AGBIOS DATABASE

(http://www.agbios.com/dbase.php?action=ShowProd&data=MON-%D8%D8531-6+x+MON-%D81445-2)

MON-ØØ531-6, MON-ØØ757-7 (MON531/757/1076)

Host Organism / Variety Gossypium hirsutum L. (Cotton) Bollgard®
Trait Resistance to lepidopteran pests including,
but not limited to, cotton bollworm, pink
bollworm, tobacco budworm.
Trait Introduction Method Agrobacterium tumefaciens-mediated plant
transformation.
Proposed Use Production of cotton for fibre,
cottonseed and cottonseed meal for
livestock feed, and cottonseed oil for
human consumption.
Company Information Monsanto Company

Summary of Regulatory Approvals

Country Environment Food and/or Feed Food Feed Marketing
Argentina 1998 1998 1998
Australia 1996 1996 1996
Brazil 2005 2005 2005
Canada 1996 1996
China 2004 2004
European Union 2005
India 2002
Japan 1997 1997 1997
Korea 2003 2004
Mexico 1997 1997 1997
Philippines 2004 2004
South Africa 1997 1997 1997
United States 1995 1995
Click on the country name for country-specific contact and regulatory information.
Notes
Canada Not grown in Canada. Not subject to variety registration.
Australia Approval for line 531; Date shown is the date of latest approval.
China Approval for line 531
Argentina Approval for line 531;
Mexico Approval for line 531; Date shown is the date of latest approval.
South Africa Approval for line 531
Philippines Approval for line 531 only.
European Union Line 531 notified as an existing product on 18 April 2005.
Brazil Approval for line 531 only
Introduction
Cotton lines 531, 757, and 1076 (trademark Bollgard®, Ingard®) were developed through specific
genetic modifications to be resistant to major caterpillar pests of cotton. The transgenic cotton lines
express a modified gene (cry1Ac) that encodes an insecticidal crystalline Cry1Ac delta-endotoxin
protein, derived from the soil bacterium Bacillus thuringiensis subsp. kurstaki strain HD73.
Insecticidal activity is caused by the selective binding of Cry1Ac protein to specific sites localized on
the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific
pores are formed that disrupt midgut ion flow and thereby cause gut paralysis and eventual death
due to bacterial sepsis. Delta-endotoxins, such as the Cry1Ac protein expressed in cotton lines 531,
757, and 1076, exhibit highly selective insecticidal activity against a narrow range of lepidopteran
insects such as cotton bollworm, tobacco budworm and pink bollworm. The specificity of action is
directly attributable to the presence of specific receptors in the target insects. There are no
receptors for delta-endotoxins of B. thuringiensis on the surface of non-lepidopteran insect guts or
mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these
proteins.

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 plants. 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).

Another selectable marker gene, aad (3"(9)-O-aminoglycoside adenylyltransferase (AAD)), was also
inserted into the host genome. This gene, which was not expressed in the plants, was used during
the development process to select for bacterial colonies that had been transformed with
recombinant plasmid DNA.

Unless otherwise indicated, the information presented here is based on documentation pertaining to
cotton line 531.
Summary of Introduced Genetic Elements
Code Name Type Promoter, Terminator Copies Form
other
cry1Ac Cry1Ac delta- IR double enhanced 3' poly(A) signal from >=1 Truncated; Line 757:
endotoxin (Bacillus CaMV 35S soybean alpha subunit 1 complete T-DNA
thuringiensis subsp. of the beta-conglycinin and 1 paritial T-DNA
kurstaki (Btk)) gene insertion event
aad 3"(9)-O-aminoglycoside SM bacterial Not expressed in
adenylyltransferase promoter plant tissues
neo neomycin SM nopaline synthase >=1 Native
phosphotransferase (nos) from A.
II (Escherichia coli) tumefaciens
Characteristics of Gossypium hirsutum L. (Cotton)
Center of Origin Reproduction Toxins Allergenicity
Believed to originate in Generally self-pollinating, but can be cross-pollinating Gossypol in
Meso-America (Peruvian- in the presence of suitable insect pollinators (bees). In cottonseed
Ecuadorian-Bolivian the U.S., compatible species include G. hirsutum, G. meal.
region). barbadense, and G. tomentosum.
Donor Organism Characteristics
Latin Name Gene Pathogenicity
Bacillus cry1Ac Although target insects are susceptible to oral doses of Bt proteins, there is no
thuringiensis evidence of toxic effects in laboratory mammals or birds given up to 10 µg protein / g
subsp. kurstaki body wt. There are no significant mammalian toxins or allergens associated with the
host organism.
Modification Method
The cotton lines 531, 757, and 1076 were produced by Agrobacterium-mediated transformation of
the cotton (Gossypium hirsutum) line L. cv Coker C312 with plasmid PV-GHBKO4. Plasmid PV-
GHBK04 contained the following elements: the 0.4 kb oriV fragment from the RK2 plasmid fused to
the 3.4 kb segment of pBR322 allowing maintenance in Escherichia coli and in Agrobacterium
tumefaciens. This was fused to the 360 bp DNA fragment from pTiT37 plasmid, which contained the
nopaline T-DNA right border.

