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Petition for the Determination of Nonregulated Status for Increased Ear Biomass

MON 87403 Maize

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

August 1, 2014

Revised September 12, 2014

USDA Petition Number #14-213-01p

OECD Unique Identifier: MON-87403-1

Monsanto Petition Number: CR262-14U1

Submitted by:

John M. Cordts, M.S., M.B.A.

Monsanto Company

800 North Lindbergh Blvd.

St. Louis, MO 63167

Phone: (314) 694-4831

Fax: (314) 694-3080

E-mail: John.M.Cordts@monsanto.com

Prepared by:

D. Anstrom, M.S., T. Bhakta, Ph.D., P. Chinnadurai, M.S., M.Phil.,

J.M. Cordts, M.S., M.B.A., T. Edrington, Ph.D., G.E. Frierdich, B.S., DABT,

M. Leibman, Ph.D., K. Nemali, Ph.D., E. Park, Ph.D., K. Skottke, Ph.D.,

G.B. Tilton, Ph.D., M. Wang, Ph.D., E.G. Webb, M.S.

Contributors and/or Principal Investigators:

P. Asiimwe, Ph.D., G. Brown, B.S., C. Garnaat, M.S., J. Hart, M.S.,

K. Howard, Ph.D., K. Klug, M.S., D. Kovalic, Ph.D., K.W. Lawry, M.S.,

M. McPherson, Ph.D., S.G. Riordan, B.S., B. Sammons, Ph.D., A. Silvanovich, Ph.D.,

S.B. Storrs, Ph.D., Q. Tian, M.D., Ph.D., R. Wang, M.S., J.M. Ward., Ph.D.,

T. Werk, M.S., J. Whitsel, M.S., Y. Yan, Ph.D.
Monsanto Company CR262-14U1 2 of 260

RELEASE OF INFORMATION

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

the regulatory process. Monsanto understands that the USDA complies with the

provisions of the Freedom of Information Act (FOIA). In the event the USDA receives a

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

information in this petition, Monsanto expects that, in advance of the release of the

document(s), USDA will, pursuant to Executive Order 12600, the Privacy Act and other

applicable authorities, 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 CBI-deleted 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. Monsanto also understands that when deemed complete, a copy

of the petition may be posted to the USDA-APHIS BRS website or other U.S.

government websites (e.g., www.regulations.gov). Except in accordance with the

foregoing, Monsanto does not authorize the release, publication or other distribution of

this information without Monsanto's prior notice and consent.

© 2014 Monsanto Company. All Rights Reserved.

This document is protected under national and international copyright law and treaties.

This document and any accompanying material are for use only by the regulatory

authority to which it has been submitted by Monsanto Company and its affiliates,

collectively “Monsanto Company,” and only in support of actions requested by Monsanto

Company. Any other use, copying, or transmission, including internet posting, of this

document and the materials described in or accompanying this document, without prior

consent of Monsanto Company, is strictly prohibited; except that Monsanto Company

hereby grants such consent to the regulatory authority where required under applicable

law or regulation. The intellectual property, information and materials described in or

accompanying this document are owned by Monsanto Company, which has filed for or

been granted patents on those materials. By submitting this document and any

accompanying materials, Monsanto Company does not grant any party or entity any right

or license to the information, material or intellectual property described or contained in

this submission.
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. 

Ml l^M^ 

John M . Cordts, M.S., M.B.A. 

Regulatory Affairs Manager 

Address: 

Monsanto Company 

800 North Lindbergh Blvd., Mail Stop C3SD 

St. Louis, MO 63167 

Tel: (314) 694-4831 

Fax: (314) 694-3080 

Monsanto Company CR262-14U1 3 of260 
Monsanto Company CR262-14U1 4 of 260

EXECUTIVE SUMMARY

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

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 maize product, MON 87403, any

progeny derived from crosses between MON 87403 and conventional maize, and any

progeny derived from crosses of MON 87403 with biotechnology-derived maize that

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

Product Description

Monsanto Company has developed biotechnology-derived maize MON 87403 that has

increased ear biomass at an early reproductive phase (R1) compared to conventional

control maize. MON 87403 was produced through insertion of the coding region of the

full-length Arabidopsis thaliana ATHB17 gene through Agrobacterium-mediated

transformation. ATHB17 is a member of the HD-Zip family of plant transcription

factors, which are proteins that bind to specific DNA sequences and regulate gene

expression. The HD-Zip family of proteins is found broadly across plant species and

specific HD-Zip proteins have been shown to play an important role in the modulation of

plant growth and development. The HD-Zip family consists of four subfamilies and

ATHB17 is a member of the class II subfamily. HD-Zip II proteins form either

homodimers or heterodimers with other HD-Zip II proteins within the same subfamily

and function as repressors of gene expression. In MON 87403, maize-specific splicing of

the ATHB17 transcript results in a truncated protein, ATHB17Δ113, which is missing the

first 113 N-terminal amino acids that are expressed in Arabidopsis thaliana. The

ATHB17∆113 protein retains the ability to form homo- and hetero-dimers and bind to

target DNA sequences like the full-length protein. The ATHB17∆113 protein is,

however, unable to function as a transcriptional repressor because the protein lacks a

functional repression domain. By a dominant-negative mechanism, ATHB17∆113 can

alter the activity of endogenous maize HD-Zip II proteins, which are predominantly

expressed in ear tissue. Thus, the ATHB17∆113 protein likely modulates HD-Zip IIregulated pathways in the ear, which leads to increased ear growth at the early

reproductive phase. This increased ear growth is associated with increased partitioning

of dry matter (photosynthate) from the source (vegetative) tissue to the sink (ear) tissue in

MON 87403 compared to control plants.

Early reproductive stages in maize are a critical period of maize growth at which the

maximum ear biomass (sink size) is determined by a combination of genetics and
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environmental conditions. Dry matter (photosynthate) produced by the plant during

reproductive stages is allocated to the ear for its growth after the sink size is determined.

Thus, ear biomass, which is set during early reproductive stages, is considered an

important determinant of reproductive success and a larger ear biomass at early

reproductive stages is associated with increased grain yield at harvest. Consistent with

this, multiple years of field testing showed that MON 87403 out-yielded its comparators

at a majority of locations tested.

MON 87403 will be combined with other deregulated biotechnology-derived traits

through traditional breeding methods to create commercial products with increased yield

opportunity as well as protection against maize pests and tolerance to multiple herbicides.

These next generation combined-trait maize products will continue to offer broader

grower choice and continued pest control durability. Adoption of improved maize

hybrids with increased yield opportunity that results in incremental increases in national

average grain yield can positively impact production, exports and economic welfare.

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

Food and Feed Safety of MON 87403 Compared to Conventional Maize

The data and information presented in this petition demonstrate MON 87403 is

agronomically, phenotypically, and compositionally comparable to commercially

cultivated maize, with the exception of the introduced trait. Moreover, the data and

information presented demonstrate MON 87403 is not expected to pose an increased

plant pest risk, including weediness, compared to commercially cultivated maize. The

food, feed, and environmental safety of MON 87403 was confirmed based on multiple,

well-established lines of evidence:

 Maize does not possess any of the attributes commonly associated with weeds,

has a history of safe consumption, and serves as an appropriate basis of

comparison for MON 87403.

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

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

 Extensive evaluation of the truncated ATHB17Δ113 protein expressed in

MON 87403 confirms that it is unlikely to be a toxin or allergen.

 A compositional assessment supports the conclusion that MON 87403 grain and

forage is compositionally equivalent to grain and forage of conventional maize.

 An extensive evaluation of MON 87403 phenotypic and agronomic characteristics

and environmental interactions demonstrates MON 87403 has no increased plant

pest risk potential compared to conventional maize.

 An assessment of potential impact to non-target organisms (NTOs) including

organisms beneficial to agriculture and endangered species indicates that
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MON 87403 is not expected to have an effect on other organisms compared to

conventional maize under normal agricultural practices.

 Evaluation of the agronomic and phenotypic characteristics of MON 87403, using

current maize cultivation and management practices, leads to the conclusion that

deregulation of MON 87403 would not have an effect on maize agronomic

practices.

Maize is a Familiar Crop Lacking Weedy Characteristics

Maize is grown extensively throughout the world, and is the largest cultivated crop

followed by wheat (Triticum sp.) and rice (Oryza sativa L.) in total global production. In

the U.S., maize is grown in almost all states and is the largest crop grown in terms of

acreage planted and net value. Maize has been studied extensively, and the

domestication of maize can be traced back to approximately 10,000 years ago in southern

Mexico.

Because of the importance of maize in agriculture, plant breeders continuously strive to

improve commercial maize yields. Initial improvements in maize yield were due to the

domestication of varieties with desirable traits like larger ear biomass. Maize ears

became larger over time during this domestication. During the hybrid era (1939 to

present), commercial maize yield in the U.S. increased nearly six-fold with an average

99 kg ha-1 increase every year. The major factor that contributed to yield increase during

this era is a favorable response of hybrids to increased plant population density which

resulted in an increase in the number of ears per hectare. Whereas increased planting

densities resulted in a net increase in grain yields, they resulted in a decrease in ear size at

the individual plant level. Commercial varieties with MON 87403 can provide increased

yield opportunity as this trait increases ear biomass during early reproductive stages at

production planting densities.

Maize is not listed as a weed in the major literature references on weeds, nor is it present

on the lists of noxious weed species published by the federal government (7 CFR

part 360). In addition, maize has been grown throughout the world without any report

that it is a serious weed. Maize is poorly suited to survive without human assistance and

is not capable of surviving as a weed due to past selection in the domestication of maize.

During domestication of maize, traits often associated with weediness, such as seed

dormancy, a dispersal mechanism, or the ability to establish reproducing populations

outside of cultivation, have not been selected. Similarly, the history of hybrid breeding in

the U.S. does not indicate there are any changes in the characteristics of maize that would

change the weediness profile of the crop. Although maize seed can overwinter in a

rotation with soybeans or other crops, mechanical and chemical measures are routinely

used to control maize volunteers. Some populations of wild annual and perennial species

that could hybridize with maize, such as hybrids containing MON 87403, are known to

exist in the U.S., however key differences in several factors such as flowering time,

geographical separation, and development timings make natural crosses in the U.S.

highly unlikely.
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Conventional Maize MPA640B and EXP257 are Appropriate Comparators to

MON 87403

Conventional control materials developed for use as comparators in safety assessment

studies were based on the appropriate fit for various studies and seed availability. The

conventional control materials included the original transformation line (LH244) and

LH244 crossed to two conventional lines (LH287 and LH295) to create F1 starting

control materials. Both MPA640B (LH244 × LH287) and EXP257 (LH244 × LH295)

were used as controls in molecular characterization studies. MPA640B was used as the

conventional control in compositional analysis studies and in phenotypic, agronomic and

environmental interactions assessments. Where appropriate, commercial hybrid maize

materials (reference hybrids) were also used to establish a range of variability or

responses representative of commercial maize in the U.S.

Molecular Characterization Verified the Integrity and Stability of the Inserted DNA

in MON 87403

MON 87403 was developed through Agrobacterium-mediated transformation of maize

immature embryos from inbred line LH244 utilizing plasmid PV-ZMAP5714.

PV-ZMAP5714 is approximately 11.7 kb in size and contains three cassettes: one

T-DNA, delineated by Left and Right Border regions, contains the ATHB17 expression

cassette, the plasmid backbone contains the cp4 epsps selectable marker cassette, and the

aadA expression cassette. PV-ZMAP5714 employs a tandem T-DNA approach to

generate marker-free plants. In this tandem T-DNA approach, a single right border and a

single left border were used to achieve separate, unlinked insertion of the T-DNA as well

as the cp4 epsps selectable marker gene located in the plasmid backbone. After initial

selection of transformants for glyphosate tolerance, the cp4 epsps cassette was segregated

away by conventional breeding and molecular analysis was used to identify plants

containing only the intended T-DNA (and not the cp4 epsps).

Characterization of the DNA insert in MON 87403 was conducted using a combination

of sequencing, PCR, and bioinformatics. The results of this characterization demonstrate

that MON 87403 contains one copy of the intended transfer DNA (T-DNA) containing

the ATHB17 expression cassette that is stably integrated at a single locus and is inherited

according to Mendelian principles over multiple generations. These conclusions are

based on several lines of evidence:

 Molecular characterization of MON 87403 by Next Generation Sequencing and

Junction Sequence Analysis (NGS/JSA) demonstrated that MON 87403 contained

a single intended DNA insert. These whole-genome sequence analyses provided

a comprehensive assessment of MON 87403 to determine the presence and

identity of sequences derived from PV-ZMAP5714 and demonstrated that

MON 87403 contained a single T-DNA insert with no detectable backbone

sequences.

 Directed sequencing (locus-specific PCR, DNA sequencing and analyses)

performed on MON 87403 was used to determine the complete sequence of the
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single DNA insert from PV-ZMAP5714, the adjacent flanking DNA, and the 5'

and 3' insert-to-flank junctions. This analysis confirmed that the sequence and

organization of the inserted DNA is identical to the corresponding region in the

PV-ZMAP5714 T-DNA. Furthermore, the genomic organization at the insertion

site was assessed by comparing the sequences flanking the T-DNA insert in

MON 87403 to the sequence of the insertion site in conventional maize. This

analysis determined that no major DNA rearrangement occurred at the insertion

site in MON 87403 upon DNA integration.

 Generational stability analysis by NGS/JSA demonstrated that the single

PV-ZMAP5714 T-DNA insert in MON 87403 has been maintained through five

breeding generations, thereby confirming the stability of the T-DNA in

MON 87403.

 Segregation analysis corroborates the insert stability demonstrated by NGS/JSA

and independently establishes the nature of the T-DNA as a single chromosomal

locus.

Taken together, the characterization of the genetic modification in MON 87403

demonstrates that a single copy of the intended T-DNA was stably integrated at a single

locus of the maize genome and that no plasmid backbone sequences are present in

MON 87403.

Data Confirms ATHB17Δ113 Protein Safety

Characterization of the introduced protein(s) in a biotechnology-derived crop is important

to establishing food, feed, and environmental safety and a multistep approach was used to

characterize and assess the safety of the ATHB17Δ113 protein expressed in MON 87403

resulting from the genetic modification. The expression level of the ATHB17Δ113

protein in selected tissues of MON 87403 was determined and exposure to humans and

animals through diet was evaluated. In addition, the donor organism for the

ATHB17Δ113 protein coding sequence, Arabidopsis thaliana, is ubiquitous in the

environment and is not commonly known for human or animal pathogenicity or

allergenicity.

Assessing the safety of the ATHB17∆113 protein requires a consideration of both the

hazard associated with the protein and the potential exposure to it. Data presented

demonstrate that the expression of this protein in maize grain is below the limit of

detection (LOD) and is extremely low in other tissues tested, hence exposure to

ATHB17∆113 protein is negligible. Bioinformatic searches using the ATHB17∆113

amino acid sequence as the query identified homologous sequences from several different

food plants, including soybean, rice, maize, tomato, potato, orange, papaya, grape, and

cruciferous vegetables. Overall the protein sequence identity of ATHB17∆113 to

homologs in these species ranges from ~58-83%, with the highest identity to the

homologs in the Brassica species Brassica rapa (a species including crops such as turnip

and napa cabbage) and Brassica oleracea (a species including crops such as cabbage and

Brussels sprouts). The amino acid sequence alignment between ATHB17∆113 and its
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food crop homologs spans the length of the ATHB17∆113 protein. Thus ATHB17∆113

shares sequence identity and structural similarity with proteins present in plants currently

consumed, establishing that humans and animals are exposed to this class of proteins and

that no adverse effects have been attributed to this class of proteins.

Bioinformatics analysis also determined that the ATHB17Δ113 protein lacks structural

similarity to known allergens, gliadins, glutenins, or protein toxins. Testing also showed

that the ATHB17Δ113 protein is rapidly digested in pepsin and pancreatin suggesting

that the negligible amount of protein expressed is further reduced by proteolysis during

ingestion thereby reducing the already negligible exposure. Additionally, a mouse

gavage study demonstrated no acute oral toxicity with a No Observable Adverse Effect

Level (NOAEL) for ATHB17Δ113 of 1335 mg/kg, the highest dose tested. Undetectably

low expression levels in grain coupled with rapid digestibility and no evidence of any

toxic or allergenic concerns supports the conclusion that consumption of the

ATHB17Δ113 protein from MON 87403 or its progeny poses no meaningful risk to

human and animal health or an increased plant pest risk.

MON 87403 is Compositionally Equivalent to Conventional Maize

Compositional analysis was conducted on grain and forage of MON 87403 grown at

eight sites representative of typical agricultural regions for maize production in the U.S.

in 2012. The evaluation of MON 87403 followed considerations relevant to the

compositional quality of maize as defined by a 2002 OECD consensus document. Grain

samples were analyzed for levels of nutrients including proximates, carbohydrates by

calculation, fiber, amino acids, fatty acids, minerals, and vitamins. The anti-nutrients

analyzed in grain included phytic acid and raffinose. Secondary metabolites analyzed in

grain included furfural, ferulic acid, and p-coumaric acid. Forage samples were analyzed

for levels of proximates, carbohydrates by calculation, fiber, and minerals. In total, 78

different components were assayed (nine in forage and 69 in grain).

Of those 78 components, 14 fatty acids, sodium, and furfural had more than 50% of

observations below the assay limit of quantitation (LOQ) and were excluded from

statistical analysis. Moisture in grain and forage was measured for conversion of

components to dry weight, but was not statistically analyzed. Therefore, 60 components

were statistically analyzed. The statistical comparisons were based on compositional data

combined across all field sites. Statistically significant differences were identified at the

5% level (α = 0.05). The compositional data from the reference hybrids were combined

across all field sites to calculate a 99% tolerance interval for each component to estimate

the natural variability of each component in maize.

Of the 60 components statistically assessed for MON 87403, none of the components

showed a significant difference between MON 87403 and the conventional control.

These results support the overall conclusion that MON 87403 was not a major contributor

to variation in component levels in maize grain and forage and confirmed the

compositional equivalence of MON 87403 to the conventional control in levels of these

components. These results support the overall food and feed safety and lack of plant pest

risk of MON 87403.
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MON 87403 Does Not Change Maize 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, 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, and environmental interaction assessment of MON 87403 included

genetically similiar conventional controls as comparators. This evaluation used a weight

of evidence approach and considered statistical differences between MON 87403 and the

conventional control with respect to reproducibility, magnitude, and directionality. The

observations were taken on plants not treated with herbicides in order to evaluate only the

impact of the introduced trait in MON 87403. Comparison to a range of commercial

references grown concurrently established the range of natural variability for maize, and

provided a context from which to further evaluate any statistical differences.

Characteristics assessed included: seed dormancy and germination, pollen morphology,

plant phenotypic observations and environmental interaction evaluations. The

phenotypic, agronomic, and environmental interaction assessment demonstrated that

MON 87403 is comparable to the conventional control. Thus, MON 87403 is not

expected to have increased weediness or plant pest potential compared to conventional

maize.

Seed dormancy and germination characterization indicated that MON 87403 seed had

dormancy and germination characteristics similar to seed of the conventional control. In

particular, the lack of hard seed, a well recognized seed characteristic associated with

weediness, supports a conclusion of no increased weediness of MON 87403 compared to

the conventional control. For pollen characteristic assessments, there were no

statistically significant differences (α=0.05) detected between MON 87403 and the

conventional control for pollen viability and diameter, and no visual differences in

general pollen morphology were observed.

The field evaluation of phenotypic, agronomic, and environmental characteristics also

supports the conclusion that MON 87403 is not likely to have increased weediness or

plant pest potential compared to conventional maize. Data were collected at 13 sites in

the U.S. during 2012 to evaluate phenotypic and agronomic characteristics of

MON 87403 compared to the conventional control and four reference hybrids. These 13

sites provided a diverse range of environmental and agronomic conditions representative

of commercial maize production areas in North America. In the combined-site analysis,

no statistically significant differences were detected between MON 87403 and the

conventional control for 12 of 13 characteristics including early stand count, days to 50%

pollen shed, days to 50% silking, stay green rating, plant height, dropped ear count, stalk

lodged plants, root lodged plants, final stand count, grain moisture, test weight and yield.

One significant difference was detected for ear height in the combined-site analysis.
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MON 87403 had increased ear height (111.1 vs. 107.5 cm) compared to the conventional

control. However, the mean value of MON 87403 for ear height was within the reference

range for this characteristic Ear height is not a reported weediness characteristic and

whereas large increases in ear height might be expected to increase stalk lodging, there

was no increase in stalk lodging in these trials. In addition, a small change in ear height

would not be expected to change agronomic practices. Thus, the measured phenotypic

characteristics of MON 87403 were not altered in terms of pest/weed potential compared

to conventional maize.

In an assessment of abiotic stress, no differences in the range of responses were observed

between MON 87403 and the conventional control for any of the 143 comparisons of

plant response to abiotic stressors. In an assessment of disease responses, no differences

in the range of responses were observed between MON 87403 and the conventional

control for any of the 176 comparisons. Finally, in an assessment of arthropod damage,

no differences in the range of responses were observed between MON 87403 and the

conventional control for any of the 150 comparisons for plant damage caused by

arthropods.

In summary, the phenotypic, agronomic, and environmental interaction data were

evaluated to characterize MON 87403, and to assess whether the trait introduced in

MON 87403 alter the plant pest potential compared to conventional maize. The

evaluation, using a weight of evidence approach, considered the reproducibility,

magnitude, and direction of detected differences between MON 87403 and the

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

Results from the phenotypic, agronomic, and environmental interactions assessment

indicated that MON 87403 does not possess enhanced weediness characteristics,

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

characteristics that would confer a plant pest risk compared to conventional maize.

MON 87403 Will Not Negatively Affect NTOs Including Those Beneficial to

Agriculture

An evaluation of the impacts of MON 87403 on non-target organisms (NTOs) is a

component of the plant pest risk assessment. Because MON 87403 does not possess

pesticidal activity, all organisms that interact with MON 87403 are considered to be

NTOs. Data from 2012 U.S. phenotypic and agronomic studies and observational data on

environmental interactions such as plant-disease interactions, arthropod damage and

arthropod abundance, were collected at select sites for MON 87403 and conventional

controls. Results from these extensive studies support conclusions of no adverse impacts

to non-target arthropod populations and no changes to plant-disease interactions.

The biochemical information and experimental data for evaluation of MON 87403

included molecular characterization, ATHB17113 protein safety assessment, data from

the environmental interactions assessment, demonstration of compositional equivalence

to conventional maize, and demonstration of agronomic and phenotypic equivalence to

conventional maize (with the exception of presence of ATHB17∆113 protein in

MON 87403). Taken together, these data support the conclusion that MON 87403 has no
Monsanto Company CR262-14U1 12 of 260

reasonable mechanism to harm NTOs, nor does it pose an additional risk to threatened

and endangered species compared to the cultivation of conventional maize.

Deregulation of MON 87403 is Not Expected to Have Effects on Maize Agronomic

Practices

An assessment of current maize agronomic practices was conducted to determine whether

the cultivation of MON 87403 has the potential to impact current maize agronomic

practices. Maize fields are typically highly managed areas that are dedicated to grain

and/or forage production.

MON 87403 was developed to provide increased ear biomass at an early reproductive

stage in maize hybrids through expression of the ATHB17Δ113 protein. As such, there

are no additional phenotypes associated with expression of the intended T-DNA in this

product. As phenotypic evaluations, evaluations of stress responses, and pest/disease

susceptibility showed no differences between MON 87403 and reference hybrids no

changes are anticipated in crop rotations, tillage practices, planting practices, fertility

management, weed and disease management, and volunteer management from the

introduction of MON 87403.

MON 87403 is similar to conventional maize in its agronomic, phenotypic,

environmental, and compositional characteristics and has naturally occurring levels of

protection against pests and diseases comparable to and typical of conventional

commercial maize hybrids. Based on this assessment, the introduction of MON 87403 is

not expected to result in changes or impacts to current maize agronomic practices.

Conclusion

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

MON 87403 is not expected to be a plant pest. Therefore, Monsanto Company requests a

determination from APHIS that MON 87403 and any progeny derived from crosses

between MON 87403 and conventional maize or deregulated biotechnology-derived

maize be granted nonregulated status under 7 CFR Part 340.
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TABLE OF CONTENTS

RELEASE OF INFORMATION.........................................................................................2

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

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

TABLE OF CONTENTS...................................................................................................13

LIST OF TABLES.............................................................................................................17

LIST OF FIGURES ...........................................................................................................20

ABBREVIATIONS AND DEFINITIONS........................................................................22

I. RATIONALE FOR THE DEVELOPMENT OF MON 87403 .............................24

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

7 CFR § 340.6 ...............................................................................................................24

I.B. Rationale for the Development of MON 87403..................................................24

I.C. Submissions to Other Regulatory Agencies........................................................26

I.C.1. Submission to FDA ..................................................................................26

I.C.2. Submissions to Foreign Government Agencies .......................................27

II. THE BIOLOGY OF MAIZE .................................................................................28

II.A. Maize as a Crop ...............................................................................................28

II.B. Characteristics of the Recipient Plant..............................................................29

II.C. Maize as a Test System in Product Safety Assessment...................................29

III. DESCRIPTION OF THE GENETIC MODIFICATION ......................................31

III.A. The Plasmid Vector PV-ZMAP5714...............................................................31

III.B. Description of the Transformation System......................................................32

III.C. The ATHB17 Coding Sequence and the ATHB17 Protein (T-DNA)..............32

III.D. Regulatory Sequences......................................................................................35

III.E. T-DNA Borders ...............................................................................................35

III.F. Genetic Elements Outside of the T-DNA Borders ..........................................35

IV. CHARACTERIZATION OF THE GENETIC MODIFICATION........................39

IV.A. Determining the Number and Identity of DNA Inserts in MON 87403..........43

IV.A.1. Next Generation Sequencing of MON 87403 and Conventional

Control Genomic DNA..........................................................................................43

IV.A.2. Characterization of insert number in MON 87403 using

Bioinformatic Analysis ..........................................................................................44

IV.B. Organization and Sequence of the Insert and Adjacent DNA in

MON 87403...................................................................................................................52

IV.C. Sequencing of the MON 87403 Insertion Site.................................................54
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IV.D. Determination of Insert Stability over Multiple Generations of

MON 87403...................................................................................................................56

IV.E. Inheritance of the Genetic Insert in MON 87403 ............................................57

IV.F. Characterization of the Genetic Modification Summary and

Conclusion.....................................................................................................................61

V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE

ATHB17Δ113 PROTEIN PRODUCED IN MON 87403 .................................................62

V.A. Identity and Function of the ATHB17Δ113 Protein from MON 87403 .........62

V.A.1. Sequence Identity of the ATHB17Δ113 Protein from

MON 87403 ...........................................................................................................62

V.A.2. Structure and Function of the ATHB17Δ113 Protein from

MON 87403 ...........................................................................................................64

V.B. Characterization and Equivalence of ATHB17Δ113 Protein from

MON 87403...................................................................................................................64

V.C. Expression Levels of ATHB17Δ113 Protein in MON 87403.........................65

V.D. Assessment of Potential Allergenicity of the ATHB17Δ113 Protein .............68

V.E. Safety Assessment Summary of ATHB17Δ113 Protein in MON 87403........68

V.E.1. The Donor Organism has a History of Safe Use ..................................68

V.E.2. ATHB17Δ113 Protein Belongs to a Common Class of Plant

Proteins ..............................................................................................................69

V.E.3. ATHB17Δ113 Protein in MON 87403 is Not Homologous to

Known Allergens or Toxins...................................................................................69

V.E.4. ATHB17Δ113 Protein in MON 87403 is Susceptible to Pepsin

and Pancreatin Digestion .......................................................................................70

V.E.5. ATHB17Δ113 Protein in MON 87403 is Not Acutely Toxic ..............70

V.E.6. Human and Animal Exposure to the ATHB17Δ113 Proteins..............70

V.F. ATHB17Δ113 Protein Characterization and Safety Conclusion.....................71

VI. COMPOSITIONAL ASSESSMENT OF MON 87403.........................................73

VI.A. Compositional Equivalence of MON 87403 Grain and Forage to

Conventional Maize ......................................................................................................74

VI.B. Compositional Assessment of MON 87403 Conclusion.................................92

VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT....................................................................................93

VII.A. Characteristics Measured for Assessment ...................................................93

VII.B. Interpretation of Phenotypic and Environmental Interaction Data ..............98

VII.B.1. Interpretation of Criteria for Detected Differences ..............................98

VII.B.2. Interpretation of Vigor and Environmental Interactions Data............101
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VII.C. Comparative Assessments of the Phenotypic, Agronomic, and

Environmental Interaction Characteristics of MON 87403 ........................................101

VII.C.1. Seed Dormancy and Germination Characteristics..............................101

VII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions

Characteristics......................................................................................................104

VII.C.3. Pollen Characteristics .........................................................................115

VII.D. Conclusions for Phenotypic, Agronomic, and Environmental

Interactions Evaluation................................................................................................117

VIII. U.S. AGRONOMIC PRACTICES ...................................................................118

VIII.A. Introduction................................................................................................118

VIII.B. Overview of U.S. Maize Production..........................................................118

VIII.B.1. Maize Production...............................................................................118

VIII.C. Management of Insect Pests.......................................................................120

VIII.D. Management of Diseases and Other Pests .................................................120

VIII.E. Weed Management ....................................................................................120

VIII.F. Crop Rotation Practices in Maize ..............................................................120

VIII.G. Maize Volunteer Management...................................................................121

VIII.H. Stewardship of MON 87403 ......................................................................121

VIII.I. Impact of the Introduction of MON 87403 on Agricultural Practices.......122

IX. PLANT PEST ASSESSMENT............................................................................123

IX.A. Characteristics of the Genetic Insert and Expressed Product ........................123

IX.A.1. Genetic Insert......................................................................................123

IX.B. Expression and Characterization of Gene Product ........................................124

IX.B.1. Protein Safety and Expression Levels ................................................124

IX.C. Compositional Characteristics.......................................................................124

IX.D. Phenotypic, Agronomic, and Environmental Interaction

Characteristics .............................................................................................................125

IX.D.1. Seed Dormancy and Germination.......................................................125

IX.D.2. Plant Growth and Development .........................................................125

IX.D.3. Response to Abiotic Stressors ............................................................126

IX.D.4. Pollen Morphology and Viability.......................................................126

IX.E. Impact to Non-target Organisms Including Those Beneficial to

Agriculture or Threatened and Endangered Species ...................................................126

IX.F. Weediness Potential of MON 87403.............................................................127

IX.G. Potential for Pollen Mediated Gene Flow and Introgression ........................127

IX.G.1. Hybridization with Cultivated Maize .................................................128
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IX.G.2. Hybridization with Wild Annual Species of Zea mays subsp.

mexicana ............................................................................................................132

IX.G.3. Hybridization with the Wild Perennial Species of Subgenus

Tripsacum ............................................................................................................132

IX.G.4. Transfer of Genetic Information to Species with which Maize

Cannot Interbreed (Horizontal Gene Flow) .........................................................132

IX.H. Potential Impact on Maize Agronomic Practices ..........................................133

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

Conventional Maize ....................................................................................................133

IX.J. Summary of Plant Pest Assessments.............................................................134

X. ADVERSE CONSEQUENCES OF INTRODUCTION .....................................135

REFERENCES ................................................................................................................136

APPENDICES .................................................................................................................146

Appendix A: USDA Notifications and Permits...............................................................147

Appendix B: Mode of Action of ATHB17∆113 Protein in MON 87403........................153

Appendix C: Overview, Materials, Methods, and Supplementary Results for

Molecular Analyses of MON 87403................................................................................167

Appendix D: Materials, Methods and Results for Characterization of

ATHB17Δ113 Protein Produced in MON 87403............................................................184

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

ATHB17Δ113 Protein in MON 87403 ............................................................................193

Appendix F: Materials and Methods for Compositional Analysis of MON 87403

Maize Grain and Forage...................................................................................................196

Appendix G: Materials, Methods, and Individual Site Results for Seed Dormancy

and Germination Assessment of MON 87403.................................................................212

Appendix H: Materials, Methods, and Individual Site Results from Phenotypic,

Agronomic, and Environmental Interaction Assessment of MON 87403 under

Field Conditions...............................................................................................................218

Appendix I: Materials and Methods for Pollen Morphology and Viability

Assessment.......................................................................................................................257
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LIST OF TABLES

Table I-1. Differences in R1 Ear Weight between MON 87403 and the

Conventional Control in 2012 U.S. Field Trials1...............................................................26

Table III-1. Summary of Genetic Elements in Plasmid PV-ZMAP5714 .........................36

Table IV-1. Summary of Genetic Elements in MON 87403 ............................................45

Table IV-2. Unique Junction Sequence Class Results......................................................50

Table IV-3. Junction Sequence Classes Detected.............................................................56

Table IV-4. Segregation of the Expression Cassette During the Development of

MON 87403 .......................................................................................................................60

Table V-1. Summary of ATHB17Δ113 Protein Levels in Tissues from

MON 87403 Grown in 2012 United States Field Trials....................................................67

Table VI-1. Summary of Maize Grain Protein and Amino Acids for MON 87403,

Conventional Control, and Conventional References........................................................76

Table VI-2. Summary of Maize Grain Total Fat and Fatty Acids for MON 87403,

Conventional Control, and Conventional References........................................................80

Table VI-3. Summary of Maize Grain Carbohydrates by Calculation and Fiber for

MON 87403, Conventional Control, and Conventional References .................................82

Table VI-4. Summary of Maize Grain Ash and Minerals for MON 87403,

Conventional Control, and Conventional References........................................................83

Table VI-5. Summary of Maize Grain Vitamins for MON 87403, Conventional

Control, and Conventional References ..............................................................................85

Table VI-6. Summary of Maize Grain Anti-nutrients and Secondary Metabolites

for MON 87403, Conventional Control, and Conventional References............................87

Table VI-7. Summary of Maize Forage Proximates, Fiber, and Minerals for

MON 87403, Conventional Control, and Conventional References .................................88

Table VI-8. Literature and ILSI Database Ranges for Components in Maize

Forage and Grain................................................................................................................90

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

Characteristics Evaluated in U.S. Field Trials, and Laboratory Studies............................95

Table VII-2. Germination Characteristics of MON 87403 and the Conventional

Control .............................................................................................................................103

Table VII-3. Field Phenotypic Evaluation Sites for MON 87403 during 2012..............106

Table VII-4. Combined-Site Comparison of MON 87403 to Conventional

Control for Phenotypic and Agronomic Characteristics During 2012.............................107

Table VII-5. Summary of Qualitative Environmental Interactions Assessments

during 2012 ......................................................................................................................110

Table VII-6. Combined-Site Comparison of CEW and ECB Damage to

MON 87403 Compared to the Conventional Control during 2012 .................................112
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Table VII-7. Summary of Arthropod Abundance Assessments and Detected

Differences during 2012 ..................................................................................................113

Table VII-8. Pollen Characteristics of MON 87403 Compared to the

Conventional Control during 2012 ..................................................................................116

Table VIII-1. Maize Production in the U.S., 2008-2013 ................................................119

Table IX-1. Summary of Published Literature on Maize Cross Pollination...................129

Table A-1. USDA Notifications and Permits Approved for MON 87403 and

Status of Trials Planted ....................................................................................................148

Table B-1. Combined Site Analysis of R1 Ear Weight, R1 Stover Weight, Total

Plant Weight, and Partitioning in MON 87403 and the Conventional Control from

2012 U.S. Field Trials......................................................................................................161

Table C-1. Sequencing (NGS) Conducted for MON 87403 and Control Genomic

DNA.................................................................................................................................179

Table C-2. Summary of NGS Data for the Conventional Control DNA Sample

Spiked with PV-ZMAP5714............................................................................................180

Table D-1. Summary of the Tryptic Masses Identified for the MON 87403 -

produced ATHB17Δ113 using MALDI-TOF MS...........................................................187

Table D-2. Molecular Weight Comparison Between the MON 87403- and

E. coli-produced ATHB17Δ113 Proteins........................................................................191

Table E-1. ATHB17Δ113 Extraction Methods

1

for Tissue Samples..............................194

Table F-1. Conventional Commercial Reference Maize Hybrids ...................................196

Table F-2. Re-expression Formulas for Statistical Analysis of Composition Data........206

Table G-1. Starting Seed of MON 87403, Conventional Control and Commercial

Maize Reference Hybrids Used in Dormancy Assessment .............................................214

Table G-2. Dormancy and Germination Characteristics of MON 87403 and the

Conventional Control Seed Produced at each of the Three Field Sites ...........................215

Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental

Interaction Assessment ....................................................................................................220

Table H-2. Field and Planting Information.....................................................................222

Table H-3. Data Missing or Excluded from Analysis.....................................................231

Table H-4. Individual Site Analysis of Phenotypic Characteristics of MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials ..................................233

Table H-5. Abiotic Stressor Evaluations Using a Categorical Scale for

MON 87403 and the Conventional Control in 2012 U.S. Field Trials............................238

Table H-6. Disease Damage Evaluations Using a Categorical Scale for

MON 87403 and the Conventional Control in 2012 U.S. Field Trials............................239

Table H-7. Arthropod Damage Evaluations Using a Categorical Scale for

MON 87403 and the Conventional Control in 2012 U.S. Field Trials............................240
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Table H-8. Individual Site Analysis: Corn Earworm Damage and European Corn

Borer Damage to MON 87403 Compared to the Conventional Control in 2012

U.S. Field Trials...............................................................................................................241

Table H-9. Individual Site Analysis: Arthropod Abundance in Sticky Trap

Samples Collected from MON 87403 Compared to the Conventional Control in

2012 U.S. Field Trials......................................................................................................242

Table H-10. Individual Site Analysis: Arthropod Abundance in Visual Counts

from MON 87403 Compared to the Conventional Control in 2012 U.S. Field

Trials ................................................................................................................................250

Table I-1. Starting Seed for Pollen Morphology and Viability Assessment....................258
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LIST OF FIGURES

Figure III-1. Circular Map of PV-ZMAP5714 .................................................................33

Figure III-2. Schematic of the Development of MON 87403...........................................34

Figure IV-2. Junctions and Junction Sequences ...............................................................42

Figure IV-3. Two Unique Junction Sequence Classes are Produced by the

Insertion of a Single Plasmid Region.................................................................................42

Figure IV-4. Schematic Representation of the Insert and Flanking Sequences in

MON 87403 .......................................................................................................................47

Figure IV-5. Breeding History of MON 87403 .................................................................48

Figure IV-6. Junction Sequences Detected by NGS/JSA .................................................51

Figure IV-7. Overlapping PCR Analysis Across the Insert in MON 87403 ....................53

Figure IV-8. PCR Amplification of the MON 87403 Insertion Site.................................55

Figure IV-9. Breeding Path for Generating Segregation Data for MON 87403...............59

Figure V-1. Insert map of MON 87403 and schematic diagram of ATHB17

transcription, mRNA processing, and predicted translation ..............................................63

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

Interpretation Methods.......................................................................................................99

Figure VIII-1. Planted Maize Acres by County in the U.S. in 2012...............................119

Figure B-1. Maize developmental stages.........................................................................154

Figure B-2. US maize yields from 1866 to 2013 in bushels per acre ..............................154

Figure B-3. Relationship between ear biomass during early reproductive stage and

kernel number at harvest..................................................................................................156

Figure B-4: Domain structure of ATHB17......................................................................158

Figure B-5: Model for dominant negative mechanism of ATHB17∆113 .......................159

Figure B-6. Ear growth and ear partitioning in MON 87403 and the control at

different developmental stages in a greenhouse ..............................................................162

Figure B-7. Summary of mode-of-action of ATHB17Δ113 protein in MON 87403

maize................................................................................................................................163

Figure C-1. Sequencing and Sequence Selection ...........................................................169

Figure C-2. Junctions and Junction Sequences...............................................................170

Figure C-3. Two Unique Junction Sequence Classes are Produced by the

Insertion of a Single Plasmid Region...............................................................................172

Figure D-1. MALDI-TOF MS Coverage Map of the MON 87403-produced

ATHB17Δ113 ..................................................................................................................187

Figure D-2. Western Blot Analysis of MON 87403- and E. coli-produced

ATHB17Δ113 Proteins....................................................................................................190
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Figure D-3. Molecular Weight and Purity Analysis of the MON 87403-produced

ATHB17Δ113 Protein......................................................................................................192

Figure I-1. General Morphology of Pollen from MON 87403, the Conventional

Control, and Commercially Available Conventional Reference Materials under

200X Magnification.........................................................................................................259
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ABBREVIATIONS AND DEFINITIONS1

~ approximately

AA amino acid

ADF acid detergent fiber

ANOVA analysis of variance

APHIS Animal and Plant Health Inspection Service

ATHB17 a member of the class II family of homeodomain-leucine zipper (HDZip) transcription factors

ATHB17Δ113 the protein expressed from the ATHB17 gene in MON 87403

BLASTX Basic Local Alignment Search Tool

B-Left Border

Region

DNA region from Agrobacterium tumefaciens containing the left

border sequence

B-Right Border

Region

DNA region from Agrobacterium tumefaciens containing the right

border sequence

BSA bovine serum albumin

CaMV cauliflower mosaic virus

CEW corn earworm

CFR Code of Federal Regulations

CTAB cetyltrimethylammonium bromide

DAS days after sowing

DEEM-FCID Dietary Exposure Evaluation Model - Food Commodity Intake

Database

DTT dithiothreitol

DWCF dry weight conversion factor

dwt dry weight of tissue

E. coli Escherichia coli

ECB European corn borer

EDTA ethylenediaminetetraacetic

ELISA enzyme-linked immunosorbent assay

EPA Environmental Protection Agency

FA fatty acid

FDA Food and Drug Administration

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FMOC fluorenylmethyl chloroformate

Gb gigabase

GC gas chromatography

GE genetically engineered

GLP Good Laboratory Practice

GRIN Germplasm Resources Information Network

HD homeodomain

HD-Zip homeodomain leucine zipper class family of transcription factors

HPLC high-performance liquid chromatography

1 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 CR262-14U1 23 of 260

HRP horseradish peroxidase

ICP inductively coupled plasma

IgG immunoglobulin G

ILSI-CCDB International Life Sciences Institute Crop Composition Database

JSA junction sequence analysis

JSC junction sequence class

LB loading buffer

LOD limit of detection

LOQ limit of quantitation

LZ leucine zipper

MALDI TOF MS matrix assisted laser desorption /ionization - time of flight mass

spectrometry

MIXED

a statistical tool developed by the SAS Institute that uses a mixedmodel ANOVA to analyze data

MMT million metric tons

MOE margin of exposure

MW molecular weight

n number of samples

NDF neutral detergent fiber

NFDM non-fat dry milk

NGS next generation sequencing

NHANES National Health and Nutrition Examination Survey

NOAEL no observable adverse effect level

OECD Organisation for Economic Co-operation and Development

OGTR Office of the Gene Technology Regulator (Australia)

OPA o-phthalaldehyde

OSL over season leaf

OSR over season root

PBST phosphate-buffered saline with Tween- 20

PCR polymerase chain reaction

PMSF phenylmethylsulfonyl fluoride

ppm parts per million

PRESS predicted residual sum of squares

PVP polyvinylpyrrolidone

QC quality control

RT-PCR reverse transcription polymersae chain reaction

SD standard deviation

SDS sodium dodecyl sulfate

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE standard error

SGF simulated gastric fluid

SIF simulated intestinal fluid

SOP standard operating procedure

TDF total dietary fiber

TFA trifluoroacetic acid

UTR untranslated region

v/v volume to volume ratio

UV ultraviolet

w/v weight to volume ratio
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I. RATIONALE FOR THE DEVELOPMENT OF MON 87403

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 (U.S.)

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 maize product, MON 87403, any

progeny derived from crosses between MON 87403 and conventional maize, and any

progeny derived from crosses of MON 87403 with biotechnology-derived maize that

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

I.B. Rationale for the Development of MON 87403

Monsanto Company has developed biotechnology-derived maize MON 87403 that has

increased ear biomass at an early reproductive stage (R1) compared to conventional

control maize. MON 87403 was produced through insertion of the coding region of the

full-length Arabidopsis thaliana ATHB17 gene through Agrobacterium-mediated

transformation. The ATHB17 protein is a member of the HD-Zip family of plant

transcription factors, which are proteins that bind to specific DNA sequences and regulate

gene expression. The HD-Zip family of proteins is found broadly across plant species

and specific HD-Zip proteins have been shown to play an important role in the

modulation of plant growth and development. The HD-Zip family consists of four

subfamilies and ATHB17 is a member of the class II subfamily. HD-Zip II proteins form

either homodimers or heterodimers with other HD-Zip II proteins and function as

repressors of gene expression. In MON 87403, maize-specific splicing of the ATHB17

transcript results in a truncated protein, ATHB17Δ113, which is missing the first 113 Nterminal amino acids that are expressed in Arabidopsis. The ATHB17∆113 protein

retains the ability to form homo- and hetero-dimers and bind to target DNA sequences

like the full-length protein. ATHB17∆113 is, however, unable to function as a

transcriptional repressor because the protein lacks a functional repression domain. By a

dominant-negative mechanism, ATHB17∆113 can alter the activity of endogenous maize

HD-Zip II proteins, which are predominantly expressed in ear tissue. Thus, the

ATHB17∆113 protein likely modulates HD-Zip II-regulated pathways in the ear, which

leads to increased ear growth at an early reproductive stage (R1). This increased ear

growth is associated with increased partitioning of dry matter (photosynthate) from the

source (vegetative) tissue to the sink (ear) tissue in MON 87403 compared to

conventional control plants. Supplemental information on the function of the
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ATHB17∆113 protein in MON 87403 is described in Appendix B: and is also published

in Rice et al. (2014).

Maize is one of the largest U.S. crops based on acreage and quantity harvested each year.

In 2013 maize was planted on 35.48 million ha in the United States (USDA-FAS 2014).

Because of its importance, plant breeders continuously strive to improve commercial

maize yield. Initial improvements in maize yield were due to the domestication of

varieties with desirable traits like larger ear biomass. Maize ears became larger over time

during the domestication era (University of Utah 2014). During the hybrid era (1939 to

present), commercial maize yield in the U.S. increased nearly six-fold with an average

99 kg ha-1 increase every year (Lee and Tollenaar 2007). The major factor that

contributed to yield increase during the hybrid era is a favorable response of hybrids to

increased plant population density which resulted in an increase in the number of ears per

hectare (Bruns and Abbas 2003). However, at the individual plant level, yield increase

was associated with a decrease in ear size. Commercial varieties with MON 87403 can

provide increased yield opportunity as this trait increases ear biomass during early

reproductive stages.

Because of the expected increase in human population growth over time, increased

agricultural output will be required to come from increased productivity (i.e., yield per

unit area) as opposed to an increase in area under production (OECD-FAO 2008).

Agricultural biotechnology provides tools to help address the increasing demand for food

and feed due to this population growth. Augmenting gains in yield opportunity from

biotechnology as well as with continual gains from breeding would have a positive

impact on the U.S. economy. For example, increasing the average rate of yield gain by

just 1 bu/ac/year, over and above historical average yield gains for the next ten years,

would have a net economic impact of $16B USD (Leibman et al. 2014).

Early reproductive stages in maize have been identified as a crucial phase during maize

growth when a determination of maximum ear size is set based on plant genetics and

environmental conditions (Borrás and Westgate 2006; Jones et al. 1996). Dry matter

produced by the plant during reproductive stages is allocated to the ear for its growth

(Ritchie et al. 1997) after the sink size is determined. Thus increase in ear size that is set

during early reproductive stages is considered an important determinant of reproductive

success (Borrás et al. 2004; Zaidi et al. 2003). Published literature suggests that a larger

ear size at early reproductive stages can result in increased kernels per hectare (Fisher and

Palmer 1983; Severini et al. 2011) thus potentially increasing agronomic benefit to

farmers.

Efficacy of MON 87403 was demonstrated by directly comparing its R1 (silking stage)

ear weight to a conventional control with the same genetic background. Ear weight is the

total weight of the primary ear including the husk, shank, cob, silk and ovules at the R1

stage. MON 87403 and the control were grown at 13 field locations within U.S. maize

production regions in 2012. MON 87403 and the control were planted at each site in

randomized complete block designs with four replications. Measurements were collected

from all of the plants at the R1 stage in a 1 m length of the designated row. Statistical

comparisons were made between MON 87403 and the control across all 13 sites
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(combined-site analyses). The level of statistical significance for all statistical

comparisons was predetermined to be 5% (α=0.05).

There was a statistically significant increase in R1 ear weight between MON 87403 and

the control in the combined site analysis (Table I-1). The R1 ear weight in MON 87403

was 11.7% higher than the control (Table I-1).

Table I-1. Differences in R1 Ear Weight between MON 87403 and the Conventional

Control in 2012 U.S. Field Trials1

Characteristic (units) MON 87403

(Mean ± SE)

Control

(Mean ± SE)

Change (%) p-value

R1 ear weight2(g) 144.50 (±8.47) 129.30 (±8.13) 11.7 0.004*

1 Locations included in the combined-site analysis: Jackson, Arkansas; Vermilion, Illinois; Warren, Illinois;

Boone, Indiana; Greene, Iowa; Jefferson, Iowa; Pawnee, Kansas; (2 sites) Polk, Nebraska; York, Nebraska;

Perquimans, North Carolina; Berks, Pennsylvania; Lehigh, Pennsylvania.

2

R1 ear weight is the dry weight of the entire primary ear at silking stage from all plants in the 1 m

sampling.

*Denotes statistical difference between MON 87403 and the control (α = 0.05). N = 51.

In summary, MON 87403 provides a potential benefit to growers and the food and feed

industry by conferring an increase in yield opportunity for maize.

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 plant incorporated

protectants (PIPs), the Environmental Protection Agency (EPA). Deregulation of

MON 87403 by USDA constitutes only one component of the overall regulatory

oversight and review of this product. As a practical matter, MON 87403 cannot be

released and marketed until FDA and USDA have completed their reviews and

assessments under their respective jurisdictions. As MON 87403 does not contain a PIP,

no submission will be made to the EPA.

I.C.1. Submission to FDA

MON 87403 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 accordance with this policy, Monsanto will

be submitting a food/feed safety and nutritional assessment summary document to FDA

in the near future.
Monsanto Company CR262-14U1 27 of 260

I.C.2. Submissions to Foreign Government Agencies

Consistent with our commitments to the Excellence Through Stewardship®(ETS)

Program2, Monsanto intends to obtain import approvals from all key maize import

markets with functioning regulatory systems prior to commercial release of hybrids

containing MON 87403.

® Excellence Through Stewardship is a registered trademark of Excellence Through Stewardship,

Washington, DC.

2

http://www.excellencethroughstewardship.org/.
Monsanto Company CR262-14U1 28 of 260

II. THE BIOLOGY OF MAIZE

The Organisation for Economic Co-operation and Development (OECD) Consensus

Document on the biology of maize (OECD 2003) provides key information on:

- general description of maize biology, including taxonomy and morphology and use

of maize as a crop plant

- agronomic practices in maize cultivation

- geographic centers of origin

- reproductive biology

- cultivated maize as a volunteer weed

- inter-species/genus introgression into relatives and interactions with other

organisms

- a summary of the ecology of maize

Additional information on the biology and uses of maize can also be found on the

Australian Government Department of Health and Ageing (Office of the Gene

Technology Regulator) web site (OGTR 2008), and in the USDA-ARS GRIN database

(USDA-ARS 2013).

To support the evaluation of the plant pest potential of MON 87403 relative to

conventional maize, additional information regarding several aspects of maize biology

can be found elsewhere in this petition. This includes: agronomic practices for maize in

Section VIII; volunteer management of maize in Section VIII.H; and inter-species/genus

introgression potential in Section IX.G.

II.A. Maize as a Crop

Maize is grown in nearly all areas of the world and is the largest cultivated crop in the

world followed by wheat (Triticum sp.) and rice (Oryza sativa L.) in total global metric

ton production. In 2012, maize was planted globally on ~174 million hectares (ha) with a

total grain production of an estimated 854 million metric tons (MMT) (USDA-FAS

2013). The top five production regions in 2012 were: USA (274 MMT), China (208

MMT), Brazil (73 MMT), EU-27 (55 MMT), and Argentina (27 MMT) (USDA-FAS

2013). In the U.S., maize is grown in almost every state and in 2012, its production value

of over $77 billion was the highest of any crop (USDA-NASS 2013a).

In industrialized countries maize has two major uses: (1) as animal feed in the form of

grain, forage or silage; and (2) as a raw material for wet- or dry-milled processed

products such as high fructose maize syrup, oil, starch, glucose, dextrose and ethanol.

By-products of the wet- and dry- mill processes are also used as animal feed. These

processed products are used as ingredients in many industrial applications and in human

food products. Most maize produced in industrialized countries is used as animal feed or

for industrial purposes, but maize remains an important food staple in many developing

regions, especially sub-Saharan Africa and Central America, where it is frequently the

mainstay of human diets (Morris 1998).
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Maize is a very familiar plant that has been rigorously studied due to its use as a staple

food/feed and the economic opportunity it brings to growers. The domestication of maize

likely occurred in southern Mexico between 7,000 and 10,000 years ago (Goodman

1988). While the putative progenitor species of maize have not been recovered, it is

likely that teosinte played an important role in contributing to the genetic background of

maize. Although grown extensively throughout the world, maize is not considered a

persistent weed or a plant that is difficult to control. Maize, as we know it today, cannot

survive in the wild because the female inflorescence (the ear) is covered by a husk

thereby restricting seed dispersal, it has no seed dormancy, and is a poor competitor in an

unmanaged ecosystem. The transformation from a wild, weedy species to one dependent

on humans for its survival most likely evolved over a long period of time through plant

breeding by the indigenous inhabitants of the Western Hemisphere. Today, virtually all

maize varieties grown in the U.S. are hybrids, a production practice that started in the

1930’s (Wych 1988). Maize hybrids are developed and used based on the positive yield

increases and plant vigor associated with heterosis, also known as hybrid vigor (Duvick

1999).

Conventional plant breeding results in desirable characteristics in a plant through the

unique combination of genes already present in the plant. However, there is a limit to

genetic diversity with conventional plant breeding. Biotechnology, as an additional tool

to conventional breeding, offers access to greater genetic diversity than conventional

breeding alone, resulting in expression of highly desirable traits that are profitable to

growers.

II.B. Characteristics of the Recipient Plant

The transformation for MON 87403 was conducted with inbred maize line LH244, a

patented maize line assigned to Holden’s Foundation Seeds LLC in 2001

(U.S. Patent #6,252,148). LH244 is a medium season yellow dent maize line with a Stiff

Stalk background that is best adapted to the central regions of the U.S. corn belt. LH244

was initiated from a single cross of LH197 × LH199 followed by a backcross to LH197.

The F2 combination ((LH197 × LH197) × LH199) was then selfed and used in the

development of LH244.

Following transformation of immature LH244 embryos, a single transformed plant was

selected and self-crossed to increase seed supplies. A homozygous inbred line was

developed though further self-crossing and selection and was then used to produce other

lines which were used for product testing, safety assessment studies, and commercial

hybrid development.

