GM Crop Database

Database Product Description

BXN
Host Organism
Gossypium hirsutum (Cotton)
Trait

Herbicide tolerant, oxynil.

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
Australia 2002 2002
Canada 1996 1996 View
Japan 1997 1998 1997
Mexico 1996 1996
United States 1994 1994 1994

Introduction Expand

Transgenic cotton (Gossypium hirsutum L.) line BXN™ was developed using recombinant DNA techniques to be tolerant of herbicides in the oxynil family, principally bromoxynil (tradename Buctril®). The oxynil family of herbicides is active against dicotyledonous plants by blocking electron flow during the light reaction of photosynthesis. This transgenic line was produced by inserting a gene 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
bxn nitrilase HT Native
nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens Native

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

The BXN™ cotton line was created by Agrobacterium-mediated transformation in which the transfer-DNA (T-DNA) contained the gene encoding the enzyme nitrilase from the soil bacterium Klebsiella pneumoniae subsp. ozaenae. For expression in plants, all but 11 bp of the 5’ and 96 bp of the 3’ non-translated regions of the gene were used. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII) from the Tn5 transposon of E. coli, strain K12.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from BXN™ cotton demonstrated the presence of one or two sites of integration of the T-DNA within the host genome. There was no evidence of integration of plasmid DNA sequences outside of the T-DNA region.

Expressed Material

The nitrilase enzyme was expressed at very low levels in unprocessed whole cottonseed, cottonseed hulls, and cottonseed meal, which are used in livestock feed. It was estimated that the enzyme comprised about 0.0006% of seed protein, which itself makes up a very small proportion of total meal protein. Refined cottonseed oil, which is the major cotton product consumed by humans, did not contain any detectable amounts of nitrilase enzyme. This result was expected as refined oils are generally free of DNA and protein.

Environmental Safety Considerations Expand

Field Testing

Transgenic BXN™ cotton was field tested in the United States at a total of 57 sites in 13 states. Field tests were also conducted in Argentina, Bolivia, and South Africa in accordance with national regulatory requirements. Based on data collected from these trials, it was concluded that this transgenic cotton line 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 line BXN™ is 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. 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 line BXN™ and its potential hybrid offspring. 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.
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

The novel proteins, nitrilase and NPTII, are not known to have any toxic properties on any nontarget organisms. The lack of known toxicity for these proteins and the low levels of their 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

This transgenic cotton line has no novel phenotypic characteristics that would extend its 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 BXN™ cotton 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 remove and destroy DNA and protein. The refined cottonseed oil from these lines was tested for the presence of nitrilase and NPTII, which were undetectable. It was concluded that there would be no dietary exposure to the novel proteins expressed in these cotton lines.

Nutritional Data

Compositional comparison of cottonseed oil, cottonseed and meal from BXN™ cotton 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.

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 the transgenic cotton line 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 BXN™ cotton to constitute a source of allergens is extremely low. In addition, the enzyme nitrilase does not have the properties of known allergens, such as glycosylation, protease stability, and presence in food at high concentrations. Also, an amino acid sequence homology search against public domain databases did not reveal any homologies between known protein allergens or toxins and the nitrilase enzyme.

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.

The BXN™ cotton line was genetically engineered to express tolerance to oxynil herbicides, 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. The modified cotton line BXN™ contains the bxn gene for oxynil tolerance, and allows farmers to use oxynil herbicides for weed control in the cultivation of cotton.

The bxn gene 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 encoding gene was introduced into the cotton genome using Agrobacterium-mediated transformation, and the bacterial form of the enzyme expressed in this transgenic cotton line functions the same as that found in monocot plants such as corn, wheat and barley.

Transgenic BXN™ cotton was field tested in the United States at a total of 57 sites in 13 states. Field tests were also conducted in Argentina, Bolivia and South Africa in accordance with national regulatory requirements. Based on data from these trials, it was concluded that the transformed cotton line did not exhibit weedy characteristics, and would not negatively affect beneficial or non-target organisms. The transgenic BXN™ cotton line was 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 BXN cotton may freely hybridize with other G. hirsutum varieties.

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 BXN™ line was tested for the presence of the nitrilase protein, which was undetectable. It was concluded that there was little potential for human dietary exposure to the novel protein expressed in this transgenic cotton line.

Compositional comparison of cottonseed oil, cottonseed and meal from BXN™ cotton 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 BXN™ 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 BXN™ cotton to constitute a source of allergens is therefore extremely low. In addition, the nitrilase enzyme does not display the properties of known allergens, such as glycosylation and protease stability. Further, an amino acid sequence comparison did not reveal any homologies between known protein allergens or toxins and the nitrilase enzyme. These data, along with the presence of the nitrilase protein in monocot food sources such as corn and barley, supported the conclusion that the nitrilase enzyme, and thus BXN™ cotton, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Australia New Zealand Food Authority Canadian Food Inspection Agency, Livestock Feeds Section Office of Food Biotechnology, Health Canada 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 Friday, March 26, 2010