Petition for the Determination of Nonregulated Status for

Insect-Resistant and Herbicide-Tolerant 4114 Maize

We submit this petition under 7 CFR §340.6 to request that the Administrator, Animal and Plant 

Health Inspection Service, make a determination that the article should no longer be regulated 

under 7 CFR §340.

Submitting Company:

Pioneer Hi-Bred International, Inc.

7100 NW 62nd Avenue

PO Box 1014

Johnston, IA 50131-1014

Submitted by:

Natalie Weber, Registration Manager

Pioneer Hi-Bred International, Inc.

DuPont Agricultural Biotechnology

DuPont Experimental Station, E353/305C

PO Box 80353

Wilmington DE 19880-0353

Telephone: 302-695-8160

Fax: 302-695-3075

Prepared by:

Natalie Weber, Jennifer Tallman, Tracy Rood, Kent Brink, John Larsen, Sally Catron, Stephanie 

Kadlicko-Stare, Trevor Jones, Bonnie Hong, Lacy Elmore, and Holly Hettinger

OECD Unique Identifier: DP-ØØ4114-3

Submission date: August 25, 2011

Revised Submission date: November 9, 2011

Revision to the latest date: December 3, 2012

NO CBI
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4114 Maize

Table of Contents

List of Tables ................................................................................................................................... 4

List of Figures.................................................................................................................................. 7

Release of Information ................................................................................................................. 10

Certification................................................................................................................................... 11

Summary ....................................................................................................................................... 12

Abbreviations, Acronyms, and Definitions................................................................................... 16

1. Rationale for the Development of 4114 Maize................................................................. 19

1-A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR §340.6.... 19

1-B. Similarity of 4114 Maize to Previously Approved 1507 and 59122 Maize........................... 19

1-C. Purpose and Need for 4114 Maize....................................................................................... 21

1-D. Prior Environmental Release and Submissions to Other Regulatory Agencies ................... 23

1-E. Maize Crop Cultivation in the U.S. and Usage ...................................................................... 23

2. Introduced Trait, Development of the Transgenic Line, and Characterization

of Insertion and Expressed Products.................................................................................... 28

2-A. The Biology of Maize ............................................................................................................ 28

2-B. Description of DNA Used for Transformation and Intended Phenotype of 

Introduced Proteins...................................................................................................................... 28

2-C. Description of Transformation, Selection, and Breeding ..................................................... 38

2-D. Molecular Characterization of the Insertion in 4114 Maize ................................................ 43

2-E. Inheritance and Stability of the Inserted DNA...................................................................... 75

2-F. Concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT in 4114 Maize ............................. 77

3. Potential Allergenicity and Toxicity of 4114 Maize........................................................... 91

3-A. Allergenicity of Maize........................................................................................................... 91

3-B. Assessment of the Allergenicity of the Introduced Proteins in 4114 Maize........................ 92

3-C. Assessment of the Toxicity of the Proteins Expressed in 4114 Maize ................................. 94

3-D. Overall Conclusions on the Potential Allergenicity and Toxicity of 4114 Maize.................. 98

4. Comparative Assessment of 4114 Maize.........................................................................100

4-A. Compositional Assessment................................................................................................. 101

4-B. Agronomic Performance and Field Observations............................................................... 124
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4-C. Overall Conclusions to Comparative Assessment of 4114 Maize ...................................... 141

5. Potential Environmental Impact of the Introduction of 4114 Maize ................................143

5-A. Potential for 4114 Maize to Have Altered Disease and Pest Susceptibilities

or to Become Weedy or Invasive................................................................................................ 143

5-B. Potential for Gene Flow Between 4114 Maize and Sexually Compatible 

Wild Relatives.............................................................................................................................. 144

5-C. Potential Environmental Impact of the Introduced Proteins in 4114 Maize ..................... 148

6. Overall Conclusions........................................................................................................164

Appendix 1. 4114 Maize USDA Release Permits and Notifications and Planted Acreage......165

Appendix 2. Equivalency of the Proteins Produced in 4114 Maize to 

Those Produced in 1507 and 59122 Maize...........................................................................168

2.1 Overview and Summary....................................................................................................... 168

2.2 Western Blot Analysis of the Introduced Proteins .............................................................. 168

2.3 Conclusions.......................................................................................................................... 169

2.4 Experimental Methods......................................................................................................... 174

Appendix 3. Materials and Methods for Genetic Characterization of 4114 Maize................177

3.1. Southern Blot Characterization of 4114 Maize................................................................... 177

3.2. Test Material ....................................................................................................................... 177

3.3. Control Material.................................................................................................................. 177

3.4. Reference Material ............................................................................................................. 177

3.5. Identification of the 4114 Maize Plants Used for Southern Blot Analysis.......................... 178

3.6. Genomic DNA Extraction .................................................................................................... 179

3.7. Quantification of Genomic DNA ......................................................................................... 179

3.8. Digestion of DNA for Southern Blot Analyses..................................................................... 179

3.9. Electrophoretic Separation and Southern Transfer............................................................ 180

3.10. DNA Probe Labeling for Southern Blot Hybridization....................................................... 180

3.11. Probe Hybridization and Visualization.............................................................................. 180

3.12. Stripping of Probes and Subsequent Hybridizations ........................................................ 181

Appendix 4. Materials and Methods for Segregation Analysis of Five Generations

of 4114 Maize.....................................................................................................................182

4.1. Greenhouse Experimental Design ...................................................................................... 182

4.2. Planting and Leaf Sample Collection................................................................................... 182

4.3. Genotypic Analysis.............................................................................................................. 182

4.4. Phenotypic Analysis ............................................................................................................ 183
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4.5. Statistical Analysis............................................................................................................... 183

Appendix 5. Materials and Methods for Determination of Cry1F, Cry34Ab1, Cry35Ab1,

and PAT Protein Concentrations.........................................................................................184

5.1. Field Trial Experimental Design........................................................................................... 184

5.2. Plant Material Collection, Shipping, Processing, and Storage............................................ 184

5.3. Protein Concentration Determination................................................................................ 186

5.4. Statistical Analysis............................................................................................................... 188

Appendix 6. Materials and Methods for Nutrient Composition...........................................192

6.1. Field Trial and Experimental Design.................................................................................... 192

6.2. Plant Material Collection, Shipping, Processing, and Storage............................................ 192

6.3. Nutrient Composition Analyses.......................................................................................... 193

6.4. Statistical Analyses.............................................................................................................. 198

Appendix 7. Materials and Methods for Analysis of Agronomic Characteristics and

Germination Evaluation......................................................................................................202

7.1. Germination and Dormancy Evaluation.............................................................................. 202

7.2. Agronomic Characteristics Evaluation ................................................................................ 205

Appendix 8. Field Insect and Disease Observations ............................................................214

References .........................................................................................................................241

List of Tables

Table 1. Regulatory History of 1507, 59122, and 1507x59122 Maize in the U.S. ....................... 20

Table 2. Description of Genetic Elements in the T-DNA Region of PHP27118............................ 32

Table 3. Generations and Comparators Used for Analysis of 4114 Maize .................................. 41

Table 4. Description of DNA Probes Used for Southern Blot Hybridization................................ 46

Table 5. Predicted and Observed Hybridizing Bands on Southern Blots; Bcl I Digest................. 50

Table 6. Predicted and Observed Hybridizing Bands on Southern Blots; Hind III Digest ............ 57

Table 7. Summary of Genotypic and Phenotypic Results for Segregating 4114 Maize............... 76
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List of Tables (continued)

Table 8. Maize Growth Stage Descriptions.................................................................................. 78

Table 9. Mean Concentrations of Transgenic Proteins in 4114 Maize........................................ 78

Table 10. Cry1F Concentrations (ng/mg Tissue Dry Weight)....................................................... 81

Table 11. Cry34Ab1 Concentrations (ng/mg Tissue Dry Weight)................................................ 83

Table 12. Cry35Ab1 Concentrations (ng/mg Tissue Dry Weight)................................................ 85

Table 13. PAT Concentrations (ng/mg Tissue Dry Weight).......................................................... 87

Table 14. Summary of 4114 Maize Protein Concentrations as a Ratio of 

1507, 59122, and/or 1507x59122 Maize...................................................................................... 89

Table 15. Proximates in Maize Grain ......................................................................................... 104

Table 16. Vitamins in Maize Grain ............................................................................................. 106

Table 17. Minerals in Maize Grain ............................................................................................. 108

Table 18. Fatty Acids in Maize Grain.......................................................................................... 111

Table 19. Total Amino Acids in Maize Grain .............................................................................. 115

Table 20. Key Anti-Nutrients in Maize Grain ............................................................................. 119

Table 21. Key Secondary Plant Metabolites in Maize Grain...................................................... 120

Table 22. Proximates and Minerals in Maize Forage................................................................. 122

Table 23. Description of Seed Germination Conditions ............................................................ 126

Table 24. Summary of Warm Germination Test Results............................................................ 127

Table 25. Summary of Cold Germination Test Results.............................................................. 127

Table 26. Summary of Diurnal Germination Test Results.......................................................... 127

Table 27. Tetrazolium Chloride (TZ) Testing of Non-Germinated Seed..................................... 128

Table 28. Site Locations for 2010 Field Trial Experiments......................................................... 131
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List of Tables (continued)

Table 29. Agronomic Characteristics Measured........................................................................ 133

Table 30. Experiment A: Summary of Agronomic Performance of 4114 Maize 

Across Eleven Locations.............................................................................................................. 135

Table 31. Experiment B: Summary of Agronomic Characteristics of 4114 Maize 

Across Six Locations.................................................................................................................... 138

Table 32. 4114 Maize Estimated Margins of Exposure to Non-Target Organisms (Cry1F) ....... 154

Table 33. 4114 Maize Estimated Margins of Exposure to Non-Target Organisms 

(Cry34/35Ab1)............................................................................................................................. 156

Table 2.1. Primary and Secondary Antibody Descriptions Used for 

Qualitative Western Blot Analysis .............................................................................................. 176

Table 5.1. Maize Growth Stage Descriptions............................................................................. 185

Table 6.1. Methods for Compositional Analysis of 4114 Maize ................................................ 193

Table 7.1. Description of Germination Test Classifications....................................................... 203

Table 7.2. Maize Growth Stage Descriptions............................................................................. 207

Table 8.1. Observations of Insects Present and Comparison 

Between 4114 Maize and Control .............................................................................................. 215

Table 8.2. Observations of Diseases Present and Comparison 

Between 4114 Maize and Control .............................................................................................. 231
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List of Figures

Figure 1. U.S. Maize Usage by Segment, 2010............................................................................. 24

Figure 2. U.S. Maize Fed by Animal Group, 1993-2010 ............................................................... 26

Figure 3. Schematic Diagram of Plasmid PHP27118.................................................................... 30

Figure 4. Schematic Diagram of the T-DNA Region from Plasmid PHP27118 ............................. 31

Figure 5. Deduced Amino Acid Sequence of the Cry1F Protein................................................... 36

Figure 6. Deduced Amino Acid Sequence of the Cry34Ab1 Protein............................................ 36

Figure 7. Deduced Amino Acid Sequence of the Cry35Ab1 Protein ........................................... 37

Figure 8. Deduced Amino Acid Sequence of the PAT Protein .................................................... 37

Figure 9. Schematic Diagram of the Development of 4114 Maize.............................................. 39

Figure 10. Breeding Diagram for 4114 Maize and Generations Used for Analyses .................... 40

Figure 11. Plasmid Map of PHP27118.......................................................................................... 44

Figure 12. Map of PHP27118 T-DNA............................................................................................ 45

Figure 13. Map of the Insertion in 4114 Maize............................................................................ 47

Figure 14. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Bcl I Digested DNA with ubiZM1 Promoter and ubiZM1 Intron Probes....................................... 51

Figure 15. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Bcl I Digested DNA with cry1F and ORF25 Terminator Probes..................................................... 52

Figure 16. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Bcl I Digested DNA with cry34Ab1 and pinII Terminator Probes.................................................. 53

Figure 17. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Bcl I Digested DNA with TA Peroxidase Promoter and cry35Ab1 Probes..................................... 54

Figure 18. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Bcl I Digested DNA with 35S Promoter, pat, and 35S Terminator Probes ................................... 55
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List of Figures (continued)

Figure 19. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Hind III Digested DNA with ubiZM1 Promoter and ubiZM1 Intron Probes .................................. 58

Figure 20. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Hind III Digested DNA with cry1F and ORF25 Terminator Probes................................................ 59

Figure 21. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Hind III Digested DNA with cry34Ab1 and pinII Terminator Probes............................................. 60

Figure 22. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Hind III Digested DNA with TA Peroxidase Promoter and cry35Ab1 Probes................................ 61

Figure 23. Southern Blot Analysis of the T3 Generation of 4114 Maize; 

Hind III Digested DNA with 35S Promoter, pat, and 35S Terminator Probes............................... 62

Figure 24. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with cry1F Probe ................................................................... 64

Figure 25. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with cry34Ab1 Probe ............................................................ 65

Figure 26. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with cry35Ab1 Probe ............................................................ 66

Figure 27. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with pat Probe ...................................................................... 67

Figure 28. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with LB Probe........................................................................ 69

Figure 29. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with spc Probe....................................................................... 70

Figure 30. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with tet Probe ....................................................................... 71

Figure 31. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with virG Probe ..................................................................... 72

Figure 32. Southern Blot Analysis of the T2, T3, BC3F1*3 and BC3F2*2 Generations 

of 4114 Maize; Bcl I Digested DNA with RB Probe ....................................................................... 73
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List of Figures (continued)

Figure 33. Map of Locations for Agronomic Data Collection for 4114 Maize ........................... 131

Figure 34. Maize Planted Acres by County in the U.S................................................................ 146

Figure 2.1. Western Blot Comparison of Cry1F Protein............................................................. 170

Figure 2.2. Western Blot Comparison of Cry34Ab1 Protein...................................................... 171

Figure 2.3. Western Blot Comparison of Cry35Ab1 Protein...................................................... 172

Figure 2.4. Western Blot Comparison of PAT Protein ............................................................... 173
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Release of Information

Pioneer Hi-Bred International, Inc. (Pioneer) is submitting the information in this petition for 

review by USDA as part of the regulatory process. By submitting this information, Pioneer 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; USDA complies with the provisions of 

FOIA and USDA’s implementation regulations (7 CFR Part 1.4); and this information is 

responsive to the specific request. Except in accordance with the Freedom of Information Act, 

Pioneer does not authorize the release, publication or other distribution of this information 

(including website posting) without Pioneer’s prior notice or consent.

© 2011 Pioneer Hi-Bred, International, A DuPont Business. All Rights Reserved.

This document is protected under copyright law. This document is for use only by USDA-APHIS and its 

contractors, and only in support of actions requested by Pioneer. Any other use of this material, without 

prior written consent of Pioneer, is strictly prohibited. By submitting this petition, Pioneer does not

grant any party or entity any right or license to the information or intellectual property described in this 

document.
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Certification

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

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

includes all relevant data and information known to the petitioners, which are favorable and

unfavorable to the petition.

Date

Natalie Weber, Registration Manager

Pioneer Hi-Bred International, Inc.

DuPont Agricultural Biotechnology

DuPont Experimental Station, E353/305C

PO Box 80353

Wilmington DE 19880-0353

Telephone: 302-695-8160

Fax: 302-695-3075
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4114 Maize

Summary

Pioneer Hi-Bred International, Inc., a DuPont Business (Pioneer) is submitting a Petition for 

Determination of Nonregulated Status for insect-resistant and herbicide-tolerant maize event 

DP-ØØ4114-3, hereafter referred to as 4114 maize. Maize line 4114 was developed by Pioneer. 

Pioneer requests a determination from USDA - Animal and Plant Health Inspection Service 

(APHIS) that 4114 maize and any crosses of this line with other nonregulated Zea mays no 

longer be considered regulated articles under 7 CFR §340. 

4114 maize produces the Cry proteins Cry1F, Cry34Ab1, and Cry35Ab1, as well as the herbicidetolerance protein PAT. The Cry1F protein confers resistance to certain lepidopteran pests, 

including European corn borer (Ostrinia nubilalis), a major maize pest. This protein and its 

associated genetic elements are identical to those in DAS-Ø15Ø7-1 maize (hereafter referred to 

as 1507 maize), which was deregulated by USDA, registered by EPA, and reviewed by FDA in 

2001. The Cry34Ab1 and Cry35Ab1 proteins together comprise an active binary insecticidal 

crystal protein that confers resistance to corn rootworm pests, including western corn 

rootworm (Diabrotica virgifera virgifera), also a major maize pest. This binary protein and the 

associated genetic elements are identical to those in DAS-59122-7 maize (hereafter referred to 

as 59122 maize), which was deregulated by USDA in 2005, registered by EPA since 2005, and 

reviewed by FDA in 2004. Finally, the PAT protein confers tolerance to the herbicidal active 

ingredient glufosinate-ammonium at current labeled rates. This protein is identical to the 

protein found in a number of approved events across several different crops that are currently 

in commercial use, including 1507 and 59122 maize; maize containing the PAT protein has been 

commercially grown in the U.S. since 1996. 1507 maize, 59122 maize, and the breeding stack of 

the two lines, 1507x59122 maize, were jointly developed by Pioneer and Dow AgroSciences and 

are now licensed broadly across the seed industry. In 2010, commercial products containing 

1507x59122 maize were grown on approximately 14 million acres or approximately 16% of U.S. 

maize acres.

4114 maize is a new transformation event that, if deregulated, will provide an alternative to the 

breeding stack combination of two previously approved events: 1507 maize, which expresses 

the Cry1F and PAT proteins, and 59122 maize, which expresses the Cry34Ab1, Cry35Ab1, and 

PAT proteins. The 1507x59122 maize breeding stack combination was reviewed and registered 

by EPA in 2005. 4114 maize will provide similar insect resistance and herbicide tolerance to 

that of 1507x59122 maize. In 4114 maize, the genes for the Cry1F, Cry34Ab1, Cry35Ab1, and 

PAT proteins are contained on a single transformation construct and have been integrated at a 

single locus in the genome; this is in contrast to the 1507x59122 maize where the insertions for 

the events are located at two unlinked loci. As a new event with all genes located at a single 

locus, 4114 maize will be bred more efficiently into new product offerings for growers that are 

customized to their local insect protection and agronomic needs. Efficient breeding of multiple 

traits in single commercial maize products is becoming more important as growers demand 

more complex products, including multiple modes of action for lepidopteran and corn 

rootworm insect resistance and tolerance to one or more classes of herbicides. 
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4114 Maize

4114 maize was developed by Agrobacterium-mediated transformation using the DNA plasmid 

vector PHP27118. The T-DNA region of PHP27118 contains the cry1F, cry34Ab1, cry35Ab1, and 

pat genes. These gene cassettes are identical to those found in 1507 and 59122 maize, based 

on sequencing of the 4114 maize insertion, and the translated proteins were found to be 

identical. Furthermore, western blot analysis demonstrated similar molecular weight and 

immunoreactivity of the proteins. Therefore, identity of the Cry1F, Cry34Ab1, Cry35Ab1, and 

PAT proteins in 4114 maize to those in 1507 and 59122 maize was confirmed. 

Molecular characterization of the 4114 maize insertion by Southern blot analysis confirmed 

that a single, intact copy of the T-DNA of PHP27118 has been inserted into the maize genome 

and that the insertion is stable during the traditional breeding process. Southern blot analysis 

verified the absence of plasmid backbone DNA. Segregation analysis of five breeding 

generations of 4114 maize confirmed the stability and Mendelian inheritance of the insertion.

The concentrations of the introduced Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were 

measured in a number of 4114 maize tissues and were compared to the concentrations in the 

respective 1507, 59122, and/or 1507x59122 maize tissues. In general, these comparisons 

revealed that 4114 maize tissues have similar or lower concentrations of the introduced 

proteins than those of 1507, 59122, and/or 1507x59122 maize. Exceptions were Cry1F and 

Cry34Ab1 concentrations in senescent tissue and also Cry1F concentrations in pollen, where 

concentrations in 4114 maize were higher. In general, senescent tissue can contain a variable 

degree of extractable protein and likely accounts for the variability in the comparison. Overall, 

these comparisons indicate that any previously conducted safety studies that used 1507, 

59122, and 1507x59122 maize are applicable to 4114 maize. Safety studies that considered 

exposure to the Cry1F protein from 1507 or 1507x59122 pollen would require the 

reassessment of results for 4114 maize using the higher concentration.

The safety of the introduced proteins in 4114 maize has been previously evaluated by 

regulatory agencies in the U.S., as referenced above, and there is a history of safe use and 

exposure. The Cry1F, Cry34Ab1, and Cry35Ab1 proteins were derived from Bacillus 

thuringiensis and the PAT protein was derived from Streptomyces viridochromogenes. B. 

thuringiensis and S. viridochromogenes are naturally occurring soil bacteria and are not 

pathogenic; therefore, animals and humans are regularly exposed to these organisms and their 

components without adverse consequences. 

The potential for allergenicity and toxicity of 4114 maize was evaluated by examining the 

allergenic potential of maize as a crop and by assessing the allergenic and toxic potential of the 

Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. Maize is not a common allergenic food and the 

modification in 4114 maize is not expected to alter the allergenic potential of maize. The Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins have been assessed previously for 1507 and 59122 

maize and have been determined to be unlikely to be potential allergens or toxins to humans 

and animals. Previous assessments of these proteins included bioinformatic analyses, 

digestibility studies, and acute protein toxicity studies and are relevant for the assessment of 
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4114 maize. Updated bioinformatic analyses support the original conclusions that the Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins are unlikely to be allergens or toxins. These data 

support the conclusion that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in 4114 maize 

are therefore safe for the food and feed supply. These data have been submitted to EPA to 

confirm the safety of these proteins and will be submitted to the FDA for their food and feed 

safety assessment of 4114 maize.

Comprehensive agronomic performance assessments for 4114 maize were conducted in 

replicated field studies at a total of 17 locations in the U.S. and Canada. These assessments 

were conducted independently of the intended effects of the introduced traits (i.e., trait 

efficacy) so that appropriate comparisons would be made to conventional maize and any 

unintended effects due to the 4114 maize insertion would be evaluated. The following 

characteristics were measured: early population, final population, time to silking, time to 

pollen shed, pollen viability (measured at six locations), seedling vigor, stalk lodging, root 

lodging, stay green, disease incidence, insect damage, plant height, ear height, and yield

(measured at 11 locations). Seed germination and dormancy data were also collected in 

laboratory experiments. Analysis of these data showed no statistically significant differences 

between 4114 maize and control maize lines, indicating the agronomic comparability of 4114 

maize to conventional maize. In addition, 4114 maize has been field tested since 2006 in the 

U.S. and Puerto Rico. All releases in the U.S. have occurred under field permits and 

notifications granted by USDA - APHIS. All field trials of 4114 maize were observed for naturally 

occurring insects or diseases, and no unexpected differences between 4114 maize and control 

were observed. Together, these data support the conclusion that 4114 maize is unlikely to 

pose a greater plant pest risk than conventional maize. 

Extensive nutrient composition analyses of grain (60 analytes) and forage (nine analytes) were

conducted to compare the composition of 4114 maize to that of a control maize line and eight

commercial maize varieties. These analyses were used to evaluate any changes in the levels of 

key nutrients, anti-nutrients and secondary metabolites. Based on the results of the 

compositional evaluation, the grain and forage of 4114 maize are comparable to commercially 

available maize and there would be no significant impact on raw or processed maize 

commodities. Along with the agronomic data included in this petition, compositional 

comparability is a general indicator that 4114 maize will not exhibit unexpected effects with 

respect to plant pest risk. 

The potential environmental impact of the introduction of 4114 maize considered three 

primary areas: the potential for 4114 maize to become weedy or invasive; the potential for 

gene flow to sexually compatible wild relatives; and the potential impacts of the introduced 

Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. Comparative analyses indicated that 4114 maize 

was comparable to conventional maize with respect to the nutrient composition and agronomic 

characteristics measured and independently of the introduced traits. In general, maize does 

not possess weediness characteristics and is not considered a weedy or invasive species. 

Therefore, 4114 maize does not exhibit any characteristics that would indicate it is any more 

likely than conventional maize to become a weed or plant pest. The potential for gene flow 
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4114 Maize

examined maize pollination biology and the hybridization potential and geographic overlap of 

maize wild relatives. While maize does possess some pollination characteristics favorable to 

gene flow, the distribution of wild relative populations are limited in the U.S. and there is low 

fitness or sterility of hybrids; therefore, it is unlikely that the inserted DNA in 4114 maize would 

be introgressed significantly into these wild relative populations. 

As referenced earlier, 4114 maize contains the same introduced proteins as those present in 

1507, 59122, and/or 1507x59122 maize and has protein concentrations that are similar to or 

lower than these lines; therefore, previously conducted environmental safety studies for these 

lines were relevant for evaluating the environmental impact of 4114 maize cultivation. Based 

on the expected environmental exposure and the sufficient margin of safety of the proteins 

from 4114 maize, these environmental studies indicated that there is a low risk of the Cry1F 

and Cry34/35Ab1 proteins to non-target orders. A review of multiple field studies conducted 

for 1507, 59122, and/or 1507x59122 maize provide support that the abundance of non-target 

organisms would not be impacted by the cultivation of 4114 maize. In addition, certain 

threatened and endangered Lepidoptera and Coleoptera are unlikely to be adversely impacted 

from 4114 maize cultivation based on the lack of habitat overlap. Nutritional and toxicological 

studies on 1507, 59122, and/or 1507x59122 maize also support that 4114 maize will not 

adversely impact non-target vertebrates. Therefore, it is unlikely that 4114 maize will pose a 

risk to non-target organisms including beneficial, threatened and endangered species, as well as 

non-target vertebrates including birds, mammals, and humans. In support of this conclusion, 

1507 maize has been commercially available in the U.S. since the 2003 growing season and 

59122 and 1507x59122 maize have been available in the U.S. since 2006 with no negative 

safety or environmental effects. Taken together, it is unlikely that 4114 maize will pose a plant 

pest risk or impact non-target organisms in the environment.

In conclusion, based on the data contained herein, Pioneer requests that APHIS grant the 

request for a determination of nonregulated status for 4114 maize and any crosses of this line 

with other nonregulated Zea mays.
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Abbreviations, Acronyms, and Definitions

~ approximately 

%DB percent dry basis

1507 maize maize line containing the DAS-Ø15Ø7-1 event

1507x59122 maize maize line containing the breeding stack DAS-Ø15Ø7-1xDAS-59122-7

32D78 Pioneer® commercial hybrid line

3394 Pioneer® commercial hybrid line

33G26 Pioneer® commercial hybrid line

33J24 Pioneer® commercial hybrid line

34A15 Pioneer® commercial hybrid line

34M94 Pioneer® commercial hybrid line

34P88 Pioneer® commercial hybrid line

35K02 Pioneer® commercial hybrid line

35T06 Pioneer® commercial hybrid line

35T36 Pioneer® commercial hybrid line

36M28 Pioneer® commercial hybrid line

37H24 Pioneer® commercial hybrid line

37Y12 Pioneer® commercial hybrid line

38B85 Pioneer® commercial hybrid line

4114 maize maize line containing the DP-ØØ4114-3 event 

59122 maize maize line containing the DAS-59122-7 event

AACC American Association of Cereal Chemists

ADF acid detergent fiber 

AOAC Association of Official Analytical Chemists

AOSA Association of Official Seed Analysis

APHIS Animal and Plant Health Inspection Service of USDA

Bcl I restriction enzyme from Bacillus caldolyticus

Bt Bacillus thuringiensis

bp base pair 

CaMV cauliflower mosaic virus

CCUR Center for Crops Utilization Research

CFR Code of Federal Regulations

CI confidence interval

CMH Cochran-Mantel-Haenszel test

cry1F gene encoding the Cry1F protein from Bacillus thuringiensis var. aizawai

Cry1F protein encoded by the cry1F gene from Bacillus thuringiensis var. aizawai

cry34Ab1 gene encoding the Cry34Ab1 protein from Bacillus thuringiensis strain PS149B1

Cry34Ab1 protein encoded by the cry34Ab1 gene from Bacillus thuringiensis strain PS149B1

cry35Ab1 gene encoding the Cry35Ab1 protein from Bacillus thuringiensis strain PS149B1

Cry35Ab1 protein encoded by the cry35Ab1 gene from Bacillus thuringiensis strain PS149B1

CS compound symmetry

CT threshold cycle

df degrees of freedom

DI deionized

DIG digoxygenin 

DNA deoxyribonucleic acid 

DT50 time for 50% dissipation
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Abbreviations, Acronyms, and Definitions (continued)

E. coli Escherichia coli 

EEC expected environmental concentration

E score expectation score 

ELISA enzyme linked immunosorbent assay 

EPA Environmental Protection Agency 

EUP experimental use permit

FAME fatty acid methyl ester

FARRP Food Allergy Research and Resource Program

FDA Food and Drug Administration 

FDA CFSAN Food and Drug Administration’s Center for Food Safety and Applied Nutrition

FDR false discovery rate 

FOIA Freedom of Information Act

GC/FID gas chromatography with flame ionization detection

GLMM Generalized Linear Mixed Model

GNAT GCN 5-related N-acetyltransferases

Hae III Restriction enzyme from Haemophilus aegyptius

HFCS high-fructose corn syrup

Hind III Restriction enzyme from Haemophilus influenzae

HPLC high performance liquid chromatography

HRP horseradish peroxidase 

ICP-OES inductively coupled plasma optical emission spectroscopy

IgE immunoglobulin E 

kb kilobase pair 

kDa kilodalton 

KR Kenward-Roger

LB Left Border of the T-DNA

LC50 lethal concentration at which 50% of a test population are affected

LLOQ lower limit of quantification 

LS-Mean Least Squares Mean

LTP lipid transfer protein

ML maximum likelihood

MOE margin of exposure

MS mass spectrometry

NCBI National Center for Biotechnology Information 

NCGA National Corn Growers Association

NDF neutral detergent fiber

NOEC no observed effect concentration

OD optical density 

OECD Organisation for Economic Co-operation and Development 

ORF open reading frame

pat

gene encoding the PAT (phosphinothricin acetyltransferase) protein from 

Streptomyces viridochromogenes

PAT phosphinothricin acetyltransferase protein from Streptomyces viridochromogenes

PBST phosphate buffered saline plus Tweena-20

 

a

Registered trademark of ICI Americas, Inc. 
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Abbreviations, Acronyms, and Definitions (continued)

PCR polymerase chain reaction 

PH09B Pioneer proprietary maize inbred

PH1B5 Pioneer proprietary maize inbred

PHNAR Pioneer proprietary maize inbred

PHR03 Pioneer proprietary maize inbred

PHTFE Pioneer proprietary maize inbred

PHWWE Pioneer proprietary maize inbred; used for initial transformation to produce 4114 

maize

PIR Protein Information Resource

PR pathogenesis-related

PRF Protein Research Foundation

pinII proteinase inhibitor II 

RB Right Border of the T-DNA

REML Residual Maximum Likelihood

SAS Statistical Analysis Software

SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis 

SGF simulated gastric fluid

SIF simulated intestinal fluid

spc spectinomycin resistance gene

ssp. subspecies

T-DNA portion of the Agrobacterium transformation plasmid between the Left and

Right Borders that is expected to be transferred to the plant

tet tetracycline resistance gene

TZ tetrazolium chloride

ubiZM1 Zea mays polyubiquitin gene

UPLC ultra performance liquid chromatography

USDA United States Department of Agriculture 

USDA-APHIS BRS United States Department of Agriculture-Animal and Plant Health Inspection Service, 

Biotechnology Regulatory Services

USDA-ERS Economic Research Service of the U.S. Department of Agriculture

USDA-NASS National Agricultural Statistics Service of the U.S. Department of Agriculture

USDA-NCRS United States Department of Agriculture - Natural Resources Conservations Service

UTR untranslated region

UV ultraviolet

var. variety

wwPDB Worldwide Protein Data Bank

* Abbreviations of units of measurement and of physical and chemical quantities are used according to the 

standard format described in Instructions to Authors in the Journal of Biological Chemistry (http://www.jbc.org/)
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1. Rationale for the Development of 4114 Maize

4114 maize (OECD Unique Identifier DP-ØØ4114-3) is a new event that has been transformed

with a single genetic construct containing each of the proteins found in the previously approved

maize events, DAS-Ø15Ø7-1 (1507 maize; Cry1F and PAT proteins) and DAS-59122-7 (59122 

maize; Cry34Ab1, Cry35Ab1, and PAT proteins) and has been developed to provide an 

alternative to the 1507x59122 maize breeding stack for more complex stack combinations. 

4114 maize is not intended to be a stand-alone commercial product and will be combined with 

other approved events using conventional breeding to create stacked products with multiple 

modes of action for control of pest insects and with tolerance to one or more classes of 

herbicides. As part of a complex breeding stack, 4114 maize will have similar insect resistance 

and herbicide tolerance benefits as those containing the combination of 1507 and 59122 maize, 

and will have added breeding advantages over the available 1507x59122 maize. 1507, 59122, 

and 1507x59122 maize contain familiar traits and are currently licensed broadly across the seed 

industry; in 2010, commercial products containing 1507x59122 maize were grown on 

approximately 14 million acres or approximately 16% of U.S. maize acres (GfK Kynetec, 2010).

Maize is the largest crop grown in the U.S. in terms of acreage and net value. Maize has 

multiple downstream uses for feed, fuel, and food that are significant for the U.S. and global 

supply. The introduction of new maize trait offerings that meet grower needs, such as stacked 

products containing 4114 maize, is critical to help keep pace with increasing maize demand in 

the U.S. and globally.

1-A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR §340.6

The Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture

(USDA) has responsibility, under the Plant Protection Act (7 U.S.C. 7701-7772) and the Plant 

Quarantine Act (7 U.S.C. 151-167), to prevent the introduction or dissemination of plant pests 

into or within the U.S. Part 340 regulates introduction of organisms altered or produced 

through genetic engineering which are plant pests or for which there is a reason to believe are 

plant pests. The APHIS regulations at 7 CFR §340.6 provide that an applicant may petition APHIS 

to evaluate submitted data on the genetically engineered crop to determine that a regulated 

article does not present a plant pest risk and therefore should no longer be regulated.

Pioneer Hi-Bred International, Inc. is submitting data for genetically engineered insect-resistant 

and herbicide-tolerant 4114 maize and requests a determination from USDA - APHIS that event 

DP-ØØ4114-3 and any crosses with other Zea mays no longer be considered regulated articles 

under 7 CFR §340.

1-B. Similarity of 4114 Maize to Previously Approved 1507 and 59122 Maize

As mentioned earlier, 4114 maize is a new event with all genes at a single locus that will provide 

an alternative to 1507x59122 maize. 4114 maize contains each of the trait proteins found in 
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the previously approved 1507 maize (Cry1F and PAT proteins) and 59122 maize (Cry34Ab1, 

Cry35Ab1, and PAT proteins). 

Both 1507 and 59122 maize and their combination 1507x59122 maize are fully approved by 

U.S. regulatory agencies (Table 1). 1507, 59122, and 1507x59122 maize products have been 

commercially available in the U.S. since 2003, 2006, and 2006, respectively. 4114 maize has the 

same introduced genetic material and proteins as 1507x59122 maize, and has similar 

agronomic properties, protein expression, and efficacy to the previously approved events; 

therefore, portions of previous regulatory data and analyses of 1507, 59122 and 1507x59122 

are relevant to 4114 maize and will be cited in this petition.

Furthermore, 1507, 59122, and 1507x59122 maize were jointly developed by Pioneer and Dow 

AgroSciences; these commercial lines contain familiar traits and are currently licensed broadly 

across the seed industry. In 2010, commercial products containing 1507x59122 maize were 

grown on approximately 14 million acres, which represents approximately 16% of U.S. maize 

acres (GfK Kynetec, 2010).

Table 1. Regulatory History of 1507, 59122, and 1507x59122 Maize in the U.S.

Product Agency Approval Datea

1507 maize

USDA August 14, 2001

FDA May 18, 2001b

EPA August 10, 2001; expires September 30, 2015

59122 maize

USDA October 7, 2005

FDA October 4, 2004b

EPA August 31, 2005; expires September 30, 2015

1507x59122 maize

USDA Not Applicablec

FDA Not Applicablec

EPA October 27, 2005; expires September 30, 2015

a

Information accessed on U.S. regulatory agency websites (EPA, 2010a; EPA, 2010b; EPA, 2011a; FDA, 2001; FDA, 

2004; USDA-APHIS, 2001; USDA-APHIS, 2005).

b

Indicates completion date of biotechnology consultation.

c

No review of 1507x59122 maize was required by USDA or FDA as the approved individual events were combined 

by conventional breeding. 
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1-C. Purpose and Need for 4114 Maize

Through the use of 4114 maize in new stacked product offerings, there will be similar insect 

resistance and herbicide tolerance benefits as those of 1507x59122 maize. Additionally, there 

are added breeding advantages of using a single transformation event in these products over 

the available 1507x59122 maize breeding stack, as described further below.

Benefits of Insect Resistance and Herbicide Tolerance of 4114 Maize

The use of 4114 maize in commercial products will provide similar insect and weed control 

benefits to those of 1507x59122 maize. 4114 maize contains the same Cry1F, Cry34Ab1, and 

Cry35Ab1 proteins as 1507x59122 maize and will provide growers with a simple, inexpensive, 

highly effective, and environmentally benign means of controlling lepidopteran and corn 

rootworm insects, including European corn borer (Ostrinia nubilalis (Hubner)) and western corn 

rootworm (Diabrotica virgifera virgifera). Both European corn borer and the corn rootworm 

species complex (Diabrotica ssp.) are major maize insect pests throughout the U.S.; monetary 

losses resulting from feeding damage and insect control for each pest exceed $1 billion each 

year (Gray et al., 2009; Ostlie et al., 2002).

Similar to the benefits of 1507 and 59122 maize, the use of 4114 maize containing the Cry1F,

Cry34Ab1, and Cry35Ab1 proteins has the potential to offer effective control of maize insect 

pests and a reduction in the use of highly toxic agricultural pesticide chemicals to control these 

insect pests (EPA, 2010a; EPA, 2010b). The Cry1F, Cry34Ab1, and Cry35Ab1 proteins also offer 

crop yield advantage under insect pressure (EPA, 2010a; EPA, 2010b). In general, for corn 

borer- and corn rootworm-protected maize varieties, a 5% yearly average yield advantage 

compared to conventional maize has been realized in the U.S. since these varieties were 

cultivated (Brookes and Barfoot, 2009; Brookes and Barfoot, 2010). 

In contrast to the chemical pesticides used to control insect pests, the Cry1F, Cry34Ab1, and 

Cry35Ab1 proteins do not pose risks to humans or to the environment (EPA, 2010a; EPA, 

2010b). As a result, 4114 maize would offer similar benefits as 1507 and 59122 maize -- 

increased worker safety and grower profits through reductions in the use of a number of highly 

toxic and expensive chemical pesticide control programs (EPA, 2010a; EPA, 2010b). In general, 

farm income has increased as a result of use of insect-protected maize (Brookes and Barfoot, 

2010).

In addition, 4114 maize also contains the PAT protein that confers tolerance to the herbicidal 

active ingredient glufosinate-ammonium at current labeled rates. Glufosinate was registered as 

an herbicide in 1993 in the U.S. and is currently under re-review at EPA (CERA, 2002; EPA, 

2011b). Glufosinate-ammonium tolerance will allow growers to proactively manage weed 

populations and, in a proper herbicide rotation program, delay the development of adverse 

populations of weeds.
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Benefits of 4114 Maize as a Replacement Option for 1507x59122 Maize

As discussed previously, 4114 maize will provide an alternative to 1507x59122 maize in new 

complex stacked product offerings. Many maize products will be stacked through traditional 

breeding with multiple conventional and genetically modified traits to meet evolving grower 

needs of insect, weed, disease, and abiotic stress management. As a single event replacing a 

breeding stack with separate unlinked segregating events, 4114 maize will potentially benefit 

growers by reducing the development time for new products and by increasing product 

offerings that meet grower and trait durability needs.

The option to use 4114 maize, which contains all genes at a single breeding locus, will reduce 

the number of breeding loci over 1507x59122 maize, thus increasing the speed at which new 

products will be available to growers. This is because the complexity and expense of breeding 

multiple traits or events into one maize product increases with each breeding locus added. For 

example, the effort required to breed two transgenic loci into an inbred line is double the effort 

required for one transgenic locus. Furthermore, each trait locus must be homozygous in an 

inbred line and as more loci are combined, the proportion of plants that are homozygous for 

each locus becomes smaller, resulting in more seed discard during the breeding process. 

With the ability to efficiently create stacked products containing additional traits, both 

transgenic and native, there is an opportunity to enhance product durability and to provide 

custom offerings that better address grower regional needs. Growers are requiring 

sophisticated stacked products that include insect resistance (i.e., Bacillus thuringiensis or Bt

traits) and herbicide tolerance, as well as native traits such as disease tolerance, drought 

tolerance, and higher yields. For Bt traits, the maize seed industry is transitioning from 

products with a single mode of action to products with multiple modes of action, in order to 

extend the durability of the traits that many growers rely on to manage pests. Therefore, it is 

important that multiple native and transgenic traits -- including Bt traits with different modes of 

action -- are bred into a single maize product to efficiently meet the needs of growers. 4114 

maize, as one component of future Bt breeding stack products with multiple modes of action, 

will provide benefits to growers in the form of multi-trait products with enhanced durability

and more diverse germplasm suited for diverse growing regions.

4114 maize is expected to be more efficiently bred into a wide variety of maize genetic 

backgrounds and with additional traits, thus giving growers more choice in products that offer

in-plant insect protection and that are customized to their local growing areas and agronomic 

needs. Ultimately, these offerings will allow growers to continue to increase their overall farm 

productivity to meet the food security needs of a growing global population.
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1-D. Prior Environmental Release and Submissions to Other Regulatory Agencies

As a new transformation event, 4114 maize is regulated by the United States Department of 

Agriculture-Animal and Plant Health and Inspection Service, Biotechnology Regulatory Services 

(USDA-APHIS BRS) and the United States Environmental Protection Agency (EPA). 4114 maize 

falls within the scope of the Food and Drug Administration’s (FDA) policy statement concerning 

regulation of food products derived from new plant varieties, including those developed by 

recombinant DNA techniques.

4114 maize has been extensively field tested in the U.S. and Puerto Rico since 2006 in over 180 

separate plantings as authorized by the USDA-APHIS permits and notifications listed in 

Appendix 1. No Experimental Use Permit (EUP) applications for 4114 maize were submitted to 

the EPA. 4114 maize has been grown in small-scale field tests under the 10-acre per pest EUP 

exemption 40 CFR §172.3(c)(1). 

An application for a seed increase registration for 4114 maize was submitted to EPA on April 18, 

2011. Seed increase registrations are used by EPA to authorize breeding and testing of events 

such as 4114 maize that will not be sold commercially without being stacked with other insect 

resistant traits. Upon EPA registration, 4114 maize will have county and national acreage 

limitations imposed by the EPA and will be prohibited from commercial use on its own. Future 

commercial breeding stacks of 4114 maize with other EPA-regulated Plant-Incorporated 

Protectant events are subject to EPA’s regulatory authority and will require full registrations 

prior to commercialization.

A voluntary safety and nutritional assessment of 4114 maize will also be submitted to the FDA’s 

Center for Food Safety and Applied Nutrition (FDA CFSAN) in the third quarter of 2011.

Regulatory submissions will be made in key U.S. maize export markets. These countries include 

Canada, Japan, Mexico, Taiwan, South Korea, and China. A full commercial launch of any 

products containing 4114 maize will only occur after obtaining all necessary authorizations in 

the U.S. and key import countries with functioning regulatory processes.

1-E. Maize Crop Cultivation in the U.S. and Usage

Maize is the largest crop grown in the U.S. in terms of acreage and net value. Maize has 

multiple downstream uses for feed, fuel, and food that are significant for U.S. and global 

supply. In 2010, over 12 billion bushels of maize were produced in the U.S. from approximately 

81 million harvested acres with a crop value of $65.97 billion (NCGA, 2011; USDA-NASS, 2011). 

The U.S. is a major global exporter of maize at approximately 55% of the total trade market 

(NCGA, 2011). The largest maize U.S. export markets in 2009-2010 were Japan, Mexico, South 

Korea, Taiwan, Egypt, and Canada (NCGA, 2011). Exports accounted for 14.5% of the maize 

produced in 2010 (Figure 1; NCGA, 2011).
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The introduction of new maize trait offerings that meet grower needs, such as stacked products 

containing 4114 maize, is critical to help keep pace with increasing maize demand in the U.S. 

and globally. A significant portion of maize cultivated in the U.S. is genetically modified and 

contains similar insect resistant traits. In 2010, 86% of maize grown in the U.S. was genetically 

modified; insect resistant varieties accounted for approximately 63% of all maize acreage, 

which includes the percentage of insect resistant traits as well as stacked varieties (USDA-NASS, 

2010). Over the past decade, maize yields and overall production have increased, in part due to 

improvements in seed varieties and agronomic production practices (NCGA, 2011; USDA-ERS, 

2009).

Some background on maize processing and use for feed, fuel, and food industries is described 

below.

Figure 1. U.S. Maize Usage by Segment, 2010

HFCS: High-fructose corn syrup

Source: NCGA, 2011
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Maize Processing for Feed, Fuel, and Food Uses

Maize grain requires processing for some downstream uses. Wet and dry milling processes are 

used to separate grain into components for food, feed, and fuel processing (OECD, 2002). 

Wet milling starts with softening the kernel in hot water and sulfur dioxide prior to further 

fractionation and processing (OECD, 2002). Products produced in the wet milling process 

include germ meal, oil (further processed into margarine, cooking oil, baking and frying fats), 

corn gluten feed, corn gluten meal, and starch (further processed into ethanol and sweeteners) 

(OECD, 2002). 

There are several means of dry milling maize grain, but by far the most widely used process 

begins with soaking the kernel in water to remove the pericarp and germ, followed by drying 

the remaining grain fraction before additional processing (OECD, 2002). Products produced in 

the dry milling process include flour, meal, germ meal, oil, beverage and fuel ethanol, distillers 

dried solubles, flaking grits, hominy feed, and grits (OECD, 2002). Maize grain may also be 

cooked in alkali and finely ground to produce what is known as masa, which is used for tortillas 

and snack chips (OECD, 2002). 

The production of fuel ethanol typically begins with dry milling of maize grain, cooking, 

saccharification, and fermentation to produce ethanol and the by-product distiller dried grains 

or solubles (OECD, 2002).

Feed Use of Maize

The largest proportion, 38.7%, of maize produced in the U.S. is used for animal feed (Figure 1; 

NCGA, 2011).

Of the maize grain that is used for feed, the greatest percentage is consumed by beef cattle, 

followed by poultry, pork, and dairy cattle (Figure 2; NCGA, 2011). A number of different 

products from the maize plant and from grain processing may be used as feed. 

The whole maize plant or its residue from harvesting are frequently used as animal feed. 

Silage, derived from the above-ground portions of the maize plant, is an important feed 

ingredient for feedlot and dairy cattle and preserves more than 90% of nutrients (OECD, 2002). 

In 2009, approximately 6% of the U.S. maize crop was used for silage (USDA-NASS, 2010). In 

addition, stalks from harvested maize plants can be grazed by ruminants in the field (OECD, 

2002).

Maize ears, without shelling (i.e., removing the grain from the cob), can be ground directly for 

ruminant feed (OECD, 2002). When ears are shelled to remove the grain, remnant cobs can 

also be used in animal feed (OECD, 2002). Maize grain can be fed to animals with minimal 

processing and can be fed whole, rolled, ground, or steam flaked (OECD, 2002). Rolled or 
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ground grain is fed to swine and poultry (OECD, 2002). Maize grain added to pet foods is 

ground, cooked, and pelleted or extruded (OECD, 2002). 

Processed products from the milling and ethanol fermentation processes are also fed to 

livestock. A by-product of the wet milling process, corn gluten meal, is fed to ruminants, 

poultry, and swine (OECD, 2002). The ethanol fermentation process produces a co-product 

called distillers dried grains/solubles or corn gluten feed that is used as animal feed to dairy 

and beef cattle, poultry, and swine (USDA-ERS, 2009; USDA-ERS, 2010; USDA-NASS, 2007).

Figure 2. U.S. Maize Fed by Animal Group, 1993-2010

Source: NCGA, 2011
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Fuel Use of Maize

Maize is the primary feedstock used to produce ethanol in the U.S.; 36.5% of maize grain

produced is fermented into fuel ethanol (NCGA, 2011; USDA-ERS, 2010). In 2009, ethanol 

represented 8% of motor vehicle gasoline supplies and is expected to remain below 10% of 

annual gasoline use by 2015 (USDA-ERS, 2010). 

Food Use of Maize

Starch, oil, grits, bran, meal, and flour from maize wet and dry milling are primarily used in 

foods (OECD, 2002). A majority of starch is converted to sweeteners, such as corn syrup, high 

fructose corn syrup, maltodextrins, and dextrose, and also fermented into ethanol (OECD, 

2002). A significant portion of U.S. maize, 3.8%, goes to the production of high-fructose corn 

syrup as an end product (Figure 1; NCGA, 2011). Approximately 6.3% (classified in the “other” 

usage segment) comprises food purposes such as starch, sweeteners, cereal/other, and 

beverage alcohol (Figure 1; NCGA, 2011).

Starch is used for food such as bakery products/mixes, condiments, candies, and prepared 

(snack, dessert, meat) foods (CCUR, 2009). Sweeteners are used for soft drinks, candies, bakery 

products/mixes, condiments (jams, jellies, dressings), and prepared foods (CCUR, 2009). Whole

maize is consumed as popcorn, sweet corn, and alkali processed grain for tortillas and snack

chips (CCUR, 2009), though these uses comprise a very minor usage segment.
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2. Introduced Trait, Development of the Transgenic Line, and Characterization of Insertion 

and Expressed Products

Characterization of the transgenic crop variety provides additional knowledge about the genetic 

modification and the trait as a starting point for the safety assessment. Knowledge about the 

transgenic crop in these areas provides background information for the food, feed, and 

environmental safety assessments and, if applicable, can also identify certain areas of greater 

potential risk or concern. 

This section provides an overall characterization of 4114 maize and includes information about 

the maize crop, the source of the donor genetic elements, the identity and intended function of 

the expressed proteins, the development of the 4114 maize line, the structure and genetic 

stability of the DNA insertion, and the tissue-specific concentration of the expressed proteins. 

The information below provides background for safety assessments in later sections.

2-A. The Biology of Maize

Biology documents on the unmodified plant species have been published by the Organization 

for Economic Co-operation and Development (OECD) (OECD, 2003). The OECD document on 

maize provides background on the biology of Zea mays including:

 general description, including information on use of maize as a crop plant;

 taxonomic status of Zea;

 identification methods;

 center of origin / diversity and maize diversity;

 reproductive biology, including sexual and asexual reproduction;

 crosses, including intra- and inter-specific crosses and gene flow; and 

 agro-ecology, including information about cultivation, volunteers and weediness, soil 

ecology, and maize-insect interactions.

2-B. Description of DNA Used for Transformation and Intended Phenotype of Introduced 

Proteins

Identity and Source of Genetic Material in the T-DNA Region of PHP27118

4114 maize was produced by Agrobacterium tumefaciens-mediated transformation with 

PHP27118 (Figure 3), which contains the cry1F, cry34Ab1, cry35Ab1, and pat expression 

cassettes (Figure 4). A summary of the genetic elements and their position in the T-DNA region

is given in Table 2. As described earlier, 4114 maize is a new transformation event that 

contains the same gene cassettes as in previously registered 1507 and 59122 maize; these 

cassette comparisons are presented in Table 2.

The first cassette contains a truncated version of the cry1F gene from Bacillus thuringiensis var. 

aizawai. The expression of the cry1F gene is controlled by the maize polyubiquitin promoter 
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(Christensen et al., 1992), providing constitutive expression of the Cry1F protein in maize. This 

region also includes the 5’ untranslated region (UTR) and intron associated with the native 

polyubiquitin promoter. The terminator for the cry1F gene is the polyadenylation signal from 

Open Reading Frame 25 (ORF 25) of the Agrobacterium tumefaciens Ti plasmid pTi15955 

(Barker et al., 1983). 

The second cassette contains the cry34Ab1 gene isolated from Bacillus thuringiensis strain 

PS149B1, which has been codon optimized for expression in plants (Ellis et al., 2002; Herman et 

al., 2002; Moellenbeck et al., 2001). The expression of the cry34Ab1 gene is controlled by a 

second copy of the maize polyubiquitin promoter with 5’ UTR and intron (Christensen et al., 

1992). The terminator for the cry34Ab1 gene is the 3’ terminator sequence from the 

proteinase inhibitor II gene of Solanum tuberosum (pinII terminator) (An et al., 1989; Keil et al., 

1986).

