GENECTIVE SA Maize event VCO-Ø1981-5 

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COMPANY NAMES 

GENECTIVE S.A., an affiliate of KWS and VCO Participation, an affiliate of Vilmorin & 

Cie, is a company organized and existing under the laws of France, having its head 

office at 1 rue Limagrain, 63720 Chappes, France, SIREN Number 513 533 612, RCS 

Clermont-Ferrand. 

On July 1, 2008, Athenix Corp. and Vilmorin & Cie entered into an agreement for the 

development of maize tolerant to glyphosate and resistant to insects. Under this 

agreement, Athenix Corp. has licensed to Vilmorin & Cie the right to use worldwide the 

genes they discover and own for the above traits. On June 23, 2011 Vilmorin & Cie has 

transferred the above agreement to its affiliate GENECTIVE S.A. 

On November 2, 2009 Athenix Corp., a biotechnology company that develops novel 

products and technology for agricultural applications, became an affiliate of Bayer 

CropScience LP. 

GENECTIVE S.A. and Bayer CropScience are continuing the collaboration initiated in 

2008 between Vilmorin & Cie and Athenix Corp. Some of the activities described in this 

petition were undertaken before the acquisition of Athenix Corp. by Bayer CropScience 

LP and based on the agreement between Athenix Corp. and Vilmorin & Cie. 

 
GENECTIVE SA Maize event VCO-Ø1981-5 

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RELEASE OF INFORMATION 

The information in this petition is being submitted by GENECTIVE S.A. for review by the 

USDA as part of the regulatory process. By submitting this information, GENECTIVE 

S.A. does not authorize its release to any third party except to the extent it is requested 

under the Freedom of Information Act (FOIA), 5 U.S.C., Section 552 and 7 CFR 1, 

covering all or some of this information. Prior to releasing the information in this petition 

in accordance with FOIA, GENECTIVE S.A. kindly requests to be notified by the 

Agency’s FOIA office. GENECTIVE S.A. does not authorize the release, publication or 

other distribution of this information without GENECTIVE S.A.’s prior notice and consent. 

©2011 GENECTIVE S.A. All rights reserved. 

This document is property of GENECTIVE S.A. and is for use by the regulatory authority to which 

it has been submitted, and only in support of actions requested by GENECTIVE S.A. All data and 

information herein must not be used, reproduced or disclosed to third parties without the written 

consent of GENECTIVE S.A. 

GENECTIVE SA Maize event VCO-Ø1981-5 

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SUMMARY

GENECTIVE S.A. is submitting a Petition for the Determination of Non-regulated Status 

under 7 CFR 340 to USDA Animal and Plant Health Inspection Service (APHIS) for 

glyphosate-tolerant maize event VCO-Ø1981-5, any progeny, and crosses of this event 

with other non-regulated maize lines. 

Transformation event VCO-Ø1981-5 contains the stably integrated epsps grg23ace5 

gene expressing the EPSPS ACE5 protein, an improved EPSPS enzyme, which confers 

tolerance to the herbicide glyphosate. The gene was introduced into maize through 

Agrobacterium-mediated transformation. Southern blot analyses show maize event 

VCO-Ø1981-5 contains a single intact copy of the epsps grg23ace5 gene. 

The EPSPS ACE5 protein was derived using a directed evolution protein engineering 

strategy from the native GRG23 protein isolated from Arthrobacter globiformis. A total of 

ten amino acids were changed to produce EPSPS ACE5. The EPSPS ACE5 protein 

exhibits tolerance to glyphosate. The native GRG23 enzyme from Arthrobacter was 

optimized to more closely match the native maize EPSPS enzyme with regards to its 

temperature optimum activity under typical field-growing conditions. The native maize 

EPSPS enzymatic half-life is 86 hours while that of the native EPSPS GRG23 enzyme 

from Arthrobacter is 10 hours at 37oC. The modified EPSPS ACE5 has an improved 

enzymatic half-life of 65 hours, bringing it closer to that of the native maize EPSPS and 

optimizing it for typical field-growing conditions. 

The EPSPS ACE5 protein has a molecular weight of 45 kDa and is made of up of 413 

amino acids. The EPSPS ACE5 protein does not share any characteristics with 

allergens or toxins. 

Planting herbicide-tolerant maize varieties, containing event VCO-Ø1981-5, will provide 

growers with another option for weed control using glyphosate. Glyphosate is widely 

used in herbicide-tolerant maize and other agricultural production systems. In many 

instances, glyphosate replaces more harmful herbicides and simplifies weed control 

practices since the grower can rely on a single herbicide only when weed control is 

required. Herbicide-tolerant crops have allowed a rapid adoption of reduced tillage 

practices. Low-till and no-till farming is quickly dominating agricultural systems, since it 

conserves valuable topsoil, reduces fuel costs to the grower and reduces the amount of 

greenhouse gases in the environment. 

Event VCO-Ø1981-5 has been field tested since 2007 in typical maize growing regions 

of the continental US and Puerto Rico. These tests have occurred at more than 20 

locations under USDA APHIS environmental release authorizations. Data and results 

collected from these trials as well as laboratory analyses presented in this petition 

demonstrate that event VCO-Ø1981-5 maize: 

 exhibits no plant pathogenic properties; 

 is no more likely to become a weed than other maize varieties currently grown; 

 is unlikely to increase the weediness potential of any other cultivated plant or 

native wild species; 

 does not cause damage to processed agricultural commodities; and, 

 is unlikely to harm other organisms that are beneficial to agriculture. 
GENECTIVE SA Maize event VCO-Ø1981-5 

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ACRONYMS AND SCIENTIFIC TERMS 

 

ai active ingredient 

A acre 

AA amino acid 

ADF Acid Detergent Fiber 

ANOVA Analysis Of Variance 

APHIS Animal and Plant Health Inspection 

Service 

BC0N, BC1N, etc. Back cross 0,1, etc. with recurrent line 

N 

BCS Bayer CropScience 

BLASTP Basic Local Alignment Search Tool 

Protein 

bp base pairs 

bu/ac bushels/acre 

dw Dry weight 

DNA DeoxyriboNucleic Acid 

E. coli Escherichia coli 

ELISA Enzyme Linked Immuno Sorbent 

Assay 

EPSPS 5-enolpyruvylshikimate-3-phosphate 

synthase 

FAO Food and Agriculture Organization of 

the United Nations 

FDA Food and Drug Administration 

FGENESH Find GENES using Hidden Markov 

model 

FIFRA Federal Insecticide Fungicide and 

Rodenticide Act 

fw Fresh weight 

g gram 

GM Genetically Modified 

GLY glyphosate 

HRP Horseradish Peroxidase 

ID identification 

kDa kiloDalton 

kg kilogram 

L liter 

LB Left Border 

lb pound (1 pound = 0.454 kg) 

LC/MS Liquid Chromatography/Mass 

Spectroscopy 

LD50 lethal dose for 50% of animals 

LOQ Limit of Quantitation 

LOD Limit of Detection 

M million 

MALDI-TOF Matrix-Assisted Laser 

 Desorption/Ionization Time of Flight 

mg milligram 

mL milliliter 

μg microgram 

NA Not Applicable 

n/a not available 

ng nanogram 

ND Not Detectable: Below the limit of 

detection 

NDF Neutral Detergent Fiber 

nm nanometer 

nt nucleotide 

OECD Organization for Economic 

Cooperation and Development 

ORF Open Reading Frame 

PCR Polymerase Chain Reaction 

RB Right Border 

RCB Randomized Complete Block 

RM Relative Maturity 

SD Standard Deviation 

SDS-PAGE Sodium Dodecyl Sulfate 

PolyAcrylamide Gel Electrophoresis 

SGF Simulated Gastric Fluid 

ssp. Subspecies 

T0 Regenerated transformed plant 

T-DNA Transfer DNA from Agrobacterium

US United States of America 

USDA United States Department of 

Agriculture 

WHO World Health Organization 

wt Wild type 

Z. mays Zea mays, maize
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TABLE OF CONTENTS 

 

 Page

TITLE PAGE .................................................................................................................................................. 1

COMPANY NAMES ....................................................................................................................................... 2

RELEASE OF INFORMATION ...................................................................................................................... 3

CERTIFICATION ........................................................................................................................................... 4

SUMMARY .................................................................................................................................................... 5

ACRONYMS AND SCIENTIFIC TERMS....................................................................................................... 6

TABLE OF CONTENTS ................................................................................................................................ 7

LIST OF TABLES ........................................................................................................................................ 11

LIST OF FIGURES ...................................................................................................................................... 13

I. RATIONALE 

I.A. Basis for the request for determination of non-regulated status ............................... 14 

I.B. Herbicide-tolerant maize event VCO-Ø1981-5 ......................................................... 14 

I.C. Rationale for the development of event VCO-Ø1981-5 and benefits ....................... 14 

I.C.1. Glyphosate-tolerant crops ............................................................................... 14 

I.C.2. Glyphosate-tolerant event VCO-Ø1981-5 maize ............................................ 15 

I.D. Adoption of event VCO-Ø1981-5 .............................................................................. 16 

I.E. Submissions to other regulatory agencies ................................................................. 17 

II. THE BIOLOGY OF MAIZE 

II.A. Biology of maize ........................................................................................................ 18 

II.B. Characteristics of the recipient cultivar ..................................................................... 18 

III. DEVELOPMENT OF HERBICIDE-TOLERANT MAIZE EVENT VCO-Ø1981-5 

III.A. Description of the transformation system ................................................................ 19 

III.B. Parent line ................................................................................................................ 20 

III.C. Breeding diagram ..................................................................................................... 20 

III.D. Generations used for analysis ................................................................................. 20 

IV. GENETIC MATERIAL USED FOR TRANSFORMATION OF EVENT VCO-Ø1981-5 

IV.A. Construction of the plasmid used for transformation .............................................. 23 

IV.B. Description of gene and regulatory regions ............................................................ 24 

 IV.C. Identity and source of the genetic material ............................................................. 24 

V. GENETIC CHARACTERIZATION OF EVENT VCO-Ø1981-5 

V.A. Overview ................................................................................................................... 26 

V.B. Copy number and insertion ....................................................................................... 26 

V.C. Absence of vector backbone .................................................................................... 27 

V.D. The flanking regions of the inserted sequence ....................................................... 27 

V.E. Mendelian inheritance ............................................................................................... 28 

V.F. Stability across generations ...................................................................................... 29 

V.G. Conclusion ................................................................................................................ 30 
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Page 

VI. CHARACTERIZATION OF THE INTRODUCED PROTEIN 

VI.A.The EPSPS ACE5 protein ........................................................................................ 31 

VI.A.1. History and background ............................................................................... 31 

VI.A.2. Characterization of the EPSPS ACE5 protein............................................. 32 

VI.A.2.1. Biochemistry and mode of action ................................................. 32 

VI.A.2.2. History of safe use ........................................................................ 34 

VI.B. Expression of the EPSPS ACE5 protein in event VCO-Ø1981-5 .......................... 36 

VI.C. Verification of biochemical and functional equivalence .......................................... 38 

 VI.D. Summary of the food and feed safety assessment of the EPSPS ACE5 

protein ...................................................................................................................... 38 

VI.D.1. Homology search to known allergens and toxins ....................................... 38 

VI.D.2. Potential N-glycosylation sites ..................................................................... 39 

VI.D.3. In vitro stability to human simulated gastric fluid ........................................ 40 

VI.D.4. Acute toxicity study in the mouse ................................................................ 40 

 VI.E. Conclusion ............................................................................................................... 41 

VII. AGRONOMIC AND PHENOTYPIC EVALUATION 

VII.A. History of field activities ......................................................................................... 42 

VII.B. Agronomic and phenotypic evaluation ................................................................... 43 

VII.C. Agronomic performance of event VCO-Ø1981-5 .................................................. 44 

VII.C.1. Agronomic evaluation of event VCO-Ø1981-5 maize in 2008 ................... 44 

VII.C.2. Agronomic evaluation of event VCO-Ø1981-5 maize in 2009 ................... 44 

VII.C.2.1. Evaluation parameters, analysis and statistics ........................... 45 

VII.C.2.2. Field locations, preparations and conditions ............................... 47 

VII.C.2.3. Results of agronomic performance evaluation ........................... 47 

 VII.D. Biotic and abiotic stress characteristics ................................................................. 50 

VII.E. Seed dormancy evaluation ..................................................................................... 52 

VII.F. Composition analysis .............................................................................................. 53 

VII.F.1. Introduction.................................................................................................. 53 

VII.F.2. Seed genetics, field trial design and sampling ........................................... 54 

VII.F.3. Analytical methods ...................................................................................... 54 

VII.F.4. Nutritional composition ................................................................................ 54 

VII.F.4.1. Overview of the analysis .............................................................. 54 

VII.F.4.2. Analysis of proximates and minerals in maize forage ................. 55 

VII.F.4.3. Analysis of proximates in maize grain ......................................... 56 

VII.F.4.4 Analysis of fatty acids in maize grain........................................... 57 

VII.F.4.5. Analysis of amino acids in maize grain ....................................... 59 

VII.F.4.6. Analysis of vitamins in maize grain .............................................. 61 

VII.F.4.7. Analysis of minerals in maize grain ............................................. 63 

VII.F.4.8. Analysis of secondary metabolites and anti-nutrients 

 in maize grain ............................................................................... 64 

 VII.G. Conclusion .............................................................................................................. 65 
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Page 

VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTICES 

VIII.A. Environmental assessment of the introduced protein ........................................... 66 

VIII.B. Potential for gene transfer ..................................................................................... 66 

VIII.B.1. Vertical gene flow ....................................................................................... 66 

VIII.B.2. Horizontal gene flow .................................................................................. 67 

 VIII.C. Weediness potential of maize event VCO-Ø1981-5 ............................................ 68 

 VIII.D. Current agronomic practices for maize ................................................................. 68 

VIII.E. Potential impact of introduction on current agricultural practices ......................... 70 

VIII.F. Weed resistance management ............................................................................. 71 

VIII.G. Potential impact on organic or conventional farming ............................................ 72 

VIII.H. Potential impacts on raw or processed agricultural products ............................... 72 

VIII.I. Potential effects on non-target organisms .............................................................. 73 

VIII.J Potential impact on biodiversity ............................................................................... 73 

VIII.K. Conclusion ............................................................................................................. 74 

IX. STATEMENT OF GROUNDS UNFAVORABLE ............................................................. 76 

X. REFERENCES ................................................................................................................. 77 

Appendix 1.FIELD TRIAL TERMINATION REPORTS 2007-2011 ............................................ 84 

Appendix 2. MATERIALS AND METHODS 

2.A. Materials and methods for molecular characterization ..................................................... 116 

2.B. Materials and methods for protein characterization tests ................................................. 119 

2.C. Materials and methods for protein levels in plant parts and during the life cycle ............. 123 

2.D. Materials and methods for agronomic studies .................................................................. 126 

2.E. Materials and methods for seed germination studies ....................................................... 129 

2.F. Materials and methods for composition analysis .............................................................. 129 

Appendix 3. CHARACTERIZATION OF EVENT VCO-Ø1981-5 MAIZE 

3.A. Insert characterization ....................................................................................................... 134 

 3.B. Insert stability across generations ..................................................................................... 139 

 3.C. Confirmation of the absence of vector backbone ............................................................. 140 

 3.D. In vitro enzyme assay ........................................................................................................ 143 

 3.E. Protein equivalency ........................................................................................................... 145 

 3.E.1. SDS-PAGE of EPSPS ACE5 ..................................................................................... 145 

 3.E.2. MALDI-TOF mass spectrophotometry protein identification ..................................... 146 

 3.E.3. Western blot analysis ................................................................................................. 147 

 3.E.4. Enzymatic activity ....................................................................................................... 148 

 3.E.5. N-terminal sequencing ............................................................................................... 148 

 3.E.6. Glycosylation assay ................................................................................................... 149 

 3.F. Protein digestibility in simulated gastric fluid ..................................................................... 150 
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Page 

Appendix 4. HERBICIDE RESISTANCE AND PRODUCT STEWARDSHIP 

4.A. Evolution of herbicide resistant weeds ............................................................................ 153 

4.B. Managing herbicide resistant weeds ............................................................................... 155 

4.C. Glyphosate resistant weeds ............................................................................................. 158 

4.D. Characteristics of glyphosate herbicides ........................................................................ 158 

4.E. Stewardship of glyphosate-tolerant event VCO-Ø1981-5 maize .................................... 159 

 4.F. Customer outreach ........................................................................................................... 159 

 4.G. Additional customer support ............................................................................................ 160 

4.H. Monitoring of effectiveness of the stewardship plan ....................................................... 160 

Appendix 5. REFERENCES - APPENDICES 1-4 ................................................................... 161 

 
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LIST OF TABLES 

Page 

Table 1. Enzymatic half-life of EPSPS enzymes ...................................................................... 15 

Table 2. Percent EPSPS ACE5 enzymatic activity after 30 min incubation ............................ 16 

Table 3. Generations used for analysis of event VCO-Ø1981-5 ............................................. 21 

Table 4. Genetic elements in the T-DNA of plasmid pAG3541 ................................................ 25 

Table 5. Segregation data for the progeny of event VCO-Ø1981-5 ........................................ 29 

Table 6. Glyphosate binding affinities for EPSPS GRG23, EPSPS ACE5 and maize 

EPSPS proteins .......................................................................................................... 34 

Table 7. Risk group classification of Arthrobacter globiformis ................................................. 35 

Table 8. Sample collection per growth stage ........................................................................... 36 

Table 9. EPSPS ACE5 expression in different tissues ............................................................ 37 

Table 10. Biochemical analyses for demonstrating protein equivalence ................................... 38 

Table 11. Results of bioinformatics analyses ............................................................................ 39 

Table 12. Summary of field trial activities ................................................................................... 42 

Table 13. Summary of field trial activities outside the US ......................................................... 43 

Table 14. Maize hybrids tested in agronomic evaluations in 2009 ............................................ 45 

Table 15. Traits evaluated in the 2009 field trials ...................................................................... 46 

Table 16. Vegetative characteristics ........................................................................................... 48 

Table 17. Reproductive parameters ........................................................................................... 49 

Table 18. Emergence .................................................................................................................. 50 

Table 19. Disease rating scales for ecological evaluations ....................................................... 50 

Table 20. Disease ratings .......................................................................................................... 51 

Table 21. Ecological stressors .................................................................................................... 51 

Table 22. Seed germination ........................................................................................................ 53 

Table 23. Proximates and minerals in event VCO-Ø1981-5 forage ......................................... 55 

Table 24. Proximates in event VCO-Ø1981-5 grain ................................................................... 56 

Table 25. Fatty acids in event VCO-Ø1981-5 grain ................................................................... 58 

Table 26. Amino acids in event VCO-Ø1981-5 grain ................................................................. 59 

Table 27. Vitamins in event VCO-Ø1981-5 grain ....................................................................... 62 

Table 28. Values for total tocopherols ........................................................................................ 62 

Table 29. Minerals in event VCO-Ø1981-5 grain ....................................................................... 63 

Table 30. Secondary metabolites and anti-nutrients in event VCO-Ø1981-5 grain .................. 64 

Table 31. History of maize production ........................................................................................ 68 
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Page 

Table 32. 2009 Field trial location details ................................................................................. 128 

Table 33. Analytical methods for maize forage ........................................................................ 130 

Table 34. Analytical methods for maize grain .......................................................................... 131 

Table 35. Description of DNA probes used in Southern analysis ............................................ 135 

Table 36. Predicted and observed hybridization band sizes for insert copy number and 

insert integrity ............................................................................................................ 135 

Table 37. Comparison of EPSPS ACE5 peptides analyzed by MALDI-MS/MS ...................... 146 

Table 38. Enzymatic activity of maize-expressed EPSPS ACE5 protein ................................ 148 

Table 39. N-terminal amino acid sequence of maize- and E.coli-produced EPSPS 

ACE5 protein ........................................................................................................... 148 
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LIST OF FIGURES

Page 

Figure 1. EPSPS ACE5 enzymatic activity under various temperature conditions .................. 16 

Figure 2. Process for the development of event VCO-Ø1981-5 ............................................... 20 

Figure 3. Breeding diagram for event VCO-Ø1981-5 ................................................................ 22 

Figure 4. Plasmid map of pAG3541 carrying the epsps grg23ace5 gene ................................ 23 

Figure 5. Diagram of the T-DNA region in plasmid pAG3541 ................................................... 24 

Figure 6. Map of the T-DNA insertion site of event VCO-Ø1981-5 .......................................... 27 

Figure 7. EPSPS functions in the shikimate pathway ............................................................... 32 

Figure 8. Transformation vector pAG3541 for event VCO-Ø1981-5 ....................................... 117 

Figure 9. Plasmid map of pAX3541 ......................................................................................... 118 

Figure 10. Map of 2009 agronomic field trial locations .............................................................. 128 

Figure 11. Map of T-DNA insertion site of event VCO-Ø1981-5 .............................................. 134 

Figure 12. Determination of insert copy number using probe 3 (epsps grgace5 gene) ........... 136 

Figure 13. Analysis of insert integrity using probe 1 (ScUbi4 promoter) and probe 2

(intron) ....................................................................................................................... 137 

Figure 14. Analysis of insert integrity using probe 3 (epsps grgace5 gene) and probe 4 

(35S terminator) ....................................................................................................... 138 

Figure 15. Insert stability across generations ........................................................................... 139

Figure 16. Map of plasmid pAG3541 with vector backbone probes ......................................... 140 

Figure 17. Absence of the transformation plasmid components (oriT, aad, tetA, and tetR 

probes) ...................................................................................................................... 141

Figure 18. Absence of transformation plasmid components (virC, virG, and virB probes) ...... 142 

Figure 19. Kinetic characterization of EPSPS ACE5 protein .................................................... 143 

Figure 20. SDS-PAGE ............................................................................................................... 145 

Figure 21. Western blot analysis of microbial-expressed and plant-expressed EPSPS 

ACE5 protein ............................................................................................................. 147 

Figure 22. Glycosylation analysis of E. coli- and plant-expressed EPSPS ACE5 protein ........ 149 

Figure 23. SDS-PAGE gel of EPSPS ACE5 protein digested in a standardized SGF ............. 150 

Figure 24. Western blot of EPSPS ACE5 protein digested in a standardized SGF ................. 151 

Figure 25. Timeline of the development of herbicide resistant weeds classified by 

herbicide mode of action ........................................................................................... 154 
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I. RATIONALE 

I.A. Basis for the request for determination of non-regulated status 

The United States Department of Agriculture (UDSA) Animal and Plant Health Inspection 

Services (APHIS) is responsible for protection of the US agricultural infrastructure 

against noxious pests and weeds. Under the Plant Protection Act (7 USC § 7701-7772) 

APHIS considers plants altered or produced by genetic engineering as restricted articles 

under 7 CFR 340 , which cannot be released into the environment without appropriate 

approvals. APHIS provides that petitions may be filed under 7 CFR §340.6 to evaluate 

data to determine that a particular regulated article does not present a plant pest risk. 

Should APHIS determine that the submitted article does not present a plant pest risk, the 

article may be deregulated and released without further restrictions. 

I.B. Herbicide-tolerant maize event VCO-Ø1981-5 

GENECTIVE S.A. has developed, in collaboration with Athenix Corp., now an affiliate of 

Bayer CropScience LP, herbicide-tolerant maize event VCO-Ø1981-5. Event VCOØ1981-5 maize plants express an improved 5-enolpyruvylshikimate-3-phosphate 

synthase (EPSPS) protein, EPSPS ACE5, which confers tolerance to the common 

herbicide glyphosate. 

I.C. Rationale for the development of event VCO-Ø1981-5 and benefits 

I.C.1. Glyphosate-tolerant crops 

N-phosphonomethylglycine, commonly referred to as glyphosate, is an important 

commercial herbicide. Glyphosate inhibits plant, fungal, and most bacterial EPSPS 

enzymes and thus prevents the shikimate pathway from producing chorismate (the 

precursor of many essential plant metabolites). As plant cells depend on the 

biosynthesis of aromatic amino acids for growth, herbicide sprays containing glyphosate 

can affect plant cells by effectively blocking the shikimate pathway. Because the 

shikimate pathway is not present in animals, glyphosate has a favorable toxicology 

profile and has become a very common non-selective herbicide (Franz et al., 1997). 

Glyphosate-tolerant crops have been commercially available since the mid-1990s, 

demonstrating a robust history of safe use (OECD, 1999; Gianessi, 2008) and their 

benefits have been well established (Nolte and Young, 2002; Cedeira and Duke, 2006; 

Gianessi, 2008). Post-emergent spray herbicides decrease farming costs by limiting the 

growth of weeds competing for water, soil nitrogen and nutrients. Herbicide sprays also 

minimize field labor costs associated with removing weeds during the growing season. In 

many instances, glyphosate replaces more harmful herbicides and simplifies weed 

control practices since the grower can rely on a single herbicide only when weed control 

is required. Historically, conventional maize production has utilized tillage (field 

ploughing) and multiple herbicide inputs (i.e., pre-season burndown, pre-emergent and 

post-emergent treatments). Herbicide-tolerant crops have allowed a rapid adoption of 

reduced tillage practices. Low-till and no-till farming is quickly dominating agricultural 

systems, since it conserves valuable topsoil, reduces fuel costs to the grower and 

reduces the amount of greenhouse gases in the environment (Brooks and Barfoot, 

2011). All of these advantages have led to the widespread adoption of herbicide-tolerant 
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crops in modern agriculture with glyphosate-tolerant crops being the most widely-used of 

these technologies. 

I.C.2. Glyphosate-tolerant event VCO-Ø1981-5 maize 

Event VCO-Ø1981-5 has been produced with an expression cassette containing the 

epsps grg23ace5 gene allowing for the synthesis of the EPSPS ACE5 protein. The 

intended effect of the modification is to confer tolerance to the herbicide glyphosate to 

maize and providing another option to simplifying weed control for growers. 

The native EPSPS GRG23 protein isolated from the source organism, Arthrobacter 

globiformis, was modified to produce the EPSPS ACE5 protein using a directed 

evolution protein engineering strategy to more closely match it to the native maize 

EPSPS enzyme with regards to its temperature optimum under typical field-growing 

conditions. 

The performance of an enzyme in planta can be directly related to the stability of that 

enzyme under field conditions. Frequently, commercial crops encounter hot weather 

conditions that can lead to enzyme denaturation and loss of enzymatic activity. 

Particularly when engineering a plant to be tolerant to herbicide spray treatment, it is 

critical that the enzyme functions under extreme environmental conditions. 

The resulting tolerance to increased temperatures is illustrated with the following 

analysis. EPSPS ACE5, native EPSPS GRG23, and the native maize EPSPS enzymes 

were incubated at 37°C (to approximate extreme heat conditions potentially experienced 

in the field) for time periods ranging from 2 to 72 hours, and then the percentage of 

activity remaining was compared to protein incubated at 4°C in order to calculate the 

enzymatic half-life at 37°C. By this analysis (Table 1), the modified EPSPS ACE5 

enzymatic half-life was greatly improved, bringing it closer to the native maize EPSPS 

temperature optimum under usual environmental conditions. 

Table 1. Enzymatic half-life of EPSPS enzymes 

EPSPS ACE5 EPSPS GRG23 Maize EPSPS 

Enzyme half-life 

@ 37°C 65 hours 10 hours 86 hours 

The temperature stability of EPSPS ACE5 protein was further evaluated by examining 

the enzymatic stability of the protein by carrying out 30 min incubations at various 

temperatures that mimic industrial processing conditions for maize. Enzymatic activity 

was monitored by way of a real-time kinetic assay, which links the release of phosphate, 

a product of the EPSPS reaction, to the generation of a fluorescent substrate. A control 

sample without EPSPS ACE5 protein was assayed and baseline activity was subtracted 

from all recorded values. The average rate (relative fluorescent units per second) 

generated from samples incubated at 4°C minus the average rate of the buffer control 

was considered to represent 100% enzymatic activity. EPSPS ACE5 activity remained 

relatively stable after incubation at 25°C and 37°C, as observed above, but dropped to 

near zero after 30 minutes of incubation at 65°C and 95°C (Figure 1, Table 2). 
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Figure 1. EPSPS ACE 5 enzymatic activity under various temperature conditions 

Table 2. Percent EPSPS ACE5 enzymatic activity after 30 min incubation 

Sample 

EPSPS ACE5 activity (%) 

4°C 25°C 37°C 65°C 95°C 

EPSPS ACE5 protein 100.00 96.72 99.67 0.55 0.33 

I.D. Adoption of event VCO-Ø1981-5 

US farmers have readily adopted genetically modified crops since their commercial 

introduction in 1996. Genetically modified maize is third to cotton and soybean in terms 

of the percentage of acres planted to genetically modified crops in the US. In 2011, 88% 

of all maize planted was genetically modified, with 23% being herbicide-tolerant only and 

49% being stacked maize varieties expressing both insect control and herbicide 

tolerance traits; the remaining being insect control only. More than 90% of the herbicidetolerant maize was planted to glyphosate-tolerant maize. The adoption of herbicidetolerant maize, which had been slower in previous years, has accelerated, reaching 72% 

of US maize acreage in 2011 (ERS USDA, 2011). 

The introduction of herbicide-tolerant crops, such as glyphosate and glufosinate 

ammonium-tolerant crops has brought many advantages to the grower, such as reduced 

crop injury, broader spectrum of weed control, more environmentally favorable profile 

and less herbicide carry-over. These tools, including event VCO-Ø1981-5 maize, will 

continue to provide the grower with greater flexibility and ease of use in their weed 

management systems. It is anticipated that event VCO-Ø1981-5 maize hybrids will be 

cultivated across the US Corn Belt without extending the current maize growing area. 
GENECTIVE SA Maize event VCO-Ø1981-5 

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I.E. Submissions to other regulatory agencies 

Food and Drug Administration 

Event VCO-Ø1981-5 maize is within the scope of the 1992 US FDA policy statement 

concerning regulation of products derived from new plant varieties, including those 

developed through biotechnology (FDA, 1992). In compliance with this policy, a food and 

feed safety and nutritional assessment summary for event VCO-Ø1981-5 will be 

submitted to the US FDA. 

In 2009, Athenix Corp., now an affiliate of Bayer CropScience LP, submitted an Early 

Food Safety Evaluation to FDA. Their review was completed with no further questions in 

October, 2010 (FDA, 2010). 

Foreign Governments 

GENECTIVE S.A. or its local subsidiary as required by local authorities intends to submit 

dossiers to request import of event VCO-Ø1981-5 maize to the proper regulatory 

authorities of foreign countries that have regulatory processes in place. These may 

include submissions to the relevant Regulatory Authorities in Canada, EU, Japan, and 

others. Event VCO-Ø1981-5 maize has been, or is currently, in field trials in maize 

growing regions in the US, Canada and the European Union. 
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II. THE BIOLOGY OF MAIZE

II.A. The biology of maize 

Maize is a member of the Maydeae tribe of the grass family, Poaceae. 

Family: Poaceae 

Subfamily: Panicoideae

(Western Hemisphere) Genus: Zea

Species: Zea mays L. (maize) 

 Subspecies: Zea mays subsp. mays (L.) Iltis (maize, 2n = 20) 

The OECD consensus document on the biology of Zea mays ssp. mays (maize) (OECD, 

2003) provides a thorough review of the following aspects of maize biology, taxonomy, 

history of use, and cultivation: 

 General description (taxonomic status, morphology, use as a crop plant); 

 Agronomic practices; 

 Center of origin; 

 Reproductive biology; 

 Intra- and inter-species crosses and gene flow; 

 Cultivation (volunteers, weediness potential, soil ecology and interactions with 

insects). 

II.B. Characteristics of the recipient cultivar

The recipient maize line for generating event VCO-Ø1981-5 was Hi-II. Event VCOØ1981-5 was generated by the transfer of the T-DNA from plasmid pAG3541 into Hi-II 

via Agrobacterium-mediated transformation. 

The Hi-II hybrid line was obtained from the crossing of two separate lines Hi-IIA and HiIIB (Armstrong et al., 1991). The resulting embryos of the hybrid are used as target 

tissue for maize transformation. Hi-IIA and Hi-IIB are partially inbred lines selected out of 

a cross between maize inbred lines A188 and B73. Hi-IIA and Hi-IIB were obtained from 

Maize Genetics COOP Stock Center (Urbana, IL, USA). 
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III. DEVELOPMENT OF HERBICIDE-TOLERANT CORN EVENT VCO-Ø1981-5

III.A. Description of the transformation system 

Maize event VCO-Ø1981-5 was generated using a standard disarmed Agrobacterium

mediated transformation protocol (Hiei and Komari, 1997). The presence and integrity of 

pAG3541 into the Agrobacterium strain was verified by Southern hybridization (data not 

shown). The verified Agrobacterium strain was then used to transform the hybrid maize 

line Hi-II (Maize Genetics Cooperation Stock Center) (Figure 2). 

Cells from the dent type hybrid maize line Hi-II were transformed by the transfer of the TDNA region of plasmid vector pAG3541 containing the epsps grg23ace5 gene. Immature 

embryos of Hi-II corn were excised at approximately eight days after pollination and 

infected with Agrobacterium tumefaciens strain LBA4404 containing plasmid pAG3541. 

After three days of co-cultivation on solid culture media, the embryos were transferred to 

media containing the antibiotic timentin to eliminate Agrobacterium from the system post 

transformation (Cheng et al., 1998). 

Only the T-DNA existing between the right and left border (RB and LB) sequences is 

integrated into the maize genome. The DNA regions outside of the T-DNA borders such 

as the bacterial antibiotic resistant marker genes and vir genes are not typically 

transferred. The bacterial antibiotic resistant marker genes are required for the 

preparation of the transformation vector. The vir genes are required for the production of 

the T-DNA transfer complex (De la Riva et al., 1998). 

