DD1997-21: Determination of the Safety of Cotton Lines With Roundup Ready™ Genes (Gossypium hirsutum L.)
This Decision Document has been prepared to explain the regulatory decision reached under the guidelines Dir95-03 Guidelines for the Assessment of Plants with Novel Traits as Livestock Feed and Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.
The Canadian Food Inspection Agency (CFIA), specifically the Feed Section of the Animal Health and Production Division, and advice from the Plant Biosafety Office, CFIA has evaluated information submitted by Monsanto Canada. Cotton lines were derived from plants that were transformed with genes which conferred tolerance to glyphosate, the active ingredient in Roundup herbicide and resistance to kanamycin as a selectable marker. CFIA has determined that feed derived from these plants with novel traits should not pose a concern to environmental or livestock feed safety.
Feed use, as cottonseed and meal, of the glyphosate tolerant cotton lines 1445 and 1698, is therefore authorized. Any other G. hirsutum lines and intra-specific hybrids resulting from the same transformation event are also authorized, 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.
Table of Contents
- Brief Identification of the Plants with Novel Traits (PNT's)
- Background Information
- Description of the Novel Glyphosate Tolerance
- Assessment Criteria for Environmental Safety
- Nutritional Assessment Criteria as Livestock Feed
- Regulatory Decision
Designation(s) of the PNT: Roundup Ready™ Cotton, line 1445, line 1698
Applicant: Monsanto Canada Inc.
Plant Species: Cotton (Gossypium hirsutum) L.
Novel Traits: Novel tolerance to glyphosate, the active ingredient of Roundup® herbicide, expressed by the Roundup-Ready™ genes; kanamycin (antibiotic) resistance.
Trait Introduction Method: Agrobacterium tumefaciens-mediated transformation.
Proposed Use of PNT's: Production of cotton for fibre, cottonseed and cottonseed meal for livestock feed, and cottonseed oil for human consumption. These materials will be grown outside Canada, in the usual production areas for cotton. Cottonseed and cottonseed meal will be imported into Canada for livestock feed use only.
Monsanto Canada Inc. has developed cotton lines, derived from the variety Coker 312, which expresses their proprietary Roundup-Ready™ genes. These confer novel tolerance to glyphosate, the active ingredient of Roundup® herbicide, which can control or suppress economically important weeds in cotton production.
The development of the glyphosate tolerant cotton lines was accomplished with recombinant DNA technology. Two genes from a bacterial source (the Roundup- Ready™ genes), were inserted into the Coker 312 cotton variety. One gene imparts reduced sensitivity to Roundup® at the biochemical site of the herbicide's activity and the other expresses an enzyme that enables the plant to degrade glyphosate. The expression of both genes is directed to the chloroplasts, the site of the herbicide activity, by the addition of genetic coding sequences of a plant derived transit peptide. These genes were introduced along with a gene conferring resistance to the antibiotic, kanamycin. The antibiotic resistance gene is of no agricultural interest; the trait was used to select transformed from non-transformed plants during the development phase.
Field tests for the glyphosate tolerant cotton lines took place at 65 locations throughout the mainland United States and Puerto Rico. The lines have received complete approval for commercialization in the United States. Health Canada has determined that cottonseed oil derived from this cotton is substantially equivalent to that derived from currently commercialized cotton (December 19, 1996).
Monsanto has provided data on the identity of the glyphosate tolerant cotton lines, a detailed description of the modification method, data and information on the gene insertion sites, copy numbers and levels of expression in the plant, the role of the inserted genes and regulatory sequences in donor organisms, and full nucleotide sequences. The novel proteins were identified, characterized, and compared to the original bacterial proteins, including an evaluation of their potential toxicity to livestock. Relevant scientific publications were also supplied.
Agronomic characteristics such as plant vigour, growth, morphology, germination and flowering, were compared to those of unmodified cotton counterparts. Stress adaptation was evaluated, including susceptibility to various pests and pathogens.