The third gene cassette contains the cry35Ab1 gene, also isolated from Bacillus thuringiensis

strain PS149B1, which has been codon optimized for expression in plant (Ellis et al., 2002; 

Herman et al., 2002; Moellenbeck et al., 2001). The expression of the cry35Ab1 gene is 

controlled by the Triticum aestivum (wheat) peroxidase promoter and leader sequence (Hertig 

et al., 1991). The terminator for the cry35Ab1 gene is a second copy of the pinII terminator (An 

et al., 1989; Keil et al., 1986).

The fourth and final gene cassette contains the phosphinothricin acetyl transferase (pat) gene 

from Streptomyces viridochromogenes, which has been codon optimized for expression in 

plants. Expression of the pat gene is controlled by the promoter and terminator regions from 

the Cauliflower Mosaic Virus (CaMV) 35S transcript (Franck et al., 1980; Odell et al., 1985; 

Pietrzak et al., 1986).
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Figure 3. Schematic Diagram of Plasmid PHP27118

Schematic diagram of plasmid PHP27118 with genetic elements indicated. Plasmid size is 

54910 bp.

virC1

virC2

virG

virB

cry1F

cry34Ab1

cry35Ab1

pat

spc

tetR

tetA

trf A ctl

ubiZM1 Intron

Right Border

Left Border

ubiZM1 Promoter

TA Peroxidase Promoter

CaMV 35S Promoter

colE1 ori

Col E1 ori

oriV

ORF25 Terminator

pinII Terminator

CaMV 35S Terminator

ubiZM1 5’UTR

cos

cos

ubiZM1 Promoter

ubiZM1 5’UTR

ubiZM1 Intron

pinII Terminator

oriT
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Figure 4. Schematic Diagram of the T-DNA Region from Plasmid PHP27118

Schematic diagram of the T-DNA indicating the cry1F, cry34Ab1, cry35Ab1, and pat genes along 

with their respective regulatory elements. The size of the T-DNA is 11978 bp.

cry1F

cry34Ab1

cry35Ab1

pat

ubiZM1 Intron

ubiZM1 Intron

Right Border Left Border

ubiZM1 Promoter

ubiZM1 Promoter TA Peroxidase Promoter

CaMV 35S Promoter

ORF25 Terminator

pinII Terminator

pinII Terminator

CaMV 35S Terminator

ubiZM1 5’UTR

ubiZM1 5’UTR
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Table 2. Description of Genetic Elements in the T-DNA Region of PHP27118

Comparison 

to Approved 

Event

Location on 

PHP27118 

T-DNA

(bp position)

Genetic 

Element

Size 

(bp) Description

1 to 25 Right Border 25 T-DNA Right Border region from Ti 

plasmid of Agrobacterium tumefaciens

26 to 43 Ti Plasmid 

Region 18 Non-functional sequence from Ti plasmid of Agrobacterium tumefaciens

44 to 114 Polylinker 

Region 71 Region required for cloning genetic elements

cry1F gene 

cassette in 

4114 maize and 

1507 maize

115 to 1014 ubiZM1 

Promoter 900

Promoter region from Zea mays

polyubiquitin gene (Christensen et al., 

1992)

1015 to 1097 ubiZM1 5’ UTR 83

5’ untranslated region from Zea mays

polyubiquitin gene (Christensen et al., 

1992)

1098 to 2107 ubiZM1 Intron 1010

Intron region from Zea mays

polyubiquitin gene (Christensen et al., 

1992)

2108 to 2129 Polylinker 

Region 22 Region required for cloning genetic elements

2130 to 3947 cry1F Gene 1818 Truncated version of the cry1F gene from 

Bacillus thuringiensis var. aizawai

3948 to 3992 Polylinker 

Region 45 Region required for cloning genetic elements

3993 to 4706 ORF 25 

Terminator 714

Terminator sequence from the

Agrobacterium tumefaciens pTi15955 

ORF 25 (Barker et al., 1983)

4707 to 4765 Polylinker 

Region 59 Region required for cloning genetic elements
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4114 Maize

Table 2. Description of Genetic Elements in the T-DNA Region of PHP27118 (continued)

Comparison 

to Approved 

Event

Location on 

PHP27118 

T-DNA

(bp position)

Genetic 

Element

Size 

(bp) Description

cry34Ab1 gene 

cassette in 

4114 maize and 

59122 maize

4766 to 5665 ubiZM1 

Promoter 900

Promoter region from Zea mays

polyubiquitin gene 

(Christensen et al., 1992)

5666 to 5748 ubiZM1 5’ UTR 83

5’ untranslated region from Zea mays

polyubiquitin gene (Christensen et al., 

1992)

5749 to 6758 ubiZM1 Intron 1010

Intron region from Zea mays

polyubiquitin gene (Christensen et al., 

1992)

6759 to 6786 Polylinker 

Region 28 Region required for cloning genetic elements

6787 to 7158 cry34Ab1 Gene 372

Codon-optimized version of the cry34Ab1 

gene encoding the 14 kDa deltaendotoxin parasporal crystal protein 

from the nonmotile strain PS149B1 of 

Bacillus thuringiensis

(Ellis et al., 2002; Herman et al., 2002; 

Moellenbeck et al., 2001)

7159 to 7182 Polylinker 

Region 24 Region required for cloning genetic elements

7183 to 7492 pinII 

Terminator 310

Terminator region from Solanum 

tuberosum proteinase inhibitor II gene 

(An et al., 1989; Keil et al., 1986)

7493 to 7522 Polylinker 

Region 30 Region required for cloning genetic elements
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4114 Maize

Table 2. Description of Genetic Elements in the T-DNA Region of PHP27118 (continued)

Comparison 

to Approved 

Event

Location on 

PHP27118 

T-DNA

(bp position)

Genetic 

Element

Size 

(bp) Description

cry35Ab1 gene 

cassette in 

4114 maize and 

59122 maize

7523 to 8820 TA Peroxidase 

Promoter 1298

Promoter from Triticum aestivum 

(wheat) peroxidase including leader 

sequence (Hertig et al., 1991)

8821 to 8836 Polylinker 

Region 16 Region required for cloning genetic elements

8837 to 9988 cry35Ab1 1152

Codon-optimized version of the cry35Ab1 

gene encoding a 44 kDa delta-endotoxin 

parasporal crystal protein from the 

nonmotile strain PS149B1 of Bacillus 

thuringiensis 

(Ellis et al., 2002; Herman et al., 2002; 

Moellenbeck et al., 2001)

9989 to 10012 Polylinker 

Region 24 Region required for cloning genetic elements

10013 to 

10322

pinII 

Terminator 310

Terminator region from Solanum 

tuberosum proteinase inhibitor II gene 

(An et al., 1989; Keil et al., 1986)

10323 to 

10367

Polylinker 

Region 45 Region required for cloning genetic elements

pat gene 

cassette in 

4114 maize, 

1507 maize and 

59122 maize

10368 to 

10897

CaMV

35S Promoter 530

35S promoter from Cauliflower Mosaic 

Virus (Franck et al., 1980; Odell et al., 

1985; Pietrzak et al., 1986)

10898 to 

10916

Polylinker 

Region 19 Region required for cloning genetic elements

10917 to 

11468 pat Gene 552

Codon-optimized phosphinothricin 

acetyltransferase gene from 

Streptomyces viridochromogenes.

11469 to 

11488

Polylinker 

Region 20 Region required for cloning genetic elements

11489 to 

11680

CaMV35S 

Terminator 192

35S terminator from Cauliflower Mosaic 

Virus (Franck et al., 1980; Pietrzak et al.,

1986)

11681 to 

11874

Polylinker 

Region 194 Region required for cloning genetic elements

11875 to 

11953

Ti Plasmid 

Region 79 Non-functional sequence from Ti plasmid of Agrobacterium tumefaciens

11954 to 

11978 Left Border 25 T-DNA Left Border region from Ti plasmid of Agrobacterium tumefaciens
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4114 Maize

Activity and Function of Expressed Proteins in 4114 Maize

The insertion of the cry1F gene in 4114 maize confers resistance to plant damage by certain 

lepidopteran pests. The Cry1F protein is comprised of 605 amino acids and has a molecular 

weight of approximately 68 kDa (Figure 5). The Cry1F protein expressed in 4114 maize is 

identical to the one expressed in 1507 maize that has been deregulated by USDA, registered by 

EPA, and reviewed by FDA in 2001 (Table 1). 

The cry34Ab1 and cry35Ab1 genes, isolated from the common soil bacterium Bacillus 

thuringiensis strain PS149B1, produce the proteins Cry34Ab1 and Cry35Ab1. The Cry34Ab1

protein is 123 amino acid residues in length and has a molecular weight of approximately 14 

kDa (Figure 6). The full-length Cry35Ab1 protein has a length of 383 amino acids and a 

molecular weight of approximately 44 kDa (Figure 7). The Cry34Ab1 and Cry35Ab1 proteins 

together comprise an active insecticidal crystal protein (i.e., the binary Cry34/35Ab1 protein), 

that confers resistance to certain corn rootworm pests. This binary protein is identical to the 

one expressed in 59122 maize deregulated by USDA in 2005, registered by EPA since 2005, and 

reviewed by FDA in 2004 (Table 1). 

Cry proteins (i.e., delta-endotoxins), including the Cry1F and Cry34/35Ab1 proteins expressed 

in 4114 maize, act by selectively binding to specific sites localized on the lining of the midgut of 

susceptible insect species (de Maagd et al., 2003; Schnepf et al., 1998). Following binding, 

pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to 

bacterial sepsis (Bravo et al., 2007). The Cry1F and Cry34/35Ab1 proteins are lethal only when 

eaten by the larvae of certain lepidopteran or coleopteran insects, respectively, and their 

specificity of action can be attributed to the presence of their respective binding sites in the 

unique environment of the target insect midgut that is required for their activity (Chambers et 

al., 1991; de Maagd et al., 2003; Ellis et al., 2002; Hua et al., 2001; Moellenbeck et al., 2001). 

There are no binding sites for the delta-endotoxins of B. thuringiensis on the surface of 

mammalian intestinal cells and the mammalian digestive system environment does not support 

the steps required for toxicity of these proteins, therefore, livestock animals and humans are 

not susceptible to these proteins (Siegel, 2001).

The pat gene expresses the phosphinothricin acetyl transferase enzyme (PAT) that confers 

tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides. The 

PAT protein is 183 amino acids residues in length and has a molecular weight of approximately 

21 kDa (Figure 8). This protein is identical to the protein found in a number of approved events

across several different crops that are currently in commercial use, including 1507 and 59122 

maize; maize containing the PAT protein (e.g., T25) has been commercially grown in the U.S. 

since 1996. 

Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, 

called glutamine synthetase, which is involved in the synthesis of glutamine. Glutamine 

synthetase is also involved in ammonia detoxification. Due to its similarity to glutamate, 

glufosinate blocks the activity of glutamine synthetase, resulting in reduced glutamine levels 
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4114 Maize

and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell 

membrane disruption and cessation of photosynthesis resulting in plant death. The PAT 

protein expressed in 4114 maize acetylates glufosinate to N-acetylglufosinate. This action

prevents the inhibition of glutamine synthetase and therefore the plant is able to survive 

applications of herbicides containing glufosinate at current labeled rates.

1 MENNIQNQCV PYNCLNNPEV EILNEERSTG RLPLDISLSL TRFLLSEFVP

51 GVGVAFGLFD LIWGFITPSD WSLFLLQIEQ LIEQRIETLE RNRAITTLRG

101 LADSYEIYIE ALREWEANPN NAQLREDVRI RFANTDDALI TAINNFTLTS

151 FEIPLLSVYV QAANLHLSLL RDAVSFGQGW GLDIATVNNH YNRLINLIHR

201 YTKHCLDTYN QGLENLRGTN TRQWARFNQF RRDLTLTVLD IVALFPNYDV

251 RTYPIQTSSQ LTREIYTSSV IEDSPVSANI PNGFNRAEFG VRPPHLMDFM

301 NSLFVTAETV RSQTVWGGHL VSSRNTAGNR INFPSYGVFN PGGAIWIADE

351 DPRPFYRTLS DPVFVRGGFG NPHYVLGLRG VAFQQTGTNH TRTFRNSGTI

401 DSLDEIPPQD NSGAPWNDYS HVLNHVTFVR WPGEISGSDS WRAPMFSWTH

451 RSATPTNTID PERITQIPLV KAHTLQSGTT VVRGPGFTGG DILRRTSGGP

501 FAYTIVNING QLPQRYRARI RYASTTNLRI YVTVAGERIF AGQFNKTMDT

551 GDPLTFQSFS YATINTAFTF PMSQSSFTVG ADTFSSGNEV YIDRFELIPV

601 TATLE*

Figure 5. Deduced Amino Acid Sequence of the Cry1F Protein

Deduced amino acid sequence from translation of the cry1F gene from plasmid PHP27118. The Cry1F 

protein is 605 amino acids in length, has a molecular mass of approximately 68 kDa, and is identical to 

the one expressed in 1507 maize. The asterisk (*) indicates the translational stop codon.

1 MSAREVHIDV NNKTGHTLQL EDKTKLDGGR WRTSPTNVAN DQIKTFVAES

51 NGFMTGTEGT IYYSINGEAE ISLYFDNPFA GSNKYDGHSN KSQYEIITQG

101 GSGNQSHVTY TIQTTSSRYG HKS*

Figure 6. Deduced Amino Acid Sequence of the Cry34Ab1 Protein

Deduced amino acid sequence from translation of the cry34Ab1 gene from plasmid PHP27118. The 

Cry34Ab1 protein is 123 amino acids in length, has a molecular mass of approximately 14 kDa, and is 

identical to the one expressed in 59122 maize. The asterisk (*) indicates the translational stop codon. 
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4114 Maize

1 MLDTNKVYEI SNHANGLYAA TYLSLDDSGV SLMNKNDDDI DDYNLKWFLF

51 PIDDDQYIIT SYAANNCKVW NVNNDKINVS TYSSTNSIQK WQIKANGSSY

101 VIQSDNGKVL TAGTGQALGL IRLTDESSNN PNQQWNLTSV QTIQLPQKPI

151 IDTKLKDYPK YSPTGNIDNG TSPQLMGWTL VPCIMVNDPN IDKNTQIKTT

201 PYYILKKYQY WQRAVGSNVA LRPHEKKSYT YEWGTEIDQK TTIINTLGFQ

251 INIDSGMKFD IPEVGGGTDE IKTQLNEELK IEYSHETKIM EKYQEQSEID

301 NPTDQSMNSI GFLTITSLEL YRYNGSEIRI MQIQTSDNDT YNVTSYPNHQ

351 QALLLLTNHS YEEVEEITNI PKSTLKKLKK YYF*

Figure 7. Deduced Amino Acid Sequence of the Cry35Ab1 Protein

Deduced amino acid sequence from translation of the cry35Ab1 gene from plasmid PHP27118. The 

Cry35Ab1 protein is 383 amino acids in length, has a molecular mass of approximately 44 kDa, and is 

identical to the one expressed in 59122 maize. The asterisk (*) indicates the translational stop codon. 

1 MSPERRPVEI RPATAADMAA VCDIVNHYIE TSTVNFRTEP QTPQEWIDDL

51 ERLQDRYPWL VAEVEGVVAG IAYAGPWKAR NAYDWTVEST VYVSHRHQRL

101 GLGSTLYTHL LKSMEAQGFK SVVAVIGLPN DPSVRLHEAL GYTARGTLRA

151 AGYKHGGWHD VGFWQRDFEL PAPPRPVRPV TQI*

Figure 8. Deduced Amino Acid Sequence of the PAT Protein

Deduced amino acid sequence from translation of the pat gene from plasmid PHP27118. The PAT 

protein is 183 amino acids in length and has a molecular mass of approximately 21 kDa. This protein is 

identical to the protein found in a number of approved events across several different crops that are 

currently in commercial use. The asterisk (*) indicates the translational stop codon. 
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4114 Maize

Equivalency of Expressed Proteins in 4114 Maize and Previously Approved Events

The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins expressed in 4114 maize are identical to the 

proteins expressed in previously approved 1507, 59122, and/or 1507x59122 maize. This 

equivalency was established using the following criteria:

 The same gene expression cassettes (promoters, protein coding sequences, and 

terminators) were used in 4114 maize as in 1507 and 59122 maize, so all genetic 

elements are identical (Table 2).

 The DNA insertion in 4114 maize was sequenced and the translated amino acid 

sequences of the encoded Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were 

determined. The translated amino acid sequences were compared and found to be 

identical to the amino acid sequences of those proteins in 1507 and 59122 maize 

(Figures 5 through 8). Therefore, the proteins in 4114 maize are also expected to be 

identical to the proteins in the breeding stack of 1507x59122 maize.

 Western blot analysis demonstrated the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins 

in 4114 maize migrate with equivalent molecular weight and similar relative 

immunoreactivity to the proteins expressed in 1507x59122 maize, indicating 

equivalency (Appendix 2).

 Bioequivalency of the expressed Cry1F and Cry34/35Ab1 proteins was established via 

side-by-side efficacy testing of 4114 maize with 1507x59122 maize in the field (data not 

shown; submitted to EPA).

Therefore, previously submitted safety data for Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins 

are also applicable for 4114 maize. 

2-C. Description of Transformation, Selection, and Breeding

Pioneer proprietary inbred line PHWWE was transformed with plasmid PHP27118 to produce 

4114 maize. Immature embryos of maize (Zea mays L.) line PHWWE were aseptically removed 

from the developing caryopsis nine to eleven days after pollination and inoculated with 

Agrobacterium tumefaciens strain LBA4404 containing plasmid PHP27118, essentially as 

described in Zhao et al., 2001. Agrobacterium tumefaciens strain LBA4404 is a disarmed strain 

that does not contain tumor-inducing factors, however with the inclusion of plasmid PHP27118

the strain will contain factors (i.e., the vir genes) that enable the transfer of the T-DNA region to 

the inoculated host plant. After three to six days of embryo and Agrobacterium co-cultivation 

on solid culture medium with no selection, the embryos were then transferred to a medium 

without herbicide selection but containing carbenicillin for selection against Agrobacterium. 

After three to five days on this medium, embryos were then transferred to selective medium 

that was stimulatory to maize somatic embryogenesis and contained bialaphos for selection of 

cells expressing the pat transgene. The medium also contained carbenicillin to select against 

any remaining Agrobacterium. After six to eight weeks on the selective medium, healthy, 

growing calli that demonstrated resistance to bialaphos were identified. 
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4114 Maize

Plants that were regenerated from transformation and tissue culture (designated T0 plants) 

were selected for further characterization by molecular analyses, herbicide and insect efficacy, 

and agronomic evaluations. Refer to Figure 9 for a schematic overview of the transformation 

and event development process for 4114 maize.

The subsequent breeding of 4114 maize proceeded as indicated in Figure 10 to produce specific 

generations for the characterization and assessments conducted, as well as for the 

development of commercial lines. Table 3 indicates the breeding generations used for each of 

the analyses described in this submission.

Assembly of plasmid PHP27118 containing cry1F, cry34Ab1, cry35Ab1, and pat gene 

cassettes in the T-DNA

↓

Transformation of Pioneer proprietary inbred

PHWWE line using Agrobacterium tumefaciens strain LBA4404 with PHP27118

↓

Selection of transformation events based on tolerance to bialaphos

Regeneration of T0 plants

↓

Molecular characterization and evaluation of T0 plants

↓

Nursery evaluation of agronomic performance and insect/herbicide efficacy of subsequent 

generations

↓

Multiple location herbicide/insect efficacy and agronomic trials

↓

Selection of event DP-ØØ4114-3 as the commercial candidate

↓

Continued field and laboratory studies to support product registration

Continued breeding and testing of 4114 maize for product development

Figure 9. Schematic Diagram of the Development of 4114 Maize
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4114 Maize

Figure 10. Breeding Diagram for 4114 Maize and Generations Used for Analyses

The breeding steps to produce the generations used for characterization, assessment, and the 

development of commercial lines are shown schematically. Pioneer proprietary inbred PHWWE was 

used for transformation to produce 4114 maize. Other Pioneer proprietary inbreds PH1B5, PHR03, 

PHTFE, PHNAR, and PH09B were used in crossing and backcrossing steps.
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4114 Maize

Table 3. Generations and Comparators Used for Analysis of 4114 Maize

Analysis Generation Comparators

Experimental 

Control and 

Reference Lines

Genetic Characterization 

and Absence of 

Backbone DNA

(Section 2-D)

T2, T3, BC3F1*3, 

BC3F2*2 Not applicable

Pioneer®

proprietary maize 

inbreds PH09B and

PHWWE

Stability and Inheritance

of the DNA Insertion

(Section 2-E)

F1*1, BC2F1*1, BC3F1*1, 

BC2F1*2, BC3F1*2 Not applicable Not applicable

Concentrations of Cry1F, 

Cry34Ab1, Cry35Ab1, 

and PAT (Section 2-F)

F1*5 Not applicablea 1507, 59122, and 

1507x59122 maizea

Compositional 

Assessment (Section 4-A)

Grain and forage from 

F1*5 PHNARxPHTFE

Pioneer® hybrids

34M94, 33G26, 

33J24, 3394, 38B85, 

37Y12, 34A15, and 

34P88

Germination and 

Dormancy (Agronomic 

Performance) 

(Section 4-B)

F1*5 PHNARxPHTFE Pioneer® hybrids

32D78 and 34P88

Field Trial Evaluations 

(Agronomic 

Performance)

(Section 4-B)

F1*5 PHNARxPHTFE

Pioneer® hybrids

34M94, 33G26, 

33J24, 3394, 38B85, 

37Y12, 34A15, 

34P88, 37H24, 

36M28, 35T06, 

35T36, and 35K02

a

To determine actual protein concentrations in 4114 maize, no comparator lines were used. However, 

comparisons were made to the protein concentrations of 1507, 59122, and 1507x59122 maize lines in the same 

experiment to determine the applicability of previously conducted safety studies to 4114 maize. 
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4114 Maize

Selection of Comparators, Experimental Controls, and Reference Lines for 4114 Maize

For the genetic characterization of the 4114 maize insertion, Pioneer proprietary maize inbreds 

were used as experimental controls (Table 3). These controls were selected because they 

represented the genetics of the maize lines that were crossed into the 4114 maize generations 

analyzed (Figure 10). Because certain probes were used that contained sequences in the 

endogenous maize genome, these controls containing the representative genetics eliminated 

background hybridization not related to the 4114 maize insertion.

For stability and inheritance of the DNA insertion, no comparators, controls, or reference lines 

were used or needed to conduct the analysis. For the measurement of concentrations of the 

expressed proteins in 4114 maize, no comparators were used, however 1507, 59122, and 

1507x59122 maize reference lines were used to determine if 4114 maize protein 

concentrations were comparable to these lines. These comparisons were important to 

determine if previously conducted safety studies were applicable to the safety assessment of 

4114 maize.

In the comparative assessments (i.e., compositional analysis and agronomic performance), a 

control maize line was used (Table 3). For these analyses, the control comparator line had a 

genetic background approximately 99% similar to that of the 4114 maize generation used (i.e.,

near-isoline), but did not go through the transformation process. 

In addition, non-transgenic Pioneer commercial maize hybrid lines, as listed in Table 3, were 

used to obtain tolerance intervals for compositional and agronomic comparisons. These 

Pioneer commercial maize hybrids were chosen to represent a wide range of non-genetically 

modified varieties that would normally be planted commercially. These tolerance intervals 

represent the normal range of variation of the maize crop for compositional analytes and 

agronomic characteristics; they help to further determine the comparability of 4114 maize to 

conventional maize, if any statistical differences were observed.
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4114 Maize

2-D. Molecular Characterization of the Insertion in 4114 Maize

Molecular characterization of the inserted DNA evaluates the integrity of the introduced 

cassettes and provides a confirmation that the elements of the expression cassettes are intact. 

The inserted DNA is also evaluated over several generations to confirm its stability through 

traditional breeding methods.

The DNA insertion in 4114 maize was characterized by Southern blot analysis to evaluate the 

copy number, integrity, and stability of the inserted cry1F, cry34Ab1, cry35Ab1, and pat

cassettes. As described earlier in Section 2-C, 4114 maize was produced by Agrobacteriummediated transformation with plasmid PHP27118. The T-DNA region of PHP27118 contains 

four cassettes: cry1F, cry34Ab1, cry35Ab1, and pat. The cry1F cassette contains the ubiZM1

promoter and intron, cry1F gene, and ORF25 terminator. The cry34Ab1 cassette contains a 

second copy of the ubiZM1 promoter and intron, cry34Ab1 gene, and pinII terminator. The 

cry35Ab1 cassette contains the TA peroxidase promoter, cry35Ab1 gene, and pinII terminator, 

and the pat cassette contains the CaMV 35S promoter, pat gene, and CaMV 35S terminator. All 

probes used for the analysis are indicated on the schematic maps of PHP27118 and the 

PHP27118 T-DNA region (Figures 11 and 12, respectively) and outlined in Table 4. Plasmid 

PHP27118 was used as a positive control for probe hybridization and to verify fragment sizes 

internal to the T-DNA. The integration pattern of the insertion in 4114 maize was investigated 

using Southern blot analysis with Bcl I digested genomic DNA from individual plants of the T3 

generation to determine copy number and Hind III digested genomic DNA to determine 

insertion integrity. Copy number and integrity of each genetic element were determined using 

probes specific to each of the genetic elements present in the PHP27118 T-DNA region (Table 4, 

Figure 12). The stability of the insertion was analyzed using Bcl I digested genomic DNA from 

individual plants of the T2, T3, BC3F1*3, and BC3F2*2 generations (Section 2; Figure 10 and Table 

3). Probes specific to each of the gene regions of PHP27118 T-DNA were used to confirm the 

stability of the insertion (Table 4, Figure 12). In addition, probes to the plasmid backbone 

region of PHP27118 located outside of the T-DNA region (Table 4, Figure 11) were used to show 

that these regions were not transferred to 4114 maize. Materials and methods for the 

molecular characterization of 4114 maize are described in Appendix 3.

Based on the Southern blot analyses described below, it was determined that a single, intact 

PHP27118 T-DNA was inserted into the genome of 4114 maize as diagramed in the insertion 

map (Figure 13) and that no region from the backbone of plasmid PHP27118 was inserted. In 

addition, these results confirmed the stability of the DNA insertion in 4114 maize across four 

breeding generations.
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4114 Maize

Figure 11. Plasmid Map of PHP27118

Schematic map of PHP27118 indicating Bcl I and Hind III restriction enzyme sites with base pair 

positions. The Right Border and Left Border regions flank the T-DNA (Figure 12) that is expected to be 

transferred during Agrobacterium-mediated transformation. Plasmid backbone probe locations are 

indicated by boxes within the DNA map. A: RB probe; B: LB probe; C: spc probe; D: tet probe; E: virG

probe.

PHP27118

54910 bp

virC1

virC2

virG

virB

cry1F

cry34Ab1

cry35Ab1

pat

spc

tetR

tetA

trf A ctl

ubiZM1 Intron

Right Border

Left Border

ubiZM1 Promoter

TA Peroxidase Promoter

CaMV 35S Promoter

colE1 ori

Col E1 ori

oriV

ORF25 Terminator

pinII Terminator

CaMV 35S Terminator

ubiZM1 5’UTR

cos

cos

HindIII (78)

Hin dIII (3969)

HindIII (11738)

HindIII (36877)

HindIII (47940)

HindIII (48849)

Hin dIII (49970)

HindIII (51485)

Bcl I (2546)

Bcl I (12212)

Bcl I (18243)

Bcl I (19795)

Bcl I (38557)

Bcl I (40721)

Bcl I (43315)

Bcl I (44899)

Bcl I (47667)

BclI (48038)

Bcl I (50460)

ubiZM1 Promoter

ubiZM1 5’UTR

ubiZM1 Intron

pinII Terminator

Bcl I (13742)

oriT

B

C

D

E
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4114 Maize

Figure 12. Map of PHP27118 T-DNA

Schematic map of T-DNA from PHP27118 indicating Bcl I and Hind III restriction enzyme sites and the 

cry1F, cry34Ab1, cry35Ab1 and pat coding and regulatory regions. The T-DNA size is 11978 bp. The 

probe locations are indicated by boxes below the DNA map and are identified below:

Number Probe Name

1 ubiZM1 promoter

2 ubiZM1 intron

3 cry1F

4 ORF25 terminator

5 cry34Ab1

6 pinII terminator

7 TA peroxidase promoter

8 cry35Ab1

9 35S promoter

10 pat

11 35S terminator

cry1F

cry34Ab1

cry35Ab1

pat

ubiZM1 Intron

ubiZM1 Intron

Right Border Left Border

ubiZM1 Promoter

ubiZM1 Promoter

TA Peroxidase Promoter

CaMV 35S Promoter

ORF25 Terminator pinII Terminator

CaMV 35S Terminator

ubiZM1 5’UTR

ubiZM1 5’UTR

Bcl I (2546)

HindIII (78)

HindIII (3969)

HindIII (11738)

pinII Terminator

1 2 3 4 1 2 5 6 7 8 6 9 10 11
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4114 Maize

Table 4. Description of DNA Probes Used for Southern Blot Hybridization

Probe Name Genetic

Element

Figure

Probe

Position on 

PHP27118 T-DNA

(bp to bp) a

Position on 

PHP27118

(bp to bp) b

Length

(bp)

cry1Fccry1F gene Figure 12

Probe 3

2133 to 3030

3038 to 3945

2133 to 3030

3038 to 3945

898

908

cry34Ab1 cry34Ab1 gene Figure 12

Probe 5

6817 to 7133 6817 to 7133 317

cry35Ab1ccry35Ab1 gene Figure 12

Probe 8

8837 to 9303

9408 to 9979

8837 to 9303

9408 to 9979

467

572

pat pat gene

Figure 12

Probe 10 10904 to 11451 10904 to 11451 548

ubiZM1 

promoter

ubiZM1 promoter Figure 12

Probe 1

150 to 1008 (copy 1)

4801 to 5659 (copy 2)

150 to 1008 (copy 1)

4801 to 5659 (copy 2) 859

ubiZM1 intron ubiZM1 5’ UTR 

and intron

Figure 12

Probe 2

1020 to 2100 (copy 1)

5671 to 6751 (copy 2)

1020 to 2100 (copy 1)

5671 to 6751 (copy 2) 1081

ORF25 

terminator ORF25 terminator Figure 12Probe 4

4003 to 4703 4003 to 4703 701

pinII 

terminator pinII terminator Figure 12Probe 6

7235 to 7468 (copy 1)

10065 to 10298 (copy 2)

7235 to 7468 (copy 1)

10065 to 10298 (copy 2) 234

TA peroxidase 

promoterc

TA peroxidase 

promoter

Figure 12

Probe 7

7523 to 8415

8416 to 8813

7523 to 8415

8416 to 8813

893

398

35S promoter CaMV 35S 

promoter

Figure 12

Probe 9

10383 to 10900 10383 to 10900 518

35S 

terminator

CaMV 35S 

terminator

Figure 12

Probe 11 11482 to 11692 11482 to 11692 211

LB

Plasmid backbone 

adjacent to T-DNA 

Left Border

Figure 11

Probe B

N/Ad12003 to 12348 346

spc

Spectinomycin 

resistance gene

Figure 11

Probe C

N/Ad13158 to 13932 775

tetc Tetracycline 

resistance gene

Figure 11

Probe D

N/Ad 19007 to 19545

19651 to 20108

539

458

virG virG gene Figure 11

Probe E

N/Ad39334 to 40077 744

RB

Plasmid backbone 

adjacent to T-DNA 

Right Border

Figure 11

Probe A

N/Ad54476 to 54865 390

a

The probe position is based on the PHP27118 T-DNA map (Figure 12).

b

The probe position is based on the PHP27118 plasmid map (Figure 11).

c

The cry1F, cry35Ab1, TA peroxidase promoter, and tet probes each are comprised of two non-overlapping 

labeled fragments that are combined in the respective hybridization solutions.

d

N/A = Not Applicable; these elements are not present in the PHP27118 T-DNA region.
Pioneer Hi-Bred International, Inc. Page 47 of 258

4114 Maize

Figure 13. Map of the Insertion in 4114 Maize

Schematic map of the insertion in 4114 maize based on Southern blot analysis. The flanking maize 

genome is represented by the horizontal dotted line. A single, intact copy of the PHP27118 T-DNA 

integrated into the maize genome. Bcl I and Hind III restriction sites are indicated with the sizes of 

observed fragments on Southern blots shown below the map in base pairs (bp). The locations of 

restriction enzyme sites outside the Right and Left Borders are not shown to scale.

cry1F

cry34Ab1

cry35Ab1

pat

ubiZM1 Intron

ubiZM1 Intron

Right Border Left Border

ubiZM1 Promoter

ubiZM1 Promoter

TA Peroxidase Promoter

CaMV 35S Promoter

ORF25 Terminator pinII Terminator

CaMV 35S Terminator

ubiZM1 5’UTR

ubiZM1 5’UTR

Bcl I

Hind III

Hind III 

Hind III 

pinII Terminator

PHP27118 T-DNA

Bcl I ~3100 bp >8600 bp

Hind III 3891 bp 7769 bp
Pioneer Hi-Bred International, Inc. Page 48 of 258

4114 Maize

Copy Number of the DNA Insertion in 4114 Maize

The restriction enzyme Bcl I has a single recognition site within the PHP27118 T-DNA (Figure 12) 

and provides information about the number of copies of the T-DNA that are integrated in the 

genome of 4114 maize. Hybridization with probes specific to the genetic elements of the 

PHP27118 T-DNA would indicate the number of copies of each element found in 4114 maize, as 

shown by the number of hybridizing bands. The site for Bcl I is located at bp 2546 of the T-DNA 

(Figure 12) and is predicted to yield fragments of greater than approximately 2500 bp and 

greater than approximately 9400 bp for a single inserted T-DNA (Table 5). The band of greater 

than 2500 bp will hybridize to the ubiZM1 promoter and intron and the cry1F hybridization 

probes (Table 5, Figure 12). Probes specific to the following elements will hybridize to the 

fragment of greater than 9400 bp: the ubiZM1 promoter and intron, the TA peroxidase 

promoter, CaMV 35S promoter, cry1F, cry34Ab1, cry35Ab1, pat, ORF25 terminator, pinII 

terminator, and CaMV 35S terminator (Figure 12). As the Bcl I restriction enzyme site is within 

the cry1F gene and between the duplicate copies of the ubiZM1 promoter and intron regions, 

probes for the cry1F gene, the ubiZM1 promoter and ubiZM1 intron will hybridize to both

fragments (Table 5, Figure 12). Therefore, the appearance of two bands for these probes 

indicates a single copy of the T-DNA. Although there are two copies of the pinII terminator in 

the T-DNA, both are located on the same Bcl I fragment and will thus yield a single band (Table 

5, Figure 12). A single band for the remaining probes indicates a single copy of those genetic 

elements.

Hybridization of Bcl I-digested genomic DNA from 4114 maize with the ubiZM1 promoter and 

intron probes resulted in a band of approximately 3100 bp and a band of greater than 8600 bp 

(Table 5, Figure 14). Based on the expected sizes for the genomic fragments, the band of 

approximately 3100 bp is derived from the Right Border or 5’ end of the DNA insertion and the 

band of greater than 8600 bp is the band expected to be greater than 9400 bp and is derived 

from the Left Border or 3’ end of the 4114 maize insertion. A band of approximately 4800 bp 

was observed in the 4114 maize plants and the PHWWE control maize with both probes (Table 

5, Figure 14). This band was determined to be due to hybridization of the ubiZM1 promoter 

and intron probes to the endogenous copy of these elements found in the native maize genome 

based upon its presence in the non-transformed PHWWE control maize line. A similar but 

slightly lower band is also seen in the non-transgenic PH09B control maize line. The presence 

of the two expected bands with the ubiZM1 promoter and intron probes, and no additional 

insertion-derived bands, indicates that there is a single copy of the PHP27118 T-DNA in 4114 

maize.

The Bcl I-digested PHP27118 plasmid lanes did not produce the expected size bands and 

produced two bands above the 8600 bp marker band; these bands were characteristic of an 

undigested plasmid and indicated that the plasmid DNA was likely methylated (Table 5, Figure 

14). Sensitivity of the Bcl I enzyme to bacterial plasmid DNA methylation is well known and 

there is a bacterial Dam methylase recognition sequence in the site (

5’GATC3’) (New England 

Biolabs, 2002). In all Bcl I sites (recognition sequence 5’TGATCA3’) on the plasmid, the central 

adenine will be methylated, blocking digestion by Bcl I. The PHP27118 plasmid used was 
Pioneer Hi-Bred International, Inc. Page 49 of 258

4114 Maize

prepared in a dam+strain of E. coli and thus all Bcl I sites would be methylated and would not 

digest as expected. Therefore on all Southern blots with Bcl I-digested plasmid, only probe 

hybridization was confirmed. 

Hybridization of the Bcl I-digested DNA with the cry1F probe also resulted in the same two 

bands of approximately 3100 bp and greater than 8600 bp (Table 5, Figure 15). Two bands are 

expected with the cry1F probe due to the location of the Bcl I restriction site within the cry1F 

gene (Figure 12), and the presence of these two bands indicates there is a single copy of the 

cry1F gene in 4114 maize. 

When hybridized to the ORF25 terminator probe, the same blot resulted in a single band of 

greater than 8600 bp (Table 5, Figure 15). As the ORF25 terminator element is located on the 3’ 

or Left Border side of the Bcl I site in the PHP27118 T-DNA (Figure 12), this demonstrates that 

the greater than 8600 bp band is indeed associated with the elements on this portion of the 

T-DNA.

The same band of greater than 8600 bp was observed when the Bcl I Southern blot was 

hybridized with the cry34Ab1 and pinII terminator probes (Table 5, Figure 16), the TA 

peroxidase promoter and cry35Ab1 probes (Table 5, Figure 17), and the 35S promoter, pat, and 

35S terminator probes (Table 5, Figure 18). Although there are two copies of the pinII 

terminator in the PHP27118 T-DNA, both copies are located on the same Bcl I fragment so that 

they result in a single hybridizing band on the Southern blot. The presence of single bands with 

all of these elements confirms that there is a single copy of the PHP27118 T-DNA inserted in 

4114 maize.

When taken together with the restriction map of the PHP27118 T-DNA (Figure 12), the 

presence of single bands with the ORF25 terminator, cry34Ab1, pinII terminator, TA peroxidase 

promoter, cry35Ab1, 35S promoter, pat, and 35S terminator probes, along with the expected 

two bands for the ubiZM1 promoter, ubiZM1 intron, and cry1F probes, demonstrate that there 

is a single inserted PHP27118 T-DNA in 4114 maize. 

Additionally, Bcl I digestion of the 4114 maize insertion provides an event-specific hybridization 

pattern when certain probes are used. If the hybridization pattern is identical between 

different plants and generations, this demonstrates the 5’ and 3’ regions of the DNA insertion 

are stable during the breeding process and is discussed later in this section.
Pioneer Hi-Bred International, Inc. Page 50 of 258

4114 Maize

Table 5. Predicted and Observed Hybridizing Bands on Southern Blots; Bcl I Digest

Probe Figure

Predicted Fragment 

Size from PHP27118

T-DNA (bp)a

Predicted Fragment 

Size from Plasmid 

PHP27118 (bp)b

Observed Fragment 

Size in 4114 Maize 

(bp)c

ubiZM1 promoter 14 >9400 (border)d, e

>2500 (border)d, e

9666

6996

>8600

~3100

~4800*

ubiZM1 intron 14 >9400 (border) d, e

>2500 (border) d, e

9666

6996

>8600

~3100

~4800*

cry1F 15, 14 >9400 (border)d, f

>2500 (border)d,f

9666

6996

>8600

~3100

ORF25 terminator 15 >9400 (border)d9666 >8600

cry34Ab1 16, 23 >9400 (border)d9666 >8600

pinII terminator 16 >9400 (border)d, g 9666 >8600

TA peroxidase 

promoter

17 >9400 (border)d9666 >8600

cry35Ab1 17, 24 >9400 (border)d9666 >8600

35S promoter 18 >9400 (border)d9666 >8600

pat 18, 25 >9400 (border)d9666 >8600

35S terminator 18 >9400 (border)d9666 >8600

LB 28 N/Ah1530 (25139)i No hybridization

spc 29 N/Ah1530 (25139)i No hybridization

tet 30 N/Ah1552 (25139)i No hybridization

virG 31 N/Ah2164 (11063)i No hybridization

RB 32 N/Ah6996 (3503)i No hybridization

Note: An asterisk (*) and gray shading indicates the designated band is due to hybridization to endogenous 

sequences. These bands were identified in the maize control lines that were analyzed.

a

Predicted size for hybridization in genomic DNA samples is based on the map of the T-DNA from PHP27118 (Figure 12). 

Border fragment sizes are rounded to the nearest 100 bp.

b

Predicted size is based on the plasmid map of PHP27118 (Figure 11) and is the size expected for Bcl I digestion of an 

unmethylated plasmid. The methylated PHP27118 used in this study did not digest with Bcl I and therefore did not 

yield these fragments.

c Observed fragment sizes are approximated from the DIG-labeled DNA Molecular Weight Marker VII fragments on the 

Southern blots. Due to the inability to determine exact sizes on the blot, all approximated values are rounded to the 

nearest 100 bp.

d

Border fragments are those in which one restriction site is in the inserted T-DNA and the other site is located in the 

flanking genomic DNA, providing a fragment of unique size for a given insertion. Border fragment sizes are rounded to 

the nearest 100 bp.

e

There are two copies of the ubiZM1 promoter and intron elements, located on either side of the Bcl I site, so both 

fragments will hybridize to these probes.

f

The Bcl I site is located within the cry1F gene, so both fragments hybridize to the cry1F probe.

g

Both copies of the pinII terminator in the PHP27118 T-DNA are located on the same Bcl I fragment.

h N/A=Not Applicable; these elements are not found on the PHP27118 T-DNA.

I

The fragment size in parenthesis is for digestion of PHP27118 with Hind III, which was included in Lanes 15 and 16 of 

certain Bcl I blots to show that the plasmid can be cut by a restriction enzyme that is not sensitive to methylation.
Pioneer Hi-Bred International, Inc. Page 51 of 258

4114 Maize

Figure 14. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA 

with ubiZM1 Promoter and ubiZM1 Intron Probes 

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the ubiZM1 

promoter and ubiZM1 intron probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded 

per lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate 

gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control

2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control

3 DIGVII marker 10 DIGVII marker

4 PH09B control 11 PH09B control

5 PHWWE control 12 PHWWE control

6 4114 maize plant 20 (T3 generation) 13 4114 maize plant 20 (T3 generation)

7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation)

ubiZM1 Promoter ubiZM1Intron

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4114 Maize

Figure 15. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA 

with cry1F and ORF25 Terminator Probes

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry1F 

and ORF25 terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per 

lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene 

copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are indicated 

adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control

2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control

3 DIGVII marker 10 DIGVII marker

4 PH09B control 11 PH09B control

5 PHWWE control 12 PHWWE control

6 4114 maize plant 20 (T3 generation) 13 4114 maize plant 20 (T3 generation)

7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation)

cry1F

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Pioneer Hi-Bred International, Inc. Page 53 of 258

4114 Maize

Figure 16. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA 

with cry34Ab1 and pinII Terminator Probes

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the 

cry34Ab1 and pinII terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded 

per lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate 

gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control

2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control

3 DIGVII marker 10 DIGVII marker

4 PH09B control 11 PH09B control

5 PHWWE control 12 PHWWE control

6 4114 maize plant 20 (T3 generation) 13 4114 maize plant 20 (T3 generation)

7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation)

cry34Ab1 pinII Terminator

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1 2 3 4 5 6 7 8 9 10 11 12 13 14
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4114 Maize

Figure 17. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA 

with TA Peroxidase Promoter and cry35Ab1 Probes

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the TA 

peroxidase promoter and cry35Ab1 probes. Approximately 3 - 4 μg of genomic DNA was digested and 

loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated 

approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 8 1 copy PHP27118 + PH09B control

2 3 copy PHP27118 + PHWWE control 9 3 copy PHP27118 + PHWWE control

3 DIGVII marker 10 DIGVII marker

4 PH09B control 11 PH09B control

5 PHWWE control 12 PHWWE control

6 4114 maize plant 20 (T3 generation) 13 4114 maize plant 20 (T3 generation)

7 4114 maize plant 22 (T3 generation) 14 4114 maize plant 22 (T3 generation)

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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4114 Maize

Figure 18. Southern Blot Analysis of the T3 Generation of 4114 Maize; Bcl I Digested DNA 

with 35S Promoter, pat, and 35S Terminator Probes

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the 35S 

promoter, pat, and 35S Terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and 

loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated 

approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 12 PHWWE control

2 3 copy PHP27118 + PHWWE control 13 4114 maize plant 20 (T3 generation)

3 DIGVII marker 14 4114 maize plant 22 (T3 generation)

4 PH09B control 15 1 copy PHP27118 + PH09B control

5 PHWWE control 16 3 copy PHP27118 + PHWWE control

6 4114 maize plant 20 (T3 generation) 17 DIGVII marker

7 4114 maize plant 22 (T3 generation) 18 PH09B control

8 1 copy PHP27118 + PH09B control 19 PHWWE control

9 3 copy PHP27118 + PHWWE control 20 4114 maize plant 20 (T3 generation)

10 DIGVII marker 21 4114 maize plant 22 (T3 generation)

11 PH09B control

1 2 3 4 5 6 7 8 9 10 11 12 13 14

35S Promoter pat 

15 16 17 18 19 20 21

35S Terminator 

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4114 Maize

Integrity of the DNA Insertion in 4114 Maize

The restriction enzyme Hind III was used to confirm the integrity of the PHP27118 T-DNA 

insertion in 4114 maize. There are three Hind III sites within the PHP27118 T-DNA: one site at 

bp 78, one after the cry1F gene at bp 3969, and one at bp 11738 just inside the Left Border of 

the T-DNA (Figure 12). An internal fragment of 3891 bp is expected to hybridize to the cry1F 

gene probe (Table 6, Figure 12), and another internal fragment of 7769 bp is expected to

hybridize to the probes for the ORF25 terminator, cry34Ab1 gene, pinII terminator, cry35Ab1 

gene, TA peroxidase promoter, pat gene and the CaMV 35S promoter and terminator regions 

(Table 6, Figure 12). Although there are two copies of the pinII terminator in the PHP27118 

T-DNA, both copies are found on the same Hind III fragment so the probe would hybridize to 

the single 7769 bp internal fragment. Because the central Hind III restriction enzyme site is 

located between the duplicate copies of the ubiZM1 promoter and intron regions, probes for 

these elements are expected to hybridize to both fragments (Table 6, Figure 12). The absence 

of any insert derived bands other than the expected bands provides a strong indication that the 

T-DNA is intact and was not truncated upon insertion. The Hind III plasmid positive control run 

alongside the 4114 maize DNA samples was used to confirm that the sizes of the observed 

bands were identical to these internal fragments.

Hybridization of Hind III-digested genomic DNA from 4114 maize with the ubiZM1 promoter 

and intron probes resulted in the expected bands of 3891 bp and 7769 bp (Table 6, Figure 19). 

An additional band of approximately 6400 bp was observed in the 4114 plants and the PHWWE 

control maize with both probes (Table 6, Figure 19). This band resulted from the hybridization 

of the ubiZM1 promoter and intron probes to the endogenous copy of these elements in the 

native maize genome based upon its presence in the PHWWE control maize line. Hybridization 

with the cry1F probe resulted in the expected band of 3891 bp in the 4114 maize plants (Table 

6, Figure 20). As expected, the 7769 bp band was observed in the 4114 maize plants with the 

ORF25 terminator probe (Table 6, Figure 20), with the cry34Ab1 and pinII terminator probes 

(Table 6, Figure 21), the TA peroxidase promoter and cry35Ab1 probes (Table 6, Figure 22), and 

the 35S promoter, pat, and 35S terminator probes (Table 6, Figure 23). The presence of the 

expected size bands that were identical to the internal bands from the PHP27118 plasmid with 

all of these elements, and no other insertion-derived bands, indicate that the PHP27118 T-DNA 

inserted intact in 4114 maize and there were no truncated copies of the T-DNA.
Pioneer Hi-Bred International, Inc. Page 57 of 258

4114 Maize

Table 6. Predicted and Observed Hybridizing Bands on Southern Blots; Hind III Digest

Probe Figure

Predicted Fragment 

Size from PHP27118

T-DNA (bp)a

Predicted Fragment 

Size from Plasmid 

PHP27118 (bp)b

Observed Fragment 

Size in 4114 Maize 

(bp)c

ubiZM1 promoter 19

7769d

3891d

7769

3891

7769e

3891e

~6400*

ubiZM1 intron 19

7769d

3891d

7769

3891

7769e

3891e

~6400*

cry1F 20 3891 3891 3891e

ORF25 terminator 20 7769 7769 7769e

cry34Ab1 21 7769 7769 7769e

pinII terminator 21 7769f7769 7769e

TA peroxidase 

promoter

22 7769 7769 7769e

cry35Ab1 22 7769 7769 7769e

35S promoter 23 7769 7769 7769e

pat 23 7769 7769 7769e

35S terminator 23 7769 7769 7769e

Note: An asterisk (*) and gray shading indicates the designated band is due to hybridization to endogenous 

sequences. These bands were identified in the maize control lines that were analyzed.

a

Predicted size for hybridization in genomic DNA samples is based on the map of the T-DNA from PHP27118 

(Figure 12).

b

Predicted size is based on the plasmid map of PHP27118 (Figure 11).

c

Observed fragment sizes are approximated from the DIG-labeled DNA Molecular Weight Marker VII fragments 

on the Southern blots. Due to the inability to determine exact sizes on the blot, all approximated values are 

rounded to the nearest 100 bp.

d

There are two copies of the ubiZM1 promoter and intron elements, located on either side of the central Hind III 

site, so both fragments will hybridize to these probes.

e

Size is same as predicted based on equal migration as the control plasmid fragment on the Southern blot.

f

Both copies of the pinII terminator in the PHP27118 T-DNA are located on the same Hind III fragment.
Pioneer Hi-Bred International, Inc. Page 58 of 258

4114 Maize

Figure 19. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hind III Digested DNA 

with ubiZM1 Promoter and ubiZM1 Intron Probes

DNA isolated from individual plants of 4114 maize was digested with Hind III and hybridized to the 

ubiZM1 promoter and ubiZM1 intron probes. Approximately 3 - 4 μg of genomic DNA was digested and 

loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated 

approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation)

2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation)

3 PH09B control 10 PH09B control

4 PHWWE control 11 PHWWE control

5 DIGVII marker 12 DIGVII marker 

6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control

7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control

8 12

ubiZM1Promoter ubiZM1Intron

1 2 3 4 5 6 7 9 10 11 13 14

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4114 Maize

Figure 20. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hind III Digested DNA 

with cry1F and ORF25 Terminator Probes

DNA isolated from individual plants of 4114 maize was digested with Hind III and hybridized to the cry1F 

and ORF25 terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded per 

lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate gene 

copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are indicated 

adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation)

2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation)

3 PH09B control 10 PH09B control

4 PHWWE control 11 PHWWE control

5 DIGVII marker 12 DIGVII marker 

6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control

7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control

cry1F ORF25 Terminator

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4114 Maize

Figure 21. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hind III Digested DNA 

with cry34Ab1 and pinII Terminator Probes

DNA isolated from individual plants of 4114 maize was digested with Hind III and hybridized to the 

cry34Ab1 and pinII terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and loaded 

per lane. The gene copy number controls included plasmid PHP27118 at the indicated approximate 

gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation)

2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation)

3 PH09B control 10 PH09B control

4 PHWWE control 11 PHWWE control

5 DIGVII marker 12 DIGVII marker 

6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control

7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control

cry34Ab1 pinII Terminator

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4114 Maize

Figure 22. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hind III Digested DNA 

with TA Peroxidase Promoter and cry35Ab1 Probes

DNA isolated from individual plants of 4114 maize was digested with Hind III and hybridized to the TA 

peroxidase promoter and cry35Ab1 probes. Approximately 3 - 4 μg of genomic DNA was digested and 

loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated 

approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb). Note: Blotches on the TA 

peroxidase panel are due to non-specific chemiluminescence from the detection process and are not 

due to hybridization of the probe to maize DNA.