The remaining portion consisted of two genes engineered for plant expression, the cry1Ac and the
NPTII encoding neo gene. The cry1Ac gene was modified for optimal expression in plants and
contained part of the 5' end of the cry1Ab gene with a portion of the cry1Ac gene. Expression of the
modified cry1Ac gene was regulated by cauliflower mosaic virus (CaMV) 35S promoter with a
duplicated enhancer region and the nontranslated region of the soybean alpha subunit of the beta-
conglycin gene which provided the mRNA polyadenylation signals (7S 3’ terminator sequence).

The transformation plasmid also contained the aad gene isolated from E. coli bacterial transposon
Tn7, which encodes the enzyme aminoglycoside adenyltransferase (AAD) that confers resistance to
the antibiotics spectinomycin and streptomycin. The aad gene was under the control of its own
bacterial promoter and terminator and was included in the construct as a marker to allow for
selection of bacteria containing PV-GHBK04 prior to transformation of the plant cells. The aad gene
has no plant regulatory sequences and was not expressed in plant tissues.

The neo gene was located downstream of the aad gene and its expression was regulated using the
CaMV 35S promoter and the non-translated region of the 3’ region of the nopaline synthase gene
(nos) from the pTiT37 plasmid of A. tumefaciens strain T37.
Characteristics of the Modification
The Introduced DNA
The inserted cry1Ac gene encoded an insecticidal protein similar to a full length Cry1Ac protein with
some minor changes arising from the design of the gene. The encoded Cry1Ac plant produced
protein was 99.4% homologous to the native protein from B. thuringiensis subsp. kurstaki HD73.

Southern blot analyses of genome DNA from line 531 demonstrated that two copies of the T-DNA
insert were integrated in a head-to-tail arrangement. One T-DNA insert contained a full-length
cry1Ac gene and the NPTII encoding gene, and the second insert contained an inactive 3’ portion of
the cry1Ac gene. The two inserts were linked and segregated as a single locus. Similar analyses
demonstrated that the ori322 region, present in PV-GHBK04, was not transferred into the 531
genome. The aad gene was present but was not expressed since it was under the control of a
bacterial promoter.

Genetic Stability of the Introduced Trait

The stability of the inserted genes for lines 531 and 1076 was demonstrated over 4 generations.
Stability for line 757 was tested and confirmed across 2 generations.

Expressed Material
Cry1Ac protein expression levels in each of the three transgenic cotton lines were
quantitated using enzyme linked immunosorbent assay (ELISA). Mean Cry1Ac expression
in line 531 was 1.56 µg protein/g and 0.86 µg protein/g fresh weight in leaf and seed
tissue respectively. In the whole plant the Cry1Ac protein averaged 0.044 µg protein/g
fresh weight, (25 µg/ plant) for a total of about 1.44 g/ acre. Expression varied
approximately 3 fold over the growing season, with the levels peaking late in the season.
Expression of the Cry1Ac protein was higher in line 757. Average values were 12.6 µg protein/g
fresh weight and 9.9 µg protein/g fresh weight in leaves and seeds respectively. Expression varied
less than 3 fold over the season and peaked early in the season in leaf tissue. Whole plant amounts
were 1.1 µg protein/g fresh weight (200 µg/plant) for a total of 12.2 g/ acre. Line 1076 produced
the cry1Ac protein at an average of 12.2 µg protein/g fresh weight and 12.7 µg protein/g fresh
weight in leaves and seed respectively. Gene expression over the growing season varied less than 5
fold, with the highest concentration occurring early in the season. Whole plant totals were 1.1 µg
protein/g fresh weight (389 µg/plant) for a total of 23.3 g/ acre. (Calculations per acre based on
60000 plants/acre). Expression in pollen and nectar was negligible in all three lines.

