Monsanto Company 12-CT-244U 1 of 620 

Petition for the Determination of Nonregulated Status for Dicamba and 

Glufosinate-Tolerant Cotton MON 88701 

The undersigned submits this petition under 7 CFR § 340.6 to request that the 

Administrator make a determination that the article should not be regulated under 

7 CFR Part 340. 

July 2, 2012 

(Revised September 10, 2012) 

OECD Unique Identifier: MON-887Ø1-3 

Monsanto Petition Number: 12-CT-244U 

Submitted by:

Marianne Malven, M.S. 

Monsanto Company 

800 North Lindbergh Blvd. 

St. Louis, MO 63167 

Phone: (314) 694-2225 

Fax: (314) 694-8414 

E-mail: marianne.malven@monsanto.com 

Prepared by:

S.M. Arackal, M.S., B.A. Comstock, B.S., D. Chandu, Ph.D., A.E. Deffenbaugh, B.S., 

S.R. Eskelsen, Ph.D., D. Howard, B.S., M. Malven, M.S., J.K. Soteres, Ph.D. 

Contributors and/or Principal Investigators:

E. Bell, Ph.D., S.L. Bollman, Ph.D., P.L. Bommireddy, Ph.D., L. Burzio, Ph.D., B. Chen, 

Ph.D., K.S. Crowley, Ph.D., R. Frazier, M.S., A. Galadima, Ph.D., C.W. Garnaat, M.S., 

Xin Gu, Ph.D., J.M. Harrison, M.Sc., M.A., J.L. Hart, M.S., R.A. Hernan, Ph.D., 

R. Heeren, B.S., T. Kaempfe, B.S., H.T. Kang, M.S., M. Koch, Ph.D., K.R. Lawry, M.S., 

T.C. Lee, Ph.D., S.L. Lemke, Ph.D., K.J. Mathis, K.E. Niemeyer, M.S., L.F. Ralston, 

Ph.D., A. Silvanovich, Ph.D., M.B. Spaur, B.S., Q. Tian, M.D., Ph.D., R. Wang, M.S. 
Monsanto Company 12-CT-244U 2 of 620 

RELEASE OF INFORMATION

Monsanto is submitting the information in this petition for review by the USDA as part of 

the regulatory process. By submitting this information, Monsanto does not authorize its 

release to any third party. In the event the USDA receives a Freedom of Information Act 

request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of this 

information, Monsanto expects that, in advance of the release of the document(s), USDA 

will provide Monsanto with a copy of the material proposed to be released and the 

opportunity to object to the release of any information based on appropriate legal 

grounds, e.g., responsiveness, confidentiality, and/or competitive concerns. Monsanto 

understands that a copy of this information may be made available to the public in a 

reading room and upon individual request as part of a public comment period. Except in 

accordance with the foregoing, Monsanto does not authorize the release, publication or 

other distribution of this information (including website posting) without Monsanto's 

prior notice and consent. 

©2012 Monsanto Company. All Rights Reserved. 

This document is protected under copyright law. This document is for use only by the 

regulatory authority to which it has been submitted by Monsanto Company and only in 

support of actions requested by Monsanto Company. Any other use of this material, 

without prior written consent of Monsanto, is strictly prohibited. By submitting this 

document, Monsanto does not grant any party or entity any right to use or license the 

information or intellectual property described in this document. 

 
CERTIFICATION 

The undersigned certifies that, to the best knowledge and belief of the undersigned, this 

petition includes all information and views on which to base a determination, and that it 

includes all relevant data and information known to the petitioner that are unfavorable to 

the petition. 

Marianne Malven, M.S. 

Regulatory Affairs Manager 

Address: 

Monsanto Company 

800 North Lindbergh Blvd., C3SD 

St. Louis, MO 63167 

Tel: (314) 694-2225 

Fax: (314) 694-8414 

Monsanto Company 12-CT-244U 30f620 
Monsanto Company 12-CT-244U 4 of 620 

EXECUTIVE SUMMARY 

The Animal and Plant Health Inspection Service (APHIS) of the United States 

Department of Agriculture (USDA) has responsibility under the Plant Protection Act 

(Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772) to prevent the 

introduction and dissemination of plant pests into the U.S. APHIS regulation 

7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data 

to determine that a particular regulated article does not present a plant pest risk and no 

longer should be regulated. If APHIS determines that the regulated article does not 

present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction 

of the article. 

Monsanto Company is submitting this request to APHIS for a determination of 

nonregulated status for the new biotechnology-derived cotton product, MON 88701, any 

progeny derived from crosses between MON 88701 and conventional cotton, and any 

progeny derived from crosses of MON 88701 with biotechnology-derived cotton that 

have previously been granted nonregulated status under 7 CFR Part 340. 

Product Description

Monsanto Company has developed dicamba and glufosinate-tolerant cotton, 

MON 88701, which will allow in-crop applications of dicamba herbicide for the control 

of broadleaf weeds from preemergence to seven days preharvest and glufosinate 

herbicide for broad spectrum weed control from emergence through early bloom growth 

stage. MON 88701 provides a wider dicamba window of application beyond the current 

preplant cotton uses and glufosinate application rates and timings that are equivalent to 

current commercial glufosinate-tolerant cotton. The combination of these two unique 

herbicide modes-of-action provides an effective weed management system for cotton 

production. Dicamba provides effective control of over 95 annual and biennial weed 

species, and suppression of over 100 perennial broadleaf and woody plant species. 

Glufosinate, a broad-spectrum contact herbicide, provides nonselective control of 

approximately 120 broadleaf and grass weeds. Additionally, dicamba and glufosinate 

provide control of herbicide-resistant weeds, including glyphosate-resistant biotypes of 

Palmer amaranth (Amaranthus palmeri), marestail (Conyza canadensis), common 

ragweed (Ambrosia artemisiifolia), giant ragweed (Ambrosia trifida) and waterhemp 

(Amaranthus tuberculatus). 

MON 88701 contains a demethylase gene from Stenotrophomonas maltophilia that 

expresses a dicamba mono-oxygenase (DMO) protein to confer tolerance to dicamba 

herbicide. DMO protein rapidly demethylates dicamba to the herbicidally inactive 

metabolite 3,6-dichlorosalicylic acid (DCSA). DCSA has been previously identified as a 

metabolite of dicamba in cotton, soybean, livestock, and soil. Monsanto will request a 

registration from U.S. EPA for the expanded use of dicamba on MON 88701, an increase 

in the dicamba residue tolerance for cottonseed, the establishment of a tolerance for 

cotton gin by-products, and the inclusion of DCSA in the residue definitions for both 

cottonseed and gin by-products. No other revisions to the dicamba pesticide residue 

tolerances are necessary including animal products such as meat, eggs, and milk. 
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Furthermore, the use of dicamba on MON 88701 does not present any new environmental 

exposure scenarios not previously evaluated and deemed acceptable by U.S. EPA. 

MON 88701 also contains a bialaphos resistance (bar) gene from Streptomyces 

hygroscopicus that expresses the phosphinothricin N-acetyltransferase (PAT) protein to 

confer tolerance to glufosinate herbicide. PAT (bar)

1

 protein acetylates the free amino 

group of glufosinate to produce non-herbicidal N-acetyl glufosinate, a well known 

metabolite in glufosinate-tolerant plants. The use pattern and rate of glufosinate on 

MON 88701 will follow the existing glufosinate-tolerant cotton uses outlined on the 

glufosinate herbicide label. The glufosinate residues in MON 88701 treated with 

commercial glufosinate rates are below the established pesticide residue tolerances for 

both cottonseed and gin by-products. Therefore, Monsanto will not seek any changes in 

the glufosinate label or the established tolerances for its use on MON 88701 cotton. 

MON 88701 will be combined, through traditional breeding methods, with other 

deregulated herbicide-tolerant (e.g., glyphosate-tolerant) events. The in-crop use of 

dicamba and glufosinate herbicides, in addition to glyphosate herbicide, provides 

improved weed management options in cotton to control a broad spectrum of grass and 

broadleaf weed species and effective control of weeds resistant to several herbicide 

families. Successful integration of MON 88701 into glyphosate-tolerant cotton systems 

will provide: 1) an opportunity for an efficient, effective weed management system for 

hard-to-control and herbicide-resistant weeds; 2) a flexible system for two additional 

herbicide modes-of-action for in-crop application in current cotton production systems as 

recommended by weed science experts to manage future weed resistance development; 

3) an option to delay or prevent further resistance to glyphosate and other critically 

important cotton herbicides; in particular, herbicides in the acetolactate synthase inhibitor 

(ALS) and protoporphyrinogen oxidase inhibitor (PPO) class of chemistry; 4) crop safety 

to dicamba, glufosinate, and glyphosate; and 5) additional weed management tools to 

enhance weed management systems necessary to maintain yield and quality to meet the 

growing needs of the food, feed, and industrial markets. 

Data and Information Presented Confirms the Lack of Plant Pest Potential and the 

Food and Feed Safety of MON 88701 Compared to Conventional Cotton

The data and information presented in this petition demonstrate MON 88701 is 

agronomically, phenotypically, and compositionally comparable to commercially 

cultivated cotton, with the exception of its tolerances to both dicamba and glufosinate. 

Moreover, the data presented demonstrate MON 88701 is unlikely to pose an increased 

plant pest risk, including weediness, or adverse environmental impact, compared to 

commercially cultivated cotton. The food, feed, and environmental safety of 

MON 88701 was confirmed based on multiple, well-established lines of evidence: 

 

1

 PAT (bar) indicates the PAT protein encoded by the bar gene isolated from S. hygroscopicus. The pat

gene from S. viridochromogenes also encodes a PAT protein that confers glufosinate tolerance. 
Monsanto Company 12-CT-244U 6 of 620 

 Cotton is a familiar crop that does not possess any of the attributes commonly 

associated with weeds, and has a history of safe usage and consumption. 

 A detailed molecular characterization of the inserted DNA demonstrated a single, 

intact copy of the T-DNA insert in a single locus within the cotton genome. 

 Extensive evaluation of the proteins expressed in MON 88701, dicamba monooxygenase (MON 88701 DMO) and phosphinothricin acetyltransferase [PAT 

(bar)], confirmed they are unlikely to be toxins or allergens. In addition, PAT 

proteins are in several other commercially-available crops that have been 

reviewed and previously deregulated by USDA, including those in cotton, corn, 

soy, canola, sugarbeet, and rice. 

 A compositional assessment of cottonseed confirmed that MON 88701 is 

compositionally equivalent to commercially cultivated cotton. 

 An extensive evaluation of phenotypic, agronomic, and plant mapping 

characteristics, as well as environmental interactions of MON 88701, 

demonstrated no increased plant pest potential compared to commercially 

cultivated cotton. 

 An assessment of potential impact on non-target organisms (NTOs) indicated that, 

under anticipated agricultural conditions, MON 88701 is unlikely to have adverse 

effects on these organisms compared to commercially cultivated cotton. 

 Evaluation of MON 88701 using current agronomic management practices for 

cotton concluded that deregulation of MON 88701 is not likely to impact cotton 

agronomic practices or land use, with the exception of the expanded window of 

dicamba application. 

Cotton is a Familiar Crop Lacking Weedy Characteristics 

Cotton, as a commodity crop, has a longstanding history of cultivation; its by-products, 

including processed fractions, also have a history of safe use and consumption. Cotton is 

grown in 17 states across the southern U.S. and in over 80 countries world-wide. In 

2011, U.S. growers planted approximately 14.7 million acres of cotton. 

The commercial cotton species in the U.S. (Gossypium hirsutum and Gossypium 

barbadense L. Merr.) do not exhibit weedy characteristics as defined by USDA, and 

neither invade established ecosystems, nor outcross to weedy relatives. Cotton is not 

listed as a weed in major weed references, nor is it present on the lists of noxious weed 

species distributed by the federal government (7 CFR Part 360). Cotton does not possess 

any of the attributes commonly associated with weeds, such as long persistence of the 

seed in the soil, ability to disperse, invade, or become a dominant species in new or 

diverse landscapes, or the ability to compete well with native vegetation. It is recognized 

that in some agricultural systems, cotton can volunteer in a subsequent rotational crop. 

However, volunteers are easily controlled through tillage or the use of appropriate 
Monsanto Company 12-CT-244U 7 of 620 

herbicides with diverse modes-of-action (e.g., ALS inhibitor, chloroacetamide, EPSPS, 

PPO inhibitor, PSI disruption, PSII inhibitor, synthetic auxin, and tubulin inhibitor 

classes). Specificity studies using the aforementioned herbicides as potential substrates 

for MON 88701 DMO showed similar injury levels for MON 88701 compared to the 

conventional control, indicating that these herbicides do not serve as a substrate for 

MON 88701 DMO at commercial application rates. Additionally, the specificity of 

PAT (bar) has been established in the published scientific literature. Therefore, 

herbicides effective for control of volunteer conventional cotton can still be used to 

control MON 88701 volunteers. 

In the continental U.S., wild populations of Gossypium species and some feral 

populations of cultivated variants of G. hirsutum exist, but these species able to cross 

with cultivated cotton are not known to exist in cotton growing areas. Importantly, 

MON 88701 would not be expected to confer a selective advantage to, or enhance the 

pest potential of, progeny resulting from such a cross if it were to occur, and could easily 

be controlled through current agronomic practices used to control conventional cotton. 

Thus, with environmental and biological limitations and varying chemical and agronomic 

practices available in the areas with wild and/or feral populations, there is limited 

probability for MON 88701 or any Gossypium species to outcross with wild or feral 

plants. 

Conventional Cotton Coker 130 is an Appropriate Comparator to MON 88701

Cotton variety Coker 130 is the near isogenic line to MON 88701 and was used as the 

conventional cotton comparator to support the safety assessment of MON 88701. 

MON 88701 and the near isogenic conventional cotton control Coker 130 have similar 

genetic backgrounds with the exception of the dmo and bar expression cassettes; thus, the 

effect of the dmo and bar expression cassettes and the expressed MON 88701 DMO and 

PAT (bar) proteins could be evaluated. 

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

in MON 88701

MON 88701 was developed through Agrobacterium-mediated transformation of 

hypocotyls from cotton variety Coker 130 utilizing vector PV-GHHT6997. 

PV-GHHT6997 contains one T-DNA that is delineated by Left and Right Border regions. 

The T-DNA contains the dmo and bar expression cassettes. The dmo expression cassette 

is regulated by the PC1SV promoter, the TEV 5′ leader sequence, and the E6 3′

untranslated region. The chloroplast transit peptide CTP2 directs transport of the 

MON 88701 DMO protein to the chloroplast and is derived from CTP2 target sequence 

of the Arabidopsis thaliana shkG gene. The bar expression cassette is regulated by the 

e35S promoter, the Hsp70 leader, and the nos 3′ untranslated region. After 

transformation, self pollination and segregation were used to select those plants 

containing a single homozygous copy of the T-DNA, including both the dmo and bar

expression cassettes, resulting in the selection of MON 88701. 
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Molecular characterization determined that MON 88701 contains one copy of the T-DNA 

at a single integration locus and all genetic elements are present. These data also 

demonstrated that MON 88701 does not contain detectable backbone sequences from the 

plasmid vector. The complete DNA sequence of the insert and adjacent genomic DNA 

sequences in MON 88701 confirmed the integrity of the inserted dmo and bar expression 

cassettes and identified the 5′ and 3′ insert to flank DNA junctions. Molecular 

characterization analysis also demonstrated that the insert in MON 88701 has been 

maintained over five consecutive generations of breeding, thereby confirming the 

stability of the insert. Furthermore, results from segregation analyses show inheritance 

and stability of the insert were as expected across multiple generations, which 

corroborates the molecular insert stability analysis determination that the MON 88701 

T DNA resides at a single chromosomal locus within the cotton genome. 

Data Confirms MON 88701 DMO and PAT (bar) Protein Safety

A multistep approach was used to characterize and assess the safety of the MON 88701 

DMO and PAT (bar) proteins expressed in MON 88701 resulting from the genetic 

modification. The expression levels of the MON 88701 DMO and PAT (bar) proteins in 

selected tissues of MON 88701 were determined. An assessment of the allergenic 

potential of the MON 88701 DMO and PAT (bar) proteins supports the conclusion that 

neither protein poses a significant allergenic risk to humans or animals. In addition, the 

donor organisms for the MON 88701 DMO and PAT (bar) protein coding sequences, 

Stenotrophomonas maltophilia and Streptomyces hygroscopicus, respectively, are 

ubiquitous in the environment and are not commonly known for human or animal 

pathogenicity or allergenicity. Bioinformatics analysis determined that the MON 88701 

DMO and PAT (bar) proteins lack structural similarity to known allergens, gliadins, 

glutenins, or protein toxins. The MON 88701 DMO and PAT (bar) proteins are rapidly 

digested in simulated gastrointestinal fluids and neither protein demonstrates acute oral 

toxicity in mice at the levels tested. Hence, the consumption of the MON 88701 DMO 

and PAT (bar) proteins from MON 88701 or its progeny poses no meaningful risk to the 

environment or human and animal health. 

MON 88701 is Compositionally Equivalent to Conventional Commercial Cotton 

Detailed compositional analyses were conducted in accordance with OECD guidelines to 

assess whether levels of key nutrients and anti-nutrients in MON 88701 cottonseed were 

comparable to levels in the conventional control, Coker 130, and several commercial 

reference cotton varieties. These compositional comparisons were made by analyzing 

cottonseed harvested from eight U.S. field sites in which MON 88701 was treated with 

dicamba and glufosinate, with the conventional control, and a range of commercial 

reference varieties that were grown concurrently in the same field trial. Compositional 

comparisons of MON 88701 not treated with dicamba or glufosinate herbicides were also 

conducted to further support the assessment of MON 88701 traits. The commercial 

reference varieties used to establish a range of natural variability for key nutrients and 

anti-nutrients have a history of safe consumption. Nutrients assessed in this analysis 

included proximates (ash, carbohydrates, and calories by calculation, moisture, protein, 

and fat), fibers (ADF, crude fiber, NDF, and TDF), amino acids (18 components), fatty 
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acids (C8-C22), minerals (calcium, copper, iron, magnesium, manganese, phosphorus, 

potassium, sodium, and zinc) and vitamin E. The anti-nutrients assessed in this analysis 

included gossypol and the cyclopropenoid fatty acids dihydrosterculic, malvalic, and 

sterculic. 

Combined-site analyses were conducted to determine if there were any statisticallysignificant differences (5% level of significance) between MON 88701 and the 

conventional control cottonseed samples. Significant differences noted from the 

combined-site statistical comparison were assessed using considerations relevant to the 

safety and nutritional quality of MON 88701 when compared to the conventional control. 

Considerations used to assess the relevance of each combined-site statistically significant 

difference included: 1) the relative magnitude of the difference in the mean values of 

nutrient and anti-nutrient components between MON 88701 and the conventional control; 

2) whether the MON 88701 component mean value is within the range of natural 

variability of that component as represented by the 99% tolerance interval of the 

commercial reference varieties grown concurrently in the same trial; 3) evaluation of the 

reproducibility of the statistical (p < 0.05) combined-site component differences at 

individual sites; and 4) an assessment of the differences within the context of natural 

variability of commercial cotton composition published in the scientific literature and in 

the International Life Sciences Institute (ILSI) Crop Composition Database. 

Based on these criteria, the observed differences were not meaningful to food and feed 

safety or nutritional value, and led to the conclusion that MON 88701 is compositionally 

equivalent to commercially cultivated cotton that has a history of safe consumption. 

These results support the overall food and feed safety of MON 88701. 

MON 88701 Does Not Change Cotton Plant Pest Potential or Environmental 

Interactions

Plant pest potential of a biotechnology-derived crop is assessed from the basis of 

familiarity that the USDA recognizes as an important underlying concept in risk 

assessment. The concept of familiarity is based on the fact that the biotechnologyderived plant is developed from a conventional plant hybrid or variety whose biological 

properties and plant pest potential are well known. Familiarity considers the biology of 

the plant, the introduced trait(s), the receiving environment, and the interactions among 

these factors. This provides a basis for comparative risk assessment between a 

biotechnology-derived plant and the conventional control. Thus, the phenotypic, 

agronomic, plant mapping, and environmental interaction assessment of MON 88701 

included the parental conventional control as a comparator. This evaluation used a 

weight-of-evidence approach and considered statistical differences between MON 88701 

and the conventional control with respect to reproducibility, magnitude, and 

directionality. The observations were taken on plants not treated with dicamba or 

glufosinate, in order to evaluate the impact of the introduced traits in MON 88701. To 

further support the trait assessment, similar supplemental observations were also 

conducted on the agronomic system that includes MON 88701 treated with dicamba and 

glufosinate herbicides. Comparison to a range of commercial reference varieties 

established the range of natural variability for cotton, and provided a context from which 
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to further evaluate any statistical differences. Characteristics assessed included: seed 

dormancy and germination, pollen morphology, plant phenotypic observations, plant 

mapping, and environmental interaction evaluations conducted in the field. The 

phenotypic, agronomic, and environmental interaction assessment demonstrated that 

MON 88701 is comparable to conventional cotton. Thus, MON 88701 is unlikely to 

have increased weediness or plant pest potential compared to commercially cultivated 

cotton. 

Seed dormancy and germination characterization demonstrated that MON 88701 

cottonseed had germination characteristics similar to cottonseed of the conventional 

control. In particular, the lack of hard seed, a well-accepted characteristic of weediness 

affecting seed germination, supports a conclusion of no increased weediness of 

MON 88701 when compared to the conventional control. Additionally, there were no 

statistically significant (5% level of significance) differences observed between 

MON 88701 and the conventional control for pollen viability and diameter, and no visual 

differences in general pollen morphology were observed. Collectively, these results 

support the conclusion that MON 88701 is not likely to exhibit increased plant pest 

potential compared to commercially cultivated cotton. 

The field evaluation of phenotypic, agronomic, plant mapping, and environmental 

interaction characteristics of MON 88701 also support the conclusion that MON 88701 is 

not likely to have an increased plant pest potential compared to commercially cultivated 

cotton. The evaluations were conducted at 26 replicated field sites across the U.S. cotton 

producing region. These assessments included plant growth and development 

characteristics, including cotton plant mapping evaluations at harvest, as well as 

observations for plant responses to abiotic stressors and plant-disease and plant-arthropod 

interactions. The observed phenotypic characteristics were similar between MON 88701 

and the conventional control. 

In a combined-site analysis of plant growth and development characteristics, data showed 

no statistically significant differences (5% level of significance) between MON 88701 

and the conventional control for stand count at 14 and 30 days after planting (DAP), final 

stand count, number of nodes above white flower at one of three observations, seed 

cotton yield, immature seed per boll, weight per boll, micronaire, fiber elongation, fiber 

uniformity, and fiber length. The mean values for MON 88701 were statistically 

different from the conventional control for eight parameters in the combined-site 

analysis. MON 88701 had shorter plants at 30 DAP and harvest, an increased number of 

nodes above white flower at two observations, a lower seed index, increased seed per 

boll, increased mature seeds per boll, and increased fiber strength. However, the mean 

values of MON 88701 were within the range of values observed for the commercial 

reference varieties for each of the characteristics listed above. Therefore, none of these 

differences were considered biologically meaningful in terms of increased plant pest 

potential of MON 88701 compared to commercially cultivated cotton. 

Plant mapping is a process commonly used by cotton agronomists and breeders to 

quantify growth and development parameters of a cotton plant, including boll retention. 

Plant mapping parameters, which include delineation of boll position and spatial retention 
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of bolls, are used to measure crop productivity and are influenced by abiotic and biotic 

stressors. In the combined-site analysis of plant mapping parameters, no statistically 

significant differences were detected between MON 88701 and the conventional control 

for number of mainstem nodes, number of nodes to first fruiting branch, total number of 

bolls per plant, number of vegetative bolls per plant, percent retention of first-position 

bolls, and percent first-position bolls. One statistically significant difference was 

detected between MON 88701 and the conventional control in the combined-site 

analysis. The mean value for first-position bolls per plant was higher for MON 88701 

than the conventional control. However, the mean value of the number of first-position 

MON 88701 bolls was within the range of the commercial reference varieties. Thus, 

MON 88701 is similar to commercially cultivated cotton varieties and unlikely to have 

increased plant pest potential, increased weediness, or an adverse environmental impact 

compared to commercially cultivated cotton. 

In an individual site assessment of abiotic stress response and disease damage, no 

differences were observed between MON 88701 and the conventional control for any of 

the 296 comparisons for the assessed abiotic stressors or for any of the 299 comparisons 

for the assessed diseases among all observations at the 26 sites. In an assessment of 

arthropod-related damage, no differences were detected between MON 88701 and the 

conventional control for any of the 288 comparisons for the assessed arthropods. The 

lack of significant biological differences in plant responses to abiotic stress, disease 

damage, and arthropod-related damage for MON 88701 support the conclusion that the 

introduction of the dicamba and glufosinate tolerance traits are unlikely to result in 

increased plant pest potential or an altered environmental impact from MON 88701 

compared to commercially cultivated cotton. 

In an assessment of pest- and beneficial-arthropod abundance, no statistically significant 

differences (5% level of significance) were detected between MON 88701 and the 

conventional control for 173 out of 178 comparisons (including 89 arthropod-pest and 89 

beneficial-arthropod comparisons) among the multiple collections conducted during the 

season at five geographically diverse sites. For the five detected differences in arthropod 

abundance, two were arthropod pests (stink bugs and tarnished plant bugs) and three were 

beneficial arthropods (Nabis spp. and Orius spp.). The differences detected in pest- and 

beneficial-arthropod abundance were small in magnitude and were not consistent with 

other collections at the individual sites or across the sites. Consequently, it is concluded 

that the differences in pest- and beneficial-arthropod abundance are not indicative of a 

consistent plant response associated with MON 88701 and are not biologically 

meaningful in terms of increased plant pest potential or an altered environmental impact 

from MON 88701 compared to commercially cultivated cotton. 

Field evaluations of phenotypic, agronomic, and plant mapping characteristics of 

MON 88701 treated with dicamba and glufosinate herbicides were also conducted to 

further support the assessment of MON 88701 traits. Data were collected from field 

trials conducted at eleven sites within the U.S. cotton-producing region. These 

assessments included plant growth and development characteristics, as well as plant 

mapping evaluations at harvest. The phenotypic, agronomic, and plant mapping 

assessments demonstrated that herbicide-treated MON 88701 is not different than the 
Monsanto Company 12-CT-244U 12 of 620 

conventional control, which further supports that MON 88701, whether treated or not 

with dicamba and glufosinate, is unlikely to have an altered plant pest potential compared 

to commercially cultivated cotton. 

In summary, the phenotypic, agronomic, plant mapping and environmental interaction 

data were evaluated to characterize MON 88701, and to assess whether the introduction 

of the traits in MON 88701 alters the plant pest potential compared to conventional 

cotton. The evaluation, using a weight-of-evidence approach, considered the 

reproducibility, magnitude, and direction of detected differences between MON 88701 

and the conventional control, and comparison to the range of the commercial reference 

varieties. Results from the phenotypic, agronomic, plant mapping, and environmental 

interactions assessment indicated that MON 88701 does not possess weedy 

characteristics, increased susceptibility or tolerance to specific abiotic stress, diseases, or 

arthropods, or characteristics that would confer a plant pest risk or a significant 

environmental impact compared to commercially cultivated cotton.

MON 88701 Will Not Adversely Affect NTOs 

Evaluation of the impacts of a biotechnology-derived crop on non-target organisms 

(NTOs) is a component of the plant pest risk assessment. Since MON 88701 does not 

possess pesticidal activity, all organisms that interact with MON 88701 are considered to 

be NTOs. The environmental assessment demonstrated that the presence of the dicamba 

and glufosinate-tolerance traits in MON 88701 did not alter plant-arthropod interactions, 

including beneficial arthropods, or alter disease susceptibility compared to the 

conventional control. In addition, plant mapping data, which is utilized to determine crop 

productivity in relation to abiotic and biotic stresses affecting yield, demonstrated that 

both MON 88701 plots treated and not treated with dicamba and glufosinate herbicides 

each had only a single significant difference from the conventional control, an increased 

number of first-position bolls that was within the range of the commercial reference 

varieties. From these data it can be concluded that both MON 88701 plants treated and 

not treated with dicamba and glufosinate responded to stressors in a similar manner. 

The biochemical information and experimental data for evaluation of MON 88701 

included molecular characterization, MON 88701 DMO and PAT (bar) safety 

assessments, the history of environmental exposure to mono-oxygenases (the class of 

enzymes to which MON 88701 DMO belongs) and the PAT proteins in several 

commercial glufosinate-tolerant events, information from the environmental interaction 

assessment, demonstration of compositional equivalence to conventional cotton, and 

demonstration of agronomic and phenotypic equivalence to conventional cotton. Overall, 

these data support the conclusion that MON 88701 has no reasonable mechanism for 

harm to NTOs and does not pose any additional risk to NTOs compared to commercially 

cultivated cotton. 

The potential for outcrossing and gene introgression from MON 88701 to sexually 

compatible species in the U.S. is unlikely, since the only known wild Gossypium species 

related to cultivated cotton do not grow in areas where cotton is cultivated, cotton pollen 

movement by wind is limited due to it is large and sticky nature, and several studies have 
Monsanto Company 12-CT-244U 13 of 620 

demonstrated that cross-pollination, even in the presence of high pollinator activity is 

limited by distance. Furthermore, should cross-pollination occur, MON 88701 and its 

progeny are not expected to exhibit a significant environmental impact because, as 

described above, evaluations have shown that the presence of the dicamba and 

glufosinate-tolerance traits are not likely to enhance weediness or plant-pest potential. 

Therefore, the environmental consequence of pollen transfer from MON 88701 to other 

Gossypium species is considered negligible. 

Deregulation of MON 88701 is Not Likely to Impact Cotton Agronomic Practices or 

Land Use

Cotton fields are typically highly managed agricultural areas that are dedicated to crop 

production for many years. Cultivation of MON 88701 would not be expected to differ 

from typical cotton cultivation, with the sole exception of an expanded window of 

dicamba application, due to the presence of the dicamba-tolerance trait in MON 88701. 

As glufosinate is already utilized within the U.S. cotton-growing areas, no change in 

agronomic practices or land use would occur with the cultivation of MON 88701 and the 

presence of the glufosinate-tolerance trait. MON 88701 likely would be used in common 

rotations on land currently used for agricultural purposes. As demonstrated, MON 88701 

is similar to commercially cultivated cotton in its agronomic, phenotypic, ecological, and 

compositional characteristics, and has comparable levels of resistance to insects, diseases, 

and abiotic stresses as compared to commercial cotton. Therefore, the introduction of 

MON 88701 into the existing cotton system is not expected to have a significant impact 

on current cultivation and pest management practices for cotton. The adoption of 

MON 88701 into glyphosate-tolerant cotton systems will provide growers with two 

additional herbicide modes-of-action and the means to control broadleaf weeds, including 

hard-to-control and herbicide-resistant broadleaf weeds, and will help preserve 

conservation tillage practices by providing growers with an additional weed management 

tool. Based on these considerations, MON 88701 is not likely to impact agronomic 

practices or land use, with the exception of the expanded application window of dicamba. 

Conclusion

Based on the data and information presented in this petition, it is concluded that 

MON 88701 is not likely to be a plant pest. Therefore, Monsanto Company requests a 

determination from USDA-APHIS that MON 88701 and any progeny derived from 

crosses between MON 88701 and conventional Gossypium cotton species or deregulated 

biotechnology-derived cotton be granted nonregulated status under 7 CFR Part 340. 
Monsanto Company 12-CT-244U 14 of 620 

TABLE OF CONTENTS 

CERTIFICATION .............................................................................................................3

EXECUTIVE SUMMARY ...............................................................................................4

TABLE OF CONTENTS ................................................................................................14

LIST OF TABLES ...........................................................................................................19

LIST OF FIGURES .........................................................................................................27

ABBREVIATIONS AND DEFINITIONS .....................................................................29

I. RATIONALE FOR THE DEVELOPMENT OF MON 88701 ...............................33

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR § 340.6 ...............................................................................................................33

I.B. Rationale for the Development of Dicamba and Glufosinate-Tolerant 

Cotton – MON 88701 ....................................................................................................33

I.C. Submissions to Other Regulatory Agencies ..........................................................36

I.C.1. Submission to FDA .....................................................................................36

I.C.2. Submission to EPA .....................................................................................36

I.C.3. Submissions to Foreign Government Agencies ..........................................37

II. THE BIOLOGY OF COTTON ................................................................................38

II.A. Cotton as a Crop ..................................................................................................38

II.B. Characteristics of the Recipient Plant ..................................................................39

II.C. Cotton as a Test System in Product Safety Assessment ......................................39

III. DESCRIPTION OF THE GENETIC MODIFICATION ....................................40

III.A. PV-GHHT6997 ...................................................................................................40

III.B. Description of the Transformation System .........................................................40

III.C. The dmo Coding Sequence and the MON 88701 DMO Protein ........................44

III.D. The bar Coding Sequence and PAT (bar) Protein .............................................44

III.E. Regulatory Sequences .........................................................................................44

III.F. T-DNA Borders ...................................................................................................44

III.G. Genetic Elements Outside of the T-DNA Borders .............................................45

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION .....................50

IV.A. Insert and Copy Number of T-DNA in MON 88701 .........................................57

IV.A.1. T-DNA Probes 1 and 5 ............................................................................57

IV.A.2. T-DNA Probes 2 and 4 ............................................................................58

IV.A.3. T-DNA Probe 3 ........................................................................................58

IV.B. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GHHT6997 Backbone Sequences in MON 88701 .................................................63
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IV.B.1. Backbone Probes 6, 7, and 8 ....................................................................63

IV.C. Organization and Sequence of the Insert and Adjacent Genomic DNA in 

MON 88701 ...................................................................................................................65

IV.D. PCR and DNA Sequence Analyses to Examine the MON 88701 

Insertion Site ..................................................................................................................67

IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 88701 ..........................................................................................69

IV.E.1. T-DNA Probe 2 and 4 ..............................................................................69

IV.F. Inheritance of the Genetic Insert in MON 88701 ...............................................72

IV.G. Genetic Modification Characterization Conclusion ...........................................76

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE 

MON 88701 DMO AND PAT (bar) PROTEINS PRODUCED IN MON 88701 ........77

V.A. Identity and Function of the MON 88701 DMO and PAT (bar) Proteins 

from MON 88701 ..........................................................................................................77

V.A.1. Mode-of-Action of DMO and MON 88701 DMO ...................................77

V.A.2. Mode-of-Action of PAT Proteins..............................................................81

V.B. Characterization and Equivalence of MON 88701 DMO and PAT (bar) 

Proteins from MON 88701 ............................................................................................82

V.C. Expression Levels of MON 88701 DMO and PAT (bar) Proteins in 

MON 88701 ...................................................................................................................83

V.C.1. Expression Levels of MON 88701 DMO Protein .....................................84

V.C.2. Expression Levels of PAT (bar) Protein ...................................................86

V.D. Assessment of Potential Allergenicity of the MON 88701 DMO and 

PAT (bar) Proteins ........................................................................................................88

V.D.1. Assessment of Potential Allergenicity of the MON 88701 DMO 

Protein ....................................................................................................................88

V.D.2. Assessment of Potential Allergenicity of the PAT (bar) Protein ..............88

V.E. Safety Assessment Summary of MON 88701 DMO and PAT (bar) 

Proteins in MON 88701 ................................................................................................89

V.E.1. MON 88701 DMO Donor Organism, History of Safe Use, and 

Specificity ..............................................................................................................89

V.E.2. PAT (bar) Donor Organism, History of Safe Use, and Specificity ...........92

V.E.3. MON 88701 DMO and PAT (bar) Proteins in MON 88701 are Not 

Homologous to Known Allergens or Toxins .........................................................94

V.E.4. MON 88701 DMO and PAT (bar) Proteins in MON 88701 are 

Labile in in vitro Digestion Assays ........................................................................94

V.E.5. MON 88701 DMO and PAT (bar) Proteins in MON 88701 are Not 

Acutely Toxic .........................................................................................................95
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V.E.6. Human and Animal Exposure to the MON 88701 DMO and 

PAT (bar) Proteins .................................................................................................95

V.F. MON 88701 DMO and PAT (bar) Protein Characterization and Safety 

Conclusion .....................................................................................................................96

VI. COMPOSITIONAL ASSESSMENT OF MON 88701 .........................................98

VI.A. Compositional Equivalence of MON 88701 Cottonseed to Conventional 

Cotton ............................................................................................................................99

VI.A.1 Nutrient Levels in Cottonseed ................................................................100

VI.A.2. Anti-Nutrient Levels in Cottonseed .......................................................105

VI.B. Compositional Assessment of MON 88701 Conclusion ..................................134

VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT ...............................................................................136

VII.A. Characteristics Measured for Assessment ......................................................136

VII.B. Interpretation of Phenotypic and Environmental Interaction Data .................142

VII.B.1. Interpretation of Detected Differences Criteria ....................................142

VII.C. Comparative Assessments of the Phenotypic, Agronomic, Plant 

Mapping, and Environmental Interaction Characteristics of MON 88701 .................145

VII.C.1. Seed Dormancy and Germination Characteristics ................................145

VII.C.2. Field Phenotypic, Agronomic, Plant Mapping, and 

Environmental Interactions Characteristics .........................................................148

VII.C.3. Pollen Characteristics ...........................................................................164

VII.D. Conclusions for Phenotypic, Agronomic, Plant Mapping, and 

Environmental Interactions Evaluation .......................................................................166

VIII. U.S. AGRONOMIC PRACTICES.....................................................................167

VIII.A. Introduction ...................................................................................................167

VIII.B. Overview of U.S. Cotton Production .............................................................168

VIII.B.1. Cotton Production................................................................................168

VIII.B.2. Cotton Seed Production .......................................................................175

VIII.C. Production Management Considerations .......................................................176

VIII.C.1. Pre-Season ...........................................................................................176

VIII.C.2. Planting and Early Season ...................................................................177

VIII.C.3. Mid- to Late-Season ............................................................................178

VIII.C.4. Preharvest and Harvest ........................................................................179

VIII.D. Management of Insect and Other Pests .........................................................179

VIII.E. Management of Diseases ...............................................................................182

VIII.F. Weed Management .........................................................................................183
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VIII.F.1. Methods of Weed Control in Cotton....................................................187

VIII.F.2. Herbicide Resistant Weeds in Cotton ..................................................204

VIII.G. Introduction of Dicamba and Glufosinate-Tolerant Cotton - 

MON 88701 .................................................................................................................206

VIII.G.1. MON 88701 Product Concept .............................................................206

VIII.G.2. Dicamba and Glufosinate Usage in MON 88701 ...............................206

VIII.G.3. MON 88701 in Combination with Glyphosate-Tolerant Cotton 

Systems ................................................................................................................209

VIII.G.4. MON 88701 as a Weed Resistance Management Tool ......................211

VIII.G.5. Introduction of Dicamba and Glufosinate-Tolerant Cotton - 

MON 88701 - Conclusion ....................................................................................212

VIII.H. Crop Rotation Practices in Cotton .................................................................215

VIII.H.1. Cotton Volunteer Management ...........................................................228

VIII.I. Stewardship of MON 88701 ...........................................................................231

VIII.J. Impact of the Introduction of MON 88701 on Agricultural Practices ............232

IX. ENVIRONMENTAL ANALYSIS ........................................................................233

IX.A. Introduction ......................................................................................................233

IX.B. Plant Pest Assessment of MON 88701 Insert and Expressed Proteins ............233

IX.B.1. Characteristics of the Genetic Insert and Expressed Protein .................234

IX.B.2. Compositional Characteristics ...............................................................235

IX.B 3. Phenotypic, Agronomic, Plant Mapping, and Environmental 

Interaction Characteristics ...................................................................................235

IX.C. Weed Potential of MON 88701 ........................................................................238

IX.D. Potential for Pollen Mediated Gene Flow and Introgression ...........................239

IX.D.1. Hybridization with Cultivated Cotton ....................................................239

IX.D.2. Hybridization with Wild and Feral Gossypium species .........................241

IX.D.3. Transfer of Genetic Information to Species with which Cotton 

Cannot Interbreed (Horizontal Gene Flow) .........................................................242

IX.E. Potential Impact on Cotton Agronomic Practices ............................................242

IX.F. Conventional Breeding with Other Biotechnology-derived or 

Conventional Cotton ....................................................................................................243

IX.G. Summary of Plant Pest Assessments ................................................................244

X. ADVERSE CONSEQUENCES OF INTRODUCTION .......................................245

REFERENCES ...............................................................................................................246

APPENDICES ................................................................................................................262

Appendix A: Notifications .............................................................................................263
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Appendix B: Materials, Methods, and Results for Molecular Analyses of 

MON 88701 .....................................................................................................................266

Appendix C: Protein Reaction Products, Materials, Methods, and Results 

for Characterization of MON 88701 DMO and PAT (bar) Proteins Produced 

in MON 88701, and Substrate Specificity ....................................................................273

Appendix D: Materials and Methods Used for the Analysis of the Levels of 

MON 88701 DMO and PAT (bar) Proteins in MON 88701 .......................................332

Appendix E: Materials, Methods, and Individual Site Results for 

Compositional Analysis of MON 88701 Cottonseed ...................................................337

Appendix F: Materials, Methods, and Individual Site Results for Seed 

Dormancy and Germination Assessment of MON 88701 ..........................................526

Appendix G: Materials, Methods, Dicamba and Glufosinate Treated Results, 

and Individual Site Results from the Phenotypic, Agronomic, Plant Mapping, 

and Environmental Interaction Assessment of MON 88701 under Field 

Conditions .......................................................................................................................532

Appendix H: Materials and Methods for Pollen Morphology and Viability 

Assessment ......................................................................................................................595

Appendix I: Herbicide Resistance ...............................................................................599

 
Monsanto Company 12-CT-244U 19 of 620 

LIST OF TABLES 

Table III-1. Summary of Genetic Elements in PV-GHHT6997 ..................................46

Table IV-1. Summary Chart of the Expected DNA Segments Based on 

Hybridizing Probes and Restriction Enzymes Used in MON 88701 Analysis ...........54

Table IV-2. Summary of Genetic Elements in MON 88701 ........................................55

Table IV-3. Segregation of the T-DNA During the Development of 

MON 88701: 1:1 Segregation ..........................................................................................75

Table IV-4. Segregation of the T-DNA During the Development of 

MON 88701: 1:2:1 Segregation.......................................................................................75

Table V-1. Herbicides Applied to MON 88701 and Conventional Control ...............81

Table V-2. Summary of MON 88701 DMO Protein Levels in Tissues from 

MON 88701 Grown in 2010 U.S. Field Trials ...............................................................85

Table V-3. Summary of PAT (bar) Protein Levels in Tissues from 

MON 88701 Grown in 2010 U.S. Field Trials ...............................................................87

Table VI-1. Summary of Differences (p<0.05) for the Comparison of 

Cottonseed Component Levels for MON 88701 vs. Conventional Control ..............107

Table VI-2. Statistical Summary of CombinedSite Cottonseed Nutrients for MON 88701 vs. Conventional Control ......................123

Table VI-3. Statistical Summary of Combined-Site Cottonseed Antinutrients for MON 88701 vs. Conventional Control ..................................................131

Table VI-4. Literature and ILSI Ranges for Components in Cottonseed ...............132

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in United States Field Trials and Laboratory 

Studies .............................................................................................................................138

Table VII-2. Combined-Site Comparison of MON 88701 to Conventional 

Control for Germination Characteristics ....................................................................147

Table VII-3. Study 1 and Study 2 Data Location Summary .....................................149

Table VII-4. Field Phenotypic Evaluation Sites for MON 88701 during 2010........151

Table VII-5. Study 1 Combined-Site Comparison of MON 88701 to 

Conventional Control during 2010 for Phenotypic and Agronomic 

Characteristics................................................................................................................153

Table VII-6. Study 2 Combined-Site Comparison of MON 88701 to 

Conventional Control during 2010 for Phenotypic and Agronomic 

Characteristics................................................................................................................155

Table VII-7. Study 2 Combined-Site Comparison of MON 88701 to 

Conventional Control during 2010 for Plant Mapping Characteristics ...................157
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Table VII-8. Study 1 Summary of Qualitative Environmental Interactions 

Assessments Including MON 88701 Response to Abiotic Stress, Disease, and 

Arthropod Damage during 2010...................................................................................160

Table VII-9. Study 1 Combined-Site Comparison of MON 88701 to 

Conventional Control during 2010 for Assessment of Thrips Damage ....................160

Table VII-10. Study 1 Combined-Site Comparison of MON 88701 to 

Conventional Control during 2010 for Quantitative Assessment of Heliothine 

Damage............................................................................................................................161

Table VII-11. Study 2 Summary of Qualitative Environmental Interactions 

Assessments Including MON 88701 Response to Abiotic Stress and Disease 

and Arthropod-related Damage during 2010 ..............................................................163

Table VII-12. MON 88701 Compared to the Conventional Control during 

2010 for Pollen Characteristics .....................................................................................166

Table VIII-1. Cotton Production in the U.S., 2000-20101 ..........................................173

Table VIII-2. U.S. Cotton Production by Region and State in 20101 .......................174

Table VIII-3. Insect Losses in Cotton in U.S. in 20101 ..............................................181

Table VIII-4. Common and Scientific Names of Weeds Referred to in this 

Petition ............................................................................................................................184

Table VIII-5. Common Weeds in Cotton Production in the Southeast Region 

of the U.S.1,2 ....................................................................................................................185

Table VIII-6. Common Weeds in Cotton Production in the Midsouth Region 

of the U.S.1,2 ....................................................................................................................186

Table VIII-7. Common Weeds in Cotton Production in the Southwest 

Region of the U.S.1,2 .......................................................................................................186

Table VIII-8. Common Weeds in Cotton Production in the West Region of 

the U.S.1,2 .........................................................................................................................187

Table VIII-9. Herbicide Applications Registered for Use in Cotton in 20101 .........192

Table VIII-10. Grass Weed Species Control Ratings to Preplant Burndown 

Herbicides in Cotton ......................................................................................................194

Table VIII-11. Grass Weed Species Control Ratings to Preplant, 

Preemergence and Postemergence Herbicides in Cotton ...........................................195

Table VIII-12. Broadleaf Weed Species Control Ratings to Preplant 

Burndown Herbicides in Cotton ...................................................................................198

Table VIII-13. Broadleaf Weed Species Control Ratings to Preplant, 

Preemergence and Postemergence Herbicides in Cotton - Part I .............................199

Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, 

Preemergence and Postemergence Herbicides in Cotton – Part II ...........................201

Table VIII-15. Common Weeds in Cotton and Weed Resistance to Herbicide 

Modes of Action in the U.S.1

 .........................................................................................205
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Table VIII-16. Anticipated Weed Management Recommendations for 

MON 88701 Combined with Glyphosate-Tolerant Cotton Systems for MO, 

AR, TN, AL, FL, GA, NC, SC, VA, LA, MS and eastern TX1

 ...................................208

Table VIII-17. Anticipated Weed Management Recommendations for 

MON 88701 Combined with Glyphosate-Tolerant Cotton Systems for 

western TX, NM, KS, OK, CA, and AZ 1

 .....................................................................209

Table VIII-18. Responses of Common Broadleaf Weeds to Dicamba and 

Glufosinate Compared to Labeled Postemergence Herbicides in Cotton 

Production – Part I ........................................................................................................213

Table VIII-19. Responses of Common Broadleaf Weeds to Dicamba and 

Glufosinate Compared to Labeled Postemergence Herbicides in Cotton 

Production – Part II .......................................................................................................214

Table VIII-20. Rotational Practices in the U.S. Following Cotton Production .......217

Table VIII-21. Rotational Practices Following Cotton Production in the 

Southeast Region ............................................................................................................218

Table VIII-22. Rotational Practices Following Cotton Production in the 

Midsouth Region ............................................................................................................221

Table VIII-23. Rotational Practices Following Cotton Production in the 

Southwest Region ...........................................................................................................223

Table VIII-24. Rotational Practices Following Cotton Production in the 

West Region ....................................................................................................................226

Table VIII-25. Herbicides and Application Timing for Control of Volunteer 

Cotton in Labeled Rotational Crops1

 ...........................................................................230

Table IX-1. Summary of Published Literature on Cotton Cross Pollination ..........241

Table A-1. USDA Notifications and Permits Approved for MON 88701 and 

Status of Trials Conducted under These Notifications ...............................................264

Table B-1. Hybridization Conditions of Utilized Probes ...........................................269

Table C-1. Summary of MON 88701 DMO Protein Identity and Equivalence ......280

Table C-2. Summary of the Tryptic Masses1 Identified for the MON 88701 

DMO Protein Using MALDI-TOF MS ........................................................................284

Table C-3. Comparison of Immunoreactive Signals Between MON 88701 

DMO and E. coli-produced MON 88701 DMO Proteins ...........................................289

Table C-4. Molecular Weight Comparison Between the MON 88701 DMO 

and E. coli-produced MON 88701 DMO Proteins Based on SDS-PAGE .................291

Table C-5. MON 88701 DMO Functional Activity Assay .........................................297

Table C-6. Herbicides Tested in Exogenous Specificity Herbicide Tolerance 

Greenhouse Trials ..........................................................................................................297

Table C-7. Herbicide Tolerance Trials Injury Ratings .............................................299

Table C-8. Compounds Used in Specificity In Vitro Experiments ...........................300
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Table C-9. Summary of MON 88701-produced PAT (bar) Protein Identity 

and Equivalence .............................................................................................................309

Table C-10. N-Terminal Sequence of the MON 88701-produced PAT (bar) 

Protein .............................................................................................................................311

Table C-11. Summary of the Tryptic Masses1 Identified for the MON 88701-

produced PAT (bar) Protein Using MALDI-TOF MS ...............................................314

Table C-12. Comparison of Immunoreactive Signals Between MON 88701- 

and E. coli-produced PAT (bar) Proteins ....................................................................319

Table C-13. Molecular Weight Comparison Between the MON 88701- and 

E. coli-produced PAT (bar) Proteins Based on SDS-PAGE .......................................321

Table C-14. PAT (bar) Functional Activity ................................................................327

Table D-1. MON 88701 DMO Protein Extraction Methods for Tissue 

Samples ...........................................................................................................................333

Table D-2. PAT (bar) Protein Extraction Methods for Tissue Samples ..................333

Table E-1. Commercial Reference Varieties ..............................................................337

Table E-2. Re-expression Formulas for Statistical Analysis of Composition 

Data .................................................................................................................................346

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................348

Table E-4. Statistical Summary of Site ARTI Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................356

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................357

Table E-6. Statistical Summary of Site GACH Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................365

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................366

Table E-8. Statistical Summary of Site KSLA Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................374

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................375

Table E-10. Statistical Summary of Site LACH Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................383

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients 

for MON 88701 (Treated) vs. Conventional Control ..................................................384

Table E-12. Statistical Summary of Site NCBD Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................392

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................393
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Table E-14. Statistical Summary of Site NMLC Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................401

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................402

Table E-16. Statistical Summary of Site SCEK Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................410

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for 

MON 88701 (Treated) vs. Conventional Control ........................................................411

Table E-18. Statistical Summary of Site TXPL Cottonseed Antinutrients for MON 88701 (Treated) vs. Conventional Control .................................419

Table E-19. Summary of Differences (p<0.05) for the Comparison of 

Cottonseed Component Levels for MON 88701 (Not Treated) vs. 

Conventional Control ....................................................................................................426

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................440

Table E-21. Statistical Summary of Combined-Site Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................448

Table E-22 Literature and ILSI Ranges for Components in Cottonseed ................449

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................451

Table E-24. Statistical Summary of Site ARTI Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................459

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................460

Table E-26. Statistical Summary of Site GACH Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................468

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................469

Table E-28. Statistical Summary of Site KSLA Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................477

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................478

Table E-30. Statistical Summary of Site LACH Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................486

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................487

Table E-32. Statistical Summary of Site NCBD Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................495

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................496
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Table E-34. Statistical Summary of Site NMLC Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................504

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................505

Table E-36. Statistical Summary of Site SCEK Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................513

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for 

MON 88701 (Not Treated) vs. Conventional Control.................................................514

Table E-38. Statistical Summary of Site TXPL Cottonseed Antinutrients for MON 88701 (Not Treated) vs. Conventional Control ..........................522

Table F-1. Starting Seed of MON 88701, Conventional Control and 

Commercial Cotton Reference Varieties Used in Dormancy Assessment ................526

Table F-2. Comparison of MON 88701 to the Conventional Control for 

Dormancy and Germination Characteristics of Cottonseed Produced at Each 

of Three Sites ..................................................................................................................529

Table G-1. Starting Seed for Study 1 ..........................................................................534

Table G-2. Starting Seed for Study 2 ..........................................................................536

Table G-3. Study 1 Field and Planting Information ..................................................538

Table G-4. Study 2 Field and Planting Information ..................................................539

Table G-5. Study 1 Data Missing or Excluded from Analysis ..................................554

Table G-6. Study 2 Data Missing or Excluded from Analysis ..................................555

Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and 

Development Characteristics - of MON 88701 Not Treated with Dicamba or 

Glufosinate Herbicides Compared to the Conventional Control ..............................556

Table G-8. Study 1 - Individual Site Phenotypic Comparison – Seed 

Characteristics - of MON 88701 Not Treated with Dicamba or Glufosinate 

Herbicides Compared to the Conventional Control. ..................................................559

Table G-9. Study 1 - Individual Site Phenotypic Comparison – Boll and 

Fiber Characteristics - of MON 88701 Not Treated with Dicamba or 

Glufosinate Herbicides Compared to the Conventional Control ..............................560

Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and 

Development Characteristics - of MON 88701 Not Treated with Dicamba or 

Glufosinate Herbicides Compared to the Conventional Control ..............................561

Table G-11. Study 2 - Individual Site Phenotypic Comparison – Seed 

Characteristics - of MON 88701 Not Treated with Dicamba or Glufosinate 

Herbicides Compared to the Conventional Control ...................................................564

Table G-12. Study 2 - Individual Site Phenotypic Comparison – Boll and 

Fiber Characteristics - of MON 88701 Not Treated with Dicamba or 

Glufosinate Herbicides Compared to the Conventional Control ..............................565
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Table G-13. Study 2 - Combined-Site Phenotypic Comparison - Growth and 

Development Characteristics - of MON 88701 Treated with Dicamba and 

Glufosinate Herbicides Compared to the Conventional Control ..............................567

Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and 

Development Characteristics - of MON 88701 Treated with Dicamba and 

Glufosinate Herbicides Compared to the Conventional Control ..............................568

Table G-15. Study 2 - Individual Site Phenotypic Comparison – Seed 

Characteristics - of MON 88701 Treated with Dicamba and Glufosinate 

Herbicides Compared to the Conventional Control ...................................................571

Table G-16. Study 2 - Individual Site Phenotypic Comparison – Boll and 

Fiber Characteristics - of MON 88701 Treated with Dicamba and 

Glufosinate Herbicides Compared to the Conventional Control ..............................572

Table G-17. Study 2 - Individual Site Phenotypic Comparison – Plant 

Mapping - of MON 88701 Not Treated with Dicamba or Glufosinate 

Herbicides Compared to the Conventional Control ...................................................574

Table G-18. Study 2 – Combined-Site Phenotypic Comparison – Plant 

Mapping - of MON 88701 Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control ......................................................................576

Table G-19. Study 2 - Individual Site Phenotypic Comparison – Plant 

Mapping - of MON 88701 Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control ......................................................................577

Table G-20 Study 1 – Qualitative Assessment of Plant Response to Abiotic 

Stressors - MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control ......................................................................579

Table G-21. Study 1 – Qualitative Assessment of Disease Damage - 

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared 

to the Conventional Control ..........................................................................................580

Table G-22. Study 1 – Qualitative Assessment of Arthropod-related Damage 

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control ......................................................................581

Table G-23. Study 1 - Individual Site Analysis: Quantitative Assessment of 

Thrips Damage - MON 88701 Not Treated with Dicamba or Glufosinate 

Herbicides Compared to the Conventional Control ...................................................582

Table G-24. Study 1 - Individual Site Analysis: Quantitative Assessment of 

Heliothine Damage - MON 88701 Not Treated with Dicamba or Glufosinate 

Herbicides Compared to the Conventional Control ...................................................583

Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not 

Treated with Dicamba or Glufosinate Herbicides Compared to the 

Conventional Control ....................................................................................................584

Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 

Not Treated with Dicamba or Glufosinate Herbicides Compared to the 

Conventional Control ....................................................................................................588
Monsanto Company 12-CT-244U 26 of 620 

Table G-27 Study 2 – Qualitative Assessment of Plant Response to Abiotic 

Stressors - MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control ......................................................................591

Table G-28. Study 2 – Qualitative Assessment of Disease Damage of 

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared 

to the Conventional Control ..........................................................................................592

Table G-29. Study 2 – Qualitative Assessment of Arthropod-related Damage 

-MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control ......................................................................593

Table H-1. Starting Seed for Pollen Morphology and Viability Assessment ...........597

Table I-1. Management Recommendations for Control of Dicamba-, 

Glufosinate- and Other Selected Synthetic Auxin-Resistant Weeds .........................609
Monsanto Company 12-CT-244U 27 of 620 

LIST OF FIGURES 

Figure III-1. Circular Map of PV-GHHT6997 Showing Probes 1-8 ..........................42

Figure III-2. Schematic of the Development of MON 88701 ......................................43

Figure III-3. Deduced Amino Acid Sequence of the MON 88701 DMO 

Protein ...............................................................................................................................49

Figure III-4. Deduced Amino Acid Sequence of the PAT (bar) Protein ....................49

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in 

MON 88701 .......................................................................................................................53

Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number 

of T-DNA in MON 88701: Probes 1 and 5 ....................................................................60

Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number 

of T-DNA in MON 88701: Probes 2 and 4 ....................................................................61

Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number 

of T-DNA in MON 88701: Probe 3 ................................................................................62

Figure IV-5. Southern Blot Analysis to Determine the Presence or Absence 

of PV-GHHT6997 Backbone Sequences in MON 88701: Probes 6, 7, and 8 ............64

Figure IV-6. Overlapping PCR Analysis across the Insert in MON 88701 ...............66

Figure IV-7. PCR Amplification of the MON 88701 Insertion Site in 

Conventional Control ......................................................................................................68

Figure IV-8. Breeding History of MON 88701 .............................................................70

Figure IV-9. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 88701: Probes 2 and 4 .................................................................71

Figure IV-10. Breeding Path for Generating Segregation Data for 

MON 88701 .......................................................................................................................74

Figure V-1. Three Components of the DMO Oxygenase System ...............................78

Figure V-2. Dicamba and Potential Endogenous Substrates Tested through 

In Vitro Experiments with DMO ....................................................................................80

Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data 

Interpretation Methods .................................................................................................143

Figure VIII-1. Planted Upland Cotton Acres by County in the U.S. in 2010 ...........171

Figure VIII-2. Planted Pima Cotton Acres by County in the U.S. in 2010 ...............172

Figure C-1. Forms of DMO Protein and Their Relation to the Wild-Type 

DMO Protein ..................................................................................................................276

Figure C-2. MALDI-TOF MS Coverage Map of the MON 88701 DMO 

Protein .............................................................................................................................285

Figure C-3. Western Blot Analysis of MON 88701 DMO and E. coliproduced MON 88701 DMO Proteins..........................................................................288
Monsanto Company 12-CT-244U 28 of 620 

Figure C-4. Molecular Weight and Purity Analysis of the MON 88701 DMO 

Protein .............................................................................................................................292

Figure C-5. Glycosylation Analysis of the MON 88701 DMO Protein ....................295

Figure C-6. UPLC Separation of Dicamba (DCB) and DCSA in Five 

Substrate Analysis ..........................................................................................................303

Figure C-7. E. coli-produced DMO Conversion of Endogenous Substrates ...........304

Figure C-8. UPLC Separation of Dicamba (DCB) and DCSA in Bridging 

Analysis ...........................................................................................................................305

Figure C-9. E. coli-produced MON 88701 DMO Conversion of o-Anisic Acid ......306

Figure C-10. MALDI-TOF MS Coverage Map of the MON 88701-produced 

PAT (bar) Protein ...........................................................................................................315

Figure C-11. Western Blot Analysis of the MON 88701- and E. coli -

produced PAT (bar) Proteins ........................................................................................318

Figure C-12. Molecular Weight and Purity Analysis of the MON 88701-

produced PAT (bar) Protein .........................................................................................322

Figure C-13. Glycosylation Analysis of the MON 88701-produced PAT (bar) 

Protein .............................................................................................................................325

Figure I-1. Weed Resistance to Various Herbicide Families1 ...................................604
Monsanto Company 12-CT-244U 29 of 620 

ABBREVIATIONS AND DEFINITIONS2

Symbol or Abbrev. Definition 

~ Approximately 

α-Cyano 

2,4-D 

2,4-DB 

α-Cyano-4-hydroxycinnamic acid 

2,4-Dichlorophenoxyacetic acid 

2,4-DB = 4-(2,4-dichlorophenoxy)butyric acid 

AA Amino Acid 

AAbA α-aminobutyric acid 

ADF Acid Detergent Fiber 

a.e. 

a.i. 

ALS 

acid equivalent 

active ingredient 

acetolactate synthase inhibitor 

APHIS Animal and Plant Health Inspection Service of the United States 

Department of Agriculture 

bar 

BIO 

Bialaphos Resistance Gene from Streptomyces hygroscopicus 

Biotechnology Industry Organization 

BLOCKS A database of amino acid motifs found in protein families 

BLOSUM Blocks Substitution Matrix, used to score similarities between 

pairs of distantly related protein or nucleotide sequences 

BSA Bovine Serum Albumin 

CFR Code of Federal Regulations 

CHT Ceramic hydroxyapatite 

CoA Coenzyme A 

COA Certificate of Analysis 

CTAB Hexadecyltrimethylammonium bromide 

DAP Days After Planting 

Da Dalton 

dCTP Deoxycytidine triphosphate 

DEAE- DiethylaminoethylDHB 2,5-dihydroxybenzoic acid 

DCSA 3,6-dichlorosalicylic acid 

DDI 

DGA 

Daily Dietary Intake 

Diglycolamine 

dicamba 3,6-dichloro-2-methoxybenzoic acid 

dmo Mono-oxygenase gene from Stenotrophomonas maltophilia 

DMO Dicamba mono-oxygenase 

DNA 

DSMA 

Deoxyribonucleic acid 

Disodium methanearsonate 

DTNB 5,5’-dithio-bis (2-nitrobenzoic acid) 

DTT Dithiothreitol 

dw Dry weight 

DWCF Dry weight conversion factor 

 

2

 Alred, G.J., C.T. Brusaw, and W.E. Oliu. 2003. Handbook of Technical Writing, 7th edn., pp. 2-7. 

Bedford/St. Martin's, Boston, MA. 
Monsanto Company 12-CT-244U 30 of 620 

ECL Enhanced Chemiluminescence 

E. coli Escherichia coli 

E.coli-produced 

MON 88701 DMO 

DMO protein produced from E. coli with the same sequence as 

MON 88701 DMO 

ELISA Enzyme-linked Immunosorbent Assay 

EPA Environmental Protection Agency 

E-Score 

ETS 

Expectation score 

Excellence Through StewardshipSM 

FA Fatty Acid 

FARRP Food Allergy Research and Resource Program 

FASTA Algorithm used to find local high scoring alignments between a 

pair of protein or nucleotide sequences 

FDA Food and Drug Administration (U.S.) 

FFDCA Federal Food, Drug and Cosmetic Act (U.S.) 

FT Flow through 

fw Fresh weight 

glufosinate butanoic acid, 2-amino-4-(hydroxymethylphosphinyl) 

GLP Good Laboratory Practice 

ha hectare 

HPLC High Performance Liquid Chromatography 

HRP Horseradish Peroxidase 

HU Hemagglutinating Unit 

ILSI 

ISO 

International Life Sciences Institute 

International Organization for Standardization 

kb Kilobase 

kDa Kilodalton 

kg Kilogram 

LB Laemmli buffer 

LOD Limit of Detection 

LOQ Limit of Quantitation 

MALDI-TOF-MS Matrix Assisted Laser Desorption Ionization - Time of Flight 

Mass Spectrometry 

μg 

μl 

Microgram 

Microliter 

mg 

mic 

Milligram 

micronaire 

MOE 

MON 87708 

Margin of Exposure 

Dicamba-tolerant soybean developed by Monsanto Company 

MON 88701 DMO 

MRL 

MSMA 

DMO protein produced in MON 88701 

Maximum Residue Levels 

Monosodium methanearsonate 

MW Molecular Weight 

MWCO Molecular Weight Cutoff 

N-acetyl glufosinate 2-acetamido-4-methylphosphinico-butanoic acid 

NADH Nicotinamide adenine dinucleotide 
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NCBI National Center for Biotechnology Information at the National 

Institutes of Health, Bethesda, MD, USA 

NDF Neutral Detergent Fiber 

NFDM Non-fat Dried Milk 

NOAEL No Observable Adverse Effect Level 

OECD Organisation for Economic Co-operation and Development 

ORF Open Reading Frame 

OSL Overseason Leaf 

p Probability from PRESS 

PAT Phosphinothricin N-acetyltransferase 

PAT (bar) PAT protein produced by the bar gene 

PBS Phosphate Buffered Saline 

PBST Phosphate Buffered Saline containing Tween-20 

PCR Polymerase Chain Reaction 

PI 

PPA 

Prediction Interval 

Plant Protection Act (7 U.S.C. § 7701-7772) 

ppm 

PPO 

parts per million 

protoporphyrinogen oxidase inhibitor 

PPT Phosphinothricin 

PRESS Predicted Residual Sum of Squares 

PRT_2011 GenBank protein database, 181.0 (Released December 18, 2010) 

PTH Phenylthiohydantoin 

PVDF Polyvinylidene difluoride 

PVP Polyvinyl pyrrolidone

RBD Refined, Bleached, and Deodorized 

RED Reregistration Eligibility Decision 

RT 

SCST 

Room temperature 

Society of Commercial Seed Technologists 

SDS Sodium Dodecyl Sulfate 

SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis 

SE Standard Error 

SGF Simulated Gastric Fluid 

S. hygroscopicus Streptomyces hygroscopicus 

SIF Simulated Intestinal Fluid 

Sinapinic Acid 3,5-dimethoxy-4-hydroxycinnamic acid 

S. maltophilia Stenotrophomonas maltophilia 

SOP Standard Operating Procedure 

TBA Tris-borate buffer with L-ascorbic acid 

TBS Tris Buffered Saline 

TCEP Tris(2-carboxyethyl)phosphine 

T-DNA Transfer DNA 

TDF 

tex 

Total Dietary Fiber 

Grams of 1000 meters of fiber 

TFA Trifluoroacetic Acid 

TFE 2,2,2,-trifluoroethanol 

TIU Trypsin Inhibitor Unit 
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Tm Melting temperature 

TNB 5-thio-nitrobenzoate 

TOX_2011 Toxin protein sequence database (Release date February 18, 

2011) 

V volts 

v/v volume to volume ratio 

w/v weight to volume ratio 

 
Monsanto Company 12-CT-244U 33 of 620 

I. RATIONALE FOR THE DEVELOPMENT OF MON 88701 

I.A. Basis for the Request for a Determination of Nonregulated Status under 

7 CFR § 340.6 

The Animal and Plant Health Inspection Service (APHIS) of the United States 

Department of Agriculture (USDA) has responsibility, under the Plant Protection Act 

(Title IV Pub. L. 106-224, 114 Stat. 438, 7 U.S.C. § 7701-7772), to prevent the 

introduction and dissemination of plant pests into the U.S. APHIS regulation 

7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data 

to determine that a particular regulated article does not present a plant pest risk and no 

longer should be regulated. If APHIS determines that the regulated article does not 

present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction 

of the article. 

Monsanto Company is submitting this request to APHIS for a determination of 

nonregulated status for the new biotechnology-derived cotton product, MON 88701, any 

progeny derived from crosses between MON 88701 and conventional cotton, and any 

progeny derived from crosses of MON 88701 with biotechnology-derived cotton that 

have previously been granted nonregulated status under 7 CFR Part 340. 

I.B. Rationale for the Development of Dicamba and Glufosinate-Tolerant Cotton – 

MON 88701 

Biotechnology derived cotton and the introduction of glyphosate-tolerant cotton systems 

permit in-crop application of agricultural herbicides containing the active ingredient 

glyphosate for effective weed control. The value of glyphosate-tolerant cotton systems 

has been demonstrated by the significant growth in the number of glyphosate-tolerant 

acres planted since introduction of this technology in 1997. Today, more than 75% of all 

cotton acres grown in the U.S. are glyphosate-tolerant (USDA-NASS, 2010). The 

glyphosate-tolerant systems deliver effective broad spectrum weed control, provides 

flexibility of application timing, increased adoption of reduced tillage practices, and has 

resulted in increased grower income (Carpenter and Gianessi, 2001; Hurley et al., 2009). 

Additionally, the glyphosate-tolerant systems provide incremental environmental 

benefits, including reduced overall herbicide usage (Brookes and Barfoot, 2012; 

Carpenter and Gianessi, 2001). Furthermore, glyphosate, as concluded by the U.S. EPA 

(1993), has a favorable safety profile. Continued use of glyphosate-tolerant cotton 

systems will maintain effective and familiar weed control management practices that are 

fully compatible with all current tillage and land management practices, including 

conservation tillage practices. Growth of conservation tillage in the U.S. was greatly 

accelerated with the introduction of glyphosate-tolerant crops in large part because of the 

broad spectrum postemergence control offered by glyphosate (Price et al., 2011). The 

benefits associated with conservation tillage, include reduced soil erosion, reduced fuel 

and labor costs, and conservation of soil moisture (CTIC, 2011). 

As with all herbicides used in agriculture, there is potential for weeds to develop 

resistance to frequent and continual use of the same herbicide over an extended time 
Monsanto Company 12-CT-244U 34 of 620 

period (Powles, 2008). Plant populations can develop resistance to a herbicide due to the 

selection of individuals that carry altered genetic codes producing alleles that can render 

those individuals tolerant to the lethal effects of a herbicide. Weed populations with 

confirmed herbicide-resistance are listed on the International Survey of Resistant Weeds 

website (www.weedscience.org). Without effective weed management practices in 

agricultural systems, herbicide resistance in weeds can become a limiting factor in crop 

production. As with many agricultural use herbicides, glyphosate has documented cases 

of weed resistance. While there have been thirteen confirmed glyphosate-resistant weeds 

in the U.S. (Heap, 2012a), glyphosate still effectively controls more than 160 weed 

species (Roundup WeatherMax® herbicide label, EPA Reg. No.524-537) and remains an 

extremely valuable tool for U.S. cotton crop production. Studies have shown that 

resistance can be postponed, contained, and managed through good management 

practices. One of the management practices most often recommended by 

University/Cooperative Extension Service and industry is the use of multiple herbicide 

modes-of-action. Simultaneously using multiple herbicides with different modesof-action significantly reduces the probability of weeds developing resistance to any or 

all of the applied herbicides (Beckie and Reboud, 2009; Powles et al., 1996). Other weed 

management recommendations include the use of multiple herbicide modes-of-action in 

sequence and the inclusion of mechanical or cultural weed management practices, in 

addition to the use of a herbicide. 

Monsanto Company has developed dicamba and glufosinate-tolerant cotton, 

MON 88701, which will allow in-crop applications of dicamba (3,6-dichloro-2-

methoxybenzoic acid) herbicide for the control of broadleaf weeds from preemergence to 

seven days preharvest and glufosinate herbicide for broad spectrum weed control from 

emergence through early bloom growth stage. MON 88701 provides dicamba tolerance 

that allows for the in-crop application of dicamba beyond the current preplant uses in 

cotton and also provides glufosinate tolerance equivalent to current commercial 

glufosinate-tolerant cotton events. The combination of the two herbicides’ distinct 

modes-of-action provides an effective weed management system. Dicamba provides 

effective control of over 95 annual and biennial weed species, and suppression of over 

100 perennial broadleaf and woody plant species (BASF, 2008) (EPA Reg. No. 7969-

137) and glufosinate is a broad-spectrum contact herbicide that provides nonselective 

control of about 120 broadleaf and grass weeds (Bayer CropScience, 2011) (EPA Reg. 

No. 264-829). Additionally, dicamba and glufosinate each provide control of many 

herbicide-resistant weeds, including glyphosate-resistant biotypes of Palmer amaranth 

(Amaranthus palmeri), marestail (Conyza Canadensis), common ragweed (Ambrosia 

artemisiifolia), giant ragweed (Ambrosia trifida) and waterhemp [Amaranthus 

tuberculatus). Weeds that are hard-to-control using glyphosate (See Roundup 

WeatherMax® label (U.S. EPA Reg. No. 524-537) for a listing], generally require a 

higher rate and/or application at a smaller growth stage in order to consistently achieve 

commercially acceptable control. To date, only four species with known dicambaresistant biotypes (i.e., common hempnettle, Galeopsis tetrahit; kochia, Kochia scoparia; 

prickly lettuce, Lactuca serriola; and wild mustard, Sinapis arvensis) and one species 

 

®Roundup and WeatherMax are registered trademarks of Monsanto Technology, LLC. 
Monsanto Company 12-CT-244U 35 of 620 

with a known glufosinate-resistant biotype (i.e., Italian ryegress, Lolium multiflorum) 

have been identified in North America (Heap, 2012b; 2012c). Known resistant weed 

populations to dicamba and glufosinate are primarily found in the western U.S. and, thus, 

are not present in the major cotton geographies. See Appendix I for additional details. 

MON 88701 will be combined, through traditional breeding methods, with other 

approved herbicide-tolerant (e.g., glyphosate-tolerant) events. The opportunity for incrop use of dicamba and glufosinate herbicides, in addition to glyphosate herbicide, 

provides new weed management options in cotton to control a broad spectrum of grass 

and broadleaf weed species and effective control of weeds resistant to several herbicide 

families. Successful integration of MON 88701 into glyphosate-tolerant cotton systems 

will provide: 1) an opportunity for an efficient, effective weed management system for 

hard-to-control and herbicide-resistant weeds; 2) a flexible system for two additional incrop herbicide modes-of-action in current cotton production practices as recommended 

by weed science experts to manage future weed resistance development; 3) an option to 

delay or prevent further resistance to glyphosate and other critically important cotton 

herbicides, in particular herbicides in the ALS and PPO class of chemistry; 4) crop safety 

to dicamba, glufosinate and glyphosate; and 5) additional weed management tools to 

enhance weed management systems necessary to maintain yield and quality to meet the 

growing needs of fiber, food, and feed. 

MON 88701 contains a demethylase gene from Stenotrophomonas maltophilia that 

expresses a dicamba mono-oxygenase (DMO) protein to confer tolerance to dicamba 

herbicide and a bialaphos resistance (bar) gene from Streptomyces hygroscopicus that 

expresses the phosphinothricin N-acetyltransferase (PAT) protein to confer tolerance to 

glufosinate herbicide. DMO protein rapidly demethylates dicamba to the herbicidally 

inactive metabolite 3,6-dichlorosalicylic acid (DCSA), a well known metabolite of 

dicamba in conventional cotton, soybean, livestock, and soil (FAO-WHO, 2011a; 2011b; 

U.S. EPA, 2009). Monsanto will request a registration from U.S. EPA for the expanded 

use of dicamba on MON 88701 cotton, an increase in the dicamba residue tolerance for 

cottonseed, the establishment of a tolerance for cotton gin by-products, and the inclusion 

of DCSA in the residue definitions for cottonseed and gin by-products. No other 

revisions to the dicamba pesticide residue tolerances are necessary, including those for 

animal products such as meat, eggs, and milk. Furthermore, the use of dicamba on 

MON 88701 does not present any new environmental exposure scenarios not previously 

evaluated and deemed acceptable by EPA. 

PAT (bar) protein acetylates the free amino group of glufosinate to produce nonherbicidal N-acetyl glufosinate, a well known metabolite in glufosinate-tolerant plants 

(OECD, 2002a). The use pattern and rate of glufosinate on MON 88701 will follow the 

existing glufosinate-tolerant cotton uses outlined on the glufosinate herbicide labels and 

the glufosinate residues in MON 88701 treated with commercial glufosinate rates are 

below the established pesticide residue tolerances established by U.S. EPA for both 

cottonseed and gin by-products (40 CFR § 180.473). Therefore, Monsanto will not 

pursue any changes in the glufosinate labels or the established tolerances for its use on 

MON 88701 cotton. 
Monsanto Company 12-CT-244U 36 of 620 

I.C. Submissions to Other Regulatory Agencies 

Under the Coordinated Framework for Regulation of Biotechnology (CFR) (USDAAPHIS, 1986), the responsibility for regulatory oversight of biotechnology-derived crops 

falls primarily on three U.S. agencies: U.S. Food and Drug Administration (FDA), the 

United States Department of Agriculture (USDA), and in the case of herbicide-tolerant 

products, the Environmental Protection Agency (EPA). A request for deregulation of 

MON 88701 made to USDA constitutes only one component of the overall regulatory 

oversight and review of this product. As a practical matter, MON 88701 cannot be 

released and marketed until FDA and USDA have completed their reviews and 

assessments under their respective jurisdictions. Additionally, EPA must complete its 

review and assessments prior to approving the use and allowable residues of dicamba on 

MON 88701. 

I.C.1. Submission to FDA

MON 88701 falls within the scope of the 1992 FDA policy statement concerning 

regulation of products derived from new plant varieties, including those developed 

through biotechnology (U.S. FDA, 1992). In compliance with this policy, Monsanto has 

initiated a consultation with the FDA (BNF No. 135) on the food and feed safety and 

compositional assessment of MON 88701. Monsanto submitted a safety and nutritional 

assessment summary document to the FDA in April 2012. 

 I.C.2. Submission to EPA 

The safety of dicamba use on many crops, including cotton, was reviewed by the 

Environmental Protection Agency (EPA) as part of the food, feed, and environmental 

safety reassessment in 2006 (U.S. EPA, 2009). Dicamba can currently be applied to 

cotton in the U.S. as a pre-plant application, at least 21 days prior to planting. The 

tolerance of MON 88701 to dicamba facilitates a wider window of application on cotton, 

allowing pre-emergence application of the herbicide up to the day of crop emergence and 

post-emergence in-crop applications through seven days pre-harvest. Monsanto will 

request a registration from U.S. EPA for the expanded use of dicamba on MON 88701, 

an increase in the dicamba residue tolerance from 0.2 ppm to 3 ppm for cottonseed, the 

establishment of a tolerance of 70 ppm for cotton gin by-products, and the inclusion of 

DCSA in the residue definitions for cottonseed and gin by-products. No other revisions 

to dicamba pesticide residue tolerances are needed including animal products such as 

meat, eggs, or milk. 

The existing 0.2 ppm pesticide residue tolerance for cottonseed supporting the current 

registered uses of dicamba on cotton (40 CFR § 180.227) is for the combined residues of 

parent dicamba and its metabolite 5-hydroxy dicamba. Cotton gin by-products, a 

ruminant feed supplement, have no established dicamba tolerance. Studies have shown 

that the proposed use of dicamba on MON 88701 cotton results in total residue 

concentrations of parent dicamba and its metabolites, including DCSA and 5-hydroxy 

dicamba, are less than 3 ppm for cottonseed and less than 70 ppm for gin by-products. 
Monsanto Company 12-CT-244U 37 of 620 

The safety of glufosinate use on many crops, including cotton, was reviewed by the 

Environmental Protection Agency (EPA) as part of the food, feed, and environmental 

safety reassessment in 2000 (U.S. EPA, 2003). In addition, glufosinate has been used 

over-the-top of glufosinate-tolerant crops since 1995 with no significant adverse effects 

reported. Glufosinate is currently labeled for in-crop application on glufosinate-tolerant 

cotton from emergence through early bloom growth stage (Bayer CropScience, 2011). 

The use pattern and rate of glufosinate on MON 88701 will follow the existing 

glufosinate-tolerant cotton uses outlined on the glufosinate herbicide label. Furthermore, 

glufosinate residues in MON 88701 treated with glufosinate are below the EPAestablished residue tolerances of 4.0 ppm and 15.0 ppm for cottonseed and gin byproducts, respectively (U.S. EPA, 2003) (40 CFR § 180.473). Both of these tolerances 

include the combined residues of parent glufosinate and its metabolites N-acetyl 

glufosinate and 3-methylphosphinico-propionic acid. Currently glufosinate is undergoing 

reregistration at EPA with the Reregistration Eligibility Decision (RED) expected by the 

end of 2013 (U.S. EPA, 2008). It is likely that EPA will affirm the safety and efficacy of 

glufosinate and approve its continued use in the marketplace upon completion of the 

reregistration process. Therefore, Monsanto will not pursue any changes in the 

glufosinate label, use pattern, or the established tolerances for its use on MON 88701 

cotton. 

I.C.3. Submissions to Foreign Government Agencies 

To support commercial introduction of MON 88701 in the U.S., regulatory submissions 

will be made to countries that import significant quantities of cotton or its processed 

fractions from the U.S. These will include submissions to a number of foreign 

government regulatory authorities, including: Japan’s Ministry of Agriculture, Forestry, 

and Fisheries and the Ministry of Health, Labour, and Welfare; the Canadian Food 

Inspection Agency; Health Canada; the Intersectoral Commission for Biosafety of 

Genetically Modified Organisms, Mexico; the Korea Food and Drug Administration; and 

the Rural Development Administration of Korea, as well as to regulatory authorities in 

other cotton importing countries with functioning regulatory systems. As appropriate, 

notifications will be made to countries that import significant quantities of cotton and 

cotton products that do not have a formal regulatory review process for biotechnologyderived crops. 
Monsanto Company 12-CT-244U 38 of 620 

II. THE BIOLOGY OF COTTON 

The Organisation for Economic Co-operation and Development Consensus Document 

(OECD, 2008) on the biology of cotton (Gossypium spp.) provides key information on: 

- general description of cotton biology, including taxonomy and 

morphology and use of cotton as a crop plant 

- agronomic practices in cotton cultivation 

- geographic centers of origin 

- reproductive biology 

- inter-species/genus introgression into relatives and interactions 

with other organisms 

- summary of the ecology of cotton 

Additional information on the biology and growth and development of cotton is available 

in the literature (Kohel and Lewis, 1984; OGTR, 2008; Smith and Cothren, 1999). 

To support the evaluation of the plant pest potential of MON 88701 relative to 

conventional cotton, additional information regarding several aspects of cotton biology 

can be found elsewhere in this petition. This includes: agronomic practices for cotton in 

Section V.III; volunteer management of cotton in Sections VIII.H and IX.C; and interspecies/genus introgression potential in Section IX.D. 

II.A. Cotton as a Crop 

Cotton belongs to the genus Gossypium that currently has approximately 50 species 

which are widely cultivated in tropical and subtropical regions around the world (OECD, 

2008; Percival et al., 1999). There are four cultivated species that were domesticated 

independently, two of which account for greater than 95% of world cotton production. 

Gossypium hirsutum (often called upland, American, Mexican, or Acala cotton) accounts 

for 90% and Gossypium barbadense (often called extra long-staple, Pima, and Egyptian 

cotton) accounts for 5% of world cotton production. Due to the utility of the fibers for 

the production of textiles, human selection pressure on cotton has altered the plant from 

essentially perennial shrubs or trees with small impermeable seeds and sparse hairs to a 

compact annual row crop, yielding large, easily germinating seeds with white, thick, 

long, and strong fibers (Brubaker et al., 1999). 

The four cultivated species, which are widely cultivated across the entire globe, are 

comprised of two diploid species G. arboretum and G. herbaceum, which evolved from 

Africa and the Middle East, and two allotetraploid species G. barbadense and G. 

hirsutum, which evolved in the Americas (Brubaker et al., 1999). 

Improved modern varieties of G. hirsutum and G. barbadense are currently cultivated in 

the southern U.S., with G. barbadense grown primarily in the western states of Arizona, 

California, New Mexico, and Texas; and G. hirsutum produced throughout the 17 states 

comprising the U.S. cotton growing region, commonly referred to as the cotton belt. 

G. hirsutum comprises the vast majority of U.S. cotton production with nearly 11 million 
Monsanto Company 12-CT-244U 39 of 620 

acres planted and 18 million bales harvested, whereas G. barbadense varieties accounted 

for approximately 200,000 acres and half a million bales in 2010 (USDA-NASS, 2011e). 

Commercial cotton, including G. hirsutum and G. barbadense, has a long history of 

agricultural production (Lee, 1984; USDA-AMS, 2001; USDA-NASS, 2012c). Extralong staple lint from G. barbadense is segregated and classed separately from G. 

hirsutum and is sold at a premium (USDA-AMS, 2001). However, cottonseed and 

cottonseed by-products (e.g., oil and meal) are not generally distinguished by species 

(OECD, 2008; USDA-FAS, 2005). 

II.B. Characteristics of the Recipient Plant 

The G. hirsutum cotton variety used as the recipient for the DNA insertion to create 

MON 88701 was Coker 130, a non-transgenic, conventional, upland inbred variety 

developed by Coker Pedigreed Seed Co., commercialized in 1990 in the U.S. (Bowman 

et al., 2006). 

II.C. Cotton as a Test System in Product Safety Assessment 

Coker 130 was used as the near isogenic, conventional parental cotton comparator 

(referred to in this petition as the conventional control) in the safety assessment of 

MON 88701. MON 88701 and the conventional control have similar genetic 

backgrounds with the exception of the T-DNA, thus, the effect of the T-DNA and the 

expressed MON 88701 DMO and PAT (bar) proteins could be assessed. In addition, 

commercial cotton varieties (referred to in this consultation document as commercial 

reference varieties) were used as reference materials to establish ranges of natural 

variability representative of commercial cotton varieties. The commercial reference 

varieties used at each field trial location were selected based on their availability and 

agronomic fit for the respective geographic region. 
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III. DESCRIPTION OF THE GENETIC MODIFICATION 

MON 88701 was developed through Agrobacterium tumefaciens-mediated 

transformation of cotton tissues from Coker 130 variety utilizing plasmid vector 

PV-GHHT6997. This section describes the plasmid vector, the donor gene, and the 

regulatory elements used in the development of MON 88701, as well as the deduced 

amino acid sequence of the MON 88701 DMO protein and PAT (bar) protein produced 

in MON 88701. In this section, transfer DNA (T-DNA) refers to DNA that is transferred 

to the plant during transformation. An expression cassette is comprised of sequences to 

be transcribed and the regulatory elements necessary for the expression of those 

sequences. 

III.A. PV-GHHT6997 

PV–GHHT6997 was used in the transformation of cotton to produce MON 88701 and its 

plasmid map is shown in Figure III-1. The elements included in this plasmid vector are 

described in Table III-1. PV- GHHT6997 is approximately 9.4 kb and contains one 

T-DNA that is delineated by Left Border and Right Border regions. The T-DNA contains 

the dmo and bar expression cassettes. The dmo expression cassette is regulated by the 

peanut chlorotic streak caulimovirus (PC1SV) promoter, the tobacco etch virus (TEV) 5′

leader sequence, and the 3′ untranslated sequence of the E6 gene from Gossypium 

barbadense. The chloroplast transit peptide CTP2 directs transport of the DMO protein 

to the chloroplast in MON 88701 and is derived from the CTP2 target sequence of the 

Arabidopsis thaliana shkG gene (Herrmann, 1995; Klee et al., 1987). The bar expression 

cassette is regulated by the e35S promoter from the 35S RNA of cauliflower mosaic virus 

(CaMV), the heat shock protein 70 (Hsp70) leader, and the nopaline synthase (nos)

3′ untranslated region. 

The backbone region of PV–GHHT6997, located outside of the T-DNA, contains two 

origins of replication for maintenance of plasmid vector in bacteria (oriV and 

ori-pBR322), a bacterial selectable marker gene (aadA), and a coding sequence for 

repressor of primer (rop) protein for maintenance of plasmid vector copy number in 

Escherichia coli (E. coli). A description of the genetic elements and their prefixes (e.g., 

B-, P-, L-, TS-, CS-, T-, and OR-) in PV-GHHT6997 is provided in Table III-1. 

III.B. Description of the Transformation System 

MON 88701 was developed through Agrobacterium-mediated transformation of 

PV-GHHT6997 (Figure III-1) into cotton hypocotyls, based on published methods 

(Duncan, 2010; Duncan and Ye, 2011). In summary, hypocotyl segments were excised 

from dark grown seedlings of germinated Coker 130 seed. After co-culturing with the 

Agrobacterium3 carrying the vector, the hypocotyl segments were placed on a sequence 

of media for callus growth containing carbenicillin and cefotaxime to inhibit the growth 

of excess Agrobacterium and glufosinate to inhibit growth of untransformed cells. The 

 

3 Agrobacterium strain used contained a disarmed Ti plasmid. 
Monsanto Company 12-CT-244U 41 of 620 

somatic embryos developing on the culture medium were then placed on medium that 

contained plant growth regulators conducive to shoot regeneration, but no antibiotics or 

glufosinate. Rooted plants (R0) with normal phenotypic characteristics were selected and 

transferred to soil for growth and further assessment. 

The R0 plants generated through the Agrobacterium-mediated transformation were selfpollinated to produce R1 seed. R0 and R1 plants were evaluated for tolerance to dicamba 

and glufosinate and screened for the presence of the T-DNA (dmo and bar expression 

cassettes) and absence of plasmid vector backbone (oriV). Subsequently, the dmo and

bar homozygous positive R1 plant was self-pollinated to give rise to R2 plants. 

Homozygous positive R2 plants containing only a single T-DNA insertion, were 

identified by a combination of analytical techniques including dicamba and glufosinate 

sprays, polymerase chain reaction (PCR), and Southern blot analysis, resulting in 

production of dicamba and glufosinate-tolerant cotton MON 88701. MON 88701 was 

selected as the lead event based on superior phenotypic characteristics and its molecular 

characteristics. Studies on MON 88701 were initiated to further characterize the genetic 

insertion and the expressed proteins, and to establish the food, feed, and environmental 

safety relative to conventional cotton. The major steps involved in the development of 

MON 88701 are depicted in Figure III-2. 

 
Monsanto Company 12-CT-244U 42 of 620 

Probe Start Position (bp) End Position (bp) Total Length (~kb) 

1 1 1310 1.3 

2 1223 2241 1.0 

3 2142 3252 1.1 

4 3153 3914 0.8 

5 3832 4625 0.8 

6 4626 6282 1.7 

7 6204 7708 1.5 

8 7630 9379 1.8 

Figure III-1. Circular Map of PV-GHHT6997 Showing Probes 1-8 

A circular map of PV-GHHT6997 used to develop MON 88701 is shown. 

PV-GHHT6997 contains a single T-DNA. Genetic elements and restriction sites (in 

bold) used in Southern analyses (with positions relative to the first base pair of the 

plasmid vector) are shown on the exterior of the map. The probes used in the Southern 

analyses are shown on the interior of the map and listed in the table. 

 
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Figure III-2. Schematic of the Development of MON 88701 

Transformed hypocotyl tissue from Coker 130 with PV-GHHT6997 in

Agrobacterium tumefaciens containing a disarmed Ti plasmid 

Selected transformants and generated rooted shoots from the 

transformed hypocotyl tissue

Identified MON 88701 as lead candidate and further evaluated its 

progeny in laboratory and field assessments for insert integrity, dicamba 

and glufosinate tolerances and agronomic performance 

Assembled Agrobacterium binary plasmid vector PV-GHHT6997 and 

transferred to Agrobacterium tumefaciens strain ABI 

Evaluated the transformed plants for tolerance to dicamba and glufosinate 

and screened the transformed plants for the presence of the T-DNA (dmo

and bar expression cassettes) and absence of backbone (oriV) 

Selected homozygous plants were identified by dicamba and glufosinate 

sprays, polymerase chain reaction (PCR), and Southern blot analysis 
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III.C. The dmo Coding Sequence and the MON 88701 DMO Protein 

The dmo expression cassette encodes a ~39 kDa MON 88701 DMO precursor protein 

consisting of a single polypeptide of 416 amino acids (Figure III-3). The dmo coding 

sequence is the codon optimized coding sequence from Stenotrophomonas maltophilia

that encodes the DMO protein (Herman et al., 2005; Wang et al., 1997). The presence of 

MON 88701 DMO protein confers dicamba tolerance. 

III.D. The bar Coding Sequence and PAT (bar) Protein 

The bar expression cassette encodes a ~21 kDa PAT (bar) protein consisting of a single 

polypeptide of 183 amino acids (Thompson et al., 1987) (Figure III-4). The bar coding 

sequence is from Streptomyces hygroscopicus and encodes the phosphinothricin 

N-acetyltransferase (PAT) protein (Thompson et al., 1987). The presence of PAT (bar) 

protein confers glufosinate tolerance. 

III.E. Regulatory Sequences 

The dmo coding sequence in MON 88701 is under the regulation of the PC1SV promoter, 

the TEV 5′ leader, and the E6 3′ untranslated region. The PC1SV promoter is the 

promoter for the Full-Length Transcript (FLt) of peanut chlorotic streak caulimovirus 

(Maiti and Shepherd, 1998) that directs transcription in plant cells. The TEV leader is the 

5′ untranslated region from the tobacco etch virus (Niepel and Gallie, 1999) and is 

involved in regulating gene expression. The chloroplast transit peptide CTP2 directs 

transport of the DMO protein to the chloroplast in MON 88701 and is derived from the 

CTP2 target sequence of the Arabidopsis thaliana shkG gene (Herrmann, 1995; Klee et 

al., 1987). The E6 3′ non-translated region is the 3′ untranslated region from the E6 gene 

of Gossypium barbadense encoding a fiber protein, which functions to direct 

polyadenylation of the mRNA (John, 1996). 

The bar coding sequence in MON 88701 is under the regulation of the e35S promoter, 

the Hsp70 leader, and the nos 3′ untranslated region. The e35S promoter is the promoter 

for the 35S RNA of cauliflower mosaic virus (CaMV) (Odell et al., 1985), containing the 

duplicated enhancer region (Kay et al., 1987) that directs transcription in plant cells. The 

Hsp70 leader is the 5′ untranslated region from the DnaK gene from Petunia hybrida

(Rensing and Maier, 1994; Winter et al., 1988) and is involved in regulating gene 

expression. The nos 3′ untranslated region is the 3′ untranslated region from the nopaline 

synthase (nos) gene of Agrobacterium tumefaciens encoding NOS and directs 

polyadenylation of the mRNA (Bevan et al., 1983; Fraley et al., 1983). 

III.F. T-DNA Borders 

PV-GHHT6997 contains Right Border and Left Border regions (Figure III-1 and 

Table III-1), which were derived from Agrobacterium tumefaciens plasmids. The border 

regions each contain a 24-25 bp nick site that is the site of DNA exchange during 

transformation (Barker et al., 1983; Depicker et al., 1982; Zambryski et al., 1982). The 

border regions separate the T-DNA from the plasmid backbone region and are involved 

in the efficient transfer of T-DNA into the cotton genome. 
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III.G. Genetic Elements Outside of the T-DNA Borders 

Genetic elements that exist outside of the T-DNA border regions are those that are 

essential for the maintenance or selection of PV-GHHT6997 in bacteria. The origin of 

replication, oriV, is required for the maintenance of the plasmid in Agrobacterium and is 

derived from the broad host plasmid RK2 (Stalker et al., 1981). The origin of replication, 

ori-pBR322, is required for the maintenance of the plasmid in E. coli and is derived from 

the plasmid vector pBR322 (Sutcliffe, 1979). Coding sequence rop encodes the repressor 

of primer (ROP) protein which is necessary for the maintenance of plasmid copy number 

in E. coli (Giza and Huang, 1989). The selectable marker aadA is a bacterial promoter 

and coding sequence for an enzyme from transposon Tn7 that confers spectinomycin and 

streptomycin resistance (Fling et al., 1985) in E. coli and Agrobacterium during 

molecular cloning. Because these elements are outside the border regions, they are not 

expected to be transferred into the cotton genome. The absence of detectable backbone 

sequence in MON 88701 has been confirmed by Southern blot analyses (See Section IV–

B). 
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Table III-1. Summary of Genetic Elements in PV-GHHT6997 

Genetic 

Element

Location in 

Plasmid 

Vector (bp) Function (Reference)

T-DNA

B1-Right 

Border 

Region

1-331 

DNA region from Agrobacterium tumefaciens containing 

the Right Border sequence used for transfer of the T-DNA 

(Depicker et al., 1982; Zambryski et al., 1982) 

Intervening 

Sequence 332-433 Sequence used in DNA cloning 

P2-PC1SV 434-866 

Promoter from the Full-Length Transcript (FLt) of peanut 

chlorotic streak caulimovirus (PC1SV) that directs 

transcription in plant cells (Maiti and Shepherd, 1998) 

Intervening 

Sequence 867-872 Sequence used in DNA cloning 

L3-TEV 873-1004 

5′ UTR leader sequence from the RNA of tobacco etch virus 

(TEV) (Niepel and Gallie, 1999) that is involved in 

regulating gene expression 

Intervening 

Sequence 1005 Sequence used in DNA cloning 

TS4-CTP2 1006-1233 

Targeting sequence of the ShkG gene from Arabidopsis 

thaliana encoding the EPSPS transit peptide region that 

directs transport of the protein to the chloroplast (Herrmann, 

1995; Klee et al., 1987) 

CS5-dmo 1234-2256 

Codon optimized coding sequence for the dicamba 

mono-oxygenase (DMO) protein of Stenotrophomonas 

maltophilia that confers dicamba tolerance (Herman et al., 

2005; Wang et al., 1997) 

Intervening 

Sequence 2257-2310 Sequence used in DNA cloning 

T6-E6 2311-2625 

3′ UTR sequence of the E6 gene from Gossypium 

barbadense (cotton) encoding a fiber protein involved in 

early fiber development (John, 1996) that directs 

polyadenylation of mRNA 

Intervening 

Sequence 2626-2637 Sequence used in DNA cloning 

P-e35S 2638-3249 

Promoter from the 35S RNA of cauliflower mosaic virus 

(CaMV) (Odell et al., 1985) containing the duplicated 

enhancer region (Kay et al., 1987) that directs transcription 

in plant cells 

Intervening 

Sequence 3250-3252 Sequence used in DNA cloning 

 
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Table III-1. Summary of Genetic Elements in PV-GHHT6997 (continued) 

Genetic 

Element

Location in 

Plasmid 

Vector (bp) Function (Reference)

L-Hsp70 3253-3348 

5′ UTR leader sequence of the DnaK gene from Petunia

hybrida that encodes heat shock protein 70 (HSP70) 

(Rensing and Maier, 1994; Winter et al., 1988) that is 

involved in regulating gene expression 

Intervening 

Sequence 3349-3354 Sequence used in DNA cloning 

CS-bar 3355-3906 

Coding sequence for the phosphinothricin 

N-acetyltransferase (PAT) protein of Streptomyces 

hygroscopicus that confers glufosinate tolerance (Thompson 

et al., 1987) 

Intervening 

Sequence 3907-3911 Sequence used in DNA cloning 

T-nos 3912-4164 

3′ UTR sequence of the nopaline synthase (nos) gene from 

Agrobacterium tumefaciens pTi encoding NOS that directs 

polyadenylation (Bevan et al., 1983; Fraley et al., 1983) 

Intervening 

Sequence 4165-4183 Sequence used in DNA cloning 

B-Left 

Border 

Region

4184-4625 

DNA region from Agrobacterium tumefaciens containing 

the Left Border sequence used for transfer of the T-DNA 

(Barker et al., 1983) 

Plasmid Vector Backbone 

Intervening 

Sequence 4626-4711 Sequence used in DNA cloning 

OR7-oriV 4712-5108 

Origin of replication from the broad host range plasmid 

RK2 for maintenance of plasmid in Agrobacterium (Stalker 

et al., 1981) 

Intervening 

Sequence 5109-6616 Sequence used in DNA cloning 

CS-rop 6617-6808 

Coding sequence for repressor of primer protein from the 

ColE1 plasmid for maintenance of plasmid copy number in 

E. coli (Giza and Huang, 1989) 

Intervening 

Sequence 6809-7235 Sequence used in DNA cloning 

OR-oripBR322 7236-7824 Origin of replication from plasmid pBR322 for maintenance 

of plasmid in E. coli (Sutcliffe, 1979) 

 
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Table III-1. Summary of Genetic Elements in PV-GHHT6997 (continued) 

Genetic 

Element

Location in 

Plasmid 

Vector (bp) Function (Reference)

Intervening 

Sequence 7825-8354 Sequence used in DNA cloning 

aadA 8355-9243 

Bacterial promoter, coding sequence, and 3′ UTR for an 

aminoglycoside-modifying enzyme, 

3'(9)-O-nucleotidyltransferase from the transposon Tn7

(Fling et al., 1985) that confers spectinomycin and 

streptomycin resistance 

Intervening 

Sequence 9244-9379 Sequence used in DNA cloning 

1

B, Border 

2

P, Promoter 

3

L, Leader 

4

TS, Targeting Sequence 

5

CS, Coding Sequence 

6

T, Transcription Termination Sequence 

7

OR, Origin of Replication 

 
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 1 MAQVSRICNG VQNPSLISNL SKSSQRKSPL SVSLKTQQHP RAYPISSSWG

 51 LKKSGMTLIG SELRPLKVMS SVSTACMLTF VRNAWYVAAL PEELSEKPLG 

101 RTILDTPLAL YRQPDGVVAA LLDICPHRFA PLSDGILVNG HLQCPYHGLE 

151 FDGGGQCVHN PHGNGARPAS LNVRSFPVVE RDALIWIWPG DPALADPGAI 

201 PDFGCRVDPA YRTVGGYGHV DCNYKLLVDN LMDLGHAQYV HRANAQTDAF 

251 DRLEREVIVG DGEIQALMKI PGGTPSVLMA KFLRGANTPV DAWNDIRWNK 

301 VSAMLNFIAV APEGTPKEQS IHSRGTHILT PETEASCHYF FGSSRNFGID 

351 DPEMDGVLRS WQAQALVKED KVVVEAIERR RAYVEANGIR PAMLSCDEAA 

401 VRVSREIEKL EQLEAA 

Figure III-3. Deduced Amino Acid Sequence of the MON 88701 DMO Protein 

The amino acid sequence of the MON 88701 DMO precursor protein was deduced from 

the full-length coding nucleotide sequence present in PV-GHHT6997 (See Table III-1 for 

more detail). The chloroplast transit peptide (CTP2) and the first 76 amino acids of the 

precursor protein are underlined. CTP2 targets MON 88701 DMO protein to the 

chloroplast. The CTP2 is cleaved in the chloroplast producing the mature 349 amino acid 

MON 88701 DMO protein that begins with the valine at position 68 (See Appendix C.1). 

The double underline shows the nine amino acids from CTP2 that are at the N-terminus 

of the mature MON 88701 protein. 

 1 MSPERRPADI RRATEADMPA VCTIVNHYIE TSTVNFRTEP QEPQEWTDDL 

 51 VRLRERYPWL VAEVDGEVAG IAYAGPWKAR NAYDWTAEST VYVSPRHQRT 

101 GLGSTLYTHL LKSLEAQGFK SVVAVIGLPN DPSVRMHEAL GYAPRGMLRA 

151 AGFKHGNWHD VGFWQLDFSL PVPPRPVLPV TEI 

Figure III-4. Deduced Amino Acid Sequence of the PAT (bar) Protein 

The amino acid sequence of the MON 88701-produced PAT (bar) protein was deduced 

from the full-length coding nucleotide sequence present in PV-GHHT6997 (See 

Table III-1 for more detail). 
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IV. CHARACTERIZATION OF THE GENETIC MODIFICATION 

Characterization of the DNA insert in MON 88701 was conducted by Southern blot, 

PCR, and DNA sequence analyses. The results of this characterization demonstrate that 

MON 88701 contains a single copy of the dmo and bar expression cassettes and lacks 

plasmid backbone; the T-DNA is stably integrated at a single locus and is inherited 

according to Mendelian principles over multiple generations. These conclusions were 

based on several lines of evidence: 1) Southern blot analyses assayed the entire cotton 

genome for the presence of the T-DNA and absence of the plasmid backbone sequences 

derived from PV-GHHT6997, and demonstrated that only a single copy of the T-DNA 

was inserted at a single genomic site and that the insert is stably inherited; 2) DNA 

sequence analyses to determine the exact sequence of the inserted DNA and the DNA 

sequences flanking the 5′ and 3′ ends of the insert, allowing a comparison to the T-DNA 

sequence in the plasmid vector to confirm that only the expected sequences were 

integrated; 3) DNA sequences flanking the 5′ and 3′ ends of the insert were compared to 

the sequence of the insertion site in conventional cotton to identify any rearrangements 

that occurred at the insertion site during transformation. Taken together, the 

characterization of the genetic modification demonstrates that a single copy of the 

T-DNA was stably integrated at a single locus of the cotton genome and that no plasmid 

backbone sequences are present in MON 88701. 

Southern blot analyses were used to determine the copy number and insertion sites of the 

integrated DNA as well as the presence or absence of plasmid vector backbone 

sequences. The Southern blot strategy was designed to ensure that all potential 

transgenic segments would be identified. The entire cotton genome was assayed with 

probes that spanned the complete plasmid vector to detect the presence of the insert as 

well as confirm the absence of any plasmid vector backbone sequences. This was 

accomplished by using probes that were not more than 2.5 kb in length to ensure a high 

level of sensitivity. This high level of sensitivity was demonstrated for each blot by 

detection of a positive control added at 0.1 copies per genome equivalent. Two sets of 

restriction enzymes were specifically chosen to fully characterize the T-DNA and detect 

any potential fragments of the T-DNA and backbone sequences. The restriction enzyme 

sets were chosen such that each enzyme set cleaves once within the inserted T-DNA and 

at least once within the known DNA flanking the 5′ or 3′ end of the insert. As a 

consequence, at least one segment containing a portion of the insert with the adjacent 5′

flanking DNA generated by one set of the enzyme(s) is of a predictable size and overlaps 

with another predictable size segment containing a portion of the insert with the adjacent 

3′ flanking DNA generated by another set of the enzyme(s). This two-set enzyme design 

ensures that the entire insert is identified in a predictable hybridization pattern. This 

strategy also maximizes the possibility of detecting an insertion elsewhere in the genome 

that could be overlooked if that band co-migrated on the gel with an expected band. 

To determine the number of copies and insertion sites of the T-DNA, and the presence or 

absence of the plasmid vector backbone sequences, duplicated samples that consisted of 

equal amounts of digested DNA were run on the agarose gel. One set of samples was run 

for a longer period of time (long run) than the second set (short run). The long run allows 

for greater resolution of large molecular weight DNA, whereas the short run allows for 
Monsanto Company 12-CT-244U 51 of 620 

retaining the small molecular weight DNA on the gel. The molecular weight markers on 

the left of the figures were used to estimate the sizes of the bands present in the long run 

lanes of the Southern blots, and the molecular weight markers on the right of the figures 

were used to estimate the sizes of bands present in the short run lanes of the Southern 

blots (Figure IV-2 through Figure IV-5). Any minor discrepancies between the 

molecular weight marker and the genomic DNA samples are likely due to differences in 

the migration rate of DNA during agarose gel electrophoresis caused by differences in 

salt concentration, base composition, or sequences of DNA (Elder and Southern, 1983; 

Sambrook and Russell, 2001). Southern blot analyses determined that a single copy of 

the T-DNA was inserted at a single locus of the cotton genome, and no additional genetic 

elements, including backbone sequences, from PV-GHHT6997 were detected in 

MON 88701. 

The PCR and DNA sequence analyses complement the Southern analyses. PCR and 

DNA sequence analyses performed on MON 88701 determined the complete DNA 

sequence of the insert and flanking genomic DNA sequences in MON 88701, confirmed 

the predicted organization of the genetic elements within the insert, and determined the 

sequences flanking the insert. In addition, DNA sequence analyses confirmed that each 

genetic element (except for the border regions) in the insert is intact and the sequence of 

the insert is identical to the corresponding sequence in PV-GHHT6997 (Figures IV-6 and 

IV-7). Furthermore, genomic organization at the MON 88701 insertion site was 

determined by comparing the sequence flanking the 5′ and 3′ ends of the insert to the 

sequence of the insertion site in conventional cotton. 

The stability of the T-DNA present in MON 88701 across multiple generations was 

demonstrated by Southern blot fingerprint analysis (Figure IV-9). Genomic DNA from 

five generations of MON 88701 (Figure IV-8) was digested with one of the enzyme sets 

used for the insert and copy number analyses and was hybridized with two probes that 

detect restriction segments that encompass the entire insert. This fingerprint strategy 

consists of two insert segments each containing its adjacent genomic DNA that assesses 

not only the stability of the insert, but also the stability of the DNA directly adjacent to 

the insert. 

Segregation analysis was conducted to determine the inheritance and stability of the 

T-DNA insert in MON 88701. Results from this analysis demonstrated that the 

inheritance and stability of the insert was as expected across multiple generations 

(Figure IV-8, Table IV-3, and Table IV-4), which corroborates the molecular insert 

stability analysis and establishes the genetic behavior of the T-DNA at a single 

chromosomal locus. 

The Southern blot analyses confirmed that the T-DNA reported in Figure IV-1 represents 

the only detectable insert in MON 88701. A circular map of PV-GHHT6997 annotated 

with the probes used in the Southern blot analysis is presented in Figure III-1 and the 

genetic elements within the MON 88701 insert are summarized in Table IV-2. A linear 

map depicting restriction sites within the insert as well as within the DNA immediately 

flanking the insert in MON 88701 is shown in Figure IV-1. Based on the plasmid map 

and the linear map of the insert, a table summarizing the expected DNA segments for 
Monsanto Company 12-CT-244U 52 of 620 

Southern analyses is presented in Table IV-1. The results from the Southern blot 

analyses are presented in Figure IV-2 through Figure IV-5. PCR amplification of the 

MON 88701 insert and the insertion site in the conventional control for DNA sequence 

analysis are shown in Figure IV-6 and Figure IV-7, respectively. The generations used in 

the generational stability analysis are depicted in the breeding history shown in 

Figure IV-8 and the results from the generational stability analysis are presented in 

Figure IV-9. The breeding path for generating the segregation data is shown in 

Figure IV-10 and the results for the segregation analysis are presented in Table IV-3 and 

IV-4. Materials and methods used for the characterization of the insert in MON 88701 

are found in Appendix B. 
Monsanto Company 12-CT-244U 

Figure IV-1. Schematic Representation of the Insert and Flanking DNA in MON 88701 A linear map of the insert and DNA flanking the insert in MON 88701 is shown. Angled arrows indicate the 

T-DNA and the beginning of the flanking DNA. Identified on the linear map are genetic elements within the insof the restriction enzymes used in the Southern analyses with positions relative to the first base pair of the DNA 

in this map. The relative sizes and locations of the T-DNA probes and the expected sizes of restriction fragmen

lower portion of the scheme. This schematic diagram is not drawn to scale. Locations of genetic elements a

approximate. Probes are also shown in Figure III-1. r1Superscript in Left Border Region indicates that the seq

was truncated compared to the sequences in PV–GHHT6997. 
Monsanto Company 12-CT-244U 

Table IV-1. Summary Chart of the Expected DNA Segments Based on Hybridizing Probes and RestrictMON 88701 Analysis 

Southern Blot Analysis T-DNA 

Figure IV-2 IV-3 IV-4 

Probe(s) Used 1,5 2,4 3 

Probing Target Digestion enzyme Expected Band Sizes on each Southern Blot

PV-GHHT6997 Pci I ~6.2 kb ~3.2 kb ~6.2 kb ~6.2 kb 

Probe Templates1 N/A ~1.3 kb ~0.8 kb ~1.0 kb ~0.8 kb ~~2 

MON 88701 

Bcl I ≥3.1 kb ~2.4 kb 

≥3.1 kb 

~2.4 kb

≥3.1 kb 

~2.4 kb 

Ssp I ~3.4 kb ~1.2 kb ~3.4 kb ~1.2 kb ~3.4 kb 

1Probe template spikes were used as positive hybridization controls in Southern blot analyses when multiple probes wer

simultaneously. 

2’ ~~’ indicates that probe template was not used. 
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Table IV-2. Summary of Genetic Elements in MON 88701 

Genetic Element 

Location in 

Sequence (bp) Function (Reference) 

5′ Flank 1-1126 Cotton genomic DNA 

Intervening 

Sequence 

1127-1219 Sequence used in DNA cloning 

P1-PC1SV 1220-1652 Promoter from the Full-Length Transcript 

(FLt) of peanut chlorotic streak caulimovirus 

(PC1SV) that directs transcription in plant 

cells (Maiti and Shepherd, 1998) 

Intervening 

Sequence 

1653-1658 Sequence used in DNA cloning 

L2-TEV 1659-1790 5′ UTR leader sequence from the RNA of 

tobacco etch virus (TEV) (Niepel and Gallie, 

1999) that is involved in regulating gene 

expression 

Intervening 

Sequence 

1791 Sequence used in DNA cloning 

TS3-CTP2 1792-2019 Targeting sequence of the ShkG gene from 

Arabidopsis thaliana encoding the EPSPS 

transit peptide region that directs transport of 

the protein to the chloroplast (Herrmann, 

1995; Klee et al., 1987) 

CS4-dmo 2020-3042 Codon optimized coding sequence for the 

dicamba mono-oxygenase (DMO) protein of 

Stenotrophomonas maltophilia that confers 

dicamba tolerance (Herman et al., 2005; 

Wang et al., 1997) 

Intervening 

Sequence 

3043-3096 Sequence used in DNA cloning 

T5-E6 3097-3411 3′ UTR sequence of the E6 gene from 

Gossypium barbadense (cotton) encoding a 

fiber protein involved in early fiber 

development (John, 1996) that directs 

polyadenylation of mRNA 

Intervening 

Sequence 

3412-3423 Sequence used in DNA cloning 

P-e35S 3424-4035 Promoter from the 35S RNA of cauliflower 

mosaic virus (CaMV) (Odell et al., 1985) 

containing the duplicated enhancer region 

(Kay et al., 1987) that directs transcription in 

plant cells 

 
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Table IV-2. Summary of Genetic Elements in MON 88701 (continued) 

Genetic Element 

Location in 

Sequence 

(bp) Function (Reference)

Intervening 

Sequence 

4036-4038 Sequence used in DNA cloning 

L-Hsp70 4039-4134 5′ UTR leader sequence of the DnaK gene 

from Petunia hybrida that encodes heat 

shock protein 70 (HSP70) (Rensing and 

Maier, 1994; Winter et al., 1988) that is 

involved in regulating gene expression 

Intervening 

Sequence 

4135-4140 Sequence used in DNA cloning 

CS-bar 4141-4692 Coding sequence for the phosphinothricin 

N-acetyltransferase (PAT) protein of 

Streptomyces hygroscopicus that confers 

glufosinate tolerance (Thompson et al., 1987) 

Intervening 

Sequence 

4693-4697 Sequence used in DNA cloning 

T-nos 4698-4950 3′ UTR sequence of the nopaline synthase 

(nos) gene from Agrobacterium tumefaciens

pTi encoding NOS that directs 

polyadenylation (Bevan et al., 1983; Fraley et 

al., 1983) 

Intervening 

Sequence 

4951-4969 Sequence used in DNA cloning 

B6-Left Border

Region r1 4970-5231 DNA region from Agrobacterium 

tumefaciens containing the Left Border 

sequence used for transfer of the T-DNA 

(Barker et al., 1983) 

3′ Flank 5232-6369 Cotton genomic DNA 

1

P, Promoter 

2

L, Leader 

3

TS, Targeting Sequence 

4

CS, Coding Sequence 

5

T, Transcription Termination Sequence 

6

B, Border 

r1Superscript in Left Border Region indicates that the sequence in MON 88701 was truncated 

compared to the sequences in PV-GHHT6997.
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IV.A. Insert and Copy Number of T-DNA in MON 88701 

The numbers of copies and insertion sites of the T-DNA sequences in the cotton genome 

were evaluated by digesting MON 88701 and conventional control genomic DNA 

samples with the restriction enzyme Bcl I or the restriction enzyme Ssp I and hybridizing 

Southern blots with probes that span the T-DNA (Figure III-1). Each restriction digest is 

expected to produce a specific banding pattern on the Southern blots (Table IV-1). Any 

additional copies and/or integration sites would be detected as additional bands on the 

blots. 

The restriction enzyme Bcl I cleaves once within the inserted T-DNA and within the 

known genomic DNA flanking the 3′ end of the insert (Figure IV-1). Therefore, if 

T-DNA sequences were present as a single copy at a single integration site in 

MON 88701, the digestion with Bcl I was expected to generate two border segments with 

expected sizes of ≥3.1 kb and ~2.4 kb (Figure IV-1 and Table IV-1). The restriction 

enzyme Ssp I cleaves once within the inserted T-DNA and within the known genomic 

DNA flanking the 5′ and 3′ ends of the insert (Figure IV-1). If T-DNA sequences were 

present as a single copy at a single integration site in MON 88701, the digestion with 

Ssp I was expected to generate two border segments with expected sizes of ~3.4 kb and 

~1.2 kb (Figure IV-1 and Table IV-1). 

The Southern blots were hybridized with T-DNA probes that collectively span the entire 

inserted DNA sequence (Figures III-1 and IV-1, Probe 1, Probe 2, Probe 3, Probe 4, and 

Probe 5). Conventional control genomic DNA digested with the restriction enzyme Bcl I 

and spiked with either probe templates and/or digested PV-GHHT6997 DNA served as 

positive hybridization controls. The positive hybridization control was spiked at 

approximately 0.1 and 1.0 copies of genome equivalents to demonstrate sufficient 

sensitivity of the Southern blot. Conventional control genomic DNA digested with the 

appropriate restriction enzymes was used as a negative control. The results of these 

analyses are shown in Figure IV-2 through Figure IV-4. 

IV.A.1. T-DNA Probes 1 and 5 

Conventional control genomic DNA digested with Bcl I (Figure IV-2, Lane 1 and Lane 8) 

or with Ssp I (Figure IV-2, Lane 3 and Lane 10) and simultaneously hybridized with 

Probe 1 and Probe 5 (Figures III-1 and IV-1) produced no detectable hybridization bands 

as expected for the negative control in the reported exposure shown in Figure IV-2. In a 

longer exposure of the blot, faint endogenous hybridization bands were present in both 

the Bcl I digest and the Ssp I digest in the conventional control genomic DNA (data not 

shown). Conventional control genomic DNA digested with Bcl I and spiked with probe 

templates of Probe 1 and Probe 5 (Figure III-1) produced the expected bands at ~1.3 kb 

and ~0.8 kb (Figure IV-2, Lane 5 and Lane 6). Conventional control genomic DNA 

digested with Bcl I and spiked with the PV-GHHT6997 DNA, previously digested with 

the restriction enzyme Pci I (Figure III-1), produced two bands at ~6.2 kb and ~3.2 kb 

(Figure IV-2, Lane 7), as expected. Detection of the positive controls indicates that the 

probes hybridized to their target sequences. 
Monsanto Company 12-CT-244U 58 of 620 

MON 88701 DNA digested with Bcl I and simultaneously hybridized with Probe 1 and 

Probe 5 (Figures III-1 and IV-1) produced the expected bands at ~3.5 kb and ~2.4 kb 

(Figure IV-2, Lane 2 and Lane 9) which is consistent with the expected ≥3.1 kb and 

~2.4 kb bands (Figure IV-1 and Table IV-1), respectively. MON 88701 DNA digested 

with the restriction enzyme Ssp I and hybridized with Probe 1 and Probe 5 (Figures III-1 

and IV-1) produced two bands at ~3.4 kb and ~1.2 kb (Figure IV-2, Lane 4 and Lane 11), 

as expected. 

The results presented in Figure IV-2 indicate that the sequences covered by Probe 1 and 

Probe 5 reside at a single detectable locus of integration in MON 88701. 

IV.A.2. T-DNA Probes 2 and 4 

Conventional control genomic DNA digested with Bcl I (Figure IV-3, Lane 1 and Lane 8) 

or with Ssp I (Figure IV-3, Lane 3 and Lane 10) and simultaneously hybridized with 

Probe 2 and Probe 4 (Figures III-1 and IV-1) produced no detectable hybridization bands 

as expected for the negative control. Conventional control genomic DNA digested with 

Bcl I and spiked with probe templates of Probe 2 and Probe 4 (Figure III-1) produced the 

expected bands at ~1.0 kb and ~0.8 kb (Figure IV-3, Lane 5 and Lane 6). Conventional 

control genomic DNA digested with Bcl I and spiked with the PV-GHHT6997 DNA, 

previously digested with the restriction enzyme Pci I (Figure III-1), produced one band at 

~6.2 kb (Figure IV-3, Lane 7), as expected. Detection of the positive controls indicates 

that the probes hybridized to their target sequences. 

MON 88701 DNA digested with Bcl I and simultaneously hybridized with Probe 2 and 

Probe 4 (Figures III-1 and IV-1) produced the expected bands at ~3.5 kb and ~2.4 kb 

(Figure IV-3, Lane 2 and Lane 9), which is consistent with the expected ≥3.1 kb and 

~2.4 kb bands (Figure IV-1 and Table IV-1), respectively. MON 88701 DNA digested 

with the restriction enzyme Ssp I and hybridized with Probe 2 and Probe 4 (Figures III-1 

and IV-1) produced two bands at ~3.4 kb and ~1.2 kb (Figure IV-3, Lane 4 and Lane 11), 

as expected. 

The results presented in Figure IV-3 indicate that the sequences covered by Probe 2 and 

Probe 4 reside at a single detectable locus of integration in MON 88701. 

IV.A.3. T-DNA Probe 3 

Conventional control DNA digested with Bcl I (Figure IV-4, Lane 1 and Lane 7) or with 

Ssp I (Figure IV-4, Lane 3 and Lane 9) and hybridized with Probe 3 (Figures III-1 and 

IV-1) produced endogenous hybridization signals that were present in all lanes 

(Figure IV-4, Lane 1 through Lane 10). The same hybridization band was produced in 

conventional control and MON 88701 DNA lanes when digested with the same enzyme. 

When digested with Bcl I and hybridized with Probe 3 hybridization bands of ~1.9 kb and 

~1.7 kb were produced with conventional control genomic DNA and MON 88701 DNA 

(Figure IV-4, Lane 1, Lane 2, and Lane 5 through Lane 8). When digested with Ssp I and 

hybridized with Probe 3, a hybridization band of ~2.5 kb was produced with conventional 

control genomic DNA and MON 88701 DNA (Figure IV-4, Lane 3, Lane 4, Lane 9, and 
Monsanto Company 12-CT-244U 59 of 620 

Lane 10). Since these bands are present in both control and test substances, these signals 

are considered to be weak hybridization of probes to endogenous E6 sequences and are not 

specific to the inserted DNA in MON 88701. 

Conventional control genomic DNA digested with Bcl I and spiked with the 

PV-GHHT6997 DNA, previously digested with the restriction enzyme Pci I 

(Figure III-1), produced one band at ~6.2 kb (Figure IV-4, Lane 5 and Lane 6), as 

expected. Detection of the spiked controls indicates that the probe hybridized to its target 

sequence. 

MON 88701 DNA digested with Bcl I and hybridized with Probe 3 (Figures III-1 and 

IV-1) produced two expected bands at ~3.5 kb and ~2.4 kb, which is consistent with the 

expected ≥3.1 kb and ~2.4 kb bands (Figure IV-1 and Table IV-1), and is in addition to 

the endogenous hybridization bands discussed above (Figure IV-4, Lane 2 and Lane 8). 

The ~3.5 kb band is less intense than the ~2.4 kb band. The difference in band intensity 

is likely due to hybridization of a smaller portion of Probe 3 to the ~3.5 kb fragment. The 

~3.5 kb band represents the 5′ end of the inserted DNA and the adjacent DNA flanking 

the 5′ end of the insert; this correlates with the expected border fragment size of ≥3.1 kb. 

The ~2.4 kb band represents the 3′ end of the inserted DNA and the adjacent DNA 

flanking the 3′ end of the insert. MON 88701 DNA digested with Ssp I (Figure IV-4, 

Lane 4 and Lane 10, Figure IV-1, and Table IV-1) and hybridized with Probe 3 produced 

one expected band at ~3.4 kb in addition to the endogenous hybridization bands 

discussed above. The ~3.4 kb band represents the 5′ end of the inserted DNA and the 

adjacent DNA flanking the 5′ end of the insert. 

The results presented in Figure IV-4 indicate that the sequence covered by Probe 3 

resides at a single detectable locus of integration in MON 88701. 
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Figure IV-2. Southern Blot Analysis to Determine Insert and Copy Number of 

T-DNA in MON 88701: Probes 1 and 5 

The blot was simultaneously hybridized with two 32P-labeled probes that span a portion 

of the T-DNA sequence (Figure III-1, Probe 1 and Probe 5). Each lane contains 

approximately 10 μg of digested genomic DNA. Arrows denote the size of the DNA, in 

kilobase pairs, obtained from 1 Kb DNA Extension Ladder on the ethidium bromide 

stained gel. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Bcl I) 

2 MON 88701 (Bcl I) 

3 Conventional Control (Ssp I) 

4 MON 88701 (Ssp I) 

5 Conventional Control (Bcl I) spiked with Probe 1 and Probe 5 template [~1.0 genome 

equivalent] 

6 Conventional Control (Bcl I) spiked with Probe 1 and Probe 5 template [~0.1 genome 

equivalent] 

7 Conventional Control (Bcl I) spiked with PV-GHHT6997 (Pci I) [~1.0 genome equivalent] 

8 Conventional Control (Bcl I) 

9 MON 88701 (Bcl I) 

10 Conventional Control (Ssp I) 

11 MON 88701 (Ssp I) 
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Figure IV-3. Southern Blot Analysis to Determine Insert and Copy Number of 

T-DNA in MON 88701: Probes 2 and 4 

The blot was simultaneously hybridized with two 32P-labeled probes that span a portion 

of the T-DNA sequence (Figure III-1, Probe 2 and Probe 4). Each lane contains 

approximately 10 μg of digested genomic DNA. Arrows denote the size of the DNA, in 

kilobase pairs, obtained from 1 Kb DNA Extension Ladder on the ethidium bromide 

stained gel. Lane designations are as follows: 

Lane Description 

1 Conventional Control (Bcl I) 

2 MON 88701 (Bcl I) 

3 Conventional Control (Ssp I) 

4 MON 88701 (Ssp I) 

5 Conventional Control (Bcl I) spiked with Probe 2 and Probe 4 template [~1.0 genome 

equivalent] 

6 Conventional Control (Bcl I) spiked with Probe 2 and Probe 4 template [~0.1 genome 

equivalent] 

7 Conventional Control (Bcl I) spiked with PV-GHHT6997 (Pci I) [~1.0 genome equivalent] 

8 Conventional Control (Bcl I) 

9 MON 88701 (Bcl I) 

10 Conventional Control (Ssp I) 

11 MON 88701 (Ssp I) 
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Figure IV-4. Southern Blot Analysis to Determine Insert and Copy Number of 

T-DNA in MON 88701: Probe 3 

The blot was hybridized with a 32P-labeled probe that spans a portion of the T-DNA 

sequence (Figure III-1, Probe 3). Each lane contains approximately 10 μg of digested 

genomic DNA. Arrows denote the size of the DNA, in kilobase pairs, obtained from 

1 Kb DNA Extension Ladder on the ethidium bromide stained gel. Lane designations are 

as follows: 

Lane 

1 Conventional Control (Bcl I) 

2 MON 88701 (Bcl I) 

3 Conventional Control (Ssp I) 

4 MON 88701 (Ssp I) 

5 Conventional Control (Bcl I) spiked with PV–GHHT6997 (Pci I) [~1.0 genome equivalent] 

6 Conventional Control (Bcl I) spiked with PV–GHHT6997 (Pci I) [~0.1 genome equivalent] 

7 Conventional Control (Bcl I) 

8 MON 88701 (Bcl I) 

9 Conventional Control (Ssp I) 

10 MON 88701 (Ssp I) 
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IV.B. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GHHT6997 Backbone Sequences in MON 88701 

To determine the presence or absence of the PV-GHHT6997 backbone sequences, 

MON 88701 and conventional control genomic DNA were digested with the restriction 

enzyme Bcl I or restriction enzyme Ssp I, and hybridized with the three backbone probes 

that collectively span the entire backbone sequences (Figure III-1, Probe 6, Probe 7, and 

Probe 8). If backbone sequences are present in MON 88701, then probing with backbone 

probes should result in hybridizing bands. Conventional control genomic DNA digested 

with the restriction enzyme Bcl I and spiked with probe templates or with digested 

PV-GHHT6997 DNA served as positive hybridization controls. The positive 

hybridization control was spiked at approximately 0.1 and 1.0 copies of genome 

equivalents to demonstrate sufficient sensitivity of the Southern blot. Conventional 

control genomic DNA digested with the appropriate restriction enzymes was used as a 

negative control. The results of these analyses are shown in Figure IV-5. 

IV.B.1. Backbone Probes 6, 7, and 8 

Conventional control DNA digested with Bcl I (Figure IV-5, Lane 1 and Lane 10) or the 

restriction enzyme Ssp I (Figure IV-5, Lane 3 and Lane 12) and hybridized with Probe 6, 

Probe 7, and Probe 8 (Figure III-1) produced no detectable hybridization bands as 

expected for the negative control. 

Conventional control genomic DNA digested with Bcl I and spiked with probe templates 

of Probe 7 and Probe 8 (Figure III-1) produced the expected bands at ~1.5 kb and ~1.8 kb 

(Figure IV-5, Lane 5 and Lane 6). Conventional control genomic DNA digested with 

Bcl I and spiked with probe template of Probe 6 (Figure III-1) produced the one expected 

band at ~1.7 kb (Figure IV-5, Lane 7 and Lane 8). Conventional control DNA digested 

with Bcl I and spiked with the PV-GHHT6997 DNA, previously digested with the 

restriction enzyme Pci I (Figure III-1), produced two bands at ~6.2 kb and ~3.2 kb 

(Figure IV-5, Lane 9), as expected. Detection of the positive controls indicates that the 

probe hybridized to its target sequence.

MON 88701 DNA digested with Bcl I (Figure IV-5, Lane 2 and Lane 11) or the 

restriction enzyme Ssp I (Figure IV-5, Lane 4 and Lane 13) and hybridized with Probes 6, 

7, and 8 produced no detectable bands.

The results presented in Figure IV-5 indicate that MON 88701 contains no detectable 

backbone sequences covered by Probes 6, 7, and 8. 

 
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Figure IV-5. Southern Blot Analysis to Determine the Presence or Absence of 

PV-GHHT6997 Backbone Sequences in MON 88701: Probes 6, 7, and 8 

The blot was hybridized with three 32P-labeled probes that span the plasmid vector 

backbone sequences (Figure III-1, Probes 6, 7, and 8). Each lane contains approximately 

10 μg of digested genomic DNA. Arrows denote the size of the DNA, in kilobase pairs, 

obtained from λ DNA/Hind III fragments on the ethidium bromide stained gel. Lane 

designations are as follows: 

Lane Description 

1 Conventional Control (Bcl I) 

2 MON 88701 (Bcl I) 

3 Conventional Control (Ssp I) 

4 MON 88701 (Ssp I) 

5 Conventional Control (Bcl I) spiked with Probe 7 and Probe 8 template [~1.0 genome 

il ] 6 Conventional Control (Bcl I) spiked with Probe 7 and Probe 8 template [~0.1 genome 

il ] 7 Conventional Control (Bcl I) spiked with Probe 6 template [~1.0 genome equivalent] 

8 Conventional Control (Bcl I) spiked with Probe 6 template [~0.1 genome equivalent] 

9 Conventional Control (Bcl I) spiked with PV-GHHT6997 (Pci I) [~1.0 genome equivalent] 

10 Conventional Control (Bcl I) 

11 MON 88701 (Bcl I) 

12 Conventional Control (Ssp I) 

13 MON 88701 (Ssp I) 

 
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IV.C. Organization and Sequence of the Insert and Adjacent Genomic DNA in 

MON 88701 

The organization and sequence of the elements within the MON 88701 insert was 

confirmed by DNA sequence analysis. PCR primers were designed with the intent to 

amplify three overlapping DNA amplicons that span the entire length of the insert and the 

associated DNA flanking the 5′ and 3′ ends of the insert (Figure IV-6). The amplified 

PCR products were subjected to DNA sequence analyses. This analysis determined that 

the DNA sequence of the MON 88701 insert is 4105 bp long (Table IV-2) and is identical 

to the corresponding T-DNA sequence of PV-GHHT6997 as described in Table III-1. 
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Figure IV-6. Overlapping PCR Analysis across the Insert in MON 88701 

PCR was performed on both conventional control genomic DNA and MON 88701 genomic DNA 

using three pairs of primers to generate overlapping PCR fragments from MON 88701 for 

sequence analysis. Approximately five microliters of each of the PCR reactions was loaded on 

the gel. The expected product size for each amplicon and an illustration of the insert in 

MON 88701 is provided at the bottom of the figure. Arrows on the agarose gel photograph 

denote the size of the DNA, in kilobase pairs, obtained from 1 Kb DNA ladder on the ethidium 

bromide stained gel. Lane designations are as follows: 

Lane Description

1 1 Kb DNA Ladder 

2 Conventional Control 

3 MON 88701 

4 No template DNA control 

5 Conventional Control 

6 MON 88701 

7 PV–GHHT6997 

8 No template DNA control 

9 Conventional Control 

10 MON 88701 

11 No template DNA control 

12 1 Kb DNA Ladder 
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IV.D. PCR and DNA Sequence Analyses to Examine the MON 88701 Insertion Site 

PCR and sequence analyses were performed on genomic DNA extracted from 

MON 88701 and the conventional control to examine the MON 88701 insertion site. The 

PCR was performed with a forward primer specific to the genomic DNA sequence 

flanking the 5′ end of the insert paired with a reverse primer specific to the genomic DNA 

sequence flanking the 3′ end of the insert (Figure IV-7). The amplified PCR product 

from the conventional control was subjected to DNA sequence analysis. Alignments 

between the conventional control sequence obtained from this analysis and the sequences 

immediately flanking the 5′ and 3′ end of the MON 88701 insert were separately 

performed to determine the integrity and genomic organization of the insertion site in 

MON 88701. The alignment analyses indicated a 123 base pair deletion from the 

conventional genomic DNA occurred upon T-DNA insertion in MON 88701. Minor 

deletions and/or insertions of DNA due to double-strand break repair mechanisms in the 

plant during Agrobacterium-mediated transformation process are not uncommon

(Salomon and Puchta, 1998). 
Monsanto Company 12-CT-244U 68 of 620 

Figure IV-7. PCR Amplification of the MON 88701 Insertion Site in Conventional 

Control 

PCR was performed on both conventional control genomic DNA and MON 88701 

genomic DNA, using Primer A specific to the 5′ flanking sequence and Primer B specific 

to the 3′ flanking sequence of the insert in MON 88701, to generate DNA fragments for 

sequence analysis. The insertion site in the conventional control (top) and MON 88701 

(bottom) are illustrated at the bottom of the figure. Approximately five microliters of 

each of the PCR reactions were loaded on the gel. Arrows on the agarose gel photograph 

denote the size of the DNA, in kilobase pairs, obtained from 1 Kb DNA Ladder on the 

ethidium bromide stained gel. Lane designations are as follows: 

Lane Description

1 1 Kb DNA Ladder 

2 Conventional Control 

3 MON 88701 

4 No template DNA control 

5 1 Kb DNA Ladder 

0.5

1.0

1.6

2.0

0.5

1.0

1.6

2.0

1 2 3 4 5

Primer A Primer B

Primer A

B-Left Border Region r1

L-TEV

CS-dmo

T-E6

P-PC1SV

5' Flank 3' Flank TS-CTP2 P-e35S L-Hsp70 CS-bar T-nos

Primer B
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IV.E. Southern Blot Analysis to Examine Insert Stability in Multiple Generations of 

MON 88701 

In order to demonstrate the stability of the insert in MON 88701, Southern blot analysis 

was performed using genomic DNA extracted from leaf tissues from five breeding 

generations of MON 88701. For reference, the breeding history of MON 88701 is 

presented in Figure IV-8. The specific generations tested are indicated in the legend of 

Figure IV-8. The R3 generation was used for the molecular characterization analyses 

shown in Figure IV-2 through Figure IV-5. To analyze insert stability, four samples from 

four additional generations of MON 88701 were evaluated by Southern blot analysis and 

compared to the R3 generation. Genomic DNA, isolated from each of the selected 

generations of MON 88701, was digested with the restriction enzyme Bcl I and 

simultaneously hybridized with Probe 2 and Probe 4 (Figures III-1 and IV-1), which was 

designed to detect both fragments generated by the Bcl I digest. Any instability 

associated with the insert would be detected as extra bands within the fingerprint on the 

Southern blot. The Southern blot has the same controls as described in Section IV.A.2. 

