DD1998-24: Determination of the Safety of the Crop Development Centre's 'CDC Triffid', a Flax (Linum usitatissimum L.) Variety Tolerant to Soil Residues of Triasulfuron and Metsulfuron-methyl

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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 Dir94-10 The Biology of Linum usitatissimum L. (Flax), and the guidelines Dir95-03 Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits.

The Canadian Food Inspection Agency (CFIA), specifically the Plant Biotechnology Office and the Feed Section of the Plant Products Division, with input from the Plant Health Risk Assessment Unit, CFIA, has evaluated information submitted by the Crop Development Centre regarding CDC Triffid. This plant was transformed with genes conferring tolerance to soil residues of the herbicides triasulfuron and metsulfuron- methyl, and resistance to the antibiotic kanamycin and production of nopaline as selectable markers. The CFIA has determined that this plant with novel traits (PNT) should pose no concerns with respect to environmental safety, the safety of livestock consuming feed derived from the PNT, and is considered substantially equivalent to flax by-products currently approved as livestock feed.

Unconfined release into the environment and livestock feed use of CDC Triffid is therefore authorized. Also, any other L. usitatissimum lines and intra-specific hybrids resulting from the same transformation event, and all their descendants, may be released provided that: no inter-specific crosses are performed; the intended use of the plants is the same; and that it is known following thorough characterization that such plants do not display any additional novel traits and are substantially equivalent to currently grown flax in terms of their potential environmental impact and livestock feed safety.

Table of Contents

  1. Brief Identification of the Plants with Novel Traits (PNT's)
  2. Background Information
  3. Description of the Novel Traits
    1. Tolerance to Soil Residues of Triasulfuron and Metsulfuron-methyl
    2. Kanamycin Resistance
    3. Nopaline Synthase
    4. Other novel DNA sequences
    5. Development Method
    6. Stability of Insertion of the Traits
  4. Assessment Criteria for Environmental Safety
    1. Potential of the PNT to Become a Weed of Agriculture or Become Invasive of Natural Habitats
    2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
    3. Altered Pest Potential
    4. Potential Impact on Non-Target Organisms
    5. Potential impact on biodiversity
  5. Nutritional Assessment Criteria for Use as Livestock Feed
    1. Nutritional Composition of PNT
    2. Antinutritional Factors
  6. Regulatory Decision

I. Brief Identification of the Plants with Novel Traits (PNT's)

Designation(s) of the PNT: CDC Triffid (experimental designation, FP967)

Applicant: Crop Development Centre of the University of Saskatchewan

Plant Species: Linum usitatissimum L. (flax, linseed)

Novel Traits: Tolerance to soil residues of triasulfuron and metsulfuron-methyl; kanamycin (antibiotic) resistance; production of nopaline.

Trait Introduction Method: Agrobacterium-mediated transformation.

Proposed Use of PNT's: For cultivation in soils containing residues of triasulfuron and metsulfuron-methyl in areas of Canada where flax is usually cultivated. This flax is to be grown for the extraction of linseed oil, and for animal feed (linseed meal).

II. Background Information

The Crop Development Centre of the University of Saskatchewan (CDC) has developed a flax line tolerant to soil residues of triasulfuron and metsulfuron-methyl which may result from a previous year's application of the products at labelled rates. Soil residues of these sulfonylurea herbicides, which are registered for use in Western Canada to control broadleaf weeds in wheat (triasulfuron and metsulfuron-methyl) and barley (metsulfuron-methyl), may persist at biologically active rates for several years subsequent to their use. Rotational crop options are restricted during this period of time as commercially unacceptable injury to many crops, including flax, may occur. Only crops such as wheat, oats and barley can be grown in these soils in the season following application of these herbicides or the land must be summer-fallowed. Flax may not be grown until 22 to 34 months after the use of either metsulfuron methyl or triasulfuron. Neither of these sulfonylurea herbicides, nor any other sulfonylurea herbicide is registered for use on flax. While it has been reported that flax may be tolerant to low, postemergent rates of certain sulfonylurea herbicides, researchers have recently reported severe injury to flax (cv. Norlin) when these post emergent sprays were applied (Wall, D.A. and Kenaschuk, E.O. 1996. Flax tolerance to thifensulfuron and tribenuron. Can. J. Plant Sci. 76:899-905).

The flax described herein, designated as CDC Triffid, was developed by the CDC to be cultivated the year following the use of triasulfuron or metsulfuron-methyl to provide an alternative to both the continuous cropping of wheat and barley on these soils and to summer-fallowing during this time.

