Cotton lines 531, 757, and 1076 (trademark Bollgard®, Ingard®) were developed through specific genetic modifications to be resistant to major caterpillar pests of cotton. The transgenic cotton lines express a modified gene (cry1Ac) that encodes an insecticidal crystalline Cry1Ac delta-endotoxin protein, derived from the soil bacterium Bacillus thuringiensis subsp. kurstaki strain HD73. Insecticidal activity is caused by the selective binding of Cry1Ac protein to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause gut paralysis and eventual death due to bacterial sepsis. Delta-endotoxins, such as the Cry1Ac protein expressed in cotton lines 531, 757, and 1076, exhibit highly selective insecticidal activity against a narrow range of lepidopteran insects such as cotton bollworm, tobacco budworm and pink bollworm. The specificity of action is directly attributable to the presence of specific receptors in the target insects. There are no receptors for delta-endotoxins of B. thuringiensis on the surface of non-lepidopteran insect guts or mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

An antibiotic resistance marker gene (neo) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of these plants. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the neo gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Another selectable marker gene, aad (3"(9)-O-aminoglycoside adenylyltransferase (AAD)), was also inserted into the host genome. This gene, which was not expressed in the plants, was used during the development process to select for bacterial colonies that had been transformed with recombinant plasmid DNA.

Unless otherwise indicated, the information presented here is based on documentation pertaining to cotton line 531.

The cotton lines 531, 757, and 1076 were produced by Agrobacterium-mediated transformation of the cotton (Gossypium hirsutum) line L. cv Coker C312 with plasmid PV-GHBKO4. Plasmid PV-GHBK04 contained the following elements: the 0.4 kb oriV fragment from the RK2 plasmid fused to the 3.4 kb segment of pBR322 allowing maintenance in Escherichia coli and in Agrobacterium tumefaciens. This was fused to the 360 bp DNA fragment from pTiT37 plasmid, which contained the nopaline T-DNA right border.

The remaining portion consisted of two genes engineered for plant expression, the cry1Ac and the NPTII encoding neo gene. The cry1Ac gene was modified for optimal expression in plants and contained part of the 5' end of the cry1Ab gene with a portion of the cry1Ac gene. Expression of the modified cry1Ac gene was regulated by cauliflower mosaic virus (CaMV) 35S promoter with a duplicated enhancer region and the nontranslated region of the soybean alpha subunit of the beta-conglycin gene which provided the mRNA polyadenylation signals (7S 3’ terminator sequence).

The transformation plasmid also contained the aad gene isolated from E. coli bacterial transposon Tn7, which encodes the enzyme aminoglycoside adenyltransferase (AAD) that confers resistance to the antibiotics spectinomycin and streptomycin. The aad gene was under the control of its own bacterial promoter and terminator and was included in the construct as a marker to allow for selection of bacteria containing PV-GHBK04 prior to transformation of the plant cells. The aad gene has no plant regulatory sequences and was not expressed in plant tissues.

The neo gene was located downstream of the aad gene and its expression was regulated using the CaMV 35S promoter and the non-translated region of the 3’ region of the nopaline synthase gene (nos) from the pTiT37 plasmid of A. tumefaciens strain T37.

The Introduced DNA
The inserted cry1Ac gene encoded an insecticidal protein similar to a full length Cry1Ac protein with some minor changes arising from the design of the gene. The encoded Cry1Ac plant produced protein was 99.4% homologous to the native protein from B. thuringiensis subsp. kurstaki HD73.

Southern blot analyses of genome DNA from line 531 demonstrated that two copies of the T-DNA insert were integrated in a head-to-tail arrangement. One T-DNA insert contained a full-length cry1Ac gene and the NPTII encoding gene, and the second insert contained an inactive 3’ portion of the cry1Ac gene. The two inserts were linked and segregated as a single locus. Similar analyses demonstrated that the ori322 region, present in PV-GHBK04, was not transferred into the 531 genome. The aad gene was present but was not expressed since it was under the control of a bacterial promoter.

Genetic Stability of the Introduced Trait
The stability of the inserted genes for lines 531 and 1076 was demonstrated over 4 generations. Stability for line 757 was tested and confirmed across 2 generations.

