DD1996-15: Determination of Environmental Safety of Dekalb Canada Inc.'s Glufosinate Ammonium-Tolerant Corn Line DLL25

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Supplement to Decision Document DD96-15

Issued: 1996-10

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 Dir94-11 The Biology of Zea mays L.(Corn/Maize).

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biosafety Office of the Plant Health and Production Division, has evaluated information submitted by Dekalb Canada Inc. regarding the glufosinate ammonium-tolerant corn line DLL25 and derived corn hybrids. They have determined that these plants with novel traits do not present altered environmental interactions when compared to currently commercialized corn varieties.

Unconfined release into the environment of DLL25 is therefore authorized. Any other Zea mays lines and intra-specific hybrids resulting from the same transformation event, or transformed with the same genetic construct, and all their descendants, may also be released, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and that the resulting lines are substantially equivalent to currently grown corn, in terms of their potential environmental impact.

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: DLL25

Applicant: Dekalb Canada Inc.

Plant Species: Corn (Zea mays L.)

Novel Traits: Glufosinate ammonium (herbicide) tolerance

Trait Introduction Method: Microprojectile bombardment

Proposed Use of PNT's: Cultivation as hybrid grain corn for livestock feed and human consumption. These materials will not be grown outside the usual production area for corn in Canada.

II. Background Information

Dekalb Genetics Co. has developed a corn line tolerant to glufosinate ammonium, a broad spectrum non-residual herbicide. This Z. mays line, referred to as DLL25 in the present document, will allow the use of glufosinate ammonium as a post-emergence herbicide, thus providing an alternative for weed control in corn production, and reducing reliance on soil-incorporated herbicides.

The development of DLL25 was based on recombinant DNA technology, by the introduction of a bacterial gene into a line of Z. mays. This gene codes for phosphinothricin acetyltransferase, an enzyme that inactivates glufosinate ammonium through acetylation, thus conferring tolerance to glufosinate ammonium.

DLL25 has been field tested under confined conditions in 1994, 1995 and 1996, in the Canadian province of Ontario.

Dekalb Canada Inc. has provided data on the identity of DLL25, a detailed description of the modification method and the isolation of transformants, data and information on donor organisms, the genes and regulatory sequences, inserted sequences and the insertion site, 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 mode of action, its levels of expression in various tissues, potential effects on plant pathways, toxicity to non-target organisms and allergenicity.

The following agronomic characteristics were compared to those of unmodified Z. mays counterparts: test weight, dropped ears, stalk lodging, barrenness, stay green, seedling vigor, plant height, ear height, growing day units to 50% pollen shed, growing degree day units to 50% silk emergence, silk delay, green snap, maturity, yield, grain moisture, disease and insect susceptibilities.

The 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 DLL25 codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. It has extremely high substrate specificity.
  • The PAT gene was originally isolated from Streptomyces hygroscopicus, an aerobic soil actinomycete. The gene was slightly modified to conform with plant codon usage, which did not result in any modification to the expressed protein. The PAT enzyme is naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature.
  • The gene is linked to a well characterized constitutive promoter. Expression levels were quantified in leaves, roots, stalk, tassel, cob, husk, kernels, silk and pollen. Highest levels were found in leaves (1.0 - 4.6 mg/g of proteins (f.w.), corresponding to 0.1 - 0.46% of total soluble protein), and no PAT was detected in either silks, pollen or mature seeds.
  • Studies showed that the enzyme was heat and pH labile, and was inactivated within one minute when subjected to typical mammalian stomach and intestinal conditions.
  • The gene nucleotide sequence and the enzyme amino acid sequence were provided. The nucleotide sequence showed no significant homology to toxins or allergens entered in the GenBank DNA database.

2. Development Method

  • The Zea mays genotype A188xB73, a cross between two publicly available inbred lines, was transformed through microprojectile bombardment of embryogenic cell cultures.
  • The original transformant was then crossed with Dekalb's elite inbred lines.

