GM Crop Database

Database Product Description

LLCotton25 (ACS-GHØØ1-3)
Host Organism
Gossypium hirsutum (Cotton)
Trait
Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for fibre, livestock feed, and human consumption.

Product Developer
Bayer CropScience (Aventis CropScience(AgrEvo))

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Argentina 2014 2014
Australia 2006 2006 2006
Brazil 2008 2008 2008
Canada 2004 2004 View
China 2006 2006
Colombia 2008 2010
European Union 2008 2008
Japan 2004 2006
Korea 2005 2005
Malaysia 2017 2017
Mexico 2006 2006
South Africa 2011 2011
Taiwan 2015
United States 2003 2003 2003

Introduction Expand

LLCotton25 was developed to allow the use of glufosinate ammonium as a weed control option in cotton production.  This genetically engineered cotton line expresses a protein, phosphinothricin-acetyl-transferase (PAT) that confers tolerance to the active ingredient L-phosphinothricin in glufosinate ammonium. The expression of PAT is due to the introduction of the bar gene. This gene was isolated from Streptomyces hygroscopius, a gram-positive soil bacterium.
Glufosinate ammonium is a post-emergence, broad-spectrum contact herbicide and plant dessicant.  L-Phosphinothricin was first isolated as bialaphos, an antibiotic synthesized during the fermentation of Streptomyces hygroscopicus or S.viridochromogenes.   In herbicidal formulations it is a component of glufosinate ammonium, which is chemically synthesized.  Glufosinate ammonium is a racemic mixture of L-phosphinothricin, the herbicidal active moiety, and its D-enantiomer.  L-Phosphinothricin is structurally similar to glutamate, the substrate of glutamine synthetase (GS), an enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia.  L-phosphinothricin inhibits the activity of GS irreversibly by binding to its active sites.  The inhibition of GS caused by the application of glufosinate ammonium to plants results in the accumulation of ammonia, the reduction in the levels of glutamine, and the inhibition of photosynthesis, all of which results in the death of the plant.  Plants transformed with the bar gene express the enzyme phosphinothricin-acetyl-transferase (PAT) which acetylates L-phosphinothricin into a non- phytotoxic metabolite (N-acetyl-L-glufosinate).

LLCotton25 was developed by introducing the bialaphos resistance (bar) gene into the cotton variety ‘Coker 312,’ using Agrobacterium-mediated plant transformation.  The development of LLCotton25 has allowed the use of glufosinate ammonium as an alternative herbicide for weed control in cotton.  This may help reduce the incidence of herbicide resistant biotypes by adding an additional class of herbicides in weed management systems.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
bar phosphinothricin N-acetyltransferase HT CaMV 35S A. tumefaciens nopaline synthase (nos) 3'-untranslated region

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
Streptomyces hygroscopicus bar S. hygroscopicus is ubiquitous in the soil and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

LLCotton25 was produced by Agrobacterium-mediated transformation of plant cells from the cotton variety “Coker 312”. The plasmid vector pGSV71 used for transformation contained the following: the gene coding for glufosinate ammonium tolerance (bar gene), the promoter region (P35S) from the cauliflower mosaic virus, and the 3’ untranslated end of the nopaline synthase gene (3’ nos). The transformation was achieved by culturing cotton tissue, excised between the hypocotyl and the radicle of three-day old cotton seedlings, with a culture of A. tumefaciens harbouring the Ti plasmid pGV3000 and the plasmid vector pGSV71. Explants from this culture were then regenerated to whole plants using tissue culture techniques. Transformed plants expressing the bar gene were selected with glufosinate ammonium.

Characteristics of the Modification Expand

The Introduced DNA:  Southern blot analysis and Polymerase Chain Reaction (PCR) amplification of the genomic DNA of LLCotton25 demonstrated one site of integration of a single copy of the T-DNA of pGSV71. Southern blot analysis also confirmed the integrity of the bar gene along with its promoter and terminator sequences and, that no sequences of the vector backbone were integrated into the genome of LLCotton25. The P35S promoter and the 3’ nos terminator sequences were derived from plant pathogenic organisms. Data from observations on several generations of LLCotton25 confirmed that these non-coding sequences did not cause diseases in the plants.

Genetic Stability of the Trait:  The stability of the bar gene, along with its promoter and terminator sequences (collectively termed the “bar gene cassette”), were assessed over multiple generations of conventional breeding at several locations, and in different genetic backgrounds. The results of genomic DNA blot analysis confirmed the stable inheritance of the bar gene cassette. Mendelian segregation data confirmed the stable inheritance of a single integrated gene.