IV.E.1. T-DNA Probe 2 and 4 

Conventional control genomic DNA digested with restriction enzyme Bcl I and 

simultaneously hybridized with Probe 2 and Probe 4 (Figures III-1 and IV-1) produced 

no hybridization signals (Figure IV-9, Lane 1) as expected for the negative control. 

Conventional control genomic DNA digested with Bcl I and spiked with the 

PV-GHHT6997 DNA, previously digested with the restriction enzyme Pci I (Figure III-1 

and Table IV-1), produced one expected band at ~6.2 kb (Figure IV-9, Lane 2). 

Conventional control genomic DNA digested with Bcl I and spiked with probe templates 

of Probe 2 and Probe 4 produced the expected bands at ~1.0 kb and ~0.8 kb (Figure IV-9, 

Lane 3 and Lane 4). Detection of the positive controls indicates that the probes 

hybridized to their target sequences. 

MON 88701 genomic DNA digested with Bcl I and hybridized with Probe 2 and Probe 4 

(Figures III-1 and IV-1) is expected to produce a Southern fingerprint with two bands at 

~3.5 kb and ~2.4 kb (Figure IV-1 and Table IV-1). Southern fingerprints produced from 

multiple generations (Figure IV-9, Lane 5, Lanes 7-9) of MON 88701 are consistent with 

the one produced from the fully characterized generation R3 (Figure IV-3, Lane 2 and 

Lane 9, and Figure IV-9, Lane 6), indicating that MON 88701 contains one copy of the 

T-DNA insert that is stable across multiple generations. 
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Figure IV-8. Breeding History of MON 88701 

R0 corresponds to the original transformed cotton plant.  designates self-pollination. 

The R3 generation was used for the molecular characterization and commercial 

development of MON 88701. The R2, R3, R4, R5, and R6 generations of MON 88701 

were used to analyze the stability of the insert across generations. The R5 generation was 

used for protein expression in tissues other than seed and for agronomic, phenotypic, and 

environmental interaction analyses. The R6 generation was used for protein expression in 

seed and for composition analysis. 
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Figure IV-9. Southern Blot Analysis to Examine Insert Stability in Multiple 

Generations of MON 88701: Probes 2 and 4 

The blot was simultaneously hybridized with two 32P-labeled probes that span a portion 

of the T-DNA sequence (Figure III-1, Probe 2 and Probe 4). Each lane contains 

approximately 10 μg of digested genomic DNA. Arrows denote the size of the DNA, in 

kilobase pairs, obtained from 1Kb DNA Extension Ladder on the ethidium bromide 

stained gel. Lane designations are as follows: 

Lane 

1 Conventional control (Bcl I) 

2 Conventional control (Bcl I) spiked with PV-GHHT6997 (Pci I) [~1.0 genome equivalent] 

3 Conventional control (Bcl I) spiked with Probe 2 and Probe 4 template [~1.0 genome 

equivalent] 

4 Conventional control (Bcl I) spiked with Probe 3 and Probe 4 template [~0.1 genome 

equivalent] 

5 MON 88701 (R2) (Bcl I) 

6 MON 88701 (R3) (Bcl I) 

7 MON 88701 (R4) (Bcl I) 

8 MON 88701 (R5) (Bcl I)) 

9 MON 88701 (R6) (Bcl I) 
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IV.F. Inheritance of the Genetic Insert in MON 88701 

The MON 88701 T-DNA resides at a single locus within the cotton genome and is 

inherited according to Mendelian principles of inheritance. During development of 

MON 88701, phenotypic and genotypic segregation data were recorded to assess the 

inheritance and stability of the MON 88701 T-DNA using Chi-square (χ

2

) analysis over 

several generations. The χ

2 analysis is based on comparing the observed segregation ratio 

to the expected segregation ratio according to Mendelian principles. 

The MON 88701 breeding path for generating pollinated segregation data is described in 

Figure IV-10. The transformed R0 plant was self-pollinated to generate R1 seed. The 

segregating R1 generation was assessed using Real-Time TaqMan analysis for the dmo

coding region. A single homozygous positive R1 plant was selected and self-pollinated to 

give rise to R2 plants that were self-pollinated to produce R3 seed. Phenotypic and 

genotypic assays confirmed the lack of insert segregation in these self-pollinated 

generations. 

Homozygous positive R3 plants were crossed to a Monsanto proprietary cotton inbred, 

which does not contain the dmo or bar coding sequence, via traditional breeding 

techniques to produce hemizygous F1 seed. The F1 plants, hemizygous for the dicamba 

and glufosinate tolerant trait, were crossed with a Monsanto proprietary cotton inbred, 

which does not contain the dmo or bar coding sequence, to produce BC1F1 seed. The 

BC1F1 generation was assessed using a glufosinate herbicide application to select for 

plants containing the MON 88701 T-DNA. The plants that survived the herbicide 

application were confirmed to be hemizygous for the MON 88701 T-DNA using an 

event-specific End-Point TaqMan analysis. The hemizygous BC1F1 plants were 

self-pollinated to produce the BC1F2 plants. For the BC1F2 generation, the plants were 

assessed using a glufosinate herbicide application and the surviving plants were assessed 

using an event-specific End-Point TaqMan analysis for the MON 88701 T-DNA. 

The inheritance of the MON 88701 T-DNA was assessed in the R1, BC1F1, and BC1F2

generations. At the BC1F1 generation, the MON 88701 T-DNA was predicted to 

segregate at a 1:1 ratio (hemizygous: homozygous negative) according to Mendelian 

inheritance principles. At the R1 and BC1F2 generations, the MON 88701 T-DNA was 

predicted to segregate at a 1:2:1 ratio 

(homozygous positive: hemizygous: homozygous negative) according to Mendelian 

inheritance principles. 

A Chi-square (χ

2

) analysis was used to compare the observed segregation ratios of the 

MON 88701 T-DNA to the expected ratios. The Chi-square (χ

2

) analysis used the 

statistical program R Version 2.12.0 (2010-10-15). 

The Chi-square was calculated as: 

χ 2

 = ∑ [( | o – e | )2 / e] 
Monsanto Company 12-CT-244U 73 of 620 

where o = observed frequency of the genotype or phenotype and e = expected frequency 

of the genotype or phenotype. The level of statistical significance was predetermined to 

be 5% (α = 0.05). 

The results of the χ

2

 analysis of the MON 88701 segregating progeny are presented in 

Table IV-3 and Table IV-4. The χ

2

 value in the BC1F1 generation indicated no 

statistically significant difference between the observed and expected 1:1 segregation 

ratio (hemizygous: homozygous negative) of the MON 88701 T-DNA. The χ

2

 value for 

the R1 and BC1F2 generations indicated no statistically significant difference between the 

observed and expected 1:2:1 segregation ratio 

(homozygous positive: hemizygous: homozygous negative) of MON 88701 T-DNA. 

These results support the conclusion that the MON 88701 T-DNA resides at a single 

locus within the cotton genome and is inherited according to Mendelian principles of 

inheritance. These results are also consistent with the molecular characterization data 

indicating that MON 88701 contains a single intact copy of the dmo and bar expression 

cassettes inserted at a single locus in the cotton genome. 
Monsanto Company 12-CT-244U 

Figure IV-10. Breeding Path for Generating Segregation Data for MON 88701 *Chi-square analysis was conducted on segregation data from the R1, BC1F1, and BC1F2 generations (bolded tex

†The cotton line used in the cross that did not contain the dmo or bar genes is a Monsanto proprietary cotton inbr =Self- Pollinated 
Monsanto Company 12-CT-244U 

Table IV-3. Segregation of the T-DNA During the Development of MON 88701: 1:1 Segregation 

 1:1 Segregation 

Generation Total Plants 

Observed # 

Plants 

Hemizygous 

Observed # 

Plants 

Homozygous 

Negative 

Expected # 

Plants 

Hemizygous 

Expected # 

Plants

Homozygous 

Negative 

χ 2 Probability2

BC1F1

1 261 123 138 130.5 130.5 0.862 0.3532 

1 Segregation was evaluated using a glufosinate herbicide application followed by End-Point TaqMan analysis for the MON 88701 inse2 Chi-square analysis was performed to analyze the segregation ratios (p 

≤ 0.05).

Table IV-4. Segregation of the T-DNA During the Development of MON 88701: 1:2:1 Segregation 

1:2:1 Segregation 

Generation 

Total 

Plants 

Observed # 

Plants 

Homozygous 

Positive 

Observed # 

Plants 

Hemizygous 

Observed # 

Plants 

Homozygous 

Negative 

Expected # 

Plants

Homozygous 

Positive

Expected # 

Plants 

Hemizygous

Expected # 

Plants

Homozygou

Negative 

R1

1 173 33 99 41 43.25 86.50 43.25 

BC1F2

2 118 36 56 26 29.50 59.00 29.50 

1 Segregation was evaluated using Real-Time TaqMan analysis for the dmo coding region. 2 Segregation was evaluated using a glufosinate herbicide application followed by End-Point TaqMan analysis for the MON 88701 inse3 Chi-square analysis was performed to analyze the segregation ratios (p 

≤ 0.05). 
Monsanto Company 12-CT-244U 76 of 620 

IV.G. Genetic Modification Characterization Conclusion 

Molecular characterization of MON 88701 by Southern blot analyses confirmed that the 

T-DNA was inserted into the cotton genome at a single locus containing one copy of the 

dmo and bar expression cassettes. No backbone DNA sequences from PV-GHHT6997 

were detected in MON 88701. 

PCR and DNA sequence analyses performed on MON 88701 and the conventional 

control determined the following: the complete DNA sequence of the insert and the DNA 

sequences flanking the 5′ and 3′ ends of the insert in MON 88701; the organization of the 

genetic elements within the insert; and the 5′ and 3′ insert-to-genomic DNA junctions. 

The PCR and DNA sequence analysis also determined the DNA sequence at the insertion 

site in the conventional control and identified a rearrangement (123 base pair deletion) 

that occurred at the insertion site in MON 88701. Minor deletions and/or insertions of 

DNA due to double-strand break repair mechanisms in the plant during 

Agrobacterium-mediated transformation process are not uncommon (Salomon and 

Puchta, 1998). 

Southern blot analysis of multiple MON 88701 generations demonstrated that the 

inserted DNA has been stably maintained through five generations of breeding, thereby, 

confirming the stability of the insert. Results from segregation analyses show inheritance 

and stability of the insert was as expected across multiple generations, which 

corroborates the molecular insert stability analysis and establishes the genetic behavior of 

the T-DNA in MON 88701 at a single chromosomal locus. 
Monsanto Company 12-CT-244U 77 of 620 

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE 

MON 88701 DMO AND PAT (bar) PROTEINS PRODUCED IN MON 88701 

Characterization of the introduced protein(s) in a biotechnology-derived crop is important 

to establishing food, feed, and environmental safety. As described in Section IV, 

MON 88701 contains dmo and bar expression cassettes that, when transcribed and 

translated, result in the expression of the MON 88701 DMO and PAT (bar) proteins, 

respectively. This section summarizes: 1) the identity and function of the MON 88701 

DMO and PAT (bar) proteins produced in MON 88701; 2) the demonstration of 

equivalence between the plant-produced and E. coli-produced proteins, which were used 

in various protein safety studies; 3) the expression levels of the MON 88701 DMO and 

PAT (bar) proteins in MON 88701 plant tissues; 4) the assessment of the potential 

allergenicity of the MON 88701 DMO and PAT (bar) proteins produced in MON 88701; 

and 5) the food, feed, and environmental safety assessment of the MON 88701 DMO and 

PAT (bar) proteins produced in MON 88701. The data support a conclusion that these 

two proteins produced in MON 88701 are safe for the environment and human or animal 

consumption based on several lines of evidence summarized below. These data were 

supplied to FDA for their evaluation in consultation BNF No. 135 on the food and feed 

safety and compositional assessment of MON 88701. 

V.A. Identity and Function of the MON 88701 DMO and PAT (bar) Proteins from 

MON 88701 

V.A.1. Mode-of-Action of DMO and MON 88701 DMO 

Wild-type DMO was initially purified from Stenotrophomonas maltophilia (S. 

maltophilia) strain DI-6, isolated from soil at a dicamba manufacturing plant (Krueger et 

al., 1989). DMO is an enzyme that catalyzes the demethylation of dicamba to the nonherbicidal compound DCSA and formaldehyde (Chakraborty et al., 2005). DMO is a 

Rieske-type non-heme iron oxygenase, that is part of a three component system 

comprised of a reductase, a ferredoxin, and a terminal oxygenase, in this case the DMO. 

These three proteins work together in a redox system similar to many other oxygenases to 

transport electrons from nicotinamide adenine dinucleotide (NADH) to oxygen and 

catalyze the demethylation of an electron acceptor substrate, in this case dicamba 

(Behrens et al., 2007). This three component redox system is presented in Figure V-1. 
Monsanto Company 12-CT-244U 78 of 620 

Figure V-1. Three Components of the DMO Oxygenase System 

The crystal structure of a DMO has been solved (D'Ordine et al., 2009; Dumitru et al., 

2009) and shows that the DMO monomers contain a Rieske [2Fe-2S] cluster domain and 

a non-heme iron center domain typical of all Rieske-type mono-oxygenases (Ferraro et 

al., 2005). To catalyze the demethylation of dicamba, electrons transferred from NADH 

are shuttled through an endogenous reductase and ferredoxin to the terminal DMO. The 

electrons are received by the Rieske [2Fe-2S] cluster on one DMO monomer and 

transferred to the non-heme iron center at the catalytic site of an adjacent monomer 

(D'Ordine et al., 2009; Dumitru et al., 2009), where it reductively activates oxygen to 

catalyze the final demethylation of dicamba. As a result of the reaction, 

3,6-dichlorosalicylic acid (DCSA) and formaldehyde are formed. DCSA is a known 

cotton, soybean, soil, and livestock metabolite whose safety has been evaluated by the 

EPA (FAO-WHO, 2011a; 2011b; U.S. EPA, 2009). Formaldehyde is found naturally in 

many plants at levels up to several hundred ppm (Adrian-Romero et al., 1999). An 

assessment of the safety and potential effects of the DMO reaction products is provided 

in Appendix C.1. 

V.A.1.1. Description of MON 88701 DMO 

DMO is targeted to chloroplasts for co-localization with the endogenous reductase and 

ferredoxin enzymes that supply electrons for the DMO demethylation reaction as 

described by Behrens et al. (2007). In the construction of the plasmid vector used in the 

development of MON 88701, PV-GHHT6997, a transit peptide coding sequence (CTP2, 

Table IV-2) was joined to the dmo coding sequence; this coding sequence results in the 

production of a precursor protein consisting of the DMO protein and an additional 76 

amino acids at the N-terminus of the protein. These additional amino acids correspond to 

the chloroplast transit peptide (CTP) from Arabidopsis thaliana EPSPS (CTP2), which is 

incorporated to improve the targeting of the precursor protein to the chloroplast 

(Herrmann, 1995; Klee et al., 1987). Typically, transit peptides are precisely removed 

from the precursor protein following delivery to the targeted plastid (Della-Cioppa et al., 

1986) resulting in the full-length protein. However, there are examples in the literature of 

alternatively processed forms of a protein targeted to a plant’s chloroplast (Behrens et al., 

2007; Clark and Lamppa, 1992). Such alternative processing is observed with the 

MON 88701 DMO protein produced in MON 88701. 

Reductase

(oxidized)

Flavin

Reductase

(reduced)

Ferredoxin

(reduced)

[2Fe-2S]

Ferredoxin

(oxidized)

NADH

+

H+

NAD+

Oxygenase

(oxidized)

[2Fe-2S]

Fe+2

Oxygenase

(reduced)

O2 O OH

OCH3

Cl

Cl

O OH

OH

Cl

Cl

3,6 DCSA

Dicamba

+ O

H H

Dicamba

Mono-Oxygenase

Dicamba

Mono-Oxygenase

Formaldehyde
Monsanto Company 12-CT-244U 79 of 620 

Analysis of cottonseed extracts from MON 88701 determined that the expressed protein 

had an apparent molecular weight of 39.5 kDa and corresponded to the DMO protein 

with nine amino acids on the N-terminus originating from the EPSPS chloroplast transit 

peptide. Except for the 9 amino acids derived from the CTP2 and an additional leucine at 

position two, the MON 88701 DMO protein has an identical sequence to the wild-type 

DMO protein from the DI-6 strain of S. maltophilia (Herman et al., 2005). The 

differences in the amino acid sequence between the wild-type DMO protein and 

MON 88701 DMO protein are not expected to have an effect on structure, activity, or 

specificity because the N-terminus and position two are sterically distant from the 

catalytic site (D'Ordine et al., 2009; Dumitru et al., 2009). The DMO protein produced in 

MON 88701 is hereinafter referred to as MON 88701 DMO protein. Accordingly, the 

DMO protein produced from E. coli with the same sequence as MON 88701 DMO is 

referred to as E. coli-produced MON 88701 DMO protein. 

As described previously the active form of DMO is a trimer (Chakraborty et al., 2005; 

Dumitru et al., 2009). For MON 88701 DMO to be functionally active and confer 

dicamba tolerance to MON 88701, a trimeric structure is required. The activity of 

MON 88701 DMO was confirmed during characterization (Section V.B and 

Appendix C). 

V.A.1.2. Specificity of MON 88701 DMO 

The substrate specificity of MON 88701 DMO was evaluated to understand potential 

interactions DMO may have with potential substrates present in MON 88701 cotton. The 

literature indicates the specificity of DMO for dicamba is due to the specific interactions 

that occur at the catalytic site (D'Ordine et al., 2009; Dumitru et al., 2009). Dicamba 

interacts with amino acids in the catalytic site of DMO through both the carboxylate 

moiety and the chlorine atoms of dicamba, which are primarily involved in orienting the 

substrate in the catalytic site. These chlorine atoms are required for catalysis (D'Ordine 

et al., 2009; Dumitru et al., 2009). Given the limited existence of chlorinated compounds 

with structures similar to dicamba in plants and other eukaryotes (Wishart, 2010; Wishart 

et al., 2009), it is unlikely that MON 88701 DMO will catalyze the conversion of other 

endogenous substrates. 

The potential for MON 88701 DMO to metabolize endogenous plant substrates was 

evaluated through in vitro experiments using a purified N-terminal histidine tagged DMO 

that was identical to wild-type DMO, except for a histidine tag at the N-terminus added to 

aid in protein purification. A comparison of DMO versions is shown in Appendix C, 

Figure C-1. A set of potential endogenous substrates was selected for evaluation based 

on structural similarity of the compounds to dicamba and their presence in cotton, corn, 

and soybean (Buchanan et al., 2000; Janas et al., 2000; Lege et al., 1995; Schmelz et al., 

2003). The potential substrates tested were o-anisic acid (2-methoxybenzoic acid), 

vanillic acid (4-hydroxy-3-methoxybenzoic acid), syringic acid 

(3,5-dimethoxy-4-hydroxybenzoic acid), ferulic acid [3-(4-hydroxy-3-methoxy-phenyl) 

prop-2-enoic acid] and sinapic acid [3-(4-hydroxy-3,5-dimethoxyphenyl)prop-2-enoic 

acid] (Figure V-2). The assay mixture included NADH, reductase, ferredoxin and DMO. 

Dicamba was first used as a positive control to demonstrate that the assay system was 
Monsanto Company 12-CT-244U 80 of 620 

functional. The disappearance of potential substrates and the formation of potential 

oxidation products were monitored using LC-UV and LC-MS (Appendix C). None of the 

tested substrates, except dicamba, were metabolized by the histidine tagged DMO in 

these in vitro experiments. To assess whether MON 88701 DMO protein has the same 

specificity as the histidine tagged DMO used in the in vitro experiments, the 

E. coli-produced MON 88701 DMO protein (i.e., lacking a histidine), shown to be 

equivalent to the plant produced MON 88701 DMO protein (Section V.B), was incubated 

with o-anisic acid, the endogenous compound that has the greatest structural similarity to 

dicamba. Again dicamba was used as a positive control to demonstrate the assay system 

was functional. This analysis demonstrated that o-anisic acid was not metabolized by the 

E. coli-produced MON 88701 DMO protein (i.e., lacking a histidine), but dicamba was. 

These results indicate that DMO, including the MON 88701 DMO protein, is specific for 

dicamba as a substrate (See Section V.E.1.3 and Appendix C.3.2 for additional details). 

Figure V-2. Dicamba and Potential Endogenous Substrates Tested through In Vitro

Experiments with DMO 

The arrow indicates methyl group removed by DMO. 

The possibility that MON 88701 DMO can metabolize exogenous substrates was tested 

through in vivo greenhouse experiments. In addition to dicamba, nine other herbicides, 

representing eight families with distinct modes-of-action, some of which are approved for 

use in cotton, were tested with MON 88701 and the conventional control (Table V-1). 

Each herbicide was applied at two spray rates that are representative of potential 

commercial rates needed to control broadleaf weeds. Herbicides were applied 

preemergence or at the 2 to 5 leaf plant growth stage and plants were scored with a visual 

rating based on the amount of injury observed. Across all of the herbicides tested, 

MON 88701 and the conventional control were similar in their level of injury, indicating 

that these herbicides do not serve as a substrate for MON 88701 DMO (Appendix C.3.). 

Cl

Cl OCH3

COOH

Dicamba

OCH3

CH=CHCOOH

OH

OCH3 H3CO

CH=CHCOOH

OH

OCH3

COOH

0-Anisic acid Vanillic acid Syringic acid ferulic acid sinapic acid

OCH3

COOH

OH

OCH3 H3CO

COOH

OH

Dicamba o-Anisic Acid Vanillic Acid Syringic Acid Ferulic Acid Sinapic Acid
Monsanto Company 12-CT-244U 81 of 620 

Table V-1. Herbicides Applied to MON 88701 and Conventional Control 

Herbicide Active 

Ingredient Herbicide Chemical Family (Mode-of-Action)1

Dicamba Benzoic (Synthetic Auxin) 

2,4-D Phenoxycarboxylic acid (Synthetic Auxin) 

2,4-DB Phenoxycarboxylic acid (Synthetic Auxin) 

Acetochlor Chloroacetamide (Inhibition of VLCFAs) 

Atrazine Triazine (Inhibition of Photosynthesis at Photosytem II) 

Oxyfluorfen Diphenylether (Inhibition of PPO) 

Halosulfuron Sulfonylurea (Inhibition of ALS) 

Trifluralin Dinitroaniline (Microtubule Assembly Inhibition) 

Paraquat Bipyridilium (Photosystem I electron diversion) 

Glyphosate Glycine (Inhibition of EPSP synthase) 

1

 (HRAC, 2009) 

2 2,4-D = 2,4-Dichlorophenoxyacetic acid; 2,4-DB = 4-(2,4-dichlorophenoxy) butyric acid. 

V.A.2. Mode-of-Action of PAT Proteins 

The mode-of-action for PAT protein has been extensively assessed, as numerous 

glufosinate-tolerant products including those in cotton, corn, soy, canola, sugarbeet and 

rice have been reviewed by the FDA and several other regulatory agencies (ILSI-CERA, 

2011; OECD, 1999a; 2002a). PAT, including the PAT (bar) protein produced in 

MON 88701, is an enzyme classified as an acetyltransferase which acetylates glufosinate 

to produce non-herbicidal N-acetyl glufosinate. Glufosinate is a racemic mixture of the 

D- and L- forms of phosphinothricin, though only the L-form has herbicidal activity. The 

herbicidal activity of glufosinate results from the binding of L-phosphinothricin to 

glutamine synthetase (OECD, 1999b; 2002a). Glutamine synthetase is responsible for 

the assimilation of ammonia generated during photorespiration. The binding of 

L-phosphinothricin to glutamine synthetase results in the inactivation of glutamine 

synthetase and a subsequent toxic build-up of ammonia within the plant, resulting in 

death of the plant (Manderscheid and Wild, 1986; OECD, 1999b; 2002a; Wild and 

Manderscheid, 1984). 

The PAT (bar) protein produced in MON 88701 acetylates the free amine group of Lphosphinothricin form of glufosinate to produce non-herbicidal N-acetyl glufosinate. 

The acetylated glufosinate is unable to bind to glutamine synthetase and therefore does 

not disrupt photorespiration and avoids the build-up of ammonia. Therefore, the 

production of PAT (bar) protein in MON 88701 confers glufosinate herbicide tolerance 

through this mechanism. 

V.A.2.1. Description of PAT (bar) 

Phosphinothricin N-acetyltransferase (PAT) proteins conferring tolerance to glufosinate 

herbicide (2-amino-4-(hydroxymethylphosphinyl) butanoic acid) have been isolated from 

two separate species of Streptomyces, S. hygroscopicus (Thompson et al., 1987) and S. 

viridochromogenes (Wohlleben et al., 1988). The PAT protein isolated from 
Monsanto Company 12-CT-244U 82 of 620 

S. hygroscopicus is encoded by the bar gene, and the PAT protein isolated from 

S. viridochromogenes is encoded by the pat gene. These PAT proteins are made up of 

183 amino acids with 85% identity at the amino acid level. Based on previous studies 

(Wehrmann et al., 1996) that have extensively characterized PAT proteins produced from 

bar and pat genes, OECD recognizes both proteins to be equivalent with regard to 

function and safety (OECD, 1999b). In addition, EPA has issued a tolerance exemption 

for PAT protein regardless of the encoding gene (U.S. EPA, 1997). The safety of PAT 

proteins present in biotechnology-derived crops has been extensively assessed (Hérouet 

et al., 2005; ILSI-CERA, 2011). 

The PAT protein produced in MON 88701 is from the bar gene, and for clarity, the PAT 

protein produced in MON 88701 will be referred to as PAT (bar). Analysis of cottonseed 

extracts from MON 88701 determined that the expressed protein corresponded to the 

183 amino acid polypeptide, resulting in a 24.1 kDa PAT (bar) protein. The activity of 

the PAT (bar) protein purified from MON 88701 cottonseed was confirmed during 

characterization (Appendix C.4.). 

V.A.2.2. PAT (bar) Specificity 

The PAT proteins, including PAT (bar), are highly specific for glufosinate in the 

presence of acetyl-CoA (Thompson et al., 1987; Wehrmann et al., 1996). While the 

herbicidal activity of glufosinate comes from the L-amino acid form, other L-amino acids 

are unable to be acetylated by PAT protein and competition assays containing 

glufosinate, high concentrations of other amino acids and PAT showed no inhibition of 

glufosinate acetylation (Wehrmann et al., 1996). Furthermore, L-glutamate, an analogue 

of glufosinate, also showed no inhibition of glufosinate acetylation in competition assays 

(Wehrmann et al., 1996). In addition, the PAT (bar) protein has more than 30-fold 

higher affinity towards L-phosphinothricin over other analogues (Thompson et al., 1987). 

Thus, the PAT (bar) protein has high substrate specificity for L-phosphinothricin, the 

herbicidal component of glufosinate, and it is unlikely to affect the metabolic system of 

MON 88701 cotton. Numerous glufosinate-tolerant products including those in cotton, 

corn, soy, canola, sugarbeet, and rice have been reviewed with no concerns identified 

(ILSI-CERA, 2011). 

V.B. Characterization and Equivalence of MON 88701 DMO and PAT (bar) 

Proteins from MON 88701 

The safety assessment of crops derived through biotechnology includes characterization 

of the physicochemical and functional properties of the protein(s) produced from the 

inserted DNA, and confirmation of the safety of the protein(s). For the safety data 

generated using E. coli-produced protein(s) to be applied to plant-produced protein(s), the 

equivalence of the plant- and E. coli-produced proteins must be assessed. For 

MON 88701 the physicochemical and functional characteristics of the MON 88701 DMO 

and MON 88701-produced PAT (bar) proteins were determined and each was shown to 

be equivalent to its respective E. coli-produced protein. A summary of the analytical 

results for each protein are shown below and the details of the materials, methods, and 

results are described in Appendix C. 
Monsanto Company 12-CT-244U 83 of 620 

The MON 88701 DMO protein purified from cottonseed of MON 88701 was 

characterized and the equivalence of the physicochemical and functional properties 

between the MON 88701 DMO and the E. coli-produced MON 88701 DMO proteins was 

established using a panel of analytical tests: 1) the identity could not be confirmed by 

N-terminal sequence analysis; however, MALDI-TOF MS analysis of peptides derived 

from tryptic digested MON 88701 DMO established the N-terminal sequence of 

MON 88701 DMO; 2) MALDI-TOF MS analysis yielded peptide masses consistent with 

the expected peptide masses from the theoretical trypsin digest of the MON 88701 DMO 

sequence; 3) MON 88701 DMO protein was detected on a western blot probed with 

antibodies specific for DMO protein and the immunoreactive and physiochemical 

properties of the MON 88701 DMO and E. coli-produced MON 88701 DMO proteins 

were shown to be equivalent; 4) the electrophoretic mobility and apparent molecular 

weight of the MON 88701 DMO and E. coli-produced MON 88701 DMO proteins were 

shown to be equivalent; 5) glycosylation status of MON 88701 DMO and 

E. coli-produced MON 88701 DMO proteins were determined to be equivalent; and 

6) functional activity of the MON 88701 DMO and the E. coli-produced MON 88701 

DMO proteins were demonstrated to be equivalent. 

The MON 88701-produced PAT (bar) protein purified from cottonseed of MON 88701 

was characterized and the equivalence of the immunoreactive and physicochemical 

characteristics and functional activity between the MON 88701- and the E. coli-produced 

PAT (bar) proteins was established using a panel of analytical tests: 1) N-terminal 

sequence analysis of the MON 88701-produced PAT (bar) protein established identity; 

2) MALDI-TOF MS analysis yielded peptide masses consistent with the expected 

peptide masses from the theoretical trypsin digest of the MON 88701-produced 

PAT (bar) sequence; 3) MON 88701-produced PAT (bar) protein was detected on a 

western blot probed with antibodies specific for PAT (bar) protein and the 

immunoreactive properties of the MON 88701-produced and E. coli-produced PAT (bar) 

proteins were shown to be equivalent; 4) the electrophoretic mobility and apparent 

molecular weight of the MON 88701-produced and E. coli-produced PAT (bar) proteins 

were shown to be equivalent; 5) glycosylation status of MON 88701- and 

E. coli-produced MON 88701 PAT (bar) proteins were determined to be equivalent; and 

6) functional activity of the MON 88701- and E. coli-produced PAT (bar) proteins were 

demonstrated to be equivalent. 

Taken together, these data provide a detailed characterization of the MON 88701 DMO 

and PAT (bar) proteins and establish their respective equivalence to E. coli-produced 

MON 88701 DMO protein and E. coli-produced PAT (bar) protein. This equivalence 

justifies the use of the E. coli-produced proteins as test subtances in the protein safety 

studies. 

V.C. Expression Levels of MON 88701 DMO and PAT (bar) Proteins in 

MON 88701 

MON 88701 DMO and PAT (bar) protein levels in various tissues of MON 88701 

relevant to the risk assessment were determined by a validated enzyme-linked 

immunosorbent assay (ELISA). Tissues of MON 88701 were collected from four 
Monsanto Company 12-CT-244U 84 of 620 

replicate plots planted in a randomized complete block field design during the 2010 

growing season from the following eight field sites in the U.S.: Arkansas (ARTI), 

Georgia (GACH), Kansas (KSLA), Louisiana (LACH), North Carolina (NCBD), New 

Mexico (NMLC), South Carolina (SCEK), and Texas (TXPL). MON 88701 plots were 

treated at the 3-5 leaf stage with glufosinate herbicide at the label rate (0.5 lbs active 

ingredient [a.i.]/acre) and at the 6-10 leaf stage with dicamba herbicide at the proposed 

label rate (0.5 lbs acid equivalent [a.e.]/acre). The field sites were representative of 

cotton-producing regions suitable for commercial production. Seed, pollen, root, and 

overseason leaf (OSL-1 through OSL-4) tissue samples were collected from each 

replicated plot at all field sites. 

V.C.1. Expression Levels of MON 88701 DMO Protein 

MON 88701 DMO protein levels were determined in all seven tissue types. The results 

obtained from ELISA are summarized in Table V-2 and the details of the materials and 

methods are described in Appendix D. Due to a limited amount of tissue, moisture 

content was not measured for pollen; therefore, pollen is reported on a fresh weight (fw) 

basis only. MON 88701 DMO protein levels in MON 88701 across tissue types ranged 

from <LOD to 410 μg/g dw. The mean MON 88701 DMO protein levels were 

determined across eight sites, with the exception of OSL-1 (7 sites) and OSL-4 (7 sites). 

Samples <LOD were not included in mean determinations. The mean MON 88701 DMO 

protein levels were highest in leaf (ranging from OSL-2 and OSL-3 at 240 μg/g dw, 

OSL-4 at 230 μg/g dw to OSL-1 at 180 μg/g dw), followed by root at 43 μg/g dw, seed at 

21 μg/g dw, and pollen at 14 μg/g fw. 
Monsanto Company 12-CT-244U 85 of 620 

Table V-2. Summary of MON 88701 DMO Protein Levels in Tissues from 

MON 88701 Grown in 2010 U.S. Field Trials 

Tissue1

Development 

Stage2

Days 

After 

Planting 

(DAP) 

MON 88701 

DMO 

Mean (SD) 

Range 

(μg/g fw)3

MON 88701 

DMO 

Mean (SD) 

Range 

(μg/g dw)4

LOQ/LOD5

(μg/g fw) 

 

OSL-1 2-4 leaf 14-25 27 (7.6) 180 (52) 0.168/0.313 

 13 – 42 110 – 280 

 

OSL-2 4-7 leaf 25-37 41 (12) 240 (69) 0.168/0.313 

 19 – 65 110 – 380 

OSL-3 9 leaf - Full flower 35-99 52 (17) 

24 – 97 

240 (75) 

91 – 410 

0.168/0.313 

 

OSL-4 Cutout – Full 

flower 

70-121 57 (18) 

0.70 – 91 

230 (59) 

2.8 – 310 

0.168/0.313 

 

Root 50% open flower – 

Full flower 

62-99 14 (3.7) 

8.2 – 21 

43 (12) 

26 – 72 

0.136/0.313 

 

Pollen 50% open flower – 

Full Flower 

68-99 14 (28) 

0.31 – 110 

NA (NA) 

NA 

0.043/0.125 

 

Seed Maturity 148-183 20 (4.6) 21 (5.0) 0.059/0.313 

 8.2 – 29 8.9 – 33 1

OSL= overseason leaf. Seed = black seed (ginned and delinted). 

2

The crop development stage each tissue was collected (Ritchie et al., 2007). 3

Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of 

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and 

maximum values) were calculated for each tissue across all sites (n=32, except OSL-3 n=31 due to one 

sample <LOD, OSL-1 and OSL-4 n=28 due to missed sample collections, and pollen n=29 due to two 

samples expressing <LOD and one being inconclusive). 

4

Protein levels are expressed as μg/g on a dry weight (dw) basis. The dry weight values were calculated by 

dividing the μg/g fw by the dry weight conversion factors obtained from moisture analysis data. NA= Not 

Applicable. 

5

LOQ=limit of quantitation; LOD=limit of detection.
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V.C.2. Expression Levels of PAT (bar) Protein 

PAT (bar) protein levels were determined in all seven tissue types. The results obtained 

from ELISA are summarized in Table V-3 and the details of the materials and methods 

are described in Appendix D. Due to a limited amount of tissue, moisture content was 

not measured for pollen; therefore, pollen is reported on a fresh weight (fw) basis only. 

PAT (bar) protein levels in MON 88701 across tissue types ranged from <LOQ to 

10 μg/g dw. The mean PAT (bar) protein levels were determined across eight sites, with 

the exception of OSL-1 (7 sites) and OSL-4 (7 sites). Samples <LOD were not included 

in mean determinations. The mean PAT (bar) protein levels were highest in seed at 

6.6 μg/g dw, followed by leaf (ranging from OSL-2 at 6.4 μg/g dw to OSL-4 at 3.2 μg/g 

dw), root at 1.8 μg/g dw, and pollen at 0.56 μg/g fw. 
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Table V-3. Summary of PAT (bar) Protein Levels in Tissues from MON 88701 

Grown in 2010 U.S. Field Trials 

Tissue1 

Development 

Stage2 

Days 

After 

Planting 

(DAP) 

PAT (bar) 

Mean (SD) 

Range 

(μg/g fw)3 

PAT (bar) 

Mean (SD) 

Range 

(μg/g dw)4 

LOD/LOQ5 

(μg/g fw) 

 

OSL-1 2-4 leaf 14-25 0.84 (0.21) 5.5 (1.5) 0.162/0.188 

 0.46 – 1.4 3.7 – 9.1 

 

OSL-2 4-7 leaf 25-37 1.1 (0.26) 6.4 (1.4) 0.162/0.188 

 0.68 – 1.6 3.8 – 9.4 

OSL-3 9 leaf – Full flower 35-99 1.0 (0.34) 4.8 (2.0) 0.162/0.188 

 0.34 – 1.7 1.3 – 10 

OSL-4 Cutout – Full 

flower 

70-121 0.78 (0.29) 

0.42 – 1.7 

3.2 (1.2) 

2.0 – 6.7 

0.162/0.188 

 

Root 50% open flowerFull flower 62-99 0.56 (0.18) 

0.27 – 0.89 

1.8 (0.75) 

0.93 – 3.3 

0.096/0.188 

 

Pollen 50% open flower – 

Full flower 

68-99 0.56 (0.24) 

0.27 – 0.90 

NA (NA) 

NA 

0.021/0.188 

 

Seed Maturity 148-183 6.1 (0.95) 6.6 (1.1) 0.032/0.188 

 4.8 – 8.8 5.2 – 9.6 1

OSL= overseason leaf. Seed = black seed (ginned and delinted). 

2

The crop development stage each tissue was collected (Ritchie et al., 2007). 3

Protein levels are expressed as the arithmetic mean and standard deviation (SD) as microgram (μg) of 

protein per gram (g) of tissue on a fresh weight basis (fw). The means, SD, and ranges (minimum and 

maximum values) were calculated for each tissue across all sites (n=32, except OSL-1 n=28 due to missed 

sample collections, OSL-4 n=27 due to missed sample collections and one sample expressing <LOD, 

OSL-3 n=31 due to one sample expressing <LOD, and pollen n=6 due to 26 samples expressing <LOQ). 

4

Protein levels are expressed as μg/g on a dry weight (dw) basis. The dry weight values were calculated by 

dividing the μg/g fw by the dry weight conversion factors obtained from moisture analysis data. NA= Not 

Applicable. 

5

LOQ=limit of quantitation; LOD=limit of detection. 

 
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V.D. Assessment of Potential Allergenicity of the MON 88701 DMO and PAT (bar) 

Proteins 

Assessing the potential allergenicity of the expressed proteins is less relevant to 

MON 88701 since only cottonseed oil and linters from cotton are used in food 

applications, which have undetectable or negligible amounts of total protein (Reeves and 

Weihrauch, 1979; Sims et al., 1996). Nonetheless, the allergenic potential of 

MON 88701 DMO and PAT (bar) proteins was assessed by comparing the biochemical 

characteristics of these introduced proteins to biochemical characteristics of known 

allergens (Codex Alimentarius, 2009). A protein is not likely to be associated with 

allergenicity if: 1) the protein is from a non-allergenic source; 2) the protein represents a 

very small portion of the total plant protein; 3) the protein does not share structural 

similarities to known allergens based on the amino acid sequence; and 4) the protein is 

rapidly digested in mammalian gastrointestinal systems. 

V.D.1. Assessment of Potential Allergenicity of the MON 88701 DMO Protein 

MON 88701 DMO has been assessed for its potential allergenicity according to the safety 

assessment guidelines described above, and conclusions were as follows. 

1) MON 88701 DMO originates from S. maltophilia, an organism that has not 

been reported to be a source of known allergens. 

2) MON 88701 DMO represents no more than 0. 008% of the total protein in the 

cottonseed of MON 887014

. Therefore, the MON 88701 DMO protein represents 

a very small portion of the total protein in the cottonseed of MON 88701 and due 

to the harsh conditions used in cottonseed processing is most likely absent in the 

oil and linters that are used for food production. 

3) Bioinformatics analyses demonstrated that the MON 88701 DMO does not 

share amino acid sequence similarities with known allergens and, therefore, is 

highly unlikely to contain immunologically cross-reactive allergenic epitopes. 

4) In vitro digestive fate experiments conducted with the MON 88701 DMO 

demonstrate that the proteins are rapidly digested in simulated gastric fluid (SGF) 

and in simulated intestinal fluid (SIF). 

Taken together, these data support the conclusion that MON 88701 DMO does not pose a 

significant allergenic risk. 

V.D.2. Assessment of Potential Allergenicity of the PAT (bar) Protein 

The non-allergenic nature of PAT (bar) protein is established in the scientific literature 

(Hérouet et al., 2005) and by the tolerance exemption set by U.S. EPA (1997). 

 

4

 % protein = (Mean level of protein expression (μg/g)/ Mean dry weight of total protein in seed μg/g) x 

100 % 
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Furthermore, the safety of PAT proteins, including the PAT (bar) protein produced in 

MON 88701, has been assessed extensively by regulatory agencies in 11 different 

countries for more than 38 biotechnology-derived events in eight different species (ILSICERA, 2011). In addition, potential allergenicity of PAT (bar) protein produced in 

MON 88701 has been assessed according to the safety assessment guidelines described 

above, and conclusions were as follows. 

1) PAT (bar) originates from S. hygroscopicus, an organism that has not been 

reported to be a source of known allergens. 

2) PAT (bar) represents no more than 0. 002% of the total protein in the 

cottonseed of MON 88701.5

 Therefore, the PAT (bar) protein represents a very 

small portion of the total protein in the cottonseed of MON 88701 and due to the 

harsh conditions used in cottonseed processing is most likely absent in the oil and 

linters that are used for food production. 

3) Bioinformatics analyses demonstrated that the PAT (bar) does not share amino 

acid sequence similarities with known allergens and, therefore, is highly unlikely 

to contain immunologically cross-reactive allergenic epitopes. 

4) In vitro digestive fate experiments conducted with the PAT (bar) demonstrate 

that the proteins are rapidly digested in simulated gastric fluid (SGF) and in 

simulated intestinal fluid (SIF). 

Taken together, these data support the conclusion that PAT (bar) does not pose a 

significant allergenic risk. 

V.E. Safety Assessment Summary of MON 88701 DMO and PAT (bar) Proteins in 

MON 88701 

Characterization of the introduced protein(s) in a biotechnology-derived crop product is 

important to establishing its food, feed, and environmental safety. This section 

summarizes: 1) the functionality of MON 88701 DMO and PAT (bar); 2) the 

characterization of MON 88701 DMO and PAT (bar); 3) the levels of MON 88701 DMO 

and PAT (bar) in plant tissues; 4) assessment of the potential allergenicity of 

MON 88701 DMO and PAT (bar); and 5) the food, feed, and environmental safety 

assessment of MON 88701 DMO and PAT (bar). The data support a conclusion that 

MON 88701 is safe for the environment and human or animal consumption based on 

several lines of evidence, all of which are summarized below. 

V.E.1. MON 88701 DMO Donor Organism, History of Safe Use, and Specificity 

Numerous factors have been considered in the safety assessment of MON 88701 DMO, 

which include but are not limited to donor organism safety, the safety of mono-

 

5

 % protein = (Mean level of protein expression (μg/g)/ Mean dry weight of total protein in seed μg/g) x 

100 % 
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oxygenases, and MON 88701 DMO protein specificity. A comprehensive food, feed, and 

environmental safety assessment of the MON 88701 DMO was conducted. The results 

are summarized below, along with the conclusions reached from the assessment. 

V.E.1.1. The dmo Donor Organism is Safe 

The dmo gene is derived from the bacterium Stenotrophomonas maltophilia (Palleroni 

and Bradbury, 1993). S. maltophilia is ubiquitous in the environment and is found 

associated with the rhizosphere of plants. S. maltophilia can be found in a variety of 

foods and feeds, and is widespread in the home environment (Berg et al., 1999; Denton 

and Kerr, 1998; Echemendia, 2010). Exposure to S. maltophilia is incidental to its 

presence in food. It has been isolated from “ready to eat” salads, vegetables, frozen fish, 

milk, and poultry (Qureshi et al., 2005; Ryan et al., 2009). S. maltophilia can be found in 

healthy individuals without causing any harm to human health (Denton et al., 1998) and 

infections caused by S. maltophilia are extremely uncommon (Cunha, 2010). Strains 

have been found in the transient flora of hospitalized patients as a commensal organism 

(Echemendia, 2010) and, similar to the indigenous bacteria of the gastrointestinal tract, 

S. maltophilia can be an opportunistic pathogen (Berg, 1996). As such, S. maltophilia is 

of low virulence in immuno-compromised patients where a series of risk factors (severe 

debilitation, the presence of indwelling devices such as ventilator tubes or catheters, for 

prolonged periods of time and prolonged courses of antibiotics) must occur for 

colonization by S. maltophilia in humans (Ryan et al., 2009). Therefore, infections by 

S. maltophilia almost exclusively occur in hospital settings, in which case they are only 

present in a minimal percentage of infections (Ryan et al., 2009). Finally, S. maltophilia

has not been reported to be source of allergens. 

The ubiquitous presence of S. maltophilia in the environment, the presence in healthy 

individuals without causing infections, the incidental presence in foods without any 

adverse safety reports, and the lack of reported allergenicity establishes the safety of the 

donor organism. 

V.E.1.2. MON 88701 DMO Protein Belongs to a Common Class of MonoOxygenases 

MON 88701 DMO is classified as an oxygenase. Oxygenases are enzymes that 

incorporate one or two oxygen atoms into substrates and are widely distributed in many 

universal metabolic pathways (Harayama et al., 1992). Within this large enzymatic class 

are mono-oxygenases that incorporate a single oxygen atom as a hydroxyl group with the 

concomitant production of water and oxidation of NAD(P)H (Harayama et al., 1992). 

Non-heme iron oxygenases, where iron is involved in the catalytic site, are an important 

class of oxygenases. Within this class are Rieske oxygenases, which contain a Rieske 

iron-sulfur [2Fe-2S] cluster. All Rieske non-heme iron oxygenases contain two catalytic 

domains, a non-heme iron domain (nh-Fe) that is a site of oxygen activation, and a 

Rieske [2Fe-2S] domain (Ferraro et al., 2005). MON 88701 DMO belongs to this class 

of oxygenases which are found in diverse phyla ranging from bacteria to plants (Ferraro 

et al., 2005; Schmidt and Shaw, 2001). 
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As discussed previously, the crystal structure of a DMO has been solved (D'Ordine et al., 

2009; Dumitru et al., 2009). The crystallography results demonstrated that, similar to all 

Rieske non-heme iron oxygenases, DMO contains two catalytically important and highly 

conserved domains; a mononuclear non-heme iron domain (nh-Fe) that is a site of 

oxygen activation, and a Rieske [2Fe-2S] domain (D'Ordine et al., 2009; Dumitru et al., 

2009; Ferraro et al., 2005). The amino acids binding the non-heme iron and those that 

constitute the Rieske [2Fe-2S] domain in the DMO protein are also highly conserved in 

these plant proteins, as is their spatial orientation (D'Ordine et al., 2009; Ferraro et al., 

2005). Rieske domains are ubiquitous in numerous bacterial and plant proteins like the 

iron-sulfur protein of the cytochrome bc1 complex, chloroplast cytochrome b6/f complex, 

and choline mono-oxygenases (Breyton, 2000; Darrouzet et al., 2004; Gray et al., 2004; 

Hibino et al., 2002; Rathinasabapathi et al., 1997; Russell et al., 1998). The presence of 

two conserved domains, a Rieske [2Fe-2S] domain and a mononuclear iron domain, 

suggests that all Rieske type non-heme iron oxygenases share the same reaction 

mechanism, by which the Rieske domain transfers electrons from the ferredoxin to the 

mononuclear iron to allow catalysis (Chakraborty et al., 2005; Dumitru et al., 2009; 

Ferraro et al., 2005). The structure and mechanistic homologies are further evidence of 

the evolutionary relatedness of all Rieske non-heme iron oxygenases to each other (Nam 

et al., 2001; Rosche et al., 1997; Werlen et al., 1996). Additionally, a FASTA alignment 

search of publicly available databases using the MON 88701 DMO protein sequence as a 

query yielded homologous sequences from many different species, predominantly 

bacteria, with amino acid sequence identity ranging up to approximately 42%. 

Alignments of MON 88701 DMO with plant proteins revealed homologous oxygenases 

present in crops such as canola (Brassica napus), corn (Zea mays), pea (Pisum sativum), 

rice (Orysa sativa), and soy (Glycine max), which were determined to have sequence 

identities up to approximately 27%. The highest homology was observed to proteins that 

are involved in chlorophyll metabolism. Chlorophyllide A oxygenase (Accession 

number: ACG42449) is Rieske-type oxygenase that is required for the formation of 

chlorophyll b, which is present in all plants (Tanaka et al., 1998). Pheophorbide A 

oxygenase (Accession number: ABD60316) is also a Rieske-type oxygenase that plays a 

key role in the overall regulation of chlorophyll degradation in plants (Rodoni et al., 

1997). Pheophorbide A oxygenase is constitutively present in all green tissues and, at 

slightly lower levels, in etiolated and non-photosynthetic tissues including seeds (Yang et 

al., 2004). As a Rieske-type oxygenase, Pheophorbide A oxygenase is expected to have 

high degree of secondary and tertiary structure homology to similar structural elements in 

DMO as described above. The presence of these conserved structural domains in these 

plant proteins is further evidence that exposure to a structural homolog of 

MON 88701 DMO has occurred through consumption of these crops. 

Therefore, MON 88701 DMO shares sequence identity and many catalytic domain 

structural similarities with a wide variety of oxygenases present in bacteria and plants 

currently widely prevalent in the environment and consumed, establishing that animals 

and humans are extensively exposed to these types of enzymes. 
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V.E.1.3. DMO Catalyzes a Specific Enzyme Reaction 

DMO converts dicamba to DCSA. This demethylation is very specific to dicamba, where 

both the carboxylate moiety and the chlorine atoms help position the substrate at the 

active site of the enzyme (D'Ordine et al., 2009; Dumitru et al., 2009). Crystallography 

studies of the substrate in the active site demonstrated that these chlorines function as 

steric “handles” that position the substrate in the proper orientation in the binding pocket 

(Dumitru et al., 2009). Potential substrates abundant in cotton (o-anisic acid, vanillic 

acid, syringic acid, ferulic acid and sinapic acid) that are structurally similar to dicamba, 

were not metabolized by an E. coli-produced N-terminal histidine DMO. In addition, E. 

coli-produced MON 88701 DMO did not metabolize o-anisic acid, the endogenous 

compound that has the greatest structural similarity to dicamba. These laboratory tests 

indicate that DMO, including MON 88701 DMO protein, is specific for dicamba (Section 

V.A.1.2). Given the limited amount of chlorinated metabolites with structures similar to 

dicamba in plants and other eukaryotes (Wishart, 2010; Wishart et al., 2009), it is 

unlikely that MON 88701 DMO will catalyze the conversion of other endogenous 

substrates. Therefore, the activity of the enzyme is specific for dicamba while it 

maintains many structural properties common to oxygenases that are ubiquitous to all 

organisms with a history of safe consumption. 

V.E.2. PAT (bar) Donor Organism, History of Safe Use, and Specificity 

The safety of PAT (bar) protein is established in the scientific literature (Hérouet et al., 

2005) and by the tolerance exemption set by the EPA (U.S. EPA, 1997). In addition, the 

safety of PAT proteins, including the PAT (bar) protein produced in MON 88701, has 

been assessed extensively by regulatory agencies in 11 different countries for more than 

38 biotechnology-derived events in eight different species (ILSI-CERA, 2011). The 

PAT (bar) protein expressed in MON 88701 has the same functional activity as the PAT 

proteins in all commercially available products that provide glufosinate tolerance in 

several crops, including cotton, corn, soybean, and canola. The lack of any documented 

reports of adverse effects of glufosinate tolerant crops since their introduction in 1995 

(Duke and Powles, 2009) further demonstrates the safety of PAT (bar) protein. 

Numerous factors have been considered in the safety assessment of PAT (bar), which 

include, but are not limited to, donor organism safety, the history of safe use, and PAT 

protein specificity. 

V.E.2.1. The bar Donor Organism is Safe 

S. hygroscopicus is a saprophytic, soil-borne bacterium with no known safety issues. 

Streptomyces species are widespread in the environment and present no known allergenic 

or toxicity issues (Kämpfer, 2006; Kutzner, 1981) though human exposure is quite 

common (Goodfellow and Williams, 1983). S. hygroscopicus is not considered 

pathogenic to plants, humans or other animals (Cross, 1989; Goodfellow and Williams, 

1983; Locci, 1989). The history of safe use of S. hygroscopicus is discussed previously 

(Hérouet et al., 2005), and this organism has been extensively reviewed during the 
Monsanto Company 12-CT-244U 93 of 620 

deregulation of several glufosinate-tolerant events with no safety or allergenicity issues 

identified. 

The ubiquitous presence of S. hygroscopicus in the environment, the widespread human 

exposure without any adverse safety or allergenicity reports, and the successive reviews 

resulting from the deregulation of several glufosinate-tolerant events with no safety or 

allergenicity issues identified establishes the safety of the donor organism. 

V.E.2.2. PAT Protein has a History of Safe Use 

The PAT (bar) protein expressed in MON 88701 is identical to the wild-type protein 

produced in S. hygroscopicus and is analogous to the PAT proteins in commercially 

available glufosinate-tolerant products in several crops including cotton, corn, soybean, 

and canola. Based on studies characterizing the kinetic and chemical mechanisms of 

PAT proteins (Wehrmann et al., 1996), OECD recognizes PAT proteins produced from 

different genes to be equivalent with regard to function and safety (OECD, 1999b). 

The safety of PAT protein present in biotechnology-derived crops has been extensively 

assessed (ILSI-CERA, 2011) and in 1997 a tolerance exemption was issued for PAT 

proteins by U.S. EPA (U.S. EPA, 1997). This exemption was based on a safety 

assessment that included rapid digestion in simulated gastric fluids, lack of significant 

homology to known toxins and known allergens, and lack of toxicity in an acute oral 

mouse gavage study. Numerous glufosinate-tolerant products including those in corn, 

soy, canola, sugarbeet and rice have been reviewed by the USDA and FDA with no 

concerns identified. Further, a comprehensive study on the safety of PAT proteins 

present in biotechnology-derived crops (Hérouet et al., 2005) demonstrated structural 

similarity only with other acetyltransferases known to not cause adverse effects after 

consumption, lack of sequence homology to know allergens and toxins, lack of 

glycosylation sites, rapid degradation in gastric and intestinal fluids, and no adverse 

effects in mice treated with high doses of PAT proteins. Hérouet et al. concluded that 

there is a reasonable certainty of no harm resulting from the inclusion of PAT proteins in 

human food or animal feed (2005). 

The history of safe use of PAT is supported by the lack of any documented reports of 

adverse effects related to this protein since the introduction of glufosinate-tolerant crops 

in 1995 (Duke and Powles, 2009). Since then, approvals have been issued by regulatory 

agencies of 11 different countries for the environmental release of greater than 38 

transformation events, including 8 different species of plants expressing the PAT protein 

(ILSI-CERA, 2011). 

V.E.2.3. PAT (bar) Catalyzes a Specific Enzyme Reaction 

The mode-of-action for PAT protein has been extensively assessed, as numerous 

glufosinate-tolerant products, including those in corn, soy, canola, sugarbeet, and rice, 

have been reviewed by the FDA and several other regulatory agencies (ILSI-CERA, 

2011; OECD, 1999b; 2002a). PAT, including the PAT (bar) protein produced in 

MON 88701, is an enzyme classified as an acetyltransferase which acetylates glufosinate 
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to produce non-herbicidal N-acetyl glufosinate. Glufosinate is a racemic mixture of the 

D- and L- forms of phosphinothricin. The herbicidal activity of glufosinate results from 

the binding of L-phosphinothricin to glutamine synthetase (OECD, 1999b; 2002a). 

Glutamine synthetase is responsible for the assimilation of ammonia generated during 

photorespiration. The binding of L-phosphinothricin to glutamine synthetase results in 

the inactivation of glutamine synthetase and a subsequent toxic build-up of ammonia 

within the plant, resulting in death of the plant (Manderscheid and Wild, 1986; OECD, 

1999b; 2002a; Wild and Manderscheid, 1984). 

The PAT (bar) protein produced in MON 88701 acetylates the free amine group of Lphosphinothricin form of glufosinate to produce non-herbicidal N-acetyl glufosinate. 

The acetylated glufosinate is unable to bind to glutamine synthetase and therefore does 

not disrupt photorespiration and avoids the build-up of ammonia. Therefore, the 

production of PAT (bar) protein in MON 88701 confers glufosinate herbicide tolerance 

through this mechanism. 

The PAT proteins, including PAT (bar), are highly specific for glufosinate in the 

presence of acetyl-CoA (Thompson et al., 1987; Wehrmann et al., 1996). While the 

herbicidal activity of glufosinate comes from the L-amino acid form, other L-amino acids 

are unable to be acetylated by PAT protein and competition assays containing 

glufosinate, high concentrations of other amino acids and PAT showed no inhibition of 

glufosinate acetylation (Wehrmann et al., 1996). Furthermore, L-glutamate, an analogue 

of glufosinate, also showed no inhibition of glufosinate acetylation in competition assays 

(Wehrmann et al., 1996). In addition, the PAT (bar) protein has more than 30-fold 

higher affinity towards L-phosphinothricin over other plant analogues (Thompson et al., 

1987). Thus, the PAT (bar) protein has high substrate specificity for L-phosphinothricin, 

the herbicidal component of glufosinate, and is unlikely to affect the metabolic system of 

MON 88701 cotton. Numerous glufosinate-tolerant products, including those in corn, 

soy, canola, sugarbeet, and rice have been reviewed with no concerns identified (ILSICERA, 2011). 

V.E.3. MON 88701 DMO and PAT (bar) Proteins in MON 88701 are Not 

Homologous to Known Allergens or Toxins 

Bioinformatics analyses were performed to assess the allergenic potential, toxicity, or 

biological activity of MON 88701 DMO and PAT (bar). The analysis demonstrated that 

neither protein shares amino acid sequence similarities with known allergens, gliadins, 

glutenins, or protein toxins which could have adverse effects to human or animal health 

(Section V.D). 

V.E.4. MON 88701 DMO and PAT (bar) Proteins in MON 88701 are Labile in in 

vitro Digestion Assays 

MON 88701 DMO and PAT (bar) were readily digestible in simulated gastric fluid 

(SGF) and simulated intestinal fluid (SIF). Rapid degradation of the MON 88701 DMO 

and PAT (bar) proteins in SGF and SIF makes it highly unlikely that either protein would 
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be absorbed in the small intestine and have any adverse effects on human or animal 

health. 

V.E.5. MON 88701 DMO and PAT (bar) Proteins in MON 88701 are Not Acutely 

Toxic 

Acute oral toxicology studies were conducted with MON 88701 DMO and PAT (bar) 

proteins individually. Results indicate that neither MON 88701 DMO or PAT (bar) 

caused any adverse effects in mice, with No Observable Adverse Effect Levels 

(NOAELs) for MON 88701 DMO at 283 mg/ kg bw and PAT (bar) at 1086 mg/kg bw, 

respectively, the highest doses tested. 

V.E.6. Human and Animal Exposure to the MON 88701 DMO and PAT (bar) 

Proteins 

Cottonseed is not consumed by humans because the majority of commercial cotton 

varieties contain the anti-nutrients gossypol and cyclopropenoid fatty acids. The primary 

human food currently produced from cottonseed is refined, bleached, and deodorized 

(RBD) oil, and to a smaller extent, linters. RBD oil contains undetectable amounts of 

protein (Reeves and Weihrauch, 1979); therefore, oil produced from MON 88701 will 

contain extremely low levels of MON 88701 DMO and PAT (bar) proteins. Linters are 

an industrial by-product of ginning, and can be consumed as a highly processed product 

composed of nearly pure (i.e., >99%) cellulose (NCPA, 2002; Nida et al., 1996). 

Cottonseed RBD oil and linters are processed fractions that contain undetectable or 

negligible amounts of protein there is minimal, if any, dietary exposure to MON 88701 

DMO and PAT (bar) proteins from consumption of foods derived from MON 88701. 

Therefore, MOE values were not calculated for the MON 88701 DMO or PAT (bar) 

proteins. Furthermore, the safety of PAT (bar) has been extensively assessed (Hérouet et 

al., 2005), several glufosinate-tolerant crops that produce PAT proteins have been 

reviewed by FDA and other regulatory agencies (ILSI-CERA, 2011) and in 1997 a 

tolerance exemption was issued for PAT proteins by U.S. EPA (1997). 

Estimated exposure of MON 88701 DMO and PAT (bar) proteins in animal feed were 

evaluated by calculating an estimate of daily dietary intake (DDI) for dairy cows. 

Exposure was calculated for the worst-case scenario, which assumes: 1) the source of 

cottonseed in the diet is cottonseed meal; 2) cottonseed meal is only derived from 

MON 88701 and contains no other cottonseed sources; 3) the protein expression level is 

the maximum expression level measured for each protein; and 4) no loss of protein due to 

heat. The maximum daily amount of MON 88701 DMO or PAT (bar) proteins 

consumed from MON 88701 would be for the dairy cow and would be 0.00043 g/kg of 

body weight for MON 88701 DMO and 0.000124 g/kg of body weight for PAT (bar). 

These values represent 0.007 and 0.002% of protein consumed, respectively. These very 

small levels of exposure of animals to MON 88701 DMO and PAT (bar) in their feed, in 

addition to the above mentioned safety data for both MON 88701 DMO and PAT (bar), 

support the conclusion that there is no risk to animal health when MON 88701 DMO or 

PAT (bar) are present in their diets. 
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V.F. MON 88701 DMO and PAT (bar) Protein Characterization and Safety 

Conclusion 

MON 88701 DMO is a Rieske-type mono-oxygenase that catalyzes the O-demethylation 

of the herbicide dicamba and has homologs in bacteria and plants that share many of the 

typical structural and functional characteristics of these types of oxygenases, while 

maintaining specificity for its substrate. The physicochemical characteristics of the 

MON 88701 DMO protein were determined and equivalence between MON 88701 DMO 

and E. coli-produced MON 88701 DMO proteins was demonstrated. This equivalence 

justifies the use of the E. coli-produced MON 88701 DMO as a test substances in the 

protein safety studies. Expression studies using ELISA demonstrated that MON 88701 

DMO was expressed at levels ranging from <LOD to 410 μg/g dw, representing a low 

percentage of the total protein. An assessment of the allergenic potential of the 

MON 88701 DMO protein supports the conclusion that the MON 88701 DMO protein 

does not pose a significant allergenic risk. In addition, the donor organism for the 

MON 88701 DMO coding sequence, S. maltophilia, is ubiquitous in the environment and 

is not commonly known for human or animal pathogenicity or allergenicity. The 

MON 88701 DMO protein lacks structural similarity to allergens, toxins or other proteins 

known to have adverse effects on mammals. The MON 88701 DMO protein is rapidly 

digested in simulated digestive fluids and demonstrates no oral toxicity in mice at the 

level tested. Based on the above information, the consumption of the MON 88701 DMO 

protein from MON 88701 or its progeny is considered safe for humans and animals 

PAT (bar) protein is an acetyltransferase that catalyzes the acetylation of the herbicide 

glufosinate. The PAT (bar) protein expressed in MON 88701 is analogous to the PAT 

proteins in all commercially available products that provide glufosinate tolerance in 

several crops including cotton, corn, soybean, and canola. PAT proteins, including the 

PAT (bar) protein isolated from MON 88701 have been previously characterized, and the 

safety of crops expressing these proteins has been well established. The data and 

information provided in this section further confirms the food and feed safety of the 

PAT (bar) protein in MON 88701. The physicochemical characteristics of the PAT (bar) 

protein were determined and equivalence between MON 88701-produced and 

E. coli-produced PAT (bar) proteins was demonstrated. This equivalence justifies the 

use of the E. coli-produced PAT (bar) as a test substance in the protein safety studies. 

Expression studies using ELISA demonstrated that MON 88701-produced PAT (bar) was 

expressed at levels ranging from <LOD to 10 μg/g dw, representing a low percentage of 

the total protein. An assessment of the allergenic potential of the PAT (bar) protein 

supports the conclusion that the PAT (bar) protein does not pose a significant allergenic 

risk. In addition, the donor organism for the PAT (bar) coding sequence, 

S. hygroscopicus, is ubiquitous in the environment and is not commonly known for 

human or animal pathogenicity, or allergenicity. The PAT (bar) protein lacks structural 

similarity to allergens, toxins or other proteins known to have adverse effects on 

mammals. The PAT (bar) protein is rapidly digested in simulated digestive fluids and 

demonstrates no oral toxicity in mice at the level tested. Based on the above information, 

the consumption of the PAT (bar) protein from MON 88701 or its progeny is considered 

safe for humans and animals. 
Monsanto Company 12-CT-244U 97 of 620 

The protein safety data presented herein support the conclusion that food and feed 

products containing MON 88701 or derived from MON 88701 are as safe as cotton 

products currently on the market for human and animal consumption. 
Monsanto Company 12-CT-244U 98 of 620 

VI. COMPOSITIONAL ASSESSMENT OF MON 88701 

Safety assessments of biotechnology-derived crops follow the comparative safety 

assessment process (Codex Alimentarius, 2009) in which the composition of grain and/or 

other raw agricultural commodities of the biotechnology-derived crop is compared to the 

appropriate conventional control that has a history of safe use. Compositional 

assessments are performed using the principles and analytes outlined in the OECD 

consensus document for cotton composition (OECD, 2009). 

A recent review of compositional assessments conducted according to OECD guidelines 

that encompassed a total of seven biotechnology-derived crop varieties, nine countries 

and eleven growing seasons concluded that incorporation of biotechnology-derived 

agronomic traits has had little impact on natural variation in crop composition. Most 

compositional variation is attributable to growing region, agronomic practices and 

genetic background (Harrigan et al., 2010). Compositional quality, therefore, implies a 

very broad range of endogenous levels of individual constituents. Numerous scientific 

publications have further documented the extensive variability in the concentrations of 

crop nutrients and anti-nutrients that reflect the influence of environmental and genetic 

factors as well as extensive conventional breeding efforts to improve nutrition, 

agronomics and yield (Reynolds et al., 2005). This observation extends to publications 

specific to cotton (Berberich et al., 1996; Hamilton et al., 2004; Nida et al., 1996). 

Compositional equivalence between biotechnology-derived and conventional crops 

supports an “equal or increased assurance of the safety of foods derived from genetically 

modified plants” (OECD, 2002b). The OECD consensus document on considerations for 

new varieties of cotton emphasize quantitative measurements of key nutrients and known 

anti-nutrients (OECD, 2009). This is based on the premise that such comprehensive and 

detailed analyses will most effectively discern any compositional changes that imply 

potential safety and nutritional concerns (e.g., anti-nutritional). Levels of the components 

in the seed of the biotechnology-derived crop are compared to: 1) corresponding levels in 

a conventional comparator, the genetically similar conventional line, grown concurrently, 

under the same field conditions; and 2) natural ranges generated from an evaluation of 

commercial reference varieties grown concurrently and from data published in the 

scientific literature. The comparison to data published in the literature places any 

potential differences between the assessed crop and its comparator in the context of the 

well-documented variation in the concentrations of crop nutrients and anti-nutrients. 

This section provides analyses of concentrations of key nutrients and anti-nutrients of 

cottonseed from MON 88701 treated with both dicamba and glufosinate compared to the 

conventional control grown and harvested under the same conditions, as appropriate. The 

analyses of concentrations of key nutrients and anti-nutrients of cottonseed from 

MON 88701 that was not treated with either dicamba or glufosinate are presented in 

Appendix E as supplemental information. In addition, conventional commercial cotton 

reference varieties were included in the composition analyses to establish a range of 

natural variability for each analyte, defined by a 99% tolerance interval. The production 

of materials for the compositional analyses used field designs to allow accurate 

assessments of compositional characteristics over a range of environmental conditions 
Monsanto Company 12-CT-244U 99 of 620 

under which MON 88701 is expected to be grown. The field trial design parameters 

included a sufficient number of trial sites to allow adequate exposure to the variety of 

conditions cotton plants typically encounter in nature. Field sites were replicated with an 

adequate number of plants sampled, and the methods of analysis were sufficiently 

sensitive and specific to detect variations in the components measured to allow 

statistically rigorous analyses. The information provided in this section also addresses 

the relevant factors in Codex Plant Guidelines, Section 4, paragraphs 44 and 45 for 

compositional analyses (Codex Alimentarius, 2009). 

VI.A. Compositional Equivalence of MON 88701 Cottonseed to Conventional 

Cotton 

Compositional analyses comparing MON 88701 treated with dicamba and glufosinate 

herbicides to the conventional control variety (Coker 130) and conventional commercial 

reference varieties demonstrated that MON 88701 is compositionally equivalent to 

conventional cotton. Samples of acid-delinted cottonseed were collected from 

MON 88701 and the conventional control grown in a 2010 U.S. field production. Nine 

unique conventional cotton varieties, known as reference substances, were included 

across all sites of the field production with four varieties per site to provide data on 

natural variability of each compositional component analyzed. The field production was 

conducted at eight sites: Arkansas (ARTI), Georgia (GACH), Kansas (KSLA), Louisiana 

(LACH), North Carolina (NCBD), New Mexico (NMLC), South Carolina (SCEK) and, 

Texas (TXPL). The sites were planted in a randomized complete block design with four 

blocks per site. All cotton plants, including MON 88701, the conventional control, and 

the reference varieties, were grown under normal agronomic field conditions for their 

respective geographic regions, including maintenance pesticides as needed. In addition, 

MON 88701 plots were treated at the 3-5 leaf stage with glufosinate herbicide at the label 

rate (0.5 lbs a.i./acre), and at the 6-10 leaf stage with dicamba herbicide at the label rate 

(0.5 lbs a.e./acre). 

Compositional analyses were conducted to assess whether levels of key nutrients and 

anti-nutrients in MON 88701 were equivalent to levels in the conventional control and 

comparable to the composition of conventional commercial reference varieties. A 

description of nutrients and anti-nutrients present in cotton is provided in the OECD 

consensus document on compositional considerations for cottonseed (OECD, 2009). 

Nutrients assessed in this analysis included proximates (ash, calories and carbohydrates 

by calculation, fat, moisture, and protein), acid detergent fiber (ADF), neutral detergent 

fiber (NDF), crude fiber (CF), total dietary fiber (TDF), amino acids (AA, 18 

components), fatty acids (FA, C8-C22), minerals (calcium, copper, iron, magnesium, 

manganese, phosphorus, potassium, sodium and zinc), and vitamin E. Methods used in 

the assessments of nutrients and anti-nutrients are found in Appendix E. In all, 65 

different analytical components were measured. Due to statistical constraints, in order to 

proceed with the statistical analysis of any component in this study, at least 50% of the 

observed values for that analyte needed to be greater than the assay limit of quantitation 

(LOQ). Of the 65 components measured, 13 had more than 50% of the observations 

below the assay LOQ and were excluded from statistical analysis. Therefore, 52 

components were statistically assessed using a mixed-model analysis of variance method. 
Monsanto Company 12-CT-244U 100 of 620 

Values for all components were expressed on a dry weight basis with the exception of 

moisture, expressed as percent fresh weight, and fatty acids, expressed as percent of total 

FA. 

For MON 88701, nine sets of statistical comparisons to the conventional control were 

conducted. One comparison was based on compositional data combined across all eight 

field sites (the combined-site analysis) and eight separate comparisons to the 

conventional control were conducted on data from each of the eight individual field sites. 

Statistically significant differences were identified at a 5% level of significance (p<0.05). 

Compositional data from the conventional commercial reference varieties, grown 

concurrently in the same trial as MON 88701 and the conventional control, Coker 130, 

were combined across all sites and used to calculate a 99% tolerance interval for each 

component to define the natural variability in cotton varieties that have a history of safe 

consumption. 

For the combined-site analysis, statistically significant differences (p < 0.05) in nutrient 

and anti-nutrient components were evaluated further using considerations relevant to the 

safety and nutritional quality of MON 88701 when compared to the conventional control. 

The evaluation included: 1) the relative magnitude of the significant difference in the 

mean values of nutrient and anti-nutrient components of MON 88701 compared to the 

conventional control; 2) whether the MON 88701 component mean values were within 

the range of natural variability of that component as represented by the 99% tolerance 

interval of commercial conventional reference varieties grown concurrently in the same 

trial; 3) analyses of the reproducibility of the significant combined-site component 

differences at individual sites; and 4) assessing the combined-site statistically significant 

differences and reproducible individual site significant differences within the context of 

natural variability of commercial cottonseed composition published in the scientific 

literature and/or in the International Life Sciences Institute Crop Composition Database 

(ILSI, 2011) (See Table VI-4). Statistical summaries of nutrients and anti-nutrients for 

individual sites are found in Appendix E. 

This analysis provides a comprehensive comparative assessment of the levels of key 

nutrients and anti-nutrients in cottonseed of MON 88701 and the conventional control 

discussed in the context of natural variability in composition of commercial cotton. 

Results of the comparison indicate that the composition of the cottonseed of 

MON 88701 is equivalent to that of conventional cotton. 

Compositional results from MON 88701 plots treated with dicamba and glufosinate label 

rates are summarized in the following subsections. Similar results were obtained for 

MON 88701 plots that were not treated with either dicamba or glufosinate, which are 

provided as additional information in Appendix E. 

VI.A.1 Nutrient Levels in Cottonseed 

In the combined-site analysis of nutrient levels in cottonseed, the following components 

had no statistically significant differences (p<0.05) in mean values between MON 88701 

and the conventional control: one proximate (protein), one type of fiber (crude fiber), 15 
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amino acids (alanine, aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, 

leucine, lysine, phenylalanine, serine, threonine, tryptophan, tyrosine, and valine), seven 

fatty acids (16:0 palmitic acid, 16:1 palmitoleic acid, 18:0 stearic acid, 18:1 oleic acid, 

18:3 linolenic acid, 20:0 arachidic acid, and 22:0 behenic acid), and four minerals 

(copper, iron, phosphorus, and sodium) (Table VI-1 and VI-2). 

The components that had significant differences in mean values between MON 88701 

and the conventional control in the combined-site analysis were: five proximates (ash, 

calories, carbohydrates, moisture, and total fat), three types of fiber (ADF, NDF, and 

TDF), three amino acids (arginine, methionine and proline), two fatty acids (14:0 

myristic acid and 18:2 linoleic acid), five minerals (calcium, magnesium, manganese, 

potassium, and zinc) and vitamin E (Table VI-1). 

The statistically significant differences in nutrients were further evaluated using the four 

previously described considerations relevant to the safety and nutritional quality of 

MON 88701 when compared to the conventional control: 

1) All nutrient component differences observed in the combined-site statistical 

analysis, whether reflecting increased or decreased MON 88701 mean values with 

respect to the conventional control, were 14.09% or less. The relative magnitudes 

of the differences were: 0.66 to 5.00% for proximates, 4.08 to 5.72% for fibers, 

2.61 to 4.82% for amino acids, 0.69 to 2.69% for fatty acids, 4.94 to 14.09% for 

minerals and 6.70% for vitamin E. 

2) With the exception of methionine, mean values for all significantly different 

nutrient components from the combined-site analysis of MON 88701 were within 

the 99% tolerance interval established from the conventional commercial 

reference varieties grown concurrently in the same trial. 

3) Assessment of the reproducibility of the combined-site differences at the eight 

individual sites showed significant differences for: NDF, methionine, proline and 

18:2 linoleic acid at one site; carbohydrates, total fat, ADF, manganese and zinc at 

two sites; TDF, arginine, 14:0 myristic acid, potassium, and vitamin E at three 

sites; magnesium at four sites, ash at six sites and calcium at seven sites. 

Moisture and calories were not affected at any site. With the exception of 

methionine, arginine, and zinc, all individual site mean values of MON 88701 for 

all nutrient components with significant differences were within the 99% 

tolerance interval established from the conventional commercial reference 

varieties grown concurrently in the same trial. 

4) All combined-site mean values and individual mean values of MON 88701 for all 

nutrient components, including those that were significantly different, were within 

the context of the natural variability of commercial cotton composition as 

published in the scientific literature and/or available in the ILSI Crop 

Composition Database (ILSI, 2011). 
Monsanto Company 12-CT-244U 102 of 620 

Five of the 19 cottonseed nutrient statistically significant differences between 

MON 88701 and the conventional control that were observed in the combined-site data 

analysis were attributable to small differences in proximates (ash, carbohydrates, total fat 

expressed as % dw, calories expressed as Kcal/100g dw, and moisture expressed as % 

fw). For ash, calories, and total fat the relative magnitude of the differences between the 

mean value for MON 88701 and the conventional control were all small increases (5.00% 

for ash, 0.66% for calories and 3.71% for total fat). The differences for carbohydrates 

and moisture between the mean value for MON 88701 and the conventional control were 

both small decreases (2.60% for carbohydrates and 4.51% for moisture). All of the 

nutrient mean values for MON 88701 observed in the combined-site analysis for 

proximates were within the 99% tolerance interval established by conventional 

commercial reference varieties grown concurrently in the same trial. Except for ash, 

significant differences for most proximate mean values between MON 88701 and the 

conventional control were not consistently observed among individual sites. There were 

no significant differences at any of the individual sites for calories or moisture. Total fat 

was increased at two sites ranging from 6.74 to 8.46% and carbohydrates were decreased 

at two sites, with decreases ranging from 4.33 to 5.08%. Although ash was increased in 

MON 88701 when compared to the conventional control at six sites, increases ranged 

from 4.95 to 11.50%, which was less than the variability for the control samples (range 

3.46 to 4.29, a relative difference of 24.0%, Table VI-1). Overall, observed differences 

in proximate values between MON 88701 and the conventional control were not 

considered to be meaningful from a food and feed safety or nutritional perspective 

because the magnitudes of combined-site differences ranged only from 0.66% to 5.00%, 

most were not consistently reproduced across the individual sites, and the mean 

MON 88701 combined-site values were within the 99% tolerance interval established by 

conventional commercial reference varieties grown concurrently in the same trial, and 

were within the context of the natural variability of commercial cotton composition as 

published in the scientific literature and/or available in the ILSI Crop Composition 

Database (ILSI, 2011). 

Three of the 19 cottonseed nutrient statistically significant differences between 

MON 88701 and the conventional control observed in the combined-site data analysis 

were attributable to small differences in fiber (ADF, NDF, and TDF all expressed as % 

dw). All relative magnitudes of the differences for fiber between the mean values for 

MON 88701 and the conventional control were small decreases (4.94% for ADF, 5.72% 

for NDF and 4.08% for TDF). All of the nutrient mean values for MON 88701 observed 

in the combined-site analysis for fiber were within the 99% tolerance interval established 

by conventional commercial reference varieties grown concurrently in the same trial. 

Significant differences for fiber mean values between MON 88701 and the conventional 

control were not consistently observed among individual sites. TDF and ADF were 

decreased at three and two sites, respectively, with decreases ranging from 4.55 to 8.15% 

for TDF and 9.27 to 9.86% for ADF. NDF was significantly different at one site with a 

small decrease of 7.40%. Overall, observed differences in fiber values between 

MON 88701 and the conventional control were not considered to be meaningful from a 

food and feed safety or nutritional perspective because they were small, not consistently 

reproduced across the individual sites, and the mean MON 88701 combined-site values 
Monsanto Company 12-CT-244U 103 of 620 

were within the 99% tolerance interval established by conventional commercial reference 

varieties grown concurrently in the same trial, and were within the context of the natural 

variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

Three other combined-site nutrient statistically significant differences between 

MON 88701 and the conventional control observed in the combined-site analysis were 

attributed to small differences in amino acids (arginine, methionine, and proline; 

expressed as % dw). For both arginine and proline, the relative magnitude of the 

differences between the mean values for MON 88701 and the conventional control were 

small decreases (3.80% for arginine and 2.61% for proline). Methionine was increased 

4.82% when MON 88701 was compared to the conventional control. With the exception 

of methionine, the nutrient mean values for MON 88701 observed in the combined-site 

analysis for amino acids were within the 99% tolerance interval established by 

conventional commercial reference varieties grown concurrently in the same trial. The 

combined-site mean value for methionine was within the context of natural variation of 

methionine found in commercial cotton as published in the scientific literature or as 

found in the ILSI Crop Composition Database (ILSI, 2011). Significant differences for 

amino acid mean values between MON 88701 and the conventional control were not 

consistently observed at all eight individual sites. Arginine and proline were decreased at 

three sites and one site, respectively, with decreases ranging from 6.10 to 8.35% for 

arginine and 6.16% for proline. Methionine was increased 12.03% at only one site. 

Overall, observed differences in amino acid values between MON 88701 and the 

conventional control were not considered to be meaningful from a food and feed safety or 

nutritional perspective because they were small in magnitude, not consistently 

reproduced across the individual sites, and with the exception of methionine, the mean 

MON 88701 combined-site values were within the 99% tolerance interval established by 

conventional commercial reference varieties grown concurrently in the same trial. In 

addition, all MON 88701 amino acid values were within the context of the natural 

variability of commercial cotton composition as published in the scientific literature or 

available in the ILSI Crop Composition Database (ILSI, 2011). 

Two of the combined-site nutrient statistically significant differences between 

MON 88701 and the conventional control were attributed to the fatty acids 14:0 myristic 

acid and 18:2 linoleic acid (expressed as % total FA). The relative magnitudes of the 

differences between the mean fatty acid values for MON 88701 and the conventional 

control in the combined-site analysis were small decreases (2.69% for 14:0 myristic acid 

and 0.69% for 18:2 linoleic acid). The nutrient mean values for MON 88701 observed in 

the combined-site analysis for both 14:0 myristic acid and 18:2 linoleic acid were within 

the 99% tolerance interval established by conventional commercial reference varieties 

grown concurrently in the same trial. Significant differences for fatty acid mean values 

between MON 88701 and the conventional control were not consistently observed among 

individual sites. 14:0 myristic acid was decreased at three sites while 18:2 linoleic acid 

was decreased at one site with differences ranging from 4.43 to 8.36% for 14:0 myristic 

acid and 1.93% for 18:2 linoleic acid. Overall, observed differences in fatty acid values 

between MON 88701 and the conventional control were not considered to be meaningful 
Monsanto Company 12-CT-244U 104 of 620 

from a food and feed safety and nutritional perspective because they were small, not 

consistently reproduced across the individual sites, and the mean MON 88701 values 

were within the 99% tolerance interval established by conventional commercial reference 

varieties grown concurrently in the same trial and were within the context of the natural 

variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

Five of the 19 cottonseed nutrient statistically significant differences between 

MON 88701 and the conventional control observed in the combined-site analysis were 

attributed to small differences in minerals (calcium, magnesium, and potassium expressed 

as % dw and manganese and zinc expressed as mg/kg dw). For calcium, magnesium, 

potassium, and manganese, the relative magnitudes of the differences between the mean 

values for MON 88701 and the conventional control were increases of 14.09% for 

calcium, 5.63% for magnesium, 9.20% for manganese, and 4.94% for potassium. The 

relative magnitude of the difference for zinc between the mean value for MON 88701 and 

the conventional control was a decrease of 6.39%. All of the nutrient mean values for 

MON 88701 observed in the combined-site analysis for minerals were within the 99% 

tolerance interval established by conventional commercial reference varieties grown 

concurrently in the same trial. Except for calcium, significant differences for mineral 

mean values between MON 88701 and the conventional control were not consistently 

observed among individual sites. Calcium was significantly different at seven sites, with 

increases ranging from 6.92 to 22.70%; this was less than the variability observed for the 

control samples (range 0.091 to 0.18, a relative difference of 97.8%, Table VI-1). 

Magnesium, potassium, and manganese were significantly different at four, three, and 

two sites, respectively, with increases ranging from 5.54 to 9.36% for magnesium, 8.01 to 

16.37% for potassium and from 16.52 to 20.59% for manganese. Zinc was significantly 

different at two sites, with decreases ranging from 7.68 to 17.66%. Overall, observed 

differences in mineral values between MON 88701 and the conventional control were not 

considered to be meaningful from a food and feed safety or nutritional perspective 

because they were small in magnitude, not consistently reproduced across the individual 

sites (with the exception of calcium), and the mean MON 88701 combined-site values 

were within the 99% tolerance interval established by conventional commercial reference 

varieties grown concurrently in the same trial and within the context of the natural 

variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

One other nutrient difference observed in the combined-site analysis between 

MON 88701 and the conventional control was attributed to vitamin E (expressed as 

mg/kg dw). The relative magnitude of the difference between the mean vitamin E value 

for MON 88701 and the conventional control in the combined-site analysis was a small 

increase of 6.70%. The nutrient mean value for MON 88701 observed in the combinedsite analysis for vitamin E was within the 99% tolerance interval established by 

conventional commercial reference varieties grown concurrently in the same trial. 

Significant differences for vitamin E mean values between MON 88701 and the 

conventional control were not consistently observed among individual sites, with 
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significant increases ranging from 7.78 to 13.28% observed at three sites. Overall, the 

observed difference in the vitamin E values between MON 88701 and the conventional 

control in the combined-site analysis were not considered to be meaningful from a food 

and feed safety and nutritional perspective because they were 13.28% or less, not 

consistently reproduced across the individual sites, and the mean MON 88701 values 

were within the 99% tolerance interval established by conventional commercial reference 

varieties grown concurrently in the same trial, and were within the context of the natural 

variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

In summary, except for calcium and ash, statistical analyses found no consistent 

differences between the levels of nutrient components in cottonseed from MON 88701 

and the conventional control. Differences were observed for calcium and ash in 

combined-site analyses and most individual sites, but the magnitudes of differences for 

these nutrients were less than the variability for the control samples, and values were 

within the range of natural variability for cottonseed. These findings support the 

conclusion of compositional equivalence of MON 88701 to conventional cotton. 

VI.A.2. Anti-Nutrient Levels in Cottonseed 

Cottonseed was analyzed for five anti-nutrients and in the combined-site analysis the 

following components had no significant differences (p<0.05) in mean values between 

MON 88701 treated with dicamba and glufosinate and the conventional control: two 

cyclopropenoid fatty acids (malvalic and sterculic) (Table VI-3). The components that 

showed statistically significant differences in mean values between MON 88701 and the 

conventional control were: one cyclopropenoid fatty acid (dihydrosterculic), free 

gossypol, and total gossypol (Table VI-1). 

The statistically significant differences in anti-nutrients were further evaluated using the 

four previously described considerations relevant to the safety and nutritional quality of 

MON 88701 when compared to the conventional control: 

1) All anti-nutrient component differences observed in the combined-site statistical 

analysis, which reflected an increase in MON 88701 mean values with respect to 

the conventional control, were small in magnitude. The relative magnitude of the 

differences for dihydrosterculic acid, free gossypol, and total gossypol were 

9.59%, 6.23%, and 6.75%, respectively. 

2) Mean values for all significantly different anti-nutrient components from the 

combined-site analysis of MON 88701 were within the 99% tolerance interval 

established from the conventional commercial reference varieties grown 

concurrently in the same trial. 

3) Assessment of the reproducibility of the combined-site differences at the eight 

individual sites showed significant differences for: dihydrosterculic at one site; 

free gossypol at two sites; and total gossypol at three sites. All individual site 

mean values of MON 88701 for all anti-nutrient components with significant 
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differences were within the 99% tolerance interval established from the 

conventional commercial reference varieties grown concurrently in the same trial. 

4) All combined-site mean values of MON 88701 for all anti-nutrient components, 

including those that were significantly different, were within the context of the 

natural variability of commercial cotton composition as published in the scientific 

literature and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

The three cottonseed anti-nutrient differences between MON 88701 and the conventional 

control observed in the combined-site data analysis were attributed to small differences in 

one cyclopropenoid fatty acid (dihydrosterculic; expressed as % total fatty acid), free 

gossypol, and total gossypol (expressed as % dw). For dihydrosterculic acid, free 

gossypol, and total gossypol, the relative magnitude of the differences between the mean 

values for MON 88701 and the conventional control were increases of 9.59% for 

dihydrosterculic acid, 6.23% for free gossypol, and 6.75% for total gossypol. These antinutrient differences between MON 88701 and the conventional control observed in the 

combined-site analysis were within the 99% tolerance interval established by 

conventional commercial reference varieties grown concurrently in the same trial. 

Significant differences for the three anti-nutrient mean values between MON 88701 and 

the conventional control were not consistently observed across all eight individual sites. 

Dihydrosterculic acid, free gossypol, and total gossypol were significantly different at 

one, two, and three sites respectively, with an increase of 28.35% for dihydrosterculic 

acid, and increases ranging from 12.69 to 22.32% for free gossypol and 9.54 to 15.53% 

for total gossypol. Overall, observed differences in anti-nutrient values between 

MON 88701 and the conventional control were not considered to be meaningful from a 

food and feed safety or nutritional perspective because they were generally small, not 

consistently reproduced across the individual sites, and the mean MON 88701 values 

were within the 99% tolerance interval established by conventional commercial reference 

varieties grown concurrently in the same trial and within the context of the natural 

variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

In summary, statistical analyses found no consistent statistically significant differences 

between the levels of anti-nutrient components in cottonseed from MON 88701 and the 

conventional control and mean values for anti-nutrients were within the range of natural 

variability for cottonseed. These findings supported the conclusion of compositional 

equivalence of MON 88701 to conventional cotton. 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MOConventional Control 

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed Proximate (% dw)Ash 4.31 4.11 5.00 0.001 3.77 - 4.74 Calories Kcal/100g 498.50 495.24 0.66 0.013 482.46 - 517.Carbohydrates 44.64 45.83 -2.60 <0.001 41.40 - 48.8Moisture (% fw) 7.15 7.48 -4.51 0.005 5.93 - 9.67 Total Fat 23.14 22.31 3.71 0.001 19.79 - 26.7Cottonseed Fiber (% dw)Acid Detergent Fiber 25.27 26.58 -4.94 0.002 23.26 - 27.7Neutral Detergent Fiber 30.73 32.59 -5.72 <0.001 25.13 - 34.4
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed Fiber (% dw)Total Dietary Fiber 39.44 41.12 -4.08 <0.001 36.91 - 42.1Cottonseed Amino Acid (% dw)Arginine 3.03 3.15 -3.80 0.002 2.33 - 3.60 Methionine 0.40 0.38 4.82 0.026 0.35 - 0.46 Proline 1.00 1.03 -2.61 0.037 0.82 - 1.21 Cottonseed Fatty Acid (% Total FA)14:0 Myristic 0.77 0.79 -2.69 0.009 0.66 - 0.95 18:2 Linoleic 55.77 56.15 -0.69 0.026 54.24 - 58.2
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed MineralCalcium (% dw) 0.15 0.13 14.09 <0.001 0.10 - 0.22 Magnesium (% dw) 0.40 0.38 5.63 <0.001 0.35 - 0.44 Manganese (mg/kg dw) 12.81 11.73 9.20 0.001 10.18 - 14.8Potassium (% dw) 1.12 1.07 4.94 0.021 0.98 - 1.24 Zinc (mg/kg dw) 37.58 40.14 -6.39 0.005 27.31 - 46.7Cottonseed Vitamin (mg/kg dw)Vitamin E 140.14 131.33 6.70 <0.001 86.23 - 179.3Cottonseed Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 0.14 9.59 0.003 0.11 - 0.19 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed Gossypol (% dw)Free Gossypol 0.94 0.89 6.23 0.016 0.80 - 1.18 Total Gossypol 1.04 0.97 6.75 <0.001 0.84 - 1.24 Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 7 SitesCalcium (% dw) Site ARTI 0.15 0.12 22.70 0.010 0.14 - 0.16 Calcium (% dw) Site GACH 0.13 0.11 17.57 <0.001 0.13 - 0.13 Calcium (% dw) Site KSLA 0.20 0.18 14.74 0.007 0.19 - 0.22 Calcium (% dw) Site NCBD 0.15 0.14 6.92 0.007 0.14 - 0.15 Calcium (% dw) Site NMLC 0.15 0.13 16.83 0.003 0.14 - 0.15 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 88701 

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 7 SitesCalcium (% dw) Site SCEK 0.11 0.091 17.98 0.027 0.10 - 0.11 Calcium (% dw) Site TXPL 0.16 0.14 15.31 <0.001 0.16 - 0.16 Cottonseed Proximate (% dw) - 6 SitesAsh Site GACH 4.53 4.21 7.56 <0.001 4.45 - 4.57 Ash Site KSLA 4.53 4.29 5.64 0.027 4.25 - 4.66 Ash Site LACH 4.35 4.12 5.56 0.013 4.23 - 4.47 Ash Site NCBD 4.34 4.14 4.95 0.033 4.29 - 4.40 Ash Site SCEK 4.11 3.74 9.95 0.010 3.99 - 4.28 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Proximate (% dw) - 6 SitesAsh Site TXPL 3.85 3.46 11.50 0.001 3.77 - 3.92 Cottonseed Fatty Acid (% Total FA) - 5 Sites18:0 Stearic Site ARTI 2.68 2.51 6.70 0.019 2.65 - 2.72 18:0 Stearic Site LACH 2.68 2.52 6.04 0.001 2.64 - 2.73 18:0 Stearic Site NCBD 2.50 2.34 6.85 0.036 2.39 - 2.64 18:0 Stearic Site NMLC 2.51 2.64 -5.13 <0.001 2.47 - 2.56 18:0 Stearic Site TXPL 2.35 2.46 -4.67 0.006 2.30 - 2.43 Cottonseed Mineral - 4 SitesMagnesium (% dw) Site GACH 0.41 0.38 6.92 <0.001 0.40 - 0.41 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 4 SitesMagnesium (% dw) Site KSLA 0.43 0.40 6.85 0.002 0.41 - 0.43 Magnesium (% dw) Site SCEK 0.39 0.36 9.36 0.005 0.37 - 0.41 Magnesium (% dw) Site TXPL 0.35 0.34 5.54 0.003 0.35 - 0.37 Cottonseed Fiber (% dw) - 3 SitesTotal Dietary Fiber Site KSLA 38.32 40.14 -4.55 0.034 37.62 - 38.7Total Dietary Fiber Site LACH 39.82 43.35 -8.15 0.002 39.02 - 40.8Total Dietary Fiber Site NMLC 39.16 41.10 -4.73 0.016 37.46 - 40.4Cottonseed Amino Acid (% dw) - 3 SitesArginine Site GACH 2.95 3.21 -8.35 0.008 2.87 - 3.02 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Amino Acid (% dw) - 3 SitesArginine Site KSLA 3.02 3.28 -7.87 0.013 2.95 - 3.10 Arginine Site NMLC 3.48 3.71 -6.10 0.005 3.42 - 3.60 Cottonseed Fatty Acid (% Total FA) - 3 Sites14:0 Myristic Site KSLA 0.68 0.72 -5.33 0.007 0.66 - 0.71 14:0 Myristic Site NCBD 0.68 0.75 -8.36 0.002 0.66 - 0.70 14:0 Myristic Site NMLC 0.93 0.98 -4.43 0.001 0.92 - 0.95 Cottonseed Mineral - 3 SitesPotassium (% dw) Site GACH 1.21 1.12 8.01 <0.001 1.17 - 1.24 Potassium (% dw) Site SCEK 1.13 1.02 10.88 0.042 1.11 - 1.17 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 3 SitesPotassium (% dw) Site TXPL 1.01 0.87 16.37 0.004 0.98 - 1.06 Cottonseed Vitamin (mg/kg dw) - 3 SitesVitamin E Site GACH 151.03 140.12 7.78 0.025 148.34 - 154.Vitamin E Site LACH 169.88 149.96 13.28 0.001 163.34 - 175.Vitamin E Site TXPL 114.39 103.66 10.35 0.033 107.81 - 118.Cottonseed Gossypol (% dw) - 3 SitesTotal Gossypol Site KSLA 1.13 1.01 12.00 0.049 1.00 - 1.24 Total Gossypol Site NMLC 0.92 0.80 15.53 0.026 0.84 - 0.97 Total Gossypol Site SCEK 1.17 1.07 9.54 0.017 1.13 - 1.23 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Proximate (% dw) - 2 SitesCarbohydrates Site SCEK 46.56 48.67 -4.33 0.031 45.10 - 47.4Carbohydrates Site TXPL 44.03 46.39 -5.08 0.010 42.73 - 45.9Total Fat Site NCBD 23.04 21.59 6.74 0.024 21.89 - 23.7Total Fat Site SCEK 25.65 23.65 8.46 0.019 24.23 - 26.7Cottonseed Fiber (% dw) - 2 SitesAcid Detergent Fiber Site ARTI 24.81 27.53 -9.86 0.007 24.44 - 25.2Acid Detergent Fiber Site LACH 25.72 28.35 -9.27 0.005 24.16 - 27.0Cottonseed Amino Acid (% dw) - 2 SitesPhenylalanine Site GACH 1.40 1.49 -5.89 0.039 1.37 - 1.43 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Amino Acid (% dw) - 2 SitesPhenylalanine Site KSLA 1.44 1.53 -5.88 0.025 1.40 - 1.46 Cottonseed Fatty Acid (% Total FA) - 2 Sites16:0 Palmitic Site LACH 24.48 24.04 1.81 0.014 24.37 - 24.516:0 Palmitic Site SCEK 24.74 24.39 1.43 0.029 24.59 - 24.916:1 Palmitoleic Site NCBD 0.46 0.48 -3.88 0.019 0.44 - 0.47 16:1 Palmitoleic Site NMLC 0.53 0.54 -2.27 0.014 0.52 - 0.53 18:3 Linolenic Site ARTI 0.14 0.13 11.92 0.012 0.14 - 0.15 18:3 Linolenic Site NMLC 0.16 0.14 8.12 0.009 0.15 - 0.16 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 2 SitesIron (mg/kg dw) Site NCBD 43.21 48.04 -10.05 0.025 41.96 - 44.4Iron (mg/kg dw) Site TXPL 60.47 79.02 -23.47 0.039 56.94 - 66.5Manganese (mg/kg dw) Site GACH 13.41 11.51 16.52 0.003 12.79 - 14.1Manganese (mg/kg dw) Site TXPL 10.91 9.04 20.59 0.007 10.18 - 11.3Zinc (mg/kg dw) Site NCBD 40.79 49.54 -17.66 0.006 40.28 - 41.3Zinc (mg/kg dw) Site NMLC 45.63 49.43 -7.68 0.009 44.12 - 46.7Cottonseed Gossypol (% dw) - 2 SitesFree Gossypol Site KSLA 1.07 0.95 12.69 0.014 1.03 - 1.10 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Gossypol (% dw) - 2 SitesFree Gossypol Site NMLC 0.85 0.69 22.32 0.011 0.83 - 0.88 Statistical Differences Observed in One SiteCottonseed Proximate (% dw)Protein Site TXPL 29.43 28.48 3.33 0.017 29.06 - 30.1Cottonseed Fiber (% dw)Crude Fiber Site KSLA 16.43 17.67 -7.04 0.019 16.06 - 17.2Neutral Detergent Fiber Site TXPL 29.75 32.12 -7.40 0.006 28.74 - 30.5Cottonseed Amino Acid (% dw)Alanine Site LACH 1.07 1.03 3.73 0.030 1.00 - 1.11 Aspartic Acid Site GACH 2.31 2.45 -6.03 0.019 2.24 - 2.36 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in One SiteCottonseed Amino Acid (% dw)Glutamic Acid Site GACH 4.57 4.96 -7.95 0.010 4.35 - 4.77 Isoleucine Site GACH 0.90 0.94 -4.21 0.034 0.90 - 0.91 Leucine Site GACH 1.51 1.58 -4.32 0.024 1.49 - 1.54 Lysine Site LACH 1.26 1.18 7.01 0.023 1.17 - 1.31 Methionine Site LACH 0.42 0.38 12.03 0.013 0.37 - 0.44 Proline Site GACH 0.98 1.05 -6.16 0.033 0.97 - 0.99 Threonine Site GACH 0.85 0.90 -5.14 0.049 0.83 - 0.88 Tryptophan Site SCEK 0.35 0.38 -6.70 0.023 0.33 - 0.38 
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in One SiteCottonseed Amino Acid (% dw)Tyrosine Site GACH 0.80 0.84 -4.30 0.037 0.79 - 0.82 Valine Site GACH 1.21 1.26 -4.19 0.017 1.19 - 1.23 Cottonseed Fatty Acid (% Total FA)18:1 Oleic Site LACH 14.70 14.29 2.89 0.021 14.48 - 15.018:2 Linoleic Site LACH 55.53 56.63 -1.93 0.001 55.15 - 55.920:0 Arachidic Site LACH 0.31 0.29 6.78 0.033 0.31 - 0.32 22:0 Behenic Site ARTI 0.14 0.15 -9.92 0.008 0.13 - 0.14 Cottonseed MineralSodium (% dw) Site KSLA 0.022 0.0080 178.30 0.020 0.019 - 0.02
Monsanto Company 12-CT-244U 

Table VI-1. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MConventional Control (continued)

Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in One SiteCottonseed Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid Site GACH 0.15 0.12 28.35 0.022 0.14 - 0.16 1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 was treated with dicamba and glufosinate. 3Mean = least-square mean. 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.31 (0.11) 4.11 (0.11) 0.21 (0.052) 0.094, 0.32 0.001 

(3.77 - 4.74) (3.34 - 5.00) (-0.49 - 0.61) 

Calories (Kcal/100g) 498.50 (1.65) 495.24 (1.71) 3.26 (1.29) 0.70, 5.82 0.013 

(482.46 - 517.46) (487.70 - 512.65) (-14.30 - 18.37) 

Carbohydrates 44.64 (0.56) 45.83 (0.57) -1.19 (0.32) -1.82, -0.56 <0.001 

(41.40 - 48.89) (42.14 - 50.30) (-5.19 - 2.45) 

Moisture (% fw) 7.15 (0.26) 7.48 (0.27) -0.34 (0.11) -0.56, -0.11 0.005 

(5.93 - 9.67) (6.15 - 9.19) (-1.82 - 0.79) 

Protein 27.91 (0.77) 27.79 (0.77) 0.13 (0.31) -0.53, 0.78 0.685 

(22.71 - 31.47) (23.53 - 31.27) (-1.99 - 3.73) 

Total Fat 23.14 (0.31) 22.31 (0.33) 0.83 (0.26) 0.32, 1.34 0.001 

(19.79 - 26.78) (20.71 - 25.20) (-2.89 - 3.86) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued)

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.27 (0.34) 26.58 (0.35) -1.31 (0.35) -2.06, -0.57 0.002 

(23.26 - 27.74) (22.08 - 29.58) (-5.42 - 1.77) 

Crude Fiber 18.17 (0.37) 18.54 (0.38) -0.38 (0.32) -1.02, 0.27 0.246 

(15.97 - 21.66) (16.06 - 21.70) (-3.36 - 4.75) 

Neutral Detergent Fiber 30.73 (0.51) 32.59 (0.53) -1.86 (0.41) -2.68, -1.05 <0.001 

(25.13 - 34.42) (28.87 - 35.89) (-6.95 - 1.16) 

Total Dietary Fiber 39.44 (0.39) 41.12 (0.41) -1.68 (0.36) -2.45, -0.91 <0.001 

(36.91 - 42.13) (39.05 - 44.37) (-5.34 - 1.09) 

Amino Acid (% dw)Alanine 1.06 (0.020) 1.05 (0.020) 0.0026 (0.0091) -0.017, 0.022 0.775 

(0.91 - 1.14) (0.88 - 1.17) (-0.13 - 0.12) 

Arginine 3.03 (0.10) 3.15 (0.10) -0.12 (0.033) -0.19, -0.049 0.002 

(2.33 - 3.60) (2.41 - 3.77) (-0.47 - 0.39) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued) 

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.39 (0.062) 2.41 (0.062) -0.015 (0.027) -0.072, 0.042 0.575 

(1.94 - 2.64) (1.92 - 2.74) (-0.29 - 0.29) 

Cystine 0.41 (0.0091) 0.40 (0.0094) 0.0096 (0.0070) -0.0043, 0.023 0.174 

(0.32 - 0.47) (0.31 - 0.46) (-0.063 - 0.082) 

Glutamic Acid 4.76 (0.13) 4.84 (0.14) -0.079 (0.072) -0.23, 0.077 0.295 

(3.80 - 5.38) (3.66 - 5.70) (-0.78 - 0.79) 

Glycine 1.10 (0.020) 1.09 (0.020) 0.0014 (0.011) -0.021, 0.024 0.896 

(0.93 - 1.19) (0.91 - 1.20) (-0.13 - 0.14) 

Histidine 0.74 (0.019) 0.75 (0.019) -0.0014 (0.0073) -0.017, 0.014 0.854 

(0.58 - 0.85) (0.61 - 0.84) (-0.062 - 0.091) 

Isoleucine 0.91 (0.018) 0.92 (0.018) -0.0066 (0.0079) -0.023, 0.010 0.421 

(0.75 - 1.01) (0.77 - 1.03) (-0.077 - 0.096) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued)

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.53 (0.032) 1.54 (0.032) -0.0018 (0.013) -0.029, 0.026 0.892 

(1.29 - 1.70) (1.28 - 1.69) (-0.14 - 0.16) 

Lysine 1.24 (0.025) 1.23 (0.025) 0.0069 (0.015) -0.026, 0.039 0.658 

(1.05 - 1.38) (1.06 - 1.39) (-0.11 - 0.15) 

Methionine 0.40 (0.0079) 0.38 (0.0084) 0.018 (0.0081) 0.0023, 0.035 0.026 

(0.35 - 0.46) (0.32 - 0.46) (-0.066 - 0.12) 

Phenylalanine 1.43 (0.039) 1.46 (0.039) -0.022 (0.014) -0.052, 0.0084 0.144 

(1.14 - 1.66) (1.15 - 1.66) (-0.18 - 0.19) 

Proline 1.00 (0.029) 1.03 (0.029) -0.027 (0.012) -0.052, -0.0018 0.037 

(0.82 - 1.21) (0.81 - 1.25) (-0.12 - 0.10) 

Serine 1.08 (0.025) 1.09 (0.026) -0.0036 (0.015) -0.035, 0.028 0.807 

(0.90 - 1.23) (0.86 - 1.24) (-0.18 - 0.16) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued) 

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.87 (0.016) 0.86 (0.016) 0.0057 (0.0083) -0.012, 0.023 0.504 

(0.74 - 0.94) (0.73 - 0.95) (-0.10 - 0.10) 

Tryptophan 0.41 (0.0092) 0.42 (0.0095) -0.0061 (0.0066) -0.019, 0.0071 0.361 

(0.33 - 0.52) (0.37 - 0.52) (-0.081 - 0.078) 

Tyrosine 0.81 (0.017) 0.81 (0.018) -0.0011 (0.0083) -0.019, 0.017 0.898 

(0.67 - 0.92) (0.67 - 0.91) (-0.074 - 0.12) 

Valine 1.21 (0.027) 1.23 (0.027) -0.012 (0.011) -0.036, 0.012 0.296 

(1.00 - 1.40) (1.00 - 1.40) (-0.090 - 0.12) 

Fatty Acid (% Total FA)14:0 Myristic 0.77 (0.030) 0.79 (0.031) -0.021 (0.0071) -0.036, -0.0060 0.009 

(0.66 - 0.95) (0.71 - 0.98) (-0.077 - 0.047) 

16:0 Palmitic 23.95 (0.30) 23.80 (0.30) 0.15 (0.076) -0.016, 0.31 0.073 

(22.34 - 25.28) (22.69 - 25.05) (-0.68 - 0.76) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued)

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.50 (0.0094) 0.50 (0.0094) 0.0022 (0.0038) -0.0060, 0.010 0.572 

(0.44 - 0.54) (0.45 - 0.54) (-0.025 - 0.039) 

18:0 Stearic 2.54 (0.058) 2.47 (0.058) 0.068 (0.036) -0.0091, 0.14 0.079 

(2.29 - 2.85) (2.15 - 2.76) (-0.16 - 0.24) 

18:1 Oleic 15.10 (0.26) 14.96 (0.26) 0.14 (0.070) -0.0049, 0.29 0.057 

(14.15 - 16.45) (14.06 - 16.44) (-0.48 - 0.75) 

18:2 Linoleic 55.77 (0.39) 56.15 (0.40) -0.39 (0.16) -0.72, -0.053 0.026 

(54.24 - 58.22) (54.04 - 57.93) (-1.42 - 0.80) 

18:3 Linolenic 0.18 (0.022) 0.17 (0.022) 0.011 (0.0068) -0.0038, 0.025 0.136 

(0.14 - 0.34) (0.12 - 0.30) (-0.0073 - 0.052) 

20:0 Arachidic 0.29 (0.0086) 0.28 (0.0087) 0.0044 (0.0047) -0.0057, 0.015 0.364 

(0.23 - 0.32) (0.23 - 0.32) (-0.027 - 0.046) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued) 

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.15 (0.0051) 0.15 (0.0051) -0.0035 (0.0029) -0.0098, 0.0029 0.260 

(0.12 - 0.19) (0.13 - 0.21) (-0.049 - 0.032) 

MineralCalcium (% dw) 0.15 (0.0093) 0.13 (0.0093) 0.018 (0.0022) 0.013, 0.023 <0.001 

(0.10 - 0.22) (0.081 - 0.19) (-0.012 - 0.038) 

Copper (mg/kg dw) 8.90 (0.70) 8.93 (0.70) -0.025 (0.16) -0.34, 0.29 0.875 

(5.22 - 11.91) (5.40 - 11.92) (-2.59 - 1.29) 

Iron (mg/kg dw) 67.21 (4.40) 71.33 (4.48) -4.12 (2.74) -9.96, 1.71 0.153 

(41.96 - 83.17) (45.03 - 95.10) (-38.15 - 12.79) 

Magnesium (% dw) 0.40 (0.0083) 0.38 (0.0084) 0.021 (0.0032) 0.015, 0.028 <0.001 

(0.35 - 0.44) (0.33 - 0.44) (-0.036 - 0.054) 

Manganese (mg/kg dw) 12.81 (0.47) 11.73 (0.48) 1.08 (0.28) 0.48, 1.68 0.001 

(10.18 - 14.81) (8.61 - 14.11) (-1.95 - 2.54) 
Monsanto Company 12-CT-244U 

Table VI-2. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 vs. Conventiona(continued)

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.72 (0.031) 0.72 (0.031) 0.0081 (0.0067) -0.0053, 0.021 0.230 

(0.56 - 0.84) (0.54 - 0.87) (-0.087 - 0.11) 

Potassium (% dw) 1.12 (0.028) 1.07 (0.028) 0.053 (0.020) 0.0089, 0.097 0.021 

(0.98 - 1.24) (0.79 - 1.27) (-0.12 - 0.27) 

Sodium (% dw) 0.034 (0.0095) 0.029 (0.0096) 0.0045 (0.0046) -0.0053, 0.014 0.346 

(0.018 - 0.12) (0.0053 - 0.10) (-0.065 - 0.030) 

Zinc (mg/kg dw) 37.58 (2.01) 40.14 (2.02) -2.57 (0.77) -4.22, -0.91 0.005 

(27.31 - 46.74) (28.22 - 52.95) (-11.57 - 3.27) 

Vitamin (mg/kg dw)Vitamin E 140.14 (9.87) 131.33 (9.88) 8.80 (2.07) 4.39, 13.22 <0.001 

(86.23 - 179.34) (91.78 - 162.98) (-6.54 - 26.36) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 was treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table VI-3. Statistical Summary of Combined-Site Cottonseed Anti-nutrients for MON 88701 vs. Conven

Difference (MON 88701 minus Control)

Analytical Component 

(Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0034) 0.14 (0.0037) 0.013 (0.0044) 0.0044, 0.022 0.003 

(0.11 - 0.19) (0.11 - 0.17) (-0.026 - 0.068) 

Malvalic Acid 0.39 (0.015) 0.37 (0.016) 0.013 (0.015) -0.016, 0.043 0.371 

(0.20 - 0.55) (0.26 - 0.49) (-0.16 - 0.16) 

Sterculic Acid 0.22 (0.0067) 0.21 (0.0072) 0.0067 (0.0081) -0.0096, 0.023 0.412 

(0.13 - 0.29) (0.17 - 0.27) (-0.085 - 0.078) 

Gossypol (% dw)Free Gossypol 0.94 (0.037) 0.89 (0.037) 0.055 (0.020) 0.012, 0.099 0.016 

(0.80 - 1.18) (0.68 - 1.20) (-0.086 - 0.20) 

Total Gossypol 1.04 (0.037) 0.97 (0.037) 0.066 (0.017) 0.031, 0.10 <0.001 

(0.84 - 1.24) (0.74 - 1.10) (-0.021 - 0.23) 

1dw = dry weight; FA = fatty acid. 2MON 88701 was treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 132 of 620 

Table VI-4. Literature and ILSI Ranges for Components in Cottonseed 

Cottonseed Tissue Components1 Literature Range2 ILSI Range3

Cottonseed Nutrients 

Proximates (% dw) 

Ash 3.87 – 5.29a

; 3.7 – 4.2d 3.761 – 5.342 

Carbohydrates by calculation 45.28 – 53.62a

 39.0 – 53.6 

Calories by calculation 

(Kcal/100g)

471.34 – 506.95a

 Not available 

Moisture (% fw) 2.25 – 7.49a

 2.3 – 9.9 

Protein 24.54 – 30.83a

; 21.2 – 25.9b 21.48 – 32.97 

Total Fat 17.37 – 25.16a

; 14.4 – 16.9d 17.201 – 27.292 

 

Fiber (% dw) 

Acid Detergent Fiber 21.10 – 34.8a

; 37.6 – 40.5d 19.74 – 38.95 

Neutral Detergent Fiber 32.92 – 45.83a

; 50.0 – 53.6d 25.56 – 51.87 

Crude Fiber 13.85 – 17.94a

 13.86 – 23.10

Total Dietary Fiber not available 33.69 – 47.55 

Amino Acids (% total AA) (% dw) 

Alanine 4.16 – 4.41a

; 3.6 – 4.2b 0.80 – 1.22 

Arginine 11.28 – 12.51 a

; 10.9 – 12.3b 2.06 – 3.72 

Aspartic acid 9.73 – 9.99 a

; 8.8 – 9.5b 1.82 – 2.94 

Cystine/Cysteine 1.60 – 1.92 a; 2.3 – 3.4b 0.35 – 0.56 

Glutamic acid 20.76 – 21.61 a

; 20.5 – 22.4 b 3.91 – 6.72 

Glycine 4.44 – 4.58 a

; 3.8 – 4.5 b 0.83 – 1.32 

Histidine 3.00 – 3.12 a; 2.6 – 2.8 b 0.57 – 0.91 

Isoleucine 3.10 – 3.67 a

; 3.0 – 3.4 b 0.62 – 1.05 

Leucine 6.27 – 6.65 a

; 5.5 – 6.1 b 1.14 – 1.86 

Lysine 4.85 – 5.37 a; 4.2 – 4.6 b 0.94 – 1.46 

Methionine 1.46 – 1.88 a

; 1.3 – 1.8 b 0.30 – 0.47 

Phenylalanine 5.56 – 5.77 a

; 5.0 – 5.6 b 1.02 – 1.72 

Proline 4.06 – 4.28 a; 3.1 – 4.0 b 0.75 – 1.23 

Serine 4.45 – 4.86 a

; 3.9 – 4.4 b 0.91 – 1.35 

Threonine 3.26 – 3.59 a; 2.8 – 3.2b 0.55 – 0.92 

Tryptophan 0.97 – 1.21 a; 1.0 – 1.4 b 0.194 – 0.319 

Tyrosine 2.65 – 2.92 a

; 2.8 – 3.3 b 0.53 – 0.84 

Valine 4.76 – 5.14 a

; 4.3 – 4.7 b 0.87 – 1.49 

 

Fatty Acids (% total FA) 

8:0 Caprylic not available not available 

10:0 Capric not available not available 

12:0 Lauric not available not available 

14:0 Myristic 0.55 – 2.40a

; 0.6 – 1.5b 0.455 – 2.400 

14:1 Myristoleic not available not available 

15:0 Pentadecanoic 0.050 – 0.17a 0.103 – 0.481 

15:1 Pentadecenoic not available not available 

16:0 Palmitic 21.23 – 27.9 a

; 17.6 – 24.8 b 15.11 – 27.90 

16:1 Palmitoleic 0.55 – 1.16 a

 0.464 – 1.190 

17:0 Heptadecanoic not available 0.092 – 0.119 

 
Monsanto Company 12-CT-244U 133 of 620 

Table VI-4. Literature and ILSI Ranges for Components in Cottonseed (continued) 

Cottonseed Tissue Components1 Literature Range2 ILSI Range3

17:1 Heptadecenoic not available not available 

18:0 Stearic 1.99 – 3.11 a; 2.0 – 2.5 b 0.20 – 3.11 

18:1 Oleic 13.90 – 20.10 a

; 15.0 – 20.7 b 12.8 – 25.3 

18:2 Linoleic 46.00 – 56.88 a

 46.0 – 59.4 

18:3 Gamma Linolenic 0.050 – 0.25 a

 0.097 – 0.232 

18:3 Linolenic 0.050 – 0.25 a 0.11 – 0.35 

20:0 Arachidic 0.25 – 0.33 a

 0.186 – 0.414 

20:1 Eicosenoic not available 0.095 – 0.098 

20:2 Eicosadienoic not available not available 

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.13 – 0.17 a

 0.104 – 0.295 

 

Vitamins (mg/kg fw) (mg/kg dw)

Vitamin E 99 – 224c

 70.825 – 197.243 

 

Minerals (% dw) 

Calcium 0.10 – 0.33a 0.10323 – 0.32581 

Copper (mg/kg dw) 3.54 – 11.14a 3.13 – 24.57 

Iron (mg/kg dw) 40.58 – 56.54 a

 36.71 – 318.38 

Magnesium 0.37 – 0.46 a

 0.34709 – 0.49312 

Manganese (mg/kg dw) 11.06 – 18.31 a 10.69 – 21.96 

Phosphorus 0.60 – 0.84 a

 0.48254 – 0.99157 

Potassium 0.98 – 1.24 a

 0.98345 – 1.44835 

Sodium 0.0054 – 0.74 a 0.01118 – 0.73548 

Zinc (mg/kg dw) 30.21 – 47.75 a

 27.0 – 59.5 

 

Cottonseed Anti-Nutrients 

Gossypol, Total (% dw) 0.57 – 1.42a

; 0.55 – 0.77d 0.547 – 1.522 

Gossypol, Free (% dw) 0.53 – 1.20a 0.454 – 1.399 

 

Cyclopropenoid Fatty Acids 

(% total FA)

 

Dihydrosterculic 0.13 – 0.24 a

 0.075 – 0.310 

Malvalic 0.33 – 0.58 a

 0.229 – 0.759 

Sterculic 0.21 – 0.56 a

 0.190 – 0.556 

 1

fw=fresh weight; dw=dry weight 

2

Literature range references; a(Hamilton et al., 2004); b(Lawhon et al., 1977); c(Smith and Creelman, 2001); d

(Bertrand et al., 2005). 

3

(ILSI, 2011). 
Monsanto Company 12-CT-244U 134 of 620 

VI.B. Compositional Assessment of MON 88701 Conclusion 

Detailed analyses were conducted on nutrient and anti-nutrient levels in MON 88701 

cottonseed from plants treated with dicamba and glufosinate, reported above, and plants 

not treated with dicamba or glufosinate (Appendix E). Component levels for 

MON 88701 were compared to levels in the conventional control. The analytes evaluated 

are consistent with those identified by the OECD as important to understanding the safety 

and nutrition of new varieties of biotechnology-derived cotton (OECD, 2009). These 

compositional comparisons were made by analyzing the acid-delinted cottonseed 

harvested from plants grown at each of eight field sites in the U.S. during the 2010 field 

season. Composition analyses of all samples, conducted in accordance with OECD 

guidelines, were performed for nutrients including proximates (ash, carbohydrates, and 

calories by calculation, moisture, protein, and fat), fibers (ADF, CF, NDF, and TDF), 

amino acids, fatty acids (C8-C22), minerals (calcium, copper, iron magnesium, 

manganese, phosphorus, potassium, sodium, and zinc), and vitamin E. The anti-nutrients 

assessed in this analysis included total and free gossypol and cyclopropenoid fatty acids 

(dihydrosterculic, malvalic, and sterculic). These analyses also included measurements 

of the same nutrients and anti-nutrients in conventional commercial cotton varieties, 

known as reference varieties, to provide data on natural variability of each compositional 

component analyzed. All cotton plants including MON 88701, the conventional control, 

and the conventional commercial reference varieties were treated with maintenance 

pesticides as necessary throughout the growing season. In addition, MON 88701 plots 

were treated at the 3-5 leaf stage with glufosinate herbicide at the label rate (0.5 lbs 

a.i./acre) and at the 6-10 leaf stage with dicamba herbicide at the label rate (0.5 lbs 

a.e./acre). 

For MON 88701 compared to the conventional control, the combined-site analysis of 

cottonseed showed no statistically significant differences (p<0.05) between nutrient and 

anti-nutrient components of MON 88701 and the control for 30 (57.7%) of the 52 mean 

value comparisons. Cottonseed nutrient component differences included mean values for 

five proximates (ash, calories, carbohydrates, moisture, and total fat), three types of fiber 

(ADF, NDF, and TDF), three amino acids (arginine, methionine, and proline), two fatty 

acids (14:0 myristic acid and 18:2 linoleic acid), five minerals (calcium, magnesium, 

manganese, potassium and zinc), and vitamin E. Cottonseed anti-nutrient component 

differences included mean values for dihydrosterculic acid, free and total gossypol. All 

nutrient and anti-nutrient component differences observed in the combined-site statistical 

analysis, whether reflecting increased or decreased MON 88701 mean values with respect 

to the conventional control, were 14.09% or less. Mean values for all significantly 

different nutrient and anti-nutrient components from the combined-site analysis of 

MON 88701, with the exception of methionine, were within the 99% tolerance interval 

established from the conventional, commercial reference varieties grown concurrently in 

the same trial. All combined-site mean values, including methionine, and individual site 

mean values of MON 88701 for all nutrient and anti-nutrient components were within the 

context of the natural variability of commercial cotton composition as published in the 

scientific literature and/or available in the ILSI Crop Composition Database (ILSI, 2011). 
Monsanto Company 12-CT-244U 135 of 620 

Overall, for MON 88701 mean component values observed to be significantly different 

from those of the conventional control, the differences with the control were generally 

shown to be of small relative magnitudes. All MON 88701 mean component values in 

the combined-site analysis, with the exception of methionine, were within the 99% 

tolerance interval established from the conventional commercial references varieties 

grown concurrently and at the same field sites. All combined-site mean values including 

methionine and individual site mean values of MON 88701 for all nutrient and antinutrient components were within the context of the natural variability of commercial 

cotton composition as published in the scientific literature and/or available in the ILSI 

Crop Composition Database (ILSI, 2011). 

For MON 88701 treated with dicamba and glufosinate, compared to the conventional 

control, most of the combined-site differences were not reproducible among the 

individual sites, with the exception of ash and calcium; however, all of the combined-site 

component values were within the range of values reported in the scientific literature 

and/or in the ILSI Crop Composition Database. Additionally, the concentrations of key 

nutrients and anti-nutrients of cottonseed from MON 88701 that was not treated with 

dicamba or glufosinate were also analyzed (See Appendix E). Results from this analysis 

were similar to those of the dicamba and glufosinate treated analysis. Based on the 

results of this composition analysis, it is concluded that cottonseed from MON 88701 is 

compositionally equivalent to conventional cotton and therefore the food and feed safety 

and nutritional quality of this product is comparable to that of the commercially 

cultivated cotton. 

Conventional cotton processing is described in Section II of this document. The 

processing of MON 88701 is not expected to be any different from that of conventional 

cotton. As described in this section, detailed compositional analyses of key components 

of MON 88701 have been performed and have demonstrated that MON 88701 is 

compositionally equivalent to conventional cotton. Additionally, the mode of action of 

the MON 88701 DMO and PAT (bar) proteins, as described in Section V.A., is well 

understood, and there is no reason to expect interactions with important nutrients or 

known anti-nutrients that are present in cotton. Therefore, when MON 88701 and its 

progeny are used on a commercial scale as a source of food or feed, these products are 

not expected to be different from the equivalent foods or feeds originating from 

commercially cultivated cotton. 
Monsanto Company 12-CT-244U 136 of 620 

VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL 

INTERACTIONS ASSESSMENT 

This section provides a comparative assessment of the phenotypic, agronomic, and 

environmental interaction characteristics of MON 88701 compared to the conventional 

control. The data support a conclusion that MON 88701 is not meaningfully different 

from the conventional control with the exception of the dicamba and glufosinatetolerance traits, and therefore is no more likely to pose a plant pest risk or have a 

significant environmental impact compared to conventional cotton. These conclusions 

are based on the results of multiple evaluations from laboratory and field experiments. 

Phenotypic, agronomic, and environmental interaction characteristics of MON 88701 

were evaluated in a comparative manner to assess plant pest potential. These assessments 

included evaluation of seed germination characteristics, plant growth and development 

characteristics, pollen characteristics, observations of plant responses to abiotic stress, 

and plant-disease and plant-arthropod interactions. Results from these assessments 

demonstrate that MON 88701 does not possess: a) increased weediness characteristics; b) 

increased susceptibility or tolerance to specific abiotic stressors, diseases, or arthropods; 

or c) characteristics that would confer a plant pest risk or a significant environmental 

impact compared to the conventional control. 

VII.A. Characteristics Measured for Assessment 

In the phenotypic, agronomic, and environmental interactions assessment of 

MON 88701, data were collected to evaluate altered plant pest potential. A detailed 

description of the regulated article phenotype is requested as part of the petition for 

determination of nonregulated status in 7 CFR § 340.6 including differences from the 

unmodified recipient organism that would “substantiate that the regulated article is 

unlikely to pose a greater plant pest risk than unmodified organism from which it was 

derived.” As part of the characterization of MON 88701, data were collected to provide a 

detailed description of the phenotypic, agronomic, and environmental interactions 

characteristics of MON 88701. 

The plant characterization of MON 88701 encompassed five general data categories: 

1) seed germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive 

development (including pollen characteristics); 4) plant mapping; 5) plant response to 

abiotic stress and interactions with diseases and arthropods. An overview of the 

characteristics assessed is presented in Table VII-1. 

The phenotypic, agronomic, and environmental interactions data were evaluated from a 

basis of familiarity (OECD, 1993) and were comprised of a combination of field and 

laboratory studies conducted by scientists who are familiar with the production and 

evaluation of cotton. In each of these assessments, MON 88701 was compared to a 

conventional control, Coker 130, which has a genetic background similar to MON 88701, 

but does not possess the dicamba and glufosinate-tolerance traits. In addition, multiple 

commercial reference varieties developed through conventional selection and breeding 

(See Appendices F-H and Tables F-1, G-1, G-2, and H-1) were included to provide a 
Monsanto Company 12-CT-244U 137 of 620 

range of comparative values that are representative of the variability in existing 

commercial cotton varieties for each characteristic. Data collected for the various 

characteristics from the commercial reference varieties provides context for interpreting 

experimental results. 
Monsanto Company 12-CT-244U 138 of 620 

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in United States Field Trials and Laboratory Studies 

Data 

Category

Characteristics 

measured 

(associated 

section where 

discussed)1

Evaluation timing (setting 

of evaluation)2

Evaluation description 

(measurement endpoints)

Seed 

germination, 

dormancy, and 

emergence 

Normal 

germinated 

(VII.C.1.) 

Day 4 and 12 (20/30oC) 

(laboratory) 

Percentage of seed producing 

seedlings exhibiting normal 

developmental characteristics 

Abnormal 

germinated 

(VII.C.1.) 

Day 12 (20/30°C) 

(laboratory) 

Percentage of seed producing 

seedlings that could not be 

classified as normal germinated 

Germinated 

(VII.C.1.) 

Day 4, 12, and 18 (10, 20, 

30, 10/20 and 10/30°C) 

(laboratory) 

Percentage of seed that had 

germinated normally and 

abnormally 

Dead 

(VII.C.1.) 

Day 4 and 12 (10, 20, 30, 

10/20, 10/30, and 20/30°C); 

Day 18 (10, 20, 30, 10/20 

and 10/30°C) (laboratory) 

Percentage of seed that had visibly 

deteriorated and become soft to the 

touch (also included non-viable 

hard and nonviable firm-swollen 

seed) 

Viable hard 

(VII.C.1.) 

Day 12 (20/30°C); Day 18 

(10, 20, 30, 10/20 and 

10/30°C) (laboratory) 

Percentage of seed that did not 

imbibe water and remained hard to 

the touch (viability determined by a 

tetrazolium test2

) 

Viable firmswollen 

(VII.C.1.) 

Day 12 (20/30°C); Day 18 

(10, 20, 30, 10/20 and 

10/30°C) (laboratory) 

Percentage of seed that imbibed 

water and were firm to the touch but 

did not germinate (viability 

determined by a tetrazolium test3

) 

Stand count 

(VII.C.2.1.) 

Approximately 14 and 30 

DAP (Field) 

Number of emerged plants in two 

rows, standardized to 20 ft rows 

Final stand count 

(VII.C.2.1.) 

Within approximately 7 

days of harvest (Field) 

Number of plants in two rows, 

standardized to 20 ft rows 

Vegetative 

Growth 

Plant vigor 

(VII.C.2.1.) 

Approximately 14 and 30 

DAP (Field) 

Rated on a 1-9 scale, where 1 = 

excellent, 5 = average, and 9 = poor 

vigor 

Plant height (cm) 

(VII.C.2.1.) 

Approximately 30 DAP and 

within approximately 7 days 

of harvest (Field) 

Distance from cotyledonary node (0 

node) to the uppermost terminal bud 

on 10 plants from two rows 

Nodes above 

white flower 

(NAWF) 

(VII.C.2.1.) 

Three weekly observations 

starting approximately 7 

days after first flower 

(Field) 

Number of nodes from upper most 

first-position white flower to the 

terminal bud on 10 plants from two 

rows 

 
Monsanto Company 12-CT-244U 139 of 620 

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in United States Field Trials and Laboratory Studies 

(continued) 

Data 

Category

Characteristics 

Measured 

(associated 

section where 

discussed) 

Evaluation timing (setting 

of evaluation)

Evaluation description 

(measurement endpoints)

Reproductive 

Development 

Seedcotton yield 

(kg/ha) 

(VII.C.2.1.) 

At harvest (Field) Hand harvested all seedcotton from 

two rows 

Seed index (g per 

100 seed) 

(VII.C.2.1.) 

Post harvest (Field) Mass of 100 ginned, fuzzy seed 

Total seed per boll 

(VII.C.2.1.) 

Post harvest (Field) Average number of seeds per boll 

calculated from a 50-boll sample 

Mature seed per 

boll (VII.C.2.1.) 

Post harvest (Field) Average number of mature seed in a 

boll calculated from a 50-boll 

sample 

Immature seed per 

boll (VII.C.2.1.) 

Post harvest (Field) Average number of immature seed 

in a boll calculated from a 50-boll 

sample 

Boll weight (g) 

(VII.C.2.1.) 

Post harvest (Field) Average mass of a single boll 

calculated from a 50-boll sample 

Fiber micronaire 

(mic units) 

(VII.C.2.1.) 

Post harvest (Field) Measure of fiber fineness and 

maturity (expressed in 

dimensionless micronaire (mic) 

units) 

Fiber elongation 

(%) (VII.C.2.1.) 

Post harvest (Field) Measure of the tensile-elastic 

behavior of the fiber. It is a 

measure of how much the fibers 

stretch before they tear. 

Fiber strength 

(g/tex) (VII.C.2.1.) 

Post harvest (Field) Force in grams required to break a 

bundle of fibers one tex unit in size. 

One tex is the mass in grams of 

1,000 meters of fiber. 

Fiber length (cm) 

(VII.C.2.1.) 

Post harvest (Field) Average length of the longer half of 

combed fibers 

Fiber uniformity 

(%) (VII.C.2.1.) 

Post harvest (Field) Ratio between the mean length and 

the longer half mean length of fibers 

Pollen viability 

(VII.C.3.) 

Flowering (laboratory) Percentage of viable pollen; viable 

pollen stains purple due to the 

presence of vital cytoplasmic 

content 

Pollen 

morphology 

(VII.C.3.) 

Flowering (laboratory) Diameter (μm) of viable pollen 

grains and observations 

 
Monsanto Company 12-CT-244U 140 of 620 

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in United States Field Trials and Laboratory Studies 

(continued) 

Data 

Category

Characteristics 

measured 

(associated 

section where 

discussed) 

Evaluation timing (setting 

of evaluation) 

Evaluation description 

(measurement endpoints)

Plant Mapping 

Characteristics 

Number of 

mainstem nodes 

per plant 

(VII.C.2.2.) 

At harvest (Field) Number of mainstem nodes from 

cotyledonary node (node 0) to 

uppermost terminal meristem on 10 

plants per plot 

Number of nodes 

to first fruiting 

branch per plant 

(VII.C.2.2.) 

At harvest (Field) Number of nodes from 

cotyledonary node (node 0) up to 

first fruiting branch on 10 plants per 

plot 

Number of firstposition bolls 

(total, normal & 

abnormal) per 

plant 

(VII.C.2.2.) 

At harvest (Field) Number of bolls at first position on 

fruiting branches off of mainstem 

on 10 plants per plot 

Number of 

vegetative bolls 

per plant 

(VII.C.2.2.) 

At harvest (Field) Number of vegetative bolls on 10 

plants per plot 

Total bolls per 

plant (VII.C.2.2.) 

Post harvest (Field) Sum of first-position bolls, secondposition bolls, and vegetative bolls 

per plant on 10 plants per plot 

Retention of firstposition bolls (%) 

(VII.C.2.2.) 

Post harvest (Field) Calculated first-position bolls 

relative to number of fruiting 

branches on the mainstem 

First-position bolls 

(%) (VII.C.2.2.) 

Post harvest (Field) Calculated first-position bolls per 

plant relative to total bolls per plant 

 
Monsanto Company 12-CT-244U 141 of 620 

Table VII-1. Phenotypic, Agronomic, and Environmental Interaction 

Characteristics Evaluated in United States Field Trials and Laboratory Studies 

(continued) 

Data 

Category

Characteristics 

measured 

(associated 

section where 

discussed) 

Evaluation timing (setting 

of evaluation) 

Evaluation description 

(measurement endpoints) 

Plantenvironmental 

interactions 

Plant response to 

abiotic stress 

(VII.C.2.3.)

Four times per growing 

season – approximately 30, 

60, 90 and 120 DAP (Field) 

Qualitative assessment of each plot, 

with rating on a 0-9 scale, where 0 

= no symptoms and 9 = severe 

symptoms 

Disease damage 

(VII.C.2.3.)

Four times per growing 

season – approximately 30, 

60, 90 and 120 DAP (Field) 

Qualitative assessment of each plot, 

with rating on a 0-9 scale, where 0 

= no symptoms and 9 = severe 

symptoms 

Arthropod-related 

damage (VII.C.2.3.)

