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32138 SPT Maintainer No CBI 

Petition for the Determination of Nonregulated Status for Maize 

32138 SPT Maintainer Used in the Pioneer Seed Production Technology 

(SPT) Process 

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., A DuPont Company 

DuPont Experimental Station 

Route 141 & Henry Clay Road 

PO Box 80353 

Wilmington, DE 19880-0353 

Telephone: 302-695-8160 

Fax: 302-695-3075 

Prepared by: 

Padma Commuri, Natalie Weber, Tracy Rood, Rod Townsend 

OECD Unique Identifier: DP-32138-1 

June 18, 2009 

NO CBI 
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32138 SPT Maintainer No CBI 

Release of Information 

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

review by the 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 FOIA, Pioneer does not 

authorize the release, publication, or other distribution of this information (including website 

posting) without Pioneer’s prior notice and consent. 

Pioneer authorizes the posting of the petition to the BRS Website at such time when the Pest 

Risk Assessment and the Environmental Assessment are published in the Federal Register for 

public comment. 

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32138 SPT Maintainer No CBI 

Summary 

Pioneer Hi-Bred International, Inc. (Pioneer), a DuPont Company, has developed a process 

called ‘Seed Production Technology’ (SPT) that facilitates large-scale, in-house production of 

male-sterile maize lines that are used as female inbred parents for subsequent hybrid seed 

production. Hybrid maize seed production requires crossing two inbred parent lines to produce 

hybrid seed sold to growers. The female inbred parent must be prevented from shedding pollen 

to avoid self- pollination that reduces hybrid seed quality and compromises hybrid yield potential. 

SPT offers a number of advantages over other approaches to controlling male-fertility in female 

inbred parents, such as detasseling (physical removal of the pollen-producing tassels) or 

cytoplasmic male-sterility (CMS). The SPT process requires no detasseling, works across all 

germplasm types, increases seed yields and seed quality, and produces F1 hybrid plants that are 

fully fertile. The female inbred parent seed produced using this technology does not contain the 

32138 SPT insertion and consequently, hybrid seed produced using these male-sterile female 

inbred parents does not contain the 32138 SPT insertion and hence are non-transgenic for SPT. 

Pioneer’s SPT process is based on a transgenic maize event DP-32138-1, hereafter referred to 

as ‘32138 SPT maintainer’. The 32138 SPT maintainer is used as a pollinator to propagate the 

seed of male-sterile female inbred parent lines. The 32138 SPT maintainer is neither a 

commercial product on its own nor part of a commercial product; there is no intent for the 32138 

SPT maintainer seed to enter commercial grain channels. The transgenic 32138 SPT maintainer 

is currently grown under USDA-APHIS permits on Company-controlled parent seed production 

fields that are reproductively isolated from other maize fields. In the future, 32128 SPT 

maintainer will be grown on a total of less than 5,000 acres/year in the U.S. Rather than 

obtaining a USDA-APHIS permit each year for these 32138 SPT maintainer acres, Pioneer is 

requesting that USDA-APHIS deregulate the 32138 SPT maintainer to facilitate its use in the 

Company’s inbred seed production process. 

In addition, Pioneer is supplying data to show that progeny derived from the SPT process are 

consistently and reliably non-transgenic for SPT genes. In order to address questions regarding 

the movement of progeny seed and grain in international commerce, we request that APHIS 

make a finding that SPT progeny lack the 32138 SPT insertion and are therefore non-transgenic 

for SPT. 

The 32138 SPT maintainer was generated by Agrobacterium-mediated transformation of a 

genetically male-sterile (ms45/ms45) maize line with a plasmid designated PHP24597. Plasmid 

PHP24597 contains three gene expression cassettes essential for the functioning of the SPT 

system: Ms45, zm-aa1, and DsRed2(Alt1)1

.

1) Ms45 is a maize gene that encodes the MS45 protein required for the production of fertile 

pollen and is controlled by a maize anther preferred promoter 5126. Mutations in the Ms45 gene 

result in male-sterile plants when in a homozygous recessive (ms45/ms45) state. A single copy 

of the Ms45 gene can restore fertility in the male-sterile ms45/ms45 genetic background. 

2) zm-aa1 is a maize gene that encodes the ZM-AA1 -amylase protein and is controlled by the 

Pg47 maize promoter. The -amylases belong to a family of glycosyl-hydrolases catalyzing 

hydrolysis of (1-4)--D-glucosidic linkages in polysaccharide molecules, such as starch. 

Expression of ZM-AA1 -amylase in developing pollen results in starch hydrolysis and depletion 

of starch reserves, and renders the pollen infertile2

. 

1

 A variant of Clontech’s (www.clontech.com) DsRed protein, isolated from a marine coral, Discosoma sp. 2

 Pollen infertility refers to the inability of pollen to germinate and fertilize the ovule. 
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32138 SPT Maintainer No CBI 

3) DsRed2 (Alt1) is a marker gene that encodes a variant of the red fluorescent protein (DsRed2) 

with one alteration (Alt1) and is controlled by the Ltp2 barley promoter. The expression of 

DsRed2 protein in seed imparts a pinkish-red coloration to the aleurone layer of maize seeds 

allowing for visual identification and efficient automated separation of 32138 SPT maintainer seed 

from yellow seed that are non-transgenic for SPT. 

The expressed MS45 protein restores fertility in transgenic 32138 SPT maintainer by enabling 

pollen production. However the Ms45 gene in 32138 SPT maintainer is hemizygous (Ms45/-) and 

as a result, only one half of the pollen produced contains the Ms45 gene. That half of the pollen 

also contains the linked zm-aa1 gene, so the expressed -amylase enzyme degrades starch, 

rendering the transgenic (Ms45/zm-aa1/DsRed2(Alt1)) pollen infertile. The remaining half of the 

pollen is non-transgenic for SPT, fertile, and carries the endogenous recessive ms45 gene. 

Therefore, when 32138 SPT maintainer is used as a pollinator to propagate the seed of malesterile female inbreds that are non-transgenic for SPT, the derived progeny retain their malesterility genotype (ms45/ms45) and do not contain the 32138 SPT insertion and are therefore 

non-transgenic for SPT. Hence F1 commercial hybrids produced using these male-sterile 

progeny do not contain the 32138 SPT insertion and are also non-transgenic for SPT. 

Molecular characterization of the DNA insertion in event DP-32138-1 confirmed that a single, 

intact copy of the PHP24597 T-DNA containing a single copy of each of the gene expression 

cassettes was inserted into the maize genome. Southern blot analysis confirmed the absence of 

any plasmid backbone sequences. The integrity and stability of the inserted PHP24597 T-DNA 

were verified across three breeding generations. 

While it is expected and intended that 32138 SPT maintainer will not enter commercial grain 

channels, Pioneer evaluated the food and feed safety of 32138 SPT maintainer seed. Both MS45 

and ZM-AA1 proteins are encoded by maize gene sequences and have no biologically significant 

homologies to known toxins or allergens. The DsRed2 protein is the only protein not derived from 

a maize gene sequence expressed in the 32138 SPT maintainer. A New Protein Consultation 

(NPC) safety package for the DsRed2 protein was submitted to FDA on October 11, 2006. 

Bioinformatic analyses revealed no biologically significant homologies to known or putative 

protein allergens or toxins. The DsRed2 protein is not glycosylated and is hydrolyzed rapidly 

within 30 seconds in simulated gastric fluid. There was no evidence of acute toxicity in mice for 

the DsRed2 protein at a dose of 1860 mg of protein per kg of body weight. Therefore, based on 

the expression levels of this protein in maize seed, a wide margin of safety exists for human and 

animal consumption of the DsRed2 protein. 

The 32138 SPT maintainer has been field tested in the U.S. since 2005 under permit and 

notification granted by USDA-APHIS. Comprehensive agronomic performance and ecological 

assessments of the 32138 SPT maintainer were conducted in replicated field studies in the U.S. 

during the 2007 growing season. Agronomic characteristics such as emergence, seedling vigor, 

plant height, ear height, stalk lodging, root lodging, stay green, time to silking and pollen shed, 

disease incidence, and insect damage were measured. Field trials of 32138 SPT maintainer 

were observed for naturally occurring disease or insect biotic stressors. In laboratory 

experiments, information was also collected on seed germination, dormancy, and the duration of 

pollen viability. Analysis of agronomic and ecological data showed no biologically meaningful 

differences between 32138 SPT maintainer and the inbred control maize, indicating no plant pest 

characteristics for 32138 SPT maintainer. Likewise, assessment of the ecological data detected 

no biologically meaningful differences between 32138 SPT maintainer and the inbred control 

maize lines indicative of a selective advantage that would result in 32138 SPT maintainer having 

an increased invasiveness potential for natural habitats. 

The data support a conclusion of agronomic comparability of 32138 SPT maintainer to 

conventional inbred maize with respect to the lack of increased weediness and plant pest 

potential. In addition, based on the compositional evaluation conducted in accordance with the 
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OECD consensus document on compositional considerations for new varieties of maize, 32138 

SPT maintainer seed was found to be compositionally equivalent to seed from conventional 

maize inbreds. 

Since the 32138 SPT maintainer will not be commercially available, there will be no impact on the 

existing hybrid maize agronomic and production practices, market dynamics, and grower choice. 

The use of the SPT process in Pioneer hybrid seed production will have no impacts on organic or 

conventional farming or on raw or processed agricultural commodities, especially since it will be 

cultivated on a limited acreage (<5,000 acres) and reproductively isolated from other maize fields. 

There will be no impact on threatened or endangered species because the proteins encoded by 

the 32138 SPT insertion are not known to be toxic. Any exposure would be limited to only those 

organisms in or near 32138 SPT maintainer fields. In addition, 32138 SPT maintainer will be 

grown on existing agricultural land, controlled and managed by Pioneer. No new land will be put 

into agricultural production because of the deregulation of 32138 SPT maintainer. 

Based on the safety assessment of 32138 SPT maintainer, we conclude that there will be no 

adverse effects on animal or human health. The 32138 SPT maintainer is not intended to enter 

the commercial food or feed supply, however, Pioneer has taken necessary steps to evaluate the 

food and feed safety of any small quantities of 32138 SPT maintainer seed and vegetative 

material that may be disposed of as animal feed. Pioneer has submitted New Protein 

Consultations to the FDA for both the DsRed2 and ZM-AA1 proteins encoded by the 32138 SPT 

insertion.

Within this petition, Pioneer has supplied data on the genetic efficiency and stability of the SPT 

system (on 2.5 million progeny seeds), detailed information on the accuracy of the mechanical 

color sorting of 32138 SPT maintainer, a molecular analysis to confirm absence of the 32138 SPT 

insertion in the SPT progeny, and quality system information on handling of SPT seed and inbred 

parent seed production practices. Pioneer requests that USDA-APHIS make a finding that the 

SPT process, as described and operated by Pioneer, consistently gives rise to progeny that lack 

the 32138 SPT insertion and which are, therefore, non-transgenic for SPT. 

The genetic efficiency of the SPT system (i.e., the reproducibility of the production of male-sterile 

progeny that lack the 32138 SPT insertion when using 32138 SPT maintainer as a pollinator) is 

greater than 99.999%. Expression of the DsRed2 protein in the 32138 SPT maintainer seed 

enables automated seed sorting using high speed commercial optical sorters to separate SPT 

seed from non-SPT seed (seed that lacks the 32138 SPT insertion). The accuracy of the 

optimized seed sorters to detect and separate fluorescent red 32138 SPT transgenic seed from 

yellow seed (non-transgenic for SPT) is >99.95%. Hence the combined genetic and mechanical 

efficiency of the SPT process is >99.9999995%. 

Based on the data contained herein, Pioneer requests USDA-APHIS to make a determination 

that 32138 SPT maintainer and crosses of maize lines containing this event with non-regulated 

maize lines no longer be considered regulated articles under 7CFR 340. 

Furthermore, in order to address questions regarding the movement of 32138 SPT maintainerderived progeny seed and grain in international commerce, Pioneer is requesting that USDAAPHIS make an additional finding that the SPT process, as described and operated by Pioneer, 

consistently and reliably produces progeny that lack the 32138 SPT insertion and are therefore 

non-transgenic for SPT. 
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Abbreviations, Acronyms, and Definitions 

~ Approximately 

5126 promoter Maize anther-preferred promoter (Cigan and Albertsen, 1997) 

32138 SPT 

maintainer Maize line containing the DP-32138-1 event 

32138 SPT 

insertion 

The DNA from plasmid PHP24597 that has been integrated into the 

genome of the 32138 SPT maintainer.

ADF Acid detergent fiber 

AOSA Association of Official Seed Analysts 

APHIS Animal and Plant Health Inspection Service 

Brittle-1 Amyloplast-targeting transit peptide from the maize brittle-1 gene 

bp Base pair 

CaMV Cauliflower Mosaic Virus 

cDNA Complementary DNA 

cM Centimorgan 

CMS Cytoplasmic male-sterility 

Da Dalton 

dam DNA adenine methylase 

DIG Digoxygenin 

DP-32138-1 Event DP-32138-1, also known as 32138 SPT maintainer

DNA Deoxyribonucleic acid 

DsRed2(Alt1) A specific fluorescent marker gene; a variant of Clontech’s DsRed gene 

DsRed2 A specific fluorescent protein expressed by DsRed2(Alt1) gene 

E. coli Escherichia coli 

E score Expectation score 

ELISA Enzyme linked immunosorbent assay 

ERS Economic Research Service, the primary source of economic and 

research information in the U.S. Department of Agriculture 

FDA Food and Drug Administration 

FDR False discovery rate 

Genetic 

efficiency of SPT 

system 

Using the SPT maintainer as a pollinator, the reproducibility of the 

production of male-sterile progeny that lack the 32138 SPT insertion. 

GRAS Generally recognized as safe 

HRP Horseradish peroxidase 

IgG Immunoglobulin G 

ILSI International Life Sciences Institute 

Inbred 705 A Pioneer elite inbred line 

IPM Integrated Pest Management 

In2-1 Terminator sequence from maize In2-1 gene 

kb Kilobase pair 

kDa Kilodalton 

LB Left Border 

LC/MS Liquid chromatography/mass spectrometry 

LLOQ Lower limit of quantitation 

LOD Limit of detection 
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Abbreviations, Acronyms, and Definitions (continued) 

Ltp2 Promoter from barley lipid transfer protein gene 

Mb Megabase 

Ms45 A specific (dominant) male-fertility gene in maize 

MS45 A specific protein expressed by the Ms45 gene in maize 

ms45 A mutant allele (recessive) of the Ms45 gene in maize 

MALDI-MS Matrix assisted laser desorption ionization mass spectrometry 

NCBI National Center for Biotechnology Information 

NDF Neutral detergent fiber 

OD Optical density 

OECD Organisation for Economic Co-operation and Development 

PAT Phosphinothricin acetyltransferase 

PVDF Polyvinylidene fluoride 

PCR Polymerase chain reaction 

Pg47 A pollen-specific polygalacturonase gene from maize 

pinII Proteinase inhibitor II from Solanum tuberosum

RB Right Border 

RFP Red fluorescent protein 

RFU Relative fluorescent unit 

RNA Ribonucleic acid 

Roguing Removal of undesirable off-types or atypical plants after a systematic 

evaluation of seed production fields. 

SOP Standard operating procedure 

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis 

Sorter accuracy 

The accuracy of mechanical seed color sorter to remove any transgenic 

32138 SPT-maintainer maize seed from non-transgenic male-sterile 

progeny maize 

SPT Seed Production Technology 

SPT system A genetic system comprising a transgenic 32138 SPT maintainer used to 

increase non-transgenic male-sterile female inbred parent seed 

SPT process A seed production process comprising the SPT system combined with 

color-based seed sorting to insure genetic purity 

SPT progeny Male-sterile female inbred seed derived from using the 32138 SPT 

maintainer as a pollen source and also the commercial hybrid grain 

derived thereafter from using the SPT process 

TSS Tissue-specific sterility 

USDA United States Department of Agriculture 

UTR Untranslated region 

zm-aa1 A specific -amylase gene in maize

ZM-AA1 A specific -amylase protein expressed in 32138 SPT maintainer and 

comprised of maize endogenous protein sequences from the Brittle-1 

transit peptide and the ZM-AA1 protein. 

zm-bt-1 A transit peptide gene sequence from the maize brittle-1 gene 

*Abbreviations of units of measurement and of physical and chemical quantities are done according 

to the standard format described in Instructions to Authors in the Journal of Biological Chemistry 

(http://www.jbc.org/).. 
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Table of Contents

Title Page 1 

Release of Information 2 

Certification 3 

Summary 4 

Abbreviations, Acronyms and Definitions 7

Table of Contents 9

List of Tables 13 

List of Figures 15

I. Introduction to Seed Production Technology (SPT) 18

I-A. Advantages of the Pioneer SPT Process 18 

I-B. Maize Hybrid Seed Production 18 

I-C. Male-Fertility Control in Hybrid Maize Production 20 

I-D. Description of the SPT Process 23 

I-E. Request for a Determination of Nonregulated Status under 7CFR §340.6 27 

I-F. Submissions to Other Regulatory Agencies 27 

I-G. Generation of 32138 SPT Maintainer 28 

I-H. Brief Description of Genes 29 

II. The Biology of Maize 34 

III. Method of Development of 32138 SPT Maintainer 35

III-A. Characterization and Generation of a Male-Sterile (ms45/ms45) Recipient Maize 35 

III-B. Description of the Transformation System 35 

III-C. Selection of Comparators for 32138 SPT Maintainer 36 

IV. Donor Genes and Regulatory Sequences 39

IV-A. Plasmid PHP24597 Used in Transformation 39 

IV-B. Identity and Source of Genetic Material in the T-DNA of PHP24597 43 

V. Genetic Characterization of 32138 SPT Maintainer 44 

V-A. Molecular Analysis Overview 44 

V-B. Characterization of 32138 SPT maintainer: Copy Number and Insertion Integrity 50 

V-C. Stability of the Insertion across Generations 65 

V-D. Plasmid Backbone DNA Analysis 74 

V-E. Inheritance of the Traits in 32138 SPT Maintainer 80 

V-F. Molecular Characterization of 32138 SPT Maintainer: Summary and Conclusions 82 

VI. Characterization of the Introduced Proteins 83

VI-A. The MS45 Protein 83 

VI-B. The ZM-AA1 Protein 85 

VI-C. The DsRed2 Protein 86 

VI-D. Expression of MS45 in 32138 SPT Maintainer 87 

VI-E. Expression of ZM-AA1 and DsRed2 in 32138 SPT Maintainer 88 

VI-F. Safety Assessment of the MS45 Protein 92 

VI-G. Safety Assessment of the ZM-AA1 Protein 93 

VI-H. Characterization of the DsRed2 Protein Expressed in 32138 SPT Maintainer 96 

VI-I. Summary of the Food and Feed Safety Assessment for the DsRed2 Protein 97 
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Table of Contents (continued)

VII. Agronomic Performance and Ecological Observations 99 

VII-A. Germination and Dormancy Evaluations 99 

VII-B. Field Trial Evaluations 101 

VII-C. Ecological Observations 107 

VII-D. Conclusions on Agronomic Performance and Ecological Observations 107 

VIII. Compositional Assessment 108 

VIII-A. Proximates 109 

VIII-B. Fatty Acids 110 

VIII-C.Total Amino Acids 113 

VIII-D. Vitamins and Minerals 116 

VIII-E. Key Anti-nutrients 119 

VIII-F. Key Secondary Plant Metabolites in Control and 32138 SPT Maintainer 120 

VIII-G.Conclusions on Compositional Assessment of Seed from 32138 SPT Maintainer 121 

IX. Potential Environmental Impact Assessment of the Seed Production Technology 

Process 122 

IX-A. Limited Use of and Exposure to the 32138 SPT Maintainer 122 

IX-B. Environmental Assessment of the Components of the 32138 SPT Maintainer: MS45, 

ZM-AA1, and DsRed2 Proteins 125 

IX-C. Fate of Transgenic DNA in Humans and Animals 126 

IX-D. Weediness Potential of 32138 SPT Maintainer 126 

IX-E. Gene Flow Assessment 127 

IX-F. Current Agronomic Practices for Cultivation of Maize Inbred Parent Seed 127 

IX-G. Potential Impact of 32138 SPT Maintainer on Agronomic Practices for 

 Cultivation of Inbred Maize Parent Seed 129 

IX-H. Potential Impact on Organic or Conventional Farming 130 

IX-I. Potential Impacts on Raw or Processed Agricultural Commodities 130 

IX-J. Potential Impact on Biodiversity 130 

IX.K. Overall Environmental and Agronomic Practices Conclusions 131 

IX-L. Non-Transgenic Status of SPT Derived Progeny 131 

X. Adverse Consequences of Introduction of the SPT System 133 

XI. References 134 

XII. Appendices 142 

Appendix 1. Other Male-Fertility Control Methods Used in Maize 143

1.1. Chemically Induced Male-Sterility 143 

1.2. Biological Male-Sterility 143 

1.3. Male-Sterility Systems Developed Using Biotechnology 144 

Appendix 2. Genetic Efficiency and Stability of the SPT System 145 

2.1. Study 1 145 

2.2. Study 2 147 

2.3. Conclusions 147 

Appendix 3. Mechanical Color Sorting of the 32138 SPT Maintainer: Process and 

Facilities 148 

3.1. Sorter Calibration and Optimization 150 

3.2. Conclusions 150 
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Table of Contents (continued) 

Appendix 4. Molecular Analysis to Confirm the Absence of the 32138 SPT Insertion in 

 SPT Progeny 151

Appendix 5. Overall Efficiency of the SPT Process 153 

Appendix 6. Inbred Parent Seed Production Practices Using the SPT 154 

6. Planting and Isolation 154 

6.2. Roguing 154 

6.3. Harvesting 154 

6.4. Sorting, Drying, and Shelling of Maize Ears 154 

6.5. Optical Seed Sorter Calibration 154 

6.6. Seed Conditioning, Treatment and Packaging 155 

6.7. Record Maintenance 155 

Appendix 7. Potential for Linkage Break between the Cassettes of the 32138 SPT 

 Insertion and Consequences for the SPT process...................................156 

7.1. Genetic Recombination Mechanisms 156 

7.2. Detection of Possible Scenarios 157 

7.3. Conclusions 157 

Appendix 8. Materials and Methods for Molecular Characterization of 32138 SPT 

Maintainer 161 

8.1. 32138 SPT Maintainer Material 161 

8.2. Control Material 161 

8.3. Reference Material 161 

8.4. Genomic DNA Extraction 161 

8.5. Quantitation of Genomic DNA 161 

8.6. Phenotypic Identification of 32138 SPT Maintainer Seed 162 

8.7. Digestion of DNA for Southern Blot Analyses 162 

8.8. Electrophoretic Separation and Southern Transfer 162 

8.9. DNA Probe Labeling for Southern Blot Hybridization 163 

8.10. Probe Hybridization and Visualization 163 

8.11. Stripping of Probes and Subsequent Hybridizations 163 

Appendix 9. Materials and Methods for the Determination of MS45, ZM-AA1 and 

 DsRed2 Protein Concentrations in 32138 SPT Maintainer 164 

9.1. Storage and Processing of Maize Tissue Samples 164 

9.2. Protein Extraction from Processed Maize Tissues for MS45 Western Blot Analysis 164 

9.3. Preparation of MS45 Standard Solutions for Western Blot Analysis 165 

9.4. SDS-PAGE Analysis 165 

9.5. Western Blot Analysis 165 

9.6. Protein Extraction from Processed Maize Tissues for Determination of 

 ZM-AA1Protein Concentration 165 

9.7. Determination of ZM-AA1 Protein Concentration 165 

9.8. Protein Extraction from Processed Maize Tissues for DsRed2 Fluorometric 

 Analysis 166 

9.9. DsRed2 Fluorometric Assay – Experimental Design 166 

9.10. Calculations for Determining Tissue Concentration of DsRed2 Protein 167 
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Table of Contents (continued)

Appendix 10. Equivalency of Plant Derived and Microbially Expressed DsRed2 Protein 168 

10.1. Method for Purification of DsRed2 Protein from 32138 SPT Maintainer 168 

10.2. Method for Purification of DsRed2 Protein Expressed in E. coli 168 

10.3. SDS-PAGE Analysis Method 169 

10.4. Western Blot Analysis Method 169 

10.5. Method for N-Terminal Amino Acid Sequence Analysis 169 

10.6. Method for LC/MS Identification of Tryptic Peptides 170 

10.7. Method for Protein Glycosylation Analysis 170 

10.8. Results of SDS-PAGE and Western Analyses of DsRed2 Protein 171 

10.9. Results of N-terminal Amino Acid Determination 172 

10.10.Results of the LC/MS Identification of Tryptic Peptides of DsRed2 173 

10.11.Results of the Protein Glycosylation Analysis of DsRed2 Protein 174 

Appendix 11. Description of Statistical Analyses 175 

11.1. Trait Inheritance Data 175 

11.2. Agronomic Data 175 

11.3. Nutrient Composition Data 176 

Appendix 12. USDA Field Trials of 32138 SPT Maintainer 178 

Appendix 13. Ecological Observations of 32138 SPT Maintainer 179 
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List of Tables

Table 1. Components of the 32138 SPT Insertion 28 

Table 2. Description of Genetic Elements in the T-DNA Region of Plasmid PHP24597 41 

Table 3. Description of DNA Probes Used for Southern Hybridization 47 

Table 4. Predicted and Observed Hybridizing Bands on Southern Blots with Bmt I

 Restriction Enzyme Digest 52 

Table 5. Predicted and Observed Hybridizing Bands on Southern Blots with EcoR I

 Restriction Enzyme Digest 59 

Table 6. Predicted and Observed Hybridizing Bands on Southern Blots with BamH I and 

BamH I/Hind III Restriction Enzyme Digests 66 

Table 7a. Comparison of Observed and Expected Segregation Ratios for 32138 SPT 

 Maintainer Based on Phenotypic Analysis (Seed Color) 81 

Table 7b. Comparison of Observed and Expected Segregation Ratios for 32138 SPT 

 Maintainer Based on Genotypic Analysis (Event-Specific PCR) 81 

Table 8. Concentration of ZM-AA1 Protein Measured in 32138 SPT Maintainer 90 

Table 9. Concentration of DsRed2 Protein Measured in 32138 SPT Maintainer 91 

Table 10. Summary of Germination Results for 32138 SPT Maintainer 100 

Table 11. Field Agronomic Characteristics Measured 103 

Table 12. Agronomic Performance of 32138 SPT Maintainer across Six Locations 105 

Table 13. Proximates in the Seed of Control and 32138 SPT Maintainer 109 

Table 14. Major Fatty Acids in the Seed of Control and 32138 SPT Maintainer 111 

Table 15. Total Amino Acids in the Seed of Control and 32138 SPT Maintainer 114 

Table 16. Vitamins in the Seed of Control and 32138 SPT Maintainer 117 

Table 17. Minerals in the Seed of Control and 32138 SPT Maintainer 118 

Table 18. Key Anti-nutrients in the Seed of Control and 32138 SPT Maintainer 119 

Table 19. Key Secondary Plant Metabolites in the Seed of Control and 32138 SPT 

 Maintainer 120 

Table 20. 32138 SPT maintainer Acres Relative to Total Maize Production in the U.S. 123 

Appendix 1 

Table 1. Transgenic Male-Sterility Systems De-regulated by USDA-APHIS 144 

Appendix 2 

Table 1. Genetic Efficiency of the SPT System across Five Generations 146 

Table 2. Genetic Efficiency of the SPT System across Five Genetic Backgrounds 146 

Table 3. Genetic Efficiency of the SPT Process under Open-Pollinated Field Conditions 147 
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Table of Contents (continued)

Appendix 4 

Table 1. Molecular Analysis to Confirm the Absence of the 32138 SPT Insertion in SPT 

Progeny 152 

Appendix 7 

Table 1. Representative Scenarios of Recombination in the 32138 SPT Insertion and 

Traceability in the SPT Process 159 

Appendix 10 

Table 1. N-terminal Amino Acid Sequence of the Microbially Expressed and Maize 

 32138 SPT Maintainer derived DsRed2 protein 173 

Table 2. Identified Peptides of the Microbially Expressed and 32138 SPT Maintainer 

 Derived DsRed2 Proteins Using LC/MS Analysis 173 

Appendix 13 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 179 

Table 2. Disease Stressor Comparison between 32138 SPT Maintainer and Control 185 
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List of Figures

Figure 1. Stages in the Production of Hybrid Maize Seed 19 

Figure 2. Schematic Diagram of the Basis for SPT 22 

Figure 3. Schematic of Hybrid Maize Seed Production Using the SPT Process 25 

Figure 4. Propagation of 32138 SPT Maintainer 26 

Figure 5. Propagation of Non-Transgenic Male-Sterile Female Inbred Parent Lines 26 

Figure 6. Schematic Diagram of the 32138 SPT Insertion 28 

Figure 7. Maize Tassels Showing the Male-Sterile Mutant Phenotype (ms45/ms45) and Male 

 Fertile Restored Phenotype (Ms45/ms45) 30 

Figure 8. Cross Section of Maize Anther (A) and Stages in the Pollen Development (B) 31 

Figure 9. Potassium Iodide Starch Staining of Pollen Grains from 32138 SPT Maintainer 32 

Figure 10. Maize Seed Expressing DsRed2 (A & B) and Showing Red Fluorescence (C) 33 

Figure 11. Schematic of the Development of 32138 SPT Maintainer 37 

Figure 12. Breeding Diagram for 32138 SPT Maintainer, and Generations Used for Analyses 38 

Figure 13. Schematic Diagram of the T-DNA Region from Plasmid PHP24597 39 

Figure 14. Schematic Diagram of Plasmid PHP24597 40 

 Figure 15. Plasmid Map of PHP24597 with Location of Restriction Enzyme Sites and Probes 

 Used for Southern Analyses 45 

 Figure 16. Map of T-DNA Region from Plasmid PHP24597 with Location of Restriction Enzyme 

 Sites and Probes Used for Southern Analyses 46 

Figure 17. Map of the Insertion in 32138 SPT Maintainer 48 

Figure 18. Genetic Backgrounds of Generations Used for 32138 SPT Maintainer 

 Southern Analysis 49 

Figure 19. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 Bmt I Digested DNA with 5126 Promoter and Ms45 Probes 53 

Figure 20. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 Bmt I Digested DNA with Pg47 Promoter and zm-bt1 Probes 54 

Figure 21. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 Bmt I Digested DNA with zm-aa1 and In2-1 Terminator Probes 55 

Figure 22. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 Bmt I Digested DNA with 35S Enhancer and Ltp2 Promoter Probes 56 

Figure 23. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 Bmt I Digested DNA with DsRed2(Alt1) and pinII Terminator Probes 57 

Figure 24. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 EcoR I Digested DNA with 5126 Promoter and Ms45 Probes 60 

Figure 25. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 EcoR I Digested DNA with Pg47 Promoter and zm-bt1 Probes 61 
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32138 SPT Maintainer No CBI 

List of Figures (continued)

Figure 26. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 EcoR I Digested DNA with zm-aa1 and In2-1 Terminator Probes 62 

Figure 27. Southern Blot Analysis of the T1S1 Generation of Maize 32138 SPT Maintainer, 

 EcoR I Digested DNA with 35S Enhancer and Ltp2 Promoter Probes 63 

Figure 28. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

 EcoR I Digested DNA with DsRed2(Alt1) and pinII Terminator Probes 64 

Figure 29. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 

 SPT Maintainer: BamH I Digest and Ms45 Probe 67 

Figure 30. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: BamH I Digest and zm-aa1 Probe 68 

Figure 31. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: BamH I Digest and DsRed2(Alt1) Probe 69 

Figure 32. Southern Blot Analysis of the T1S1 and BC3 Generations of Maize32138 SPT 

 Maintainer: BamH I/Hind III Digest and DsRed2(Alt1) Probe 70 

Figure 33. Southern Blot Analysis of the BC4 Generation of 32138 SPT Maintainer: 

 BamH I Digest and Ms45 Probe 71 

Figure 34. Southern Blot Analysis of the BC4 Generation of 32138 SPT Maintainer: 

 BamH I Digest and zm-aa1 Probe 72 

Figure 35. Southern Blot Analysis of the BC4 Generation of 32138 SPT Maintainer: 

 BamH I Digest and DsRed2(Alt1) Probe 73 

Figure 36. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: EcoR I Digest and spc Probe 75 

Figure 37. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: EcoR I Digest and tet Probe 76 

Figure 38. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: EcoR I Digest and virG Probe 77 

Figure 39. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: EcoR I Digest and LB Probe 78 

Figure 40. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

 Maintainer: EcoR I Digest and RB Probe 79 

Figure 41. Deduced Amino Acid Sequence of the Full-Length MS45 Protein 83 

Figure 42A. Western Blot Showing the Anther and Stage-Specific Expression of MS45 Protein 

 During Pollen Development 84 

Figure 42B. Western Blot Showing the Endogenous Glycosylation of MS45 Protein in the 

 Anthers 84 

Figure 43. Deduced Amino Acid Sequence of the ZM-AA1 Protein 85 

Figure 44. Illustration of Mode of Action of -amylase 86 

Figure 45. Deduced Amino Acid Sequence of the DsRed2 Protein 86 

Figure 46. Western Blot Analysis of the MS45 Protein in Anthers (Panel A & B) and in Different 

 Tissues (Panel C) of the 32138 SPT Maintainer 89 

Figure 47. Identification of Agronomic Field Trials for 32138 SPT Maintainer 102 
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List of Figures (continued)

Appendix 2 

Figure 1. Appearance of Seed Mixture under Normal and Fluorescent Illumination 145 

Appendix 3 

Figure 1. Satake ScanMaster II Color Sorter 149 

Figure 2. Seed Passing Through Multiple Channels of a Seed Sorter 149 

Figure 3. Pioneer SPT Maintainer Conditioning Facility (Color Sort A) 150 

Appendix 7 

Figure 1. Aberrant Gene Recombination in the SPT Insert—A Few Possible Scenarios 158 

Appendix 10 

Figure 1. SDS PAGE and Western Blot Analysis of Microbially Expressed and 32138 

 SPT Maintainer Derived DsRed2 protein 172 

Figure 2. Glycosylation Analysis of the Microbially Expressed and 32138 SPT Maintainer 

 Derived DsRed2 Proteins 174 

 
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32138 SPT Maintainer No CBI 

I. Introduction to Seed Production Technology (SPT) 

I-A. Advantages of the Pioneer SPT Process

Maize plants bear both male flowers (located on the tassel) and female flowers (located on the 

ear), and plants can both self-pollinate and cross-pollinate. Hybrid maize seed production 

requires crossing of two inbred parent lines to produce the F1 hybrid seed sold to growers. In the 

hybrid seed production field, the female inbred seed parent must be prevented from shedding 

pollen to avoid self- or sib-pollination that reduces seed quality and compromises yield potential. 

In order to produce hybrid seed, it is first necessary to increase the seed of the inbred parents 

(Figure 1). Pioneer has developed a novel process called Seed Production Technology (SPT) 

that facilitates the increase of large quantities of male-sterile female inbred parent seed. Fertility 

is completely restored in F1 hybrids plants generated using the male-sterile female inbred parent 

seed generated using SPT. This technology offers the following advantages over other 

approaches to maize fertility control: 

 The need to remove pollen bearing tassels from female inbred parent lines by hand or 

mechanical detasseling is eliminated resulting in: 

 higher quality seed products for growers 

 increased female inbred parent seed yields 

 increased production efficiency and reduced costs 

 increased worker safety 

 The process works across all germplasm types so it can be used to produce any maize 

hybrid. 

 Increasing male-sterile female inbred lines is a simple process achieved via cross-pollination 

with a transgenic 32138 SPT maintainer. 

I-B. Maize Hybrid Seed Production 

Hybrid maize (Zea mays L.) is grown on more than 80 million acres in the United States (USDANational Agricutural Statistics Service (NASS), 2008), making it the largest producer of hybrid 

maize. The seed used to produce hybrid maize is generated by crossing two unique inbred 

parental lines referred to as the ‘male inbred parent’ (the plant that is the pollinator) and ‘female 

inbred parent’ (the plant on which the seed is produced). 

For inbred parent seed increase, male and female inbred parental lines are grown separately and 

allowed to self-pollinate in Pioneer fields that are reproductively isolated from other maize plants 

in order to maintain genetic purity and increase the quantity of the inbred parents (Figure 1). In 

order to produce hybrid seed, male and female inbred parent lines are inter-planted in adjacent 

rows (e.g., 2 rows male, 4 rows female, 2 rows male) in reproductively isolated contractor fields 

and allowed to open pollinate. 

To produce pure hybrid seed, the male inbred parent must cross pollinate the female inbred 

parent and the female inbred parent must be prevented from self-pollinating. The most common 

practice to accomplish this is to cut off the pollen-bearing tassels (i.e., detasseling) on the female 

inbred parent (Figure 1). 

In the hybrid seed production fields, the ears of the detasseled female inbred parent can only be 

fertilized with pollen from the male inbred parent planted in adjacent rows. The male inbred 

parent is removed from the fields before the ears reach maturity. Thus, the seed produced on the 
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32138 SPT Maintainer No CBI 

female seed parent will be uniformly hybrid. The F1 hybrid seed is harvested, cleaned, 

conditioned, and bagged for sale to growers. When planted in growers’ fields, the hybrid seed 

will produce a uniform F1 crop. The grain harvested from growers’ fields is used for feed and 

fuel production, processed into food products and industrial materials, or exported.

Figure 1. Stages in the Production of Hybrid Maize Seed 

Inbred 

Male Parent

Inbred

Female 

Parent

Parent Seed Increase

Commercial Hybrid Seed Production

Harvest F1 Hybrid seed 

Commercial Maize Grain Production 

Pioneer Fields Contractor Fields Seed Sales

Hybrid seed planted 

Grain harvested for commercial use

Grower Fields

<5,000 acres/yr

Detasseling of the Female Inbred Parent

Sold to Growers

Inbred 

Male Parent

Inbred

Female 

Parent

Parent Seed Increase

Commercial Hybrid Seed Production

Harvest F1 Hybrid seed 

Commercial Maize Grain Production 

Pioneer Fields Contractor Fields Seed Sales

Hybrid seed planted 

Grain harvested for commercial use

Grower Fields

<5,000 acres/yr

Detasseling of the Female Inbred Parent

Sold to Growers
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32138 SPT Maintainer No CBI 

I-C. Maize Fertility Control in Hybrid Maize Production 

I-C.1. Detasseling

The physical removal of the pollen-producing tassels (detasseling) has been the most widely 

used method of preventing female inbred parent self-pollination since maize hybrids were first 

produced in the 1920s (Wych, 1988). Less common or experimental methods include the 

application of chemical sterilants, the use of biological systems based on cytoplasmic malesterility, and systems to control male-fertility using biotechnology (Refer to Appendix 1 for 

additional information on these systems). 

Detasseling can be accomplished manually or mechanically with tassel-cutting or tassel-pulling 

machines. Thousands of temporary workers are employed to detassel maize hybrid seed 

production fields. The detasseling process is not entirely reliable, and on occasion a female plant 

will escape complete detasseling. For example, natural variation in plant development can result 

in the emergence of tassels after the detasseling process is complete or a mechanical detasseler 

can fail to remove a tassel completely. Incomplete detasseling results in pollen shed by the 

female inbred parent plant and self-pollination. When this occurs, seed of the female inbred 

parent is harvested along with the hybrid seed which results in reduced seed quality, lowered trait 

purity, and lost yield potential. Mechanical detasseling is faster than hand detasseling, but 

causes more damage to the plants and results in up to 40% reduction in seed yield (Wych, 1988). 

Today, a combination of mechanical and hand detasseling is often used; no form of detasseling is 

entirely satisfactory. Therefore, a need exists for an alternative method of pollen control that is 

safe, efficient, and highly effective. 

I-C.2. Genetic Male-Sterility

Male-sterility in maize refers to the failure to produce functional pollen. Male-sterile maize plants 

can neither self-pollinate, nor sib-pollinate neighboring plants and can serve as female inbred 

parents in the production of hybrid maize seed. Male-sterility traits display either nuclear (also 

known as genetic) or cytoplasmic inheritance (Refer to Appendix 1 for details). 

Genetic male or pollen sterility in maize was first described by Eyster (1921) that it is due to a 

single recessive gene, designated as ms. Since then, many different genetic male-sterility loci 

have been identified in maize. The chromosome number and map position of nearly all presently 

identified male-sterility genes are well characterized (Neuffer et al., 1997). Sterility phenotype is 

determined by a single gene and the allele for male-sterility is recessive (ms), although a small 

number of dominant male-sterile alleles have been described (Neuffer et al., 1997). In maize, a 

male-sterility plant trait is expressed only by maintenance of a homozygous recessive (ms/ms) 

condition for the fertility gene. 

Limitations to the Use of Homozygous Recessive (ms/ms) Lines as Male-Sterile Parents 

Increase of male-sterile (ms/ms) inbred parent seed is not possible via self-pollination as the 

homozygosity of the recessive gene (ms) results in male-sterility. 

One way to increase the seed of the male-sterile inbred parent is to cross-pollinate male-sterile 

plants (ms/ms) with male-fertile plants which are heterozygous for the male-sterility gene 

(Ms/ms). The progeny from such a cross-breeding are approximately fifty percent male-sterile 

(ms/ms) and fifty percent male-fertile (Ms/ms). Since the male-sterile seed can not be separated 

from the male-fertile seed, this is not a practical way to increase the male-sterile parent seed. 

Recovery and maintenance of pure male-sterile (ms/ms) inbred populations using traditional 

breeding methods requires: a) additional maize lines for the maintenance of the male-sterility trait, 
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32138 SPT Maintainer No CBI 

and b) several intermediate crosses and selection methods that are time consuming. Therefore, it 

is not practical for use in commercial hybrid maize production. 

Thus, a need exists for a convenient and effective process for generating male-sterile inbred 

female parent lines. 

I-C.3. Basis for the Development of Seed Production Technology 

Section I-C.2 described how cross-pollinating the male-sterile (ms/ms) plants with heterozygous 

(Ms/ms) male-fertile plants yields a mix of male-sterile and male-fertile genotypes. However, if all 

pollen containing the fertility restoration gene (Ms) could be eliminated from the heterozygous 

male parent (Ms/ms) thereby ensuring pollination of male-sterile (ms/ms) parent only with pollen 

carrying the recessive ms allele, the male-sterile progeny would retain their homozygous (ms/ms) 

condition and could be used as male-sterile female inbred parent in hybrid seed production. This 

is the basis for the Pioneer Seed Production Technology (SPT) process utilizing a transgenic 

32138 SPT maintainer (Figure 2). 

Pioneer’s novel SPT process generates male-sterile female inbred parent lines that maintain the 

homozygous recessive state (ms/ms) without any intermediate crosses. Male-sterile female 

inbred parent seed can be multiplied, while maintaining the homozygous recessive state of the 

male-sterility gene and providing for full restoration of fertility in the F1 hybrid plants grown in 

commercial fields. 
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32138 SPT Maintainer No CBI 

Figure 2. Schematic Diagram of the Basis for SPT 

‡ = Represents 32138 SPT maintainer. 

The SPT construct containing three gene cassettes [Ms45, zm-aa1, and DsRed2(Alt1)] was 

inserted into a male-sterile maize line that is homozygous recessive for ms45 (ms45/ms45) 

shown as ms/ms in Figure 2. The SPT insertion was integrated in the genome at a locus that 

segregates independently from the endogenous ms45 locus (data not shown). Therefore, 32138 

SPT maintainer is homozygous for the ms45 recessive allele and hemizygous for the 

recombinant Ms45 gene from the SPT insertion, indicated as Ms/- in Figure 2. A single copy of 

Ms45 gene from the 32138 SPT insertion restores fertility in the male-sterile (ms45/ms45) line 

(Figure 7B). 32138 SPT maintainer sheds two different types of pollen in a 1:1 ratio: a) nontransgenic fertile pollen (with the ms45 allele) that do not contain the SPT insertion, and b) 

transgenic infertile* pollen (with both the ms45 allele and the transgenic Ms45 allele) that contain 

the 32138 SPT insertion (Figure 5). The transgenic pollen carrying Ms45 are rendered infertile 

due to the -amylase gene (Refer to Sections 1-D and 1-H.2 for more details) in the SPT 

insertion (Figure 9). 

Therefore, homozygous recessive (ms45/ms45) male-sterile female inbred parent seed can be 

increased by utilizing the pollen from an isogenic SPT maintainer (Figure 3, Step II and Figure 5). 

*= Pollen infertility refers to the inability of pollen to germinate and fertilize the ovule.

ms/ms X ms/ms; Ms /-

ms/ms

• 100 % Male Sterile

• Homozygous Recessive (ms/ms) 

• Do Not Contain the SPT insertion 

PROGENY =

‡

Pollen containing 

the “Ms” fertility 

gene are rendered 

infertile using 

biotechnology

ms/ms X ms/ms; Ms /-

ms/ms

• 100 % Male Sterile

• Homozygous Recessive (ms/ms) 

• Do Not Contain the SPT insertion 

PROGENY =

‡

Pollen containing 

the “Ms” fertility 

gene are rendered 

infertile using 

biotechnology
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32138 SPT Maintainer No CBI 

I-D. Description of the SPT Process 

Seed Production Technology is a process rather than a trait or a product. The SPT process 

works across all germplasm, requires no detasseling, and produces hybrid seed that are fully 

fertile. The male-sterile female inbred parent increase process is entirely controlled by Pioneer 

and the total acreage in the US planted to the transgenic 32138 SPT maintainer and SPT derived 

male-sterile female inbred parent seed increase each year will be less than 5,000 acres. 

32138 SPT Maintainer 

The SPT process uses a transgenic 32138 SPT maintainer in an ms45/ms45 genetic background 

(Figure 3, Step I). The 32138 insertion in SPT maintainer contains three gene expression 

cassettes essential for the functioning of the SPT system: Ms45, zm-aa1, and DsRed2(Alt1) 

(Figure 6). 

The 32138 SPT maintainer is homozygous for the endogenous ms45 recessive mutant allele and 

hemizygous for the SPT insertion (i.e., ms45/ms45; SPT/-) and therefore contains a single copy 

of the Ms45 gene. Expression of a single copy of Ms45 gene in the ms45/ms45 genetic 

background restores male-fertility and enables pollen production in the 32138 SPT maintainer. 

The 32138 SPT maintainer sheds two different types of pollen in a 1:1 ratio: a) fertile, does not 

contain SPT insertion, and b) infertile, does contain 32138 SPT insertion (Figure 5). Pollen 

containing the 32138 SPT insertion is rendered infertile due to the action of ZM-AA1 -amylase 

protein. Expression of ZM-AA1 -amylase in 32138 SPT maintainer pollen results in the 

depletion of starch (Figure 9) and depriving the SPT pollen of the energy reserves required for 

successful pollen germination and fertilization. Hence, any pollen containing the 32138 SPT 

insertion is made infertile. 