The development of CDC Triffid was based on recombinant DNA technology and Agrobacterium-mediated transformation. An altered acetolactate synthase enzyme (ALS) from Arabidopsis thaliana was integrated into the genomic DNA of flax to confer tolerance to chlorosulfuron. Two other genes were also inserted: one conferring resistance to kanamycin, the other coding for the enzyme nopaline synthase. Both of these traits were used to select successful transformants in vitro.

CDC Triffid was field tested in Canada, under confined conditions, in Saskatchewan, Manitoba and Alberta from 1989 to 1995.

The CDC has submitted data and information on the identity of CDC Triffid, a description of the modification method, information on the stability of the insertion of the genes, activity of ALS in CDC Triffid compared to its non-modified counterpart cv. Norlin, molecular characterization of the kanamycin resistance gene, and levels of expression of the kanamycin resistance and nopaline synthase genes. The potential toxicity and the allergenicity of the novel proteins were assessed. Agronomic characteristics such as seed production, time to maturity and plant height were compared to those of the unmodified flax cv. Norlin, its closest counterpart.

The Plant Biotechnology Office of the Plant Health and Production Division, with input from the Plant Health Risk Assessment Unit, on behalf of the Plant Protection Division, CFIA, has reviewed the information submitted by the CDC for the determination of environmental safety, based on the following assessment criteria as described in the regulatory directive Dir94-08: Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits:

  • 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
  • potential impact on biodiversity

The Feed Section of the Plant Health and Production Division, CFIA, has also reviewed the information submitted by the CDC based on the following assessment criteria for determining safety and efficacy of livestock feed, as described in the regulatory directive Dir95-03: Guidelines for the Assessment of Livestock Feed from Plants with Novel Traits:

  • potential impact on livestock
  • potential impact on livestock nutrition

III. Description of the Novel Traits

1. Tolerance to Soil Residues of Triasulfuron and Metsulfuron-methyl

  • Sulfonylurea herbicides, such as triasulfuron and metsulfuron-methyl, target and bind to the enzyme acetolactate synthase (ALS) thereby inhibiting the biosynthesis of the branched chain amino acids valine, leucine and isoleucine and resulting in the accumulation of toxic levels of alpha-ketoglutarate.
  • In addition to its native ALS gene, CDC Triffid contains an als gene from a chlorsulfuron tolerant line of A. thaliana. This variant als gene differs from the wild type A. thaliana gene by one nucleotide and the resulting ALS enzyme differs by one amino acid from the wild type ALS enzyme. The inserted als gene is linked to its native promoter and terminator.
  • Enzyme extracts from CDC Triffid exhibited a slightly higher ALS activity compared to its non-modified counterpart cv. Norlin. Whereas the statistical significance of this higher activity could not be verified, it may be expected due to the presence of at least two additional copies of the als gene in CDC Triffid.

2. Kanamycin Resistance

  • Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell.
  • The kanamycin-resistance gene, nptII, isolated from the bacterium Escherichia coli, codes for an enzyme, neomycin phosphotransferase II (nptII) that phosphorylates kanamycin. This prevents kanamycin from binding to ribosomes and renders the cells resistant to the antibiotic. Plant cells that have been co-cultured with A. tumefaciens are plated onto a growth medium containing kanamycin. Those plant cells that have been successfully transformed and thus contain the kanamycin-resistance gene, are able to grow on this medium.
  • The nptII gene is linked to a constitutive promoter. The CDC has demonstrated, using PCR analysis, that the inserted nptII gene was unaltered when compared to the native E. coli gene. The gene product, nptII, was detected in seeds, cotyledons and leaves of CDC Triffid.
  • nptII is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids.

3. Nopaline Synthase

  • Nopaline synthase is an enzyme which catalyses the synthesis of nopaline, an opine which is formed as the result of the condensation of the amino acid arginine and alpha-ketoglutaric acid. When wild-type A. tumefaciens infects a host plant, the opine synthase gene present on the T-DNA region of the Ti plasmid of the bacterium directs infected host cells to synthesize an opine, such as nopaline. The type of opine produced is specific to the particular strain of A. tumefaciens. Opines are metabolized as a source of carbon and nitrogen only by a bacterium possessing a Ti-plasmid and the gene specific for catabolism of the particular opine.
  • The nopaline synthase gene, nos, is linked to its native promoter which is known to be constitutive with a slightly stronger expression in roots.
  • Levels of nopaline were quantified at two growth stages in CDC Triffid: seedling and pre-flowering, and was detected in roots, stems and leaves. Expression in roots ranged from 0.05 to 0.63 mg of nopaline/g tissue (fresh weight) at the seedling stage, and 0.07 to 0.47 mg/g (f.w.) at pre-flowering; in stems, at 0.01 to 0.33 mg/g (f.w.) at the seedling stage, and 0.02 to 0.12 mg/g (f.w.) at pre-flowering; and leaves, at 0.05 to 2.4 mg/g (f.w.) at the seedling stage, and 0.23 to 0.72 mg/g (f.w.) at pre-flowering. Nopaline was not detected in seeds.
  • A sequence homology search for nopaline synthase revealed similarity only to other opine synthases and no similarity was found to known sequenced toxins or allergens.
  • This trait was introduced to permit the identification of transformed plant embryos. Nopaline was detected in successfully transformed plant embryos.