3. Stable Integration into the Plant's Genome

  • The provided data showed that gene integration occurred at only one insertion site. The insertion site was very well characterized.
  • An antibiotic resistance marker, driven by a bacterial promoter, was used for selection in bacteria. Although this gene was partially integrated into the plant's genome, it is not expressed and the plant is not resistant to the antibiotic.
  • The novel gene was stably inherited over twelve generations.

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 Dekalb Canada Inc. on the reproductive and survival biology of DLL25-derived corn hybrids, and determined that vegetative vigor, flowering period, time to maturity, seed production, and disease and insect susceptibilities were within the normal range of expression of characteristics in unmodified Z. mays counterparts. DLL25 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 Dekalb that there is no reason to believe that line DLL25 would behave differently than corn counterparts in its interactions with the environment.

The biology of corn (Zea mays), described in Dir94-11, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. Corn does not possess the potential to become weedy due to traits such as lack of seed dormancy, the non-shattering aspect of corn cobs, and poor competitive ability of seedlings.

According to the information provided by Dekalb, DLL25 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. Tolerance to this herbicide will not, in itself, render corn weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified. Tolerance to glufosinate ammonium will not render corn weedy, since this herbicide is not presently used in crop rotation cycles involving corn. Glufosinate ammonium tolerant corn volunteer plants can easily be managed by mechanical means or by the use of other available herbicides.

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. Some canola (Brassica napus) varieties modified to express glufosinate ammonium tolerance are now registered in Canada. Should glufosinate ammonium tolerant corn and canola be grown in rotation, volunteers would not be controlled with this herbicide. Agricultural extension personnel, in both the private and public sectors, should therefore promote careful management practices for growers who use any of these herbicide tolerant crops.

The above considerations, together with the fact that the novel trait has no intended effect on weediness or invasiveness, led CFIA to conclude that DLL25 has no altered weed or invasiveness potential compared to currently commercialized corn varieties.

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

The biology of corn, as described in Dir94-11, indicates that there are no wild relatives in Canada that can hybridize with Zea mays.

CFIA therefore concludes that gene flow from DLL25 to corn relatives will not occur in Canada.

3. Altered Plant Pest Potential

The intended effect of the novel trait is unrelated to plant pest potential, and corn is not a plant pest in Canada (Dir94-11). In addition, agronomic characteristics of the DLL25-derived corn hybrids were shown to be within the range of values displayed by currently commercialized corn hybrids, and indicate that the growing habit of corn was not inadvertently altered.

CFIA has therefore determined that DLL25 does 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 possess proteolytic or heat stability. A search of the GenBank DNA sequence database revealed no significant homology with the known toxins or allergens entered in that database.

Based on the above, and on the agronomic properties of DLL25-derived corn hybrids, 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

DLL25 has no novel phenotypic characteristics which would extend its use beyond the current geographic range of corn production in Canada. Since corn does not outcross to wild relatives in Canada, there will be no transfer of novel traits to unmanaged environments.

CFIA has therefore concluded that the potential impact on biodiversity of this corn line is equivalent to that of currently commercialized corn varieties.

V. Regulatory Decision

Based on the review of data and information submitted by Dekalb 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 DLL25.

If at any time, Dekalb Canada Inc. becomes aware of any information regarding risk to the environment, or risk to animal or human health, that could result from release of these materials in Canada, or elsewhere, Dekalb Canada Inc. must immediately provide such information to CFIA. On the basis of such new information, CFIA may re-evaluate the potential impact of the release and re-evaluate its decision.

Unconfined release into the environment of DLL25 is therefore authorized. Any other Zea mays lines and intra-specific hybrids resulting from the same transformation event, or transformed with the same genetic construct, and all their descendants, may also be released, provided no inter-specific crosses are performed, provided the intended use is similar, provided it is known following thorough characterization that these plants do not display any additional novel traits and that the resulting lines are substantially equivalent to currently grown corn, in terms of their potential environmental impact.

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