J.R. Simplot Company JRS01 Page 1 of 74

Petition for Determination of Nonregulated Status for InnateTM Potatoes with 

Low Acrylamide Potential and Reduced Black Spot Bruise: Events E12 and E24 

(Russet Burbank); F10 and F37 (Ranger Russet); J3, J55, and J78 (Atlantic); G11 

(G); H37and H50 (H)

We submit this petition under 7 CFR 340.6 to request that the Administrator make a determination that 

the articles should not be regulated under 7 CFR Part 340.

Submitted by Pete Clark Ph.D.

J.R. Simplot Company

Prepared by Pete Clark Ph.D.* and Susan Collinge Ph.D.

J.R. Simplot Company

Submitted on March 1, 2013

J.R. Simplot Company Petition JRS01

OECD Unique identifiers:

SPS-F10-7

SPS-F37-7

SPS-E12-8

SPS-E24-2

SPS-J3-4

SPS-J55-2

SPS-J78-7

SPS-G11-9

SPS-H37-9

SPS-H50-4

J. R. Simplot Company Contributors:

Jingsong Ye Ph.D., Troy Weeks Ph.D., Hua Yan Ph.D., Craig Richael Ph.D., Roshani Shakya Ph.D., Rekha 

Chawla Ph.D., Hui Duan Ph.D., Pete Clark Ph.D.,

and Susan Collinge Ph.D.

*Corresponding Author:

Pete Clark Ph.D.

J.R. Simplot Company

5369 West Irving Street

Boise, ID 83706

Tel.: (208) 780-6066

This Document (Including Appendices) Does Not Contain CBI

J.R. Simplot Company JRS01 Page 3 of 74

Release of Information

J.R. Simplot Company is submitting the information in this assessment for review by the USDA as part of 

the regulatory process. By submitting this information, J.R. Simplot Company does not authorize its 

release to any third party except to the extent it is requested and required under the Freedom of 

Information Act, 5 U.S.C., paragraph 552; USDA complies with the provisions of FOIA and USDA’s 

implementation regulations (7 CFR Part 1.4). Except in accordance with the Freedom of Information act, 

J.R. Simplot Company does not authorize the release, publication or other distribution of this 

information without J.R. Simplot Company’s prior notice and consent.
J.R. Simplot Company JRS01 Page 4 of 74

Table of Contents

Abbreviations, Acronyms, and Definitions ...................................................................................................7

Summary .......................................................................................................................................................9

1. Rationale for Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise ...................12

1.1. Basis for Determination of Nonregulated Status .......................................................................12

1.2. Rationale for Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise ...........13

1.3. Benefits of Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise ..............14

1.4. Submissions to Other Regulatory Agencies................................................................................15

1.5. Conclusions: Rationale for Development of Potatoes with Low Acrylamide Potential and 

Reduced Black Spot Bruise......................................................................................................................15

2. The Biology of Potato..........................................................................................................................15

2.1. History of Potato.........................................................................................................................15

2.2. Use of the Potato as Food and Feed in the USA.........................................................................16

2.3. Taxonomy of the Genus Solanum...............................................................................................16

2.4. Genetics of Potato ......................................................................................................................17

2.5. Potato Growth and Life Cycle .....................................................................................................17

2.6. Seed Propagation........................................................................................................................18

2.7 Variety Development..................................................................................................................18

2.8. Recipient Potato Varieties..........................................................................................................20

2.9. Typical Agronomic Practices.......................................................................................................20

2.10. Pollination and Outcrossing........................................................................................................20

2.11. Wild potatoes in the U.S.............................................................................................................21

2.12. Weediness ..................................................................................................................................23

2.13. Conclusions: The Biology of Potato ...........................................................................................23

3. Method of Development: Description of Marker-Free DNA Transformation ....................................24

3.1. Introduction................................................................................................................................24

3.2. Parental Varieties .......................................................................................................................25

3.3. Plasmid used for Transformation ...............................................................................................26

3.4. Backbone Portion of pSIM1278..................................................................................................28

3.5. The Transfer DNA (DNA insert) of the Vector ............................................................................28

3.6. Open Reading Frames Associated with the DNA Insert .............................................................30

3.7. Conclusions of Methods of Development: Description of Marker-Free DNA Transformation..30

4. Characteristics of Transferred DNA and Gene Regulation .................................................................31

4.1. Description and History of the Modification..............................................................................31

4.2. Verification of Insert Integrity and Number of Inserted Sequences..........................................32

4.3. Selection of Backbone-free Plants..............................................................................................37

4.4. Flanking Regions of Inserted Sequences....................................................................................37

4.5. Stability of Inserted DNA ............................................................................................................38

4.6. Event-Specific PCR ......................................................................................................................38

4.7. Conclusions: Characteristics of transferred DNA and Gene Regulation.....................................38

5. Expression of the Gene Products........................................................................................................39

6. Efficacy and Tissue-Specificity of Gene Silencing................................................................................40

7. Agronomic Performance, Disease, and Efficacy of Traits...................................................................41

7.1. Agronomy and Phenotypic Evaluation .......................................................................................42

7.2. Disease susceptibility and polyphenol oxidase ..........................................................................44

7.3. Disease Incidence: Storage Studies............................................................................................46

7.4. Disease Incidence: Late Blight and Bacterial Soft Rot ................................................................46
J.R. Simplot Company JRS01 Page 5 of 74

7.5. Evidence of Trait Efficacy: Reducing Sugars ...............................................................................46

7.6. Evidence of Trait Efficacy: Black Spot Bruise .............................................................................47

7.7. Evidence of Trait Efficacy: Reduced Asparagine and Acrylamide..............................................47

7.8. Survival in Fields .........................................................................................................................49

7.9. Conclusion on Agronomic Performance, Disease, and Efficacy of Traits...................................49

8. Compositional assessment .................................................................................................................50

8.1. Proximates, Vitamins, Minerals, and Glycoalkaloids..................................................................50

8.2. Allergenicity of InnateTM Potatoes ..............................................................................................55

8.3. Conclusions of Compositional Assessment ................................................................................56

9. Environmental Assessment of Introduction .......................................................................................56

9.1. Potential for Gene Transfer and Outcrossing.............................................................................57

9.2. Inter-Species Pollination and Hybridization Potential................................................................57

9.3. Weediness Potential...................................................................................................................58

9.4. Endangered Species Considerations and Impact on Biodiversity...............................................58

9.5. Effects on Current Agronomic Practices in Potato .....................................................................59

9.6. Unintended or Non-target Effects..............................................................................................59

9.7. Summary of the Environmental Assessment of Introduction ....................................................59

10. Crop Introduction and the Potato Industry ....................................................................................60

10.1. Industry Need for Potatoes with Reduced Acrylamide Potential...............................................60

10.2. Potato uses and exports.............................................................................................................61

10.3. Submissions to Other Regulatory Agencies................................................................................61

10.4. Impact on the Organic Market ...................................................................................................61

10.5. History of Biotech Potatoes........................................................................................................62

10.6. Stewardship of InnateTM Potatoes ..............................................................................................62

11. Conclusions: Determination of Nonregulated Status for InnateTM Potatoes.................................63

12. Statement of Grounds Unfavorable................................................................................................65

13. Certification.....................................................................................................................................66

14. Record of Field Test Reports...........................................................................................................67

15. Literature Cited ...............................................................................................................................68
J.R. Simplot Company JRS01 Page 6 of 74

List of Tables

Table 1. InnateTM events and their OECD unique identifiers......................................................................10

Table 2. Recipient Potato Material .............................................................................................................20

Table 3. Example of Agronomic Inputs for Two Potato Variety Classes.....................................................20

Table 4. Genetic elements of the pSIM1278 backbone..............................................................................28

Table 5. Genetic elements of the DNA insert of pSIM1278, from Left Border site to Right Border site....29

Table 6. Summary of Down-Regulated Genes in Different Tissues............................................................40

Table 7. Summary of Agronomic Observations ..........................................................................................43

Table 8. Differences in Asparagine and Acrylamide Between Controls and Events...................................48

Table 9. Traits Incorporated into InnateTM Potatoes...................................................................................50

Table 10. Statistical Differences in Composition Data: Events F10, F37, E12 and E24...............................52

Table 11. Statistical Differences in Composition Data: Events J3, J55, J78, G11, H37 and H50 .................53

Table 12. Glycoalkaloids (mg/100 g)1 in Ranger Russet, Russet Burbank, Atlantic, G, and H Varieties......55

Table 13. Field release notifications...........................................................................................................67

List of Figures

Figure 1. Potato Variety Development Schematic......................................................................................19

Figure 2. Potato Production in the United States.......................................................................................22

Figure 3. Vector pSIM1278 .........................................................................................................................27

Figure 4. Silencing Approach Using the DNA Insert of pSIM1278 ..............................................................31

Figure 5. Diagram of DNA Inserts................................................................................................................33

Figure 6. Biosynthesis of GLN and ASN in plants .......................................................................................49

List of Appendices

Appendix 1: ...............................................................................................Characterization of Inserted DNA

Appendix 2: .................................................................. Evidence for the Absence of Vector Backbone DNA

Appendix 3: .................................................................................Evidence for Stability of the Inserted DNA

Appendix 4: ......................................................................... Junction Analysis and Event-Specific Detection

Appendix 5: ....................................................................... Efficacy and Tissue-Specificity of Gene Silencing

Appendix 6: ..................................................................................Field Performance and Tuber Evaluations

Appendix 7: ....................................................................................................... Black Spot Bruise Tolerance

Appendix 8: ................................................................................................................. Disease Susceptibility

Appendix 9: ..............................................................................................................Compositional Analyses

Appendix 10: .......................................................................................................... Post-Harvest Monitoring
J.R. Simplot Company JRS01 Page 7 of 74

Abbreviations, Acronyms, and Definitions

Abbreviation Definition

AGP pAgp-derived probe used in DNA gel blot hybridization

ALA Alanine

A. tumefaciens Agrobacterium tumefacians

ARG Arginine

ASN Asparagine

ASN1 Asn1 gene-derived probe used in DNA gel blot hybridization

Asn1 Asparagine synthetase-1 gene

ASP Aspartic acid

Backbone DNA DNA associated with vector backbone

chs Chalcone synthase gene

cwt/A Unit of measure equal to 100lbs/ acre

CYS Cysteine

DNA Deoxyribonucleic acid

DNA insert The DNA sequence from pSIM1278 located between the LB and RB intended to be 

integrated into the potato genome

EB Ethidium bromide

fASN1 Fragment of the Asn1 gene

FDA Food & Drug Administration

G0 First generation greenhouse-grown tuber seed

G1 First generation field-grown tuber seed

G2 Second generation field-grown tuber seed

G3 Third generation field-grown tuber seed

GBS pGbss-derived probe used in DNA gel blot hybridization

GLN Glutamine

GLU Glutamic acid

GLY Glycine

GM Genetically modified

gus -glucuronidase gene

Hd Restriction enzyme Hind III

HIS Histidine

IPD Inter-genebank Potato Database

ipt Isopentyltransferase gene – produces cytokinin hormones associated with plant 

growth and development

ILE Isoleucine

LB Left Border (a 25-base pair sequence) similar to A. tumefaciens T-DNA border

Left Border side The region of the DNA insert in the vicinity of the Left Border, which may either 

extend up to the cleavage site in the Left Border or be truncated at a position of 

between a single base pair and 100s of base pairs downstream from that cleavage 

site

Left Border site The 25-base pair sequence defined as Left Border, similar to A. tumefaciens T-DNA
J.R. Simplot Company JRS01 Page 8 of 74

Abbreviations, Acronyms, and Definitions (Continued)

Abbreviation Definition

LEU Leucine

LYS Lysine

MAFF Japan Ministry of Agriculture, Forestry, and Fisheries

MET Methionine

MHLW Japan Ministry of Health, Labor and Welfare

Non-coding DNA DNA not coding for RNA that is translated into protein

ORFs Open reading frames

pAgp Promoter of the ADP glucose pyrophosphorylase gene

PCR Polymerase chain reaction

pGbss Promoter of the granule-bound starch synthase gene

PHE Phenylalanine

PhL Phospholrylase-L gene

PPO Ppo5 gene-derived probe used in DNA gel blot hybridization

Ppo5 Polyphenol oxidase-5 gene

Pro Functionally-active promoter

PRO Proline

R1 Water dikinase R1 gene

RB Right Border (a 25-base pair sequence) similar to A. tumefaciens T-DNA border

Right Border side The region of the DNA insert in the vicinity of the Right Border, which may either 

extend up to the cleavage site in the Right Border or be truncated at a position of 

between a single base pair and 100s of base pairs upstream from that cleavage site

Right Border site The 25-base pair sequence defined as Right Border, similar to A. tumefaciens T-DNA

RNA Ribonucleic acid

RNAi RNA interference

SER Serine

Sol t 1 42 kDa allergen known as patatin

Somaclonal

variation

Genetic and/ or phenotypic variation among clonally propagated plants of a single 

donor clone; generated by tissue culture and other forms of vegetative propagation

TPS True potato seed

T-DNA Transfer DNA from A. tumefaciens delineated by left and right border sequences

THR Threonine

TRP Tryptophan

TYR Tyrosine

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

VAL Valine
J.R. Simplot Company JRS01 Page 9 of 74

Summary

The J. R. Simplot Company has pioneered a new approach that marks a significant and vital advance in 

plant breeding. Simplot’s InnateTM technologies allow us to transform plants with plant DNA . In the 

present petition, InnateTM technologies have been used to transform potato plants with non-coding

potato DNA (DNA not coding for RNA that is translated into protein). We seek nonregulated status of 

potato plants transformed using our InnateTM technologies and submit the evidence that these plants 

should not be classified as “Regulated articles” as defined under 7 CFR 340.

To explain, Simplot’s InnateTM technologies allow researchers to isolate genetic elements from any plant 

genome, rearrange them, or link them together in desired permutations, and introduce them back into 

the genome. More specifically, in these events, the genomic DNA comes from potato or wild potato (a 

group of related plant species that are sexually-compatible with potato). We incorporate no viral 

markers, and no vector backbone sequences, into the plant genome. 

As demonstrated in the data presented in this petition, the transformed cultivated potato plants are not 

parasitic, and our data show no increase in pest potential compared to the controls. Furthermore, the 

expression of the integrated genetic elements results in traits highly sought-after in the potato industry, 

namely: reduced black spot bruise, low asparagine, and lower levels of reducing sugars. One key 

consequence of these beneficial traits is that the harvested potatoes contain less acrylamide when 

cooked than untransformed, commercially available potatoes.

Among other things, we have used InnateTM technologies to address two critical potato quality issues: 

(1) large amounts of asparagine, a non-essential free amino acid that is rapidly oxidized to form 

acrylamide upon frying or baking; and (2) susceptibility to enzymatic browning and discoloration, which 

happens when polyphenol oxidase leaks out from the damaged plastids of bruised potatoes. In the 

cytoplasm, the enzyme oxidizes phenols, which then rapidly polymerize to produce dark pigments. 

Browning is also triggered non-enzymatically as a consequence of the partial degradation of starch into 

glucose and fructose. When heated, these reducing sugars react with amino acids, such as asparagine, 

through the Maillard reaction to produce a variety of desired compounds contributing to flavor, aroma, 

and browning, but also acrylamide.

Because our InnateTM technologies effectively accelerate the process of conventional crossing, it allows 

desired changes in traditional varieties to occur much faster than is currently possible, while maintaining 

the desired characteristics of the original parent plant. For potatoes, InnateTM technologies are

particularly attractive because potatoes are notorious for having a high degree of heterozygosity, 

suffering from inbreeding depression, and limited predominantly to clonal propagation. These inherent 

factors significantly hinder and prevent us from commercially introducing quality, sought-after traits into 

this valuable crop. Today’s traditional breeding techniques simply result in random genomic 

rearrangements and trait segregation, and do not allow for the simultaneous addition of multiple 

desired traits.

To illustrate this point, there currently are no varieties available that produce tubers with low 

acrylamide potential, reduced black spot bruise and reduced sugars, while displaying all other traits 

important to the food industry. Therefore, instead of attempting to develop new varieties, the J. R. 

Simplot Company improved the quality of five existing potato varieties by transforming them with a 

non-coding transfer DNA that silences the genes related to expression of black spot bruise, asparagine, 
J.R. Simplot Company JRS01 Page 10 of 74

and reducing sugars in tubers. Our proprietary plant expression cassettes, included in the DNA insert, 

are derived from the potato genome.

This DNA insert comprises two expression cassettes and was inserted into our pSIM1278 transformation 

vector. The first cassette comprises fragments of both the asparagine synthetase-1 gene (Asn1) and the 

polyphenol oxidase-5 gene (Ppo5), arranged as inverted repeats between the Agp promoter of the ADP 

glucose pyrophosphorylase gene (Agp) and the Gbss promoter of the granule-bound starch synthase 

gene (Gbss) and results in silencing of both the Ppo5 and Asn1 genes. The second cassette is comprised 

of fragments of the promoters of the starch associated gene (R1) and the phosphorylase-L gene (PhL), 

operably linked to the same Agp and Gbss promoters as the first cassette. The function of the second 

cassette is to silence the promoters of the starch associated gene (R1) and the phosphorylase-L gene 

(PhL). Importantly, in keeping with our InnateTM technologies, these expression cassettes contain potato 

genomic DNA, e.g. DNA from either the selected plant species or from a plant that is sexually compatible 

with the selected plant species. 

We transformed five different varieties including Ranger Russet, Russet Burbank, Atlantic, variety G, and 

variety H with pSIM1278, and identified transformed shoots using polymerase chain reaction (PCR) 

rather than markers. We also performed numerous tests and confirmed that none of the plants 

contained vector backbone DNA. Transformed plants were propagated to produce the InnateTM events. 

Ten of these events were selected for detailed analyses: F10 and F37 for Ranger Russet, E12, and E24 for 

Russet Burbank, J3, J55, and J78 for Atlantic, G11 for variety G, and H37 and H50 for variety H. See 

Table 1 for their OECD unique identifiers.

Table 1. InnateTM events and their OECD unique identifiers

Event number OECD Unique Identifier

F10 SPS-F10-7

F37 SPS-F37-7

E12 SPS-E12-8

E24 SPS-E24-2

J3 SPS-J3-4

J55 SPS-J55-2

J78 SPS-J78-7

G11 SPS-G11-9

H37 SPS-H37-9

H50 SPS-H50-4

Extensive analyses of the various events demonstrated that the achievement of the desired modified 

traits was enabled by targeted gene silencing. Simplot found there to be reduced RNA (transcript) levels 

of the four targeted genes in tubers, which was found to be associated with: 

(1) 69 – 78 percent reductions in free-asparagine;

(2) Black spot bruise tolerance; and

(3) Lower levels of reducing sugars.