A fluorescent color marker (DsRed2(Alt1)) gene is linked to the other two SPT genes (Ms45 and

zm-aa1) and it confers a pinkish red phenotype to any seed expressing the 32138 SPT insertion 

(Figures 4 and 10). The DsRed2 protein exhibits a high fluorescent intensity and emits a strong 

red fluorescence under appropriate illumination so seeds containing the SPT insertion can be 

readily detected and separated from the progeny seeds that do not contain the 32138 SPT 

insertion in a highly efficient manner using mechanical color sorting (Figure 3, Color Sort A and B; 

Refer to Appendix 3). 

Step I: 32138 SPT Maintainer Seed Increase 

32138 SPT maintainer seed is replicated via self-pollination (Figure 3 Step I and Figure 4). The 

SPT insertion is transmitted maternally. Upon self-pollination, 32138 SPT maintainer produces 

two different types of seed (yellow: pinkish red) in a 1:1 ratio (Figures 4 and 10). Yellow seed is 

non-transgenic for SPT (does not contain the 32138 SPT insertion). Pinkish red seed that 

fluoresces a bright red color under appropriate illumination is transgenic for SPT (contains the 

32138 SPT insertion). 

 

Color Sort A 

Self-pollinated 32138 SPT maintainer seed containing a mixture of yellow and pinkish red (SPT 

maintainer) seed in a 1:1 ratio is passed thru mechanical color sorters twice (Refer to Appendix 3 

for details) to separate the 32138 SPT maintainer seed from the yellow seed (Figure 3, Color Sort 

A and Figure 4). Color sorters detect the fluorescent red 32138 SPT maintainer seed and 

separate it from the yellow/non-transgenic for SPT seed. Pure seed of 32138 SPT maintainer is 

then available for the propagation of non-transgenic male-sterile female inbred parent seed 

(Figure 3, Step I). Therefore, color sort A is for the purification of 32138 SPT maintainer seed. 

Yellow seed collected from color sort A will be discarded (Refer to Appendix 3 for more details). 
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32138 SPT Maintainer No CBI 

Step II: Male-Sterile Female Inbred Parent Seed Increase 

For seed increase of male-sterile (ms45/ms45) female inbred parent, 32138 SPT maintainer will 

be planted in either a 2:2 or 4:4 or 4:2 planting pattern (male-sterile female: 32138 SPT 

maintainer rows). Fertile pollen (that does not contain the 32138 SPT insertion and carry the 

ms45 recessive allele) from the 32138 SPT maintainer plants will cross-pollinate and fertilize 

nearly isogenic male-sterile (ms45/ms45) female inbred parent plants (Figure 3, Step II and 

Figure 5). The inbred seed produced on the male-sterile female inbred plants will maintain their 

homozygous recessive (ms45/ms45) state and genetic purity. All male-sterile progeny seed 

harvested will be yellow in color and will not contain the 32138 SPT insertion (Figure 5 and Refer 

to Appendix 4 for more details). 

Color Sort B 

The SPT system is highly genetically efficient (Refer to Appendix 4 and 5) and virtually all of the 

fertile pollen from 32138 SPT maintainer should be non-transgenic for SPT and therefore all of 

the male-sterile inbred progeny seed made using the pollen from 32138 SPT maintainer should 

also be non-transgenic for SPT. The purpose of the color sort B is to ensure the purity of the 

male-sterile female inbred parent by removing any seeds of 32138 SPT maintainer. Each batch 

of male-sterile female inbred parent seed is passed twice through the color sorter (Figure 3, Color 

Sort B and Figure 5). This step helps to assure that no seed of the 32138 SPT maintainer will be 

planted in the F1 hybrid seed fields that produce seed sold to growers (Figure 3, Step III). 

Steps III. Commercial Maize Hybrid Seed Production 

Commercial maize hybrid seed production (Figure 3, Steps III) using the male-sterile progeny 

seed derived using SPT technology will take place normally. The purity of the male-sterile female 

inbred progeny derived from the SPT process is insured by passing the seed twice through the 

mechanical color sorters (Refer to Appendix 3 for details on the color sorter). Therefore, the F1 

hybrid seed harvested and sold to growers for commercial grain production is non-transgenic for 

SPT and the resulting F1 hybrid plants are fully fertile. 

Steps IV. Commercial Maize Grain Production 

Commercial maize grain production using the F1seed generated using the male-sterile female 

inbred parent will take place normally. The F1 hybrid plants grown in the growers’ fields are nontransgenic for SPT and therefore, the grain harvested from growers’ fields is also non-transgenic 

for SPT (Figure 3, Step IV). 

 
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32138 SPT Maintainer No CBI Figure 3. Schematic of Hybrid Maize Seed Production Using the SPT Process 

Step I.SPT Maintainer Seed Increase Step II.Female Inbred Parent Seed IncreaseStep III.Commercial Hybrid Seed Production Step IV.Commercial Maize Grain Production 

SPT maintainer line interplanted with male sterile female inbred parent line.

Male-sterile female inbred parent

Other elite inbred parent 

F1 Hybrid (100% Fertile) 

Sold to Growers

Pioneer Controlled , <5,000 Acres Contractor Fields Grower FieldsHybrid seed planted Grain harvested for commercial use SPT maintainer plants are destroyed after pollination. Male sterile female parent seed harvested is non-transgenic for SPT. Seed increase by self-pollination

SPT Maintainer Line

Male-Sterile Female Inbred Parent

Color Sort AColor sorting twice to separate SPT maintainer (red) seed from the yellow seed.

Color Sort B 

Color sorting twice to remove any SPT maintainer kernels

Transgenic for SPTNon-transgenic for SPT

Step I.SPT Maintainer Seed Increase Step II.Female Inbred Parent Seed IncreaseStep III.Commercial Hybrid Seed Production Step IV.Commercial Maize Grain Production 

SPT maintainer line interplanted with male sterile female inbred parent line.

Male-sterile female inbred parent

Other elite inbred parent 

F1 Hybrid (100% Fertile) 

Sold to Growers

Pioneer Controlled , <5,000 Acres Contractor Fields Grower FieldsHybrid seed planted Grain harvested for commercial use SPT maintainer plants are destroyed after pollination. Male sterile female parent seed harvested is non-transgenic for SPT. Seed increase by self-pollination

SPT Maintainer Line

Male-Sterile Female Inbred Parent

Color Sort AColor sorting twice to separate SPT maintainer (red) seed from the yellow seed.

Color Sort B 

Color sorting twice to remove any SPT maintainer kernels

Transgenic for SPTNon-transgenic for SPT
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32138 SPT Maintainer No CBI 

Figure 4. Propagation of 32138 SPT Maintainer (Figure 3, Step I) 

Figure 5. Propagation of Non-Transgenic Male-Sterile Female Inbred Parent Lines 

(Figure 3, Step II) 
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32138 SPT Maintainer No CBI 

I-E. 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. 

In order to facilitate the use of the SPT process in hybrid seed production, Pioneer is 

submitting data on transgenic 32138 SPT maintainer and is requesting APHIS determine that 

32138 SPT maintainer and its crosses with non-regulated maize lines no longer be 

considered regulated articles under 7CFR 340. 

Furthermore, in order to address questions regarding the movement of progeny seed and 

grain in international commerce, Pioneer is requesting that USDA-APHIS make an additional 

finding that the SPT process, as described and operated by Pioneer, consistently and reliably 

produces progeny that lack the 32138 SPT insertion and are therefore non-transgenic for 

SPT. 

I-F. Submissions to Other Regulatory Agencies 

Small amounts of the transgenic 32138 SPT maintainer may enter into feed channels via 

signed contractual agreements. Therefore, a New Protein Consultation (NPC) for the 

DsRed2 protein color marker was submitted to FDA on October 11, 2006. An NPC for the 

ZM-AA1 protein was submitted to FDA on June 18, 2009. 

The 32138 SPT maintainer is not a commercial product. The SPT process generates 

progeny seed that do not contain the 32138 SPT insertion and hence are non-transgenic for 

SPT genes. Export approvals are not required for grain harvested from hybrid seed that was 

generated using the SPT process. 
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32138 SPT Maintainer No CBI 

I-G. Generation of the 32138 SPT Maintainer 

A non-transgenic male-sterile maize line homozygous recessive (ms45/ms45) for Ms45 locus 

was transformed with the SPT insertion (Figure 6) to produce the 32138 SPT maintainer. 

The SPT insertion contains gene cassettes for Ms45, zm-aa1, and DsRed2(Alt1) (Table 1). 

The 32138 SPT insertion was integrated in the genome at a locus that segregates 

independently from the endogenous ms45 locus (data not shown). Therefore, the 32138 

SPT maintainer is homozygous for the ms45 recessive allele and hemizygous for the Ms45 

gene from the 32138 SPT insertion. 

Figure 6. Schematic Diagram of the 32138 SPT Insertion 

Table 1. Components of the 32138 SPT Insertion

Gene Ms45 zm-aa1 DsRed(Alt1) 

Source Zea mays L. Zea mays L. Discosoma sp. 

Promoter Anther preferred 

5126

Pollen preferred 

Pg47 

Endosperm preferred 

Ltp2

Gene Product MS45 protein -Amylase DsRed2 fluorescent 

protein 

Function Restores fertility 

in ms45/ms45

background 

Makes pollen 

infertile by 

depletion of 

starch 

Serves as a screenable 

marker for identification 

and separation of 

transgenic SPT seeds 

from non-SPT seeds 

Ms45 zm-aa1 DsRed(Alt1) 
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32138 SPT Maintainer No CBI 

I-H. Brief Description of Genes

I-H.1. Maize Male-Fertility Gene Ms45 

The Ms45 gene is one of the many nuclear fertility genes that have been identified and 

isolated in maize using activator transposon tagging (Albertsen et al., 1993). Mutations in the 

Ms45 gene have been shown to cause male-sterility (Albertsen et al., 1993). Expression of 

the Ms45 gene is localized to anthers and the MS45 protein is produced in the tapetum. 

Mutations in Ms45 are typically recessive and homozygous mutants are male-sterile (the 

mutant Ms45 allele is designated ms45). A plant that is homozygous for ms45 (i.e., 

ms45/ms45) is male-sterile and produces no pollen (Figure 7A). In both inbred parent 

increase and hybrid seed production, it is necessary to maintain the homozygous recessive 

state of the female inbred parent in order to maintain male-sterility. However, due to the 

recessive nature of the mutation, any normal maize line carrying the Ms45 allele, when used 

as a male inbred parent for hybrid seed production, will restore fertility to the hybrid (Figure 

7B). Similarly, male-fertility can be restored by a single copy of the Ms45 gene transformed 

into a homozygous recessive plant (ms45/ms45). Hence, transformations of the ms45/ms45

male-sterile plants with the SPT insertion containing a single copy of the Ms45 gene will 

restore fertility (Figure 7B). 

Anther Morphology 

In maize, the anther wall is composed of four cell layers, the epidermis, the endothecium, the 

middle layer and the innermost layer, the tapetum (Figure 8A). The cell layers of the anther 

surround the locule that contains the sporogenous cells which undergo meiosis to form a 

tetrad or quartet of microspores encased in a callose sheath. During anther development, a 

callose sheath is dissolved by the action of callase. As a result the microspores are released 

as free floating cells into the locule and formation of the outer cell wall, called the exine takes 

place. Later, the inner microspore cell wall or intine is produced. The microspores 

subsequently go through two mitotic divisions, fill with starch, and ultimately become mature 

pollen (Figure 8B). One of the proposed functions of the tapetum is that it is involved in 

furnishing precursors during the development of the exine (Bedinger, 1992). Homozygous 

recessive mutations of the Ms45 gene result in male-sterility due to a lack of, or incomplete 

exine formation on the microspores. Consequently, the microspores of ms45 mutants break 

down shortly after the early to mid-uninucleate stages of development and pollen is not 

produced. 

Anther-Specific Expression of Ms45 

The expression of Ms45 gene has been characterized and is confined to anthers in the 

developing maize tassels (Cigan et al., 2001; Unger et al., 2002). Specifically, the MS45 

protein is localized to the tapetal cell layers of the anthers and is first detected during the 

tetrad stage, attains its highest level in the early uninucleate stage and quickly drops off 

shortly after the start of the mid-uninucleate stage of microsporogenesis (Cigan et al., 2001; 

Unger et al., 2002; Figure 8B). 
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32138 SPT Maintainer No CBI 

Figure 7. Maize Tassels Showing the Male-Sterile Mutant Phenotype (ms45/ms45) and 

Male-Fertile Restored Phenotype (Ms45/ms45) 
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32138 SPT Maintainer No CBI 

Figure 8. Cross-Section of Maize Anther (A) and Stages in the Pollen Development (B) 

Yellow box in Figure 8B identifies the stages for MS45 protein expression (Cigan et al., 

2001).
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32138 SPT Maintainer No CBI 

I-H.2. Maize -Amylase Gene zm-aa1

The maize -amylase zm-aa1 gene in 32138 SPT maintainer was isolated from a cDNA 

library of the developing maize embryo and endosperm at ten days after pollination. The 

endogenous zm-aa1 gene is predominantly expressed in the scutellum tissues of the 

geminating seed;; minimal expression of zm-aa1 (<0.25% of the levels in the scutellum) is 

observed in endosperm, stalk, leaf and root. Endogenous zm-aa1 expression was not 

observed in pollen (internal data not shown. 

Alpha-amylases belong to a family of glycosyl-hydrolases catalyzing hydrolysis of (1-4)--Dglucosidic linkages in polysaccharide molecules, such as starch. By linking the zm-aa1 gene 

to a maize pollen-preferred promoter, Pg47, and a transit peptide sequence from the maize 

brittle-1 gene that targets the protein to the amyloplast, the ZM-AA1 protein in 32138 SPT 

maintainer is expressed in the developing pollen grain resulting in the depletion of starch and 

depriving the pollen of the energy reserves required for successful pollen germination and 

fertilization. Hence, any pollen containing the SPT insertion will be infertile. 

The transgenic 32138 SPT maintainer is homozygous for the ms45 recessive mutation and 

hemizygous for the SPT insertion (i.e., ms45/ms45:SPT/-). Therefore, 50% of the pollen 

produced by the 32138 SPT maintainer is transgenic but infertile since it contains the 32138 

SPT insertion (i.e., positive for -amylase) and 50% is non-transgenic and fertile, since it 

lacks the 32138 SPT insertion (i.e., negative for -amylase). Figure 9 shows pollen from the 

32138 SPT maintainer stained with potassium iodide. There is equal distribution of pollen 

that contains starch (dark purple) and pollen that lacks starch (unstained). 

Figure 9. Potassium Iodide Starch Staining of Pollen Grains from 32138 SPT 

Maintainer 
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32138 SPT Maintainer No CBI 

I-H.3 Discosoma sp. (coral) Color Marker Gene DsRed2(Alt1)

The DsRed2(Alt1) gene is the only non-maize gene sequence utilized in the generation of the 

32138 SPT maintainer. DsRed2 is a variant of Clontech’s (www.clontech.com; WassonBlader, 2001) original “living color” red fluorescent protein (DsRed), isolated from a marine 

coral, Discosoma sp. The Alt1 suffix denotes that this version includes a single base pair 

substitution, made during cloning, that does not change the protein sequence. The DsRed2 

protein belongs to a family of red fluorescent proteins that are used as vital color markers for 

a variety of applications in biological systems. 

 

The DsRed2 protein expressed from the DsRed2(Alt1) gene under the control of the barley 

Ltp2 promoter turns seeds containing the SPT insert a pink color that is visible to the naked 

eye (Figure 10A & B). The DsRed2 protein exhibits a high fluorescent intensity and emits a 

strong red fluorescence at a wavelength of 583 nm. When seeds expressing the DsRed2 

protein are appropriately illuminated (Figure 10C), DsRed2 is efficiently excited and 

fluoresces a bright red color. Therefore, seeds containing the 32138 SPT insertion can be 

readily detected with high sensitivity using appropriate illumination and optical filter sets, and 

separated from non-SPT seed in a highly efficient manner using mechanical color sorting 

(see Appendix 3 for more details). 

Figure 10. Maize Seed Expressing DsRed2 (A & B) and Showing Red Fluorescence (C)
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32138 SPT Maintainer No CBI 

II. The Biology of Maize 

Refer to the OECD Consensus Document on the Biology of Zea mays subsp. mays (Maize), 

2003, for information pertaining to the following aspects of maize biology: 

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. 
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32138 SPT Maintainer No CBI 

III. Method of Development of 32138 SPT Maintainer 

III-A. Characterization and Generation of a Male-Sterile (ms45/ms45) 

Recipient Maize 

The maize recipient line utilized for transformation and generation of 32138 SPT maintainer is 

designated as Hi-II(ms45). The Hi-II line was converted to male-sterile Hi-II(ms45) by 

backcrossing the ms45 allele into Hi-II (Cigan et al., 2001). 

Hi-II maize has been used for maize transformation for a number of years because of its high 

transformability and good culturability. Hi-II is a derivative of the A188 and B73 maize parent 

lines which are publicly available from the University of Minnesota and Iowa State University, 

respectively. Hi-II is approximately 50:50 of A188 and B73 parental lines (Armstrong, et al., 

1991). The material was developed to have a higher regeneration potential. 

In order to generate a male-sterile (ms45/ms45) recipient Hi-II maize line, a male-sterile 

female that was homozygous for an ms45 mutant Ac excision allele, ms45’-9301 (ms45), was 

pollinated using pollen bulked from Hi-II maize plants (Armstrong, 1994), resulting in the 

introgression (over four generations) of the ms45 allele into Hi-II maize germplasm. The 

resultant source material for transformation consisted of embryos segregating for ms45. This 

allowed for transformation directly into a homozygous ms45 background (ms45/ms45) and 

provided the opportunity to directly test gene complementation in the T0 generation (fertility of 

the regenerated plants). 

III-B. Description of the Transformation System 

Plasmid PHP24597 (containing the Ms45, zm-aa1, and DsRed2(Alt1) gene expression 

cassettes) was used to generate 32138 SPT maintainer through Agrobacterium mediated 

transformation. Refer to Section IV for the detailed description of the plasmid. 

Immature embryos of maize Hi-II(ms45), were aseptically removed from the developing 

caryopsis 9 to 10 days after pollination and infected with Agrobacterium tumefaciens strain 

LBA4404 containing plasmid PHP24597, essentially as described in Zhao et al., 2001. After 

6 days of embryo and Agrobacterium co-cultivation on solid culture medium with no selection, 

the embryos were transferred to maintenance (no selection) medium that was stimulatory to 

maize somatic embryogenesis. The maintenance medium contained carbenicillin to kill any 

remaining Agrobacterium. 

After approximately two weeks on the maintenance medium, healthy, growing calli that 

demonstrated red fluorescence were identified, indicating presence of the T-DNA from 

plasmid PHP24597. The putative transgenic calli were continually transferred to fresh 

maintenance medium for further growth until the start of the regeneration process. 

The embryonic calli were regenerated into whole transgenic plants and transferred to the 

greenhouse. The regenerated plants (designated T0 plants) were screened for Ms45 gene 

complementation and ms45/ms45 transgenic samples were used for molecular analysis for 

transgene copy number and the absence of Agrobacterium backbone DNA by PCR. Positive 

plants were further advanced to generate 32138 SPT maintainer. Refer to Figure 11 for a 

schematic diagram of the development process for 32138 SPT maintainer and Figure 12 for a 

breeding diagram. 
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32138 SPT Maintainer No CBI 

III-C. Selection of Comparators for 32138 SPT Maintainer 

To ensure accurate assessment of the impact of transgene insertion on various 

characteristics of 32138 SPT maintainer, a proper selection of the comparator plants is 

important. Depending on the study, two types of maize lines were used as comparators: 

inbred control maize and reference inbred maize (Figure 12). 

The inbred control maize plants have a genetic background similar to that of 32138 SPT 

maintainer but lack the transgenic insert. For most analyses, the near-isoline inbred 705 was 

used as the control. Hi-II(ms45) and Hi-II lines were used as additional negative controls for 

Southern blot analyses. For the Western blot analysis, male-sterile progeny derived from 

32138 SPT maintainer were used as a negative control. 

Non-transgenic maize reference inbreds were used to help determine the normal variation 

seen in inbred maize and to develop the statistical tolerance intervals for the nutrient 

composition and agronomic assessments. For Western blot analysis, near-isoline inbred 705 

was used as a reference. 

 
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32138 SPT Maintainer No CBI 

Figure 11. Schematic of the Development of 32138 SPT Maintainer 

Identification and selection of Ms45, zm-aa1, and DsRed2(Alt1) gene 

constructs for SPT system functionality 

Assembly of plasmid PHP24597 containing Ms45, zm-aa1, and 

DsRed2(Alt1) gene cassettes 

Co-cultivation of immature embryos of Hi-II(ms45) with Agrobacterium 

tumefaciens strain LBA4404 with PHP24597 plasmid 

Selection and regeneration of red fluorescing embryonic calli 

Regeneration of T0 maize plants and screen for fertility 

Molecular analysis for transgene copy number and the absence of 

Agrobacterium backbone DNA by PCR 

Crossing and backcrossing for product development to generate SPT 

maintainer 

Selection of 32138 as the lead maize SPT maintainer 

Field and laboratory studies to support product registration 

Generation of SPT maize maintainers in different genetic backgrounds 
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32138 SPT Maintainer No CBI 

Figure 12. Breeding Diagram for 32138 SPT Maintainer, and Generations Used for 

Analyses 

Analysis 

Relevant 

Section of 

Petition 

32138 SPT 

Maintainer 

Generation Used 

Control(s) Used 

Molecular 

characterization 

V-A, B, C and 

D T1S1, BC4, BC3, Inbred 705, Hi-II, Hi-II(ms45) 

Mendelian 

inheritance of 

the traits 

V-E BC3S2F1, 

BC3S4, and BC4 N/A 

Detection of 

MS45 by 

Western blotting 

VI-D BC1S2 

Inbred 705 and non-transgenic 

male-sterile (ms45/ms45) 

progeny inbred derived from 

transgenic 32138 SPT maintainer 

ZM-AA1 and 

DsRed2 protein 

concentration 

VI-E BC3S2 Inbred 705 

Seed 

germination / 

dormancy 

VII-A BC3S5 Inbred 705 

Field agronomic 

characteristics VII-B BC3S2 Inbred 705 

Nutrient 

composition 

assessment 

VIII BC3S2 Inbred 705 

T0 

T1

BC0

BC1

Cross 705

Backcross

705 inbred

BC2

BC3

BC4

T1S1

BC1S1

BC3S1

BC2S1

BC1S2

Tester

BC3S3

BC3S2F1

(Recipient Line is Hi-II(ms45))

BC3S2

Cross with 

BC3S4

Backcross

705 inbred

Backcross

705 inbred

Backcross

705 inbred

 =Self

BC3S5





 

    

T0 

T1

BC0

BC1

Cross 705

Backcross

705 inbred

BC2

BC3

BC4

T1S1

BC1S1

BC3S1

BC2S1

BC1S2

Tester

BC3S3

BC3S2F1

(Recipient Line is Hi-II(ms45))

BC3S2

Cross with 

BC3S4

Backcross

705 inbred

Backcross

705 inbred

Backcross

705 inbred

 =Self

BC3S5





 

    





 

    
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32138 SPT Maintainer No CBI 

IV. Donor Genes and Regulatory Sequences 

IV-A. Plasmid PHP24597 Used in Transformation 

Maize (Zea mays L.) event DP-32138-1 was produced by Agrobacterium-mediated 

transformation with plasmid PHP24597 (Figure 14). The T-DNA region of this plasmid is 

represented schematically in Figure 13. A summary of the genetic elements and their 

positions on the T-DNA are described in Table 2. 

Figure 13. Schematic Diagram of the T-DNA Region from Plasmid PHP24597 

Schematic diagram of the T-DNA indicating the Ms45 gene region, zm-aa1 gene, 

and the DsRed2(Alt1) gene along with their respective regulatory elements. The 

size of the T-DNA is 9950 bp. 

DsRed2 (Alt1)

Ms45 Genomic

zm-bt1 Transit Peptide zm-aa1

CaMV 35S Enhancer

Left Border Right Border

5126 Promoter

Ltp2 Promoter

Pg47 Promoter

In 2-1 Terminator

pinII Terminator
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32138 SPT Maintainer No CBI 

Figure 14. Schematic Diagram of Plasmid PHP24597

Schematic diagram of plasmid PHP24597 with genetic elements indicated. 

Plasmid size is 52869 bp. 

virB

virC1

virC2

virG

tetR

tetA

ctl

rep

spc

DsRed2 (Alt1)

Ms45 Genomic

zm-bt1 Transit Peptide

zm-aa1

CaMV 35S Enhancer

Left Border

Right Border

5126 Promoter

Ltp2 Promoter

Pg47 Promoter

oriV

colE1 ori

oriT

colE1 ori

In2-1 Terminator

cos

cos

pinII Terminator

trfA
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32138 SPT Maintainer No CBI 

Table 2. Description of Genetic Elements in the T-DNA Region of Plasmid PHP24597

Location 

on T-DNA 

(base pair 

position) 

Genetic 

Element 

Size 

(base 

pairs) 

Description 

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

Agrobacterium tumefaciens

26 to 177 Ti Plasmid 

Region 152 Non-functional sequence from Ti plasmid of A. 

tumefaciens

178 to 203 Polylinker 

Region 26 Region required for cloning genetic elements 

204 to 706 5126 

Promoter 503 Maize anther-preferred 5126 promoter (Cigan and Albertsen, 1997) 

707 to 2658 

Ms45 

Genomic 

Region 

1952 

Maize genomic DNA including the Ms45

coding sequence and associated 3’ 

untranslated region as indicated below: 

Exon 1 bp 708 to 1086 

Intron 1 bp 1087 to 1215 

Exon 2 bp 1216 to 1499 

Intron 2 bp 1500 to 1596 

Exon 3 bp 1597 to 1764 

Intron 3 bp 1765 to 1850 

Exon 4 bp 1851 to 2258 

3’ UTR bp 2384 to 2658 

(Albertsen et al., 1993; Albertsen et al., 1995) 

2659 to 

2730 

Polylinker 

Region 72 Region required for cloning genetic elements 

2731 to 

5466 

Pg47

Promoter 2736 

Promoter from the maize, pollen 

polygalacturonase (Pg47) gene (Allen and 

Lonsdale, 1993) 

5467 to 

5468 

Polylinker 

Region 2 Region required for cloning genetic elements 

5469 to 

5693 

zm-bt1 

Transit 

Peptide 

225 Amyloplast-targeting transit peptide from the 

maize brittle-1 gene (Sullivan et al., 1991) 

5694 to 

6956 zm-aa1 Gene 1263 Maize -amylase gene 

6957 to 

7033 

Polylinker 

Region 77 Region required for cloning genetic elements 

7034 to 

7377 

In2-1 

Terminator 344 Terminator sequence from the maize In2-1gene (Hershey and Stoner, 1991) 

7378 to 

7411 

Polylinker 

Region 34 Region required for cloning genetic elements 

7412 to 

7849 

CaMV 35S 

Enhancer 438 

Enhancer region from the Cauliflower Mosaic 

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

al., 1985; Odell et al., 1988). 
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32138 SPT Maintainer No CBI 

Table 2. Description of Genetic Elements in the T-DNA Region of Plasmid PHP24597 

(continued) 

Location 

on T-DNA 

(base pair 

position) 

Genetic 

Element 

Size 

(base 

pairs) 

Description 

7850 to 

7905 

Polylinker 

Region 56 Region required for cloning genetic elements 

7906 to 

8761 

Ltp2

Promoter 856 Promoter from barley lipid transfer protein (Ltp2) gene (Kalla et al., 1994) 

8762 to 

8809 

Polylinker 

Region 48 Region required for cloning genetic elements 

8810 to 

9487 

DsRed2(Alt1)

Gene 678 

Modified DsRed2 gene (Wasson-Blader T, 

2001) with internal BstE II restriction site 

removed 

9488 to 

9528 

Polylinker 

Region 41 Region required for cloning genetic elements 

9529 to 

9839 

pinII 

Terminator 311 

Terminator region from Solanum tuberosum

proteinase inhibitor II gene (Keil et al., 1986; 

An et al., 1989). 

9840 to 

9872 

Polylinker 

Region 33 Region required for cloning genetic elements 

9873 to 

9925 

Ti Plasmid 

Region 53 Non-functional sequence from Ti plasmid of A. 

tumefaciens

9926 to 

9950 Left Border 25 T-DNA Left Border region from Ti plasmid of 

Agrobacterium tumefaciens
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32138 SPT Maintainer No CBI 

IV-B. Identity and Source of Genetic Material in the T-DNA of PHP24597 

The T-DNA of plasmid PHP24597 (Figure 13) contains three gene cassettes. 

The Ms45 Cassette

The first cassette contains the Ms45 Genomic sequence, which is a region of maize genomic 

DNA comprising the Ms45 gene and associated 3’ untranslated region (Albertsen et al., 1993; 

Albertsen et al., 1995). The Ms45 gene includes four exons with three introns that are 

removed by splicing. Expression of the MS45 protein encoded by the Ms45 gene in the 

anther tapetum is required for the production of fertile male pollen by the maize plant (Cigan 

et al., 2001). 

The expression of the Ms45 gene is controlled by the maize anther-specific 5126 promoter 

(Cigan and Albertsen, 1997). The terminator for the Ms45 gene is the endogenous Ms45 3’ 

untranslated region sequence from the maize genome, which is included in the Ms45

genomic sequence (Albertsen et al., 1993). The full-length MS45 protein is comprised of 412 

amino acids and has a molecular weight of approximately 47 kDa (Figure 41). 

The zm-aa1 Cassette

The second cassette contains a truncated maize -amylase gene (zm-aa1) that encodes the 

ZM-AA1 protein. The zm-aa1 gene sequence was truncated to remove the native N-terminal 

transit peptide sequence and was replaced by sequence from the zm-bt1 gene (brittle-1)

encoding the Brittle-1 transit peptide for targeting the ZM-AA1 protein to amyloplasts (Sullivan 

et al., 1991). The ZM-AA1 protein prevents accumulation of starch in the nascent pollen 

grain, thus preventing the pollen from developing and germinating normally. The complete 

translation product, including transit peptide, comprises 495 amino acids and has a molecular 

weight of approximately 54 kDa (Figure 43). Following processing of the Brittle-1 transit 

peptide the primary ZM-AA1 protein observed is 445 amino acids in length and has a 

molecular weight of approximately 49 kDa (Figure 43). 

The expression of the zm-aa1 gene and attached transit peptide is controlled by the Pg47

promoter, which is the 5’ regulatory region from the maize polygalacturonase (Pg47) gene 

(Allen and Lonsdale, 1993). The terminator for the zm-aa1 gene is the 3’ terminator 

sequence from the maize In2-1 gene (Hershey and Stoner, 1991). 

The DsRed2(Alt1) Cassette 

The third cassette contains the DsRed2(Alt1) gene. The DsRed2(Alt1) gene is a modified 

version of the original DsRed2 gene (Wasson-Blader, 2001) in which an internal BstE II 

restriction site was removed without altering the amino acid sequence of the expressed 

protein. The expression of the DsRed2(Alt1) gene is controlled by the barley lipid transfer 

protein (Ltp2) promoter, which provides aleurone-preferred transcription of the gene (Kalla et 

al., 1994). Located 5’ to the Ltp2 promoter is the enhancer region from the cauliflower 

mosaic virus (CaMV 35S enhancer) (Franck et al., 1980; Odell et al., 1985 and 1988). The 

terminator for the DsRed2(Alt1) gene is the 3’ terminator sequence from the proteinase 

inhibitor II gene of Solanum tuberosum (pinII terminator) (Keil et al., 1986; An et al., 1989). 

The DsRed2(Alt1) gene encodes the DsRed2 protein. Expression of the DsRed2 protein in 

the aleurone layer of the maize seed produces a red coloration in seeds containing the DNA 

insertion, allowing for separation and identification of seed containing the 32138 SPT 

insertion during seed sorting. The full-length DsRed2 protein has a length of 225 amino acids 

and a molecular weight of approximately 26 kDa (Figure 45). 
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32138 SPT Maintainer No CBI 

V. Genetic Characterization of 32138 SPT Maintainer 

V-A. Molecular Analysis Overview 

To characterize the DNA insertion in 32138 SPT maintainer, Southern blot analysis was 

conducted. The 32138 SPT maintainer was generated using Agrobacterium-mediated 

transformation with plasmid PHP24597 (Figure 15). The T-DNA from PHP24597 (Figure 16) 

has been inserted in 32138 SPT maintainer and contains three gene cassettes. The first 

cassette is the Ms45 cassette comprised of the anther-specific 5126 promoter and the Ms45

gene with associated 3’ untranslated region (Ms45 Genomic). The second is the zm-aa1

cassette comprised of the Pg47 promoter, the zm-bt1 transit peptide, the zm-aa1 gene, and 

the In2-1 terminator. The third cassette is the DsRed2(Alt1) cassette containing the CaMV 

35S enhancer, the Ltp2 promoter, the DsRed2(Alt1) gene and the pinII terminator. Individual 

plants of the T1S1 generation were analyzed to determine the copy number of each of the 

genetic elements inserted into 32138 SPT maintainer and to verify that the integrity of the 

PHP24597 T-DNA was maintained upon integration. The analysis confirmed a single, intact 

T-DNA of PHP24597 has been inserted into the maize genome to produce 32138 SPT 

maintainer (Section V-B). 

Stability of the 32138 SPT insertion during traditional maize breeding procedures was 

confirmed using Southern blot analysis. The analysis was conducted on two generations, 

T1S1 and BC3, and verified that the insertion remained intact and stably integrated as 

demonstrated by identical hybridization patterns in the two generations. A third generation 

(BC4) was also analyzed by Southern blot analysis, which confirmed the same stable, eventspecific hybridization pattern as exhibited by the T1S1 and BC3 generations. These results 

confirmed the stability of the insertion in 32138 SPT maintainer across multiple breeding 

generations (Section V-C). 

Both the T1S1 and BC3 generations were analyzed for plasmid sequences from PHP24597 

outside of the T-DNA, i.e. regions not intended for transformation. The analysis confirmed 

the absence of backbone sequences in 32138 SPT maintainer (Section V-D). 

Genomic DNA from leaf material of the Hi-II(ms45), Hi-II, and inbred 705 were used as 

negative controls for all Southern blot analyses to determine which hybridizing bands 

originated from the endogenous maize genome. Many of the elements in the 32138 SPT 

insertion are derived from the maize genome and probes would be expected to hybridize to 

sequences in the endogenous maize genome as well as the 32138 SPT insertion. Plasmid 

PHP24597 was used as a positive control for probe hybridization and to verify fragment sizes 

internal to the T-DNA of PHP24597. All probes used for the analysis are indicated on the 

schematic maps of PHP24597 plasmid and T-DNA (Figures 15 and 16, respectively) and 

outlined in Table 3. 

Based on these analyses, a schematic map of the insertion region in 32138 SPT maintainer 

was determined and is presented in Figure 17. 

Detailed descriptions of the generations analyzed and the methods used for Southern blot 

analysis are further described in Appendix 8.
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32138 SPT Maintainer No CBI 

Figure 15. Plasmid Map of PHP24597 with Location of Restriction Enzyme Sites and 

Probes Used for Southern Analyses 

Schematic map of plasmid PHP24597 indicating EcoR I and BamH I restriction 

enzyme sites with base pair positions. The Right Border and Left Border regions 

flank the T-DNA (Figure 16) that is expected to be transferred during 

Agrobacterium-mediated transformation. The location of the probes used for 

Southern analysis is indicated as lettered boxes inside the plasmid map. A: RB 

probe; B: LB probe; C: spc probe; D: virG probe; E: tet probe. 

PHP24597

52869 bp

virB

virC1

virC2

virG

tetR

tetA

ctl rep

spc

Ms45 Genomic

zm-bt1 Transit Peptide

zm-aa1

CaMV 35S Enhancer

Left Border

Right Border

5126 Promoter

Ltp2 Promoter

Pg47 Promoter

oriV

colE1 ori

oriT

colE1 ori

In2-1 Terminator

cos

cos

Eco RI (191)

Eco RI (7407)

EcoRI (9869)

EcoRI (15489)

EcoRI (39263)

Eco RI (42147)

Eco RI (42559

EcoRI (43870)

Eco RI (45268)

BamHI (1456)

BamHI (4271)

BamHI (6773)

BamHI (36017)

Bam HI (37358)

BamHI (39217)

BamHI (40153)

BamHI (40353)

BamHI (45499)

pinII Terminator

DsRed2 (Alt1)

-

-

1

BamHI (8792)

C

D

E

PHP24597

52869 bp

virB

virC2

vir

tetR

tetA

ctl rep

spc

Ms45 Genomic

zm-bt1 Transit Peptide

zm-aa1

CaMV 35S Enhancer

Left Border

Right Border

5126 Promoter

Ltp2 Promoter

Pg47 Promoter

oriV

colE1 ori

oriT

colE1 ori

In2-1 Terminator

cos

cos

Eco RI (191)

Eco RI (7407)

EcoRI (9869)

EcoRI (15489)

EcoRI (39263)

Eco RI (42147)

Eco RI (42559

EcoRI (43870)

Eco RI (45268)

BamHI (1456)

BamHI (4271)

BamHI (6773)

BamHI (36017)

Bam HI (37358)

BamHI (39217)

BamHI (40153)

BamHI (40353)

BamHI (45499)

pinII Terminator

DsRed2 (Alt1)

-

-

1

BamHI (8792)

A

B

The locations of additional enzymes used for analysis are given below: 

Enzyme Locations (bp) 

Bmt I 8503, 36160, 38359, 39502, 45917 

Hind III 179, 34837, 45900, 46809, 47930, 49445 
Pioneer Hi-Bred Page 46 of 188 

32138 SPT Maintainer No CBI 

Figure 16. Map of T-DNA Region from Plasmid PHP24597 with Location of Restriction 

Enzyme Sites and Probes Used for Southern Analyses

Schematic map of T-DNA from PHP24597 indicating restriction enzyme sites for 

BamH I, Bmt I, EcoR I, and Hind III and the Ms45, zm-aa1, and DsRed2(Alt1) 

coding and regulatory regions. T-DNA size is 9950 bp. The locations of the 

probes used are shown as numbered boxes below the map and are identified 

below: 

DsRed2 (Alt1)

Ms45 Genomic

zm-aa1

CaMV 35S Enhancer

Right Border Left Border

5126 Promoter

Ltp2 Promoter

Pg47 Promoter

In2-1 Terminator

pinII Terminator

zm-bt1 Transit Peptide

Bmt I (8503)

Hind III (179)

EcoR I (191)

EcoR I (7407)

EcoR I (9869)

BamH I (1456)

BamH I (4271)

BamH I (6773)

BamH I (8792)

1 2 3 4 5 6 7 8 9 10

Number Probe Name 

1 5126 promoter 

2 Ms45 

3 Pg47 promoter

4 zm-bt1 

5 zm-aa1 

6 In2-1 terminator 

7 35S enhancer 

8 Ltp2 promoter

9 DsRed2(Alt1)

10 pinII terminator
Pioneer Hi-Bred Page 47 of 188 

32138 SPT Maintainer No CBI 

Table 3. Description of DNA Probes Used for Southern Hybridization 

Probe Name Genetic 

Element 

Figure 

Probe 

Position on 

PHP24597 

T-DNA 

(bp to bp)1

Position on 

PHP24597 

Plasmid 

(bp to bp)2

Length 

(bp) 

5126 

promoter 5126 promoter Figure 16 

probe 1 304 to 706 304 to 706 403 

Ms453 Ms45 Genomic 

region

Figure 16 

probe 2 

707 to 1426 

1427 to 2258 

2263 to 2658 

707 to 1426 

1427 to 2258 

2263 to 2658 

720 

832 

396 

Pg47

promoter

Pg47 promoter 

(3’ region)

Figure 16 

probe 3 5028 to 5451 5028 to 5451 424 

zm-bt1 zm-bt1 transit 

peptide 

Figure 16 

probe 4 5469 to 5693 5469 to 5693 225 

zm-aa14 zm-aa1 gene Figure 16 

probe 5 

5701 to 6333 

6334 to 6935 

5701 to 6333 

6334 to 6935 

603 

602 

In2-1 

terminator In2-1 terminator Figure 16 

probe 6 7049 to 7377 7049 to 7377 329 

35S enhancer CaMV 35S 

enhancer region 

Figure 16 

probe 7 7427 to 7846 7427 to 7846 420 

Ltp2 promoter Ltp2 promoter Figure 16 

probe 8 7906 to 8759 7906 to 8759 854 

DsRed2(Alt1) DsRed2(Alt1)

gene 

Figure 16 

probe 9 8810 to 9487 8810 to 9487 678 

pinII

terminator pinII terminator Figure 16 

probe 10 9582 to 9815 9582 to 9815 234 

RB

Plasmid 

backbone 

adjacent to 

T-DNA Right 

Border 

Figure 15 

probe A N/A5 52436 to 

52825 390 

LB

Plasmid 

backbone 

adjacent to 

T-DNA Left 

Border 

Figure 15 

probe B N/A 9975 to 10320 346 

spc Spectinomycin 

resistance gene

Figure 15 

probe C N/A 11130 to 

11904 775 

virG virG gene Figure 15 

probe D N/A 37294 to 

38037 744 

Tet6 Tetracycline 

resistance gene 

Figure 15 

probe E N/A 

16983 to 

17521 

17627 to 

18084 

539 

458 

 

Footnotes: 

1. The probe position is based on the PHP24597 T-DNA map (Figure 16). 

2. The probe position is based on the PHP24597 plasmid map (Figure 15). 

3. The Ms45 probe is comprised of three non-overlapping labeled fragments that are combined in the hybridization 

solution. 

4. The zm-aa1 probe is comprised of two non-overlapping labeled fragments that are combined in the 

hybridization solution. 

5. Not Applicable as this element is not located in the PHP24597 T-DNA region. 

6. The tet probe is comprised of two non-overlapping labeled fragments that are combined in the hybridization 

solution. 
Pioneer Hi-Bred Page 48 of 188 

32138 SPT Maintainer No CBI 

Figure 17. Map of the Insertion in 32138 SPT Maintainer 

Schematic map of the insertion in 32138 SPT Maintainer based on Southern blot 

analysis. The flanking maize genome is represented by the horizontal dotted line 

on either side of the PHP24597 T-DNA insertion. A single, intact copy of the 

PHP24597 T-DNA integrated into the maize genome. Bmt I, EcoR I, BamH I, 

and Hind III restriction enzyme sites are indicated with the observed fragments 

on Southern blots shown below the map. The dashed vertical line in the BamH I 

box below(~1500bp) indicates the BamH I restriction enzyme site that appears to 

be partially blocked from cutting on the Southern blots (Figures 31 and 35). The 

locations of restriction enzyme sites outside the Right and Left Borders are not 

shown to scale. Note: Complete Southern blot analysis was conducted with 

BamH I/Hind III and with each of the probes and confirmed the intact regions of 

the PHP24597 T-DNA (data not shown). 

DsRed2 (Alt1)

Ms45 Genomic

zm-aa1

CaMV 35S Enhancer

Right Border Left Border

5126 Promoter

Ltp2 Promoter

Pg47 Promoter

In2-1 Terminator

pinII Terminator

zm-bt1 Transit Peptide

EcoR I

Bmt I

BamH I ~3800 bp

>8600 bp

2815 bp 2502 bp 2019 bp

7216 bp 2462 bp

~4600 bp

>8600 bp

BamH I/Hind III 1277 bp 2815 bp ~2600 bp 2502 bp 2019 bp

~1500 bp

PHP24597 T-DNA

Bmt I

Hind III

EcoR I

EcoR I

EcoR I

BamH I

BamH I

BamH I BamH I

Bmt I Bmt I BamH I BamH I

BamH I

Hind III
Pioneer Hi-Bred Page 49 of 188 

32138 SPT Maintainer No CBI 

Figure 18. Genetic Backgrounds of Generations Used for 32138 SPT Maintainer 

Southern Analysis

Breeding diagram with the three generations used for Southern analysis shown 

in black font. The maize genetic backgrounds of each generation are further 

explained (gray boxes with arrows) to understand the degree of hybridization 

pattern uniformity between individuals. In several cases, depending on the 

probes used, differences were observed between individuals of the T1S1 

generation because of the segregating genetic backgrounds. 

T0 

T1

BC0

BC1

Cross 

705

Backcross

705

BC2

BC3

BC4

T1S1

BC1S1

BC3S1

BC4S1

BC2S1

BC1S2

Tester

BC3S3

BC3S2F1

(Recipient 

Line is 

Hi-II(ms45))

BC3S2

Cross with 

BC3S4





 

   



Backcross

705

Backcross

705

Backcross

705

BC3S5



Segregation of 705 and Hi-II genetic 

backgrounds in T1S1; Differences in 

endogenous genetic background 

present between individual 32138 

maize plants

Increase in 705 genetic background 

with each round of backcrossing in 

BC3 and BC4; Nearly uniform 705 

endogenous genetic background 

between individual 32138 maize plants
Pioneer Hi-Bred Page 50 of 188 

32138 SPT Maintainer No CBI 

V-B. Characterization of 32138 SPT Maintainer: Copy Number and Insertion 

Integrity 

The integration pattern of the insertion in 32138 SPT maintainer was investigated with Bmt I 

digestion to determine copy number and with EcoR I digestion to determine insertion 

integrity. Southern blots containing samples from the T1S1 generation of 32138 SPT 

maintainer were hybridized with several probes to confirm copy number and integrity of each 

genetic element. The 5126 promoter and Ms45 probes were used to characterize the Ms45

cassette (Table 3, Figure 16). The Pg47 promoter, zm-bt1, zm-aa1, and In2-1 terminator 

probes were used to characterize the zm-aa1 cassette (Table 3, Figure 16). Probes to the 

35S enhancer, Ltp2 promoter, DsRed2(Alt1), and pinII terminator elements were used to 

characterize the DsRed2(Alt1) cassette (Table 3, Figure 16). 