After approximately two weeks of culture, the embryos were transferred to selection 

media containing glyphosate. Transformed callus tolerant to glyphosate were identified 

and transferred to fresh selection media. Embryogenic callus was ultimately regenerated 

into whole plants and transferred to the greenhouse for further analysis (Vande Berg et 

al., 2008). The regenerated plants (designated as T0) were evaluated further for 

tolerance to glyphosate spray and through molecular techniques for the presence of the 

gene of interest. 

Figure 2 represents a schematic diagram of the steps involved in the development of 

event VCO-Ø1981-5 up to the molecular characterization. 
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Figure 2. Process for the development of event VCO-Ø1981-5 

Isolation and optimization of the gene of tolerance to glyphosate, epsps grg23ace5. 

↓

Preparation of the gene construct for maize transformation (pAG3541). 

↓

Disarmed Agrobacterium-mediated transformation of immature maize embryos. 

↓

Selection of transformed callus on glyphosate containing media. 

↓

Regeneration of T0 maize plants. 

↓

Evaluation of T0 events for tolerance to glyphosate in the greenhouse. 

↓

Molecular characterization of events and field evaluation of BC0B plants. 

III.B. Parent line 

Event VCO-Ø1981-5 was generated by the transfer of the T-DNA from plasmid 

pAG3541 into Hi-II via Agrobacterium-mediated transformation. 

Following recovery from glyphosate selection during the plant transformation process, 

the T0 plantlet was transplanted into a germination soil mix and grown in a temperaturecontrolled greenhouse. At flowering, the plant was pollinated with inbred B110 

(Committee for Agricultural Development, Iowa State University), and the seed were 

harvested and dried. These back-cross zero (BC0B, Figure 3) seeds were then planted 

in a winter nursery in Puerto Rico in 2007/2008. 

III.C. Breeding diagram 

The breeding diagram of event VCO-Ø1981-5 maize is shown in Figure 3. 

III.D. Generations used for analysis 

The generations used for regulatory studies to analyze maize event VCO-Ø1981-5 are 

described in Table 3. 
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Table 3. Generations used for analysis of event VCO-Ø1981-5 

Study Letter code Generation Page # in 

petition 

Insert characterization (copy #, stability 

within generation) B BC1B 26 

Absence of vector backbone A BC1B 27 

Mendelian inheritance D BC0A, BC1A, 

BC2A, BC3A 28 

Structural stability (across generations) C BC0B, BC1B, 

BC2B, BC1B2

29 

Protein expression E BC0BS2B 36 

Agronomic performance (2008) F BC1B 44 

Agronomic performance, biotic and 

abiotic stressors (2009) G BC0BS2B, 

BC0BS2CH 44, 50 

Field emergence H BC0BS2B, 

BC0BS2CH 50 

Warm and cold germination I BC1B1S2 52 

Composition J BC0BS2B 53 
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Figure 3. Breeding diagram for event VCO-Ø1981-5 

 

F1BC0B [C] Cross to B109 inbred (B1)

BC0BS1

BC0BS2



F1[BC1B1] 

BC1B1

Backcross 

to B109 

inbred 

BC2B1[C]



Negative 

Segregant 

(homozygous)

Positive 

Segregant 

(homozygous)

Cross to 

B110

Backcross to 

B109 inbred

BC0BS2B 

(non-transgenic 

near-isogenic 

hybrid) 

[E, G, H. J]

BC0BS2B 

(positive 

VCO-Ø1981-5 

hybrid) 

[E, G, H. J] 

Cross to AAX3 

inbred 

F1[BC0A] [D]

BC1A [D]

Backcross to 

AAX3 inbred 

Backcross to 

AAX3 inbred 

Event VCO-Ø1981-5 /T0 (Hi-II)

BC0BS2CH 

(positive 

VCO-Ø1981-5 

hybrid) 

[G, H] 

BC0BS2CH 

(non-transgenic 

near-isogenic 

hybrid) 

[G, H] 

BC3A [D]

Backcross to 

AAX3 inbred 



BC1B1S1



BC1B1S2 

[I]

BC2A [D] Segregating into:

Backcross to B110 inbred (B)

 BC1B [A, B, C, F]

Cross to 

B116 inbred

Cross to B110 

inbred (B2) 

F1[BC0B2]

BC1B2 [C]

Backcross to 

B110 inbred 

Cross to 

B116 inbred

Cross to CH01 

inbred

Cross to CH01 

inbred

x; cross 

; selfing

[A, B, ...]; letter code corresponding to regulatory studies listed in Table 3 
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IV. GENETIC MATERIAL USED FOR TRANSFORMATION OF EVENT VCO-Ø1981-5

IV.A. Construction of the plasmid used for transformation 

A synthesized coding region comprising a maize chloroplast transit peptide from 

acetohydroxyacid synthase (Fang et al., 1992) and the sequence encoding the epsps 

grg23ace5 gene was generated. It was then subcloned downstream from a constitutive 

plant promoter (Ubiquitin4 promoter from Saccharum officinarum L.) (Albert and Wei, 

2003) and upstream from a plant terminator sequence (3’ non-coding sequence of 35S 

gene from Cauliflower mosaic virus) (Gardner et al., 1981). The promoter-coding region -

terminator fragment from this intermediate plasmid was subcloned into plasmid pSB11 

(Japan Tobacco, Inc.) to create plasmid pAX3541 (Appendix 2, Figure 9). The plasmid 

was mobilized into Agrobacterium tumefaciens strain LBA4404, which also harbors the 

plasmid pSB1 (Japan Tobacco, Inc.), using triparental mating and plating on media 

containing spectinomycin, streptomycin, tetracycline and rifampicin. The cointegrate 

product of pSB1 and pAX3541 resulted in the final plasmid, pAG3541 (Figure 4). 

Figure 4. Plasmid map of pAG3541 carrying the epsps grg23ace5 gene 
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IV.B. Description of gene and regulatory sequences 

Event VCO-Ø1981-5 was produced by disarmed Agrobacterium-mediated 

transformation using the plasmid pAG3541. This plasmid contains the 3730 bp T-DNA 

region (Figure 5) including the epsps grg23ace5 gene expression cassette. 

The T-DNA of plasmid pAG3541 contains a single gene cassette including a modified 

version of the 5-enolpyruvylshikimate-3-phosphate synthase nucleotide sequence from 

Arthrobacter globiformis (Schouten et al., 2010) (epsps grg23ace5 gene coding region) 

that encodes the EPSPS ACE5 enzyme. Arthrobacter species are part of the Grampositive coryneform bacteria considered one of the major groups of aerobic soil bacteria 

and are found ubiquitously in soil and other natural habitats (Conn, 1948; Conn and 

Dimmick, 1947, Mulder et al., 1966). 

Regulatory sequences are comprised of the ScUbi4 promoter, the ScUbi4 intron, the 

mAHAS chloroplast transit peptide, and the 35S terminator from Cauliflower mosaic 

virus. 

Figure 5. Diagram of the T-DNA region in plasmid pAG3541 

IV.C. Identity and source of the genetic material 

A detailed list of the genetic and regulatory elements, their position within the T-DNA and 

their origin is provided in Table 4. 
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Table 4. Genetic elements in the T-DNA of plasmid pAG3541 

Genetic element Size 

(bp) 

Gene bank 

accession 

number 

Vector 

nucleotide 

location 

Description 

Right border 25 AB027254.1 18,085-18,109 

DNA sequence of right border sequence of 

nopaline type T-DNA derived from plasmid pTiT37. 

Used as the initiation point of T-DNA transfer from 

Agrobacterium tumefaciens to the plant genome 

(Zambryski et al., 1980; Komari et al., 1996). 

Intervening 

sequence 73 NA 18,110-18,182 Sequences used in DNA cloning. 

Promoter from 

sugarcane 

ubiquitin-4 gene 

364 AF093504 18,183-18,546 

Promoter region of the ubiquitin-4 gene from 

Saccharum officinarum L. (sugarcane) (Albert and 

Wei, 2003). 

5’ untranslated 

region (UTR) 79 NA 18,547-18,625 

5’ untranslated region of the ubiquitin-4 gene from 

Saccharum officinarum L. (sugarcane)(Albert and 

Wei, 2003). 

Intron from 

sugarcane 

ubiquitin-4 gene 

1358 NA 18,626-19,983 

Intron region of the ubiquitin-4 gene from 

Saccharum officinarum L. (sugarcane)(Albert and 

Wei, 2003). 

Intervening 

sequence 9 NA 19,984-19,992 Sequences used in DNA cloning. 

Maize AHAS 

chloroplast 

transit peptide 

198 X63553.1 19,993-20,190 

N-terminal chloroplast transit peptide sequence 

derived from the Zea mays L. (maize) 

acetohydroxyacid synthase (ahas) gene (Fang et 

al., 1992). The chloroplast transit peptide allows the 

expressed protein to be transported to the 

chloroplast. 

Intervening 

sequence 9 NA 20,191-20,199 Sequences used in DNA cloning. 

epsps grg23ace5

gene 1242 NA 20,200-21,441 

Coding sequence of the modified 5-

enolpyruvylshikimate-3-phosphate synthase from 

Arthrobacter globiformis (Schouten et al., 2010) 

Intervening 

sequence 8 NA 21,442-21,449 Sequences used in DNA cloning. 

35S CaMV 

terminator 270 V00140 21,450-21,719 

Terminator region of the 35S transcript of the 

cauliflower mosaic virus, which terminates mRNA 

transcription and induces polyadenylation (Gardner 

et al., 1981). 

Intervening 

sequence 70 NA 21,720-21,789 Sequences used in DNA cloning. 

Left border 25 AB027254.1 21,790-21,814 

DNA sequence of left border sequence from Ti 

plasmid pTiA6. Defines the termination point of TDNA transfer from A. tumefaciens to the plant 

genome (Thomashow, et al., 1980; Komari et al., 

1996). 
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V. GENETIC CHARACTERIZATION OF EVENT VCO-Ø1981-5 

V.A. Overview 

Event VCO-Ø1981-5 was generated using Agrobacterium-mediated transformation of a 

3730 bp sized T-DNA fragment originating from vector plasmid pAG3541. The epsps 

grg23ace5 gene expression cassette located on the T-DNA was inserted into event 

VCO-Ø1981-5 as a single copy intact insert. Molecular characterization verified that 

functional components of the expression cassette were inserted in the expected order. 

The insert has been shown to be stably inherited through four generations using both 

molecular (Southern blots) and phenotypic Mendelian segregation analysis that tracked 

tolerance to glyphosate. Southern blot analysis has confirmed the absence of the 

transformation vector components outside of the T-DNA borders. These results clearly 

indicate that the trait is inherited as expected for a single locus. 

V.B. Copy number and insertion 

The number of copies of the inserted transgene was investigated by Southern blot 

analysis. Genomic DNA samples of event VCO-Ø1981-5 and appropriate control 

samples were digested with the restriction enzymes HindIII and NdeI independently 

(Figure 6). Each of these restriction enzymes cuts one time within the T-DNA region. 

When hybridized with the epsps grg23ace5 gene probe (Figure 6, probe #3), the 

resulting number of bands indicates the insert copy number within the maize genome. 

Both digests, with HindIII and NdeI, probed with the epsps grg23ace5 gene sequence 

produced a single band, indicating that a single copy of the insert is present in event 

VCO-Ø1981-5 (Appendix 3, Figure 12). 

Southern blot analysis was also conducted to determine insert integrity. Maize genomic 

DNA from event VCO-Ø1981-5 and appropriate non-transgenic controls) was digested 

with a combination of HindIII and EcoRI, and independently with MfeI. The locations of 

these restriction enzyme sites are shown in Figure 6. A set of four independent probes of 

the major genetic elements (ScUbi4 promoter, ScUbi4 intron, epsps grg23ace5 gene, 

and 35S terminator; Figure 6, probes 1-4, respectively) was used to confirm the integrity 

of the expression cassette. The results indicate that the T-DNA segment that contains 

the epsps grg23ace5 expression cassette is intact and the functional components were 

inserted in the expected order. 

The predicted sizes of hybridization products for these analyses are shown in Figure 6. 

A description of the probes and their sizes is provided in Appendix 3, Table 34. 

Predicted and observed hybridization fragments are described in Appendix 3, Table 35. 

Southern blots are presented in Appendix 3, Figures 12-14. 
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Figure 6. Map of the T-DNA insertion site of event VCO-Ø1981-5 

Note: The T-DNA size in plasmid pAG3541 is 3730 bp; it is 3692 bp after insertion into the Hi-II 

genome (Section V.D below). 

V.C. Absence of vector backbone 

Southern blot analysis was conducted to verify the absence of the transformation 

plasmid components outside of the transferred T-DNA region.

Southern blot analysis results (Appendix 3, Figures 17 and 18) indicate that none of the 

vector probes hybridized to genomic DNA of event VCO-Ø1981-5 confirming the 

absence of vector backbone. The same probes, however, did show hybridization with 

the plasmid vector control on each blot indicating that if the vector sequences were 

inadvertently transferred to event VCO-Ø1981-5, they would have been detected in this 

analysis. 

V.D. The flanking regions of the inserted sequence 

The maize genomic flanking regions of the insertion in event VCO-Ø1981-5 were 

identified using a PCR based approach that includes the Genome WalkerTM strategy, 

followed by a bioinformatics analysis of the obtained sequences. 

The sequences surrounding the insertion site have been identified by sequencing 2 Kb 

for the 5’ flanking region and 1.7 Kb for the 3’ flanking region. These sequences allowed 

for mapping the insertion site of event VCO-Ø1981-5 to the maize chromosome 1. 

Comparison of the sequences from event VCO-Ø1981-5 with the Hi-II original sequence 

showed that only a short deletion (21 bp) in the maize genome was created by the 
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insertion of event VCO-Ø1981-5. The insertion process also created some short 

deletions at the extremities of the T-DNA borders - 22 bp and 16 bp on the right and left 

borders, respectively, which resulted in the insert being 3692 bp in size (T-DNA fragment 

originating from vector plasmid pAG3541 is 3730 bp). However, all regulatory sequences 

and the epsps grg23ace5 gene were found to be inserted intact. This was also 

confirmed by Southern blot analysis as discussed in section V.B. above. 

Bioinformatics analysis of the insertion region revealed the presence of a sequence 

potentially coding for an Acanthoscurrin-homolog protein. The insertion of the T-DNA 

seems to have occurred in the 5’ untranslated region (UTR) of this gene, which has 

probably prevented its transcription in event VCO-Ø1981-5. Acanthoscurrin-2 and 

Acanthoscurrin-homolog transcripts have been found in maize (Yang et al., 2006), 

sorghum (Tanaka et al., 2008) and rice (Paterson et al., 2009). Acanthoscurrin has been 

described as an antimicrobial peptide (Lorenzini et al., 2003). The insertion, however, 

did not produce any phenotypic changes in event VCO-Ø1981-5 maize, nor were any 

differences in agronomic performance or disease incidence observed that would indicate 

a potential effect on the expression of an antimicrobial peptide. In addition, one can 

expect that the Acanthoscurrin-2 protein is still expressed in maize event VCO-Ø1981-5, 

since the Acanthoscurrin-2 and Acanthoscurrin-homolog proteins are encoded by two 

different genes in maize (Alexandrov et al., 2009; Yang et al., 2006). 

Bioinformatics analysis also identified 12 potential open reading frames (ORFs, defined 

here as a sequence between two STOP codons) created by the T-DNA insertion in 

event VCO-Ø1981-5. No homologies with known allergen sequences were identified 

using a short (8) amino acid search algorithm when these potential ORFs were analyzed 

for their putative allergenicity. Using the 80 AA sliding window (> 35% homology) 

approach, two areas of potential homology were identified (AllergenOnline Database 

Version 11; http://www.allergenonline.org/databasefasta.shtml). However, these 

sequences are also present and are potential ORFs in the native maize genome, They 

were not a result of the insertion present in event VCO-Ø1981-5. No new ORFs were 

created by the insertion. No significant homologies with known toxins or other harmful 

proteins were found, when ORFs were analyzed against publically available databases 

using the BLASTP algorithm. All potential matches were manually inspected for 

homologies with known toxins. Moreover, it is highly unlikely that the identified genetic 

sequences would generate any translatable mRNA, since no START codons were found 

upstream. 

V.E. Mendelian inheritance 

Insert stability was confirmed using phenotypic Mendelian segregation analysis. For 

these experiments, the back-cross zero plants (BC0B) were crossed with the nonparental AAX3 inbred, to produce four successive generations: BC0A, BC1A, BC2A, 

BC3A (Figure 3). 

Progeny plants were sprayed with glyphosate to identify plants that inherited the epsps 

grg23ace5 gene and assess the segregation ratio in each generation. Positive 

segregants that survived the spray (little or no leaf damage) were scored as “tolerant”, 

while negative segregants did not survive the spray and were scored as “sensitive” 

(Table 5). All plants were evaluated two weeks after spraying. A segregation ratio of 1:1, 

or 50% tolerant and 50% sensitive, was expected in each generation if the epsps 

grg23ace5 gene was inserted at a single locus. 
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Observed segregation patterns were compared to the expected patterns and these data 

were compared using a chi-squared (CHI2) distribution analysis, as follows: 2 = Σ [(|o – 

e|)2/e], where o = observed frequency of tolerance, and e = expected frequency of 

tolerance. A CHI2 value of ≥ 0.05 was treated as the cutoff for statistical support of a 1:1 

segregation in each generation, and this value was exceeded for each of the 

segregation analysis groups. The results of this analysis (Table 5) are consistent with 

the molecular results (see section V.F.) and confirm the stable inheritance of a single 

copy of epsps grg23ace5 gene into the progeny of event VCO-Ø1981-5. 

Table 5. Segregation data for the progeny of event VCO-Ø1981-5 

Generation 

used 

Number 

of plants 

Observed 

tolerant 

Observed 

sensitive

Tolerant 

(%)

CHI2 test 

(p-value) 

BC0A 28 12 16 42.9 0.450 

BC1A 153 78 75 51.0 0.808 

BC2A 58 29 29 50 1.000 

BC3A 74 38 36 51.4 0.816 

p0.05 in the CHI2 test indicates no significant difference from expected ratio 

V.F. Stability across generations 

Stability was determined using molecular and phenotypic methodologies. The molecular 

methodology was based on Southern blot analysis, where gel banding patterns were 

predicted and observed using specific restriction enzymes and genetic probes. The 

phenotypic methodology evaluated the inheritance of the epsps grg23ace5 gene through 

successive breeding generations. The tolerance to glyphosate application on plants 

grown under field conditions for event VCO-Ø1981-5 was used to evaluate the 

segregation ratios of this event (see section V.E.). 

Southern blot analysis was conducted on multiple generations of event VCO-Ø1981-5 

progeny to evaluate the stability of the T-DNA insertion site. Genomic DNA isolated from 

leaf material of event VCO-Ø1981-5 plants from four breeding generations resulting from 

crosses with non-transgenic inbred line B110 (BC0B, BC1B, BC2B, and BC1B2) and 

negative controls were digested with the restriction enzyme HindIII, which as noted 

earlier, cuts once within the T-DNA region. When hybridized with probe 3, specific for the 

epsps grg23ace5 gene coding region, genomic DNA from event VCO-Ø1981-5 digested 

with HindIII produces a single band 4040 bp in size, since one of the restriction sites is 

outside of the T-DNA, but within the maize genomic DNA (Figure 6). Plasmid pAG3541 

was included as a hybridization control. All four generations analyzed showed an 

identical hybridization pattern producing the same single ~4000 bp band (Appendix 3, 

Figure 15). If the genetic insert was unstable within the maize genome through 

successive breeding of the event, one would expect to detect changes in the banding 

pattern produced. The data thus indicates a stable insertion site in event VCO-Ø1981-5 

across four generations. 
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V.G. Conclusion 

Results of the molecular characterization (Bernard and MacIntosh, 2011a) of event 

VCO-Ø1981-5 indicate that the event contains a single insert containing the epsps 

grg23ace5 gene expression cassette. The results demonstrated that the T-DNA 

segment that contains the epsps grg23ace5 gene expression cassette is intact and the 

functional components are in the expected order. The insert has been shown to be 

stably inherited in two different genetic backgrounds and through four generations using 

molecular analysis and Mendelian segregation analysis that tracked phenotypic 

tolerance to glyphosate. Southern blot analysis has confirmed the absence of the 

transformation vector components outside of the T-DNA borders. Those results clearly 

indicate that the trait is inherited as expected for a single locus. Genomic flanking 

sequences of 2 Kb and 1.7 Kb for the 5’ and 3’ junctions, respectively, of the insertion in 

event VCO-Ø1981-5 were identified using a PCR based approach. Bioinformatics 

analysis did not identify any homologies with known allergen sequences created by the 

insertion. 
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VI. CHARACTERIZATION OF THE INTRODUCED PROTEIN 

VI.A. The EPSPS ACE5 protein 

Maize event VCO-Ø1981-5 expresses an improved 5-enolpyruvyl-3-shikimate 

phosphate synthase (EPSPS) of bacterial origin, the EPSPS ACE5 protein, to confer 

tolerance to the herbicide glyphosate. The EPSPS ACE5 protein was derived from the 

native EPSPS GRG23 protein isolated from Arthrobacter globiformis using a directed 

evolution protein engineering strategy. The native EPSPS GRG23 enzyme from 

Arthrobacter was optimized by changing a total of ten amino acids to produce EPSPS 

ACE5, which more closely matches the native maize EPSPS enzyme. The changes 

made to create the EPSPS ACE5 protein did not alter its enzymatic profile with the 

exception of having greater temperature stability of the enzymatic activity under 

environmental conditions, yet still remaining sensitive to industrial processing 

temperatures and rapidly digesting in simulated gastric fluid. Therefore, EPSPS ACE5 

protein is expected to have the same safety profile as other previously deregulated 

EPSPS proteins. The safety of EPSPS ACE5 protein was also evaluated as part of an 

Early Food Safety Evaluation submitted to FDA in 2009. FDA completed their review 

with no further questions in October, 2010 (FDA, 2010). 

This section of the petition provides a compilation of the protein safety data (Bernard and 

MacIntosh, 2011b) for EPSPS ACE5 protein as it is expressed in maize event VCOØ1981-5. 

VI.A.1. History and background 

EPSPS enzymes are required for plant amino acid biosynthesis. This EPSPS enzymatic 

reaction is very important in plant biology, as it is a non-branching step in the shikimate 

pathway, leading to the biosynthesis of a large number of aromatic plant metabolites, 

including essential aromatic amino acids (phenylalanine, tyrosine, and tryptophan), 

tetrahydrofolate, ubiquinone and vitamin K (Franz et al., 1997). It has been estimated 

that 35% or more of the plant dry mass is made up of molecules derived from the 

shikimate pathway (Franz et al., 1997). 

In addition to plants, EPSPS enzymes are also found in prokaryotic systems, and 

glyphosate is toxic to most bacterial species. However, certain bacteria are tolerant to 

glyphosate, and it has been found that the EPSPS enzymes isolated from these bacteria 

often have a high tolerance to glyphosate. Furthermore, genes encoding glyphosatetolerant EPSPS enzymes have been transferred to recipient organisms, including plants, 

by means of recombinant DNA technology to confer glyphosate tolerance (OECD, 1999; 

Nolte et al., 2002; Cedeira et al., 2006; Gianessi, 2008; Vande Berg et al., 2008). Indeed 

a number of crops (e.g., maize, soybean, canola, alfalfa, etc.) expressing glyphosate 

tolerance have been granted non-regulated status (USDA-APHIS, 2011) and are 

commercially available in several countries (Biosafety Clearinghouse, 2011). EPSPS 

ACE5 is another example of a bacterial EPSPS that confers glyphosate tolerance in a 

plant system (Peters et al., 2010, Schouten et al., 2010). 

EPSPS enzymes have a long history of safe use as they have been expressed in GM 

maize, cotton, soybean, canola, and sugarbeet and represent a large proportion of the 

total US acreage. According to Brookes and Barfoot (2009), 91% of the total US 

soybean crop in 2007, was planted to glyphosate-tolerant varieties, first introduced in 
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1996. Glyphosate-tolerant maize and cotton were first introduced in 1997 and 10 years 

later were planted on 52% and 70% of the US maize and cotton acres, respectively 

(Brookes and Barfoot, 2009). There have been no identified safety or environmental 

concerns raised for the glyphosate-tolerant technology, regardless of the crop in which 

they are expressed. 

VI.A.2. Characterization of the EPSPS ACE5 protein 

VI.A.2.1. Biochemistry and mode of action 

The EPSPS enzymatic reaction is very important in plant biology, as it is a nonbranching step in the shikimate pathway and the reaction mechanism has been very 

well-characterized. EPSPS enzymes catalyze the formation of 5-enolpyruvylshikimate-3-

phosphate (EPSP) by linking phosphoenolpyruvate (PEP) to shikimate 3-phosphate 

(S3P) at a hydroxyl position on the S3P ring. During the reaction, PEP is hydrolyzed and 

an inorganic phosphate molecule (Pi) is released (Herrmann and Weaver, 1999). The 

final product of the shikimate pathway is chorismate (Figure 7), which is utilized for the 

biosynthesis of a large number of aromatic plant metabolites, including essential 

aromatic amino acids (phenylalanine, tyrosine, and tryptophan), tetrahydrofolate, 

ubiquinone and vitamin K (Franz et al., 1997). The amino acid phenylalanine is further 

metabolized to produce several compounds needed for plant growth, including 

anthocyanins and lignin. 

Figure 7. EPSPS functions in the shikimate pathway 

The enzymatic function of EPSPS ACE5 protein was confirmed by carrying out 

enzymatic assays with purified E.coli-produced EPSPS ACE5 protein. An enzymatic 

assay was developed to quantify the production of inorganic phosphate (Pi) by EPSPS 

enzymes. The assay used enzyme coupling and resulted in the generation of a highly 

fluorescent product (Vazquez et al., 2003) (see Appendix 3, Section 3.D.). 
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Glyphosate inhibition of EPSPS enzymes 

N-phosphonomethylglycine, commonly referred to as glyphosate, is an important 

commercial herbicide. Glyphosate inhibits plant, fungal, and most bacterial EPSPS 

enzymes and thus prevents the shikimate pathway from producing chorismate (the 

precursor of many essential plant metabolites). As plant cells depend on the 

biosynthesis of aromatic amino acids for growth, herbicide sprays containing glyphosate 

can affect plant cells by effectively blocking the shikimate pathway. Because the 

shikimate pathway is not present in humans and animals, glyphosate has a favorable 

toxicology profile and has become a very common non-selective herbicide (Franz et al.,

1997). 

Glyphosate is a structural analog of PEP and competes with PEP for binding in the 

active site of EPSPS enzymes (Schonbrunn et al., 2001). This mode of action is 

supported by enzymatic and structural studies. Treatment of plant cells with glyphosate 

leads to substantial accumulation of shikimate-5-phosphate (Amrhein et al., 1980), and 

inhibits the incorporation of radiolabeled shikimate-3-phosphate into phenylalanine, 

tyrosine or tryptophan (Hollander and Amrhein, 1980). X-ray crystal studies have 

demonstrated that glyphosate is bound by EPSPS at the site normally occupied by PEP, 

and traps the enzyme in a non-productive state (Schonbrunn et al., 2001). It is important 

to note that for plant EPSPS enzymes including maize, the binding affinity for glyphosate 

is much tighter than the binding affinity for PEP (Vande Berg et al., 2008). Thus, 

relatively low concentrations of glyphosate in the plant cell are capable of effectively 

shutting down the shikimate pathway and producing cell death. 

Enzymatic function in the presence of glyphosate 

For a heterologous EPSPS enzyme to function well in plants, it is essential that it be 

similar to the native plant EPSPS in terms of binding affinity for the natural substrate 

phosphoenolpyruvate (PEP). However, a glyphosate-tolerant EPSPS must also possess 

very low affinity for glyphosate so that it can bind PEP and retain enzymatic activity. 

The potential for EPSPS ACE5 to provide glyphosate tolerance in plants was evaluated 

by carrying out enzymatic assays in the presence and absence of glyphosate. Using the 

in vitro enzyme assay described in Appendix 3, the EPSPS activity of EPSPS ACE5 was 

measured over a range of PEP concentrations. The maize EPSPS and native EPSPS 

GRG23 were assayed alongside for comparison, and the Km and Ki values for the 

enzymes are shown in Table 6 (Peters et al., 2010; Schouten et al., 2010). While all 

three enzymes bind PEP with similar affinity, the native EPSPS GRG23 and EPSPS 

ACE5 proteins bind only weakly to glyphosate (Ki is over 900-fold higher than Km) and 

therefore will preferentially bind PEP even at high glyphosate concentrations (Peters et 

al., 2010; Schouten et al., 2010). This suggests that EPSPS ACE5 expression in maize 

can substantially reduce glyphosate-induced toxicity. In contrast, the maize EPSPS 

enzyme binds to glyphosate with 90-fold greater affinity than PEP, and is therefore 

inhibited by much lower concentrations of the herbicide. It should be noted that the Km

value for EPSPS ACE5 is similar to published literature values for other bacterial EPSPS 

enzymes (Barry et al., 1997; Priestman et al., 2005; Funke et al., 2006). 
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Table 6. Glyphosate binding affinities for EPSPS GRG23, EPSPS ACE5, and maize 

EPSPS proteins 

Enzyme Km(PEP) 

μM 

Ki 

μM Ki/Km

EPSPS GRG23 11 9,525 869 

EPSPS ACE5 16.6 14,700 919 

Maize EPSPS 18 0.2 0.01 

VI.A.2.2. History of safe use 

Recipient crop – Zea mays 

Zea mays, the crop that was transformed with the expression cassette allowing the 

synthesis of an improved bacterial epsps gene is a crop widely used for food and feed 

that is unlikely to cause any pathogenic, toxic, allergenic or other adverse effects for 

humans or animals. Maize is a member of the Maydeae tribe of the grass family, which 

requires the help of man to disperse its seeds for survival. A large portion of the maizeproduced in the US is utilized for animal feeds and biofuels, but maize is also processed 

for inclusion in many foodstuffs (e.g., high fructose corn syrup, corn grits, corn meal, 

etc.). A comprehensive review of Zea mays, including detailed information on the 

biology, taxonomy, identification, diversity, reproductive characteristics and ecology, was 

published by OECD (2003). 

Source organism and the epsps grg23ace5 gene 

DNA is the basis for all life, with the same nucleic acids and structure, and as such is 

generally recognized as safe. Humans and animals are exposed to DNA in everything 

they consume. The addition of a transgene, such as epsps grg23ace5, to maize is 

indistinguishable from any other DNA that is present in their diet. 

The epsps grg23ace5 gene is an optimized form of the native epsps gene (Schouten et 

al., 2010) sourced from the common soil bacterium Arthrobacter globiformis. 

Arthrobacter species are part of the Gram-positive coryneform bacteria considered one 

of the major groups of aerobic soil bacteria and are found ubiquitously in soil, water and 

other natural habitats (Conn, 1948; Conn and Dimmick, 1947; Mulder et al., 1966). In 

addition to their prevalence in soil and water, Arthrobacter spp. (including A. globiformis) 

have been shown to be associated with the naturally occurring microflora of various 

produce including broccoli (Padaga et al., 2000), strawberries (Krimm et al., 2005), and 

sugarbeets (Bugbee et al., 1975). Arthrobacter is one of the only organisms that can 

grow in the presence of - and reduce - hexavalent chromium (Lower, 1998). 

A. globiformis works synergistically with Streptomyces to degrade agricultural pesticides 

such as organophosphate insecticide diazinon and also has the individual ability to 

degrade herbicides like glyphosate and pentachlorophenols (Lower, 1998; Microbewiki, 

2011). Arthrobacteria are weakly motile and are nonsporulating. Most Arthrobacter

species are obligate aerobes. As the upper layers of soil that they inhabit change oxygen 

concentration frequently, some Arthrobacter species, including A. globiformis, have 

developed adaptive oxygen independent growth strategies (Lower, 1998). 
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Arthrobacter sp. are not classified in many countries and are assigned the lowest risk 

group in others (Table 7). Arthrobacter strains are assigned biosafety level 1 (BSL1) for 

exposure control and personal protection. Human infections have been described for 

other Arthrobacter species, but not for A. globiformis (Mages et al., 2008). There are no 

known effects on animals. 

Table 7. Risk group classification of Arthrobacter globiformis 

U.S.A 

Not classified. NIH Guidelines for Research Involving Recombinant DNA Molecules, Appendix 

B: Classification of human etiologic agents on the basis of hazard, January 

2011.http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.pdf 

Canada Not classified. Pathogen Safety Data Sheets, Public Health Agency of Canada. 

http://www.phac-aspc.gc.ca/lab-bio/res/psds-ftss/index-eng.php

Belgium Not classified. Revised lists of pathogens and their corresponding class of biological risk, 

Belgian Biosafety Server. http://www.biosafety.be/RA/Class/ClassBEL.html

Switzerland 

Risk group 1. Classification of Organisms According to Risk Presented to People and the 

Environment. Bacteria (2005) and Viruses (2004). Swiss Department of the Environment, 

Transport, Energy and Communications. 

http://www.bafu.admin.ch/biotechnologie/01744/01753/index.html?lang=en

France 

Not classified. Principles de Classement et Guides Officiels de la Commission de Génie 

Génétique, France, January 2000. 

ftp://trf.education.gouv.fr/pub/rechtec/commis/genetique/principe/guide.pdf

Germany 

Risk group 1. Bundesanstalt für Arbeitsschutz und Arbeitsmedizin, Technische Regeln für 

Biologische Arbeitstoffe (TRBA) http://www.baua.de/cln_137/de/Themen-von-A-Z/BiologischeArbeitsstoffe/TRBA/TRBA.html

UK Not classified. HSE Advisory Committee on Dangerous Pathogens, The Approved List of 

Biological Agents, April 2004. http://www.hse.gov.uk/pubns/misc208.pdf

Singapore 

Not classified. List of biological agents and toxins, Ministry of Health, Singapore, November 

2009. http://www.biosafety.moh.gov.sg/home/uploadedFiles/Common/List_of_Biological_Agents_

and_Toxins.pdf. Division into Schedules based on risk groups as defined by the WHO and other 

factors such as the potential to be weaponized, etc. 

