GM Crop Database

Database Product Description

31807/31808
Host Organism
Gossypium hirsutum (Cotton)
Trait
Resistance to lepidopteran insects; oxynil herbicide tolerance, including bromoxynil.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for fibre, livestock feed, and human consumption.

Product Developer
Calgene Inc.

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1998
Japan 1999 1999 1998 View
United States 1998 1998 1997

Introduction Expand

Transgenic cotton lines 31807 and 31808 were developed using recombinant DNA techniques to be both resistant to major caterpillar pests of cotton and to herbicides in the oxynil family, principally bromoxynil (tradename Buctril®). These 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 (B.t.k) 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 31807 and 31808, 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 mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

These cotton lines contain a second gene (bxn) that encodes for a bacterial enzyme, nitrilase, from Klebsiella pneumoniae. The nitrilase enzyme hydrolyses the herbicides ioxynil and bromoxynil into non-phytotoxic compounds. Specifically, the nitrilase enzyme breaks down bromoxynil into 3,5 dibromo-4-hydroxybenzoic acid (DBHA), eliminating its herbicidal activity.

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 transgenic lines. 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).

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
cry1Ac Cry1Ac delta-endotoxin IR CaMV 35S Truncated
bxn nitrilase HT chimera of the 35S promoter and the promoter from a mannopine synthase gene from A. tumefaciens Native
nptII neomycin phosphotransferase II SM bacterial promoter Not expressed in plant tissues

Characteristics of Gossypium hirsutum (Cotton) Expand

Center of Origin Reproduction Toxins Allergenicity

Believed to originate in Meso-America (Peruvian-Ecuadorian-Bolivian region).

Generally self-pollinating, but can be cross-pollinating in the presence of suitable insect pollinators (bees). In the U.S., compatible species include G. hirsutum, G. barbadense, and G. tomentosum.

Gossypol in cottonseed meal.

Cotton is not considered to be allergenic, although there are rare, anecdotal reports of allergic reactions in the literature.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Bacillus thuringiensis subsp. kurstaki cry1Ac

Although target insects are susceptible to oral doses of Bt proteins, there is no evidence of toxic effects in laboratory mammals or bird given up to 10 µg protein / g body wt. There are no significant mammalian toxins or allergens associated with the host organism.

Modification Method Expand

These bioengineered cotton (Gossypium hirsutum L.) lines were produced by Agrobacterium-mediated transformation in which the transfer-DNA (T-DNA) region of the bacterial tumour inducing (Ti) plasmid was modified to contain DNA sequences encoding Cry1Ac protein, a nitrilase enzyme, and the NPTII enzyme.

The nucleotide sequence of the cry1Ac gene was modified by site-directed mutagenesis to contain plant-preferred codons in order to maximize protein expression in plant cells. Expression of the cry1Ac gene was controlled by including promoter sequences from the cauliflower mosaic virus (CaMV) 35S transcript. The bxn gene was under the control of a chimera of the CaMV 35S promoter and the promoter from a mannopine synthase gene from A. tumefaciens. Expression of the neo gene was also regulated by cauliflower mosaic virus 35S promoter.

Characteristics of the Modification Expand

Expressed Material

The expression levels of Cry1Ac, nitrilase, and NPTII were quantitated by Western immunoblot analysis of samples of cottonseed and meal. The estimated concentrations of the three proteins in seed were 2.5 ppm, 0.25 ppm and 2.5 ppm, respectively, on a fresh weight basis. For samples of meal, the values were 2.25 ppm, 1.4 ppm and 13.5 ppm for Cry1Ac, nitrilase, NPTII, respectively. None of these proteins were detected in samples of oil from cotton lines 31807 and 31808 at a detection limit of 0.01 ppm.

Environmental Safety Considerations Expand

Field Testing

The transgenic cotton lines 31807 and 31808 were field tested in the United States and it was concluded that they did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.

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 31807 and 31808 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 the wild 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 introduced genes were not expected to confer an ecological advantage to transgenic cotton lines 31807 and 31808 and their potential hybrid offspring. Resistance to specific lepidopteran insects, tolerance to bromoxynil herbicide and kanamycin resistance will not render cotton weedy or invasive of natural habitats since none of the reproductive or growth characteristics have been modified. Since both lines were to be cultivated in a managed agricultural system there would be very little selective pressure for these plants to become weeds. It was concluded that it was unlikely that the traits would confer any selective advantage to the cotton lines 31807 and 31808.

There are no specific problems with cotton as a weed. Cottonseed may remain in the field after harvesting and germinate under favourable 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 other than bromoxynil.

Secondary and Non-Target Adverse Effects

It was concluded that the genes inserted into the transgenic cotton lines 31807 and 31808 would not result in any deleterious effects or significant impacts on nontarget organisms, with the exception of the target lepidopteran caterpillars, including those that are recognized as beneficial to agriculture and those that are recognized as threatened or endangered in the United States.