Expressed Material
Cry1Ac protein expression levels in each of the three transgenic cotton lines were quantitated using enzyme linked immunosorbent assay (ELISA). Mean Cry1Ac expression in line 531 was 1.56 µg protein/g and 0.86 µg protein/g fresh weight in leaf and seed tissue respectively. In the whole plant the Cry1Ac protein averaged 0.044 µg protein/g fresh weight, (25 µg/ plant) for a total of about 1.44 g/ acre. Expression varied approximately 3 fold over the growing season, with the levels peaking late in the season. Expression of the Cry1Ac protein was higher in line 757. Average values were 12.6 µg protein/g fresh weight and 9.9 µg protein/g fresh weight in leaves and seeds respectively. Expression varied less than 3 fold over the season and peaked early in the season in leaf tissue. Whole plant amounts were 1.1 µg protein/g fresh weight (200 µg/plant) for a total of 12.2 g/ acre. Line 1076 produced the cry1Ac protein at an average of 12.2 µg protein/g fresh weight and 12.7 µg protein/g fresh weight in leaves and seed respectively. Gene expression over the growing season varied less than 5 fold, with the highest concentration occurring early in the season. Whole plant totals were 1.1 µg protein/g fresh weight (389 µg/plant) for a total of 23.3 g/ acre. (Calculations per acre based on 60000 plants/acre). Expression in pollen and nectar was negligible in all three lines.

Expression levels for the NPTII protein in line 531 were 3.14 µg protein /g fresh weight and 2.45 µg protein/g fresh weight in leaf and seed tissues respectively. Expression varied about 2 fold over the season. In line 757 NPTII expression was 6.9 µg protein/g fresh weight and 3.3 µg protein/g fresh weight in leaves and seeds respectively. For line 1076, the NPTII expression was 16.3 µg protein/g fresh weight and 7.9 µg protein/g fresh weight in leaves and seed respectively. Gene expression varied 2 to 3 fold over the growing season for line 757 and line 1076.

Field Testing
The transgenic cotton lines 531, 757 and 1076 were field tested in the United States (1990-1994). Agronomic characteristics such as yield, boll size, plant vigour, growth, morphology, germination and flowering, were compared to those of unmodified cotton counterparts and were shown to be within the range of non-transformed cotton lines. Stress adaptation was evaluated, including susceptibility to various pests and pathogens. Susceptibilities to diseases such as bacterial blight, boll rot, Fusarium, Phymatotricum root rot, and verticillium wilt were unchanged. Processing qualities of cotton lint such as micronaire, length, strength and elongation were compared. The observed variability was within the range of inherent variability of cotton varieties and was not attributed to the inserted genes. Reports demonstrated that the cotton lines 531, 757 and 1076 did not exhibit weedy characteristics and had no effect on nontarget organisms or the general environment relative to conventional cotton varieties.

Outcrossing
Cotton (Gossypium hirsutum) is mainly a self-pollinating plant, but insects, especially bumblebees and honeybees, also routinely transfer pollen. The pollen is heavy and sticky and the range of natural crossing is limited. Outcrossing rates of up to 28% to other cotton cultivars have been observed under field conditions and decline rapidly with distance from the pollen source. Given proximity and the availability of insects as pollen vectors, transgenic cotton lines 531, 757 and 1076 are likely to hybridize with other cotton varieties.

In the United States, compatible species include G. hirsutum (wild or under cultivation), G. barbadense (cultivated Pima cotton), and G. tomentosum. There are no wild relatives or wild populations of cotton in Canada that can naturally hybridize with G. hirsutum. It was reported that gene movement from G. hirsutum to G. barbadense may be possible given suitable conditions, while gene transfer to G. tomentosum is less probable due to chromosomal incompatibility and non-synchronous flowering periods. Overall, the probability of gene transfer in unmanaged ecosystems is unlikely due to the relatively isolated distribution of Gossypium species, different breeding systems, and genome incompatibility. Hybrids resulting from artificial crosses between cotton and wild species are generally sterile, unstable and of poor fitness

Weediness Potential
The Cry1Ac or NPTII encoding genes were not expected to confer an ecological advantage to transgenic cotton lines 531, 757 and 1076 and their potential hybrid offspring. Resistance to kanamycin and specific lepidopteran insects will not render cotton weedy or invasive of natural habitats since none of the reproductive or growth characteristics have been modified. Field trial reports indicated that traits which may confer a selective advantage, such as increase in volunteers from seed, regrowth from stubble, or increase in seed dormancy in cotton lines 531, 757 and 1076 were equivalent to non-modified varieties.

There are no specific problems with cotton as a weed. Cottonseed may remain in the field after harvesting and germinate under favorable conditions. Seeds may also survive mild and dry winters. Suitable treatments for any volunteers in the next crop include cultivation and the use of herbicides.

Secondary and Non-Target Adverse Effects
It was concluded that the introduction of the Cry1Ac and NPTII encoding genes into transgenic cotton lines 531, 757 and 1076 would not result in any deleterious effects or significant impacts on nontarget organisms, including those that are recognized as beneficial to agriculture and those that are recognized as threatened or endangered in the United States and Canada.