EPSPS ACE5 protein 

EPSPS ACE5 is an improved glyphosate-tolerant EPSPS enzyme. It was derived using 

a directed evolution protein engineering strategy from the native EPSPS GRG23 protein 

isolated from Arthrobacter globiformis (Peters et al., 2010; Schouten et al., 2010). A total 

of ten amino acids were changed to produce the EPSPS ACE5 protein, which is 97.6% 

identical and 98.5% homologous at the amino acid level to the originally isolated EPSPS 

GRG23 enzyme. 

As reviewed by ILSI (ILSI, 2010), several publicly available documents issued by 

regulatory authorities indicate that similar EPSPS protein family members expressed in 

glyphosate-tolerant crops are safe. The results of studies summarized in this petition are 

consistent with the published information, indicating that maize containing the EPSPS 

ACE5 protein can be safely used as food or feed. 
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Chloroplast transit peptide 

Many nuclear-encoded proteins in plants are localized into the chloroplast to perform key 

metabolic functions, including amino acid biosynthesis and photosynthesis (Keegstra 

and Cline, 1999; Bruce, 2000). Included in this set of chloroplast-localized proteins are 

the enzymes that function in the shikimate pathway (Mousdale and Coggins, 1985). 

Thus, it is imperative that glyphosate-tolerant EPSPS enzymes become targeted to 

chloroplasts to function more directly in the shikimate pathway and confer more robust 

tolerance following glyphosate applications. 

Chloroplast translocation is facilitated by addition of an N-terminal extension (referred to 

as a transit peptide) that guides the newly-translated proteins from the cytoplasm to the 

chloroplast outer membrane. Following translocation inside the chloroplast, the transit 

peptide is cleaved to yield the mature protein. The chloroplast transit peptide selected for 

the EPSPS ACE5 protein was cloned from the region upstream of the maize 

acetohydroxy acid synthase (AHAS) gene. The cleavage of the transit peptide was 

inferred by the verification of mature protein molecular weight and the N-terminal 

sequence of the EPSPS ACE5 protein isolated from event VCO-Ø1981-5 maize 

(Schouten, 2011). 

VI.B. Expression of the EPSPS ACE5 protein in event VCO-Ø1981-5 

The protein expression levels were determined by validated enzyme-linked 

immunosorbant assay (ELISA, Appendix 2, Section 2.C.) for various tissues of event 

VCO-Ø1981-5 and the non-transgenic near-isogenic control planted at three locations in 

Iowa during the 2009 growing season (Stauffer, 2011). At each location, three replicated 

plots of event VCO-Ø1981-5 and the non-transgenic control (non-transgenic nearisogenic hybrid) were planted using a randomized complete block field design. The 

plants were grown under conditions representative of commercial corn production in 

Iowa. Table 8 lists the growth stages and the plant tissue types collected. 

Table 8. Sample collection per growth stage 

Plant Tissue 

 

# of plants per location and growth stage 

V4 V8 R1 R4 R6 

Leaf 6 3 3 3 3 

Root 6 3 3 3 3 

Whole Plant - 3 3 3 3 

Pollen - - 3 - - 

Grain - - - - 3 

V4 (vegetative 4-leaf stage), V8 (vegetative 8-leaf stage), 

R1 (reproductive silking stage), R4 (reproductive kernel dough stage), 

R6 (physiological maturity) 
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The EPSPS ACE5 protein was detected at various life stages and in all tissues of event 

VCO-Ø1981-5 plants except in grain (Table 9). In general, the expression levels of the 

EPSPS ACE5 protein in leaf were highest at the V4 and V8 stages, dropped off 

approximately 3-fold at the R1 and R4 stages, and dropped to under the limit of 

detection (LOD) at R6. The protein expression levels in root were about 30% of leaf at 

the V4 stage and dropped to below the LOD at the reproductive growth stages (R1, R4, 

and R6). Whole plant expression levels of the EPSPS ACE5 protein were very similar to 

those of leaf, with the exception of the V4 stage as no whole plant samples were 

collected for that stage. Protein expression in the pollen was low to undetectable, and 

expression in grain was undetectable. All values for control tissue (leaf, root, whole 

plant, pollen and grain) were either 0.0 ng EPSPS ACE5 protein/mg dw or below the 

LOD, which is provided in Table 9. 

Expression levels were in the tens of nanograms per milligram of dry tissue weight at the 

V4 and V8 stages, when the plant is undergoing rapid growth, and very low in the later 

reproductive stages when the plant’s energy is going towards development of the grain 

and pollen. The epsps grg23ace5 gene is driven by a constitutive promoter (Ubiquitin 4 

promoter from sugarcane) and EPSPS ACE5 protein expression was detected in all 

tissues evaluated except for grain. 

Table 9. EPSPS ACE5 expression in different tissues 

Tissue Growth 

Stage 

Amount of protein (ng /mg dw) Number of 

Samples 

<LOD/Total 

Number of 

Samples Average1 Range1 LOD 

Leaf V4 17.77 7.47 - 24.86 

1.34 

0/18 

 V8 18.59 8.63 - 24.01 0/9 

 R1 4.75 2.95 - 7.63 0/9 

 R4 6.20 1.40 - 8.70 0/9 

 R6 0.002 0.00 6.53 9/9 

Root V4 6.77 1.72 - 14.97 

1.57 

0/18 

 V8 2.63 0.00 - 4.54 2/9 

 R1 0.00 0.00 - 1.86 8/9 

 R4 0.00 0.00 9/9 

 R6 0.00 0.00 5.32 9/9 

Whole Plant V8 12.69 5.42 - 21.50 

0.92 

0/9 

 R1 1.95 0.00 - 4.45 3/9 

 R4 3.48 1.09 - 6.93 0/9 

 R6 0.00 0.00 4.31 9/9 

Pollen R1 0.00 0.00 - 9.60 6.43 8/9 

Grain R6 0.00 0.00 1.79 9/9 

1 across locations 

2 any values below the LOD are represented by a value of zero 
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VI.C. Verification of biochemical and functional equivalence 

The EPSPS ACE5 protein was produced in E. coli for use in studies to investigate the 

potential toxicity and allergenicity of the protein, since it is not feasible to produce an 

adequate amount of the protein for these studies from event VCO-Ø1981-5 plants. 

Therefore, it was necessary to demonstrate the equivalence between the E. coliproduced EPSPS ACE5 protein and the plant-produced protein in order to utilize the 

safety data. The E. coli-produced and plant-produced EPSPS ACE5 protein was 

compared, using the following six biochemical tests listed in Table 10. 

Table 10. Biochemical analyses for demonstrating protein equivalence 

Equivalence criteria Methodology 

Molecular weight Protein mobility in SDS-PAGE 

Peptide mass identification MALDI-TOF mass spectroscopy 

Immunoreactivity Western blot analysis 

Sequence comparison N-terminal sequencing 

Biological activity Enzymatic activity 

Glycosylation profile Glycosylation analysis 

These biochemical tests each confirmed the equivalency of the EPSPS ACE5 protein 

from E. coli- and plant-produced sources (Schouten, 2011). The analytical methods used 

are described in Appendix 2. The results are shown in Appendix 3. 

VI.D. Summary of the food and feed safety assessment of the EPSPS ACE5 

protein 

VI.D.1. Homology searches to known allergens and toxins 

One aspect of the protein safety assessment evaluates the amino acid similarity 

between the newly introduced protein and known allergens and toxins (Codex, 2003). 

These bioinformatics analyses are conducted in silico using different publically available 

protein databases. 

Many different genomes have been sequenced over the last decade providing a vast 

resource of protein sequences and tools for analyzing sequence data. Several different 

databases are publicly available that contain thousands of protein sequences (e.g.,

Uniprot-Swissprot, 2011; National Center for Biotechnology Information, 2011; Protein 

Informatics Resource, 2011). Search algorithms are used to compare the newly 

introduced protein with database proteins to classify and determine the relatedness to 

known protein families. A publicly available, curated genetic database of known protein 

allergen sequences, AllergenOnline (2011), has been developed allowing for 

characterization of the allergenic potential of unknown proteins in a consistent manner. 

This database (Version 11.0) contains a comprehensive list of unique proteins of known 

and putative allergenic proteins (food, airway, venom/salivary and contact) (Accessed 

March 31, 2011). Bioinformatic algorithms, FASTA and BLASTP, were utilized to 

evaluate the level of similarity of the EPSPS ACE5 protein to known allergens and 

toxins, respectively (Bernard and MacIntosh, 2011c). 
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The amino acid sequence of the EPSPS ACE5 protein was assessed for similarity to 

protein sequences in the National Center for Biotechnology Information Protein dataset 

by BLASTP bioinformatics analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi). The EPSPS 

ACE5 protein is highly homologous to other 5-enolpyruvylshikimate-3-phosphate 

synthase (EPSPS) enzymes. The top 100 hits from this search are all members of this 

class of enzymes (EC 2.5.1.19) and share greater than 50% protein sequence homology 

with EPSPS ACE5. All other resulting sequence matches, with an expectancy value of 

1.0 or less, were manually inspected for sequences with known toxic or anti-nutritional 

effects as indicated in the EPA Code of Federal Regulations document 40 CFR 725.421. 

The EPSPS ACE5 protein exhibits no significant homology at >35% to known toxins or 

other harmful proteins (Table 11). 

Three different similarity searches were conducted (Table 11): 

 The entire EPSPS ACE5 protein sequence was compared to the toxin 

sequences using the BLASTP search algorithm. No homologies were identified; 

 Contiguous EPSPS ACE5 peptides of 80 amino acids were compared to the 

allergen sequences using the FASTA search algorithm. No significant 

homologies were identified; 

 The EPSPS ACE5 protein sequence was screened for eight contiguous amino 

acid identity matches with known or putative allergens. No matches were 

identified. 

Table 11. Results of bioinformatics analyses 

Type of in silico search Results Purpose 

Full-length BLASTP No hits at > 35% identity Toxins 

80-mer FASTA No hits at > 35% identity Allergens 

8-mer exact match No matches Allergens 

VI.D.2. Potential N-glycosylation sites 

Glycosylation analysis was performed to determine if maize-produced EPSPS ACE5 

was post-translationally modified by the addition of carbohydrate moieties. Both 

microbial- and maize-derived EPSPS ACE5 protein was separated by SDS-PAGE along 

with horseradish peroxidase (positive control protein) and soybean trypsin inhibitor 

(negative control protein). Gels were stained with Gelcode glycoprotein staining kit 

(Thermo Fisher Scientific, Rockford, IL) according to manufacturer’s instructions. 

Glycoproteins were detected as bright magenta bands on the gel. The same gel was 

then stained with Imperial Coomassie Blue stain for detection of all proteins loaded on 

the gel (Appendix 3, Figure 23). 

No glycosylation was detected for either the microbial-produced or maize-produced 

EPSPS ACE5 protein. The glycoprotein positive control, horseradish peroxidase, stained 

a bright magenta color while the glycoprotein negative control, soybean trypsin inhibitor, 

showed only very faint light pink color. These results show that neither maize- nor 

microbial-produced EPSPS ACE5 protein have been modified by the addition of 

detectable levels of carbohydrates. 
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VI.D.3. In vitro stability to human simulated gastric fluid 

The resistance to enzymatic digestion of EPSPS ACE5 protein was analyzed using 

biochemical methods (Lautraite, 2011). The E. coli-produced EPSPS ACE5 protein was 

tested for stability in human simulated gastric fluid (SGF) with pepsin at pH 1.2 for 

incubation times ranging from 0.5 to 60 minutes using a standard pepsin digestion 

method (Thomas et al., 2004). 

Test protein and reference protein (horseradish peroxidase and ovalbumin) solutions 

were incubated with human SGF (pH 1.2) at approximately 37°C and samples were 

taken for analysis at time-points of 0, 0.5, 2, 5, 10, 20, 30 and 60 minutes. The resultant 

protein solutions were analysed for presence of the test protein and potential stable 

protein fragments by Coomassie blue-stained SDS-PAGE and western blot (Appendix 3, 

Figures 24 and 25). 

The EPSPS ACE5 protein was degraded very rapidly in human simulated gastric fluid, 

within 30 seconds of incubation, in presence of pepsin, at pH 1.2. Both reference 

proteins were digested as expected: HRP was rapidly digested and OVA was slowly 

digested (data not shown). 

With such rapid SGF digestion, it is unlikely the EPSPS ACE5 protein could have any 

adverse effects on humans or animals that consume event VCO-Ø1981-5 maize. 

Moreover, EPSPS ACE5 protein was not detected in grain samples (Section VI.B.) 

further limiting any potential dietary exposure to humans. 

It should be noted here that, while the optimized EPSPS ACE5 protein provides greater 

temperature stability under environmental conditions, the enzymatic activity remains 

sensitive to industrial processing temperatures (Section VI.A.2.1.) and its digestibility in 

SGF is comparable to that of other plant- expressed EPSPS proteins. 

VI.D.4. Acute toxicity study in the mouse 

To assess the toxic potential of the EPSPS ACE5 protein and confirm the safety 

assessment from data already collected (i.e., bioinformatics analysis, potential 

glycosylation sites, in vitro digestibility, etc.) an acute oral toxicity study was conducted 

in mice (Arulnesan, 2008). Two groups of CD-1 mice were tested with purified microbialproduced protein. Group 1 consisted of 3 male and 3 female mice that were each 

administered the negative buffer control. Group 2 consisted of 5 male and 5 female mice 

that were each administered EPSPS ACE5 protein at a dose of 2000 mg/kg. Both the 

negative buffer control and the EPSPS ACE5 protein were suspended in 3% methyl 

cellulose and administered by oral gavage at a dose volume of 25 ml/kg. The animals 

were observed for 14 days after dosing and body weights were recorded on Day 1 (prior 

to dosing), Day 7, Day 14 and Day 15, just prior to necropsy. 

No mortality or evidence of toxicity were observed post dosing or during the 14-day 

observation period in any of the animals. Animals in each group gained body weight by 

the end of the study. After the animals were sacrificed (Day 15), they were submitted for 

gross necropsy and no gross pathological findings were observed. Based on these 

results, there is no evidence of toxicity when EPSPS ACE5 is administered orally at a 

dose level of 2000 mg/kg. 
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VI.E. Conclusion 

EPSPS enzymes, a common class of enzymes found in plant, bacterial, and fungal 

species, have a long history of safe use. The EPSPS ACE5 protein was derived from the 

native EPSPS GRG23 protein isolated from Arthrobacter globiformis using a directed 

evolution protein engineering strategy. The native EPSPS GRG23 enzyme from 

Arthrobacter was optimized to produce the EPSPS ACE5 protein, which more closely 

matches the native maize EPSPS enzyme with regards to the temperature optimum for 

its activity under typical environmental conditions. The optimized EPSPS ACE5 protein 

provides greater temperature stability under environmental conditions but remains 

sensitive to industrial processing temperatures. A significant amount of safety and 

characterization data for the EPSPS ACE5 protein has been generated, which indicates 

this protein is not toxic or allergenic. The enzyme was isolated from a common soil 

bacterium, which is not known as a source of allergens or toxins. The recipient crop, Zea 

mays, is a crop widely used for food and feed and is unlikely to have any pathogenic, 

toxic, allergenic or other adverse effects in humans or animals. 

The enzyme kinetics of the native maize EPSPS, the native EPSPS GRG23, and the 

EPSPS ACE5 proteins were evaluated and the Km and Ki values for the enzymes 

determined. While all three enzymes bind PEP with similar affinity, the native EPSPS 

GRG23 and EPSPS ACE5 proteins bind only weakly to glyphosate (Ki is over 900-fold 

higher than Km) and therefore will preferentially bind PEP even at very high glyphosate 

concentrations. This suggests that EPSPS ACE5 protein expression in maize can 

substantially reduce glyphosate-induced toxicity. In contrast, the native maize EPSPS 

enzyme binds to glyphosate with 90-fold tighter affinity than PEP, and is therefore 

inhibited by much lower concentrations of the herbicide. It should be noted that the Km

value for the EPSPS ACE5 protein is similar to published literature values for other 

bacterial EPSPS enzymes. 

The EPSPS ACE5 protein has no significant sequence homology to known allergens or 

toxins. It is readily digestible when subjected to simulated gastric fluid resulting in 

complete degradation in less than 30 seconds and has not been glycosylated in planta. 

This enzyme is completely inactivated by a thirty-minute treatment at 65°C, indicating 

that the EPSPS ACE5 protein would be inactivated with typical heat processing or 

cooking procedures. The protein expression level in various tissues of event VCOØ1981-5 plants harvested over an entire growing season, were determined and the 

pattern of expression was as expected given the genetic elements of the transformation 

cassette (e.g., expression in maize grain was below the LOD). Additionally, an acute 

toxicity study has been conducted with the EPSPS ACE5 protein used as the test article 

in mice. The toxicity study was conducted using the maximum allowable total dose (e.g.,

2000 mg/kg) of a highly pure starting material and indicated no evidence of toxicity. In 

conclusion, the data described in this section indicate that the EPSPS ACE5 protein is 

likely safe for consumption and would pose little or no risk to human or animal health. 
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VII. AGRONOMIC AND PHENOTYPIC EVALUATION 

VII.A. History of field activities 

Event VCO-Ø1981-5 has been field tested in typical maize growing regions of the US 

and in winter nurseries in Puerto Rico since 2007. Table 12 lists the field trials and 

associated USDA notifications (see Appendix 1 for field trial reports from 2007-2009). 

Table 13 provides a list of field trials conducted in Europe and Canada. 

Table 12. Summary of field trial activities 

USDA 

notification #

Effective 

date Type of trial

Number of 

locations 

planted

State

07-283-104n 12/03/2007 Efficacy 1 PR 

08-059-104n 04/08/2008 Efficacy 1 IA 

08-067-108n 03/20/2008 Efficacy 1 PR 

08-073-108n 04/242008 Efficacy 1 IA 

08-105-109n 05/08/2008 Efficacy 1 NE 

08-254-111n 09/26/2008 Breeding 1 PR 

08-280-103n 10/23/2008 Breeding 1 PR 

08-309-103n 11/10/2008 Breeding 1 PR 

09-047-108n 02/24/2009 Breeding 1 PR 

09-091-101n 04/23/2009 Regulatory 19 IL, IN, SD, IA, 

NE, MN, WI 

09-086-104n 04/14/2009 Efficacy 2 IA, IN 

09-215-106n 08/22/2009 Breeding 1 PR 

09-341-105n 12/11/2009 Breeding 1 PR 

10-085-109n* 04/26/2010 Regulatory 3 IA, MN, WI 

10-071-113n* 04/01/2010 Breeding 1 PR 

11-013-102n* 02/11/2011 Breeding 1 PR 

*; reports pending 
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Table 13. Summary of field trial activities outside the US 

Field trial 

reference #

Effective 

date Type of trial

Number of 

locations 

planted

Year of 

planting Country

B/ES/09/01 03/13/2009 Efficacy 1 2009 Spain 

B/ES/09/01 04/07/2009 Efficacy 1 2009 Spain 

B/CZ/09/04 04/28/2010* Regulatory 3 2010 Czech 

Republic 

B/SK/10/03 04/16/2010* Regulatory 2 2010 Slovak 

Republic 

B/ES/10/47 05/17/2010 Regulatory 1 2010 Spain 

B/ES/10/47 04/29/2010 Regulatory 1 2010 Spain 

B/ES/10/47 05/12/2010 Regulatory 2 2010 Spain 

B/RO/10/04 06/14/2010* Regulatory 1 2011 Romania 

B/CZ/09/04 04/28/2010* Regulatory 3 2011 Czech 

Republic 

B/SK/10/03 04/16/2010* Regulatory 3 2011 Slovak 

Republic 

B/ES/11/16 03/14/2011 Regulatory 3 2011 Spain 

B/ES/11/16 04/04/2011 Regulatory 1 2011 Spain 

B/ES/11/16 06/24/2011 Regulatory 2 2011 Spain 

11-VMC1-

426-COR 04/15/2011 Regulatory 2 2011 Canada 

*Valid for five years 

VII.B. Agronomic and phenotypic evaluation 

Maize lines containing the epsps grg23ace5 gene have been shown to be tolerant to 

applications of the broad-spectrum herbicide glyphosate. Transformation events were 

first tested under greenhouse and field environments to assess the level of tolerance to 

glyphosate. As part of the evaluation process, events were grown under field conditions 

to compare plant morphology and growth characteristics to the non-transgenic nearisogenic hybrid (non-transgenic control). Agronomic performance evaluations were 

conducted on event VCO-Ø1981-5 as compared to the non-transgenic control. These 

evaluations included both multi-location field trials and laboratory experiments aimed at 

determining if the GM crop has unanticipated effects that would render it phenotypically 

different from the appropriate non-transgenic control. While these field trials focused on 

agronomic performance, regular and frequent observations on non-target effects to 

weeds, insects and disease susceptibility were also evaluated. 

Field trials in 2007 and 2008 were primarily focused on event selection and only a brief 

summary of the agronomic data from a single location in 2008 is provided here. 

Additional agronomic evaluations were carried out in several different locations in the 

2009 growing season. The agronomic evaluations were conducted by scientists with 
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expertise in the production and evaluation of maize. Laboratory testing for germination 

under warm and cold conditions was also performed. 

In 2009, a total of 17 field sites were utilized across six US states to collect agronomic 

data along with samples for other regulatory studies. The morphological and growth 

characteristics of event VCO-Ø1981-5 were evaluated in two genetic backgrounds, and 

compared to their respective non-transgenic near-isogenic hybrids (non-transgenic 

controls). Hybrids were characterized under diverse environmental and growing 

conditions representative of typical maize production in North America. 

VII.C. Agronomic performance of event VCO-Ø1981-5 

VII.C.1. Agronomic evaluation of event VCO-Ø1981-5 maize in 2008 

The field trial conducted in 2008 was located in Polk County, Iowa, USA. Agronomic 

practices used were representative for maize production in highly productive soils in 

Central Iowa. As the test event is a segregating plant from the first backcross generation 

(BC1B, Figure 3), the plots were sprayed with a glyphosate solution (5lbs glyphosate per 

gallon or 600 g/L) to eliminate the negative segregants. The sprayer output rate was 

calibrated to apply 10.7 gallons of glyphosate solution per acre (100 L/ha) at a speed of 

3.6 mph (5.8 km/h). 

Parameters for the 2008 field trial focused on event selection and evaluated seedling 

and germination qualities, vegetative and reproductive parameters, and grain quality. 

The agronomic characteristics evaluated were: days to emergence, 50% pollen shed, 

50% silking, 50% black layer, ear and plant height, ear girth and length, number of 

kernel rows/ear, kernels/row, kernel weight, pollen weight, and number of tassel 

branches. Four replicates were grown at a single location in 2008. 

Overall, event VCO-Ø1981-5 performed similarly to the non-transgenic control. All data 

comparisons between event VCO-Ø1981-5 and the non-transgenic control were carried 

out using a two-sided Dunnett’s analysis (Moore and McCabe, 1999). For all 13 of the 

agronomic traits no biologically significant differences were observed between the two 

groups. Event VCO-Ø1981-5 plants were phenotypically indistinguishable from the nontransgenic control in every measurement taken in the trial. 

VII.C.2. Agronomic evaluation of VCO-Ø1981-5 maize in 2009 

For the 2009 field trials, the back-cross zero (BC0B) generation was selfed two times to 

produce BC0BS2 (Figure 3). In order to evaluate agronomic performance characteristics 

of Event VCO-Ø1981-5 as compared to an appropriate non-transgenic control, two testcrosses were made and seed bulked for multiple location analysis in 2009 (Bernard and 

MacIntosh, 2011d). The test lines shown in Table 14 are maize hybrids of two genetic 

backgrounds: B116 (Committee for Agricultural Development, Iowa State University) and 

CH01 (private inbred line) containing the GM event or the non-transgenic near-isogenic 

comparator (non-transgenic control). Each of these hybrids was tested at each of the 17 

separate locations. 
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Table 14. Maize hybrids tested in agronomic evaluations in 2009 

VII.C.2.1. Evaluation parameters, analysis and statistics 

All trials were conducted using a randomized complete block design, with three 

replicated plots of each entry per location. Weed control was limited to conventional and 

cultural practices (hand hoeing); no broad-spectrum herbicides, such as glyphosate or 

glufosinate, were allowed except as a pre-plant or pre-emergence herbicide. 

Typical agronomic evaluations used for maize were conducted (Table 15). Means for the 

event were compared to the appropriate non-transgenic control using a two-sided 

Dunnett’s test with alpha = 0.05 (Moore and McCabe, 1999). The standard deviation and 

range of values collected across all locations are also reported (Tables 16 and 17). 

Hybrid Tested Pedigree of the hybrids 

Event VCO-Ø1981-5 hybrid BC0BS2 VCO-Ø1981-5 X B116 (BC0BS2B) 

Non-transgenic near-isogenic hybrid BC0BS2 negative segregant X B116 

(BC0BS2B) 

Event VCO-Ø1981-5 hybrid BC0BS2 VCO-Ø1981-5 X CH01 (BC0BS2CH) 

Non-transgenic near-isogenic hybrid BC0BS2 negative segregant X CH01 

(BC0BS2CH) 
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Table 15. Traits evaluated in the 2009 field trials 

General 

Characteristic Trait Growth Stage Description Scale 

Emergence Emergence V2 - V4 Plant emergence 

Percentage of plants 

to emerge after 

germination 

Vegetative 

characteristics 

Plant height Maturity

Five plants per plot. 

Measure from ground to 

base of flag leaf. 

Measured in inches 

Ear height Maturity

Five plants per plot. 

Measure from ground to 

point of attachment of 

primary ear. 

Measured in inches 

Final stand count Preharvest 

Total number of plants in 

middle two rows. Number of plants 

Stalk lodging Preharvest Plants broken below ear. Percentage of total 

Root lodging Preharvest 

Plants leaning at soil 

surface greater than 30° 

from vertical. 

Percentage of total 

Grain moisture Harvest Moisture content of 

harvested, shelled grain. Percent moisture 

Grain weight Harvest Total raw weight of grain 

harvested. Pounds per plot 

Reproductive 

parameters 

Days to 50% 

pollen shed 

Pollenshed 

Number of days from 

planting to the day 50% 

of plants are shedding 

pollen. 

Number of days 

Days to 50% 

silking Silking 

Days from planting to 

the day 50% of the 

plants have silks 

emerged from the 

primary ear. 

Number of days 

Dropped ears Preharvest 

Ears on the ground, no 

longer attached to the 

stalk. 

Percentage of total 

Barrenness Preharvest Plants with no ears. Percentage of total 

Yield Harvest Yield corrected to 15% 

moisture. Bushels per acre 

Evaluation Details for Certain Parameters 

Pollination Data: Plots were evaluated daily during the pollination and tassel formation. 

Plots were considered at 50% pollen shed when 50% of the plants in each plot had 

dehisced anthers. Similarly, plots were recorded to be at 50% silking when 50% of the 

plants had emerged silks on the primary ear. 

Plant and Ear Heights: Plant and ear heights were measured according to maize 

growing stage. Plant heights were measured from ground level to the base of the flag 

leaf. Ear heights were measured from the ground level to the base of the primary ear 

shank. 
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VII.C.2.2. Field locations, preparations and conditions 

A total of 17 field sites were utilized in 2009 across six US states to collect agronomic 

data along with samples for other regulatory studies, such as compositional analysis. 

The locations include diverse environments within the major maize growing regions of 

the US. Off-season locations in Puerto Rico were used for breeding purposes (Table 

12). The agronomic practices were representative of the location in, which the field trials 

were conducted. 

VII.C.2.3. Results of agronomic performance evaluation

Vegetative and reproductive characteristics 

As outlined in Table 15, a range of different vegetative and reproductive agronomic 

characteristics were measured across the 17 locations in the 2009 field trials. Ranges, 

standard deviations and p-values were recorded and calculated across all the locations 

(Tables 16 and 17). 

With only minor exceptions, the evaluation of vegetative agronomic characteristics 

showed no statistical differences between event VCO-Ø1981-5 and its non-transgenic 

near-isogenic comparator (non-transgenic control). The grain moisture was slightly 

higher for the non-transgenic control than event VCO-Ø1981-5, but the ranges were 

large and overlapping, and the standard deviations also overlapped. In the B116 genetic 

background, differences were measured in the final stand count where the nontransgenic control had slightly less plants as compared to event VCO-Ø1981-5, but for 

the CH01 background there were no statistical differences in stand count. 

There were no statistically significant differences in any of the reproductive 

measurements, with the exception of barrenness for CH01 background, but the 

numerical values were so low that these statistics are not meaningful (e.g., the range is 

many times larger than the mean value). 
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Table 16. Vegetative characteristics

Agronomic 

characteristic 

(unit) 

Genetic 

background 

VCO-Ø1981-5 

hybrid 

Non-transgenic 

control 

Plant height 

(inches) 

B116 

116.9  14.7 113.5  14.2 Mean  St Dev 

75.4-123.3 71.3-122.1 Range 

0.7918 p-value 

CH01 

110.4  12.1 106.8  13.5 Mean  St Dev 

77.1-128.7 63.8-130.7 Range 

0.7632 p-value 

Ear height 

(inches) 

B116 

63.3  7.7 59.9  6.9 Mean  St Dev 

34.7-65.7 33.2-61.1 Range 

0.4040 p-value 

CH01 

56.8  6.7 52.2  7.4 Mean  St Dev 

35.6-58.3 22.2-54.5 Range 

0.2095 p-value 

Final stand count 

(# plants) 

B116 

48.8  8.1 43.9  9.2 Mean  St Dev 

39.7-72 34.7-72 Range 

0.0067 p-value 

CH01 

48.1  7.7 45.6  9.6 Mean  St Dev 

38.0-72 36.3-72 Range 

0.1797 p-value 

Stalk lodging 

(% of total) 

B116 

3.5  3.0 3.1  4.2 Mean  St Dev 

0-10.0 0-12.9 Range 

0.6302 p-value 

CH01 

4.5  3.9 6.0  7.3 Mean  St Dev 

0-12.3 0-23.5 Range 

0.4118 p-value 

Root lodging 

(% of total) 

B116 

12.4  21.9 9.1  21.6 Mean  St Dev 

0-83.3 0-83.3 Range 

0.4992 p-value 

CH01 

2.0  4.0 0.8  2.0 Mean  St Dev 

0-15.4 0-8.1 Range 

0.1632 p-value 

Grain moisture 

(moisture in % fw) 

B116 

25.3  3.6 27.1  5.3 Mean  St Dev 

17.8-31.0 19.0-36.7 Range 

0.0923 p-value 

CH01 

21.7  3.9 24.2  4.6 Mean  St Dev 

16.5-29.2 14.4-29.5 Range 

0.0318 p-value 

Grain weight 

(pounds per plot) 

B116 

19.5  5.4 18.2  4.7 Mean  St Dev 

5.2-26.4 5.5-26.8 Range 

0.2292 p-value 

CH01 

19.9  5.2 18.6  5.8 Mean  St Dev 

6.2-27.5 3.9-28.0 Range 

0.3662 p-value 
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Table 17. Reproductive parameters 

Agronomic 

characteristic 

Genetic 

background 

VCO-Ø1981-5 

hybrid 

Nontransgenic 

control 

Days to 50% 

pollen shed 

(# days) 

B116 

72.6  8.6 73.3  8.5 Mean  St Dev 

59.0-94.0 59.0-92.7 Range 

0.6978 p-value 

CH01 

72.3  8.4 72.7  8.8 Mean  St Dev 

58.0-92.7 57.3-93.7 Range 

0.7365 p-value 

Days to 50% 

silking 

(# days) 

B116 

74.6  8.8 75.0  8.2 MeanSt Dev 

59.7-96.3 59.3-94.7 Range 

0.8087 p-value 

CH01 

72.6  9.1 72.6  9.2 Mean  St Dev 

57.7-95.3 56.7-95.7 Range 

0.9003 p-value 

Dropped ears 

(% of total) 

B116 

0.1  0.3 0.2  0.7 Mean  St Dev 

0-0.7 0-2.6 Range 

0.5282 p-value 

CH01 

0.4  1.0 0.2  0.5 Mean  St Dev 

0-3.5 0-2.0 Range 

0.2648 p-value 

Barrenness 

(% of total) 

B116 

1.3  2.5 2.2  3.5 Mean  St Dev 

0-8.9 0-11.8 Range 

0.2497 p-value 

CH01 

1.0  1.0 4.0  6.8 Mean  St Dev 

0-5.5 0-25.1 Range 

0.0167 p-value 

Yield 

(bushels per 

acre) 

B116 

143.0  45.4 130.7  39.4 Mean  St Dev 

40.8-209.5 42.8-219.9 Range 

0.1584 p-value 

CH01 

150.4  38.9 137.9  40.1 Mean  St Dev 

58.7-224.5 31.8-208.0 Range 

0.1896 p-value 

Emergence 

Emergence data from the 2009 field trials are shown in Table 18. While the ranges for 

percent emergence overlap well between event VCO-Ø1981-5 and the non-transgenic 

control, means for the non-transgenic control are slightly lower. These differences in 

emergence may be attributed to the seed quality of the non-transgenic control. Since 

there was considerably more event VCO-Ø1981-5 seed than non-transgenic control 

seed produced in the winter nursery in Puerto Rico, the highest quality seed could be 

selected for the transgenic plots. For the non-transgenic comparator (non-transgenic 

control), some lower quality seed had to be used, in order to have sufficient seed for all 

17 field locations, which may have impacted the emergence values. At 12 of the 17 

locations, the emergence was numerically higher for event VCO-Ø1981-5 seeds, while 5 

of the locations had split data (i.e., one background showed higher or equal germination 

than the non-transgenic control, and the other background showed lower germination 

than the non-transgenic control). The emergence was numerically lower than typically 
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(Tang et al., 2000) observed (65-78%), regardless of the genetic background or the 

presence or absence of the herbicide tolerance trait. Despite the statistically significant 

differences , the ranges for the percent emergence for the VCO-Ø1981-5 hybrids in both 

B116 and CH01 backgrounds (55.7 – 96.4 and 49.3-100) overlapped with the ranges for 

the respective non-transgenic controls (46.1-82.1 and 52.6-85.2). For all ranges, the 

minimum and maximum values were also spread out over a wide range of values. The 

same observation applies to the standard deviations, as they are relatively high in 

relation to their respective means and thus indicate that values had a wide distribution. 