Eight of the selected events contained both expression cassettes. Two events, J78 and G11, had only 

the silencing cassette for the Asn1 and Ppo5 genes integrated into their genomes and produced tubers 

with successfully down-regulated expression of the Asn1 and Ppo5 genes. The effective reduction of 
J.R. Simplot Company JRS01 Page 11 of 74

asparagine found in events J78 and G11, resulting from silencing of the Asn1 and Ppo5 genes results in 

significantly reduced acrylamide (66 to 70%), making these commercially valuable potato varieties even 

without silencing the starch associated genes (R1) and phosphorylase-L (PhL).

Ultimately, we demonstrate that the InnateTM tubers exhibited the desired traits which can lead to lower 

acrylamide levels in cooked potatoes and, importantly, we showed that the transformed tubers were 

otherwise substantially equivalent to untransformed control tubers.

The propagation of commercial potato varieties through cloning of seed potatoes mitigates concerns 

about increased weediness or pest potential such as seed dispersal, survival outside of cultivation, or 

outcrossing. One of the most important potato varieties, Russet Burbank, produces few flowers and is 

male sterile. Other factors limiting outcrossing include the tendency for most fertile varieties to be selfpollinated, an inability to attract honey bees because they lack nectar, the pollen transfer range is

limited to about 20 meters, and true seeds would be unlikely to grow into mature potatoes since potato 

seeds are not saved and propagated in the typical farming operation. If potatoes were grown from true 

potato seed, the offspring would be so diverse that they would not be useful as commercial potatoes. In 

addition, potatoes are not known to escape from commercial fields or show weediness potential. Wild 

potato varieties are rare in the United States and for the most part geographically isolated from 

commercial production areas, further reducing concerns about cross-pollination with wild species.

The modifications described in this petition were intended to enhance the quality, not agronomic 

characteristics, of potato by inactivating four native genes. Planting, cultivation, management and 

harvesting techniques were not affected by the incorporated traits. The InnateTM varieties are likely to be 

planted in areas that are already growing potatoes, and would not result in a significant expansion of 

acres. The modifications described in this petition are highly unlikely to increase the weediness or 

invasiveness of potato because the incorporated traits (reduced free-asparagine, black spot bruise 

tolerance, and reduced sugars) do not play a role in survival, dispersal, or ecological competitiveness. 

Field trials over multiple years with the 10 events did not provide any evidence for altered growth 

characteristics such as accelerated tuber sprouting, increased plant vigor, increased tuber set, or 

delayed senescence. No new pesticides are expressed, and no natural defense mechanisms activated, 

and thus modified potatoes are unlikely to display enhanced weediness or pesticidal properties. 

Therefore, the potential to impact insects and other non-target organisms, weed or disease 

susceptibility, endangered species or biodiversity is negligible for InnateTM potatoes.

We therefore seek nonregulated status for these events based on the weight of evidence demonstrating

their safety when compared to untransformed potatoes. The technologies presented in this petition

and the resultant transformed plants and their tuber products satisfy the requirements for nonregulated

status. The transformed plants and subsequent traits described in this petition should not be 

considered plant pests. In conclusion, plants displaying the incorporated traits which have resulted from 

the addition of well characterized, non-coding regions from potato or wild potato are as safe as 

untransformed potatoes based on all of the data contained in this submission. The J.R. Simplot 

Company requests a determination from APHIS that the 10 potato events described in this petition, and 

any progeny derived from these events, are granted nonregulated status and would no longer be 

considered regulated articles under 7 CFR Part 340.

The genetic services and research and development methods and associated genetic tools, disclosed in 

this petition, such as expression vectors, and the transformed plants and plant cells, and methods of 

transformation, have been trademark-branded by Simplot as InnateTM.
J.R. Simplot Company JRS01 Page 12 of 74

1. Rationale for Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise

The potato varieties were transformed using InnateTM technologies in order to address the need of the 

potato industry to improve quality by reducing expression of the enzyme responsible for black spot 

bruise and to reduce acrylamide through lowering the concentration of the reactants, namely 

asparagine and reducing sugars. Use of InnateTM technologies allows for transformation of potatoes

using genetic material that contains only non-coding regulatory regions. The desirable traits were 

simultaneously incorporated into five of the most popular potato processing varieties. Such trait 

addition into existing varieties would not be possible to achieve through traditional breeding because 

potato is tetraploid, highly heterozygous and sensitive to inbreeding depression. The resulting InnateTM

potatoes are not plant pests as verified by phenotype, Southern blot hybridization, and PCR. 

1.1. Basis for Determination of Nonregulated Status 

Part 340 Section 7 of the United States Federal Register regulates, among other things, the introduction 

of organisms and products altered or produced through genetic engineering that are plant pests or are 

believed to be plant pests. The 10 events described in this petition were transformed with DNA that 

does not alter the pest characteristics of the potatoes. All events were confirmed to be free of 

Agrobacterium and free of Agrobacterium-derived backbone DNA. In addition, the scientific evidence 

presented here shows that the inserted genetic material contains only non-coding regulatory regions 

and are as safe as untransformed potatoes.

Field evaluations demonstrated that the events displayed similar agronomic and phenotypic 

characteristics compared to their untransformed controls. This submission reviews the biosafety 

implications of all minor differences observed. The most important intentionally-incorporated trait is a 

reduced level in the free amino acid, asparagine in tubers. As predicted, we confirmed that this change 

was associated with a reduced potential to form acrylamide upon frying.

Most of the events also displayed lowered levels of the reducing sugars glucose and fructose when fresh 

or when analyzed at one month after harvest (Appendix 9. Compositional Analyses). Although reduced 

asparagine alone significantly decreases acrylamide formation, as shown in events J78 and G11, the 

lowered levels of reducing sugars further reduces acrylamide formation and limits heat-induced 

browning. The plant-derived transfer DNA used for transformation consisted of two inverted repeats. 

Each of these repeats was inserted between promoters that are predominantly active in tubers. 

Expression of the first inverted repeat triggered the degradation of transcripts for the asparagine 

synthetase-1 (Asn1) and the polyphenol oxidase-5 (Ppo5) genes. Expression of the second inverted 

repeat resulted in reduction in the starch associated gene (R1) and the phosphorylase-L gene (PhL)

transcripts in tubers. The events presented in this submission all contained at least the fragment of the

DNA insert carrying the Asn1/Ppo5 gene silencing cassette. 

The 10 events produced by transformation of 5 potato varieties with the specified non-coding transfer 

DNA are well characterized and safe as determined by agronomic and compositional evaluations, when 

compared to the untransformed controls. 
J.R. Simplot Company JRS01 Page 13 of 74

1.2. Rationale for Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise

The Swedish National Food Administration and Stockholm University announced in April 2002, a 

relationship between the formation of certain blood adducts and the consumption of foods later found 

to contain acrylamide (NTP 2012). Other researchers also studied the mechanism for acrylamide 

formation in food, and the J.R. Simplot Company had also earlier predicted that acrylamide would form 

as a result of the well-known but complex Maillard browning reaction. Cooking or heat treating of foods 

that are rich in reducing sugars and amino acids may result in browning, commonly referred to as the 

Maillard reaction. This reaction occurs between sugars and amino acids, affecting changes in the color, 

flavor, functional properties and nutritional value of food (Obrien and Morrissey 1989). More 

specifically, we found acrylamide formed primarily when the amino acid asparagine along with reducing 

sugars were heated at temperatures above 120°C, as would occur during frying and baking. The 

biochemical basis of acrylamide formation was later published by Stadler et al. (2002).

Various governments responded to the news about finding acrylamide in food by providing guidance 

and surveys. Through one such survey, the FDA determined that potato products contribute 35% of the 

acrylamide exposure through diet in the US. The scientific community has since gathered additional 

information, including epidemiology and toxicology studies. Along with studies to mitigate the levels of 

acrylamide in food, the scientific community has also been working to understand the health 

implications of acrylamide consumption (NTP 2012).

Based on a recent notice (Federal Register 2009), FDA is considering issuing guidance for industry on the 

reduction of acrylamide levels in food products. The introduction of InnateTM potatoes with low 

acrylamide potential would provide potatoes that are largely indistinguishable from existing varieties. 

The reduction in asparagine and sugars using InnateTM technologies and the resulting reduction in 

acrylamide upon heating will address food industry needs with respect to the proposed FDA guidance. 

As such, this approach to lowering acrylamide could be adopted readily by the food industry if such 

guidance is issued by the FDA. In addition, litigation in the state of California as a result of Proposition 65 

(OEHHA 2011a; OEHHA 2011b) resulted in legal settlements with restaurant chains and the retail french 

fry and potato chip manufacturers regarding perceived risks from acrylamide. Some settlements 

required signs warning consumers, but in addition, some manufacturers of retail french fries and potato 

chips would be required to reduce acrylamide in their products or add warning labels. As of December 1, 

2011, some potato chip manufacturers began including a statement on packaging warning consumers of 

the presence of acrylamide and its relationship to browning in potatoes. Potato processors affected by 

these rulings in California could be motivated to adopt the low acrylamide, InnateTM potato products. 

Following the discovery of acrylamide in foods and in response to governmental concerns, many

additives were studied and some have shown effectiveness at reducing acrylamide. Some of the more 

promising options include citric acid, enzymes such as asparaginase, and amino acids that substitute for

asparagine in the Maillard reaction and therefore form products other than acrylamide when heated. 

Another approach to reducing acrylamide could be based on either traditional breeding or 

biotechnology to reduce the precursors for acrylamide. The use of biotechnology allows for specific 

deactivation of genes involved in the formation of asparagine and reducing sugars. In a crop like 

potatoes, which is highly heterozygous and suffers from inbreeding depression, the speed to develop 

such changes could take decades through traditional breeding. In contrast, biotechnology allows for 

specific changes in traditional varieties much faster, while maintaining the desired characteristics of the 

original parent.
J.R. Simplot Company JRS01 Page 14 of 74

Potato transformation with the pSIM1278 construct results in the incorporation of two silencing 

cassettes into the potato. Expression of the first cassette lowers transcript levels for the Asn1

(asparagine synthetase-1) and Ppo5 (polyphenol oxidase-5) genes and, consequently, limits (1) 

formation of the acrylamide precursor asparagine, and (2) formation of impact-induced black spot 

bruise that occurs when the enzyme polyphenol oxidase oxidizes phenols to produce dark pigments. 

The presence of black spot bruise results in lower quality and subsequent production losses during 

processing into fries or chips. A reduction in the formation of reducing sugars is accomplished by the 

down-regulated transcript levels for the PhL (phosphorylase-L) and R1 (starch associated) genes

resulting from expression of the second cassette. These traits function by slowing the conversion of 

starch to reducing sugars (glucose and fructose). Benefits include improved quality, especially relating 

to color control, and thus contributing to the desired golden brown colors required by most french fry or 

chip customers. Also, the reducing sugars react with amino acids, such as asparagine, to produce 

Maillard products including acrylamide.

1.3. Benefits of Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise

The events described here produce tubers with a greatly reduced potential to form acrylamide, thus 

addressing a potentially critical food safety issue for the potato industry, particularly in light of recent 

toxicology studies (NTP 2012). 

Many methods have been tested and research is ongoing to reduce acrylamide through process 

changes, reduction in dextrose, and additives such as asparaginase, citrate, and competing amino acids. 

The required capital expense to implement process changes throughout the potato industry would cost 

millions of dollars. In addition to the expense, these process changes have significant drawbacks 

including potentially negative flavors associated with additives such as asparaginase or citrate. Typically, 

fry manufacturers add dextrose during processing of french fries to develop the desired golden brown 

color, but dextrose also increases the formation of acrylamide through the Maillard reaction. Significant 

reductions in acrylamide occur by merely omitting dextrose from the process; however, the signature 

golden brown colors must then be developed some other way (such as though the addition of colors like

annatto). The use of alternate colors, results in an absence of the typical flavors that develop through 

those browning reactions. Another challenge with the use of additives to reduce reactants like 

asparagine will be the principle of moisture migration that occurs during frozen storage resulting in a 

return of asparagine to the surface and increased acrylamide compared with the initial testing at the 

time of processing. In contrast, the InnateTM potatoes will significantly reduce acrylamide without the 

use of new additives or process changes. The resulting foods will have the same appealing color, flavors, 

and aromas as they currently have today. 

The blackening that occurs after potatoes are bruised affects quality and recovery in processing french 

fries and chips. Potatoes that have been damaged and show black spot bruise must be trimmed or could 

be rejected before processing, resulting in quality challenges or economic loss. Management of reducing 

sugars presents significant challenges and potential financial loss because processors reject loads with 

reducing sugar content above 2%, which is approximately 20% of potatoes produced. However, 

potatoes developed with InnateTM technologies exhibit significantly less black spot bruising and lower 

reducing sugar content, which will likely have economic benefits for growers and processors.
J.R. Simplot Company JRS01 Page 15 of 74

1.4. Submissions to Other Regulatory Agencies

Simplot has initiated and will complete a consultation process with the Food and Drug Administration 

(FDA) prior to commercial distribution of tubers or products from the 10 events.

An assessment of the safety of these events will also be submitted to Health Canada and the Canadian 

Food Inspection Agency (CFIA) in 2013. Furthermore, submissions in support of approval to import 

products from the events will be made to the Japan Ministry of Health, Labor and Welfare (MHLW) and 

the Ministry of Agriculture, Forestry, and Fisheries (MAFF). Submissions are anticipated for other key 

international markets including Mexico.

1.5. Conclusions: Rationale for Development of Potatoes with Low Acrylamide Potential and 

Reduced Black Spot Bruise

In summary, there is an important need to improve the quality and reduce levels of acrylamide in fried 

and baked potatoes. Because it was not feasible to reduce acrylamide formation and black spot bruise 

in existing varieties through traditional breeding, we accomplished this goal by applying InnateTM

technologies. The varieties represented in this petition represent almost 30% of the US Potato market. 

However, about 70% of the remaining acreage and over 70 major varieties could benefit from the 

reduction in acrylamide through transformation with pSIM1278. We now seek nonregulated status for 

10 events based on the weight of evidence demonstrating their safety. We have transformed plants 

with the addition of genomic DNA from potato and wild potato where the integrated genetic material 

contains only well characterized non-coding regulatory regions that are as safe as untransformed 

potatoes.

2. The Biology of Potato

This section is based, in part, on the Organization for Economic Co-Operation and Development’s (OECD) 

series on harmonization of regulatory oversight in biotechnology no. 8, Consensus document on the 

biology of Solanum tuberosum, 1997.

This section describes potato biology and how it contributes to the effective management of the 

InnateTM potatoes. All commercial potato varieties must be propagated through cloning, effectively 

mitigating many concerns about increased pest potential such as seed dispersal, survival outside of 

cultivation, or outcrossing. 

2.1. History of Potato

The cultivated potato traces its origin to Andean and Chilean landraces developed by pre-Colombian 

cultivators. These landraces exhibit morphological and genetic diversity, and are distributed from 

western Venezuela to northern Argentina, and in southern Chile (Spooner et al. 2005). Introduced to 

Europe by Spain in 1536, the potato was subsequently conveyed by European mariners to territories and 

ports throughout the world. Once established in Europe, the potato soon became an important food 

staple and field crop. But lack of genetic diversity, due to the fact that very few varieties were initially 

introduced, left the crop vulnerable to disease. In 1845, a plant disease known as late blight, caused by 

the fungus-like oomycete pathogen Phytophthora infestans, spread rapidly through the poorer 

communities of western Ireland, resulting in the crop failures that led to the Great Irish Famine. Potato 

remains an essential crop in Europe, where per capita production is still the highest in the world, but the 
J.R. Simplot Company JRS01 Page 16 of 74

crop also made its way back over the Atlantic Ocean to North America. The industry gained greater 

importance particularly in western states after the horticulturist Luther Burbank developed the Russet 

Burbank potato in 1872. In 2011, the United States harvested 21.4 million tons of potatoes, enough to 

make it the world's fourth biggest producer (NPC 2012). Potatoes in the United States are grown in 

many states, although about half of the crop comes from Idaho and Washington. The top ten potato 

producing states of Idaho, Washington, Wisconsin, North Dakota, Colorado, Oregon, Maine, Minnesota, 

California and Michigan account for almost 88% of the United States potato crop (USDA-NASS 2010b). 

Most potatoes are harvested in July through October. Only about one third of US potatoes are

consumed fresh. Around 60% of annual output is processed into frozen products (such as frozen fries 

and wedges), chips, dehydrated potato and starch, while 6% is replanted as seed potato. Americans eat, 

on average, approximately 54 kg of potatoes per person per year. The most rapid expansion in 

production and consumption of potatoes has occurred in southern and eastern Asia. China is now the 

world's largest potato-producing country, and nearly a third of the world's potatoes are harvested in 

China and India.

2.2. Use of the Potato as Food and Feed in the USA

Harvested potatoes are either used for food (~93%), feed (<1%), industrial purposes (<1%) or as “seed” 

for planting (5%). The type of use is partially dependent on a variety of characteristics related to tuber

quality. According to the National Potato Council, approximately 50% of all tubers are used for the 

combination of frozen fries, potato chips, and shoestrings (julienne cut crispy snacks), whereas 11% are 

dehydrated and <2% are canned. An additional 28% of tubers are not processed but sold as fresh 

potatoes (NPC 2012). 

Raw potato waste products (peels, out of specification raw potatoes, or other non-processed raw potato 

products) and processed discards (french fry, hash brown, etc.) are routinely incorporated into feed 

rations at livestock feedlot operations including those owned by the J.R. Simplot Company. 

Approximately 15,000 tons of processed potato waste and 60,000 tons of raw potato waste are 

integrated into livestock feed at the J.R. Simplot Company’s Grandview, ID feedlot on an annual basis. 

The typical feed ration used for cattle finishing at J.R. Simplot Company feedlots includes 8-14% potato 

waste. One of the major benefits of using the potato waste is that it is not discarded into landfills, but 

used in a sustainable manner as part of a long-term approach to reducing waste and integrating 

sustainability with respect to the Earth’s valuable natural resources.

2.3. Taxonomy of the Genus Solanum

The Solanaceae family contains several well-known cultivated crops such as tomato (Solanum 

lycopersicum also referred to as Lycopersicon esculentum), eggplant (Solanum melogena), tobacco 

(Nicotiana tabacum), pepper (Capsicum annuum) and potato (Solanum tuberosum). Within the genus 

Solanum, over a thousand species have been recognized. Potatoes will not hybridize with non-tuber 

bearing Solanum (tomato, eggplant, etc.) species including weeds commonly found in and around 

commercial potato fields (Love 1994). 

The genus Solanum is divided into several subsections, of which the subsection potatoe contains all 

tuber-bearing potatoes. The subsection potatoe is divided into series, of which tuberosa is relevant to 

this document. Within the series tuberosa approximately 54 species of wild and cultivated potatoes are 

found. One of these is S. tuberosum.
J.R. Simplot Company JRS01 Page 17 of 74

S. tuberosum is divided into two subspecies: tuberosum and andigena. The subspecies tuberosum is the 

cultivated potato widely in use as a crop plant in, for example, North America and Europe. The 

subspecies andigena is also a cultivated species, but cultivation is restricted to Central and South 

America (Hanneman 1994).

