DD1996-11: Determination of Environmental Safety of Agrevo Canada Inc.'s Glufosinate Ammonium-Tolerant Canola Line HCN28

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Supplement to Decision Document DD96-11

Issued: 1996-05

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 Dir94-09 The Biology of Brassica napus L. (Canola/Rapeseed).

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division has evaluated information submitted by AgrEvo Canada Inc. regarding a glufosinate ammonium-tolerant canola line. They have determined that this plant with novel traits does not present altered environmental interactions when compared to currently commercialized canola varieties.

Unconfined release into the environment of HCN28 and other B. napus lines derived from it, but without the introduction of any other novel trait, is therefore considered safe.

Please note that, while determining the environmental safety of plants with novel traits is a critical step in the commercialization of these plant types, other requirements still need to be addressed, such as for Variety Registration (CFIA) and for the evaluation of feed (CFIA) and food safety (Health Canada).

Table of Contents

  1. Brief Identification of the Plant with Novel Traits (PNT)
  2. Background Information
  3. Description of the Novel Trait
    1. Glufosinate Ammonium Tolerance
    2. Development Method
    3. 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 to Be Invasive of Natural Habitats
    2. Potential for Gene Flow to 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. Regulatory Decision

I. Brief Identification of the Plant With Novel Traits (PNT)

Designation(s) of the PNT: HCN28

Applicant: AgrEvo Canada Inc

Plant Species: Canola (Brassica napus L.)

Novel Traits: Glufosinate ammonium (herbicide) tolerance

Trait Introduction Method: Agrobacterium tumefaciens-mediated transformation

Proposed Use of PNT's: Production of B. napus for seed oil for human consumption and seed oil and meal for livestock feed. These materials will not be grown outside the normal production area for canola.

II. Background Information

AgrEvo has developed a Brassica napus canola line tolerant to glufosinate ammonium, a broad spectrum non-residual herbicide. This B. napus line, referred to as HCN28 in the present document, will allow the use of glufosinate ammonium as a post-emergence herbicide, thus providing an alternative for weed control in canola production, and reducing reliance on soil-incorporated herbicides.

The development of HCN28 was based on recombinant DNA technology, by the introduction of a bacterial gene into a line of B. napus. This gene codes for phosphinothricin acetyltransferase, an enzyme that inactivates glufosinate ammonium through acetylation, thus conferring tolerance to glufosinate ammonium. It is the same as the gene inserted in HCN92, a glufosinate ammonium tolerant canola line that was authorized for unconfined release and feed use on March 10, 1995 (see DD95-01).

HCN28 has been field tested in Canada under confined conditions in Saskatchewan (1993, 95), Alberta (1994, 95), Manitoba (1995) and Ontario (1995).

AgrEvo has provided data on the identity of HCN28, a detailed description of the modification method, data and information on the gene insertion, the role of the inserted gene and of regulatory sequences in donor organisms, their molecular characterization, and full nucleotide sequences. The novel protein was identified and characterized, including its levels of expression in seed, potential toxicity to non-target organisms and allergenicity.

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, and resistance to white rust and blackleg, were compared to those of unmodified B. napus counterparts.

Canadian Food Inspection Agency (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:

  • potential of the PNT to become a weed of agriculture or to be invasive of natural habitats,
  • potential for gene-flow to wild relatives whose hybrid offspring may become more weedy or more 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, and
  • potential impact on biodiversity.

III. Description of the Novel Trait

1. Glufosinate Ammonium Tolerance

  • Phosphinothricin (L-PPT), the active ingredient of glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonia in susceptible plants within hours of application.
  • The phosphinothricin tolerance gene engineered into HCN28 codes for PPT- acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. It has extremely high substrate specificity; experimental data clearly showed that neither L-PPT's analog L-glutamic acid, D-PPT, nor any protein amino acid can be acetylated by the PAT enzyme.
  • The PAT gene was originally isolated from Streptomyces viridochromogenes, an aerobic soil actinomycete. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature.
  • The gene is linked to the same constitutive promoter as for line HCN92. Expression levels were quantified in seeds and ranged from 95 to 246 µg/mg of seed tissue; seed expression levels from HCN92 ranged from 150 to 223 µg/mg of seed tissue.
  • Studies showed that the enzyme was inactivated within one minute when subjected to typical mammalian stomach conditions.
  • The gene nucleotide sequence and the enzyme amino acid sequence were provided. The nucleotide sequence showed no significant homology the toxins or allergens entered in to GENEBANK DNA database.

2. Development Method

  • Brassica napus cultivar AC Excel was transformed using a disarmed non- pathogenic Agrobacterium tumefaciens vector; the vector contained the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease- causing genes were removed, and replaced with the gene coding for glufosinate ammonium tolerance. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was shown to be the case in HCN28.
  • The original transformant was backcrossed twice with B. napus line AC Excel; HCN28 was derived from single seed descent.

