GM Crop Database

Database Product Description

GHB614 (BCS-GHØØ2-5)
Host Organism
Gossypium hirsutum (Cotton)
Trade Name
GlyTol™ Cotton
Trait
Glyphosate herbicide tolerance.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for fibre, livestock feed, and human consumption.

Product Developer
Bayer CropScience USA LP

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Argentina 2014 2014 2012
Australia 2009
Brazil 2010 2010 2010
Canada 2008 2008
China 2010 2010 View
Colombia 2012
European Union 2011 2011
Japan 2010 2010
Korea 2010 2010
Mexico 2008 2008
Taiwan 2015 2015
United States 2008 2008 2009

Introduction Expand

Glyphosate normally exerts herbicide activity by binding and inactivating EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), an enzyme that is essential for the synthesis of proteins in plants. Cotton line GHB614 has been genetically modified (GM) for tolerance to glyphosate herbicides by expression in the plant of a modified epsps gene from corn (Z. mays), 2mepsps, which introduces two amino acid changes in the enzyme. The amino acid changes in the 2mEPSPS protein significantly lower the sensitivity to glyphosate, allowing the enzyme to continue to function in the presence of the herbicide. 

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
epsps 5-enolpyruvyl shikimate-3-phosphate synthase HT

Promoter region of the histone H4 gene from Arabidopsis thaliana.

Intron of gene II of the histone H3.III variant from A. thaliana.

3’ untranslated region of the histone H4 gene from A. thaliana.

1

double-mutated epsps gene from Z. mays

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.

Modification Method Expand

Cotton line GHB614 was developed through Agrobacterium-mediated transformation of the cotton variety Coker 312, using the transformation vector pTEM2. Cotton explants were exposed to a culture of disarmed Agrobacterium tumefaciens containing plasmid p-TEM2. After co-culture, the cotton cells were regenerated to whole plants using the appropriate regeneration media with 500 mg/L claforan to eliminate residual Agrobacterium, and then selected with glyphosate.

The shoots that developed were transferred to the greenhouse, further tested for tolerance to glyphosate, and allowed to flower and set seed. The transformation was confirmed by 2mEPSPS enzyme activity assay, by glyphosate application to leaves, and by polymerase chain reaction (PCR) and Southern blot analyses.

The transformation vector used to generate cotton line GHB614, p-TEM2, contains one gene expression cassette within the left and right border segments (T-DNA). The sequence of the 2mepsps gene is derived from the wildtype epsps gene from corn (Zea mays) with two single nucleotide mutations introduced by site directed mutagenesis. A methionine codon has been added to the N-terminal end of the 2mEPSPS protein sequence in order to restore the cleavage site of the optimized plastid transit peptide. The double mutant produces a 47 kDa protein with normal enzyme function and reduced affinity for glyphosate.

The Ph4a748At promoter and h3At intron are regulatory elements used to control expression of the 2mepsps gene in cotton and are derived from the histone H4 gene of the plant Arabidopsis thaliana. The use of these elements directs high level constitutive expression, particularly in rapidly growing plant tissues. TPotp C, encodes the optimized transit peptide derived from genes of corn and sunflower and targets the mature protein to the plastids where it is normally located in the cell. The 3’histonAt terminator from Arabidopsis thaliana corresponds to the polyadenylation signal which is essential to end transcription of the introduced gene.

Characteristics of the Modification Expand

The Introduced DNA

Genomic DNA from leaf tissue of GHB614 cotton plants (identity confirmed by PCR) was analysed using Southern blot analysis to determine the insert number, the copy number, the integrity of the inserted 2mepsps gene cassette, and evaluate the presence or absence of plasmid backbone sequences. Isolated genomic DNA samples from GHB614 cotton and conventional cotton were digested with nine different restriction enzymes, separated on agarose gels and then subjected to Southern blot analysis. To determine the insert and copy number of the introduced DNA, the separated DNA fragments were transferred to a membrane and sequentially hybridized with different radioactively labelled probes: four probes containing each single genetic element present in the p-TEM2 vector used for the transformation, and the complete T-DNA probe.

Based on a comparison of the size and pattern of observed fragments with the expected fragment sizes from digestion of genomic DNA, a single and unique site of insertion of the transgenic sequences is present in cotton line GHB614.
The organisation of the genetic elements within the insert in GHB614 cotton was further characterised using PCR analysis by amplifying three overlapping regions of DNA spanning the entire length of the insert. The PCR products generated, following PCR of genomic DNA from GHB614 cotton, were all of the expected size.