Lane Sample Lane Sample

1 4114 maize plant 20 (T3 generation) 8 4114 maize plant 20 (T3 generation)

2 4114 maize plant 22 (T3 generation) 9 4114 maize plant 22 (T3 generation)

3 PH09B control 10 PH09B control

4 PHWWE control 11 PHWWE control

5 DIGVII marker 12 DIGVII marker 

6 3 copy PHP27118 + PHWWE control 13 3 copy PHP27118 + PHWWE control

7 1 copy PHP27118 + PH09B control 14 1 copy PHP27118 + PH09B control

TA Peroxidase Promoter cry35Ab1

1 2 3 4 5 6 7 8 9 10 11 12 13 14

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4114 Maize

Figure 23. Southern Blot Analysis of the T3 Generation of 4114 Maize; Hind III Digested DNA 

with 35S Promoter, pat, and 35S Terminator Probes

DNA isolated from individual plants of 4114 maize was digested with Hind III and hybridized to the 35S 

promoter, pat, and 35S Terminator probes. Approximately 3 - 4 μg of genomic DNA was digested and 

loaded per lane. The gene copy number controls included plasmid PHP27118 at the indicated 

approximate gene copy number and 4 μg of genomic DNA. Sizes of the DIG VII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 4114 maize plant 20 (T3 generation) 12 DIGVII marker 

2 4114 maize plant 22 (T3 generation) 13 3 copy PHP27118 + PHWWE control

3 PH09B control 14 1 copy PHP27118 + PH09B control

4 PHWWE control 15 4114 maize plant 20 (T3 generation)

5 DIGVII marker 16 4114 maize plant 22 (T3 generation)

6 3 copy PHP27118 + PHWWE control 17 PH09B control

7 1 copy PHP27118 + PH09B control 18 PHWWE control

8 4114 maize plant 20 (T3 generation) 19 DIGVII marker 

9 4114 maize plant 22 (T3 generation) 20 3 copy PHP27118 + PHWWE control

10 PH09B control 21 1 copy PHP27118 + PH09B control

11 PHWWE control

35S Promoter pat 35S Terminator 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

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4114 Maize

Stability of the Insertion in 4114 Maize

Stability of the 4114 maize insertion was confirmed by performing Southern blot analysis on 

Bcl I-digested genomic DNA from individual plants of four generations of 4114 maize: T2, T3, 

BC3F1*3, and BC3F2*2. As described earlier, hybridization of Bcl I-digested DNA to the probe for 

the cry1F gene resulted in an event-specific hybridization pattern consisting of a band of 

approximately 3100 bp and a band of greater than 8600 bp (Figure 13). This hybridization 

pattern can be used to demonstrate the stability of the 5’ and 3’ region of the 4114 maize

insertion. Hybridizations with the cry34Ab1, cry35Ab1, and pat probes resulted in a single band 

of greater than 8600 bp (Figure 13), and can be used to demonstrate the stability of the 3’ 

region of the insertion. The presence of these event-specific hybridization patterns in each of 

the four generations with these probes would confirm the stability of the insertion in 4114 

maize during breeding.

Hybridization of the Bcl I-digested DNA with the cry1F probe resulted in two bands of 

approximately 3100 bp and greater than 8600 bp that were consistently observed in each of 

the four generations, indicating stability of the 5’ and 3’ regions of the 4114 maize insertion

(Table 5, Figure 24). When hybridized to the cry34Ab1 (Table 5, Figure 25), cry35Ab1 (Table 5, 

Figure 26), and pat probes (Table 5, Figure 27), a band of greater than 8600 bp was consistently 

observed in all plants from the four generations. The observation of these expected event 

specific hybridization patterns with all probes indicated that the 4114 DNA insertion was stable

during the breeding process.
Pioneer Hi-Bred International, Inc. Page 64 of 258

4114 Maize

Figure 24. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with cry1F Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the cry1F 

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 25. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with cry34Ab1 Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the 

cry34Ab1 probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene 

copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with 

Hind III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. 

Sizes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs 

(kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 26. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with cry35Ab1 Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the 

cry35Ab1 probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene 

copy number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with 

Hind III in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. 

Sizes of the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs

(kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 27 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 27. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with pat Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the pat

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 27 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Absence of Plasmid Backbone DNA in 4114 Maize

Four generations of 4114 maize (T2, T3, BC3F1*3, and BC3F2*2) were analyzed by Southern blot 

analysis for plasmid sequences from the PHP27118 plasmid backbone. Probes for genes and 

regions on the PHP27118 plasmid backbone outside the T-DNA region were used to determine 

if any plasmid backbone was inserted in 4114 maize during transformation. The spc, tet, and 

virG probes hybridize to the spectinomycin resistance, tetracycline resistance, and virG genes 

respectively (Table 4, Figure 11). The LB probe hybridizes to the plasmid backbone region 

outside the Left T-DNA border, and the RB probe hybridizes to the region outside the Right 

Border of the T-DNA of PHP27118 (Table 4, Figure 11). These probes would show whether

regions of the PHP27118 backbone outside the T-DNA was transferred into 4114 maize. 

Genomic DNA from the T2, T3, BC3F1*3, and BC3F2*2 generations was digested with Bcl I and 

hybridized to the backbone probes described above. The LB (Figure 28), spc (Figure 29), tet

(Figure 30), virG (Figure 31), and RB (Figure 32) probes showed no bands from backbone 

hybridization in the 4114 maize or control maize plants. This confirmed that no sequence from 

the PHP27118 plasmid backbone was inserted during transformation. The plasmid lanes 

showed hybridization to the undigested plasmid in the Bcl I lanes (Figures 28 through 32, Lanes 

1 and 2) and to the expected size bands for digestion with Hind III (Figures 28 through 32, Lanes 

15 and 16), thus demonstrating successful hybridization of the probes to plasmid backbone 

sequences.
Pioneer Hi-Bred International, Inc. Page 69 of 258

4114 Maize

Figure 28. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114

Maize; Bcl I Digested DNA with LB Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the LB 

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 29. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with spc Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the spc

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 30. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with tet Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the tet

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 31. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with virG Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the virG 

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Figure 32. Southern Blot Analysis of the T2, T3, BC3F1*3and BC3F2*2 Generations of 4114 

Maize; Bcl I Digested DNA with RB Probe

DNA isolated from individual plants of 4114 maize was digested with Bcl I and hybridized to the RB

probe. Approximately 3 - 6 μg of genomic DNA was digested and loaded per lane. The gene copy 

number controls included plasmid PHP27118 (digested with Bcl I in Lanes 1 and 2; digested with Hind III 

in Lanes 15 and 16) at the indicated approximate gene copy number and 4 μg of genomic DNA. Sizes of 

the DIG VII molecular weight markers are indicated adjacent to the blot image in kilobase pairs (kb).

Lane Sample Lane Sample

1 1 copy PHP27118 + PH09B control 9 4114 maize plant 37 (BC3F1*3generation)

2 3 copy PHP27118 + PHWWE control 10 4114 maize plant 39 (BC3F2*2generation)

3 DIGVII marker 11 4114 maize plant 41 (BC3F2*2generation)

4 4114 maize plant 9 (T2 generation) 12 PH09B control

5 4114 maize plant 12 (T2 generation) 13 PHWWE control

6 4114 maize plant 20 (T3 generation) 14 DIGVII marker

7 4114 maize plant 22 (T3 generation) 15 3 copy PHP27118 + PH09B control (Hind III)

8 4114 maize plant 30 (BC3F1*3generation) 16 1 copy PHP27118 + PHWWE control (Hind III)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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4114 Maize

Physical Map of the DNA Insertion in 4114 Maize

Based on the Southern blot analysis, it was determined that a single, intact PHP27118 T-DNA 

was inserted into the genome of 4114 maize. A physical map of the DNA insertion in 4114 

maize showing the restriction enzymes employed in the analysis was developed using these 

data and is presented in Figure 13.

Summary and Conclusions

Southern blot analysis was conducted on 4114 maize to confirm insertion copy number, 

integrity, and stability of the insertion. Analysis with Bcl I, examining the sequences flanking 

the DNA insertion, indicated a single copy of the PHP27118 T-DNA is present in 4114 maize. 

The Hind III analysis indicated that the PHP27118 T-DNA had inserted intact in the genome. 

Analysis with Bcl I demonstrated that the 4114 DNA insertion is stable across the T2, T3, 

BC3F1*3, and BC3F2*2generations and during the traditional breeding process, as identical 

hybridization patterns were observed in each of the four generations. In addition, analysis with 

probes for the regions of PHP27118 outside the T-DNA region demonstrated that no plasmid 

backbone sequences were incorporated into the 4114 maize genome.
Pioneer Hi-Bred International, Inc. Page 75 of 258

4114 Maize

2-E. Inheritance and Stability of the Inserted DNA

The stability of the inserted DNA during the breeding process is evaluated by examining the 

inheritance and segregation of the genes and/or traits in multiple generations. The segregation 

of these genes or traits as a single unit and as a single genetic locus will confirm that the 

inserted DNA will be predictably and stably inherited through the commercial breeding process.

For 4114 maize, the inheritance of both the DNA insertion and the herbicide-tolerance 

phenotype was evaluated to ensure stability of the traits during the plant breeding process and 

to confirm the traits were inserted at a single genetic locus. For the analyses, segregating 

generations of 4114 maize (F1*1, BC2F1*1, BC3F1*1, BC2F1*2, and BC3F1*2 generations) were 

evaluated. The breeding history of these five generations is shown in the breeding diagram 

(Section 2, Figure 10 and Table 3). These generations were selected for analysis because they 

represent a range of different crossing and backcrossing points in a typical maize breeding 

program. Chi-square analysis was conducted on the five segregating generations to determine 

if the observed segregation ratios were consistent with the expected ratios. The assay methods 

and statistical analysis for the trait inheritance data are described in Appendix 4.

The presence of the 4114 event insertion was determined by event-specific and cry1F, 

cry34Ab1, cry35Ab1, and pat gene-specific endpoint PCR analyses performed on leaf punches 

from seedlings of each generation. The herbicide-tolerance phenotype was determined by 

treating the plants with glufosinate herbicide and visually evaluating each plant for herbicide 

injury. A positive plant exhibited no herbicidal injury and a negative plant exhibited severe 

herbicide injury. 

Results from the segregation analysis are provided in Table 7. In every case, a positive plant 

tested positive for the presence of the 4114 event, the cry1F, cry34Ab1, cry35Ab1 and pat

genes, and the herbicide-tolerance phenotype, indicating the 4114 insertion segregated as a 

unit. To confirm that the inserted DNA and the herbicide-tolerance phenotype segregate 

according to Mendel’s laws of genetics for a single genetic locus, chi-square analysis was 

performed for the PCR and the herbicide-tolerance phenotype data.

No statistically significant differences were found between the observed and expected 

segregation ratios for the F1*1, BC2F1*1, BC3F1*1, and BC2F1*2 generations of 4114 maize (Table 

7), indicating that these four generations all segregated as expected based on Mendel’s laws. 

The 4114 insertion segregated as a unit and the observed ratios were indicative of an insertion 

at a single genetic locus.

The observed segregation ratio of the BC3F1*2 generation in the original analysis of 99 seedlings 

was statistically significant (p-value <0.05) compared to the expected 1:1 segregation ratio, with 

a chi-square value of 5.34 and p-value of 0.0208. Typically an observed segregation ratio would 

not be statistically different compared to a 1:1 expected ratio (50:50 based on 100 plants) if it 

fell in the range of 50:50 ± 9 (ratio of 41:59 or 59:41). The BC3F1*2 generation observed 

segregation ratio for the original sample was 38:61, only two plants outside of the acceptable 
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4114 Maize

range. In order to determine if the statistical difference was a false positive result due to 

random sampling, additional sampling of 96 seedlings from the same seed source was 

performed. For the original sample, the chance of a false positive result due to random 

sampling was 5%; if a statistical difference was found when sampling was repeated, the chance 

of a false positive result would be greatly reduced to 5% x 5% = 0.25%. This additional sampling 

would not only confirm the prior result, but would also increase the statistical power of the 

BC3F1*2 generation analysis. 

Chi-square analysis of the combined data (n=195) for this single seed source (95% confidence) 

was performed and no statistically significant difference was found between the observed 

segregation ratio and the expected segregation ratio for the BC3F1*2 generation (Table 7). The 

significant difference observed in the original BC3F1*2 generation analysis was therefore likely 

due to a false positive result associated with random sampling.

These results indicate that the inserted DNA and the herbicide-tolerance phenotype in 4114 

maize segregate predictably according to Mendel’s laws of genetics and are consistent with the 

finding that the 4114 maize insertion is at a single genetic locus. Taken together with the 

stability results described earlier for the Southern analysis, these results confirm the stability of 

the insertion through the breeding process.

Table 7. Summary of Genotypic and Phenotypic Results for Segregating 4114 Maize

4114 Maize 

Generationa

Observed Valuesb

(expected segregation = 1:1) Statistical Analysis

Positive Negative Total Chi-SquarecP-value

F1*1 52 46 98 0.367 0.545

BC2F1*1 48 52 100 0.160 0.689

BC3F1*1 47 53 100 0.360 0.549

BC2F1*2 53 47 100 0.360 0.549

BC3F1*2 87 108 195 1.62 0.1326

a

The BC3F1*2 generation analysis was conducted during two time points. The first analysis was conducted with 

99 seedlings and found to be statistically significant by Chi-square analysis. To increase the statistical power, an 

additional 96 seedlings from the same seed lot were tested. The combined results of the two time points within 

the single seed lot are shown and were not statistically different from the expected segregation ratio.

b

PCR analysis (consisting of event-specific PCR analysis to confirm the presence or absence of maize event 4114, 

and gene-specific PCR analysis to confirm the presence or absence of the cry1F, cry34Ab1, cry35Ab1, and pat

genes) and herbicide (i.e., glufosinate) tolerance analysis were conducted for each plant in each entry. All PCR 

results matched the corresponding herbicide tolerance result for each plant analyzed.

c

Degrees of freedom = 1.
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4114 Maize

2-F. Concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT in 4114 Maize

Determining the concentrations of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in various 

tissues of 4114 maize is important background information for the conduct of exposure and 

safety assessments. To determine if previous safety studies conducted for 1507 (expresses 

Cry1F and PAT), 59122 (expresses Cry34Ab1, Cry35Ab1, and PAT), or 1507x59122 maize 

(expresses Cry1F, Cry34Ab1, Cry35Ab1, and PAT) are applicable to 4114 maize, it is necessary to 

determine if the concentrations of the expressed proteins in 4114 maize are comparable to 

these commercial lines. 

In order to determine the concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT and to assess 

comparability, 4114 maize was grown side-by-side with commercial 1507, 59122, and 

1507x59122 maize lines at five commercial maize-growing sites in the U.S. and Canada during 

the 2010 growing season. The sites were located in Iowa (two sites), Illinois, Nebraska, and 

Ontario, Canada. The F1*5generation of seed (Section 2; Figure 10 and Table 3) was used 

because F1 hybrid seed is representative of seed that growers would plant in commercial maize 

fields. Four replications (from different plants) per tissue were collected from each site, for a 

total of n=20 for each tissue and time point.

Plant tissue samples were collected throughout the growing season at various growth 

developmental stages (Table 8) and processed as described in Appendix 5. Time points for 

sampling were chosen to determine the range of protein concentrations throughout the 

growing season and for their relevance to commercial maize production practices. The protein 

concentrations in R6 tissues (i.e., plant senescence and grain harvest) and R1 pollen values (i.e.,

at pollen shed) have relevance for the evaluation of impact on non-target arthropods in the 

environment. The R4 stage of the whole plant sample (i.e., forage) is the stage at which 

growers harvest plants for silage for animal feed. Grain is normally harvested at the R6 stage of 

development and is used for food and feed.

Concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT protein in plant tissue extracts were 

measured in the following tissues using specific quantitative enzyme-linked immunosorbent 

assay (ELISA) methods, as described in Appendix 5:

 Leaf—V6, V9, R1, R4, and R6

 Root—V6, V9, R1, R4, and R6

 Whole Plant (the above-ground portion of the plant, including the ear at R1 and 

R6)—V9, R1, and R6

 Pollen—R1

 Forage (the above-ground portion of the plant, including the ear)—R4

 Grain—R6

The ranges of transgenic protein mean concentrations in leaf, root, and whole-plant tissues 

over the course of the growing season, as well as the mean concentrations in pollen and root, 

are summarized in Table 9 for 4114 maize (data taken from Tables 10 through 13). The mean 
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4114 Maize

PAT concentrations in 4114 maize pollen and grain were below the lower limit of quantification 

(LLOQ) of the ELISA assay.

Table 8. Maize Growth Stage Descriptions

Growth 

Stagea Description

V6 The collar of the sixth leaf becomes visible.

V9 The collar of the ninth leaf becomes visible.

R1 Silks become visible.

R4 The material within the kernel produces a doughy consistency.

R6 Typical harvest maturity for grain (regarded as physiological maturity).

a Adapted from Ritchie et al., 2005

Table 9. Mean Concentrations of Transgenic Proteins in 4114 Maize

Tissue

Cry1F Cry34Ab1 Cry35Ab1 PAT

Range in ng/mg tissue dry weighta

Leaf 2.0 - 34 19 - 110 27 - 90 0.52 - 14

Root 3.8 - 5.5 17 - 23 6.4 - 13 0.13 - 0.65

Whole Plantb4.1 - 12 23 - 52 21 - 76 0.090 - 8.7

Pollen 35 9.2 0.34 <0.28c

Grain 3.3 24 1.1 <0.069c

a

Ranges reflect the range of means at different growth stages. 

b Whole plant ranges include forage.

c

< Lower Limit of Quantification (LLOQ); indicates the values of the sample(s) were detected below the assay 

LLOQ
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4114 Maize

For each transgenic protein, the concentrations in each 4114, 1507, 59122, and 1507x59122 

maize tissue were measured (Tables 10 through 13). The Cry1F, Cry34Ab1, and Cry35Ab1 

protein concentrations in 4114 maize in each tissue were divided by the respective protein 

concentrations in 1507, 59122, and/or 1507x59122 maize to provide an “expression ratio”

(Table 14). When the expression ratio was close to one, this indicated that 4114 maize had 

comparable expression to 1507, 59122, and/or 1507x59122 maize. If the expression ratio was 

less than one, this indicated that 4114 maize had lower expression. For values greater than 

one, expression was determined to be higher in 4114 maize.

The concentrations of Cry1F protein in 4114 maize were comparable to or lower than 

concentrations in 1507 maize and 1507x59122 maize in all tissues measured except for pollen 

(R1) and leaf (R6) tissues (Tables 10 and 14). Concentrations of Cry1F in pollen (R1) and leaf 

(R6) tissues were higher in 4114 maize.

The concentrations of Cry34Ab1 protein in 4114 maize were comparable to or lower than 

concentrations in 59122 maize and 1507x59122 maize in all tissues measured except for leaf 

(R6) tissue (Tables 11 and 14). The concentration of Cry34Ab1 in leaf (R6) tissue was higher in 

4114 maize.

The concentrations of Cry35Ab1 protein in 4114 maize were comparable to or lower than 

concentrations in 59122 maize and 1507x59122 maize in all tissues measured (Tables 12 and 

14).

The concentrations of PAT protein in 4114 maize were comparable to or greater than 

concentrations in 1507 maize and were comparable to or lower than concentrations in 59122 

maize and 1507x59122 maize, with the exception of leaf (R6) which was higher (Tables 13 and 

14). It was expected that PAT concentrations in 1507x59122 maize tissues would be greater 

than PAT concentrations in 4114, 1507, and/or 59122 maize, since 1507x59122 contains two 

copies of the pat gene (one each from 1507 and 59122 maize) and 4114, 1507, and 59122 

maize each contain one copy of the pat gene. In general, PAT protein concentrations in pollen, 

R4 root tissue, and all R6 tissues (grain, leaf, root, and whole plant) were very low and, in some 

cases, below the lower limit of quantification. 

It is noted that, in most tissues and time points, the concentrations of Cry1F, Cry34Ab1, 

Cry35Ab1 and PAT in 4114 maize differed from 1507, 59122, and/or 1507x59122 maize, even 

though the same promoter-gene combinations are present in 4114 maize as in 1507 and/or

59122 maize. Expression differences can likely be attributed to event-to-event variation and 

are generally considered minor for a biological system (i.e., 5.4-fold or less in all cases and often 

less than 2-fold).

In addition, these expression differences are not necessarily biologically meaningful. For 

example, as described fully in insect efficacy data submitted to the EPA, despite the fact that 

concentrations of Cry1F, Cry34Ab1 and Cry35Ab1 are generally lower in 4114 maize than in 

1507, 59122 and/or 1507x59122 maize, 4114 maize was equivalent to 1507 maize in efficacy 
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4114 Maize

against target lepidopteran pests. 4114 maize may be slightly less efficacious and less 

consistent in efficacy against corn rootworm compared to 1507x59122 maize. At one location 

in 2010 where corn rootworm pressure was high, 4114 maize was less efficacious than 

1507x59122 maize. However, for the remaining eight location-by-year comparisons, there 

were no significant differences in corn rootworm efficacy between 4114 and 1507x59122 

maize. Therefore, although in many cases the concentrations of Cry1F, Cry34Ab1, and 

Cry35Ab1 proteins were generally lower in 4114 maize tissues than in 1507, 59122, and/or 

1507x59122 maize, there was little to no biological impact in terms of efficacy.

As is summarized in Table 14, in most cases the concentrations of Cry1F, Cry34Ab1, Cry35Ab1, 

and PAT were generally comparable to or lower than 1507, 59122, and/or 1507x59122 maize. 

The notable exceptions were Cry1F and Cry34Ab1 in late-season R6 leaf tissue, where 

concentrations in 4114 maize were 2.3- to 5.4-fold higher than 1507, 59122, and 1507x59122 

maize, and Cry1F in pollen, where concentrations in 4114 maize were 1.4- to 1.5-fold higher 

than 1507 and 1507x59122 maize. R6 leaf tissue is collected at harvest time, when tissue has 

begun to senesce to varying degrees, and can be variable in degree of senescence and total 

extractable protein levels. This may impact the concentrations of transgenic proteins reported; 

R6 leaf tissue ratios were not representative of comparisons between 4114 maize and 1507, 

59122, and/or 1507x59122 maize in leaf tissues at other time points. In some cases, PAT 

concentrations were higher in 4114 maize as compared to 1507 maize and were also higher in 

R6 leaf for similar reasons described above; however, the concentrations of PAT overall were 

very low or below the lower limit of quantification which likely impacted ratios and the 

comparisons. The comparisons for all other tissues, with the exception of Cry1F pollen, 

demonstrate that 4114 maize tissues have similar or lower concentrations of Cry1F, Cry34Ab1,

Cry35Ab1, and PAT than 1507, 59122, and/or 1507x59122 maize tissues.

In general, these comparisons indicate any previously conducted safety studies that used 1507, 

59122, and 1507x59122 maize are applicable to 4114 maize. Safety studies that considered 

exposure to the Cry1F protein from 1507 or 1507x59122 pollen would require the 

reassessment of results for 4114 maize using the higher concentration.
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4114 Maize

Table 10. Cry1F Concentrations (ng/mg Tissue Dry Weight)

Tissue (Growth Stage) 4114 Maize 1507 Maize 1507x59122 Maize

Leaf (V6)

Meana11 18 18

Range 7.8 - 17 11 - 26 13 - 23

CIb9.4 - 13 15 - 21 15 - 21

Root (V6)

Mean 4.2 5.7 6.3

Range 0.69 - 6.0 1.1 - 7.8 3.3 - 8.7

CI 2.8 - 5.5 4.4 - 7.1 5.0 - 7.6

Leaf (V9)

Mean 9.7 13 16

Range 5.3 - 14 0.78 - 20 9.6 - 24

CI 6.8 - 13 10 - 16 13 - 18

Root (V9)

Mean 5.0 7.2 6.9

Range 1.3 - 7.5 4.2 - 12 4.2 - 11

CI 3.1 - 6.9 5.3 - 9.1 5.0 - 8.7

Whole Plant (V9)

Mean 12 16 16

Range 8.6 - 15 10 - 19 9.6 - 19

CI 10 - 13 14 - 17 15 - 18

Leaf (R1)

Mean 13 16 17

Range 7.2 - 28 10 - 23 11 - 26

CI 9.6 - 18 12 - 21 12 - 23

Pollen (R1)

Mean 35 23 24

Range 19 - 49 19 - 26 18 - 28

CI 29 - 42 19 – 27 20 - 29

Root (R1)

Mean 5.5 7.0 6.1

Range 3.9 - 7.8 5.1 - 9.9 3.9 - 7.5

CI 4.8 - 6.3 6.1 - 7.9 5.3 - 7.0

Whole Plant (R1)

Mean 9.9 14 13

Range 7.8 - 13 10 - 18 9.6 - 15

CI 9.1 - 11 13 - 15 12 - 14

Forage (R4)

Mean 7.8 8.4 9.1

Range 5.6 - 11 5.6 - 12 6.0 - 14

CI 6.4 - 9.4 7.0 - 10 7.5 - 11

Leaf (R4)

Mean 34 37 37

Range 19 - 56 25 - 49 23 - 58

CI 27 - 42 30 - 47 29 - 46

Root (R4)

Mean 3.8 4.4 5.3

Range 2.3 - 5.7 3.0 - 5.7 3.0 - 7.2

CI 2.9 - 4.6 3.6 - 5.3 4.4 - 6.2

Grain (R6)

Mean 3.3 3.2 2.8

Range 2.3 - 7.2 1.9 - 5.1 1.7 - 4.5

CI 2.8 - 4.0 2.7 - 3.8 2.4 - 3.4

Leaf (R6)

Mean 2.0 0.79 0.37

Range 0.32 - 21 <0.14c-21 <0.14c-16

CI 0.38 - 10 0.15 - 4.1 0.072 - 1.9
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4114 Maize

Table 10. Cry1F Concentrations (ng/mg Tissue Dry Weight) (continued)

Tissue (Growth Stage) 4114 Maize 1507 Maize 1507x59122 Maize

Root (R6)

Mean 3.8 5.0 4.7

Range 1.4 - 6.3 2.9 - 7.5 2.7 - 8.1

CI 3.1 - 4.8 4.1 - 6.2 3.8 - 5.8

Whole Plant (R6)

Mean 4.1 4.9 3.8

Range 2.4 - 9.4 2.2 - 8.4 2.2 - 7.4

CI 3.0 - 5.5 3.6 - 6.6 2.8 - 5.1

a Least squares means (estimated from statistical models; see Appendix 5 for additional details).

b

Statistical Confidence Interval

c

< Lower Limit of Quantification (LLOQ); indicates the values of the sample(s) were detected below the assay 

LLOQ
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4114 Maize

Table 11. Cry34Ab1 Concentrations (ng/mg Tissue Dry Weight) 

Tissue (Growth Stage) 4114 Maize 1507x59122 

Maize 59122 Maize

Leaf (V6)

Meana21 32 33

Range 17 - 25 25 - 43 24 - 46

CIb19 - 22 30 - 35 31 - 36

Root (V6)

Mean 17 29 26

Range 9.0 - 23 19 - 42 16 - 36

CI 13 - 22 24 - 34 22 - 31

Leaf (V9)

Mean 26 42 38

Range 22 - 31 32 - 54 23 - 46

CI 23 - 29 37 - 48 33-43

Root (V9)

Mean 21 36 37

Range 13 - 28 30 - 42 23 - 48

CI 18 - 24 31 - 41 31-41

Whole Plant (V9)

Mean 23 41 41

Range 18 - 26 26 - 52 26 - 54

CI 18 - 28 36 - 46 36 - 45

Leaf (R1)

Mean 50 70 75

Range 36 - 84 52 - 110 54 - 110

CI 33 - 67 53 - 87 58 - 92

Pollen (R1)

Mean 9.2 46 44

Range 4.7 - 16 35 - 67 36 - 64

CI 7.7 - 11 38 - 55 36 - 53

Root (R1)

Mean 19 29 29

Range 8.7 - 30 14 - 45 16 - 39

CI 10 - 28 20 - 38 19 - 38

Whole Plant (R1)

Mean 32 56 57

Range 24 - 42 44 - 82 50 - 76

CI 29 - 35 50 - 62 52 - 64

Forage (R4)

Mean 52 93 86

Range 36 - 64 68 - 130 56 - 120

CI 46 - 60 81 - 110 75 - 98

Leaf (R4)

Mean 110 200 190

Range 66 - 140 150 - 250 84 - 270

CI 84 - 130 180 - 220 160 - 210

Root (R4)

Mean 23 35 32

Range 7.5 - 36 18 - 51 12 - 51

CI 17 - 29 29 - 41 26 - 38

Grain (R6)

Mean 24 24 24

Range 14 - 39 14 - 42 17 - 33

CI 20 - 28 20 - 29 20 - 29

Leaf (R6)

Mean 19 6.0 7.7

Range 4.2 - 66 0.30 - 130 0.90 - 120

CI 4.4 - 82 1.4 - 26 1.8 - 34
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4114 Maize

Table 11. Cry34Ab1 Concentrations (ng/mg Tissue Dry Weight) (continued)

Tissue (Growth Stage) 4114 Maize 1507x59122 

Maize 59122 Maize

Root (R6)

Mean 18 32 37

Range 5.4 - 54 14 - 54 17 - 66

CI 14 - 24 24 - 42 28 - 49

Whole Plant (R6)

Mean 36 48 48

Range 20 - 62 26 - 88 28 - 70

CI 29 - 44 39 - 58 40 - 59

a Least squares means (estimated from statistical models; see Appendix 5 for additional details).

b

Statistical Confidence Interval
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4114 Maize

Table 12. Cry35Ab1 Concentrations (ng/mg Tissue Dry Weight) 

Tissue (Growth Stage) 4114 Maize 1507x59122 

Maize 59122 Maize

Leaf (V6)

Meana27 33 36

Range 17 - 38 25 - 50 27 - 50

CIb22 - 33 26 - 41 29 - 44

Root (V6)

Mean 13 18 16

Range 9.0 - 20 14 - 23 11 - 23

CI 11 - 15 15 - 22 14 - 19

Leaf (V9)

Mean 33 46 40

Range 28 - 39 35 - 58 35 - 47

CI 30 - 37 42 - 50 37 - 44

Root (V9)

Mean 13 17 17

Range 7.8 - 19 13 - 22 9.3 - 27

CI 10 - 17 13 - 21 13 - 21

Whole Plant (V9)

Mean 76 79 85

Range 58 - 100 52 - 100 66 - 100

CI 67 - 86 70 - 89 76 - 96

Leaf (R1)

Mean 68 71 81

Range 43 - 130 56 - 110 66 - 110

CI 56 - 83 58 - 87 67 - 100

Pollen (R1)

Mean 0.34 <0.32c0.33

Range <0.32c- 0.53 <0.32c<0.32c- 0.44

CI NAdNA NA

Root (R1)

Mean 9.2 10 11

Range 4.2 - 15 6.6 - 16 7.2 - 15

CI 7.2 - 12 7.9 - 13 8.4 - 14

Whole Plant (R1)

Mean 66 75 69

Range 44 - 100 58 - 110 54 - 98

CI 57 - 75 66 - 86 60 - 79

Forage (R4)

Mean 29 28 30

Range 18 - 52 17 - 42 20 - 52

CI 23 - 35 23 - 34 24 - 36

Leaf (R4)

Mean 90 100 100

Range 66 - 110 84 - 130 52 - 130

CI 81 - 99 95 - 110 93 - 110

Root (R4)

Mean 6.4 8.0 8.0

Range 2.1 - 12 3.9 - 14 1.1 - 13

CI 3.6 - 9.3 5.1 - 11 5.1 - 11

Grain (R6)

Mean 1.1 1.4 1.4

Range 0.54 - 2.3 0.69 - 2.0 0.75-2.3

CI 0.84 - 1.4 1.1 - 1.8 1.1 - 1.8

Leaf (R6)

Mean 72 72 59

Range 41 - 110 2.0 - 190 2.2 - 130

CI 36 - 108 36 - 110 23 - 96
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4114 Maize

Table 12. Cry35Ab1 Concentrations (ng/mg Tissue Dry Weight) (continued)

Tissue (Growth Stage) 4114 Maize 1507x59122 

Maize 59122 Maize

Root (R6)

Mean 6.9 8.9 10.0

Range 2.2 - 14 3.3 - 18 3.3 - 17

CI 3.5 - 10 5.5 - 12 6.6 - 13

Whole Plant (R6)

Mean 21 31 27

Range 13 - 54 20 - 46 13 - 64

CI 15 - 30 22 - 44 19 - 38

a Least squares means (estimated from statistical models; see Appendix 5 for additional details).

b

Statistical Confidence Interval

c

< Lower Limit of Quantification (LLOQ); indicates the values of the sample(s) were detected below the assay 

LLOQ

d Greater than 80% of samples were <LLOQ, statistical analysis not available (NA)
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4114 Maize

Table 13. PAT Concentrations (ng/mg Tissue Dry Weight) 

Tissue 

(Growth Stage) 4114 Maize 1507 Maize 1507x59122 Maize 59122 Maize

Leaf (V6)

Meana9.0 5.6 16 12

Range 4.2 - 14 3.1 - 10 7.2 - 24 8.4 - 18

CIb7.6 - 11 4.7 - 6.6 13 - 18 9.8 - 14

Root (V6)

Mean 0.44 0.29 1.7 1.1

Range 0.14 - 0.78 0.075 - 0.45 0.93 - 2.9 0.42 - 2.1

CI 0.32 - 0.62 0.21 - 0.40 1.2 - 2.3 0.78 - 1.5

Leaf (V9)

Mean 9.8 6.6 21 12

Range 4.8 - 15 2.9 - 8.4 10 - 32 4.1 - 16

CI 6.7 - 13 3.4 - 9.7 17 - 24 8.8 - 15

Root (V9)

Mean 0.65 0.36 2.2 1.5

Range 0.39 - 0.90 0.22 - 0.57 1.1 - 3.0 0.99 - 2.6

CI 0.30 - 1.00 0.013 - 0.71 1.8 - 2.5 1.1 - 1.8

Whole Plant 

(V9)

Mean 8.7 3.6 15 11

Range 6.6 - 11 3.0 - 4.2 11 - 18 8.0 - 15

CI 7.7 - 9.8 3.2 - 4.0 13 - 17 9.6 - 12

Leaf (R1)

Mean 14 6.5 27 19

Range 5.0 - 24 2.0 - 9.6 17 - 31 12 - 26

CI 9.9 - 17 2.8 - 10 23 - 31 15 - 22

Pollen (R1)

Mean <0.28c<0.28c<0.28c<0.28c

Range <0.28c<0.28c<0.28c<0.28c

CI NAdNA NA NA

Root (R1)

Mean 0.44 0.24 1.1 0.98

Range 0.30 - 0.72 0.12 - 0.48 0.60 - 2.3 0.60 - 1.6

CI 0.34 - 0.57 0.18 - 0.31 0.86 - 1.4 0.76 - 1.3

Whole Plant 

(R1)

Mean 4.9 2.4 8.4 6.7

Range 3.2 - 7.4 1.6 - 3.8 5.4 - 14 4.6 - 9.4

CI 4.0 - 5.9 1.9 - 2.9 6.9 - 10 5.5 - 8.1

Forage (R4)

Mean 1.9 0.75 3.5 2.5

Range 1.1 - 2.8 0.54 - 1.2 1.8 - 4.8 1.0 - 4.0

CI 1.4 - 2.4 0.23 - 1.3 3.0 - 4.0 2.0 - 3.0

Leaf (R4)

Mean 11 4.2 18 13

Range 5.7 - 20 2.6 - 5.5 9.6 - 26 3.6 - 22

CI 7.5 - 14 0.95 - 7.3 15 - 21 10 - 17

Root (R4)

Mean 0.16 0.13 0.62 0.33

Range <0.069c - 0.39 <0.069c - 0.33 0.30 - 1.9 <0.069c - 0.87

CI 0.10 - 0.25 0.083 - 0.20 0.40 - 0.95 0.22 - 0.51

Grain (R6)

Mean <0.069c<0.069c0.089 0.071

Range <0.069c<0.069c<0.069c - 0.45 <0.069c - 0.10

CI NA NA NA NA

Leaf (R6)

Mean 0.52 0.25 0.33 0.25

Range <0.14c - 2.0 <0.14c - 1.0 <0.14c - 2.5 <0.14c - 1.1

CI NA NA NA NA
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4114 Maize

Table 13. PAT Concentrations (ng/mg Tissue Dry Weight) (continued)

Tissue 

(Growth Stage) 4114 Maize 1507 Maize 1507x59122 Maize 59122 Maize

Root (R6)

Mean 0.13 0.13 0.41 0.41

Range <0.069c - 0.66 <0.069c - 0.39 0.078 - 1.8 0.11 - 1.6

CI 0.084 - 0.21 0.084 - 0.21 0.26 - 0.64 0.26 - 0.64

Whole Plant 

(R6)

Mean 0.090 0.073 0.18 0.13

Range <0.046c - 0.76 <0.046c - 0.28 <0.046c - 1.1 <0.046c - 0.64

CI 0.028 - 0.29 0.023 - 0.24 0.058 - 0.58 0.039 - 0.40

a Least squares means (estimated from statistical models; see Appendix 5 for additional details).

b

Statistical Confidence Interval

c

< Lower Limit of Quantification (LLOQ); indicates the values of the sample(s) were detected below the assay 

LLOQ

d Greater than 80% of samples were <LLOQ, statistical analysis not available (NA)
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Table 14. Summary of 4114 Maize Protein Concentrations as a Ratio of 1507, 59122, and/or 

1507x59122 Maize

Tissue

(Growth Stage)

Protein Concentration Ratio

4114 Maize:1507 

Maize

4114 Maize:59122 

Maize

4114 Maize:1507x59122 

Maize

Cry1F

Leaf (V6) 0.611 0.611

Leaf (V9) 0.746 0.606

Leaf (R1) 0.813 0.765

Leaf (R4) 0.919 0.919

Leaf (R6) 2.53 5.41

Root (V6) 0.737 0.667

Root (V9) 0.694 0.725

Root (R1) 0.786 0.902

Root (R4) 0.864 0.717

Root (R6) 0.760 0.809

Whole Plant (V9) 0.750 0.750

Whole Plant (R1) 0.707 0.762

Whole Plant (R6) 0.837 1.08

Pollen (R1) 1.52 1.46

Forage (R4) 0.929 0.857

Grain (R6) 1.03 1.18

Cry34Ab1

Leaf (V6) 0.636 0.656

Leaf (V9) 0.667 0.619

Leaf (R1) 0.667 0.714

Leaf (R4) 0.579 0.550

Leaf (R6) 2.32 3.17

Root (V6) 0.654 0.586

Root (V9) 0.568 0.583

Root (R1) 0.655 0.655

Root (R4) 0.719 0.657

Root (R6) 0.486 0.563

Whole Plant (V9) 0.561 0.561

Whole Plant (R1) 0.561 0.571

Whole Plant (R6) 0.750 0.750

Pollen (R1) 0.209 0.200

Forage (R4) 0.605 0.559

Grain (R6) 1.00 1.00
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Table 14. Summary of 4114 Maize Protein Concentrations as a Ratio of 1507, 59122, and/or 

1507x59122 Maize (continued)

Tissue

(Growth Stage)

Protein Concentration Ratio

4114 Maize:1507 

Maize

4114 Maize:59122 

Maize

4114 Maize:1507x59122 

Maize

Cry35Ab1

Leaf (V6) 0.750 0.818

Leaf (V9) 0.825 0.717

Leaf (R1) 0.840 0.958

Leaf (R4) 0.900 0.900

Leaf (R6) 1.22 1.00

Root (V6) 0.813 0.722

Root (V9) 0.765 0.765

Root (R1) 0.836 0.920

Root (R4) 0.800 0.800

Root (R6) 0.690 0.775

Whole Plant (V9) 0.894 0.962

Whole Plant (R1) 0.957 0.880

Whole Plant (R6) 0.778 0.677

Pollen (R1) 1.03 1.06a

Forage (R4) 0.967 1.04

Grain (R6) 0.857 0.800

PAT

Leaf (V6) 1.61 0.750 0.563

Leaf (V9) 1.48 0.817 0.467

Leaf (R1) 2.15 0.737 0.519

Leaf (R4) 2.62 0.846 0.611

Leaf (R6) 2.08 2.08 1.58

Root (V6) 1.52 0.400 0.259

Root (V9) 1.81 0.464 0.295

Root (R1) 1.83 0.449 0.400

Root (R4) 1.23 0.485 0.258

Root (R6) 1.00 0.317 0.317

Whole Plant (V9) 2.42 0.791 0.580

Whole Plant (R1) 2.04 0.731 0.583

Whole Plant (R6) 1.23 0.692 0.500

Pollen (R1) 1.00b1.00

b

1.00b

Forage (R4) 2.53 0.760 0.543

Grain (R6) 1.00b0.972 0.775

a

1507x59122 maize mean Cry35Ab1 protein concentration value was < Lower Limit of Quantification (LLOQ); therefore, the 

LLOQ was used.

b Mean PAT concentration values were < LLOQ; therefore, the LLOQ was used.
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3. Potential Allergenicity and Toxicity of 4114 Maize

The allergenicity and toxicity of 4114 maize was evaluated by examining the allergenic potential 

of maize as a crop and by assessing the allergenic and toxic potential of the introduced Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins. The proteins expressed in 4114 maize are identical to 

those in previously approved events 1507 and 59122 maize, as discussed in Section 1. As

described in Section 2, the equivalency of the proteins expressed in 4114 maize to those 

expressed in 1507 and 59122 maize was established by sequencing of the protein coding 

regions of the insertion and by western blot. These analyses demonstrated that the Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins introduced into 4114 maize are equivalent to those 

proteins expressed in 1507x59122 maize (Section 2 and Appendix 2). Therefore, previous 

safety studies conducted for 1507 and 59122 maize on the purified proteins are relevant for 

4114 maize. 

The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were derived from common soil bacteria and 

have a history of safe use in agricultural crop commodities. These proteins have been present 

in commercial maize varieties such as 1507, 59122, and 1507x59122 maize since 2003, 2006, 

and 2006, respectively; these commercial lines contain familiar traits and are currently licensed 

broadly across the seed industry and are planted on approximately 16% of U.S. maize acres (GfK 

Kynetec, 2010). In addition to these varieties the PAT protein has also been present in a 

number of other commercial crops and commercially planted in the U.S. since 1996.

The Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were previously determined to have little 

potential for toxicity or allergenicity based on bioinformatics analyses as well as toxicity and 

allergenicity studies. In this section, published or previously submitted information on the 

allergenicity and toxicity potential of these proteins is summarized and updated bioinformatics 

analyses on the proteins are included. 

3-A. Allergenicity of Maize

Although maize is widely grown worldwide with overall production at over 700 million tons per 

year and is the fourth most consumed food calorically, it is not considered a major allergenic 

food (Hefle et al., 1996; Moneret-Vautrin et al., 1998). In a few case studies, allergenic 

reactions were reported and maize allergens identified. Specifically, the 9 kDa Zea m 14 protein 

(i.e., the maize lipid transfer protein [LTP]) was identified as the major allergenic protein in 

maize (Fasoli et al., 2009; Pasini et al., 2002; Pastorello et al., 2000; Pastorello et al., 2003). 

LTPs are small proteins that facilitate the transfer of phospholipids and other lipids across 

membranes. These proteins are widely distributed throughout the plant kingdom and belong 

to the pathogenesis-related (PR) protein family (Hoffmann-Sommergruber, 2002). The Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins are neither related to LTPs nor involved in lipid transfer 

across membranes.

Because maize is not a common allergenic food, it is not expected that the genetic modification 

in 4114 maize would alter the allergenic potential of maize.
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3-B. Assessment of the Allergenicity of the Introduced Proteins in 4114 Maize

The allergenic potential of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins was previously 

assessed for 1507 and 59122 maize through a “weight-of-evidence” approach and provided 

support that the proteins were unlikely to be allergens; this evaluation of allergenicity also has 

relevance for 4114 maize because it contains the same introduced proteins. A weight-ofevidence approach takes into account a variety of relevant factors and experimental 

observations used to derive an overall assessment of the allergenic potential of the introduced 

protein, because no single factor has been recognized as the primary indicator for allergenic 

potential, and no validated animal model that is predictive of allergenic potential is available 

(Codex, 2003). The allergenicity potential assessments are therefore based on what is known 

about food allergens, including the amino acid sequence identity to known human allergens; 

physicochemical properties such as thermal stability or stability to pepsin or pancreatin 

digestion in vitro (Thomas et al., 2004); glycosylation status, and history of exposure and safety 

of the gene(s) source. 

An important part of the weight-of-evidence approach for assessing the potential allergenicity 

of proteins introduced and expressed in genetically modified plants is an amino acid sequence 

comparison of the introduced protein against the sequences of known allergens. This is 

performed to identify the degree of potential for cross-reactivity between allergenic proteins 

and the protein of interest. These evaluations employ two techniques; a search for contiguous, 

identical stretches of eight amino acid residues or longer, and an identity search using the 

FASTA35 alignment algorithm (Pearson and Lipman, 1988) to search for alignments of 80 amino 

acid residues or longer possessing a 35% sequence identity or greater (Codex, 2003; FAO/WHO, 

2001). If the introduced protein lacks both potential allergenic epitopes and identity to known 

allergens, the protein is “not a known allergen and is unlikely to be cross-reactive to known 

allergens” (Codex, 2003; Codex, 2009). 

The allergenic protein sequences used for all comparisons were from the peer-reviewed 

database from the Food Allergy Research and Resource Program (FARRP) at the Department of 

Food Science and Technology at the University of Nebraska at Lincoln (FARRP Release 11 -

January 2011; http://www.allergenonline.com), which contains 1491 entries representing both 

putative and proven food, environmental, and contact allergens as well as proteins implicated 

in celiac disease. 

Source and History of Safe Exposure to the Cry1F, Cry34Ab1, Cry35Ab1, and PAT Proteins

The Cry1F, Cry34Ab1, and Cry35Ab1 proteins were derived from Bacillus thuringiensis, which is 

a naturally-occurring, common bacterium that is not a known mammalian pathogen and is 

present in soil, dust, insects, and leaves (EPA, 1998; McClintock et al., 1995; Schnepf et al., 

1998). Some strains of B. thuringiensis have been shown to be opportunistic pathogens; 

however, this pathogenicity was not related to the Cry proteins (Hernandez et al., 1999). 

Microbial preparations of B. thuringiensis containing Cry proteins have been used safely as 
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pesticide sprays for decades, and have been deemed to pose no toxic effects to mammals (EPA, 

1998).

The PAT protein from Streptomyces viridochromogenes has a safe history of exposure to 

humans, animals, and the environment. The source organism S. viridochromogenes is 

widespread in soil and is not associated with human, animal, or plant pathogens (Hérouet et al., 

2005). Related PAT proteins are found in at least six other species of common soil bacteria, of 

which none have been reported as toxic or allergenic to humans or animals (Bartsch and Tebbe, 

1989; Hérouet et al., 2005; Kutzner, 1981). Furthermore, PAT has been a protein present in 

commercial maize events, as well as other crops, since 1996.

These proteins have also been present in commercial maize varieties such as 1507, 59122, and 

1507x59122 maize since 2003, 2006, and 2006, respectively. These commercial lines are 

currently licensed broadly across the seed industry; in 2010, commercial products containing 

1507x59122 maize were grown on approximately 14 million acres, which represents 

approximately 16% of U.S. maize acres (GfK Kynetec, 2010). Because of the broad use of 

1507x59122 maize in commercial planting and lack of reported adverse effects, there is a 

history of safe use and exposure of these proteins.

The Potential Allergenicity of the Cry1F Protein 

Previous work has established that the Cry1F protein does not have properties typically 

associated with allergens. The Cry1F protein was found to have no sequence homology with 

known allergens, to possess no N-glycosylation sites, to be rapidly degraded in simulated gastric 

fluid (SGF) and simulated intestinal fluid (SIF), and to be thermolabile (EPA, 2010a; Ladics et al., 

2006). 

The bioinformatics analysis was repeated to include protein allergens that have been added to 

the updated FARRP allergen database since the original analysis was conducted. When the 

Cry1F protein sequence was used as a query in the search for contiguous, identical stretches of 

amino acids 8 residues or greater in length, no 8 residue or greater matches to known or 

putative allergens were returned. Similarly, there were no alignments greater than 35% 

identity over 80 or greater amino acids. 

The updated bioinformatics analysis, the history of safe use of Cry1F, and studies 

demonstrating the lack of potential allergenicity, support the conclusion that the Cry1F protein 

is unlikely to be an allergen. 

The Potential Allergenicity of the Cry34Ab1 and Cry35Ab1 Proteins

Previous work has established that the Cry34Ab1 and Cry35Ab1 proteins do not have properties 

typically associated with allergens. These proteins were found to have no sequence homology 

with known allergens, to possess no N-glycosylation sites, and to be degraded in SGF (EPA, 

2010b).
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The bioinformatics analysis was repeated to include protein allergens that have been added to 

the updated FARRP allergen database since the original analyses were conducted. When the 

Cry34Ab1 and Cry35Ab1 protein sequences were used as a query in the search for contiguous, 

identical stretches of eight amino acids residues or greater in length, no eight residue or greater 

matches to known or putative allergens were returned. Similarly, there were no alignments 

that were greater than 35% identity over 80 or greater amino acids.

The updated bioinformatics analysis, in addition to the history of safe use of the Cry34Ab1 and 

Cry35Ab1 proteins and studies demonstrating the lack of potential allergenicity, support the 

conclusion that these proteins are unlikely to be allergens.

The Potential Allergenicity of the PAT Protein

Previous work has established that the PAT protein does not have properties typically 

associated with allergens (Hérouet et al., 2005). The PAT protein was found to have no 

sequence homology with known allergens, to possess no N-glycosylation sites, to be rapidly 

degraded in SGF and SIF, and to be thermolabile (Hérouet et al., 2005). 

The bioinformatics analysis of the PAT protein was repeated to take into account protein 

allergens that have been added to the updated FARRP allergen database since the original work 

took place. When the PAT protein sequence was used as a query in the search for contiguous, 

identical stretches of eight amino acids residues or greater in length, no eight contiguous amino 

acid residue or greater matches to known or putative allergens were returned. Similarly, there 

were no alignments that were greater than 35% identity over 80 or greater amino acids. 

The updated bioinformatics analysis, in addition to the history of safe use of PAT protein and 

studies demonstrating the lack of potential allergenicity support the conclusion that the PAT 

protein is unlikely to be an allergen.

3-C. Assessment of the Toxicity of the Proteins Expressed in 4114 Maize

The potential toxicity of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins was previously 

assessed for 1507 and 59122 maize by acute toxicity studies and by bioinformatic comparison 

of the protein sequences to publicly available protein sequences. These assessments provided 

evidence that the proteins are unlikely to be toxic and are also relevant for the assessment of

the proteins in 4114 maize. As described earlier, the Cry1F, Cry34Ab1, Cry35Ab1, and PAT 

proteins expressed in 4114 maize have been present in commercial maize lines such as 1507,

59122, and 1507x59122 maize for a number of years and represent a sizable portion of current 

maize acreage. To date, no known adverse reactions to these proteins have been reported. 

Updated bioinformatics analyses were conducted on the Cry1F, Cry34Ab1, Cry35Ab1, and PAT 

proteins to include proteins that have been added to public datasets since the original analyses 
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4114 Maize

were conducted. The amino acid sequences of each of the proteins were compared with 

publicly available protein sequences. Proteins most similar to those produced in 4114 maize 

were manually inspected to identify any that could be potentially toxic to humans or animals. A 

close match could be an indicator of toxicological potential of these proteins. 

To search for potential similarity to known toxins, the proteins were queried using the BLASTP 

2.2.13 algorithm against Release 183.0 (4/15/11) of the NCBI Entrez Protein dataset, which 

incorporates non-redundant entries from all GenBank and RefSeq nucleotide translations along 

with protein sequences from SWISS-PROT (http://www.expasy.org/sprot/), PIR (Protein 

Information Resource; http://pir.georgetown.edu/), PRF (Protein Research Foundation; 

http://www.prf.or.jp/index-e.html), and wwPDB (Worldwide Protein Data Bank; 

http://www.wwpdb.org/). The scoring matrix used was the default (BLOSUM62), low 

complexity filtering was turned off, and the number of alignments returned was set to the 

maximum value of 2000.