II.C. Maize as a Test System in Product Safety Assessment

Hybrid maize MPA640B (LH244 × LH287) and EXP257 (LH244 × LH295) were used

as near isogenic, conventional controls for this submission (hereafter referred to as

conventional controls), based on seed availability. As noted, one parent of each of these

control maize lines is LH244, the inbred from which MON 87403 is derived, while the

other parents (LH287 and LH295) are other maize inbreds. As such, both of these maize
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hybrids constitute relevant comparators for MON 87403. In addition, other commercial

maize hybrids (hereafter referred to as reference hybrids) were used to establish ranges of

natural variability representative of commercial maize hybrids. Reference hybrids used

at each field trial location were selected based on their availability and agronomic fit for

the respective geographic regions. Both MPA640B and EXP257 were used in molecular

characterization studies, while MPA640B was used as the conventional control in

compositional analysis and in phenotypic, agronomic and environmental interactions

assessments. Where appropriate, reference hybrids were used to establish a range of

variability or responses representative of commercial maize in the U.S.

In developing the data to support this petition, appropriate MON 87403 test materials

were generated for the molecular characterization (Sections III and IV), protein

characterization and expression analysis (Section V), compositional analysis (Section

VI), and phenotypic, agronomic and environmental interactions assessment (Section VII).

The full molecular characterization studies (NGS/JSA) were conducted with the R3

generation (Figure IV-4). Initiation of commercial breeding efforts was conducted with

the R4 generation (Figure IV-4). Protein characterization and expression analysis,

composition analysis, and phenotypic, agronomic and environmental interactions

assessment were conducted with various MON 87403 breeding generations as noted in

the Breeding Tree (Figure IV-4).
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III.DESCRIPTION OF THE GENETIC MODIFICATION

MON 87403 was developed through Agrobacterium tumefaciens-mediated

transformation of maize immature embryos from line LH244 utilizing plasmid

PV-ZMAP5714. This section describes the plasmid vector, the donor genes, and the

regulatory elements used in the development of MON 87403. In this section, transfer

DNA (T-DNA) refers to DNA that is transferred to the plant during transformation and

retained through the development of MON 87403. An expression cassette is comprised

of sequences to be transcribed and the genetic regulatory elements necessary for the

expression of those sequences.

III.A. The Plasmid Vector PV-ZMAP5714

Plasmid vector PV-ZMAP5714 was used for the transformation of maize to produce

MON 87403 and its plasmid map is shown in Figure III-1. The elements included in this

plasmid vector are described in Table III-1. PV-ZMAP5714 is approximately 11.7 kb in

length and contains three cassettes: one T-DNA element, delineated by Left and Right

Border regions, contains the ATHB17 expression cassette, and the plasmid backbone

contains the cp4 epsps selectable marker cassette and the aadA expression cassette.

PV-ZMAP5714 employs a tandem T-DNA approach to generate marker-free plants

(Huang et al. 2004). In this tandem T-DNA approach, a single right border and a single

left border were used to achieve separate, unlinked insertion of the T-DNA as well as the

cp4 epsps selectable marker gene located in the plasmid backbone. After initial selection

of transformants for glyphosate tolerance, the cp4 epsps cassette was segregated away by

conventional breeding and molecular analysis was used to identify plants containing only

the intended T-DNA (and not the cp4 epsps).

The T-DNA contains the ATHB17 expression cassette. The ATHB17 expression cassette

is regulated by the e35s/Ract1 chimeric promoter from the 35S RNA of cauliflower

mosaic virus (CaMV) and the act1 gene from Oryza sativa , the 5 ́ untranslated leader

sequence of the Cab gene from Triticum aestivum, the act1 intron from Oryza sativa, and

the 3 ́ untranslated region of heat shock protein 17 (Hsp17) of Triticum aestivum. The

plasmid backbone contains the cp4 epsps and aadA expression cassettes. The cp4 epsps

expression cassette is regulated by the act1 promoter from Oryza sativa, the act1 intron

from Oryza sativa, the CTP2 targeting sequence from Arabidopsis thaliana, and the nos

3′ untranslated region from Agrobacterium tumefaciens. The aadA expression cassette is

regulated by the bacterial promoter, and 3' untranslated region of an aminoglycosidemodifying enzyme, 3''(9)-O-nucleotidyltransferase from the transposon Tn7. During

transformation, both the T-DNA and the cp4 epsps expression cassette were inserted into

the maize genome (Section III.B). Subsequently, traditional breeding, segregation,

selection and screening were used to isolate those plants that contain the ATHB17

expression cassette (T-DNA) and do not contain the cp4 epsps and aadA expression

cassettes (backbone).

The backbone region of PV-ZMAP5714, located outside of the T-DNA, contains two

origins of replication for maintenance of the plasmid vector in bacteria (ori-V, ori-pUC),

a bacterial selectable marker gene (aadA), and a coding sequence for repressor of primer
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(ROP) protein for the maintenance of the plasmid vector copy number in Escherichia

coli (E. coli). The backbone also contains the cp4 epsps expression cassette. A

description of the genetic elements and their prefixes (e.g., B-, P-, L-, I-, CS-, T-, and

OR-) in PV-ZMAP5714 is provided in Table III-1.

III.B. Description of the Transformation System

MON 87403 was developed through Agrobacterium tumefaciens-mediated

transformation of immature maize embryos based on the method described by Sidorov

and Duncan (2009), utilizing PV-ZMAP5714. Immature embryos were excised from a

post-pollinated maize ear of LH244. After co-culturing the excised immature embryos

with Agrobacterium carrying the plasmid vector, the immature embryos were placed on

selection medium containing glyphosate and carbenicillin disodium salt in order to inhibit

the growth of untransformed plant cells and excess Agrobacterium, respectively. Once

transformed callus developed, the callus was placed on media conducive to shoot and

root development. The rooted plants (R0) with normal phenotypic characteristics were

selected and transferred to soil for growth and further assessment. As demonstrated in

this petition (Section VII), the use of disarmed Agrobacterium tumefaciens strain ABI, a

designated plant pest, as the transformation vector has not imparted plant pest

characteristics to MON 87403.

The R0 plants generated through the transformation process described above had already

been exposed to glyphosate in the selection medium and demonstrated glyphosate

tolerance. The R0 plants containing the cp4 epsps expression cassette, as well as the TDNA cassette from the plasmid vector backbone, were self-pollinated to produce R1 seed

and R1 plants (Huang et al. 2004). Subsequently, R1 plants that were positive for the

T-DNA and negative for the cp4 epsps expression cassette were identified by a

polymerase chain reaction (PCR) based analysis (Huang et al. 2004). The R1 plants

homozygous for the T-DNA were selected for further development and their progenies

were subjected to further molecular and phenotypic assessments. As is typical of a

commercial event production and selection process, hundreds of different transformation

events (regenerants) were generated in the laboratory using PV-ZMAP5714. After many

months of careful selection and evaluation of these hundreds of events in the laboratory,

greenhouse and field, MON 87403 was selected as the lead event based on superior

agronomic, phenotypic, and molecular characteristics (Prado et al. 2014). Studies on

MON 87403 were initiated to further characterize the genetic insertion and the expressed

product, and to establish the food, feed, and environmental safety relative to commercial

maize. The major steps involved in the development of MON 87403 are depicted in

Figure III-2.

III.C. The ATHB17 Coding Sequence and the ATHB17 Protein (T-DNA)

The ATHB17 coding sequence is from Arabidopsis thaliana and encodes the ATHB17

protein (Figure III-3). For additional information about the truncated ATHB17 protein

produced in MON 87403, see Section V.
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Figure III-1. Circular Map of PV-ZMAP5714

A circular map of PV-ZMAP5714 used to develop MON 87403 is shown.

PV-ZMAP5714 contains a single T-DNA. Genetic elements are shown on the exterior of

the map.
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Figure III-2. Schematic of the Development of MON 87403

1 MIKLLFTYIC TYTYKLYALY HMDYACVCMY KYKGIVTLQV CLFYIKLRVF

51 LSNFTFSSSI LALKNPNNSL IKIMAILPEN SSNLDLTISV PGFSSSPLSD

101 EGSGGGRDQL RLDMNRLPSS EDGDDEEFSH DDGSAPPRKK LRLTREQSRL

151 LEDSFRQNHT LNPKQKEVLA KHLMLRPRQI EVWFQNRRAR SKLKQTEMEC

201 EYLKRWFGSL TEENHRLHRE VEELRAMKVG PTTVNSASSL TMCPRCERVT

251 PAASPSRAVV PVPAKKTFPP QERDR

Figure III-3. Deduced Amino Acid Sequence of the ATHB17 Protein

The amino acid sequence of the ATHB17 protein was deduced from the full-length

coding nucleotide sequence present in PV-ZMAP5714. The amino-terminal 113 amino

acids (highlighted in gray) are not predicted to be translated in MON 87403 as a result of

mRNA splicing.

Transformed LH244 (a maize line for more efficient transformation)

immature embryos with PV-ZMAP5714 in Agrobacterium tumefaciens

Selected transformants containing the selectable marker (cp4 epsps

expression cassette) and generated rooted shoots from the transformed

callus tissues

Identified MON 87403 as lead event and further evaluated its progeny in

laboratory and field assessments for T-DNA insert integrity, absence of

all other vector DNA including cp4 epsps, and superior phenotypic

characteristics

Assembled Agrobacterium binary plasmid vector PV-ZMAP57147 and

transferred to Agrobacterium tumefaciens strain ABI

Evaluated by PCR and selected the transformed plants for the homozygous

presence of the T-DNA and absence of the vector backbone that includes

the cp4 epsps expression cassette

Evaluated plants for insert integrity using molecular analyses
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III.D. Regulatory Sequences

The ATHB17 coding sequence in MON 87403 is under the regulation of the e35S/Ract1

promoter, the chlorophyll a/b binding protein (CAB) leader, the Ract1 intron, and the

heat shock protein 17 (Hsp17) 3' untranslated region. The e35S/Ract1 promoter, which

directs transcription in plant cells, is a chimeric promoter consisting of the duplicated

enhancer region from the cauliflower mosaic virus 35S RNA promoter (CaMV) (Kay et

al. 1987) combined with the promoter of the act1 gene from Oryza sativa that encodes

Actin 1 (McElroy et al. 1990). The Cab leader sequence is the 5' untranslated region

from the chlorophyll a/b-binding (CAB) protein of Triticum aestivum and is involved in

regulating gene expression (Lamppa et al. 1985). The Ract1 intron is the intron from the

act1 gene from Oryza sativa (McElroy et al. 1990). The Hsp17 3' non-translated region

is the 3' untranslated region from the heat shock protein, Hsp17, of Triticum aestivum

(McElwain and Spiker 1989) that directs polyadenylation of the mRNA.

III.E. T-DNA Borders

PV-ZMAP5714 contains Left and Right Border regions (Figure III-1 and Table III-1) that

were derived from A. 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 maize genome. As demonstrated in this petition (Section VII), the use of genetic

elements from A. tumefaciens, a designated plant pest, has not imparted plant pest

characteristics to MON 87403.

III.F. 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-ZMAP5714 in bacteria and are referred

to as plasmid backbone. The origin of replication, ori V, 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-pUC, is required for the maintenance of the

plasmid in E. coli and is derived from the plasmid vector pUC (Vieira and Messing

1987). Coding sequence rop encodes the repressor of primer (ROP) protein which is

necessary for the maintenance of plasmid vector copy number in E. coli (Giza and Huang

1989). The backbone also contains the cp4 epsps expression cassette that codes for the

CP4 EPSPS protein (conferring tolerance to glyphosate) that was used as the selectable

marker during transformation (Huang et al. 2004). The cp4 epsps expression cassette is

regulated by the Ract1 promoter from Oryza sativa, the Ract1 intron from Oryza sativa,

the CTP2 targeting sequence from Arabidopsis thaliana, and the nos 3′ untranslated

region from Agrobacterium tumefaciens. The absence of the backbone and other

unintended plasmid sequence in MON 87403 was confirmed by sequencing and

bioinformatic analyses (see Section IV.A).
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Table III-1. Summary of Genetic Elements in Plasmid PV-ZMAP5714

Genetic Element Location in Plasmid Function (Reference)

T-DNA

B

1

-Right Border

Region

1-357 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 358-375 Sequence used in DNA cloning

P

2

-e35S/Ract1 376-1556 Chimeric promoter consisting of the duplicated

enhancer region from the cauliflower mosaic

virus 35S RNA promoter (CaMV) (Kay et al.

1987) combined with the promoter of the act1

gene from Oryza sativa that encodes Actin 1

(McElroy et al. 1990) that directs transcription

in plant cells

Intervening Sequence 1557-1561 Sequence used in DNA cloning

L

3

-Cab 1562-1622 5' UTR leader sequence from chlorophyll a/bbinding (CAB) protein of Triticum aestivum

(wheat) that is involved in regulating gene

expression (Lamppa et al. 1985)

Intervening Sequence 1623-1638 Sequence used in DNA cloning

I

4

-Ract1 1639-2118 Intron and flanking UTR sequence of the act1

gene from Oryza sativa (rice) encoding rice

Actin 1 protein. This sequence is involved in

regulating gene expression (McElroy et al.

1990)

Intervening Sequence 2119-2130 Sequence used in DNA cloning

CS5-ATHB17 2131-2958 Coding sequence of the ATHB17 gene from

Arabidopsis thaliana encoding a member of

the class II homeodomain-leucine zipper gene

family (HD-Zip II) that is thought to act as a

transcription factor (Ariel et al. 2007)

Intervening Sequence 2959-2971 Sequence used in DNA cloning

T

6

-Hsp17 2972-3181 3' UTR sequence from a heat shock protein,

Hsp17, of Triticum aestivum (wheat)

(McElwain and Spiker 1989) that directs

polyadenylation of the mRNA

Intervening Sequence 3182-3234 Sequence used in DNA cloning

B-Left Border Region 3235-3676 DNA region from Agrobacterium tumefaciens

containing the left border sequence used for

transfer of the T-DNA (Barker et al. 1983)
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Table III-1 (continued). Summary of Genetic Elements in Plasmid PV-ZMAP5714

Genetic Element Location in Plasmid Function (Reference)

Backbone

Intervening Sequence 3677-3682 Sequence used in DNA cloning

P-Ract1 3683-4603 Promoter and leader of the act1 gene from

Oryza sativa (rice) encoding the rice Actin 1

protein (McElroy et al. 1990) that directs

transcription in plant cells

I-Ract1 4604-5081 Intron and flanking UTR sequence of the act1

gene from Oryza sativa (rice) encoding rice

Actin 1 protein (McElroy et al. 1990). This

sequence is involved in regulating gene

expression

Intervening Sequence 5082-5090 Sequence used in DNA cloning

TS7-CTP2 5091-5318 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)

CS-cp4 epsps 5319-6686 Coding sequence of the aroA gene from

Agrobacterium sp. strain CP4 encoding the

CP4 EPSPS protein that provides glyphosate

tolerance (Barry et al. 2001); (Padgette et al.

1996)

Intervening Sequence 6687-6701 Sequence used in DNA cloning

T-nos 6702-6954 3' UTR sequence of the nopaline synthase

(nos) gene from Agrobacterium tumefaciens

pTi encoding NOS (Bevan et al. 1983; Fraley

et al. 1983), that directs polyadenylation of the

mRNA

Intervening Sequence 6955-7005 Sequence used in DNA cloning

OR8-ori V 7006-7402 Origin of replication from the broad host range

plasmid RK2, used for maintenance of plasmid

in Agrobacterium (Stalker et al. 1981)

Intervening Sequence 7403-8910 Sequence used in DNA cloning

CS-rop 8911-9102 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 9103-9529 Sequence used in DNA cloning

OR-ori-pUC 9530-10118 Origin of replication from plasmid pUC for

maintenance of plasmid in E. coli (Vieira and

Messing 1987)
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Table III-1 (continued). Summary of Genetic Elements in Plasmid PV-ZMAP5714

Genetic Element Location in Plasmid Function (Reference)

Intervening Sequence 10119-10648 Sequence used in DNA cloning

aadA 10649-11537 Bacterial promoter, coding sequence, and 3'

UTR for an aminoglycoside-modifying

enzyme, 3''(9)-O-nucleotidyltransferase from

the transposon Tn7 (Fling et al. 1985). This

sequence confers spectinomycin and

streptomycin resistance

Intervening Sequence 11538-11673 Sequence used in DNA cloning

1B, Border

2

P, Promoter

3 L, Leader

4

I, Intron

5CS, Coding Sequence

6 T, Transcription Termination Sequence

7 TS,Targeting Sequence

8 OR, Origin of Replication
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IV.CHARACTERIZATION OF THE GENETIC MODIFICATION

Characterization of the DNA insert in MON 87403 was conducted using a combination

of sequencing, PCR, and bioinformatics. The results of this characterization demonstrate

that MON 87403 contains one copy of the intended transfer DNA (T-DNA) containing

the ATHB17 expression cassette that is stably integrated at a single locus and is inherited

according to Mendelian principles over multiple generations. These conclusions are

based on several lines of evidence:

1. Molecular characterization of MON 87403 by Next Generation Sequencing and

Junction Sequence Analysis (NGS/JSA) demonstrated that MON 87403 contained a

single intended DNA insert. These whole-genome sequence analyses provided a

comprehensive assessment of MON 87403 to determine the presence and identity of

sequences derived from PV-ZMAP5714 (DuBose et al. 2013; Kovalic et al. 2012),

demonstrating that MON 87403 contained a single T-DNA insert with no detectable

backbone sequences.

2. Directed sequencing (locus-specific PCR, DNA sequencing and analyses) performed

on MON 87403 was used to determine the complete sequence of the single DNA

insert from PV-ZMAP5714, the adjacent flanking DNA, and the 5' and 3' insert-toflank junctions. This analysis confirmed that the sequence and organization of the

inserted DNA is identical to the corresponding region in the PV-ZMAP5714

T-DNA. Furthermore, the genomic organization at the insertion site was assessed by

comparing the sequences flanking the T-DNA insert in MON 87403 to the sequence

of the insertion site in conventional maize. This analysis determined that no major

DNA rearrangement occurred at the insertion site in MON 87403 upon DNA

integration.

3. Generational stability analysis by NGS/JSA demonstrated that the single

PV-ZMAP5714 T-DNA insert in MON 87403 has been maintained through five

breeding generations, thereby confirming the stability of the T-DNA in MON 87403.

4. Segregation analysis corroborates the insert stability demonstrated by NGS/JSA and

independently establishes the nature of the T-DNA as a single chromosomal locus.

Taken together, the characterization of the genetic modification in MON 87403

demonstrates that a single copy of the intended T-DNA was stably integrated at a single

locus of the maize genome and that no plasmid backbone sequences are present in

MON 87403.

A schematic representation of the NGS/JSA methodology and the basis of the

characterization using NGS/JSA and PCR sequencing are illustrated in Figure IV-1

below. Appendix C provides an additional overview of these techniques, their use in

DNA characterization in crop plants and the materials and methods.
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Figure IV-1. Molecular Characterization using Sequencing and Bioinformatics

Genomic DNA from the test and the conventional control was sequenced using

technology that produces a set of short, randomly distributed sequence reads (each

approximately 100 bp long) that comprehensively cover both genomes (Step 1).

Utilizing these genomic sequence reads, bioinformatics search tools were used to select

all sequence reads that were significantly similar to the transformation plasmid (Step 2)

for use in read mapping to determine the presence/absence of backbone sequences and

Junction Sequence Analysis (JSA) bioinformatics to determine the insert and copy

number (Step 3). Overlapping PCR products are also produced which span any inserts

and their wild type locus (Step 4 and Step 5 respectively); these overlapping PCR

products are sequenced to allow for detailed characterization of the inserted DNA and

insertion site(s).

The NGS/JSA method characterized the genomic DNA from MON 87403 and the

conventional control using short (~100 bp) randomly distributed sequence fragments

(sequencing reads) generated in sufficient number to ensure comprehensive coverage of

the sample genomes. It has previously been demonstrated that 75× coverage of the

soybean genome is adequate to provide comprehensive coverage and ensure detection of

inserted DNA (Kovalic et al. 2012) and similarly 75× coverage provides comprehensive

coverage of the maize genome (Clarke and Carbon 1976). To confirm sufficient

sequence coverage of the genome, the 100-mer sequence reads are analyzed to determine

the coverage of a known single-copy endogenous gene, this demonstrates the depth of

coverage (the median number of times each base of the genome is independently

sequenced). The level of sensitivity of this method was demonstrated by detection of a

positive control spiked at 1 and 1/10th copy-per-genome equivalent, thus confirming the

Step 1: Next GenerationSequencing (NGS) of

genomic DNA samples. A collection of 100-mer

sequences are generatedwhich comprehensively

cover the test andcontrol sample genomes

Step 2: Selection of all 100-mers containing

sequence significantly similar to that of the

transformation plasmid

Step 3: Bioinformatic analysis to detect and

characterize all selected100-mersequences

originating from the transgenic insertions

Step 4: Directed sequencing across the insertion

from the 5' flank to the 3' flank

Step 5: Directedsequencing across the wildtype

insertion site

1) Presence or absence of backbone insertions:

No unintendedbackbone sequences

detected (Read mapping)

2) Insert and copy number determined:

Junctionsequence pairs detected (JSA)

3) Exact sequence of insert(s) is determined

4) Organization and intactness of genetic

elements in the insert sequence is confirmed

5) Integrity andorganization of insertionsite(s)

Experimental Stage Resultant MolecularCharacterization

NGS/JSA

Directed

Sequencing
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method’s ability to detect any sequences derived from the transformation plasmid.

Bioinformatics analysis was then used to select sequencing reads that contained

sequences similar to the transformation plasmid, and these were analysed in depth to

determine the number and the identity of sequence in the DNA inserts. NGS/JSA was

run on all 5 generations of MON 87403 samples and the conventional controls. Results

of NGS/JSA are shown in Sections IV.A and IV.D.

The number of DNA inserts was determined by analyzing sequence reads for novel

junctions, while the identity of the inserted DNA and absence of backbone was assessed

by sequence read mapping. The junctions of the DNA insert and the flanking DNA are

unique for each insertion (Kovalic et al. 2012). An example is shown in Figure IV-2.

Therefore, insertion sites can be recognized by analyzing for sequence reads containing

such junctions.

Each insertion will produce two unique junction sequence classes characteristic of the

genomic locus, one at the 5' end of the insert (Figure IV-3, named junction sequence class

A, or JSC-A, in this case) and one at the 3' end of the insert (junction sequence class B,

JSC-B) (Kovalic et al. 2012).
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Figure IV-2. Junctions and Junction Sequences

Depicted above are five example junction sequences formatted and labeled to indicate the

plasmid/flanking DNA portions of the sequences and with the junction point indicated

(plasmid DNA is shown in bold, underlined text and flank DNA is shown in plain text).

Junctions are detected by examining the NGS data for sequences having portions of

plasmid sequences that span less than the full read. A group of junction sequences which

share the same junction point and common flanking sequence is called a Junction

Sequence Class (or JSC).

Figure IV-3. Two Unique Junction Sequence Classes are Produced by the Insertion

of a Single Plasmid Region

A schematic representation of a single DNA insertion within the genome showing the

inserted DNA, the 5' and 3' flanks (depicted as areas bounded by dotted lines), and the

two distinct regions spanning the junctions between inserted DNA and flanking DNA

(shaded boxes). The group of ~100-mer sequences in which each read contains

sequences from both the DNA insert and the adjacent flanking DNA at a given junction is

called a Junction Sequence Class. In this example, two distinct junction sequence classes

(in this case: Class A at the 5' end and Class B at the 3' end) are represented.

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGGAT

Plasmid DNA FlankingDNA

Junction

5’ Flank DNA insert 3’ Flank

Insert Junction Regions

Junction Sequences: Class A

Junction Sequences: Class B
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By evaluating the number of unique junction classes detected, the number of insertion

sites of the plasmid sequence can be determined. For a single insert, two junction

sequence classes are expected, each originating from one end of the insert, both

containing portions of plasmid DNA insert and flanking sequence.

Directed sequencing (locus-specific PCR and DNA sequencing analyses, Figure IV-1,

step 5) complements the NGS/JSA analyses. Sequencing of the insert and flanking

genomic DNA determined the complete sequence of the insert and flanks. This analysis

evaluates if the sequence of the insert is identical to the corresponding sequence from the

T-DNA in PV-ZMAP5714, if each genetic element in the insert is intact, if the T-DNA

sequence is inserted as a single copy, and further confirms that no vector backbone or

other unintended plasmid sequences were present within the T-DNA insert in

MON 87403. Results are described in Sections IV.B and IV.C; methods are presented in

Appendix C: .

The stability of the T-DNA present in MON 87403 across multiple generations was

evaluated by NGS/JSA analyses as described above. This information was used to

determine the number and identity of insertion sites. For a single insert, two junction

sequence classes are expected; each one originates from either end of the insert, both

containing portions of DNA insert and flanking sequence. All integrated sequences are

expected to align to the T-DNA region of the plasmid. Results are described in

Section IV.D.; methods are presented in Appendix C: .

Segregation analysis of the T-DNA was conducted to determine the inheritance and

stability of the insert in MON 87403. Segregation analysis corroborates the insert

stability demonstrated by NGS/JSA and independently establishes the genetic behavior of

the T-DNA. Results and methods are described in Section IV.E.

IV.A. Determining the Number and Identity of DNA Inserts in MON 87403

The number of insertion sites of PV-ZMAP5714 DNA in MON 87403 was assessed by

performing NGS/JSA on MON 87403 genomic DNA. A plasmid map of PV-ZMAP5714

is shown in Figure III-1. Table IV-1 provides descriptions of the genetic elements

present in MON 87403. A schematic representation of the insert and flanking sequences

in MON 87403 is shown in Figure IV-4. For full details on materials and methods see

Appendix C.

IV.A.1. Next Generation Sequencing of MON 87403 and Conventional

Control Genomic DNA

Genomic DNA from five generations of MON 87403 and the appropriate conventional

control (Figure IV-5) was isolated from seed and prepared for sequencing according to

the manufacturer’s protocol (Illumina, TruSeq library protocol. For material and method

details see Appendix C: ). These genomic DNA libraries were used to generate short

(~100 bp) randomly distributed sequence fragments (sequencing reads) of the maize

genome (see Figure IV-1, Step 1).
Monsanto Company CR262-14U1 44 of 260

To demonstrate sufficient sequence coverage the 100-mer sequence reads were analyzed

by mapping all reads to a known single-copy endogenous gene (Zea mays pyruvate

decarboxylase (pdc3), GenBank accession version: AF370006.2). The analysis of

sequence coverage plots showed that the depth of coverage (i.e., the median number of

times any base of the genome is expected to be independently sequenced) was 75× or

greater for the five generations of MON 87403 (R3, R4, R5, R4F1 and R5F1) and the

conventional control. It has previously been demonstrated that 75× coverage of the

soybean genome is adequate to provide comprehensive coverage and ensure detection of

inserted DNA (Kovalic et al. 2012) and similarly 75× coverage provides comprehensive

coverage of the maize genome (Clarke and Carbon 1976).

To demonstrate the method’s ability to detect any sequences derived from the

PV-ZMAP5714 transformation plasmid, a sample of conventional control DNA spiked

with PV-ZMAP5714 DNA at 1 and 1/10th genome equivalent was analyzed by NGS and

bioinformatics. The level of sensitivity of this method was demonstrated to a level of

1 genome equivalent, 100% nucleotide identity was observed over 100% of

PV-ZMAP5714 (Table C-2). This result demonstrates that all nucleotides of the

transformation plasmid are observed by the sequencing and bioinformatic assessments

performed. Also, observed coverage was adequate (Clarke and Carbon 1976) at a level

1/10th genomic equivalent (99.97% coverage at 98.83% identity for the 1/10th genome

equivalent spiked control sample, Table C-2) and, hence, a detection level of at most

1/10th genome equivalent was achieved for the plasmid DNA sequence assessment.

IV.A.2. Characterization of insert number in MON 87403 using Bioinformatic

Analysis

The number of insertion sites of DNA from PV-ZMAP5714 in MON 87403 was assessed

by performing NGS/JSA on MON 87403 genomic DNA using the R3 generation (Figure

IV-5).
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Table IV-1. Summary of Genetic Elements in MON 87403

Genetic Element1 Location in Sequence2Function (Reference)

5' Flanking DNA 1-1345 DNA sequence flanking the 5'

end of the insert

B

3

-Right Border Regionr1 1346-1369 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 1370-1387 Sequence used in DNA

cloning

P

4

-e35S/Ract1 1388-2568 Chimeric promoter consisting

of the duplicated enhancer

region from the cauliflower

mosaic virus 35S RNA

promoter (CaMV) (Kay et al.

1987) combined with the

promoter of the act1 gene from

Oryza sativa that encodes

Actin 1 (McElroy et al. 1990)

that directs transcription in

plant cells

Intervening Sequence 2569-2573 Sequence used in DNA

cloning

L

5

-Cab 2574-2634 5' UTR leader sequence from

chlorophyll a/b-binding (CAB)

protein of Triticum aestivum

(wheat) that is involved in

regulating gene expression

(Lamppa et al. 1985)

Intervening Sequence 2635-2650 Sequence used in DNA

cloning

I

6

-Ract1 2651-3130 Intron and flanking UTR

sequence of the act1 gene from

Oryza sativa (rice) encoding

Actin 1 protein. This sequence

is involved in regulating gene

expression (McElroy et al.

1990)
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Table IV-1 (continued). Summary of Genetic Elements in MON 87403

Genetic Element1 Location in Sequence2Function (Reference)

Intervening Sequence 3131-3142 Sequence used in DNA

cloning

CS7-ATHB17 3143-3970 Coding sequence of the

ATHB17 gene from

Arabidopsis thaliana encoding

a member of the class II

homeodomain-leucine zipper

gene family (HD-Zip II) that is

thought to act as a transcription

factor (Ariel et al. 2007)

Intervening Sequence 3971-3983 Sequence used in DNA

cloning

T

8

-Hsp17 3984-4193 3' UTR sequence from a heat

shock protein, Hsp17, of

Triticum aestivum (wheat)

(McElwain and Spiker 1989)

that directs polyadenylation of

the mRNA

Intervening Sequence 4194-4246 Sequence used in DNA

cloning

B-Left Border Regionr1 4247-4477 DNA region from

Agrobacterium tumefaciens

containing the left border

sequence used for transfer of

the T-DNA (Barker et al.

1983)

3' Flanking DNA 4478-5744 DNA sequence flanking the 3'

end of the insert

1Although flanking sequences and intervening sequences are not functional genetic elements;

they comprise a portion of the sequence.

2Numbering refers to the sequence of the insert in MON 87403 and adjacent DNA.

3B, Border

4

P, Promoter

5L, Leader Sequence

6

I, Intron Sequence

7CS, Coding Sequence

8T, Transcriptional Terminator

r1Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was

truncated compared to the sequences in PV-ZMAP5714.
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Figure IV-4. Schematic Representation of the Insert and Flanking Sequences in MON 87403

DNA derived from T-DNA of PV-ZMAP5714 integrated in MON 87403. Right-angled arrows indicate the ends of the integrated

T-DNA and the beginning of the flanking sequence. Genetic elements within the insert are identified on the map. This schematic

diagram is drawn to scale, the exact coordinates of every element is shown in Table IV-1.

r1Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was truncated compared to the sequences in

PV-ZMAP5714.
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Figure IV-5. Breeding History of MON 87403

R0 corresponds to the transformed plant, F# is the filial generation,  designates

self-pollination.

1Generation used for molecular characterization

2 Generations used to confirm insert stability

3 Generation used for commercial development of MON 87403

4 Generation used for agronomic/phenotypic and compositional analysis studies

LH244 R0

LH244 R1

LH244 R2

LH244 R3

1,2

LH244 R4

2,3

LH244 R5

2 R5F1

(LH244 x LH287)

2,4

R4F1

(LH244 x LH295)

2
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IV.A.2.1. Selection of Sequence Reads Containing Sequence of the

PV-ZMAP5714

PV-ZMAP5714 was transformed into the parental variety LH244 to produce

MON 87403. Consequently, any DNA inserted into MON 87403 will consist of

sequences that are similar to the PV-ZMAP5714 DNA sequence. Therefore, to fully

characterize the DNA from PV-ZMAP5714 inserted in MON 87403, it is sufficient to

completely analyze only the sequence reads that have similarity to the transformation

plasmid (Figure IV-1, Step 2).

Using established criteria (described in the materials and methods, Appendix C: ),

sequence reads similar to the transformation plasmid were selected from MON 87403 and

the conventional control sequence datasets and were then used as input data for

bioinformatic junction sequence analysis.

IV.A.2.2. Determination of the Insert Number

The NGS/JSA method described above used the entire plasmid sequence as a query to

determine the DNA insertion site number. Any inserted transformation plasmid

sequence, regardless of origin, either T-DNA or backbone, can be identified by mapping

reads to the transformation plasmid sequence while the number of inserted DNA

molecules can be determined using JSA. Therefore unlike the traditional Southern blot

analysis that separately hybridizes T-DNA or backbone probes, in NGS/JSA the

determination of the T-DNA insert number and of the absence of backbone or unintended

sequences are simply represented by the identification of sequence reads that match the

transformation plasmid, the determination of the overall insert number in the genome

followed by determination of the exact identity of any DNA insert using directed

sequencing and sequence analysis.

By evaluating the number of unique junction classes, the number of DNA insertion sites

can be determined (Figure IV-1, Step 3). If MON 87403 contains a single T-DNA insert,

two junction sequence classes (JSCs), each containing portions of T-DNA sequence and

flanking sequence, will be detected.

To determine the insert number in MON 87403, the selected sequence reads described

above were analyzed using JSA (Kovalic et al. 2012). JSA uses bioinformatic analysis to

find and classify partially matched reads characteristic of the ends of insertions. The

number of resultant unique JSCs were determined by this analysis and are shown in Table

IV-2.
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Table IV-2. Unique Junction Sequence Class Results

Sample

Junction Sequence

Classes Detected

MON 87403 2

LH244 0

The location and orientation of the junction sequences relative to the T-DNA insert

determined for MON 87403 (as described in Section IV.B) are illustrated in Figure IV-6.

As shown in the figure, there are two junction sequence classes identified in MON 87403.

Junction Sequence Class A and Class B (JSC-A and JSC-B) both contain the T-DNA

border sequence joined to genomic flanking sequence, indicating that they represent the

sequences at the junctions of the intended T-DNA insert and genomic flanking sequence.

The presence of two, and only two, junction sequence classes (joining T-DNA border and

flanking sequences) indicate this single pair of JSCs likely arises from the insertion of the

intended PV-ZMAP5714 T-DNA at a single locus in the genome of MON 87403. JSC-A

represents the junction of the T-DNA Left Border sequence to the 5' flank and JSC-B

represents the junction of the T-DNA Right Border sequence to the 3' flank. Complete

alignment of the JSCs to the full flank/insert sequence confirms that both of these JSCs

originate from the same locus of the MON 87403 genome and are linked by contiguous,

known and expected DNA that makes up the single insert.

IV.A.2.3. Determination of the Inserted DNA Identity

To determine the identity of inserted DNA, all selected sequences decribed in section

IV.A.2.1 were mapped to the transformation plasmid sequence. While thousands of

sequence reads from the R3 generation mapped to the plasmid T-DNA sequence, only

four reads mapped to the plasmid backbone. From this result it was determined that

MON 87403 does not contain any sequence from the transformation plasmid backbone.

Based on the comprehensive NGS/JSA study, it was concluded that MON 87403 contains

one T-DNA inserted into a single locus, as shown in Figure IV-6 and is devoid of

backbone sequence. The identity of the DNA insert was confirmed by the sequencing

and analysis of overlapping PCR products from this locus as described below in

Section IV.B., which showed that the DNA insert contained only the T-DNA elements

from the plasmid.
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Figure IV-6. Junction Sequences Detected by NGS/JSA

Linear map of MON 87403 illustrating the relationship of the detected junction sequences to the insert locus. The individual junction

sequences detected by JSA are illustrated as stacked bars.

r1Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was truncated compared to the sequences in PV-ZMAP5714.
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IV.B. Organization and Sequence of the Insert and Adjacent DNA in MON 87403

The organization of the elements within the DNA insert and the adjacent genomic DNA was

assessed using directed DNA sequence analysis (refer to Figure IV-1, Step 5). PCR primers

were designed to amplify three overlapping regions of the MON 87403 genomic DNA that span

the entire length of the insert (Figure IV-7). The amplified PCR products were subjected to

DNA sequencing analyses. The results of this analysis confirm that the MON 87403 insert is

3,132 bp and that each genetic element within the T-DNA is intact compared to the

transformation plasmid PV-ZMAP5714, with the exception of the border regions. The border

regions both contain small terminal deletions with the remainder of the inserted border regions

being identical to the sequence in PV-ZMAP5714. The sequence and organization of the insert

was also shown to be identical to the corresponding T-DNA of PV-ZMAP5714, confirming that

a single copy of the T-DNA was inserted as intended. This analysis also shows that only T-DNA

elements (described in Table IV-1) were present. Moreover, the result, together with the

conclusion of single DNA insert detected by NGS/JSA, demonstrated that no PV-ZMAP5714

backbone elements are present in MON 87403.
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Figure IV-7. Overlapping PCR Analysis Across the Insert in MON 87403

PCR was performed on both conventional control genomic DNA and MON 87403 genomic

DNA using three pairs of primers to generate overlapping PCR fragments from MON 87403 for

sequencing analysis. To verify the PCR products, a portion of each PCR was loaded on a

1% (w/v) agarose gel and visualized by ethidium bromide staining. The expected product size

for each amplicon is provided in the illustration. Lane designations are as follows:

Lane Lane

1 1 Kb Plus DNA Ladder 7 PV-ZMAP5714

2 MON 87403 8 Conventional Control

3 No Template Control 9 MON 87403

4 Conventional Control 10 No Template Control

5 MON 87403 11 Conventional Control

6 No Template Control 12 1 Kb Plus DNA Ladder

Arrows next to the agarose gel photograph denote the size of the DNA, in kilobase pairs, obtained

from the 1 Kb DNA Plus Ladder (Invitrogen, Grand Island, NY) on the ethidium bromide stained

gel.

r1 Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was truncated compared

to the sequences in PV-ZMAP5714.

1 2 3 4 5 6 7 8 9 10 11 12

12

3

2

1.6

1

0.2

12

3

2

1.6

1

0.2

Product A Product B Product C
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IV.C. Sequencing of the MON 87403 Insertion Site

PCR and sequence analysis were performed on genomic DNA extracted from the conventional

control to examine the insertion site in conventional maize (refer to Figure IV-1, Step 6). The

PCR was performed with one primer specific to the genomic DNA sequence flanking the 5' end

of the MON 87403 insert paired with a second primer specific to the genomic DNA sequence

flanking the 3' end of the insert (Figure IV-8). A sequence comparison between the PCR product

generated from the conventional control and the sequence generated from the 5' and 3' flanking

sequences of MON 87403 indicates that 149 bases of maize genomic DNA were deleted during

integration of the T-DNA. The remainder of the flanks in MON 87403 are identical to the

conventional control. Such changes are common during plant transformation and these changes

presumably resulted from double-stranded break repair mechanisms in the plant during

Agrobacterium-mediated transformation process (Salomon and Puchta 1998).
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Figure IV-8. PCR Amplification of the MON 87403 Insertion Site

PCR analysis was performed to evaluate the insertion site. PCR was performed on conventional

control DNA using Primer A, specific to the 5' flanking sequence, and Primer B, specific to the

3′ flanking sequence of the insert in MON 87403. The DNA generated from the conventional

control PCR was used for sequencing analysis. This illustration depicts the MON 87403

insertion site in the conventional control (upper panel) and the MON 87403 insert (lower panel).

To verify the PCR products, a portion of each PCR was loaded on the gel. Lane designations are

as follows:

Lane

1 1 Kb Plus DNA Ladder

2 Conventional Control

3 No template DNA control

Arrows denote the size of the DNA, in kilobase pairs, obtained from the 1 kb DNA Plus Ladder (Invitrogen, Grand

Island, NY) on the ethidium bromide stained gel.

r1Superscript in Left and Right Border Regions indicate that the sequence in MON 87403 was truncated compared to

the sequences in PV-ZMAP5714.

Primer A

Primer A Primer B

Primer B

1 2 3

12

3

2

1.6

1

0.2
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IV.D. Determination of Insert Stability over Multiple Generations of MON 87403

In order to demonstrate the stability of the T-DNA present in MON 87403 through multiple

generations, NGS/JSA analysis was performed using DNA obtained from five breeding

generations of MON 87403. The breeding history of MON 87403 is presented in Figure IV-5,

and the specific generations tested are indicated in the figure legend. The MON 87403 R3

generation was used for the molecular characterization analyses discussed in Sections IV.AIV.C. and shown in Figure IV-5. To assess stability, four additional generations were evaluated

by NGS/JSA analysis as previously described in Section IV.A, and compared to the fully

characterized R3 generation. The conventional controls used for the generational stability

analysis included LH244, which included similar background genetics to the R3, R4 and the R5

generations and represents the original transformation line; LH244 × LH295, a hybrid with

similar background genetics to the R4F1 hybrid; and LH244 × LH287, a hybrid with similar

background genetics to the R5F1 hybrid. Genomic DNA isolated from each of the selected

generations of MON 87403 and conventional control was used for NGS/JSA analysis.

To determine the insert number in the MON 87403 generations, the sequences selected as

described in Section IV.A.2.1 were analyzed using JSA (Kovalic et al. 2012). Table IV-3 shows

the number of resultant JSCs containing PV-ZMAP5714 DNA sequence determined by this

analysis.

Table IV-3. Junction Sequence Classes Detected

Sample

Junction Sequence

Classes Detected

MON 87403 (R3) 2

MON 87403 (R4) 2

MON 87403 (R4F1) 2

MON 87403 (R5) 2

MON 87403 (R5F1) 2

LH244 0

LH244 × LH295 0

LH244 × LH287 0

Alignment of the JSCs from each of the assessed MON 87403 generations (R4, R5, R4F1, and

R5F1) to the full flank/insert sequence and JSCs determined for the MON 87403 R3 generation,

confirms that the pair of JSCs originates from the same region of the MON 87403 genome and is

linked by contiguous, known and expected DNA sequence. This single identical pair of JSCs is

observed as a result of the insertion of PV-ZMAP5714 T-DNA at a single locus in the genome of

MON 87403. The consistency of these JSC data across all generations tested demonstrates that

this single locus was stably maintained throughout the MON 87403 breeding process.

These results demonstrate that the MON 87403 single integration locus was maintained through

several generations of breeding MON 87403; thereby confirming the stability of the insert.
Monsanto Company CR262-14U1 57 of 260

Based on this comprehensive sequence data and bioinformatic analysis (NGS/JSA), it is

concluded that MON 87403 contains a single and stable T-DNA insertion.

IV.E. Inheritance of the Genetic Insert in MON 87403

The MON 87403 T-DNA resides at a single locus within the maize genome and therefore should

be inherited according to Mendelian principles of inheritance. During development of lines

containing MON 87403, genotypic segregation data were recorded to assess the inheritance and

stability of the MON 87403 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 87403 breeding path for generating segregation data is described in Figure IV-9. The

transformed R0 plant was self-pollinated to generate R1 seed. An individual plant homozygous

for the MON 87403 T-DNA was identified in the R1 segregating population via an End-Point

TaqManPCR assay.

The homozygous positive R1 plant was self-pollinated to give rise to R2 seed. The R2 plants

were self-pollinated to produce R3 seed. The R3 plants were self-pollinated to produce R4 seed.

R4 plants homozygous for the MON 87403 T-DNA were crossed via traditional breeding

techniques to a Monsanto proprietary recurrent parent that does not contain the ATHB17 coding

sequence to produce hemizygous R4F1 seed. The R4F1 plants were crossed with the recurrent

parent to produce BC1F1 seed. The BC1F1 generation was tested for the presence of the T-DNA

by End-Point TaqMan PCR to select for hemizygous MON 87403 plants. BC1F1 plants

hemizygous for MON 87403 T-DNA were crossed with the recurrent parent to produce the

BC2F1 plants. BC2F1 plants hemizygous for MON 87403 T-DNA were crossed with the

recurrent parent to produce the BC3F1 plants.

The inheritance of the MON 87403 T-DNA was assessed in the BC1F1, BC2F1, and BC3F1

generations. At the BC1F1, BC2F1, and BC3F1 generations, the MON 87403 T-DNA was

predicted to segregate at a 1:1 ratio (hemizygous positive: homozygous negative) according to

Mendelian inheritance principles.

A Pearson’s chi-square (χ

2

) analysis was used to compare the observed segregation ratios of the

ATHB17 coding sequence to the expected ratios.

The Chi-square was calculated as:

χ

2

= ∑ [( | o – e | )

2

/ e]

 TaqMan is a registered trademark of Roche Molecular Systems, Inc.
Monsanto Company CR262-14U1 58 of 260

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 segregating progeny of MON 87403 are presented in Table

IV-4. The χ

2

value in the BC1F1, BC2F1, and BC3F1 generations indicated no statistically

significant difference between the observed and expected 1:1 segregation ratio

(hemizygous positive: homozygous negative) of MON 87403 T-DNA. These results support the

conclusion that the MON 87403 T-DNA resides at a single locus within the maize genome and is

inherited according to Mendelian principles of inheritance. These results are also consistent with

the molecular characterization data indicating that MON 87403 contains a single intact copy of

the ATHB17 expression cassette inserted at a single locus in the maize genome.
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Figure IV-9. Breeding Path for Generating Segregation Data for MON 87403

Chi-square analysis was conducted on segregation data from BC1F1, BC2F1, and BC3F1

generations (bolded text).

TI: Trait Integration: Replacement of genetic background of MON 87403 by recurrent

background except inserted gene.

RP: Recurring parent

BC: Back-Cross.

: Self- Pollinated.

LH244 R1

Breeding path continued

LH244 R4

LH244 R3

LH244 R2







Transformed LH244 R0 Plant



R4F1(LH244 R4 x RP)

TI:BC1F1

TI:BC2F1

x RP

x RP

x RP

TI:BC3F1

x RP

(Expected segregation 1:1)

(Hemizygous positive: Homozygous negative)

(Expected segregation 1:1)

(Hemizygous positive: Homozygous negative)

(Expected segregation 1:1)

(Hemizygous positive: Homozygous negative)
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Table IV-4. Segregation of the Expression Cassette During the Development of MON 87403

Generation Number of

plants

Observed

Positives

Observed

Negatives

Expected

Positives

Expected

Negatives χ

2

Probability

BC1F1 180 88 92 90.00 90.00 0.09 0.766

BC2F1 178 82 96 89.00 89.00 1.10 0.294

BC3F1 181 101 80 90.50 90.50 2.44 0.119
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IV.F. Characterization of the Genetic Modification Summary and

Conclusion

Molecular characterization of MON 87403 by NGS/JSA and directed sequencing

demonstrated that a single copy of the intended transfer DNA (T-DNA) containing the

ATHB17 expression cassette from PV-ZMAP5714 was integrated into the maize genome

at a single locus. These analyses also showed no PV-ZMAP5714 backbone elements

were present in the event.

Directed sequence analyses performed on MON 87403 confirmed the organization and

intactness of the full T-DNA and all expected elements within the insert, with the

exception of incomplete Right and Left Border sequences that do not affect the

functionality of the ATHB17 expression cassette. Analysis of the T-DNA insertion site in

maize showed that the 5' and 3' genomic DNA flanking the T-DNA insert in MON 87403

are identical to the conventional control, except for a 149 base pair deletion of genomic

DNA at the insertion site in MON 87403. This deletion is not expected to affect food or

feed safety.

Generational stability analysis by NGS/JSA demonstrated that the T-DNA in

MON 87403 was maintained through five breeding generations, thereby confirming the

stability of the insert. Results from segregation analyses show heritability and stability of

the insert occurred as expected across multiple generations, which corroborates the

molecular insert stability analysis and establishes the genetic behavior of the T-DNA in

MON 87403 at a single chromosomal locus.
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V. CHARACTERIZATION AND SAFETY ASSESSMENT OF THE

ATHB17Δ113 PROTEIN PRODUCED IN MON 87403

Characterization of the introduced protein(s) in a biotechnology-derived crop is important

to establishing food, feed, and environmental safety. As described in Sections IV and V,

MON 87403 contains an ATHB17 expression cassette that, when transcribed and

translated, results in the expression of the ATHB17Δ113 protein.

This section summarizes: 1) the identity and function of the ATHB17Δ113 protein

produced in MON 87403; 2) assessment of equivalence between the plant-produced and

E. coli-produced proteins; 3) the level of the ATHB17Δ113 protein in plant tissues from

MON 87403; 4) assessment of the potential allergenicity of the ATHB17Δ113 protein

produced in MON 87403; and 5) the food and feed safety assessment of the

ATHB17Δ113 protein produced in MON 87403. The data support a conclusion that the

ATHB17Δ113 protein produced in MON 87403 is safe for human or animal consumption

based on several lines of evidence summarized below.

V.A. Identity and Function of the ATHB17Δ113 Protein from MON 87403

V.A.1. Sequence Identity of the ATHB17Δ113 Protein from MON 87403

As described in Section IV, the ATHB17 gene is derived from Arabidopsis thaliana.

Compared to the wild type sequence of the ATHB17 protein, the amino acid sequence of

the ATHB17-encoded protein expressed in MON 87403 lacks 113 amino acids due to

alternative mRNA splicing, and has thus been designated ATHB17∆113. The ATHB17

expression cassette in MON 87403 includes the ATHB17 coding sequence and the

I-Ract1 intron (Table IV-1). Typically, pre-mRNA processing includes mRNA splicing,

which consists of the removal of the introns from pre-mRNA transcripts and ligation of

exons to produce mature mRNA (Simpson and Filipowicz 1996). To determine the

sequence of the mature ATHB17 mRNA transcript in MON 87403, total RNA was

extracted from leaf tissue and then PolyA+ RNA was enriched. mRNA transcripts of

ATHB17 were amplified by RT-PCR and then analyzed by sequence analysis. In

MON 87403, the ATHB17 pre-mRNA is spliced to remove the majority of the I-Ract1

intron as well as a portion of the ATHB17 gene (Figure V-1). In order to predict the

amino acid sequence of the protein produced from the ATHB17 mRNA, a Basic Local

Alignment Search Tool (BLASTX) search was performed to compare the ATHB17

mRNA consensus sequence against the GenBank_Protein_Preferred protein sequence

database. The top result of this homology search was the Arabidopsis thaliana ATHB17

protein sequence (GenBank ID 179876107). The first methionine (M) amino acid in the

BLASTX alignment provides a predicted translation start site for the ATHB17-derived

protein in MON 87403, and results in the protein ATHB17∆113, which is lacking the

N-terminal 113 amino acids found in wild type ATHB17 (Figure V-2). To confirm the

presence of the ATHB17Δ113 protein in MON 87403, western blot analysis was

conducted on MON 87403 with an anti-ATHB17 antibody. Western blots revealed the

presence of an immunogenic band corresponding to the expected molecular weight of the

predicted ATHB17∆113 protein (Figure D-2).
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Figure V-1. Insert map of MON 87403 and schematic diagram of ATHB17

transcription, mRNA processing, and predicted translation

The nucleotide sequence of the mature ATHB17 mRNA was determined using RT-PCR

followed by Sanger sequencing. The mRNA sequence that codes for the ATHB17

protein in MON 87403 was determined. This analysis allowed for the identification of

the splice junction in MON 87403 ATHB17 mRNA. The maize splicing of MON 87403

ATHB17 mRNA results in the excision of the I-Ract1 element (splice junction site 2662)

and a portion of the CS-ATHB17 coding region (splice junction site 3417). The observed

CS-ATHB17 coding region is predicted to produce a version of theATHB17 protein in

MON 87403 without the first 113 amino acids at the N-terminus compared to the

sequence of ATHB17 found in Arabidopsis (the translation initiation site is denoted by

an *).

ATHB17 mRNA

2662

Spliced

Region

ATHB17Δ113

Translation

Transcription & mRNA Processing

3417

*
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1 MNRLPSSEDG DDEEFSHDDG SAPPRKKLRL TREQSRLLED SFRQNHTLNP KQKEVLAKHL

61 MLRPRQIEVW FQNRRARSKL KQTEMECEYL KRWFGSLTEE NHRLHREVEE LRAIKVGPTT

121 VNSASSLTMC PRCERVTPAA SPSRAVVPVP AKKTFPPQER DR

Figure V-2. Predicted Amino Acid Sequence of the Protein Produced by

MON 87403 ATHB17 mRNA

The consensus sequence of MON 87403 mature ATHB17 mRNA was used in a BLASTX

2.2.23 search of the GenBank_Protein_Preferred database. The predicted amino acid

sequence of MON 87403 ATHB17∆113, beginning with the first methionine amino acid

in the top BLAST result is shown above.

V.A.2. Structure and Function of the ATHB17Δ113 Protein from

MON 87403

The Arabidopsis thaliana ATHB17 protein is a member of the HD-Zip family of

transcription factors. In plants, the family of HD-Zip proteins is further segregated into

four distinct subfamilies designated as I, II, III, and IV, based upon the sequences of

conservative structural domains and motifs that convey DNA specificity and

physiological function (Ariel et al. 2007). As described in Section V.A.1, expression of

the Arabidopsis thaliana ATHB17 gene in MON 87403 results in production of the

ATHB17Δ113 protein, which consists of a single polypeptide chain of 162 amino acids

and has a predicted molecular weight of ~22 kDa. HD-Zip subfamily II members are

characterized by highly conserved domains that include a homeodomain (HD) that

recognizes a 9 bp DNA sequence CAAT(C/G)ATTG (Sessa et al. 1993), a leucine-zipper

(LZ) domain responsible for protein dimerization, and a repression domain (Rice et al.

2014). ATHB17Δ113 retains the HD and LZ domains found in wild-type ATHB17 but

lacks the repression domain.

V.B. Characterization and Equivalence of ATHB17Δ113 Protein from

MON 87403

The safety assessment of crops derived through biotechnology includes characterization

of the physicochemical and functional properties of the protein produced from the

inserted DNA, and confirmation of the safety of the protein. For safety data generated

using E. coli-produced protein to be applied to plant-produced protein, the equivalence of

the plant- and E. coli-produced proteins must be assessed. As reported in Section V.C,

the expression level of the ATHB17Δ113 protein in MON 87403 is very low in all tissues

evaluated. Due to this low level expression, the MON 87403-produced ATHB17Δ113

protein was not isolated to a high level of purity, but instead limited amounts of the

protein were enriched in leaf extract to a purity of approximately 3% (Appendix D: ).

Characterization of this preparation enabled the demonstration of equivalence between

the MON 87403-produced ATHB17Δ113 protein and the E. coli-produced

ATHB17Δ113 protein. A summary of these analytical results are shown below and the

details of the materials, methods, and results are described in Appendix D: .

The ATHB17Δ113 protein purified from leaf tissue of MON 87403 was characterized

and equivalence of the physicochemical properties between the MON 87403-produced
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and E. coli-produced proteins was established using a panel of analytical techniques

(Appendix D: ), including: 1) SDS-PAGE analysis, to establish equivalence of apparent

molecular weight between the MON 87403-produced and E. coli-produced

ATHB17Δ113 proteins; 2) western blot analysis with antibodies specific for the

ATHB17Δ113 protein to establish immunoreactive equivalence between the

MON 87403-produced and E. coli-produced ATHB17Δ113 proteins; and

3) MALDI TOF MS analysis of MON 87403-produced ATHB17Δ113 to establish

protein identity.