2.4. Genetics of Potato

The basic chromosome number in the genus Solanum is twelve. S. tuberosum subsp. tuberosum can be 

diploids (2n=2x=24) or tetraploids (2n=4x=48). The diploids have a limited range in parts of South 

America, while the tetraploids are the most commonly cultivated all over the world. How tetraploidy 

originated in potato is unclear. The cultivated S. tuberosum subsp. tuberosum can be either an 

autotetraploid (doubling of the chromosomes of a diploid species) or an allotetraploid (doubling of the 

chromosomes of a diploid hybrid between two related species).

While nearly all diploid species are self-incompatible, the cultivated tetraploid S. tuberosum subsp. 

tuberosum is capable of self-pollination (selfing). Plaisted (1980) has shown that under field conditions 

selfing is most likely for tetraploid S. tuberosum, with 80-100 percent of the seeds formed due to selfing. 

Conner and Dale (1996) collected outcrossing data from several field experiments with genetically 

modified potatoes, performed in New Zealand, the United Kingdom and Sweden. In each study, the 

outcrossing rate was zero when receiving plants were separated by more than 20 meters from the 

genetically modified ones. Although many Solanum species are fertile, it appears that a large number of 

the tetraploid cultivated S. tuberosum subsp. tuberosum cultivars have reduced fertility. Events of three 

of five varieties in this petition produce abundant flowers and pollen. However, Russet Burbank and the 

H varieties and events are sterile and have no outcrossing potential.

2.5. Potato Growth and Life Cycle

Potatoes grown in both commercial and organic farming operations go through the following five

general growth stages of development (Dwelle 2003): 1) Sprouts develop from eyes on seed tubers, 

grow upwards, and emerge from the soil. Roots initiate development at the base of the emerging 

sprouts, 2) Vegetative growth begins. Leaves and branch stems develop from aboveground nodes. Roots 

and stolons develop at belowground nodes, 3) Tubers begin to form at stolon tips, but do not enlarge. 

Flowering is initiated at the end of this stage, 4) Tuber cells expand with the accumulation of water, 

nutrients, and carbohydrates, and 5) Vines turn yellow and lose leaves, tuber growth slows and vines 

die. Tuber maturation is completed as tuber skins set.

In greater detail, the potato life cycle begins with certified seed potatoes, which are cut into pieces 

containing at least one eye and are typically planted in the field from February through May. Plants will 

establish by forming vegetative tissues, including roots, leaves, stems, and stolons. Tuber formation 

begins approximately 30-60 days after planting. Tubers are derived from lateral underground buds that 

develop at the base of the main stem, when kept underground. These develop into stolons due to 

diagravitropical growth. When conditions are favorable for tuber initiation, the elongation of the stolon 

stops, and cells located in the pith and the cortex of the apical region of the stolon first enlarge and then 

divide longitudinally. The combination of these processes results in the swelling of the subapical part of 

the stolon. During enlargement, tubers become the largest nutrient sink of the potato plant storing large

amounts of carbohydrates (mainly starch) and also significant amounts of protein. Furthermore, potato

tubers decrease their general metabolic activity and as such behave as typical storage sinks. Potato 

tubers of varieties such as Ranger Russet, Russet Burbank, Atlantic, G, and H are harvested from 90 to 
J.R. Simplot Company JRS01 Page 18 of 74

160 days after planting, which may vary with production area and marketing conditions. Typical 

potatoes are about 20-24% dry matter, of which approximately 60-80% consists of starch. After potato 

vines die back, the tuber skins thicken and harden which provides greater protection to tubers during 

harvest and blocks entry of pathogens. Although dormancy is defined as the absence of visible growth, 

dormant tuber meristems are metabolically active. In general, rates of many cellular processes such as 

respiration, transcription, and translation are suppressed during dormancy and non-dividing, dormant 

tuber meristems are arrested in the G-1 phase (a period prior to the synthesis of DNA in mitosis). 

Activation of cell metabolism during early spring triggers the development of sprouts from the eyes of 

the primary tuber.

2.6. Seed Propagation 

Potato is clonally propagated, which means that tubers rather than seeds are used for planting. A single 

potato plant produces approximately 12 tubers, therefore it takes multiple years to propagate a new 

potato variety and generate sufficient amounts of tuber seed needed for commercialization. A typical 

seed bulk-up program would progress as follows. In the initial year, propagation in tissue culture would

produce about 100 plants, and then these plants are grown in a greenhouse to produce thousands of 

mini-tubers, also called “nuclear seed”. In the second year, mini-tubers are planted at field sites with 

the lowest possible incidence of diseases and pests, producing generation-1 (G1) seed. Those G1 tubers 

are grown in the third year to increase the amount of tubers, producing G2 seed. In the fourth, fifth, and 

sixth years, bulk-up continues with commercial seed often representing G3, G4, and G5 tubers 

respectively.

2.7 Variety Development

Potato varieties take many years to develop (see Figure 1). The decision to establish a new variety is 

based on many factors such as need in the market place, potential consumer acceptance, and pest 

tolerance or resistance. Potato varieties do not have a high frequency of introduction and 

discontinuation compared to some other crops such as field corn or soybeans. Since potatoes are 

clonally propagated, there is a reduced risk of varietal dilution due to cross pollination.
J.R. Simplot Company JRS01 Page 19 of 74

Figure 1. Potato Variety Development Schematic
J.R. Simplot Company JRS01 Page 20 of 74

2.8. Recipient Potato Varieties

The potato varieties chosen for modification all represent significant value to the potato industry and 

relatively large percentage of the overall acreage (Table 2). 

Table 2. Recipient Potato Material

Variety Usage Percent of Seed Acreage in US1

Russet Burbank Fries 22.0

Ranger Russet Fries 6.3

Atlantic Chips 2.6

G Chips 2.0

H Chips 0.7

1

2009 data for seed potatoes: http://www.nationalpotatocouncil.org/NPC/resources_statistics.cfm

2.9. Typical Agronomic Practices

Examples of typical agronomic practices for a successful potato crop are described in Table 3.

Table 3. Example of Agronomic Inputs for Two Potato Variety Classes

Russet Varieties Round White Varieties

Planting Date April 1 to May 10 April 1 to May 10

Planting Rate 15,000 - 18,000 seed pc or 17 – 23 cwt/A 17,000 – 19,000 seed pc or 19 – 24 cwt/A

Row Spacing 34-36” between rows 34-36” between rows 

Seed Spacing 10-12” within row 8-10” within row

Fertilizer For 600 cwt/A yields and optimum soil 

test levels:

250 lb N – 100 lb P2O5 – 330 lb K2O per 

acre

For 600 cwt/A yields and optimum soil test 

levels:

250 lb N – 100 lb P2O5 – 330 lb K2O per 

acre

Yield/Plant 2-4 lb 2-4 lb

Yield/Acre 400-700 cwt/A 400-700 cwt/A

Harvest Date September 1 to October 15 August 1 (early season varieties only) to 

October 15

2.10. Pollination and Outcrossing

Potato is clonally propagated, which means that tubers rather than seeds are used for planting. 

Harvested tubers are either used for food (~90%), feed (<5%), industrial purposes (<2%) or as “seed” for 

planting (5%). Therefore pollination to produce seed is not a factor in major commercial potato 

production, e.g., if InnateTM potatoes were planted in close proximity to organic potatoes, any cross 

pollination would not impact the organic tubers.

Some of the most important potato varieties including Russet Burbank, produce few flowers and are 

male sterile. The variety H produces few if any flowers and exhibits male infertility. Many cultivars 

flower less than wild material, and flowers that do form often drop after pollination resulting in no berry 

formation. Flowering has no impact on tuber development.

Approximately 80% of seed produced by fertile varieties is derived from self-pollination (Plaisted 1980). 

Cross-pollination is typically mediated by certain insects including bumblebees (Bombus spp.) that 
J.R. Simplot Company JRS01 Page 21 of 74

typically don’t travel much more than 3 kilometers, rather than wind pollination (OECD 1997). 

Additionally, other bee species such as honey bees (Apis mellifera) are not pollinators of potatoes since 

potato flowers do not contain ample nectar (OECD 1997). Field evaluations in New Zealand, the United 

Kingdom and Sweden demonstrated through genetic testing that outcrossing rates were zero when

receiving plants were separated by more than 20 meters from genetically modified plants (Conner and 

Dale 1996). Additionally, potato pollen grains are extremely temperature sensitive. Pallais et al. (1988) 

showed that pollen viability decreased by 30 to 70% when exposed to temperatures of 30°C for up to 30 

minutes.

2.11. Wild potatoes in the U.S. 

This discussion is based on data in the U.S. Potato Genebank and on a monogram published by the 

American Society of Plant Taxonomists entitled Wild Potatoes (Solanum section Petota; Solanaceae) of 

North and Central America (Spooner et al. 2004). The USDA maintains the US Potato Genebank and 

participates in the Association of Potato Inter-genebank Collaborators (APIC), which has produced a 

global inventory of wild potato genetic resources available on the internet, the Inter-genebank Potato 

Database (IPD 2011), which includes the U.S. Potato Genebank records. Many of the IPD records from

the U.S. were obtained by John Bamberg and associates (including Spooner) during a decade of 

collecting wild potatoes in the Southwest U.S. (Bamberg et al. 2003). 

The only two wild potato species that grow within the borders of the USA, and for which specimens exist 

in gene banks, include the tetraploid species S. fendleri (recently reclassified as S. stoloniferum; 

however, some sources, including the IPD, still use the S. fendleri designation) and the diploid species S.

jamesii (Bamberg et al. 2003; IPD 2011; Bamberg and del Rio 2011a; Bamberg and del Rio 2011b; 

Spooner et al. 2004). Love (1994) reported that a third species, S. pinnatisectum, is also a native species 

in the USA. However, Spooner et al. (2004) determined that what was previously thought to be S. 

pinnatisectum was in fact S. jamesii. Through more than 10 years of field work and assessments of 

existing records, Bamberg et al. (2003) and Spooner et al. (2004) established the presence of only these 

two species, S. fendleri and S. jamesii, in the U.S. These researchers also attempted to verify previously 

recorded locations, and through this process, updated the maps of current known locations of these 

species, providing latitude and longitude locations for each documented population (Bamberg et al.

2003) and distribution maps (Spooner et al. 2004). These species mostly reside in dry forests, scrub 

desert, and sandy areas at altitudes of 5,000 to 10,000 feet, well isolated from most commercial 

production areas (Bamberg and del Rio 2011a).

While there is some overlap between the acreage used for commercial production and occurrence of 

wild species on a county level, the majority of the potato production in the United States is not in wild 

potato zones (Figure 2). However, there is a possibility that a few wild potato plants may be growing 

near potato fields (Love 1994). Spooner et al. (2004) describe S. jamesii habitat in the U.S. as among 

boulders on hillsides, sandy alluvial stream bottoms, in gravel along trails or roadways, rich organic soil 

of alluvial valleys, sandy fallow fields, grasslands, juniper-pinyon scrub deserts, oak thicket, coniferous 

and deciduous forests at elevations between 4,500 to 9,400 feet. They describe S. fendleri habitat 

similarly, and at elevations between 4700 to 11,200 feet. The risk of hybridization between commercial 

varieties and wild species is low because of the lack of geographical overlap and the fact that potatoes 

are grown from tuber seed. Furthermore, studies discussed in Conner and Dale (1996) indicate that a 

separation of 20 meters will be sufficient to prevent outcrossing between wild and commercial 

potatoes. 
J.R. Simplot Company JRS01 Page 22 of 74

Love (1994) evaluated the risk associated with growing transgenic potatoes in Canada and the USA and 

concluded that, given the number and potency of barriers to hybridization and more specifically to 

introgression and stabilization, the only sound conclusion is that gene introgression into wild Solanum

species will not occur under natural conditions in these geographies. Therefore, according to Love 

(1994), potato gene movement from commercial fields to wild potato species would not occur.

Figure 2. Potato Production in the United States

(County Information is for states with wild potato populations (Hijmans and Spooner 2001)

Based on conversations with Bamberg, Love (1994) reported that no one has ever reported finding 

hybrids between native and cultivated potatoes in the U.S., although gene transfer has been 

accomplished using special laboratory techniques (Love 1994). Love concluded that, based on the 

barriers that exist (including geographic isolation as described previously), endosperm imbalances, and 

multiple ploidy levels, natural hybridization is highly unlikely, and gene introgression (cross hybridization 

over multiple generations) is impossible or at least highly improbable (Love 1994). More recently, the 

US EPA has concluded that, based on its review of the scientific literature, successful gene introgression 

between native and cultivated potatoes in the U.S. is virtually excluded due to constraints of 

geographical isolation and other barriers to natural hybridization. These barriers include incompatible 

(unequal) endosperm balance numbers that lead to endosperm failure and embryo abortion, multiple 

ploidy levels and incompatible mechanisms that do not express reciprocal genes to allow fertilization to 

proceed. No natural hybrids have been observed between these species and cultivated potatoes in the 

U.S. (US EPA 2011). Based upon these biological barriers to hybridization, it is unlikely that inter-species 

pollination would occur with the InnateTM potatoes that are the subject of this petition.
J.R. Simplot Company JRS01 Page 23 of 74

2.12. Weediness

Standard growing practices for potatoes make it highly unlikely that potatoes would persist in a field 

from one crop cycle to the next. Where potatoes are a rotation crop, as is often the case, other crops 

such as alfalfa, corn or wheat would be grown following potatoes. In that situation, any potatoes left in 

the field would be eliminated by tilling, field preparations with herbicides, and harsh winters. In the rare 

event that potatoes survived mixed in with another crop, they have to face the same challenges during 

the next year. 

Potatoes are typically grown on a 3 year minimum rotation to minimize soil-borne disease buildup such 

as white mold, pink rot, Pythium leak, Verticillium wilt, and powdery scab (Hopkins et al. 2003). 

Potatoes left to grow in the field are not desired as they can harbor disease, have no protection with 

fungicide, and could compete with the desired rotation crop the following year. For these reasons, it is 

standard practice for potato growers to monitor and control such growth, either with herbicide or 

tillage. Soil is disturbed greatly during potato growth and harvest operations, therefore requiring tillage 

prior to planting the rotation crop. Typically a broad spectrum pre-emerge herbicide is applied in the 

rotation crop to control a wide variety of weeds. Potato growers rarely leave the ground fallow 

following potato to maximize economic return. If potatoes grew inadvertently in the field left fallow, 

they would not be protected with insecticide or fungicide and would be susceptible to insects such as 

Colorado potato beetle and diseases such as early blight, further decreasing the chance of survival and 

reproduction.

Outside of cultivation, potato seedlings from tubers have difficulty establishing themselves as they 

cannot compete effectively with other plants (Love 1994). Potato is not known as a colonizer of 

unmanaged ecosystems. In climax vegetation, potatoes are not able to compete with other species such 

as grasses, trees and shrubs (CFIA 1996), virtually eliminating any chance for them to become feral. 

Furthermore, potatoes generally do not cause toxic effects to other plants or animals (including nontarget organisms) making pest potential negligible.

Although almost all growers use tubers for planting, there is a small market for “true potato seed” (TPS). 

TPS is seed that is produced as a result of pollination. The seed is produced inside fruits that resemble 

small tomatoes on the potato vine. The major disadvantage of TPS is that it segregates for numerous 

traits because potato is highly heterozygous and each seed produced is potentially a new variety. When 

TPS is used for planting, harvested tubers will be variable in quality. TPS plants take longer to establish 

themselves and set tubers, resulting in lower yield compared to plants grown from tuber seed. Some 

growers in tropical areas use TPS because it is easy to ship and free of pathogens (Dwelle 2003). Love 

(1994) reported that botanical seed (TPS, not tubers or “seed potatoes”) can survive and germinate for 

periods of time in excess of seven years however, it is unlikely that plants from TPS would persist for the 

reasons stated above.

2.13. Conclusions: The Biology of Potato

The propagation of commercial potato varieties through cloning mitigates concerns about increased 

pest potential such as seed dispersal, survival outside of cultivation, or outcrossing. Potatoes are rarely 

grown from true potato seed because the offspring would show such extreme diversity they would not 

be useful as commercial potatoes. In addition, potatoes are not known to escape from commercial 

fields or show weediness potential. Wild potato varieties are rare in the United States and 
J.R. Simplot Company JRS01 Page 24 of 74

geographically isolated from commercial production areas, further reducing concerns about crosspollination with wild species.

An understanding of the biology of potatoes shows the extremely difficult challenges associated with 

simultaneously incorporating multiple traits through traditional breeding. Thus, potatoes benefit greatly 

by the use of InnateTM technologies which provide a superior alternative to breeding, through the 

addition of non-coding DNA, and result in new potato varieties that are not plant pests.

3. Method of Development: Description of Marker-Free DNA Transformation

3.1. Introduction

Simplot’s InnateTM technologies comprise many aspects of plant biology all working together to produce 

traits of interest in transformed plants. Specifically, the potatoes were modified to reduce asparagine 

and black spot bruise. Along with the potato derived expression cassettes, are promoters and spacers, 

also from the potato genome, all creating the gene silencing system. The targeted genes are then 

combined with the backbone elements of pSIM1278, to create the vector that is inserted into 

Agrobacterium. Potato plants are transformed by Agrobacterium, resulting in the incorporation of the 

expression cassettes that result in the desired traits of interest. A part of this process is the selection of 

potatoes to receive the new technology, and in this case, we chose some of the most prominent 

varieties for processing chips and fries. The pSIM1278 acts by effectively reducing asparagine which 

leads to reduced acrylamide levels in cooked potatoes, as well as reducing black spot bruise incidence. 

Description of the Transformation System 

The C58-derived Agrobacterium strain AGL1 was developed by precisely deleting the transfer DNA of the 

hyper-virulent plasmid pTiBo542 (Lazo et al. 1991). Transformed plants were grown on media containing 

the antibiotic timentin, which prevents survival of Agrobacterium, thus selecting for plants that are free 

of Agrobacterium. At the conclusion of the selection process, all Agrobacterium has been removed, no 

antibiotics remain, and the result is that genomic DNA from the potato or sexually compatible species 

have been added to the new plants.

Transformation Method

Stock plants were maintained in magenta boxes with 40 ml half-strength M516 medium containing 3% 

sucrose and 2 g/l gelrite (propagation medium). Potato internode segments of four to six mm were cut 

from four-week old plants, infected with the Agrobacterium AGL1 strain carrying pSIM1278, and 

transferred to tissue culture media containing 3% sucrose and 6 g/l agar (co-cultivation medium). 

Infected explants were transferred, after two days, to M404 medium containing 3% sucrose, 6 g/l agar 

and 150 mg/l timentin to eliminate Agrobacterium (hormone-free medium). Details of the methods are 

described in Richael et al. (2008).

