GM Crop Database

Database Product Description

281-24-236 (DAS-24236-5)
Host Organism
Gossypium hirsutum (Cotton)
Trait

Insect resistant, Lepidoptera.

Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for fibre, livestock feed, and human consumption.

Product Developer
DOW AgroSciences LLC

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Brazil 2009 2009 2009
Canada 2005 2005 View
Colombia 2016
Japan 2005
Mexico 2004 2004
Taiwan 2015
United States 2004 2004 2004

Introduction Expand

The cotton line 281-24-236 was developed to resist attack by lepidopteran insects pests and has demonstrated resistance to tobacco budworm (Heliothis virescens), beet armyworm (Spodoptera exigua), soybean looper (Pseudoplusia includens), and cotton bollworm (Helicoverpa zea). This line produces, within its tissues, a synthetic version of the insecticidal protein Cry1F, a delta-endotoxin, originally derived from Bacillus thuringiensis var.aizawai. Cry proteins act by binding to specific sites located in the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt ion flow in the midgut, thereby causing paralysis and death. Cry1F is insecticidal only to lepidopteran insects, and the specificity of action is directly attributable to the presence of specific binding sites in these insects. The epithelium of mammalian intestinal cells do not possess binding sites for delta-endotoxins of B. thuringiensis, therefore humans and livestock animals are not susceptible to the damaging effects of these proteins.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
cry1F cry1F delta-endotoxin IR mannopine synthase (d mas 2') promoter from pTi15955 four copies of the octopine synthase (4OCS) enhancer from pTiAch5 3' polyadenylation signal from ORF25 (Agrobacterium tumefaciens) 1 functional; Cry1F active insecticidal core and non-toxic portions of the Cry1Ab1 and Cry1Ca3 proteins. Sequence modified for optimal in planta expression.
pat phosphinothricin N-acetyltransferase SM ubiquitin (ubi) ZM (Zea mays) promoter and the first exon and intron 3' polyadenylation signal from ORF25 (Agrobacterium tumefaciens) 1 functional; 1 partial, non-expressed; Altered coding sequence for optimal expression in plant cells.

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 var. aizawai cry1F While target insects are susceptible to oral doses of Bt proteins, no evidence of toxic effects in laboratory mammals or birds.
Streptomyces viridochromogenes pat

S. viridochromogenes is ubiquitous in the soil. It exhibits very slight antimicrobial activity, is inhibited by streptomycin, and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

The cotton line 281-24-236 was produced by Agrobacterium-mediated transformation of plant cells from the cotton variety ‘Germain’s Acala GC510.’ The pAGM281 plasmid was used for the transformation. It contained the cry1F gene, coding for a full length chimeric Cry1F protein (delta-endotoxin) which confers Lepidopteran insect resistance; a mannopine synthase promoter containing four copies of the octopine synthase enhancer ((4OCS)delta-mas2’) from A. tumefaciens strain LBA 4404 pTi15955; and a bi-directional terminator (ORF25polyA) from the same A. tumefaciens strain as the promoter. The pAGM281 plasmid also contained a synthetic version of the pat gene, coding for glufosinate ammonium tolerance, used as a selectable marker. The expression of the pat gene was under the control of a Zea mays ubiquitin promoter (UbiAm1). The plasmid backbone, derived from plasmid Rk2, contained an erythromycin resistance gene to allow the selection of bacteria containing pAGM281.

Successful transformants were detected as those tolerant to glufosinate ammonium. Resistance to lepidopteran insects was tested by conducting a bioassay using leaf discs from the successful transformants. Leaf discs were fed to the larvae of cotton bollworm, a target lepidopteran pest. The successful event was designated 281-24-236 and was subsequently introgressed into the elite genotype ‘PSC355,’ a cultivar with a broad adaptation to the southern United States.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of the genomic DNA from line 281-24-236 indicated the incorporation of single copies of the cry1F and pat genes, along with their respective regulatory sequences. Further analysis demonstrated a second, partial copy of the pat gene together with a second copy of the UbiZm1 promoter; however, this pat gene fragment was not associated with its terminator, ORF25 Poly A. No protein expression was detected from the gene fragment. Evidence was also provided to show that none of the plasmid backbone sequences, including the erythromycin gene, were incorporated into the genomic DNA.