Considering the wide variation observed for all the entries tested, regardless of genetic 

background or presence/absence of the introduced trait, the statistically significant 

differences based on the p-values are therefore not biologically significant. 

Table 18. Emergence 

VII.D. Biotic and abiotic stress characteristics 

Specific observations were recorded during the conduct of the 2009 agronomic 

performance field trials regarding ecological characteristics of event VCO-Ø1981-5 

(BC0BS2 in B116 and CH01 backgrounds, Figure 3) and their respective non-transgenic 

controls. The rating scales for ecological evaluations are listed in Table 19. Late season 

disease ratings are summarized in Table 20 and the severity of a specific stressor is 

shown in Table 21. No differences between the event VCO-Ø1981-5 entries and the 

non-transgenic controls were identified. 

Table 19. Disease rating scales for ecological evaluations 

General 

characteristic Trait Growth Stage Description Scale 

Ecological 

interactions 

Late season 

disease rating Maturity Rating for disease pressure 

% of leaf area with 

symptoms: 

1 = no visible symptoms 

2 = <1% of leaf area 

3 = 1-5% of leaf area 

4 = 6-20% of leaf area 

5 = 21-50% of leaf area 

6 = 51-75% of leaf area 

7 = 76-90% of leaf area 

8 = 91-99% of leaf area 

9 = plant is dead 

Insect, disease, 

weed and abiotic 

stressors 

Over 

season 

Visual 

observations Descriptive 

Agronomic 

characteristic 

(unit) 

Generation/Genetic 

background 

VCO-Ø1981-5 

hybrid 

Non-transgenic 

control 

Emergence 

(%) 

BC0BS2B/B116 

77.8  12.4 65.4  10.6 Mean  SD 

55.7-96.4 46.1-82.1 Range 

0.001 p-value 

BC0BS2CH/CH01 

76.6  13.9 68.9  11.5 Mean  SD 

49.3-100 52.6-85.2 Range 

0.0052 p-value 
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Table 20. Disease ratings 

Agronomic 

characteristic 

(unit) 

Genetic 

background VCO-Ø1981-5 hybrid Non-transgenic control 

Late season 

disease rating 

(Scale 1-9) 

B116 

4 4 Mean 

2-6 2-6 Range 

0.8032 p-value 

CH01 

4 4 Mean 

2-6 2-6 Range 

0.7805 p-value 

Table 21. Ecological stressors 

Stressor State County1 Severity2

Difference to 

non-transgenic 

control 

Insects 

Fall armyworm 

Spodoptera fugiperda IL Clinton Mild No difference 

Japanese beetles 

Popillia japonica

IL Clinton Mild No difference 

WI Walworth Mild No difference 

European corn borer 

Ostrinia nubilalis

IL Stark Very mild No difference 

MN Stearns Mild No difference 

WI Walworth Mild No difference 

Aphids 

Acyrthosiphon pisum MN Stearns Mild No difference 

Grasshoppers 

Melanoplus spp 

MN Stearns Mild No difference 

Northern corn rootworm 

Diabrotica barberi

IA Kossuth Moderate No difference 

IA Wright Mild No difference 

Black cutworm 

Agrotis ipsilon IA Wright Mild No difference 

Common stalk borer 

Papaipema nebris WI Walworth Mild No difference 

Western corn rootworm 

Diabrotica virgifera virgifera WI Walworth Moderate No difference 

Diseases 

Gray leaf spot 

IL Clinton Mild No difference 

IL Stark Very mild No difference 

MN Stearns Mild No difference 

IA Jasper Mild No difference 
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Table 21. Ecological stressors (continued) 

Gray leaf spot 

IA Wright Mild No difference 

IA Scott Mild No difference 

NE Valley(1) Mild No difference 

NE Valley(2) Mild No difference 

Common rust 

IL Stark-=8 Mild to 

moderate No difference 

IA Scott Mild No difference 

NE Valley(1) Mild No difference 

NE Valley(2) Mild No difference 

WI Walworth Mild No difference 

Northern leaf blight IA Kossuth Mild No difference 

Northern corn leaf spot IA Webster Mild No difference 

Anthracnose IA Webster Moderate No difference 

Common smut IA Wright Mild No difference 

Abiotic 

Excess moisture 

IL Clinton Severe No difference 

IN Parke Moderate No difference 

IA Scott Mild No difference 

Nitrogen deficiency IA Wright Mild No difference 

1 three replications per location 

2 based on visual, non-quantitative observations related to crop growth and development 

VII.E. Seed dormancy evaluation 

Seed dormancy and germination analysis were conducted using standard laboratory 

assays. Seeds of event VCO-Ø1981-5 and the non-transgenic control used in the 

germination studies were produced in the greenhouse and had a slightly different 

genetic background from the seed used to generate the emergence data from the 2009 

field trials (BC1B1S2, Figure 3). The warm germination test was conducted in 

accordance with the Association of Official Seed Analysts “Rules for Testing Seed” 

(AOSA, 2010). 

The tetrazolium test was performed on the resulting ungerminated seeds at the end of 

each test to determine the percent non-viable seed. The germination and viability results 

provided below (Table 22) indicate that the germination and dormancy characteristics of 

event VCO-Ø1981-5 are not different as compared to the non-transgenic control. 
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Table 22. Seed germination 

Line 

Warm germination Cold germination 

Mean % 

germination 

% Nonviable seed Mean % 

germination 

% Nonviable seed 

Event VCO-Ø1981-5 99.6 0.4 97.0 3.0 

Non-transgenic control 99.6 0.4 93.0 7.0 

The laboratory studies show a high level of germination, with equivalent germination for 

the event VCO-Ø1981-5 and the non-transgenic control (99.6%) under warm conditions. 

A small difference in germination, 97% and 93%, respectively, was seen under cold 

conditions. 

In conclusion, the observations and data collected from all the agronomic field trials 

conducted to evaluate agronomic performance and environmental effects found that 

event VCO-Ø1981-5 maize is equivalent to other maize hybrids with only a few minor 

differences. Event VCO-Ø1981-5 maize did not exhibit any plant pest characteristics. 

VII.F. Composition analysis 

VII.F.1. Introduction 

Compositional analyses establish the nutritional value, and can identify any potential 

unintended effects of the transformation process on the plant composition, by measuring 

key nutrients, anti-nutrients and secondary plant metabolites. To evaluate the nutritional 

quality of event VCO-Ø1981-5, composition evaluations were carried out from field 

samples collected from five locations in three states in 2009 in the US (BC0BS2, Figure 

3). As part of the evaluation of new maize varieties and to support the safety 

assessment, a range of compositional parameters were tested for event VCO-Ø1981-5 

and compared to a non-transgenic near-isogenic hybrid (also referred to as the nontransgenic or negative control) and three commercial maize hybrids, representing the 

natural variation found in maize hybrids. The commercial hybrids were selected for their 

adaption to major US maize growing regions. Hybrids Ag5539 (RM: 105 days) and 

Ag58036 (RM: 103) are well adapted to the upper Midwest region. Hybrid Ag7584 (RM: 

113) is well adapted to the lower Midwest. Hybrids Ag58036 and Ag7584 are currently 

sold in the US. Hybrid AgR5539 was last sold in 2010. 

Detailed compositional analyses of 71 analytes were conducted for forage and grain 

samples of event VCO-Ø1981-5, a non-transgenic control and three reference hybrid 

varieties using standard methods. The analytes evaluated are the standard parameters 

by which maize varieties are assessed and are outlined in the OECD Consensus 

Document on Compositional Considerations for New Varieties of Maize (Zea mays) 

(OECD, 2002). 

Results are presented and compared to the ranges of the references material grown, 

harvested, and analyzed simultaneously with the test material and to the published 
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values (ILSI, 2007) for nutritional content of maize forage (Table 23) and grain (Tables 

24-29). 

VII.F.2. Seed genetics, field trial design, and sampling 

Seeds of event VCO-Ø1981-5 and the non-transgenic control had the same genetic 

background in order to better compare the nutritional data: BC0BS2 x B116 and 

BC0BS2 VCO-Ø1981-5 x B116. 

Plant material derived from event VCO-Ø1981-5 and the non-transgenic control was 

produced and collected at five field locations across the US Corn Belt in 2009. The 

reference hybrids (AgR5539, AgR7584, and AgR58036) were handled the same as the 

event VCO-Ø1981-5 hybrids and their respective non-transgenic control. Data from the 

reference hybrids is used in conjunction with published data to help determine normal 

ranges and variability within the measured parameters. 

VII.F.3. Analytical methods 

The analytical methods were AOAC, AACC and AOCS International Methods or 

published methods as detailed in Appendix 2, Section 2.F. 

VII.F.4. Nutritional composition 

VII.F.4.1. Overview of the analysis 

Detailed compositional analysis was conducted in accordance with the OECD 

Consensus Document on Compositional Considerations for New Varieties of Maize (Zea 

mays) (OECD, 2002; Bernard and MacIntosh, 2011e). Compositional analyses of forage 

samples included protein, fat, acid detergent fiber (ADF), neutral detergent fiber (NDF), 

crude fiber, ash, calcium, phosphorus, and carbohydrates. Compositional analyses of 

grain samples included protein, fat, ADF, NDF, crude fiber, ash, carbohydrates, fatty 

acids, amino acids, vitamins, minerals, key anti-nutrients, and secondary metabolites. 

.

Data means, standard deviations, ranges and p-values were determined for the 

compositional data. Data were analyzed using JMP 8 software (SAS Institute, Cary, NC) 

and the equation Yij = U + Ti + Lj + LTij + eij. For each analyte an ANOVA model was fit 

using the site and line main effects and the site by line interaction. P-values (≤0.05%) 

were not reported where the means were below the LOD, or where missing data made 

the effect non-testable. When data points were at or below the LOQ, the LOQ value was 

used to calculate the averages, standard deviations and data ranges. The range of 

determined values for each of the analytes for the reference lines is also reported. 
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VII.F.4.2. Analysis of proximates and minerals in maize forage 

The levels of proximates were measured in forage samples of event VCO-Ø1981-5, the 

non-transgenic control, and three reference hybrids. The results are reported in Table 

23. No statistically significant differences were observed between event VCO-Ø1981-5 

and non-transgenic control (p-values were >0.05). 

Table 23. Proximates and minerals in event VCO-Ø1981-5 forage 

Analyte 

(% of dry weight) 

VCO-Ø1981-5 

hybrid 

Nontransgenic 

control 

Range 

Reference 

hybrids Published1

Protein 

Mean ± SD 6.1 ± 1.3 6.2 ± 1.6 

Range 3.9-8.3 3.4-8.9 3.6-9.1 3.9-11.6 

p-value 0.640 

Fat 

Mean ± SD 2.0 ± 0.3 2.1 ± 0.6 

Range 1.4-2.7 1.1-3.2 1.3-3.5 0.3-4.1 

p-value 0.499 

Acid detergent 

fiber 

Mean ± SD 34.3 ± 3.6 32.2 ± 3.1 

Range 27.8-43.0 23.0-35.6 19.1-41.0 18.8-47.4 

p-value 0.168 

Neutral 

detergent fiber 

Mean ± SD 55.5 ± 4.1 52.9 ± 4.6 

Range 49.5-64.6 40.8-61.0 34.4-61.8 26.4-57.9 

p-value 0.125 

Crude fiber 

Mean ± SD 28.0 ± 3.8 26.6 ± 1.6 

Range 22.8-38.3 23.6-30.6 14.8-32.5 n/a 

p-value 0.234 

Ash 

Mean ± SD 5.6 ± 0.8 5.4 ± 0.7 

Range 4.4-7.0 4.7-7.0 3.9-8.0 1.5-6.8 

p-value 0.236 

Calcium 

Mean ± SD 0.25 ± 0.04 0.25 ± 0.03 

Range 0.18-0.35 0.21-0.30 0.14-0.38 0.07-0.58 

p-value 0.935 

Phosphorus 

Mean ± SD 0.19 ± 0.04 0.19 ± 0.04 

Range 0.14-0.27 0.12-0.26 0.12-0.29 0.09-0.32 

p-value 0.629 

Carbohydrates 

(calculated) 

Mean ± SD 86.3 ± 1.9 86.3 ± 2.2 

Range 83.5-89.3 82.2-90.4 81.0-89.9 80.8-92.1 

p-value 0.928 1 ILSI (2006) Crop Composition Database (Version 3.0) 

n/a; not available 
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VII.F.4.3. Analysis of proximates in maize grain 

The levels of proximates were measured in grain samples of event VCO-Ø1981-5, the 

non-transgenic control, and three reference hybrids. The results are reported in Table 

24. No statistically significant differences were observed between event VCO-Ø1981-5 

and the non-transgenic control (p-values were >0.05). 

Table 24. Proximates in event VCO-Ø1981-5 grain 

Analyte 

(% of fresh weight) 

VCO-Ø1981-5 

hybrid 

Nontransgenic 

control 

Range 

Reference 

hybrids Published1

Moisture 

Mean ± SD 12.6 ± 1.6 13.0 ± 2.1 

Range 10.1-15.0 10.2-17.5 9.7-15.8 6.9-21.4 

p-value 0.297 

Protein 

Mean ± SD 10.3 ± 1.2 11.0 ± 1.2 

Range 7.1-11.8 7.4-12.3 5.6-13.0 6.7-14.7 

p-value 0.227 

Fat 

Mean ± SD 4.4 ± 0.9 4.4 ± 0.8 

Range 2.6-5.3 2.8-5.4 2.5-7.0 2.5-5.3 

p-value 0.990 

Acid detergent 

fiber 

Mean ± SD 3.5 ± 0.7 3.3 ± 0.7 

Range 2.3-4.4 2.0-4.1 2.3-4.2 1.8-11.3 

p-value 0.154 

Neutral 

detergent fiber 

Mean ± SD 12.0 ± 1.0 11.9 ± 1.1 

Range 10.6-14.4 10.1-14.3 7.4-15.0 6.2-20.6 

p-value 0.716 

Crude fiber 

Mean ± SD 2.8 ± 0.4 2.8 ± 0.6 

Range 1.5-3.4 1.1-3.3 1.4-3.5 1.4-3.3 

p-value 0.562 

Total dietary 

fiber 

Mean ± SD 9.9 ± 0.8 10.1 ± 0.7 

Range 8.5-11.4 8.6-11.6 7.5-11.1 9.0-35.3 

p-value 0.289 

Starch 

Mean ± SD 68.2 ± 1.6 67.0 ± 1.7 

Range 66.1-70.8 64.8-71.0 64.1-74.5 26.5-73.8 

p-value 0.090 

Ash 

Mean ± SD 1.3 ± 0.06 1.3 ± 0.04 

Range 1.2-1.4 1.2-1.4 1.1-1.7 0.6-6.3 

p-value 0.373 

Carbohydrates 

(calculated) 

Mean ± SD 84.0 ± 1.8 83.3 ± 1.8 

Range 82.0-88.7 81.9-88.5 79.3-90.5 77.4-89.5 

p-value 0.282 1 ILSI (2006) Crop Composition Database (Version 3.0) 
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VII.F.4.4. Analysis of fatty acids in maize grain 

The levels of twenty-four fatty acids were measured in event VCO-Ø1981-5 grain, the 

non-transgenic control, and three reference hybrids. The levels of the following fatty 

acids were below the level of quantitation for the assays used (data not shown): caprylic 

(C8:0), capric (C10:0), myristic (C14:0), myristoleic (C14:1), pentadecanoic (C15:0), 

pentadecenoic (C15:1), heptadecanoic (17:0), heptadecenoic (C17:1), gamma linolenic 

(C18:3), eicosadienoic (C20:2), eicosatrienoic (C20:3), arachidonic (C20:4), and erucic 

(C22:1). Results for those fatty acids that were measurable are shown in Table 25. In the 

majority of fatty acids, for which levels could be evaluated, no statistically significant 

differences were observed between event VCO-Ø1981-5 and the non-transgenic control 

(p-values were ≥0.05). 

A statistical difference was observed for the analyte linoleic acid. The calculated means 

for both the event VCO-Ø1981-5 and the non-transgenic control were slightly above the 

data ranges for the three commercial varieties and the ILSI composition database. 

However, event VCO-Ø1981-5 values were lower than the non-transgenic control and 

closer to the commercial and published data ranges. For the non-essential fatty acid 

analytes palmitic and eicosenoic acid, the p-values determined showed a statistical 

difference in the calculated means; however, the standard deviations and ranges 

overlap, and the means and data ranges fell within the reference and published data 

ranges for those analytes. Therefore, it is unlikely that these differences indicate any 

biological significance. 
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Table 25. Fatty acids in event VCO-Ø1981-5 grain 

Analyte 

(% Total fatty acids) 

VCO-Ø1981-5 

hybrid 

Non-transgenic 

control 

Range 

Reference 

hybrids Published1

Lauric 

(C12:0) 

Mean ± SD 0.06 ± 0.6 0.06 ± 0.5 

Range 0-0.15 0-0.130 0-0.18 ND-0.3 

p-value 0.802 

Palmitic 

(C16:0) 

Mean ± SD 11.04 ± 0.16 10.80 ± 0.26 

Range 10.65-11.25 10.40-11.32 8.36-12.62 8.57-17.46 

p-value 0.0162

Palmitoleic 

(C16:1) 

Mean ± SD 0.15 ± 0.01 0.14 ± 0.01 

Range 0.13-0.16 0.13-0.16 0-0.14 0.11-0.26 

p-value 0.182 

Stearic 

(C18:0) 

Mean ± SD 1.99 ± 0.18 2.05 ± 0.15 

Range 1.67-2.29 1.76-2.25 1.77-2.70 1.02-2.86 

p-value 0.058 

Oleic 

(C18:1) 

Mean ± SD 19.88 ± 1.16 19.06 ± 0.99 

Range 18.39-21.79 17.55-20.93 21.91-33.36 17.4-38.5 

p-value 0.076 

Linoleic 

(C18:2) 

Mean ± SD 64.76 ± 1.30 65.79 ± 1.20 

Range 62.37-66.34 63.42-67.70 51.61-62.64 47.7-65.6 

p-value 0.0122

Linolenic 

(C18:3) 

Mean ± SD 1.12 ± 0.11 1.16 ± 0.11 

Range 0.97-1.29 1.00-1.37 0.950-1.60 0.77-2.25 

p-value 0.111 

Arachidic 

(C20:0) 

Mean ± SD 0.44 ± 0.03 0.44 ± 0.21 

Range 0.41-0.49 0.40-0.47 0.38-0.58 0.34-0.57 

p-value 0.650 

Eicosenoic 

(C20:1) 

Mean ± SD 0.21 ± 0.01 0.20 ± 0.01 

Range 0.20-0.24 0.18-0.23 0.23-0.35 0.17-0.45 

p-value 0.0252

Behenic 

(C22:0) 

Mean ± SD 0.09 ± 0.09 0.06 ± 0.08 

Range 0-0.18 0-0.17 0-0.20 0.11-0.29 

p-value 0.258 

Lignoceric 

(C24:0) 

Mean ± SD 0.24 ± 0.03 0.23 ± 0.03 

Range 0.20-0.30 0.19-0.33 0-0.28 0.14-0.23 

p-value 0.704 1 ILSI (2006) Crop Composition Database (Version 3.0) 

2 p ≤ 0.05 indicates a statistical difference 
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VII.F.4.5. Analysis of amino acids in maize grain 

The levels of essential and non-essential amino acids were measured in grain samples 

of event VCO-Ø1981-5, the non-transgenic control, and three reference hybrids. The 

results are reported in Table 26. In the majority of amino acids evaluated, no statistically 

significant differences were observed between event VCO-Ø1981-5 and the nontransgenic control (p-values were >0.05). 

For the analytes arginine, phenylalanine, and tyrosine the p-values showed a statistical 

difference in the calculated means; however, the ranges and standard deviations 

overlap, and the means and data ranges fell within the reference and published data 

ranges for these analytes. Therefore, it is unlikely that these differences indicate any 

biological significance. 

Table 26. Amino acids in event VCO-Ø1981-5 grain 

Analyte 

(% dry weight) 

VCO-Ø1981-5 

hybrid 

Non-transgenic 

control 

Range 

Reference 

hybrids Published1

Alanine 

Mean ± SD 0.796 ± 0.111 0.865 ± 0.112 

Range 0.527-0.934 0.529-1.003 0.378-1.097 0.489-1.20 

p-value 0.164 

Arginine 

Mean ± SD 0.395 ± 0.034 0.429 ± 0.038 

Range 0.307-0.445 0.334-0.479 0.267-0.524 0.119-0.637 

p-value 0.0482

Aspartic acid 

Mean ± SD 0.749 ± 0.099 0.789 ± 0.099 

Range 0.478-0.907 0.521-0.906 0.381-0.998 0.335-0.963 

p-value 0.344 

Cystine 

Mean ± SD 0.128 ± 0.031 0.132 ± 0.022 

Range 0.070-0.173 0.071-0.165 0.071-0.216 0.125-0.325 

p-value 0.690 

Glutamic acid 

Mean ± SD 2.125 ± 0.297 2.320 ± 0.291 

Range 1.379-2.489 1.465-2.699 0.986-2.963 0.965-3.12 

p-value 0.153 

Glycine 

Mean ± SD 0.389 ± 0.030 0.400 ± 0.024 

Range 0.312-0.432 0.349-0.439 0.276-0.447 0.184-0.498 

p-value 0.429 

Histidine 

Mean ± SD 0.311 ± 0.030 0.330 ± 0.036 

Range 0.239-0.353 0.252-0.407 0.176-0.363 0.137-0.416 

p-value 0.200 

Isoleucine 

Mean ± SD 0.374 ± 0.047 0.411 ± 0.047 

Range 0.251-0.432 0.273-0.472 0.192-0.494 0.179-0.568 

p-value 0.083 

Leucine 

Mean ± SD 1.316 ± 0.210 1.465 ± 0.211 

Range 0.778-1.584 0.843-1.687 0.534-1.802 0.654-2.100 

p-value 0.093 
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Table 26. Amino acids in event VCO-Ø1981-5 grain (continued) 

Analyte 

(% of dry weight)

VCO-Ø1981-5 

hybrid 

Non-transgenic 

control

Range 

Reference 

hybrids Published1

Lysine 

Mean ± SD 0.305 ± 0.036 0.314 ± 0.044 0.197-0.418 0.172-0.597 Range 0.257-0.372 0.260-0.378 

p-value 0.524 

Methionine 

Mean ± SD 0.128 ± 0.033 0.132 ± 0.019 

Range 0.075-0.184 0.077-0.148 0.065-0.200 0.129-0.326 

p-value 0.653 

Phenylalanine 

Mean ± SD 0.525 ± 0.075 0.587 ± 0.081 

Range 0.334-0.622 0.352-0.676 0.236-0.702 0.244-0.809 

p-value 0.0502

Proline 

Mean ± SD 0.957 ± 0.132 1.021 ± 0.110 

Range 0.630-1.132 0.689-1.134 0.460-1.177 0.462-1.34 

p-value 0.207 

Serine 

Mean ± SD 0.497 ± 0.056 0.523 ± 0.060 

Range 0.345-0.560 0.361-0.600 0.272-0.644 0.254-0.728 

p-value 0.171 

Threonine 

Mean ± SD 0.372 ± 0.038 0.391 ± 0.037 

Range 0.273-0.420 0.289-0.441 0.225-0.445 0.231-0.666 

p-value 0.253 

Tryptophan 

Mean ± SD 0.075 ± 0.008 0.076 ± 0.005 

Range 0.060-0.087 0.063-0.082 0.050-0.097 0.027-0.090 

p-value 0.656 

Tyrosine 

Mean ± SD 0.234 ± 0.036 0.282 ± 0.081 

Range 0.165-0.281 0.157-0.441 0.135-0.337 0.103-0.534 

p-value 0.0082

Valine 

Mean ± SD 0.509 ± 0.061 0.539 ± 0.050 

Range 0.351-0.578 0.394-0.595 0.275-0.629 0.266-0.723 

p-value 0.222 1 ILSI (2006) Crop Composition Database (Version 3.0) 

2 p ≤ 0.05 indicates a statistical difference 
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VII.F.4.6. Analysis of vitamins in maize grain 

The levels of vitamins were measured in grain samples of event VCO-Ø1981-5, the nontransgenic control, and three reference hybrids. The results are reported in Table 27. For 

the majority of vitamins evaluated, no statistically significant differences were observed 

between event VCO-Ø1981-5 and the non-transgenic control (p-values were >0.05). 

For the analytes beta carotene, vitamin B3, and total tocopherols, the p-values 

determined showed a statistical difference in the calculated means; however, the 

standard deviations and data ranges overlapped. 

For total tocopherols, the means and the standard deviations are calculated on total 

tocopherols determined for each genotype from three replicates for each of the five field 

locations. The ranges reported in Table 27 are the minimum and the maximum results 

reported for the three replicates in all locations. As shown in Table 28 below, the 

standard deviation ranges overlap for the three entries, even though the mean for the 

non-transgenic control is below the value for the Mean-SD (26.5) of VCO-Ø1981-5 

event. All means fell within the reference and published data ranges for these analytes. 

Therefore, it is unlikely that these differences indicate any biological significance. 
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Table 27. Vitamins in event VCO-Ø1981-5 grain

Analyte 

(mg/kg of dry weight) 

VCO-Ø1981-5 

hybrid 

Non-transgenic 

control 

Range 

Reference 

hybrids Published1

Beta 

carotene 

Mean ± SD 4.79 ± 1.30 3.97 ± 0.71 

Range 2.50-6.42 3.11-5.43 2.50-15.62 0.53-17.03 

p-value 0.00722

Vitamin B1 

Mean ± SD 3.45 ± 0.62 3.24 ± 0.36 

Range 2.53-4.46 2.69-4.03 2.41-4.27 2.51-40.0 

p-value 0.221 

Vitamin B2 

Mean ± SD 0.91 ± 0.05 0.94 ± 0.10 <0.9 (all 

below 

LOQ) 

Range 0.90-1.10 0.90-1.26 0.8-2.36 

p-value not calculated3

Folic acid 

Mean ± SD 0.83 ± 0.18 0.85 ± 0.16 

Range 0.43-1.06 0.57-1.08 0.46-1.19 0.15-1.32 

p-value 0.741 

Vitamin B3 

(Niacin) 

Mean ± SD 23.84 ± 4.90 19.64 ± 3.90 12.50-

38.20 Range 13.30-32.51 14.41-26.87 14.11-39.91

p-value 0.00522

Vitamin B5 

Mean ± SD 5.54 ± 0.48 5.30 ± 0.67 

Range 4.56-6.31 4.16-6.20 4.23-10.12 n/a 

p-value 0.201 

Vitamin B6 

Mean ± SD 3.45 ± 1.09 3.06 ± 1.40 

Range 1.74-5.46 0.33-4.97 1.34-7.09 3.68-11.32 

p-value 0.494 

Total 

tocopherols 

Mean ± SD 37.7 ± 11.25 25.8 ± 8.81 

Range 20.8-58.6 17.6-50.9 22.4-65.30 4.1-41.7 

p-value <0.000122

 1 ILSI (2006) Crop Composition Database (Version 3.0) 2 p ≤ 0.05 indicates a statistical difference 3 p value could not be calculated because there was no variability recorded for the test hybrid 

 n/a; not available 

Table 28. Values for total tocopherols 

 Mean Mean - SD Mean + SD MIN MAX 

VCO-Ø1981-5 37.7 26.5 49.0 20.8 58.6 

Non-transgenic 

control 25.8 17.0 34.6 17.6 50.9 

Reference 

Hybrids 45.2 34.2 56.2 22.4 65.3 
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VII.F.4.7. Analysis minerals in maize grain 

The levels of minerals were measured in grain samples of event VCO-Ø1981-5, the nontransgenic control, and three reference hybrids. The results are reported in Table 29. No 

statistically significant differences were observed between event VCO-Ø1981-5 and the 

non-transgenic control (p-values were ≥0.05) in the majority of minerals evaluated. 

For the analytes calcium, copper, and potassium the p-values determined showed a 

statistical difference in the calculated means. However, for potassium the standard 

deviations and data ranges overlapped for event VCO-Ø1981-5 hybrids and the nontransgenic control, and both data ranges fell within the reference and published data 

ranges for these analytes. For copper and calcium, the standard deviations and data 

ranges overlapped for the event VCO-Ø1981-5 and the non-transgenic control, and the 

means and data ranges from both entries overlapped those generated from the 

reference hybrids. The data ranges for all 5 entries fell within the published data ranges 

for copper and calcium. Taken together, it is unlikely that these small differences indicate 

any biological significance. 

Table 29. Minerals in event VCO-Ø1981-5 grain 

Analyte 

(mg/kg of dry weight) 

VCO-Ø1981-5 

hybrid 

Nontransgenic 

control 

Range 

Reference 

hybrids Published1

Calcium 

Mean ± SD 52.0 ± 10.8 60.0 ± 11.1 

Range 34.4-70.0 39.0-76.0 29.0-66.0 21.6-163.1 

p-value 0.000122

Copper 

Mean ± SD 1.6 ± 0.4 1.3 ± 0.3 

Range 0.8-2.3 0.6-1.7 0.6-1.6 0.8-7.1 

p-value 0.000522

Iron 

Mean ± SD 22.7 ± 5.2 22.4 ± 2.6 

Range 16.6-39.2 17.9-27.5 11.6-27.3 10.4-49.1 

p-value 0.811 

Magnesium 

Mean ± SD 1346 ± 107 1396 ± 150 

Range 1196-1550 1115-1591 895-1602 788-1940 

p-value 0.338 

Phosphorus 

Mean ± SD 2222 ± 188 2285 ± 180 

Range 1939-2569 2024-2536 1780-3084 1606-5330 

p-value 0.485 

Potassium 

Mean ± SD 3446 ± 317 3314 ± 284 

Range 2974-3840 2893-3773 2892-4252 2710-6030 

p-value 0.04622

Sodium 

Mean ± SD 2.7 ± 2.5 1.7 ± 0.7 

Range 0.6-10.7 0.6-3.4 0.6-7.7 1.0-731.5 

p-value 0.140 

Zinc 

Mean ± SD 23.0 ± 2.8 23.9 ± 3.5 

Range 17.9-26.8 16.6-31.8 15.6-28.4 6.5-33.8 

p-value 0.316 1 ILSI (2006) Crop Composition Database (Version 3.0) 2 p ≤ 0.05 indicates a statistical difference calculated. 
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VII.F.4.8. Analysis of secondary metabolites and anti-nutrients in maize grain 

The levels of key secondary metabolites and known anti-nutrients were measured in 

grain samples of event VCO-Ø1981-5, the non-transgenic control, and three reference 

hybrids. The results are reported in Table 30. The levels of the furfural measured below 

the level of quantitation for the assay (data not shown). In all but one of the metabolites 

evaluated, no statistically significant differences were observed between event VCOØ1981-5 and the non-transgenic control (p-values were >0.05). 

For the analyte ferulic acid, the p-value showed a statistical difference in the calculated 

means of event VCO-Ø1981-5 and the non-transgenic control; however, the standard 

deviations and data ranges overlapped and the level of ferulic acid was lower in event 

VCO-Ø1981-5. The data ranges of both entries overlapped with the data range of the 

reference hybrids and were well within the range of the published values. Therefore, it is 

unlikely that this difference indicates any biological significance. 

Table 30. Secondary metabolites and anti-nutrients in event VCO-Ø1981-5 grain 

Analyte 

(% of dry weight unless 

noted otherwise) 

VCO-Ø1981-5 

hybrid 

Nontransgenic 

control 

Range 

Reference 

hybrids Published1

SECONDARY METABOLITES 

Ferulic acid Mean ± SD 0.272 ± 0.035 0.318 ± 0.034 

Range 0.206-0.332 0.276-0.385 0.175-0.309 0.09-0.390 

p-value 0.00012

p-Coumaric 

acid 

Mean ± SD 0.020 ± 0.005 0.021 ± 0.003 

Range 0.010-0.028 0.017-0.027 0.015-0.045 0.009-0.043 

p-value 0.410 

Inositol 

Mean ± SD 0.011 ± 0.003 0.010 ± 0.003 

Range 0.007-0.017 0.006-0.016 0.007-0.022 0.009-0.377 

p-value 0.237 

ANTI-NUTRIENTS 

Raffinose 

Mean ± SD 0.309 ± 0.082 0.279 ± 0.050 

Range 0.174-0.455 0.208-0.379 0.080-0.323 0.053-0.264 

p-value 0.075 

Phytic acid 

Mean ± SD 0.840 ± 0.122 0.864 ± 0.073 

Range 0.686-1.09 0.719-0.994 0.598-1.479 0.111-1.370 

p-value 0.569 

Trypsin 

inhibitor 

(TIU/mg dry 

weight)3

Mean ± SD 2.79 ± 0.64 2.91 ± 0.63 

1.05-4.55 1.45-7.18 Range 1.58-3.53 1.48-3.68 

p-value 0.325 

1 ILSI (2006) Crop Composition Database (Version 3.0) 

2 p ≤ 0.05 indicates a statistical difference calculated 3 TIU = trypsin inhibitor units 
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VII.G. Conclusion 

Event VCO-Ø1981-5 has been grown in the field under USDA notifications since 2007. 