Due to the low quantity and purity of the enriched MON 87403-produced ATHB17Δ113,

direct assessment of the ATHB17Δ113 N-terminal sequence, specific DNA binding

activity, and glycosylation status were not feasible to be conducted for the

MON 87403-produced ATHB17Δ113 protein. Instead, these characteristics of

ATHB17Δ113 in MON 87403 were assessed indirectly. The N-terminal sequence of the

E. coli-produced ATHB17Δ113 protein was directly analyzed and shown to match the

N-terminus predicted by the RT-PCR analysis of MON 87403 ATHB17 RNA as detailed

in Section V.A.1. An SDS-PAGE assessment demonstrated that the apparent molecular

weights of the MON 87403-produced and E. coli-produced ATHB17∆113 were

equivalent, which supports a conclusion that the N-termini are equivalent (Figure D-2).

Analysis of the DNA binding activity of the E. coli-produced ATHB17Δ113

demonstrated that the protein specifically bound to the target DNA sequence

CAAT(C/G)ATTG, as expected. While functional activity could not be directly

measured for the isolated MON 87403-produced ATHB17Δ113 protein, the activity

measured for E. coli-producted ATHB17∆113 is consistent with the mechanism of action

for ATHB17∆113 in MON 87403.

There are no predicted N-terminal targeting or signal sequences in the ATHB17Δ113

amino acid sequence, which is expected due to the truncation of the N-terminus of the

protein. Thus, although the ATHB17Δ113 protein sequence contains a consensus

potential N-glycosylation sequence (NHT, starting at amino acid position 45), it lacks the

N-terminal signal sequence required for transport to the endoplasmic reticulum, the site

of N-glycosylation (Pattison and Amtmann 2009; Vitale and Denecke 1999). In total,

this supports a conclusion that the MON 87403-produced ATHB17Δ113 protein is not

glycosylated. This is similar to the CP4-EPSPS protein, which contains potential

N-glycosylation sites but has been shown to not be glycosylated (Harrison et al. 1996).

Taken together, these data provide a characterization of the MON 87403-produced

ATHB17Δ113 protein and establish its equivalence to the E. coli-produced

ATHB17Δ113 protein.

V.C. Expression Levels of ATHB17Δ113 Protein in MON 87403

ATHB17Δ113 protein levels in various tissues of MON 87403 relevant to the risk

assessment were determined by a validated enzyme-linked immunosorbent assay

(ELISA). Tissues of MON 87403 were collected from four replicate plots planted in a

randomized complete block field design during the 2012 growing season from the

following five field sites in The United States: Jackson County, Arkansas
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(site code ARNE); Story County, Iowa (site code IALL); Jefferson County, Iowa

(site code IARL); Pawnee County, Kansas (site code KSLA) and Lehigh County,

Pennsylvania (site code PAGR). The field sites were representative of maize-producing

regions suitable for commercial production. OSL13, OSR14, forage, and grain tissue

samples were collected from each replicated plot at all field sites.

Determining the protein expression levels serves to characterize MON 87403 and the

levels are then used to assess food, feed and environmental exposure. Protein expression

levels themselves do not provide information relevant to the assessment of the plant pest

potential of MON 87403. Therefore, it is deemed appropriate to determine the

ATHB17Δ113 protein levels for OSL1, OSR1, forage, and grain tissue samples, which

were collected from each replicated plot at all field sites. Leaf and root tissues are

distinct above and below ground plant tissues that may be important to environmental

exposure. Forage and grain levels were determined to evaluate food and feed exposure in

humans and animals and are required for margin of exposure calculations for both.

The ELISA results obtained for each sample were averaged across the five sites and are

summarized in Table V-1. The details of the materials and methods are described in

Appendix E. The individual ATHB17Δ113 protein levels in MON 87403 across all

samples analyzed from all sites ranged from <LOD (<0.00028 μg/g dwt)

to 0.017 μg/g dwt. The mean ATHB17Δ113 protein level among all tissue types was

highest in OSL1 at 0.014 μg/g dwt and lowest in grain at <LOD μg/g dwt.

3 Over season leaf 1

4 Over season root 1
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Table V-1. Summary of ATHB17Δ113 Protein Levels in Tissues from MON 87403

Grown in 2012 United States Field Trials

Tissue Type1 Development

Stage2

Mean(SD)

Range

(μg/g dwt)

3

LOQ/LOD4

(μg/g dwt)

OSL1 V3-V4 0.014 (0.0020) 0.00109/0.00049

0.0096 – 0.017

OSR1 V3-V4 0.0023 (0.0016) 0.00078/0.00065

0.00083 – 0.0058

Forage R5 0.0018 (0.00064) 0.00078/0.00063

0.0011 – 0.0035

Grain R6 <LOD (N/A) 0.00156/0.00028

N/A – N/A

1OSL= over season leaf

OSR= over season root

2The crop development stage each tissue was collected.

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 dry weight basis (dwt). The means, SD, and ranges

(minimum and maximum values) were calculated for each tissue across all sites. (n=20, except

OSR1 where n=16 due to four samples resulting in inconclusive levels)

4LOQ=limit of quantitation; LOD=limit of detection.
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V.D. Assessment of Potential Allergenicity of the ATHB17Δ113 Protein

The allergenic potential of an introduced protein is assessed by comparing the

biochemical characteristics of the introduced protein to biochemical characteristics of

known allergens (Codex Alimentarius 2009). Using a weight of evidence approach, 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 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 does not show resistance to pepsin digestion. The

ATHB17Δ113 protein has been assessed for its potential allergenicity according to these

safety assessment guidelines.

1) The ATHB17Δ113 protein originates from Arabidopsis thaliana an organism that

has not been reported to be a source of known allergens.

2) The ATHB17Δ113 protein level in grain is < the limit of detection of the assay

and, therefore, conservatively is less than 0.0000003% of the total protein in the

grain that could be consumed from MON 87403 maize.

3) Bioinformatics analyses demonstrated that the ATHB17Δ113 protein does not

share amino acid sequence similarities with known allergens and, therefore, is

highly unlikely to contain immunologically cross-reactive allergenic epitopes.

4) Finally, in vitro digestive fate experiments conducted with the ATHB17Δ113

protein demonstrate that the protein is rapidly digested by pepsin in simulated

gastric fluid (SGF) and in simulated intestinal fluid (SIF).

Taken together, these data support the conclusion that the ATHB17Δ113 protein does not

pose a significant allergenic risk to humans.

V.E. Safety Assessment Summary of ATHB17Δ113 Protein in MON 87403

V.E.1. The Donor Organism has a History of Safe Use

The Arabidopsis genus is a member of the mustard (Brassicaceae) family, which includes

many well known food crops including cabbage, cauliflower, radish, turnip and

canola/rapeseed. The most well known member of the Arabidopsis genus is a small

annual flowering plant Arabidopsis thaliana (L.) which is also known under the common

name of mouse-ear cress (Meyerowitz and Somerville 1994). Arabidopsis thaliana offers

important advantages for basic research in genetics and molecular biology due to the

small size of its sequenced genome, short life cycle, well developed classical genetics,

and many naturally occurring mutants. Arabidopsis thaliana thus serves as an excellent

model system for studying complex processes in plants.

Arabidopsis thaliana is not purposely consumed as a food source by humans. Camelina

sativa, an emerging oilseed crop, is reported to be the cultivated species most closely

related to Arabidopsis (Flannery et al. 2006). Camelina sativa leaves are consumed as

fresh greens by humans in the country of Georgia (Facciola 1998).
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Arabidopsis thaliana is generally not considered an allergenic or toxic source organism.

Although Arabidopsis thaliana contains homologs of proteins previously described as

allergens in other plant species (e.g., germins, lipid transfer protein, profilins, and small

molecular weight calcium binding proteins), no Arabidopsis proteins have been reported

in a peer-reviewed database of known allergens (FARRP 2013). One case of

occupational asthma has been reported in a laboratory worker due to exposure to

Arabidopsis pollen (Yates et al. 2008). In a recent internal bioinformatic assessment,

none of the HD-Zip family of proteins in Arabidopsis, including the ATHB17 protein,

showed any homology to any known allergens, toxins, or any other protein with known

adverse effects.

V.E.2. ATHB17Δ113 Protein Belongs to a Common Class of Plant Proteins

The Arabidopsis thaliana HB17 (ATHB17) gene encodes a member of the HD-Zip

protein family of transcription factors (Ariel et al. 2007). The HD-Zip protein family is

characterized by the presence of a leucine zipper (LZ) domain adjacent to the C-terminus

of a homeodomain (HD) (Mukherjee et al. 2009; Ruberti et al. 1991). These two

domains, HD and LZ, are individually present in transcription factors found across

eukaryotic organisms, however such combination of HD and LZ domains in a single

transcription factor is unique to plants; HD-Zip genes are found in all plant groups, but

not in animals or fungi (Ariel et al. 2007). A bioinformatic search for homeobox genes

(i.e. genes encoding proteins containing a HD) performed across several plant genomes

(Mukherjee et al. 2009) yielded a comprehensive list of HD-Zip family members present

in several plant species. It identified 48 HD-Zip genes in the Arabidopsis genome,

47 genes in the rice genome, and 70 genes in the maize genome. The HD-Zip family

comprises 63% of all homeobox genes in maize and 40% - 60% of the homeobox genes

in other plant species, making it the most abundant class of homeobox genes in many

plant species.

Bioinformatic searches using the ATHB17∆113 amino acid sequence as the query

identify homologous sequences from several different plant species, including the food

crops soybean, rice, corn tomato, potato, orange, papaya, grape, and cruciferous

vegetables. Overall the protein sequence identity of ATHB17∆113 to homologs in these

species range from ~58-83%, with the highest identity to the homologs in the Brassica

species Brassica rapa (a species including common food crops such as turnip and napa

cabbage) and Brassica oleracea (a species including common food crops such as cabbage

and Brussels sprouts). The amino acid sequence alignment between ATHB17∆113 and

its food crop homologs spans the length of the ATHB17∆113 protein. Thus

ATHB17∆113 shares sequence identity and structural similarity with proteins present in

plants currently consumed, establishing that humans and animals are exposed to this class

of proteins and that no adverse effects have been attributed to this class of proteins.

V.E.3. ATHB17Δ113 Protein in MON 87403 is Not Homologous to Known

Allergens or Toxins

Bioinformatics analyses were performed to assess the potential for allergenicity, toxicity,

or biological activity of ATHB17Δ113. The analysis demonstrated that ATHB17Δ113
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protein does not share amino acid sequence similarity with known allergens, gliadins,

glutenins, or protein toxins which could have adverse effects to human or animal health.

V.E.4. ATHB17Δ113 Protein in MON 87403 is Susceptible to Pepsin and

Pancreatin Digestion

ATHB17Δ113 was readily digestible by pepsin in simulated gastric fluid and pancreatin

in simulated intestinal fluid. Rapid degradation of ATHB17Δ113 by pepsin and

pancreatin makes it highly unlikely that ATHB17Δ113 in MON 87403 would be

absorbed in the digestive tract and have any adverse effects on human or animal health.

V.E.5. ATHB17Δ113 Protein in MON 87403 is Not Acutely Toxic

An acute oral toxicology study was conducted with the ATHB17Δ113 protein. Results

indicated that ATHB17Δ113 did not cause any adverse effects in mice, with a No

Observable Adverse Effect Level (NOAEL) of 1335 mg/kg body weight, the highest dose

tested. As described below, this NOAEL (i.e., known safe dose) is approximately one

billion times (1 x 109) higher than a conservative estimate of high end exposure through

dietary consumption of MON 87403.

V.E.6. Human and Animal Exposure to the ATHB17Δ113 Proteins

A common approach used to assess potential health risks for potentially toxic materials is

to calculate a Margin of Exposure (MOE) between the lowest NOAEL from an

appropriate animal toxicity study and an estimate of human exposure. Since no evidence

of mammalian toxicity has been reported for ATHB17Δ113, and the expression levels in

grain are extremely low (2.5 x 10-4g/g fwt5), dietary risk assessments would normally

not be considered necessary. Nevertheless, a dietary risk assessment was still conducted

for this protein in order to provide further assurances of safety by calculating a MOE

between the NOAEL (1335 mg/kg body wt/day) for the ATHB17Δ113 protein in a

mouse acute oral toxicity study (Section V.E.5 and 95th percentile consumption estimate

of acute dietary exposure determined using the Dietary Exposure Evaluation Model -

Food Commodity Intake Database (DEEM-FCID) (U.S. EPA 2013). DEEM-FCID

utilizes food consumption data from the National Health and Nutrition Examination

Survey (NHANES) conducted in 2003-2008 (Included: vegetable maize, popcorn, and

field maize (flour, meal, bran, and starch). Based on levels of expressed protein on a

fresh weight basis, 95th percentile exposure to ATHB17Δ113 for the general U.S.

population was estimated to be 0.51 ng/kg body weight (bw). For non-nursing infants in

the U.S., the most highly exposed sub-population, 95th percentile exposure to

ATHB17Δ113 was estimated to be 1.21 ng/kg bw. For the ATHB17Δ113 protein, MOEs

for acute dietary intake were estimated to be 2.6 x 109and 1.1  109for the general

5

Fresh weight basis was calculated by multiplying the dry weight value of 2.8 x 10-4 μg/gram (Section

V.C) by a dry weight correction factor of 0.882 to account for 11.8% moisture content in the grain.
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population and non-nursing infants, respectively. The MOEs reflect that a human would

have to eat millions of kilograms of maize in one day to achieve exposures to the

expressed proteins in MON 87403 that were not toxic to mice, which would be a physical

impossibility. These very large MOEs further indicate that there is no meaningful risk to

human health from dietary exposure to the ATHB17Δ113 protein produced by

MON 87403.

Actual MOEs will likely be much higher because the exposure estimates utilized are

conservative (95th percentile, assume 100% market penetration of MON 87403) and as

described in Section V.E.4., ATHB17Δ113 is rapidly digested, further minimizing

exposures. In addition, there are a number of steps in the processing of maize to make

food ingredients, including high temperature treatments, hydrolyses, soaking in slightly

acidic water, and drying that can denature a protein. Changes in temperature, pH, and

physical disruptions associated with food processing and cooking/preparation generally

lead to loss of protein structure and functionality (Hammond and Jez 2011). Like other

proteins, the ATHB17Δ113 protein in MON 87403 is expected to be similarly susceptible

to denaturation when exposed to high temperatures, pH extremes, and digestive

environments encountered during processing and cooking of foods containing

MON 87403. Thus, there are likely to be significantly lower exposures to the

functionally active form of this protein through consumption of MON 87403 than the

already negligible levels estimated above.

The potential ATHB17Δ113 protein exposure to animals from consumption of

MON 87403 in feeds was evaluated by calculating an estimate of daily dietary intake

(DDI) and relating that value to total daily dietary protein intake. The highest percentage

of ATHB17Δ113 protein was in the lactating dairy cow, which was 0.000001% (g/g) of

the total daily dietary protein intake (6.8 x 10-8 g of ATHB17Δ113/kg bw divided by 5.9

g/kg bw total daily dietary protein intake for lactating dairy cow). The percentage of the

ATHB17Δ113 protein consumed as part of the daily protein intake for chickens and pigs

is much less than for the lactating dairy cow. Therefore, in the maximal intake scenario,

poultry, swine and lactating dairy cattle would be consuming 0.000001% (g/g) or less of

their total protein as ATHB17Δ113 protein from MON 87403 maize.

In summary, there is no significant risk to human and animal health associated with

dietary exposure to the ATHB17Δ113 protein in food and feed products derived from

MON 87403.

V.F. ATHB17Δ113 Protein Characterization and Safety Conclusion

The data and information provided in this section address the questions important to the

food and feed safety of the ATHB17Δ113 protein in MON 87403, including the potential

allergenicity and toxicity. To summarize, ATHB17∆113 belongs to the HD-Zip class of

plant transcription factors, and has high amino acid sequence homology to HD-Zip

proteins encoded in several common food crops. The donor organism for the

ATHB17Δ113 coding sequence, Arabidopsis thaliana, is ubiquitous in the environment

and not commonly known for human or animal pathogenicity, or allergenicity. The

physicochemical characteristics of the ATHB17Δ113 protein from MON 87403 have
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been characterized and shown to be equivalent to those of E. coli-produced

ATHB17Δ113, which was used for the described safety studies. The expression level of

ATHB17Δ113 protein in selected tissues of MON 87403 were determined and shown to

be below the limit of detection of a sensitive assay in grain. An assessment of the

allergenic potential of the protein supports the conclusion that the ATHB17Δ113 protein

does not pose a meaningful safety risk to humans and animals or an allergenic risk to

humans. The ATHB17Δ113 protein lacks structural similarity to allergens, toxins or

other proteins known to have adverse effects on mammals. In addition, the

ATHB17Δ113 protein is rapidly digested in simulated digestive fluids and demonstrated

no oral toxicity in mice at a test level that corresponds to a very large margin of exposure

when considering the protein is undetectable in grain. Based on the above information,

the consumption of the ATHB17Δ113 protein from MON 87403 or its progeny is

considered safe for humans and animals.

The protein safety data presented herein support the conclusion that food and feed

products containing MON 87403 or derived from MON 87403 are as safe as maize

currently on the market for human and animal consumption.
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VI.COMPOSITIONAL ASSESSMENT OF MON 87403

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 are 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 maize composition (OECD 2002b).

A recent review of compositional assessments conducted according to OECD guidelines

which 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). Numerous scientific publications have further

documented the extensive variability in the concentrations of crop nutrients, antinutrients, and secondary metabolites that reflect the influence of environmental and

genetic factors as well as extensive conventional breeding efforts to improve nutrition,

agronomics, and yield (Harrigan et al. 2010; Harrigan et al. 2009; Ridley et al. 2011;

Zhou et al. 2011)

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 2002a). OECD consensus documents on compositional

considerations for new crop varieties emphasize quantitative measurements of essential

nutrients and known anti-nutrients. These quantitative measurements effectively discern

any compositional changes that imply potential nutritional or safety (e.g., anti-nutritional)

concerns. Levels of the components in grain and/or other raw agricultural commodities

of the biotechnology-derived crop product are compared to: 1) corresponding levels in a

conventional comparator, a genetically similar conventional line, grown concurrently,

under similar field conditions, and 2) natural ranges generated from an evaluation of

commercial reference hybrids 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, anti-nutrients, and

secondary metabolites.

This section provides analyses of concentrations of key nutrients, anti-nutrients, and

secondary metabolites of MON 87403 compared to that of a conventional control grown

and harvested under similar conditions, as appropriate. In addition, conventional

commercial maize reference varieties (hereafter refered to as reference hybrids) were

included in the composition analyses to establish a range of natural variability for each

component, defined by the 99% tolerance interval. The production of materials for

compositional analyses used a sufficient variety of field trial sites, robust field designs

(randomized complete block design with four blocks), and sensitive analytical methods

that allow accurate assessments of compositional characteristics over a range of

environmental conditions under which MON 87403 is expected to be grown.
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VI.A. Compositional Equivalence of MON 87403 Grain and Forage to

Conventional Maize

Grain and forage samples were collected from MON 87403, a conventional control

maize, and a total of 17 different reference hybrids (Table F-1) grown in the United

States during a 2012 field production. The reference hybrids were included in the

composition analyses to provide data on the natural variability for each component. The

field production was conducted at eight sites. The field sites were planted in a

randomized complete block design with four blocks per site. MON 87403, the

conventional control, and reference hybrids were grown in areas of the United States that

were typical for maize production and under normal agronomic field conditions for their

respective geographic regions.

The evaluation of MON 87403 followed considerations relevant to the compositional

quality of maize as defined by the OECD consensus document (OECD 2002b). Grain

samples were analyzed for levels of nutrients including proximates, carbohydrates by

calculation, fiber, amino acids, fatty acids, minerals, and vitamins. The anti-nutrients

analyzed in grain included phytic acid and raffinose. Secondary metabolites analyzed in

grain included furfural, ferulic acid, and p-coumaric acid. Forage samples were analyzed

for levels of proximates, carbohydrates by calculation, fiber, and minerals. In total, 78

different components were assayed (nine in forage and 69 in grain).

Of those 78 components, 14 fatty acids (caprylic, capric, lauric, myristic, myristoleic,

pentadecanoic, pentadecenoic, palmitoleic, heptadecanoic, heptadecenoic, gamma

linolenic, eicosadienoic, eicosatrienoic, and arachidonic acids), sodium, and furfural had

more than 50% of observations below the assay limit of quantitation (LOQ) and were

excluded from statistical analysis. Moisture in grain and forage was measured for

conversion of components to dry weight, but was not statistically analyzed. Therefore,

60 components were statistically analyzed.

The statistical comparison of MON 87403 and the conventional control was based on

compositional data combined across all field sites. Statistically significant differences

were identified at the 5% level (α = 0.05). The compositional data from the reference

hybrids were combined across all field sites to calculate a 99% tolerance interval for each

component to estimate the natural variability of each component in maize.

A statistically significant difference between MON 87403 and the conventional control

does not necessarily imply biological relevance from a food and feed safety perspective.

Therefore, statistically significant differences observed are typically evaluated in the

context of natural variability to determine whether a detected difference indicates a

biologically relevant compositional change. However, in this study, no significant

differences between MON 87403 and the conventional control were observed in any of

the measured components. Measurements of grain nutrients, including protein, amino

acids (18 components), total fat, fatty acids (22 components), carbohydrates by

calculation, fiber (3 components), ash, minerals (9 components), vitamins (7

components), antinutrients (phytic acid and raffinose), and secondary metabolites

(furfural, ferulic acid, and p-coumaric acid) demonstrated that values in MON 87403
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were not statistically significantly different from those in the conventional control (Table

VI-1 to Table VI-6). Forage component levels, including ash, protein, total fat,

carbohydrates by calculation, fiber (ADF and NDF), and minerals (calcium and

phosphorus) were also not statistically significantly different between MON 87403 and

the conventional control (Table VI-7). International Life Sciences Institute Crop

Composition Database (ILSI-CCDB) and published literature values for all analytes are

provided in Table VI-8.

The compositional analysis provided a comprehensive comparative assessment of the

levels of key nutrients, anti-nutrients, and secondary metabolites in maize grain and

forage of MON 87403 and the conventional control. The lack of any statistically

significant differences between MON 87403 and the conventional control demonstrated

that MON 87403 was not a major contributor to variation in nutrient, antinutrient, or

secondary metabolite component levels in maize grain or forage and confirmed the

compositional equivalence of MON 87403 to the conventional control in levels of these

components.
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Table VI-1. Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and Conventional

References

Difference (Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

Protein 10.13 (0.34) 10.15 (0.34) (8.58 - 12.63) 4.01 -0.016 (0.14) 0.911 -0.16

8.41 - 11.90 8.49 - 12.50 7.72, 12.67

Alanine 0.77 (0.030) 0.77 (0.030) (0.63 - 0.99) 0.36 0.00036 (0.012) 0.975 0.05

0.63 - 0.91 0.62 - 0.98 0.55, 1.01

Arginine 0.48 (0.0093) 0.48 (0.0093) (0.43 - 0.61) 0.13 -0.00007 (0.0051) 0.988 -0.01

0.41 - 0.53 0.41 - 0.54 0.41, 0.59

Aspartic Acid 0.63 (0.018) 0.63 (0.018) (0.56 - 0.79) 0.21 0.00058 (0.0080) 0.943 0.09

0.54 - 0.72 0.52 - 0.74 0.52, 0.78

Cystine/Cysteine 0.21 (0.0041) 0.21 (0.0041) (0.17 - 0.26) 0.06 0.0017 (0.0032) 0.589 0.83

0.18 - 0.23 0.18 - 0.24 0.16, 0.25

Glutamic Acid 1.85 (0.077) 1.86 (0.077) (1.50 - 2.47) 0.91 -0.0056 (0.033) 0.870 -0.30

1.48 - 2.26 1.43 - 2.34 1.26, 2.52
Monsanto Company CR262-14U1 77 of 260

Table VI-1 (continued). Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and

Conventional References

Difference (Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

Glycine 0.37 (0.0065) 0.37 (0.0065) (0.32 - 0.44) 0.08 0.00016 (0.0036) 0.966 0.04

0.32 - 0.41 0.33 - 0.42 0.32, 0.44

Histidine 0.28 (0.0068) 0.28 (0.0068) (0.22 - 0.35) 0.09 0.0016 (0.0034) 0.648 0.55

0.24 - 0.33 0.24 - 0.33 0.20, 0.35

Isoleucine 0.36 (0.014) 0.36 (0.014) (0.28 - 0.44) 0.17 -0.00044 (0.0061) 0.944 -0.12

0.29 - 0.43 0.28 - 0.46 0.26, 0.46

Leucine 1.27 (0.059) 1.27 (0.059) (0.98 - 1.65) 0.71 0.0015 (0.026) 0.956 0.12

0.98 - 1.58 0.93 - 1.64 0.81, 1.73

Lysine 0.27 (0.0048) 0.27 (0.0048) (0.23 - 0.33) 0.07 0.0013 (0.0027) 0.625 0.50

0.24 - 0.30 0.23 - 0.31 0.24, 0.31

Methionine 0.21 (0.0063) 0.20 (0.0063) (0.16 - 0.26) 0.08 0.00071 (0.0028) 0.803 0.35

0.17 - 0.23 0.17 - 0.25 0.15, 0.26
Monsanto Company CR262-14U1 78 of 260

Table VI-1 (continued). Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and

Conventional References

Difference (Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

Phenylalanine 0.52 (0.022) 0.52 (0.022) (0.42 - 0.65) 0.24 0.0024 (0.0093) 0.801 0.47

0.41 - 0.63 0.40 - 0.64 0.36, 0.68

Proline 0.95 (0.030) 0.95 (0.030) (0.73 - 1.12) 0.43 0.0032 (0.015) 0.830 0.34

0.80 - 1.10 0.76 - 1.18 0.64, 1.17

Serine 0.44 (0.015) 0.45 (0.015) (0.37 - 0.58) 0.18 -0.0068 (0.0072) 0.356 -1.51

0.35 - 0.51 0.37 - 0.55 0.34, 0.57

Threonine 0.36 (0.0098) 0.35 (0.0098) (0.31 - 0.44) 0.11 0.0025 (0.0043) 0.570 0.69

0.30 - 0.40 0.30 - 0.41 0.28, 0.43

Tryptophan 0.077 (0.0015) 0.077 (0.0015) (0.067 - 0.096) 0.01 -0.00019 (0.00085) 0.827 -0.25

0.063 - 0.086 0.069 - 0.084 0.064, 0.093

Tyrosine 0.41 (0.016) 0.41 (0.016) (0.34 - 0.55) 0.20 0.00043 (0.0081) 0.959 0.10

0.32 - 0.48 0.32 - 0.52 0.30, 0.53
Monsanto Company CR262-14U1 79 of 260

Table VI-1 (continued). Summary of Maize Grain Protein and Amino Acids for MON 87403, Conventional Control, and

Conventional References

Difference (Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value % Relative6

Valine 0.46 (0.013) 0.46 (0.013) (0.38 - 0.57) 0.17 -0.00092 (0.0061) 0.880 -0.20

0.38 - 0.52 0.39 - 0.56 0.35, 0.57

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.

6The relative magnitude of the difference in mean values between MON 87403 and the control, expressed as a percent of the control.
Monsanto Company CR262-14U1 80 of 260

Table VI-2. Summary of Maize Grain Total Fat and Fatty Acids for MON 87403, Conventional Control, and Conventional

References

Difference

(Test minus Control)

Component

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Total Fat (% dwt)13.56 (0.072) 3.54 (0.072) (2.49 - 4.70) 0.98 0.026 (0.060) 0.682

3.10 - 3.90 3.11 - 4.09 1.93, 5.49

16:0 Palmitic612.77 (0.14) 12.68 (0.14) (9.90 - 13.12) 2.99 0.097 (0.072) 0.219

12.27 - 13.50 10.59 - 13.59 7.90, 14.94

18:0 Stearic 2.04 (0.022) 2.06 (0.022) (1.46 - 2.42) 0.46 -0.025 (0.019) 0.206

1.87 - 2.14 1.82 - 2.28 1.05, 2.72

18:1 Oleic 21.84 (0.30) 21.77 (0.30) (21.13 - 34.04) 7.59 0.070 (0.19) 0.716

20.77 - 23.46 20.50 - 28.08 14.77, 38.25

18:2 Linoleic 61.23 (0.30) 61.39 (0.30) (51.19 - 62.88) 6.25 -0.15 (0.16) 0.372

59.60 - 62.78 57.12 - 63.36 45.88, 70.60

18:3 Linolenic 1.31 (0.014) 1.30 (0.014) (0.78 - 1.48) 0.24 0.012 (0.010) 0.267

1.22 - 1.40 1.16 - 1.40 0.69, 1.67
Monsanto Company CR262-14U1 81 of 260

Table VI-2 (continued). Summary of Maize Grain Total Fat and Fatty Acids for MON 87403, Conventional Control, and

Conventional References

Difference

(Test minus Control)

Component

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

20:0 Arachidic 0.45 (0.012) 0.45 (0.012) (0.30 - 0.52) 0.14 -0.0017 (0.0034) 0.634

0.41 - 0.52 0.40 - 0.54 0.26, 0.55

20:1 Eicosenoic 0.20 (0.0030) 0.20 (0.0030) (0.19 - 0.30) 0.05 -0.0020 (0.0017) 0.243

0.18 - 0.22 0.18 - 0.23 0.14, 0.33

22:0 Behenic 0.16 (0.0081) 0.16 (0.0081) (0.055 - 0.23) 0.13 0.0029 (0.0045) 0.544

0.062 - 0.21 0.069 - 0.20 0, 0.26

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.

6Expressed as % total fatty acid. Prefix numbers refer to number of carbon atoms and number of carbon-carbon double bonds in the fatty acid

molecule; 16:0 means sixteen carbon atoms and zero double bonds. Numbers are not included in text discussion for reasons of clarity. The

following fatty acids with more than 50% of observations below the assay LOQ were excluded from statistical analysis: caprylic acid,

capric acid, lauric acid, myristic acid, myristoleic acid, pentadecanoic acid, pentadecenoic acid, palmitoleic acid, heptadecanoic acid,

heptadecenoic acid, gamma linolenic acid, eicosadienoic acid, eicosatrienoic acid, and arachidonic acid.
Monsanto Company CR262-14U1 82 of 260

Table VI-3. Summary of Maize Grain Carbohydrates by Calculation and Fiber for MON 87403, Conventional Control, and

Conventional References

Difference

(Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Carbohydrates by 84.98 (0.34) 84.98 (0.34) (82.55 - 86.97) 3.84 0.0021 (0.12) 0.986

Calculation 83.09 - 86.73 82.77 - 86.61 81.80, 87.71

Acid Detergent Fiber 3.63 (0.068) 3.62 (0.068) (2.52 - 4.42) 1.49 0.0087 (0.062) 0.893

3.16 - 4.07 2.91 - 4.40 2.36, 4.43

Neutral Detergent Fiber 9.47 (0.19) 9.41 (0.19) (6.86 - 12.18) 3.48 0.063 (0.16) 0.696

8.11 - 10.67 7.53 - 11.01 5.32, 12.85

Total Dietary Fiber 13.04 (0.15) 12.95 (0.15) (9.83 - 17.30) 4.19 0.098 (0.22) 0.657

11.53 - 14.70 11.50 - 15.69 10.05, 15.51

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.
Monsanto Company CR262-14U1 83 of 260

Table VI-4. Summary of Maize Grain Ash and Minerals for MON 87403, Conventional Control, and Conventional References

Difference

(Test minus Control)

Component

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Ash (% dwt)11.31 (0.026) 1.33 (0.026) (1.02 - 1.53) 0.42 -0.017 (0.019) 0.371

1.09 - 1.52 1.10 - 1.51 1.08, 1.60

Calcium (% dwt) 0.0037 (0.00021) 0.0037 (0.00021) (0.0011 - 0.0058) 0.00 0.00006 (0.00006) 0.321

0.0026 - 0.0049 0.0025 - 0.0049 0.0011, 0.0059

Copper (mg/kg dwt) 1.57 (0.11) 1.55 (0.11) (0.92 - 6.11) 1.87 0.023 (0.12) 0.852

1.13 - 3.35 1.04 - 2.91 0.29, 3.17

Iron (mg/kg dwt) 19.33 (0.82) 19.60 (0.82) (14.66 - 25.54) 8.09 -0.27 (0.21) 0.240

15.60 - 24.15 15.81 - 23.90 10.87, 27.03

Magnesium (% dwt) 0.12 (0.0033) 0.12 (0.0033) (0.093 - 0.14) 0.04 -0.00010 (0.0013) 0.939

0.10 - 0.14 0.096 - 0.14 0.092, 0.15

Manganese (mg/kg dwt) 6.17 (0.31) 6.14 (0.31) (4.02 - 9.46) 3.99 0.029 (0.11) 0.787

4.36 - 7.85 4.55 - 8.55 2.59, 10.23
Monsanto Company CR262-14U1 84 of 260

Table VI-4 (continued). Summary of Maize Grain Ash and Minerals for MON 87403, Conventional Control, and

Conventional References

Difference

(Test minus Control)

Component

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Phosphorus (% dwt) 0.30 (0.0068) 0.30 (0.0068) (0.24 - 0.36) 0.10 -0.0017 (0.0041) 0.683

0.25 - 0.33 0.24 - 0.34 0.24, 0.38

Potassium (% dwt) 0.33 (0.0076) 0.33 (0.0076) (0.27 - 0.42) 0.10 -0.0023 (0.0044) 0.611

0.30 - 0.40 0.30 - 0.40 0.23, 0.42

Zinc (mg/kg dwt) 20.52 (0.83) 20.98 (0.83) (15.56 - 30.10) 10.65 -0.46 (0.30) 0.165

16.30 - 25.20 16.49 - 27.14 9.09, 32.95

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.
Monsanto Company CR262-14U1 85 of 260

Table VI-5. Summary of Maize Grain Vitamins for MON 87403, Conventional Control, and Conventional References

Difference

(Test minus Control)

Component (mg/kg dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Vitamin A (-Carotene) 1.16 (0.039) 1.14 (0.039) (0.48 - 2.85) 0.49 0.025 (0.023) 0.319

0.89 - 1.37 0.88 - 1.37 0, 3.10

Vitamin B1 (Thiamin) 3.48 (0.13) 3.52 (0.13) (2.54 - 4.99) 1.41 -0.046 (0.042) 0.277

2.72 - 4.19 2.82 - 4.23 1.73, 5.12

Vitamin B2 (Riboflavin) 1.83 (0.059) 1.71 (0.059) (1.35 - 2.35) 1.00 0.12 (0.053) 0.057

1.48 - 2.43 1.22 - 2.22 1.25, 2.22

Vitamin B3 (Niacin) 16.47 (0.78) 16.59 (0.78) (12.77 - 30.15) 14.27 -0.12 (0.46) 0.788

12.74 - 21.74 12.61 - 26.88 7.36, 30.18

Vitamin B6 (Pyridoxine) 7.11 (0.17) 6.89 (0.17) (4.66 - 8.80) 3.51 0.22 (0.19) 0.255

5.50 - 10.63 5.85 - 9.36 4.51, 8.98

Vitamin B9 (Folic Acid) 0.39 (0.016) 0.39 (0.016) (0.22 - 0.77) 0.20 0.00065 (0.0086) 0.940

0.27 - 0.50 0.28 - 0.47 0.038, 0.69
Monsanto Company CR262-14U1 86 of 260

Table VI-5 (continued). Summary of Maize Grain Vitamins for MON 87403, Conventional Control, and Conventional

References

Difference

(Test minus Control)

Component (mg/kg dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Vitamin E (-Tocopherol) 17.87 (0.65) 18.33 (0.65) (8.68 - 25.90) 8.20 -0.47 (0.24) 0.095

14.70 - 20.76 15.03 - 23.23 2.50, 27.12

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.
Monsanto Company CR262-14U1 87 of 260

Table VI-6. Summary of Maize Grain Anti-nutrients and Secondary Metabolites for MON 87403, Conventional Control, and

Conventional References

Difference

(Test minus Control)

Component

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Anti-nutrient (% dwt1)

Phytic Acid 0.89 (0.017) 0.87 (0.017) (0.68 - 1.15) 0.40 0.023 (0.016) 0.164

0.70 - 1.04 0.67 - 1.07 0.68, 1.18

Raffinose 0.22 (0.012) 0.23 (0.012) (0.062 - 0.35) 0.14 -0.0030 (0.0041) 0.487

0.14 - 0.30 0.17 - 0.31 0.00088, 0.40

Secondary Metabolite (μg/g dwt)

Ferulic Acid 2262.60 (61.27) 2213.54 (61.27) (1381.65 - 2990.97) 1350.33 49.06 (48.06) 0.341

1833.71 - 2587.97 1344.15 - 2694.48 827.07, 3473.40

p-Coumaric Acid 216.73 (5.25) 212.58 (5.25) (103.35 - 383.41) 86.91 4.15 (5.09) 0.441

160.59 - 242.25 173.81 - 260.72 6.62, 433.65

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.
Monsanto Company CR262-14U1 88 of 260

Table VI-7. Summary of Maize Forage Proximates, Fiber, and Minerals for MON 87403, Conventional Control, and

Conventional References

Difference

(Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Ash 4.00 (0.35) 4.14 (0.35) (2.05 - 7.98) 4.10 -0.14 (0.11) 0.199

2.17 - 5.87 2.14 - 6.24 0.67, 7.56

Carbohydrates by 86.98 (0.43) 86.76 (0.43) (82.09 - 90.15) 6.01 0.22 (0.28) 0.449

Calculation 84.01 - 90.21 83.73 - 89.74 81.04, 92.37

Protein 7.08 (0.27) 7.19 (0.27) (4.14 - 10.27) 4.92 -0.12 (0.19) 0.548

5.60 - 8.92 4.42 - 9.34 3.56, 10.69

Total Fat 1.96 (0.12) 1.98 (0.12) (0.62 - 3.18) 2.41 -0.023 (0.12) 0.851

0.92 - 2.89 0.53 - 2.94 0.81, 3.33

Acid Detergent Fiber 23.01 (0.89) 22.44 (0.89) (16.01 - 37.25) 12.85 0.57 (0.83) 0.513

16.18 - 33.12 17.33 - 30.17 17.89, 28.94

Neutral Detergent Fiber 36.83 (1.06) 37.23 (1.06) (27.09 - 54.66) 26.57 -0.40 (1.33) 0.768

28.67 - 44.62 29.77 - 56.33 30.85, 44.85
Monsanto Company CR262-14U1 89 of 260

Table VI-7 (continued). Summary of Maize Forage Proximates, Fiber, and Minerals for MON 87403, Conventional Control, and

Conventional References

Difference

(Test minus Control)

Component (% dwt)1

MON 87403

Mean (S.E.)2

Range

Control

Mean (S.E.)

Range

Conventional

Reference (Range)3

Tolerance Interval4

Control Range

Value5

Mean

(S.E.) p-Value

Calcium 0.22 (0.015) 0.23 (0.015) (0.12 - 0.40) 0.17 -0.013 (0.0083) 0.150

0.12 - 0.34 0.16 - 0.33 0.10, 0.36

Phosphorus 0.16 (0.0058) 0.16 (0.0058) (0.10 - 0.30) 0.09 0.0011 (0.0049) 0.828

0.12 - 0.22 0.13 - 0.22 0.045, 0.30

1dwt = dry weight.

2Mean (S.E.) = least-square mean (standard error).

3Range is the minimum and maximum raw values for the conventional reference maize hybrids.

4With 95% confidence, interval contains 99% of the values expressed in the population of conventional reference maize hybrids.

Negative limits set to zero.

5Maximum value minus minimum value for the control maize hybrid.
Monsanto Company CR262-14U1 90 of 260

Table VI-8. Literature and ILSI Database Ranges for Components in Maize Forage

and Grain

Grain Tissue Components1 Literature Range2ILSI Range3

Grain Nutrients

Proximates (% dwt)

Ash 1.17 – 2.01a; 1.27 – 1.63b0.616 – 6.282

Carbohydrates by calculation 81.31 – 87.06a; 82.10 – 85.98b77.4 – 89.5

Fat, total 2.95 – 4.40a; 3.18 – 4.23b1.742 – 5.900

Protein 8.27 – 13.33a; 9.17 – 12.19b6.15 – 17.26

Fiber (% dwt)

Acid detergent fiber 1.82 – 4.48a; 1.83 – 3.39b1.82 – 11.34

Neutral detergent fiber 6.51 –12.28a; 6.08 – 10.36b5.59 – 22.64

Total dietary fiber 10.65 – 16.26a; 10.57 – 14.56b9.01 – 35.31

Amino Acids (% dwt)

Alanine 0.60 – 1.04a; 0.68 – 0.96b0.44 - 1.39

Arginine 0.34 – 0.52a; 0.34 – 0.50b0.12 - 0.64

Aspartic acid 0.52 – 0.78a; 0.59 – 0.76b0.33 – 1.21

Cystine 0.19 – 0.26a; 0.20 – 0.26b0.13 – 0.51

Glutamic acid 1.54 – 2.67a; 1.71 – 2.44b0.97 – 3.54

Glycine 0.33 – 0.43a; 0.33 – 0.42b0.18 – 0.54

Histidine 0.25 – 0.37a; 0.27 – 0.34b0.14 – 0.43

Isoleucine 0.30 – 0.48a; 0.32 – 0.44b0.18 – 0.69

Leucine 1.02 – 1.87a; 1.13 – 1.65b0.64 – 2.49

Lysine 0.26 – 0.33a; 0.28 – 0.31b0.17 – 0.67

Methionine 0.17 – 0.26a; 0.16 – 0.30b0.12 – 0.47

Phenylalanine 0.43 – 0.72a; 0.45 – 0.63b0.24 – 0.93

Proline 0.74 – 1.21a; 0.78 – 1.11b0.46 – 1.63

Serine 0.39 – 0.67a; 0.43 – 0.60b0.24 – 0.77

Threonine 0.29 – 0.45a; 0.31 – 0.39b0.22 – 0.67

Tryptophan 0.047 – 0.085a; 0.042 – 0.070b0.027 – 0.215

Tyrosine 0.13 – 0.43a; 0.12 – 0.41b0.10 – 0.64

Valine 0.42 – 0.62a; 0.45 – 0.58b0.27 – 0.86

Fatty Acids (% Total FA)

16:0 Palmitic 8.80 – 13.33a; 9.84 – 12.33b7.94 – 20.71

18:0 Stearic 1.36 – 2.14 a; 1.30 – 2.10b1.02 – 3.40

18:1 Oleic 19.50 – 33.71a; 19.59 – 29.13b17.4 – 40.2

18:2 Linoleic 49.31 – 64.70a; 56.51 – 65.65b36.2 – 66.5

18:3 Linolenic 0.89 – 1.56a; 1.03 – 1.38b0.57 – 2.25

20:0 Arachidic 0.30 – 0.49a; 0.30 – 0.41b0.279 – 0.965

20:1 Eicosenoic 0.17 – 0.29a; 0.17 – 0.27b0.170 – 1.917

22:0 Behenic 0.069 – 0.28a; 0.059 – 0.18b0.110 – 0.349

Minerals

Calcium (% dwt) 0.0030 – 0.0083c0.00127 – 0.02084

Copper (mg/kg dwt) 0.85 – 3.54c0.73 – 18.50

Iron (mg/kg dwt) 10.58 – 30.97c10.42 – 49.07

Magnesium (% dwt) 0.085 – 0.15c0.0594 – 0.194

Manganese (mg/kg dwt) 3.67 – 9.39c1.69 – 14.30

Phosphorous (% dwt) 0.25 – 0.38c0.147 – 0.533

Potassium (% dwt) 0.29 – 0.47c0.181 – 0.603

Zinc (mg/kg dwt) 16.67 – 31.38c6.5 – 37.2
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Table VI-8 (continued). Literature and ILSI Database Ranges for Components in

Maize Forage and Grain

Grain Tissue Components1 Literature Range2ILSI Range3

Vitamins (mg/kg dwt)

Folic acid 0.19 – 0.35a; 0.23 – 0.42b0.147 – 1.464

Vitamin A [–Carotene] 122 – 4740d

0.19 – 46.81

Vitamin B1 [Thiamine] 2.33 – 4.17a; 2.71 – 4.33b1.26 – 40.00

Vitamin B2 [Riboflavin] 0.94 – 2.42a; 1.64 – 2.81b0.50 – 2.36

Vitamin B3 [Niacin] 15.07 – 32.38a; 13.64 – 42.06b10.37 – 46.94

Vitamin B6 [Pyridoxine] 4.93 – 7.53a; 4.97 – 8.27b3.68 – 11.32

Vitamin E [–Tocopherol] 5.96 – 18.44a

; 2.84 – 15.53b1.537 – 68.672

Grain Anti–Nutrients (% dwt)

Phytic acid 0.69 – 1.09a; 0.60 – 0.94b0.111 – 1.570

Raffinose 0.079 – 0.22a; 0.061 – 0.15b0.020 – 0.320

Grain Secondary Metabolites (g/g dwt)

Ferulic acid 1205.75 – 2873.05a; 1011.40 – 2539.86b291.9 – 3885.8

p–Coumaric acid 94.77 – 327.39a; 66.48 – 259.68b53.4 – 576.2

Forage Tissue Components1 Literature Range2ILSI Range3

Forage Nutrients

Proximates (% dwt)

Ash 2.67 – 8.01a; 4.59 – 6.90b1.527 – 9.638

Carbohydrates by calculation 81.88 – 89.26a; 84.11 – 87.54b76.4 – 92.1

Fat, total 1.28 – 3.62a; 0.20 – 1.76b0.296 – 4.570

Protein 5.80 – 10.24a; 5.56 – 9.14b3.14 – 11.57

Fiber (% dwt)

Acid detergent fiber 19.11 – 30.49a; 20.73 – 33.39b16.13 – 47.39

Neutral detergent fiber 27.73 – 49.62a; 31.81 – 50.61b20.29 – 63.71

Minerals (% dwt)

Calcium 0.12 – 0.33a; 0.21 – 0.41b0.07139 – 0.57679

Phosphorous 0.090 – 0.26a; 0.13 – 0.21b0.09362 – 0.37041

1

dwt=dry weight; FA = fatty acids.

2Literature range references: aUS and bChile (Harrigan et al. 2009), c

France (Ridley et al. 2011), d(Safawo

et al. 2010).

3

ILSI range is from ILSI Crop Composition Database, 2011 [Accessed 9 January 2013] (ILSI 2011).
Monsanto Company CR262-14U1 92 of 260

VI.B. Compositional Assessment of MON 87403 Conclusion

Compositional analysis was conducted on grain and forage of MON 87403 grown at

eight sites in a 2012 field production in the United Sates that are representative of typical

agricultural regions for maize production. The compositional analysis, based on the

OECD consensus document for maize, included measurement of nutrients, anti-nutrients

and secondary metabolites in conventional reference hybrids to provide data on the

natural variability of each compositional component analyzed.

Of the 60 components statistically assessed for MON 87403, none of the components

showed a significant difference between MON 87403 and the conventional control.

These results support the overall conclusion that MON 87403 was not a major contributor

to variation in component levels in maize grain and forage and confirmed the

compositional equivalence of MON 87403 to the conventional control in levels of these

components.
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VII. PHENOTYPIC, AGRONOMIC, AND ENVIRONMENTAL

INTERACTIONS ASSESSMENT

This section provides a comparative assessment of the phenotypic, agronomic, and

environmental interaction characteristics of MON 87403 compared to the conventional

control. The data support a conclusion that MON 87403 is not meaningfully different

from the conventional control with the exception of the increased ear biomass trait, and

therefore, is not expected to pose a plant pest risk compared to conventional maize.

These conclusions are based on the results of multiple evaluations from laboratory and

field assessments.

Phenotypic, agronomic, and environmental interaction characteristics of MON 87403

were evaluated in a comparative manner to assess plant pest potential. These assessments

included evaluation of seed germination characteristics, plant growth and development

characteristics, observations of plant responses to abiotic stress, plant-disease and plantarthropod interactions, and pollen characteristics. Results from these assessments

demonstrate that MON 87403 does not possess a) increased weediness characteristics;

b) increased susceptibility or tolerance to specific abiotic stresses, diseases, or

arthropods; or c) characteristics that would confer a plant pest risk compared to the

conventional control.

VII.A. Characteristics Measured for Assessment

In the phenotypic, agronomic, and environmental interactions assessment of

MON 87403, 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. That description includes

differences from the unmodified recipient organism that would “substantiate that the

regulated article is unlikely to pose a greater plant pest risk than the unmodified organism

from which it was derived.” As part of the characterization of MON 87403, data were

collected to provide a detailed description of the phenotypic, agronomic, and

environmental interaction characteristics of MON 87403. A subset of these data were

included in an evaluation of specific characteristics related to altered plant pest potential

(e.g., seed dormancy, lodging, ear drop, and environmental interactions data).

The plant characterization of MON 87403 encompassed six general data categories: 1)

seed germination, dormancy, and emergence; 2) vegetative growth; 3) reproductive

development (including pollen characteristics); 4) lodging and seed retention on the

plant; 5) plant response to abiotic stress and interactions with diseases and arthropods;

and 6) intended phenotype (increased ear biomass). 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 maize. In each of these assessments, MON 87403 was compared to an

appropriate conventional control that had a genetic background similar to MON 87403

but did not possess increased ear biomass trait. In addition, multiple commercial maize

hybrids developed through conventional breeding and selection (see Appendices G, H,
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and I and Tables G-1, H-1, and I-1) were included to provide a range of comparative

values for each characteristic that are representative of the variability in existing

commercial maize hybrids. Data collected for the various characteristics from the

commercial reference hybrids provided context for interpreting experimental results.
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Table VII-1. Phenotypic, Agronomic, and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials, and Laboratory Studies

Data category

Characteristics

measured

(associated section

where discussed)

Evaluation timing1

(Setting of evaluation)

Evaluation description

(measurement endpoints)

Germination,

dormancy, and

emergence

Normal germinated

(VII.C.1)

Day 4 and 7:

20/30°C (Laboratory)

Percentage of seed producing seedlings

exhibiting normal developmental

characteristics

Abnormal

germinated

(VII.C.1)

Day 7:

20/30°C (Laboratory)

Percentage of seed producing seedlings

that could not be classified as normal

germinated

Germinated

(VII.C.1)

Day 4, Day 7, and

Day 12:

5, 10, 20, 30, 10/20 and

10/30°C (Laboratory)

Percentage of seed that had germinated

(both normally or abnormally)

Dead

(VII.C.1)

Day 4 and 7:

5, 10, 20, 30, 10/20,

10/30, and 20/30°C.

Day 12:

5, 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 non-viable firm-swollen seed)

Viable hard

(VII.C.1)

Day 7:

20/30°C

Day 12:

5, 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 firm-swollen

(VII.C.1)

Day 7:

20/30°C

Day 12:

5, 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 test2)

Early stand count

(VII.C.2.1)

V2 – V51growth stage

(Field)

Number of emerged plants in two rows

Vegetative

growth

Final stand count

(VII.C.2.1)

Pre-harvest (Field) Number of plants in two rows

Plant vigor

(VII.C.2.1)

V2 – V51growth stage

(Field)

Rated on a 1-9 scale, where 1 =

excellent vigor and 9 = poor vigor

Stay green

(VII.C.2.1)

Maturity1(Field) Rated on a 1-9 scale, 1 = 90 to 100%; 2

= 80-89%; 3 = 70-79%; 4 = 60-69%; 5

= 50-59%; 6 = 40-49%; 7 = 30-39%; 8

= 20-29%; and 9 = 0 – 19% green

tissue

Ear height

(VII.C.2.1)

R1 – Maturity1(Field) Distance from the soil surface at the

base of the plant to the ear attachment

node on five plants per plot

Plant height

(VII.C.2.1)

R1 – Maturity1(Field) Distance from the soil surface at the

base of the plant to the flag leaf collar

on five plants per plot
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Table VII-1 (continued). Phenotypic, Agronomic and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

Data category

Characteristics

measured

Evaluation timing1

(Setting of evaluation)1 Evaluation description

Reproductive

growth

Days to 50% pollen

shed

(VII.C.2.1)

Pollen shed1(Field) Days from planting until 50% of

the plants have begun to shed

pollen

Days to 50% silking

(VII.C.2.1)

Silking1(Field) Days from planting until 50% of

the plants have multiple silks

exposed

Pollen viability

(VII.C.3)

Tasseling (Laboratory) Percentage of viable pollen based

on pollen grain staining

characteristics

Pollen morphology

(VII.C.3)

Tasseling (Laboratory) Diameter of viable pollen grains

and visual observations

Grain moisture

(VII.C.2.1)

Harvest (Field) Percentage moisture of harvested

shelled grain

Test weight

(VII.C.2.1)

Harvest (Field) Test weight (kilogram/hecto liter)

of harvested shelled grain

Yield

(VII.C.2.1)

Harvest (Field) Calculated in Mg/ha, adjusted to

15.5% grain moisture content

Lodging and

Seed retention

Stalk lodged plants

(VII.C.2.1)

Pre-harvest (Field) Number of plants per plot broken

below the ear

Root lodged plants

(VII.C.2.1)

Pre-harvest (Field) Number of plants per plot leaning

at the soil surface at >30° from the

vertical

Dropped ears

(VII.C.2.1)

Pre-harvest (Field) Number of mature ears dropped

from plants

Environmental

interactions

Abiotic stress

response

(VII.C.2.2.1)

Four times during growing

season (Field)

Qualitative assessment of each

plot, with categorical scale of

increasing severity (none, slight,

moderate, severe)

Disease damage

(VII.C.2.2.1)

Four times during growing

season (Field)

Qualitative assessment of each

plot, with categorical scale of

increasing severity (none, slight,

moderate, severe)

Arthropod damage

(VII.C.2.2.1)

Four times during growing

season (Field)

Qualitative assessment of each

plot, with categorical scale of

increasing severity (none, slight,

moderate, severe)

Stalk rot disease

(VII.C.2.2.1)

Harvest (Field) Qualitative assessment of each

plot, with categorical scale of

increasing severity (none, slight,

moderate, severe)

Ear/kernel rot

disease

(VII.C.2.2.1)

Harvest (Field) Qualitative assessment of each

plot, with categorical scale of

increasing severity (none, slight,

moderate, severe)
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Table VII-1 (continued). Phenotypic, Agronomic and Environmental Interaction

Characteristics Evaluated in U.S. Field Trials and Laboratory Studies

Characteristics

measured

Evaluation timing1

(Setting of evaluation)1 Evaluation description

Environmental

interactions

Corn earworm

damage

(VII.C.2.2.2)

R5 growth stage1(Field) Quantitative assessment on 10

plants per plot by examining ears

for damaged area using a plastic

film grid (each grid cell =

0.5 cm2)

European corn borer

damage

(VII.C.2.2.2)

R6 growth stage1(Field) Quantitative assessment on 10

plants per plot by counting

number of feeding galleries and

total length of feeding galleries

in each stalk

Arthropod

abundance

(VII.C.2.2.2)

Five collection times during

growing season (Field)

Quantitative assessment of

arthropod abundance via sticky

traps collections and visual

counts

Intended

Phenotype

R1 ear biomass

(VII.C.2.1)

R1 Growth Stage1(Field) Dry weight in grams of whole

ears from 1 m in length of row

1

Plant growth stages were determined using descriptions and guidelines outlined in Corn Growth and

Development (Ritchie et al. 1997).