The novel proteins, Cry1Ac, nitrilase and NPTII expressed in cotton lines 31807 and 31808 are not known to have any toxic effects on any nontarget organisms. The lack of known toxicity for these proteins and their low levels of expression in plant tissue suggest no potential for deleterious effects on beneficial organisms such as bees and earthworms. Both nitrilase and NPTII act on a narrow range of substrates making it unlikely that these compounds would react with other substances to produce novel toxic compounds in organisms that consume these transgenic cotton lines.

Impact on Biodiversity

The transgenic cotton lines 31807 and 31808 had no novel phenotypic characteristics that would extend their use beyond the current geographic range of cotton production. 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 31807 and 31808 to species in unmanaged environments was not significant.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Only refined cottonseed oil and cellulose from processed linters of cottonseed are 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. It is generally accepted that the refined oil does not contain protein. The refining process for cottonseed oil includes heat, solvent and alkali treatments that remove and destroy DNA and protein. The refined cottonseed oil from these lines was tested for the presence of Cry1Ac, nitrilase and NPTII, which were undetectable at a detection limit of 0.01 ppm. It was concluded that there would be no dietary exposure to the novel proteins expressed in these cotton lines.

Nutritional Data

Compositional comparisons of cottonseed oil, cottonseed and meal from lines 31808 and 31807 were made to the same materials from commercial non-transgenic cotton. Parameters measured included proximate analysis (moisture, crude fat/oil, protein, and ash) of cottonseed meal; fibre analysis (crude fibre, acid detergent, and neutral detergent fibres) of cottonseed; fatty acid composition of refined cottonseed oil; and amino acid profile of cottonseed meal. No significant differences between the transgenic lines and the traditionally bred lines were observed for any of these parameters.

Toxicity

Cottonseed contains a high number of anti-nutritional factors and the raw seed is unsuitable for monogastric animals. Gossypol has numerous toxic effects on mammals, including heart damage and heart failure, such that whole cottonseed or cottonseed meal is not used in human foods and the presence of gossypol limits its use in animal feed. However, removal or inactivation of gossypol during processing enables the use of some cottonseed meal in feed for fish, poultry and pigs.

The cyclopropenoid fatty acids, sterculic acid (C-17), dihydrosterculic acid (C-19) and malvalic acid (C-18), are unique fatty acids common in cotton and produce undesirable biological effects, including the inhibition of biodesaturation of stearic to oleic acid, affecting phospholipid biosynthesis.

The levels of gossypol and cyclopropenoid fatty acids in transgenic cotton lines were measured and found to be within the normal range of variation reported for traditionally bred cotton cultivars.

Allergenicity

Refined cottonseed oil and cellulose from linters are devoid of protein and, given that most allergens are proteins, their consumption is unlikely to result in an allergic reaction. Therefore the potential for cottonseed oil or linters from cotton lines 31807 and 31808 to constitute a source of allergens is extremely low.

Abstract Collapse

Cotton (Gossypium hirsutum L.) is primarily grown for its seed bolls that produce fibres used in numerous textile products. The major producers of cotton seed and lint are China, the United States, India, Pakistan, Brazil, Uzbekistan and Turkey.

About two thirds of the harvested cotton crop is seed, which is separated from the lint during ginning. The cotton seed is crushed to produce cottonseed oil, cottonseed cake (meal) and hulls. Cottonseed oil is used primarily as cooking oil, in shortening and salad dressing, and is used extensively in the preparation of snack foods such as crackers, cookies and chips. The meal and hulls are an important protein concentrate for livestock, and may also serve as bedding and fuel. Linters, or fuzz, which are not removed in ginning, are used in felts, upholstery, mattresses, twine, wicks, carpets, surgical cottons, and in industrial products such as rayon, film, shatterproof glass, plastics, sausage skins, lacquers, and cellulose explosives.

Effective weed management is critical to cotton production. The removal of weeds early in the growing season extremely important in cotton production. Young cotton seedlings grow slowly early in the season and are not very competitive. Early weed pressure has detrimental effects on final yield. Weeds can also be detrimental later in the growing season; weeds can interfere with harvesting, and can result in a reduction in lint quality due to trash or staining. Precautions such as pre-plant tillage or herbicide application are common approaches for reducing weed competition, as are multiple herbicide treatments. The broadleaf weeds are the most difficult to control because there are few herbicide options available. Many producers will use as many as four herbicides per year in an effort to control weeds.

Tobacco budworm (Heliothis virescens), pink bollworm (Pectinophora gossypiella), and cotton bollworm (Helicoverpa zea) are three of the most destructive pests in cotton. In the United States alone the combined costs of control and yield loss attributed to these pests is up to $476 million per year. In Egypt, China and Brazil, pink bollworm commonly causes cotton losses of up to 20 percent.