Expressed Material:  The PAT protein was expected to be expressed in all tissues of the LLCotton25 plant since the bar gene was linked to a constitutive promoter (P35S from cauliflower mosaic virus). The PAT protein was quantitatively determined from LLCotton25 plants grown at various locations in the U.S.A. Plant tissues (i.e., roots, leaves, stems and pollen) and seed and fibre fractions (cleaned seed, fuzzy seed, and lint) were analyzed using the enzyme linked immunoabsorbant assay technique (ELISA). The concentrations of PAT protein were determined on a fresh weight basis. In plant tissues other than the seed and lint, PAT protein averaged 7.97 μg/g in roots, 59.2 μg/g in leaves, 38.8 μg/g in stems, and 19.2 μg/g in pollen. The average expression of PAT protein in cleaned seed was 127 μg/g and 69.9 μg/g in fuzzy seed. PAT protein was detected in all fractions of the seed (i.e., hulls, solvent extracted meal, toasted meal). The fibre components (i.e., lint coat and lint fractions) contained less than 1.5% of the total PAT protein expressed in the plant. There was a negligible amount of PAT protein expressed in the oil.

Environmental Safety Considerations Expand

Field Testing:  LLCotton25 was field tested in the United States and Puerto Rico at 43 locations over three years (1999-2001). These trials were conducted to evaluate the agronomic performance, crop quality, and plant pest potential of LLCotton25. Seed and lint yield and quality data, and evaluations of stand counts, seedling vigour, plant height, height to node ratio, leaf morphology, and plant maturity supported the conclusion that LLCotton25 was as safe to grow as other cotton varieties and did not demonstrate the potential to become a plant health risk.

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 LLCotton25 were to be grown in proximity with other cotton cultivars, and sufficient insect pollinators were present and active, the possibility would exists for introgression of the bar gene into these cultivars.

In the United States, species genetically compatible with LLCotton25 include G. hirsutum (wild or under cultivation), G. barbadense (cultivated Pima cotton), and G. tomentosum. There have been reports of outcrossing from G. hirsutum to G. barbadense under suitable conditions, such as the presence of suitable 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. hirstum 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 LLCotton25 other than tolerance to glufosinate ammonium. Tolerance to glufosinate ammonium will not render LLCotton25 weedy or invasive of natural habitats since none of the reproductive or growth characteristics of this line have been modified. In the event of the formation of herbicide tolerant hybrids, there would no competitive advantage conferred on any hybrid progeny in the absence of sustained glufosinate ammonium use. The herbicide tolerant plants could be controlled by cultural, or mechanical means, or by using herbicides other than glufosinate ammonium. The same treatments would apply to cotton volunteers appearing in a following crop.

LLCotton25 did not exhibit any increased tendency towards weediness, compared to its unmodified parental line, as demonstrated by the nonsignificant differences in parameters such as seed germination, seedling vigour, time to first bloom, and plant maturity.

Secondary and Non-target Adverse Effects:  Field trial observations of LLCotton25 revealed no negative effects on nontarget organisms; no alterations in the population levels of beneficial insects and birds were observed. LLCotton25 did not display any altered susceptibility to diseases such as Rhizoctonia sp. and Phymototricum sp. leading to the conclusion that its plant pest potential (e.g., increased potential to harbour a disease or become an alternate host) was not changed. A similar conclusion was reached with regard to susceptibility to insect pests such as budworm, boll weevil, thrips, cotton aphids, plant bugs, whiteflies, cutworms, stinkbug, and cotton bollworm. Data on germination, seedling vigour, plant growth and morphology, and agronomic performance indicated no significant differences among LLCotton25, its unmodified parental line, and other cotton cultivars, leading to the conclusion that LLCotton25 would not be expected to grow in new habitats and displace other species. The PAT enzyme is expressed at very low levels in the plant tissues, has a high substrate specificity to L-phosphinothricin, is rapidly degraded in simulated gastric and intestinal fluid conditions, and does not have any sequence similarities to known allergens or toxins. This enzyme is also present in the environment since it is produced by species of the soil bacterium Streptomyces, with no known adverse effects on humans or animals. This information led to the determination that LLCotton25 will not result in altered impacts on non-target organisms, compared to conventional cotton varieties.