2 Viability of hard and firm-swollen seed were determined by a tetrazolium test (AOSA/SCST 2010).
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VII.B. Interpretation of Phenotypic and Environmental Interaction Data

Plant pest risk assessments for biotechnology-derived crops are comparative assessments,

and are considered from a basis of familiarity. The concept of familiarity is based on the

fact that the biotechnology-derived plant is developed from a well-characterized

conventional crop whose biological properties and plant pest potential are well-known.

Familiarity considers the biology of the crop, the introduced trait, 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 maize was the basis for

selecting appropriate endpoints and estimating the range of responses that would be

considered typical for maize. As such, MON 87403 was compared to the conventional

control in the assessment of phenotypic, agronomic, and environmental interaction

characteristics. An overview of the characteristics assessed is presented in Table VII-1.

A subset of the data relating to well-understood weedy characteristics (e.g., seed

dormancy, pre-harvest seed loss characteristics, and lodging) was used to assess whether

there was an increase in weediness potential of MON 87403 compared to conventional

maize. Evaluation of environmental interaction characteristics (e.g., plant abiotic stress,

plant-disease, and plant-arthropod interactions) was also considered in the plant pest

assessment. Prior to analysis, the overall dataset was evaluated for possible evidence of

biologically-relevant changes and unexpected plant responses. No unexpected

observations or issues were identified. Based on all of the data collected, an assessment

was made to determine if MON 87403 could be expected to pose an increased plant pest

risk compared to conventional maize.

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

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. 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 crop’s pest/weed potential.

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 change for the crop in terms of plant pest 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

combined-site 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 Reference Hybrids 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

characteristic is assessed relative to the range of variation of the reference hybrids

included in the study (e.g., reference range).

Hazard identification and

risk assessment on

difference

Outside variation for crop? No

Yes

No

Yes

Adverse in terms of plant

pest/weed 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 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

change for the crop in

terms of plant pest/weed

potential

Step 5

Step 6

Step 1
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Step 4 - Evaluate Differences in the Context of the Crop

If the mean value of the characteristic for a biotechnology-derived crop is outside the

variation of the reference hybrids in the study, the mean value of the biotechnologyderived crop is assessed relative to known values common for the crop (e.g., published

values).

Step 5 - Relevance of Differences to Plant Pest/Weed Potential

If the mean value of the characteristic for a biotechnology-derived crop is outside the

range of values common for the crop, the difference is then assessed for whether or not it

is meaningful in terms of plant pest/weed potential.

Step 6 - Conduct Risk Assessment on Identified Hazard

If an adverse effect (hazard) is identified, a 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.
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VII.B.2. Interpretation of Vigor and Environmental Interactions Data

For the qualitative assessments of vigor, abiotic stress response, disease damage, and

arthropod damage, the biotechnology-derived crop and the conventional control are

considered different in susceptibility or tolerance if the range of injury symptoms of each

did not overlap across all four replications. Any observed differences are assessed for

biological significance in the context of the range of the reference hybrids, and for

consistency in other observation times and sites. Differences that are not consistently

observed in multiple environments are not considered to be biologically meaningful in

terms of plant pest potential.

Quantitative assessments of corn earworm and European corn borer damage are analyzed

within individual sites and pooled across sites in a combined-site analysis. Statistically

significant differences detected between the biotechnology-derived crop and the

conventional control are evaluated using the method outlined in Figure VII-1.

Quantitative assessments of arthropod abundance are only analyzed within each

individual site. Statistically significant differences between the biotechnology-derived

crop and the conventional control were assessed for biological significance in the context

of the range of the reference hybrids, and for consistency with other collection times and

collection sites and in the context of pest potential. Differences that are not consistently

detected in multiple environments are not considered biologically meaningful in terms of

plant pest potential.

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

Environmental Interaction Characteristics of MON 87403

This section provides the results of comparative assessments conducted in replicated

laboratory and/or multi-site field experiments to provide a detailed phenotypic,

agronomic, and environmental interactions description of MON 87403. The

characteristics for MON 87403 evaluated in these assessments included: seed dormancy

and germination characteristics (Section VII.C.1), plant phenotypic, agronomic, 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 G, H, and I.

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 with 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). Information on germination and dormancy

characteristics is therefore useful when assessing a plant for increased weediness

potential. To assess germination characteristics, standardized germination assays are

available and routinely used. The Association of Official Seed Analysts (AOSA), an

internationally recognized seed testing organization, recommends a temperature range of

20/30 °C as optimal for testing the germination and dormancy characteristics of maize

seed (AOSA 2012b; a). Additional temperature regimes were also evaluated.
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A comparative assessment of seed germination and dormancy characteristics was

conducted on MON 87403 and the conventional control. The seed lots used for the

germination testing of MON 87403 (selfed F2 grain), the conventional control, and the

reference hybrids (4 per site, 9 unique across all locations) were produced in replicated

field trials during 2012 in Story County, Iowa (IA), Warren County, Illinois (IL), and

Lehigh County, Pennsylvania (PA). These geographic areas represent a broad range of

environmental conditions for maize production for this product. The experiments were

arranged as separate split-plot experiments with four replications for each temperature

regime.

Descriptions of the evaluated germination and dormancy characteristics and the timing of

the evaluations for all temperature regimes are listed in Table VII-1. Additional details

on the materials and experimental methods used in this evaluation are presented in

Appendix G: .

In the combined-site analysis, in which data were pooled across the three seed production

sites, no statistically significant differences (α=0.05) were detected between MON 87403

and the conventional control for any characteristic at the AOSA temperature regime

(20/30 °C), or at the temperature regimes of 5, 10, 20, 30, 10/20, 10/30 °C (Table VII-2).

In addition, no hard seed were observed at any temperature.

The germination and dormancy characteristics evaluated were used to assess MON 87403

in the context of plant pest/weed potential. The results of this assessment, particularly the

lack of increased hard seed, and no changes in other germination and dormancy

characteristics, support the conclusion that the introduction of increased ear biomass trait

is not expected to result in increased plant pest/weed potential compared to conventional

maize.
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Table VII-2. Germination Characteristics of MON 87403 and the Conventional

Control

Temperature

(°C)

Assessment

Category

Mean % (S.E.)1 Reference

Range2

MON 87403 Control

5 Germinated 5.7 (0.90) 6.2 (1.24) 2.3 – 9.5

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 4.1 (0.60) 3.5 (0.69) 1.5 – 6.1

Viable Firm-Swollen 90.3 (1.21) 90.3 (1.15) 86.8 – 94.8

10 Germinated 95.0 (0.73) 94.0 (0.79) 95.0 – 99.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 1.8 (0.58) 2.0 (0.51) 0.5 - 3.6

Viable Firm-Swollen 3.2 (0.66) 4.0 (0.98) 0.0 – 3.8

203 Germinated 99.3 (0.26) 99.1 (0.23) 96.3 - 99.8

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 0.7 (0.26) 0.9 (0.23) 0.3 – 3.7

Viable Firm- Swollen† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

30 Germinated 99.4 (0.23) 99.6 (0.19) 97.5 - 99.8

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 0.6 (0.23) 0.4 (0.19) 0.3 - 2.5

Viable Firm- Swollen† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

10/20 Germinated 99.4 (0.19) 99.5 (0.19) 96.0 - 100.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 0.4 (0.15) 0.5 (0.19) 0.0 – 3.8

Viable Firm- Swollen 0.2 (0.11) 0.0 (0.00) 0.0 - 0.5

10/30 Germinated 99.6 (0.19) 99.5 (0.19) 97.0 – 100.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 0.4 (0.19) 0.5 (0.19) 0.0 - 3.0

Viable Firm- Swollen† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

20/30 Normal Germinated 98.9 (0.31) 98.8 (0.32) 95.6 – 99.8

(AOSA) Abnormal Germinated 0.8 (0.25) 0.7 (0.22) 0.0 - 2.0

Viable Hard† 0.0 (0.00) 0.0 (0.00) 0.0 - 0.0

Dead 0.3 (0.13) 0.5 (0.23) 0.3 – 2.5

Viable Firm- Swollen 0.1 (0.08) 0.0 (0.00) 0.0 - 0.1

Note: The experimental design was a split-plot with four replications.

No statistically significant differences were detected between MON 87403 and the

conventional control (α =0.05) using ANOVA.

1MON 87403 and the conventional control values represent means with standard error

(S.E.) in parentheses. N = 12. In some instances, the total percentage of both

MON 87403 and the conventional control did not equal 100% due to numerical rounding

of the means.

2Reference range is the minimum and maximum mean values observed among the 9

commercially available reference hybrids.

3

Three replicates of the reference Midland Phillips 799 were used from sites ILMN and

PAGR.

†No statistical comparison could be made due to lack of variability in the data.
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VII.C.2. Field Phenotypic, Agronomic, and Environmental Interactions

Characteristics

Phenotypic and agronomic characteristics, and environmental interactions were evaluated

under field conditions as part of the plant characterization assessment of MON 87403.

These data were developed to provide USDA-APHIS with a detailed description of

MON 87403 relative to the conventional control and reference hybrids. 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 87403 and the

conventional control that may impact its plant pest/weed potential. Specific

characteristics that are related to weediness, (e.g., lodging and ear drop), were used to

assess whether there is a potential increase in weediness of MON 87403 compared to

conventional maize. Environmental interactions including plant response to abiotic

stress, disease damage, arthropod-related damage, and arthropod abundance were also

assessed as an indirect indicator of changes to MON 87403 and are also considered in the

plant pest assessment.

The results of the assessments of agronomic and phenotypic characteristics demonstrated

that the introduction of the increased ear biomass trait did not meaningfully alter the plant

pest/weed potential of MON 87403 compared to conventional maize. Furthermore, the

lack of meaningful differences in plant response to abiotic stress, disease damage,

arthropod-related damage, and arthropod abundance also support the conclusion that the

introduction of the increased ear biomass trait is not expected to result in increased plant

pest/weed potential for MON 87403 compared to conventional maize.

VII.C.2.1. Field Phenotypic and Agronomic Characteristics

Data were collected at 13 sites (Table VII-3) in the U.S. during 2012 to evaluate

phenotypic and agronomic characteristics of MON 87403 compared to the conventional

control. These 13 sites provided a diverse range of environmental and agronomic

conditions representative of commercial maize production areas in North America. The

experimental design at each site was a randomized complete block with four replications.

At all sites, MON 87403, the conventional control, and four reference hybrids were

evaluated. A total of 21 unique reference hybrids were evaluated among the 13 sites

(Table H-1). The planted plot dimensions varied between sites due to variability in

available planting equipment (Table H-2). All plots of MON 87403, the conventional

control, and the reference hybrids at each site were uniformly managed in order to assess

whether the introduction of the increased ear biomass maize trait altered the phenotypic

and agronomic characteristics of MON 87403 compared to the conventional control.

Descriptions of the evaluated phenotypic characteristics and the timing of the evaluations

are listed in Table VII-1. The materials, methods, details concerning the timing of

phenotypic assessments, and detailed results of the individual-site data comparisons are

presented and discussed in Appendix H (Table H-4). The results of the combined site

analyses are summarized below.

In the combined-site analysis, no statistically significant differences were detected

between MON 87403 and the conventional control for 12 of 13 characteristics including
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early stand count, days to 50% pollen shed, days to 50% silking, stay green rating, plant

height, dropped ear count, stalk lodged plants, root lodged plants, final stand count, grain

moisture, test weight and yield (Table VII-4). One significant difference was detected for

ear height in the combined-site analysis. MON 87403 had increased ear height (111.1 vs.

107.5 cm) compared to the conventional control. However, the mean value of

MON 87403 for ear height was within the reference range for this characteristic and a

small ear height difference would not be expected to influence plant weediness. Thus,

the measured phenotypic characteristics of MON 87403 were not altered in terms of

pest/weed potential compared to conventional maize (Figure 1, step 2, “no” answer) and

(Figure 1, step 3, “no” answer).

Plant vigor data were summarized as ranges within individual sites. MON 87403 and the

conventional control were considered different if the range of vigor values did not

overlap across all four replications. There were no differences observed between

MON 87403 and the conventional control in the plant vigor ratings across sites (Table H4).

The phenotypic and agronomic characteristics evaluated in this study were used to

provide a detailed description of MON 87403 compared to the conventional control. A

subset of these characteristics was used to assess the weediness of MON 87403. The

results of the agronomic and phenotypic assessment demonstrate that there were no

changes indicating altered weediness/pest potential of MON 87403 compared to the

conventional control. Thus, the introduction of the increased ear biomass trait is not

expected to result in increased plant pest/weed potential from MON 87403 compared to

conventional maize.

Evaluation of Intended Phenotypic Characteristic:

In other specifically designed experiments, ear biomass data were collected at 13 sites in

the U.S. during 2012 to evaluate the intended phenotype of MON 87403 compared to the

conventional control (Table VII-1). These 13 sites provided a diverse range of

environmental and agronomic conditions representative of commercial maize production

areas in North America. The experimental design at each site was a randomized

complete block with four replications. At all sites, MON 87403 and the conventional

control were evaluated. The harvested area within each plot was 1 m in length. All plots

of MON 87403 and the conventional control were uniformly managed in order to assess

whether the introduction of the increased ear biomass maize trait demonstrated the

intended phenotypic characteristic. Descriptions of the evaluated phenotypic

characteristic and the timing of the evaluation are listed in Table VII-1).

As noted previously (Section I.B), in the combined-site analysis, a statistically significant

difference was detected between MON 87403 and the conventional control for increased

ear biomass (Table I-1). MON 87403 had increased ear biomass (mean 144.5 g/m of row

vs. 129.3 g/m of row) compared to the conventional control thereby demonstrating the

intended phenotype.
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Table VII-3. Field Phenotypic Evaluation Sites for MON 87403 during 2012

Site Code County, State

ARNE Jackson, Arkansas

IABG Greene, Iowa

IARL Jefferson, Iowa

ILCX Vermilion, Illinois

ILMN Warren, Illinois

INSH Boone, Indiana

KSLA Pawnee, Kansas

NCBD Perquimans, North Carolina

NEDC Butler, Nebraska

NESH Polk, Nebraska

NEYO York, Nebraska

PAGR Lehigh, Pennsylvania

PAHM Berks, Pennsylvania
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Table VII-4. Combined-Site Comparison of MON 87403 to Conventional Control for Phenotypic and Agronomic

Characteristics During 2012

Mean (S.E.)1 Reference Range2

Phenotypic Characteristic (units) MON 87403 Control Min Max

Early stand count (#/plot) 83.0(0.92) 83.6 (0.90) 75.4 96.8

Days to 50% pollen shed 63.5 (0.67) 63.0 (0.65) 56.4 71.3

Days to 50% silking 63.9 (0.65) 64.0 (0.70) 56.0 68.8

Stay green rating (1-9 scale) 5.3 (0.36) 5.2 (0.35) 1.0 9.0

Ear height (cm) 111.1 (2.24)* 107.5 (2.25) 86.9 133.2

Plant height (cm) 240.5 (3.69) 239.5 (3.48) 184.2 260.3

Dropped ears (#/plot) 1.2 (0.45) 0.9 (0.36) 0.0 6.1

Stalk lodged plants (#/plot) 2.4 (0.58) 2.2 (0.53) 0.0 10.8

Root lodged plants (#/plot) 0.7 (0.26) 0.5 (0.17) 0.0 1.0

Final stand count (#/plot) 72.5 (0.72) 72.0 (0.70) 63.8 76.0

Grain moisture (%) 19.1 (0.61) 19.0 (0.58) 14.0 28.7

Test weight (kg/hl) 72.6 (0.69) 72.7 (0.67) 67.9 79.2

Yield (Mg/ha) 10.1 (0.45) 10.3 (0.52) 6.3 14.3

* Indicates statistical difference between MON 87403 and the conventional control (α =0.05).

Note: The experimental design was a randomized complete block with four replicates per site.

1

Test and control values represent means with standard error in parentheses. N = 51.

2 Reference range is calculated from the minimum and maximum mean values from among 21 unique reference materials.
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VII.C.2.2. Environmental Interaction Characteristics

USDA-APHIS considers the environmental interaction of the biotechnology-derived crop

compared to its conventional control to determine the potential for increased plant pest

characteristics. Evaluations of environmental interactions were conducted as part of the

plant characterization for MON 87403. In the 2012 U.S. field trials conducted to

evaluate the phenotypic and agronomic characteristics of MON 87403, data were also

collected on plant response to abiotic stress (e.g., drought, wind, nutrient deficiency, etc.),

disease damage, arthropod-related damage, and arthropod abundance (Tables VII-5

through VII-7 and H-5 through H-10). These data were used to assess plant pest potential

(Section IX) compared to the conventional control. The results of the field evaluations

showed that the increase ear biomass trait did not unexpectedly alter the assessed

environmental interactions of MON 87403 compared to the conventional control. The

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

damage, arthropod-related damage, and arthropod abundance support the conclusion that

the introduction of the increased ear biomass trait is not expected to result in increased

plant pest potential from MON 87403 compared to commercial maize.

VII.C.2.2.1. Qualitative Environmental Interactions Assessment

Plant responses to abiotic stressors, disease damage, and arthropod damage were assessed

at natural levels, i.e., no artificial infestation or imposed abiotic stress; therefore these

levels typically varied between observations at a site and among sites. Plant responses to

abiotic stress, disease damage, and arthropod damage data were collected from each plot

using a categorical scale (none, slight, moderate, and severe) 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 stressors, disease damage, and arthropod damage

symptoms potentially occurring across the season and across sites. These data were

categorical and therefore were summarized and not subjected to ANOVA. For a

particular stressor, all comparisons of the range of responses for MON 87403 to the range

of responses for the conventional control across all observation times and sites are

reported.

Descriptions of the evaluated environmental interactions characteristics and the timing of

the evaluations are listed in Table VII-1. The materials, methods, additional details

concerning the qualitative environmental interactions assessments, and detailed results of

the qualitative data comparisons are presented and discussed in Appendix H (Tables H-5

through H-7).

In the qualitative assessment, no differences in the range of responses were observed

between MON 87403 and the conventional control for any of the 143 comparisons of

plant response to abiotic stressors, including cold, drought, flood, frost, hail, heat,

nutrient deficiency, soil compaction, sunscald, and wind (Tables VII-5 and H-5).

Additionally, no differences in the range of responses were observed between

MON 87403 and the conventional control for any of the 176 comparisons for plant

damage caused by diseases, including anthracnose, bacterial leaf spot, ear rot, eyespot,

Fusarium sp., Goss’s bacterial wilt, gray leaf spot, leaf blight, maize rough dwarf virus,
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Pythium sp., Rhizoctonia sp., rust, seedling blight, smut, stalk rot, and Stewart’s bacterial

wilt (Tables VII-5 and H-6). Finally, no differences in the range of responses were

observed between MON 87403 and the conventional control for any of the 150

comparisons for plant damage caused by arthropods, including aphid, armyworm,

billbug, cutworm, corn earworm, corn flea beetle, rootworm beetle, European corn borer,

grasshopper, Japanese beetle, sap beetle, spider mite, stink bug, and wireworm adult

(Tables VII-5 and H-7).

The lack of differences observed between MON 87403 and the conventional control for

plant responses to abiotic stressors, disease damage, and arthropod-related damage in

multiple environments across the U.S. supports the conclusion that the introduction of the

increased ear biomass trait is not expected to cause a biologically meaningful change in

terms of plant pest potential compared to the conventional control (See Section VII.B.2).
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Table VII-5. Summary of Qualitative Environmental Interactions Assessments

during 2012

Note: The experimental design was a randomized complete block with four replicates per

site.

No differences were observed between MON 87403 and the conventional control during

any observation for damage caused by any of the assessed stressors.

1MON 87403 and the conventional control were considered different in susceptibility or

tolerance if the range of injury symptoms across four replications did not overlap

between MON 87403 and the conventional control.

Stressor

Number of observations

across all sites

Number of observations

with no differences

between MON 87403 and

the conventional control

across all sites1

Abiotic stressors 143 143

Disease damage 176 176

Arthropod-related damage 150 150

Total 469 469
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VII.C.2.2.2. Quantitative Environmental Interactions Assessment

Quantitative arthropod assessments on corn earworm (CEW: Helicoverpa zea) damage,

European corn borer (ECB: Ostrinia nubilalis) damage, and arthropod abundance were

conducted at four sites (Table VII-3: IABG, NEYO, NCBD, and PAHM). CEW and

ECB damage was assessed once during the growing season at each site. Arthropod

abundance was assessed from collections performed five times during the growing season

at each site using sticky traps and visual counts.

Descriptions of the evaluated environmental interactions characteristics and the timing of

the evaluations are listed in Table VII-1. The materials, methods, additional details

concerning the specific arthropod damage assessments, sticky traps, and visual counts

with detailed results of the individual-site data comparisons are presented and discussed

in Appendix H (Tables H-8 through H-10). The results of the combined-site analysis are

summarized below.

In the combined-site analysis, no statistically significant differences (α=0.05) were

detected between MON 87403 and the conventional control for plant damage caused by

CEW or ECB (Table VII-6). Considering this lack of significant differences, the results

indicate no changes in susceptibility or resistance of MON 87403 to these common maize

pests (See Figure VII-1, Step 2, answer “no”).

In an assessment of arthropod abundance from sticky traps, no statistically significant

differences were detected between MON 87403 and the conventional control for 130 out

of 144 comparisons among the collections at the four sites (Tables VII-7 and H-9). The

mean abundance values for MON 87403 were within the respective ranges of commercial

reference hybrids for seven of the 14 detected differences. For the remaining seven

differences, the mean abundance values from MON 87403 were outside the reference

range; however, these differences were not consistently detected across collections or

sites. Thus, these differences were not indicative of a consistent response associated with

the trait and are not considered biologically meaningful in terms of increased pest

potential of MON 87403 compared to the conventional control (See Section VII.B.2)

In an assessment of arthropod abundance from visual counts, no statistically significant

differences were detected between MON 87403 and the conventional control for 61 out

of 66 comparisons among the collections at the four sites (Tables VII-7 and H-10). The

mean abundance values for MON 87403 were within the respective ranges of commercial

reference hybrids for three of the five detected differences. For the remaining two

differences, the mean abundance values from MON 87403 were outside the reference

range; however, these differences were not consistently detected across collections or

sites. Thus, these differences were not indicative of a consistent response associated with

the trait and are not considered biologically meaningful in terms of increased pest

potential of MON 87403 compared to the conventional control (See Section VII.B.2).
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Table VII-6. Combined-Site Comparison of CEW and ECB Damage to MON 87403 Compared to the Conventional Control

during 2012

Pest Arthropod Assessment

Timing Damage assessment

Mean (S.E.)1

Reference

range

2 MON 87403 Control

Corn earworm

(H. zea)

R5 Damage area of 10 plants per plot (cm2) 1.8 (0.41) 2.7 (0.78) 0.0 – 5.0

European corn borer

(O. nubilalis)

R6 Number of stalk galleries of 10 plants per

plot

0.3 (0.12) 0.3 (0.11) 0.0 – 1.2

European corn borer

(O. nubilalis)

R6 Stalk gallery length (cm) of 10 plants per

plot

1.3 (0.57) 1.4 (0.50) 0.0 – 4.7

Note: The experimental design was a randomized complete block with four replicates per site.

No statistically significant differences were detected between MON 87403 and the conventional control (α=0.05) using ANOVA.

1 MON 87403 and the conventional control values represent means with standard error in parentheses. N = 16.

2 Reference range is calculated from the minimum and maximum mean values from among 11 unique reference materials across all

sites (IABG, NCBD, NEYO, and PAHM).
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Table VII-7. Summary of Arthropod Abundance Assessments and Detected Differences during 2012

Summary of Statistical Comparisons1 Summary of Detected Differences2

Arthropod

Abundance

Assessment

Number

of sites

Number of

comparisons

across sites

Number of

comparisons

where no

differences

were detected Arthropod Site

Collection

Number

Within

reference

range?

Consistently

detected

across

collections

or sites?

Sticky Traps 4 144 130 Lacewings IABG 1 Yes No

Lacewings NEYO 3 Yes No

Lacewings NEYO 5 No No

Macro-parasitic

hymenoptera PAHM 4 No No

Micro-parasitic

hymenoptera NCBD 2 Yes No

Aphids NEYO 5 Yes No

Corn flea beetles IABG 3 & 5 No No

Corn rootworm

beetles NEYO 3 Yes No

Thrips NCBD 2 Yes No

Thrips NEYO 2 & 4 No No

Thrips NEYO 5 Yes No

Thrips PAHM 3 No No

Visual Counts 4 66 61 Minute pirate bugs PAHM 4 No No

Corn rootworm

beetles PAHM 1 No No

Sap beetles NCBD 5 Yes No

Sap beetles NEYO 3 Yes No

Shining flower

beetles NCBD 2 Yes No
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1Quantitative arthropod abundance assessments were statistically analyzed at α=0.05 using ANOVA. Lack of sufficient arthropod

abundance precluded statistical comparisons between MON 87403 and the conventional control for additional 92 comparisons (sticky

traps) and 171 comparisons (visual counts); however, descriptive statistics were provided for these comparisons in Appendix H

(Tables H-9 and H-10).

2Nineteen statistically significant differences were detected. These differences are further discussed in Section VII.C.2.2 using the

approach outlined in Section VII.B.2.
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VII.C.3. Pollen Characteristics

USDA-APHIS considers the potential for gene flow and introgression of the

biotechnology-derived trait into sexually compatible plants and wild relatives to

determine 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 87403. In addition, morphological

characterization of pollen produced by MON 87403 and the conventional control is

relevant to the plant pest risk assessment because it adds to the detailed description of the

phenotype of MON 87403 compared to the conventional control.

The viability and morphology of pollen collected from MON 87403 compared to that of

the conventional control were assessed. Pollen was collected from MON 87403, the

conventional control, and four commercial references (Table I-1) grown under similar

agronomic conditions at the field site in Story County, Iowa, a geographic area that

represents environmentally relevant conditions for maize production for this product.

The trial was arranged in a randomized complete block design with four replications.

Once all plants across the replications reached the flowering stage, pollen was collected

from three non-systematically selected plants per plot and stained for assessment.

Descriptions of the evaluated pollen viability and morphology characteristics and the

timing of the evaluations are listed in Table VII-1. The details of the materials and

experimental methods used in this evaluation are presented in Appendix I: .

No statistically significant differences (α=0.05) were detected between MON 87403 and

the conventional control for percent viable pollen or pollen grain diameter (Table VII-8).

Furthermore, no visual differences in general pollen morphology were observed between

MON 87403 and the conventional control (Figure I-1).

The pollen characterization data contribute to the detailed phenotypic description of

MON 87403 compared to the conventional control. Based on the assessed

characteristics, the results support a conclusion that neither pollen viability nor

morphology of MON 87403 were altered compared to conventional maize.
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Table VII-8. Pollen Characteristics of MON 87403 Compared to the Conventional

Control during 2012

Pollen

Characteristic

(unit)

Mean (S.E.)1

Reference Range2

MON 87403 Control

Viability3(%) 97.8 (0.70) 98.7 (0.36) 97.3 – 99.3

Diameter4(μm) 81.8 (1.20) 84.6 (2.06) 80.5 – 83.7

Note: No significant differences were detected between the MON 87403 and the

conventional control (α=0.05).

1 MON 87403 and the conventional control values represent means with standard error

(S.E.) in parentheses. N=4.

2 Reference range is the minimum and maximum mean value observed among the four

reference hybrids.

3

Evaluated from three sub-samples per replication at 40X magnification.

4

Evaluated from 10 representative viable pollen grains per replication at 200X

magnification.
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VII.D. Conclusions for Phenotypic, Agronomic, and Environmental

Interactions Evaluation

Comparative plant characterization data between a biotechnology-derived crop and the

conventional control are interpreted in the context of contributions to increased plant pest

potential as assessed by USDA-APHIS. Under the framework of familiarity,

characteristics for which no differences are detected support a conclusion of no increased

plant pest potential of the biotechnology-derived crop compared to the conventional crop.

Ultimately, a weight of evidence approach that considers all characteristics and data is

used for the overall risk assessment of differences and their significance.

An extensive and robust set of agronomic, phenotypic, and environmental interactions

data, including specific weedy characteristics, were used to assess whether the

introduction of the increased ear biomass trait altered the plant pest potential of

MON 87043 compared to the conventional control, considered within the context of the

variation among the reference hybrids. These assessments included six general data

categories: 1) seed germination, dormancy, and emergence; 2) vegetative growth; 3)

reproductive development (including pollen characteristics); 4) lodging and seed

retention on the plant; 5) plant response to abiotic stress and interactions with diseases

and arthropods; and 6) intended phenotype (increased ear biomass). Within these data

categories, data relevant to understanding specific characteristics associated with

weediness were also assessed to determine whether there was a potential increase in

weediness of MON 87403 compared to conventional maize.

Results from these assessments comparing MON 87403 and the conventional control

demonstrate that MON 87403 does not possess: 1) increased weediness characteristics;

2) increased susceptibility or tolerance to specific abiotic stress, diseases, or arthropods;

or 3) characteristics that would confer a plant pest risk compared to conventional maize.

Therefore, based on the results of multiple assessments discussed above and presented in

the appendices, the weight of evidence indicates that MON 87403 is not meaningfully

different from conventional maize with the exception of the increased ear biomass trait

and is not expected to pose a plant pest risk compared to conventional maize.
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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 of

deregulation on agricultural and cultivation practices must be considered. This section

provides a summary of current agronomic practices in the U.S. and North America for

producing maize, and is included in this petition as a baseline to assess possible impacts

to agricultural practices due to the cultivation of MON 87403. Discussions include maize

production, plant growth and development, general management practices during the

season, management of weeds, insects and diseases, maize rotational crops, and volunteer

management. Information presented in the previous section demonstrated that

MON 87403 is no more susceptible to diseases or pests than commercially cultivated

maize. Additionally, data presented in this section show that MON 87403 is not expected

to pose a plant pest risk compared to conventional maize. MON 87403 provides an

increased ear biomass at the R1 reproductive stage derived from the ATHB17∆113

protein and hence an increased yield opportunity. Thus, there are no expected changes to

the inputs needed for MON 87403, and no expected impacts to agronomic practices

employed for production of maize compared to the current situation.

VIII.B. Overview of U.S. Maize Production

VIII.B.1. Maize Production

The U.S., China, Brazil, Ukraine, and Argentina are the top five countries producing

maize globally (USDA-FAS 2014). As noted in Section I.B, maize (Zea mays L.) is the

largest crop grown in the U.S. in terms of acreage planted (95.4 million acres in 2013)

(Table VIII-1) (USDA-NASS 2014c), exceeding soybean and wheat with acreages of

76.5 (USDA-NASS 2014b) and 56.2 million acres (USDA-NASS 2014a), respectively.

The value of maize reached $62.7 billion in the United States in 2013, exceeding

soybeans and wheat with values of $41.8 and $14.4 billion, respectively (USDA-NASS

2013a). The principal uses of maize are feed and residual, ethanol fuel, export, and highfructose corn syrup (Capehart et al. 2012).

The planting of 95.4 million acres of maize in 2013 was down 1.8 million acres from

2012 (Table VIII-1). Much of that production occurs in upper Midwest states (Figure

VIII-1). Of the 2013 acreage planted, approximately 87.7 million acres were harvested

for grain and 6.3 million acres were harvested for silage (USDA-NASS 2013b). Total

production was approximately 13.9 billion bushels with an average yield of 158.8 bushels

per acre (Figure VIII-1). The value of maize grain production in the U.S. has ranged

from $46.7 to $77.4 billion in the past 6 years (Table VIII-1).
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Table VIII-1. Maize Production in the U.S., 2008-2013

Year

Acres

Planted

(x 1,000)

Acres

Harvested

(x 1,000)

Production

(x 1,000 $)

Production

(x 1,000 bushels)

Yield

(bushels/acre)

2013 95,365 87,668 62,716,048 13,925,147 158.8

2012 97,155 87,375 74,330,610 10,780,296 123.4

2011 91,936 83,989 76,939,603 12,359,612 147.2

2010 88,192 81,446 64,643,295 12,446,865 152.8

2009 86,382 79,490 46,734,066 13,091,862 164.7

2008 85,982 78,570 49,312,615 12,091,648 153.9

Source: (USDA-NASS 2013b)

Figure VIII-1. Planted Maize Acres by County in the U.S. in 2012

Source: (USDA-NASS 2012)
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VIII.C. Management of Insect Pests

Monsanto summarized major issues associated with the management of insect pests in its

petition for nonregulated status for Corn Rootworm-Protected Maize MON 87411

(Petition #13-290-01p) (Monsanto 2013). None of the information on this subject has

changed in any substantive way and is incorporated here by reference (Monsanto 2013)

(Section IX.D). In brief, insect pests continue to cause damage to maize and are

commonly addressed by insecticide treatment of seeds, soil, or over-the-top application

of insecticides, or use of a number of crop rotation or integrated pest management

practices.

MON 87403 was developed to increase the yield opportunity of commercial maize

hybrids and has no unique pest control attributes. Thus, no changes to insect pest control

practices are expected from use of MON 87403.

VIII.D. Management of Diseases and Other Pests

Monsanto summarized major issues associated with the management of diseases and

other pests in its petition for nonregulated status for Corn Rootworm-Protected Maize

MON 87411 (Petition #13-290-01p) (Monsanto 2013). None of the information on this

subject has changed in any substantive way and is incorporated here by reference

(Monsanto 2013) (Section IX.E). Briefly, management of diseases and pests of maize are

important to protecting the yield of harvested grain. Disease and pest incidence varies

from year to year and growers may choose to use pesticides or a variety of management

practices to control problem diseases or pests.

Environmental observations in field studies have demonstrated no apparent impact of

MON 87403 on diseases of maize (Section VII.C.2). Therefore, no changes in current

disease management practices are anticipated from the introduction of MON 87403.

VIII.E. Weed Management

Monsanto summarized major issues associated with the management of weeds in its

Petition for nonregulated status for corn rootworm-protected MON 87411 (Petition

#13-290-01p) (Monsanto 2013). None of the information on this subject has changed in

any substantive way and is incorporated here by reference (Monsanto 2013)

(Section IX.F). Briefly, weed control in maize is essential to optimizing yield because

weeds compete with maize for light, nutrients, and moisture.

MON 87403 was developed to increase the yield opportunity of commercial maize

hybrids and has no unique herbicide tolerance traits. Thus, no changes to herbicide use

or weed resistance management practices are expected from use of MON 87403.

VIII.F. Crop Rotation Practices in Maize

Crop rotation is a well-established farming practice and a useful management tool for

maize production. Crop rotations are used to diversify farm income, spread labor

requirements throughout the year, and spread the crop loss risk associated with weather
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and pest damage across two or more crops. In terms of soil and pest management,

rotations are used to 1) manage weed, insect, and disease pests, 2) reduce soil erosion by

wind and water, 3) maintain or increase soil organic matter, 4) provide biologically fixed

nitrogen when legumes are used in the rotation, and 5) manage excess nutrients (Singer

and Bauer 2009). Studies in U.S. corn belt states indicate maize yield is about 10-15%

higher in maize grown following soybean than maize grown following maize (Singer and

Bauer 2009). Despite the many benefits of crop rotations, crop price fluctuations, input

costs, rental agreements, government price supports, weather, choice of farming system

and on-farm resources, and other factors all contribute to decisions regarding crop

rotations. Market conditions such as U.S. government-mandated ethanol use and record

high commodity maize prices have increased the demand for maize grain and resulted in

increases in maize acreage in recent years (Singer and Bauer 2009; USDA-NASS 2013b).

Introduction of MON 87403 is not, however, expected to impact crop rotation practices

any more so than current biotechnology-derived products available to growers.

VIII.G. Maize Volunteer Management

Volunteer maize commonly occurs in rotational crops in the season following cultivation

of conventional or biotechnology-derived maize. Viable grain is not produced on the

approximately 8% of U.S. maize acres that are cultivated for the production of silage, and

volunteer maize plants typically do not occur in the rotational crops that follow maize

harvested as silage. In the warmer climates of the Southeast and Southwest, volunteer

maize is rare because maize grain remaining after harvest is likely to germinate in the fall

and the resulting plants can usually be controlled by tillage or by freezing temperatures in

the winter. In northern maize-growing regions, volunteer maize does not always occur in

rotational crops because of seed decomposition over the winter, efficient harvest

procedures, and tillage prior to planting rotational crops. None of the agronomic or

phenotypic data collected for MON 87403 (Section VIII.C), including data regarding

germination and seed dormancy, indicates that MON 87403 is any more likely to

volunteer than existing commercial maize hybrids.

Management of volunteer maize in rotational crops involves minimizing or reducing the

potential for volunteers through practices that include: 1) adjusting harvest equipment to

minimize the amount of grain lost in the field; 2) planting hybrids that reduce the extent

of ear drop; 3) choosing hybrids with superior stalk strength and reduced lodging; and 4)

practicing no-till production to significantly reduce the potential for volunteer growth in

rotational crops. If volunteer maize does occur in subsequent crops, pre-plant tillage, incrop cultivation and the use of selective herbicides are very effective management tools.

Introduction of MON 87403 is not expected to impact maize volunteer management

practices any more so than existing commercial maize hybrids.

VIII.H. Stewardship of MON 87403

Monsanto develops effective products and technologies that deliver value to growers and

conserve resources that agriculture depends on, and is committed to assuring that its

products and technologies are safe and environmentally responsible. Monsanto

demonstrates this commitment by implementing product stewardship processes
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throughout the lifecycle of a product and by participation in the Excellence Through

Stewardship®(ETS) Program (BIO 2010). ETS 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 2013) 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 87403 in all key maize importing countries

with functioning regulatory systems to assure global compliance and support the flow of

international trade. These actions will be consistent with the Biotechnology Industry

Organization Policy on Product Launch (BIO 2010). Monsanto continues to monitor

other countries that are key importers of maize 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.

Monsanto also commits to industry best practices on seed quality assurance and control

to ensure the purity and integrity of MON 87403 seed. As with all of Monsanto’s

products, before commercializing MON 87403 products in any country, the appropriate

detection methods will be made available to maize producers, processors, and buyers.

VIII.I. Impact of the Introduction of MON 87403 on Agricultural Practices

MON 87403 has been developed to provide an increased yield opportunity in maize

hybrids that will be grown in the U.S. The introduction of MON 87403 is not expected to

have major impacts on current agronomic, cultivation and management practices for

maize. No changes are anticipated in crop rotations, tillage practices, planting practices,

fertility management, weed and disease management, and volunteer management from

the introduction of MON 87403.

MON 87403 has been shown to be comparable to conventional maize in its

compositional, phenotypic, and agronomic characteristics (Sections VI and VII). When

introgressed into existing biotechnology-derived maize hybrids that contain insect

protection and herbicide tolerance traits, MON 87403 is expected to continue to provide

benefits to growers, that include reduced use of insecticides , increased yield protection

and opportunity, water conservation , and increased worker safety.
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IX.PLANT PEST ASSESSMENT

This section provides a brief review and assessment of the plant pest potential of

MON 87403 and its impact on agronomic practices and the environment. USDA-APHIS

has responsibility, under the Plant Protection Act (PPA) (7 U.S.C. § 7701-7772), to

prevent the introduction and dissemination of plant pests into the U.S. 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 should

no longer 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.

According to PPA, the definition of “plant pest” includes the living stage of any of the

following, or a similar article that can directly or indirectly injure, damage, or cause

disease in any plant or plant product: (A) a protozoan; (B) a nonhuman animal; (C) a

parasitic plant; (D) a bacterium; (E) a fungus; (F) a virus or viroid; or (G) an infectious

agent or other pathogens (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 article in question is not

expected to pose a plant pest risk. Information in this petition related to plant pest risk

characteristics includes: 1) mode-of-action and changes to plant metabolism; 2)

composition; 3) expression and characteristics of the gene product; 4) potential for

weediness of the regulated article; 5) impacts to NTOs; 6) disease and pest

susceptibilities; 7) impacts on agronomic practices; and 8) impacts on the weediness of

any other plant with which it can interbreed, as well as the potential for gene flow. Using

the assessment above, the data and analysis presented in this petition lead to a conclusion

that MON 87403 is not expected to be a plant pest, and therefore should no longer be

subject to regulation under 7 CFR § 340.

IX.A. Characteristics of the Genetic Insert and Expressed Product

IX.A.1. Genetic Insert

As described in Section III, MON 87403 was developed by Agrobacterium-mediated

transformation of maize embryos using plasmid vector PV-ZMAP5714. Characterization

of the DNA insert in MON 87403 was conducted using a combination of sequencing,

PCR, and bioinformatics methods. The results of this characterization demonstrate that

MON 87403 contains one copy of the intended T-DNA containing the ATHB17

expression cassette that is stably integrated at a single locus and is inherited according to

Mendelian principles over multiple generations. These methods also confirmed that no

vector backbone or other unintended plasmid sequences are present in MON 87403.

Additionally, the genomic organization at the insertion site was assessed by comparing

the sequences flanking the T-DNA insert in MON 87403 to the sequence of the insertion

site in conventional maize. This analysis determined that no major DNA rearrangement

occurred at the insertion site in MON 87403 upon DNA integration.
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IX.B. Expression and Characterization of Gene Product

IX.B.1. Protein Safety and Expression Levels

The safety and expression of the ATHB17Δ113 protein is detailed in Section V.

Expression levels were determined from four tissue types from trials conducted in 2012

in the United States and are presented in Section V.C. The expression in the various

tissues ranged from <LOD (<0.00028 μg/g dw) to 0.017 μg/g dw. ATHB17Δ113

represents a very small percentage of the total protein in maize seed (no more than

0.001%). The ATHB17Δ113 protein originates from Arabidopsis thaliana, and no

Arabidopsis proteins have been reported in the peer-reviewed database of known

allergens (FARRP 2013). Futhermore, bioinformatic assessments show that the

ATHB17Δ113 protein does not show homology to any known allergens, toxins, or any

other protein with known adverse effects. These assessments also showed that the amino

acid sequence of the ATHB17Δ113 protein shares 58%-83% sequence identity to

proteins present in a variety of food crops including corn, rice, sorghum, cruciferous

vegetables, tomato, potato, papaya, orange and grape, indicating that ATHB17∆113 is

very similar to proteins that have a robust history of safe use. Finally, the protein is

rapidly digested in simulated gastric fluid and simulated intestinal fluid assays

(Section V.D) and a mouse gavage study demonstrated no acute oral toxicity with a

NOAEL for ATHB17Δ113 of 1335 mg/kg, further supporting a conclusion that this

protein is safe for consumption.

IX.C. Compositional Characteristics

Compositional comparisons based on OECD guidance were presented in Section VI to

assess whether levels of nutrients, anti-nutrients, and secondary metabolites in grain and

forage derived from MON 87403 are comparable to levels in the conventional control

and several reference hybrids for which there is an established history of safe

consumption. Nutrients assessed in this analysis included proximates, carbohydrates by

calculation, acid detergent fiber, neutral detergent fiber, total dietary fiber, minerals,

amino acids, and vitamins. Anti-nutrients assessed in grain included phytic acid and

raffinose. Secondary metabolites assessed in grain included furfural, ferulic acid, and pcoumaric acid. Forage samples were assessed for levels of proximates, fiber, minerals

(calcium and phosphorus), and carbohydrates by calculation. In all, 78 different

components were assayed (nine in forage and 69 in grain). Of those 78 components, 14

fatty acids, sodium, and furfural had more than 50% of observations below the assay limit

of quantitation (LOQ) and were excluded from statistical analysis. Moisture in grain and

forage was measured for conversion of components to dry weight, but was not

statistically analyzed. Therefore, 60 components were statistically analyzed.

A combined-site analysis of the data was conducted to determine statistically significant

differences (at 5%) between MON 87403 and the conventional control. The biological

significance of differences from the data was reviewed using considerations relevant to

food and feed safety and nutritional quality. These considerations included: 1) a

determination of the mean differences of nutrient and anti-nutrient components of

MON 87403 and the conventional control; 2) consideration of the mean differences
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between MON 87403 and the conventional control in the context of the range of values

for the conventional control, 3) comparison of the mean differences between MON 87403

and the conventional control to variation in conventional maize as estimated by in-study

reference hybrid values and assessing whether the mean values for MON 87403 were

within 99% tolerance intervals, literature values, and/or ILSI-CCDB values. These

evaluations of variation within the conventional control and conventional maize,

including reference hybrids, are important as crop composition is known to be influenced

by environment and germplasm.

The compositional analysis provided a comprehensive comparative assessment of the

levels of key nutrients, anti-nutrients, and secondary metabolites in maize grain and

forage of MON 87403 and the conventional control. Of the 60 components statistically

assessed there were no statistically significant differences in any component. These

results support the overall conclusion that MON 87403 was compositionally equivalent to

the conventional control.

IX.D. Phenotypic, Agronomic, and Environmental Interaction

Characteristics

An extensive set of comparative plant characterization data were used to assess whether

the introduction of an increased ear biomass trait altered the plant pest potential of

MON 87403 compared to the conventional control (Section VII). Phenotypic,

agronomic, and environmental interaction characteristics of MON 87403 were evaluated

and compared to those of the conventional control. As described previously, these

assessments included: seed dormancy and germination characteristics; agronomic and

plant phenotypic characteristics; observations for abiotic stress response, disease damage,

arthropod-related damage, arthropod abundance, and pollen characteristics. Results from

all phenotypic, agronomic, and environmental interaction assessments demonstrated that

MON 87403 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 87403 is no more likely to pose a plant pest risk than conventional maize.

IX.D.1. Seed Dormancy and Germination

A comparative assessment of seed germination and dormancy characteristics was

conducted on MON 87403 and the conventional control. The results of this assessment,

particularly the lack of increased hard seed, and no changes in other germination and

dormancy characteristics, support the conclusion that the introduction of MON 87403 is

not expected to result in increased plant pest/weed potential compared to conventional

maize.

IX.D.2. Plant Growth and Development

Evaluations of plant growth and development characteristics in the field are useful for

assessing potential weediness characteristics such as stalk and root lodging. Phenotypic

characteristics such as early stand count, days to 50% pollen shed and silking, stay green,
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ear height, plant height, dropped ears, stalk lodged plants, root lodged plants, final stand

count, grain moisture, test weight, R1 ear biomass, and yield were assessed. In the

combined-site analysis in which the data were pooled among the sites, no statistically

significant differences, except for ear height, were detected between MON 87403 and the

conventional control for any of the assessed characteristics. Thus, the phenotypic and

agronomic characteristics of MON 87403 were not altered in terms of pest/weed potential

compared to conventional maize.

IX.D.3. Response to Abiotic Stressors

No differences were observed during comparative field observations between

MON 87403 and the conventional control with respect to responses to abiotic stressors

such as drought, mineral and nutrient toxicity, or temperature stress. The lack of

significant differences in the MON 87403 response to abiotic stress supports the

conclusion that the introduction of the ATHB17Δ113 protein is unlikely to result in

increased pest/weed potential compared to conventional maize.

IX.D.4. Pollen Morphology and Viability

Evaluations of pollen morphology and viability from field-grown plants provide useful

information in a plant pest assessment as it relates to the potential for gene flow to, and

possible introgression of a biotechnology-derived trait into sexually-compatible plants

and wild relatives. No statistically significant differences were detected between

MON 87403 and the conventional control for percent viable pollen or pollen grain

diameter. Additionally, no visual differences in general pollen morphology were

observed between MON 87403 and the conventional control. Based on the assessed

characteristics, the results support a conclusion that neither pollen viability nor

morphology of MON 87403 were altered and therefore do not contribute to an increased

pest/weed potential compared to conventional maize.

IX.E. Impact to Non-target Organisms Including Those Beneficial to

Agriculture or Threatened and Endangered Species

Data from 2012 U.S. phenotypic and agronomic studies and observational data on

environmental interactions such as plant-disease interactions, arthropod damage and

arthropod abundance, were collected at select sites for MON 87403 and conventional

controls. Results from these extensive studies support conclusions of no adverse impacts

to non-target arthropod populations and no changes to plant-disease interactions.

As also noted, the ATHB17Δ113 protein belongs to a common class of plant protein

transcription factors (HD-Zip) and shares protein sequence homology with a number of

common food crops that are consumed widely by humans and animals without noted

allergenic or toxic effects. Bioinformatics analyses also assessed the potential for

allergenicity, toxicity, or biological activity of ATHB17Δ113. Those analyses

demonstrated that ATHB17Δ113 protein does not share amino acid sequence similarity

with known allergens, gliadins, glutenins, or protein toxins which could have adverse

effects to human or animal health. Additionally, a mouse gavage study using
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ATHB17Δ113 protein also supports a conclusion of safety from consumption of

MON 87403 by vertebrate organisms. Taken together, these data, along with the

compositional equivalence data noted above (Section VI.A), support the conclusion that

MON 87403 has no reasonable mechanism for harm to NTOs, or impact on threatened

and endangered species compared to cultivation of conventional maize.

IX.F. Weediness Potential of MON 87403

Maize is not listed as a weed in the major weed references (Crockett 1977; University of

Montana 2011), nor is it present on the lists of noxious weed species distributed by the

federal government (7 CFR § 360). In addition, maize has been grown throughout the

world without any report that it is a serious weed. During domestication of maize, traits

often associated with weediness, such as, seed dormancy, a seed dispersal mechanism, or

the ability to form reproducing populations outside of cultivation, have not been selected.

Even if individual kernels of maize were distributed within a field or along transportation

routes from the fields to storage or processing facilities, sustainable volunteer maize

populations are not found growing in fence rows, ditches, or road sides. Maize is poorly

suited to survive without human assistance and is not capable of surviving as a weed

(Galinat 1988; Keeler 1989).

In comparative studies between MON 87403 and a conventional control, phenotypic,

agronomic and environmental interaction data were evaluated (Section VII) for changes

that would impact the plant pest potential, in particular, plant weediness potential.

Results of these evaluations show that there is no fundamental difference between

MON 87403 and the conventional control for traits potentially associated with weediness.

The only difference noted related to ear height and that characteristic is not known to

contribute to weediness. Furthermore, comparative field observations between

MON 87403 and its conventional control and their response to abiotic stressors indicated

no differences and, therefore, no increased weediness potential. Collectively, these

findings support the conclusion that MON 87403 has no increased weed potential

compared to conventional maize and it is no more likely to become a weed than

conventional maize.

IX.G. Potential for Pollen Mediated Gene Flow and Introgression

Pollen mediated gene flow is a process whereby one or more genes successfully integrate

into the genome of a recipient plant. Introgression 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 relative humidity, and field architecture. Because gene introgression is a

natural biological process, it does not constitute an environmental risk in and of itself.

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 will result in increased plant pest potential.

The potential for gene introgression from MON 87403 is discussed below.
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IX.G.1. Hybridization with Cultivated Maize

Maize morphology fosters cross pollination, therefore, high levels of pollen mediated

gene flow can occur in this species. In addition, researchers recognize that (1) the

amount of gene flow that occurs can be high because of open pollination; (2) the percent

gene flow can vary by population, hybrid or inbred; (3) the level of gene flow decreases

with greater distance between the source and recipient plants; (4) environmental factors

affect the level of gene flow; (5) maize pollen is viable for a short period of time under

field conditions; (6) maize produces ample pollen over an extended period of time; and,

(7) maize is almost exclusively wind pollinated.

Based on several studies conducted on the extent of pollen mediated gene flow between

maize fields, results were found to vary depending on the experimental design,

environmental conditions, and detection method, as expected. In general, the percent of

gene flow diminished with increasing distance from the source field, generally falling

below 1% at distances >200 m (~660 feet) (Table IX-1). This information is useful for

managing gene flow during maize breeding, seed production, identity preservation or

other applications; in addition, it forms the basis for the USDA-APHIS performance

standards for maize. All testing and production of regulated MON 87403 seed or grain

have been conducted under USDA notification according to these standards. Gene flow

from fields planted with MON 87403 to other maize would not be of concern because of

the lack of potential to cause harm to humans and to the environment.
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Table IX-1. Summary of Published Literature on Maize Cross Pollination

Pollinator

Distance

(m)

Reported

Outcrossing

(%)

Comments Country Reference

~1 28.6 Frequencies of outcrossing by distance. Three year study.

Pollen source was a yellow dent and the pollen recipient

was a white sweet maize.

USA (Jones and Brooks 1950)

25 14.2

75 5.8

125 2.3

200 1.2

300 0.5

400 0.2

500 0.2

100 0.01 Frequencies of outcrossing by distance and pollen

viability. Two year study. A purple gene marker was

utilized to measure pollen mobility.

Mexico (Luna et al. 2001)

150 0.00

200 0.01

300 0.00

400 0.00

1 9.7-19.0 Frequency of outcrossing by distance. Conducted over

three years and three sites. Single male and female per

location.

Canada (Ma et al. 2004)

5 1.3-2.6

10 0.7-2.0

14 0.3-0.6

19 0.4

24 0-0.3

28 0.1-0.5

33 0-0.3

36 0-0.1
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Table IX-1 (continued). Summary of Published Literature on Maize Cross Pollination

Pollinator

Distance

(m)

Reported

Outcrossing

(%)

Comments Country Reference

24-32 0.01-0.7 Frequencies of outcrossing by distance. One pollen donor and 7

pollen recipients had different relative maturities. The pollen

donor had the genetic markers P1-rr and R1-nj. Pollination of

the pollen recipient caused the female with typically a yellow

kernel to produce a kernel with a purple coloration. Conducted

over two years/two sites. Values reported here are from one

site.

USA (Halsey et al.

60-62 0.01-0.2 2005)

123-125 0.001-0.08

244-254 0-0.02

486-500 0-0.005

743-745 0-0.002

1 17.0-29.9 Frequency of outcrossing by distance. Pollination was

quantified by measuring outcrossing from a transgenic hybrid

plot to a conventional grain production field. A combination of

three marker genes were utilized to detect outcrosses: y1 (seed

color gene), Bt and glyphosate tolerance. Two years/two sites.

USA (Goggi et al.

2006)

10 1.5-2.5

35 0.4

100 0.03-0.05

150 0.01-0.03

200 0.007-0.03

250 0.002-0.03

~1 3-13 The main objective of the study was to compare a PCR based

method to measure outcrossing rates determined by phenotypic

analysis. Four Bt hybrids and a single non-Bt hybrid were used

as a pollen donor and reciepient, respectively. Conducted in

one year/one site.

Spain (Pla et al. 2006)

2 0.2-10

5 0.1-2.3

10 0.2-3.7

20 0.1-0.8

40 0-0.7

80 0.1-0.2
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Table IX-1 (continued). Summary of Published Literature on Maize Cross Pollination

Pollinator

Distance

(m)

Reported

Outcrossing

(%)

Comments Country Reference

2 34.9 Frequency of outcrossing (expressed as % GM DNA) by

distance. The study was conducted in a large farm scale

evaluation across the UK. Values reported here are

maximum raw values

UK (Weekes et al.

5 9.9 2007)

10 12.2

20 8.2

25 4

50 5.9

150 5.4

200 0.24

52 0.009 Frequency of outcrossing by distance. Outcrossing was

measured using the occurrence of yellow kernels in 13

white kernel maize fields.

Switzerland (Bannert and

85 0.015 Stamp 2007)

105 0.003

125 0.01

149 0.016

150 0.007

200 0.009

287 0.005

371 0.008

402 0.005

458 0.0002

4125 0.006

4440 0.0005
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IX.G.2. Hybridization with Wild Annual Species of Zea mays subsp. mexicana

For gene flow to occur by normal sexual transmission, the following conditions must

exist: (1) the two parents must be sexually compatible; (2) there must be overlapping

flowering times; and (3) a suitable factor (such as wind or insects) must be present and

capable of transferring pollen between the two parents.