In 2009, a total of 17 field sites across six US states were utilized to collect agronomic 

data. Hybrids were characterized under diverse environmental and growing conditions, 

which were representative for maize production across North America. Overall, these 

evaluations indicate that event VCO-Ø1981-5 does not show any unexpected changes 

in plant morphology as compared to conventional maize. Where small statistically 

significant differences were observed, they appeared unrelated to the introduced trait. 

The agronomic performance data indicate no biologically meaningful differences 

between event VCO-Ø1981-5 and the non-transgenic control (Bernard and MacIntosh, 

2011d). 

As part of the 2009 agronomic performance field trials, abiotic and biotic stress factors 

were also evaluated for event VCO-Ø1981-5 and compared to the non-transgenic 

control. Event VCO-Ø1981-5 did not differ in response to abiotic stressors. No 

differences between the event VCO-Ø1981-5 and the non-transgenic control were 

observed for disease occurrence or severity and response to insect pressure. 

The germination test performed in the laboratory showed a high level of germination for 

event VCO-Ø1981-5 seeds. Under warm conditions, germination was equivalent to that 

of the non-transgenic control (99.6%), while there were only small differences between 

event VCO-Ø1981-5 and the non-transgenic control seeds under cold conditions, 97% 

and 93%, respectively. 

Forage and grain samples of event VCO-Ø1981-5 were analyzed for their nutrient 

composition and compared to a non-transgenic control. To place the results in context of 

normal ranges of variability, the ranges for three commercial reference hybrids were 

determined. Published data ranges were provided as additional points of reference. 

Plant material was collected from five locations in 2009, analyzed using standards 

methods and statistically evaluated. A few statistically significant differences were noted 

for some analytes. However, the ranges and standard deviations overlapped for event 

VCO-Ø1981-5 and the non-transgenic control and none of the values determined were 

distinctly different from the normal range of maize variability for any particular analyte. 

Overall, event VCO-Ø1981-5 is comparable in nutrient composition to the nontransgenic control, reference hybrids and the publish data ranges. 

In conclusion, no biologically meaningful differences between event VCO-Ø1981-5 and 

the non-transgenic control were identified with regards to its agronomic performance and 

nutrient composition. 
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VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTICES 

VIII.A. Environmental assessment of the introduced protein 

The EPSPS ACE5 protein was derived using a directed evolution strategy from the 

native EPSPS GRG23 protein and is 97.6% identical and 98.5% homologous at the 

amino acid level. The EPSPS GRG23 protein was isolated from Arthrobacter 

globiformis, a common soil bacterium. Arthrobacter species are part of the Gram-positive 

coryneform bacteria considered one of the major groups of aerobic soil bacteria and are 

found ubiquitously in soil and other natural habitats (Conn, 1948; Conn and Dimmick, 

1947, Mulder et al., 1966). The EPSPS ACE5 protein is a glyphosate-tolerant 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme. EPSPS enzymes are 

found commonly in nature in microorganisms such as bacteria and fungi and in plants. 

The EPSPS ACE5 protein has no amino acid homology to proteins with known toxic or 

allergenic effects (Bernard and MacIntosh, 2011c). It is unlikely that the EPSPS ACE5 

protein when expressed in maize will pose a safety risk to the environment. 

EPSPS enzymes are found throughout the plant kingdom and are required for plant 

amino acid biosynthesis. In addition to plants, EPSPS enzymes are also found in 

prokaryotic systems, and glyphosate is toxic to most bacterial species. However, certain 

bacteria are tolerant to glyphosate, and it has been found that the EPSPS enzymes 

isolated from these bacteria often have a high tolerance to glyphosate. Furthermore, 

genes encoding glyphosate-tolerant EPSPS enzymes have been transferred to recipient 

organisms, including plants, to confer glyphosate tolerance (OECD, 1999; Nolte and 

Young, 2002; Cedeira and Duke, 2006; Gianessi, 2008; Vande Berg et al., 2008). 

To date, EPSPS enzymes conferring glyphosate tolerance have been expressed in 

maize, cotton, soybean, canola, and sugarbeets and represent a large proportion of the 

US acreage. According to Brookes and Barfoot (2009), 91% of the total US soybean 

crop in 2007 was planted to glyphosate-tolerant varieties, first introduced in 1996. 

Glyphosate-tolerant maize and cotton were first introduced in 1997 and 10 years later 

were planted on 52% and 70% of the US maize and cotton acres, respectively (Brookes 

and Barfoot, 2009). Also in this time period, other herbicide-tolerant crops have been 

introduced into the marketplace (i.e., expressing the phosphinothricin acetyl transferase 

[PAT] protein). There have been no identified environmental concerns raised for 

herbicide-tolerant traits or the glyphosate-tolerant technology specifically, regardless of 

the crop in which they are expressed. 

VIII.B. Potential for gene transfer 

Maize is a highly domesticated crop that requires human intervention to produce high 

yielding results. Maize pollen does not drift large distances and the seed is heavy and 

not wind-born (Pleasants et al., 2001). Thus maize volunteers are rarely found outside of 

maize cultivation areas. 

VIII.B.1. Vertical gene flow 

Gene flow from maize to its wild relatives such as teosinte is not of particular concern in 

the US because the non-cultivated species are geographically restricted and occur only 

in Mexico and Central America. Teosinte species have co-existed with cultivated 

varieties of maize in these regions for thousands of years, yet they remain genetically 
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distinct with only a few cases of introgression (Baltazar et al., 2005). While isolated 

populations of teosinte used to exist in the US in Florida and Texas, there have been no 

reports of these in the last 25 years (EPA, 2001). Some teosinte species are grown in 

botanical gardens, but pollen flow from these sources is highly unlikely (EPA, 2001). 

The closest known relative of maize is Tripsacum, a genus of sixteen species, of which 

three can be found in the US: T. dactyloides, T. floridanum and T. lanceolatum. It is 

theorized that Tripsacum may be a progenitor of domesticated maize. Crosses between 

Zea mays L. subspecies (ssp.) mays and T. dactyloides are feasible, but only through 

human intervention and with difficulty. Moreover, the progeny of such crosses are 

frequently sterile or unstable (CFIA, 1994). However, maize breeders view Tripsacum as 

a good source for genes that could impart improved environmental traits, such as 

drought tolerance, and pest and disease resistance (OECD, 2003). The potential for Z. 

mays L. (ssp.) mays to introgress genes into T. dactyloides under field conditions was 

determined to be ‘extremely remote’ by the EPA (2001). 

Weed spectrum shifts or glyphosate resistant weeds have been identified, but the 

incidence still remains low as compared to other herbicides (e.g., ALS herbicides). To 

date, 13 weed species have been identified as resistant to glyphosate in the US or 

Canada, and 21 weed species globally (Brookes and Barfoot, 2011; Weed Science, 

2011). Glyphosate provided growers unprecedented weed control in crops such as 

soybean and cotton, where chemical weed control options are limited. Consequently, 

rapid grower adoption of glyphosate-tolerant crops led to heavy selection pressure for 

glyphosate-resistant weeds. In light of the increased frequency of weed resistance, 

proactive integrated weed management systems are being promoted and recommended 

by the industry and being adopted by growers. 

Taken together, it is unlikely that event VCO-Ø1981-5 would be found outside of maize 

cultivation areas or would introgress into wild relatives. Even if there were a rare 

introgression of the herbicide-tolerant trait from event VCO-Ø1981-5 into a wild maize 

relative, event VCO-Ø1981-5 has no selective advantage since glyphosate is just one of 

many different broad-spectrum herbicides that are commercially available. Likewise, 

management practices should be utilized to prevent, delay and/or minimize the 

development of glyphosate resistance in weeds. 

VIII.B.2. Horizontal gene flow 

The potential for horizontal gene transfer from plants to soil bacteria has been studied 

for many years with no such occurrences being observed in nature or in the field (EPA, 

2001; Connor et al., 2003) and only a few examples observed under laboratory or 

greenhouse conditions (De Vries et al., 2001; Kay et al., 2002). Event VCO-Ø1981-5 

was developed to optimize expression in plants and not in bacteria, therefore even if 

such a genetic transfer did occur, expression in a bacterial strain, using the sugar cane 

ubiquitin 4 promoter, would not be expected to function in bacteria. The potential for 

event VCO-Ø1981-5 to be transferred beyond the typical maize cultivation regions by 

horizontal gene flow is negligible. 
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VIII.C. Weediness potential of maize event VCO-Ø1981-5 

In the U.S., maize is not listed as a weed in the major weed references nor is it listed as 

a noxious weed species by the Federal Government, and maize has been grown 

throughout the world without any report that it is a serious weed. Maize has lost the 

ability to survive in the wild due to its long process of domestication and requires human 

intervention to disseminate its seeds. Although maize from a previous crop can 

sometimes overwinter and germinate, it cannot persist as a weed. Volunteers are 

common in many agronomic systems, but they can be controlled easily with mechanical 

or chemical methods (OECD, 2003). 

Event VCO-Ø1981-5 has been evaluated carefully for agronomic performance and seed 

germination characteristics under field and laboratory conditions and did not show any 

biologically meaningful differences when compared to the non-transgenic control 

(Section VII.). The laboratory study yielded a level of germination for event VCO-Ø1981-

5 equivalent to that of the non-transgenic control under warm conditions and only small 

differences under cold conditions (Section VII.E.). Differences in field emergence during 

the trials conducted in 2009 may be attributed to a difference in seed quality for event 

VCO-Ø1981-5 and the non-transgenic control (Section VII.C.2.3.). Overall, none of the 

agronomic parameters measured indicate that event VCO-Ø1981-5 has any more 

potential for weediness than maize varieties currently in the market. 

Transformation event VCO-Ø1981-5 is tolerant to the broad-spectrum herbicide 

glyphosate only and remains sensitive to other herbicides registered for use on weeds in 

maize and other rotational crops. Any volunteers of event VCO-Ø1981-5 are as easily 

controlled with mechanical means or chemical treatments as other maize varieties used 

in current agricultural systems. 

VIII.D. Current agronomic practices for maize 

Maize is grown all across the US, with the majority (>80%) in the Midwest, from western 

New York to Nebraska and from the Canadian border south through Oklahoma and the 

northern panhandle of Texas. A recent history of maize planted acres is provided in 

Table 31 along with the yield, price and overall value of maize production (USDA-NASS, 

2011a). It is anticipated that event VCO-Ø1981-5 maize hybrids will be cultivated across 

the US Corn Belt without extending the current maize growing area. 

Table 31. History of maize production 

Year Planted (all 

purposes) 1 Harvested 1 Yield 2 Production3 Price per unit 4

Value of 

production 5

2010 88,192 81,446 152.8 12,446,865 3.83 47,671,493 

2009 86,482 79,620 164.9 13,130,632 3.70 48,588,665 

2008 85,982 78,570 153.9 12,091,648 4.06 49,312,615 

2007 93,527 86,520 150.7 13,037,875 4.20 54,666,959 

2006 78,327 70,638 149.1 10,531,123 3.04 32,083,011 

2005 81,779 75,117 147.9 11,112,187 2.00 22,194,287 

2004 80,929 73,631 160.3 11,805,581 2.06 24,377,913 

1 - thousand acres, 2 – bushel/acre, 3 - thousand bushels, 4 - dollars/bushel, 5 - thousand dollars 
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US growers produced the largest corn crop on record in 2009, 13.1 billion bushels at a 

value $48.6 billion (USDA-NASS, 2011a). Slightly less than half of all maize production 

is utilized for animal feeds, about 17% is exported and about 10% utilized for food (e.g., 

sweeteners, cereal, starch, high fructose corn syrup, and alcohol (NCGA, 2011). These 

segments have been relatively stable over the last 10 years. The fastest growing 

segment is for ethanol production, growing to nearly 5 billion bushels in 2010 up from 

just under 1 billion bushels in 2002. The growth of maize production is at least partially 

explained by improved technology, such as biotechnology, which boosts agricultural 

productivity providing US producers a competitive edge globally. 

In 2011, roughly 72% of the total maize acreage was planted with herbicide-tolerant 

maize in the US, out of approximately 87.8M total maize grain acres (USDA-NASS, 

2011b). Of the 61.5 M acres planted with herbicide-tolerant varieties in 2011, 23% had 

only the herbicide-tolerant trait, while 49% were maize varieties stacked with both insect 

control and herbicide tolerance traits (USDA-NASS, 2011b). More than 90% of the 

herbicide-tolerant maize was planted to glyphosate-tolerant maize, with the balance 

planted to LibertyLink®1 maize and non-GM Clearfield®2 technology. Event VCO-Ø1981-

5 will offer a new opportunity to US growers. It will be made available to them through 

our affiliate in the US and through licenses to third parties. It is difficult to predict the 

adoption rate of this new event compared to what is available on the market today and it 

is expected to be adopted by the market as a replacement product. As such, it is unlikely 

that the availability of this new event will significantly increase the maize acreage planted 

with by glyphosate-tolerant maize. 

Herbicides were applied to 98% of the maize planted acreage in 2010 (USDA-NASS, 

2011c). Glyphosate has surpassed atrazine as the most widely applied herbicide with 

66% of the planted acreage being treated (USDA-NASS, 2011c). Atrazine was applied 

to 61% of the planted acres. These were followed closely by s-metolachlor and 

acetochlor, at 25% of the planted maize acreage reported. 

Weeds cause significant yield reductions in maize by competing for light, water and 

nutrients, requiring careful management by producers. Weeds can also bring insect and 

fungal infestations. Many different weed species are found in maize growing regions, 

including perennials, annuals, grasses, broadleaf and sedge types. Early season weeds 

are perhaps the most damaging when the maize crop is becoming established. 

Reductions in maize yield are directly proportional to the amount of weeds present 

(Gianessi et al., 2002). Prior to the 1960s, crop rotation, cultivation and tillage were the 

primary methods to control weeds in maize production fields. As herbicide use became 

widespread in the following decade, low and no-till farming were embraced and today 

almost every maize acre receives an herbicide treatment. 

Weed management brings the best results when herbicides are applied on smaller 

weeds, early in the growing season. The uses of pre-emergent herbicides, such as 

atrazine, continue as a common weed management practice, even with the advent of 

herbicide-tolerant crops. Introduction of herbicide-tolerant crops, such as glyphosateand glufosinate-tolerant crops has brought many advantages to the grower; better crop 

tolerance, broader spectrum of weed control, more environmentally favorable profile and 

more favorable crop rotation opportunities. These new tools, including event VCO-

 

1

 LibertyLink is a registered trademark of Bayer 

2

 Clearfield is a registered trademark of BASF Corporation 
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Ø1981-5 maize, will continue to provide the grower with greater flexibility and ease of 

use in their weed management systems. 

VIII.E. Potential impact of introduction on current agricultural practices 

Weed management is a significant aspect of successful maize production for US 

growers. Until the advent of herbicide-tolerant maize, growers used a range of different 

herbicides pre-plant, pre- and post-emergence to manage weeds. Herbicide programs in 

maize can vary due to the geographic area, weed spectrum, and rotational versus 

continuous maize. Growers have traditionally relied upon triazine products in continuous 

maize where potential for carryover of the residual materials would not be a concern. 

Several weeds, however, have developed resistance to the triazines (LeBaron, 1991). 

Adverse weather conditions also reduce the effect of the triazines and other soil applied 

herbicides. Sulfonylurea and other herbicides are used to control grass and broadleaf 

weeds post-emergence in maize. In general, maize often receives a soil applied 

herbicide and a follow-up post-emergence application. Due to potential crop injury, 

rotational concerns and weed competition, multiple herbicide applications applied postemergence are not widely used in maize. Also, many products are used in combination 

as premixes or as tank mixes, to widen the spectrum of control. The reasons for this are 

to prevent maize injury, reduce weed pressure on the crop, and reduce rotational 

restrictions to soybeans or other legumes. Perennials are difficult to control because 

they propagate by seed and/or underground plant parts. Control of these diverse species 

requires the use of multiple herbicide families and multiple applications, making weed 

management complex and difficult. 

Aside from the material cost associated with traditional weed control programs in maize, 

there are the additional costs of fuel and time required to apply these herbicides. 

Utilization of event VCO-Ø1981-5 maize will facilitate fewer herbicide applications by 

offering broad-spectrum post-emergence weed control with a single chemical, 

glyphosate. Further, glyphosate is a suitable tank-mix partner for many selective 

herbicides making possible one pass weed control even where hard to control weeds 

may be present in the larger weed population. The ability to control weeds with a single 

broad-spectrum herbicide, or as a tank mix, allows for no- or minimum-till maize 

cropping systems. Reducing tillage has the effect of reducing fuel costs simply by 

reduction of equipment passes. No- or minimum-till maize cropping systems also have 

the added benefit of minimizing soil disturbance providing numerous advantages for soil 

and environmental quality (e.g., greater carbon sequestration, less soil and nutrient runoff, soil moisture retention, etc.). 

It is not surprising therefore that US maize growers have rapidly embraced glyphosatetolerant maize products, now reported to be more than 70% of all maize-planted acres, 

based on the ease of use and excellent human, animal and environmental safety profiles 

of glyphosate. A grower utilizing herbicide-tolerant maize minimizes the need for multiple 

herbicide applications with more toxic herbicides, since weeds are controlled through 

precisely timed applications. The herbicide, in the herbicide-tolerant system, is only 

applied when weed pressure requires management, causing no crop damage to the 

herbicide-tolerant maize. 

The potential impact of the introduction of event VCO-Ø1981-5 on current agricultural 

practices will not be significantly different than for the previously deregulated herbicidetolerant maize lines. Growers have more than 15 years of experience with herbicide-
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tolerant maize products. Aside from the tolerance to glyphosate herbicides that ease 

weed control, event VCO-Ø1981-5 maize will require the same management as any 

other maize variety. Cultivation practices for insect and nutrient management will not be 

any different. 

No data has been generated for event VCO-Ø1981-5 that indicates an increase in 

fitness or weediness characteristics that would provide a competitive advantage or 

extend maize production beyond the current production areas. There have been no 

noted effects on insect or disease susceptibilities during multiple field trials. US 

agriculture has utilized herbicide-tolerant crops over the past 15 years and the cultivation 

of event VCO-Ø1981-5 maize will not change current agronomic practices. 

VIII.F. Weed resistance management 

Growers have had to manage resistance in weeds and insects since the advent of 

biological and chemical control measures. Theoretically, regular use of any pesticide 

could lead to resistance, but growers have ample options using a variety of techniques 

to prevent or delay the development of resistant weeds in maize fields. 

Resistance management of event VCO-Ø1981-5 will be no different than the 

management practices used with other maize varieties. While glyphosate-tolerant maize 

provides the potential for a single weed control herbicide, there are many other effective 

herbicides in the marketplace that can and should be used in conjunction with 

glyphosate. Some of additional techniques for weed resistance management include 

crop rotation, rotating herbicide modes-of-action, tank mixing herbicides with different 

modes-of-action, and mechanical weed control. 

Weed scientists agree that adopting and implementing best management practices that 

reduce weed resistance to herbicides is critical (Boerboom and Owen, 2006). A 

diversified and detailed integrated weed management plan not only improves overall 

weed control, it provides additional benefits such as improving the overall level and 

consistency of weed control, adding flexibility in scheduling applications and reducing 

the risk of yield loss due to weed competition. 

Ideally, integrated weed management should utilize all available tools including 

herbicides in a well-balanced program, as the lower the diversity of weed control tools, 

the higher the risk of selecting resistant biotypes becomes. To ensure diversification is 

maintained in weed control methods, growers are encouraged to keep detailed records 

of weed management practices for each field. Integrated weed management guidelines 

promote an economically viable, environmentally sustainable and socially acceptable 

weed control program, which is fully detailed in Appendix 4. 

The highlights of an integrated weed management include: 

1) Correctly identify weeds and look for trouble areas within field to identify resistance 

indicators; 

2) Rotate crops; 

3) Start the growing season with clean fields; 

4) Rotate herbicide modes of action by using multiple modes of action during the 

growing season and apply no more than two applications of a single herbicide mode 

of action to the same field in a two-year period. One method to accomplish this is to 

rotate herbicide-tolerant trait systems; 
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5) Apply recommended rates of herbicides to actively growing weeds at the correct time 

with the right application techniques; 

6) Control any weeds that may have escaped the herbicide application; 

7) Thoroughly clean field equipment between fields. 

Glyphosate-tolerant event VCO-Ø1981-5 volunteers can be managed through crop 

rotation, using many different herbicides, and/or mechanical techniques. Volunteers can 

also be managed in continuous corn with mechanical methods, applications of burndown 

herbicides prior to crop emergence, or through different herbicide-tolerant maize 

systems, such as glufosinate-tolerant maize. 

Today, growers have a wide range of weed control options. Information on weed 

resistance management will be provided to growers to assist them in making practical 

and useful choices to prevent, delay and/or minimize weed resistance development 

when using event VCO-Ø1981-5. 

VIII.G. Potential impact on organic or conventional farming 

Glyphosate-tolerant maize varieties have been in use for more than 15 years and the 

introduction of event VCO-Ø1981-5 maize will not change the potential impact on 

organic farming from the current agricultural situation. Organic and conventional growers 

may choose not to plant or sell glyphosate-tolerant maize. These growers will still be 

able to produce non-transgenic maize and will be able to coexist with biotechnology 

maize producers as they do now. Techniques have long been practiced to allow 

coexistence by minimizing or preventing cross-pollination. For maize, this is used for 

example when growing specialty corn, such as waxy or sweet, to keep the quality of 

products that can be affected by conventional maize pollination. Isolation distances 

between fields help to limit the effects of pollen flow. In addition to spatial isolation, 

growers can use reproductive isolation to minimize or eliminate cross-pollination (i.e., 

plant varieties of different maturity groups) or stagger planting dates for temporal 

isolation. Perhaps most importantly, growers need to communicate openly with each 

other in order to continue and maintain the historical practice of coexistence. 

VIII.H. Potential impacts on raw or processed agricultural products 

Data submitted in this petition in support of event VCO-Ø1981-5 maize with regards to 

agronomic performance evaluations, disease and insect susceptibility, and 

compositional analysis indicate no biological differences from the non-transgenic control 

that would be expected to cause a direct or indirect plant pest effect on raw or processed 

agricultural products. While the EPSPS ACE5 protein was the result of genetic 

modification to improve enzymatic activity under field conditions, thereby making it more 

similar to the native maize EPSPS protein, the introduced amino acid changes did not 

impact the EPSPS ACE5 protein stability under industrial conditions that are typically 

found in maize wet and dry milling processes. In addition, Athenix Corp., now an affiliate 

of Bayer CropScience LP, submitted an Early Food Safety Evaluation to FDA in 2009 

and FDA completed their review with no further questions in October, 2010 (FDA, 2010). 
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VIII.I. Potential effects on non-target organisms 

Maize production requires a highly managed environment in order to produce acceptable 

commercial level yields. Weeds and insect pests must be managed to keep below 

economic thresholds in order to prevent any loss of yield. Agronomic performance and 

environmental effects studies have been conducted across a wide range of 

environments with no adverse effects on non-target organisms. 

Potential exposure to maize event VCO-Ø1981-5 would not have adverse effects to nontarget organisms because the EPSPS ACE5 protein has no insecticidal or other 

pesticidal properties and as such does have target organisms. Time to bloom and other 

reproductive aspects are the same for event VCO-Ø1981-5 as for the non-transgenic 

control, therefore impacts to pollinators are not expected and have not been observed. 

Since the EPSPS ACE5 protein is expressed in planta, and the protein environmentally 

degrades as observed for all EPSPS enzymes, there is minimal exposure to non-target 

organisms. Agronomic performance evaluations, disease and insect susceptibility, and 

compositional analysis of event VCO-Ø1981-5 indicate no biological differences from the 

non-transgenic control maize. Furthermore, field trial reports prepared for the 

notifications listed in Table 11 (Section VII.) did not indicate any differences in insect 

pests or beneficial insect population occurrences (Appendix 1). As maize event VCOØ1981-5 and conventional maize are not different with respect to their phenotypic and 

agronomic characteristics and ecological interactions (except for the introduced trait), it 

can be concluded that the impact of event VCO-Ø1981-5 on non-target organisms will 

not be different from conventional corn. 

EPSPS proteins and, specifically, the EPSPS ACE5 protein, have no known toxic or 

allergenic properties (Bannon et al, 2008; Delaney et al., 2008; Bernard and MacIntosh, 

2011c). An acute oral toxicity study in mice did not cause any toxicity, mortality or 

morbidity (Arulnesan, 2008). EPSPS ACE5 protein digested very quickly in simulated 

gastric fluids, did not match any toxic or allergenic protein sequences using a 

bioinformatics analysis and no glycosylation was detected (Bernard and MacIntosh, 

2011b; Bernard and MacIntosh, 2011c; Shouten, 2011). A detailed compositional 

analysis of event VCO-Ø1981-5 forage and grain was conducted, and only minor 

statistical differences were noted as compared to the non-transgenic control maize 

(Bernard and MacIntosh, 2011e). However, almost all of those differences were within 

the normal published ranges for various maize composition analytes. Glyphosatetolerant EPSPS enzymes have been previously assessed for safety and are widely used 

in commercially available maize products in production today (USDA, 2011). EPSPS 

GRG23, from which EPSPS ACE5 protein was derived, is a naturally occurring enzyme 

isolated from a common soil organism. No additional exposure risks are anticipated from 

this protein expressed in event VCO-Ø1981-5 maize. 

VIII.J. Potential impact on biodiversity 

Herbicide-tolerant crop systems have improved high production agriculture by providing 

valuable weed control without crop damage. Adoption of low or no till farming facilitated 

by glyphosate-tolerant technology further reduces disruptions to the soil environment. 

Plant expressed EPSPS ACE5 protein also limits environmental exposure and as noted 

above, no adverse effects on non-target organisms have been identified. 
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No increased weediness potential has been observed during the evaluation of event 

VCO-Ø1981-5. There are no expectations that an unconfined release of event VCOØ1981-5 should lead to increases in weediness of other sexually compatible species. 

The evaluation of event VCO-Ø1981-5 indicated no adverse effects on non-target 

organisms common to the agricultural ecosystem. Furthermore, glyphosate-tolerant 

maize varieties have been used in agricultural production for many years with no 

adverse effects reported. There is no reason to anticipate that this new event, with a 

similar mode of action, would cause negative effects to biodiversity. Glyphosate use and 

crop production practices are not expected to change significantly with the market 

introduction of event VCO-Ø1981-5, therefore there should be no indirect or cumulative 

impact on biodiversity. The effects of glyphosate on contamination of soil, water, and air 

are minimal (Cedeira and Duke, 2006); therefore, negative effects on soil microflora, 

aquatic organisms, arthropods, and mammals are not anticipated when the herbicide is 

applied according to label instructions. 

VIII.K. Conclusion 

Regulated field trials of event VCO-Ø1981-5 have been conducted since 2007. The 

agronomic performance data, collected during the 2009 season, indicate no biologically 

meaningful differences between event VCO-Ø1981-5 and the non-transgenic control. 

Ecological observations, conducted at the same time on a variety of stressors (e.g.,

insects, diseases, abiotic), also showed no differences between event VCO-Ø1981-5 

and the non-transgenic control regardless whether the stressor was measured to be mild 

to moderate in severity. 

EPSPS enzymes are found throughout the plant kingdom, are required for plant amino 

acid biosynthesis and the corresponding transgenes have been expressed in maize, 

cotton, soybean, canola, and sugarbeet and represent a large proportion of the US 

acreage for the last 15 years, without any reports of adverse effects. There have been 

no identified environmental concerns raised for herbicide-tolerant traits or the 

glyphosate-tolerant technology specifically, regardless of the crop in which they are 

expressed. 

Maize has been grown throughout the world without any report that it is a serious weed. 

Gene flow concerns are minimal, since teosinte is not found in the US or Canada and 

crosses with other wild relatives of maize, while possible, are difficult to produce and 

generally cause instability or sterility in the offspring. While glyphosate resistance has 

been found, it is easily managed using traditional methods such as other herbicides and 

crop rotation. Information on weed resistance management will be provided to growers 

to assist them in making practical and useful choices to prevent, delay and/or minimize 

weed resistance when planting event VCO-Ø1981-5 maize varieties. 

Given the extensive experience growers have with herbicide-tolerant crops, there is no 

expectation that the introduction of event VCO-Ø1981-5 would alter current agronomic 

practices. The potential impact on organic farming should not change from the current 

agricultural situation, since techniques allowing coexistence are well established. 

Perhaps the most important measure to continue and maintain the historical practice of 

coexistence, is for growers to communicate openly. Given the results demonstrating that 

event VCO-Ø1981-5 is nutritionally equivalent to non-transgenic maize there should also 

be no impacts to raw or processed agricultural products. Agronomic performance 

evaluations, disease and insect susceptibility, and compositional analysis of event 
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VCO-Ø1981-5 showed no biological differences to the non-transgenic control that might 

indicate a direct or indirect effect on non-target organisms including beneficial, 

threatened or endangered species. These results also support the lack of impacts on 

biodiversity. Glyphosate effects on contamination of soil, water and air are minimal. 

Furthermore, the EPSPS ACE5 protein has no known toxic or allergenic properties that 

could impact non-target organisms. 
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IX. STATEMENT OF GROUNDS UNFAVORABLE 

GENECTIVE S.A. knows of no study data and/or observations associated with 

glyphosate-tolerant event VCO-Ø1981-5 maize that will result in adverse environmental 

consequences for its introduction. Previously deregulated glyphosate-tolerant maize 

lines have been grown safely for more than 10 years with no adverse effects being 

observed. The only biologically relevant phenotypic difference between event VCOØ1981-5 and conventional maize is the expression of the EPSPS ACE5 protein, which 

provides tolerance to the application of glyphosate. Planting glyphosate-tolerant event 

VCO-Ø1981-5 maize will provide growers with another choice for using glyphosatetolerant technology. 

 
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X. REFERENCES 

AOSA, 2010. Association of Official Seed Analysts. Rules for Testing Seed 

http://www.aosaseed.com/publications.htm#2010 AOSA Rules for Testing Seed 

Albert, H. and Wei, H. 2003. Promoter of the sugarcane UBI4 gene. US Patent 

6,638,766. 

Alexandrov N.N., Brover V.V., Freidin S., Troukhan M.E., Tatarinova T.V., Zhang H., 

Swaller T.J., Lu Y.P., Bouck J., Flavell R.B., and Feldmann K.A. 2009. Insights 

into corn genes derived from large-scale cDNA sequencing. Plant Mol Biol

69:179-94. 

Allergen Online, 2011. http://www.allergenonline.org 

Amrhein, N., Deus, B., Gehrke, P., and Steinrucken, H.C. 1980. The site of the inhibition 

of the shikimate pathway by glyphosate: II. Plant Physiol 66:830-834. 

Armstrong C., Green C.E., and Phillips R.L. 1991. Development and availability of 

germplasm with high Type II culture formation response. MNL 65:92-93. 

Arulnesan, N., 2008. Single dose acute oral toxicity study of Athenix GRG23ace5 protein 

(GRG23ACE5-108) in CD-1 mice. Project No: 204217. Nucro-Technics, 

Scarborough, Ontario, Canada. Athenix Corp. unpublished report. 

Baltazar M.B., de Jesús Sánchez-Gonzalez J., de la Cruz-Larios L., and Schoper J.B. 