4. Other novel DNA sequences

Genes conferring resistance to the antibiotics ampicillin, carbenicillin and spectinomycin were present in an E. coli vector used as an intermediary during the cloning of the target sequences to be transferred to flax. These three antibiotic resistance genes were under the control of a bacterial promoter which is active only in bacteria and not active in plants. These genes are therefore not expected to be expressed in CDC Triffid. Portions of Hind III fragments 10 and 23 (Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M., and Schell, J. 1983. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO 2:2143-2150.) from the nopaline Ti plasmid pTiC58 were also incorporated.

5. Development Method

  • The flax cv. Norlin was transformed using the disarmed A. tumefaciens Ti-plasmid vector pGV3850. The vector contained the T-DNA region of an A. tumefaciens plasmid from which oncogenic regions had been deleted and replaced with the mutant als gene and the kanamycin resistance gene.
  • Successful transformants were selected in vitro, on the basis of expression of kanamycin resistance and presence of nopaline. These were subsequently grown on medium containing chlorsulfuron to confirm the expression of the inserted chlorsulfuron-tolerant als gene.

6. Stability of Insertion of the Traits

Data were obtained from the literature (Mc Sheffrey, S. A., Mc Hughen, A., and Devine, M. D. 1992. Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum). Theoretical and Applied Genetics 84:480-486.) showing that the genes inserted into CDC Triffid were integrated in at least two unlinked loci, with a possible third locus exhibiting partial linkage to one of the other two. The expression of the genes remained stable after eight generations of selfing CDC Triffid plants.

IV. Assessment Criteria for Environmental Safety

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

The biology of flax (Linum usitatissimum L.), described in Dir94-10: The Biology of Linum usitatissimum L. (Flax), states that unmodified plants of this species are not invasive of unmanaged habitats in Canada, nor are they considered a weed.

CFIA evaluated information submitted by the CDC, on the reproductive and survival biology of CDC Triffid, and determined that growth habit, vegetative vigour, and seed yield, were substantially equivalent to its non-modified counterpart. No genes for cold tolerance or winter hibernation were inserted in CDC Triffid.

As described in Dir94-10, flax may appear in fields where the previous year's flax crop was harvested late resulting in seed pod shattering and escape of seed to the ground. As flax is a poor competitor, the presence of volunteers in a competitive succeeding crop such as cereals or canola would not be expected to cause significant yield loss. Flax volunteers can, however, cause difficulties as volunteers still green at harvest may interfere with crop harvesting and causing grain storage problems (Flax Council of Canada. 1996. Growing Flax: Production, Management and Diagnostic Guide. 3rd edition (Flax Council of Canada, 465 - 167 Lombard Avenue, Winnipeg, Manitoba, R3B 0T6).). CDC Triffid may also volunteer in soils where it is intended to be grown (i.e. in soils containing residues of triasulfuron and metsulfuron-methyl). Since CDC Triffid does not possess any novel traits that would confer increased competitiveness, these volunteers would not be expected to differ in their seriousness as a weed compared with other non-transformed flax varieties. Some suppression of CDC Triffid volunteers, as well as volunteers of other flax varieties can be provided with the use of herbicides that contain dichlorprop or dicamba or with a recently registered herbicide containing quinclorac.

The above considerations, together with the fact that the novel traits have no intended effects on weediness or invasiveness, led the CFIA to conclude that CDC Triffid does not possess altered weed or invasiveness potential compared to currently commercialized flax.

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

The biology of flax, as described in Dir94-10, indicates that there are no wild relatives in Canada that can freely hybridize with L. usitatissimum. The CFIA therefore concludes that gene flow from CDC Triffid to wild relatives is not possible in Canada.

Flax is predominantly a self-pollinating species, with a rate of natural outcrossing ranging from 0 to 5%, and this species possess does not possess any mechanism for wind or insect pollination. Thus gene flow to other L. usitatissimum is expected to occur at low frequency, if at all.

3. Altered Pest Potential

The intended effects of the novel traits are unrelated to plant pest potential, and flax is not a plant pest in Canada (Dir94-10). Furthermore, agronomic characteristics of CDC Triffid were shown to be within the range of values displayed by currently commercialized flax, which suggests that CDC Triffid has not been inadvertently altered. The CFIA has therefore determined that CDC Triffid does not display any altered pest potential.