Maize and annual teosinte (Zea mays subsp. mexicana), are genetically compatible, windpollinated and teosinte pollen can pollinate maize silks when in close proximity to each

other, e.g. in areas of Mexico and Guatemala (Wilkes 1972). Maize crosses with

teosinte; however, teosinte is not present in the U.S. other than as an occasional botanical

garden specimen or small feral populations in Florida, Alabama, and Maryland. In

experimental studies where maize and teosinte species were planted together, very low

hybridization rates were observed for maize and Zea mays subsp. mexicana (Baltazar et

al. 2005; Ellstrand et al. 2007). Differences in factors such as flowering time,

geographical distribution, and development factors make natural crosses in the U.S.

highly unlikely.

IX.G.3. Hybridization with the Wild Perennial Species of Subgenus

Tripsacum

In contrast with maize and teosinte, which hybridize under certain conditions, it is only

with extreme difficulty and special techniques that maize and the related perennial

species, Tripsacum dactyloides (gamma grass) hybridize. Furthermore, hybrids of the

cross are male sterile, even after several backcrosses to maize (Russell and Hallauer

1980).

A single species, Tripsacum floridanum (Florida gamma grass), found in the extreme

southern Florida counties of Miami-Dade, Collier and Monroe has been categorized as a

threatened species by the state of Florida and listed on the USDA Natural Resources

Conservation Service (USDA-NRCS) database (USDA-NRCS 2012). Another species,

Tripsacum dactyloides (Eastern gamma grass), found primarily throughout the eastern

U.S., has been categorized as endangered in Massachusetts and Pennsylvania, and as

threatened in New York (USDA-NRCS 2013). However, given the level of difficulty for

natural hybridization between species of Tripsacum and Zea as mentioned above, the

occurrence of T. floridanum primarily in both highly urbanized and non-agricultural,

swampy areas of the state where commercial maize is not typically grown, as well as the

preference of T. dactyloides for wet habitats where hybrid maize production would not

occur, it is very unlikely there would be any impact on this species due to the

introduction of MON 87403.

IX.G.4. Transfer of Genetic Information to Species with which Maize Cannot

Interbreed (Horizontal Gene Flow)

Monsanto is aware of no reports confirming the transfer of genetic material from maize to

other species with which maize cannot interbreed. The probability for horizontal gene

flow to occur is judged to be exceedingly small. Even if it were to occur, the
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consequences would be negligible since the ATHB17Δ113 protein is similar to other HDZip proteins found in maize that are not known to have meaningful toxicity to humans

and NTOs. Similar to the situation with transfer of the protein genes, the likelihood of

horizontal transfer of the ATHB17Δ113 coding sequence from MON 87403 is also

exceedingly small. The consequence of such transfer, given the known lack of toxicity of

these classes of proteins would also be expected to be inconsequential. In either case, the

presence of this coding sequence would not be expected to increase the pest/weed

potential of the recipient species.

IX.H. Potential Impact on Maize Agronomic Practices

An assessment of current maize agronomic practices was conducted to determine whether

the cultivation of MON 87403 has the potential to impact current maize management

practices (Section VIII). Maize fields are typically highly managed agricultural areas that

are dedicated to crop production. Other than the specific insertion of the ATHB17

coding sequence that provides the potential for increased ear biomass at an early

reproductive phase and hence increased yield opportunity, MON 87403 is similar to other

high-yielding maize hybrids available commercially in the U.S.

The data presented demonstrate that MON 87403 is similar to commercially cultivated

maize in its agronomic, phenotypic, ecological, and compositional characteristics, and

has levels of resistance to insect pests and diseases comparable to other commercially

cultivated maize. Based on this assessment, the introduction of MON 87403 is not likely

to impact current U.S. maize agronomic or cultivation practices or lead to an increased

plant pest potential compared to other maize hybrids widely available to growers.

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

Conventional Maize

Numerous biotechnology-derived maize products have been deregulated or are under

consideration for deregulation by USDA-APHIS. Once deregulated, MON 87403 may

be bred with these deregulated maize products, as well as with conventional maize,

creating new improved hybrids. APHIS has determined that none of the individual

biotechnology-derived maize products it has previously deregulated displays increased

plant pest characteristics. APHIS has also concluded that progeny derived from crosses

of these deregulated maize products with conventional or previously deregulated maize

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 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 biotechderived maize product across multiple generations; 2) stability of each of the introduced

traits is continually and repeatedly assessed as new combined-trait hybrids are created by

plant breeders and tested over multiple seasons prior to commercialization; 3) combinedtrait products are developed using conventional breeding that has been safely used for

thousands of years to generate new varieties (Steiner et al. 2013; WHO 1995); 4)

worldwide organizations, such as World Health Organization, Food and Agriculture

Organization/ World Health Organization, International Seed Federation, CropLife
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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;

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 distinct from those present in the single event biotech

products (Pilacinski et al. 2011).

Therefore, based on the considerations above and the conclusion that MON 87403 is no

more likely to pose a plant pest risk than commercially cultivated maize, it can be

concluded that any progeny derived from crosses between MON 87403 and conventional

maize or deregulated biotechnology-derived maize are no more likely to pose a plant risk

than commercially cultivated maize.

IX.J. Summary of Plant Pest Assessments

A plant pest, as defined in the PPA, is the living stage of any of the following that can

directly or indirectly injure, damage, or cause disease in any plant or plant product: (A) a

protozoan; (B) a nonhuman animal; (C) a parasitic plant; (D) a bacterium; (E) a fungus;

(F) a virus or viroid; (G) an infectious agent or other pathogen, or (H) any article similar

to or allied with any of the articles specified in the preceding subparagraphs (7 U.S.C. §

7702[14]). Characterization data presented in Sections III through VII and Section IX of

this petition confirm that MON 87403, with the exception of the increased ear biomass at

R1 phenotype, is not fundamentally different from conventional maize, in terms of plant

pest potential. Monsanto is not aware of any study results or observations associated

with MON 87403 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 87403 compared to conventional maize, followed by a

risk assessment on detected differences. The plant pest risk assessment in this petition

was based on the following lines of evidence: 1) insertion of a single functional copy of

the ATHB17Δ113 cassette; 2) characterization and safety of the expressed products; 3)

compositional equivalence of MON 87403 forage and grain compared to a conventional

control; 4) phenotypic, agronomic, and environmental characteristics demonstrating no

increased plant pest potential compared to conventional maize; 5) negligible risk to

NTOs, including organisms beneficial to agriculture; 6) familiarity with maize as a

cultivated crop and 7) no greater likelihood to impact agronomic practices, cultivation

practices, or the management of weeds, diseases and insects, than conventional maize.

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

conventional maize and previously deregulated biotechnology-derived maize,

MON 87403 is not expected to be a plant pest. Results also support a conclusion of no

increased weediness potential of MON 87403 compared to conventional maize.

Therefore, Monsanto Company requests a determination from USDA-APHIS that

MON 87403 and any progeny derived from crosses between MON 87403 and other

commercial maize be granted nonregulated status under 7 CFR Part 340.
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X. ADVERSE CONSEQUENCES OF INTRODUCTION

Monsanto knows of no study results or observations associated with MON 87403

indicating that there would be adverse consequences from its introduction. MON 87403

produces a truncated ATHB17Δ113 protein, which has been fully characterized and its

safety has been thoroughly assessed in this submission. As demonstrated by field test

results and laboratory tests, the only phenotypic differences between MON 87403 and

conventional maize are related to an increased ear biomass phenotype at the R1 growth

stage.

The data and information presented in this petition demonstrate that MON 87403 is

unlikely to pose an increased plant pest risk compared to conventional maize. This

conclusion is reached based on multiple lines of evidence developed from a detailed

characterization of the product compared to conventional maize, followed by risk

assessment on detected differences. The characterization evaluations included molecular

analyses, which confirmed the insertion of one copy of the intended DNA containing the

ATHB17Δ113 cassette that is stably integrated at a single locus and is inherited

according to Mendelian principles over multiple generations.

Analysis of key nutrients, anti-nutrients, and secondary metabolites of MON 87403

demonstrate that MON 87403 is compositionally equivalent to conventional maize. The

phenotypic evaluations, including an assessment of seed germination and dormancy

characteristics, plant growth and development characteristics, pollen characteristics,

ecological interaction characteristics, and environmental interactions also indicated

MON 87403 is unchanged compared to conventional maize. There is no indication that

MON 87403 would have an adverse impact on beneficial or non-target organisms,

including threatened or endangered species. Therefore, based on the lack of increased

pest potential compared to conventional maize, the risks for humans, animals, and other

NTOs from MON 87403 are negligible.

The introduction of MON 87403 will not adversely impact cultivation practices or the

management of weeds, diseases, and insects in maize production systems. Farmers

familiar with commercial maize hybrids will be advised to continue to employ the same

crop rotational practices, weed control practices and/or volunteer control measures

currently in place for these products.
Monsanto Company CR262-14U1 136 of 260

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APPENDICES
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Appendix A: USDA Notifications and Permits

Field trials of MON 87403 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 87403 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 2012 -

2014 seasons, are still in preparation. A list of trials conducted under USDA notifications

or permits and the status of the final reports for these trials are provided in Table A-1.

.
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Table A-1. USDA Notifications and Permits Approved for MON 87403 and Status

of Trials Planted

Field Trial

Year USDA No. Effective Date Trial Status

Release

State Sites

2007 07-126-102n 6/8/2007 Submitted HI 2

07-126-102n 6/8/2007 Submitted HI 1

08-036-109n 3/6/2008 Submitted IA 9

2008 08-036-112n 3/21/2008 Submitted IL 4

IN 2

08-036-115n 3/20/2008 Submitted IL 6

08-036-117n 3/6/2008 Submitted KS 3

08-106-111rm 6/30/2008 Submitted HI 1

08-330-103rm 2/27/2009 Submitted IL 8

09-034-101n 3/5/2009 Submitted IA 8

09-035-102n 3/25/2009 Submitted KS 2

2009 OH 4

09-035-103n 3/25/2009 Submitted IA 1

IL 1

09-142-101rm 8/31/2009 Submitted HI 1

09-266-101n 10/23/2009 Submitted HI 1

09-317-101rm 2/12/2010 Submitted CA 5

IA 12

IL 11

IN 3

KS 10

OH 6

10-054-140n 3/24/2010 Submitted KS 13

10-056-112n 3/26/2010 Submitted NE 15

2010 10-063-105rm 4/17/2010 Submitted HI 2

10-067-112n 4/7/2010 Submitted AR 1

IL 3

MO 1

OH 1

WI 1

10-067-113n 4/7/2010 Submitted IA 9

IL 6

IN 1

10-068-104n 4/8/2010 Submitted IA 6

IL 7

OH 5

10-068-116n 4/8/2010 Submitted IA 4

IL 2

IN 3

KS 1

2011 NE 2

10-068-117n 4/8/2010 Submitted IL 2

NE 1

10-069-121n 4/9/2010 Submitted IL 1

10-078-109n 4/18/2010 Submitted IA 1

10-123-102n 6/2/2010 Submitted HI 3

10-131-103rm 6/29/2010 Submitted HI 2

10-264-101n 10/20/2010 Submitted HI 1
Monsanto Company CR262-14U1 149 of 260

10-131-103rm 6/29/2010 Submitted HI 1

10-264-101n 10/20/2010 Submitted HI 1

10-322-103n 12/20/2010 Submitted HI 2

10-350-104rm 1/18/2011 Submitted HI 1

10-350-109rm 2/15/2011 Submitted CA 3

IA 6

IL 8

IN 1

KS 6

OH 6

TX 1

10-351-114rm 3/15/2011 Submitted CA 2

IA 9

IL 7

IN 1

KS 5

OH 2

11-012-106n 2/6/2011 Submitted KS 5

MS 1

2011 11-012-107n 2/6/2011 Submitted IA 5

11-014-103n 2/6/2011 Submitted IL 3

11-034-115n 3/5/2011 Submitted CA 5

KS 5

11-040-108n 3/11/2011 Submitted IA 2

IL 3

IN 1

11-040-109n 3/11/2011 Submitted NE 13

11-041-101n 3/18/2011 Submitted KS 14

11-041-126n 3/11/2011 Submitted MS 1

11-045-102n 3/16/2011 Submitted IL 8

11-045-104n 3/16/2011 Submitted OH 6

TX 1

11-046-101rm 5/18/2011 Submitted HI 2

11-048-102n 3/19/2011 Submitted IL 11

IN 1

11-048-110n 3/19/2011 Submitted IA 11

11-066-102n 4/6/2011 Submitted HI 1

11-067-109n 4/7/2011 Submitted IL 2

OH 5

11-068-101n 4/7/2011 Submitted KS 4

11-068-104n 4/8/2011 Submitted IL 6

11-068-106n 4/7/2011 Submitted IA 6

11-097-105n 4/20/2011 Submitted IL 1

11-123-104rm 9/1/2011 Submitted HI 2

11-154-110n 7/3/2011 Submitted HI 1

11-154-111n 7/3/2011 Submitted HI 1

11-291-108rm 2/15/2012 Submitted IA 3

IL 2

IN 1

2012 KS 21

11-305-104rm 3/1/2012 Submitted HI 2

11-320-102rm 3/15/2012 Submitted IA 9

IL 4
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IN 1

KS 3

NE 11

SD 3

11-322-101n 12/18/2011 Submitted HI 2

11-322-102n 12/18/2011 Submitted HI 2

11-326-105rm 3/22/2012 Submitted IA 12

IL 23

11-342-104rm 12/23/2011 Submitted HI 1

IL 1

12-032-117rm 6/1/2012 Submitted HI 1

PR 1

12-058-101n 3/28/2012 Submitted MN 6

WI 7

12-059-120n 3/28/2012 Submitted IL 1

NE 1

2012 12-061-105n 3/31/2012 Submitted IA 1

IL 1

NC 1

NE 1

PA 1

12-062-111n 4/1/2012 Submitted AR 1

IA 1

IL 3

IN 1

KS 1

MS 1

OH 1

PA 1

12-065-109n 4/4/2012 Submitted IL 3

MO 1

NE 3

12-072-104n 4/11/2012 Submitted TX 2

12-074-110n 4/13/2012 Submitted IA 1

12-125-106rm 9/1/2012 Submitted HI 1

PR 2

12-143-104n 6/20/2012 Submitted HI 4

12-214-105rm 12/1/2012 Submitted HI 1

12-251-101n 10/7/2012 Submitted AR 1

IL 1

KS 1

NE 1

12-143-104n 6/20/2012 Submitted HI 2

12-214-104rm 12/1/2012 Submitted HI 1

12-312-103n 12/7/2012 Submitted HI 2

12-312-106rm 3/1/2013 Submitted HI 1

12-312-109rm 3/7/2013 Submitted PR 1

2013 12-320-109rm 3/15/2013 Submitted IA 19

SD 4

TN 1

12-320-114rm 3/15/2013 In Progress IL 28

IN 1

KS 8
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MS 1

12-320-125rm 3/15/2013 In Progress

KS 7

NE 7

13-037-101rm 6/1/2013 In Progress HI 2

13-037-102rm 6/1/2013 In Progress HI 2

13-037-104rm 6/1/2013 In Progress PR 1

13-037-105rm 6/1/2013 In Progress HI 1

13-037-106rm 6/1/2013 In Progress HI 2

PR 1

13-039-102n 3/8/2013 Submitted HI 3

13-044-101rm 3/7/2013 Submitted HI 1

13-052-105n 3/23/2013 Submitted IA 2

MN 9

SD 1

2013 13-059-103n 3/30/2013 Submitted IL 1

13-066-105n 4/6/2013 Submitted AR 1

IA 1

IL 1

LA 1

13-119-103n 5/29/2013 In Progress HI 2

PR 1

13-120-102rm 9/1/2013 In Progress HI 1

PR 1

13-120-103rm 9/1/2013 In Progress HI 1

PR 1

13-120-104rm 9/1/2013 In Progress HI 1

13-120-105rm 9/1/2013 In Progress HI 1

13-120-106rm 9/1/2013 In Progress HI 1

13-120-107rm 9/1/2013 In Progress HI 1

13-220-103n 9/7/2013 Submitted PR 1

13-301-101n 12/4/2013 In Progress HI 1

13-213-101rm 12/1/2013 In Progress HI 1

13-213-102rm 12/1/2013 In Progress HI 1

13-213-104rm 12/1/2013 In Progress HI 1

13-213-105rm 12/1/2013 In Progress HI 1

13-213-106rm 12/1/2013 In Progress HI 1

13-297-103rm 3/1/2014 In Progress HI 1

13-297-106rm 3/1/2014 In Progress HI 1

13-297-108rm 3/1/2014 In Progress HI 2

13-297-109rm 3/1/2014 In Progress HI 1

13-301-101n 12/4/2013 In Progress HI 3

2014 13-305-101rm 3/1/2014 In Progress IL 1

13-305-102rm 3/1/2014 In Progress IL 23

IN 1

13-305-103rm 3/1/2014 In Progress IL 2

13-305-105rm 3/1/2014 In Progress IL 17

IN 1

13-305-106rm 3/1/2014 In Progress KS 10

NE 8

13-305-108rm 3/1/2014 In Progress KS 8

NE 6

13-305-109rm 3/1/2014 In Progress KS 8

NE 6
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13-305-113rm 3/1/2014 In Progress IA 24

MS 1

TN 2

13-305-114rm 3/1/2014 In Progress IA 15

13-305-115rm 3/1/2014 In Progress IL 1

NE 1

2014 13-305-116rm 3/1/2014 In Progress IA 17

14-031-117rm 6/1/2014 In Progress HI 1

14-038-103n 3/9/2014 In Progress IL 1

MI 1

MN 11

NE 1

14-065-101n 4/5/2014 In Progress IL 1

NE 1

14-065-102n 4/5/2014 In Progress IL 1

NE 1

Grand

Total 849
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Appendix B: Mode of Action of ATHB17∆113 Protein in MON 87403

Grain yield results from a sequential growth and development process - first the plant

grows in the vegetative phase and produces photosynthetic tissue, followed by flowering,

the production of seeds, and finally seed filling and maturation (Figure B-2).

MON 87403 was produced through insertion of the coding region of the full-length

ATHB17 gene through Agrobacterium-mediated transformation into maize. ATHB17 is a

member of the HD-Zip family of proteins that are plant transcription factors; i.e. proteins

that bind to specific DNA sequences and are essential for regulation of gene expression.

This family of proteins is found broadly across plant species (Ariel et al, 2007) and is

thought to play an important role in the regulation of plant growth and development.

Expression of ATHB17 in MON 87403 results in increased biomass of the ear at the early

reproductive phase which increases yield opportunity in maize. The purpose of this

appendix is to describe further details on the mechanism of action of ATHB17 in

MON 87403.

Maize (Zea mays subsp. mays) is one of the world’s most important cereal crops with a

global production forecast of ~960 million MT in 2013-14. The United States is the

leading maize producing country in the world (USDA-FAS, 2014). In 2013 maize was

planted on 35.48 million ha in the United States (USDA-FAS, 2014). Improvements in

crop yield have been a primary focus of conventional breeding. Maize breeders have

been extremely successful at improving grain yields in the last 70 years (Figure B-2).

Commercial maize yield in the United States increased nearly six-fold in the last 70 years

of genetic improvement, with an approximately 99 kg ha-1 increase every year from 1939

(Lee and Tollenaar, 2007). This continuous yield increase was due to many factors

including genetic improvement. Yield has increased despite the fact that the ear size per

plant has declined (Bruns and Abbas, 2003). Since MON 87403 results in increased ear

biomass, it has the potential to address some of the challenges faced by maize breeders in

their efforts to continue to improve yield and the efficiency of maize production.
Monsanto Company CR262-14U1 154 of 260

Figure B-1. Maize developmental stages. Vegetative stages are identified based on the

number of visible leaf collars. Note the ear with silks at the R1 stage. Reproductive

stages are mainly characterized by kernel development as shown in the insert. By the R6

stage, kernels attain physiological maturity. (Nafziger 2012)

Figure B-2. US maize yields from 1866 to 2013 in bushels per acre (compiled from

USDA-NASS, 2014).

B.1. Maize growth stages, source and sink tissues:

In order to understand the mode of action of ATHB17 in maize, a review of maize

developmental stages and the impact of partitioning on reproductive tissue development
Monsanto Company CR262-14U1 155 of 260

is needed. Maize vegetative growth begins with germination and seedling emergence

(VE, Figure B-1) and ends when ‘tassel’ or male inflorescence has completely emerged

from the plant (VT, Figure B-1). Pollen from anthers on the tassel is shed normally after

the VT stage. Ear tissue is initiated at a node by approximately the V5 growth stage and

the ear grows rapidly as the plant approaches the flowering stage. The reproductive

growth stage in maize starts with R1 when silks (receptors of pollen) are visible on the

ear (female inflorescence), and ends at R6 or physiological maturity (Ritchie et al., 1997).

At the R1 stage- ovules on the ear are completely developed, and after pollination they

will mature during reproductive growth stages as grain for harvest (Figure B-1).

Vegetative tissue (mostly leaves) is the primary source tissue for production of

reproductive tissue biomass. The ear is considered the major sink tissue during

reproductive stages in maize (Ritchie et al., 1997). Sink tissues lack the ability to

produce biomass but accumulate the biomass that is produced by the source tissue (Ho,

1988).

B.2. Partitioning of assimilates to sink tissue in maize:

The transport and distribution of photosynthetic assimilates produced by the source tissue

to sink tissue is referred to as ‘partitioning’ (Gifford and Evans, 1981). As the maize ear

grows, it generates the driving force needed to transport assimilates produced by the

source tissue to the sink tissue (Tang and Boyer, 2013). Increased ear partitioning

involves preferential accumulation of biomass in the ear tissue relative to the other

growing vegetative organs.

Grain yield in maize is a function of total assimilates (biomass) produced by the source

tissue and the fraction of produced assimilates partitioned to the sink or ear tissue (Lee

and Tollenaar, 2007). Thus, increased partitioning to the ear can result in higher yield

regardless of an associated increase in total assimilates produced by the plant. The

increased assimilate partitioning to the ear during the R1 stage has been one of the factors

that has contributed to yield increase in North American maize hybrids (Echarte et al.,

2004).

B.3. Sink size during early reproductive stage can affect grain yield in maize:

The size of the sink (ear) during the early reproductive stages can have a profound effect

on grain yield in maize (Lizaso et al., 2007). In maize, the maximum sink size is

determined during early reproductive stages, and is influenced both by the plant’s

genetics and by prevailing environmental conditions (Jones et al., 1996; Borras and

Westgate, 2006). An increase in ear biomass at early reproductive stages can result in

larger kernel number at harvest (Fisher and Palmer, 1983; Severini et al, 2011) (Figure

B-3) and is considered an important determinant of reproductive success (Zaidi et al,

2003). Thus, the early reproductive stages are crucial phases in maize development, and

a greater ear biomass during the early reproductive stages provides increased yield

opportunity in maize.
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Figure B-3. Relationship between ear biomass during early reproductive stage and

kernel number at harvest

et al., 2011).

B.4. Grain yield improvemen

achieved by selection and

The genetic changes that

breeding were typically achieved through the se

encoding transcriptional regulator proteins (Doebley et al., 2006). For example, selection

for maize plants with single stalk and short branches tipped by ears during the

domestication of maize from teosinte indirectly

of the tb1 gene, which encodes a transcription factor (Doebley

examples of indirect selection for specific variants during conventional breeding can be

found in crops like rice, wheat, tomato

expression of a transcription factor using agricultural biotechnology is an extension

conventional breeding and

the introduction of new genetic elements that use or modify existing pathways in the

plant.

B.5. ATHB17 is a member of the HD

HD-Zip proteins are a family of plant

sequences and regulate gene expression. This family of proteins

plant species and is thought to play an important role in

and development (Ariel et al,

subfamilies, or classes designated as I, II, III, and IV (Ariel et al, 2007

CR262-14U1

. Relationship between ear biomass during early reproductive stage and

kernel number at harvest (graph made from the data on dent corn in Table 1,

mprovement through domestication and breeding has been

election and safe use of transcriptional regulator proteins:

The genetic changes that have resulted in yield improvement through conventional

breeding were typically achieved through the selection and safe use of plant genes

encoding transcriptional regulator proteins (Doebley et al., 2006). For example, selection

for maize plants with single stalk and short branches tipped by ears during the

domestication of maize from teosinte indirectly resulted in selection of specific variants

gene, which encodes a transcription factor (Doebley et al.,

examples of indirect selection for specific variants during conventional breeding can be

found in crops like rice, wheat, tomato and cauliflower (Doebley et al.,

expression of a transcription factor using agricultural biotechnology is an extension

and provides an opportunity to further enhance crop yields through

the introduction of new genetic elements that use or modify existing pathways in the

ATHB17 is a member of the HD-Zip family of transcription factors

Zip proteins are a family of plant transcription factors which bind to specific DNA

sequences and regulate gene expression. This family of proteins is found broadly across

and is thought to play an important role in the regulation of plant growth

and development (Ariel et al, 2007). The HD-Zip family comprises four distinct

bfamilies, or classes designated as I, II, III, and IV (Ariel et al, 2007

156 of 260

. Relationship between ear biomass during early reproductive stage and

(graph made from the data on dent corn in Table 1, Severini

reeding has been largely

roteins:

resulted in yield improvement through conventional

lection and safe use of plant genes

encoding transcriptional regulator proteins (Doebley et al., 2006). For example, selection

for maize plants with single stalk and short branches tipped by ears during the

resulted in selection of specific variants

et al., 2006). Other

examples of indirect selection for specific variants during conventional breeding can be

et al., 2006). Thus

expression of a transcription factor using agricultural biotechnology is an extension of

an opportunity to further enhance crop yields through

the introduction of new genetic elements that use or modify existing pathways in the

actors:

which bind to specific DNA

is found broadly across

the regulation of plant growth

Zip family comprises four distinct

bfamilies, or classes designated as I, II, III, and IV (Ariel et al, 2007). All HD-Zip
Monsanto Company CR262-14U1 157 of 260

proteins are characterized by the presence of a homeodomain (HD) with an immediately

adjacent leucine zipper (LZ) domain. HD-Zip proteins have been shown to dimerize with

members of their subfamily (as homo- or heterodimers), but not with other subfamilies of

HD-Zip proteins to bind to target DNA sequences (Sessa et al., 1993). Many HD-Zip

proteins have been shown to function as repressors of gene expression (Sessa et al., 1993;

Steindler et al., 1999; Ohgishi et al., 2001; Henriksson et al. 2005), including down

regulating the transcription of genes within the HD-Zip family (Ohgishi et al., 2001;

Sorin et al., 2009). Transcriptional repressors are proteins that bind to DNA sites to

prevent RNA polymerase from initiating transcription (Lewin, 2000).

ATHB17 is a member of the class II subfamily. The protein consists of 275 amino acids

and contains five domains; a homeodomain (HD), a leucine zipper (LZ), an N-terminal

domain, a repression domain and a C-terminal domain. It has been shown that HD-Zip II

proteins in Arabidopsis recognize a 9 bp DNA sequence CAAT(G/C)ATTG (Sessa et al

1993) which is distinct from the consensus sequence for other HD-Zip proteins. In maize

18 HD-Zip II genes have been identified through a systematic bioinformatic analysis

(Zhao et al., 2011). Characterization of ATHB17 and of the maize HD-Zip II proteins

tested showed that they are all able to function as transcriptional repressors (Rice et al.,

2014).

Based on the reported literature, HD-Zip II proteins have diverse functions throughout

plant growth and development. For example, overexpression of several HD-Zip II genes

has been shown to increase shade avoidance (or avoiding competition for light from

neighboring plants) responses (ex: elongation to intercept light) in Arabidopsis (Ciarbelli

et al., 2008; Sawa et al., 2002; Sorin et al., 2009; Steindler et al., 1999). In addition,

multiple Arabidopsis lines over-expressing ATHB17 suggested a role in regulating

photosynthetic capacity (Hymus et al., 2013). Interestingly, some HD-Zip II proteins are

involved in regulation of reproductive growth and development (Meijer et al 1997).

In the following sections, the mode-of-action of ATHB17 expression in MON 87403 is

described through an assessment of the molecular and phenotypic characteristics of

MON 87403.

B.6. MON 87403 expresses a truncated ATHB17 protein:

MON 87403 was produced through insertion of the coding region of the full-length

ATHB17 gene into maize through Agrobacterium-mediated transformation. Western blot

analysis of leaf extracts revealed that MON 87403 and other ATHB17-transformed maize

line express a truncated ATHB17 protein of ~20 kDa, in comparison to the predicted size

~32 kDa (Rice et al., 2014). Sequence analysis of the ATHB17 transcript produced in

MON 87403 confirms that a truncated transcript is produced, and as a result of splicing is

predicted to encode a version of the ATHB17 protein that lacks the first 113 amino acids

(ATHB17∆113) corresponding to an expected molecular weight of ~22kDa. The

domains that comprise ATHB17 are shown in Figure B-4, which also depicts the

truncation of the repression domain in the ATHB17∆113 protein produced in

MON 87403.
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Figure B-4: Domain structure of ATHB17. ATHB17 contains the characteristic

Homeodomain (HD) and Leucine Zipper (LZ) domains of HD-Zip family members. HD

is required for DNA binding. LZ domain is responsible for homodimerization and

hetero-dimerization with other HD-Zip II proteins. A repression domain is present

upstream of the HD. ATHB17 protein expressed in MON 87403 lacks the first 113

amino acids resulting in truncation of the repression domain.

B.7. ATHB17∆113 retains the functional binding properties of ATHB17:

The subcellular localization of ATHB17∆113 was evaluated in maize protoplasts. The

results indicated that ATHB17∆113 is transported into the nucleus, consistent with

activity as a transcription factor (Rice et al., 2014). In vitro DNA binding studies showed

that like other HD-Zip II proteins, ATHB17∆113 binds efficiently to consensus DNA

targets for class I and class II HD-Zips, with slightly higher affinity for the class II

binding sites (Rice et al., 2014). Studies conducted to identify maize proteins that

interact with ATHB17 identified several HD-Zip II proteins but did not identify HD-Zip

proteins from other classes. (Rice et al., 2014). The results show that ATHB17∆113

retains DNA binding and protein-protein interaction (ability to form homo- and heterodimers) properties that are characteristic of the full-length protein.

B.8. ATHB17∆113 relieves repression of HD-Zip II proteins:

Since ATHB17Δ113 lacks a large portion of the repression domain of ATHB17, it would

not be expected to act as a transcriptional repressor. To determine whether

ATHB17Δ113 protein can act as a transcriptional repressor in maize, an in-vitro assay

system was established that allows for detection of repression of reporter gene expression

in maize protoplasts. The results showed that the repression activity observed when fulllength ATHB17 was added was not observed when ATHB17Δ113 was added. Based on

these results, it was concluded that ATHB17Δ113 lacks the ability to act as a

transcriptional repressor (Rice et al., 2014).

Although ATHB17Δ113 does not function as a repressor, the protein retains dimerization

and DNA binding properties (Rice et al., 2014). Therefore, the likely action of

ATHB17Δ113 expressed in maize is to attenuate the activity of endogenous HD-Zip II

proteins through a dominant-negative mechanism. The dominant-negative mechanism

can occur either through formation of non-functional homodimers that compete for DNA

binding sites, or formation of less active heterodimers through competition for DNA

binding (see Figure B-5).

To evaluate the ability of ATHB17∆113 to act as a dominant negative regulator, the

ability of ATHB17∆113 to relieve repression activity of the full-length ATHB17 protein
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was examined using the maize protoplast system described above

reporter gene expression caused by the full

relieved as increasing amounts of

suggests that ATHB17Δ113

protein on gene repression.

Because ATHB17∆113 can form heterodimers with maize HD

factors and bind to the same DNA sequence, ATHB17

dominant negative regulator of endogenous maize HD

Experiments were conducted in

al., 2014). Dose-dependent relief of the HD

amounts of ATHB17113 was observed for all tested HD

suggest that ATHB17∆113 protein

proteins, that repress transcription

sites. Thus, it is likely that ATHB17

pathways associated with maize HD

Figure B-5: Model for dominant negative mechanism of ATHB17

ATHB17 is expressed as

domain. Maize endogenous HD

ATHB17113 can interact with endogenous HD

ATHB17113 and endogenous HD

for DNA binding resulting in altered target expression due

repression. In this view, only one repression domain

only one of the two repression domains

CR262-14U1

was examined using the maize protoplast system described above. The repression of the

ter gene expression caused by the full-length ATHB17 protein was gradually

relieved as increasing amounts of ATHB17Δ113 were added (Rice et al., 2014).

ATHB17Δ113 has the ability to counteract the effect of the full

ne repression.

∆113 can form heterodimers with maize HD-Zip II transcriptional

factors and bind to the same DNA sequence, ATHB17∆113 could be expected to act as a

dominant negative regulator of endogenous maize HD-Zip II transcription facto

Experiments were conducted in maize leaf protoplasts to evaluate this possibility (Rice et

dependent relief of the HD-Zip II repression activity by increasing

113 was observed for all tested HD-Zip II proteins

∆113 protein can act as a regulator of endogenous maize HD

that repress transcription of genes with promoters containing class

Thus, it is likely that ATHB17∆113 has the potential to affect activities and

pathways associated with maize HD-Zip II proteins.

: Model for dominant negative mechanism of ATHB17∆113

ATHB17 is expressed as a truncated protein in maize lacking part of the repression

Maize endogenous HD-Zip IIs function as transcriptional repressors.

113 can interact with endogenous HD-Zip IIs to form

113 and endogenous HD-Zip IIs, or ATHB17113 homodimers can compete

for DNA binding resulting in altered target expression due to inability to cause active

In this view, only one repression domain is on each face of the DNA so that

only one of the two repression domains is visible (Rice et al., 2014).

159 of 260

The repression of the

length ATHB17 protein was gradually

added (Rice et al., 2014). This

has the ability to counteract the effect of the full-length

Zip II transcriptional

∆113 could be expected to act as a

Zip II transcription factors.

leaf protoplasts to evaluate this possibility (Rice et

II repression activity by increasing

II proteins. These results

can act as a regulator of endogenous maize HD-Zip II

promoters containing class II binding

fect activities and

∆113

truncated protein in maize lacking part of the repression

IIs function as transcriptional repressors.

to form heterodimers of

113 homodimers can compete

to inability to cause active

is on each face of the DNA so that
Monsanto Company CR262-14U1 160 of 260

B.9. ATHB17∆113 likely modulates HD-Zip IIs through a dominant-negative

mechanism to impact reproductive growth:

Based on the molecular mechanism proposed above, it is likely that expression of

ATHB17∆113 impacts maize ear growth through interaction with functions of

endogenous maize HD-Zip II proteins. HD-Zip II proteins are involved in many

processes related to reproductive development (Zuniga-Mayo et al., 2012). For example,

a knock-out of a HD-Zip II gene in Arabidopsis showed no altered phenotypes during the

vegetative growth phase but showed altered reproductive and fruit development

compared to the wild type (Zuniga-Mayo et al., 2012).

To investigate whether the HD-Zip II proteins might be involved in the regulation of ear

growth in maize, the expression patterns of HD-Zip II genes were evaluated in two

different maize hybrids (Rice et al., 2014). The results showed that HD-Zip II genes

were expressed in all sampled maize tissue types, as well as across developmental stages.

There were eight HD-Zip II genes that were predominantly expressed in the ear tissue,

suggesting that they might be actively involved in the regulation of plant reproductive or

ear growth (Rice et al., 2014).

Our analysis indicates that the likely action of ATHB17Δ113 expressed in maize is to

attenuate the activity of endogenous HD-Zip II proteins through a dominant-negative

mechanism. As several HD-Zip II genes were predominantly expressed in the ear tissue,

we believe that ATHB17∆113 protein in maize likely modulates HD-Zip II regulated

pathways in the ear, leading to changes in ear growth.

B.10. MON 87403 maize has increased ear biomass during the early reproductive

stages:

Based on the role of specific HD-Zip II proteins in growth and development (Ciarbelli et

al., 2008; Zuniga-Mayo et al., 2012; Bou-Torrent et al., 2012), expression patterns of

HD-Zip II genes in maize during the V16-R1 stages, and likely action of ATHB17Δ113

through a dominant-negative mechanism, a hypothesis was developed that the

MON 87403 event in maize results in increased ear biomass at the R1 stage compared to

a conventional maize hybrid. The ear biomass at the R1 growth stage was measured in

MON 87403 and conventional control plants which were grown at 13 field locations

within the maize production regions of the United States in 2012. Characteristics

measured included stover or vegetative biomass and ear biomass at the R1 growth stage

(~60-70 days after planting). Stover and ear biomass per 1 meter length of row were

determined by cutting plants at the ground level and measuring the dry weight of the

vegetative material and top-most ear, respectively. Total biomass per 1 meter length of

row was calculated as the sum of stover and ear biomass. From these measurements,

biomass partitioning to the ear was calculated as the ratio of ear biomass to total biomass

(Marcelis, 1996). Statistical comparisons were made between MON 87403 and the

control across all 13 sites (combined-site analyses) for the above characteristics.

Statistical analysis indicated no significant differences in the R1 stover biomass and the

R1 total biomass between MON 87403 and the control. There was a statistically
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significant increase in R1 ear biomass (p < 0.05) in MON 87403 compared to the control

in the combined site analysis (Table B-1). The R1 ear biomass in MON 87403 was

approximately 12% higher than the control. Moreover, partitioning to the ear was

significantly increased in MON 87403 compared to the control. These findings support

that ATHB17∆113 protein in maize regulates ear growth.

Table B-1. Combined Site Analysis of R1 Ear Weight, R1 Stover Weight, Total

Plant Weight, and Partitioning in MON 87403 and the Conventional Control from

2012 U.S. Field Trials

Characteristic (units) MON 87403

(Mean ± SE)

Control

(Mean ± SE)

Change (%) p-value

R1 ear biomass (g) 144.50 (±8.47) 129.30 (±8.13) 11.7 0.004*

R1 stover biomass (g) 768.13 (±20.57) 772.32 (±20.45) -0.5 0.791

R1 total plant biomass (g) 912.63 (±25.47) 901.62 (±26.02) 1.2 0.563

Partitioning to ear 0.16 (±0.0064) 0.14 (±0.0058) 10.0 0.001*

Locations included in the combined-site analysis: Jackson, Arkansas; Vermilion, Illinois; Warren, Illinois;

Boone, Indiana; Greene, Iowa; Jefferson, Iowa; Pawnee, Kansas; (2 sites) Polk, Nebraska; York, Nebraska;

Perquimans, North Carolina; Berks, Pennsylvania; Lehigh, Pennsylvania. (Partitioning to ear = R1 ear

biomass / R1 total plant biomass). All measurements are dry weight per 1 meter length of row.

*Denotes a statistically significant difference between MON 87403 and the control (α = 0.05)

In MON 87403, the expression of the ATHB17113 transcription factor results in an

increase of ear biomass and partitioning to ear at the R1 growth stage. Increased ear

biomass and improvement in partitioning to the ear during the R1 stage in maize are traits

that have been improved through breeding (Echarte et al., 2004). Thus, increasing ear

biomass through introduction of the ATHB17113 transcription factor in maize is similar

to genetic improvements that have been achieved through conventional breeding. The

increased ear biomass at the R1 growth stage can provide the opportunity for a yield

advantage (Fisher and Palmer, 1983; Zaidi et al., 2003; Severini et al, 2011).

Ear biomass advantage in MON 87403 occurs during early ear development and

accumulates through the silking stage

A greenhouse study was conducted to confirm field results and to assess the growth stage

when an ear biomass advantage is seen in MON 87403 compared to the control. Plants

were grown in pots under optimal growing conditions in a greenhouse. Plants were

sampled at 4 stages; V16 (late vegetative), VT (tasseling), early R1, and late R1 (silking).

The ear biomass of MON 87403 was approximately 18% greater (29.4 g vs. 25.0 g) than

that of the control at the late R1 stage (Figure B-6). Statistical analysis indicated that ear

biomass was significantly greater in MON 87403 than the control at early R1 and late R1

stages. Throughout the time course during which biomass was measured (V16 – late R1),

ears of MON 87403 plants accumulated approximately 24 % more biomass (26.8 g vs.

21.6 g), on average, than the ears of control plants. In addition to supporting the ear

biomass advantage in MON 87403 compared to the control that was observed in the field
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trial, these results also suggest that the increased early ear biomass of MON 87403

accumulates gradually over the period from V16 to late R1.

Figure B-6. Ear growth and ear partitioning in MON 87403 and the control at

different developmental stages in a greenhouse. Symbol ‘*’ indicates statistical

significance at a level of 5% (α = 0.05). (A) Ear biomass (dry weight) (B) Ear

partitioning (ear dry weight/total dry weight). “DAS” is days after sowing.

Similar to ear biomass advantage, partitioning to ear was significantly higher in

MON 87403 compared to the control at the early and the late R1 growth stages (6).

These data support the ear biomass advantage in MON 87403 compared to the control

was due to increased partitioning to the ear, similar to the findings observed in the field

trial.

Conclusion:

Based on the studies described above, the ATHB17Δ113 protein can bind to target DNA

sequences but lacks transcriptional repression activity. By a dominant-negative

mechanism, ATHB17Δ113 protein in MON 87403 maize can alter the activity of

endogenous HD-Zip IIs. HD-Zip IIs have been shown to play a role in reproductive

growth in Arabidopsis (Ciarbelli et al., 2008; Zuniga-Mayo et al., 2012; Bou-Torrent et

al., 2012). In maize, several HD-Zip IIs are predominantly expressed in ear tissue. The

ATHB17∆113 protein likely modulates HD-Zip II-regulated pathways in the ear, leading

to changes in ear growth. The result is an increase in ear partitioning and ear biomass at

the R1 growth stage in MON 87403 maize. A larger ear at the R1 growth stage increases

V16 (49 DAS)

VT (51 DAS)

Early R1 (56 DAS)

Late R1 (61 DAS)

E

ar Bio

m

ass (g/pla

nt)

0

5

10

15

20

25

30

35

MON 87403

Control

V16 (49 DAS)

VT (51 DAS)

Early R1 (56 DAS)

Late R1 (61 DAS)

E

ar P

artitio

nin

g

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

MON 87403

Control

*

*

*

*

A B
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yield opportunity in MON

MON 87403 is summarized

Figure B-7. Summary of

maize

CR262-14U1

yield opportunity in MON 87403 maize (Leibman et al., 2014). The mode of action of

zed in Figure B-7.

. Summary of mode-of-action of ATHB17Δ113 protein in MON

163 of 260

(Leibman et al., 2014). The mode of action of

action of ATHB17Δ113 protein in MON 87403
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Appendix C: Overview, Materials, Methods, and Supplementary Results for

Molecular Analyses of MON 87403

C.1. NGS/JSA Overview

Safety assessments of biotechnology-derived crops include a detailed molecular

characterization of the inserted DNA sequence and its location within the genome (Codex

Alimentarius 2009). Typically, molecular characterization has relied on Southern blot

analysis to establish locus and copy number along with targeted sequencing of

polymerase chain reaction products spanning any inserted DNA to complete the

characterization process. With the advent of next-generation sequencing (Shendure and

Ji 2008; Zhang et al. 2011), improvements in sequencing technologies have enabled

alternative methods for molecular characterizations which do not require Southern blot

analysis. Next-Generation Sequencing and Junction Sequence Analysis bioinformatics

(NGS/JSA) utilizes sequencing (both next-generation technologies and traditional

methods) and bioinformatics to produce characterizations equivalent to those achieved by

current Southern blot-based methods.

There are multiple advantages to using next-generation sequencing and bioinformatics,

most notably the robustness, simplicity and consistency of the method compared with

Southern blot studies, which require customized experimental design for every

transformation event. The new sequencing-based method overcomes many technical

challenges inherent in Southern blot analyses (e.g., false positive hybridization bands

resulting from incomplete digestion or star activity (Wei et al. 2008)) and the need for

radioactive 32P-labeled probes. This new method provides higher reproducibility,

because it is less dependent on complex lab based procedures. The method described

here is essentially identical for all transformation events and it robustly establishes

molecular characteristics of genetically engineered crops (Kovalic et al. 2012).

Additionally, similar techniques are being used to characterize transgene integration sites

and insert molecular anatomy in mammalian systems (DuBose et al. 2013; Zhang et al.

2012).

Method Synopsis

Molecular characterization of the inserted DNA and associated native flanking sequences

consists of a multistep approach to determine:

 the number of insertion sites;

 the presence/absence plasmid backbone;

 insert copy number at each insertion site;

 DNA sequence of each inserted DNA;

 sequence of the native locus at each insertion site.
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Additionally, current methods also establish a description of any genetic rearrangements

that may have occurred at the insertion site as a consequence of transformation.

Generational stability analysis, which demonstrates the stable heritability of inserted

DNA sequences over a number of breeding generations, is also routinely conducted.

The first step of the molecular characterization, determination of number of insert sites, is

conducted using a combination of next-generation sequencing technologies (NGS) and

Junction Sequence Analysis (JSA) bioinformatics (DuBose et al. 2013; Kovalic et al.

2012). A schematic representation of the basis of the characterization, including the

NGS/JSA methodology and the directed sequencing, is presented in Figure C-1 (Kovalic

et al. 2012).

Genomic DNA from the transformation event and the conventional control are used to

generate short (~100 bp) randomly distributed sequence fragments (sequencing reads) in

sufficient numbers to ensure comprehensive coverage of the genomes (Shendure and Ji

2008) (Figure C-1, box 1). Sufficient numbers of sequence fragments are obtained

(≥75× genome coverage) to comprehensively cover the genomes of the sequenced

samples (Ajay et al. 2011; Clarke and Carbon 1976; Wang et al. 2008). A previous study

with a variety of transformation events demonstrate that 75× coverage of the genome is

adequate to provide comprehensive coverage and ensure detection of inserted DNA,

producing results equivalent to Southern blot analysis (Kovalic et al. 2012).

Notwithstanding known biases in next-generation sequencing techniques, including the

Illumina sequencing by synthesis method employed here (Minoche et al. 2011), it has

previously been established experimentally that given deep next-generation sequencing, it

is possible to achieve comprehensive coverage of complex genomes that form the

foundation for accurate whole genome studies (Ajay et al. 2011; Wang et al. 2008).

To confirm sufficient sequence coverage in both the transformation event and the control,

the 100 bp sequence reads are analyzed to determine the coverage of a known singlecopy endogenous gene, this analysis demonstrates coverage at ≥75× median depth in

each sample. Furthermore, in order to confirm the method’s ability to detect any

sequences derived from the transformation plasmid, plasmid DNA is spiked into

conventional control DNA at a single copy genome equivalent ratio and 1/10 copy

genome equivalent ratio. This analysis demonstrates that any portion of the plasmid may

be detected at a single copy per genome level and 1/10 copy genome equivalent level,

which is adequate sensitivity to observe any inserted fragment.

Also of note is that although the method presented here provides 75× or greater coverage

of the genomes under study, accurate assembly of complete genome sequences for the

transformation event and conventional control is not technically possible using currently

available sequence assembly tools. This is due to the nature of the sequences generated in

this study, short reads of a single short insert length (Miller et al. 2010), in addition to

limitation on available sequence assembly algorithms (Zhang et al. 2011). The sequences

generated with this method represent datasets sufficient for achieving precise molecular

characterization of transformed DNA in transformation events where reference to a

template sequence (plasmid DNA) is utilized for comparison (Kovalic et al. 2012).
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Figure C-1. Sequencing and Sequence Selection

Genomic DNA from the test and control material were sequenced using Illumina HiSeq/TruSeq

technology (Illumina, Inc.) that produces large numbers of short sequence reads approximately

100 bp in length. Sufficient numbers of these sequence fragments were obtained to

comprehensively cover the genomes of each sample at ≥75× median coverage. Using these

genome sequence reads, bioinformatics search tools were used to select all sequence reads that

are significantly similar (as defined in the text) to the transformation plasmid. Only the selected

sequence reads were used in further bioinformatics analysis to determine the insert number by

detecting and characterizing all junction sequences and the presence or absence of the plasmid

backbone sequences by lack of detectable sequences, including the use of suitable controls for

experimental comprehensiveness and sensitivity.

Using bioinformatics tools, the sequence reads that are derived from the plasmid vector

are selected for further analysis out of the comprehensive genomic sequence dataset

produced from the transformation event. To determine the insert number, the known

sequence of the transformation vector plasmid is used as a query sequence in the

bioinformatics analysis to search for and select the sequences that contain any portion of

sequence of the plasmid. The DNA sequencing reads with a match to the query sequence

having an e-value of 1 × 10−5or less and having a match length of at least 30 bases with

at least 96.7% sequence identity are collected. The results of a parameter optimization

study that systematically evaluated many different potential parameter sets established

these selection criteria as providing the best possible combination of sensitivity and

specificity.

The number of DNA inserts is determined by analyzing the selected sequences for novel

junctions. The junctions of the DNA insert and flanking DNA are unique for each
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insertion and an example is shown in Figure C-2 below (Kovalic et al. 2012). Therefore,

insertion sites can be recognized by analyzing for sequence reads containing such

junctions. Each insertion will produce two unique junction sequence classes

characteristic of the genomic locus, with one at the 5' end of the insert, in this case named

Junction Sequence Class A (JSC-A), and similarly one at the 3' end of the insert, JSC-B

(as illustrated in Figure 3 from Kovalic et al., 2012). By evaluating the number and the

sequences of all unique junction classes detected, the T-DNA copy number and the

number of insertion sites of the plasmid sequence can be determined. For a single insert,

two junction sequence classes are expected, one each originating from either end of the

insert, both containing portions of T-DNA and flanking sequence.

The identity of inserted DNA was confirmed by mapping of sequence reads. The

selected sequences, a subset of which are junction sequences, represent plasmid sequence

integrated into the genome of the transformation event. These sequences were compared

to the transformation plasmid to determine which region(s), T-DNA or backbone, of the

transformation plasmid was (were) integrated during transformation.

Figure C-2. Junctions and Junction Sequences

Depicted above are five example junction sequences formatted and labeled to indicate the

plasmid/flanking DNA portions of the sequences and with the junction point indicated (plasmid

DNA is shown in bold, underlined text and flank DNA is shown in plain text). Junctions are

detected by examining the NGS data for sequences having portions of plasmid sequences that

span less than the full read. Detected junctions are typically characteristic of plasmid insertions

in the genome. A group of junction sequences which share the same junction point and common

flanking sequence (as shown above) is called a Junction Sequence Class (or JSC).

The next step in the molecular characterization is confirmation of the integrity of the

insert and flanking sequence of the native locus at the insertion site. This analysis is

conducted using directed sequencing, locus-specific PCR and DNA sequencing analyses,

which complements the NGS/JSA analyses, and is common to both the Southern-based

and the NGS/JSA characterization methods. Directed sequencing (locus-specific PCR

and DNA sequencing analyses) of the transformation event determines the complete

sequence of the insert and flanks. This confirms that the sequence of the insert is

identical to the corresponding sequence in the plasmid vector and if each genetic element

in the insert is intact. Furthermore, the genomic organization at the insertion site is

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGT

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGG

ATCCATGTAGATTTCCCGGACATGAAGCCAAGAACTAGGAAACGACGACGTGTCGGGAT

Plasmid DNA FlankingDNA

Junction
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assessed by comparing the insert and flanking sequence to the sequence of the insertion

site in conventional control genome.

Finally, the stability of the T-DNA across multiple generations is evaluated by NGS/JSA

analyses. Genomic DNA from multiple generations of the transformation event is

assayed for the number and sequences of all unique junction classes, as well as the

identity of the inserted sequence, as described above. This information is used to

determine the number and identity of insertion sites. For a single T-DNA insert, two

junction sequence classes are expected, both containing portions of T-DNA and flanking

sequence (Figure C-2), with one each originating from either end of the insert (Figure C3). All the integrated sequences align to the T-DNA portion of the plasmid. In the case

of an event where a single locus is stably inherited over multiple generations, two

identical junction sequence classes are expected in all the generations tested and all the

integrated sequences align to the T-DNA portion of the plasmid.
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Figure C-3. Two Unique Junction Sequence Classes are Produced by the Insertion

of a Single Plasmid Region

A schematic representation of a single DNA insertion within the genome showing the inserted

DNA, the 5' and 3' flanks (depicted as areas bounded by dotted lines), and the two distinct regions

spanning the junctions between inserted DNA and flanking DNA (shaded boxes). The group of

~100-mer sequences in which each read contains sequences from both the DNA insert and the

adjacent flanking DNA at a given junction is called a Junction Sequence Class. In this example,

two distinct junction sequence classes (in this case: Class A at the 5’ end and Class B at the

3’ end) are represented.

5’ Flank DNA insert 3’ Flank

Insert Junction Regions

Junction Sequences: Class A

Junction Sequences: Class B
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C.2. Materials and Methods

C.2.1. Test Substance

The test substance in this study was MON 87403. Genomic DNA for use in this study

was extracted from tissue listed in the table below.

Generation Seed ORION6ID

R3 11346584

R4 11346578

R5 11346585

R4F1 11346581

R5F1 11346575

6 ORION is a proprietary database used at Monsanto Company to track Regulatory plant samples.
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C.2.2. Control Substance

The control substance is the conventional maize variety LH244, which has similar

genetic background as the test substances. Genomic DNA for use in this study was

extracted from tissue listed in the table below.

Control Substance Generations Seed ORION ID

LH244 R3, R4, R5 11264747

LH244 x LH295 R4F1 (hybrid) 11266870

LH244 x LH287 R5F1 (hybrid) 11333170

C.2.3. Reference Substance

The reference substance was plasmid vector PV-ZMAP5714, which was used to develop

MON 87403. Whole plasmid served as a positive control for sequencing and

bioinformatic analyses. The identity of the reference plasmid was confirmed by

restriction enzyme digestion prior to the study. Documentation of the confirmation of the

plasmid vector identity was archived with the raw data. Appropriate molecular size

markers from commercial sources were used for size estimations on agarose gels. The

unique identity of the molecular weight markers was documented in the raw data.

C.2.4. Characterization of Test, Control, and Reference Substances

The seed for the test and control substances used in this study were obtained from

Monsanto Trait Development. The synthesis records for these materials are located in the

MIDAS7 system. The identities of the test substance and the conventional control

substance were confirmed by the sequencing in the study. No certificates of analysis

(COA) or verification of identity (VOI) certificates were generated for these materials.

The Study Director reviewed the chain of custody documentation to confirm the identity

of the test and control substances prior to the use of these materials in the study.

Test, control and reference DNA substance were considered stable during storage if they

yielded interpretable signals in sequencing experiments and/or did not appear visibly

degraded on the stained gels.

7 MIDAS is a proprietary database used at Monsanto Company to track plant synthesis records.
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C.2.5. Genomic DNA Isolation

For sequencing library construction and PCR reactions, genomic DNA was isolated from

seed tissues of the test and control substances. First the seeds were decontaminated by

vigorously agitating them by hand for 30 seconds with 0.05% (v/v) Tween-20, followed

by a tap water rinse. The seeds were then vigorously agitated with 0.5% (w/v) NaOCl,

allowed to stand for one minute at room temperature, and rinsed with tap water. The

seeds were then vigorously agitated with 1% (v/v) HCl, allowed to stand for one minute

at room temperature, and rinsed with tap water. The 1% (v/v) HCl rinse was repeated

one time, and then the seeds were rinsed with distilled water and placed in a drying oven

at 80°C to dry. The dried seeds were ground to a fine powder in a Harbil paint shaker.