The PCR products generated from GHB614 cotton genomic DNA were subject to DNA sequencing to further confirm the organisation of genetic elements within the insert, as well as to determine the 5’ and 3’ insert-to-genomic DNA junctions, and the complete DNA sequence of the inserted DNA and adjacent genomic DNA regions. The sequence determination indicated that the size of the inserted DNA in GHB614 cotton is 3978 base pairs (bp) and the arrangement of the genetic elements within the insert is identical to the corresponding transformation vector, pTEM2. In addition to the insert sequence, 214 bp of Right Border flanking sequence (3’ end of insert) and 738 bp of Left Border flanking sequence (5’ end of insert) were found to be completely identical to the cotton genomic sequences present at the integration site before transformation.

Stability and Inheritance

For breeding purposes and further evaluation of inheritance, various backcrosses were performed and evaluated in the greenhouse for segregation and glyphosate resistance. For the segregation analysis, data from a Chi-square test of inheritance were used to determine the heritability and stability of the new trait. All Chi-square values indicate no significant differences between observed and expected genetic ratios across all tested generations of GHB614 cotton. These results are consistent with a single site of insertion for the 2mepsps gene expression cassette.
To determine the stability of the inserted DNA, Southern blot analyses were done using genomic DNA isolated from multiple generations of GHB614 cotton (T3, T4, T5 and T6). For these analyses, DNA samples from leaf tissue representing each generation were digested and probed to detect two integration fragments corresponding to ~4850 bp and ~9100 bp. In all tested samples, the expected 5’ and 3’ integration fragments were present. These results are consistent with bands detected in other Southern analyses of GHB614 cotton and confirm the stability of the insert across multiple generations of breeding.

Expressed Material

Concentrations of 2mEPSPS protein were determined in samples of fuzzy cottonseed collected from nine field trial locations in the southern United States. At each site, six plots were planted with transgenic cotton and three plots planted with the non-transgenic control. Three of the transgenic plots were sprayed three times with glyphosate herbicide at the level of 840 g/hectare, and three transgenic plots were untreated. Because the cottonseed (fuzzy seed) had been ginned but not delinted, it could not be ground into a homogeneous material. A procedure was developed to effectively remove the lint and the associated seed coat. This created two fractions, which were designated ‘kernel’ and ‘lint coat’. These fractions were analysed separately for 2mEPSPS protein and total extractable protein and the respective values added to give values for the fuzzy seed as received from the field.

The 2mEPSPS protein was found in all fractions of transgenic fuzzy seed (kernel and lint coat). As expected, more than 99.5% of the novel protein was found in the kernel samples. The lint coat generally contained less than 0.5% of the 2mEPSPS protein, and some samples were below the limit of detection. The levels of 2mEPSPS protein varied between different trial sites and between treatments with glyphosate. On a fresh weight basis, the 2mEPSPS protein content in fuzzy seed of GHB614 cotton, not sprayed with glyphosate, ranged from about 15.8 µg/g to 25.5 µg/g fresh weight, with an overall average value of 19.2 ±3.1 µg/g. On a fresh weight basis, the fuzzy seed from GHB614 cotton plants, sprayed with a conventional herbicide regime, contained 2mEPSPS protein in the range 16.2 µg/g to 30.5 µg/g, with an overall average value of 21.2 ± 4.0 µg/g. Using the average values for the amount of novel protein in unsprayed and sprayed fuzzy seed relative to the amount of crude protein, the 2mEPSPS protein comprised an average of 0.0093% ± 0.0018% and 0.0100% ± 0.0019% of the total crude protein respectively.

Environmental Safety Considerations Expand

Field Testing

Field studies were designed to compare agronomic performance of the transformed GlyTol cotton event GHB614, with the non-transformed Coker 312 counterpart. Data from agronomic trials were taken from 17 locations in 5 states over the 2004 and 2005 growing seasons. Studies were conducted in geographic regions of the southern United States representative of the regions in which nearly 94% of the total upland cotton production occurs. A comparison of 18 agronomic characteristics across eight locations in 2004 and nine locations in 2005 compared plant growth and plant mapping data taken by field agronomists to evaluate the growth and development of the plant. Plant mapping data was taken to evaluate the potential reproductive success of the cotton plant. The number of bolls, first position bolls, plant height, and total number of nodes are all key parameters for cotton production as it impacts the value, maturity, and development of the cotton fiber. Plant height and height to node ratio was calculated from these parameters as it is used as an indication of insufficient herbicide tolerance. These parameters are also used to determine the application timing of plant growth regulators (PGR) used to manage the height of the cotton plant which improves mechanical harvesting efficiency. Morphology ratings were taken to monitor for irregular plant development in key portions of the plant such as the leaves, flowers, and bolls. Fertility of the plant was measured in rating the number of embryos (seed) in the boll, their weight and the size of the fruit developed to house them. Fertility ratings were also taken to evaluate the fertility of the flower by the amount of pollen present and dehiscence.