The cry1F gene is a synthetic, plant-optimized gene that codes for Cry1F, a full-length chimeric version of Cry1Fa2, originally from Bacillus thuringiensis var. aizawai. In this synthetic version, the first 694 amino acids are from the toxic portion of Cry1Fa2; the remaining portion consists of C-terminal sequences from Cry1Ca3 (B.t. var. aizawai PS81I), and Cry1Ab1 (B.t. var. berliner 1715). These last two fragments are part of the C-terminal portion of the crystal protein that is cleaved by proteases in the lepidopteran mid-gut. The molecular weight of the plant-expressed, protease-cleaved Cry1F protein is 65kDa, as confirmed by Western blot analysis.

Genetic Stability of the Trait

The stability of the inserted insect resistance trait in line 281-24-236 was assessed within and across generations using Southern blot analysis. First generation plants, generated from backcrosses with one of the parental lines, segregated in a 1:1 ratio of resistant to non-resistant plants. The progeny resulting from the self-pollination of these first generation plants segregated in a 3:1 ratio of resistant to non-resistant plants. These results were not significantly different from the expected Mendelian segregation ratios and confirmed the stable inheritance of a single dominant trait.

Expressed Material

The levels of Cry1F and PAT protein in the tissues and cottonseed processed products of event 281-24-236 cotton were quantified using enzyme-linked immunosorbent assay (ELISA) methods. Samples for analysis were obtained from field trials at 6 different locations in 2001. The average levels of Cry1F protein, on a dry matter basis, were 6.46–7.67 ng/mg in leaves, 5.04 ng/mg in squares, 5.71 ng/mg in flowers, 4.02 ng/mg in bolls, 5.13 ng/mg in seeds, and 3.3 ng/mg in cottonseed (fresh-weight basis). The average level of Cry1F in pollen was below the level of quantification (0.09 ng/mg). The concentration of Cry1F in hulls was 0.22 ng/mg, and was not detected in toasted meal, or refined cottonseed oil.

The average levels of PAT protein were considerably lower than those of the Cry1F protein. Average levels of PAT protein, on a dry matter basis, were 0.21 – 0.43 ng/mg in leaves, 0.51 ng/mg in squares, 0.22 ng/mg in bolls, 0.47 ng/mg in seeds, and 0.42 ng/mg in cottonseed (fresh-weight basis). The average level of Cry1F in pollen (0.09 ng/mg) was also below the level of quantification, and neither was it detectable in refined cottonseed oil.

Environmental Safety Considerations Expand

Field Testing

The cotton line 281-24-236 was field tested in the United States from 2000 to 2002, and in Puerto Rico in 2000-01 for a total of 63 location-years. These trials were conducted to evaluate agronomic performance and crop quality, and to determine the plant pest risk potential of 281-24-236. Comparisons were made between the modified line and one of its parental lines, PSC355. While there were small but significant differences in vegetative growth parameters, these were within the observed range of commercial cultivars. Also observed were small but significant differences in some reproductive parameters (e.g., percent retention of bolls) and fibre quality. These slight differences would not be expected to confer weediness characteristics to line 281-24-236. The susceptibility of line 281-24-236 to cotton diseases, such as seed rot, Fusarium and Verticilium wilts, and boll rot, was evaluated and determined similar to that of the parental line PSC355. Results from these field trials demonstrate that the growth, agronomic performance and disease susceptibility of line 281-24-236 is similar to that of conventional cotton. This cotton line is therefore not expected to become a plant pest risk, neither in terms of weediness, nor in becoming a more suitable host for plant diseases.

Outcrossing

Cotton (G. hirsutum) is mainly a self-pollinating plant, but pollen is also routinely transferred by insects, particularly bumblebees and honey bees. The pollen is heavy and sticky and the range of natural crossing is limited. Outcrossing rates of up to 28% to other cotton cultivars grown directly adjacent to the pollen source have been observed under field conditions when sufficient insect pollinators have been present. The rate of outcrossing declines rapidly with increased distance from the pollen source. Results of outcrossing research trials in the United States have shown that pollen movement decreases rapidly at 12 metres from the pollen source. Nevertheless, certified seed cotton growers in the United States must maintain an isolation distance of 202 metres between fields. If the cotton line 281-24-236 were to be grown in proximity with other cotton cultivars, and sufficient insect pollinators were present and active, the cry1F gene could possibly introgress into these cultivars. In the event of the formation of hybrids, there would no competitive advantage conferred on any hybrid progeny in the absence of infestations by Lepidopteran insect species.
In the United States, species genetically compatible with 281-24-236 cotton include G. hirsutum (wild or under cultivation), G. barbadense (cultivated Pima cotton), and G. tomentosum. Outcrossing from G. hirsutum to G. barbadense is possible under suitable conditions, such as the presence of pollinators. G. thurberi is another wild species found in the United States, but it is genetically incompatible with G. hirsutum. While G. hirsutum and G. tomentosum are genetically compatible (both possess the AADD genome), the possibility of gene transfer is unlikely due to the non-synchronous flowering periods (day for G. hirsutum and night for G. tomentosum), and lack of common pollinators. Populations of wild Gossypium species occur in southern Florida (G. hirsutum), Hawaii (G. tomentosum), Puerto Rico and the U.S. Virgin Islands. In other cotton growing areas of the world, species that could intercross with G. hirsutum include G. mustelinum in Brazil, and G. lanceolatum in Mexico.