Expression levels for the NPTII protein in line 531 were 3.14 µg protein /g fresh weight and 2.45 µg
protein/g fresh weight in leaf and seed tissues respectively. Expression varied about 2 fold over the
season. In line 757 NPTII expression was 6.9 µg protein/g fresh weight and 3.3 µg protein/g fresh
weight in leaves and seeds respectively. For line 1076, the NPTII expression was 16.3 µg protein/g
fresh weight and 7.9 µg protein/g fresh weight in leaves and seed respectively. Gene expression
varied 2 to 3 fold over the growing season for line 757 and line 1076.
Environmental Safety Considerations
Field Testing
The transgenic cotton lines 531, 757 and 1076 were field tested in the United States (1990-1994).
Agronomic characteristics such as yield, boll size, plant vigour, growth, morphology, germination
and flowering, were compared to those of unmodified cotton counterparts and were shown to be
within the range of non-transformed cotton lines. Stress adaptation was evaluated, including
susceptibility to various pests and pathogens. Susceptibilities to diseases such as bacterial blight,
boll rot, Fusarium, Phymatotricum root rot, and verticillium wilt were unchanged. Processing
qualities of cotton lint such as micronaire, length, strength and elongation were compared. The
observed variability was within the range of inherent variability of cotton varieties and was not
attributed to the inserted genes. Reports demonstrated that the cotton lines 531, 757 and 1076 did
not exhibit weedy characteristics and had no effect on nontarget organisms or the general
environment relative to conventional cotton varieties.

Outcrossing
Cotton (Gossypium hirsutum) is mainly a self-pollinating plant, but insects, especially bumblebees
and honeybees, also routinely transfer pollen. The pollen is heavy and sticky and the range of
natural crossing is limited. Outcrossing rates of up to 28% to other cotton cultivars have been
observed under field conditions and decline rapidly with distance from the pollen source. Given
proximity and the availability of insects as pollen vectors, transgenic cotton lines 531, 757 and 1076
are likely to hybridize with other cotton varieties.

In the United States, compatible species include G. hirsutum (wild or under cultivation), G.
barbadense (cultivated Pima cotton), and G. tomentosum. There are no wild relatives or wild
populations of cotton in Canada that can naturally hybridize with G. hirsutum. It was reported that
gene movement from G. hirsutum to G. barbadense may be possible given suitable conditions, while
gene transfer to G. tomentosum is less probable due to chromosomal incompatibility and non-
synchronous flowering periods. Overall, the probability of gene transfer in unmanaged ecosystems
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 Cry1Ac or NPTII encoding genes were not expected to confer an ecological advantage to
transgenic cotton lines 531, 757 and 1076 and their potential hybrid offspring. Resistance to
kanamycin and specific lepidopteran insects will not render cotton weedy or invasive of natural
habitats since none of the reproductive or growth characteristics have been modified. Field trial
reports indicated that traits which may confer a selective advantage, such as increase in volunteers
from seed, regrowth from stubble, or increase in seed dormancy in cotton lines 531, 757 and 1076
were equivalent to non-modified varieties.

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

Secondary and Non-Target Adverse Effects

It was concluded that the introduction of the Cry1Ac and NPTII encoding genes into transgenic
cotton lines 531, 757 and 1076 would not result in any deleterious effects or significant impacts on
nontarget organisms, including those that are recognized as beneficial to agriculture and those that
are recognized as threatened or endangered in the United States and Canada.

The novel proteins, Cry1Ac and NPTII, expressed in these cotton lines 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 expression in plant tissue suggest no potential for deleterious effects on beneficial
organisms such as bees and earthworms.

The insecticidal protein was shown to be active on only lepidopteran species, even when fed at
levels that were well above the concentrations that were effective on target species. Host range
specificity was similar for the native and synthetic proteins. Tobacco hornworm was the most
sensitive species and tobacco budworm, corn earworm and European corn borer were relatively less
sensitive. The test system included six non-target species representing the orders Coleoptera,
Diptera, Orthoptera and Homoptera (boll weevil, Southern corn rootworm, Colorado potato beetle,
yellow fever mosquito, German cockroach and green peach aphid).

Dietary toxicity studies were performed using the microbial protein on beneficial insects (honeybee,
ladybird beetle, green lacewing larvae and parasitic wasp). No effect was observed on non-target
insects at concentrations more than 100 fold higher than those used to control target insects.

Acute oral and short-term studies were undertaken with albino mice and bobwhite quail. After 8
days there were no observable effects on mice fed the insecticidal protein at the highest dose of
4,000 mg/kg body weight. Raw cottonseed was fed at up to 100,000 ppm to bobwhite quail with no
observed effects. These observations were expected, as the novel proteins are rapidly inactivated in
simulated mammalian stomach fluids by enzymatic degradation and pH-mediated proteolysis. The
proteins expressed in transgenic cotton lines were shown to be equivalent to the original microbial
proteins produced by the common soil B. thuringiensis subsp. kurstaki bacteria.