Evaluation of plant mapping data and crop development data showed no significant differences in the GlyTol cotton event GHB614 and the non-transgenic Coker 312 counterpart. Findings across locations show that GlyTol cotton is similar for maturity and yield to the nontransformed counterpart in both Coker 312 and commercial cotton varieties. No differences were noted in the morphology of the plants when compared across testing locations. All plants with the GlyTol cotton event GHB614 and their non-transgenic Coker 312 counterparts appeared to develop normally, with no abnormalities noted in field observations.

Seed Dormancy

Seed dormancy was evaluated in the GlyTol cotton event GHB614 to ensure that seed dormancy was not affected by the transformation of the Coker 312 germplasm or the production of the 2mEPSPS protein. Seeds were collected from the 2005 efficacy trials of transformation event GHB614 in six locations to test for effects to the seed dormancy. No significant differences were seen in germination between the transgenic and nontransgenic seed produced from the 2005 plot sites. Normal variation was detected between those which were planted immediately after harvest, and those which were stored for six months. This indicates that transformation event GHB614 does not increase seed dormancy and therefore does not contribute to the weediness of the transformed cotton plant through increased seed dormancy.

Potential Plant Pest Risk

In addition to agronomic performance, composition of the seed was evaluated for any potential plant pest risks to current cotton production in the United States. Composition data provided information on gossypol, antinutrient levels, and other toxicant contents. The overall conclusion is that there are no agronomically meaningful differences between the transformed GlyTol cotton event GHB614 and non-transformed cotton varieties evaluated. The resulting conclusion is that there are no new agronomic plant pest risks from the introduction of GlyTol cotton.

Potential for Increased Weediness

In the United States, cotton (G. hirsutum) is not a weed pest and has no sexually compatible weedy relatives except perhaps G. tomentosum in Hawaii where there is no commercial cotton production. There is limited probability that Glytol cotton event GHB614 or any Gossypium species containing GlyTol cotton event GHB614 would become a weed problem. From the agronomic and phenotypic data, there were no consistent significant differences in germination, dormancy, phenotypic or plant morphological characteristics between the transgenic GlyTol cotton event GHB614 and the conventional near isogenic line Coker 312 that would impact plant pest or noxious weed potential. Based on these data there was no evidence to suggest that GlyTol cotton has a higher likelihood to become a weed than conventional cotton. There were no instances in which volunteer monitoring after harvest revealed any differences in survival or persistence relative to other cotton varieties.

Potential Impact on Non-Target Organisms

Compositional analysis on the plants containing 2mEPSPS protein indicated no significant changes in the overall gossypol content of the plants or antinutrient levels between GlyTol cotton event GHB614 and the non-transgenic counterpart. This indicates that the transformed cotton is no more toxic than its non-transgenic counterpart. Composition findings are reinforced by visual field observations made by cooperators conducting field evaluations of the GlyTol cotton plants. Cooperators visually monitored all plots for differences in beneficial insect populations and types for each trial, as well as birds, pollinators, and other wildlife species. No reports of differences in populations for any of these non-target organisms were made by cooperators making these observations.

Food and/or Feed Safety Considerations Expand

Compositional Analysis

The main purpose of compositional studies is to determine if any unexpected changes in composition have occurred to the food and to establish its nutritional adequacy. In the case of cottonseed, the key components that should be considered in the comparison include protein, fat, carbohydrate, amino acids, fatty acids, vitamins, minerals and the antinutrients, gossypol and cyclopropenoid fatty acids. Compositional analyses were done on fuzzy seed collected from GHB614 cotton and the non-GM counterpart, Coker 312, grown in field trials typical of commercial agricultural production.