Genomic DNA was extracted using a hexadecyltrimethylammonium bromide (CTAB)

extraction protocol. Briefly, 16 ml CTAB buffer (1.5% (w/v) CTAB, 75 mM Tris HCl

(pH 8.0), 100 mM EDTA (pH 8.0), 1.05 M NaCl, and 0.75% (w/v) PVP) and RNase A

was added to ground seed tissue. The samples were incubated at ~65 for 60 minutes

with intermittent mixing. The samples were cooled to room temperature and subjected to

three rounds of chloroform:isoamyl alcohol (24:1) extraction. Approximately 1.6 ml of

10% CTAB solution (10% (w/v) CTAB and 0.7 M NaCl) was added to the samples,

mixed by inversion and extracted once with chloroform:isoamyl alcohol (24:1).

Approximately 15 ml of CTAB precipitation buffer (1% (w/v) CTAB, 50 mM Tris HCl

(pH 8.0), 10 mM EDTA (pH 8.0)) was added to the samples, mixed by inversion, and

allowed to stand at room temperature for 50-70 minutes. Following centrifugation to

pellet the DNA, the samples were dissolved in high salt TE buffer (10 mM Tris HCl (pH

8.0), 1 mM EDTA (pH 8.0), 1 M NaCl). The DNA was precipitated with 3 M sodium

acetate (pH 5.2) and 100% (v/v) ethanol. The DNA pellets were washed with 70%

ethanol, air dried and resuspended in TE buffer (10 mM Tris HCl, 1 mM EDTA,

pH 8.0). All extracted DNA was stored in a 4°C refrigerator.

C.2.6. DNA Quantification

PV-ZMAP5714 DNA and extracted genomic DNA were quantified using a QubitTM

Fluorometer (Invitrogen, Carlsbad, CA). For directed sequencing, genomic DNA was

quantified using a NanodropTM Spectrophotometer (Thermo Scientific, Wilmington, DE)

according to manufacturer’s instructions.

C.2.7. Agarose Gel Electrophoresis

After quantification, approximately 0.5-1 μg of the extracted DNA for NGS/JSA

sequencing library construction was run on a 1% (w/v) agarose gel to check the quality.

C.2.8. Shearing of DNA

Approximately 1 μg of DNA from the test, control and reference substances were sheared

using a Covaris S-220 ultrasonicator. The DNA was diluted to ~ 20 ng/μl in Buffer EB

(Qiagen Inc., Valencia, CA) and fragmented using the following settings to create

approximately 325 bp fragments with 3' or 5' overhangs: duty cycle of 10; peak incident
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power of 175; intensity of 5.0, 200 bursts per cycle, in the frequency sweeping mode at

~ 6°C for 80 seconds for test and control DNA or 60 seconds for reference DNA.

C.2.9. Bioanalyzer Analysis

One microliter of sheared genomic DNA was diluted 1:10 in Buffer EB and run on a

DNA High Sensitivity chip on an Agilent 2100 Bioanalyzer to check the quality of the

shearing. After preparing the chip according to the manufacturer’s instructions, 1 μl of

each diluted DNA sample or water was added to individual wells and the chip was run on

the Bioanalyzer using the dsDNA, High Sensitivity Assay reagents.

C.2.10. Paired End Library Preparation

Paired end genomic DNA libraries were prepared for the test, control, and reference

substances using the Illumina TruSeq DNA Sample Preparation Kit (Illumina, San Diego,

CA) according to the manufacturer’s instructions for the low-throughput procedure with

the following exception: a Sage Science Pippin Prep DNA Size Selection system (Sage

Science Inc., Beverly, MA) was used to size select the DNA fragments instead of agarose

gel electrophoresis.

First, the 3' and 5' overhangs of the DNA fragments generated by the shearing process

were converted into blunt ends by adding 10 μl of Illumina Resuspension Buffer and

40 μl of Illumina End Repair mix to each sample and mixing thoroughly by pipette.

Then the libraries were incubated for 30 minutes at 30°C. The end-repaired samples

were purified using AMPure XP beads (Beckman Coulter, Inc., Brea, CA) and

resuspended in 17.5 μl of Illumina Resuspension Buffer.

Fifteen microliters of each library was transferred to a new tube for adenylation, which

adds a single adenosine nucleotide to the 3' ends of the blunt fragments. Then 2.5 μl of

Illumina Resuspension Buffer and 12.5 μl of Illumina A-Tailing Mix were added to each

library and mixed thoroughly by pipetting. The libraries were incubated for 30 minutes

at 37°C. After incubation, 2.5 μl each of individual adapter index, Illumina Resuspension

Buffer, and Illumina DNA Ligase Mix was immediately added to each tube, and mixed

thoroughly by pipetting to begin ligation of each library. The libraries were incubated for

10 minutes at 30°C. Then 5 μl of Illumina Stop Ligase Buffer was added to each tube

and mixed thoroughly by pipetting to stop the ligation reaction. Next, another

AMPure XP bead cleanup was performed on the libraries which were then resuspended

in 32.5 μl of Illumina Resuspension Buffer prior to size selection.

The libraries were run on the Sage Science Pippin Prep Size Selection system using

2% gel cassettes according to the manufacturer’s instructions. Ten microliters of loading

solution were added to 30 μl of each of the purified libraries and mixed thoroughly by

pipetting. Forty microliters of Marker B was loaded in the cassette well designated for

the reference sample, and 40 μl of each DNA library was loaded in the remaining wells

for analysis. After elution of the desired size range (~445 bp) of DNA fragments, the

DNA sample in the elution chamber of the cassette was removed from the cassette by

pipette and transferred into PCR strip tubes.
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After removal from the Pippin Prep, the libraries were again put through the AMPure XP

bead cleanup procedure and resuspended in 22.5 μl of Illumina Resuspension Buffer.

Twenty microliters of the resuspended library was added to five microliters of Illumina

PCR Master Mix and 25 μl of Illumina PCR Primer Cocktail and mixed thoroughly by

pipetting. The DNA fragments were enriched through PCR using the following cycling

conditions: 1 cycle at 98°C for 30 seconds; 10 cycles at 98°C for 10 seconds, 60°C for

30 seconds, 72°C for 30 seconds; 1 cycle at 72°C for 5 minutes. Following PCR

amplification, a final AMPure XP bead cleanup was performed on the libraries which

were resuspended in 32.5 μl of Illumina Resuspension Buffer. Finally, 1 μl of each DNA

library was diluted 1:10 in Buffer EB for running in a DNA High Sensitivity chip on an

Agilent 2100 Bioanalyzer as described above. All purified library DNA was stored in a

-20°C freezer.

C.2.11. Next-Generation Sequencing

The library samples described above were sequenced by The Genome Analysis Center

(TGAC, Monsanto) using Illumina HiSeq technology that produces short sequence reads

(~100 bp long). Sufficient numbers of these sequence fragments were obtained

(≥75x genome coverage) to comprehensively cover the entire genomes of the test event,

the conventional control and the spiked-in control (Kovalic et al. 2012). Sequencing runs

performed by the TGAC passed standard QC criteria. No sequence data in this study

failed to meet these QC acceptance criteria.

C.2.12. Junction Sequence Analysis Bioinformatics

High-throughput sequence reads were enriched by mapping to the PV-ZMAP5714

transformation plasmid sequence using the local alignment software BlastAll (V2.2.21) in

order to collect all reads that were sourced from the plasmid as well as reads with

sequences representing integration point. All collected reads were further refined by

removing sequencing artefacts of sequencing adapters, redundant reads and low quality

read ends. All quality refined reads were then used to identify junction points with

custom developed bioinformatics tools as detailed below. All significant junctions are

reported for both the test and the control samples. All software versions were

documented in the archived data package and the software versions which were used in

this study have been archived.

Sequencing Read Enrichment

The transformation plasmid PV-ZMAP5714 sequence was used as reference to find all

reads that were either fully matched to the insert plasmid fragments or partially matched

as junction sequences. The sequence used was obtained from the MEGA8system. A

junction sequence is characterized by a combination of transformation plasmid sequence

8 MEGA is a proprietary database used at Monsanto Company to track sequences and annotations.
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and flanking sequence that is likely to be host genome flanking sequence or any other coinserted sequence, or a discontinuity in plasmid sequence caused by duplication or

deletion. Local alignment with BlastAll (V2.2.21) was performed to collect all

sequencing reads with an E-score of less than 1e-5 and at least 30 bases match of greater

than 96.7% identity to the transformation plasmid (Kovalic et al. 2012). Both reads of

the paired-end sequences were collected in all cases.

Read Quality Refinement

In order to identify all duplicate read pairs, a high quality segment (bases 3-42) of all

collected pairs was compared to all others with short sequence alignment software

(Bowtie v.0.12.3) allowing up to 1 mismatch. If multiple read pairs were matched at both

paired reads, such read pairs were deemed redundant and only the best quality pair of

reads was kept for further analysis.

Computer software Novoalign (v.2.06.09) was used to remove any adapter sequences at

either end of the sequencing reads. Low quality read ends (with phred scores of 12 or

lower) were trimmed. Only reads of 30 bases or longer after adapter and quality

trimming were collected. A custom developed Perl script "farm_gen_sm_bucket.pl" was

used to perform read enrichment and read quality refinement as described above.

Junction Detection

Enriched and quality refined reads of both test and control samples were aligned against

the whole PV-ZMAP5714 transformation plasmid sequence in order to detect junction

sequences using custom developed Perl script "farm_blast_map.pl". Reads with partial

match to the transformation plasmid of at least 30 bases match and 96.7% identity were

collected as potential junction sequences and their match cutoff position on the plasmid

were noted (Kovalic et al. 2012). The collected reads were also aligned against the

genomic sequence collection of the host genome in order to remove junction reads

sourced from the plant endogenous homologues. Custom developed Perl script

“junctions_by_bn.pl” was used to map reads and to identify the junction position on the

transformation plasmid and their supporting junction reads. For each junction position,

all supporting junction reads were aligned at the 30 plasmid bases proximal to the

junction position. The remaining bases of these reads were sorted to show the alignment

and the consensus of the flanking junction sequences past the junction point.

Effective Sequencing Depth Determination

A single copy locus (Zea mays pyruvate decarboxylase (pdc3), GenBank accession

version: AF370006.2) was selected from the Zea mays genome and used to determine the

effective sequence depth coverage. All reads with at least 30 bases match and

96.7% identity were considered as reads sourced from this locus. A custom developed

Perl script “farm_match_reads.pl” was used to perform such alignment and calculate the

actual depth distribution at this locus. The analysis showed that pdc3 displayed 75× or

greater median coverage for each sample, as listed in Table C-1.
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Table C-1. Sequencing (NGS) Conducted for MON 87403 and Control Genomic

DNA

Sample

Total Nucleotides

(Gb)

Effective Median Depth of

Coverage (x-fold)

LH244 264.5 106x

LH244 x LH295 296.1 108x

LH244 x LH287 294.7 115x

R3 246.4 113x

R4 280.7 110x

R4F1 310.1 120x

R5 281.5 113x

R5F1 311.0 121x

For each sample the raw data produced are presented in terms of total nucleotide number.

Effective depth of coverage is determined by mapping and alignment of all raw data to a well

known single copy locus within the maize genome (pdc3). The median effective depths of

coverage are shown for all samples.

Positive Spike-in Controls and Experimental Limit of Detection

To produce “spike-in” positive control samples for sequencing, plasmid DNA libraries

were created as described above and then diluted to 1 and 1/10 maize genome equivalents

(representation of the plasmid DNA at concentrations equivalent to single copy or

1/10 copy per genome) before pooling with samples produced from the control materials

(as described above). At 1 genome equivalent, 100% nucleotide identity was observed

over 100% of PV-ZMAP5714 (Table C-2). This result demonstrates that all nucleotides

of the transformation plasmid are observed by the sequencing and bioinformatic

assessments performed. Also, observed coverage was adequate (Clarke and Carbon

1976) at a level of at least 1/10th genomic equivalent (98.83% coverage at

99.97% identity for the 1/10th genome equivalent spiked control sample) and, hence, a

detection level of at most 1/10th genome equivalent was achieved for the plasmid DNA

sequence assessment.
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Table C-2. Summary of NGS Data for the Conventional Control DNA Sample

Spiked with PV-ZMAP5714

1 Extent of coverage is calculated as the percent of all PV-ZMAP5714 bases observed in the sequencing of

the spike-in samples:

Extent of coverage =

number of spike in bases detected

total length (bp) of spike in plasmid × 100

2

Percent identity of coverage is calculated as the percent of all PV-ZMAP5714 bases observed in the

sequencing of the spike-in samples:

Percent identity of coverage

=

number of identical bases (spike in vs. plasmid sequence) detected

total length (bp) of spike in plasmid detected × 100

C.2.13. PCR and DNA Sequence Analyses to Examine the Insert and Flanking

Sequences in MON 87403

Overlapping PCR products, denoted as Product A, Product B, and Product C (Figure IV7) were generated that span the insert and adjacent 5′ and 3′ flanking DNA sequences in

MON 87403. For each fragment generation experimental conditions were chosen to

successfully produce on-target amplifications. These products were analyzed to

determine the nucleotide sequence of the insert in MON 87403, as well as that of the

DNA flanking the 5′ and 3′ ends of the insert.

The PCR analyses for Product A, Product B, and Product C were each conducted using

84 ng of genomic DNA template in a 50 l reaction volume. The reaction contained a

final concentration of 0.5 M of each primer and 1x concentration of Phusion High

Fidelity PCR Master Mix with HF Buffer (Thermo Scientific).

The amplification of Product A and Product B were performed under the following

cycling conditions: 1 cycle at 98C for 30 seconds; 35 cycles at 98C for 30 seconds,

65C for 30 seconds, 72C for 2 minutes 30 seconds; 1 cycle at 72C for 5 minutes.

The amplification of Product C was performed under the following cycling conditions: 1

cycle at 98C for 30 seconds; 35 cycles at 98C for 30 seconds, 72C for 30 seconds,

72C for 2 minutes; 1 cycle at 72C for 5 minutes.

1/10th copy Spike 1 copy Spike

Extent of coverage1of

PV-ZMAP5714

98.83% 100%

Percent identity of coverage2of

PV-ZMAP5714

99.97% 100%
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Aliquots of each PCR product were separated on an 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 Exo-SAP IT (Affymetrix,

Santa Clara, CA) and a MinElute PCR Purification Kit (Qiagen, Inc., Valencia, CA)

according to the manufacturer’s instructions and quantified using the Nanodrop

spectrophotometer according to the manufacturer’s instructions. 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 sequences from multiple sequencing

reactions performed on the overlapping PCR products. This consensus sequence was

aligned to the PV-ZMAP5714 sequence to determine the integrity and organization of the

integrated DNA and the 5′ and 3′ insert-to-flank DNA junctions in MON 87403.

C.2.14. PCR and DNA Sequence Analyses to Examine the Integrity of the DNA

Insertion Site in MON 87403.

To examine the MON 87403 T-DNA insertion site in control maize, PCR and sequence

analyses were performed on genomic DNA from the conventional control maize LH244.

The primers used in this analysis were designed from the DNA sequences flanking the

insert in MON 87403. 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 84 ng of genomic DNA template in a 50 l

reaction volume. The reaction contained a final concentration of 0.5 M of each primer

and 1x concentration of Phusion High Fidelity PCR Master Mix with HF Buffer (Thermo

Scientific). The amplification was performed under the following cycling conditions: 1

cycle at 98C for 30 seconds; 35 cycles at 98C for 20 seconds, 71C for 20 seconds,

72C for 2 minutes; 1 cycle at 72C for 5 minutes.

A small aliquot of each PCR product was separated on an 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 conventional control LH244 was

purified using Exo-SAP IT (Affymetrix, Santa Clara, CA) and a MinElute PCR

Purification Kit (Qiagen, Inc., Valencia, CA) according to the manufacturer’s instructions

and quantified using the Nanodrop spectrophotometer according to the manufacturer’s

instructions. The purified PCR product was sequenced using multiple primers, including

primers used for PCR amplification. All sequencing was performed by TGAC using

BigDye terminator chemistry (Applied Biosystems).

A consensus sequence was generated by compiling sequences from multiple sequencing

reactions performed on the verified PCR product. This consensus sequence was aligned

to the 5′ and 3′ sequences flanking the MON 87403 insert to determine the integrity and

organization of the insertion site.
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Appendix D: Materials, Methods and Results for Characterization of

ATHB17Δ113 Protein Produced in MON 87403

D.1. Characterization of ATHB17Δ113 Protein in MON 87403

D.1.1. Materials

The MON 87403-produced ATHB17Δ113 protein was purified from lyophilized leaf

(lot 11347255) of MON 87403. The MON 87403-produced ATHB17Δ113 protein was

stored in a -80 oC freezer in a buffer solution containing 20 mM NaPO4, pH 7.4, 0.5 M

NaCl, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic (EDTA), 2 μM

leupeptin, 2 μM E-64 and 1% IGEPAL CA-630.

The E. coli-produced ATHB17Δ113 protein (lot 11380003) was used as the reference

substance. The ATHB17Δ113 protein reference substance was generated from cell paste

produced by large-scale fermentation of E. coli containing the pMON109241 expression

plasmid. The coding sequence for ATHB17 contained on the expression plasmid

(pMON109241) was confirmed prior to and after fermentation. Records pertaining to the

production of the E. coli-produced ATHB17Δ113 protein are archived under Orion

lot 11359538 in the Monsanto Regulatory Archives.

D.1.2. ATHB17Δ113 Protein Purification

The ATHB17Δ113 protein was purified from lyophilized leaf (lot# 11347255) of

MON 87403. The purification procedure was not performed under a Good Laboratory

Practice (GLP) protocol, however, all procedures were documented and, where

applicable, Standard Operation Procedures were followed. The ATHB17Δ113 was

purified from an extract of lyophilized leaf using a combination of techniques, including

anionic exchange and immunoaffinity chromatography. The purification procedure is

briefly described below.

ATHB17Δ113 was enriched from lyophilized MON 87403 leaf tissue. Approximately

1 kg of lyophilized tissue was resuspended in a total of 44 L of chilled extraction buffer

(100 mM NaPO4, pH 7.4, 100 mM NaCl, 5 mM DTT, 2 mM MgCl2, 1 mM EDTA, 1 mM

benzamidine-HCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μM leupeptin, 2 μM

pepstatin A, 2 μM E-64, 2 μM bestatin, 10U/ml benzonase and 1% IGEPAL CA-630)

and homogenized with an Ultra-Turrax T-50 dispersing instrument (IKA Work Inc.,

Wilmington, NC) equipped with a medium grind probe at 6400 rpm for 30 seconds. The

suspension was then transferred to 4°C and ~560 g of poly(vinylpolypyrrolidone)

equilibrated in 4 L of extraction buffer, and 1.25 kg of Amberlite IRA-402 resin

equilibrated in 4 L of extraction buffer were added yielding concentrations of ~1% (w/v)

and ~2.5 (w/v), respectively, in a final volume of 52 L (approximately 1:50 tissue to

buffer ratio). The extraction slurry was incubated with stirring at 4°C for 2 hours before

the addition of 1 kg of Celpure P100 filter agent (Imerys Filtration Minerals, San Jose,

CA). The extraction slurry was then filtered using a 14-inch plate and frame filter press

(Ertel Alsop, Kingston, NY) with MicroMedia cellulose and diatomaceous earth filter

sheets (Ertel Alsop). The filtrate was chased through the filter press with 75 L of chase
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buffer (1 mM DTT, 1 mM EDTA, 1 mM PMSF, 2 μM leupeptin, 2 μM pepstatin A,

2 μM E-64, 2 μM bestatin and 1% IGEPAL CA-630). The eluting chase filtrate was

combined with the extraction filtrate until a total volume of 100 L was reached and the

remaining chase filtrate was discarded.

The resulting clarified extract solution was loaded onto a 2.2 L (7 cm x 20 cm)

Sulfopropyl-Sepharose Fast Flow cation exchange resin (GE Healthcare, Piscataway, NJ)

column pre-equilibrated with Equilibration Buffer (20 mM NaPO4, pH 7.4, 1 mM DTT,

1 mM EDTA, 1 mM MgCl2 and 1% IGEPAL CA-630). The loaded column was washed

with Buffer A (20 mM NaPO4, pH 7.4, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin and

2 μM E-64) and the bound ATHB17Δ113 was eluted using a 4-step salt gradient of

50 mM, 500 mM, 600 mM and 1.0 M NaCl in Buffer A. Elution fractions containing the

MON 87403-produced ATHB17Δ113 protein were identified by western blot analysis

and pooled. The ATHB17Δ113 in these fractions was precipitated by the addition of

(NH4)2SO4 at a final saturation of 60% followed by overnight incubation on an orbital

shaker at 4°C. Following overnight incubation, the precipitated ATHB17Δ113 was

pelleted by centrifugation and the resulting supernatants were discarded.

The resulting precipitated MON 87403-produced ATHB17Δ113 protein was immediately

resuspended in 240 ml of Immuno Equilibration Buffer (20 mM NaPO4, pH 7.4, 0.5 M

NaCl, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin, 2 μM E-64 and 1% IGEPAL CA-630).

The resuspended solution was clarified by centrifugation and the resulting supernatant

was loaded onto a 1.0 ml (1.3 cm x 1.0 cm) Protein G Fast Flow (GE Healthcare) column

to which a monoclonal anti-ATHB17 antibody had been conjugated. The loaded column

was washed with successive 10 column volume washes of Immuno Wash Buffer (20 mM

NaPO4, pH 7.4, 0.1 M NaCl, 1 mM DTT, 1 mM EDTA, 2 μM leupeptin and 2 μM E-64),

Immuno Wash Buffer with 1.0 M NaCl and Immuno Wash Buffer with 10% propylene

glycol. The bound ATHB17Δ113 was then eluted with IgG Elution Buffer (Thermo

Scientific, Rockford, IL) containing 2 μM leupeptin and 2 μM E-64. Elution fractions

containing the MON 87403-produced ATHB17Δ113 protein were identified by western

blot analysis. The ATHB17Δ113 in these fractions was further concentrated by the

addition of 25 μl of UNOsphere S cation exchange support resin (Bio-Rad, Hercules,

CA), pre-equilibrated in IgG Elution Buffer, to each fraction. Following overnight

incubation at 4°C with shaking, the ATHB17Δ113 bound resin was pelleted by

centrifugation and the resulting supernatant was discarded. The resulting resin pellets

were washed with 15 ml of Immuno Wash Buffer followed by centrifugation and

combined in a filter paper spin cup microfuge tube (Thermo Scientific). The bound

ATHB17Δ113 was then eluted with Immuno Equilibration Buffer. Elution fractions

containing the MON 87403-produced ATHB17Δ113 protein were identified by western

blot analysis and pooled.

The final buffer composition of the sample was 20 mM NaPO4, pH 7.4, 0.5 M NaCl,

1 mM DTT, 1 mM EDTA, 2 μM leupeptin, 2 μM E-64 and 1% IGEPAL CA-630. The

purified MON 87403-produced ATHB17Δ113 protein was aliquoted, assigned

lot 11361368 and stored at in a -80°C freezer.
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D.1.3. MALDI-TOF Tryptic Mass Map Analysis

D.1.3.1. Methods

MALDI-TOF tryptic mass fingerprint analysis was used to confirm the identity of the

MON 87403-produced ATHB17Δ113 protein. The MON 87403-produced

ATHB17Δ113 protein was diluted in 1× loading buffer (LB; 62 mM Tris-HCl, 5% (v/v)

2-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate (SDS), 0.005% (w/v) bromophenol

blue, 10% (v/v) glycerol, pH 6.8). The sample was heated to 99°C for 5 minutes and

loaded at ~150 ng in four lanes of a pre-cast Tris-glycine (4-20 %) polyacrylamide

gradient mini-gel (Invitrogen). Pre-stained MW standards (Precision Plus Protein

Standards, Bio-Rad) were loaded on the gel for molecular weight reference. Following

electrophoresis, proteins were briefly fixed in 40% (v/v) methanol, 7% (v/v) acetic acid

and stained for 16 hours with Brilliant Blue G-Colloidal stain (Sigma-Aldrich). Gels

were briefly destained in 10% (v/v) acetic acid, 25% (v/v) methanol followed by 3 hours

in 25% (v/v) methanol. Each ~22 kDa band was excised and destained with 40% (v/v)

methanol/10% (v/v) acetic acid. The excised bands were incubated in 100 mM

ammonium bicarbonate for 1 hour and treated with 10 mM DTT at 37°C for 2 hours

followed by incubation for 20 minutes with 10 mM iodoacetic acid in the dark. The

excised bands were then washed 3 times with 25 mM ammonium bicarbonate, dried

using vacuum centrifugation and rehydrated with 20 μl of 20 μg/ml trypsin (Promega,

Madison, WI). After 1 hour, excess liquid was removed and the excised bands were

incubated overnight at 37°C in 40 μl of 10% (v/v) acetonitrile in 25 mM ammonium

bicarbonate. The excised bands were then sonicated for 5 minutes and the resulting

extract was transferred to new microcentrifuge tube labeled Extract 1. The excised bands

were then extracted two more times, each with 30 μl of a 50% (v/v) acetonitrile,

0.1% (v/v) trifluoroacetic acid (TFA) solution and sonicated for 5 min. These two

extracts were combined with the first extract and dried using vacuum centrifugation. The

extracts were solubilized in 5 μl of 50% (v/v) acetonitrile, 0.1% TFA and sonicated for

5 min. Extract was spotted to wells on an analysis plate and mixed with

α-cyano-4-hydroxycinnamic acid (α-Cyano, Thermo Scientific). Test samples were

analyzed using AB Sciex TOF/TOF 5800 MS in reflector positive ion mode. Signal-toNoise was set to ≥10. Only monoisotopic ions were assigned in a mass list. A plate

model/default calibration was performed using TOF/TOF calibration mixture standards

(AB Sciex, Foster City, CA). The samples in α-Cyano matrix were analyzed in the 500

to 5000 Da range. The mass spectra were searched against the ATHB17Δ113 protein

sequence using Mascot and ProteinPilot protein identification tools. Search parameter

criteria included: Peptide Mass Tolerance ± 0.5 Da, Fixed Modifications- Carboxymethyl

(C). Peptide mass fingerprint was generated by Mascot and ProteinPilot. All matching

masses were tallied and a coverage map was generated for the mass fingerprint.

D.1.3.2. Results of MALDI-TOF Tryptic Mass Map Analysis

Peptide mass fingerprint analysis is a standard technique used for confirming the identity

of proteins. The identity of the MON 87403-produced ATHB17Δ113 protein was

confirmed by MALDI-TOF MS analysis of peptide fragments produced by the trypsin

digestion of the MON 87403-produced ATHB17Δ113 protein.
Monsanto Company CR262-14U1 187 of 260

There were 8 unique peptides identified that corresponded to the expected masses

(Table D-1). The identified masses were used to assemble a peptide map of the

ATHB17Δ113 protein (Figure D-1). The experimentally determined coverage of the

ATHB17Δ113 protein was 50% (81 out of 162 amino acids). This analysis confirms the

identity of MON 87403-produced ATHB17Δ113 protein.

Table D-1. Summary of the Tryptic Masses Identified for the MON 87403 -

produced ATHB17Δ113 using MALDI-TOF MS

Experimental

Mass1 Calculated Mass2 Diff.3 Fragment4

Sequence5

873.46 873.43 0.03 154-160 TFPPQER

878.47 878.45 0.02 37-43 LLEDSFR

1001.56 1001.53 0.03 153-160 KTFPPQER

1218.65 1218.61 0.04 66-74 QIEVWFQNR

1374.67 1374.63 0.04 93-103 WFGS...ENHR

1378.68 1378.68 0.00 33-43 EQSR...DSFR

2224.17 2223.99 0.18 116-135 VGPT...RCER

2358.02 2357.95 0.07 4-25 LPSS...APPR

1 Only experimental masses that matched calculated masses are listed in the table.

2 The calculated mass is the relative molecular mass calculated from the matched peptide

sequence.

3 The calculated difference between the experimental mass and the calculated mass.

4

Fragment numbering is based on the predicted N-terminus of the protein.

5

For peptide matches greater than nine amino acids in length the first 4 residues and last 4

residues are shown separated by dots (...).

001 MNRLPSSEDG DDEEFSHDDG SAPPRKKLRL TREQSRLLED SFRQNHTLNP

051 KQKEVLAKHL MLRPRQIEVW FQNRRARSKL KQTEMECEYL KRWFGSLTEE

101 NHRLHREVEE LRAIKVGPTT VNSASSLTMC PRCERVTPAA SPSRAVVPVP

151 AKKTFPPQER DR

Figure D-1. MALDI-TOF MS Coverage Map of the MON 87403-produced

ATHB17Δ113

The amino acid sequence of the MON 87403-produced ATHB17Δ113 protein was deduced from

the ATHB17 gene present in MON 87403 and RT-PCR analysis of MON 87403 ATHB17 RNA.

Boxed regions correspond to peptides that were identified from the MON 87403-produced

ATHB17Δ113 protein sample using MALDI-TOF MS. In total, 50% (81 out of 162 amino acids)

of the expected protein sequence was identified.
Monsanto Company CR262-14U1 188 of 260

D.1.4. Western Blot Analysis-Immunoreactivity

D.1.4.1. Methods

Quantitative western blot analysis was used to confirm the identity of the MON 87403

produced ATHB17Δ113 protein, determine the concentration of MON 87403 produced

ATHB17Δ113 in the enriched sample, and to assess the equivalence of the

immunoreactivity of the MON 87403 produced and E. coli-produced ATHB17Δ113

proteins.

MON 87403-produced and E. coli-produced ATHB17Δ113 proteins were diluted into

1× LB and heated to 99°C for 7 minutes. The E. coli-produced ATHB17Δ113 was used

to prepare a standard curve ranging between ~0.5 and ~3.0 ng (purity corrected). The

E. coli-produced ATHB17Δ113 protein and MON 87403-produced ATHB17Δ113

protein (two dilutions of 1:40 and 1:80 in duplicate) were loaded onto a pre-cast

Tris-glycine (4-20%) polyacrylamide mini-gel (Invitrogen). Precision Plus ProteinTM

Molecular Dual color Standards (Bio-Rad) were loaded on the gel in parallel for

molecular weight reference and to verify electrotransfer of the proteins to the membrane.

Following electrophoresis at a constant voltage, proteins were electrotransferred to a

nitrocellulose membrane (Invitrogen, Grand Island, NY).

The membrane was blocked with 5% non-fat dry milk (NFDM) in 1 phosphate buffered

saline containing 0.05% (v/v) Tween-20 (PBST) and incubated with a polyclonal antiATHB17Δ113 antibody (lot G858502B) at a dilution of 1:4000 in 1% NFDM in PBST

for 1 hour at RT. After washing with PBST, the membrane was next incubated with

horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Thermo Scientific)

at a dilution of 1:8,000 in 1% NFDM in PBST for 30 minutes at RT and washed again,

with PBST. Immunoreactive bands were detected using the ECLTM detection system (GE

Healthcare) and Amersham Hyperfilm (GE Healthcare). The film was developed using a

Konica SRX-101A automated film processor (Konica Minolta Medical & Graphic, Inc.,

Tokyo, Japan).

The prominent immunoreactive band in each lane, representing ATHB17Δ113, was

quantified. Quantification of the bands on the blot was performed on a GS 800

densitometer with the supplied Quantity One® software (Bio-Rad). The concentration of

MON 87403-produced ATHB17Δ113 in the sample was calculated as the mean of the

concentrations of MON 87403-produced ATHB17Δ113 in each lane relative to the

standard curve.

D.1.4.2. Results of ATHB17Δ113 Protein Immunoreactivity Equivalence

Western blot analysis was conducted using rabbit anti-ATHB17Δ113 polyclonal

antibodies to 1) assess the identity of MON 87403-produced ATHB17Δ113, 2) determine

the concentration of the MON 87403-produced ATHB17Δ113 in the enriched sample

from the leaf tissue of MON 87403; and 3) assess the relative immunoreactivity of the

MON 87403 produced and the E. coli-produced ATHB17Δ113 proteins.
Monsanto Company CR262-14U1 189 of 260

The results demonstrated that immunoreactive bands with the same electrophoretic

mobility were present in lanes loaded with the MON 87403-produced (Figure D-2, lanes

8-11) or the E. coli-produced (Figure D-2, lanes 2-7) ATHB17Δ113 protein. As

expected, the signal intensity increased with increasing load amounts of the

MON 87403-produced and E. coli-produced ATHB17Δ113 proteins, supporting

identification of MON 87403-produced ATHB17Δ113 protein. Quantitative western blot

analysis was conducted to determine the concentration of MON 87403-produced

ATHB17Δ113 in the enriched sample. The concentration of the MON 87403-produced

ATHB17Δ113 protein was determined to be 0.008 mg/ml. Because the

MON 87403-produced and the E. coli-produced ATHB17Δ113 proteins were both

immunoreactive to an anti-ATHB17Δ113 antibody, the MON 87403-produced and

E. coli-produced ATHB17Δ113 proteins were determined to have equivalent

immunoreactivity.
Monsanto Company CR262-14U1 190 of 260

Figure D-2. Western Blot Analysis of MON 87403- and E. coli-produced

ATHB17Δ113 Proteins

Aliquots of the MON 87403-produced ATHB17Δ113 and the E. coli-produced

ATHB17Δ113 proteins were subjected to SDS-PAGE and electrotransferred to a

nitrocellulose membrane. Proteins were detected using anti- ATHB17Δ113 antibodies as

the primary antibodies. Immunoreactive bands were visualized using HRP-conjugated

secondary antibodies and an ECL system. The molecular weights (kDa) of the standards

are shown on the left. The 10 minute exposure is shown. Lanes 1 and 12 were cropped

from the image. Lane designations are as follows.

Lane Sample Amount (ng) Dilution

1 Precision Plus ProteinTM Standards - -

2 E. coli-Produced ATHB17Δ113 0.4 -

3 E. coli-Produced ATHB17Δ113 0.7 -

4 E. coli-Produced ATHB17Δ113 1.1 -

5 E. coli-Produced ATHB17Δ113 1.4 -

6 E. coli-Produced ATHB17Δ113 1.8 -

7 E. coli-Produced ATHB17Δ113 2.1 -

8 MON 87403-produced ATHB17Δ113 - 1:80

9 MON 87403-produced ATHB17Δ113 - 1:80

10 MON 87403-produced ATHB17Δ113 - 1:40

11 MON 87403-produced ATHB17Δ113 - 1:40

12 Precision Plus ProteinTM Standards - -

kDa

250.0

100.0

75.0

50.0

37.0

25.0

20.0

15.0

10.0

150.0

2 3 4 5 6 7 8 9 10 11
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D.1.5. Apparent Molecular Weight and Purity Estimation using SDS-PAGE

D.1.5.1. Methods

MON 87403-produced and E. coli-produced ATHB17Δ113 proteins were diluted into

1× LB and heated to 95°C for 5 min. The MON 87403-produced ATHB17Δ113 protein

was loaded in duplicate at ~8.0, ~12.0, and ~16.0 ng, based on the ATHB17Δ113 protein

concentration, onto a pre-cast (4-20%) polyacrylamide mini-gel (Invitrogen). The

E. coli-produced ATHB17Δ113 protein was loaded at ~4 ng, based on the purity

corrected ATHB17Δ113 protein concentration, in a single lane. Broad Range Molecular

Weight Standards (Bio-Rad) were prepared and loaded on the gel at ~90 ng. Following

electrophoresis at a constant voltage, proteins were stained using a ProteoSilverTM Silver

Stain Kit (Sigma-Aldrich, St. Louis, MO) according to manufacturer’s recommended

protocol. Analysis of the gel was performed using a Bio-Rad GS-800 densitometer

supplied with Quantity One software (version 4.6.7). Apparent MW and purity were

reported as an average of all six lanes containing the MON 87403-produced

ATHB17Δ113 protein.

D.1.5.2. Results of ATHB17Δ113 Protein Molecular Weight Equivalence

The MON 87403-produced ATHB17Δ113 protein (Figure D-3, lanes 3-8) migrated to the

same position on the gel as the E. coli-produced ATHB17Δ113protein (Figure D-3, lane

2) and the apparent MW was calculated to be 22.4 kDa (Table D-2). Because the

experimentally determined apparent MW of the MON 87403-produced ATHB17Δ113

protein was within the pre-set acceptance limits for equivalence (Table D-2), the

MON 87403-produced and E. coli-produced ATHB17Δ113 proteins were determined to

have equivalent apparent molecular weights.

The purity of the MON 87403-produced ATHB17Δ113 protein was calculated based on

the six loads on the gel (Figure D-3, lanes 3-8). The average purity was determined to be

3%.

Table D-2. Molecular Weight Comparison Between the MON 87403- and

E. coli-produced ATHB17Δ113 Proteins

Apparent MW

of MON 87403-produced

ATHB17Δ113 Protein (kDa)

Apparent MW

of E. coli-produced

ATHB17Δ113 Protein

1

(kDa)

Preset Acceptance

Limits for the

MON 87403-produced

ATHB17Δ113 Protein

2

(kDa)

22.4 22.2 22.2 – 23.5

1As reported on the Certificate of Analysis for lot 11380003

2 Calculated lower and upper bounds for one future assay based on two-tailed 95% prediction

interval derived from apparent MW determinations for E. coli-produced ATHB17Δ113.
Monsanto Company CR262-14U1 192 of 260

Figure D-3. Molecular Weight and Purity Analysis of the MON 87403-produced

ATHB17Δ113 Protein

Aliquots of the MON 87403-produced ATHB17Δ113 and the E. coli-produced

ATHB17Δ113 proteins were subjected to SDS-PAGE and the gel was stained with silver

stain. The molecular weights (kDa) of the standards are shown on the left. Lane 10 was

cropped from the image. Lane designations are as follows:

Lane Sample Amount (ng)

1 Broad Range MW Standards 90

2 E. coli-produced ATHB17Δ113 4

3 MON 87403-produced ATHB17Δ113 8

4 MON 87403-produced ATHB17Δ113 8

5 MON 87403-produced ATHB17Δ113 12

6 MON 87403-produced ATHB17Δ113 12

7 MON 87403-produced ATHB17Δ113 16

8 MON 87403-produced ATHB17Δ113 16

9 Broad Range MW Standards 90

10 Blank -

1 2 3 4 5 6 7 8 9

kDa

200.0

116.3

97.4

66.2

45.0

31.0

21.5

14.4

6.5

ATHB17Δ113
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Appendix E: Materials and Methods Used for the Analysis of the Levels of

ATHB17Δ113 Protein in MON 87403

E.1. Materials

Over season leaf (OSL1), over season root (OSR1), forage, and grain tissue samples from

MON 87403 were harvested from five field sites in The United States during the 2012

growing season from starting seed lot 11332602. E coli-produced ATHB17Δ113

(lot 11280374) was used as the analytical reference standard.

E.2. Characterization of the Materials

The identity of MON 87403 was confirmed by conducting MON 87403 event-specific

polymerase chain reaction (PCR) analyses on the starting seed.

E.3. Field Design and Tissue Collection

Field trials were initiated during the 2012 planting season to generate tissues of

MON 87403 at various maize growing locations in The United States. OSL1, OSR1,

forage, and grain, tissue samples from the following field sites were analyzed: Jackson

County, Arkansas (site code ARNE); Story County, Iowa (site code IALL); Jefferson

County, Iowa (site code IARL); Pawnee County, Kansas (site code KSLA) and Lehigh

County, Pennsylvania (site code PAGR). At each site, four replicated plots of plants

containing MON 87403 were planted using a randomized complete block field design.

Tissue samples were collected from each replicated plot at all field sites. See Table V-1

for detailed descriptions of when the samples were collected.

E.4. Tissue Processing and Protein Extraction

All tissue samples harvested were shipped to Monsanto’s processing facility and were

ground by the Monsanto Sample Management Team to facilitate protein extraction. All

ground tissue samples were then lyophilized by the Monsanto Sample Management

Team. The lyophilized tissue samples were stored in a -80C freezer until transferred on

dry ice to the analytical facility.

The ATHB17Δ113 protein was extracted from maize tissues as described in Table E-1.

The protein extracts were aliquoted and stored frozen in a -80°C freezer until analysis.
Monsanto Company CR262-14U1 194 of 260

Table E-1. ATHB17Δ113 Extraction Methods

1

for Tissue Samples

Sample Type Tissue-to-Buffer

Ratio

Extraction Buffer

Leaf2/Grain/Root3/Forage 1:100 1 × TB + 0.1% BSA4

1ATHB17Δ113 protein was extracted from each tissue by adding the appropriate volume of

extraction buffer, beads, and shaking in a Harbil mixer (Harbil Industries Inc., Compton, CA).

The extracted samples were clarified using centrifugation and a serum filter.

2Over season leaf (OSL1).

3Over season root (OSR1).

4

0.1 M Tris, 0.1 M Na2B4O7, 0.005 M MgCl2, 0.05% (v/v) Tween-20 and 0.1 % (w/v) BSA.

E.5. ATHB17Δ113 Antibodies

Mouse monoclonal antibody (Lot G-869055) specific for the ATHB17Δ113 protein was

purified using Protein G column. The concentration of the purified antibody was

determined to be 3.79 mg/ml by spectrophotometric methods. The purified antibody was

stored in a phosphate buffered saline (0.001 M KH2PO4, 0.01 M Na2HPO4,

0.137 M NaCl, 0.0027 M KCl and 0.05 % NaN3).

Goat polyclonal antibodies specific for the ATHB17Δ113 protein were purified using

affinity chromatography with a Protein G column. The concentration of the purified

antibodies was determined to be 10.72 mg/ml by spectrophotometric methods. The

purified antibodies (lot G-869057) were stored in a phosphate buffered saline

(0.001 M KH2PO4, 0.01 M Na2HPO4, 0.137 M NaCl, 0.0027 M KCl and 0.05% NaN3).

The goat polyclonal antibodies were coupled with biotin (Thermo Fisher Scientific,

Waltham, MA) according to the manufacturer’s instructions and assigned lot G-869059.

The detection reagent was Poly-HRP conjugate (Thermo Fisher Scientific).

E.6. ATHB17Δ113 ELISA Method

Mouse anti-ATHB17Δ113 capture antibody was diluted into a coating buffer

(0.015 M Na2CO3 and 0.035 M NaHCO3) and immobilized onto 96-well microtiter plates

at 1 g/ml followed by incubation in a 4C refrigerator for ≥12 hours. Prior to each step

in the assay, plates were washed with 1 × phosphate buffered saline containing

0.05 % (v/v) Tween 20. Plates were blocked with the addition of 300 μl per well of

Blocker Casein in PBS (Thermo Fisher Scientific, catalog number 37528) for

60 to 90 minutes at 37C. ATHB17Δ113 protein standard or sample extract was added at

200 l per well and incubated for 60 to 65 minutes at 37C. Biotinylated goat

anti-ATHB17Δ113 antibodies were added at 200 l per well and incubated for

60 to 65 minutes at 37C. Poly-HRP conjugate was added at 200 l per well and
Monsanto Company CR262-14U1 195 of 260

incubated for 30 to 35 minutes at 37C. Plates were developed by adding 200 l per well

of horseradish peroxidase substrate, 3,3',5,5'-tetramethylbenzidine (Kirkegaard & Perry,

Gaithersburg, MD). The enzymatic reaction was terminated by the addition of 100 l per

well of 6 M H3PO4. Quantification of the ATHB17Δ113 protein was accomplished by

interpolation from an ATHB17Δ113 protein standard curve that ranged from

0 - 0.5 ng/ml.

E.7. Data Analyses

ELISA plates were analyzed on a SPECTRAmax Plus 384 (Molecular Devices,

Sunnyvale, CA) microplate spectrophotometer, using a dual wavelength detection

method. Protein concentrations were determined by optical absorbance at a wavelength

of 450 nm with a simultaneous reference reading of 620 nm. Data reduction analyses

were performed using Molecular Devices SOFTmax PRO GxP version 5.4 software.

Absorbance readings and protein standard concentrations were fitted with a fiveparameter logistic curve fit. Following the interpolation from the standard curve, the

amount of protein (ng/ml) in the tissue was reported on a “g/g dwt” basis for data that

were greater than or equal to the LOQ. This conversion utilized a sample dilution factor

and a tissue-to-buffer ratio. Microsoft Excel 2007 (Microsoft, Redmond, WA) was used

to calculate the protein levels in tissues. The sample means, standard deviations (SDs),

and ranges were also calculated using Microsoft Excel 2007. All protein expression

levels were rounded to two significant figures.

Any test substance extract that resulted in unexpectedly negative or positive results by

ELISA analysis were 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. Samples that were not confirmed to be either positive or negative

were reported as inconclusive and omitted from all calculations.
Monsanto Company CR262-14U1 196 of 260

Appendix F: Materials and Methods for Compositional Analysis of MON 87403

Maize Grain and Forage

Compositional comparisons between MON 87403 and the conventional control maize

hybrid were performed using the principles and analytes outlined in the OECD consensus

documents for maize composition (OECD 2002). These principles are accepted globally

and have been employed previously in assessments of maize products derived through

biotechnology. The compositional assessment was conducted on grain and forage

samples harvested from a single growing season conducted in the United States during

2012 under typical agronomic practices.

F.1. Materials

Harvested grain and forage from MON 87403, a conventional control (MPA640B) that

has similar genetic background to that of MON 87403, and conventional, commercial

reference maize hybrids were compositionally assessed. The reference hybrids are listed

in Table F-1.

Table F-1. Conventional Commercial Reference Maize Hybrids

Material Name Seed Lot # Field Site Codes

Burrus 645 11227210 ILMN, NESH

Gateway 4148 11273005 ILMN, PAGR

Gateway 6158 11273006 ARNE, INSH

H-9180 11226704 IARL

Lewis 6442 11226558 NESH

Lewis 7007 11226559 IALL, IARL, KSLA

LG2540 11226898 PAGR

11266730 ARNE, IARL, ILMN, INSH

LG2620 11226861 KSLA

Midland Phillips 799 11226703 ARNE, ILMN, INSH, NESH, PAGR

Mycogen 2M746 11226705 IALL

NC+ 4443 11226700 ARNE

NC+ 5220 11226701 IALL, KSLA

Phillips 713 11300072 KSLA

Phillips 717 11300073 IALL, NESH

Stewart S588 11226918 PAGR

Stewart S602 11226919 IARL

Stine 9724 11298951 INSH
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F.2. Characterization of the Materials

The identities of MON 87403, the conventional control, and reference hybrids were

confirmed prior to use in the compositional assessment.

F.3. Field Production of the Samples

Grain and forage samples from MON 87403, the conventional control, and the reference

hybrids were collected from eight replicated sites in United States during the 2012

growing season. The field sites were located in: Jackson, Arkansas (ARNE); Story, Iowa

(IALL); Jefferson, Iowa (IARL); Warren, Illinois (ILMN); Boone, Indiana (INSH);

Pawnee, Kansas (KSLA); Polk, Nebraska (NESH); and Lehigh, Pennsylvania (PAGR).

Starting seeds were planted in a randomized complete block design with four plots for

each of MON 87403, the conventional control, and the reference hybrids. The production

was conducted under normal agronomic field conditions for their respective geographic

regions that are typical areas for maize production in the United States.

Forage was collected at early dent (R5) and grain was collected at physiological maturity.

Forage samples were shipped on dry ice and grain was shipped at ambient temperature

from the field sites to Monsanto Company (Saint Louis, Missouri). Subsamples were

ground to a powder, stored in a freezer set to maintain -20°C located at Monsanto

Company (Saint Louis, Missouri). Subsamples were shipped on dry ice to Covance

Laboratories Inc. (Madison, Wisconsin) for compositional analysis.

F.4. Summary of Analytical Methods

Nutrients analyzed in this study included moisture, ash, protein, total fat, carbohydrates

by calculation, acid detergent fiber (ADF), neutral detergent fiber (NDF), total dietary

fiber (TDF), amino acids (18 components), fatty acids (22 components), minerals

(calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and

zinc) and vitamins [β-carotene (referred to as vitamin A), B1, B2, B6, E (α-tocopherol),

niacin, and folic acid], in the grain, and moisture, ash, protein, total fat, carbohydrates by

calculation, ADF, NDF, calcium and phosphorus in the forage. The anti-nutrients

assessed in grain included phytic acid and raffinose. Secondary metabolites assessed in

grain included furfural, ferulic acid, and p-coumaric acid.

All compositional analyses were performed at Covance Laboratories, Inc. (Madison,

Wisconsin). Methods for analysis were based on internationally-recognized procedures

and literature publications. Brief descriptions of the methods utilized for the analyses are

described below.

F.4.1. 2-Furaldehyde

The ground samples were extracted with 4% trichloroacetic acid and injected directly on

a high-performance liquid chromatography system for quantitation of free furfurals by

ultraviolet detection (Albala-Hurtado, et al. 1997). The limit of quantitation was

0.500 ppm.
Monsanto Company CR262-14U1 198 of 260

Reference Standard:

ACROS Organics, 2 Furaldehyde, 99.5%, Lot Number A0296679

F.4.2. 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 was primarily cellulose and lignin and insoluble protein

complexes remained in the Ankom filter bag, and was determined gravimetrically

(Goering and Van Soest 1970; Komarek, et al. 1993). The limit of quantitation was

0.100%.

F.4.3. Amino Acid Composition

The following 18 amino acids were analyzed:

 Total alanine  Total lysine

 Total arginine  Total methionine

 Total aspartic acid (including asparagine)  Total phenylalanine

 Total cystine (including cysteine)  Total proline

 Total glutamic acid (including glutamine)  Total serine

 Total glycine  Total threonine

 Total histidine  Total tryptophan

 Total isoleucine  Total tyrosine

 Total leucine  Total valine

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at

approximately 106-118oC. Phenol was added to the 6N hydrochloric acid to prevent

halogenation of tyrosine. Cystine and cysteine were 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 approximately 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 2012d; Barkholt and Jensen 1989; Henderson and Brooks 2010; Henderson, et

al. 2000; Schuster 1988). The limit of quantitation for this study was 0.100 mg/g.
Monsanto Company CR262-14U1 199 of 260

Reference Standards:

Component Manufacturer Lot No. Purity (%)

L-Alanine Sigma-Aldrich BCBC5470 99.8

L-Arginine Monohydrochloride Sigma-Aldrich 1361811 100

L-Aspartic Acid Sigma-Aldrich BCBB9274 100.6

L-Cystine Sigma-Aldrich 1451329 100

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 BCBC6907 99.9

L-Lysine Monohydrochloride Sigma-Aldrich 1362380 100.2

L-Methionine Sigma-Aldrich 1423807 99.9

L-Phenylalanine Sigma-Aldrich BCBC5774 100

L-Proline Sigma-Aldrich 1414414 99.7

L-Serine Sigma-Aldrich 1336081 99.9

L-Threonine Sigma-Aldrich 1402329 100

L-Tyrosine Sigma-Aldrich BCBC2417 100

L-Valine Sigma-Aldrich 1352709 100

L-Tryptophan Sigma-Aldrich BCBB1284 99.8

F.4.4. Ash

All organic matter was driven off when the samples were ignited at approximately 550oC

in a muffle furnace for at least 5 hours. The remaining inorganic material was

determined gravimetrically and referred to as ash (AOAC 2012h). The limit of

quantitation was 0.100%.

F.4.5. Vitamin A (Beta Carotene)

The samples were saponified and extracted with hexane. The samples were then injected

on a reverse phase high-performance liquid chromatography (HPLC) system with

ultraviolet light detection. Quantitation was achieved with a linear regression analysis

(AOAC 2012a; Quackenbush 1987). The limit of quantitation was 0.0200 mg/100g.

Reference Standard:

Manufacturer Analyte Lot No. Purity (%)

Sigma-Aldrich Beta Carotene 091M1417V 98.2*

* Based on E1%=2280 for Lambda Maximum of 478 to 479 nm in Hexane.

F.4.6. Carbohydrate

The total carbohydrate level was calculated by difference using the fresh weight-derived

data and the following equation (USDA 1973):

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)
Monsanto Company CR262-14U1 200 of 260

The limit of quantitation was calculated as 0.100%.

F.4.7. Fat by Acid Hydrolysis

The samples were hydrolyzed with hydrochloric acid. The fat was extracted using ether

and hexane. The extracts were dried down and filtered through a sodium sulfate column.

The remaining extracts were then evaporated, dried, and weighed (AOAC 2012k; l). The

limit of quantitation was 0.100%.

F.4.8. Fat by Soxhlet Extraction

The samples were weighed into a cellulose thimble containing sodium sulfate and dried

to remove excess moisture. Pentane was dripped through the samples to remove the fat.

The extract was then evaporated, dried, and weighed (AOAC 2012m; n). The limit of

quantitation was 0.100%.

F.4.9. Fatty Acids

The following 22 fatty acids were analyzed:

 8:0 Caprylic  18:0 Stearic

 10:0 Capric  18:1 Oleic

 12:0 Lauric  18:2 Linoleic

 14:0 Myristic  18:3 gamma-Linolenic

 14:1 Myristoleic  18:3 Linolenic

 15:0 Pentadecanoic  20:0 Arachidic

 15:1 Pentadecenoic  20:1 Eicosenoic

 16:0 Palmitic  20:2 Eicosadienoic

 16:1 Palmitoleic  20:3 Eicosatrienoic

 17:0 Heptadecanoic  20:4 Arachidonic

 17:1 Heptadecenoic  22:0 Behenic

The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The

saponification mixture was methylated with 14% boron trifluoride in methanol. The

resulting methyl esters 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 2009b; a). The limit of quantitation was 0.00400%.
Monsanto Company CR262-14U1 201 of 260

Reference Standards:

Manufacturer Lot No. Component Weight

(%)

Purity

(%)

JY10-W MA7-W

Nu-Chek Prep

GLC Reference

Standard

Covance 1

Covance 2

JY10-W

MA7-W

Methyl Octanoate 3.0 1.25 99.7

Methyl Decanoate 3.25 1.25 99.6

Methyl Laurate 3.25 1.25 99.8

Methyl Myristate 3.25 1.25 99.8

Methyl Myristoleate 1.0 1.25 99.5

Methyl Pentadecanoate 1.0 1.25 99.6

Methyl Pentadecenoate 1.0 1.25 99.4

Methyl Palmitate 10.0 15.75 99.8

Methyl Palmitoleate 3.0 1.25 99.7

Methyl Heptadecanoate 1.0 1.25 99.6

Methyl 10-

Heptadecenoate 1.0 1.25 99.5

Methyl Stearate 7.0 14.00 99.8

Methyl Oleate 10.0 15.75 99.8

Methyl Linoleate 10.0 15.75 99.8

Methyl Gamma

Linolenate 1.0 1.25 99.4

Methyl Linolenate 3.0 1.25 99.5

Methyl Arachidate 2.0 1.25 99.8

Methyl 11-Eicosenoate 2.0 1.25 99.6

Methyl 11-14

Eicosadienoate 1.0 1.25 99.5

Methyl 11-14-17

Eicosatrienoate 1.0 1.25 99.5

Methyl Arachidonate 1.0 1.25 99.4

Methyl Behenate 1.0 1.25 99.8

F.4.10. Vitamin B9 (Folic Acid)

The samples were hydrolyzed in a potassium phosphate buffer with the addition of

ascorbic acid to protect the folic acid during autoclaving. Following hydrolysis by

autoclaving, the samples were treated with a chicken-pancreas enzyme and incubated

approximately 18 hours to liberate the bound folic acid. The amount of folic acid was

determined by comparing the growth response of the samples, using the bacteria

Lactobacillus casei, with the growth response of a folic acid standard. This response was

measured turbidimetrically (AOAC 2012p; q; Infant Formula Council 1985). The limit

of quantitation was 0.0600 μg/g.