One of the most important parameters to monitor when performing similarity searches is the 

expectation score, or E score. This score represents the probability that a particular alignment 

is due to random chance and can be used to evaluate the significance of an alignment. The 

calculated E score depends on the overall length of the aligned sequences (including inserted 

gaps), the number of identical and conserved residues within the alignment, and the size of the 

database (Baxevanis, 2005; Pearson and Lipman, 1988). When examining an alignment 

between two protein sequences, a very small E score is more likely to reflect a true similarity, 

while a large E score is more likely to be produced by chance and therefore less biologically 

relevant.

A cutoff expectation (E) value of 1.0 was used to generate biologically meaningful similarity 

between the protein of interest and proteins in the NCBI datasets. Although a statistically 

significant sequence similarity generally requires a match with an expectation value less than 

0.01, a cutoff of E < 1.0 insures that proteins with even limited similarity will not be overlooked 

in the search (Pearson, 2000). 

The Potential Toxicity of the Cry1F Protein 

Previous work has established that the Cry1F protein does not have properties typically 

associated with toxins. Cry1F was found to lack sequence homology with known toxins and was 

not found to be acutely toxic in mice (EPA, 2010a).

In the updated bioinformatics analysis, the Cry1F protein sequence returned 544 protein 

accessions with an E score less than 1.0. Six accessions possessed virtually complete identity 

(E = 0); the top scoring alignment was to the originally isolated Cry1F protein (Chambers et al., 

1991) at 99% identity while the remaining 5 accessions were to closely related Cry1F-like 

proteins from Bacillus thuringiensis. Of the remaining 538 alignments returned, 465 are from 

the soil bacterium Bacillus thuringiensis, including 19 accessions classified as hypothetical or 

unnamed. These proteins are known by a variety of descriptions (parasporal crystal protein, 
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4114 Maize

crystal protein, insecticidal protein, pesticidal crystal protein, insecticidal delta-endotoxin, 

crystalline entomocidal protoxin, crystal protein, insecticidal toxin, and delta-endotoxin, among 

others), and represent a class of highly specific insect toxins with a well characterized mode of 

action that are considered safe for humans and animals (Bravo et al., 2007). Twenty-nine 

additional matches are to synthetic constructs, consisting primarily of transformation vectors 

containing intact, truncated, or chimeric versions of the Cry proteins mentioned above. The 

remaining 44 accessions represent Cry-related protein sequences from other Bacillus families, 

or translations of nucleotide sequences generated during genome sequencing projects. None 

of the protein sequences returned by the BLASTP search identified safety concerns that might 

arise from the expression of Cry1F in genetically modified plants.

The updated bioinformatics analysis, in addition to the history of safe use of Cry1F and studies 

demonstrating the lack of potential toxicity, support the conclusion that the Cry1F protein is 

unlikely to be a toxin to humans or animals. 

The Potential Toxicity of the Cry34Ab1 and Cry35Ab1 Proteins

Previous work has established that the Cry34Ab1 and Cry35Ab1 proteins do not have properties 

typically associated with toxins. The Cry34Ab1 and Cry35Ab1 proteins were found to not be 

acutely toxic in mice (EPA, 2010b; Juberg et al., 2009). Furthermore, earlier submitted 

bioinformatic analysis showed the proteins did not share sequence homology with any known 

protein toxins (EPA, 2010b).

In the updated bioinformatics analysis, the Cry34Ab1 protein sequence returned 41 protein 

accessions with an expectation (E) score less than 1.0. The top 16 accessions are derived from 

Bacillus thuringiensis (Bt). The two highest scoring accessions had identities of 100%; one is 

listed as a “13.6 kDa insecticidal crystal protein” while the other is annotated as a “Cry34Ab1-

like” protein. The fourteen other accessions possess a variety of descriptions (crystal protein, 

insecticidal crystal protein, Cry34Ab1-like protein, among others), and represent a class of 

highly specific insect toxins with a well characterized mode of action that are considered safe 

for humans and animals (Bravo et al., 2007). 

The next highest scoring sequence (31% sequence identity) is to a recently added (March 2011) 

putative protein translation from the Streptomyces whole genome sequencing project (RefSeq 

accession ZP_08234223) that is described as an “aegerolysin”; this sequence has a significant 

match to the Pfam aegerolysin domain (PF06355) (NCBI, 2011). Sequences from these types of 

projects are not peer reviewed for annotation accuracy; while they may possess domain 

similarities to certain classes of proteins, they often have unconfirmed function (NCBI, 2009; 

NCBI, 2011). In addition, this match is not surprising given that three Cry34Ab1-like protein 

sequences were used as a scaffold to define the Pfam aegerolysin domain (NCBI, 2011).

Aegerolysins were originally isolated from edible mushrooms and are associated with 

primordial and fruiting body formation; these proteins have also been isolated from bacteria 

and plants (Berne et al., 2002; Berne et al., 2009). Only certain fungal aegerolysins have been 
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shown to have hemolytic properties (Berne et al., 2002). While proteins may be described as 

aegerolysins and share the aegerolysin domain, many of these proteins possess diverse 

biological functions unrelated to hemolytic properties (Berne et al., 2009). The few known 

hemolytic fungal aegerolysins include Aa-Pri1 from Agrocybe aegerita (i.e., edible poplar or 

chestnut mushroom; Fernandez and Labarère, 1997), pleurotolysin A and ostreolysin from 

Pleurotus ostreatus (i.e., edible oyster mushroom; Berne et al., 2005; Sakurai et al., 2004), and 

Asp-hemolysin from Aspergillus fumigatus (Ebina et al., 1994). A CLUSTALW alignment was 

used to further compare the similarity of Cry34Ab1 and the ZP_08234223 accession with these 

sequences. Both Cry34Ab1 and the ZP_08234223 accession show very limited identity (13-20%) 

to any of these known fungal toxins, while the toxin sequences are more homologous to each 

other (41-80% identity). Furthermore, none of these known fungal toxins appeared in the 

Cry34Ab1 BLAST search. Therefore, it is unlikely that Cry34Ab1 possesses the toxic properties 

of the known fungal aegerolysins.

Of the remaining 24 sequences, 10 represent insecticidal crystal proteins from non-Bt species, 

while 12 others are annotated as hypothetical or predicted proteins derived from the 

translations of nucleotide sequences generated during genome sequencing projects. The two 

remaining sequences are low significance matches, one annotated as a beta-Nacetylglucosaminidase and the other a lysophospholipase Plb1. None of the protein sequences 

returned by the BLASTP search identified safety concerns that might arise from the expression 

of Cry34Ab1 protein in genetically modified plants.

The Cry35Ab1 protein sequence returned 45 protein accessions with an expectation (E) score 

less than 1.0. Nine accessions possessed an E score of 0; all 9 were derived from Bacillus 

thuringiensis. The top scoring alignment, a “43.8 kDa insecticidal crystal protein”, displayed 

100% identity over the entire length of the Cry35Ab1 sequence. There were an additional 

seven accessions from Bacillus thuringiensis, and 19 accessions displaying similarity to the 

mosquitocidal Bin toxins from Lysinibacillus sphaericus (synonym: Bacillus sphaericus) and

Bacillus cereus (Baumann et al., 1988; Baumann et al., 1991; reviewed by Federici et al., 2003). 

Of the remaining 10 sequences, six sequences of low significance (E=0.053-0.45) represent 

translations of nucleotide sequences generated during genome sequencing projects and four 

accessions are described as hypothetical proteins. None of the protein sequences returned by 

the BLASTP search identified safety concerns that might arise from the expression of the 

Cry35Ab1 protein in genetically modified plants.

The updated bioinformatics analysis, in addition to the history of safe use of the Cry34Ab1 and 

Cry35Ab1 proteins and studies demonstrating the lack of potential toxicity, support the 

conclusion that these proteins are unlikely to be toxins to humans or animals. 

The Potential Toxicity of the PAT Protein

Previous work has established that the PAT protein does not have properties typically 

associated with toxins. PAT was found to lack sequence homology with known toxins and were 

found not to be acutely toxic in mice (Hérouet et al., 2005). 
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In the updated bioinformatics analysis, the PAT protein sequence returned 1752 protein 

accessions with an expectation (E) score less than 1.0. The highest scoring alignments (E = 1 x 

10-105 and 1 x 10-88, respectively) were attributed to PAT proteins from Streptomyces 

viridochromogenes (100% identity over the entire 183 amino acid length of the query 

sequence) and Streptomyces hygroscopicus (84% identity to the query sequence). Both of 

these proteins have undergone extensive safety evaluations and have been deemed safe for 

expression in transgenic plants (Hérouet et al., 2005). Seven other accessions represented 

sequences derived from these two bacterial strains. 

Twelve of the alignments were to synthetic constructs, consisting primarily of transformation 

vectors containing portions of the PAT proteins mentioned above. Of the remaining 1731 

sequences, 518 were annotated as phosphinothricin acetyltransferases. This bacterial enzyme 

deactivates the non-selective herbicide glufosinate, which is a potent inhibitor of glutamine 

synthesis (Dröge-Laser et al., 1994) and is widely used for the generation of glufosinate-tolerant

transgenic plants. Another 861 alignments are with sequences characterized as known or 

putative acetyltransferases, N-acetyltransferases, or GCN5 or GNAT acetyltransferases, based 

upon the possession of an acetyltransferase domain present in the PAT query sequence. One 

hundred and thirty-seven other accessions were classified as acetyltransferase related 

sequences including sortases, acyltransferases, antibiotic or toxin resistance proteins, 

ribosomal-protein-alanine acetyltransferases, or histone acetyltransferases. Ten other 

accessions were annotated as ArsR family transcriptional regulators, containing both the 

Arsenical Resistance Operon Repressor domain as well as the acetyltransferase domain. 

Fourteen accessions described as possessing a range of functionalities were returned; all of 

these sequences contained additional domains responsible for their annotations. Lastly, 191 of 

the accessions returned were classified as hypothetical, predicted, putative, unnamed, or 

unknown proteins. None of the protein sequences returned by the BLASTP search identified 

any safety concerns from the expression of PAT protein in genetically modified plants.

The updated bioinformatics analysis, in addition to the history of safe use of the PAT protein 

and studies demonstrating the lack of potential toxicity, support the conclusion that this 

protein is unlikely to be a toxin.

3-D. Overall Conclusions on the Potential Allergenicity and Toxicity of 4114 Maize

The potential for allergenicity and toxicity of 4114 maize was evaluated by examining the 

allergenic potential of maize as a crop and by assessing the allergenic and toxic potential of the 

Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins. Maize is not a common allergenic food and the 

modification in 4114 maize is not expected to alter the allergenic potential of maize. The Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins have been assessed previously for 1507 and 59122 

maize and have been determined to be unlikely to be potential allergens or toxins to humans 

and animals. Previous assessments of these proteins included bioinformatic analyses,

digestibility, N-glycosylation studies, acute protein toxicity studies, and in some cases, heat 

stability studies; these studies are relevant for the assessment of the proteins in 4114 maize.
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Updated bioinformatic analyses support the original conclusions that the Cry1F, Cry34Ab1, 

Cry35Ab1, and PAT proteins are unlikely to be allergens or toxins. Additionally, 1507x59122 

maize has been commercially available in the U.S. since the 2006 growing season with no 

reports of allergenicity or toxicity. These data support the conclusion that the Cry1F, Cry34Ab1, 

Cry35Ab1, and PAT proteins in 4114 maize are unlikely to be potential allergens or toxins to 

humans and animals, and therefore support the food and feed safety of these proteins. These 

data have been provided to EPA in support of the safety of these proteins and also will be 

submitted to the FDA for the food and feed safety assessment of 4114 maize.
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4. Comparative Assessment of 4114 Maize

In order to assess the safety of 4114 maize relative to conventional maize, a comparative 

assessment was conducted that was designed to be independent of the intended effects of the

insect-resistant and herbicide-tolerant traits. The comparative assessment uses nontransformed (i.e., conventional) comparators for compositional and agronomic comparisons 

and will establish the safety of 4114 maize relative to varieties that have a history of safe use in 

the environment and as food and feed. The objective is to determine if the modified organism 

presents any new or greater risks relative to its comparator, or whether it can be used 

interchangeably with its comparator without negatively affecting human and animal health and

the environment in which it is grown. The compositional and agronomic analyses will highlight 

any differences between 4114 maize and its comparators in order to determine if 4114 maize is 

as safe as other lines that have a history of safe use. The studies were designed to eliminate 

the intended effects of the introduced traits (i.e., trait efficacy) so that appropriate comparisons

would be able to be made between 4114 maize and conventional maize and unintended effects 

due to the 4114 maize insertion would also be evaluated. Therefore, the receiving environment 

was managed to minimize insect and weed pressure in the evaluations.

To ensure an accurate comparative evaluation of the transgenic plant, a proper selection of 

comparator plants is important. As described earlier in Section 2, non-transgenic near-isoline 

maize lines were selected as comparators for 4114 maize in these analyses, since they had a 

similar genetic background (i.e., approximately 99% similar). Statistical comparisons were 

made to these comparators to determine if there were any significant differences. In order to 

determine if any observed statistical differences were indicative of differences with 

conventional maize varieties, a statistical tolerance interval was established from nontransgenic commercial maize lines for the compositional and agronomic comparisons. These 

commercial lines are typical of those grown in maize-growing regions; represent a wide range 

of varieties that would normally be planted commercially; and represent the normal range of 

variation of the maize crop. In addition, for the compositional assessment, publicly available 

information was gathered on the range of natural variation of maize analyte concentrations

(i.e., literature range). If the measured values of 4114 maize fell within the statistical tolerance 

interval or the literature range, then these measured values would be considered comparable 

to conventional maize.

The compositional and agronomic comparative assessments of 4114 maize are discussed 

further below. These analyses indicated that 4114 maize was comparable to conventional 

maize with respect to the compositional analytes and agronomic characteristics measured and 

independently of the intended effects of the insect-resistant and herbicide-tolerant traits. 

Overall, these analyses indicate that 4114 maize is as safe as conventional maize varieties and 

does not pose a greater risk than conventional maize varieties in food, feed, and the 

environment.
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4114 Maize

4-A. Compositional Assessment

Compositional analysis of 4114 maize grain and forage was used to evaluate any changes in the 

levels of key nutrients, anti-nutrients, and secondary metabolites compared to the nontransformed, near-isoline control. For this analysis, grain was selected because of its use in 

both food and feed, and forage was selected for its use as feed. These analyses provided an 

indication whether 4114 maize is as nutritious as conventional maize. The U.S. FDA will review 

the details of the compositional analyses as a component of the food and feed safety 

assessment of 4114 maize.

Comprehensive compositional analyses were performed on grain and forage from 4114 maize 

and a near-isoline control grown in 2010 at six field locations in the U.S. and Canada (see 

Section 4-B; Table 28; Experiment B). These sites are representative of commercial maizegrowing areas. The F1*5

generation of seed (Section 2; Figure 10 and Table 3) was planted

because F1 hybrid seed is representative of seed that growers would plant in commercial maize 

fields. The near-isoline control plants had a genetic background approximately 99% similar to 

that of the 4114 maize generation used, but did not go through the transformation process. 

Although 4114 maize contains traits that are efficacious against certain lepidopteran and corn 

rootworm target pests and glufosinate-ammonium tolerance, each field trial site was managed 

to maintain a relatively insect-free and weed-free environment. Evaluation in this environment 

ensured that compositional characteristics measured would be independent of the intended 

effects of the insect-resistant and herbicide-tolerant traits; therefore, these evaluations would 

appropriately compare 4114 maize to conventional maize and also assess unintended effects of 

the transgene. Composition was not impacted by the intended effects of the traits and 

therefore could be used to assess comparability of 4114 maize to conventional maize. 

Each location utilized a randomized complete block design containing four blocks. Each block 

contained 4114 maize and the control maize planted in two-row plots. One forage sample (subsample of 3 whole plants) was collected at the R4 growth stage from 4114 maize and the 

control maize from each of the four blocks. Forage samples were obtained by cutting the aerial 

portion of the plants from the root system approximately 1 inch (2.5 cm) above the soil surface 

after secondary or tertiary ears with exposed silks were removed. The plants (including the 

primary ear) were then cut into sections approximately 3 inches (7.5 cm) or less, and 

approximately 1/3 of the total sample was collected in a pre-labeled plastic-lined cloth bag. 

One grain sample (equal to five pooled ears) was collected at the R6 growth stage from 4114 

maize and the control maize from each of the four blocks. All samples were collected from 

impartially selected, healthy individual plants that had previously been self-pollinated.

Forage and grain were also collected from non-modified commercial maize hybrids (reference 

hybrids) in two separate experiments. Pioneer® hybrids 34M94, 33G26, 33J24, and 3394 were 

analyzed in 2003 at six field locations in maize-growing areas of North America (Bagley, IA; York, 

NE; Chula, GA; New Holland, OH; Larned, KS and Hereford, PA). Pioneer® hybrids 38B85, 37Y12, 

34A15, and 34P88 were analyzed in 2007 at six field locations in maize-growing areas of North 
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4114 Maize

America (Tallahassee, FL; York, NE; Germansville, PA; Richland, IA; Larned, KS and Branchton, 

Ontario). These Pioneer commercial products were chosen to represent a wide range of nongenetically modified varieties that would normally be planted commercially. 

For the reference hybrid trials, each location utilized a randomized complete block design 

containing three blocks. Each block contained two-row plots. Procedures for planting, 

harvesting, processing, and compositional analysis of the reference hybrid trials were similar to 

those employed for the trials containing near-isoline control and 4114 maize. Composition data 

collected from the reference hybrids was used to help determine the normal variation for the 

measured analytes in commercial maize.

The analytes for compositional assessment were selected considering the OECD consensus 

document on compositional considerations for new varieties of maize (OECD, 2002). 

Compositional analyses of grain samples included protein, fat, acid detergent fiber (ADF), 

neutral detergent fiber (NDF), ash, carbohydrates, fatty acids, amino acids, vitamins and 

minerals, key anti-nutrients, and key secondary metabolites. Compositional analyses of forage 

samples included protein, fat, ADF, NDF, ash, carbohydrates, calcium, and phosphorus.

Statistical analysis of nutrient composition data was conducted to test for differences in the 

analyte mean values between 4114 maize and the control. (For details of the statistical 

methodology, refer to Appendix 6). When numerous analytes are being evaluated on the same 

samples, controlling false positive outcomes is important. Since the introduction of the false 

discovery rate (FDR) approach in the mid-1990’s, it has been widely employed across a number 

of scientific disciplines, including genomics, ecology, medicine, plant breeding, epidemiology, 

dairy science, and signal/image processing (e.g., Pawitan et al., 2005; Spelman and Bovenhuis, 

1998). A false positive outcome occurs when an analyte mean of the transgenic line is deemed 

significantly different from the analyte mean of the control line, when in fact the two means are 

not different. If one uses a 5% type I error rate for each analyte, then the number of false 

positives increases as the number of analytes increase. In order to help manage the false 

positive rate, the false discovery rate (FDR) method of Benjamini and Hochberg was applied to 

account for making multiple comparisons (Benjamini and Hochberg, 1995; Westfall et al., 

1999). P-values were adjusted accordingly, resulting in the false positive rate being held to 5%. 

Both adjusted and non-adjusted P-values are reported for the analyses that follow. A

significant difference between the mean of 4114 maize and that of the control line was 

established with an FDR-adjusted P-value <0.05.

Using the data obtained from the reference hybrids, a statistical tolerance interval was 

calculated to contain, with 95% confidence, 99% of the values contained in the population of 

commercial maize. This statistical tolerance interval and the combined range of values for each 

analyte from the published literature, where available, provided further context for 

interpretation of the composition results for 4114 maize. 4114 maize analyte ranges that fell 

within the tolerance interval and/or combined literature range for that analyte were considered 

to be within the range of normal variability of commercial maize hybrids.
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4114 Maize

Key Nutrients in Maize Grain

As described in Section 1-E, maize grain is used in the U.S. for food, feed, and fuel. 

Approximately 38.7% of maize grown in the U.S. is used domestically for animal feed (primarily 

beef, poultry, pork and dairy) because of its high nutrient value and relative low cost (NCGA, 

2011). Approximately 10.1% of maize grain is used for food products, with 3.8% going to high 

fructose corn syrup, and 6.3% comprising beverage alcohol, cereal, other sweeteners, and 

starch (NCGA, 2011). A significant portion of maize is used for fuel ethanol (36.5%) with the 

remainder being exported (14.5%) (NCGA, 2011).

Proximates in Maize Grain

Proximates were analyzed in 4114 maize and near-isoline control grain. Results are shown in 

Table 15. No statistically significant differences were observed between the 4114 maize and 

control mean values for crude protein, crude fat, crude fiber, acid detergent fiber (ADF), neutral 

detergent fiber (NDF), and carbohydrates (adjusted P-values > 0.05). A statistically significant 

difference was observed between the 4114 maize and control mean values for ash, however all 

of the individual values were within the tolerance interval.

In conclusion, analysis of proximates in maize grain demonstrates that 4114 maize is 

comparable to near-isoline and reference maize hybrids.
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4114 Maize

Table 15. Proximates in Maize Grain

Analyte (% Dry Weight) Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Crude Protein

Meanb10.2 10.4

6.59 - 13.5 6.00 - 17.3

Rangec8.92 - 11.4 7.32 - 11.7

CId9.54 - 10.7 9.78 - 10.9

Adjusted P-Valuee0.789

P-Valuef0.455

Crude Fat

Mean 4.81 4.68

1.45 - 5.75 2.47 - 5.90

Range 4.28 - 5.73 3.88 - 5.67

CI 4.47 - 5.17 4.35 - 5.04

Adjusted P-Value 0.679

P-Value 0.177

Crude Fiber

Mean 2.57 2.48

0.941 - 3.73 0.490 -

5.50

Range 1.36 - 3.38 1.34 - 3.16

CI 2.35 - 2.80 2.27 - 2.70

Adjusted P-Value 0.789

P-Value 0.520

ADF

Mean 3.83 4.01

1.43 - 5.73 1.82 - 11.3

Range 3.24 - 4.48 3.53 - 5.16

CI 3.56 - 4.12 3.72 - 4.32

Adjusted P-Value 0.679

P-Value 0.0965

NDF

Mean 10.4 10.4

5.75 - 20.6 5.59 - 22.6

Range 9.39 - 11.3 9.58 - 11.2

CI 9.97 - 10.8 9.93 - 10.8

Adjusted P-Value 0.908

P-Value 0.824

Ash

Mean 1.33 1.44

0.531 - 2.16 0.616 -

6.28

Range 1.12 - 1.52 1.30 - 1.60

CI 1.29 - 1.38 1.39 - 1.48

Adjusted P-Value 0.0498g

P-Value 0.00298

Carbohydrates

Mean 83.7 83.6

80.3 - 89.7 77.4 - 89.5

Range 82.5 - 84.9 82.2 - 85.7

CI 83.2 - 84.2 83.1 - 84.1

Adjusted P-Value 0.908

P-Value 0.758

a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 

1982; Watson, 1987).

b Least squares mean

c Range denotes the lowest and highest individual value across locations.

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f Non-adjusted P-value

g

Statistically significant difference, adjusted P-value < 0.05
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4114 Maize

Vitamins and Minerals in Maize Grain

Maize is a nutritional source of vitamins and minerals for both humans and animals; therefore 

vitamins and minerals were measured in 4114 maize and near-isoline control grain. Based on 

OECD guidance, the following vitamins were analyzed: beta-carotene, vitamin B1 (thiamin), 

vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B6 (pyridoxine), vitamin B9 (folic acid), and 

vitamin E (α-tocopherol) (OECD, 2002). The following minerals were also analyzed, based on 

OECD guidance: calcium, copper, iron, magnesium, phosphorus, potassium, sodium and zinc 

(OECD, 2002).

Vitamin results are shown in Table 16 and mineral results are shown in Table 17. No 

statistically significant differences were observed between the 4114 maize and control mean 

values for any of the vitamins analyzed (adjusted P-values were > 0.05). For the minerals, there 

were no statistical differences for all of the analytes except phosphorus and potassium, 

however all individual data points were within the respective tolerance intervals.

In conclusion, vitamin and mineral analyses of maize grain demonstrate that 4114 maize is 

comparable to near-isoline and reference maize hybrids.
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4114 Maize

Table 16. Vitamins in Maize Grain

Analyte

(mg/kg Dry Weight)

Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Beta-Carotene

Meanb16.4 18.1

0 - 68.3 0.190 -

46.8

Rangec7.27 - 26.2 11.7 - 26.4

CId12.4 - 21.7 13.7 - 24.0

Adjusted P-Valuee0.679

P-Valuef0.267

Vitamin B1 

(Thiamine)

Mean 1.97 2.20

0.414 -

6.64 1.26 - 40.0

Range <1.80g - 2.92 <1.80g - 3.28

CI 1.44 - 2.70 1.62 - 2.99

Adjusted P-Value 0.679

P-Value 0.168

Vitamin B2 

(Riboflavin)

Mean <0.900g<0.900g

NCh 0.250 -

5.60

Range <0.900g<0.900g

CI NAi NAi

Adjusted P-Value NAi

P-Value NAi

Vitamin B3 

(Niacin)

Mean 13.9 13.8

0 - 51.7 9.30 - 70.0

Range 10.6 - 18.0 11.7 - 16.6

CI 12.8 - 15.1 12.7 - 15.0

Adjusted P-Value 0.908

P-Value 0.786

Vitamin B6 

(Pyridoxine)

Mean 4.68 4.29

1.83 - 11.1 3.68 - 11.3

Range 2.35 - 6.88 2.50 - 6.36

CI 3.60 - 5.90 3.26 - 5.46

Adjusted P-Value 0.772

P-Value 0.404

Vitamin B9

(Folic Acid)

Mean 0.872 0.792

0 - 2.30 0.147 –

683

Range 0.181 - 1.91 0.0369 - 1.55

CI 0.656 - 1.09 0.577 - 1.01

Adjusted P-Value 0.789

P-Value 0.523
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4114 Maize

Table 16. Vitamins in Maize Grain (continued)

Analyte

(mg/kg Dry Weight)

Control

(n=24)

4114 maize

(n=24)

Tolerance

Interval

Literature

Rangea

Vitamin E

(α-Tocopherol)

Mean 5.47 5.68

2.18 - 28.2 1.50 - 68.7

Range 2.09 - 13.3 2.14 - 14.8

CI 3.06 - 9.75 3.18 - 10.1

Adjusted P-Value 0.908

P-Value 0.672

a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 

2002; Watson, 1982; Watson, 1987).

b Least squares mean

c Range denotes the lowest and highest individual value across locations

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f Non-adjusted P-value

g <Lower limit of quantification (LLOQ); indicates that sample value(s) were detected below the assay LLOQ. 

Sample results which were below the LLOQ were assigned a value equal to the LLOQ for statistical analysis

h Tolerance interval not calculated (NC)

i Statistical analysis not available (NA)
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4114 Maize

Table 17. Minerals in Maize Grain

Analyte (% Dry Weight)

Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Calcium

Meanb0.00325 0.00335

0.00127 -

0.00902

0.00127 -

0.100

Rangec0.00270 - 0.00399 0.00251 - 0.00448

CId0.00288 - 0.00367 0.00296 - 0.00378

Adjusted P-Valuee0.679

P-Valuef0.308

Copper

Mean 0.0000645 0.0000653

0 -

0.000662

0.0000730 -

0.00185

Range <0.0000625g 

-

0.0000869

<0.0000625g -

0.0000876

CI NAhNAh

Adjusted P-Value NAh

P-Value NAh

Iron

Mean 0.00168 0.00166

0.000857 -

0.00269

0.000100 -

0.0100

Range 0.00146 - 0.00228 0.00147 - 0.00210

CI 0.00156 - 0.00181 0.00155 - 0.00179

Adjusted P-Value 0.908

P-Value 0.775

Magnesium

Mean 0.133 0.135

0.0381 -

0.195 0.0594 - 1.00

Range 0.103 - 0.155 0.112 - 0.157

CI 0.121 - 0.146 0.123 - 0.149

Adjusted P-Value 0.679

P-Value 0.251

Phosphorus

Mean 0.289 0.311

0.127 -

0.472 0.147 - 0.750

Range 0.258 - 0.334 0.296 - 0.330

CI 0.278 - 0.301 0.298 - 0.324

Adjusted P-Value 0.0115i

P-Value 0.000353

Potassium

Mean 0.387 0.416

0.194 -

0.687 0.181 - 0.720

Range 0.343 - 0.441 0.385 - 0.478

CI 0.373 - 0.401 0.401 - 0.431

Adjusted P-Value 0.0498i

P-Value 0.00306

Sodium

Mean 0.0000582 0.0000628

0 - 0.00207 0 - 0.150

Range <0.0000625g 

-

0.000489

<0.0000625g -

0.000171

CI 0.0000409 -

0.0000828

0.0000447 -

0.0000882

Adjusted P-Value 0.908

P-Value 0.714
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4114 Maize

Table 17. Minerals in Maize Grain (continued)

Analyte (% Dry Weight) Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Zinc

Mean 0.00174 0.00174

0.00104 -

0.00271

0.000650 -

0.00372

Range 0.00150 - 0.00203 0.00149 - 0.00201

CI 0.00162 - 0.00188 0.00161 - 0.00188

Adjusted P-Value 0.946

P-Value 0.919

a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 

2002; Watson, 1982; Watson, 1987).

b Least squares mean

c Range denotes the lowest and highest individual value across locations.

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f Non-adjusted P-value

g <Lower limit of quantification (LLOQ); indicates that sample value(s) were detected below the assay LLOQ. 

Sample results which were below the LLOQ were assigned a value equal to the LLOQ for statistical analysis.

h Statistical analysis not available (NA)

i 

 Statistically significant difference, adjusted P-value < 0.05
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4114 Maize

Fatty Acids in Maize Grain

The oil in maize consists mostly of five major fatty acids: palmitic (C16:0), stearic (C18:0), oleic

(C18:1), linoleic (C18:2), and linolenic (C18:3) (OECD, 2002). Other fatty acids are present, but 

usually fall below 1% of total fatty acids (OECD, 2002). The complete fatty acid profile was 

measured in seed samples derived from 4114 maize, and compared with corresponding values 

from samples of control maize.

Results of the fatty acid analysis are shown in Table 18. The concentrations of the following 

fatty acids were below the lower limit of quantification for both 4114 maize and control and are 

not reported in Table 18: Caprylic Acid (C8:0); Capric Acid (C10:0); Lauric Acid (C12:0); Myristic 

Acid (C14:0); Myrisoleic Acid (C14:1); Pentadecanoic Acid (C15:0); Pentadecenoic Acid (C15:1); 

Heptadecenoic Acid (C17:1); γ- Linolenic Acid (C18:3); Nonadecanoic Acid (C19:0); 

Eicosatrienoic Acid (C20:3); Arachidonic Acid (C20:4); Heneicosanoic Acid (C21:0); Erucic Acid 

(C22:1); and Tricosanoic Acid (C23:0). 

A small but statistically significant difference (adjusted P-value was < 0.05) was observed in the 

concentration of eicosenoic acid between 4114 maize and control samples, however all 

individual data points for 4114 maize were within the tolerance interval. No statistically 

significant differences were observed between the 4114 maize and near-isoline control for any 

of the other fatty acid mean values (adjusted P-values were > 0.05).

In conclusion, fatty acid analysis of maize grain demonstrates that 4114 maize is comparable to 

near-isoline and reference maize hybrids.
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4114 Maize

Table 18. Fatty Acids in Maize Grain

Analyte (% Total Fatty Acids)a Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangeb

Palmitic Acid 

(C16:0)

Meanc14.1 14.2

5.51 - 18.4 7.00 - 20.7

Ranged13.2 - 15.6 13.1 - 16.2

CIe13.6 - 14.5 13.7 - 14.7

Adjusted P-Valuef0.789

P-Valueg0.518

Palmitoleic Acid 

(C16:1)

Mean 0.124 0.139

0 - 0.337 0 - 1.00

Range 0.0948 - 0.144 0.125 - 0.156

CI NAhNA

Adjusted P-Value NA

P-Value NA

Heptadecanoic 

Acid (C17:0)

Mean 0.0940 0.100

0 - 0.189 0 - 0.111

Range 0.0940 - 0.0940 0.100 - 0.100

CI NA NA

Adjusted P-Value NA

P-Value NA

Heptadecadienoic 

Acid (C17:2)

Mean 0.307 0

NCiNRj

Range 0.307 - 0.307 0 – 0

CI NA NA

Adjusted P-Value NA

P-Value NA

Stearic Acid 

(C18:0)

Mean 1.60 1.60

0.566 - 4.67 0 - 4.00

Range 1.45 - 2.07 1.37 - 2.14

CI 1.46 - 1.75 1.47 - 1.75

Adjusted P-Value 0.908

P-Value 0.810

Oleic Acid (C18:1)

Mean 22.6 22.1

10.4 - 65.6 17.4 - 50.0

Range 20.7 - 25.8 20.3 - 23.8

CI 21.5 - 23.8 21.0 - 23.2

Adjusted P-Value 0.0817

P-Value 0.00629

Linoleic Acid 

(C18:2)

Mean 60.1 60.5

30.4 - 81.7 34.0 - 70.0

Range 54.9 - 62.9 56.3 - 63.5

CI 58.4 - 61.8 58.8 - 62.2

Adjusted P-Value 0.679

P-Value 0.203

(9,15) Isomer of 

Linoleic Acid 

(C18:2)

Mean 0 0.249

0 - 6.92 NR

Range 0 - 0 0.240 - 0.258

CI NA NA

Adjusted P-Value NA

P-Value NA
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4114 Maize

Table 18. Fatty Acids in Maize Grain (continued)

Analyte (% Total Fatty Acids)a Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangeb

Linolenic Acid 

(C18:3)

Mean 0.787 0.770

0 - 3.34 0 - 2.25

Range 0.410 - 1.22 0.305 - 1.12

CI 0.576 - 1.08 0.563 - 1.05

Adjusted P-Value 0.908

P-Value 0.697

Arachidic Acid 

(C20:0)

Mean 0.392 0.394

0.159 -

0.849 0 - 2.00

Range 0.343 - 0.505 0.347 - 0.481

CI 0.359 - 0.428 0.361 - 0.430

Adjusted P-Value 0.789

P-Value 0.518

Eicosenoic Acid 

(C20:1)

Mean 0.235 0.265

0.213 -

0.370

0 - 1.92

Range 0.204 - 0.255 0.229 - 0.336

CI 0.224 - 0.246 0.253 - 0.278

Adjusted P-Value 0.00472k

P-Value <0.0001

Eicosadienoic Acid 

(C20:2)

Mean 0.275 0

0 - 0.351l0 - 0.533

Range 0.275 - 0.275 0 – 0

CI NA NA

Adjusted P-Value NA

P-Value NA

Behenic Acid 

(C22:0)

Mean 0 0.291

0 - 0.566 0 - 0.500

Range 0 - 0 0.291 - 0.291

CI NA NA

Adjusted P-Value NA

P-Value NA
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4114 Maize

Table 18. Fatty Acids in Maize Grain (continued)

Analyte (% Total Fatty Acids)a Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangeb

Lignoceric Acid 

(C24:0)

Mean 0.240 0.233

0 - 0.675 0 - 0.500

Range 0.185 - 0.318 0.211 - 0.276

CI 0.205 - 0.280 0.200 - 0.273

Adjusted P-Value 0.908

P-Value 0.796

a

The concentrations of the following fatty acids were below the lower limit of quantification for both 4114 maize 

and control and are not reported here: Caprylic Acid (C8:0); Capric Acid (C10:0); Lauric Acid (C12:0); Myristic Acid 

(C14:0); Myrisoleic Acid (C14:1); Pentadecanoic Acid (C15:0); Pentadecenoic Acid (C15:1); Heptadecenoic Acid 

(C17:1); γ- Linolenic Acid (C18:3); Nonadecanoic Acid (C19:0); Eicosatrienoic Acid (C20:3); Arachidonic Acid 

(C20:4); Heneicosanoic Acid (C21:0); Erucic Acid (C22:1); and Tricosanoic Acid (C23:0).

b Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 

2002; Watson, 1982; Watson, 1987).

c Least squares mean

d Range denotes the lowest and highest individual value across locations.

e 95% Confidence Interval

f False Discovery Rate (FDR) adjusted P-value

g Non-adjusted P-value

h

Statistical analysis not available (NA)

i

A tolerance interval could not be calculated (NC)

j

 Analyte ranges were not reported (NR) in the published literature references

k

 Statistically significant difference, adjusted P-value < 0.05

l

 No tolerance interval could be calculated for this analyte, and the listed interval is the min/max range.
Pioneer Hi-Bred International, Inc. Page 114 of 258

4114 Maize

Total Amino Acids in Maize Grain

Maize grain is generally a good source of essential and non-essential amino acids for most 

domestic animal species. Total levels of 18 amino acids were measured in 4114 maize and 

near-isoline control grain. 

Results are shown in Table 19. No statistically significant differences were observed between 

the 4114 maize and near-isoline control maize for any of the amino acid mean values (adjusted 

P-values were > 0.05).

In conclusion, total amino acid analysis of maize grain demonstrates that 4114 maize is 

comparable to near-isoline and reference maize hybrids.
Pioneer Hi-Bred International, Inc. Page 115 of 258

4114 Maize

Table 19. Total Amino Acids in Maize Grain

Analyte (% Dry Weight)

Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Alanine

Meanb0.739 0.757

0.491 - 1.09 0.439 -

1.39

Rangec0.604 - 0.840 0.460 - 0.886

CId0.676 - 0.797 0.695 - 0.813

Adjusted P-Valuee0.772

P-Valuef0.398

Arginine

Mean 0.434 0.438

0.253 -

0.551

0.119 -

0.640

Range 0.362 - 0.492 0.364 - 0.514

CI 0.408 - 0.463 0.411 - 0.466

Adjusted P-Value 0.908

P-Value 0.707

Aspartic Acid

Mean 0.680 0.701

0.442 -

0.947

0.335 -

1.21

Range 0.562 - 0.751 0.460 - 0.813

CI 0.634 - 0.724 0.657 - 0.743

Adjusted P-Value 0.679

P-Value 0.260

Cystine

Mean 0.193 0.194

0.136 -

0.418

0.0800 -

0.514

Range 0.157 - 0.253 0.144 - 0.252

CI 0.168 - 0.220 0.169 - 0.222

Adjusted P-Value 0.946

P-Value 0.931

Glutamic Acid

Mean 1.93 1.96

1.17 - 2.88 0.965 -

3.54

Range 1.59 - 2.24 1.21 - 2.31

CI 1.78 - 2.07 1.82 - 2.10

Adjusted P-Value 0.789

P-Value 0.546

Glycine

Mean 0.372 0.377

0.249 -

0.485

0.184 -

0.539

Range 0.318 - 0.442 0.305 - 0.445

CI 0.346 - 0.400 0.351 - 0.406

Adjusted P-Value 0.789

P-Value 0.461

Histidine

Mean 0.293 0.303

0.180 -

0.362

0.137 -

0.434

Range 0.257 - 0.342 0.230 - 0.426

CI 0.271 - 0.317 0.280 - 0.328

Adjusted P-Value 0.679

P-Value 0.274

Isoleucine

Mean 0.339 0.349

0.143 -

0.587

0.179 -

0.710

Range 0.288 - 0.388 0.229 - 0.408

CI 0.312 - 0.364 0.323 - 0.374

Adjusted P-Value 0.679

P-Value 0.251
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4114 Maize

Table 19. Total Amino Acids in Maize Grain (continued)

Analyte (% Dry Weight) Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Leucine

Mean 1.26 1.31

0.659 - 1.95 0.642 - 2.49

Range 1.02 - 1.49 0.775 - 1.56

CI 1.13 - 1.37 1.19 - 1.42

Adjusted P-Value 0.679

P-Value 0.206

Lysine

Mean 0.293 0.288

0.112 -

0.551

0.0500 -

0.668

Range 0.224 - 0.349 0.213 - 0.350

CI 0.256 - 0.335 0.252 - 0.330

Adjusted P-Value 0.789

P-Value 0.474

Methionine

Mean 0.179 0.182

0.0724 -

0.490 0.100 - 0.468

Range 0.144 - 0.231 0.136 - 0.230

CI 0.156 - 0.206 0.158 - 0.209

Adjusted P-Value 0.916

P-Value 0.846

Phenylalanine

Mean 0.523 0.554

0.298 -

0.693 0.244 - 0.930

Range 0.443 - 0.611 0.370 - 0.666

CI 0.474 - 0.568 0.508 - 0.596

Adjusted P-Value 0.679

P-Value 0.121

Proline

Mean 0.899 0.926

0.454 - 1.64 0.462 - 1.63

Range 0.763 - 1.01 0.624 - 1.11

CI 0.828 - 0.964 0.858 - 0.990

Adjusted P-Value 0.679

P-Value 0.235

Serine

Mean 0.500 0.518

0.266 -

0.683 0.235 - 0.910

Range 0.421 - 0.554 0.352 - 0.745

CI 0.459 - 0.545 0.475 - 0.564

Adjusted P-Value 0.679

P-Value 0.308

Threonine

Mean 0.361 0.373

0.176 -

0.578 0.224 - 0.666

Range 0.318 - 0.402 0.284 - 0.438

CI 0.341 - 0.381 0.354 - 0.392

Adjusted P-Value 0.679

P-Value 0.165

Tryptophan

Mean 0.0634 0.0639

0.00876 -

0.127

0.0271 -

0.215

Range 0.0367 - 0.0726 0.0377 - 0.0773

CI 0.0563 - 0.0692 0.0569 - 0.0696

Adjusted P-Value 0.908

P-Value 0.806
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Table 19. Total Amino Acids in Maize Grain (continued)

Analyte (% Dry Weight) Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Tyrosine

Mean 0.263 0.257

0.0707 -

0.505 0.103 - 0.790

Range 0.199 - 0.333 0.163 - 0.367

CI 0.240 - 0.289 0.234 - 0.282

Adjusted P-Value 0.792

P-Value 0.561

Valine

Mean 0.460 0.473

0.159 -

0.749 0.210 - 0.855

Range 0.393 - 0.507 0.334 - 0.542

CI 0.428 - 0.488 0.443 - 0.500

Adjusted P-Value 0.679

P-Value 0.187

a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 

2002; Watson, 1982; Watson, 1987).

b Least squares mean

c Range denotes the lowest and highest individual value across locations.

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f 

 Non-adjusted P-value
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Key Anti-nutrients in Maize Grain

Maize grain contains several key anti-nutrients: raffinose, phytic acid, and trypsin inhibitor 

(OECD, 2002). Raffinose is a low molecular weight oligosaccharide that is non-digestible, 

creating flatulence from consumption, but typically can be removed through processing of 

maize (OECD, 2002). In addition, phytic acid chelates mineral nutrients including calcium, 

magnesium, potassium, iron and zinc, rendering them unavailable to monogastric animals. 

Trypsin inhibitor can interfere with the digestion of proteins, resulting in decreased animal 

growth.

Levels of key anti-nutrients were measured in 4114 maize and near-isoline control grain. 

Results are shown in Table 20. No statistically significant differences were observed between 

the mean values for 4114 maize and near-isoline control for any of the anti-nutrient measured 

(adjusted P-values were > 0.05).

In conclusion, anti-nutrient analysis of maize grain demonstrates that 4114 maize is comparable 

to near-isoline control maize and reference maize hybrids.
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Table 20. Key Anti-Nutrients in Maize Grain

Analyte

(% Dry Weight or as Indicated)

Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Phytic Acid

Meanb0.998 1.02

0.418 - 1.41 0.111 - 1.57

Rangec0.842 - 1.17 0.827 - 1.52

CId0.917 - 1.09 0.941 - 1.11

Adjusted P-Value

e

0.789

P-Valuef0.542

Raffinose

Mean 0.0823 0.108

0 - 0.398 0.0200 - 0.320

Range <0.0800g 

-

0.210

<0.0800g -

0.236

CI 0.0503 - 0.134 0.0670 - 0.173

Adjusted P-Value 0.679

P-Value 0.0826

Trypsin 

Inhibitor

(TIU/mg)h

Mean 2.67 2.58

1.60 - 4.89 1.09 - 7.18

Range 1.45 - 4.68 1.70 - 3.48

CI 2.24 - 3.18 2.16 - 3.07

Adjusted P-Value 0.908

P-Value 0.694

a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 

2002; Watson, 1982; Watson, 1987).

b Least squares mean

c Range denotes the lowest and highest individual value across locations.

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f Non-adjusted P-value

g

<Lower limit of quantification (LLOQ); indicates that sample value(s) were detected below the assay LLOQ. 

Sample results which were below the LLOQ were assigned a value equal to the LLOQ for statistical analysis.

h

Abbreviation: TIU = trypsin inhibitor units 
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Key Secondary Plant Metabolites in Maize Grain

Secondary plant metabolites are neither nutrients nor anti-nutrients, but can be analyzed as 

indicators of the absence of unintended effects of the genetic modification on metabolism 

(OECD, 2002). Characteristic plant metabolites in maize are the phenolic acids: ferulic acid, 

furfural, and p-coumaric acid.

Ferulic acid, furfural, and p-coumaric acid were measured in 4114 maize and near-isoline 

control grain. Results are shown in Table 21. No statistically significant differences were 

observed between the mean values for 4114 maize and near-isoline control for any of the 

secondary plant metabolites measured (adjusted P-values were > 0.05).

In conclusion, metabolite analysis of maize grain demonstrates that 4114 maize is comparable 

to near-isoline control maize and reference maize hybrids.

Table 21. Key Secondary Plant Metabolites in Maize Grain

Analyte

(% Dry Weight)

Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

p-Coumaric 

Acid

Meanb0.0183 0.0186

0.000341 -

0.0387 0.003 - 0.0576

Rangec0.0120 - 0.0276 0.0151 - 0.0249

CId0.0159 - 0.0210 0.0162 - 0.0214

Adjusted P-Valuee0.789

P-Valuef0.546

Furfural

Mean <0.000100g<0.000100g

NCh0 - 0.000634

Range <0.000100g<0.000100g

CI NA NA

Adjusted P-Value NA

P-Value NA

Ferulic Acid

Mean 0.255 0.266

0.0553 - 0.309 0.0200 - 0.389

Range 0.199 - 0.299 0.227 - 0.292

CI 0.243 - 0.268 0.253 - 0.280

Adjusted P-Value 0.679

P-Value 0.231

a Literature ranges are taken from published literature for maize (Codex, 1996; Codex, 2005; ILSI, 2006; OECD, 

2002; Watson, 1982; Watson, 1987).

b Least squares mean

c Range denotes the lowest and highest individual value across sites.

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f Non-adjusted P-value

g <Lower Limit of Quantification (LLOQ); Indicates that the values of the sample or samples were detected below 

the assay’s LLOQ. Sample results which were below the LLOQ were assigned a value equal to the LLOQ for 

statistical analysis.

h A tolerance interval could not be calculated (NC).
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4114 Maize

Key Nutrients in Maize Forage

Maize silage is an important feed ingredient for feedlot cattle and dairy cattle. In the U.S., 

approximately 10% of the maize crop is harvested as forage. The whole maize plant contains 

about one and one-half times the nutrients of the grain, and the ensiling process preserves 

more than 90% of the nutrients (OECD, 2002).

Proximates and Minerals in Maize Forage

Crude protein, crude fat, crude fiber, ADF, NDF, ash, carbohydrates, and the minerals calcium 

and phosphorus were measured in 4114 maize and near-isoline control forage.

Results are shown in Table 22. For each analyte measured, there was no statistical difference 

between 4114 maize and the near-isoline control maize (adjusted P-values were > 0.05).

In conclusion, proximate and mineral analysis of maize forage demonstrated that 4114 maize 

forage is comparable to near-isoline and reference maize forage.
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Table 22. Proximates and Minerals in Maize Forage

Analyte (% Dry Weight)

Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Crude Protein

Meanb8.93 9.10

3.35 - 14.6 3.14 - 15.9

Rangec5.82 - 11.0 5.72 - 11.0

CId7.52 - 10.0 7.75 - 10.1

Adjusted P-Valuee0.679

P-Valuef0.297

Crude Fat

Mean 3.30 3.26

1.17 - 3.98 0.296 - 6.70

Range 1.97 - 4.89 1.95 - 5.60

CI 2.63 - 4.15 2.60 - 4.10

Adjusted P-Value 0.938

P-Value 0.881

Crude Fiber

Mean 21.8 21.2

13.4 - 32.6 19.0 - 62.8

Range 15.6 - 27.6 17.6 - 25.1

CI 19.9 - 23.9 19.3 - 23.3

Adjusted P-Value 0.789

P-Value 0.524

ADF

Mean 28.8 27.6

17.8 - 42.0 16.1 - 47.4

Range 21.7 - 38.2 20.1 - 34.6

CI 25.3 - 32.9 24.2 - 31.4

Adjusted P-Value 0.679

P-Value 0.215

NDF

Mean 47.4 47.5

30.8 - 71.4 20.3 - 63.7

Range 39.5 - 57.4 41.2 - 52.8

CI 44.0 - 51.1 44.1 - 51.2

Adjusted P-Value 0.966

P-Value 0.966

Ash

Mean 4.98 4.87

0.773 - 8.52 1.30 - 10.5

Range 3.27 - 7.09 3.50 - 6.36

CI 4.19 - 5.92 4.10 - 5.79

Adjusted P-Value 0.760

P-Value 0.374

Carbohydrates

Mean 82.8 82.7

76.7 - 90.9 76.4 - 92.1

Range 78.7 - 87.6 80.1 - 86.6

CI 81.2 - 84.4 81.2 - 84.3

Adjusted P-Value 0.939

P-Value 0.896

Calcium

Mean 0.259 0.216

0 - 0.563 0.0714 - 0.600

Range 0.143 - 0.654 0.0878 -

0.375

CI 0.195 - 0.344 0.162 - 0.287

Adjusted P-Value 0.679

P-Value 0.156
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4114 Maize

Table 22. Proximates and Minerals in Maize Forage (continued)

Analyte (% Dry Weight) Control

(n=24)

4114 maize 

(n=24)

Tolerance

Interval

Literature

Rangea

Phosphorus

Mean 0.274 0.289

0.0198 - 0.465 0.0936 - 0.550

Range 0.191 - 0.369 0.213 - 0.367

CI 0.243 - 0.308 0.257 - 0.326

Adjusted P-Value 0.679

P-Value 0.115

a Literature ranges are taken from published literature for maize (ILSI, 2006; Watson, 1982).

b Least squares mean

c Range denotes the lowest and highest individual value across locations.

d 95% Confidence Interval

e False Discovery Rate (FDR) adjusted P-value

f Non-adjusted P-value

Conclusions on Compositional Assessment of 4114 maize

Extensive nutritional compositional analyses of grain and forage were conducted to evaluate 

the composition of 4114 maize as compared to a non-transgenic near-isoline control. 

Compositional analysis of 4114 maize was used to evaluate any changes in the levels of key 

nutrients, anti-nutrients or secondary metabolites.

Compositional analyses of grain included crude protein, crude fat, crude fiber, acid detergent 

fiber (ADF), neutral detergent fiber (NDF), ash, carbohydrates, fatty acids, total amino acids, key 

anti-nutrients, and key secondary metabolites. Compositional analyses of forage included 

crude protein, crude fat, crude fiber, ADF, NDF, ash, carbohydrates, calcium, and phosphorus. 

In total, data from 69 different analytical components (60 in grain, nine in forage) were 

analyzed. While statistical differences were observed in some of these analytes between 4114 

maize and its near-isoline control, all values for 4114 maize were within the ranges for the 

commercial maize lines (i.e., tolerance intervals) or within published literature ranges for maize. 

Therefore, based on these analyses, the grain and forage of 4114 maize are considered to be 

comparable to conventional maize with respect to nutrient composition. Furthermore, the 

presence of 4114 maize would not be expected to impact raw or processed maize commodities.

In conclusion, 4114 maize is comparable to conventional maize with respect to nutrient 

composition.
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4114 Maize

4-B. Agronomic Performance and Field Observations

Agronomic evaluations were conducted on 4114 maize and conventional and/or control maize. 

These evaluations form the basis to determine whether 4114 maize is agronomically 

comparable to conventional maize and they provide an appropriate scientific determination 

whether 4114 maize is no more likely to pose a plant pest risk when compared to conventional 

maize.

The agronomic evaluations were based on both laboratory experiments and replicated, 

multi-site field trials conducted by agronomists and scientists who are considered experts in the 

production and evaluation of maize. To evaluate the agronomic characteristics of 4114 maize, 

data were collected on representative characteristics that influence reproduction, crop survival, 

and potential weediness. In each of these assessments, 4114 maize was compared to a 

near-isoline control (i.e., approximately 99% similar) that did not go through the transformation 

process. In each experiment, the agronomic characteristics of 4114 maize were comparable to 

the control or commercial comparators.