Some of the probes used to characterize the 32138 SPT insertion are highly homologous to 

sequences found in the maize genome. The 5126 promoter, Ms45, Pg47 promoter, zm-bt1, 

zm-aa1, and In2-1 terminator probes all hybridized to sequences in both control and 32138 

SPT maintainer genomic DNA in addition to the PHP24597 T-DNA. These bands are 

identified by their presence in inbred control maize samples and are thus not part of the TDNA insertion. They are indicated in Tables 4 and 5 by asterisks (*) and gray shading. As 

expected, based on the breeding diagram presented in Figure 18, the hybridization pattern 

due to the maize background varied between individual plants of the T1S1 generation of 

32138 SPT maintainer depending on the probe used. In addition, differences with some 

probes were also detected in inbred control maize lines. Therefore, in order to ensure correct 

identification of bands due to the maize background and not due to the 32138 SPT insertion, 

all appropriate inbred control maize lines (inbred 705, Hi-II, and Hi-II (ms45) were included on 

these blots). The bands indicated as endogenous in Tables 4 and 5 were identified by their 

presence in at least one of the maize control plants. 

Based on the Southern blot analyses as discussed below, it was determined that a single, 

intact PHP24597 T-DNA has been inserted into the genome of 32138 SPT maintainer as 

diagramed on the insertion map (Figure 17). 

V-B.1. Copy Number 

Copy number of the integrated elements in 32138 SPT maintainer was investigated using 

Bmt I digestion, as there is a single restriction enzyme site in the PHP24597 T-DNA at base 

pair (bp) position 8503 (Figure 16). Any additional Bmt I sites would fall in the flanking 

genome regions outside the T-DNA confirming the number of copies of the elements. 

Hybridization with the probes from each cassette, except for the Ltp2 promoter probe, would 

indicate the number of copies of each element found in 32138 SPT maintainer based on the 

number of hybridizing bands (e.g., one hybridizing band indicates one copy of the element). 

The Bmt I site is located within the Ltp2 promoter element, so two hybridizing bands would be 

expected with this probe for a single T-DNA insertion. Predicted and observed fragment 

sizes for 32138 SPT maintainer with Bmt I are provided in Table 4. The Bmt I Southern blot 

analysis confirmed a single copy of each of the elements and the expected arrangement of 

these elements in the 32138 SPT insertion. 

Ms45 Cassette:

A single copy of the Ms45 cassette from PHP24597 was inserted into 32138 SPT maintainer. 

The 5126 promoter and Ms45 probes were hybridized to Bmt I-digested genomic DNA from 

individual 32138 SPT maintainer plants of the T1S1 generation (Table 4, Figure 19). Both of 

the probes hybridized to the same single fragment of greater than 8600 bp (Table 4, Figure 

19), indicating a single copy insertion with the expected arrangement of genetic elements on 

the inserted fragment in 32138 SPT maintainer. The 5126 promoter and Ms45 probes are 
Pioneer Hi-Bred Page 51 of 188 

32138 SPT Maintainer No CBI 

homologous to elements endogenous to the maize genome and therefore each probe also 

hybridized to bands in inbred control maize samples. 

zm-aa1 Cassette:

Likewise, a single copy of the zm-aa1 cassette was inserted into 32138 SPT maintainer. The 

four genetic elements comprising this cassette - the Pg47 promoter, zm-bt1, zm-aa1, and

In2-1 terminator - were used as probes to determine number of copies inserted. The probes 

of this cassette are homologous to elements endogenous to the maize genome and therefore 

each probe also hybridized to bands in inbred control maize samples. In the case of the 

Pg47 promoter probe, the 32138-specific band co-migrated with a strong endogenous band 

and is not visible as a separate band (Figure 20). Each of the zm-bt1, zm-aa1, and In2-1 

terminator probes hybridized to the same single fragment of greater than 8600 bp (Table 4, 

Figures 20 and 21) as seen with the 5126 promoter and Ms45 probes (Table 4, Figure 19), 

indicating a single copy insertion with the expected arrangement of these genetic elements 

on the inserted fragment in 32138 SPT maintainer. The Pg47 promoter element is likely 

present as a single copy, based on the presence of the same band of greater than 8600 bp 

for both the Ms45 and zm-bt1 elements that flank the Pg47 promoter in the PHP24597 

T-DNA (Table 4, Figures 19 and 20). A single, intact copy of the Pg47 promoter element with 

the expected arrangement was confirmed by sequencing of the 32138 SPT insertion. These 

hybridizations and sequence data confirm that a single copy of the zm-aa1 cassette was 

inserted in 32138 SPT maintainer in the expected arrangement on the T-DNA. 

DsRed2(Alt1) Cassette:

Similar to the data on the other cassettes, a single copy of the DsRed2(Alt1) cassette was 

inserted into 32138 SPT maintainer. The four elements comprising this cassette – the 35S 

enhancer, Ltp2 promoter, DsRed2(Alt1) gene and pinII terminator - were hybridized to Bmt Idigested DNA. The 35S enhancer, DsRed2(Alt1) gene and pinII terminator probes each 

hybridized to a single fragment of greater than 8600 bp (Table 4, Figures 22 and 23), 

indicating a single copy insertion while the Ltp2 promoter probe hybridized to two fragments, 

both greater than 8600 bp (Table 4, Figure 22), also confirming a single copy insertion. The 

presence of two bands hybridizing to the Ltp2 promoter probe is expected due to the location 

of the Bmt I restriction site within the Ltp2 promoter element, and also indicates the presence 

of only one copy of the inserted T-DNA. Comparison of the two bands on the Ltp2 promoter 

blot with the other probes of this cassette indicates that the lower band with the Ltp2

promoter probe is the same band associated with the 35S enhancer element and the upper 

band is the same associated with the DsRed2(Alt1) gene and pinII terminator (Figures 22 and 

23). These hybridizations confirm the DsRed2(Alt1) cassette was inserted as a single copy in 

the expected arrangement on the T-DNA. 

This Southern blot analysis demonstrates that the 32138 SPT insertion contains a single 

copy of each of the elements and that each of the cassettes inserted as expected based on 

the map of the PHP24597 T-DNA. A final map of the insertion in 32138 SPT maintainer 

based on the analysis with Bmt I is presented in Figure 17.
Pioneer Hi-Bred Page 52 of 188 

32138 SPT Maintainer No CBI 

Table 4. Predicted and Observed Hybridizing Bands on Southern Blots 

 with Bmt I Restriction Enzyme Digest 

Probe Restriction 

Enzyme Figure

Predicted Fragment 

Size from PHP24597

T-DNA1

 (bp) 

Predicted 

Fragment Size 

from Plasmid 

PHP245972

 (bp)

Observed Fragment 

Size in 32138 SPT 

maintainer 3 (bp) 

>8600 

5126 promoter Bmt I 19 >8500 (border)4 15455 ~7800* 

~2700* 

>8600 

Ms45 Bmt I 19 >8500 (border) 15455 ~5200* 

Obscured by 

endogenous bands 

Pg47 promoter Bmt I 20 >8500 (border) 15455 3 bands >8600* 

2 bands 6100 – 8600*

2 bands 4900 – 6100*

2 bands 3600 – 4900*

>8600 

zm-bt1 Bmt I 20 >8500 (border) 15455 ~4200* 

>8600 

zm-aa1 Bmt I 21 >8500 (border) 15455 

3 bands >8600* 

3 bands 6100 – 8600*

2 bands 4900 – 6100*

2 bands 3600 – 4900*

~3000* 

~2700* 

~2500*5

~1500* 

>8600 

In2-1 

terminator Bmt I 21 >8500 (border) 15455 

>8600* 

~8000* 

~4700* 

~3200* 

35S enhancer Bmt I 22 >8500 (border) 15455 >8600 

Ltp2 promoter Bmt I 22 >8500 (border) 

>1400 (border) 

15455 

27657 

>8600 

>8600 

DsRed2(Alt1) Bmt I 23 >1400 (border) 27657 >8600 

pinII terminator Bmt I 23 >1400 (border) 27657 >8600 

Note: An asterisk (*) and gray shading indicates the designated band is due to probe hybridization to endogenous 

maize genome sequences, as determined by the presence of the same band in both 32138 SPT maintainer and 

inbred control maize lanes. Certain endogenous bands may be difficult to discern on a printed copy but are visible 

on the original film. Not all endogenous bands are the same in all samples due to genomic differences in varieties 

used in the breeding process. 

Footnotes: 

1. Predicted fragment sizes for 32138 SPT maintainer are based on the map of the PHP24597 T-DNA (Figure 16). 

2. Predicted fragment sizes for hybridization to samples containing the plasmid positive control are based on the 

PHP24597 plasmid map as shown in Figure 15. 

3. Observed fragment sizes are considered approximate from these analyses and are based on the indicated 

sizes of the DIG-labeled DNA Molecular Weight Marker VII fragments on the Southern blots. Due to 

incorporation of DIG molecules for visualization, the marker fragments typically run approximately 5-10% 

larger than their actual indicated molecular weight. The sizes of fragments not corresponding directly to 

plasmid fragments are rounded to the nearest 100 bp. 

4. Minimum fragment size predicted based on an intact insertion of the T-DNA from PHP24597 (Figure 16). 

Fragment size is rounded to the nearest 100 bp. 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. 

5. The ~2500 bp endogenous band is seen in only one of the DP-32138-1 plants and the Hi-II control plants. 
Pioneer Hi-Bred Page 53 of 188 

32138 SPT Maintainer No CBI 

Figure 19. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

Bmt I Digested DNA with 5126 Promoter and Ms45 Probes 

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5126 Promoter Ms45

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer (T1S1 

generation) and of inbred control maize (inbred 705, Hi-II, and Hi-II (ms45)) was digested with 

Bmt I and hybridized to the 5126 promoter and Ms45 probes. Probes used are indicated 

below each panel. Approximately 4 g of genomic DNA was digested and loaded per lane. 

Positive control lanes included plasmid PHP24597 at approximately one gene copy and 4 μg 

of inbred control maize (inbred 705) DNA. Sizes of the DIGVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb). An arrow indicates the 32138-

specific band. 

Note: The hybridization banding pattern is different between the two Hi-II plants with the 

5126 promoter probe (Lanes 4 and 5) due to variability of the maize genetic background in 

this control. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II) 15 Control (Hi-II) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 Control (Hi-II(ms45)) 17 Control (Hi-II(ms45)) 

8 32138 SPT maintainer / plant 4 18 32138 SPT maintainer / plant 8 

9 32138 SPT maintainer / plant 5 19 32138 SPT maintainer / plant 9 

10 32138 SPT maintainer / plant 6 20 32138 SPT maintainer / plant 10 
Pioneer Hi-Bred Page 54 of 188 

32138 SPT Maintainer No CBI 

Figure 20. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

Bmt I Digested DNA with Pg47 Promoter and zm-bt1 Probes 

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Pg47 Promoter zm-bt1

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer 

(T1S1 generation) and of inbred control maize (inbred 705, Hi-II, and Hi-II(ms45)) was 

digested with Bmt I and hybridized to the Pg47 promoter and zm-bt1 probes. Probes used 

are indicated below each panel. Approximately 4 g of genomic DNA was digested and 

loaded per lane. Positive control lanes included plasmid PHP24597 at approximately one 

gene copy and 4 μg of inbred control maize (inbred 705) DNA. Sizes of the DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). An arrow 

indicates the 32138-specific band. 

Note: The arrow in the Pg47 promoter panel indicates the putative location of the 32138-

specific band based on hybridization with flanking element probes (Figure 19, Ms45 and 

Figure 20 zm-bt1), but the band itself is obscured by the strong endogenous band of the 

same size. Sequence data confirmed a single copy of this element as described in Section 

V-B1. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II) 15 Control (Hi-II(ms45)) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 Control (Hi-II(ms45)) 17 32138 SPT maintainer / plant 1 

8 32138 SPT maintainer / plant 8 18 32138 SPT maintainer / plant 2 

9 32138 SPT maintainer / plant 9 19 32138 SPT maintainer / plant 3 

10 32138 SPT maintainer / plant 10 20 DIGVII marker 
Pioneer Hi-Bred Page 55 of 188 

32138 SPT Maintainer No CBI 

Figure 21. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

Bmt I Digested DNA with zm-aa1 and In2-1 Terminator Probes

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zm-aa1 In2-1 Terminator

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer (T1S1 

generation) and of inbred control maize (inbred 705, Hi-II, and Hi-II(ms45)) was digested with 

Bmt I and hybridized to the zm-aa1 and In2-1 terminator probes. Probes used are indicated 

below each panel. Approximately 4 g of genomic DNA was digested and loaded per lane. 

Positive control lanes included plasmid PHP24597 at approximately one gene copy and 4 μg 

of inbred control maize (inbred 705) DNA. Sizes of the DIGVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb). An arrow indicates the 32138-

specific band. 

Note: The hybridization pattern is different between individuals of the Hi-II(ms45) maize 

(Lanes 6 and 7) and 32138 SPT maintainer (Lanes 8 through 10) with the zm-aa1 probe due 

to variability in the maize genetic background. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II) 15 Control (Hi-II(ms45)) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 Control (Hi-II(ms45)) 17 32138 SPT maintainer / plant 1 

8 32138 SPT maintainer / plant 8 18 32138 SPT maintainer / plant 2 

9 32138 SPT maintainer / plant 9 19 32138 SPT maintainer / plant 3 

10 32138 SPT maintainer / plant 10 20 DIGVII marker 
Pioneer Hi-Bred Page 56 of 188 

32138 SPT Maintainer No CBI 

Figure 22. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

Bmt I Digested DNA with 35S Enhancer and Ltp2 Promoter Probes 

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35S Enhancer Ltp2 Promoter

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer (T1S1 

generation) and of inbred control maize (inbred 705, Hi-II, and Hi-II(ms45)) was digested with 

Bmt I and hybridized to the 35S enhancer and Ltp2 promoter probes. Probes used are 

indicated below each panel. Approximately 4 g of genomic DNA was digested and loaded 

per lane. Positive control lanes included plasmid PHP24597 at approximately one gene copy 

and 4 μg of inbred control maize (inbred 705) DNA. Sizes of the DIGVII molecular weight 

markers are indicated adjacent to the blot image in kilobase pairs (kb). 

An arrow indicates the 32138-specific band in the 35S Enhancer panel. Two arrows indicate 

the 32138-specific bands in the Ltp2 promoter panel. 

Note: In Lanes 1 and 11, the PHP24597 plasmid band is faint and migrates above the 8.6 kb 

marker. This band is observed on the original X-ray film. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II(ms45)) 15 Control (Hi-II) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 32138 SPT maintainer / plant 1 17 Control (Hi-II(ms45)) 

8 32138 SPT maintainer / plant 2 18 32138 SPT maintainer / plant 4 

9 32138 SPT maintainer / plant 3 19 32138 SPT maintainer / plant 5 

10 DIGVII marker 20 32138 SPT maintainer / plant 6 
Pioneer Hi-Bred Page 57 of 188 

32138 SPT Maintainer No CBI 

Figure 23. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer 

Bmt I Digested DNA with DsRed2(Alt1) and pinII Terminator Probes 

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DsRed2 pinII Terminator

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer 

(T1S1 generation) and of inbred control maize (inbred 705, Hi-II, and Hi-II(ms45)) was 

digested with Bmt I and hybridized to the DsRed2(Alt1) and pinII terminator probes. Probes 

used are indicated below each panel. Approximately 4 g of genomic DNA was digested and 

loaded per lane. Positive control lanes included plasmid PHP24597 at approximately one 

gene copy and 4 μg of inbred control maize (inbred 705) DNA. Sizes of the DIGVII molecular 

weight markers are indicated adjacent to the blot image in kilobase pairs (kb). An arrow 

indicates the 32138-specific band. 

Note: In Lanes 1 and 11, the PHP24597 plasmid band is faint and migrates above the 8.6 kb 

marker. This band is observed on the original X-ray film. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II(ms45)) 15 Control (Hi-II(ms45)) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 32138 SPT maintainer / plant 1 17 32138 SPT maintainer / plant 1 

8 32138 SPT maintainer / plant 2 18 32138 SPT maintainer / plant 2 

9 32138 SPT maintainer / plant 3 19 32138 SPT maintainer / plant 3 

10 DIGVII marker 20 DIGVII marker 
Pioneer Hi-Bred Page 58 of 188 

32138 SPT Maintainer No CBI 

V-B.2. Insertion Integrity 

EcoR I digestion was used to verify that the inserted T-DNA containing the Ms45, zm-aa1,

and DsRed2(Alt1) cassettes was complete and intact in maize 32138 SPT maintainer. There 

are three EcoR I sites in the PHP24597 T-DNA (base pair positions 191, 7407, and 9869; 

Figure 16). Two internal fragments, one of 7216 bp and one of 2462 bp, would be expected 

to hybridize to each of the probes and would confirm the integrity of all three cassettes in the 

insertion. The probes of the Ms45 and zm-aa1 cassettes would be expected to hybridize to 

the 7216 bp band, as these cassettes are contained on this internal fragment. The probes of 

the DsRed2(Alt1) cassette would be expected to hybridize to the internal 2462 bp band 

containing this cassette. Expected and observed fragment sizes with EcoR I are given in 

Table 5. This analysis described below confirmed that the T-DNA was inserted intact and as 

expected in 32138 SPT maintainer. 

As stated previously, the 5126 promoter, Ms45, Pg47 promoter, zm-bt1, zm-aa1, and In2-1 

terminator probes are homologous to elements endogenous to the maize genome and 

therefore each probe hybridized to bands in inbred control maize samples. These hybridizing 

bands from the endogenous maize genome are indicated in Table 5 by asterisks (*) and gray 

shading. 

The 5126 promoter and Ms45 probes for the Ms45 cassette hybridized to a single insertderived band of 7216 bp that matched the plasmid control band (Table 5, Figure 24). 

Similarly, three of the probes for the zm-aa1 cassette (Pg47 promoter, zm-bt1, and In2-1 

terminator) hybridized to the same internal 7216 bp band (Table 5, Figures 25 and 26). The 

zm-aa1 probe hybridized to an endogenous band of a similar size as the 7216 bp internal 

band, thus obscuring the ability to determine if the 32138-specific band was present on the 

Southern blot (Figure 26). The zm-aa1 cassette is likely intact based on the presence of the 

7216 bp band for the two probes flanking the zm-aa1 gene (zm-bt1 and In2-1 terminator) 

(Figures 25 and 26). As further support of the integrity of the zm-aa1 cassette, an intact copy 

was confirmed by sequencing of the 32138 SPT insertion (data not shown). The probes for 

the DsRed2(Alt1) cassette (35S enhancer, Ltp2 promoter, DsRed2(Alt1), and pinII terminator) 

hybridized to the expected internal band of 2462 bp (Table 5, Figures 27 and 28). The size of 

the band for each probe was confirmed by hybridization to the PHP24597 plasmid fragment 

corresponding to the T-DNA (Lanes 1 and 11, Figures 24 through 28). In some cases, the 

hybridization to the plasmid fragment is not clearly visible on a printed copy but can be seen 

on the film exposure of the Southern blot. Because these probes hybridized to the internal 

fragments of the predicted sizes, the insertion in 32138 SPT maintainer was determined to be 

intact and as expected based on the PHP24597 T-DNA map. A schematic map of the 32138 

SPT insertion based on this analysis is presented in Figure 17. 
Pioneer Hi-Bred Page 59 of 188 

32138 SPT Maintainer No CBI 

Table 5. Predicted and Observed Hybridizing Bands on Southern Blots with EcoR I 

Restriction Enzyme Digest 

Probe Restriction 

Enzyme Figure 

Predicted 

Fragment Size 

from 

PHP245971 (bp)

Predicted 

Fragment 

Size from 

PHP24597 

T-DNA2 (bp)

Observed Fragment Size in 

Maize 32138 SPT Maintainer3 

(bp) 

72164 5126

promoter EcoR I 24 7216 7216 

2 bands >8600* 

72164

Ms45 EcoR I 24 7216 7216 

>8600* 

72164

Pg47

promoter EcoR I 25 7216 7216 3 bands >8600* 

~7400* 

~5600* 

72164

zm-bt1 EcoR I 25 7216 7216 >8600* 

~8600* 

~3300* 

Obscured by endogenous bands

zm-aa1 EcoR I 26 7216 7216 

2 bands >8600* 

2 bands 4900 – 8600* 

4 bands 3600 – 4900* 

~4300*5

2 bands 2800 – 3600* 

4 bands 1950 – 2800* 

~1700* 

72164

In2-1 

terminator EcoR I 26 7216 7216 

2 bands >8600* 

~8600* 

~7400* 

~3400* 

35S enhancer EcoR I 26 2462 2462 24624

Ltp2 promoter EcoR I 26 2462 2462 24624

DsRed2(Alt1) EcoR I 28 2462 2462 24624

pinII 

terminator EcoR I 28 2462 2462 24624

Note: An asterisk (*) and gray shading indicates the designated band is due to probe hybridization to endogenous 

maize genome sequences, as determined by the presence of the same band in both 32138 and inbred control maize 

lanes. Certain endogenous bands may be difficult to discern on a printed copy but are visible on the original film. 

Not all endogenous bands are the same in all samples due to genomic differences in varieties used in the breeding 

process. 

Footnotes: 

1. Predicted fragment sizes for hybridization to samples containing the plasmid positive control are based on the 

PHP24597 plasmid map as shown in Figure 15. 

2. Predicted fragment sizes for maize 32138 SPT maintainer are based on the map of the PHP24597 T-DNA as 

shown in Figure 16. 

3. Observed fragment sizes are considered approximate from these analyses and are based on the indicated sizes 

of the DIG-labeled DNA Molecular Weight Marker VII fragments on the Southern blots. Due to incorporation of 

DIG molecules for visualization, the marker fragments typically run approximately 5-10% larger than their actual 

indicated molecular weight. The sizes of fragments not corresponding directly to plasmid fragments are rounded 

to the nearest 100 bp. 

4. Observed fragment size is the same as the predicted fragment size based on equivalent migration on the 

Southern blots. 

5. The ~4300 bp endogenous band is seen in only one of the DP-32138-1 plants and one of the Hi-II control plants. 
Pioneer Hi-Bred Page 60 of 188 

32138 SPT Maintainer No CBI 

Figure 24. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

EcoR I Digested DNA with 5126 Promoter and Ms45 Probes 

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5126 Promoter Ms45

Genomic DNA isolated from leaf tissue from individual plants of maize SPT maintainer (T1S1 

generation) and of inbred control maize (inbred 705, Hi-II, and Hi-II(ms45)) was digested with 

EcoR I and hybridized to the 5126 promoter and Ms45 probes. Probes used are indicated 

below each panel. Approximately 4 g of genomic DNA was digested and loaded per lane. 

Positive control lanes included plasmid PHP24597 at approximately one gene copy and 4 μg 

of control maize (inbred 705) DNA. Sizes of the DIGVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb). An arrow indicates the 32138-

specific band. 

Note: The hybridization banding pattern is different between the two Hi-II(ms45) plants with 

the Ms45 probe (Lanes 16 and 17) due to variability of the maize genetic background in this 

control. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II) 15 Control (Hi-II) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 Control (Hi-II(ms45)) 17 Control (Hi-II(ms45)) 

8 32138 SPT maintainer / plant 4 18 32138 SPT maintainer / plant 8 

9 32138 SPT maintainer / plant 5 19 32138 SPT maintainer / plant 9 

10 32138 SPT maintainer / plant 6 20 32138 SPT maintainer / plant 10 
Pioneer Hi-Bred Page 61 of 188 

32138 SPT Maintainer No CBI 

Figure 25. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

EcoR I Digested DNA with Pg47 Promoter and zm-bt1 Probes 

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Pg47 Promoter zm-bt1

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer (T1S1 

generation) and of control maize (inbred 705, Hi-II, and Hi-II(ms45)) was digested with EcoR I 

and hybridized to the Pg47 promoter and zm-bt1 probes. Probes used are indicated below 

each panel. Approximately 4 g of genomic DNA was digested and loaded per lane. 

Positive control lanes included plasmid PHP24597 at approximately one gene copy and 4 μg 

of control maize (inbred 705) DNA. Sizes of the DIGVII molecular weight markers are 

indicated adjacent to the blot image in kilobase pairs (kb). An arrow indicates the 32138-

specific band. 

Note: In Lane 1 and 11, the PHP24597 plasmid band is faint and migrates between the 6.1 

and 7.4 kb markers. This band is observed on the original X-ray film. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II) 15 Control (Hi-II(ms45)) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 Control (Hi-II(ms45)) 17 32138 SPT maintainer / plant 1 

8 32138 SPT maintainer / plant 8 18 32138 SPT maintainer / plant 2 

9 32138 SPT maintainer / plant 9 19 32138 SPT maintainer / plant 3 

10 32138 SPT maintainer / plant 10 20 DIGVII marker 
Pioneer Hi-Bred Page 62 of 188 

32138 SPT Maintainer No CBI 

Figure 26. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

EcoR I Digested DNA with zm-aa1 and In2-1 Terminator Probes 

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zm-aa1 In2-1 Terminator

Genomic DNA isolated from leaf tissue from individual plants of 32138 maize (T1S1 

generation) and of control maize (705, Hi-II, and Hi-II(ms45)) was digested with EcoR I and 

hybridized to the zm-aa1 and In2-1 terminator probes. Probes used are indicated below each 

control lanes included plasmid PHP24597 at approximately one gene copy and 4 μg of 

control maize (705) DNA. Sizes of the DIG VII molecular weight markers are indicated 

adjacent to the blot image in kilobase pairs (kb). An arrow indicates the 32138-specific band. 

Note: The arrow in the zm-aa1 probe panel indicates the putative location of the 7216 bp 

internal band based on hybridization with flanking element probes (Figure 25, zm-bt1 and 

Figure 26, In2-1 terminator), but the band itself is obscured by strong endogenous bands of 

the same size. Sequence data confirmed an intact copy of the zm-aa1 cassette as described 

in Section V-B2. The hybridization pattern between the 32138 plants with the zm-aa1 probe 

is different due to variability in the maize genetic background (Lanes 8 through 10). 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II) 15 Control (Hi-II(ms45)) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 Control (Hi-II(ms45)) 17 32138 SPT maintainer / plant 1 

8 32138 SPT maintainer / plant 8 18 32138 SPT maintainer / plant 2 

9 32138 SPT maintainer / plant 9 19 32138 SPT maintainer / plant 3 

10 32138 SPT maintainer / plant 10 20 DIG VII marker 
Pioneer Hi-Bred Page 63 of 188 

32138 SPT Maintainer No CBI 

Figure 27. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

EcoR I Digested DNA with 35S Enhancer and Ltp2 Promoter Probes. 

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35S Enhancer Ltp2 Promoter

Genomic DNA isolated from leaf tissue from individual plants of 32138 maize (T1S1 

generation) and of control maize (705, Hi-II, and Hi-II(ms45)) was digested with EcoR I and 

hybridized to the 35S enhancer and Ltp2 promoter probes. Probes used are indicated below 

each panel. Approximately 4 g of genomic DNA was digested and loaded per lane. 

Positive control lanes included plasmid PHP24597 at approximately one gene copy and 4 μg 

of control maize (705) DNA. Sizes of the DIG VII molecular weight markers are indicated 

adjacent to the blot image in kilobase pairs (kb). An arrow indicates the 32138-specific band. 

Note: In Lane 1, the PHP24597 plasmid band is faint and migrates between the 1.95 and 2.8 

kb markers. This band is observed on the original X-ray film. 

Lane Sample Lane Sample 

1 control (705) + 1 copy of PHP24597 11 control (705) + 1 copy of PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 control (705) 13 control (705) 

4 control (Hi-II) 14 control (Hi-II) 

5 control (Hi-II(ms45)) 15 control (Hi-II) 

6 control (Hi-II(ms45)) 16 control (Hi-II(ms45)) 

7 32138 maize / plant 1 17 control (Hi-II(ms45)) 

8 32138 maize / plant 2 18 32138 maize / plant 4 

9 32138 maize / plant 3 19 32138 maize / plant 5 

10 DIG VII marker 20 32138 maize / plant 6 
Pioneer Hi-Bred Page 64 of 188 

32138 SPT Maintainer No CBI 

Figure 28. Southern Blot Analysis of the T1S1 Generation of 32138 SPT Maintainer, 

EcoR I Digested DNA with DsRed2(Alt1) and pinII Terminator Probes 

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DsRed2 pinII Terminator

Genomic DNA isolated from leaf tissue from individual plants of 32138 SPT maintainer 

(T1S1 generation) and of control maize (inbred 705, Hi-II, and Hi-II(ms45)) was digested with 

EcoR I and hybridized to the DsRed2(Alt1) and pinII terminator probes. Probes used are 

indicated below each panel. Approximately 4 g of genomic DNA was digested and loaded 

per lane. Positive control lanes included plasmid PHP24597 at approximately one gene copy 

and 4 μg of control maize (inbred 705) DNA. Sizes of the DIG VII molecular weight markers 

are indicated adjacent to the blot image in kilobase pairs (kb). 

An arrow indicates the 32138-specific band. 

Lane Sample Lane Sample 

1 Control (inbred 705) + 1 copy of 

PHP24597 11 Control (inbred 705) + 1 copy of 

PHP24597 

2 DIGVII marker 12 DIGVII marker 

3 Control (inbred 705) 13 Control (inbred 705) 

4 Control (Hi-II) 14 Control (Hi-II) 

5 Control (Hi-II(ms45)) 15 Control (Hi-II(ms45)) 

6 Control (Hi-II(ms45)) 16 Control (Hi-II(ms45)) 

7 32138 SPT maintainer / plant 1 17 32138 SPT maintainer / plant 1 

8 32138 SPT maintainer / plant 2 18 32138 SPT maintainer / plant 2 

9 32138 SPT maintainer / plant 3 19 32138 SPT maintainer / plant 3 

10 DIG VII marker 20 DIG VII marker 
Pioneer Hi-Bred Page 65 of 188 

32138 SPT Maintainer No CBI 

V-C. Stability of the Insertion across Generations 

Stability of the 32138 SPT insertion was confirmed by using Southern blot analysis on three 

generations: T1S1, BC3, and a segregating BC4 generation. BamH I restriction digestion 

was selected for characterization of these generations; the sites are indicated on the maps of 

the PHP24597 plasmid and T-DNA (Figures 15 and 16, respectively). Four BamH I sites are 

located within the T-DNA of PHP24597 at base pair positions 1456, 4271, 6773, and 8792 

(Figure 16). This digestion provides a unique Southern hybridization pattern at the Right and 

Left Borders to confirm the stability of the insertion between the T1S1, BC3, and BC4 

generations. 

These hybridizations, as discussed below, confirmed the stability of the insertion in 32138 

SPT maintainer across these three generations. The Southern blot analysis for BamH I, 

described below, is presented in a schematic map of the 32138 SPT insertion (Figure 17). 

T1S1 and BC3 Generation Analysis

BamH I analysis was conducted on individual plants of the T1S1 and BC3 generations of 

32138 SPT maintainer and from the control maize lines. The Ms45, zm-aa1, and 

DsRed2(Alt1) gene probes were used to confirm the genetic stability across the two 

generations. Both the Ms45 and zm-aa1 probes are highly identical or similar to the genes 

found in the endogenous maize genome, and therefore hybridized to a number of bands of 

varying sizes in the control plant lines. These hybridizing endogenous bands were identified 

in 32138 SPT maintainer samples based on their presence in the control maize lines and are 

identified in Table 6 by an asterisk (*) and gray shading. 

A band of greater than 1500 bp and one of 2815 bp would be expected at the Right Border of 

the 32138 SPT insertion with the Ms45 probe and would confirm the stability of the Right 

Border flanking region (Table 6). All T1S1 and BC3 samples of 32138 SPT maintainer were 

consistent with the plasmid controls and showed the two bands attributed to the insertion at 

approximately 3800 bp and 2815 bp as expected (Figure 29). The 2815 bp band migrated 

equivalently with the plasmid control (Figure 29). This hybridization result confirms the 

stability of the Right Border flanking region of the 32138 SPT insertion across the two 

generations. 

The zm-aa1 probe would be expected to hybridize with two internal bands of 2019 bp and 

2502 bp, confirming sites internal to the T-DNA (Table 6). As expected, bands of 2019 bp 

and 2502 bp were observed and migrated equivalently with the plasmid control (Figure 30). 

These observed bands from the 32138 SPT insertion were consistent between the T1S1 and 

BC3 generations (Figure 30), confirming the stability of the T-DNA insertion. 

At the Left Border of the 32138 SPT insertion, a band of greater than 1200 bp would be 

expected with the DsRed2(Alt1) probe and would confirm the stability of the Left Border 

flanking region (Table 6). One band of approximately 4600 bp and a second faint band at 

approximately 6100 bp were visible in all samples of the 32138 SPT maintainer plants on the 

Southern blot (Figure 31). The second faint 6100 bp band was determined to be from 

partially blocked digestion at the BamH I site in the genomic DNA flanking the insertion, 

which may be due to methylation or secondary structure of DNA surrounding this site. In 

order to clarify the partial digestion of the BamH I site, a BamH I/Hind III double digestion was 

conducted and was hybridized with the DsRed2(Alt1) probe (Figure 32). A single band of 

approximately 2600 bp was observed in all T1S1 and BC3 samples of 32138 SPT maintainer. 

The presence of only one band in the double digestion (Figure 32) confirms the previous 

conclusions of a single insertion of the DsRed2(Alt1) gene in 32138 SPT maintainer. The 

BamH I and BamH I/Hind III Southern blots both showed consistent banding patterns for all 

samples of 32138 SPT maintainer from both generations, further confirming the conservation 
Pioneer Hi-Bred Page 66 of 188 

32138 SPT Maintainer No CBI 

of the Left Border of the T-DNA insertion and the equivalence of the insertion between the 

two generations analyzed. 

BC4 Generation Analysis

Similar hybridization patterns to the T1S1 and BC3 generations were also observed for the 

segregating BC4 generation. The BC4 generation was also analyzed using BamH I digestion 

and hybridization with the three gene probes, Ms45, zm-aa1, and DsRed2(Alt1). For those 

individual maize 32138 SPT maintainer plants that contained the 32138 SPT insertion, the 

Ms45 probe hybridized to a band of approximately 3800 bp and an internal band of 2815 bp 

due to the 32138 SPT insertion (Table 6, Figure 33) similar to the T1S1 and BC3 generations 

discussed above (Figure 29). As with the T1S1 and BC3 generations, the zm-aa1 probe also 

hybridized to the two expected internal bands of 2019 bp and 2502 bp due to the 32138 SPT 

insertion (Table 6, Figure 34). The DsRed2(Alt1) probe also hybridized to two bands, one at 

4600 bp and a faint one at 6100 bp (Table 6, Figure 35), which were also observed in the 

T1S1 and BC3 generations (Figure 31). These results indicated that the 32138 SPT insertion 

remained stable during the typical maize breeding process. 

Table 6. Predicted and Observed Hybridizing Bands on Southern Blots with BamH I 

and BamH I/Hind III Restriction Enzyme Digests 

Probe Restriction 

Enzyme(s) Figure

Predicted 

Fragment Size 

from 

PHP24597 T-DNA1

(bp) 

Predicted 

Fragment Size 

from Plasmid 

PHP245972

(bp) 

Observed Fragment 

Size 3 in 32138 SPT 

maintainer 

(bp) 

~3800 

28155

Ms45 BamH I 29, 33 >1500 (border) 4

2815 

2815 

8826 ~7000* 

~2700* 

25025

20195

zm-aa1 BamH I 30, 33 2019 

2502 

2019 

2502 multiple bands >2800*6

1-2 bands 1950-2800*6

3 bands <1950*6

DsRed2(Alt1) BamH I 30, 34 >1200 (border) 27225 ~6100 (faint) 

~4600 

DsRed2(Alt1) BamH I/ 

Hind III 32 >1200 (border) 26045 ~2600 

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 and are not due to the 32138 

SPT insertion. 

Footnotes: 

1. Predicted size for hybridization in genomic DNA samples is based on the map of the T-DNA from PHP24597 

(Figure 16). Border fragment sizes are rounded to the nearest 100 bp. 

2. Predicted size is based on the plasmid map of PHP24597 (Figure 15). 

3. 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. 

4. 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. 

5. Observed size is same as expected size due to equivalent migration to the plasmid band. 

6. Not all bands present across T1S1, BC3, and BC4 generations. These bands represent a summary of 

observations in these generations. 
Pioneer Hi-Bred Page 67 of 188 

32138 SPT Maintainer No CBI 

Figure 29. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: BamH I Digest and Ms45 Probe 

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Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with BamH I 

and hybridized to the Ms45 probe. Approximately 4 μg of genomic DNA was digested and 

loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 4 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). Arrows 

indicate the 32138-specific bands. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 DIGVII marker 12 32138 SPT maintainer BC3 (plant 22-Red) 

3 Inbred 705 control 13 32138 SPT maintainer BC3 (plant 23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (plant 24-Red) 

5 Hi-II(ms45) control 15 Hi-II control 

6 Hi-II control 16 Hi-II(ms45) control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Inbred 705 control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 DIGVII marker 

9 32138 SPT maintainer T1S1 (plant 6-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 7-Red) 
Pioneer Hi-Bred Page 68 of 188 

32138 SPT Maintainer No CBI 

Figure 30. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: BamH I Digest and zm-aa1 Probe 

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Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with BamH I 

and hybridized to the zm-aa1 probe. Approximately 4 μg of genomic DNA was digested and 

loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 4 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). Arrows 

indicate the 32138-specific bands. 

Note: The 2019 bp band expected in both plasmid control and 32138 SPT maintainer 

samples is faint, but is observed on the original X-ray film.

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 DIGVII marker 12 32138 SPT maintainer BC3 (plant 22-Red) 

3 Inbred 705 control 13 32138 SPT maintainer BC3 (plant 23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (plant 24-Red) 

5 Hi-II(ms45) control 15 Hi-II control 

6 Hi-II control 16 Hi-II(ms45) control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Inbred 705 control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 DIGVII marker 

9 32138 SPT maintainer T1S1 (plant 6-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 7-Red) 
Pioneer Hi-Bred Page 69 of 188 

32138 SPT Maintainer No CBI 

Figure 31. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: BamH I Digest and DsRed2(Alt1) Probe 

kb

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*

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*

Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with BamH I 

and hybridized to the DsRed2(Alt1) probe. Approximately 4 μg of genomic DNA was 

digested and loaded per lane. Positive control lanes include plasmid PHP24597 at the 

indicated approximate gene copy numbers and 4 μg of control maize DNA. Sizes of the DIG 

VII molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Note: In lanes 1 and 19, the PHP24597 plasmid band is faint and migrates above the 8.6 kb 

marker. This band is observed on the original X-ray film. An asterisk indicates the faint band 

observed at ~6100 bp. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 DIGVII marker 12 32138 SPT maintainer BC3 (T22-Red) 

3 Inbred 705 control 13 32138 SPT maintainer BC3 (T23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (T24-Red) 

5 Hi-II(ms45) control 15 Hi-II control 

6 Hi-II control 16 Hi-II(ms45) control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Inbred 705 control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 DIGVII marker 

9 32138 SPT maintainer T1S1 (plant 5-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 6-Red) 
Pioneer Hi-Bred Page 70 of 188 

32138 SPT Maintainer No CBI 

Figure 32. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: BamH I/Hind III Digest and DsRed2(Alt1) Probe 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with BamH 

I/Hind III and hybridized to the DsRed2(Alt1) probe. Approximately 4 μg of genomic DNA 

was digested and loaded per lane. Positive control lanes include plasmid PHP24597 at the 

indicated approximate gene copy numbers and 4 μg of control maize DNA. Sizes of the DIG 

VII molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (T21-Red) 

2 DIGVII marker 12 32138 SPT maintainer BC3 (T22-Red) 

3 Inbred 705 control 13 32138 SPT maintainer BC3 (T23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (T24-Red) 

5 Hi-II(ms45) control 15 Hi-II control 

6 Hi-II control 16 Hi-II(ms45) control 

7 32138 SPT maintainer T1S1 (T3-Red) 17 Inbred 705 control 

8 32138 SPT maintainer T1S1 (T4-Red) 18 DIGVII marker 

9 32138 SPT maintainer T1S1 (T5-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (T6-Red) 
Pioneer Hi-Bred Page 71 of 188 

32138 SPT Maintainer No CBI 

Figure 33. Southern Blot Analysis of the BC4 Generation of 32138 SPT Maintainer: 

BamH I Digest and Ms45 Probe 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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Genomic DNA isolated from leaf tissue of individual plants of the BC4 generation of 32138 

SPT maintainer and of the controls was digested with BamH I and hybridized to the Ms45

probe. The BC4 generation was segregating for the 32138 SPT insertion. Approximately 5 

μg of genomic DNA was digested and loaded per lane. Positive control lanes include plasmid 

PHP24597 at the indicated approximate gene copy numbers and 5 μg of control maize DNA. 

Sizes of the DIG VII molecular weight markers are indicated adjacent to the blot image in 

kilobases (kb). Arrows indicate the 32138-specific bands. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Hi-II(ms45) control 16 32138 SPT maintainer BC4 / plant 71 

2 1 copy PHP24597 + Hi-II(ms45) control 17 32138 SPT maintainer BC4 / plant 72 

3 DIGVII marker 18 32138 SPT maintainer BC4 / plant 73 

4 Hi-II(ms45) control 19 32138 SPT maintainer BC4 / plant 74 

5 Inbred 705 control 20 32138 SPT maintainer BC4 / plant 75 

6 blank 21 32138 SPT maintainer BC4 / plant 76 

7 32138 SPT maintainer BC4 / plant 61 22 32138 SPT maintainer BC4 / plant 77 

8 32138 SPT maintainer BC4 / plant 62 23 32138 SPT maintainer BC4 / plant 78 

9 32138 SPT maintainer BC4 / plant 63 24 32138 SPT maintainer BC4 / plant 79 

10 32138 SPT maintainer BC4 / plant 65 25 32138 SPT maintainer BC4 / plant 80 

11 32138 SPT maintainer BC4 / plant 66 26 Hi-II control 

12 32138 SPT maintainer BC4 / plant 67 27 Hi-II control 

13 32138 SPT maintainer BC4 / plant 68 28 DIGVII marker 

14 32138 SPT maintainer BC4 / plant 69 29 1 copy PHP24597 + Inbred 705 control 

15 32138 SPT maintainer BC4 / plant 70 30 3 copy PHP24597 + Inbred 705 control 
Pioneer Hi-Bred Page 72 of 188 

32138 SPT Maintainer No CBI 

Figure 34. Southern Blot Analysis of the BC4 Generation of 32138 SPT Maintainer: 

BamH I Digest and zm-aa1 Probe 

Genomic DNA isolated from leaf tissue of individual plants of the BC4 generation of 32138 

SPT maintainer and of the controls was digested with BamH I and hybridized to the zm-aa1 

probe. The BC4 generation was segregating for the 32138 SPT insertion. Approximately 5 

μg of genomic DNA was digested and loaded per lane. Positive control lanes include plasmid 

PHP24597 at the indicated approximate gene copy numbers and 5 μg of control maize DNA. 

Sizes of the DIG VII molecular weight markers are indicated adjacent to the blot image in 

kilobases (kb). Arrows indicate the 32138-specific bands. 

Note: The 2019 bp band expected in both plasmid control and 32138 SPT maintainer 

samples is faint in this figure, but was observed on the original X-ray film. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Hi-II(ms45) control 16 32138 SPT maintainer BC4 / plant 71 

2 1 copy PHP24597 + Hi-II(ms45) control 17 32138 SPT maintainer BC4 / plant 72 

3 DIGVII marker 18 32138 SPT maintainer BC4 / plant 73 

4 Hi-II(ms45) control 19 32138 SPT maintainer BC4 / plant 74 

5 Inbred 705 control 20 32138 SPT maintainer BC4 / plant 75 

6 Blank 21 32138 SPT maintainer BC4 / plant 76 

7 32138 SPT maintainer BC4 / plant 61 22 32138 SPT maintainer BC4 / plant 77 

8 32138 SPT maintainer BC4 / plant 62 23 32138 SPT maintainer BC4 / plant 78 

9 32138 SPT maintainer BC4 / plant 63 24 32138 SPT maintainer BC4 / plant 79 

10 32138 SPT maintainer BC4 / plant 65 25 32138 SPT maintainer BC4 / plant 80 

11 32138 SPT maintainer BC4 / plant 66 26 Hi-II control 

12 32138 SPT maintainer BC4 / plant 67 27 Hi-II control 

13 32138 SPT maintainer BC4 / plant 68 28 DIGVII marker 

14 32138 SPT maintainer BC4 / plant 69 29 1 copy PHP24597 + Inbred 705 control 

15 32138 SPT maintainer BC4 / plant 70 30 3 copy PHP24597 + Inbred 705 control 
Pioneer Hi-Bred Page 73 of 188 

32138 SPT Maintainer No CBI 

Figure 35. Southern Blot Analysis of the BC4 Generation of 32138 SPT Maintainer: 

BamH I Digest and DsRed2(Alt1) Probe 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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*

Genomic DNA isolated from leaf tissue of individual plants of the BC4 generation of 32138 

SPT maintainer and of the controls was digested with BamH I and hybridized to the 

DsRed2(Alt1) probe. The BC4 generation was segregating for the 32138 SPT insertion. 

Approximately 5 μg of genomic DNA was digested and loaded per lane. Positive control 

lanes include plasmid PHP24597 at the indicated approximate gene copy numbers and 5 μg 

of control maize DNA. Sizes of the DIG VII molecular weight markers are indicated adjacent 

to the blot image in kilobases (kb). 

Note: An asterisk indicates the faint band observed at ~6100 bp. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Hi-II(ms45) control 16 32138 SPT maintainer BC4 / plant 71 

2 1 copy PHP24597 + Hi-II(ms45) control 32138 SPT maintainer BC4 / plant 72 

3 DIGVII marker 18 32138 SPT maintainer BC4 / plant 73 

4 Hi-II(ms45) control 19 32138 SPT maintainer BC4 / plant 74 

5 Inbred 705 control 20 32138 SPT maintainer BC4 / plant 75 

6 Blank 21 32138 SPT maintainer BC4 / plant 76 

7 32138 SPT maintainer BC4 / plant 61 22 32138 SPT maintainer BC4 / plant 77 

8 32138 SPT maintainer BC4 / plant 62 23 32138 SPT maintainer BC4 / plant 78 

9 32138 SPT maintainer BC4 / plant 63 24 32138 SPT maintainer BC4 / plant 79 

10 32138 SPT maintainer BC4 / plant 65 25 32138 SPT maintainer BC4 / plant 80 

11 32138 SPT maintainer BC4 / plant 66 26 Hi-II control 

12 32138 SPT maintainer BC4 / plant 67 27 Hi-II control 

13 32138 SPT maintainer BC4 / plant 68 28 DIGVII marker 

14 32138 SPT maintainer BC4 / plant 69 29 1 copy PHP24597 + Inbred 705 control 

15 32138 SPT maintainer BC4 / plant 70 30 3 copy PHP24597 + Inbred 705 control 
Pioneer Hi-Bred Page 74 of 188 

32138 SPT Maintainer No CBI 

V-D. Plasmid Backbone DNA Analysis 

Both the T1S1 and BC3 generations of 32138 SPT maintainer were analyzed by Southern 

blot analysis for plasmid sequences from the PHP24597 plasmid backbone. Probes for 

genes and regions on the PHP24597 plasmid backbone, i.e. outside of the T-DNA region 

were used to determine if any plasmid backbone was inserted into 32138 SPT maintainer as 

a result of transformation. The spc, tet, and virG probes hybridize to the spectinomycin 

resistance, tetracycline resistance, and virG genes, respectively (Table 3, Figure 15). The LB 

probe hybridizes to the region outside the Left T-DNA Border, and the RB probe hybridizes to 

the region outside the Right Border of the T-DNA of PHP24597 (Table 3, Figure 15). These 

probes would confirm if any region of the backbone outside the T-DNA was transferred into 

32138 SPT maintainer. Based on the analysis as described below, 32138 SPT maintainer 

does not contain plasmid backbone sequences from PHP24597. 

