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office European Commission Scientific Committee on Plants European Commission: Community Register of GM Food and Feed Food Standards Australia New Zealand Health Canada, Office of Food Biotechnology Japanese Biosafety Clearing House, Ministry of Environment Office of the Gene Technology Regulator THE COMMISSION OF THE EUROPEAN COMMUNITIES U.S. Department of Agriculture, Animal and Plant Health Inspection Service U.S. Food and Drug Administration U.S.Department of Agriculture, Animal and Plant Health Inspection Service

This record was last modified on Thursday, February 26, 2015