Field observations were made to assess responses of 4114 maize and control maize to naturally 

occurring insects and diseases in a wide variety of environmental conditions. In each case, 4114 

maize demonstrated no unexpected responses to insects or diseases as compared to the 

control plants.

Based on the analyses described below, 4114 maize is comparable to conventional maize and 

will not pose a greater plant pest risk or increased weed potential when compared to 

conventional maize.

Germination and Dormancy Evaluations

In order to evaluate germination and dormancy, seeds from the F1*5 generation (Section 2; 

Figure 10 and Table 3) of 4114 maize were tested for germination assays under warm, cold, and 

diurnal conditions (Table 23). The F1*5generation of seed was used because F1 hybrid seed is 

representative of seed that growers would plant in commercial maize fields. A near-isoline 

control was used for comparison. In addition, two commercial maize lines, Pioneer® hybrids

32D78 and 34P88, were evaluated in the study to establish a reference range for germination 

and dormancy evaluations but were not included in the statistical analysis. This reference 

range provided context for any statistical differences observed in the comparisons; if the values 

for 4114 maize fell within this reference range, it indicated that 4114 maize was comparable to 

conventional maize lines.

Each germination test contained eight replicates of 50 seeds each of 4114 maize, near-isoline 

control, and two commercial lines. The “Rules for Testing Seeds”, published by the Association 

of Official Seed Analysis, were used as guidelines for the germination methods and 

interpretation of results (AOSA, 2007). Each replicate was placed between sheets of moist 

germination toweling, rolled up, and placed in a growth chamber set to the appropriate test 
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4114 Maize

conditions as specified in Table 23. At the end of each germination test, each seed was defined 

as either germinated or non-germinated. A given seed was classified as germinated if some or 

all of the essential structures necessary to produce a normal plant under favorable conditions 

had emerged, or non-germinated if none of these structures had emerged. Germination rates 

were reported as a percentage of germinating seed as follows: (number of germinated 

seeds/total seeds)*100. The results are presented in Tables 24, 25, and 26. 

Non-germinated seed were further defined into three categories:

 hard (i.e., did not absorb water);

 imbibed (i.e., absorbed water but did not show signs of growth during the germination 

test); or

 dead (i.e., absorbed water, did not show signs of growth during the germination test, 

and displayed distinct signs of decay such as an extremely soft interior that did not hold 

shape under gentle pressure). 

Non-germinated seeds that were classified as hard or imbibed were further evaluated for 

potential dormancy using a standard Tetrazolium Chloride (TZ) test (AOSA, 2005). The TZ test 

was developed as a color test to evaluate seed viability. The color phase of the test occurs 

when the colorless testing solution consisting of water and 2,3,5-triphenyltetrazolium chloride 

is added to the seed. Once this solution penetrates into living cells, the TZ solution is reduced 

to a reddish (pink), water-insoluble compound. The absence or presence of variation in color 

characteristics within the tissues allows for recognition and location of living tissues within the 

embryo structure. Dead seed were excluded from the TZ test, due to the possibility of a false 

positive result from bacterial growth. The counts for each class of non-germinated seed and 

the corresponding TZ test result are presented in Table 27. In non-germinated hard seed, 

potential dormancy may be indicated if the seed is viable (i.e., positive TZ test); however, seed 

dormancy is not commonly observed in maize.

Germination rates in 4114 maize under warm, cold, and diurnal growing conditions were 

comparable to those of control maize under corresponding growing conditions. No potentially 

dormant seed were identified using the TZ test.

The data provided here support the conclusion that 4114 maize is comparable to conventional 

maize with respect to germination and potential dormancy.
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4114 Maize

Table 23. Description of Seed Germination Conditions

Warm germination test

 Simulated optimal maize growth conditions

 Continuous setting of 25°C and 90% relative humidity for 10 days

 Germinated seed counted after 10 days

Cold germination test

 Simulated early spring planting conditions in Midwestern U.S.

 Continuous setting of 10°C and 90% relative humidity for 10 days, 

followed by three days at a continuous setting of 25°C and 90% 

relative humidity 

 Germinated seed counted after 13 days

Diurnal germination test

 Simulated daily weather conditions in Midwestern U.S.

 Cyclical setting of 10°C and 90% relative humidity for 16 hours 

and then 25°C and 90% relative humidity for 8 hours, repeated 

daily for 10 days

 Germinated seed counted after 10 days
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Table 24. Summary of Warm Germination Test Results

Statistic 4114 

Maize

Control 

Maize

Reference 

Range

Frequency 396/400 395/399

96.0% - 100%

Mean 99.0% 99.0%

Range 96.0% -

100% 96.0% - 100%

P-valuea1.00

a

P-value was determined using Fisher’s exact test for germination frequencies.

Table 25. Summary of Cold Germination Test Results

Statistic 4114 

Maize

Control 

Maize

Reference 

Range

Frequency 371/398 383/399

86.0% - 98.0%

Mean 93.2% 96.0%

Range 86.0% -

98.0% 91.8% - 100%

LS-Mean 93.2% 96.0%

SEM 1.26% 0.982%

P-valuea0.0865

a P-value was determined using GLMM-based statistical test for mean germination rates.

Table 26. Summary of Diurnal Germination Test Results

Statistic 4114 Maize Control 

Maize

Reference 

Range

Frequency 395/400 395/399

96.0% - 100% Mean 98.8% 99.0%

Range 94.0% - 100% 96.0% - 100%

P-valuea1.00

a P-value was determined using Fisher’s exact test for germination frequencies
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Table 27. Tetrazolium Chloride (TZ) Testing of Non-Germinated Seed

Germination 

Test Maize Line

Total NonGerminated Seed

TZ Test of Non-Germinated Seed NonGerminated 

Seed Not 

Tested with TZ

(i.e. Dead Seed)

Result: Viable Result: 

Non-Viable

Hard Imbibed Dead Hard Seed Imbibed 

Seed

Hard or 

Imbibed Seed

Warm

4114 Maize 0 4 0 0 0 4 0

Control Maize 0 3 1 0 0 3 1

32D78 (Reference) 0 1 0 0 0 1 0

34P88 (Reference) 0 6 0 0 0 6 0

Cold

4114 Maize 0 27 0 0 7 20 0

Control Maize 0 16 0 0 1 15 0

32D78 (Reference) 0 31 0 0 1 30 0

34P88 (Reference) 0 15 0 0 2 13 0

Diurnal

4114 Maize 0 5 0 0 2 3 0

Control Maize 0 4 0 0 4 0 0

32D78 (Reference) 0 1 0 0 0 1 0

34P88 (Reference) 0 5 0 0 1 4 0
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Field Trial Evaluations

Agronomic data were collected from the F1*5generation of 4114 maize and near-isoline 

control maize within two experiments (denoted A and B) that were conducted at 17 total field 

locations in 2010 (Table 28; Figure 33). For sites listed in the same location (e.g., sites 3 and 12; 

Table 28), the field trials were conducted independently. The F1*

5

generation of seed (Section 

2; Figure 10 and Table 3) was used because F1 hybrid seed is representative of seed that 

growers would plant in commercial maize fields. The trial locations provided a range of 

environmental and agronomic conditions representative of the major maize-growing regions of 

the U.S. and Canada, where commercial production of 4114 maize is expected. Agronomic 

parameters observed for Experiments A and B are provided in Table 29. The data collected 

were identical for both experiments, except yield was only collected in Experiment A (11 

locations) and pollen viability was only collected in Experiment B (6 locations) (Table 29). 

Details of the methods used are presented in Appendix 7. 

Although 4114 maize contains traits that are efficacious against certain lepidopteran and corn 

rootworm target pests and glufosinate-ammonium tolerance, efficacy of the traits was not 

assessed in these agronomic evaluations. As with the compositional assessments, each field 

trial site was managed to maintain a relatively insect-free and weed-free environment in order 

to ensure that agronomic characteristics measured would be independent of the intended

effects of the insect-resistant and herbicide-tolerant traits; therefore, these evaluations would 

appropriately compare 4114 maize to conventional maize and also assess unintended effects of 

the transgene. For example, yield or insect damage scores were not impacted by the intended 

effects of the traits and therefore could be used to assess comparability of 4114 maize to 

conventional maize. 

Agronomic data were also collected from non-modified commercial maize hybrids (reference 

hybrids) in three separate experiments. Pioneer® hybrids 34M94, 33G26, 33J24, and 3394 were 

analyzed in 2003 at six field locations in maize-growing areas of North America (Bagley, IA; York, 

NE; Chula, GA; New Holland, OH; Larned, KS and Hereford, PA). Pioneer® hybrids 38B85, 37Y12, 

34A15, and 34P88 were analyzed in 2007 at six field locations in maize-growing areas of North 

America (Tallahassee, FL; York, NE; Germansville, PA; Richland, IA; Larned, KS and Branchton, 

Ontario). Pioneer® hybrids 37H24, 36M28, 35T06, 35T36, 35K02, and 34P88 were analyzed in 

2009 at four field locations in the maize-growing areas of Chile. These Pioneer commercial 

products were chosen to represent a wide range of non-genetically modified varieties that 

would normally be planted commercially. 

For the reference hybrid trials, each location utilized a randomized complete block design 

containing three blocks. Each block contained two-row plots. Procedures for planting and 

collecting data from the reference hybrid trials were similar to those employed for the trials 

containing near-isoline control and 4114 maize. Agronomic data collected from the reference 

hybrids was used to help determine the normal range of variation for the agronomic 

characteristics in commercial maize.
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4114 Maize

A statistical analysis of agronomic data was conducted to test for differences in the mean 

values between the 4114 maize and the near-isoline control (see Appendix 7 for statistical 

model). When numerous comparisons are being made, it is important to control the rate of 

false-positive results. Since the introduction of the false discovery rate (FDR) approach in the 

mid-1990’s, it has been widely employed across a number of scientific disciplines, including 

genomics, ecology, medicine, plant breeding, epidemiology, dairy science, and signal/image 

processing (e.g., Pawitan et al., 2005; Spelman and Bovenhuis, 1998). A false positive result 

occurs when two means are deemed significantly different when, in fact, they are not. If one 

uses a 5% type I error rate for each agronomic characteristic measured, then the number of 

false positives increases as the number of characteristics increase. In order to help manage the 

false positive rate, the FDR method of Benjamini and Hochberg was applied to account for 

making multiple comparisons (Benjamini and Hochberg, 1995; Westfall et al., 1999). P-values 

were adjusted accordingly. This resulted in the false positive rate being held to 5%. Both 

adjusted and unadjusted P-values are provided for the agronomic data. In each experiment, a 

significant difference between the mean of 4114 maize and that of the control line was 

established with an FDA-adjusted P-value of less than 0.05.

Using the data obtained from the reference hybrids, a statistical tolerance interval was 

calculated to contain, with 95% confidence, 99% of the values contained in the population of 

commercial maize. This statistical tolerance interval provided further context for interpretation 

of the agronomic results for 4114 maize. The agronomic characteristic measurements for 4114 

maize that fell within the tolerance interval for that characteristic were considered to be within 

the range of normal variability of commercial maize hybrids.
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Table 28. Site Locations for 2010 Field Trial Experiments

Map Site 

Number Country Location

Experiment A: 2010 Field Trials

1

U.S.

Sheridan, IN

2 Deerfield, MI

3 Richland, IA

4 Rochelle, IL

5 Bagley, IA

6 Seymour, IL

7 York, NE

8 Carlyle, IL

9 Wyoming, IL

10 Atlantic, IA

11 Geneva, MN

Experiment B: 2010 Field Trials

12

U.S.

Richland, IA

13 Wyoming, IL

14 Geneva, MN

15 York, NE

16 Canada Branchton, ON

17 Thorndale, ON

1

2

3, 12

5

6

7, 15

8

Canada

United States

9, 13

10

11, 14

4

16

17

Figure 33. Map of Locations for Agronomic Data Collection for 4114 Maize
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Experiment A contains data from 11 field site locations planted in the U.S. during the 2010 

growing season (Table 28, Figure 33). The purpose of Experiment A was to evaluate and 

compare the agronomic characteristics and yield of 4114 maize with a near-isoline control. 

Each site consisted of a randomized complete block design with four blocks, with each block 

containing 4114 maize and the control maize. Procedures employed to control the introduction 

of experimental bias in this study were as follows: non-systematic selection of trial areas and 

plot areas within each site, randomization of the maize entries within each block, and uniform 

maintenance treatments across each plot area.

The following agronomic characteristics were evaluated in all four blocks: early population, 

seedling vigor, time to silking, time to pollen shed, plant height, ear height, stalk lodging, root 

lodging, final population, stay green, disease incidence, insect damage, and yield. Descriptions 

of the characteristics and their measurement are found in Table 29.

The results of Experiment A are summarized in Table 30. No statistically significant differences 

were observed between 4114 maize and control maize values for all characteristics measured. 

Based on the data obtained in this study, agronomic characteristics and yield were comparable 

between 4114 maize and the control maize. 
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Table 29. Agronomic Characteristics Measured

General

Characteristic

Characteristic 

Measured

Evaluation 

Timinga Data Description Scale

Germination/

Emergence

Early Population V2-V4 Number of plants emerged 

per plot

Actual count per 

plot

Seedling Vigor V2-V4

Visual estimate of average 

vigor of emerged plants per 

plot

From 1-9, where 

1=short plants 

with small leaves, 

and 9=tall plants 

with large leaves

Vegetative 

Parameters

Plant Height R4 Height from the soil surface 

to the tip of the tassel Height in cm

Ear Height R4

Height from the soil surface 

to the base of the primary 

ear

Height in cm

Stalk Lodging R6

Visual estimate of percent 

of plants in the plot with 

stalks broken below the 

primary ear

0 to 100%

Root Lodging R6

Visual estimate of percent 

of plants in the plot leaning 

approximately 30° or more

in the first 2 feet (0.6m) 

above the soil surface

0 to 100%

Final Population R6 The number of plants 

remaining per plot

Actual count per 

plot

Stay Green R6 Overall plant health

Ranging from 1-9 

where 1=no visible 

green tissue; 

5=approximately 

50% green tissue 

remaining; 9=very 

green 

approximately 

90% or greater 

green tissue 

remaining
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Table 29. Agronomic Characteristics Measured (continued)

General 

Characteristic

Characteristic 

Measured

Evaluation 

Timinga Data Description Scale

Reproductive 

Parameters

Time to Silking Approximately 

50% silking

From the time of planting 

until approximately 50% of 

the plants have emerged 

silks

Number of 

Accumulated Heat 

Units (AHU)

Time to Pollen 

Shed

Approximately 

50% pollen 

shed

From the time of planting 

until approximately 50% of 

the plants have tassels 

shedding pollen

Number of 

Accumulated Heat

Units (AHU)

Yield Approximately 

R6

Harvest weight per area 

adjusted to 13% moisture 

content

Bushels per acre

Pollen Viabilityb During pollen 

shed

Pollen Shape

Percentage of 

pollen grains with 

collapsed walls at 

0, 30, 60, and 120 

minutes

Pollen Color

Percentage of 

pollen grains with 

intense yellow 

color at 0, 30, 60, 

and 120 minutes

Pest Response

Disease 

Incidence

Approximately

R6

Visual estimate of foliar 

disease incidence

Ranging from 1-9 

where 1=poor 

disease resistance 

or high infection; 

9=best disease 

resistance or low 

infection

Insect Damage Approximately

R6

Visual estimate of insect 

damage

Ranging from 1-9 

where 1=poor 

insect resistance 

or high damage; 

9=best insect 

resistance or low 

damage

a

Refer to Ritchie et al., 2005 for a description of maize growth stages.

b

Pollen viability has been correlated to pollen shape and color (Luna et al., 2001)
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Table 30. Experiment A: Summary of Agronomic Performance of 4114 Maize Across Eleven 

Locations

Agronomic TraitaControl Maize 4114 Maize Tolerance Intervalb

Early 

Population

(Count)

Meanc56 56

41 - 60

Ranged37 - 60 40 - 60

CIe53 - 58 53 - 58

Adjusted P-Valuef0.823

P-Valueg0.690

Final 

Population

(Count)

Mean 54 55

34 - 60

Range 39 - 60 38 - 63

CI 51 - 57 51 - 57

Adjusted P-Value 0.823

P-Value 0.823

Time to Silking

(Accumulated 

Heat Units)

Mean 1370 1370

841 - 1790

Range 1020 - 1590 1020 - 1640

CI 1270 - 1480 1270 - 1480

Adjusted P-Value 0.823

P-Value 0.563

Time to Pollen 

Shed

(Accumulated 

Heat Units)

Mean 1410 1410

855 - 1810

Range 1100 - 1620 1100 - 1670

CI 1310 - 1500 1310 - 1500

Adjusted P-Value 0.823

P-Value 0.759

Seedling Vigor

(1-9 Scale)

Mean 7 7

3 - 9

Range 4 - 9 4 - 9

CI 6 - 8 6 - 8

Adjusted P-Value 0.823

P-Value 0.614

Stalk Lodging

(%)

Mean 2 2

0 - 20i

Range 0 - 20 0 - 20

CI NAhNA

Adjusted P-Value 0.823

P-Value 0.386

Root Lodging

(%)

Mean 6 6

0 - 8

i

Range 0 - 45 0 - 45

CI NA NA

Adjusted P-Value 0.823

P-Value 0.485

Stay Green

(1-9 Scale)

Mean 5 5

1 - 9

Range 1 - 9 1 - 9

CI 3 - 6 3 - 6

Adjusted P-Value 0.823

P-Value 0.121
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Table 30. Experiment A: Summary of Agronomic Performance of 4114 Maize Across Eleven 

Locations (continued)

Agronomic Trait Control Maize 4114 Maize Tolerance Interval

Disease 

Incidence

(1-9 Scale)

Mean 7 7

1 – 9

Range 5 - 9 5 - 9

CI 7 - 8 7 - 8

Adjusted P-Value 0.823

P-Value 0.749

Insect Damage

(1-9 Scale)

Mean 8 8

2 – 9

Range 6 - 9 5 - 9

CI NA NA

Adjusted P-Value 0.823

P-Value 0.792

Plant Height

(cm)

Mean 292 294

112 – 389

Range 232 - 323 234 - 325

CI 277 - 307 279 - 309

Adjusted P-Value 0.823

P-Value 0.196

Ear Height

(cm)

Mean 121 120

39 – 167

Range 80 - 162 71 - 153

CI 106 - 136 105 - 135

Adjusted P-Value 0.823

P-Value 0.449

Yield

(bushels per 

acre)

Mean 174 176

NCj

Range 68 - 257 105 - 240

CI 149 - 199 151 - 201

Adjusted P-Value 0.823

P-Value 0.632

a Refer to Table 29 for descriptions of each agronomic characteristic measured.

b Tolerance Intervals were calculated from the non-modified commercial maize lines evaluated. These intervals 

contain 99% of the values with 95% confidence level unless otherwise noted.

c Least squares mean

d Range denotes the lowest and highest individual value across sites.

e 95% Confidence Interval

f False Discovery Rate (FDR) adjusted P-value

g Non-adjusted P-value

h Statistical analysis was not available (NA)

i No tolerance interval could be calculated; these values are minimum/maximum ranges

j No tolerance interval could be calculated (NC)
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4114 Maize

Experiment B contains data from six field site locations planted in the U.S. and Canada during 

the 2010 growing season (Table 28, Figure 33). The purpose of Experiment B was to evaluate 

and compare the agronomic and pollen characteristics of 4114 maize with a near-isoline 

control.

Each site included a randomized block design containing four blocks, with each block containing 

a plot of 4114 maize and one plot of control maize. Procedures employed to control the 

introduction of experimental bias in this study were as follows: non-systematic selection of trial 

areas and plot areas within each site, randomization of the maize entries within each block, and 

uniform maintenance treatments across each plot area.

The following agronomic characteristics were measured: early population, final population, 

time to silking, time to pollen shed, pollen viability, seedling vigor, stalk lodging, root lodging, 

stay green, disease incidence, insect damage, plant height, and ear height. Descriptions of the 

characteristics and their measurement are found in Table 29. 

Results of Experiment B are summarized in Table 31. No statistically significant differences 

were observed between 4114 maize and control maize values for all characteristics measured.

Based on the data obtained in this study, agronomic characteristics and yield were comparable 

between 4114 maize and the control maize. 
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4114 Maize

Table 31. Experiment B: Summary of Agronomic Characteristics of 4114 Maize Across Six 

Locations

Agronomic CharacteristicaControl Maize 4114 Maize Tolerance Intervalb

Early Population

(count)

Meanc57 58

41 – 60

Ranged50 - 59 42 - 60

CIe53 - 59 55 - 60

Adjusted P-Valuef0.354

P-Valueg0.0596

Final Population

(count)

Mean 55 57

34 – 60

Range 38 - 59 46 - 60

CI 49 - 58 52 - 59

Adjusted P-Value 0.225

P-Value 0.0125

Time to Silking 

(accumulated heat units)

Mean 1380 1390

841 – 1790

Range 1300 - 1490 1300 - 1590

CI 1320 - 1450 1320 - 1450

Adjusted P-Value 0.494

P-Value 0.367

Time to Pollen Shed 

(accumulated heat units)

Mean 1420 1430

855 – 1810

Range 1350 - 1510 1350 - 1620

CI 1350 - 1480 1360 - 1490

Adjusted P-Value 0.494

P-Value 0.384

Pollen Viability – Shapeh

(% of collapsed pollen)

0 minutes

Mean 3 2

0 – 65

Range 0 - 80 0 - 70

CI 0 - 13 0 - 12

Adjusted P-Value 0.440

P-Value 0.244

Pollen Viability - Shape

(% of collapsed pollen)

30 minutes

Mean 67 55

0 – 100

Range 15 - 100 20 - 90

CI 32 - 94 21 - 87

Adjusted P-Value 0.354

P-Value 0.0786

Pollen Viability - Shape

(% of collapsed pollen)

60 minutes

Mean 88 91

4 – 100

Range 40 - 100 50 - 100

CI 63 - 100 67 - 100

Adjusted P-Value 0.494

P-Value 0.348

Pollen Viability - Shape

(% of collapsed pollen)

120 minutes

Mean 98 97

37 – 100

Range 85 - 100 85 - 100

CI NAiNA

Adjusted P-Value 0.602

P-Value 0.502
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Table 31. Experiment B: Summary of Agronomic Characteristics of 4114 Maize Across Six 

Locations (continued)

Agronomic Characteristic Control Maize 4114 Maize Tolerance Interval

Pollen Viability - Colorh

(% of intense yellow 

pollen)

0 minutes

Mean 2 2

0 - 84

Range 0 – 80 0 - 70

CI 0 – 12 0 - 10

Adjusted P-Value 0.376

P-Value 0.123

Pollen Viability - Color

(% of intense yellow 

pollen)

30 minutes

Mean 57 50

0 - 100

Range 15 - 100 20 - 90

CI 22 - 89 16 - 84

Adjusted P-Value 0.440

P-Value 0.235

Pollen Viability - Color

(% of intense yellow 

pollen)

60 minutes

Mean 82 86

14 - 100

Range 40 - 100 55 - 100

CI 50 - 99 56 - 100

Adjusted P-Value 0.376

P-Value 0.155

Pollen Viability - Color

(% of intense yellow 

pollen)

120 minutes

Mean 95 97

56 - 100

Range 85 - 100 85 - 100

CI NA NA

Adjusted P-Value 0.376

P-Value 0.151

Seedling Vigor

(1-9 scale)

Mean 8 7

3 - 9

Range 6 - 9 5 - 9

CI NA NA

Adjusted P-Value 0.354

P-Value 0.0522

Stalk Lodging

(%)

Mean 0 0

0 - 20j

Range 0 - 4 0 - 5

CI NA NA

Adjusted P-Value NA

P-Value NA

Root Lodging

(%)

Mean 0 0

0 - 8

j

Range 0 - 2 0 - 1

CI NA NA

Adjusted P-Value NA

P-Value NA

Stay Green

(1-9 scale)

Mean 4 4

1 - 9

Range 1 - 7 1 - 7

CI 2 - 6 3 - 6

Adjusted P-Value 0.735

P-Value 0.729
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Table 31. Experiment B: Summary of Agronomic Characteristics of 4114 Maize Across Six 

Locations (continued)

Agronomic Characteristic Control Maize 4114 Maize Tolerance Interval

Disease Incidence

(1-9 scale)

Mean 8 8

1 – 9

Range 5 - 9 5 - 9

CI NA NA

Adjusted P-Value 0.735

P-Value 0.735

Insect Damage

(1-9 scale)

Mean 8 8

2 – 9

Range 7 - 9 7 - 9

CI NA NA

Adjusted P-Value 0.735

P-Value 0.731

Plant Height

(cm)

Mean 285 287

112 – 389

Range 256 - 324 265 - 313

CI 268 - 302 270 - 304

Adjusted P-Value 0.494

P-Value 0.333

Ear Height

(cm)

Mean 113 115

39 – 167

Range 82 - 150 79 - 145

CI 95 - 130 97 - 132

Adjusted P-Value 0.376

P-Value 0.167

a Refer to Table 29 for descriptions of each agronomic characteristic measured. 

b Tolerance Intervals were calculated from the non-modified commercial maize lines evaluated. These intervals 

contain 99% of the values with 95% confidence level unless otherwise noted.

c Least squares mean

d Range denotes the lowest and highest individual value across sites.

e 95% Confidence Interval

f False Discovery Rate (FDR) adjusted P-value

g Non-adjusted P-value 

h

 Pollen viability has been correlated to pollen shape and color (Luna et al., 2001)

i

Statistical analysis was not available (NA)

j

No tolerance interval could be calculated; these values are minimum/maximum ranges
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4114 Maize

Field Insect and Disease Observations

As discussed in Section 1, 4114 maize has been field tested in the U.S. and Puerto Rico since 

2006, as authorized by USDA-APHIS permits and notifications. For each trial, a survey of the 

naturally occurring insects and diseases and any unexpected differences in the response of 

4114 maize as compared to the control line (near-isoline and/or conventional maize lines) were 

recorded by experienced plant breeders and field staff at least every four weeks. A summary of 

these surveys for each trial and any differences seen between 4114 maize and control lines are 

presented in Appendix 8. These observations provide a means to determine if 4114 maize will 

respond differently from conventional maize lines to insects or diseases in the environment.

In every case, 4114 maize did not exhibit any unexpected responses to naturally occurring 

insects or diseases. These results support the conclusion that 4114 maize is comparable to 

control maize lines with similar genetics or to conventional maize lines with respect to insect or 

disease response. 

Conclusions on Agronomic Performance and Field Observations

4114 maize was observed in laboratory experiments and at 17 field locations in the U.S. and 

Canada to measure agronomic parameters. These experiments and field studies evaluate the 

characteristics of maize over a broad range of environmental conditions that represent regions 

where 4114 maize will be grown. The agronomic parameters measured are characteristic traits 

for reproduction, survival, and potential weediness.

The agronomic data showed no significant differences between 4114 maize and control maize 

(near-isoline controls and/or commercial maize lines) with respect to early population, 

vegetative growth, reproductive parameters, yield, and pest responses. These data support the 

conclusion that 4114 maize is agronomically comparable to conventional maize.

Observations from U.S. and U.S. territory field trials showed no unexpected differences in the 

response of 4114 maize and control maize to naturally occurring insects and diseases. These 

results support the conclusion that 4114 maize is comparable to control maize lines with similar 

genetics and/or to conventional maize lines.

Based on these analyses, 4114 maize is comparable to conventional maize and will not pose a 

greater plant pest risk or increased weed potential than conventional maize.

4-C. Overall Conclusions to Comparative Assessment of 4114 Maize

A comparative assessment was conducted to determine if 4114 maize would present any new 

or greater risks relative to maize varieties that have a history of safe use in the environment 

and as food and feed. This comparative assessment included compositional and agronomic 

analyses with appropriate comparators to determine comparability to conventional maize. 

These comparative analyses were designed to eliminate the effects of the introduced insect-
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resistant and herbicide-tolerant traits in 4114 maize, so that an appropriate comparison to 

conventional maize could be made and any unintended effects that may be due to the 4114 

maize insertion could be evaluated.

Extensive nutrient composition analyses of grain and forage were conducted to compare the 

composition of 4114 maize to controls. These analyses were used to evaluate any changes in 

the levels of key nutrients, anti-nutrients and secondary metabolites. Based on the results of 

the compositional evaluation, the grain and forage of 4114 maize are as safe as commercially 

available maize and there would be no significant impact on raw or processed maize 

commodities.

A comprehensive agronomic evaluation for 4114 maize was conducted. The agronomic 

evaluations were based on both laboratory experiments and replicated, multi-site field trials. 

Data were collected on representative characteristics that influence reproduction, crop survival, 

and potential weediness. Field observations included observed responses of 4114 maize and 

control maize to naturally occurring insects and diseases in a wide variety of environmental 

conditions. These data support the conclusion that, independent of the introduced insectresistant and herbicide-tolerant traits, 4114 maize is comparable to conventional maize and is 

unlikely to pose a greater plant pest risk than conventional maize. 

In conclusion, these analyses indicated that 4114 maize was comparable to conventional maize 

with respect to the compositional analytes and agronomic characteristics measured. 4114 

maize differs from conventional maize only in its herbicide tolerance and its efficacy against 

certain lepidopteran and corn rootworm pests. Overall, these comparative assessments

indicate that 4114 maize is as safe as conventional maize varieties and does not pose a greater 

risk than conventional maize varieties in food, feed, and the environment.
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4114 Maize

5. Potential Environmental Impact of the Introduction of 4114 Maize

The potential environmental impact of a transgenic plant needs to be considered in the context 

of the characteristics of the recipient crop, the introduced trait, and the environment in which it 

will be introduced (OECD, 1993). Knowledge in each of these areas will provide background on 

which a risk or safety assessment can be made about the environmental release of the 

transgenic plant (OECD, 1993). In particular, weediness, gene transfer or flow, and trait effects 

are particular issues that may be relevant to evaluating the new transgenic line and its safety 

(OECD, 1993).

In order to evaluate the potential environmental impact of the introduction of 4114 maize, the 

potential for 4114 maize to become weedy or invasive, the potential for gene flow to sexually

compatible wild relatives, and the potential impacts of the introduced proteins (Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT) were considered. As described further below, in each case, it is 

not expected that 4114 maize will adversely impact the environment with respect to these 

considerations.

5-A. Potential for 4114 Maize to Have Altered Disease and Unintended Pest Susceptibilities

or to Become Weedy or Invasive

In evaluating the potential for 4114 maize to become more weedy or invasive than 

conventional maize, general maize biology was considered. Maize is a cultivated annual plant 

that generally cannot survive temperatures below freezing and is typically grown in temperate 

regions (OECD, 2003). Maize is not classified as a weed, is not on the U.S. federal or state 

noxious weed lists, and possesses few characteristics of notably successful weeds (Baker, 1974; 

Keeler, 1989; USDA-NRCS, 2011). Therefore, the natural characteristics of maize do not 

indicate a high potential for weediness or invasiveness.

A comparative assessment of 4114 maize was conducted to determine if the DNA insertion 

altered the nutritive or agronomic characteristics of maize. Compositional and agronomic 

comparison data were collected on 4114 maize in multiple location field trials as described in 

Section 4; these analyses were designed to be independent of the introduced traits so that an 

appropriate comparison to conventional maize could be made and any unintended effects of 

the 4114 maize insertion could be assessed. These analyses showed that 4114 maize was 

comparable to conventional maize in composition, except for the introduced proteins, and was 

comparable in agronomics. In the agronomic analyses, a number of characteristics were 

measured, including certain ones that may be indicative of weediness: germination and 

emergence (germination rate, early population, seedling vigor); reproductive (time to silking, 

time to pollen shed, pollen viability, yield); vegetative (final population stalk lodging, root 

lodging, stay green, plant height, ear height); and pest response (disease incidence, insect 

damage). Characteristics related to seed germination, seed production, reproductive time and 

vegetative competitiveness have been identified with successful weeds (Baker, 1974); changes 

to these parameters relative to the conventional variety could indicate a change in the 

potential weediness of a crop. 4114 maize was comparable to conventional maize in each of 
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4114 Maize

these characteristics, indicating that 4114 maize is unlikely to become more weedy or invasive 

than conventional maize.

In addition, 4114 maize has been field tested since 2006 in multiple locations that provide a 

range of environmental conditions and also include regions representative of maize cultivation 

in the U.S. These fields were frequently monitored by expert growers for the incidence of 

diseases and insects and the effect of these on 4114 maize and control plants. In all cases, no 

unexpected differences were observed between 4114 maize and the control comparators. 

In summary, 4114 maize is unlikely to become more weedy or invasive than conventional maize 

when cultivated. Compositional and agronomic comparisons indicate no unexpected effects of 

the presence of the introduced proteins that alter the nutritional composition and weediness 

potential of maize. No unexpected differences were detected between 4114 maize and control 

maize in response to insects and diseases. Furthermore, the expression of the introduced 

proteins (Cry1F, Cry34Ab1, Cry35Ab1, PAT) is unlikely to increase the potential of 4114 maize to 

become weedy. 

5-B. Potential for Gene Flow Between 4114 Maize and Sexually Compatible Wild Relatives

The potential for gene flow between a transgenic crop and its sexually compatible wild relatives 

is assessed through several factors. One factor includes the potential for pollen flow and 

outcrossing to occur significantly outside the cultivated field. Other factors include the overlap 

of the wild relative geographic distribution with the region of transgenic crop cultivation and 

the possibility of genetic compatibility between the crop and the relative. Finally, to determine 

the potential for widespread introgression of the trait into wild relative populations, whether 

the trait itself alters weediness characteristics and whether the wild relative is a noxious weed 

is considered.

4114 maize will be cultivated similarly to other commercial and conventional maize varieties; 

therefore, it is appropriate to examine maize pollination biology, regions of maize cultivation in 

the U.S. and the geographic distribution of sexually compatible wild relatives in order to 

determine the potential for gene flow. The regions of maize cultivation in the U.S. and the 

genetic compatibility and geographic distribution of sexually compatible wild relatives of maize, 

within the genera Zea and Tripsacum, are discussed further below. Based on this information, 

there is low potential for gene flow between 4114 maize and its wild relatives of the genera Zea

and Tripsacum in the U.S.

The potential for the insertion in 4114 maize to become widespread in wild relative populations 

is also unlikely. The insertion, as discussed in Section 5-A, does not make 4114 maize more 

weedy than conventional cultivated maize; furthermore, none of the sexually compatible wild 

relatives are listed as noxious weeds. 
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4114 Maize

Pollination Biology of Maize and Impact on Gene Flow

Maize is almost entirely cross-fertilizing and its pollen is typically wind dispersed (OECD, 2003); 

millions of pollen grains are produced per plant (Jarosz et al., 2003). Despite pollination 

characteristics that are favorable for pollen flow, other factors make it highly unlikely that 

viable maize pollen will travel significantly outside of the cultivated field. Pollen viability is 

reduced in a matter of hours under high temperature and low humidity (Aylor, 2004). Studies 

also indicate that the majority of maize pollen is unlikely to be dispersed significant distances 

outside the originating field (Jarosz et al., 2003). Numerous studies show the majority (84-92%) 

of pollen grains travel less than five meters (Pleasants et al., 2001), with nearly all (>99.75%) 

pollen traveling less than 100 meters (Byrne and Fromherz, 2003; Matsuo et al., 2004; Sears 

and Stanley-Horn, 2000). Therefore, the potential of cross-pollination between cultivated 

maize and its wild relatives will be highest where the wild relatives grow near or adjacent to 

areas of cultivation. Therefore, the geographic range of wild relatives and the regions of maize 

cultivation are one critical factor in determining the potential for gene flow.

Regions of Maize Cultivation in the U.S.

Field maize is a major crop worldwide, but represents the largest crop grown in the U.S. It is 

grown in most states, with production concentrated in the Heartland region (including Illinois, 

Iowa, Indiana, eastern portions of South Dakota and Nebraska, western Kentucky and Ohio, and 

the northern two-thirds of Missouri). Iowa and Illinois are the top maize-producing states and 

typically account for slightly more than one-third of the U.S. crop (USDA-ERS, 2009). Figure 34 

indicates acres planted in the U.S. by county (USDA-NASS, 2011).

Additional maize varieties include popcorn and sweet corn, both of which are minor crops 

compared to field maize (OECD, 2002). While the range of cultivation of popcorn and sweet 

maize include the entire U.S., in total all acreage represents less than 1% of the acreage of field 

corn in 2007 (USDA-NASS, 2009).
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4114 Maize

Figure 34. Maize Planted Acres by County in the U.S.

Source: USDA-NASS, 2011
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4114 Maize

Taxonomic Classification of Maize and Related Wild Relatives

Taxonomically, maize (Zea mays L.) is a member of the Maydeae tribe of the grass family, 

Poaceae (OECD, 2003). Teosinte, within the genus Zea, and the genus Tripsacum are the 

closest relatives to maize taxonomically. The genus Tripsacum is also included in the Maydeae

tribe (OECD, 2003). Annual teosintes are grouped into the species Zea mays, although there is 

some dispute of this classification based on characteristics that prevent a high degree of 

introgression (OECD, 2003). Annual teosintes have been further classified into the subspecies 

Zea mays ssp. mexicana and Zea mays ssp. parviglumis (OECD, 2003). In contrast, perennial 

teosintes are classified as different species altogether: Zea perennis and Zea diploperennis

(OECD, 2003). Both annual and perennial teosintes are considered the closest wild relatives of 

cultivated maize (OECD, 2003). Perennial plants of the genus Tripsacum are considered the 

next closest relatives of maize (OECD, 2003). Neither the Zea genus nor the Tripsacum genus 

are listed as noxious weeds on the federal or state noxious weed lists (USDA-NRCS, 2011).

Potential for Gene Flow with the Genus Zea

Both annual and perennial teosintes are normally confined to the tropical and subtropical 

regions of Mexico, Honduras, Guatemala, and Nicaragua (Iltis, 2011). In the U.S., sparsely 

dispersed introduced populations of annual teosintes Zea mexicana (synonym: Zea mays ssp. 

mexicana) and Zea mays ssp. parviglumis have been reported in Florida, Maryland, and 

Alabama (USDA, 2011). Also, an isolated population of Zea perennis (perennial teosinte) has 

been introduced in South Carolina (USDA, 2011). While maize can hybridize with these species 

under natural conditions, there is incompatibility between some maize populations and certain 

types of teosinte that results in low fitness of some hybrids and prevents a high rate of 

introgression (OECD, 2003). Together with the very limited geographic range of the teosinte 

population in the U.S., the probability of gene flow from cultivated maize fields to these wild 

relatives is very low. 

Potential for Gene Flow with the Genus Tripsacum

Plants of the genus Tripsacum are mostly found in Mexico, Central, and South America (OECD, 

2003). Three of these species (T. dactyloides, T. floridanum, and T. lanceolatum) exist as native 

species populations in the continental U.S.; and two species (T. fasciculatum and T. latifolium) 

were introduced in Puerto Rico (USDA, 2011). T. dactyloides occurs throughout the eastern half 

of the U.S. T. lanceolatum occurs in Arizona and New Mexico (USDA, 2011) and T. floridanum is 

native to southern Florida (USDA, 2011). Although it is extremely difficult, Tripsacum species (T. 

dactyloides, T. floridanum, and T. lanceolatum) can be crossed with maize; however, hybrids 

have a high degree of sterility and are genetically unstable (OECD, 2003). Successful crosses of 

maize with Tripsacum species have been made experimentally, however such crosses are not 

known to occur in the wild (OECD, 2003). Therefore, gene flow between cultivated maize and 

relatives of the genus Tripsacum is highly unlikely.
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4114 Maize

Conclusions on the Potential for Gene Flow between 4114 Maize and Wild Relatives

The potential for gene flow between maize and relatives of the genera Zea and Tripsacum is 

very low. While wild native or introduced populations of these genera occur where maize is 

cultivated, limited geographic range and low fitness or sterility of hybrids prevent successful 

gene flow. Furthermore, none of these wild relatives are considered to be noxious weeds and 

4114 maize does not exhibit greater potential for weediness as determined from agronomic 

comparisons to conventional maize. Therefore, any incidental gene flow between 4114 maize 

and its wild relatives would not transform maize wild relatives into more weedy species, nor 

would the introduced trait be introgressed widely in wild relative populations. 

5-C. Potential Environmental Impact of the Introduced Proteins in 4114 Maize

4114 maize is comparable in agronomic characteristics and compositional characteristics to 

conventional maize except for the presence of the introduced proteins, Cry1F, Cry34Ab1, 

Cry35Ab1, and PAT. Therefore, one aspect of assessing the potential for the environmental 

impact of 4114 maize cultivation is to consider the potential risk of the introduced proteins to 

non-target organisms; this risk is quantified by examining the environmental exposure and the 

potential hazard of the introduced proteins to non-target organisms. As described earlier, the 

Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins in 4114 maize are identical to those in previously 

approved 1507 and/or 59122 maize, which USDA and EPA determined would not adversely 

impact the environment (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and 

Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). 

The impact of the PAT protein from 4114 maize on the environment is expected to be 

negligible. The PAT protein is not toxic or allergenic and its food, feed, and environmental 

safety is well known and documented (CERA, 2011; Hérouet et al., 2005; OECD, 1999). 

Furthermore, the PAT protein was evaluated for 1507 and 59122 maize and is not expected to 

have any significant impact on non-target organisms and threatened and endangered species 

(USDA-APHIS, 2001; USDA-APHIS, 2005). 

The Cry1F and the binary Cry34Ab1 and Cry35Ab1 proteins (i.e., the Cry34/35Ab1 protein) are 

known to have insecticidal activity against certain specific lepidopteran species and corn 

rootworm species, respectively. The specificity of this activity to target insects has been well 

established, and there is low risk of hazard to non-target orders, avian species, and mammals, 

as determined previously by USDA and EPA for 1507 and 59122 maize and by EPA for 

1507x59122 maize (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 

2000; USDA-APHIS, 2001; USDA-APHIS, 2005). With the exception of Cry1F protein 

concentrations in pollen, the concentrations of Cry1F and Cry34/35Ab1 in 4114 maize tissues 

are generally similar to or lower than 1507, 59122, and 1507x59122 maize, as shown in Section 

2-F. Therefore, the previously conducted environmental safety studies for 1507, 59122 and 

1507x59122 maize are relevant for evaluating the environmental impact of 4114 maize. The 

concentration of Cry1F protein in 4114 maize pollen is approximately 1.5 times that of 1507 or 

1507x59122 maize. Despite this increase, the laboratory tests still indicate a sufficient margin 
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of safety for the expected environmental exposure to the Cry1F protein from 4114 maize 

pollen.

Similar to the previous conclusions for 1507 and 59122 maize, the cultivation of 4114 maize is 

unlikely to have adverse effects on non-target arthropods, including threatened and 

endangered arthropods, and non-target vertebrates, including birds, mammal, and humans. In 

support of this conclusion, 1507x59122 maize has been commercially available in the U.S. since 

the 2006 growing season with no negative safety or environmental effects.

Environmental Exposure and Environmental Persistence

One aspect of evaluating the potential environmental impact of the introduced proteins

considers the probability and degree to which non-target organisms will be exposed to the 

proteins. Organisms can be exposed to the proteins in several ways. Animals and insects can 

feed on different parts of the plant and could be exposed to the proteins directly. Also, as the 

plant tissue residue degrades in the agricultural field, soil dwelling organisms may be exposed 

to these proteins if they persist in soil and aquatic organisms could be exposed from water 

runoff. Predatory insects may also be exposed to the Cry1F and Cry34/35Ab1 proteins from 

prey insects that feed on 4114 maize (i.e., tri-trophic interactions), if these proteins accumulate 

in the prey insects. 

For soil dwelling or aquatic organisms, protein exposure is highly dependent on protein 

degradation and dissipation in soils. Most Cry proteins do not persist or accumulate in soil over 

time (Clark et al., 2005; Icoz and Stotzky, 2008). Similarly, the soil dissipation of Cry1F and 

Cry34/35Ab1 proteins is very rapid. In data previously provided for 1507 and 59122 maize, the 

time required for 50% of the Cry1F protein activity to dissipate in soil (DT50) is 3.13 days and the 

DT50 for Cry34/35Ab1 proteins is approximately 3.2 days (Hunst and Rood, 2004; Shanahan and 

Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). Later published work concluded that 

the Cry1F protein activity dissipated even more quickly in soil; it took less than one day for 50% 

of the activity to dissipate (Herman et al., 2001). Additionally, no Cry1F protein was detected in 

soil sampled from fields following three years of successive cultivation of 1507 maize (Shan et 

al., 2008). Lack of accumulation in soil would limit the amount of protein present in field water 

runoff. Because of the rapid dissipation of activity in soil and the low potential for the proteins 

to accumulate in soil, exposure to Cry1F and Cry34/35Ab1 from soil would not be significant, 

nor would these proteins accumulate in aquatic habitats.

For protein exposure through tri-trophic interactions, the degree of persistence and 

accumulation of these proteins in the prey insect (i.e., bioaccumulate) will limit overall 

exposure to predatory insects. Proteins, in general, do not have the chemical characteristics 

needed to accumulate (Spacie et al., 1995; USGS, 2010). Lack of accumulation and short halflife has been confirmed for certain Cry proteins in prey species (Li and Romeis, 2010; Lundgren 

and Wiedenmann, 2005; Meissle and Romeis, 2009; Obrist et al., 2005; Romeis and Meissle, 

2011), there have been no studies using validated and robust ELISA or western blot methods 

that have indicated bioaccumulation of Cry proteins in prey foods. Exposure to Cry proteins via 
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4114 Maize

prey is a less significant route of exposure than direct feeding on plant material (e.g., pollen). 

Therefore, predatory insects are unlikely to be significantly exposed to Cry1F or 

Cry34/Cry35Ab1 proteins through tri-tropic interactions.

Potential environmental exposure of the introduced proteins can be estimated to determine 

the significance of laboratory safety testing conducted. The mean concentrations of the Cry1F, 

Cry34Ab1, and Cry35Ab1 proteins were measured in 4114 maize plant tissues (Section 2-F) and 

were used to determine or estimate an expected environmental concentration (EEC). In certain 

instances, the protein concentration in a particular tissue was used directly to determine the 

EEC, depending on the feeding habits or the preferred habitat of the non-target organism. In 

other cases, estimations were made to determine the EEC. EEC can be estimated by 

considering the protein concentrations in the plant tissue consumed or contacted and the 

amount of plant matter present in the environment. Certain factors can limit environmental 

exposure and can be used to refine estimations. Degree of pollen dispersal and deposition 

were considered for pollen consumption by certain non-target organisms. For EEC in soil and 

aquatic environments, field planting density and residual field plant matter were used to guide 

numerical estimates. As general consideration, the lack of overlap between geographic range 

or preferred habitat of the wildlife species to regions of maize cultivation would further limit 

potential environmental exposure. 

In summary, non-target organisms can be exposed to proteins from 4114 maize through direct 

consumption of plant tissue. In general, indirect exposure via soil and via tri-trophic 

interactions is not expected to be significant. In order to determine the significance of 

laboratory safety testing on non-target organisms, the potential environmental exposure for 

4114 maize was estimated from the mean Cry1F and Cry34/35Ab1 protein concentrations. 

Potential environmental exposure was considered in determining the potential impact on nontarget organisms. 
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4114 Maize

Potential Hazard of the Introduced Proteins on Non-Target Organisms

In order to determine the potential hazard of the Cry1F and Cry34/35Ab1 proteins may have on 

non-target organisms, early-tier laboratory studies using purified protein were conducted on 

representative species of interest and surrogate species. These species were selected to 

provide an evaluation of the effects of Cry1F and Cry34/35Ab1 on different families and orders 

of organisms. Surrogate species are typically selected because they are amenable to the 

laboratory setting, are environmentally sensitive and representative of the agroecosystem, and 

can be used to predict potential impacts on related non-target organisms, including beneficial, 

threatened, or endangered species (Romeis et al., 2011). 

Early-tier testing is typically done to simulate higher than expected environmental exposure, 

typically 10-fold (Rose, 2007). The 10-fold margin of exposure also accounts for interspecies 

variability (Romeis et al., 2011). The EPA has concluded that if there is no significant hazard 

(i.e., greater than 50% adverse effect) observed at these unrealistically high laboratory 

exposures, then the risk of hazard at environmentally relevant concentrations is very low (Rose, 

2007). Only when the early-tier hazard study detects an effect, should the next tier study be 

triggered (Romeis et al., 2008).

Early-tier laboratory testing was conducted for previously approved 1507 and 59122 maize 

(EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and Stauffer, 2000; USDA-APHIS, 

2001; USDA-APHIS, 2005). These studies include representative and surrogate species in order 

to predict the potential impacts of the Cry1F and Cry34/35Ab1 proteins on non-target 

organisms in the environment. These studies were considered in determining the potential 

hazard of the proteins and were used to determine the overall potential impact of 4114 maize 

on non-target organisms in the environment.

Potential Impact of the Cry1F and Cry34/35Ab1 Proteins on Non-Target Organisms

As determined for previously approved events 1507 and 59122 maize, the Cry1F and 

Cry34/35Ab1 proteins target certain specific lepidopteran species and corn rootworm species, 

respectively. In addition, the lack of adverse effects of these proteins on non-target organisms 

was confirmed through laboratory assays and field assessments. Based on these safety studies 

for 1507 and 59122 maize, the Cry1F and Cry34/35Ab1 proteins are not expected to adversely 

affect other invertebrates and all vertebrate organisms, including non-target birds, mammals, 

and humans, because of the high specificity of these insecticidal proteins to certain insect 

orders. 

Many of the previously conducted safety studies for 1507 and 59122 maize are relevant for 

evaluating the environmental impact of 4114 maize, because the concentrations of Cry1F and 

Cry34/35Ab1 in 4114 maize tissues are generally similar to or lower than 1507, 59122, and 

1507x59122 maize. One exception is the slight increase in Cry1F levels in pollen. Despite this 

increase, the laboratory tests still indicate a sufficient margin of safety for expected 

environmental concentration (EEC) to the Cry1F protein from 4114 maize pollen. Therefore, as 
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4114 Maize

with 1507 and 59122 maize, the margin of safety of the Cry1F and Cry34/35Ab1 proteins 

indicated that 4114 maize would not adversely affect non-target organisms and the 

environment.

An overview of specificity of the proteins and an overview of the potential impact of the 

proteins on non-target organisms is discussed further below. The safety studies conducted on 

the purified Cry1F and Cry34/35Ab1 proteins were used to assess the potential hazard on a 

number of representative non-target species (Tables 32 and 33). The potential exposure to the 

proteins was calculated by estimating the EEC. Margins of exposure for 4114 maize were 

calculated by dividing the no observable effect concentration (NOEC) or the lethal 

concentration (LC50) from the previously conducted safety studies for 1507 and 59122 maize by 

the EEC of each protein in 4114 maize. Both the potential hazard and the potential exposure 

were used to conclude the overall potential impact the proteins from 4114 maize would have 

on non-target organisms.

Specificity to Target Insect Pests

The insecticidal Cry1F and Cry34/35Ab1 proteins have been assessed by laboratory studies to 

evaluate the specificity of these proteins to target insects. In general, only insect species within 

a given order and closely related families are susceptible to a given insecticidal protein. 

Extensive studies have determined that Cry1F has specificity for certain lepidopteran target 

pests. Cry1F is EPA labeled for protection against European corn borer (Ostrinia nubilalis), fall 

armyworm (Spodoptera frugiperda), corn earworm (Helicoverpa zea), western bean cut worm 

(Striacosta albicosta Smith), black cutworm (Agrotis ipsilon), lesser corn stalk borer 

(Elasmopalpus lignosellus), southwestern corn borer (Diatraea grandiosella), and sugarcane 

borer (Diatraea saccharalis) (EPA, 2011).

Similarly, studies have confirmed the specificity of Cry34Ab1 and Cry35Ab1 for certain corn 

rootworm target pests. Cry34/35Ab1 is EPA labeled for protection against northern corn 

rootworm (Diabrotica barberi), western corn rootworm (Diabrotica virgifera virgifera), and 

Mexican corn rootworm (Diabrotica virgifera zeae) (EPA, 2011). Cry34/35Ab1 also has activity 

for southern corn rootworm (Diabrotica undecimpunctata howardi) as determined by use in 

bioassays (EPA, 2010b).