The novel proteins, Cry1Ac and NPTII, expressed in these cotton lines are not known to have any toxic properties on any nontarget organisms. The lack of known toxicity for these proteins and the low levels of expression in plant tissue suggest no potential for deleterious effects on beneficial organisms such as bees and earthworms.

The insecticidal protein was shown to be active on only lepidopteran species, even when fed at levels that were well above the concentrations that were effective on target species. Host range specificity was similar for the native and synthetic proteins. Tobacco hornworm was the most sensitive species and tobacco budworm, corn earworm and European corn borer were relatively less sensitive. The test system included six non-target species representing the orders Coleoptera, Diptera, Orthoptera and Homoptera (boll weevil, Southern corn rootworm, Colorado potato beetle, yellow fever mosquito, German cockroach and green peach aphid).

Dietary toxicity studies were performed using the microbial protein on beneficial insects (honeybee, ladybird beetle, green lacewing larvae and parasitic wasp). No effect was observed on non-target insects at concentrations more than 100 fold higher than those used to control target insects.

Acute oral and short-term studies were undertaken with albino mice and bobwhite quail. After 8 days there were no observable effects on mice fed the insecticidal protein at the highest dose of 4,000 mg/kg body weight. Raw cottonseed was fed at up to 100,000 ppm to bobwhite quail with no observed effects. These observations were expected, as the novel proteins are rapidly inactivated in simulated mammalian stomach fluids by enzymatic degradation and pH-mediated proteolysis. The proteins expressed in transgenic cotton lines were shown to be equivalent to the original microbial proteins produced by the common soil B. thuringiensis subsp. kurstaki bacteria.

Impact on Biodiversity
The transgenic cotton lines 531, 757 and 1076 had no novel phenotypic characteristics that would extend their use beyond the current geographic range of cotton production. Since there is no occurrence of wild relatives of cotton in Canada, there will be no transfer of novel traits to unmanaged environments. Similarly, as the risk of gene transfer to wild relatives in the United States is very remote, it was determined that the risk of transferring genetic traits from cotton lines 531, 757 and 1076 to species in unmanaged environments was insignificant.

Dietary Exposure
Refined cottonseed oil and cellulose from processed linters of cottonseed are the only cotton products consumed by humans. Processed linters are essentially pure cellulose (>99%) and are subjected to heat and solvent treatment that would be expected to remove and destroy DNA and protein. Similarly, the refining process for cottonseed oil includes heat, solvent and alkali treatments that would remove and destroy DNA and protein. Typically, cottonseed oils are pooled and blended together and it is anticipated that the oil from 531, 757, and 1076 cottonseed will not be handled or treated any differently than other cottonseed oils. The genetic modification in these cotton lines will not result in any change in the consumption pattern for this product. As the introduced gene products are not detectable in the refined oil produced from transgenic cotton, there is no anticipated human exposure to these proteins based on normal consumption patterns.

Nutritional Data
Compositional comparison of cottonseed from lines 531, 757 and 1076 was made to commercial non-transgenic cottonseed. The cottonseed used for compositional analyses was taken from four to six trial sites. The compositional analyses of cottonseed included proximates (crude protein, crude fat, crude fibre, ash and gross energy), amino acid composition, fatty acids profile, aflatoxins and levels of tocopherols.

The concentrations of protein, oil, carbohydrate and ash were the same for transgenic cotton lines 531, 757 and 1076 and the control parent line Coker 312 line. Line 1076 contained slightly higher carbohydrate and slightly lower oil concentrations than the control line, but both were within the normal range for cottonseed. Fatty acid concentration was within the normal published range for cottonseed. Additional analyses of composite samples of cottonseed products (raw meal, toasted meal, kernel, refined oil) from each line showed that the products from the transgenic cotton lines were similar in composition to the control line. Feed studies of a four week rat feeding trial showed no difference in weight gain of animals fed diets containing 10% raw cottonseed meal from line 531 vs. Coker 312.

The analysis of the fatty acid composition of refined oil from 531 and 757 cotton did not reveal any significant differences with the parent, non-transgenic variety and was within the normal range reported for cottonseed oils. In addition, the levels of alpha-tocopherol in refined oil from transgenic and control lines were similar. The consumption of refined oil from 531 and 757 cottonseed will have no significant impact on the nutritional quality of the food supply.

Toxicity
Toxicity of cotton lines was assessed by analysis of naturally occurring toxins in cotton and an evaluation of the novel proteins Cry1Ac and NPTII. Toxicant analyses in cottonseed and refined oil evaluated levels of gossypol and cyclopropenoid fatty acids (sterculic and malvalic acid).