Impact on Biodiversity
The transgenic cotton lines 531, 757 and 1076 had no novel phenotypic characteristics that would
extend their use beyond the current geographic range of cotton production. Since there is no
occurrence of wild relatives of cotton in Canada, there will be no transfer of novel traits to
unmanaged environments. Similarly, as the risk of gene transfer to wild relatives in the United
States is very remote, it was determined that the risk of transferring genetic traits from cotton lines
531, 757 and 1076 to species in unmanaged environments was insignificant.
Food and/or Feed Safety Considerations
Dietary Exposure
Refined cottonseed oil and cellulose from processed linters of cottonseed are the only cotton
products 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 and
protein. Similarly, the refining process for cottonseed oil includes heat, solvent and alkali treatments
that would remove and destroy DNA and protein. Typically, cottonseed oils are pooled and blended
together and it is anticipated that the oil from 531, 757, and 1076 cottonseed will not be handled or
treated any differently than other cottonseed oils. The genetic modification in these cotton lines will
not result in any change in the consumption pattern for this product. As the introduced gene
products are not detectable in the refined oil produced from transgenic cotton, there is no
anticipated human exposure to these proteins based on normal consumption patterns.

Nutritional Data
Compositional comparison of cottonseed from lines 531, 757 and 1076 was made to commercial
non-transgenic cottonseed. The cottonseed used for compositional analyses was taken from four to
six trial sites. The compositional analyses of cottonseed included proximates (crude protein, crude
fat, crude fibre, ash and gross energy), amino acid composition, fatty acids profile, aflatoxins and
levels of tocopherols.

The concentrations of protein, oil, carbohydrate and ash were the same for transgenic cotton lines
531, 757 and 1076 and the control parent line Coker 312 line. Line 1076 contained slightly higher
carbohydrate and slightly lower oil concentrations than the control line, but both were within the
normal range for cottonseed. Fatty acid concentration was within the normal published range for
cottonseed. Additional analyses of composite samples of cottonseed products (raw meal, toasted
meal, kernel, refined oil) from each line showed that the products from the transgenic cotton lines
were similar in composition to the control line. Feed studies of a four week rat feeding trial showed
no difference in weight gain of animals fed diets containing 10% raw cottonseed meal from line 531
vs. Coker 312.

The analysis of the fatty acid composition of refined oil from 531 and 757 cotton did not reveal any
significant differences with the parent, non-transgenic variety and was within the normal range
reported for cottonseed oils. In addition, the levels of alpha-tocopherol in refined oil from transgenic
and control lines were similar. The consumption of refined oil from 531 and 757 cottonseed will
have no significant impact on the nutritional quality of the food supply.

Toxicity
Toxicity of cotton lines was assessed by analysis of naturally occurring toxins in cotton and an
evaluation of the novel proteins Cry1Ac and NPTII. Toxicant analyses in cottonseed and refined oil
evaluated levels of gossypol and cyclopropenoid fatty acids (sterculic and malvalic acid).

Cotton is known for the production of anti-nutritional factors and untreated raw seed is unsuitable
as livestock feed for monogastric animals. The transgenic and parental lines were assayed for the
presence of potential toxins, including gossypol, dihydrosterculic acid, sterculic acid, malvalic acid
and aflatoxins B1, B2, G1 and G2. At detection thresholds of 0.002% or 1 ppb, respectively, neither
free gossypol nor any of the four aflatoxins were detected in the oil from transgenic cottonseed.
Similarly, the respective levels of the cyclopropenoid fatty acids (dihydrosterculic, sterculic and
malvalic) were statistically identical in cottonseed samples from transgenic and control lines.

The deduced amino acid sequences of both Cry1Ac and NPTII were compared to the amino acid
sequence of known protein toxins. No significant similarities were found.
Allergenicity
Refined cottonseed oil and cellulose from linters are devoid of detectable protein and their
consumption is unlikely to result in an allergic reaction since most allergens are proteins. Therefore
the potential for cottonseed oil or linters from transgenic cotton lines to constitute a source of
allergens is extremely low.