Although Agrobacterium is effective in cleaving at the Right Border (RB) site, it often failed to fully 

release the DNA insert from its plasmid vector by also cutting at the Left Border (LB) site (Gelvin 2003). 

Consequently, some infected plant cells received the DNA insert itself as well as additional vector 

backbone sequences containing the backbone marker gene for cytokinin. These cells started expressing 

the isopentenyltransferase (ipt) gene resulting in production of the plant hormone cytokinin, which 

commonly regulates growth and development processes in plants.
J.R. Simplot Company JRS01 Page 25 of 74

After one month, the infected explants were transferred to fresh medium lacking any synthetic 

hormones and incubated in a Percival growth chamber under a 16 hr photoperiod at 24oC where they 

started to form shoots. Many shoots expressed the ipt gene and displayed a cytokinin overproduction 

phenotype; these shoots were discarded and not considered for further analyses. PCR genotyping 

demonstrated that about 0.3 to 1.5% of the remaining shoots contained at least part of the DNA insert

while lacking the ipt gene. Details on ipt-based marker-free plant transformation were published by

Richael et al. (2008).

The process of eliminating Agrobacterium started two days after explant infection. For this purpose, 

tissues were subjected to the antibiotic timentin (150 mg/l) until proven to be free of live 

Agrobacterium. Proof was obtained by incubating stem fragments of transformed events on nutrient 

broth-yeast extract (NBY medium) for 2 weeks at 28OC (repeated twice). In accordance with 7 CFR Part 

340, transformed plants were transported and planted in the field only when free of live Agrobacterium.

The potato events were confirmed to be free of Agrobacterium-derived vector backbone DNA by the 

following three methods: 1) If plants had the negative selectable isopentenyl isomerase (ipt) marker 

gene in the vector backbone, they were discarded. Plants with ipt gene expression would have stunted 

phenotypes, abnormal leaves, or the inability to root due to the cytokinin overproduction caused by ipt

expression; 2) Absence of the backbone DNA was confirmed with Southern blot hybridization; 3) PCR 

was used to confirm no fragments of the backbone DNA were present. 

3.2. Parental Varieties

Russet Burbank. Luther Burbank developed this variety in the early 1870s. Plants are vigorous and 

continue vine growth throughout the season. Stems are thick, prominently angled and finely mottled. 

Leaflets are long to medium in width and light to medium green in color. The blossoms are few, white 

and not fertile. The cultivar is tolerant to common scab but is susceptible to Fusarium and Verticillium

wilts, leafroll and net necrosis and virus Y. Plants require conditions of high and uniform soil moisture 

and controlled nitrogen fertility to produce tubers free from knobs, pointed ends and dumbbells. Jellyend and sugar-end develop in tubers when plants are subjected to stress. The tubers produced are large 

brown-skinned and white-fleshed, display good long-term storage characteristics, and represent the 

standard for excellent baking and processing quality. The variety is sterile and widely grown in the 

Northwest and Midwest, especially for the production of french fries.

Ranger Russet. This full season variety was released in 1991. Ranger Russet is more resistant than 

Russet Burbank to Verticillium wilt, viruses X and Y, leafroll and net necrosis, and Fusarium dry rot. It is 

highly resistant to hollow heart. Plants are large and upright to spreading. Stems are thick, green that

can be light brownish to light purple in full sun. Leaves are large, broad and medium green. Flowers are 

abundant and produce viable pollen. Buds are green with reddish-purple base and pedicel and moderate 

amount of short pubescence. Corolla is medium large, red-purple color and anthers are bright yellow. It 

produces high yields of good quality, high specific gravity tubers that are long and slightly flattened, and 

well suited for baking and processing into french fries. Tubers are susceptible to common scab and black 

spot bruise. Ranger Russet matures earlier than Russet Burbank and would be considered a mediumlength storage variety. The variety is fertile and mainly grown in the Northwest, especially for the 

production of french fries.

Atlantic. Plants are moderately large, with thick, upright stems, and slightly swollen, sparsely pubescent 

nodes. Leaves are bright, medium green, smooth, and moderately pubescent with prominent wings, 

large asymmetrical primary leaflets and numerous secondary and tertiary leaflets. Flowers are profuse 
J.R. Simplot Company JRS01 Page 26 of 74

with green, awl-shaped, pubescent calyx lobes, pale lavender corolla, orange anthers and abundant, 

viable pollen. The cultivar is tolerant to scab and Verticillium wilt, resistant to pinkeye, highly resistant to 

Race A of golden nematode, virus X, tuber net necrosis, and shows some resistance to black spot bruise. 

Tubers are susceptible to internal heat necrosis, particularly in sandy soils in warm, dry seasons. Hollow 

heart in the larger diameter tubers (diameter > 4 inches) can be serious in some growing areas. Tubers 

are oval to round with light to heavy scaly netted skin, moderately shallow eyes, and white flesh. Tuber 

dormancy is medium-long. With high yield potential, high specific gravity and uniform tuber size and 

shape, Atlantic is the standard variety for chipping from the field or from very short-term storage (Webb 

et al. 1978). The variety is fertile and mainly grown in the Northeast and Southeast, especially for the 

production of chips.

Variety G. Mid-season chipping variety with medium size tubers, round shape, shallow eyes, smooth 

slightly netted skin, white flesh, and high solids. It can be used as a fresh crop variety through mid-term 

storage. It generally has low defects but can display pressure bruise late in the storage season. It 

appears to be fairly resistant to PVY, is tolerant to common scab, and is confirmed to have resistance to 

the golden nematode Globadera rostochiensis Ro1. It has moderate flowering with white flowers which 

are both male and female fertile.

Variety H. Mid to late season chipping variety with large round to oval tubers, somewhat flattened 

shape, slightly indented eyes, smooth tan skin, pale yellow flesh, and has low to medium solids relative 

to other chipping varieties. It is generally used as a mid to late season storage variety. It is resistant to 

tuber early blight but susceptible to common scab. It has sparse white flowers and has no male fertility 

and very low female fertility.

3.3. Plasmid used for Transformation

The plasmid pSIM1278 is a binary transformation vector consisting of two parts: vector backbone and 

the DNA insert (Figure 3). The backbone section contains well-characterized bacterial origins of 

replication from plasmids pVS1 and pBR322, and the nptIII gene for bacterial resistance to kanamycin 

(see Table 4 for details of the backbone of pSIM1278). An expression cassette comprising the 

Agrobacterium ipt gene preceded by the polyubiquitin (Ubi7) promoter (GenBank accession no. 

U26831.1) and followed by the Ubi3 terminator was introduced as a 2.6-kb SacII fragment into the 

vector backbone (Garbarino and Belknap 1994). The DNA insert consists of the genetic elements 

described in Table 5. 
J.R. Simplot Company JRS01 Page 27 of 74

Figure 3. Vector pSIM1278

The vector backbone region, on the left, starts at position 9,957-bp and ends at 19,468-bp (= 9,512-bp). 

The backbone DNA consists mainly of bacterial DNA and is intended only to support maintenance of the 

DNA insert prior to plant transformation. The DNA insert region, including flanking Border sequences, is 

on the right from 19,469-bp to 19,660 and from 1-bp to 9,956, which is a total of 10,148-bp. 
J.R. Simplot Company JRS01 Page 28 of 74

3.4. Backbone Portion of pSIM1278

The purpose of the backbone portion of pSIM1278 is to maintain the DNA insert in bacteria; it is not 

intended for transfer to plant cells. The various elements of the backbone are described in Table 4.

Table 4. Genetic elements of the pSIM1278 backbone

Genetic Element Origin Intended Function Other 

effects on 

plant 

Genbank Accession 

Number

Start-End 

Point in 

pSIM1278

(bp)

Reference

SacII restriction site S. tuberosum Site for connection of Ubi7 

promoter with LB flanking 

sequence.

None AJ272136.1 19,219-

19,224

Polyubiquitin

promoter (Ubi7)

including the coding 

sequence for a 76-

amino-acid potato 

ubiquitin monomer 

(UBQmon)

S. tuberosum

var. Ranger 

Russet

Drives expression of the ipt

backbone marker gene

None U26831.1 17,479-

19,218

Isopentenyl 

transferase (ipt) 

gene

Agrobacterium

tumefaciens

condensation of AMP and 

isopentenylpyrophosphate 

to form isopentenyl-AMP, a 

cytokinin

Cytokinin 

formation

NC_002377.1 16,744-

17,466

Smigocki 

and 

Owens 

1988

Terminator of the 

ubiquitin-3 gene 

(tUbi3)

S. tuberosum Terminate ipt gene 

transcription

None GP755544.1 16,038-

16,392

Garbarino 

and 

Belknap 

1994

Neomycin 

phosphotransferase 

III (nptIII) gene

E. coli Aminoglycoside 

phosphotransferase

None FJ362602.1 15,048-

15,842

Courvalin 

et al. 1977

Origin of replication 

for pBR322 (pBR322 

ori)

E. coli Start position for plasmid 

replication in bacterial cells

None J01784.1 14,477-

14,757

*

(pBR322 bom) E. coli pBR322 region for 

replication in E. coli

None J01749.1 14,077-

14,337

*

pVS1 replicon 

( pVS1Rep)

Pseudomonas 

fluorescens 

plasmid pVS1

pVS1 region for replication 

in Agrobacterium

None AJ537514.1 (4,501-

5,501)

12,667-

13,667

*

pVS1 partitioning 

protein StaA (PVS1 

Sta)

Pseudomonas 

fluorescens 

plasmid pVS1

pVS1 stability None AJ537514.1 (6,095-

7,095)

11,074-

12,074

*

overdrive Agrobacterium

tumefaciens

Enhances cleavage at the 

Right Border site

None K00549.1 (103-132) 9,963-

9,992

*

*: see: http://www.cambia.org/daisy/cambia/585.html - (General structure map of pCAMBIA vectors)

3.5. The Transfer DNA (DNA insert) of the Vector

The transfer DNA (DNA insert) consists of non-coding genetic elements described in Table 5. The DNA 

insert or a functional part thereof, is the only part of vector pSIM1278 that is present in selected 

transformed events.
J.R. Simplot Company JRS01 Page 29 of 74

Table 5. Genetic elements of the DNA insert of pSIM1278, from Left Border site to Right Border site

Genetic Element Origin Intended Function Genbank 

Accession 

Number

Start-End 

Point in 

pSIM1278

Reference

1. Left Border (LB) site1Synthetic Site for secondary cleavage to 

release single-stranded DNA insert

from pSIM1278

AY5665552

(bases 1-25)

19,469 –

19,493

van Haaren

et al. 1989

2. DNA flanking the LB sequence S. tuberosum

var. Ranger 

Russet

Supports secondary cleavage at LB AY5665552

(bases 26-

187)

19,494 –

19,655

3. KpnI restriction site S. tuberosum Site for connection of DNA insert

with LB flanking sequence.

AF393847.1 19,656 –1

4. Promoter for the ADP glucose 

pyrophosphorylase gene (pAgp), 1st 

copy

S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of Asn1 and Ppo5, 

especially in tubers

HM363752 2-2,261

5. Fragment of the asparagine 

synthetase-1 (Asn1) gene (1st copy

antisense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (9) double stranded 

RNA that triggers the degradation of 

Asn1 transcripts to impair 

asparagine formation

HM363759 2,262-2,666 Chawla et 

al. 20123

6. 3’-untranslated sequence of the 

polyphenol oxidase-5 gene (Ppo5) 

(1st copy, in antisense orientation)

S. verrucosum Generates with (8) double stranded 

RNA that triggers the degradation of 

Ppo5 transcripts to block black spot 

bruise development

HM363754 2,667-2,810

7. Spacer-1 S. tuberosum

var. Ranger 

Russet

Sequence between the 1st inverted 

repeats

HM363753 2,817-2,973

8. 3’-untranslated sequence of the 

polyphenol oxidase-5 gene (Ppo5) 

(2nd copy, in sense orientation)

S. verrucosum Generates (6) double stranded RNA 

that triggers the degradation of 

Ppo5 transcripts to block black spot 

bruise development

HM363754 2,974-3,117

9. Fragment of the asparagine 

synthetase-1 (Asn1) gene (2nd copy, 

in sense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (5) double stranded 

RNA that triggers the degradation of 

Asn1 transcripts to impair 

asparagine formation

HM363759 3,118-3,523 Chawla et 

al. 20123

10. Promoter for the granule-bound 

starch synthase (pGbss) gene (1st

copy, convergent orientation relative 

to the 1st copy of pAgp)

S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of Asn1 and Ppo5, 

especially in tubers

HM363755 3,530-4,215

11. pAgp, 2nd copy S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of the promoters of PhL 

and R1, especially in tubers

HM363752 4,232-6,491

12. Fragment of promoter for the 

potato phosphorylase-L (pPhL) gene 

(1st copy, in antisense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (16) double stranded 

RNA that triggers the degradation of 

PhL transcripts to limit the 

formation of reducing sugars 

through starch degradation

HM363758 6,492-7,000

13. Fragment of promoter for the 

potato R1 gene (pR1) (1st copy, in 

antisense orientation)

 S. tuberosum 

 var. Ranger 

 Russet

Generates with (15) double stranded 

RNA that triggers the degradation of 

R1 transcripts to limit the formation 

of reducing sugars through starch 

degradation

HM363757 7,001-7,532

14. Spacer-2 S. tuberosum

var. Ranger 

Russet

Sequence between the 2nd inverted 

repeat

HM363756 7,539-7,796
J.R. Simplot Company JRS01 Page 30 of 74

Table 5 continued. Genetic elements of the DNA insert of pSIM1278, from Left Border site to Right 

Border site

Genetic Element Origin Intended Function Genbank 

Accession 

Number

Start-End 

Point in 

pSIM1278

Reference

15. Fragment of promoter for the 

potato R1 gene (pR1) (2nd copy, in 

sense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (13) double 

stranded RNA that triggers the 

degradation of R1 transcripts to 

limit the formation of reducing 

sugars through starch degradation

HM363757 7,797-8,328

16. Fragment of promoter for the 

potato phosphorylase-L (pPhL) gene 

(2nd copy, in sense orientation)

S. tuberosum

var. Ranger 

Russet

Generates with (12) double 

stranded RNA containing this 

sequence will trigger the 

degradation of PhL transcript to 

limit the formation of reducing 

sugars through starch degradation

HM363758 8,329-8,837

17. pGbss (2nd copy, convergent 

orientation relative to the 2nd copy 

of pAgp)

S. tuberosum

var. Ranger 

Russet

One of the two convergent 

promoters that drives expression of 

an inverted repeat containing 

fragments of the promoters of PhL 

and R1, especially in tubers

HM363755 8,838-9,761

18. SacI restriction site S. tuberosum Site for connection of DNA insert 

with RB flanking sequence.

AF143202 9,76 –

9,767

19. DNA flanking the RB sequence S. tuberosum

var. Ranger 

Russet 

Supports primary cleavage at RBLike siteAY5665552

(bases 231-391)

9,77 –

9,931

20. Right Border (RB) sequence1Synthetic Site for primary cleavage to release 

single stranded DNA insert from 

pSIM1278

AY5665552

(bases 392-416)

9,932 -

9,956

van 

Haaren et 

al. 1989

1

The LB and RB sequences (25-bp each) were synthetically designed to be similar to and function like T-DNA borders from Agrobacterium 

tumefaciens.

2

GenBank Accession AY566555 was revised to clarify the sources of DNA for the Border regions.

3

ASN1 described as genetic elements 5 and 9 is referred to as StAst1 in Chawla et al. 2012.

3.6. Open Reading Frames Associated with the DNA Insert

Open reading frames (ORFs) are long sequences of DNA (a few hundred base pairs) that start with an 

ATG triplet and end with a stop codon. ORFs may encode part or all of a protein if flanked by 

appropriate regulatory sequences.

The full 10,148-bp DNA insert, from the cleavage site in the Left Border element to the cleavage site in 

the Right Border element contains fragments associated with an ORF of the Asn1 gene and the trailer 

associated with an ORF of the Ppo5 gene. These inverted repeats produce a pool of variably-sized RNAs 

that trigger gene silencing and are not translated into protein when transcribed. 

3.7. Conclusions of Methods of Development: Description of Marker-Free DNA Transformation

A description of InnateTM technologies outlines the plant biological systems all working together to 

create the plants. These include trait identification, design of vectors, incorporation of vectors into 

Agrobacterium, recipient potato variety selection, transforming plants, and confirmation that the new 

potatoes contain the expected DNA inserts. The InnateTM methods allowed us to insert the desired 

traits into potato varieties using non-coding DNA into potato and develop new potato events that are 

not plant pests. 
J.R. Simplot Company JRS01 Page 31 of 74

4. Characteristics of Transferred DNA and Gene Regulation

The potato events all produce potatoes that are silenced for specific genes. The silencing methods are

part of Simplot’s InnateTM technologies for reducing gene expression. This patented method provides 

the framework for reducing the expression of genes that ultimately reduce black spot bruise, 

asparagine, sugars, and acrylamide in potatoes. A brief introduction to gene silencing is given here, 

describing how similar methods are found in nature. In general, the inserted DNA contains silencing 

cassettes that, when expressed, generate variably-sized and unprocessed transcripts. These transcripts

trigger the degradation of mRNAs that would normally code for an enzyme, like asparagine synthetase. 

This results in much reduced levels of the targeted “silenced” enzymes. 

All potato events were analyzed by DNA gel blot analyses to determine the structure and copy number 

of the integrated DNA insert sequences and to confirm the absence of vector backbone sequences. 

These studies were carried out as parts of the characterization and biosafety assessment of the events. 

In addition, molecular characterization was used to determine the sequence of the junctions flanking 

the DNA insert and show stability of the inserted DNA. Sequencing information of the junctions 

provided the basis for developing event-specific PCR tests for all events. 

4.1. Description and History of the Modification

Mechanism for Gene Silencing

The silencing approach applied here to down-regulate the transcript levels of several genes is illustrated 

in Figure 4. Two copies of a DNA segment comprising fragments of four targeted genes are inserted, as 

an inverted repeat, between two convergent promoters that are predominantly active in tubers. Plants 

containing the silencing cassette produce a diverse and unpolyadenylated array of RNA molecules in 

tubers that dynamically and vigorously down-regulate the intended target genes. The size of the RNA 

molecules is generally smaller than the distance between the two promoters according to Yan et al.

2006.

Figure 4. Silencing Approach Using the DNA Insert of pSIM1278

The expression of an inverted repeat is a regulatory system that has evolved naturally as a consequence 

of the duplication of genes and regulatory elements. One example relates to a 27-kb region of the 
J.R. Simplot Company JRS01 Page 32 of 74

soybean genome containing two perfectly repeated and inverted clusters of three chalcone synthase 

(Chs) genes (Tuteja et al. 2009). This results in the silencing of all Chs gene family members through the 

formation of double-stranded RNA. In this example, the double-stranded RNA initiates gene silencing, 

and is an example of naturally occurring inhibition of a metabolic pathway, which functions specifically 

in one tissue while allowing expression in other parts of the plant.

