DD1996-16: Determination of Environmental Safety of Plant Genetic Systems Inc.'s (PGS) Male Sterile Corn (Zea mays L.) Line MS3

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Issued: 1998-03

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 Plant Genetic Systems Inc. (PGS). This information is in regard to the male sterile transgenic corn line MS3 for a novel corn hybridization system, InVigor™. CFIA has determined that this plant with novel traits does not present altered environmental interactions when compared to currently commercialized corn varieties in Canada.

Unconfined release into the environment of MS3 is therefore authorized. Any other Zea mays lines and intraspecific 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, and 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 the evaluation of feed safety (CFIA) and food safety (Health Canada) and for Variety Registration (CFIA).

Table of Contents

  1. Brief Identification of the Plant with Novel Traits (PNT)
  2. Background Information
  3. Description of the Novel Traits
    1. Nuclear Male Sterility
    2. Glufosinate Ammonium Tolerance
    3. Development Method
    4. 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 be Invasive of Natural Habitats
    2. Potential for Gene Flow to Wild 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: MS3; InVigor™ system

Applicant: Plant Genetic Systems Canada Inc. (PGS)

Plant Species: Zea mays L.

Novel Traits: Male sterility; glufosinate ammonium (herbicide) tolerance

Trait Introduction Method: Electroporation of immature embryos

Proposed Use of PNT's: Production of Zea mays hybrids for livestock and human consumption. These materials will not be grown outside the normal production area for corn in Canada.

II. Background Information

Plant Genetic Systems Inc. has developed a novel Zea mays hybridization system. This system involves the use of line MS3, a male sterile and herbicide tolerant line that does not produce viable pollen grains. When this line is crossed with an inbred line, the progeny is one hundred per cent true hybrid. Of these hybrids, 50% will be herbicide tolerant and male sterile. Even so, all plants within a stand should be well pollinated and produce seeds, since corn (an efficient outcrosser) is wind pollinated and the male fertile plants produce large quantities of pollen. The male sterility gene is the same as that found in the rapeseed (Brassica napus) lines MS1 and MS8 authorized for unconfined release by CFIA on April 28 1995 and October 21 1996, as explained in Decision Documents DD95-04 and DD96-17.

The development of line MS3 was based on recombinant DNA technology by the introduction of bacterial genes into the Zea mays inbred line H99. Nuclear male sterility results from the localized production of an RNAse (barnase) in a specific anther cell layer at a specific stage in anther development. Tolerance to the herbicide glufosinate ammonium results from the expression of a gene coding for phosphinothricin acetyltransferase, an enzyme that inactivates glufosinate ammonium through acetylation. Herbicide tolerance was introduced as a field selection trait to obtain 100% hybrid seed.

This line was not tested under confined conditions in Canada. It was tested since 1992 in the USA (Iowa, Illinois, Hawaï), Belgium, France, Chile and Argentina.

PGS has submitted data to CFIA on the identity of MS3: detailed descriptions of the modification method, data and information on the inserted DNA and the gene insertion site, the role of the inserted genes in donor organisms, the role of regulatory sequences in donor organisms, their molecular characterization and nucleotide sequences.

The novel proteins were identified and characterized, including their mode of action, potential toxicity to non-target organisms, potential for allergenicity, and levels of expression in the plant. A number of relevant scientific publications were also supplied.

Agronomic characteristics such as seed germination, vegetative vigor, time to maturity, time to tassel emergence, time to and process of silk extrusion, appearance of female inflorescences, male and female fertility, seed set, seed size and shape, cob size and yield parameters were compared to those of unmodified Z. mays counterparts. Stress adaptation was evaluated, including susceptibilities to various pests and pathogens of Z. mays under normal corn production practices and to herbicides other than glufosinate ammonium.