Nine field trials were conducted in 2005 at sites representing primary cotton-growing regions of the south-eastern United States. At each test site, six plots of transgenic event GHB614 cotton and three non-transgenic plots of Coker 312 were planted. Three of the six plots containing GHB614 cotton were sprayed three times with glyphosate herbicide (0.75 pounds active ingredient/acre). Compositional analysis of the cottonseed samples included proximates (protein, fat, ash and moisture), acid detergent fibre (ADF), neutral detergent fibre (NDF), minerals (calcium, iron, magnesium, phosphorus, potassium and zinc), amino acids, fatty acids, vitamin E (alpha tocopherol) and carbohydrates by calculation. In addition, three known anti-nutrients found in cotton (gossypol, cyclopropenoid fatty acids and phytic acid) were analysed. Methods of analysis were based on internationally recognised procedures (e.g. AOAC International methods) or other published methods.

No differences of biological significance were observed between GHB614 cotton and its conventional counterpart. Statistically significant differences in some of the key constituents were noted, however the differences observed were minor, and in each case the levels observed were within the range of literature values reported for conventional cotton varieties. The composition of cotton is known to vary significantly with the site, agricultural conditions and season of production, and differences reported here most likely reflect normal biological variability. Food from GHB614 cotton is therefore considered to be compositionally equivalent to food from conventional cotton varieties.

Potential Toxicity and Allergenicity

In terms of its potential toxicity and allergenicity, it is worth noting that the 2mEPSPS protein has been evaluated previously as the novel protein present in glyphosate-tolerant corn line GA21 which has been approved for food and feed use in numerous countries. It is therefore likely to have been widely distributed in corn based foods. This modified enzyme is derived from the native EPSPS enzyme in maize (99.5% amino acid homology), and is closely related to other EPSPS enzymes from plants and microorganisms which are natural constituents of human diets.
Bioinformatic studies with the 2mEPSPS protein sequence has confirmed the absence of any significant amino acid sequence similarity to known protein toxins or allergens.

Two independent acute oral toxicity studies using purified 2mEPSPS protein have been conducted by different applicants. In the study conducted by Monsanto for GA21 maize, the modified EPSPS (2mEPSPS) protein was administered by a single oral gavage dose to ten male and ten female CD-1 mice, at target doses of 5, 15 and 50 mg/kg bodyweight. In this study, this corresponded to actual doses of 3.7, 11.8 and 45.6 mg/kg respectively. The results of the study showed no statistically significant differences between test and control treatment groups in mean body weights, cumulative weight gains or food consumption in either males or females at any treatment level. The study concluded that there was no evidence of toxicity in mice following a single oral dose of 45.6 mg/kg modified EPSPS (2mEPSPS) protein. In the study conducted by Bayer for GHB614 cotton, the 2mEPSPS protein was administered by a single oral gavage dose of 2000 mg protein/kg bodyweight to 5 female OF1 mice. A second group of female mice received the same dose of bovine serum albumin as a negative control. All animals were observed for clinical signs daily for fifteen days and body weights were measured weekly. At termination, all animals were subjected to necropsy including macroscopic examination. There were no clinical signs, mortalities or treatment related effects on bodyweight in female OF1 mice observed during this study. Based on these findings, it was concluded that no oral toxicity was demonstrated in mice at a very high dose of 2000 mg/kg bodyweight.

One of the criteria for assessing potential allergenicity is to determine the stability of novel proteins in conditions that simulate human digestion. Proteins that are rapidly degraded in such conditions are considered less likely to be involved in eliciting an allergic response. The 2mEPSPS protein was subjected to digestibility studies using simulated human gastric fluid (SGF) containing pepsin and simulated intestinal fluid (SIF) containing porcine pancreatin, which is a mixture of enzymes including amylase, trypsin, lipase, ribonuclease and protease. In SGF (with pepsin), there was no full length or partially degraded 2mEPSPS protein observed at 30 seconds and at subsequent time points. In SIF, the 2mEPSPS protein band was only faintly visible after scanning the gel even at time zero. At all subsequent incubation times, there was no full length or partially degraded 2mEPSPS protein observed.

The weight of evidence shows that the 2mEPSPS protein is not toxic and unlikely to be allergenic in humans.

Links to Further Information Expand

Brazil Ministry of Science and Technology - MCT The National Biosafety Technical - CTNBio Executive Secretary Canadian Food Inspection Agency, Animal Feed Division Colombian Agricultural Institute European Food Safety Authority European Food Safety Authority (EFSA) Food Standards Australia New Zealand Japan (MAFF) U.S. Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Wednesday, October 21, 2015