Data to support the suitability of cottonseed and cottonseed meal from the cotton lines as livestock feed was provided. Proximate analyses including crude protein, crude fat, crude fibre, ash, moisture and gross energy were supplied. Since the genetic modification effects an enzyme in the biosynthetic pathway for aromatic amino acids, the amino acid profiles of the transformed cotton lines were provided.
The Feed Section of the Animal Health and Production Division, and advice from the Plant Biosafety Office has reviewed the above information. The following assessment criteria, as described in the regulatory directives Dir94-08 and Dir95-03 were used to determine the suitability as livestock feed and the environmental safety of feed from these plants with novel traits:
- potential impact on livestock
- potential impact on livestock nutrition
- potential impact of the PNT's or their gene products on non-target species, including humans
- potential for the PNT's to become plant pests,
- potential of the PNT's to become weeds of agriculture or to be invasive of natural habitats
- potential impact on biodiversity
- potential for gene-flow to wild relatives whose hybrid offspring may become more weedy or more invasive
- A genetic construct containing 2 genes which confer tolerance to glyphosate, the active ingredient in Roundup® herbicide, was used to transform line 1445. A similar construct containing only one of the two genes was used to transform line 1698
- The CP4 EPSPS gene expresses a bacterial version from Agrobacterium tumefaciens strain CP4, of 5-enol-pyruvlshikimate-3-phosphate synthase, a plant enzyme involved in the shikimate biochemical pathway for the production of aromatic amino acids. Glyphosate inhibits the native plant EPSPS, thus blocking the shikimate pathway and halting the production of these necessary amino acids. The CP4-EPSPS is a glyphosate tolerant version of the enzyme with high catalytic activity. Under glyphosate selection, the addition of the bacterial, glyphosate tolerant enzyme will permit the normal production of aromatic amino acids.
- The second gene, gox, is derived from Achromobacter sp., a ubiquitous soil bacteria species which expresses an enzyme that degrades glyphosate by conversion to aminomethylphosphonic acid (AMPA) and glyoxylate. Although line 1495 had been transformed with a vector containing both of the Roundup Ready™ genes, Southern analysis indicated that the gox gene had not been inserted into the plants genome. Cotton line 1698 was transformed with a vector lacking the gox gene. The absence of gox gene expression was confirmed in both lines by ELISA.
- A plant-derived coding sequence expressing a chloroplast transit peptide was co-introduced with each of the glyphosate resistance genes. The chloroplast transit peptides facilitates the import of the protein products from the glyphosate resistance genes into the chloroplast, which is the location of the shikimate pathway and the site of action for glyphosate.
- The CP4-EPSPS gene with the chloroplast transit peptide coding sequence is linked to a constitutive promoter. Expression of the enzyme averaged 0.082 µg/mg fresh weight (f.w) in the seed and 0.052 µg/mg (f.w) in the leaf for line 1445. For line 1698 expression of the enzyme in the seed averaged 0.205 µg/mg (f.w) and 0.031 µg/mg (f.w) in the leaf.
- The plant expressed enzyme showed no significant protein sequence homology to a database of known toxins or allergens assembled from the Swissprot, Genepept, and Pir protein. It is common in nature and no toxic or allergenic effects would be expected to occur. Data was presented which showed that the CP4 EPSPS enzyme is rapidly inactivated by heat and by digestion in simulated mammalian gastric fluid.
- Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thereby disrupting normal protein synthesis and killing the bacterial cell.
- The kanamycin-resistance gene, isolated from the bacterium E. coli, codes for Neomycin phosphotransferase type II (NPT II), an enzyme that phosphorylates kanamycin, thereby preventing it from binding to ribosomes and rendering the cells resistant to the antibiotic.
- The gene is linked to a constitutive promoter. Expression levels for the NPT II protein in line 1445 averaged 0.045 µg/mg (f.w) in the leaf and 0.0067 µg/mg (f.w) in the seed. For line 1698 NPT II expression averaged 0.311 µg/mg (f.w) in the leaf and 0.204 µg/mg (f.w) in the seed.