Weediness Potential

Cultivated Gossypium hirsutum is not typically considered a weed species in the United States or other countries, but it is listed as a potential weed in southern Florida. Although the cotton plant is cultivated as an annual crop, it behaves as a perennial plant in undisturbed environments and suitable climatic conditions. Cotton does not tolerate cold conditions thereby limiting its overwintering potential to southern Florida, Hawaii, and Puerto Rico.

No competitive advantage was conferred to the cotton line 281-24-236 other than the production of the insecticidal protein Cry1F. The levels of expression of the PAT protein in this cotton line do not confer tolerance to herbicidal applications of glufosinate ammonium. The purpose of introducing the pat gene was to provide a mechanism for selection of transformants. The cotton line 281-24-236 was not expected to become weedy or invasive of natural habitats since none of the reproductive or growth characteristics of this line were substantially modified. While there were observed significant differences in some vegetative growth characteristics (number of vegetative branches and vegetative bolts) and reproductive parameters (percent retention of bolls), these were within the ranges observed in conventional cotton varieties. Seed dormancy, germination, and seedling establishment, were not significantly altered in 281-24-236, compared to the parental line. The reproductive potential, in terms of number of seeds per boll, was also not significantly different in this cotton line.

Secondary and Non-Target Adverse Effects

The impact of 281-24-236 cotton on non-target organisms was investigated. Ecological toxicity studies were conducted with representative terrestrial and aquatic species that could be inadvertently affected by Cry1F. Non-target organisms included insects other than lepidopterans (e.g., honeybees, ladybird beetles, green lacewings, parasitic wasps), soil arthropods (collembola), earthworms, and representative terrestrial and aquatic species such as mice, bobwhite quail, rainbow trout and Daphnia magna. Toxicological effects were determined from feeding studies. The bobwhite quail and rainbow trout were fed Cry1F as expressed in cottonseed meal. The remaining studies were conducted with the full-length chimeric Cry1F from a microbial source (Pseudomonas fluorescens). Data were provided to substantiate the equivalency between the plant and microbial Cry1F, in terms of biochemistry and bioactivity. Results of these feeding studies showed no observable adverse effects that could be attributed to the Cry1F protein.

Cry1F protein in cotton line 281-24-236 was shown to be efficacious against the target lepidopteran pests. Insecticidal effects on non-target lepidopterans, such as the monarch butterfly, although not directly assessed in the field, were not expected given the very low expression of the Cry protein in the pollen. Based on the results from dietary studies of monarch butterfly larvae with Cry1F protein, the estimated concentration of Cry1F necessary for a 50% reduction in larval growth is 450,000 times the concentration in pollen from line 281-24-236. This, combined with the low volume of pollen produced, would result in very little risk to non-target lepidopterans, such as the monarch butterfly. While cotton is mostly self-pollinated, any cross-pollination would be achieved by non-lepidopteran insects, such as bees, which are unaffected by Cry proteins.

Based on this information, it was concluded that, compared to conventional cotton varieties, the cultivation of line 281-24-236 would pose no greater risk to interacting organisms, with the exception of specific lepidopteran insect pests of cotton.

Impact on Biodiversity

The cotton line 281-24-236 did not exhibit any novel phenotypic characteristics that would increase its survival in either unmanaged habitats or in areas outside of the current geographical range of cotton production. Since the potential of gene transfer to wild relatives in the United States is quite minimal, the risk of transferring the insect resistance trait to species in unmanaged habitats is insignificant. In countries (e.g., Brazil, Mexico) where hybrids could form with wild relatives of cotton, the introgression of the insect resistance trait into these wild species would confer no other advantage than increased resistance to attack by certain Lepidopteran insects.

