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cotton event 531, which produces the Cry1Ac insect-control protein and the NPTII
selectable marker protein. Therefore, Bollgard II cotton produces two proteins for
effective control of the major lepidopteran insect pests of cotton, including the cotton
bollworm, tobacco budworm, pink bollworm, and armyworm. Bollgard II cotton also
produces the -D-glucuronidase (GUS) marker protein. In addition, Bollgard II cotton
provides a more effective insect resistance management program compared to single gene
products.
Bollgard cotton has been grown globally on more than 32 million acres since commercial
introduction in 1996 (James, 2002). The primary benefits that have resulted from the use
of Bollgard cotton are reduced insecticide use, improved control of target insect pests,
improved yield, reduced production costs, and improved profitability for cotton growers
(Edge et al., 2001; Carpenter and Gianessi, 2001; Betz et al., 2000; Economic Research
Service/USDA, 2000; Falck-Zepeda et al., 1998; Falck-Zepeda et al., 2000; Fernandez-
Cornjeo and McBride, 2000; Klotz-Ingram et al., 1999; Traxler and Falck-Zepeda, 1999;
Xia et al., 1999). With the addition of a second insect protection protein, Bollgard II cotton
provides increased control of cotton bollworm, as well as certain secondary insect pests of
cotton, including armyworm (U.S. EPA, 2002). Furthermore, along with the other
components of Monsantos insect resistance management program, combining the
Cry2Ab2 and Cry1Ac proteins in a single product provides an additional tool to delay the
development of insect resistance to Cry proteins in cotton.
The Cry2Ab2 protein produced in Bollgard II cotton event 15985 is derived from the
naturally occurring soil bacterium Bacillus thuringiensis (B.t.). Microbial formulations of
Bacillus thuringiensis, which include the Cry2A class of proteins, have been registered in
numerous countries worldwide and have been safely used for control of lepidopteran insect
pests for more than 40 years (Lthy et al., 1982; Baum et al., 1999; IPCS, 1999; Betz et al.,
2000). B.t. microbial formulations have been shown to be specific to the target insect pests
and do not have deleterious effects to non-target organisms such as beneficial insects, birds,
fish, and mammals, including humans (U.S. EPA, 1988; U.S. EPA, 1998). Therefore, there is
a history of safe dietary and occupational exposure to Cry proteins derived from B.t.,
including those of the Cry2A class.
The GUS protein present in Bollgard II cotton was used as a marker to facilitate the
selection of Cry2Ab2-producing plants. The GUS protein served no other purpose and has
cotton, which produces the Cry1Ac insect control protein, has been adopted
broadly by growers since its commercial introduction in 1996, as it provides effective
protection from feeding damage by lepidopteran insect pests such as tobacco budworm,
pink bollworm, and cotton bollworm (Carpenter and Gianessi, 2001). Bollgard cotton
growers typically apply significantly less insecticide to control these pests, realize higher
yields, and achieve greater profitability compared to growers using conventional cotton
varieties (Fernandez-Cornejo and McBride, 2000). Bollgard cotton has been grown on
more than 32 million acres globally since it was introduced in the United States in 1996
(James, 2002). The food, feed, and environmental safety of Bollgard cotton has been
reviewed (Hamilton et al., 2002; Monsanto, 2002).
The introduction of Bollgard II cotton, producing both the Cry1Ac and Cry2Ab2 proteins,
is expected to expand the range of benefits to both growers and the environment. Bollgard
II cotton provides equivalent or increased control of the major insect pests of cotton
(tobacco budworm, pink bollworm, and cotton bollworm) compared to Bollgard cotton,
with additional control of secondary lepidopteran insect pests such as beet and fall
armyworm. Combining the Cry2Ab protein with the Cry1Ac protein in Bollgard cotton
will also provide an additional tool to delay the development of resistance since these two
protein classes have different modes of action (Crickmore et al., 1998). In general, if the
second insecticidal protein is sufficiently different in its mechanism of action from the first,
and is itself highly efficacious against the target pest species, then insects would need to
develop two distinct modes of resistance to survive both proteins, which is highly unlikely.