Genomic DNA from individuals of the T1S1 and BC3 generations was digested with EcoR I 

and hybridized to the backbone probes described above. For each of the probes, no 

hybridization to any of the 32138 SPT maintainer individuals was observed (Figures 36 

through 38). In each case, the plasmid positive control hybridized to the probe (Figures 36 

through 40, Lanes 1 and 19), indicating successful hybridization of the probes. The lack of 

hybridization in 32138 SPT maintainer confirms that the backbone of plasmid PHP24597 was 

not inserted during transformation. 
Pioneer Hi-Bred Page 75 of 188 

32138 SPT Maintainer No CBI 

Figure 36. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: EcoR I Digest and spc Probe 

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize lines was digested with 

EcoR I and hybridized to the spc probe. Approximately 4 μg of genomic DNA was digested 

and loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 4 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (T21-Red) 

2 Inbred 705 control 12 32138 SPT maintainer BC3 (T22-Red) 

3 Hi-II(ms45) control 13 32138 SPT maintainer BC3 (T23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (T24-Red) 

5 Hi-II control 15 DIGVII marker 

6 DIGVII marker 16 Hi-II control 

7 32138 SPT maintainer T1S1 (T3-Red) 17 Hi-II(ms45) control 

8 32138 SPT maintainer T1S1 (T4-Red) 18 Inbred 705 control 

9 32138 SPT maintainer T1S1 (T6-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (T7-Red) 
Pioneer Hi-Bred Page 76 of 188 

32138 SPT Maintainer No CBI 

Figure 37. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: EcoR I Digest and tet Probe 

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Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with EcoR I 

and hybridized to the tet probe. Approximately 3 μg of genomic DNA was digested and 

loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 3 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Note: The lines across Lanes 4 through 6 and Lanes 7 through 14 are due to scratches on 

the blot membrane. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 Inbred 705 control 12 32138 SPT maintainer BC3 (plant 22-Red) 

3 Hi-II(ms45) control 13 32138 SPT maintainer BC3 (plant 23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (plant 24-Red) 

5 Hi-II control 15 DIGVII marker 

6 DIGVII marker 16 Hi-II control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Hi-II(ms45) control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 Inbred 705 control 

9 32138 SPT maintainer T1S1 (plant 5-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 6-Red) 
Pioneer Hi-Bred Page 77 of 188 

32138 SPT Maintainer No CBI 

Figure 38. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: EcoR I Digest and virG Probe 

kb

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Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with EcoR I 

and hybridized to the virG probe. Approximately 4 μg of genomic DNA was digested and 

loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 4 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 Inbred 705 control 12 32138 SPT maintainer BC3 (plant 22-Red) 

3 Hi-II(ms45) control 13 32138 SPT maintainer BC3 (plant 23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (plant 24-Red) 

5 Hi-II control 15 DIGVII marker 

6 DIGVII marker 16 Hi-II control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Hi-II(ms45) control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 Inbred 705 control 

9 32138 SPT maintainer T1S1 (plant 6-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 7-Red) 
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32138 SPT Maintainer No CBI 

Figure 39. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: EcoR I Digest and LB Probe

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Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer control maize lines was digested with EcoR I 

and hybridized to the LB probe. Approximately 3 μg of genomic DNA was digested and 

loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 3 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Note: A dark spot across Lanes 3 through 5 is an artifact of the blot hybridization and not 

due to specific hybridization. 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 Inbred 705 control 12 32138 SPT maintainer BC3 (plant 22-Red) 

3 Hi-II(ms45) control 13 32138 SPT maintainer BC3 (plant 23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (plant 24-Red) 

5 Hi-II control 15 DIGVII marker 

6 DIGVII marker 16 Hi-II control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Hi-II(ms45) control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 Inbred 705 control 

9 32138 SPT maintainer T1S1 (plant 5-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 6-Red) 
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32138 SPT Maintainer No CBI 

Figure 40. Southern Blot Analysis of the T1S1 and BC3 Generations of 32138 SPT 

Maintainer: EcoR I Digest and RB Probe

Genomic DNA isolated from leaf tissue from individual plants grown from pinkish red seed 

from two generations of 32138 SPT maintainer and control maize was digested with EcoR I 

and hybridized to the RB probe. Approximately 3 μg of genomic DNA was digested and 

loaded per lane. Positive control lanes include plasmid PHP24597 at the indicated 

approximate gene copy numbers and 3 μg of control maize DNA. Sizes of the DIG VII 

molecular weight markers are indicated adjacent to the blot image in kilobases (kb). 

Lane Sample Lane Sample 

1 3 copy PHP24597 + Inbred 705 control 11 32138 SPT maintainer BC3 (plant 21-Red) 

2 Inbred 705 control 12 32138 SPT maintainer BC3 (plant 22-Red) 

3 Hi-II(ms45) control 13 32138 SPT maintainer BC3 (plant 23-Red) 

4 Hi-II(ms45) control 14 32138 SPT maintainer BC3 (plant 24-Red) 

5 Hi-II control 15 DIGVII marker 

6 DIGVII marker 16 Hi-II control 

7 32138 SPT maintainer T1S1 (plant 3-Red) 17 Hi-II(ms45) control 

8 32138 SPT maintainer T1S1 (plant 4-Red) 18 Inbred 705 control 

9 32138 SPT maintainer T1S1 (plant 5-Red) 19 3 copy PHP24597 + Inbred 705 control 

10 32138 SPT maintainer T1S1 (plant 6-Red) 
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32138 SPT Maintainer No CBI 

V-E. Inheritance of the Traits in 32138 SPT Maintainer 

Chi-square analysis of trait inheritance data from three different generations (BC3S2F1, 

BC3S4, and BC4) was performed to determine the heritability and stability of the insertion in 

32138 SPT maintainer. The breeding history of the three generations evaluated for 

Mendelian inheritance is described in Figure 12. The BC3S2F1, BC3S4, and BC4 

generations of 32138 SPT maintainer were expected to segregate 1:1 for the trait. Stability 

and inheritance of the insertion in 32138 SPT maintainer was evaluated using phenotypic and 

genotypic analyses. 

Seed from BC3S2F1, BC3S4, and BC4 generations were visually sorted for kernel color (red 

versus yellow) phenotype using a Leica3

 KL 2500 LCD halogen cold light source. The red 

kernel phenotype is imparted due to the expression of DsRed2(Alt1) gene and the 

corresponding protein DsRed2. Yellow kernel color confirms the absence of the 

DsRed2(Alt1) gene expression. As described in Sections V-B and V-C, Southern blot 

analysis confirmed the integrity and stability of the T-DNA insertion in 32138 SPT maintainer, 

and thus the evaluation of the DsRed2(Alt1) gene expression also correlates to the presence 

of the intact T-DNA. 

Color sorted seed were planted separately and all the plants that emerged were analyzed for 

the presence of the 32138 SPT insertion (scored as positive) or absence of the insertion 

(scored as negative) using event-specific Polymerase Chain Reaction (PCR). Results of 

phenotypic and genotypic analyses are summarized in Tables 7a and 7b, respectively. Upon 

comparison of phenotypic and genotypic analysis, the results of the phenotypic analysis for 

each individual plant corresponded to the event-specific PCR results with one exception. 

One event-specific PCR negative plant from the BC3S2F1 generation was scored as a red 

kernel (Table 7b). This is ascribed to an error in the color assignment during the manual 

color sorting process as the negative PCR result was confirmed by three replicate samples. 

To confirm that the insertion in 32138 SPT maintainer segregated according to Mendel’s laws 

of genetics, chi-square analysis was performed on the observed and the expected trait 

segregation ratios based on the results from the phenotypic and genotypic evaluation (Tables 

7a and 7b). All P-values were greater than 0.05, indicating no statistically significant 

differences between the observed and the expected frequencies for inheritance of the trait in 

three generations of 32138 SPT maintainer, confirming Mendelian inheritance of the 

introduced trait. Details of the statistical methodology can be found in Appendix 11. 

In summary, the Mendelian inheritance and the stability of the insertion have been 

demonstrated in three generations of self- and cross- pollinated 32138 SPT maintainer. 

These results are consistent with the Southern blot analysis (Sections V-B and V-C) that 

confirmed insertion of a single intact copy of the T-DNA at a single locus in 32138 SPT 

maintainer. 

3

 Registered trademark of Schott Corporation 
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32138 SPT Maintainer No CBI 

Table 7a. Comparison of Observed and Expected Segregation Ratios for 32138 SPT 

Maintainer Based on Phenotypic Analysis (Seed Color) 

Seed Color 

Observed Expected 

ChiGeneration # of Seeds Square 

Analyzed 

Red Yellow Red Yellow P-Value 

BC3S2F1 260 127 133 130 130 0.7098 

BC3S4 275 136 139 137.5 137.5 0.8564 

BC4 100 52 48 50 50 0.6892 

Table 7b. Comparison of Observed and Expected Segregation Ratios for 32138 SPT 

Maintainer Based on Genotypic Analysis (Event-Specific PCR)

# of Plants 

Emerged1 Event-Specific PCR Results 

Observed Expected 

ChiSquare Generation Pinkish 

Red 

Seed 

Yellow 

Seed Positive Negative Positive Negative P-Value 

BC3S2F1 126 130 1252 1312 128 128 0.7077 

BC3S4 127 135 127 135 131 131 0.6211 

BC4 52 48 52 48 50 50 0.6892 

Footnotes: 

1. All red and yellow seed (from Table 7a) were planted. The numbers of plants in this column represent actual 

numbers of red and yellow plants that emerged. 

2. Numbers do not correlate with the number of plants that emerged. This is ascribed to an error in the color 

assignment during the visual color sorting process as the negative PCR result was confirmed by three replicate 

samples.
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V-F. Molecular Characterization of 32138 SPT Maintainer: Summary and 

Conclusions 

Southern blot analysis was conducted to characterize the DNA insertion in 32138 SPT 

maintainer. The analysis confirmed that a single, intact PHP24597 T-DNA was inserted into 

the maize genome to produce 32138 SPT maintainer. A single copy of each of the Ms45, 

zm-aa1, and DsRed2(Alt1) expression cassettes was present and the integrity of the 

PHP24597 T-DNA was maintained. The analysis confirmed the stability of the insertion in 

32138 SPT maintainer across the T1S1, BC3, and BC4 generations, thus confirming stability 

of inheritance during traditional breeding procedures. In addition, Southern blot analysis 

verified the absence of plasmid backbone sequences in 32138 SPT maintainer.

Chi-square analysis of trait inheritance data from three different generations (BC3S2F1, 

BC3S4, and BC4) was performed to determine the heritability and stability of the insertion in 

32138 SPT maintainer. This analysis confirmed the Mendelian inheritance of the 32138 SPT 

insertion. 
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32138 SPT Maintainer No CBI 

VI. Characterization of the Introduced Proteins 

VI-A. The MS45 Protein 

VI-A.1. Identity of the MS45 Protein 

The Ms45 gene, one of the many nuclear fertility genes identified in maize that contribute to 

the male-fertility phenotype, and the gene has been isolated using an activator-transposon 

tagging approach (Albertsen et al., 1993). Ms45 is an endogenous maize gene, tightly 

expressed at particular developmental stages in anthers. Expression of the MS45 protein 

encoded by the Ms45 gene in the anther tapetum is required for the production of fertile 

pollen by the maize plant (Cigan et al., 2001). The full-length MS45 protein is comprised of 

412 amino acids and has a molecular weight of approximately 47 kDa (Figure 41). 

Figure 41. Deduced Amino Acid Sequence of the Full-Length MS45 Protein 

 1 MEKRNLQWRR GRDGIVQYPH LFFAALALAL LVADPFGLSP LAEVDYRPVK 

 51 HELAPYGEVM GSWPRDNASR LRRGRLEFVG EVFGPESIEF DLQGRGPYAG 

101 LADGRVVRWM GEEAGWETFA VMNPDWSEEV CANGVNSTTR KQHEKEEFCG 

151 RPLGLRFHGE TGELYVADAY YGLMVVGQSG GVASSVAREA DGDPIRFAND 

201 LDVHRNGSVF FTDTSMRYSR KDHLNILLEG EGTGRLLRYD PETSGVHVVL 

251 KGLVFPNGVQ ISEDHQFLLF SETTNCRIMR YWLEGPRAGE VEVFANLPGF 

301 PDNVRSNGRG QFWVAIDCCR TPAQEVFAKR PWLRTLYFKF PLSLKVLTWK 

351 AARRMHTVLA LLDGEGRVVE VLEDRGHEVM KLVSEVREVG RKLWIGTVAH 

401 NHIATIPYPL ED 

VI-A.2. Stage-Specific Expression of MS45 Protein during Pollen Development 

In order to evaluate and further confirm the anther-specific and stage-specific expression of 

the MS45 protein, anther samples from conventional maize inbred 705 were collected at 

targeted stages of development between the Pollen Mother Cell (PMC) and binucleate pollen 

and analyzed by western blot analysis (Figure 42A). As expected, the maximum expression 

of MS45 protein was observed during quartet (tetrad) to uninucleate stages (Figure 42A, 

lanes 5-10). A bacterially produced non-glycosylated form of MS45 protein was used as a 

reference standard protein (Figure 42A, lanes 2-4) and migrated at a slightly lower molecular 

weight on SDS-PAGE gels than the endogenous maize MS45 protein. The shift in the 

expected molecular weight (47 kDa) of the endogenous MS45 protein in anther tissues was 

confirmed to be due to glycosylation as shown in Figure 42B. 

VI-A.3. Intended Function of Ms45 Gene in the 32138 SPT Maintainer 

The Ms45 gene from the 32138 SPT insertion complements the ms45 male-sterile allele and 

restores fertility in the transgenic 32138 SPT maintainer. Male-sterile female inbred plants 

are homozygous for ms45 (i.e., ms45/ms45) and produce no pollen (Figure 7A). Male-fertility 

can be restored by a single copy of the Ms45 gene transformed into a homozygous recessive 

male-sterile plant (ms45/ms45). Hence, transformation of the ms45/ms45 genotype with the 

32138 SPT insertion containing a single copy of the Ms45 gene restores fertility (Figure 7B) 

in the transgenic 32138 SPT maintainer. 
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32138 SPT Maintainer No CBI 

Figure 42A. Western Blot Showing Anther and Stage-Specific Expression of MS45 

Protein during Pollen Development. Anther samples from maize inbred line 705 

were collected and analyzed as described in Appendix 9.2-9.5. 

 MWM=Molecular weight marker. 

-Uninucleate

49

38

1 2 3 4 5 6 7 8 9 10 

62 

kDa

49

38

MWM

200ng/ml

100ng/ml

1st Quartet

Meiotic to Quartet

Quartet to Early Nucleate

Mid to Late Uninucleate

2nd Mitotic

MS45 Protein 

Standard

Microsporogenesis

Anther Developmental Stages 

Early to Mid

50ng/ml

-Uninucleate

49

38

1 2 3 4 5 6 7 8 9 10 

62 

kDa

49

38

MWM

200ng/ml

100ng/ml

1st Quartet

Meiotic to Quartet

Quartet to Early Nucleate

Mid to Late Uninucleate

2nd Mitotic

MS45 Protein 

Standard

Microsporogenesis

Anther Developmental Stages 

Early to Mid

50ng/ml

Figure 42B. Western Blot Showing Endogenous Glycosylation of MS45 Protein in 

Anthers. Approximately 18 anthers were extracted in 0.2 ml buffer containing 0.5% 

SDS and 1% ß-mercaptoethanol. The extract was centrifuged and the protein extract 

was incubated with either the extraction buffer (Control), or 500 units each of Nglycosidase F (PNGase F) or endoglycosidase H (Endo H) (New England Biolabs, Inc.) 

at 37o

C for 2 h. A shift in the mobility due to the endogenous glycosylation of MS45 

protein was examined by Western blotting using an MS45 polyclonal antibody R3976. 

 MWM=Molecular Weight Marker 

1 2 3

kD

MWM Control

+ PNGaseF+ Endo H

1 2 3

kD

MWM Control

+ PNGaseF+ Endo H

Control

+ PNGaseF+ Endo H
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VI-B. The ZM-AA1 Protein 

VI-B.1. Identity of the ZM-AA1 Protein 

The endogenous zm-aa1 gene that encodes ZM-AA1 maize -amylase protein (EC 3.2.1.1) 

is expressed predominantly in the scutellum of germinating seed and minimal expression is 

observed in the developing endosperm. No expression of the endogenous zm-aa1 gene is 

detected in pollen (internal data not shown). The zm-aa1 sequence in plasmid PHP24597 

was isolated from a maize cDNA library prepared from combined tissue of the developing 

embryo and endosperm at ten days after pollination. The zm-aa1 gene sequence was 

truncated to remove the native N-terminal transit peptide sequence and was replaced by 

sequence from the zm-bt1 gene (brittle-1) encoding the Brittle-1 transit peptide for targeting 

the ZM-AA1 protein to amyloplasts (Sullivan et al., 1991). Figure 43 shows the deduced 

amino acid sequence from translation of the zm-bt1 transit peptide sequence linked to the 

zm-aa1 gene region from plasmid PHP24597. The complete translation product, including 

the transit peptide (underlined region) is 495 amino acids in length and has a molecular 

weight of approximately 54 kDa. Following processing of the Brittle-1 transit peptide the 

primary ZM-AA1 protein observed is 445 amino acids in length and has a molecular weight of 

approximately 49 kDa. The observed 445 amino acid ZM-AA1 protein in 32138 SPT 

maintainer is comprised of maize endogenous protein sequences from the Brittle-1 transit 

peptide and the ZM-AA1 protein. 

Figure 43. Deduced Amino Acid Sequence of the ZM-AA1 Protein

 1 MAATMAVTTM VTRSKESWSS LQVPAVAFPW KPRGGKTGGL EFPRRAMFAS

 51 VGLNVCPGVP AGRDPREPDP KVVRAACGLV QAQVLFQGFN WESCKQQGGW 

101 YNRLKAQVDD IAKAGVTHVW LPPPSHSVSP QGYMPGRLYD LDASKYGTAA 

151 ELKSLIAAFH GRGVQCVADI VINHRCAEKK DARGVYCIFE GGTPDDRLDW 

201 GPGMICSDDT QYSDGTGHRD TGEGFAAAPD IDHLNPRVQR ELSAWLNWLR 

251 SDAVGFDGWR LDFAKGYSPA VARMYVESTG PPSFVVAEIW NSLSYSGDGK 

301 PAPNQDQCRQ ELLDWTRAVG GPAMAFDFPT KGLLQAGVQG ELWRLRDSSG 

351 NAAGLIGWAP EKAVTFVDNH DTGSTQKLWP FPSDKVMQGY AYILTHPGVP 

401 CIFYDHMFDW NLKQEISTLS AIRARNGIRA GSKLRILVAD ADAYVAVVDE 

451 KVMVKIGTRY GVSSVVPSDF HPAAHGKDYC VWEKASLRVP AGRHL 

The underlined region represents the Brittle-1 transit peptide (75 amino acids). The arrow () 

following the serine residue at position 50 indicates the primary transit peptide processing site 

observed in vivo in 32138 SPT maintainer.

VI-B.2. Intended Function of ZM-AA1 in the 32138 SPT Maintainer 

Starch molecules are glucose polymers linked together by -1,4 and -1,6 glucosidic bonds 

(Figure 44). -amylases belong to a family of glycosyl-hydrolases (1,4--D-Glucan 

glucanohydrolase; EC 3.2.1.1) catalyzing hydrolysis of (1-4)--D-glucosidic linkages in 

polysaccharide molecules, such as starch. By acting at random locations along the starch 

chain, -amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose, 

maltose and/or, glucose. Expression of ZM-AA1 in the developing pollen of 32138 SPT 

maintainer results in the depletion of starch and deprivation of the energy reserves required 

for pollen germination and fertilization, thereby resulting in pollen infertility. 
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Figure 44. Illustration of the Mode of Action of -Amylase 

VI-C. The DsRed2 Protein 

VI-C.1. Identity of DsRed2 Protein 

Red fluorescent protein, known commercially as DsRed, was originally isolated from the 

coral-like anemone Discosoma species by Matz et al. (1999), who referred to the protein as 

drFP583. DsRed2 is a variant of the original red fluorescent protein (DsRed), modified with 

six point mutations (http://www.clontech.com/products/detail.asp?product_id=157257&tabno=2). These 

mutations improve the solubility of DsRed2 by reducing its tendency to form aggregates. The 

DsRed2 protein is encoded by theDsRed2(Alt1) gene, a modified version of the original 

DsRed2 gene (Wasson-Blader, 2001) in which an internal BstE II restriction enzyme 

recognition site was removed without altering the amino acid sequence of the expressed 

protein. Figure 45 below shows the deduced amino acid sequence from translation of the 

DsRed2(Alt1) gene from plasmid PHP24597. The DsRed2 protein is 225 amino acids in 

length with an approximate molecular weight of 26 kDa. 

Figure 45. Deduced Amino Acid Sequence of the DsRed2 Protein 

 1 MASSENVITE FMRFKVRMEG TVNGHEFEIE GEGEGRPYEG HNTVKLKVTK 

 51 GGPLPFAWDI LSPQFQYGSK VYVKHPADIP DYKKLSFPEG FKWERVMNFE 

101 DGGVATVTQD SSLQDGCFIY KVKFIGVNFP SDGPVMQKKT MGWEASTERL 

151 YPRDGVLKGE THKALKLKDG GHYLVEFKSI YMAKKPVQLP GYYYVDAKLD 

201 ITSHNEDYTI VEQYERTEGR HHLFL 

VI-C.2. Intended Function of DsRed2 Protein in the 32138 SPT Maintainer 

The DsRed2 protein belongs to a family of red fluorescent proteins which are members of a 

larger group of proteins in several Anthozoa species (this class includes corals, anemones 

and sea pens). The unique feature of fluorescent proteins is their ability to enter an excited 

state and emit light of a certain wavelength (i.e. fluoresce) upon absorption of ultraviolet or 

visible light. Application of the DsRed protein as a visual selection marker for highthroughput seed sorting has been previously demonstrated in Arabidopsis (Stuitje et al., 

2003). The DsRed2 protein provides a very effective means of tracking transgenic maize 

seed expressing this protein and is used as a quality assurance color marker that allows 

monitoring of seeds to ensure seed purity. 

6

5

4

3 2

1

-amylase sites of action

1-4- - D-glucosidic linkages 

 -1,4  -1,6 Glucan 

and/or

and/or -amylase

6

5

4

3 2

1

-amylase sites of action

1-4- - D-glucosidic linkages 

 -1,4  -1,6 Glucan 

and/or

and/or -amylase
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VI-D. Expression of MS45 in 32138 SPT Maintainer 

The 32138 SPT maintainer contains the Ms45, zm-aa1 and DsRed2(Alt1) genes encoding 

the MS45, ZM-AA1 and DsRed2 proteins, respectively. The transcription of the Ms45 gene is 

under the control of the maize anther-specific 5126 promoter (Cigan and Albertsen, 1997). A 

quantitative enzyme linked immunosorbent assay (ELISA) for the MS45 protein was not 

developed due to marginal solubility of MS45 in the compatible ELISA buffers. Hence, the 

MS45 protein was analyzed using Western blot analysis. 

 

In order to facilitate collection of anther samples at the targeted developmental stages 

(Chang and Neuffer, 1989), greenhouse tissue samples from BC1S2 (Figure 12) generation 

were collected and used for Western blot analysis of the MS45 protein. A non-transgenic 

male-sterile (ms45/ms45) female inbred derived from transgenic 32138 SPT maintainer was 

used as a negative control, and a male-fertile inbred 705 was used as a ‘reference’ (positive 

control) for MS45 detection. Developing anther samples were collected at the following 

developmental stages: pollen mother cell, meiosis, quartet to early uninucleate, mid 

uninucleate, mid to late uninucleate, late uninucleate, binucleate (2nd mitosis) and starch 

filling (see Figure 8B). The developmental stage of the anthers was determined based on the 

staging scheme of Chang and Neuffer (1989). Western blot analysis of the MS45 protein 

utilized two samples of four anthers each for each stage. 

As expected, the MS45 protein was only detected in anthers and not in the other tissues 

analyzed (Figure 46). The MS45 protein was detected in both 32138 SPT maintainer (test) 

and the male-fertile inbred 705 (reference) during the late meiosis-quartet stage to the early 

uninucleate stage of microspore development. The anther-expressed MS45 migrated at a 

higher molecular weight than the microbially expressed protein, due to the observed 

glycosylation of the endogenous MS45 protein (Figure 42B). MS45 protein was undetectable 

in any of the negative control male-sterile (ms45/ms45) female inbred samples. Some nonspecific cross reactivity of MS45 antibodies with other plant proteins at lower molecular 

weights (~30kDa) was observed in the male-sterile (ms45/ms45) female inbred negative 

control and male-fertile inbred 705 positive control samples. The MS45 protein was not 

consistently observed in all of the 32138 SPT maintainer and male-fertile 705 materials 

sampled due to a narrow window of expression for the MS45 protein and also due to the 

rapid progress of the microspore development from one stage to the next (within hours) 

making it difficult to obtain a single exact stage of development. 

Based on the Western blot analysis, detectable levels of the MS45 protein in both 32138 SPT 

maintainer and inbred 705 reference line are limited to the anthers. 
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VI-E. Expression of ZM-AA1 and DsRed2 in 32138 SPT Maintainer

Concentrations of the ZM-AA1 and DsRed2 proteins were measured in tissue samples 

collected from a replicated field study grown at six field locations in the United States in 2007. 

Three replicated samples per tissue per location were collected for 32138 SPT maintainer 

and one sample per tissue per location was collected for the male-fertile inbred 705 control 

maize. Growth stages indicated are as described by Ritchie et al. (1993). Tissue samples 

from 32138 SPT maintainer and inbred controls were analyzed from the following tissues and 

growth stages: leaf (V9, R1, R4, R6), whole plant (V9, R1, R6), pollen (R1), forage (R4), and 

seed (R6). Forage refers to all above ground material of the plant, i.e. ears and all vegetative 

material except the roots. The concentrations of ZM-AA1 and DsRed2 in different tissues of 

32138 SPT maintainer were quantified using ELISA and fluorometric assays, respectively 

(see Appendix 9 for the details of the methodology). 

Results of the ZM-AA1 protein concentration measurements are presented in Table 8. The 

endogenous ZM-AA1 protein was not detected in any of the inbred 705 control tissues (data 

not presented). In the 32138 SPT maintainer, as expected the ZM-AA1 protein was highest 

in the pollen (Table 8), with values 16 to 400 fold higher as compared to the other tissues. 

Low levels of ZM-AA1 protein concentration was detected in the leaf, whole plant, pollen, 

forage and seed tissues. The tissue expression pattern may be due to the influence of the 

CaMV35S enhancer (Odell et al., 1988) located immediately downstream of the zm-aa1

expression cassette (See Figures 14 and 15). Of the different tissues analyzed, the lowest 

concentrations of ZM-AA1 protein were found in the seed tissue (Table 8), and were 

approximately 400 fold less than the protein levels measured in pollen. In leaf and whole 

plant tissue samples, the ZM-AA1 protein concentrations decreased as the plants matured 

and were below the limit of quantitation by the R6 stage of development. 

Results of the DsRed2 protein concentration measurements are presented in Table 9. As 

expected, the DsRed2 protein concentrations were found to be highest in the 32138 SPT 

maintainer seed. The DsRed2 was also detected in leaf, whole plant and forage tissues. 

The DsRed2 protein was below the limit of quantitation in the pollen tissue. This expression 

pattern may be due to the influence of CaMV35S enhancer located upstream of the Ltp2

promoter in the DsRed2(Alt1) expression cassette (Figures 14 and 15). As expected, the 

protein concentration of DsRed2 was below the limit of quantitation for all inbred 705 control 

tissues (data not shown). 

These results demonstrate that, in the 32138 SPT maintainer, the MS45 protein is confined to 

anthers. In addition, the ZM-AA1 protein is detected at the highest concentration in the 

pollen, and the DsRed2 protein is detected at the highest concentration in the seed tissue. 
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32138 SPT Maintainer No CBI 

Figure 46. Western Blot Analysis of the MS45 Protein in Developing Anthers (Panel A 

& B) and in Different Tissues (Panel C) of the 32138 SPT Maintainer

The expression of MS45 was evaluated in multiple tissues from: a) 32138 SPT 

maintainer (test), b) non-transgenic male-sterile (ms45/ms45) female inbred 

negative control and c) non-transgenic male-fertile (Ms45/Ms45) reference inbred 

705. No sample was loaded in the lane marked “Blank” in panel C. See 

Appendix 9.1-9.5 for details on the sample extraction, SDS-PAGE, and Western 

Blot analysis. 

kDa

62

49

38

kDa

62

49

38

kDa

62

49

38

MS45 Protein

Standard

Late

Uninucleate

Binucleate

Mitosis

Starch 

Filling

Stage

200 ng/ml

100 ng/ml

50 ng/ml

Test

Control

Reference

Test

ControlReference

Test

Control

Reference

Test

Control

Reference

MS45 Protein

Standard

Pollen 

Mother 

Cell

Meiosis

Quartet to

Early

Nucleate

Mid

Nucleate

Whole 

Plant-V9

Whole 

Plant-R1 Pollen Seed

A

B

C MS45 Protein

Standard100 ng/ml 200 ng/ml

50 ng/ml

Test

Control

Reference

Test

Control

ReferenceTest

Control

Reference

200 ng/ml

100 ng/ml

50 ng/ml

Test

Control

Reference

Test

Control

Reference

Test

Blank

Reference

Test

Control

Reference

Test = 32138 SPT Maintainer

Control = ms45/ms45

Reference = Inbred 705

kDa

62

49

38

kDa

62

49

38

kDa

62

49

38

MS45 Protein

Standard

Late

Uninucleate

Binucleate

Mitosis

Starch 

Filling

Stage

200 ng/ml

100 ng/ml

50 ng/ml

Test

Control

Reference

Test

ControlReference

Test

Control

Reference

Test

Control

Reference

MS45 Protein

Standard

Pollen 

Mother 

Cell

Meiosis

Quartet to

Early

Nucleate

Mid

Nucleate

Whole 

Plant-V9

Whole 

Plant-R1 Pollen Seed

A

B

C MS45 Protein

Standard100 ng/ml 200 ng/ml

50 ng/ml

Test

Control

Reference

Test

Control

ReferenceTest

Control

Reference

200 ng/ml

100 ng/ml

50 ng/ml

Test

Control

Reference

Test

Control

Reference

Test

Blank

Reference

Test

Control

Reference

kDa

62

49

38

kDa

62

49

38

kDa

62

49

38

MS45 Protein

Standard

Late

Uninucleate

Binucleate

Mitosis

Starch 

Filling

Stage

200 ng/ml 200 ng/ml

100 ng/ml 100 ng/ml

50 ng/ml 50 ng/ml

Test

Control Control

Reference Reference

Test

Control ControlReference Reference

Test

Control Control

Reference Reference

Test

Control Control

Reference Reference

MS45 Protein

Standard

Pollen 

Mother 

Cell

Meiosis

Quartet to

Early

Nucleate

Mid

Nucleate

Whole 

Plant-V9

Whole 

Plant-R1 Pollen Seed

A

B

C MS45 Protein

Standard100 ng/ml 200 ng/ml

50 ng/ml

Test

Control

Reference

Test

Control

ReferenceTest

Control

Reference

200 ng/ml

100 ng/ml

50 ng/ml

Test

Control

Reference

Test

Control

Reference

Test

Blank

Reference

Test

Control

Reference

Test = 32138 SPT Maintainer

Control = ms45/ms45

Reference = Inbred 705
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32138 SPT Maintainer No CBI 

Table 8. Concentration of ZM-AA1 Protein Measured in 32138 SPT Maintainer 

Footnotes: 

1. Ritchie et al., 1993 

2. Lower Limit of Quantitation (LLOQ) for the assay and tissue type 

3. Range denotes the lowest and highest individual value across sites 

4. NA=not applicable; all samples below LLOQ 

ng/mg Tissue Dry Weight 

Tissue Growth 

Stage1

LLOQ2

Expression 

in 32138 SPT 

Maintainer 

(Mean  SD) 

Range of 

Expression3

Number of 

Samples 

<LLOQ/ 

Number of 

samples 

Analyzed 

V9 7.3  3.0 2.6 -12.0 0/18 

R1 6.3  2.1 3.0 – 10.0 0/18 

R4 2.8  1.8 <0.28 – 6.6 1/18 

Leaf 

R6 

0.28 

NA4

 NA4 18/18 

V9 11.0 3.2 3.4 – 15.0 0/18 

Whole Plant R1 3.8  0.89 2.0- 5.8 0/18 

R6 

0.092 

NA4

 NA4 18/18 

Pollen R1 0.55 180.0  52.0 110.0 - 260.0 0 /13 

Forage R4 0.092 1.7  0.73 0.12 – 3.0 0/18 

Seed R6 0.14 0.44  0.23 0.14 – 0.84 0/18 
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Table 9. Concentration of DsRed2 Protein Measured in 32138 SPT Maintainer 

Footnotes: 

1. Ritchie et al., 1993 

2. Lower Limit of Quantitation (LLOQ) for the assay and tissue type 

3. Range denotes the lowest and highest individual value across sites 

4. NA=not applicable; all samples below LLOQ 

ng/mg Tissue Dry Weight 

Tissue Growth 

Stage1

LLOQ2

Expression 

in 32138 SPT 

Maintainer 

(Mean  SD) 

Range of 

Expression3

Number of 

Samples 

<LLOQ/ 

Number of 

Samples 

Analyzed 

V9 87  15 <72 - 120 2/18 

R1 160  28 110 - 220 0/18 

R4 210  75 <72 - 360 2/18 

Leaf 

R6 

72 

NA4

 NA4 18/18 

V9 80  9.1 60 - 96 0/18 

Whole Plant R1 77  7.5 64 - 92 0/18 

R6 

36 

40  5.7 <36 - 52 8/18 

Pollen R1 72 NA4 NA4 13/13 

Forage R4 36 60  8.1 44 - 76 0/18 

Seed R6 36 410  110 300 - 770 0/18 
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VI-F. Safety Assessment of the MS45 Protein 

The Ms45 gene is a native maize fertility gene that encodes the MS45 protein. The MS45 

protein is normally present in maize in developing anthers (Cigan et al., 2001). Expression of 

MS45 protein in the 32138 SPT maintainer is targeted to the developing anthers where it 

complements the naturally occurring male-sterile ms45 mutation in the endogenous Ms45

gene. As compared to the male-fertile inbred 705, the levels of MS45 protein in 32138 SPT 

maintainer appear slightly elevated on Western blots (Figure 46). However, neither the 

endogenous nor the recombinant MS45 protein is expressed in the mature pollen, seed, or 

any other tissues (Section VI-D). Therefore there is no increase in exposure to the MS45 

protein from 32138 SPT maintainer. 

Bioinformatic analysis was conducted to evaluate the potential allergenicity of the MS45 

protein. The MS45 protein sequence was compared to a database of 1313 known and 

putative allergen sequences comprising the FARRP8 dataset at the University of Nebraska 

(updated 1/2008). Potential identities between the MS45 protein sequence and proteins in 

the allergen database were evaluated with the FASTA34 sequence alignment tool using the 

default parameters. A 35% or greater identity threshold over any 80 or greater amino acid 

length sequence between the query protein and an allergen was used to indicate the 

potential for cross-reactivity (FAO/WHO, 2001; Codex, 2003). No identity matches of  35% 

over  80 residues were observed. In addition, protein sequences were evaluated for any 8 

or greater contiguous identical amino acid matches to the allergens contained in the 

database noted above. There were no 8 or greater contiguous identical amino acid matches 

observed. 

The native MS45 protein was found to be glycosylated (Figure 42B). Based on the similar 

mobility of MS45 protein from 32138 SPT maintainer and the reference line (Figure 46), it is 

likely that the MS45 protein in 32138 SPT maintainer is also glycosylated. Based on its 

glycosylation status the MS45 protein in the 32138 SPT maintainer is no more likely to be an 

allergen than the native MS45, and there is no risk of increased allergenic potential. 

To search for potential similarity to known toxins, the MS45 sequence was queried using the 

BLASTP 2.2.13 algorithm against Release 165.0 (4/15/08) of the Genpept “nr” dataset, which 

incorporates non-redundant entries from all Genbank nucleotide translations along with 

protein sequences from SWISS-PROT (http://www.expasy.org/sprot/), PIR 

(http://pir.georgetown.edu/), PRF (http://www4.prf.or.jp/en/), and PDB 

(http://www.wwpdb.org/). Because of the large number of automated conceptual translations 

derived from genomic sequences, protein sequences from the RefSeq collection 

(http://www.ncbi.nlm.nih.gov/RefSeq/) were excluded from the search using the filtering 

statement "protein_all [filter] NOT srcdb_refseq [prop]” to delimit the results returned. A cutoff 

expectation (E) value of 1.0 was used to generate biological meaningful similarity between 

the proteins 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 scoring matrix used was the default (BLOSUM62), and the 

number of alignments returned was set to the maximum value (2000). 

The MS45 BLASTP search returned 509 protein sequences with an E score of less than 1.0. 

Five of the accessions returned by the searches displayed complete significance (E = 0) and 

represent identical or very closely related male-fertility protein sequences from maize, rice, 

and wheat. Two hundred and sixty of the remaining 504 accessions represented bacterial, 

plant, and animal derived sequences described as strictosidine synthases, 

gluconolactonases, or hemomucins, and 194 of the accessions returned were described as 

hypothetical, predicted, putative, unknown, or unnamed proteins. The remaining 50 

sequence alignments represent five small groups classified as adipocyte plasma membrane-
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32138 SPT Maintainer No CBI 

associated proteins, ABC transporters, diisopropyl fluorophosphatases, senescence marker 

proteins, or NHL repeat proteins. The functionalities attributed to the aligning accessions 

were often based upon possession of one or more conserved domains within the aligned 

sequence. None of the protein sequences returned by the BLASTP search identified safety 

concerns that might arise from the expression of MS45 in genetically modified plants. 

Based on this information, Pioneer determined that the MS45 protein is unlikely to cause an 

allergic reaction in humans or be toxic in humans or animals.

VI-G. Safety Assessment of the ZM-AA1 Protein

The -amylase expressed in 32138 maintainer is derived from two endogenous maize gene 

sequences that encode the ZM-AA1 protein, and the Brittle-1 transit peptide required for 

directing the ZM-AA1 protein into the amyloplasts. Endogenous zm-aa1 gene is 

predominantly expressed in the scutellum tissues of the germinating seed and minimally 

expressed (<0.25% of the levels in the scutellum) in endosperm, stalk, leaf and root (internal 

data not shown). Expression of the endogenous zm-aa1 in the scutellum tissues 

corresponds well with the literature evidence that -amylases are required during cereal seed 

germination are required for the hydrolysis of endosperm starch into metabolizable sugars, 

which provide the energy for the growth of roots and shoots (Akazawa and Hara-Mishimura, 

1985; Beck and Ziegler, 1989). 

Germinating maize seeds, known as sprouts, are extensively used in Latin America in the 

production of alcoholic beverages, for example, Chicha (maize beer), Sora, Napú, Fubá, 

Champuz, Acupe, and Tesgüino (Lorence-Quiñones et al., 1999). Chicha is the most 

important traditional fermented beverage in Latin America and is made by germinating maize 

seed to produce amylase required for starch conversion. Chicha has been consumed by the 

Andean Indians for centuries. Sprouted maize seed has been increasingly used in bread and 

tortilla products (see, for example, http://www.foodforlife.com) and has been explored as a 

feed supplement (Naga, 1986). In addition, livestock feeding of maize grain that has 

sprouted from moisture exposure is an accepted practice (Heiniger, 1999). Therefore, there 

is a previous history of exposure and safe use of ZM-AA1 -amylase endogenous to the 

germinating maize seed. 

The -amylases are ubiquitous enzymes present in many organisms including plants and 

insects (Campos et al., 1989; Raimbaud et al., 1989; Silva et al., 1999; Cristofoletti et al., 

2001). The -amylases from multiple sources including plants, fungi and bacteria have a 

long history of safe consumption in foods. Purified microbial -amylases are commonly used 

in the modern food processing industry (Pariza and Johnson, 2001) and these enzymes have 

a long history of safe use. Additionally their safety has been verified by an array of laboratory 

studies (see, for example, Landry et al., 2003). 

In plants, -amylases aid in the hydrolysis of transient starch in the leaves, which occurs 

during the dark cycle of photosynthesis, and are also required for the hydrolysis of storage 

starch during seed germination or tuber sprouting. Multiple forms of -amylases exist in 

maize, and are expressed in the endosperm and other tissues (Chao and Scandalios, 1971; 

James et al., 2007). Amylases are reported to occur endogenously in the pollen of numerous 

plants (Castro and Clement, 2007; Bhadula and Sawhney, 1989), including maize, where 

starch hydrolysis is associated with pollen germination (Brewbaker, 1971; Agarwala et al., 

1981; Wakhle et al., 1983). A recent work of Castro and Clement (2007) demonstrated that 

the amylase activity in Lilium pollen is attributed, in part, to -amylase protein. 

Maize has a long history of safe food and feed use for humans and animals. Maize is not 

considered a common allergenic food (Hefle et al., 1996; Moneret-Vautrin et al., 1998),
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32138 SPT Maintainer No CBI 

although in a few case studies, allergenic reactions were reported and maize allergens 

identified (Pastorello et al., 2000, 2003; Pasini et al., 2002; Weichel et al., 2006b). The ZMAA1 protein, however, was not identified as an allergenic protein in maize (Weichel et al., 

2006a). 

There is only one -amylase protein currently included in the peer-reviewed FARRP8 

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 

(Release 8 - January 2008). It is derived from Aspergillus oryzae and has been reported to 

be one of the most often described causes of allergenic Baker’s asthma (Blanco et al., 1991; 

Houba et al., 1998). The ZM-AA1 -amylase did not show significant similarity to the 

Aspergillus -amylase in the bioinformatics screen noted below. The US FDA has granted 

GRAS status to cereal and bacterial-derived -amylase-containing preparations and permits 

the use of flour containing -amylase from Aspergillus oryzae. 

There has been one report suggesting an association of barley amylase with allergic 

reactions to flour (Sandiford et al., 1994). However, there are insufficient data to prove a 

cause and effect relationship, so the finding has been judged as inconclusive. For this 

reason, barley and other cereal -amylases are not included in the AllergenOnline database. 

Bioinformatic analysis was conducted to evaluate the potential allergenicity of the ZM-AA1 

protein sequence. The ZM-AA1 complete translation product, including the Brittle-1 transit 

peptide is 495 amino acids in length (420 amino acids of the ZM-AA1 protein itself plus 75 

additional amino acids (residues 1-75) contributed by the maize Brittle-1 transit peptide used 

to target the protein to amyloplasts) and was compared to a database of 1313 known and 

putative allergen sequences comprising the FARRP8 database. Potential identities between 

the ZM-AA1 protein sequence and proteins in the allergen database were evaluated with the 

FASTA34 sequence alignment tool using the default parameters. A 35% or greater identity 

threshold over any 80 or greater amino acid length sequence between the query protein and 

an allergen was used to indicate the potential for cross-reactivity (FAO/WHO, 2001; Codex, 

2003). No identity matches of  35% over  80 residues were observed. In addition, protein 

sequences were evaluated for any 8 or greater contiguous identical amino acid matches to 

the allergens contained in the database noted above. There were no 8 or greater contiguous 

identical amino acid matches observed. 

Based on this information, Pioneer determines that ZM-AA1 protein is unlikely to cause an 

allergic reaction in humans or to be toxins in humans or animals.

To search for potential similarity to known toxins, -amylase sequence was queried using the 

BLASTP 2.2.13 algorithm against Release 165.0 (4/15/08) of the Genpept “nr” dataset, which 

incorporates non-redundant entries from all Genbank nucleotide translations along with 

protein sequences from SWISS-PROT (http://www.expasy.org/sprot/), PIR 

(http://pir.georgetown.edu/), PRF (http://www4.prf.or.jp/en/), and PDB 

(http://www.wwpdb.org/). Because of the large number of automated conceptual translations 

derived from genomic sequences, protein sequences from the RefSeq collection 

(http://www.ncbi.nlm.nih.gov/RefSeq/) were excluded from the search using the filtering 

statement "protein_all [filter] NOT srcdb_refseq [prop]” to delimit the results returned. A cutoff 

expectation (E) value of 1.0 was used to generate biological meaningful similarity between 

the proteins 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 scoring matrix used was the default (BLOSUM62), and the 

number of alignments returned was set to the maximum value (2000). 
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32138 SPT Maintainer No CBI 

The ZM-AA1 BLASTP search returned 1893 protein sequences with an E score of less than 

1.0. Seventeen of the accessions returned by the searches displayed complete significance 

(E = 0) and represent very closely related -amylases from rice, maize, and banana. A total 

of 741 additional protein sequences were identified as -amylases from various bacterial, 

archaebacterial, and eukaryotic species. Five of the sequences returned aligned primarily 

with the Brittle-1 transit peptide portion of the -amylase sequence. The remaining 1130 

sequences represented a variety of protein sequences that are functionally related to 

-amylases and contain one or more well-characterized glycosyl hydrolase domains. None 

of the protein sequences returned by the BLASTP search identified safety concerns that 

might arise from the expression of ZM-AA1 in genetically modified plants. 