Along with the non-target assessments described below, the specificity of the Cry1F and 

Cry34/35Ab1 proteins to the lepidopteran insects and corn rootworm species, respectively,

provides support that these proteins are unlikely to pose a risk to unrelated non-target 

organisms in the environment.
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4114 Maize

Potential Impact on Soil Dwelling Non-Target Organisms

The potential hazard of the Cry1F and Cry34/35Ab1 proteins on two representative soil 

dwelling species, springtail and earthworm, was previously assessed for 1507 and 59122 maize 

though laboratory assays that used purified protein (EPA, 2010a; EPA, 2010b; Hunst and Rood, 

2004; Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). The results from 

the laboratory assays are given in Tables 32 and 33. Based on the calculation used by Hunst 

and Rood (2004) for 59122 maize, the EEC of the proteins in soil from 4114 maize was 

estimated based on factors such as planting density, dry weight of a maize plant, and mean 

protein concentrations in senescent (R6) maize tissue (Tables 32 and 33). None of the soil 

dwelling species tested showed significant adverse effects from the proteins and the margins of 

exposure were greater than 22-fold (Tables 32 and 33, Representative Soil Dwelling Species). 

Furthermore, as described earlier, soil dwelling non-target organisms are unlikely to be exposed 

to significant concentrations of the Cry1F and Cry34/35Ab1 proteins in soil, as realistic 

environmental exposures are expected to be significantly lower based on protein degradation 

and lack of accumulation in soil. 

Taking together the potential protein hazard and potential exposure in soil, soil dwelling nontarget invertebrates are unlikely to be impacted by the Cry1F and Cry34/35Ab1 proteins at the 

expected environmental concentrations for 4114 maize. 
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4114 Maize

Table 32. 4114 Maize Estimated Margins of Exposure to Non-Target Organisms (Cry1F)

Organism: Common 

Name

Species

(Order)

Cry1F Test 

Material

Expected 

Environmental 

Concentration 

(EEC) from 4114 

Maize

Results

Margin of 

Exposure 

(MOE)a

Representative Soil Dwelling Species

Springtail

Folsomia candida

(Collembola)

Purified protein in 

yeast diet

0.041 mg/kgbLC50 and NOEC 

>12.5 mg/kg 

diet

>300

Earthworm

Eisenia fetida

(Haplotaxida)

Purified protein in 

soil

0.041 mg/kgbLC50 and NOEC 

>2.5 mg/kg dry 

soil

>60

Representative Coleopteran Species

Ladybird beetle

Hippodamia convergens

(Coleoptera)

Family: Coccinellidae

Purified protein in 

honey diet

35 μg/g Cry1F in 

pollenc

LC50 >480 ppm 

diet

>13

Representative Lepidopteran Species

Monarch butterfly 

Danaus plexippus

(Lepidoptera)

Purified protein in 

artificial diet

0.5 ng/mg Cry1F 

from pollen 

deposition on 

milkweed host 

plantd

LC50 > 10 ppm 

(10,000 

ng/mL); 

NOEC < 10 ppm

20

Representative Arthropod Species

Parasitic Hymenoptera

Nasonia vitripennis

(Hymenoptera)

Family: Pteromalidae

Purified protein in 

honey diet

35 μg/g Cry1F in 

pollenc

LC50 >320 ppm 

diet

>9

Green lacewing

Chrysoperla carnea

(Neuroptera)

Family: Chrysopidae

Purified protein in 

moth egg diet

35 μg/g Cry1F in 

pollenc

LC50 >480 ppm 

diet

>13

Honeybee

Apis mellifera

(Hymenoptera)

Family: Apidae

Purified protein in 

sucrose solution

35 μg/g Cry1F in 

pollenc

NOEC >640 ng 

per larvaee

>9d
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4114 Maize

Table 32. 4114 Maize Estimated Margins of Exposure to Non-Target Organisms (Cry1F)

(continued)

Organism: Common 

Name

Species

(Order)

Cry1F Test 

Material

Expected 

Environmental 

Concentration 

(EEC) from 4114 

Maize

Results

Margin of 

Exposure 

(MOE)a

Representative Arthropod Species

Water flea

Daphnia magna

(Cladocera)

Family: Daphniidae

Purified protein 

dissolved in water

0.0046 μg/ml Cry1FfLC50 and NOEC 

>100 mg/L

>21000

a

The margin of exposure is calculated by dividing the NOEC (or LC50) by the EEC

b For 4114 maize, the concentration of Cry1F protein in soil can be calculated based on estimating the amount of 

protein from senescent tissue in a field. Senescent tissue mean concentration is for R6 whole plant tissue as 

presented in Section 2-F. A modified version of the calculation used by Hunst and Rood (2004) was used to 

calculate the concentration of protein in soil. New assumptions were used for this calculation; plants per acre 

was increased to 30,000, based on current average maize plant population density (Farnham, 2001; Nielsen and 

Thomison, 2002) and the weight of the maize plant was now based on dry weight, since protein concentration is 

based on dry weight (Nguyen and Jehle, 2007). Protein in soil: (30,000 plants/acre x 300 g/plant (dry weight) x 

4.1 μg/g Cry1F R6 whole plant tissue)/9.08 x 105kg soil/acre (Hunst and Rood, 2004). This number is 0.041 

mg/kg soil.

c Mean concentration as reported in Section 2-F.

d This number is calculated based on a number of metrics: the weight of an average pollen grain is 250 ng 

(Fonseca et al., 2003); the high pollen deposition rate in a maize field is 1000 pollen grains/cm2 (Pleasants et al., 

2001); the approximate weight of a milkweed leaf (17.7 mg/cm2); and the amount of Cry1F per pollen grain 

based on the mean concentration as presented in Section 2-F and the weight of a pollen grain (0.0088 ng

Cry1F/grain). The EEC = [0.0088 ng Cry1F/pollen grain X 1000 pollen grains/cm2]/17.7 mg/cm2.

e The MOE assumes that honey bee larvae will ingest 2 mg of pollen (dividing the final number by a factor of two). 

Taking this behavioral component into consideration, the tested concentration would be equivalent to an MOE 

of 9. However, if the pollen consumption is not factored in, the tested concentration would be equivalent to an 

MOE of 18. 

f

Estimate based on the protein from 4114 senescent maize tissue moving into a neighboring aquatic 

environment, using worst-case assumptions modified from the GENEEC model and Raybould and Vlachos (2011), 

as follows: 1) assume 10% of senescent maize tissue from a 10 hectare field will be deposited into a 1 hectare x 

2 meter deep pond (20,000,000 L); 2) assume there are 30,000 plants per acre (average plant population density, 

as above), which is equivalent to 74,100 plants per hectare (1 hectare = 2.47 acres); 3) assume the dry weight of 

a maize plant is 300 g, since protein concentrations are expressed in dry weight, as above. Cry1F concentration 

in a pond = [4.1 μg/g (dry weight) X 74,100 plants/ha X 300 g/plant]/20,000,000L. Based on these assumptions, 

the concentration of Cry1F that would enter a pond under worst-case assumptions is 0.0046 μg/ml. The 

additional contribution of Cry1F protein via aerial deposition of maize pollen is negligible (low part per billion).
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4114 Maize

Table 33. 4114 Maize Estimated Margins of Exposure to Non-Target Organisms (Cry34/35Ab1)

Organism: Common 

Name

Species

(Order)

Cry34/35Ab1 

Test Material

Expected 

Environmental 

Concentration 

(EEC) from 4114 

Maize

Results

Margin of 

Exposure 

(MOE)a

Representative Soil Dwelling Species

Springtail

Folsomia candida

(Collembola)

Purified protein in 

yeast diet

0.56 mg/kg in soilbLC50 and NOEC 

>12.7 mg/kg 

diet

>22

Earthworm

Eisenia fetida

(Haplotaxida)

Purified protein in 

soil

0.56 mg/kg in soilbLC50 and NOEC 

>25.3 mg/kg dry 

soilc

>45

Representative Coleopteran Species

Ladybird beetle

Hippodamia convergens

(Coleoptera)

Family: Coccinellidae

Purified protein in 

sugar water diet

9.54 μg/g 

Cry34/35Ab1 in 

pollend

LC50 and NOEC 

>280 μg/mL diet

>29

Ladybird beetle

Coleomegilla maculata

(Coleoptera)

Family: Coccinellidae

Purified protein in 

artificial diet

9.54 μg/g 

Cry34/35Ab1 in 

pollend

LC50> 901 ppm

EC50 < 901ppm

94

Representative Arthropod Species

Parasitic Hymenoptera

Nasonia vitripennis

(Hymenoptera)

Family: Pteromalidae

Purified protein in 

sugar water diet

9.54 μg/g 

Cry34/35Ab1 in 

pollend

LC50 >280 μg/mL 

diet

>29

Green lacewing

Chrysoperla carnea

(Neuroptera)

Family: Chrysopidae

Solution of purified 

protein in moth 

egg diet

9.54 μg/g 

Cry34/35Ab1 in 

pollend

LC50 and NOEC 

>280 ppm diet

>29

Honeybee

Apis mellifera

(Hymenoptera)

Family: Apidae

Purified protein in 

sucrose solution

9.54 μg/g 

Cry34/35Ab1 in 

pollend

NOEC >5600 ng 

per larvaee

>294f
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4114 Maize

Table 33. 4114 Maize Estimated Margins of Exposure to Non-Target Organisms (Cry34/35Ab1)

(continued)

Organism: Common 

Name

Species

(Order)

Cry34/35Ab1 

Test Material

Expected 

Environmental 

Concentration 

(EEC) from 4114 

Maize

Results

Margin of 

Exposure 

(MOE)a

Representative Arthropod Species

Water flea

Daphnia magna

(Cladocera)

Family: Daphniidae

Purified protein 

dissolved in water

0.0634 μg/ml 

Cry34/35Ab1f

LC50 and NOEC 

>100 mg/L

>1500

a The MOE is calculated by dividing the NOEC (or LC50) by the EEC

b For 4114 maize, the concentration of Cry34/35Ab1 protein in soil can be calculated based on estimating the 

amount of protein from senescent tissue in a field. Senescent tissue concentration is the sum of the mean 

concentrations of Cry34Ab1 and Cry35Ab1 in R6 whole plant tissue as presented in Section 2-F. A modified 

version of the calculation used by Hunst and Rood (2004) was used to calculate the concentration of protein in 

soil. New assumptions were used for this calculation; plants per acre was increased to 30,000, based on current 

average maize plant population density (Farnham, 2001; Nielsen and Thomison, 2002) and the weight of the 

maize plant was now based on dry weight, since protein concentration is based on dry weight (Nguyen and 

Jehle, 2007). Protein in soil: [30,000 plants/acre x 300 g/plant (dry weight) x (36 μg/g Cry34Ab1 + 21 μg/g 

Cry35Ab1 R6 whole plant tissue)]/9.08 x 105kg soil/acre. This number is 0.56 mg/kg soil.

c The final concentration of the experiment was previously reported as 76 mg/kg (EPA, 2010b); however, the 

original calculation was based on 76 mg in 3 kg of soil; therefore, the final concentration in the soil was 25.3 

mg/kg.

d This concentration is the sum of the individual protein mean concentrations as reported in Section 2-F. 

Cry34Ab1 expression in pollen is 9.2 μg/g and Cry35Ab1 expression is 0.34 μg/g.

e The MOE assumes that honey bee larvae will ingest 2 mg of pollen (dividing the final number by a factor of two). 

Taking this behavioral component into consideration, the tested concentration would be equivalent to an MOE 

of 294. However, if the pollen consumption is not factored in, the tested concentration would be equivalent to 

an MOE of 587. 

f Estimate based on the protein from 4114 senescent maize tissue moving into a neighboring aquatic 

environment, using worst-case assumptions modified from the GENEEC model and Raybould and Vlachos (2011), 

as follows: 1) assume 10% of senescent maize tissue from a 10 hectare field will be deposited into a 1 hectare x 

2 meter deep pond; 2) assume there are 30,000 plants per acre (average plant population density, as above), 

which is equivalent to 74,100 plants per hectare (1 hectare = 2.47 acres); 3) assume the dry weight of a maize 

plant is 300 g, since protein concentrations are expressed in dry weight, as above. Cry34/35Ab1 concentration 

in a pond = [(36 μg/g Cry34Ab1 + 21 μg/g Cry35Ab1) (dry weight) X 74,100 plants/ha X 300 g/plant]/20,000,000L.

Based on these assumptions, the concentration of Cry34/35Ab1 protein that would enter a pond under worstcase assumptions is 0.0634 μg/ml. The additional contribution of Cry34/35Ab1 protein via aerial deposition of 

maize pollen is negligible (low part per billion).
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4114 Maize

Potential Impact on Non-Target Coleoptera

Toxicity of the Cry1F and Cry34/35Ab1 protein on representative coleopteran species was 

evaluated for both 1507 and 59122 maize (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; 

Shanahan and Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). Hippodamia convergens

was tested with the Cry1F and Cry34/35Ab1 proteins (Tables 32 and 33, respectively). In 

addition, Coleomegilla maculata was also tested for Cry34/35Ab1 (Table 33). The results from 

the laboratory assays are given in Tables 32 and 33. The EEC of the proteins is based on the 

mean concentrations of the proteins in 4114 maize pollen (Section 2-F). None of the 

coleopteran species tested showed significant adverse effects from the proteins and the 

margins of exposure were greater than 13-fold (Tables 32 and 33, Representative Coleopteran 

Species).

In addition, the EPA reviewed data from two studies that provided additional evidence for lack 

of impact of Cry34/35Ab1 on non-target Coleoptera. In one study, the toxicity of the purified 

Cry34/35Ab1 protein to carabid beetles (Poecilus cupreus) was assessed (EPA, 2010b). No 

statistically significant differences were observed in the populations fed Cry34/35Ab1 and the 

control. This study confirmed that, at field concentrations, there would be no impact of the 

proteins on carabid beetles. In the second study, the possibility of impact of the Cry34/35Ab1 

protein in predator-prey feeding interactions was examined (EPA, 2010b). There was no effect 

on ladybird beetle (Coleomegilla maculata) larvae that were consuming aphids that had been 

feeding on maize plants expressing the Cry34/35Ab1 protein. Because levels of the 

Cry34/35Ab1 protein in 4114 maize pollen are comparable to or lower than those of 59122 

maize, as described in Section 2-F, these studies and their conclusions are relevant to 4114 

maize.

Based on these studies, non-target coleopteran species are unlikely to be impacted by the 

Cry1F and Cry34/35Ab1 proteins at the expected environmental concentrations for 4114 maize.

Potential Impact on Non-Target Lepidoptera

Due to the established specificity of the Cry1F protein for Lepidoptera, the potential impact of

1507 maize on a representative lepidopteran species of conservation interest, the monarch 

butterfly (Danaus plexippus), was studied. The data indicated that 1507 maize would not pose 

a risk to monarch butterfly larvae (EPA, 2010a; Shanahan and Stauffer, 2000; USDA-APHIS, 

2001).

The most likely route of exposure of non-target Lepidoptera to transgenic proteins from 4114 

maize would be via pollen deposition on host plants (i.e., milkweed) in maize fields and in field 

margins. Weed control practices make the likelihood of having host plants growing within the 

confines of the maize field remote; therefore, the most realistic exposure of monarch butterfly 

to maize pollen would be via incidental ingestion of maize pollen on milkweed plants located in 

field margins. The density of maize pollen deposition within maize fields and field margins has 
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4114 Maize

been extensively studied (Pleasants et al., 2001) and these values can be used to estimate the 

EEC for monarch butterfly to maize pollen. Pollen deposition rates were reported to be 1000 

(high in-field), 171 (in-field average), and 63 (field edge) pollen grains/cm2(Pleasants et al., 

2001). In order to calculate the EEC and margin of exposure, the 1000 grains/cm2rate was used 

(Table 32); however, the more realistic case of pollen deposition would be the value at the field 

edge. The EEC was calculated based on a number of metrics: the weight of an average pollen 

grain; a high pollen deposition rate in a maize field; the approximate weight of a milkweed leaf 

per cm2; and the amount of Cry1F per pollen grain based on the concentration as presented in 

Section 2-F (Table 32).

The results of the monarch butterfly study are provided in Table 32. Based on the

concentration tested in the study and the EEC, the margin of exposure was calculated to be 20-

fold (Table 32).

Furthermore, a laboratory feeding study was conducted that exposed monarch butterfly larvae 

to 1507x59122 maize pollen, containing both the Cry1F and Cry34/35Ab1 proteins, at up to 

1600 pollen grains/cm2on milkweed leaves (EPA, 2010b). The results of the feeding study 

showed no reduction in the survival, weight gain, development, and leaf consumption of the 

larvae and no sub-chronic effects were seen (EPA, 2010b). The pollen deposition was higher 

than the maximum expected pollen deposition rate described above and the assay also 

contained the Cry1F and Cry34/35Ab1 proteins; therefore, the results of this study would be 

applicable to assessment of 4114 maize. Based on these studies, non-target lepidopteran 

species are unlikely to be impacted by incidental exposure to the Cry1F and Cry34/35Ab1 

proteins from 4114 maize pollen at the expected pollen deposition rates.

Potential Impact on Other Non-Target Arthropods

Laboratory assays for four representative arthropods of the orders Hymenoptera (parasitic 

hymenoptera, Nasonia vitripennis, and honeybee, Apis mellifera), Neuroptera (green lacewing, 

Chrysoperla carnea), and Cladocera (Daphnia magna) were also conducted using purified Cry1F 

and Cry34/35Ab1 proteins at doses exceeding expected environmental exposures (Tables 32

and 33). These studies were submitted for 1507 and 59122 maize and supported the 

conclusion that Cry1F and Cry34/35Ab1 exposure does not have an adverse impact on nontarget, beneficial arthropods (EPA, 2010a; EPA, 2010b; Hunst and Rood, 2004; Shanahan and 

Stauffer, 2000; USDA-APHIS, 2001; USDA-APHIS, 2005). The results from these assays are

provided in Table 32 and 33. For three of the species tested (parasitic hymenoptera, green 

lacewing, and honeybee), the EEC of the proteins was based on the concentrations of the 

proteins in 4114 maize pollen (Section 2-F). For Daphnia magna, the potential EEC in an aquatic 

environment was estimated from a modified GENEEC model, which was used to make worstcase estimates for the amount of protein that could move into pond water from a neighboring 

field (Raybould and Vlachos, 2011). None of the other arthropod species tested showed 

significant adverse effects from the proteins and the margins of exposure were greater than 9-

fold (Tables 32 and 33, Representative Arthropod Species).
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4114 Maize

In addition, to further test the impacts of Cry34/35Ab1 on non-target arthropods, EPA reviewed 

an additional study on the minute pirate bug (Orius insidiosus) in the order Hymenoptera (EPA, 

2010b). EPA concluded that at field concentrations, it is unlikely that Cry34/35Ab1 would have 

any adverse effects on this insect.

These studies have relevance for 4114 maize, expressing the Cry1F and Cry34/Cry35Ab1 

proteins. Based on the conclusions of these studies, it is unlikely that 4114 maize would have 

an adverse impact on non-target, beneficial arthropods.

Field Monitoring for Effects of Cry1F and Cry34/35Ab1 on Non-Target Arthropods

In addition to early tier laboratory studies, multiple field studies have been conducted to 

investigate the potential effects of cultivating transgenic maize hybrids containing an insectresistant protein on non-target arthropod abundance. One field study reviewed by USDA 

showed that 1507 maize expressing the Cry1F protein had no effect on beneficial arthropod 

abundance (Shanahan and Stauffer, 2000; USDA-APHIS, 2001). A second three-year field study

showed no impact on abundance of several different non-target organisms, including 

detritovores, herbivores, and predatory arthropods, by comparing fields of 1507 maize with 

near-isoline control fields (Higgins et al., 2009). 

Similar field studies were also conducted for 59122 maize and were reviewed by different U.S. 

regulatory agencies; results indicated that 59122 maize expressing the Cry34/35Ab1 proteins 

had no effect on beneficial arthropod abundance (EPA, 2010b; Hunst and Rood, 2004; USDAAPHIS, 2005). A three-year field study was evaluated by the EPA to determine the impact of 

59122 maize, expressing the Cry34/Cry35Ab1 proteins, and 1507x59122 maize, expressing both 

Cry1F and Cry34/35Ab1 proteins, on non-target arthropods after continuous cropping at three 

locations in Iowa, Nebraska, and Indiana (EPA, 2010b). Similarly, the results showed no impact 

on non-target arthropod populations between fields of the control maize and 59122 and 

1507x59122 maize hybrids (EPA, 2010b). The EPA concluded in their assessment that there is 

negligible potential for long-term adverse effects on non-target arthropods after continuous 

cultivation of both a maize line containing the Cry34/Cry35Ab1 proteins and a stacked line that 

includes the Cry34/35Ab1 and Cry1F proteins (EPA, 2010b). 

Results from these field studies confirm laboratory testing and support the hypothesis that 

1507 and 59122 maize are unlikely to adversely impact non-target arthropods occurring in 

maize fields. Because of comparable Cry1F and Cry34/35Ab1 protein expression, 4114 maize is 

not expected to adversely impact beneficial non-target arthropods in fields. 
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4114 Maize

Potential Impact of 4114 Maize on Threatened and Endangered Arthropods

The impact of 4114 maize on threatened and endangered arthropods can also be evaluated by 

comparing to previous assessments of 1507 and 59122 maize. These assessments concluded 

that threatened and endangered species would unlikely be impacted by the cultivation of either 

maize line (EPA, 2010a; EPA, 2010b; USDA-APHIS, 2001; USDA-APHIS, 2005).

As of July 2011, 60 insect species are listed on the US Fish and Wildlife Service website as 

threatened or endangered (US FWS, 2011). Twenty-one of these insects are in the order 

Lepidoptera and 15 are in Coleoptera, with the remaining representing insects of other orders. 

Almost all of the species of Lepidoptera and Coleoptera currently listed were added prior to the 

deregulation of 1507 and 59122 maize, except for one coleopteran species, the Salt Creek Tiger 

Beetle (Cicindela nevadica lincolniana; Family: Carabidae) (US FWS, 2010; US FWS, 2011).

For 1507 maize, USDA considered the impact of the Cry1F protein on two lepidopteran species, 

the Karner blue butterfly (Lycaeides melissa samuelis) and Mitchell’s satyr butterfly (US FWS, 

2011; USDA-APHIS, 2001). USDA concluded that both species would not be expected to be 

present in or close to maize fields; therefore it is unlikely there would be any impact of 1507 

maize cultivation (USDA-APHIS, 2001). For 59122 maize, USDA considered the impact on one 

primary threatened and endangered coleopteran species, the American burying beetle (US 

FWS, 2011; USDA-APHIS, 2005). From review of preferred habitats, this beetle was unlikely to 

be found in active maize fields and therefore would not be exposed significantly to the 

Cry34/35Ab1 protein from 4114 maize (USDA-APHIS, 2005).

For other lepidopteran and corn rootworm resistant maize events, EPA has made similar 

conclusions for the Karner blue butterfly and the American burying beetle and has not 

identified any new threatened or endangered species that would be impacted by cultivation 

(EPA, 2009; EPA, 2010b; EPA, 2010c). Furthermore, EPA examined the habitats of the other 

threatened and endangered insect species in the orders Diptera, Hemiptera, Odonata and 

Orthoptera and found that they primarily occupy dune, meadow or prairie, or open forest 

habitats and are not closely associated with row crop production, often times due to the 

specificity of the habitat of their host plants (EPA, 2009; EPA, 2010b; EPA, 2010c).

As mentioned earlier, only one coleopteran species, the Salt Creek Tiger Beetle (Cicindela 

nevadica lincolniana; Family: Carabidae), was added in 2005 to the threatened and endangered 

insect species list (US FWS, 2010; US FWS, 2011). This beetle is found in Nebraska (Lancaster 

and Saunders counties), the third largest state for maize cultivation (US FWS, 2010; USDA-NASS, 

2010). However, the beetle’s critical habitats have been characterized as non-vegetated stream 

banks or edges that are in saline or freshwater wetlands and the beetles prefer to be within a 

few meters of these habitats (US FWS, 2010). Wetland areas, stream edges, or saline wetlands 

are unlikely to be planted with maize. In addition, the beetle family is Carabidae, which is 

unrelated to those of corn rootworm species (Chrysomelidae). Therefore, it is highly unlikely 

that this beetle will be exposed to the Cry34/35Ab1 protein from 4114 maize or will be 

impacted due to the specificity of the Cry34/35Ab1 protein to corn rootworm species.
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4114 Maize

Similar to the conclusions for 1507 and 59122 maize, it is unlikely that threatened and 

endangered species such as the Karner blue and Mitchell’s satyr butterflies and the American 

burying beetle would be impacted by 4114 maize cultivation. Based on critical habitat and the 

Cry34/35Ab1 target insect specificity, it is also unlikely that the Salt Creek Tiger Beetle would be 

adversely affected. Furthermore, the target specificity of the introduced proteins and the lack 

of habitat overlap with regions of maize cultivation also support the conclusion that 4114 maize 

would not adversely impact the other listed threatened and endangered insects.

Potential Impact on Non-Target Vertebrate Species

Wildlife in and around maize fields are of interest due to potential exposure to 4114 maize 

grain and plant residue. A wide variety of toxicology studies conducted over the past 40 years 

have established the safety of the microbial preparations of B. thuringiensis, including their 

expressed insecticidal Cry proteins (Betz et al., 2000). As discussed in Section 3, the 

allergenicity and toxicity of the Cry1F, Cry34Ab1, and Cry35Ab1 proteins has been assessed and 

all three are unlikely to be potential allergens or toxins to humans or animals. Based on quail 

and mouse toxicological studies for Cry1F, both EPA and USDA concluded that the protein was 

unlikely to have an adverse effect on vertebrates, including non-target birds, mammals, and 

humans (EPA, 2010a; USDA-APHIS, 2001). EPA and USDA made similar conclusions for 

Cry34Ab1 and Cry35Ab1 on the basis of poultry, mouse, and rainbow trout toxicological studies 

(EPA, 2010b; USDA-APHIS, 2005). Providing additional evidence for the lack of toxicity of the 

Cry1F and Cry34/35Ab1 proteins, recently published rodent feeding studies showed no 

toxicological effects of diets containing 1507 and 1507x59122 maize grain (Appenzeller et al., 

2009; MacKenzie et al., 2007). Additional feeding studies for laying hens, swine, beef cattle, 

and dairy cows with diets containing 1507 and 59122 maize have all shown no differences in 

nutritional equivalency from diets containing conventional maize (Faust et al., 2007; Huls et al., 

2008; Jacobs et al., 2008; Scheideler et al., 2008; Sindt et al., 2007; Stein et al., 2009).

Results from these studies are relevant for 4114 maize, which expresses the Cry1F, Cry34Ab1, 

and Cry35Ab1 proteins, and provide evidence that 4114 maize is unlikely to have adverse 

effects on non-target vertebrate species, including birds, mammals, and humans.
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4114 Maize

Conclusions for the Environmental Impact of the Introduced Proteins in 4114 Maize

The environmental impact of 4114 maize focused on the introduced Cry1F and Cry34/35Ab1 

proteins and considered the potential hazard and the level of environmental exposure to nontarget organisms. Due to the presence of the same proteins and similar or lower protein 

expression, the previously conducted environmental safety studies for 1507, 59122, and 

1507x59122 maize were relevant to the assessment of 4114 maize. As previously established 

for 1507 and 59122 maize, the Cry1F and Cry34/Cry35Ab1 proteins have insecticidal activity 

against lepidopteran and corn rootworm species, and there is low risk of hazard to non-target 

orders. The low hazard to several representative non-target organisms was confirmed in the 

laboratory at protein concentrations greater than those expected in the environment and the 

potential environmental exposure to the Cry1F and Cry34/35Ab1 proteins was determined. 

Based on this analysis, there is a sufficient margin of exposure for the proteins in the 

environment and, therefore, the cultivation of 4114 maize is unlikely to adversely impact nontarget organisms. Likewise, multiple field studies have also confirmed that the abundance of 

non-target organisms under field conditions is not impacted by maize expressing the Cry1F and 

Cry34/35Ab1 proteins. Similar to the conclusions for 1507 and 59122 maize, the cultivation of 

4114 maize is unlikely to have adverse effects on non-target arthropods, including threatened 

and endangered arthropods, and non-target vertebrates, including birds, mammal, and 

humans. 
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4114 Maize

6. Overall Conclusions

In conclusion, based on the data contained herein, Pioneer requests that APHIS grant the 

request for a determination of nonregulated status for 4114 maize and any crosses of this line 

with other nonregulated Zea mays. 4114 maize was modified to produce the Cry1F, Cry34Ab1, 

Cry35Ab1, and PAT proteins; these proteins and associated genetic elements in 4114 maize are 

identical to those in previously approved 1507, 59122, and 1507x59122 maize. In addition, the 

Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins are unlikely to be allergens or toxins and are safe 

for the food and feed supply. 

4114 maize contains all the gene cassettes that express the Cry1F, Cry34Ab1, Cry35Ab1, and 

PAT proteins at a single genetic locus. Furthermore, molecular characterization confirmed that 

4114 maize contains a single copy, intact insertion that is stable and segregates according to 

Mendel’s laws of genetics. 

Nutrient composition and agronomic comparative assessments were conducted to determine if 

4114 maize would present any new or greater risks relative to maize varieties that have a 

history of safe use in the environment and as food and feed. These analyses indicated that 

4114 maize is as safe as conventional maize varieties and does not pose a greater risk than 

conventional maize varieties in food, feed, and the environment.

It is unlikely that 4114 maize will pose a plant pest risk or impact non-target organisms in the 

environment. Maize is not considered a weed, and 4114 maize does not exhibit any 

characteristics that would indicate it is any more likely than conventional maize to become a 

weed or plant pest. Because sexually compatible wild relative populations are limited in the 

U.S. and there is low potential for gene flow, it is unlikely that the inserted DNA in 4114 maize 

would be introgressed significantly into these wild relative populations. Based on the evidence 

supporting the environmental safety of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins, it is 

unlikely that 4114 maize will pose a risk to non-target organisms including beneficial, 

threatened and endangered species, as well as non-target vertebrates including birds, 

mammals, and humans.

In support for the safety of 4114 maize, 1507 maize has been commercially available in the U.S. 

since the 2003 growing season and 59122 and 1507x59122 maize have been available in the 

U.S. since 2006 with no adverse food, feed, or environmental effects. Maize products 

containing 1507x59122 maize were grown on more than 14 million acres in 2010.
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4114 Maize

Appendix 1. 4114 Maize USDA Release Permits and Notifications and Planted Acreage

Plantings through May 25, 2011 are listed

Year of 

Planting

Permit Name Permit 

Valid Date State Number of Counties Where 4114 Maizea was Planted Acreage

2006 06-019-03R 5/2/2006 PR 2 0.010

06-019-04R 5/3/2006 HI 1 0.014

2007

06-019-03R 5/2/2006 CA 1 0.009

PR 1 0.039

06-019-04R 5/3/2006 HI 1 0.039

07-040-101rm 5/3/2007

CA 1 0.009

HI 1 0.146

IA 2 0.072

IL 4 0.092

IN 1 0.030

MN 1 0.030

MO 1 0.020

NE 1 0.020

PR 2 0.110

WI 1 0.020

2008

07-040-101rm 5/3/2007 HI 1 0.020

PR 1 0.130

08-014-111n 4/16/2008

CA 1 0.050

IA 2 0.230

IL 4 0.270

IN 2 0.160

MN 1 0.012

MO 1 0.060

NE 2 0.110

PR 2 1.510

WI 1 0.070

08-014-131n 3/31/2008 HI 1 0.280

08-095-105n 5/14/2008

IA 1 0.070

IL 2 0.060

MN 1 0.014

NE 1 1.077

OK 1 0.050

TX 1 0.040

2009

08-014-111n 4/16/2008 PR 1 0.001

09-013-108n 4/1/2009

IA 5 0.750

IL 6 0.370

IN 2 0.280

MO 1 0.114

NE 1 0.186

09-016-103n 2/15/2009 IL 1 0.032

NE 1 0.023
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4114 Maize

4114 Maize USDA Release Permits and Notifications and Planted Acreage (continued)

Year of 

Planting Permit Name Permit Valid Date State Number of Counties Where 4114 Maizea was Planted Acreage

2009

09-035-109n 3/17/2009

AR 1 0.018

CO 1 0.057

DE 1 0.020

GA 1 0.129

HI 1 0.128

KS 1 0.018

MN 1 0.134

PR 3 2.980

TN 1 0.018

WI 1 0.098

09-264-102n 10/21/2009 HI 1 0.064

PR 2 2.370

2010

09-264-102n 10/21/2009 HI 1 0.083

PR 1 0.281

10-015-106n 3/1/2010b

IA 3 0.090

IL 3 0.080

IN 1 0.011

MI 1 0.011

MN 1 0.034

NE 1 1.057

10-050-115n 3/9/2010b NE 1 0.016

10-052-101n 3/19/2010b

AR 1 0.017

CA 1 0.200

CO 2 0.060

GA 1 1.006

HI 1 0.630

IA 9 2.500

IL 6 2.210

IN 4 0.920

KS 1 0.224

MI 1 0.286

MN 2 0.310

MO 3 0.540

NE 1 0.619

PA 1 0.186

PR 4 1.020

SD 1 0.039

TN 1 0.068

WI 1 0.372

10-118-105n 5/6/2010bIL 1 0.186

10-281-101n 10/30/2010bPR 1 0.069

10-284-101n 11/16/2010bPR 2 1.470
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4114 Maize

4114 Maize USDA Release Permits and Notifications and Planted Acreage (continued)

Year of 

Planting Permit Name Permit Valid Date State Number of Counties Where 4114 Maizea was Planted Acreage

2011

11-040-123n 3/17/2011bPR 2 0.404

10-281-101n 10/30/2010bTN 1 0.104

10-284-101n 11/16/2010b HI 1 0.030

PR 1 0.684

11-019-109n 3/1/2011b

IA 1 0.023

IL 2 0.046

NE 1 0.023

OK 1 0.023

TX 1 0.003

11-040-123n 3/17/2011b

CO 1 0.049

GA 1 0.260

HI 1 0.698

IA 7 2.880

IL 6 4.019

IN 5 2.114

KS 1 0.654

MI 1 0.554

MN 2 2.137

MO 3 1.182

NE 1 0.847

SD 1 0.498

TN 1 0.041

WI 2 0.518

11-040-124n 3/17/2011b HI 1 0.033

IA 1 0.002

a 4114 maize is also referred to as DP-004114-3 and EA-2244.04.1.14 in USDA final reports.

b Final field permit test reports not yet due to USDA.
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4114 Maize

Appendix 2. Equivalency of the Proteins Produced in 4114 Maize to Those Produced in 

1507 and 59122 Maize

2.1. Overview and Summary

As discussed earlier in Section 2, the introduced cry1F, cry34Ab1, cry35Ab1, and pat gene 

cassettes in 4114 maize are identical to those cassettes in 1507 and/or 59122 maize. Based on 

the DNA sequence of the genes, the translated protein sequences are also identical. Prior 

protein safety studies for 1507 and 59122 maize were conducted on the Cry1F, Cry34Ab1, 

Cry35Ab1, and PAT proteins and are described in Section 3. These protein safety studies 

confirmed that none of the proteins are potential allergens or toxins. In order to verify the 

applicability and relevance of these protein safety studies for 4114 maize, the identity and 

equivalency of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins should be established. 

In order to verify the identity of the introduced proteins in 4114 maize and the equivalency to 

the proteins in 1507 and/or 59122 maize, western blot analysis of the Cry1F, Cry34Ab1, 

Cry35Ab1, and PAT proteins was conducted in sample extracts of 4114 maize and 1507x59122 

maize. The western blot analysis demonstrated that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT 

proteins derived from 4114 maize have equivalent molecular weights and immunoreactivity to 

the proteins expressed in 1507x59122 maize. In all four western blots, the near-isoline control 

showed no immunoreactive proteins. 

As described in Section 2, the western blot analysis in conjunction with other analyses 

confirmed that the proteins in 4114 maize are the same as those of 1507 and/or 59122 maize; 

therefore, prior protein safety studies conducted for 1507 and/or 59122 maize on Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT are relevant to the safety assessment of 4114 maize. 

2.2. Western Blot Analysis of the Introduced Proteins

Cry1F

Western blot analysis using anti-Cry1F antibodies (Figure 2.1) demonstrated that the Cry1F 

protein derived from 4114 maize (Lane 3) had the same molecular weight and relative 

immunoreactivity as the Cry1F protein derived from 1507x59122 maize (Lane 4). Both protein 

samples migrated as two bands of approximately 60 kDa and 62 kDa in size. The double 

banding pattern was expected because plant-derived Cry1F protein can be partially degraded 

by plant proteases to a smaller, more stable truncated protein, therefore appearing as two 

bands (the larger intact protein and the smaller truncated protein). Relative amounts of the 

two bands can vary from sample to sample. The Cry1F standard protein included on the blot 

(Lane 2) consisted of the truncated form of the protein and migrated at the expected 

approximately 60 kDa.
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4114 Maize

Cry34Ab1

Western blot analysis using anti-Cry34Ab1 antibodies (Figure 2.2) demonstrated that the 

Cry34Ab1 protein derived from 4114 maize (Lane 3) had the same molecular weight and 

relative immunoreactivity as the Cry34Ab1 protein derived from 1507x59122 maize (Lane 4), 

with each sample migrating at approximately 14 kDa. Similarly, the Cry34Ab1 standard protein 

(Lane 2) migrated at approximately 14 kDa and appeared to be equivalent to the plant-derived 

proteins.

Cry35Ab1

Western blot analysis using anti-Cry35Ab1 antibodies (Figure 2.3) demonstrated that the 

Cry35Ab1 protein derived from 4114 maize (Lane 3) had the same molecular weight and 

relative immunoreactivity as the Cry35Ab1 protein derived from 1507x59122 maize (Lane 4). 

Both protein samples migrated as two bands of approximately 40 kDa and 44 kDa in size. Like 

the Cry1F protein, the Cry35Ab1 protein can be partially degraded to a more stable truncated 

protein by plant proteases, and the relative amounts of each protein can vary from sample to 

sample. The Cry35Ab1 standard protein (Lane 2) consisted of the truncated form of the protein 

and migrated at the expected approximately 40 kDa.

PAT Protein

Western blot analysis using anti-PAT antibodies (Figure 2.4) demonstrated that the PAT protein 

derived from 4114 maize (Lane 3) had the same molecular weight and relative 

immunoreactivity as the PAT protein derived from 1507x59122 maize (Lane 4), with each 

sample migrating at approximately 21 kDa. The PAT standard protein (Lane 2) migrated at the 

same molecular weight as the plant-derived PAT proteins (approximately 21 kDa). 

2.3. Conclusions

The western blot analyses demonstrated that the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins 

derived from 4114 maize have equivalent molecular weights and immunoreactivity to the 

proteins expressed in 1507x59122 maize. In all four western blots, the near-isoline control 

showed no immunoreactive proteins. These data, in addition to the identical DNA sequences 

encoding the proteins, demonstrates equivalency of the transgenic proteins in 4114 maize to 

those in 1507 and/or 59122 maize. Therefore, prior safety studies conducted on Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT for 1507 and/or 59122 maize are applicable to 4114 maize.
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4114 Maize

 1 2 3 4 5 

Lane Sample ID

1 Molecular Weight Marker

2 Cry1F Standard (~4 ng)

3

Protein Extract of R1 Leaf from 4114 Maize 

(undiluted)

4

Protein Extract of R1 Leaf from 1507x59122 

Maize (undiluted)

5

Protein Extract of R1 Leaf from Near-Isoline 

Control Maize (undiluted)

Figure 2.1. Western Blot Comparison of Cry1F Protein 

Plant-derived Cry1F protein can be partially degraded by plant proteases to a smaller, more stable 

truncated protein, therefore appearing as two bands (~60 kDa and ~62 kDa). The Cry1F standard 

protein consisted of the truncated protein only (~60 kDa).

Cry1F

kDa

188 

62

49

38

28

18

14

6

3
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4114 Maize

 1 2 3 4 5 

Lane Sample ID

1 Molecular Weight Marker

2 Cry34Ab1 Standard (~10 ng)

3

Protein Extract of R1 Leaf from 4114 Maize 

(1:2 dilution)

4

Protein Extract of R1 Leaf from 1507x59122 

Maize (1:3 dilution)

5

Protein Extract of R1 Leaf from Near-Isoline 

Control Maize (undiluted)

Figure 2.2. Western Blot Comparison of Cry34Ab1 Protein

Plant-derived and standard Cry34Ab1 proteins migrated at the expected mass of ~14 kDa.

Cry34Ab1

kDa

188 

62

49

38

28

18

14

6

3

3
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4114 Maize

 1 2 3 4 5 

Lane Sample ID

1 Molecular Weight Marker

2 Cry35Ab1 Standard (~10 ng)

3

Protein Extract of R1 Leaf from 4114 Maize 

(1:2 dilution)

4

Protein Extract of R1 Leaf from 1507x59122 

Maize (1:2 dilution)

5

Protein Extract of R1 Leaf R1 from Near-Isoline 

Control Maize (undiluted)

Figure 2.3. Western Blot Comparison of Cry35Ab1 Protein

Plant-derived Cry35Ab1 protein can be partially degraded by plant proteases to a smaller, more stable 

truncated protein, therefore appearing as two bands (~40 kDa and ~44 kDa). The Cry35Ab1 standard 

protein consisted of the truncated protein only (~40 kDa).

Cry35Ab1

kDa

188

62

49

38

28

18

14

6

3

3
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 1 2 3 4 5 

Lane Sample ID

1 Molecular Weight Marker

2 PAT Standard (~4 ng)

3

Protein Extract of R1 Leaf from 4114 Maize 

(undiluted)

4

Protein Extract of R1 Leaf from 1507x59122 

Maize (1:2 dilution)

5

Protein Extract of R1 Leaf from Near-Isoline 

Control Maize (undiluted)

Figure 2.4. Western Blot Comparison of PAT Protein

Plant-derived and standard PAT proteins showed the expected mass of ~21 kDa. 

PAT

kDa

188

62

49

38

28

18

14

6

3

3
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2.4. Experimental Methods

2.4.1. Experimental Design

One maize leaf tissue sample from each of 4114 maize, 1507x59122 maize, and the control 

maize was extracted and evaluated using western blot analysis for expression of the Cry1F, 

Cry34Ab1, Cry35Ab1, and PAT proteins.

2.4.2. Sample Collection, Processing, and Storage

Maize leaf tissue samples were collected at the R1 growth stage (Ritchie et al., 2005). One 

sample from each of 4114 maize, 1507x59122 maize, and the control maize were collected and 

processed as described in Appendix 5. A representative leaf sub-sample from each maize line 

was weighed at a target weight of 10 mg + 5% into an individual 1.2 ml tube, assigned a unique 

identification number, and stored in a temperature-monitored freezer at ≤ -10 °C until prepared 

for extraction.

2.4.3. Sample Extraction and Preparation for SDS-PAGE

Two chilled (2-8 °C) 5/32” chrome balls and 0.6 ml of chilled (2-8 °C) extraction buffer (PBST: 

phosphate buffered saline, pH 7.4, with 0.05% Tweenb-20) were added to each tube, and the 

tubes were covered securely with strip caps. The samples were extracted for 30 seconds at a 

setting of 1500 strokes per minute on a Geno/Grinderc(SPEX CertiPrep, Inc., Metuchen, NJ). 

The extracts were then centrifuged for 10 minutes at a setting of 4000 rpm at 4 °C and the 

supernatants were transferred to new tubes.

The samples were diluted as needed in PBST to adjust for relative band intensity in the western 

blots; the 4114 maize extract was not diluted for the PAT and Cry1F western blots but was 

diluted 1:2 for the Cry34Ab1 and Cry35Ab1 western blots, and the 1507x59122 maize extract 

was not diluted for the Cry1F western blot but was diluted 1:3 for the Cry34Ab1 western blot 

and 1:2 for the Cry35Ab1 and PAT western blots.

2.4.4. Preparation of Protein Standard Solutions

The Cry1F and PAT protein standards were each diluted to a concentration of 200 ng/ml in 

PBST, and the Cry34Ab1 and Cry35Ab1 protein standards were each diluted to a concentration 

of 500 ng/ml in PBST.

 

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2.4.5. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

All sample extracts and protein standard solutions were prepared for electrophoresis by adding 

25% NuPAGEd4X LDS (Invitrogen Corporation, Carlsbad, CA) and 10% 10X reducing buffer 

containing dithiothreitol (DTT) (Invitrogen), heated for approximately seven minutes at 95 C, 

and then loaded onto two NuPAGE Novex 4-12% Bis-Tris 12-well gels (Invitrogen). Samples 

were loaded at 20 l/well and SeeBluedPre-Stained Standard (Invitrogen) was loaded at 10 

l/well.

Electrophoresis was conducted with a XCell SureLockd Mini-Cell electrophoresis unit 

(Invitrogen) with NuPAGE MES SDS running buffer (Invitrogen) at a constant voltage of 200V for 

approximately 40-44 minutes.

2.4.6. Western Blot Analysis

After electrophoresis, gels were removed from the gel cassette and proteins were 

electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen) for 

approximately seven minutes using an iBlotd module (Invitrogen). Following transfer, the PVDF 

membrane blots were blocked with a 5% non-fat dry milk (NFDM)/PBST solution for 

approximately 30 minutes at 20-25 C, followed by an incubation of approximately 1 hour at 

20-25 °C in a specific monoclonal primary antibody (Table 2.1), which was diluted 1:2000 in 1%

NFDM/PBST. The blots were then washed with PBST three times, for at least 10 minutes per 

wash. Following the third wash, the blots were incubated in a secondary antibody (Table 2.1;

Promega U.S., Madison, WI), which was conjugated to the enzyme horseradish peroxidase 

(HRP) and diluted 1:10,000 in 1% NFDM/PBST, for approximately 1 hour at 20-25 °C, after which 

the blots were washed with PBST three times for at least 10 minutes per wash.

The blots were developed using SuperSignale West Dura Extended Duration Substrate detection 

kit (Pierce Biotechnology, Inc., Rockford, IL) followed by image capture using the Kodak Image 

Station 4000R Pro imaging system (Carestream Health, Inc., Rochester, NY). The resulting 

images were evaluated for similarities between 4114 maize and 1507x59122 maize.

 

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Table 2.1. Primary and Secondary Antibody Descriptions Used for Qualitative Western Blot 

Analysis

Target 

Protein Primary Antibody Secondary Antibody

Cry1F Cry1F Monoclonal Antibody 

205A62.1 

Anti-Mouse IgG HRP Conjugate 

(Promega)

Cry34Ab1 Cry34Ab1 Monoclonal Antibody 

1E1.G6

Anti-Mouse IgG HRP Conjugate 

(Promega)

Cry35Ab1 Cry35Ab1 Monoclonal Antibody 

8B5.1A10 

Anti-Mouse IgG HRP Conjugate 

(Promega)

PAT PAT Monoclonal Antibody 22G6 Anti-Mouse IgG HRP Conjugate 

(Promega)
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Appendix 3. Materials and Methods for Genetic Characterization of 4114 Maize

3.1. Southern Blot Characterization of 4114 Maize

Southern blot analysis was conducted to characterize the DNA insertion in 4114 maize. 

Individual plants of the T2, T3, BC3F1*3, and BC3F2*2generations were analyzed by Southern 

blot to determine the number of each of the genetic elements of the expression cassette that 

were inserted and to verify that the integrity of the PHP27118 T-DNA was maintained upon 

integration. The integration patterns of the insertion in 4114 maize were investigated with Bcl I 

and Hind III restriction enzymes. Southern blot analysis was conducted on individual plants of 

the four generations to confirm stability of the insertion across generations and to verify the 

absence of backbone sequences from plasmid PHP27118.

3.2. Test Material

Seeds from the T2, T3, BC3F1*3, and BC3F2*2generations of 4114 maize were planted and leaf 

tissue harvested from individual plants was used for genomic DNA extraction.

3.3. Control Material

Seeds from the unmodified Pioneer maize proprietary inbreds PHWWE and PH09B were 

planted and leaf tissue harvested from individual plants was used for genomic DNA extraction.

3.4. Reference Material

Plasmid DNA from PHP27118 was prepared from E. coli (Invitrogen, Carlsbad, CA) and was used 

as a positive control for Southern blot analysis to verify probe hybridization and to verify sizes 

of fragments internal to the plasmid. The plasmid stock was a copy of the plasmid used for 

transformation to produce 4114 maize and was digested with restriction enzymes to confirm 

the plasmid map. The probes used in this study were derived from plasmid PHP27118 or from a 

plasmid containing equivalent genetic elements.

The Bcl I recognition sequence contains a Dam methylase recognition sequence (

5’GATC3’) (New 

England Biolabs, 2002). The PHP27118 plasmid used in this analysis was prepared in a dam+

strain of E. coli and thus the central adenine residue in all Bcl I sites (recognition sequence 

5’TGATCA3’) was methylated and did not digest as expected (Pioneer data not shown). 

Therefore, the Bcl I-digested plasmid served only as a positive control to demonstrate probe 

hybridization and not to provide any band size data. As Dam methylase is specific to bacteria 

and not found in maize plants, maize genomic DNA will be digested normally by Bcl I. Plasmid 

PHP27118 digested with Hind III was included on some of the Bcl I Southern blots to show that 

the plasmid used in this analysis was of sufficient quality and cut properly when digested with 

an enzyme that is not sensitive to methylation.
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DNA molecular weight markers for gel electrophoresis and Southern blot analysis were used to 

determine approximate molecular weights. For Southern analysis, DNA Molecular Weight 

Marker VII, digoxigenin (DIG) labeled (Roche, Indianapolis, IN), was used as a size standard for 

hybridizing fragments. ΦX174 RF DNA/Hae III Fragments (Invitrogen, Carlsbad, CA) was used as 

a molecular weight standard to determine sufficient migration and separation of the fragments 

on the gel.

3.5. Identification of the 4114 Maize Plants Used for Southern Blot Analysis

Phenotypic analysis of 4114 maize plants and control plants was carried out by the use of 

lateral flow devices able to detect the Cry1F, Cry34Ab1 and PAT proteins to confirm the 

absence or presence of these proteins in material used for Southern blot analysis.

Leaf extracts were prepared by grinding leaf punches to homogeneity in 400 μl of EB2 

extraction buffer (Envirologix, Inc., Portland, ME). Lateral flow devices (Envirologix) were 

placed in the homogenate and allowed to develop. After incubation, the results were read 

from the lateral flow devices. A single stripe indicated a negative result and a double stripe 

indicated the sample was positive for the Cry1F, Cry34Ab1 or PAT proteins.

Genotypic analysis of the 4114 maize and control maize plants was carried out by real-time

polymerase chain reaction (PCR) using assays specific for the DNA insertion. A leaf sample was 

taken from each test and control plants for event-specific PCR analysis. DNA was extracted 

from each leaf sample using the Extract-N-AmpfPlant PCR kit using the described procedure 

(Sigma-Aldrich, St. Louis, MO).

Real-time PCR was performed on each DNA sample utilizing an ABI PRISMg7500 Fast Real-Time 

PCR System (Applied Biosystems, Inc., Foster City, CA). TaqMangprobe (Applied Biosystems, 

Inc.) and primer sets (Integrated DNA Technologies, Coralville, IA) were designed to detect 

target sequences from the insertion in 4114 maize. In addition, a second TaqMan probe and 

primer set for a reference maize endogenous gene was used to confirm the presence of 

amplifiable DNA in each reaction. The assay analysis consisted of real-time PCR determination 

of qualitative positive/negative calls. The extracted DNA was assayed using TaqMan Universal 

PCR Master Mix, No AmpEraseg UNG (Applied Biosystems, Inc.). Initial incubation was at 95C 

for 10 minutes followed by 40 cycles as follows: 95C for 15 seconds, 60C for one minute.

Positive or negative determination for each plant was based on comparison of the threshold 

cycle (CT) of the insertion target PCR to that of the maize endogenous reference target. If the 

event-specific and endogenous PCR targets amplified above CT, then the plant was scored as 

positive for the 4114 event. If the endogenous target amplified and the event target did not, 

then the plant was scored as negative.

 

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A subset of 4114 maize plants that were identified as containing the inserted DNA and 

expressing the Cry1F, Cry34Ab1, and PAT proteins from the four generations described 

previously were selected for Southern blot analysis.

3.6. Genomic DNA Extraction

Genomic DNA was extracted from leaf tissue harvested from individual test and control plants. 