Four times per growing 

season – approximately 30, 

60, 90 and 120 DAP (Field) 

Qualitative assessment of each plot, 

with rating on a 0-9 scale, where 0 

= no symptoms and 9 = severe 

symptoms 

Thrips damage 

assessment 

(VII.C.2.3.)

Three assessments at 

approximately 14, 21 and 28 

DAP (Field) 

Specific quantitative assessment of 

thrips from 10 plants in each plot 

using a 0-5 scale, where 0 = no 

thrips or visible damage and 5 = 

numerous thrips or severe damage 

from thrips 

Heliothine damage 

assessment 

(VII.C.2.3.)

Four assessments at 

approximately 45, 60, 75 

and 90 DAP (Field) 

Percent damage (number of 

damaged fruiting bodies divided by 

total number of fruiting bodies) and 

number live larvae on the top 7 

nodes of 10 plants in each plot 

Arthropod 

abundance 

(VII.C.2.3.)

Four collections at 

approximately 30, 60, 90 

and 120 DAP (Field) 

Number of pest and beneficial 

arthropods 

1

All cottonseed was from mature open bolls. 

2

Cotton plant growth stages were determined using descriptions and guidelines outlined in Cotton Growth 

and Development (Ritchie et al., 2007). 

3

Viability of hard and firm-swollen seed were determined by a tetrazolium test (AOSA, 2007). 
Monsanto Company 12-CT-244U 142 of 620 

VII.B. Interpretation of Phenotypic and Environmental Interaction Data 

Plant pest risk assessments for biotechnology-derived crops are comparative assessments. 

Familiarity provides a basis from which the potential environmental impact of a 

biotechnology-derived plant can be evaluated. The concept of familiarity is based on the 

fact that the biotechnology-derived plant is developed from a well-characterized 

conventional plant variety. Familiarity considers the biology of the crop, the introduced 

trait(s), the receiving environment and the interaction of these factors, and provides a 

basis for comparative environmental risk assessment between a biotechnology-derived 

plant and its conventional counterpart. 

Expert knowledge and experience with conventionally bred cotton was the basis for 

selecting appropriate endpoints and estimating the range of responses that would be 

considered typical for cotton. As such, MON 88701 was compared to the conventional 

control, Coker 130, in the assessment of measured characteristics. An overview of the 

characteristics assessed is presented in Table-VII-1. Evaluation of environmental 

interaction characteristics (e.g., plant abiotic stress, plant-disease, and plant-arthropod 

interactions) was also considered in the plant pest assessment. Based on all of the data 

collected, an assessment was made to determine if MON 88701 is likely to pose an 

increased plant pest risk compared to commercial cotton. Prior to analysis, the overall 

dataset was evaluated for possible evidence of biologically relevant changes and an 

unexpected plant response. No unexpected observations or issues were identified. 

VII.B.1. Interpretation of Detected Differences Criteria 

Comparative plant characterization data between a biotechnology-derived crop and the 

conventional control are interpreted in the context of contributions to increased plant 

pest/weed potential as assessed by APHIS. Under the framework of familiarity, 

characteristics for which no differences are detected support a conclusion of no increased 

plant pest/weed potential of the biotechnology-derived crop compared to the conventional 

crop. Characteristics for which differences are detected are considered in a step-wise 

method (Figure VII-1) or in a similar fashion. All detected differences for a 

characteristic are considered in the context of whether or not the difference would 

increase the plant pest/weed potential of the biotechnology-derived crop. Ultimately, a 

weight-of-evidence approach considering all characteristics and data is used for the 

overall risk assessment of differences and their significance. In detail, Figure VII-1 

illustrates the stepwise assessment process employed: 

 
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Figure VII-1. Schematic Diagram of Agronomic and Phenotypic Data 

Interpretation Methods 

Note: A “no” answer at any step indicates that the characteristic does not contribute to a biological or 

environmental change for the crop in terms of plant pest/weed potential and subsequent steps are not 

considered. If the answer is “yes” or “uncertain” the subsequent step is considered. 

Steps 1 and 2 - Evaluate Detected Statistically Significant Differences 

Data on each measured characteristic are statistically analyzed, where appropriate, within 

each individual site and in a combined-site analysis, in which the data are pooled among 

sites. All statistically significant differences are evaluated and considered in the context 

of a change in plant pest/weed potential. Differences detected in individual site analyses 

that are not detected when data across multiple environments are pooled in the combinedsite analysis are considered not biologically meaningful in terms of plant pest/weed 

potential and, therefore, are not further considered in subsequent steps. Any difference 

detected in the combined-site analysis is further assessed. 

Step 3 - Evaluate differences in the context of commercial reference varieties included 

in the Study 

If a difference for a characteristic is detected in the combined-site analysis across 

multiple environments, then the mean value of the biotechnology-derived crop for the 

Hazard identification and 

risk assessment on 

difference 

Outside variation for crop? No 

Yes 

No 

Yes 

Adverse in terms of plant pest 

potential? 

Yes 

Outside variation of study references? 

No 

Not adverse; the direction 

or magnitude of the 

detected difference in the 

measured characteristic 

does not contribute to a 

biological or 

environmental change for 

the crop in terms of plant 

pest/weed potential 

Yes 

Statistical differences detected 

in combined-site analysis? 

No 

Differences detected in the combined-site 

and individual site analyses are evaluated

Step 2 

Step 3 

Step 4 

The measured

characteristic does not 

contribute to a biological 

or environmental change 

for the crop in terms of 

plant pest/weed potential 

Step 5 

Step 6 

Step 1 
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characteristic is assessed relative to the range of variation of the commercial reference 

varieties included in the study (e.g., reference range). 

Step 4 - Evaluate Differences in the Context of the Crop 

If the mean value of the characteristics for a biotechnology-derived crop is outside the 

variation of the commercial reference varieties included in the study, the mean value of 

the biotechnology-derived crop is assessed relative to known values common for the crop 

(e.g., published values). 

Step 5 - Relevance of Difference to Plant Pest/Weed Potential 

If the mean value of the characteristics for a biotechnology-derived crop is outside the 

range of values common for the crop, the detected difference for the characteristic is then 

assessed for whether or not it is adverse in terms of plant pest/weed potential. 

Step 6 - Conduct Risk Assessment on Identified Hazard 

If an adverse effect (hazard) is identified, risk assessment on the difference is conducted. 

The risk assessment considers contributions to enhanced plant pest/weed potential of the 

crop itself, the impact of differences detected in other measured characteristics, and 

potential for and effects of trait introgression into any populations growing outside of 

cultivated environments or into a sexually-compatible species. 

VII.B.1.1. Interpretation of Vigor and Environmental Interactions Data 

For the qualitative assessments of vigor and abiotic stress response, disease damage, and 

arthropod damage, the biotechnology-derived crop and conventional control were 

considered different in plant response ratings if the range of values or injury symptoms 

did not overlap between the biotechnology-derived crop and the conventional control 

across all four replications. Any observed differences between the biotechnology-derived 

crop and conventional control were assessed for biological significance in the context of 

the range of the commercial reference varieties, and consistency in other observation 

times and sites. Differences that are not consistently observed at other 

observations/collections and sites are considered not biologically meaningful in terms of 

plant pest potential or an adverse environmental impact. 

Quantitative assessments of arthropod damage were analyzed within individual sites and 

pooled across sites in a combined-site analysis. Statistically significant differences 

detected between the biotechnology-derived crop and conventional control were 

evaluated using the method outlined in Figure VII-1. 

 

Quantitative assessments of arthropod abundance were only analyzed within each 

individual site. Statistically significant differences between the biotechnology-derived 

crop and conventional control were assessed for biological significance in the context of 

the range of the commercial reference varieties, and for consistency with other collection 

times and collection sites. Differences that are not consistently detected at other times 
Monsanto Company 12-CT-244U 145 of 620 

and sites are considered not biologically meaningful in terms of plant pest potential or an 

adverse environmental impact. 

VII.C. Comparative Assessments of the Phenotypic, Agronomic, Plant Mapping, 

and Environmental Interaction Characteristics of MON 88701 

This section provides the results of comparative assessments conducted in replicated 

laboratory and multi-site field experiments to provide a detailed phenotypic, agronomic, 

plant mapping, and environmental interaction description of MON 88701. The 

MON 88701 characteristics evaluated in these assessments included: seed dormancy and 

germination characteristics (Section VII.C.1.), plant phenotypic, plant mapping, and 

environmental interaction observations under field conditions (Section VII.C.2.), and 

pollen characteristics (Section VII.C.3). Additional details for each assessment are 

provided in Appendices F through H. 

VII.C.1. Seed Dormancy and Germination Characteristics 

USDA-APHIS considers the potential for weediness to constitute a plant pest factor 

(7 CFR § 340.6). Seed germination and dormancy mechanisms vary among species and 

their genetic basis tends to be complex. Seed dormancy (e.g., hard seed) is an important 

characteristic that is often associated with plants that are considered weeds (Anderson, 

1996; Lingenfelter and Hartwig, 2007). Cotton does not exhibit significant levels of seed 

dormancy as this characteristic has been removed through selection and conventional 

breeding (Christiansen and Moore, 1959). To assess germination characteristics, 

standardized germination assays are routinely used. The Association of Official Seed 

Analysts (AOSA), an internationally recognized seed testing organization, recommends a 

temperature range of alternating 20/30°C as optimal for testing the germination 

characteristics of cottonseed (AOSA, 2007; 2010a; 2010b; AOSA/SCST, 2010). 

Comparative assessments of seed dormancy and germination characteristics were 

conducted on MON 88701 and the conventional control. In addition, nine unique 

commercial reference varieties were included to provide a range of comparative values 

that are representative of existing variability in commercial cotton varieties. The seed 

lots for MON 88701, the conventional control, and the commercial reference varieties 

were produced in three replicated field trials during 2010 located in Arkansas (ARPR), 

North Carolina (NCME), and Texas (TXPL). These geographic areas represent 

environmentally relevant conditions for cotton production. In addition to the AOSA 

recommended temperature range of 20/30°C, seed was tested at five additional 

temperature regimes of 10, 20, 30, alternating 10/20, and alternating 10/30°C to assess 

seed germination properties. The details of the materials, experimental methods, and 

germination data from all of the individual production sites are presented in Appendix F. 

In the combined-site analysis, in which the data were pooled from the three individual 

sites, no statistically significant differences (5% level of significance) were detected 

between MON 88701 and the conventional control for any characteristic at the AOSA 

temperature regime (alternating 20o

C/30oC), or at the temperature regimes of 10oC, 20oC, 

alternating 10o

C/20oC, or alternating 10oC/30oC (Table VII-2). MON 88701 had a 
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significantly higher percentage of germinated seed (96.7 vs. 94.4, respectively) and lower 

percent dead seed (3.3 vs. 5.6, respectively) than the conventional control at 30oC. These 

differences were small in magnitude, not observed at other temperatures and the mean 

values of percent germinated and dead seed for MON 88701 were within the range of 

commercial reference varieties. Therefore, the differences in percent germinated and 

dead seed at 30o

C are not considered to be biologically meaningful in terms of altered 

dormancy or germination characteristics (See Figure VII- 1 Step 3, answer “no”). 

The dormancy and germination characteristics evaluated were used to assess MON 88701 

in the context of plant pest risk. The results of this assessment, particularly the fact that 

no hard seed were observed at any temperature, support the conclusion that there are no 

seed germination characteristic differences between MON 88701 and the conventional 

control. Thus, the introduction of the dicamba and glufosinate-tolerant traits into cotton 

is not likely to result in increased plant pest potential, increased weediness, or an altered 

environmental impact from MON 88701 compared to commercially cultivated cotton. 
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Table VII-2. Combined-Site Comparison of MON 88701 to Conventional Control 

for Germination Characteristics 

Temperature 

Regime

Germination

Category

Mean % (S.E.)1

MON 887012 Control Reference Range (%)3

10 °C Germinated 32.8 (6.3) 34.8 (4.7) 14.2 - 58.0

Viable Hard 0.0 (0.0) 0.3 (0.2) 0.2 - 23.8

Dead 22.8 (3.8) 22.1 (3.3) 7.7 - 22.8

Viable Firm-Swollen 44.3 (7.3) 42.8 (5.9) 29.0 - 66.5

20 °C Germinated 95.7 (0.7) 95.3 (1.2) 88.5 - 97.8

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 - 4.0

Dead 4.3 (0.7) 4.7 (1.2) 2.0 - 9.0

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0 - 2.0

30 °C Germinated 96.7 (0.8)* 94.4 (1.2) 90.5 - 97.8

Viable Hard† 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0

Dead 3.3 (0.8)* 5.6 (1.2) 2.3 - 9.5

Viable Firm-Swollen† 0.0 (0.0) 0.0 (0.0) 0.0 - 0.0

10/20 °C Germinated 94.4 (1.3) 92.0 (1.4) 64.3 - 91.3

Viable Hard 0.0 (0.0) 0.1 (0.1) 0.1 - 17.5

Dead 4.3 (0.7) 6.6 (1.2) 5.0 - 10.3

Viable Firm-Swollen 1.3 (1.0) 1.3 (0.6) 0.0 - 21.3

10/30 °C Germinated 95.7 (1.0) 95.1 (1.5) 90.8 - 95.5

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 - 2.0

Dead 4.3 (1.0) 4.9 (1.5) 4.3 - 7.8

Viable Firm-Swollen 0.0 (0.0) 0.0 (0.0) 0.0 - 1.5

20/30 °C Normal Germinated 89.6 (1.9) 88.0 (2.9) 80.8 - 92.8

(AOSA)4 Abnormal Germinated 4.8 (1.2) 6.0 (1.3) 2.0 - 6.3

Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 - 4.8

Dead 5.6 (0.9) 6.0 (2.0) 4.0 - 10.8

Viable Firm-Swollen 0.1 (0.1) 0.0 (0.0) 0.0 - 3.8

Note: The experimental design was a split-plot with four replications (n = 12) and statistical analysis 

consisted of an analysis of variance (ANOVA) model. 

*Statistically significant differences detected (α=0.05) between MON 88701 and the conventional control. 1

S.E. = Standard Error. 

2

In some instances, the total percentage of MON 88701 did not equal 100% due to numerical rounding of 

the means. 

3

Minimum and maximum means determined from among the commercial reference varieties. 

4

AOSA recommended. 

†No statistical comparison could be made due to lack of variability in the data.
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VII.C.2. Field Phenotypic, Agronomic, Plant Mapping, and Environmental 

Interactions Characteristics 

Phenotypic, agronomic, and plant mapping characteristics, and environmental 

interactions were evaluated under field conditions as part of the plant characterization 

assessment of MON 88701. These data were developed to provide USDA-APHIS with a 

detailed description of MON 88701 relative to the conventional control and commercial 

reference varieties. According to 7 CFR § 340.6, as part of the petition to seek 

deregulation, a petitioner must submit “a detailed description of the phenotype of the 

regulated article.” This information is being provided to assess whether there are 

phenotypic differences between MON 88701 and the conventional control that may 

impact its plant pest/weed potential. Environmental interactions were also assessed as an 

indirect indicator of phenotypic changes to MON 88701 compared to the same 

comparators described above and are also considered in the plant pest assessment. 

The results of the assessment of agronomic, phenotypic, and plant mapping 

characteristics demonstrated that the introduction of the dicamba and glufosinatetolerance traits did not meaningfully alter the weediness of MON 88701 compared to the 

conventional control. Furthermore, the lack of meaningful differences in plant response 

to abiotic stress, disease damage, arthropod-related damage, and pest- and beneficialarthropod abundance also support the conclusion that the introduction of the dicamba and 

glufosinate-tolerance traits are not likely to result in increased plant pest potential, 

increased weediness, or an adverse environmental impact from MON 88701 compared to 

the conventional control. 

VII.C.2.1. Field Phenotypic and Agronomic Characteristics 

Two field studies were conducted during 2010 to evaluate phenotypic and agronomic 

characteristics of MON 88701 compared to the conventional control. One study was 

designed to collect phenotypic and environmental interaction data, while the other was 

designed for the collection of plant mapping data and tissue samples for expression and 

compositional analyses. Field sites in both studies were planted in randomized complete 

block designs with four replicates per site. The sites were selected to provide a diverse 

range of environmental and agronomic conditions representative of commercial cotton 

production areas in North America (Table VII-3). All plots of MON 88701, the 

conventional control, and the commercial reference varieties at each site were uniformly 

managed in order to assess whether the introduction of the dicamba and glufosinatetolerance traits altered the phenotypic and agronomic characteristics of MON 88701 

compared to the conventional control. Both studies included MON 88701 that was not 

treated with dicamba or glufosinate herbicides to assess the effects of the traits on the 

plant. 

Study 1 was conducted at 15 sites in the U.S. (Table VII-3). MON 88701, the 

conventional control, and four commercial reference varieties (three conventional 

reference varieties and one glyphosate-tolerant reference variety) were evaluated at each 
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site. Across sites, a total of 11 commercial reference varieties (Table G-1) were 

evaluated. 

An additional study, Study 2, was conducted at 11 sites in the U.S. (Table VII-3). 

MON 88701, the conventional control, and four conventional reference varieties were 

evaluated at each site. Across sites a total of eight unique commercial reference varieties 

were evaluated. This study was designed for collection of plant mapping data, as well as, 

the production of tissues for the expression and compositional analyses discussed above 

in Sections V.C. and VI, respectively. Study 2 generated plant mapping information and 

data across test locations treated and not treated with dicamba or glufosinate herbicides in 

Study 2 (See section VII.C.2.2, C.2.3.2 and G.12.3), allowing for assessment of 

MON 88701 under the agronomic system that it is expected to be used. 

Results from Study 1 and Study 2 are presented in the following sections: 

Table VII-3. Study 1 and Study 2 Data Location Summary 

Data/Results1 Study 1 Study 2 Petition Location 

Not treated phenotypic characteristics   Section VII.C.2.1 

Not treated plant mapping  Section VII.C.2.2 

Not treated environmental interactions   Section VII.C.2.3 

Treated phenotypic characteristics  Appendix G.12.3 

Treated plant mapping  Appendix G.13.2 

Treated environmental characteristics 

through plant mapping 

 Section VII.C.2.3; 

Appendix G.13.2 

1

Not treated = not treated with dicamba or glufosinate herbicides; treated = treated with dicamba and 

glufosinate herbicides. 

All plant, seed, and fiber characteristic data, except for plant vigor (qualitative data), 

were statistically analyzed within each site (i.e., individual site analysis) and in a 

combined-site analysis in which the data were pooled across all sites within a study. The 

reference range was determined from the minimum and maximum mean values from the 

commercial reference varieties to provide phenotypic characteristic values representative 

of commercial cotton varieties. 

For the assessment of plant vigor MON 88701 and the conventional control were 

considered different in plant response rating if the range of values did not overlap 

between the MON 88701 and the conventional control across all four plot replications. 

Any observed differences between MON 88701 and the conventional control were 

assessed for biological significance in the context of the range of the commercial 

reference varieties, and for consistency in other observations and sites. Differences that 
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are not consistently observed at other observations and sites are considered not 

biologically meaningful in terms of plant pest potential or an adverse environmental 

impact. 

A description of the evaluated phenotypic characteristics and the designated 

developmental stages when evaluations occurred are listed in Table VII-1. The results 

from Study 1 and 2 from combined-site analyses of MON 88701 plots not treated with 

either dicamba or glufosinate compared to the conventional control are presented in the 

following sub-sections. The results of these studies demonstrate that the introduction of 

the dicamba and glufosinate-tolerance traits did not alter MON 88701 compared to the 

conventional control in terms of weediness. The individual site data comparisons and 

methods and detailed results of the supplemental evaluations of MON 88701 plots that 

were treated with dicamba and glufosinate in Study 2 are presented and discussed in 

Appendix G. 
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Table VII-4. Field Phenotypic Evaluation Sites for MON 88701 during 2010 

Study 1 Locations1

 Study 2 Locations2

County, State Site Code County, State Site Code 

 

Jackson, AR ARAU Crittenden, AR ARPR 

Crittenden, AR ARPR Desha, AR ARTI 

Desha, AR ARTI Tift, GA GACH 

Tift, GA GACH Twiggs, GA GAJE 

Twiggs, GA GAJE Pawnee, KS KSLA 

Pawnee, KS KSLA Rapides, LA LACH 

Rapides, LA LABU Perquimans, NC NCBD 

Rapides, LA LACH Caswell, NC NCME 

Perquimans, NC NCBD Dona Ana, NM NMLC 

Caswell, NC NCME Barnwell, SC SCEK 

Dona Ana, NM NMGA Hale, TX TXPL 

Dona Ana, NM NMLC 

Barnwell, SC SCEK 

Hale, TX TXPL 

San Patricio, TX TXPO 

 

Note: Field trials at all sites were conducted under USDA Notification number 10-071-101n. 

1

MON 88701 was not treated with dicamba or glufosinate herbicides in Study 1. 

2

Study 2 included plots not treated with dicamba or glufosinate herbicides and plots treated with 

dicamba and glufosinate herbicides. 

 

VII.C.2.1.1. Field Phenotypic and Agronomic Characteristics of MON 88701 – 

Study 1

Vigor ratings were collected from each plot using a 1-9 scale, where 1 is outstanding 

plant vigor and 9 is poorest plant vigor. Since vigor data are categorical (qualitative), the 

data were not statistically analyzed. There were no differences observed between 

MON 88701 and the conventional control in plant vigor (Table G-7) at 14 and 30 days 

after planting (DAP) for 29 of 30 comparisons from all sites. At ARPR at 30 days after 

planting, MON 88701 had lower plant vigor than the conventional control (ranges of 4.0-

4.0 vs. 2.0-3.0, respectively), but was within the range of the commercial reference 

varieties. Since only one difference (out of 30) was identified and it fell within the 

reference range, MON 88701 is unlikely to have increased plant pest/weed potential or an 

adverse environmental impact compared to the conventional control (See Section 

VII.B.1.1.). 
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In the combined-site analysis (Table VII-5), no statistically significant differences were 

detected between MON 88701 and the conventional control for stand count at 14 and 30 

DAP, final stand count, number of nodes above white flower at observation 1, seedcotton 

yield, number of immature seed per boll, weight per boll, fiber micronaire, fiber 

elongation, fiber uniformity, and fiber length. The following statistically significant 

differences were detected in the combined-site analysis. MON 88701 plants were shorter 

at 30 DAP (18.3 vs. 19.7 cm) and at harvest (109.8 vs. 116.4 cm), had increased nodes 

above white flower at observation 2 (6.0 vs. 5.7) and observation 3 (4.9 vs. 4.6), a 

decreased seed index (9.8 vs. 10.5 g per 100 fuzzy seed), increased total seed per boll 

(29.0 vs. 27.4), increased mature seeds per boll (22.6 vs. 19.7), and increased fiber 

strength (31.8 vs. 31.0 g/tex) compared to the conventional control. However, the mean 

values of MON 88701 were within the range of the commercial reference varieties for the 

eight characteristics listed above. Thus, MON 88701 is unlikely to have increased plant 

pest/weed potential or an adverse environmental impact compared to the conventional 

control. (See Figure VII-1, Step 3, answer “no”). 
Monsanto Company 12-CT-244U 

Table VII-5. Study 1 Combined-Site Comparison of MON 88701 to Conventional Control during 2010 Agronomic Characteristics 

Phenotypic Characteristic (units)

MON 88701 Control Refer

Mean (SE)2 Mean (SE) Minimum 

Stand Count at 14 DAP3 (# in 2 rows per plot) 146.0 (4.3) 152.4 (4.2) 96.2 Stand Count at 30 DAP (# in 2 rows per plot) 131.8 (5.5) 137.7 (5.5) 86.7 

Final Stand Count at harvest (# in 2 rows per plot) 125.2 (5.9) 128.9 (6.0) 88.2 

Plant Height at 30 DAP (cm) 18.3 (1.2)* 19.7 (1.2) 8.3 

Plant Height at harvest (cm) 109.8 (3.8)* 116.4 (4.2) 84.4 

Nodes Above White Flower: (# of nodes at observation 1) 6.9 (0.2) 6.7 (0.2) 5.8 

 (# of nodes at observation 2) 6.0 (0.2)* 5.7 (0.2) 5.1 

 (# of nodes at observation 3) 4.9 (0.3)* 4.6 (0.3) 3.7 

Seedcotton Yield (kg/ha) 2937.8 (153.7) 2869.9 (156.0) 2107.0 

Seed Index (g per 100 fuzzy seed) 9.8 (0.2)* 10.5 (0.1) 8.9 

Total Seed per Boll (# per boll) 29.0 (0.4)* 27.4 (0.3) 26.4 

Mature Seed per Boll (# per boll) 22.6 (0.7)* 19.7 (0.6) 11.8 Immature Seed per Boll (# per boll) 6.4 (0.5) 7.7 (0.5) 3.4 

Weight per Boll (g) 4.8 (0.1) 4.8 (0.1) 4.2 

Fiber Micronaire (mic units)4 4.6 (0.1) 4.5 (0.1) 4.0 

Fiber Elongation (%) 6.0 (0.1) 6.0 (0.1) 4.8 Fiber Strength (g/tex) 31.8 (0.2)* 31.0 (0.1) 30.7 

Fiber Uniformity (%) 84.0 (0.1) 83.7 (0.1) 83.7 

Fiber Length (cm) 2.8 (0.0) 2.9 (0.0) 2.8 

* Indicates a statistically significant difference (

α=0.05) between MON 88701 and the conventional control (n = 60). 1 Reference range = Minimum and maximum mean values across all 15 sites and eleven references from the Study 1 field trial. 2 SE = standard error.

3DAP = days after planting. 

4 Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).
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VII.C.2.1.2. Field Phenotypic and Agronomic Characteristics of MON 88701 – 

Study 2

Vigor ratings were collected from each plot using a 1-9 scale, where 1 is outstanding 

plant vigor and 9 is poorest plant vigor. Due to the non-specific nature of the scale used, 

the data were not statistically analyzed. There were no differences between MON 88701 

and the conventional control in plant vigor (Table G-10) at 14 and 30 DAP for 22 of 22 

comparisons from all sites. Therefore, the lack of differences in plant vigor supports a 

conclusion that MON 88701 is unlikely to have increased plant pest/weed potential or an 

adverse environmental impact compared to the conventional control (See Section 

VII.B.1.1.). 

In the combined-site analysis of MON 88701 (Table VII-6), no statistically significant 

differences were detected between MON 88701 and the conventional control for stand 

count at 14 and 30 DAP, final stand count at harvest, nodes above white flower 

observations 1 and 3, seedcotton yield, immature seed per boll, weight per boll, fiber 

micronaire, fiber elongation, fiber uniformity, and fiber length. The following 

statistically significant differences were detected in the combined-site analysis. 

MON 88701 plants were shorter than the conventional control at the 30 DAP (18.0 vs. 

19.2 cm) and at harvest (96.1 vs. 105.0 cm), had increased nodes above white flower at 

observation 2 (5.5 vs. 5.2), had a decreased seed index (9.4 vs. 10.7 g/100 seed), had 

increased total seed per boll (29.1 vs. 27.0 seed) and increased mature seed per boll (23.3 

vs. 20.1), and had increased fiber strength as compared to the conventional control (30.9 

vs. 30.2 g/tex). However, the mean values of MON 88701 were within the reference 

range for the seven characteristics listed above. Thus, MON 88701 is unlikely to have 

increased plant pest/weed potential or an adverse environmental impact compared to the 

conventional control. (See Figure VII-1, Step 3, answer “no”). 

Results of the supplemental evaluation of MON 88701, described above, under the 

agronomic system in which it is expected to be used (i.e., MON 88701 treated with 

dicamba and glufosinate herbicides) are provided in Appendix G and further demonstrate 

that MON 88701 is unlikely to have increased plant pest/weed potential or an adverse 

environmental impact compared to the conventional control (See G.12.3). 

VII.C.2.1.3. Field Phenotypic and Agronomic Characteristics – Conclusion 

The results of the agronomic and phenotypic assessments on MON 88701 from Study 1 

and Study 2 demonstrate that the introduction of the dicamba and glufosinate-tolerant 

traits did not alter MON 88701 compared to the conventional control relating to plant 

pest/weed potential. Additionally, agronomic and phenotypic assessments of 

MON 88701 treated with dicamba and glufosinate herbicides were also comparable to the 

conventional control. Thus, the introduction of the dicamba and glufosinate-tolerant 

traits into cotton is not likely to result in increased plant pest potential, increased 

weediness or an altered environmental impact from MON 88701 compared to 

commercially cultivated cotton. 
Monsanto Company 12-CT-244U 

Table VII-6. Study 2 Combined-Site Comparison of MON 88701 to Conventional Control during 2010 Agronomic Characteristics Phenotypic Characteristic (units)

MON 887011 Control Refer

Mean (SE)3 Mean (SE) Minimum 

Stand Count at 14 DAP4 (# in 2 rows per plot) 150.5 (4.2) 155.0 (4.4) 108.4 Stand Count at 30 DAP (# in 2 rows per plot) 149.4 (3.9) 152.8 (4.0) 105.8 

Final Stand Count at harvest (# in 2 rows per plot) 146.3 (4.0) 150.5 (4.3) 110.5 

Plant Height at 30 DAP (cm) 18.0 (1.1)* 19.2 (1.1) 11.4 

Plant Height at harvest (cm) 96.1 (4.2)* 105.0 (4.9) 85.2 

Nodes Above White Flower: (# of nodes at observation 1) 6.6 (0.2) 6.4 (0.2) 6.0 

 (# of nodes at observation 2) 5.5 (0.3)* 5.2 (0.3) 4.8 

 (# of nodes at observation 3) 4.1 (0.2) 3.8 (0.2) 3.2 

Seedcotton Yield (kg/ha) 3,334.1 (210.2) 3,164.1 (210.8) 2,181.7 

Seed Index (g per 100 fuzzy seed) 9.4 (0.2)* 10.7 (0.2) 9.4 

Total Seed per Boll (# per boll) 29.1 (0.4)* 27.0 (0.4) 26.1 

Mature Seed per Boll (# per boll) 23.3 (0.7)* 20.1 (0.8) 14.6 Immature Seed per Boll (# per boll) 5.8 (0.6) 6.9 (0.6) 2.7 

Weight per Boll (g) 4.9 (0.1) 4.8 (0.1) 4.5 Fiber Micronaire (mic units)5 4.7 (0.1) 4.6 (0.1) 4.2 Fiber Elongation (%) 6.1 (0.1) 6.2 (0.1) 5.6 Fiber Strength (g/tex) 30.9 (0.2)* 30.2 (0.2) 30.7 

Fiber Uniformity (%) 83.6 (0.2) 83.4 (0.2) 82.8 

Fiber Length (cm) 2.8 (0.0) 2.8 (0.0) 2.7 

* Indicates a statistically significant difference (

α=0.05) between MON 88701 and the conventional control (n = 44). 1 MON 88701 plots were not treated with dicamba or glufosinate. 2Reference range = Minimum and maximum mean values across all 11 sites and eight references from the Study 2 field trial. 

3 SE = standard error.

4 DAP = days after planting. 

5 Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).
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VII.C.2.2. Plant Mapping Characteristics 

Plant mapping is a process commonly used by agronomists and breeders to quantify 

growth and development parameters of a cotton plant, including boll retention (Kerby et 

al., 2010; Plant and Kerby, 1995). Plant mapping parameters are used to measure crop 

productivity and are influenced by abiotic and biotic stressors. Plant mapping 

characteristics (Table VII-7) were evaluated under field conditions to provide USDAAPHIS with a detailed description of MON 88701 boll retention and distribution relative 

to the conventional control and commercial reference varieties, and to consider 

differences in context of pest/weed potential. 

In addition to the methods discussed in Section VII.C.2.1, 10 plants from each plot in 

Study 2 were mapped at harvest for the number of mainstem nodes, number of nodes to 

the first fruiting branch, total number of bolls (sum of first-position, second-position and 

vegetative bolls), total number of first-position bolls, and total number of vegetative 

bolls. The percent of first-position bolls relative to total bolls and percent retention of 

first-position bolls on mainstem fruiting branches were calculated from plant mapping 

data. The combined-site statistical analysis comparing MON 88701 not treated with 

either dicamba or glufosinate to the conventional control is summarized below. Results 

of the individual site data comparisons are presented in Appendix G.13.1. Also the 

experimental methods and detailed results from the supplemental analyses comparing 

MON 88701 treated with dicamba and glufosinate herbicides to the conventional control 

are presented and discussed in Appendix G.13.2. 

In the combined-site analysis of plant mapping parameters (Table VII-7), no statistically 

significant differences were detected between MON 88701 and the conventional control 

for number of mainstem nodes per plant, number of nodes to first fruiting branch, total 

number of bolls per plant, vegetative bolls per plant, percent retention of first-position 

bolls and percent first-position bolls (relative to total bolls). The mean value for firstposition bolls per plant was higher in MON 88701 than the conventional control (5.2 vs. 

4.6) (Table VII-7). However, the mean value for first-position bolls per plant was within 

the reference range. Furthermore, similar results of the plant mapping evaluation of 

dicamba and glufosinate-treated MON 88701, the agronomic system in which 

MON 88701 is expected to be used, were observed (See Appendix G.13.2; Table G-18). 

Thus, MON 88701 is unlikely to have increased plant pest/weed potential or an adverse 

environmental impact compared to the conventional control. (See Figure VII-1, Step 3, 

answer “no”). 
Monsanto Company 12-CT-244U 

Table VII-7. Study 2 Combined-Site Comparison of MON 88701 to Conventional Control during 201

Characteristics 

Phenotypic Characteristic (units)

MON 887011 Control Refer

Mean (SE)3 Mean (SE) Minimum 

Mainstem Nodes (# per plant) 18.1 (0.4) 18.2 (0.4) 16.0 

Nodes to First Fruiting Branch (# per plant) 5.2 (0.2) 5.5 (0.2) 4.2 

Total Bolls4 (# per plant) 9.8 (0.6) 9.0 (0.7) 8.6 

Total First-Position Bolls (# per plant) 5.2 (0.3)* 4.6 (0.3) 2.9 

Total Vegetative Bolls (# per plant) 2.0 (0.6) 1.9 (0.6) 0.7 

% Retention of First-Position Bolls (per plant) 42.1 (2.5) 38.6 (2.6) 21.2 

% First-Position Bolls relative to total bolls (per plant) 57.5 (2.2) 56.5 (2.1) 36.0 

* Indicates a statistically significant difference (

α=0.05) between MON 88701 and the conventional control (n = 44). 1 MON 88701 plots were not treated with dicamba or glufosinate. 

2 Reference range = Minimum and maximum mean values among eight conventional commercial reference varieties. 3 SE = standard error.

4 Total Bolls = number of first-position bolls + number of second-position bolls + number of vegetative bolls.
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VII.C.2.3. Environmental Interaction Characteristics 

USDA-APHIS considers the environmental interactions of the biotechnology-derived 

crop compared to its conventional counterpart to determine the potential for increased 

plant pest characteristics. Evaluations of environmental interactions were conducted as 

part of the plant characterization for MON 88701. In the 2010, US field trials conducted 

for evaluation of phenotypic and agronomic characteristics of MON 88701, data were 

also collected on plant response to abiotic stress (i.e., drought, wind, nutrient deficiency, 

etc.), disease damage, arthropod-related damage, and arthropod abundance (Appendix G; 

Tables G-20 through G-29). These data were used as part of the environmental analysis 

(Section IX) to assess plant pest potential and provide an indication of potential effects of 

MON 88701 on non-target organisms (NTOs) compared to the conventional control. The 

results of the field evaluations showed that the dicamba and glufosinate-tolerance traits 

did not unexpectedly alter the assessed environmental interactions of MON 88701 

compared to the conventional control. The lack of significant biologically meaningful 

differences in plant response to abiotic stress, disease damage, arthropod-related damage, 

and pest- and beneficial-arthropod abundance supports the conclusion that the 

introduction of the dicamba and glufosinate-tolerance traits are unlikely to result in 

increased plant pest potential or an altered environmental impact from MON 88701 

compared to commercial cotton. 

VII.C.2.3.1. Study 1 Environmental Interactions of MON 88701 

MON 88701 was compared to the conventional control for qualitative and quantitative 

environmental interactions in Study 1 (See Section VII.C.2.1.). Qualitative assessments 

were conducted at 15 sites and included plant response to abiotic stressors, disease 

damage, and arthropod damage. The assessments were conducted four times during the 

growing season on all plots (4 time points x 4 plot replications = 16 data points per 

assessment). The first assessment was made at approximately 30 days after planting 

and the three subsequent assessments at approximately 30 day intervals thereafter. 

Plant response to abiotic stressors, disease damage, and arthropod damage were assessed 

at natural levels (no artificial infestation or imposed abiotic stress); therefore these levels 

typically varied between observations at a site and among sites. Plant response to abiotic 

stress, and disease damage and arthropod damage data were collected from each plot 

using a 0 – 9 scale of increasing severity of observed damage for each stressor. This 

scale was utilized to allow for the evaluation of the wide variety of potential abiotic 

stressor, disease damage, and arthropod damage symptoms potentially occurring across 

the season and across sites. Due to the non-specific nature of the scale used, the data 

were not statistically analyzed but rather were placed into one of the following 

categories: none (0), slight (1-3), moderate (4-6), or severe (7-9). MON 88701 and 

conventional control cotton were considered different in plant response to stressors if the 

range of injury symptoms across all four replications did not overlap between 

MON 88701 and the conventional control. Any observed differences between the 

MON 88701 and conventional control were assessed for biological significance in the 

context of the range of the commercial reference varieties, and for consistency in other 

observations and sites. 
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In addition to the qualitative assessment, quantitative arthropod assessments were 

conducted at five sites and included thrips damage, heliothine damage, and pest- and 

beneficial-arthropod abundance. Thrips damage was assessed three times (approximately 

14, 21, and 28 DAP) during the growing season, heliothine damage was assessed four 

times (approximately 45, 60, 75, and 90 DAP) during the growing season, and arthropod 

abundance was assessed from collections performed four times (approximately 30, 60, 

90, and 120 DAP) during the growing season. 

Thrips damage was quantitatively assessed in each plot from 10 randomly selected plants 

using the arthropod-specific 0–5 rating scale of increasing severity. Heliothine damage 

was assessed quantitatively by recording the total number of fruiting bodies (flower buds, 

flowers, and bolls), number of damaged fruiting bodies and number of live larvae on the 

top 7 nodes from 10 randomly selected plants of each plot. These numerical data along 

with the quantitative arthropod abundance data were subjected to statistical analysis. 

VII.C.2.3.1.1. Qualitative Assessment Results - Study 1 

In an individual site assessment of qualitative data (Tables VII-8, G-20 through G-22), 

no differences were observed between MON 88701 and the conventional control for any 

of the 169 comparisons for plant response to abiotic stressors, including compaction, 

drought/dry, flood, hail, heat, nutrient deficiency, wet soil/ excess precipitation, and 

wind damage. Also, no differences were observed between MON 88701 and the 

conventional control for any of the 170 comparisons for the assessed diseases, including 

anthracnose, Ascochyta leafblight, bacterial blight, boll rot, cotton leaf rust, damping off, 

Fusarium wilt, leaf spots, Pythium, reniform nematode, Rhizoctonia, root-knot 

nematode, Thielaviopsis, and Verticillium wilt. Finally, no differences were observed 

between MON 88701 and the conventional control for any of the 159 comparisons for 

the assessed arthropods, including aphids, beet armyworms, cut worms, fall armyworms, 

fleahoppers, grasshoppers, heliothines, southern corn rootworm beetles, soybean 

loopers, spider mites, stink bugs, tarnished plant bugs, thrips, and whiteflies. Since no 

differences were observed between MON 88701 and the conventional control for plant 

response to abiotic stressors, disease damage, and arthropod-related damage in multiple 

environments, the assessed results support the conclusion that MON 88701 is unlikely to 

have increased plant pest/weed potential or an adverse environmental impact compared 

to the conventional control (See Section VII.B.1.1.). 

 
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Table VII-8. Study 1 Summary of Qualitative Environmental Interactions 

Assessments Including MON 88701 Response to Abiotic Stress, Disease, and 

Arthropod Damage during 2010 

 

Stressor 

Number of 

Observations 

Across All Sites 

Number of Observations 

with No Observed 

Differences Between 

MON 88701 and the 

Conventional Control 

Abiotic stressors 169 169 

Disease damage 170 170 

Arthropod-related damage 159 159 

Total 498 498 

Note: The experimental design was a randomized complete block with four replications (n = 60). 

VII.C.2.3.1.2. Quantitative Assessment Results - Study 1 

In the combined-site analysis of thrips damage data (Table VII-9), no statistically 

significant differences were detected between MON 88701 and the conventional control. 

There were no biological differences in thrips damage that would contribute to increased 

pest potential of MON 88701 compared to the conventional control (See Section 

VII.B.1.1.). 

Table VII-9. Study 1 Combined-Site Comparison of MON 88701 to Conventional 

Control during 2010 for Assessment of Thrips Damage 

Observation MON 88701 (SE)1

 Control (SE) Reference range 

 

1 0.5 (0.1) 0.4 (0.1) 0.0 – 1.2 

2 0.1 (0.0) 0.1 (0.0) 0.0 – 0.2 

3 0.0 (0.0) 0.1 (0.0) 0.0 – 0.5 

 

Note: The experimental design was a randomized complete block design with four replications (n 

= 20). No statistically significant differences (α=0.05) were detected between MON 88701 and 

the conventional control. 

1

SE= standard error. 

In the combined-site analysis of heliothine damage data (Table VII-10), no statistically 

significant differences were detected between MON 88701 and the conventional control 

for percent damaged fruiting bodies and the number of live larvae. Thus, there is no 

biological difference in heliothine damage that would contribute to increased pest 

potential of MON 88701 compared to the conventional control (See Section VII.B.1.1.).
Monsanto Company 12-CT-244U 161 of 620 

Table VII-10. Study 1 Combined-Site Comparison of MON 88701 to Conventional 

Control during 2010 for Quantitative Assessment of Heliothine Damage 

 Percent Damaged Fruiting Bodies # of Live Larvae1

Observation MON 88701 

(SE)2

Control 

(SE) 

Reference 

Range 

MON 88701

(SE) 

Control 

(SE) 

Reference 

Range 

1 2.8 (0.9) 1.8 (0.8) 0.0 – 8.7 0.1 (0.1) 0.0 (0.0) 0.0 – 0.2 

2 5.3 (3.0) 6.3 (2.8) 1.2 – 28.1 0.2 (0.1) 0.1 (0.0) 0.0 – 0.5 

3 3.7 (0.7) 2.6 (0.5) 1.2 – 5.2 0.1 (0.0) 0.1 (0.0) 0.0 – 0.1 

4 6.3 (1.9) 6.9 (1.8) 2.6 – 12.1 0.1 (0.0) 0.1 (0.0) 0.0 – 0.3 

 

Note: No statistically significant differences (α=0.05) were detected between MON 88701 and 

conventional control (n = 20). 

1

 Number of immature heliothines. 

2

 SE = standard error. 

For arthropod abundance, a total of 178 comparisons were made between MON 88701 

and the conventional control for the following pest- and beneficial-arthropods: aphids, 

cabbage loopers, fall armyworms, fleahoppers, heliothines, southern armyworms, stink 

bugs, tarnished plant bugs, thrips, white flies, big eyed bugs, braconids, lacewings, 

ladybird beetles, Damsel bugs, Orius spp., and spiders (Araneae) (Tables G-25 and G26). No statistically significant differences were detected between MON 88701 and the 

conventional control for 173 out of 178 comparisons, including 89 pest arthropod 

comparisons and 89 beneficial arthropod comparisons. 

The five differences detected between MON 88701 and the conventional control included 

two differences for pest arthropods and three differences for beneficial arthropods 

(Tables G-25 and G-26). In the pest arthropod assessment, MON 88701 had lower 

abundance than the conventional control for stink bugs (0.3 vs. 1.8 per plot) and for 

tarnished plant bugs (0.5 vs. 2.0 per plot) in Collection 4 at the LABU site. For tarnished 

plant bugs, the mean abundance value for MON 88701 was within the reference range. 

For stink bugs, the mean abundance value for MON 88701 was outside the reference 

range. However, the statistical differences detected in stink bugs abundance were not 

consistent across collections or sites (Table G-25). 

In the beneficial arthropod assessment, MON 88701 had increased abundance compared 

to the conventional control (Table G-26) for Damsel bugs in Collection 2 at the GACH 

site (6.0 vs. 2.3). MON 88701 had lower abundance than the conventional control for 

Orius spp. in Collection 2 (0.0 vs. 1.5 per plot) and collection 3 (0.5 vs. 2.8 per plot) at 

the ARAU site. The mean abundance value for MON 88701 was within the reference 

ranges for the differences detected for Damsel bugs The mean abundance values for 

Orius spp. in Collection 2 and collection 3 at the ARAU site were outside their respective 

reference range. However, the differences detected for Orius spp. were not consistently 

detected across collections or sites (Table G-26). 
Monsanto Company 12-CT-244U 162 of 620 

Since the arthropod differences detected were not consistently observed at other 

collections and sites, MON 88701 is unlikely to have increased plant pest/weed potential 

or an adverse environmental impact compared to the conventional control (See Section 

VII.B.1.1.). 

VII.C.2.3.2. Study 2 Environmental Interactions of MON 88701

MON 88701 was compared to the conventional control for qualitative environmental 

interactions in Study 2 (See section VII.C.2.1.). Only qualitative assessments were 

conducted at all 11 sites and included plant response to abiotic stressors, arthropod 

damage, and disease damage. The observations of plant response to abiotic stressors, 

disease damage, and arthropod damage were performed four times during the growing 

season at each site on all plots (4 replications). The first observation was made at 

approximately 30 days after planting and the three subsequent observations at 

approximately 30 day intervals thereafter. (Section VII.C.2.3.1). 

VII.C.2.3.2.1 Qualitative Assessment Results - Study 2 

In an individual site assessment for Study 2 qualitative data (Table VII-11, G-27, G-28 

and G-29), no differences were observed between MON 88701 and the conventional 

control for any of the 127 comparisons for plant response to abiotic stressors, including 

compaction, drought (dry), flood, hail damage, heat, nutrient deficiency, wet soil (excess 

precipitation), and wind damage. Also, no differences were observed between 

MON 88701 and the conventional control for any of the 129 comparisons for the 

assessed diseases, including anthracnose, ascochyta leaf blight, bacterial blight, boll rot, 

cotton leaf rust, damping off, Fusarium wilt, leaf spots, Pythium, reniform nematode, 

Rhizoctonia, root-knot nematode, thielaviopsis, and Verticillium wilt. Finally, no 

differences were observed between MON 88701 and the conventional control for any of 

the 129 comparisons for the assessed arthropods, including aphids, beet armyworms, 

cabbage loopers, cut worms, fall armyworms, fleahoppers, grasshoppers, heliothines, 

southern corn rootworm beetle, soybean loopers, spider mites, stink bugs, tarnished 

plant bugs, thrips and white flies. Since no differences were observed between 

MON 88701 and the conventional control for plant response to abiotic stressors, disease 

damage, and arthropod-related damage in multiple environments, the assessed results are 

similar to those in Study 1 and support the conclusion that the biotechnology-derived 

traits in MON 88701 are unlikely to have increased plant pest/weed potential or an 

adverse environmental impact compared to the conventional control (See Section 

VII.B.1.1.). 

 
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Table VII-11. Study 2 Summary of Qualitative Environmental Interactions 

Assessments Including MON 88701 Response to Abiotic Stress and Disease and 

Arthropod-related Damage during 2010 

Stressor 

Number of 

Observations 

Across All Sites 

Number of Observations 

with No Observed 

Differences Between 

MON 88701 and the 

Conventional Control 

Abiotic stressors 127 127 

Disease damage 129 129 

Arthropod-related damage 129 129 

Total 385 385 

Note: The experimental design was a randomized complete block with four replications (n = 

60 

VII.C.2.3.2.2. Plant Mapping as an Indicator of Plant Response to Environmental 

Stress 

Final boll retention and distribution, as reflected in the plant mapping data, can provide 

an indication of the effect that abiotic and biotic stressors had on a cotton plant because 

squares and early bolls tend to abort if the plant experiences stress (Guinn, 1982; Kerby 

et al., 2010; University of California, 1996). For example, if plants experienced severe 

stress during early flowering, this could result in fewer bolls on the lowermost fruiting 

branches compared to unstressed plants. If plant map results are similar between two 

cotton lines this usually indicates that plants responded to stress in a similar manner. 

Within a study location and based on the proximity of plots within a location, it can be 

concluded that all plots would be subjected to similar stressors. 

As previously indicated, there were no differences in plant mapping parameters between 

MON 88701 not treated with dicamba or glufosinate herbicides and the conventional 

control that would be indicative of a differential plant response to abiotic or biotic 

stressors (Study 2, Section VII.C.2.2, Table VII-7). Similar results were observed for 

MON 87701 plots treated with dicamba and glufosinate (Table G-18). Thus, since all 

Study 2 plots would be subjected to similar stressors and since MON 88701 treated and 

not treated with dicamba or glufosinate herbicides had similar plant map results, each 

compared to the conventional control (See Tables VII-7 and G-18); it can be concluded 

that both responded to stressors in a similar manner. Results showed that only the mean 

number of first-position bolls was significantly different in both comparisons of 

MON 88701 not treated compared to the conventional control (5.2 vs. 4.6, respectively) 

and MON 88701 treated compared to the conventional control (5.2 vs. 4.6, respectively). 

Both of the mean values of the number of first-position bolls in MON 88701 were within 

the reference range. Therefore, these data support the conclusion that the biotechnologyderived traits in MON 88701 are unlikely to have increased plant pest potential, increased 
Monsanto Company 12-CT-244U 164 of 620 

weediness, or an adverse environmental impact compared to commercially cultivated 

cotton. 

VII.C.2.3.3. Conclusions - Qualitative and Quantitative Environmental Interactions

The results of the qualitative and quantitative data of MON 88701 from Study 1 and 

qualitative data from Study 2 showed that the dicamba and glufosinate-tolerance traits did 

not unexpectedly alter the assessed environmental interactions of MON 88701 compared 

to the conventional control. The lack of significant biological differences in plant 

responses to abiotic stress, disease damage, arthropod-related damage, thrips damage, 

heliothine damage, and pest- and beneficial-arthropod abundance for MON 88701 

supports the conclusion that the introduction of the dicamba and glufosinate-tolerance 

traits are unlikely to result in increased plant pest potential, increased weediness or an 

altered environmental impact from MON 88701 compared to commercially cultivated 

cotton, irrespective of whether or not dicamba and glufosinate herbicide treatments were 

applied. 

VII.C.3. Pollen Characteristics 

USDA-APHIS considers the potential for gene flow and introgression of the 

biotechnology-derived trait(s) into other cotton varieties and wild relatives to assess the 

potential for increased weedy or invasive characteristics of the receiving species. Pollen 

morphology and viability information are pertinent to this assessment and, therefore, 

were assessed for MON 88701. In addition, characterization of pollen produced by 

MON 88701 and the conventional control is relevant to the plant pest risk assessment 

because it adds to the detailed description of the phenotype of MON 88701 compared to 

the conventional control. 

The purpose of this evaluation was to assess the morphology and viability of pollen 

collected from MON 88701 compared to that of the conventional control. Pollen was 

collected from MON 88701, the conventional control, and four commercial reference 

varieties grown under similar agronomic conditions in Crittenden County, Arkansas 

(ARPR). The field trial was arranged in a randomized complete block design with four 

replications. Five flowers (subsamples) were collected from each plot; pollen was 

extracted from each flower and stained with Alexander’s stain (Alexander, 1980). Pollen 

viability was evaluated for each subsample and pollen grain diameter was evaluated for 

ten representative viable pollen grains per subsample. General morphology of the pollen 

was observed for each subsample. MON 88701 was compared to the conventional 

control for percentage viable pollen and pollen diameter. A reference range was 

calculated from the minimum and maximum mean values of the commercial reference 

varieties to provide pollen viability and pollen diameter values representative of 

commercial cotton (See Appendix H). 

No statistically significant differences (α=0.05) were detected between MON 88701 and 

the conventional control for percentage viable pollen or pollen grain diameter (Table VII12). Furthermore, no visual differences in general pollen morphology were observed 

between MON 88701 and the conventional control. These results demonstrate that the 
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introduction of the dicamba and glufosinate-tolerance traits did not alter the overall 

morphology or pollen viability of MON 88701 compared to the conventional control (See 

Figure VII-1, Step 2, answer “no”). The pollen characterization data contribute to the 

detailed phenotypic description of MON 88701 compared to the conventional control. 

The results support an overall conclusion that MON 88701 is not different than the 

conventional control in terms of plant pest or weed characteristics and is no more likely 

to pose a plant pest risk than commercially cultivated cotton. 
Monsanto Company 12-CT-244U 166 of 620 

Table VII-12. MON 88701 Compared to the Conventional Control during 2010 for 

Pollen Characteristics 

 MON 88701 

Conventional 

Control Reference Range1

Pollen Characteristic Mean (S.E.)2 Mean (S.E.) Minimum Maximum 

 

Viability (%) 97.8 (0.46) 97.9 (0.40) 96.0 98.2 

Diameter(μm) 95.2 (1.49) 92.2 (1.47) 94.0 95.2 

 

The experimental design was a randomized complete block design with four replications (n = 4). 

No significant differences were detected between MON 88701 and the conventional control 

(α=0.05) using analysis of variance (ANOVA). 

1 Reference ranges = Minimum and maximum values of four commercial reference varieties. 

2

 S.E. = Standard Error. 

VII.D. Conclusions for Phenotypic, Agronomic, Plant Mapping, and Environmental 

Interactions Evaluation 

Domesticated cotton lacks characteristics that are commonly associated with plants that 

are considered weeds (e.g. seed dormancy, seed dispersal mechanisms, ability to compete 

with and displace native vegetation). An extensive and robust set of information and data 

were used to assess whether the introduction of the dicamba and glufosinate-tolerant 

traits altered the plant pest potential of MON 88701 compared to the conventional 

control. These assessments included five general data categories: 1) seed dormancy and 

germination characteristics; 2) agronomic and phenotypic characteristics; 3) plant 

mapping characteristics; 4) observations of abiotic stress response, disease damage, 

arthropod related damage, and pest- and beneficial-arthropod abundance; and 5) pollen 

characteristics. Results from these assessments comparing MON 88701 and the 

conventional control demonstrate that MON 88701 does not possess weedy 

characteristics, increased susceptibility or tolerance to specific abiotic stressors, diseases, 

or arthropods, or characteristics that would confer a plant pest risk or significant 

environmental impact compared to conventional cotton. Therefore, based on the results 

of multiple assessments discussed above, MON 88701 is comparable to commercially 

cultivated cotton, and is no more likely to pose a plant pest/weediness risk or have a 

significant environmental impact. 
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VIII. U.S. AGRONOMIC PRACTICES 

VIII.A. Introduction 

As part of the plant pest assessment required by 7 CFR § 340.6(c)(4), impacts to 

agricultural and cultivation practices must be considered. This section provides a 

summary of current agronomic practices in the U.S. for producing cotton and is included 

in this petition as a baseline to assess possible impacts to agricultural practices due to the 

cultivation of MON 88701. Discussions include cotton production, seed production, 

plant growth and development, general management practices during the season, 

management of insects, diseases and weeds, cotton rotational crops, and volunteer cotton 

management. Information presented in the previous section demonstrated that 

MON 88701 is no more susceptible to diseases or pests than commercially cultivated 

cotton. Additionally, data presented in Section VII show that, with the exception of 

tolerances to both dicamba and glufosinate herbicides, MON 88701 is phenotypically 

equivalent to commercially cultivated cotton. Thus, there are no changes to the inputs 

needed for MON 88701, and no likely impacts to the majority of the agronomic practices 

employed for the production of cotton. Agronomic practices that maybe influenced from 

the deregulation of MON 88701 are discussed. 

Cotton production in the U.S. is limited primarily by climate. Cotton is a woody, warmseason perennial plant that is planted in 17 states across the southern U.S. Aside from 

temperature, the most influential climatic factor impacting cotton agronomic practices is 

moisture. Rainfall requirements and patterns are a major determinant of the cotton 

production practices adopted in both dryland and irrigated cotton. The length of the 

season may vary between cotton production regions, but the production cycle and 

production practices used are fairly consistent among geographic regions and between the 

upland and Pima cotton types. Proper seedbed preparation, appropriate variety selection, 

appropriate planting dates and plant population, and good integrated pest management 

practices are important for optimizing the yield potential and economic returns of cotton. 

Annual and perennial weeds are a serious problem and must be managed in order to 

maximize cotton yield and quality. Weeds compete with cotton for water, nutrients, and 

light, resulting in reduced cotton lint yields and lint quality when left uncontrolled. Weed 

species in cotton vary from region to region and from state to state, but the economic 

thresholds of cotton require some form of weed management practice on all cotton 

acreage. Weed management practices include mechanical tillage, crop rotations, cultural 

practices (e.g., planting clean seed, cleaning tillage and harvesting equipment), and 

herbicide application. Numerous selective herbicides are utilized for preplant, 

preemergence, and postemergence control of annual and perennial weeds in cotton. 

Approximately 97% of the cotton acreage in the U.S. receives a herbicide application. 

Herbicide-tolerant cotton is currently grown on 78% of U.S. cotton acres (Brookes and 

Barfoot, 2012) and glyphosate-tolerant cotton weed control systems have become the 

standard program for weed management in cotton since commercial introduction of 

glyphosate-tolerant cotton in 1997 (Brookes and Barfoot, 2012). Glyphosate-tolerant 

cotton has facilitated the growth of conservation tillage systems in cotton production 

which has resulted in reduced soil erosion, reduced fuel and labor costs, improved water 
Monsanto Company 12-CT-244U 168 of 620 

quality, and conserved soil moisture. Herbicides can replace the need for preplant tillage 

for weed control in no-tillage production systems. Insect pests, diseases, and nematodes 

are also common and continuous threats to cotton production and integrated pest 

management programs must be implemented to prevent yield losses due to these pests. 

Volunteer cotton (i.e., cotton plants that have germinated and emerged unintentionally in 

a subsequent crop) is not considered a significant problem in rotational crops primarily 

because mechanical and chemical control methods are available to manage the occasional 

volunteer cotton plant. Preplant tillage generally destroys volunteer cotton plants prior to 

planting rotational crops. Volunteer cotton is generally more of a problem in no-till 

cotton because of the lack of preplant tillage, but herbicides are available for control of 

volunteer cotton in rotational crops. Given that MON 88701 is agronomically, 

phenotypically, and ecologically comparable to commercially cultivated cotton, the 

introduction of MON 88701 in the cotton production system is expect to have no impact 

on the management of cotton volunteer plants in rotational crops such as corn, soybean, 

sorghum, and wheat. The numerous control measures that are effective on conventional 

and glyphosate-tolerant volunteer plants will continue to be effective on volunteer 

MON 88701 plants if they arise. See Section VIII.H.1 for additional information on 

control of MON 88701 volunteers. 

As shown in Sections VI and VII, with the exception of the tolerances to both dicamba 

and glufosinate herbicides, no biologically meaningful differences were observed in 

composition, phenotype, or environmental interactions between MON 88701 and 

commercially cultivated cotton. Moreover, herbicide-tolerant cotton is currently grown 

on 78% of U.S. cotton acres (Brookes and Barfoot, 2012). Therefore, it is anticipated 

that commercialization of MON 88701 in the U.S. is not likely to impact current cotton 

cultivation and/or agronomic practices, beyond the intended benefits of effective 

management of common and troublesome weeds, including herbicide-resistant weeds.

VIII.B. Overview of U.S. Cotton Production 

VIII.B.1. Cotton Production 

The majority of the value of the producer’s cotton crop is based on the quality and 

quantity of the lint produced, and with the exception of contracted acres for planting seed 

production. Little consideration is given by growers to the disposition of the cottonseed 

and its by-products. Most of the world’s cotton production (116.40 million bales 

annually) is grown in China (30.5 million bales), India (26.4 million bales), United States 

(18.1 million bales), Pakistan (8.6 million bales) and Brazil (9.0 million). Figures are 

from the 2010/2011 cotton season (USDA-FAS, 2012). In 2010/2011, the U.S. supplied 

over 14 million bales of the world’s cotton exports, accounting for approximately 40% of 

the total world export market for cotton (USDA-FAS, 2011). China, Bangladesh, 

Indonesia, and Turkey are major importers of cotton. The largest customers for U.S. 

cotton are Asian countries and Mexico, due to the prevalence of textile manufacturing 

(NCCA, 2010). Cottonseed production currently results in approximately 10% of the 

world’s oilseed production (USDA-FAS, 2010), and is exceeded by soybean (58%) and 

rapeseed (13%). 
Monsanto Company 12-CT-244U 169 of 620 

Gossypium hirsutum (upland cotton) cultivars account for more than 90% of the world’s 

annual cotton crop and 97% of the U.S. cotton production (Smith and Cothren, 1999; 

USDA-NASS, 2011e). G. barbadense, known as extra-long staple, Pima, or Egyptian 

cotton, is also grown in the U.S, which accounts for approximately 3% of the acreage in 

the U.S. (USDA-NASS, 2012c). The long, strong, fine fibers produced by Pima are ideal 

for specialized uses, but due to the geographic limitation for optimum production it is 

economically less viable than the G. hirsutum cultivars in the U.S. Pima cotton requires a 

longer growing season than upland cotton, and production is limited to the Southwestern 

states. 

Cotton is a crop that produces two commodities: fiber and seed. The modern cotton gin 

has enhanced the value of cotton commodities by separating the fiber from the seed and 

by removing foreign matter, while preserving the inherent qualities of the fiber and seed 

(Smith and Cothren, 1999). The fiber is the more valuable product of the crop, normally 

accounting for approximately 85% of the value. For every 100 pounds of fiber produced 

by the cotton plant, it also produces about 162 pounds of cottonseed (NCCA, 2010). 

Cottonseed is crushed for oil and meal used in both food products and in livestock feed. 

Cotton (Gossypium spp.) is grown in the U.S. across southern states where the climate is 

warmer and the season is longer (Figures VIII-1 and VIII-2). The total U.S. cotton 

acreage in the past 10 years has varied from approximately 9.15 to 15.77 million planted 

acres, with the lowest acreage recorded in 2009 and the highest in 2001 (Table VIII-1). 

Average cotton yields have varied from 632 to 879 pounds per acre over this same time 

period. Total annual cotton production ranged from 12.19 to 23.89 million bales (480 

pounds/bale) over the past ten years. The variations observed in cotton acreage and 

production is driven by current market conditions, rather than agronomic considerations. 

According to data from USDA-NASS (USDA-NASS, 2011b), cotton was planted on 

approximately 11 million acres in the U.S. in 2010, producing approximately 18 million 

bales of cotton (Table VIII-1). The value of cotton production reached $7.32 billion in 

the U.S. in 2010 (USDA-NASS, 2011b). 

U.S. cotton production is divided into the following four major cotton growing regions, 

which span the southern and southwestern states: Southeast region (AL, FL, GA, NC, SC, 

and VA), Midsouth region (AR, LA, MS, MO, and TN), Southwest region (KS, NM, OK, 

and TX), and West region (AZ and CA) (Table VIII-2). Cotton planting and production 

figures for these regions in 2010 are shown in Table VIII-2 and discussed below (USDANASS, 2011e). Approximately 5.6 million acres of cotton were planted in Texas, 

representing about 51% of the total U.S. cotton acres. Texas produced 8.1 million bales 

(480 pounds/bale) of cotton, which represents approximately 44% of the U.S. cotton 

production. The second largest production state for cotton was Georgia with 

approximately 12% of U.S. cotton production. Average cotton yields across the four 

cotton growing regions ranged from 727 to 1416 pounds cotton lint per acre, with the 

highest yields in the West with full irrigation, and the lowest yields in areas such as 

Alabama, Oklahoma, and Texas, where little to no irrigation is employed (Table VIII-2). 

The average cotton yield across all regions is 821 pounds cotton lint per acre. The value 

of the cotton lint production among the four regions ranged from $0.86 billion in the 

West region to $3.35 billion in the Southwest region. The total value of the cottonseed 
Monsanto Company 12-CT-244U 170 of 620 

production in the U.S. in 2010 was $1 billion with the value among the regions ranging 

from $134 million in the West region to $461 million in the Southwest region. 
Monsanto Company 12-CT-244U 171 of 620 

Figure VIII-1. Planted Upland Cotton Acres by County in the U.S. in 2010 

(USDA-NASS, 2012a) 

 
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Figure VIII-2. Planted Pima Cotton Acres by County in the U.S. in 2010 

(USDA-NASS, 2012b) 
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Table VIII-1. Cotton Production in the U.S., 2000-20101 

Year 

Acres 

Planted 

(×1000) 

Acres 

Harvested 

(×1000) 

Average 

Yield 

(lbs/acre) 

Total Production 

(480 lb bales) 

Value 

(billions $) 

 

2010 10,973 10,707 821 18,314,500 7.318 

2009 9,150 7,691 777 12,187,500 3.788 

2008 9,471 7,569 813 12,815,300 3.021 

2007 10,872 10,489 879 19,206,900 5.653 

2006 15,274 12,732 814 21,587,800 5.013 

2005 14,245 13,803 831 23,890,200 5.695 

2004 13,659 13,057 855 23,250,700 4.853 

2003 13,480 12,003 730 18,255,200 5.517 

2002 13,958 12,417 665 17,208,600 3.777 

2001 15,769 13,828 705 20,302,800 3.122 

2000 15,517 13,053 632 17,188,300 4.260 

 1

 (USDA-NASS, 2011b)
Monsanto Company 12-CT-244U 

Table VIII-2. U.S. Cotton Production by Region and State in 20101 

Region/State

Acres Planted 

(thousands)

Acres Harvested

(thousands)

Average Yield

(pounds/acre)

Total Production

(thousand bales)

Cotton Lint $ 

Value (thousands)

Cot

Value 

Southeast RegionAlabama 340 337 684 480 199,066 

Florida 92 89 809 150 54,792 

Georgia 1,330 1,320 811 2,230 926,966 

North Carolina 550 545 854 970 338,957 South Carolina 202 201 872 365 136,656 Virginia 83 82 685 117 46,051 

Region Totals 2,597 2,574 804 4312 1,702,488 1

Midsouth Region

Arkansas 545 540 1,049 1,180 395,914 

Louisiana 255 250 864 450 174,960 Mississippi 420 415 983 850 308,856 Missouri 310 308 1,068 685 226,214 Tennessee 390 387 843 680 275,482 Region Totals 1,920 1,900 971 3,845 1,381,426 2

Southwest Region

Kansas 51 49 784 80 34,675 

New Mexico 50 49 1,084 110 46,721 Oklahoma 285 270 738 415 180,276 

Texas 5,567 5,367 723 8,082 3,083,472 4

Region Totals 5,953 5,734 727 8,687 3,345,144 4West

Arizona 198 196 1,460 595 246384 

California 306 303 1,388 876 610042 Region Totals 504 499 1,416 1,471 856,426 1

U.S. Total 10,973 10,707 821 18,315 7,317,704 1,0

1 (USDA-NASS, 2011e; 2011c) 
Monsanto Company 12-CT-244U 175 of 620 

VIII.B.2. Cotton Seed Production 

Standardized seed production practices are responsible for maintaining high-quality seed 

stocks, which is essential for U.S. agriculture. The value of seed quality (including 

genetic purity, vigor, and absence of weed seed, seed-borne diseases, and inert materials, 

such as dirt) are a major factor impacting crop yield potential. States developed seed 

laws and certification agencies to ensure that purchasers who received certified seed 

could be assured that the seed met established seed quality standards (Bradford, 2006). 

The federal government passed the U.S. Federal Seed Act of 1939 to recognize seed 

certification and the establishment of official certifying agencies. Regulations first 

adopted in 1969 under the Federal Seed Act recognize land history, field isolation, and 

varietal purity standards for foundation, registered, and certified seed. Under 

international agreements such as the Organisation for Economic Co-operation and 

Development (OECD) system, the U.S. and other countries mutually recognize minimum 

seed quality standards (Bradford, 2006). The Association of Official Seed Certifying 

Agencies (AOSCA) represents state and private seed certification organizations in the 

U.S., and includes international member countries in North and South America, 

Australia, and New Zealand. 

Cotton seed is separated into four seed classes: 1) breeder; 2) foundation; 3) registered; 

and 4) certified (AOSCA, 2012). Breeder seed is seed directly controlled by the 

originating or sponsoring plant breeding organization or firm. Foundation seed is firstgeneration seed increased from breeder seed and is handled in a manner to maintain 

specific levels of varietal purity and identity. Registered seed is the progeny of 

foundation seed that is handled to maintain satisfactory varietal purity and identity. 

Certified seed is the progeny of breeder, foundation or registered seed, and is typically 

two generations removed from foundation seed. While not all cotton seed sold to 

growers is officially certified, commercial cotton seed sold and planted for typical cotton 

production is produced predominately to meet or exceed certified seed standards. This 

section of the petition will provide a broad overview of the practices used in producing 

certified seed. 

The majority of the cotton seed is produced in Texas with significant quantities produced 

in Arizona, Arkansas, California, and Mississippi (McDonald and Copeland, 1997). The 

entire seed production process at the majority of the seed companies uses International 

Organization for Standardization (ISO) certification standards and therefore include 

internal and external audits (ISO, 2009). ISO standards ensure desirable characteristics 

of seeds and services, such as quality, safety, reliability, and efficiency. The ISO 

standards represent an international consensus on good management practices with the 

aim of ensuring that the organization can consistently deliver excellent products or 

services. The standards must meet the customer’s requirements, applicable seed 

regulatory requirements, and continually improve the process and process control 

systems (ISO, 2009). Agronomic practices for producing cotton seed are similar to 

commercial cotton production. However, increased management is needed in certain 

agronomic practices (e.g., fertility, water management, cultivation, use of plant growth 
Monsanto Company 12-CT-244U 176 of 620 

regulators, etc.) to produce seed with high quality, high germination rates, and high 

genetic purity. 

After harvest and the ginning and delinting processes, commercially certified cotton seed 

must meet state and federal seed standards and labeling requirements. AOSCA standards 

for certified cotton seed are as follows: 98% pure seed (minimum), 2% inert matter 

(maximum), 0.02% weed seed (maximum), 0.3% other crop seeds (maximum), and 70% 

germination (minimum) (AOSCA, 2009). The cotton seed industry historically sets a 

minimum of 80% germination for labeling purposes. State seed certification standards 

vary slightly from state to state and can be more restrictive than the seed standards of 

AOSCA. 

When deregulated, MON 88701 seed will be produced in the same manner as 

commercially certified cotton seed, such that it will meet all state and federal seed 

standards and labeling requirements. 

VIII.C. Production Management Considerations 

VIII.C.1. Pre-Season 

Production decisions regarding crop rotation, tillage system, soil fertility, variety 

selection, and row spacing need to be made well in advance of planting the cotton crop. 

Many of the decisions in this area are made prior to or immediately after harvest of the 

previous crop. The rotation of cotton with other crops should be an integral part of a 

farm management program. Ideally, cotton should be rotated with other crops on a 

regular basis to maintain soil productivity and reduce the incidence of various weeds, 

insect pests or diseases (Hake et al., 1996d). However, production costs, relative rate of 

return, and the current market conditions will dictate which crops to rotate with cotton or 

whether to grow continuous cotton. See Section VIII.H for additional details on crop 

rotation practices in cotton. 

Tillage has been an integral part of production agriculture and is synonymous with 

seedbed preparation. The primary purposes of preplant tillage are to incorporate residue 

from the previous crop, reduce wheel traffic compaction from the previous season, 

improve water filtration and soil aeration, control weeds, loosen the soil for root 

penetration, and provide a suitable environment for the planting and germination of 

cottonseed (Hake et al., 1996d). Decreased profitability in cotton production, as well as 

soil erosion concerns, have increased interest in conservation tillage systems. The 

benefits of conservation tillage or no-till systems are well documented and include 

reduced soil erosion, reduced fuel and labor costs, and conservation of soil moisture 

(CTIC, 2011). 

Maintaining optimum crop nutrition is critical in achieving high yields and quality in 

cotton. Pre-season soil test results for nitrogen, phosphorus, and potassium plus 

determination of pH, together with previous cropping and fertilization history determine 

the fertilizer and liming needs for the upcoming cotton crop. In the Southwest and West 

regions, monitoring soil salinity is of additional importance because cotton is most 
Monsanto Company 12-CT-244U 177 of 620 

sensitive to sodium and salts during the germination and seedling growth stage (Hake et 

al., 1996d). Soil salinity will severely delay emergence, which can make the plants more 

vulnerable to seedling disease. 

Yield potential has generally been the most important factor considered by growers in 

variety selection (Smith and Cothren, 1999). Growers also need to consider fiber 

properties (e.g., length, strength, micronaire, etc.), cold tolerance, seedling vigor, heat 

tolerance, leaf hairiness, insect and disease resistance, maturity, and a number of other 

factors. Cotton varieties are classified into three maturity groups: short-, medium-, or 

long-season varieties (Smith and Cothren, 1999). More determinate plants are planted in 

the short season northern portions of the cottonbelt and longer-season or more 

indeterminate varieties planted in the south. Growers in areas of western Texas and 

Oklahoma have tended to select ‘stripper’ or ‘stormproof’ varieties which produce a boll 

that is more resistant against yield loss under storm and hail conditions. “Picker” cotton 

varieties grown in the high plains of Texas produce large open bolls and are susceptible 

to yield loss from seasonally strong thunderstorm activity. Growers are advised to plant 

three or four varieties to reduce the risk of planting the entire farm to a poor-yielding 

variety or using traits that do not add value to their cropping system (NCCA, 2007). 

VIII.C.2. Planting and Early Season 

The yield potential of a cotton crop is determined in the first 30 to 40 days after seed is 

placed in the ground (Deterling and El-Zik, 1982). Planting date management is an 

important element in achieving early fruit set, and establishing a strong yield potential 

(Smith and Cothren, 1999). Cotton should be planted into prepared seedbeds that are 

firm, warm, and moist. Cotton specialists recommend planting cotton when soil 

temperatures at seeding depth are at 64° F or higher at 8 a.m. for three consecutive days, 

with a favorable five-day forecast (Deterling and El-Zik, 1982; Smith and Cothren, 

1999). Under favorable conditions, emergence can occur anywhere from 5 to 15 days 

following planting. Once emerged, the cotton plant goes through a period of slow growth 

and development before entering phases of rapid vegetative and reproductive growth. 

The growth of the cotton plant is temperature dependent and growth ceases when the 

average daily temperature falls below 60° F (Hake et al., 1996b). It is during this early 

period of slow development that cotton must be protected from damaging weed, insect, 

and disease pests to prevent yield losses (Hake et al., 1996b; Smith and Cothren, 1999). 

When planting is delayed significantly due to time constraints or weather conditions, 

growers are advised to switch to more determinate (short-season) type varieties. Planting 

good quality seed with a germination of 85% or higher is also important for establishing a 

good and uniform stand of cotton (Deterling and El-Zik, 1982). The single most 

important practice for minimizing damage from seedling diseases is selection of highquality planting seed (Smith and Cothren, 1999). Most cottonseed sold commercially is 

treated with a fungicide to protect the germinating seed and seedlings from seed- and 

soil-borne pathogens (Smith and Cothren, 1999).

Plant population management contributes toward early fruiting, good fruit retention, and 

improved earliness of crop maturity (Smith and Cothren, 1999). Seeding rates vary 
Monsanto Company 12-CT-244U 178 of 620 

across the cotton growing region of the U.S. The seeding range will vary depending 

upon row-spacing, soil classification, available moisture, and overall environmental 

conditions. Cotton has the ability to compensate in response to row spacing and plant 

populations. However, higher plant densities tend to cause cotton plants to grow taller, 

develop more vegetative growth, and create more shading within the canopy (Smith and 

Cothren, 1999). These characteristics can result in delayed fruiting, alter the 

reproductive/vegetative balance, and decrease fruit retention. Conversely, low 

populations can delay overall plant maturity, allow sunlight to penetration through the 

canopy contributing to more weeds, and result in insufficient structure to produce 

adequate fruit which can influence overall harvestable yields. 

VIII.C.3. Mid- to Late-Season 

After early development, the next critical stage in the development of a cotton crop is 

rapid vegetative growth that includes the initiation of the first ‘squares.’ These floral 

buds develop into the subsequent fruiting forms called bolls. Fruiting development 

generally begins with the formation of fruiting branches on nodes four through eight 

(Deterling and El-Zik, 1982). After the accumulation of 40 to 60 days following 

emergence, the first square becomes visible, which is normally five to eight weeks after 

planting, depending on the area and temperature (Deterling and El-Zik, 1982; Hake et al., 

1996c). Approximately 85% of the total bolls that are harvested come from squares set 

during the first four to five weeks of squaring (Deterling and El-Zik, 1982). Therefore, it 

is critical to properly manage cotton during this period to maximize yields. 

Management practices, such as water management, plant nutrition management, and 

weed, disease, and insect control are critical during this reproductive growth phase. To 

maximize yield fruiting square and resulting boll retention is critical, especially the first 

bolls set on the plant. The first three ‘positions’ on each reproductive branch are the key 

sites for fruiting and will account for the vast majority of the plant’s yield (Deterling and 

El-Zik, 1982). Further, the first-position, or squares nearest the main stem, will account 

for over 50% of the total lint produced per plant. The second-position squares account 

for another one-third or more of the harvest, while squares further out on each 

reproductive branch produce 15% or less of the final number of mature bolls harvested 

that contribute to yield (Deterling and El-Zik, 1982). 

Most growers or crop consultants currently use a number of measurements during 

midseason to monitor and manage cotton plant growth. Although each will not be 

discussed in detail here, the grower may monitor any one or more of the following 

parameters: 1) plant height; 2) number of mainstem nodes; 3) node number of first 

fruiting branch; 4) total number of fruiting branches; 5) height-to-node ratios; and 6) 

square or fruit retention (Hake et al., 1996c). These parameters are commonly referred to 

as plant mapping. In general, as cotton is a perennial grown as an annual the cotton 

grower is seeking to favor reproductive growth at the expense of vegetative growth. This 

transition from vegetative to reproductive growth influences crop maturity and season 

length. Available options to influence cotton plant growth include the use of a plant 

growth regulator such as mepiquat chloride, fertility management (primarily nitrogen and 

potassium), and water management. Also, weed management and insect pest control are 
Monsanto Company 12-CT-244U 179 of 620 

important at mid-season, both of which can dramatically decrease both square and 

immature boll retention. 

As the end of the growing season approaches, the yield is established and management 

efforts shift to protecting the crop yield and quality. The stage in cotton when vegetative 

growth ceases is generally referred to as “cut-out” (Hake et al., 1996a). When the nodes 

above the first-position white flower decline to four or five, cut-out has been reached. 

This is also the point at which the last effective bloom, which could contribute to yield, is 

on the plant. The timing of cut-out is critical for both yield and quality of cotton. If cutout occurs too early, due to environment and management practices, the crop may not 

take full advantage of the available season. Late cut-out is often associated with poor 

early-season fruit retention and results in delayed maturity and harvest. 

VIII.C.4. Preharvest and Harvest 

The complete defoliation or desiccation of leaf tissue in preparation for harvest is a 

necessity with harvesting (Hake et al., 1996a). Leaves not only interfere with harvesting, 

but contribute to trash and moisture content, which influences ginning, cleaning, and 

overall quality of cotton lint. Effective defoliation is an essential step in the overall 

process of harvesting high quality cotton lint with the grower seeking to accomplish a 

complete, quick and efficient defoliation by chemical means. Defoliation attempts to 

speed up and control the natural process of senescence. An additional objective of 

defoliation is to kill or desiccate weeds that can reduce harvest efficiency, contribute to 

the weed seed bank, and reduce both the quality and value of the lint because of staining 

by vegetation (University of Georgia, 2012). Successful defoliation in cotton depends on 

a number of factors including: 1) plant-water status; 2) nitrogen fertility status; 3) 

weather conditions; and 4) the chemical defoliant(s) (Smith and Cothren, 1999). 

Defoliation should begin as early as possible by balancing the realistic yield potential of 

the crop with the need to preserve quality, schedule harvesting equipment, and minimize 

losses from weathering. Defoliation normally occurs when approximately 60% of the 

bolls are open (Smith and Cothren, 1999). Cotton is then harvested mechanically using 

pickers or strippers. 

VIII.D. Management of Insect and Other Pests 

Insect and mite pests are a common and continuous threat to cotton production in all 

regions of the U.S., leading to decreased yield and quality. Generally, fewer than 25 

insect pests are considered persistent problems causing economic losses in cotton (Smith 

and Cothren, 1999). The susceptibility of cotton plants to insect pests varies across and 

within the various production regions. Insect and mite pests affect cotton production by 

decreasing yield and reducing quality. Nearly every phenological stage of cotton is 

susceptible to injury by one or more insect pests during the growing season. Therefore, 

cotton fields must be monitored regularly to detect the presence of insect pests. The 

susceptibility of cotton plants to economic yield losses from insect pests is influenced by 

pest population density, timing of infestations as related to plant phenology, local 

environmental conditions, and agronomic practices (Smith and Cothren, 1999). 
Monsanto Company 12-CT-244U 180 of 620 

Numerous insect species are observed in cotton fields across the U.S., but only a few are 

considered of economic importance. Yield loss and treatment costs for the most common 

insect pests in cotton in 2010 are shown in Table VIII-3. These data are estimates 

collected from surveys of county agents, extension specialists, private consultants, and 

research entomologists. Insect damage resulted in yield losses of approximately 986 

thousand bales of cotton in 2010 or a 3.9% yield loss which represented an average loss 

of $22.56 per acre. The lepidopteran pests, bollworm/budworm, caused the greatest yield 

reductions followed by stink bugs and lygus insects, both of which are piercing and 

sucking insects. Thrips infested more acres in 2010 than any other insect in cotton. 

However, this insect ranked sixth in yield reductions, due to the damage occurring early 

in the growing season before the development of fruiting structures. 

Successful and economical management of insect pests in cotton is accomplished through 

an integrated pest management approach of variety selection and implementation of 

cultural, biological, and chemical strategies (University of Georgia, 2011). Preplant 

tillage and crop rotation are important agronomic or cultural practices utilized to reduce 

insect populations prior to planting cotton. Other agronomic practices are utilized to 

promote early maturity and reduce that period of time the crop is susceptible to insect and 

mite pests, and to increase the probability that an acceptable yield can be produced before 

insect pest densities exceed economic threshold levels (Smith and Cothren, 1999). 

Nematodes are another serious pest in cotton and have the potential to cause significant 

loss of yield, reduction in fiber quality, and crop maturity. Yield losses in cotton from 

nematodes exceed $400 million annually in the U.S (NCCA, 2007). Management 

decisions for controlling nematodes must be made prior to or at planting since few 

control options are available during the season. 
Monsanto Company 12-CT-244U 

Table VIII-3. Insect Losses in Cotton in U.S. in 20101 Insect Pest 

% Yield 

Reduction 

Cotton Acres 

Infested 

Cotton 

Acres 

Treated 

Treatment 

Cost 

($/Acre) 

Cotton Bales 

Lost 

Bollworm/Budworm 1.186 8,148,844 2,113,842 2.54 263,902 Stink bugs 0.724 6,712,988 2,782,462 3.45 162,397 Lygus spp. 0.677 5,932,835 2,458,413 6.86 191,826 Cotton Fleahopper 0.362 4,487,032 2,357,727 2.79 81,048 Aphids 0.286 7,133,029 1,270,253 1.39 60,377 Thrips 0.200 10,165,601 3,469,195 2.32 45,964 Spider mites 0.199 3,522,479 885,684 1.75 57,189 Fall Armyworm 0.199 2,762,701 203,109 0.20 41,256 Clouded Plant Bugs 0.024 614,569 213,683 0.14 6,465 Silverleaf Whitefly 0.020 508,430 111,902 0.81 4,935 Cutworms 0.003 487,946 543,570 0.19 699 Grasshoppers 0.001 1,293,128 53,300 0.02 373 Beet Armyworm 0.001 967,552 52,222 0.06 146 Loopers 0.001 735,998 9,160 0.01 197 Saltmarsh caterpillar 0.001 689,127 3,400 0.00 140 Banded winged whitefly 0.000 483,273 - 0.00 0 Southern Armyworms 0.000 242,500 - 0.00 0 Boll Weevil 0.000 115,470 25,920 0.01 0 Pink Bollworm 0.000 97,725 - 0.00 0 Cotton Leaf Perforator 0.000 14,988 14,988 0.00 0 European cornborer 0.000 0 - 0.00 0 Other Insects (1-4) 0.023 649,594 26,648 0.01 68,906 Total 3.906 22.56 985,821 1(Williams, 2010). 
Monsanto Company 12-CT-244U 182 of 620 

VIII.E. Management of Diseases 

Disease management is essential in cotton production to achieve optimum yields and 

economic returns. Plant pathologists estimate that diseases cause annual losses in cotton 

production of 1.8 million bales or a yield reduction of approximately 9.0 % in the U.S. 

(Blasingame et al., 2008). Seedling diseases, fungal wilts, root rots, and foliar diseases 

constitute the major disease complex in cotton (Smith and Cothren, 1999). Yield losses 

are often underestimated because most of the diseases are caused by soil-borne pathogens 

that attack the roots and cause little reduction of the plant size or change to the crop 

canopy (Smith and Cothren, 1999). These types of infestation can result in yield losses 

of as much as 20% without any awareness of the root infections by soil-borne pathogens. 

The major seedling disease complex (i.e., Pythium spp., Rhizoctonia spp., Fusarium spp., 

and Thielaviopsis spp.) are caused by fungal pathogens and are generally classified as 

seed-borne pathogens that occur on or in seed prior to planting and soil-borne pathogens 

that reside in soil (Smith and Cothren, 1999). The soil-borne pathogens are the most 

important causes of seedling disease and the most difficult to control. Verticillium wilt 

and Fusarium wilt are the two major fungal wilt diseases causing losses in cotton 

production. The pathogens penetrate root tips and enter the xylem vessels of the cotton 

plant and the plants subsequently develop the characteristics of wilt symptoms. 

Phymatotrichum root rot, macrophomina root rot, agrobacterium root rot and root gall are 

the primary diseases attacking roots of older cotton plants (Smith and Cothren, 1999). 

The foliar diseases are caused by pathogens that infect leaves, stems, bolls, and 

occasionally seedling roots. Bacterial blight, boll rot, fungal leaf spots, fungal boll rots, 

viran and mycoplasmal make up the primary foliar diseases. 

An integrated management system is the best means of controlling diseases in cotton. 

This includes agronomic and cultural practices (i.e., fertility, water management, crop 

rotation), use of resistant varieties, applications of fungicides and bactericides, and 

applications of biocontrol agents (Smith and Cothren, 1999). The single most important 

practice for minimizing damage from seedling diseases is selection of high-quality 

planting seed that has minimal seed coat damage and has been assessed for germination 

and vigor (Smith and Cothren, 1999). Seedbed conditions that encourage rapid 

germination and emergence will minimize seedling disease losses (NCCA, 2007). 

Selection of varieties with satisfactory levels of resistance is also an important step in the 

control of certain other diseases. Various cultural practices such as crop rotation, proper 

fertility and water management, clean tillage systems, early planting, eliminating weeds 

which are host plants to the pathogen, and practices that increase decomposition of crop 

residues can reduce the severity of diseases (Smith and Cothren, 1999). Fungicides are 

used to protect seeds and seedlings from seed- and soil-borne pathogens during their first 

few weeks of growth. Commercial cottonseed is normally treated and planted with a 

mixture of chemical fungicides applied to control these soil borne pathogens (Smith and 

Cothren, 1999). Fungicides are also used to prevent epidemics of foliar diseases when 

they approach economically damaging levels. An average of three fungicide treatments 

were made to cotton in 2007 (USDA-ERS, 2012a). Foliar fungicides are applied to 

approximately 2% of the cotton acreage (USDA-NASS, 2008). 
Monsanto Company 12-CT-244U 183 of 620 

VIII.F. Weed Management 

Weed control in cotton is essential to maximize both cotton fiber yield and quality. In 

contrast to other crops, including corn and soybean, cotton emergence and above ground 

growth is relatively slow during the first few weeks after planting. The slow early growth 

of cotton does not permit the crop to aggressively compete against weed species that 

often grow more rapidly (Smith and Cothren, 1999). This is especially true under cool 

weather or adverse growing conditions which often prevail after cotton is planted. The 

extent or degree to which weeds interfere with cotton growth and yield is dependent on 

the species, densities, duration, and environmental conditions. For example, a single 

common cocklebur over 30 row-feet can reduce cotton yields by 8.85%, while a single 

prickly sida plant at the same density reduces cotton yields by only 0.26% (Smith and 

Cothren, 1999). Weed-crop competition studies have demonstrated that the control of 

weeds during the first four to eight weeks after cotton planting is critical as weeds 

compete against the crop for water, nutrients, light, and other resources necessary for 

growth (Smith and Cothren, 1999). Although late-season infestations may not impact 

yield, they reduce harvesting efficiency, contribute to the weed seed bank, and lower the 

lint grade (Vargas et al., 1996). Weeds can also have an impact on cotton diseases and 

insect management because certain weed species can be a host for Rhizoctonia and 

Verticillium wilt and harbor insects such as lygus bugs. 

The occurrence and frequency of individual weed species in cotton vary greatly between 

and within each state and geographical growing region. Cultural and chemical control 

practices can cause shifts in the composition of weed populations (Smith and Cothren, 

1999). Weed populations are affected over time by edaphic (soil-rated) factors, 

reproductive ability, control methods, cropping sequences, herbicide regimes, herbicideresistance, climatic changes, and other environmental situations (Smith and Cothren, 

1999). The most common weeds in cotton are not necessarily the most troublesome 

weeds. The degree of importance depends on the interference to cotton growth and yield, 

reduction in lint quality, and expense of control (Smith and Cothren, 1999). The proper 

identification of the weed species, especially in the immature stages of growth is essential 

to the development of an effective weed management program. Table 4 lists the common 

and species names of all weeds referred to in this petition. Tables VIII-5 through VIII-8 

provide summaries of the most common weeds in cotton for each of the four major cotton 

growing regions (Southeast, Midsouth, Southwest, and West). Barnyardgrass, crabgrass, 

pigweed spp. (including Palmer amaranth), morningglory spp., common cocklebur, and 

common lambsquarters are common annual weed species in almost all cotton growing 

regions. Johnsongrass, bermudagrass and nutsedge are common perennial weed species. 

Weed species of the Solanaceae family, such as the nightshade spp. and groundcherry, 

are more common in the Southwest and West regions. Palmer amaranth, morningglory 

spp., and nutsedge spp. are not only common in cotton, but are frequently reported as 

some of the most troublesome or serious weed species in cotton (Webster et al., 2009). 
Monsanto Company 12-CT-244U 184 of 620 

Table VIII-4. Common and Scientific Names of Weeds Referred to in this Petition 

Common Name Scientific Name Common Name Scientific Name 

 

Annual bluegrass Poa annua Little barley Hordeum pusillum 

Barnyardgrass Echinochloa crus-galli Jimsonweed Datura stramonium 

Bermudagrass Cynodon dactylon Johnsongrass Sorghum halepense 

Bindweed, field Convolvulus arvensis Junglerice Echinochloa colona 

Black nightshade 

(Eastern) Solanum ptychanthum Large Crabgrass Digitaria sanguinalis 

Broadleaf signalgrass Urochloa platyphylla Morningglory spp Ipomoea spp. 

Browntop millet Urochloa ramosa Mustard spp. Brassica spp. 

Buttercup Ranunculus spp. Nightshade, hairy Solanum physalifolium 

Carolina geranium Geranium carolinianum Nightshade, 

silverleaf 

Solanum 

elaeagnifolium 

Chickweed Stellaria media Nutsedge spp. Cyperus spp. 

Citronmelon Citrullus lanatus Palmer amaranth Amaranthus palmeri 

Common cocklebur Xanthium strumarium Pigweed spp. Amaranthus spp 

Common hempnettle Galeopsis tetrahit Prickly lettuce Lactuca serriola 

Common lambsquarters Chenopodium album Prickly sida Sida spinosa 

Common ragweed Ambrosia artemisiifolia Purple nutsedge Cyperus rotundus 

Crabgrass spp. Digitaria spp. Purslane, common Portulaca oleracea 

Crowfootgrass Dactyloctenium 

aegyptium Purslane, horse Trianthema portulacastrum 

Cupgrass, southwestern Eriochloa acuminata Red rice Oryza punctata 

Curly dock Rumex crispus Redweed Melochia corchorifolia 

Cutleaf eveningprimrose Oenothera laciniata Russian thistle Salsola tragus 

Devil's claw Proboscidea louisianica Sandbur Cenchrus spp. 

Fall panicum Panicum dichotomiflorum Shepard’s purse Capsella bursa-pastoris 

Florida beggarweed Desmodium tortuosum Sicklepod Senna obtusifolia 

Florida pusley Richardia scabra Smartweed spp. Polygonum spp. 

Foxtail spp. Setaria faberi Smellmellon Cucumis melo 

Giant foxtail Setaria spp. Sprangletop, red Leptochloa panicea 

Giant ragweed Ambrosia trifida Spreading dayflower Commelina diffusa 

Goosegrass Eleusine indica Spurge spp. Euphorbia spp. 

Groundcherry spp. Physalis spp. Spurred anoda Anoda cristata 

Henbit Lamium amplexicaule Sunflower Helianthus annuus 

Hemp sesbania Sesbania herbacea Texas blueweed Helianthus ciliaris 

Horseweed (marestail) Conyza canadensis Texas millet Urochloa texana 

Italian ryegrass Lolium multiflorum Texas panicum Urochloa reptans 

Kochia Kochia scoparia Tropical spiderwort Tradescantia ohiensis 

Velvetleaf Abutilon theophrasti 
Monsanto Company 12-CT-244U 185 of 620 

Table VIII-4. Common and Scientific Names of Weeds Referred to in this Petition 

(continued) 

Common Name Scientific Name 

Virginia pepperweed Lepidium virginicum 

Volunteer corn Zea Mays 

Volunteer peanut Arachis hypogaea 

Common waterhemp Amaranthus rudis 

Tall waterhemp Amaranthus tuberculatus 

Wild lettuce Lactuca canadensis 

Wild mustard Sinapis arvensis 

Wild radish Raphanus raphanistrum 

Woolyleaf bursage Ambrosia grayi 

Yellow nutsedge Cyperus esculentus 

Table VIII-5. Common Weeds in Cotton Production in the Southeast Region of the 

U.S.1,2 

Crabgrass spp.(6) 

Morningglory spp.(6) 

Prickly sida(5) 

Florida pusley(4) 

Nutsedge spp.(4) 

Sicklepod(4) 

Broadleaf signalgrass(3) 

Goosegrass(3) 

Pigweed spp.(3) 

Common cocklebur(2) 

Common lambsquarters(2) 

Common ragweed(2) 

Florida beggarweed(2) 

Palmer amaranth(2) 

Texas millet(2) 

Bermudagrass(1) 

Crowfootgrass(1) 

Horseweed (marestail)(1) 

Jimsonweed(1) 

Johnsongrass(1) 

Smartweed spp.(1) 

Spurge spp.(1) 

Volunteer peanut(1) 

1

OK data (Webster et al., 2009). 

2

Number provided in parenthesis is the number of states out of the six total states (AL, FL, GA, NC, SC, 

and VA) in the Southeast region reporting each weed as one of the ten most common weeds. 

   
Monsanto Company 12-CT-244U 186 of 620 

Table VIII-6. Common Weeds in Cotton Production in the Midsouth Region of the 

U.S.1,2

Morningglory spp.(5) 

Broadleaf 

signalgrass(4) 

Crabgrass spp.(4) 

Nutsedge spp.(4) 

Prickly sida(4) 

Spurge spp.(4) 

Pigweed spp.(3) 

Velvetleaf(3) 

Barnyardgrass(2) 

Horseweed 

(marestail)(2) 

Johnsongrass(2) 

Palmer amaranth(2) 

Bermudagrass(1) 

Browntop millet(1) 

Common cocklebur(1) 

Cutleaf eveningprimrose(1) 

Goosegrass(1) 

Hemp sesbania(1) 

Henbit(1) 

Spurred anoda(1)

1

AR, LA & MO data (Webster et al., 2009); MS & TN data (Webster et al., 2005). 

2

Number provided in parenthesis is the number of states out of the five total states (AR, LA, MS, MO, & 

TN) in the Midsouth region reporting each weed as one of the ten most common weeds. 

Table VIII-7. Common Weeds in Cotton Production in the Southwest Region of the 

U.S.1,2

Johnsongrass(4) 

Nutsedge spp.(4) 

Cocklebur, common(3) 

Palmer amaranth(3) 

Silverleaf Nightshade 

(3) 

Common 

lambsquarters(2) 

Large Crabgrass(2) 

Devil's claw(2) 

Morningglory spp.(2) 

Mustard spp.(2) 

Pigweed spp.(2) 

Russian thistle(2) 

Barnyardgrass(1) 

Bermudagrass(1) 

Bindweed, field (1) 

Foxtail spp.(1) 

Groundcherry spp.(1) 

Kochia(1) 

Horseweed 

(marestail)(1) 

Shepard’s purse(1) 

Smartweed(1) 

Smellmelon(1) 

Spurred anoda(1) 

Sprangletop, red(1) 

Sunflower(1) 

Texas blueweed(1) 

Texas millet(2) 

Velvetleaf(1) 

Woolyleaf bursage(1) 

1

OK data (Webster et al., 2009); KS – Stewart Duncan, Ph.D., Kansas State University – Personal 

Communication November, 2010; NM –Jamshid Ashigh, Ph.D., New Mexico State University – Personal 

Communication November, 2010; TX –Wayne Keeling, Ph.D. and Gaylon Morgan, Ph.D., Texas A&M 

University - Personal Communications November, 2010. 

2

Number provided in parenthesis is the number of states out of the four total states (OK, KS, TX, & NM) in 

the Southwest region reporting each weed as one of the ten most common weeds. 

 
Monsanto Company 12-CT-244U 187 of 620 

Table VIII-8. Common Weeds in Cotton Production in the West Region of the 

U.S.1,2

Barnyardgrass(2) 

Morningglory spp.(2) 

Sprangletop(2) 

Bermudagrass(1) 

Bindweed, field(1) 

Cupgrass, 

southwestern(1) 

Groundcherry spp.(1) 

Lambsquarters, 

common(1) 

Johnsongrass(1) 

Junglerice(1) 

Nutsedge spp.(1) 

Pigweed spp.(1) 

Nightshade, black(1) 

Nightshade, hairy(1) 

Nightshade, silverleaf(1) 

Palmer amaranth(1) 

Purslane, common(1) 

Purslane, horse(1) 

Volunteer corn(1) 1.

1

Source:AZ – Bill McCloskey, Ph.D., University of Arizona – Personal Communication, November, 2010; 

CA – Steven Wright, Ph.D., University of California - Personal Communication November, 2010. 

2

Number provided in parenthesis is the number of states out of the two total states (AZ & CA) in the West 

Region reporting each weed as one of the ten most common weeds.

VIII.F.1. Methods of Weed Control in Cotton 

Weeds in cotton are controlled through the integrated use of various cultural, mechanical, 

and chemical methods (Hake et al., 1996d). Crop rotation, or the lack of rotation, in 

conjunction with other weed control methods, can play an important factor on the weed 

spectrum and drastically impact weed populations (Smith and Cothren, 1999). 

Historically, mechanical tillage and hand hoeing were the most important tools in cotton 

weed control. Current weed management practices include as many as five tillage 

operations in conventional tillage systems and two or three tillage operations in mulch 

tillage, or no-till systems (USDA-ERS, 2012b). Approximately, 38% of the total cotton 

acres are post-plant cultivated and within conventional tillage systems, over 50% cotton 

acres are cultivated for weed control (USDA-ERS, 2012b). 

The use of chemical methods for weed control began to develop in cotton in the 1940s 

and 1950s with the discovery and development of several selective herbicides (Buchanan, 

1992). Dinoseb, chloropropham, dalapon, and diuron were developed and used in cotton. 

Despite the increased use of herbicides in the late 1950s, less than 10% of the total U.S. 

cotton acreage received a herbicide treatment. However, herbicide use rapidly 

accelerated in the 1960s as a series of more selective herbicides were introduced into the 

market. These herbicides provided good weed control with less cotton injury than most 

products used a decade earlier. These products included trifluralin, DSMA/MSMA, 

prometryn, and fluometuron. These herbicides, representing different chemical families 

and modes-of-action, are still widely used today. Additional herbicides were introduced 

during the 1970s that were efficient, effective, and relatively economical on a wide range 

of weed species. Glyphosate was introduced in the early 1970s and quickly became one 

of the most effective herbicides for nonselective spot treatments for control of 

johnsongrass and other weeds (Buchanan, 1992). Glyphosate was also an effective 

burndown treatment within no-till cotton production. The use of dinoseb for broadleaf 

weed control was halted in 1987 with the suspension of the registration by the 

Environmental Protection Agency (McWhorter and Bryson, 1992). Registrations were 

also discontinued for dinitramine, flurachloralin, profluralin, dalapon, dipropetryn, and 
Monsanto Company 12-CT-244U 188 of 620 

perfluidone. Numerous additional selective herbicides for grass and broadleaf weed 

control were introduced in the 1980s in cotton including fluazifop, metolachlor, 

oxyfluorfen, and sethoxydim. However, the use of these products does not equal the 

acreage treated with the herbicides which were discontinued (Buchanan, 1992). By the 

mid 1980s, there were 33 herbicides and herbicide combinations applied in cotton 

(Buchanan, 1992). The greatest use of herbicides on a per-acre basis was in the Midsouth 

which averaged 5.7 herbicide applications per acre each year. During the 1990s, the 

herbicides lactofen, bromoxynil, clethodim, clomazone, quizalifop, and pyrithiobac were 

introduced for use in cotton. 

The first biotechnology-derived herbicide-tolerant cotton became available in 1995 and 

provided tolerance to bromoxynil (Stalker et al., 1996). Approximately 50,000 acres of 

bromoxynil-tolerant cotton were planted the year of introduction and approximately 2500 

growers planted 200,000 acres of bromoxynil-tolerant cotton in 1996 (Smith and 

Cothren, 1999). The second herbicide-tolerant cotton product, the first generation 

glyphosate-tolerant cotton, was introduced in 1997. Glyphosate-tolerant cotton in 

combination with glyphosate herbicide became the standard program for weed 

management in cotton. The first generation glyphosate-tolerant weed control system in 

cotton provided postemergence control of a broad spectrum of weeds with excellent 

early-season crop safety (Wilcut et al., 2003). Glyphosate-tolerant cotton expanded the 

grower’s options for weed management and made the mechanics of weed control much 

easier, more convenient, and less expensive (Carpenter and Gianessi, 2001; Wilcut et al., 

2003). This system also provided a better fit into no-till and reduced-tillage systems, 

resulting in an increase in conservation tillage systems in cotton (Baldwin and Baldwin, 

2002; Carpenter and Gianessi, 2001). Glyphosate could be applied postemergence to 

glyphosate-tolerant cotton from emergence through the four-leaf stage. After the four 

leaf stage and up to layby (canopy closure in the row), glyphosate had to be applied as a 

post-directed spray between the crop rows to minimize contact with the cotton plants to 

prevent potential crop injury. 

In 2003, glyphosate-tolerant cotton was planted on approximately 59% of the cotton 

acres in the U.S. (USDA-NASS, 2003). Glyphosate was the most widely used herbicide 

in cotton in terms acres treated (USDA-NASS, 2004). However, cotton growers 

continued to use a variety of herbicides with various modes-of-action in glyphosatetolerant cotton. Trifluralin and pendimethalin were used on nearly half of the U.S. cotton 

acreage for small seeded grass and broadleaf weed control. Various substituted urea 

herbicides (diuron, prometryn, fluometuron and linuron) were also used on 50% of the 

U.S. cotton acreage (USDA-NASS, 2004). The soil residual activity of these herbicides 

on a number of weed species provided additional season-long control of continuously 

germinating weeds in glyphosate-tolerant cotton systems (Askew et al., 2002; Wilcut et 

al., 2003). Other herbicide products representing additional modes-of-action, including 

carfentrazone, MSMA, pyrithiobac and metolachlor, were also used on cotton ranging 

from four to 11% of the acres (USDA-NASS, 2002). 

In 2006, a second generation glyphosate-tolerant product was introduced providing 

increased tolerance to glyphosate in the reproductive stages of cotton. This allowed for 

an expanded window for over-the-top applications of glyphosate in cotton. Glyphosate 
Monsanto Company 12-CT-244U 189 of 620 

can be applied over-the top in second generation glyphosate-tolerant cotton from 

emergence up to 7 days prior to harvest. With this additional application flexibility, 

growers were able to more effectively manage weeds in cotton using over-the-top 

applications as opposed to post-directed or hooded sprayer applications with previous 

glyphosate-tolerant varieties. In addition, foliar insecticides could be combined with 

glyphosate in a single application during the season for secondary pests such as thrips, 

aphids, and plant bugs. Mepiquat chloride, a plant growth regulator commonly used in 

cotton production to reduce vegetative growth and increase fruit retention, could also be 

applied with glyphosate in a single application. In 2010, approximately 78% of the 

cotton acreage was planted to herbicide-tolerant cotton, which was nearly all glyphosatetolerant (Brookes and Barfoot, 2012). 

The third herbicide-tolerant cotton product, glufosinate-tolerant cotton, was introduced in 

2003. Only 3% was planted to glufosinate-tolerant cotton in 2010 (USDA-ERS-FAS, 

2010). Approximately 50% of the acres planted to cotton varieties containing both 

herbicide- and insect-tolerant traits (USDA-NASS, 2011a). 

Table VIII-9 provides a summary of the herbicide applications registered for use in 

cotton in 2010, the data are discussed below. Herbicides are used on essentially all 

(99+%) cotton acres in the U.S. (Monsanto Company, 2011). A total of 32.8 million 

pounds of herbicide active ingredient were applied in cotton in 2010. Glyphosate was the 

predominate herbicide used in cotton with 19.6 million pounds active ingredient being 

applied on 91% of the acres. The number of glyphosate applications, on glyphosatetolerant cotton, average approximately 2.4 applications per year at an average rate of 2.0 

pounds of glyphosate active ingredient per acre per crop year (Monsanto, 2011). 

Dinitroanaline herbicides (pendimethalin and trifluralin) were applied on 53% of the 

cotton acres. Diuron (18%), flumioxazin (16%), metolachlor (16%), pyrithiobac (15%), 

fomesafen (13%), and 2,4-D (13%) were also frequently used herbicides in cotton 

(Monsanto Company, 2011). 

According to USDA-ERS (2012a) statistics, growers make on average a total of four 

herbicide applications in cotton during the growing season. Approximately 16-19% of 

the growers utilizing the latest glyphosate-tolerant cotton varieties applied a fall herbicide 

application to control weeds prior to planting cotton depending on their crop rotation 

(Prince et al., 2011). Approximately 53-97% of the growers applied spring burndown 

treatments in glyphosate-tolerant cotton, which consisted of predominately glyphosate 

and/or synthetic auxins (2,4-D, dicamba). 

Dicamba is currently labeled for use in cotton, although dicamba use is limited because 

applications are restricted to early preplant only, due to cotton injury. Before planting 

cotton, a minimum accumulation of one inch of rainfall or overhead irrigation must occur 

and a waiting interval of 21 days is required per 0.25 lbs acid equivalent (a.e.) or less. 

Dicamba-treated acres have increased in cotton primarily because it is a leading herbicide 

recommendation for glyphosate-resistant marestail (horseweed) in the Midsouth region 

(McClelland et al., 2006). 
Monsanto Company 12-CT-244U 190 of 620 

Glufosinate may be used for weed control in non-glufosinate-tolerant cotton when 

applied with a hood sprayer in-crop to avoid contact with cotton plants. Glufosinate can 

also be applied in glufosinate-tolerant cotton from emergence up to the early bloom 

growth stage. 

Approximately 15, 39, and 42% of growers made 1, 2, and 3 in-crop applications of 

glyphosate in continuous cotton, respectively (Prince et al., 2011). Although glyphosate 

is used extensively in glyphosate-tolerant cotton, non-glyphosate herbicides with 

different modes-of-action are also utilized to provide residual weed control, improve the 

control of certain weed species, extend weed control, and/or control resistant weeds. The 

use of herbicides with different modes-of-action is an effective practice to reduce the 

potential risk of weeds developing resistance to glyphosate or other herbicide modes-ofaction. Approximately 49-76% of growers applied non-glyphosate herbicides prior to 

planting, at planting, or postemergence in glyphosate-tolerant cotton in 2010 depending 

on cropping system (Prince et al., 2011). The non-glyphosate herbicides were ALS 

inhibitors (trifloxysulfuron, pyrithiobac), photosystem II inhibitors (prometryn, 

fluometuron, diuron), mitosis inhibitors (metolachlor), PPO inhibitors (flumioxazin, 

fomesafen), and synthetic auxins (2,4-D, dicamba). 

Weed management in conventional cotton varieties is very similar. The major difference 

in the herbicide programs is that alternative postemergence herbicides or herbicide tank 

mixtures are applied in place of glyphosate as in-crop post applications. Glyphosate can 

still be applied alone or in combinations with other herbicides in preplant burndown or 

preharvest applications in conventional cotton. A herbicide or combination of herbicides 

(trifluralin, pendimethalin, fluometuron, fomesafen, flumioxazin) is generally applied at 

planting for residual grass and broadleaf weed control. Generally, at least two in-crop 

post applications are made for control of emerged weeds during the growing season. 

Pyrithiobac, trifloxysulfuron, prometryn, clethodim, and sethoxydim are some of the 

more common herbicides used post in cotton. In addition, a layby application of one or 

more herbicides is applied such as diuron, MSMA, prometryn, or trifloxysulfuron. 

Tables VIII-10 through VIII-14 provide a summary of the control ratings of common 

weed species to various herbicides and herbicide combinations in cotton. These tables 

list only the most commonly used herbicides or herbicide treatments in cotton production 

and control ratings are for non-glyphosate-resistant weeds. Seldom would one field or 

farm have all weed species, but they generally have a mixture of grass and broadleaf 

weed species. These ratings are utilized to facilitate the selection of a herbicide program 

for the cotton crop, which offers the best overall control of the weed species. 

Dinitroanalines (trifluralin and pendimethalin) provide effective control of most listed 

annual grasses, but only certain broadleaf species. Postemergence treatments of 

quizalofop, fluazifop, sethoxydim and clethodim are effective on the annual grasses and 

perennial grasses listed such as johnsongrass and bermudagrass, but provide no control of 

the broadleaf species. On the other hand, preemergence or postemergence applications of 

fluometuron or pyrithiobac provide good control of many broadleaf weeds and poor or no 

control of most grasses. In-crop applications of glyphosate and glufosinate provide good 

to excellent control of a broad spectrum of annual grass and broadleaf weeds. However, 
Monsanto Company 12-CT-244U 191 of 620 

glyphosate provides more effective control of perennial weeds such as bermudagrass, 

johnsongrass, and nutsedge species as compared to glufosinate. In addition, glyphosate 

combinations are the most effective herbicide treatments for silverleaf nightshade, Texas 

blueweed, and woolyleaf bursage, which are problem weeds in the Southwest region. 

Post-directed layby applications of MSMA in combination with diuron, flumioxazin, or 

prometryn and the premix combination of prometryn/trifloxysulfuron provide broad 

spectrum weed control. Due to the broad range of weed species present in cotton, 

multiple treatments and/or combinations of herbicides are used to achieve effective 

season-long weed control in cotton. 
Monsanto Company 12-CT-244U 

Table VIII-9. Herbicide Applications Registered for Use in Cotton in 20101 Herbicide Chemical Family 

Mode of Action (MOA) 

Cotton Acres 

Treated (%) 

Cotton Acres 

Treated per 

MOA (%) 

Quantity 

Applied (100

lbs a.i. 2) 

Glyphosate Glycine EPSPS inhibitor 91 91 19,602 Pendimethalin Dinitroanaline Microtubule 

inhibitor 

18 

53 1,584 

Trifluralin Dinitroanaline 35 3,554 Diuron Urea 

PSII inhibitor 

18 

34 

1,527 

Prometyrn Triazine 8 650 Fluometuron Urea 7 619 Linuron Urea <1 31 Carfentrazone Triazolinone 

PPO inhibitor 

1 

30 

2 

Flumioxazin N-phenylphthalimide 16 114 Fomesafen Diphenylether 13 342 Lactofen Diphenylether <1 1 Oxyfluorfen Diphenylether <1 6 Pyraflufen Phenylpyrazole <1 <1 2,4-D Phenoxy 

Synthetic Auxin 

13 

21 

891 

2,4-DB Phenoxy <1 1 Dicamba Benzoic acid 8 192 
Monsanto Company 12-CT-244U 

Table VIII-9. Herbicide Applications Registered for Use in Cotton in 20101(continued) Herbicide Chemical Family 

Mode of Action (MOA) 

Percent of 

Cotton Acres 

Treated 

Percent of 

Cotton Acres 

Treated per 

MOA 

Quantity 

Applied 

(1000 lbs 

a.i.2) 

Pyrithiobac Benzoate 

ALS inhibitor 

15 

20 

72 

Thifensulfuron Sulfonylurea <1 <1

Tribenuron Sulfonylurea <1 <1 Trifloxysulfuron Sulfonylurea 4 3 Acetochlor Chloroacetamide Long-chain fatty 

acid inhibitor 

<1 16 47 

Metolachlor Chloroacetamide 16 1,766 Paraquat Bipyridylium Photosystem-I- electron diverter 10 10 547 

Glufosinateammonium Phosphinic acid Glutamine synthesis inhibitor 8 8 535 MSMA Organoarsenical Cell membrane disruption 6 6 747 Clethodim Cyclohexanedione 

ACCase inhibitor 

<1 

1 

6 

Fluazifop Aryloxyphenoxy- propionate <1 1 Diflufenzopyr Semicarbazone Auxin transport <1 <1 <1 Clomazone Isoxazolidinone Diterpene synthesis inhibitor <1 <1 10 Total 99.4 1(Monsanto, 2011). 2a.i.= active ingredient. 
Monsanto Company 12-CT-244U 

Table VIII-10. Grass Weed Species Control Ratings to Preplant Burndown Herbicides in Cotton 

 Common Grass Weeds1

Product AB2 BG2 CG2 GFT4 GG2 IRG2 JGs2 LB2 RR4 2,4-D N N N 0 N N N N 0 Glufosinate 

63 - 83 - 83 83 93 73 - 

Glufosinate + 2,4-D or dicamba 

64 - 74 84 - 54 - 74 74 

Glyphosate E F E 8 E G G-E E 8 Glyphosate + 2,4-D E F G-E 8 G-E G G E 8 Glyphosate + dicamba E F G-E 8 G-E G G E 8 Glyphosate + carfentrazone or pyraflufen 

E F E - E G G-E E - 

Glyphosate + diuron E F G - G F F-G E - Glyphosate + thifensulfuron/ tribenuron E F E - E G G-E E - Glyphosate + flumioxazin E F E 8 E G G-E E 8 Paraquat G-E P F-G 8 F-G F P G 7 Paraquat + diuron E P G - G F-G P G-E - 

1Weed Species: AB = annual bluegrass, BG = bermudagrass, CG = crabgrass, GFT = giant foxtail, GG = goosegrass, IRG = Italian 

johnsongrass, LB = little barley, RR = red rice, SB = sandbur, TP = Texas millet (Texas panicum), VC = volunteer corn. 2(University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-80% control

N = < 30% control. 

3 (MSU, 2012). Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control. 4 (University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control. 
Monsanto Company 12-CT-244U 

Table VIII-11. Grass Weed Species Control Ratings to Preplant, Preemergence and Postemergence Herbi Common Grass Weeds & Nutsedge1Product BYG2 BSG3 CG3 CFG3 CPG4 FT3 GG3 JR5 JGs3 ST4 TP3 BG3 JPreplant Incorporated OnlyPendimethalin

9

G

E

E

C

E

E

C

E - G

N

Trifluralin

9

G

E

E

C

E

E

C

E

C

G

N

Preemergence

Pendimethalin - F

G

G

C

G

G

C

G - F

N

Clomazone - E

E

G - E

E - G - F P-F

Fluometuron

7

P F-G F-G - F-G

F - P - P

N

Diuron

7

P F-G F-G

C - F

C

P

N

P

N

Fomesafen - F-G F-G - - - - - - - F

N 

Pyrithiobac

6

P

P - - P P-F - F-G - N

N

Postemergence Residual Control

Metolachlor - F-G

E

E

C

E

E

C

F

C P-F

N

Pyrithiobac - P

P - - P P-F - F - N

N

Trifloxsulfuron - P

P

P - P

P - P - P

N
Monsanto Company 12-CT-244U 

Table VIII-11. Grass Weed Species Control Ratings to Preplant, Preemergence and Postemergence H(continued)  Common Grass Weeds & Nutsedge1Product BYG2 BSG3 CG3 CFG3 CPG4 FT3 GG3 JR4 JGs3 ST4 TP3 BG3 Postemergence Over-The-TopQuizalofop 8 G

G

G - E

G - E - G

G

Fluazifop

7 G-E

G

F

C

E

G

C G-E

C

G

G 

Sethoxydim 8 E G-E F-G

C

E G-E

C G-E

C

E

F

Clethodim 8 E G-E

G

C

E G-E

C

E

C

E

G 

MSMA - P

P

P

P - P

N

P

N N-P

N

Fluometuron - P P-F P-F - - P-F - P - N

N

Pyrithiobac

2

N

N

N

N N-P N-P

N

P

N

N

N 

Trifloxsulfuron

7

N

P

N

N N-P N-P

N

F

N N-P

N

Glyphosate

9

E

E

E

C

E

E

C

E

C

E

F 

Glyphosate + Pyrithiobac

9

E

E

E - E

E - E - E

F 

Glyphosate + Trifloxsulfuron - E

E

E - E

E - E - E

F 

Glufosinate 8 G

G

G

C

G

P

C

G

C

G

N
Monsanto Company 12-CT-244U 

Table VIII-11. Grass Weed Species Control Ratings to Preplant, Preemergence and Postemergence H(continued)  Common Grass Weeds & Nutsedge1Product BYG2 BSG3 CG3 CFG3 CPG4 FT3 GG3 JR4 JGs3 ST4 TP3 BG3 Postemergence Directed – LaybyMSMA - F

F

F

P

F

F

N

F

N

P

N

Diuron + MSMA

9

G

G F-G - F-G F-G - F-G - F

N

Prometryn + MSMA

9 F-G F-G F-G - F-G F-G - F-G - F

N

Flumioxazin + MSMA

9

F

F

F - F

F - F - P-F

N

Prometryn/ trifloxysulfuron

+MSMA

9 F-G F-G F-G - F-G F-G - F-G - F

N

1Weed species: BYG = barnyardgrass, BSG = broadleaf signalgrass, CG = crabgrass, CFG = crowsfootgrass, CPG = cupg

GG = goosegrass, JR = junglerice, JGs = seedling johnsongrass, ST = sprangletop, TP = Texas panicum (Texas millet), B= rhizome johnsongrass, NSy = yellow nutsedge, and NSp = purple nutsedge. 

2(University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control. 3(University of Georgia, 2012).Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-8

60% control; N = < 30% control. 

4 (University of California, 2012). Ratings Key: C = control, P = partial control, N = no control, - = no information. 
Monsanto Company 12-CT-244U 

Table VIII-12. Broadleaf Weed Species Control Ratings to Preplant Burndown Herbicides in Cotton 

 Common Broadleaf Weeds1

Product CD2 BC3 CG3 CW3 CM3 CLP3 HB3 MT3 PA3 SA3 V2,4-D 9 G F P F E P-F G-E F F-G GGlufosinate4 - - 8 10 - 7 6 9 - - Glufosinate + 2,4-D or dicamba2 8 10 8 10 - 8 10 9 9 Glyphosate 7 G-E P-F E G-E P-F G-E G-E E G 

Glyphosate + 2,4-D 9 E F-G E E E E E E Glyphosate + dicamba 9 E G E E G E E E - GGlyphosate + carfentrazone or 

pyraflufen - G-E F-G E E F E G-E E G Glyphosate + diuron - G-E G E G-E F-G E G-E E G Glyphosate + thifensulfuron / tribenuron nuron - G-E G-E E G-E F E G-E E - Glyphosate + flumioxazin 7 G-E G E E F-G E G-E E - GParaquat 5 E G-E E F F G-E P-F F-G F-G Paraquat + diuron - E E E G G-E E F-G G-E F-G 1Weed Species: CD = curly dock, BC = buttercup, CG = Carolina geranium, CW = chickweed, CM = citronmelon, CLP = 

henbit, MT = marestail, PA = Palmer amaranth, SA spurred anoda, VP = Virginia pepperweed, WL = wild lettuce, WR = w

2 (University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control. 3(University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-8

60% control; N = < 30% control. 

4 (MSU, 2012). Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control.
Monsanto Company 12-CT-244U 

Table VIII-13. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence HPart I 

 Common Broadleaf Weeds1

Product CB2 DC3 FB2 FP2 GC4 HS2 NS4 JW2 LQ2 MG2 PA2 

Preplant Incorporated Pendimethalin N N P E N N N N G-E P G Trifluralin N N P E N N N N G-E P G Preemergence Pendimethalin N N P F-G N N N N G P P-F Clomazone F F F-G F-G - F - G G P-F N-P Fluometuron F-G F-G G-E F-G - P - G G-E G F Diuron F F G P-F C P C G G-E F F-G Fomesafen G F-G P P - P - - E P-F E Pyrithiobac N-P F-G G F - P - F-G G F G-E Postemergence Residual Control Metolachlor P N P-F G - P - - F P G Pyrithiobac N-P G G F - P - F-G G F G-E Trifloxsulfuron - G F-G P-F - - - - - - P-F 
Monsanto Company 12-CT-244U 

Table VIII-13. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence H– Part I (continued) 

 Common Broadleaf Weeds1

Product CB2 DC3 FB2 FP2 GC4 HS2 NS3 JW2 LQ2 MG2 

Postemergence Over-The-TopQuizalofop

N

N

N

N - N - N

N

N

Fluazifop

N

N

N

N

N

N

N

N

N

N

Sethoxydim

N

N

N

N

N

N

N

N

N

N

Clethodim

N

N

N

N

N

N

N

N

N

N

MSMA

E P-F

E N-P

P - P

P

P P-F

Fluometuron F-G F-G

G P-F - - - G

G

G 

Pyrithiobac 

G

G

G N-P

P G-E

C

E

N

G

Trifloxsulfuron G-E

G G-E

P - - - N

G

G 

Glyphosate 

E

E

E P-G

C P-F

C

E

G F-G

Glyphosate + Pyrithiobac

E

E

E P-G - G-E - E

G G-E

Glyphosate + Trifloxsulfuron

E

E

E P-G - - - E

E

E

Glufosinate

E G-E

G

F

C - C

E

E

E 

Postemergence Directed – Layby

MSMA

E P-F

E

P

P

N

P

F P-F

F

Diuron + MSMA

E G-E

E

F - P-F - G

G G-E

Prometryn + MSMA

E G-E

E

F - P-F - G

G G-E

Flumioxazin + MSMA

E G-E

E F-G - - - E F-G

E 

Prometryn/ trifloxysulfuron + MSMA

E G-E

E

F - - - G G-E

E

1Weed species: CB = common cocklebur, DC = devil’s claw, FB = Florida beggarweed, FP = Florida pusley, GC = ground cherry, H

nightshade, JW = jimsonweed, LG = Common lambsquarters, MG = morningglory species, PA = Palmer amaranth, PW = pigweed spe

2(University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-80% control; P 

= < 30% control 

3Personal communications with Dr. Wayne Keeling, Texas A & M University - 2011, Weed control ratings key: E = 90% or better 

control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control.

4 (University of California, 2012). Ratings Key: C = control, P = partial control, N = no control, - = no information.
Monsanto Company 12-CT-244U 

Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence – Part II 

 Common Broadleaf Weeds1

Product PL2 RW2 RdW2 SP2 SG2 SN3 SW2 TB3 TSW2 VL4 Preplant IncorporatedPendimethalin E N N N N N N N N 0 Trifluralin E N N N N N N N N 0 PreemergencePendimethalin G N N N N N N N N 0 Clomazone G-E G G-E P N N E N F 10 Fluometuron E E E G P-F N G N F 3 Diuron E G G-E F F N G N P-F 7 Fomesafen G G - P - N - N N 1 Pyrithiobac G N-P G-E P-F G N G N P 85 Postemergence Residual Control Metolachlor G P - P P-F N - N E - Pyrithiobac G N-P G-E P G N G N P - Trifloxsulfuron - - - P-F - N - N - - 
Monsanto Company 12-CT-244U 

Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence H– Part II (continued) 

 Common Broadleaf Weeds1

Product PL2 RW2 RdW2 SP2 SG2 SN3 SW2 TB3 TSW2 

Postemergence Over-The-TopQuizalofop

N

N

N

N

N

N

N

N

N

Fluazifop

N

N

N

N

N

N

N

N

N

Sethoxydim

N

N

N

N

N

N

N

N

N

Clethodim

N

N

N

N

N

N

N

N

N

MSMA P-F P-F

N P-F

N P-F N-P

N

P 

Fluometuron F-G

G F-G F-G P-F

N F-G

N

P 

Pyrithiobac 

F

P - P-F F-G

P

G

N

F

Trifloxsulfuron - G

G

E - P

G

N P-F 

Glyphosate F-G

E

E

E

G

E

G

G P-G

Glyphosate + Pyrithiobac

G

E

E

E

G

E

E

G

G 

Glyphosate + Trifloxsulfuron

G

E - E

G

E

E

G P-G 

Glufosinate F-G

E - E F-G F-G

G F-G P-F 
Monsanto Company 12-CT-244U 

Table VIII-14. Broadleaf Weed Species Control Ratings to Preplant, Preemergence and Postemergence H– Part II (continued) 

 Common Broadleaf Weeds1

Product PL2 RW2 RdW2 SP2 SG2 SN3 SW2 TB3 TSW2 

Postemergence Directed – LaybyMSMA P-F

F

N

F

N

N

P

N

F 

Diuron + MSMA

G

E G-E G-E

G

F

F

N

G

Prometryn + MSMA F-G

E

G G-E

G

F

F

N F-G

Flumioxazin + MSMA

G G-E - G-E

G F-G

G

N G-E

Prometryn/ trifloxysulfuron + MSMA - E - E - F - N F-G

1Weed species: PL = purslane, RW = Common ragweed, RdW = redweed, SP = sicklepod, SG = Spurge, SN = silve

smartweed, TB = Texas blueweed, TSW = tropical spiderwort, VL = velvetleaf, WB = woolyleaf bursage. 2(University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-8

60% control; N = < 30% control.

3Personal communications with Dr. Wayne Keeling, Texas A & M University - 2011, Weed control ratings key: E = 90% 

80% to 90% control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control. 

4 (MSU, 2012). Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control. 5 (University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control. 
Monsanto Company 12-CT-244U 204 of 620 

VIII.F.2. Herbicide Resistant Weeds in Cotton 

Table VIII-15 provides a summary of the common weeds in cotton that have biotypes 

reported resistant to the various herbicide modes-of-action in the U.S. To date there are 

only two species with biotypes confirmed to be resistant to dicamba in the U.S. after over 

40 years of use – kochia and prickly lettuce (Heap, 2012c). Additionally, a population of 

lambsquarters has been confirmed as resistant to dicamba in New Zealand, and in 

Canada, common hempnettle and wild mustard have been confirmed as resistant, for a 

total of five species worldwide with confirmed resistance to dicamba. Currently in the 

U.S., six grass species and eight broadleaf species have been confirmed to have resistance 

to glyphosate. Dicamba provides good to excellent control of all eight of these broadleaf 

species. None of these broadleaf weed biotypes have been shown to have populations 

that are resistant to both glyphosate and dicamba. The first species in the U.S. with a 

biotype resistant to glufosinate was recently confirmed in a glyphosate-resistant Italian 

Ryegrass population (Avila-Garcia and Mallory-Smith, 2011). Additionally, a population 

of goosegrass from Malaysia has been confirmed resistant to glufosinate (Seng et al., 

2010). Thus, there are a total of two species worldwide with biotypes that have 

resistance to glufosinate. A discussion regarding the usefulness of MON 88701 in 

management of herbicide resistant weeds can be found in Section VIII.G., and the 

potential for development of dicamba and glufosinate resistance in weeds following the 

introduction of MON 88701 can be found in Appendix I. 
Monsanto Company 12-CT-244U 205 of 620 

Table VIII-15. Common Weeds in Cotton and Weed Resistance to Herbicide Modes 

of Action in the U.S.1 

   Mode of Action 

Weed Species

ACCase Inhibitors 

ALS Inhibitors 

Chloroacetamides

Dinitroanilines

Glycines

Organoarsenicals

Photosystem II 

Inhibitors 

Thiocarbamates

Ureas & Amides

Synthetic Auxins

Bipyridiliums

PPO Inhibitors

Glutamine 

Synthase Inhibitors

Annual Grasses 

Barnyardgrass X X X X X 

Crabgrass spp. (large, 

smooth) X X 

Foxtail spp. (giant, green) X X X X 

Italian ryegrass X X X X X 

Goosegrass X X X X X 

 Junglerice X 

Annual Broadleaves 

Black nightshade 

(Eastern) X X 

Common cocklebur X X 

Common purslane X X 

Common ragweed X X X X 

Horseweed (marestail) X X X X X 

Jimsonweed X 

Lambsquarters X X 

Palmer amaranth X X X X 

Prickly sida X 

Pigweed spp. (redroot, 

smooth, Powell, 

waterhemp) X X X X X 

Russian thistle X 

Smartweed spp. 

(Pennsylvania, 

ladysthumb) X 

Sunflower X 

Velvetleaf X 

Perennial Grasses 

Johnsongrass X X X X 

Perennial Broadleaves 

Field bindweed X 

1

(Heap, 2012d)
Monsanto Company 12-CT-244U 206 of 620 

VIII.G. Introduction of Dicamba and Glufosinate-Tolerant Cotton - MON 88701 

VIII.G.1. MON 88701 Product Concept 

Monsanto has developed herbicide-tolerant cotton, MON 88701, which will offer 

growers cotton varieties that are tolerant to both dicamba and glufosinate herbicides. 

Herbicide tolerances to dicamba and glufosinate were developed due to the benefits 

associated with these herbicides, including: the ability to control glyphosate-resistant and 

hard-to-control weeds with two unique modes-of-action and the familiarity growers have 

with these herbicides. Since dicamba is currently labeled for only preplant applications 

in cotton, MON 88701 will facilitate a wider window of application for dicamba in cotton 

by allowing preemergence applications of dicamba up to the day of crop emergence, as 

well as postemergence in-crop applications up to seven days preharvest. MON 88701 

will provide the ability for in-crop postemergence applications of glufosinate from 

emergence up to the early bloom growth stage, which is the same as the current 

application timing for glufosinate-tolerant cotton. MON 88701 will be combined with 

glyphosate-tolerant cotton utilizing traditional breeding techniques. This combination of 

herbicide-tolerance traits will allow the use of dicamba, glufosinate, and glyphosate 

herbicides in an integrated weed management program to control a broad spectrum of 

grass and broadleaf weed species in cotton. These herbicides will provide three distinct 

modes-of-action for use in conjunction with other herbicide active ingredients and 

modes-of-action for an effective weed resistance management program in cotton. 

Dicamba will offer improved in-crop postemergence control of glyphosate’s difficult-tocontrol broadleaf weeds, including Florida pusley, hemp sesbania, lambsquarters, 

morningglory species, prickly sida, purslane, and Pennsylvania smartweed (Table VIII16). Glufosinate will offer improved control of certain broadleaf weeds, including 

lambsquarters, morningglory species, and velvetleaf, as compared to glyphosate. 

Dicamba and glufosinate will also offer effective control options for glyphosate-resistant 

broadleaf weed biotypes, including glyphosate-resistant biotypes of Palmer amaranth, 

marestail, common ragweed, giant ragweed, and waterhemp. Additionally, dicamba and 

glufosinate will offer an effective control option for broadleaf species resistant to other 

herbicide classes (e.g., ALS and PPO chemistries). 

VIII.G.2. Dicamba and Glufosinate Usage in MON 88701 

The current labeled use of dicamba in cotton is limited to early preplant application. 

Significant restrictions exist in cotton for preplant application of dicamba, including a 

maximum application rate of 0.25 lbs a.e. per acre, a 21-day interval between application 

and planting cotton, and a minimum of one inch of rainfall or overhead irrigation before 

planting cotton to avoid cotton injury (BASF, 2008). To support the introduction of 

MON 88701, Monsanto will be submitting an application to U.S. EPA to amend 

Registration Number 524-582, a DGA salt formulation, to allow preemergence and incrop postemergence dicamba applications to MON 88701. If approved, growers would 

be authorized to apply dicamba alone or in mixtures with glyphosate, glufosinate, or 
Monsanto Company 12-CT-244U 207 of 620 

other herbicides for preplant or postemergence in-crop applications on MON 88701. 

Pending EPA registration, dicamba would be authorized to be applied up to 1.0 lb a.e. per 

acre prior to planting, up to the emergence of cotton, and postemergence in-crop 

applications, up to 0.5 lbs a.e. per acre each, could be applied up through seven days prior 

to harvest. These application rates are well within the dicamba rates applied to other 

crops, such as corn and sugarcane (BASF, 2008). Maximum application amounts for 

dicamba will be established for total preplant/preemergence applications and in-crop 

applications with the combined total not to exceed 2.0 lbs a.e. per acre of dicamba per 

year for all applications. Based on the dicamba label requested by Monsanto, aerial 

applications of dicamba will not be allowed on MON 88701. 

Glufosinate is currently labeled for preplant and in-crop applications in cotton varieties 

designated as glufosinate-tolerant (Bayer CropScience, 2011). No changes to the 

glufosinate product labels will be necessary to permit broadcast in-crop applications of 

glufosinate to MON 88701. Glufosinate can also be applied as a burndown treatment 

prior to planting or prior to emergence of any conventional or non-glufosinate herbicidetolerant cotton varieties. Directed postemergence applications are also permitted in nonglufosinate-tolerant varieties, provided no herbicide contacts the cotton foliage. Once 

MON 88701 is available, growers will be able to apply glufosinate alone or tank-mixed 

with dicamba for preplant or postemergence in-crop applications on MON 88701. 

Application rates and timings for glufosinate alone will be the same as currently labeled 

for glufosinate use in glufosinate-tolerant varieties (i.e., from emergence up to the early 

bloom stage at 0.402 to 0.530 lbs a.i./acre, seasonal maximum of 1.59 lbs a.i. per acre) 

(Bayer CropScience, 2011). 

The expected use patterns for dicamba and glufosinate on MON 88701 will vary across 

U.S. cotton growing regions. This variability is dictated by the environment and weed 

spectrum variations across these regions. The recommendations for the Midsouth and 

Southeast regions are shown in (Table VIII-16). In these regions, conventional tillage 

planted acres are expected to receive a single in-crop application per season of dicamba at 

0.5 lbs a.e. per acre and conservation tillage or no-tillage acres are expected to receive 

two applications (one preplant application at 0.375 lbs a.e. per acre and one in-crop 

application at 0.50 lbs a.e. per acre). All acres in this region where glyphosate-resistant 

weeds are present, regardless of tillage, are expected to receive a single in-crop 

application of glufosinate as 0.53 lbs a.i. per acre. For the remaining acres where 

glyphosate-resistant weeds are not present, glyphosate will likely be used for control of 

late-emerging weeds. Dicamba and glufosinate use in eastern Texas, is expected to be 

similar to that described for the Midsouth and Southeast regions. 