Therefore, Bollgard II cotton, containing both the Cry1Ac and Cry2Ab proteins, provides
added protection against the risk of resistance developing in the primary target insect
species and is expected to extend the effectiveness of this technology for the grower and
prolong the overall benefits already documented for Bollgard cotton.
In conclusion, lepidopteran insect pests -- cotton bollworm, tobacco budworm, and pink
bollworm -- are the main insect pest problem in cotton production. Bollgard cotton,
producing the insecticidal protein Cry1Ac, has been widely adopted by growers because of
its efficacy against these pests and demonstrated environmental and economic benefits.
The introduction of Bollgard II cotton, producing both the Cry1Ac and Cry2Ab2 proteins,
will expand the range of benefits. Furthermore, Bollgard II cotton, in combination with
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other components of the insect resistance management program, is expected to significantly
delay the development of insect resistance.
The following sections describe the molecular characterization of the inserted DNA, the
levels of the Cry2Ab2 and GUS proteins, the safety assessment of the Cry2Ab2 and GUS
proteins, the compositional analyses of cottonseed, cottonseed oil and cottonseed meal
alone and for Bollgard II cotton compared to other cotton varieties and the environmental
risk assessment of Bollgard II cotton.
Molecular Characterization of Bollgard II Cotton
Bollgard II cotton event 15985 was generated by re-transformation of cotton meristems of
Bollgard cotton event 531, variety DP50B. A particle acceleration plant transformation
procedure was used to insert the cry2Ab2 insect control coding sequence and the uidA
marker coding sequence into the Bollgard cotton genome. The purified plasmid vector,
PV-GHBK11, is a 8.7Kb high copy number based plasmid containing well-characterized
DNA elements for selection and replication of the plasmid in bacteria (Figure 1). The
purified, linear DNA was inserted into the Bollgard cotton genome. The linear plasmid
fragment only contains two plant gene expression cassettes, each using separate controlling
DNA elements essential for production in the cotton plant cells and does not contain the
nptII selectable marker gene or origin of replication. The first cassette contains a copy of
the cry2Ab2 gene encoding the B.t. insecticidal protein Cry2Ab2 and the second cassette
contains the uidA gene encoding the -D-glucuronidase (GUS) marker protein to facilitate
selection of Cry2Ab2-producing plants. The GUS protein serves no other purpose and has
no known insect control properties.
The cry2Ab2 and uidA genes are both under the regulation of the enhanced cauliflower
mosaic virus 35S promoter (e35S) (Odell et al., 1985) and the 3 untranslated region of the
nopaline synthase gene (NOS 3) from Agrobacterium tumifaciens, which provides the
signal for mRNA polyadenylation. The e35S promoter driving the cry2Ab2 gene is also
fused to the 5 untranslated leader sequence from the petunia heat shock protein 70
(HSP70) and the chloroplast transit peptide from the Arabidopsis thaliana 5-enolpyruvyl
shikimate-3-phosphate synthase gene (CPT2), which is used to direct the protein to the
chloroplasts.
Integration of DNA into cotton germlings was detected by histochemical staining for GUS
protein activity in vascular tissue. Non-transformed tissue was removed and growth of
meristems containing the introduced DNA was promoted. The resulting seed from these
plants was screened for the production of Cry2Ab2 protein.
The molecular characterization of Bollgard II cotton demonstrated that there is one DNA
cry2Ab2 insert. The single DNA insert in Bollgard II cotton event 15985 contains one
copy of the cry2Ab and uidA gene cassettes from the linear DNA PV-GHBK11 used for
transformation containing:
August 2003 5
the complete cry2Ab coding region and cassette, although the restriction site
following the NOS 3 polyadenylation sequence in the cassette is not present;
and
the complete uidA coding region and cassette, except that 260 bp of the 5 end
of the enhanced CaMV 35S promoter is not present; however, the truncated
promoter is functional as demonstrated by production of the GUS protein.
Sequencing of the DNA inserted into Bollgard II cotton confirmed the molecular details
above. PCR and DNA sequencing verified the 5 and 3 ends of the insert and confirmed
that the DNA flanking the insert was native to cotton. Production of the full-length
Cry2Ab2 and GUS proteins was confirmed by western blot analysis.