2005. Pollination between maize and teosinte: an important determinant of gene 

flow in Mexico. Theor Appl Genet 110: 519–526 

Bannon, G., Astwood, J.D., Dobert, R.C. and Fuchs, R.L. 2008. Biotechnology and 

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APPENDIX 1 

Field Trial Termination Reports 2007-2009 

 
Athenix Corporation ATX-07-16 1 07-283-104n 

USDA Field Termination Report

USDA Notification/Permit Number: 07-283-104n 

Applicant Internal Number: ATX-07-16 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; corn 

Lines: 5234-5283, 5814-5816, 5766-5767, 5878-5886, 5764-5765, 5835-5869, 6022-6031, 

6058-6059, 6038-6039, 6054-6057, 6103-6110, 6012-6021, 6135, 6136, 5402-5423, 

5768-5784, 5090-5225, 5513-5562, 6046-6053, 6129-6131, 6040-6045, 6111-6128, 

6148, 6149, 6032-6036, 6062, 5898-5922, 5563-5628, 1-100, 5946-5964, 6065-6094, 

6132, 6133, 6145, 6146, 6096-6100, 6147, 6342, 6343, 6353-6370, 6402, 6403, 6419-

6433, 6507-6522, 6598, 6599, 5685-5705, 5872-5877, 5424, 5425, 5427, 5436, 5443, 

5445, 6663-6667, 6676-6684, 6671, 6672, 6742, 6743, 6371-6373, 6404-6418, 6441-

6463, 6536-6560, 6622-6634, 6650-6652, 6304, 6306, 6307, 6750-6752, 6845-6916, 

6933-6971, 6978-6993, 7045-7048, 6972, 6994-7013, 6791-6792, 6831, 6923-6925, 

6794-6797, 6832-6844, 6926-6932, 7014, 5980-5982, 6001-6011

Section I. Site Release Information:

Trial sites were requested for one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 1 Juana Diaz / PR 2.00 12/28/2007 4/22/2008 

Section II. List of characteristic observations collected on the regulatory compliance InSeason Forms approximately every four weeks during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated, insects and 

environment:

a Plant Morphological - Observations 

b Plant Disease - Observations 

c Difference in Insect Occurrence - Observations 

d Unusual Occurrences During Flowering Stage -Observations 

e Unusual Occurrences During Pesticide Applications -Observations 

f Difference in Environmental Conditions - Observations 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-16 2 07-283-104n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Juana Diaz Co., PR : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized environmental 

releases or compliance incidents 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010 

Maize event VCO-Ø1981-5 

Page 86 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-17 1 08-059-104n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-059-104n 

Applicant Internal Number: ATX-07-17 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; corn 

Lines: 5122, 6041, 6113-6117, 6127, 6033, 6035, 6062, 6068, 6078, 6081, 6091, 6093, 

6342, 6359, 6421, 6423, 6427-6430, 6508, 6513, 6514, 6516, 6521, 6598, 6407-6418, 

6447-6455, 6537-6560, 6626-6634, 6845-6916, 6933-6972, 6978-6993, 7045-7047, 

7010, 7004, 7006, 7091-7101, 7148-7157, 7176-7183, 7214-7241, 7311-7317, 7613-

7620, 7725-7727, 7754, 7622-7649, 7691-7693, 7722-7724, 7751-7753, 7792-7793, 

7391-7400, 7433-7452, 7518-7535, 7610-7612, 7049, 7795, 7869-7873, 7728-7729, 

7874-7879, 7884-7886 

Section I. Site Release Information:

Trial sites were requested for one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Scott / IA 0.54 5/06/2008 11/28/2008 

Section II. List of characteristic observations collected on the regulatory compliance InSeason Forms approximately every four weeks during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated, insects and 

environment:

a Plant Morphological - Observations 

b Plant Disease - Observations 

c Difference in Insect Occurrence - Observations 

d Unusual Occurrences During Flowering Stage -Observations 

e Unusual Occurrences During Pesticide Applications -Observations 

f Difference in Environmental Conditions - Observations 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-17 2 08-059-104n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Scott Co., IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010

Maize event VCO-Ø1981-5 

Page 88 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-18 1 08-067-108n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-067-108n 

Applicant Internal Number: ATX-07-18 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047 

Section I. Site Release Information:

Trial sites were requested for one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 1st Finca Florida-Santa Isabel / PR 0.33 4/25/2008 7/21/2008 

1 2nd Finca Florida-Santa Isabel / PR 0.43 8/08/2008 10/27/2008 

2 plantings same location 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated.:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, tasseling, seed production, etc. similar for both transgenic 

and non-transgenic plants? 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-18 2 08-067-108n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Santa Isabel Co., PR (1st planting) : 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

1. Santa Isabel Co., PR (2nd planting): 

a. Cooperator reported no unusual differences in plant disease. 

b. Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c. Cooperator noted no unusual plant growth observations. 

d. Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010

Maize event VCO-Ø1981-5 

Page 90 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-19 1 08-073-108n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-073-108n 

Applicant Internal Number: ATX-07-19 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; corn 

Lines: 5122, 6041, 6113-6117, 6127, 6033, 6035, 6062, 6068, 6078, 6081, 6091, 6093, 

6342, 6359, 6421, 6423, 6427-6430, 6508, 6513, 6514, 6516, 6521, 6598, 6407-6418, 

6447-6455, 6537-6560, 6626-6634, 6845-6916, 6933-6972, 6978-6993, 7045-7047, 

7010, 7004, 7006, 7091-7101, 7148-7157, 7176-7183, 7214-7241, 7311-7317, 7613-

7620, 7725-7727, 7754, 7622-7649, 7691-7693, 7722-7724, 7751-7753, 7792-7793, 

7391-7400, 7433-7452, 7518-7535, 7610-7612, 7049, 7795, 7869-7873, 7728-7729, 

7874-7879, 7884-7886, 7887, 7888 

Section I. Site Release Information:

Trials sites were requested for three (3) locations: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Scott / IA N/A Not Planted N/A 

2 Madison / IA N/A Not Planted N/A 

3 Polk / IA 1.86 6/16/2008 12/07/2008 

Section II. List of characteristic observations collected on the regulatory compliance InSeason Forms approximately every four weeks during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated, insects and 

environment:

a Plant Morphological - Observations 

b Plant Disease - Observations 

c Difference in Insect Occurrence - Observations 

d Unusual Occurrences During Flowering Stage -Observations 

e Unusual Occurrences During Pesticide Applications -Observations 

f Difference in Environmental Conditions - Observations 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-19 2 08-073-108n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Scott Co., IA: Not Planted. 

2. Madison Co., IA : Not Planted 

3. Polk Co., IA : 

a. Cooperator reported no observed differences in plant morphology. 

b. Cooperator reported no unusual differences in plant disease. 

c. Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d. Cooperator noted no unusual growth characteristics during flowering stage. 

e. Cooperator reported no unusual interactions after pesticide applications. 

f. Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010 

Maize event VCO-Ø1981-5 

Page 92 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-23 1 08-105-109n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-105-109n 

Applicant Internal Number: ATX-07-23 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047

Section I. Site Release Information:

Trial sites were requested for two (2) locations: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 1 Washington / NE 0.2 6/03/2008 09/26/2008 

2 2 York / NE N/A Not Planted N/A 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, growth, flowering, seed production, etc. similar for both 

transgenic and non-transgenic plants? 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-23 2 08-105-109n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Washington Co., NE (1st planting) : 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

2. York Co., NE : Not Planted 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010 

Maize event VCO-Ø1981-5 

Page 94 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-24 1 08-254-111n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-254-111n 

Applicant Internal Number: ATX-07-24 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047

Section I. Site Release Information:

Trial sites were requested for one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 1st Santa Isabel / PR 0.87 12/10/2008 4/08/2009 

1 2nd Santa Isabel / PR * 0.65 7/27/2009 10/21/2009 

* 2nd planting at same location was planted under Notification 08-254-111n and Notification 09-215-106n. 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, tasseling, seed production, etc. similar for both transgenic 

and non-transgenic plants? 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-24 2 08-254-111n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Santa Isabel Co., PR (1st planting) : 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

1. Santa Isabel Co., PR (2nd planting): 

a. Cooperator reported no unusual differences in plant disease. 

b. Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c. Cooperator noted no unusual plant growth observations. 

d. Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010 

Maize event VCO-Ø1981-5 

Page 96 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-25 1 08-280-103n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-280-103n 

Applicant Internal Number: ATX-07-25 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047

Section I. Site Release Information:

Trial sites were requested for one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Barranquitas / PR 0.58 11/14/2008 3/02/2009 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated.:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, tasseling, seed production, etc. similar for both transgenic 

and non-transgenic plants? 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-25 2 08-280-103n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Barranquitas Co., PR: 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010 

Maize event VCO-Ø1981-5 

Page 98 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
Athenix Corporation ATX-07-27 1 08-309-103n 

USDA Field Termination Report

USDA Notification/Permit Number: 08-309-103n 

Applicant Internal Number: ATX-07-27 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6750-6752, 6845-6916, 6933-6971, 6978-6993, 7045-7048, 6972 

Section I. Site Release Information:

Trials sites were requested for one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Juana Diaz / PR 0.33 12/18/2008 4/03/2009 

Section II. List of characteristic observations collected on the regulatory compliance InSeason Forms approximately every four weeks during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated, insects and 

environment:

a Plant Morphological - Observations 

b Plant Disease - Observations 

c Difference in Insect Occurrence - Observations 

d Unusual Occurrences During Flowering Stage -Observations 

e Unusual Occurrences During Pesticide Applications -Observations 

f Difference in Environmental Conditions - Observations 

Maize event VCO-Ø1981-5 

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Athenix Corporation ATX-07-27 2 08-309-103n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Juana Diaz Co., PR : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: May 12, 2010 

Maize event VCO-Ø1981-5 

Page 100 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
ATX-07-29 1 09-047-108n 

USDA Field Termination Report

USDA Notification/Permit Number: 09-047-108n 

Applicant Internal Number: ATX-07-29 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047

Section I. Site Release Information:

Trials were conducted at one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Santa Isabel/Corozal / PR 0.41 03/13/2009 06/19/2009 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated.:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, tasseling, seed production, etc. similar for both transgenic 

and non-transgenic plants? 

Maize event VCO-Ø1981-5 

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ATX-07-29 2 09-047-108n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Santa Isabel/(Corozal) Co., PR: 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: July 08, 2010 

Maize event VCO-Ø1981-5 

Page 102 of 163 CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION
ATX-07-34 1 09-091-101n 

USDA Field Termination Report

USDA Notification/Permit Number: 09-091-101n 

Applicant Internal Number: ATX-07-34 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047 

Section I. Site Release Information:

Trials were requested at twenty-two (22) locations: 

USDA-Notification 

Release Site No. 

City / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Bradford (Bureau Co.) / IL N/A Not Planted N/A 

2 Ivesdale (Champaign Co.) / IL 0.2 05/22/2009 10/28/2009 

3 Carlyle (Clinton Co.) / IL 0.2 06/02/2009 11/10/2009 

4 Mt Pulaski (Logan Co.) / IL 0.2 05/24/2009 10/21/2009 

5 Wyoming (Stark Co.)/ IL 0.2 05/22/2009 11/13/2009 

6 Lebanon (Boone Co.) / IN 0.4 05/30/2009 11/23/2009 

7 Rockville (Parke Co.) / IN 0.2 05/23/2009 12/03/2009 

8 Atlantic (Cass Co.) / IA 0.2 05/08/2009 10/09/2009 

9 Colfax (Jasper Co.) / IA 0.5 05/21/2009 12/07/2009 

10 Wesley (Kossuth Co.) / IA 0.2 05/19/2009 10/20/2009 

11 Winterset (Madison Co.) / IA N/A Not Planted N/A 

12 Walcott (Scott Co.) / IA 0.2 05/16/2009 11/12/2009 

13 Ottumwa (Wapello Co.) / IA 0.2 06/01/2009 07/07/2009 

14 Gowrie (Webster Co.) / IA 0.2 05/11/2009 11/04/2009 

15 Clarion (Wright Co.) / IA 0.2 05/20/2009 10/19/2009 

16 Paynesville (Stearns Co.) / MN 0.2 05/19/2009 11/23/2009 

17 Burwell (Valley Co.) / NE (site 1) 0.2 05/13/2009 11/10/2009 

18 Burwell (Valley Co.) / NE (site 2) 0.2 05/14/2009 11/07/2009 

19 Arlington (Washington Co.) / NE 0.2 05/06/2009 10/08/2009 

20 Bruce (Brookings Co.) / SD 0.2 05/21/2009 07/31/2009 

21 Delavan (Walworth Co.) / WI (site 1) 0.2 05/12/2009 11/10/2009 

22 Delavan (Walworth Co.) / WI (site 2) N/A Not Planted N/A 

Maize event VCO-Ø1981-5 

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ATX-07-34 2 09-091-101n 

Section II. List of characteristic observations collected on the regulatory compliance InSeason Forms approximately every four weeks during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated, insects and 

environment:

a Plant Morphological - Observations 

b Plant Disease - Observations 

c Difference in Insect Occurrence - Observations 

d Unusual Occurrences During Flowering Stage -Observations 

e Unusual Occurrences During Pesticide Applications -Observations 

f Difference in Environmental Conditions - Observations 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Bradford (Bureau Co.) , IL : 

Not Planted 

2. Ivesdale (Champaign Co.) , IL : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

3. Carlyle (Clinton Co.) , IL : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

4. Mt. Pulaski (Logan Co.) , IL : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Maize event VCO-Ø1981-5 

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ATX-07-34 3 09-091-101n 

5. Wyoming (Stark Co.) , IL : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

6. Lebanon (Boone Co.) , IN : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

7. Rockville (Parke Co.) , IN : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

8. Atlantic (Cass Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

9. Colfax (Jasper Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Maize event VCO-Ø1981-5 

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ATX-07-34 4 09-091-101n 

10. Wesley (Kossuth Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

11. Winterset (Madison Co.) , IA : 

Not Planted 

12. Walcott (Scott Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

13. Ottumwa (Wapello Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

14. Gowrie (Webster Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

15. Clarion (Wright Co.) , IA : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Maize event VCO-Ø1981-5 

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ATX-07-34 5 09-091-101n 

16. Paynesville (Stearns Co.) , MN : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

17. Burwell (Valley Co.) , NE (site 1) : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

18. Burwell (Valley Co.) , NE (site 2) : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

19. Arlington (Washington Co.) , NE : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

20. Bruce (Brookings Co.) , SD : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Maize event VCO-Ø1981-5 

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ATX-07-34 6 09-091-101n 

21. Delavan (Walworth Co.) , WI : 

a) Cooperator reported no observed differences in plant morphology. 

b) Cooperator reported no unusual differences in plant disease. 

c) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

d) Cooperator noted no unusual growth characteristics during flowering stage. 

e) Cooperator reported no unusual interactions after pesticide applications. 

f) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

22. Delavan (Walworth Co.) , WI : 

Not Planted 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: September 26, 2010

Maize event VCO-Ø1981-5 

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ATX-07-37 1 09-086-104n 

USDA Field Termination Report

USDA Notification/Permit Number: 09-086-104n 

Applicant Internal Number: ATX-07-37 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047 

Section I. Site Release Information:

Trials sites were requested at three (3) locations: 

USDA-Notification 

Release Site No. 

City / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Bluffton (Wells Co.) / IN 0.5 05/25/2009 11/12/2009 

2 Ames (Story Co.) / IA 1.3 05/09/2009 11/16/2009 

3 Arlington (Washington Co.) / NE N/A Not Planted N/A 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, growth, flowering, seed production, etc. similar for both 

transgenic and non-transgenic plants? 

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ATX-07-37 2 09-086-104n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Bluffton (Wells Co.) IN: 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

2. Ames (Story Co.) IA: 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: September 26, 2010

Maize event VCO-Ø1981-5 

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ATX-07-40 1 09-215-106n 

USDA Field Termination Report

USDA Notification/Permit Number: 09-215-106n 

Applicant Internal Number: ATX-07-40 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; Corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047 

Section I. Site Release Information:

Trial was conducted at one (1) location: 

USDA-Notification 

Release Site No. 

County / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 Santa Isabel / PR 0.42 4/06/2010 6/18/2010 

Planted under Notification 08-254-111n and Notification 09-215-106n. 

Section II. List of agronomic observations collected on the regulatory compliance InSeason Forms approximately once every month during the growing season between the 

transgenic plants/regulated & non-transgenic plants/non-regulated.:

a Disease: Resistance/susceptibility to disease. 

b Insects: Abundance of non-target species and resistance/susceptibility to insect 

feeding not specifically engineered for resistance. 

c Plant Growth Is plant morphology and growth similar for both transgenic and nontransgenic plants? 

d Weediness Is germination, tasseling, seed production, etc. similar for both transgenic 

and non-transgenic plants? 

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ATX-07-40 2 09-215-106n 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Santa Isabel Co., PR (1st planting) : 

a) Cooperator reported no unusual differences in plant disease. 

b) Cooperator reported no unusual differences in insect pests or beneficial insect populations. 

c) Cooperator noted no unusual plant growth observations. 

d) Cooperator reported no unusual weediness characteristics. 

No unauthorized environmental releases occurred during the testing/growing phase of this regulated test. A 

Potential Compliance Incident (PCI) did occur during the volunteer monitoring period of this regulated field 

site, soil movement reported on August 10, 2010 (BCS PCI-10-005). 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to an 

Athenix Corporation facility or other laboratories for analysis was destroyed. Unplanted seed which 

was not utilized by the cooperator was returned to Athenix Corporation for storage or destroyed. 

Preparation Date: February 7, 2011 

Maize event VCO-Ø1981-5 

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ATX-07-43 09-341-105n 

1

USDA Field Termination Report

USDA Notification/Permit Number: 09-341-105n 

Applicant Internal Number: ATX-07-43 

Applicant: Athenix Corporation 

 Research Triangle Park, NC 27709 

 

Regulated Article: Zea mays; corn 

Lines: 6845-6916, 6933-6972, 6978-6993, 7045-7047 

Section I. Site Release Information:

Trial was conducted at one (1) location: 

USDA-Notification 

Release Site No. 

City / State Acreage 

Planted 

Date 

Planted 

Date 

Terminated 

1 

 

1 Juana Diaz / PR 0.32 01/26/2010 05/05/2010 

Maize event VCO-Ø1981-5 

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ATX-07-43 2 09-341-105n 

Section II. List of characteristic observations collected on the regulatory compliance InSeason Forms during the growing season (approximately: emergence, ~60, ~90 and ~120 

Days After Planting – Crop Dependent) between the transgenic plants & non-transgenic 

plants, insects and environment:

a Overall Crop/Test Condition Observations 

b Isolation Condition Observations 

c Phenotypic Plant Characteristic (Includes Weediness) Observations 

d Plant Disease Observations 

e Insect Beneficial Observations 

f Insect Pest Observations 

g Difference in Environmental Conditions Observations 

Section III. Summarized results from the regulatory In-Season Observation Forms:

1. Juana Diaz, PR : 

a) Cooperator reported overall crop condition observations as good. 

b) Cooperator reported overall isolation condition observations as good. 

c) Cooperator noted no unusual phenotypic plant characteristics. 

d) Cooperator reported no unusual differences in plant disease. 

e) Cooperator reported no unusual differences with beneficial insects. 

f) Cooperator reported no unusual differences with pest insects. 

g) Cooperator reported no unusual differences in environmental and/or weather conditions. 

No plot damage occurred. Trial was properly conducted and completed without any unauthorized 

environmental releases or compliance incidents. 

Section IV. Plant Disposition: 

All plot locations were harvested or terminated, and all remaining material that was not sent to a 

Bayer CropScience/Athenix Corporation facility or other laboratories for analysis was destroyed. 

Unplanted seed which was not utilized by the cooperator was returned to Bayer 

CropScience/Athenix Corporation for storage or destroyed. 

Signature: Date: May 19, 2011

Randy J. Wegener 

Regulatory Compliance Specialist 

Bayer CropScience – Regulatory Affairs BioScience 

Maize event VCO-Ø1981-5 

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APPENDIX 2 

Materials and Methods – Product Characterization 
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2.A. Materials and methods for molecular characterization 

Materials 

Leaf material was generated from various backcross generations of event VCO-Ø1981-5 

with the B110 and B109 inbred lines (BC0B, BC1B, BC2B, and BC1B2) or the AAX3 

inbred line (BC0A, BC1A, BC2A, BC3A) and appropriate non-transgenic control material 

for molecular characterization. Young leaves from 10 to 20 plants were harvested from 

greenhouse grown plants. Control materials were either non-transgenic control maize of 

the BC1B generation or the B110 inbred line. 

Reference materials

DNA of plasmid pAG3541 was included on all Southern blots as a positive control for 

probe hybridization. The Agrobacterium transformation plasmid pAG3541 (Figure 8) and 

E. coli plasmid pAX3541 (Figure 9) were used when appropriate to verify expected 

hybridization product sizes. The plasmid pAX3541 was prepared as a precursor in the 

cloning process of pAG3541 and contains the entire epsps grg23ace5 gene expression 

cassette. DNA molecular weight markers were used for Southern blot analysis. A 1 Kb 

DNA Extension Ladder (Life Technologies, Carlsbad, CA) was included as a size 

standard for hybridizing fragments on each gel and represented by kb line markers in 

this report. 

Plant processing and genomic DNA extraction 

All plants produced for these analyses were grown in the greenhouse and individually 

analyzed by event specific PCR. Leaf tissue of 10 to 20 plants from PCR positive plants 

of the same generation were combined, and genomic DNA was extracted and quantified 

for use in Southern blot analyses (Dellaporta et al., 1983). 

Quantitation of genomic DNA 

The concentration of each DNA sample was measured using a Quant-iTTM PicoGreen® 

dsDNA kit (Life Technologies, Carlsbad, CA) with a spectrofluorometer following the 

manufacturer’s instructions. A Lambda DNA standard was used to calibrate the 

instrument prior to quantification. 
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Figure 8. Transformation vector pAG3541 for event VCO-Ø1981-5 

Coding sequences are shown as orange arrows and regulatory elements as a blue box. 

Restriction enzyme locations are identified for the entire vector and Southern probes (5-

10) are indicated for the vector backbone. 
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Figure 9. Plasmid map of pAX3541 

Southern blot analysis 

Southern blot analyses were performed using standard molecular biology techniques 

(Southern, 1975; Sambrook et al., 1989). Genomic DNA (15 μg) was digested with the 

appropriate restriction enzymes overnight at the optimal temperature for each enzyme. 

Digested DNA was loaded onto 0.65% agarose gels and separated electrophoretically in 

2.5X TAE (100 mM Tris-acetate, 2.5 mM EDTA pH 8.3) buffer along with DNA molecular 

weight markers. Agarose gels containing the separated genomic DNA were submerged 

in 0.25 N HCl for 10 minutes to depurinate the DNA. Depurination was followed by 

denaturation in 0.5 M NaOH and 1.5 M NaCl for 30 minutes. Finally, the gel was 

neutralized in 0.5 M Tris-HCl at pH 7.0 and 1.5 M NaCl for 30 minutes, and soaked in 

20X SSC (3M NaCl, 0.3 M sodium citrate pH 7.0) for 30 minutes. The DNA was 

transferred to a Nytran SuPerCharge nylon (Whatman, Sanford, ME) membrane and 

assembled as a part of the TurboBlotterTM (Whatman, Sanford, ME) system. The DNA 

was allowed to transfer onto the membrane overnight in a 20X SSC buffer. Following 

transfer to the membrane, the DNA was bound to the membrane using a Stratalinker UV 

Crosslinker (Stratagene, La Jolla, CA). 

DNA probe preparation 

Element-specific DNA probes were labeled with dCTP-32P via random priming using the

Ready-To-Go DNA labeling beads (GE Healthcare Bio-Sciences Corp., Piscataway, NJ). 

Two of the probes with low GC content (ScUbi4 intron and 35S terminator) were labeled 

with dTTP-32P via random priming using the RadPrime DNA (Life Technologies, 

Carlsbad, CA) labeling system. For all element-specific probes, 25-30 ng of DNA were 

used for labeling. The radiolabeled probes were added to fresh hybridization solution, 
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and the membranes were incubated overnight. Hybridization was carried out at 65°C for 

approximately 16 hours, followed by two washes each in a) 40 mM NaPO4, pH 7.0, 

1 mM EDTA, 5% SDS, 0.5% BSA (Fraction V) and b) 40 mM NaPO4, pH 7.0, 1 mM 

EDTA, 1% SDS. The membranes were then subjected to autoradiography (Kodak AR XOMAT film using a Kodak intensifying screen at -80oC). 

Confirmation of the absence of vector backbone components and insert integrity 

Southern blot analysis was conducted to verify the absence of the transformation 

plasmid components outside of the transferred T-DNA region and the integrity of the 

insert. Genomic DNA (BC1B generation was used in the B110 inbred background), 

appropriate negative controls (both the non-transgenic control (BC1B) and the nontransgenic inbred (B110) were digested with a combination of HindIII and EcoRI, and 

independently with MfeI and NdeI. 

The probes employed were designed to hybridize to the functional components of the 

plasmid outside of the T-DNA. The Agrobacterium plasmid pAG3541 was included as a 

positive control for hybridization of the transformation plasmid components and loaded 

as genomic equivalent of 0.5, 1 and 3 copies. 

 

 2.B. Materials and methods for protein characterization tests 

Studies on potential toxicology and allergenicity for food, feed and the environment are 

conducted with purified EPSPS ACE5 protein expressed by Escherichia coli. In order to 

utilize the safety data of the proteins produced in the microorganism for the safety 

assessment of the same protein produced in a genetically modified plant, it is important 

to confirm that the protein produced in a microorganism is representative of the protein 

produced in the modified plant. The analytical tests show that the protein produced in E. 

coli is representative of EPSPS ACE5 protein produced in event VCO-Ø1981-5. 

Materials 

EPSPS ACE5 protein was produced in E. coli strain BL21-DE3 star (Life Technologies, 

Carlsbad, CA) using a T7 inducible expression plasmid with IPTG induction and was 

purified using ammonium sulfate fractionation (38-80% saturation) followed by 

hydrophobic interaction and anion exchange chromatography. The purified EPSPS 

ACE5 protein was dialyzed into phosphate buffered saline (137mM NaCl, 2.7mM KCl, 

10mM Na2HPO4/KH2PO4 pH 7.4) lyophilized, and stored at -80oC. For protein analysis, 

lyophilized protein was dissolved in ultrapure water to original volume yielding purified 

EPSPS ACE5 protein in PBS buffer for subsequent analysis. 

EPSPS ACE5 protein was purified from event VCO-Ø1981-5 leaf material in extraction 

buffer (40mM Tris-HCl, pH 8.0, 80mM NaCl, including complete protease inhibitor 

cocktail [Roche Diagnostics, Mannheim, Germany]) followed by ammonium sulfate 

fractionation (38-80% saturation), treatment with activated charcoal, and filtration 

through 0.22 micron polyester filters (Corning Life Sciences, Lowell, MA). Soluble 

extracted proteins were then further purified utilizing fast protein liquid chromatography 

(FPLC column) using hydrophobic interaction, anion exchange, and size exclusion 

chromatography. The protein fractions enriched for maize-produced EPSPS ACE5 

protein were concentrated using Biomax Ultrafree 5,000 dalton nominal molecular 

weight limit centrifugation tubes and stored at 4oC in 40mM Tris, pH 7.5, 80mM NaCl, 

10% glycerol buffer prior to analysis. 
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In vitro enzyme assay 

The function of EPSPS ACE5 protein was confirmed by carrying out enzymatic assays 

with purified E.coli-produced EPSPS ACE5 protein. An enzymatic assay was developed 

to quantify the production of inorganic phosphate (Pi) by EPSPS enzymes. The assay 

used enzyme coupling and resulted in the generation of a highly fluorescent product 

(Vazquez et al., 2003). This purified EPSPS ACE5 protein was mixed with shikimate-3-

phosphate (S3P) and phosphoenolpyruvate (PEP) and the presence of product 

(inorganic phosphate) was quantified using a fluorimeter. 

Analysis of the molecular weight comparison by SDS-PAGE 

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was 

used to separate proteins based on their molecular weight. This technique was 

employed prior to Coomassie Blue gel staining for molecular weight determination and 

mass spectroscopy analysis, as well as prior to electroblotting onto nitrocellulose or 

polyvinylidene fluoride (PVDF) membranes for western blot analysis or N-terminal 

sequencing, respectively. Proteins were prepared for SDS PAGE analysis by mixing with 

Laemmli buffer and heating for approximately 5 minutes to 100oC. Proteins were then 

loaded into the wells of NuPage 4-12% Bis-tris gels (Life Technologies, Carlsbad, CA) 

with either NuPage MOPs or MES running buffer in an XCell Surelock MiniCell 

electrophoresis chamber (Life Technologies, Carlsbad, CA). Novex Sharp prestained 

protein molecular weight markers (Life Technologies, Carlsbad, CA) were included as a 

visual reference for molecular weight determination. 

For molecular weight determination, total protein amounts of ~450 ng maize-produced 

EPSPS ACE5 and ~400 ng E. coli-produced EPSPS ACE5 were separated on a 4-12% 

Bis-Tris polyacrylamide gel in 1X MES buffer alongside prestained protein molecular 

weight marker. The separated proteins were visualized by Imperial Coomassie protein 

stain (Life Technologies, Carlsbad, CA) and photographed. 

MALDI-TOF mass spectrophotometry protein identification 

Protein bands were excised from Coomassie Blue stained SDS-Page gels and 

submitted to Alphalyse, Inc. (Palo Alto, CA) for analysis. The protein samples were 

reduced and alkylated with iodoacetamide and digested with trypsin that cleaves after 

lysine and arginine residues. The resulting peptides were concentrated on a ZipTip 

micropurification column and eluted onto an anchorchip target for analysis on a Bruker 

Autoflex III MALDI TOF/TOF instrument. The peptide mixture was analyzed in positive 

reflector mode for accurate peptide mass determination. MALDI MS/MS was performed 

on approximately 15 peptides for partial peptide sequencing. The MS and MS/MS 

spectra were combined and used for database searching using the Mascot software. 

Peptide masses and peptide fragment masses were matched against the expected 

EPSPS ACE5 sequence. 
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Western blot analysis 

Immunoreactivity was examined by western blot analysis. Purified maize- and E. coliproduced EPSPS ACE5 proteins were separated on a 4-12% Bis-Tris polyacrylamide 

gel along with protein molecular weight markers. Separated proteins were blotted onto 

nitrocellulose membrane in an XCell Surelock MiniCell (Life Technologies, Carlsbad, 

CA) system with a constant current of 30V for approximately 2 hours. Successive 

applications of primary antibody (affinity purified rabbit anti-EPSPS ACE5), and donkey 

anti-rabbit IgG linked horseradish peroxidase secondary antibody were applied. 

Electrochemiluminescencent substrate was then applied to allow visualization of 

immobilized antibody complexes by developing on x-ray film. 

Analysis of enzymatic activity of EPSPS ACE5 protein 

EPSPS ACE5 is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme, 

which releases inorganic phosphate as a by-product of its catalytic reaction. Released 

phosphate was detected by way of a real-time, coupled fluorescent kinetic assay. The 

final product of this assay was a red fluorescent marker with an excitation wavelength of 

555 nm and emission wavelength of 590 nm detectable by way of a spectrofluorometer 

(Molecular Devices). 

Samples containing both required substrates for the synthesis reaction as well as 

various dilutions of maize-produced EPSPS ACE5 samples were assayed to determine 

appropriate dilutions of the enzyme to yield a rate of product formation within the 

dynamic or linear range of the assay. Additional samples were assayed in triplicate 

containing an appropriate dilution of plant-purified EPSPS ACE5 along with specific 

reaction substrates as well as the following negative controls: buffer only, buffer plus 

substrate without enzyme, and buffer plus enzyme but without substrate. 

The enzymatic activity of E. coli-produced EPSPS ACE5 protein was analyzed following 

the same methods as described above (data not shown). 

N-terminal sequencing analysis 

Proteins were prepared for N-terminal sequencing by SDS Page separation followed by 

electroblotting onto a polyvinylidene fluoride (PVDF) membrane. Electroblotting onto 

PVDF membrane was performed exactly as described for blotting onto nitrocellulose for 

western blotting except that the PVDF membrane was equilibrated in 100% methanol for 

approximately 30 seconds prior to equilibration in transfer buffer. Proteins were 

transferred to the PVDF membrane by applying a constant charge of 30V for 120 

minutes. Blotted EPSPS ACE5 proteins were stained with Imperial Coomassie Blue for 5 

minutes and then destained for approximately 3 minutes in 50% methanol, 10% acetic 

acid until bands were clearly visible. The PVDF membrane was then washed extensively 

in ultrapure water and bands corresponding to EPSPS ACE5 were carefully excised for 

further analysis. Excised bands were packaged and shipped to Alphalyse, Inc (Palo Alto, 

CA) for sequence analysis. 

The analysis is performed on an ABI Procise 494 sequencer. The procedure determines 

the N-terminal amino acid sequence of proteins and peptides by the Edman degradation 

chemistry. The sequencing takes place on an acid-etched glass fiber disk or on a PVDF 

membrane. The Edman degradation is a cyclic procedure where amino acid residues 
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are cleaved off one at a time and identified by chromatography. There are three steps in 

the cyclic procedure. In step one the phenylisothiocyanate (PITC) reagent is coupled to 

the N-terminal amino group under alkaline conditions. In step two the N-terminal residue 

is cleaved in acidic media. In step three, the PITC coupled residue is transferred to a 

flask, converted to a PTH-residue and identified by HPLC chromatography. The next 

cycle is then started for identification of the next N-terminal residue. The cyclic process 

is not always 100% and therefore carryover from previous cycle can be observed. The 

identity of each single residue is detected by subtracting the previous chromatogram 

from the present chromatogram. 

Glycosylation Assay 

Glycosylation analysis was performed to determine if maize-expressed EPSPS ACE5 

was modified post-translation by the addition of carbohydrate moieties. Both microbialexpressed and maize-expressed EPSPS ACE5 proteins were separated by SDS-PAGE 

along with horseradish peroxidase (positive control protein) and soybean trypsin inhibitor 

(negative control protein). Gels were stained with Gelcode glycoprotein staining kit from 

Pierce Biotechnology, Inc. according to manufacturer’s instructions. The gel containing 

separated proteins was treated with periodate solution, which oxidizes cis-diol sugar 

groups in glycoproteins. The resulting aldehyde groups were detected through the 

formation of Schiff-base bonds with a proprietary reagent that produces magenta bands, 

traditionally called the periodic acid-Schiff (PAS) reagent method. Glycoproteins were 

detected as bright magenta bands on the gel. The same gel was then stained with 

Imperial Coomassie Blue stain for detection of all proteins loaded on the gel. 

Protein digestibility in simulated gastric fluid 

The E. coli-produced EPSPS ACE5 protein was evaluated for in vitro gastric digestibility 

using a standard pepsin digestion method (Thomas et al., 2004). 

Test protein and reference protein solutions were incubated with human SGF (a pepsin 

solution at pH 1.2) at approximately 37°C and samples were taken for analysis at timepoints ranging from 0 to 60 minutes. Reference proteins, horseradish peroxidase (HRP) 

and ovalbumin (OVA), known to be digested rapidly and slowly, respectively, were 

tested in parallel. 