 
Monsanto Company 12-CT-244U 208 of 620 

Table VIII-16. Anticipated Weed Management Recommendations for MON 88701 

Combined with Glyphosate-Tolerant Cotton Systems for MO, AR, TN, AL, FL, GA, 

NC, SC, VA, LA, MS and eastern TX1 

Application 

Timing 

Conventional Tillage Conservation Tillage 

(No-till or reduced till) 

No GR 

Weeds2

GR Weeds or Suspected 

GR Weeds2

No GR 

Weeds2

GR Weeds or Suspected 

GR Weeds2

Preemergence 

(burndown, at 

planting) 3

Residual Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Postemergence3 

Dicamba 

+ 

Glyphosate 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Postemergence3 Glyphosate Glufosinate Glyphosate 

Glufosinate 

+ 

Residual 1

 The anticipated use patterns represent a high-end estimate for predicting dicamba use associated with 

MON 88701 combined with glyphosate-tolerant cotton. Actual weed control practices by growers will 

vary depending on the specific weed spectrum and agronomic situation of the individual cotton field, 

specifically dicamba use could be lower especially for the preemergence and second postemergence 

applications. 2

 Recommendations for all fields in these regions will assume GR weeds are present. 3 Monsanto and academics recommend the use of soil residuals as part of a comprehensive weed resistance 

management program to ensure that two effective herbicide modes-of-action are used in cotton and to 

provide protections against additional resistance development to existing cotton herbicides.

In western Texas, New Mexico, Kansas, Oklahoma, California and Arizona, dicamba is 

expected to be utilized more extensively than glufosinate for management of hard-tocontrol and/or glyphosate-resistant weeds in MON 88701. Glufosinate is considered less 

effective on the weed spectrum under the high temperature and low humidity 

environmental conditions in these regions (Bayer CropScience, 2011). The 

recommendations for these cotton growing areas are shown in (Table VIII-17). All acres 

are expected to receive one preplant application of dicamba (0.375 lbs a.e. per acre). 

Areas with glyphosate-resistant weeds are also expected to receive two in-crop 

applications of dicamba (0. 50 lbs a.e./acre) per season, whereas areas without 

glyphosate-resistant weeds will only receive one in-crop application of dicamba (0.50 lbs 

a.e./acre). 

 
Monsanto Company 12-CT-244U 209 of 620 

Table VIII-17. Anticipated Weed Management Recommendations for MON 88701 

Combined with Glyphosate-Tolerant Cotton Systems for western TX, NM, KS, OK, 

CA, and AZ 1 

Application 

Timing 

Conventional Tillage Conservation Tillage 

(No-till or reduced till) 

No GR 

Weeds 

GR Weeds or Suspected 

GR Weeds 

No GR 

Weeds 

GR Weeds or Suspected 

GR Weeds 

Preemergence 

(burndown, at 

planting) 2

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Dicamba 

+ 

Glyphosate 

+ 

Residual 

Postemergence2 

Dicamba 

+ 

Glyphosate 

Dicamba 

+ 

Glyphosate 

Dicamba 

+ 

Glyphosate 

Dicamba 

+ 

Glyphosate 

Postemergence2 Glyphosate 

Dicamba 

+ 

Glyphosate 

Glyphosate 

Dicamba 

+ 

Glyphosate 

1 The anticipated use patterns represent a high-end estimate for predicting dicamba use associated with 

MON 88701 combined with glyphosate-tolerant cotton. Actual weed control practices by growers will 

vary depending on the specific weed spectrum and agronomic situation of the individual cotton field, 

specifically dicamba use could be lower especially for the preemergence and second postemergence 

applications. 2 Monsanto and academics recommend the use of soil residuals as part of a comprehensive weed resistance 

management program to ensure that two effective herbicide modes-of-action are used in cotton and to 

provide protections against additional resistance development to existing cotton herbicides.

VIII.G.3. MON 88701 in Combination with Glyphosate-Tolerant Cotton Systems 

With the introduction of MON 88701 into glyphosate-tolerant systems, growers will 

continue to be able to use dicamba, glufosinate, and glyphosate herbicides for preplant 

burndown, without the plant-back restrictions currently in place. Tables VIII-10 and 

VIII-12 show weed control ratings for glyphosate, glufosinate, glyphosate tank-mixed 

with dicamba, and glufosinate tank-mixed with dicamba compared to other herbicide 

regimes when applied as a preplant burndown application to common broadleaf weed 

species found in fields prior to planting cotton. Glyphosate alone provides excellent 

control of many grass species and is superior to glufosinate on many grass species. 

Therefore, glufosinate applications in MON 88701 are not expected to provide 

improvement in control of grass species in no-till systems, except where glyphosateresistant grass species may be present. Certain hard-to-control broadleaf weeds such as 

curly dock, Carolina geranium, cutleaf primrose, and wild radish, are difficult to control 

with glyphosate. Similarly, glufosinate provides unsatisfactory control of certain 
Monsanto Company 12-CT-244U 210 of 620 

broadleaf weeds such as Carolina geranium, cutleaf primrose, and henbit. Tank-mixing 

dicamba with glyphosate or glufosinate improves the control of the difficult-to-control 

weed species for both of these products. The dicamba tank mix combinations will also 

provide excellent control of glyphosate-resistant marestail and fair to good control of 

glyphosate-resistant Palmer amaranth. Dicamba will be complementary to glyphosate or 

glufosinate for preplant burndown weed control in cotton and will offer growers equal or 

superior weed control to other preplant herbicides or herbicide tank mixtures. 

MON 88701 will provide additional application flexibility with dicamba allowing 

applications up to the day of planting in cotton. In addition to complementing the weed 

control of glyphosate, dicamba and glufosinate will provide additional modes-of-action in 

the glyphosate-tolerant cotton systems to lower the potential risk of weed species 

developing resistance to glyphosate. Furthermore, dicamba and glufosinate will provide 

alternative modes-of-action for control of broadleaf weeds with populations known to be 

resistant to glycine, ALS, and PPO classes of herbicides (see Table VIII-15). 

Upon integration of MON 88701 into glyphosate-tolerant cotton systems, in-crop 

postemergence applications of dicamba, glufosinate, and glyphosate herbicides will be 

permitted in cotton production. Tables VIII-18 and VIII-19 illustrate common broadleaf 

weed responses to dicamba, glyphosate, glufosinate, and several other labeled in-crop 

over-the-top herbicide treatments in cotton. Since dicamba is not currently labeled for incrop applications in cotton, weed control ratings for dicamba were taken from labeled incrop applications of dicamba in corn for comparison purposes. Glyphosate provides 

good to excellent control of all the listed annual grasses and most of the annual broadleaf 

weeds. When compared to glufosinate, glyphosate provides better control of some of the 

annual grasses (broadleaf signalgrass, crabgrass, crowsfootgrass, foxtail, goosegrass, 

seedling johnsongrass, Texas panicum), perennial grasses (johnsongrass and 

bermudagrass), and some of the broadleaf weeds (devil’s claw, Florida beggarweed, 

Florida pusley, pigweed species, and silverleaf nightshade). However, glufosinate data 

illustrates slightly better control of some of the broadleaf weeds compared to glyphosate, 

namely lambsquarter, morningglory, and velvetleaf. Dicamba as a tank mixture will 

complement the weed control of in-crop application(s) of either glyphosate or 

glufosinate. The use of dicamba will improve control of most of the broadleaf weeds 

where either glyphosate or glufosinate provide unsatisfactory control, with the exception 

of spurge, silverleaf nightshade, Texas blueweed, tropical spiderwort, velvetleaf, and 

woolyleaf bursage. Other herbicide treatments are available when needed to provide 

effective control of these weed species. In addition, an in-crop application of dicamba in 

combination with either glyphosate or glufosinate will assist in the management of 

glyphosate-resistant weed biotypes. 

Currently, many residual and non-residual herbicides are used in combination with 

glyphosate in preplant and in-crop postemergence applications in cotton. The addition of 

dicamba and glufosinate to the system are expected to offer increased benefits over the 

current alternative herbicides as supplements to glyphosate for preplant and in-crop 

applications on MON 88701, including increased flexibility and reduced crop injury. 
Monsanto Company 12-CT-244U 211 of 620 

Since planting interval restrictions following preplant applications of dicamba in cotton 

will be removed, dicamba will have greater flexibility for preplant applications than 

current preplant applications of 2,4-D and will potentially replace some 2,4-D 

applications in cotton. The broadleaf weed control provided by dicamba and glufosinate, 

plus the crop tolerance when applied to MON 88701, will allow the potential replacement 

of some other in-crop alternative herbicides used for broadleaf weed control in cotton, 

particularly diuron, fomesafen, fluometuron, and paraquat. Considering the 

characteristics of dicamba and glufosinate from the perspective of weed control and 

compatibility with glyphosate, it is concluded that MON 88701 will complement the 

established safety and efficacy of glyphosate use in glyphosate-tolerant cotton systems. 

VIII.G.4. MON 88701 as a Weed Resistance Management Tool 

Although herbicide resistance may eventually occur in a weed species when an herbicide 

is widely used, resistance can be delayed, contained, and managed through research, 

education, and good management practices. The addition of dicamba and glufosinate 

tolerance to the glyphosate-tolerant cotton systems will provide an efficient method for 

incorporation of additional modes-of-action in the system, and reduce the potential for 

further resistance development to glyphosate, dicamba, and glufosinate, as well as other 

important cotton herbicides. Current research, conducted by Monsanto, to define the 

optimum weed management systems indicate the following: 1) in MO, AR, TN, AL, FL, 

GA, NC, SC, VA, LA, MS, and eastern TX, the recommendation will be to apply a soilactive residual herbicide followed by an in-crop early postemergence application of 

dicamba tank-mixed with glyphosate, and a residual product, followed by a late postemergence application of glufosinate tank-mixed with a residual product (Table VIII-16); 

and 2) in western TX, KS, OK, NM, AZ, and CA, the recommendation will be to apply a 

soil-active residual herbicide, followed by an in-crop postemergence application of 

dicamba tank-mixed with glyphosate at early and late postemergence (Table VIII-17). 

This will ensure the use of more than one mode-of-action against the targeted species. In 

both areas, a preplant application of dicamba tank-mixed with glyphosate may be 

recommended, in addition to the in-crop applications described above. This is not 

expected to increase selection pressure on either product since the preplant weed 

spectrum is generally different from the in-crop spectrum. 

Stewardship of dicamba and glufosinate to preserve their usefulness for growers is an 

important aspect of Monsanto’s stewardship commitment, as is discussed in Appendix I. 

Specifically, Monsanto has implemented and will continue to develop and proactively 

provide weed resistance management practices6

, and will utilize multiple methods to 

distribute technical and stewardship information to growers, academics, and grower 

advisors through a variety of communication tools. Monsanto’s Technology Use Guide 

 

6

 Weed resistance management guidelines available at http://www.weedtool.com and 

http://www.monsanto.com/weedmanagement/Pages/default.aspx
Monsanto Company 12-CT-244U 212 of 620 

(TUG) will set forth the requirements and best practices for the cultivation of 

MON 88701 including recommendations on weed resistance management practices. 

Growers purchasing products containing MON 88701 are required by the Monsanto 

Technology Stewardship Agreement (MTSA) to read and follow the TUG. Furthermore, 

Monsanto is committed to actively evaluate herbicide performance and weed efficacy on 

a continuing basis, and develop additional mitigation plans as necessary to manage 

resistance development for glyphosate, dicamba, and glufosinate. 

VIII.G.5. Introduction of Dicamba and Glufosinate-Tolerant Cotton - MON 88701 

- Conclusion 

Integration of MON 88701 into glyphosate-tolerant cotton systems will allow the use of 

dicamba, glufosinate, and glyphosate herbicides in an integrated weed management 

program to control a broad spectrum of grass and broadleaf weed species in cotton. 

These herbicides will also provide three distinct modes-of-action for an effective 

proactive and reactive weed resistance management program in cotton. Due to the crop 

safety of MON 88701 to dicamba and glufosinate, growers will be afforded two effective 

herbicide modes-of-action for in-crop control of glyphosate’s hard-to-control and 

resistant broadleaf weeds that are present in U.S. cotton production. 

Furthermore, the integration of MON 88701, along with the glyphosate-tolerant cotton 

systems, will provide growers with the ability to continue use of established cotton 

production practices including tillage systems; the same planting and harvesting 

machinery; traditional management of insects, diseases, and other pests; and many of the 

current herbicides used for weed control, including glyphosate with its established 

environmental and grower benefits. Therefore, it is anticipated that the 

commercialization of MON 88701 in the U.S. is not likely to impact current cotton 

agronomic practices, cultivation or seed production practices, beyond the intended 

benefits of more effective and improved management of common and troublesome 

weeds, including herbicide-resistant weeds. 
Monsanto Company 12-CT-244U 

Table VIII-18. Responses of Common Broadleaf Weeds to Dicamba and Glufosinate Compared to LabHerbicides in Cotton Production – Part I 

 Common Broadleaf Weeds1

Product CB2 DC3 FB2 FP2 GC4 HS2 NS3 JW2 LQ2 MG2 PA2 Postemergence Over-The-Top Dicamba 

E5 G-E 

G5 G5 C4 E5 C4 E5 E5 E5 G-E5 

MSMA E P-F E N-P P - P P P P-F P Fluometuron F-G F-G G P-F - - - G G G P-F Pyrithiobac G G G N-P P G-E C E N G F Trifloxsulfuron G-E G G-E P - - - N G G P-F Glyphosate E E E P-G C P-F C E G F-G E Glyphosate + Pyrithiobac E E E P-G - G-E - E G G-E E Glyphosate + Trifloxsulfuron E E E P-G - - - E E E E Glufosinate E G-E G F C - C E E E F-G 

1 Weed species: CB = common cocklebur, DC = devil’s claw, FB = Florida beggarweed, FP = Florida pusley, GC = gro

sesbania, NS = nightshade, JW = jimsonweed, LG = Common lambsquarters, MG = morningglory species, PA = Ppigweed species, PS = Prickly sida. 

2(University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-8

60% control; N = < 30% control. 

3 Personal communications with Dr. Wayne Keeling, Texas A & M University - 2011, Weed control ratings key: E = 90% 

80% to 90% control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control. 

4 (University of California, 2012). Ratings Key: C = control, P = partial control, N = no control, - = no information. 5 (University of Georgia, 2010). Weed control ratings key: E = Excellent control, 90% or above; G = Good control, 80% or 

control, less than 80% control; P = Poor control.
Monsanto Company 12-CT-244U 

Table VIII-19. Responses of Common Broadleaf Weeds to Dicamba and Glufosinate Compared to LabHerbicides in Cotton Production – Part II 

 Common Broadleaf Weeds1

Product PL2 RW2 RdW2 SP2 SG2 SN3 SW2 TB3 TSW2 VL4 Postemergence Over-The-Top Dicamba 

E5 E5 - E5 P6 F3 E5 F3 P5 F-G

MSMA P-F P-F N P-F N P-F N-P N P - Fluometuron F-G G F-G F-G P-F N F-G N P - Pyrithiobac F P G P-F F-G P G N F 9 Trifloxsulfuron - G G E - P G N P-F - Glyphosate F-G E E E G E G G P-G 7 Glyphosate + Pyrithiobac G E E E G E E G G - Glyphosate + Trifloxsulfuron G E - E G E E G P-G - Glufosinate F-G E - E F-G F-G G F-G P-F 10

1 Weed species: PL = purslane, RW = Common ragweed, RdW = redweed, SP = sicklepod, SG = Spurge, SN = silve

smartweed, TB = Texas blueweed, TSW = tropical spiderwort, VL = velvetleaf, WB = woolyleaf bursage. 

2 (University of Georgia, 2012). Weed control ratings key: E = 90% or better control; G = 80% to 90% control; F = 60%-8

60% control; N = < 30% control. 

3 Personal communications with Dr. Wayne Keeling, Texas A & M University, Weed control ratings key: E = 90% or be

90% control; F = 60%-80% control; P = 30% to 60% control; N = < 30% control. 

4 (MSU, 2012). Rating scale: 0-3 = none to slight, 4-6 = fair, 7-8 = good, 9-10 = excellent control. 5 (University of Georgia, 2010). Weed control ratings key: E = Excellent control, 90% or above; G = Good control, 80% or 

control, less than 80% control; P = Poor control.

6 (University of California, 2012). Ratings Key: C = control, P = partial control, N = no control, - = no information.7 (University of Arkansas, 2011). Rating Scale: 0 = no control, 10 = 100% control. 
Monsanto Company 12-CT-244U 215 of 620

VIII.H. Crop Rotation Practices in Cotton 

The rotation of cotton with other crops is an integral part of most farm management 

programs across the southern United States cotton growing region. Ideally, cotton should 

be rotated with other crops on a regular basis to maintain soil productivity and reduce the 

incidence of various weeds, insect pests, or diseases (Hake et al., 1996d). Rotating cotton 

with grass crops such as corn helps to reduce the soil inoculum level of the seedling 

disease fungi Pythium and Rhizoctonia. These seedling diseases can increase in 

continuous cotton cropping systems. Crop rotations or the lack of rotations, along with 

weed control programs used in these crops, can play an important factor on the weed 

spectrum (Smith and Cothren, 1999). In addition, the crop rotation and weed control 

programs can increase or decrease the populations of certain weed species. Production 

costs, relative rate of return, and the current market conditions will dictate which crops to 

rotate with cotton or whether to grow continuous cotton. These economic factors may 

outweigh the agronomic benefits of crop rotation. According to Sandretto and Payne 

(2006) statistics, cotton was grown in a continuous cropping system on 73% of the 

acreage in the major cotton growing states in 2003. Cotton was rotated with other row 

crops such as corn or soybean on about 20% of the acreage. 

Crop rotations for cotton vary from region to region and state to state and often within a 

state. This section provides a detailed description and quantitative assessment by state of 

the rotational cropping practices immediately following cotton production. This 

assessment accounts for about 99% of the total cotton acreage. These data are presented 

in Tables VIII-20 through VIII-24). Seventeen crops immediately follow cotton in the 

crop rotation sequence according to this assessment. In the U.S., approximately 54% of 

the cotton acres are followed by cotton in the crop rotation sequence. Corn (16%), 

soybean (8%), sorghum (8%), wheat (9%), and peanuts (4%) are the other crops most 

frequently following cotton. The other crops following cotton are 0.5% or less of the 

cotton acres. 

Grower survey data available to Monsanto (2011) for dicamba, glufosinate, and 

glyphosate herbicide usage were utilized for this assessment. For the purpose of this 

assessment, a 50% adoption rate in U.S. cotton and soybean production was assumed for 

both MON 88701 and MON 87708 (dicamba-tolerant soybean), respectively. In the 

following data tables, columns F, H, and J provide the number of acres of dicamba, 

glufosinate, glyphosate that follow cotton in the rotation crops, respectively. Columns K, 

L, and M provide the percentage of dicamba, glufosinate, and glyphosate usage in the 

total rotational crop acreage (i.e., the percentage of cotton acres where dicamba, 

glufosinate and glyphosate, respectively, are used in the subsequent crop). For the entire 

U.S. (Table VIII-20), 33.1%, 11.9%, and 75.0% of the rotational crop acreage would be 

treated with dicamba, glufosinate, and glyphosate, respectively. The percentage of 

dicamba usage in the rotation would be the highest in the Southeast region (35.8%) and 

lowest in the West region (17.9%). MO (45.6%) and OK (48.0%) would be the states 

with the highest dicamba usage in the rotation and NM (5.8%) and AZ (3.2%) the lowest. 

The percentage of glufosinate usage in the rotation would be the highest in the Southeast 

(25.7%) and Midsouth (21.4%) regions and lowest in the Southwest region (2.5%). MO 

(43.0%) and OK (45.1%) would be the states with the highest glufosinate usage in the 
Monsanto Company 12-CT-244U 216 of 620

rotation and LA (1.8%), AZ (0%), and NM (0.2%) the lowest. The percentage of 

glyphosate usage in the rotation would be the highest in the Midsouth region (89.9%) and 

lowest in the West region (37.8.0%). MO (95.3%) would have the highest glyphosate 

usage in the rotation and AZ (19.7%) the lowest. In the Southwest region where almost 

55% of the cotton is grown, 33.1%, 2.5%, and 70.4% of the rotational crop acres 

following cotton would be treated with dicamba, glufosinate, and glyphosate, 

respectively. 
Monsanto Company 12-CT-244U 

Table VIII-20. Rotational Practices in the U.S. Following Cotton Production 

A B C D E F G H I J K 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop of 

Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop5

Glyphosate 

Usage in 

Rotational 

Crop6

% Usage in Tot

A

% Acres % Acres % Acres Dicamba Gluf

United States 10,974 

Cotton Corn Soybean Sorghum Wheat Barley Peanut Sunflower Alfalfa8

Vegetables9

Dry Beans Peppers Tomatoes Onions Tobacco 

5858 1736 861 836 1025 40 432 22 47 50 0.5 8 24 6 30 

Total:10

10,974

53.4 15.8 7.8 7.6 9.3 0.4 3.9 0.2 0.4 0.5 0.005 0.1 0.2 0.06 0.3 

50 8.1 50 8.3 5.6 

 5.0  NL  NL  NL  NL  NL  NL  NL  NL  NL 

2930 141 431 69 57 Total: 3,630 

21.6 2.1 1.0 

 NL  NL  NL  NL  NL  NL  NL  NL  NL  NL  NL  NL 

1264 37 8 Total: 1,309

90.2 88.0 95.9 34.7 14.1 27.5 21.1 72.7 50 2 40.0 37.5 45.8 33.3 NL 

5284 1527 826 290 145 11 91 16 241 0.2 3 11 2 Total: 8,231 

26.7 1.3 3.9 0.6 0.5 0.02 

33.1 

11.5 0.3 0.1 11.9 

This table was developed by compiling the data from all four regional summaries (Tables VIII-21 through VIII-24). All acreages are ex

NL indicates not labeled for use. 

1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e). 2Column C is obtained by compiling the data from the four regional summaries. 3Column D is obtained by dividing Column C by Column A. 4Column E is obtained by dividing Column F by Column C; Column F is obtained by compiling the data from all four regional summar5Column G is obtained by dividing Column H by Column C; Column H is obtained by compiling the data from all four regional summa6Column I is obtained by dividing Column J by Column C; Column J is obtained by compiling the data from all four regional summarie7 Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Colu

obtained by dividing Column J Total by Column C Total. 8Newly seeded alfalfa. 

9Vegetables: Cauliflower (37k acres), lettuce (271 k acres), and broccoli (124k acres) (USDA-NASS, 2011d). 10 Totals may not be exact due to rounding. 
Monsanto Company 12-CT-244U 

Table VIII-21. Rotational Practices Following Cotton Production in the Southeast Region 

A B C D E F G H I J K 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres 

Following 

Cotton2

% 

Rotational 

Crop 

Acres3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop5

Glyphosate 

Usage in 

Rotational Crop6 % Usage in Tot

A

% Acres % Acres % Acres Dicamba Gluf

Region 2597 

Cotton Corn Soybean Peanut Tobacco 

1311 340 507 410 30 

Total:10

2597 

50.5 13.1 19.5 15.8 1.1

50 6.2 50 NL NL 

656 21 254 

Total:930

50 2.3 0.9 NL NL 

65684

Total: 668

95.1 88.3 97.8 20.9 NL 

1247 300 496 86Total: 2128 

25.2 0.8 9.8

35.8 

25.2 0.3 0.2

25.7 

AL 340 

Cotton Corn Soybean Peanut 

102 68 119 51

Total: 340 

30 20 35 15 

50 78

50 NL 

51 5 60 

Total: 115

50 28

18NL

51 1 1

Total: 54

97 97 100 30 

99 66 119 15 Total: 299

15.0 1.4 17.5 

33.9 

15.0 0.4 0.4 

15.8 

FL 92 

Cotton Corn Soybean Peanut 

46 9 5 32 

Total: 92 

50 10 5 35 

50 78

50 NL 

23 1 2

Total:26

50 28

18NL 

23 1 0.05 

Total: 24

99 788

94827 

46 7 4 9 Total: 66

25.0 0.7 2.5 28.2 

25.0 1.1 0.1 26.1 

GA 1330 

Cotton Corn Soybean Peanut 

798 133 133 266 

Total: 1330 

60 10 10 20 

50 78

50 NL 

399 9 67 

Total: 475

50 28

18NL 

399 3 1 

Total: 403

93 92 99 18 

742 122 132 48 

Total: 1044

30.0 0.7 5.0 

35.7 

30.0 0.2 0.1 

30.3 
Monsanto Company 12-CT-244U 

Table VIII-21. Rotational Practices Following Cotton Production in the Southeast Region (continued) 

A B C D E F G H I J K L 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop of 

Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop5

Glyphosate 

Usage in 

Rotational 

Crop6

% Usage in Tota

Ac

% Acres % Acres % Acres Dicamba Glufo

NC 550 

Cotton Corn Soybean Peanut Tobacco 

231 83 165 44 28 

Total: 550

42 15 30 8 5 

50 78

50NL NL 

116 3 83 

Total: 201

50 28

18NL NL 

116 2 1 

Total: 119

99 79 95 238

NL 

229 65 157 10 

Total: 461

21.0 0.5 15.0 

36.5 

21.0 0.4 0.2 

21.5 

SC 202 

Cotton Corn Soybean Peanut Tobacco 

101 30 61 8 2 

Total: 202 

50 15 30 4 1 

50 78

50 NL NL 

51 2 30 

Total: 83

50 28

18NL NL 

51 1 1 

Total:

52

100 86 98 238

101 26 59 2

Total: 188

25.0 1.1 15.0 

41.1 

25.0 0.3 0.3 

25.6 

VA 83 

Cotton Corn Soybean Peanut 

33 17 25 8 

Total: 83 

40 20 30 10 

50 78

50 NL 

17 1 12 

Total: 30

50 28

18NL

17 0.3 0.2 

Total: 

17

91 79 99 238

30 13 25 2 

Total: 70

20.0 1.4 15.0 

36.4 

20.0 0.4 0.3 

20.7 

The Southeast region summary was developed by compiling the data from all the states within the region. Column C, Column F, Coobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column Column F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Cexpressed as 1000s of acres. NL indicates not labeled for use. 1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e). 2Column C is obtained by multiplying Column A by Column D. 
Monsanto Company 12-CT-244U 

3The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and 

Specialist in cotton (Dale Monks, Auburn University; David Wright, Ph.D., University of Florida; Jared Whitaker, Ph.D., UniveJones, Ph.D., Clemson University; and Henry Wilson, Ph.D., Virginia Tech University, Personal Communications, November, 2010)

4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in 

(50%) are future market adoption estimates. 5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (

adoption estimate. 6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011). 

7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Colum

is obtained by dividing Column J Total by Column C Total. 8Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, 

herbicide/crop in the U.S. was used. 

10Totals may not be exact due to rounding. 
Monsanto Company 12-CT-244U 

Table VIII-22. Rotational Practices Following Cotton Production in the Midsouth Region 

A B C D E F G H I J K L 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop of 

Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop5

Glyphosate 

Usage in 

Rotational 

Crop6

% Usage in Tota

Ac

% Acres % Acres % Acres Dicamba Glufo

Region1920 

Cotton Wheat Corn Soybean Sorghum 

786 59 711 337 27 

Total: 9

1920 

40.9 3.0 37.0 17.6 1.4

50 6.0 10.1 50 12 

393 4 72 169 3

Total: 640

50 NL 2.1 1.0 NL 

393 15 4 

Total: 411 

99 15 85 94 45 

780 9 607 317 

12 Total: 1725 

20.5

0.23.78.80.233.3 

20.5 0.8 0.221.4 

AR 545 

Cotton Corn Soybean Sorghum 

218 164 136 27

Total: 545 

40 30 25 5 

50 78

50 128

109 16 68 3 

Total: 196

50 28

18NL

109 3 1 

Total: 114

100 788

87 458

218 128 119 12 

Total: 476

20.0 2.9 12.5 0.6 36.0 

20.0 0.6 0.320.9 

LA 255 

Cotton Corn Soybean 

0 191 64 

Total: 255 

0 75 25 

50 78

50

0 13 32

Total: 45

50 28

18

0 4 1 

Total: 4

100 78 100 

0 149 64

Total: 213

5.3 12.5 17.8 

1.5 0.3 1.8 

MS 420 

Cotton Corn Soybean 

168 189 63 

Total: 420 

40 45 15 

50 78

50 

84 13 32 

Total: 129

50 28

18

84 4 1 

Total: 88

99 98 99 

166 185 62 

Total: 414

20.0 3.2 7.530.7 

20.0 0.9 0.2 21.1 
Monsanto Company 12-CT-244U 

Table VIII-22. Rotational Practices Following Cotton Production in the Midsouth Region (continued) 

A B C D E F G H I J K L 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop of 

Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop5

Glyphosate 

Usage in 

Rotational 

Crop6 % Usage in Total R

% Acres % Acres % Acres Dicamba Glufos

MO 310 

Cotton Corn Soybean 

264 31 16 

Total: 310

85 10 5 

50 78

50 

132 2 8 

Total: 142

50 4 2 

132 1 0.3 

Total: 133

100 59 88 

264 18 14 

Total: 295

42.5 0.6 2.545.6 

42.5 0.4 0.143.0 

TN 390 

Cotton Wheat Corn Soybean 

137 59 137 59 

Total: 390 

35 15 35 15 

50 68

11 50

68 4 27 29 

Total: 128

50 NL 28

18

68 3 1 

Total: 72

97 158

93 100

132 9 127 59

Total: 327

17.5 0.9 6.9 7.5 32.8 

17.5 0.7 0.2 18.4 

The Midsouth region summary was developed by compiling the data from all the states within the region. Column C, Column F, Coobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column Column F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Cexpressed as 1000s of acres. NL indicates not labeled for use. 1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e) 2Column C is obtained by multiplying Column A by Column D. 3The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and 

Specialist in cotton (Tom Barber, Ph.D., University of Arkansas; John Kruse, Louisiana State University; Darrin Dobbs, Ph.D., MAndrea Jones, Ph.D., University of Missouri; and Larry Steckel, Ph.D., University of Tennessee, Personal Communications Novemb

4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in 

(50%) are future market adoption estimates. 5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (

adoption estimate. 6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011). 

7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Colum

is obtained by dividing Column J Total by Column C Total. 8Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, the per

herbicide/crop in the U.S. was used. 

9Totals may not be exact due to rounding. 
Monsanto Company 12-CT-244U 

Table VIII-23. Rotational Practices Following Cotton Production in the Southwest Region 

A B C D E F G H I J K 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational Crop5

Glyphosate 

Usage in 

Rotational 

Crop6 % Usage in Total 

% Acres % Acres % Acres Dicamba Glufo

Region5953 

Cotton Wheat Corn Soybean Sorghum Dry Bean Alfalfa8 

Peanuts Sunflower Peppers 

3607 765 685 17 808 0.5 18 22 22 8 

Total: 10

5953 

60.6 12.9 11.5 0.3 13.6 0.01 0.3 0.4 0.4 0.1 

50 5.3 7.0 50 8.2 NL NL NL NL NL

1804 41 48 8 66 

Total: 1967

3.8 NL 2.0 1.0 NL NL NL NL NL NL 

138 14 0.2

Total: 152

86.9 13.9 90.5 78.2 34.4 35.0 50.0 23.0 74.0 43.0

3136 106 620 13 278 0.2 9 5 16 3

Total: 4188

30.3 0.7 0.8 0.1 1.133.1 

2.3 0.2 0.003 2.5 

KS 51 

Cotton Wheat Corn Soybean Sorghum 

3 13 20 3 13

Total: 51 

5.0 25.0 40.0 5.0 25.0 

50 13 11 50 19

1 2 2 1 2

Total: 9

50 NL 29

19NL 

1 0.4 0.03 

Total: 2

100 8 88 96 57

3 1 18 2 7

Total: 31

2.5 3.3 4.2 2.5 4.717.2 

2.5 0.8 0.1 3.4 
Monsanto Company 12-CT-244U 

Table VIII-23. Rotational Practices Following Cotton Production in the Southwest Region (continued) 

A B C D E F G H I J K L 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop of 

Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop5

Glyphosate 

Usage in 

Rotational 

Crop6 % Usage in Total Ro

% Acres % Acres % Acres Dicamba Glufosi

NM 50 

Cotton Wheat Corn Sorghum Dry Beans Alfalfa8 

Peppers 

3 12 5 5 0.5 18 8 

Total: 50 

5.0 24.0 10.0 10.0 1.0 35.0 15.0 

50 69

79129

NL NL NL 

1 0.7 0.4 1 

Total: 3

0 NL 29

NL NL NL NL

0 0.1 

Total: 0.1

919

15978945935950 439

2 2 4 2 0.2 9 3 

Total: 22 

2.5 1.4 0.7 1.2 5.8 

0.2 0.2 

OK 285 

Cotton Corn Soybean Sorghum 

257 9 14 6 

Total: 285 

90 3 5 2 

50 79

50 129

128 0.6 7 0.7 

Total: 137

50 29

19NL 

128 0.2 0.1 

Total:129

99 69 75 35

254 6 11 2

Total: 273

45.0 0.2 2.5 0.248.0 

45.0 0.1 0.145.1 

TX 5567 

Cotton Wheat Corn Sorghum Peanuts Sunflower 

3346 740 651 785 22 22 

Total: 5567

60.1 13.3 11.7 14.1 0.4 0.4 

50 5 7 8 NL NL 

1673 39 45 63 

Total: 1819

0.25 NL 29

NL NL NL 

8 13 

Total: 21

86 14 91 34 239

749

2877 104 593 267 5 16 

Total: 3862

30.1 0.7 0.8 1.1

32.7 

0.2 0.2 

0.4 

The Southwest region summary was developed by compiling the data from all the states within the region. Column C, Column F, Coobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column Column F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Cexpressed as 1000s of acres. NL indicates not labeled for use. 1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e). 
Monsanto Company 12-CT-244U 

2Column C is obtained by multiplying Column A by Column D. 3The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and 

Specialist in cotton (Stewart Duncan, Ph.D., Kansas State University; Jamshid Ashigh, Ph.D., New Mexico State University; Don State University; Wayne Keeling, Ph.D., Texas A & M University; and Gaylon Morgan, Ph.D., Texas A & M University, 

November, 2010).). 4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in (50%) are future market adoption estimates. 5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (

adoption estimate for most of the Southwest region. No glufosinate usage is expected in NM and West TX. 6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011). 

7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Colum

is obtained by dividing Column J Total by Column C Total. 

8 Newly seeded alfalfa; Glyphosate usage in alfalfa (50%) is future market adoption estimate. 9Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, the per

herbicide/crop in the U.S. was used. 

10Totals may not be exact due to rounding. 
Monsanto Company 12-CT-244U 

Table VIII-24. Rotational Practices Following Cotton Production in the West Region 

A B C D E F G H I J K L 

State/ 

Total 

Cotton 

Acres1

Rotational 

Crops 

Following 

Cotton 

Rotational 

Crop 

Acres2

% 

Rotational 

Crop of 

Total 

Cotton3

Dicamba 

Usage in 

Rotational 

Crop4

Glufosinate 

Usage in 

Rotational 

Crop Option5

Glyphosate 

Usage in 

Rotational 

Crop6 % Usage in Total R

% Acres % Acres % Acres Dicamba Glufo

Region504 

Cotton Wheat Barley Alfalfa8 

Tomatoes Onions Vegetables9

153 202 40 30 24 6 50

Total: 11

504 

30.4 40.0 7.9 5.9 4.9 1.2 9.8 

50.0 6.0 4.0 NL NL NL NL 

77 12 2 

Total: 90

50.0 NL NL NL NL NL NL 

77 

Total: 77

79.0 15.0 27.0 50.0 43.0 27.0 3.0

121 30 11 15 11 2 1

Total: 190

15.2 2.4 0.3 17.9 

15.2 15.2 

AZ 198 

Wheat Barley Alfalfa8 

Vegetables9

79 40 30 50

Total: 198 

40 20 15 25 

610

410NL NL

5 2 

Total: 6 

NL NL NL NL 

Total: 0 

1510

271050 310

12 11 15 1 

Total: 39 

1.6 0.5 3.2 0.0 

CA 306 

Cotton Wheat Tomatoes Onions

153 122 24 6 

Total: 306 

50 40 8 2 

50 610

NLNL

777

Total: 84

50 NL NL NL

77 

Total: 77

79 1510

43 27

121 18 11 2

Total: 151

25.0 2.4 

27.4 

25.0

25.0 

The West region summary was developed by compiling the data from all the states within the region. Column C, Column F, Coluobtained by compiling data from all the states within this region; Column D is obtained by dividing Column C by Column A, Column Column F by Column C, Column G is obtained by dividing Column H by Column C, Column I is obtained by dividing Column J by Cexpressed as 1000s of acres. NL indicates not labeled for use. 1Cotton acreage based on 2010 planting data (USDA-NASS, 2011e). 2Column C is obtained by multiplying Column A by Column D. 
Monsanto Company 12-CT-244U 

3The rotational crop percentages in Column D are based on estimates from individual state Extension Crop Production Specialist and 

Specialist in cotton (Bill McCloskey, Ph.D., University of Arizona; and Steve Wright, Ph.D., University of California, Personal Co

2010). 4Dicamba usage data in Column E except for cotton and soybean is based on 2010 data (Monsanto Company, 2011). Dicamba usage in (50%) are future market adoption estimates. 5Glufosinate usage data in Column G except for cotton is based on 2010 (Monsanto Company, 2011). Glufosinate usage in cotton (

adoption estimate. 6Glyphosate usage data in Column I is based on 2010 data (Monsanto Company, 2011). 

7Column K is obtained by dividing Column F Total by Column C Total; Column L is obtained by dividing Column H Total by Colum

is obtained by dividing Column J Total by Column C Total. 8Newly seeded alfalfa; Glyphosate usage in alfalfa (50%) is future market adoption estimate. 9AZ acreage: cauliflower (3), lettuce (62), broccoli (7); U.S. acreage: cauliflower (35), lettuce (199), broccoli (125). 

10Since no data was reported or the survey sample size was too small for the herbicide data in the state to be statistically reliable, 

herbicide/crop in the U.S. was used.

11Totals may not be exact due to rounding. 
Monsanto Company 12-CT-244U 228 of 620 

VIII.H.1. Cotton Volunteer Management 

Volunteer cotton refers to plants that have germinated, emerged and established 

unintentionally from the previous year’s cotton crop (Roberts et al., 2002). Volunteer 

cotton plants generally come from seed that falls to the ground as a result of preharvest 

losses due to adverse weather condition or losses during the harvesting operation. 

Volunteer cotton will compete with the rotational crop and potentially cause yield loss 

and act as early host plants for pests such as spider mites and aphids (Roberts et al., 

2002). Although volunteer cotton in soybean fields can impact yield, recent studies 

indicate that other common grasses or broadleaf weeds are more problematic in soybean 

(Lee et al., 2009). The occurrence of volunteer cotton depends on the tillage after 

harvesting the crop and the severity of winters. Cotton volunteers are more frequently 

observed in conservation tillage systems where tillage is not used prior to planting. An 

integrated weed management system of tillage and herbicides has traditionally been the 

most common method of volunteer cotton control (Roberts et al., 2002). 

Mechanical tillage prior to planting is an effective and efficient method for controlling 

seedling volunteer cotton plants. This is accomplished in most soil conditions because 

the root and hypocotyls of seedling cotton are easily destroyed by the tillage process 

(Roberts et al., 2002). Any damage occurring below the cotyledons will kill the plant 

because there are no growing points from which the plant can recover. The 

disadvantages of tillage are moisture loss under arid conditions and the possibility of 

increased soil erosion. In-crop cultivation is a highly effective option for satisfactory 

control of volunteer seedlings. Where cultivation is not appropriate, the use of herbicides 

is effective in controlling volunteers. 

University weed specialists have identified numerous effective and economical herbicide 

treatments for control of volunteer cotton in the various rotational crops including cotton 

(Table VIII-25). University studies have shown that the timing of the herbicide 

application can greatly impact the effectiveness of many herbicides. Newly emerged 

cotton (up to 2- 3-leaf stage) as a volunteer is much easier to control with herbicides than 

more mature cotton (Thompson and Steckel, 2010). If the volunteer cotton plants contain 

the glyphosate-tolerance trait, the use of glyphosate alone in subsequent rotational crops 

will not control these seedlings. Similarly, volunteer cotton plants containing the 

dicamba and glufosinate-tolerant traits from MON 88701 would not be controlled with 

either dicamba or glufosinate herbicides, and alternative herbicides would be required for 

control. 

Currently both dicamba and glufosinate are labeled for use in crops rotated with cotton. 

Dicamba herbicide is labeled for weed control in soybean, corn, cotton, sorghum, wheat, 

barley, oats, millet, pasture, rangeland, asparagus, sugarcane, turf, grass grown for seed, 

conservation reserve programs, and fallow croplands. Glufosinate is used for 

postemergence weed control in canola, corn, cotton, and soybean varieties containing 

glufosinate-tolerance. It may also be applied as a preplant burndown application in 

conventional or herbicide-tolerant varieties of canola, corn, cotton, soybean, or sugarbeet. 

The herbicide control options available in rotational crops will continue to result in the 

ability to manage cotton volunteers. 
Monsanto Company 12-CT-244U 229 of 620 

Preplant burndown applications of carfentrazone, paraquat, or flumioxazin will 

effectively control emerged volunteer cotton prior to planting rotational crops (Murdock 

et al., 2002; Roberts et al., 2002). In most situations, these preplant measures are 

sufficient and no additional control measures specifically for cotton volunteers are 

required the remainder of the season. In the event these measures are not sufficient, the 

preplant treatment will generally reduce infestation levels, allowing for more effective incrop management of the remaining volunteer cotton in rotational crops. 

In emerged cotton, in-crop cultivation has been traditionally used in the subsequent 

cotton crop or other crops to effectively remove weeds and volunteer cotton plants 

between the crop rows. In reduced tillage situations, special high-residue cultivators with 

sweeps may be used to effectively lift weeds out of the soil to leave the ground cover 

undisturbed. Cotton emerging within the row can negatively impact cotton growth and 

management decisions due to increased plant population and disease susceptibility 

(Roberts et al., 2002). However, plants remaining at the end of the season can generally 

be harvested with the planted population by mechanical picking or stripping. Several 

herbicides are also available for control of volunteer cotton plants after the emergence of 

the rotational crop. In emerged cotton, applications of carfentrazone or paraquat with 

hooded sprayers or other selective equipment will effectively control volunteer plants and 

other weeds in row middles (Alford et al., 2002; Murdock et al., 2002). However, special 

precautions must be taken to ensure that these non-selective herbicides do not contact the 

cotton crop (Gray et al., 2002). Chlorimuron and imazaquin provide control of volunteer 

cotton in soybean (Clemmer et al., 2001; York et al., 2004) (Table VIII-25). Volunteer 

cotton in corn generally is not an issue because of the sensitivity of cotton to a number of 

commonly used corn herbicides (e.g., atrazine). 

 
Monsanto Company 12-CT-244U 230 of 620 

Table VIII-25. Herbicides and Application Timing for Control of Volunteer Cotton 

in Labeled Rotational Crops1 

Rotation Crop/ Herbicide Product 

Rate 

Product/Acre 

Preplant / 

Preemergent 

InCrop 

 

Cotton 

Gramoxone (paraquat) 

C

32 oz X X* 

Aim (carfentrazone) 1 oz X X* 

ET (pyraflufen) 1.5 oz X X* 

Layby Pro (linuron/diuron) 32 oz X* 

Sharpen (saflufenacil) 1.5-2.0 oz X 

Corn 

Atrazine 32 oz X X 

Callisto (mesotrione) 3 fl oz X X 

Sharpen (saflufenacil) 1.5-2.0 oz X 

Status (diflufenzopyr/dicamba) 0.75 oz X X 

2,4-D 32 oz X X 

Soybean 

Resource (flumiclorac) 8 oz X 

Gramoxone Inteon (paraquat) 32 oz X 

Sencor + Classic (metribuzin + 

chlorimuron) X 

Classic (chlorimuron) 2/3 oz X 

2,4-D 32 oz X 

Peanuts 

Gramoxone Inteon (paraquat) 32 oz X 

Classic (chlorimuron) 2/3 oz X 

Sunflower 

Paraquat 32 oz X 

Sharpen (saflufenacil/) 1.5-2.0 oz X 

Buctril 1 pt X 

Sorghum 

Gramoxone Inteon (paraquat) 32 oz X 

Sharpen (saflufenacil) 1.5-2.0 oz X 

Atrazine 32 oz X X 

Wheat 

CleanWave (aminopyralid/fluroxypyr) 14 oz X 

Buctril (bromoxynil) 1 pt X 

2,4-D 32 oz X 

Starane (fluroxypyr) 16 oz X 

Sharpen (saflufenacil) 1.5-2.0 oz X 

 1

 (Grichar et al., 2010; Keeling et al., 2009; Roberts et al., 2002; Thompson and Steckel, 2010; York et al., 

2004). 

*Hooded or selective equipment only 
Monsanto Company 12-CT-244U 231 of 620 

VIII.I. Stewardship of MON 88701 

Monsanto develops effective products and technologies and is committed to assuring that 

its products and technologies are safe and environmentally responsible. Monsanto 

demonstrates this commitment by implementing product stewardship processes 

throughout the lifecycle of a product and by participation in the Excellence Through 

StewardshipSM (ETS) Program (BIO, 2010). These policies and practices include 

rigorous field compliance and quality management systems and verification through 

auditing. Monsanto’s Stewardship Principles are also articulated in Technology Use 

Guides (Monsanto Company, 2012) and Monsanto Technology Stewardship Agreements 

that are signed by growers who utilize Monsanto branded traits, to ensure stewardship 

compliance. 

As an integral action of fulfilling this stewardship commitment, Monsanto will seek 

biotechnology regulatory approvals for MON 88701 in all key cotton import countries 

with a functioning regulatory system to assure global compliance and support the flow of 

international trade. These actions will be consistent with the Biotechnology Industry 

Organization (BIO) Policy on Product Launch (BIO, 2010). Monsanto continues to 

monitor other countries that are key importers of cotton from the U.S., for the 

development of formal biotechnology approval processes. If new functioning regulatory 

processes are developed, Monsanto will make appropriate and timely regulatory 

submissions. In addition, Monsanto actively interacts with and participates in cotton 

industry groups, such as the National Cotton Council, state grower boards, Farm Bureau, 

Cotton Inc., and trade affiliates, to obtain input on market trends to ensure awareness of 

the current key markets for whole cottonseed and cottonseed by-products. 

Monsanto also commits to industry best practices on seed quality assurance and control 

to ensure the purity and integrity of MON 88701 cottonseed. As with all of Monsanto’s 

products, before commercializing MON 88701 in any country, a MON 88701 detection 

method will be made available to cotton producers, processors, and buyers. 

The dicamba and glufosinate-tolerant cotton system, which is applying dicamba and/or 

glufosinate herbicide to MON 88701 integrated into the glyphosate cotton systems, will 

enable expanded use of dicamba herbicide in cotton production. Monsanto is seeking 

regulatory approvals with the U.S. EPA for the expanded application of dicamba 

herbicide as a weed control tool in cotton. Furthermore, Monsanto will establish 

appropriate dicamba Maximum Residue Levels (MRLs) for key cotton import countries. 

No additional regulatory approvals with U.S. EPA will be required for glufosinate 

products for use in MON 88701. 

As with all U.S. EPA registered herbicides for agricultural use, it is possible that offsite 

movement during and/or following application can occur such that non-target plants may 

be exposed to direct spray or to spray drift. Research has demonstrated that herbicide 

formulation, application equipment, and application procedures can be optimized to 

significantly reduce spray drift potential in most circumstances (Jordan et al., 2009; 

SDTF, 1997). Monsanto is addressing potential offsite movement of dicamba by seeking 

U.S. EPA registration of a low volatility dicamba formulation (DGA salt) for ground 
Monsanto Company 12-CT-244U 232 of 620 

application only. Additionally Monsanto will implement a robust stewardship program 

that will include a strong emphasis on grower and applicator training. Furthermore, 

Monsanto will consult with U.S. EPA to identify what additional measures, if any are 

necessary, to address any potential impact of off-site movement of these herbicides. 

Stewardship of dicamba and glufosinate, to preserve their usefulness for growers, is also 

an important aspect of Monsanto’s stewardship commitment. Detailed information 

regarding dicamba and glufosinate weed resistance and the usefulness of dicamba and 

glufosinate-tolerant cotton in combination with glyphosate-tolerant cotton to address 

herbicide-resistance issues is presented in Section VIII.G and Appendix I. 

VIII.J. Impact of the Introduction of MON 88701 on Agricultural Practices 

Introduction of MON 88701 is expected to have no impact on current agronomic, 

cultivation and management practices for cotton, with the exception of expanded dicamba 

application timings and more options for effective weed management. Dicamba has been 

used in corn, soybean, and small grain cropping systems since 1967. MON 88701 with 

its excellent crop tolerance to dicamba allows preemergence applications through crop 

emergence and in-crop postemergence applications up to seven days prior to harvest. 

MON 88701 will be combined with glyphosate-tolerant cotton systems utilizing 

traditional breeding techniques. Cotton containing both MON 88701 and glyphosatetolerance will allow the use of glyphosate, dicamba, and glufosinate herbicides in an 

integrated weed management program to control a broad spectrum of grasses and 

broadleaf weed species, and to sustain and complement the benefits and value of the 

glyphosate use in glyphosate-tolerant cotton systems. 

MON 88701 has been shown to be comparable to commercially cultivated cotton in its 

agronomic, phenotypic, and compositional characteristics (refer to Sections VI, VII, and 

VIII), and has the same levels of susceptibility to insect pests and diseases as commercial 

cotton. Like other herbicide-tolerant cotton, such as glyphosate-tolerant cotton that have 

been cultivated and consumed in the U.S. since 1996, dicamba and glufosinate tolerant 

cotton (MON 88701) will improve the current agricultural practices for U.S. cotton 

growers by providing two additional in-crop herbicide modes-of-action for the control of 

glyphosate’s hard-to-control and resistant broadleaf weeds, as well as weeds resistant to 

other herbicide families, thereby improving the efficiency in the U.S. cotton production 

system to maximize or maintain cotton yield potential, and help meet growing needs for 

fiber, food, and feed. 

 
Monsanto Company 12-CT-244U 233 of 620 

IX. ENVIRONMENTAL ANALYSIS 

IX.A. Introduction 

This section provides a brief review and assessment of the plant pest potential of 

MON 88701 and its impact on current agronomic practices. USDA-APHIS has 

responsibility, under the Plant Protection Act (7 U.S.C. § 7701-7772), to prevent the 

introduction and dissemination of plant pests into the U.S. APHIS regulation 

7 CFR § 340.6 provides that an applicant may petition APHIS to evaluate submitted data 

to determine that a particular regulated article does not present a plant pest risk and no 

longer should be regulated. If APHIS determines that the regulated article does not 

present a plant pest risk, the petition must be granted, thereby allowing unrestricted 

introduction of the article. 

The definition of “plant pest” in the Plant Protection Act (PPA) includes living organisms 

that could directly or indirectly injure, damage, or cause disease in any plant or plant 

product (7 U.S.C. § 7702[14]). 

The regulatory endpoint under the PPA for biotechnology-derived crop products is not 

zero risk, but rather a determination that deregulation of the regulated article is unlikely 

to pose a plant pest risk. The approach used to assess the plant pest potential of 

MON 88701 is a weight-of-evidence approach based primarily on eight lines of evidence: 

1) insertion of a single functional copy of the dmo and bar expression cassettes; 

2) characterization of MON 88701 DMO and PAT (bar) expressed in MON 88701; 

3) protein safety of MON 88701 DMO and PAT (bar); 4) compositional equivalence of 

harvested MON 88701 seed to conventional commercial cotton; 5) phenotypic, 

agronomic, plant mapping, and environmental interaction characteristics demonstrating 

no increased plant pest potential compared to commercially cultivated cotton, including 

disease and pest susceptibilities; 6) negligible risk to NTOs; 7) familiarity with cotton as 

a cultivated crop and the inherently low plant pest potential of cotton; and 8) no greater 

likelihood to impact agronomic practices, including land use, cultivation practices, or the 

management of weeds (other than the intended benefits of dicamba and glufosinate for 

weed control), diseases, and insects than commercially cultivated cotton. 

Using the aforementioned assessment, the data and analysis presented in this petition lead 

to a conclusion that MON 88701 is unlikely to be a plant pest, and therefore should no 

longer be subject to regulation under 7 CFR Part 340. 

IX.B. Plant Pest Assessment of MON 88701 Insert and Expressed Proteins 

This section summarizes the details of the genetic insert, characteristics of the genetic 

modification, and safety and expression of the MON 88701 DMO and PAT (bar) proteins 

used to evaluate the food, feed, and environmental safety of MON 88701. 
Monsanto Company 12-CT-244U 234 of 620 

IX.B.1. Characteristics of the Genetic Insert and Expressed Protein 

IX.B.1.1. Genetic Insert 

As described in Section IV, molecular analyses demonstrated that MON 88701 contains a 

single copy of the inserted T-DNA at a single integration locus. No backbone sequences 

from the PV–GHHT6997 were detected in the genome of MON 88701. In addition, data 

confirmed the organization and sequence of the insert and the stability of the insert over 

several breeding generations. 

IX.B.1.2. Mode-of-Action 

MON 88701 exhibits tolerance to the herbicide dicamba through the insertion of a 

demethylase gene from Stenotrophomonas maltophilia that encodes DMO and the 

herbicide glufosinate through the insertion of a N-acetyltransferase gene from 

Streptomyces hygroscopicus that encodes PAT. The DMO protein is a Rieske-type nonheme iron oxygenase that catalyzes the demethylation of dicamba to the non-herbicidal 

compound DCSA (Section V.A.1.). As shown in section V.A. and by Dumitru et. al. 

(2009), DMO is specific for dicamba. 

The PAT protein has been extensively assessed, as numerous glufosinate-tolerant 

products including those in cotton, corn, soy, canola, sugarbeet and rice have been 

reviewed by the USDA and several other regulatory agencies (ILSI-CERA, 2011; OECD, 

1999b; 2002a). The PAT (bar) protein produced in MON 88701 acetylates the free 

amine group of L-phosphinothricin form of glufosinate to produce non-herbicidal 

N-acetyl glufosinate (Section V.A.2.). PAT is specific for glufosinate (Thompson et al., 

1987; Wehrmann et al., 1996). 

IX.B.1.3. Protein Safety and Expression 

The safety of the expressed proteins is detailed in Section V. A history of safe use has 

been established for both MON 88701 DMO and PAT (bar) proteins (Section V.E). 

MON 88701 DMO and PAT (bar) lack structural similarity to known allergens, gliadins, 

glutenins, or protein toxins (Section V.D). MON 88701 DMO and PAT (bar) are present 

at very low levels in MON 88701 cottonseed (Section V.C.) and will constitute a small 

portion of the total protein present in feed derived from MON 88701 (Section V.E.). No 

consumption of the MON 88701 DMO or PAT (bar) proteins derived from MON 88701 

is expected for the U.S. general population at the present time given that the only foods 

produced from cottonseed are RBD oil and linters, which contain undectable and 

neligible amounts of total protein, respectively. As shown in Section V.E, 

MON 88701 DMO and PAT (bar) are readily digestible in simulated gastric and 

simulated intestinal fluids and show no oral toxicity in mice (Section V.E.). In addition, 

PAT proteins have been evaluated in several previous safety assessments with no safety 

concerns identified. 
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IX.B.2. Compositional Characteristics 

Detailed compositional analyses in accordance with OECD guidelines were conducted to 

assess whether levels of key nutrients and anti-nutrients in MON 88701, both treated and 

not treated with dicamba or glufosinate herbicides, were comparable to levels present in 

the near isogenic conventional cotton control Coker 130 and several conventional, 

commercial reference varieties (Section VI). Seed were harvested from eight individual 

sites in which MON 88701, the conventional control, and a range of commercial 

reference varieties were grown concurrently in the same field trial. The commercial 

reference varieties were used to establish a range of natural variability for the key 

nutrients and anti-nutrients in commercial cotton varieties that have a history of safe 

consumption. 

The combined-site analysis was conducted to determine statistically significant 

differences (5% level of significance) between MON 88701 treated with dicamba and 

glufosinate and the conventional control. The biological significance of difference from 

the combined-site data were reviewed using considerations relevant to food and feed 

safety and nutritional quality. These considerations included: 1) the relative magnitude 

of differences in the mean values of nutrient and anti-nutrient components of 

MON 88701 and the conventional control; 2) whether the MON 88701 component mean 

value was within the range of natural variability of commercial cotton as represented by 

the 99% tolerance interval of the commercial reference varieties grown concurrently in 

the same field trial; 3) whether the MON 88701 component mean value was within the 

context of the natural variability of commercial cotton composition as published in the 

scientific literature and/or available in the ILSI Crop Composition Database; and 

4) analyses of the reproducibility of the statistically significant combined-site component 

differences at individual sites. 

Assessment of the analytical results confirmed that the differences observed in the 

combined-site analysis were not meaningful to food and feed safety or the nutritional 

quality of MON 88701 cotton. To further support the safety assessment, similar 

compositional analyses were also conducted on cottonseed from MON 88701 not treated 

with dicamba or glufosinate herbicides. Based on the analyzed nutrient and anti-nutrient 

levels of both dicamba and glufosinate-treated and not treated MON 88701, MON 88701 

is compositionally equivalent to conventional commercial cotton and therefore the food 

and feed safety and nutritional quality of this product is comparable to that of 

commercially cultivated cotton. These results support the overall conclusion that 

MON 88701 is unlikely to be a plant pest. 

IX.B 3. Phenotypic, Agronomic, Plant Mapping, and Environmental Interaction 

Characteristics 

An extensive set of comparative plant characterization data were used to assess whether 

the introduction of the dicamba and glufosinate tolerance traits altered the plant pest 

potential of MON 88701 compared to the conventional control (Section VII). 

Phenotypic, agronomic, plant mapping, and environmental interaction characteristics of 

MON 88701 were evaluated and compared to those of the conventional control (Section 
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VII.B). As described below, these assessments included: seed dormancy and germination 

characteristics; agronomic and phenotypic characteristics; plant mapping characteristics; 

observations for abiotic stress response, disease damage, arthropod-related damage, 

arthropod abundance, and pollen characteristics. To support the trait assessment, similar 

observations were also conducted on MON 88701 treated with dicamba and glufosinate 

herbicides. Results from all phenotypic, agronomic, plant mapping, and environmental 

interaction assessments demonstrated that MON 88701 does not possess weedy 

characteristics, or increased susceptibility or tolerance to specific diseases, insects, or 

abiotic stressors compared to the conventional control. Taken together, the results of the 

analysis support a determination that MON 88701 is no more likely to pose a plant pest 

risk or have a biologically meaningful change in environmental impact than 

commercially cultivated cotton. 

IX.B.3.1. Seed Dormancy and Germination 

Seed dormancy and germination characterization demonstrated that MON 88701 seed 

had germination characteristics similar to those of the conventional control (Section 

VII.C.1). In particular, the lack of hard seed, a well-accepted characteristic often 

associated with plants that are weeds, supports a conclusion of no increased weediness or 

plant pest potential of MON 88701 compared to commercially cultivated cotton. 

IX.B.3.2. Plant Growth and Development 

Of the growth and development characteristics assessed between MON 88701 and the 

conventional control, eight significant differences were detected in a combined-site 

analysis (Section VII.C.2.1). MON 88701 observed a reduction in plant height at 30 

DAP and at harvest, increased number of nodes above white flower at observations 2 and 

3, a lower seed index, increased total and immature seed per boll, and increased fiber 

strength. The differences in mean values of MON 88701 were within the range of the 

commercial reference varieties for these characteristics. Thus, the differences in these 

parameters are not considered to be biologically meaningful in terms of increased 

weediness or plant pest potential of MON 88701 compared to commercially cultivated 

cotton. 

Plant mapping is a process commonly used to quantify growth and development 

parameters of the cotton plant, including boll retention and distribution (Section 

VII.C.2.2). Of the plant mapping characteristics assessed between MON 88701 and the 

conventional control, one significant difference was detected where MON 88701 had 

increased first-position bolls per plant compared to the conventional control. However, 

the mean value of the number of first-position bolls for MON 88701 was within the range 

of values observed for the commercial reference varieties. Thus, it is unlikely that the 

difference is biologically meaningful in the context of increased weediness of 

MON 88701 compared to commercially cultivated cotton. 
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IX.B.3.3. Response to Abiotic Stressors 

No biologically meaningful differences were observed during comparative field 

observations between MON 88701 and the conventional control in their response to 

abiotic stressors, such as compaction, drought, high winds, nutrient deficiency, etc. 

(Section VII.C.2.3). The lack of significant biologically meaningful differences in the 

MON 88701 response to abiotic stress support the conclusion that the introduction of the 

dicamba and glufosinate-tolerance traits are unlikely to result in increased weediness or 

plant pest potential compared to commercially cultivated cotton. 

IX.B.3.4. Pollen Morphology and Viability 

Evaluations of pollen morphology and viability from field-grown plants provide 

information useful in a plant pest assessment as it relates to the potential for gene flow 

and introgression of the biotechnology-derived trait(s) into other cotton varieties and wild 

relatives (Section VII.C.3). Pollen morphology and viability evaluations demonstrated 

no statistically significant differences between MON 88701 and the conventional control. 

Taken together, these comparative assessments indicate that MON 88701 is not likely to 

have increased weediness or plant pest potential compared to commercially cultivated 

cotton. 

IX.B.3.5. Interactions with Non-target Organisms 

Evaluation of MON 88701 for potential adverse impacts on NTOs is a component of the 

plant pest risk assessment. Since MON 88701 is not intended to have pesticidal activity, 

all organisms that interact with MON 88701 can be considered to be NTOs. In 2010 U.S. 

field trials, observational data on environmental interactions were collected for 

MON 88701 and the conventional control. In addition, multiple commercial reference 

varieties were included in the analysis to establish a range of natural variability for each 

characteristic among commercial cotton varieties. The environmental interactions 

assessment (Section VII.C.2.3) included data collected on plant-arthropod, plant-disease 

interactions, and plant mapping. The results of this assessment indicated that the 

presence of the dicamba and glufosinate-tolerance traits did not alter plant-arthropod 

interactions, including beneficial arthropods and arthropod pests, nor did it alter disease 

susceptibility of MON 88701 compared to conventional cotton. In addition, there were 

no differences in plant mapping parameters between MON 88701, not treated with 

dicamba or glufosinate herbicides, and the conventional control that would be indicative 

of a differential plant response to abiotic or biotic stressors. Thus, since all plots 

evaluated for plant mapping characteristics were at the same sites they would be 

subjected to similar stressors. Given that MON 88701 plants treated and not treated with 

dicamba and glufosinate herbicides had similar plant map results, it can be concluded that 

both responded to stressors in a similar manner. Therefore, these data support the 

conclusion that the biotechnology-derived traits in MON 88701, treated or not treated 

with dicamba and glufosinate herbicides, are unlikely to have increased plant pest 

potential, weediness, or an adverse environmental impact compared to commercially 

cultivated cotton. The lack of biologically meaningful differences in disease damage, 

arthropod-related damage, pest- and beneficial-arthropod abundance, and plant mapping 
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data demonstrate that the introduction of the dicamba and glufosinate-tolerance traits are 

unlikely to be biologically meaningful in terms of increased plant pest potential as 

compared to commercially cultivated cotton. 

The potential for MON 88701 to influence NTOs was evaluated using a combination of 

biochemical information and experimental data. The biochemical information and 

experimental data included molecular characterization, the MON 88701 DMO and 

PAT (bar) safety assessments, the history of environmental exposure to 

mono-oxygenases (the class of enzymes to which DMO belongs) and PAT proteins, 

results from the environmental interactions assessment described above, and the 

demonstration of compositional, agronomic and phenotypic equivalence to conventional 

cotton. Taken together, these data support the conclusion that MON 88701 is unlikely to 

adversely affect NTOs, including those beneficial to agriculture. Any effects on nontarget organisms that could potentially result from proposed changes in herbicide labels 

will be evaluated by the EPA. 

IX.C. Weed Potential of MON 88701 

Cotton is not listed as a weed in the major weed references (Crockett, 1977; Holm et al., 

1997), nor is it present on the lists of noxious weed species distributed by the federal 

government (7 CFR Part 360). United States Department of Agriculture has previously 

determined that “cotton is not considered to be a serious, principal or common weed pest 

in the U.S.” (USDA-APHIS, 1995). Commercial Gossypium species in the U.S. are not 

considered weeds and are not effective in invading established ecosystems. Cotton is not 

considered to have weedy characteristics in the U.S. and does not possess attributes 

commonly associated with weeds, such as long soil persistence, the ability to invade and 

become a dominant species in new or diverse landscapes, or the ability to compete well 

with native vegetation. It is recognized that in some agricultural systems, cotton can 

volunteer in a subsequent rotational crop. However, volunteers are easily controlled 

through tillage or use of appropriate herbicides (Alford et al., 2002; Murdock et al., 2002; 

Roberts et al., 2002). 

 

In comparative studies between MON 88701 and the conventional control, phenotypic, 

agronomic, plant mapping, and environmental interaction data were evaluated (Section 

VII) for changes that would impact the plant pest potential and in particular, plant weed 

potential. Results of these evaluations show that there is no biologically meaningful 

difference between MON 88701 and the conventional control for characteristics 

potentially associated with weediness. Furthermore, comparative field observations 

between MON 88701 and its conventional control in their response to abiotic stressors, 

such as drought, heat stress, and high winds, indicated no biologically meaningful 

differences and, therefore, no increased weed potential. Data on environmental 

interactions also indicate that MON 88701 does not confer any biologically meaningful 

increased susceptibility or tolerance to specific diseases or insect pests. Collectively, 

these findings support the conclusion that MON 88701 has no increased weediness 

compared to commercially cultivated cotton. 
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Volunteer MON 88701, like volunteer commercial cotton, would compete poorly with 

any succeeding crops and soon die, making it extremely unlikely to have any prolonged 

negative effects. Volunteer MON 88701 would also not be “extremely difficult to 

manage” because it can be controlled easily with numerous alternative herbicides and 

other mechanical means (Alford et al., 2002; Murdock et al., 2002; Roberts et al., 2002). 

IX.D. Potential for Pollen Mediated Gene Flow and Introgression 

Pollen mediated gene flow (often referred to as cross pollination) occurs when pollen of 

one plant fertilizes ovules of a second plant. Pollen mediated gene flow is affected by 

both biotic and abiotic factors such as plant biology, pollen biology/volume, plant 

phenology, overlap of flowering times, proximity of the pollen source and sink, ambient 

conditions such as temperature and humidity, and field architecture. Pollen mediated 

gene flow is a natural biological process, and therefore does not constitute an 

environmental risk in and of itself. 

Introgression is a process whereby one or more genes successfully incorporate into the 

genome of a recipient plant. Pollen mediated gene flow and gene introgression must be 

considered in the context of the transgenes inserted into the biotechnology-derived plant, 

and the likelihood that the presence of the transgenes and their subsequent transfer to 

recipient plants and plant populations will result in increased plant pest potential. The 

potential for gene flow and introgression from deregulation of MON 88701 is discussed 

in greater detail below. 

IX.D.1. Hybridization with Cultivated Cotton 

Although natural crossing can occur, cotton is normally considered to be a selfpollinating crop (Niles and Feaster, 1984). There are no morphological barriers to crosspollination based on flower structure. However, the pollen is heavy and sticky and 

transfer by wind is limited. Pollen is transferred instead by insects, in particular by 

various wild bees, bumble bees (Bombus sp.), and honeybees (Apis mellifera) (Van 

Deynze et al., 2005). Numerous studies on cotton cross-pollination have been conducted, 

and the published results, with and without supplemental pollinators, are summarized in 

Table IX-1. Recent cotton literature shows that the frequency of cross-pollination 

decreases with distance from the pollen source. McGregor (1976) traced movement of 

pollen by means of fluorescent particles and found that, even among flowers located only 

150 to 200 feet from a cotton field that was surrounded by a large number of bee colonies 

to ensure ample opportunity for transfer of pollen, fluorescent particles were detected on 

only 1.6% of the flowers. In a 1996 study with various field designs, Llewellyn and Fitt 

(1996) also found low levels of cross-pollination in cotton. At one meter from the source 

they observed cross-pollination frequencies of 0.15 to 0.4%, decreasing to below 0.3% at 

16 meters from the source. Umbeck et al. (1991) used a selectable marker to examine 

cross-pollination from a 30 x 136 meter source of biotechnology-derived cotton. Crosspollination decreased from five to less than one percent from one to seven meters, 

respectively, away from the source plot. A low level of cross-pollination (less than one 

percent) was sporadically detected at the furthest sampling distance of 25 meters. Berkey 

et al. (2002) reported that cross pollination between fields separated by a 13 foot road 
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decreased from 1.89% in the row nearest the source to zero percent in the 24th row. Van 

Deynze et al. (2005) conducted a two year study on pollen-mediated gene flow with high 

and low pollinator activity. In the presence of high pollinator activity the pollination 

frequency was 7.65% at 0.3 meters and less than 1% at greater than nine meters. 

Whereas, the pollination frequency in the presence of low pollinator activity was below 

1% at just over a meter. In a 2008 study, pollination frequencies of 5.00% and 0.00% 

were demonstrated at 1 and 8 meters, respectively (Kairichi et al., 2008). 

The potential for outcrossing and gene introgression from MON 88701 to cultivated 

cotton in the U.S. is low since cotton pollen movement by wind is limited due to it is 

large and sticky nature, and several studies have demonstrated that cross-pollination, 

even in the presence of high pollinator activity, is limited by distance. Therefore, the 

environmental consequences of pollen transfer from MON 88701 to other cotton or 

related Gossypium species is considered to be negligible. 

 
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Table IX-1. Summary of Published Literature on Cotton Cross Pollination 

Distance 

from Pollen 

Source 

(meters) 

CrossPollination 

(%) Comments Reference 

45-61 1.60% 

 Used fluorescent particles to follow 

pollinator movement in cotton fields 

over one season. 

(McGregor, 1976) 

1 0.15-0.4% Used a selectable marker to examine 

cross-pollination in the progeny of 

buffer row plants over one season. 

(Llewellyn and Fitt, 

1996) 

4 <0.08% 

16 <0.03% 

1 5% Used a selectable marker to examine 

cross-pollination from a 20 x 136 

meter source of biotechnologyderived cotton over one season. 

(Umbeck et al., 1991) 

1-25 

<1% 

5 1.89% Used herbicide bioefficacy to 

examine pollen flow between fields 

separated by a 13 foot road over one 

season. 

(Berkey et al., 2002) 10.5 0.77% 

17 0.13% 

25 0.00% 

0.3 7.65% * Used herbicide bioefficacy confirmed 

by DNA testing to measured pollenmediated gene flowing in four 

directions over 2 years. 

(Van Deynze et al., 

2005) 

>9 < 1% * 

>1 < 1% ** 

1625 0.04% ** 

1 5.00% Used ELISA strips to examine 

pollen-mediated gene flow in four 

directions from Bt source over a 

period of one season. 

2-7 2.00% (Kairichi et al., 2008) 

8 0.00% 

* High pollinator activity 

** Low pollinator activity 

IX.D.2. Hybridization with Wild and Feral Gossypium species 

Based on cytological evidence, seven genomic types, A through G, many with subtypes, 

have been identified for the genus Gossypium (Endrizzi et al., 1984). The domesticated 

species G. hirsutum and G. barbadense are allotetraploid (AADD, 2n=4x=52), while G. 

thurberi is a diploid (DD, 2n=2x=26), and G. tomentosum is an allotetraploid (AADD, 

2n=4x=52). G. tomentosum is capable of crossing with domesticated cotton to produce 

fertile offspring (Waghmare et al., 2005). However, Hawaii is the only U.S. region 

where G. tomentosum is found and domesticated cotton is not grown commercially in 

Hawaii, with the exception of potential counter-season breeding nurseries where 

appropriate isolation distances and practices are required (Wagner et al., 1990). Thus, the 

potential for gene flow to these wild relatives is limited. Importantly, MON 88701 would 

not be expected to confer a selective advantage to, or enhance the pest potential of, 

progeny resulting from such a cross if it were to occur. Any potential gene exchange 

between G. thurberi and domesticated cotton, if it were to occur, would result in triploid 
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(ADD, 3x=39), sterile plants because G. hirsutum and G. barbadense are allotetraploids 

(AADD, 2n=4x=52) and G. thurberi is a diploid (DD, 2n=2x=26). Such sterile hybrids 

have not been observed to persist in the wild. Fertile allohexaploids (6x=78) have not 

been reported in the wild. 

Only two ‘wild’ Gossypium species related to cultivated cotton are known to be present 

in the U.S., G. thurberi Todaro, which is known in Arizona (Fryxell, 1984), and feral 

populations of cultivated G. hirsutum and ‘wild’ populations of G. hirsutum are known to 

occur in South Florida and Puerto Rico (Brubaker et al., 1999). Both of these species 

would be capable of crossing with cultivated cotton, but they are not known to exist in 

cotton growing areas. Importantly, MON 88701 would not be expected to confer a 

selective advantage to, or enhance the pest potential of, progeny resulting from such 

crosses if they were to occur. 

Importantly, the environmental consequences of pollen transfer from MON 88701 to 

other cotton or related Gossypium species is considered to be negligible due to the plant 

biology and limited movement of cotton pollen, the safety of the introduced protein, and 

the lack of any selective advantage by the dicamba and glufosinate traits that might be 

conferred on a recipient plant of feral or wild cotton, or a wild relative. 

 

IX.D.3. Transfer of Genetic Information to Species with which Cotton Cannot 

Interbreed (Horizontal Gene Flow) 

Monsanto is unaware of any reports regarding the unaided transfer of genetic material 

from cotton species to other sexually-incompatible plant species. The likelihood for 

horizontal gene flow to occur is exceedingly small. Therefore, potential ecological risk 

associated with horizontal gene flow from MON 88701 due to the presence of the 

dicamba and glufosinate-tolerance traits are not expected. The consequence of horizontal 

gene flow of the dicamba and glufosinate-tolerance traits into other plants that are 

sexually-incompatible is negligible since, as data presented in this petition confirm, the 

genes and traits confer no increased plant pest potential to cotton. Thus, in the highly 

unlikely event that horizontal gene transfer were to occur, the presence of the dicamba 

and glufosinate-tolerance traits would not be expected to increase pest potential in the 

recipient species. 

IX.E. Potential Impact on Cotton Agronomic Practices 

An assessment of current cotton agronomic practices was conducted to determine 

whether the cultivation of MON 88701 has the potential to impact current cotton and 

weed management practices (Section VIII). Cotton fields are typically highly managed 

agricultural areas that are dedicated to crop production. MON 88701 is likely to be used 

in common rotations on land previously used for agricultural purposes. Certified seed 

production will continue to use well-established industry practices to deliver high quality 

seed containing MON 88701 to growers. Cultivation of MON 88701 is not expected to 

differ from typical cotton cultivation, with the exception of an expanded window of 

dicamba applications. As glufosinate is already utilized within U.S. cotton growing 

areas, no change in agronomic practices or land use would occur with the cultivation of 
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MON 88701 and the presence of the glufosinate-tolerance trait. Due to the crop safety of 

MON 88701 to dicamba and glufosinate, growers will have two herbicide modes-ofaction for in-crop control of glyphosate’s hard-to-control and resistant broadleaf weeds 

that are present in U.S. cotton production. As a result of cultivation of MON 88701 

integrated into the glyphosate-tolerant cotton systems, the number of dicamba-treated 

cotton acres will likely increase, whereas the number of glufosinate-treated cotton acres 

is expected to remain relatively static with minimal increase in use as cotton varieties 

utilizing the biotechnology-derived glufosinate-tolerance trait are currently commercially 

available and being utilized across the U.S. cottonbelt. Additionally, due to the expanded 

timing of in-crop applications to cotton, dicamba treatments will be later in the growing 

season than most current labeled dicamba uses. 

MON 88701 is similar to commercially cultivated cotton in its agronomic, phenotypic, 

ecological, and compositional characteristics, and has levels of resistance to insect pests 

and diseases comparable to commercially cultivated cotton. Based on this assessment, 

the introduction of MON 88701 is not likely to impact current U.S. cotton agronomic or 

cultivation practices, or weed management practices, other than the intended weed 

control benefits. 

IX.F. Conventional Breeding with Other Biotechnology-derived or Conventional 

Cotton 

Several biotechnology-derived cotton products have been deregulated or are under 

consideration for deregulation. Once deregulated, MON 88701 may be bred with these 

deregulated biotechnology-derived cotton products, as well as with conventional cotton, 

creating new improved varieties. APHIS has determined that none of the individual 

biotechnology-derived cotton products it has previously deregulated displays increased 

plant pest characteristics. APHIS has also concluded that any progeny derived from 

crosses of these deregulated biotechnology-derived cotton products with conventional or 

previously deregulated biotechnology-derived cotton are unlikely to exhibit new plant 

pest properties. This presumption, that combined-trait biotechnology products are 

unlikely to exhibit new characteristics that would pose new plant pest risks or potential 

environmental impacts not observed in the single event biotech product, is based upon 

several facts. Namely: 1) stability of the genetic inserts is confirmed in each approved 

biotech-derived cotton product across multiple generations (See Section IV.E for 

MON 88701 data); 2) stability of each of the introduced traits is continually and 

repeatedly assessed as new combined-trait varieties are created by plant breeders and 

tested over multiple seasons prior to commercialization; 3) combined-trait products are 

developed using the well established process of conventional breeding that has been 

safely used for thousands of years to generate new varieties (Cellini et al., 2004; NRC, 

2004; WHO, 1995); 4) worldwide organizations, such as World Health Organization, 

Food and Agriculture Organization/World Health Organization, International Seed 

Federation, CropLife International and U.S. FDA, conclude that the safety of the 

combined-trait product can be based on the safety of the parental GE events (CLI, 2005; 

FAO-WHO, 1996; ISF, 2005; U.S. FDA, 2001; WHO, 1995); and 5) practical 

applications in the field have shown that two unrelated biotechnology traits combined 

together by conventional breeding do not display new characteristics or properties 
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distinct from those present in the single event biotech products (Brookes and Barfoot, 

2012; James, 2010; Lemaux, 2008; Pilacinski et al., 2011; Sankula, 2006).

Therefore, based on the considerations above and the conclusion that MON 88701 is no 

more likely to pose a plant pest risk than commercially cultivated cotton it can be 

concluded that any progeny derived from crosses between MON 88701 and conventional 

cotton or deregulated biotechnology-derived cotton are no more likely to pose a plant risk 

than commercially cultivated cotton. 

IX.G. Summary of Plant Pest Assessments 

Plant pests are defined in the PPA as certain living organisms that can directly or 

indirectly injure, cause damage to, or cause disease to any plant or plant product (7 

U.S.C. § 7702[14]). Characterization data presented in Sections III through VII of this 

petition confirm that although MON 88701 contains the dicamba and glufosinate-tolerant 

traits, it is not different from commercially cultivated cotton in terms of pest potential in 

its phenotypic, agronomic, plant mapping, and environmental interaction characteristics. 

Monsanto is not aware of any study results or observations associated with MON 88701 

that would suggest an increased plant pest risk would result from its introduction. 

The plant pest assessment was based on multiple lines of evidence developed from a 

detailed characterization of MON 88701 compared to commercially cultivated cotton, 

followed by a risk assessment on detected differences. The risk assessment considered 

various factors, including: 1) insertion of a single functional copy of the dmo and bar

expression cassettes; 2) characterization and safety of the MON 88701 DMO and 

PAT (bar) proteins; 3) compositional equivalence of harvested MON 88701 cottonseed 

as compared to commercially cultivated cotton; 4) phenotypic, agronomic, and 

environmental interaction characteristics demonstrating no increased plant pest potential 

compared to commercially cultivated cotton; 5) negligible risk to NTOs; 6) familiarity 

with cotton as a cultivated crop and the inherently low plant pest potential of cotton; and 

7) no greater likelihood to impact agronomic practices, including land use, cultivation 

practices, or the management of weeds, diseases, and insects than commercially 

cultivated cotton, with the exception of the expanded window of dicamba application. 

Based on the data and information presented in this petition, it is concluded that, like 

conventional cotton and currently deregulated biotechnology-derived cotton, 

MON 88701 is highly unlikely to be a plant pest. Therefore, Monsanto Company 

requests a determination from APHIS that MON 88701 and any progeny derived from 

crosses between MON 88701 and other commercial cotton be granted nonregulated status 

under 7 CFR Part 340. 
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X. ADVERSE CONSEQUENCES OF INTRODUCTION 

Monsanto does not know of any results or observations associated with MON 88701 or 

the MON 88701 DMO and PAT (bar) proteins indicating that there would be an adverse 

environmental consequence from the introduction of MON 88701. MON 88701 contains 

DMO and PAT that confers dicamba and glufosinate tolerance to the cotton plant, 

respectively. As demonstrated by field results and laboratory tests, the only phenotypic 

differences between MON 88701 and conventional cotton are the tolerances to dicamba 

and glufosinate herbicides. 

The data and information presented in this petition demonstrate that MON 88701 is 

unlikely to pose an increased plant pest risk or to have an adverse environmental 

consequence compared commercially cultivated cotton. This conclusion is reached based 

on multiple lines of evidence developed from a detailed characterization of the product 

compared to commercially cultivated cotton, followed by a risk assessment on detected 

differences. The characterization evaluation included molecular analyses, which 

confirmed the insertion of a single functional copy of the dmo and bar expression 

cassettes at a single locus within the cotton genome. The amino acid sequence of the 

MON 88701 DMO and PAT (bar) proteins expressed in MON 88701 are identical to the 

amino acid sequences of the respective E. coli-produced proteins utilized in the protein 

safety studies supporting the safety of the proteins. Analyses of key nutrients and, antinutrients of MON 88701 seed demonstrate that MON 88701 is compositionally 

equivalent to commercially cultivated cotton. The phenotypic evaluations of 

MON 88701, including an assessment of seed germination and dormancy characteristics, 

plant growth and development characteristics, plant mapping parameters, pollen 

characteristics, and environmental interactions also indicated that MON 88701 is no more 

weedy than commercially cultivated cotton. There is no indication that MON 88701 

would have an adverse impact on beneficial or non-target organisms. Therefore, based 

on the lack of increased plant pest potential or adverse environmental consequences 

compared to commercially cultivated cotton, the risks for humans, animals, and other 

NTOs from introducing MON 88701 are negligible under the conditions of use. 

Successful integration of MON 88701 into glyphosate-tolerant cotton systems will 

provide growers with an opportunity for an efficient, effective weed management system 

for the management of glyphosate’s hard-to-control and resistant broadleaf weeds; 

provide a flexible system for inclusion of a second and third herbicide mode-of-action in 

cotton production practices as recommended by weed science experts to manage weed 

resistance development; and continue to provide cotton growers with effective weed 

control systems necessary for production yields to meet the growing needs of the food, 

feed, and industrial markets. 
Monsanto Company 12-CT-244U 246 of 620 

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APPENDICES 

 
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Appendix A: Notifications 

Field trials of MON 88701 have been conducted in the U.S. since 2007. The protocols 

for these trials include field performance, breeding and observation, agronomics, and 

generation of field materials and data necessary for this petition. In addition to the 

MON 88701 phenotypic assessment data, observational data on pest and disease stressors 

were collected from these product development trials. The majority of the final reports 

have been submitted to the USDA. However, some final reports, mainly from the 2011-

2012 seasons, are still in preparation. A list of trials conducted under USDA notifications 

and the status of the final reports for these trials are provided in Table A-1. 

 
Monsanto Company 12-CT-244U 264 of 620 

Table A-1. USDA Notifications and Permits Approved for MON 88701 and Status 

of Trials Conducted under These Notifications 

USDA # Effective Date # Release sites per state Trial Status

2007

07-241-107n 9/28/2007 PR-2 Submitted to USDA

2008

08-042-109n 3/12/2008 TX-2, TN-1, NC-2, MS-3, GA-4 Submitted to USDA

08-056-112n 3/26/2008 NM-2 Submitted to USDA

08-056-117n 3/26/2008

TX-3, SC-2, NC-2, MS-2, LA-1, GA-4, 

AR-1 Submitted to USDA

08-266-130n 10/19/2008 PR-3 Submitted to USDA

2009

09-058-104n 3/29/2009 CA-1 Submitted to USDA

09-065-111n 4/5/2009 AZ-5, GA-1, MS-3, SC-2, TX-4 Submitted to USDA

09-068-108n 4/8/2009

AL-1, AR-2, AZ-1, GA-1, IL-1, LA-1, 

MS-1, NC-4, NM-2, TX-1 Submitted to USDA

09-072-103n 4/8/2009 AR-1, MS-2, SC-5, TN-2, TX-5 Submitted to USDA

09-224-101n 9/21/2009 PR-2 Submitted to USDA

2010

10-054-134n 3/20/2010 TX-4 Submitted to USDA

10-059-109n 3/28/2010 GA-2, NC-9, SC-3 Submitted to USDA

10-061-102n 7/10/2010 MS-1, PR-7 Submitted to USDA

10-064-101n 4/3/2010

CA-2, GA-1, LA-1, MO-1, OK-3, SC-1, 

AR-1 Submitted to USDA

10-067-104n 4/7/2010 AZ-5, IL-1, MS-4, NM-2, PR-2, TX-10 Submitted to USDA

10-071-101n 4/9/2010

AR-4, AZ-2, GA-2, KS-1, LA-1, NC-2, 

NM-1, SC-1, TX-2 Submitted to USDA

10-071-102n 4/10/2010

AR-1, GA-1, LA-1, MS-1, NC-1, SC-1, 

TN-1, TX-2 Submitted to USDA

10-242-102n 9/29/2010 PR-2 Submitted to USDA

10-285-105n 11/11/2010 AR-1, GA-1, LA-1, NM-1 Submitted to USDA

2011

11-045-101n 3/16/2011 MS-1, PR-2 Pending

11-052-105n 3/23/2011 AL-1, FL-2, GA-9, MS-1, NC-6, SC-4 Pending

11-053-105n 3/25/2011 AR-3, LA-2, MO-2, MS-8, TN-5, TX-4 Pending

11-075-107n 4/15/2011

AL-1, AR-1, AZ-4, IL-1, LA-1, MO-1, 

MS-4, NC-1, SC-1, TX-9 Pending

11-068-103n 4/8/2011

AL-2, AR-2, AZ-1, CA-2. GA-2, LA-1, 

NC-1, NM-1, SC-1, TX-5 Pending

11-083-104n 4/23/2011 AL-1,MS-1 Pending

11-084-107n 4/24/2011 NC-1 Pending

11-091-102n 5/1/2011 TX-1 Pending
Monsanto Company 12-CT-244U 265 of 620 

Table A-1. USDA Notifications and Permits Approved for MON 88701 and Status 

of Trials Conducted under These Notifications (continued) 

USDA # Effective Date # Release sites per state Trial Status

2011 cont.

11-094-101n 5/4/2011 AZ-1 Pending

11-111-104n 5/21/2011 FL-1 Pending

11-133-103n 6/12/2011 IL-1 Pending

11-153-101n 7/2/2011 MS-1, PR-2 Pending

11-152-101n 7/1/2011 GA-1 Pending

11-199-102n 8/17/2011 PR-1 Pending

11-290-101n 11/16/2011 MS-1, PR-3 Pending

2012

12-018-101n 2/17/2012 AL-1, TX-2 Pending

12-053-110n 3/23/2012

AR-3, CA-1, GA-2, LA-2, MS-11, NC1, TN-1, TX-2 Pending

12-046-104n 3/16/2012

AL-1, AR-4, FL-1, GA-2, LA-1, NC-3, 

SC-1, TN-4, TX-2 Pending

12-051-106n 3/21/2012

AL-3, AR-3, FL-1, GA-3, MS-7, SC-1, 

TN-1, TX-5 Pending

12-051-105n 3/21/2012 GA-5, MS-4, NC-6, SC-2, TN-1, TX-5 Pending

12-046-109n 3/16/2012 AR-1, MO-5, TN-13, TX-2 Pending

12-055-101n 3/25/2012 AR-1, CA-1, SC-1, TX-1 Pending

12-068-101n 4/7/2012 CA-4 Pending

12-053-109n 3/23/2012 AL-1, NC-1, SC-1, TX-4 Pending

12-069-101n 4/8/2012 GA-9, TX-2 Pending

12-075-102n 4/14/2012 AL-1, AR-2, MS-1, NC-1, SC-1, TX-4 Pending

12-074-107n 4/13/2012 TX-1 Pending

12-081-101n 4/20/2012 AL-1, TX-2 Pending
Monsanto Company 12-CT-244U 266 of 620 

Appendix B: Materials, Methods, and Results for Molecular Analyses of 

MON 88701 

B.1. Materials 

The genomic DNA used in molecular analyses was isolated from leaf tissue of the R3

generation of MON 88701 and the conventional control (Coker 130). The leaf tissue was 

harvested from a greenhouse production in 2010. For generational stability analysis, 

genomic DNA was extracted from leaf tissue of the R2, R3, R4, R5 and R6 generations of 

MON 88701. The leaf tissue was harvested from production plan PPN-10-113. The 

reference substance, PV-GHHT6997 (Figure III-1), was used as a positive hybridization 

control in Southern blot analyses. Probe templates generated from PV-GHHT6997 were 

used as additional positive hybridization controls. As additional reference standards, the 

1 Kb DNA Extension Ladder and λ DNA/Hind III Fragments from Invitrogen (Carlsbad, 

CA) were used for size estimations on agarose gels and subsequent Southern blots. The 

1 Kb DNA Ladder from Invitrogen was used for size estimations on agarose gels for PCR 

analyses. 

B.2. Characterization of the Materials 

The identity of the source materials was verified by methods used in molecular 

characterization to confirm the presence or absence of MON 88701. The stability of the 

genomic DNA was confirmed by observation of interpretable signals from digested DNA 

samples on ethidium bromide stained agarose gels and/or specific PCR products, and the 

samples did not appear visibly degraded on the ethidium bromide stained gels. 

B.3. DNA Isolation for Southern Blot and PCR Analyses 

Genomic DNA was isolated from MON 88701 leaf tissue using a 

hexadecyltrimethylammonium bromide (CTAB) based method. Briefly, 20 ml of CTAB 

buffer (1.5% w/v CTAB, 75 mM Tris HCl, 100 mM EDTA, 1.05 M NaCl, and 

0.75% w/v PVP) and 10 mg RNase A were added to approximately 4 ml of ground leaf 

tissue and incubated at 60-70 °C for 40-50 min with intermittent mixing. Twenty 

milliliters of chloroform was added to the samples and mixed by hand for 2-3 min, then 

centrifuged at 10,300 × g for 8-10 min. The upper aqueous phase was put into a clean 

tube and the chloroform step was repeated twice. After the last chloroform step, the 

aqueous phase was put into a clean tube and the DNA was precipitated with 20 ml of 

100% v/v ethanol. The sample was centrifuged for one minute to condense the pellet, 

and then the precipitated DNA was hooked out and put into a tube with 4-6 ml of 

70% v/v ethanol to wash the DNA pellet. The samples were centrifuged at 5,100 × g for 

5 min to pellet the DNA. DNA pellets were air dried, then resuspended in 250 μl of TE 

buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). All extracted DNA was stored in a 

-20 C freezer. 

B.4. Quantification of DNA 

Genomic DNA was quantified using a QubitTM Fluorometer (Invitrogen, Carlsbad, CA). 
Monsanto Company 12-CT-244U 267 of 620 

B.5. Restriction Enzyme Digestion of DNA 

Approximately 10 μg of genomic DNA extracted from MON 88701 and conventional 

control were digested with restriction enzyme Bcl I (New England Biolabs, Inc., Beverly, 

MA) or with restriction enzyme Ssp I- HF (New England Biolabs, Inc.). All Bcl I digests 

were conducted in 10X NEBuffer 3 buffer at 50 °C in a total volume of ~500 μl with 

~50 units of restriction enzyme. All Ssp I-HF digests were conducted in 10X NEBuffer 4 

at 37 °C in a total volume of ~500 μl with ~100 units of restriction enzyme. For the 

purpose of running positive hybridization controls, ~10 μg of genomic DNA extracted 

from the conventional control was digested with the restriction enzyme Bcl I and the 

appropriate positive hybridization control(s) were added to these digests prior to loading 

the agarose gel. 

B.6. Agarose Gel Electrophoresis 

Digested DNA was resolved on ~0.8% (w/v) agarose gels. For T-DNA insert/copy 

number and plasmid vector backbone analyses, individual digests containing ~10 μg each 

of MON 88701 and conventional control genomic DNA were loaded on the same gel in a 

long run/short run format. The long run allows for greater resolution of large molecular 

weight DNA, whereas the short run allows for retaining the small molecular weight DNA 

on the gel. The positive hybridization controls were only run in the short run format. For 

the insert stability analysis, individual digests of ~10 μg each of genomic DNA extracted 

from five leaf samples from multiple generations of MON 88701 and the conventional 

control along with the positive hybridization controls were loaded on the agarose gel in a 

single run format. 

B.7. DNA Probe Preparation for Southern Blot Analyses 

Probe templates were prepared by PCR amplification using the PV-GHHT6997 DNA as 

template. The PCR products were separated on an agarose gel by electrophoresis and 

purified from the gel using QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) 

according to manufacturer’s instruction. The probe templates were designed based on the 

nucleotide composition (%GC) of the sequence in order to optimize the detection of 

DNA sequences during hybridization. When possible, probes possessing similar melting 

temperature (Tm) were combined in the same Southern blot hybridization. 

Approximately 25 ng of each probe template were radiolabeled with either [α32P] 

deoxycytidine triphosphate (dCTP) (6000 Ci/mmol) or [α32P] deoxyadenosine 

triphosphate (dATP) (6000 Ci/mmol) using RadPrime DNA Labeling System 

(Invitrogen, Carlsbad, CA) according to manufacturer’s instruction. 

B.8. Southern Blot Analyses of DNA 

Genomic DNA isolated from MON 88701 and the conventional control was digested and 

evaluated using Southern blot analyses (Southern, 1975). The PV-GHHT6997 DNA, 

previously digested with the restriction enzyme Pci I was added to conventional control 

genomic DNA digested with Bcl I to serve as positive hybridization control on each 

Southern blot. When multiple probes were hybridized simultaneously to one Southern 
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blot, the probe templates were spiked in the digested conventional control genomic DNA 

to serve as additional positive hybridization controls on the Southern blot. The DNA was 

then separated by agarose gel electrophoresis and transferred onto a nylon membrane. 

Southern blots were hybridized and washed at 55 °C, 60 °C, or 65 °C, depending on the 

calculated melting temperature (Tm) of the probes that were used. Table B-1 lists the 

radiolabeling conditions and hybridization temperatures of the probes used in this study. 

Multiple exposures of each blot were then generated using Kodak Biomax MS film 

(Eastman Kodak, Rochester, NY) in conjunction with one Kodak Biomax MS 

intensifying screen in a -80 °C freezer. 
Monsanto Company 12-CT-244U 269 of 620 

Table B-1. Hybridization Conditions of Utilized Probes 

Probe

Labeling 

Method

Element Sequence Spanned by 

DNA Probe

Probe labeled 

with dNTP 

(32P)

Hybridization/

Wash

Temperature (C)

1 RadPrime

B-Right Border, P-PC1SV, LTEV, TS-CTP2, CS-dmo 

(portion) dATP 55

2 RadPrime

TS-CTP2 (portion), CS-dmo 

(portion) dCTP 65

3 RadPrime CS-dmo (portion), T-E6, P-e35S dATP 55

4 RadPrime

P-e35S (portion), L-Hsp70, CSbar, T-nos (portion) dCTP 65

5 RadPrime

CS-bar (portion) , T-nos, BLeft Border dATP 55

6 RadPrime OR-ori V dCTP 60

7 RadPrime

CS-rop, OR-ori-pBR322 

(portion) dCTP 60

8 RadPrime OR-ori-pBR322 (portion), aadA dCTP 60

 
Monsanto Company 12-CT-244U 270 of 620 

B.9. DNA Sequence Analyses of the Insert 

Overlapping PCR products, denoted as Product A, Product B, and Product C, were 

generated that span the insert and adjacent 5′ and 3′ flanking DNA sequences in 

MON 88701. These products were analyzed to determine the nucleotide sequence of the 

insert in MON 88701, as well as that of the DNA flanking the 5′ and 3′ ends of the insert. 

The PCR analysis for Product A was conducted using ~100 ng of genomic DNA template 

in a 50 l reaction volume. The reaction volume contained either a final concentration of 

2 mM MgSO4, 0.2 M of each primer, 0.2 mM of each dNTP, and 0.02 units/μl of 

Accuprime Taq DNA Polymerase High Fidelity (Invitrogen) or a final concentration of, 

1.5 mM MgCl2, 0.2 M of each primer, 0.2 mM of each dNTP, and 0.02 units/μl of 

Phusion Hot Start II High Fidelity DNA Polymerase (Finnzymes, Espoo, Finland). The 

Phusion Hot Start II High Fidelity DNA Polymerase was used to enhance the 

amplification and sequencing of a small A-rich region located in Product A. 

The PCR analyses for Product B and Product C were each conducted using ~100 ng of 

genomic DNA template in a 50 l reaction volume containing a final concentration of 

2 mM MgSO4, 0.2 M of each primer, 0.2 mM of each dNTP, 2.5% (v/v) DMSO, and 

0.02 units/μl of Accuprime Taq DNA Polymerase High Fidelity (Invitrogen). 

The amplification of Product A using Accuprime Taq was performed under the following 

cycling conditions: 1 cycle at 94 C for 2 minutes; 35 cycles at 94 C for 15 seconds, 

59 C for 30 seconds, 68 C for 2.25 minutes; 1 cycle at 68 C for 5 minutes. The 

amplification of Product A using Phusion Hot Start II DNA Polymerase was performed 

under the following cycling conditions: 1 cycle at 98 C for 30 seconds, 35 cycles at 

98 C for 10 seconds, 64 C for 15 seconds, and 72 C for 1.25 minutes. The 

amplification of Product B was performed under the following cycling conditions: 

1 cycle at 94 C for 2 minutes; 35 cycles at 94 C for 15 seconds, 50 C for 30 seconds, 

68 C for 3 minutes; 1 cycle at 68 C for 5 minutes. The amplification of Product C was 

performed under the following cycling conditions: 1 cycle at 94 C for 2 minutes; 

35 cycles at 94 C for 15 seconds, 62 C for 30 seconds, 68 C for 2 minutes; 1 cycle at 

68 C for 5 minutes. 

A small aliquot of each PCR product was separated on a 1.0% (w/v) agarose gel and 

visualized by ethidium bromide staining to verify that the products were the expected 

size. Prior to sequencing, each verified PCR product was purified using the QIAquick 

PCR Purification Kit (Qiagen, Inc., Valencia, CA) and quantified using a Qubit 

fluorometer. The purified PCR products were sequenced using multiple primers, 

including primers used for PCR amplification. All sequencing was performed by 

Monsanto TGAC (The Genome Analysis Center) using BigDye terminator chemistry 

(Applied Biosystems, Foster City, CA). 

A consensus sequence was generated by compiling multiple sequencing reactions 

performed on the overlapping PCR products. This consensus sequence was aligned to the 
Monsanto Company 12-CT-244U 271 of 620 

PV-GHHT6997 sequence to determine the integrity and organization of the integrated 

DNA and the 5′ and 3′ insert-to-flank DNA junctions in MON 88701. 

B.10. PCR and DNA Sequence Analysis to Examine the MON 88701 Insertion Site 

To examine the MON 88701 insertion site in conventional cotton, PCR and sequence 

analyses were performed on genomic DNA from both MON 88701 and conventional 

cotton. The primers used in this analysis were designed from the DNA sequences 

flanking the insert in MON 88701. A forward primer specific to the DNA sequence 

flanking the 5′ end of the insert was paired with a reverse primer specific to the DNA 

sequence flanking the 3′ end of the insert. 

The PCR reactions were conducted using ~100 ng of genomic DNA template in a 50 l 

reaction volume containing a final concentration of 2 mM MgSO4, 0.2 M of each 

primer, 0.2 mM of each dNTP, and 0.02 units/μl of Accuprime Taq DNA Polymerase 

High Fidelity (Invitrogen). The amplification was performed under the following cycling 

conditions: 1 cycle at 94 C for 2 minutes; 35 cycles at 94 C for 15 seconds, 58 C for 

30 seconds, 68 C for 30 seconds; 1 cycle at 68 C for 5 minutes. 

A small aliquot of each PCR product was separated on a 1.2% (w/v) agarose gel and 

visualized by ethidium bromide staining to verify that the PCR products were the 

expected size prior to sequencing. Only the verified PCR product from the parental 

conventional control was purified with the QIAquick PCR Purification Kit (Qiagen) and 

quantified using a Qubit Fluorometer. The purified PCR product was sequenced using 

multiple primers, including primers used for PCR amplification. All sequencing was 

performed by the Monsanto TGAC (The Genome Analysis Center) using BigDye 

terminator chemistry (Applied Biosystems). 

A consensus sequence was generated by compiling multiple sequencing reactions 

performed on the verified PCR product. This consensus sequence was aligned to the 5′

and 3′ sequences flanking the MON 88701 insert to determine the integrity and 

organization of the insertion site. 

 
Monsanto Company 12-CT-244U 272 of 620 

References for Appendix B 

Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated 

by gel electrophoresis. Journal of Molecular Biology 98:503-517. 
Monsanto Company 12-CT-244U 273 of 620 

Appendix C: Protein Reaction Products, Materials, Methods, and Results for 

Characterization of MON 88701 DMO and PAT (bar) Proteins Produced in 

MON 88701, and Substrate Specificity 

C.1. DMO Reaction Products 

MON 88701 when treated with dicamba herbicides will yield the reaction products 3,6-

dichlorosalicylic acid (DCSA) and formaldehyde during demethylation of the herbicide. 

These products, as you will see in the text below, have either been previously deemed 

safe (DCSA) or are commonly produced in nature and at sufficiently low levels in this 

MON 88701 cropping system (formaldehyde) so as to not raise concerns with regard to 

the plant pest risk assessment for MON 88701. 

C.1.1. DCSA in MON 88701 

DCSA is a metabolite generated when dicamba herbicide is sprayed on MON 88701 

cotton and soybean and is also produced by livestock and soil whose safety has been 

evaluated by the Environmental Protection Agency (U.S. EPA, 2009; FAO-WHO, 2011). 

DCSA residue levels were measured in dicamba-treated MON 88701 to support 

Monsanto’s registration request for the inclusion of DCSA in the cottonseed and gin byproduct dicamba residue definitions. DCSA is structurally similar to salicylic acid (SA). 

Numerous studies have reported on the stress defense activities of SA, although most 

studies have looked at the protective effects of exogenously applied SA (Janda et al, 

2007). 

C.1.2. Formaldehyde in the Environment 

Formaldehyde is ubiquitous in the environment; plants and animals are constantly 

exposed to low levels already present in the environment and the atmosphere from a 

variety of biogenic (e.g., plant and animal) and anthropogenic (e.g., automotive or 

industrial emissions) sources. In water, formaldehyde dissipates through biodegradation 

to low levels in a few days (USHHS-ATSDR, 1999). Aerobic biodegradation half-lives 

are estimated to be 1-7 days for surface water and 2-14 days for ground water (U.S. EPA, 

2008). The half-life of formaldehyde in air is dependent on a number of factors (light 

intensity, temperature, and location). Through reaction with hydroxyl radical, the halflife of formaldehyde in air varies from 7 to 70 hours (U.S. EPA, 2008). The photolytic 

half-life of formaldehyde in air (e.g., in the presence of sunlight) is estimated to be 1.6-6 

hours (U.S. EPA, 2008; USHHS-ATSDR, 1999). Formaldehyde is rapidly consumed in 

the atmosphere through direct photolysis or by oxidation with hydroxyl or nitrate radicals 

(USHHS-ATSDR, 1999). 

Humans are constantly exposed to low levels of formaldehyde. Human exposure to 

formaldehyde is primarily due to indoor air exposures (USHHS-ATSDR, 1999). 

Formaldehyde is found in a variety of consumer products such as cosmetics and paints, 

often as an antimicrobial agent, and is used extensively in urea-formaldehyde “slowrelease” fertilizer formulations and adhesives (USHHS-ATSDR, 1999). Indoor 

formaldehyde air concentrations are generally significantly higher than outdoor air 
Monsanto Company 12-CT-244U 274 of 620 

concentrations (USHHS-ATSDR, 1999) as a result of combustion (cooking, heating, 

tobacco use) and the emission of formaldehyde from a variety of construction materials 

(e.g., particle board, plywood or foam insulation) as well as permanent press fabrics (e.g., 

clothing or draperies) (U.S. CPSC, 1997). Formaldehyde present in outdoor air results 

from a number of sources, and levels of formaldehyde are generally higher in urban areas 

than in rural areas (WHO-IPCS, 1989). Direct contributions of formaldehyde to the 

atmosphere (i.e., those in the form of formaldehyde itself) from man-made sources are 

present, but are generally considered to be small relative to natural sources or indirect 

production of formaldehyde in the atmosphere (WHO, 2002). 

C.1.3. Formaldehyde in MON 88701 

Formaldehyde is a metabolite when dicamba is sprayed on MON 88701 cotton. 

However, formaldehyde is not considered a relevant metabolite in the demethylation of 

dicamba by U.S. EPA. According to the guidelines published by Office of Prevention, 

Pesticides and Toxic Substances, United States Environmental Protection Agency (US 

EPA OPPTS 860.1300), the methoxy side chain that is cleaved from dicamba to form 

formaldehyde would specifically not be chosen to be labeled in a metabolism study (U.S. 

EPA, 1996). This is because it is not metabolically stable and would not be considered a 

significant moiety as it would be readily metabolized and incorporated into the 1-carbon 

pool of the plant through known pathways. Therefore, formaldehyde was not measured 

in the residue study when dicamba was applied to MON 88701. 

The maximum theoretical production of formaldehyde produced from dicamba-treated 

MON 88701 is estimated to be 6.3 mg/kg and 33 mg/kg7. This is well within the range of 

formaldehyde concentrations measured for a variety of agricultural commodities, 

including up to 60 mg/kg in fruits and vegetables (WHO-IPCS, 1989). Plants have a 

large capacity to metabolize formaldehyde naturally produced from internal processes (A. 

Hanson (2011), C.V. Griffin, Sr. Eminent Scholar, Horticulture Department, University 

of Florida, Personal Communication), and any additional amount of formaldehyde that 

could be theoretically produced in the plant by dicamba treatment in MON 88701 would 

be metabolized very quickly. Thus the incremental increase in formaldehyde over and 

above the levels already presumed to be present in the cotton plant would be small and 

transient and associated with an outdoor application of dicamba herbicide. Further, since 

current literature supports that formaldehyde is only emitted from foliage under certain 

 

7

 Calculation based an assumption that the entire 0.56 kg/ha (0.5 lb/acre a.e.) application of dicamba that is 

intercepted by the MON 88701 cotton plant at the 6-leaf or first bloom plus 15 day growth stage is 

instantaneously and completely absorbed, and then instantaneously metabolized by the DMO enzyme 

(Complete demethylation of 560 g (2.5 mol)/ ha dicamba would yield 2.5 mol/ha formaldehyde). Canopy 

closure, and thus spray interception, is estimated at 30% at the 6-leaf stage (Krutz et al., 2012), resulting in 

production of 23 g/ha formaldehyde. Canopy closure is near complete at the first bloom plus 15 day 

growth stage (Reddy et al., 2009), so no adjustment is applied. Above-ground biomass of 6-leaf plants is 

estimated to be 0.7 metric tons/ha (Ducamp et al., 2012), and the estimated maximum theoretical 

concentration is 33 mg/kg formaldehyde in planta. For dicamba applications at first bloom plus 15 day 

growth stage, the crop biomass is estimated to be 12 metric tons/ha (Boquet and Breitenbeck, 2000), and 

the estimated maximum theoretical formaldehyde concentration produced in planta is 6.3 mg/kg. 
Monsanto Company 12-CT-244U 275 of 620 

conditions (Nemecek-Marshall et al., 1995; Cojocariu et al., 2004; Cojocariu et al., 2005) 

and that emission rates are low (Nemecek-Marshall et al., 1995), little opportunity exists 

for formaldehyde to be released from MON 88701 after dicamba treatment. Therefore 

human safety concerns of formaldehyde released from dicamba-treated MON 88701 are 

considered to be negligible and the most relevant route of exposure is from repeated 

inhalation of concentrated levels associated with indoor or occupational environments. 

USHHS-NTP (2011) has already stated that there is no evidence to suggest that dietary 

intake of formaldehyde is important, despite NTP’s 12th Report on Carcinogens 

reclassifying formaldehyde as a known human carcinogen by (USHHS-NTP, 2011). In 

addition, the only human food currently produced from cottonseed is refined, bleached, 

and deodorized (RBD) oil, and to a smaller extent, linters. Therefore, the potential for 

human exposure to any formaldehyde in dicamba-treated MON 88701 cottonseed is 

highly unlikely. 

C.1.4. Conclusion 

Data from both dicamba and glufosinate-treated and not treated MON 88701 compared to 

a conventional control are available from multiple sites across the U.S., where 

agronomic, phenotypic and environmental interaction data were collected. The results of 

this assessment demonstrate no biologically meaningful difference between MON 88701 

treated with and without dicamba and glufosinate and the conventional control, and 

support a conclusion that the formation of DCSA and formaldehyde does not alter the 

weedy characteristics or increase susceptibility or tolerance to diseases, insect pests or 

abiotic stresses. Therefore, MON 88701, as cultivated, is no more likely to be a plant 

pest risk or have a biologically meaningful change in environmental impact than 

conventional cotton. 

C.2. Characterization of MON 88701 DMO Protein in MON 88701 

C.2.1 Forms of DMO 

Various forms of the DMO protein (Figure C-1) were used to establish enzyme structure, 

activity, substrate specificity and safety of the proteins in MON 88701. The wild-type 

DMO was first isolated and characterized from Stenotrophomonas maltophilia (Herman 

et al., 2005). The MON 88701 DMO protein present in MON 88701 is identical to the 

wild-type DMO, except for an additional leucine at position two and an additional nine 

amino acids at the N-terminus from the chloroplast transit peptide, CTP2 (Figure C-1). 

The E. coli-produced form of DMO is identical to the wild-type DMO, but with a 

histidine-tag on the N-terminus (Figure C-1), was used for specificity experiments. The 

differences in the amino acid sequence or the addition of N-terminal histidine tag did not 

appear to have an effect on mode-of-action, structure, functional activity, or specificity of 

DMO, as these changes are sterically distant from the catalytic domain centers involved 

in electron transport (Rieske and non-heme iron centers) and the catalytic centers for the 

dicamba substrate (D’Ordine et al., 2009; Dumitru et al., 2009). 
Monsanto Company 12-CT-244U 276 of 620 

Figure C-1. Forms of DMO Protein and Their Relation to the Wild-Type DMO 

Protein 

The diagram represents the various DMO forms described in this petition. The wild-type 

DMO form isolated from S. maltophilia was the first form sequenced (Herman et al., 

2005). The MON 88701 DMO protein has an insertion of a leucine at position 2, and the 

addition of 9 amino acids from CTP2 at the N-terminus. MON 88701 DMO was purified 

from cottonseed of MON 88701. E. coli-produced MON 88701 DMO has the same 

sequence as plant-produced MON 88701 DMO; equivalence between the two proteins 

has been demonstrated (Section V.B and Appendix C). The N-terminal histidine-tagged 

DMO was produced in E. coli and was used for in vitro specificity studies (Section 

V.A.1.2). 

C.2.2. Materials 

The MON 88701 DMO protein (lot 11299151) was purified from cottonseed of 

MON 88701 (lot 11287350). The MON 88701 DMO protein was stored in a -80 oC 

freezer in a buffer solution containing 50 mM potassium phosphate, pH 8.0, 1 mM DTT, 

1 mM benzamidine HCl, 0.1 M sodium chloride and 10% glycerol. 

The E. coli-produced MON 88701 DMO protein (lot 11300031) was used as the 

reference substance. The DMO protein reference substance was generated from cell 

paste produced by large-scale fermentation of E. coli containing the pMON136400 

expression plasmid. The coding sequence for dmo contained on the expression plasmid 

(pMON136400) was confirmed prior to and after fermentation. The E. coli-produced 

MON 88701 DMO protein was previously characterized. 

C.2.3. Description of Assay Control 

Protein MW standards (Precision Plus Protein Standards Dual color; Bio-Rad, Hercules, 

CA) were used to calibrate some SDS-PAGE gels and verify protein transfer to 

polyvinylidene difluoride (PVDF) and nitrocellulose membranes. Broad Range 

SDS-PAGE MW standards (Bio-Rad, Hercules, CA) were used to generate a standard 

curve for the apparent MW estimation. Bovine serum albumin (BSA) and 

-aminobutyric acid (AAbA) were used as hydrolysis control and internal calibration 

standard for amino acid analysis. The E. coli-produced MON 88701 DMO reference 

standard was used to construct a standard curve for the estimation of total protein 

concentration using a Bio-Rad protein assay. A phenylthiohydantoin (PTH) amino acid 

standard mixture (Applied Biosystems, Foster City, CA) was used to calibrate the 
Monsanto Company 12-CT-244U 277 of 620 

Applied Biosystems 494 Procise Sequencing System for each analysis. A peptide 

mixture (Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster City, CA) 

was used to calibrate the MALDI-TOF mass spectrometer for tryptic mass analysis. 

Transferrin (Sigma-Aldrich, St. Louis, MO) was used as positive control for 

glycosylation analysis. 

C.2.4. Protein Purification 

The MON 88701 DMO was purified from cottonseed of MON 88701. The purification 

procedure was not performed under a GLP plan; however, all procedures were 

documented on worksheets and, where applicable, SOPs were followed. The 

MON 88701 DMO protein was purified from an extract of ground cottonseed using a 

combination of ammonium sulfate precipitation, hydrophobic interaction 

chromatography, anion exchange chromatography, mixed mode ion exchange 

chromatography and size exclusion chromatography. The purification procedure is 

briefly described below. 

Approximately 1 kg of MON 88701 cottonseed expressing the DMO protein was mixed 

with ~1 kg of dry ice and ground to fine powder using a laboratory mill (model 3100, 

Perten Instruments). The ground powder was suspended in two liters of hexane (EMD 

Chemicals Inc., Gibbstown, NJ ) and filtered. This process was repeated four times in 

order to completely defat the powder. After drying overnight, the powder was ready for 

further processing. All grinding and defatting steps were done in a fume hood at room 

temperature. 

The ground powder was mixed with extraction buffer (50 mM Tris, pH 8.0, 2.0 M 

deionized urea, 0.2 M boric acid, 1.0 mM dithiothreitol (DTT), 1.0 mM 

benzamidine-HCl, 1.0 μm bestatin, 1.0 μM E-64 and Complete EDTA free protease 

inhibitor cocktail (Roche Applied Science, Indianapolis, IN)) to a final volume of 8 liters 

and incubated for 2 h at room temperature. The slurry was centrifuged at 15000 × g for 

30 min at 4 °C. The supernatant was collected and brought to 0.05% polyethyleneimine 

(PEI). The solution was stirred at ~4 °C for 30 min and then centrifuged at 15000 × g for 

30 min. The supernatant was collected and ~2.2 kg of ammonium sulfate was slowly 

added to bring the solution to 50% w/v ammonium sulfate saturation. This solution was 

stirred at ~4 °C for 2 h and the pellet was collected by centrifugation at 15000 × g for 

30 min. The pellet was resuspended in 10 liters of the resuspension buffer (50 mM 

Tris-HCl, pH 8.0, 0.35 M ammonium sulfate, 10 mM DTT, 1.0 mM benzamidine-HCl, 

1.0 μM bestatin, 1.0 μM E-64 and Complete EDTA free protease inhibitor cocktail). The 

solution was stirred in the cold room overnight and then centrifuged at 30,000  g for 1 h. 

Supernatant was collected and loaded onto a 1 liter butyl sepharose column (GE 

Healthcare) equilibrated with butyl sepharose equilibration (BSE) buffer (50 mM 

Tris-HCl, pH 8.0, 0.35 M ammonium sulfate, 1 mM DTT, 1.0 mM benzamidine-HCl, 

1.0 μM bestatin, 1.0 μM E-64 and Complete EDTA-free protease inhibitor cocktail). All 

column steps were run at room temperature. The column was washed with 5 liters BSE 

buffer. Proteins were eluted with 1 liter of buffer containing 25 mM Triethanolamine, 

pH 8.0, 100 μM dicamba, 1.0 mM DTT, 1.0 mM benzamidine-HCl, 1.0 μM bestatin, 

1.0 μM E-64 and Complete EDTA free protease inhibitor cocktail (BSEL buffer). After 
Monsanto Company 12-CT-244U 278 of 620 

eluting the proteins with 1 liter of BSEL buffer, the flow was stopped for one hour and 

then elution was continued with additional 1 liter of BSEL buffer. Both elutions were 

pooled and loaded onto a 25 ml DEAE macroprep column (Bio Rad) equilibrated with 

DEAE macroprep equilibration (DME) buffer (50 mM Tris-HCl, pH 8.0, 100 μM 

dicamba, 1.0 mM DTT, 1.0 mM benzamidine-HCl, 1.0 μM bestatin, 1.0 μM E-64 and 

Complete EDTA free protease inhibitor cocktail). All steps associated with DEAE 

macroprep were performed at ~4 °C. The column was washed with 125 ml DME buffer 

and proteins were eluted with 75 ml DME buffer containing 70 mM NaCl and then with a 

linear gradient that increased from 70 mM to 350 mM NaCl over 500 ml. Fractions 

containing MON 88701 DMO were pooled and loaded onto a 2.5 ml ceramic 

hydroxyapatite (CHT) column (Bio-Rad) equilibrated with CHT equilibration buffer 

(50 mM Tris-HCl, pH 8.0, 100 μM dicamba, 1.0 mM DTT, 1.0 mM benzamidine-HCl, 

1.0 μM bestatin, 1.0 μM E-64 and Complete EDTA free protease inhibitor cocktail ). All 

steps associated with CHT were performed at ~4 °C. Most of the MON 88701 DMO was 

found in the flow through and wash fractions. Flow through and wash fractions from 

CHT were pooled separately (Pooled FT and Pooled Wash, respectively) and reloaded on 

two separate CHT columns as follows. 

The Pooled FT was loaded onto a ~10 ml CHT column (CHT3). The column was 

washed with 50 ml of CHT equilibration buffer and step eluted using the CHT 

equilibration buffer containing 1 mM, 2 mM and 3 mM potassium phosphate, pH 8.0. 

The Pooled Wash was loaded onto a ~3 ml CHT column (CHT2). CHT2 was washed 

with ~45 ml of CHT equilibration buffer and step eluted using the CHT equilibration 

buffer containing 1 mM, 2 mM and 3 mM potassium phosphate, pH 8.0. 

Wash fractions from both the CHT2 and CHT3 chromatography runs that contained 

MON 88701 DMO were pooled and loaded onto a ~1 ml DEAE macroprep column 

equilibrated with DME buffer for concentration. The column was washed with 10 ml of 

the DME buffer and eluted with DME buffer containing 500 mM NaCl. The 

MON 88701 DMO containing fractions were pooled and loaded onto a Hi-Prep 

Sephacryl S 100 size exclusion column equilibrated at ~4 °C with 50 mM potassium 

phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 0.1 M NaCl and 10% (v/v) 

glycerol. Fractions containing MON 88701 DMO were pooled and concentrated with 

aquacide (EMD Bioscienes, Inc., La Jolla, CA) at ~4 °C to a final volume of 750 μl. 

Elution fractions (1-3 mM potassium phosphate, pH 8.0 fractions) from both the CHT2 

and CHT3 that contained MON 88701 DMO were pooled and concentrated using a 

Amicon ultra spin concentrator (Millipore, Bedford, MA) with a 10K MWCO. The 

centriprep concentrated pool was then loaded onto a Hi Prep Sephacryl S 100 size 

exclusion column equilibrated at ~4 °C with 50 mM potassium phosphate, pH 8.0, 1 mM 

DTT, 1 mM benzamidine-HCl, 0.1 mM NaCl and 10% (v/v) glycerol. Fractions 

containing MON 88701 DMO were pooled and concentrated with aquacide at ~4 °C to a 

final volume of 750 μl. 

Both aquacide concentrated samples were pooled to a final volume of 1.5 ml. The final 

buffer composition of the purified MON 88701 DMO protein was 50 mM potassium 

phosphate, pH 8.0, 1 mM DTT, 1 mM benzamidine-HCl, 100 mM sodium chloride and 
Monsanto Company 12-CT-244U 279 of 620 

10% (v/v) glycerol. This MON 88701 DMO purified from the cottonseed of 

MON 88701 was aliquoted and stored in a -80 °C freezer. 

 
Monsanto Company 12-CT-244U 280 of 620 

C.2.5. Summary of DMO Protein Identity and Equivalence 

Table C-1. Summary of MON 88701 DMO Protein Identity and Equivalence 

Analytical Test Assessment Analytical Test Outcome 

1. N-terminal sequence analysis of the 

MON 88701 DMO protein to assess 

identity 

 The identity could not be confirmed by 

N-terminal sequence analysis 

 MALDI-TOF MS1

 analysis of peptides 

derived from tryptic digested MON 88701 

DMO established the N-terminal sequence of 

MON 88701 DMO 

2. MALDI-TOF MS1 analysis of peptides 

derived from tryptic digested 

MON 88701 DMO protein to assess 

identity 

 MALDI-TOF MS1 analysis yielded peptide 

masses consistent with the expected peptide 

masses from the theoretical trypsin digest of 

the MON 88701 DMO sequence 

3. Western blot analysis using anti-DMO 

polyclonal antibodies to assess identity 

and immunoreactive equivalence 

between MON 88701 DMO and the 

E. coli-produced MON 88701 DMO 

proteins 

 MON 88701 DMO protein identity was 

confirmed using a western blot probed with 

antibodies specific for DMO protein 

 Immunoreactive properties of the 

MON 88701 DMO and the E. coli-produced 

MON 88701 DMO proteins were shown to be 

equivalent 

4. SDS-PAGE2 to assess equivalence of 

the apparent molecular weight between 

MON 88701 DMO and the 

E. coli-produced MON 88701 DMO 

proteins 

 Electrophoretic mobility and apparent 

molecular weight of the MON 88701 DMO 

and the E. coli-produced MON 88701 DMO 

proteins were shown to be equivalent 

5. Glycosylation analysis of the 

MON 88701 DMO protein to assess 

equivalence between the MON 88701 

DMO and E. coli-produced 

MON 88701 DMO proteins 

 Glycosylation status of MON 88701 DMO 

and E. coli-produced MON 88701 DMO 

proteins were shown to be equivalent 

6. DMO enzymatic activity analysis to 

assess functional equivalence between 

MON 88701 DMO and the 

E. coli-produced MON 88701 DMO 

proteins 

 Functional activity of the MON 88701 DMO 

and the E. coli-produced MON 88701 DMO 

proteins were shown to be equivalent 

1

MALDI-TOF MS = Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry 

2

SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis 

C.2.6. N-Terminal Sequencing 

C.2.6.1. Methods 

N-terminal sequencing by automated Edman degradation chemistry was carried out in an 

attempt to confirm the identity of MON 88701 DMO. 
Monsanto Company 12-CT-244U 281 of 620 

MON 88701 DMO was separated by SDS-PAGE and transferred to PVDF membrane. 

The blot was stained using Coomassie Blue R-250. The major band at ~39 kDa 

containing MON 88701 DMO was excised from the blot and was used for N-terminal 

sequence analysis. The analysis was performed for 15 cycles using automated Edman 

degradation chemistry (Hunkapiller et al., 1983) using an Applied Biosystems 494 

Procise Sequencing System equipped with 140C Microgradient system a Perkin Elmer 

Series 200 UV/VIS Absorbance Detector with Procise Control Software (version 2.1) 

for amino acid detection after each cycle. Chromatographic data were collected using 

SequencePro (version 2.1) software. A control protein, -lactoglobulin, (Applied 

Biosystems, Foster City, CA) was analyzed before and after the sequence analysis of the 

MON 88701 DMO protein to verify that the sequencer met performance criteria for 

repetitive yield and sequence identity. Identity was established if ≥ 8 amino acids, 

consistent with the predicted sequence of the N-terminus of the MON 88701 DMO, were 

observed during analysis. 

C.2.6.2. Results of the N-terminal Sequence Analysis 

N-terminal sequencing reaction was performed on MON 88701 DMO protein. The 

reaction did not yield any observable sequence presumably because the N-terminus was 

blocked. Although this analysis did not yield N-terminal sequence data, the N-terminus 

of the MON 88701 DMO protein was determined using MALDI-TOF tryptic mass map 

analysis (see Section C.2.6). 

C.2.7. MALDI-TOF Tryptic Mass Map Analysis 

C.2.7.1. Methods 

MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the 

MON 88701 DMO protein. MON 88701 DMO protein (~15 μg) was chilled in a -20 °C 

freezer for at least 10 min. The chilled protein was precipitated with 200 μl of 

95% acetone in a -20 °C freezer overnight. Precipitated protein sample was pelleted in a 

refrigerated centrifuge for at least 45 min at more than 13,000  g. The supernatant was 

carefully removed and discarded. The protein pellet was washed twice with 200 μl of 

chilled ethanol to remove residual supernatant. The pellet was dried to completion using 

a Speed Vac concentrator and resuspended in 30 μl of 40% 2,2,2,-trifluoroethanol (TFE) 

in 25 mM ammonium bicarbonate. The resuspended protein was vortexed vigorously 

and then sonicated for 5 min in a water bath. The sample was incubated at ~37 °C for 1 h 

to denature the proteins. Denatured protein sample was reduced with ~5 mM 

tris(2-carboxyethyl)phosphine (TCEP) for 1 h at ~37 °C. Reduced protein sample was 

then alkylated in the dark for 30 min at room temperature with ~10 mM iodoacetic acid. 

Additional TCEP was added to ~5 mM and the sample was incubated for 10 min at room 

temperature. The reduced and denatured test substance was mixed with 67 μl of 25 mM 

ammonium bicarbonate and 2.5 μl of trypsin solution (0.2 μg/μl in 25 mM ammonium 

bicarbonate). The tryptic digestion was allowed to proceed for 15 h at 37 °C followed by 

quenching with 1 μl of formic acid. Proteolytic peptides were dried to completion using 

Speed Vac concentrator. To solubilize the dried peptides, a solution of 50% acetonitrile, 

0.1% TFA was added and sonicated for 5 min. Aliquots from the digest were spotted to 
Monsanto Company 12-CT-244U 282 of 620 

three wells on an analysis plate. For each spot, 0.75 μl of 2, 5 dihydroxybenzoic acid 

(DHB), α-cyano-4-hydroxycinnamic acid (α-Cyano), or 

3, 5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Thermo Fisher Scientific Inc.) 

was added to one of the spots. The sample in DHB matrix was analyzed in the 300 to 

5000 Da range. Samples in α-Cyano and Sinapinic acid were analyzed in the 500 to 

5000 Da and 500 to 7000 Da range, respectively. The analysis was performed using a 

VoyagerTM DE Pro BiospectrometryTM workstation (Applied Biosystems) using Voyager 

Instrument Control Panel software (version 5.10.2) and Data Explorer data analysis 

software (version 4.0.0.0). Protonated peptide masses were monoisotopically resolved in 

reflector mode (Aebersold, 1993; Billeci and Stults, 1993). CalMix 2 was used as the 

external calibrant (Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster 

City, CA) for the analysis. GPMAW32 software (Lighthouse Data, Odense M, Denmark) 

was used to generate a theoretical trypsin digest of the MON 88701 DMO protein 

sequence. Masses within 1 Da of a monosiotopic mass were matched against the 

theoretical digest of the MON 88701 DMO sequence. All matching masses were tallied 

and a coverage map was generated for the mass fingerprint. The tryptic mass fingerprint 

coverage was considered acceptable if  40% of the protein sequence was identified by 

matching experimental masses observed for the tryptic peptide fragments to the expected 

masses for the fragments (Biron et al., 2006, Krause et al., 1999). 

C.2.7.2. Results of MALDI-TOF Tryptic Mass Map Analysis 

The identity of the MON 88701 DMO protein was confirmed by MALDI-TOF MS 

analysis of peptide fragments produced from tryptic digestion of the MON 88701 DMO 

protein. The ability to identify a protein using this method is dependent upon matching a 

sufficient number of observed tryptic peptide fragment masses with predicted tryptic 

peptide fragment masses. In general, protein identification made by peptide mapping is 

considered to be reliable if  40% of the protein sequence was identified by matching 

experimental masses observed for the tryptic peptide fragments to the expected masses 

for the fragments (Biron et al., 2006, Krause et al., 1999). 

There were 19 unique peptides identified that corresponded to the masses expected to be 

produced by tryptic digestion of the MON 88701 DMO protein (Table C-2). The 

identified masses were used to assemble a coverage map of the entire MON 88701 DMO 

protein (Figure C-2). The experimentally determined mass coverage of the MON 88701 

DMO protein was 66.5% (232 out of 349 amino acids). This analysis serves as identity 

confirmation for the MON 88701 DMO protein. 

To identify the N-terminus, the experimentally determined masses of the peptides 

produced from tryptic digestion of the MON 88701 DMO protein were examined for the 

presence of a mass that matched the theoretical mass expected from the MON 88701 

DMO protein deduced from the dmo gene present in MON 88701. A mass was identified 

that corresponded to the predicted mass of an acetylated peptide with nine amino acids 

from CTP2 followed by the MON 88701 DMO protein deduced from the dmo gene 

present in MON 88701. The additional nine amino acids of CTP2 resulted from the 

alternative processing of CTP2. Alternative processing of DMO precursor proteins has 

been observed in other dicamba-tolerant plants containing the dmo gene (Behrens et al., 
Monsanto Company 12-CT-244U 283 of 620 

2007). Hence, the MON 88701 DMO protein was designated to have an N-terminal end 

as shown in Figure C-2. 
Monsanto Company 12-CT-244U 

Table C-2. Summary of the Tryptic Masses1 Identified for the MON 88701 DMO Protein Using MALDI-T

-cyano DHB Sinapinic acid Expected Mass Diff.2 Fragment3 Sequence 

720.40 720.37 0.03 140-145 VDPAYR 833.51 833.45 833.45 0.06 108-114 SFPVVER 856.49 856.43 0.06 251-257 EQSIHSR 914.60 914.53 0.07 305-312 VVVEAIER 

 1030.58 1030.57 0.01 293-301 SWQAQALVK 

1108.61 1108.59 1108.50 0.11 176-185 ANAQTDAFDR 1275.87 1275.83 1275.73 0.14 35-45 TILDTPLALYR 1286.83 1286.70 0.13 302-312 EDKVVVEAIER 1428.84 1428.83 1428.69 0.15 218-230 GANTPVDAWNDIR 

 1470.74 1470.63 0.11 146-158 TVGGYGHVDCNYK  1501.91 1501.79 0.12 189-202 EVIVGDGEIQAALMK  1506.86 1506.73 0.13 176-188 ANAQTDAFDRLER  1577.89 1577.80 1577.73 0.16 279-292 NFGIDDPEMDGVLR  1731.92 1731.80 0.12 1-15 VMSSVSTACMLTFVR +42 Da (N-a

 1745.09 1744.99 1744.93 0.16 234-250 VSAMLNFIAVAPEGTPK 

 1994.30 1994.23 1994.03 0.27 159-175 LLVDNLMDLGHAQYVHR  2143.35 2143.12 0.23 16-34 NAWYVAALPEELSEKPLG 2398.37 2398.35 2398.09 0.28 258-278 GTHILTPETEASCHYFFGSS 2724.72 2724.31 0.41 115-139 DALIWIWPGDPALADPGAIPG

1Only experimental masses that matched expected masses are listed in the table. 2The difference between the expected mass and the first column mass. Other masses shown within a row are also within 1 Da of the ex3Position refers to amino acid residues within the predicted MON 88701 DMO sequence as depicted in Figure C-2. 

DHB = 5-dihydroxybenzoic acid matrix, 

α-cyano = 

α-cyano-4-hydroxycinnamic acid matrix, Sinapinic acid = 3, 5-dimethoxy-4-hydrox
Monsanto Company 12-CT-244U 285 of 620 

Figure C-2. MALDI-TOF MS Coverage Map of the MON 88701 DMO Protein 

The amino acid sequence of the MON 88701 DMO protein was deduced from the dmo

gene present in MON 88701. Boxed regions correspond to regions covered by tryptic 

peptides that were identified from the MON 88701 DMO protein sample using 

MALDI-TOF MS. Underlined region corresponds to the nine amino acids from CTP2 

retained at the N-terminus of the MON 88701 DMO. In total, 66.5% (232 of 349 total 

amino acids) of the expected protein sequence was covered by the identified peptides. 

 
Monsanto Company 12-CT-244U 286 of 620 

C.2.8. Western Blot Analysis-Immunoreactivity 

C.2.8.1. Methods 

Western blot analysis was performed to confirm the identity of the MON 88701 DMO 

protein purified from cottonseed of MON 88701 and to compare the immunoreactivity of 

the MON 88701 DMO and E. coli-produced MON 88701 DMO proteins. 

The MON 88701 DMO and E. coli-produced MON 88701 DMO proteins were analyzed 

concurrently on the same gel using three loadings of 0.5, 2, and 6 ng. Loadings of the 

three concentrations were made in duplicate on the gel. Aliquots of each protein were 

diluted in water and 5X Laemmli buffer (LB) containing 312 mM Tris-HCl, 25% (v/v) 

2-mercaptoethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue, 50% (v/v) 

glycerol, pH 6.8, heated at 101 °C for 3 min, and applied to a 15 well pre-cast 

Tris-glycine 4-20% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA). Pre-stained 

molecular weight markers (Precision Plus Protein Standards Dual color; Bio-Rad, 

Hercules, CA) were loaded in parallel to verify electrotransfer of the proteins to the 

membrane and to estimate the size of the immunoreactive bands observed. 

Electrophoresis was performed at a constant voltage of 150 V for 90 min. Electrotransfer 

to a 0.45 μm nitrocellulose membrane (Invitrogen, Carlsbad, CA) was performed for 

105 min at a constant voltage of 25 V. After electrotransfer, the membrane was stored 

overnight with 1 phosphate buffered saline containing 0.05% (v/v) Tween-20 (PBST) at 

4 °C. The membrane was blocked for 1 h with 5% (w/v) NFDM in PBST at room 

temperature. The membrane was then probed with a 1:5000 dilution of goat anti-DMO 

antibody (lot 11223358) in 2% NFDM in PBST for 1 h at room temperature. Excess 

antibody was removed using two 1 min washes followed by three 5 min washes with 

PBST. Finally, the membrane was probed with horseradish peroxidase (HRP)-

conjugated horse anti-goat IgG (Thermo, Rockford, IL) at a dilution of 1:10,000 in 

2% NFDM in PBST for 1 h at room temperature. Excess HRP-conjugate was removed 

using two 1 min washes and three 5 min washes with PBST. All washes were performed 

at room temperature. Immunoreactive bands were visualized using the ECL detection 

system (GE, Healthcare, Piscataway, NJ) with exposure to Amersham Hyperfilm ECL 

(GE, Healthcare, Piscataway, NJ). The film was developed using a Konica SRX-101A 

automated film processor (Konica Minolta Medical & Graphic, Inc., Tokyo, Japan). 

Quantification of the bands on the blot was performed using a Bio-Rad GS-800 

densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA) 

using the band location and volume tool. The signal intensities of the immunoreactive 

bands observed for the MON 88701 DMO and E. coli-produced MON 88701 DMO 

proteins migrating at the expected position on the blot film were quantified as “adjusted 

volume” values. The raw data was exported to a Microsoft Excel (2007) file. The 

immunoreactivity of the MON 88701 DMO and E. coli-produced MON 88701 DMO 

proteins were reported as the mean signal intensity at each amount of protein analyzed. 