Expression of Neighboring Genes

Previous studies on convergent transcription have shown that transcripts are of variable size but smaller 

than the distance between the two promoters, both in yeast (Prescott and Proudfoot 2002) and plants 

(Yan et al. 2006). In a study on convergent transcription-based silencing of the -glucuronidase (gus)

gene, no expression of neighboring genes was found (Yan et al. 2006). The RNA gel blot analyses 

presented in this petition (Appendix 5. Efficacy and Tissue-Specificity of Gene Silencing) demonstrated 

the absence of bands or smears indicative of read-through transcripts that hybridized with a probe 

derived from the entire Asn1/Ppo5 inverted repeat (see, for instance, Figure 2 of Appendix 5. Efficacy 

and Tissue-Specificity of Gene Silencing). 

Although the possibility of rare read-through transcripts cannot be excluded, it is unlikely that such 

transcripts would interfere with the expression of neighboring genes because single stranded transcripts 

are ineffective in triggering gene silencing (Yan et al. 2006). Furthermore, studies on the expression of 

an antibiotic selectable marker gene next to a convergent transcription-based silencing construct 

demonstrated that this antibiotic gene was not affected by expression of the silencing construct (Yan et 

al. 2006). In the unlikely event that read-through occurred, and lowered the expression of an adjacent 

gene in any of the 10 events, it would have been evident through change in plant phenotype. After 

extensive evaluations from multiple field trials over three years, no unexpected agronomic, phenotypic,

or biochemical differences were observed with these 10 events when compared with control varieties.

4.2. Verification of Insert Integrity and Number of Inserted Sequences

A diagram of the structure of inserts is shown in Figure 5 (see Abbreviations for most keys; AGP = pAgp, 

GBS = pGbss). Details on characterization of the inserted DNA are provided in Appendix 1. 

Characterization of Inserted DNA. Two events (J78 and G11) contain a single truncated copy of the DNA

insert with a functionally-active Asn1/Ppo5 gene silencing cassette (and part of the R1/PhL promoter 

silencing cassette that is not functionally active). 

The comprehensive molecular analyses confirmed anticipated similarities between the events of this 

petition and T-DNA integration events described elsewhere:

(1) Six events (F10, F37, E12, E24, J78, and G11) contain 1 copy (whereby “copy” implies the 

presence of at least an Asn1/Ppo5 gene silencing cassette), three events contain 2 copies (J3, 

J55, and H50), and the remaining event H37 contains 1 copy with 3 additional fragments. This 

finding indicated that the number of copies of the DNA insert is similar to that reported 

elsewhere for T-DNAs in potato (Wolters et al. 1998) and other crops such as tobacco (De Neve 

et al. 1997; De Buck et al. 1999), Arabidopsis (De Buck et al. 2009), rice (Sallaud et al. 2003; Yang 

et al. 2005), wheat (Cheng et al. 1997), and barley (Bartlett et al. 2008). Although high copy 

numbers have been linked to post-transcriptional gene silencing (Lechtenberg et al. 2003), we

found no differences in the extent and persistence of silencing activities between higher-copy 

events and the events with only one copy.

(2) Event J55 contained two linked DNA inserts positioned as an inverted repeat. Lechtenberg et al.

(2003) showed with bacterial T-DNA that the presence of a second gene copy either in tandem 
J.R. Simplot Company JRS01 Page 33 of 74

or an inverted arrangement did not result in silencing. Thus, it’s likely that the inverted linked 

DNA insert copies in J55 would not contribute to silencing and therefore, the silencing of 

targeted genes functions as intended based on the inverted repeats positioned between 

convergent promoters.

(3) Inserts are occasionally flanked by short DNA sequences that are derived from the plant genome 

or the DNA insert. These insertions appear to be part of the integration process and occur at 

rather high frequencies (Windels et al. 2003). An example of an event with such sequences

includes the 49-bp sequence between the two DNA inserts of J55. A blast search of this short 

DNA sequence using GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi? 

CMD=Web&PAGE_TYPE=BlastHome) partially matched known sequences from S. tuberosum, 

confirming that the origin was most likely from either the plant genome or the DNA insert. 

(4) Most transferred DNA inserts are shorter than the full distance between the Left and Right 

Border sequences, as shown by short deletions near the borders. Such deletions are also 

associated with T-DNA integration and hypothesized to result from double-strand break repair 

(Gheysen et al. 1991). Short deletions that did not impair the functional activity of any of the 

two silencing cassettes were found in, for instance, events F10 (a 38-bp deletion at the Right 

Border) and F37 (a 12-bp deletion at the Left Border). Large deletions at the Right Border side 

that resulted in loss of the PhL/R1 promoter silencing cassette occurred in events J78 and G11.

Figure 5. Diagram of DNA Inserts

F10 Insert:

F37 Insert:
J.R. Simplot Company JRS01 Page 34 of 74

Figure 5 (Continued). Diagram of DNA Inserts

E12 Insert:

E24 Insert:

J3 Insert:
J.R. Simplot Company JRS01 Page 35 of 74

Figure 5 (Continued). Diagram of DNA Inserts

J55 Insert:

J78 Insert:

G11 Insert: 
J.R. Simplot Company JRS01 Page 36 of 74

Figure 5 (Continued). Diagram of DNA Inserts

H37 Insert:

Diagram (4) is a small fragment containing part of the AGP promoter.
J.R. Simplot Company JRS01 Page 37 of 74

Figure 5 (Continued). Diagram of DNA Inserts

H50 Insert:

4.3. Selection of Backbone-free Plants

Agrobacterium-mediated transformation often results in transfer of “backbone” sequences that are 

vector DNA in addition to the intended sequences positioned between the Left and Right Borders of the 

plant derived DNA insert. The frequency of transfer of backbone DNA was estimated at 75% for tobacco 

(Kononov et al. 1997) and 47% to 67% for Arabidopsis (Oltmanns et al. 2010).

In contrast, all the events in this submission were confirmed to be free of vector backbone DNA

(Appendix 2. Evidence for the Absence of Vector Backbone DNA). This finding confirms previously 

published work on Simplot’s cytokinin vectors (Richael et al. 2008). 

4.4. Flanking Regions of Inserted Sequences

Extensive research was conducted to characterize the inserted DNA for all events (Appendix 1. 

Characterization of Inserted DNA). In addition, at least one junction was sequenced for all events to 

develop event-specific PCR methods.

Among the evidence supporting safety relative to the insertion site are the following: (1) most insertions 

do not disrupt gene expression because DNA integrates at random when no selection is used during 

transformation (Kim et al. 2007) and only approximately 5.5% of the potato genome is occupied by 

genes (Zhu et al. 2008); (2) insertions that do disrupt gene functions are almost always recessive (Hagio 

et al. 2002; Chiou et al. 2006; Daxinger et al. 2008), which means that change to a single chromosome 

would be unlikely to result in an observable phenotype (in contrast to most other crops, transformed 

potatoes are not self-fertilized (backcrossed) to develop events that are homozygous for the insert); and 
J.R. Simplot Company JRS01 Page 38 of 74

(3) if an unusual and undesirable phenotype occurred, that event would be selected only if equivalent or 

superior to the untransformed potato variety. 

4.5. Stability of Inserted DNA

Bacterial T-DNAs are not always stable after insertion into a plant. The estimated instability rate of

between 0.5-5.9×10-4, is associated with meiosis (Müller et al. 1987; Conner et al. 1998). Because potato 

is a vegetatively propagated crop and reproduction does not involve meiosis, DNA insertions were

expected to be stable. Nevertheless, the stability of integrated DNA inserts was confirmed both by DNA 

gel blot hybridization and by assaying G0, G1, G2, and G3 tubers for polyphenol oxidase activity

(Appendix 3. Evidence for Stability of the Inserted DNA).

It should be mentioned here that one event that is not considered in this submission for nonregulated 

status, designated as G09, was found to be unstable. About 2% of the G2 tubers of this event had 

partially lost black spot bruise tolerance (Appendix 3. Evidence for Stability of the Inserted DNA). Such

instability may have been associated with the unusually complex DNA insert organization of G09 (7 

copies of at least part of the DNA insert). While not certain that the large copy number was the cause of 

instability, it would be recommended to select events with the minimum number of copies needed to 

express the desired traits. For example, it would be desirable to select future events with relatively 

simple DNA insertions (2 copies or less).

Although instability is undesired and may result in a phenotypic reversion back to wild-type where

tubers lose the incorporated traits (black spot bruise tolerance, reduced acrylamide-forming potential, 

and, in some cases, reduced formation of fructose and glucose), it would not trigger any biosafety 

issues. To date, all assessments have been limited to G0, G1, G2, and G3 material. Continued testing will 

monitor phenotypic stability of the new traits.

4.6. Event-Specific PCR

As part of our stewardship and identity preservation plan as well as for internal event detection, eventspecific PCR was developed for all the events submitted for nonregulated status. The methods 

developed will be used to monitor plants and tubers in field and storage as part of quality management 

programs (Appendix 4. Junction Analysis and Event-Specific Detection).

4.7. Conclusions: Characteristics of transferred DNA and Gene Regulation

We found the 10 events to contain at least 1 full or partial copy of the DNA insert from pSIM1278 and, in 

the case of H37, three additional fragments of pSIM1278. Although the inserts were not always fulllength, therefore, each event contained at least one functional Asn1/Ppo5 gene silencing cassette. This 

means there are three main differences between these events and their untransformed counterparts: 1) 

InnateTM tubers contained less asparagine, 2) more glutamine and 3) at least some degree of black spot 

bruise tolerance. The one exception being that conventional Atlantic already displays some black spot 

bruise tolerance. All of the events were free of detectable vector backbone DNA. Reduced asparagine 

contributes to significant reductions in acrylamide after heating, in all events, even if the insert 

contained only the Asn1/Ppo5 silencing cassette and lacked the R1/PhL silencing cassette that would 

contribute to lower reducing sugars. 
J.R. Simplot Company JRS01 Page 39 of 74

All insertions represent simplex insertions into tetraploid loci. This heterozygote genotype is commonly 

referred to as AAAa, whereby “a” represents an allele carrying the insertion, and “A” stands for lack of 

the insertion. Simplex insertions are unlikely to cause expression of recessive phenotypes, which usually 

require homozygote insertions (knock-out of all “A” dominant alleles).

It is possible but not likely that a promoter element positioned within an insertion could function as a 

long-distance enhancer or promoter for a neighboring open reading frame. Even if the expression of 

open reading frames adjacent to insert promoters had occurred, it would be rare that this would result 

in a functional and unwanted protein. If an insertion interrupted the expression of an essential gene, the 

interruption would be expected to disrupt the growth of the plant or change the composition of the 

plant tissues. The combined tests for molecular structure show that the potato events have resulted 

from the addition of genetic material from a donor organism where the material is well characterized 

and contains only non-coding regulatory regions of DNA. 

5. Expression of the Gene Products 

The DNA insert used to transform these ten potato events contains two expression cassettes designed 

to lower the transcript levels of endogenous genes. The first cassette contains two fragments of the 

asparagine synthetase-1 (Asn1) gene (404-bp) and the polyphenol oxidase-5 (Ppo5) gene (143-bp), 

inserted as inverted repeats between the convergent-oriented promoters of the ADP glucose 

pyrophosphorylase (Agp) and granule-bound starch synthase (Gbss) genes. The second cassette contains

fragments of the promoters of the starch associated genes R1 (531-bp) and phosphorylase-L (PhL) (508-

bp), operably linked to the same regulatory elements used for the first cassette.

Transcript levels for the Asn1 and Ppo5 genes were reduced in the tubers, stolons, and, in some cases, 

other tissues of all potato events as expected (Appendix 5. Efficacy and Tissue-Specificity of Gene 

Silencing). It was also expected that transcript levels for the PhL and/or R1 genes would be reduced in 

tubers, stolons, and, in some cases, other tissues of the potato events F10, F37, E12, E24, J3, J55, H37 

and H50.

It is possible that these intended changes could trigger unintended effects because free asparagine and 

glutamine are signals of organic nitrogen status that regulates gene expression (Gutiérrez et al. 2008), 

while glucose stimulates the formation of abscisic acid and auxin while suppressing cytokinin production 

(Ramon et al. 2008). However, if unintended changes did occur, studies described in this petition 

demonstrated that they have no consistent impact on plant agronomics or tuber composition.

Previous studies have shown that Ppo gene silencing lowers the amount of protein to levels that are 

undetectable by Western blot analysis (Llorente et al. 2011). Similarly, silencing of the R1 gene 

diminished accumulation of a ~160kDa protein that is at least partially bound to starch granules 

(Lorberth et al. 1998). 

The inserted DNA in all 10 events included in this petition functions by silencing the genes involved with 

production of asparagine, polyphenol oxidase, and reducing sugars. By design, the events contain only 

non-coding regulatory sequences. Multiple studies included in this petition show the material to be well 

characterized and not associated with plant pest traits. 
J.R. Simplot Company JRS01 Page 40 of 74

6. Efficacy and Tissue-Specificity of Gene Silencing

The purpose of these studies was to show the effectiveness of gene silencing based on RNA expression 

as measured through Northern blot analyses. Gene silencing methods were employed to lower the 

activity of the Asn1, Ppo5, PhL, and R1 native proteins. Transcript levels rather than protein amounts 

were evaluated to link new phenotypic traits to changes at the molecular level. For this purpose, RNA 

was isolated from various tissues of the events and their untransformed counterparts and used to carry 

out Northern blot analyses (see Appendix 5. Efficacy and Tissue-Specificity of Gene Silencing, with a 

summary in Table 1). Each letter (A, P, L, R) in Table 6 indicates that silencing was confirmed, although 

the amount of silencing varied depending on the gene and tissue.

InnateTM technologies have given us the ability to selectively regulate genes in specific tissues rather 

than the entire plant. For example, strong silencing of the Asn1 gene involved in ASN (asparagine)

formation in potato tubers was desired, yet such changes in leaves and stems might adversely affect 

growth. The promoters used to drive gene silencing, the Agp promoter and the Gbss promoter, have 

been well characterized and are known to be highly active in tubers and stolons and less active in 

photosynthetically-active tissues and roots (Nakata et al. 1994; Visser et al. 1991). Therefore, silencing 

was predicted to be stronger in tubers and stolons, and less pronounced in leaves, stems, roots, and 

flowers, as shown in Table 6.

Table 6. Summary of Down-Regulated Genes in Different Tissues

Event Tubers1Stolons1Roots1Stems1Leaves1Flowers1, 2

F10 A P R A L R A P R P A A

F37 A P R A L R A P R A P A R

E12 A P L R A L R A P P A

E24 A P L R A P L R A P A

J3 A P L R A R A A P A

J55 A P L R A L R A P A

J78 A P A P A

G11 A P A P R R A

H373A P L R L R P P R

H503A P L R A P L R A P R A P R

1

A = Asn1, P = Ppo5, L = PhL, R = R1. Letters in table indicate down-regulated gene expression by tissue.

2

The partially down-regulated Asn1 gene expression might alter the amino acid composition of the flowers. Such 

effects will be limited to a reduction in ASN and an increase in GLN. Since ASN and GLN are similar non-essential 

amino acids, changes in the levels of these compounds is not expected to affect the quality of petal, nectar, and 

pollen as feed for insects or other organisms.

3

Events derived from variety H do not produce flowers.

The targeted four genes (Asn1, Ppo5, PhL, and R1) are partially silenced in tubers of most events, except 

for J78 and G11 which do not contain the cassette for silencing the PhL/R1 genes (Table 6). In addition,

the PhL gene did not appear down-regulated in tubers of the Ranger Russet events, F10 or F37. For 

some events, E24, and H50, all four genes were also down-regulated in stolons. The two events J78 and 

G11, which contain a truncated DNA insert with a functionally active Asn1/Ppo5 silencing cassette, 

produced tubers that contained reduced transcript levels for the Asn1 and Ppo5 genes only, since they 

did not contain the PhL/R1 silencing cassette. Overall, more down-regulation of the four targeted genes 

occurred in tubers and stolons than in the roots, stems, leaves, or flowers (Table 6).
J.R. Simplot Company JRS01 Page 41 of 74

Event G11, which does not contain the PhL/R1 promoter silencing cassette, displayed reduced R1 

transcript levels in leaves and stems. This unexpected change may be a consequence of DNA integration 

or somaclonal variation (Skirvin et al. 1993).

The expression studies showed that silencing was most effective in tubers and stolons, as predicted with 

the chosen promoters, Agp and Gbss. In addition, silencing was predictable based on the molecular 

characterization that showed all events contained the Asn1 and Ppo5 genes, but two events, J78 and 

G11 events, did not contain the R1 and PhL genes, and therefore did not show silencing of those genes

in tubers. 

Considering the potato events in this study, expression of the targeted genes would provide evidence 

that the desired phenotype with low asparagine, reduced black spot, and lowered reducing sugars had 

been achieved. It could provide information relevant to the plant pest risk assessment, for example, all 

InnateTM potatoes in the study had reduced or similar expression to the controls. Thus, the altered 

expression is unlikely to affect weediness or other plant pest characteristics. Also, with these events, 

the expression studies pertain to endogenous proteins rather than new proteins that could require a 

safety evaluation, such as those associated with herbicide tolerance or insect resistance. 

Additional evidence for the safety of these potato events compared to the untransformed controls is 

illustrated in the agronomy and composition studies. It can therefore be concluded that these modified 

expression levels do not impact plant agronomics or influence tuber composition. Thus, the expression 

studies provide additional evidence that the InnateTM potatoes are not plant pests and the integrated 

genetic material contains only non-coding regulatory regions that are well characterized, and are as safe

as untransformed potatoes.

7. Agronomic Performance, Disease, and Efficacy of Traits

The purpose of the agronomic trials was to confirm that the InnateTM potatoes had equivalent 

phenotypes compared with their untransformed controls when grown at multiple locations representing 

the major areas for potato production in the US. Observations throughout the growing season allowed 

for a thorough assessment of growth, disease and pest susceptibility, and measurements of tuber yield 

and quality. The field assessments aided in the selection of the events that contained the intentionally 

incorporated new traits and maintained all benefits of the conventional parent varieties. These 

assessments also made it possible to ensure that the transformations had not resulted in the 

introduction of unintended effects associated with weediness or pest-like characteristics. 

After completing the agronomic trials, potato tubers were tested for evidence of the efficacy of the 

traits, including an assessment for black spot bruise and reducing sugars. In addition, after processing 

into french fries or potato chips, the potatoes were tested for acrylamide. A brief summary of the 

acrylamide results will be presented here with the supporting details available in Appendix 9. 

Compositional Analyses.
J.R. Simplot Company JRS01 Page 42 of 74

7.1. Agronomy and Phenotypic Evaluation

This summary includes agronomic evaluations of 10 events, representing 5 varieties, at geographically 

distinct sites that represent most of the main production areas for potatoes destined for fry and chip 

production in the USA (Appendix 6. Field Performance and Tuber Evaluations). All agronomic trials 

were conducted under Biotechnology Quality Management System (BQMS) standards and USDA-APHIS 

compliance (For a complete listing of trials, see Table 13 in Section 14. Record of Field Test Reports). 