The protein incubation for the test and reference materials was made in 2 ml 

microcentrifuge tubes in a waterbath at approximately 37°C. For each test and reference 

protein solution, 80 μl was added to 1,520 μl of SGF and mixed. Samples of 200 μl were 

taken at 0.5, 2, 5, 10, 20, 30 and 60 minutes. The tubes were agitated after each 

sampling and at approximately 45 minutes. A dilution of the test protein solution at 1/10 

in phosphate buffered saline (PBS 1X, 137 mM NaCl, 2.7 mM KCl, 10 mM Phosphate 

buffer, pH 7.4) supplemented with 20% glycerol was prepared for the 10% loading 

control. As soon as samples were taken, the reaction was terminated by adding the 

200 μl sample to a tube containing 70 μl 200 mM NaHCO3 (pH 11.0). 

Additional control samples were prepared: 

- a zero minute incubation of protein (10 μl) with 'SGF without pepsin' (190 μl); 

- a zero minute incubation of the 1/10 diluted protein (10 μl) with 'SGF without pepsin' 

(190 μl) (10% loading control); 

- a 60 minutes incubation of protein (10 μl) with 'SGF without any pepsin' (190 μl); 
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- a 'time zero' sample was produced by adding the protein (10 μl) to SGF (190 μl) after 

the reaction was terminated as above; 

- a sample of SGF alone before incubation and the reaction terminated as above; 

- a sample of SGF alone after 60 minutes incubation and the reaction terminated as 

above. 

SDS-PAGE was carried out following the method of Laemmli (1970) using a BioRad 

Mini-Protean III cell (BioRad, France). Prior to running SDS-PAGE, 5 μl of 5X Leammli 

solution was added to 20 μl of digestion samples and heated for 10 minutes at more 

than 90°C before loading the gel. Samples of 15 μl were added to wells of an SDSPAGE gel (15 wells, 1 mm 10-20% gradient polyacrylamide Tris/Tricine)(BioRad, 

France). A suitable marker solution (2.5-200 kDa) was used to provide reference points 

of known molecular weights on the gel (Mark 12, Invitrogen, France). Gels were stained 

with Coomassie blue (Invitrogen). 

For western blot analysis, a polyvinylidene fluoride (PVDF) membrane was placed on 

the SDS-PAGE gel in a Tris/Glycine buffer and an electrical current applied in order to 

transfer the protein bands onto the membrane. To detect the EPSPS ACE5 protein band 

and/or its potential fragments, the membrane was incubated in the presence of a specific 

rabbit polyclonal anti-EPSPS ACE5 protein antibody. The hybridization of the antibody 

with the proteins immobilized on the membrane was revealed using a goat anti-rabbit 

polyclonal antibody coupled with a peroxidase. The hybridization bands were visualized 

using enhanced chemiluminescence (ECL) detection system (Amersham ECL detection 

system, GE Healthcare Life Sciences, France). 

2.C. Materials and methods for protein levels in plant parts and during the life 

cycle 

Materials 

The field production of test materials (maize event VCO-Ø1981-5) was initiated in the 

2009 growing season to generate test and control materials at three locations in the US 

cornbelt. The field sites were as follows: Wright County, IA, Kossuth County, IA, and 

Webster County, IA. At each site, three replicated plots of event VCO-Ø1981-5 and the 

non-transgenic control hybrid were planted using a randomized complete block design. 

For each replicate, two plants each for event VCO-Ø1981-5 and the non-transgenic 

control were collected per growth stage. Leaf, root, and whole plant samples were 

collected at five different growth stages, pollen and mature grain were collected at the 

appropriate stages: 

V4 (vegetative 4-leaf stage) (leaf, root) 

V8 (vegetative 8-leaf stage) (leaf, root, whole plant) 

R1 (reproductive silking stage) (leaf, root, whole plant, pollen) 

R4 (reproductive kernel dough stage) (leaf, root, forage) 

R6 (physiological maturity) (leaf, root, whole plant, grain) 

All samples were packed and shipped on wet ice to the Athenix processing site in 

Research Triangle Park, NC. Plants were separated into parts, labeled, and frozen at 

minus 80°C until the time of processing. 
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Previous generations of the seed planted in this trial had been characterized by PCR 

and Southern blot and shown to be positive for the epsps grg23ace5 gene sequence. 

Representative tissues from event VCO-Ø1981-5 were characterized by event-specific 

PCR after receiving plants from the field to confirm correct planting, sampling, and 

labeling. 

The control material was a non-transgenic control produced through the same breeding 

scheme as event VCO-Ø1981-5. The identities of the test and control corn were 

confirmed by event-specific PCR prior to analysis. 

The protein reference standard (batch # GRG23ACE5-0108) was produced through over 

expression in E. coli. The purity of the standard was determined to be 89.15% by SDSPAGE and densitometry. 

Tissue processing and protein extraction 

Leaf, root, and grain samples were processed by grinding in dry ice in a Grindomix 200 

(Retsch). Ground tissues were sub-sampled and lyophilized frozen under vacuum until 

dry and stored at - 80°C until protein extracts were made. Whole plant and forage 

samples were processed by coarsely chopping the tissue and then grinding in dry ice in 

a VCM12 industrial blender (Sympak). Ground tissues were sub-sampled and lyophilized 

frozen under vacuum until dry and stored at -80°C until protein extracts were made. 

Pollen samples were sub-sampled and processed by grinding with stainless steel beads 

in 1.5 ml tubes in a MiniBeadbeater-96 (Bio Spec Products Inc.). Pollen samples were 

processed immediately before protein extracts were made. 

A sub-sample of each lyophilized sample was weighed into a 1.5 ml tube containing 4-8 

stainless steel beads. Using buffer ratios of 30:1 (leaf, root, forage, whole plant, and 

grain) and 120:1 (pollen), an appropriate volume of ELISA extraction buffer was added 

to each sample. Tissues were crushed in a MiniBeadbeater-96 for approximately 2.5 

minutes at room temperature. Extracts were spun down at 4°C, diluted at 1:10, 1:25, or 

1:50 in ELISA extraction buffer, and added to the ELISA wells at a volume of 50 μl. All 

experimental samples were run in triplicate wells on the ELISA plate. 

Antibodies 

The rabbit polyclonal antibody specific for EPSPS ACE5 was purified using 

immunoaffinity chromatography. The concentration and optimal dilution for plate-coating 

were determined, and the antibody stored at -20°C prior to analysis. A goat polyclonal 

antibody specific for EPSPS ACE5 was purified using immunoaffinity chromatography. 

The antibody was conjugated to horseradish peroxidase (HRP), its concentration and 

optimal dilution for use as a detection (secondary) antibody were determined, and the 

antibody conjugate was stored at -20°C prior to analysis. 

Enzyme linked immunosorbent assay (ELISA) 

Rabbit anti-EPSPS ACE5 antibody was diluted to a concentration of 0.5 μg/ml in coat 

buffer (0.01 M phosphate buffered saline, pH 7.4), added to medical grade polystyrene 

96-well EIA/RIA plates at a volume of 50 μl/well, and incubated overnight at 4°C. Plates 

were washed in wash buffer (0.01 M phosphate buffered saline and TWEEN® 20 0.05%, 

pH 7.4) and blocked for 1 hour at room temperature in 100 μl/well block buffer (0.01 M 

phosphate buffered saline, TWEEN 20 0.05%, pH 7.4 and 1% Bovine Serum Albumin). 
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Plates were washed in wash buffer. Samples and standards were diluted to appropriate 

concentrations in extraction buffer (0.01 M phosphate buffered saline (NaCl 0.138 M; 

KCl 0.0027 M); TWEEN 20 0.55%, pH 7.4), added to the plates at 50 μl/well, and 

incubated at room temperature for 1 hour. Plates were washed in wash buffer. HRPconjugated goat anti-EPSPS ACE5 antibody was diluted 1:2000 in coat buffer and 

added to plates. Plates were incubated at room temperature for 1 hour. Plates were 

washed in wash buffer. Tetramethyl benzidine (TMB) detection substrate was added to 

plates at 100 μl/well, and incubated at room temperature for 10 minutes. 1 N HCl was 

added to plates at 100 μl/well to stop the color reaction, and plates were read on a 

spectrophotometer (Spectromax 190 microplate reader, Molecular Devices, Sunnyvale, 

CA). Quantification of EPSPS ACE5 was determined by interpolation from the standard 

curve, whose linear range was 3 to 60 ng/ml. 

ELISA validation 

The ELISA assay for EPSPS ACE5 was validated under GLP by determining the Limit of 

Detection (LOD) of the assay in each tissue (matrix). Limit of Detection is defined as a 

concentration of protein detected in a matrix, which is statistically different from a nontransgenic control. LOD was determined in the following tissue types and ages: 

lyophilized leaf (V8 and R6), root (V8 and R6), whole plant (V8 and R6), pollen, and 

grain from non-transgenic control plants. Twelve replicate samples per tissue type and 

age were run vs. the standard curve in the EPSPS ACE5 ELISA assay. 

Calculations 

The Limit of Detection (LOD) for the GRG23 ACE5 protein was calculated as follows: 

a) For each tissue, an optical density (OD) value was calculated by performing 12 

replicate measurements and determining the arithmetic mean of those 12 values. 

The replicate values were corrected for background using a buffer blank. 

b) OD values of multiple non-transgenic tissue samples were measured as 

described in step a. 

c) The arithmetic mean (average) of the tissue OD values from step b was 

calculated. 

d) The standard deviation (sd) of the tissue OD values from step c was calculated. 

e) The standard deviation from step d was multiplied by 3 (3*sd) and added to the 

average OD value from step c. 

f) The value from step e (average + 3*sd) was converted to ng EPSPS ACE5/ml 

sample volume using a linear standard curve obtained with Microsoft Excel 

software. 

g) The ng/ml value from step e was corrected for dilution (multiplied by a dilution 

factor) and converted to ng/mg dry weight of tissue (using tissue weight and total 

sample volume) to provide the LOD for EPSPS ACE5 in plant tissue. 

The value in step g. was corrected for the percent purity of the EPSPS ACE5 standard 

by multiplying by 0.8915. The resulting values represent the LOD of EPSPS ACE5 in 

each tissue. Any values from event VCO-Ø1981-5 which fell below the LOD for that 

tissue were considered to be below the LOD of the assay, and were assigned a value of 

zero for calculation purposes. 
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Statistics 

Data are presented as ng of EPSPS ACE5/mg dw tissue. SoftMax Pro software 

(Molecular Devices, Sunnyvale, CA) calculated the concentration of EPSPS ACE5 in 

each sample in micrograms/milliliter (μg/ml) by comparison to a standard curve. 

Calculations to determine ng/mg dry weight were performed as follows: 

 The concentration of EPSPS ACE5 in ng/ml was multiplied by 0.05 ml (the 

volume of extract added to each well), which gave a value for the total ng of 

EPSPS ACE5 per well. 

 The dry tissue (dw) weight, volume of buffer used to create the tissue extract, 

and the volume of extract added to the ELISA were used to determine the mg dw 

tissue added per well. 

 Finally, the total ng EPSPS ACE5 per well was divided by the total mg dw tissue 

added to the well to calculate the expression level in ng EPSPS ACE5/mg dw 

tissue. 

Means and standard deviations of expression level (ng EPSPS ACE5/mg dw) were 

determined for each growth stage per tissue type across all three locations. 

2.D. Materials and methods for agronomic studies 

Genetics of event VCO-Ø1981-5 for field trial evaluations 

Following recovery from glyphosate selection during the plant transformation protocol, 

the T0 plantlet of event VCO-Ø1981-5 was transplanted into a germination soil mix and 

grown in a temperature-controlled greenhouse (daytime temperature = 27°C; night time 

temperature = 22°C). At flowering, the plant was pollinated with inbred B110 (Committee 

for Agricultural Development, Iowa State University), and the seeds were harvested and 

dried. These backcross zero (BC0B) seeds were then planted in a winter nursery 

(2007/2008) in Puerto Rico. Positive segregants were further identified by glyphosate 

spraying and crossed with inbred B110 line to produce back-cross one (BC1B) seeds 

used for the agronomic study in 2008. For the 2009 field trials, the back-cross zero 

(BC0B) were selfed two times to produce BC0BS2. In order to evaluate agronomic 

performance characteristics of event VCO-Ø1981-5 as compared to an appropriate nontransgenic control, two test-crosses were made and seed bulked for multiple location 

analysis in 2009. 

Trial design 

All trials were conducted using a randomized complete block design, with three 

replicated plots of each entry per location for 2009. Each plot consisted of four, 30 in 

(76.2 cm) rows by 17.5 to 20 ft (5.25-6.0 meters) long. Plants were thinned prior to 

reaching the V8 leaf stage resulting in a uniform number of plants in each row. Weed 

control was limited to conventional and cultural practices (hand hoeing); no broadspectrum herbicides, such as glyphosate or glufosinate are allowed except as a preplant or pre-emergence herbicide. Data on all parameters were collected on the middle 

two rows of each four-row plot. 
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Statistics 

All data collected was analyzed using Statistix Version 8.1 (Analytical Software, 

Tallahassee, FL). Within and “across location” analyses were conducted using an 

Analysis of Variance (ANOVA) for a Randomized Block Design. Means for the event 

were compared to the appropriate non-transgenic control using a two-sided Dunnett’s 

test with alpha = 0.05 (Moore and McCabe, 1999). The standard deviation and range of 

values collected across all locations are also reported. The following equation, Yij = U + 

Ti + Lj + LTij + eij, was used for the multi-location analysis where T was the presence or 

absence of the transgene, and L was the location. 

Field Locations, Preparations and Conditions 

The field trial conducted in 2008 was located in Polk County, Iowa, USA. As event VCOØ1981-5 was in the segregating backcross 1 (BC1B) generation, the plots were sprayed 

with a glyphosate solution to identify the negative segregants. 

A total of 17 field sites were utilized in 2009 across six US states to collect agronomic 

data (Figure 10 and Table 32). The locations include diverse environments within the 

major maize growing regions of the US. Off-season locations in Puerto Rico were also 

used for breeding purposes (not included in Figure 10). Table 32 lists the state, county 

and type of trial data collected. Color markers link the trial data to the location from 

which it was collected. The agronomic practices were representative of the location in 

which the field trials were conducted. 
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Figure 10. Map of 2009 agronomic field trial locations

Table 32. 2009 Field trial location details 

State County 

Color 

Marker on 

Map 

Protocol 

Iowa 

Cass Green agronomic performance 

Jasper Green agronomic performance 

Kossuth Red agronomic performance, protein 

expression, compositional analysis 

Webster Red agronomic performance, protein 

expression, compositional analysis 

Wright Red agronomic performance, protein 

expression, compositional analysis 

Scott Green agronomic performance 

Illinois 

Clinton Green agronomic performance 

Stark Green agronomic performance 

Champaign Green agronomic performance 

Logan Green agronomic performance 

Indiana 

Parke Green agronomic performance 

Boone Blue agronomic performance and 

compositional analysis 

Nebraska 

Washington Green agronomic performance 

Valley Green agronomic performance 

Valley Blue agronomic performance and 

compositional analysis 

Minnesota Stearns Green agronomic performance 

Wisconsin Walworth Green agronomic performance 
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2.E. Materials and methods for seed germination studies 

Five replicates of 50 seeds were planted on moistened germination paper and placed at 

25°C for seven days and percent emergence was determined. The cold germination test 

was conducted in accordance with AOSA, Seed Vigor Testing Handbook (2009). Four 

replications of 50 seeds were planted on moist creped cellulose paper and covered with 

1⁄2 to 3⁄4 inch of sand, placed at 10°C for 7 days and then moved to 25°C for 4 days at 

which time percent emergence was determined. 

The tetrazolium test was performed on the resulting ungerminated seeds at the end of 

each test to determine the percent non-viable seed. Seeds were bisected longitudinally 

and placed into a 0.1% tetrazolium solution to stain. After staining the seed was 

evaluated for viability determined by the appearance of red stain on living viable tissue 

and the location of the stain. 

The equation used to calculate percent germination was: 

100 – [(number of non-viable seed / germinated seeds) x 100] = % germinated 

2.F. Materials and methods for composition analysis 

Materials 

Plant material derived from event VCO-Ø1981-5 and the non-transgenic control was 

produced and collected at five field locations across the USA corn belt in 2009 (DM Crop 

Research Group, Inc., Granger, IA 50109). The reference hybrids (AgR5539, AgR7584, 

and AgR58036) were handled identically as the GM hybrids and the non-transgenic 

controls. Data from the reference hybrids is used in conjunction with published data to 

help determine normal ranges and variability within the measured parameters. 

Field Trial Description 

The field sites were planted in a randomized block design with 3 replications per site. 

The sites included five locations across three states (Boone County, IN; Valley County, 

NE; Kossuth County, IA; Webster County, IA; and Wright County, IA). No herbicide 

sprays (conventional or glyphosate) were used post-emergence. The planting population 

and all agronomic practices were representative of those used for maize production in 

each study location. 

Pollination Procedures 

In order to produce grain, hand pollinations were conducted within each replicate. 

Location personnel made five pollinations in each row. Five separate pollinates were 

carried out per replicate. Plants were crossed within the row (sib mating), but were not 

selfed. Standard breeding procedures for hand pollination of regulated material were 

followed. 
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Sampling Procedures 

For the forage samples, one whole plant from each plot was collected at the dough (R4) 

stage. Each plant was placed in a large plastic bag and shipped on ice directly from the 

field to the laboratory for analysis (EPL Bio-Analytical Services). For the grain samples, 

two ears were harvested at physiological maturity (R6) and 150 grams of grain were 

collected from center portion of each ear for analysis. Sampled grain was shipped at 

ambient temperature to the laboratory for analysis (EPL Bio-Analytical Services). 

Analytical Methods

The analytical methods were AOAC, AACC and AOCS International Methods or 

published methods as detailed in Table 33 for maize forage and Table 34 for maize 

grain. 

Table 33. Analytical methods for maize forage 

Maize Forage 

Analyte Method 

Ash AOAC International Method 923.03, 2000. 

Carbohydrates

100 – Ash (%Dry Base) - Fat (%DB) - Protein (%DB) 

USDA, 1973. Energy Value of Food, Agriculture Handbook No. 

74, pp. 2-11. 

Crude Fat AOAC International Method 922.06, 2000. 

Moisture AOAC International Method 930.15, 2000. 

Crude Protein

Foss-Tecator, 1999. In Foss-Tecator Kjeltec 2300 Site 

Preparation, Installation, and Operating Guide, Foss-Tecator 

AB, Box 70, S-263 21 Hoganas, Sweden. 

Acid Detergent Fiber Ankom Technology, 1999. ANKOM200 Fiber Analyzer 

Operator’s Manual, Ankom Technology, 140 Turk Hill Park, 

Fairport, NY 14450. 

Neutral Detergent Fiber

Ankom Technology, 1999. ANKOM200 Fiber Analyzer 

Operator’s Manual, Ankom Technology, 140 Turk Hill Park, 

Fairport, NY 14450. 

Crude Fiber

Ankom Technology, 1999. ANKOM200 Fiber Analyzer 

Operator’s Manual, Ankom Technology, 140 Turk Hill Park, 

Fairport, NY 14450.; Ankom Technology, 2006. ANKOM2000

Fiber Analyzer Operator’s Manual, Ankom Technology, 140 

Turk Hill Park, Fairport, NY 14450. 

Minerals AOAC International Method 999.11, 2000. 
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Table 34. Analytical methods for maize grain 

Maize Grain Analytical Methods 

Analyte Method 

Ash AOAC International Method 923.03, 2000.

Carbohydrates

100 – Ash (%DryBasis) - Fat (%DB) - Protein (%DB) USDA 

(1973).Merrill A.L. and Watt B.K. “Energy Value of Foods,” 

Agriculture Handbook No. 74, pp. 2-11. 

Crude Fat

Ankom Technology, 2008. ANKOMHCl Hydrolysis System 

Operator’s Manual, Ankom Technology, 2052 O’Neil Road, 

Macedon, NY 14502; Ankom Technology (2008). ANKOMXT15

Extraction System Operator’s Manual, Ankom Technology, 

2052 O’Neil Road, Macedon, NY 14502. 

Moisture AOAC International Method 925.09, 2000.

Crude Protein

Foss-Tecator, 1999. Foss-Tecator Kjeltec 2300 Site 

Preparation, Installation, and Operating Guide, Foss-Tecator 

AB, Box 70, S-263 21 Hoganas, Sweden. 

Acid Detergent Fiber

Ankom Technology, 1999. ANKOM200 Fiber Analyzer 

Operator’s Manual, Ankom Technology, 140 Turk Hill Park, 

Fairport, NY 14450.

Neutral Detergent Fiber

Ankom Technology, 1999. ANKOM200 Fiber Analyzer 

Operator’s Manual, Ankom Technology, 140 Turk Hill Park, 

Fairport, NY 14450.

Crude Fiber

Ankom Technology, 1999. ANKOM200 Fiber Analyzer 

Operator’s Manual, Ankom Technology, 140 Turk Hill Park, 

Fairport, NY 14450; Ankom Technology, 2006. ANKOM2000

Fiber Analyzer Operator’s Manual, Ankom Technology, 140 

Turk Hill Park, Fairport, NY 14450.

Amino Acids

Liu, H. J. 1994. Determination of amino acids by precolumn 

derivatization with 6-aminoquinolyl-n-hydroxysuccinimidyl 

carbamate and ultra-performance liquid chromatography with 

ultraviolet detection. J. Chrom. A, 670:59-66; Waters Method, 

Analysis of amino acids in feeds and foods using modification 

of the accq•tag method tm for amino acid analysis. 

Tryptophan 

Rogers, S.R.; Pesti, G.M. 1990. Determination of Tryptophan 

from Feedstuffs Using Reverse Phase High-performance 

liquid chromatography. J. Micronutr Anal. 7:27-35. 

Fatty Acid AOAC International Method 939.05, 2000. 

AOCS, Ce 2-66.; AOCS, Ce 1e-91. 

Vitamin B1/B2 

(Thiamine/Riboflavin) AACC International Method 86-80, 2000. 

Vitamin E Weber, E.J., 1984. High performance liquid chromatography of 

the tocols in corn grain. JAOCS, 61:1231-1234. 

Folic Acid AACC International Method 86-47, 2000. 

Phytic Acid AOAC International Method 986.11, 2000. 

Raffinose/Inositol AACC International Method 80-04, 2000. 

Minerals AOAC International Method 999.11, 2000. 

Vitamin A (Beta-carotene) AOAC International Method 941.15, 2000. 

Niacin AACC International Method 86-51, 2000. 

Vitamin B6 (Pyridoxine) AACC International Method 86-31, 2000. 
GENECTIVE SA Maize event VCO-Ø1981-5 

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Maize Grain Analytical Methods 

Trypsin inhibitor Anonymous 1997. Trypsin Inhibitor Activity. AOCS, Ba 12-75. 

p-Coumaric Acid and 

Ferulic Acid (phenolics)

Figueroa-Espinoza, M-C, Morel, M-H, Rouau, X. 1998. Effect 

of lysine, tyrosine, cysteine, and glutathione on the oxidative 

cross-linking of feruloylated arabinoxylans by a fungal 

laccase. J. Agric. Food Chem., 46, 2583-2589; Classen, D., 

Arnason, J.T., Serratos, J.A., Lambert, J.D.H., Nozzolillo, C., 

Philogene, B.J.R., 1990. Correlation of phenolic acid content 

of maize to resistance to Sitophilus zeamais, the maize 

weevil, J. Chem. Ecol. 16: 301-315; Krygier, K., Sosulski, F., 

Hogge, L. 1982. Free, esterified, and insoluble-bound 

phenolic acids. 1. Extraction and purification procedure. J. 

Agric. Food Chem. 30: 330-334; Sosulski, F., Krygier, K., 

Hogge, L. 1982. Free, esterified, and insoluble-bound 

phenolic acids. 3. Composition of phenolic acids in cereals 

and potato flours. J. Agric. Food Chem. 30: 337-340. 

Furfural

Bredie, W.L.P., Mottram, D.S., Guy, R.C.E. 1998. Aroma 

volatiles generated during extrusion cooking of maize flour. 

J.Agric. Food. Chem. 46:1479-1487.; Buttery, R.G., Stern, 

D.J., Ling, L.C. 1994. Studies on flavor volatiles of some 

sweet corn products. J. Agric. Food. Chem. 42: 791-795. 

Pantothenic Acid AOAC International Method 945.74, 1960. 

Total Dietary Fiber AOAC International Method 991.43, 2000. 

Starch Corn Refiners Association Method A-20, 2nd Rev, 1985. 

 

Statistical Analysis 

Data means, standard deviations, ranges and p-values were determined for the 

compositional data. Data were analyzed using JMP 8 software package from SAS 

Institute, Cary NC. For each analyte an ANOVA model was fit using the site and line 

main effects and the site by line interaction. Single degree of freedom contrasts were 

used to test for differences between the experimental line (VCO-Ø1981-5) and the nontransgenic control. P-values (≤0.05%) are not reported where the means were below the 

LOD, or where missing data made the effect non-testable. When data points were at or 

below the LOQ, the LOQ value was used to calculate the averages, standard deviations 

and data ranges. 

 
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APPENDIX 3 

Characterization of event VCO-Ø1981-5 maize 
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3.A. Insert Characterization 

Transgene copy number was investigated by Southern blot analysis. Genomic DNA 

samples of event VCO-Ø1981-5 and appropriate control samples were digested with the 

restriction enzymes HindIII and NdeI independently (Figure 11). Each of these restriction 

enzymes cuts one time within the T-DNA region. When hybridized with the epsps 

grg23ace5 gene probe (Figure 11 and Table 34, probe #3), the resulting number of 

bands indicates the insert copy number within the maize genome. 

The predicted sizes for the hybridization products are shown in Figure 11. The HindIII 

cleavage site located within the maize genome, adjacent to the left border region of the 

T-DNA insert, was further identified through isolation and sequencing of the flanking 

DNA regions (data not shown). Predicted and observed results are described in Table 

35 below. Southern blots are presented in Figure 12. 

Southern blot analysis was also conducted to determine insert integrity. Maize genomic 

DNA (event VCO-Ø1981-5 and appropriate non-transgenic controls) was digested with a 

combination of HindIII and EcoRI, and independently with MfeI. The locations of these 

restriction enzyme sites are shown in Figure 11. A set of four independent probes of the 

major genetic elements, namely, ScUbi4 promoter, ScUbi4 intron, epsps grg23ace5

gene and the 35S terminator (Figure 11, Tables 35 and 36, probes # 1-4) was used to 

confirm the integrity of the expression cassette. The predicted sizes for the hybridization 

products are shown in Figure 11 and Table 36. Southern blots are presented in Figures 

13 and 14 along with the predicted and observed results. 

Figure 11. Map of the T-DNA insertion site of event VCO-Ø1981-5 
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Table 35. Description of DNA probes used in Southern analysis 

Probe N° Description Probe size (bp) 

1 ScUbi4 promoter probe 351 

2 ScUbi4 intron probe 969 

3 epsps grg23ace5 probe 988 

4 35S terminator probe 280 

5 aad probe 784 

6 tetA/tetR probe 1956 

7 oriT probe 2001 

8 virC probe 1526 

9 virG probe 925 

10 virB probe

Region probed: 8513; 

3 overlapping probes of 2985, 

2757 and 2900 bp were used 

simultaneously. 

Table 36. Predicted and observed hybridization band sizes for insert copy number 

and insert integrity 

Probe No Probe Restriction 

Enzyme 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

Figure No 

3 epsps grg23ace5 gene 

probe HindIII 4040 bp ~4000 bp 12 

3 epsps grg23ace5 gene 

probe NdeI 3327 bp ~3300 bp 12 

1 ScUbi4 promoter probe HindIII/EcoRI 3599 bp ~3600 bp 13 

1 ScUbi4 promoter probe MfeI >3537 bp ~3800 bp 13 

2 ScUbi4 intron probe HindIII/EcoRI 3599 bp ~3600 bp 13 

2 ScUbi4 intron probe MfeI >3537 bp,and 

1143 bp 

~3800 bp, 

~1100 bp 13 

3 epsps grg23ace5 gene 

probe HindIII/EcoRI 3599 bp ~3600 bp 14 

3 epsps grg23ace5 gene 

probe MfeI 1143 bp and >2904 bp 

~1100 bp, 

~4000 bp 

14 

4 35S terminator probe HindIII/EcoRI 3599 bp ~3600 bp 14 

4 35S terminator probe NdeI >3327 bp ~3400 bp 14 
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Figure 12. Determination of insert copy number using probe 3 (epsps grg23ace5

gene)

The lane numbers, sample ID (restriction enzyme), and predicted and observed fragment sizes 

are indicated in the table below. The blot was probed with epsps grg23ace5 gene (probe 3).

Lane Sample ID 

(Restriction Enzyme) 

Predicted Fragment 

Size 

Observed Fragment 

Size 

1 pAX3541 (HindIII/EcoRI) 3599 bp ~3600 bp 

2 B110 (HindIII) -- -- 

3 BC1B non-transgenic control 

(HindIII) -- -- 

4 VCO-Ø1981-5 (HindIII) 4040 bp ~4000 bp 

5 B110 (NdeI) -- -- 

6 BC1B non-transgenic control 

(NdeI) -- -- 

7 VCO-Ø1981-5 (NdeI) 3327 bp ~3500 bp 

--; no hybridization 
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Figure 13. Analysis of insert integrity using probe1 (ScUbi4 promoter) and 

 probe 2 (intron) 

The lane numbers, sample ID (restriction enzyme), and predicted and observed fragment sizes 

are presented in the table below. 

Lane 

Sample ID 

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

Lane

Sample ID 

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

1 pAX3541 

(HindIII/EcoRI) 3599 bp ~3600 bp 8 VCO-Ø1981-5 (HindIII/EcoR1) 3599 bp ~3600 bp 

2 B110 

(HindIII/EcoRI) -- -- 9 

BC1B nontransgenic 

control 

(HindIII/EcoR1) 

-- -- 

3 

BC1B nontransgenic 

control 

(HindIII/EcoRI) 

-- -- 10 B110 

(HindIII/EcoR1) -- -- 

4 VCO-Ø1981-5 

(HindIII/EcoRI) 3599 bp ~3600 bp 11 VCO-Ø1981-5 (MfeI) 

>3537 bp, 

1143 bp 

~ 3800 bp,

~1100 bp 

5 B110 (MfeI) -- -- 12 

BC1B nontransgenic 

control (MfeI) 

-- -- 

6 

BC1B nontransgenic 

control (MfeI) 

-- -- 13 B110 (MfeI) -- -- 

7 VCO-Ø1981-5 

(MfeI) >3537 bp ~3800bp 14 pAX3541 (HindIII/EcoRI) 3599 bp ~3600 bp 

--; no hybridization 
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Figure 14. Analysis of insert integrity probe 3 (epsps grg23ace5 gene) and 

 probe 4 (35S terminator) 

The lane numbers, sample ID (restriction enzyme), and predicted and observed fragment sizes 

are presented in the table below. 

Lane 

Sample ID 

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

Lane 

Sample ID 

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

1 pAX3541 

(HindIII/EcoRI) 3599 bp ~3600 bp 8 pAX3541 (HindIII/EcoRI) 3599 bp ~3600 bp

2 B110 

(HindIII/EcoRI) -- -- 9 VCO-Ø1981-5 (HindIII/EcoRI) 3599 bp ~3600 bp

3 

BC1B nontransgenic control 

(HindIII/EcoRI) 

-- -- 10

BC1B nontransgenic control 

(HindIII/EcoRI)

-- -- 

4 VCO-Ø1981-5 

(HindIII/EcoRI) 3599 bp ~3600 bp 11 B110 (HindIII/EcoRI) -- -- 

5 B110 (MfeI) -- -- 12 VCO-Ø1981-5 

(NdeI) 3327 bp ~3400 bp

6 

BC1B nontransgenic control 

(MfeI) 

-- -- 13

BC1B nontransgenic control 

(NdeI)

-- -- 

7 VCO-Ø1981-5 

(MfeI) 

1143 bp, 

>3527 bp 

~1100 bp, 

~4000bp       

--; no hybridization 
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3.B. Insert stability across generations 

Southern blot analysis was conducted on multiple generations of event VCO-Ø1981-5 

progeny to evaluate the stability of the T-DNA insertion site. Genomic DNA isolated from 

leaf material of event VCO-Ø1981-5 plants from four breeding generations resulting from 

crosses with non-transgenic inbred line B110 (BC0B, BC1B, BC2B, and BC1B2) and 

non-transgenic controls were digested with the restriction enzyme HindIII. When 

hybridized with probe 3, specific for the epsps grg23ace5 coding region, genomic DNA 

from event VCO-Ø1981-5 digested with HindIII produces a single band >3600 bp in size 

(Figure 11). The transformation plasmid pAG3541 was included as a hybridization 

control. All four generations analyzed showed an identical hybridization pattern 

producing the same single ~4000 bp band (Figure 15). 

Figure 15. Insert stability across generations 

The lane numbers, sample ID (generation) (restriction enzyme), and predicted and observed 

fragment sizes are presented in the table below. 