4. Potential Impact on Non-Target Organisms

As discussed in Section III.3, CDC Triffid produces nopaline due to expression of the nos gene. Only A. tumefaciens strains of the nopaline sub-group and some Pseudomonas species produce the enzyme necessary for the catabolism of nopaline. Since oncogenic A. tumefaciens strains utilize this compound as a carbon and nitrogen source, the presence of nopaline in soils that have been cultivated to CDC Triffid may provide an environment for enrichment of this soil bacterium. Many dicotyledonous plants (dicots) and some gymnosperms (e.g. conifers) are susceptible to infection by A. tumefaciens. Monocotyledonous plants (monocots), such as small grain cereals, are not susceptible to infection by this bacterium.

A residual effects trial was conducted by the CDC in 1995, where a monocot and a dicot crop were grown on soil cultivated the previous year to CDC Triffid and its non-modified counterpart cv. Norlin. Crown galls were not detected on the dicot crop in the year of cultivation and there were no observed differences in plant counts of the dicot crop in those plots previously cultivated to CDC Triffid when compared to cv. Norlin.

NOTE: The effect on dicot crops (e.g. canola, lentils) grown in soils previously cultivated to CDC Triffid flax has not been fully assessed. As with all other flax varieties, cultivation of CDC Triffid for more than one growing season should not be encouraged and rotation with monocots (e.g. cereals) should be advised. It is presently recommended that flax not be grown the year following a previous flax crop in order to prevent the buildup of root diseases (e.g. Melampsora lini), and a three-year period is recommended between flax crops to avoid fusarium wilt (Dir94-10). These recommendations should be judiciously followed, and monitoring for the presence of crown galls in subsequent dicotyledonous crops should also be encouraged.

5. Potential impact on biodiversity

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

The presence of nopaline in plant tissue may contribute to the enrichment of A. tumefaciens in soils where CDC Triffid has been cultivated. A. tumefaciens is a ubiquitous soil bacterium and crown gall disease is a common occurrence in many agro-ecosystems. Given this, enrichment of A. tumefaciens in soils planted to CDC Triffid should not have a negative impact on the biodiversity of the rhizosphere.

Cultivation of CDC Triffid will increase rotational cropping options in areas where triasulfuron and metsulfuron-methyl have been used. CDC Triffid should not be planted in consecutive growing seasons in a single rotation.

V. Nutritional Assessment Criteria for Use as Livestock Feed

1. Nutritional Composition of PNT

Proximate analysis data for FP967 versus cv. Norlin (control) obtained from samples from three Saskatchewan plots in 1994 were evaluated. There were no differences in protein or ether extract content between the two lines. Crude fibre was significantly higher in FP967 than in Norlin, but was within the normal range for flax. For the amino acids valine, leucine, isoleucine and arginine, data were obtained from two samples of FP967 per site from eight sites, and from two samples of cv. Norlin from one site. There were no differences between cv. Norlin and FP967 in these amino acids.

2. Antinutritional Factors

The concentration of total cyanogenic compounds (total of linamarin, linustatin, neolinustatin) measured in three samples of each line, taken from one site, showed no differences between FP967 and cv. Norlin.

VI. Regulatory Decision

Based on the review of data and information submitted by the Crop Development Centre, the Plant Biotechnology Office of the Plant Health and Production Division, CFIA, has concluded that the novel genes and their corresponding traits in CDC Triffid should not confer any characteristics that would result in intended or unintended ecological effects following unconfined release, as long as this plant is properly managed. Proper management should include discouraging the continuous cropping of CDC Triffid.

Based on the review of data submitted to the Feed Section of the Plant Health and Production Division, CFIA, concludes that CDC Triffid with its novel traits does not in itself raise any concerns regarding livestock safety or nutritional composition. Flax seeds, flax oil, and flax meal are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. CDC Triffid has been determined to be substantially equivalent to traditional flax varieties, therefore, it meets the present ingredient definition of flax seeds and derivatives and is approved for use in Canada.

Unconfined release into the environment and livestock feed use of CDC Triffid is therefore authorized. Also, any other L. usitatissimum lines and intra-specific hybrids resulting from the same transformation event, and all their descendants, may be released, provided that: no inter-specific crosses are performed; the intended use is the same; and that it is known following thorough characterization that these plants do not display any additional novel traits and, are substantially equivalent to currently grown flax, in terms of their potential environmental impact and livestock feed safety.

This bulletin is published by the Plant Health and Production Division, Canadian Food Inspection Agency. For further information, please contact the Plant Biosafety Office or the Feed Section of the Plant Health and Production Division at the following address:

Plant Health and Production Division
Canadian Food Inspection Agency
59 Camelot Drive
Nepean, Ontario, K1A 0Y9

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