The Cry1Ac and NPTII proteins are not likely to be toxic or allergenic. The potential for allergenicity
was assessed based on studies that included digestive degradation and sequence similarity to
known allergens. The plant expressed proteins Cry1Ac and NPTII showed no significant protein
sequence homology to a database of known toxins or allergens. Also, unlike known allergenic
proteins, both Cry1Ac and NPTII were rapidly degraded when exposed to simulated gastric fluids
(pH 1.2, pepsin digestion).
Links to Further Information
Australian Genetic Manipulation Advisory Committee (GMAC) [PDF Size: 34853
bytes]
GR-3: Application for commercialisation of insect-resistant cotton
Brazillian national Biosafety Technical Commission (CTNBio) [PDF Size: 101004
bytes]
Approval for commercial cultivation of insect resistant cotton.
Canadian Food Inspection Agency, Plant Biotechnology Office [PDF Size: 161311
bytes]
Decision Document 96-14: Determination of Environmental Safety of BollgardTM Insect Resistant
Cotton (Gossypium hirsutum L.)
European Commission Scientific Committee on Plants [PDF Size: 153882 bytes]
Opinion of the Scientific Committee on Plants on the genetically modified cotton line, insect-tolerant
notified by the Monsanto company (notification C/ES/96/02) (Opinion expressed by SCP on 14 July
1998)
European Commission: Community Register of GM Food and Feed [PDF Size: 15677
bytes]
Notification of the placing on the Community Register of MON-ØØØ531-6.
European Commission: DG health and Consumer Protection [PDF Size: 105981
bytes]
Opinion of the Scientific Committee on Plants on the genetically modified cotton line, insect-tolerant
notified by the Monsanto company (notification C/ES/96/02) (Opinion expressed by SCP on 14 July
1998)
Japanese Biosafety Clearing House, Ministry of Environment [PDF Size: 111906
bytes]
Outline of the biological diversity risk assessment report: Type 1 use approval for MON531
Japanese Biosafety Clearing House, Ministry of Environment [PDF Size: 178917
bytes]
Outline of the biological diversity risk assessment report: Type 1 use approval for MON757
Monsanto Company [PDF Size: 100937 bytes]
Product safety description
Office of Food Biotechnology, Health Canada [PDF Size: 47546 bytes]
NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGY INSECT RESISTANT COTTON LINES 531 and
757
Office of the Gene Technology Regulator [PDF Size: 393927 bytes]
DIR 022/2002: Commercial release of insecticidal (INGARD® event 531) cotton
U.S.Department of Agriculture, Animal and Plant Health Inspection Service [PDF
Size: 7741109 bytes]
Monsanto Co. Petition for Determination of Non-regulated Status: BollgardTM Cotton Line 531
(Gossypium hirsutum L.) with the gene from Bacillus thuringiensis subsp. kurstaki
US Food and Drug Administration [PDF Size: 127946 bytes]
Memorandum to file concerning insect-resistant cotton.
USDA-APHIS Environmental Assessment [PDF Size: 70188 bytes]
USDA/APHIS Determination on a Petition 94-308-01p of Monsanto Agricultural Company Seeking
Nonregulated Status of Lepidopteran- Resistant Cotton Lines 531, 757, 1076
References
1. Berberich, S.A., Ream, J.E., Jackson, T.L., Wood, R., Stipanovic, R., Harvey, P., Patzer, S.
& Fuchs, R.L. (1996). The composition of insect-protected cottonseed is equivalent to that
of conventional cottonseed. Journal of Agricultural and Food Chemistry 44, 365-371.
2. Betz, F.S., Hammond, B.G. & Fuchs, R.L. (2000). Safety and advantages of Bacillus
thuringiensis-protected plants to control insect pests. Regulatory Toxicology 32, 156-173.
3. Perlak, F.J., R.W. Deaton, T.A. Armstrong, R.L. Fuchs, S.R. Sims, J.T. Greenplate & D.A.
Fischoff. (1990). Insect resistant cotton plants. Biotechnology 8: 9-13.
4. US Food and Drug Administration (1994). Secondary Food Additives Permitted in Food for
Human Consumption; Food Additives Permitted in Feed and Drinking Water of Animals;
Aminoglycoside 3'-Phosphotransferase II; Final Rule. Federal Register, 59:26700-26711.
5. Wilson, F.W., H.M. Flint, W.R. Dearon, D.A. Fischoff, F.J. Perlak, T.A. Armstrong, R.L.
Fuchs, S.A. Berberich, N.J. Parks & B.R. Stapp. (1992). Resistance of cotton lines
containing a Bacillus thuringiensis toxin to pink bollworm (Lepidoptera: Gelchiidae) and
other insects. J. Econ. Entomol. 85: 75-80.

THIS RECORD WAS LAST MODIFIED ON FRIDAY, JULY 22, 2005.