Planting was carried out mechanically to facilitate subsequent harvests and to ensure that InnateTM

potatoes were kept separate from unmodified material. The agronomic evaluations relate to both field 

observations and tuber assessments, both at harvest and during storage. Table 7 shows which 

characteristics were evaluated. Some differences in data collected reflect differences in product use in 

processing, e.g., Ranger Russet and Russet Burbank used for french fries and Atlantic, G, and H are used 

in the potato chip industry.
J.R. Simplot Company JRS01 Page 43 of 74

Table 7. Summary of Agronomic Observations

Characteristic

Number of Site Years1

Ranger 

Russet

Russet 

Burbank Atlantic G H

Early Emergence 4 4 13 3 3

Final Emergence 13 4 4 3 3

Stems Per Plant 4 4 7 NA NA

Plant Vigor 8-11 11 7-8 6 5

Foliage Color 8-11 11 14-15 6 5

Leaflet Size 8-11 11 14-15 6 5

Leaflet Curl 8-11 11 14-15 6 5

Senescence 3 3 6 NA NA

Vine Size NA NA NA 4 4

Vine Maturity Rating 7 7 9-10 5 5

Flower Color 2-4 3-4 7 NA NA

Total Yield 11 11 14 3 3

% 4-6 oz. 9-10 8-10 NA NA NA

% 6-10 oz. 9-10 8-10 NA NA NA

% 10-14 oz. 9-10 8-10 NA NA NA

% >14 oz. 9-10 8-10 NA NA NA

Specific Gravity 9-10 8-10 14 3 3

High Sugar 9-10 8-10 NA NA NA

Sugar Ends 9-10 8-10 NA NA NA

Total Internal Defects 9-10 4 14 3 3

% U.S. #1 NA NA 14 3 3

% Grade B NA NA 14 3 3

% Grade A NA NA 14 3 3

% Oversize NA NA 14 3 3

% Pickouts NA NA 14 3 3

Insect Stressors 3-5 5 7 1 1

Disease Stressors 3-5 5 7 1 1

Abiotic Stressors 3-5 5 7 1 1

Incidental Stressors 8 8 9 6 6

NA = characteristic not evaluated

1

Total number of sites in 2009, 2010, and 2011 for each characteristic. Ranges indicate that not all lines of each 

variety were grown at every site.

The agronomic and ecological data that are presented in Appendix 6 (Field Performance and Tuber 

Evaluations) show that over multi-year and multi-site field trials no variety specific differences leading

to increased weed or pest potential were present. The studies were conducted with as much scientific 

rigor as possible to ensure product and trait safety. Statistical methods were employed to test for 

differences between the InnateTM varieties and appropriate controls. We used a step-wise approach to 

interpret any differences that were observed (both statistically significant and non-statistically 
J.R. Simplot Company JRS01 Page 44 of 74

different). For each comparison between a test line (event) and the appropriate conventional control, 

the following logic was employed to assess the results. First, when p-values were available, statistical 

significance was determined for each comparison. If the p-value indicated no statistical significance, it 

was unlikely that there was a difference that would be biologically meaningful or indicative of increased 

pest potential. Next, if the p-value indicated statistical significance or the data did not support 

calculation of a p-value, the mean value of the test line was compared to the combined control range, 

which represents the minimum and maximum mean values of all conventional lines included in the 

experiments. If the value for the test line was within the combined control range, it was concluded that 

the test line exhibited characteristics within the natural variation of potatoes and that the difference 

was unlikely to be biologically meaningful or indicative of increased pest potential. If the value of the 

test line was outside the combined control range, further consideration was given to the difference in 

the context of agronomic impact and increased pest potential.

The combined control range for the agronomic characteristics used a total of 319 mean values from 

conventional Ranger, Burbank, Atlantic, G, and H varieties grown as controls in the field trials. For the 

yield and grading data for the Burbank and Ranger lines, the combined control range used a total of 170 

mean values of the Burbank and Ranger conventional controls. For the yield and grading data for the 

Atlantic, G, and H lines, a total of 160 mean values of Atlantic, G, and H conventional controls were used 

to determine the combined control range.

Although some significant agronomic differences were observed between events and controls, most fell 

within the combined control range of the conventional varieties. For example, some differences in yield 

and tuber size were observed in the events, and these attributes will be investigated further in 

commercial trials. Thus, we concluded that no biologically meaningful differences that would contribute 

to increased weediness or pest potential were observed for any of the agronomic characteristics, yield 

and grading characteristics, or ecological interactions (namely plant-insect interactions, plant-disease 

interactions, and plant interactions with abiotic stressors) of the events compared to their 

untransformed controls (Appendix 6. Field Performance and Tuber Evaluations). There were no 

differences in phenotype and tuber composition (Appendix 9. Compositional Analyses) or disease 

susceptibility (Appendix 8. Disease Susceptibility). It can be concluded that the events are not different 

from their untransformed controls in terms of agronomy, tuber yield, and tuber composition. Therefore, 

the transformation of potato with pSIM1278 does not introduce characteristics that will encourage or 

enhance weediness or pest potential.

7.2. Disease susceptibility and polyphenol oxidase

Polyphenol oxidase enzymes (Ppo) are found in most organisms including animals, plants, fungi and 

bacteria. Although much is known about the molecular biology of Ppo and its role in enzymatic 

browning, little is understood about the function of Ppo-mediated browning in plant physiology. Some 

have hypothesized that Ppos represent part of the plant’s defense response against insects and 

pathogens. Considering that the present submission involves silencing of Ppo in tubers, we reviewed 

relevant literature and conducted studies relating to the disease response of the events. 

Ppos are copper metalloenzymes which oxidize mono- and o-diphenols to o-diquinones by utilizing 

molecular oxygen (Thipyapong et al. 2004). Typically, Ppo activity is latent until the enzyme is released 

by disruption of the cell structure through forces like wounding and senescence. When cell membranes 

are damaged, Ppo enzyme is released and reacts along with oxygen molecules to produce quinones
J.R. Simplot Company JRS01 Page 45 of 74

(Thipyapong et al. 2004). The production of black and brown quinones is responsible for much of the 

interest in PPO in the post-harvest physiology of many fruit and vegetable crops. 

It is common for multiple homologues or alleles to exist within species, each responsible for expression 

in different plant tissues. In potato, 6 genes encoding Ppo have been identified, tomato (Lycopersicon 

esculentum) possesses 7 PPO genes, and banana (Musa acuminata) is known to contain a Ppo family of 

at least 4 genes (Thipyapong et al. 2007; Mayer 2006). A number of researchers evaluated induced Ppo

activity and response mechanisms to biotic stressors, in particular pests and pathogens (Steffens et al.

1994). Studies with biotech tomatoes found a positive correlation between high levels of Ppo in leaf 

tissue and increased resistance to pathogens and insect pests. In one study, three lines with higher Ppo

expression were tested against the bacterial pathogen Pseudomonas syringae pv. Tomato, the causal 

agent of bacterial speck in tomato (Li and Steffens 2002). These lines showed enhanced suppression of 

disease symptoms and exhibited 15-fold fewer lesions per leaf area than controls. Although these 

results indicated a protective effect of Ppo, the mechanism for disease resistance remains unknown (Li 

and Steffens 2002). 

In potato, impacts sustained during harvest and postharvest activities induce the release of Ppo from 

cell plastids, facilitating oxidation of phenols to quinones, and resulting in negative effects on quality 

and recovery in processing french fries and chips, as well as the marketability of fresh potatoes. A family 

of 6 genes encoding Ppo exists in potato, with one gene (Ppo5) being tuber-specific and the remaining 5 

genes responsible for Ppo expression in other tissues. The tuber-specific Ppo5 gene was downregulated, resulting in reduced susceptibility to black spot bruise, as shown in the current petition for 

nonregulated status.

Researchers found that by down-regulating Ppo activity in potato e.g., see Hakimi et al. 2006, disease 

symptoms of P. infestans were reduced, apparently corresponding to an increase in the plant’s 

resistance to late blight. The authors proposed that tubers may also display enhanced disease resistance 

against certain other fungal pathogens that infect potato tubers, including Rhizoctonia (black scurf), 

Fusarium (dry rot), Spongospora (powdery scab) and Alternaria (early blight). 

Enzymatic browning is an important reaction that occurs in the fruit of the apple (Malus domestica). In a 

study conducted by Valentines et al. (2005), the roles of enzymatic browning and lignification (the 

chemical strengthening of cell walls in response to pathogenic infection) as resistance mechanisms 

against Penicillium expansum were investigated in Golden Delicious apples. A significant increase in 

decay was observed following the treatment of peeled apples with a Ppo substrate which had induced

higher Ppo activity levels. The Ppo enzyme, and particularly the browning process induced by treatment 

with Ppo substrate, may have contributed to increase decay and indicates that an overexpression of Ppo

may lead to higher level of sensitivity towards the pathogen (Valentines et al. 2005).

Some researchers have proposed that enhanced Ppo may increase resistance to disease, while others 

claim that reduced Ppo could also increase resistance. Considering that some evidence exists for a 

relationship between Ppo and diseases, we chose to test the events for several important potato 

diseases and conclude that there is no impact on disease resistance or susceptibility with regards to the 

Ppo5 gene as demonstrated in the remainder of this section.
J.R. Simplot Company JRS01 Page 46 of 74

7.3. Disease Incidence: Storage Studies

Throughout the 2009 cold storage period, pink and soft rot diseases were occasionally observed on both 

untransformed and InnateTM potatoes from the Canyon County, ID, and Bingham County, ID, field trial 

sites. In addition, Pythium leak disease was observed on some untransformed and InnateTM potatoes

from Adams County, WA, and Bingham County, ID, for the 2010 cold storage period. These observed 

disease incidences were attributed to environmental soil conditions that favored infection of tubers and 

the relatively high presence of disease within the specific field trial sites. 

7.4. Disease Incidence: Late Blight and Bacterial Soft Rot

Disease incidence was evaluated by intentionally infecting the events and their untransformed controls 

with the causal agents of late blight (Phytophthora infestans) and soft rot (Erwinia carotovora) and 

evaluating disease progression. Details regarding the following tests are included in Appendix 8. 

Disease Susceptibility.

Late Blight Testing with Tubers. Results of studies from the 2009 field trials showed an increase in late 

blight symptoms in tubers of Russet Burbank event E12 and a decrease in symptoms in Ranger Russet 

event F10 compared with their respective controls. In similar tests with multiple late blight strains

inoculated onto tubers, we found event G11 to be less susceptible, H50 more susceptible, and H37 

variable compared with controls. After seeing these mixed results in a laboratory setting, we conducted 

additional field testing in 2011, using only the Ranger Russet, Russet Burbank and Atlantic varieties. 

Using tubers from the 2011 field season, event E12 was found to be less susceptible to late blight than 

controls. In addition, Atlantic event J3 was found to be more susceptible than Atlantic controls; 

however, J3 was similar to Ranger Russet and Russet Burbank controls. Ranger Russet events F10 and 

F37 did not differ from their controls.

Late Blight Foliage Testing. In a replicated field trial in 2011, potato plants were deliberately infected 

with the causal agent of P. infestans. Of the events in that trial (E12, E24, F10, J3, J55, and J78), the only 

significant difference was that event F10 was more resistant than control. This replicated field trial 

provides evidence that InnateTM events were no more susceptible to late blight than the untransformed 

controls.

Soft Rot Testing with Tubers. A test with tubers in 2009 showed a similar response in controls and all 

events tested. Further testing in 2011 showed that Atlantic events J3 and J55 were less susceptible than 

untransformed controls. Considering both studies, we conclude that the events have similar 

susceptibility to bacterial soft rot as the controls

The studies with late blight and bacterial soft rot confirm that silencing of the target genes did not

enhance susceptibility to these common diseases. As with any new variety development, we would 

want to avoid modifications that enhance disease susceptibility. However, if any events showed slightly 

higher susceptibility to disease, it would not enhance the weediness or result in the creation of plant 

pests.

7.5. Evidence of Trait Efficacy: Reducing Sugars

Tubers contain large amounts of phosphorylated starch, some of which is degraded during storage to 

produce glucose and fructose. These reducing sugars react with amino acids to form Maillard products 
J.R. Simplot Company JRS01 Page 47 of 74

including acrylamide when heated at temperatures above 120OC. Two enzymes involved in starch 

phosphorylation are water dikinase R1 and phosphorylase-L (R1 and PhL). The partial or complete 

silencing of the associated genes in tubers can limit the accumulation of sugars, thus decreasing the 

potential to produce acrylamide.

The amount of reducing sugars was assessed by taking samples from either fresh or stored potatoes

(Appendix 9. Compositional Analyses). Many of the events had lowered levels of reducing sugars either 

at the time of harvest or after storage for 1 month (F10, F37, E12, E24, J3, and J55, J78), however, in 

most cases we did not observe significant differences after 2-5 months of storage. It was not expected 

that J78 would have lowered levels of reducing sugars because it was one of two events, J78 and G11, 

that did not contain the R1/PhL promoter silencing cassette. Tubers of the events G11, H37, and H50

contain the same amount of reducing sugars as tubers of their untransformed counterparts. The 

inability of the silencing construct to limit glucose/fructose formation in H37 and H50 may be due to the 

fact that the H variety is naturally low in glucose and fructose. Thus, we concluded that silencing of the 

promoters associated with the PhL/R1 genes effectively lowered reducing sugars near the time of 

harvest in most events but these differences were not sustained throughout storage for 2-5 months. 

These data indicate that partial silencing of the R1 and PhL genes contributes to lower reducing sugar 

levels and a lowered potential to form acrylamide.

7.6. Evidence of Trait Efficacy: Black Spot Bruise

Two different methods were utilized to measure the incidence of black spot bruise, one by catechol 

assay and the other by physically impacting tubers using a rotating drum (Appendix 7. Black Spot Bruise 

Tolerance). Tubers of events were shown to contain either very low (F37) or undetectable activity levels 

(all other events, except for Atlantic events), as visualized by a catechol assay. Atlantic events were not 

tested because Atlantic controls did not react with the catechol assay and appeared to display some

black spot bruise tolerance. The barrel bruise test indicated undetectable levels of black spot bruise in 

event E12, at least a 5-fold reduction in levels for events F10 and F37, and about 2-fold reduced levels in 

events E24, H37, and H50. The black spot bruise tolerance of event G11 showed variability depending on 

site.

7.7. Evidence of Trait Efficacy: Reduced Asparagine and Acrylamide

Silencing of the asparagine synthetase gene resulted in average reductions of 69 to 78% free asparagine 

in the potato events (Table 8). The lower levels of asparagine which combines with reducing sugars in 

the Maillard reaction to form acrylamide, results in average reductions of 58 to 72% acrylamide in fries 

and chips (Table 8). Along with the reduction in free asparagine, we have observed an increase in free 

glutamine, a direct result of silencing the gene that codes for the asparagine synthetase enzyme that 

converts glutamine to asparagine (Figure 6).
J.R. Simplot Company JRS01 Page 48 of 74

Table 8. Differences in Asparagine and Acrylamide Between Controls and Events

Variety Free Asparagine 

(ppm)

Percent Reduction 

from Control

Acrylamide (ppb)1Percent Reduction 

from Control

Ranger Control 2351 0% 371.9 0%

F10 567.2 76% 150.6 60%

F37 642.5 73% 127.3 66%

Burbank Control 2421 0% 493.8 0%

E12 618.2 74% 162.4 67%

E24 591.9 76% 158.9 68%

Atlantic Control 2268 0% 842.7 0%

J3 516.2 77% 278.7 67%

J55 502.4 78% 233.4 72%

J78 551.1 76% 251.1 70%

G Control 3406 0% 1254.2 0%

G11 794 77% 421.5 66%

H Control 2805 0% 531.2 0%

H37 877.5 69% 223.0 58%

H50 672.7 76% 174.7 67%

1

Before testing for acrylamide, Ranger Russet and Russet Burbank controls and events were made into french fries. 

Atlantic, G, and H varieties were processed into potato chips. All results were from tubers analyzed near the time 

of harvest.

Nitrogen assimilation into amino acids occurs in all organisms and involves numerous genes and 

enzymes. As the primary nitrogen transport compounds within the plant, the amino acids glutamine and 

asparagine play crucial roles for plant growth and development (McGrath and Coruzzi 1991; Urquhart 

and Joy 1981). In senescing leaves, protein hydrolysis produces ammonium (Masclaux-Daubresse et al.

2006). Therefore, toxic ammonium must be immediately re-assimilated into organic molecules through 

nitrogen cycling. First, ammonium is assimilated into the glutamine amide group. Next, glutamine 

transfers to the position of 2-oxoglutarate, yielding two molecules of glutamate caused by the concerted 

reaction of the glutamine synthetase gene (GS). Typically, the nitrogen incorporates into other amides 

and amino acids, including asparagine. The glutamine-dependent enzyme asparagine synthetase (Asn1) 

catalyzes the reaction that converts aspartate to asparagine, transferring the amide nitrogen group, and 

converting glutamine to glutamate. The asparagine biosynthesis pathway is represented in Figure 6. 

Using InnateTM technologies, the Asn1 gene is silenced in potato tubers, thereby allowing glutamine to 

retain its amide group, and to remain as glutamine in the system. This explains the reason lower 

asparagine levels in InnateTM potatoes create a higher level of glutamine. 
J.R. Simplot Company JRS01 Page 49 of 74

Figure 6. Biosynthesis of GLN and ASN in plants

Figure 6 is from New World Encyclopedia, http://www.newworldencyclopedia.org/entry/Asparagine.

7.8. Survival in Fields

It is possible that InnateTM or conventional potatoes will persist in fields for several years after planting. 

In most Northern areas, potatoes are rotated with other crops which minimizes the survival in 

subsequent years and the chance of accidental mixing in subsequent potato plantings. However, 

growers who have planted InnateTM potatoes will be instructed to eliminate any potatoes persisting in 

the field before reverting to conventional potato production. Farmers will also be instructed not to sell 

waste potatoes from InnateTM plantings to small processors unless the trade restrictions with this 

material are clearly stated and the small processors are not engaged in potato export outside of North 

America. 

After conducting field trials with potatoes, we routinely look for potatoes in the field the year following. 

We have not found any potatoes to persist in test plots from 2009 – 2011 trials after monitoring and 

destroying volunteers in 2010, 2011, and 2012 (to date as of this submission) (Summarized in Appendix 

10. Post-Harvest Monitoring). These field trials consisted of small plots that are carefully monitored by 

researchers and represent a well-controlled environment. In large tracts of land typical of commercial 

potato growing operations, there might be a greater likelihood that potatoes could be overlooked. If 

potatoes were grown repeatedly year after year on the same ground, it would be more likely for 

contamination of a subsequent potato crop to occur. Extra precautions may need to be considered in 

those instances.