Inheritance analysis of the cry2Ab2 insert conforms to the expected Mendelian segregation
pattern for a single genetic locus. The stability of the insert was demonstrated by Southern
blot over four generations of selfing and two generations of backcrossing. In addition,
progeny of Bollgard II cotton event 15985 have been field tested at multiple sites in the
U.S. since 1998. No instability of the DNA cry2Ab2 insert has been detected during
extensive field-testing and commercial seed production of Bollgard II cotton based on the
following results:
analyses of seed obtained from multi-site trials over four years showed similar levels of
the Cry2Ab2 and GUS proteins;
the production of the Cry2Ab2 protein has been confirmed by immuno-detection and/or
efficacy data under various environmental conditions and in numerous Bollgard II
cotton varieties;
the insecticidal efficacy has been maintained during the development of this product in
the U.S. and other world areas where this product will be commercialized; and
the production of the Cry2Ab2 protein has been maintained after transfer of the
cry2Ab2 gene into different varieties of cotton.
These data confirm that the Bollgard II cotton insert is stably integrated in the cotton
genome.
Cry2Ab2 and GUS Protein Levels in Bollgard II Cotton Plants
Enzyme-linked immunosorbent assays (ELISA) (Harlow and Lane, 1988) were developed
and optimized to estimate the Cry2Ab2 and GUS protein levels in cottonseed and cotton
leaf matrices. Cry2Ab2 and GUS proteins were detected in various plant tissues of
Bollgard II cotton plants during the 1998 growing season across eight locations
representative of major cotton production regions (Table 1). The Cry2Ab2 and GUS
proteins were detected in Bollgard II cotton plants but, as expected, neither protein was
detected in the parental control, Bollgard cotton, or in the non-transgenic control. The
Cry2Ab2 protein levels estimated in Bollgard II cotton leaf and seed were 23.9 and 43.2
g/g fresh weight, respectively. The mean protein levels for GUS were 106 and 58.8 g
fresh weight in leaf and cottonseed, respectively. These protein levels are low in
August 2003 6
comparison to total protein levels. Levels of the Cry2Ab2 protein were also measured in
whole plants collected at the end of the season, and in pollen. In field tests from the 1998
season, mature Bollgard II cotton plants contained an estimated 8.8 g Cry2Ab2 protein/g
fresh weight. The Cry2Ab2 protein was not detected in pollen collected from Bollgard II
cotton plants above the limit of detection of the assay (0.25 g/g fresh weight).
Safety Assessment of Cry2Ab2 and GUS Proteins in Bollgard II Cotton
Safety assessments of the Cry2Ab2 and GUS proteins produced in Bollgard II cotton event
15985 included protein characterization, demonstration of the lack of similarity to known
allergens and toxins, the long history of safe consumption of similar proteins, in vitro
digestibility, and the lack of acute oral toxicity in mice.
Cry2Ab2 is a protein derived from Bacillus thuringiensis and has also been designated
Cry2Ab2, CryIIB, CryB2 or CryIIAb (Liang and Dean, 1994; Widner and Whiteley, 1990;
Crickmore et al., 1998). In the current nomenclature scheme, Cry protein names are
assigned according to amino acid similarity to establish holotype proteins as defined by
Crickmore et al. (1998). In this nomenclature, Cry proteins with similar amino acid
sequences are grouped together. Cry proteins with the same Arabic numeral, e.g., Cry2,
share at least 45% amino acid sequence identity. Those with the Arabic numeral and upper
case letter, e.g., Cry2A, share at least 75% sequence identity. Finally, Cry proteins with
the same Arabic numeral, upper case letter and lower case letter, e.g., Cry2Ab, share
greater than 95% sequence identity.
Bacillus thuringiensis (B.t.) is a gram-positive bacterium commonly present in soil and has
been used commercially in the U.S. since 1958 in microbially derived products with
insecticidal activity (U.S. EPA, 1988). Bacillus thuringiensis subsp. kurstaki, present in
commercial microbial pest control products such as DiPel
and Crymax