- This protein is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids.
- The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database.
- The full nucleotide sequence and corresponding amino acid sequence were provided.
- A streptomycin/spectinomycin resistance marker gene was used for selection of bacterial transformants during the development process. The gene is driven by a bacterial promoter and its lack of expression in the transformed plant was confirmed by specific ELISA.
- Coker line 312 cotton 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 genes coding for Roundup® tolerance and kanamycin resistance were inserted. 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 demonstrated in the glyphosate tolerant cotton lines.
- The inserted T-DNA, including the partial genes, was well characterized by restriction enzyme analysis, for the 2 lines. The stability of the lines was demonstrated for 5 generations. Southern blot data demonstrating the stability of the CP4 EPSPS and NPT II genes was provided using DNA from the R3 and R5 generations.
1. Potential of the PNT's to Become Weeds of Agriculture or Be Invasive of Natural Habitats
Cotton (G. hirsutum) is a member of the family Malvaceae. It is a perennial species cultivated as an annual and grown in the United States, mostly in areas from Virginia southward and westward to California. Cotton is not considered a weed pest in the regions where it is grown. It is not grown in Canada as it is not adapted to environmental conditions found at these latitudes
The glyphosate tolerant cotton lines have no specific added genes for cold tolerance or winter hibernation; no overwintered plants are possible. No cultivation of these cotton lines in any location in Canada will occur.
The biology of Gossypium hirsutum, described in the submission, shows that unmodified plants of this species are not invasive of unmanaged habitats in Canada. According to the information provided by Monsanto, cotton lines carrying the Roundup™ Ready genes were determined not to be different from their counterparts in this respect.
2. Potential for Gene Flow to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
The pollen is heavy and sticky and cross pollination usually occurs in the presence of insect pollinators (bumble bees and honey bees). Cotton is normally self pollinating and the range of natural crossing among cotton plants is very limited.
Wild species of cotton (G. barbadense and G. tomentosum) occur only in arid parts of the tropics and sub-tropics. There are no wild relatives in Canada that can naturally hybridize with G. hirsutum. CFIA therefore concludes that gene flow from glyphosate tolerant lines to cotton relatives is not possible in Canada.
Cotton is not a plant pest in Canada and the intended effects of both novel traits are unrelated to plant pest potential. In addition, agronomic characteristics of the Roundup® tolerant cotton lines, were shown to be within the range of values displayed by currently commercialized cotton varieties. Susceptibilities to diseases such as: bacterial blight; boll rot; Verticillium wilt; and Ascochyta blight were unchanged, leading to the conclusion that plant pest potential was not inadvertently altered. Similarly, no differences were noted in the susceptibility of the transformed cotton lines to insect pests when compared to non-transformed cotton lines. Comparable responses between the roundup tolerant cotton lines and the controls were noted for: aphids; fleahoppers; boll weevils; tobacco budworm; cotton bollworm; and thrips.
CFIA has therefore determined that Roundup® tolerant lines do not display any altered plant pest potential.
The detailed characterization of the novel gene and resulting enzyme, as briefly summarized in Part III of the present document, has led to the conclusion that these do not result in altered toxic or allergenic properties. The enzyme is ubiquitous in microorganisms, fungi and plants, and is rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis.
Cotton is known for the production of anti-nutritional factors and untreated raw seed is unsuitable as feed for monogastric animals. The levels of the toxicant gossypol were not significantly different when the 2 lines were compared to the non-transformed controls. No significant difference in levels of the toxicant cyclopropenoid fatty acids (dihydrosterculic, sterculic and malvalic) were determined for the two lines. The four primary aflatoxins associated with cotton were not detected in either of the lines at the limits of detection.