We conclude from our assessment that the presence of zm-aa1 encoded -amylase in maize 

32138 SPT maintainer does not represent any safety concerns
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32138 SPT Maintainer No CBI 

VI-H. Characterization of the DsRed2 Protein Expressed in 32138 SPT 

Maintainer 

Protein safety assessment studies were conducted on DsRed2 protein as it is the only protein 

not derived from a maize gene sequence incorporated into 32138 SPT maintainer. In order 

to obtain sufficient protein to conduct safety assessment studies, DsRed2 protein was 

expressed and purified by using an E.coli protein expression system. Characterization of the 

protein was achieved through the determination of purity, concentration, and identity. Purity 

was determined by utilizing a sodium dodecyl sulfate polyacrylamide gel electrophoresis 

(SDS-PAGE) method. Concentration was determined by using a modified Bradford assay 

and the identity was determined by utilizing Western blot analysis, electrospray mass 

spectroscopy, tryptic peptide analysis by liquid chromatography/mass spectrometry (LC/MS), 

N-terminal amino acid sequencing, and florescence activity assay. 

Small quantities of DsRed2 protein were also purified from 32138 SPT maintainer in order to 

evaluate and establish equivalency of the DsRed2 protein derived from the E.coli expression 

system with DsRed2 protein derived from 32138 SPT maintainter. 

Equivalence of the microbially expressed and plant-derived DsRed2 protein was achieved 

using the following analyses: 

1) SDS-PAGE to confirm equivalent molecular weight; 

2) Western blot analysis to confirm equivalent molecular weight and 

immunoreactivity; 

3) N-terminal amino acid sequence analysis to determine the identity of the 

proteins; 

4) Identification of tryptic peptides by Liquid chromatography/mass spectroscopy 

(LC/MS) to confirm the identity of the proteins; 

5) Glycoprotein staining to determine potential post-translational modification 

(glycosylation). 

A detailed description of the methods used in the equivalency studies and the resulting data 

are included in Appendix 10. 

Based on the results from the above analyses, the equivalency of the DsRed2 protein 

expressed in E. coli to the DsRed2 protein expressed in planta in 32138 SPT maintainer was 

demonstrated. Therefore, the DsRed2 protein derived from the microbial expression system 

was appropriate for utilization in safety assessment studies as a substitute for DsRed2 

protein expressed in the 32138 SPT maintainer. Microbially expressed DsRed2 was used 

subsequently for in vitro and in vivo safety assessment studies summarized in Section VI-I. 
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32138 SPT Maintainer No CBI 

VI-I. Summary of the Food and Feed Safety Assessment for the DsRed2 

Protein 

A food and feed safety assessment was conducted to assess the allergenicity and toxicity 

potential of the DsRed2 protein. The data and assessment form the basis of the conclusion 

that the DsRed2 protein is unlikely to cause an allergic reaction in humans or be a toxin in 

humans or animals, and therefore is safe for human and animal consumption. A detailed 

assessment of animal and human safety of the DsRed2 protein was submitted to FDA on 

October 11, 2006 as part of New Protein Consultation 004. The conclusions of the safety 

assessment are summarized below. 

Toxicological Safety Assessment 

1) The DsRed2 protein belongs to a family of red fluorescent proteins (RFP) which are 

members of a group of fluorescent proteins identified in several Anthozoa species. 

DsRed2 is a modified variant of the original RFP isolated from a coral-like anemone 

Discosoma sp. Although no evidence could be found regarding the specific use of the 

DsRed2 protein in the food industry, a DsRed variant has been successfully used as a 

vital marker in mice. There were no observed detrimental effects (Long et al., 2005), or 

signs of toxicity (Figueiredo et al., 2008), when DsRed variants were over expressed in 

mice. 

2) Plants stably expressing DsRed variants did not show any abnormalities, indicating that 

the protein did not interfere with plant growth, development, fertility, germination or 

morphogenesis (Jach et al., 2001; Dietrich and Maiss, 2002; Wenck et al., 2003; 

Mirabella et al., 2004; Stuitje et al., 2003). 

3) No biologically relevant amino acid sequence identities were observed between known 

protein toxins and the DsRed2 protein sequence. The DsRed2 protein sequence was 

queried using the BLASTP 2.2.13 algorithm against Release 165.0 (4/15/08) of the 

Genpept “nr” dataset. Bioinformatic analyses revealed the DsRed2 to be similar to 

other colored fluorescent proteins. Additionally, no evidence of toxicity has been 

observed in studies that have been conducted using RFPs or related green fluorescent 

proteins in transgenic plants and animals (Richards et al., 2003). These data support 

the conclusion that the DsRed2 protein is unlikely to be a toxin. 

4) There was no evidence of acute toxicity in mice at a target dose of 2000 mg (equivalent 

to approximately 1860 mg of DsRed2 protein) protein preparation per kg of body 

weight. To translate this type of mouse acute toxicity dose to human exposure, based 

on expression levels of the DsRed2 protein in 32138 SPT maintainer seed, a child 

weighing 10 kg would have to consume approximately 45 kg of 32138 SPT maintainer 

and an adult weighing approximately 60 kg would have to consume about 270 kg of 

32138 SPT maintainer seed. Based on these simplistic calculations, it is clear there is 

a wide margin of safety for the DsRed2 protein, especially considering other factors 

such as the low likelihood of exposure to this protein due to its use as part of an 

internal seed production process. 
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32138 SPT Maintainer No CBI 

Allergenicity Assessment 

5) The DsRed2 protein was evaluated for its allergenicity and toxicity potential using 

FDA’s published guidance for the early food safety evaluation of new proteins in new 

plant varieties (FDA, 2006). The allergenic potential of DsRed2 was assessed by: 1) 

bioinformatic comparison of the amino acid sequence of the DsRed2 protein with 

known or putative protein allergen sequences; 2) evaluation of the stability of the 

DsRed2 protein using an in vitro gastric digestion model; and 3) assessment of the 

DsRed2 gene source and history of use or exposure. 

6) The DsRed2 amino acid sequence was compared to a database of 1313 known and 

putative allergen sequences comprising the FARRP8 dataset at the University of 

Nebraska (updated 1/2008). Potential identities between the protein sequences and 

proteins in the allergen database were evaluated with the FASTA34 sequence 

alignment tool using the default parameters. A 35% or greater identity threshold over 

any 80 or greater amino acid length sequence between the query protein and an 

allergen was used to indicate the potential for cross-reactivity (FAO/WHO, 2001, and 

Codex, 2003). No identity matches of  35% over  80 residues were observed for the 

DsRed2 protein sequences. In addition, the DsRed2 protein sequence was evaluated 

for any 8 or greater contiguous identical amino acid matches to the allergens contained 

in the database noted above. There were no 8 or greater contiguous identical amino 

acid matches observed. These data indicate the lack of both amino acid identity and 

immunologically relevant similarities between the DsRed2 protein and known or 

putative allergens. 

7) The DsRed2 parent protein was rapidly hydrolyzed (less than 30 seconds) in simulated 

gastric fluid containing pepsin at pH 1.2 as demonstrated by SDS-PAGE analysis. A 

faint band was visible near the dye front around the 3kDa molecular weight marker and 

was visible through 60 minutes. This band is likely a mix of breakdown products from 

the DsRed2 protein.

8) Finally, the DsRed2 protein is not glycosylated (refer to Appendix 10). 

Exposure Assessment 

9) The DsRed2 protein will be used only as a screenable marker that will enable efficient 

detection and removal of maize seed that express the protein. The fluorescent protein 

will therefore be contained within an internal process and is not intended to be present 

in a commercial seed product. De minimis amounts of 32138 SPT maintainer seed and 

vegetative material may be used for feed. Any unused 32138 SPT maintainer inbred 

seed or discarded seed from color sorting (Figure 3, Stage II) will be disposed of using 

established procedures for the destruction of proprietary inbred materials. Discarded 

inbred parent seed and seed by-products (e.g., husks and cobs) will not enter into the 

commodity grain supply. Pioneer procedures for disposal of unwanted material include 

devitalization followed by landfills, composting, and/or incineration. Discard maize 

seed and by-products can also be fed directly to animals, in which case, the feed 

supplier must sign a purchase agreement that the materials will be utilized for farm use 

only and cannot be delivered or sold commercially. Outdated 32138 SPT maintainer 

seed inventory (i.e., lines that are no longer used as parents in commercial hybrid seed 

products) will be disposed of using the discard procedures described above. 

Therefore, exposure to the DsRed2 protein is extremely low. Results of the safety 

assessment indicate that the DsRed2 protein is safe for animal and human 

consumption. Based on the data and information provided in this submission, we have 

determined that the DsRed2 protein is unlikely to cause an allergic reaction in humans 

or be a toxin in humans or animals. 
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VII. Agronomic Performance and Ecological Observations

Agronomic evaluations were conducted to assess the agronomic comparability of 32138 SPT 

maintainer to conventional inbred maize. This evaluation also included observed responses 

to insect and disease stressors and formed the basis for Pioneer’s conclusion that 32138 

SPT maintainer is comparable to conventional inbred maize and plant pest risk is no more 

than conventional inbred maize. 

The agronomic evaluations were based on both laboratory experiments and replicated, multisite field trials conducted by agronomists and scientists who are considered experts in the 

production and evaluation of maize. For each assessment, 32138 SPT maintainer was 

compared to null segregant near-isoline inbred that did not contain the 32138 SPT insertion. 

To evaluate the agronomic characteristics of 32138 SPT maintainer, data were collected on 

representative characteristics that influence reproduction and survival of the crop. 

VII-A. Germination and Dormancy Evaluations 

Seed is considered germinated with the uptake of water (i.e., when there is visible elongation 

of embryonic axis and visible penetration of the structures surrounding the embryo by the 

radicle. Seed dormancy is regarded as the failure of an intact viable seed to complete 

germination under favorable conditions (Bewley, 1997). In maize, dormancy is extremely 

rare. Standardized germination tests are routinely used to assess any potential seed 

dormancy, which is considered an important weediness trait (Basu et al., 2004; Anderson, 

1996; Lingenfelter and Hartwig, 2007). 

In order to test the germination and potential dormancy of the 32138 SPT maintainer, seeds 

from the BC3S5 generation (see Figure 12) were tested under 3 different germination 

conditions. The Rules for Testing Seeds published by the Association of Official Seed 

Analysts (AOSA) in 2007 were used as general guidelines for the germination method and 

interpretation of results. 

a. Warm Germination Test: This test is conducted under optimal growing conditions, 

25o

C (78oF) at 90% relative humidity for 10 days. The AOSA recommends a 

temperature range of 20-30oC as optimal for germination of corn (AOSA, 2007). 

b. Cold Germination Test: This test is conducted at 10o

C (50oF) at 90% relative 

humidity for 10 days. The cold test is a stress test that simulates early spring 

planting conditions in the Midwestern US. 

c. Diurnal Test: Germination and potential dormancy were also evaluated at alternating 

temperatures of 25o

C and 10oC (78oF and 50oF) at 90% relative humidity for 10 days. 

This test simulates daily weather conditions in the major maize growing regions in the 

U.S. The lower temperature was maintained for 16 hours and the higher temperature 

for 8 hours. 

All three tests above included four replicates of 100 seeds of each entry: i) maize 32138 SPT 

maintainer, ii) near-isoline non-transgenic inbred control, and iii) three reference inbreds 

(Pioneer proprietary inbreds used to produce current commercial hybrid seed). All the tests 

were conducted in temperature-controlled growth chambers in the dark using a rolled towel 

test method. At the end of 10 days, the numbers of seeds germinated were counted and the 

percent germination was calculated. 

 

Germination rates were reported as a percentage of germinating seed as follows: (number of 

seeds germinated /total number of seeds)*100. 
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32138 SPT Maintainer No CBI 

Means were calculated for all germination data (Table 10). Statistical tests for differences 

were conducted at the significance level of = 0.05. For more details on the statistical 

analysis, refer to Appendix 11. No statistically significant differences were detected between 

the 32138 SPT maintainer, near-isoline inbred control, and the three reference inbreds for the 

warm and cold germination tests. No statistically significant differences were detected 

between the 32138 SPT maintainer, near-isoline inbred control, and two of the three 

reference inbreds for the diurnal test. A statistically significant difference was observed for 

the diurnal test between the 32138 SPT maintainer and one of the reference inbreds used 

(reference inbred B) as this reference inbred had a higher germination rate under the diurnal 

test. 

Seeds that did not germinate in these tests were tested for viability using the Tetrazolium 

Chloride (TZ) test. The TZ test was developed as a color test for seed viability; viable but 

non-germinated seed would be an indicator of potential dormancy 

(http://www.seedlab.oscs.orst.edu/Page_Technical_Brochures/ValueTZTests.htm). All of the non-germinated 

seeds tested were non-viable (data not shown). Hence, there was no dormancy observed 

amongst any of the entries. 

Germination and dormancy characteristics of the 32138 SPT maintainer were not altered 

when compared to other inbred maize. The 32138 SPT maintainer exhibited no dormancy 

tendencies and is unlikely to present an increased potential for weediness as compared to 

the conventional inbred maize. 

Table 10. Summary of Germination Results for 32138 SPT Maintainer 

a. Warm Germination Test

Parameter 32138 SPT 

Maintainer 

Near-isoline 

Inbred 

Reference 

Inbred A 

Reference 

Inbred B 

Reference 

Inbred C 

 % Germination 

Mean 99 98.5 97.51 98.75 99.5 

Range 98 - 100 98 - 99 95 - 99 97 - 100 98 - 100 

P-value 0.549 0.153 0.752 0.441 

b. Cold Germination Test

Parameter 32138 SPT 

Maintainer 

Nearisoline 

Inbred 

Reference 

Inbred A 

Reference 

Inbred B 

Reference 

Inbred C 

% Germination

Mean 96.25 97 97.25 95.75 94.75 

Range 94 - 98 96 - 99 96 - 99 95 - 96 93 - 96 

P-value 0.565 0.437 0.723 0.322 

c. Diurnal Test

Parameter 32138 SPT 

Maintainer 

Near-isoline 

Inbred 

Reference 

Inbred A 

Reference 

Inbred B 

Reference 

Inbred C 

% Germination

Mean 96.83 93.66 97.79 99.75 97.34 

Range 92 - 100 92 - 97 93 - 100 99 - 100 95 - 99 

P-value 0.217 0.560 0.03441 0.767 

1

Statistically significant difference, P-value <0.05.
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32138 SPT Maintainer No CBI 

VII-B. Field Trial Evaluations 

The 32138 SPT maintainer has been field tested in the United States since 2005 as 

authorized by USDA permits and notifications listed in Appendix 12. The list compiles a 

number of test sites in diverse regions of the U.S. including the major crop-growing areas and 

off season nurseries in Hawaii and Puerto Rico. Agronomic data were collected to assess 

agronomic comparability as it relates to plant pest potential. 

Also, throughout the crop growing season, 32138 SPT maintainer was observed for 

unexpected differences in response to abiotic stress (e.g., drought, excess moisture, 

abnormal temperatures, etc.). Monthly observations for response to naturally occurring 

abiotic stressors indicated that 32138 SPT maintainer and near-isoline inbred controls were 

similar with respect to their response to abiotic stress. 

Agronomic data were collected from 32138 SPT maintainer and control maize grown at six 

different locations in 2007 (Figure 47). The number of locations is sufficient because 32138 

SPT maintainer will be grown on only a small number of acres (<5,000) that are under 

Pioneer control. The trial locations provide a range of environmental conditions 

representative of where 32138 SPT maintainer seed production is expected. Agronomic 

practices used to prepare and maintain each field site were determined based on the 

geographic location of each site. 

The purpose of this experiment was to evaluate the agronomic characteristics of 32138 SPT 

maintainer (Table 11) and also to collect tissue samples for measurement of ZM-AA1 and 

DsRed2 protein concentrations (Section VIE, Tables 8 and 9) and nutrient composition 

analyses (Section VIII, Tables 13-19). Seed from the BC3S3 generation was used (Figure 

12). The control plants were non-transgenic near-isoline inbred 705. 

The following agronomic characteristics were measured: early population, final population, 

seedling vigor, time to silking, time to pollen shed, stalk lodging, root lodging, stay green, 

disease incidence, insect damage, plant height, ear height, and pollen viability (i.e., shape 

and color) over time. Table 11 describes the quantitative field agronomic characteristics that 

were collected during the 2007 growing season. 

Pollen collected was evaluated using a magnifying lens for shape and color at various time 

points in order to evaluate pollen viability of 32138 SPT maintainer and inbred control maize 

over time. The correlation between visual appearance and viability of pollen is well 

documented (Luna et al., 2001). Pollen can be viable, but not fertile as seen in the case of 

50% of the pollen from 32138 SPT maintainer pollen (Figure 9). There were no 

morphological (shape, size, and color) differences between the pollen from inbred 705 control 

and fertile and infertile pollen from 32138 SPT maintainer. 

Each site employed a randomized complete block design containing four blocks, of which 

three blocks were used for analysis of agronomic characteristics. Each block was separated 

by an alley distance of at least 36 inches (in.) (0.9 meters (m)) and contained one-row plots 

planted with maize 32138 SPT maintainer or the near-isoline inbred control. The 32138 SPT 

maintainer inbred and the near-isoline inbred control were planted at a rate of approximately 

30 seeds per row. Each row was approximately 25 ft (7.6 m) in length, with seed spacing of 

approximately 10 in. (0.25 m). Spacing between rows was approximately 30 in. (0.76 m).

Each one-row plot was bordered on either side by one row of maize inbreds of different 

genetic background. Normal agronomic practices were employed throughout the growing 

season. 

A statistical analysis of agronomic data was conducted to test for differences in the mean 

values between the 32138 SPT maintainer and the near-isoline inbred control (refer to 
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32138 SPT Maintainer No CBI 

Appendix 11 for statistical model used). The false discovery rate (FDR) adjustment of Pvalues being employed by Pioneer conservatively addresses the issues associated with 

multiple comparative testing while retaining the power for detecting real differences. 

Results of the agronomic trials are summarized in Table 12. For all characteristics measured 

(early population, final population, seedling vigor, time to silking, time to pollen shed, stalk 

lodging, root lodging, stay green, disease incidence, insect damage, plant height, ear height, 

and pollen viability over time), no statistical differences in mean values were observed 

between 32138 SPT maintainer and near-isoline inbred control maize across locations 

(adjusted P-value > 0.05). These results indicate that 32138 SPT maintainer is agronomically 

comparable to near-isoline inbred control maize. 

Agronomic performance of the reference inbreds was used to help determine the normal 

variation for the agronomic characteristics measured in the inbred. Using the data obtained 

from the reference inbreds, a statistical tolerance interval was calculated to contain, with 95% 

confidence, 99% of the values contained in the inbred population. This statistical tolerance 

interval provided further context for the interpretation of the agronomic performance of 32138 

SPT maintainer. The agronomic measurements in 32138 SPT maintainer that fell within the 

tolerance interval were considered to be within the range of normal variability of maize 

inbreds. 

Figure 47. Identification of Agronomic Field Trials for 32138 SPT Maintainer 

 Red stars indicate approximate location of agronomic field trial sites 
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32138 SPT Maintainer No CBI 

Table 11. Field Agronomic Characteristics Measured 

General 

Characteristic 

Characteristic 

Measured 

Evaluation 

Timing1 Data Description Scale 

Early 

Population V2-V4 Number of plants emerged per plot 

Actual count per 

plot 

Germination / 

Emergence 

Seedling Vigor V2-V4 

Visual estimate of 

average vigor of 

emerged plants per plot 

From 1 to 9, where 

1=short plants with 

small leaves, and 

9=tall plants with 

large leaves 

Plant Height Approximately 

R6 

Height from the soil 

surface to the tip of the 

tassel 

Height in cm 

Ear Height Approximately 

R6 

Height from the soil 

surface to the base of 

the primary ear 

Height in cm 

Stalk Lodging Approximately 

R6 

Visual estimate of 

percent of plants in the 

plot with stalks broken 

below the primary ear 

0 to 100% 

Root Lodging Approximately 

R6 

Visual estimate of 

percent of plants in the 

plot leaning 

approximately 30 or 

more in the first 2 feet 

(0.6 m) above the soil 

surface. 

0 to 100% 

Final 

Population 

Approximately 

R6 

The number of plants 

remaining per plot 

Actual count per 

plot 

Vegetative 

Parameters 

Stay Green Approximately 

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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32138 SPT Maintainer No CBI 

Table 11. Field Agronomic Characteristics Measured (continued)

General 

Characteristic

Characteristic 

Measured 

Evaluation 

Timing1 Data Description Scale 

Time to Silking Approximately 

50% silking 

From the time of 

planting until 

approximately 50% of 

the plants have 

emerged silks 

Number of 

accumulated 

Growing Degree 

Units (GDU) 

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 

Growing Degree 

Units (GDU) 

Pollen Shape 

Percentage of 

pollen grains with 

collapsed walls at 

0, 30, 60 & 120 

minutes 

Reproductive 

Parameters 

Pollen Viability 

Approximately 

50% pollen 

shed 

Pollen Color 

Percentage of 

pollen grains with 

intense yellow color 

at 0, 30, 60 & 120 

minutes 

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 Ecological 

Interactions 

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 

Footnotes: 

1= Refer to Ritchie et al., 1993 for a description of maize growth stages. 
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32138 SPT Maintainer No CBI 

Table 12. Agronomic Performance of 32138 SPT Maintainer Across Six Locations 

Agronomic Characteristic1 

(Unit) Control 32138 SPT 

Maintainer 

Tolerance 

Interval 

Mean2 28 28 

Range3 26 - 30 24 - 30 

Adjusted P-value4 0.945 

Early Population 

(number of 

plants) P-value5 0.728 

8 - 26 

Mean 26 27 

Range 17 - 29 23 - 29 

Adjusted P-value 0.945 

Final Population 

(number of 

plants) P-value 0.53 

11-29 

Mean 8 8 

Range 7 - 9 5 - 9 

Adjusted P-value 0.799 

Seedling Vigor 

(1-9 scale) 

P-value 0.138 

4 - 9 

Mean 1420 1410 

Range 1330 - 1570 1340 - 1550 

Adjusted P-value 0.975 

Time to Silking 

(growing degree 

units) P-value 0.874 

1120 - 1750 

Mean 1450 1440 

Range 1350 - 1640 1340 - 1640 

Adjusted P-value 0.956 

Time to Pollen 

Shed 

(growing degree 

units) P-value 0.791 

1060 - 1880 

Mean 2 1 

Range 0 - 15 0 - 5 

Adjusted P-value 0.801 

Stalk Lodging 

(% plants 

broken below 

primary ear) P-value 0.363 

0 - 29.3 

Mean 0 0.1 

Range 0 - 0 0 - 2 

Adjusted P-value NA6

Root Lodging 

(% plants 

leaning ~30 or 

more) P-value NA 

NA 

Mean 5 4 

Range 1 - 7 2 - 7 

Adjusted P-value 0.799 

Stay Green 

(1-9 scale) 

P-value 0.276 

1 - 9 

Mean 6 6 

Range 5 - 8 5 - 8 

Adjusted P-value 0.945 

Disease 

Incidence 

(1-9 scale) P-value 0.711 

1 - 9 

Mean 6 6 

Range 5 - 8 5 - 7 

Adjusted P-value 0.945 

Insect Damage 

(1-9 scale) 

P-value 0.737 

1 - 9 

Mean 212 211 

Range 181 - 230 189 - 224 

Adjusted P-value 0.945 

Plant Height 

(cm) 

P-value 0.602 

128 - 299 
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32138 SPT Maintainer No CBI 

Table 12. Agronomic Performance of 32138 SPT Maintainer across Six Locations 

(continued)

Agronomic Characteristic (Unit) Control 32138 SPT 

Maintainer 

Tolerance 

Interval 

Mean 75 76 

Range 55 - 86 61 - 87 

Adjusted P-value 0.799 

Ear Height 

(cm) 

P-value 0.187 

40.7 - 123 

Pollen Viability (Shape) - % (0 to 100) pollen grains with collapsed walls 

Mean 10 12 

Range 0 - 90 0 - 90 

Adjusted P-value 0.799 

0 minutes 

(%) 

P-value 0.259 

0 - 94.9 

Mean 37 37 

Range 0 - 99 0 - 99 

Adjusted P-value 1 

30 minutes 

(%) 

P-value 1 

0 - 100 

Mean 71 68 

Range 30 - 100 10 - 100 

Adjusted P-value 0.739 

60 minutes 

(%) 

P-value 0.102 

0 - 100 

Mean 98 98 

Range 80 - 100 90 - 100 

Adjusted P-value 0.801 

120 minutes 

(%) 

P-value 0.363 

83.6 - 100 

Pollen Viability (Color) - % (0 to 100) pollen grains with intense yellow 

color

Mean 9 11 

Range 0 - 70 0 - 70 

Adjusted P-value 0.799 

0 minutes 

(%) 

P-value 0.275 

0 - 71.1 

Mean 35 38 

Range 0 - 90 0 - 90 

Adjusted P-value 0.801 

30 minutes 

(%) 

P-value 0.387 

0 - 100 

Mean 65 64 

Range 20 - 95 25 - 100 

Adjusted P-value 0.945 

60 minutes 

(%) 

P-value 0.741 

0 - 100 

Mean 94 93 

Range 75 - 100 60 - 100 

Adjusted P-value 0.945 

120 minutes 

(%) 

P-value 0.75 

54.3 - 100 

Footnotes: 

 1. Refer to Table 11 for descriptions of each agronomic characteristic measured. 

2. Least squares mean 

3. Range denotes the lowest and highest individual value across sites. 

4. False Discovery Rate (FDR) adjusted P-value 

5. Non-adjusted P-value 

6. Not applicable
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32138 SPT Maintainer No CBI 

VII-C. Ecological Observations 

Ecological observations (plant interactions with insects and diseases) were recorded for all 

USDA-APHIS permitted field trials of 32138 SPT maintainer during the 2005-2008 growing 

seasons. Plant breeders and field staff that are experts in the fields of plant pathology and 

entomology observed 32138 SPT maintainer and control lines at least every four weeks for 

insect and disease pressure and recorded the severity of any stressor seen. Any unexpected 

differences in response between 32138 SPT maintainer and various control lines (null 

segregants, near-isoline and/or conventional maize inbred lines) were recorded. 

The following scale was used when recording observations: 

 mild – very little disease or insect injury (<10%) visible; 

 moderate – noticeable plant tissue damage (10% to 30%); 

 severe – significant plant tissue damage (>30%). 

A summary of the insect and disease ecological observations is presented in Appendix 13. In 

every case, the severity of insect or disease stress on 32138 SPT maintainer was not 

qualitatively different from various control lines grown at the same location. These results 

support the conclusion that the ecological interactions for 32138 SPT maintainer were 

comparable to control maize lines with similar genetics or to conventional inbred maize lines. 

VII-D. Conclusions on Agronomic Performance and Ecological Observations 

32138 SPT maintainer was observed in experiments at six field locations to measure 

agronomic data. Data generated from these studies represent observations that are typically 

recorded by plant breeders and agronomists to evaluate the characteristics of maize over a 

range of environmental conditions that 32138 SPT maintainer would encounter. The 

measured characteristics provide crop biology data useful in establishing a basis to assess 

agronomic comparability and familiarity of 32138 SPT maintainer to conventional inbred 

maize in the context of ecological risk assessment. 

The agronomic data demonstrated no biologically meaningful differences between 32138 

SPT maintainer and inbred control maize (near-isoline of 32138 SPT maintainer and/or 

conventional maize inbred lines) with respect to germination/emergence, vegetative growth, 

reproductive parameters, yield, and ecological interactions. These data support the 

conclusion that 32138 SPT maintainer is comparable in agronomic characteristics to 

conventional inbred maize. 

Monthly observations of all USDA-permitted field trials for responses of 32138 SPT 

maintainer to naturally occurring insect and disease stressors demonstrated no unexpected 

differences from inbred control maize. In addition, assessment of the ecological data 

detected no biologically significant differences between 32138 SPT maintainer and inbred 

control maize lines indicative of a selective advantage that would result in increased weed 

potential or plant pest risk for 32138 SPT maintainer. 
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32138 SPT Maintainer No CBI 

VIII. Compositional Assessment 

Compositional analysis of maize 32138 SPT maintainer was used to evaluate any changes in 

the levels of key nutrients, anti-nutrients, and secondary metabolites compared to the nearisoline inbred control and two reference inbreds. Along with agronomic data, compositional 

similarity is a general indicator that the 32138 SPT maintainer will not exhibit unexpected 

effects with respect to plant pest risk. 

Comprehensive compositional analyses were performed on seed from 32138 SPT maintainer 

and a non-transgenic near-isoline inbred control (inbred 705), grown in 2007 at six field 

locations in maize growing areas of the Midwest (Figure 47, see Section VII-B for details on 

field trials). 

Seed samples from were also collected from two non-transgenic, self-pollinated maize 

inbreds (reference inbreds) grown in 2007 at the same six field locations as the 32138 SPT 

maintainer (Figure 47). The reference inbreds were planted, harvested, processed, and 

analyzed using the same methods as those employed for the near-isoline inbred control and 

maize 32138 SPT maintainer. Compositional analysis of the reference inbreds was used to 

help determine the normal variation for the measured analytes in inbred seed. Using the data 

obtained from the reference inbreds, a statistical tolerance interval was calculated to contain, 

with 95% confidence, 99% of the values contained in the inbred population. This statistical 

tolerance interval provided further context for interpretation of the composition results for 

32138 SPT maintainer. The analyte ranges in 32138 SPT maintainer that fell within the 

tolerance interval were considered to be within the range of normal variability of maize 

inbreds. 

The compositional assessment was conducted in accordance with the OECD consensus 

document on compositional considerations for new varieties of maize (OECD, 2002). 

Compositional analyses included protein, fat, acid detergent fiber (ADF), neutral detergent 

fiber (NDF), ash, carbohydrates, fatty acids, amino acids, vitamins and minerals, key antinutrients (raffinose, phytic acid and trypsin inhibitor) and key secondary metabolites (furfural, 

ferulic acid, and p-coumaric acid). 

Statistical analysis of nutrient composition data was conducted to test for differences in the 

analyte mean values between the 32138 SPT maintainer and the near-isoline inbred control 

(for details on the statistical methodology used, refer to Appendix 11). Pioneer applied False 

Discovery Rate (FDR) adjustment of p-values in order to conservatively address the issues 

associated with multiple comparative testing while retaining the power for detecting real 

differences. 
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32138 SPT Maintainer No CBI 

VIII-A. Proximates 

Proximates were analyzed in 32138 SPT maintainer and near-isoline inbred control maize 

seed. Results are shown in Table 13. No statistically significant differences were observed 

between 32138 SPT maintainer and inbred control maize mean values for fat, acid detergent 

fiber (ADF), neutral detergent fiber (NDF), ash and carbohydrates (adjusted P-values > 0.05). 

A statistically significant difference was observed for crude protein (adjusted P-value < 0.05) 

however the individual protein values were within the tolerance interval. 

In conclusion, analysis of proximates demonstrates that 32138 SPT maintainer is comparable 

to near-isoline and reference maize inbreds. 

Table 13. Proximates in the Seed of Control and 32138 SPT Maintainer 

Analyte (% Dry Weight) Control 32138 SPT 

Maintainer 

Tolerance

Interval 

Mean1 10.7 11.4 

Range2 9.59-11.3 9.00-12.4 

Adjusted P-value3 0.0116

Crude Protein 4

5

P-value 0.0014 

7.55-13.8 

Mean 3.14 3.36 

Range 2.03 - 3.61 2.47-4.25 

Adjusted P-value 0.352 

Fat 

P-value 0.234 

1.91-4.38 

Mean 3.76 3.81 

Range 2.82-4.59 2.92-4.66 

Adjusted P-value 0.777 

ADF 

P-value 0.697 

0-8.12 

Mean 9.88 10.0 

Range 7.79-12.4 6.31 – 14.8 

Adjusted P-value 0.87 

NDF 

P-value 0.816 

4.35 – 27.4

Mean 1.36 1.49 

Range 1.02 – 1.54 1.23-1.79 

Adjusted P-value 0.127 

Ash 

P-value 0.0559 

0.559 - 2.80

Mean 84.7 83.8 

Range 83.9-87.0 82.4-86.8 

Adjusted P-value 0.0732 

Carbohydrates 

P-value 0.0247 

80.7-89.1 

Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations. 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. The analyte values were transformed for statistical analysis. Means and confidence intervals have been 

back transformed to the data scale. 
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32138 SPT Maintainer No CBI 

VIII-B. Fatty Acids 

Concentrations of 29 fatty acids were measured in 32138 SPT maintainer and near-isoline 

inbred control maize seed. Levels of 16 fatty acids were below the limit of quantitation for the 

assay: caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), 

myristoleic acid (C14:1), pentadecanoic acid (C15:0), pentadecenoic acid (C15:1), 

heptadecadienoic acid (C17:2), -linolenic acid (C18:3), nonadecanoic acid (C19:0), 

eicosadienoic acid (C20:2), eicosatrienoic acid (C20:3), arachidonic acid (C20:4), 

heneicosanoic acid (C21:0), erucic acid (C22:1) and tricosanoic acid (C23:0). Therefore, no 

statistical analyses were conducted on these fatty acids and data are not shown. 

Results of the fatty acid analysis are presented in Table 14. No statistically significant 

differences (adjusted P-values > 0.05) were observed between the 32138 SPT maintainer 

and near-isoline inbred control maize for the fatty acid mean values for palmitic acid (C16:0), 

palmitoleic acid (C16:1), heptadecenoic acid (C17:1), stearic acid (C18:0), linoleic acid 

(C18:2), and eicosenoic acid (C20:1). Statistically significant differences (adjusted P-values 

< 0.05), were observed for heptadecanoic acid (C17:0), oleic acic (C18:1), and linolenic acid 

(C18:3), arachidic acid (C20:0), behenic acid (C22:0) and lignoceric acid (C24:0), however 

the individual values for these fatty acids were within the respective tolerance interval. 

In conclusion, fatty acid analysis demonstrates that 32138 SPT maintainer is comparable to 

near-isoline and reference maize inbreds. 
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32138 SPT Maintainer No CBI 

Table 14. Major Fatty Acids in the Seed of Control and 32138 SPT Maintainer 

Analyte (% Total Fatty Acids) Control 32138 SPT 

Maintainer 

Tolerance

Interval

Mean1 11.0 11.2 

Range2 10.4 - 11.5 10.5 - 12.1 

Adjusted P-value3 0.111 

Palmitic acid 

(C16:0) 

P-value 0.0467 

6.62 - 15.3

Mean 0.195 0.183 

Range 0 - 0.395 0.124 - 0.343 

Adjusted P-value 0.588 

Palmitoleic acid 

(C16:1) 

P-value 0.467 

0 - 0.319

Mean 0.182 0.166 

Range 0.166 - 

0.211 0.147 - 0.202 

Adjusted P-value 0.0114

Heptadecanoic acid 

(C17:0) 

P-value 0.000835 

0 - 0.263 

Mean 0.125 0.104 

Range 0 - 0.147 0 – 0.110 

Adjusted P-value NA 

Heptadecenoic acid 

(C17:1) 

P-value NA 

0 – 0.1206

Mean 2.17 2.06 

Range 1.97 - 2.37 1.89 - 2.34 

Adjusted P-value 0.0558 

Stearic acid 

(C18:0) 

P-value 0.0177 

1.04 - 3.31

Mean 19.0 20.9 

Range 17.5 - 20.8 18.9 - 23.3 

Adjusted P-value 0.00005714

Oleic acid 

(C18:1) 

P-value 0.000000394 

6.75 - 45.3

Mean 56.9 56.9 

Range 53.2 - 59.5 52.9 - 60.2 

Adjusted P-value 0.985 

Linoleic acid5

(C18:2) 

P-value 0.985 

27.1 - 72.7

Mean 2.67 2.32 

Range 2.44 - 3.07 2.01 - 2.61 

Adjusted P-value 0.0002854

Linolenic acid 

(C18:3) 

P-value 0.00000399 

1.62 - 3.42

Mean 0.407 0.445 

Range 0.366 - 

0.467 0.384 - 0.533 

Adjusted P-value 0.01164

Arachidic acid 

(C20:0) 

P-value 0.00146 

0.179 - 

0.778 

Mean 0.307 0.278 

Range 0.281 - 

0.350 0.258 - 0.312 

Adjusted P-value 0.131 

Eicosenoic acid 5

(C20:1) 

P-Value 0.0635 

0.0899 - 

0.656 

Mean 0.431 0.370 

Range 0.362 - 

0.558 0.290 - 0.504 

Adjusted P-value 0.01164

Behenic acid 

(C22:0) 

P-value 0.00176 

0.241 - 

0.856 
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32138 SPT Maintainer No CBI 

Table 14. Major Fatty Acids in the Seed of Control and 32138 SPT Maintainer 

(continued) 

Analyte (% Total Fatty Acids) Control 32138 SPT 

Maintainer 

Tolerance

Interval

Mean 0.582 0.423 

Range 0.475 - 0.772 0.302 - 0.654 

Adjusted P-value 0.001614

Lignoceric acid 5

(C24:0) 

P-value 0.0000668 

0.152 - 

1.00 

Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. Analyte values were transformed for statistical analysis. Means and confidence intervals have been 

back transformed to the data scale. 

 6. No tolerance interval could be calculated; these values are minimum/maximum ranges
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32138 SPT Maintainer No CBI 

VIII-C. Total Amino Acids 

Concentrations of 20 amino acids were measured in 32138 SPT maintainer and near-isoline 

inbred control maize seed. Results are presented in Table 15. No statistically significant 

differences (adjusted P-values > 0.05) were observed between 32138 SPT maintainer and 

near-isoline inbred control maize for the amino acid mean values for cystine, lysine, 

threonine, isoleucine, histidine, valine, leucine, arginine, phenylalanine, glycine, glutamic 

acid, proline, serine, and tyrosine. Statistically significant differences (adjusted P-values < 

0.05), were observed for methionine, tryptophan, alanine, and aspartic acid, however the 

individual values for these amino acids were within the respective tolerance intervals. 

In conclusion, total amino acid analysis demonstrates that 32138 SPT maintainer is 

comparable to near-isoline and reference maize inbreds
Pioneer Hi-Bred Page 114 of 188 

32138 SPT Maintainer No CBI 

Table 15. Total Amino Acids in the Seed of Control and 32138 SPT Maintainer 

Analyte (% Dry Weight) Control 32138 SPT 

Maintainer 

Tolerance

Interval

Mean1 0.238 0.208 

Range2 0.182 - 0.297 0.170 - 0.257 

Adjusted P-value3 0.0133 Methionine 4

P-value5 0.00211 

0 - 0.459 

Mean 0.211 0.201 

Range 0.128 - 0.269 0.132 - 0.277 

Adjusted P-value 0.572 Cystine 

P-value 0.450 

0.0199 - 

0.376 

Mean 0.317 0.314 

Range 0.215 - 0.417 0.274 - 0.405 

Adjusted P-value 0.870 Lysine5

P-value 0.815 

0.131 - 0.509

Mean 0.103 0.0880 

Range 0.0860 - 0.126 0.0771 - 0.0991 

Adjusted P-value 0.0163 Tryptophan 4

P-value 0.00281 

0.0234 - 

0.171 

Mean 0.366 0.381 

Range 0.282 - 0.399 0.302 - 0.419 

Adjusted P-value 0.318 

Threonine 

P-value 0.188 

0.294 - 0.475

Mean 0.396 0.411 

Range 0.330 - 0.422 0.326 - 0.455 

Adjusted P-value 0.321 Isoleucine5

P-value 0.195 

0.287 - 0.498

Mean 0.304 0.313 

Range 0.235 - 0.344 0.243 - 0.338 

Adjusted P-value 0.437 

Histidine5

P-value 0.317 

0.211 - 0.401

Mean 0.509 0.536 

Range 0.420 - 0.537 0.443 - 0.579 

Adjusted P-value 0.0957 

Valine5

P-value 0.037 

0.397 - 0.644

Mean 1.41 1.50 

Range 1.25 - 1.52 1.26 - 1.71 

Adjusted P-value 0.0957 Leucine 

P-value 0.0357 

0.994 - 1.82

Mean 0.437 0.451 

Range 0.330 - 0.494 0.383 - 0.515 

Adjusted P-value 0.472 Arginine 

P-value 0.350 

0.175 - 0.694
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32138 SPT Maintainer No CBI 

Table 15. Total Amino Acids in the Seed of Control and 32138 SPT Maintainer 

(continued) 

Analyte (% Dry Weight) Control 32138 SPT 

Maintainer 

Tolerance

Interval 

Mean 0.559 0.596 

Range 0.468 - 0.612 0.477 - 0.685 

Adjusted P-value 0.119 Phenylalanine 

P-value 0.0508 

0.400 - 0.722

Mean 0.396 0.403 

Range 0.295 - 0.448 0.330 - 0.438 

Adjusted P-value 0.635 Glycine5

P-value 0.525 

0.256 - 0.537

Mean 0.823 0.883 

Range 0.714 - 0.865 0.801 - 0.971 

Adjusted P-value 0.0313 Alanine 4

P-value 0.00726 

0.629 - 1.05 

Mean 0.744 0.811 

Range 0.628 - 0.824 0.743 - 0.936 

Adjusted P-value 0.0332 Aspartic Acid 4

P-value 0.00859 

0.513 - 0.960

Mean 2.23 2.39 

Range 1.80 - 2.40 1.77 - 2.63 

Adjusted P-value 0.100 Glutamic Acid5

P-value 0.0395 

1.70 - 2.85 

Mean 0.955 0.989 

Range 0.883 - 1.01 0.852 - 1.08 

Adjusted P-value 0.209 Proline 

P-value 0.114 

0.679 - 1.26 

Mean 0.498 0.523 

Range 0.366 - 0.540 0.364 - 0.594 

Adjusted P-value 0.326 Serine 

P-value 0.207 

0.373 - 0.646

Mean 0.252 0.268 

Range 0.188 - 0.303 0.199 - 0.349 

Adjusted P-value 0.352 Tyrosine 

P-value 0.232 

0.127 - 0.377

 

 Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. The analyte values were transformed for statistical analysis. Means and confidence intervals have been 

back transformed to the data scale. 
Pioneer Hi-Bred Page 116 of 188 

32138 SPT Maintainer No CBI 

VIII-D. Vitamins and Minerals 

Vitamins and minerals were measured in 32138 SPT maintainer and near-isoline inbred 

control maize seed. Based on OECD guidance, the following vitamins were analyzed: betacarotene, vitamin B1 (thiamin), vitamin B2 (riboflavin), vitamin B6 (pyridoxine), vitamin B3 

(niacin), vitamin B9 (folic acid) and -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 presented in Table 17. The 

level of vitamin B2 (riboflavin) was below the limit of quantitation for the assay and therefore 

no statistical analysis was conducted on this analyte. No statistically significant differences 

(adjusted P-values > 0.05) were observed between the 32138 SPT maintainer and nearisoline inbred control maize mean values for beta-carotene, vitamin B1 (thiamin), vitamin B6 

(pyridoxine), vitamin B9 (folic acid), vitamin B5 (pantothenic acid), -tocopherol, copper, 

phosphorus, and sodium. Statistically significant differences (adjusted P-values < 0.05), were 

observed for vitamin B3, calcium, iron, magnesium, potassium, and zinc, however the 

individual values for these analytes were within the respective tolerance interval. 

In conclusion, vitamin and mineral analyses demonstrate that 32138 SPT maintainer is 

comparable to near isoline and reference maize inbreds.
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32138 SPT Maintainer No CBI 

Table 16. Vitamins in the Seed of Control and 32138 SPT Maintainer 

Analyte(% Dry Weight) Control 32138 SPT 

Maintainer 

Tolerance

Interval

Mean1 6.44 6.79 

Range2 <2.506

- 11.1 <2.506- 13.8 

Adjusted P-value3 0.709 

Betacarotene5

P-value 0.599 

0 - 27.0 

Mean 2.96 3.47 

Range <1.806 - 4.43 2.84 - 3.93 

Adjusted P-value 0.127 

Vitamin B1 

(Thiamin) 

P-value 0.0571 

1.39 - 6.14 

Mean <0.9006

 <0.9006

Range <0.9006

 <0.9006

Adjusted P-value NA 

Vitamin B2 

(Riboflavin) 

P-value NA 

NA7 

Mean 6.52 7.03 

Range 4.52 - 10.1 5.39 - 8.84 

Adjusted P-value 0.376 

Vitamin B65

(Pyridoxine) 

P-value 0.257 

0 - 14.7

Mean 18.3 15.4 

Range 12.7 - 23.4 12.4 - 21.3 

Adjusted P-value 0.01634

Vitamin B3 

(Niacin) 

P-value 0.00281 

8.99 - 61.3

Mean 0.754 0.778 

Range 0.540 - 1.11 0.495 - 1.08 

Adjusted P-value 0.820 

Folic acid5

P-value 0.746 

0 - 2.33 

Mean 8.54 9.81 

Range 3.81 - 17.7 3.05 - 22.3 

Adjusted P-value 0.387 -tocopherol5

P-value 0.269 

0 - 16.5 

Mean 5.25 5.44 

Range 3.57-7.41 4.89-6.16 

Adjusted P-value 0.754 

Vitamin B5 

Pantothenic 

acid (mg/kg) P-value 0.671 

0.600-10.1 

 

Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. The analyte values were transformed for statistical analysis. Means and confidence intervals have been back 

transformed to the data scale. 

6. < Lower Limit of Quantitation (LLOQ); indicates that the values of the sample or samples were detected 

below the assay LLOQ 

7. Not Applicable 
Pioneer Hi-Bred Page 118 of 188 

32138 SPT Maintainer No CBI 

Table 17. Minerals in the Seed of Control and 32138 SPT Maintainer 

Analyte (% Dry Weight) Control 32138 SPT 

Maintainer 

Tolerance

Interval

Mean1 0.00295 0.00340 

Range2 0.00233 - 0.00382 0.00252 - 0.00505 

Adjusted P-value3 0.0116

Calcium 4

5

P-value 0.00121 

0 - 0.0127 

Mean 0.0000494 0.0000913 

Range <0.00006256

- 

0.000205 

<0.00006256 - 

0.000161 

Adjusted P-value 0.0539 

Copper5

P-value 0.0167 

0 - 0.000373

Mean 0.00167 0.00183 

Range 0.00139 - 0.00197 0.00154 - 0.00209 

Adjusted P-value 0.01734 Iron5

P-value 0.00322 

0.000721 - 

0.00290 

Mean 0.124 0.144 

Range 0.0907 - 0.156 0.119 - 0.163 

Adjusted P-value 0.0193 Magnesium 4

5

P-value 0.00386 

0.0303 - 0.178

Mean 0.345 0.366 

Range 0.245 - 0.409 0.319 - 0.412 

Adjusted P-value 0.173 Phosphorus 

P-value 0.0905 

0.0807 - 0.506

Mean 0.321 0.362 

Range 0.255 - 0.380 0.322 - 0.416 

Adjusted P-value 0.011

Potassium 4

P-value 0.00071 

0.191 - 0.446

Mean 0.0000104 0.0000510 

Range <0.00006256 - 

0.000139 

<0.00006256 - 

0.000281 

Adjusted P-value 0.0957 

Sodium5

P-value 0.0353 

0.0000625 - 

0.0005477

Mean 0.00245 0.00282 

Range 0.00202 - 0.00330 0.00228 - 0.00322 

Adjusted P-value 0.01164

Zinc 4

5

P-value 0.00157 

0.000482 - 

0.00426 

Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. The analyte values were transformed for statistical analysis. Means and confidence intervals have been 

back transformed to the data scale. 