The immunoreactivity of the MON 88701 DMO and E. coli-produced MON 88701 DMO 

proteins were considered equivalent if the overall mean of the immunoreactive signal of 

the MON 88701 DMO protein was within ± 35% of the overall mean of immunoreactive 

signal of the E. coli-produced MON 88701 DMO protein. 
Monsanto Company 12-CT-244U 287 of 620 

C.2.8.2. Results of MON 88701 DMO Protein Immunoreactivity Equivalence 

Western blot analysis was conducted using goat anti-DMO polyclonal antibodies to 

1) assess the identity of the MON 88701 DMO protein isolated from the cottonseed of 

MON 88701 and 2) determine the relative immunoreactivity of the MON 88701 DMO 

and the E. coli-produced MON 88701 DMO proteins. The results demonstrated that the 

anti-DMO antibodies recognized the MON 88701 DMO protein that migrated to the same 

position on the blot as the E. coli-produced MON 88701 DMO protein (Figure C-3). 

Furthermore, the immunoreactive signal increased with increasing amounts of 

MON 88701 DMO protein loaded. Two other bands, one migrating at ~75 kDa and the 

other at ~17 kDa were also observed. These bands were prominent in lanes with higher 

load amounts (Figure C-3, Lanes 3-6), and may represent products of aggregation and 

degradation of DMO, respectively. 

Densitometric analysis was conducted to compare the immunoreactivity of MON 88701 

DMO and E. coli-produced MON 88701 DMO proteins. The mean signal intensity 

(OD  mm

2

) from the MON 88701 DMO bands and from the E. coli-produced 

MON 88701 DMO bands at each amount of protein analyzed was calculated and then 

overall mean signal intensity was calculated (Table C-3). The immunoreactivity was 

considered equivalent if the overall mean signal intensity of all MON 88701 DMO 

protein bands was within ±35% of the overall mean signal intensity of E. coli-produced 

MON 88701 DMO protein bands across all loading levels. 

The overall mean signal intensity of the E. coli-produced MON 88701 DMO bands was 

6.500 OD × mm2

, and the overall mean signal intensity of the MON 88701 DMO bands 

was 4.440 OD × mm2

. Because overall mean signal intensity of the MON 88701 DMO 

protein bands was between 4.225 and 8.775 (between -35% and +35% of the 

E. coli-produced MON 88701 DMO bands), the MON 88701 DMO and E. coli-produced 

MON 88701 DMO proteins were determined to have equivalent immunoreactivity. 
Monsanto Company 12-CT-244U 288 of 620 

Figure C-3. Western Blot Analysis of MON 88701 DMO and E. coli-produced 

MON 88701 DMO Proteins 

Aliquots of the MON 88701 DMO protein and the E. coli-produced MON 88701 DMO 

protein were subjected to SDS-PAGE and electrotransferred to a nitrocellulose 

membrane. The membrane was incubated with anti-DMO antibodies and 

immunoreactive bands were visualized using an ECL system (GE Healthcare, 

Piscataway, NJ). Approximate molecular weights (kDa) are shown on the left. Lanes 

loaded with molecular weight markers were cropped, and lanes were renumbered relative 

to the original gel loading. The 6 min exposure is shown. Lane designations are as 

follows: 

Lane Sample Amount (ng) 

1 E. coli-produced MON 88701 DMO protein 0.5 

2 E. coli-produced MON 88701 DMO protein 0.5 

3 E. coli-produced MON 88701 DMO protein 2 

4 E. coli-produced MON 88701 DMO protein 2 

5 E. coli-produced MON 88701 DMO protein 6 

6 E. coli-produced MON 88701 DMO protein 6 

7 MON 88701 DMO protein 0.5 

8 MON 88701 DMO protein 0.5 

9 MON 88701 DMO protein 2 

10 MON 88701 DMO protein 2 

11 MON 88701 DMO protein 6 

12 MON 88701 DMO protein 6 

 
Monsanto Company 12-CT-244U 289 of 620 

Table C-3. Comparison of Immunoreactive Signals Between MON 88701 DMO and 

E. coli-produced MON 88701 DMO Proteins 

Mean Signal intensity from 

MON 88701 DMO

(OD × mm2) 

Mean Signal intensity from 

E. coli-produced 

MON 88701 DMO

(OD × mm2) 

Preset Acceptance limits for 

MON 88701 DMO1 

(OD × mm2) 

4.440 6.500 4.225 – 8.775 

1

The acceptance limits for MON 88701 DMO are based on the interval between +35% (6.500 × 1.35) and 

-35% (6.500 × 0.65) of the overall mean of the E. coli-produced MON 88701 DMO signal intensity across 

six loads. 

 
Monsanto Company 12-CT-244U 290 of 620 

C.2.9. Molecular Weight and Purity Estimation using SDS-PAGE 

C.2.9.1. Methods 

MON 88701 DMO and E. coli-produced MON 88701 DMO proteins were mixed with 

5X LB and diluted with water to a final total protein concentration of 0.1 μg/μl. 

Molecular Weight Standards, Bio-Rad broad range (Hercules, CA) were diluted to a final 

total protein concentration of 0.9 g/l. The MON 88701 DMO was analyzed in 

duplicate at 0.5, 1, and 1.5 μg protein per lane. The E. coli-produced MON 88701 DMO 

reference standard was analyzed at 0.5 μg total protein in a single lane. The samples 

were loaded onto a 10-well pre-cast Tris glycine 4-20% (w/v) polyacrylamide gradient 

mini-gel (Invitrogen, Carlsbad, CA) and electrophoresis was performed at a constant 

voltage of 125 V for 90 min. Proteins were fixed by placing the gel in a solution of 

40% (v/v) methanol and 7% (v/v) acetic acid for 25 min, stained for ~16 h with Brilliant 

Blue G-Colloidal stain (Sigma-Aldrich, St. Louis, MO). Gels were destained once for 

30 sec with a solution containing 10% (v/v) acetic acid and 25% (v/v) methanol, and with 

25% (v/v) methanol for a total of 6 h. Analysis of the gel was performed using a Bio-Rad 

GS-800 densitometer with the supplied Quantity One software (version 4.4.0, Hercules, 

CA). The apparent MW of each observed band was estimated from a standard curve 

generated by the Quantity One software which was based on the MWs of the markers and 

their migration distance on the gel. To determine purity, all visible bands within each 

lane were quantified using Quantity One software. Apparent MW and purity were 

reported as an average of all six lanes containing the MON 88701 DMO. 

C.2.9.2. Results of MON 88701 DMO Protein Molecular Weight Equivalence 

The molecular weight and purity of the MON 88701 DMO protein were determined to be 

39.5 kDa and 97%, respectively. To assess molecular weight (MW) and purity, the 

MON 88701 DMO protein was subjected to SDS-PAGE. The gel was stained with 

Brilliant Blue G Colloidal stain and analyzed by densitometry (Figure C-4). 

E. coli-produced MON 88701 DMO protein was loaded in a single lane for reference 

(Figure C-4, Lane 2). The MON 88701 DMO protein (Figure C-4, Lanes 3-8) had an 

apparent MW of 39.5 kDa (Table C-4). The apparent MW of the E. coli-produced MON 

88701 DMO protein as reported on its Certificate of Analysis was 38.7 kDa (Table C-4). 

Because the apparent MW of MON 88701 DMO protein was within the preset 

acceptance limits for equivalence (Table C-4), the MON 88701 DMO and 

E. coli-produced MON 88701 DMO proteins were determined to have equivalent 

apparent MWs. 

The purity of the MON 88701 DMO protein was calculated based on the six loads on the 

gel (Figure C-4, Lanes 3 to 8). The average purity was determined to be 97%. 
Monsanto Company 12-CT-244U 291 of 620 

Table C-4. Molecular Weight Comparison Between the MON 88701 DMO and 

E. coli-produced MON 88701 DMO Proteins Based on SDS-PAGE 

Apparent MW of MON 88701 

DMO Protein1

(kDa) 

Apparent MW of 

E. coli-Produced MON 88701 

DMO Protein2 

(kDa) 

Preset Acceptance 

Limits for 

MON 88701 DMO3

(kDa) 

39.5 38.7 38.5-39.7 

1

The reported value is the mean molecular weight across all six loads. 2

The molecular weight of the E. coli-produced MON 88701 DMO protein as reported on its Certificate of 

Analysis. 

3

See Section C.6. 

 
Monsanto Company 12-CT-244U 292 of 620 

Figure C-4. Molecular Weight and Purity Analysis of the MON 88701 DMO 

Protein 

Aliquots of the MON 88701 DMO and the E. coli-produced MON 88701 DMO proteins 

were separated by SDS-PAGE and then stained with Brilliant Blue G-Colloidal stain. 

Approximate molecular weights are shown on the left and correspond to the markers 

loaded in Lanes 1 and 9. Empty lane was partially cropped. Lane designations are as 

follows: 

Lane Sample Amount (μg) 

1 Broad Range Molecular Weight Markers 4.5 

2 E. coli-produced MON 88701 DMO protein 0.5 

3 MON 88701 DMO protein 0.5 

4 MON 88701 DMO protein 0.5 

5 MON 88701 DMO protein 1 

6 MON 88701 DMO protein 1 

7 MON 88701 DMO protein 1.5 

8 MON 88701 DMO protein 1.5 

9 Broad Range Molecular Weight markers 4.5 
Monsanto Company 12-CT-244U 293 of 620 

C.2.10. Glycosylation Analysis 

C.2.10.1. Methods 

Glycosylation analysis was used to determine whether the MON 88701 DMO was posttranslationally modified with covalently bound carbohydrate moieties. Aliquots of the 

MON 88701 DMO protein, the E. coli-produced MON 88701 DMO (negative control) 

and the positive control, transferrin (Sigma-Aldrich, St Louis, MO), were each diluted 

with water and brought to 1X LB. These samples were heated at ~101 °C for 3 min. The 

MON 88701 DMO, the E. coli- produced MON 88701 DMO and transferrin were loaded 

at approximately 50 and 100 ng per lane on a Tris-glycine 10 well 4-20% polyacrylamide 

gradient mini-gel (Invitrogen, Carlsbad, CA). Precision Plus Protein Dual color 

Standards (Bio-Rad, Hercules, CA) were also loaded to verify electrotransfer of the 

proteins to the membrane and as markers for molecular weight. Electrophoresis was 

performed at a constant voltage of 150 V for 90 min. Electrotransfer to a 0.45 μm PVDF 

membrane (Invitrogen, Carlsbad, CA) was performed for 90 min at a constant voltage of 

25 V. 

Carbohydrate detection was performed directly on the PVDF membrane at room 

temperature using the Amersham ECL glycoprotein Detection Module (GE, Healthcare, 

Piscataway, NJ). With this module, carbohydrate moieties of proteins were oxidized with 

sodium metaperiodate and then biotinylated with biotin-X-hydrazide. The biotinylated 

proteins can be detected on the blot by addition of streptavidin conjugated to HRP for 

luminol-based detection using ECL reagents (GE, Healthcare, Piscataway, NJ) and with 

subsequent exposure to Amersham Hyperfilm (GE, Healthcare). The film was developed 

using a Konica SRX-101A automated film processor (Konica Minolta Medical & 

Graphic, Inc., Tokyo, Japan). 

An identical blot run in parallel to that used for the glycosylation analysis was stained to 

visualize the proteins present on the membrane. Proteins were stained for 30 sec to 2 min 

using Coomassie Brilliant Blue R-250 staining solution (Bio-Rad, Hercules, CA) and 

then destained with 1X Coomassie Brilliant Blue R-250 destaining solution (Bio-Rad) for 

5 min. After washing with water, the blot was dried and scanned using Bio-Rad GS-800 

densitometer with the supplied Quantity One software (version 4.4.0). 

C.2.10.2. Results of Glycosylation Analysis 

Some eukaryotic proteins are post-translationally modified by the addition of 

carbohydrate moieties (Rademacher et al., 1988). To test whether DMO protein was 

glycosylated when expressed in the cottonseed of MON 88701, the MON 88701 DMO 

protein was analyzed using an ECL Glycoprotein Detection Module (GE, Healthcare, 

Piscataway, NJ). Transferrin, a glycosylated protein, was used as a positive control in the 

assay. To assess equivalence of the MON 88701 DMO and E. coli-produced 

MON 88701 DMO proteins, the E. coli-produced MON 88701 DMO protein was also 

analyzed. The positive control was clearly detected at expected molecular weight 

(~80 kDa) and the band intensity increased with increasing concentration (Figure C-5, 

Panel A, Lanes 1-2). In contrast, signals were not observed in the lanes containing the 
Monsanto Company 12-CT-244U 294 of 620 

MON 88701 DMO or E. coli-produced MON 88701 DMO proteins at the expected 

molecular weight for the MON 88701 DMO protein (Figure C-5 Panel A, Lanes 7-8 and 

Lanes 4-5, respectively). To assess that sufficient MON 88701 DMO and 

E. coli-produced MON 88701 DMO proteins were present for glycosylation analysis, a 

second membrane (with identical loadings and transfer times) was stained with 

Coomassie Blue R250 for protein detection (Figure C-5 Panel B). Both the MON 88701 

DMO and E. coli-produced MON 88701 DMO proteins were clearly detected (Figure C5 Panel B, Lanes 7-8 and Lanes 4-5, respectively). These data indicate that the 

glycosylation status of MON 88701 DMO and E. coli-produced MON 88701 DMO 

proteins are equivalent and that neither is glycosylated. 
Monsanto Company 12-CT-244U 295 of 620 

Figure C-5. Glycosylation Analysis of the MON 88701 DMO Protein 

Aliquots of the transferrin (positive control), E. coli-produced MON 88701 DMO protein 

and MON 88701 DMO protein were subjected to SDS-PAGE and electrotransferred to 

PVDF membranes. Panel A corresponds to detection of the labeled carbohydrate 

moieties, where present, using the ECL-based system with exposure to Hyperfilm. A 

6 min exposure is shown. Panel B corresponds to Coomassie Brilliant Blue R250 

staining of an equivalent blot to confirm the presence of proteins. The signal was 

captured using a Bio-Rad GS-800 with Quantity One software (version 4.4.0). 

Approximate molecular weights (kDa) correspond to the Precision Plus, dual color 

markers (used to verify transfer and MW). Lanes loaded with molecular weight markers 

were partially cropped, and lanes were renumbered relative to the original gel loading. 

Arrows indicate the expected migration MON 88701 DMO protein. Lane designations 

are as follows: 

Lane Sample Amount (ng) 

1 Transferrin (positive control) 50 

2 Transferrin (positive control) 100 

3 Empty - 

4 E. coli-produced MON 88701 DMO (negative control) 50 

5 E. coli-produced MON 88701 DMO (negative control) 100 

6 Empty - 

7 MON 88701 DMO 50 

8 MON 88701 DMO 100 
Monsanto Company 12-CT-244U 296 of 620 

C.2.11. Functional Activity Analysis 

C.2.11.1. Methods 

The specific activity of MON 88701 DMO and E. coli-produced MON 88701 DMO was 

determined by quantifying the conversion of 3,6-dichloro-2-methoxybenzoic acid 

(dicamba) to 3,6-dichlorosalicylic acid (DCSA) via HPLC (Agilent Technologies 1100 

series, Santa Clara, CA) separation and fluorescence detection (Agilent Technologies 

1200 series, G1321A). Each assay reaction contained 25 mM potassium phosphate, 

pH 7.2, 3.4 μg ferredoxin, 3.4 μg reductase, 0.5 mM FeSO4, 10 mM MgCl2, 0.7 mM 

NADH, 0.3 mM dicamba, 2 μl (42.48 U/ml) of formaldehyde dehydrogenase and either 

2.9 μg MON 88701 DMO or 3 μg his-DMO as an assay positive control. The reactions 

were performed in PCR tubes (Sorenson, Salt Lake City, UT) and incubated at 30 °C for 

15 min. Reactions (200 μl) were initiated by the addition of dicamba and quenched with 

the addition of 50 μl of 5% H2SO4. Reactions were then filtered using Whatman 

Anotop 10 filters (0.2 μm, GE healthcare), and 40 μl was transferred to a HPLC sample 

vial (200 μl, Agilent) for analysis. Twenty-five microliters of the filtered reaction was 

injected onto a Phenomenex® Synergi 4 μm C18/ODS Hydro-RP column (150 × 4.6 mm 

ID, Torrance, CA). The mobile phase consisted of solvent A (21.5 mM phosphoric acid) 

and solvent B (100% acetonitrile) running at 1.5 ml/min. DCSA was eluted from the 

column using a linear gradient from 90% to 40% solvent A for the first 14 min, followed 

by a step to 10% solvent A for 1 min and then re-equilibration at 90% solvent A for 

10 min before the next injection. DCSA was monitored by the detection of fluorescent 

emission at 424 nm (excitation 306 nm) and quantified relative to a standard curve of 

DCSA generated using 0.1, 0.3, 0.6, 0.9, 1.2, 2.4, and 4.8 nmol/250 μl. Chromatographic 

data were collected using AtlasTM 2003 software (Thermo Fisher Scientific Inc). The 

specific activity was calculated based on the amount of purity corrected MON 88701 

DMO protein added to the reaction mixture and expressed as nmol of DCSA produced 

per minute per mg of MON 88701 DMO protein (nmol  min-1 × mg-1). 

C.2.11.2. Results of Functional Activity 

The functional activities of the MON 88701 DMO and E. coli-produced MON 88701 

DMO proteins were determined by quantifying the conversion of dicamba to DCSA 

using HPLC separation and fluorescence detection. In this assay, protein-specific activity 

is expressed as nmol DCSA × minute-1 × mg-1 of DMO. 

The experimentally-determined specific activities for the MON 88701 DMO and 

E. coli-produced MON 88701 DMO proteins are presented in Table C-5. The specific 

activities of MON 88701 DMO and E. coli-produced MON 88701 DMO proteins were 

5.48 and 7.23 nmol DCSA × minute-1 × mg-1 of DMO, respectively. Because the mean 

specific activities of the MON 88701-produced and E. coli-produced MON 88701 DMO 

proteins fall within the preset acceptance criterion (Table C-5), the MON 88701 DMO 

and E. coli-produced MON 88701 DMO proteins were determined to have equivalent 

functional activity. 
Monsanto Company 12-CT-244U 297 of 620 

Table C-5. MON 88701 DMO Functional Activity Assay 

MON 88701 DMO1 

(nmol DCSA × minute-1 × mg-1) 

E. coli-produced 

MON 88701 DMO1 

(nmol DCSA × minute-1 × mg-1) 

Preset Acceptance Limits 

for MON 88701 DMO2 

(nmol DCSA × minute-1 × mg-1) 

5.48 ± 1.3 7.23 ± 2.1 1.69-20.74 

1

Value refers to mean and standard deviation calculated based on n = 5. 

2

See Section C.6.

C.3. Substrate Specificity of MON 88701 DMO Protein 

C.3.1. Exogenous Specificity Herbicide Tolerance - Greenhouse Analysis 

C.3.1.1. Materials 

MON 88701 (lots IG2000000439645080059904 and IG2000000371459002138624) and 

the near isogenic conventional control, Coker 130, (lots IG2000000025726392598528, 

IG2000000025726407540736, and IG2000000025726372937728) were grown in 

greenhouses during 2010 and 2011. At the 2-5 leaf growth stage or pre-emergent, 

MON 88701 and the near isogenic conventional control, Coker 130, were sprayed with 

different herbicides. The herbicides tested are listed in Table C-6. 

Table C-6. Herbicides Tested in Exogenous Specificity Herbicide Tolerance 

Greenhouse Trials 

 

 

 Harness and Roundup WeatherMax are registered trademarks of Monsanto Technology LLC. All other 

trademarks are the property of their respective owners. 

Manufacturer/ 

Retailer 

Herbicide Herbicide 

Formulation Lot Number 

 

Albaugh 2,4-DB Butyrac 200 HPR-0404-14987-F 

BASF dicamba Clarity® KIH-0702-18134-F 

Dow atrazine Atrazine AGT-0804-19336-F 

Dow trifluralin Treflan® MB231656T7 

Dow oxyfluorfen Goal® 2XL EWP-0107-11628-F 

Helena 2,4-D 2,4-D Amine 4 RUD-0502-15805-F 

Monsanto acetochlor Harness® MUS-0704-18520-F 

Monsanto halosulfuron Permit® MUS-0405-15154-F

Monsanto glyphosate Roundup WeatherMax® MUS-0905-19887-F 

Syngenta paraquat Gramoxone® GTA-0606-17421 

 
Monsanto Company 12-CT-244U 298 of 620 

C.3.1.2. Exogenous Specificity Herbicide Tolerance Greenhouse Method 

MON 88701 and the near isogenic conventional control, Coker 130, were planted in pots 

containing Redi-earth® and Osmocote® 14-14-14 slow release fertilizer or Peters® 20-

20-20 fertilizer. There were 10 replicate pots and one plant per replicate of MON 88701 

and the conventional control for each herbicide and rate tested. The pots were randomly 

placed in a greenhouse and grown under normal agronomic conditions for cotton (relative 

humidity 10-70%, temperature 21-34 ̊C, 14 hour photoperiod, and watering as needed). 

At pre-emergence or when the plants were at the 2-5 leaf growth stage, the replicates 

were treated with a single herbicide and rate (Table 1). Two different application rates of 

each herbicide were applied to different replicate sets (Table 2). Based on the U.S. 

herbicide labeled rates, the rates for the experiments were chosen and then adjusted for 

use on cotton and for the optimal growing conditions in the greenhouse in order to 

achieve approximately 40 to 80% injury. Twenty to 22 days after application, all plants 

were rated for percent injury. Ratings were based on visual assessment of chlorosis, 

necrosis, malformation, stunting, and biomass reduction with 0 being no visible injury 

and 100 being completely dead. All 10 replicate ratings were averaged. 

C.3.1.3. Results of Herbicide Tolerance Greenhouse Trials 

MON 88701 demonstrated reduced injury ratings for dicamba, but similar injury ratings, 

and therefore similar levels of susceptibility as the near isogenic conventional control, 

Coker 130, for the remaining 9 herbicides tested (Table C-7). 

 
Monsanto Company 12-CT-244U 

Table C-7. Herbicide Tolerance Trials Injury Ratings 

Injury Observations 

(days after 

application) 

Inju

Formulation Manufacturer Herbicide1

Labeled Rate Range 

(g/ha)2

Rates Applied 

(g/ha)2

Control4

Average (Rang

Clarity® BASF dicamba 140-2242 (a.e.) 

561 (a.e.) 

1120 (a.e.) 22 

87 (80-90) 92 (85-95)

2,4-D Amine 4 Helena 2,4-D 140-2242 (a.e.) 

280 (a.e.) 561 (a.e.) 22 

86 (80-90) 96 (90-99) 

Butyrac® 200 Albaugh 2,4-DB 130-1682 (a.e.) 

280 (a.e.) 561 (a.e.) 22 

79 (70-85) 96 (90-99) 

Gramoxone® Syngenta paraquat 280-1120 (a.e.) 

561 (a.e.) 841 (a.e.) 20 

88 (85-95) 95 (90-98) 

Harness® Monsanto acetochlor 930-4485 (a.i.) 

4485 (a.i.) 6732 (a.i.) 21 

70 (30-100) 84 (40-100) 

Atrazine Dow atrazine 1100-3800 (a.i.) 

1682 (a.i.) 3364 (a.i.) 21 

28 (20-40) 48 (20-95) 

Treflan® Dow trifluralin 560-2242 (a.i.) 

4485 (a.i.) 6732 (a.i.) 21 

2 (0-5) 

4 (0-10) 

Roundup WeatherMax® Monsanto glyphosate 280-4162 (a.e.) 

75 (a.e.) 

240 (a.e.) 21 

9 (5-20) 

46 (35-60) 

Goal® 2XL Dow oxyfluorfen 280-2242 (a.i.) 

561 (a.i.) 841 (a.i.) 21 

37 (25-25) 46 (30-80) 

Permit® Monsanto halosulfuron 36-140 (a.i.) 

75 (a.i.) 

200 (a.i.) 21 

48 (40-55) 59 (50-65) 

1Herbicides applied pre-emergent were acetochlor, atrazine, and trifluralin. All other herbicides were applied when the plants were at the 2-5 leaf grow2a.e. = acid equivalent; a.i. = active ingredient. Each herbicide contains the active ingredient directly or the salt form of the active ingredient. W

application, the salt form is calculated back to the acid that is the active ingredient and therefore called acid equivalent. Each labeled rate is for cereasince these herbicides are not labeled to be sprayed on cotton or are labeled for cotton only as a pre-plant treatment. Based on the labeled rates, the r

chosen and then adjusted for use in-crop on cotton and for the optimal growing conditions in the greenhouse. 3Injury ratings were determined by visual inspection of each plant. Ratings were based on visual assessment of chlorosis, necrosis, malformation, stu

0 percent = no visual adverse effects and 100 percent = completely dead. 4Control plants were near isogenic conventional cotton control Coker 130. Reported average and range of 10 replicate plants. 5Reported average and range of 10 replicate plants.
Monsanto Company 12-CT-244U 300 of 620 

C.3.2. In Vitro Endogenous Specificity Experiments 

C.3.2.1 Materials 

The DMO protein used in the endogenous specificity in vitro experiments was generated 

in Escherichia coli with a histidine-tag at the N-terminus and has an identical amino acid 

sequence to MON 88701 DMO with the exception of the lack of leucine at the second 

position and the nine amino acids from CTP2 (Figure C-1). The compounds tested and 

standards used in the in vitro experiments are listed in Table C-8. 

Table C-8. Compounds Used in Specificity In Vitro Experiments 

C.3.2.2. In Vitro Specificity Experiments Enzymatic Reaction Mixture Method 

The reaction of E. coli-produced DMO with different compounds evaluated as potential 

substrates was carried out using similar reaction conditions described in the 

characterization portion of this appendix (Appendix C.2.10.). The compounds 

(Table C-8) were combined with E. coli-produced DMO at 0.2 and/or 0.012 mM. The 

concentrations tested ensured adequate reaction conditions in terms of the substrate for 

the detection of product formation or disappearance of substrate. 

C.3.2.3. In Vitro Experiments Liquid Chromatography Separation Method 

The reaction mixture was separated by Ultra Performance Liquid Chromatography 

(UPLC) using an ACQUITY UPLC BEH C18 Column containing 1.7 μm Bridged Ethyl 

Hybrid (BEH) particles and an ACQUITY BEH C18 VanGuard Pre-column. The 

Manufacturer/ 

Retailer Compound 

Common 

Name 

Lot/Product 

Number 

 

Compounds Tested: 

Aldrich 2-methoxybenzoic acid o-anisic acid A0230443

Chem Service 3,6-dichloro-2-methoxybenzoic acid dicamba 341-9143

Sigma 2,4-dichlorophenoxyacetic acid 2,4-D D7299-100G

Sigma 3-(4-hydroxy-3,5-

dimethoxyphenyl)prop-2-enoic acid 

sinapic acid D7927-1G

Fluka 3,5-dimethoxy-4-hydroxybenzoic acid syringic acid 86230

Fluka 4-hydroxy-3-methoxybenzoic acid vanillic acid 94770

Fluka 3-(4-hydroxy-3-methoxy-phenyl)prop2-enoic acid ferulic acid 46278 

Compounds Used as Standards:

Monsanto 3,6-dichlorosalicylic acid DCSA GLP-0603-16959-T 

Riedel-de Haen 2,4-dichlorophenol 2,4-DCP 35811 

 
Monsanto Company 12-CT-244U 301 of 620 

column was heated to 40°C. The tested substrates and potential oxidative by-products 

were monitored by ACQUITY UPLC photodiode array (PDA) with wavelength range 

from 200nm to 320nm with 1.2nm resolution. The chromatography was performed at 

0.25ml/min and directed to the mass spectrometer following the separation. Both mobile 

phase A (water) and solvent B (acetonitrile) contained 0.1% v/v formic acid. Gradients 

used were substrate-specific: 

 The gradient for dicamba was run from 40 to 50% solvent B in 3min, 50 to 100% 

solvent B in 0.1 min and then kept at 100% solvent B for 1min before returning to 

40% solvent B in 0.1 min. 

 The gradient for 2,4-D was run from 40 to 45% solvent B in 6min, held at 45% 

solvent B for 1min, 45 to 100% solvent B in 0.1 min, and then held at 100% 

solvent B for 0.5 min before returning to 40% solvent B in 0.1 min. 

 The gradient for ferulic acid, o-anisic acid, sinapic acid, syringic acid, and vanillic 

acid were run from 0 to 100% solvent B in 4 min and then held at 100% solvent B 

for 1 min before returning to 0% solvent B in 0.1 min.

Five microliters injection of each sample was used for UPLC analysis where the 

disappearance of the potential substrate was monitored, and a 50 μl injection was used for 

UPLC analysis where formation of potential oxidative by-products was monitored. 

C.3.2.4. In Vitro Experiments Mass Spectrometry Detection Method 

Elution from the UPLC column (C.3.2.3) flowed directly to a Waters Micro Q-TOF mass 

spectrometer. The parameters used for the mass determination were: negative mode, 

capillary voltage of 2800 V, sample cone voltage of 26 V for all analytes with the 

exception of 2,4-D and 2,4-DCP, which was 10 V. The extraction cone was 1.5 V. The 

source temperature was 150 °C and the desolvation temperature was 390 °C. The 

desolvation gas flow was 500 L/hour. Scan time was 0.76 seconds and inter-scan delay 

was 0.1 seconds. The m/z range used was specific to each substrate and product. The 

m/z range for dicamba and DCSA was from 160 to 225 from 0 to 4 minutes. The m/z at 

175, which is the fragment ion of dicamba, was used as a detection method for dicamba. 

This fragment ion of dicamba gave better sensitivity than the parent ion. The m/z at 205 

or 207 was used to detect DCSA. The m/z range for 2,4-D and 2,4-DCP was from 160-

164 or 160-225, dependent on the specific experiment from 0 to 6 minutes. The m/z 

range for all other acids is from 120 to 230 within 4 minutes. 

C.3.2.5. Results of In Vitro Experiments with Endogenous Cotton Compounds 

The reaction of dicamba with E. coli-produced DMO has been well characterized 

utilizing an in vitro enzymatic assay that monitors the formation of DCSA by LC-MS, 

which allows for the detection of the product with high sensitivity. Both the substrate 

and reaction products can be detected by LC-UV and LC-MS after separation by UPLC 

(Figure C-6). 
Monsanto Company 12-CT-244U 302 of 620 

Compounds structurally similar to dicamba and found in cotton, soybean and corn were 

used as potential substrates to determine if these compounds could be metabolized by 

DMO (Table C-8). The compounds tested were syringic acid, o-anisic acid, vanillic acid, 

ferulic acid, and sinapic acid. Mass spectrometry scans were taken from 120 m/z to 250 

m/z to cover the range of all potential oxidation products formed by DMO. Standard 

reaction conditions of dicamba with a histidine tagged E. coli-produced DMO were used 

as a positive control. LC-MS data demonstrated that there are no additional peaks 

formed when reactions of each compound incubated with histidine tagged E. coliproduced DMO and without histidine tagged E. coli-produced DMO are compared 

(Figure C-7) (dicamba m/z 205, 2, 4-D m/z 163, ferulic acid m/z 175, o-anisic acid m/z 

137, sinapic acid m/z 209, syringic acid m/z 183, and vanillic acid m/z 153). There were 

no peaks observed at the respective masses for the predicted reaction products of each 

compound incubated with histidine tagged E. coli-produced DMO, indicating these 

compounds are not catabolized by DMO. 

To assess whether MON 88701 DMO protein has the same specificity as the histidine 

tagged DMO used in the in vitro experiments, the E. coli-produced MON 88701 DMO 

protein (i.e., lacking a histidine tag), shown to be equivalent to the plant produced 

MON 88701 DMO protein (Section V.B), was incubated with o-anisic acid, the 

endogenous compound that has the greatest structural similarity to dicamba. Again 

dicamba was used as a positive control to demonstrate the assay system was functional 

(Figure C-8). This analysis demonstrated that o-anisic acid was not metabolized by the 

E. coli-produced MON 88701 DMO protein (i.e., lacking a histidine tag), but dicamba 

was (Figures C-8 and C-9). These results indicate that DMO, including the MON 88701 

DMO protein, is specific for dicamba as a substrate. 

 
Monsanto Company 12-CT-244U 303 of 620 

Figure C-6. UPLC Separation of Dicamba (DCB) and DCSA in Five Substrate 

Analysis 

Dicamba and DCSA were separated by UPLC and detected by UV absorbance using a Photo 

Diode Array (PDA) and mass spectrometry (MS). 
Monsanto Company 12-CT-244U 

Figure C-7. E. coli-produced DMO Conversion of Endogenous Substrates Endogenous substrates, as well as dicamba and 2,4-D, were incubated with E. coli-produced DMO and the formation of products and 

was monitored by LC-MS (top two chromatograms) and LC-UV (bottom two chromatograms) for a positive control (dicamba 

endogenous compound:, sinapic acid(c), ferulic acid (d), anisic acid (e), syringic acid (f), and vanillic acid (g). For each experimen

made with (+E. coli-produced DMO, upper) and without (-E. coli-produced DMO, lower). The red line indicates the migration of th

the case of dicamba) in each chromatogram. 

8,107,108 G4 Vanillic

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

Dicamba-02-12-09-1126 1: TOF MS E

3.1

2.48 2.77 3.28 3.44 3.57 3.73 3.17 2.96

Dicamba-02-12-09-1130 1: TOF MS E

3.6

2.48 3.61 3.78 2.77 3.49

2.99

T

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

2.0e+2

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

2.0e+2

Dicamba-02-12-09-1126 3: Diode A

2.68 Range: 2.49

Dicamba-02-12-09-1130 3: Diode A

2.68 Range: 2.58

2.38

+DMO+DMO-DMO-DMO

8,107,108 G5 Syringic

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

Dicamba-02-12-09-1175 1: TOF MS ES183

1.24e3

2.76

2.51

2.68 3.03 3.47

Dicamba-02-12-09-1179 1: TOF MS ES183

1.05e3

2.51 2.77

2.30 2.68 3.01 3.52

Time

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

2.0e+2

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

2.0e+2

Dicamba-02-12-09-1175 3: Diode Array 

2.67 Range: 2.417e+2

2.78

Dicamba-02-12-09-1179 3: Diode Array 

2.67 Range: 2.419e+2

2.42 2.78

+DMO+DMO-DMO-DMO

8,107,108 G8 Anistic

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

Dicamba-02-12-09-1148 1: TOF MS ES137

1.54e3

3.45

3.19

2.78

3.33 3.71

Dicamba-02-12-09-1152 1: TOF MS ES137

2.12e3

3.45

3.19

2.65

3.32

Time

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

1.0e+22.0e+2

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

1.0e+2

Dicamba-02-12-09-1148 3: Diode Array 

3.09 Range: 1.661e+2

2.44 2.94

Dicamba-02-12-09-1152 3: Diode Array 

3.09 Range: 2.097e+2

2.44

+DMO

+DMO

-DMO

-DMO

8,107,108 G7 Ferrulic 

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

2.50 2.75 3.00 3.25 3.50 3.75

% 0

100

Dicamba-02-12-09-1103 1: TOF MS ES179

1.89e3

2.70

3.02

2.86

Dicamba-02-12-09-1107 1: TOF MS ES179

2.20e3

2.70

3.00

Di b 02 12 09 1103 3 Di d A

Time

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

2.0e+2

2.50 2.75 3.00 3.25 3.50 3.75

AU

0.0

2.0e+2

Dicamba-02-12-09-1103 3: Diode Array

2.91 Range: 2.866e+

2.99

Dicamba-02-12-09-1107 3: Diode Array

2.91 Range: 2.978e+

2.39

2.99

+DMO+DMO-DMO-DMO

8,107,108 B2 DCB

2.00 2.50 3.00

% 0

100

2.00 2.50 3.00

% 0

100

D icam ba-02-12-09-1023 1: TO F MS ES205

4.56e3

2.54

D icam ba-02-12-09-1027 1: TO F MS ES205

9 9

2.64

2.04

2.38

2.81 2.87 3.38 3.09

Tim e

2.00 2.50 3.00

AU

0.0

1.0e+1

2.00 2.50 3.00

AU

0.0

1.0e+12.0e+1

D icam ba-02-12-09-1023 3: D iode Array 

2.66 R ange: 2.288e+1

2.44

D icam ba-02-12-09-1027 3: D iode Array 

2.65 R ange: 1.837e+1

+DMO-DMO

+DMO-DMO

3,6‐DCSA

3,5‐DCSA

DCB

T im e

3.00 4.00 5.00

AU

0.0

5.0e+ 1

3.00 4.00 5.00

AU

0.0

5.0e+ 1

x 1 6 3 .7 3 R ange: 9 .014 e + 1

3.38 4.33

D ica m b a-0 2 -1 2 -09 -10 7 0 3 : D io d e A rray 

x16 3.73 R ange: 8.997e+ 1

3.38 4.33

8,107,108 C 3 2,4-D

3.00 4.00 5.00

% 0

100

3.00 4.00 5.00

% 0

100

D ica m b a-0 2 -1 2 -09 -10 7 0 1: T O F M S E S - 

163

9 4

3.83

3.49 3.55

3.96 4.43

4.51 5.26

D ica m b a-0 2 -1 2 -09 -10 7 4 1: T O F M S E S - 

163

8 9

3.86

3.07 3.30

3.90 4.43

5.12 5.23

+DMO

+DMO

-DMO

-DMO

2,4‐DCP

8,107,108 H6 Sinapic 

2.00 2.50 3.00 3.50

% 0

100

2.00 2.50 3.00 3.50

% 0

100

Dicamba-02-12-09-1197 1: TOF MS 

2.38

2.19 2.13

1.93

2.49 2.71 3.12 3.17 3.54

Dicamba-02-12-09-1202 1: TOF MS 

2.36 2.52

2.29

2.26

1.94 1.83

2.78

3.063.25

3.45

T

2.00 2.50 3.00 3.50

AU

0.0

2.0e+2

2.00 2.50 3.00 3.50

AU

0.0

2.0e+2

Dicamba-02-12-09-1197 3: Diode A

2.15 Range: 2.3

1.69

2.82

2.32

Dicamba-02-12-09-1202 3: Diode A

2.15 Range: 2.50

1.78

2.82

2.33

2.66 3.10 3.20

+DMO

+DMO

-DMO

-DMO

e

a. Dicamba (DCB) b. 2,4-D c. Sinapic Acid

d. Ferulic Acid e. Anisic Acid f. Syringic Acid g. Vanillic Acid
Monsanto Company 12-CT-244U 305 of 620 

Figure C-8. UPLC Separation of Dicamba (DCB) and DCSA in Bridging Analysis 

Dicamba and DCSA were separated by UPLC and detected by UV absorbance using a Photo 

Diode Array (PDA) and mass spectrometry (MS). 
Monsanto Company 12-CT-244U 306 of 620 

Figure C-9. E. coli-produced MON 88701 DMO Conversion of o-Anisic Acid 

o-anisic acid was incubated with E. coli-produced MON 88701 DMO and the formation 

of predicted oxidative product and the disappearance of o-anisic acid was monitored by 

LC-UV (A chromatograms) and LC-MS (B and C chromatograms). o-Anisic acid was 

included in a reaction mixture made with (+DMO, upper) and without (-DMO, lower) 

MON 88701 DMO. The dotted line indicates the solvent delay between the UV and MS 

detectors as they are connected in series. 

 
Monsanto Company 12-CT-244U 307 of 620 

C.4. Characterization of PAT (bar) Protein in MON 88701 

C.4.1. Materials 

The MON 88701-produced PAT (bar) protein (lot 11295997) was purified from 

cottonseed of MON 88701 (lot 11287350). The MON 88701-produced PAT (bar) 

protein was stored in a -80 oC freezer in a buffer solution containing 50 mM Tris-HCl, 

pH 7.5, 0.16 M sodium chloride and 20% glycerol. 

The E. coli-produced PAT (bar) protein (lot 11270310) was used as the reference 

substance. The PAT (bar) protein reference substance was generated from cell paste 

produced by large-scale fermentation of E. coli containing the pMON106653 expression 

plasmid. The coding sequence for bar contained on the expression plasmid 

(pMON106653) was confirmed prior to and after fermentation. The E. coli-produced 

PAT (bar) protein was previously characterized. 

C.4.2. Description of Assay Control 

Protein MW standards (Precision Plus Protein Standards Dual color; Bio-Rad, Hercules, 

CA) were used to calibrate some SDS-PAGE gels and verify protein transfer to 

polyvinylidene difluoride (PVDF) and nitrocellulose membranes. Broad Range 

SDS-PAGE molecular weight standards (Bio-Rad, Hercules, CA) were used to generate a 

standard curve for the apparent MW estimation. Bovine serum albumin (BSA) and 

-aminobutyric acid (AAbA) were used as hydrolysis control and internal calibration 

standard for amino acid analysis. A phenylthiohydantoin (PTH) amino acid standard 

mixture (Applied Biosystems, Foster City, CA) was used to calibrate the Applied 

Biosystems 494 Procise Sequencing System for each analysis. A peptide mixture 

(Sequazyme Peptide Mass Standards kit, Applied Biosystems, Foster City, CA) was used 

to calibrate the MALDI-TOF mass spectrometer for tryptic mass analysis. Transferrin 

(Sigma-Aldrich, St. Louis, MO) was used as positive control for glycosylation analysis. 

C.4.3. PAT (bar) Protein Purification 

The plant-produced PAT (bar) protein was purified from cottonseed of MON 88701. 

The purification procedure was not performed under a GLP plan; however, all procedures 

were documented on worksheets and, where applicable, SOPs were followed. The plantproduced PAT (bar) was purified from an extract of ground cottonseed using a 

combination of dye affinity chromatography and anionic exchange chromatography. The 

purification procedure is briefly described below. 

Approximately 1 kg of cottonseed of MON 88701 was ground to fine powder using a 

laboratory mill (model 3100, Perten Instruments). The ground powder was suspended in 

4 liters of hexane (EMD Chemicals Inc., Gibbstown, NJ ) and filtered 3 times in order to 

defat the powder. After drying overnight, the powder was ready for further processing. 

All grinding and defatting steps were done in a fume hood at room temperature. 
Monsanto Company 12-CT-244U 308 of 620 

A portion (200 g) of the defatted powder was extracted with 2 liters of 20 mM Tris-HCl, 

pH 7.5, and the solids were removed by centrifugation at 25,000  g for 20 min. The 

decanted solution was treated with 15 ml of 1 M CaCl2 solution to precipitate some 

proteins and centrifuged at 25,000  g for 20 min to remove the precipitated proteins. 

The soluble portion (~1450 ml), containing the PAT (bar) protein, was batch absorbed 

onto 20 ml of reactive brown 10 agarose (Sigma-Aldrich, St. Louis, MO) equilibrated 

with 200 ml of 20 mM Tris-HCl, pH 7.5. The reactive brown 10 agarose was centrifuged 

at 1000  g for 2 min and the resin, after decanting the supernatant, was transferred to a 

column. To remove unbound proteins, reactive brown 10 agarose was washed with 80 ml 

of 20 mM Tris-HCl buffer, pH 7.5, followed by 120 ml of 20 mM Tris-HCl buffer, 

pH 7.5, 1.5 M NaCl. Finally, the column was rinsed with 120 ml of 20 mM Tris-HCl 

buffer, pH 7.5. The PAT (bar) protein was then eluted, with 80 ml of 1 mM acetyl CoA 

in 20 mM Tris-HCl, pH 7.5. The eluted PAT (bar) protein was loaded onto a 1 ml Q 

Sepharose Fast Flow (GE Healthcare) column, equilibrated with 10 ml of 20 mM 

Tris-HCl, pH 7.5, using an automated chromatography system (AKTA, GE Healthcare). 

The Q Sepharose Fast Flow column was washed with 20 ml of 20 mM Tris-HCl, pH 7.5, 

0.1 M NaCl and consecutive step wise elution using 0.2 M and 0.5 M NaCl in 20 mM 

Tris-HCl, pH 7.5 to a total volume of 23 ml was conducted. Fractions containing 

PAT (bar) protein were pooled (8 ml) and concentrated to a volume of 1170 μl using a 

centrifugal filter (Ultracel 10K; Millipore, Billerica, MA; Molecular Weight Cutoff 

(MWCO) of 10 kDa). Buffer was added to the concentrated sample to bring the final 

volume to 2 ml and the final buffer composition to 50 mM Tris-HCl, pH 7.5, 0.16 M 

NaCl, and 20% (v/v) glycerol. This PAT (bar) protein purified from the cottonseed of 

MON 88701 was aliquoted and stored in a -80 °C freezer. 

 
Monsanto Company 12-CT-244U 309 of 620 

C.4.4. Summary of PAT (bar) Protein Identity and Equivalence 

Table C-9. Summary of MON 88701-produced PAT (bar) Protein Identity and 

Equivalence 

Analytical Test Assessment 

Section 

Cross 

Reference Analytical Test Outcome 

1. N-terminal sequence analysis of 

the MON 88701-produced 

PAT (bar) protein to assess 

identity 

VI.C.3.1.  The identity was confirmed by 

N-terminal sequence analysis 

2. MALDI-TOF MS1 analysis of 

peptides derived from tryptic 

digested MON 88701-produced 

PAT (bar) protein to assess 

identity 

VI.C.3.2.  MALDI-TOF MS1

 analysis yielded 

peptide masses consistent with the 

expected peptide masses from the 

theoretical trypsin digest of the 

MON 88701 PAT (bar) sequence 

3. Western blot analysis using antiPAT (bar) polyclonal 

antibodies to assess identity and 

immunoreactive equivalence 

between MON 88701- and the 

E. coli-produced PAT (bar) 

proteins 

VI.C.3.3.  MON 88701-produced PAT (bar) 

protein identity was confirmed using a 

western blot probed with antibodies 

specific for PAT protein 

 Immunoreactive properties of the 

MON 88701- and the E. coli-produced 

PAT (bar) proteins were shown to be 

equivalent 

4. SDS-PAGE2 to assess 

equivalence of the apparent 

molecular weight between 

MON 88701- and the 

E. coli-produced PAT (bar) 

proteins 

VI.C.3.4.  Electrophoretic mobility and apparent 

molecular weight of the MON 88701- 

and the E. coli-produced PAT (bar) 

proteins were shown to be equivalent 

5. Glycosylation analysis of the 

PAT (bar) protein to assess 

equivalence between the 

MON 88701- and the 

E. coli-produced PAT (bar) 

proteins 

VI.C.3.5.  Glycosylation status of MON 88701- 

and the E. coli-produced PAT (bar) 

proteins were shown to be equivalent 

6. PAT (bar) enzymatic activity 

analysis to assess functional 

equivalence between 

MON 88701- and the 

E. coli-produced PAT (bar) 

proteins 

VI.C.3.6.  Functional activity of the MON 88701- 

and the E. coli-produced PAT (bar) 

proteins were shown to be equivalent 

1

 MALDI-TOF MS = Matrix-assisted laser desorption/ionization time-of-flight mass 

spectrometry 

2

 SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis 
Monsanto Company 12-CT-244U 310 of 620 

C.4.5. N-Terminal Sequencing 

C.4.5.1. Methods 

N-terminal sequencing, carried out by automated Edman degradation chemistry, was used 

to confirm the identity of the MON 88701-produced PAT (bar). 

One aliquot of MON 88701-produced PAT (bar) was used for N-terminal sequence 

analysis. The analysis was performed for 15 cycles. using automated Edman degradation 

chemistry (Hunkapiller et al., 1983). An Applied Biosystems 494 Procise Sequencing 

System equipped with 140C Microgradient system a Perkin Elmer Series 200 UV/VIS 

Absorbance Detector with Procise Control Software (version 2.1) was used for amino 

acid detection after each cycle. Chromatographic data were collected using SequencePro 

(version 2.1) software. A control protein, -lactoglobulin, (Applied Biosystems, Foster 

City, CA) was analyzed before and after the sequence analysis of the MON 88701-

produced PAT (bar) protein to verify that the sequencer met performance criteria for 

repetitive yield and sequence identity. Identity was established if ≥ 8 amino acids, 

consistent with the predicted sequence of the N-terminus of the MON 88701-produced 

PAT (bar), were observed during analysis. 

C.4.5.2. Results of the N-terminal Sequence Analysis 

N-terminal sequencing of the first 15 amino acids was performed on 

MON 88701-produced PAT (bar). The expected sequence for the PAT (bar) protein 

deduced from the bar gene present in MON 88701 was observed. The data obtained 

correspond to the deduced PAT (bar) protein beginning at amino acid positions 2 and 3 

(Table C-10, Experimental Sequence 1 and 2, respectively). The N-terminal methionine 

residue in the PAT (bar) protein was not observed. This result is expected as removal of 

the N-terminal methionine, catalyzed by methionine aminopeptidase, is common in many 

organisms and has no effect on protein structure or activity (Arfin and Bradshaw, 1988; 

Bradshaw et al., 1998; Polevoda and Sherman, 2000). Hence, the sequence information 

confirms the identity of the PAT (bar) protein isolated from the cottonseed of 

MON 88701. 
Monsanto Company 12-CT-244U 311 of 620 

Amino acid 

residue # from 

the N-terminus

→ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Expected 

Sequence

→ M S P E R R P A D I R R A T E A

 │ │ │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental 

Sequence 1

→ - S P E R R P A D I R R A T E A

 │ │ │ │ │ │ │ │ │ │ │ │ │

Experimental 

Sequence 2

→ - - P E R X X A D I X X X T E -

Table C-10. N-Terminal Sequence of the MON 88701-produced PAT (bar) Protein 

The expected amino acid sequence of the N-terminus of PAT (bar) protein was deduced 

from the bar coding region present in MON 88701. The experimental sequences 

obtained from the MON 88701-produced PAT (bar) protein were compared to the 

expected sequence. The single letter IUPAC-IUB amino acid code is M, methionine; 

S, serine; P, proline; E, glutamic acid; R, arginine; A, alanine; D, aspatic acid; 

I, isoleucine; and T, threonine. X indicates that the residue was not identifiable; 

(-) indicates the residue was not observed. 
Monsanto Company 12-CT-244U 312 of 620 

C.4.6. MALDI-TOF Tryptic Mass Map Analysis 

C.4.6.1. Methods 

MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the 

MON 88701-produced PAT (bar) protein. MON 88701-produced PAT (bar) protein was 

subjected to SDS-PAGE and the gel was stained using Brilliant Blue G Colloidal stain. 

Each ~25 kDa band was excised and transferred to a microcentrifuge tube. The gel slices 

were destained with 40% (v/v) methanol/ 10% (v/v) acetic acid and washed in 100 mM 

ammonium bicarbonate and then, to reduce the protein in each, gel slices were incubated 

in 100 μl of 10 mM DTT at ~37 °C for 1 h. The protein was then alkylated in the dark 

for 20 min with 100 μl of 20 mM iodoacetic acid and washed three times for 15-20 min 

each with 200 μl of 25 mM ammonium bicarbonate. Gel slices were dried with a 

Speed-Vac® concentrator (Thermo Fisher Scientific, Waltham, MA) and then rehydrated 

with 20 μl of trypsin solution (20 μg/ml). After 1.25 h, excess liquid was removed and 

the gel was incubated overnight at ~37.5 °C in 40 μl of 10% acetonitrile in 25 mM 

ammonium bicarbonate. Gel slices were sonicated for 5 min to further elute proteolytic 

fragments. The resulting extracts were transferred to new microcentrifuge tubes labeled 

Extract 1 and dried using Speed-Vac concentrator. The gel slices were re-extracted twice 

with 30 μl of a 60% acetonitrile, 0.1% trifluoroacetic acid, 0.1% β-octylglucopyranoside 

solution and sonicated for 5 min. Both extracts were pooled into a new tube labeled 

Extract 2 and dried with a Speed-Vac concentrator. A solution (20 μl) of 0.1% 

trifluoroacetic acid (TFA) was added to all Extract 1 and 2 tubes and the samples were 

dried to completion via vacuum centrifugation. To solubilize the extracts, 5 μl of 50% 

acetonitrile, 0.1% trifluoroacetic acid was added to Extract 1 tube and 10 μl of 50% 

acetonitrile, 0.1% trifluoroacetic acid was added to Extract 2 tube and all were sonicated 

for 5 min. Each extract was spotted to three wells on an analysis plate. For each extract, 

0.75 μl of 2, 5 dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid 

(α-Cyano), or 3, 5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid) (Thermo Fisher 

Scientific Inc.) was added to one of the spots. The samples in DHB matrix were analyzed 

in the 300 to 5000 Da range. Samples in α-Cyano and sinapinic acid were analyzed in 

the 500 to 5000 Da and 500 to 7500 Da range, respectively. The analysis was performed 

using a VoyagerTM DE Pro BiospectrometryTM workstation (Applied Biosystems) using 

Voyager Instrument Control Panel software (version 5.10.2) and Data Explorer data 

analysis software (version 4.0.0.0). Protonated peptide masses were monoisotopically 

resolved in reflector mode (Aebersold, 1993; Billeci and Stults, 1993). CalMix 2 was 

used as the external calibrant (Sequazyme Peptide Mass Standards kit, Applied 

Biosystems, Foster City, CA) for the analysis. GPMAW32 software (Lighthouse Data, 

Odense M, Denmark) was used to generate a theoretical trypsin digest of the PAT (bar) 

protein sequence. Masses within 1 Da of a monosiotopic mass were matched against the 

theoretical digest of the PAT (bar) sequence. All matching masses were tallied and a 

coverage map was generated for the mass fingerprint. The tryptic mass fingerprint 

coverage was considered acceptable if  40% of the protein sequence was identified by 

matching experimental masses observed for the tryptic peptide fragments to the expected 

masses for the fragments (Biron et al., 2006, Krause et al., 1999). 
Monsanto Company 12-CT-244U 313 of 620 

C.4.6.2. Results of MALDI-TOF Tryptic Mass Map Analysis 

The identity of the MON 88701-produced PAT (bar) protein was also confirmed by 

MALDI-TOF MS analysis of peptide fragments produced from tryptic digestion of the 

MON 88701-produced PAT (bar) protein. The ability to identify a protein using this 

method is dependent upon matching a sufficient number of observed tryptic peptide 

fragment masses with predicted tryptic peptide fragment masses. In general, protein 

identification made by peptide mapping is considered to be reliable if  40% of the 

protein sequence was identified by matching experimental masses observed for the 

tryptic peptide fragments to the expected masses for the fragments (Biron et al., 2006, 

Krause et al., 1999). 

There were 10 unique peptides identified that corresponded to the masses expected to be 

produced by tryptic digestion of the PAT (bar) protein (Table C-11). The identified 

masses were used to assemble a coverage map of the entire MON 88701-produced 

PAT (bar) protein (Figure C-10). The experimentally determined mass coverage of the 

MON 88701-produced PAT (bar) protein was 84.7% (155 out of 183 amino acids). This 

analysis serves as additional identity confirmation for the MON 88701-produced 

PAT (bar) protein. 
Monsanto Company 12-CT-244U 

Table C-11. Summary of the Tryptic Masses1 Identified for the MON 88701-produced PAT (bar) Protein 

MS 

-cyano DHB Sinapinic acid Expected 

Mass Diff.2 Fragment3 Sequence Extract 1 Extract 2 Extract 1 Extract 2 Extract 1 Extract 2 

879.65 879.46 0.19 113-120 SLEAQGFK

1144.65 1144.75 1144.84 1144.56 0.09 136-145 MHEALGYAP

1403.93 1404.03 1404.12 1404.18 1403.79 0.14 100-112 TGLGSTLYTHL

1523.02 1523.13 1523.14 1523.19 1522.93 1522.86 0.16 121-135 SVVAVIGLPNDP

1843.07 1843.18 1843.27 1842.98 1843.19 1842.85 0.22 38-52 TEPQEPQEWTDD

1859.06 1859.22 1859.22 1858.98 1859.18 1858.86 0.20 81-96 NAYDWTAESTVY

2391.45 2391.64 2391.20 0.25 57-78 YPWLVAEVDGEVAGI

2676.67 2676.64 2676.88 2676.35 0.32 55-78 ERYPWLVAEVDGEVAG

2840.62 2840.32 0.30 13-37 ATEADMPAVCTIVNHY

3353.14 3353.36 3353.17 3353.48 3352.73 0.41 155-183 HGNWHDVGFWQLDFSLPV

1Only experimental masses that matched expected masses are listed in the table. 2The difference between the expected mass and the first column mass. Other masses shown within a row are also within 1 Da of the ex3Position refers to amino acid residues within the predicted PAT (bar) sequence as depicted in Figure C-10. 

DHB = 5-dihydroxybenzoic acid matrix, 

α-cyano = 

α-cyano-4-hydroxycinnamic acid matrix, Sinapinic acid = 3, 5-dimethoxy-4-hydrox
Monsanto Company 12-CT-244U 315 of 620 

Figure C-10. MALDI-TOF MS Coverage Map of the MON 88701-produced 

PAT (bar) Protein 

The amino acid sequence of the PAT (bar) protein was deduced from the bar gene 

present in MON 88701. Boxed regions correspond to regions covered by tryptic peptides 

that were identified from the MON 88701-produced PAT (bar) protein sample using 

MALDI-TOF MS. In total, 84.7% (155 out of 183 amino acids) of the expected protein 

sequence was covered by the identified peptides. 

 
Monsanto Company 12-CT-244U 316 of 620 

C.4.7. Western Blot Analysis-Immunoreactivity 

C.4.7.1. Methods 

Western blot analysis was performed to confirm the identity of the PAT (bar) protein 

purified from cottonseed of MON 88701 and to compare the immunoreactivity of the 

MON 88701- and E. coli-produced proteins. 

The MON 88701- and E. coli-produced PAT (bar) proteins were analyzed concurrently 

on the same gel using three loadings of 2, 4, and 6 ng. Loadings of the three 

concentrations were made in duplicate on the gel. Aliquots of each protein were diluted 

in water and 5X Laemmli buffer (LB) containing 312 mM Tris-HCl, 25% (v/v) 

2-mercaptoethanol, 10% (w/v) SDS, 0.025% (w/v) bromophenol blue, 50% (v/v) 

glycerol, pH 6.8, heated at ~96 °C for 4 min, and applied to a 15 well pre-cast 

Tris-glycine 4-20% polyacrylamide gradient gel (Invitrogen, Carlsbad, CA). Pre-stained 

moelcular weight markers (Precision Plus Protein Standards Dual color; Bio-Rad, 

Hercules, CA) were loaded in parallel to verify electrotransfer of the proteins to the 

membrane and to estimate the size of the immunoreactive bands observed. 

Electrophoresis was performed at a constant voltage of 150 V for 85 min. Electrotransfer 

to a 0.45 μm PVDF membrane (Invitrogen, Carlsbad, CA) was performed for 60 min at a 

constant current of 200 mA. After electrotransfer, the membrane was blocked for 1 h 

with 10% (w/v) non-fat dried milk (NFDM) in 1X phosphate buffered saline containing 

0.05% (v/v) Tween-20 (PBST). The membrane was then probed with a 1:2000 dilution 

of goat anti-PAT (bar) antibody (lot G863803) in 5% NFDM in PBST for 1 h at room 

temperature. Excess antibody was removed using three 10 min washes with PBST. 

Finally, the membrane was probed with horseradish peroxidase (HRP)-conjugated horse 

anti-goat IgG (Thermo, Rockford, IL) at a dilution of 1:10,000 in 5% NFDM in PBST for 

1 h at room temperature. Excess HRP-conjugate was removed using three 10 min washes 

with PBST. All washes were performed at room temperature. Immunoreactive bands 

were visualized using the ECL detection system (GE, Healthcare, Piscataway, NJ) with 

exposure to Amersham Hyperfilm ECL (GE, Healthcare, Piscataway, NJ). The film was 

developed using a Konica SRX-101A automated film processor (Konica Minolta Medical 

& Graphic, Inc., Tokyo, Japan). 

Quantification of the bands on the blot was performed using a Bio-Rad GS-800 

densitometer with the supplied Quantity One software (version 4.4.0, Hercules, CA) 

using the lane selection and contour tool. The signal intensities of the immunoreactive 

bands observed for the MON 88701- and E. coli-produced proteins migrating at the 

expected position on the blot film were quantified as “contour quantity” values. The raw 

data was exported to a Microsoft Excel (2007) file. The immunoreactivity of the 

MON 88701- and E. coli-produced PAT (bar) proteins were reported as the mean signal 

intensity at each amount of protein analyzed. The immunoreactivity of the MON 88701- 

and E. coli-produced PAT (bar) proteins were considered equivalent if the overall mean 

of the immunoreactive signal of the MON 88701-produced protein was within ± 35% of 

the overall mean of immunoreactive signal of the E. coli-produced PAT (bar) protein. 
Monsanto Company 12-CT-244U 317 of 620 

C.4.7.2. Results of PAT (bar) Protein Immunoreactivity Equivalence 

Western blot analysis was conducted using goat anti- PAT (bar) polyclonal antibodies to 

1) assess the identity of the PAT (bar) protein isolated from the cottonseed of 

MON 88701 and 2) to determine the relative immunoreactivity of the MON 88701- and 

the E. coli-produced PAT (bar) proteins. The results demonstrated that the 

anti-PAT (bar) antibodies recognized the MON 88701-produced PAT (bar) protein that 

migrated to an identical position on the blot as the E. coli-produced PAT (bar) protein 

(Figure C-11). Furthermore, the immunoreactive signal increased with increasing 

amounts of PAT (bar) protein loaded. 

Densitometric analysis was conducted to compare the immunoreactivity of 

MON 88701- and the E. coli-produced PAT (bar) proteins. The mean signal intensity 

(OD  mm

2

) from the MON 88701-produced PAT (bar) bands and from the 

E. coli-produced PAT (bar) bands at each amount of protein analyzed was calculated and 

then overall mean signal intensity was calculated (Table C-12). The immunoreactivity 

was considered equivalent if the overall mean signal intensity of all 

MON 88701-produced PAT (bar) protein bands was within ±35% of the overall mean 

signal intensity of all E. coli-produced PAT (bar) protein bands. 

The overall mean signal intensity of the E. coli-produced PAT (bar) bands was 4.669 

OD × mm2, and the overall mean signal intensity of the MON 88701-produced PAT (bar) 

bands was 4.167 OD × mm2

. Because overall mean signal intensity of the 

MON 88701-produced PAT (bar) protein bands was between 3.035 and 6.303 OD × mm2

(between -35% and +35% of the E. coli-produced PAT (bar) bands), the 

MON 88701-produced and E. coli-produced PAT (bar) proteins were determined to have 

equivalent immunoreactivity. 

 
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Figure C-11. Western Blot Analysis of the MON 88701- and E. coli -produced 

PAT (bar) Proteins 

Aliquots of the MON 88701-produced PAT (bar) protein and the E. coli-produced 

PAT (bar) protein were subjected to SDS-PAGE and electrotransferred to a PVDF 

membrane. The membrane was incubated with anti-PAT (bar) antibodies and 

immunoreactive bands were visualized using an ECL system (GE Healthcare, 

Piscataway, NJ). Approximate molecular weights (kDa) are shown on the left. Lanes 

loaded with molecular weight markers were cropped, and lanes were renumbered relative 

to the original gel loading. The 1 min exposure is shown. Lane designations are as 

follows: 

Lane Sample Amount (ng) 

1 E. coli-produced PAT (bar) protein 2 

2 E. coli-produced PAT (bar) protein 2 

3 E. coli-produced PAT (bar) protein 4 

4 E. coli-produced PAT (bar) protein 4 

5 E. coli-produced PAT (bar) protein 6 

6 E. coli-produced PAT (bar) protein 6 

7 Empty - 

8 MON 88701-produced PAT (bar) protein 2 

9 MON 88701-produced PAT (bar) protein 2 

10 MON 88701-produced PAT (bar) protein 4 

11 MON 88701-produced PAT (bar) protein 4 

12 MON 88701-produced PAT (bar) protein 6 

13 MON 88701-produced PAT (bar) protein 6 

 
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Table C-12. Comparison of Immunoreactive Signals Between MON 88701- and 

E. coli-produced PAT (bar) Proteins 

Mean Signal intensity from 

MON 88701-produced 

PAT (bar)

(OD × mm2) 

Mean Signal intensity from 

E. coli-produced 

PAT (bar)

(OD × mm2) 

Preset Acceptance limits 

for MON 88701-produced 

PAT (bar)

1 

(OD × mm2) 

4.167 4.669 3.035 – 6.303 

1

The acceptance limits for the MON 88701-produced PAT (bar) are based on the interval between +35% 

(4.669 × 1.35) and -35% (4.669 × 0.65) of the overall mean of the E. coli-produced PAT (bar) signal 

intensity across all loads. 

 
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C.4.8. Molecular Weight and Purity Estimation using SDS-PAGE 

C.4.8.1. Methods 

MON 88701- and E. coli-produced PAT (bar) proteins were mixed with 5X LB and 

diluted with water to a final total protein concentration of 0.136 μg/μl. Molecular Weight 

Standards, Bio-Rad broad range (Hercules, CA) were diluted to a final total protein 

concentration of 0.9 g/l. The MON 88701-produced PAT (bar) was analyzed in 

duplicate at 1, 2, and 3 μg protein per lane. The E. coli-produced PAT (bar) reference 

standard was analyzed at 1 μg total protein in a single lane. The samples were loaded 

onto a 10-well pre-cast Tris glycine 4-20% polyacrylamide gradient mini-gel (Invitrogen, 

Carlsbad, CA) and electrophoresis was performed at a constant voltage of 150 V for 

95 min. Proteins were fixed by placing the gel in a solution of 40% (v/v) methanol and 

7% (v/v) acetic acid for 30 min, stained for 16.25 h with Brilliant Blue G-Colloidal stain 

(Sigma-Aldrich, St. Louis, MO). Gels were destained once for 30 to 45 sec with a 

solution containing 10% (v/v) acetic acid and 25% (v/v) methanol, and four times for 2 h 

each (for a total of 8 h) with 25% (v/v) methanol. Analysis of the gel was performed 

using a Bio-Rad GS-800 densitometer with the supplied Quantity One software (version 

4.4.0, Hercules, CA). The apparent MW of each observed band was estimated from a 

standard curve generated by the Quantity One software which was based on the MWs of 

the markers and their migration distance on the gel. To determine purity, all visible bands 

within each lane were quantified using Quantity One software. Apparent MW and purity 

were reported as an average of all six lanes containing the MON 88701-produced 

PAT (bar). 

C.4.8.2. Results of PAT (bar) Protein Molecular Weight Equivalence 

The molecular weight and purity of the PAT (bar) protein was determined to be 24.1 kDa 

and 99%, respectively. To assess apparent molecular weight (MW) and purity, the 

MON 88701-produced PAT (bar) protein was subjected to SDS-PAGE. The gel was 

stained with Brilliant Blue G Colloidal stain and analyzed by densitometry (Figure C-12). 

E. coli-produced PAT (bar) protein was loaded in a single lane for reference (Figure C12, Lane 2). The MON 88701-produced PAT (bar) protein (Figure C-12, Lanes 3-8) had 

an apparent MW of 24.1 kDa (Table C-13). The apparent MW of the E. coli-produced 

PAT (bar) protein as reported on its Certificate of Analysis was 25.0 kDa (Table C-8). 

Because the apparent MW of MON 88701-produced PAT (bar) protein was within the 

preset acceptance limits (Table C-13), the MON 88701-produced and E. coli-produced 

PAT (bar) proteins were determined to have equivalent apparent MWs. 

The purity of the MON 88701-produced PAT (bar) protein was calculated based on the 

six loads on the gel (Figure C-12, Lanes 3-8). The average purity was determined to be 

more than 99%. 
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Table C-13. Molecular Weight Comparison Between the MON 88701- and 

E. coli-produced PAT (bar) Proteins Based on SDS-PAGE 

Apparent Molecular Weight 

of MON 88701-Produced 

PAT (bar) Protein1 

(kDa) 

Apparent Molecular Weight 

of E. coli-Produced 

PAT (bar) Protein2 

(kDa) 

Preset Acceptance Limits 

for MON 88701-

produced PAT (bar)

3

 

(kDa) 

24.1 25.0 23.9-25.4 

1

The reported value is the mean molecular weight across all six loads. 2

The molecular weight of the E. coli-produced PAT (bar) protein as reported on its Certificate of Analysis. 3

See Section C.6. 
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Figure C-12. Molecular Weight and Purity Analysis of the MON 88701-produced 

PAT (bar) Protein 

Aliquots of the MON 88701-produced and the E. coli-produced PAT (bar) proteins were 

subjected to SDS-PAGE and then stained with Brilliant Blue G-Colloidal stain. 

Approximate molecular weights are shown on the left and correspond to the markers 

loaded in Lanes 1 and 9. Empty lane was partially cropped. Lane designations are as 

follows: 

Lane Sample Amount (μg) 

1 Broad Range Molecular Weight Markers 4.5 

2 E. coli-produced PAT (bar) protein 1 

3 MON 88701-produced PAT (bar) protein 1 

4 MON 88701-produced PAT (bar) protein 1 

5 MON 88701-produced PAT (bar) protein 2 

6 MON 88701-produced PAT (bar) protein 2 

7 MON 88701-produced PAT (bar) protein 3 

8 MON 88701-produced PAT (bar) protein 3 

9 Broad Range Molecular Weight markers 4.5 

 
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C.4.9. Glycosylation Analysis 

C.4.9.1. Methods 

Glycosylation analysis was used to determine whether the MON 88701-produced 

PAT (bar) was post-translationally modified with covalently bound carbohydrate 

moieties. Aliquots of the MON 88701-produced PAT (bar) protein, the E. coli-produced 

PAT (bar) (negative control) and the positive control, transferrin (Sigma-Aldrich, 

St. Louis, MO), were each diluted with water and brought to 1X LB. These samples were 

heated at ~102 °C for 4 min. The MON 88701-produced PAT (bar), the E. coli-produced 

PAT (bar) and transferrin were loaded at approximately 50 and 100 ng per lane on a 

Tris-glycine 10 well 4-20% polyacrylamide gradient mini-gel (Invitrogen, Carlsbad, CA). 

Precision Plus Protein Dual color Standards (Bio-Rad, Hercules, CA) were also loaded to 

verify electrotransfer of the proteins to the membrane and as markers for molecular 

weight. Electrophoresis was performed at a constant voltage of 150 V for 90 min. 

Electrotransfer to a 0.45 μm PVDF membrane (Invitrogen, Carlsbad, CA) was performed 

for 60 min at a constant current of 200 mA. 

Carbohydrate detection was performed directly on the PVDF membrane at room 

temperature using the Amersham ECL glycoprotein Detection Module (GE, Healthcare, 

Piscataway, NJ). With this module, carbohydrate moieties of proteins were oxidized with 

sodium metaperiodate and then biotinylated with biotin-X-hydrazide. The biotinylated 

proteins can be detected on the blot by addition of streptavidin conjugated to HRP for 

luminol-based detection using ECL reagents (GE, Healthcare, Piscataway, NJ) and with 

subsequent exposure to Amersham Hyperfilm (GE, Healthcare). The film was developed 

using a Konica SRX-101A automated film processor (Konica Minolta Medical & 

Graphic, Inc., Tokyo, Japan). 

An identical blot run in parallel to that used for the glycosylation analysis was stained to 

visualize the proteins present on the membrane. Proteins were stained for 30 sec to 2 min 

using Coomassie Brilliant Blue R-250 staining solution (Bio-Rad, Hercules, CA) and 

then destained with 1X Coomassie Brilliant Blue R-250 destaining solution (Bio-Rad) for 

more than 5 min. After washing with water, the blot was dried and scanned using BioRad GS-800 densitometer with the supplied Quantity One software (version 4.4.0). 

C.4.9.2. Results of Glycosylation Analysis 

Some eukaryotic proteins are post-translationally modified by the addition of 

carbohydrate moieties (Rademacher et al., 1988). To test whether PAT (bar) protein was 

glycosylated when expressed in the cottonseed of MON 88701, the 

MON 88701-produced PAT (bar) protein was analyzed using an ECL Glycoprotein 

Detection Module (GE, Healthcare, Piscataway, NJ). Transferrin, a glycosylated protein, 

was used as a positive control in the assay. To assess equivalence of the MON 88701- 

and E. coli-produced PAT (bar) proteins, the E. coli-produced PAT (bar) protein was 

also analyzed. The positive control was clearly detected at the expected molecular 

weight (~80 kDa) and the band intensity increased with increasing concentration 

(Figure C-13, Panel A, Lanes 1-2). In contrast, signals were not observed in the lanes 
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containing the MON 88701- or E. coli-produced protein at the expected molecular weight 

for the PAT (bar) protein (Figure C-13 Panel A, Lanes 7-8 and Lanes 4-5, respectively). 

To assess whether the MON 88701- and E. coli-produced PAT (bar) proteins were 

loaded appropriately for glycosylation analysis, a second membrane (with identical 

loadings and transfer times) was stained with Coomassie Blue R-250 for protein detection 

(Figure C-13 Panel B). Both the MON 88701- and E. coli-produced PAT (bar) proteins 

were clearly detected (Figure C-13 Panel B, Lanes 7-8 and Lanes 4-5, respectively). 

These data indicate that the glycosylation status of MON 88701-produced PAT (bar) 

protein and E. coli-produced PAT (bar) protein are equivalent and that neither is 

glycosylated. 

 
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Figure C-13. Glycosylation Analysis of the MON 88701-produced PAT (bar) 

Protein 

Aliquots of the transferrin (positive control), E. coli-produced PAT (bar) protein and 

MON 88701-produced PAT (bar) protein were subjected to SDS-PAGE and 

electrotransferred to PVDF membranes. Panel A corresponds to detection of labeled 

carbohydrate moieties, where present, using the ECL-based system with exposure to 

Hyperfilm. A 7 min exposure is shown. Panel B corresponds to Coomassie Blue R-250 

staining of an equivalent blot to confirm the presence of proteins. The signal was 

captured using a Bio-Rad GS-800 with Quantity One software (version 4.4.0). 

Approximate molecular weights (kDa) correspond to the Precision Plus, dual color 

markers (used to verify transfer and MW). Lanes loaded with molecular weight markers 

were cropped, and lanes were renumbered relative to the original gel loading. Arrows 

indicate the expected migration of PAT (bar) protein. Lane designations are as follows: 

Lane Sample Amount (ng) 

1 Transferrin (positive control) 50 

2 Transferrin (positive control) 100 

3 Empty - 

4 E. coli-produced PAT (bar) (negative control) 50 

5 E. coli-produced PAT (bar) (negative control) 100 

6 Empty - 

7 MON 88701-produced PAT (bar) 50 

8 MON 88701-produced PAT (bar) 100 

 
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C.4.10. Functional Activity Analysis 

C.4.10.1. Methods 

PAT (bar) catalyzes the reaction of phosphinothricin (PPT) with acetyl CoA to form 

acetyl PPT and free CoA. To assess functional activity of PAT (bar), the amount of CoA 

released during the reaction can be monitored using the reduction of 5,5’-dithio-bis 

(2-nitrobenzoic acid) (DTNB) by CoA to form the colorimetric reagent 

5-thio-nitrobenzoate (TNB) (Wehrmann et al., 1996). 

Prior to functional activity analysis, both MON 88701- and E. coli-produced PAT (bar) 

proteins were diluted to a purity corrected concentration of 1 ng/μl with a 50 mM Tris, 

pH 7.5, and 0.5 mM EDTA buffer. Assays for both proteins were conducted using five 

replicates. The reaction mixtures containing 2 mM acetyl CoA, 1 mM DTNB, 50 mM 

Tris, pH 7.8, and 0.5 mM EDTA with or without 1 mM phosphinothricin were pre 

incubated at ~30 °C for 10-60 min. The reactions were then initiated by the addition of 

10 ng of PAT (bar) enzyme. The reaction rate was monitored in each well at 412 nm and 

~30 °C using a plate reader in one minute intervals for 30 min. A response curve was 

prepared using 3.9 μM to 250 μM β-mercaptoethanol in 1 mM DTNB, 50 mM Tris, 

pH 7.8, and 0.5 mM EDTA. The response curve was generated only to verify assay 

conditions and instrument performance. The initial assay results are reported as the mean 

velocity of the reaction of PAT (bar) (generated by the KC4 software, Power Wave Xi, 

Bio Tek, Richmond, VA) and expressed as min-1. The specific activities of the 

MON 88701- and E. coli-produced PAT (bar) proteins were then calculated using the 

molar absorptivity of product released during the assay, TNB (13,600 M-1  cm

-1

 or 

13.6 μmol-1  ml). Specific activity is expressed as μmol of TNB released per minute 

per mg of PAT (bar) (μmol min-1 × mg-1). Calculations of the specific activities were 

performed using Microsoft Excel (2007). 

C.4.10.2. Results of Functional Activity 

The functional activities of the MON 88701- and E. coli-produced PAT (bar) proteins 

were assessed using a colorimetric assay that measures PAT (bar) catalyzed release of 

coenzyme A (CoA) from acetyl-CoA upon transfer of an acetyl-group to 

phosphinothricin. In this assay, protein-specific activity is expressed as 

μmol × minute-1 × mg-1 of PAT enzyme. 

The experimentally determined specific activities for the MON 88701- and 

E. coli-produced PAT (bar) proteins are presented in Table C-14. The specific activities 

of MON 88701- and E. coli-produced PAT (bar) proteins were 36.4 and 

46.2 μmol × minute-1 × mg-1 of PAT (bar), respectively. Because the specific activities 

of the MON 88701-produced and E. coli-produced PAT (bar) proteins fall within the 

preset acceptance criterion (Table C-14), the MON 88701- and E. coli-produced 

PAT (bar) proteins were determined to have equivalent functional activity. 
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Table C-14. PAT (bar) Functional Activity 

MON 88701-produced 

PAT (bar)

1 

(μmol × minute-1 × mg-1) 

E. coli-produced 

PAT (bar)

1 

(μmol × minute-1 × mg-1) 

Preset Acceptance Limits 

for 

MON 88701-produced 

PAT (bar)

2 

(μmol × minute-1 × mg-1) 

36.4 ± 1.3 46.2 ± 2.1 30.17 - 51.70 

1

Value refers to mean and standard deviation calculated based on n = 5. 

2

See Section C.6.

C.5. Substrate Specificity of PAT (bar) Protein Produced in MON 88701 

The PAT proteins, including PAT (bar) protein, were demonstrated to be highly specific 

for glufosinate in the presence of acetyl-CoA. Since the specificity of PAT proteins has 

been well established in literature, and due to the lack of any documented reports of nonspecific effects of PAT proteins since the introduction of glufosinate tolerant crops in 

1995, in-house experiments were not conducted to further demonstrate substrate 

specificity of PAT (bar) protein isolated from MON 88701. 

C.6. Prediction Intervals as Acceptance criteria 

Acceptance criteria (acceptance limits) based on prediction intervals were used to assess 

the equivalence of the MON 88701-produced and E. coli-produced proteins for apparent 

MW and functional activity. A prediction interval is an estimate of an interval in which a 

randomly selected future observation from a population will fall, with a certain degree of 

confidence, given what has already been observed (Hahn and Meeker, 1991a; b); i.e., 

prediction intervals are generated based on the statistical analysis of the existing data. 

Data obtained from multiple assays of E. coli-produced protein conducted under GLP 

guidelines were used for this purpose. 

To generate the 95% prediction interval (PI), the mean and standard deviation of the data 

from several assays were calculated. The number of assays used to calculate the mean 

and the number of future assays (one for equivalence studies) were used in the following 

formula to generate the PI: 

Xഥ േ ݎሺ1 െ ߙ ; ݉, ݊ሻ ሺݏሻ

r(1-; m, n) is estimated using the formula given below: 

ݎሺଵିఈ;௠,௡ሻ ؆ ݐሺଵି.଴ହ/ሺଶ௠ሻ; ௡ିଵሻඨ1൅ 

1

݊
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Where Xҧis mean of the replicate assays; s is standard deviation of the replicates;  is 

the level of confidence; n is the number of assays used to generate the mean; and m is the 

number of future assays (one for equivalence studies). The t-value is the 

100(1-.05/(2m))th percentile from Student’s t-distribution with n-1 degrees of freedom. 

With 95% confidence, all m future values of the assay will fall within this interval (Hahn 

and Meeker, 1991a; b). If the assay means do not appear to have been derived from a 

normal distribution, but the logarithms of the raw values do follow a normal distribution, 

then prediction intervals may be applied to the logarithms of the raw values (Hahn and 

Meeker, 1991a; b). 

 
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Appendix D: Materials and Methods Used for the Analysis of the Levels of 

MON 88701 DMO and PAT (bar) Proteins in MON 88701 

D.1. Materials 

Seed, over season leaf (OSL-1-4), root, and pollen tissue samples from dicamba and 

glufosinate-treated MON 88701 were harvested from eight field sites in the U.S. during 

the 2010 growing season from starting seed lot 11268129, with the exception of OSL-1 

(7 sites) and OSL-4 (7 sites). MON 88701 plots were treated at the 3-5 leaf stage with 

glufosinate herbicide at the label rate (0.5 lbs a.i./acre) and at the 6-10 leaf stage with 

dicamba herbicide at the label rate (0.5 lbs a.e./acre). E. coli-produced MON 88701 

DMO (lot 11293429) and PAT (bar) protein (lot 11270310) were used as the analytical 

reference standards. 

D.2. Characterization of the Materials 

The identity of MON 88701 was confirmed by conducting MON 88701 event specific 

polymerase chain reaction (PCR) analyses on the harvested seed from each site. Any 

seed sample and its associated tissues for which three or more pools out of four tested 

unexpectedly during PCR verification were not analyzed in this study. 

D.3. Field Design and Tissue Collection 

Field trials were initiated during the 2010 planting season to generate MON 88701 seed, 

OSL-1-4, root, and pollen samples at various cotton growing locations in the U.S. The 

tissue samples from the following field sites were analyzed: Arkansas (ARTI), Georgia 

(GACH), Kansas (KSLA), Louisiana (LACH), North Carolina (NCBD), New Mexico 

(NMLC), South Carolina (SCEK) and Texas (TXPL). These field sites were 

representative of cotton producing regions suitable for commercial production. At each 

site, four replicated plots of plants containing MON 88701 were planted using a 

randomized complete-block field design. Seed, over season leaf (OSL-1-4), root, and 

pollen samples were collected from each replicated plot at all field sites, except OSL-1 at 

site TXPL and OSL-4 at site LACH. See Tables V-4 and V-5 for detailed descriptions of 

when the samples were collected. 

D.4. Tissue Processing and Protein Extraction 

Tissue samples were shipped to Monsanto Company (St. Louis, Missouri), and were 

prepared by the Monsanto Sample Management Team. The prepared tissue samples were 

stored in a -80° C freezer until transferred on dry ice to the analytical facility. 

D.4.1. MON 88701 DMO Protein 

MON 88701 DMO protein was extracted from tissue samples as described in Table D-1. 

MON 88701 DMO was extracted from over season leaf (OSL-1-4) and root tissues 

samples with the appropriate amount of Tris borate buffer with 0.5% (w/v) bovine serum 

albumin (1 × TB + 0.5% BSA) [0.1 M Tris, 0.1 M Na2B4O7, 0.05 M MgCl2, 0.05% (v/v) 
Monsanto Company 12-CT-244U 333 of 620 

Tween 20 at pH 7.8, 0.5% (w/v) BSA]. MON 88701 DMO was extracted from pollen 

and seed tissues with the appropriate amount of phosphate buffered saline (PBS) with 

Tween 20 (1 × PBST). Extractions were done using 8 1/4′′ chrome-steel beads, and 

shaking in a Harbil mixer (Fluid Management, Wheeling, Illinois). Insoluble material 

was removed from all tissue extracts using a serum filter (Fisher Scientific, Pittsburgh, 

PA) The extracts were aliquoted and stored frozen in a -80 °C freezer until ELISA 

analysis. 

Table D-1. MON 88701 DMO Protein Extraction Methods for Tissue Samples 

Sample Type Tissue-to-Buffer Ratio Extraction Buffer 

Leaf1 1:100 1 × TB + 0.5% BSA

Root 1:100 1 × TB + 0.5% BSA

Pollen 1:100 1 × PBST

Seed 1:100 1 × PBST

 1

Over- season leaf (OSL-1, OSL-2, OSL-3, and OSL-4).

D.4.2. PAT (bar) Protein 

PAT (bar) protein was extracted from tissue samples as described in Table D-2. 

PAT (bar) was extracted from over season leaf (OSL-1-4) and root tissues samples with 

the appropriate amount of Tris borate buffer with L-ascorbic acid (1× TBA) [0.1 M Tris, 

0.1 M Na2B4O7, 0.05 M MgCl2, 0.05% (v/v) Tween 20 at pH 7.8, 0.2% (w/v) L-ascorbic 

acid]. PAT (bar) was extracted from pollen and seed tissues with the appropriate amount 

of 1 × PBST. Extractions were done using 8 1/4′′ chrome-steel beads, and shaking in a 

Harbil mixer. Insoluble material was removed from all tissue extracts using a serum 

filter. The extracts were aliquoted and stored frozen in a -80 °C freezer until ELISA 

analysis. 

Table D-2. PAT (bar) Protein Extraction Methods for Tissue Samples 

Sample Type Tissue-to-Buffer Ratio Extraction Buffer 

Leaf1 1:100 1× TBA

Root 1:100 1× TBA

Pollen 1:100 1× PBST

Seed 1:100 1× PBST

 1

Over- season leaf (OSL-1, OSL-2, OSL-3, and OSL-4)

 
Monsanto Company 12-CT-244U 334 of 620 

D.5. Protein Antibodies 

D.5.1. MON 88701 DMO Protein 

Goat polyclonal antibodies specific for the DMO protein were purified using Protein G 

affinity chromatography. The concentration of the purified IgG was determined to be 

8.1 mg/ml by spectrophotometric methods. The purified antibody was stored in 1× PBS. 

Protein G-affinity purified goat polyclonal anti-DMO antibodies were coupled with biotin 

(Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s instructions. 

The detection reagent was NeutrAvidin (Thermo Fisher Scientific, Rockford, IL) 

conjugated to horseradish peroxidase (HRP). 

D.5.2. PAT (bar) Protein 

Goat polyclonal PAT (bar)-specific IgG was purified by Protein G-affinity 

chromatography followed by PAT (bar) antigen affinity chromatography. The 

concentration of the purified IgG was determined to be 3.6 mg/ml by spectrophotometric 

methods. The purified antibody was stored in 1× PBS. 

Protein G-affinity purified goat polyclonal anti-PAT (bar) antibodies were coupled with 

biotin (Thermo Fisher Scientific, Rockford, IL) according to the manufacturer’s 

instructions. The detection reagent was NeutrAvidin (Thermo Fisher Scientific, 

Rockford, IL) conjugated to horseradish peroxidase (HRP). 

D.6. Protein ELISA Method 

D.6.1. MON 88701 DMO Protein 

Goat anti-DMO antibodies were diluted in coating buffer (15 mM Na2CO3, 35 mM 

NaHCO3, and 150 mM NaCl) to a final concentration of 5 μg/ml and immobilized onto 

96 well microtiter plates followed by incubation in a 4° C refrigerator for > 8 hours. 

Prior to each step in the assay, plates were washed with 1× PBST. Plates were blocked 

with the addition of 200 μl per well of blocking buffer, Blocker Casein (Thermo Fisher 

Scientific, Rockford, IL) in Tris Buffered Saline (TBS) for 60 to 70 minutes at room 

temperature (RT). DMO protein standard or sample extract was added at 100 μl per well 

and incubated for 60 to 65 minutes at 37° C. Biotinylated goat anti-DMO antibodies 

prepared in 1× Tris-borate buffer with 10% Blocker Casein in TBS were added at 100 μl 

per well and incubated for 60 to 65 minutes at 37° C. NeutrAvidin HRP conjugate was 

added at 100 μl per well and incubated for 30 to 35 minutes at 37° C. Plates were 

developed by adding 100 μl per well of substrate, 3,3',5,5' tetramethyl benzidine (TMB; 

Kirkegaard & Perry, Gaithersburg, MD). The enzymatic reaction was terminated by the 

addition of 100 μl per well of 3 M H3PO4. Quantification of the DMO protein was 

accomplished by interpolation from a DMO protein standard curve that ranged from 

0.313 – 10 ng/ml. 
Monsanto Company 12-CT-244U 335 of 620 

D.6.2. PAT (bar) Protein 

Affinity purified goat anti PAT (bar) antibodies were diluted in coating buffer (15 mM 

Na2CO3, 35 mM NaHCO3, and 150 mM sodium chloride) to a final concentration of 

4 μg/ml and immobilized onto 96 well microtiter plates, followed by incubation in a 4° C 

refrigerator for >12 h. Prior to each step in the assay, plates were washed with 1× PBST. 

Plates were blocked with the addition of 200 μl per well of blocking buffer 

(1× PBST+1% BSA) for 60 to 70 minutes at 37° C. PAT (bar) protein standard or 

sample extract was added at 100 μl per well and incubated for 60 to 70 minutes at 37° C. 

Biotinylated goat anti-PAT (bar) antibodies diluted in 1 × PBST + 0.1% BSA were added 

at 100 μl per well and incubated for 60 to 70 minutes at 37° C. NeutrAvidin HRP 

conjugate was added at 100 μl per well and incubated for 60 to 70 minutes at 37° C. 

Plates were developed by adding 100 μl per well of TMB substrate. The enzymatic 

reaction was terminated by the addition of 100 μl per well of 3 M H3PO4. Quantification 

of the PAT (bar) protein was accomplished by interpolation from a PAT (bar) protein 

standard curve that ranged from 0.625 – 20 ng/ml. 

D.7. Moisture Analysis 

Tissue moisture content was determined using an IR-200 Moisture Analyzer (Denver 

Instrument Company, Arvada, CO). A homogeneous tissue-specific site pool (TSSP) 

was prepared consisting of samples of a given tissue type grown at a given site. The 

average percent moisture for each TSSP was calculated from triplicate analyses. A TSSP 

Dry Weight Conversion Factor (DWCF) was calculated as follows: 

DWCF  ൌ 1 െ ൬

Mean% TSSP Moisture

100 ൰

The DWCF was used to convert protein levels assessed on a μg/g fresh weight (fw) basis 

into levels reported on a μg/g dry weight (dw) basis using the following calculation: 

Protein Level in Dry Weight  ൌ  Protein Level Fresh Weight

DWCF

Due to a limited amount of tissue, pollen was not analyzed for moisture content. 

Therefore, no dry weight calculation was performed and pollen was reported on a μg/g 

fresh weight (fw) basis only. 

The protein levels (ng/ml) that were reported to be less than or equal to the limit of 

detection (LOD) or less than the limit of quantitation (LOQ) on a fresh weight basis were 

not reported on a dry weight basis. 

D.8. Data Analyses 

All MON 88701 DMO and PAT (bar) ELISA plates were analyzed on a SPECTRAmax 

Plus 384 (Molecular Devices, Sunnyvale, CA) microplate spectrophotometer, using a 

dual wavelength detection method. All protein concentrations were determined by 

optical absorbance at a wavelength of 450 nm with a simultaneous reference reading of 
Monsanto Company 12-CT-244U 336 of 620 

620-650 nm. Data reduction analyses were performed using Molecular Devices 

SOFTmax PRO GxP version 5.0.1 software. Absorbance readings and protein standard 

concentrations were fitted with a four-parameter logistic curve fit. Following the 

interpolation from the standard curve, the amount of protein (ng/ml) in the tissue was 

converted to a μg/g fw basis for data that were greater than or equal to the LOQ. This 

conversion utilized a sample dilution factor, and tissue-to-buffer ratio. The protein values 

in μg/g fw were also converted to μg/g dw by applying the DWCF (except pollen). 

Microsoft Excel 2007 (Microsoft, Redmond, WA) was used to calculate the protein levels 

in all cotton tissues. The sample means, standard deviations, and ranges were also 

calculated by Microsoft Excel 2007. All protein expression levels were rounded to two 

significant figures. 

Any MON 88701 sample extracts that resulted in an unexpectedly negative result by 

ELISA analysis was re extracted twice for the protein of interest and re analyzed by 

ELISA to confirm the results. Samples with confirmed unexpected results were omitted 

from all calculations. 
Monsanto Company 12-CT-244U 337 of 620 

Appendix E: Materials, Methods, and Individual Site Results for Compositional 

Analysis of MON 88701 Cottonseed 

E.1. Materials 

Cottonseed from MON 88701 (Seed Lot Number 11268129) treated with dicamba and 

glufosinate (T) and MON 88701 not treated with dicamba or glufosinate (NT) and the 

conventional control (Seed Lot Number 11268128) was evaluated. The conventional 

control has background genetics similar to that of MON 88701 but does not contain either 

the dicamba mono-oxygenase (DMO) or phosphinothricin N-acetyltransferase (PAT) 

proteins. The commercial reference varieties were nine conventional cotton varieties 

(Table E-1). 

Table E-1. Commercial Reference Varieties 

Material Name Seed Lot No. Field Sites1

 

SG 125 11266155 ARTI, SCEK, NCBD, TXPL 

DP 435 11266762 ARTI, NCBD, TXPL 

DP 5415 11266157 ARTI, LACH, KSLA 

FM 989 10001810 ARTI, LACH, GACH 

Delta Opal 11266158 SCEK, GACH, NCBD 

Atlas 11266765 SCEK, TXPL, KSLA, NMLC 

ST 474 11266156 GACH, LACH, NCBD, NMLC, SCEK 

DP 565 11266764 GACH, LACH, KSLA, NMLC 

NM 1517-99 11268233 TXPL, KSLA 

 

1

Field sites described in Section VII.A. 

E.2. Characterization of the Materials 

The identities of MON 88701(T) and MON 88701(NT), the conventional control, and 

commercial reference varieties were confirmed by verifying the chain of custody 

documentation prior to analysis. To further confirm the identities of MON 88701(T) and 

MON 88701(NT), the conventional control, and commercial reference varieties, eventspecific polymerase chain reaction (PCR) analyses were conducted on the harvested, 

acid-delinted cottonseed from each site to confirm the presence or absence of the 

MON 88701 event. 

E.3. Field Production of the Samples 

Cottonseed samples were collected from MON 88701(T) and MON 88701(NT) and the 

conventional control Coker 130 grown in a 2010 U.S. field production. Four different 

conventional cotton varieties, known as reference substances, were included at each site 

of the field production to provide data on natural variability of each compositional 
Monsanto Company 12-CT-244U 338 of 620 

component analyzed. The field production was conducted at eight sites: Arkansas 

(ARTI), Georgia (GACH), Kansas (KSLA), Louisiana (LACH), North Carolina 

(NCBD), New Mexico (NMLC), South Carolina (SCEK) and Texas (TXPL). The sites 

were planted in a randomized complete block design with four blocks per site. 

MON 88701 plots were treated at the 3-5 leaf stage with glufosinate herbicide at the label 

rate (0.5 lbs a.i. /acre) and at the 6-10 leaf stage with dicamba herbicide at the label rate 

(0.5 lbs a.e./acre). T/C/R substances were grown under normal agronomic field 

conditions for their respective geographic regions. Cottonseed samples were harvested 

and ginned from all plots and shipped at ambient temperature to Monsanto Company (St. 

Louis, Missouri). The samples were acid-delinted and a subsample was obtained from 

each for compositional analyses. These subsamples were ground and stored in a freezer 

set to maintain 20°C until their shipment on dry ice to Covance Laboratories Inc. 

(Madison, Wisconsin) for analysis. The label on the samples shipped listed the protocol 

(study) number, tissue type, material name, storage conditions, and a unique sample ID 

number.

E.4. Summary of Analytical Methods 

Harvested, acid-delinted cottonseed samples were analyzed by Covance Laboratories Inc. 

Upon receipt, the samples were stored in a freezer set to maintain -20 °C until their use. 

Nutrients assessed in this analysis included proximates (ash, fat, moisture, protein, and 

carbohydrates and calories by calculation), acid detergent fiber (ADF), neutral detergent 

fiber (NDF), crude fiber (CF), total dietary fiber (TDF), amino acids (AA), fatty acids 

(C8-C22), minerals (Ca, Cu, Fe, Mg, Mn, P, K, Na, Zn) and vitamin E (α-tocopherol). 

Anti-nutrients analyzed included gossypol and cyclopropenoid fatty acids (CPFA). 

E.4.1. Acid Detergent Fiber 

The ANKOM2000 Fiber Analyzer automated the process of removal of proteins, 

carbohydrates, and ash. Fats and pigments were removed with an acetone wash prior to 

analysis. The fibrous residue that is primarily cellulose, lignin, and insoluble protein 

complexes remained in the Ankom filter bag, and were determined gravimetrically. 

(Komarek, et al., 1993; USDA, 1970). The results were reported on fresh weight basis. 

The limit of quantitation was 0.100%. 

 
Monsanto Company 12-CT-244U 339 of 620 

E.4.2. Amino Acid Composition 

The following 18 amino acids were analyzed: 

Total threonine Total aspartic acid (including asparagine) 

Total serine Total tyrosine 

Total phenylalanine Total glutamic acid (including glutamine) 

Total proline Total histidine 

Total glycine Total lysine 

Total alanine Total arginine 

Total valine Total tryptophan 

Total isoleucine Total methionine 

Total leucine Total cystine (including cysteine) 

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at 

approximately 106-110oC. Phenol was added to the 6N hydrochloric acid to prevent 

halogenation of tyrosine. Cystine and cysteine are converted to S-2-

carboxyethylthiocysteine by the addition of dithiodipropionic acid. Tryptophan was 

hydrolyzed from proteins by heating at approximately 110oC in 4.2N sodium 

hydroxide for 20 hours. The samples were analyzed by HPLC after pre-injection 

derivatization. The primary amino acids were derivatized with o-phthalaldehyde (OPA) 

and the secondary amino acids are derivatized with fluorenylmethyl chloroformate 

(FMOC) before injection. (AOAC, 2011a; Barkholt and Jensen, 1989; Henderson and 

Brooks, 2010; Henderson, et al., 2000; Schuster, 1988). The results were reported on 

fresh weight basis. The limit of quantitation was 0.100 mg/g. 

Reference Standards: 

Component Manufacturer Lot No. Purity (%) 

 

L-Alanine Sigma-Aldrich 1440397 99.9 

L-Arginine Monohydrochloride Sigma-Aldrich 1361811 100 

L-Aspartic Acid Sigma-Aldrich BCBB9274 100.6 

L-Cystine Sigma-Aldrich 1418036 99.9 

L-Glutamic Acid Sigma-Aldrich 1423805 100.2 

Glycine Sigma-Aldrich 1119375 100 

L-Histidine Monohydrochloride Monohydrate Sigma-Aldrich BCBB1348 99.9 

L-Isoleucine Sigma-Aldrich 1423806 100 

L-Leucine Sigma-Aldrich BCBB1733 98.6 

L-Lysine Monohydrochloride Sigma-Aldrich 1362380 100.2 

L-Methionine Sigma-Aldrich 1423807 99.9 

L-Phenylalanine Sigma-Aldrich BCBB9200 100 

L-Proline Sigma-Aldrich 1414414 99.7 

L-Serine Sigma-Aldrich 1336081 99.9 

L-Threonine Sigma-Aldrich 1402329 100 

L-Tryptophan Sigma-Aldrich BCBB1284 99.8 

L-Tyrosine Sigma-Aldrich BCBB5393 99.5 

L-Valine Sigma-Aldrich 1352709 100 

 
Monsanto Company 12-CT-244U 340 of 620 

E.4.3. Ash 

The sample was placed in an electric furnace at 550 °C and ignited. The nonvolatile 

matter remaining was quantified gravimetrically and calculated to determine percent ash 

(AOAC, 2011b). The limit of quantitation was 0.100%. 

E.4.4. Calories 

Calories were calculated using the Atwater factors with the fresh weight-derived data and 

the following equation: 

calories (Kcal/100g) = (4 × % protein) + (9 × % fat) + (4 × % carbohydrates) 

The limit of quantitation was calculated as 2.00 Kcalories/100g on a fresh weight basis 

(Code of Federal Regulation, Title 21, Part 101.9, pp. 24-25). 

E.4.5. Carbohydrates 

The total carbohydrate level was calculated by difference using the fresh weight-derived 

data and the following equation: 

% carbohydrates = 100% - (% protein + % fat + % moisture + % ash) 

The results were reported on fresh weight basis (USDA, 1973). The limit of quantitation 

was 0.100%. 

E.4.6. Crude Fiber 

Crude fiber was quantitated as the loss on ignition of dried residue remaining after 

digestion of the sample with 1.25% sulfuric acid and 1.25% sodium hydroxide solutions 

under specific conditions (AOAC, 2011c). The results were reported on fresh weight 

basis. The limit of quantitation was 0.100%. 

E.4.7. Fat by Soxhlet Extraction

The sample was weighed into a cellulose thimble containing sodium sulfate and dried to 

remove excess moisture. Pentane was dripped through the sample to remove the fat. The 

extract was then evaporated, dried, and weighed (AOAC, 2011d; e). The results were 

reported on fresh weight basis. The limit of quantitation was 0.100%. 
Monsanto Company 12-CT-244U 341 of 620 

E.4.8. Cyclopropenoid Fatty Acids 

The total lipid fraction was extracted from the sample using chloroform and methanol. A 

portion of the lipid fraction was then saponified with a mild alkaline hydrolysis. The free 

fatty acids were extracted with ethyl ether and hexane. The free fatty acids were then 

converted to their phenacyl derivatives with 2-bromoacetophenone. The derivatives were 

quantitated on a high-performance liquid chromatography system equipped with an 

ultraviolet detector. The amount of malvalic, sterculic and dihydrosterculic acids were 

determined by comparison to an external calibration curves of similarly derivatized 

reference standards (Wood, 1986). The results were expressed on a fresh weight basis. 

The limit of quantitation was 50.0 μg/g. 

Reference Standards: 

 Monsanto, Malvalic Acid, 100%, Lot Number GLP-0208-12964-A 

 Monsanto, Sterculic Acid, 99%, Lot Number GLP-0208-12963-A 

 Monsanto, Dihydrosterculic, 98%, Lot Number GLP-0311-14467-A 

E.4.9. Fatty Acids 

The lipid was extracted and saponified with 0.5 N methanolic sodium hydroxide, and 

methylated with 14% boron trifluoride in methanol. The resulting methyl esters of the 

fatty acids were extracted with heptane containing an internal standard. The methyl 

esters of the fatty acids were analyzed by gas chromatography using external standards 

for quantitation (AOCS, 1997; 2001; 2009a; c). The results were reported on fresh 

weight basis. The limit of quantitation was 0.0200%. 

 
Monsanto Company 12-CT-244U 342 of 620 

Reference Standards: 

Component Lot Number Component Weight 

(%) 

Purity 

(%) 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 1 

JY20-U 

Methyl Octanoate 16.66 99.6 

Methyl Decanoate 16.66 99.6 

Methyl Laurate 16.66 99.8 

Methyl Myristate 16.66 99.8 

Methyl Palmitoleate 16.66 99.7 

Methyl Linolenate 16.66 99.5 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 2 

AU16-U 

Methyl Arachidate 33.33 99.6 

Methyl 11-Eicosenoate 33.33 99.5 

Methyl Arachidonate 33.33 99.6 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 3 

J28-U 

Methyl Myristoleate 12.5 99.5 

Methyl Pentadecanoate 12.5 99.6 

Methyl 10-Pentadecenoate 12.5 99.5 

Methyl Heptadecanoate 12.5 99.6 

Methyl 10-Heptadecenoate 12.5 99.5 

Methyl 11-14 Eicosadienoate 12.5 99.6 

Methyl Behenate 12.5 99.8 

Methyl 11-14-17 

Eicosatrienoate 12.5 99.5 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 3 

F15-V 

Methyl Myristoleate 12.5 99.5 

Methyl Pentadecanoate 12.5 99.6 

Methyl 10-Pentadecenoate 12.5 99.5 

Methyl Heptadecanoate 12.5 99.6 

Methyl 10-Heptadecenoate 12.5 99.5 

Methyl 11-14 Eicosadienoate 12.5 99.6 

Methyl Behenate 12.5 99.8 

Methyl 11-14-17 

Eicosatrienoate 12.5 99.5 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 4 

MA30-U 

Methyl Palmitate 27.0 99.6 

Methyl Stearate 19.0 99.5 

Methyl Oleate 27.0 99.8 

Methyl Linoleate 27.0 99.8 

Nu-Chek Prep GLC 

Reference Standard 

Hazelton No. 4 

JA31-V 

Methyl Palmitate 27.0 99.7 

Methyl Stearate 19.0 99.7 

Methyl Oleate 27.0 99.8 

Methyl Linoleate 27.0 99.8 

Nu-Chek Prep Methyl 

Gamma Linolenate U-63M-M18-U Not applicable Not applicable >99 

Nu-Chek Prep Methyl 

Gamma Linolenate U-63M-N2-U Not applicable Not applicable >99 

Nu-Chek Prep 

Methyl Tridecanoate N-13M-F16-V Not applicable Not applicable >99 

Nu-Chek Prep 

Methyl Tridecanoate N-13M-MA25-T Not applicable Not applicable >99 

 
Monsanto Company 12-CT-244U 343 of 620 

E.4.10. Free and Total Gossypol 

For free gossypol, the sample was extracted with aqueous acetone. The solution was then 

filtered and the free gossypol was reacted with aniline. For total gossypol analysis, the 

sample was extracted using a complexing reagent containing acetic acid, 

3-amino-1-propanol, and dimethylformamide. The solution was then filtered and the 

total gossypol was reacted with aniline. For both analyses, the dianilinogossypol was 

quantitated spectrophotometrically using a standard curve (AOCS, 2011a; b) The results 

were reported on fresh weight basis. The limit of quantitation was 0.00200%. 

Reference Standard: 

 Sigma-Aldrich, Gossypol, 97.7%, Lot Number 059K4046 

E.4.11. ICP Emission Spectrometry 

The sample was dried, precharred, and ashed overnight in a muffle furnace set to 

maintain 500 C. The ashed sample was re-ashed with nitric acid, treated with 

hydrochloric acid, taken to dryness, and put into a solution of 5% hydrochloric acid. The 

amount of each element was determined at appropriate wavelengths by comparing the 

emission of the unknown sample, measured on the inductively coupled plasma 

spectrometer, with the emission of the standard solutions (AOAC, 2011f; g). The results 

were reported on fresh weight basis. 

Inorganic Ventures Reference Standards and Limits of Quantitation

Mineral Lot Numbers

Concentration 

(μg/mL)

Limit of 

Quantitation 

(ppm) 

Calcium E2-MEB360079MCA, E2-MEB360081 200, 1000 20.0

Copper E2-MEB360079MCA, E2-MEB360080MCA 2.00, 10.0 0.500

Iron E2-MEB360079MCA, E2-MEB360082 10.0, 50.0 2.00

Magnesium E2-MEB360079MCA, E2-MEB360080MCA 50.0, 250 20.0

Manganese E2-MEB360079MCA, E2-MEB360080MCA 2.00, 10.0 0.300

Phosphorus E2-MEB360079MCA, E2-MEB360081 200, 1000 20.0

Potassium E2-MEB360079MCA, E2-MEB360081 200, 1000 100

Sodium E2-MEB360079MCA, E2-MEB360081 200, 1000 100

Zinc E2-MEB360079MCA, E2-MEB360080MCA 10.0, 50.0 0.400

 
Monsanto Company 12-CT-244U 344 of 620 

E.4.12. Moisture 

The sample was dried in a vacuum oven at approximately 100 C to a constant weight. 

The moisture weight loss was determined and converted to percent moisture (AOAC, 

2011h; i). The results were reported on fresh weight basis. The limit of quantitation was 

0.100%. 

E.4.13. Neutral Detergent Fiber, Enzyme Method 

The ANKOM2000 Fiber Analyzer automated the process of the removal of proteins, 

carbohydrates, and ash. The fats and pigments were removed with an acetone wash prior 

to analysis. Hemicellulose, cellulose, lignin, and insoluble protein fraction were left in 

the filter bag and determined gravimetrically (AACC, 1999; Komarek et al., 1994; 

USDA, 1970). The results were reported on fresh weight basis. The limit of quantitation 

was 0.100%. 

E.4.14. Protein 

The protein and other organic nitrogen in the sample were converted to ammonia by 

digesting the sample with sulfuric acid containing a catalyst mixture. The acid digest was 

made alkaline. The ammonia was distilled and then titrated with a previously 

standardized acid. The percent nitrogen was calculated and converted to equivalent 

protein using the factor 6.25 (AOAC, 2011j; k; AOCS, 2009a) The results were reported 

on fresh weight basis. The limit of quantitation was 0.100%. 

E.4.15. Total Dietary Fiber 

Duplicate samples were gelatinized with -amylase and digested with enzymes to break 

down starch and protein. Ethanol was added to each sample to precipitate the soluble 

fiber. The sample was filtered, and the residue was rinsed with ethanol and acetone to 

remove starch and protein degradation products and moisture. Protein content was 

determined for one of the duplicates; ash content was determined for the other. The total 

dietary fiber in the sample was calculated using protein and ash values (AOAC, 2011j). 

The results were reported on fresh weight basis. The limit of quantitation was 1.00%. 

E.4.16. Vitamin E 

The sample was saponified to break down any fat and release vitamin E. The saponified 

mixture was extracted with ethyl ether and then quantified by high-performance liquid 

chromatography using a silica column (Cort, et al., 1983; McMurray, et al., 1980; Speek, 

et al., 1985). The results were reported on fresh weight basis. The limit of quantitation 

was 0.500 mg/100g. 

Note: Alpha tocopherol is part of a mixed standard which also includes beta, delta, and 

gamma isomers. The reference standard material for those isomers may contain small 

amounts of alpha tocopherol. All reference standards that contributed to the alpha 

tocopherol concentration are listed below. 
Monsanto Company 12-CT-244U 345 of 620 

Reference Standards: 

 USP, Alpha Tocopherol, 98.9%, Lot Number N0F068 

 Acros Organics, D-gamma-Tocopherol, 99.4%, A0083534 

 Sigma-Aldrich, (+)-delta-Tocopherol, 92%, 090M1916V 

E.5. Data Processing and Statistical Analysis 

After compositional analyses were performed, data spreadsheets containing individual 

values for each analysis were sent to Monsanto Company (St. Louis, Missouri) for 

review. Data were then transferred to Certus International (Chesterfield, MO) where they 

were converted into the appropriate units and statistically analyzed. The formulas that 

were used for re-expression of composition data for statistical analysis are listed in 

Table E-2. 
Monsanto Company 12-CT-244U 346 of 620 

Table E-2. Re-expression Formulas for Statistical Analysis of Composition Data 

Component From (X) To Formula1

Proximates (excluding Moisture 

and Calories), Fiber, Gossypol % fw % dw X/d

Calories Kcal/100g fw Kcal/100g dw X/d

Copper, Iron, Manganese, Zinc ppm fw mg/kg dw X/d

Calcium, Magnesium, 

Phosphorus, Potassium, Sodium ppm fw % dw X/(104

d)

Vitamin E mg/100g fw mg/kg dw 10(X/d)

Amino Acids (AA) mg/g fw % dw X/(10d)

Sterculic, Malvalic, and 

Dihydrosterculic Acids 2 μg/g fw % fw X/104

Fatty Acids (FA) % fw % Total FA

(100)Xj/X, 

for each FAj

where X is 

over all the 

FA

1

d is the fraction of the sample that is dry matter.

2

Sterculic Acid, Malvalic Acid and Dihydrosterculic Acid were first converted to % fw 

as an intermediate step for final re-expression as % Total FA.

In order to complete a statistical analysis for a compositional component in this study, at 

least 50% of the values for a component had to be greater than the assay limit of 

quantitation (LOQ). Components with more than 50% of observations below the assay 

LOQ were excluded from summaries and analysis. The following 13 components with 

more than 50% of the observations below the assay LOQ were excluded: 8:0 caprylic 

acid, 10:0 capric acid, 12:0 lauric acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 

15:1 pentadecenoic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic acid, 18:3 gammalinolenic acid, 20:1 eicosenoic acid; 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, and 

20:4 arachidonic acid. 

If less than 50% of the observations for a component were below the LOQ, individual 

analyses that were below the LOQ were assigned a value equal to one-half the LOQ. In 

this study 187 values for 22:0 behenic acid were assigned a value of 0.010% fw and 187 

values for sodium were assigned a value of 50.00 ppm fw. 

The data were assessed for potential outliers using a studentized PRESS residuals 

calculation. A PRESS residual is the difference between any value and its value 

predicted from a statistical model that excludes the data point. The studentized version 

scales these residuals so that the values tend to have a standard normal distribution when 

outliers are absent. Thus, most values are expected to be between  3. Extreme data 

points that are also outside of the  6 studentized PRESS residual range are considered 

for exclusion, as outliers, from the final analyses. One sodium value from the 

commercial control at the ARTI site and one sodium value from a commercial reference 
Monsanto Company 12-CT-244U 347 of 620 

at the ARTI site were extreme data points that were outside the  6 studentized PRESS 

residual range and were removed from the statistical analysis. 

All cottonseed components were statistically analyzed using a mixed model analysis of 

variance. The eight replicated field sites were analyzed individually and as a combined 

data set. Individual site analysis mean comparison tests were not conducted on site ARTI 

sodium content because only one Coker 130 replicate was available at that site. 

Analyses of the combined replicated sites were performed using model (1). 

(1) Yijk = U + Ti + Lj + B(L)jk + LTij + eijk, 

where Yijk = unique individual observation, U = overall mean, Ti = substance effect, 

Lj = random site effect, B(L)jk = random block within site effect, 

LTij = random site by substance interaction effect, and eijk = residual error. 

Individual sites were also analyzed separately. Individual site analyses were performed 

using model (2). 

(2) Yij = U + Ti + Bj + eij, 

where Yij = unique individual observation, U = overall mean, Ti = substance effect, 

Bj = random block effect, and eij = residual error. 

Pairwise comparisons between the test and control materials were defined within the 

ANOVA and tested using t-tests. The variability from the ANOVA was used to compute 

the standard error of the difference and to conduct the t-tests for the comparisons. 

For each compositional component, a range of observed values and a 99% tolerance 

interval were calculated. A tolerance interval is an interval that one can claim, with a 

specified degree of confidence, contains at least a specified proportion, p, of an entire 

sampled population for the parameter measured. The calculated tolerance intervals are 

expected to contain, with 95% confidence, 99% of the quantities expressed in the 

population of conventional cotton. Each tolerance interval estimate was based upon the 

average observation for each unique reference material. Because negative quantities are 

not possible, negative calculated lower tolerance bounds were set to zero. 

SAS® (Version 9.2) software was used to generate all summary statistics and perform all 

analyses. 

Report tables present p-values from SAS as either <0.001 or the actual value truncated to 

three decimal places. 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Convent

 Difference (MON 88701 minus Control)

Analytical Component (Unis)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.62 (0.087) 4.74 (0.12) -0.13 (0.15) -0.55, 0.29 0.440 

(4.51 - 4.74) (4.49 - 5.00) (-0.49 - 0.047) 

Calories (Kcal/100g) 495.41 (3.00) 488.30 (3.88) 7.11 (3.90) -3.73, 17.94 0.142 

(487.88 - 504.08) (487.70 - 494.60) (4.92 - 16.38) 

Carbohydrates 45.08 (0.58) 46.35 (0.81) -1.27 (0.96) -3.94, 1.39 0.254 

(43.42 - 46.31) (45.03 - 47.37) (-3.95 - -0.33) 

Moisture (% fw) 7.10 (0.27) 7.63 (0.35) -0.53 (0.35) -1.49, 0.43 0.197 

(6.71 - 7.58) (7.32 - 7.40) (-0.69 - -0.36) 

Protein 27.53 (0.24) 27.04 (0.33) 0.49 (0.41) -0.65, 1.63 0.297 

(27.16 - 28.11) (26.97 - 27.11) (0.12 - 0.65) 

Total Fat 22.76 (0.59) 21.50 (0.78) 1.26 (0.81) -0.98, 3.50 0.193 

(21.32 - 24.40) (21.15 - 22.89) (0.69 - 3.25) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 24.81 (0.32) 27.53 (0.45) -2.71 (0.55) -4.23, -1.20 0.007 

(24.44 - 25.20) (26.57 - 28.49) (-3.98 - -2.13) 

Crude Fiber 18.33 (0.90) 19.47 (1.20) -1.14 (1.26) -4.64, 2.36 0.417 

(15.97 - 20.56) (19.33 - 19.85) (-3.36 - -0.40) 

Neutral Detergent Fiber 31.27 (0.79) 32.89 (1.06) -1.61 (1.13) -4.75, 1.53 0.227 

(29.99 - 32.89) (30.67 - 34.42) (-4.43 - 0.095) 

Total Dietary Fiber 40.85 (1.06) 41.67 (1.50) -0.82 (1.83) -5.91, 4.27 0.678 

(39.82 - 42.13) (40.50 - 42.84) (-0.70 - 1.09) 

Amino Acid (% dw)Alanine 1.05 (0.015) 1.02 (0.018) 0.029 (0.017) -0.018, 0.077 0.161 

(1.01 - 1.09) (0.99 - 1.05) (0.011 - 0.036) 

Arginine 3.00 (0.052) 3.02 (0.073) -0.018 (0.084) -0.25, 0.21 0.840 

(2.86 - 3.07) (2.89 - 3.13) (-0.098 - -0.032) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.41 (0.045) 2.32 (0.060) 0.087 (0.065) -0.093, 0.27 0.252 

(2.29 - 2.48) (2.19 - 2.42) (-0.031 - 0.10) 

Cystine 0.40 (0.010) 0.37 (0.014) 0.034 (0.017) -0.015, 0.082 0.124 

(0.38 - 0.42) (0.35 - 0.39) (0.014 - 0.074) 

Glutamic Acid 4.82 (0.099) 4.51 (0.14) 0.31 (0.17) -0.15, 0.78 0.134 

(4.61 - 5.07) (4.34 - 4.67) (0.11 - 0.27) 

Glycine 1.10 (0.020) 1.08 (0.026) 0.027 (0.028) -0.052, 0.11 0.397 

(1.05 - 1.14) (1.03 - 1.11) (-0.015 - 0.021) 

Histidine 0.74 (0.016) 0.74 (0.023) 0.0028 (0.026) -0.071, 0.076 0.919 

(0.71 - 0.76) (0.71 - 0.77) (-0.0019 - -0.00095) 

Isoleucine 0.89 (0.018) 0.91 (0.024) -0.010 (0.025) -0.079, 0.058 0.696 

(0.87 - 0.92) (0.88 - 0.93) (-0.027 - -0.0076) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.53 (0.027) 1.50 (0.037) 0.029 (0.042) -0.086, 0.14 0.524 

(1.47 - 1.55) (1.44 - 1.55) (-0.0060 - 0.032) 

Lysine 1.22 (0.044) 1.23 (0.058) -0.0029 (0.062) -0.18, 0.17 0.965 

(1.15 - 1.27) (1.19 - 1.26) (-0.041 - -0.016) 

Methionine 0.39 (0.015) 0.35 (0.021) 0.041 (0.025) -0.029, 0.11 0.181 

(0.35 - 0.43) (0.35 - 0.36) (-0.014 - 0.087) 

Phenylalanine 1.43 (0.027) 1.41 (0.038) 0.016 (0.045) -0.11, 0.14 0.737 

(1.36 - 1.45) (1.34 - 1.48) (-0.027 - 0.022) 

Proline 0.97 (0.021) 1.00 (0.028) -0.027 (0.030) -0.11, 0.057 0.417 

(0.95 - 1.00) (0.98 - 1.02) (-0.027 - -0.024) 

Serine 1.11 (0.022) 1.03 (0.031) 0.089 (0.038) -0.016, 0.19 0.079 

(1.07 - 1.19) (0.99 - 1.06) (0.011 - 0.096) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.88 (0.012) 0.85 (0.017) 0.029 (0.021) -0.028, 0.087 0.230 

(0.85 - 0.90) (0.82 - 0.88) (-0.0045 - 0.033) 

Tryptophan 0.41 (0.0062) 0.42 (0.0087) -0.012 (0.011) -0.042, 0.017 0.306 

(0.40 - 0.42) (0.40 - 0.44) (-0.041 - 0.0060) 

Tyrosine 0.82 (0.015) 0.79 (0.021) 0.030 (0.026) -0.042, 0.10 0.313 

(0.79 - 0.84) (0.76 - 0.82) (0.0011 - 0.028) 

Valine 1.19 (0.021) 1.21 (0.027) -0.018 (0.026) -0.090, 0.055 0.537 

(1.14 - 1.23) (1.17 - 1.24) (-0.030 - -0.016) 

Fatty Acid (% Total FA)14:0 Myristic 0.77 (0.0085) 0.78 (0.012) -0.0049 (0.013) -0.041, 0.031 0.723 

(0.76 - 0.79) (0.77 - 0.78) (-0.029 - 0.0018) 

16:0 Palmitic 25.16 (0.10) 24.98 (0.14) 0.18 (0.18) -0.31, 0.67 0.360 

(25.01 - 25.28) (24.92 - 25.05) (0.028 - 0.094) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.53 (0.0052) 0.52 (0.0073) 0.017 (0.0089) -0.0082, 0.041 0.137 

(0.52 - 0.54) (0.52 - 0.52) (-0.00060 - 0.017) 

18:0 Stearic 2.68 (0.026) 2.51 (0.036) 0.17 (0.045) 0.044, 0.29 0.019 

(2.65 - 2.72) (2.45 - 2.57) (0.083 - 0.22) 

18:1 Oleic 14.81 (0.11) 14.68 (0.15) 0.12 (0.17) -0.34, 0.59 0.501 

(14.46 - 15.08) (14.58 - 14.70) (-0.24 - 0.18) 

18:2 Linoleic 54.73 (0.22) 55.31 (0.31) -0.59 (0.37) -1.62, 0.45 0.189 

(54.24 - 55.29) (55.26 - 55.38) (-0.45 - 0.037) 

18:3 Linolenic 0.14 (0.0021) 0.13 (0.0030) 0.015 (0.0035) 0.0056, 0.025 0.012 

(0.14 - 0.15) (0.12 - 0.14) (0.0064 - 0.021) 

20:0 Arachidic 0.31 (0.0079) 0.29 (0.011) 0.023 (0.011) -0.0087, 0.054 0.115 

(0.31 - 0.32) (0.27 - 0.31) (0.0032 - 0.046) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.14 (0.0026) 0.15 (0.0033) -0.015 (0.0031) -0.024, -0.0065 0.008 

(0.13 - 0.14) (0.15 - 0.16) (-0.019 - -0.012) 

MineralCalcium (% dw) 0.15 (0.0035) 0.12 (0.0050) 0.028 (0.0061) 0.011, 0.045 0.010 

(0.14 - 0.16) (0.12 - 0.13) (0.024 - 0.035) 

Copper (mg/kg dw) 9.66 (0.34) 9.64 (0.41) 0.018 (0.38) -1.03, 1.06 0.963 

(9.23 - 10.15) (8.79 - 9.79) (-0.57 - 0.58) 

Iron (mg/kg dw) 75.27 (5.63) 80.76 (7.78) -5.49 (8.79) -29.91, 18.93 0.566 

(72.55 - 77.65) (72.89 - 87.72) (-15.17 - 2.25) 

Magnesium (% dw) 0.41 (0.0076) 0.40 (0.010) 0.0099 (0.011) -0.020, 0.040 0.413 

(0.40 - 0.42) (0.38 - 0.41) (0.0077 - 0.016) 

Manganese (mg/kg dw) 13.27 (0.63) 11.50 (0.89) 1.77 (1.06) -1.18, 4.73 0.171 

(12.58 - 13.63) (11.34 - 11.55) (1.03 - 1.95) 
Monsanto Company 12-CT-244U 

Table E-3. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.83 (0.012) 0.84 (0.016) -0.015 (0.018) -0.066, 0.036 0.450 

(0.82 - 0.84) (0.82 - 0.87) (-0.027 - 0.0054) 

Potassium (% dw) 1.18 (0.029) 1.11 (0.040) 0.071 (0.047) -0.059, 0.20 0.204 

(1.16 - 1.20) (1.06 - 1.15) (0.028 - 0.14) 

Sodium (% dw) 0.027 (0.0038) ND6 

(0.023 - 0.029) (0.013 - 0.013) 

Zinc (mg/kg dw) 37.08 (1.65) 40.81 (2.03) -3.72 (1.87) -8.92, 1.47 0.117 

(35.15 - 38.88) (38.63 - 40.71) (-3.48 - -3.41) 

Vitamin (mg/kg dw)Vitamin E 147.20 (2.44) 136.55 (3.46) 10.65 (4.23) -1.10, 22.40 0.065 

(144.99 - 149.40) (133.79 - 139.31) (6.47 - 15.60) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ6Not determined due to insufficient number of observations for the control.
Monsanto Company 12-CT-244U 

Table E-4. Statistical Summary of Site ARTI Cottonseed Anti-nutrients for MON 88701 (Treated) vs. Co

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0073) 0.15 (0.010) 0.0022 (0.013) -0.033, 0.037 0.867 

(0.14 - 0.16) (0.14 - 0.15) (-0.0021 - -0.00026) 

Malvalic Acid 0.37 (0.016) 0.36 (0.023) 0.0088 (0.025) -0.061, 0.078 0.742 

(0.34 - 0.39) (0.33 - 0.37) (-0.031 - 0.032) 

Sterculic Acid 0.21 (0.014) 0.20 (0.020) 0.013 (0.023) -0.052, 0.078 0.605 

(0.19 - 0.22) (0.19 - 0.20) (-0.0076 - 0.032) 

Gossypol (% dw)Free Gossypol 0.91 (0.035) 0.82 (0.049) 0.090 (0.060) -0.076, 0.26 0.207 

(0.80 - 1.02) (0.80 - 0.84) (-0.035 - 0.10) 

Total Gossypol 0.97 (0.034) 0.93 (0.044) 0.042 (0.044) -0.081, 0.16 0.399 

(0.89 - 1.04) (0.94 - 0.98) (0.0013 - 0.10) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. Co

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.53 (0.044) 4.21 (0.047) 0.32 (0.034) 0.23, 0.41 <0.001 

(4.45 - 4.57) (4.12 - 4.23) (0.31 - 0.34) 

Calories (Kcal/100g) 497.72 (2.28) 496.55 (2.63) 1.16 (3.48) -7.79, 10.12 0.751 

(489.91 - 504.20) (494.57 - 498.27) (-6.91 - 8.49) 

Carbohydrates 44.91 (0.59) 45.84 (0.68) -0.94 (0.91) -3.27, 1.39 0.348 

(43.42 - 46.94) (44.64 - 47.09) (-3.23 - 1.14) 

Moisture (% fw) 6.98 (0.15) 7.23 (0.18) -0.25 (0.23) -0.85, 0.35 0.328 

(6.42 - 7.33) (6.99 - 7.48) (-1.06 - 0.12) 

Protein 27.41 (0.33) 27.30 (0.37) 0.11 (0.37) -0.83, 1.06 0.770 

(26.87 - 27.78) (26.45 - 28.21) (-0.43 - 0.96) 

Total Fat 23.14 (0.46) 22.67 (0.53) 0.47 (0.70) -1.34, 2.28 0.536 

(21.58 - 24.46) (22.26 - 23.02) (-1.16 - 1.93) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.69 (0.52) 27.52 (0.60) -1.83 (0.79) -3.86, 0.20 0.068 

(25.04 - 26.77) (26.81 - 28.13) (-3.09 - -0.87) 

Crude Fiber 19.27 (0.68) 19.92 (0.78) -0.64 (1.00) -3.21, 1.92 0.547 

(17.10 - 20.64) (18.70 - 21.18) (-1.60 - -0.54) 

Neutral Detergent Fiber 31.47 (0.90) 33.92 (1.04) -2.44 (1.38) -5.98, 1.09 0.135 

(29.71 - 34.04) (32.79 - 35.89) (-5.13 - -1.40) 

Total Dietary Fiber 39.64 (0.86) 41.11 (1.00) -1.46 (1.32) -4.85, 1.93 0.318 

(37.72 - 41.91) (39.89 - 42.04) (-4.32 - -0.44) 

Amino Acid (% dw)Alanine 1.04 (0.019) 1.10 (0.022) -0.059 (0.025) -0.12, 0.0050 0.064 

(1.02 - 1.05) (1.06 - 1.17) (-0.13 - -0.016) 

Arginine 2.95 (0.061) 3.21 (0.067) -0.27 (0.064) -0.43, -0.10 0.008 

(2.87 - 3.02) (3.07 - 3.46) (-0.47 - -0.14) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.31 (0.043) 2.45 (0.047) -0.15 (0.044) -0.26, -0.035 0.019 

(2.24 - 2.36) (2.36 - 2.60) (-0.29 - -0.054) 

Cystine 0.40 (0.012) 0.40 (0.013) -0.0089 (0.018) -0.054, 0.036 0.636 

(0.38 - 0.42) (0.38 - 0.43) (-0.052 - 0.011) 

Glutamic Acid 4.57 (0.098) 4.96 (0.11) -0.39 (0.099) -0.65, -0.14 0.010 

(4.35 - 4.77) (4.77 - 5.21) (-0.63 - -0.17) 

Glycine 1.08 (0.020) 1.13 (0.023) -0.052 (0.026) -0.12, 0.014 0.099 

(1.06 - 1.12) (1.09 - 1.20) (-0.13 - 0.011) 

Histidine 0.73 (0.013) 0.76 (0.014) -0.029 (0.012) -0.061, 0.0030 0.067 

(0.68 - 0.76) (0.75 - 0.78) (-0.022 - -0.020) 

Isoleucine 0.90 (0.010) 0.94 (0.012) -0.040 (0.014) -0.075, -0.0042 0.034 

(0.90 - 0.91) (0.92 - 0.97) (-0.074 - -0.012) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.51 (0.022) 1.58 (0.024) -0.068 (0.021) -0.12, -0.013 0.024 

(1.49 - 1.54) (1.52 - 1.65) (-0.14 - -0.030) 

Lysine 1.24 (0.018) 1.23 (0.020) 0.0073 (0.024) -0.055, 0.069 0.775 

(1.21 - 1.28) (1.22 - 1.25) (0.0016 - 0.032) 

Methionine 0.40 (0.017) 0.42 (0.019) -0.025 (0.024) -0.086, 0.036 0.337 

(0.36 - 0.43) (0.37 - 0.46) (-0.057 - -0.011) 

Phenylalanine 1.40 (0.030) 1.49 (0.033) -0.088 (0.032) -0.17, -0.0064 0.039 

(1.37 - 1.43) (1.41 - 1.61) (-0.18 - -0.033) 

Proline 0.98 (0.020) 1.05 (0.022) -0.065 (0.022) -0.12, -0.0075 0.033 

(0.97 - 0.99) (1.03 - 1.09) (-0.097 - -0.032) 

Serine 1.03 (0.031) 1.12 (0.035) -0.090 (0.039) -0.19, 0.010 0.069 

(0.96 - 1.11) (1.08 - 1.20) (-0.18 - 0.011) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.85 (0.015) 0.90 (0.017) -0.046 (0.018) -0.092, -0.00006 0.049 

(0.83 - 0.88) (0.87 - 0.95) (-0.10 - -0.0034) 

Tryptophan 0.42 (0.011) 0.42 (0.013) 0.0013 (0.017) -0.043, 0.046 0.942 

(0.39 - 0.45) (0.39 - 0.44) (-0.044 - 0.041) 

Tyrosine 0.80 (0.011) 0.84 (0.013) -0.036 (0.013) -0.069, -0.0030 0.037 

(0.79 - 0.82) (0.81 - 0.89) (-0.068 - -0.015) 

Valine 1.21 (0.018) 1.26 (0.019) -0.053 (0.015) -0.091, -0.014 0.017 

(1.19 - 1.23) (1.23 - 1.32) (-0.090 - -0.022) 

Fatty Acid (% Total FA)14:0 Myristic 0.78 (0.0068) 0.77 (0.0079) 0.0029 (0.010) -0.024, 0.030 0.793 

(0.76 - 0.79) (0.76 - 0.78) (-0.026 - 0.018) 

16:0 Palmitic 24.40 (0.14) 24.12 (0.16) 0.28 (0.20) -0.24, 0.80 0.227 

(24.27 - 24.66) (23.78 - 24.45) (-0.11 - 0.56) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.52 (0.0058) 0.51 (0.0067) 0.011 (0.0089) -0.012, 0.034 0.268 

(0.51 - 0.54) (0.50 - 0.52) (-0.012 - 0.025) 

18:0 Stearic 2.54 (0.032) 2.43 (0.037) 0.11 (0.046) -0.0057, 0.23 0.058 

(2.45 - 2.67) (2.37 - 2.46) (0.066 - 0.21) 

18:1 Oleic 14.64 (0.098) 14.39 (0.11) 0.25 (0.13) -0.078, 0.58 0.107 

(14.39 - 14.84) (14.06 - 14.61) (-0.0098 - 0.34) 

18:2 Linoleic 55.81 (0.24) 56.59 (0.28) -0.78 (0.35) -1.67, 0.12 0.075 

(55.04 - 56.24) (56.02 - 57.32) (-1.39 - 0.12) 

18:3 Linolenic 0.15 (0.0066) 0.15 (0.0076) 0.0076 (0.010) -0.018, 0.033 0.481 

(0.15 - 0.16) (0.14 - 0.15) (0.00091 - 0.013) 

20:0 Arachidic 0.29 (0.0098) 0.28 (0.011) 0.013 (0.013) -0.021, 0.047 0.376 

(0.26 - 0.31) (0.26 - 0.29) (0.0016 - 0.015) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.14 (0.0026) 0.14 (0.0030) -0.00014 (0.0037) -0.0097, 0.0095 0.971 

(0.14 - 0.15) (0.14 - 0.15) (-0.0017 - 0.0028) 

MineralCalcium (% dw) 0.13 (0.0015) 0.11 (0.0018) 0.019 (0.0023) 0.013, 0.025 <0.001 

(0.13 - 0.13) (0.11 - 0.11) (0.014 - 0.024) 

Copper (mg/kg dw) 8.51 (0.26) 8.21 (0.30) 0.30 (0.39) -0.70, 1.31 0.473 

(8.02 - 9.13) (7.48 - 8.64) (-0.49 - 1.13) 

Iron (mg/kg dw) 75.42 (3.80) 78.00 (4.39) -2.58 (5.81) -17.51, 12.35 0.675 

(70.35 - 79.72) (75.01 - 80.40) (-8.31 - 4.71) 

Magnesium (% dw) 0.41 (0.0052) 0.38 (0.0054) 0.026 (0.0034) 0.018, 0.035 <0.001 

(0.40 - 0.41) (0.37 - 0.39) (0.021 - 0.031) 

Manganese (mg/kg dw) 13.41 (0.31) 11.51 (0.34) 1.90 (0.37) 0.95, 2.85 0.003 

(12.79 - 14.14) (10.81 - 11.75) (1.18 - 2.39) 
Monsanto Company 12-CT-244U 

Table E-5. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.78 (0.011) 0.76 (0.012) 0.018 (0.0082) -0.0028, 0.040 0.076 

(0.75 - 0.81) (0.75 - 0.79) (0.0052 - 0.029) 

Potassium (% dw) 1.21 (0.012) 1.12 (0.013) 0.090 (0.010) 0.064, 0.12 <0.001 

(1.17 - 1.24) (1.10 - 1.13) (0.075 - 0.11) 

Sodium (% dw) 0.022 (0.0045) 0.017 (0.0052) 0.0049 (0.0069) -0.013, 0.023 0.515 

(0.019 - 0.027) (0.013 - 0.022) (-0.0020 - 0.014) 

Zinc (mg/kg dw) 39.10 (0.67) 39.55 (0.75) -0.45 (0.83) -2.58, 1.68 0.610 

(37.41 - 40.18) (38.49 - 40.84) (-1.35 - 0.51) 

Vitamin (mg/kg dw)Vitamin E 151.03 (2.27) 140.12 (2.63) 10.90 (3.47) 1.97, 19.83 0.025 

(148.34 - 154.95) (133.64 - 145.15) (3.19 - 18.52) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-6. Statistical Summary of Site GACH Cottonseed Anti-nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0067) 0.12 (0.0077) 0.033 (0.010) 0.0069, 0.059 0.022 