Other Considerations

In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in the United States have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including 281-24-236 maize, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The genetic modification of 281-24-236 cotton would not be expected to result in any change in the consumption pattern of cotton products. The seed, oil and lint fraction from this cotton line did not exhibit any novel characteristics that would alter the consumption pattern. This was confirmed by comparison of the levels of proximates and fatty acids in line 281-24-236 to null plants and conventional cotton cultivars. Only refined cottonseed oil and cellulose from processed linters of cottonseed are consumed by humans. There were no detectable proteins in the oil from line 281-24-236. Processed linters are composed of pure (>99%) cellulose and are treated with heat and solvent that would be expected to remove and destroy any residual protein.

Nutritional and Compositional Data

The nutritional components of 281-24-236 cottonseed, kernels, toasted meal, hulls and oil were determined analytically and compared to those of the non-transgenic control (a null segregant resulting from several backcrosses of 281-24-236 with both parental lines). Cottonseed analysis included proximates (crude protein, crude fibre, acid detergent fibre, neutral detergent fibre, total fat, ash, and moisture), minerals, amino acids, and fatty acids, and anti-nutrients (cyclopropenoid fatty acids, gossypol [total and free], aflatoxins). Oil analysis included crude fat, crude protein, moisture, fatty acids, anti-nutrients and anti-oxidants. Kernels, toasted meal, and hulls were analyzed as appropriate for the particular fraction. Other than small differences in the levels of some components in cotton seed, which were determined to be biologically insignificant, and most likely due to genotypic and environmental effects, the analyses demonstrated that the composition of 281-24-236 cottonseed is comparable to that of the non-transgenic control and seed of other commercial cotton varieties. The composition of cottonseed oil was also comparable to the non-transgenic control and commercial cottonseed oil. Anti-nutrient levels in 281-24-236 were similar to those of the non-transgenic control and to values published in the literature.

Toxicity and Allergenicity

The potential for toxicity and allergenicity of the Cry1F and PAT proteins in line 281-24-236 was assessed from the following data and information: results of searches for amino acid sequence similarity between the Cry1F and PAT proteins and known toxins and allergens; analysis for possible glycosylation of Cry1F; in-vitro digestibility studies using simulated gastric fluid; assessment of heat stability; and acute oral toxicity studies with mice.

Both the Cry1F and PAT proteins showed no amino acid sequence similarity to known allergens. Both of these proteins were degraded within one minute of exposure to simulated gastric fluid and were no longer detectable by either SDS-PAGE or Western blot analysis. The Cry1F protein was no longer biologically active after exposure to temperatures greater than 75°C. No post-translational glycosylation was detected in cotton-expressed Cry1F protein. Levels of natural toxicants in cotton (e.g., gossypol) were not significantly different in 281-24-236 cotton compared to the non-transgenic control and conventional cotton varieties.

Results of feeding studies on mammals, birds, soil invertebrates, aquatic organisms, and non-target insects showed no observable adverse effects of either the microbially expressed Cry1F or the protein expressed in cotton tissues. Generally, for most of the species tested, there was at least a 5-fold safety margin between the lowest observed effect level and the highest estimated concentration to which these species might be exposed. A 3-fold safety margin was determined for honeybees. An additional non-target insect feeding study was requested by the Environmental Protection Agency (EPA) of the United States as a condition of registration. This study was conducted with the minute pirate bug, an important predatory insect found in cotton fields.

Cry proteins in Bt biopesticide formulations have a long history of safe use, with no evidence of adverse effects, other than insecticidal activity towards lepidopteran insect pests. The Cry1F protein also was expressed at very low levels in the various tissues of 281-24-236 cotton. On the basis of this information, and the results of the various safety studies and sequence homology investigations, a determination was made that 281-24-236 cotton did not demonstrate potential for toxicity or allergenicity compared to conventional cotton varieties.

Abstract Collapse

Cotton (Gossypium hirsutum L.) was grown commercially in over 80 countries with a combined production of 44.2 million metric tonnes of cotton seed and 24.8 million metric tonnes of cotton lint in 2006. The major producers of cotton seed and lint were China, the United States, India, Pakistan, Brazil, Uzbekistan and Turkey. Cotton is primarily grown for its seed bolls that produce fibres used in numerous textile products.

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 a cooking oil, in shortening, margarine 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.