7.9. Conclusion on Agronomic Performance, Disease, and Efficacy of Traits

Transformation of potato with the DNA insert of pSIM1278 did not alter the agronomic characteristics of 

potatoes, and yielded the expected changes of reduced back spot bruise, lower levels of reducing sugars 

and reduced acrylamide potential (see Table 9). Reducing sugars were lowered slightly in most events, 

and would not be expected to be diminished in the events that did not contain the silencing construct. 

Significantly reduced asparagine contributed to reduced levels of acrylamide in fries and chips. The 

studies with late blight and bacterial soft rot confirm that silencing of the target genes did not enhance 
J.R. Simplot Company JRS01 Page 50 of 74

susceptibility to these common diseases. However, if any events showed slightly higher susceptibility to 

disease, it would not enhance the weediness or result in the creation of plant pests.

Table 9. Traits Incorporated into InnateTM Potatoes

Event Reduced 

Acrylamide 

Potential

Black Spot Bruise Tolerance Reduced Accumulation of Glucose and 

Fructose

F10 Yes Yes Yes

F37 Yes Yes Yes

E12 Yes Yes Yes

E24 Yes Yes Yes

J3 Yes PPO assay indicated some resistance in

the Original Variety

Yes

J55 Yes PPO assay indicated some resistance in 

the Original Variety

Yes

J78 Yes PPO assay indicated some resistance in 

the Original Variety

Yes

G11 Yes Yes ( Variable depending on site) No

H37 Yes Yes No

H50 Yes Yes No

Although some significant agronomic differences were observed between events and controls, most fell 

within the combined control range of the conventional varieties. For example, some differences in yield 

and tuber size were observed in events, and these attributes will be investigated further in commercial 

trials. Thus, we concluded that no biologically meaningful differences that would contribute to 

increased weediness or pest potential were observed for any of the agronomic characteristics, yield and 

grading characteristics, or ecological interactions (namely plant-insect interactions, plant-disease 

interactions, and plant interactions with abiotic stressors) of the events compared to their 

untransformed controls. It can be concluded that events are not different from their untransformed 

controls in terms of agronomy, tuber yield, and tuber composition. Therefore, the transformation of 

potato with pSIM1278 does not introduce characteristics that will encourage or enhance weediness or 

pest potential.

8. Compositional assessment

The compositional assessment studies, evaluating proximates, vitamins, amino acids, minerals, sugars, 

and glycoalkaloids, were conducted on InnateTM potatoes to 1) show equivalence to the untransformed 

controls, 2) compare the InnateTM potatoes to literature ranges, where applicable; and 3) show that 

there were no changes in potential toxins. Ultimately, results of the composition studies showed food 

safety equivalence between the events and the untransformed controls.

8.1. Proximates, Vitamins, Minerals, and Glycoalkaloids

These analyses were conducted to confirm that composition of events remained within the normal 

levels for potato and would have equivalent food quality, feed quality, and safety when compared to 

untransformed potatoes (Appendix 9. Compositional Analyses). We also tested for the effectiveness of 

gene silencing and expected to find lower amounts of asparagine and, in most cases, lower amounts of 

glucose and fructose compared to untransformed controls. The compositional assessments determined 

the amounts of 1) moisture, protein, total fat, ash, crude fiber, carbohydrate, and calories; 2) vitamins 
J.R. Simplot Company JRS01 Page 51 of 74

B6, B3, and C; 3) minerals copper, magnesium, and potassium; 4) glycoalkaloids; 5) free amino acids; 6) 

total amino acids; and 7) reducing sugars for tubers collected from events grown in potato-growing 

areas of the United States. For all the nutrients listed above, the goal was to demonstrate that events

contained equivalent amounts compared with the untransformed controls. Along with agronomic data, 

the compositional analyses helped determine whether the InnateTM potatoes exhibited compositional 

changes that might impact plant pest risk or food and feed safety. 

Statistical analyses of nutrient composition were conducted to test for differences in the analyte mean 

values between each of the events and their untransformed controls. A complete description of 

statistical methods can be found in Appendix 9. Compositional Analyses. Either the P-value or adjusted 

P-values are provided, whereby differences < 0.05 were considered statistically significant. Further 

context for data interpretation was provided through the use of tolerance intervals and published 

literature. 

The following varieties, all grown in the various field trial sites, were used to calculate tolerance 

intervals: Snowden, Chieftain, Red Norland, Ida Rose, Russet Burbank, Ranger Russet, Atlantic, G, and H. 

The tolerance intervals were calculated to contain, with 95% confidence, 99% of the values contained in 

the population. This statistical tolerance interval and the combined range of values for each analyte 

available from the published literature were used to interpret the composition results. In interpreting 

the data, emphasis was placed on the analyte means; means that fell within the tolerance interval 

and/or combined literature range for the analyte were considered to be within the normal variability of 

commercial potato varieties.

Results of all significant differences between events and controls are summarized in Table 10 for Ranger 

Russet and Russet Burbank and in Table 11 for Atlantic, G, and H varieties. Most of these differences; 

reduced free ASN and total ASP + ASN, increased free GLN and total GLU + GLN, reduced acrylamide, 

and lowered reducing sugars were expected because of the intended gene silencing. Other significant 

differences included decreased free VAL (J3, J55), decreased free LYS (H37), increased free ARG (F37), 

increased total LYS (G11), increased total PRO (G11), increased Vitamin C (F10, F37), increased niacin 

(F10, F37), decreased pyridoxine (J3, J55), decreased sucrose (E12, E24), and increased sucrose (H37).

Both the intended changes and these observed differences fell within the tolerance intervals or

combined literature values, and therefore were considered to be within normal ranges for potatoes.

It’s important to note that for events E12 and E24, the free ASN levels were not statistically different

from controls at the adjusted p < .05 level. However, these events showed a 74 to 76% reduction in free 

ASN. Such large differences were sometimes not statistically significant, which was a direct result of 

using the correction for False Discovery Rate (Benjamini and Hochberg 1995). These reductions were of 

practical significance and are considered similarly to the other events regarding this trait. 

Note that free amino acids would not normally be part of a compositional assessment, however, they 

were included because the quantity of free asparagine is important to show the efficacy of silencing the 

Asn1 gene for the purpose of reducing acrylamide. Many of the free amino acids are found at such low 

levels in potatoes that values barely exceed the detection limit. For the purpose of assessing the 

nutritional equivalence of the events, we recommend that the focus should be on comparing total 

amino acids to controls. 

The most important changes between InnateTM tubers and their untransformed controls relates to 

reduction of the amino acid asparagine (ASN) and reducing sugars. Reduced amounts of free ASN and 
J.R. Simplot Company JRS01 Page 52 of 74

reducing sugars in the InnateTM potatoes resulted in lowered acrylamide after heat processing compared 

to the conventional potatoes (see Tables 10 and 11 as well as Appendix 9. Compositional Analyses). 

Table 10. Statistical Differences in Composition Data: Events F10, F37, E12 and E24

Variety Event Attribute Difference 

Expected?

Comments

Ranger Russet F10 Decreased Free ASN Yes

Increased Free GLN Yes

Decreased Total ASP+ASN Yes

Increased Total GLU+GLN Yes

Decreased Reducing Sugars Yes Glucose and Fructose

Reduced Acrylamide Yes

Increased Niacin (B3) No Within TI and CLR

Increased Vitamin C No Within TI and CLR

Ranger Russet F37 Decreased Free ASN Yes

Increased free ASP Yes

Increased Free GLN Yes

Decreased Total ASP+ASN Yes

Increased Total GLU+GLN Yes

Decreased Reducing Sugars Yes Glucose and Fructose

Reduced Acrylamide Yes

Increased Vitamin C No Within TI and CLR

Increased Niacin (B3) No Within TI and CLR

Increased free ARG No Within TI and CLR

Russet Burbank E12 Decreased Free ASN Yes 74% reduced, not significant

Decreased Total ASN+ASP Yes

Increased Total GLU+GLN Yes

Decreased Reducing Sugars Yes Glucose and Fructose

Reduced Acrylamide Yes

Decreased Sucrose

Reduced Acrylamide

No

Yes

Within TI and CLR

Russet Burbank E24 Decreased Free ASN Yes 76% reduced, not significant

Decreased Total ASP+ASN Yes

Increased Total GLU+GLN Yes

Decreased Reducing Sugars

Decreased Sucrose

Reduced Acrylamide

Yes

No

Yes

Glucose and Fructose

Reduced Acrylamide Yes Within TI and CLR

Decreased Sucrose

Reduced Acrylamide

No

Yes

Within TI and CLR

TI = tolerance interval

CLR = combined literature range
J.R. Simplot Company JRS01 Page 53 of 74

Table 11. Statistical Differences in Composition Data: Events J3, J55, J78, G11, H37 and H50

Variety Event Attribute Difference 

Expected?

Comments

Atlantic J3 Decreased Free ASN Yes

Decreased Reducing Sugars Yes Glucose and Fructose

Reduced Acrylamide Yes

Decreased Pyridoxine (B6) No Within TI and CLR

Decreased Free VAL No Within TI and CLR

Atlantic J55 Decreased Free ASN Yes

Decreased Reducing Sugars Yes Glucose and Fructose

Reduced Acrylamide Yes

Decreased Pyridoxine (B6) No Within TI and CLR

Decreased Free VAL No Within TI and CLR

Atlantic J78 Decreased Free ASN Yes Adjusted P = .0548

Decreased Reducing Sugars No Glucose and Fructose

Reduced Acrylamide Yes

Variety G G11 Decreased Free ASN Yes

Increased Free GLN Yes

Decreased Total ASP+ASN Yes

Increased Total GLU+GLN Yes

Reduced Acrylamide Yes

Increased Total LYS No Within TI and CLR

Increased Total PRO No Within TI and CLR

Variety H H37 Decreased Free ASN Yes

Increased Free GLN Yes

Decreased Total ASP+ASN Yes

Increased Total GLU+GLN Yes

Increased Sucrose No Within TI and CLR

Reduced Acrylamide Yes

Decreased Free LYS No Within TI and CLR

Variety H H50 Decreased Free ASN Yes

Increased Free GLN Yes

Decreased Total ASP+ASN Yes

Increased Total GLU+GLN Yes

Reduced Acrylamide Yes

TI = tolerance interval

CLR = combined literature range

For the compositional assessment, all events were compared with their respective controls mostly at the 

time of harvest. In addition, the potatoes were tested for reducing sugars and acrylamide, following 

storage (Appendix 9. Compositional Analyses, Storage Studies: Reducing Sugars and Acrylamide). 

Many of the events had lowered levels of reducing sugars either at the time of harvest or after storage 

for 1 month (F10, F37, E12, E24, J3, and J55, J78), however, in most cases we did not observe significant 

differences after 2 – 5 months of storage. Thus, we concluded that silencing of the promoters 
J.R. Simplot Company JRS01 Page 54 of 74

associated with the PhL/R1 genes effectively lowered reducing sugars near the time of harvest but these 

differences were not sustained throughout storage for 2-5 months.

During storage of up to 7 months, acrylamide levels in most events were consistently lower (p < .0001) 

than controls, although there were exceptions with the G and H varieties. In event G11, we observed a 

mean of 2,050 ppb compared with 1,769 ppb for the control after 1 month of storage. This was unusual 

and throughout the rest of the study, acrylamide levels in event G11 were always much lower than 

controls (46 to 58% reduction at months 5 and 7). For variety H, acrylamide levels were always lower in 

events H37 or H50, but not statistically significant in months 5 and 7. Overall, the modifications from 

insertion of pSIM1278 resulted in dramatic reductions in acrylamide that persisted throughout the 

typical storage periods. 

This research also confirmed that the events are as safe as controls and did not contain increased levels 

of natural glycoalkaloids, which are toxins commonly found in Solanaceous crops, including potato. The 

principal glycoalkaloids in potatoes are -solanine and -chaconine. For food safety purposes, an upper 

limit for glycoalkaloid content of 20mg per 100g of potato is generally accepted.

The InnateTM potato events F10, F37, E12, E24, J3, J55, J78, H37, H50 and their corresponding 

untransformed controls had mean glycoalkaloid ranges from 5.7 to 11.3 mg/ 100g (Table 12); all below 

the 20mg/ 100g safety limit described by Sinden (1987). For one event, E24, the range of values 

extended to 33.81, beyond the desired limit of 20 mg/ 100 g. Although a cause was not identified, this 

could be from mis-handling and excessive exposure to light (Percival 1999). Since the mean value was 

close to the control, we would not expect that glycoalkaloid production was related to the modification. 

The InnateTM potato event G11 had mean glycoalkaloids of 20.3 mg/100g and untransformed controls 

contained 19.8 mg/100g (Table 12). The higher mean levels of glycoalkaloids in the G event and 

untransformed controls is attributed to additional handling at the storage sites and thus, increased 

exposure to light and temperature flux (Percival 1999). Based on the lack of differences between the 

events and controls, there would not be a safety issue as a result of the introduction of the reduced 

acrylamide potential and low black spot bruise traits in InnateTM potatoes. All glycoalkaloid levels were 

within the normal ranges for potato, when compared with the tolerance intervals or combined literature 

ranges.
J.R. Simplot Company JRS01 Page 55 of 74

Table 12. Glycoalkaloids (mg/100 g)1 in Ranger Russet, Russet Burbank, Atlantic, G, and H Varieties 

Variety Total Glycoalkaloids (mg/100 g)

Mean Range P-Value TI CLR2

Ranger Control 6.704 3.06-10.23

0.000-33.05 3.20-210.4 F10 6.979 2.84-9.51 0.8285

F37 6.805 4.28-9.42 0.9812

Burbank Control 7.404 2.74-18.59

0.000-33.05 3.20-210.4 E12 5.678 1.69-10.53 0.1707

E24 6.845 1.45-33.81 0.8174

Atlantic Control 6.274 2.09-11.40

0.000-33.05 3.20-210.4 J3 5.723 1.62-11.67 0.2393

J55 5.864 1.83-11.12 0.4833

J78 6.084 1.65-14.27 0.7198

G Control 19.82 6.17-44.40

2.830-94.46 2.20-154.20

G11 20.32 5.81-55.3 0.9153

H Control 10.65 5.96-21.07

H37 10.76 5.41-17.25 1.470- 303.5 2.20-154.2 0.8951

H50 11.30 6.23-22.81 0.4379

1

TI = Tolerance Interval, CLR = Combined Literature Range. P-values indicating significant differences with controls would be

underlined.

2

Literature ranges from Kozukue et al. (2008). 

We would not expect to find novel amino acid sequences or proteins in the events because the 

expression cassettes consist only of non-coding DNA. There are two expression cassettes in the 

transferred DNA insert. One comprises an inverted repeat of fragments of asparagine synthetase-1 gene 

and the polyphenol oxidase-5 gene between the Agp and Gbss promoters. The other one is comprised of 

fragments of the promoter of the starch associated gene (R1) and the phosphorylase-L gene linked to 

the same Agp and Gbss promoters as the first cassette. The pool of mRNA produced by these two 

cassettes from either a fragment of promoters or a part of genes is unlikely to be further processed to 

form any amino acids or proteins. Instead, all mRNA produced by the DNA insert is processed using RNAi 

machinery in plant cells to silence the targeted genes: destroy mRNA of the targeted genes (Asn1, PPO) 

and prevent translation to form active gene products such as a protein. Therefore no changes to amino 

acid sequences or proteins are found with InnateTM potatoes. 

8.2. Allergenicity of InnateTM Potatoes

The potato events described in this petition have the same composition compared to untransformed 

controls. Therefore, issues with allergies would not be expected above what individuals with potato 

allergies would normally have. Some allergies have been detected in children as a result of patatin 

contained in potatoes.

Patatin is a storage glycoprotein that displays lipase activity and makes up about 40% of the soluble 

protein in tubers (Mignery et al. 1988). Allergic reactions to processed potatoes are considered to be 
J.R. Simplot Company JRS01 Page 56 of 74

very uncommon and have been reported for children only (De Swert et al. 2002; De Swert et al. 2007), 

with patatin (Sol t 1) identified as the major allergen involved in this reaction (Astwood et al. 2000).

Because potato protein naturally contains a relatively large proportion of patatin, those who are allergic 

to potatoes would avoid them and changes to patatin levels would not alter that behavior.

8.3. Conclusions of Compositional Assessment

The intentional changes in ASN and reducing sugars accomplished through transformation of various 

potato varieties with the DNA insert of pSIM1278 resulted in lower acrylamide levels in french fries and 

potato chips. These modifications did not alter the quality of potato as food because (1) altered ASN and 

GLN levels are still within the normal range for potato, and (2) any impact on nutritional quality would 

be minimal from the observed differences in the levels of ASN, ASP, GLU and GLN because as nonessential amino acids they can be synthesized by the body. Unintentional changes in the amounts of 

specific amino acids, vitamin C, pyridoxine and vitamin B3 were minor and inconsistent among varieties, 

while resulting in levels that fell within normal ranges for potato. While one of the varieties (G) had 

higher levels of glycoalkaloids than expected, the events were no different from untransformed controls

and were within the literature ranges reported for potato varieties. Therefore, it can be concluded that

tubers from the events are as safe for consumption in food and feed as the untransformed controls. 

9. Environmental Assessment of Introduction

After a thorough battery of studies to investigate the weediness potential and food and feed safety of 

InnateTM potatoes, the evidence submitted demonstrates that the InnateTM technologies used to 

introduce native traits is unlikely to harm humans, animals, or the environment.

The results of the composition analyses as described in Appendix 9. Compositional Analyses show 

overwhelming evidence of a lack of differences between InnateTM potatoes and the untransformed 

controls when tested for proximates, minerals, and glycoalkaloids.

The agronomic trials confirmed that the InnateTM potatoes had similar phenotypes compared with the 

untransformed controls when grown at multiple locations representing the major areas for potato 

production in the US. Observations throughout the growing season allowed for a thorough assessment 

of growth, ecological interactions including potential altered susceptibility to insects and diseases, and 

measurements of tuber yield and quality. The field assessments aided in selection of the most viable 

plants that contained the intentionally incorporated new traits and maintained all benefits of the 

conventional parent varieties (from among a larger number of events). Additionally, the field studies 

were used to ensure that the transformations had not resulted in the introduction of unintended effects 

associated with weediness or increased pest potential. 

After completing the agronomic trials, potato tubers were tested for evidence of the efficacy of the 

traits, including an assessment for black spot bruise and reducing sugars. In addition, after processing 

into french fries or potato chips, the potatoes were tested for acrylamide. 

Proper assessment of a biotech crop includes testing that identifies any potential negative impact on the 

environment. The concept of familiarity has been widely used to describe the interactions that a 

transformed plant may have in nature by considering the biology of the crop, the introduced trait, the 

receiving environment, and the interactions with all of these factors (Hokanson et al. 1999). This 

concept allows regulatory decision-makers to draw upon past experience with introduction of plants 
J.R. Simplot Company JRS01 Page 57 of 74

into the environment and compare genetically engineered plants to their non-engineered counterparts. 