Reference Standard:

USP, Folic acid, 98.9%, Lot Number Q0G151
Monsanto Company CR262-14U1 202 of 260

F.4.11. Minerals / ICP Emission Spectrometry

The following nine minerals were analyzed:

 Calcium  Phosphorus

 Copper  Potassium

 Iron  Sodium

 Magnesium  Zinc

 Manganese

The samples were dried, precharred, and ashed overnight in a muffle furnace set to

maintain 500C. The ashed samples were 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 samples, measured on the inductively coupled plasma

spectrometer, with the emission of the standard solutions (AOAC 2012s; o). The limits

of quantitation were as follows:

Inorganic Ventures Reference Standards and Limits of Quantitation:

Mineral Lot Numbers

Concentration

(μg/mL) LOQ (ppm)

Calcium F2-MEB453071MCA, F2-MEB453073 200, 1000 20.0

Copper F2-MEB453071MCA, F2-MEB453072MCA 2.00, 10.0 0.500

Iron F2-MEB453071MCA, F2-MEB453074 10.0, 50.0 2.00

Magnesium F2-MEB453071MCA, F2-MEB453072MCA 50.0, 250 20.0

Manganese F2-MEB453071MCA, F2-MEB453072MCA 2.00, 10.0 0.300

Phosphorus F2-MEB453071MCA, F2-MEB453073 200, 1000 20.0

Potassium F2-MEB453071MCA, F2-MEB453073 200, 1000 100

Sodium F2-MEB453071MCA, F2-MEB453073 200, 1000 100

Zinc F2-MEB453071MCA, F2-MEB453072MCA 10.0, 50.0 0.400

F.4.12. Moisture

The samples were dried in a vacuum oven at approximately 100C. The moisture weight

loss was determined and converted to percent moisture (AOAC 2012r; j). The limit of

quantitation was 0.100%.

F.4.13. Neutral Detergent Fiber

The ANKOM2000 Fiber Analyzer automated the process of the removal of protein,

carbohydrate, and ash. Fats and pigments were removed with an acetone wash prior to

analysis. Hemicellulose, cellulose, lignin and insoluble protein fraction were left in the
Monsanto Company CR262-14U1 203 of 260

filter bag and determined gravimetrically (AACC 1998; Komarek, et al. 1994; USDA

1970). The limit of quantitation was 0.100%.

F.4.14. Vitamin B3 (Niacin)

The samples were hydrolyzed with sulfuric acid and the pH was adjusted to remove

interferences. The amount of niacin was determined by comparing the growth response

of the samples, using the bacteria Lactobacillus plantarum, with the growth response of a

niacin standard. This response was measured turbidimetrically (AOAC 2012i; p). The

limit of quantitation was 0.300 μg/g.

Reference Standard:

USP, Niacin, 99.8%, Lot Number J0J235

F.4.15. p-Coumaric Acid and Ferulic Acid

The ground samples were extracted with methanol followed by alkaline hydrolysis and

buffering prior to injection on an analytical HPLC system for quantification of

p-coumaric acid and ferulic acid by ultra violet (UV) detection (Hagerman and Nicholson

1982). The limit of quantitation for the p-coumaric acid and ferulic acid was 33.3 ppm.

Reference Standards:

Component Manufacturer Lot No. Purity (%)

p-Hydroxycinnamic Acid

(p-Coumaric Acid)

Sigma-Aldrich 091M1197V 99.6

4-Hydroxy-3-

methoxycinnamic Acid

(Ferulic Acid)

4-Hydroxy-3-

methoxycinnamic Acid

(Ferulic Acid)

ACROS Organics

ACROS Organics

A0261354

A0294716

99.4

99.4

F.4.16. Phytic Acid

The samples were extracted using hydrochloric acid and sonication, purified using a

silica based anion exchange column, concentrated and injected onto a high-performance

liquid chromatography (HPLC) system with a refractive index detector (Lehrfeld 1989;

Lehrfeld 1994). The limit of quantitation was 0.100%.

Reference Standard:

Sigma-Aldrich, Phytic Acid Sodium Salt Hydrate, 97.9%, Lot Number BCBH8701V
Monsanto Company CR262-14U1 204 of 260

F.4.17. Protein

The protein and other organic nitrogen in the samples were converted to ammonia by

digesting the samples 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. Instrumentation was used to automate the digestion, distillation and

titration processes. The percent nitrogen was calculated and converted to equivalent

protein using the factor 6.25 (AOAC 2012g; f). The limit of quantitation was 0.100%.

F.4.18. Raffinose

Sugars in the samples were extracted with a 50:50 water:methanol solution. Aliquots

were taken, dried under inert gas, and then reconstituted with a hydroxylamine

hydrochloride solution in pyridine containing phenyl-β-D-glucopyranoside as the internal

standard. The resulting oximes were converted to silyl derivatives by treatment with

hexamethyldisilazane and trifluoracetic acid treatment, and then analyzed by gas

chromatography using a flame ionization detector (Brobst 1972; Mason and Slover

1971). The limit of quantitation was 0.0500%.

Reference Standard:

Sigma-Aldrich, D-(+)-Raffinose pentahydrate, 99.6%, Lot Number 019K1156

F.4.19. 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 2012c).

The limit of quantitation was 1.00%.

F.4.20. Vitamin B1 (Thiamine Hydrochloride)

The samples were autoclaved under weak acid conditions to extract the thiamine. The

resulting solutions were incubated with a buffered enzyme solution to release any bound

thiamine. The solutions were purified on a cation-exchange column. Aliquots were

reacted with potassium ferricyanide to convert thiamine to thiochrome. The thiochrome

was extracted into isobutyl alcohol, measured on a fluorometer, and quantitated by

comparison to a known standard (AOAC 2012b; t; u). The limit of quantitation was

0.010 mg/100g.

Reference Standard:

USP, Thiamine Hydrochloride, 99.7%, Lot Number P0K366
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F.4.21. Vitamin B2 (Riboflavin)

The samples were hydrolyzed with dilute hydrochloric acid and the pH was adjusted to

remove interferences. The amount of riboflavin was determined by comparing the

growth response of the samples, using the bacteria Lactobacillus rhamnosus, with the

growth response of multipoint riboflavin standards. The growth response was measured

turbidimetrically (AOAC 2012e; p; c). The limit of quantitation was 0.200 μg/g.

Reference Standard:

USP, Riboflavin, 99.7%, Lot Number N1J079

The United States Pharmacopeia, Twenty-Ninth Revision, p. 1913, United States

Pharmacopeial Convention, Inc., Rockville, MD (2005).

F.4.22. Vitamin B6 (Pyridoxine Hydrochloride)

The samples were hydrolyzed with dilute sulfuric acid in the autoclave and the pH was

adjusted to remove interferences. The amount of pyridoxine was determined by

comparing the growth response of the samples, using the yeast Saccharomyces

cerevisiae, with the growth response of a pyridoxine standard. The response was

measured turbidimetrically. Results were reported as pyridoxine hydrochloride (AOAC

2012v; Atkins, et al. 1943). The limit of quantitation was 0.0700 μg/g.

Reference Standard:

USP, Pyridoxine hydrochloride, 99.8%, Lot Number Q0G409

F.4.23. Vitamin E (α-Tocopherol)

The samples were saponified to break down any fat and release vitamin E. The

saponified mixtures were extracted with ethyl ether and then quantitated by highperformance liquid chromatography using a silica column (Cort, et al. 1983; McMurray,

et al. 1980; Speek, et al. 1985). The limit of quantitation was 0.00500 mg/g.

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.

Reference Standard:

Manufacturer Component Lot No. Purity (%)

USP Alpha Tocopherol O0K291 99

Acros Organics D-gamma-Tocopherol A0083534 99.3

Sigma-Aldrich (+)-δ-Tocopherol 090M1916V 92
Monsanto Company CR262-14U1 206 of 260

F.5. Data Processing and Statistical Analysis

After compositional analyses were performed, data spreadsheets containing individual

values for each analysis were sent to Monsanto Company for review. Data were then

transferred to Certus International, Inc., where they were converted into the appropriate

units and statistically analyzed. The following formulas were used for re-expression of

composition data for statistical analysis (Table F-2):

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

Component From (X) To Formula1

Proximates (excluding Moisture),

Fiber, Anti-nutrients % fwt % dw X/d

Amino Acids (AA) mg/g fwt % dw X/(10d)

Secondary Metabolites ppm fwt μg/g dw X/d

Copper, Iron, Manganese, Zinc ppm fwt mg/kg dw X/d

Calcium, Magnesium, Phosphorus,

Potassium ppm fwt % dw X/(104

d)

Vitamins B2, B3, B6, B9 μg/g fwt mg/kg dw X/d

Vitamin A, Vitamin B1 mg/100g fwt mg/kg dw 10X/d

Vitamin E mg/g fwt mg/kg dw 103X/d

Fatty Acids (FA) % fwt % Total FA

(100)Xj/X, for

each FAj where X

is over all the FA

1

‘X’ is the individual sample value; d is the fraction of the sample that is dry matter.

In order to complete a statistical analysis for a compositional constituent in this

compositional assessment, at least 50% of all the values for an analyte in grain or forage

had to be greater than the assay limit of quantitation (LOQ). Analytes with more than

50% of observations below the assay LOQ were excluded from summaries and analysis.

The following 16 analytes in grain with more than 50% of observations below the assay

LOQ were excluded from statistical analysis: 8:0 caprylic acid, 10:0 capric acid, 12:0

lauric acid, 14:0 myristic acid, 14:1 myristoleic acid, 15:0 pentadecanoic acid, 15:1

pentadecenoic acid, 16:1 palmitoleic acid, 17:0 heptadecanoic acid, 17:1 heptadecenoic

acid, 18:3 gamma linolenic acid, 20:2 eicosadienoic acid, 20:3 eicosatrienoic acid, 20:4

arachidonic acid, sodium, and furfural.

Otherwise, individual results below the LOQ were assigned a value equal to one-half the

quantitation limit. Forty-five observations for 22:0 behenic acid and one value for

calcium were assigned a value equal to one-half of the LOQ (0.002% fwt and 10.00 ppm

fwt, respectively).

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
Monsanto Company CR262-14U1 207 of 260

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 ranges are considered

for exclusion, as outliers, from the final analyses. Eleven results had PRESS residual

values outside of the  6 range.

Of the eleven flagged values, only the copper values from two conventional references

were removed from further analysis as outliers. The remaining values were not removed

because they were not extreme values or they were deemed sufficiently close to

neighboring values to lack sufficient evidence for removal.

The outlier test procedure was reapplied to the remaining copper data to detect potential

outliers that were masked in the first analysis. One copper value from a commercial

reference was identified as a potential outlier, but the value was not an extreme value and

was not removed as an outlier.

Maize compositional components were statistically analyzed using a mixed-model

analysis of variance with the SAS MIXED procedure.

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.

For each component analysis, individual mean comparison tests of MON 87403 vs.

conventional control were conducted.

A range of observed values from the reference hybrids was determined for each

analytical component. Additionally, data from the reference hybrids were used to

develop 99% tolerance intervals. 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.

For each compositional component analyzed, two-sided 99% tolerance intervals were

calculated that are expected to contain, with 95% confidence, 99% of the quantities

expressed in the population of reference hybrids. Each estimate was based upon the

average of all observations per unique reference hybrid. Because negative quantities are

not possible, negative calculated lower tolerance bounds were set to zero.
Monsanto Company CR262-14U1 208 of 260

References for Appendix F

AACC. 1998. Approved methods of the American Association of Cereal Chemists.

American Association of Cereal Chemists, Inc., St. Paul, Minnesota.

Albala-Hurtado, S., M.T. Veciana-Nogues, M. Izquierdo-Pulido and M.C. Vidal-Carou.

1997. Determination of free and total furfural compounds in infant milk formulas by

high-performance liquid chromatography. Journal of Agricultural and Food Chemistry

45:2128-2133.

AOAC. 2012a. AOAC official method 2005.07: β·carotene in supplements and raw

materials reversed-phase high pressure liquid chromatographic method. Association of

Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012b. AOAC official method 942.23: Thiamine (Vitamin B1) in human and pet

foods. Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012c. AOAC official method 985.29: Total dietary fiber in foods. Association

of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012d. 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. 2012e. AOAC official method 940.33: Riboflavin (Vitamin B2) in vitamin

preparations. Association of Analytical Communities International, Gaithersburg,

Maryland.

AOAC. 2012f. AOAC official method 979.09: Protein in grains. Official Methods of

Analysis of AOAC International, Association of Analytical Communities International,

Gaithersburg, Maryland.

AOAC. 2012g. AOAC official method 955.04: Nitrogen (total) in fertilizers Kjeldahl

method. Official Methods of Analysis of AOAC International, Association of Analytical

Communities International, Gaithersburg, Maryland.

AOAC. 2012h. AOAC official method 923.03: Ash of flour. Official Methods of

Analysis of AOAC International, Association of Analytical Communities International,

Gaithersburg, Maryland.

AOAC. 2012i. AOAC official method 944.13: Niacin and niacinamide (nicotinic acid

and nicotinamide) in vitamin preparations. Association of Analytical Communities

International, Gaithersburg, Maryland.

AOAC. 2012j. AOAC official method 926.08: Loss on drying (moisture) in cheese.

Association of Analytical Communities International, Gaithersburg, Maryland.
Monsanto Company CR262-14U1 209 of 260

AOAC. 2012k. AOAC official method 922.06: Fat in flour. Official Methods of Analysis

of AOAC International, Association of Analytical Communities International,

Gaithersburg, Maryland.

AOAC. 2012l. AOAC official method 954.02: Fat (crude) or ether extract in pet food.

Official Methods of Analysis of AOAC International, Association of Analytical

Communities International, Gaithersburg, Maryland.

AOAC. 2012m. 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. 2012n. 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. 2012o. AOAC official method 985.01: Metals and other elements in plants and

pet foods. Official Methods of Analysis of AOAC International, Association of

Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012p. AOAC official method 960.46: Vitamin assays. Association of Analytical

Communities International, Gaithersburg, Maryland.

AOAC. 2012q. AOAC official method 992.05: Total folate (Pteroylglutamic acid) in

infant formula. Association of Analytical Communities International, Gaithersburg,

Maryland.

AOAC. 2012r. 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. 2012s. 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. 2012t. AOAC official method 953.17: Thiamine (Vitamin B1) in grain products.

Association of Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012u. AOAC official method 957.17: Thiamine (Vitamin B1). Association of

Analytical Communities International, Gaithersburg, Maryland.

AOAC. 2012v. AOAC official method 961.15: Vitamin B6 (pyridoxine, pyridoxal,

pyridoxamine) in food extracts. Association of Analytical Communities International,

Gaithersburg, Maryland.

AOCS. 2009a. Preparation of methyl esters of fatty acids. Method Ce 2-66, American Oil

Chemists' Society, Champaign, Illinois.
Monsanto Company CR262-14U1 210 of 260

AOCS. 2009b. Determination of saturated, cis-monounsaturated, and cis-polyunsaturated

fatty acids in marine and other oils containing long chain polyunsaturated fatty acids

(PUFAs) by capillary GLC3. Official Methods and Recommended Practices of the

American Oil Chemists' Society, 5th Edition. Method Ce 1i-07, American Oil Chemists'

Society, Champaign, Illinois.

Atkins, L., A.S. Schultz, W.L. Williams and C.N. Frey. 1943. Yeast microbiological

methods for determination of vitamins. Industrial and Engineering Chemistry, Analytical

Edition 15:141-144.

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.

Brobst, K.H. 1972. Gas-liquid chromatography of trimethylsilyl derivatives. Pages 3-8 in

Methods in Carbohydrate Chemistry. Volume 6. Academic Press, New York, New York.

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.

Goering, H.K. and P.J. Van Soest. 1970. Forage fiber analyses (apparatus, reagents,

procedures, and some applications). Agriculture Handbook No. 379. U.S. Department of

Agriculture, Agricultural Research Service, Washington, D.C.

Hagerman, A.E. and R.L. Nicholson. 1982. High-performance liquid chromatographic

determination of hydroxycinnamic acids in the maize mesocotyl. Journal of Agricultural

and Food Chemistry 30:1098-1102.

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.

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.

Infant Formula Council. 1985. Methods of analysis for infant formulas. Infant Formula

Council, Atlanta, Georgia.

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 feeds. National

Conference on Forage Quality, Evaluation, and Utilization, American Society of
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Agronomy, Inc., Crop Science Society of America, Inc., Soil Society of America, Inc.,

Madison, Wisonsin.

Lehrfeld, J. 1989. High-performance liquid chromatography analysis of phytic acid on a

pH-stable macroporous polymer column. Cereal Chemistry 66:510-515.

Lehrfeld, J. 1994. HPLC separation and quantitation of phytic acid and some inositol

phosphates in foods: Problems and solutions. Journal of Agricultural and Food Chemistry

42:2726-2731.

Mason, B.S. and H.T. Slover. 1971. A gas chromatographic method for the determination

of sugars in foods. Journal of Agricultural and Food Chemistry 19:551-554.

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.

OECD. 2002. Consensus document on compositional considerations for new varieties of

maize (Zea mays): Key food and feed nutrients, anti-nutrients and secondary plant

metabolites. OECD ENV/JM/MONO (2002)25. Organisation of Economic Co-Operation

and Development, Paris, France.

Quackenbush, F.W. 1987. Reverse phase HPLC separation of cis and trans-carotenoids

and its application to -carotenes in food materials. Journal of Liquid Chromatography

10:643-654.

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.

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.

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74. US Department of Agriculture, Agricultural Research Service, Washington, D.C.
Monsanto Company CR262-14U1 212 of 260

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

Dormancy and Germination Assessment of MON 87403

G.1. Materials

Seed germination and dormancy characteristics were assessed on seed from MON 87403,

the conventional control, and reference hybrids produced in replicated field trials during

2012 at the following sites: Story County, Iowa (IALL); Warren County, Illinois (ILMN);

and Lehigh County, Pennsylvania (PAGR).

G.2. Characterization of the Materials

The identities of the MON 87403 and the conventional control starting seed were verified

by event-specific polymerase chain reaction (PCR) analyses. During the growing season,

the field planting order of MON 87403 and the conventional control plots was confirmed

by event-specific PCR analyses. Chain-of-custody documentation for all starting seed for

this dormancy and germination study was maintained from harvest through shipment to

the performing laboratory with the use of packaging labels and plant sample transfer

forms.

G.3. Germination Testing Facility and Experimental Methods

Germination and dormancy evaluations were conducted at BioDiagnostics, Inc. in River

Falls, WI. The principal investigator was qualified to conduct seed germination and

dormancy testing consistent with the standards established by the Association of Official

Seed Analysts (AOSA), a seed trade association and Society of Commercial Seed

Technologists (SCST) (AOSA/SCST 2010; AOSA, 2012a; 2012b).

The seed lots (Selfed F2 grain) of MON 87403, the conventional control, and four

reference hybrids from each location were tested under seven different temperature

regimes. Seven germination chambers were used in the study and each chamber was

maintained dark under one of the following seven temperature regimes: constant

temperature of approximately 5, 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 8 hours.

The temperature inside each germination chamber was monitored and recorded

throughout the duration of the study.

Approximately 100 seeds each of MON 87403, the conventional control, and the

reference hybrids were placed on pre-moistened germination towels. Additional premoistened germination towels were placed on top of the seed. All rolled germination

towels were labeled and placed into an appropriately labeled bucket. Each bucket within

a temperature regime represented a replicate per site. There were 4 reps per site for a

total of 12 buckets for each temperature regime. Each bucket contained 1 towel per

entry. Buckets were then placed in the appropriate germination chambers. Each

temperature regime constituted a separate split-plot experiment with four replications. A

description of each germination characteristic evaluated and the timing of evaluations are
Monsanto Company CR262-14U1 213 of 260

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., alternating 20/30 °C) was assessed periodically during the study for normally

germinated, abnormally germinated, hard (viable and nonviable), dead, and firm swollen

(viable and nonviable) seed as defined by AOSA guidelines (AOSA, 2012a; 2012b).

AOSA only provides guidelines for testing seed under optimal temperatures, whereas

additional temperature regimes were included to test diverse environmental conditions.

Therefore, each rolled germination towel in the additional temperature regimes (i.e., 5,

10, 20, 30, alternating 10/20, and 10/30 °C) was assessed periodically during the study

for germinated, hard (viable and nonviable), dead, and firm swollen (viable and

nonviable) seed. Because temperature extremes could affect the development of

seedlings, AOSA standards were not applied and no distinction was made between

normal or abnormal germinated seed. Therefore, any seedling with a radical of 1 mm or

more was classified as germinated.

The calculation of percent seed in each assessment category was based on the actual

number of seeds evaluated (e.g., 99 or 100). Across temperature regimes, the total

number of seeds evaluated from each germination towel was approximately 100.

Within both AOSA and the additional temperature regimes, hard and firm-swollen seeds

remaining at the final evaluation date were subjected to a tetrazolium (Tz) test for

evaluation of viability according to AOSA standards (AOSA/SCST 2010). The number

of nonviable hard and nonviable firm-swollen seed was added to the number of dead seed

counted on all collection dates to determine the total percent dead seed. Total counts for

percent viable hard and viable firm-swollen seed were determined from the Tz test.

G.4. Statistical Analysis

An analysis of variance was conducted using SAS®version 9.3 (SAS 2010) according to

a split-plot design (production site as the whole plot and starting seed material as the subplot) with four replications. MON 87403 was compared to the conventional control for

dormancy and germination characteristics of seed 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 dormancy and 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% (α=0.05). MON 87403 was not

statistically compared to the reference hybrids, nor were comparisons made across

temperature regimes. The minimum and maximum mean values were determined from

the reference materials across the study sites (i.e., reference range) representative of

maize hybrids. Results from the combined-site analysis are presented in Table VII-2.

G.5. Individual-Site Seed Dormancy and Germination Analysis

In the individual site analyses, no statistically significant differences were detected at

sites IALL and ILMN. Two statistically significant differences were detected between
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MON 87403 and the conventional control at the PAGR site for the measured

characteristics abnormal germinated seed and viable firm-swollen seed. MON 87403 had

significantly fewer abnormal germinated seed (0.3% vs. 1.3%) and more viable firmswollen seed (0.3% vs. 0.0%) than the conventional control at the 20/30 °C. Statistically

significant differences between MON 87403 and the conventional control for both

germination characteristics in the individual site analyses were not detected in the

combined site analysis (Table VII-2) and are unlikely to be biologically meaningful in

terms of increased pest/weed potential (See Figure VII-1, Step 2, answer “no”).

Table G-1. Starting Seed of MON 87403, Conventional Control and Commercial

Maize Reference Hybrids Used in Dormancy Assessment

Site1 Material Type Material Name Phenotype Material ID

IALL Control MPA640B Conventional 11354876

IALL Reference Lewis 7007 Conventional 11354877

IALL Reference Mycogen 2M746 Conventional 11354878

IALL Reference NC+ 5220 Conventional 11354879

IALL Reference Phillips 717 Conventional 11354880

IALL Test MON 87403 Increased Ear Biomass

Maize 11354881

ILMN Control MPA640B Conventional 11354882

ILMN Reference Gateway 4148 Conventional 11354883

ILMN Reference Midland Phillips 799 Conventional 11354884

ILMN Reference Burrus 645 Conventional 11354885

ILMN Reference LG2540 Conventional 11354886

ILMN Test MON 87403 Increased Ear Biomass

Maize 11354887

PAGR Control MPA640B Conventional 11354888

PAGR Reference Gateway 4148 Conventional 11354889

PAGR Reference Midland Phillips 799 Conventional 11354890

PAGR Reference Stewart S588 Conventional 11354891

PAGR Reference LG2540 Conventional 11354892

PAGR Test MON 87403 Increased Ear Biomass

Maize 11354893

1

IALL = Story County, Iowa, ILMN = Warren County, Illinois, and PAGR = Lehigh County, Pennsylvania.
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Table G-2. Dormancy and Germination Characteristics of MON 87403 and the Conventional Control Seed Produced at each

of the Three Field Sites

IALL1ILMN1PAGR1

Temperature Assessment Mean % (S.E.)2 Mean % (S.E.)2 Mean % (S.E.)2

(°C) Category MON 87403 Control MON 87403 Control MON 87403 Control

5 Germinated 8.8 (0.95) 10.8 (1.80) 4.5 (1.04) 5.0 (1.29) 3.8 (1.44) 2.8 (0.85)

Viable Hard 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 3.8 (0.48) 2.3 (1.11) 4.0 (0.71) 3.3 (0.75) 4.5 (1.76) 5.0 (1.47)

Viable Firm Swollen 87.5 (1.32) 87.0 (2.12) 91.5 (1.55) 91.8 (1.31) 91.8 (2.87) 92.3 (1.65)

10 Germinated 93.8 (1.18) 92.5 (1.94) 97.0 (0.58) 95.5 (0.87) 94.3 (1.44) 94.0 (0.91)

Viable Hard 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 1.5 (1.50) 1.8 (1.11) 1.0 (0.41) 1.8 (0.85) 3.0 (0.71) 2.5 (0.87)

Viable Firm Swollen 4.8 (1.38) 5.8 (2.63) 2.0 (0.71) 2.8 (1.18) 2.8 (1.03) 3.5 (0.87)

203 Germinated 100.0 (0.00) 99.8 (0.25) 99.3 (0.25) 99.0 (0.41) 98.8 (0.63) 98.5 (0.29)

Viable Hard 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 0.0 (0.00) 0.3 (0.25) 0.8 (0.25) 1.0 (0.41) 1.3 (0.63) 1.5 (0.29)

Viable Firm Swollen 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0(0.00) †0.0 (0.00)

30 Germinated 99.8 (0.25) 99.8 (0.25) 99.8 (0.25) 99.5 (0.50) 98.8 (0.48) 99.5 (0.29)

Viable Hard 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 0.3 (0.25) 0.3 (0.25) 0.3 (0.25) 0.5 (0.50) 1.3 (0.48) 0.5 (0.29)

Viable Firm Swollen 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)
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Table G-2 (continued). Dormancy and Germination Characteristics of MON 87403 and the Conventional Control Seed

Produced at each of the Three Field Sites

IALL1ILMN1PAGR1

Temperature Assessment Mean % (S.E.)2 Mean % (S.E.)2 Mean % (S.E.)2

(°C) Category MON 87403 Control MON 87403 Control MON 87403 Control

10/20 Germinated 99.5 (0.29) 100.0 (0.00) 99.5 (0.50) 99.8 (0.25) 99.3 (0.25) 98.8 (0.25)

Viable Hard 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 0.3 (0.25) 0.0 (0.00) 0.3 (0.25) 0.3 (0.25) 0.8 (0.25) 1.3 (0.25)

Viable Firm Swollen 0.3 (0.25) 0.0 (0.00) 0.3 (0.25) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

10/30 Germinated 99.5 (0.29) 99.5 (0.29) 99.8 (0.25) 99.8 (0.25) 99.5 (0.50) 99.3 (0.48)

Viable Hard 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 0.5 (0.29) 0.5 (0.29) 0.3 (0.25) 0.3 (0.25) 0.5 (0.50) 0.8 (0.48)

Viable Firm Swollen 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

20/30 Normal Germinated 99.5 (0.50) 100.0 (0.00) 98.0 (0.41) 98.3 (0.25) 99.3 (0.48) 98.3 (0.63)

(AOSA) Abnormal Germinated 0.5 (0.50) 0.0 (0.00) 1.5 (0.29) 0.8 (0.25) 0.3 (0.25)* 1.3 (0.48)

Viable Hard 0.0 (0.00)†0.0 (0.00) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

Dead 0.0 (0.00) 0.0 (0.00) 0.5 (0.29) 1.0 (0.41) 0.3 (0.25) 0.5 (0.50)

Viable Firm- Swollen 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.3 (0.25)* 0.0 (0.00)

Note: The experimental design was a split-plot with four replications.

*Indicates a statistically significant differences between MON 87403 and the conventional control (α=0.05) using ANOVA.

1

Site codes are as follows: IALL = Story County, Iowa, ILMN = Warren County, Illinois and PAGR = Lehigh County, Pennsylvania.

2MON 87403 and the conventional control values represent means with standard error (S.E.) in parentheses. N = 4. In some instances, the total percentage of both MON 87403

and the conventional control did not equal 100% due to numerical rounding of the means.

3Three replicates of the reference Midland Phillips 799 were used from sites ILMN and PAGR.

†No statistical comparison could be made due to lack of variability in the data.
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References for Appendix G

AOSA/SCST (2010). Tetrazolium testing handbook. Ithaca, New York, Association of

Official Seed Analysts and the Society of Commercial Seed Technologists.

Association of Official Seed Analysts. 2012a. AOSA Rules for testing seeds. Volume 1.

Principles and Procedures. Association of Official Seed Analysts, Ithaca, NY.

Association of Official Seed Analysts. 2012b. AOSA Rules for testing seeds. Volume 4.

Seedling Evaluation. Association of Official Seed Analysts, Ithaca, NY.

SAS. (2010). SAS/STAT software version 9.3. SAS Institute, Inc., Cary, North Carolina.
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Appendix H: Materials, Methods, and Individual Site Results from Phenotypic,

Agronomic, and Environmental Interaction Assessment of MON 87403 under

Field Conditions

H.1. Materials

Agronomic, phenotypic, and environmental interaction characteristics were assessed for

MON 87403, the conventional control, and 21 reference hybrids grown under similar

agronomic conditions. Four reference hybrids were planted per site (Table H-1).

H.2. Characterization of the Materials

The presence or absence of the MON 87403 event in the starting seed of MON 87403 and

the conventional control was verified by event-specific polymerase chain reaction (PCR)

analyses. No molecular analyses were performed on the reference starting seed.

H.3. Field Sites and Plot Design

Field trials were established in 2012 at 13 sites that provided a range of environmental

and agronomic conditions representative of U.S. maize growing regions (Table VII-3).

The Principal Investigator at each site was familiar with the growth, production, and

evaluation of maize characteristics.

At all sites, seed of MON 87403, the conventional control, and four reference hybrids

were planted in a randomized complete block design with four replications. At all sites

except IABG, ILCX, NEDC, NEYO, NCBD, and PAHM, each replicated plot consisted

of 8 rows of maize spaced approximately 0.76 m apart and approximately 6 m long.

Phenotypic and qualitative environmental interaction data were targeted to be collected

from rows 4 and 5. The remaining rows were used for other purposes.

At sites: IABG, NEYO, NCBD, and PAHM each replicated plot consisted of 16 rows of

maize spaced approximately 0.76 - 0.96 m apart and approximately 6 m long.

Phenotypic and qualitative environmental interaction data were targeted to be collected

from rows 2 and 3. Rows 6 and 8 were targeted to collect arthropod samples using sticky

traps (rows 8 and 10 at NCBD site). Rows 9, 10, 11, and 12 were targeted for visual

counts of arthropod abundance. Rows 13 and 14 were targeted to assess plant damage

caused by corn earworm (Helicoverpa zea) and European corn borer (Ostrinia nubilalis).

The remaining rows were used for other purposes.

At sites: ILCX, and NEDC, each replicated plot consisted of 6 rows of maize spaced

approximately 0.76 m apart and approximately 6 m long. Phenotypic and qualitative

environmental interaction data were collected from rows 2 and 3. The remaining rows

were used for other purposes.

H.4. Planting and Field Operations

Planting information, soil description, and cropping history of the trial area are listed in

Table H-2. Prior to planting, the Principal Investigator at each site prepared the plot area
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with a proper seed bed according to local agronomic practices, including tillage,

fertilization, 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 practices

were applied as necessary. Maintenance operations were performed uniformly across all

plots.
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Table H-1. Starting Seed for Phenotypic, Agronomic, and Environmental

Interaction Assessment

Site

Code1 Material Name

Regulatory Lot

Number Phenotype T/C/R2

All

MON 87403 11332602 Increased Ear

Biomass Maize T

MPA640B311332601 Conventional C

ARNE

Gateway 6158 11273006 Conventional R

Midland Phillips 799 11226703 Conventional R

NC+ 4443 11226700 Conventional R

LG2540 11266730 Conventional R

IABG

Gateway 6158 11273006 Conventional R

LG2620 11226861 Conventional R

Burrus 645 11227210 Conventional R

Legacy L7671 11226598 Conventional R

IARL

Lewis 7007 11226559 Conventional R

H-9180 11226704 Conventional R

Stewart S602 11226919 Conventional R

LG2540 11266730 Conventional R

ILCX

Gateway 4148 11273005 Conventional R

Mycogen 2M746 11226705 Conventional R

NC+ 4443 11226700 Conventional R

LG2548 11266731 Conventional R

ILMN

Gateway 4148 11273005 Conventional R

Midland Phillips 799 11226703 Conventional R

Burrus 645 11227210 Conventional R

LG2540 11266730 Conventional R

INSH

Gateway 6158 11273006 Conventional R

Midland Phillips 799 11226703 Conventional R

Stine 9724 11298951 Conventional R

LG2540 11266730 Conventional R

KSLA

Lewis 7007 11226559 Conventional R

LG2620 11226861 Conventional R

NC+ 5220 11226701 Conventional R

Phillips 713 11300072 Conventional R

NCBD

Pioneer 32T16 11226579 Conventional R

Mycogen 2M746 11226705 Conventional R

Stewart S588 11226918 Conventional R

Legacy L7671 11226598 Conventional R
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Table H-1 (continued). Test, Control, and Reference Starting Seed

Site

Code1 Material Name

Regulatory Lot

Number Phenotype T/C/R2

NEDC

Gateway 4148 11273005 Conventional R

LG2620 11226861 Conventional R

Stewart S588 11226918 Conventional R

Phillips 717 11300073 Conventional R

NESH

Lewis 6442 11226558 Conventional R

Midland Phillips 799 11226703 Conventional R

Burrus 645 11227210 Conventional R

Phillips 717 11300073 Conventional R

NEYO

Pioneer 32B81 11226578 Conventional R

Mycogen 2M746 11226705 Conventional R

Burrus 645 11227210 Conventional R

LG2540 11266730 Conventional R

PAGR

Gateway 4148 11273005 Conventional R

Midland Phillips 799 11226703 Conventional R

Stewart S588 11226918 Conventional R

LG2540 11226898 Conventional R

PAHM

Gateway 4148 11273005 Conventional R

Midland Phillips 799 11226703 Conventional R

Stewart S588 11226918 Conventional R

LG2540 11226898 Conventional R

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA;

ILCX = Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee

County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE;

NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA.

2 T/C/R = Test/Control/Reference.

3 MPA640B = LH244+LH287.
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Table H-2. Field and Planting Information

Site1

Planting

Date2

Harvest

Date2

Approximate

Planting Rate

(seeds/m)

Approximate

Plot Size

(m × m)

Rows

per Plot Soil Type % OM3

Previous Crop

2011

ARNE 05/15/2012 9/20/2012 7.2 6.1 × 6.1 8 Sandy Loam 1.3 Soybean

IABG 05/09/2012 10/05/2012 7.2 6.1 × 12.2 16 Loam 4.0 Soybean

IARL 05/11/2012 10/11/2012 7.2 6.1 × 6.1 8 Silt Clay Loam 3.4 Soybean

ILCX 05/11/2012 09/24/2012 7.2 6.2 × 4.6 6 Silt Clay Loam 3.1 Soybean

ILMN 05/10/2012 10/01/2012 6.9 6.2 × 6.3 8 Silt Clay Loam 4.5 Soybean

INSH 05/16/2012 11/01/2012 7.2 6.1 × 6.1 8 Silt Loam 1.8 Soybean

KSLA 05/11/2012 09/20/2012 8.2 6.1 × 6.1 8 Silt Loam 2.6 Sorghum

NCBD 05/11/2012 09/20/2012 6.6 6.1 × 15.4 16 Sandy Loam 2.6 Cotton

NEDC 05/05/2012 09/10/2012 7.2 6.2 × 4.6 6 Silt Loam 2.6 Soybean

NESH 05/09/2012 09/25/2012 7.2 6.2 × 6.1 8 Silt Loam 1.5 Wheat

NEYO 05/09/2012 10/09/2012 7.2 6.1 × 12.2 16 Silt Loam 3.0 Soybean

PAGR 05/18/2012 10/10/2012 7.2 6.1 × 6.1 8 Loam 2.4 Soybeans

PAHM 05/19/2012 10/22/2012 8.2 6.1 × 12.2 16 Sandy Loam 1.6 Vegetables4

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County,

IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE;

NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA.

2

Planting and Harvest Date = mm/dd/yyyy.

3 % OM = Percent Organic Matter.

4 Vegetables = peppers, tomatoes, potatoes, cabbage, maize.
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H.5. Phenotypic Observations

The description of the characteristics measured and the designated developmental stages

where observations occurred are listed in Table VII-1.

H.6. Environmental Observations

Environmental interactions (i.e., interactions between the crop plants and their receiving

environment) were used to characterize MON 87403 by evaluating plant response to

abiotic stressors, disease damage, and arthropod-related damage using qualitative

methods described in Section H.7. In addition, specific arthropod damage (corn earworm

damage and European corn borer) and arthropod abundance were evaluated using the

quantitative methods described in Section H.8.

H.7. Plant Response to Abiotic Stress, Disease Damage, and Arthropod-Related

Damage

MON 87403 and the conventional control were evaluated at all sites for plant response to

abiotic stressors, disease damage, and arthropod damage. A target of three abiotic

stressors, three diseases, and three arthropod pests were evaluated four times during the

following four crop developmental stages: V6–V8; V12-VT; R1-R3; and R5-R6.

Abiotic stressor, disease damage and arthropod damage observations were collected from

each plot using the categorical scale of increasing severity listed below:

Category Severity of plant damage

None No symptoms observed

Slight Symptoms not damaging to plant development (e.g., minor feeding

or minor lesions); mitigation likely not required

Moderate Intermediate between slight and severe; likely requires mitigation

Severe Symptoms damaging to plant development (e.g., stunting or death);

mitigation unlikely to be effective

Method used for selecting stressors at each field site:

1. Prior to each data collection, maize was surveyed in proximity to the study area or the

border rows of the study for abiotic stressors (e.g., drought), diseases (e.g., gray

leaf spot), and arthropod damage (e.g., corn flea beetle).

2. The Principal Investigator chose three abiotic stressors, three diseases, and three

arthropod species that are actively causing damage for subsequent evaluation in

the study plots. The Principal Investigators 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.
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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 “none”.

As indicated above, the Principal Investigator 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 maize during the given observation period. Therefore, the

type of abiotic stressors, diseases, and arthropod pests assessed varied between

observations at a site and between sites.

In addition, ear and kernel rot disease and stalk rot disease were evaluated at harvest (R6

growth stage) using the above categorical scale. Ear and kernel rot disease data were

collected by evaluating five non-systematically selected ears (one per plant) from each

plot. The husks were pulled back and each ear was examined for disease. To evaluate

stalk rot, five non-systematically selected stalks in each plot were cut longitudinally. The

stalks were then examined for disease.

H.8. Arthropod Abundance

Specific arthropod (corn earworm and European corn borer) damage and arthropod

abundance were assessed quantitatively from observations/collections performed at

IABG, NEYO, NCBD, and PAHM sites.

Corn earworm damage was evaluated at R5 growth stage by examining ears from ten

plants (5 consecutive plants per row) in each plot. The husks were pulled back and each

ear was examined for corn earworm damage using a plastic film grid (size of each grid

0.5 cm2). Damage (cm2) per plant was calculated as the total number of grid cells

matching the damage area multiplied by 0.5 (each grid cell = 0.5 cm2).

European corn borer damage was evaluated at R6 growth stage by examining ten plants

(5 consecutive plants per row) in each plot. Damage was assessed by splitting each of ten

plants and counting the number of feeding galleries per plant and length of feeding

gallery (cm.) in each stalk.

Arthropods were collected using yellow sticky traps five times during the growing season

at the following intervals: late vegetative – VT, R1, R2, R3, and R4 growth stage. Sticky

traps (two per plot) were deployed in rows 6 and 8 of each plot at the approximate

midpoint between the ground level and the top of the plant canopy for all arthropod

collections. At each specified collection, traps were deployed for approximately 7 days.

Sticky traps were then sent to the Kansas State University, Manhattan, Kansas, for

arthropod identification and enumeration. For pre-processing, four sticky traps from

each sampling date at each site were non-systematically selected to prepare a list of pest

and beneficial arthropods for enumeration. A maximum of twelve arthropods were

enumerated for each collection. From the selected samples, up to six pest and six

beneficial arthropods were counted based on their abundance and likelihood of their

presence in maize. Thus, the suite of arthropods assessed often varied between
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collections from a site and between sites due to differences in temporal activity and

geographical distribution of arthropod taxa.

Five visual counts were conducted during the growing season at approximately VT-R1,

R1, R2, R3, and R4-R5 from five non-systematically selected plants per plot to collect

abundance data per plot. Visual counts were made by examining the stalk, the leaf blade,

the leaf collar, the ear tip, the silk, and the tassel of each plant.

H.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 data summarization or statistical

analysis as indicated Section H-10.

H.10. Statistical Analysis

Plant Growth and Development

Plant vigor data were summarized but not subjected to an analysis of variance (ANOVA);

due to the qualitative and subjective nature of the rating. MON 87403 was considered

different from the conventional control in vigor if the ranges of vigor of MON 87403 did

not overlap with the range of vigor of the conventional control across all replications.

Any observed differences between the MON 87403 and conventional control were

further assessed in the context of the range of the commercial reference materials, and for

consistency at other sites.

An ANOVA was conducted according to a randomized complete block design using

SAS® (SAS Software Release 9.3 (TS1M0), 2010) to compare MON 87403 and the

conventional control for the phenotypic characteristics listed in Table VII-1, with the

exception of plant vigor. The level of statistical significance was predetermined to be 5%

(α = 0.05). Comparisons of MON 87403 and the conventional control were conducted

within site (individual site analysis) and in a combined-site analysis, in which the data

were pooled across sites. MON 87403 and the conventional control materials were not

statistically compared to the commercial reference materials. The reference range for

each measured phenotypic characteristic was determined from the minimum and

maximum mean values from the 21 conventional commercial reference maize hybrids

planted among the sites. Data excluded from the study and the reasons for their exclusion

are listed in Table H-3.
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H.11. Environmental Interaction Data

The environmental interaction data (i.e., plant response to abiotic stressors, disease

damage, and arthropod damage) are categorical and were not subjected to ANOVA.

MON 87403 and conventional control were considered different in susceptibility or

tolerance if the range of injury symptoms of each did not overlap across all four

replications. Any observed differences were further assessed in the context of the range

of the reference materials, and for consistency at other sites.

An ANOVA was conducted according to a randomized complete block design using

SAS® (SAS Software Release 9.3 (TS1M0), 2010) for corn earworm damage, European

corn borer damage, and arthropod abundance. The level of statistical significance was

predetermined to be 5% (α = 0.05). MON 87403 was compared to the conventional

control at each site (individual-site analysis) for corn earworm damage, European corn

borer damage, and the arthropod abundance. Additionally, corn earworm damage and

European corn borer damage data were pooled across sites (combined-site analysis) for

statistical comparison between MON 87403 and the conventional control. Minimum and

maximum mean values were calculated for corn earworm damage and European corn

borer damage from 11 conventional commercial reference maize hybrids that were

included at IABG, NEYO, NCBD, and PAHM sites. The reference range for arthropod

abundance evaluated from a given collection and site was determined from the minimum

and maximum mean values collected from the conventional commercial reference maize

hybrids at the site.

For the arthropod abundance data, statistical analyses and significance testing of

differences between MON 87403 and the conventional control materials were only

performed for the arthropods present in sufficient numbers to estimate the material mean

arthropod counts and the variation of the means. An inclusion criterion was established

where a given arthropod must have an average count per plot per collection time (across

all materials) of ≥ 1.

Data excluded from the study and the reasons for their exclusion are listed in Table H-3.

H.12. Individual Field Site Plant Growth, Development, and Environmental

Interactions Results and Discussion

H.12.1. Plant Growth Development

In the individual-site analysis, a total of 14 statistically significant differences were

detected out of 155 comparisons between MON 87403 and the conventional control

(Table H-4). These differences were distributed among eight of the 13 phenotypic

characteristics. MON 87403 had higher early stand count than the conventional control

at the ILMN site (82.8 vs. 79.8 plants). MON 87403 reached days to 50% pollen shed

later than the conventional control at the PAGR site (68.8 vs. 65.8 days). MON 87403

reached days to 50% silking earlier than the conventional control at the NESH site (68.8

vs. 71.5 days) but reached days to 50% silking later than the conventional control at the

PAGR site (69.3 vs. 66.8 days). MON 87403 had higher stay green (more green tissue)
Monsanto Company CR262-14U1 227 of 260

than the conventional control at the NESH site (9.0 vs. 8.0 rating). MON 87403 had

higher final stand count than the conventional control at the NESH site (77.5 vs. 74.8

plants). MON 87403 had lower test weight than the conventional control at the NCBD

site (70.2 vs. 71.9 kg/hL). MON 87403 had a lower yield than the conventional control at

the ILMN site (15.5 vs. 17.0 Mg/ha), at the NCBD site (8.7 vs. 10.0 Mg/ha), and at the

PAGR site (12.1 vs. 13.9 Mg/ha). The statistical differences between MON 87403 and

the conventional control detected in the individual-site analyses for early stand count,

days to 50% pollen shed, days to 50% silking, stay green, final stand count, test weight,

and yield were not detected in the combined-site analysis. Thus, the differences detected

for these phenotypic characteristics at individual sites do not indicate a consistent

response associated with the trait and are unlikely to be biologically meaningful, in terms

of increased pest/weed potential of MON 87403 compared to conventional control

(Figure VII-1, step 2, “no” answer). MON 87403 had increased ear height compared to

the conventional control at the ILMN site (125.3 vs. 119.9 cm), NEDC site (121.8 vs.

111.4 cm), NESH site (124.3 vs. 118.2 cm) and PAHM site (89.3 vs. 78.0 cm). While

statistical differences were detected for ear height in both the individual site and the

combined-site analyses, the mean values of MON 87403 for ear height in the combinedsite analysis were within the range of values for commercial maize reference hybrids.

Therefore, these differences in ear height were not indicative of a consistent plant

response associated with the trait and are unlikely to be biologically meaningful in terms

of increased plant pest/weed potential of MON 87403 compared to conventional control

(Figure VII-1, step 3, “no” answer).

In individual site assessments of plant vigor, MON 87403 and the conventional control

were considered different if the range of values did not overlap between MON 87403 and

the conventional control across all four replications. There were no differences observed

between MON 87403 and the conventional control in plant vigor (Table H-4).

Plant Response to Abiotic Stressor, Disease Damage, and Arthropod-related Damage:

In the individual-site assessment, no differences were observed between MON 87403 and

the conventional control for any of the 143 comparisons for the assessed abiotic stressors,

including cold, drought, flood, frost, hail, heat, nutrient deficiency, soil compaction,

sunscald, and wind (Table H-5).

In the individual-site assessment, no differences were observed between MON 87403 and

the conventional control for any of the 176 comparisons for the assessed diseases,

including anthracnose, bacterial leaf spot, ear rot, eyespot, Fusarium sp., Goss’s bacterial

wilt, gray leaf spot, leaf blight, maize rough dwarf virus, northern leaf spot, Pythium sp.,

Rhizoctonia sp., rust, seedling blight, smut, stalk rot, and Stewart’s bacterial wilt

(Table H-6).

In the individual-site assessment, no differences were observed between MON 87403 and

the conventional control for any of the 150 comparisons for the assessed arthropods,

including aphids, armyworms, bean leaf beetles, billbugs, cutworms, corn earworms, corn

flea beetles, corn rootworm beetles, European corn borers, grasshoppers, Japanese
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beetles, June beetles, sap beetles, slugs, spider mites, stink bugs, and wireworm beetles

(Table H-7).

Corn Earworm and European Corn Borer Damage:

In the individual-site analysis, no statistically significant difference was detected out of

12 comparisons between MON 87403 and the conventional control for corn earworm and

European corn borer among all observations at all four sites (Table H-8).

Sticky Trap:

A total of 144 statistical comparisons were made between MON 87403 and the

conventional control for arthropod abundance involving the following arthropods: aphids,

billbugs, corn flea beetles, corn rootworm beetles, delphacid planthoppers, grasshoppers,

lacewings, ladybird beetles, leafhoppers, macro-parasitic hymenoptera, micro-parasitic

hymenoptera, minute pirate bugs, damsel bugs, sap beetles, seedcorn beetles, spiders,

syrphid flies, tachinid flies, tarnished plant bugs, thrips and click beetles (Table H-9).

Lack of sufficient arthropod abundance precluded statistical comparisons between

MON 87403 and the conventional control for 92 additional comparisons; however,

descriptive statistics were provided for these comparisons.

No statistically significant differences were detected between MON 87403 and the

conventional control for 130 out of 144 comparisons. The mean abundance of lacewings

was higher in MON 87403 than the conventional control in Collection 1 (2.0 vs. 0.3 per

plot) at the IABG site. The mean abundance of lacewings was lower in MON 87403 than

the conventional control in Collection 3 (2.5 vs. 8.0 per plot) and higher than the

conventional control in Collection 5 (3.0 vs. 0.8 per plot), at the NEYO site. The mean

abundance of macro-parasitic hymenoptera was lower in MON 87403 than the

conventional control in Collection 4 (0.3 vs. 3.3 per plot) at the PAHM site. The mean

abundance of micro-parasitic hymenoptera was higher in MON 87403 than the control in

Collection 2 (21.0 vs. 8.3 per plot) at the NCBD site. The mean abundance of aphids was

higher in MON 87403 than the conventional control in Collection 5 (22.5 vs. 13.8 per

plot) at the NEYO site. The mean abundance of corn flea beetles was lower in

MON 87403 than the conventional control in Collection 3 (0.0 vs. 1.5 per plot) at the

IABG site and higher in MON 87403 than the conventional control in Collection 5 (1.3

vs. 0.3 per plot) at the IABG site. The mean abundance of corn rootworm beetles was

lower in MON 87403 than the conventional control in Collection 3 (23.0 vs. 34.3 per

plot) at the NEYO site. The mean abundance of thrips was higher in MON 87403 than

the conventional control in Collection 2 (32.5 vs. 18.5 per plot) at the NCBD site, in

Collection 2 (183.0 vs. 71.5 per plot), Collection 4 (134.3 vs. 60.3 per plot), and

Collection 5 (138.0 vs. 62.3 per plot) at the NEYO site and Collection 3 (9.7 vs. 2.8 per

plot) at the PAHM site.

The mean abundance values for MON 87403 were within the reference ranges for all

differences detected in arthropod abundance with the exception of the difference detected

for lacewing abundance in Collection 5 at the NEYO site (MON 87403 mean = 3.0 per

plot; reference range = 0.5 – 2.8 per plot), macro-parasitic hymenoptera abundance in
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Collection 4 at the PAHM site (MON 87403 mean = 0.3 per plot; reference range = 1.5 –

2.3 per plot), corn flea beetles abundance in Collection 3 (MON 87403 mean = 0.0 per

plot; reference range = 1.8 – 2.0 per plot) and Collection 5 (MON 87403 mean = 1.3 per

plot; reference range = 2.0 – 3.8 per plot) at the IABG site, thrips abundance in

Collection 2 (MON 87403 mean = 183.0 per plot; reference range = 66.8 – 159.0 per

plot) and Collection 4 (MON 87403 mean = 134.3 per plot; reference range = 56.3 –

125.0 per plot) at the NEYO site, and in Collection 3 at the PAHM site (MON 87403

mean = 9.7 per plot; reference range = 2.3 – 4.8 per plot). However, these differences

were not consistently detected across collections or sites.

Thus, these differences in lacewings, macro-parasitic hymenoptera, micro-parasitic

hymenoptera, aphids, corn flea beetles, corn rootworms, and thrips were not indicative of

a consistent response associated with the trait and are not considered biologically

meaningful in terms of increased pest potential of MON 87403 compared to conventional

maize (Section VII.B.2).

Visual Counts:

A total of 66 statistical comparisons were made between MON 87403 and the

conventional control for arthropod abundance involving the following arthropods: antlike flower beetles, corn flea beetles, Japanese beetles, lacewing adults, lacewing larvae,

ladybird beetle adults, ladybird beetle larvae, minute pirate bugs, corn rootworm beetles,

sap beetles, shining flower beetles, spiders, stink bugs and click beetles (Table H-10).

Lack of sufficient arthropod abundance precluded statistical comparisons between

MON 87403 and the conventional control for 171 additional comparisons; however, the

descriptive statistics were provided for these comparisons.

No statistically significant differences were detected between MON 87403 and the

conventional control for 61 out of 66 comparisons. The mean abundance of minute pirate

bugs was lower in MON 87403 than the conventional control in Collection 4 (0.0 vs. 1.0

per plot) at the PAHM site. The mean abundance of corn rootworm beetles was lower in

MON 87403 than the conventional control in Collection 1 (0.3 vs. 2.0 per plot) at the

PAHM site. The mean abundance of sap beetles was lower in MON 87403 than the

conventional control in Collection 5 (1.3 vs. 4.5 per plot) at the NCBD site and

Collection 3 (1.0 vs. 4.5 per plot) at the NEYO site. The mean abundance of shining

flower beetles was lower in MON 87403 than the conventional control in Collection 2

(7.0 vs. 12.3 per plot) at the NCBD site.

The mean abundance values for MON 87403 were within the reference ranges for all

differences detected in arthropod abundance with the exception of the difference detected

for minute pirate bug abundance in Collection 4 at the PAHM site (MON 87403 mean =

0.0 per plot; reference range = 0.5 – 3.7 per plot) and corn rootworm beetle abundance in

Collection 1 at the PAHM site (MON 87403 mean = 0.3 per plot; reference range = 1.0 –

2.0 per plot). However, these differences were not consistently detected across

collections or sites.
Monsanto Company CR262-14U1 230 of 260

Thus these differences in minute pirate bugs, corn rootworm beetles, sap beetles and

shining flower beetles were not indicative of a consistent response associated with the

trait and are not considered biologically meaningful in terms of increased pest potential

of MON 87403 compared to conventional maize (Section VII.B.2).
Monsanto Company CR262-14U1 231 of 260

Table H-3. Data Missing or Excluded from Analysis

Site Code1 Material Name Material Type Plots Characteristics Reason for Exclusion

All All All All

Data was both collected & analyzed for

Anthesis Silking Interval, but not reported

for combined site or individual site

analysis.

Not relevant to the mode of action for

this trait.

IALL,

ILCY,

ILPH,

ILRD,

OHTR,

MSST,

ILWY

All All All All phenotypic and environmental

interactions data.

Extreme weather conditions (strong

wind or drought) that caused extensive

damage.

ILCX All All 401thru

407

All phenotypic and environmental

interactions data.

Drought stress

PAHM

Midland Phillips 799

MON 87403

Gateway 4148

Midland Phillips 799

Reference

Test

Reference

Reference

106, 206,

306, 406 Arthropod data collection Planting error

PAHM

NEDC

IABG

MON 87403

Burrus 645

Test

Reference

206, 407,

405, 401

First sub-sample of ear height and/or

plant height are missing.