Tobacco budworm (Heliothis virescens), pink bollworm (Pectinophora gossypiella), and cotton bollworm (Helicoverpa zea) are three of the most destructive pests of cotton. These insects cause damage to squares and bolls (tobacco budworm, cotton bollworm) and staining of lint (pink bollworm). Species such as the soybean looper (Pseudoplusia includens) and beet armyworm (Spodoptera exigua) cause feeding damage to leaves. Insect damage to the plant also increases susceptibility to diseases (e.g., boll rot), and ultimately results in the losses of yield and crop quality. In the United States alone the combined costs of control and yield loss attributed to these pests is approximately $476 million per year. In Egypt, China and Brazil, pink bollworm commonly causes cotton losses of up to 20 percent.

Methods of insect control in cotton include the use of insecticides and integrated pest management methods, such as scouting and the use of economic thresholds, prior to application. In 2004, 64% of the cotton acreage in the United States was treated with insecticides; those most commonly used were malathion, acephate and aldicarb. However, the effectiveness of many insecticides has been reduced due to the development of resistance in some insect pests, such as the tobacco budworm.

The cotton line 281-24-236 was genetically engineered to resist attack from Lepidopteran insect pests such as the tobacco budworm, cotton bollworm, beet armyworm, and soybean looper. This insect resistance is conferred by the cry1F gene, originally isolated from the common soil bacterium Bacillus thuringiensis (Bt) var. aizawai. The cry1F gene produces the insect control protein Cry1F, a delta-endotoxin, in the plant tissues. Cry proteins, of which Cry1F 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. Cry1F 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 cry1F gene was introduced into the cotton line ‘Germain’s Acala GC510’ by Agrobacterium-mediated transformation.

The cotton line 281-24-236 was field tested in the United States from 2000 to 2002, and in Puerto Rico in 2000-01. Agronomic characteristics such as yield, boll size, plant vigour, growth, morphology, germination and flowering were found to be within the range for commercial cotton lines. Susceptibility to diseases was not altered compared to conventional cotton varieties. The cotton line 281-24-236 did not demonstrate morphological or growth characteristics such as those observed in weedy and invasive plant species.

The effect of the cultivation of line 281-24-236 on non-target organisms was investigated during field testing. Non-lepidopteran insects were not detrimentally affected by this cotton line; this was not unexpected, given the insecticidal specificity of the Cry proteins. Insecticidal effects on the monarch butterfly, a lepidopteran, although not directly assessed in the field, were not expected given the very low expression of the Cry protein in the pollen. Based on the results from dietary studies of monarch butterfly larvae with Cry1F protein, the estimated concentration of Cry1F necessary for a 50% reduction in larval growth is several hundred thousand-fold the concentration in pollen from line 281-24-236. This, combined with the low volume of pollen produced, would result in very little risk to non-target lepidopterans, such as the monarch butterfly. While cotton is mostly self-pollinated, any cross-pollination would be achieved by non-lepidopteran insects, such as bees, which are unaffected by Cry proteins. Other non-target organisms, such as arthropods, mammals, birds, and aquatic organisms were not negatively affected by the cultivation of line 281-24-236. It was concluded that, compared to conventional cotton varieties, the cultivation of line 281-24-236 would pose no greater risk to interacting organisms, with the exception of specific lepidopteran insect pests of cotton.

The potential for introgression of the insect resistance trait from line 281-24-236 cotton into other cotton plants, or to wild relatives of cotton, was investigated. 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) and G. barbadense (cultivated Pima cotton), and is genetically compatible with 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 genomic incompatibility. Assuming proximity, synchronicity of flowering and presence of insects, the cotton line 281-24-236 could freely hybridize with other G. hirsutum varieties and wild plants. In the event of the formation of hybrids, there would no competitive advantage conferred on any hybrid progeny in the absence of infestations by Lepidopteran insect species.
The food and livestock safety of the cotton line 281-24-236 was based on: the equivalence of the Cry1F protein to the bacterially produced protein which has a history of safe use as Bt insecticidal treatments; the lack of amino acid sequence homology of both the Cry1F and PAT proteins to known toxins and allergens; results of feeding studies with mammals, birds, aquatic organisms and non-target arthropods, demonstrating the lack of toxicity of the Cry1F protein to organisms other than lepidopteran insects; the rapid digestibility of the proteins in simulated gastric fluids; the very low levels of expression of both novel proteins in all plant tissues, including seed and oil; and the equivalence in nutritional components and anti-nutrients, of the seed and oil compared to conventional cotton cultivars.

Links to Further Information Expand


This record was last modified on Friday, August 4, 2017