3. Stable Integration into the Plant's Genome

  • The provided data showed that there was no incorporation of any coding region from outside the T-DNA borders and that gene integration occurred at only one insertion site.
  • HCN28 is at least four generations removed from the original transformant.

IV. Assessment Criteria for Environmental Safety

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

CFIA evaluated data submitted by AgrEvo on the reproductive and survival biology of HCN28, and determined that vegetative vigor, flowering period, time to maturity, seed production, and overwintering were within the normal range of expression of characteristics in unmodified B. napus counterparts. HCN28 has no specific added genes for cold tolerance or winter hibernation. Based on the molecular characterization of the plants and their agronomic performance, CFIA concurs with AgrEvo that there is no reason to believe that line HCN28 would behave differently than HCN92 in its interactions with the environment.

The biology of B. napus, described in Dir94-09, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information provided by AgrEvo, HCN28 was determined not to be different from its counterpart in this respect. No competitive advantage was conferred to glufosinate ammonium-tolerant plants, other than tolerance to glufosinate ammonium.

Glufosinate ammonium is not used in normal crop rotation cycles, and resistance is therefore not an issue of concern in weed management control. Glufosinate-resistant B. napus volunteer plants can easily be managed by mechanical means and other available chemicals used to control B. napus.

The above considerations, together with the fact that the novel trait has no intended effect on weediness or invasiveness, led CFIA to conclude that HCN28 has no altered weed or invasiveness potential compared to currently commercialized canola varieties.

NOTE: A longer term concern, if there is general adoption of several different crop and specific herbicide weed management systems, is the potential development of crop volunteers with a combination of novel resistances to different herbicides. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, agricultural extension personnel, in both the private and public sectors, should promote careful management practices for growers who use these herbicide tolerant crops, to minimize the development of multiple resistance.

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

Brassica napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of the species B. napus (oilseed rape, Polish canola, turnip, rutabaga), B. juncea (brown mustard, Indian mustard), B. carinata (Ethiopian mustard), B. nigra (black mustard), Diplotaxis muralis (sand rocket, stinking wall rocket), Raphanus raphanistrum (wild radish), and Erucastrum gallicum (dog mustard) (Dir 94-09). Studies show that introgression of the herbicide tolerance gene is most likely to occur with B. rapa, the other major canola species and an occasional weed of cultivated land especially in the eastern provinces of Canada.

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 would only occur in managed ecosystems where glufosinate ammonium is used for broad spectrum weed control, e.g., in the cultivation of plant cultivars developed to exhibit glufosinate ammonium tolerance and in which glufosinate ammonium is used to control weeds. As with glufosinate ammonium-tolerant B. napus, these herbicide tolerant individuals, should they arise, would be easily controlled using mechanical and other available chemical means. Hybrids, if they developed, could potentially result in the loss of glufosinate ammonium as a tool to control these species. This, however, can be minimized by the use of sound crop management practices.

The above considerations led CFIA to conclude that gene flow from HCN28 to canola relatives is possible, but would not result in increased weediness or invasiveness of these relatives.

3. Altered Plant Pest Potential

The intended effect of the novel trait is unrelated to plant pest potential, and Brassica napus is not a plant pest in Canada (Dir94-09). In addition, agronomic characteristics of HCN28, including Albugo candida (white rust) and Leptosphaeria maculens (blackleg) resistance, were shown to be within the range of values displayed by currently commercialized B. napus varieties, leading to the conclusion that plant pest potential was not inadvertently altered.

CFIA has therefore determined that HCN28 did not display any altered pest potential.

4. Potential Impact on Non-Target Organisms

The PAT enzyme is rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis. It does not contain potential glycosylation sites nor does it possess proteolytic or heat stability, indicating that it is not a likely allergen. A search of the GENEBANK DNA sequence database revealed no significant homology with the toxins or allergens entered in that database.

Based on the above, and on the agronomic properties of HCN28, CFIA has determined that the unconfined release of this line will not result in altered impacts on interacting organisms, including humans, compared with currently commercialized counterparts.

5. Potential Impact on Biodiversity

HCN28 has no novel phenotypic characteristics which would extend its use beyond the current geographic range of canola production in Canada. Since outcross species are only found in disturbed habitats, transfer of novel traits would not impact unmanaged environments.

CFIA has therefore concluded that the potential impact on biodiversity of HCN28 is equivalent to that of currently commercialized canola lines.

V. Regulatory Decision

Based on the review of data and information submitted by AgrEvo Canada Inc., CFIA has concluded that neither the novel gene, nor its resulting gene product and associated novel trait, confer any intended or unintended ecological advantage to HCN28. Should these traits be transferred through outcrossing to related plants, these would not result in any ecological advantage.

Unconfined release into the environment of HCN28 and other B. napus lines derived from it, but without the introduction of any other novel trait, is therefore considered safe.

Please note that, while determining the environmental safety of plants with novel traits is a critical step in the commercialization of these plant types, other requirements still need to be addressed, such as for Variety Registration (CFIA) and for the evaluation of feed (CFIA) and food safety (Health Canada).

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