The tissue was pulverized in tubes containing grinding beads using a Geno/Grinderh(SPEX 

CertiPrep, Inc., Metuchen, NJ) instrument and the genomic DNA was isolated using a ureabased procedure (modification from Chen and Dellaporta, 1994). Approximately 1 gram of 

ground tissue per sample was extracted with 5 ml Urea Extraction Buffer (7 M urea, 0.34 M 

NaCl, 0.05 M Tris-HCl, pH 8.0, 0.02 M EDTA, 1% N-lauroylsarcosine) for 15-18 minutes at 37C, 

followed by two extractions with phenol/chloroform/isoamyl alcohol (25:24:1) and one 

extraction with water saturated chloroform. The DNA was precipitated from the aqueous 

phase by the addition of 1/10 volume of 3 M NaOAc (pH 5.2) and 1 volume of isopropyl alcohol, 

followed by centrifugation to pellet the DNA. After washing the pellet twice with 70% ethanol, 

the DNA was dissolved in 0.5 ml distilled water and treated with 10 g ribonuclease A for 15 

minutes at 37C. The sample was then washed with 70% ethanol. After drying, the DNA was 

re-dissolved with 0.5 ml distilled water and stored at 4C.

3.7. Quantification of Genomic DNA

Following extraction, the DNA samples were quantified on a spectrofluorometer using 

PicoGreenireagent (Molecular Probes, Inc., Eugene, OR) following a standard procedure. The 

DNA was also visualized on an agarose gel to confirm quantification values from the PicoGreen 

analysis and to determine DNA quality.

3.8. Digestion of DNA for Southern Blot Analyses 

Genomic DNA samples extracted from selected 4114 maize and control maize plants were 

digested with restriction enzymes following a standard procedure. Approximately 3 to 6 g of 

genomic DNA was digested using 50 units of enzyme according to manufacturer’s

recommendations. The digestions were carried out at 37C for approximately three hours, 

followed by ethanol precipitation with 1/10 volume of 3 M NaOAc (pH 5.2) and 2 volumes of 

100% ethanol. After incubation at ≤-5C and centrifugation, the DNA was allowed to dry and 

then re-dissolved in TE buffer (10mM Tris, 1 mM EDTA, pH 7.5). The reference plasmid, 

PHP27118, was spiked into a control plant DNA sample in an amount equivalent to 

approximately one or three gene copies per maize genome and digested with the same enzyme 

to serve as a positive control for probe hybridization and to verify sizes of fragments internal to 

the plasmid on the Southern blot.

 

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3.9. Electrophoretic Separation and Southern Transfer

Following restriction enzyme digestion, the resultant DNA fragments were electrophoretically 

separated by size through an agarose gel. A molecular weight standard [ΦX174 RF DNA/HaeIII 

Fragments (Invitrogen)] was used to determine sufficient migration and separation of the 

fragments on the gel. DIG labeled DNA Molecular Weight Marker VII (Roche), which is visible 

after DIG detection as described below, was used to determine hybridizing fragment size on the 

Southern blots.

Agarose gels containing the separated DNA fragments were depurinated, denatured, and 

neutralized in situ, and transferred to a nylon membrane in 20x SSC buffer (3M NaCl, 0.3 M 

sodium citrate) using the method as described for the TURBOBLOTTERjRapid Downward 

Transfer System (Whatman, Inc., Piscataway, NJ). The DNA was then bound to the membrane 

by UV crosslinking (Stratalinker, Stratagene, La Jolla, CA).

3.10. DNA Probe Labeling for Southern Blot Hybridization

Probes for the cry1F, cry34Ab1, cry35Ab1 and pat genes were used to detect genes within the 

T-DNA insertion. Probes for the ubiZM1 promoter, ubiZM1 5’ UTR and intron region, ORF25 

terminator, pinII terminator, TA peroxidase promoter, and CaMV 35S promoter and terminator 

regions were used to detect regulatory regions within the T-DNA insertion. To determine 

whether PHP27118 backbone was incorporated during T-DNA insertion, the Right Border (RB) 

and Left Border (LB) backbone probes were used to analyze the backbone regions directly 

outside the T-DNA borders, and probes for the spectinomycin resistance (spc), tetracycline 

resistance (tet), and virG genes were used to confirm absence of these genes and other 

portions of the backbone. DNA fragments of the probe elements were generated by PCR from 

plasmid PHP27118 or a plasmid with equivalent elements using specific primers. PCR 

fragments were electrophoretically separated on an agarose gel, excised and purified using a 

gel purification kit (Qiagen, Valencia, CA). DNA probes were generated from these fragments 

by PCR that incorporated a DIG labeled nucleotide, [DIG-11]-dUTP, into the fragment. PCR 

labeling of isolated fragments was carried out according to the procedures supplied in the PCR 

DIG Probe Synthesis Kit (Roche).

3.11. Probe Hybridization and Visualization

Labeled probes were hybridized to the target DNA on the nylon membranes for detection of 

the specific fragments using the procedures essentially as described for DIG Easy Hyb solution

(Roche). After stringent washes, the hybridized DIG-labeled probes and DIG-labeled DNA 

standards were visualized using CDP-Star Chemiluminescent Nucleic Acid Detection System 

with DIG Wash and Block Buffer Set (Roche). Blots were exposed to X-ray film for one or more 

time points to detect hybridizing fragments and to visualize molecular weight standards bound 

to the nylon membrane. Images were digitally captured by detection with the Luminescent 

 

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Image Analyzer LAS-3000 (Fujifilm Medical Systems, Stamford, CT). Digital images were 

compared to original X-ray film exposures as verification for use in this submission. The sizes of 

detected bands were documented for each digest and each probe.

3.12. Stripping of Probes and Subsequent Hybridizations

Following hybridization and detection, membranes were stripped of DIG-labeled probe to 

prepare the blot for subsequent re-hybridization to additional probes. Membranes were rinsed 

briefly in distilled, de-ionized water and then stripped in a solution of 0.2 M NaOH and 0.1% SDS 

at 37°C with constant shaking. The membranes were then rinsed in 2x SSC and used directly for 

subsequent hybridizations. The alkali-based stripping procedure effectively removes probes 

labeled with the alkali-labile DIG.
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Appendix 4. Materials and Methods for Segregation Analysis of Five Generations 

of 4114 Maize

Five generations of 4114 maize were evaluated using polymerase chain reaction (PCR) analyses 

and herbicide tolerance testing to confirm Mendelian inheritance of the genotype and 

phenotype.

4.1. Greenhouse Experimental Design

Five separate generations (F1*1, BC2F1*1, BC3F1*1, BC2F1*2, and BC3F1*2) of 4114 maize were 

planted and grown in a greenhouse under standard maize production environmental 

conditions. Leaf punch samples were collected from each generation and analyzed using PCR 

analyses specific for the event DP-ØØ4114-3, cry1F gene, cry34Ab1 gene, cry35Ab1 gene, and 

pat gene. After sample collection, all plants were treated with a broadcast application of 

glufosinate and then visually evaluated for herbicide resistance. 

4.2. Planting and Leaf Sample Collection 

Maize seeds, approximately 126 for each generation, were planted in separate cell-divided flats 

and grown in a greenhouse using typical greenhouse procedures. Ten days after planting, each 

generation was thinned to a final population of approximately 100 plants. 

When plants were at approximately the V2 growth stage (the growth stage when the collar of 

the second leaf is visible) and prior to herbicide application, leaf samples were collected from 

each plant. The samples each consisted of one leaf punch distributed into an individual bullet 

tube and placed on dry ice until they were transferred to a freezer (≤ -10°C) for storage. 

Individual plants and corresponding leaf punch samples were uniquely labeled to allow a given 

sample to be traced back to the originating plant.

After the data from the original entries were analyzed, it was determined that additional plants 

for the BC3F1*2 generation were needed to verify the original result and increase the statistical 

power. An additional 126 seeds from same BC3F1*2 seed source and 91 seeds from a second 

BC3F1*2 generation seed source were planted and sampled following the previously outlined 

procedures. No thinning was performed on the plants from the second seed source before leaf 

punch samples were collected. 

4.3. Genotypic Analysis

Leaf punch samples were analyzed using event-specific PCR analysis to confirm the presence or 

absence of event DP-ØØ4114-3 as well as gene-specific PCR analysis to confirm the presence or 

absence of the cry1F, cry34Ab1, cry35Ab1, and pat genes. 
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4.4. Phenotypic Analysis

After sample collection, all plants were treated with a tank mixture containing Ignitek280 SL 

herbicide (Bayer CropScience AG, Monheim am Rhein, Germany) and ammonium sulfate. The 

Ignite 280 SL herbicide contained 24.5% glufosinate (by weight) in the form of 

glufosinate-ammonium, equivalent to 2.34 pounds glufosinate active ingredient per gallon (0.28 

kilograms glufosinate active ingredient per liter). The tank mix was applied at an approximate 

rate of 22 fluid ounces per acre (1.6 liters per hectare) and the spray volume was 21.6 gallons 

per acre (202 liters per hectare). 

Each plant was visually evaluated 7 days after herbicide application for the presence or absence 

of herbicide injury, and was identified as presenting either an herbicide-tolerant phenotype 

(plant exhibited no herbicidal injury) or an herbicide-susceptible phenotype (plant exhibited 

severe herbicide injury). 

For the additional BC3F1*2 generation plants Ignite 280 SL herbicide was applied with a spray 

volume of 19.7 gallons per acre (184 L/ha) without the addition of ammonium sulfate and 

plants were visually evaluated for herbicide injury 4 days after herbicide application.

4.5. Statistical Analysis

A chi-square analysis (95%) was performed on the segregation results of each 4114 maize 

generation to compare the observed segregation ratio to the expected segregation ratio (1:1). 

 

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Appendix 5. Materials and Methods for Determination of Cry1F, Cry34Ab1, Cry35Ab1, and 

PAT Protein Concentrations

Concentrations of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were determined in

various tissues of 4114 maize using enzyme-linked immunosorbent assays (ELISA). Plant tissues 

were collected from 4114 maize plants from a 2010 field trial and analyzed as described further 

below.

5.1. Field Trial Experimental Design

4114 maize plants of the F1*5 generation were grown at five sites in 2010 located in commercial 

maize growing regions of North America, with four sites in the United States and one site in 

Canada [Bagley, IA; Atlantic, IA; Wyoming, IL; York, NE; and Branchton, ON].

Each field site utilized a randomized complete block design with 4114 maize, 1507 maize, 59122 

maize, 1507x59122 maize, and the control maize planted in two-row plots within four replicate 

blocks. Approximately 30 seeds were planted in each 25 ft (7.6 m) row resulting in a seed 

spacing of approximately 10 inches (25 cm). Row spacing was approximately 30 inches (76 cm) 

and every two-row plot was bordered on either side by one row of commercial maize of similar 

maturity. In addition, blocks were separated by an alley distance of at least 36 inches (0.9 m) 

and each site was surrounded by a minimum of four external border rows. In order to ensure 

grain purity, ear shoots of test and control plants were covered prior to silk emergence and the 

primary ear was self-pollinated by hand. To control experimental bias in this study, the 

following procedures were utilized: non-systematic selection of trial and plot areas within each 

site, randomization of maize entries within each block, and uniform maintenance across blocks 

in each field site. 

5.2. Plant Material Collection, Shipping, Processing, and Storage

5.2.1. Tissue Collection 

Leaf, root, whole plant, pollen, forage, and grain samples for protein concentration analysis 

were collected from impartially selected, healthy, representative plants from each entry. Each 

sample was placed on dry ice after collection before being transferred to a freezer (≤ -10 °C) for 

storage until shipment. 

The following tissues were collected with the plant growth stages indicated in parentheses: leaf 

(V6, V9, R1, R4, and R6), root (V6, V9, R1, R4, and R6), whole plant (V9, R1, and R6), pollen (R1), 

forage (R4), and grain (R6). Plant growth stages are explained in Table 5.1 (Ritchie et al., 2005).
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Table 5.1. Maize Growth Stage Descriptions

Growth 

Stagea Descriptiona

V6 The collar of the sixth leaf becomes visible

V9 The collar of the ninth leaf becomes visible

R1 The silks become visible

R4 The material within the kernel produces a doughy 

consistency

R6 The typical harvest maturity for grain; regarded as 

physiological maturity

a

Growth stages are as described in Ritchie et al., 2005

Leaf

Four leaf samples (one sample per plant) were each collected from 4114 maize, 1507 maize, 

59122 maize, and 1507x59122 maize and one sample was collected from the control maize at 

each site. Samples were obtained from the youngest healthy leaf that had emerged at least 8 

inches (20 cm) from the whorl. 

Root

Four root samples (one sample per plant) were each collected from 4114 maize, 1507 maize, 

59122 maize, and 1507x59122 maize and one sample was collected from the control maize at 

each site. Samples were obtained by cutting a circle 10-15 inches (25-38 cm) in diameter 

around the base of the plant to a depth of 7-9 inches (18-23 cm). The root ball was removed 

from the soil, shaken to remove excess soil, and thoroughly cleaned with water. A

representative sub-sample of root tissue was collected.

Whole Plant

Following the collection of all other samples for a respective growth stage, whole plant samples 

were collected from the remaining aerial portion of the plants. Four whole plant samples (one 

sample equals one plant) were each collected from 4114 maize, 1507 maize, 59122 maize, and 

1507x59122 maize and one sample was collected from the control maize at each site. Samples 

were obtained by cutting the plants from the root system approximately 1 inch (2.5 cm) above 

the soil surface line. The plants were chopped into sections less than 3 inches (7.5 cm) in 

length.
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Pollen

Four pollen samples (one sample per plant) were each collected from 4114 maize, 1507 maize, 

59122 maize, and 1507x59122 maize and one sample was collected from the control maize at 

each site. The tassel selected for sampling had half to three-quarters of the main spikes 

shedding and was bagged no more than one day prior to collection. Samples were obtained by 

shaking or tapping the bagged tassel to dislodge the pollen. 

Forage

Following the collection of R4 leaf and root samples, forage samples were collected from the 

remaining aerial portion of the plants. Four forage samples (one sample equals one plant) were 

each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and one 

sample was collected from the control maize at each site. All plants used for sampling 

contained self-pollinated primary ears. Any secondary or tertiary ears with exposed silks were 

removed prior to sampling. Samples were obtained by cutting the plants from the root system 

approximately 1 inch (2.5 cm) above the soil surface line. The plants were chopped into 

sections less than 3 inches (7.5 cm) in length.

Grain

Four grain samples (one sample equals the grain from one self pollinated primary ear per plant) 

were each collected from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize and 

one sample was collected from the control maize at each site. The grain samples were 

obtained by husking and shelling each ear. A representative sub-sample of 15 kernels from 

each ear was collected. Husks and cobs were retained for inclusion with the R6 whole plant 

samples.

5.2.2. Sample Shipping, Processing, and Storage

All tissue samples were shipped frozen and stored at ≤ -5 °C. Whole plant and forage samples 

were coarsely homogenized. Leaf, root, whole plant, pollen, forage, and grain samples were 

lyophilized under vacuum until dry. Following lyophilization, leaf, root, whole plant, forage, and 

grain samples were finely homogenized and stored frozen until analysis.

5.3. Protein Concentration Determination

Concentrations of the Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins were determined using 

specific quantitative ELISA methods. 

5.3.1. Protein Extraction

Analytical samples were weighed into 1.2-ml tubes at target weights (±5%) of 10 mg for leaf; 20 

mg for root and grain; 30 mg for forage, and whole plant; and 5 mg for pollen. Each sample 

analyzed for protein concentrations was extracted with 0.6 ml of chilled PBST buffer (Phosphate 
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Buffered Saline, pH 7.45 and 0.05% Tweenl-20). Following centrifugation, supernatants were 

removed, diluted, and analyzed.

5.3.2. Cry1F ELISA Procedure

The Cry1F ELISA method utilized a sequential ELISA format to determine the concentration of 

the Cry1F protein in sample extracts. The Cry1F ELISA kit employed was obtained from 

Envirologix, Inc. (Portland, Maine). Standards (analyzed in triplicate wells) and diluted sample 

extracts (analyzed in duplicate wells) were incubated in a plate pre-coated with a Cry1F-specific 

antibody. Following incubation, unbound substances were washed from the plate. A different 

Cry1F-specific antibody conjugated to the enzyme horseradish peroxidase (HRP) was added to 

the plate and incubated. The unbound substances were washed from the plate. Detection of 

the bound Cry1F-antibody complex was accomplished by the addition of substrate, which 

generated a colored product in the presence of HRP. The reaction was stopped with an acid 

solution and the optical density (OD) of each well was determined using a plate reader. An 

average of the results from duplicate wells was used to determine the concentration of the 

Cry1F protein in ng/mg sample weight.

5.3.3. Cry34Ab1 ELISA Procedure

A similar procedure was used as for the Cry1F ELISA, except a Cry34Ab1-specific antibody was 

used on the pre-coated plate and a second Cry34Ab1-specific antibody conjugated to HRP was 

used. The Cry34Ab1 ELISA kit employed was obtained from Envirologix, Inc.

5.3.4. Cry35Ab1 ELISA Procedure

A similar procedure was used as for the Cry1F ELISA, except a Cry35Ab1-specific antibody was 

used on the pre-coated plate and a second Cry35Ab1-specific antibody conjugated to HRP was 

used. The Cry35Ab1 ELISA kit employed was obtained from Acadia BioScience, LLC (Portland, 

Maine).

5.3.5. PAT ELISA Procedure

A similar procedure was used as for the Cry1F ELISA, except a PAT-specific antibody was used 

on the pre-coated plate and a second PAT-specific antibody conjugated to HRP was used. The 

PAT ELISA kit employed was obtained from Envirologix, Inc.

5.3.6. Calculations for Determining Protein Concentrations

SoftMaxmPro GxP (Molecular Devices, Sunnyvale, CA) software was used to perform the 

calculations required to convert OD values obtained by the microtiter plate reader to protein 

concentration values.

 

l

Registered trademark of ICI Americas, Inc.
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Standard Curve

A standard curve was included on each ELISA plate. The equation for the standard curve was 

generated by the software, which used a quadratic fit to relate the mean OD values obtained 

for the standards to the respective standard concentration (ng/ml).

The quadratic regression equation was applied as follows:

y = Cx2+ Bx + A

Where x = known standard concentration and y = respective mean absorbance value (OD)

Sample Concentration

Interpolation of the sample concentration (ng/ml) was accomplished by solving for x in the 

above equation using values for A, B, and C determined by the standard curve. 

Sample concentration (ng/ml) =  

2C

B B - 4C A -sampleOD

2

 

The sample concentration values were adjusted for a dilution factor expressed as 1:N by 

multiplying the interpolated concentration by N.

Adjusted sample concentration values obtained from SoftMax Pro GxP software were 

converted from ng/ml to ng/mg sample weight as follows:

ng/mg Sample Weight = ng/ml x Extraction Volume (ml)/Sample Weight (mg)

Lower Limit of Quantification (LLOQ)

The LLOQ, in ng/mg sample weight, was calculated as follows:

LLOQ = Reportable Assay LLOQ x Extraction Volume

Sample Target Weight

5.4. Statistical Analysis

Statistical analyses were conducted using SASnsoftware, Version 9.2 (SAS Institute, Inc., Cary, 

NC) to estimate mean concentrations of Cry1F, Cry34Ab1, Cry35Ab1, and PAT proteins derived 

from 4114 maize, 1507 maize, 59122 maize, and 1507x59122 maize. 

 

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Registered trademark of Molecular Devices Corporation

n

Registered trademark of SAS Institute, Inc.
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5.4.1. Processing of Data

Transformation

Protein concentrations were first subjected to a natural logarithm transformation “ln(y)” before 

statistical analyses. Residuals were examined for validation of the normality and homogeneous 

variance assumptions. When these assumptions were questionable, other types of transforms 

or none were considered. The following proteins and tissue types did not undergo “ln(y)” 

transformation because the original data achieved model validation assumptions. 

 Cry1F in leaf (V9), root (R4, V6, and V9), and whole plant (V9)

 Cry34Ab1 in leaf (R1 and R4), root (R1, R4, and V6), and whole plant (V9) 

 Cry35Ab1 in leaf (R4 and R6), root (R4 and R6), and grain (R6) 

 PAT in leaf (R1, R4 and V9), forage (R4), and root (V9)

If the protein concentration underwent “ln(y)” transformation, the statistical analyses were 

conducted based on the transformed data. The estimated mean values and the confidence 

limits were then back-transformed to the original data scale for reporting purposes.

Partial LLOQ Sample Values

For a given protein in a given tissue, the number of samples below the LLOQ determined 

whether statistical analysis was conducted. The following rules were implemented: 

If < 80% of samples for each entry were below the LLOQ, then the statistical model considering 

LLOQ was utilized to conduct statistical analysis.

If ≥ 80% of samples for a single entry within the study were below the LLOQ, then statistical 

analysis was not conducted. 

5.4.2. Statistical Models

Default Model

For a given protein concentration in a given tissue, data were analyzed using the following 

linear mixed model:
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yijk = μi + lj + rk(j) + (μl)ij + εijk Model 1

lj ~ iid N(0, σ2

Site), rk(j) ~ iid N(0,σ2Rep), (μl)ij ~ iid N(0, σ2Ent×Site), and εijk ~ iid N(0, σ2Error)

Where μi denotes the mean of the ith entry (fixed effect), lj denotes the effect of the jth site (random 

effect), rk(j) denotes the effect of the k

th block within the jth site (random effect), (μl)ij denotes the 

interaction between the entries and sites (random effect) and εijk denotes the effect of the plot 

assigned the ith entry in the kth block of the jth site (random effect or residual). Notation ~iid N(0, σ2

a)

indicates random variables that are identically independently distributed (iid) as normal with zero 

mean and variance σ

2

a.

The residual maximum likelihood (REML) estimation procedure was utilized to generate 

estimates of variance components and entry means. The estimated means are known as least 

squares means (LS-Means). 

SAS PROC MIXED was utilized to fit Model 1 and to generate LS-Means, 95% confidence 

intervals. 

Model Considering LLOQ

For Cry1F in leaf (R6), PAT in root (R4 and R6), and whole plant (R6), <80% of sample values 

were detected below the assay LLOQ. Sample results below the LLOQ were treated as leftcensored observations at the respective assay LLOQ value. Data were analyzed using the 

following linear mixed model:

yijk = μi + lj + rk(j) + (μl)ij + εijk Model 2

lj ~ iid N(0, σ2

Site), rk(j) ~ iid N(0,σ2Rep), (μl)ij ~ iid N(0, σ2Entry×Site), εijk ~ iid N(0, σ2Error)

Where μi denotes the mean of the ith entry (fixed effect), lj denotes the effect of the jth site (random 

effect), rk(j) denotes the effect of the k

th block within the jth site (random effect), (μl)ij denotes the 

interaction between the entries and sites (random effect) and εijk denotes the residual for the 

observation obtained from the plot assigned to the ith entry in the kth block of the jth site. 

Model 2 can also be written as:

yijk = μijk + εijk = μi + lj + rk(j) + (μl)ij + εijk

Where μijk denotes the mean of yijk. The conditional likelihood for each observation, given the 

random effects, was formulated according to the status of the observation (i.e. observed or leftcensored) (Thiébaut and Jacqmin-Gadda, 2004):
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

















  



 



left censored

c

P y c

observed

y

P y y

f y

ijk

ijk

ijk ijk

ijk ijk

ijk

( ) ( )

( ) ( )

( | )

*

*













Where  denotes the vector of all random effects, denotes the standard normal density function,

 denotes the standard normal cumulative distribution function, y

*

ijk denotes the observed sample 

value of yijk, and c denotes the assay LLOQ value. 

The conditional likelihood function was a product of all individual conditional likelihoods, and the 

marginal likelihood function was formed when the conditional likelihood function was integrated 

over all random effects. 

The maximum likelihood (ML) procedure was then used to generate estimates of variance 

components and entry means (i.e. LS-Means). 

SAS PROC NLMIXED was utilized to fit Model 2 and generate LS-Means and 95% confidence 

intervals. 

5.4.3. Reported Statistics

For each expressed trait protein in each tissue that was statistically analyzed, entry LS-Mean 

(back-transformed, if needed), range, and 95% confidence interval (back-transformed, if 

needed) (labeled as Mean, Range, and CI, respectively) are provided in Section 2; Tables 10-13. 

In these tables, descriptive statistics (arithmetic means and ranges) are reported for proteins 

and tissues that were not statistically analyzed using mixed model analyses due to insufficient 

above-LLOQ samples.

The Cry1F, Cry34Ab1, and Cry35Ab1 protein mean concentrations in 4114 maize in each tissue 

were divided by the respective protein mean concentrations in 1507, 59122, and/or 

1507x59122 maize to provide an “expression ratio” (Section 2; Table 14). When the expression 

ratio was close to one, this indicated that 4114 maize had comparable expression to 1507, 

59122, and/or 1507x59122 maize. If the expression ratio was less than one, this indicated that 

4114 maize had lower expression. For values greater than one, expression was determined to 

be higher in 4114 maize. 
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Appendix 6. Materials and Methods for Nutrient Composition

Nutrient composition was determined in 4114 maize using plants from a 2010 field trial and 

analyzed as described further below.

6.1. Field Trial and Experimental Design

4114 maize plants of the F1*5 generation were grown at six sites in 2010 in commercial maize 

growing regions of North America, with four sites in the U.S. and two sites in Canada [Richland, 

IA; Wyoming, IL; Geneva, MN; York, NE; Branchton, ON Canada; and Thorndale, ON Canada].

Each field site utilized a randomized complete block design with 4114 maize and the control 

maize planted in two-row plots within four replicate blocks. Approximately 30 seeds were 

planted in each 25 ft (7.6 m) row resulting in a seed spacing of approximately 10 inches (25 cm). 

Row spacing was approximately 30 inches (76 cm) and every two-row plot was bordered on 

either side by one row of commercial maize of similar maturity. In addition, blocks were 

separated by an alley distance of at least 36 inches (0.9 m) and each site was surrounded by a 

minimum of four external border rows. Maintenance fertilizer, herbicides, and pesticides were 

applied uniformly to 4114 maize and the control maize at each site, as needed. In order to 

ensure grain purity for compositional analyses, ear shoots of test and control plants were 

covered prior to silk emergence and the primary ear was self-pollinated by hand. To control 

experimental bias in this study, the following procedures were utilized: non-systematic 

selection of trial and plot areas within each site, randomization of maize entries within each 

block, and uniform maintenance across blocks in each field site. 

6.2. Plant Material Collection, Shipping, Processing, and Storage

6.2.1. Tissue Collection

All samples were collected from impartially selected, healthy individual plants. All control 

maize samples were collected prior to collection of 4114 maize samples. All forage and grain 

samples were assigned unique sample identification numbers that described the sample by site, 

entry, block, sample number, and tissue type. Samples were placed on dry ice within thirty 

minutes of collection and were maintained in coolers on wet or dry ice and/or in the freezers 

until shipment. 

Forage

One forage sample (composite of 3 whole plants) was collected from 4114 maize and the 

control maize from each of the four blocks. Any secondary or tertiary ears with exposed silks 

were removed prior to sampling. Forage samples were obtained by cutting the aerial portion of 

the plants from the root system approximately 1 inch (2.5 cm) above the soil surface line. The 

plants (including ears) were then cut into sections approximately 3 inches (7.5 cm) or less, and 

approximately 1/3 of the total sample was collected in a pre-labeled plastic-lined cloth bag. 
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Grain

One grain sample (five pooled ears equaled one sample) was collected from 4114 maize and 

the control maize from each of the four blocks at typical harvest maturity. Each ear was husked 

and shelled, and the grain was collected into a pre-labeled plastic-lined cloth bag. 

After collection, forage and grain samples were shipped from each field site to EPL BioAnalytical Services (EPL-BAS) for processing and nutrient composition analysis. Each sample 

was labeled by site, entry, block, sample number, tissue type, and growth stage. A unique 

sample identification number was also included on each label. 

6.2.2. Sample Shipping, Processing, and Storage

After collection, forage and grain samples were shipped and stored frozen at -20 °C. Samples 

were lyophilized, ground, and homogenized before nutrient composition analysis.

6.3. Nutrient Composition Analyses

Nutrient composition analyses of maize forage and grain were conducted and included the 

analysis methods described in Table 6.1.

Table 6.1. Methods for Compositional Analysis of 4114 Maize

Nutritional Analyte Method

Moisture in forage 

and grain

The analytical procedure for moisture determination was based on a method 

published by the Association of Official Analytical Chemists (AOAC). Samples 

were assayed to determine the percentage of moisture by gravimetric 

measurement of weight loss after drying in a forced air oven (forage) or 

vacuum oven (grain). 

Ash in forage and 

grain

The analytical procedure for ash determination was based on a method 

published by the AOAC. Samples were analyzed to determine the percentage 

of ash by gravimetric measurement of the weight loss after ignition in a 

muffle furnace. 

Crude protein in 

forage and grain

The analytical procedure for crude protein determination utilized an 

automated Kjeldahl technique based on a method provided by the 

manufacturer of the titrator unit (Foss-Tecator). Ground samples were 

digested in the presence of a catalyst. The digestate was then distilled and 

titrated with a Foss-Tecator Kjeltec titrator unit.
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Table 6.1. Methods for Compositional Analysis of 4114 Maize (continued)

Nutritional Analyte Method

Crude fat in forage 

and grain

The analytical procedure for crude fat determination was based on methods 

provided by the manufacturer of the hydrolysis and extraction apparatus 

(Ankom Technology). Samples were hydrolyzed with 3N hydrochloric acid at 

90 °C for 60 minutes. The hydrolysates were extracted with a petroleum 

ether/ethyl ether/ethyl alcohol solution at 90 °C for 60 minutes. The ether 

extracts were evaporated and the fat residue remaining determined 

gravimetrically.

Carbohydrate in 

forage and grain

The carbohydrate content in maize grain on a dry weight basis was calculated 

using a formula obtained from the United States Department of Agriculture 

“Energy Value of Foods,” in which the percent dry weight of crude protein, 

crude fat, and ash was subtracted from 100%.

Crude fiber in forage 

and grain

The analytical procedure for crude fiber determination was based on 

methods provided by the manufacturer of the extraction apparatus (Ankom 

Technology). Samples were analyzed to determine the percentage of crude 

fiber by digestion and solubilization of other materials present.

Neutral detergent 

fiber in forage and 

grain

The analytical procedure for neutral detergent fiber (NDF) determination was 

based on a method provided by the manufacturer of the extraction apparatus 

(Ankom Technology). Samples were analyzed to determine the percentage of 

NDF by digesting with a neutral detergent solution, sodium sulfite, and alpha 

amylase. The remaining residue was dried and weighed to determine the 

NDF content.

Acid detergent fiber in 

forage and grain

The analytical procedure for acid detergent fiber (ADF) determination was 

based on a method provided by the manufacturer of the extraction apparatus 

(Ankom Technology). Samples were analyzed to determine the percentage of 

ADF by digesting with an acid detergent solution and washing with reverse 

osmosis water. The remaining was dried and weighed to determine the ADF 

content.

Minerals in forage and 

grain

The analytical procedure for the determination of minerals is based on 

methods published by the AOAC and CEM Corporation. The maize forage 

minerals determined were calcium and phosphorus. The maize grain 

minerals determined were calcium, copper, iron, magnesium, manganese, 

phosphorus, potassium sodium, and zinc. The samples were digested in a 

microwave based digestion system and the digestate was diluted using 

deionized water. Samples were analyzed by inductively coupled plasma 

optical emission spectroscopy (ICP-OES).

Tryptophan in grain

The analytical procedure for tryptophan determination was based on an 

established lithium hydroxide hydrolysis procedure with reverse phase ultra 

performance liquid chromatography (UPLC) with ultraviolet (UV) detection 

published by the Journal of Micronutrient Analysis. 
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Table 6.1. Methods for Compositional Analysis of 4114 Maize (continued)

Nutritional Analyte Method

Cystine and 

methionine in grain

The analytical procedure for cystine and methionine determination was based 

on methods obtained from Waters Corporation, AOAC, and Journal of 

Chromatography A. The procedure converts cystine to cysteic acid and 

methionine to methionine sulfone, after acid oxidation and hydrolysis, to the 

6-aminoquinolyl-N-hydroxysuccinimidyl carbamate derivatives which are then 

analyzed by reverse phase UPLC with UV detection. 

Additional amino acids 

in grain

Along with tryptophan, cystine, and methionine, 15 additional amino acids 

were determined. The analytical procedure for analysis of these amino acids 

was based on methods obtained from Waters Corporation and the Journal of 

Chromatography A. The procedure converts the free acids, after acid 

hydrolysis, to the 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate 

derivatives, which are analyzed by reverse phase UPLC with UV detection. 

Fatty acids in grain

The analytical procedure for determination of fatty acids was based on 

methods published by the AOAC and American Oil Chemist Society (AOCS). 

The procedure converts the free acids, after ether extraction and base 

hydrolysis, to the fatty acid methyl ester (FAME) derivatives, which are 

analyzed by gas chromatography with flame ionization detection (GC/FID). 

Results are reported as percent total fatty acids but presented in the raw data 

as %DB.

Thiamine (vitamin B1) 

and Riboflavin 

(vitamin B2) in grain

The analytical procedure for the determination of Thiamine (Vitamin B1) and 

Riboflavin (Vitamin B2) was based on a method published by the American 

Association of Cereal Chemists (AACC). The samples were extracted with 10% 

acetic acid/4.3% trichloroacetic acid solution. A 50 fold dilution was 

performed then the samples were analyzed by reverse phase HPLC tandem 

mass spectrometry (MS/MS). 

Niacin (vitamin B3) in 

grain

The analytical procedure for the determination of Niacin (vitamin B3) was 

based on a method published by the AACC. Niacin (vitamin B3) was extracted 

from the sample by adding deionized (DI) water and autoclaving. A tube 

array was prepared using three different dilutions of the samples. This tube 

array was inoculated with Lactobacillus plantarum and allowed to incubate 

for approximately 18 to 22 hours. After incubation, the bacterial growth was 

determined using a spectrophotometer at an absorbance of 660 nm. The 

absorbance readings were compared to a standard curve generated using 

known concentrations of nicotinic acid.
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Table 6.1. Methods for Compositional Analysis of 4114 Maize (continued)

Nutritional Analyte Method

Pantothenic acid 

(vitamin B5) in grain

The analytical procedure for the determination of pantothenic acid (Vitamin 

B5) was based on a method from the AOAC. Pantothenic acid (Vitamin B5) 

content was determined using a microbiological assay. Pantothenic acid 

(Vitamin B5) was extracted from the sample by an acetic acid buffer solution, 

consisting of acetic acid adjusted to a pH of 5.65 with sodium hydroxide, and 

autoclaving the samples. A tube array was prepared using three different 

dilutions of the samples. This tube array was inoculated with Lactobacillus 

plantarum and allowed to incubate for approximately 18-22 hours. After 

incubation, the bacterial growth was determined using a spectrophotometer 

at an absorbance of 660 nm. The absorbance readings were compared to a 

standard curve generated using known concentrations of D-pantothenic acid 

hemicalcium salt.

Pyridoxine (vitamin 

B6) in grain

The analytical procedure for the determination of pyridoxine (Vitamin B6) 

was based on a method from the AACC. Pyridoxine (Vitamin B6) was 

determined using a microbiological assay. Pyridoxine (Vitamin B6) was 

extracted from the sample by adding sulfuric acid and autoclaving. The pH 

was adjusted and a tube array was prepared using four different dilutions of 

the samples. This tube array was inoculated with Saccharomyces cerevisiae

and allowed to incubate for approximately 18-22 hours. After incubation, the 

microbial growth was determined using a spectrophotometer at an 

absorbance of 600 nm. The absorbance readings were compared to a 

standard curve generated using known concentrations of pyridoxine 

hydrochloride.

Total folate as folic 

acid (vitamin B9) in 

grain

The analytical procedure for determination of total folate as folic acid was 

based on a microbiological assay published by the AACC. Samples were 

hydrolyzed and digested by protease and amylase enzymes to release the 

folate from the grain. A conjugase enzyme was used to convert the naturally 

occurring folypolyglutamates. An aliquot of the extracted folates was mixed 

with a folate and folic acid free microbiological growth medium. The mixture 

was inoculated with Lactobacillus casei. The total folate content was 

determined by measuring the turbidity of the Lactobacillus casei growth 

response in the sample and comparing it to the turbidity of the growth 

response with folic acid standards.

Tocopherols 

(vitamin E) in grain

The analytical procedure for determination of tocopherols (Vitamin E) was 

based on methods from the Journal of the American Oil Chemists’ Society and 

Analytical Sciences. Alpha, beta, delta, and gamma-tocopherols were 

extracted with hot hexane and the extracts were analyzed by normal phase 

HPLC with fluorescence detection.

Beta-carotene in grain

The analytical procedure for determination of beta-carotene was based on a 

method published by the AOAC. Fat-soluble pigments from the ground maize 

grain were extracted and determined spectrophotometrically and expressed 

as carotene.
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Table 6.1. Methods for Compositional Analysis of 4114 Maize (continued)

Nutritional Analyte Method

Trypsin inhibitor in 

grain

The analytical procedure for the determination of trypsin inhibitor was based 

on a method published by the AOCS. Trypsin inhibitor was extracted with 

sodium hydroxide. Trypsin was added and reacted with trypsin inhibitor. The 

amount of trypsin present in the sample was measured using a 

spectrophotometer, and the amount of inhibitor was calculated based on how 

much trypsin remained.

Furfural in grain

The analytical procedure for the determination of furfural was based on 

methods published in the Journal of Agricultural and Food Chemistry. Ground 

maize grain was analyzed for furfural content by reverse phase HPLC with UV 

detection. 

p-Coumaric and ferulic

acid in grain

The analytical procedure for the determination of p-coumaric and ferulic acids 

was developed based on methods published in Journal of Agricultural and 

Food Chemistry and The Journal of Chemical Ecology. Ground maize grain was 

analyzed to determine the amounts of p-coumaric acid and ferulic acid by 

separating the total content of phenolic acids using reverse phase HPLC and 

UV detection.

Phytic acid in grain

The analytical procedure for the determination of phytic acid was based on a 

method published by the AOAC. The samples were analyzed to determine the 

amount of phytic acid by extracting the phytic acid with dilute hydrochloric 

acid (HCl) and isolating it using an aminopropyl silica solid phase extraction 

column. Once isolated and eluted, the phytic acid was analyzed for elemental 

phosphorus by ICP-OES. 
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6.4. Statistical Analyses

Statistical analyses were conducted using SASosoftware, Version 9.2 (SAS Institute, Inc., Cary, 

NC) to evaluate and compare the nutrient composition of forage and grain derived from 4114 

maize and the control maize.

6.4.1. Processing of Data

Fatty Acids

For some fatty acid analytes, absolute sample values were detected below the assay lower limit 

of quantification (LLOQ). When sample values for each fatty acid analyte were converted from 

an absolute value to a relative proportion (percentage of total fatty acids), sample results below 

the LLOQ were reported as zero to reflect a negligible proportion. However, these zeros were 

not “true” zeros, but were some unknown small positive values. Therefore, these “zero” sample 

values were treated as missing values during subsequent statistical analysis. 

Data Transformation

A natural logarithmic “ln(y)” transformation was performed for the raw data of all analytes 

before statistical analyses. For each analyte, residuals were examined for validation of the 

normality and homogeneous variance assumptions. 

Residual distributions skewed to the left for the following analytes after “ln(y)” transformation 

and therefore, either no transformation, a cubic “(y)3” transformation, a square “(y)2” 

transformation, or a square root “√y” transformaon was performed to the raw data instead: 

 Vitamin B9 - No transformation was performed.

 Crude protein in both forage and grain, and tryptophan and valine in grain- A 

cubic “(y)3” transformation was performed.

 NDF, alanine, aspartic acid, glutamic acid, isoleucine, leucine, phenylalanine, 

proline, and threonine - A square “(y)2” transformation was performed.

 Vitamin B6 – A square root “√y” transformaon was performed.

The model assumptions were reasonably satisfied for these analytes after the moreappropriate transformation or non-transformation.

For a given analyte, the same type of transformation or non-transformation was used for all 

statistical analyses and comparisons performed. The statistical results were then backtransformed to the original data scale for reporting purposes. 

 

o

Registered trademark of SAS Institute, Inc.
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6.4.2. Statistical Analyses

For a given analyte, the number of samples below the assay LLOQ value determined whether a 

statistical analysis was conducted. The following rules were implemented: 

 If, for each entry, <80% of samples were below the LLOQ, then the analysis was 

conducted. 

 If, for a single entry, ≥80% of samples were below the LLOQ, then the analysis was

not conducted. 

If, for a given analyte, a statistical analysis was not conducted due to insufficient data, the 

logistic regression likelihood-ratio test was used to test if the proportion of assay values below 

the LLOQ was different between 4114 maize and the control maize.

Default Model

For a given analyte, data were analyzed using the following linear mixed model:

yijk = μi + lj + rk(j) + (μl)ij + εijk Model 1

lj ~ iid N(0, σ2

Site), rk(j) ~ iid N(0,σ2Rep), (μl)ij ~ iid N(0, σ2Ent×Site), and εijk ~ iid N(0, σ2Error)

Where μi denotes the mean of the i

th entry (fixed effect), lj denotes the effect of the jth site (random effect), 

rk(j) denotes the effect of the kth block within the jth site (random effect), (μl)ij denotes the interaction between 

the entries and sites (random effect) and εijk denotes the effect of the plot assigned the ith entry in the kth block 

of the jth site (random effect or residual). Notation ~iid N(0, σ2

a) indicates random variables that are identically 

independently distributed (iid) as normal with zero mean and variance σ

2

a.

The residual maximum likelihood (REML) estimation procedure was utilized to generate 

estimates of variance components and entry means. The estimated means are known as least 

squares means (LS-Means). The statistical comparison was conducted by testing for differences 

in LS-Means between 4114 maize and the control maize. The approximated degrees of 

freedom for the statistical test were derived by the Kenward-Roger (KR) method (Kenward and 

Roger, 1997).

SAS PROC MIXED was utilized to fit Model 1 and to generate LS-Means, 95% confidence 

intervals, and statistical comparisons (P-values). By default, the variance components in Model 

1 are all constrained to be non-negative. When the estimated value of σ

2

Ent×Site is zero, the KR 

method pools degrees of freedom for the interaction term with the degrees of freedom for 

residuals. Consequently, the degrees of freedom for the statistical test could be larger than 

what was expected under the original experimental design. In order to stabilize the degrees of 

freedom across all analytes, effect (μl)ij in Model 1 was combined with εijk and the compound 

symmetry (CS) structure was used to model the corresponding residual variance structure. This 

approach allows σ

2

Ent×Site to take negative values without affecting the degrees of freedom

(Littell et al., 2006). 
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4114 Maize

Model Considering LLOQ

For the analytes sodium, vitamin B1, vitamin B5, and raffinose, <80% of sample values were 

detected below the assay LLOQ. Sample results below the LLOQ were treated as left-censored 

observations at the respective assay LLOQ value. Data were analyzed using the following linear 

mixed model:

yijk = μi + lj + rk(j) + (μl)ij + εijk Model 2

lj ~ iid N(0, σ2

Site), rk(j) ~ iid N(0,σ2Rep), (μl)ij ~ iid N(0, σ2Entry×Site), εijk ~ iid N(0, σ2Error)

Where μi denotes the mean of the i

th entry (fixed effect), lj denotes the effect of the jth site (random effect),

rk(j) denotes the effect of the k

th block within the jth site (random effect), (μl)ij denotes the interaction between 

the entries and sites (random effect) and εijk denotes the residual for the observation obtained from the plot 

assigned to the i

th entry in the kth block of the jth site. Notation ~iid N(0, σ2

a) indicates random variables that 

are identically independently distributed (iid) as normal with zero mean and variance σ

2

a.

Model 2 can also be written as:

yijk = μijk + εijk = μi + lj + rk(j) + (μl)ij + εijk

Where μijk denotes the mean of yijk, The conditional likelihood for each observation, given the random effects, 

was formulated according to the status of the observation (observed or left-censored) (Thiébaut and JacqminGadda, 2004): 



















  



 



left censored

c

P y c

observed

y

P y y

f y

ijk

ijk

ijk ijk

ijk ijk

ijk

( ) ( )

( ) ( )

( | )

*

*













Where  denotes the vector of all random effects, denotes the standard normal density function,  denotes 

the standard normal cumulative distribution function, y

*

ijk denotes the observed sample value of yijk, and c

denotes the assay LLOQ value. 

The conditional likelihood function was a product of all individual conditional likelihoods, and the marginal 

likelihood function was formed when the conditional likelihood function was integrated over all random 

effects. 

The maximum likelihood procedure was then used to generate estimates of variance 

components and entry means (LS-Means). The statistical comparison was conducted by testing 

for difference in LS-Means between 4114 maize and the control maize. The approximated 

degrees of freedom for the statistical test were derived by the KR method. 

SAS PROC NLMIXED was utilized to fit Model 2 and generate LS-Means, 95% confidence 

intervals, and statistical comparisons (P-values). 
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FDR Adjustment

The FDR method of Benjamini and Hochberg (Benjamini and Hochberg, 1995; Westfall et al., 

1999) was applied as a post-hoc procedure to account for multiple comparisons due to multiple 

compositional analytes, and P-values were adjusted accordingly. The FDR adjustment was 

conducted for each set of comparisons. A significant difference was identified if an adjusted 

P-value <0.05. 

SAS PROC MULTTEST was utilized to provide adjusted P-values.

6.4.3. Interpretations of Statistical Results

Where a statistically significant difference (adjusted P-value <0.05) was identified for a given 

analyte in the analysis between 4114 maize and the control maize, the respective range of 

individual values from 4114 maize was compared to a tolerance interval. Tolerance intervals 

containing 99% of the values for corresponding analytes of the conventional maize population 

with 95% confidence level (Graybill, 1976) were derived from data collected under previous 

studies. In each of these studies, four non-modified commercially available maize lines were 

grown at six sites in North America, and were harvested, processed, and analyzed using 

methods similar to those employed in this nutrient composition study for 4114 maize. The 

selected maize varieties represent the non-modified maize population with a history of safe 

use, and the selected environments (site and year combinations) represent maize growth under 

a range of environmental conditions (i.e., soil type, temperature, precipitation, and irrigation) 

and maize maturity group zones similar to the sites used in the 4114 maize agronomic study. 

Ranges containing individual values outside the tolerance interval for a given analyte were then 

compared to the respective literature range obtained from published literature (Codex, 1996; 

Codex, 2005; ILSI, 2006; OECD, 2002; Watson, 1982; Watson, 1987).

6.4.4. Reported Statistics

For each analyte that was statistically analyzed using mixed model analysis, entry LS-Mean 

(back-transformed, if needed), range, and 95% confidence interval (back-transformed, if 

needed) (labeled as Mean, Range, and CI, respectively) are provided in Section 4; Tables 15-22. 

Both the non-adjusted P-values and FDR-adjusted P-values (labeled as P-Value and Adjusted PValue, respectively) are provided for comparisons between 4114 maize and the control maize. 

For each analyte, a tolerance interval (Section 4; Tables 15-22) and a literature range (Section 4; 

Tables 15-22), if available, are provided. 

In Section 4, Tables 15-22, descriptive statistics (arithmetic means and ranges) are reported for 

analytes that were not statistically analyzed using mixed model analysis. Note: for fatty acid 

analytes, means and ranges were calculated based on assay values above the LLOQ. When all 

fatty acid samples values were below the LLOQ for a given entry, means and ranges were 0 and 

0-0, respectively, and were not reported. 
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Appendix 7. Material and Methods for Analysis of Agronomic Characteristics and 

Germination Evaluation

7.1. Germination and Dormancy Evaluation

4114 maize and two commercial Pioneer® hybrid maize reference lines (32D78 and 34P88) 

were evaluated for germination rates under warm, cold, and diurnal growing conditions.

7.1.2. Experimental Design

Three separate germination tests (warm, cold, and diurnal) were conducted. For a given 

germination test, approximately 400 seed from each of 4114 maize, the control maize, 32D78

maize, and 34P88 maize were evaluated. The seed from each maize line were arranged into 

eight individual replicates, with approximately 50 seed per replicate. No broken or damaged 

seed were included in any of the germination tests. To control experimental bias in this study, 

the following procedures were utilized: randomization of each sample replication within the 

growth chamber and uniform maintenance of environmental conditions across all entries and 

replicates within the growth chamber.

7.1.3. Germination Tests

The warm germination test simulated optimal maize growth conditions. Each replicate was 

placed between sheets of moist germination toweling and rolled up. The rolls were then placed 

in a dark growth chamber at a continuous setting of 25 °C and 90% relative humidity for 10 

days. After 10 days, the number of germinated seed in each replicate was counted.

The cold germination test simulated early spring planting conditions in the midwestern United 

States. Each replicate was placed between sheets of moist germination toweling and rolled up. 

The rolls were then placed in a dark growth chamber at a continuous setting of 10 °C and 90% 

relative humidity for 10 days, followed by three days at a continuous setting of 25 °C and 90% 

relative humidity. After 13 days, the number of germinated seed in each replicate was counted.

The diurnal germination test simulated daily weather conditions in the midwestern United 

States. Each replicate was placed between sheets of moist germination toweling and rolled up. 

The rolls were then placed in a growth chamber at a cyclical setting of 10 °C and 90% relative 

humidity for 16 hours and then 25 °C and 90% relative humidity for 8 hours, repeated daily for 

10 days. After 10 days, the number of germinated seed in each replicate was counted.

7.1.4. Seed Evaluation

Classification of Germinated and Non-Germinated Seed

At the end of each germination test, each seed was defined as either germinated or nongerminated. A given seed was classified as germinated if any of the essential structures 
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necessary to produce a normal plant under favorable conditions had emerged, or 

non-germinated if these structures had not emerged. 

Non-germinated seed were further defined into three categories: hard (i.e., did not absorb 

water), imbibed (i.e., absorbed water but did not show signs of growth during the germination 

test), or dead (i.e., absorbed water, did not show signs of growth during the germination test, 

and displayed distinct signs of decay such as an extremely soft interior that did not hold shape 

under gentle pressure). 

Descriptions of all germination classifications are summarized in Table 7.1.

Table 7.1. Description of Germination Test Classifications

Germination 

Classification Description

Germinated Seed Visible emergence of any structures necessary to produce a 

healthy plant.

NonGerminated 

Seed

Hard

No emergence of structures necessary to produce a healthy 

plant. Seed did not absorb water during the germination 

test.

Imbibed

No emergence of structures necessary to produce a healthy 

plant. Seed did absorb water during the germination test but 

showed no signs of growth.

Dead

No emergence of structures necessary to produce a healthy 

plant. Seed did absorb water during the germination test but 

showed no signs of growth. Seed displayed distinct signs of 

decay (i.e., extremely soft interior that did not hold shape 

under gentle pressure).
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Evaluation of Non-Germinated Seed Viability

Germinated seed were considered viable. Non-germinated seed classified as dead were 

considered non-viable and no further assessments of viability were conducted. 

Non-germinated seed classified as hard or imbibed were further evaluated for viability using a 

tetrazolium chloride (TZ) test (AOSA, 2005). In non-germinated hard seed, potential dormancy 

can be indicated by seed viability; however, seed dormancy is not commonly observed in maize.

The TZ test was conducted as follows: seeds were bisected longitudinally and placed in a 

separate 50 ml vial for each entry, then stained with 1% TZ solution (prepared by dissolving 10 g 

of 2,3,5-triphenyltetrazolium chloride into one liter of water) for approximately two hours at 

25 °C. Any living cells were stained a reddish-pink color by the TZ solution, allowing 

identification of viable tissues. Seed with staining patterns indicative of 100% viable tissue in 

the essential seed structures (i.e., radical, embryo axis, plumule, and coleoptiles), were 

considered viable. Seed with staining patterns (or non-staining) indicative of less than 100% 

viable tissue in the essential seed structures were considered to be non-viable.

7.1.5. Statistical Analysis

Statistical analyses of germination data were conducted to evaluate the germination rate of 

seed derived from 4114 maize compared to the germination rate of seed derived from the 

control maize. Statistical analyses were conducted separately for each of the three germination 

tests (warm, cold, and diurnal) using SASpsoftware, Version 9.2 (SAS Institute, Inc., Cary, NC).

For a given germination test, when the minimum value of the expected count of total 

non-germinated seed derived from either 4114 maize or the control maize was less than five, a 

Fisher’s exact test was conducted to compare total germination frequencies. A significant 

difference was identified if the P-value < 0.05. SAS PROC FREQ was utilized to conduct Fisher’s 

exact tests.

For a given germination test, if the expected count of total non-germinated seed derived from 

both 4114 maize and the control maize was five or more, then a Generalized Linear Mixed 

Model (GLMM) assuming binomial distribution with the “logit” link function was utilized to 

analyze the data. Maximum likelihood method with Laplace approximation (SAS Institute Inc., 

2008; Vonesh, 1996) was utilized to estimate and compare mean germination rates between 

4114 maize and the control maize. A significant difference was identified if the P-value < 0.05. 