The Plant Biosafety Office of the Plant Health and Production Division, 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 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 Traits

1. Nuclear Male Sterility

  • The male sterility gene codes for the barnase ribonuclease (RNAse). Male sterility results from the production of this enzyme at a specific stage during anther development in the tapetum cell layer of the anther. The RNAse affects RNA production, disrupting normal cell functioning and arresting early anther development.
  • The gene is linked to an anther-specific promoter, and the enzyme was detected only in early stages of development of the tapetum cell layer of anthers. It was not detected in the other plant tissues tested: leaves, immature kernels, roots, dry and germinating seeds.
  • The full nucleotide sequence of the gene was provided. Barnase is a small single-domain protein, containing no disulfide bonds, metalion cofactors or other non-peptide components. It unfolds completely into an inactive form when heated. When subjected to comparative analyses using the FASTDB algorithm of Intelligenetics with three databases of polypeptide sequences, the enzyme amino acid sequence did not show significant homology with other proteins present in the databases, other than with ribonucleases from other bacilli. No resemblance with potential toxins or allergens was observed.
  • The barnase gene was isolated from Bacillus amyloliquefaciens, a common soil bacterium frequently used as a source for industrial enzymes. The enzyme is therefore naturally occurring in the soil. More generally, ribonucleases are very commonly found in various organisms including bacteria and plants.
  • The gene and its associated regulatory sequences are the same as those of the canola (B. napus) lines MS1 and MS8 that were authorized for unconfined release by CFIA on April 28, 1995 and October 21, 1996 (please see DD95-04 and DD96-17).

2. Glufosinate Ammonium Tolerance

  • Phosphinothricin (PPT), the active ingredient of the herbicide 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 MS3 codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. PAT has extremely high substrate specificity for L-PPT and dimethylphosphinothricin (DMPT), but cannot acetylate L-PPT's analog L-glutamic acid, D-PPT, nor any protein amino acid.
  • Expression levels of PAT were estimated through the quantification of RNA transcripts: 0.05 pg/µg of RNA was found in leaves and immature kernels, while none was detected in roots, dry and germinating seeds. A spectrophotometric assay showed no evidence for the presence of PAT in MS3 kernels.
  • The gene was isolated from Streptomyces hygroscopicus, an aerobic soil actinomycete. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature.
  • A plant derived coding sequence expressing a chloroplast transit peptide was co-introduced with the gene. This peptide facilitates the import of the newly translated enzyme into chloroplasts.
  • The nucleotide sequence of the gene was provided. When subjected to comparative analyses using the FASTDB algorithm of Intelligenetics with three databases of polypeptide sequences, the enzyme amino acid sequence did not show significant homology with other proteins present in the databases, except with other phosphinothricin acetyltransferases originating from different organisms. No resemblance with potential toxins or allergens was observed.

3. Development Method

  • The Zea mays inbred line H99 was transformed by electroporation of immature embryos in the presence of a preparation of linearized plasmids. One plasmid carried the genes of interest (male sterility and herbicide tolerance), while the other plasmid, called helper plasmid, carried genes useful at the development stage (e.g. a bacterial marker gene, not expressed in plants).

4. Stable Integration into the Plant's Genome

  • The data provided showed that there was incorporation of three copies of the plasmid carrying the genes of interest, and a partial copy of the helper plasmid, all inserted at a single insertion site. None of the coding sequences of the helper plasmid are expressed in MS3.
  • Segregation was predictable over at least six generations observed and showed that transformation resulted in integration at one single dominant locus.
  • Comparisons between the original transgenic plant and plants from four and six generations away showed no difference in the gene integration pattern.

IV. Assessment Criteria for Environmental Safety

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

CFIA evaluated data submitted by PGS on the reproductive and survival biology of MS3 and derived 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. MS3 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 PGS that there is no reason to believe that line MS3 and derived hybrids would behave differently than corn counterparts in their 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 PGS, MS3 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 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 MS3 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 MS3 derived hybrids to corn relatives is not possible 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 MS3 and 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 MS3 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 databases for polypeptide sequences revealed no significant homology with the known toxins or allergens entered in these databases.

Based on the above, and on the agronomic properties of MS3 and derived 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

MS3 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 PGS, CFIA has concluded that neither the novel genes, nor their resulting gene products and associated novel traits, confer any intended or unintended ecological advantage to MS3.

If at any time, PGS 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, PGS 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 MS3 is therefore authorized. Any other Zea mays lines and intraspecific 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, and 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).

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