(0.14 - 0.16) (0.11 - 0.13) (0.021 - 0.050) 

Malvalic Acid 0.37 (0.027) 0.32 (0.031) 0.044 (0.038) -0.055, 0.14 0.304 

(0.26 - 0.45) (0.31 - 0.34) (-0.052 - 0.12) 

Sterculic Acid 0.21 (0.011) 0.18 (0.013) 0.030 (0.015) -0.0072, 0.068 0.092 

(0.18 - 0.25) (0.18 - 0.20) (-0.0018 - 0.050) 

Gossypol (% dw)Free Gossypol 0.85 (0.017) 0.86 (0.019) -0.016 (0.020) -0.068, 0.037 0.474 

(0.83 - 0.88) (0.85 - 0.90) (-0.055 - 0.028) 

Total Gossypol 0.97 (0.019) 0.96 (0.020) 0.019 (0.017) -0.026, 0.063 0.324 

(0.93 - 1.01) (0.90 - 0.99) (-0.012 - 0.033) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.53 (0.063) 4.29 (0.071) 0.24 (0.078) 0.040, 0.44 0.027 

(4.25 - 4.66) (4.21 - 4.33) (0.042 - 0.34) 

Calories (Kcal/100g) 496.63 (2.75) 495.83 (3.16) 0.80 (3.87) -9.15, 10.75 0.844 

(492.91 - 499.03) (492.30 - 504.10) (-8.23 - 6.50) 

Carbohydrates 44.10 (0.71) 44.23 (0.82) -0.14 (1.09) -2.94, 2.67 0.905 

(42.20 - 44.98) (42.53 - 45.14) (-0.77 - 2.45) 

Moisture (% fw) 7.02 (0.17) 7.36 (0.19) -0.34 (0.20) -0.86, 0.18 0.153 

(6.63 - 7.36) (7.17 - 7.62) (-0.73 - 0.19) 

Protein 28.42 (0.62) 28.82 (0.72) -0.40 (0.93) -2.80, 1.99 0.681 

(26.95 - 30.82) (28.58 - 29.04) (-1.63 - -0.87) 

Total Fat 22.96 (0.55) 22.62 (0.64) 0.34 (0.81) -1.75, 2.43 0.692 

(22.34 - 23.50) (21.87 - 24.18) (-1.58 - 1.56) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 24.04 (0.55) 24.01 (0.61) 0.027 (0.65) -1.64, 1.70 0.968 

(23.86 - 24.16) (22.08 - 25.22) (-1.08 - 1.77) 

Crude Fiber 16.43 (0.24) 17.67 (0.28) -1.24 (0.37) -2.19, -0.29 0.019 

(16.06 - 17.24) (17.49 - 17.88) (-1.69 - -0.24) 

Neutral Detergent Fiber 28.04 (0.88) 30.20 (1.01) -2.15 (1.21) -5.27, 0.97 0.136 

(25.13 - 30.18) (28.87 - 32.60) (-2.93 - 0.63) 

Total Dietary Fiber 38.32 (0.42) 40.14 (0.49) -1.83 (0.63) -3.46, -0.19 0.034 

(37.62 - 38.75) (39.32 - 41.35) (-3.74 - -0.57) 

Amino Acid (% dw)Alanine 1.04 (0.011) 1.06 (0.012) -0.022 (0.014) -0.058, 0.014 0.175 

(1.02 - 1.05) (1.02 - 1.10) (-0.048 - -0.00010) 

Arginine 3.02 (0.053) 3.28 (0.060) -0.26 (0.069) -0.43, -0.082 0.013 

(2.95 - 3.10) (3.10 - 3.43) (-0.34 - -0.14) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.38 (0.037) 2.50 (0.043) -0.11 (0.053) -0.25, 0.022 0.083 

(2.32 - 2.46) (2.39 - 2.64) (-0.26 - -0.073) 

Cystine 0.43 (0.010) 0.42 (0.012) 0.0076 (0.016) -0.033, 0.048 0.651 

(0.41 - 0.44) (0.41 - 0.43) (-0.017 - 0.015) 

Glutamic Acid 4.82 (0.12) 5.07 (0.14) -0.25 (0.18) -0.71, 0.21 0.219 

(4.58 - 5.22) (4.86 - 5.43) (-0.78 - -0.017) 

Glycine 1.08 (0.018) 1.11 (0.021) -0.029 (0.027) -0.099, 0.040 0.330 

(1.06 - 1.12) (1.07 - 1.18) (-0.11 - -0.0024) 

Histidine 0.75 (0.013) 0.77 (0.014) -0.024 (0.011) -0.052, 0.0035 0.074 

(0.73 - 0.77) (0.74 - 0.80) (-0.045 - 0.0022) 

Isoleucine 0.92 (0.014) 0.95 (0.015) -0.027 (0.014) -0.063, 0.0091 0.112 

(0.91 - 0.95) (0.91 - 0.98) (-0.062 - 0.0079) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.54 (0.020) 1.58 (0.022) -0.043 (0.021) -0.097, 0.011 0.098 

(1.51 - 1.57) (1.52 - 1.65) (-0.083 - -0.0065) 

Lysine 1.22 (0.017) 1.25 (0.019) -0.033 (0.021) -0.087, 0.021 0.178 

(1.21 - 1.23) (1.19 - 1.30) (-0.073 - 0.027) 

Methionine 0.39 (0.015) 0.39 (0.017) 0.0021 (0.019) -0.046, 0.050 0.915 

(0.37 - 0.42) (0.34 - 0.44) (-0.017 - 0.027) 

Phenylalanine 1.44 (0.022) 1.53 (0.025) -0.090 (0.029) -0.16, -0.016 0.025 

(1.40 - 1.46) (1.45 - 1.58) (-0.13 - -0.049) 

Proline 1.01 (0.018) 1.07 (0.020) -0.060 (0.027) -0.13, 0.0090 0.075 

(0.98 - 1.03) (1.03 - 1.12) (-0.12 - -0.042) 

Serine 1.08 (0.029) 1.11 (0.034) -0.031 (0.045) -0.15, 0.083 0.513 

(1.03 - 1.18) (1.06 - 1.20) (-0.17 - 0.0023) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.86 (0.013) 0.88 (0.015) -0.022 (0.019) -0.071, 0.028 0.317 

(0.84 - 0.88) (0.83 - 0.92) (-0.053 - 0.0050) 

Tryptophan 0.42 (0.0061) 0.42 (0.0070) -0.0028 (0.0090) -0.026, 0.020 0.771 

(0.42 - 0.43) (0.42 - 0.43) (-0.016 - 0.011) 

Tyrosine 0.80 (0.014) 0.84 (0.016) -0.039 (0.019) -0.087, 0.0096 0.094 

(0.78 - 0.83) (0.79 - 0.87) (-0.074 - -0.015) 

Valine 1.21 (0.017) 1.26 (0.019) -0.048 (0.023) -0.11, 0.010 0.087 

(1.19 - 1.23) (1.21 - 1.30) (-0.085 - -0.027) 

Fatty Acid (% Total FA)14:0 Myristic 0.68 (0.0087) 0.72 (0.0096) -0.038 (0.0090) -0.061, -0.015 0.007 

(0.66 - 0.71) (0.71 - 0.73) (-0.049 - -0.016) 

16:0 Palmitic 22.61 (0.089) 22.73 (0.10) -0.12 (0.13) -0.46, 0.21 0.394 

(22.34 - 22.84) (22.69 - 22.78) (-0.11 - 0.093) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.50 (0.0057) 0.49 (0.0062) 0.0067 (0.0059) -0.0086, 0.022 0.312 

(0.48 - 0.52) (0.49 - 0.50) (-0.0046 - 0.021) 

18:0 Stearic 2.31 (0.031) 2.20 (0.036) 0.11 (0.047) -0.016, 0.23 0.075 

(2.29 - 2.32) (2.15 - 2.28) (0.039 - 0.17) 

18:1 Oleic 14.69 (0.11) 14.83 (0.13) -0.14 (0.17) -0.59, 0.31 0.454 

(14.51 - 14.88) (14.74 - 14.99) (-0.48 - 0.14) 

18:2 Linoleic 57.84 (0.19) 57.78 (0.22) 0.059 (0.29) -0.67, 0.79 0.843 

(57.49 - 58.22) (57.65 - 57.93) (-0.44 - 0.19) 

18:3 Linolenic 0.18 (0.0027) 0.17 (0.0030) 0.0066 (0.0033) -0.0018, 0.015 0.100 

(0.18 - 0.19) (0.17 - 0.18) (0.0051 - 0.010) 

20:0 Arachidic 0.23 (0.0029) 0.24 (0.0034) -0.0021 (0.0045) -0.014, 0.0094 0.651 

(0.23 - 0.24) (0.23 - 0.24) (-0.0063 - 0.0087) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.13 (0.0030) 0.13 (0.0035) -0.0051 (0.0046) -0.017, 0.0067 0.318 

(0.12 - 0.14) (0.13 - 0.14) (-0.0084 - 0.0036) 

MineralCalcium (% dw) 0.20 (0.0060) 0.18 (0.0065) 0.026 (0.0061) 0.010, 0.042 0.007 

(0.19 - 0.22) (0.17 - 0.19) (0.013 - 0.038) 

Copper (mg/kg dw) 10.08 (0.54) 11.01 (0.61) -0.93 (0.71) -2.75, 0.89 0.246 

(8.74 - 11.06) (10.09 - 11.33) (-2.59 - 0.69) 

Iron (mg/kg dw) 78.87 (5.50) 74.39 (6.35) 4.48 (8.40) -17.11, 26.07 0.616 

(74.42 - 82.99) (72.65 - 76.27) (3.93 - 8.73) 

Magnesium (% dw) 0.43 (0.0033) 0.40 (0.0038) 0.027 (0.0046) 0.015, 0.039 0.002 

(0.41 - 0.43) (0.39 - 0.40) (0.015 - 0.041) 

Manganese (mg/kg dw) 13.34 (0.29) 12.56 (0.33) 0.78 (0.44) -0.35, 1.90 0.135 

(12.74 - 13.60) (11.96 - 13.28) (-0.53 - 1.64) 
Monsanto Company 12-CT-244U 

Table E-7. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Treated) vs. Conventiona

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.79 (0.0090) 0.78 (0.010) 0.0090 (0.011) -0.019, 0.037 0.440 

(0.75 - 0.82) (0.77 - 0.79) (-0.024 - 0.029) 

Potassium (% dw) 1.09 (0.010) 1.08 (0.012) 0.018 (0.015) -0.020, 0.056 0.281 

(1.08 - 1.11) (1.05 - 1.10) (-0.0048 - 0.063) 

Sodium (% dw) 0.022 (0.0028) 0.0080 (0.0032) 0.014 (0.0042) 0.0033, 0.025 0.020 

(0.019 - 0.025) (0.0054 - 0.013) (0.0098 - 0.016) 

Zinc (mg/kg dw) 40.79 (1.18) 42.00 (1.37) -1.21 (1.81) -5.85, 3.44 0.533 

(37.59 - 43.87) (40.59 - 43.50) (-5.91 - 3.27) 

Vitamin (mg/kg dw)Vitamin E 93.11 (2.67) 92.34 (2.91) 0.76 (2.71) -6.20, 7.72 0.789 

(86.23 - 100.03) (91.78 - 95.85) (-6.54 - 4.18) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-8. Statistical Summary of Site KSLA Cottonseed Anti-nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.010) 0.12 (0.012) 0.026 (0.014) -0.011, 0.063 0.129 

(0.11 - 0.18) (0.12 - 0.13) (-0.0087 - 0.054) 

Malvalic Acid 0.45 (0.030) 0.37 (0.034) 0.083 (0.039) -0.018, 0.18 0.088 

(0.34 - 0.55) (0.33 - 0.39) (-0.024 - 0.15) 

Sterculic Acid 0.23 (0.014) 0.20 (0.016) 0.031 (0.019) -0.017, 0.079 0.159 

(0.18 - 0.28) (0.19 - 0.21) (-0.023 - 0.078) 

Gossypol (% dw)Free Gossypol 1.07 (0.027) 0.95 (0.030) 0.12 (0.033) 0.036, 0.20 0.014 

(1.03 - 1.10) (0.86 - 1.05) (0.051 - 0.20) 

Total Gossypol 1.13 (0.033) 1.01 (0.038) 0.12 (0.047) 0.00016, 0.24 0.049 

(1.00 - 1.24) (1.00 - 1.02) (0.0061 - 0.23) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. Co

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.35 (0.047) 4.12 (0.047) 0.23 (0.066) 0.066, 0.39 0.013 

(4.23 - 4.47) (4.06 - 4.15) (0.11 - 0.41) 

Calories (Kcal/100g) 494.11 (3.01) 494.75 (3.01) -0.64 (4.19) -10.89, 9.61 0.883 

(482.46 - 501.83) (490.27 - 498.67) (-14.30 - 4.88) 

Carbohydrates 45.92 (0.79) 47.84 (0.79) -1.91 (1.11) -4.64, 0.81 0.136 

(44.17 - 48.89) (46.77 - 50.30) (-5.19 - 1.91) 

Moisture (% fw) 6.70 (0.26) 6.98 (0.26) -0.28 (0.36) -1.17, 0.62 0.478 

(6.46 - 6.94) (6.15 - 7.40) (-0.90 - 0.79) 

Protein 27.44 (0.56) 25.80 (0.56) 1.64 (0.80) -0.31, 3.59 0.084 

(27.06 - 27.92) (23.53 - 27.85) (0.074 - 3.73) 

Total Fat 22.27 (0.64) 22.25 (0.64) 0.018 (0.90) -2.18, 2.22 0.984 

(19.79 - 23.86) (21.29 - 23.02) (-2.89 - 1.18) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.72 (0.45) 28.35 (0.45) -2.63 (0.63) -4.18, -1.08 0.005 

(24.16 - 27.08) (27.81 - 29.58) (-5.42 - -0.73) 

Crude Fiber 18.73 (0.66) 19.62 (0.66) -0.89 (0.93) -3.15, 1.38 0.376 

(17.75 - 19.77) (18.46 - 20.54) (-1.79 - 0.17) 

Neutral Detergent Fiber 33.12 (0.65) 34.05 (0.65) -0.93 (0.42) -1.97, 0.10 0.070 

(32.24 - 34.42) (32.61 - 35.84) (-1.65 - 0.23) 

Total Dietary Fiber 39.82 (0.49) 43.35 (0.49) -3.53 (0.69) -5.21, -1.85 0.002 

(39.02 - 40.86) (42.33 - 44.37) (-5.34 - -1.47) 

Amino Acid (% dw)Alanine 1.07 (0.018) 1.03 (0.018) 0.038 (0.014) 0.0050, 0.072 0.030 

(1.00 - 1.11) (1.00 - 1.06) (0.00025 - 0.082) 

Arginine 2.96 (0.073) 2.98 (0.073) -0.017 (0.084) -0.22, 0.19 0.849 

(2.64 - 3.13) (2.89 - 3.13) (-0.25 - 0.15) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.37 (0.050) 2.30 (0.050) 0.074 (0.062) -0.077, 0.22 0.278 

(2.17 - 2.49) (2.25 - 2.40) (-0.12 - 0.22) 

Cystine 0.41 (0.016) 0.38 (0.016) 0.026 (0.019) -0.021, 0.074 0.222 

(0.38 - 0.46) (0.36 - 0.44) (-0.024 - 0.082) 

Glutamic Acid 4.78 (0.13) 4.52 (0.13) 0.27 (0.16) -0.13, 0.67 0.151 

(4.32 - 5.26) (4.37 - 4.75) (-0.15 - 0.79) 

Glycine 1.11 (0.020) 1.06 (0.020) 0.057 (0.025) -0.0034, 0.12 0.060 

(1.02 - 1.18) (1.04 - 1.09) (-0.024 - 0.14) 

Histidine 0.74 (0.020) 0.72 (0.020) 0.019 (0.014) -0.014, 0.052 0.206 

(0.68 - 0.78) (0.67 - 0.76) (0.013 - 0.029) 

Isoleucine 0.90 (0.018) 0.88 (0.018) 0.020 (0.020) -0.028, 0.068 0.355 

(0.84 - 0.97) (0.87 - 0.90) (-0.028 - 0.096) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.51 (0.026) 1.48 (0.026) 0.039 (0.028) -0.030, 0.11 0.217 

(1.40 - 1.58) (1.44 - 1.52) (-0.035 - 0.12) 

Lysine 1.26 (0.024) 1.18 (0.024) 0.083 (0.027) 0.016, 0.15 0.023 

(1.17 - 1.31) (1.12 - 1.23) (0.021 - 0.15) 

Methionine 0.42 (0.017) 0.38 (0.017) 0.045 (0.013) 0.013, 0.077 0.013 

(0.37 - 0.44) (0.32 - 0.42) (0.020 - 0.075) 

Phenylalanine 1.41 (0.027) 1.39 (0.027) 0.015 (0.033) -0.066, 0.095 0.668 

(1.28 - 1.47) (1.36 - 1.43) (-0.077 - 0.094) 

Proline 1.00 (0.015) 0.98 (0.015) 0.017 (0.022) -0.036, 0.071 0.459 

(0.94 - 1.04) (0.95 - 1.02) (-0.039 - 0.093) 

Serine 1.07 (0.022) 1.03 (0.022) 0.038 (0.030) -0.036, 0.11 0.257 

(0.99 - 1.15) (1.01 - 1.06) (-0.047 - 0.12) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.87 (0.016) 0.84 (0.016) 0.027 (0.012) -0.0036, 0.057 0.074 

(0.80 - 0.90) (0.82 - 0.86) (-0.021 - 0.060) 

Tryptophan 0.41 (0.014) 0.39 (0.014) 0.019 (0.018) -0.026, 0.063 0.347 

(0.36 - 0.46) (0.38 - 0.43) (-0.024 - 0.078) 

Tyrosine 0.80 (0.018) 0.79 (0.018) 0.010 (0.021) -0.042, 0.063 0.643 

(0.72 - 0.84) (0.76 - 0.81) (-0.045 - 0.058) 

Valine 1.21 (0.024) 1.18 (0.024) 0.026 (0.029) -0.044, 0.096 0.397 

(1.11 - 1.29) (1.17 - 1.19) (-0.054 - 0.12) 

Fatty Acid (% Total FA)14:0 Myristic 0.74 (0.013) 0.75 (0.013) -0.012 (0.018) -0.057, 0.032 0.523 

(0.71 - 0.76) (0.73 - 0.78) (-0.032 - 0.0064) 

16:0 Palmitic 24.48 (0.091) 24.04 (0.091) 0.44 (0.13) 0.12, 0.75 0.014 

(24.37 - 24.55) (23.92 - 24.16) (0.21 - 0.61) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.51 (0.0035) 0.50 (0.0035) 0.0090 (0.0049) -0.0030, 0.021 0.116 

(0.50 - 0.52) (0.49 - 0.51) (-0.0051 - 0.020) 

18:0 Stearic 2.68 (0.018) 2.52 (0.018) 0.15 (0.026) 0.089, 0.22 0.001 

(2.64 - 2.73) (2.49 - 2.57) (0.11 - 0.24) 

18:1 Oleic 14.70 (0.095) 14.29 (0.095) 0.41 (0.13) 0.084, 0.74 0.021 

(14.48 - 15.01) (14.13 - 14.53) (0.16 - 0.72) 

18:2 Linoleic 55.53 (0.14) 56.63 (0.14) -1.09 (0.20) -1.59, -0.60 0.001 

(55.15 - 55.99) (56.52 - 56.72) (-1.42 - -0.63) 

18:3 Linolenic 0.15 (0.0042) 0.15 (0.0042) 0.0063 (0.0059) -0.0082, 0.021 0.327 

(0.14 - 0.17) (0.13 - 0.15) (-0.0073 - 0.019) 

20:0 Arachidic 0.31 (0.0054) 0.29 (0.0054) 0.020 (0.0071) 0.0022, 0.037 0.033 

(0.31 - 0.32) (0.29 - 0.30) (0.016 - 0.023) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.14 (0.0039) 0.14 (0.0039) 0.00029 (0.0055) -0.013, 0.014 0.959 

(0.14 - 0.15) (0.14 - 0.15) (-0.0046 - 0.0027) 

MineralCalcium (% dw) 0.12 (0.0039) 0.12 (0.0039) 0.0047 (0.0050) -0.0075, 0.017 0.381 

(0.11 - 0.13) (0.12 - 0.12) (-0.012 - 0.015) 

Copper (mg/kg dw) 8.48 (0.30) 8.70 (0.30) -0.22 (0.42) -1.26, 0.82 0.621 

(7.86 - 9.49) (8.11 - 9.14) (-1.27 - 0.99) 

Iron (mg/kg dw) 76.74 (4.19) 68.59 (4.19) 8.15 (4.72) -3.40, 19.69 0.134 

(73.99 - 83.17) (66.28 - 70.38) (3.68 - 12.79) 

Magnesium (% dw) 0.41 (0.0065) 0.39 (0.0065) 0.021 (0.0093) -0.0018, 0.043 0.065 

(0.39 - 0.44) (0.38 - 0.41) (-0.014 - 0.054) 

Manganese (mg/kg dw) 13.13 (0.37) 12.87 (0.37) 0.26 (0.52) -1.03, 1.54 0.642 

(11.92 - 13.79) (12.31 - 13.87) (-1.95 - 1.16) 
Monsanto Company 12-CT-244U 

Table E-9. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.75 (0.014) 0.71 (0.014) 0.036 (0.019) -0.012, 0.083 0.113 

(0.71 - 0.80) (0.69 - 0.73) (-0.023 - 0.11) 

Potassium (% dw) 1.18 (0.022) 1.17 (0.022) 0.0099 (0.029) -0.061, 0.081 0.742 

(1.16 - 1.22) (1.12 - 1.27) (-0.087 - 0.064) 

Sodium (% dw) 0.023 (0.0043) 0.015 (0.0043) 0.0078 (0.0060) -0.0069, 0.022 0.242 

(0.021 - 0.024) (0.0053 - 0.027) (-0.0031 - 0.017) 

Zinc (mg/kg dw) 33.97 (0.93) 35.74 (0.93) -1.77 (1.32) -4.99, 1.45 0.227 

(31.68 - 37.84) (35.10 - 37.09) (-5.41 - 2.55) 

Vitamin (mg/kg dw)Vitamin E 169.88 (2.48) 149.96 (2.48) 19.92 (3.48) 11.40, 28.43 0.001 

(163.34 - 175.33) (148.96 - 152.67) (14.16 - 26.36) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-10. Statistical Summary of Site LACH Cottonseed Anti-nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.011) 0.13 (0.011) 0.019 (0.016) -0.019, 0.057 0.271 

(0.13 - 0.19) (0.12 - 0.14) (-0.0082 - 0.068) 

Malvalic Acid 0.39 (0.035) 0.35 (0.035) 0.032 (0.049) -0.088, 0.15 0.535 

(0.33 - 0.53) (0.31 - 0.38) (-0.047 - 0.16) 

Sterculic Acid 0.22 (0.016) 0.20 (0.016) 0.020 (0.023) -0.037, 0.076 0.432 

(0.19 - 0.29) (0.17 - 0.21) (-0.011 - 0.076) 

Gossypol (% dw)Free Gossypol 0.86 (0.026) 0.81 (0.026) 0.056 (0.036) -0.033, 0.14 0.175 

(0.81 - 0.92) (0.78 - 0.84) (0.0028 - 0.13) 

Total Gossypol 0.93 (0.025) 0.90 (0.025) 0.033 (0.036) -0.055, 0.12 0.395 

(0.90 - 1.00) (0.82 - 0.94) (-0.015 - 0.086) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.34 (0.057) 4.14 (0.064) 0.20 (0.070) 0.024, 0.39 0.033 

(4.29 - 4.40) (3.94 - 4.26) (0.13 - 0.35) 

Calories (Kcal/100g) 497.98 (1.61) 491.80 (1.86) 6.18 (2.46) -0.15, 12.51 0.053 

(491.46 - 501.77) (488.93 - 494.48) (-3.01 - 10.35) 

Carbohydrates 44.40 (0.59) 44.36 (0.68) 0.044 (0.87) -2.20, 2.29 0.961 

(43.84 - 45.31) (43.65 - 45.15) (-1.31 - 0.79) 

Moisture (% fw) 9.18 (0.22) 8.96 (0.23) 0.22 (0.19) -0.26, 0.70 0.287 

(8.64 - 9.67) (8.59 - 9.19) (-0.050 - 0.48) 

Protein 28.24 (0.61) 29.84 (0.70) -1.60 (0.85) -3.78, 0.59 0.119 

(26.53 - 29.33) (29.62 - 30.42) (-1.99 - -0.53) 

Total Fat 23.04 (0.30) 21.59 (0.34) 1.45 (0.46) 0.28, 2.63 0.024 

(21.89 - 23.76) (21.03 - 22.21) (-0.32 - 2.22) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.91 (1.12) 26.31 (1.29) -0.40 (1.71) -4.80, 3.99 0.822 

(24.26 - 27.74) (24.72 - 28.08) (-1.88 - -0.096) 

Crude Fiber 17.01 (0.29) 16.93 (0.33) 0.086 (0.38) -0.90, 1.08 0.831 

(16.31 - 17.78) (16.30 - 17.90) (-0.12 - 0.31) 

Neutral Detergent Fiber 31.08 (1.03) 31.14 (1.19) -0.057 (1.58) -4.11, 4.00 0.972 

(29.23 - 32.66) (30.85 - 31.49) (-1.62 - 1.16) 

Total Dietary Fiber 38.51 (0.55) 39.52 (0.64) -1.01 (0.85) -3.19, 1.16 0.285 

(36.91 - 39.40) (39.05 - 39.86) (-0.90 - -0.088) 

Amino Acid (% dw)Alanine 1.11 (0.020) 1.10 (0.023) 0.011 (0.031) -0.068, 0.091 0.727 

(1.07 - 1.14) (1.05 - 1.14) (-0.0051 - 0.026) 

Arginine 3.08 (0.052) 3.20 (0.060) -0.12 (0.080) -0.33, 0.080 0.178 

(2.97 - 3.20) (3.11 - 3.27) (-0.19 - -0.031) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.50 (0.053) 2.55 (0.061) -0.045 (0.081) -0.25, 0.16 0.604 

(2.43 - 2.58) (2.46 - 2.63) (-0.042 - 0.0096) 

Cystine 0.43 (0.018) 0.42 (0.021) 0.016 (0.028) -0.056, 0.088 0.594 

(0.40 - 0.46) (0.39 - 0.46) (-0.051 - 0.055) 

Glutamic Acid 4.71 (0.13) 5.08 (0.15) -0.37 (0.20) -0.88, 0.14 0.120 

(4.64 - 4.79) (4.85 - 5.40) (-0.66 - -0.19) 

Glycine 1.11 (0.019) 1.11 (0.022) 0.0013 (0.029) -0.072, 0.075 0.964 

(1.06 - 1.16) (1.08 - 1.14) (-0.015 - 0.023) 

Histidine 0.76 (0.013) 0.76 (0.015) 0 (0.019) -0.049, 0.049 0.999 

(0.73 - 0.79) (0.74 - 0.79) (-0.032 - 0.052) 

Isoleucine 0.96 (0.018) 0.96 (0.020) -0.00058 (0.020) -0.053, 0.051 0.978 

(0.90 - 1.00) (0.93 - 0.97) (-0.030 - 0.040) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.60 (0.023) 1.60 (0.027) -0.0061 (0.035) -0.097, 0.085 0.869 

(1.55 - 1.65) (1.55 - 1.63) (-0.025 - 0.023) 

Lysine 1.25 (0.029) 1.26 (0.033) -0.010 (0.043) -0.12, 0.10 0.819 

(1.17 - 1.36) (1.22 - 1.29) (-0.060 - 0.077) 

Methionine 0.40 (0.0093) 0.41 (0.011) -0.011 (0.014) -0.048, 0.026 0.477 

(0.38 - 0.42) (0.40 - 0.42) (-0.046 - 0.016) 

Phenylalanine 1.48 (0.027) 1.52 (0.031) -0.043 (0.041) -0.15, 0.063 0.342 

(1.44 - 1.54) (1.45 - 1.56) (-0.058 - -0.012) 

Proline 1.03 (0.022) 1.09 (0.025) -0.065 (0.029) -0.14, 0.0098 0.075 

(0.98 - 1.11) (1.07 - 1.12) (-0.094 - -0.010) 

Serine 1.11 (0.027) 1.13 (0.032) -0.017 (0.042) -0.12, 0.090 0.696 

(1.11 - 1.13) (1.09 - 1.19) (-0.087 - 0.028) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.90 (0.014) 0.89 (0.016) 0.0096 (0.021) -0.045, 0.064 0.669 

(0.88 - 0.92) (0.86 - 0.92) (-0.0050 - 0.015) 

Tryptophan 0.41 (0.021) 0.45 (0.024) -0.039 (0.032) -0.12, 0.045 0.285 

(0.40 - 0.44) (0.39 - 0.52) (-0.081 - 0.024) 

Tyrosine 0.84 (0.012) 0.84 (0.014) 0.0015 (0.018) -0.045, 0.047 0.937 

(0.81 - 0.87) (0.82 - 0.87) (-0.026 - 0.039) 

Valine 1.26 (0.025) 1.30 (0.027) -0.038 (0.021) -0.091, 0.016 0.130 

(1.17 - 1.31) (1.24 - 1.32) (-0.067 - -0.010) 

Fatty Acid (% Total FA)14:0 Myristic 0.68 (0.0074) 0.75 (0.0086) -0.063 (0.011) -0.091, -0.034 0.002 

(0.66 - 0.70) (0.74 - 0.76) (-0.077 - -0.044) 

16:0 Palmitic 22.89 (0.12) 23.10 (0.14) -0.21 (0.18) -0.68, 0.25 0.286 

(22.47 - 23.15) (23.07 - 23.15) (-0.68 - 0.079) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.46 (0.0047) 0.48 (0.0053) -0.018 (0.0055) -0.033, -0.0044 0.019 

(0.44 - 0.47) (0.47 - 0.49) (-0.025 - -0.0092) 

18:0 Stearic 2.50 (0.037) 2.34 (0.043) 0.16 (0.057) 0.015, 0.31 0.036 

(2.39 - 2.64) (2.32 - 2.38) (0.011 - 0.22) 

18:1 Oleic 15.04 (0.12) 14.70 (0.14) 0.35 (0.19) -0.14, 0.84 0.127 

(14.58 - 15.26) (14.51 - 14.83) (-0.17 - 0.75) 

18:2 Linoleic 56.95 (0.23) 57.19 (0.26) -0.24 (0.35) -1.13, 0.66 0.528 

(56.35 - 57.88) (57.01 - 57.46) (-1.12 - 0.80) 

18:3 Linolenic 0.31 (0.012) 0.29 (0.014) 0.028 (0.018) -0.018, 0.074 0.178 

(0.27 - 0.34) (0.27 - 0.30) (-0.0012 - 0.052) 

20:0 Arachidic 0.28 (0.0065) 0.28 (0.0075) 0.0046 (0.0099) -0.021, 0.030 0.663 

(0.26 - 0.30) (0.27 - 0.28) (-0.024 - 0.024) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.16 (0.0056) 0.15 (0.0065) 0.011 (0.0086) -0.011, 0.033 0.258 

(0.15 - 0.19) (0.15 - 0.16) (-0.0044 - 0.032) 

MineralCalcium (% dw) 0.15 (0.0014) 0.14 (0.0017) 0.0095 (0.0022) 0.0039, 0.015 0.007 

(0.14 - 0.15) (0.14 - 0.14) (0.0056 - 0.013) 

Copper (mg/kg dw) 6.82 (0.36) 6.91 (0.41) -0.084 (0.54) -1.48, 1.31 0.883 

(5.81 - 7.58) (6.64 - 7.19) (-1.38 - 0.53) 

Iron (mg/kg dw) 43.21 (1.00) 48.04 (1.15) -4.83 (1.53) -8.75, -0.90 0.025 

(41.96 - 44.44) (45.03 - 50.87) (-6.43 - -2.22) 

Magnesium (% dw) 0.41 (0.010) 0.40 (0.012) 0.011 (0.016) -0.030, 0.052 0.529 

(0.40 - 0.43) (0.37 - 0.44) (-0.036 - 0.049) 

Manganese (mg/kg dw) 14.12 (0.36) 13.83 (0.42) 0.29 (0.56) -1.14, 1.72 0.622 

(13.57 - 14.81) (13.65 - 14.11) (-0.54 - 0.52) 
Monsanto Company 12-CT-244U 

Table E-11. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.64 (0.020) 0.66 (0.023) -0.020 (0.031) -0.099, 0.059 0.548 

(0.61 - 0.67) (0.59 - 0.71) (-0.087 - 0.068) 

Potassium (% dw) 1.06 (0.019) 1.08 (0.022) -0.022 (0.029) -0.097, 0.052 0.471 

(1.04 - 1.09) (1.03 - 1.16) (-0.12 - 0.032) 

Sodium (% dw) 0.11 (0.0084) 0.099 (0.0096) 0.0074 (0.011) -0.022, 0.036 0.539 

(0.068 - 0.12) (0.094 - 0.10) (-0.031 - 0.030) 

Zinc (mg/kg dw) 40.79 (1.30) 49.54 (1.50) -8.75 (1.98) -13.84, -3.66 0.006 

(40.28 - 41.37) (44.04 - 52.95) (-11.57 - -3.76) 

Vitamin (mg/kg dw)Vitamin E 169.03 (3.66) 156.99 (4.23) 12.04 (5.60) -2.34, 26.43 0.084 

(163.57 - 179.34) (151.55 - 162.98) (8.84 - 16.38) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-12. Statistical Summary of Site NCBD Cottonseed Anti-nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.14 (0.0093) 0.13 (0.011) 0.012 (0.014) -0.024, 0.048 0.422 

(0.13 - 0.15) (0.12 - 0.14) (0.0062 - 0.024) 

Malvalic Acid 0.36 (0.040) 0.37 (0.046) -0.017 (0.056) -0.16, 0.13 0.773 

(0.20 - 0.43) (0.36 - 0.41) (0.0058 - 0.056) 

Sterculic Acid 0.22 (0.024) 0.22 (0.028) -0.0024 (0.037) -0.098, 0.093 0.951 

(0.13 - 0.27) (0.21 - 0.23) (0.0078 - 0.040) 

Gossypol (% dw)Free Gossypol 0.94 (0.026) 0.90 (0.030) 0.038 (0.039) -0.063, 0.14 0.374 

(0.91 - 0.97) (0.81 - 0.95) (-0.024 - 0.097) 

Total Gossypol 1.12 (0.067) 1.09 (0.077) 0.037 (0.10) -0.22, 0.30 0.731 

(1.07 - 1.18) (1.08 - 1.10) (-0.0038 - 0.050) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.16 (0.087) 4.27 (0.10) -0.11 (0.13) -0.45, 0.22 0.434 

(3.77 - 4.38) (4.20 - 4.39) (-0.093 - 0.13) 

Calories (Kcal/100g) 496.46 (2.64) 492.81 (3.05) 3.66 (4.04) -6.73, 14.04 0.406 

(486.87 - 500.48) (490.52 - 494.31) (-7.44 - 9.96) 

Carbohydrates 42.09 (0.46) 42.60 (0.53) -0.50 (0.70) -2.31, 1.30 0.504 

(41.40 - 43.69) (42.14 - 43.05) (-1.65 - 1.09) 

Moisture (% fw) 6.59 (0.25) 7.28 (0.28) -0.70 (0.32) -1.53, 0.13 0.082 

(5.93 - 7.28) (6.63 - 7.75) (-1.82 - -0.34) 

Protein 31.15 (0.13) 31.18 (0.15) -0.025 (0.19) -0.52, 0.47 0.902 

(30.63 - 31.47) (31.00 - 31.27) (-0.65 - 0.46) 

Total Fat 22.59 (0.54) 21.95 (0.62) 0.64 (0.83) -1.48, 2.77 0.471 

(20.62 - 23.58) (21.42 - 22.22) (-1.60 - 2.16) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 24.46 (0.51) 26.40 (0.59) -1.94 (0.79) -3.96, 0.080 0.056 

(23.26 - 25.45) (25.76 - 27.10) (-3.08 - -1.80) 

Crude Fiber 17.90 (0.74) 17.71 (0.84) 0.20 (0.94) -2.23, 2.63 0.841 

(17.33 - 18.57) (16.06 - 20.78) (-2.21 - 1.44) 

Neutral Detergent Fiber 29.73 (0.87) 32.83 (1.00) -3.09 (1.32) -6.49, 0.31 0.066 

(27.53 - 32.00) (31.58 - 34.49) (-6.95 - -0.41) 

Total Dietary Fiber 39.16 (0.71) 41.10 (0.75) -1.94 (0.55) -3.36, -0.53 0.016 

(37.46 - 40.44) (39.09 - 43.00) (-3.61 - -0.88) 

Amino Acid (% dw)Alanine 1.11 (0.011) 1.13 (0.013) -0.019 (0.014) -0.054, 0.015 0.212 

(1.10 - 1.13) (1.09 - 1.17) (-0.044 - 0.0067) 

Arginine 3.48 (0.049) 3.71 (0.053) -0.23 (0.048) -0.35, -0.10 0.005 

(3.42 - 3.60) (3.67 - 3.77) (-0.34 - -0.17) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.60 (0.033) 2.66 (0.037) -0.062 (0.043) -0.17, 0.048 0.206 

(2.55 - 2.64) (2.58 - 2.74) (-0.11 - -0.031) 

Cystine 0.43 (0.013) 0.44 (0.015) -0.010 (0.020) -0.061, 0.040 0.620 

(0.39 - 0.47) (0.43 - 0.45) (-0.063 - 0.038) 

Glutamic Acid 5.30 (0.087) 5.46 (0.10) -0.16 (0.13) -0.50, 0.18 0.285 

(5.24 - 5.38) (5.29 - 5.70) (-0.46 - -0.0085) 

Glycine 1.16 (0.014) 1.18 (0.015) -0.021 (0.016) -0.063, 0.020 0.247 

(1.14 - 1.19) (1.17 - 1.20) (-0.037 - -0.013) 

Histidine 0.82 (0.010) 0.83 (0.012) -0.0065 (0.015) -0.044, 0.031 0.675 

(0.80 - 0.85) (0.83 - 0.84) (-0.026 - 0.024) 

Isoleucine 0.97 (0.017) 0.98 (0.018) -0.011 (0.012) -0.041, 0.019 0.397 

(0.94 - 1.01) (0.94 - 1.03) (-0.038 - 0.021) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.65 (0.021) 1.66 (0.023) -0.0069 (0.022) -0.063, 0.049 0.763 

(1.62 - 1.70) (1.63 - 1.69) (-0.039 - 0.0098) 

Lysine 1.33 (0.019) 1.36 (0.022) -0.030 (0.022) -0.087, 0.027 0.232 

(1.26 - 1.38) (1.32 - 1.39) (-0.058 - -0.0056) 

Methionine 0.43 (0.012) 0.40 (0.014) 0.033 (0.018) -0.014, 0.081 0.129 

(0.40 - 0.46) (0.38 - 0.41) (-0.013 - 0.071) 

Phenylalanine 1.61 (0.026) 1.61 (0.028) -0.0044 (0.022) -0.061, 0.052 0.850 

(1.56 - 1.66) (1.60 - 1.66) (-0.038 - 0.015) 

Proline 1.14 (0.027) 1.18 (0.031) -0.040 (0.039) -0.14, 0.060 0.353 

(1.09 - 1.21) (1.10 - 1.25) (-0.080 - -0.015) 

Serine 1.17 (0.026) 1.20 (0.030) -0.028 (0.040) -0.13, 0.075 0.512 

(1.15 - 1.23) (1.16 - 1.24) (-0.069 - -0.0042) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.92 (0.015) 0.93 (0.017) -0.0072 (0.021) -0.062, 0.048 0.747 

(0.89 - 0.94) (0.91 - 0.94) (-0.024 - 0.0027) 

Tryptophan 0.46 (0.014) 0.44 (0.016) 0.019 (0.020) -0.032, 0.071 0.382 

(0.43 - 0.52) (0.43 - 0.45) (-0.019 - 0.076) 

Tyrosine 0.87 (0.014) 0.89 (0.015) -0.012 (0.014) -0.048, 0.024 0.440 

(0.85 - 0.92) (0.86 - 0.91) (-0.039 - 0.0089) 

Valine 1.32 (0.024) 1.34 (0.026) -0.021 (0.019) -0.070, 0.029 0.329 

(1.26 - 1.40) (1.31 - 1.40) (-0.048 - 0.0048) 

Fatty Acid (% Total FA)14:0 Myristic 0.93 (0.0046) 0.98 (0.0054) -0.043 (0.0071) -0.062, -0.025 0.001 

(0.92 - 0.95) (0.97 - 0.98) (-0.060 - -0.037) 

16:0 Palmitic 24.19 (0.088) 24.11 (0.10) 0.083 (0.13) -0.26, 0.43 0.562 

(24.02 - 24.42) (23.89 - 24.34) (-0.32 - 0.33) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.53 (0.0022) 0.54 (0.0025) -0.012 (0.0033) -0.021, -0.0037 0.014 

(0.52 - 0.53) (0.53 - 0.54) (-0.019 - -0.0082) 

18:0 Stearic 2.51 (0.020) 2.64 (0.021) -0.14 (0.016) -0.18, -0.094 <0.001 

(2.47 - 2.56) (2.61 - 2.70) (-0.15 - -0.095) 

18:1 Oleic 16.21 (0.067) 16.21 (0.076) 0.0024 (0.088) -0.22, 0.23 0.979 

(16.03 - 16.40) (16.10 - 16.35) (-0.11 - 0.24) 

18:2 Linoleic 54.32 (0.084) 54.29 (0.097) 0.029 (0.13) -0.30, 0.36 0.833 

(54.30 - 54.33) (54.04 - 54.50) (-0.18 - 0.30) 

18:3 Linolenic 0.16 (0.0019) 0.14 (0.0022) 0.012 (0.0029) 0.0043, 0.019 0.009 

(0.15 - 0.16) (0.14 - 0.15) (0.0078 - 0.014) 

20:0 Arachidic 0.30 (0.0052) 0.31 (0.0060) -0.011 (0.0080) -0.031, 0.0095 0.225 

(0.29 - 0.30) (0.28 - 0.32) (-0.025 - 0.014) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.16 (0.0065) 0.19 (0.0075) -0.023 (0.0099) -0.049, 0.0021 0.065 

(0.16 - 0.17) (0.17 - 0.21) (-0.049 - -0.0086) 

MineralCalcium (% dw) 0.15 (0.0029) 0.13 (0.0034) 0.021 (0.0042) 0.011, 0.032 0.003 

(0.14 - 0.15) (0.12 - 0.13) (0.0081 - 0.031) 

Copper (mg/kg dw) 11.35 (0.15) 11.75 (0.17) -0.40 (0.22) -0.97, 0.18 0.134 

(11.11 - 11.91) (11.46 - 11.92) (-0.76 - 0.060) 

Iron (mg/kg dw) 63.88 (3.33) 64.62 (3.84) -0.74 (5.08) -13.80, 12.32 0.890 

(60.27 - 66.59) (63.58 - 66.45) (-6.18 - 2.76) 

Magnesium (% dw) 0.39 (0.0053) 0.37 (0.0061) 0.019 (0.0081) -0.0015, 0.040 0.062 

(0.38 - 0.41) (0.36 - 0.38) (0.0045 - 0.036) 

Manganese (mg/kg dw) 12.93 (0.23) 12.90 (0.26) 0.029 (0.34) -0.86, 0.92 0.936 

(12.73 - 13.13) (12.00 - 13.47) (-0.47 - 1.13) 
Monsanto Company 12-CT-244U 

Table E-13. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Treated) vs. Conventio(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.77 (0.010) 0.79 (0.012) -0.020 (0.016) -0.060, 0.021 0.264 

(0.74 - 0.80) (0.78 - 0.80) (-0.035 - 0.019) 

Potassium (% dw) 1.10 (0.016) 1.11 (0.018) -0.0086 (0.017) -0.052, 0.035 0.636 

(1.05 - 1.14) (1.07 - 1.14) (-0.020 - -0.0045) 

Sodium (% dw) 0.021 (0.0034) 0.013 (0.0039) 0.0075 (0.0052) -0.0057, 0.021 0.203 

(0.019 - 0.022) (0.0054 - 0.023) (-0.0044 - 0.016) 

Zinc (mg/kg dw) 45.63 (0.60) 49.43 (0.69) -3.80 (0.92) -6.16, -1.44 0.009 

(44.12 - 46.74) (47.66 - 50.87) (-5.64 - -0.92) 

Vitamin (mg/kg dw)Vitamin E 114.29 (2.04) 112.18 (2.36) 2.11 (3.12) -5.90, 10.12 0.528 

(107.78 - 119.15) (107.02 - 115.99) (-5.75 - 12.13) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-14. Statistical Summary of Site NMLC Cottonseed Anti-nutrients for MON 88701 (Treated) Control 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.16 (0.0079) 0.14 (0.0092) 0.019 (0.012) -0.012, 0.050 0.178 

(0.14 - 0.18) (0.12 - 0.15) (-0.0065 - 0.041) 

Malvalic Acid 0.34 (0.023) 0.29 (0.026) 0.054 (0.035) -0.035, 0.14 0.177 

(0.30 - 0.38) (0.26 - 0.31) (-0.0093 - 0.074) 

Sterculic Acid 0.20 (0.018) 0.18 (0.020) 0.018 (0.027) -0.051, 0.087 0.531 

(0.18 - 0.23) (0.17 - 0.19) (-0.012 - 0.032) 

Gossypol (% dw)Free Gossypol 0.85 (0.026) 0.69 (0.030) 0.15 (0.040) 0.052, 0.26 0.011 

(0.83 - 0.88) (0.68 - 0.70) (0.14 - 0.18) 

Total Gossypol 0.92 (0.026) 0.80 (0.030) 0.12 (0.040) 0.022, 0.23 0.026 

(0.84 - 0.97) (0.74 - 0.87) (0.060 - 0.18) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.11 (0.095) 3.74 (0.095) 0.37 (0.10) 0.12, 0.62 0.010 

(3.99 - 4.28) (3.38 - 3.98) (0.25 - 0.61) 

Calories (Kcal/100g) 511.69 (2.44) 503.38 (2.44) 8.31 (3.45) -0.13, 16.75 0.052 

(505.01 - 517.46) (499.09 - 512.65) (-7.65 - 18.37) 

Carbohydrates 46.56 (0.54) 48.67 (0.54) -2.11 (0.75) -3.95, -0.27 0.031 

(45.10 - 47.48) (47.50 - 49.59) (-3.20 - -0.23) 

Moisture (% fw) 6.73 (0.17) 7.08 (0.17) -0.35 (0.19) -0.81, 0.12 0.119 

(6.27 - 7.13) (6.63 - 7.37) (-0.89 - 0.030) 

Protein 23.70 (0.42) 23.92 (0.42) -0.22 (0.49) -1.43, 0.98 0.669 

(22.71 - 24.70) (23.56 - 24.61) (-0.85 - 0.64) 

Total Fat 25.65 (0.44) 23.65 (0.44) 2.00 (0.63) 0.46, 3.54 0.019 

(24.23 - 26.78) (22.92 - 25.20) (-0.97 - 3.86) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.95 (0.44) 27.04 (0.44) -1.09 (0.60) -2.56, 0.37 0.118 

(24.75 - 26.52) (26.24 - 27.74) (-1.75 - -0.099) 

Crude Fiber 19.72 (0.80) 19.13 (0.80) 0.59 (1.13) -2.17, 3.36 0.617 

(17.98 - 21.66) (16.91 - 21.70) (-2.59 - 4.75) 

Neutral Detergent Fiber 31.34 (0.67) 33.60 (0.67) -2.27 (0.95) -4.59, 0.049 0.053 

(29.42 - 32.89) (32.74 - 35.52) (-6.10 - -0.44) 

Total Dietary Fiber 39.72 (0.62) 41.87 (0.62) -2.14 (0.88) -4.29, 0.0015 0.050 

(38.66 - 40.44) (40.16 - 43.29) (-3.56 - -0.63) 

Amino Acid (% dw)Alanine 0.96 (0.022) 0.94 (0.022) 0.017 (0.029) -0.055, 0.089 0.583 

(0.91 - 1.00) (0.88 - 0.97) (-0.020 - 0.033) 

Arginine 2.52 (0.088) 2.59 (0.088) -0.063 (0.10) -0.31, 0.18 0.556 

(2.33 - 2.74) (2.41 - 2.71) (-0.18 - 0.021) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.08 (0.054) 2.07 (0.054) 0.0087 (0.070) -0.16, 0.18 0.904 

(1.94 - 2.20) (1.92 - 2.18) (-0.085 - 0.096) 

Cystine 0.36 (0.017) 0.35 (0.017) 0.0082 (0.018) -0.036, 0.052 0.666 

(0.32 - 0.41) (0.31 - 0.39) (-0.018 - 0.024) 

Glutamic Acid 4.14 (0.13) 4.10 (0.13) 0.039 (0.18) -0.40, 0.47 0.833 

(3.80 - 4.40) (3.66 - 4.40) (-0.11 - 0.14) 

Glycine 0.99 (0.022) 0.98 (0.022) 0.016 (0.031) -0.061, 0.093 0.627 

(0.93 - 1.04) (0.91 - 1.02) (-0.0040 - 0.033) 

Histidine 0.64 (0.021) 0.64 (0.021) 0.0012 (0.025) -0.060, 0.062 0.961 

(0.58 - 0.70) (0.61 - 0.66) (-0.053 - 0.033) 

Isoleucine 0.81 (0.023) 0.82 (0.023) -0.0053 (0.030) -0.078, 0.068 0.865 

(0.75 - 0.88) (0.77 - 0.83) (-0.077 - 0.054) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.36 (0.034) 1.36 (0.034) 0.0086 (0.046) -0.10, 0.12 0.858 

(1.29 - 1.46) (1.28 - 1.40) (-0.053 - 0.064) 

Lysine 1.12 (0.027) 1.11 (0.027) 0.0024 (0.025) -0.059, 0.064 0.925 

(1.05 - 1.18) (1.06 - 1.17) (-0.018 - 0.026) 

Methionine 0.38 (0.016) 0.33 (0.016) 0.041 (0.020) -0.0093, 0.091 0.093 

(0.35 - 0.42) (0.32 - 0.35) (0.017 - 0.077) 

Phenylalanine 1.22 (0.033) 1.23 (0.033) -0.0097 (0.039) -0.11, 0.086 0.811 

(1.14 - 1.31) (1.15 - 1.27) (-0.075 - 0.043) 

Proline 0.87 (0.026) 0.87 (0.026) 0.0028 (0.026) -0.060, 0.066 0.916 

(0.82 - 0.92) (0.81 - 0.93) (-0.035 - 0.034) 

Serine 0.98 (0.028) 0.96 (0.028) 0.019 (0.033) -0.062, 0.10 0.587 

(0.90 - 1.04) (0.86 - 1.03) (-0.0099 - 0.042) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.79 (0.019) 0.77 (0.019) 0.016 (0.023) -0.041, 0.073 0.518 

(0.74 - 0.83) (0.73 - 0.81) (0.0053 - 0.039) 

Tryptophan 0.35 (0.0093) 0.38 (0.0093) -0.025 (0.0085) -0.046, -0.0048 0.023 

(0.33 - 0.38) (0.37 - 0.40) (-0.040 - 0.0010) 

Tyrosine 0.72 (0.020) 0.71 (0.020) 0.0042 (0.025) -0.058, 0.066 0.873 

(0.67 - 0.78) (0.67 - 0.74) (-0.033 - 0.040) 

Valine 1.07 (0.029) 1.07 (0.029) 0.0056 (0.039) -0.090, 0.10 0.889 

(1.00 - 1.14) (1.00 - 1.10) (-0.084 - 0.049) 

Fatty Acid (% Total FA)14:0 Myristic 0.70 (0.011) 0.73 (0.011) -0.028 (0.012) -0.057, 0.00046 0.052 

(0.67 - 0.72) (0.72 - 0.75) (-0.049 - 0.0062) 

16:0 Palmitic 24.74 (0.086) 24.39 (0.086) 0.35 (0.12) 0.049, 0.65 0.029 

(24.59 - 24.94) (24.07 - 24.59) (0.17 - 0.61) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.48 (0.0056) 0.48 (0.0056) -0.0075 (0.0076) -0.026, 0.011 0.361 

(0.47 - 0.49) (0.47 - 0.49) (-0.020 - 0.018) 

18:0 Stearic 2.78 (0.036) 2.67 (0.036) 0.11 (0.050) -0.015, 0.23 0.076 

(2.75 - 2.85) (2.58 - 2.76) (0.0076 - 0.18) 

18:1 Oleic 14.40 (0.11) 14.46 (0.11) -0.059 (0.15) -0.43, 0.31 0.706 

(14.15 - 14.68) (14.42 - 14.49) (-0.33 - 0.19) 

18:2 Linoleic 55.54 (0.18) 55.87 (0.18) -0.33 (0.25) -0.94, 0.29 0.242 

(55.18 - 55.96) (55.61 - 56.29) (-1.11 - 0.13) 

18:3 Linolenic 0.15 (0.0017) 0.15 (0.0017) 0.0044 (0.0023) -0.0012, 0.0099 0.103 

(0.15 - 0.16) (0.14 - 0.15) (-0.00022 - 0.011) 

20:0 Arachidic 0.29 (0.0070) 0.30 (0.0070) -0.0046 (0.0099) -0.029, 0.020 0.656 

(0.27 - 0.31) (0.29 - 0.30) (-0.027 - 0.019) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.14 (0.0033) 0.14 (0.0033) -0.0014 (0.0046) -0.013, 0.0099 0.765 

(0.13 - 0.14) (0.13 - 0.14) (-0.0030 - 0.00006) 

MineralCalcium (% dw) 0.11 (0.0040) 0.091 (0.0040) 0.016 (0.0056) 0.0025, 0.030 0.027 

(0.10 - 0.11) (0.081 - 0.095) (0.0059 - 0.023) 

Copper (mg/kg dw) 5.82 (0.24) 5.64 (0.24) 0.18 (0.32) -0.61, 0.97 0.593 

(5.22 - 6.30) (5.40 - 5.85) (-0.28 - 0.55) 

Iron (mg/kg dw) 63.78 (4.68) 73.46 (4.68) -9.68 (4.30) -20.21, 0.84 0.065 

(59.75 - 67.62) (63.01 - 89.93) (-22.31 - -1.16) 

Magnesium (% dw) 0.39 (0.0090) 0.36 (0.0090) 0.033 (0.0080) 0.014, 0.053 0.005 

(0.37 - 0.41) (0.34 - 0.37) (0.014 - 0.044) 

Manganese (mg/kg dw) 11.39 (0.51) 9.72 (0.51) 1.67 (0.72) -0.080, 3.42 0.058 

(10.88 - 11.68) (8.61 - 11.03) (0.65 - 2.27) 
Monsanto Company 12-CT-244U 

Table E-15. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Treated) vs. ConventionControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.67 (0.022) 0.63 (0.022) 0.035 (0.018) -0.0093, 0.080 0.100 

(0.64 - 0.71) (0.58 - 0.68) (0.0036 - 0.072) 

Potassium (% dw) 1.13 (0.031) 1.02 (0.031) 0.11 (0.043) 0.0051, 0.22 0.042 

(1.11 - 1.17) (0.88 - 1.08) (0.046 - 0.23) 

Sodium (% dw) 0.022 (0.0033) 0.015 (0.0033) 0.0070 (0.0046) -0.0043, 0.018 0.180 

(0.018 - 0.027) (0.012 - 0.023) (0.0039 - 0.013) 

Zinc (mg/kg dw) 29.14 (0.82) 30.08 (0.82) -0.94 (0.82) -2.96, 1.08 0.297 

(27.31 - 31.57) (28.22 - 31.74) (-2.85 - 1.07) 

Vitamin (mg/kg dw)Vitamin E 162.17 (1.77) 158.20 (1.77) 3.97 (2.50) -2.16, 10.10 0.164 

(158.92 - 165.82) (153.15 - 162.63) (2.38 - 7.55) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-16. Statistical Summary of Site SCEK Cottonseed Anti-nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0087) 0.15 (0.0087) 0.0014 (0.012) -0.029, 0.031 0.914 

(0.12 - 0.18) (0.14 - 0.15) (-0.023 - 0.043) 

Malvalic Acid 0.41 (0.029) 0.43 (0.029) -0.022 (0.041) -0.12, 0.078 0.607 

(0.33 - 0.52) (0.39 - 0.46) (-0.11 - 0.13) 

Sterculic Acid 0.23 (0.014) 0.24 (0.014) -0.011 (0.020) -0.060, 0.038 0.607 

(0.19 - 0.28) (0.22 - 0.25) (-0.060 - 0.060) 

Gossypol (% dw)Free Gossypol 1.10 (0.029) 1.13 (0.029) -0.030 (0.027) -0.096, 0.035 0.303 

(1.05 - 1.18) (1.06 - 1.20) (-0.086 - 0.00085) 

Total Gossypol 1.17 (0.025) 1.07 (0.025) 0.10 (0.031) 0.026, 0.18 0.017 

(1.13 - 1.23) (1.05 - 1.10) (0.074 - 0.13) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 3.85 (0.051) 3.46 (0.051) 0.40 (0.072) 0.22, 0.57 0.001 

(3.77 - 3.92) (3.34 - 3.61) (0.24 - 0.58) 

Calories (Kcal/100g) 498.01 (2.54) 494.42 (2.54) 3.59 (3.47) -4.90, 12.07 0.340 

(489.04 - 502.78) (489.10 - 500.98) (-1.24 - 13.67) 

Carbohydrates 44.03 (0.48) 46.39 (0.48) -2.36 (0.64) -3.92, -0.79 0.010 

(42.73 - 45.99) (45.65 - 47.07) (-3.73 - -0.88) 

Moisture (% fw) 6.88 (0.21) 7.47 (0.21) -0.59 (0.29) -1.30, 0.13 0.090 

(6.32 - 7.37) (7.11 - 7.79) (-1.47 - 0.18) 

Protein 29.43 (0.24) 28.48 (0.24) 0.95 (0.29) 0.24, 1.66 0.017 

(29.06 - 30.14) (28.09 - 28.77) (0.38 - 1.82) 

Total Fat 22.71 (0.48) 21.70 (0.48) 1.01 (0.65) -0.58, 2.61 0.169 

(20.94 - 23.59) (20.71 - 22.88) (0.15 - 2.88) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.53 (0.37) 25.53 (0.37) 0.0023 (0.41) -1.01, 1.01 0.995 

(25.31 - 25.83) (24.51 - 26.91) (-1.08 - 0.91) 

Crude Fiber 17.93 (0.38) 18.10 (0.38) -0.17 (0.45) -1.26, 0.92 0.716 

(17.17 - 18.84) (17.35 - 19.63) (-1.48 - 1.07) 

Neutral Detergent Fiber 29.75 (0.41) 32.12 (0.41) -2.38 (0.58) -3.80, -0.96 0.006 

(28.74 - 30.56) (30.49 - 33.05) (-4.31 - -0.26) 

Total Dietary Fiber 39.54 (0.62) 40.47 (0.62) -0.93 (0.72) -2.69, 0.83 0.245 

(38.76 - 40.86) (39.15 - 42.09) (-3.24 - 0.56) 

Amino Acid (% dw)Alanine 1.07 (0.019) 1.05 (0.019) 0.022 (0.026) -0.042, 0.086 0.438 

(1.05 - 1.10) (0.97 - 1.10) (-0.040 - 0.12) 

Arginine 3.25 (0.074) 3.25 (0.074) -0.0020 (0.10) -0.26, 0.25 0.985 

(3.15 - 3.33) (2.94 - 3.49) (-0.34 - 0.39) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.51 (0.047) 2.45 (0.047) 0.060 (0.067) -0.10, 0.22 0.407 

(2.43 - 2.55) (2.26 - 2.62) (-0.092 - 0.29) 

Cystine 0.41 (0.015) 0.40 (0.015) 0.0068 (0.017) -0.034, 0.047 0.697 

(0.39 - 0.43) (0.36 - 0.45) (-0.030 - 0.073) 

Glutamic Acid 4.94 (0.13) 5.02 (0.13) -0.072 (0.19) -0.53, 0.39 0.714 

(4.73 - 5.14) (4.41 - 5.32) (-0.44 - 0.73) 

Glycine 1.12 (0.022) 1.11 (0.022) 0.0062 (0.031) -0.070, 0.082 0.849 

(1.07 - 1.15) (1.03 - 1.19) (-0.073 - 0.12) 

Histidine 0.77 (0.018) 0.76 (0.018) 0.017 (0.026) -0.046, 0.079 0.538 

(0.73 - 0.81) (0.71 - 0.82) (-0.062 - 0.091) 

Isoleucine 0.95 (0.013) 0.93 (0.013) 0.013 (0.017) -0.027, 0.054 0.457 

(0.93 - 0.97) (0.89 - 0.97) (-0.023 - 0.051) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.58 (0.026) 1.55 (0.026) 0.028 (0.037) -0.064, 0.12 0.486 

(1.52 - 1.61) (1.45 - 1.64) (-0.072 - 0.16) 

Lysine 1.26 (0.026) 1.24 (0.026) 0.024 (0.037) -0.067, 0.12 0.537 

(1.21 - 1.33) (1.19 - 1.33) (-0.11 - 0.13) 

Methionine 0.40 (0.021) 0.39 (0.021) 0.016 (0.029) -0.056, 0.088 0.605 

(0.37 - 0.44) (0.32 - 0.44) (-0.066 - 0.12) 

Phenylalanine 1.51 (0.034) 1.48 (0.034) 0.026 (0.048) -0.092, 0.14 0.614 

(1.46 - 1.55) (1.36 - 1.61) (-0.14 - 0.19) 

Proline 1.04 (0.024) 1.04 (0.024) 0.0028 (0.033) -0.079, 0.084 0.935 

(0.99 - 1.11) (1.01 - 1.11) (-0.12 - 0.10) 

Serine 1.11 (0.031) 1.11 (0.031) 0.00043 (0.044) -0.11, 0.11 0.992 

(1.06 - 1.13) (0.97 - 1.17) (-0.087 - 0.16) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.89 (0.016) 0.86 (0.016) 0.032 (0.022) -0.022, 0.086 0.197 

(0.86 - 0.92) (0.80 - 0.91) (-0.026 - 0.10) 

Tryptophan 0.42 (0.013) 0.43 (0.013) -0.0094 (0.018) -0.054, 0.035 0.626 

(0.40 - 0.44) (0.38 - 0.47) (-0.070 - 0.030) 

Tyrosine 0.84 (0.017) 0.81 (0.017) 0.031 (0.024) -0.028, 0.089 0.245 

(0.82 - 0.86) (0.74 - 0.87) (-0.048 - 0.12) 

Valine 1.26 (0.030) 1.23 (0.030) 0.024 (0.042) -0.079, 0.13 0.586 

(1.21 - 1.29) (1.16 - 1.29) (-0.046 - 0.12) 

Fatty Acid (% Total FA)14:0 Myristic 0.86 (0.010) 0.84 (0.010) 0.017 (0.013) -0.016, 0.050 0.246 

(0.84 - 0.88) (0.81 - 0.87) (-0.010 - 0.047) 

16:0 Palmitic 23.15 (0.14) 23.01 (0.14) 0.13 (0.16) -0.27, 0.53 0.443 

(22.72 - 23.62) (22.86 - 23.25) (-0.27 - 0.76) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.48 (0.0058) 0.46 (0.0058) 0.016 (0.0072) -0.0018, 0.034 0.070 

(0.47 - 0.49) (0.45 - 0.47) (0.0040 - 0.039) 

18:0 Stearic 2.35 (0.025) 2.46 (0.025) -0.11 (0.028) -0.18, -0.047 0.006 

(2.30 - 2.43) (2.40 - 2.52) (-0.16 - -0.097) 

18:1 Oleic 16.34 (0.078) 16.16 (0.078) 0.18 (0.11) -0.083, 0.45 0.143 

(16.22 - 16.45) (15.86 - 16.44) (-0.14 - 0.53) 

18:2 Linoleic 55.42 (0.21) 55.58 (0.21) -0.15 (0.29) -0.87, 0.56 0.616 

(54.97 - 55.95) (55.18 - 56.13) (-1.16 - 0.77) 

18:3 Linolenic 0.17 (0.0056) 0.17 (0.0056) 0.0057 (0.0079) -0.014, 0.025 0.497 

(0.17 - 0.18) (0.16 - 0.17) (0.00091 - 0.011) 

20:0 Arachidic 0.28 (0.0055) 0.28 (0.0055) -0.0041 (0.0078) -0.023, 0.015 0.618 

(0.27 - 0.28) (0.28 - 0.29) (-0.0061 - -0.0022) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.16 (0.0015) 0.16 (0.0015) 0.0020 (0.0020) -0.0029, 0.0068 0.362 

(0.16 - 0.16) (0.16 - 0.16) (-0.00054 - 0.0042) 

MineralCalcium (% dw) 0.16 (0.0016) 0.14 (0.0016) 0.021 (0.0023) 0.015, 0.027 <0.001 

(0.16 - 0.16) (0.13 - 0.14) (0.018 - 0.023) 

Copper (mg/kg dw) 10.49 (0.15) 9.98 (0.15) 0.51 (0.22) -0.023, 1.04 0.057 

(10.09 - 10.87) (9.58 - 10.41) (0.12 - 1.29) 

Iron (mg/kg dw) 60.47 (5.67) 79.02 (5.67) -18.55 (7.06) -35.82, -1.28 0.039 

(56.94 - 66.50) (67.45 - 95.10) (-38.15 - -0.95) 

Magnesium (% dw) 0.35 (0.0045) 0.34 (0.0045) 0.019 (0.0040) 0.0087, 0.028 0.003 

(0.35 - 0.37) (0.33 - 0.34) (0.0082 - 0.024) 

Manganese (mg/kg dw) 10.91 (0.34) 9.04 (0.34) 1.86 (0.48) 0.70, 3.03 0.007 

(10.18 - 11.37) (8.83 - 9.54) (0.64 - 2.54) 
Monsanto Company 12-CT-244U 

Table E-17. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Treated) vs. Convention(continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.58 (0.0099) 0.57 (0.0099) 0.0036 (0.011) -0.022, 0.030 0.742 

(0.56 - 0.61) (0.54 - 0.60) (-0.0098 - 0.018) 

Potassium (% dw) 1.01 (0.023) 0.87 (0.023) 0.14 (0.033) 0.062, 0.22 0.004 

(0.98 - 1.06) (0.79 - 0.93) (0.073 - 0.27) 

Sodium (% dw) 0.024 (0.010) 0.047 (0.010) -0.023 (0.014) -0.058, 0.012 0.161 

(0.019 - 0.027) (0.019 - 0.090) (-0.065 - 0.0062) 

Zinc (mg/kg dw) 34.10 (0.45) 34.96 (0.45) -0.86 (0.64) -2.44, 0.71 0.227 

(33.36 - 35.30) (33.70 - 35.89) (-2.31 - 1.61) 

Vitamin (mg/kg dw)Vitamin E 114.39 (2.75) 103.66 (2.75) 10.73 (3.90) 1.20, 20.26 0.033 

(107.81 - 118.39) (93.92 - 109.90) (6.69 - 14.40) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-18. Statistical Summary of Site TXPL Cottonseed Anti-nutrients for MON 88701 (Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.16 (0.0047) 0.16 (0.0047) -0.0044 (0.0066) -0.021, 0.012 0.533 

(0.14 - 0.17) (0.15 - 0.17) (-0.026 - 0.021) 

Malvalic Acid 0.41 (0.020) 0.47 (0.020) -0.054 (0.029) -0.12, 0.017 0.112 

(0.33 - 0.47) (0.44 - 0.49) (-0.16 - 0.0068) 

Sterculic Acid 0.23 (0.013) 0.26 (0.013) -0.026 (0.018) -0.070, 0.017 0.189 

(0.18 - 0.27) (0.25 - 0.27) (-0.085 - 0.024) 

Gossypol (% dw)Free Gossypol 0.98 (0.024) 0.93 (0.024) 0.054 (0.033) -0.028, 0.14 0.157 

(0.91 - 1.06) (0.91 - 0.95) (-0.025 - 0.13) 

Total Gossypol 1.06 (0.024) 1.01 (0.024) 0.053 (0.022) -0.00013, 0.11 0.050 

(1.03 - 1.11) (0.97 - 1.05) (-0.021 - 0.092) 

1dw = dry weight; FA = fatty acid. 2MON 88701 plants were treated with dicamba and glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 420 of 620 

E.5. Composition of Cottonseed Not Treated with Dicamba or Glufosinate 

E.5.1. Nutrient Levels in Cottonseed Not Treated with Dicamba or Glufosinate 

In the combined-site analysis of nutrient levels in cottonseed, the following components had no 

significant differences (p<0.05) in mean values between MON 88701 not treated with dicamba 

or glufosinate and the conventional control: one proximate (protein), 17 amino acids (alanine, 

aspartic acid, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, 

phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), eight fatty acids (16:0 

palmitic acid, 16:1 palmitoleic acid, 18:0 stearic acid, 18:1 oleic acid, 18:2 linoleic acid, 18:3 

linolenic acid, 20:0 arachidic acid, and 22:0 behenic acid), and four minerals (copper, iron, 

phosphorus, and sodium) (Tables E-19 and E-20). 

The nutrient components that had significant differences in mean values between MON 88701 

and the conventional control in the combined-site analysis were: five proximates (ash, calories, 

carbohydrates, moisture, and total fat), four types of fiber (ADF, crude fiber, NDF, and TDF), 

one amino acid (arginine), one fatty acid (14:0 myristic acid), five minerals (calcium, 

magnesium, manganese, potassium, and zinc) and vitamin E (Table E-19). 

The significant differences in nutrients were further evaluated using considerations relevant to 

the safety and nutritional quality of MON 88701 when compared to the conventional control: 

All nutrient component differences observed in the combined-site statistical analysis, whether 

reflecting increased or decreased MON 88701 mean values, with respect to the conventional 

control, were 16.14% or less. The relative magnitude of the differences were as follows: 1.04 to 

6.45% for proximates; 3.35 to 4.12% for fibers; 3.82% for arginine; 2.39% for 14:0 myristic 

acid; 5.48 to 16.14% for minerals; and 5.84% for vitamin E. 

1) All mean values for all significantly different nutrient components from the combinedsite analysis of MON 88701 were within the 99% tolerance interval established from the 

conventional commercial reference varieties grown concurrently in the same trial. 

2) Assessment of the reproducibility of the combined-site differences at the eight individual 

sites showed statistically significant differences for: calories, carbohydrates, total fat, 

crude fiber, and NDF at one site; moisture, ADF, TDF, arginine and zinc at two sites; 

14:0 myristic, potassium, and vitamin E at three sites; ash and magnesium at four sites, 

manganese at five sites and calcium at 7 sites. With the exception of potassium, arginine 

and zinc, each at a single site, all individual site mean values of MON 88701 for all 

nutrient components with significant differences were within the 99% tolerance interval 

established from the conventional commercial reference varieties grown concurrently in 

the same trial. The control values for arginine and zinc were also outside the tolerance 

interval at the individual sites where the MON 88701 value was outside the tolerance 

interval.

3) With the exception of calories, combined-site mean values and individual site mean 

values of MON 88701 for all nutrient components including those that were significantly 

different were within the context of the natural variability of commercial cotton 
Monsanto Company 12-CT-244U 421 of 620 

composition as published in the scientific literature and/or available in the ILSI Crop 

Composition Database (ILSI, 2011). 

Five of the 17 cottonseed nutrient statistically significant differences between MON 88701 and 

the conventional control observed in the combined-site data analysis were attributable to small 

differences in proximates (ash, carbohydrates expressed as % dw, calories expressed as Kcal/100g 

% dw, moisture expressed as % fw and total fat expressed as % dw). For ash, calories, and total 

fat, the relative magnitudes of the differences between the mean values for MON 88701 and the 

conventional control were all small increases (4.77% for ash, 1.04% for calories and 5.37% for 

total fat). The relative magnitudes of differences for mean values of carbohydrates and moisture 

between MON 88701 and the conventional control were both small decreases; 2.96% for 

carbohydrates and 6.45% for moisture. All of the nutrient mean values for MON 88701 

observed in the combined-site analysis for proximates were within the 99% tolerance interval 

established by conventional commercial reference varieties grown concurrently in the same trial. 

Significant differences for proximate mean values between MON 88701 and the conventional 

control were not consistently observed among sites. Ash was significantly different at four 

individual sites, with relative magnitudes of differences ranging from 4.34 to 11.0%. Moisture 

was decreased at two sites, with relative magnitude of differences ranging from 8.51 to 11.58%, 

carbohydrates had a relative decrease of 4.71% at one site. Both calories and total fat had small 

increases at one site with 1.30% for calories and 6.90% for total fat. Overall, observed 

differences in proximate values between MON 88701 and the conventional control were not 

considered to be meaningful from a food and feed safety or nutritional perspective because they 

were small, not consistently reproduced across the individual sites, and the mean MON 88701 

combined-site values were within the 99% tolerance interval established by conventional 

commercial reference varieties grown concurrently in the same trial and within the context of the 

natural variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

Four of the 17 cottonseed nutrient differences between MON 88701 and the conventional control 

observed in the combined-site data analysis were attributable to small differences in fiber (ADF, 

crude fiber, NDF, and TDF, all expressed as % dw). All relative magnitudes of the differences 

between the mean value for MON 88701 and the conventional control were small decreases 

(3.93% for ADF, 4.12% for crude fiber, 3.56% for NDF, and 3.35% for TDF). All of the 

nutrient mean values for MON 88701 observed in the combined-site analysis for fiber were 

within the 99% tolerance interval established by conventional commercial reference varieties 

grown concurrently in the same trial. Significant differences for fiber mean values between 

MON 88701 and the conventional control were not consistently observed among individual sites. 

ADF and TDF were significantly different at two sites, with small decreases ranging from 8.50 

to 8.91% for ADF and 5.21 to 6.83% for TDF. Crude fiber and NDF were significantly different 

at one site, with a small decrease of 5.46% for crude fiber and 5.66% for NDF. Overall, 

observed differences in fiber values between MON 88701 and the conventional control were not 

considered to be meaningful from a food and feed safety or nutritional perspective because they 

were small, not consistently reproduced across the individual sites, and the mean MON 88701 

combined-site values were within the 99% tolerance interval established by conventional 

commercial reference varieties grown concurrently in the same trial and within the context of the 
Monsanto Company 12-CT-244U 422 of 620 

natural variability of commercial cotton composition as published in the scientific literature 

and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

One of the cottonseed nutrient differences between MON 88701 and the conventional control 

observed in the combined-site analysis was attributed to a small difference in one amino acid 

(arginine, expressed as % dw). The relative magnitude of the difference between the mean value 

for MON 88701 and the conventional control was a small decrease of 3.82%. The mean arginine 

value for MON 88701 observed in the combined-site analysis was within the 99% tolerance 

interval established by conventional commercial reference varieties grown concurrently in the 

same trial. Significant differences for arginine mean values between MON 88701 and the 

conventional control were not consistently observed among individual sites, with small decreases 

ranging from 5.36 to 8.48% observed at two sites. Overall, observed differences in arginine 

values between MON 88701 and the conventional control were not considered to be meaningful 

from a food and feed safety or nutritional perspective because they were small, not consistently 

reproduced across the individual sites, and the mean MON 88701 combined-site values was 

within the 99% tolerance interval established by conventional commercial reference varieties 

grown concurrently in the same trial or within the context of the natural variability of 

commercial cotton composition as published in the scientific literature and/or available in the 

ILSI Crop Composition Database (ILSI, 2011). 

One of the cottonseed nutrient differences between MON 88701 and the conventional control 

observed in the combined-site analysis was attributed to the fatty acid 14:0 myristic acid 

(expressed as % total FA). The relative magnitude of the difference between the mean fatty acid 

value for MON 88701 and the conventional control in the combined-site analysis was a small 

decrease of 2.39%. The mean 14:0 myristic acid value for MON 88701 observed in the 

combined-site analysis was within the 99% tolerance interval established by conventional 

commercial reference varieties grown concurrently in the same trial. Significant differences for 

14:0 myristic acid mean values between MON 88701 and the conventional control were not 

consistently observed among individual sites, with three sites with small decreases ranging from 

3.08 to 4.79%. Overall, observed differences in 14:0 myristic acid values between MON 88701 

and the conventional control were not considered to be meaningful from a food and feed safety 

and nutritional perspective because they were small, not consistently reproduced across the 

individual sites, and the mean MON 88701 combined-site value was within the 99% tolerance 

interval established by conventional commercial reference varieties grown concurrently in the 

same trial and within the context of the natural variability of commercial cotton composition as 

published in the scientific literature and/or available in the ILSI Crop Composition Database 

(ILSI, 2011). 

Five of the 17 cottonseed nutrient differences between MON 88701 and the conventional control 

observed in the combined-site data analysis were attributable to small differences in minerals 

(calcium, magnesium, and potassium expressed as % dw and manganese and zinc expressed as 

mg/kg dw). For calcium, magnesium, manganese and potassium, the relative magnitude of the 

differences between the mean values for MON 88701 and the conventional control were all 

increases (14.72% for calcium, 5.81% for magnesium, 16.14% for manganese and 5.48% for 

potassium). The difference for zinc between the mean value for MON 88701 and the 

conventional control was a decrease of 5.81%. All of the nutrient mean values for MON 88701 
Monsanto Company 12-CT-244U 423 of 620 

observed in the combined-site analysis for minerals were within the 99% tolerance interval 

established by conventional commercial reference varieties grown concurrently in the same trial. 

With the exception of calcium and manganese, significant differences for mineral mean values 

between MON 88701 and the conventional control were not consistently observed among 

individual sites. Although calcium was significantly different at seven sites, with increases 

ranging from 9.33 to 24.41%, the variability observed for the test (mean range 0.10 to 0.21% 

dw) and control samples (mean range 0.081 to 0.19% dw) were very similar (Table E-20). 

Manganese was significantly different at five sites with increased differences ranging from 12.88 

to 29.26%. Magnesium was significantly different at four sites with relative increases in 

MON 88701, ranging from 5.69 to 8.36%, and potassium was significantly increased at three 

sites, with relative increases ranging from 8.76 to 18.36%. Zinc was significantly different at 

two sites with small decreases in relative magnitude of differences ranging from 8.98 to 16.23%. 

Overall, observed differences in mineral values between MON 88701 and the conventional 

control were not considered to be meaningful from a food and feed safety or nutritional 

perspective because they were generally small, not consistently reproduced across individual 

sites (with the exception of calcium), and the mean MON 88701 combined-site values were 

within the 99% tolerance interval established by conventional, commercial reference varieties 

grown concurrently in the same trial or within the context of the natural variability of 

commercial cotton composition as published in the scientific literature and/or available in the 

ILSI Crop Composition Database (ILSI, 2011). 

The relative magnitude of the difference between the mean vitamin E value for MON 88701 

and the conventional control in the combined-site analysis was a small increase of 5.84%. The 

mean vitamin E value for MON 88701 observed in the combined-site analysis was within the 

99% tolerance interval established by conventional commercial reference varieties grown 

concurrently in the same trial. The significant difference for the vitamin E mean value between 

MON 88701 and the conventional control was not consistently observed among individual sites, 

with significant increases ranging from 6.54 to 11.36% observed at three sites. Overall, the 

observed difference in the vitamin E value between MON 88701 and the conventional control 

was not considered to be meaningful from a food and feed safety and nutritional perspective 

because it was small, not consistently reproduced across the individual sites, and the mean 

MON 88701 combined-site value was within the 99% tolerance interval established by 

conventional commercial reference varieties grown concurrently in the same trial and was within 

the context of the natural variability of commercial cotton composition as published in the 

scientific literature and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

In summary, with the exception of calcium and manganese, statistical analyses found no 

consistent differences between the levels of nutrient components in cottonseed from MON 88701 

and the conventional control. Differences were observed for calcium and manganese in 

combined analyses and most individual sites, but the magnitudes of differences for this nutrient 

was less than the variability for the control samples, and values were within the range of natural 

variability for cottonseed. 

These findings support the conclusion of compositional equivalence of MON 88701 to 

conventional cotton. 
Monsanto Company 12-CT-244U 424 of 620 

E.5.2. Anti-Nutrient Levels in Cottonseed Not Treated with Dicamba or Glufosinate 

Cottonseed was analyzed for five anti-nutrients, namely: dihydrosterculic acid, malvalic acid, 

sterculic acid, free gossypol, and total gossypol. Out of these five anti-nutrients, in the 

combined-site analysis of MON 88701 not treated with dicamba or glufosinate and the 

conventional control, malvalic and sterculic acids, as well as free gossypol, did not show any 

significant differences (p<0.05) in their mean values (Table E-21). In the combined-site analysis 

dihydrosterculic acid and total gossypol were significantly different (Table E-2). 

The significant differences in anti-nutrients were further evaluated using considerations relevant 

to the safety and nutritional quality of MON 88701 when compared to the conventional control: 

1) All anti-nutrient component differences observed in the combined-site statistical analysis, 

which reflected an increase in MON 88701 mean values with respect to the conventional 

control, were 12.64% or less. The relative magnitudes of the differences for dihydrosterculic 

acid and total gossypol were 12.64% and 6.26%, respectively. 

2) Mean values for all significantly different anti-nutrient components from the combined-site 

analysis of MON 88701 were within the 99% tolerance interval established from the 

conventional commercial reference varieties grown concurrently in the same trial. 

3) Assessment of the reproducibility of the combined-site differences at the eight individual 

sites showed significant differences for: dihydrosterculic and total gossypol at only one site. 

All individual site mean values of MON 88701 for both anti-nutrient components with 

significant differences were within the 99% tolerance interval established from the 

conventional commercial reference varieties grown concurrently in the same trial. 

4) All combined-site mean values and individual site mean values of MON 88701 for all antinutrient components including those that were significantly different were within the context 

of the natural variability of commercial cotton composition as published in the scientific 

literature and/or available in the ILSI Crop Composition Database (ILSI, 2011). 

The two cottonseed anti-nutrient differences between MON 88701 and the conventional control 

observed in the combined-site data analysis were attributed to small differences in one 

cyclopropenoid fatty acid (dihydrosterculic; expressed as % total fatty acid) and total gossypol 

(expressed as % dw). The relative magnitude of the differences between the mean values for 

MON 88701 and the conventional control were increases of 12.64% for dihydrosterculic acid 

and 6.26% for total gossypol. These anti-nutrient mean values for MON 88701 observed in the 

combined-site analysis were within the 99% tolerance interval established by conventional 

commercial reference varieties grown concurrently in the same trial. Significant differences for 

the two anti-nutrient mean values between MON 88701 and the conventional control were not 

consistently observed among individual sites. Both dihydrosterculic acid and total gossypol 

were significantly different at only one site with an increase of 33.11% for dihydrosterculic acid 

and an increase of 7.99% for total gossypol. Overall, observed differences in anti-nutrient values 

between MON 88701 and the conventional control were not considered to be meaningful from a 

food and feed safety or nutritional perspective because they were generally small, not 
Monsanto Company 12-CT-244U 425 of 620 

consistently reproduced across the individual sites, and the mean MON 88701 values were 

within the 99% tolerance interval established by conventional commercial reference varieties 

grown concurrently in the same trial and within the context of the natural variability of 

commercial cotton composition as published in the scientific literature and/or available in the 

ILSI Crop Composition Database (ILSI, 2011). 

In summary, statistical analyses found no consistent statistically significant differences between 

the levels of anti-nutrient components in cottonseed from MON 88701 and the conventional 

control and mean values for anti-nutrients were within the natural variability found for 

cottonseed. These findings supported the conclusion of compositional equivalence of 

MON 88701 to conventional cotton. 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for M(Not Treated) vs. Conventional Control 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed Proximate (% dw)Ash 4.30 4.11 4.77 0.002 3.76 - 4.88 Calories (Kcal/100g) 500.37 495.24 1.04 <0.001 487.62 - 511Carbohydrates 44.47 45.83 -2.96 <0.001 41.07 - 48.8Moisture (% fw) 7.00 7.48 -6.45 <0.001 5.81 - 9.07 Total Fat 23.51 22.31 5.37 <0.001 20.99 - 25.5Cottonseed Fiber (% dw)Acid Detergent Fiber 25.53 26.58 -3.93 0.009 23.30 - 30.4Crude Fiber 17.78 18.54 -4.12 0.020 14.54 - 20.7
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for M(Not Treated) vs. Conventional Control (continued) 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed Fiber (% dw)Neutral Detergent Fiber 31.43 32.59 -3.56 0.005 28.05 - 37.2Total Dietary Fiber 39.75 41.12 -3.35 0.001 36.22 - 43.2Cottonseed Amino Acid (% dw)Arginine 3.03 3.15 -3.82 0.002 2.31 - 3.62 Cottonseed Fatty Acid (% Total FA)14:0 Myristic 0.77 0.79 -2.39 0.018 0.66 - 0.95 Cottonseed MineralCalcium (% dw) 0.15 0.13 14.72 <0.001 0.10 - 0.21 Magnesium (% dw) 0.40 0.38 5.81 <0.001 0.35 - 0.45 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued) 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in Combined-Site AnalysisCottonseed MineralManganese (mg/kg dw) 13.63 11.73 16.14 <0.001 10.59 - 17.4Potassium (% dw) 1.13 1.07 5.48 0.012 0.99 - 1.32 Zinc (mg/kg dw) 37.81 40.14 -5.81 0.009 27.60 - 46.0Cottonseed Vitamin (mg/kg dw)Vitamin E 139.01 131.33 5.84 0.002 87.22 - 184.Cottonseed Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 0.14 12.64 <0.001 0.12 - 0.19 Cottonseed Gossypol (% dw)Total Gossypol 1.03 0.97 6.26 <0.001 0.84 - 1.52 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued) 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 7 SitesCalcium (% dw) Site ARTI 0.15 0.12 24.41 0.007 0.14 - 0.16 Calcium (% dw) Site GACH 0.13 0.11 17.80 <0.001 0.13 - 0.13 Calcium (% dw) Site KSLA 0.20 0.18 10.11 0.032 0.18 - 0.21 Calcium (% dw) Site NCBD 0.15 0.14 9.33 0.002 0.15 - 0.15 Calcium (% dw) Site NMLC 0.15 0.13 15.03 0.006 0.14 - 0.15 Calcium (% dw) Site SCEK 0.11 0.091 21.62 0.013 0.10 - 0.12 Calcium (% dw) Site TXPL 0.16 0.14 17.83 <0.001 0.16 - 0.17 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued) 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Fatty Acid (% Total FA) - 5 Sites18:0 Stearic Site ARTI 2.65 2.51 5.54 0.035 2.59 - 2.71 18:0 Stearic Site LACH 2.61 2.52 3.54 0.014 2.58 - 2.64 18:0 Stearic Site NCBD 2.53 2.34 7.94 0.021 2.49 - 2.57 18:0 Stearic Site NMLC 2.52 2.64 -4.70 <0.001 2.49 - 2.56 18:0 Stearic Site TXPL 2.31 2.46 -6.14 0.001 2.26 - 2.34 Cottonseed Mineral - 5 SitesManganese (mg/kg dw) Site ARTI 14.86 11.50 29.26 0.034 13.28 - 17.4Manganese (mg/kg dw) Site GACH 14.26 11.51 23.89 <0.001 13.82 - 15.0
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 5 SitesManganese (mg/kg dw) Site KSLA 14.18 12.56 12.88 0.013 13.48 - 14.8Manganese (mg/kg dw) Site SCEK 12.44 9.72 28.04 0.008 10.59 - 13.8Manganese (mg/kg dw) Site TXPL 11.38 9.04 25.84 0.002 10.73 - 12.8Cottonseed Proximate (% dw) - 4 SitesAsh Site GACH 4.39 4.21 4.34 0.003 4.24 - 4.50 Ash Site NCBD 4.33 4.14 4.56 0.043 4.20 - 4.48 Ash Site SCEK 4.10 3.74 9.72 0.011 3.91 - 4.23 Ash Site TXPL 3.84 3.46 11.00 0.001 3.76 - 3.98 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Fatty Acid (% Total FA) - 4 Sites18:3 Linolenic Site ARTI 0.14 0.13 9.88 0.022 0.14 - 0.14 18:3 Linolenic Site NCBD 0.36 0.29 25.90 0.008 0.34 - 0.38 18:3 Linolenic Site NMLC 0.16 0.14 9.67 0.004 0.15 - 0.16 18:3 Linolenic Site SCEK 0.16 0.15 6.70 0.005 0.15 - 0.16 Cottonseed Mineral - 4 SitesMagnesium (% dw) Site GACH 0.41 0.38 8.36 <0.001 0.40 - 0.43 Magnesium (% dw) Site KSLA 0.42 0.40 5.69 0.004 0.41 - 0.43 Magnesium (% dw) Site SCEK 0.38 0.36 7.25 0.017 0.36 - 0.41 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Mineral - 4 SitesMagnesium (% dw) Site TXPL 0.36 0.34 7.43 <0.001 0.35 - 0.37 Cottonseed Fatty Acid (% Total FA) - 3 Sites14:0 Myristic Site KSLA 0.69 0.72 -4.64 0.013 0.66 - 0.70 14:0 Myristic Site NCBD 0.71 0.75 -4.79 0.024 0.69 - 0.73 14:0 Myristic Site NMLC 0.95 0.98 -3.08 0.008 0.94 - 0.95 Cottonseed Mineral - 3 SitesPotassium (% dw) Site GACH 1.22 1.12 8.76 <0.001 1.18 - 1.24 Potassium (% dw) Site SCEK 1.14 1.02 11.89 0.031 1.11 - 1.19 Potassium (% dw) Site TXPL 1.03 0.87 18.36 0.002 0.99 - 1.09 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Vitamin (mg/kg dw) - 3 SitesVitamin E Site GACH 149.29 140.12 6.54 0.046 142.84 - 153Vitamin E Site LACH 167.00 149.96 11.36 0.002 158.95 - 173Vitamin E Site NCBD 174.64 156.99 11.24 0.025 165.41 - 184Cottonseed Proximate (% dw) - 2 SitesMoisture (% fw) Site GACH 6.39 7.23 -11.58 0.015 6.19 - 6.64 Moisture (% fw) Site SCEK 6.48 7.08 -8.51 0.019 6.16 - 6.98 Cottonseed Fiber (% dw) - 2 SitesAcid Detergent Fiber Site ARTI 25.07 27.53 -8.91 0.010 24.53 - 25.4Acid Detergent Fiber Site LACH 25.94 28.35 -8.50 0.008 25.56 - 26.3
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Fiber (% dw) - 2 SitesTotal Dietary Fiber Site LACH 41.09 43.35 -5.21 0.016 39.95 - 42.2Total Dietary Fiber Site NMLC 38.30 41.10 -6.83 0.003 37.12 - 39.3Cottonseed Amino Acid (% dw) - 2 SitesArginine Site KSLA 3.00 3.28 -8.48 0.009 2.88 - 3.05 Arginine Site NMLC 3.51 3.71 -5.36 0.008 3.34 - 3.62 Lysine Site KSLA 1.19 1.25 -5.16 0.028 1.16 - 1.22 Lysine Site LACH 1.25 1.18 6.25 0.035 1.23 - 1.27 Cottonseed Fatty Acid (% Total FA) - 2 Sites18:2 Linoleic Site LACH 56.04 56.63 -1.03 0.027 55.71 - 56.3
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in More than One Individual SiteCottonseed Fatty Acid (% Total FA) - 2 Sites18:2 Linoleic Site NCBD 56.04 57.19 -2.01 0.021 55.76 - 56.3Cottonseed Mineral - 2 SitesZinc (mg/kg dw) Site NCBD 41.50 49.54 -16.23 0.009 39.45 - 43.0Zinc (mg/kg dw) Site NMLC 44.99 49.43 -8.98 0.004 44.56 - 46.0Statistical Differences Observed in One SiteCottonseed Proximate (% dw)Calories (Kcal/100g) Site NCBD 498.21 491.80 1.30 0.048 496.22 - 499Carbohydrates Site TXPL 44.20 46.39 -4.71 0.014 43.36 - 44.5Total Fat Site NCBD 23.08 21.59 6.90 0.022 22.78 - 23.3
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued)

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in One SiteCottonseed Fiber (% dw)Crude Fiber Site KSLA 16.71 17.67 -5.46 0.047 16.10 - 17.3Neutral Detergent Fiber Site LACH 32.12 34.05 -5.66 0.003 30.20 - 33.9Cottonseed Amino Acid (% dw)Aspartic Acid Site KSLA 2.35 2.50 -5.91 0.038 2.32 - 2.37 Glutamic Acid Site GACH 4.69 4.96 -5.45 0.041 4.51 - 4.98 Histidine Site KSLA 0.73 0.77 -6.41 0.006 0.69 - 0.76 Isoleucine Site KSLA 0.90 0.95 -5.13 0.017 0.88 - 0.94 Leucine Site KSLA 1.51 1.58 -4.68 0.017 1.47 - 1.55 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued) 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in One SiteCottonseed Amino Acid (% dw)Phenylalanine Site KSLA 1.42 1.53 -6.88 0.014 1.37 - 1.45 Tyrosine Site KSLA 0.79 0.84 -5.94 0.045 0.76 - 0.80 Cottonseed Fatty Acid (% Total FA)18:1 Oleic Site NCBD 15.29 14.70 4.04 0.026 15.01 - 15.522:0 Behenic Site ARTI 0.14 0.15 -6.75 0.029 0.14 - 0.15 Cottonseed Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid Site GACH 0.16 0.12 33.11 0.012 0.14 - 0.17 Sterculic Acid Site GACH 0.23 0.18 24.51 0.028 0.20 - 0.24 
Monsanto Company 12-CT-244U 

Table E-19. Summary of Differences (p<0.05) for the Comparison of Cottonseed Component Levels for MON 88701 (Not Treated) vs. Conventional Control (continued) 

 Mean Difference 

(MON 88701 minus Control)

Analytical Component (Units)1

MON 887012

Mean3

Control4

Mean

Mean Difference 

(% of Control)

Significance

(p-Value)

MON 8870

Range

Statistical Differences Observed in One SiteCottonseed Gossypol (% dw)Free Gossypol Site NMLC 0.86 0.69 23.91 0.008 0.76 - 0.95 Total Gossypol Site TXPL 1.09 1.01 7.99 0.009 1.03 - 1.16 1dw = dry weight; fw = fresh weight; FA = fatty acid. 2Test refers to MON 88701 (Not Treated). These plants were not treated with dicamba or glufosinate. 3Mean = least-square mean. 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not TreateControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.30 (0.11) 4.11 (0.11) 0.20 (0.052) 0.084, 0.31 0.002 

(3.76 - 4.88) (3.34 - 5.00) (-0.42 - 0.79) 

Calories (Kcal/100g) 500.37 (1.65) 495.24 (1.71) 5.13 (1.29) 2.57, 7.69 <0.001 

(487.62 - 511.92) (487.70 - 512.65) (-5.97 - 15.41) 

Carbohydrates 44.47 (0.56) 45.83 (0.57) -1.36 (0.32) -1.99, -0.73 <0.001 

(41.07 - 48.81) (42.14 - 50.30) (-5.09 - 1.56) 

Moisture (% fw) 7.00 (0.26) 7.48 (0.27) -0.48 (0.11) -0.71, -0.26 <0.001 

(5.81 - 9.07) (6.15 - 9.19) (-1.45 - 0.15) 

Protein 27.71 (0.77) 27.79 (0.77) -0.075 (0.31) -0.73, 0.58 0.810 

(22.49 - 31.29) (23.53 - 31.27) (-2.53 - 4.39) 

Total Fat 23.51 (0.31) 22.31 (0.33) 1.20 (0.26) 0.69, 1.71 <0.001 

(20.99 - 25.54) (20.71 - 25.20) (-1.21 - 3.21) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.53 (0.34) 26.58 (0.35) -1.05 (0.35) -1.79, -0.30 0.009 

(23.30 - 30.43) (22.08 - 29.58) (-4.02 - 5.70) 

Crude Fiber 17.78 (0.37) 18.54 (0.38) -0.76 (0.32) -1.41, -0.12 0.020 

(14.54 - 20.73) (16.06 - 21.70) (-5.57 - 3.82) 

Neutral Detergent Fiber 31.43 (0.51) 32.59 (0.53) -1.16 (0.41) -1.97, -0.35 0.005 

(28.05 - 37.27) (28.87 - 35.89) (-4.66 - 6.42) 

Total Dietary Fiber 39.75 (0.39) 41.12 (0.41) -1.38 (0.36) -2.15, -0.61 0.001 

(36.22 - 43.22) (39.05 - 44.37) (-4.41 - 2.04) 

Amino Acid (% dw)Alanine 1.05 (0.020) 1.05 (0.020) -0.0034 (0.0091) -0.023, 0.016 0.714 

(0.88 - 1.15) (0.88 - 1.17) (-0.076 - 0.11) 

Arginine 3.03 (0.10) 3.15 (0.10) -0.12 (0.033) -0.19, -0.050 0.002 

(2.31 - 3.62) (2.41 - 3.77) (-0.37 - 0.26) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.39 (0.062) 2.41 (0.062) -0.022 (0.027) -0.079, 0.035 0.422 

(1.95 - 2.69) (1.92 - 2.74) (-0.28 - 0.20) 

Cystine 0.40 (0.0091) 0.40 (0.0094) -0.0018 (0.0070) -0.016, 0.012 0.793 

(0.33 - 0.49) (0.31 - 0.46) (-0.074 - 0.086) 

Glutamic Acid 4.71 (0.13) 4.84 (0.14) -0.13 (0.072) -0.29, 0.025 0.093 

(3.79 - 5.57) (3.66 - 5.70) (-1.01 - 0.57) 

Glycine 1.09 (0.020) 1.09 (0.020) -0.0061 (0.011) -0.029, 0.017 0.577 

(0.92 - 1.19) (0.91 - 1.20) (-0.11 - 0.088) 

Histidine 0.74 (0.019) 0.75 (0.019) -0.0076 (0.0073) -0.023, 0.0079 0.312 

(0.58 - 0.84) (0.61 - 0.84) (-0.069 - 0.064) 

Isoleucine 0.91 (0.018) 0.92 (0.018) -0.0087 (0.0079) -0.026, 0.0082 0.291 

(0.76 - 1.01) (0.77 - 1.03) (-0.074 - 0.074) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.53 (0.032) 1.54 (0.032) -0.0080 (0.013) -0.036, 0.020 0.544 

(1.28 - 1.68) (1.28 - 1.69) (-0.095 - 0.14) 

Lysine 1.23 (0.025) 1.23 (0.025) -0.0019 (0.015) -0.034, 0.031 0.904 

(1.03 - 1.37) (1.06 - 1.39) (-0.084 - 0.14) 

Methionine 0.39 (0.0079) 0.38 (0.0084) 0.011 (0.0081) -0.0049, 0.027 0.167 

(0.33 - 0.44) (0.32 - 0.46) (-0.081 - 0.088) 

Phenylalanine 1.43 (0.039) 1.46 (0.039) -0.026 (0.014) -0.056, 0.0044 0.088 

(1.13 - 1.63) (1.15 - 1.66) (-0.14 - 0.11) 

Proline 1.02 (0.029) 1.03 (0.029) -0.014 (0.012) -0.039, 0.011 0.246 

(0.78 - 1.16) (0.81 - 1.25) (-0.13 - 0.10) 

Serine 1.08 (0.025) 1.09 (0.026) -0.0031 (0.015) -0.035, 0.028 0.834 

(0.93 - 1.28) (0.86 - 1.24) (-0.14 - 0.14) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.87 (0.016) 0.86 (0.016) 0.0033 (0.0083) -0.014, 0.021 0.694 

(0.73 - 0.95) (0.73 - 0.95) (-0.062 - 0.086) 

Tryptophan 0.41 (0.0092) 0.42 (0.0095) -0.0092 (0.0066) -0.022, 0.0041 0.171 

(0.34 - 0.50) (0.37 - 0.52) (-0.099 - 0.11) 

Tyrosine 0.81 (0.017) 0.81 (0.018) -0.0030 (0.0083) -0.021, 0.015 0.718 

(0.68 - 0.88) (0.67 - 0.91) (-0.076 - 0.074) 

Valine 1.21 (0.027) 1.23 (0.027) -0.013 (0.011) -0.037, 0.011 0.257 

(0.98 - 1.38) (1.00 - 1.40) (-0.10 - 0.15) 

Fatty Acid (% Total FA)14:0 Myristic 0.77 (0.030) 0.79 (0.031) -0.019 (0.0071) -0.034, -0.0036 0.018 

(0.66 - 0.95) (0.71 - 0.98) (-0.087 - 0.041) 

16:0 Palmitic 23.93 (0.30) 23.80 (0.30) 0.13 (0.076) -0.035, 0.29 0.116 

(22.30 - 25.45) (22.69 - 25.05) (-0.48 - 0.63) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.50 (0.0094) 0.50 (0.0094) -0.00052 (0.0038) -0.0087, 0.0077 0.894 

(0.45 - 0.55) (0.45 - 0.54) (-0.024 - 0.024) 

18:0 Stearic 2.52 (0.058) 2.47 (0.058) 0.045 (0.036) -0.032, 0.12 0.228 

(2.16 - 2.93) (2.15 - 2.76) (-0.24 - 0.26) 

18:1 Oleic 15.05 (0.26) 14.96 (0.26) 0.094 (0.070) -0.055, 0.24 0.196 

(14.05 - 16.29) (14.06 - 16.44) (-0.54 - 1.00) 

18:2 Linoleic 55.84 (0.39) 56.15 (0.40) -0.31 (0.16) -0.64, 0.023 0.065 

(54.22 - 58.48) (54.04 - 57.93) (-1.65 - 0.73) 

18:3 Linolenic 0.18 (0.022) 0.17 (0.022) 0.013 (0.0068) -0.0017, 0.027 0.078 

(0.11 - 0.38) (0.12 - 0.30) (-0.032 - 0.11) 

20:0 Arachidic 0.29 (0.0086) 0.28 (0.0087) 0.0044 (0.0047) -0.0057, 0.015 0.365 

(0.23 - 0.32) (0.23 - 0.32) (-0.049 - 0.042) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.15 (0.0051) 0.15 (0.0051) -0.0010 (0.0029) -0.0074, 0.0053 0.730 

(0.12 - 0.17) (0.13 - 0.21) (-0.057 - 0.027) 

MineralCalcium (% dw) 0.15 (0.0093) 0.13 (0.0093) 0.019 (0.0022) 0.014, 0.024 <0.001 

(0.10 - 0.21) (0.081 - 0.19) (0.0037 - 0.039) 

Copper (mg/kg dw) 8.94 (0.70) 8.93 (0.70) 0.015 (0.16) -0.30, 0.33 0.925 

(5.02 - 12.15) (5.40 - 11.92) (-2.19 - 1.72) 

Iron (mg/kg dw) 72.43 (4.40) 71.33 (4.48) 1.10 (2.74) -4.74, 6.94 0.693 

(41.73 - 109.70) (45.03 - 95.10) (-20.63 - 27.89) 

Magnesium (% dw) 0.40 (0.0083) 0.38 (0.0084) 0.022 (0.0032) 0.016, 0.028 <0.001 

(0.35 - 0.45) (0.33 - 0.44) (-0.015 - 0.055) 

Manganese (mg/kg dw) 13.63 (0.47) 11.73 (0.48) 1.89 (0.28) 1.29, 2.50 <0.001 

(10.59 - 17.47) (8.61 - 14.11) (-0.84 - 5.26) 
Monsanto Company 12-CT-244U 

Table E-20. Statistical Summary of Combined-Site Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.71 (0.031) 0.72 (0.031) -0.0035 (0.0067) -0.017, 0.0099 0.605 

(0.58 - 0.87) (0.54 - 0.87) (-0.078 - 0.072) 

Potassium (% dw) 1.13 (0.028) 1.07 (0.028) 0.059 (0.020) 0.015, 0.10 0.012 

(0.99 - 1.32) (0.79 - 1.27) (-0.13 - 0.31) 

Sodium (% dw) 0.026 (0.0095) 0.029 (0.0096) -0.0035 (0.0046) -0.013, 0.0064 0.466 

(0.0053 - 0.082) (0.0053 - 0.10) (-0.085 - 0.026) 

Zinc (mg/kg dw) 37.81 (2.01) 40.14 (2.02) -2.33 (0.77) -3.99, -0.67 0.009 

(27.60 - 46.04) (28.22 - 52.95) (-13.50 - 1.99) 

Vitamin (mg/kg dw)Vitamin E 139.01 (9.87) 131.33 (9.88) 7.68 (2.07) 3.26, 12.09 0.002 

(87.22 - 184.47) (91.78 - 162.98) (-7.82 - 32.93) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2Test refers to MON 88701 (Not Treated). These plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-21. Statistical Summary of Combined-Site Cottonseed Anti-nutrients for MON 88701 (Not TreatControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0034) 0.14 (0.0037) 0.017 (0.0044) 0.0086, 0.026 <0.001 

(0.12 - 0.19) (0.11 - 0.17) (-0.011 - 0.058) 

Malvalic Acid 0.39 (0.015) 0.37 (0.016) 0.019 (0.015) -0.011, 0.048 0.210 

(0.24 - 0.50) (0.26 - 0.49) (-0.11 - 0.19) 

Sterculic Acid 0.22 (0.0067) 0.21 (0.0072) 0.0099 (0.0081) -0.0064, 0.026 0.229 

(0.12 - 0.27) (0.17 - 0.27) (-0.075 - 0.093) 

Gossypol (% dw)Free Gossypol 0.93 (0.037) 0.89 (0.037) 0.041 (0.020) -0.0031, 0.084 0.065 

(0.76 - 1.10) (0.68 - 1.20) (-0.14 - 0.27) 

Total Gossypol 1.03 (0.037) 0.97 (0.037) 0.061 (0.017) 0.026, 0.096 <0.001 

(0.84 - 1.52) (0.74 - 1.10) (-0.028 - 0.44) 

1dw = dry weight; FA = fatty acid. 2Test refers to MON 88701 (Not Treated). These plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 449 of 620 

Table E-22 Literature and ILSI Ranges for Components in Cottonseed 

Cottonseed Tissue Components1 Literature Range2 ILSI Range3

Cottonseed Nutrients 

Proximates (% dw) 

Ash 3.87 – 5.29a; 3.7 – 4.2d 3.761 – 5.342 

Carbohydrates by calculation 45.28 – 53.62a 39.0 – 53.6 

Calories by calculation 

(Kcal/100g) 

471.34 – 506.95a Not available 

Moisture (% fw) 2.25 – 7.49a 2.3 – 9.9 

Protein 24.54 – 30.83a; 21.2 – 25.9b 21.48 – 32.97 

Total Fat 17.37 – 25.16a; 14.4 – 16.9d 17.201 – 27.292 

 

Fiber (% dw) 

Acid Detergent Fiber 21.10 – 34.8a

; 37.6 – 40.5d 19.74 – 38.95 

Neutral Detergent Fiber 32.92 – 45.83a

; 50.0 – 53.6d 25.56 – 51.87 

Crude Fiber 13.85 – 17.94a

 13.86 – 23.10

Total Dietary Fiber not available 33.69 – 47.55

 

Amino Acids (% total AA) (% dw) 

Alanine 4.16 – 4.41a; 3.6 – 4.2b 0.80 – 1.22 

Arginine 11.28 – 12.51 a; 10.9 – 12.3b 2.06 – 3.72 

Aspartic acid 9.73 – 9.99 a; 8.8 – 9.5b 1.82 – 2.94 

Cystine/Cysteine 1.60 – 1.92 a; 2.3 – 3.4b 0.35 – 0.56 

Glutamic acid 20.76 – 21.61 a; 20.5 – 22.4 b 3.91 – 6.72 

Glycine 4.44 – 4.58 a; 3.8 – 4.5 b 0.83 – 1.32 

Histidine 3.00 – 3.12 a; 2.6 – 2.8 b 0.57 – 0.91 

Isoleucine 3.10 – 3.67 a; 3.0 – 3.4 b 0.62 – 1.05 

Leucine 6.27 – 6.65 a; 5.5 – 6.1 b 1.14 – 1.86 

Lysine 4.85 – 5.37 a; 4.2 – 4.6 b 0.94 – 1.46 

Methionine 1.46 – 1.88 a; 1.3 – 1.8 b 0.30 – 0.47 

Phenylalanine 5.56 – 5.77 a; 5.0 – 5.6 b 1.02 – 1.72 

Proline 4.06 – 4.28 a; 3.1 – 4.0 b 0.75 – 1.23 

Serine 4.45 – 4.86 a; 3.9 – 4.4 b 0.91 – 1.35 

Threonine 3.26 – 3.59 a; 2.8 – 3.2b 0.55 – 0.92 

Tryptophan 0.97 – 1.21 a; 1.0 – 1.4 b 0.194 – 0.319 

Tyrosine 2.65 – 2.92 a; 2.8 – 3.3 b 0.53 – 0.84 

Valine 4.76 – 5.14 a; 4.3 – 4.7 b 0.87 – 1.49 

 

Fatty Acids (% total FA) 

8:0 Caprylic not available not available 

10:0 Capric not available not available 

12:0 Lauric not available not available 

14:0 Myristic 0.55 – 2.40a; 0.6 – 1.5b 0.455 – 2.400 

14:1 Myristoleic not available not available 

15:0 Pentadecanoic 0.050 – 0.17a 0.103 – 0.481 

15:1 Pentadecenoic not available not available 

16:0 Palmitic 21.23 – 27.9 a; 17.6 – 24.8 b 15.11 – 27.90 

16:1 Palmitoleic 0.55 – 1.16 a 0.464 – 1.190 

17:0 Heptadecanoic not available 0.092 – 0.119 

 
Monsanto Company 12-CT-244U 450 of 620 

Table E-22. Literature and ILSI Ranges for Components in Cottonseed (continued) 

Cottonseed Tissue Components1 Literature Range2 ILSI Range3

17:1 Heptadecenoic not available not available 

18:0 Stearic 1.99 – 3.11 a; 2.0 – 2.5 b 0.20 – 3.11 

18:1 Oleic 13.90 – 20.10 a; 15.0 – 20.7 b 12.8 – 25.3 

18:2 Linoleic 46.00 – 56.88 a 46.0 – 59.4 

18:3 Gamma Linolenic 0.050 – 0.25 a 0.097 – 0.232 

18:3 Linolenic 0.050 – 0.25 a 0.11 – 0.35 

20:0 Arachidic 0.25 – 0.33 a 0.186 – 0.414 

20:1 Eicosenoic not available 0.095 – 0.098 

20:2 Eicosadienoic not available not available 

20:3 Eicosatrienoic not available not available

20:4 Arachidonic not available not available

22:0 Behenic 0.13 – 0.17 a 0.104 – 0.295 

 

Vitamins (mg/kg fw) (mg/kg dw)

Vitamin E 99 – 224c 70.825 – 197.243 

 

Minerals (% dw) 

Calcium 0.10 – 0.33a 0.10323 – 0.32581 

Copper (mg/kg dw) 3.54 – 11.14a 3.13 – 24.57 

Iron (mg/kg dw) 40.58 – 56.54 a 36.71 – 318.38 

Magnesium 0.37 – 0.46 a 0.34709 – 0.49312 

Manganese (mg/kg dw) 11.06 – 18.31 a 10.69 – 21.96 

Phosphorus 0.60 – 0.84 a 0.48254 – 0.99157 

Potassium 0.98 – 1.24 a 0.98345 – 1.44835 

Sodium 0.0054 – 0.74 a 0.01118 – 0.73548 

Zinc (mg/kg dw) 30.21 – 47.75 a 27.0 – 59.5 

 

Cottonseed Anti-Nutrients 

Gossypol, Total (% dw) 0.57 – 1.42a; 0.55 – 0.77d 0.547 – 1.522 

Gossypol, Free (% dw) 0.53 – 1.20a 0.454 – 1.399 

 

Cyclopropenoid Fatty Acids 

(% total FA)

 

Dihydrosterculic 0.13 – 0.24 a 0.075 – 0.310 

Malvalic 0.33 – 0.58 a 0.229 – 0.759 

Sterculic 0.21 – 0.56 a 0.190 – 0.556 

 1

fw=fresh weight; dw=dry weight 

2

Literature range references; a(Hamilton, et al., 2004); b(Lawhon, et al., 1977); c(Smith and Creelman, 2001); d

(Bertrand, et al., 2005). 

3

(ILSI, 2011).
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. Co

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.71 (0.087) 4.74 (0.12) -0.030 (0.15) -0.45, 0.39 0.850 

(4.58 - 4.88) (4.49 - 5.00) (-0.42 - 0.39) 

Calories (Kcal/100g) 499.07 (3.00) 488.30 (3.88) 10.76 (3.90) -0.069, 21.59 0.050 

(493.81 - 501.81) (487.70 - 494.60) (7.21 - 13.11) 

Carbohydrates 44.29 (0.58) 46.35 (0.81) -2.06 (0.96) -4.72, 0.60 0.097 

(43.36 - 45.15) (45.03 - 47.37) (-2.98 - -1.67) 

Moisture (% fw) 7.08 (0.27) 7.63 (0.35) -0.55 (0.35) -1.51, 0.41 0.184 

(6.14 - 7.86) (7.32 - 7.40) (-1.26 - -0.37) 

Protein 27.39 (0.24) 27.04 (0.33) 0.35 (0.41) -0.79, 1.48 0.442 

(26.65 - 28.02) (26.97 - 27.11) (-0.32 - 0.91) 

Total Fat 23.62 (0.59) 21.50 (0.78) 2.12 (0.81) -0.12, 4.36 0.058 

(22.57 - 24.08) (21.15 - 22.89) (1.18 - 2.93) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.07 (0.32) 27.53 (0.45) -2.45 (0.55) -3.97, -0.94 0.010 

(24.53 - 25.48) (26.57 - 28.49) (-3.34 - -1.42) 

Crude Fiber 17.00 (0.90) 19.47 (1.20) -2.47 (1.26) -5.97, 1.03 0.121 

(14.54 - 18.91) (19.33 - 19.85) (-2.18 - -0.94) 

Neutral Detergent Fiber 31.02 (0.79) 32.89 (1.06) -1.86 (1.13) -5.01, 1.28 0.175 

(29.94 - 32.82) (30.67 - 34.42) (-3.04 - -0.73) 

Total Dietary Fiber 39.70 (1.06) 41.67 (1.50) -1.97 (1.83) -7.06, 3.12 0.343 

(36.22 - 42.86) (40.50 - 42.84) (-4.27 - -4.04) 

Amino Acid (% dw)Alanine 1.03 (0.015) 1.02 (0.018) 0.0099 (0.017) -0.037, 0.057 0.591 

(1.00 - 1.05) (0.99 - 1.05) (0.0033 - 0.028) 

Arginine 2.93 (0.052) 3.02 (0.073) -0.086 (0.084) -0.32, 0.15 0.362 

(2.82 - 3.03) (2.89 - 3.13) (-0.21 - 0.068) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.30 (0.045) 2.32 (0.060) -0.024 (0.065) -0.20, 0.16 0.729 

(2.23 - 2.36) (2.19 - 2.42) (-0.085 - 0.077) 

Cystine 0.38 (0.010) 0.37 (0.014) 0.016 (0.017) -0.032, 0.064 0.406 

(0.36 - 0.40) (0.35 - 0.39) (0.0017 - 0.039) 

Glutamic Acid 4.52 (0.099) 4.51 (0.14) 0.0076 (0.17) -0.46, 0.47 0.965 

(4.29 - 4.73) (4.34 - 4.67) (0.057 - 0.11) 

Glycine 1.07 (0.020) 1.08 (0.026) -0.0049 (0.028) -0.084, 0.074 0.872 

(1.04 - 1.10) (1.03 - 1.11) (-0.015 - 0.029) 

Histidine 0.71 (0.016) 0.74 (0.023) -0.021 (0.026) -0.094, 0.053 0.474 

(0.66 - 0.74) (0.71 - 0.77) (-0.042 - 0.034) 

Isoleucine 0.88 (0.018) 0.91 (0.024) -0.029 (0.025) -0.098, 0.040 0.304 

(0.82 - 0.93) (0.88 - 0.93) (-0.054 - 0.0021) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.48 (0.027) 1.50 (0.037) -0.017 (0.042) -0.13, 0.098 0.700 

(1.41 - 1.55) (1.44 - 1.55) (-0.071 - 0.045) 

Lysine 1.20 (0.044) 1.23 (0.058) -0.032 (0.062) -0.21, 0.14 0.633 

(1.04 - 1.31) (1.19 - 1.26) (-0.016 - -0.0053) 

Methionine 0.38 (0.015) 0.35 (0.021) 0.027 (0.025) -0.043, 0.097 0.347 

(0.36 - 0.40) (0.35 - 0.36) (0.036 - 0.050) 

Phenylalanine 1.39 (0.027) 1.41 (0.038) -0.022 (0.045) -0.15, 0.10 0.649 

(1.34 - 1.43) (1.34 - 1.48) (-0.10 - 0.067) 

Proline 0.97 (0.021) 1.00 (0.028) -0.023 (0.030) -0.11, 0.061 0.490 

(0.89 - 1.03) (0.98 - 1.02) (-0.044 - 0.0082) 

Serine 1.05 (0.022) 1.03 (0.031) 0.022 (0.038) -0.083, 0.13 0.590 

(1.02 - 1.07) (0.99 - 1.06) (0.011 - 0.078) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.85 (0.012) 0.85 (0.017) -0.00041 (0.021) -0.058, 0.057 0.985 

(0.82 - 0.86) (0.82 - 0.88) (-0.028 - 0.040) 

Tryptophan 0.41 (0.0062) 0.42 (0.0087) -0.0085 (0.011) -0.038, 0.021 0.468 

(0.41 - 0.42) (0.40 - 0.44) (-0.033 - 0.012) 

Tyrosine 0.80 (0.015) 0.79 (0.021) 0.0015 (0.026) -0.071, 0.074 0.957 

(0.76 - 0.83) (0.76 - 0.82) (-0.034 - 0.043) 

Valine 1.17 (0.021) 1.21 (0.027) -0.034 (0.026) -0.11, 0.039 0.263 

(1.12 - 1.23) (1.17 - 1.24) (-0.059 - -0.0050) 

Fatty Acid (% Total FA)14:0 Myristic 0.78 (0.0085) 0.78 (0.012) -0.0013 (0.013) -0.037, 0.035 0.927 

(0.76 - 0.80) (0.77 - 0.78) (-0.0043 - 0.0026) 

16:0 Palmitic 25.06 (0.10) 24.98 (0.14) 0.075 (0.18) -0.41, 0.56 0.692 

(24.85 - 25.45) (24.92 - 25.05) (0.012 - 0.41) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.54 (0.0052) 0.52 (0.0073) 0.018 (0.0089) -0.0064, 0.043 0.107 

(0.52 - 0.55) (0.52 - 0.52) (0.016 - 0.024) 

18:0 Stearic 2.65 (0.026) 2.51 (0.036) 0.14 (0.045) 0.015, 0.26 0.035 

(2.59 - 2.71) (2.45 - 2.57) (0.10 - 0.26) 

18:1 Oleic 14.82 (0.11) 14.68 (0.15) 0.13 (0.17) -0.33, 0.60 0.475 

(14.65 - 14.99) (14.58 - 14.70) (-0.050 - 0.37) 

18:2 Linoleic 54.83 (0.22) 55.31 (0.31) -0.49 (0.37) -1.52, 0.55 0.263 

(54.28 - 55.22) (55.26 - 55.38) (-1.09 - -0.074) 

18:3 Linolenic 0.14 (0.0021) 0.13 (0.0030) 0.013 (0.0035) 0.0030, 0.023 0.022 

(0.14 - 0.14) (0.12 - 0.14) (0.0051 - 0.019) 

20:0 Arachidic 0.31 (0.0079) 0.29 (0.011) 0.015 (0.011) -0.017, 0.046 0.266 

(0.28 - 0.32) (0.27 - 0.31) (0.0037 - 0.012) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.14 (0.0026) 0.15 (0.0033) -0.010 (0.0031) -0.019, -0.0016 0.029 

(0.14 - 0.15) (0.15 - 0.16) (-0.018 - -0.0043) 

MineralCalcium (% dw) 0.15 (0.0035) 0.12 (0.0050) 0.030 (0.0061) 0.013, 0.047 0.007 

(0.14 - 0.16) (0.12 - 0.13) (0.024 - 0.039) 

Copper (mg/kg dw) 9.81 (0.34) 9.64 (0.41) 0.17 (0.38) -0.88, 1.21 0.680 

(9.20 - 10.96) (8.79 - 9.79) (-0.45 - 0.41) 

Iron (mg/kg dw) 86.87 (5.63) 80.76 (7.78) 6.11 (8.79) -18.31, 30.53 0.525 

(72.55 - 103.10) (72.89 - 87.72) (0.83 - 10.40) 

Magnesium (% dw) 0.43 (0.0076) 0.40 (0.010) 0.028 (0.011) -0.0019, 0.058 0.059 

(0.41 - 0.45) (0.38 - 0.41) (0.013 - 0.037) 

Manganese (mg/kg dw) 14.86 (0.63) 11.50 (0.89) 3.36 (1.06) 0.41, 6.32 0.034 

(13.28 - 17.47) (11.34 - 11.55) (2.51 - 3.29) 
Monsanto Company 12-CT-244U 

Table E-23. Statistical Summary of Site ARTI Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.84 (0.012) 0.84 (0.016) -0.0071 (0.018) -0.058, 0.044 0.717 

(0.80 - 0.87) (0.82 - 0.87) (-0.027 - 0.012) 

Potassium (% dw) 1.22 (0.029) 1.11 (0.040) 0.12 (0.047) -0.013, 0.25 0.067 

(1.16 - 1.32) (1.06 - 1.15) (0.092 - 0.11) 

Sodium (% dw) 0.017 (0.0038) ND6 

(0.0054 - 0.030) (0.013 - 0.013) 

Zinc (mg/kg dw) 39.24 (1.65) 40.81 (2.03) -1.56 (1.87) -6.76, 3.63 0.449 

(35.79 - 45.91) (38.63 - 40.71) (-2.84 - -2.25) 

Vitamin (mg/kg dw)Vitamin E 141.82 (2.44) 136.55 (3.46) 5.27 (4.23) -6.49, 17.02 0.281 

(132.11 - 146.82) (133.79 - 139.31) (-7.20 - 12.36) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ6Not determined due to insufficient number of observations for the control.
Monsanto Company 12-CT-244U 

Table E-24. Statistical Summary of Site ARTI Cottonseed Anti-nutrients for MON 88701 (Not Treated) vControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0073) 0.15 (0.010) 0.0079 (0.013) -0.027, 0.043 0.567 

(0.14 - 0.18) (0.14 - 0.15) (-0.011 - 0.0068) 

Malvalic Acid 0.37 (0.016) 0.36 (0.023) 0.011 (0.025) -0.058, 0.081 0.674 

(0.33 - 0.43) (0.33 - 0.37) (-0.0096 - 0.012) 

Sterculic Acid 0.23 (0.014) 0.20 (0.020) 0.030 (0.023) -0.035, 0.094 0.272 

(0.17 - 0.27) (0.19 - 0.20) (-0.013 - 0.022) 

Gossypol (% dw)Free Gossypol 0.84 (0.035) 0.82 (0.049) 0.020 (0.060) -0.15, 0.19 0.749 

(0.78 - 0.92) (0.80 - 0.84) (-0.021 - -0.0027) 

Total Gossypol 0.93 (0.034) 0.93 (0.044) 0.0038 (0.044) -0.12, 0.13 0.934 

(0.87 - 1.04) (0.94 - 0.98) (-0.0059 - 0.063) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.39 (0.044) 4.21 (0.047) 0.18 (0.034) 0.095, 0.27 0.003 

(4.24 - 4.50) (4.12 - 4.23) (0.12 - 0.25) 

Calories (Kcal/100g) 501.28 (2.28) 496.55 (2.63) 4.73 (3.48) -4.23, 13.69 0.232 

(498.88 - 503.43) (494.57 - 498.27) (2.32 - 6.61) 

Carbohydrates 44.81 (0.59) 45.84 (0.68) -1.03 (0.91) -3.36, 1.30 0.306 

(44.28 - 45.20) (44.64 - 47.09) (-1.89 - -0.36) 

Moisture (% fw) 6.39 (0.15) 7.23 (0.18) -0.84 (0.23) -1.43, -0.24 0.015 

(6.19 - 6.64) (6.99 - 7.48) (-0.97 - -0.57) 

Protein 27.03 (0.33) 27.30 (0.37) -0.27 (0.37) -1.21, 0.67 0.493 

(26.24 - 28.02) (26.45 - 28.21) (-1.02 - 0.63) 

Total Fat 23.77 (0.46) 22.67 (0.53) 1.10 (0.70) -0.71, 2.91 0.180 

(23.35 - 24.21) (22.26 - 23.02) (0.40 - 1.47) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 26.12 (0.52) 27.52 (0.60) -1.40 (0.79) -3.43, 0.63 0.136 

(24.62 - 27.38) (26.81 - 28.13) (-3.01 - 0.58) 

Crude Fiber 18.88 (0.68) 19.92 (0.78) -1.04 (1.00) -3.60, 1.53 0.346 

(17.57 - 19.90) (18.70 - 21.18) (-2.85 - 1.20) 

Neutral Detergent Fiber 32.42 (0.90) 33.92 (1.04) -1.49 (1.38) -5.03, 2.05 0.327 

(30.60 - 34.01) (32.79 - 35.89) (-1.93 - 0.94) 

Total Dietary Fiber 39.71 (0.86) 41.11 (1.00) -1.39 (1.32) -4.79, 2.00 0.339 

(37.96 - 42.04) (39.89 - 42.04) (-3.36 - 0.94) 

Amino Acid (% dw)Alanine 1.06 (0.019) 1.10 (0.022) -0.034 (0.025) -0.098, 0.030 0.234 

(1.02 - 1.10) (1.06 - 1.17) (-0.076 - 0.029) 

Arginine 3.06 (0.061) 3.21 (0.067) -0.16 (0.064) -0.32, 0.0098 0.060 

(2.96 - 3.18) (3.07 - 3.46) (-0.28 - -0.062) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.39 (0.043) 2.45 (0.047) -0.066 (0.044) -0.18, 0.047 0.192 

(2.30 - 2.49) (2.36 - 2.60) (-0.11 - 0.00069) 

Cystine 0.41 (0.012) 0.40 (0.013) 0.0020 (0.018) -0.043, 0.047 0.913 

(0.38 - 0.43) (0.38 - 0.43) (-0.032 - 0.050) 

Glutamic Acid 4.69 (0.098) 4.96 (0.11) -0.27 (0.099) -0.52, -0.016 0.041 

(4.51 - 4.98) (4.77 - 5.21) (-0.26 - -0.23) 

Glycine 1.11 (0.020) 1.13 (0.023) -0.016 (0.026) -0.083, 0.050 0.559 

(1.06 - 1.14) (1.09 - 1.20) (-0.055 - 0.033) 

Histidine 0.76 (0.013) 0.76 (0.014) -0.00082 (0.012) -0.033, 0.031 0.949 

(0.73 - 0.78) (0.75 - 0.78) (-0.018 - 0.0081) 

Isoleucine 0.95 (0.010) 0.94 (0.012) 0.00070 (0.014) -0.035, 0.036 0.961 

(0.91 - 0.97) (0.92 - 0.97) (-0.012 - 0.011) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.56 (0.022) 1.58 (0.024) -0.019 (0.021) -0.074, 0.037 0.425 

(1.51 - 1.60) (1.52 - 1.65) (-0.049 - 0.0079) 

Lysine 1.26 (0.018) 1.23 (0.020) 0.029 (0.024) -0.033, 0.091 0.286 

(1.19 - 1.29) (1.22 - 1.25) (-0.040 - 0.062) 

Methionine 0.40 (0.017) 0.42 (0.019) -0.019 (0.024) -0.080, 0.042 0.464 

(0.38 - 0.42) (0.37 - 0.46) (-0.081 - 0.035) 

Phenylalanine 1.45 (0.030) 1.49 (0.033) -0.040 (0.032) -0.12, 0.041 0.259 

(1.41 - 1.49) (1.41 - 1.61) (-0.12 - 0.0089) 

Proline 1.03 (0.020) 1.05 (0.022) -0.021 (0.022) -0.078, 0.036 0.393 

(0.97 - 1.10) (1.03 - 1.09) (-0.065 - 0.010) 

Serine 1.08 (0.031) 1.12 (0.035) -0.040 (0.039) -0.14, 0.061 0.357 

(1.02 - 1.14) (1.08 - 1.20) (-0.055 - 0.023) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.88 (0.015) 0.90 (0.017) -0.017 (0.018) -0.063, 0.029 0.395 

(0.85 - 0.91) (0.87 - 0.95) (-0.040 - 0.016) 

Tryptophan 0.41 (0.011) 0.42 (0.013) -0.016 (0.017) -0.061, 0.028 0.389 

(0.39 - 0.42) (0.39 - 0.44) (-0.042 - 0.030) 

Tyrosine 0.84 (0.011) 0.84 (0.013) -0.0011 (0.013) -0.034, 0.032 0.936 

(0.82 - 0.85) (0.81 - 0.89) (-0.036 - 0.027) 

Valine 1.24 (0.018) 1.26 (0.019) -0.020 (0.015) -0.059, 0.018 0.235 

(1.19 - 1.28) (1.23 - 1.32) (-0.040 - 0.011) 

Fatty Acid (% Total FA)14:0 Myristic 0.77 (0.0068) 0.77 (0.0079) -0.0036 (0.010) -0.030, 0.023 0.745 

(0.76 - 0.79) (0.76 - 0.78) (-0.026 - 0.022) 

16:0 Palmitic 24.27 (0.14) 24.12 (0.16) 0.15 (0.20) -0.37, 0.67 0.494 

(24.04 - 24.70) (23.78 - 24.45) (-0.20 - 0.26) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.51 (0.0058) 0.51 (0.0067) -0.0013 (0.0089) -0.024, 0.022 0.886 

(0.50 - 0.52) (0.50 - 0.52) (-0.0059 - 0.013) 

18:0 Stearic 2.50 (0.032) 2.43 (0.037) 0.070 (0.046) -0.050, 0.19 0.193 

(2.45 - 2.54) (2.37 - 2.46) (0.039 - 0.16) 

18:1 Oleic 14.51 (0.098) 14.39 (0.11) 0.12 (0.13) -0.20, 0.45 0.374 

(14.39 - 14.63) (14.06 - 14.61) (0.00041 - 0.47) 

18:2 Linoleic 56.07 (0.24) 56.59 (0.28) -0.52 (0.35) -1.41, 0.38 0.197 

(55.90 - 56.24) (56.02 - 57.32) (-1.16 - -0.12) 

18:3 Linolenic 0.14 (0.0066) 0.15 (0.0076) -0.0076 (0.010) -0.033, 0.018 0.485 

(0.11 - 0.15) (0.14 - 0.15) (-0.0064 - 0.0089) 

20:0 Arachidic 0.29 (0.0098) 0.28 (0.011) 0.015 (0.013) -0.019, 0.049 0.309 

(0.26 - 0.30) (0.26 - 0.29) (0.0032 - 0.042) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.15 (0.0026) 0.14 (0.0030) 0.0075 (0.0037) -0.0021, 0.017 0.100 

(0.14 - 0.16) (0.14 - 0.15) (0.0012 - 0.013) 

MineralCalcium (% dw) 0.13 (0.0015) 0.11 (0.0018) 0.020 (0.0023) 0.014, 0.026 <0.001 

(0.13 - 0.13) (0.11 - 0.11) (0.016 - 0.024) 

Copper (mg/kg dw) 8.67 (0.26) 8.21 (0.30) 0.46 (0.39) -0.54, 1.47 0.288 

(8.13 - 9.20) (7.48 - 8.64) (0.055 - 1.72) 

Iron (mg/kg dw) 74.17 (3.80) 78.00 (4.39) -3.83 (5.81) -18.76, 11.10 0.539 

(59.77 - 87.01) (75.01 - 80.40) (-20.63 - -0.99) 

Magnesium (% dw) 0.41 (0.0052) 0.38 (0.0054) 0.032 (0.0034) 0.023, 0.040 <0.001 

(0.40 - 0.43) (0.37 - 0.39) (0.026 - 0.034) 

Manganese (mg/kg dw) 14.26 (0.31) 11.51 (0.34) 2.75 (0.37) 1.80, 3.70 <0.001 

(13.82 - 15.04) (10.81 - 11.75) (2.07 - 3.10) 
Monsanto Company 12-CT-244U 

Table E-25. Statistical Summary of Site GACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.78 (0.011) 0.76 (0.012) 0.012 (0.0082) -0.0088, 0.034 0.192 

(0.75 - 0.81) (0.75 - 0.79) (0.00079 - 0.021) 

Potassium (% dw) 1.22 (0.012) 1.12 (0.013) 0.098 (0.010) 0.072, 0.12 <0.001 

(1.18 - 1.24) (1.10 - 1.13) (0.087 - 0.11) 

Sodium (% dw) 0.022 (0.0045) 0.017 (0.0052) 0.0051 (0.0069) -0.013, 0.023 0.497 

(0.0053 - 0.039) (0.013 - 0.022) (-0.0019 - 0.026) 

Zinc (mg/kg dw) 38.62 (0.67) 39.55 (0.75) -0.93 (0.83) -3.06, 1.20 0.311 

(37.11 - 40.86) (38.49 - 40.84) (-2.50 - 1.19) 

Vitamin (mg/kg dw)Vitamin E 149.29 (2.27) 140.12 (2.63) 9.17 (3.47) 0.24, 18.10 0.046 

(142.84 - 153.55) (133.64 - 145.15) (-2.30 - 15.25) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-26. Statistical Summary of Site GACH Cottonseed Anti-nutrients for MON 88701 (Not Treated) Control 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.16 (0.0067) 0.12 (0.0077) 0.039 (0.010) 0.012, 0.065 0.012 

(0.14 - 0.17) (0.11 - 0.13) (0.020 - 0.058) 

Malvalic Acid 0.41 (0.027) 0.32 (0.031) 0.087 (0.038) -0.011, 0.19 0.072 

(0.36 - 0.43) (0.31 - 0.34) (0.085 - 0.11) 

Sterculic Acid 0.23 (0.011) 0.18 (0.013) 0.045 (0.015) 0.0071, 0.082 0.028 

(0.20 - 0.24) (0.18 - 0.20) (0.039 - 0.059) 

Gossypol (% dw)Free Gossypol 0.91 (0.017) 0.86 (0.019) 0.044 (0.020) -0.0089, 0.096 0.085 

(0.85 - 0.95) (0.85 - 0.90) (0.036 - 0.091) 

Total Gossypol 0.98 (0.019) 0.96 (0.020) 0.024 (0.017) -0.020, 0.068 0.221 

(0.93 - 1.03) (0.90 - 0.99) (-0.010 - 0.061) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. Co

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.46 (0.063) 4.29 (0.071) 0.16 (0.078) -0.038, 0.37 0.091 

(4.40 - 4.55) (4.21 - 4.33) (0.071 - 0.23) 

Calories (Kcal/100g) 500.94 (2.75) 495.83 (3.16) 5.11 (3.87) -4.84, 15.06 0.244 

(494.73 - 507.94) (492.30 - 504.10) (0.76 - 15.41) 

Carbohydrates 44.42 (0.71) 44.23 (0.82) 0.19 (1.09) -2.62, 2.99 0.870 

(42.57 - 46.25) (42.53 - 45.14) (-0.94 - 0.041) 

Moisture (% fw) 7.08 (0.17) 7.36 (0.19) -0.29 (0.20) -0.81, 0.23 0.213 

(6.51 - 7.47) (7.17 - 7.62) (-0.85 - 0.13) 