Lane Sample ID Predicted Fragment 

Size 

Observed Fragment 

Size 

1 pAG3541 (NdeI) 5261 bp ~5300 bp 

2 B110 (HindIII) -- -- 

3 BC0B non-transgenic control (HindIII) -- -- 

4 VCO-Ø1981-5 Event (BC0B) (HindIII) 4040 bp ~4000 bp 

5 VCO-Ø1981-5 Event (BC1B) (HindIII) 4040 bp ~4000 bp 

6 VCO-Ø1981-5 Event (BC2B) (HindIII) 4040 bp ~4000 bp 

7 VCO-Ø1981-5 Event (BC1B2) (HindIII) 4040 bp ~4000 bp 

--; no hybridization 
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3.C. Confirmation of the absence of vector backbone 

DNA (event VCO-Ø1981-5 and appropriate non-transgenic controls) was digested with a 

combination of HindIII and EcoRI, and independently with MfeI and NdeI. The locations 

of these restriction enzyme sites are shown in Figures 11 and 16. The probes employed 

were designed to hybridize to the functional components of the plasmid outside of the TDNA (Probe 5: aad, Probe 6: tetR/tetA, Probe 7: oriT, Probe 8: virC, Probe 9: virG, and 

Probe 10: virB; Figure16 and Table 33). The Agrobacterium plasmid pAG3541 was 

included as a positive control for hybridization of the transformation plasmid components 

and loaded as genomic equivalent of 0.5, 1 and 3 copies (Figures 17 and 18). The 

tetA/tetR probe hybridizes with a 6700 bp fragment in the control and the aad probe 

hybridizes with a 5300 bp fragment in the control, while the oriT probe hybridizes with a 

14,000 bp fragment. Southern blot analysis results are shown in Figures 17 and 18. 

Figure 16. Map of plasmid pAG3541 with vector backbone probes 

Southern Probe # Description 

5 aad probe

6 tetA/tetR probe

7 oriT probe 

8 virC probe

9 virG probe

10 virB probe
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Figure 17. Absence of the transformation plasmid components (oriT, aad, tetA, 

and tetR probes) 

The lane numbers, sample ID (restriction enzyme), and predicted and observed fragment sizes 

are indicated in the table below. Brackets indicate the copy number related to maize genomic 

equivalent. 

Lane 

Sample ID 

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

Lane 

Sample ID

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

1 VCO-Ø1981-5 

(HindIII/EcoR1) -- -- 9 pAG3541 

(NdeI) [3 copies]

5261 bp, 

6699 bp 

~5300 bp, 

~6700 bp 

2 

BC1B nontransgenic control 

(HindIII/EcoR1) 

-- -- 10 pAG3541 

(NdeI) [1 copy] 

5261 bp, 

6699 bp 

~5300 bp, 

~6700 bp 

3 B110 

(HindIII/EcoR1) -- -- 11 pAG3541 

(NdeI) [0.5 copy]

5261 bp, 

6699 bp 

~5300 bp, 

~6700 bp 

4 VCO-Ø1981-5 

(HindIII/EcoR1) -- -- 12 VCO-Ø1981-5 (HindIII/EcoR1) -- -- 

5 

BC1B nontransgenic control 

(HindIII/EcoR1) 

-- -- 13 

BC1B nontransgenic control 

(HindIII/EcoR1) 

-- -- 

6 pAG3541 

(NdeI) [0.5 copy*] 13,994 bp ~14,000 

bp 14 B110 (HindIII/EcoR1) -- -- 

7 pAG3541 

(NdeI) [1 copy] 13,994 bp ~14,000 

bp 15 VCO-Ø1981-5 (MfeI) -- -- 

8 

pAG3541 

(NdeI) [3 copies] 13,994 bp ~14,000 

bp 16 

BC1B nontransgenic control 

(MfeI) 

-- -- 

--; no hybridization 

*; considered as genome equivalent 
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Figure 18. Absence of the transformation plasmid components (virC, virG, and 

virB probes) 

The lane numbers, sample ID (restriction enzyme), and predicted and observed fragment sizes 

are presented in the table below. Brackets indicate the copy number related to maize genomic 

equivalent. 

Lane 

Sample ID 

(Restriction 

Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

Lane Sample ID 

(Restriction Enzyme) 

Predicted 

Fragment 

Size 

Observed 

Fragment 

Size 

1 B110 

(HindIII/EcoR1) -- -- 14 BC1B non-transgenic control (MfeI) -- -- 

2 

BC1 nontransgenic control 

(HindIII/EcoR1) 

-- -- 15 VCO-Ø1981-5 (MfeI) -- -- 

3 VCO-Ø1981-5 

(HindIII/EcoR1) -- -- 16 pAG3541 

(NdeI) [0.5 copy] 11,174 bp ~11,500 bp 

4 B110 (MfeI) -- -- 17 pAG3541 (NdeI) [1 copy] 11,174 bp ~11,500 bp 

5 

BC1B nontransgenic control 

(MfeI) 

-- -- 18 pAG3541 

(NdeI) [3 copies] 11,174 bp ~11,500 bp 

6 VCO-Ø1981-5 

(MfeI) -- -- 19 pAG3541 (NdeI) [1 copy] 11,174 bp ~11,500 bp 

7 pAG3541 

(NdeI) [0.5 copy*] 

11,174 bp, 

13,994 bp 

~11,500 bp, 

~14,000 bp 20 pAG3541 

(NdeI) [3 copies] 11,174 bp ~11,500 bp 

8 pAG3541 

(NdeI) [1 copy] 

11,174 bp, 

13,994 bp 

~11,500 bp, 

~14,000 bp 21 B110 (HindIII/EcoR1) -- -- 

9 pAG3541 

(NdeI) [3 copies] 

11,174 bp, 

13,994 bp 

~11,500 bp, 

~14,000 bp 22 BC1B non-transgenic 

control (HindIII/EcoR1) -- -- 

10 B110 

(HindIII/EcoR1) -- -- 23 VCO-Ø1981-5 (HindIII/EcoR1) -- -- 

11 

BC1B nontransgenic control 

(HindIII/EcoR1) 

-- -- 24 B110 (MfeI) -- -- 

12 VCO-Ø1981-5 

(HindIII/EcoR1) -- -- 25 BC1B non-transgenic control (MfeI) -- -- 

13 B110 (MfeI) -- -- 26 VCO-Ø1981-5 (MfeI) -- -- 

--; no hybridization 

*; considered as genome equivalent 
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3.D. In vitro enzyme assay

The function of EPSPS ACE5 protein was confirmed by carrying out enzymatic assays 

with purified E.coli-produced EPSPS ACE5 protein. An enzymatic assay was developed 

to quantify the production of inorganic phosphate (Pi) by EPSPS enzymes. The assay 

used enzyme coupling and resulted in the generation of a highly fluorescent product 

(Vazquez et al., 2003). This purified EPSPS ACE5 protein was mixed with shikimate-3-

phosphate (S3P) and phosphoenolpyruvate (PEP) and the presence of product 

(inorganic phosphate) was quantified using a fluorimeter. As anticipated, addition of 

EPSPS ACE5 protein led to the steady-state production of inorganic phosphate (Figure 

19), while control reactions without EPSPS ACE5, PEP or S3P did not generate a 

fluorescent signal. 

Figure 19. Kinetic characterization of EPSPS ACE5 protein 

Panel A 

 

Panel A: A scatter plot of EPSPS ACE5 (designated as GRG23ace5 in the above plot) enzymatic 

activity as a function of the concentration of PEP at different glyphosate concentrations. The 

apparent Michaelis – Menten binding constant for PEP [Km (app)] and maximal rate (Vmax) were 

calculated from the half-maximal and maximal rates, respectively.
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Panel B 

Panel B: The Km (app) was measured at glyphosate concentrations ranging from 0 to 8 mM, and 

was plotted against the glyphosate concentration to derive the Ki for the enzyme (designated as 

GRG23ace5 in the above plot) (x intercept= 14,700 μM glyphosate). 
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3.E. Protein equivalency

3.E.1. SDS-PAGE of EPSPS ACE5 

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was 

used to separate proteins based on their molecular weight. 

The dominant protein bands for both the maize- and E. coli-produced EPSPS ACE5 

preparations migrated at a rate consistent with a molecular weight of approximately 

45,000 Daltons when compared to the molecular weight markers (Figure 20). This data 

indicates that the molecular weight of event VCO-Ø1981-5 maize partially purified 

EPSPS ACE5 protein is consistent with both the molecular weight of the E. coli -

produced protein and the expected molecular weight of 44.3 kDa. 

Figure 20. SDS PAGE 

Lane # Sample ID 

1 Novex Sharp Prestained Protein Marker 

2 ~ 450 ng EPSPS ACE5 (maize expressed) 

3 ~ 400 ng EPSPS ACE5 (microbial-produced) 

4 Blank 
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3.E.2. MALDI-TOF Mass spectrophotometry protein identification

Peptides confirmed by MS/MS sequencing are indicated in Table 37 and were mapped 

onto the expected EPSPS ACE5 amino acid sequence. These matching peptides 

constitute 45% coverage of the microbial-expressed EPSPS ACE5 protein and 42% 

coverage of the maize-derived protein when mapped onto the expected EPSPS ACE5 

amino acid sequence. 

The degree of coverage for both microbial-expressed EPSPS ACE5 protein and the 

maize derived EPSPS ACE5 protein confirms their identity and indicates that both 

proteins were expressed as intended. 

Table 37. Comparison of EPSPS ACE5 peptides analyzed by MALDI MS/MS 

Residue # 

Observed 

plant-derived 

protein mass 

Observed 

bacterialderived 

protein mass 

Theoretical 

Mass 

(calculated)

Sequence 

1-12 1361.67 1 1361.68 1 1360.70 METDRLVIPGSK 

27-44 1848.00 1848.02 1847.02 GTSVLVRPLVSA 

87-98 1231.63 1231.65 1230.68 FLPPFVAAGQGK 

87-108 2364.20 ND 2363.23 FLPPFVAAGQGKFTVDGSEQLR 

99-108 1151.55 1151.56 1150.56 FTVDGSEQLR 

99-109 1307.63 1307.64 1306.66 FTVDGSEQLRR 

109-120 1433.85 1433.87 1432.87 RRPLRPVVDGIR 

110-120 1277.75 1277.76 1276.77 RPLRPVVDGIR 

172-188 1973.06 1 1973.09 1 1972.08 VKIPNPVSQPYLTMTLR 

174-188 1745.88 1 1745.90 1 1744.92 IPNPVSQPYLTMTLR 

189-210 2356.06 1 2356.09 2355.08 MMRDFGIETSTDGATVSVPPGR 

192-210 1905.89 1905.92 1904.91 DFGIETSTDGATVSVPPGR 

192-214 2397.14 2397.17 2396.16 DFGIETSTDGATVSVPPGRYTAR 

215-237 2419.14 2419.17 2418.15 RYEIEPDASTASYFAAASAVSGR 

216-237 2263.02 2263.06 2262.04 YEIEPDASTASYFAAASAVSGR 

335-348 1630.72 1 1630.74 1 1629.76 TLGVQTDVGHDWMR 

349-358 1084.54 1084.54 1083.55 IYPSTPHGGR 

367-377 1207.65 1 1207.66 1 1206.68 IAMAFSILGLR 

392-402 1319.62 1319.63 1318.63 TFPGFFDYLGR 

1; Peptide identified following correction of observed mass for the oxidation of methionine. 

ND = not detected 
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3.E.3. Western blot analysis 

As shown in Figure 21, defined bands of the expected molecular weight of approximately 

45,000 Daltons were present for all three lanes containing partially purified maizeproduced EPSPS ACE5 protein (Lanes 2, 3, and 4) and all three lanes containing E. 

coli-produced EPSPS ACE5 protein (Lanes 5, 6, and 7). The rabbit anti-EPSPS ACE5 

antibody used in this analysis has been previously shown to be specific only for EPSPS 

ACE5 protein, and therefore immunoreactivity of both EPSPS ACE5 proteins confirms 

their identity. 

Figure 21. Western blot analysis of microbial-expressed and plant-expressed 

EPSPS ACE5 protein 

Lane # Sample ID 

1 Novex Sharp Prestained Protein Marker 

2 ~ 0.45 ng EPSPS ACE5 (maize-expressed) 

3 ~ 0.90 ng EPSPS ACE5 (maize-expressed) 

4 ~ 1.80 ng EPSPS ACE5 (maize-expressed) 

5 ~ 0.40 ng EPSPS ACE5 (E. coli -expressed) 

6 ~ 0.80 ng EPSPS ACE5 (E. coli -expressed) 

7 ~ 1.60 ng EPSPS ACE5 (E. coli -expressed) 
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3.E.4. Enzymatic activity 

Maize-expressed EPSPS ACE5 protein showed enzymatic activity only in the presence 

of its substrates as shown in Table 38. This result is comparable to the activity observed 

for E. coli-produced EPSPS ACE5 protein (data not shown). 

Table 38. Enzymatic activity of maize-expressed EPSPS ACE5 protein 

Sample Identification Average Rate (RFU/sec) 

Buffer Control 1.17 

Buffer + PEP 2.614 

Buffer + Event VCO-Ø1981-5 leaf 1.985 

Buffer + Event VCO-Ø1981-5 leaf + PEP 17.662 

RFU; relative fluorescence units 

PEP; phosphoenolpyruvate 

3.E.5. N-terminal sequencing 

The N-terminal analysis of maize- and E. coli-produced EPSPS ACE5 protein was 

consistent with the expected sequence (Table 39). The parentheses around the 

methionine for the maize-derived EPSPS ACE5 protein sequence indicate low signal 

strength for that cycle. 

Table 39. N-terminal amino acid sequence of maize- and E. coli-produced 

EPSPS ACE5 protein 

Expected sequence M E T D R L V I P G S K S I T N 

Maize-expressed 

EPSPS ACE5 sequence (M)E T D R L V I P G S K S I T N 

Microbial-expressed 

EPSPS ACE5 sequence M E T D R L V I P G S K S I T N 
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3.E.6. Glycosylation assay 

No glycosylation was detected for either the microbial- or maize-expressed EPSPS 

ACE5 protein. As shown in Figure 22, the glycoprotein positive control, horseradish 

peroxidase, stained a bright magenta color while the glycoprotein negative control, 

soybean trypsin inhibitor, showed only very faint light pink color. These results show that 

neither maize- nor microbial-expressed EPSPS ACE5 protein have been modified by the 

addition of detectable levels of carbohydrates. 

Figure 22. Glycosylation analysis of E. coli- and plant-expressed EPSPS ACE5 

protein 

Lane # Sample ID 

1 Novex Sharp Prestained Protein Marker 

2 blank 

3 ~ 4μg microbial expressed EPSPS ACE5 

4 blank 

5 ~ 4.5μg maize expressed EPSPS ACE5 

6 blank 

7 5 μg Soybean Trypsin Inhibitor (negative control)

8 5 μg Horseradish Peroxidase (positive control) 

9 blank 

10 blank 
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3.F. Protein digestibility in simulated gastric fluid 

 

It was shown that the pepsin was active, and that the two reference proteins, HRP and 

OVA, were rapidly and slowly digested, respectively. The results of the reference 

proteins are in line with the results obtained in an international ring trial organized by 

ILSI (Thomas et al., 2004). These quality control procedures confirm that the study 

procedures and reagents were adequate to detect the rate of digestion of proteins in this 

SGF study. 

The EPSPS ACE5 protein was degraded very rapidly with no residual protein visible at 

30 seconds of incubation with SGF, in presence of pepsin, at pH 1.2. There was no 

significant digestion at pH 1.2 in the absence of pepsin, showing that digestion requires 

pepsin. 

Figure 23. SDS-PAGE gel of EPSPS ACE5 protein digested in a standardized SGF 
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Figure 24. Western blot of EPSPS ACE5 protein digested in a standardized SGF 
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APPENDIX 4 

Herbicide Resistance and Product Stewardship 
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4.A. Evolution of herbicide resistant weeds

Herbicides are the most economical, effective and reliable method of weed control in 

most crop production systems. Herbicides act by targeting and inhibiting specific plant 

biochemical processes or pathways. The process of specific activity is termed “mode of 

action” (MOA). Herbicides are classified into groups based on their MOA (HRAC, 2011). 

During the past several decades, diversity in weed control methods has been declining. 

Consolidation of agriculture has occurred at all levels including combining smaller farms 

to form larger farms. The resulting economic pressures have led to the selection of the 

most profitable crops and have driven the adoption of monocultures. Tillage, a key 

cultural practice contributing to a diversified weed management program, has also been 

severely reduced through the adoption of conservation tillage systems such as no-till 

and minimum tillage to combat the widespread problem of soil erosion (Anderson, 1996). 

Weed control in the absence of complementary cultural control practices has resulted in 

the use of herbicides as the only weed control tactic. With this decline in use of 

alternative weed control methods, extensive use of herbicides with a single MOA has not 

only resulted in weed shifts but also high selection pressure for herbicide resistant 

weeds. Plants have the ability to adapt to ensure survival, which includes adapting to 

survive an herbicide application. The development of herbicide resistance is a function of 

time and exposure and also the genetic capability of the weed population present in a 

field (HRAC, 2011). 

Herbicide resistance is the naturally-occurring inheritable ability of some weed biotypes 

within a given population to survive an herbicide treatment that should, under normal use 

conditions, effectively control that weed population (HRAC, 2011). 

The first herbicide resistant weed was identified in 1964 (HRAC, 2011). An increase in 

the number of documented herbicide resistant weeds began a steep incline after the 

ALS inhibiting herbicides were introduced in the 1980’s. ALS herbicides inhibit the plant 

enzyme acetolactate synthase (ALS) and provide effective control of many grass and 

broadleaf weed species (Anderson, 1996; Whaley et al., 2007). ALS herbicides were 

available for a broad number of crops for both postemergence and residual weed 

control. Farming practices shifted, as use of ALS inhibitors enable a reduction in the 

amount of tillage needed for weed control benefitting soil conservation efforts. The lack 

of diversified weed control methods lead to the selection of populations of ALS 

herbicide-resistant weed species or biotypes. 

There are more than 40 weed species resistant to the ALS class of chemistry in the US 

today and 112 ALS-resistant weed species reported worldwide (Heap, 2011). Virtually all 

waterhemp (Amaranthus rudis) is considered by university weed scientists to be 

resistant to ALS inhibiting herbicides, resulting in the conclusion that ALS inhibiting 

herbicides are considered “obsolete” technology for weed control in soybean (Nordby et

al., 2007). In addition to weeds resistant to ALS inhibiting herbicides, resistance has also 

developed to many other herbicide modes of action as evident in Figure 26 (Heap, 

2011). 
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Figure 25. Timeline of the development of herbicide resistant weeds classified by 

herbicide mode of action 

In addition to the increasing frequency of weed resistance, there has been a steady 

decline in herbicide discovery (Duke, 2005). We must maintain the utility of the current 

herbicides in the marketplace to continue to enable the economic and environmental 

advancements in agriculture that have been enabled by chemical weed control. The use 

of detailed, diversified integrated weed management plans will be needed to deter 

resistance and to continue to enable conservation tillage practices, fewer herbicide 

applications, and the use of herbicides with more favorable environmental profiles. 

 
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4.B. Managing herbicide resistant weeds

Ideally integrated weed management should utilize all available tools including 

herbicides in a well-balanced program as the lower the diversity of weed control tools, 

the higher the risk of selecting a resistant biotype becomes. To ensure diversification is 

maintained in weed control methods, we also encourage growers to keep detailed 

records of weed management practices for each field. The following are our integrated 

weed management guidelines to promote an economically viable, environmentally 

sustainable, and socially acceptable weed control program:

1. Know your weeds, know your fields

Today’s herbicides control a broad spectrum of weed species, minimizing the 

importance of weed identification to a grower. However, identification of weed 

species will help identify an herbicide program that works best for every acre. 

Equally important is for the grower to understand the weed pressure and history 

within each field. Problematic areas like difficult-to-control weeds or dense weed 

populations should be closely monitored. There are several indications for a 

grower to consider with weed escapes to identify resistant weeds. 

Resistance indicators 

 The field has been sprayed repeatedly with the same herbicide (mode of 

action), particularly if there was no mode of action diversity in the weed 

management system; 

 A patch of weeds occurs in the same area year after year and is 

spreading; 

 Many weed species are managed, but one particular weed species is no 

longer controlled. For example, following a glyphosate application, 

actively growing marestail can still be seen, in the absence of other 

weeds; 

 Surviving weeds of the problem species may be in a patch where some 

are dead and some exhibit variable symptoms, but all are approximately 

the same age. 

2. Crop rotation

Crop rotation is one of the most important factors in an Integrated Weed 

Management (IWM) program. Crop rotation adds weed management diversity 

through the inherent use of herbicides with different modes of action. In addition, 

crops vary in their ability to compete for sunlight, water and nutrients with weeds. 

Different planting times and seedbed preparation techniques can lead to a variety 

of cultural methods, which employ diversity in a weed management program. 

Reliance on a monoculture crop leads to weed population shifts of fewer weed 

species but at overall higher densities of such weeds, which increases the 

selection pressure for herbicide resistant weeds. 

3. Start with clean fields 

Yields can be significantly reduced by early season weed competition. Proper 

tillage or the use of a burndown herbicide program should be used to control all 

emerged weeds prior to planting. Not only does the control of weeds prior to 

planting aid in the ease of planting, it also eliminates weed competition for soil 

moisture, light and nutrients. 
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Regardless of the tillage system (conventional, minimal, or no-till), a pre or early 

post-emergent soil-applied residual herbicide should be a part of every weed 

control program. A soil-applied herbicide provides residual weed control allowing 

the crop to get a head start. Residual herbicides minimize the weed pressure and 

allow a wider post-emergent herbicide application window. Generally, soil-applied 

herbicides can be included in the burndown herbicide program for residual weed 

control on no-till acres. A residual herbicide also introduces another mode of 

action into weed resistance management programs (Nordby et al., 2007). 

4. Rotate herbicide modes of action 

There are three key factors in using herbicides to promote good resistant weed 

management: 

 

 Use multiple modes of action during the growing season 

The use of multiple modes of action during the growing season increases the 

diversity within the weed control program by reducing the selection pressure of a 

single mode of action. A planned two pass herbicide (pre- followed by postemergence) program implements multiple modes of action in weed management 

systems for delaying weed resistance. 

Apply no more than two applications of a single herbicide mode of action to the 

same field in a two-year period 

Repeated, successive use of herbicides with the same mode of action increases 

the likelihood that resistant plants will reproduce and become dominant in the 

population. The best way to manage resistant weeds is to prevent them from 

spreading or populating. Herbicide-resistant weeds become problematic due to 

overuse of a single herbicide mode of action. To preserve an herbicide’s efficacy, 

maintain its use, and reap its benefits, growers should not use more than two 

applications of a single herbicide mode of action on the same field in a two-year 

period (Boerboom and Owen, 2006). In addition, rotating crops generally allows 

additional modes of actions to be used in a weed management program. 

 

 Rotate herbicide-tolerant trait systems 

To ensure the viability of all traits for the future, rotate the herbicide tolerant trait 

used in each field each year to increase the chemical diversity used in each field. 

5. Correct herbicide application

Product efficacy can be influenced by a multitude of factors. Ensuring correct use 

rates, weed growth stage and crop growth stage, and application technique will 

maximize weed control (Boerboom and Owen, 2006). 

Apply to actively growing weeds 

Herbicides provide peak performance when applied to actively growing weeds. 

Weeds that are actively growing absorb more herbicide. Conditions that provide 

peak growing environment for weeds are adequate soil moisture, optimal soil 

nutrients and temperature, and sunlight. 
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Timing 

The use of pre-emergent residual herbicides will provide key control of early 

season weeds that result in the greatest crop yield reduction and open a wider 

application window for post-emergence applications. Post-emergence herbicides 

should be applied after crop emergence when weeds are within the growth stage 

range specified on the label for optimal performance. Applying post-emergence 

herbicides to smaller weeds increases crop yield again by eliminating early 

season weed competition. 

Application technique 

Herbicides differ in the optimal application technique. Read and follow all label 

instructions to ensure proper application technique is achieved. Factors affecting 

weed control include: spray coverage, carrier volume, application speed, 

adjuvants, and tankmix partners. 

Product rate 

The rate listed on the product label has been researched and tested by 

manufacturers and university researchers to provide the optimal control of the 

weeds at the height/growth stage listed on the label. The application of an 

herbicide at a rate less than listed on the label can result in insufficient control 

and will have a significant impact on the immediate weed control and therefore 

the weed seed bank by allowing partially controlled weeds to reproduce and set 

seed. 

6. Control weed escapes 

Weeds that escape the herbicide applications should be controlled to reduce 

weed seed production. A grower should consider spot herbicide applications, row 

wicking, cultivation or hand removal of weeds to improve weed management for 

the subsequent growing seasons. 

7. Clean equipment

To prevent the spread of herbicide-resistant weeds and potentially introduce new 

invasive weeds on to the farm, avoid moving equipment that has not been 

thoroughly cleaned. 

 
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4.C. Glyphosate resistant weeds

There are currently 13 glyphosate-resistant weeds in the United States. These weeds 

include palmer amaranth (Amaranthus palmeri), marestail (Conyza canadensis), 

waterhemp (Amaranthus rudis), giant ragweed (Ambrosia trifida), common ragweed 

(Ambrosia artemisifolia), Johnsongrass (Sorghum halepense), Italian ryegrass (Lolium 

multiflorum), hairy fleabane (Conyza bonariensis), jungle rice (Echinochloa colona), 

annual bluegrass (Poa annua), kochia (Kochia scoparia), goosegrass (Eleusine indica), 

and rigid ryegrass (Lolium rigidum). There are an additional 8 glyphosate resistant 

weeds that can be found in other parts of the world (Heap, 2011). 

Although the list of herbicide resistant weeds continues to grow, corn growers still have 

many effective conventional chemical options for weed control both with soil-applied 

residual herbicides and post-emergence herbicides. The various modes-of-action 

available to a corn grower include triazines, photosystem II inhibitors, chloroacetamides, 

HPPD’s (4-hydroxyphenyl-pyruvate-dioxygenase), PPO’s (protoporphyrinogen oxidase), 

dinitroanilines, growth regulators, and ALS inhibitors. The conventional chemistry 

options for weed control in corn offer a grower multiple choices when rotating herbicide 

modes of action even when growing continuous corn. In addition to conventional 

chemistry, glufosinate (a glutamine synthease inhibitor) and the LibertyLink trait system 

gives growers the option to continue to use a herbicide-tolerant trait system and rotate 

herbicide MOA’s for weed resistance management. 

4.D. Characteristics of glyphosate herbicide 

Glyphosate is a non-selective, broad spectrum systemic herbicide introduced to the 

marketplace in the 1970’s. Glyphosate can be formulated in multiple ways: glyphosate 

isopropylamine salt (Roundup®3), glyphosate trimethylsulfonium salt (Touchdown®4), or 

glyphosate diammonium salt (Touchdown4®4 or Touchdown Pro®4). Glyphosate is the 

only member of the glycine herbicide family. Glyphosate inhibits the biosysnthesis of the 

aromatic amino acids in the shikimic acid pathway by inhibiting the enzyme 5-

enolpyruvylshikimate-3-phosphate synthase (EPSPS) (Anderson, 1996; Vencill, 2002). 

Glyphosate is labeled for the control of 113 annual broadleaf and grass weeds and 

additional 62 perennial weeds (Roundup Weathermax®3 label 2006). Glyphosate is likely 

the most broad spectrum herbicide available today for weed control in row crops. The 

effectiveness of glyphosate is well established; around 70% of the corn acres planted 

were herbicide-tolerant in 2010 (National Academies, 2010). 

 

 

3

 Roundup and Roundup Weathermax are registered trademarks of Monsanto Company 

4

 Touchdown, Touchdown4, and Touchdown Pro are registered trademarks of Syngenta Group Company 
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4.E. Stewardship of glyphosate-tolerant event VCO-Ø1981-5 maize 

Maintaining the sustainability of this technology is of high importance and the adoption of 

a life-cycle approach to product stewardship is a must. This means that appropriate 

stewardship principles are applied at every stage of development of such products, from 

research to product discontinuation. Our commitment to stewardship extends to 

corporate relationships and to the application of these stewardship and quality 

assurance standards that are required in those relationships. This commitment is also 

proven through all third party agreements related to these products, which include the 

following: 

“We are committed to apply the proper stewardship for these products and 

expects those with whom we contract to handle material containing our 

technology in an appropriate manner. This includes without limitation adherence 

to the stewardship and quality assurance provisions described in this 

Agreement”. 

All the teams involved in the development of such products in our organization as well as 

our subcontractors are committed to apply the stewardship guidelines and are aware of 

the procedures derived from these guidelines in order to communicate the appropriate 

information within the team matrix to correctly and rapidly respond to issues that may 

develop from the handling of such technologies. Field development and market support 

teams are provided with the corresponding tools necessary to support and help growers 

as a local and direct contact for any questions they may have and, which are related to 

our technologies with regards to product performance, product integrity or any eventual 

impacts on human and environmental health and safety. 

The procedures derived from these guidelines are audited regularly to check the 

conformity between procedures and their applications on site. Any non-conformity will be 

promptly corrected. 

4.F. Customer outreach 

We have a commitment to stewardship on all of our products, including herbicidetolerant trait (HTT) technology. We strive to provide best management practices of HTT 

technology, which includes integrated weed management to our customers. Education of 

integrated weed management is the only practical method for its success. Education 

starts internally with our own field development, technical service, and seed salesmen. 

Externally, we collaborate with key influencers to help growers understand the long term 

economic viability of integrated weed management. Those key influencers include 

university extension agents, agronomists, consultants, and local retail seed and 

chemical salesmen. In addition, we directly provide the integrated weed management 

message to growers through grower meetings, trade shows, and web and mail 

communications. 

A Technology Use Agreement or similar agreement will be developed and provided to 

each grower at the time of seed purchase. By signing the agreement, the grower will 

agree to best management strategies that are indicated in the agreement. The 

agreement will contain company contact information including a website for the best 
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management practices and product information. In addition, a toll free hotline for growers 

to obtain live technical product support will be provided. We are committed to our 

stewardship principles and procedures to communicate appropriate information to 

rapidly respond to any issue that may develop. 

Growers may also contact the seed company for product support. The seed company 

name and contact information will be provided on the label of each bag of seed sold. 

Each grower purchase of event VCO-Ø1981-5 maize will be recorded by seed company 

partners. This information will be provided to us to maintain a database of all growers 

utilizing products derived from event VCO-Ø1981-5. This database could be used to 

disseminate updated stewardship information. 

4.G. Additional customer support 

Product information 

There are a number of ways that a grower can obtain product information. The product 

label is the formal legal method of communicating directions for use of a herbicide. 

Screening for Herbicide Resistance 

Currently, confirmation of weed resistance is commonly conducted by collecting seed of 

suspected resistant plants. Those seeds are replanted in a greenhouse environment 

and sprayed with various rates of the herbicide to which resistance is suspected. The 

survival of the weeds confirms resistance. 

4.H. Monitoring of effectiveness of the stewardship plan 

Each grower purchase of event VCO-01981-5 maize will be recorded by the individual 

seed companies selling this event. This information will be provided to us to enable us to 

maintain a database of all growers utilizing event VCO-01981-5 products. We regularly 

utilize market research surveys to determine market share and adoption of technology. 

Seed company partners will have direct contact with growers and will be able to provide 

feedback to us regarding the stewardship effectiveness. Our field representatives, or 

those from our affiliates or licensees will also interact with growers and will be a source 

of information. 

We will continue to support ongoing efforts to understand weed resistance to herbicides 

and to apply learning to product labels and provide information to growers. 

 
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APPENDIX 5 

References Appendices 1-4 
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References 

Anderson, W.P. 1996. Weed Science: Principles and Applications. West Publishing, St. 

Paul, MN. pg. 128, 219-220, 256-257. 

AOSA, 2009. Association of Official Seed Analysts. Seed Vigor Testing Handbook. 

http://www.aosaseed.com/publications.htm#Vigor 

Boerboom, C. and M. Owen. 2006. The Glyphosate, Weeds, and Crop Series: Facts 

About Glyphosate-Resistant Weeds. www.glyphosateweedscrops.org. 

Dellaporta, S.L., Wood, J. and. Hicks, J.B, 1983. A plant DNA minipreparation: version II. 

Plant Mol Biol Reporter 1:19-21. 

Duke, S. O. 2005. Taking stock of herbicide-resistant crops ten years after introduction. 

Pest Management Science 61: 211-218. 

Heap, I. 2009. The International Survey of Herbicide Resistant Weeds. 

www.weedscience.com accessed September 15, 2009. 

Herbicide Resistance Action Committee (HRAC). 2011. www.hracglobal.com 

Laemmli U.K. 1970. Cleavage of structural proteins during the assembly of the head of 

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Moore DS and McCabe GP. 1999. Introduction to the Practice of Statistics, 3rd Ed. New 

York: WH Freeman. 

Nordby D., B. Hartzler, and K. Bradley. 2007. The Glyphosate, Weeds, and Crop Series: 

Biology and Management of Waterhemp. www.glyphosateweedscrops.org. 

National Academies 2010. http://www7.nationalacademies.org/ocga/testimony/t_Herbici

de_Resistant_Weeds_in_GE_Crops.asp 

Sambrook, J., Fritsch, E.F., and Maniatis T. 1989. Molecular Cloning, A Laboratory 

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Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated 

by gel electrophoresis J Mol Biol 98:503-517. 

Thomas K., Aalbers M., Bannon G.A., Bartels M., Dearman R.J., Esdaile D.J., Fu T.J., 

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S.C., Rice E.A., Privalle L.S., Steiner H. Y.,Teshima R., van Ree R., 

Woolhiser M., and Zawodnyk J. 2004. A multi-laboratory evaluation of a 

common in vitro pepsin digestion assay protocol used in assessing the safety 

of novel proteins. Reg Tox and Pharm, 39: 87-98. 
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Vencill, W.K., ed. 2002. Herbicide Handbook: Eighth Edition. Weed Science Society of 

America, Lawrence KS. pg. 213-234, 267-268. 

Whaley C.M., H.P. Wilson, and J.H. Westwood. 2007. A new mutation in plant Als 

confers resistance to five classes of Als-inhibiting herbicides. Weed Science

55: 83-90.