More insecticides are applied to conventionally grown cotton than to any other single crop. Each year cotton producers around the world use nearly $2.6 billion worth of pesticides, which include pesticides such as aldicarb, phorate, methamidophos and endosulfan. Cotton pests, such as the tobacco budworm, have developed some resistance to many of the insecticides used to control them. In regions where insecticide-resistant populations have developed budworm damage can reduce yields by 29%, despite an average of six insecticide applications each growing season.

The cotton lines 31807 and 31808 were genetically engineered to resist attack from the major lepidopteran pests of cotton and to tolerate herbicides in the oxynil family, including bromoxynil and ioxynil. Oxynil herbicides act by blocking electron flow during the light reaction of photosynthesis, inhibiting cellular respiration in dicotyledonous plants. Oxynil herbicides applied at rates recommended for effective weed control are toxic to conventional cotton varieties. These transgenic cotton lines contain the bxn gene for oxynil tolerance, and the cry1Ac gene from Bacillus thuringiensis, which codes for the insect control protein Cry1Ac, a delta-endotoxin. These genes were introduced into the cotton genome by Agrobacterium-mediated transformation.

The Cry1Ac protein produced by the transgenic cotton is almost identical to that found in nature and in commercial Bt spray formulations. Cry proteins, of which Cry1Ac is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry1Ac is insecticidal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

The bxn gene, conferring tolerance to oxynil compounds, was isolated from the bacterium Klebsiella pneumoniae subspecies ozaenae and codes for the enzyme nitrilase, which hydrolyses ioxynil and bromoxynil into non-toxic compounds. The nitrilase enzyme in cotton lines 31807 and 31808 is a bacterial version of an enzyme that is widespread in nature, found in monocot plants such as corn, wheat and barley.

The transgenic cotton lines 31807 and 31808 were field tested in the United States. Based on data from these trials it was concluded that the transformed cotton lines 31807 and 31808 did not exhibit weedy characteristics and had no effect on non-target organisms or the general environment. The transformed lines were not expected to impact on threatened or endangered species.

Cotton plants are primarily self-pollinating, but insects, especially bumblebees and honeybees, also distribute cotton pollen. Cotton can cross-pollinate with compatible species including G. hirsutum (wild or under cultivation), G. barbadense (cultivated Pima cotton), and G. tomentosum. Overall, the probability of gene transfer to wild species in unmanaged ecosystems is low due to the relatively isolated distribution of Gossypium species, different breeding systems, and genome incompatibility. Assuming proximity, synchronicity of flowering and availability of insects, transgenic cotton lines 31087 and 31808 may freely hybridize with other G. hirsutum varieties.

Regulatory authorities in the United States have mandatory requirements for developers of Bt cotton to implement specific Insect Resistant Management (IRM) Programs. The potential for Bt-resistant insect populations to develop increases as acreages planted with transgenic Bt cotton hybrids expand. Hence, these IRM programs are designed to reduce this potential and prolong the effectiveness of plant-expressed Bt toxins, and the microbial Bt spray formulations that contain these same toxins.

Human consumption of cotton products is limited to refined cottonseed oil and cellulose from processed linters of cottonseed. Processed linters are essentially pure cellulose, and are subjected to heat and solvent treatment that would be expected to remove and destroy DNA. It is generally accepted that the refined oil does not contain protein as the refining process includes heat, solvent and alkali treatments that would remove and destroy any DNA and protein present. The refined cottonseed oil from the transgenic lines was tested for the presence of the nitrilase and Cry1Ac proteins, which were undetectable. It was concluded that there was little potential for human dietary exposure to the novel proteins expressed in these transgenic cotton lines.

Compositional comparison of cottonseed oil, cottonseed and meal from lines 31807 and 31808 was made to the same materials from commercial non-transgenic cotton. Parameters measured included proximate analysis (moisture, crude fat/oil, protein, and ash) of cottonseed meal; fibre analysis (crude fibre, acid detergent, and neutral detergent fibres) of cottonseed; fatty acid composition of refined cottonseed oil; and amino acid profiles of cottonseed meal. No significant differences between the transgenic lines and the traditionally bred lines were observed for any of these parameters. It was also determined that the transgenic cotton contained levels of gossypol and cyclopropenoid fatty acids, toxic substances naturally found in cotton, within the normal range of variation reported for conventional cotton cultivars.

Refined cottonseed oil and cellulose from linters are devoid of protein and, given that most allergens are proteins, their consumption is unlikely to result in an allergic reaction. The potential for cottonseed oil or linters from 31807 or 31808 cotton to constitute a source of allergens is therefore extremely low. The presence of the nitrilase protein in monocot food sources such as corn and barley further supported the conclusion that the nitrilase enzyme posed little risk of toxicity. It was concluded that the nitrilase and Cry1Ac proteins, and thus 31807 and 31808 cotton, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Health Canada Novel Foods U.S.Department of Agriculture, Animal and Plant Health Inspection Service US Food and Drug Administration USDA-APHIS Environmental Assessment

This record was last modified on Tuesday, February 24, 2015