Impact on Biodiversity:  The genetic modification that resulted in LLCotton25 did not confer any novel phenotypic characteristics that would increase its survival in either unmanaged habitats, or in areas outside the current geographic 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 herbicide tolerance trait to species in unmanaged habitats is insignificant.

Food and/or Feed Safety Considerations Expand

Dietary Exposure:  The genetic modification of LLCotton25 would not be expected to result in any change in the consumption pattern of cotton products. LLCotton25 did not express any novel characteristics with regard to cotton seed, oil, and the lint fraction that would alter the consumption pattern. This was confirmed by comparison of the levels of these fractions, and those of the proximates, and fatty acids in LLCotton25 to the unmodified parent and conventional cotton cultivars.

Only refined cottonseed oil and cellulose from processed linters of cottonseed are consumed by humans. 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. PAT protein was detected in linters from LLCotton25; this protein would also be expected to be removed and during heat and solvent treatment. Cottonseed oil for human consumption is highly refined and does not contain detectable levels of protein. The PAT protein was not detected in either crude oil, or the more refined, deodorized oil. The consumption of these cotton fractions would therefore not be expected to result in an increase in exposure to PAT protein.

Nutritional and Compositional Data:  The nutritional components of LLCotton25 cottonseed were determined analytically and compared to those of its parental line, ‘Coker 312.’ These components included proximates [moisture, ash, fat (ether extract), crude protein, crude fibre, acid detergent fibre (ADF), neutral detergent fibre (NDF), and carbohydrates], amino acids, fatty acids, minerals (calcium, phosphorus, iron, magnesium, potassium, and zinc), and vitamin E. Lint samples were analyzed for crude protein, fat, ash, crude fibre, ADF and NDF. The nutritional composition of the products of cottonseed processing (linters, delinted seed, meal, toasted meal, hull, crude oil and deodorized oil) was also determined, as appropriate for the particular fraction. The data from the nutritional analyses demonstrated that the composition of LLCotton25 cottonseed is comparable to that of the nonmodified parental line ‘Coker 312,’ and seed of other commercial cotton varieties. The composition of the refined (deodorized) oil fraction was also comparable to Coker 312 and commercial cottonseed oil.

The levels of the anti-nutritional factors phytic acid, gossypol (total and free), and cyclopropenoid fatty acids, were determined in cottonseed of LLCotton25 and Coker 312. The levels of these anti-nutrients in LLCotton25 were comparable to those of Coker 312, and to values published in the literature, except for levels of free gossypol, which were higher in both LLCotton25 and its parental line Coker 312. The higher level of gossypol was therefore attributed to genotype, and not to any unintended effect of the genetic modification in LLCotton25.

The feeding performance of LLCotton25 was assessed in a 33-day broiler chicken study. Treatments also included the parental line, and a conventional cotton variety. Cottonseed meal comprised 10% of the total ration fed to the broilers. No differences in animal performance, (weight gain, feed efficiency), feed consumption, and chilled carcass weight were observed among the treatments.

Potential Allergenicity and Toxicity:  The potential for toxicity and allergenicity of LLCotton25 was investigated using the following data and information: comparison of the amino acid sequence of the PAT to known toxins and allergens and the assessment potential glycosylation sites using several public databases (e.g., SwissProt, trEMBL, GeneSequ-Prot); citations from published and unpublished safety studies using the PAT protein (i.e., ex vivo digestibility using pig and cattle gastric fluids, in vitro digestibility using simulated gastric and intestinal fluid conditions, assessment of heat sensitivity, acute intravenous and subchronic oral toxicity studies in rodents); and results from a feeding study of broiler chickens using with LLCotton25 cottonseed meal.

The PAT protein in LLCotton25, as expressed by the bar gene, showed no amino acid sequence similarity with known toxins and allergens, and no potential glycosylation sites. The protein was rapidly digested under simulated gastric fluid conditions but demonstrated some resistance to heat degradation. The results of acute and subchronic rodent studies showed no adverse effects of the PAT protein, thereby addressing the concern with regard to the heat stability. The broiler chicken feeding study using cottonseed meal (10% of the total ration) also showed no adverse effects that could be attributed to the PAT protein in LLCotton25.

The PAT protein was expressed at very low levels in LLCotton25 cottonseed and was virtually absent from the oil. This fact, along with negative results of the various safety studies and sequence homology investigations, led to the conclusion that LLCotton25 did not demonstrate any potential for toxicity and 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.