6. < Lower Limit of Quantitation (LLOQ); indicates that the values of the sample or samples were detected 

below the assay LLOQ 

7. No tolerance interval could be calculated; these values are minimum/maximum ranges.
Pioneer Hi-Bred Page 119 of 188 

32138 SPT Maintainer No CBI 

VIII-E. Key Anti-nutrients 

Maize seed contains several key anti-nutrients: raffinose, phytic acid and trypsin inhibitor 

(OECD, 2002). Raffinose is a low molecular weight carbohydrate that is non-digestible. 

Phytic acid binds most of the phosphorus in maize, which results in reduced bioavailability of 

phosphorus for non-ruminant animals. 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 32138 SPT maintainer and near-isoline inbred 

control seed. Results are shown in Table 18. No statistically significant difference (adjusted 

P-value > 0.05) was observed between the mean values for 32138 SPT maintainer and nearisoline inbred control maize for trypsin inhibitor. Statistically significant differences (adjusted 

P-values < 0.05), were observed for raffinose and phytic acid, however the individual values 

were within the respective tolerance interval. 

In conclusion, anti-nutrient analysis demonstrates that 32138 SPT maintainer is comparable 

to near isoline inbred control maize and reference maize inbreds. 

Table 18. Key Anti-nutrients in the Seed of Control and 32138 SPT Maintainer 

Analyte 

(% Dry Weight or as 

Indicated) 

Control 32138 SPT 

Maintainer 

Tolerance 

Interval

Mean1 0.227 0.141 

Range2 <0.08005- 0.303 <0.08005 - 0.292 

Adjusted P-value3 0.0116 Raffinose 4

P-value 0.00175 

0 - 0.339 

Mean 1.04 0.841 

Range 0.395 - 1.60 0.470 - 1.08 

Adjusted P-value 0.0499 Phytic acid 4

P-value 0.0144 

0 - 2.19 

Mean 3.45 2.78 

Range 2.17 - 4.21 2.02 - 3.76 

Adjusted P-value 0.0539 

Trypsin 

Inhibitor 

(TIU6

/mg) P-value 0.0164 

1.16 - 5.17 

Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. <Lower Limit of Quantitation (LLOQ); indicates that the values of the sample or samples were detected 

below the assay LLOQ 

6. Trypsin Inhibitor Units 
Pioneer Hi-Bred Page 120 of 188 

32138 SPT Maintainer No CBI 

VIII-F. Key Secondary Plant Metabolites in Control and 32138 SPT Maintainer 

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 furfural and phenolic acids 

(ferulic acid and p-coumaric acid). 

Furfural, p-coumaric acid and ferulic acid were measured in 32138 SPT maintainer and nearisoline inbred control maize seed. Results are shown in Table 19. The level of furfural was 

below the limit of quantitation for the assay and therefore no statistical analysis was 

conducted on this analyte. Statistically significant differences were observed between the 

mean values for 32138 SPT maintainer and near-isoline control maize for p-coumaric acid 

and ferulic acid (adjusted P-values < 0.05). However, the individual values for these analytes 

were within the respective tolerance interval. 

In conclusion, metabolite analysis demonstrates that 32138 SPT maintainer is comparable to 

near-isoline control maize. 

Table 19. Key Secondary Plant Metabolites in the Seed of Control and 32138 SPT 

Maintainer 

Analyte 

(% Dry Weight) Control 32138 SPT 

Maintainer 

Tolerance 

Interval

Mean1 <0.0001007 <0.0001007

Range2 <0.0001007 <0.0001007

Adjusted P-value3 NA 

Furfural 

P-value NA 

NA6 

Mean 0.0277 0.0230 

Range 0.0210 - 0.0399 0.0149 - 0.0385

Adjusted P-value 0.01164

p-Coumaric 

acid5

P-value 0.00151 

0.00421 - 0.0411

Mean 0.147 0.174 

Range 0.120 - 0.194 0.110 - 0.245 

Adjusted P-value 0.03074 Ferulic acid5

P-value 0.00635 

0.00769 - 0.354 

Footnotes: 

1. Least squares mean 

2. Range denotes the lowest and highest individual value across locations 

3. False Discovery Rate (FDR) adjusted P-value 

4. Statistically significant difference, adjusted P-value < 0.05 

5. The analyte values were transformed for statistical analysis. Means and confidence intervals have been 

back transformed to the data scale. 

6. Not applicable. 

7. < Lower Limit of Quantitation (LLOQ); indicates that the values of the sample or samples were 

detected below the assay LLOQ 
Pioneer Hi-Bred Page 121 of 188 

32138 SPT Maintainer No CBI 

VIII-G. Conclusions on Compositional Assessment of Seed from 32138 SPT 

Maintainer 

Extensive nutritional compositional analysis of 32138 SPT maintainer was conducted to 

evaluate the composition as compared to a non-transgenic near isoline inbred control and 

two conventional maize inbreds. In total, data from 59 different analytical components are 

presented here. Compositional analysis of 32138 SPT maintainer was used to evaluate any 

changes in the levels of key nutrients, anti-nutrients or secondary metabolites. 

Compositional analyses included protein, fat, acid detergent fiber (ADF), neutral detergent 

fiber (NDF), ash, carbohydrates, fatty acids, total amino acids, minerals and vitamins, key 

anti-nutrients (raffinose, phytic acid and trypsin inhibitor) and key secondary metabolites 

(furfural, ferulic acid and p-coumaric acid). Based on the compositional evaluation, 32138 

SPT maintainer is comparable to inbred maize with respect to nutrient composition. The 

detected statistical differences between 32138 SPT maintainer and near-isoline inbred 

control maize likely reflect the natural variability of the components in the inbreds since the 

levels of all the individual analytes for 32138 SPT maintainer are within the 99% tolerance 

intervals for inbred maize. 
Pioneer Hi-Bred Page 122 of 188 

32138 SPT Maintainer No CBI 

IX. Potential Environmental Impact Assessment of the Seed 

Production Technology Process 

IX-A. Limited Use of and Exposure to the 32138 SPT Maintainer 

The 32138 SPT maintainer will be grown on very small acreages (Sections I-A and I-D) and 

the Company’s SPT process for hybrid seed production is extremely effective in virtually 

eliminating any transgenic seed of 32138 SPT maintainer from commercial seed (Section I-D 

and Appendices 2-4). The following are some of the unique use and exposure limiting 

attributes of 32138 SPT maintainer: 

 32138 SPT maintainer is not a commercial product. Unlike other transgenic 

events that are deregulated and placed into commerce, 32138 maintainer will not be 

a commercial product, nor will the 32138 SPT insertion be present in commercial 

seed sold to growers. Cultivation of the 32138 SPT maintainer will be conducted 

entirely under Pioneer’s control, by Company employees on Company-controlled 

land, as a part of Pioneer’s SPT process for hybrid seed production. Specifically, the 

32138 SPT maintainer will be increased in different genetic backgrounds (Figure 3, 

Step I) and then used in parent (inbred) seed production (Figure 3, Step II). As a 

consequence of the SPT process, the parent seed derived from Step II (Figure 3) of 

the process does not inherit any SPT genes and is not transgenic. Consequently, 

when those parents are used to produce F1 hybrid seed (Figure 3, Step III), no 

32138 SPT insertion is present in the hybrid progeny seed that enters into commerce 

or the commercial maize grain produced by growers (Figure 3, Step IV). Since 

parent seed and hybrid seed that enter commerce does not contain the 32138 SPT 

insertion it need not be considered in any assessment of environmental impact. 

 The amount of 32138 SPT maintainer cultivated is low. Pioneer will implement 

the SPT process for the generation of female inbred parents incrementally. However, 

if all female inbred parents for Pioneer hybrid seed production were eventually 

produced using SPT (maximum internal use scenario), 32138 SPT maintainer would 

be grown on <5,000 acres each year in the U.S. A small portion of these acres will 

be used for crossing the 32138 SPT insertion into Pioneer elite inbred parents and 

increasing these SPT maintainers by self-pollination (Figure 3, Step I). The larger 

portion of these acres will be used for female inbred parent seed production (Figure 

3, Step II). These two steps combined represent a total of <0.006% of the cultivated 

maize acres in the U.S. (Table 20). 

In the future, Pioneer might license the SPT system to third parties. If the SPT 

system were adopted across the entire U.S. seed industry (maximum industry use 

scenario), the total annual U.S. acreage planted to 32138 SPT maintainer would still 

not exceed 20,000 acres (Table 20). 
Pioneer Hi-Bred Page 123 of 188 

32138 SPT Maintainer No CBI 

Table 20. 32138 SPT maintainer Acres Relative to Total Maize Production in the 

U.S. 

Footnotes: 

1. Source: USDA-NASS, 2008 

2. Assumes that 100% of Pioneer maize hybrid production uses the SPT process 

3. Assumes that 100% of maize hybrid seed production in the U.S. uses the SPT process 

 32138 SPT maintainer sheds virtually no fertile transgenic pollen. The 32138 

SPT maintainer produces virtually no fertile transgenic pollen and so does not pass 

any SPT genes when used as a pollinator. As discussed in Section I-D, 50% of the 

pollen produced by 32138 SPT maintainer lack the SPT insertion and is fertile, 

whereas, the other 50% is transgenic, but infertile. Since 32138 SPT maintainer 

does not shed fertile pollen that is transgenic for SPT, there is virtually no risk of gene 

flow to other maize crops or wild or weedy maize relatives (vertical gene flow). 

 32138 SPT maintainer seed is easily identified and removed. As part of the SPT 

process, color sorting is used to identify seed containing 32138 SPT insertion 

(Appendix 3 describes the accuracy of this color sorting process.) All seeds of the 

32138 SPT maintainer contain a color marker protein (DsRed2) that causes the seed 

to fluoresce a bright red color when illuminated by an appropriate excitation light 

source. When the SPT process is used to increase the 32138 SPT maintainer 

(Figure 3, Step I), 50% of the seed harvested after self-pollinating the 32138 SPT 

maintainer is transgenic. Color sorting is used to identify and separate the transgenic 

red fluorescent 32138 SPT maintainer seed from yellow seed that does not contain 

SPT derived DNA (Figure 3, Color Sort A). 

Parent inbred seed harvested in Step II (Figure 3) of the process does not contain 

the 32138 SPT insertion. However, a second color sorting step (Figure 3, Color Sort 

B) is performed for quality management to prevent any low-level presence of 

transgenic SPT seed in parent seed used for commercial seed production. 

Pioneer has considered unlikely scenarios whereby the genetic components of the 

SPT system might fail, for example, in the case of rare genetic recombination events 

where the DsRed2(Alt1) gene becomes de-coupled from the Ms45 and zm-aa1 malesterility gene cassettes. As described in Appendix 7, the occurrence of genetic 

recombination mechanisms affecting the linkage of the cassettes of the 32138 SPT 

insertion are rare and would not be expected to occur based on the biological 

mechanisms or series of events that would need to occur for heritability. In the case 

that a linkage break did occur between the cassettes of the 32138 SPT insertion, 

Pioneer has considered the impact on resulting progeny and the overall SPT 

process. After considering each possible scenario and the phenotypes that result, 

Pioneer concludes that the progeny seed or plants would be easily identified, 

removed, or not propagated further. 

Current maize planting in the U.S.1

 87,327,000 acres 

Estimated 32138 SPT maintainer acres required 

by Pioneer (maximum internal use scenario)2 

<5,000 acres 

(<0.006% of total U.S. maize 

acres) 

Estimated 32138 SPT maintainer acres required 

by industry for (maximum industry use 

scenario)3

<20,000 acres 

(<0.023% of total U.S. maize 

acres) 
Pioneer Hi-Bred Page 124 of 188 

32138 SPT Maintainer No CBI 

 32138 SPT maintainer is genetically, mechanically, and reproductively 

contained. The SPT process employs a combination of genetic, mechanical, and 

reproductive measures that effectively contain the 32138 SPT maintainer within the 

Pioneer seed production process. The efficiency of the genetic containment is 

discussed in Appendix 2 and the accuracy of mechanical color sorting is discussed in 

Appendix 3. The combined efficiency of the genetic containment (i.e., the frequency 

with which the 32138 SPT insertion is not inherited through pollen) and mechanical 

color sorting accuracy (i.e., the accuracy of mechanical seed sorting to remove any 

transgenic 32138 SPT maintainer seed from non-transgenic male-sterile progeny 

maize parent seed) is calculated to be >99.9999995% (Appendix 5). In addition, 

inbred parent seed production is conducted under reproductive containment (spatial, 

temporal, etc.) to ensure genetic purity of the inbred parents (Appendix 6). 

 32138 SPT maintainer seed will be handled separately from other parent seed. 

All 32138 SPT maintainer seed (Figure 3, Step I) will be processed and conditioned 

(cleaned, sized, and treated) in a facility dedicated to exclusively handling this seed, 

according to specially developed protocols that minimize any potential for cross 

contamination of non-transgenic seed with transgenic 32138 SPT maintainer seed 

(Appendix 6). After color sort A, 32138 SPT maintainer seed will be dyed a blue 

color to distinguish it from any other maize seed and facilitate separate handling. In 

addition, 32138 SPT maintainer seed will be bagged in bags clearly marked 

“Maintainer.”

 De minimis amounts of 32138 SPT maintainer seed and vegetative material may 

be used for feed. Any unused 32138 SPT maintainer inbred seed or discarded 

seed from color sorting (Figure 3, Stage II) will be disposed of using established 

procedures for the destruction of proprietary inbred materials. Seed companies take 

precautions to insure that inbred parent lines are not misappropriated by third parties; 

special procedures have been established for the disposal of such material. 

Discarded parent seed and seed by-products (e.g., husks and cobs) will not enter 

into the commodity grain supply. Pioneer procedures for disposal of unwanted 

material include landfills, composting, and incineration. Discard maize seed and byproducts can also be fed directly to animals, in which case, the animal feeder must 

sign a purchase agreement that the materials will be utilized for farm use only and 

cannot be delivered or sold commercially. Outdated 32138 SPT maintainer seed 

inventory (i.e., lines that are no longer used as parents in commercial hybrid seed 

products) will be disposed of using the discard procedures described above. 
Pioneer Hi-Bred Page 125 of 188 

32138 SPT Maintainer No CBI 

IX-B. Environmental Assessment of the Components of the 32138 SPT 

Maintainer: MS45, ZM-AA1, and DsRed2 Proteins 

A very small percentage of the maize acreage will be planted to 32138 SPT maintainer 

(<5,000 acres), (Figure 3, Steps I and II), so there is minimal environmental exposure to the 

SPT gene expression products: MS45, -amylase, and DsRed2 proteins. Two of the 

proteins, MS45 and -amylase are derived from endogenous maize gene sequences that 

have no amino acid sequence similarities to protein toxins or allergens. The DsRed2 protein 

is from a non-food source, but does not have properties of protein toxins or allergens. 

Seed products derived from the SPT process do not contain the 32138 SPT insertion 

(Appendix 2). Therefore, there is no exposure to the SPT gene expression products MS45, 

-amylase, and DsRed2 proteins from the end of Step II of the SPT process, onwards (Figure 

3). 

Ms45 is an endogenous maize fertility gene expressing the MS45 protein in the developing 

anthers. Expression of MS45 protein in the developing anthers of 32138 SPT maintainer 

complements the naturally occurring male-sterile ms45/ms45 mutation in the endogenous 

Ms45 gene. MS45 protein is normally present in developing maize anthers and maize has an 

established history of exposure and safe use. Therefore, there is no novel environmental 

exposure to the MS45 protein, since the expression is targeted to the anther in the 32138 

SPT maintainer. 

The ZM-AA1 protein in 32138 SPT maintainer is comprised of maize endogenous protein 

sequences from the Brittle-1 transit peptide and the ZM-AA1 protein. The -amylases are 

widely distributed in nature and are produced by plants, animals, and microorganisms. 

Multiple known forms of -amylases exist in maize and are expressed in the endosperm and 

other tissues (Chao and Scandalios, 1971; James et al., 2007). Amylases are reported to 

occur endogenously in the pollen of numerous plants including maize (Brewbaker, 1971; 

Agarwala et al., 1981; Wakhle et al. 1983). In the 32138 SPT maintainer, the zm-aa1 gene is 

expressed in the developing pollen grains and expressed at very low-levels in seed, leaves, 

and whole plant due to incomplete tissue specificity of the promoter used. These tissues may 

represent novel routes of environmental exposure for ZM-AA1 to wildlife. Organisms directly 

ingesting fresh plant material would be regarded as pests (e.g., European corn borer, corn 

rootworms, etc.), and would be purposely controlled using appropriate IPM (Integrated Pest 

Management) practices, as described in Section IX-F.3. Other non-pest organisms may also 

incidentally consume plant material (i.e. pollen or decaying crop residue). However, as 

shown in Table 8, expression of ZM-AA1 in whole plant and leaf tissue greatly diminishes to 

<0.28 ng/mg tissue dry weight by the R6 stage of maize development (Table 8), therefore 

exposure of decomposers to ZM-AA1 in crop residue is negligible. Pollinivorous insects 

inhabiting 32138 SPT maintainer-containing fields (<0.023% of maize acreage in the U.S.) 

may be exposed to elevated levels of ZM-AA1. However as previously described, -

amylases are ubiquitous enzymes present in many organisms including plants and insects 

(Campos et al., 1989; Raimbaud et al., 1989; Silva et al., 1999; Cristofoletti et al., 2001). 

Based on their endogenous nature, exposure to these enzymes and their byproducts is 

continuous for wildlife ingesting plant material (e.g., pollen from maize). Finally, the limited 

number of acres to be planted with 32138 SPT maintainer greatly reduces any exposure to 

populations of pollinivorous insects. 

The DsRed2 protein is the only protein not derived from a maize gene sequence used in the 

32138 SPT maintainer. DsRed2 belongs to a family of red fluorescent proteins that are used 

as in vivo color markers for a variety of applications in biological systems. The utility of the 

DsRed2 protein marker and other DsRed variants was demonstrated in both 

monocotyledonous and dicotyledonous plant species, (Jach et al., 2001; Dietrich and Maiss, 

2002; Wenck et al., 2003; Mirabella et al., 2004; Stuitje et al. 2003). Plants stably 
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transformed with DsRed did not show any abnormalities, indicating that the protein did not 

interfere with plant growth, development, fertility, germination or morphogenesis (Jach et al., 

2001; Wenck et al. 2003). 

As described earlier, the DsRed2 fluorescent protein is used as a color marker to facilitate 

identification and separation of 32138 SPT maintainer seeds. Seed expressing the DsRed2 

protein will be contained within the Company-controlled parent inbred seed production 

process. The DsRed2 protein is not present in the parent seed product derived from the 

32138 SPT maintainer (see Appendix 3) or in commercially sold hybrid seed products, 

therefore exposure to the DsRed2 protein in food and feed is unlikely. Exposure of wildlife 

such as birds, rodents, or beneficial arthropods in or near fields where the crop is grown to 

the DsRed2 protein will be very limited. 

 

The DsRed2 protein has been extensively used as an in vivo marker of gene expression. An 

extensive search of published literature revealed no reports of adverse effects that would 

suggest a risk to human health and the environment. For example, there were no observed 

detrimental effects (Long et al. 2005) or signs of toxicity (Figueiredo et al., 2008), when 

DsRed variants were over-expressed in mice. Furthermore, transfected cell lines from an 

arachnid (Ixodes scapularis) expressing DsRed2 also exhibited no apparent adverse effects 

(Kurtti et al., 2008). Based on the data and information provided in the safety assessment for 

the DsRed2 protein (Section VI-I), we have determined that the DsRed2 protein is not likely 

to be allergenic or toxic to humans, and is also unlikely to cause adverse effects to other 

organisms which might possibly be exposed in the field. Therefore, with the combination of 

the safety assessment for DsRed2 and the very limited environmental exposure to DsRed2 

protein in the seed and other tissues of 32138 SPT maintainer, we conclude that there will be 

no significant adverse environmental consequences. 

We conclude that there is no potential risk to the environment due to the expression of the 

SPT genes in the 32138 SPT maintainer, or the deployment of the Pioneer SPT process. We 

anticipate no adverse effects on humans, domesticated animals or other organisms, including 

threatened and endangered species as a result of consuming seed or other tissues from 

32138 SPT maintainer. 

IX-C. Fate of Transgenic DNA in Humans and Animals 

In general, SPT derived DNA from 32138 SPT maintainer will not be consumed by humans or 

animals because the 32138 SPT insertion will not be present in the commodity grain. De 

minimis amounts of discard grain of 32138 SPT maintainer and vegetative material may be 

fed to animals, but the material will not enter the food/feed commodity stream. In the very 

unlikely event that any SPT derived DNA is consumed by humans, the acid environment of 

the stomach rapidly degrades DNA. DNA is degraded within 30 seconds when it was mixed 

with human saliva and hydrochloric acid in a simulation of the conditions in the human 

stomach (Mercer et al. 1999). DNA from the 32138 SPT insertion is equivalent to other DNA 

consumed in the normal diets. 

IX-D. Weediness Potential of 32138 SPT Maintainer 

Various characteristics that might impart weediness to 32138 SPT Maintainer were evaluated 

(Section VII). Compared to the inbred control maize, no differences were seen in the 

characteristics such as seed germination, emergence, seedling vigor, disease and insect 

susceptibility. Assessment of these data detected no biologically significant differences 

between 32138 SPT maintainer and inbred control maize. Furthermore, post-harvest 

monitoring of field trial plots containing 32138 SPT maintainer showed no differences in 

survivability or persistence of 32138 SPT maintainer as compared to conventional maize. 
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These results support a conclusion of no increased weediness potential of 32138 SPT 

maintainer. 

In general maize is not considered a weed. Maize seed cannot be disseminated without 

human intervention, nor can it readily survive from one growing season to the next. During 

the domestication of maize, traits associated with weediness such as seed dormancy, 

efficient dispersal mechanisms, or the ability to establish reproductive populations outside of 

cultivation, have not been selected. Maize is not listed on the noxious weed list distributed by 

the federal government (7CFR part 360). Commercial maize varieties in the U.S. are not 

effective in invading established ecosystems (CFIA, 1994). Based on the weediness 

potential evaluation, 32138 SPT maintainer has no additional weediness potential and is 

comparable to any other conventional maize. 

IX-E. Gene Flow Assessment 

IX-E.1. Vertical Transfer of the Introduced Genetic Material 

Gene flow can occur to the same or a closely related species (vertical) through pollen 

dissemination. Maize is an open pollinated monoecious plant that produces abundant pollen, 

up to 50 million pollen grains per plant (Miller 1985). The 32138 SPT maintainer produces 

two types of pollen in 1:1 ratio: i) fertile, non-transgenic for SPT (do not contain the 32138 

SPT insertion) and ii) infertile, transgenic for the 32138 SPT insertion. The occurrence of 

occasional red fluorescent seeds in batches of parent seed produced using SPT, before color 

sorting (Appendix 2) suggest that a minute fraction of the fertile pollen may carry SPT genes. 

Even in the extremely rare instance that another maize variety or another species was 

successfully pollinated by transgenic SPT pollen, the resulting plants would be unable to pass 

along the SPT genes, since any transgenic pollen produced would be infertile. 

Consequently, the risk of vertical gene flow of SPT genes from the 32138 SPT maintainer is 

essentially zero. 

IX-E.2. Horizontal Transfer of the Introduced Genetic Material 

There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to 

microbes (Nap et al., 1992; Redenbaugh et al., 1994). Because of the universality and the 

identical chemical make-up of DNA in all organisms, there is no reason to believe an 

organism is more likely to take-up a transgene than another gene. Many genomes have 

been sequenced from bacteria that are closely associated with plants (e.g., Agrobacterium

and Rhizobium), and there is no evidence that these organisms contain genes derived from 

plants (Kaneko et al., 2002; Wood et al., 2001). Where sequence data indicate that 

horizontal gene transfer may have occurred, these events are estimated to occur on an 

evolutionary time scale on the order of millions of years (Koonin et al., 2001; Brown, 2003). 

In addition, transgene DNA promoters and coding sequences are optimized for plant 

expression and not bacterial expression, and it is therefore very unlikely that a protein 

corresponding to the transgene would be produced. The amount of 32138 SPT maintainer 

maize discard seed and by-products fed directly to animals is very minimal. Even if such a 

transfer were to take place and protein produced, the DNA and protein would not present a 

human health or plant pest risk. The gene encoding DsRed2 protein is already found in 

nature, and the Ms45 and zm-aa1 gene sequences are endogenous to maize. 

IX-F. Current Agronomic Practices for Cultivation of Maize Inbred Parent Seed 

Because the 32138 SPT maintainer will only be grown on a small number of Companycontrolled acres to increase maintainer seed (Figure 3, Step I) and as a pollinator for malesterile female inbred parent seed (Figure 3, Step II), the scope of the agronomic practices 

assessment can be limited to maize parent inbred seed production. 
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In general, agronomic practices for maize parent inbred seed production are similar to 

practices used in commercial grain production (Wych, 1988). However, a parent inbred seed 

production field is monitored and managed more intensively than a typical grain production 

field in order to obtain the best possible yield of high quality, genetically pure seed. Seed 

production fields are carefully isolated from other fields and rogued (removal of off-type 

plants) to maintain genetic purity. A seed production field usually requires more agronomic 

inputs (fertilizers, herbicides, etc.) than a typical grain field. This is due to both the agronomic 

characteristics of inbred lines, which are typically less robust than hybrids, as well as the high 

value of quality parent inbred seed (Beck, 2002). 

Maize for seed production is typically grown as a row crop, with an average row width of 

about 30 inches. In the Corn Belt, planting usually occurs in late-April to late-May, later than 

the typical hybrid grain production, to avoid early season stress. Harvesting of inbred seed 

generally occurs from late August to mid-October at higher seed moisture and usually 

happens earlier than the harvest of grain. The inbred ears are harvested at higher seed 

moistures (approximately 32% vs. 15 – 20% for grain) than with grain in order to avoid seed 

loss and to maintain optimum seed quality. A higher percentage of seed production acres 

are irrigated than grain production acres due to the value of seed, the reduced vigor of 

inbreds, their less well developed root systems, and to help ensure predictable nicking 

(synchronization of pollen production and silking). 

IX-F.1. Fertilizer Use

Fertilizer use in seed production fields is calibrated for maximum yield and quality of seed. In 

the U.S., fertilizer use is generally quite similar to grain production fields except for nitrogen 

which is typically less than for grain production fields. The amount of nitrogen applied is 

proportional to the expected yield potential, which is lower for inbreds as compared to 

hybrids. Phosphate, potash, and to a lesser extent, sulfur, are added to soils in order to 

ensure the best seed yield and quality. Micronutrients (magnesium, boron, zinc, 

molybdenum) may also be supplemented if the soil tests indicate a need. 

IX-F.2. Weed Control Practices 

Control of weeds in seed production fields is critical because weeds compete with maize for 

water, nutrients, and sunlight and reduce crop yields. In particular, inbred lines used in seed 

production often have low vigor, and do not compete well with aggressive weeds. Weed 

species such as giant foxtail, barnyard grass and pigweed can reduce maize yields by up to 

13, 35, and 50% respectively (Bosnic and Swanton, 1997; Fausey et al. 1997; Knake and 

Slife, 1965). Later in the process, weeds can interfere with harvesting, cleaning and 

conditioning of the seed crop. Weeds can also act as hosts to insects and diseases that may 

reduce seed yield and/or quality. 

Pioneer must manage a wide array of broadleaf and grass weeds simultaneously and also 

consider the effect of the herbicide on the inbred line. A combination of weed management 

strategies is generally used: tillage, scouting, herbicide application (pre-emergence and postemergence). The combination that Pioneer selects to control weeds depends on factors such 

as: weed spectrum, level of infestation, soil type, cropping system, weather, time, cost and 

labor availability. 

IX-F.3. Insect Control Practices 

Insect infestations can lead to a reduction in quality and quantity of seed in a seed production 

field. Insect control in seed production fields are usually achieved via insecticides (seed 

treatments, granular and/or foliar). Intensive Integrated Pest Management (IPM) programs 
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are practiced in order to scout for insects and determine if and when an insecticide may be 

needed. 

IX-F.4. Disease Control Practices 

In order to produce high yield and high quality seed, scouting and monitoring is needed to 

make sure plant pathogens do not spread. Inbreds are less vigorous and more susceptible to 

disease pressure than hybrids. Thus, foliar fungicides for diseases are more likely to be 

applied in seed production compared to hybrid grain production. 

IX-F.5. Crop Rotation Practices 

Approximately 60% of commercial hybrid maize is grown in rotation following soybean, with 

another 13% in rotation with other row crops and small grains (USDA-ERS ARMS, 2005). A 

greater percentage of maize seed production fields are typically rotated, especially in the 

Corn Belt, as crop rotation aids in the management of diseases, insects and weeds and 

increases organic matter and soil fertility. Importantly, for seed production fields, crop 

rotation also aids genetic purity, as there will be no volunteer corn from the previous year. 

IX-G. Potential Impact of 32138 SPT Maintainer on Agronomic Practices for 

Cultivation of Inbred Maize Parent Inbred Seed 

The introduction of the SPT process in hybrid seed production is expected to result in 

significant benefits. Using 32138 SPT maintainer to eliminate detasseling will increase worker 

safety by significantly reducing the number of seasonal workers, especially high school 

students, exposed to the hazards of working around heavy farm machinery. Use of 32138 

SPT maintainer in female inbred parent seed production will also reduce fossil fuel use and 

soil compaction, as there will be no need to run heavy detasseling machinery across seed 

fields. Since detasseling damages plants and reduces plant health, seed yields will be 

increased by using the SPT process, resulting in a potential reduction in acres needed for 

parent inbred seed production. The high efficiency of the SPT process will improve seed 

quality resulting in a fewer batches of seed that fail to meet quality standards and better 

products for growers containing fewer off-types. 

Because the SPT genes are not present in hybrid seed sold to farmers, there will be no 

potential impacts on agronomic practices for the cultivation of commercial (hybrid) maize. 

The 32138 SPT maintainer will only be grown on a small number of Pioneer-controlled acres 

during the increase and parent inbred seed production phases. Therefore, the scope of the 

agronomic practices assessment is limited to maize parent inbred seed production practices. 

No changes in cultivation or management practices such as planting times, row spacing, 

irrigation amount and type, tillage intensity, or crop residue management are anticipated with 

Pioneer’s use of the 32138 SPT maintainer in the production of female inbred parents. The 

32138 SPT maintainer is comparable to conventional maize inbreds in phenotypic, 

agronomic, ecological and compositional characteristics. The only exceptions are the pinkish 

red aleurone color conferred by the DsRed protein and the reduced amount of fertile pollen 

that is shed. The pinkish red aleurone color does not confer any differences in the seed 

structure and function, so there is no potential agronomic impact. Although half the SPT 

maintainer pollen is infertile, maize produces such an abundance of pollen, about 14 to 50 

million pollen grains per plant (Miller 1985), that there will be enough fertile pollen to insure 

complete fertilization of parent inbred seed plants. The 32138 SPT maintainer has the same 

level of resistance to insects and diseases as other inbreds and does not carry any novel 

tolerances to herbicides, so pest management practices are unaltered. 
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We conclude that there will be no adverse impacts on the cultivation of maize parent inbred 

seed due to the deregulation of 32138 SPT maintainer. As described above, there will be a 

number of benefits arising from the elimination of detasseling, including improved worker 

safety, reduced fuel use and soil compaction as well as improved seed yields and improved 

hybrid seed quality. 

IX.G.1. Potential Impact on Insect, Disease and Weed Control Practices 

The use of the SPT process in hybrid seed production will not cause changes and/or shifts in 

the insect and weed populations in maize. The 32138 SPT maintainer is agronomically 

comparable to conventional parent inbred lines and does not carry herbicide tolerance or 

insect resistance traits. Pioneer will not have to make any adjustments to the Company’s 

existing insect, disease and weed control practices. 

IX.G.2. Potential Impact on Volunteer Management 

We do not expect changes in volunteer management practices. Data from a warm and cold 

and diurnal germination study (Section VII-A) demonstrated that germination of 32138 SPT 

maintainer has not changed and no dormancy characteristics are displayed. In general, 

maize grows as a volunteer in the year following cultivation in the U.S. and only then under 

favorable environmental conditions. Volunteers, if they germinate, do not compete well with 

the succeeding crop and can easily be controlled by mechanical tillage or with herbicides. 

IX.G.3. Potential Impact on Crop Rotation Practices 

The 32138 SPT maintainer is agronomically no different than conventional maize parent 

inbred lines and hence no changes in the crop rotation are expected. Pioneer will continue to 

choose rotational crops based on internal practices in our breeding nurseries and parent 

inbred seed fields. 

IX-H. Potential Impact on Organic or Conventional Farming 

Because 32138 SPT maintainer is not a commercial product, and there will be no 32138 SPT 

insertion derived DNA in seed sold to growers, there will be no potential impacts on organic 

or conventional farming. 

IX-I. Potential Impacts on Raw or Processed Agricultural Commodities

Because 32138 SPT maintainer is not a commercial product, and since there will be no 

detectable SPT genes or SPT proteins in seed sold to growers or grain harvested by 

growers, there should be no impacts on raw or processed agricultural commodities. 

IX-J. Potential Impact on Biodiversity 

The 32138 SPT maintainer does not have an increased weediness potential and cultivation 

should not lead to increased weediness of other sexually compatible relatives since 32138 

SPT maintainer does not produce viable transgenic pollen. There would be no impact on any 

organisms including threatened or endangered species that came into contact with transgenic 

sterile SPT pollen because the SPT proteins are not known to be toxic. Importantly, any 

exposure would be limited to only those organisms in or near SPT maintainer fields. In 

addition, 32138 SPT maintainer will be grown on existing agricultural land, controlled and 

managed by Pioneer. No new land will be put into agricultural production because of the 

deregulation of 32138 SPT maintainer. 
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IX-K. Overall Environmental and Agronomic Practices Conclusions 

It is appropriate to conduct a limited environmental and agronomic practices assessment for 

32138 SPT maintainer because it will be used within Pioneer as a part of the Company’s SPT 

process for hybrid seed production. Even if the SPT system is widely licensed and used for 

all female inbred seed increases across the entire seed industry, the total acreage will still not 

exceed 20,000 acres (<0.023% of the maize acres in the U.S.) 

A thorough characterization of 32138 SPT maintainer was performed, including molecular 

analysis, protein detection analysis, phenotypic and ecological evaluation, and nutrient 

composition evaluation. Assessment of the data generated supports the conclusion of no 

increased plant pest potential, phenotypic comparability, and familiarity as they relate to 

ecological risk assessment. 

No environmental effects due to the expression of Ms45 and zm-aa1 -amylase gene 

sequences introduced in 32138 SPT maintainer are expected due to the previous history of 

exposure and safe use. Based on the safety assessment the DsRed2 protein is not likely to 

be allergenic or toxic to humans, and is also unlikely to cause adverse effects to other 

organisms which might possibly be exposed in the field. Likewise, there is no impact on 

public health and safety expected due to the DNA introduced in 32138 SPT maintainer. 

The 32138 SPT maintainer has been shown to be agronomically and ecologically similar to 

conventional maize inbred parent lines that have no weedy tendencies and are non-invasive 

in natural habitats. No differences were seen in characteristics such as seed germination 

and dormancy, emergence, seedling vigor, plant height, lodging, stay green, time to 

flowering, pollen viability, disease incidence and insect damage. Assessment of these data 

detected no biologically significant differences between 32138 SPT maintainer and inbred 

control maize indicative of a selective advantage that would result in increased weediness or 

outcrossing potential. On the basis of these data, it is concluded that there is no increased 

plant pest potential as a result of the use of 32138 SPT maintainer in seed production. 

Because 32138 SPT maintainer is not a commercial product, there is no significant impact 

expected on raw or processed agricultural commodities or organic or conventional farming. 

We do not expect any changes in the current agronomic practices for inbred or hybrid seed 

production, beyond the advantages of eliminating the need to detassel female inbred parent 

seed plants. We anticipate no changes in the level of usage of insecticides or herbicides. 

The use of transgenic 32138 SPT maintainer in Pioneer’s hybrid seed production process 

results in no adverse environmental impact. SPT technology advances agricultural 

sustainability by facilitating a more efficient hybrid production and the availability of a greater 

number of improved hybrids to growers to help achieve higher yields per acre. 

IX-L. Non-Transgenic Status of SPT Derived Progeny 

Pioneer is aware of the scrutiny of transgenic products in international commerce by the grain 

trade and importing countries as well as the various approvals and labeling requirements for 

transgenic foods in different jurisdictions. 

Pioneer is requesting APHIS find that the SPT process, as described and operated by 

Pioneer, consistently and reliably gives rise to progeny seed that lack the 32138 SPT 

insertion and are not transgenic. This will help us to address questions regarding the 

movement of seed and grain in international commerce. 

As discussed in Appendices 2-5, Pioneer has demonstrated that the overall SPT process is 

robust and highly efficient at generating progeny that lack the SPT genes. The genetic 
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efficiency of the SPT system was demonstrated to be >99.999% (Appendix 2) and the 

accuracy of the mechanical color sorting process was >99.95% (Appendix 3). The 

expression of the DsRed2 protein and corresponding red fluorescence of the seed are 

reliable and accurate markers to detect the presence of the 32138 SPT insertion (Appendix 

3). The combined efficiency of the genetic containment (i.e., the frequency with which the 

32138 SPT insertion is not inherited through pollen) and mechanical color sorting accuracy 

(i.e., the accuracy of mechanical seed sorting to remove any 32138 SPT maintainer seed 

from male-sterile progeny maize parent inbred parent seed) is calculated to be 

>99.9999995% (Appendix 5). In addition, enhanced quality management procedures will be 

used for parent inbred parent seed production to provide further checks and control on the 

system (Appendix 6). Combined, these procedures will effectively ensure the generation of 

progeny seed that do not contain the 32138 SPT insertion, even in the very rare event of 

recombination within the 32138 SPT insertion (Appendix 7). In view of this evidence, Pioneer 

asserts that progeny derived using the SPT process are non-transgenic and any 

environmental impacts from SPT progeny will be no different from the environmental impact 

of any other commercially grown maize. 
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X. Adverse Consequences of Introduction 

Pioneer Hi-Bred International, Inc. is unaware of any information indicating that 32138 SPT 

maintainer may pose a greater plant pest risk than conventional maize. There are no 

adverse environmental consequences anticipated with its introduction and use in the 

Company’s SPT process for hybrid seed production. Thus, we make the statement 

“unfavorable information: NONE,” and on the basis of the substantial benefits that the SPT 

process brings to hybrid seed production (reduced labor and fuel costs, reduced soil 

compaction, and improved worker safety and yield/quality of inbred seed), Pioneer requests 

that 32138 SPT maintainer be granted non-regulated status under 7 CFR Part 340.

. 
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XII. Appendices†

 1-13 

†= Data in the Appendices 2-7 were generated in response to the requests made by 

USDA-APHIS in their letter to Pioneer, dated 14 Sept, 2007.
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Appendix 1 

Other Male-Fertility Control Methods Used in Maize 

1.1. Chemically Induced Male-Sterility 

A number of chemicals (e.g., auxins, antiauxins, halogenated aliphatic acids, gibberellins, 

arsenicals and ethepon) applied as a foliar spray prior to flowering have been investigated as 

sterilants in commercial production of hybrid maize seed (McRae, 1985) as well as coating 

tassels with resin to prevent pollen release (Newlin,1971). One product called DetasselorTM

(Rohm & Haas; Zeneca) became commercially available in 1990. However, there has been 

little commercial use of chemical sterilants because of factors such as a failure to obtain 

complete pollen sterility, variation in response based on the genotype and environment as 

well as the challenges of accurate delivery of the chemicals and a narrow window of 

application. Furthermore, some chemicals reduced seed yields in excess of 20% because of 

damage to the female flower (Newhouse et al., 1996). 

1.2. Biological Male-Sterility 

Male-sterility describes a phenotype in which tassels are formed but no viable pollen is shed, 

or the pollen grains do not function normally during fertilization. Male-sterile maize plants can 

neither self-pollinate, nor cross-pollinate neighboring plants and therefore are very useful in 

the production of hybrid maize since they eliminate the need for detasseling. Cytoplasmic 

male-sterility (CMS) has been widely used, and nuclear male-sterility systems have also been 

investigated for use in hybrid maize production (Wych, 1988). Nuclear male-sterility is 

described in detail under section I-C.2. 

CMS results from a specific interaction between a plant’s nuclear and mitochondrial 

genomes. The CMS characteristic is inherited exclusively through the female maize plant, 

since only the female provides cytoplasm to the fertilized maize seed. CMS plants can be 

fertilized with pollen from a male inbred parent, but sometimes fertility may not be restored 

completely. CMS produced seed is usually blended with seed of the same hybrid produced 

by detasseling to insure that an adequate amount of pollen is available for fertilization in the 

grower’s field and to contribute to cytoplasmic diversity. Cytoplasmic male-sterility does not 

work in all types of maize germplasm, depending upon the presence or absence of nuclear 

restoration genes, thereby restricting genetic combinations available for use in hybrid seed 

production. Furthermore, the CMS based male-sterility can break down and fail, particularly 

under stressful environmental conditions, resulting in some level of pollen production by the 

male-sterile lines and reduced hybrid seed quality. 

Major investments of time and resources are required to backcross a male-sterility inducing 

cytoplasm into those elite female inbred parent lines where CMS will work so these females 

may be used for production of commercial hybrids. A major challenge is increasing seed of 

the male-sterile female inbred parent since self-pollination is not possible. Thus, CMS lines 

must be multiplied by repeated crossing to a specific maintainer that has the same genetics 

as the CMS line, but carries a normal fertile cytoplasm. 

Several CMS types (e.g., CMS-T, CMS-S, and CMS-C) have been used for commercial 

hybrid maize seed production. The CMS-T type was widely used for more than a decade 

and 70 to 90% of the hybrid maize grown in the United States carried T-cytoplasm. When the 

Race T of the Southern maize leaf blight epiphytotic swept the United States in 1970, plants 

that carried T-type cytoplasm were more susceptible to the blight than plants carrying normal 

cytoplasm (Havey, 2004). This prompted a re-evaluation of the use of CMS-T and limited its 

use in hybrid seed production (Wych 1988 and Kaul 1988). CMS-C and CMS-S are in 

somewhat common use today. 
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1.3. Male-Sterility Systems Developed using Biotechnology 

Research has been underway to develop alternatives to the conventional methods of 

inducing male-sterility in maize. For example, transgenic dominant sterility systems have 

been described and developed that rely on the introduction of cytocidal genes regulated by 

anther-specific promoters to confer male-sterility (Albertsen et al., 1993; Greenland et al., 

1997; Unger et al., 2001, 2002; and Havey 2004). 

Pioneer previously developed a male-sterility system (Tissue-Specific Sterility, or TSS) based 

on the DNA adenine methylase (dam) gene from E.coli that disrupts normal pollen 

development when placed under control of an anther-specific promoter. The dam gene is 

linked to a selectable marker PAT (Phosphinothricin Acetyltransferase) gene conferring 

tolerance to the herbicidal active ingredient glufosinate-ammonium. When a TSS transgenic 

female inbred parent is crossed to a normal parent of the same genotype during female 

inbred parent increase, 50% of the progeny plants are male-sterile and herbicide tolerant and 

50% are male-fertile and herbicide susceptible. During hybrid production, these male-fertile 

plants can be eliminated by glufosinate treatment as they lack herbicide tolerance gene. The 

surviving plants are male-sterile and serve as the female inbred parent in hybrid production 

with normal male inbred parents. Resulting hybrid plants are 50% transgenic, male-sterile 

and herbicide tolerant and 50% male-fertile and herbicide sensitive. This 1:1 blend of malefertile plants and male-sterile plants results in enough pollen to insure all plants are fully 

pollinated in the grower’s field. 

Another approach uses barnase, an extracellular ribonuclease (RNAse) from Bacillus 

amyloliquefaciens, to induce sterility. Placed under the control of an anther-specific 

promoter, the barnase gene blocks pollen development by interfering with RNA production 

resulting in a male-sterile plant. Barnase is also linked to the PAT selectable marker and 

male-sterile female inbred parents are selected by herbicide treatment. A variation of the 

barnase technology uses a transgenic fertility restorer line homozygous for barstar, the B. 

amyloliquefaciens intracellular inhibitor of barnase for use on the male inbred parent. When 

the transgenic barnase male-sterile line and the barstar fertility restorer line are crossed, 

barstar binds to and inhibits barnase, resulting in restoration of fertility. Hybrids derived from 

the barnase/barstar system are fully fertile (50% because they lack the barnase gene and 

50% because of the presence of the barstar gene that inhibits barnase). 

Both the TSS and barnase approaches to male-sterility involve added cost and complexity, 

such as the need to remove non-transgenic plants by herbicide application. Moreover the 

product is a mixture of transgenic and non-transgenic hybrid seed. Neither the TSS or 

barnase systems have been adopted in commercial hybrid maize seed production. 

USDA-APHIS has deregulated several transgenic male-sterility systems in various crops 

(Table 1). 

Table 1. Transgenic Male-Sterility Systems Deregulated by USDA-APHIS. 

Crop Event(s) Developer Petition No. Mechanism 

Maize MS3 Plant Genetic Systems 95-228-01p barnase 

Chicory RM3-3, RM3-4, RM3-6 Bejo 97-148-01p barnase 

Maize 676, 678, 680 Pioneer Hi-Bred 97-342-01p dam (TSS) 

Rapeseed MS8 x RF3 AgrEvo 98-278-01p barnase/barstar

Maize MS6 AgrEvo 98-349-01p 

(95-228-01p) barnase 

Rapeseed MS1, RF1/RF2 Aventis 01-206-01p barnase/barstar
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Appendix 2 

Genetic Efficiency and Stability of the SPT System 

To confirm the genetic efficiency of the SPT system, Pioneer conducted a study to measure 

the efficiency and stability of the SPT system across several generations and in different 

genetic backgrounds, under hand-pollination conditions (Study 1) and under open-pollinated 

field conditions (Study 2). This is important since Pioneer is asking USDA-APHIS to concur 

that the SPT process reliably gives rise to progeny that are non-transgenic or lack the 32138 

SPT insertion. 

The SPT system is highly genetically efficient, so virtually all of the fertile pollen from the 

32138 SPT maintainer should lack the 32138 SPT insertion, so therefore virtually all of the 

harvested female inbred seed should also be non-transgenic for SPT. 