SAS PROC GLIMMIX was utilized to implement this type of analysis.

Let yij represent the number of germinated seed in the j

th replicate (each replicate contained a 

total of 50 seed) of the i

th entry, j = 1, 2, ..., 8. yij ~ Binomal (nij, ij), where nij denotes the total 

 

p

Registered trademark of SAS Institute, Inc.
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number of seed in the j

th replicate of the ith entry, and ij denotes the probability of a seed 

being germinated in the jth replicate of the ith entry. The “logit” link function was expressed as:

logit( ) log

1

ij

ij ij

ij



 



 

        

Which transformed the parameter ij on the unit scale into a parameter on the linear predictor 

scale, denoted as ij. Logit link is default for binomial data.

The GLMM on the linear predictor scale was:

ij = i + rj(i)

Where μi denotes the mean response for the i

th entry (fixed effect) and rj(i) denotes the effect of the 

j

th replicate within the ith entry (random effect nested within fixed effect). For this model, it was 

assumed that random effects rj(i) ~ N(0, 2

R) were independently and identically distributed of each 

other. 

Reported statistics for each germination test included descriptive statistics (total germination 

frequency, mean, and range of individual replicate germination rates) for 4114 maize and the 

control maize, and P-values for statistical comparisons in Section 4; Tables 24-26. When GLMM 

was utilized, estimated mean germination rate and standard error of the estimate were also 

provided (labeled as LS-Mean and SEM, respectively) for 4114 maize and the control maize

Section 4; Tables 24-26. For each germination test, the range of germination rates for 

individual replicates across reference maize lines (labeled as Reference Range) is provided

Section 4; Tables 24-26.

7.2. Agronomic Characteristics Evaluation

Agronomic characteristics were evaluated in 4114 maize using plants from two experiments in 

2010 and analyzed as described below.
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7.2.1. Field Trial and Experimental Design

4114 maize plants of the F1*5 generation were grown in two experiments at a total of 17 sites in 

2010, with 15 sites in the U.S. and two sites in Canada. For those sites in the same location 

(e.g., 3 and 12), the field trials were conducted independently of each other.

Site Country Location

Experiment A: 2010 Field Trials

1

U.S.

Sheridan, IN

2 Deerfield, MI

3 Richland, IA

4 Rochelle, IL

5 Bagley, IA

6 Seymour, IL

7 York, NE

8 Carlyle, IL

9 Wyoming, IL

10 Atlantic, IA

11 Geneva, MN

Experiment B: 2010 Field Trials

12

U.S.

Richland, IA

13 Wyoming, IL

14 Geneva, MN

15 York, NE

16 Canada Branchton, ON

17 Thorndale, ON

Each field site utilized a randomized complete block design with 4114 maize and the control 

maize planted in two-row plots within four replicate blocks. Approximately 30 seeds were 

planted in each 25 ft (7.6 m) row. Row spacing was approximately 30 inches (76 cm) and every 

two-row plot was bordered on either side by one row of commercial maize of similar maturity. 

In addition, blocks were separated by an alley distance of at least 36 inches (0.9 m) and each 

site was surrounded by a minimum of four external border rows. Maintenance fertilizer, 

herbicides, and pesticides were applied uniformly to 4114 maize and the control maize at each 

site, as needed. In plots where grain yield was collected, plants were allowed to open-pollinate. 

To control experimental bias in this study, the following procedures were utilized: 

non-systematic selection of trial and plot areas within each site, randomization of maize entries 

within each block, and uniform maintenance across blocks in each field site. 
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7.2.2. Agronomic Data Collection

Agronomic characteristics were recorded for each maize line within all blocks at each site. 

Descriptions of maize growth stages are provided in Table 7.2. 

Table 7.2. Maize Growth Stage Descriptions

Growth 

Stagea Descriptiona

V2 The stage when the collar of the second leaf becomes 

visible.

V4 The stage when the collar of the fourth leaf becomes 

visible.

V6 The stage when the collar of the sixth leaf becomes visible.

V7 The stage when the collar of the seventh leaf becomes 

visible.

V9 The stage when the collar of the ninth leaf becomes 

visible.

R1 The stage when silks become visible.

R4 The stage when the material within the kernel produces a 

doughy consistency.

R6 Typical grain harvest would occur. This stage is regarded as 

physiological maturity.

a

 Growth stages as described in Ritchie et al., 2005

The following characteristics were recorded:

Early Population

The total number of plants emerged per plot was determined between V2 and V4 growth 

stages. 

Seedling Vigor

Seedling vigor was visually estimated per plot when plants were between V2 and V4 growth 

stages using a 1-9 scale, with 1 corresponding to poor vigor (short plants with small, thin leaves) 

and 9 corresponding to good vigor (tall plants with large, robust leaves). 
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Time to Silking

The number of accumulated heat unitsq was calculated per plot from the planting date to the 

date when approximately 50% of plants had produced silks, except for two sites, which did not 

record 50% silking date for 4114 maize in Blocks 2, 3, and 4; and the control maize in Block 4.

Time to Pollen Shed

The number of accumulated heat units was calculated per plot from the planting date to the 

date when approximately 50% of plants had shed pollen, except for one site which did not 

record the dates to 50% pollen shed. 

Pollen Viability

Pollen viability was only assessed at six of the 17 sites, with four sites in the U.S. and two sites 

in Canada. Pollen viability has been correlated to pollen shape and color (Luna et al., 2001). 

Pollen viability per plot was assessed indirectly by evaluating pollen shape and color when 

approximately 50% of the plants had shed pollen. The percentage of pollen grains with 

collapsed wallsrand intense yellow colors were recorded at four time points (0 minutes, 30 

minutes, 60 minutes, and 120 minutes).

Plant Height

Plant height was measured in centimeters from the soil surface to the tip of the tassel for ten 

plants per plot at the R4 growth stage, except for two field sites which measured plant height at 

the R4 or R5 growth stages. 

Ear Height

Ear height was measured in centimeters from the soil surface to the base of the primary ear for 

ten plants per plot at the R4 growth stage, except for two field sites which measured ear height 

at the R4 or R5 growth stages. 

Stalk Lodging

Stalk lodging severity was visually estimated per plot as the percentage of plants broken below 

the primary ear at the R6 growth stage. 

 

q

Heat units = ((maximum temperature + minimum temperature)/2) – 50 °F 

If the maximum temperature was greater than 86 °F then 86 °F was used. If the minimum temperature was less 

than 50 °F, 50 °F was used. Heat units were calculated for each growing day and summed to give a total 

accumulated heat unit value. If a daily heat unit was negative, 0 (zero) was assigned. 

r

Percentage of collapsed pollen compared to spherical pollen. 

s

Percentage of pollen with intense yellow color compared to pollen white in color. 
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Root Lodging

Root lodging severity was visually estimated per plot as the percentage of plants leaning 

approximately 30 degrees or more in the first 2 feet (0.6 m) above the soil surface at the R6 

growth stage. 

Final Population

The total number of remaining plants per plot was recorded at the R6 growth stage. Previously 

sampled plants were included in the final population total. 

Stay Green

Overall plant health was visually estimated per plot at the R6 growth stage using a 1 to 9 scale, 

with 1 corresponding to no visible green tissue remaining, 5 corresponding to approximately 

50% green tissue remaining, and 9 corresponding to approximately ≥90% green tissue 

remaining. 

Disease Incidence

Foliar disease incidence was visually estimated per plot at the R6 growth stage using a 1-9 scale, 

with 1 corresponding to poor disease resistance (high infection) and 9 corresponding to 

excellent disease resistance (low infection).

Insect Damage

Insect damage was visually estimated per plot at the R6 growth stage using a 1-9 scale, with 1 

corresponding to poor insect resistance (high damage) and 9 corresponding to excellent insect 

resistance (low damage). 

Yield

Yield was only assessed at 11 of the 17 sites in the U.S. during the 2010 growing season. Maize 

grain was collected from each plot at typical harvest maturity (R6 growth stage). Yield was 

determined based on the weight (kg) and moisture content (%) of the grain. The following 

formulas were used to determine yield:

Grain weights from each plot were adjusted to 0% moisture content.

Grain(dry weight) (kg) = Grain(fresh weight) (kg) – (Grain(fresh weight) (kg) * % actual moisture)

Example: Grain(fresh weight) = 9.66 kg, Actual Moisture = 21.5%

Grain(dry weight) = 9.66 kg – (9.66 kg * 21.5%) = 7.58 kg
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The grain dry weights were then adjusted to 15.5% moisture content.

Grain(@ 15.5% moisture) (kg) = Grain(dry weight) (kg) / (1 – 15.5% moisture)

Example: Grain(dry weight) = 7.58 kg

Grain(@ 15.5% moisture) = 7.58 kg / (1 -15.5%) = 8.98 kg

The grain weights at 15.5% moisture were then converted to bu/A.

Yield (bu/A) = Grain(@ 15.5% moisture) (kg) * 43560 ft2

/A

Plot Area (ft2) * 25.4 kg/bu

Example: Grain(@ 15.5% moisture) = 8.98 kg, Plot Area = 125 ft2

Yield (bu/A) = 8.98 kg * 43560 ft2

/A = 123 bu/A 125 ft2

* 25.4 kg/bu

7.2.3. Statistical Methods

Statistical analyses were conducted separately for two studies to evaluate and compare 

agronomic characteristics of 4114 maize and the control maize using SAS software, Version 9.2 

(SAS Institute, Inc. Cary, NC).

7.2.4. Processing of Data

Plant and Ear Height

Plant height and ear height were measured on ten plants per plot. Plot average (average of ten 

plants) was treated as the response. 

Transformation

Pollen viability (color and shape) at 0, 30, and 60 minutes, were percentage data. They were 

converted to proportion data. Early population and final population were binomial count data. 

They were also converted to proportion data according to:

y= number of stand count / max (target seedling rate, which is 60. Maximum stand count 

across plots which could be more or less than 60)
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Proportion data was subjected to the arcsine square-root transformation,

 z y  arcsin( )

where y denotes proportion value, and z denotes transformed value.

The transformed data met statistical assumptions of mixed model analyses, namely normality 

and constant variance assumptions. The statistical results were then transformed back to the 

original scale. 

7.2.5. Statistical Analyses

In one of the two experiments, greater than 80% of data values for root lodging and stalk 

lodging were at a uniform value (i.e., 0% lodging); therefore, the analysis was not conducted for 

these two characteristics. 

Mixed Model Analysis

For a given agronomic characteristic, data were analyzed using the following linear mixed 

model:

yijk = μi + lj + rk(j) + (μl)ij + εijk Model 1

lj ~ iid N(0, σ2

Site), rk(j) ~ iid N(0,σ2Rep), (μl)ij ~ iid N(0, σ2Ent×Site), and εijk ~ iid N(0, σ2Error)

where μi denotes the mean of the ith entry (fixed effect), lj denotes the effect of the j

th site (random 

effect), rk(j) denotes the effect of the k

th block within the jth site (random effect), (μl)ij denotes the 

interaction between the entries and sites (random effect) and εijk denotes the effect of the plot 

assigned the ith entry in the k

th block of the jth site (random effect or residual). Notation ~iid N(0, σ2

a)

indicates random variables that are identically independently distributed (iid) as normal with zero 

mean and variance σ

2

a.

The residual maximum likelihood (REML) estimation procedure was utilized to generate 

estimates of variance components and entry means. The estimated means are known as least 

squares means (LS-means). The statistical comparison was conducted by testing for difference 

in LS-means for contrasts of interest. The approximated degrees of freedom for statistical test 

were derived by Kenward-Roger (KR) method (Kenward and Roger, 1997). 

SAS PROC MIXED was utilized to fit Model 1 and generate LS-means and 95% confidence 

intervals. The same procedure also provided statistical comparisons (P-values). By default, the 

variance components in Model 1 are all constrained to be non-negative. When the estimated 

value of σ

2

Ent×Site is zero, the KR method pools degrees of freedom for the interaction term with 

the degrees of freedom for residuals. Consequently, the degrees of freedom for the statistical 

test could be drastically larger than what was expected under the original experimental design. 
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In order to make the degrees of freedom remain stable across all agronomic characteristics, 

effect (μl)ij in Model 1 was combined with εijk and the compound symmetry (CS) structure was 

used to model the corresponding residual variance structure. This approach allows σ

2

Ent×Site to 

take negative values without affecting the degrees of freedom (Littell et al., 2006). However, 

expanding the parameter space sometimes can cause the model to fail to converge. In one of 

the two studies, for time to silking, the model with CS structure failed to converge and 

parameters were estimated using the default setting. 

Generalized Cochran-Mantel-Haenszel (CMH) Test

In one agronomic study the characteristics insect damage, stalk lodging, and root lodging had 

very discrete values. In the other agronomic study, the characteristics disease incidence, insect 

damage, seedling vigor, and pollen viability (color and shape) at 120 minutes, had very discrete 

values. This violated the normality assumption in Model 1; hence, it was not feasible to apply 

the mixed model approach on these characteristics. Instead, comparisons of entries were 

based on the generalized CMH test.

The generalized CMH test was developed specifically for stratified nominal-by-ordinal 

contingency tables (Agresti, 2002; Koch et al., 1990). It compares entries (a nominal variable) 

based on their values (recorded on an ordinal scale) while controlling for location (the 

stratifying variable). The test’s P-values can be directly interpreted as testing for the difference 

between the arithmetic means for each entry, because the data values were used as the scores 

in the generalized CMH test.

Note that when applying the generalized CMH test, the block-nested-within-location effect and 

the location-by-entry effect in the design were ignored. This does not present any issues 

because for these agronomic characteristics, the block-nested-within-location effect and the 

location-by-entry effect are often negligible compared with the location effect. 

SAS PROC FREQ was used to perform the generalized CMH test.

7.2.6. FDR (False Discovery Rate) Adjustment

The FDR method of Benjamini and Hochberg (Benjamini and Hochberg, 1995; Westfall et al., 

1999) was applied as a post-hoc procedure to account for multiple comparisons due to multiple 

agronomic characteristics and P-values were adjusted accordingly. The FDR adjustment was 

conducted for results. A significant difference was identified if an adjusted P-value <0.05.

SAS PROC MULTTEST was utilized to provide adjusted P-values.

7.2.7. Interpretation of Statistical Results

Where a statistically significant difference (adjusted P-value <0.05) was identified for a given 

agronomic characteristic, the respective range of individual values was compared to a tolerance 
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interval. Tolerance intervals containing 99% of the values for corresponding characteristics of 

the conventional maize population with 95% confidence level (Graybill, 1976) were derived 

from data collected under multiple historical studies. In those studies, a total of eight nonmodified commercial maize lines were grown in a total of twelve sites in North America and a 

total of six non-modified commercial maize lines were grown in a total of four sites in Chile 

under normal agronomic practices. The selected maize lines represent a non-modified maize 

population with a history of safe use, and the selected environments (site and year 

combinations) represent maize growth under a wide range of environmental conditions (i.e.,

soil type, temperature, precipitation, and irrigation) and maize maturity group zones similar to 

the sites used in these 4114 maize agronomic studies.

7.2.8. Reported Statistics

For each agronomic characteristic that was statistically analyzed using mixed model analysis, 

entry LS-mean (back-transformed, if needed), range, and 95% confidence interval of the mean 

(labeled as Mean, Range, and CI, respectively) are provided in Section 4; Tables 30 and 31. Both 

the non-adjusted P-values and the FDR-adjusted P-values (labeled as P-Value and Adjusted 

P-Value, respectively) are provided for statistical comparisons. For each agronomic 

characteristic, a tolerance interval, if available, is provided for the results in Section 4; Tables 30 

and 31. In the tables (Section 4; Tables 30 and 31), descriptive statistics (arithmetic means and 

ranges) are reported for characteristics that were not statistically analyzed using mixed model 

analysis.
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Appendix 8. Field Insect and Disease Observations

4114 maize has been field tested in the U.S. and Puerto Rico since 2006, as authorized by USDAAPHIS permits and notifications (Appendix 1). For each trial, a survey of the naturally occurring 

insects and diseases and any unexpected differences in the response of 4114 maize as 

compared to the control line (near-isoline and/or conventional maize lines) were recorded by 

experienced plant breeders and field staff at least every four weeks. The plant breeders and 

field staff were familiar with plant pathology and entomology and also recorded the severity of 

any insect or disease in the field. These observations provide a means to determine if 4114 

maize will respond differently from conventional maize lines to insects or diseases in the 

environment. 

A summary of the naturally-occurring insects present in the fields and any unexpected 

differences seen between 4114 maize and control lines is presented in Table 8.1. A summary of 

diseases present in the fields is presented in Table 8.2. 

The following scale was used to evaluate 4114 maize and control lines (Tables 8.1 and 8.2; 

“Range of Severity in 4114 Maize”):

 Mild – very little disease or insect injury (<10%) visible

 Moderate – noticeable plant tissue damage (10% to 30%)

 Severe – significant plant tissue damage (>30%)

In every case, the 4114 maize did not exhibit any unexpected responses to naturally-occurring

insects or diseases as compared to control line.

 
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Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control

Year of 

Planting Permit Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2006

06-019-03R PR Guayama Fall armyworm (Spodoptera frugiperda) mild to moderate no

Corn earworm (Helicoverpa zea) mild no

06-019-04R HI Kauai

Corn earworm (Helicoverpa zea) mild to severe no

Rose beetle (Adoretus sinicus) mild no

Leafhopper (Cicadellidae) mild to moderate no

Thrips (Frankliniella spp.) mild no

Spider mites (Tetranychidae) mild no

06-019-03R PR Salinas

Corn earworm (Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild, moderate no

Aphids (Aphididae) mild no

2007 07-040-101rm

CA Yolo Painted ladies (Vanessa spp.) mild no

HI Kauai

Corn earworm (Helicoverpa zea) mild no

Aphids (Aphididae) mild no

Leafhopper (Cicadellidae) mild no

Rose beetle (Adoretus sinicus) mild no

Fall armyworm (Spodoptera frugiperda) mild no

IA

Linn

Corn rootworm (Diabrotica spp.) mild to moderate no

Japanese beetle (Popillia japonica) mild no

European corn borer (Ostrinia nubilalis) moderate no

Polk

Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no

Corn earworm (Helicoverpa zea) mild no

Bean leaf beetles (Cerotoma trifurcata) mild no

IL

Bureau European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no

Champaign Corn rootworm (Diabrotica spp.) mild no

Grasshoppers (Orthoptera) mild no

McDonough Corn rootworm (Diabrotica spp.) mild to moderate no
Pioneer Hi-Bred International, Inc. Page 216 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2007 07-040-101rm

IL

McDonough European corn borer (Ostrinia nubilalis) mild to moderate no

Ogle Corn rootworm (Diabrotica spp.) mild no

Japanese beetle (Popillia japonica) mild no

IN Tipton Corn rootworm (Diabrotica spp.) mild no

MN Blue Earth Aphids (Aphididae) mild no

Corn rootworm (Diabrotica spp.) mild no

PR

Juana Diaz Corn earworm (Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Salinas Corn earworm (Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild to moderate no

WI Rock Corn rootworm (Diabrotica spp.) moderate no

Aphids (Aphididae) mild no

NE York

European corn borer (Ostrinia nubilalis) mild no

Lady beetles (Coccinellidae) mild no

Aphids (Aphididae) mild to moderate no

Corn rootworm (Diabrotica spp.) mild to moderate no

Spider mites (Tetranychidae) mild no

Lacewings (Neuroptera) mild no

2008

07-040-101rm

PR Salinas

Corn earworm (Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Corn planthoppers (Delphacidae) mild no

HI Kauai

Leafhopper (Cicadellidae) mild no

Spider mites (Tetranychidae) mild to moderate no

Corn earworm (Helicoverpa zea) moderate no

08-014-111n

CA Yolo Painted ladies (Vanessa spp.) mild no

IN

Gibson Japanese beetle (Popillia japonica) mild no

Tipton Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no
Pioneer Hi-Bred International, Inc. Page 217 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2008 08-014-111n

IA

Polk European corn borer (Ostrinia nubilalis) mild no

Japanese beetle (Popillia japonica) mild no

Linn Japanese beetle (Popillia japonica) mild to moderate no

European corn borer (Ostrinia nubilalis) mild no

NE

Nance

European corn borer (Ostrinia nubilalis) mild no

Lady beetles (Coccinellidae) mild no

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

Grasshoppers (Orthoptera) mild no

Stink bug (Pentatomidae) mild no

York

European corn borer (Ostrinia nubilalis) mild to moderate no

Lady beetles (Coccinellidae) mild to moderate no

Stink bug (Pentatomidae) mild no

Beetles (Coleoptera) mild to moderate no

Grasshoppers (Orthoptera) mild to moderate no

Aphids (Aphididae) mild to moderate no

Corn rootworm (Diabrotica spp.) mild to moderate no

Lacewings (Neuroptera) mild no

MN Blue 

Earth

European corn borer (Ostrinia nubilalis) mild to moderate no

Corn rootworm (Diabrotica spp.) moderate no

PR

Juana 

Diaz

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Corn earworm (Helicoverpa zea) mild to moderate no

Aphids (Aphididae) severe no

Salinas

Fall armyworm (Spodoptera frugiperda) mild to severe no

Corn earworm (Helicoverpa zea) mild to severe no

Cornsilk fly (Euxesta stigmatias) mild no

Aphids (Aphididae) mild no

Grasshoppers (Orthoptera) mild to moderate no
Pioneer Hi-Bred International, Inc. Page 218 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2008

08-014-111n

IL

Bureau Corn rootworm (Diabrotica spp.) mild to moderate no

Champaign Corn rootworm (Diabrotica spp.) mild no

McDonough Japanese beetle (Popillia japonica) mild no

Corn rootworm (Diabrotica spp.) mild no

Ogle Japanese beetle (Popillia japonica) severe no

Corn rootworm (Diabrotica spp.) severe no

WI Rock

Japanese beetle (Popillia japonica) mild no

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

Grasshoppers (Orthoptera) mild no

08-014-131n HI Kauai

Leafhopper (Cicadellidae) mild no

Corn earworm (Helicoverpa zea) mild to moderate no

Spider mites (Tetranychidae) mild to moderate no

Rose beetle (Adoretus sinicus) mild no

Thrips (Frankliniella spp.) mild no

Leafhopper (Cicadellidae) mild no

08-095-105n

OK Caddo

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Green June beetles (Cotinus nitida) mild no

Corn earworm (Helicoverpa zea) moderate no

IL

Clinton

Corn earworm (Helicoverpa zea) mild no

Fall armyworm (Spodoptera frugiperda) mild no

Japanese beetle (Popillia japonica) mild no

Stark

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

European corn borer (Ostrinia nubilalis) mild no

IA Jefferson

European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no

Corn earworm (Helicoverpa zea) mild no
Pioneer Hi-Bred International, Inc. Page 219 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2008 08-095-105n

MN Freeborn

Aphids (Aphididae) mild no

European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no

NE York

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

Grasshoppers (Orthoptera) mild no

Corn earworm (Helicoverpa zea) mild to moderate no

TX Wharton

Corn earworm (Helicoverpa zea) mild no

Corn stalk borers (Pyralidae) mild no

Fall armyworm (Spodoptera frugiperda) mild no

2009

08-014-111n PR Salinas

Fleahoppers (Miridae) moderate to severe no

Corn earworm (Helicoverpa zea) mild to severe no

Corn sap beetle (Carpophilus spp.) severe no

Fall armyworm (Spodoptera frugiperda) moderate no

09-013-108n

IL

Bureau Corn rootworm (Diabrotica spp.) mild no

Japanese beetle (Popillia japonica) mild no

Fulton

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

Grasshoppers (Orthoptera) mild no

McDonough European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no

IN

Gibson Japanese beetle (Popillia japonica) mild no

Tipton

Japanese beetle (Popillia japonica) mild no

Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no

IA Dallas

Bean leaf beetles (Cerotoma trifurcata) mild no

European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no
Pioneer Hi-Bred International, Inc. Page 220 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2009

09-013-108n

IA

Dallas

Aphids (Aphididae) mild no

Lady beetles (Coccinellidae) mild no

Stink bug (Pentatomidae) mild no

Kossuth Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

Linn

Corn earworm (Helicoverpa zea) mild no

Japanese beetle (Popillia japonica) mild no

Corn earworm (Helicoverpa zea) mild no

Polk Beetles (Coleoptera) mild no

Aphids (Aphididae) mild no

NE York

European corn borer (Ostrinia nubilalis) mild no

Corn blotch leafminers (Agromyza parvicornis) mild no

Western bean cutworm (Richia albicosta) mild no

Corn earworm (Helicoverpa zea) mild no

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

09-035-109n

AR Crittenden

Corn earworm (Helicoverpa zea) mild to severe no

European corn borer (Ostrinia nubilalis) severe no

Fall armyworm (Spodoptera frugiperda) severe no

CO Weld Spider mites (Tetranychidae) mild no

GA Grady

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Aphids (Aphididae) moderate no

Spider mites (Tetranychidae) moderate no

HI Kauai

Leafhopper (Cicadellidae) mild no

Spider mites (Tetranychidae) mild no

Rose beetle (Adoretus sinicus) mild no

Aphids (Aphididae) mild no

Cornsilk fly (Euxesta stigmatias) mild no
Pioneer Hi-Bred International, Inc. Page 221 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2009 09-035-109n

HI Kauai

Corn sap beetle (Carpophilus spp.) mild no

Beet armyworm (Spodoptera exigua) mild no

Rose beetle (Adoretus sinicus) mild no

Corn earworm (Helicoverpa zea) mild no

MN Blue 

Earth

European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

PR

Guayama

Fall armyworm (Spodoptera frugiperda) mild to severe no

Corn earworm (Helicoverpa zea) mild to moderate no

Aphids (Aphididae) moderate to severe no

Corn sap beetle (Carpophilus spp.) mild no

Juana 

Diaz

Fall armyworm (Spodoptera frugiperda) mild to severe no

Corn earworm (Helicoverpa zea) mild to moderate no

Corn sap beetle (Carpophilus spp.) mild to severe no

Cornsilk fly (Euxesta stigmatias) mild to severe no

Aphids (Aphididae) mild to severe no

Spider mites (Tetranychidae) moderate no

Corn planthoppers (Delphacidae) mild no

Leafhopper (Cicadellidae) mild no

Fleahoppers (Miridae) moderate no

Whiteflies (Aleyrodidae) moderate no

Salinas

Corn earworm (Helicoverpa zea) mild to severe no

Fall armyworm (Spodoptera frugiperda) mild to severe no

Fleahoppers (Miridae) mild to severe no

Cornsilk fly (Euxesta stigmatias) moderate no

TN Obion

European corn borer (Ostrinia nubilalis) mild no

Southwestern corn borer (Diatraea grandiosella) mild no

Grasshoppers (Orthoptera) mild no
Pioneer Hi-Bred International, Inc. Page 222 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2009

09-035-109n

TN Obion Fall armyworm (Spodoptera frugiperda) mild no

Corn earworm (Helicoverpa zea) mild no

WI Rock

Aphids (Aphididae) mild no

Japanese beetle (Popillia japonica) mild no

Corn rootworm (Diabrotica spp.) mild to moderate no

09-264-102n

HI Kauai

Corn earworm (Helicoverpa zea) mild no

Beetles (Coleoptera) mild no

Corn flea beetle (Chaetocnema pulicaria) mild no

Whiteflies (Aleyrodidae) mild no

Leafhopper (Cicadellidae) mild no

Lacewings (Neuroptera) mild no

Wasps (Hymenoptera) mild no

Spider mites (Tetranychidae) mild no

Aphids (Aphididae) mild no

Lady beetles (Coccinellidae) mild no

PR

Juana 

Diaz

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Aphids (Aphididae) mild to moderate no

Spider mites (Tetranychidae) mild no

Corn earworm (Helicoverpa zea) mild no

Corn sap beetle (Carpophilus spp.) mild no

Cornsilk fly (Euxesta stigmatias) mild no

Salinas

Fall armyworm (Spodoptera frugiperda) mild to severe no

Grasshoppers (Orthoptera) mild no

Corn planthoppers (Delphacidae) mild to moderate no

Corn earworm (Helicoverpa zea) mild to moderate no

Corn sap beetle (Carpophilus spp.) mild to severe no

Cornsilk fly (Euxesta stigmatias) mild to moderate no

Spider mites (Tetranychidae) mild to severe no
Pioneer Hi-Bred International, Inc. Page 223 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2009

09-264-102n PR Salinas Aphids (Aphididae) mild to severe no

09-016-103n

IL Stark Aphids (Aphididae) mild no

European corn borer (Ostrinia nubilalis) mild no

NE York Black cutworms (Agrotis ipsilon) mild no

European corn borer (Ostrinia nubilalis) mild no

2010

09-264-102n

HI Kauai

Thrips (Frankliniella spp.) mild no

Leafhopper (Cicadellidae) mild no

Fall armyworm (Spodoptera frugiperda) mild no

Lacewings (Neuroptera) mild no

Lady beetles (Coccinellidae) mild no

Corn earworm (Helicoverpa zea) mild no

Spider mites (Tetranychidae) mild no

Aphids (Aphididae) mild no

Wasps (Hymenoptera) mild no

PR Salinas

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Spider mites (Tetranychidae) mild to moderate no

Leafhopper (Cicadellidae) mild to moderate no

Corn sap beetle (Carpophilus spp.) mild to moderate no

Cornsilk fly (Euxesta stigmatias) moderate no

Corn earworm (Helicoverpa zea) mild to moderate no

Aphids (Aphididae) mild to moderate no

Corn planthoppers (Delphacidae) moderate no

10-050-101n NE Keith

Corn rootworm (Diabrotica spp.) mild no

Grasshoppers (Orthoptera) mild no

Corn earworm (Helicoverpa zea) mild no

10-052-101n AR Crittenden

Fall armyworm (Spodoptera frugiperda) moderate to severe no

Corn earworm (Helicoverpa zea) moderate to severe no

Southwestern corn borer (Diatraea grandiosella) moderate to severe no
Pioneer Hi-Bred International, Inc. Page 224 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-052-101n

AR Crittenden European corn borer (Ostrinia nubilalis) moderate to severe no

CA Yolo Spider mites (Tetranychidae) mild to moderate no

CO Philips

Grasshoppers (Orthoptera) mild no

Corn rootworm (Diabrotica spp.) mild no

Corn earworm (Helicoverpa zea) mild no

Weld Spider mites (Tetranychidae) mild no

GA Grady

Aphids (Aphididae) mild no

Fall armyworm (Spodoptera frugiperda) mild no

Spider mites (Tetranychidae) mild no

Aphids (Aphididae) mild no

HI Kauai

Aphids (Aphididae) mild no

Corn earworm (Helicoverpa zea) mild no

Spider mites (Tetranychidae) mild no

Fall armyworm (Spodoptera frugiperda) mild no

Beet armyworm (Spodoptera exigua) mild no

Lacewings (Neuroptera) mild no

Thrips (Frankliniella spp.) mild no

Leafhopper (Cicadellidae) mild no

Lady beetles (Coccinellidae) mild no

IL

Bureau Japanese beetle (Popillia japonica) mild no

Champaign Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no

Fulton

Grasshoppers (Orthoptera) mild no

Lady beetles (Coccinellidae) mild no

Corn rootworm (Diabrotica spp.) mild no

Stink bugs (Pentatomidae) mild no

Aphids (Aphididae) mild no
Pioneer Hi-Bred International, Inc. Page 225 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect

Overall Severity in 

Field

Unexpected 

Difference in 

Comparison?

2010 10-052-101n

IL

La Salle Japanese beetle (Popillia japonica) mild no

McDonough

Lady beetles (Coccinellidae) mild no

Grasshoppers (Orthoptera) mild no

Corn rootworm (Diabrotica spp.) mild no

Ogle Corn rootworm (Diabrotica spp.) mild to severe no

Japanese beetle (Popillia japonica) severe no

IN Gibson Japanese beetle (Popillia japonica) mild noCorn rootworm (Diabrotica spp.) mild no

Tipton Corn rootworm (Diabrotica spp.) mild no

IA

Bremer Corn earworm (Helicoverpa zea) mild no

Corn earworm (Helicoverpa zea) mild no

Dallas

Bean leaf beetles (Cerotoma trifurcata) mild no

Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no

Lady beetles (Coccinellidae) mild no

Fall armyworm (Spodoptera frugiperda) mild no

Delaware Corn earworm (Helicoverpa zea) mild no

Japanese beetle (Popillia japonica) mild no

Kossuth

Lady beetles (Coccinellidae) mild no

Corn rootworm (Diabrotica spp.) mild no

Lady beetles (Coccinellidae) mild no

Linn Corn earworm (Helicoverpa zea) mild no

Japanese beetle (Popillia japonica) mild no

Madison

Bean leaf beetles (Cerotoma trifurcata) mild no

Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no

Corn rootworm (Diabrotica spp.) mild no
Pioneer Hi-Bred International, Inc. Page 226 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-052-101n

IA

Madison Lady beetles (Coccinellidae) mild no

Fall armyworm (Spodoptera frugiperda) mild no

Polk

Corn earworm (Helicoverpa zea) mild no

Japanese beetle (Popillia japonica) mild no

Beetles (Coleoptera) mild no

Corn earworm (Helicoverpa zea) mild no

Van Buren Corn rootworm (Diabrotica spp.) mild no

KS Finney Flea beetle (Galerucinae) mild no

Cucumber beetle (Chrysomelidae) mild no

MI Gratiot

Western bean cutworm (Richia albicosta) mild no

Corn rootworm (Diabrotica spp.) mild No

Lady beetles (Coccinellidae) mild No

MN Blue Earth European corn borer (Ostrinia nubilalis) mild No

Corn rootworm (Diabrotica spp.) mild No

MO Scott

European corn borer (Ostrinia nubilalis) mild No

Fall armyworm (Spodoptera frugiperda) mild No

Grasshoppers (Orthoptera) mild No

NE York

Corn rootworm (Diabrotica spp.) mild No

European corn borer (Ostrinia nubilalis) mild No

Western bean cutworm (Loxagrotis albicosta) mild No

PA Lebanon European corn borer (Ostrinia nubilalis) mild No

Fall armyworm (Spodoptera frugiperda) mild No

PR Guayama

Corn sap beetle (Carpophilus spp.) mild to severe No

Cornsilk fly (Euxesta stigmatias) mild to severe No

Fall armyworm (Spodoptera frugiperda) mild to severe No

Grasshoppers (Orthoptera) mild No

Corn planthoppers (Delphacidae) mild to moderate No

Corn earworm (Helicoverpa zea) mild to severe No
Pioneer Hi-Bred International, Inc. Page 227 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-052-101n

PR

Guayama

Aphids (Aphididae) mild to severe no

Fall armyworm (Spodoptera frugiperda) mild to severe no

Corn sap beetle (Carpophilus spp.) mild to severe no

Spider mites (Tetranychidae) mild to severe no

Corn flea beetle (Chaetocnema pulicaria) mild no

Juana 

Diaz

Cornsilk fly (Euxesta stigmatias) mild to severe no

Aphids (Aphididae) mild to severe no

Spider mites (Tetranychidae) mild to moderate no

Corn planthoppers (Delphacidae) mild to moderate no

Spider mites (Tetranychidae) moderate to severe no

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Grasshoppers (Orthoptera) mild to moderate no

Corn earworm (Helicoverpa zea) mild to moderate no

Corn sap beetle (Carpophilus spp.) mild to severe no

Thrips (Frankliniella spp.) mild to moderate no

Salinas

Fall armyworm (Spodoptera frugiperda) moderate no

Aphids (Aphididae) mild to moderate no

Corn planthoppers (Delphacidae) moderate no

Cornsilk fly (Euxesta stigmatias) mild to moderate no

Corn sap beetle (Carpophilus spp.) mild to moderate no

Santa 

Isabel

Fall armyworm (Spodoptera frugiperda) mild no

Corn planthoppers (Delphacidae) mild no

Corn sap beetle (Carpophilus spp.) moderate no

Spider mites (Tetranychidae) moderate no

TN Obion

Fall armyworm (Spodoptera frugiperda) mild no

European corn borer (Ostrinia nubilalis) mild no

Southwestern corn borer (Diatraea grandiosella) mild no

Corn earworm (Helicoverpa zea) mild no
Pioneer Hi-Bred International, Inc. Page 228 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010

10-052-101n

TN Obion Grasshoppers (Orthoptera) mild no

WI Rock

Corn rootworm (Diabrotica spp.) mild no

Grasshoppers (Orthoptera) mild no

Japanese beetle (Popillia japonica) mild no

10-118-105n IL Gallatin Japanese beetle (Popillia japonica) mild no

10-281-101n

HI Kauai

Leafhopper (Cicadellidae) mild no

Spider mites (Tetranychidae) mild no

Corn earworm (Helicoverpa zea) mild no

PR Salinas

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Corn planthoppers (Delphacidae) mild to moderate no

Grasshoppers (Orthoptera) mild no

Cornsilk fly (Euxesta stigmatias) mild to moderate no

Corn sap beetle (Carpophilus spp.) mild to moderate no

Aphids (Aphididae) mild to moderate no

Corn earworm (Helicoverpa zea) mild to moderate no

Spider mites (Tetranychidae) mild to moderate no

10-284-101n

HI Kauai

Corn earworm (Helicoverpa zea) mild no

Spider mites (Tetranychidae) mild no

Leafhopper (Cicadellidae) mild no

Lacewings (Neuroptera) mild no

PR Salinas

Corn sap beetle (Carpophilus spp.) mild to severe no

Corn earworm (Helicoverpa zea) mild to severe no

Cornsilk fly (Euxesta stigmatias) mild to severe no

Spider mites (Tetranychidae) mild to severe no

Corn planthoppers (Delphacidae) mild to severe no

Chinch bugs (Lygaeidae) moderate no

Thrips (Frankliniella spp.) mild to severe no

Lady beetles (Coccinellidae) mild no
Pioneer Hi-Bred International, Inc. Page 229 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010

10-284-101n PR

Salinas

Leafhopper (Cicadellidae) mild no

Lacewings (Neuroptera) mild no

Fall armyworm (Spodoptera frugiperda) mild to severe no

Aphids (Aphididae) mild to severe no

Grasshoppers (Orthoptera) mild to moderate no

Guayama

Corn planthoppers (Delphacidae) mild to moderate no

Spider mites (Tetranychidae) moderate no

Corn earworm (Helicoverpa zea) moderate no

Fall armyworm (Spodoptera frugiperda) moderate to severe no

Corn sap beetle (Carpophilus spp.) moderate no

Thrips (Frankliniella spp.) moderate no

Cornsilk fly (Euxesta stigmatias) moderate no

Fall armyworm (Spodoptera frugiperda) mild to moderate no

Aphids (Aphididae) mild to moderate no

Spider mites (Tetranychidae) moderate no

Grasshoppers (Orthoptera) moderate no

10-015-106n

IL

Champaign Fall armyworm (Spodoptera frugiperda) mild no

Corn earworm (Helicoverpa zea) mild no

Clinton

Corn earworm (Helicoverpa zea) mild no

Western bean cutworm (Richia albicosta) mild no

Grasshoppers (Orthoptera) mild no

Japanese beetle (Popillia japonica) mild no

Stark

Corn rootworm (Diabrotica spp.) mild no

Aphids (Aphididae) mild no

European corn borer (Ostrinia nubilalis) mild no

IA Guthrie

Grasshoppers (Orthoptera) mild no

European corn borer (Ostrinia nubilalis) mild no

Western bean cutworm (Richia albicosta) mild no
Pioneer Hi-Bred International, Inc. Page 230 of 258

4114 Maize

Table 8.1. Observations of Insects Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Insect Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-015-106n

IA

Jefferson

Corn rootworm (Diabrotica spp.) mild no

European corn borer (Ostrinia nubilalis) mild no

Corn earworm (Helicoverpa zea) mild no

Shelby

European corn borer (Ostrinia nubilalis) mild no

Corn earworm (Helicoverpa zea) moderate no

Aphids (Aphididae) mild to moderate no

Grasshoppers (Orthoptera) mild no

MI Lenawee Corn earworm (Helicoverpa zea) mild no

Aphids (Aphididae) mild no

MN Freeborn Aphids (Aphididae) mild no

European corn borer (Ostrinia nubilalis) mild no

NE York

Corn earworm (Helicoverpa zea) mild no

European corn borer (Ostrinia nubilalis) mild no

Grasshoppers (Orthoptera) mild no

Corn rootworm (Diabrotica spp.) mild no

Beetles (Coleoptera) mild no

Picnic beetles (Nitidulidae) mild no

Aphids (Aphididae) mild no

2011

10-284-101n PR Guayama

Fall armyworm (Spodoptera frugiperda) mild to severe no

Aphids (Aphididae) mild to severe no

Spider mites (Tetranychidae) mild to severe no

Corn planthoppers (Delphacidae) mild to moderate no

Cornsilk fly (Euxesta stigmatias) mild to severe no

Thrips (Frankliniella spp.) mild to severe no

Corn earworm (Helicoverpa zea) mild to severe no

Corn sap beetle (Carpophilus spp.) moderate no

11-040-123n PR Guayama Corn planthoppers (Delphacidae) mild to moderate no

Thrips (Frankliniella spp.) mild to moderate no
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4114 Maize

Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2006 06-019-04R HI Kauai Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

2007 07-040-

101rm

CA Yolo Common smut (Ustilago zeae) mild no

IA

Linn Gray leaf spot (Cercospora zeae-maydis) mild no

Polk

Stewart's wilt (Pantoea stewartii) mild to moderate no

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

Common corn rust (Puccinia sorghi) mild to moderate no

Common smut (Ustilago zeae) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Southern corn rust (Puccinia polysora) mild to moderate no

Anthracnose (Colletotrichum graminicola) mild no

IL

Bureau Common corn rust (Puccinia sorghi) mild no

Champaign Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

McDonough Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Northern corn leaf blight (Exserohilum turcicum) moderate no

Ogle

Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

IN Tipton

Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild to severe no

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

MO Saline Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Southern corn rust (Puccinia polysora) mild no

WI Rock Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

NE York Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no
Pioneer Hi-Bred International, Inc. Page 232 of 258

4114 Maize

Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Stressor Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2008 08-014-111n

IN

Gibson Northern corn leaf blight (Exserohilum turcicum) mild no

Tipton

Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

IA

Polk

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Southern corn rust (Puccinia polysora) mild no

Crazy top (Sclerophthora macrospora) mild no

Linn Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

NE

Nance

Common corn rust (Puccinia sorghi) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Common smut (Ustilago zeae) mild to moderate no

Crazy top (Sclerophthora macrospora) moderate no

York Common corn rust (Puccinia sorghi) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

MO Saline Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

IL

Bureau

Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Anthracnose (Colletotrichum graminicola) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Champaign

Gray leaf spot (Cercospora zeae-maydis) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

McDonough Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no
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Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2008

08-014-111n WI Rock

Common corn rust (Puccinia sorghi) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) moderate no

Northern corn leaf blight (Exserohilum turcicum) mild no

Gibberella stalk rot (Gibberella zeae) mild no

08-095-105n

OK Caddo Common smut (Ustilago zeae) mild no

IL

Clinton Gray leaf spot (Cercospora zeae-maydis) mild no

Stark

Anthracnose (Colletotrichum graminicola) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

IA Jefferson Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

NE York

Common corn rust (Puccinia sorghi) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) mild no

Corn eyespot (Aureobasidium zeae) mild no

2009 09-013-108n IL

Bureau

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Douglas

Gray leaf spot (Cercospora zeae-maydis) mild to severe no

Northern corn leaf blight (Exserohilum turcicum) mild to severe no

Common corn rust (Puccinia sorghi) mild no

Southern corn rust (Puccinia polysora) mild no

Anthracnose (Colletotrichum graminicola) mild no

Fulton

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

McDonough Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

IN Gibson Gray leaf spot (Cercospora zeae-maydis) mild to moderate no
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Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2009 09-013-108n

IN

Gibson Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

Southern corn rust (Puccinia polysora) mild to moderate no

Tipton

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

IA

Dallas

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Southern corn rust (Puccinia polysora) mild no

Kossuth

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Corn eyespot (Aureobasidium zeae) mild no

Common corn rust (Puccinia sorghi) mild to moderate no

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

Common smut (Ustilago zeae) mild to moderate no

Linn Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

Polk

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Common smut (Ustilago zeae) mild no

Common corn rust (Puccinia sorghi) mild no

Corn eyespot (Aureobasidium zeae) mild no

NE York

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Common corn rust (Puccinia sorghi) mild to moderate no

Common smut (Ustilago zeae) mild to moderate no

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

MO Saline Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

09-035-109n AR Crittenden Common corn rust (Puccinia sorghi) mild to moderate no
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Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2009

09-035-109n

AR Crittenden

Southern corn rust (Puccinia polysora) mild to moderate no

Northern corn leaf blight (Exserohilum turcicum) moderate no

Gray leaf spot (Cercospora zeae-maydis) mild to severe no

GA Grady

Northern corn leaf blight (Exserohilum turcicum) mild no

Southern corn leaf blight (Bipolaris maydis) mild no

Common corn rust (Puccinia sorghi) mild no

HI Kauai

Maize chlorotic mottle machlomovirus moderate no

Aspergillus ear and kernel rot (Aspergillus spp.) mild no

Fusarium (Fusarium spp.) mild no

MN Blue Earth Northern corn leaf blight (Exserohilum turcicum) mild no

TN Obion

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Southern corn rust (Puccinia polysora) mild no

Brown Spot (Physoderma maydis) mild no

WI Rock

Anthracnose (Colletotrichum graminicola) mild no

Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Anthracnose (Colletotrichum graminicola) mild no

09-264-102n HI Kauai

Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

Maize chlorotic mottle machlomovirus mild no

09-016-103n IL Stark Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

2010 09-264-102n HI Kauai

Maize chlorotic mottle machlomovirus mild no

Common smut (Ustilago zeae) mild no

Maize mosaic rhabdovirus mild no
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Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010

09-264-102n HI Kauai Maize dwarf mosaic potyvirus mild no

Common corn rust (Puccinia sorghi) mild no

10-050-101n

NE Keith Common corn rust (Puccinia sorghi) mild no

AR Crittenden

Diplodia ear rot (Stenocarpella maydis) mild to moderate no

Fusarium ear mold (Fusarium spp.) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) moderate no

Southern corn rust (Puccinia polysora) moderate no

Southern corn leaf blight (Bipolaris maydis) moderate no

GA Grady

Northern corn leaf blight (Exserohilum turcicum) mild to moderate no

Common corn rust (Puccinia sorghi) mild no

Southern corn leaf blight (Bipolaris maydis) mild to moderate no

Southern corn rust (Puccinia polysora) mild to moderate no

Common smut (Ustilago zeae) moderate no

HI Kauai

Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

Northern corn leaf spot (Bipolaris zeicola) mild no

IL

Bureau

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Common corn rust (Puccinia sorghi) mild no

Champaign

Gray leaf spot (Cercospora zeae-maydis) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Southern corn rust (Puccinia polysora) mild no

Common corn rust (Puccinia sorghi) mild no

Fulton

Gray leaf spot (Cercospora zeae-maydis) mild no

Anthracnose (Colletotrichum graminicola) mild no

Northern corn leaf spot (Bipolaris zeicola) mild no

La Salle Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no
Pioneer Hi-Bred International, Inc. Page 237 of 258

4114 Maize

Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-052-101n

IL

La Salle Common corn rust (Puccinia sorghi) mild no

McDonough

Gray leaf spot (Cercospora zeae-maydis) mild no

Common smut (Ustilago zeae) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Ogle Common corn rust (Puccinia sorghi) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

IN

Gibson Gray leaf spot (Cercospora zeae-maydis) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Shelby

Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Tipton Northern corn leaf blight (Exserohilum turcicum) mild no

White Common corn rust (Puccinia sorghi) mild no

IA

Bremer Common corn rust (Puccinia sorghi) mild no

Dallas

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Delaware Gray leaf spot (Cercospora zeae-maydis) mild no

Kossuth

Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

Linn Common smut (Ustilago zeae) mild no

Common corn rust (Puccinia sorghi) mild no

Madison

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Gray leaf spot (Cercospora zeae-maydis) moderate no

Polk Northern corn leaf blight (Exserohilum turcicum) mild to moderate no
Pioneer Hi-Bred International, Inc. Page 238 of 258

4114 Maize

Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-052-101n

IA Polk

Common corn rust (Puccinia sorghi) mild to moderate no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

Common smut (Ustilago zeae) moderate no

Van Buren Common smut (Ustilago zeae) mild no

KS Finney Common corn rust (Puccinia sorghi) mild no

MI Gratiot

Common smut (Ustilago zeae) mild no

Stewart's wilt (Pantoea stewartii) mild no

Common corn rust (Puccinia sorghi) mild no

MN

Blue Earth Northern corn leaf blight (Exserohilum turcicum) mild no

Common smut (Ustilago zeae) mild no

Kandiyohi

Common corn rust (Puccinia sorghi) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

MO Scott

Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

NE York

Common smut (Ustilago zeae) mild no

Common corn rust (Puccinia sorghi) mild no

Goss's wilt (Clavibacter michiganensis) mild no

Common smut (Ustilago zeae) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

PA

Lebanon Gray leaf spot (Cercospora zeae-maydis) mild no

Juana Diaz Northern corn leaf blight (Exserohilum turcicum) mild no

Common smut (Ustilago zeae) mild no

TN Obion

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Southern corn rust (Puccinia polysora) mild no

Brown spot (Physoderma maydis) mild no
Pioneer Hi-Bred International, Inc. Page 239 of 258

4114 Maize

Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010

10-052-101n

TN Obion Common corn rust (Puccinia sorghi) mild no

WI Rock

Northern corn leaf blight (Exserohilum turcicum) mild no

Common smut (Ustilago zeae) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Corn eyespot (Aureobasidium zeae) mild no

Common corn rust (Puccinia sorghi) mild no

10-118-105n IL Gallatin Northern corn leaf blight (Exserohilum turcicum) mild no

Gray leaf spot (Cercospora zeae-maydis) mild to moderate no

10-281-101n

HI Kauai Common smut (Ustilago zeae) mild no

Common corn rust (Puccinia sorghi) mild no

PR Salinas

Maize stripe tenuivirus mild no

Corn leaf blight (Pleosporaceae) mild no

Common corn rust (Puccinia sorghi) mild no

10-284-101n

HI Kauai Common smut (Ustilago zeae) mild no

Maize stripe tenuivirus mild no

PR

Salinas

Common corn rust (Puccinia sorghi) mild no

Maize stripe tenuivirus mild no

Corn leaf blight (Pleosporaceae ) mild no

Guayama Maize stripe tenuivirus mild no

Southern corn leaf blight (Bipolaris maydis) mild no

10-015-106n

IN Clinton

Anthracnose (Colletotrichum graminicola) mild no

Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

IL

Champaign Diplodia ear rot (Stenocarpella maydis) mild no

Clinton Gray leaf spot (Cercospora zeae-maydis) mild no

Northern corn leaf blight (Exserohilum turcicum) mild no

Stark Gray leaf spot (Cercospora zeae-maydis) mild no
Pioneer Hi-Bred International, Inc. Page 240 of 258

4114 Maize

Table 8.2. Observations of Diseases Present and Comparison Between 4114 Maize and Control (continued)

Year of 

Planting

Permit 

Number State County Disease Overall Severity in Field

Unexpected 

Difference in 

Comparison?

2010 10-015-106n

IL Stark

Anthracnose (Colletotrichum graminicola) mild no

Common corn rust (Puccinia sorghi) mild no

IA

Guthrie

Eye spot of corn (Aureobasidium zeae) mild no

Gibberella stalk rot (Gibberella zeae) mild no

Common smut (Ustilago zeae) mild no

Jefferson

Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

Southern corn leaf blight (Bipolaris maydis) mild no

Eye spot of corn (Aureobasidium zeae) mild no

Shelby

Gray leaf spot (Cercospora zeae-maydis) mild no

Common corn rust (Puccinia sorghi) mild no

Common smut (Ustilago zeae) mild no

MI Lenawee Common corn rust (Puccinia sorghi) mild no

MN Freeborn

Northern corn leaf blight (Exserohilum turcicum) mild no

Common corn rust (Puccinia sorghi) mild no

NE York

Common corn rust (Puccinia sorghi) mild no

Gray leaf spot (Cercospora zeae-maydis) mild no
Pioneer Hi-Bred International, Inc. Page 241 of 258

4114 Maize

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Section 1

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NCGA (2011) 2011 World of Corn. National Corn Growers Association, 

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Linear Model, Duxbury Press, Boston, pp 270-275

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Hochberg, eds, Multiple Comparisons and Multiple Tests Using SAS, SAS Institute Inc, Cary, pp 

13-40

Appendix 8

No references cited in this section.