Effective weed management is critical to cotton production. Weed control is more difficult in cotton than in other row crops, such as corn or soybean; cotton grows slowly early in the growing season and is thus less competitive with weeds. Competition from weeds has negative impacts on crop establishment, and ultimately, on crop yield. Weeds can also be detrimental later in the growing season, interfering with harvesting, and resulting in a reduction in lint quality due to trash and possible staining. Weed control strategies include pre-plant tillage, crop rotation, and the use of herbicides. Annual grasses and some small seed broadleaf weeds typically are controlled with preplant incorporated herbicides (e.g., trifluralin, norflurazon, pendimethalin) while most broadleaf species are controlled with postemergence herbicides (e.g., fluometuron, pendimethalin, pyrithiobac sodium). Crop rotation (e.g., with soybean) prevents the build-up of problem weeds, and herbicide resistant biotypes by allowing the use of different herbicides.
The cotton line LLCotton25 was developed to allow the use of glufosinate ammonium (trade name Liberty®), as a weed control option in cotton production. The herbicidal mode of action of glufosinate ammonium is related to the activity of glutamine synthetase (GS), the enzyme required for the synthesis of the amino acid glutamine. L-phosphinothricin, the active ingredient of glufosinate ammonium, is a structural analog of glutamate, and acts as a competitive inhibitor. After application of the herbicide, L-phosphinothricin competes with glutamine for its active sites on GS. The results of the inhibition of GS are an accumulation of ammonia in the plant, a reduction in the synthesis of glutamine, and an inhibition of photosynthesis. This causes the death of plant cells, and eventually, the entire plant. This genetically engineered cotton line LLCotton25 contains the bar gene, which codes for the production of the enzyme phosphinothricin acetyl-transferase (PAT). This enzyme acetylates glufosinate ammonium, rendering it inactive in the plant. The expression of the bar gene in LLCotton25 allows it to survive the otherwise lethal application of glufosinate ammonium. The bar gene was isolated from Streptomyces hygroscopius, a gram-positive soil bacterium.
LLCotton25 was developed by Agrobacterium-mediated transformation of the cotton variety ‘Coker 312’ with a plasmid vector containing the bar gene. Whole plants were treated with glufosinate ammonium and successful transformants were detected by selecting plants that had not exhibited the phytotoxic effects of glufosinate ammonium
LLCotton25 was field tested in the United States and Puerto Rico at 43 locations over three years (1999-2001). The data from these field trials showed that LLCotton25 did not differ significantly, in terms of plant morphology, growth, agronomic performance, and susceptibility to diseases and pests, from the nontransformed parent ‘Coker 312.’ LLCotton25 did not exhibit any increased tendency towards weediness, compared to the unmodified parental line. This was demonstrated by nonsignificant differences in parameters such as seed germination, seedling vigour, time to first bloom, and plant maturity, which affect the reproductive fitness and competitiveness of a plant. The impact on beneficial and nontarget organisms from the cultivation of LLCotton25 was similar to that of its unmodified parental line and conventional cotton cultivars. These results also led to the conclusion that the cultivation of LLCotton25 would not be expected to impact 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) 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, LLCotton25 could freely hybridize with other G. hirsutum varieties and wild plants.

The food and livestock safety of LLCotton25 was established based on: the fact that PAT protein constitutes a small amount of the total protein in LLCotton25, so there is little dietary exposure; the determination that the introduced gene and the novel protein are unlikely to be toxic or allergenic; and, in vitro, in vivo and other safety studies (e.g., intravenous and feeding studies) conducted in the laboratory using PAT protein. The nutritional equivalence of LLCotton25, compared to its untransformed parent line, was demonstrated by the analysis of key nutrients, including: protein, crude fat, ash, crude fibre, ADF, NDF, amino acid, fatty acids, minerals, vitamin E, as well as anti-nutrients. The equivalence of LLCotton25 to conventional cotton, in terms of livestock feed, was confirmed in feeding studies conducted with broiler chickens.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biosafety Office Comissão Técnica Nacional de Biossegurança - CTNBio (Brazil) European Food Safety Authority Food Standards Australia New Zealand Health Canada Novel Foods Japanese Biosafety Clearing House, Ministry of Environment Office of the Gene Technology Regulator, Australia U.S. Department of Agriculture, Animal and Plant Health Inspection Service U.S. Food and Drug Administration USDA-APHIS Petition

This record was last modified on Monday, August 7, 2017