Feeding trials were conducted on: rats with 5-10% raw cottonseed meal included in the diet; bobwhite quail with 10% raw cottonseed meal included in the diet; and catfish with 20% cottonseed meal included in the diet. No significant difference in weight gain, feed conversion or gross necroscopy was observed when diets containing Roundup® tolerant cottonseed meal were compared to diets containing raw cottonseed meal from the control cotton line.
Based on the above, CFIA has determined that, when compared with currently commercialized counterparts, the use of the Roundup® tolerant cotton lines as livestock feed will not result in altered impacts on interacting organisms, including humans.
The CFIA has determined that the cotton lines will have no impact on biodiversity. The lines have no novel phenotypic characteristics which would extend their use beyond the current geographic range of cotton production into Canada. Since cotton does not outcross to wild relatives in Canada, there will be no transfer of novel traits to unmanaged environments.
Cottonseed contains a high number of anti-nutritional factors and the raw seed is unsuitable for monogastric animals. Processing serves to lower the levels of endogenous toxins and renders the cottonseed meal suitable for livestock feed. The levels of total gossypol in the seeds of line 1445 were significantly higher than in the Coker 312 control. Further studies with isolines derived from glyphosate tolerant line 1445 showed no tendency to higher gossypol levels compared to the Coker 312 controls. The study authors concluded that this indicated that elevated gossypol levels were not associated with the glyphosate tolerant trait. Total gossypol in the seeds of line 1698 was significantly lower than the Coker 312 control. Levels of gossypol in the raw seed for both lines were well within the reported range for cotton. Gossypol was below the level of detection in cottonseed oil from the transformed lines and the control. Anthocyanin, flavonoid and tannin in the transformed lines were within acceptable levels for cotton and within the range of the controls for the Roundup tolerant lines. The levels of the cyclopropenoid fatty acids, malvalic, sterculic and dihydrosterculic acids were found to be within the normal range for cotton.
Seed from the two PNT lines from six U.S. locations was shown to be substantially equivalent to the control Coker 312 line in concentration of oil, carbohydrate and ash. Protein concentration in both transformed lines was slightly but significantly higher than the control lines, although the amounts were within the normal range for cottonseed. The company has suggested that minor differences in nutrient values in the transformed lines may reflect the large variability of the parental strain. Despite differences in protein concentration, the amino acid profile was the same for the transformed and control lines. The crude fat content and fatty acid profile were the same for the Roundup® tolerant and control line and were within the normal published range for cottonseed. Additional analyses of composite samples of cottonseed products (full fat flour, toasted meal and refined oil) from each line showed that these products from the PNT lines were similar in composition to the control line. Therefore, the introduction of the novel trait into G. hirsutum would not likely result in any secondary effects affecting the composition or nutritional quality of the cultivar.
Based on the review of data and information submitted by the Monsanto Company, and thorough comparisons of the Roundup® tolerant cotton lines with unmodified cotton counterparts, the Feed Section of the Plant Products Division has concluded that the novel genes and their corresponding traits do not in themselves raise any concerns regarding the safety or nutritional composition of the transformed lines. Cottonseed and cottonseed meal are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. As Roundup® tolerant cotton has been assessed and found to be substantially equivalent to traditional cotton varieties, with respect to safety and nutritional quality, Roundup® tolerant lines and their products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada. As none of the Roundup® tolerant cotton lines will be grown in Canada and the seed cannot overwinter, the release of the feed as meal or seed into the environment should have no intended or unintended effect.
Feed use, as cottonseed and meal of the glyphosate tolerant cotton lines 1445 and 1698, is therefore authorized. Any other G. hirsutum lines and intra-specific hybrids resulting from the same transformation event are also authorized, 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.
This bulletin is published by the Plant Health and Production Division. For further information, please contact the Plant Biosafety Office or the Feed Section at:
Plant Biosafety Office
Plant Health and Production Division
Plant Products Directorate
59 Camelot Drive, Nepean
Ontario, K1A 0Y9
Animal Health and Production Division
Animal Products Directorate
59 Camelot Drive, Nepean
Ontario, K1A 0Y9
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