The transgenic SPT seed contains the DsRed2 gene cassette tightly linked to the Ms45 and 

zm-aa1 male-sterility cassettes. Expression of the DsRed2 protein imparts a pinkish red 

color to seed containing the 32138 SPT insertion and causes the seed to emit a strong red 

fluorescence when appropriately illuminated. Therefore, expression of the DsRed2 protein 

serves as a highly sensitive visible marker for seed containing the 32138 SPT insertion and it 

enables SPT seed to be identified and separated from non-SPT seed. 

2.1. Study 1 

Hand-pollinated crosses were made between 32138 SPT maintainer (used as a pollen donor, 

or pollinator) and female maize lines that were non-transgenic for SPT. The seed from the 

female lines was hand harvested and examined for the presence of any transgenic SPT 

seed. 

The harvested seed was manually sorted using a light box, Leica KL2500 light systems 

(Schott Glas, Mainz, Germany) equipped with a fluorescent excitation filter to look for red 

fluorescing and hence transgenic SPT seed. As shown in Figure 1A (Appendix 2), pinkish 

red transgenic SPT seed could be distinguished from yellow seed (non-transgenic for SPT) 

under normal light (Appendix 2-Figure 1A) and fluoresced a bright red when viewed under 

fluorescent illumination (Appendix 2-Figure 1B). 

Figure 1. Appearance of Seed Mixture under Normal and Fluorescent Illumination 

Arrow shows the 32138 SPT maintainer seed amongst the yellow seed. 

Fluorescent Illumination (540nm)

A

Normal Illumination

B
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In order to assess the efficiency and stability of the SPT system, this study was conducted 

within a single genotypic background over six generations (Appendix 2-Table 1), and across 

five different elite inbred backgrounds designated A-E (Appendix 2-Table 2). 

Table 1. Genetic Efficiency of the SPT System across Five Generations

† = {100 – (# of red fluorescing seed/ total # of seed) x100} 

Table 2. Genetic Efficiency of the SPT System across Five Genetic Backgrounds 

† = {100 – (# of red fluorescing seed/ total # of seed) x100} 

Generation Total no. 

of Seed 

No. of Red 

Fluorescing 

Seed 

No. of Yellow 

Seed 

Genetic 

Efficiency†

(%) 

T0 362 0 362 100 

T1 19,012 0 19,012 100 

T2 86,126 2 86,124 99.998 

T3 213,689 4 213,685 99.998 

T4 437,298 3 437,295 99.999 

T5 348,909 1 348,908 99.999 

Total 1,105,396 10 1,105,386 >99.999 

Generation Genetic 

Background

Total no. 

of Seed 

No. of Red 

Fluorescing 

Seed 

No. of 

Yellow 

Seed 

Genetic 

Efficiency† 

(%) 

A 84,178 0 84,178 100 

T3 

B 129,511 4 129,507 99.997 

C 130,606 0 130,606 100 

T4 D 38,421 0 38,421 100 

B 320,520 3 320,517 99.999 

E 78,354 1 78,353 99.999 

T5 

B 218,303 0 218,303 100 

Total 999,893 8 999,885 >99.999 
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2.2. Study 2 

Genetically male-sterile (ms45/ms45) plants were open pollinated in the field with transgenic 

32138 SPT maintainer pollen (Step II-Figure 3). More than 380,000 seeds were harvested 

and subjected to machine color sorting using optimal conditions and procedures (Appendix 2-

Table 3). The harvested seed was expected to be all non-transgenic for SPT (do not contain 

the 32138 SPT insertion) and hence are yellow in color. In the first pass, five red fluorescing 

seeds were identified and removed. In the second pass of the remaining seed, no additional 

red fluorescing seeds were found, confirming that the first pass identified all of the SPT seed. 

Table 3. Genetic Efficiency of the SPT Process under Open-Pollinated Field 

Conditions 

 

Footnotes: 

1. Total number of seeds was estimated based on the known test weight value for inbred 

used and the amount of seed (in kg) that passed thru the sorter. 

2. N/A= not applicable 

2.3. Conclusions 

These studies demonstrate that the SPT process generates progeny seed that are nontransgenic for SPT in a highly stable and efficient manner. In the first experiment (using 

hand-pollinated seed), the SPT system was efficient and stable across six generations and 

across five different genetic backgrounds. In the second experiment (open-pollinated field 

conditions), only five out of 380,005 seeds were fluorescing red, indicating that the genetic 

efficiency of the SPT system was 99.999% in the field. Taking into account the results from 

both the studies (1 & 2), genetic efficiency of the SPT system to deliver the progeny that are 

non-transgenic for SPT was never less than 99.997% and overall was >99.999%. 

Pass # Total no. 

of seed1 

No. of Red 

Fluorescing 

Seed 

No. of 

Yellow seed 

Genetic 

Efficiency 

1 380,005 5 380,000 99.999% 

2 380,000 0 380,000 N/A2
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Appendix 3 

Mechanical Color Sorting of the 32138 SPT Maintainer: Process and Facilities 

Mechanical color sorting is an integral component of the SPT process, and takes place in 

Pioneer seed conditioning facilities. 

As described (Appendix 2), the DsRed2 gene is tightly genetically linked to the Ms45 and zmaa1 genes in the 32138 SPT maintainer and serves as a visible marker for seed containing 

the 32138 SPT insertion. The DsRed2 protein emits a strong red fluorescence when 

appropriately illuminated (Appendix 2-Figure 1B). Therefore, seeds containing the 32138 

SPT insertion can be readily detected and separated from seeds that lack the 32138 SPT 

insertion in a highly efficient manner using mechanical color sorters equipped with 

appropriate optical filter sets. 

At two important steps in the SPT process (Figure 3, Steps I & II) color sorters are used to 

separate yellow seed (non-transgenic for SPT) from red flourescing transgenic SPT seed 

(Figure 3, Color Sort A & B). 

Color sorting is accomplished using Satake ScanMaster II series, 40-channel sorters (Satake 

Corporation, Houston, TX, USA) (Appendix 3-Figure 1). Each sorter is equipped with an 

excitation light source (Satake P/N 0912360660, green), optical band pass filters [Satake P/N 

4676200110] and a Satake background filter (P/N 4661840160) to enable color sorting of 

maize seeds based on DsRed2 fluorescence. The sorter is equipped with multiple high 

resolution charge coupled device imaging systems that view each seed from the front and 

back. Seed can pass through the sorter at rates of up to 5,000 kg per hour (Appendix 3-

Figure 2). 

Color Sort A, which takes place in Pioneer’s dedicated SPT maintainer conditioning facility 

(Appendix 3-Figure 3), is designed to separate and collect the transgenic 32138 SPT 

maintainer seed for use in female inbred parent seed increase (Figure 3, Step I). In Color 

Sort A, seed is expected to be segregating 1:1 for the SPT genes, and therefore 50% of the 

seed will be yellow and 50% will be red fluorescent. The harvested seed is sorted twice (two 

passes). After the first pass, the red fluorescent transgenic SPT seed is collected and re-run 

through the sorter, in order to make sure all yellow (non-SPT) seed is removed. 

Color Sort B, which takes place in Pioneer’s inbred seed conditioning facilities, is a quality 

control check to identify and remove any low-levels of unwanted fluorescent red 32138 SPT 

maintainer seed from female inbred parent seed (Figure 3, Step II). In Color Sort B, seed is 

expected to be virtually free of fluorescent red (non-transgenic for SPT) seed. The harvested 

seed is sorted twice (two passes) to identify and remove any adventitious fluorescent red 

transgenic SPT seed. 
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32138 SPT Maintainer No CBI 

Figure 1. Satake ScanMaster II Color Sorter 

Figure 2. Seed Passing Through Multiple Channels of a Seed Sorter 
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Figure 3. Pioneer SPT Maintainer Conditioning Facility (Color Sort A) 

3.1. Sorter Calibration and Optimization 

Prior to using the mechanical sorters, it is important to optimize their settings and confirm 

their accuracy. In other words, determine the accuracy with which the mechanical sorters 

correctly identify and sort a known (i.e. a representative and very low) amount of transgenic 

SPT (fluorescent red) seed from large batches of yellow seed that is non-transgenic for SPT. 

First, the optimal settings for sorting SPT seed were determined experimentally. More than 3 

tons (>3000 kg or >6720 lbs) of maize seed was passed through the sorter in multiple 

batches. Each batch contained an average of 51,000 seeds that were non-transgenic for 

SPT physically mixed with a pre-determined amount (0.05%) of transgenic 32138 SPT 

maintainer seed. Sorter parameters (e.g., feed rate) were adjusted so that all transgenic 

32138 SPT maintainer seeds were consistently separated from each batch of seed. 

The accuracy of the sorter at the optimal settings identified was then evaluated by passing 

through another 50 batches of seed non-transgenic for SPT mixed with 0.05% transgenic 

32138 SPT maintainer (fluorescent red) seeds and determining the machine’s ability to detect 

and separate transgenic 32138 SPT maintainer (red fluorescing seed) from the non-SPT 

(yellow) seed. The number of red fluorescing 32138 SPT maintainer seed detected in each 

run (comprising of two passes) were counted and recorded. Sorter accuracy was calculated 

as the percentage of transgenic 32138 SPT maintainer seed detected by the sorter relative to 

a known number of transgenic 32138 SPT maintainer seed added. Sorter accuracy was 

recorded after two passes and confirmed as >99.95% (data not shown). 

3.2. Conclusions 

Mechanical color sorters provide a fast, efficient and accurate way of identifying and sorting 

transgenic SPT seed from the seed that is non-transgenic for SPT. Under the optimal 

settings identified, the accuracy of the color sorters to detect and separate fluorescing 

transgenic SPT seed was determined to be at >99.95%. 

Color 

Sorters
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Appendix 4 

Molecular Analysis to Confirm the Absence of the 32138 SPT Insertion in SPT 

Progeny

In addition to the phenotypic studies described in Appendix 2, Pioneer also conducted a 

molecular experiment to confirm the absence of the 32138 SPT insertion in the male-sterile 

progeny derived from the SPT system. Pioneer is requesting that USDA-APHIS find that 

progeny from the SPT process lack the 32138 SPT insertion and are therefore nontrasngenic for SPT. 

As previously discussed (Appendix 2), the SPT system is highly genetically efficient. Nearly 

all of the functional pollen from 32138 SPT maintainer should not contain the SPT insertion, 

and therefore, as described in Appendix 2, >99.999% of the seed made using the pollen from 

the SPT maintainer do not contain the SPT insertion. For the seeds used in the molecular 

experiment described below, a transgenic 32138 SPT maintainer (used as a pollen donor or 

pollinator) was crossed to a male-sterile ms45/ms45 maize line non-transgenic for SPT. The 

seed from the male-sterile line was hand harvested and passed twice through an optical 

sorter to identify and separate any red fluorescent SPT transgenic seed from the nonfluorescent, non-transgenic SPT progeny seed (Appendix 2). 

 

Non-fluorescent (yellow) seeds (15,000 seed) were randomly selected from a sorted 

population and planted in the greenhouse. Two leaf samples (punches) were collected from 

each seedling and genomic DNA was extracted from each leaf sample. Each genomic DNA 

sample was analyzed using Real-Time PCR assays, for the three introduced SPT genes from 

the T-DNA region of plasmid PHP24597 (Figure 13): Ms45, zm-aa1, and DsRed2(Alt1). The 

three gene assays on the samples were run in independent PCR reactions. Nearly every 

plant was analyzed in duplicate, with ten samples giving low DNA results in the first round of 

analysis and 25 samples in the second round of analysis (Appendix 4-Table 1). In total, all 

15,000 plants were analyzed at least once in the Real-Time PCR analysis. 

In summary for both rounds of analysis, 14,998 plants tested negative for all three gene PCR 

assays (Appendix 4-Table 1). Positive and negative control samples gave the expected 

results during each run (data not shown). Two of the tested plants had unexpected results. 

One progeny maize plant was negative for the Ms45 and DsRed2(Alt1) PCR assays, but 

tested positive in both rounds of the analysis for the zm-aa1 PCR assay. A second plant that 

had been negative for all assays in the first round of analysis was positive for the 

DsRed2(Alt1) assay and negative for the zm-aa1 and Ms45 assays in the second PCR 

round. 
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Table 1. Results of Qualitative Real-time PCR Analysis 

Analysis 

Round1

Total 

Samples 

Analyzed 

Negative 

for All 

PCR 

Assays 

Low DNA 

Result 

(No 

Sample)2

Low 

DNA 

Result 

(Other)3

Positive 

for 

zm-aa1

PCR4

Positive for 

DsRed2(Alt1)

PCR5

1 15,000 14,989 4 6 1 0 

2 15,000 14,973 21 4 1 1 

Footnotes: 

1. Two individual leaf samples were collected from each plant and DNA was extracted from each 

sample and analyzed by Real-Time PCR. 

2. Missing leaf punch or cracked extraction tube caused absence of DNA sample. 

3. No reason determined for low DNA result. 

4. Negative for Ms45 and DsRed2(Alt1). 

5. Negative for Ms45 and zm-aa1. 

The single plant that was positive for the zm-aa1 but negative for the Ms45 and DsRed2(Alt1)

in the both the first and second round of PCR assays was investigated further to determine 

the origin of the zm-aa1 gene. Sequencing of the DNA insertion in this plant demonstrated 

that the positive zm-aa1 PCR assay was the result of the presence of a different T-DNA 

source plasmid that contained a similar zm-aa1 cassette and was not from plasmid 

PHP24597. Therefore, the zm-aa1 positive result was attributed to the presence of a single 

seed containing a different T-DNA and was not due to the transmission of the 32138 SPT 

insertion to the male-sterile progeny. 

The plant that was positive for only the DsRed2(Alt1) PCR assay in the second round of 

analysis was attributed to sample contamination by DNA positive for DsRed2(Alt1), as the 

same plant was negative for all three assays in the first round of analysis and negative for the 

zm-aa1 and Ms45 PCR assays in the second round. Duplicate leaf punches were prepared 

independently and it was possible that contamination could have occurred during extraction 

or during the individual set-up of the DsRed2(Alt1) assay. This sample was positive for the 

maize endogenous reference gene in all PCR assays in the first round of analysis, indicating 

that the DNA sample was adequate and it was not a false negative result. The negative 

results for the Ms45 and zm-aa1 assays in both rounds of analysis of this plant also indicate 

the absence of the PHP24597 T-DNA insertion. 

Conclusions 

Based on the two independent rounds of PCR screening, none of the 15,000 male-sterile 

progeny plants originating from 32138 SPT maintainer event DP-32138-1 contained cloned 

genes derived from plasmid PHP24597. This demonstrates the extremely high efficiency of 

the SPT system in preventing the transfer of the 32138 SPT insertion to the male-sterile 

maize progeny. 
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32138 SPT Maintainer No CBI 

Appendix 5 

Overall Efficiency of the SPT Process 

In order to calculate the overall efficiency of the SPT Process, it is necessary to take into 

account both the genetic efficiency and the mechanical sorting accuracy. 

Genetic efficiency is defined as the ability of the 32138 SPT maintainer to prevent 

transmission of the 32138 SPT insertion to progeny seed when the 32138 SPT maintainer is 

used as a pollen source (pollinator). As shown in Appendix 2, under both hand-pollinated 

and open-pollinated field conditions, the genetic efficiency of the SPT system was never less 

than 99.997% and overall was >99.999%. 

Mechanical color sorting accuracy is defined as the ability of the mechanical color sorters to 

accurately identify and separate red fluorescing seed from yellow seed. As demonstrated in 

Appendix 3, the mechanical color sorting accuracy was determined to be >99.95%. 

Embedded in this accuracy is the fact that the DsRed2 protein is an accurate and reliable 

color marker for determining the presence of 32138 SPT insertion, which was demonstrated 

in Appendix 4. 

The overall efficiency of the SPT process can be calculated by combining the genetic 

efficiency and mechanical sorter accuracy: 

The genetic efficiency of the SPT system is >99.999%. 

The mechanical sorter accuracy is >99.95%. 

This means that of the possible <0.001% of the SPT progeny that might contain the 

32138 SPT insertion (i.e. red fluorescing seed), the color sorter will accurately identify 

and sort these with >99.95% accuracy, or will only fail <0.05% of inaccuracy to separate 

red fluorescing seed from yellow seed. 

Therefore, the overall efficiency of the SPT process is 100% - (0.001% × 0.05%) or 

>99.9999995%. 
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32138 SPT Maintainer No CBI 

Appendix 6 

Inbred Parent Seed Production Practices Using the SPT 

As part of its normal inbred parent inbred seed production process, Pioneer adheres to many 

practices designed to produce high-quality, genetically pure inbred seed. With the use of the 

SPT process, additional procedures will be put in place to ensure that seeds containing the 

32138 SPT insertion are not inadvertently carried forward into hybrid seed production or 

commercial seed. 

6.1. Planting and Isolation 

 All 32138 SPT maintainer inbred seed will be uniquely dyed with a blue color.

 All seed production will meet minimum isolation distances required by government 

regulations for certification of seed products.

 For male-sterile female inbred parent increase, 32138 SPT maintainer will be planted 

in either a 2:2 or 4:4 or 4:2 planting pattern (male-sterile female: 32138 SPT 

maintainer rows). The first row and the last row must be 32138 SPT maintainer 

(pollinator) rows.

6.2. Roguing (Removal of Off-Types in the Field) 

 Roguing needs are determined and documented according to inbred parent corn 

seed production location and upon visual inspection for off types by experienced and 

qualified field personnel. 

 Any off-type plants (rogues) identified will be destroyed. 

i. Male-sterile rows: Any fertile plants detected in the male-sterile rows will be 

destroyed. 

ii. 32138 SPT maintainer rows: Any sterile plants detected in the maintainer 

rows will be destroyed. 

6.3. Harvesting 

 32138 SPT maintainer and male-sterile inbreds will be harvested and kept 

separately. 

6.4. Sorting, Drying, and Shelling of Maize Ears 

 All off-type ears will be removed at sorting for both 32138 SPT maintainer and malesterile lines. 

 For quality control purposes, sorting, drying and shelling of 32138 SPT maintainer 

inbreds will be done in a small lot handling system. 

6.5. Optical Seed Sorter Calibration 

 The calibration of the seed sorter (Sataki ScanMaster II, Satake Corporation, 

Houston, TX, USA) will be verified daily before use according to the manufacture’s 

instructions for maintaining the equipment and to specifications described in a 

Pioneer standard operating procedure (SOP). 

 Calibration records will be maintained locally and archived. 
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32138 SPT Maintainer No CBI 

6.6. Seed Conditioning, Treatment, and Packaging 

 32138 SPT maintainer (Color Sort A): 

 32138 SPT maintainer seed will be conditioned in a building dedicated exclusively to 

conditioning SPT maintainers. 

 All 32138 SPT maintainer seed will be color sorted using two sequential sorting 

steps. Records from each production run will be archived. 

 The reject portion of the maintainer shall be discarded using Regulatory Compliance 

ISO 9000 discard procedures and shall not be used for any other purpose. 

 Following the second color sorting process of SPT materials, samples will be 

collected for a purity check. 

 All 32138 SPT maintainer inbreds will be dyed with a blue color. 

 Parent corn seed designated as MAINTAINER will be packaged in bags clearly 

marked MAINTAINER and labeled per instructions.

Male-Sterile Progeny (Color Sort B):

 All SPT male-sterile progeny will be color sorted twice to remove 32138 SPT 

maintainer (red fluorescent) seed. 

 Following color sorting, a sample will be collected for purity check. 

 If an 32138 SPT maintainer seed is detected, the seed lot will be color sorted again. 

 Results will be recorded and archived. 

 To monitor the efficacy of the SPT process, discarded seed will be collected and 

analyzed further for the presence of any maintainer seed. Results will be recorded 

and archived. 

 Any discard seed shall be destroyed using Regulatory Compliance ISO 9000 

procedures and shall not be used for any other purpose. 

6.7. Record Maintenance 

 All records will be retained for a minimum of 5 years. 

 Records will be available for audit upon written request. 
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32138 SPT Maintainer No CBI 

Appendix 7 

Potential for Linkage Break between the Cassettes of the 32138 SPT Insertion 

and Consequences for the SPT Process 

Pioneer has demonstrated that the SPT process is extremely stable and efficient in ensuring 

that the 32138 SPT insertion is not transmitted during the seed production process. 

However, there are potential scenarios, described below, under which the genetic 

components of the SPT system might become decoupled (i.e. linkage break between the 

cassettes via genetic recombination). These occurrences are rare and would not be 

expected to occur based on the biological mechanisms or series of events that would need to 

occur for heritability. If a linkage break between the cassettes does occur in the 32138 SPT 

insertion the effects of such occurrences could be detected and these seed eliminated from 

the seed supply, thus not compromising the efficiency of the SPT process. 

7.1. Genetic Recombination Mechanisms 

The possibility of a linkage break between the gene cassettes of the 32138 SPT insertion in 

32138 SPT maintainer would be rare. Research on meiotic recombination, crossover rates 

based on genomic location, and somatic recombination are summarized below and indicate 

that such occurrences resulting in linkage break are rare. 

The insertion in 32138 SPT maintainer is maintained in the hemizygous state because of 

-amylase expression. As discussed previously, pollen containing the insertion will be nonfunctional because of the -amylase enzyme activity and, therefore, only pollen without the 

insertion will be successful in pollination. Because 32138 SPT maintainer plants can only be 

hemizygous (SPT/-) for the insertion, the likelihood of a linkage break by meiotic 

recombination mechanisms within the T-DNA insertion is rare. In plants, meiotic 

recombination occurs between regions of identical homology on the chromosomes that pair 

and form chiasmata (Hamant et al., 2006). Furthermore, strict pairing of chromosomes and 

chiasmata formation occurs because of the protein complexes and machinery involved. This 

strict pairing of chromosomes has been observed in allopolyploid species and is evidence of 

the tightness of these conserved meiotic mechanisms; only identical chromosomes will pair 

and form chiasmata and genetically-related non-identical chromosomes (homeologous 

chromosomes) will not pair with each other (Hamant et al., 2006). The lack of a homologous 

region for pairing in the 32138 SPT insertion would make crossovers within the insertion rare; 

any recombination that might occur would have to take place via illegitimate recombination 

mechanisms. 

Furthermore, the 32138 SPT insertion is located near a centromeric region of the maize 

genome. Centromeric regions have a low level of recombination that has been confirmed by 

molecular mapping as well as by microscopic examination of maize chromosomes (Anderson 

et al., 2003). Based on the chromosomal location of the 32138 SPT insertion and positions 

of nearby mapping markers, in general, the average frequency of meiotic recombination in 

this region was calculated to be approximately 0.09 cM/Mb, 10-fold lower than the average 

maize meiotic recombination frequency of 1 cM/Mb (Fengler et al., 2007; personal 

communication, K. Fengler). However, based on the above discussion, hemizygosity of the 

insertion would eliminate the possibility of meiotic crossover via legitimate recombination 

mechanisms, thus these numbers are applicable only to surrounding genomic regions and 

not representative of recombination within the 32138 SPT insertion. 

Recombination in somatic cells may have an impact on linkage within the 32138 SPT 

insertion, but would also be expected to be extremely rare. The primary concern for such 

recombination would be a double-strand break repair in genomic DNA that could potentially 
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32138 SPT Maintainer No CBI 

become heritable (Puchta, 2005). Somatic recombination mechanisms occur primarily as a 

result of DNA damage and double-strand break repair (Puchta, 2005; Schuermann et al., 

2005). In general, these repair mechanisms rely on template information from homologous 

regions located at an allele on a sister chromatid (allelic recombination), elsewhere on the 

chromosome (interchromasomal recombination), or elsewhere in the genome (ectopic 

recombination) (Puchta, 2005). Based on scientific research, rates of allelic and ectopic 

recombination are similar and have been estimated to be about 10-3 to 10-5 in tobacco 

transformants with double-strand breaks induced experimentally via an active maize Ac

transposon element or a rare cutting enzyme (Shalev and Levy, 1997; Puchta, 1999; Puchta, 

2005). In these same experiments, the rarity of somatic recombination was also shown in the 

absence of induced double-stranded breaks, since no changes to the transgenic insertion 

were detected (Shalev and Levy, 1997; Puchta, 1999). Repair from non-homologous regions 

can also occur, creating repair junctions that have been altered from the original region 

(Salomon and Puchta, 1998; Puchta, 2005). Non-homologous recombination mechanisms 

have also been studied via the induction of double-strand breaks (Salomon and Puchta, 

1998), so the frequency of these types of repairs is not well known in the endogenous 

genome. Further, the probability of a somatic recombination event occurring in the 32138 

SPT maintainer and being inherited is rare, since it requires the chance of a double-stranded 

break repair within a small pool of cells of the flower meristem 

In addition, transposons (Ac and Ds elements) have been implicated in maize genome 

breakage, recombination and rearrangements (Ralston et al., 1989; Dooner and Belachew, 

1991; Weil and Wessler, 1993; Zhang and Peterson, 1999). In general, chromosomal 

changes are associated with linked elements and the presence of active Ac elements 

(Ralston et al., 1989; Dooner and Belachew, 1991; Weil and Wessler, 1993; Zhang and 

Peterson, 1999). While there is evidence from these studies that these elements are points 

of chromosomal breaks, recombination, and rearrangement if all the necessary elements are 

present, any rearrangements that may occur in the 32138 insertion in maize 32138 SPT 

maintainer would not likely result in a linkage break within the insertion. Transposon-like 

elements are not present within the 32138 SPT insertion but may be present in the 

neighboring genome and any rearrangements that may occur would move the insertion as a 

unit, thus not affecting the linkage of the gene cassettes. 

7.2. Description of Few Possible Scenarios 

Given this body of research and knowledge about meiotic recombination, somatic 

recombination, and transposon rearrangement, the rare possibility exists for a linkage break 

within the 32138 SPT insertion. The impact of several possible scenarios has been assessed 

to determine the ability to detect recombination of the SPT insertion (Appendix 7-Table 1). In 

most cases, deletion of one or two of the three SPT genes (Ms45, zm-aa1 and DsRed2) from 

a single break (Appendix 7-Figure 1Scenarios 1 and 2) gives rise to seed of the wrong color 

that will be removed during sorting, or to plants with phenotypes opposite to those expected 

(i.e., male-fertile or male-sterile) that will be removed after routine inspection as part of quality 

control practices (Appendix 7-Table 1). In the extraordinarily rare occurrence of two breaks 

between each of the cassettes deleting zm-aa1 (Appendix 7-Figure 1 Scenario 3), the 

proportion of seed observed on each ear in the sorting process would indicate an aberrant 

plant genotype and would be removed from use (Appendix 7-Table 1). In the case of the 

deletion of the Ms45 and DsRed2 genes, the zm-aa1 gene from the SPT insertion would 

likely be passed on to the next generation, however the progeny would be sterile and not 

propagated further (Appendix 7-Table 1). 

7.3. Conclusions 

The occurrence of genetic recombination mechanisms affecting the linkage of the cassettes 

of the 32138 SPT insertion are rare and would not be expected to occur based on the 
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32138 SPT Maintainer No CBI 

biological mechanisms or series of events that would need to occur for heritability. In the 

case that a linkage break did occur between the cassettes of the 32138 SPT insertion, 

Pioneer has considered the impact on resulting progeny and the overall SPT process. After 

considering a few representative possible scenarios and the resultant phenotypes, Pioneer 

concludes that in these scenarios the progeny seed or plants would most likely be identified, 

removed, or not propagated further. 

Figure 1. Aberrant Gene Recombination in the SPT Insert—A Few Possible Scenarios

MS45 -amylase DsRed2

MS45 -amylase DsRed2

MS45 -amylase DsRed2

Ms45 MS45 -amylase zm-aa1 DsRed2(Alt1) DsRed2

MS45 -amylase DsRed2

MS45 -amylase DsRed2

Ms45

Ms45

zm-aa1

zm-aa1

DsRed2(Alt1)

DsRed2(Alt1)

Scenario 1

Scenario 2

Scenario 3

MS45 -amylase DsRed2

MS45 -amylase DsRed2

MS45 -amylase DsRed2

Ms45 MS45 -amylase zm-aa1 DsRed2(Alt1) DsRed2

MS45 -amylase DsRed2

MS45 -amylase DsRed2

Ms45

Ms45

zm-aa1

zm-aa1

DsRed2(Alt1)

DsRed2(Alt1)

Scenario 1

Scenario 2

Scenario 3
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32138 SPT Maintainer No CBI 

Seed Increase of SPT maintainer by 

Self-Pollination 

Seed Increase of the Male-Sterile Female 

Inbred Parent using SPT Maintainer as a 

Pollinator (in the Case of a Recombination 

Event in the SPT Pollen). 

Scenario 

# 

Gene(s) 

Deleted 

Will Color Sort A 

be Able to 

Detect and 

Discard the 

Seed? 

Impact on 32138 SPT 

maintainer Seed 

Increase 

(Step I, Figure 3) 

Will Color Sort 

B be Able to 

Detect and 

Discard the 

Seed? 

Impact on Male-Sterile Female 

Inbred Parent Seed Increase 

(Step II, Figure 3) and 

Commercial Hybrid Seed 

Production (Step III, Figure 3) 

Impact on 

Commercial Maize 

Seed and Grain 

Production 

(Step III & IV, 

Figure 3) 

DsRed2(Alt1) Yes 

None 

Will be discarded as 

yellow seed during 

Color Sort A

No 

Yellow seed planted in female 

inbred parent rows (Step III, 

Figure 3), but is an obvious offtype plant (fertile) that will be 

removed (rougued) before pollen 

shed 

None 

1 

Ms45 + zmaa1 No 

None 

Pinkish red color seed 

may be planted in 

maintainer rows but is 

an obvious off-type 

(male-sterile) that will 

be removed (rougued). 

Yes 

None 

The red fluorescing seed will be 

removed during Color Sort B and 

will not enter Hybrid Seed 

Production Fields (Step III, 

Figure 3). 

None 

Ms45 No 

None 

Pinkish red color seed 

may be planted in 

maintainer rows but is 

an obvious off-type 

(male-sterile) that will 

be removed (rougued). 

Yes 

None 

The red fluorescing seed will be 

removed during Color Sort B and 

will not enter Hybrid Seed 

Production Fields (Step III, 

Figure 3). 

None 

2 

zm-aa1+ 

DsRed2(Alt1) Yes 

None 

Will be discarded as 

yellow seed during 

Color Sort A 

No 

Yellow seed planted in female 

inbred parent rows (Step III, 

Figure 3), but is an obvious offtype plant (fertile) that will be 

removed (rougued) before pollen 

shed 

None 

Table 1. Representative Scenarios of Recombination within the 32138 SPT Insertion and Traceability in the SPT Process 
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Seed Increase of SPT maintainer by 

Self-Pollination 

Seed Increase of the Male-Sterile Female 

Inbred Parent using SPT Maintainer as a 

Pollinator (in the Case of a 

Recombination Event in the SPT Pollen). 

4Two recombination events would be required to delete Ms45 and DsRed2(Alt1) while retaining -amylase, which would be extraordinarily rare 

Scenario 

# 

Gene(s) 

Deleted 

Will Color Sort 

A be Able to 

Detect and 

Discard the 

Seed? 

Impact on 32138 SPT 

maintainer Seed 

Increase 

(Step I, Figure 3) 

Will Color 

Sort B be 

Able to 

Detect and 

Discard the 

Seed? 

Impact on Male-Sterile Female 

Inbred Parent Seed Increase 

(Step II, Figure 3) and 

Commercial Hybrid Seed 

Production (Step III, Figure 3) 

Impact on 

Commercial 

Grain Production 

(Step IV, Figure 3) 

zm-aa14 No 

Pinkish Red seed may be 

planted in maintainer rows. 

Upon self-pollination, instead 

of 50:50 (red:yellow) 

segregation, it will result in 

75:25 (red : yellow) seed. 

Incorrect ratio can be 

detected during visual 

inspection of 32138 SPT 

maintainer ears prior to Color 

Sort A (Appendix 6.4) and 

ears will be discarded.

Yes None The red fluorescing seed will be 

removed during Color Sort B 

None 

3 

Ms45 +

DsRed2(Alt1)4 Yes 

None 

Will be discarded as yellow 

seed

No 

None

Pollen containing the gene 

deletions will be infertile due to -

amylase and therefore virtually no 

transmission of transgene to 

male- sterile female inbred seed 

and hybrid seed (Steps II and III 

Figure 3). 

None 

Table 1. Representative Scenarios of Recombination within the SPT Insertion and Traceability in the SPT Process (continued) 
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32138 SPT Maintainer No CBI 

Appendix 8 

Materials and Methods for Molecular Characterization of 32138 SPT Maintainer

To characterize the DNA insertion in 32138 SPT maintainer, Southern blot analysis was 

conducted. Individual plants of the T1S1 generation were analyzed to determine the copy 

number of each of the genetic elements inserted and to verify the integrity of the PHP24597 

T-DNA. The insertion in 32138 SPT maintainer was characterized using Bmt I, BamH I,

BamH I/Hind III, and EcoR I restriction enzyme digestion. Southern blot analysis was 

conducted on individual plants of two generations, T1S1 and BC3, to confirm insert stability 

across generations and to verify the absence of backbone sequences from plasmid 

PHP24597. An additional generation, BC4, was analyzed to verify insertion stability and to 

confirm Mendelian segregation of the insertion (discussed in Section V-E). All probes used 

for the analysis are indicated on the schematic maps of plasmid PHP24597 and its T-DNA 

(Section V, Figures 15 and 16, respectively) and outlined in Section V, Table 3. 

8.1. 32138 SPT Maintainer Material 

Seeds from the T1S1, BC3, and BC4 generations (Figure 12—breeding diagram) of 32138 

SPT maintainer were planted and leaf tissue harvested from individual plants was used for 

genomic DNA extraction. For the T1S1, red (i.e. seeds containing the 32138 SPT insertion) 

and yellow seeds were planted and only the plants from the pinkish red seed were used for 

analysis. For the BC4 generation, both red and yellow seeds (a segregating population) were 

planted and analyzed (Shown in Figures 33, 34, and 35). 

8.2. Control Material 

Seeds from the unmodified lines of Hi-II, Hi-II (ms45), and inbred 705 were planted and leaf 

tissue harvested from individual plants was used for genomic DNA extraction. These control 

DNA samples were used as negative controls to help interpret hybridization results since the 

5126 promoter, Ms45, Pg47 promoter, zm-bt1, zm-aa1, and In2-1 terminator probes crosshybridize with endogenous maize sequences. 

8.3. Reference Material 

Plasmid DNA from PHP24597 was amplified in E. coli and was used as a positive control for 

Southern analysis to verify probe hybridization and to verify sizes of internal fragments. The 

plasmid stock was a copy of the plasmid used for Agrobacterium-transformation experiments 

to produce 32138 SPT maintainer and was digested with restriction enzymes to confirm the 

plasmid map. The probes used in this study (Table 3) were derived from plasmid PHP24597 

or from a plasmid containing equivalent genetic elements. 

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. X174 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. 

8.4. Genomic DNA Extraction 

Genomic DNA was extracted from leaf tissue harvested from individual plants as described 

above. The tissue was pulverized in tubes containing grinding beads using a Geno/GrinderTM 

(SPEX CertiPrep, Inc., Metuchen, NJ) instrument and the genomic DNA isolated using a 
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32138 SPT Maintainer No CBI 

urea-based procedure (modification from Chen and Dellporta, 1994). Approximately 1 gram 

of ground tissue 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 12-30 minutes at 37C, 

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 TE buffer (10mM Tris, 1 mM EDTA, pH 7.5) and 

treated with 10 g Ribonuclease A for 15 minutes at 37C. The sample was extracted once 

with phenol:chloroform:isoamyl alcohol (25:24:1) and once with water saturated chloroform, 

followed by precipitation with isopropyl alcohol and washing with 70% ethanol. After drying, 

the DNA was re-dissolved with 0.5 mL TE buffer and stored at 4C. 

8.5. Quantitation of Genomic DNA 

Following extraction, the DNA was quantified on a spectrofluorometer using PicoGreen® 

reagent (Molecular Probes, Inc., Eugene, OR) following a standard procedure. The DNA was 

also visualized on an agarose gel to confirm quantitation values from the PicoGreen® 

analysis and to determine DNA quality. 

8.6. Phenotypic Identification of 32138 SPT Maintainer Seed 

Phenotypic analysis of 32138 SPT maintainer was carried out by visual observation of the 

seed prior to planting. The color of the seed was determined and documents as either red 

(containing the 32138 SPT insertion) or yellow (not containing the 32138 SPT insertion) as an 

indication of the seed and plant phenotype. 

A preliminary Southern blot analysis of DNA isolated from all 32138 SPT maintainer plants 

was used to confirm the presence of the 32138 SPT insertion and the Ms45, ZM-AA1, and 

DsRed2(Alt1) genes. Methods for this preliminary characterization are described below. For 

the T1S1 and BC3 generations, the final Southern blot analysis was carried out on a subset 

of 32138 SPT maintainer plants. 

8.7. Digestion of DNA for Southern Blot Analyses 

Genomic DNA samples extracted from selected 32138 SPT maintainer and inbred control 

maize plants were digested with restriction enzymes following a standard procedure. 

Approximately 2 g of genomic DNA was digested in a volume of 100 L using 50 units of 

enzyme according to manufacturer’s recommendations. The digestions were carried out at 

37C for 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 4C and centrifugation, the DNA 

was allowed to dry and re-dissolved in TE buffer. The reference plasmid, PHP24597, 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 internal fragments on the 

Southern blot. 

8.8. Electrophoretic Separation and Southern Transfer 

Following restriction enzyme digestion, the DNA fragments produced were electrophoretically 

separated by size through an agarose gel and a molecular weight standard [X174 RF 

DNA/Hae III Fragments (Invitrogen)] was used to determine sufficient migration and 

separation of the fragments on the gel. DIG labeled DNA Molecular Weight Marker VII 

(Roche), visible after DIG detection as described below, was used to determine hybridizing 

fragment size on the Southern blots. 
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32138 SPT Maintainer No CBI 

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 TURBOBLOTTERTM Rapid Downward 

Transfer System (Schleicher & Schuell, Keene, NH). Following transfer to the membrane, 

the DNA was bound to the membrane by UV crosslinking (Stratalinker, Stratagene, La Jolla, 

CA).

8.9. DNA Probe Labeling for Southern Blot Hybridization 

Probes homologous to the 5126 promoter, Ms45, Pg47 promoter, zm-bt1, zm-aa1, In2-1

terminator, 35S enhancer, Ltp2 promoter, DsRed2(Alt1), and pinII terminator were used to 

detect genes and elements within the insertion (Table 3). Backbone probes (spc, tet, virG, 

LB, and RB) for the PHP24597 plasmid were used to verify absence of plasmid backbone 

DNA in 32138 SPT maintainer (Table 3). DNA fragments of the probe elements were 

generated by PCR from plasmid PHP24597 (Figure 15) 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). 

8.10. Probe Hybridization and Visualization 

The DNA fragments bound to the nylon membrane were detected as discrete bands when 

hybridized to a labeled probe. 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 DIGlabeled 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. Images were then digitally captured 

by detection with the Luminescent 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 report. The sizes of detected bands were documented for each digest and 

each probe. 

8.11. 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 N NaOH and 

1.0% SDS at 40°C with constant shaking. The membranes were then rinsed in 2x SSC and 

either used directly for subsequent hybridizations or stored at 4°C or -20°C for later use. The 

alkali-based stripping procedure effectively removes probes labeled with the alkali-labile DIG.
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32138 SPT Maintainer No CBI 

Appendix 9 

Materials and Methods for the Determination of MS45, ZM-AA1 and DsRed2 

Protein Concentrations in 32138 SPT Maintainer 

The MS45 protein levels in 32138 SPT maintainer was visualized using Western blot analysis 

and the concentration of ZM-AA1 and DsRed2 proteins were determined using an enzyme 

linked immunosorbent assays (ELISA) and a fluorometric assay, respectively. 

9.1 Storage and Processing of Maize Tissue Samples 

Upon receipt, all plant tissue samples were stored in temperature-monitored freezers at <-

10°C. Forage samples were coarsely homogenized on dry ice using a Stephan VCM 12 

(Stephan Machinery Singapore Pte Ltd, Singapore) blender for approximately 2 minutes and 

sub-sampled. All samples were lyophilized at <-12°C under vacuum until dry. The 

lyophilization time varied between 18 to 72 hours depending on the sample size and tissue 

type. Forage and seed tissues were finely ground for approximately 60 seconds using a 

GenoGrinder (BT&C/OPS Diagnostics, Metuchen, NJ, USA). 

Two samples of four anthers each were collected for each developmental stage from plants 

derived from the test, control, and reference lines and used for protein extraction. Between 

lyophilization and grinding, samples were stored frozen in temperature-monitored freezers at 

<-10°C.

Tissue samples, with the exception of developing anther samples for Western blot analysis, 

were weighed into 1.2 ml tubes at the following target weights (+/- 5%); 5 mg for pollen, 10 

mg for leaf, 20 mg for grain and 30 mg for whole plant and stalk tissue. Developing anther 

samples were transferred to a 1.2 ml tube (4 anthers per stage per tube) without weighing 

and were kept frozen <-10°C until analysis. 

Inbred seed samples derived from BC1S2 and BC3S2 generations of 32138 SPT maintainer 

consisted of red and yellow seed in ~1:1 ratio. Each seed sample was color sorted by hand 

into red and yellow seeds and the purity of red kernel sample was confirmed by a 

fluorescence assay using a light box. All kernel samples remained frozen during the color 

sorting process. Yellow kernel samples were used as negative controls for MS45 Western 

blot analysis. Red kernel samples (32138 SPTmaintainer) were lyophilized under vacuum 

until dry, and were finely ground and stored frozen until analysis. 

9.2 Protein Extraction from Processed Maize Tissues for MS45 Western Blot 

Analysis

 

For MS45 western blot analysis, all samples were extracted in 600 μl of 1X LDS sample 

buffer (prepared using the following ratio of 65% PBST, 25% 4X LDS sample buffer and 10% 

10X reducing buffer containing DTT) with the addition of two 5/32” steel balls per tube in a 

genogrinder for a single 30 sec cycle at a setting of 1500 strokes per minute. Extracts were 

centrifuged until separated in a refrigerated centrifuge and supernatants were prepared for 

electrophoresis. 
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32138 SPT Maintainer No CBI 

9.3. Preparation of MS45 Standard Solution for Western Blot Analysis 

MS45 protein standard was diluted to the following concentrations; 200, 100, and 50 ng/ml in 

1XLDS sample buffer prepared using the following ratio of 65% PBST, 25% 4XLDS sample 

buffer and 10% 10X reducing buffer containing DTT. 

9.4. SDS-PAGE Analysis 

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed 

using a 4-12% Bis-Tris Gels (Invitrogen, #NP0322). Samples that were heated for 5-15 

minutes at 70C then stored on either ice, or kept frozen were briefly warmed (less than 2 

minutes) at 70C prior to loading. Samples were loaded at 20 l /well and SeeBlue®

 Prestained Standard (Invitrogen, #LC5625) was loaded at 10 l/well. Electrophoresis was 

conducted using a XCell SureLockTM Mini-Cell electrophoresis unit (Invitrogen, #EI0001) with 

NuPAGE®

 MES SDS Running Buffer (Invitrogen, #NP0002) and a constant voltage setting of 

200V for approximately 30 min or until the dye front neared the bottom of the gel. 

9.5. Western Blot Analysis 

Following SDS-PAGE, gels were removed from the gel cassette and electrophoretically 

transferred to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) for approximately 7 

minutes using an iBlot Module (Invitrogen Catalog # IB1001) according to manufacturer’s 

instructions. Following the transfer, the PVDF membrane blots were blocked with a 5% low 

fat milk/PBST solution followed by at least 60 minute at room temperature or overnight at 4oC 

incubation with a primary antibody diluted in 1% low fat milk/PBST solution (mouse 

monoclonal antibody 6B3.F7 lot# 090104 diluted at 1:2,500). The blots were washed with 

PBST at least three times, with each wash for at least 10 minutes and then incubated for at 

least 60 minutes with the secondary antibody conjugated to horseradish peroxidase (HRP) 

from Promega, (Anti-mouse IgG-HRP conjugate diluted 1:25,000. The unbound secondary 

antibody-HRP conjugate was removed with additional 3 washes of at least 10 minutes each 

in PBST. 

The blots were developed using a chemiluminescent detection kit (Thermo Scientific, IL, 

USA) followed by detection using a Kodak 4000R Pro Imaging system utilizing the Kodak 

Molecular Imaging Software. Developed blots were evaluated for the presence or absence of 

bands with the expected migration. 

9.6. Protein Extraction from Processed Maize Tissues for Determination of 

ZM-AA1 Protein Concentration 

Each sample was extracted with 600 microliters (μl) of chilled (2-8C) buffer solution which is 

comprised of 50 millimolar (mM) HEPES, 150 mM NaCl, 0.15% Tween-20, 0.5 mM CaCl2. 

0.5% polyethylene glycol and 5 mM Sodium metabisulfite. Two 5/32” steel balls were added 

to each tube, and the samples were homogenized with a single 30 second cycle at a setting 

of 1500 strokes per minute using a SPEX Certiprep GenoGrinder (BT&C/OPS Diagnostics). 

Following centrifugation in Jouan GR-422 model centrifuge at a setting of 4,000 rpm and 4o

C 

for 10 min supernatants were removed, diluted and analyzed for ZM-AA1 protein 

concentrations using specific enzyme linked immunosorbent assay (ELISA). 

9.7. Determination of ZM-AA1 Protein Concentration 

The ZM-AA1 ELISA utilized a “sandwich” Enzyme-Linked ImmunoSorbent Assay (ELISA) for 

the determination of the concentration of ZM-AA1. In this assay, standards and samples 

were incubated in stabilized plates that have been pre-coated with a ZM-AA1-specific 

antibody. Unbound substances were washed from the plate and the bound ZM-AA1 was 
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32138 SPT Maintainer No CBI 

incubated with a different ZM-AA1-specific antibody that had been conjugated to the enzyme 

horseradish peroxidase (HRP). After about one hour of incubation, unbound substances 

were washed off from the plate. The detection of the ZM-AA1-antibody complex was 

accomplished by the addition of a substrate that generated a colored product in the presence 

of HRP. The reaction was stopped after 30 minutes with 1 N Hydrochloric Acid and the 

optical density of each well was determined using a Molecular Devices plate reader 

(Molecular Devices Corporation, Sunnyvale, CA) with a wavelength setting of 450 nm minus 

650 nm. SoftMax Pro software5

 was used to perform the calculations that generated the 

quadratic fit of the standard curve and converted the sample optical density (OD) values to 

ZM-AA1 protein concentration values. The mean duplicate well values in ng/ml were used in 

the calculation of the reported ZM-AA1 concentration of each sample (ng/mg dry weight). A 

characterized sample extract (QCE) was included on each plate as a control for data 

acceptance. 