Cotton is known for the production of anti-nutritional factors and untreated raw seed is unsuitable as livestock feed for monogastric animals. The transgenic and parental lines were assayed for the presence of potential toxins, including gossypol, dihydrosterculic acid, sterculic acid, malvalic acid and aflatoxins B1, B2, G1 and G2. At detection thresholds of 0.002% or 1 ppb, respectively, neither free gossypol nor any of the four aflatoxins were detected in the oil from transgenic cottonseed. Similarly, the respective levels of the cyclopropenoid fatty acids (dihydrosterculic, sterculic and malvalic) were statistically identical in cottonseed samples from transgenic and control lines.

The deduced amino acid sequences of both Cry1Ac and NPTII were compared to the amino acid sequence of known protein toxins. No significant similarities were found.

Allergenicity
Refined cottonseed oil and cellulose from linters are devoid of detectable protein and their consumption is unlikely to result in an allergic reaction since most allergens are proteins. Therefore the potential for cottonseed oil or linters from transgenic cotton lines to constitute a source of allergens is extremely low.

The Cry1Ac and NPTII proteins are not likely to be toxic or allergenic. The potential for allergenicity was assessed based on studies that included digestive degradation and sequence similarity to known allergens. The plant expressed proteins Cry1Ac and NPTII showed no significant protein sequence homology to a database of known toxins or allergens. Also, unlike known allergenic proteins, both Cry1Ac and NPTII were rapidly degraded when exposed to simulated gastric fluids (pH 1.2, pepsin digestion).

GR-3: Application for commercialisation of insect-resistant cotton

Approval for commercial cultivation of insect resistant cotton.

Decision Document 96-14: Determination of Environmental Safety of BollgardTM Insect Resistant Cotton (Gossypium hirsutum L.)

Opinion of the Scientific Committee on Plants on the genetically modified cotton line, insect-tolerant notified by the Monsanto company (notification C/ES/96/02) (Opinion expressed by SCP on 14 July 1998)

Notification of the placing on the Community Register of MON-ØØØ531-6.

Opinion of the Scientific Committee on Plants on the genetically modified cotton line, insect-tolerant notified by the Monsanto company (notification C/ES/96/02) (Opinion expressed by SCP on 14 July 1998)

Outline of the biological diversity risk assessment report: Type 1 use approval for MON531

Outline of the biological diversity risk assessment report: Type 1 use approval for MON757

Resolution 01247: Authorizing the commercial cultivation of Bollgard(r) cotton (in Spanish).

Resolution 02708: Authorizing importation for use in food and/or feed.

Product safety description

NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGY INSECT RESISTANT COTTON LINES 531 and 757

DIR 022/2002: Commercial release of insecticidal (INGARD® event 531) cotton

Monsanto Co. Petition for Determination of Non-regulated Status: BollgardTM Cotton Line 531 (Gossypium hirsutum L.) with the gene from Bacillus thuringiensis subsp. kurstaki

Memorandum to file concerning insect-resistant cotton.

USDA/APHIS Determination on a Petition 94-308-01p of Monsanto Agricultural Company Seeking Nonregulated Status of Lepidopteran- Resistant Cotton Lines 531, 757, 1076

Adamczyk,J.J.; Greenberg,S.M.; Armstrong,J.S.; Mullins,W.J.; Braxton,L.B.; Lassiter,R.B.; Siebert,M.W. (2008). Evaluations of Bollgard®, Bollgard II®, and Widestrike® technologies against beet and fall armywork larvae (Lepidoptera: noctuidae). Florida Entomologist 91(4): 531-536. [View abstract]

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Perlak, F.J., R.W. Deaton, T.A. Armstrong, R.L. Fuchs, S.R. Sims, J.T. Greenplate & D.A. Fischoff. (1990). Insect resistant cotton plants. Biotechnology 8: 9-13.

US Food and Drug Administration (1994). Secondary Food Additives Permitted in Food for Human Consumption; Food Additives Permitted in Feed and Drinking Water of Animals; Aminoglycoside 3'-Phosphotransferase II; Final Rule. Federal Register, 59:26700-26711.

Wilson, F.W., H.M. Flint, W.R. Dearon, D.A. Fischoff, F.J. Perlak, T.A. Armstrong, R.L. Fuchs, S.A. Berberich, N.J. Parks & B.R. Stapp. (1992). Resistance of cotton lines containing a Bacillus thuringiensis toxin to pink bollworm (Lepidoptera: Gelchiidae) and other insects. J. Econ. Entomol. 85: 75-80.

Berberich, S.A., Ream, J.E., Jackson, T.L., Wood, R., Stipanovic, R., Harvey, P., Patzer, S. & Fuchs, R.L. (1996). The composition of insect-protected cottonseed is equivalent to that of conventional cottonseed. Journal of Agricultural and Food Chemistry 44, 365-371.