Electronic data collection error
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Table H-3 (continued). Data Missing or Excluded from Study

Site Code1 Material Name Material Type Plots Characteristics Reason for Exclusion

NESH Midland Phillips 799 Reference 203 Test Weight Data collection error

PAGR MON 87403 Test 204 Early Stand Count Statistical outlier

NCBD Legacy L7671 Reference 207 Moisture & Yield Data collection error

ARNE

NEYO

LG2540

Mycogen 2M746 Reference

301,

103,

203,

204,

304,

403

Days to 50% Pollen Fewer than 10 plants shed pollen.

PAGR

INSH

Stewart S588

LG2540 Reference

102,

202,

206

Phenotypic data, stalk rot and ear rot

Plants were 10% below final stand

count for site

IABG All All All Environmental interaction evaluation # 4

(Other) Improper selection of stressor

NEYO All All All

Environmental interaction evaluation # 3

(2 arthropod, 1 abiotic and1 disease

stressor)

Missing data for five stressors

INSH All All All Environmental interaction evaluation # 3

(Drought) Missing data for 8 stressors

KSLA All All All Insect stressor observation 2 (velvet bean

caterpillar) Improper selection of stressor

Note: The study also included additional test entry not relevant to the objectives of this report, which was included in the statistical analysis input file.

However, no statistical comparisons were made using this additional test entry.

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren

County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk

County, NE; NEYO = York County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA.
Monsanto Company CR262-14U1 233 of 260

Table H-4. Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional

Control in 2012 U.S. Field Trials

Phenotypic Characteristics (units)

Plant Vigor (1-9 scale)2 Early stand count (#/plot) Days to 50% pollen shed

Range Mean (S.E.)3 Mean (S.E.)3

Site

Code1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 3 – 4 3 – 4 83.5 (0.96) 83.5 (0.87) 54.5 (0.87) 53.5 (0.65)

IABG 1 – 2 2 – 3 82.5 (2.87) 84.5 (0.87) 65.0 (0.00) 65.0 (0.00)

IARL 3 3 – 5 83.3 (2.43) 79.0 (2.68) 61.0 (0.00) 61.3 (0.48)

ILCX 1 1 82.7 (1.33) 83.0 (0.58) 60.3 (0.33) 60.7 (0.33)

ILMN 1 – 2 1 – 3 82.8 (0.63)* 79.8 (0.48) 63.0 (0.00) †63.0 (0.00)

INSH 2 – 4 3 – 5 81.3 (0.48) 82.0 (1.08) 65.0 (1.08) 63.5 (0.87)

KSLA 2 2 94.0 (1.22) 95.0 (1.63) 57.5 (0.96) 57.0 (0.58)

NCBD 3 – 4 3 72.3 (2.50) 72.8 (1.70) 59.3 (0.25) 59.8 (0.25)

NEDC 2 2 89.0 (1.35) 86.5 (0.65) 71.0 (0.00) 70.5 (0.50)

NESH 2 2 88.5 (1.19) 92.8 (2.59) 68.0 (0.00) 68.0 (0.00)

NEYO 1 1 78.3 (1.55) 79.3 (1.11) 68.0 (0.00) 68.0 (0.00)

PAGR 3 – 4 1 – 8 73.0 (1.00) 80.0 (1.83) 68.8 (0.75)* 65.8 (0.75)

PAHM 2 – 4 2 – 4 86.0 (2.20) 88.5 (0.87) 63.0 (0.71) 63.0 (0.41)
Monsanto Company CR262-14U1 234 of 260

Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional

Control in 2012 U.S. Field Trials

Phenotypic Characteristics (units)

Days to 50% silking Stay-green rating (1-9 scale) Ear height (cm)

Mean (S.E.)3 Mean (S.E.)3 Mean (S.E.)3

Site

Code1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 55.3 (1.18) 54.0 (0.58) 4.3 (0.25) 4.5 (0.50) 136.1 (3.99) 133.1 (3.66)

IABG 63.8 (0.63) 63.8 (0.25) 8.0 (0.41) 7.8 (0.75) 98.7 (1.66) 104.6 (2.52)

IARL 60.5 (0.50) 60.5 (0.50) 3.5 (0.29) 4.0 (0.41) 96.4 (4.00) 91.7 (4.85)

ILCX 64.0 (1.00) 66.0 (0.00) 3.3 (1.45) 3.3 (0.67) 108.6 (0.92) 105.1 (3.64)

ILMN 63.0 (0.00) †63.0 (0.00) 4.3 (0.25) 4.3 (0.25) 125.3 (1.28)* 119.9 (1.26)

INSH 66.0 (1.35) 64.5 (1.19) 4.0 (0.00) 4.0 (0.00) 107.2 (6.34) 108.9 (3.69)

KSLA 58.5 (0.50) 59.0 (0.58) 1.0 (0.00) 1.0 (0.00) 100.5 (3.36) 95.8 (3.40)

NCBD 59.3 (0.25) 59.5 (0.29) 6.3 (0.25) 6.3 (0.25) 127.1 (5.15) 128.7 (3.14)

NEDC 70.0 (0.58) 71.0 (0.00) 7.8 (0.48) 8.0 (0.41) 121.8 (3.99)* 111.4 (2.40)

NESH 68.8 (0.75)* 71.5 (1.66) 9.0 (0.00)* 8.0 (0.00) 124.3 (1.17)* 118.2 (1.74)

NEYO 68.0 (0.00) 68.3 (0.25) 6.0 (0.00) 5.5 (0.29) 113.2 (3.21) 103.6 (1.90)

PAGR 69.3 (0.75)* 66.8 (0.75) 2.0 (0.41) 1.5 (0.29) 95.9 (6.04) 98.8 (7.60)

PAHM 64.0 (0.71) 64.3 (0.63) 8.5 (0.29) 8.5 (0.29) 89.3 (5.39)* 78.0 (1.95)
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Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional

Control in 2012 U.S. Field Trials

Phenotypic Characteristics (units)

Plant height (cm) Dropped ears (#/plot) Stalk lodged plants (#/plot)

Mean (S.E.)3 Mean (S.E.)3 Mean (S.E.)3

Site

Code1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 265.0 (2.39) 268.6 (0.83) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

IABG 257.1 (3.96) 257.3 (3.51) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

IARL 213.5 (4.32) 205.7 (3.93) 0.3 (0.25) 0.0 (0.00) 1.8 (1.75) 0.0 (0.00)

ILCX 209.5 (9.34) 205.3 (5.74) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00) 0.0 (0.00)

ILMN 265.4 (2.74) 269.5 (3.41) 0.0 (0.00) 0.0 (0.00) 0.5 (0.29) 1.0 (0.58)

INSH 223.4 (8.86) 230.2 (5.59) 3.8 (0.63) 2.3 (0.48) 10.3 (3.64) 11.3 (2.02)

KSLA 213.1 (8.54) 212.8 (4.04) 0.0 (0.00) †0.0 (0.00) 0.0 (0.00) †0.0 (0.00)

NCBD 264.7 (6.83) 261.1 (3.03) 8.5 (4.33) 7.3 (3.20) 0.5 (0.29) 0.3 (0.25)

NEDC 257.8 (4.28) 251.8 (2.14) 0.0 (0.00) 0.5 (0.29) 3.5 (1.55) 3.8 (1.31)

NESH 258.8 (2.49) 249.2 (4.03) 0.0 (0.00) †0.0 (0.00) 0.3 (0.25) 0.8 (0.25)

NEYO 265.3 (2.60) 260.1 (0.87) 0.0 (0.00) †0.0 (0.00) 0.3 (0.25) 0.3 (0.25)

PAGR 214.4 (6.66) 224.4 (4.12) 2.0 (0.00) 1.0 (0.41) 7.5 (1.85) 6.3 (2.39)

PAHM 210.8 (7.96) 209.3 (5.87) 1.0 (0.71) 1.0 (0.41) 6.0 (1.78) 4.8 (1.03)
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Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional

Control in 2012 U.S. Field Trials

Phenotypic Characteristics (units)

Root lodged plants (#/plot) Final stand count (#/plot) Grain moisture (%)

Mean (S.E.)3 Mean (S.E.)3 Mean (S.E.)3

Site

Code1 MON 87403 Control MON 87403 Control MON 87403 Control

ARNE 0.3 (0.25) 0.0 (0.00) 75.3 (0.25) 75.0 (0.00) 16.1 (0.32) 16.1 (0.14)

IABG 0.8 (0.75) 1.3 (0.75) 72.3 (1.55) 72.3 (1.11) 16.6 (0.10) 17.5 (0.66)

IARL 4.3 (1.89) 1.8 (1.03) 74.8 (0.75) 73.5 (0.96) 17.4 (0.13) 17.7 (0.17)

ILCX 0.3 (0.33) 0.3 (0.33) 76.0 (0.00) †76.0 (0.00) 25.9 (0.58) 26.4 (1.22)

ILMN 2.3 (1.93) 1.8 (1.44) 75.3 (0.25) 75.5 (0.29) 20.9 (1.55) 19.1 (1.18)

INSH 0.8 (0.48) 1.0 (0.71) 71.5 (1.04) 70.8 (1.89) 19.3 (0.27) 19.5 (0.48)

KSLA 0.0 (0.00) †0.0 (0.00) 76.8 (1.60) 76.3 (0.25) 16.4 (0.34) 16.0 (0.32)

NCBD 0.0 (0.00) †0.0 (0.00) 68.8 (1.60) 68.8 (1.89) 19.5 (0.25) 19.6 (0.21)

NEDC 0.0 (0.00) †0.0 (0.00) 75.8 (0.63) 73.8 (1.31) 15.5 (0.27) 15.4 (0.55)

NESH 0.0 (0.00) 0.8 (0.48) 77.5 (1.26)* 74.8 (1.44) 16.7 (0.60) 16.4 (0.27)

NEYO 0.0 (0.00) †0.0 (0.00) 61.5 (0.65) 60.0 (1.41) 15.7 (0.13) 15.7 (0.14)

PAGR 0.0 (0.00) †0.0 (0.00) 63.8 (0.63) 65.8 (0.75) 30.6 (0.46) 29.4 (1.09)

PAHM 0.0 (0.00) †0.0 (0.00) 73.8 (0.25) 74.5 (0.65) 19.9 (0.52) 20.2 (0.46)
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Table H-4 (continued). Individual Site Analysis of Phenotypic Characteristics of MON 87403 Compared to the Conventional

Control in 2012 U.S. Field Trials

Phenotypic Characteristics (units)

Test weight (kg/hl) Yield (Mg/ha)

Mean (S.E.)3 Mean (S.E.)3

Site

Code1 MON 87403 Control MON 87403 Control

ARNE 74.6 (0.69) 74.5 (0.27) 10.2 (0.17) 10.7 (0.17)

IABG 75.8 (0.36) 77.4 (0.46) 7.5 (1.35) 8.8 (1.19)

IARL 75.4 (0.95) 75.7 (0.84) 7.6 (0.51) 6.4 (0.75)

ILCX 72.7 (0.66) 71.8 (2.82) 7.9 (1.38) 4.9 (1.41)

ILMN 70.1 (0.35) 71.4 (0.61) 15.5 (0.44)* 17.0 (0.44)

INSH 71.1 (0.34) 70.6 (0.86) 10.5 (0.88) 11.1 (0.53)

KSLA 78.4 (0.33) 77.8 (0.57) 14.0 (0.41) 13.5 (0.46)

NCBD 70.2 (0.64)* 71.9 (0.73) 8.7 (0.64)* 10.0 (0.54)

NEDC 74.8 (0.53) 74.7 (0.37) 7.0 (0.12) 7.7 (0.11)

NESH 76.4 (0.38) 75.3 (0.13) 5.0 (0.16) 4.6 (0.56)

NEYO 76.0 (0.40) 75.8 (0.27) 13.3 (0.47) 12.9 (0.40)

PAGR 59.3 (0.38) 60.3 (0.98) 12.1 (0.73)* 13.9 (0.86)

PAHM 68.6 (0.50) 68.2 (0.26) 11.7 (0.44) 11.0 (0.34)

Note: The experimental design was a randomized complete block with four replicates per site.

* Indicates statistically significant difference between MON 87403 and the conventional control (α =0.05) using ANOVA.

†

Indicates p-values could not be generated due to lack of variability in the data.

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX = Vermilion County, IL; ILMN = Warren County, IL;

INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD = Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York

County, NE; PAGR = Lehigh County, PA; PAHM = Berks County, PA.

2 Data were not subjected to statistical analysis. Plant vigor rating range (minimum - maximum); the range of plant vigor ratings for the references is as follows: ARNE

1– 4; IABG 1 – 3; IARL 2 – 4; ILCX 1; ILMN 1 – 3; INSH 3 – 4; KSLA 2; NCBD 3 – 4; NEDC 2; NESH 2; NEYO 1; PAGR 1 – 8; PAHM 2 – 5

3 MON 87403 and the conventional control values represent means with standard error in parentheses. N = 4, except where noted in Table H-5.
Monsanto Company CR262-14U1 238 of 260

Table H-5. Abiotic Stressor Evaluations Using a Categorical Scale for MON 87403

and the Conventional Control in 2012 U.S. Field Trials

Abiotic Stressor Number of Observations

across Sites1

Number of Observations where No

Differences were Observed between

MON 87403 and the Conventional

Control

Total 143 143

Cold 2 2

Drought236 36

Flood37 7

Frost 2 2

Hail 9 9

Heat 27 27

Nutrient deficiency 24 24

Soil compaction 4 4

Sunscald 4 4

Wind 28 28

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87403 and the conventional control during any observation for damage

caused by any of the assessed abiotic stressors. Data were not subjected to statistical analysis.

Observational data collected at four crop development stages: V6–V8; V12-VT; R1-R3; and R5-R6.

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX =

Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD

= Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE;

PAGR = Lehigh County, PA; PAHM = Berks County, PA.

2

Includes dryness, dry/heat.

3

Includes wet soil.
Monsanto Company CR262-14U1 239 of 260

Table H-6. Disease Damage Evaluations Using a Categorical Scale for MON 87403

and the Conventional Control in 2012 U.S. Field Trials

Disease Number of Observations

across Sites1

Number of Observations where No

Differences were Observed between

MON 87403 and the Conventional

Control

Total 176 176

Anthracnose 8 8

Bacterial leaf spot 2 2

Ear rot219 19

Eyespot 6 6

Fusarium sp. 8 8

Goss’s bacterial wilt 11 11

Gray leaf spot 23 23

Leaf blight324 24

Maize rough dwarf virus 3 3

Northern leaf spot 7 7

Pythium sp. 2 2

Rhizoctonia sp. 2 2

Rust412 12

Seedling blight 2 2

Smut 15 15

Stalk rot517 17

Stewart’s bacterial wilt 15 15

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87403 and the conventional control during any observation for damage

caused by any of the assessed diseases. Data were not subjected to statistical analysis.

Observational data collected at four crop development stages: V6–V8; V12-VT; R1-R3; and R5-R6.

Additional assessments of ear rot disease and stalk rot disease were made on 5 plant/plots at harvest.

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX =

Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD

= Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE;

PAGR = Lehigh County, PA; PAHM = Berks County, PA.

2 Assessed on 5 non-systematically selected plants. At ARNE, ILCX, ILMN, INSH, NEDC, and NESH ear rot data

were collected both on a per plot basis and on 5 plant/plots.

3

Includes northern and southern.

4

Includes common and southern.

5 Assessed on 5 non-systematically selected plants. At ILCX, NEDC, NESH, and PAGR stalk rot data were collected

both on a per plot basis and on 5 plant/plots.
Monsanto Company CR262-14U1 240 of 260

Table H-7. Arthropod Damage Evaluations Using a Categorical Scale for

MON 87403 and the Conventional Control in 2012 U.S. Field Trials

Arthropod

Number of

Observations

across Sites1

Number of Observations

where No Differences were

Observed between

MON 87403 and the

Conventional Control

Total 150 150

Aphids (Aphididae) 5 5

Armyworms (Noctuidae)224 24

Bean leaf beetles (Cerotoma trifurcata) 2 2

Billbugs (Sphenophorus parvulus) 2 2

Cutworms (Noctuidae)310 10

Corn earworms (Helicoverpa zea) 13 13

Corn flea beetles (Chaetocnema pulicaria)

4

7 7

Corn rootworm beetles (Diabrotica sp.) 14 14

European corn borers (Ostrinia nubilalis) 17 17

Grasshoppers (Melanoplus spp.) 20 20

Japanese beetles (Popillia japonica) 7 7

June beetles (Scarabaeidae) 2 2

Sap beetles (Nitidulidae) 6 6

Slugs5(Gastropoda) 1 1

Southwestern corn borer (Diatraea grandiosella) 2 2

Spider mites (Tetranychus sp.) 5 5

Stink bugs (Pentatomidae) 11 11

Western bean cutworm (Richia albicosta) 1 1

Click beetles (Elateridae)61 1

Note: The experimental design was a randomized complete block with four replicates per site.

No differences were observed between MON 87403 and the conventional control during any observation for damage

caused by any of the assessed arthropods. Data were not subjected to statistical analysis.

Observational data collected at four crop development stages: V6–V8; V12-VT; R1-R3; and R5-R6.

1

Site code: ARNE = Jackson County, AR; IABG = Greene County, IA; IARL = Jefferson County, IA; ILCX =

Vermilion County, IL; ILMN = Warren County, IL; INSH = Boone County, IN; KSLA = Pawnee County, KS; NCBD

= Perquimans County, NC; NEDC = Butler County, NE; NESH = Polk County, NE; NEYO = York County, NE;

PAGR = Lehigh County, PA; PAHM = Berks County, PA.

2

Includes beet armyworm and fall armyworm

3

Includes black cutworm

4

Includes flea beetle

5

Slugs are not arthropods but are one of the common stressor in Maize.

6 Wireworm adults
Monsanto Company CR262-14U1 241 of 260

Table H-8. Individual Site Analysis: Corn Earworm Damage and European Corn

Borer Damage to MON 87403 Compared to the Conventional Control in 2012 U.S.

Field Trials

Pest Damage assessment Site1

Mean (S.E.)2

MON 87403 Control

Corn earworm

(H. zea)

3

Damage area of 10 plants per

plot (cm2)

IABG 0.6 (0.12) 1.2 (0.25)

NCBD 3.4 (0.85) 6.4 (2.01)

NEYO 2.9 (0.38) 3.3 (0.44)

PAHM 0.4 (0.19) 0.1 (0.06)

European corn borer

(O. nubilalis)

4

Number of stalk galleries of

10 plants per plot IABG 0.0 (0.00) 0.0 (0.03)

NCBD 0.1 (0.08) 0.2 (0.09)

NEYO 0.0 (0.03) 0.1 (0.03)

PAHM 1.0 (0.27) 1.0 (0.25)

European corn borer

(O. nubilalis)

4

Stalk gallery length (cm) of

10 plants per plot IABG 0.0 (0.00) 0.1 (0.08)

NCBD 0.6 (0.39) 1.3 (0.64)

NEYO 0.1 (0.10) 0.2 (0.10)

PAHM 4.6 (1.27) 4.0 (1.04)

Note: The experimental design was a randomized complete block with four replicates per site.

1

Site code: IABG = Greene, IA; NCBD = Perquimans, NC; NEYO = York, NE; PAHM = Berks, PA.

2 MON 87403 and the conventional control values represent means with standard error in parentheses.

3 Damage assessment for H. zea was conducted at R5 growth stage.

4 Damage assessments for O. nubilalis were conducted at R6 growth stage.
Monsanto Company CR262-14U1 242 of 260

Table H-9. Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403 Compared to

the Conventional Control in 2012 U.S. Field Trials

Spiders (Araneae) Ladybird beetles (Coccinellidae) Lacewings (Chrysopidae)

Beneficial Arthropod Beneficial Arthropod Beneficial Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG — — — 0.0 (0.00)† 0.3 (0.25) 0.0 – 3.0 2.0 (0.41)* 0.3 (0.25) 1.0 – 3.3

NCBD 0.3 (0.25)† 1.0 (1.00) 0.5 – 1.0 1.0 (0.41) 1.5 (0.87) 1.0 – 2.0 — — —

NEYO 0.0 (0.00)† 0.3 (0.25) 0.3 – 0.5 0.3 (0.25)† 0.0 (0.00) 0.3 – 2.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 1.3 (0.48) 0.0 – 0.7 12.3 (2.40) 14.5 (0.87) 11.3 – 25.5 — — —

2 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.3 (0.25) 0.3 – 1.5 1.8 (0.48) 2.3 (1.11) 2.5 – 4.0

NCBD 0.3 (0.25)† 0.5 (0.29) 0.3 – 1.3 5.0 (1.08) 5.3 (1.65) 5.5 – 6.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NEYO 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5 0.3 (0.25)† 0.0 (0.00) 0.3 – 1.0 0.8 (0.75)† 0.5 (0.29) 0.0 – 1.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 7.3 (4.06) 4.3 (1.84) 2.5 – 11.5 — — —

3 IABG 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3 — — — 0.0 (0.00)† 0.3 (0.25) 0.0 – 1.0

NCBD 0.5 (0.29)† 0.5 (0.29) 0.0 – 0.8 4.5 (0.96) 3.0 (0.71) 2.8 – 4.5 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3

NEYO 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 2.5 (1.04)* 8.0 (1.73) 1.0 – 3.3

PAHM 2.0 (0.58)† 0.3 (0.25) 0.0 – 0.5 1.7 (0.67) 2.8 (1.38) 2.5 – 4.3 — — —

4 IABG — — — 0.0 (0.00)† 0.3 (0.25) 0.3 – 1.5 2.3 (0.85) 0.5 (0.29) 3.5 – 6.3

NCBD 0.5 (0.29)† 0.5 (0.29) 0.0 – 0.8 6.0 (1.47) 6.0 (0.82) 3.0 – 7.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NEYO — — — — — — 1.5 (0.65) 3.3 (1.97) 0.5 – 2.8

PAHM 1.3 (0.88)† 0.5 (0.29) 0.0 – 0.8 2.3 (1.20) 0.8 (0.25) 0.3 – 2.7 — — —

5 IABG — — — 0.3 (0.25) 0.5 (0.50) 0.3 – 2.8 5.3 (2.29) 6.5 (2.53) 5.5 – 9.3

NCBD 1.0 (0.41)† 1.0 (0.41) 0.3 – 1.8 2.0 (1.41) 1.5 (0.65) 0.5 – 3.5 — — —

NEYO 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5 — — — 3.0 (0.41)* 0.8 (0.48) 0.5 – 2.8

PAHM — — — 0.0 (0.00)† 0.5(0.50) 0.5 – 2.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3
Monsanto Company CR262-14U1 243 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials

Macro-parasitic hymenoptera Micro-parasitic hymenoptera Damsel bugs5(Nabidae)

Beneficial Arthropod Beneficial Arthropod Beneficial Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG 6.5 (1.19) 4.5 (1.04) 6.0 – 9.0 15.0 (2.12) 12.0 (3.58) 13.3 – 18.0 — — —

NCBD 0.5 (0.50)† 0.0 (0.00) 0.0 – 0.3 33.3 (1.93) 44.5 (1.32) 50.8 – 61.8 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8

NEYO 2.0 (0.91) 0.8 (0.25) 1.5 – 1.8 15.3 (2.14) 13.3 (2.06) 14.0 – 19.8 — — —

PAHM 0.7 (0.33)† 1.0 (0.41) 0.0 – 1.8 24.7 (2.33) 27.0 (4.80) 22.0 – 37.5 — — —

2 IABG 7.3 (3.20) 4.8 (0.85) 5.3 – 11.0 20.8 (1.44) 17.8 (4.11) 15.5 – 20.8 — — —

NCBD 0.8 (0.48)† 0.3 (0.25) 0.0 – 1.5 21.0 (6.47)* 8.3 (1.80) 14.3 – 25.8 — — —

NEYO 0.3 (0.25) 1.0 (0.58) 0.5 – 1.8 15.5 (2.60) 12.3 (2.50) 15.5 – 24.0 — — —

PAHM 1.0 (1.00) 1.3 (0.95) 1.0 – 2.3 19.3 (1.67) 25.5 (6.64) 16.0 – 29.8 — — —

3 IABG 3.8 (0.75) 3.3 (1.11) 3.3 – 5.3 11.3 (2.10) 10.5 (5.12) 12.8 – 15.5 — — —

NCBD — — — 18.8 (2.95) 19.8 (2.78) 13.5 – 26.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NEYO 2.3 (1.11) 2.3 (0.75) 1.8 – 3.0 15.5 (1.85) 13.5 (1.19) 12.5 – 25.3 — — —

PAHM 1.3 (0.33) 2.0 (0.41) 0.3 – 2.8 25.3 (5.49) 23.5 (6.22) 21.0 – 29.3 — — —

4 IABG 6.0 (0.91) 5.3 (0.95) 3.0 – 6.5 19.5 (1.50) 15.0 (7.75) 10.5 – 21.5 — — —

NCBD — — — 17.8 (1.49) 17.0 (0.91) 9.0 – 22.8 — — —

NEYO 1.8 (0.75) 3.8 (1.65) 2.0 – 2.8 12.3 (3.12) 12.5 (4.21) 10.5 – 12.0 — — —

PAHM 0.3 (0.33)* 3.3 (1.03) 1.5 – 2.3 51.0 (12.74) 32.0 (6.47) 27.0 – 47.5 — — —

5 IABG 2.0 (0.71) 2.5 (0.50) 1.5 – 2.3 5.8 (1.25) 9.0 (3.51) 7.5 – 10.5 — — —

NCBD 0.5 (0.29)† 0.3 (0.25) 0.0 – 0.5 14.5 (2.33) 13.5 (3.23) 10.0 – 15.5 — — —

NEYO 0.8 (0.48) 1.0 (0.41) 1.5 – 2.0 12.8 (4.70) 7.5 (1.26) 7.5 – 14.0 — — —

PAHM 2.0 (0.58) 3.5 (0.50) 3.5 – 5.0 37.0 (10.97) 29.3 (5.28) 32.5 – 41.0 — — —
Monsanto Company CR262-14U1 244 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials

Minute pirate bugs (Anthocoridae) Syrphid flies (Syrphidae) Aphids (Aphididae)

Beneficial Arthropod Beneficial Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG 0.8 (0.48)† 1.0 (0.41) 0.3 – 1.0 — — — 0.0 (0.00)† 0.0 (0.00) 0.3 – 1.5

NCBD — — — 1.3 (0.75) 1.5 (0.50) 1.3 – 4.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NEYO — — — 0.8 (0.48)† 0.8 (0.48) 0.3 – 1.0 — — —

PAHM 0.7 (0.67)† 0.5 (0.29) 0.0 – 2.3 4.0 (1.15) 6.8 (1.84) 3.5 – 6.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.0

2 IABG 1.0 (0.58) 2.0 (1.08) 1.3 – 3.5 — — — — — —

NCBD — — — 6.0 (1.00) 4.3 (1.31) 3.5 – 6.5 — — —

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 — — — — — —

PAHM 1.0 (0.58) 1.3 (0.48) 1.0 – 1.5 5.3 (2.33) 6.0 (2.74) 4.0 – 10.3 — — —

3 IABG 0.8 (0.25) 1.0 (0.41) 0.8 – 1.3 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.5 (0.29)† 0.3 (0.25) 0.3 – 1.0

NCBD — — — 11.5 (2.40) 7.5 (2.40) 9.3 – 16.3 — — —

NEYO — — — 0.5 (0.29) 0.5 (0.50) 0.8 – 2.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.5

PAHM 2.0 (0.58) 2.0 (0.71) 1.3 – 4.0 2.3 (1.86) 1.8 (0.48) 1.8 – 4.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

4 IABG 0.5 (0.29) 0.8 (0.25) 0.8 – 3.3 — — — 1.0 (0.71) 1.3 (0.95) 1.0 – 2.8

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 22.3 (5.95) 14.8 (2.78) 13.3 – 22.8 — — —

NEYO — — — 1.8 (0.75) 0.8 (0.75) 1.5 – 2.8 1.3 (0.25) 1.0 (0.71) 1.0 – 2.3

PAHM 0.3 (0.33)† 0.3 (0.25) 0.0 – 1.0 9.3 (2.96) 8.3 (1.38) 8.0 – 14.5 — — —

5 IABG 1.5 (0.65) 0.8 (0.48) 0.8 – 4.0 1.5 (0.96)† 0.5 (0.50) 0.5 – 1.5 1.3 (0.48) 3.5 (1.26) 2.0 – 2.5

NCBD 0.8 (0.25)† 0.3 (0.25) 0.3 – 0.8 72.5 (17.50) 49.0 (6.77) 62.5 – 102.8 0.5 (0.50)† 0.5 (0.29) 0.5 – 1.5

NEYO — — — 0.8 (0.48) 0.8 (0.25) 1.3 – 2.0 22.5 (3.28)* 13.8 (3.07) 14.5 – 32.0

PAHM 0.3 (0.33)† 0.8 (0.48) 0.0 – 1.3 6.7 (1.33) 4.0 (1.41) 5.0 – 12.0 — — —
Monsanto Company CR262-14U1 245 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials

Billbugs (Curculionidae) Corn flea beetles (Chaetocnema sp.) Corn rootworm beetles (Diabrotica sp.)

Pest Arthropod Pest Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG — — — — — — 2.3 (0.63) 1.5 (0.65) 0.8 – 1.8

NCBD — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 — — —

NEYO — — — 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.8 1.0 (0.41) 0.8 (0.48) 1.3 – 2.8

PAHM — — — 6.0 (1.73) 6.0 (2.16) 5.3 – 13.8 0.7 (0.33) 1.3 (1.25) 0.7 – 3.5

2 IABG — — — 1.5 (0.29) 2.8 (1.03) 2.5 – 5.0 2.8 (0.75) 1.8 (0.75) 0.5 – 2.8

NCBD 0.5 (0.50)† 0.5 (0.29) 0.5 – 1.3 — — — 0.3 (0.25)† 0.0 (0.00) 0.0 - 0.8

NEYO — — — 0.5 (0.29)† 0.8 (0.48) 0.5 – 1.0 9.3 (0.63) 9.8 (3.07) 7.8 – 13.8

PAHM — — — 7.7 (1.45) 4.3 (1.25) 5.0 – 19.3 1.0 (0.58)† 0.5 (0.29) 0.3 – 1.0

3 IABG — — — 0.0 (0.00)* 1.5 (0.65) 1.8 – 2.0 0.8 (0.25) 0.5 (0.50) 1.0 – 2.0

NCBD — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NEYO — — — — — — 23.0 (3.00)* 34.3 (3.75) 15.0 – 27.8

PAHM — — — 17.7 (1.76) 16.5 (2.72) 10.5 – 24.5 1.0 (0.00)† 1.0 (0.41) 0.0 – 2.0

4 IABG — — — 1.0 (0.71) 1.0 (0.71) 0.8 – 3.8 1.3 (0.63) 0.3 (0.25) 0.3 – 2.0

NCBD — — — 7.0 (2.27) 1.8 (0.48) 6.8 – 15.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NEYO — — — — — — 5.3 (1.03) 9.0 (2.48) 2.3 – 7.5

PAHM — — — 16.3 (2.19) 7.3 (1.93) 6.0 – 34.3 0.3 (0.33)† 1.0 (0.58) 0.0 – 1.0

5 IABG — — — 1.3 (0.25)* 0.3 (0.25) 2.0 – 3.8 0.5 (0.29)† 0.3 (0.25) 0.3 – 1.3

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 18.3 (2.84) 11.3 (3.12) 21.3 – 29.3 — — —

NEYO — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.3 – 0.5

PAHM — — — 6.3 (2.73) 4.3 (0.63) 4.0 – 11.0 — — —
Monsanto Company CR262-14U1 246 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials

Delphacid planthoppers (Delphacidae) Grasshoppers (Orthoptera) Leafhoppers (Cicadellidae)

Pest Arthropod Pest Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG — — — — — — 8.3 (0.63) 7.5 (1.89) 8.5 – 14.0

NCBD 24.0 (3.49) 28.8 (6.70) 27.3 – 41.0 — — — 43.5 (5.81) 50.0

(10.06) 43.0 – 70.5

NEYO — — — — — — 1.8 (0.48) 0.8 (0.75) 0.8 – 1.5

PAHM — — — — — — 16.3 (1.20) 12.8 (3.33) 14.0 – 33.5

2 IABG — — — — — — 8.8 (2.10) 10.3 (1.11) 7.5 – 11.3

NCBD — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 30.3 (4.82) 24.3 (2.46) 21.8 – 53.3

NEYO — — — — — — 1.0 (0.41)† 1.0 (0.41) 0.3 – 1.0

PAHM — — — 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.5 10.3 (3.71) 11.8 (2.02) 9.0 – 26.8

3 IABG — — — — — — 2.3 (0.63) 4.5 (0.50) 2.3 – 3.8

NCBD 2.8 (1.38) 1.3 (0.63) 0.5 – 2.3 — — — 46.5 (6.28) 37.3 (2.72) 35.8 – 59.3

NEYO — — — — — — 1.0 (0.71)† 1.3 (0.63) 0.0 – 1.5

PAHM — — — 0.3 (0.33)† 1.0 (0.41) 0.0 – 0.8 17.0 (4.58) 15.8 (4.39) 7.0 – 26.0

4 IABG — — — — — — 3.8 (0.75) 6.5 (1.04) 2.8 – 6.8

NCBD 1.3 (0.25)† 0.8 (0.48) 0.5 – 1.3 — — — 24.0 (2.12) 24.0 (2.58) 21.3 – 37.0

NEYO — — — — — — 1.3 (0.95)† 1.0 (0.41) 0.5 – 1.5

PAHM — — — 0.7 (0.67)† 0.0 (0.00) 0.0 – 1.0 12.3 (1.45) 13.5 (4.33) 6.5 – 26.5

5 IABG — — — — — — 4.5 (1.19) 2.5 (0.65) 2.0 – 6.0

NCBD — — — — — — 16.3 (2.66) 21.0 (3.11) 15.0 – 22.3

NEYO — — — — — — 0.8 (0.48) 0.5 (0.29) 0.0 – 2.3

PAHM — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 10.7 (2.85) 9.3 (2.56) 6.5 – 24.0
Monsanto Company CR262-14U1 247 of 260

Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials

Sap beetles (Nitidulidae) Seedcorn beetles (Stenolophus sp.) Tachinid flies (Tachinidae)

Pest Arthropod Pest Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG 0.0 (0.00)† 0.8 (0.48) 0.8 – 1.5 — — — — — —

NCBD — — — — — — — — —

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 — — — — — —

PAHM — — — — — — — — —

2 IABG 2.0 (0.71)† 0.5 (0.50) 0.0 – 1.0 — — — — — —

NCBD — — — — — — — — —

NEYO 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8 — — — 0.5 (0.50)† 0.0 (0.00) 0.0 – 0.3

PAHM — — — — — — — — —

3 IABG 1.0 (0.58)† 0.5 (0.29) 0.0 – 1.3 — — — — — —

NCBD — — — 0.3 (0.25)† 0.5 (0.50) 0.0 – 0.3 — — —

NEYO 1.0 (0.71) 0.5 (0.29) 0.5 – 1.5 — — — — — —

PAHM — — — — — — — — —

4 IABG 2.0 (1.68) 1.5 (0.87) 0.8 – 2.3 — — — — — —

NCBD — — — — — — — — —

NEYO 0.8 (0.48) 1.0 (0.00) 1.0 – 3.0 — — — — — —

PAHM — — — — — — — — —

5 IABG 0.5 (0.50) 0.3 (0.25) 1.0 – 1.5 — — — — — —

NCBD — — — — — — — — —

NEYO 1.3 (0.75) 2.5 (1.04) 0.3 – 2.0 — — — — — —

PAHM — — — — — — — — —
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Table H-9 (continued). Individual Site Analysis: Arthropod Abundance in Sticky Trap Samples Collected from MON 87403

Compared to the Conventional Control in 2012 U.S. Field Trials

Thrips (Thripidae) Tarnished plant bugs (Lygus sp.) Click beetle5(Elateridae)

Pest Arthropod Pest Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG 31.5 (11.59) 41.5 (7.79) 26.8 – 50.8 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.5 — — —

NCBD 50.5 (10.90) 40.5 (5.01) 37.5 – 76.3 — — — 2.0 (0.71) 3.8 (1.11) 2.0 – 5.0

NEYO 4.8 (2.25) 1.0 (0.58) 4.0 – 8.0 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.0 — — —

PAHM 12.0 (5.77) 8.8 (5.01) 9.5 – 13.5 1.7 (0.88) 0.0 (0.00) 0.8 – 2.5 — — —

2 IABG 86.3 (23.97) 79.3 (27.29) 40.3 – 95.5 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3 — — —

NCBD 32.5 (5.85)* 18.5 (4.03) 20.8 – 34.5 — — — 3.0 (0.41) 2.8 (1.31) 3.0 – 7.5

NEYO 183.0 (54.68)* 71.5 (13.76) 66.8 – 159.0 — — — — — —

PAHM 1.3 (0.88) 2.5 (1.04) 1.8 – 6.7 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.5 — — —

3 IABG 38.5 (4.50) 39.0 (18.18) 24.0 – 130.0 — — — — — —

NCBD 39.3 (6.30) 29.8 (6.17) 46.0 – 59.3 — — — 2.0 (0.71) 0.8 (0.25) 2.0 – 3.0

NEYO 319.3 (102.23) 207.3

(58.63)

171.8 –

511.0 — — — — — —

PAHM 9.7 (4.26)* 2.8 (2.14) 2.3 – 4.8 — — — — — —

4 IABG 95.5 (6.96) 139.3

(82.00) 44.8 – 167.8 — — — — — —

NCBD 33.8 (5.27) 26.5 (2.90) 27.8 – 85.8 — — — 0.8 (0.25) 0.8 (0.48) 0.8 – 3.3

NEYO 134.3 (36.50)* 60.3 (14.02) 56.3 – 125.0 — — — — — —

PAHM 11.0 (5.29) 7.5 (3.30) 6.0 – 12.3 — — — 0.0 (0.00)† 0.8 (0.48) 0.0 – 0.5

5 IABG 30.0 (14.57) 15.3 (2.87) 17.8 – 41.5 — — — — — —

NCBD 33.3 (5.50) 44.3 (15.58) 18.5 – 42.8 — — — 1.3 (0.25) 0.8 (0.25) 1.5 – 2.3

NEYO 138.0 (10.12)* 62.3 (15.52) 102.3 –

173.8 — — — — — —

PAHM 1.7 (0.88) 1.3 (0.48) 0.7 – 3.5 0.7 (0.67)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5
Monsanto Company CR262-14U1 249 of 260

Note: The experimental design was a randomized complete block with four replicates per site.

*

Indicates a statistically significant difference between MON 87403 and the conventional control (α = 0.05) using ANOVA.

†

Indicates p-values could not be generated where the taxa did not meet inclusion criteria. Among taxa that did not meet inclusion criteria, centipedes, millipedes, and corn

earworms data are not presented due to low probability of capture on sticky traps.

(—)A dash indicates data not available.

1 Arthropods were enumerated at five crop development stages: Collection 1 = late vegetative – VT growth stage; Collection 2 = R1 growth stage; Collection 3 = R2 growth stage;

Collection 4 = R3 growth stage; Collection 5 = R4 growth stage.

2

Site code: IABG = Greene, IA; NCBD = Perquimans, NC; NEYO = York, NE; PAHM = Berks, PA.

3 MON 87403 and the conventional control values represent means with standard error in parentheses. N = 4 (IABG, NCBD, and NEYO sites); N= 3 (PAHM site).

4 Reference range is calculated from the minimum and maximum mean values from among reference materials at each site.

5 Click beetle = wireworm adults; Damsel bug = Nabis bug
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Table H-10. Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the

Conventional Control in 2012 U.S. Field Trials

Ant-like flower beetles (Anthicidae) Click beetles (Elateridae) Corn flea beetles (Chaetocnema sp.)

Pollen Feeder Pest Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NCBD — — — 1.0 (0.71) 2.3 (1.31) 2.0 – 4.3 0.5 (0.29)† 0.0 (0.00) 0.3 – 2.0

NEYO — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM — — — — — — 8.7 (3.53) 6.5 (2.40) 3.0 – 8.5

2 IABG 1.0 (0.41) 1.8 (0.75) 1.3 – 5.0 — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NCBD — — — 1.5 (0.87) 0.5 (0.29) 0.5 – 2.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 1.0

NEYO — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM — — — — — — 9.3 (2.73) 9.3 (3.64) 6.5 – 9.5

3 IABG 2.0 (1.00) 2.3 (1.03) 1.8 – 4.3 — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NCBD — — — 0.8 (0.25)† 0.3 (0.25) 0.3 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NEYO — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM — — — — — — 6.7 (4.06) 6.3 (1.65) 8.8 – 11.0

4 IABG 3.8 (0.85) 3.5 (0.87) 3.5 – 4.5 — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NCBD — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 0.5 (0.29)† 0.0 (0.00) 0.0 – 1.0

NEYO — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM — — — — — — 3.3 (0.88) 3.3 (1.11) 4.0 – 8.3

5 IABG 1.0 (0.71)† 0.8 (0.48) 0.5 – 1.3 — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NCBD — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 1.0 (0.71) 0.5 (0.29) 0.5 – 3.0

NEYO — — — — — — 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM — — — — — — 3.3 (1.33) 3.8 (2.25) 3.7 – 4.8
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Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the

Conventional Control in 2012 U.S. Field Trials

Japanese beetles (Scarabaiedae) Lacewing adults (Chrysopidae) Lacewing larvae (Chrysopidae)

Pest Arthropod Beneficial Arthropod Beneficial Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.5 (0.29)† 0.5 (0.29) 0.3 – 0.5

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.5 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3

2 IABG 0.8 (0.75)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 1.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 1.0 (0.41) 0.5 – 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.5 0.3 (0.33)† 0.0 (0.00) 0.0 – 0.7

3 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8

NCBD 0.5 (0.50)† 0.0 (0.00) 0.0 – 0.5 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3 0.5 (0.50)† 0.5 (0.29) 0.0 – 0.3

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.5 (0.50) 0.0 – 0.3

4 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.5 (0.29)† 0.3 (0.25) 0.0 – 0.5

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.0 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.3

5 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.3 – 1.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0
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Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the

Conventional Control in 2012 U.S. Field Trials

Ladybird beetles adult (Coccinellidae) Ladybird beetles Larvae (Coccinellidae) Minute pirate bugs (Anthocoridae)

Beneficial Arthropod Beneficial Arthropod Beneficial Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.5

NCBD 0.0 (0.00)† 0.5 (0.50) 0.3 – 1.3 0.0 (0.00)† 0.5 (0.29) 0.3 – 1.3 2.3 (1.03) 2.5 (1.32) 1.8 – 4.8

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 1.0 (0.58) 2.5 (1.32) 2.3 – 6.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 6.7 (1.67) 9.5 (2.40) 8.5 – 11.7

2 IABG 0.3 (0.25)† 0.5 (0.29) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 6.3 (1.65) 4.5 (0.96) 3.0 – 11.0

NCBD 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.8 1.3 (0.75) 2.0 (0.58) 0.5 – 2.3 1.0 (0.41) 1.8 (0.63) 0.8 – 3.8

NEYO 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 2.7 (0.33) 3.5 (1.76) 0.8 – 3.7 0.3 (0.33)† 0.8 (0.48) 0.0 – 0.5 5.7 (2.03) 6.0 (2.20) 5.3 – 8.0

3 IABG 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.8 (0.75) 2.5 (0.96) 2.0 – 4.0

NCBD 0.5 (0.29)† 0.3 (0.25) 0.0 – 1.0 1.3 (0.95)† 0.8 (0.25) 0.5 – 1.0 1.0 (1.00)† 0.3 (0.25) 0.3 – 1.8

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 0.5 (0.50) 0.0 – 1.3 1.7 (0.33) 1.8 (0.25) 1.3 – 2.0 8.7 (4.67) 5.8 (1.55) 6.3 – 11.5

4 IABG 0.5 (0.29)† 0.3 (0.25) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 2.3 (1.44) 2.5 (1.55) 1.5 – 3.3

NCBD 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.3 0.5 (0.50)† 0.5 (0.50) 0.5 – 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.8 0.7 (0.67)† 2.0 (0.71) 0.3 – 1.5 0.0 (0.00)* 1.0 (0.41) 0.5 – 3.7

5 IABG 0.0 (0.00)† 0.3 (0.25) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.5 (0.87) 1.0 (0.58) 0.8 – 2.5

NCBD 0.0 (0.00)† 0.5 (0.50) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.5 (0.29) 0.0 – 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.3 (0.33)† 0.3 (0.25) 0.0 – 0.5 0.7 (0.33)† 0.8 (0.48) 0.5 – 1.0 1.0 (0.58) 1.8 (0.85) 1.3 – 5.5
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Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the

Conventional Control in 2012 U.S. Field Trials

Corn rootworm beetles (Chrysomelidae) Sap beetles (Nitidulidae) Shining flower beetles (Phlacridae)

Pest Arthropod Pest Arthropod Pollen Feeder

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control MON 87403 Control

1 IABG — — — 0.5 (0.29)† 0.3 (0.25) 0.3 – 0.8 — — —

NCBD 1.0 (0.58) 1.3 (0.75) 1.0 – 2.0 4.3 (3.92) 4.5 (2.87) 2.5 – 5.3 5.0 (0.91) 6.0 (0.71) 3.8 – 6.8

NEYO 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.5 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 — — —

PAHM 0.3 (0.33)* 2.0 (0.71) 1.0 – 2.0 4.3 (0.67) 8.0 (1.08) 1.8 – 8.5 1.3 (0.88) 3.5 (1.55) 1.7 – 3.0

2 IABG 3.0 (1.29) 2.5 (0.65) 1.5 – 6.0 4.5 (0.96) 9.5 (3.97) 8.8 – 19.0 — — —

NCBD 0.3 (0.25)† 0.5 (0.50) 0.0 – 1.0 6.3 (5.27) 7.3 (3.59) 4.3 – 9.8 7.0 (3.19)* 12.3 (1.70) 6.8 – 10.5

NEYO 1.0 (0.41) 1.5 (0.65) 1.3 – 2.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 — — —

PAHM 0.3 (0.33) 1.3 (0.63) 0.7 – 1.8 2.7 (0.33) 4.3 (0.75) 2.5 – 6.0 2.3 (0.33) 1.8 (0.75) 1.0 – 2.0

3 IABG 0.5 (0.29)† 1.0 (0.41) 0.5 – 1.3 4.8 (1.03) 2.8 (0.85) 3.5 – 7.5 — — —

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.5 (0.87) 2.5 (1.19) 1.5 – 3.8 1.5 (0.65) 1.0 (0.00) 1.0 – 3.0

NEYO 3.8 (1.75) 6.8 (1.03) 1.8 – 6.0 1.0 (0.00)* 4.5 (0.96) 0.3 – 1.3 — — —

PAHM 1.0 (0.58)† 0.0 (0.00) 0.0 – 1.0 2.3 (2.33) 1.0 (1.00) 1.0 – 3.3 2.0 (1.53) 1.0 (0.41) 0.3 – 2.7

4 IABG 1.3 (0.95) 0.8 (0.48) 0.8 – 2.3 3.3 (1.38) 7.5 (1.89) 4.3 – 9.0 — — —

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.3 (0.25)† 0.3 (0.25) 0.3 – 1.3 0.8 (0.25)† 0.0 (0.00) 0.0 – 2.0

NEYO 0.8 (0.25)† 1.3 (0.48) 0.3 – 1.0 2.0 (1.08) 0.8 (0.48) 0.3 – 2.3 — — —

PAHM 0.0 (0.00)† 0.8 (0.48) 0.0 – 0.5 1.3 (0.88) 1.8 (0.75) 1.5 – 4.0 0.0 (0.00)† 0.5 (0.29) 0.0 – 1.3

5 IABG 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 5.3 (2.72) 5.0 (1.78) 1.8 – 5.8 — — —

NCBD 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 1.3 (0.48)* 4.5 (1.19) 1.0 – 5.5 0.8 (0.48)† 0.3 (0.25) 0.0 – 0.8

NEYO — — — 0.0 (0.00)† 1.0 (0.71) 0.0 – 1.0 — — —

PAHM 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 1.3 (0.67) 1.5 (0.65) 1.5 – 4.8 1.0 (1.00)† 0.5 (0.29) 0.0 – 0.3
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Table H-10 (continued). Individual Site Analysis: Arthropod Abundance in Visual Counts from MON 87403 Compared to the

Conventional Control in 2012 U.S. Field Trials

Spiders (Araneae) Stink bugs (Pentatomidae)

Beneficial Arthropod Pest Arthropod

Mean (S.E.)3

Reference

range4

Mean (S.E.)3

Reference

range4

Coll.1Site2 MON 87403 Control MON 87403 Control

1 IABG 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NCBD 4.8 (1.25) 3.0 (0.91) 3.5 – 4.5 0.8 (0.48)† 1.3 (0.48) 0.0 – 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.0

PAHM 0.7 (0.33) 1.5 (0.50) 0.0 – 2.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

2 IABG 1.3 (0.75)† 0.0 (0.00) 0.0 – 1.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NCBD 3.8 (0.75) 2.8 (1.18) 3.5 – 5.0 1.5 (0.50)† 0.5 (0.29) 0.3 – 1.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 1.7 (0.88) 2.3 (1.44) 0.7 – 2.0 0.3 (0.33)† 0.0 (0.00) 0.0 – 0.3

3 IABG 1.5 (0.50) 0.8 (0.25) 0.8 – 1.0 0.3 (0.25)† 0.3 (0.25) 0.3 – 0.5

NCBD 3.0 (1.22) 3.8 (1.11) 4.3 – 6.0 0.3 (0.25)† 0.3 (0.25) 0.0 – 0.5

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.8 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

PAHM 4.0 (1.53) 3.5 (2.22) 2.5 – 3.7 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

4 IABG 0.5 (0.29) 0.8 (0.25) 0.8 – 2.3 1.0 (0.71)† 0.5 (0.29) 0.0 – 1.0

NCBD 2.5 (0.65) 3.0 (1.29) 2.8 – 5.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 2.0 (0.58) 1.5 (0.65) 1.0 – 3.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.3

5 IABG 0.3 (0.25)† 0.0 (0.00) 0.3 – 0.3 0.3 (0.25)† 0.0 (0.00) 0.0 – 0.3

NCBD 3.0 (0.71) 2.3 (0.48) 2.0 – 3.3 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

NEYO 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.0

PAHM 2.7 (0.33) 2.8 (0.75) 1.5 – 5.0 0.0 (0.00)† 0.0 (0.00) 0.0 – 0.5
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Note: The experimental design was a randomized complete block with four replicates per site.

*

Indicates a statistically significant difference between MON 87403 and the conventional control (α = 0.05) using ANOVA.

†

Indicates p-values could not be generated where the taxa did not meet inclusion criteria

(—) A dash indicates data not available.

1 Arthropods were enumerated at five crop development stages: Collection 1 = VT-R1 growth stage; Collection 2 = R1 growth stage; Collection 3 = R2 growth stage; Collection 4

= R3 growth stage; Collection 5 = R4-R5 growth stage.

2

Site code: IABG = Greene, IA; NCBD = Perquimans, NC; NEYO = York, NE; PAHM = Berks, PA.

3 MON 87403 and the conventional control values represent means with standard error in parentheses. N = 4.

4 Reference range is calculated from the minimum and maximum mean values from among reference materials at each site.
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References for Appendix H

SAS Software Release 9.3 (TS1M0). 2010. Copyright 2002-2010 by SAS Institute, Inc.,

Cary, North Carolina.
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Appendix I: Materials and Methods for Pollen Morphology and Viability

Assessment

I.1. Plant Production

MON 87403, the conventional control, and reference hybrids were grown under similar

agronomic conditions in a field trial in Story County, Iowa (Table I-1). The trial was

arranged in a randomized complete block design with four replications. Each plot

consisted of eight rows approximately 6 m in length.

I.2. Flower Collection and Pollen Sample Preparation

Tassel bags were placed on three non-systematically selected plants during pollen shed.

The following morning, pollen was collected from three plants per plot and transferred to

a uniquely labeled tube. Pollen collected from each plant in a plot represented a

subsample. Within approximately 30 minutes of collection, Alexander’s stain solution

(Alexander, 1980), in a 1:5 dilution with distilled water, was added to each tube (at least

2:1 (v/v) stain to pollen) to fix and stain the pollen, rendering the pollen non-viable. The

tubes were closed and the contents shaken until thoroughly mixed. Subsamples were

placed on wet ice immediately after pollen collection and maintained under those

conditions until receipt at the performing laboratory.

I.3. Data Collection

Slides were prepared by aliquoting suspended pollen/stain solution onto a slide. Pollen

characteristics were assessed under an Olympus©9 BX53 light microscope equipped with

an Olympus© DP72 digital color camera. The microscope and camera were connected to

a computer running Microsoft Windows XP®10 and installed with an Olympus©cellSens

(version 1.4.1) software.

I.3.1. Pollen Viability

When pollen grains were exposed to the staining solution, viable pollen grains stained red

to purple due to the presence of living cytoplasmic content. Non-viable pollen grains

stained light blue to green or colorless, and the shape appeared round to collapsed

depending on the degree of hydration. For each pollen sample, the number of viable and

non-viable pollen grains was counted from a random field of view under the microscope.

A minimum of 100 pollen grains were counted for each of the three subsamples per plot.

Mean pollen viability for each replicate was calculated from the subsamples as shown in

Table VII-8.

 Olympus Corporation.

 Windows XP is a registered trademark of Microsoft Corporation.
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I.3.2. Pollen Diameter

For a single predetermined subsample per plot, pollen grain diameter was measured along

two perpendicular axes for 10 representative pollen grains per replication. Mean pollen

diameter for each replicate was calculated from the total of 20 diameter measurements as

shown in Table VII-8.

I.3.3. General Pollen Morphology

General pollen morphology of MON 87403, the conventional control, and the reference

hybrids was observed Figure I-1.

I.4. Statistical Analysis

An analysis of variance was conducted according to a randomized complete block design

using SAS©(SAS, 2012). The level of statistical significance was predetermined to be

5% (α=0.05). MON 87403 was compared to the conventional control material for

percent viable pollen and pollen grain diameter. MON 87403 and conventional control

were not statistically compared to the reference hybrids. Minimum and maximum mean

values were calculated for each characteristic from the four reference hybrids. General

pollen morphology was qualitative; therefore, no statistical analysis was conducted on

these observations.

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

Material Name Material Type Phenotype Monsanto Lot Number

MPA640B Control Conventional 11332601

Lewis 7007 Reference Conventional 11226559

Mycogen 2M746 Reference Conventional 11226705

NC+ 5220 Reference Conventional 11226701

Phillips 717 Reference Conventional 11300073

MON 87403 Test Increased Ear Biomass

Maize 11332602
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Figure I-1. General Morphology of Pollen from MON 87403, the Conventional

Control, and Commercially Available Conventional Reference Materials under

200X Magnification

The maize pollen samples were stained with Alexander’s stain diluted 1:5 with distilled

water. Viable pollen grains stained red to purple, while non-viable pollen grains stained

blue to green and the shape appeared round to collapsed depending on the degree of

hydration.
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References for Appendix I

Alexander, M.P. 1980. A versatile stain for pollen fungi, yeast and bacteria. Stain

Technology 55: 13-18.

SAS. 2012. SAS/STAT software version 9.3. SAS Institute, Inc., Cary, North Carolina.