A quantitative range for the assay was 0.23 ng/ml to 5.5 ng/ml. The lower limit of quantitation 

(LLOQ) in ng/mg dry weight for each tissue was based on extraction volume (l) to weight 

ratios, the limit of quantitation for the ELISA in ng/ml, and the dilutions used for analysis. The 

sample LLOQs on a ng/mg dry weight basis for ZM-AA1 were 0.28ng/mg dry weight for leaf; 

0.092 ng/mg dry weight for whole plant and forage; 0.55 ng/mg dry weight for pollen; and 

0.14ng/mg dry weight for seed. 

9.8. Protein Extraction from Processed Maize Tissues for DsRed2 Fluorometric 

Analysis 

Lyophilized plant tissue samples were weighed into 1.2 ml microtiter tubes at the following 

target weights: pollen 5 mg, leaf 10 mg, grain 10 mg, stalk 20 mg, and whole plant 20 mg. 

Each sample was extracted in 0.8 ml Assay Buffer (1X PBST pH 7.45, 25% StabilZyme 

Select6

 Buffer) and centrifuged until separated. Following centrifugation, the supernatants 

were removed to new tubes and diluted as applicable. 

9.9. DsRed2 Fluorometric Assay – Experimental Design 

Each standard sample (analyzed in triplicate wells on each assay plate), tissue sample 

(analyzed in duplicate wells), and quality control (QC) sample (see description below for QC 

samples) was dispensed into a separate assay plate well, with each well containing a final 

volume of 300 l. Assay Buffer was added to unoccupied wells and the plate was analyzed 

within 30 minutes of plating using a fluorometer at 563 nm excitation, 600 nm emission with a 

590 nm cutoff filter. Each well was read six times, and the fluorometer output was presented 

as a mean Relative Fluorescence Unit (RFU) value per well. SoftMax7

 Pro software was 

used to perform the calculations required to convert the RFU values obtained by the 

fluorometer to protein concentration values. 

The DsRed2 fluorometric assay was validated for quantification of DsRed2 protein in 

lyophilized maize tissues by an assessment of the following: specificity (cross-reactivity and 

matrix effects), accuracy and precision (fortification recovery and assay ruggedness), 

extraction efficiency, and sample dilution agreement. Minimum dilutions were established 

based on the outcome of the dilution agreement, matrix effects, and fortification recovery 

results. In addition, a database was established for standard curve quality control purposes. 

Control of experimental bias was achieved through use of replicate testing. Quality Control 

(QC) samples were included in the analysis to monitor assay performance during the 

validation testing, and consisted of diluted standard (Assay Buffer fortified with DsRed2 at 4.0 

5

 Registered trademark of Molecular Devices Corporation. Sunnyvale CA

6

 StabilZyme Select is a registered trademark of SurModics, Inc. 

7

 Registered trademark of Molecular Devices Corporation.
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32138 SPT Maintainer No CBI 

and 0.5 g/ml, hereafter referred to as QC1 and QC2, respectively) and diluted extracts of a 

DsRed2-positive grain sample (referred to as QCE). Information gathered on the QCE 

samples was used to help establish a baseline value and range for potential use as part of 

the acceptance criteria for the DsRed2 assay. The DsRed2 fluorometric assay demonstrated 

an acceptable level of specificity, accuracy, precision, and extraction efficiency for the 

quantification of DsRed2 protein from lyophilized maize tissues. 

9.10. Calculations for Determining Tissue Concentration of DsRed2 Protein 

a. Standard Curve 

A standard curve was included on each assay plate. The equation for the standard 

curve was generated by the software, which used a quadratic fit to relate the mean 

RFUs obtained from the standards to the respective standard concentrations in 

nanograms per milliliter (ng/ml). 

The quadratic regression equation was applied as follows: y = Cx2

 + Bx + A 

Where x = known standard concentration and y = respective mean Relative 

Fluorescence Unit (RFU). 

b. Sample Concentration 

Interpolation of the sample concentration (g/ml) was solved using the values for A, 

B, and C that were determined for the standard curve. 

Sample concentration (g/ml) = 	 


2C

B B - 4C A -sample RFU

2

 

e.g.: given curve parameters of A = 0.143, B = 0.00625, C= -0.00000399 and a sample RFU = 0.249, 

Sample concentration = 	 
	 


2 	 
 0.00000399

0.00625 0.00625 4 0.00000399 0.143 0.249 2



     = 17 g/ml 

c. Adjustment for Dilution Factor 

Concentration values were adjusted for a dilution factor expressed as 1:N by 

multiplying the interpolated concentration by N. 

 

e.g.: given a dilution factor of 1:8 and an interpolated concentration of 0.63 g/ml, 

Adjusted concentration = 8 * 0.63 g/ml = 5.0 g/ml 

 
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Appendix 10 

Equivalency of Plant Derived and Microbially Expressed DsRed2 Protein

10.1. Method for Purification of the DsRed2 Protein from 32138 SPT Maintainer 

Approximately 50 g of ground seed derived from 32138 SPT maintainer was homogenized in 

250 ml Buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM sodium chloride and EDTAfree Complete Protease Inhibitor Cocktail [Roche Applied Science, Indianapolis, IN]). 

Particulate material in the resulting slurry was removed by filtering through four layers of 

cheese cloth. The extract was further clarified by centrifugation for 20 minutes at 30,500 g

and filtration through 0.45 m membrane. 

Proteins in the extract were subjected to ammonium sulfate fractionation (45-65% saturation). 

The precipitated protein was resuspended in Buffer B (50 mM Tris, pH 8.0, 50 mM NaCl, 0.8 

M ammonium sulfate and 1 mM EDTA). The sample was then applied to a Phenyl 

Sepharosec

 column (Cat. # 17-1355-01, GE Healthcare Bio-Sciences, Piscataway, NJ). The 

bound protein was eluted from the column with a linear ammonium sulfate gradient mixed 

with Buffer B and Buffer C (50 mM Tris, pH 8.0, 50 mM NaCl and 1 mM EDTA). The peak 

fractions containing DsRed2 protein were pooled and the buffer of the sample was 

exchanged to Buffer D (50 mM Tris, pH 8.5, 50 mM NaCl and 1 mM EDTA). The resulting 

sample was loaded on to a Mono Q8

 column (Cat. # 17-5166-01, GE Healthcare BioSciences). The bound protein was eluted from the column with a linear NaCl gradient 

obtained by mixing Buffer D with Buffer E (50 mM Tris, pH 8.5, 1 M NaCl and 1 mM EDTA). 

DsRed2 protein fractions were pooled and diluted with Buffer F (25 mM MES, pH 5.9, 50 mM 

NaCl and 1 mM EDTA). Proteins in the resulting sample were separated by a Mono S8

column (Cat. # 17-5168-01, GE Healthcare Bio-Sciences). After buffer exchange into Buffer 

D, the DsRed2 protein sample was loaded back to the Mono Q8 column for further purification 

as described above. The purified protein was stored at -80o

C with or without concentration 

by Nanosep9

 3K concentrators (Pall Corporation, NY). 

10.2. Method for Purification of the DsRed2 Protein Expressed in E. coli

The microbial DsRed2 protein was expressed in E. coli strain BL21 (DE3) as a fusion protein 

containing a His-thioredoxin tag and was purified using an immobilized metal affinity column 

(TALON10 Metal Affinity resin, Clontech, Cat. # 635503,1290 Terra Bella Avenue, Mountain 

View, CA 94043, USA). After the fusion protein was eluted from the TALON column, the tag 

was cleaved in solution with thrombin (Calbiochem, Cat. # 605195, EMD Biosciences, Inc.). 

Thrombin cleavage resulted in two additional N-terminal amino acid residues (glycine and 

serine) that are not part of the DsRed2 protein. Following thrombin cleavage, the protein was 

concentrated and buffer exchanged by tangential flow filtration. Thrombin was removed by 

chromatography on HiTrapb

 SP Sepharose11 HP columns (Cat. # 17-1151-01, GE Healthcare 

Bio-Sciences, Piscataway, NJ). Diafiltration was used to remove the cleaved fusion tag and 

exchange the buffer to 50 mM ammonium bicarbonate, pH 7.5. The purified DsRed2 protein 

solution was lyophilized and the resulting powder was stored at -80C. 

8

 Registered trademark of GE Healthcare Bio-Sciences AB 

9

 Registered trademark of Pall Corporation 

10 Registered trademark of Clontech 11 Registered trademark of GE Healthcare Bio-Sciences AB
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10.3. SDS-PAGE Analysis Method 

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by 

first mixing DsRed2 protein samples with Laemmli sample buffer (Gradipore Limited, 22 

Rodborough Road, Frenchs Forest NSW 2086, Australia) containing 100 mM dithiothreitol 

and heating the solution to 100°C for approximately five minutes. The prepared protein 

samples were loaded into a 10-20% gradient Tris-HCl Ready Gel12 (Bio-Rad Laboratories, 

Inc., CA ). PageRuler13 Prestained Protein Ladder (#SM0671; Fermentas, Inc., Maryland, 

USA) molecular weight markers were loaded into the gel to provide a visual estimate of 

molecular weight. Electrophoresis was conducted using the Ready Gel Cell system (Bio-Rad 

Laboratories, Inc) with Tris-glycine running buffer (Gradipore Limited) at a constant 150 volts 

(V) for ~60 minutes or until the dye front neared the bottom of the gel. 

Upon completion of electrophoresis, the gels were removed from the gel cassette and 

washed three times with deionized water for approximately five minutes each. The gels were 

then stained for approximately 60 minutes with GelCode14 Coomassie Blue stain reagent 

(Pierce Biotechnology, Inc., IL), and rinsed with deionized water at least four times for 

approximately ten minutes each, or until the background of the gel was clear. 

10.4. Western Blot Analysis Method 

Following SDS-PAGE, the resulting gel was soaked in Transfer buffer (48 mM Tris-HCl, pH 

8.6, 39 mM glycine, 0.0375% sodium dodecyl sulfate, and 20% methanol) for approximately 

20 minutes. A polyvinylidene difluoride (PVDF, Bio-Rad Laboratories, Inc.) membrane was 

briefly placed in 100% methanol, followed by immersion in Transfer buffer for 10-15 minutes. 

A Trans-Blot15 SD Semi-Dry Electrophoretic Transfer Cell system (Bio-Rad Laboratories, Inc.) 

was used to transfer proteins from the gel to the membrane at 10 V for ~45 minutes. 

Following protein transfer, the membrane was then washed three times for five minutes each 

in Classic buffer (50 mM Tris-HCl, pH 7.0, 500 mM NaCl, 0.5% Tween16-20), and then 

blocked by incubating in phosphate-buffered saline solution with Tween-20 (PBST: 8.1 mM 

phosphate buffer, pH 7.4, 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween-20) containing 5% 

(w/v) non-fat dry milk for 60 minutes. The blocked membrane was washed three times for 

five minutes each in Classic buffer, and then incubated for 60 minutes with a DsRed rabbit 

polyclonal primary antibody (Clontech, Cat. #632496) diluted 1: 5,000 in PBST containing 5% 

non-fat dry milk. The unbound antibody was rinsed from the membrane with three washes of 

Classic buffer for five minutes each. The membrane was then incubated for 60 minutes with 

a secondary antibody (anti-rabbit IgG horseradish peroxidase conjugate) diluted 1:10,000 in 

Classic buffer. The membrane was then washed three times with Classic buffer for five 

minutes each and developed with Pierce Super Signal14 West Pico Chemiluminescent 

Substrate (Pierce Biotechnology, Inc., 3747 N. Meridian Road, Rockford, IL 61105, USA) 

according to the manufacturer’s instructions. The molecular weight of the recognized protein 

band was estimated using PageRuler Prestained Protein Ladder (#SM0671; Fermentas). 

10.5. Method for N-Terminal Amino Acid Sequence Analysis 

The 32138 SPT maintainer derived DsRed2 protein sample was separated by SDS-PAGE 

and then electrophoretically transferred to a PVDF membrane as previously described. The 

resulting blot was stained with Ponceau S solution (Sigma-Aldrich, St. Louis, MO) to visualize 

the protein band. The DsRed2 band was excised and sent to Crop Genetics (DuPont 

12 Registered trademark of Bio-Rad Laboratories, Inc. 13 Registered trademark of Fermentas, Inc. 14 Registered trademark of Pierce Biotechnology, Inc. 15 Registered trademark of Bio-Rad Laboratories, Inc. 16 Registered trademark of ICI Americas, Inc.
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32138 SPT Maintainer No CBI 

Experimental Station) for Edman N-terminal amino acid sequencing with the Procise17 494 

cLC analyzer (Applied Biosystems, Inc., Foster City, CA) equipped with an online high 

performance liquid chromatography system. 

The microbially expressed DsRed2 protein sample was sent to Crop Genetics (E. I. du Pont 

de Nemours and Company, DuPont Experimental Station, Wilmington, DE 19880, USA) as 

lyophilized powder. The protein was resuspended in water and transferred to PVDF 

membrane using ProSorb18 Inserts (Applied Biosystems, Foster City CA 94404, USA) and 

washed with 200 l 0.1% TFA in water. The membrane was removed and used for Edman 

N-terminal amino acid sequencing with the Procise17 494 LC analyzer (Applied Biosystems) 

equipped with an online high performance liquid chromatography system. 

10.6. Method for LC/MS Identification of Tryptic Peptides 

Following SDS-PAGE, the 32138 SPT maintainer derived DsRed2 protein was visualized by 

staining with Coomassie Blue. The bands (~27 kDa and ~18 kDa) were then excised from 

the gel. The gel slices were digested with trypsin for approximately 14 hours at 37°C. Tryptic 

peptides were analyzed using a Thermo Fisher Scientific LCQ19 ion trap mass spectrometer 

(Thermo Fisher Scientific, Inc., USA) equipped with an Agilent 1100 HPLC (Agilent, 5301 

Stevens Creek Blvd, Santa Clara, CA) and a New Objective PicoView20 ESI source (New 

Objective, 2 Constitution Way, Woburn, MA 01801, USA). The sample was injected into an 

Agilent 1100 HPLC nanoflow system, separated on a C18 capillary column, and eluted at a 

flow rate of 200 nl/min directly into the mass spectrometer using a reverse phase gradient. 

Peptides were identified by analyzing the tandem MS spectra of the peptides using Turbo 

SEQUEST software. Peptide matches were made by comparing identified peptides with the 

expected DsRed2 peptides as determined by in silico trypsin cleavage of the protein 

sequence. In addition, the identified peptides were screened to detect a blocked N-terminal 

sequence. 

In the case of microbially expressed DsRed2 protein, following SDS-PAGE and Coomassie 

Blue staining, the gel slice containing the sample was excised, placed in an Eppendorf tube, 

and digested with trypsin for approximately 14 hours at 37°C. Tryptic peptides were analyzed 

using a ThermoFisher Scientific LCQ21 Classic ion trap mass spectrometer (Thermo Fisher 

Scientific, Inc.) equipped with an Agilent 1100 HPLC (Agilent, Santa Clara, CA) and a New 

Objective PicoView22 ESI source (New Objective, Woburn, MA). The sample was injected 

into an Agilent 1100 HPLC nanoflow system, separated on a C18 capillary column and eluted 

at a flow rate of 200 nl/min directly into the mass spectrometer using a reverse phase 

gradient. Peptides were identified by analyzing the tandem MS spectra of the peptides using 

Turbo SEQUEST software. The peptide matches were made by comparison to the DsRed2 

peptides as determined by in silico trypsin cleavage of the protein sequence. Allowances 

were made for the modifications to the peptides caused by oxidation of methionine (observed 

value is 15.999 Da greater than the theoretical value). 

10.7. Method for Protein Glycosylation Analysis 

A GelCode glycoprotein staining kit (Pierce Biotechnology, Inc.) was used according to the 

manufacturer’s instructions to determine whether the DsRed2 proteins was glycosylated. 

The microbially expressed and the maize derived DsRed2 proteins, a positive control protein 

(horseradish peroxidase), and a negative control protein (soybean trypsin inhibitor) were run 

17 Registered trademark of Applied Biosystems 18 Registered trademark of Applied Biosystems 19 Registered trademark of Thermo Fisher Scientific, Inc. 20 Registered trademark of New Objective 21 Registered trademark of ThermoFisher Scientific Inc. 22 Registered trademark of New Objective
Pioneer Hi-Bred Page 171 of 188 

32138 SPT Maintainer No CBI 

by SDS-PAGE as described in the above methodology. Following electrophoresis, the gel 

was fixed with 50% methanol for 30 minutes and washed with 3% acetic acid. The gel was 

then incubated with oxidizing solution for 15 minutes and washed three times with 3% acetic 

acid. The gel was incubated with GelCode glycoprotein staining reagent (Pierce 

Biotechnology, Inc.) for 15 minutes and then incubated in reducing reagent. The gel was 

then extensively washed with 3% acetic acid and deionized water. Glycoproteins were 

detected as magenta colored bands on the gel. Following glycoprotein detection, the gel was 

scanned and the image was captured electronically. The same gel was then stained with 

Coomassie Blue, as described previously, to visualize all protein bands. 

10.8. Results of SDS-PAGE and Western Analyses of DsRed2 Protein 

SDS-PAGE analysis indicated that the partially purified 32138 SPT maintainer derived 

DsRed2 protein (Appendix 10-Figure 1, lane 2) and the microbially expressed DsRed2 

protein (Appendix 10-Figure 1, lane 3) samples had predominant bands migrating at 

approximately 27 kDa. For both the 32138 SPT maintainer derived DsRed2 protein and the 

microbially expressed DsRed2 protein, a minor band migrating at approximately 18 kDa was 

detected. A faint band migrating at approximately 10 kDa was also detected in the 

microbially expressed and 32138 SPT maintainer derived DsRed2 proteins. The 18 kDa and 

10 kDa minor bands were likely truncated DsRed2 fragments resulted from partial 

fragmentation of the protein during denaturation. 

Western blot analysis of the 32138 SPT maintainer derived and microbially expressed 

DsRed2 proteins resulted in detection of a major band migrating at approximately 27 kDa. 

For both the 32138 SPT maintainer derived DsRed2 protein and the microbially expressed 

DsRed2 protein, a minor band migrating at approximately 18 kDa was also recognized by the 

antibody, suggesting that the band was a truncated DsRed2 fragment. 

In conclusion, DsRed2 proteins from the two sources demonstrated equivalency in SDSPAGE and Western analyses. 
Pioneer Hi-Bred Page 172 of 188 

32138 SPT Maintainer No CBI 

10.9. Results of N-terminal Amino Acid Determination 

Analysis by LC/MS following trypsin digestion of the 32138 SPT maintainer derived DsRed2 

protein indicated that the first methionine residue (M) was cleaved off and the second 

residue, alanine (A), was exposed. LC/MS analysis also demonstrated that this alanine 

residue was acetylated, confirming a blocked N-terminus of the protein. Therefore, the actual 

N-terminal sequence of the 32138 SPT maintainer derived DsRed2 protein was found to be 

ASSENVITEFMR (Appendix 10-Table 1). As expected, N-terminal sequencing analysis 

indicated that the microbially expressed DsRed2 had two additional N-terminal amino acid 

residues (glycine and serine) resulting from thrombin cleavage of the His-thioredoxin fusion 

tag used for protein expression and purification. Therefore, after accounting for the missing 

N-terminal methionine residue of the 32138 SPT maintainer derived DsRed2 and the two 

additional N-terminal amino acid residues of the microbially expressed DsRed2, the 

N-terminal sequences of the microbially expressed and 32138 SPT maintainer derived 

DsRed2 proteins matched the expected sequence of the DsRed2 protein. 

. 

Lane Sample ID 

1 Fermentas PageRuler Prestained Protein Ladder 

2 DsRed2 Derived from 32138 SPT Maintainer (~1 μg for SDS-PAGE and 

~100ng for the Western Blotting 

3 DsRed2 Derived from Microbial Expression System, 

 (~1 μg for SDS-PAGE and ~100ng for the Western Blotting) 

SDS-PAGE Western Blot

Figure 1. SDS PAGE and Western Blot Analysis of Microbially Expressed and 

32138 SPT Maintainer Derived DsRed2 protein

1 2 3 1 2 3

kDa

170 

130

95

72

55

43

34

26

17

11

kDa

170 

130

95

72

55

43

34

26

17

11

SDS-PAGE Western Blot

Figure 1. SDS PAGE and Western Blot Analysis of Microbially Expressed and 

32138 SPT Maintainer Derived DsRed2 protein

1 2 3 1 2 3

SDS-PAGE Western Blot

Figure 1. SDS PAGE and Western Blot Analysis of Microbially Expressed and 

32138 SPT Maintainer Derived DsRed2 protein

1 2 3 1 2 3

kDa

170 

130

95

72

55

43

34

26

17

11

kDa

170 

130

95

72

55

43

34

26

17

11
Pioneer Hi-Bred Page 173 of 188 

32138 SPT Maintainer No CBI 

Table 1. N-terminal Amino Acid Sequence of the Microbially Expressed and 32138 SPT 

Maintainer derived DsRed2 protein. 

10.10. Results of the LC/MS Identification of Tryptic Peptides of DsRed2 

LC/MS analysis of the trypsin digest of the 32138 SPT maintainer derived and microbially 

expressed DsRed2 proteins identified 11 and 9 peptides, respectively, that matched the 

expected tryptic peptides of the DsRed2 protein, resulting in coverage of 67% and 51% of the 

DsRed2 amino acid sequence (Appendix 10-Table 2). The 32138 SPT maintainer derived 

DsRed2 protein and the microbially expressed DsRed2 protein each possessed two of the 

identified peptides with an oxidized methionine residue. The 18 kDa minor band from the 

32138 SPT maintainer derived DsRed2 protein sample was also analyzed by LC/MS 

following trypsin digestion. Seven peptides (87 amino acid residues) were identified 

matching the DsRed2 protein sequence (data not presented), indicating that this band was a 

truncated fragment derived from the DsRed2 protein 

Table 2. Identified Peptides of the Microbially Expressed and 32138 SPT Maintainer 

Derived DsRed2 Proteins Using LC/MS Analysis 

Footnotes: 

1. The N-terminal glycine and serine residues are not a part of the DsRed2 sequence but remain following 

thrombin cleavage of the His-thioredoxin fusion tag used for protein expression and purification of the 

microbially expressed DsRed2. 

2. ND is an abbreviation for not detected 

3. Peptide identified following correction for alanine acetylation (addition of 42.0370 Da to theoretical mass) 

4. Peptide identified following correction for methionine oxidation (addition of 15.995 Da to theoretical mass) 

Amino 

Acid 

Residue # 

Microbially Expressed 

DsRed2 

32138 SPT Maintainer Derived 

DsRed2 

GS1+1-13 GSMASSENVITEFMR ND 2

2-13 ND ASSENVITEFMR 3

18-45 ND MEGTVNGHEFEIEGEGEGRPYEGHNTVK 

18-36 MEGTVNGHEFEIEGEGEGR 4 ND 

37-45 PYEGHNTVK ND 

71-83 ND VYVKHPADIPDYK 

75-83 HPADIPDYK ND 

84-95 KLSFPEGFKWER KLSFPEGFKWER 

122-138 ND VKFIGVNFPSDGPVMQK4

124-138 FIGVNFPSDGPVMQK ND 

139-149 ND KTMGWEASTER4

140-149 TMGWEASTER 4 ND 

150-158 ND LYPRDGVLK 

154-163 ND DGVLKGETHK 

167-178 LKDGGHYLVEFK LKDGGHYLVEFK 

185-198 KPVQLPGYYYVDAK KPVQLPGYYYVDAK 

199-216 ND LDITSHNEDYTIVEQYER 

Actual

32138 SPT Maintainer Derived

Microbially Expressed

M -A -S-S-E-N -V -I -T -E -F-M -R 

A -S-S-E -N -V -I -T -E -F-M -R 

G -S-M -A -S-S-E-N -V -I -T -E -F-M -R 

Actual

32138 SPT Maintainer Derived

Microbially Expressed

M -A -S-S-E-N -V -I -T -E -F-M -R 

A -S-S-E -N -V -I -T -E -F-M -R 

G -S-M -A -S-S-E-N -V -I -T -E -F-M -R 
Pioneer Hi-Bred Page 174 of 188 

32138 SPT Maintainer No CBI 

10.11. Results of the Protein Glycosylation Analysis of DsRed2 Protein 

Glycosylation was not detected for either the 32138 SPT maintainer derived or microbially 

expressed DsRed2 proteins (Appendix 10-Figure 2). The glycoprotein positive control 

(horseradish peroxidase) was clearly visible as a magenta colored band and the negative 

control (soybean trypsin inhibitor) was not visible after staining. 

Figure 2. Glycosylation Analysis of the Microbially Expressed and 32138 SPT 

Maintainer Derived DsRed2 protein 

Lane Sample ID 

1 Fermentas PageRuler Prestained Protein Ladder 

2 Negative Control, Soybean Trypsin Inhibitor (1.5 μg) 

3 Positive Control, Horseradish Peroxidase (1.5 μg) 

4 DsRed2 Derived from Microbial Expression System, 

 (~1.5 μg) 

5 DsRed2 Derived from 32138 SPT Maintainer (1.5 μg) 

a This gel was stained with the glycoprotein staining kit

b This gel was stained with the glycoprotein staining kit followed by staining with 

Coomassie Blue

kD

170 

130

95

72

55

43

34

26

17

11

Glycoprotein Staining a Total Protein Staining b

kD

170 

130

95

72

55

43

34

26

17

11

1 2 3 4 5 1 2 3 4 5 

a This gel was stained with the glycoprotein staining kit

b This gel was stained with the glycoprotein staining kit followed by staining with 

Coomassie Blue

kD

170 

130

95

72

55

43

34

26

17

11

Glycoprotein Staining a Total Protein Staining b

kD

170 

130

95

72

55

43

34

26

17

11

1 2 3 4 5 1 2 3 4 5 
Pioneer Hi-Bred Page 175 of 188 

32138 SPT Maintainer No CBI 

 Appendix 11 

Description of Statistical Analyses 

11.1. Trait Inheritance Data (Section V-E, Table 7) 

Based on Mendel’s segregation law, the expected segregation ratios in Table 7 were tested 

by the statistic: 

2 = [	(o – e)2 / e] 

where o = observed frequency of the genotype, 

e = expected frequency of the genotype, and 

For the binomial case, chi-squared values are calculated by 

2 = 	 [(o – e)2

 / e] 

= (nobs(pos) – nexp(pos))

2

 / nexp(pos) + 

(nobs(neg) – nexp(neg))

2

 / nexp(neg)

where n is the total of the positive (nobs(pos)) and negative (nobs(neg)) frequencies. 

2 follows a chi-squared distribution with one degree of freedom (df) for binomial cases 

(Agresti, 2002). 

2

 values > 3.84 (2(df=1)) have probability p< 0.05, indicate a significant difference between 

observed and expected frequencies. 

11.2. Agronomic Data (Section VII-B) 

Agronomic data (Table 12) were analyzed using a linear mixed model designed to account 

for the design effects of location and blocks within location. The linear mixed model assumed 

the entries were a fixed effect while the locations, blocks within locations and the entry by 

location interaction were random effects. 

Early population, seedling vigor, time to silking, time to pollen shed, pollen viability, stalk 

lodging, root lodging, final population, stay green, disease incidence and insect damage data 

were analyzed using the following linear mixed model: 

yijk = i + 
j + rk(j) + ()ij + ijk 


j ~ iid N(0, 2

Loc), rk(j) ~ iid N(0,2Rep), ()ij ~ iid N(0,2Loc×Ent), and ijk ~ iid 

N(0,2

plot) 

Where i denotes the mean of the ith entry (fixed effect), 
j denotes the effect of the jth

site (random effect), rk(j) denotes the effect of the kth block within the jth site (random 

effect), ()ij denotes the interaction between the entries and sites (random effect) 

and ijk 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(0,2

a) indicates random variables that are 

identically independently distributed (iid) as normal with zero mean and variance 2

a. 
Pioneer Hi-Bred Page 176 of 188 

32138 SPT Maintainer No CBI 

Plant height and ear height data were analyzed using the following linear mixed model: 

yijkl = i + 
j + rk(j) + ()ij + ijk + ijkl 

ÿj ~ iid N(0,2

Loc), rk(j) ~ iid N(0,2Rep), ()ij ~ iid N(0,2Loc×Ent), ijk ~ iid 

N(0,2

plot), and ijkl ~ iid N(0,2observations) 

Where i denotes the mean of the ith entry (fixed effect), 
j denotes the effect of the jth site 

(random effect), rk(j) denotes the effect of the kth block within the jth site (random effect), ()ij

denotes the interaction between the ith entries and jth sites (random effect), ijk denotes the 

effect of the plot assigned the ith entry in the kth block of the jth site (random plot), and ijkl 

denotes the effect of the plant assigned the lth plant in the ith entry in the kth block of the jth site 

(observational error). 

A significant difference between the mean of 32138 SPT maintainer and the means of nearisoline control was established with a false discovery rate (FDR)-adjusted P-value <0.05. 

When numerous comparisons are being made, it is important to control the rate of false 

positive results. Since the introduction of the 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., 2006). 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. Pioneer's adjustment of pvalues using the FDR method is appropriate for the statistical analysis of agronomic and 

nutrient composition data because the adjustment controls the chance of declaring "false 

significant differences" when numerous comparisons are conducted in a single study. In 

particular, the FDR method offers a potential gain in power compared to other multiplicity 

adjustment procedures (Bonferroni, Holm’s, Hochberg’s step-up, etc.) (Westfall, 2006), and 

the potential for an increase in power is larger when more of the hypotheses are non-true. 

11.3. Nutrient Composition Data (Section VIII-A through VIII-F) 

Data were analyzed using a linear mixed model designed to account for the design effects of 

location and blocks within location. The linear mixed model assumes the entries are a fixed 

effect while the locations, blocks within locations and the entry by location interaction are 

random effects. A significant difference between the mean of 32138 SPT maintainer and the 

means of the near-isoline control was established with an FDR-adjusted P-value <0.05 for 

each analyte. 

Composition data presented in Tables 13-19 were analyzed using the following linear mixed 

model: 

yijk = i

 + j + rk(j) + ()ij + ijk 

j

 ~ iid N(0,2

Loc), rk(j) ~ iid N(0,2Rep), ()ij ~ iid N(0,2Loc×Ent), and ijk ~ iid N(0,2plot) 

Where i denotes the mean of the ith entry (fixed effect), 
j denotes the effect of the jth site 

(random effect), rk(j) denotes the effect of the kth block within the jth site (random effect), ()ij

denotes the interaction between the entries and sites (random effect) and ijk denotes the 
Pioneer Hi-Bred Page 177 of 188 

32138 SPT Maintainer No CBI 

effect of the plot assigned the ith entry in the kth block of the jth site (random effect or residual). 

Notation ~iid N(0,2

a) indicates random variables that are identically independently distributed 

(iid) as normal with zero mean and variance 2

a. 
Pioneer Hi-Bred Page 178 of 188 

32138 SPT Maintainer No CBI 

Appendix 12 

USDA Field Trials of 32138 SPT Maintainer 1, 2 

Year of 

Planting Permit Name Permit Valid Date State 

# of Counties 

Where 32138 

SPT Maintainer 

was Planted 

Acreage3

2005 05-024-04r 5/25/2005 HI 1 0.02 

2006 05-024-04r 5/25/2005 HI 1 0.083 

IA 1 0.025 2006 06-019-03R 5/2/2006 PR 1 0.022 

2006 06-019-04R 5/3/2006 HI 1 0.35 

2007 06-019-03R 5/2/2006 PR 1 0.003 

2007 06-019-04R 5/3/2006 HI 1 0.032 

HI 2 0.364 

IL 3 0.058 

IA 4 0.742 

MN 1 0.023 

NE 1 0.035 

2007 07-040-101rm 5/3/2007 

PR 1 0.101 

HI 2 5.448 2008 07-040-101rm 5/3/2007 PR 1 0.6 

IL 2 0.0459 

IA 1 0.002 

KS 1 0.046 2008 08-011-101n4

 4/16/2008 

NE 1 2.819 

2008 08-011-102n4 4/2/2008 OK 1 0.046 

IL 1 0.046 

2008 08-014-105n IA 1 0.022 4 4/16/2008 

PR 1 0.138 

2008 08-014-124n4 3/17/2008 HI 1 1.003 

HI 2 11.926 

2008 08-023-101n IA 1 0.011 4 3/1/2008 

WA 1 0.69 

2009 08-014-105n4 4/16/2008 PR 1 0.06 

2009 08-014-124n4 3/17/2008 HI 1 0.068 

2009 09-012-101n4 3/1/2009 HI 1 0.443 

2009 09-013-107n4 4/1/2009 IA 2 0.01 

 HI 1 0.023 2009 09-035-107n4

 3/19/2009 WI 1 0.006 

Footnotes: 

1. Plantings through June 5, 2009 are listed. 

2. In USDA final reports, 32138 SPT Maintainer is called E6611.32.1.38. 

3. Total acreage for all 32138 SPT Maintainer (acreage includes non-transgenic 32138 progeny plants) 

4. Final field test report not yet due to USDA. 
Pioneer Hi-Bred Page 179 of 188 

32138 SPT Maintainer No CBI 

Appendix 13 

Ecological Observations of 32138 SPT Maintainer 

Key to “Range of severity in 32138 SPT Maintainer” in Appendix 13-Tables 1 and 2: 

mild – very little disease or insect injury (<10%) visible; 

moderate – noticeable plant tissue damage (10% to 30%); 

severe – significant plant tissue damage (>30%). 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 

Year of 

Planting 

Permit 

number State County Stressor 

Range of 

Severity in 

32138 SPT 

maintainer 

Difference 

with Control? 

Corn earworm 

(Helicoverpa zea) mild no 

Leafhopper 

(Cicadellidae) mild no 

Rose beetle (Adoretus 

sinicus) mild no 

2005 05-024-04r HI Kauai 

Beet armyworm 

(Spodoptera exigua) 

mild to 

moderate no 

Corn earworm 

(Helicoverpa zea) mild no 

Leafhopper 

(Cicadellidae) 2006 05-024-04r HI Kauai mild no 

Rose beetle (Adoretus 

sinicus) mild no 

Corn flea beetle 

(Chaetocnema 

pulicaria) 

mild no 

Corn earworm 

(Helicoverpa zea) mild no 

European corn borer 

(Ostrinia nubilalis) mild no 

Western bean cutworm 

(Richia albicosta) mild no 

Fall armyworm 

(Spodoptera frugiperda) mild no 

IA Polk 

Corn rootworm 

(Diabrotica spp.) mild no 

Fall armyworm 

(Spodoptera frugiperda) 

mild to 

severe 

no 

2006 06-019-03R 

PR Salinas Corn earworm 

(Helicoverpa zea) mild no 

Corn earworm 

(Helicoverpa zea) mild no 

Leafhopper 

(Cicadellidae) 2006 06-019-04R HI Kauai mild no 

Rose beetle (Adoretus 

sinicus) mild no 

Fall armyworm 

(Spodoptera frugiperda) moderate no 

2007 06-019-03R PR Salinas Corn earworm 

(Helicoverpa zea) mild no 
Pioneer Hi-Bred Page 180 of 188 

32138 SPT Maintainer No CBI 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 

(continued) 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

maintainer 

Difference 

with Control? 

Corn earworm 

(Helicoverpa zea) mild no

Leafhopper 

(Cicadellidae) mild no

Rose beetle 

(Adoretus sinicus) mild no

Spidermite 

(Tetranychidae) mild no

2007 06-019-04R HI Kauai 

Leafhopper 

(Cicadellidae) mild no

Leafhopper 

(Cicadellidae) mild no

Corn earworm 

(Helicoverpa zea) mild no

Spidermite 

(Tetranychidae) mild no

Green lacewing

(Chrysoperla carnea) mild no 

Syrphid Flies 

(Syrphidae) mild no 

Honolulu

Spidermite 

(Tetranychidae) mild no

Green lacewing

(Chrysoperla carnea) mild no 

Leafhopper 

(Cicadellidae) mild no

Corn earworm 

(Helicoverpa zea) mild no

Syrphid Flies 

(Syrphidae) mild no 

Spidermite 

(Tetranychidae) mild no

HI 

Kauai 

Aphids (Aphididae) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

Bureau European corn borer 

(Ostrinia nubilalis) mild no 

Leafhopper 

(Cicadellidae) mild no

Clinton Japanese beetle 

(Popillia japonica) mild no

Western bean 

cutworm (Richia 

albicosta) 

mild no 

2007 07-040-101rm 

IL 

Stark 

European corn borer 

(Ostrinia nubilalis) mild no 
Pioneer Hi-Bred Page 181 of 188 

32138 SPT Maintainer No CBI 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 

(continued) 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

Cutworms 

(coleopteran) mild no 

European corn borer 

(Ostrinia nubilalis) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

Leafhopper 

(Cicadellidae) mild no

Green 

Aphids (Aphididae) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

European corn borer 

(Ostrinia nubilalis) mild no 

Leafhopper 

(Cicadellidae) mild no

Aphids (Aphididae) mild no 

Jefferson

Corn earworm 

(Helicoverpa zea) mild no

Japanese beetle (Popillia 

japonica) mild no

Corn rootworm 

(Diabrotica spp.) Linn mild no 

European corn borer 

(Ostrinia nubilalis) mild no 

Beetles (Coleoptera) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

Aphids (Aphididae) mild no 

Corn earworm 

(Helicoverpa zea) mild no 

2007 07-040-101rm IA 

Polk 

European corn borer 

(Ostrinia nubilalis) mild no 
Pioneer Hi-Bred Page 182 of 188 

32138 SPT Maintainer No CBI 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 

(continued)

Year of 

Planting Permit Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

European corn borer 

(Ostrinia nubilalis) mild no 

MN Freeborn Aphids (Aphididae) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

European corn borer 

(Ostrinia nubilalis) mild no 

Western bean cutworm 

(Richia albicosta) 

mild to 

moderate no 

Leafhopper 

(Cicadellidae) mild no

Corn earworm 

(Helicoverpa zea) mild no

Picnic beetle 

(Glischrochilus 

quadrisignatus)

mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

Fall armyworm 

(Spodoptera frugiperda) mild no 

Beetles (Coleoptera) mild no 

Butterflies (Lepidoptera) mild no 

NE York 

Western bean cutworm 

(Richia albicosta) mild no 

Fall armyworm 

(Spodoptera frugiperda) mild no 

2007 07-040-101rm 

PR Guayama Corn earworm 

(Helicoverpa zea) mild no

Corn earworm 

(Helicoverpa zea) mild no

Rose beetle (Adoretus 

sinicus) Honolulu mild no 

Common thrips 

(Thripidae) mild no 

Leafhopper 

(Cicadellidae) mild no

Spidermite 

(Tetranychidae) mild no 

HI 

Kauai 

Corn earworm 

(Helicoverpa zea) mild no

Fall armyworm 

(Spodoptera frugiperda) mild no 

2008 07-040-101rm 

PR Salinas Corn earworm 

(Helicoverpa zea) mild no
Pioneer Hi-Bred Page 183 of 188 

32138 SPT Maintainer No CBI 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 

 (continued) 

Year of 

Planting

Permit Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

Fall armyworm 

(Spodoptera frugiperda) mild no 

Clinton Japanese beetle (Popillia 

japonica) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

IL 

Stark European corn borer 

(Ostrinia nubilalis) mild no 

IA Polk Japanese beetle (Popillia 

japonica) mild no 

KS Pawnee European corn borer 

(Ostrinia nubilalis) mild no 

Corn rootworm 

(Diabrotica spp.) mild no 

Aphids (Aphididae) mild no 

Grasshoppers 

(Orthoptera) mild no 

2008 08-011-101n 

NE York 

European corn borer 

(Ostrinia nubilalis) mild no 

2008 08-011-102n OK Caddo Fall armyworm 

(Spodoptera frugiperda) mild no 

Japanese beetle (Popillia 

japonica) IL Ogle Corn rootworm 

(Diabrotica spp.) 

mild no 

IA Polk European corn borer 

(Ostrinia nubilalis) mild no 

Fall armyworm 

(Spodoptera frugiperda) mild no 

2008 08-014-105n 

PR Juana 

Diaz Corn earworm 

(Helicoverpa zea) mild no 

Spidermite 

(Tetranychidae) mild no 

Leafhopper 

(Cicadellidae) mild no 

Japanese beetle (Popillia 

japonica) mild no 

2008 08-014-124n HI Kauai 

Rose beetle (Adoretus 

sinicus) mild no 
Pioneer Hi-Bred Page 184 of 188 

32138 SPT Maintainer No CBI 

Table 1. Insect Stressor Comparison between 32138 SPT Maintainer and Control 

 (continued) 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

Spidermite 

(Tetranychidae) mild no 

Kauai 

Leafhopper 

(Cicadellidae) mild no

Leafhopper 

(Cicadellidae) mild no

HI 

Honolulu 

Common thrips 

(Thripidae) mild no 

IA Polk Japanese beetle 

(Popillia japonica) mild no 

2008 08-023-101n 

WA Franklin No Disease N/A no 

N/A= not applicable 
Pioneer Hi-Bred Page 185 of 188 

32138 SPT Maintainer No CBI 

Table 2. Disease Stressor Comparison between 32138 SPT Maintainer and Control 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

Aspergillus ear and 

kernel rot 

(Aspergillus sp.) 

mild no 

Fusarium 

(Fusarium sp.) mild no 2005 05-024-04r HI Kauai 

Gibberella ear rot 

(Gibberella zeae) mild no 

2006 05-024-04r HI Kauai No disease N/A no 

Stewart's seedling 

wilt (Pantoea 

stewartii) 

mild to 

severe 

no 

Common smut 

(Ustilago zeae) 

mild to 

severe 

no 

Common corn rust 

(Puccinia sorghi) mild no 

Northern corn leaf 

blight (Exserohilum 

turcicum) 

mild to 

moderate no 

Gray leaf spot 

(Cercospora zeaemaydis) 

mild to 

moderate no 

IA Polk 

Southern corn rust 

(Puccinia polysora) mild no 

2006 06-019-03R 

PR Salinas No disease N/A no 

2006 06-019-04R HI Kauai No disease N/A no 

2007 06-019-03R PR Salinas No disease N/A no 

Common smut 

(Ustilago zeae) mild no 

2007 06-019-04R HI Kauai Common corn rust 

(Puccinia sorghi) mild no 
Pioneer Hi-Bred Page 186 of 188 

32138 SPT Maintainer No CBI 

Table 2. Disease Stressor Comparison between 32138 SPT Maintainer and Control 

 (continued) 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

Honolulu Common corn rust 

(Puccinia sorghi) mild no 

HI 

Kauai Common smut 

(Ustilago zeae) mild no 

Bureau 

Common corn rust 

(Puccinia sorghi) mild no 

Root rot 

(Phytopthora spp.) mild no 

Seedling Blight 

(Pythium Spp) mild no

Gray leaf spot 

(Cercospora zeaemaydis) 

mild no 

Clinton 

Common corn rust 

(Puccinia sorghi) mild no 

Gray leaf spot 

(Cercospora zeaemaydis) 

mild no 

IL 

Stark Anthracnose 

(Colletotrichum 

graminicola) 

mild no 

Common smut 

(Ustilago zeae) mild no 

Common corn rust 

(Puccinia sorghi) mild no Green 

Gray leaf spot 

(Cercospora zeaemaydis) mild no 

Northern corn leaf 

blight (Exserohilum 

turcicum) 

mild no 

Jefferson 

Common corn rust 

(Puccinia sorghi) mild no 

IA 

Linn 

Gray leaf spot 

(Cercospora zeaemaydis) 

mild no 

Northern corn leaf 

blight (Exserohilum 

turcicum) 

mild no 

Stewart's seedling 

wilt (Pantoea 

stewartii) 

mild to 

moderate no 

Common smut 

(Ustilago zeae) mild no 

Common corn rust 

(Puccinia sorghi) mild no 

Gray leaf spot 

(Cercospora zeaemaydis) 

mild to 

moderate no 

Anthracnose 

(Colletotrichum 

graminicola) 

mild no 

2007 07-040-101rm 

IA Polk 

Southern corn rust 

(Puccinia polysora) mild no 
Pioneer Hi-Bred Page 187 of 188 

32138 SPT Maintainer No CBI 

Table 2. Disease Stressor Comparison between 32138 SPT Maintainer and Control 

(continued) 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

Northern corn leaf 

blight (Exserohilum 

turcicum) 

moderate no 

IA Polk 

Gray leaf spot 

(Cercospora zeaemaydis) 

moderate no 

Common smut 

(Ustilago zeae) mild no 

MN Freeborn Common corn rust 

(Puccinia sorghi) mild no 

Common corn rust 

(Puccinia sorghi) mild no 

NE York Gray leaf spot 

(Cercospora zeaemaydis) 

mild to 

moderate no 

2007 2007-040-

101rm 

PR Guayama No Disease N/A no 

Honolulu Common corn rust 

(Puccinia sorghi) mild no

Leafhopper 

(Cicadellidae) mild no

Spidermite 

(Tetranychidae) mild no 

HI 

Kauai 

Corn earworm 

(Helicoverpa zea) mild no

Fall armyworm 

(Spodoptera 

frugiperda) 

mild no 

2008 07-040-101rm 

PR Salinas 

Corn earworm 

(Helicoverpa zea) mild no

Clinton 

Gray leaf spot 

(Cercospora zeaemaydis) mild no 

IL 

Stark Common corn rust 

(Puccinia sorghi) mild no 

IA Polk No Disease N/A no 

KS Pawnee Common corn rust 

(Puccinia sorghi) mild no 

2008 08-011-101n 

NE York Eyespot 

(Aureobasidium zeae) mild no 

2008 08-011-102n OK Caddo No Disease N/A no 

IL Ogle No Disease N/A no 

2008 IA Polk No Disease N/A no 08-014-105n 

PR Juana 

Diaz No Disease N/A no 
Pioneer Hi-Bred Page 188 of 188 

32138 SPT Maintainer No CBI 

 Table 2. Disease Stressor Comparison between 32138 SPT Maintainer and Control 

(continued) 

Year of 

Planting 

Permit 

Number State County Stressor 

Range of 

Severity in 

32138 SPT 

Maintainer 

Difference 

with 

Control? 

2008 08-014-124n HI Kauai No Disease N/A no 

Kauai No Disease N/A no 

HI 

Honolulu No Disease N/A no 

IA Polk No Disease N/A no 

2008 08-023-101n 

WA Franklin No Disease N/A no 

N/A=not applicable