Protein 27.36 (0.62) 28.82 (0.72) -1.47 (0.93) -3.86, 0.93 0.176 

(26.05 - 28.45) (28.58 - 29.04) (-2.53 - -0.58) 

Total Fat 23.76 (0.55) 22.62 (0.64) 1.13 (0.81) -0.96, 3.22 0.222 

(22.48 - 25.18) (21.87 - 24.18) (0.42 - 3.21) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 24.37 (0.55) 24.01 (0.61) 0.36 (0.65) -1.31, 2.03 0.601 

(23.30 - 25.67) (22.08 - 25.22) (-0.26 - 1.22) 

Crude Fiber 16.71 (0.24) 17.67 (0.28) -0.96 (0.37) -1.91, -0.015 0.047 

(16.10 - 17.37) (17.49 - 17.88) (-1.54 - -0.51) 

Neutral Detergent Fiber 28.65 (0.88) 30.20 (1.01) -1.54 (1.21) -4.66, 1.57 0.259 

(28.05 - 29.04) (28.87 - 32.60) (-3.72 - -0.82) 

Total Dietary Fiber 38.55 (0.42) 40.14 (0.49) -1.59 (0.63) -3.22, 0.044 0.054 

(37.44 - 39.47) (39.32 - 41.35) (-2.29 - -1.13) 

Amino Acid (% dw)Alanine 1.03 (0.011) 1.06 (0.012) -0.035 (0.014) -0.071, 0.00096 0.054 

(1.02 - 1.04) (1.02 - 1.10) (-0.061 - 0.0081) 

Arginine 3.00 (0.053) 3.28 (0.060) -0.28 (0.069) -0.45, -0.10 0.009 

(2.88 - 3.05) (3.10 - 3.43) (-0.37 - -0.059) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.35 (0.037) 2.50 (0.043) -0.15 (0.053) -0.28, -0.012 0.038 

(2.32 - 2.37) (2.39 - 2.64) (-0.27 - -0.047) 

Cystine 0.39 (0.010) 0.42 (0.012) -0.032 (0.016) -0.073, 0.0083 0.096 

(0.36 - 0.42) (0.41 - 0.43) (-0.074 - 0.011) 

Glutamic Acid 4.68 (0.12) 5.07 (0.14) -0.40 (0.18) -0.86, 0.062 0.076 

(4.63 - 4.71) (4.86 - 5.43) (-0.72 - -0.18) 

Glycine 1.07 (0.018) 1.11 (0.021) -0.047 (0.027) -0.12, 0.023 0.144 

(1.05 - 1.08) (1.07 - 1.18) (-0.11 - 0.016) 

Histidine 0.73 (0.013) 0.77 (0.014) -0.050 (0.011) -0.078, -0.022 0.006 

(0.69 - 0.76) (0.74 - 0.80) (-0.069 - -0.031) 

Isoleucine 0.90 (0.014) 0.95 (0.015) -0.049 (0.014) -0.085, -0.013 0.017 

(0.88 - 0.94) (0.91 - 0.98) (-0.074 - -0.017) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.51 (0.020) 1.58 (0.022) -0.074 (0.021) -0.13, -0.020 0.017 

(1.47 - 1.55) (1.52 - 1.65) (-0.095 - -0.013) 

Lysine 1.19 (0.017) 1.25 (0.019) -0.065 (0.021) -0.12, -0.010 0.028 

(1.16 - 1.22) (1.19 - 1.30) (-0.084 - -0.024) 

Methionine 0.39 (0.015) 0.39 (0.017) 0.0063 (0.019) -0.041, 0.054 0.747 

(0.38 - 0.41) (0.34 - 0.44) (-0.053 - 0.049) 

Phenylalanine 1.42 (0.022) 1.53 (0.025) -0.11 (0.029) -0.18, -0.031 0.014 

(1.37 - 1.45) (1.45 - 1.58) (-0.14 - -0.013) 

Proline 1.00 (0.018) 1.07 (0.020) -0.065 (0.027) -0.13, 0.0039 0.059 

(0.96 - 1.03) (1.03 - 1.12) (-0.094 - -0.00058) 

Serine 1.07 (0.029) 1.11 (0.034) -0.044 (0.045) -0.16, 0.071 0.373 

(1.05 - 1.07) (1.06 - 1.20) (-0.14 - 0.017) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.84 (0.013) 0.88 (0.015) -0.036 (0.019) -0.086, 0.014 0.121 

(0.82 - 0.86) (0.83 - 0.92) (-0.062 - 0.021) 

Tryptophan 0.42 (0.0061) 0.42 (0.0070) -0.0058 (0.0090) -0.029, 0.017 0.551 

(0.40 - 0.44) (0.42 - 0.43) (-0.024 - 0.012) 

Tyrosine 0.79 (0.014) 0.84 (0.016) -0.050 (0.019) -0.098, -0.0013 0.045 

(0.76 - 0.80) (0.79 - 0.87) (-0.076 - -0.0034) 

Valine 1.20 (0.017) 1.26 (0.019) -0.058 (0.023) -0.12, 0.00007 0.050 

(1.15 - 1.23) (1.21 - 1.30) (-0.076 - -0.0020) 

Fatty Acid (% Total FA)14:0 Myristic 0.69 (0.0087) 0.72 (0.0096) -0.033 (0.0090) -0.056, -0.010 0.013 

(0.66 - 0.70) (0.71 - 0.73) (-0.050 - -0.020) 

16:0 Palmitic 22.54 (0.089) 22.73 (0.10) -0.19 (0.13) -0.52, 0.15 0.208 

(22.30 - 22.75) (22.69 - 22.78) (-0.48 - 0.057) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.48 (0.0057) 0.49 (0.0062) -0.0079 (0.0059) -0.023, 0.0073 0.239 

(0.47 - 0.50) (0.49 - 0.50) (-0.015 - 0.0026) 

18:0 Stearic 2.25 (0.031) 2.20 (0.036) 0.048 (0.047) -0.074, 0.17 0.356 

(2.16 - 2.34) (2.15 - 2.28) (-0.066 - 0.16) 

18:1 Oleic 14.65 (0.11) 14.83 (0.13) -0.18 (0.17) -0.62, 0.27 0.356 

(14.23 - 14.99) (14.74 - 14.99) (-0.54 - 0.0051) 

18:2 Linoleic 58.08 (0.19) 57.78 (0.22) 0.30 (0.29) -0.43, 1.04 0.335 

(57.39 - 58.48) (57.65 - 57.93) (-0.26 - 0.73) 

18:3 Linolenic 0.18 (0.0027) 0.17 (0.0030) 0.0014 (0.0033) -0.0070, 0.0099 0.684 

(0.17 - 0.18) (0.17 - 0.18) (-0.0031 - 0.0059) 

20:0 Arachidic 0.23 (0.0029) 0.24 (0.0034) -0.0038 (0.0045) -0.015, 0.0077 0.435 

(0.23 - 0.24) (0.23 - 0.24) (-0.011 - 0.0067) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.13 (0.0030) 0.13 (0.0035) -0.0068 (0.0046) -0.019, 0.0049 0.196 

(0.12 - 0.14) (0.13 - 0.14) (-0.019 - 0.0086) 

MineralCalcium (% dw) 0.20 (0.0060) 0.18 (0.0065) 0.018 (0.0061) 0.0022, 0.034 0.032 

(0.18 - 0.21) (0.17 - 0.19) (0.0037 - 0.021) 

Copper (mg/kg dw) 10.86 (0.54) 11.01 (0.61) -0.15 (0.71) -1.97, 1.67 0.842 

(9.15 - 12.15) (10.09 - 11.33) (-2.19 - 1.26) 

Iron (mg/kg dw) 90.85 (5.50) 74.39 (6.35) 16.46 (8.40) -5.13, 38.05 0.107 

(74.66 - 109.70) (72.65 - 76.27) (1.19 - 27.33) 

Magnesium (% dw) 0.42 (0.0033) 0.40 (0.0038) 0.023 (0.0046) 0.011, 0.035 0.004 

(0.41 - 0.43) (0.39 - 0.40) (0.015 - 0.026) 

Manganese (mg/kg dw) 14.18 (0.29) 12.56 (0.33) 1.62 (0.44) 0.50, 2.74 0.013 

(13.48 - 14.84) (11.96 - 13.28) (0.20 - 2.14) 
Monsanto Company 12-CT-244U 

Table E-27. Statistical Summary of Site KSLA Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.76 (0.0090) 0.78 (0.010) -0.014 (0.011) -0.042, 0.014 0.251 

(0.75 - 0.78) (0.77 - 0.79) (-0.020 - -0.0075) 

Potassium (% dw) 1.11 (0.010) 1.08 (0.012) 0.029 (0.015) -0.0087, 0.067 0.104 

(1.08 - 1.12) (1.05 - 1.10) (0.020 - 0.030) 

Sodium (% dw) 0.015 (0.0028) 0.0080 (0.0032) 0.0070 (0.0042) -0.0039, 0.018 0.159 

(0.0054 - 0.022) (0.0054 - 0.013) (0.0062 - 0.016) 

Zinc (mg/kg dw) 40.63 (1.18) 42.00 (1.37) -1.37 (1.81) -6.01, 3.27 0.482 

(36.90 - 42.39) (40.59 - 43.50) (-6.60 - 0.49) 

Vitamin (mg/kg dw)Vitamin E 94.05 (2.67) 92.34 (2.91) 1.71 (2.71) -5.26, 8.67 0.556 

(87.22 - 99.35) (91.78 - 95.85) (-2.10 - 7.57) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-28. Statistical Summary of Site KSLA Cottonseed Anti-nutrients for MON 88701 (Not Treated) vControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.14 (0.010) 0.12 (0.012) 0.017 (0.014) -0.020, 0.054 0.296 

(0.13 - 0.15) (0.12 - 0.13) (0.00032 - 0.031) 

Malvalic Acid 0.41 (0.030) 0.37 (0.034) 0.044 (0.039) -0.056, 0.15 0.308 

(0.35 - 0.45) (0.33 - 0.39) (-0.012 - 0.094) 

Sterculic Acid 0.21 (0.014) 0.20 (0.016) 0.012 (0.019) -0.036, 0.060 0.552 

(0.19 - 0.22) (0.19 - 0.21) (-0.019 - 0.030) 

Gossypol (% dw)Free Gossypol 1.00 (0.027) 0.95 (0.030) 0.055 (0.033) -0.029, 0.14 0.151 

(0.96 - 1.03) (0.86 - 1.05) (-0.030 - 0.13) 

Total Gossypol 1.09 (0.033) 1.01 (0.038) 0.075 (0.047) -0.046, 0.20 0.172 

(1.05 - 1.12) (1.00 - 1.02) (0.073 - 0.11) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.27 (0.047) 4.12 (0.047) 0.16 (0.066) -0.0069, 0.32 0.057 

(4.14 - 4.40) (4.06 - 4.15) (0.081 - 0.25) 

Calories (Kcal/100g dw) 498.08 (3.01) 494.75 (3.01) 3.33 (4.19) -6.92, 13.58 0.457 

(493.35 - 504.84) (490.27 - 498.67) (-3.41 - 14.57) 

Carbohydrates 45.34 (0.79) 47.84 (0.79) -2.50 (1.11) -5.22, 0.23 0.066 

(44.46 - 46.08) (46.77 - 50.30) (-5.09 - -1.23) 

Moisture (% fw) 6.44 (0.26) 6.98 (0.26) -0.54 (0.36) -1.43, 0.35 0.190 

(5.81 - 7.10) (6.15 - 7.40) (-1.45 - 0.11) 

Protein 27.41 (0.56) 25.80 (0.56) 1.62 (0.80) -0.33, 3.56 0.088 

(26.70 - 28.07) (23.53 - 27.85) (-1.15 - 4.39) 

Total Fat 22.96 (0.64) 22.25 (0.64) 0.71 (0.90) -1.49, 2.90 0.462 

(22.01 - 24.43) (21.29 - 23.02) (-0.67 - 3.15) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.94 (0.45) 28.35 (0.45) -2.41 (0.63) -3.96, -0.86 0.008 

(25.56 - 26.33) (27.81 - 29.58) (-4.02 - -1.48) 

Crude Fiber 17.47 (0.66) 19.62 (0.66) -2.15 (0.93) -4.42, 0.12 0.059 

(14.96 - 19.14) (18.46 - 20.54) (-5.57 - 0.12) 

Neutral Detergent Fiber 32.12 (0.65) 34.05 (0.65) -1.93 (0.42) -2.96, -0.89 0.003 

(30.20 - 33.94) (32.61 - 35.84) (-2.42 - -1.25) 

Total Dietary Fiber 41.09 (0.49) 43.35 (0.49) -2.26 (0.69) -3.94, -0.58 0.016 

(39.95 - 42.26) (42.33 - 44.37) (-4.41 - -0.26) 

Amino Acid (% dw)Alanine 1.06 (0.018) 1.03 (0.018) 0.028 (0.014) -0.0052, 0.062 0.084 

(1.02 - 1.07) (1.00 - 1.06) (0.012 - 0.047) 

Arginine 3.00 (0.073) 2.98 (0.073) 0.018 (0.084) -0.19, 0.22 0.836 

(2.92 - 3.09) (2.89 - 3.13) (-0.13 - 0.16) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.38 (0.050) 2.30 (0.050) 0.075 (0.062) -0.076, 0.23 0.269 

(2.35 - 2.45) (2.25 - 2.40) (-0.040 - 0.18) 

Cystine 0.38 (0.016) 0.38 (0.016) -0.0067 (0.019) -0.054, 0.041 0.741 

(0.35 - 0.40) (0.36 - 0.44) (-0.038 - 0.031) 

Glutamic Acid 4.67 (0.13) 4.52 (0.13) 0.15 (0.16) -0.25, 0.55 0.384 

(4.57 - 4.83) (4.37 - 4.75) (-0.18 - 0.36) 

Glycine 1.10 (0.020) 1.06 (0.020) 0.039 (0.025) -0.022, 0.099 0.168 

(1.07 - 1.12) (1.04 - 1.09) (0.012 - 0.076) 

Histidine 0.73 (0.020) 0.72 (0.020) 0.010 (0.014) -0.023, 0.044 0.477 

(0.68 - 0.77) (0.67 - 0.76) (-0.015 - 0.062) 

Isoleucine 0.91 (0.018) 0.88 (0.018) 0.029 (0.020) -0.019, 0.077 0.186 

(0.88 - 0.93) (0.87 - 0.90) (0.017 - 0.056) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.52 (0.026) 1.48 (0.026) 0.045 (0.028) -0.024, 0.11 0.158 

(1.50 - 1.54) (1.44 - 1.52) (0.013 - 0.079) 

Lysine 1.25 (0.024) 1.18 (0.024) 0.074 (0.027) 0.0069, 0.14 0.035 

(1.23 - 1.27) (1.12 - 1.23) (0.023 - 0.14) 

Methionine 0.40 (0.017) 0.38 (0.017) 0.028 (0.013) -0.0036, 0.060 0.073 

(0.38 - 0.42) (0.32 - 0.42) (-0.0027 - 0.064) 

Phenylalanine 1.43 (0.027) 1.39 (0.027) 0.038 (0.033) -0.043, 0.12 0.296 

(1.41 - 1.46) (1.36 - 1.43) (-0.0091 - 0.090) 

Proline 1.03 (0.015) 0.98 (0.015) 0.045 (0.022) -0.0079, 0.099 0.082 

(1.02 - 1.05) (0.95 - 1.02) (0.0012 - 0.10) 

Serine 1.06 (0.022) 1.03 (0.022) 0.025 (0.030) -0.049, 0.099 0.445 

(1.04 - 1.08) (1.01 - 1.06) (0.0023 - 0.049) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.87 (0.016) 0.84 (0.016) 0.023 (0.012) -0.0073, 0.053 0.112 

(0.83 - 0.89) (0.82 - 0.86) (0.0022 - 0.034) 

Tryptophan 0.40 (0.014) 0.39 (0.014) 0.0098 (0.018) -0.035, 0.054 0.610 

(0.39 - 0.43) (0.38 - 0.43) (-0.033 - 0.041) 

Tyrosine 0.81 (0.018) 0.79 (0.018) 0.023 (0.021) -0.029, 0.075 0.320 

(0.81 - 0.82) (0.76 - 0.81) (-0.0051 - 0.050) 

Valine 1.23 (0.024) 1.18 (0.024) 0.047 (0.029) -0.023, 0.12 0.152 

(1.20 - 1.26) (1.17 - 1.19) (0.0063 - 0.080) 

Fatty Acid (% Total FA)14:0 Myristic 0.73 (0.013) 0.75 (0.013) -0.020 (0.018) -0.065, 0.024 0.312 

(0.69 - 0.77) (0.73 - 0.78) (-0.087 - 0.041) 

16:0 Palmitic 24.26 (0.091) 24.04 (0.091) 0.21 (0.13) -0.099, 0.53 0.145 

(23.95 - 24.55) (23.92 - 24.16) (-0.13 - 0.63) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.50 (0.0035) 0.50 (0.0035) 0.00039 (0.0049) -0.012, 0.012 0.939 

(0.50 - 0.51) (0.49 - 0.51) (-0.011 - 0.012) 

18:0 Stearic 2.61 (0.018) 2.52 (0.018) 0.089 (0.026) 0.025, 0.15 0.014 

(2.58 - 2.64) (2.49 - 2.57) (0.026 - 0.15) 

18:1 Oleic 14.46 (0.095) 14.29 (0.095) 0.17 (0.13) -0.16, 0.50 0.252 

(14.26 - 14.65) (14.13 - 14.53) (-0.27 - 0.52) 

18:2 Linoleic 56.04 (0.14) 56.63 (0.14) -0.58 (0.20) -1.08, -0.092 0.027 

(55.71 - 56.35) (56.52 - 56.72) (-0.91 - -0.30) 

18:3 Linolenic 0.15 (0.0042) 0.15 (0.0042) 0.0015 (0.0059) -0.013, 0.016 0.802 

(0.14 - 0.16) (0.13 - 0.15) (-0.012 - 0.014) 

20:0 Arachidic 0.29 (0.0054) 0.29 (0.0054) 0.0028 (0.0071) -0.015, 0.020 0.710 

(0.27 - 0.31) (0.29 - 0.30) (-0.022 - 0.024) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.15 (0.0039) 0.14 (0.0039) 0.0070 (0.0055) -0.0064, 0.020 0.247 

(0.13 - 0.16) (0.14 - 0.15) (-0.0068 - 0.027) 

MineralCalcium (% dw) 0.13 (0.0039) 0.12 (0.0039) 0.0092 (0.0050) -0.0030, 0.021 0.115 

(0.12 - 0.14) (0.12 - 0.12) (0.0045 - 0.017) 

Copper (mg/kg dw) 8.24 (0.30) 8.70 (0.30) -0.47 (0.42) -1.51, 0.57 0.311 

(7.69 - 8.79) (8.11 - 9.14) (-1.27 - 0.086) 

Iron (mg/kg dw) 77.97 (4.19) 68.59 (4.19) 9.38 (4.72) -2.16, 20.93 0.093 

(69.22 - 98.27) (66.28 - 70.38) (-0.024 - 27.89) 

Magnesium (% dw) 0.41 (0.0065) 0.39 (0.0065) 0.020 (0.0093) -0.0022, 0.043 0.068 

(0.41 - 0.42) (0.38 - 0.41) (0.0091 - 0.029) 

Manganese (mg/kg dw) 13.87 (0.37) 12.87 (0.37) 0.99 (0.52) -0.29, 2.28 0.106 

(13.02 - 14.71) (12.31 - 13.87) (-0.84 - 2.08) 
Monsanto Company 12-CT-244U 

Table E-29. Statistical Summary of Site LACH Cottonseed Nutrients for MON 88701 (Not TreateControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.72 (0.014) 0.71 (0.014) 0.0034 (0.019) -0.044, 0.051 0.867 

(0.68 - 0.73) (0.69 - 0.73) (-0.011 - 0.021) 

Potassium (% dw) 1.15 (0.022) 1.17 (0.022) -0.023 (0.029) -0.093, 0.048 0.461 

(1.13 - 1.18) (1.12 - 1.27) (-0.13 - 0.026) 

Sodium (% dw) 0.023 (0.0043) 0.015 (0.0043) 0.0074 (0.0060) -0.0072, 0.022 0.261 

(0.0054 - 0.029) (0.0053 - 0.027) (-0.0081 - 0.024) 

Zinc (mg/kg dw) 34.21 (0.93) 35.74 (0.93) -1.54 (1.32) -4.75, 1.68 0.287 

(32.91 - 35.63) (35.10 - 37.09) (-2.57 - -1.04) 

Vitamin (mg/kg dw)Vitamin E 167.00 (2.48) 149.96 (2.48) 17.04 (3.48) 8.52, 25.55 0.002 

(158.95 - 173.30) (148.96 - 152.67) (9.99 - 20.70) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2Test refers to MON 88701 (Not Treated). These plants were not treated with dicamb3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-30. Statistical Summary of Site LACH Cottonseed Anti-nutrients for MON 88701 (Not Treated) vControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.16 (0.011) 0.13 (0.011) 0.030 (0.016) -0.0078, 0.069 0.099 

(0.14 - 0.19) (0.12 - 0.14) (-0.0041 - 0.055) 

Malvalic Acid 0.41 (0.035) 0.35 (0.035) 0.059 (0.049) -0.062, 0.18 0.278 

(0.36 - 0.50) (0.31 - 0.38) (0.011 - 0.19) 

Sterculic Acid 0.23 (0.016) 0.20 (0.016) 0.029 (0.023) -0.028, 0.085 0.260 

(0.21 - 0.27) (0.17 - 0.21) (-0.0031 - 0.093) 

Gossypol (% dw)Free Gossypol 0.84 (0.026) 0.81 (0.026) 0.031 (0.036) -0.057, 0.12 0.420 

(0.76 - 0.90) (0.78 - 0.84) (-0.046 - 0.12) 

Total Gossypol 0.93 (0.025) 0.90 (0.025) 0.035 (0.036) -0.052, 0.12 0.363 

(0.89 - 1.01) (0.82 - 0.94) (-0.028 - 0.18) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.33 (0.057) 4.14 (0.064) 0.19 (0.070) 0.0083, 0.37 0.043 

(4.20 - 4.48) (3.94 - 4.26) (0.038 - 0.36) 

Calories (Kcal/100g) 498.21 (1.61) 491.80 (1.86) 6.41 (2.46) 0.078, 12.75 0.048 

(496.22 - 499.62) (488.93 - 494.48) (3.24 - 10.35) 

Carbohydrates 43.25 (0.59) 44.36 (0.68) -1.11 (0.87) -3.36, 1.13 0.259 

(41.07 - 44.88) (43.65 - 45.15) (-3.08 - 1.23) 

Moisture (% fw) 8.67 (0.22) 8.96 (0.23) -0.29 (0.19) -0.76, 0.19 0.184 

(7.98 - 9.07) (8.59 - 9.19) (-0.61 - -0.010) 

Protein 29.35 (0.61) 29.84 (0.70) -0.49 (0.85) -2.68, 1.70 0.592 

(27.82 - 31.29) (29.62 - 30.42) (-2.05 - 0.88) 

Total Fat 23.08 (0.30) 21.59 (0.34) 1.49 (0.46) 0.32, 2.66 0.022 

(22.78 - 23.39) (21.03 - 22.21) (0.72 - 2.17) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 26.21 (1.12) 26.31 (1.29) -0.10 (1.71) -4.50, 4.30 0.955 

(23.72 - 30.43) (24.72 - 28.08) (-2.57 - 5.70) 

Crude Fiber 17.82 (0.29) 16.93 (0.33) 0.90 (0.38) -0.094, 1.88 0.067 

(17.57 - 18.04) (16.30 - 17.90) (-0.33 - 1.52) 

Neutral Detergent Fiber 32.76 (1.03) 31.14 (1.19) 1.61 (1.58) -2.44, 5.67 0.353 

(31.18 - 37.27) (30.85 - 31.49) (-0.26 - 6.42) 

Total Dietary Fiber 38.49 (0.55) 39.52 (0.64) -1.03 (0.85) -3.21, 1.14 0.277 

(37.06 - 40.31) (39.05 - 39.86) (-2.58 - -1.10) 

Amino Acid (% dw)Alanine 1.13 (0.020) 1.10 (0.023) 0.027 (0.031) -0.052, 0.11 0.418 

(1.06 - 1.15) (1.05 - 1.14) (-0.075 - 0.11) 

Arginine 3.10 (0.052) 3.20 (0.060) -0.10 (0.080) -0.31, 0.10 0.253 

(2.92 - 3.22) (3.11 - 3.27) (-0.30 - 0.011) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.56 (0.053) 2.55 (0.061) 0.013 (0.081) -0.19, 0.22 0.876 

(2.35 - 2.67) (2.46 - 2.63) (-0.28 - 0.14) 

Cystine 0.42 (0.018) 0.42 (0.021) 0.0075 (0.028) -0.064, 0.079 0.798 

(0.38 - 0.49) (0.39 - 0.46) (-0.073 - 0.086) 

Glutamic Acid 4.90 (0.13) 5.08 (0.15) -0.18 (0.20) -0.69, 0.33 0.405 

(4.39 - 5.18) (4.85 - 5.40) (-1.01 - 0.18) 

Glycine 1.13 (0.019) 1.11 (0.022) 0.020 (0.029) -0.054, 0.093 0.522 

(1.07 - 1.17) (1.08 - 1.14) (-0.063 - 0.062) 

Histidine 0.77 (0.013) 0.76 (0.015) 0.015 (0.019) -0.034, 0.064 0.466 

(0.75 - 0.80) (0.74 - 0.79) (0.0060 - 0.024) 

Isoleucine 0.95 (0.018) 0.96 (0.020) -0.0094 (0.020) -0.061, 0.043 0.661 

(0.91 - 1.00) (0.93 - 0.97) (-0.028 - 0.022) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.63 (0.023) 1.60 (0.027) 0.024 (0.035) -0.067, 0.11 0.533 

(1.56 - 1.68) (1.55 - 1.63) (-0.065 - 0.088) 

Lysine 1.32 (0.029) 1.26 (0.033) 0.057 (0.043) -0.054, 0.17 0.243 

(1.27 - 1.37) (1.22 - 1.29) (-0.0085 - 0.086) 

Methionine 0.42 (0.0093) 0.41 (0.011) 0.0089 (0.014) -0.028, 0.046 0.557 

(0.39 - 0.44) (0.40 - 0.42) (-0.0049 - 0.019) 

Phenylalanine 1.49 (0.027) 1.52 (0.031) -0.026 (0.041) -0.13, 0.079 0.548 

(1.42 - 1.55) (1.45 - 1.56) (-0.14 - 0.055) 

Proline 1.08 (0.022) 1.09 (0.025) -0.013 (0.029) -0.088, 0.063 0.684 

(1.06 - 1.13) (1.07 - 1.12) (-0.057 - 0.032) 

Serine 1.16 (0.027) 1.13 (0.032) 0.032 (0.042) -0.076, 0.14 0.483 

(1.05 - 1.22) (1.09 - 1.19) (-0.14 - 0.13) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.92 (0.014) 0.89 (0.016) 0.029 (0.021) -0.026, 0.083 0.237 

(0.87 - 0.95) (0.86 - 0.92) (-0.050 - 0.079) 

Tryptophan 0.44 (0.021) 0.45 (0.024) -0.016 (0.032) -0.099, 0.067 0.642 

(0.41 - 0.50) (0.39 - 0.52) (-0.099 - 0.11) 

Tyrosine 0.85 (0.012) 0.84 (0.014) 0.0095 (0.018) -0.036, 0.055 0.618 

(0.83 - 0.87) (0.82 - 0.87) (-0.0099 - 0.043) 

Valine 1.26 (0.025) 1.30 (0.027) -0.036 (0.021) -0.090, 0.017 0.140 

(1.21 - 1.32) (1.24 - 1.32) (-0.074 - -0.0017) 

Fatty Acid (% Total FA)14:0 Myristic 0.71 (0.0074) 0.75 (0.0086) -0.036 (0.011) -0.065, -0.0069 0.024 

(0.69 - 0.73) (0.74 - 0.76) (-0.065 - -0.013) 

16:0 Palmitic 23.40 (0.12) 23.10 (0.14) 0.30 (0.18) -0.16, 0.76 0.152 

(23.11 - 23.69) (23.07 - 23.15) (0.26 - 0.62) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.47 (0.0047) 0.48 (0.0053) -0.0094 (0.0055) -0.023, 0.0047 0.147 

(0.46 - 0.48) (0.47 - 0.49) (-0.024 - 0.0016) 

18:0 Stearic 2.53 (0.037) 2.34 (0.043) 0.19 (0.057) 0.041, 0.33 0.021 

(2.49 - 2.57) (2.32 - 2.38) (0.11 - 0.26) 

18:1 Oleic 15.29 (0.12) 14.70 (0.14) 0.59 (0.19) 0.11, 1.08 0.026 

(15.01 - 15.50) (14.51 - 14.83) (0.18 - 1.00) 

18:2 Linoleic 56.04 (0.23) 57.19 (0.26) -1.15 (0.35) -2.04, -0.25 0.021 

(55.76 - 56.31) (57.01 - 57.46) (-1.65 - -0.71) 

18:3 Linolenic 0.36 (0.012) 0.29 (0.014) 0.074 (0.018) 0.028, 0.12 0.008 

(0.34 - 0.38) (0.27 - 0.30) (0.048 - 0.11) 

20:0 Arachidic 0.30 (0.0065) 0.28 (0.0075) 0.023 (0.0099) -0.0021, 0.049 0.065 

(0.29 - 0.30) (0.27 - 0.28) (0.013 - 0.031) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.16 (0.0056) 0.15 (0.0065) 0.0093 (0.0086) -0.013, 0.031 0.327 

(0.16 - 0.17) (0.15 - 0.16) (-0.00007 - 0.015) 

MineralCalcium (% dw) 0.15 (0.0014) 0.14 (0.0017) 0.013 (0.0022) 0.0072, 0.018 0.002 

(0.15 - 0.15) (0.14 - 0.14) (0.0067 - 0.018) 

Copper (mg/kg dw) 6.59 (0.36) 6.91 (0.41) -0.31 (0.54) -1.71, 1.08 0.587 

(5.37 - 7.35) (6.64 - 7.19) (-0.067 - 0.19) 

Iron (mg/kg dw) 44.35 (1.00) 48.04 (1.15) -3.69 (1.53) -7.62, 0.23 0.060 

(41.73 - 46.45) (45.03 - 50.87) (-9.14 - -0.44) 

Magnesium (% dw) 0.41 (0.010) 0.40 (0.012) 0.010 (0.016) -0.031, 0.051 0.545 

(0.40 - 0.42) (0.37 - 0.44) (-0.015 - 0.055) 

Manganese (mg/kg dw) 14.78 (0.36) 13.83 (0.42) 0.95 (0.56) -0.48, 2.38 0.148 

(13.36 - 15.70) (13.65 - 14.11) (0.97 - 1.97) 
Monsanto Company 12-CT-244U 

Table E-31. Statistical Summary of Site NCBD Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.64 (0.020) 0.66 (0.023) -0.021 (0.031) -0.10, 0.058 0.521 

(0.60 - 0.66) (0.59 - 0.71) (-0.078 - 0.072) 

Potassium (% dw) 1.04 (0.019) 1.08 (0.022) -0.045 (0.029) -0.12, 0.030 0.183 

(1.03 - 1.06) (1.03 - 1.16) (-0.13 - -0.0056) 

Sodium (% dw) 0.074 (0.0084) 0.099 (0.0096) -0.026 (0.011) -0.055, 0.0031 0.069 

(0.061 - 0.082) (0.094 - 0.10) (-0.038 - -0.020) 

Zinc (mg/kg dw) 41.50 (1.30) 49.54 (1.50) -8.04 (1.98) -13.13, -2.95 0.009 

(39.45 - 43.05) (44.04 - 52.95) (-13.50 - -2.09) 

Vitamin (mg/kg dw)Vitamin E 174.64 (3.66) 156.99 (4.23) 17.65 (5.60) 3.27, 32.04 0.025 

(165.41 - 184.47) (151.55 - 162.98) (7.48 - 32.93) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-32. Statistical Summary of Site NCBD Cottonseed Anti-nutrients for MON 88701 (Not Treated) vControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0093) 0.13 (0.011) 0.023 (0.014) -0.013, 0.059 0.167 

(0.12 - 0.18) (0.12 - 0.14) (-0.0060 - 0.032) 

Malvalic Acid 0.36 (0.040) 0.37 (0.046) -0.018 (0.056) -0.16, 0.13 0.760 

(0.26 - 0.45) (0.36 - 0.41) (-0.11 - 0.039) 

Sterculic Acid 0.23 (0.024) 0.22 (0.028) 0.010 (0.037) -0.085, 0.11 0.791 

(0.16 - 0.27) (0.21 - 0.23) (-0.055 - 0.044) 

Gossypol (% dw)Free Gossypol 0.94 (0.026) 0.90 (0.030) 0.044 (0.039) -0.057, 0.14 0.314 

(0.88 - 0.99) (0.81 - 0.95) (-0.00010 - 0.18) 

Total Gossypol 1.21 (0.067) 1.09 (0.077) 0.12 (0.10) -0.14, 0.38 0.287 

(1.05 - 1.52) (1.08 - 1.10) (0.021 - 0.44) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. C

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.31 (0.087) 4.27 (0.10) 0.042 (0.13) -0.29, 0.38 0.761 

(4.25 - 4.38) (4.20 - 4.39) (-0.0052 - 0.087) 

Calories (Kcal/100g) 495.87 (2.64) 492.81 (3.05) 3.06 (4.04) -7.32, 13.45 0.482 

(487.62 - 499.09) (490.52 - 494.31) (-5.97 - 7.23) 

Carbohydrates 42.30 (0.46) 42.60 (0.53) -0.30 (0.70) -2.10, 1.50 0.688 

(41.59 - 43.70) (42.14 - 43.05) (-0.90 - 1.56) 

Moisture (% fw) 7.03 (0.25) 7.28 (0.28) -0.25 (0.32) -1.08, 0.58 0.471 

(6.78 - 7.42) (6.63 - 7.75) (-0.92 - 0.15) 

Protein 30.79 (0.13) 31.18 (0.15) -0.39 (0.19) -0.89, 0.11 0.103 

(30.68 - 30.89) (31.00 - 31.27) (-0.59 - -0.31) 

Total Fat 22.62 (0.54) 21.95 (0.62) 0.67 (0.83) -1.46, 2.79 0.455 

(20.99 - 23.29) (21.42 - 22.22) (-1.21 - 1.54) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 26.43 (0.51) 26.40 (0.59) 0.030 (0.79) -1.99, 2.05 0.971 

(25.17 - 27.78) (25.76 - 27.10) (-0.16 - 1.44) 

Crude Fiber 17.75 (0.74) 17.71 (0.84) 0.040 (0.94) -2.39, 2.47 0.967 

(16.63 - 18.67) (16.06 - 20.78) (-2.11 - 2.09) 

Neutral Detergent Fiber 30.87 (0.87) 32.83 (1.00) -1.96 (1.32) -5.36, 1.44 0.199 

(29.16 - 32.29) (31.58 - 34.49) (-4.66 - 0.72) 

Total Dietary Fiber 38.30 (0.71) 41.10 (0.75) -2.81 (0.55) -4.22, -1.39 0.003 

(37.12 - 39.32) (39.09 - 43.00) (-4.03 - -1.98) 

Amino Acid (% dw)Alanine 1.11 (0.011) 1.13 (0.013) -0.019 (0.014) -0.054, 0.015 0.211 

(1.09 - 1.13) (1.09 - 1.17) (-0.044 - 0.023) 

Arginine 3.51 (0.049) 3.71 (0.053) -0.20 (0.048) -0.32, -0.075 0.008 

(3.34 - 3.62) (3.67 - 3.77) (-0.23 - -0.12) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.63 (0.033) 2.66 (0.037) -0.033 (0.043) -0.14, 0.076 0.469 

(2.52 - 2.69) (2.58 - 2.74) (-0.091 - 0.069) 

Cystine 0.43 (0.013) 0.44 (0.015) -0.0077 (0.020) -0.058, 0.043 0.713 

(0.41 - 0.46) (0.43 - 0.45) (-0.022 - 0.0095) 

Glutamic Acid 5.31 (0.087) 5.46 (0.10) -0.15 (0.13) -0.49, 0.19 0.309 

(5.08 - 5.57) (5.29 - 5.70) (-0.50 - 0.18) 

Glycine 1.16 (0.014) 1.18 (0.015) -0.018 (0.016) -0.060, 0.023 0.309 

(1.11 - 1.19) (1.17 - 1.20) (-0.023 - 0.0094) 

Histidine 0.82 (0.010) 0.83 (0.012) -0.010 (0.015) -0.048, 0.027 0.510 

(0.79 - 0.84) (0.83 - 0.84) (-0.026 - 0.014) 

Isoleucine 0.97 (0.017) 0.98 (0.018) -0.0018 (0.012) -0.032, 0.028 0.885 

(0.94 - 1.01) (0.94 - 1.03) (-0.014 - 0.017) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.64 (0.021) 1.66 (0.023) -0.015 (0.022) -0.071, 0.041 0.519 

(1.57 - 1.67) (1.63 - 1.69) (-0.024 - 0.035) 

Lysine 1.31 (0.019) 1.36 (0.022) -0.045 (0.022) -0.10, 0.012 0.097 

(1.29 - 1.33) (1.32 - 1.39) (-0.057 - -0.0086) 

Methionine 0.42 (0.012) 0.40 (0.014) 0.021 (0.018) -0.026, 0.069 0.295 

(0.39 - 0.44) (0.38 - 0.41) (-0.016 - 0.048) 

Phenylalanine 1.58 (0.026) 1.61 (0.028) -0.026 (0.022) -0.083, 0.030 0.287 

(1.50 - 1.63) (1.60 - 1.66) (-0.034 - 0.013) 

Proline 1.12 (0.027) 1.18 (0.031) -0.057 (0.039) -0.16, 0.043 0.200 

(1.07 - 1.16) (1.10 - 1.25) (-0.13 - 0.055) 

Serine 1.20 (0.026) 1.20 (0.030) -0.0068 (0.040) -0.11, 0.097 0.872 

(1.11 - 1.28) (1.16 - 1.24) (-0.055 - 0.063) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.92 (0.015) 0.93 (0.017) -0.0029 (0.021) -0.058, 0.052 0.896 

(0.86 - 0.95) (0.91 - 0.94) (-0.00072 - 0.037) 

Tryptophan 0.44 (0.014) 0.44 (0.016) -0.0052 (0.020) -0.057, 0.046 0.803 

(0.43 - 0.44) (0.43 - 0.45) (-0.020 - 0.0087) 

Tyrosine 0.87 (0.014) 0.89 (0.015) -0.018 (0.014) -0.054, 0.018 0.250 

(0.84 - 0.88) (0.86 - 0.91) (-0.029 - 0.011) 

Valine 1.29 (0.024) 1.34 (0.026) -0.044 (0.019) -0.093, 0.0056 0.071 

(1.25 - 1.32) (1.31 - 1.40) (-0.078 - -0.019) 

Fatty Acid (% Total FA)14:0 Myristic 0.95 (0.0046) 0.98 (0.0054) -0.030 (0.0071) -0.048, -0.012 0.008 

(0.94 - 0.95) (0.97 - 0.98) (-0.032 - -0.026) 

16:0 Palmitic 24.15 (0.088) 24.11 (0.10) 0.044 (0.13) -0.30, 0.39 0.757 

(24.06 - 24.37) (23.89 - 24.34) (-0.23 - 0.47) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. Control (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.53 (0.0022) 0.54 (0.0025) -0.0080 (0.0033) -0.017, 0.00054 0.060 

(0.53 - 0.53) (0.53 - 0.54) (-0.014 - -0.0036) 

18:0 Stearic 2.52 (0.020) 2.64 (0.021) -0.12 (0.016) -0.17, -0.082 <0.001 

(2.49 - 2.56) (2.61 - 2.70) (-0.14 - -0.12) 

18:1 Oleic 16.13 (0.067) 16.21 (0.076) -0.085 (0.088) -0.31, 0.14 0.382 

(16.01 - 16.29) (16.10 - 16.35) (-0.23 - -0.072) 

18:2 Linoleic 54.43 (0.084) 54.29 (0.097) 0.14 (0.13) -0.19, 0.47 0.326 

(54.22 - 54.63) (54.04 - 54.50) (-0.11 - 0.59) 

18:3 Linolenic 0.16 (0.0019) 0.14 (0.0022) 0.014 (0.0029) 0.0065, 0.021 0.004 

(0.15 - 0.16) (0.14 - 0.15) (0.0032 - 0.020) 

20:0 Arachidic 0.30 (0.0052) 0.31 (0.0060) -0.0075 (0.0080) -0.028, 0.013 0.389 

(0.29 - 0.31) (0.28 - 0.32) (-0.026 - 0.022) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.17 (0.0065) 0.19 (0.0075) -0.020 (0.0099) -0.046, 0.0053 0.097 

(0.16 - 0.17) (0.17 - 0.21) (-0.057 - -0.00078) 

MineralCalcium (% dw) 0.15 (0.0029) 0.13 (0.0034) 0.019 (0.0042) 0.0082, 0.030 0.006 

(0.14 - 0.15) (0.12 - 0.13) (0.015 - 0.024) 

Copper (mg/kg dw) 11.38 (0.15) 11.75 (0.17) -0.37 (0.22) -0.95, 0.20 0.157 

(11.09 - 11.56) (11.46 - 11.92) (-0.77 - 0.018) 

Iron (mg/kg dw) 67.95 (3.33) 64.62 (3.84) 3.33 (5.08) -9.74, 16.39 0.541 

(61.15 - 83.28) (63.58 - 66.45) (-2.69 - -1.19) 

Magnesium (% dw) 0.38 (0.0053) 0.37 (0.0061) 0.0076 (0.0081) -0.013, 0.028 0.386 

(0.37 - 0.39) (0.36 - 0.38) (-0.0069 - 0.021) 

Manganese (mg/kg dw) 13.23 (0.23) 12.90 (0.26) 0.33 (0.34) -0.55, 1.22 0.377 

(12.92 - 13.61) (12.00 - 13.47) (-0.55 - 1.20) 
Monsanto Company 12-CT-244U 

Table E-33. Statistical Summary of Site NMLC Cottonseed Nutrients for MON 88701 (Not Treated) vs. CControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.76 (0.010) 0.79 (0.012) -0.031 (0.016) -0.071, 0.0099 0.109 

(0.75 - 0.78) (0.78 - 0.80) (-0.046 - -0.0099) 

Potassium (% dw) 1.11 (0.016) 1.11 (0.018) -0.00071 (0.017) -0.045, 0.043 0.968 

(1.09 - 1.12) (1.07 - 1.14) (-0.044 - 0.029) 

Sodium (% dw) 0.017 (0.0034) 0.013 (0.0039) 0.0041 (0.0052) -0.0092, 0.017 0.465 

(0.0054 - 0.023) (0.0054 - 0.023) (-0.0019 - 0.018) 

Zinc (mg/kg dw) 44.99 (0.60) 49.43 (0.69) -4.44 (0.92) -6.80, -2.08 0.004 

(44.56 - 46.04) (47.66 - 50.87) (-6.30 - -3.03) 

Vitamin (mg/kg dw)Vitamin E 115.90 (2.04) 112.18 (2.36) 3.72 (3.12) -4.29, 11.73 0.285 

(113.71 - 119.14) (107.02 - 115.99) (0.18 - 7.09) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-34. Statistical Summary of Site NMLC Cottonseed Anti-nutrients for MON 88701 (Not Treated) Control 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.16 (0.0079) 0.14 (0.0092) 0.022 (0.012) -0.0097, 0.053 0.136 

(0.14 - 0.18) (0.12 - 0.15) (-0.0016 - 0.056) 

Malvalic Acid 0.33 (0.023) 0.29 (0.026) 0.039 (0.035) -0.050, 0.13 0.308 

(0.24 - 0.38) (0.26 - 0.31) (-0.070 - 0.13) 

Sterculic Acid 0.19 (0.018) 0.18 (0.020) 0.0079 (0.027) -0.061, 0.077 0.780 

(0.12 - 0.24) (0.17 - 0.19) (-0.075 - 0.079) 

Gossypol (% dw)Free Gossypol 0.86 (0.026) 0.69 (0.030) 0.17 (0.040) 0.063, 0.27 0.008 

(0.76 - 0.95) (0.68 - 0.70) (0.12 - 0.27) 

Total Gossypol 0.88 (0.026) 0.80 (0.030) 0.083 (0.040) -0.019, 0.19 0.091 

(0.84 - 0.92) (0.74 - 0.87) (-0.028 - 0.18) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. Co

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 4.10 (0.095) 3.74 (0.095) 0.36 (0.10) 0.12, 0.61 0.011 

(3.91 - 4.23) (3.38 - 3.98) (0.20 - 0.79) 

Calories (Kcal/100g) 508.96 (2.44) 503.38 (2.44) 5.58 (3.45) -2.87, 14.02 0.157 

(506.18 - 511.92) (499.09 - 512.65) (-4.16 - 9.98) 

Carbohydrates 47.15 (0.54) 48.67 (0.54) -1.52 (0.75) -3.36, 0.32 0.090 

(46.17 - 48.81) (47.50 - 49.59) (-3.27 - -0.78) 

Moisture (% fw) 6.48 (0.17) 7.08 (0.17) -0.60 (0.19) -1.07, -0.14 0.019 

(6.16 - 6.98) (6.63 - 7.37) (-1.04 - -0.17) 

Protein 23.61 (0.42) 23.92 (0.42) -0.31 (0.49) -1.52, 0.89 0.547 

(22.49 - 24.37) (23.56 - 24.61) (-1.23 - 0.78) 

Total Fat 25.13 (0.44) 23.65 (0.44) 1.48 (0.63) -0.061, 3.02 0.057 

(24.62 - 25.54) (22.92 - 25.20) (-0.15 - 2.28) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 25.58 (0.44) 27.04 (0.44) -1.46 (0.60) -2.93, 0.0044 0.050 

(24.19 - 26.37) (26.24 - 27.74) (-3.49 - 0.082) 

Crude Fiber 19.49 (0.80) 19.13 (0.80) 0.36 (1.13) -2.41, 3.12 0.763 

(18.47 - 20.73) (16.91 - 21.70) (-3.23 - 3.82) 

Neutral Detergent Fiber 32.88 (0.67) 33.60 (0.67) -0.73 (0.95) -3.04, 1.59 0.472 

(31.85 - 34.31) (32.74 - 35.52) (-3.28 - 0.90) 

Total Dietary Fiber 41.46 (0.62) 41.87 (0.62) -0.40 (0.88) -2.55, 1.74 0.661 

(40.03 - 43.22) (40.16 - 43.29) (-3.26 - 2.04) 

Amino Acid (% dw)Alanine 0.93 (0.022) 0.94 (0.022) -0.011 (0.029) -0.083, 0.061 0.724 

(0.88 - 0.98) (0.88 - 0.97) (-0.059 - 0.10) 

Arginine 2.51 (0.088) 2.59 (0.088) -0.075 (0.10) -0.32, 0.17 0.482 

(2.31 - 2.67) (2.41 - 2.71) (-0.20 - 0.26) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.03 (0.054) 2.07 (0.054) -0.037 (0.070) -0.21, 0.13 0.610 

(1.95 - 2.12) (1.92 - 2.18) (-0.17 - 0.20) 

Cystine 0.36 (0.017) 0.35 (0.017) 0.0031 (0.018) -0.041, 0.047 0.868 

(0.33 - 0.38) (0.31 - 0.39) (-0.026 - 0.066) 

Glutamic Acid 3.96 (0.13) 4.10 (0.13) -0.14 (0.18) -0.57, 0.30 0.473 

(3.79 - 4.14) (3.66 - 4.40) (-0.61 - 0.47) 

Glycine 0.96 (0.022) 0.98 (0.022) -0.023 (0.031) -0.10, 0.054 0.494 

(0.92 - 1.00) (0.91 - 1.02) (-0.087 - 0.088) 

Histidine 0.64 (0.021) 0.64 (0.021) -0.00060 (0.025) -0.062, 0.061 0.981 

(0.58 - 0.68) (0.61 - 0.66) (-0.058 - 0.064) 

Isoleucine 0.81 (0.023) 0.82 (0.023) -0.0090 (0.030) -0.082, 0.064 0.774 

(0.76 - 0.85) (0.77 - 0.83) (-0.067 - 0.074) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.34 (0.034) 1.36 (0.034) -0.014 (0.046) -0.13, 0.099 0.770 

(1.28 - 1.42) (1.28 - 1.40) (-0.071 - 0.14) 

Lysine 1.08 (0.027) 1.11 (0.027) -0.031 (0.025) -0.092, 0.031 0.269 

(1.03 - 1.12) (1.06 - 1.17) (-0.066 - 0.049) 

Methionine 0.36 (0.016) 0.33 (0.016) 0.030 (0.020) -0.020, 0.080 0.188 

(0.33 - 0.41) (0.32 - 0.35) (-0.012 - 0.088) 

Phenylalanine 1.20 (0.033) 1.23 (0.033) -0.029 (0.039) -0.12, 0.066 0.480 

(1.13 - 1.25) (1.15 - 1.27) (-0.10 - 0.098) 

Proline 0.86 (0.026) 0.87 (0.026) -0.012 (0.026) -0.074, 0.051 0.665 

(0.78 - 0.89) (0.81 - 0.93) (-0.080 - 0.077) 

Serine 0.94 (0.028) 0.96 (0.028) -0.013 (0.033) -0.094, 0.068 0.710 

(0.93 - 0.96) (0.86 - 1.03) (-0.11 - 0.098) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.78 (0.019) 0.77 (0.019) 0.0049 (0.023) -0.052, 0.062 0.840 

(0.73 - 0.82) (0.73 - 0.81) (-0.028 - 0.086) 

Tryptophan 0.36 (0.0093) 0.38 (0.0093) -0.019 (0.0085) -0.040, 0.0015 0.063 

(0.34 - 0.38) (0.37 - 0.40) (-0.038 - 0.010) 

Tyrosine 0.71 (0.020) 0.71 (0.020) -0.0059 (0.025) -0.068, 0.056 0.822 

(0.68 - 0.74) (0.67 - 0.74) (-0.035 - 0.073) 

Valine 1.05 (0.029) 1.07 (0.029) -0.013 (0.039) -0.11, 0.082 0.751 

(0.98 - 1.11) (1.00 - 1.10) (-0.10 - 0.11) 

Fatty Acid (% Total FA)14:0 Myristic 0.71 (0.011) 0.73 (0.011) -0.013 (0.012) -0.042, 0.015 0.305 

(0.69 - 0.74) (0.72 - 0.75) (-0.033 - 0.013) 

16:0 Palmitic 24.54 (0.086) 24.39 (0.086) 0.15 (0.12) -0.15, 0.44 0.278 

(24.37 - 24.64) (24.07 - 24.59) (-0.12 - 0.56) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.49 (0.0056) 0.48 (0.0056) 0.0028 (0.0076) -0.016, 0.021 0.726 

(0.47 - 0.50) (0.47 - 0.49) (-0.016 - 0.024) 

18:0 Stearic 2.79 (0.036) 2.67 (0.036) 0.12 (0.050) -0.0034, 0.24 0.054 

(2.72 - 2.93) (2.58 - 2.76) (0.033 - 0.24) 

18:1 Oleic 14.44 (0.11) 14.46 (0.11) -0.019 (0.15) -0.39, 0.35 0.901 

(14.05 - 14.68) (14.42 - 14.49) (-0.40 - 0.20) 

18:2 Linoleic 55.63 (0.18) 55.87 (0.18) -0.25 (0.25) -0.86, 0.37 0.366 

(55.18 - 56.00) (55.61 - 56.29) (-0.66 - 0.24) 

18:3 Linolenic 0.16 (0.0017) 0.15 (0.0017) 0.0098 (0.0023) 0.0042, 0.015 0.005 

(0.15 - 0.16) (0.14 - 0.15) (0.0036 - 0.014) 

20:0 Arachidic 0.31 (0.0070) 0.30 (0.0070) 0.012 (0.0099) -0.013, 0.036 0.281 

(0.30 - 0.31) (0.29 - 0.30) (0.00039 - 0.024) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued) 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.14 (0.0033) 0.14 (0.0033) -0.0010 (0.0046) -0.012, 0.010 0.836 

(0.13 - 0.15) (0.13 - 0.14) (-0.015 - 0.015) 

MineralCalcium (% dw) 0.11 (0.0040) 0.091 (0.0040) 0.020 (0.0056) 0.0058, 0.033 0.013 

(0.10 - 0.12) (0.081 - 0.095) (0.0064 - 0.030) 

Copper (mg/kg dw) 5.65 (0.24) 5.64 (0.24) 0.015 (0.32) -0.78, 0.80 0.965 

(5.02 - 6.58) (5.40 - 5.85) (-0.43 - 0.73) 

Iron (mg/kg dw) 67.74 (4.68) 73.46 (4.68) -5.73 (4.30) -16.25, 4.80 0.231 

(60.53 - 81.81) (63.01 - 89.93) (-14.73 - 3.89) 

Magnesium (% dw) 0.38 (0.0090) 0.36 (0.0090) 0.026 (0.0080) 0.0063, 0.045 0.017 

(0.36 - 0.41) (0.34 - 0.37) (-0.00067 - 0.046) 

Manganese (mg/kg dw) 12.44 (0.51) 9.72 (0.51) 2.73 (0.72) 0.97, 4.48 0.008 

(10.59 - 13.87) (8.61 - 11.03) (-0.44 - 5.26) 
Monsanto Company 12-CT-244U 

Table E-35. Statistical Summary of Site SCEK Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.64 (0.022) 0.63 (0.022) 0.0061 (0.018) -0.039, 0.051 0.751 

(0.59 - 0.72) (0.58 - 0.68) (-0.040 - 0.054) 

Potassium (% dw) 1.14 (0.031) 1.02 (0.031) 0.12 (0.043) 0.015, 0.23 0.031 

(1.11 - 1.19) (0.88 - 1.08) (0.027 - 0.31) 

Sodium (% dw) 0.018 (0.0033) 0.015 (0.0033) 0.0028 (0.0046) -0.0085, 0.014 0.562 

(0.0054 - 0.025) (0.012 - 0.023) (-0.018 - 0.013) 

Zinc (mg/kg dw) 29.14 (0.82) 30.08 (0.82) -0.94 (0.82) -2.96, 1.08 0.297 

(27.60 - 30.85) (28.22 - 31.74) (-2.60 - 1.99) 

Vitamin (mg/kg dw)Vitamin E 159.04 (1.77) 158.20 (1.77) 0.84 (2.50) -5.29, 6.97 0.749 

(154.81 - 162.27) (153.15 - 162.63) (-7.82 - 7.76) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-36. Statistical Summary of Site SCEK Cottonseed Anti-nutrients for MON 88701 (Not Treated) vControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.15 (0.0087) 0.15 (0.0087) 0.0062 (0.012) -0.024, 0.036 0.632 

(0.15 - 0.16) (0.14 - 0.15) (-0.0031 - 0.012) 

Malvalic Acid 0.43 (0.029) 0.43 (0.029) -0.0044 (0.041) -0.10, 0.096 0.918 

(0.40 - 0.48) (0.39 - 0.46) (-0.026 - 0.015) 

Sterculic Acid 0.23 (0.014) 0.24 (0.014) -0.011 (0.020) -0.060, 0.038 0.596 

(0.21 - 0.25) (0.22 - 0.25) (-0.020 - -0.0016) 

Gossypol (% dw)Free Gossypol 1.06 (0.029) 1.13 (0.029) -0.064 (0.027) -0.13, 0.0010 0.052 

(1.03 - 1.10) (1.06 - 1.20) (-0.14 - 0.012) 

Total Gossypol 1.14 (0.025) 1.07 (0.025) 0.069 (0.031) -0.0076, 0.15 0.069 

(1.06 - 1.23) (1.05 - 1.10) (-0.00076 - 0.18) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. Conven

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Proximate (% dw)Ash 3.84 (0.051) 3.46 (0.051) 0.38 (0.072) 0.20, 0.56 0.001 

(3.76 - 3.98) (3.34 - 3.61) (0.20 - 0.63) 

Calories (Kcal/100g) 500.56 (2.54) 494.42 (2.54) 6.14 (3.47) -2.35, 14.63 0.127 

(499.03 - 501.60) (489.10 - 500.98) (-0.76 - 12.43) 

Carbohydrates 44.20 (0.48) 46.39 (0.48) -2.18 (0.64) -3.75, -0.62 0.014 

(43.36 - 44.54) (45.65 - 47.07) (-3.71 - -1.24) 

Moisture (% fw) 6.86 (0.21) 7.47 (0.21) -0.61 (0.29) -1.32, 0.099 0.079 

(6.30 - 7.24) (7.11 - 7.79) (-0.89 - -0.29) 

Protein 28.77 (0.24) 28.48 (0.24) 0.30 (0.29) -0.42, 1.01 0.348 

(28.28 - 29.48) (28.09 - 28.77) (-0.49 - 0.74) 

Total Fat 23.19 (0.48) 21.70 (0.48) 1.49 (0.65) -0.097, 3.09 0.061 

(22.75 - 23.37) (20.71 - 22.88) (0.36 - 2.63) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fiber (% dw)Acid Detergent Fiber 24.53 (0.37) 25.53 (0.37) -1.00 (0.41) -2.01, 0.0086 0.051 

(23.82 - 25.07) (24.51 - 26.91) (-2.23 - -0.13) 

Crude Fiber 17.12 (0.38) 18.10 (0.38) -0.98 (0.45) -2.07, 0.11 0.070 

(16.76 - 17.54) (17.35 - 19.63) (-2.09 - -0.10) 

Neutral Detergent Fiber 30.70 (0.41) 32.12 (0.41) -1.42 (0.58) -2.84, 0.0015 0.050 

(30.40 - 31.02) (30.49 - 33.05) (-2.03 - 0.031) 

Total Dietary Fiber 40.66 (0.62) 40.47 (0.62) 0.19 (0.72) -1.57, 1.95 0.797 

(38.95 - 42.48) (39.15 - 42.09) (-1.42 - 1.70) 

Amino Acid (% dw)Alanine 1.05 (0.019) 1.05 (0.019) 0.0027 (0.026) -0.061, 0.067 0.920 

(1.04 - 1.09) (0.97 - 1.10) (-0.061 - 0.069) 

Arginine 3.15 (0.074) 3.25 (0.074) -0.10 (0.10) -0.36, 0.15 0.355 

(3.10 - 3.24) (2.94 - 3.49) (-0.37 - 0.18) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Aspartic Acid 2.47 (0.047) 2.45 (0.047) 0.027 (0.067) -0.14, 0.19 0.702 

(2.42 - 2.55) (2.26 - 2.62) (-0.20 - 0.19) 

Cystine 0.41 (0.015) 0.40 (0.015) 0.0059 (0.017) -0.035, 0.046 0.734 

(0.39 - 0.44) (0.36 - 0.45) (-0.019 - 0.050) 

Glutamic Acid 4.95 (0.13) 5.02 (0.13) -0.068 (0.19) -0.53, 0.39 0.728 

(4.85 - 5.11) (4.41 - 5.32) (-0.48 - 0.57) 

Glycine 1.11 (0.022) 1.11 (0.022) -0.0046 (0.031) -0.081, 0.071 0.886 

(1.08 - 1.14) (1.03 - 1.19) (-0.086 - 0.050) 

Histidine 0.74 (0.018) 0.76 (0.018) -0.013 (0.026) -0.075, 0.050 0.635 

(0.70 - 0.77) (0.71 - 0.82) (-0.047 - 0.034) 

Isoleucine 0.92 (0.013) 0.93 (0.013) -0.010 (0.017) -0.051, 0.031 0.569 

(0.89 - 0.94) (0.89 - 0.97) (-0.058 - 0.022) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Leucine 1.55 (0.026) 1.55 (0.026) 0.00049 (0.037) -0.091, 0.092 0.989 

(1.52 - 1.58) (1.45 - 1.64) (-0.090 - 0.10) 

Lysine 1.22 (0.026) 1.24 (0.026) -0.016 (0.037) -0.11, 0.075 0.680 

(1.17 - 1.26) (1.19 - 1.33) (-0.066 - 0.018) 

Methionine 0.37 (0.021) 0.39 (0.021) -0.018 (0.029) -0.090, 0.053 0.553 

(0.33 - 0.40) (0.32 - 0.44) (-0.068 - 0.051) 

Phenylalanine 1.48 (0.034) 1.48 (0.034) 0.00099 (0.048) -0.12, 0.12 0.984 

(1.47 - 1.52) (1.36 - 1.61) (-0.13 - 0.11) 

Proline 1.05 (0.024) 1.04 (0.024) 0.012 (0.033) -0.069, 0.094 0.724 

(1.02 - 1.08) (1.01 - 1.11) (-0.074 - 0.076) 

Serine 1.12 (0.031) 1.11 (0.031) 0.0077 (0.044) -0.10, 0.12 0.867 

(1.07 - 1.17) (0.97 - 1.17) (-0.11 - 0.14) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Amino Acid (% dw)Threonine 0.88 (0.016) 0.86 (0.016) 0.020 (0.022) -0.034, 0.073 0.407 

(0.87 - 0.90) (0.80 - 0.91) (-0.042 - 0.075) 

Tryptophan 0.41 (0.013) 0.43 (0.013) -0.013 (0.018) -0.058, 0.032 0.502 

(0.39 - 0.43) (0.38 - 0.47) (-0.079 - 0.051) 

Tyrosine 0.82 (0.017) 0.81 (0.017) 0.015 (0.024) -0.043, 0.074 0.546 

(0.81 - 0.85) (0.74 - 0.87) (-0.042 - 0.074) 

Valine 1.26 (0.030) 1.23 (0.030) 0.029 (0.042) -0.074, 0.13 0.513 

(1.19 - 1.38) (1.16 - 1.29) (-0.072 - 0.15) 

Fatty Acid (% Total FA)14:0 Myristic 0.83 (0.010) 0.84 (0.010) -0.013 (0.013) -0.046, 0.020 0.358 

(0.81 - 0.85) (0.81 - 0.87) (-0.063 - 0.019) 

16:0 Palmitic 23.24 (0.14) 23.01 (0.14) 0.22 (0.16) -0.18, 0.62 0.219 

(22.99 - 23.40) (22.86 - 23.25) (0.045 - 0.54) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)16:1 Palmitoleic 0.47 (0.0058) 0.46 (0.0058) 0.0043 (0.0072) -0.013, 0.022 0.575 

(0.45 - 0.48) (0.45 - 0.47) (-0.017 - 0.019) 

18:0 Stearic 2.31 (0.025) 2.46 (0.025) -0.15 (0.028) -0.22, -0.083 0.001 

(2.26 - 2.34) (2.40 - 2.52) (-0.24 - -0.090) 

18:1 Oleic 16.14 (0.078) 16.16 (0.078) -0.021 (0.11) -0.29, 0.25 0.852 

(16.04 - 16.22) (15.86 - 16.44) (-0.22 - 0.31) 

18:2 Linoleic 55.63 (0.21) 55.58 (0.21) 0.050 (0.29) -0.67, 0.77 0.868 

(55.38 - 55.91) (55.18 - 56.13) (-0.74 - 0.44) 

18:3 Linolenic 0.16 (0.0056) 0.17 (0.0056) -0.0030 (0.0079) -0.022, 0.016 0.714 

(0.14 - 0.18) (0.16 - 0.17) (-0.032 - 0.012) 

20:0 Arachidic 0.26 (0.0055) 0.28 (0.0055) -0.018 (0.0078) -0.037, 0.00061 0.055 

(0.24 - 0.28) (0.28 - 0.29) (-0.049 - -0.0036) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Fatty Acid (% Total FA)22:0 Behenic 0.16 (0.0015) 0.16 (0.0015) 0.0019 (0.0020) -0.0030, 0.0067 0.385 

(0.15 - 0.16) (0.16 - 0.16) (-0.0028 - 0.0060) 

MineralCalcium (% dw) 0.16 (0.0016) 0.14 (0.0016) 0.024 (0.0023) 0.019, 0.030 <0.001 

(0.16 - 0.17) (0.13 - 0.14) (0.018 - 0.033) 

Copper (mg/kg dw) 10.33 (0.15) 9.98 (0.15) 0.35 (0.22) -0.18, 0.88 0.156 

(9.96 - 10.53) (9.58 - 10.41) (-0.45 - 0.83) 

Iron (mg/kg dw) 69.52 (5.67) 79.02 (5.67) -9.50 (7.06) -26.76, 7.77 0.227 

(59.77 - 92.17) (67.45 - 95.10) (-14.73 - -2.93) 

Magnesium (% dw) 0.36 (0.0045) 0.34 (0.0045) 0.025 (0.0040) 0.015, 0.035 <0.001 

(0.35 - 0.37) (0.33 - 0.34) (0.015 - 0.032) 

Manganese (mg/kg dw) 11.38 (0.34) 9.04 (0.34) 2.34 (0.48) 1.17, 3.51 0.002 

(10.73 - 12.83) (8.83 - 9.54) (1.35 - 4.00) 
Monsanto Company 12-CT-244U 

Table E-37. Statistical Summary of Site TXPL Cottonseed Nutrients for MON 88701 (Not Treated) vs. CoControl (continued)

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

MineralPhosphorus (% dw) 0.58 (0.0099) 0.57 (0.0099) 0.0062 (0.011) -0.020, 0.032 0.582 

(0.58 - 0.59) (0.54 - 0.60) (-0.020 - 0.032) 

Potassium (% dw) 1.03 (0.023) 0.87 (0.023) 0.16 (0.033) 0.079, 0.24 0.002 

(0.99 - 1.09) (0.79 - 0.93) (0.061 - 0.30) 

Sodium (% dw) 0.018 (0.010) 0.047 (0.010) -0.029 (0.014) -0.063, 0.0062 0.091 

(0.0054 - 0.024) (0.019 - 0.090) (-0.085 - 0.0040) 

Zinc (mg/kg dw) 34.14 (0.45) 34.96 (0.45) -0.82 (0.64) -2.39, 0.76 0.250 

(33.08 - 35.14) (33.70 - 35.89) (-2.58 - 0.54) 

Vitamin (mg/kg dw)Vitamin E 110.33 (2.75) 103.66 (2.75) 6.67 (3.90) -2.86, 16.20 0.137 

(103.52 - 114.05) (93.92 - 109.90) (-6.38 - 20.14) 

1dw = dry weight; fw = fresh weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 

Table E-38. Statistical Summary of Site TXPL Cottonseed Anti-nutrients for MON 88701 (Not Treated) vControl 

 Difference (MON 88701 minus Control)

Analytical Component (Units)1

MON 887012 

Mean (S.E.)3 

(Range)

Control4

Mean (S.E.) 

(Range)

Mean (S.E.) 

(Range)

95% 

Confidence Interval

Significan

(p-Value

Cyclopropenoid Fatty Acid (% Total FA)Dihydrosterculic Acid 0.16 (0.0047) 0.16 (0.0047) -0.0020 (0.0066) -0.018, 0.014 0.777 

(0.16 - 0.17) (0.15 - 0.17) (-0.0083 - 0.0048) 

Malvalic Acid 0.42 (0.020) 0.47 (0.020) -0.046 (0.029) -0.12, 0.025 0.161 

(0.40 - 0.44) (0.44 - 0.49) (-0.072 - -0.018) 

Sterculic Acid 0.23 (0.013) 0.26 (0.013) -0.025 (0.018) -0.068, 0.019 0.216 

(0.21 - 0.25) (0.25 - 0.27) (-0.043 - 0.0078) 

Gossypol (% dw)Free Gossypol 0.98 (0.024) 0.93 (0.024) 0.052 (0.033) -0.030, 0.13 0.170 

(0.94 - 1.05) (0.91 - 0.95) (0.0089 - 0.14) 

Total Gossypol 1.09 (0.024) 1.01 (0.024) 0.081 (0.022) 0.028, 0.13 0.009 

(1.03 - 1.16) (0.97 - 1.05) (0.053 - 0.11) 

1dw = dry weight; FA = fatty acid. 2 MON 88701 plants were not treated with dicamba or glufosinate. 3Mean (S.E.) = least-square mean (standard error). 4Control refers to the non-biotechnology derived, conventional control (Coker 130). 5With 95% confidence, interval contains 99% of the values expressed in the population of commercial substances. Negativ
Monsanto Company 12-CT-244U 523 of 620 

References for Appendix E 

AACC. 1999. Official method 32-20. American Association of Cereal Chemists, St. Paul, 

Minnesota. 

AOAC. 2011a. AOAC official method 988.15: Tryptophan in foods and food and feed 

ingredients. Official Methods of Analysis of AOAC International, Association of 

Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011b. AOAC official method 923.03: Ash of flour. Official Methods of 

Analysis of AOAC International, Association of Analytical Communities International, 

Gaithersburg, Maryland. 

AOAC. 2011c. AOAC official method 962.09: Fiber (crude) in animal feed and pet food. 

Association of Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011d. AOAC official method 948.22: Fat (crude) in nuts and nut products. 

Official Methods of Analysis of AOAC International, Association of Analytical 

Communities International, Gaithersburg, Maryland. 

AOAC. 2011e. AOAC official method 960.39: Fat (crude) or ether extract in meat. 

Official Methods of Analysis of AOAC International, Association of Analytical 

Communities International, Gaithersburg, Maryland. 

AOAC. 2011f. AOAC official method 984.27: Calcium, copper, iron, magnesium, 

manganese, phosphorus, potassium, sodium, and zinc in infant formula. Official Methods 

of Analysis of AOAC International, Association of Analytical Communities 

International, Gaithersburg, Maryland. 

AOAC. 2011g. AOAC official method 985.01: Metals and other elements in plants and 

pet foods. Association of Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011h. AOAC official method 925.09: Solids (total) and loss on drying 

(moisture) in flour. Official Methods of Analysis of AOAC International, Association of 

Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011i. AOAC official method 926.08: Loss on drying (moisture) in cheese. 

Association of Analytical Communities International, Gaithersburg, Maryland. 

AOAC. 2011j. AOAC official method 955.04: Nitrogen (total) in fertilizers. Official 

Methods of Analysis of AOAC International, Association of Analytical Communities 

International, Gaithersburg, Maryland. 

AOAC. 2011k. AOAC official method 979.09: Protein in grains. Official Methods of 

Analysis of AOAC International, Association of Analytical Communities International, 

Gaithersburg, Maryland. 
Monsanto Company 12-CT-244U 524 of 620 

AOCS. 1997. Preparation of methyl esters of fatty acids. Method Ce 2-66, American Oil 

Chemists' Society, Champaign, Illinois. 

AOCS. 2001. Determination of fatty acids in edible oils and fats by capillary GLC. 

Method Ce 1e-91, American Oil Chemists' Society, Champaign, Illinois. 

AOCS. 2009a. Official Methods and Recommended Practices of the American Oil 

Chemists' Society. Method Ce 1i-07, American Oil Chemists' Society, Champaign, 

Illinois. 

AOCS. 2009b. Nitrogen-ammonia-protein modified Kjeldahl method titanium dioxide + 

copper sulfate catalyst. Official Methods and Recommended Practices of the American 

Oil Chemists' Society, 5th Edition. Method Ac 4-91, American Oil Chemists' Society, 

Champaign, Illinois. 

AOCS. 2009c. Direct methylation of lipids in foods for the determination of total fat, 

saturated, cis-monounsaturated, cis-polyunsaturated, and trans fatty acids by gas 

chromatography. Method Ce 1k-09, American Oil Chemists' Society, Champaign, 

Illinois. 

AOCS. 2011a. Total gossypol. Method Ba 8-78, American Oil Chemists' Society, 

Champaign, Illinois. 

AOCS. 2011b. Free gossypol. Method Ba 7-58, American Oil Chemists' Society, 

Champaign, Illinois. 

Barkholt, V. and A.L. Jensen. 1989. Amino acid analysis determination of cysteine plus 

half-cystine in proteins after hydrochloric acid hydrolysis with a disulfide compound as 

additive. Analytical Biochemistry 177:318-322. 

Bertrand, J.A., T.Q. Sudduth, A. Condon, T.C. Jenkins and M.C. Calhoun. 2005. Nutrient 

content of whole cottonseed. Journal of Dairy Science 88:1470-1477. 

Cort, W., T.S. Vicente, E.H. Waysek and B.D. Williams. 1983. Vitamin E content of 

feedstuffs determined by high-performance liquid chromatographic fluorescence. Journal 

of Agriculture and Food Chemistry 31:1330-1333. 

Hamilton, K.A., P.D. Pyla, M. Breeze, T. Olson, M. Li, E. Robinson, S.P. Gallagher, R. 

Sorbet and Y. Chen. 2004. Bollgard II cotton: Compositional analysis and feeding studies 

of cottonseed from insect-protected cotton (Gossypium hirsutum L.) producing the 

Cry1Ac and Cry2Ab2 proteins. Journal of Agricultural and Food Chemistry 52: 6969-

6976. 

Henderson, J.W. and A. Brooks. 2010. Improved amino acid methods using Agilent 

ZORBAX Eclipse Plus C18 columns for a variety of Agilent LC instrumentation and 

separation goals. Agilent Technologies, Inc., Wilmington, Delaware. 
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Henderson, J.W., R.D. Ricker, B.A. Bidlingmeyer and C. Woodward. 2000. Rapid, 

accurate, sensitive, and reproducible HPLC analysis of amino acids. Agilent 

Technologies, Inc., Wilmington, Delaware. 

ILSI. 2011. Crop Composition Database, Version 4.2. International Life Sciences 

Institute, Washington, D.C. http://www.cropcomposition.org/. 

Komarek, A.R., J.B. Robertson and P.J. Van Soest. 1993. A comparison of methods for 

determining ADF using the Filter Bag Technique versus conventional filtration. Journal 

of Animal Science 72:114. 

Komarek, A.R., J.B. Robertson and P.J. Van Soest. 1994. Comparison of the filter bag 

technique to conventional filtration in the Van Soest NDF analysis of 21 foods. National 

Conference on Forage Quality, Evaluation, and Utilization, American Society of 

Agronomy, Inc., Crop Science Society of America, Inc., Soil Society of America, Inc., 

Madison, Wisonsin. 

Lawhon, J.T., C.M. Cater and K.F. Mattil. 1977. Evaluation of the food use potential of 

sixteen varieties of cottonseed. Journal of the American Oil Chemists' Society 54:75-80. 

McMurray, C.H., W.J. Blanchflower and D.A. Rice. 1980. Influence of extraction 

techniques on determination of Tocopherol in animal feedstuffs. Journal of the 

Association of Official Analytical Chemists 63:1258-1261. 

Schuster, R. 1988. Determination of amino acids in biological pharmaceutical plant and 

food samples by automated precolumn derivatization and high-performance liquid 

chromatography. Journal of Chromatography 431:271-284. 

Smith, C.W. and R.A. Creelman. 2001. Vitamin E concentration in upland cotton seeds. 

Crop Science 41:577-579. 

Speek, A.J., J. de Schrijver and W.H.P. Schreurs. 1985. Vitamin E composition of some 

seed oils as determined by high-performance liquid chromatography with fluorometric 

detection. Journal of Food Science 50:121-124. 

USDA. 1970. Forage fiber analyses (apparatus, reagents, procedures, and some 

applications). Agricultural Handbook No. 379. U.S. Department of Agriculture, 

Washington, DC. 

USDA. 1973. Energy value of foods: Basis and derivation. Agricultural Handbook No. 

74. US Department of Agriculture, Agricultural Research Service, Washington, D.C. 

Wood, R. 1986. High-performance liquid chromatography analysis of cyclopropene fattyacids. Biochemical Archives 2:63-72. 
Monsanto Company 12-CT-244U 526 of 620 

Appendix F: Materials, Methods, and Individual Site Results for Seed 

Dormancy and Germination Assessment of MON 88701 

F.1. Materials 

Seed dormancy and germination characteristics were assessed on seed from MON 88701, 

the conventional control, and commercial reference varieties produced in 2010 field trials 

at the following sites: Crittenden County, Arkansas (ARPR); Caswell County, North 

Carolina (NCME); and Hale County, Texas (TXPL) in 2010 (Table VII-3). The field 

trial at each site was established in a randomized complete block design with four 

replications. The seed from MON 88701, the conventional control, and the commercial 

reference varieties were harvested from all four replicated plots at each of the three field 

sites. 

Table F-1. Starting Seed of MON 88701, Conventional Control and Commercial 

Cotton Reference Varieties Used in Dormancy Assessment 

Site1 Material Material Type Phenotype Sample ID 

 

ARPR Coker 130 Control Conventional 11268128 

ARPR MON 88701 Test DGT Cotton1

 11268129 

ARPR SG 125 Reference Conventional 11266155 

ARPR DP 565 Reference Conventional 11266764 

ARPR ST 474 Reference Conventional 11266156 

ARPR DP 5415 Reference Conventional 11266157 

NCME Coker 130 Control Conventional 11268128 

NCME MON 88701 Test DGT Cotton 11268129 

NCME DP 435 Reference Conventional 11266762 

NCME Delta Opal Reference Conventional 11266158 

NCME SG 125 Reference Conventional 11266155 

NCME FM 989 Reference Conventional 10001810 

TXPL Coker 130 Control Conventional 11268128 

TXPL MON 88701 Test DGT Cotton 11268129 

TXPL Atlas Reference Conventional 11266765 

TXPL DP 435 Reference Conventional 11266762 

TXPL SG 125 Reference Conventional 11266155 

TXPL NM 1517-99 Reference Conventional 11268233 

 

1

DGT Cotton = Dicamba and glufosinate-tolerant cotton. 

 
Monsanto Company 12-CT-244U 527 of 620 

F.2. Characterization of the Materials 

For the MON 88701, the parental conventional control, and the commercial reference 

varieties starting seed lots, the presence or absence of the MON 88701 insert was 

confirmed by event-specific polymerase chain reaction analyses. 

F.3. Germination Testing Facility and Experimental Methods 

Seed dormancy and germination evaluations were conducted at BioDiagnostics, Inc. in 

River Falls, WI. The principal investigator was qualified to conduct seed dormancy and 

germination testing consistent with the standards established by the Association of 

Official Seed Analysts (AOSA), a seed trade association (AOSA, 2010a; b; 

AOSA/SCST, 2010). 

Seed lots of MON 88701, the conventional control, and four commercial reference 

varieties were produced from each of three field sites and tested under six different 

temperature regimes. Each temperature regime constituted a different experiment (i.e., 

no comparisons were made between temperature regimes). Six germination chambers 

were maintained dark under one of the following temperature regimes: constant 

temperature of approximately 10, 20, or 30 °C, or alternating temperatures of 

approximately 10/20, 10/30, or 20/30 °C. The alternating temperature regimes were 

maintained at the lower temperature for 16 hours and the higher temperature for eight 

hours. The temperature inside each germination chamber was monitored and recorded 

every 15 minutes throughout the duration of the assessment. Prior to the study, the 

starting seed were treated uniformly with the commercial seed treatment fungicides 

mefenoxam and fludioxonil at labeled rates. Approximately 100 cottonseeds were placed 

on the germination towels using a vacuum planting system. Two additional premoistened germination towels were placed on top of the cottonseed. The set was rolled 

up and secured with a rubber band. All rolled germination towels were labeled and then 

placed into an appropriately labeled bucket. One germination towel was prepared for 

each of the six starting cottonseed entries from each individual site, all of which were 

placed into a single bucket. A bucket was prepared for each site and replicaton for a total 

of 12 buckets per temperature regime. Buckets were placed in the appropriate 

germination chambers. 

A description of each germination characteristic evaluated and the timing of evaluations 

are presented in Table VII-1. The types of data collected depended on the temperature 

regime. Each rolled germination towel in the AOSA-recommended temperature regime 

(i.e., 20/30°C) was evaluated periodically during the study for normal germinated, 

abnormal germinated, viable hard, dead, and viable firm-swollen seed as defined by 

AOSA guidelines (AOSA, 2010a; b; AOSA/SCST, 2010). AOSA only provides 

guidelines (AOSA, 2010a) for testing seed under optimal temperatures (20/30°C); 

however, additional temperature regimes were included to test a range of temperature 

conditions. Each rolled germination towel in the additional temperature regimes (i.e., 10, 

20, 30, 10/20, and 10/30°C) was evaluated periodically for germinated, viable hard, dead, 

and viable firm-swollen seed. Emergence and/or development of essential structures of 

seedlings that otherwise would be categorized as “normal germinated” under optimal 
Monsanto Company 12-CT-244U 528 of 620 

temperature conditions may not be so at non-optimal temperatures. Therefore, for the 

additional temperature regimes, no distinction was made between normal and abnormal 

germinated seed. 

F.4. Statistical Analysis 

An analysis of variance was conducted using SAS Version 9.2 (SAS, 2008) according to 

a split-plot design with four replications. MON 88701 was compared to the conventional 

control for dormancy and germination characteristics of cottonseed produced within each 

site (i.e., individual site analysis) and in a combined-site analysis in which the data were 

pooled across all three sites. The seed germination characteristics analyzed included 

percent germinated seed, percent viable hard seed, percent dead seed, and percent viable 

firm-swollen seed. The percent germinated seed were categorized as either normal 

germinated or abnormal germinated for the AOSA temperature regime. The level of 

statistical significance was predetermined to be 5% (=0.05). MON 88701 was not 

statistically compared to the reference varieties, nor were comparisons made across 

temperature regimes. The minimum and maximum mean values were determined from 

the reference materials across all sites to provide a range of values (i.e., reference range) 

representative of commercial cotton varieties. Results from the combined-site analysis 

are presented in Table VII-2. 

F.5. Individual Site Seed Dormancy and Germination Analyses 

In the individual site analyses, no statistically significant differences were detected 

between MON 88701 and the parental conventional control for any of the measured 

characteristics (i.e., percent germinated, viable hard, dead, or viable firm-swollen seed) in 

any temperature regime for seed produced at the ARPR and NCME sites. Three 

statistically significant differences in total were detected between MON 88701 and the 

conventional control for cottonseed produced at the TXPL site (Table F-2). At TXPL, 

MON 88701 had fewer dead seed than the conventional control at 10/20°C (4.5% vs. 

9.5%); MON 88701 had more germinated seed than the conventional control at 20/30°C 

(92.5% vs. 84.0%); and, MON 88701 had fewer abnormal germinated seed than the 

conventional control at 20/30°C (2.5% vs. 7.3%). Statistically significant differences 

between MON 88701 and the conventional control for germination characteristics in the 

individual site analyses were not consistently detected across temperature regimes or the 

individual sites. While some statistically significant differences were detected in the 

combined-site analysis, these statistical differences were within the range of values 

expected for the commercial reference varieties. Thus, MON 88701 is unlikely to have 

increased plant pest/weed potential or an adverse environmental impact compared to the 

conventional control. (See Figure VII-1, Schematic Diagram of Agronomic and 

Phenotypic Data Interpretation Methods, step 3, answer “no”). 

 

 SAS is a registered trademark of SAS Institute, Inc. 
Monsanto Company 12-CT-244U 

Table F-2. Comparison of MON 88701 to the Conventional Control for Dormancy and Germination CharCottonseed Produced at Each of Three Sites  ARPR1 NCME1 Temperature Germination Mean % (S.E.)2 Mean % (S.E.)2 Regime Category MON 88701 Control MON 88701 Control3 MON 10 °C Germinated 49.5 (13.5) 41.8 (11.2) 22.0 (7.2) 24.7 (4.2)  Viable Hard 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.7 (0.7)  Dead 16.0 (5.0) 15.0 (3.6) 32.0 (6.5) 26.7 (3.9)  Viable Firm Swollen 34.5 (15.7) 43.0 (13.7) 46.0 (12.2) 48.0 (5.8) 520 °C Germinated 96.0 (0.7) 96.8 (0.5) 94.5 (1.6) 97.3 (0.9)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)  Dead 4.0 (0.7) 3.3 (0.5) 5.5 (1.6) 2.7 (0.9)  Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 30 °C Germinated 99.0 (0.7) 97.0 (0.9) 95.8 (1.7) 93.7 (1.9)  Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0)  Dead 1.0 (0.7) 3.0 (0.9) 4.3 (1.7) 6.3 (1.9)  Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 
Monsanto Company 12-CT-244U 

Table F-2. Comparison of MON 88701 to the Conventional Control for Dormancy and GerminatioCottonseed Produced at Each of Three Sites (continued)  ARPR 1 NCME 1 TXPL Temperature Germination Mean % (S.E.)2 Mean % (S.E.)2 Mean Regime Category MON 88701 Control MON 88701 Control3 MON 10/20 °C Germinated 98.0 (0.7) 95.3 (1.1) 90.3 (2.5) 90.3 (3.9) 95.0 ( Viable Hard 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0.0) 0.0 (0

 Dead 2.0 (0.7) 4.3 (0.9) 6.5 (0.6) 6.0 (2.5) 4.5 (0 Viable Firm Swollen 0.0 (0.0) 0.3 (0.3) 3.3 (2.9) 3.7 (1.5) 0.5 (0

10/30 °C Germinated 97.3 (0.8) 97.8 (0.9) 93.8 (2.7) 96.7 (0.9) 96.0 (

 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

 Dead 2.8 (0.8) 2.3 (0.9) 6.3 (2.7) 3.3 (0.9) 4.0 (1 Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

20/30 °C Normal Germinated 92.0 (1.9) 94.5 (0.3) 84.2 (4.6) 84.7 (3.1) 92.5 (

(AOSA) Abnormal Germinated 3.8 (1.4) 2.5 (0.3) 8.0 (2.6) 9.0 (3.0) 2.5 (0 Viable Hard 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 0.0 (0

 Dead 4.3 (1.5) 3.0 (0.6) 7.5 (2.0) 6.3 (0.9) 5.0 (0 Viable Firm Swollen 0.0 (0.0) 0.0 (0.0) 0.3 (0.3) 0.0 (0.0) 0.0 (0

Note: The experimental design for the germination test was a split-plot with four replications and statistical analysis consisted o(ANOVA) model. *Statistically significant differences detected (

α = 0.05) between MON 88701 and the conventional control (n = 11). 

1 In some instances, the total percentage of MON 88701 or the control did not equal 100% due to numerical rounding of the means. 2 SE = Standard Error. 3Control – The NCME site only had 3 reps across all temperature regimes compared to 4 reps for other sites and treatments
Monsanto Company 12-CT-244U 531 of 620 

References for Appendix F 

AOSA. 2010. AOSA Rules for testing seeds Vol. 1: Principles and procedures. 

Association of Official Seed Analysts, Ithaca, New York. 

AOSA. 2010. AOSA Rules for testing seeds Vol. 4: Seedling evaluation. Association of 

Official Seed Analysts, Ithaca, New York. 

AOSA/SCST. 2010. Tetrazolium testing handbook. Association of Official Seed Analysts 

and the Society of Commercial Seed Technologists, Ithaca, New York. 

 
Monsanto Company 12-CT-244U 532 of 620 

Appendix G: Materials, Methods, Dicamba and Glufosinate Treated Results, 

and Individual Site Results from the Phenotypic, Agronomic, Plant Mapping, 

and Environmental Interaction Assessment of MON 88701 under Field 

Conditions 

G.1. Materials 

Data were collected from two different studies during 2010 to evaluate phenotypic, 

agronomic, and environmental interaction characteristics. In Study 1, MON 88701 not 

treated with dicamba or glufosinate herbicides was evaluated against the conventional 

control (Table G-1). In Study 2, MON 88701 not treated with dicamba or glufosinate 

herbicides and MON 88701 treated with dicamba- and glufosinate herbicides were both 

evaluated against the conventional control (Table G-2). Assessments were conducted on 

the agronomic system that includes MON 88701 treated with dicamba and glufosinate 

herbicides to support the assessment of MON 88701. In Study 1, a total of 11 cotton 

reference varieties were evaluated among the sites (Table G-1). Seven of the commercial 

varieties were conventional and four of the commercial varieties were tolerant to 

glyphosate herbicide. Three conventional reference varieties and one glyphosate-tolerant 

reference variety were grown at each location. In Study 2, a total of 8 commercial 

conventional reference varieties were evaluated among the sites. Four commercial 

conventional reference varieties were planted per site (Table G-2). 

G.2. Characterization of the Materials 

The presence or absence of the MON 88701 insert in the starting seed of MON 88701 

and the conventional control were confirmed by event-specific polymerase chain reaction 

analyses. 

G.3. Field Sites and Plot Design 

Field sites in both studies (Table VII-3) are representative of commercial cotton growing 

areas and are distributed across a geographical area to include a variety of agronomic 

practices, soils and climatic factors. The researchers at each field site were familiar with 

the growth, production, and evaluation of cotton characteristics. The starting seed in each 

study were planted at each site in a randomized complete-block design with four 

replications. 

Data from Study 1 were collected at fifteen field sites in the U.S. during 2010 (Table VII3). Each plot (Table G-3) at the ARAU (Arkansas), ARPR (Arkansas), GACH 

(Georgia), LABU (Louisiana), and SCEK (South Carolina) consisted of 12 rows on 0.91 

to 1.02 meter centers and rows were 9.1 meters long. In general, rows 2 and 3 were 

designated for the collection of following data: phenotypic, plant response to abiotic 

stress, disease damage, and arthropod damage. Rows 5 and 7 were designated for the 

collection of arthropod samples. Rows 9 and 10 were designated for the collection of 

damage data from thrips and heliothine spp. Rows 1, 4, 6, 8, 11, and 12 were used as 

buffer rows. 
Monsanto Company 12-CT-244U 533 of 620 

Each plot (Table G-3) at the ARTI (Arkansas), GAJE (Georgia), KSLA (Kansas), LACH 

(Louisiana), NCBD (North Carolina), NCME (North Carolina), NMGA (New Mexico), 

NMLC (New Mexico), TXPL (Texas), and TXPO (Texas) was 4 rows, row spacing of 

0.76 to 1.02 meters and rows were 6.1 meters long. In general, rows 2 and 3 were 

designated for the collection of the following data: phenotypic, plant response to abiotic 

stress, disease damage, and arthropod damage. Rows 1 and 4 were used as buffer rows. 

Data from Study 2 were collected at eleven field sites in the U.S. during 2010 (Section 

VII, Table VII-3). Each plot (Table G-4) was eight rows, row spacing was 0.76 to 1.02 

meters and rows were 6 meters long. Rows 2 and 3 were designated for the collection of 

phenotypic, plant mapping and environmental interaction data. 

G.4. Planting and Field Operations 

Field and planting information for Study 1 and Study 2 are listed in Tables G-3 and G-4, 

respectively. Prior to planting, the Field Cooperator at each field site prepared the plot 

area with a proper seed bed according to local agronomic practices, including tillage, 

fertilization and pH adjustment, and pest management. During the growing season, all 

plots were assessed for agronomic conditions and pest populations, including pest 

arthropods, diseases, and weeds. Fertilizer, irrigation, agricultural chemicals and other 

management treatments were applied as necessary. All maintenance operations 

mentioned above were performed uniformly across all plots at a site. 
Monsanto Company 12-CT-244U 534 of 620 

Table G-1. Starting Seed for Study 1 

Site1 Material Name Phenotype2

Regulatory

Lot Number

All MON 88701 DGT 11268129

All Coker 130 Control 11268128

ARTI Phytogen Phy315RF Glyphosate-Tolerant 11266967

ARTI Delta Opal Conventional 11266158

ARTI DP435 Conventional 11266762

ARTI ST474 Conventional 11266156

LABU Bayer FM9058F Glyphosate-Tolerant 11266968

LABU DP565 Conventional 11266764

LABU SG125 Conventional 11266155

LABU ST474 Conventional 11266156

NCME Nex Gen NG3410RF Glyphosate-Tolerant 11266969

NCME DP5415 Conventional 11266157

NCME Delta Opal Conventional 11266158

NCME DP435 Conventional 11266762

GAJE Bayer FM9058F Glyphosate-Tolerant 11266968

GAJE DP493 Conventional 11266763

GAJE DP565 Conventional 11266764

GAJE SG125 Conventional 11266155

NCBD Phytogen Phy315RF Glyphosate-Tolerant 11266967

NCBD ST474 Conventional 11266156

NCBD DP5415 Conventional 11266157

NCBD Delta Opal Conventional 11266158

TXPO Bayer FM9058F Glyphosate-Tolerant 11266968

TXPO DP435 Conventional 11266762

TXPO DP493 Conventional 11266763

TXPO DP565 Conventional 11266764

TXPL All tex patriotRF Glyphosate-Tolerant 11266966

TXPL SG125 Conventional 11266155

TXPL ST474 Conventional 11266156

TXPL DP5415 Conventional 11266157

ARPR Nex Gen NG3410RF Glyphosate-Tolerant 11266969

ARPR ST474 Conventional 11266156

ARPR DP493 Conventional 11266763

ARPR SG125 Conventional 11266155

ARAU Phytogen Phy315RF Glyphosate-Tolerant 11266967

ARAU Delta opal Conventional 11266158

ARAU DP435 Conventional 11266762

ARAU ST474 Conventional 11266156

SCEK Bayer FM9058F Glyphosate-Tolerant 11266968

SCEK SG125 Conventional 11266155

SCEK ST474 Conventional 11266156

SCEK DP5415 Conventional 11266157

 
Monsanto Company 12-CT-244U 535 of 620 

Table G-1. Starting Seed for Study 1 (continued) 

Site1 Material Name Phenotype2

Regulatory

Lot Number

GACH Nex Gen NG3410RF Glyphosate-Tolerant 11266969

GACH Delta opal Conventional 11266158

GACH DP435 Conventional 11266762

GACH DP493 Conventional 11266763

LACH Phytogen Phy315RF Glyphosate-Tolerant 11266967

LACH DP5415 Conventional 11266157

LACH ST474 Conventional 11266156

LACH DP435 Conventional 11266762

KSLA All tex patriotRF Glyphosate-Tolerant 11266966

KSLA Delta opal Conventional 11266158

KSLA DP435 Conventional 11266762

KSLA DP493 Conventional 11266763

NMLC Nex Gen NG3410RF Glyphosate-Tolerant 11266969

NMLC DP565 Conventional 11266764

NMLC SG125 Conventional 11266155

NMLC ST474 Conventional 11266156

NMGA All tex patriotRF Glyphosate-Tolerant 11266966

NMGA DP5415 Conventional 11266157

NMGA Delta opal Conventional 11266158

NMGA DP435 Conventional 11266762

1

 Sites - ARTI = Desha County, Arkansas; LABU = Rapides County, Louisiana; NCME = Caswell County, 

North Carolina; GAJE = Twiggs County, Georgia; NCBD = Perquimans County, North Carolina; TXPO = 

San Patricio County, Texas; TXPL = Hale County, Texas; ARPR = Crittenden County, Arkansas; ARAU = 

Jackson County, Arkansas; SCEK = Barnwell County, South Carolina; GACH = Tift County, Georgia; 

LACH = Rapides County, Louisiana; KSLA = Pawnee County, Kansas; NMLC = Dona Ana County, New 

Mexico; NMGA = Dona Ana County, New Mexico. 

2 Phenotype abbreviations: DGT = dicamba and glufosinate-tolerant. 

 
Monsanto Company 12-CT-244U 536 of 620 

Table G-2. Starting Seed for Study 2 

Site1

 Material Name2 Phenotype3 

Regulatory Lot 

Number 

 

All Coker 130 Conventional 11268128 

ARPR SG125 Conventional 11266155 

ARPR DP 565 Conventional 11266764 

ARPR ST 474 Conventional 11266156 

ARPR DP 5415 Conventional 11266157 

ARTI SG125 Conventional 11266155 

ARTI DP 5415 Conventional 11266157 

ARTI DP 435 Conventional 11266762 

ARTI FM 989 Conventional 10001810 

GACH DP 565 Conventional 11266764 

GACH ST 474 Conventional 11266156 

GACH FM 989 Conventional 10001810 

GACH Delta Opal Conventional 11266158 

GAJE SG125 Conventional 11266155 

GAJE ST 474 Conventional 11266156 

GAJE DP 5415 Conventional 11266157 

GAJE DP 435 Conventional 11266762 

KSLA DP 565 Conventional 11242914 

KSLA DP 5415 Conventional 11266157 

KSLA Atlas Conventional 11266765 

KSLA NM 1517-99 Conventional 11268233 

LACH DP 565 Conventional 11266764 

LACH ST 474 Conventional 11266156 

LACH DP 5415 Conventional 11266157 

LACH FM 989 Conventional 10001810 

NCBD SG125 Conventional 11266155 

NCBD ST 474 Conventional 11266156 

NCBD DP 435 Conventional 11266762 

NCBD Delta Opal Conventional 11266158 

NCME SG125 Conventional 11266155 

NCME DP 435 Conventional 11266762 

NCME FM 989 Conventional 10001810 

NCME Delta Opal Conventional 11266158 

 

 
Monsanto Company 12-CT-244U 537 of 620 

Table G-2. Starting Seed for Study 2 (continued)

Site1

 Material Name2 Phenotype3 

Regulatory 

Lot Number 

 

NMLC DP 565 Conventional 11266764 

NMLC ST 474 Conventional 11266156 

NMLC Atlas Conventional 11266765 

NMLC NM 1517-99 Conventional 11268233 

SCEK SG125 Conventional 11266155 

SCEK ST 474 Conventional 11266156 

SCEK Delta Opal Conventional 11266158 

SCEK Atlas Conventional 11266765 

TXPL SG125 Conventional 11266155 

TXPL DP 435 Conventional 11266762 

TXPL Atlas Conventional 11266765 

TXPL NM 1517-99 Conventional 11268233 

All MON 88701 (U) DGT 11268129 

All MON 88701 (S) DGT 11268129 

 

1

 ARPR= Crittenden County, Arkansas; ARTI = Desha County, Arkansas; GACH = Tift County, Georgia; 

GAJE = Twiggs County, Georgia; KSLA = Pawnee County, Kansas; LACH = Rapides County, Louisiana; 

NCBD = Perquimans County, North Carolina; NCME = Caswell County, North Carolina; NMLC = Dona 

Ana County, New Mexico; SCEK = Barnwell County, South Carolina; TXPL = Hale County, Texas. 2

 U = unsprayed, S = sprayed 

3

DGT = dicamba and glufosinate-tolerant. 
Monsanto Company 12-CT-244U 

Table G-3. Study 1 Field and Planting Information Site Planting Date1 

Planting Rate 

(seeds/m) Plot Size (m × m)2 Rows/plot Soil Texture 

% Organic Matter 

ARAU 5/25/2010 16 9.1 × 0.97 12 sandy loam 1.0 ARPR 5/19/2010 16 9.1 × 0.91 12 silt loam 0.9 ARTI 5/24/2010 16 6.1 × 0.97 4 silt loam 2.2 GACH 6/03/2010 16 9.1 × 0.91 12 loamy sand 1.0 GAJE 6/25/2010 16 6.1 × 0.91 4 loamy sand 0.8 KSLA 6/02/2010 16 6.1 × 0.76 4 loam 2.1 LABU 5/20/2010 16 9.1 × 1.02 12 silt loam 0.6 LACH 5/22/2010 16 6.1 × 1.02 4 very fine sandy 0.8 NCBD 5/21/2010 16 6.1 × 0.97 4 loamy sand 1.6 NCME 6/15/2010 16 6.1 × 0.76 4 loamy sand 0.9 NMGA 5/20/2010 16 6.1 × 0.97 4 sandy loam 1.0 NMLC 5/19/2010 16 6.1 × 0.97 4 loam 1.0 SCEK 5/22/2010 16 9.1 × 1.02 12 loamy sand 1.5 TXPL 5/25/2010 16 6.1 × 1.02 4 clay loam 0.5 TXPO 5/21/2010 16 6.1 × 0.76 4 sandy clay loam 1.1 1 Month-day-year. 2 Length × width. 
Monsanto Company 12-CT-244U 

Table G-4. Study 2 Field and Planting Information Site Code Planting Date1 

Planting Rate 

(seeds/m) Plot Size (m × m)2 Rows/plot Soil Texture 

% Organic Matter 

ARPR 5/25/2010 16 6 × 0.91 8 Clay 1.3 

ARTI 5/24/2010 16 6 × 0.97 8 Silt loam 1.6 

GACH 5/26/2010 16 6 × 0.91 8 Loamy sand 1.0 

GAJE 6/25/2010 16 6 × 0.91 8 Loamy sand 0.8 

KSLA 6/02/2010 16 6 × 0.76 8 Loam 3.1 

LACH 5/21/2010 16 6 x 1.02 8 Silt loam 0.6 

NCBD 5/27/2010 16 6 x 0.97 8 Loam 2.2 

NCME 6/11/2010 16 6 x 0.76 8 Loamy sand 1.0 

NMLC 5/17/2010 16 6 x 0.97 8 Sandy loam 1.0 

SCEK 5/19/2010 16 6 x 1.02 8 Loamy sand 1.3 

TXPL 5/25/2010 16 6 x 1.02 8 Clay loam 0.5 

1 Month-day-year. 2 Length × width. 
Monsanto Company 12-CT-244U 540 of 620 

G.5. Phenotypic Observations 

In both Study 1 and Study 2, the description of the characteristics measured and the 

designated developmental stages when observations occurred are listed in Table VII-1. 

G.6. Environmental Observations 

In both Study 1 and Study 2, environmental interactions (i.e., interactions between crop 

plants and their receiving environment) were used to characterize MON 88701 by 

evaluating plant response to abiotic stress, disease damage, and arthropod-related damage 

using qualitative methods described in section G.7. In addition, pest damage and pestand beneficial-arthropod abundance were evaluated in Study 1 using the quantitative 

methods described in the following sections (G.7 and G.8). 

G.7. Plant Response to Abiotic Stress, Disease Damage, and Arthropod-Related 

Damage 

In Study 1 and Study 2, plots containing MON 88701 not treated with dicamba and 

glufosinate herbicides and the conventional control were evaluated qualitatively at all 

sites for differences in plant response to abiotic stress, disease damage, and arthropodrelated damage. Three abiotic stressors, three diseases, and three arthropod pests were 

evaluated four times during the growing season at the following intervals: 

Observation 1: approximately 30 days after planting (DAP)

Observation 2: approximately 60 DAP 

Observation 3: approximately 90 DAP 

Observation 4: approximately 120 DAP 

Method used for selecting stressors at each field site: 

1. Prior to each data collection, cotton was surveyed in proximity to the study area or the 

border rows of the study for abiotic stressors (e.g., drought), diseases (e.g., Alternaria 

black spot), and arthropod damage (e.g., thrips). 

2. Cooperators chose three abiotic stressors, three diseases, and three arthropod species 

that are actively causing damage for subsequent evaluation in the study plots. 

Cooperators were requested to select additional stressors if present. 

3. If fewer than three abiotic stressors, diseases, or arthropod species were present, the 

cooperator chose additional abiotic stressors, diseases, and arthropod species that are 

known to commonly occur in that geographical region and cause damage at the study 

site at that time. 

4. All plots at a site were rated for the same abiotic stressors, diseases, and arthropod 

pests at a given observation, even if that selected stressor was not present in some or 

all of the plots. 

5. If a selected stressor was not present, the cooperator recorded the rating as “0” (= 

none). 
Monsanto Company 12-CT-244U 541 of 620 

As indicated above, the researcher at each field site chose abiotic stressors, diseases, and 

arthropod pests that were either actively causing plant injury in the study area or were 

likely to occur in cotton during the given observation period. Therefore, abiotic stressors, 

diseases, and arthropod pests assessed often varied between observations at a site and 

between sites. Qualitative plant response to abiotic stress and disease damage and 

arthropod-related damage observations were collected from each plot using a continuous 

0 – 9 scale of increasing severity (in Study 2, qualitative abiotic and biotic stressor were 

not evaluated on plots with MON 88701 treated with dicamba and glufosinate 

herbicides). Data were collected numerically and then placed into one of the following 

categories for reporting purposes: 

Rating Severity of plant damage 

0 none (no symptoms observed) 

1 – 3 slight (symptoms not damaging to plant development) 

4 – 6 moderate (intermediate between slight and severe) 

7 – 9 severe (symptoms damaging to plant development) 

 

In Study 1, a quantitative assessment for differences in thrips and heliothine damage on 

MON 88701 and the conventional control plants was conducted at ARAU, ARPR, 

GACH, LABU, and SCEK. 

Thrips damage was assessed quantitatively from rows 9 and 10 of each plot from 10 

randomly selected plants using the arthropod-specific 0 – 5 rating scales of increasing 

severity listed below. Damage was rated at approximately 14, 21, and 28 DAP. 

Heliothine damage was assessed from rows 9 and 10 of each plot. Visual observations 

were conducted at 45, 60, 75 and 90 DAP to record total number of fruiting bodies 

(flower buds, flowers and bolls), number of damaged fruiting bodies and number live 

larvae on the top 7 nodes from 10 randomly selected plants. 

Rating Severity of plant damage 

0 No Thrips or damage visible 

1 Few thrips present; no brownish tinge along the edges of leaves and silvering 

on the underside of leaves 

2 Numerous thrips present; newest leaves show only a slight brownish tinge 

along the edges of leaves and some silvering on the underside of some leaves 

3 Numerous thrips present; newest leaves show considerable browning along 

the edges of leaves and some silvering on the underside of most leaves 

4 Numerous thrips present; extensive silvering of leaves with some curling of 

leaves 

5 Numerous thrips present; extensive silvering of leaves, leaves often curl 

upwards and the plant is generally ragged in appearance 
Monsanto Company 12-CT-244U 542 of 620 

G.8. Arthropod Abundance 

Pest and beneficial arthropods were collected at the ARAU, ARPR, GACH, LABU, and 

SCEK sites four times during the growing season at the following intervals: 

Collection 1: 30 DAP 

Collection 2: 60 DAP 

Collection 3: 90 DAP 

Collection 4: 120 DAP 

Arthropods were collected using a vertical beat sheet sampling method (Drees and Rice, 

1985). The beat sheet was approximately 0.91 × 0.91 m, constructed of a stiff material 

and had a collection trough at the bottom. The sheet was placed in a designated row and 

the collecting trough was positioned near the base of the plants. Plants were shaken 

vigorously along the length of the beat sheet to dislodge arthropods from the plants. This 

sample constituted a subsample. Two subsamples were collected form both rows 5 and 7 

for a total of 4 subsamples per plot. The subsamples collected within the same row were 

at least 1.5 m apart. The four sub-samples were combined into one pre-labeled container 

and placed on freezer ice packs and sent to a laboratory to be enumerated. 

A maximum of the five pest and five beneficial arthropods were enumerated for each 

collection. For each individual collection (e.g., Collection 1, ARPR site), four randomly 

selected samples were examined to determine presence and relative abundance of up to 

five pest- and beneficial-arthropods to be enumerated for that particular collection and 

site. Thus, the suite of pest- and beneficial-arthropods assessed often varied between 

collections from a site and between sites due to differences in temporal activity and 

geographical distribution of arthropod taxa. 

G.9. Data Assessment 

Experienced scientists familiar with the experimental design and evaluation criteria were 

involved in all components of data collection, summarization, and analysis. Study 

personnel assessed that measurements were taken properly, data were consistent with 

expectations based on experience with the crop, and the experiment was carefully 

monitored. Prior to analysis, the overall dataset was evaluated for evidence of 

biologically relevant changes and for possible evidence of an unexpected plant response. 

Any unexpected observations or issues during the trials that would impact the trial 

objectives were noted. Data were then subjected to categorical or statistical analysis as 

indicated G.10 (categorical analysis) and G.11 (statistical analysis). 

G.10. Environmental Interactions Evaluation Criteria for Qualitative Data 

The following data were categorical and not statistically analyzed: plant vigor at 14 DAP, 

plant vigor at 30 DAP, plant response to abiotic stress, disease damage and arthropod 

damage. MON 88701 and the conventional control were considered different in plant 

response rating if the range of vigor or stressor values did not overlap between the 
Monsanto Company 12-CT-244U 543 of 620 

MON 88701 and the conventional control across all four replications. Any observed 

differences between the MON 88701 and the conventional control were assessed for 

biological significance in the context of the range of the commercial reference materials, 

and for consistency in other observation times and sites. Differences that are not 

consistently observed at other times and sites are considered not biologically meaningful 

in terms of plant pest potential or an adverse environmental impact. 

G.11. Statistical Analysis 

Analysis of variance was conducted on the quantitative data according to a randomized 

complete block design using SAS Version 9.2 (SAS, 2008). The level of significance 

was predetermined to be =0.05. In separate analysis, MON 88701 not treated with 

dicamba or glufosinate herbicides from Study 1, MON 88701 not treated with dicamba or 

glufosinate herbicides from Study 2, and MON 88701 treated with dicamba and 

glufosinate herbicides were each compared to the conventional control within each site 

(individual site analysis) and in a combined-site analysis. In both Study 1 and Study 2, 

no statistical comparisons were made between MON 88701 and the reference varieties. 

The reference range for each characteristic analyzed across sites was determined from the 

minimum and maximum mean values from the reference cotton varieties planted among 

the sites, within a study. 

MON 88701 not treated with dicamba or glufosinate herbicides from Study 1 was 

statistically compared to the conventional control within each site (individual site 

analysis) and in a combined-site analysis, in which the following data were pooled across 

sites: stand count at 14 DAP, stand count at 30 DAP, final stand count at harvest, plant 

height at 30 DAP, plant height at harvest, nodes above white flower (3 observations), 

seed cotton yield, seed index, total seed per boll, total mature seed per boll, total 

immature seed per boll, boll weight, fiber micronaire, fiber elongation, fiber strength, 

fiber uniformity, fiber length, thrips damage and percent heliothine damaged fruiting 

bodies and number of live heliothine larvae. Pest and beneficial arthropod abundance 

data were statistically analyzed only within individual observations/ collections and sites 

due to the variation in temporal activity and geographical distribution of the taxa. The 

reference range for pest- and beneficial-arthropod abundance and damage of each 

arthropod evaluated from a given collection/observation and site was determined from 

the minimum and maximum mean abundance or damage values collected from the 

reference varieties at the site. Data excluded from Study 1 (Table G-5) and the reasons 

for their exclusion are listed in the study file. Exclusion of these data did not adversely 

affect the quality of the study. 

MON 88701 not treated with dicamba or glufosinate herbicide and MON 88701 treated 

with dicamba and glufosinate herbicides from Study 2 were each compared to the 

conventional control within each site (individual site analysis) and in a combined-site 

analysis, in which the following data were pooled across sites: stand count at 14 DAP, 

stand count at 30 DAP, final stand count, plant height at 30 DAP, plant height at harvest, 

 

 SAS is a registered trademark of the SAS Institute. 
Monsanto Company 12-CT-244U 544 of 620 

nodes above white flower (3 observations), seed cotton yield, number of mainstem nodes 

per plant, number of nodes to first fruiting branch, total number of bolls per plant, total 

number of first-position bolls per plant, total number of vegetative bolls per plant, percent 

retention of first-position bolls, percent first-position bolls of total bolls per plant, seed 

index, total seed per boll, total mature seed per boll, total immature seed per boll, weight 

per boll, fiber micronaire, fiber elongation, fiber strength, fiber uniformity, and fiber 

length. Data excluded from Study 2 (Table G-6) and the reasons for their exclusion are 

listed in the study file. Exclusion of these data did not adversely affect the quality of the 

study. 

G.12. Phenotypic Results from 2010 - Results and Discussion 

The individual site data will be reported in three separate comparisons: Study 1 - 

MON 88701 not treated with dicamba or glufosinate herbicides vs. the conventional 

control; Study 2 - MON 88701 not treated with dicamba or glufosinate herbicides vs. the 

conventional control; and, Study 2 - MON 88701 treated with dicamba and glufosinate 

herbicides vs. the conventional control. The agronomic system that includes MON 88701 

treated with dicamba and glufosinate herbicides was assessed to support the assessment 

of MON 88701. 

G.12.1. Individual Phenotypic Characteristics - Results and Discussion for Study 1 

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

In the individual site analysis of MON 88701 data, a total of 59 statistically significant 

differences were detected out of a total of 285 comparisons made between MON 88701 

and the conventional control (Tables G-7, G-8 and G-9). At LACH (Table G-7), 

MON 88701 had a lower stand count (plants per plot) than the conventional control at 14 

DAP (100.8 vs. 127.8), 30 DAP (101.8 vs. 125.0) and at harvest (63.3 vs. 73.5). At 

GAJE, the final stand count was higher for MON 88701 than the control (167.5 vs. 156.3 

plants/plot). Plants of MON 88701 were shorter (cm) than the conventional control at 30 

DAP at LACH (12.4 vs. 14.5), NCBD (20.0 vs. 24.5) and NMLC (6.7 vs. 8.3). Plants of 

MON 88701 were shorter (cm) than the control at harvest at GAJE (87.1 vs. 98.6), KSLA 

(129.9 vs. 142.0), LACH (120.5 vs. 140.5), NCBD (104.8 vs. 111.0), and TXPL (44.9 vs. 

48.4). Plants of MON 88701 had more nodes above white flower at observation 1 at 

KSLA (5.1 vs. 4.4), NMLC (8.4 vs. 7.4) and TXPL (5.0 vs. 4.5). Plants of MON 88701 

had more nodes above white flower at observation 2 at ARPR (7.1 vs. 6.6) and KSLA 

(4.2 vs. 3.2). Plants of MON 88701 had more nodes above white flower at observation 3 

at KSLA (4.6 vs. 3.5), SCEK (5.2 vs. 4.6) and TXPO (6.4 vs. 5.7). Plants of MON 88701 

a higher seedcotton yield (Kg/ha) than the conventional control at ARAU (2912.9 vs. 

2166.8) and at GAJE (2320.4 vs. 1843.1). Plants of MON 88701 (Table G-8) had a lower 

seed index (grams of 100 fuzzy seed) than the conventional control ARPR (9.4 vs. 10.3), 

ARTI (10.1 vs. 10.9), GAJE (8.3 vs. 9.8), KSLA (11.8 vs. 12.5), LABU (9.4 vs. 10.5), 

LACH (8.8 vs. 9.8), NCBD (9.0 vs. 9.8), NCME (9.7 vs. 10.6), and NMGA (10.6 vs. 

11.4). Plants of MON 88701 had more total seed per boll than the conventional control at 

ARAU (31.6 vs. 29.6), ARPR (29.4 vs. 27.3), KSLA (31.2 vs. 28.1), NCME (31.7 vs. 

27.5), NMGA (34.4 vs. 30.3) and NMLC (31.0 vs. 25.9). Plants of MON 88701 had 

more mature seed per boll than the control at ARPR (27.3 vs. 21.7), ARTI (20.2 vs. 17.6), 
Monsanto Company 12-CT-244U 545 of 620 

KSLA (29.9 vs. 26.6), NCME (28.4 vs. 22.1), NMGA (32.9 vs. 28.8), and NMLC (26.5

vs. 21.6). Plants of MON 88701 had fewer immature seed per boll than the conventional 

control at ARPR (2.1 vs. 5.7), ARTI (5.7 vs. 9.0), and SCEK (6.5 vs. 11.1). Plants of 

MON 88701 (Table G-9) had lower boll weight (grams per boll) than the conventional 

control at ARTI (4.1 vs. 4.8) and GACH (4.2 vs. 4.5) and a higher boll weight at NMLC 

(5.1 vs. 4.6). Plants of MON 88701 had higher fiber micronaire (mic units) than the 

conventional control at NMGA (5.1 vs. 4.9) and TXPL (4.9 vs. 4.5). Plants of 

MON 88701 had greater fiber elongation (%) than the conventional control at ARAU (6.8 

vs. 6.2) and lower fiber elongation at NCBD (5.7 vs. 6.6). Plants of MON 88701 had 

greater fiber strength (g/tex) than the conventional control at KSLA (31.5 vs. 30.3), 

NCBD (32.6 vs. 30.9), NMLC (31.2 vs. 30.0), and TXPL (34.1 vs. 32.2). Plants of 

MON 88701 had greater fiber uniformity than the conventional control at TXPL (85.4 vs. 

84.1%) and shorter cotton fiber at the NCME (2.8 vs. 2.9 cm). 

The statistical differences detected in the individual site analyses for stand count at 14 

DAP, stand count at 30 DAP, final stand count at harvest, nodes above white flower at 

observation 1, seedcotton yield, immature seed per boll, boll weight, micronaire, fiber 

elongation, fiber uniformity, and fiber length were not detected in the combined-site 

analysis. Therefore, the differences between MON 88701 and the conventional control 

were not indicative of a consistent plant response and MON 88701 is unlikely to have 

increased plant pest/weed potential or an adverse environmental impact compared to the 

conventional control (See Figure VII-1, Schematic Diagram of Agronomic and 

Phenotypic Data Interpretation Methods, step 2, answer “no”). While statistical 

differences were detected in the combined-site analysis for plant height at 30 DAP at 

three sites, plant height at harvest at five sites, nodes above white flower observation 2 at 

two sites, and nodes above white flower observation 3 at three sites, seed index at nine 

sites, total seed per boll at six sites, and mature seed per boll at six sites and fiber strength 

at four sites, the assessed phenotypic value of MON 88701 for these phenotypic 

characteristics were within their respective reference range. Therefore, the differences 

between MON 88701 and the conventional control were not indicative of a consistent 

plant response and MON 88701 is unlikely to have increased plant pest/weed potential or 

an adverse environmental impact compared to the conventional control (See Figure VII1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods, step 

3, answer “no”). 

G.12.2. Individual Phenotypic Characteristics - Results and Discussion for Study 2 

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

In the individual site analysis for MON 88701 data, 43 statistically significant differences 

were detected out of 209 comparisons between MON 88701 and the conventional control 

(Table G-10, G-11 and G-12). At 30 DAP, plants of MON 88701 were shorter (cm) than 

the conventional control at KSLA (15.7 vs. 18.3), LACH (13.5 vs. 15.9), and NCME 

(17.2 vs. 19.7 cm). At harvest, plants of MON 88701 were shorter (cm) than the 

conventional control at GAJE (77.4 vs. 92.6), KSLA (113.5 vs. 127.0 cm), LACH (154.4 

vs. 180.0), and NCME (71.4 vs. 85.0). Plants of MON 88701 had more nodes above 

white flower than the conventional control at observation 1 at GACH (5.9 vs. 5.3), at 
Monsanto Company 12-CT-244U 546 of 620 

observation 2 at NCBD (4.8 vs. 3.9) and TXPL (5.5 vs. 5.1) and at observation 3 at 

GACH (4.1 vs. 3.6) and KSLA (3.7 vs. 2.5). Plants of MON 88701 had higher 

seedcotton yield than the conventional control at KSLA (4,487.0 vs. 3,726.5 kg/ha) and 

NMLC (1,938.4 vs. 1,479.3 kg/ha). Plants of MON 88701 had a lower seed index (gram 

of 100 seed) than the conventional control at ARPR (8.8 vs. 10.0), ARTI (9.9 vs. 12.0), 

GAJE (8.5 vs. 10.4), KSLA (11.7 vs. 12.8), LACH (9.4 vs. 10.3), NCBD (8.8 vs. 10.5), 

NCME (8.8 vs. 10.2), SCEK (8.5 vs. 9.8), and TXPL (9.9 vs. 11.0). Plants of 

MON 88701 had more total seed per boll than the conventional control at ARPR (26.8 vs. 

23.9), KSLA (28.4 vs. 24.9), and NMLC (33.8 vs. 30.7). Plants of MON 88701 had more 

mature seed per boll than the conventional control at ARPR (24.2 vs. 15.9), ARTI (23.7

vs. 18.0), GAJE (15.0 vs. 11.6), and KSLA (25.9 vs. 22.7). Plants of MON 88701 had 

fewer immature seed per boll than the conventional control at ARPR (2.6 vs. 8.0) and 

ARTI (4.6 vs. 8.9). Plants of MON 88701 had greater weight per boll than the 

conventional control at KSLA (5.8 vs. 5.3 g/boll) and NMLC (5.7 vs. 5.1 g/boll). Plants 

of MON 88701 had higher fiber micronaire than the conventional control at NMLC (4.9 

vs. 4.7 mic units), lower fiber elongation at ARPR (5.0 vs. 5.7 %), higher fiber strength 

(g/tex) at KSLA (30.4 vs. 29.4), NMLC (30.1 vs. 27.3), and TXPL (32.0 vs. 31.0). Plants 

of MON 88701 had higher fiber uniformity (%) than the conventional control at KSLA 

(84.2 vs. 83.3) and NMLC (83.4 vs. 81.1 %) and shorter fiber length (cm) at ARPR (2.8 

vs. 2.9) and NCBD (2.8 vs. 2.9). 

The statistical differences detected in the individual site analysis for nodes above white 

flower (observations 1 and 3), seedcotton yield, immature seed per boll, weight per boll, 

fiber micronaire, fiber elongation, fiber uniformity, and fiber length were not detected in 

the combined-site analysis. Therefore, the differences between MON 88701 and the 

conventional control were not indicative of a consistent plant response and MON 88701 

is unlikely to have increased plant pest/weed potential or an adverse environmental 

impact compared to the conventional control (See Figure VII-1, Schematic Diagram of 

Agronomic and Phenotypic Data Interpretation Methods, step 2, answer “no”). Although 

statistical differences were detected for plant height at 30 DAP at three sites, plant height 

at harvest at four sites, nodes above white flower observation 2 at two sites, seed index at 

nine sites, total seed per boll at three sites, and mature seed per boll at four sites and fiber 

strength at three sites, the assessed values of MON 88701 for the combined-site analysis 

were within their respective reference range. Therefore, the differences between 

MON 88701 and the conventional control were not indicative of a consistent plant 

response and MON 88701 is unlikely to have increased plant pest/weed potential or an 

adverse environmental impact compared to the conventional control (See Figure VII-1, 

Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods, step 3, 

answer “no”). 

G.12.3. Combined-site and Individual Site Phenotypic Characteristics - Results and 

Discussion for Study 2 - MON 88701 Treated with Dicamba and Glufosinate 

Herbicides 

To support the assessment of MON 88701, these assessments were also conducted on the 

agronomic system that includes MON 88701 treated with dicamba and glufosinate 
Monsanto Company 12-CT-244U 547 of 620 

herbicides. In the combined-site analysis (Table G-13), no statistically significant 

differences were detected between MON 88701 treated with dicamba and glufosinate 

herbicides and the conventional control for stand count at 14 DAP, stand count at 30 

DAP, stand count at harvest, number of nodes above white flower observation 3, 

seedcotton yield, immature seed per boll, weight per boll, fiber micronaire, fiber 

elongation, fiber uniformity and fiber length. Therefore, the lack of differences in the 

above characteristics supports a conclusion that MON 88701 is unlikely to have increased 

plant pest/weed potential or an adverse environmental impact compared to the 

conventional control (See Figure VII-1, Schematic Diagram of Agronomic and 

Phenotypic Data Interpretation Methods, step 2, answer “no”). The following 

statistically significant differences were detected in the combined-site analysis. Plants of 

MON 88701 were shorter (cm) than the conventional control at 30 DAP (18.1 vs. 19.2) 

and at harvest (98.4 vs. 105.0). Plants of MON 88701 had a higher number of nodes 

above white flower at observation 1 (6.7 vs. 6.4) and at observation 2 (5.6 vs. 5.2), lower 

seed index (9.5 vs. 10.7 g per 100 fuzzy seed), more seed per boll (28.5 vs. 27.0), more 

mature seed per boll (22.8 vs. 20.1), and, increased fiber strength (31.2 vs. 30.2 g/tex). 

However, the mean values for the above characteristics were within the reference range. 

Thus, MON 88701 is unlikely to have increased plant pest/weed potential or an adverse 

environmental impact compared to the conventional control. 

In the individual site analysis for MON 88701 plant growth and development data (Table 

G-14 – G-16), a total of 41 statistically significant differences were detected out of a total 

of 209 comparisons made between treated MON 88701 and the conventional control. 

Plants of MON 88701 were shorter than the conventional control at 30 DAP at GAJE 

(23.2 vs. 26.0 cm). Plants of MON 88701 were shorter (cm) than the conventional 

control at harvest at GAJE (81.7 vs. 92.6), KSLA (113.6 vs. 127.0), LACH (164.8 

vs.180.0) and NCME (75.7 vs. 85.0). Plants of MON 88701 had more nodes above white 

flower at observation 1 than the conventional control at LACH (8.6 vs. 7.6) and TXPL 

(6.2 vs. 5.7), Plants of MON 88701 had more nodes above white flower at observation 2 

than the conventional control at GAJE (6.1 vs. 5.7), NCBD (5.7 vs. 3.9) and TXPL (5.7 

vs. 5.1). Plants of MON 88701 had more nodes above white flower at observation 3 than 

the conventional control at ARTI (4.9 vs. 4.7), KSLA (3.4 vs. 2.5), and NCBD (3.8 vs. 

3.0). Plants of MON 88701 had lower seedcotton yield than the conventional control at 

GACH (4,107.9 vs. 4,471.5 kg/ha) and higher at NMLC (2,048.0 vs. 1,479.3 kg/ha). 

Plants of MON 88701 had a lower seed index (g/100 fuzzy seed) than the conventional 

control at ARPR (9.1 vs. 10.0), ARTI (9.6 vs. 12.0), GACH (9.0 vs. 9.7), GAJE (8.1 vs. 

10.4), LACH (9.5 vs. 10.3), NCBD (9.2 vs. 10.5), NCME (8.9 vs. 10.2), SCEK (8.5 vs. 

9.8), and TXPL (9.9 vs. 11.0). Plants of MON 88701 had more total seed per boll than 

the conventional control at the ARPR (26.1 vs. 23.9) and GACH (29.7 vs. 27.4). Plants 

of MON 88701 had more mature seed per boll than the conventional control at ARPR 

(23.2 vs. 15.9), ARTI (22.5 vs. 18.0) and GACH (27.2 vs. 23.4). Plants of MON 88701 

had fewer immature seed per boll than the conventional control at ARPR (2.9 vs. 8.0) and 

ARTI (5.3 vs. 8.9). Plants of MON 88701 had higher fiber micronaire than the control at 

NMLC (4.9 vs. 4.7 mic units) and higher fiber strength (g/tex) at GACH (31.0 vs. 29.5), 

KSLA (30.7 vs. 29.4), LACH (31.3 vs. 29.9), NMLC (29.3 vs. 27.3), SCEK (30.7 vs. 

30.0) and the TXPL (33.0 vs. 31.0). Plants of MON 88701 had higher fiber uniformity 
Monsanto Company 12-CT-244U 548 of 620 

(%) than the conventional control at KSLA (84.3 vs. 83.3) and lower at SCEK (82.5 vs. 

83.9). Plants of MON 88701 had shorter fiber length (cm) at SCEK (2.7 vs. 2.8). 

The statistical differences detected in the individual site analysis for nodes above white 

flower observations 3 at three locations, seedcotton yield at two locations, immature seed 

per boll at two locations, fiber micronaire at one location, fiber uniformity at two 

locations and fiber length at one location were not detected in the combined-site analysis 

(Table G-13). Therefore, the differences between MON 88701 and the conventional 

control were not indicative of a consistent plant response and MON 88701 is unlikely to 

have increased plant pest/weed potential or an adverse environmental impact compared to 

the conventional control. (See Figure VII-1, Schematic Diagram of Agronomic and 

Phenotypic Data Interpretation Methods, step 3, answer “no”). Although statistical 

differences were detected for plant height at 30 DAP at one site, plant height at harvest at 

four sites, nodes above white flower observation 1 at two sites, nodes above white flower 

observation 2 at three sites, seed index at nine sites, total seed per boll at two sites, and 

mature seed per boll at three sites and fiber strength index at 6 sites, the assessed values 

of MON 88701 for the combined-site analysis were within their respective reference 

range. Therefore, the differences between MON 88701 and the conventional control 

were not indicative of a consistent plant response and MON 88701 is unlikely to have 

increased plant pest/weed potential or an adverse environmental impact compared to the 

conventional control (See Figure VII-1, Schematic Diagram of Agronomic and 

Phenotypic Data Interpretation Methods, step 3, answer “no”). 

G.13. Plant Mapping Results and Discussion 

G.13.1. Individual Site Plant Mapping Results for MON 88701 Not Treated with 

Dicamba or Glufosinate Herbicides - Results and Discussion. 

In the individual site analysis, 14 statistically significant differences were detected out of 

77 comparisons between MON 88701 and the control (Table G-17). Plants of 

MON 88701 had more mainstem nodes compared to the conventional control at ARTI 

(20.9 vs. 19.8) and a fewer number of nodes to the first fruiting branch at GACH (6.5 vs. 

7.9) and GAJE (5.0 vs. 5.8). Plants of MON 88701 had fewer total bolls per plant 

compared to the conventional control at KSLA (19.1 vs. 20.6) and more total bolls at 

NCME (6.8 vs. 4.2) and NMLC (6.7 vs. 4.5). Plants of MON 88701 had more firstposition bolls per plant compared to the conventional control at NCME (3.5 vs. 2.5), 

NMLC (4.4 vs. 3.0), and TXPL (7.8 vs. 7.1) and more vegetative bolls per plant at 

NCME (1.0 vs. 0.1). Plants of MON 88701 retained a higher percentage (%) of firstposition bolls per plant compared to the conventional control at the NCME (35.6 vs. 

23.9) and NMLC (30.1 vs. 21.3) and a higher percent of first-position bolls per plant 

relative to total bolls per plant at GAJE (67.4 vs. 61.8) and SCEK (73.2 vs. 65.7). 

The statistical differences (Table G-17) detected between MON 88701 and the control in 

the individual site analysis for total mainstem nodes per plant at one location, the number 

of nodes per plant to the first fruiting branch at two sites, total bolls per plant at three 

sites, total vegetative bolls per plant at one site, percent first-position boll retention per 

plant at two sites and percent first-position bolls per plant relative to total bolls per plant 
Monsanto Company 12-CT-244U 549 of 620 

at two sites were not detected in the combined-site analysis (Table VII-6). Therefore, the 

differences between MON 88701 and the conventional control were not indicative of a 

consistent plant response and MON 88701 is unlikely to have increased plant pest/weed 

potential or an adverse environmental impact compared to the conventional control (See 

Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation 

Methods, step 2, answer “no”). Although statistical differences were detected between 

the MON 88701 and the conventional control for total first-position bolls at three sites 

(Table G-17), the assessed values of MON 88701 for the combined-site analysis were 

within their respective reference range. Therefore, the differences between MON 88701 

and the conventional control were not indicative of a consistent plant response and 

MON 88701 is unlikely to have increased plant pest/weed potential or an adverse 

environmental impact compared to the conventional control (See Figure VII-1, Schematic 

Diagram of Agronomic and Phenotypic Data Interpretation Methods, step 3, answer 

“no”). 

G.13.2. Combined-site and Individual Site Plant Mapping Results for MON 88701 

Treated with Dicamba and Glufosinate Herbicides - Results and Discussion. 

To support the assessment of MON 88701, plant mapping assessments were also 

conducted on the agronomic system that includes MON 88701 treated with dicamba and 

glufosinate herbicides. In the combined-site analysis (Table G-18) of plant mapping 

parameters, no statistically significant differences were detected between MON 88701 

and the conventional control for total mainstem nodes per plant, the number of nodes per 

plant to the first fruiting branch, total bolls per plant, total vegetative bolls per plant, 

percent first-position boll retention per plant and percent first-position bolls per plant 

relative to total bolls per plant. Therefore, the lack of differences in the above 

characteristics supports a conclusion that MON 88701 is unlikely to have increased plant 

pest/weed potential or an adverse environmental impact compared to the conventional 

control (See Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data 

Interpretation Methods, step 2, answer “no”). Plants of MON 88701 had a significantly 

higher number of first-position bolls per plant compared to the conventional control (5.2 

vs. 4.6). However, the mean values of MON 88701 were within the reference range. 

Thus, MON 88701 is unlikely to have increased plant pest/weed potential or an adverse 

environmental impact compared to the conventional control. (See Figure VII-1, 

Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods, step 3, 

answer “no”). 

In the individual site analysis, 15 statistically significant differences were detected out of 

77 comparisons between MON 88701 treated with dicamba and glufosinate herbicides 

compared to the conventional (Table G-19). Plants of MON 88701 had more mainstem 

nodes per plant compared to the conventional control at NCBD (16.5 vs. 14.8) and a 

fewer number of nodes to the first fruiting branch at GACH (6.5 vs. 7.9) and GAJE (5.1 

vs. 5.8). Plants of MON 88701 had a higher number of total bolls per plant compared to 

the conventional control at NCME (7.1 vs. 4.2), NMLC (7.7 vs. 4.5) and TXPL (14.3 vs. 

11.3) and a higher number of first-position bolls per plant at NCBD (6.5 vs. 5.5), NCME 

(3.4 vs. 2.5), NMLC (4.7 vs. 3.0) and TXPL (8.1 vs. 7.1). Plants of MON 88701 had 
Monsanto Company 12-CT-244U 550 of 620 

more vegetative bolls per plant compared to the convention control at NCME (1.1 vs. 

0.1) and less at SCEK (0.3 vs. 0.7). Plants of MON 88701 retained a higher percent of 

first-position bolls compared to the control at NCME (33.7 vs. 23.9) and NMLC (32.2 vs. 

21.3) and had a higher percentage of first-position bolls per plant relative to total bolls 

per plant at the GACH (45.4 vs. 36.6). 

The statistical differences detected in the individual site analysis (Table G-19) for total 

mainstem nodes per plant, the number of nodes per plant to the first fruiting branch, total 

bolls per plant, total vegetative bolls per plant, percent first-position boll retention per 

plant and percent first-position bolls per plant relative to total bolls per plant were not 

detected in the combined-site analysis (Table G-18). Therefore, the differences between 

MON 88701 and the conventional control were not indicative of a consistent plant 

response and MON 88701 is unlikely to have increased plant pest/weed potential or an 

adverse environmental impact compared to the conventional control (See Figure VII-1, 

Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods, step 2, 

answer “no”). Although statistical differences were detected for the total number of firstposition bolls at four sites (Table G-19), the assessed values of MON 88701 for the 

combined-site analysis were within their respective reference range. Therefore, the 

differences between MON 88701 and the conventional control were not indicative of a 

consistent plant response and MON 88701 is unlikely to have increased plant pest/weed 

potential or an adverse environmental impact compared to the conventional control (See 

Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation 

Methods, step 3, answer “no”). 

G.14. Individual Site Environmental Interactions - Results and Discussion 

G.14.1. Individual Site Environmental Interactions - Results and Discussion – 

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides – Study 1 

(Qualitative Data Assessment) 

In an individual site assessment, no differences were observed between MON 88701 and 

the conventional control for any of the 169 comparisons for the assessed abiotic 

stressors, including compaction, drought/dry, flood, hail, heat, nutrient deficiency, wet 

soil/excess precipitation, and wind damage (Table G-20). 

In an individual site assessment, no differences were observed between MON 88701 and 

the control for any of the 170 comparisons for the assessed diseases, including 

anthracnose, Ascochyta leaf blight, bacterial blight, boll rot, cotton leaf rust, damping off, 

Fusarium wilt, leaf spots, Pythium, reniform nematode, Rhizoctonia, root-knot nematode, 

Thielaviopsis, and Verticillium wilt (Table G-21) 

In an individual site assessment, no differences were observed between MON 88701 and 

the control for any of the 159 comparisons for the assessed arthropod stressors, 

including aphids, beet armyworms, cut worms, fall armyworms, fleahoppers, 
Monsanto Company 12-CT-244U 551 of 620 

grasshoppers, heliothines, southern corn rootworm beetles, soybean loopers, spider 

mites, stink bugs, tarnished plant bugs, thrips, and white flies (Table G-22). 

(Quantitative Data Assessment) 

Thrips Damage 

A total of 15 thrips damage comparisons (three observation events × 5 sites) were made 

between MON 88701 and the conventional control in the individual site analysis (Table 

G-23). Of these comparisons, no numerical differences were observed for 10 

comparisons for which p-values could not be generated due to lack of variability in the 

data. Four of the remaining five comparisons were not significantly different. 

MON 88701 had significantly less damage from thrips in observation 3 at the ARPR site 

(0.1 vs. 0.3). However, there was no significant difference between MON 88701 and the 

conventional control for Observation 3 in the combined-site analysis (Table VII-8). 

Therefore, the lack of difference for 14 comparisons and the one site/observation 

difference between MON 88701 and the conventional control were not indicative of a 

consistent plant response and MON 88701 is unlikely to have increased plant pest/weed 

potential or an adverse environmental impact compared to the conventional control (See 

Figure VII-1, Schematic Diagram of Agronomic and Phenotypic Data Interpretation 

Methods, step 2, answer “no”). 

Heliothines Damage 

A total of 40 heliothine damage comparisons (4 observations × 5 sites) were made 

between MON 88701 and the conventional control in the individual site analysis (Table 

G-24). Of these comparisons, no numerical differences were observed for three 

comparisons for which p-values could not be generated due to lack of variability in the 

data. For the remaining 37 comparisons, no statistically significant differences were 

detected between MON 88701 and the control for 35 out of 37 comparisons (Table G24). Two statistically significant differences were detected between MON 88701 and the 

parental control. Plants of MON88701 had fewer heliothine damage fruiting bodies 

compared to the conventional control in Observation 4 at ARAU (8.7 vs. 15.1%), and 

more live larvae Observation 1 at GACH (0.5 vs. 0.1). Although the above statistical 

differences were detected, the assessed values of MON 88701 were not significantly 

different than the control in the combined-site analysis (Table VII-9). Therefore, the lack 

of difference for 38 comparisons and the two site/observation differences between 

MON 88701 and the conventional control were not indicative of a consistent plant 

response, and MON 88701 is unlikely to have increased plant pest/weed potential or an 

adverse environmental impact compared to the conventional control (See Figure VII-1, 

Schematic Diagram of Agronomic and Phenotypic Data Interpretation Methods, step 2, 

answer “no”). 

Arthropod Abundance 
Monsanto Company 12-CT-244U 552 of 620 

A total of 178 comparisons were made between MON 88701 and the conventional 

control for arthropod abundance involving the following pest- and beneficial-arthropods: 

aphids, cabbage loopers, fall armyworms, fleahoppers, heliothines, southern armyworms, 

stink bugs, tarnished plant bugs, thrips, white flies, big eyed bugs, braconids, Damsel 

bugs, lacewings, ladybird beetles, Orius spp and Araneae (spiders) (Tables G-25 and G26). No statistically significant differences were detected between MON 88701 and the 

conventional control for 173 out of 178 comparisons, including 89 pest arthropod 

comparisons and 89 beneficial arthropod comparisons. The five differences detected 

between MON 88701 and the conventional control included two differences for pest 

arthropods and three differences for beneficial arthropods. 

At collection 4 at LABU, the abundance of stink bugs per plot in MON 88701 was lower 

compared to the conventional control (0.3 vs. 1.8 per plot) and lower for tarnished plant 

bugs (0.5 vs. 2.0). For tarnished plant bugs, the mean abundance value for MON 88701 

was within the reference ranges for the differences detected. For stink bugs, the mean 

abundance value for MON 88701 was outside the reference range for the difference 

detected. Since the two differences mentioned above were not consistently detected in 

multiple environments, these data support a conclusion that the differences are considered 

not biologically meaningful in terms of plant pest potential or an adverse environmental 

impact (See Section VII.B.1.1., Interpretation of Environmental Interactions Data). 

The abundance of Damsel bugs per plot MON 88701 was higher compared to the 

conventional control in Collection 2 at GACH (6.0 vs, 2.3) and lower for Orius spp. in 

Collection 2 (0.0 vs. 1.5 per plot) and collection 3 (0.5 vs. 2.8 per plot) at the ARAU site. 

The mean abundance value for MON 88701 was within the reference range for the 

difference detected for Damsel bugs. The mean abundance values for Orius spp. in 

Collection 2 and collection 3 at the ARAU site were outside their respective reference 

range. However, the differences detected for Orius spp. were not consistently detected 

across collections or sites (Table G-26). Thus, the detected differences in beneficial 

arthropod abundance were not indicative of a consistent response associated with 

MON 88701 and are not considered biologically meaningful in terms of an adverse 

environmental impact of MON 88701 compared to conventional cotton (See Section 

VII.B.1.1., Interpretation of Environmental Interactions Data).

G.14.2. Individual Site Environmental Interactions - Results and Discussion – 

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides – Study 2 

In an individual site assessment, no differences were observed between MON 88701 and 

the control for any of the 127 comparisons for the assessed abiotic stressors, including 

compaction, drought, dry, flood, hail damage, heat, nutrient deficiency, wet soil, excess 

precipitation, and wind damage (Table G-27). 

In an individual site assessment, no differences were observed between MON 88701 and 

the control for any of the 129 comparisons for the assessed diseases, including 

anthracnose, Ascochyta leaf blight, bacterial blight, boll rot, cotton leaf rust, damping 
Monsanto Company 12-CT-244U 553 of 620 

off, Fusarium wilt, leaf spots, Pythium, reniform nematode, Rhizoctonia, root-knot 

nematode, Thielaviopsis, and Verticillium wilt (Table G-28). 

In an individual site assessment, no differences were observed between MON 88701 and 

the control for any of the 129 comparisons for the assessed arthropod stressors, 

including aphids, beet armyworms, cabbage loopers, cut worms, fall armyworms, 

fleahoppers, grasshoppers, heliothines, southern corn rootworm beetle, soybean loopers, 

spider mites, stink bugs, tarnished plant bugs, thrips, and white flies (Table G-29). 

These results support a conclusion that MON 88701 would not confer a plant pest risk or 

significant environmental impact compared to conventional cotton (See Section 

VII.B.1.1., Interpretation of Environmental Interactions Data). 
Monsanto Company 12-CT-244U 

Table G-5. Study 1 Data Missing or Excluded from Analysis Site Material name Material type Plots Characteristics Reason for exclusion AZME All All All All 

Site was dropped due to low stand comaintenance, and non-reliable data cocooperator. 

All All All All 

Nodes above cracked boll observations 1, 2,and 3 

Nodes above white flower data were growth and development trend relevaadvancement towards cutout. 

ARAU and LABU All All All Arthropod collection 1 Neonate soybean looper identification 

because of the size. 

LABU All All All Arthropod collection 3 Beneficial species from collection 2 w

collection 3 as there were no benefici

KSLA All All All 14 D stand counts 

Stand counts taken on 6/16 were dropwere taken again on 6/23 from all fourows with good count were chosen fo

KSLA All All All 14 D plant vigor Vigor was taken at 6/16 and 6/23 but 

because it was taken 21 days after pla

NCBD Coker 130 Control 403 Final stand count Stand count was poor in these plots a

were destroyed by the lightning strike

NCBD Coker 130 Control 403 Seed cotton yield A couple feet in the plot were destroyNCME DP 435 and DP 5415 Reference 206, 305, 406 Stand count at 30 DAP and final stand count Stand count was poor in these plots b

germplasm. 

NCME DP 5415 Reference 406 Seedcotton yield Yield was very low due to poor stand TXPL All All All Nodes above cracked boll observations 4 This was extra collection and not nee
Monsanto Company 12-CT-244U 

Table G-6. Study 2 Data Missing or Excluded from Analysis Site Material name Material type Plots Characteristics Reason for exclusion ARTI SG125 Reference 106 Plant mapping Plant mapping data sheet was mispTXPO All All All All Plot area was destroyed by lightninALL All All All 

Nodes above cracked boll and days of planning to first cracked boll date 

Nodes above white flower data wgrowth and development trends radvancement towards cutout. Repnot have added any value. 

ALL All All All Days of planting to first flower date Data on three observations on nod

sufficient in proving data on crop Repetition of summary would not 

ARTI GACH LACH NCME 

FM 989 Reference All ALL 

Due to poor germination and stanFM 989 will be excluded from aland reporting. Germination ranged target stand counts. 
Monsanto Company 12-CT-244U 

Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and Development Characteristics Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control 

 Phenotypic Characteristics 

Site

Stand count at 14 DAP 

(# per ~6 m) 

Stand count at 30 DAP 

 (# per ~6 m) Final stand count Plant vigor at 14 DAP1 

MON 88701 

(SE)3 Control (SE) 

MON 88701 

(SE) Control (SE) 

MON 88701 

(SE) Control (SE) MON 88701 Control 

ARAU 165.7 (10.0) 182.2 (12.0) 159.3 (7.3) 174.0 (11.1) 154.2 (9.0) 181.0 (4.9) 3.0-4.0 2.0-3.0 ARPR 179.8 (3.2) 185.3 (4.1) 179.7 (3.2) 183.5 (4.7) 178.3 (3.5) 181.8 (4.7) 5.0-6.0 6.0-6.0 ARTI 173.0 (6.5) 175.5 (5.0) 171.5 (6.4) 173.5 (5.3) 171.0 (6.4) 172.5 (4.9) 1.0-1.0 1.0-1.0 GACH 170.7 (2.7) 171.7 (4.0) 168.8 (2.3) 167.7 (3.9) 166.8 (2.9) 168.5 (3.6) 3.0-4.0 3.0-4.0 GAJE 178.8 (3.3) 182.8 (5.8) 168.5 (4.9) 160.8 (3.3) 167.5 (1.8)* 156.3 (4.9) 1.0-2.0 1.0-1.0 KSLA 127.8 (2.8) 141.5 (3.2) 74.5 (2.5) 74.0 (0.7) 70.3 (2.3) 70.3 (0.8) 2.0-2.0 2.0-2.0 LABU 147.5 (1.0) 155.7 (3.3) 146.2 (1.3) 160.0 (4.1) 94.3 (1.8) 91.8 (0.7) 1.0-3.0 2.0-3.0 LACH 100.8 (3.0)* 127.8 (1.1) 101.8 (2.6)* 125.0 (2.6) 63.3 (0.9)* 73.5 (4.7) 2.0-3.0 2.0-3.0 NCBD 117.3 (2.3) 125.3 (6.8) 106.8 (5.0) 116.5 (7.5) 106.5 (2.7) 115.3 (8.8) 3.0-3.0 2.0-3.0 NCME 95.8 (22.8) 105.0 (11.8) 90.5 (23.0) 99.8 (12.3) 90.5 (21.6) 97.0 (12.2) 3.0-6.0 3.0-6.0 NMGA 116.5 (17.3) 116.0 (12.8) 70.0 (0.0) 70.0 (0.0) 70.3 (0.5) 70.8 (0.6) 1.0-2.0 2.0-2.0 NMLC 122.5 (6.7) 116.0 (18.8) 70.0 (0.0) 70.0 (0.0) 70.8 (0.9) 71.8 (1.0) 2.0-2.0 1.0-2.0 SCEK 182.7 (4.3) 188.2 (3.2) 183.3 (3.4) 189.3 (3.1) 179.2 (2.6) 181.3 (3.1) 2.0-3.0 2.0-3.0 TXPL 163.0 (3.8) 151.0 (15.9) 142.8 (8.3) 155.5 (9.5) 157.8 (1.9) 146.8 (13.9) 1.0-1.0 1.0-1.0 TXPO 147.8 (13.3) 162.3 (12.1) 143.3 (9.1) 145.5 (13.0) 137.0 (6.1) 151.0 (5.4) 1.0-1.0 1.0-1.0 
Monsanto Company 12-CT-244U 

Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and Development Characteristics Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued)  Phenotypic Characteristics Site 

Plant height at 30 DAP 

(cm) 

Plant height before harvest

(cm) 

Nodes above white flower 

(obs. 1) 

Nodes above white flower 

(obs. 2) 

N

MON 88701 

(SE) 

Control 

(SE) 

MON 88701 

(SE) Control (SE)

MON 88701 

(SE) Control (SE) 

MON 88701 

(SE) Control (SE) 

M

ARAU 30.9 (0.5) 33.7 (1.2) 159.3 (2.9) 168.8 (8.8) 7.7 (0.1) 7.6 (0.2) 7.1 (0.3) 7.1 (0.1) 6ARPR 29.1 (1.5) 31.8 (2.6) 126.9 (3.6) 134.4 (3.1) 7.7 (0.2) 7.2 (0.2) 7.1 (0.1)* 6.6 (0.2) 6ARTI 29.5 (0.6) 29.4 (0.3) 131.9 (2.9) 139.2 (4.8) 8.5 (0.1) 8.4 (0.1) 7.5 (0.2) 7.1 (0.2) 4GACH 28.2 (0.4) 29.7 (1.4) 84.6 (2.6) 88.1 (2.0) 5.2 (0.1) 5.0 (0.2) 3.4 (0.2) 3.1 (0.3) GAJE 27.0 (0.8) 27.9 (0.7) 87.1 (3.7)* 98.6 (5.3) 7.3 (0.6) 7.7 (0.5) 6.0 (0.2) 5.9 (0.1) 3KSLA 17.5 (1.4) 19.4 (0.8) 129.9 (3.5)* 142.0 (5.5) 5.1 (0.2)* 4.4 (0.2) 4.2 (0.5)* 3.2 (0.2) 4LABU 15.8 (0.8) 17.2 (0.7) 150.2 (3.7) 161.2 (6.4) 7.5 (0.2) 7.6 (0.4) 7.0 (0.2) 6.6 (0.2) 7LACH 12.4 (0.6)* 14.5 (0.6) 120.5 (3.2)* 140.5 (9.8) 8.2 (0.2) 8.5 (0.1) 7.8 (0.1) 7.6 (0.1) 6NCBD 20.0 (1.8)* 24.5 (1.0) 104.8 (9.5)* 111.0 (10.5) 6.2 (0.5) 5.6 (0.5) 4.0 (0.4) 3.7 (0.6) 2NCME 16.2 (1.1) 15.7 (0.3) 90.3 (4.4) 94.7 (2.1) 4.8 (0.1) 4.8 (0.1) 3.1 (0.1) 2.9 (0.1) 2NMGA 5.0 (0.2) 5.2 (0.4) 92.3 (3.0) 96.6 (3.8) 8.6 (0.4) 8.4 (0.5) 8.7 (0.2) 8.5 (0.3) 5NMLC 6.7 (0.7)* 8.3 (0.6) 96.0 (4.6) 94.6 (2.1) 8.4 (0.5)* 7.4 (0.3) 8.2 (0.3) 8.0 (0.2) 6SCEK 8.0 (0.9) 8.4 (1.5) 98.9 (8.0) 97.8 (5.0) 5.3 (0.2) 5.0 (0.3) 5.4 (0.4) 4.9 (0.3) 5TXPL 8.0 (0.4) 8.1 (0.3) 44.9 (0.7)* 48.4 (0.8) 5.0 (0.1)* 4.5 (0.2) 4.4 (0.2) 3.9 (0.2) 2TXPO 19.9 (0.8) 22.6 (0.8) 129.7 (1.2) 129.9 (0.9) 8.8 (0.1) 8.5 (0.3) 7.0 (0.2) 6.6 (0.1) 6
Monsanto Company 12-CT-244U 

Table G-7. Study 1 - Individual Site Phenotypic Comparison – Growth and Development Characteristics Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued)  Phenotypic Characteristics Site 

Seed cotton yield 

(Kg/ha)

MON 88701 (SE) Control (SE) 

ARAU 2912.9 (231.8)* 2166.8 (250.2) ARPR 3452.4 (202.0) 3309.7 (155.9) ARTI 3444.8 (90.4) 3488.7 (87.3) GACH 2880.8 (97.8) 2875.6 (101.1) GAJE 2320.4 (42.3)* 1843.1 (256.8) KSLA 4330.4 (184.1) 4341.9(365.1) LABU 1534.1 (153.7) 1719.4 (67.8) LACH 1464.1 (245.0) 1736.0 (87.4) NCBD 3990.5 (375.5) 4112.1 (299.2) NCME 1324.7 (110.5) 1296.3 (140.9) NMGA 3459.6 (211.1) 3498.9 (165.2) NMLC 4771.7 (199.9) 4522.5 (158.1) SCEK 3625.0 (125.2) 3901.9 (143.5) TXPL 3638.4 (114.1) 3689.3 (29.6) TXPO 916.5 (115.9) 856.8 (41.5) * Indicates a statistically significant difference (

α = 0.05) between MON 88701 and conventional control (n = 4). 1Plant vigor score range (minimum-maximum). The range of plant vigor score at 14 DAP for the references is as follows: ARAU 3

GACH 3-7; GAJE 1-3; KSLA 2-4; LABU 2-5; LACH 2-5; NCBD 3-4; NCME 3-6; NMGA 1-3; NMLC 2-3; SCEK 2-4; TXPL 1-1; Trated per plot using a rating scale of 1-9 where: 1-3 is excellent vigor, 4-6 is average vigor, and 7-9 is poor vigor. 

2Plant vigor score range (minimum-maximum). The range of plant vigor score at 30 DAP for the references is as follows: ARAU 3

GACH 3-7; GAJE 2-2; KSLA 2-3; LABU 2-3; LACH 1-4; NCBD 2-4; NCME 3-7; NMGA 1-5; NMLC 1-5; SCEK 3-4; TXPL 3-3; TX

3 SE = Standard error 
Monsanto Company 12-CT-244U 

Table G-8. Study 1 - Individual Site Phenotypic Comparison – Seed Characteristics - of MON 8870Dicamba or Glufosinate Herbicides Compared to the Conventional Control. Site 

Seed index 

(g per 100 fuzzy seed) 

Total seed per boll 

(# per boll) 

Mature seed per boll 

(# per boll) 

Im

MON 88701 (SE)1 Control (SE) MON 88701 (SE) Control (SE) MON 88701 (SE) Control (SE) MON 8

ARAU 10.6 (0.4) 10.6 (0.2) 31.6 (0.3)* 29.6 (1.0) 20.5 (1.2) 19.2 (0.9) 11.ARPR 9.4 (0.2)* 10.3 (0.1) 29.4 (0.3)* 27.3 (0.9) 27.3 (0.6)* 21.7 (0.7) 2.1 ARTI 10.1 (0.2)* 10.9 (0.1) 26.0 (0.7) 26.6 (0.5) 20.2 (1.0)* 17.6 (0.4) 5.7 GACH 10.3 (0.6) 10.4 (0.5) 28.0 (0.9) 27.6 (0.7) 17.4 (0.5) 17.0 (0.8) 10.GAJE 8.3 (0.4)* 9.8 (0.2) 25.8 (0.9) 26.3 (1.3) 15.6 (0.8) 15.2 (2.0) 10.KSLA 11.8 (0.2)* 12.5 (0.2) 31.2 (1.1)* 28.1 (0.4) 29.9 (1.1)* 26.6 (0.4) 1.3 LABU 9.4 (0.2)* 10.5 (0.1) 29.5 (0.4) 27.9 (0.7) 21.2 (0.8) 18.9 (1.1) 8.4 LACH 8.8 (0.3)* 9.8 (0.3) 28.4 (0.8) 28.3 (0.7) 15.5 (1.2) 16.0 (0.7) 12.NCBD 9.0 (0.1)* 9.8 (0.3) 30.1 (1.5) 27.8 (1.7) 21.1 (3.5) 16.2 (2.1) 9.0 NCME 9.7 (0.2)* 10.6 (0.1) 31.7 (2.0)* 27.5 (0.8) 28.4 (1.0)* 22.1 (0.7) 3.4 NMGA 10.6 (0.2)* 11.4 (0.2) 34.4 (0.4)* 30.3 (0.8) 32.9 (0.6)* 28.8 (0.6) 1.6 NMLC 10.5 (1.0) 10.5 (0.6) 31.0 (0.7)* 25.9 (1.5) 26.5 (1.0)* 21.6 (1.3) 4.5 

SCEK 8.5 (0.2) 9.3 (0.2) 27.1 (0.9) 27.1 (0.8) 20.5 (0.8) 16.0 (2.1) 6.5 TXPL 11.5 (0.3) 11.5 (0.6) 28.3 (0.6) 28.0 (0.8) 25.4 (0.7) 24.5 (0.8) 2.9 TXPO 9.0 (0.2) 9.6 (0.4) 23.3 (0.2) 22.7 (0.7) 16.8 (0.8) 14.7 (0.9) 6.6 

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and conventional control (n = 4). 

1 SE = Standard error 
Monsanto Company 12-CT-244U 

Table G-9. Study 1 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON 

with Dicamba or Glufosinate Herbicides Compared to the Conventional Control 

Site 

Boll weight (g/boll) Micronaire (mic units)1 Elongation (%) Strength (g/tex) Uniformity (%) 

MON 88701 

(SE)2 

Control 

(SE) 

MON 88701 

(SE) 

Control 

(SE) 

MON 88701 

(SE) 

Control 

(SE) 

MON 88701 

(SE) 

Control 

(SE) 

MON 88701 

(SE) 

Control 

(SE) 

ARAU 4.9 (0.1) 4.9 (0.2) 3.8 (0.0) 3.6 (0.1) 6.8 (0.2)* 6.2 (0.1) 33.2 (0.5) 32.3 (0.2) 85.3 (0.1) 84.4 (0.3ARPR 4.8 (0.1) 4.6 (0.2) 5.2 (0.1) 5.0 (0.2) 5.4 (0.2) 5.2 (0.4) 29.8 (0.3) 29.8 (0.6) 83.0 (0.4) 82.4 (0.4ARTI 4.1 (0.1)* 4.8 (0.1) 4.5 (0.1) 4.7 (0.0) 5.2 (0.2) 5.5 (0.3) 32.1 (0.1) 31.0 (0.5) 84.4 (0.4) 83.6 (0.1GACH 4.2 (0.1)* 4.5 (0.1) 4.6 (0.0) 4.6 (0.1) 7.1 (0.1) 6.8 (0.3) 30.8 (0.5) 30.6 (0.5) 83.8 (0.2) 84.1 (0.5GAJE 3.6 (0.1) 4.0 (0.3) 3.9 (0.1) 3.7 (0.1) 7.1 (0.5) 6.7 (0.3) 32.2 (0.7) 32.1 (0.2) 84.0 (0.2) 83.8 (0.2KSLA 6.7 (0.2) 6.3 (0.1) 4.5 (0.0) 4.5 (0.1) 5.8 (0.3) 5.6 (0.4) 31.5 (0.4)* 30.3 (0.3) 84.1 (0.5) 83.4 (0.3LABU 4.5 (0.1) 4.7 (0.1) 4.7 (0.1) 4.7 (0.1) 5.0 (0.3) 5.4 (0.3) 32.3 (0.6) 31.2 (0.3) 84.1 (0.1) 84.1 (0.2LACH 4.1 (0.2) 4.5 (0.2) 4.4 (0.0) 4.5 (0.0) 5.5 (0.2) 6.0 (0.4) 30.5 (0.2) 29.9 (0.2) 83.7 (0.3) 83.3 (0.4NCBD 5.4 (0.2) 5.1 (0.4) 4.7 (0.0) 4.5 (0.5) 5.7 (0.3)* 6.6 (0.4) 32.6 (1.1)* 30.9 (0.8) 85.1 (0.4) 84.7 (0.4NCME 5.7 (0.3) 5.3 (0.1) 5.0 (0.0) 4.8 (0.1) 6.2 (0.1) 6.4 (0.3) 32.6 (0.2) 31.8 (0.3) 84.1 (0.4) 84.2 (0.3NMGA 5.9 (0.1) 5.8 (0.1) 5.1 (0.1)* 4.9 (0.0) 5.9 (0.2) 6.0 (0.2) 30.9 (0.2) 31.2 (0.4) 82.3 (0.8) 83.7 (0.2NMLC 5.1 (0.1)* 4.6 (0.3) 4.7 (0.1) 4.6 (0.1) 7.1 (0.2) 6.8 (0.2) 31.2 (0.3)* 30.0 (0.2) 83.5 (0.3) 83.0 (0.4

SCEK 4.1 (0.2) 4.4 (0.2) 4.7 (0.1) 4.7 (0.1) 5.1 (0.1) 5.2 (0.1) 32.1 (0.5) 31.2 (0.3) 83.3 (0.2) 83.7 (0.1TXPL 5.8 (0.1) 6.0 (0.1) 4.9 (0.1)* 4.5 (0.1) 7.2 (0.1) 7.3 (0.3) 34.1(0.1)* 32.2 (0.5) 85.4 (0.4)* 84.1 (0.4TXPO 3.3 (0.1) 3.2 (0.2) 4.6 (0.1) 4.5 (0.1) 5.0 (0.4) 4.8 (0.1) 31.0 (0.6) 31.2 (0.2) 83.8 (0.6) 83.2 (0.2

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and conventional control (n = 4). 

1 Measure of fiber fineness and maturity (expressed in dimensionless micronaire units). 2 SE = Standard error. 
Monsanto Company 12-CT-244U 

Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characterist

Not Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control 

Phenotypic Characteristic (units)

Stand count 14 DAP1

(# per plot)

Stand count at 30 DAP 

(# per plot)

Final Stand Count at 

harvest

Plant Height at 30 DAP

(cm)

MON 88701 Control MON 88701 Control MON 88701 Control MON 88701 Control 

Site Mean (SE)2 Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) ARPR 185.5 (1.9) 179.8 (2.3) 180.3 (0.9) 179.5 (2.9) 178.0 (0.4) 177.5 (3.5) 20.5 (0.6) 21.1 (0.5) ARTI 155.3 (5.9) 164.8 (7.2) 153.8 (6.3) 164.3 (7.1) 153.3 (6.5) 163.8 (7.2) 33.0 (0.2) 32.8 (0.2) GACH 131.3 (8.4) 132.8 (8.0) 129.0 (8.0) 127.8 (10.3) 128.5 (9.4) 129.5 (9.1) 25.1 (1.0) 25.8 (0.8) GAJE 179.8 (4.4) 181.3 (2.7) 166.0 (2.3) 164.0 (3.0) 162.3 (6.0) 157.3 (8.4) 23.6 (1.1) 26.0 (0.7) 

KSLA 156.5 (7.4) 162.5 (5.9) 154.8 (7.4) 158.5 (5.3) 150.5 (7.0) 150.0 (6.7) 15.7 (0.6)* 18.3 (1.2) 

LACH 98.8 (3.1) 110.3 (5.4) 101.0 (3.1) 112.5 (7.6) 92.3 (1.1) 94.8 (2.9) 13.5 (0.1)* 15.9 (0.8) NCBD 141.8 (2.3) 140.3 (3.2) 140.8 (2.2) 140.3 (3.6) 132.5 (2.5) 134.3 (2.3) 17.8 (0.9) 19.4 (0.7) 

NCME 132.0 (8.2) 148.0 (19.5) 158.5 (6.3) 160.3 (9.7) 146.3 (9.0) 166.3 (8.3) 17.2 (0.7)* 19.7 (0.9) NMLC 157.5 (7.5) 168.8 (11.9) 154.5 (7.3) 164.0 (2.4) 153.3 (6.2) 163.3 (5.8) 7.0 (0.2) 7.2 (0.2) 

SCEK 186.3 (3.7) 191.5 (4.6) 182.5 (3.6) 190.3 (3.9) 181.5 (3.0) 188.8 (3.8) 16.7 (1.1) 17.9 (1.3) 

TXPL 130.8 (3.3) 125.0 (10.4) 122.3 (7.4) 119.0 (8.8) 130.5 (3.2) 130.0 (11.3) 7.8 (0.6) 7.4 (0.5) 
Monsanto Company 12-CT-244U 

Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characteristics Not Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued) 

1st Vigor3,4 rating

2nd Vigor3,5 rating

Nodes above white flower

(obs. 1)

Nodes 

MON 88701 Control MON 88701 Control MON 88701 Control MON 8

Site Range Range Range Range Mean (SE) Mean (SE) Mean (

ARPR 4 – 6 3 - 4 4 - 5 3 - 5 5.5 (0.1) 5.7 (0.2) 3.7 (0.

ARTI 2 – 3 2 - 3 2 - 2 2 - 2 8.9 (0.3) 8.5 (0.2) 6.9 (0.

GACH 3 – 4 2 - 5 3 - 5 4 - 6 5.9 (0.2)* 5.3 (0.2) 4.7 (0.

GAJE 1 – 2 1 - 1 2 - 2 2 - 2 6.3 (0.3) 6.6 (0.5) 5.9 (0.

KSLA 2 – 5 2 - 3 2 - 3 2 - 2 4.8 (0.3) 4.6 (0.2) 4.4 (0.LACH 4 – 7 3 - 5 2 - 3 1 - 2 7.9 (0.2) 7.6 (0.1) 8.2 (0.NCBD 2 – 2 2 - 3 3 - 3 3 - 3 7.2 (0.1) 7.1 (0.5) 4.8 (0.NCME 2 – 5 1 - 5 2 - 3 2 - 3 5.1 (0.3) 5.2 (0.2) 3.2 (0.NMLC 1 – 3 2 - 2 1 - 2 1 - 1 7.4 (0.3) 7.1 (0.2) 8.1 (0.SCEK 2 – 4 3 - 6 2 - 3 3 - 3 7.5 (0.2) 7.4 (0.2) 4.8 (0.TXPL 1 -1 1 - 1 3 - 3 3 - 3 5.9 (0.2) 5.7 (0.1) 5.5 (0.

Phenotypic Characteristic (units)
Monsanto Company 12-CT-244U 

Table G-10. Study 2 - Individual Site Phenotypic Comparison – Growth and Development Characterist

Not Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued) 

Phenotypic characteristics (units)

Nodes above white flower 

(obs. 3)

Seedcotton yield

(kg/ha)

MON 88701 Control MON 88701 Control

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR 2.3 (0.2) 2.4 (0.4) 2,561.6 (191.4) 2,301.7 (173.4)ARTI 4.8 (0.1) 4.7 (0.1) 3,864.4 (250.8) 3,869.0 (112.9)

GACH 4.1 (0.1)* 3.6 (0.3) 4,424.4 (74.0) 4,471.5 (99.5)GAJE 3.9 (0.1) 3.8 (0.0) 1,750.1 (261.1) 1,548.4 (342.4)KSLA 3.7 (0.4)* 2.5 (0.4) 4,487.0 (294.6)* 3,726.5 (81.7)LACH 5.8 (0.1) 5.7 (0.1) 1,956.0 (173.0) 2,046.9 (210.4)NCBD 3.4 (0.3) 3.0 (0.3) 4,224.0 (326.0) 3,792.7 (283.0)NCME 2.0 (0.2) 1.9 (0.1) 1,508.4 (112.7) 1,610.6 (206.9)NMLC 7.5 (0.2) 7.1 (0.2) 1,938.4 (22.1)* 1,479.3 (65.6)SCEK 3.8 (0.3) 3.5 (0.1) 5,219.8 (236.4) 5,424.3 (148.4)TXPL 3.6 (0.1) 3.5 (0.3) 4,741.3 (165.8) 4,534.5 (215.6)

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and control (n = 4). 

1 DAP = Days after planting. 2 SE = Standard error. 3 Plant vigor was rated per plot using a rating scale of 1-9 where: 1-3 is excellent vigor, 4-6 is average vigor, and 7-9 is poor vigor. 4 First plant vigor score range (minimum-maximum). The range of plant vigor score at 14 DAP for the references is as follows: ARPR 

6; GAJE 1-3; KSLA 2-5; LACH 4-7; NCBD 3-3; NCME 1-6; NMLC 2-5;SCEK 4-6; TXPL 1-1. 

5 Plant vigor score range (minimum-maximum). The range of plant vigor score at 30 DAP for the references is as follows: ARPR 4-6; 

GAJE 2-2; KSLA 2-3; LACH 2-3; NCBD 3-3; NCME 2-3; NMLC 1-3; SCEK 2-3; TXPL 3-3.
Monsanto Company 12-CT-244U 

Table G-11. Study 2 - Individual Site Phenotypic Comparison – Seed Characteristics - of MON 8870Dicamba or Glufosinate Herbicides Compared to the Conventional Control 

 Seed Characteristics 

Seed index 

(g per 100 seed) 

Total seed per boll 

(# per boll) 

Mature seed per boll 

(# per boll) 

Imma

MON 88701 Control MON 88701 Control MON 88701 Control MON 887

Site Mean (SE) 1 Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SARPR 8.8 (0.2)* 10.0 (0.4) 26.8 (0.4)* 23.9 (0.7) 24.2 (0.4)* 15.9 (0.6) 2.6 (0.2ARTI 9.9 (0.3)* 12.0 (1.0) 28.4 (0.3) 26.8 (0.7) 23.7 (0.7)* 18.0 (0.6) 4.6 (0.6GACH 9.1 (0.3) 9.7 (0.3) 29.4 (0.7) 27.4 (1.1) 24.9 (1.0) 23.4 (1.1) 4.5 (1.4GAJE 8.5 (0.4)* 10.4 (0.5) 26.6 (1.1) 24.6 (0.7) 15.0 (0.5)* 11.6 (0.1) 11.6 (0.KSLA 11.7 (0.2)* 12.8 (0.3) 28.4 (1.7)* 24.9 (0.4) 25.9 (1.9)* 22.7 (1.0) 2.5 (0.6LACH 9.4 (0.2)* 10.3 (0.2) 31.4 (0.4) 28.7 (1.2) 20.7 (1.6) 17.5 (1.9) 10.7 (1.NCBD 8.8 (0.2)* 10.5 (0.2) 27.0 (0.8) 25.7 (1.3) 18.4 (1.2) 18.2 (0.9) 8.6 (0.6NCME 8.8 (0.1)* 10.2 (0.1) 28.3 (0.5) 27.6 (1.7) 25.8 (0.6) 23.0 (1.7) 2.5 (0.7NMLC 10.3 (0.2) 10.9 (0.2) 33.8 (2.0)* 30.7 (1.1) 30.7 (2.2) 28.1 (1.4) 3.2 (1.0SCEK 8.5 (0.2)* 9.8 (0.2) 28.9 (2.0) 27.3 (1.3) 20.8 (1.4) 19.1 (1.4) 8.1 (2.0TXPL 9.9 (0.3)* 11.0 (0.3) 31.4 (0.6) 30.1 (0.9) 26.7 (0.8) 25.7 (0.7) 4.7 (0.* Indicates a statistically significant difference (

α = 0.05).between MON 88701 and control (n = 0.4). 

1 SE = Standard error. 
Monsanto Company 12-CT-244U 

Table G-12. Study 2 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON with Dicamba or Glufosinate Herbicides Compared to the Conventional Control 

Weight per Boll

(g/boll)

Fiber Micronaire 

(mic units)2 Fiber Elongation (%) Fiber Streng

MON 88701 Control MON 88701 Control MON 88701 Control MON 88701 

Site Mean (SE)1 Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) ARPR 4.3 (0.1) 4.1 (0.2) 5.0 (0.1) 4.9 (0.1) 5.0 (0.3)* 5.7 (0.2) 30.8 (0.4) 3

ARTI 4.5 (0.0) 4.4 (0.1) 5.0 (0.2) 4.8 (0.1) 5.4 (0.2) 5.4 (0.2) 29.7 (0.3) 2GACH 5.0 (0.2) 4.9 (0.1) 4.8 (0.1) 4.7 (0.1) 5.9 (0.1) 6.3 (0.3) 30.4 (0.4) 2GAJE 3.6 (0.2) 3.7 (0.1) 3.9 (0.1) 3.6 (0.1) 7.5 (0.5) 7.0 (0.3) 31.1 (0.6) 3KSLA 5.8 (0.3) * 5.3 (0.1) 4.3 (0.0) 4.4 (0.1) 6.3 (0.2) 6.2 (0.3) 30.4 (0.1)* 2LACH 5.4 (0.1) 5.4 (0.3) 4.7 (0.1) 4.6 (0.1) 5.5 (0.3) 5.9 (0.3) 30.7 (0.6) 2

NCBD 4.6 (0.3) 4.7 (0.2) 4.4 (0.1) 4.3 (0.1) 5.9 (0.1) 6.0 (0.2) 32.6 (0.9) 3NCME 4.9 (0.1) 5.0 (0.3) 4.9 (0.1) 4.9 (0.1) 6.0 (0.3) 5.9 (0.4) 31.8 (0.2) 3NMLC 5.7 (0.3)* 5.1 (0.3) 4.9 (0.0)* 4.7 (0.1) 6.2 (0.1) 6.4 (0.2) 30.1 (0.3)* 2

SCEK 4.6 (0.3) 4.4 (0.4) 4.9 (0.0) 4.9 (0.0) 7.0 (0.2) 6.9 (0.2) 29.9 (0.4) 3TXPL 5.6 (0.1) 5.7 (0.1) 4.6 (0.1) 4.6 (0.1) 6.6 (0.2) 6.5 (0.2) 32.0 (0.3)* 3
Monsanto Company 12-CT-244U 

Table G-12. Study 2 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON with Dicamba or Glufosinate Herbicides Compared to the Conventional Control (continued) 

Fiber Uniformity (%) Fiber Length (cm)

MON 88701 Control MON 88701 Control

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR 83.9 (0.2) 84.6 (0.1) 2.8 (0.0)* 2.9 (0.0)

ARTI 83.6 (0.4) 83.6 (0.1) 2.7 (0.0) 2.8 (0.0)GACH 83.6 (0.4) 83.4 (0.4) 2.8 (0.0) 2.8 (0.0)GAJE 82.6 (0.9) 82.5 (0.6) 2.8 (0.1) 2.8 (0.1)KSLA 84.2 (0.2)* 83.3 (0.1) 3.1 (0.0) 3.0 (0.0)LACH 84.1 (0.3) 83.4 (0.4) 2.8 (0.0) 2.8 (0.0)

NCBD 84.8 (0.2) 84.8 (0.6) 2.8 (0.0)* 2.9 (0.0)NCME 82.6 (0.5) 83.2 (0.6) 2.7 (0.0) 2.8 (0.1)NMLC 83.4 (0.6)* 81.1 (0.7) 2.8 (0.0) 2.7 (0.1)

SCEK 83.1 (0.2) 83.9 (0.1) 2.7 (0.0) 2.8 (0.0)TXPL 83.5 (0.3) 83.9 (0.3) 2.8 (0.0) 2.8 (0.0)

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and control (n = 4). 

1 SE = Standard error. 2 Measure of fiber fineness and maturity (expressed in dimensionless micronaire units). 
Monsanto Company 12-CT-244U 

Table G-13. Study 2 - Combined-Site Phenotypic Comparison - Growth and Development Characteristics Treated with Dicamba and Glufosinate Herbicides Compared to the Conventional Control Phenotypic Characteristic (units)

MON 88701 Control Refer

Mean (SE)2 Mean (SE) Minimum 

Stand Count at 14 DAP3 (# per plot) 152.5 (4.9) 155.0 (4.4) 108.4 Stand Count at 30 DAP (# per plot) 151.1 (4.8) 152.8 (4.0) 105.8 

Final Stand Count at harvest (# per plot) 147.7 (5.0) 150.5 (4.3) 110.5 

Plant Height at 30 DAP (cm) 18.1 (1.1)* 19.2 (1.1) 11.4 

Plant Height at harvest (cm) 98.4 (4.4)* 105.0 (4.9) 85.2 

Nodes Above White Flower: (# of nodes at observation 1) 6.7 (0.2)* 6.4 (0.2) 6.0 

(# of nodes at observation 2) 5.6 (0.3)* 5.2 (0.3) 4.8 (# of nodes at observation 3) 4.0 (0.2) 3.8 (0.2) 3.2 

Seedcotton Yield (kg/ha) 3,295.5 (191.0) 3,164.1 (210.8) 2,181.7 

Seed Index (g per 100 seed) 9.5 (0.2)* 10.7 (0.2) 9.4 

Total Seed per Boll (# per boll) 28.5 (0.4)* 27.0 (0.4) 26.1 

Mature Seed per Boll (# per boll) 22.8 (0.7)* 20.1 (0.8) 14.6 Immature Seed per Boll (# per boll) 5.7 (0.6) 6.9 (0.6) 2.7 

Weight per Boll (g) 4.7 (0.1) 4.8 (0.1) 4.5 Fiber Micronaire (mic units)4 4.6 (0.1) 4.6 (0.1) 4.2 Fiber Elongation (%) 6.0 (0.1) 6.2 (0.1) 5.6 Fiber Strength (g/tex) 31.2 (0.2)* 30.2 (0.2) 30.7 

Fiber Uniformity (%) 83.5 (0.2) 83.4 (0.2) 82.8 

Fiber Length (cm) 2.8 (0.0) 2.8 (0.0) 2.7 

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and the conventional control (n = 44). 1 Reference range = Minimum and maximum mean values across all 11 sites and nine references from the Study 2 field trial. 

2 SE = Standard error. 3 DAP = days after planting. 4 Measure of fiber fineness and maturity (expressed in dimensionless micronaire units).
Monsanto Company 12-CT-244U 

Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and Development CharacterisTreated with Dicamba and Glufosinate Herbicides Compared to the Conventional Control 

Phenotypic Characteristic (units)

 Stand count at 14 DAP1

(# per plot)

Stand count at 30 DAP 

(# per plot)

Final Stand Count at 

Harvest

Plant Height at 30 DAP

(cm)

MON 88701 Control MON 88701 Control MON 88701 Control MON 88701 Control M

Site Mean (SE)2 Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) ARPR 182.0 (4.4) 179.8 (2.3) 178.8 (4.8) 179.5 (2.9) 177.0 (5.0) 177.5 (3.5) 20.7 (0.7) 21.1 (0.5) 

ARTI 169.3 (8.3) 164.8 (7.2) 167.5 (8.0) 164.3 (7.1) 166.5 (8.0) 163.8 (7.2) 33.2 (0.2) 32.8 (0.2) 1

GACH 128.0 (15.7) 132.8 (8.0) 126.0 (16.4) 127.8 (10.3) 123.5 (16.9) 129.5 (9.1) 23.9 (0.6) 25.8 (0.8) 1GAJE 186.0 (2.3) 181.3 (2.7) 170.0 (2.1) 164.0 (3.0) 171.0 (2.8) 157.3 (8.4) 23.2 (1.0)* 26.0 (0.7) 

KSLA 170.0 (1.1) 162.5 (5.9) 167.5 (3.4) 158.5 (5.3) 162.0 (2.8) 150.0 (6.7) 16.6 (0.81) 18.3 (1.2) 1

LACH 109.0 (6.4) 110.3 (5.4) 108.5 (5.7) 112.5 (7.6) 91.3 (2.7) 94.8 (2.9) 14.7 (0.4) 15.9 (0.8) 1NCBD 141.8 (2.5) 140.3 (3.2) 141.8 (2.2) 140.3 (3.6) 136.8 (3.6) 134.3 (2.3) 17.8 (0.5) 19.4 (0.7) 

NCME 133.5 (8.3) 148.0 (19.5) 152.5 (3.4) 160.3 (9.7) 155.0 (7.0) 166.3 (8.3) 18.6 (0.6) 19.7 (0.9) NMLC 166.8 (4.6) 168.8 (11.9) 168.8 (3.4) 164.0 (2.4) 160.0 (3.1) 163.3 (5.8) 7.4 (0.1) 7.2 (0.2) 

SCEK 183.5 (3.3) 191.5 (4.6) 182.8 (3.1) 190.3 (3.9) 180.3 (3.3) 188.8 (3.8) 15.6 (1.2) 17.9 (1.3) 1

TXPL 107.8 (19.8) 125.0 (10.4) 98.0 (19.1) 119.0 (8.8) 101.8 (20.0) 130.0 (11.3) 7.8 (0.4) 7.4 (0.5) 
Monsanto Company 12-CT-244U 

Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and Development CharacterisTreated with Dicamba and Glufosinate Herbicides Compared to the Conventional Control (continued) 

Phenotypic Characteristic (units)

1st Vigor3,4 rating

2nd Vigor3,5 rating

Nodes above white flower

(obs. 1)

Nodes 

MON 88701 Control MON 88701 Control MON 88701 Control MON 88

Site Range Range Range Range Mean (SE) Mean (SE) Mean (SARPR 3 – 5 3 - 4 4-5 3 - 5 5.6 (0.0) 5.7 (0.2) 3.7 (0.2

ARTI 2 – 3 2 - 3 2-4 2 - 2 8.8 (0 .1) 8.5 (0.2) 6.6 (0.2GACH 4 – 5 2 - 5 4-6 4 - 6 5.8 (0.2) 5.3 (0.2) 4.7 (0.3GAJE 1 – 1 1 - 1 2-3 2 - 2 6.3 (0.4) 6.6 (0.5) 6.1 (0.2KSLA 1 – 3 2 - 3 2-3 2 - 2 4.4 (0.3) 4.6 (0.2) 4.1 (0.4LACH 3 – 6 3 - 5 1-2 1 - 2 8.6 (0.2)* 7.6 (0.1) 8.2 (0.0NCBD 2 – 2 2 - 3 3-3 3 - 3 7.5 (0.2) 7.1 (0.5) 5.7 (0.3NCME 3 – 5 1 - 5 2-3 2 - 3 4.9 (0.2) 5.2 (0.2) 3.6 (0.3NMLC 1 - 2 2 - 2 1-1 1 - 1 7.9 (0.1) 7.1 (0.2) 8.0 (0.2SCEK 3 - 4 3 - 6 2-3 3 - 3 7.7 (0.3) 7.4 (0.2) 5.1 (0.4TXPL 1 - 1 1 - 1 3-3 3 - 3 6.2 (0.3)* 5.7 (0.1) 5.7 (0.0
Monsanto Company 12-CT-244U 

Table G-14. Study 2 - Individual Site Phenotypic Comparison – Growth and Development CharacterisTreated with Dicamba and Glufosinate Herbicides Compared to the Conventional Control (continued) 

Phenotypic characteristics (units)

Nodes above white flower 

(obs. 3)

Seedcotton yield

(kg/ha)

MON 88701 Control MON 88701 Control

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR 2.3 (0.2) 2.4 (0.4) 2,705.9 (124.4) 2,301.7 (173.4)

ARTI 4.9 (0.0)* 4.7 (0.1) 4,109.3 (266.9) 3,869.0 (112.9)GACH 4.0 (0.1) 3.6 (0.3) 4,107.9 (42.0)* 4,471.5 (99.5)GAJE 3.8 (0.0) 3.8 (0.0) 1,977.6 (341.5) 1,548.4 (342.4)KSLA 3.4 (0.1)* 2.5 (0.4) 4,182.0 (329.6) 3,726.5 (81.7)LACH 5.7 (0.1) 5.7 (0.1) 1,940.4 (182.3) 2,046.9 (210.4)NCBD 3.8 (0.3)* 3.0 (0.3) 4,025.4 (189.5) 3,792.7 (283.0)NCME 2.1 (0.2) 1.9 (0.1) 1,785.4 (181.0) 1,610.6 (206.9)NMLC 7.0 (0.7) 7.1 (0.2) 2,048.0 (74.4)* 1,479.3 (65.6)SCEK 3.8 (0.3) 3.5 (0.1) 5,264.2 (106.1) 5,424.3 (148.4)TXPL 3.7 (0.2) 3.5 (0.3) 4,307.3 (109.1) 4,534.5 (215.6)

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and control (n = 44). 

1 DAP = Days after planting. 2 SE = Standard error. 3 Plant vigor was rated per plot using a rating scale of 1-9 where: 1-3 is excellent vigor, 4-6 is average vigor, and 7-9 is poor vigor. 4 First plant vigor score range (minimum-maximum). The range of plant vigor score at 14 DAP for the references is as follows: ARPR 

6; GAJE 1-3; KSLA 2-5; LACH 4-7; NCBD 3-3; NCME 1-6; NMLC 2-5;SCEK 4-6; TXPL 1-1. 

5 Plant vigor score range (minimum-maximum). The range of plant vigor score at 30 DAP for the references is as follows: ARPR 4

GAJE 2-2; KSLA 2-3; LACH 2-3; NCBD 3-3; NCME 2-3; NMLC 1-3; SCEK 2-3; TXPL 3-3.
Monsanto Company 12-CT-244U 

Table G-15. Study 2 - Individual Site Phenotypic Comparison – Seed Characteristics - of MON 88701 T

and Glufosinate Herbicides Compared to the Conventional Control 

 Seed Characteristics 

Seed index 

(g per 100 seed) 

Total seed per boll 

(# per boll) 

Mature seed per boll 

(# per boll) 

Immatu

(# 

MON 88701 Control MON 88701 Control MON 88701 Control MON 8870

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE

ARPR 9.1 (0.2)* 10.0 (0.4) 26.1 (0.8)* 23.9 (0.7) 23.2 (1.5)* 15.9 (0.6) 2.9 (0.9)

ARTI 9.6 (0.1)* 12.0 (1.0) 27.7 (0.3) 26.8 (0.7) 22.5 (1.0)* 18.0 (0.6) 5.3 (1.0)

GACH 9.0 (0.3)* 9.7 (0.3) 29.7 (0.7)* 27.4 (1.1) 27.2 (1.4)* 23.4 (1.1) 2.5 (0.7) 

GAJE 8.1 (0.4)* 10.4 (0.5) 25.4 (1.8) 24.6 (0.7) 12.6 (0.7) 11.6 (0.1) 12.8 (1.7) 

KSLA 12.3 (0.3) 12.8 (0.3) 25.8 (0.5) 24.9 (0.4) 23.7 (0.3) 22.7 (1.0) 2.1 (0.8) 

LACH 9.5 (0.3)* 10.3 (0.2) 29.8 (1.0) 28.7 (1.2) 21.6 (2.2) 17.5 (1.9) 8.3 (1.8) 

NCBD 9.2 (0.2)* 10.5 (0.2) 28.0 (0.7) 25.7 (1.3) 19.7 (0.6) 18.2 (0.9) 8.3 (1.2) 

NCME 8.9 (0.2)* 10.2 (0.1) 29.0 (0.4) 27.6 (1.7) 23.1 (1.4) 23.0 (1.7) 5.9 (1.4) 

NMLC 10.2 (0.1) 10.9 (0.2) 31.8 (0.8) 30.7 (1.1) 29.6 (1.4) 28.1 (1.4) 2.2 (0.7) 

SCEK 8.5 (0.2)* 9.8 (0.2) 27.9 (1.3) 27.3 (1.3) 21.4 (1.5) 19.1 (1.4) 6.5 (1.9) 

TXPL 9.9 (0.4)* 11.0 (0.3) 32.2 (0.9) 30.1 (0.9) 26.5 (0.8) 25.7 (0.7) 5.8 (1.1) 

* Indicates a statistically significant difference between MON 88701 and control (p 

≤ 0.05). 
Monsanto Company 12-CT-244U 

Table G-16. Study 2 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON 

Dicamba and Glufosinate Herbicides Compared to the Conventional Control 

Fiber Boll weight 

(g/boll)

Fiber Micronaire 

(mic units)1 Fiber Elongation (%) Fiber Streng

MON 88701 Control MON 88701 Control MON 88701 Control MON 88701 

Site Mean (SE2

) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) 

ARPR 4.1 (0.2) 4.1 (0.2) 5.0 (0.0) 4.9 (0.1) 5.3 (0.1) 5.7 (0.2) 30.3 (0.7) 3ARTI 4.4 (0.1) 4.4 (0.1) 4.8 (0.0) 4.8 (0.1) 5.1 (0.1) 5.4 (0.2) 29.2 (0.4) 2GACH 4.8 (0.1) 4.9 (0.1) 4.8 (0.1) 4.7 (0.1) 5.8 (0.3) 6.3 (0.3) 31.0 (0.4)* 2GAJE 3.3 (0.2) 3.7 (0.1) 3.7 (0.2) 3.6 (0.1) 7.5 (0.1) 7.0 (0.3) 31.5 (0.2) 3KSLA 5.4 (0.2) 5.3 (0.1) 4.4 (0.0) 4.4 (0.1) 6.1 (0.1) 6.2 (0.3) 30.7 (0.3)* 2LACH 5.0 (0.2) 5.4 (0.3) 4.7 (0.2) 4.6 (0.1) 5.4 (0.1) 5.9 (0.3) 31.3 (0.3)* 2NCBD 4.7 (0.1) 4.7 (0.2) 4.3 (0.1) 4.3 (0.1) 6.2 (0.2) 6.0 (0.2) 33.9 (0.2) 3NCME 4.8 (0.1) 5.0 (0.3) 4.8 (0.1) 4.9 (0.1) 5.9 (0.2) 5.9 (0.4) 32.2 (0.7) 3NMLC 5.3 (0.2) 5.1 (0.3) 4.9 (0.1)* 4.7 (0.1) 6.1 (0.2) 6.4 (0.2) 29.3 (0.3)* 2

SCEK 4.3 (0.2) 4.4 (0.4) 4.9 (0.0) 4.9 (0.0) 6.7 (0.2) 6.9 (0.2) 30.7 (0.3)* 3TXPL 5.8 (0.1) 5.7 (0.1) 4.8 (0.1) 4.6 (0.1) 6.4 (0.3) 6.5 (0.2) 33.0 (0.4)* 3
Monsanto Company 12-CT-244U 

Table G-16. Study 2 - Individual Site Phenotypic Comparison – Boll and Fiber Characteristics - of MON 

Dicamba and Glufosinate Herbicides Compared to the Conventional Control (continued) 

Fiber Uniformity (%) Fiber Length (cm)

MON 88701 Control MON 88701 Control

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR 83.9 (0.5) 84.6 (0.1) 2.8 (0.0) 2.9 (0.0) ARTI 83.5 (0.2) 83.6 (0.1) 2.8 (0.0) 2.8 (0.0) GACH 84.3 (0.6) 83.4 (0.4) 2.8 (0.1) 2.8 (0.0) GAJE 82.6 (1.1) 82.5 (0.6) 2.8 (0.0) 2.8 (0.1) KSLA 84.3 (0.3)* 83.3 (0.1) 3.1 (0.0) 3.0 (0.0) LACH 83.7 (0.3) 83.4 (0.4) 2.8 (0.0) 2.8 (0.0) NCBD 84.4 (0.4) 84.8 (0.6) 2.8 (0.0) 2.9 (0.0) NCME 83.6 (0.2) 83.2 (0.6) 2.7 (0.0) 2.8 (0.1) NMLC 81.4 (0.6) 81.1 (0.7) 2.7 (0.0) 2.7 (0.1) 

SCEK 82.5 (0.5)* 83.9 (0.1) 2.7 (0.0)* 2.8 (0.0) TXPL 84.2 (0.4) 83.9 (0.3) 2.8 (0.1) 2.8 (0.0) 

* Indicates a statistically significant difference (

α = 0.05).between MON 88701 and control (n = 4). 

1 Measure of fiber fineness and maturity (expressed in dimensionless micronaire units). 2 SE = Standard error. 
Monsanto Company 12-CT-244U 

Table G-17. Study 2 - Individual Site Phenotypic Comparison – Plant Mapping - of MON 88701 Not Trea

Glufosinate Herbicides Compared to the Conventional Control 

Phenotypic Characteristic (units)

Total Mainstem Nodes 

 (#) 

Nodes to first fruiting 

branch (#) 

Total bolls1

(# per plant) 

Total first-position bolls 

(# per plant) 

MON 88701 Control MON 88701 Control MON 88701 Control MON 88701 Control M

Site Mean (SE)2 Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) ARPR 16.4 (0.4) 16.9 (0.5) 6.1 (0.1) 6.3 (0.2) 6.2 (0.4) 5.9 (0.6) 3.3 (0.1) 3.1 (0.3) 

ARTI 20.9 (0.7)* 19.8 (0.2) 5.4 (0.1) 5.4 (0.2) 11.3 (1.2) 9.7 (0.9) 7.2 (1.0) 6.1 (0.4) GACH 21.6 (0.6) 20.9 (0.4) 6.5 (0.5)* 7.9 (0.5) 9.9 (0.4) 9.4 (1.3) 4.1 (0.5) 3.2 (0.5) GAJE 17.7 (0.4) 17.5 (0.7) 5.0 (0.2)* 5.8 (0.2) 9.0 (0.6) 10.1 (0.6) 5.8 (0.2) 5.9 (0.3) KSLA 18.9 (0.4) 19.4 (0.6) 3.8 (0.1) 4.0 (0.3) 19.1 (0.2)* 20.6 (0.3) 4.7 (0.4) 5.2 (0.3) 

LACH 20.9 (0.4) 22.8 (1.4) 7.1 (0.6) 6.9 (0.4) 7.1 (0.8) 4.9 (1.2) 2.9 (0.5) 2.8 (0.6) 

NCBD 15.0 (0.4) 14.8 (0.6) 4.2 (0.1) 4.3 (0.2) 9.8 (0.4) 8.9 (0.5) 6.3 (0.5) 5.5 (0.3) NCME 14.5 (0.5) 15.2 (0.2) 4.5 (0.1) 4.6 (0.1) 6.8 (0.7)* 4.2 (0.2) 3.5 (0.3)* 2.5 (0.1) NMLC 19.6 (0.2) 18.9 (0.3) 4.8 (0.5) 5.0 (0.4) 6.7 (0.7)* 4.5 (0.5) 4.4 (0.3)* 3.0 (0.4) SCEK 15.8 (0.2) 15.8 (0.2) 4.6 (0.2) 4.6 (0.1) 9.7 (0.4) 10.0 (0.6) 6.9 (0.3) 6.3 (0.3) TXPL 18.3 (0.2) 18.2 (0.3) 5.7 (0.1) 5.9 (0.2) 12.4 (0.5) 11.3 (0.4) 7.8 (0.2)* 7.1 (0.1) 
Monsanto Company 12-CT-244U 

Table G-17. Study 2 - Individual Site Phenotypic Comparison – Plant Mapping - of MON 88701 Not Treate

Glufosinate Herbicides Compared to the Conventional Control (continued)

Phenotypic Characteristic (units)

% First-position boll 

retention 

% First-position bolls over 

total bolls

MON 88701 Control MON 88701 Control

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR 33.0 (1.9) 30.3 (3.7) 56.6 (3.6) 56.0 (4.4)

ARTI 45.4 (4.9) 42.3 (2.8) 63.9 (2.1) 66.8 (2.9)GACH 26.8 (2.0) 23.0 (2.6) 42.7 (5.0) 36.6 (3.2)GAJE 45.1 (1.5) 52.6 (4.9) 67.4 (2.3)* 61.8 (1.9)KSLA 32.2 (2.0) 34.0 (2.4) 24.8 (1.9) 24.9 (1.3)LACH 20.9 (2.8) 18.4 (4.8) 48.8 (1.0) 55.8 (1.9)NCBD 58.8 (4.3) 52.5 (0.9) 66.5 (4.5) 64.5 (4.0)NCME 35.6 (3.3)* 23.9 (0.8) 53.9 (2.7) 58.4 (1.7)NMLC 30.1 (2.1)* 21.3 (3.3) 69.5 (3.0) 65.8 (2.8)SCEK 62.2 (2.6) 57.0 (2.5) 73.2 (3.5)* 65.7 (1.9)TXPL 72.5 (1.4) 69.6 (1.7) 65.8 (2.0) 64.9 (2.2)

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and conventional control (n = 4). 

1 Total bolls = number of first-position bolls + number of second-position bolls + number of vegetative bolls. 2 SE = Standard error. 
Monsanto Company 12-CT-244U 

Table G-18. Study 2 – Combined-Site Phenotypic Comparison – Plant Mapping - of MON 88701 TreatGlufosinate Herbicides Compared to the Conventional Control 

Phenotypic Characteristic (units)

MON 88701 Control Refer

Mean (SE)2 Mean (SE) Minimum 

Mainstem Nodes (# per plant) 18.3 (0.3) 18.2 (0.4) 16.0 

Nodes to First Fruiting Branch (# per plant) 5.2 (0.1) 5.5 (1.1) 4.2 

Total Bolls (# per plant)3 10.1 (0.6) 9.0 (0.7) 8.6 

Total First-Position Bolls (# per plant) 5.2 (0.3)* 4.6 (0.3) 2.9 

Total Vegetative Bolls (# per plant) 2.1 (0.6) 1.9 (0.6) 0.7 % Retention First-Position Bolls (per plant) 41.7 (2.3) 38.6 (2.6) 21.2 

% First-Position Bolls of Total Bolls (per plant) 56.7 (2.1) 56.5 (2.1) 36.0 

*Indicates a statistically significant difference (

α = 0.05).between the test and control (n = 44). 1 Reference range = Minimum and maximum mean values among the commercial conventional reference varieties. 

2 SE = Standard error. 3 Total Bolls = number of first-position bolls + number of second-position bolls + number of vegetative bolls.
Monsanto Company 12-CT-244U 

Table G-19. Study 2 - Individual Site Phenotypic Comparison – Plant Mapping - of MON 88701 TreatGlufosinate Herbicides Compared to the Conventional Control 

Phenotypic Characteristic (units)

Total Mainstem Nodes 

 (#) 

Nodes to first fruiting 

branch (#) 

Total bolls1

(# per plant) 

Total first-position bolls

(# per plant) 

MON 88701 Control MON 88701 Control MON 88701 Control MON 88701 Control 

Site Mean (SE)2 Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) Mean (SE) ARPR 16.3 (0.4) 16.9 (0.5) 6.0 (0.0) 6.3 (0.2) 5.7 (0.3) 5.9 (0.6) 3.4 (0.4) 3.1 (0.3) 

ARTI 19.7 (0.1) 19.8 (0.2) 5.2 (0.1) 5.4 (0.2) 11.0 (0.3) 9.7 (0.9) 6.8 (0.3) 6.1 (0.4) GACH 21.2 (0.9) 20.9 (0.4) 6.5 (0.5)* 7.9 (0.5) 8.7 (1.6 ) 9.4 (1.3) 3.8 (0.8) 3.2 (0.5) GAJE 17.0 (0.2) 17.5 (0.7) 5.1 (0.1)* 5.8 (0.2) 9.5 (1.1) 10.1 (0.6) 5.5 (0.4) 5.9 (0.3) KSLA 19.6 (0.7) 19.4 (0.6) 3.7 (0.1) 4.0 (0.3) 20.1 (0.7) 20.6 (0.3) 4.8 (0.3) 5.2 (0.3) LACH 21.4 (0.8) 22.8 (1.4) 6.4 (0.4) 6.9 (0.4) 7.3 (1.1) 4.9 (1.2) 3.8 (0.5) 2.8 (0.6) NCBD 16.5 (0.7)* 14.8 (0.6) 4.5 (0.2) 4.3 (0.2) 10.2 (1.0) 8.9 (0.5) 6.5 (0.4)* 5.5 (0.3) NCME 15.1 (0.3) 15.2 (0.2) 4.8 (0.1) 4.6 (0.1) 7.1 (0.4)* 4.2 (0.2) 3.4 (0.3)* 2.5 (0.1) NMLC 19.4 (0.2) 18.9 (0.3) 4.8 (0.4) 5.0 (0.4) 7.7 (0.6)* 4.5 (0.5) 4.7 (0.2)* 3.0 (0.4) SCEK 16.1 (0.4) 15.8 (0.2) 4.6 (0.1) 4.6 (0.1) 9.4 (0.3) 10.0 (0.6) 6.5 (0.3) 6.3 (0.3) TXPL 18.5 (0.7) 18.2 (0.3) 5.3 (0.2) 5.9 (0.2) 14.3 (1.8)* 11.3 (0.4) 8.1 (0.2)* 7.1 (0.1) 
Monsanto Company 12-CT-244U 

Table G-19. Study 2 - Individual Site Phenotypic Comparison – Plant Mapping - of MON 88701 TreatGlufosinate Herbicides Compared to the Conventional Control (continued) 

Phenotypic Characteristic (units)

% Retention firstposition bolls (per plant) % First-position bolls of total bolls (per plant) 2

MON 88701 Control MON 88701 Control

Site Mean (SE) Mean (SE) Mean (SE) Mean (SE)ARPR 33.5 (3.7) 30.3 (3.7) 61.4 (2.6) 56.0 (4.4)

ARTI 46.7 (2.0) 42.3 (2.8) 61.6 (1.5) 66.8 (2.9)GACH 25.2 (4.2) 23.0 (2.6) 45.4 (3.7)* 36.6 (3.2)GAJE 45.6 (2.8) 52.6 (4.9) 61.4 (2.0) 61.8 (1.9)KSLA 32.0 (2.8) 34.0 (2.4) 24.5 (1.6) 24.9 (1.3)LACH 25.5 (2.0) 18.4 (4.8) 57.5 (5.2) 55.8 (1.9)NCBD 54.5 (1.4) 52.5 (0.9) 65.2 (3.1) 64.5 (4.0)NCME 33.7 (2.4)* 23.9 (0.8) 50.5 (3.3) 58.4 (1.7)NMLC 32.2 (1.5)* 21.3 (3.3) 64.8 (4.6) 65.8 (2.8)SCEK 57.5 (3.0) 57.0 (2.5) 70.0 (2.7) 65.7 (1.9)TXPL 72.1 (2.3) 69.6 (1.7) 61.0 (6.3) 64.9 (2.2)

* Indicates a statistically significant difference (

α = 0.05) between MON 88701 and conventional control (n = 4). 

1 Total bolls = number of first-position bolls + number of second-position bolls + number of vegetative bolls. 2 SE = Standard error. 
Monsanto Company 12-CT-244U 579 of 620 

Table G-20 Study 1 – Qualitative Assessment of Plant Response to Abiotic Stressors 

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared to 

the Conventional Control 

 

Note 1: The experimental design was a randomized complete block with four replications. Data were 

not subjected to statistical analysis. No differences were observed between MON 88701 and the 

conventional control during any observation of plant response to abiotic stressors. 

Abiotic Stressor 

Number of observations 

across all sites 

Number of observations where 

no differences were observed 

between MON88701 and the 

conventional control 

Total 169 169 

Compaction 4 4 

Drought/ Dry 40 40 

Flood 1 1 

Hail 6 6 

Heat 46 46 

Nutrient deficiency 22 22 

Wet soil/excess precipitation 17 17 

Wind damage 33 33 
Monsanto Company 12-CT-244U 580 of 620 

Table G-21. Study 1 – Qualitative Assessment of Disease Damage - MON 88701 Not 

Treated with Dicamba or Glufosinate Herbicides Compared to the Conventional 

Control 

Disease 

Number of 

observations 

across all sites 

Number observations where no 

differences were observed between 

MON 88701 and the conventional control 

 

Total 170 170 

Anthracnose 2 2 

Ascochyta leaf blight 2 2 

Bacterial blight 23 23 

Boll rot 26 26 

Cotton leaf rust 13 13 

Damping off 1 1 

Fusarium wilt 14 14 

Leaf spots1

 43 43 

Pythium 11 11 

Reniform nematode 1 1 

Rhizoctonia 16 16 

Root-knot nematode 6 6 

Thielaviopsis 1 1 

Verticillium wilt 11 11 

 

Note 1: The experimental design was a randomized complete block with four replications. Data were 

not subjected to statistical analysis. No differences were observed between MON 88701 and the 

conventional control during any observation of plant response to abiotic stressors. 

1

includes Septoria

 

 
Monsanto Company 12-CT-244U 581 of 620 

Table G-22. Study 1 – Qualitative Assessment of Arthropod-related Damage - 

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared to the 

Conventional Control 

Arthropod 

Number of 

observations 

across all sites 

Number observations 

where no differences 

were observed between 

MON 88701 and the 

conventional control 

Total 159 159 

Aphids (Aphididae) 31 31 

Beet armyworms (Spodoptera exigua) 2 2 

Cut worms (Noctuidae) 1 1 

Fall armyworms (Spodoptera frugiperda) 4 4 

Fleahoppers (Pseudatomoscelis seriatus) 4 4 

Grasshoppers (Acrididae) 8 8 

Heliothines (Helicoverpa zea and 

Heliothis virescens) 25 25 

Southern corn rootworm beetles 

(Diabrotica undecimpunctata howardi) 3 3 

Soybean loopers (Pseudoplusia inclunes) 2 2 

Spider mites (Tetranychus spp.) 9 9 

Stink bugs (Pentatomidae) 28 28 

Tarnished plant bugs (Lygus lineolaris) 21 21 

Thrips (Thripidae) 16 16 

White flies (Bemisia spp.) 5 5 

Note 1: The experimental design was a randomized complete block with four replications. Data were 

not subjected to statistical analysis. No differences were observed between MON 88701 and the 

conventional control during any observation of plant response to abiotic stressors. 
Monsanto Company 12-CT-244U 582 of 620 

Table G-23. Study 1 - Individual Site Analysis: Quantitative Assessment of Thrips 

Damage - MON 88701 Not Treated with Dicamba or Glufosinate Herbicides 

Compared to the Conventional Control 

Rating1 Site MON 88701 (SE)2 Control (SE) 

 

1 

ARAU 0.0 (0.0) †

 0.0 (0.0) 

ARPR 0.9 (0.1) 0.8 (0.1) 

GACH 0.0 (0.0) †

 0.0 (0.0) 

LABU 0.0 (0.0) †

 0.0 (0.0) 

SCEK 1.5 (0.2) 1.4 (0.2) 

2 

ARAU 0.1 (0.0) 0.1 (0.0) 

ARPR 0.3 (0.1) 0.2 (0.1) 

GACH 0.0 (0.0) †

 0.0 (0.0) 

LABU 0.0 (0.0) †

 0.0 (0.0) 

SCEK 0.0 (0.0) †

 0.0 (0.0) 

3 

ARAU 0.0 (0.0) †

 0.0 (0.0) 

ARPR 0.1 (0.0)* 0.3 (0.1) 

GACH 0.0 (0.0) †

 0.0 (0.0) 

LABU 0.0 (0.0) †

 0.0 (0.0) 

SCEK 0.0 (0.0) †

 0.0 (0.0) 

 

* Indicates a statistically significant difference (α = 0.05) between MON 88701 and conventional control 

(n = 4). 

† No p-values were generated due to lack of variability in the data.

1 Thrips damage observation # 1 was made at approximately 14 DAP and the two subsequent observations at 

approximately seven day intervals thereafter. 

2 SE = Standard error. 
Monsanto Company 12-CT-244U 

Table G-24. Study 1 - Individual Site Analysis: Quantitative Assessment of Heliothine Damage - MON with Dicamba or Glufosinate Herbicides Compared to the Conventional Control 

 Percent Damaged Fruiting Bodies # of Live La

Observation1 Site MON 88701 (SE)2 Control (SE) MON 88701 (SE) 1 

ARAU 3.8 (1.0) 2.0 (0.7) 0.0 (0.0) 

ARPR 0.0 (0.0) 0.1 (0.1) 0.0 (0.0) 

GACH 9.2 (2.0) 6.8 (2.5) 0.5 (0.2)* 

LABU 1.0 (0.6) 0.3 (0.3) 0.0 (0.0) 

SCEK 0.0 (0.0) 0.0 (0.0) 0.0 (0.0) 

2 

ARAU 0.2 (0.2) 0.6 (0.6) 0.0† (0.0) 

ARPR 1.6 (0.3) 2.2 (0.4) 0.1 (0.1) 

GACH 0.8 (0.5) 0.4 (0.4) 0.0 (0.0) 

LABU 2.9 (1.7) 2.6 (0.9) 0.1 (0.1) 

SCEK 21.2 (13.3) 26.0 (9.2) 0.6 (0.4) 

3 

ARAU 2.5 (1.0) 1.4 (0.6) 0.0 (0.0) 

ARPR 4.9 (0.6) 4.9 (1.4) 0.3 (0.1) 

GACH 4.1 (2.0) 3.3 (1.4) 0.1 (0.0) 

LABU 4.7 (1.1) 2.9 (0.9) 0.1 (0.1) 

SCEK 2.3 (2.3) 0.6 (0.6) 0.0 (0.0) 

4 

ARAU 8.7 (1.5)* 15.1 (2.4) 0.0 (0.0) 

ARPR 0.0† (0.0) 0.0 (0.0) 0.0† (0.0) 

GACH 1.5 (0.5) 2.8 (1.3) 0.0 (0.0) 

LABU 20.0 (3.8) 16.4 (2.4) 0.4 (0.1) 

SCEK 1.2 (0.5) 0.4 (0.4) 0.0 (0.0) 

* Indicates a statistically significant difference (α = 0.05) between MON 88701 and the conventional control (n = 4).

1 Heliothine damage observation 1 was made at approximately 45 DAP and the two subsequent observations at approximately 15 day intervals thereaf

2 SE = Standard Error. 
Monsanto Company 12-CT-244U 

Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or GlCompared to the Conventional Control  Pest Arthropod Aphids (Aphididae) Cabbage loopers (Trichoplusia ni) Fall armywormColl.1 Site MON 88701 (SE)2 Control (SE) Reference range MON 88701 (SE) Control (SE) Referencer ange MON 88701 (SE) 1 

ARAU 

−

−

− 2.0 (0.9) 2.3 (0.5) 0.8 – 1.8 

−

ARPR 0.0 (0.0) 0.0 (0.0) 0.0 – 0.5 

−

−

−

−

GACH 3358.0 (656.5) 1971.8 (419.3) 1193.5 – 5796.0 

−

−

−

−

LABU 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 1.3 (0.6) 2.0 (0.4) 0.5 – 1.8 

−

SCEK 27.3 (8.2) 20.0 (6.4) 10.3 – 31.3 

−

−

−

−

2 

ARAU 36.8 (8.4) 30.0 (4.6) 30.0 – 144.3 

−

−

−

−

ARPR 24.3 (10.0) 17.3 (5.6) 16.0 – 30.3 1.3 (0.6) 1.0 (0.4) 0.5 – 1.5 

−

GACH 6.8 (1.6) 2.5 (0.9) 2.5 – 4.5 

−

−

−

−

LABU 54.3 (12.1) 51.0 (20.1) 70.0 – 156.0 

−

−

−

−

SCEK 4713.3 (1424.1) 7440.0 (1117.1) 994.0 – 6840.0 

−

−

−

−

3 

ARAU 6.0 (1.7) 10.3 (3.7) 8.0 – 23.0 5.3 (2.3) 3.0 (1.1) 1.8 – 6.0 

−

ARPR 15.8 (8.8) 19.0 (10.5) 12.3 – 15.3 0.0 (0.0) 0.3 (0.3) 0.0 – 0.0 

−

GACH 19.3 (6.5) 19.3 (6.6) 8.3 – 10.3 3.8 (1.6) 1.3 (1.0) 1.3 – 3.8 1.5 (1.5) 

LABU 4.8 (1.1) 10.0 (3.2) 3.8 – 18.0 0.3 (0.3) 0.3 (0.3) 0.0 – 0.8 

−

SCEK 6.3 (3.0) 8.5 (1.9) 2.0 – 10.5 0.8 (0.5) 2.0 (1.2) 0.5 – 2.5 

−

4 

ARAU 4.8 (1.3) 5.8 (1.5) 1.5 – 15.8 0.8 (0.3) 0.8 (0.5) 0.0 – 0.5 

−

ARPR 3.5 (0.9) 5.5 (1.2) 1.3 – 5.0 

−

−

−

−

GACH 2.0 (1.3) 4.3 (2.3) 0.8 – 4.5 

−

−

−

−

LABU 1959.3 (303.3) 1993.8 (492.9) 795.3 – 2218.5 

−

−

−

−

SCEK 130.0 (15.9) 145.5 (38.0) 70.0 – 183.8 

−

−

−

−
Monsanto Company 12-CT-244U 

Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or GlCompared to the Conventional Control (continued)  Pest Arthropod

Fleahoppers (Pseudatomoscelis seriatus)

Heliothines (Helicoverpa zea and 

Heliothis virescens) 

Southern Ar

Coll. Site MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) 1 

ARAU 

−

−

−

−

−

−

−

ARPR 

−

−

−

−

−

−

−

GACH 0.5 (0.3) 0.5 (0.5) 0.0 – 0.5 0.3 (0.3) 0.8 (0.5) 0.0 – 0.3 

−

LABU 

−

−

−

−

−

− 0.3 (0.3) 

SCEK 

−

−

−

−

−

−

−

2 

ARAU 

−

−

− 0.8 (0.5) 0.5 (0.3) 0.3 – 1.0 

−

ARPR 

−

−

− 2.5 (1.0) 2.0 (0.9) 1.3 – 3.5 

−

GACH 

−

−

− 0.3 (0.3) 0.3 (0.3) 0.0 – 1.3 

−

LABU 0.5 (0.5) 0.0 (0.0) 0.3 – 0.8 1.3 (0.6) 0.8 (0.5) 0.3 – 1.5 

−

SCEK 3.3 (1.0) 4.8 (3.0) 1.5 – 4.5 11.8 (4.6) 6.5 (2.7) 9.5 – 18.8 

−

3 

ARAU 

−

−

− 0.8 (0.5) 2.0 (0.9) 0.3 – 2.3 

−

ARPR 

−

−

− 0.3 (0.3) 0.8 (0.5) 0.3 – 2.5 

−

GACH 

−

−

− 0.5 (0.5) 1.3 (1.3) 0.0 – 0.5 

−

LABU 

−

−

− 1.5 (0.5) 0.8 (0.5) 0.5 – 1.8 

−

SCEK 6.0 (1.8) 6.0 (1.6) 2.8 – 9.5 1.3 (0.5) 0.8 (0.3) 0.5 – 1.5 

−

4 

ARAU 

−

−

− 1.0 (0.7) 0.5 (0.3) 0.0 – 0.5 

−

ARPR 

−

−

−

−

−

−

−

GACH 0.3 (0.3) 0.0 (0.0) 0.0 – 0.0 

−

−

−

−

LABU 

−

−

− 0.0 (0.0) 0.3 (0.3) 0.0 – 0.8 

−

SCEK 2.5 (0.9) 2.3 (1.0) 1.0 – 4.8 0.3 (0.3) 0.0 (0.0) 0.0 – 0.3 

−
Monsanto Company 12-CT-244U 

Table G-25. Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or GlCompared to the Conventional Control (continued)  Pest Arthropod  Stink bugs (Pentatomidae) Tarnished plant bugs (Lygus lineolaris) ThripColl. Site MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) 1 

ARAU 

−

−

−

−

−

− 4.3 (1.7) 

ARPR 

−

−

− 2.5 (0.9) 0.5 (0.3) 0.0 – 1.0 96.8 (31.9) 

GACH 

−

−

−

−

−

− 5.0 (1.6) 

LABU 

−

−

−

−

−

− 0.3 (0.3) 

SCEK 

−

−

−

−

−

−

−

2 

ARAU 0.8 (0.5) 0.3 (0.3) 0.0 – 1.5 1.0 (0.4) 0.8 (0.5) 0.0 – 1.5 

−

ARPR 

−

−

− 1.8 (0.8) 3.3 (1.1) 2.0 – 4.0 24.0 (6.4) 

GACH 

−

−

− 0.3 (0.3) 1.5 (0.7) 0.5 – 1.0 1.3 (0.5) 

LABU 

−

−

− 1.3 (0.6) 0.8 (0.3) 0.5 – 0.8 1.0 (0.7) 

SCEK 

−

−

−

−

−

− 13.8 (2.8) 

3 

ARAU 

−

−

− 0.5 (0.5) 1.8 (0.9) 0.3 – 0.5 

−

ARPR 

−

−

− 0.0 (0.0) 0.3 (0.3) 0.3 – 2.0 2.8 (2.1) 

GACH 

−

−

−

−

−

− 24.0 (8.8) 

LABU 

−

−

−

−

−

− 1.0 (0.4) 

SCEK 

−

−

−

−

−

− 3.5 (1.8) 

4 

ARAU 1.8 (0.9) 1.3 (0.6) 0.3 – 1.5 

−

−

− 2.0 (1.4) 

ARPR 0.5 (0.3) 0.3 (0.3) 0.5 – 1.3 1.3 (1.3) 0.3 (0.3) 0.8 – 1.5 8.3 (1.7) 

GACH 

−

−

−

−

−

− 1.3 (1.0) 

LABU 0.3 (0.3)* 1.8 (0.5) 0.5 – 1.0 0.5 (0.5)* 2.0 (0.6) 0.5 – 2.0 1.5 (1.2) 

SCEK 

−

−

−

−

−

− 11.0 (3.5) 
Monsanto Company 12-CT-244U 

Table G-25 Study 1 - Abundance of Pest Arthropods - MON 88701 Not Treated with Dicamba or GlCompared to the Conventional Control (continued) 

 White flies (Bemisia spp.)

Coll. Site MON 88701 (SE) Control (SE) Reference range 1 

ARAU 

−

−

−

ARPR 0.0 (0.0) 0.3 (0.3) 0.0 – 0.5 

GACH 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 

LABU 

−

−

−

SCEK 

−

−

−

2 

ARAU 0.5 (0.3) 0.3 (0.3) 0.5 – 1.5 

ARPR 

−

−

−

GACH 1.3 (0.3) 0.5 (0.3) 1.3 – 2.3 

LABU 

−

−

−

SCEK 0.5 (0.5) 0.5 (0.5) 0.0 – 0.5 

3 

ARAU 0.0 (0.0) 0.3 (0.3) 0.0 – 0.5 

ARPR 

−

−

−

GACH 

−

−

−

LABU 

−

−

−

SCEK 

−

−

−

4 

ARAU 

−

−

−

ARPR 2.0 (0.7) 3.0 (1.4) 0.8 – 3.0 

GACH 1.5 (0.7) 0.3 (0.3) 0.3 – 1.0 

LABU 

−

−

−

SCEK 1.3 (1.0) 0.5 (0.3) 0.0 – 0.8 

Note: A dash (-) indicates arthropod not evaluated. * Indicates a statistically significant difference (

α = 0.05) between MON 88701 and the conventional control (n = 4). 

1 Arthropod collection 1 was made at approximately 30 DAP and the three subsequent collections at approximately 30 day intervals ther2 SE = Standard error. 
Monsanto Company 12-CT-244U 

Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 Not Treated with DicaHerbicides Compared to the Conventional Control  Beneficial Arthropod Big eyed bugs (Geocoris spp.) Braconids (Braconidae) Damsel bugColl.1 Site MON 88701 (SE)2 Control (SE) Reference range MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) Con1 

ARAU 

−

−

−

−

−

− 1.3 (0.5) 1.

ARPR 2.3 (0.6) 0.5 (0.5) 0.8 – 2.3 

−

−

− 0.5 (0.5) 1.

GACH 

−

−

−

−

−

− 0.8 (0.5) 0.

LABU 

−

−

− 0.0 (0.0) 0.0 (0.0) 0.0 – 0.3 

−

SCEK 0.3 (0.3) 0.0 (0.0) 0.3 – 1.0 

−

−

−

−

2 

ARAU 0.3 (0.3) 0.0 (0.0) 0.0 – 1.3 

−

−

−

−

ARPR 1.0 (0.4) 1.5 (0.5) 0.3 – 0.8 

−

−

−

−

GACH 10.5 (1.3) 11.8 (1.9) 6.8 – 16.5 

−

−

− 6.0 (2.0)* 2.

LABU 1.5 (0.7) 2.3 (1.3) 0.5 – 3.3 

−

−

− 0.5 (0.3) 0.

SCEK 0.8 (0.5) 1.3 (0.5) 1.0 – 3.3 

−

−

−

−

3 

ARAU 0.0 (0.0) 0.3 (0.3) 0.0 – 1.3 

−

−

−

−

ARPR 

−

−

−

−

−

−

−

GACH 1.0 (0.4) 2.5 (0.9) 1.5 – 4.5 

−

−

− 3.3 (1.0) 1.

LABU 

−

−

−

−

−

−

−

SCEK 7.0 (0.7) 6.0 (2.0) 5.0 – 13.0 

−

−

−

−

4 

ARAU 1.5 (0.5) 2.5 (0.7) 1.5 – 3.5 

−

−

− 1.8 (0.5) 3.

ARPR 7.5 (3.0) 4.0 (0.7) 3.0 – 7.5 

−

−

−

−

GACH 5.0 (1.6) 5.5 (3.0) 1.3 – 8.5 

−

−

− 3.3 (0.8) 4.

LABU 

−

−

− 0.5 (0.5) 2.0 (1.4) 3.0 – 8.8 

−

SCEK 27.8 (9.4) 35.0 (4.8) 16.5 – 44.3 

−

−

−

−
Monsanto Company 12-CT-244U 

Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 Not Treated with DicaHerbicides Compared to the Conventional Control (continued)  Beneficial Arthropod Lacewings (Chrysopa spp.and Hemerobius

spp.) Ladybird Beetles (Coccinellidae) 

Coll. Site MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) Control (SE) Reference range MON 88701 (SE) C1 

ARAU 

−

−

− 0.5 (0.3) 0.5 (0.3) 0.3 – 0.8 0.0 (0.0) 

ARPR 

−

−

− 2.3 (0.5) 3.3 (1.1) 1.0 – 2.5 1.8 (1.1) 

GACH 0.8 (0.8) 0.3 (0.3) 0.3 – 0.5 14.3 (4.6) 12.3 (3.9) 6.0 – 43.8 0.8 (0.5) 

LABU 

−

−

− 0.5 (0.5) 0.3 (0.3) 0.0 – 0.8 

−

SCEK 

−

−

− 0.5 (0.3) 0.8 (0.3) 0.3 – 1.3 

−

2 

ARAU 3.0 (0.4) 3.5 (0.9) 2.3 – 5.0 1.0 (0.4) 1.0 (0.7) 1.0 – 2.0 0.0 (0.0)* 

ARPR 2.5 (0.7) 3.0 (1.3) 1.8 – 4.0 9.0 (2.5) 4.8 (0.3) 5.5 – 8.5 1.0 (0.6) 

GACH 0.3 (0.3) 1.0 (0.4) 1.0 – 2.0 10.8 (2.3) 10.0 (1.4) 3.3 – 12.3 

−

LABU 

−

−

− 8.5 (1.4) 9.5 (2.4) 8.0 – 12.5 0.3 (0.3) 

SCEK 3.3 (0.8) 2.5 (1.0) 1.8 – 3.5 34.8 (6.5) 46.3 (10.8) 18.3 – 36.3 8.5 (3.1) 

3 

ARAU 2.8 (1.3) 4.0 (0.7) 1.8 – 3.5 7.3 (3.2) 10.3 (2.7) 9.3 – 18.3 0.5 (0.3)* 

ARPR 1.0 (0.4) 1.5 (0.9) 0.5 – 1.5 6.5 (1.0) 6.0 (1.8) 5.5 – 8.0 0.3 (0.3) 

GACH 3.0 (1.8) 4.0 (1.2) 2.0 – 5.5 12.0 (3.2) 11.0 (2.4) 4.8 – 10.3 

−

LABU 

−

−

−

−

−

−

−

SCEK 0.3 (0.3) 1.0 (0.4) 0.3 – 0.8 7.8 (0.9) 5.0 (0.7) 6.3 – 8.3 2.0 (1.7) 

4 

ARAU 4.3 (1.6) 2.5 (0.5) 1.8 – 3.5 10.8 (2.5) 12.0 (1.7) 9.3 – 13.0 

−

ARPR 3.3 (1.1) 3.8 (1.3) 0.5 – 4.3 6.5 (1.6) 5.3 (1.0) 2.0 – 4.0 0.0 (0.0) 

GACH 0.0 (0.0) 0.0 (0.0) 0.3 – 0.5 1.3 (0.6) 2.0 (1.1) 3.3 – 7.0 

−

LABU 0.3 (0.3) 0.3 (0.3) 0.5 – 2.3 6.3 (3.3) 3.5 (1.3) 4.0 – 8.0 3.8 (3.1) 

SCEK 1.0 (0.4) 0.3 (0.3) 0.5 – 1.8 7.5 (0.3) 5.0 (2.4) 5.0 – 10.8 3.8 (0.5) 
Monsanto Company 12-CT-244U 

Table G-26. Study 1 - Abundance of Beneficial Arthropods - MON 88701 Not Treated with Dicamba or GHerbicides Compared to the Conventional Control (continued) 

 Spiders (Araneae) 

Coll. Site MON 88701 (SE) Control (SE) Reference range 1 

ARAU 1.0 (0.4) 1.0 (0.4) 0.3 – 1.3 

ARPR 1.5 (0.5) 1.0 (0.7) 0.8 – 1.8 

GACH 1.5 (0.9) 2.5 (0.7) 0.3 – 2.0 

LABU 0.3 (0.3) 1.0 (1.0) 0.3 – 1.3 

SCEK 0.5 (0.5) 0.8 (0.3) 0.5 – 1.0 

2 

ARAU 1.0 (0.4) 1.3 (0.5) 0.3 – 2.0 

ARPR 6.0 (1.3) 4.0 (0.4) 2.5 – 5.8 

GACH 4.5 (0.3) 6.0 (1.2) 4.3 – 6.8 

LABU 5.3 (1.5) 3.0 (1.3) 2.8 – 5.3 

SCEK 2.8 (0.5) 4.3 (1.3) 1.8 – 4.3 

3 

ARAU 2.0 (0.4) 2.0 (1.1) 0.8 – 1.8 

ARPR 0.3 (0.3) 0.0 (0.0) 0.0 – 0.5 

GACH 4.0 (0.4) 2.8 (0.5) 3.8 – 4.3 

LABU 

−

−

−

SCEK 4.3 (1.0) 2.8 (0.9) 3.5 – 7.8 

4 

ARAU 3.3 (1.0) 3.5 (0.9) 3.5 – 7.3 

ARPR 1.5 (0.3) 2.0 (1.0) 0.3 – 1.8 

GACH 3.0 (0.8) 5.8 (1.8) 3.5 – 6.0 

LABU 0.8 (0.5) 0.8 (0.5) 0.3 – 1.5 

SCEK 6.5 (1.3) 6.0 (0.8) 8.0 – 12.5 

Note: A dash (-) indicates arthropod not evaluated. * Indicates a statistically significant difference (

α = 0.05) between MON 88701 and the conventional control (n = 4). 

1 Arthropod collection 1 was made at approximately 30 DAP and the three subsequent collections at approximately 30 day intervals ther2 SE = Standard error. 
Monsanto Company 12-CT-244U 591 of 620 

Table G-27 Study 2 – Qualitative Assessment of Plant Response to Abiotic Stressors 

- MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared to 

the Conventional Control 

Abiotic Stressor 

Number of 

observations 

across all sites 

Number of observations where no 

differences were observed between 

MON 88701 and the control 

Total 127 127 

Compaction 4 4 

Drought/ Dry 30 30 

Flood 1 1 

Hail Damage 6 6 

Heat 30 30 

Nutrient deficiency 10 10 

Wet soil/excess precipitation 17 17 

Wind damage 29 29 

Note 1: The experimental design was a randomized complete block with four replications. Data were 

not subjected to statistical analysis. No differences were observed between MON 88701 and the 

conventional control during any observation of plant response to abiotic stressors. 
Monsanto Company 12-CT-244U 592 of 620 

Table G-28. Study 2 – Qualitative Assessment of Disease Damage of MON 88701 

Not Treated with Dicamba or Glufosinate Herbicides Compared to the 

Conventional Control 

Disease 

Number of observations 

across all sites 

Number of observations where no 

differences were observed between 

MON 88701 and the control 

 

Total 129 129 

Anthracnose 3 3 

Ascochyta leaf blight 3 3 

Bacterial blight 14 14 

Boll rot 15 15 

Cotton leaf rust 7 7 

Damping off 1 1 

Fusarium wilt 11 11 

Leaf spots1

 36 36 

Pythium 9 9 

Reniform nematode 1 1 

Rhizoctonia 12 12 

Root-knot nematode 6 6 

Thielaviopsis 1 1 

Verticillium wilt 10 10 

 

Note 1: The experimental design was a randomized complete block with four replications. Data were 

not subjected to statistical analysis. No differences were observed between MON 88701 and the 

conventional control during any observation of plant response to abiotic stressors 

1

 Includes Alternaria and Septoria 
Monsanto Company 12-CT-244U 593 of 620 

Table G-29. Study 2 – Qualitative Assessment of Arthropod-related Damage -

MON 88701 Not Treated with Dicamba or Glufosinate Herbicides Compared to the 

Conventional Control 

Arthropod 

Number of 

observations 

across all sites 

Number of 

observations where no 

differences were 

observed between 

MON 88701 and the 

control 

Total 129 129 

Aphids (Aphididae) 24 24 

Beet armyworms (Spodoptera exigua) 1 1 

Cabbage loopers (Trichoplusia ni) 1 1 

Cut worms (Noctuidae) 3 3 

Fall armyworms (Spodoptera frugiperda) 4 4 

Fleahoppers (Pseudatomoscelis seriatus) 2 2 

Grasshoppers (Acrididae) 6 6 

Heliothines (Helicoverpa zea and 

Heliothis virescens) 23 23 

Southern corn rootworm beetles 

(Diabrotica undecimpunctata howardi) 2 2 

Soybean loopers (Pseudoplusia inclunes) 1 1 

Spider mites (Tetranychus spp.) 9 9 

Stink bugs (Pentatomidae) 21 21 

Tarnished plant bugs (Lygus lineolaris) 14 14 

Thrips (Thripidae) 16 17 

White flies (Bemisia spp.) 2 2 

Note 1: The experimental design was a randomized complete block with four replications. Data were 

not subjected to statistical analysis. No differences were observed between MON 88701 and the 

conventional control during any observation of plant response to abiotic stressors. 

 
Monsanto Company 12-CT-244U 594 of 620 

References for Appendix G

Drees, B.M. and M.E. Rice. 1985. The vertical beat sheet: A new device for sampling 

soybeans insects. Journal of Economic Entomology 78:1507-1510. 

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina. 

 
Monsanto Company 12-CT-244U 595 of 620 

Appendix H: Materials and Methods for Pollen Morphology and Viability 

Assessment 

H.1. Plant Production 

MON 88701, the conventional control, and four commercial reference varieties were 

grown under similar agronomic conditions in a field trial in Crittenden County, Arkansas 

(Table G-1; ARPR site). The trial was arranged in a randomized complete-block design 

with four replications. Each plot consisted of eight rows approximately 6 m in length. 

H.2. Flower Collection and Sample Preparation 

Five flowers, each open less than 24 hours at the time of collection, were collected from 

each plot. The pollen obtained from an individual flower comprised a subsample of the 

plot and was placed in a uniquely labeled, clean container. Six hundred μl of 

Alexander’s stain (Alexander, 1980) diluted 1:5 with distilled water was added to each 

container, and the container contents were thoroughly mixed. Containers were placed on 

wet (water) ice within 10 minutes of pollen collection. After transport to the performing 

laboratory, the pollen in the containers was allowed to stain at ambient temperatures for 

at least 20 hours. 

H.3. Data Collection 

Pollen subsamples were assessed for pollen viability, diameter, and general morphology. 

Slides were prepared by aliquoting 30 μl of suspended pollen/stain solution onto a slide. 

The slides were viewed under an Olympus BX51TRF light/fluorescence microscope with 

an Olympus DP70 digital color camera. The associated PC computer had imaging 

software for diameter measurement (I-Pro Plus version 6.2.1.491© 1993-2007, Media 

Cybernetics, Inc.) and camera software (DP Controller 1. 2. 1.108 © 2001-2003, 

Olympus Optical Co., Ltd. and DP Manager version 1, 2, 1, 107 © 2001-2003, Olympus 

Optical Co., Ltd.). 

H.3.1. Pollen Viability 

To assess pollen viability, 77 or more pollen grains were evaluated under the 40X ocular 

lens (400X total magnification) for each subsample. When exposed to the staining 

solution, viable pollen grains stained purple because of the presence of vital cytoplasmic 

content, while dead pollen grains stained clear to light blue-green. In addition, viable 

pollen grains appeared round, whereas non-viable pollen grains appeared round to 

collapsed depending on the degree of hydration. 

H.3.2. Pollen Diameter 

Pollen diameter was measured under the 40X ocular lens (400X total magnification) 

using software (Image-Pro Plus version 6.2.1.491© 1993-2007 Media Cybernetics, Inc.) 
Monsanto Company 12-CT-244U 596 of 620 

to view digital images. For each replication, pollen diameter was measured along two 

perpendicular axes for ten representative viable pollen grains. 

H.3.3. General Pollen Morphology 

General morphology of the pollen was observed for each subsample of MON 88701, the 

conventional control, and the commercial reference varieties during determination of 

pollen viability. 

H.4. Statistical Analysis 

Monsanto Statistics Technology Center performed the statistical analysis. An analysis of 

variance was conducted according to a randomized complete block design using SAS

Version 9.2 (SAS, 2008) with a significance level of 5% (p≤0.05). MON 88701 was 

compared to the conventional control for percent viable pollen and pollen grain diameter. 

MON 88701 was not statistically compared to the reference varieties. A reference range 

for each measured characteristic was determined from the minimum and maximum mean 

values from among the four commercial reference varieties. General pollen morphology 

was qualitative; therefore, no statistical analysis was conducted on these observations. 

 

SAS is a registered trademark of SAS Institute, Inc. 
Monsanto Company 12-CT-244U 597 of 620 

Table H-1. Starting Seed for Pollen Morphology and Viability Assessment 

Material Phenotype Monsanto ID

 

Coker 130 Conventional 11268128

Nex Gen NG3410RF Glyphosate-tolerant 11266969

ST474 Conventional 11266156

DP 493 Conventional 11266763

SG125 Conventional 11266155

MON 88701 Dicamba and glufosinate-tolerant 11268129

 

 
Monsanto Company 12-CT-244U 598 of 620 

References for Appendix H

Alexander, M.P. 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain 

Technology 55:13-18. 

SAS. 2008. SAS/STAT software version 9.2. SAS Institute, Inc., Cary, North Carolina. 

 
Monsanto Company 12-CT-244U 599 of 620 

Appendix I: Herbicide Resistance 

I.1. Introduction 

Based upon theory of natural selection, plant populations can develop resistance to an 

herbicide due to the selection of individuals that carry specific genes that can render those 

individuals unaffected by the typical lethal effects of an herbicide. The application of an 

herbicide to the plant does not, itself, cause a mutation in subsequent generations. 

Rather, over time, those few plant biotypes containing resistant gene(s) become dominant 

in the population with repeated use of the herbicide in the absence of other control 

methods, such as use of other herbicides and/or use of cultural control methods. The 

development of resistant populations is a possibility for all herbicides. The probability 

for resistance to develop is a function of: frequency of resistant allele(s)8

, mechanism of 

resistance, dominance or recessive nature of the resistant allele(s), relative fitness of the 

resistant biotype, and frequency or duration of herbicide use in the absence of other 

control methods (Beckie, 2006; Jasieniuk, et al.,1996; Sammons et al., 2007). The 

probability of resistance is not the same for all herbicides, with some herbicides (e.g., 

ALS and ACCase classes) exhibiting resistance more quickly than other herbicides (e.g.

auxin class, glyphosate, dinitroanilines class). 

Herbicide resistance can become a limiting factor in crop production if the resistant weed 

population cannot be controlled with other herbicides or cultural practices. In general, 

this has not been the case for any herbicide. In most crops, there are multiple herbicide 

options for growers to use. However, good management practices to delay the 

development of herbicide resistance have been identified and are being actively promoted 

by the public and private sectors (HRAC, 2010) and are being implemented by growers. 

Monsanto considers product stewardship to be a fundamental component of customer 

service and business practices. Stewardship of the dicamba and glufosinate herbicides to 

preserve their usefulness for growers is an important aspect of Monsanto’s stewardship 

commitment. Although herbicide resistance may eventually occur in weed species when 

any herbicide is widely used, resistance can be postponed, contained, and managed 

through research, education, and good management practices. These are the key 

elements of Monsanto’s approach to providing stewardship of dicamba and glufosinate 

used on MON 88701 integrated into the glyphosate-tolerant cotton systems. Monsanto 

will invest in research, and grower/retailer education and training programs to provide 

information on best practices to manage dicamba and glufosinate weed resistance in 

cotton production. This appendix provides an overview of Monsanto’s approach to the 

development of best management practices to mitigate dicamba and glufosinate weed 

resistance. Monsanto works closely with weed scientists in academia and with other 

companies to research and develop best management practices and to uniformly 

communicate such practices to growers. Evidence of this cooperative effort is the recent 

development and posting of herbicide-resistant training modules on the WSSA website 

 

8 An allele is any of several forms of a gene, usually arising through mutation, that are responsible for 

hereditary variation.
Monsanto Company 12-CT-244U 600 of 620 

(www.wssa.net) and the publication of guidelines by the Herbicide Resistance Action 

Committee (HRAC) on their website (www.hracglobal.com). 

I.2. The Herbicide Dicamba 

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is classified as a benzoic acid herbicide 

belonging to the synthetic auxin group of herbicides (HRAC, 2010). The herbicides in 

this group act as growth regulators similar to endogenous indole acetic acid (IAA) but are 

structurally diverse. The synthetic auxin group includes five chemical families (benzoic 

acid, pyridine-carboxylic acid, quinoline carboxylic acid, phenoxy-carboxylic acid and a 

separate class which includes one herbicide, benazolin ethyl). The specific site of action 

among the different synthetic auxin chemical families may be different. In addition to 

dicamba, specific herbicides in this group include 2,4-D, 2,4-DB, mecoprop, MCPA, 

clopyralid, picloram, quinclorac and several other active ingredients. Dicamba and other 

synthetic auxin herbicides are classified in Herbicide Group 4 by the Weed Science 

Society of America (HRAC, 2009). Most herbicides in this group are active on broadleaf 

weeds only, but a few have significant activity on grasses, e.g., quinclorac. Dicamba 

provides preemergence and postemergence control of over 95 annual and biennial 

broadleaf weed species and control or suppression of over 100 perennial broadleaf and 

woody species (BASF, 2008). Dicamba is not active on grass weeds and is often used in 

combination with other herbicides to provide broad spectrum weed control. 

Dicamba herbicide was commercialized in the U.S. for agricultural use in 1967 and is 

currently labeled for preemergence and/or postemergence weed control in corn, soybean, 

cotton, sorghum, small grains (wheat, barley and oats), millet, pasture, rangeland, 

asparagus, sugarcane, turf, grass grown for seed, conservation reserve program land, and 

fallow cropland, and for non-crop uses (U.S. EPA, 2009). Dicamba is sold as standalone 

formulation which can be tank-mixed with one or more active ingredients depending 

upon the crop and the weed spectrum. Dicamba is also sold as a premix formulation with 

other herbicides. 

Dicamba acts in plants by mimicking naturally-occurring plant growth hormones called 

auxins, thereby destroying tissue through uncontrolled cell division and growth (Ahrens, 

1994). Ahrens (1994) further states that dicamba has been found to affect cell wall 

integrity and nucleic acid metabolism whereas in other cases it has been found to increase 

cell wall permeability, leading to cell enlargement. At low concentrations, dicamba has 

been found to increase synthesis of DNA, RNA, and proteins, resulting in altered cell 

division and growth. At high concentrations, inhibition of cell division and growth occur. 

In general, dicamba and other synthetic auxin herbicides have been found to affect 

multiple plant physiological systems. Grossmann (2010), in a review of auxin 

herbicides, outlined a proposed mechanism and mode-of-action for auxin herbicides and 

IAA at supraoptimal endogenous concentrations in dicot plant species. The proposal was 

based upon recent identification of receptors for auxins and hormone interaction in 

signaling between auxin, ethylene, and the upregulations of abscisic acid biosynthesis 

which would account for a large part of the various auxin-herbicide-mediated responses 

that are seen in sensitive dicots. In addition, research has indicated that there is a high 
Monsanto Company 12-CT-244U 601 of 620 

level of redundancy in auxin receptors which may account for the lack of development of 

widespread resistance to this herbicide group (Walsh et al., 2006). 

Dicamba is taken up by plants through the roots, stems, and foliage (Ahrens, 1994; NPIC, 

2002). Dicamba translocates to all plant tissues but accumulates in growing tissues. 

Translocation of dicamba is typically slower in tolerant plants such as grasses compared 

to broadleaf plants. 

I.3. The Herbicide Glufosinate 

Glufosinate [2-Amino-4-(hydroxymethylphosphinyl) butanoic acid] is classified as a 

phosphinic acid herbicide belonging to the glutamine synthetase inhibitor group of 

herbicides (HRAC, 2010). Bialaphos is the only other active ingredient belonging to the 

phosphinic acid chemical family. Glufosinate and bialaphos are classified in Herbicide 

Group 10 by the Weed Science Society of America (HRAC, 2010). Glufosinate provides 

postemergence control of over 90 annual grass and broadleaf weed species and 25 

biennial and perennial grass and broadleaf weed species. 

Glufosinate was first approved for use in the U.S. in 1994 (U.S. EPA, 2008) and is 

currently labeled for non-crop uses, preplant burndown to glufosinate-tolerant and nontolerant crops and/or in-crop postemergence weed control in glufosinate-tolerant canola, 

corn, cotton, and soybean, (Bayer CropScience, 2011). Glufosinate is sold as standalone 

formulation which can be tank mixed with one or more active ingredients depending 

upon the crop and the weed spectrum. 

Glufosinate acts in plants by inhibiting the enzyme glutamine synthase, causing a toxic 

buildup of ammonia within the treated plant (Bayer, 2010). Glufosinate is a nonselective 

herbicide and has no residual activity. This herbicide has a different mode-of-action than 

the other major herbicides used in cotton. 

I.4. Herbicide-Resistant Weeds and Resistance Management Strategies 

The development of herbicide-resistant weeds is not a new phenomenon and resistance is 

not limited to certain select herbicides. In 1957, the first U.S. herbicide-resistant weed, a 

spreading dayflower biotype resistant to 2,4-D, was identified in Hawaii (Heap, 2012a). 

See Table VIII-4 for scientific names of weeds mentioned in Appendix I. Through 

November 2011, there are approximately 80 individual weed species with known 

herbicide-resistant biotypes to one or more herbicides in the U.S. For example, there are 

45 weed species resistant to ALS herbicides, 16 to ACCase inhibitors, 24 to photosystem 

II inhibitors, and 13 to glycine herbicides (Heap, 2012b). Growers have been managing 

herbicide-resistant weeds for decades with the use of alternative herbicides and/or 

cultural methods such as tillage or crop rotation. 

The occurrence of an herbicide-resistant weed biotype does not end the useful lifespan or 

preclude the effective use of the herbicide as part of an overall diversified weed 

management system. The three herbicide classes with the highest number of resistant 

species, ALS, ACCase and triazine herbicides, are still effectively used by growers today. 
Monsanto Company 12-CT-244U 602 of 620 

It is important to distinguish herbicide resistance from herbicide tolerance. A herbicide 

resistant weed is one in which there is an inherited ability of a plant to survive and 

reproduce following exposure to a dose of herbicide normally lethal to the wild type 

(WSSA, 2012). A herbicide-tolerant weed species is one that is naturally tolerant to a 

herbicide, for example a grass species is not killed by the application of a broadleaf 

herbicide (WSSA, 2012). Furthermore, certain weed species, while neither resistant nor 

tolerant, are inherently difficult to control with a particular herbicide, requiring more 

careful herbicide use and weed management practices. 

Since the first confirmed cases of herbicide resistance, research has been directed at 

determining which practices are best for managing existing resistance situations and how 

best to reduce the development of herbicide resistance. Resistance management practices 

most often recommended by University/Cooperative Extension Service (CES) and 

industry are: 1) use of multiple herbicide modes-of-action in mixture, sequence, or in 

rotation; 2) crop rotation; 3) use of cultural control measures such as tillage and time of 

planting; and 4) use of the labeled herbicide rate at the recommended timing of 

application (Gressel and Segel, 1990; Beckie, 2006). Recent research by Beckie and 

Reboud (2009) indicates that in some cases herbicide mixtures offer a better management 

option than rotating herbicides. Simultaneously using two herbicides with different 

modes-of-action, each effective on the same weed species, significantly reduce the 

probability of weeds developing resistance to either or both herbicides (Beckie and 

Reboud, 2009). Crop rotation is also an effective method for resistance management due 

to the fact that it fosters the use of additional herbicide modes-of-action and, potentially, 

use of additional cultural practices to manage weeds over time. The use of multiple 

methods of weed control in a single location is the technical basis for management 

programs to delay the development of resistance. This general concept has been referred 

to as applying “diversity” within a crop or across a crop rotation (Beckie, 2006; Powles, 

2008). 

It is generally accepted that conservation tillage practices (minimum-till and no-till) 

create environments where herbicide resistance is more likely to develop (Beckie, 2006). 

This is primarily due to selection pressure put on weeds by herbicide use due to the 

absence of tillage as a cultural weed management practice to supplement herbicide use. 

However, this is not always the case. Legere et al. (2000) found that an increase in the 

use of ACCase inhibitors in a conservation tillage system (e.g., aryloxyphenoxy 

propionates and phenylpyrazolines herbicide families) did not result in an increased 

incidence of wild oat populations resistant to ACCase inhibitors. In conclusion, 

conservation tillage practices should not be considered a primary contributing factor to 

the development of resistance in all cases. 

I.5. Characteristics of Herbicides and Herbicide Use Influencing Resistance 

While the incidence of weed resistance is often associated with repeated applications of 

an herbicide, the actual probability for the development of resistant populations is related, 

in part, to the specific herbicide active ingredient, chemical family and the herbicide 

group. Some herbicides are more prone to the development of resistance than others 

(Heap, 2012c). The graph in Figure I-1 illustrates the global instances of weed resistance 
Monsanto Company 12-CT-244U 603 of 620 

to various herbicide groups. The different slopes of observed resistance are largely due 

to the factors described above, which relate to the specific herbicide active ingredient as 

well as to the group and herbicide family and its function. 

 
Monsanto Company 12-CT-244U 604 of 620 

Figure I-1. Weed Resistance to Various Herbicide Families1

As can be seen in Figure I-1, weed resistance to the synthetic auxin group of herbicides 

has been slower to develop than for other herbicide groups even though these were the 

first synthetic herbicides discovered and used commercially. Possible reasons for this are 

discussed below. 

1

Global number of resistant biotypes 

I.6. Mechanisms of Resistance and Inheritance of Resistance 

To date, the three known basic mechanisms by which weed species develop resistance to 

a herbicide have been identified: 1) target site alteration (target site), 2) enhanced 

metabolism of the herbicides (metabolism), and 3) reduced absorption and/or 

translocation of the herbicide such that the herbicide does not get to the site of action 

within the plant cell (exclusion) (Sammons et al., 2007). 

Herbicide resistance via target site alteration is the most common resistance mechanism 

among the various herbicide groups and chemical families. It has been found that a target 

site mechanism is the most common mechanism for ALS inhibitors, ACCase inhibitors, 
Monsanto Company 12-CT-244U 605 of 620 

and triazines, but is less common for other herbicide groups, such as glyphosate (Powles 

and Yu, 2010). The most common type of target site alteration is one where amino acid 

substitution(s) occur in the protein that is the target of the herbicide such that the 

alteration prevents the binding of the herbicide to the protein and as a result the activity 

of the targeted protein is not altered and the plant grows normally. 

In the case of synthetic auxin herbicides, resistance has been speculated to be due to 

mutation(s) in genes encoding an auxin-binding protein causing reduced herbicide 

binding (Zheng and Hall, 2001; Goss and Dyer, 2003). In several studies, differential 

herbicide absorption, translocation, and metabolism were ruled out as possible 

mechanisms of resistance in kochia (Cranston et al., 2001) and in wild mustard (Zheng 

and Hall, 2001). However, current research has not presented convincing evidence for a 

single mechanism of resistance and this inability to elucidate the mechanism of resistance 

may be due to a lack of thorough understanding of the mechanism (mode) of action of 

auxin herbicides (Jasieniuk et al., 1996). Walsh et al. (2006) identified seven alleles at 

two distinct genetic loci that conferred significant resistance to picolinate auxins 

(picloram) in Arabidopsis, yet had minimal cross-resistance to 2,4-D and IAA, a naturally 

occurring plant growth regulator.. 

Multiple mechanisms for inheritance of dicamba resistance have been reported in the 

literature. Jasieniuk et al. (1995) reported results indicating that inheritance of dicamba 

resistance in wild mustard is determined by a single, completely dominant nuclear allele. 

However, Cranston et al. (2001) reported results indicating that dicamba resistance in 

kochia is determined by a quantitative trait (two or more genes). The slow development 

of weed resistance to synthetic auxin herbicides may in part be due to their proposed 

multiple sites of physiological action in plants (Jasieniuk et al., 1996) and to the 

possibility that inheritance, at least in some species, is determined by a quantitative trait 

(Cranston et al., 2001). 

Little is known about the resistance mechanisms in glufosinate-resistant biotypes. AvilaGarcia and Mallory-Smith (2011) conducted an initial set of experiments to understand 

the mechanism of resistance in the ryegrass population that was also resistant to 

glyphosate. They found that resistance was not due to an insensitive or altered target site 

and hypothesized that reduced translocation is responsible for the resistance to both 

glyphosate and glufosinate in these populations. 

 

I.7. Weeds Resistant to Dicamba and Glufosinate 

As noted earlier, like other herbicides, the use of dicamba may lead to the development of 

dicamba-resistant weed species. To date, there are four species with known resistant 

biotypes to dicamba in the U.S./Canada after over 40 years of use: common hempnettle, 

kochia, prickly lettuce, and wild mustard (Heap, 2012a). Additionally, a population of 

common lambsquarters has been confirmed to be resistant in New Zealand, for a total of 

five species worldwide with confirmed resistant biotypes to dicamba. For the synthetic 

auxin group of herbicides there exist a total of 29 species globally with biotypes having 

confirmed resistance to at least one member of this group, but only nine species in the 

U.S. and four species in Canada (Heap, 2012a). All of these populations are, except for 
Monsanto Company 12-CT-244U 606 of 620 

two (wild carrot in OH and MI, and waterhemp in NE), found in western states or 

western Canadian provinces. In some weed species, cross-resistance between different 

herbicides within the auxin group has been confirmed (plant cross-resistance to another 

herbicide as a result of exposure to a similarly acting herbicide). Therefore, 

consideration has to be given to the possibility that dicamba resistance could extend to 

some of the other broadleaf species listed as resistant to other synthetic auxin herbicides 

(Cranston et al., 2001; Jasieniuk et al., 1995; Miller et al., 2001). However, because of 

differences in sites of action among the chemistry families within this group (i.e., benzoic 

acids compared to pyridine-carboxylic acids) cross resistance between the herbicide 

groups is not a certainty (Monaco et al., 2002). 

With the introduction of MON 88701 into glyphosate-tolerant cotton systems, where 

dicamba will be applied in combination with glyphosate and glufosinate, it is important to 

note that kochia is the only broadleaf species with resistant biotypes to either synthetic 

auxins or glyphosate. However, there are no known kochia biotypes resistant to both of 

these herbicides or resistant to glufosinate. In addition, the evolution of a dicambaglyphosate resistant biotype is unlikely because dicamba, glyphosate, and/or glufosinate, 

each with a distinct mode-of-action, will likely be applied in the same season to 

MON 88701 in the glyphosate-tolerant cotton systems. If populations with resistance to 

both glyphosate and dicamba herbicides were to occur, there are other herbicide options 

for managing the weed in cotton (e.g., glufosinate, clomazone and flumioxazin) and in its 

rotational crops (e.g., atrazine and isoxaflutole in corn) (Table I-1). The glyphosateresistant kochia biotype may be found in western cotton growing areas of Texas and 

Oklahoma. 

To date there are two weed species with confirmed resistance to glufosinate: goosegrass 

in Malaysia and Italian ryegress in Oregon, U.S. (Heap, 2012d). In the case of 

goosegrass, the resistant populations evolved due to use of glufosinate in a rubber 

plantation (Seng et al, 2010). In the case of Italian ryegrass, the resistance was actually 

discovered in populations exposed to glyphosate that evolved resistance to glyphosate 

and which had not been exposed to glufosinate; exemplifying a case of cross-resistance 

(Avila-Garcia and Mallory-Smith, 2011). No resistance in a broadleaf species has been 

found to date. 

Italian ryegrass may require special consideration when designing appropriate 

management programs because of the potential for cross resistance between glyphosate 

and glufosinate to exist. Avila-Garcia and Mallory-Smith (2011) demonstrated the only 

case of glufosinate cross resistance, which developed when the populations evolved 

resistance to glyphosate. It is not known if the reverse is true, though it is possible. 

Where there are known glyphosate resistant ryegrass populations Monsanto will 

recommend not to use glufosinate to control these populations. Likewise, dicamba will 

not be an option, since it does not control grasses such as ryegrass. Other herbicides such 

as those in the ACCase or ALS classes will be recommended. It is important to note that 

ryegrass is generally a weed target in preplant burndown applications and not in the 

cotton crop itself because of the biology of the species. 
Monsanto Company 12-CT-244U 607 of 620 

I.8. Sustainable Use of Dicamba and Glufosinate as a Weed Management Option in 

Cotton 

MON 88701 will be sold only in cotton varieties that also contain other herbicide-tolerant 

traits, including glyphosate-tolerance. Cotton varieties containing both MON 88701 and 

a glyphosate-tolerant system will enable dicamba and glufosinate to be applied with 

glyphosate and/or other cotton herbicides in an integrated weed management program. 

Dicamba primarily will be used in mixtures with either glyphosate or glufosinate or in 

sequence with glyphosate or glufosinate to control a broad spectrum of grass and 

broadleaf weed species. Glyphosate and glufosinate will not be used in mixtures due to 

antagonism (i.e., glufosinate damages the leaf tissue before glyphosate gets into the plant 

and/or can be translocated to growing parts of the plant) and reduced efficacy of 

glyphosate on susceptible weed species. Dicamba and glufosinate applications on 

MON 88701 will provide effective control of glyphosate-resistant broadleaf weeds and 

improve the control of annual and perennial broadleaf weed species, some of which are 

difficult to control with glyphosate. Dicamba and glufosinate will also help delay 

development and/or combat existing weed resistance issues that can limit the use of the 

PPO- and ALS-inhibiting herbicide groups by providing additional modes-of-action for 

management of certain broadleaf species known to be prone to resistance to many of the 

current herbicide options for weed management (i.e., Amarathus spp.). Likewise, 

dicamba will help to mediate potential evolution of resistance to glufosinate in broadleaf 

species and glufosinate will do the same for the potential evolution of resistant broadleaf 

species to dicamba. Cultivation of a combined MON 88701 and glyphosate-tolerance 

trait product will foster the adoption of Integrated Pest Management (IPM) practices in 

cotton by allowing growers to continue to primarily focus on postemergence in-crop 

weed control, as they have practiced with the glyphosate-tolerant cotton systems. This 

will allow growers to delay some herbicide treatments until field scouting indicates a 

need for additional postemergence weed control which is consistent with the principles of 

IPM, and also herbicide resistance management practices. Increasing postemergence 

herbicide options in cotton is important, especially in conservation tillage situations, 

where consistency of postemergence herbicides has generally been greater than that of 

soil active residual products, which have greater degree of inconsistent weed control, and 

thus has been a factor in the adoption of conservation tillage systems in the U.S. 

Upon the integration of MON 88701 into the glyphosate-tolerant cotton systems and 

pending approval of the use of dicamba on MON 88701 by the U.S. EPA, 

preplant/preemergence applications of dicamba can be made up to 1.0 lb a.e./acre up 

through crop emergence (cracking) and in-crop postemergence applications up to 0.5 lb 

a.e./acre could be applied through 7 days preharvest, with the combined total not to 

exceed 2.0 lbs a.e. dicamba per year for all applications. Residual herbicides also will be 

recommended for use, to provide early season weed control and to supplement dicamba 

and glufosinate activity on certain hard-to-control and glyphosate-resistant weed 

biotypes, such as glyphosate-resistant Palmer amaranth where weed populations can be 

very substantial. See section I.8.1 for specific weed management recommendations.

Dicamba and glufosinate, as complementary herbicides to glyphosate, will provide new 

weed control options in cotton that strengthen the utility and sustainability of glyphosate 
Monsanto Company 12-CT-244U 608 of 620 

as a weed control tool in the glyphosate-tolerant cotton systems. Likewise, glyphosate, as 

a complementary herbicide to dicamba and glufosinate, would strengthen the utility and 

sustainability of dicamba and glufosinate as weed control tools for the combined 

MON 88701 glyphosate-tolerance trait product. 

In the event there is known or suspected presence of a dicamba-resistant or glufosinateresistant weed biotype, other options for managing the resistant biotypes are available to 

the grower. There are multiple preemergence (including soil residuals) and postemergent 

herbicide options for managing weed populations that are resistant or may potentially 

develop resistance to dicamba or glufosinate in cotton, as well for crops grown in rotation 

with cotton. These options are noted in Table I-1. 
Monsanto Company 12-CT-244U 

Table I-1. Management Recommendations for Control of Dicamba-, Glufosinate- and Other SelectResistant Weeds 

Weed Species1

Herbicide Resistant 

Biotypes 

Primary 

Crop Cotton 

Rotational Crops 

Corn Sorghum Soybeans 

Kochia 

dicamba, fluroxpyr (populations also resistant to glyphosate) 

Atrazinea Atrazinea Saflufenacila 

Clomazonea Saflufenacila Saflufenacila Clomazonea Flumioxazini Isoxaflutolea Isoxaflutolea Flumioxazina Glyphosatei Mesotrionea Mesotrionea GlyphosateaParaquati Glyphosatea Glyphosatea Paraquata

Prickly Lettuce Dicamba, 2,4 D, MCPA 

Glyphosatei Saflufenacila Saflufenacila Saflufenacila 

Paraquati Atrazinea Atrazinea Chlorimuron/metribu

Flumioxazini Carfentrazone + atrazinea Carfentrazone + atrazinea Glyphosate + imazethapyra 

Isoxaflutole + atrazinea

Isoxaflutole + atrazinea

Wild mustard 

Dicamba , 2,4 D,MCPA, picloram, dichlorprop, mecoprop

Glyphosatei Glyphosatec Glyphosatec Glyphosatec Paraquati Atrazinec Atrazinec Chlorimuronc

Primisulfuronc Primisulfuronc Chlorimuron/metribuNicosulfurond NicosulfurondHalosulfurond Halosulfurond

Field Bindweed 2,4 D 

Glyphosatei Glyphosatea Glyphosatea Glyphosatea 

Paraquati Glyphosate + imazethapyra Glyphosate + imazethapyra

Flumioxazini Glyphosate + Imazamoxa Glyphosate + Imazamoxa

Yellow Starthistlee Picloram 
Monsanto Company 12-CT-244U 

Table I-1. Management Recommendations for Control of Dicamba-, Glufosinate- and Other Selected Synt

Resistant Weeds (continued) 

Weed Species1

Herbicide 

Resistant Biotypes 

Primary 

Crop Cotton 

Rotational Crops 

Corn Sorghum Soybeans 

Spreading Dayflower 2,4 D B

p

Lambsquartersg Dicamba

 Isoxaflutolea Metribuzinb BParaquati Atrazinea Cloransulamb CFlumioxazini Saflufenacila Saflufenacila G

Glyphosateb Mesotrionea Imazamoxb S

 Bromoxynilb Glyphosateb

Goosegrass Glufosinate 

Clethodimh Clethodimh

Glyphosateh Glyphosateh Glyphosateh Glyphosateh G

pendimethalinh pendimethalinh pendimethalinh

trifluralinh trifluralinh

Italian ryegrass 

Glufosinate (populations also resistant to glyphosate) 

Metolachlor (fall applied)h

Metolachlor (fall applied)h

Metolachlor (fall applied)h

Metolachlor (fall applied)h

Clethodimh Clethodimh

Glyphosateh Glyphosateh Glyphosateh Glyphosateh G

Paraquati
Monsanto Company 12-CT-244U 

1Scientific names for each weed species can be found in Table VIII-4. aBernards et al., 2010. bLoux et al., 2010.cMSU, 2010. dKells and Stachler, 1997. ePNWE, 2010. fUniversity of Arkansas CES, 2010. gResistance to lambsquarters has only been confirmed in New Zealand. hSteckel et al., 2011 iSmith et al., 2012
Monsanto Company 12-CT-244U 612 of 620 

I.9. Stewardship of Dicamba and Glufosinate Use on MON 88701 

In order to steward the use of agricultural herbicides and herbicide-tolerant cropping 

systems such as the combined trait MON 88701 and glyphosate-tolerant cotton product, 

Monsanto has conducted investigations and worked extensively with academics and other 

herbicide manufacturers to understand and recommend best practices to manage 

herbicide resistance. These investigations have demonstrated that one of the major 

factors contributing to the development of resistant weed biotypes has been poor weed 

control management practices. The primary reasons for lack of adequate management 

includes: 1) application of herbicides at rates below those indicated on the product label 

for the weed species, and 2) sole reliance on a particular herbicide for weed control 

without the use of other herbicides or cultural control methods (Beckie, 2006; Peterson et 

al., 2007). 

I.9.1. Weed Control Recommendations 

The proposed label for dicamba use on MON 88701 is based on the maximum allowable 

use rates and patterns. Prior to launch of MON 88701 in glyphosate-tolerant cotton 

systems, Monsanto, in cooperation with academics, will conduct trials to confirm the 

optimum rate and timing for dicamba, glufosinate and glyphosate, alone and in 

combination, and other herbicides. Recommendations to growers will be developed from 

this information and will be provided in herbicide product labels, Monsanto’s 

Technology Use Guide (TUG), and in other education and training materials to be 

broadly distributed. Specifically, current research conducted by Monsanto to define the 

optimum weed management systems support use recommendations that include the 

application of dicamba and glyphosate for preemergence on conservation tillage acres 

and early postemergence in-crop applications. In some situations, a second in-crop 

application of either dicamba tank-mixed with glyphosate or glufosinate, with or without 

a soil residual will be recommended (see Section VIII.G.4 for additional details) 

These recommendations will ensure more than one mechanism of action against the 

targeted species, which is a fundamental component of a good weed resistance 

management program. These management systems, which include the use of multiple 

effective herbicide modes-of-action, will reduce the potential for further resistance 

development to glyphosate, dicamba, and glufosinate, as well as other critical cotton 

herbicides. Furthermore, the preplant weed spectrum is generally different from the incrop weed spectrum therefore multiple applications of glyphosate and dicamba are not 

expected to increase selection pressure on either herbicide. 

I.9.2. Dispersal of Technical and Stewardship Information 

Monsanto will use multiple methods to distribute technical and stewardship information 

to growers, academics and grower advisors. Monsanto’s TUG will set forth the 

requirements and best practices for the cultivation of MON 88701 including 

recommendations on weed resistance management practices. Growers who purchase 
Monsanto Company 12-CT-244U 613 of 620 

varieties containing MON 88701 will be required to enter into a limited use license with 

Monsanto and must sign and comply with the Monsanto Technology Stewardship 

Agreement (MTSA), which requires the grower to follow the TUG. 

The weed resistance management practices that will be articulated in the TUG will also 

be broadly communicated to growers and retailers in order to minimize the potential for 

the development of resistant weeds. These practices will be communicated through a 

variety of means, including direct mailings to each grower purchasing a cotton variety 

containing MON 88701, a public website9

, and reports in farm media publications. The 

overall weed resistance management program will be reinforced through collaborations 

with U.S. academics, who will provide their recommendations for appropriate 

stewardship of dicamba and glufosinate in cotton production, as well as by collaboration 

with crop commodity groups who have launched web-based weed resistance educational 

modules. Finally, Monsanto will urge growers to report any incidence of repeated nonperformance of dicamba or glufosinate on weeds in fields planted with MON 88701, and 

Monsanto will investigate cases of unsatisfactory weed control to determine the cause as 

defined in I.9. 

The EPA is the U.S. federal regulatory agency that administers the federal law governing 

pesticide sale and use under the Federal Insecticide, Fungicide and Rodenticide Act 

(FIFRA). EPA encourages pesticide manufacturers to provide growers with information 

regarding an herbicide’s mode-of-action to aid growers in planning herbicide use 

practices and to foster the adoption of effective weed resistance management practices as 

specified by EPA in Pesticide Registration (PR) Notice 2001-5 (U.S. EPA, 2001). In that 

document EPA states that “this approach to resistance management is sound and would 

be highly beneficial to pesticide manufacturers and pesticide users.” EPA approves all 

pesticide label use instructions based on its evaluation of supporting data supplied by the 

pesticide registrant or manufacturer. By approving a label, EPA has concluded that the 

product will not cause unreasonable adverse effects to the environment when used in 

accordance with the label’s directions. After EPA approves a pesticide label, it is a 

violation of federal law to use the pesticide for a use or in a manner not in accordance 

with the label directions. Monsanto incorporates EPA’s guidelines for pesticide 

resistance management labeling on its agricultural herbicide labels, and will continue to 

do so in the future. Monsanto will adopt a similar approach to pesticide resistance 

management guidance on its dicamba product labels. 

In summary, Monsanto will require weed resistance management practices through the 

MTSA and TUG for its biotechnology-derived herbicide-tolerant products, such as 

MON 88701 integrated into the glyphosate-tolerant cotton systems, and to promote these 

practices through product labeling and educational outreach efforts as an effective means 

to manage weed resistance development for both dicamba, glufosinate, and glyphosate. 

 

9

 http://www.monsanto.com/weedmanagement/Pages/default.aspx 
Monsanto Company 12-CT-244U 614 of 620 

I.9.3. Weed Resistance Management Practices 

Monsanto will provide information to growers and grower advisors on best management 

practices to delay the development of resistance to dicamba and glufosinate. The weed 

resistance management recommendations for the use of dicamba and glufosinate in 

conjunction with cotton varieties containing MON 88701 will be consistent with the 

Herbicide Resistance Action Committee’s guidelines for prevention and management of 

herbicide resistance (HRAC, 2010)10. These guidelines recommend an integrated 

approach to weed resistance management, including crop management (i.e., cover crops, 

crop rotation, etc.), cultivation techniques, and the use of multiple herbicide modes-ofaction to manage a weed population. 

In cases where resistance is confirmed for dicamba or glufosinate in cotton producing 

areas, Monsanto and University/Cooperative Extension Service (CES) personnel will 

provide recommendations for alternative herbicide control methods to growers. These 

recommendations would be made available through Monsanto supplemental labels, 

Monsanto and university publications, and internet sites to growers, consultants, retailers 

and distributors. For all existing cases of dicamba-resistant and glufosinate-resistant 

weeds in the U.S. and globally today, alternative herbicides and cultural methods are 

available to growers to effectively control these biotypes. Examples of recommended 

alternative herbicides from University/CES personnel that are applicable to weed species 

known to be resistant to glufosinate, dicamba and other synthetic auxin herbicides are 

found in Table I-1. However, these examples in Table I-1 are only a subset of product 

combinations of available cotton herbicides. 

I.10. Monsanto Weed Performance Evaluation and Weed Resistance Management 

Plan 

An important part of a weed resistance management plan is the timely acquisition of 

information regarding product performance. Monsanto has an extensive technical, sales 

and marketing presence in the cotton markets where MON 88701 will be grown. 

Through our relationships with farm advisors, key University/CES personnel, and 

growers using our seeds and traits products, Monsanto will acquire important and timely 

information regarding product performance. This will allow the timely recognition of 

performance issues that could arise related to weed resistance or other means. Field 

employees and hired consultants are trained and provided processes for responding to 

product performance inquiries. Individual performance issues that could be related to 

potential resistance are promptly handled. In addition performance inquiries are 

periodically reviewed by Monsanto for trends that could indicate the need for follow up 

action on a broad scale. 

If dicamba or glufosinate resistance is confirmed, the scientific and grower communities 

will be notified and a weed resistance mitigation plan will be implemented by Monsanto 

 

10 The Herbicide Resistance Action Committee (HRAC) is an international body founded by the 

agrochemical industry for the purpose of supporting a cooperative approach to the management of 

herbicide resistance and the establishment of a worldwide herbicide resistance database. 
Monsanto Company 12-CT-244U 615 of 620 

in cooperation with the University/CES and/or the appropriate herbicide producer . The 

mitigation plan will be designed to manage the resistant biotype through effective and 

economical weed management recommendations implemented by the grower. The scope 

and level of intensity of the mitigation plan may vary depending on a combination of the 

following factors: 1) biology and field characteristics of the weed (seed shed, seed 

dormancy, etc.), 2) importance of the weed in the agricultural system, 3) resistance status 

of the weed to other herbicides with alternate modes-of-action, and 4) availability of 

alternative control options. These factors are analyzed by Monsanto and University/CES 

personnel in combination with economic and practical management considerations to 

develop a tailored mitigation strategy. The plan considers what is technically appropriate 

for the particular weed and incorporates practical management strategies that can be 

implemented by the grower. 

After a mitigation plan is developed, Monsanto communicates the plan to the grower 

community through the use of supplemental herbicide labeling (labeling which includes 

newly approved use directions, or other instructions)11, informational fact sheets, retailer 

training programs, agriculture media and/or other means, as appropriate. 

In addition to the grower inquiry initiated process, Monsanto, alone and/or in cooperation 

with University/CES, will conduct field studies to understand the potential for weed 

resistance and weed shifts as the result of various weed management programs 

implemented for MON 88701 integrated into glyphosate-tolerant cotton systems. These 

studies will allow researchers to better track specific factors that can influence the 

development of resistance to specific weeds. 

I.11. Summary 

Development of weed resistance is a complex process that can be difficult to accurately 

predict. Multiple methods for managing weed resistance are available and no single 

option is best for all farming situations. No single agronomic practice will mitigate 

resistance for all herbicides or all weeds. As a result, weed resistance needs to be 

managed on a case-by-case basis, tailored for the particular herbicide and weed species, 

and utilize an integrated system approach to meet grower needs. Using good weed 

management principles, built upon achieving high levels of control through proper 

application rate, choice of cultural practices, and appropriate companion weed control 

products will allow dicamba and glufosinate herbicides to continue to be used effectively. 

In cases where weed populations have evolved or developed resistance to dicamba and/or 

glufosinate, effective management options are available and experience has shown that 

growers will continue to find value in using dicamba and glufosinate in their weed 

control programs. 

The key principles for effective stewardship of dicamba and glufosinate use, including 

the integration of MON 88701 in the glyphosate-tolerant cotton systems, comprise: 

 

11 Monsanto will communicate information broadly so registrants are aware of when Monsanto is not the 

registrant or provider of the chemistry,. 
Monsanto Company 12-CT-244U 616 of 620 

1) basing weed management and weed resistance management practices on local needs 

and using the tools necessary to optimize crop yield, 2) using proper rate and timing of 

application, 3) not relying solely on one herbicide weed control option across a cropping 

system, 4) responding rapidly to instances of unsatisfactory weed control, and 

5) providing up-to-date weed management and weed resistance management training. 

Overall, there is a low potential for dicamba-resistant broadleaf weed populations to arise 

from the use of dicamba applied to MON 88701 integrated into glyphosate-tolerant 

cotton systems. The reasons are as follows: 

 Dicamba will be used in combination with glyphosate and/or glufosinate in a 

majority of cropping situations, and weed recommendations will also include the 

concurrent use of residual herbicides for complementary weed control and 

different modes-of-action. These use patterns mean that there will be multiple 

modes-of-action against the major broadleaf species present in cotton production. 

This is a primary way to delay the development of resistance. 

 The development of resistance to auxin herbicides has been found to be relatively 

slow. This observation is hypothesized to be due to multiple sites of action within 

plants and evidence suggesting that resistance is determined by multiple genes 

(quantitative traits), at least in some species. 

 Only four broadleaf weed species have been confirmed to be resistant to dicamba 

in the U.S., and relatively low numbers of broadleaf species have been confirmed 

to be resistant to synthetic auxin herbicides even though dicamba has been widely 

in use for over 40 years. 

 Known resistant broadleaf populations to dicamba and other auxin herbicides are 

primarily found in the western U.S. and, thus, are not present in the major cotton 

geographies. In addition, the known dicamba-resistant biotypes are not major 

weed species present in the U.S. cotton crop. 

Likewise, the probability for weed species to evolve resistance to glufosinate as a result 

of glufosinate use in the MON88701 system is considered to be low because: 

 Two species have been confirmed to be resistant to glufosinate worldwide and 

one (ryegrass) in the US. This suggests that the frequency for resistant alleles in 

native weed populations is fairly low. 

 Known resistant populations to glufosinate herbicide within the U.S. are only 

found in Oregon, and thus, are not present in the major cotton geographies. 

 In the MON 88701 system, glufosinate will likely be used in combination with 

dicamba and in sequence with glyphosate. Residual herbicides will also be 

recommended and likely used in this cropping system. As noted above, these use 

patterns mean that there will be multiple modes-of-action against the major 

broadleaf species present in soybean production. This is a primary way to delay 

the development of resistance. 
Monsanto Company 12-CT-244U 617 of 620 

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