In the case of InnateTM potatoes, all of these factors were considered with respect to familiarity. Beyond 

the environmental factors it is also important to consider the utility of the trait. In this case, the trait 

reduces the level of acrylamide after frying, which can have a positive effect on human health while 

maintaining the typical characteristics of a potato e.g., not changing farming practices or preparation of 

potatoes for human consumption.

9.1. Potential for Gene Transfer and Outcrossing

As previously discussed, the risks associated with accidental gene transfer in potatoes through 

outcrossing would be minimal for several reasons: 1) potatoes are almost always clonally propagated 

using “seed potatoes”; 2) a high percentage of fertile potatoes will be self-pollinated and are not 

frequented by honeybees due to a lack of nectar; 3) pollen transfer tends to be limited to about 20 

meters which could be controlled easily in commercial fields; and 4) it is unlikely that true InnateTM seeds 

would grow into mature potatoes since potato seeds are not saved and propagated in the typical 

farming operation. 

In the unlikely event that outcrossing was to occur between InnateTM and untransformed plants in the 

field, the impact would be negligible to future plantings since tubers rather than seed are removed from 

the field. If seedlings did arise from an outcrossing event, establishment would be nearly impossible 

since potatoes are grown in rotation and are poor competitors compared to other crop and weed 

species (Love 1994; CFIA 1996). If seedlings were to establish after harvest, they would be easily 

identified and eliminated as part of the standard agricultural practices in potato production. In either 

case, seedlings could be identified and eliminated to prevent them from entering the commercial 

stream.

In the unlikely case of growing TPS seed near the commercial InnateTM fields, a separation of 20 meters 

would be required to avoid outcrossing with potato events (Conner and Dale 1996). 

9.2. Inter-Species Pollination and Hybridization Potential

The tetraploid species Solanum fendleri and diploid Solanum jamesii are the only wild species that grow 

within the borders of the United States (Bamberg et al. 2003; Spooner et al. 2004). These species mostly 

occur outside of the major potato growing regions in Texas, New Mexico, Utah, Colorado, and Arizona

(see Figure 2). These species reside in dry forests, scrub desert, and sandy areas at altitudes of 5,000 to 

10,000 feet, well isolated from most commercial production areas (Bamberg and del Rio 2011a). 

Therefore, pollen flow from commercial events to wild species is extremely unlikely simply because of 

the geographic location. 

All Texas records in the IPD database are from the far western part of the state in El Paso, Culbertson 

and Jeff Davis Counties, where potatoes are not grown commercially. The two S. fendleri and one of the 

S. jamesii records are from the Davis Mountains, and one of the three S. jamesii entries is from 

Guadalupe National Park (IPD 2011; National Atlas 2004b; Hall et al. 2000). 

The Colorado records in the IPD database are from mountainous locations very near the New Mexico 

border. Spooner et al. (2004) report that records of S. jamesii from natural habitats in Colorado are 

limited to the extreme southwestern and southeastern parts of the state. The San Luis Valley, a major 
J.R. Simplot Company JRS01 Page 58 of 74

potato production area, lies between the mountainous regions where the wild potatoes were found 

(Bamberg et al. 2003; National Atlas 2004a; Spooner et al. 2004). 

While there appears to be minimal, if any, overlap geographically between cultivated and wild potatoes 

in the U.S., there is a possibility that a few wild potato plants may be growing near potato fields (Love 

1994). Spooner et al. (2004) describe S. jamesii habitat in the U.S. as among boulders on hillsides, sandy 

alluvial stream bottoms, in gravel along trails or roadways, rich organic soil of alluvial valleys, sandy 

fallow fields, grasslands, juniper-pinyon scrub deserts, oak thicket, coniferous and deciduous forests at 

elevations between 4,500 to 9,400 feet. They describe S. fendleri habitat similarly, and at elevations 

between 4,700 to 11,200 feet.

Based on conversations with Bamberg, Love (1994) reported that no one has ever reported finding 

hybrids between native and cultivated potatoes in the U.S., although gene transfer has been 

accomplished using special laboratory techniques (Love 1994). Love concluded that, based on the 

barriers that exist, including geographic isolation (as described above), endosperm imbalances, and 

multiple ploidy levels, natural hybridization is highly unlikely, and introgression (cross hybridization over 

multiple generations) impossible or at least highly improbable (Love 1994). More recently, the US EPA 

has concluded that, based on its review of the scientific literature, successful gene introgression 

between native and cultivated potatoes in the U.S. is virtually excluded due to constraints of 

geographical isolation and other barriers to natural hybridization. These barriers include incompatible 

(unequal) endosperm balance numbers that lead to endosperm failure and embryo abortion, multiple 

ploidy levels and incompatible mechanisms that do not express reciprocal genes to allow fertilization to 

proceed. No natural hybrids have been observed between these species and cultivated potatoes in the 

U.S. (US EPA 2011). 

9.3. Weediness Potential

As previously described, weediness is a term used to describe the ability of a plant to become a weed 

(survive and thrive) outside of cultivation. The InnateTM modifications described in this petition are 

unlikely to increase the weediness of potato because the incorporated traits (reduced free-asparagine, 

black spot bruise tolerance, and reduced sugars) are not associated with weediness and will not help the 

plants thrive outside of cultivation. Multiple field trials of 10 events did not provide any evidence for 

altered growth characteristics such as accelerated tuber sprouting, increased plant vigor, increased 

tuber set, and delayed senescence or other key agronomic characteristics associated with survival 

outside of cultivation. Additionally, all fields were monitored for 2 years after study completion for 

volunteer activity as required by USDA compliance. Through 3 years of field studies and up-to-date 

volunteer monitoring of every site, no differences have been observed that would lead us to believe that 

the events have properties that would increase the survivability compared to conventional potatoes

(see Appendix 10. Post-Harvest Monitoring). Due to modern agricultural practices it is highly unlikely 

that potatoes would persist in a field from one crop cycle to the next, particularly since potatoes are 

typically grown as a rotational crop. 

9.4. Endangered Species Considerations and Impact on Biodiversity

Endangered Species 

Based on an assessment of threatened and endangered species in potato cropping systems, and 

considering the safety and compositional data of InnateTM potatoes, it is highly unlikely that there will be 

any effect on endangered species, the environment, or non-target organisms. Since these potatoes 
J.R. Simplot Company JRS01 Page 59 of 74

introduce nothing new to the environment, e.g., no new proteins or genes, they would have the same 

effect on endangered species as any other potatoes. InnateTM varieties are likely to be planted in areas 

that are already growing potatoes, and would not result in a significant expansion of acres. 

Natural Biodiversity 

The modifications described in this petition were intended to enhance the quality, not agronomic 

characteristics, of potato by reducing the expression of four native genes. Thus, we reduced the plant’s 

ability to develop black spot bruise, store asparagine, and convert starch to sugars. Because no new 

pesticides are expressed, and no natural defense mechanisms have been activated, the modified 

potatoes have no new properties to enhance their weediness or pest resistance. The incidence of 

disease symptoms identified during growth and storage were similar for InnateTM and untransformed 

control potatoes. The InnateTM plants show no tendency to be more invasive. Invasiveness is not a trait 

associated with any potatoes and therefore not associated with silencing the genes affecting potato 

quality. These changes in potato quality have no more impact on biodiversity and endangered species 

than any other commercial potato varieties.

9.5. Effects on Current Agronomic Practices in Potato

Field trials with multiple potato events over three different crop years have indicated that no differences 

will be needed in the agronomic practices used to grow the InnateTM potatoes. The incorporated traits 

are not expected to affect agronomic practices. All events considered for this submission were selected 

to exhibit similar phenotypic characteristics to the control varieties when grown using standard industry 

practice. There were no differences in disease susceptibility that would require additional treatments 

nor did the potatoes have different nutrient requirements that would alter fertilizer programs. Planting, 

cultivation, management and harvesting processes were not affected by the incorporated traits. (see 

Table 3 for a typical agronomic input scenario in the Northwestern United States).

9.6. Unintended or Non-target Effects

The lack of insecticidal traits in InnateTM potatoes would mean that all insects would be considered nontarget. There are a large number of insects that feed on potato leaves and other insects that feed on 

pests. Many of these insects, including the Colorado potato beetle, potato aphid, European corn borer, 

potato leafhopper, and potato psyllid are considered pests. The events have not been modified to 

display any new pesticidal activities and do not contain new pesticide-expressing genes. No differences 

were observed in the field trials for insects or other animals interacting within the potato ecosystem 

proving a lack of pest potential to other organisms. The only modifications concerned the intended 

effects of reduced transcript levels for four quality-associated genes, mainly in tubers and stolons. 

During the three seasons of field releases, the observations and measurements showed no differences 

in ecological interactions (namely plant-insect interactions, plant-disease interactions, and plant 

interactions with abiotic stressors) between the events and the control varieties (Appendix 6. Field 

Performance and Tuber Evaluations), supporting an absence of unintended effects. 

9.7. Summary of the Environmental Assessment of Introduction

The modifications described in this petition were intended to enhance the quality, not agronomic 

characteristics, of potato by inactivating four native genes. Planting, cultivation, management and 

harvesting techniques were not affected by the incorporated traits. The InnateTM varieties are likely to be 

planted in areas that are already growing potatoes, and would not result in a significant expansion of 
J.R. Simplot Company JRS01 Page 60 of 74

acres. The modification described in this petition is highly unlikely to increase the weediness or 

invasiveness of potato because the incorporated traits (reduced free-asparagine, black spot bruise 

tolerance, and reduced sugars) do not play a role in survival, dispersal, or ecological competitiveness. 

Field trials over multiple years with 10 events did not provide any evidence for altered growth 

characteristics such as accelerated tuber sprouting, increased plant vigor, increased tuber set, or 

delayed senescence. No new pesticides are expressed, and no natural defense mechanisms activated, 

and thus modified potatoes are unlikely to display enhanced weediness or pesticidal properties. 

Therefore, the potential to impact insects and other non-target organisms, weed or disease 

susceptibility, endangered species or biodiversity is negligible for InnateTM potatoes.

The data presented in this petition demonstrate that introduction of InnateTM potatoes will have a 

similar environmental impact when compared to untransformed potatoes. Use of InnateTM technologies

and the resultant transformed plants, and their tuber products, satisfy the statutory requirements for 

nonregulated status in that our transformants have no pesticidal traits and are not plant pests. To 

conclude, we feel that the plants containing these traits, are not plant pests and have resulted from the 

addition of genetic material from a donor plant where the integrated genetic material contains only 

non-coding regulatory regions, that are well characterized, and are as safe as untransformed potatoes. 

The evidence presented in this submission demonstrates that the deregulation and cultivation of 

InnateTM potatoes is highly unlikely to cause any adverse environmental or biological impacts as a result 

of their cultivation.

10. Crop Introduction and the Potato Industry

10.1. Industry Need for Potatoes with Reduced Acrylamide Potential

Based on a recent notice (Federal Register 2009), FDA is considering issuing guidance for industry on the 

reduction of acrylamide levels in food products. The introduction of InnateTM potatoes with low 

acrylamide potential would provide potatoes that are largely indistinguishable from existing varieties. 

The reduction in asparagine and sugars using InnateTM technologies and the resulting reduction in 

acrylamide upon heating will address industry needs with respect to the proposed FDA guidance. 

In addition, recent litigation in the state of California resulted in legal settlements with restaurant 

chains, and the retail french fry and potato chip manufacturers regarding acrylamide. The settlements 

required manufacturers of retail fries and potato chips to reduce the level of acrylamide in their 

products. Potato processors affected by these rulings in California could be motivated to adopt the low 

acrylamide, InnateTM potato events.

The low acrylamide potatoes will provide a safer option for all sectors of the potato processing market 

and thus should increase the demand for these InnateTM potatoes. The benefits resulting from the 

modifications could result in increased market share, particularly for Ranger Russet and Atlantic. The 

reduction of sugars and black spot bruise in Ranger Russet may increase the popularity of that variety. 
J.R. Simplot Company JRS01 Page 61 of 74

10.2. Potato uses and exports

Potatoes are grown commercially in 36 of the 50 states in the US. Total US production in 2010 was 40.4

billion lbs, with total value of $3.72 billion, planted on 1.03 million acres (NPC 2012). The commercial 

uses include 37% as frozen, 27% sold fresh, 14% chips, 8% dehydrated, 5% seed potatoes, and 1% 

canned (NPC 2012). Annual per capita consumption in the US was expected to be approximately 112 lbs 

per person in 2012 (NPC 2012). 

The states with the largest volume and sales value in 2009 were Idaho, Washington, Wisconsin, 

Colorado, Oregon, Minnesota, North Dakota, Michigan, Maine, and California (USDA-NASS 2010b). 

These data represent the combined markets including all processing and fresh varieties. 

During the 2009/10 marketing year (September-August), US exports of all potatoes and potato products 

(including starch) totaled $1.19 billion—essentially unchanged from a year earlier (USDA ERS 2010). 

Japan remained the top foreign market for U.S. potatoes with 28% of the total export value; primarily 

frozen french fries, other frozen potato products, potato flakes and granules. Japan was followed by 

Canada (24 % of export value), Mexico (11 %), South Korea (5 %), and China (4 %).

Potato exports from the U.S. in 2010 totaled 5.7 billion lbs (farm weight of potatoes) and 812 million lbs 

of fresh potatoes. Most of the processed potato exports were frozen, representing 51.4% of total farm 

weight production (USDA ERS 2010).

10.3. Submissions to Other Regulatory Agencies

Approval for these events will be sought in Canada, Japan, Mexico, and the US. These approvals should 

ensure continued trade with key export markets.

10.4. Impact on the Organic Market

Organic potatoes in 2008 represented less than 1.0% (0.7%) of total potato production with 8,273 acres 

of certified organic potatoes (USDA ERS 2011). Of the 8,273 organic acres, California, Washington, and 

Oregon were the top three organic potato producers with 3,697 acres, 1,600 acres, and 1,101 acres, 

respectively. Potato varietal selection and breeding are similar between organic and conventional 

potato production as commercial potatoes are predominantly planted from tuber seed. Risks to organic 

growers would be most likely to occur with accidental mixing of planting material or of potatoes in 

farming, transportation, or processing channels. These risks are the same as those that organic growers 

already experience when keeping their organically grown potatoes separate from conventionally grown 

potatoes. Because potatoes are clonally propagated, the risk of contaminating seed supplies through 

cross-pollination is negligible. Organic farmers routinely plant organic tuber seed material and any 

incidence of cross-pollination in production fields will not affect the harvested potatoes. Producers of 

organic true potato seed will be able to protect their seed from InnateTM material by ensuring that the 

seed production fields are sufficiently isolated from InnateTM potato fields. Potential outcrossing can be 

prevented by separating fields planted for organic TPS production by at least 20 meters from fields 

planted with InnateTM potatoes (Conner and Dale 1996). 
J.R. Simplot Company JRS01 Page 62 of 74

10.5. History of Biotech Potatoes

A review of the history of the introduction of genetically modified potatoes in 1995 in the U.S. (Thornton 

2008) indicates the importance of a careful assessment of the performance of InnateTM events, 

synchronizing approvals in North American and trading countries, and weathering public acceptance 

issues that may result from activism against food industry members that adopt the new technology. 

This history points to the importance of consumer acceptance when introducing any InnateTM crops and 

products to the market. Since the time when biotech potatoes were on the market briefly, the adoption 

of biotechnology in the food supply has increased consistently. Herbicide tolerant soybeans have 

increased from 54% to 93% of the U.S. soybean acreage (USDA-NASS 2010a) since 2000. Genetically 

modified (GM) corn production has increased from 25% of U.S. corn acreage in 2000 to 86 % of corn 

acreage in 2010 (USDA-NASS 2010a). These two crops provide significant quantities of GM-derived 

ingredients in widely marketed processed food and feed products, such as protein, oils, starches, and 

sweeteners. Many of the conventional processed potato products made from the events presented in 

this petition already contain one or more of these GM ingredients. Importantly, the low acrylamide 

potato events contain a new trait that has value to the consumer. The growing interest in improving 

food safety by reducing acrylamide in processed potatoes is expected to provide an incentive for 

enhanced consumer acceptance of these new events and the products derived from them. 

10.6. Stewardship of InnateTM Potatoes

Stewardship of Potato Events

Throughout development of the InnateTM potatoes, the Biotechnology Quality Management System 

(USDA-BRS Compliance assistance program) has served as a stewardship program to conduct field trials, 

monitor movement, and complete testing covering the duration of the regulated status of InnateTM 

potatoes. As a part of BQMS, qualified and experienced potato growers were identified, and agreements 

were established to ensure compliance and conformance requirements for growing InnateTM

potatoes. Processes were developed that included Standard Operating Procedures (SOPs) and forms 

for all critical control points to guide internal and external collaborators to conduct activities using 

InnateTM potatoes. 

The following management procedures were included for effective implementation of BQMS:

1. Document and record control

2. Resource management

3. Training external and internal associates

4. Planning and process realization

5. Conducting internal audits

6. Implement corrective / preventive actions

7. Continuous improvement of the system

8. Resolution of compliance issues

Targeted training programs were used to educate and bring awareness on handling InnateTM potatoes in 

storage, transport, planting, harvest, post-harvest, processing, and final disposition to ensure no 

InnateTM potato material entered into the food or feed channels prior to de-regulation. Both internal and 

external audits were used to verify that planned activities occurred and personnel were adhering to the 

protocols recommended by BQMS. This stewardship program includes corrective and preventive actions 
J.R. Simplot Company JRS01 Page 63 of 74

to avoid compliance and conformance incidents. While BQMS served as a quality management system 

during product development, we anticipate a full extension of such methods as outlined in the 

‘Excellence Through Stewardship®’ (BIO 2007) program.

Identity Preservation 

To help prevent the trade disruptions experienced with other potato events, international approvals will 

be pursued from key trading partner countries before the InnateTM varieties are launched commercially. 

The initial InnateTM introduction will build up slowly as seed becomes available and will be controlled 

within existing processing channels to ensure that potatoes enter only the intended markets. This will 

provide an extended period of time to assess consumer acceptance and to address grower and industry 

awareness. A limited introduction in a vertically integrated supply chain will be well controlled by 

grower and processor agreements. In this situation, conventional products will be considered “identity 

preserved” with respect to the well-controlled stewardship of the InnateTM crop and its products. As

InnateTM potato adoption increases, programs for identity preservation will be implemented as needed. 

It’s expected that development and implementation of identity preservation systems will add some cost

to the supply chain. The total costs will depend upon the type and extent of market penetration. 

Synchronized Regulatory Approvals

We intend to follow the recommended stewardship policy statement released by the Biotechnology 

Industry Organization in May 2007. 

“To help ensure the continued adoption of agricultural biotechnology globally and to continue to have 

products of agricultural biotechnology bring value to the marketplace, BIO’s Food and Agriculture 

Section supports actions that facilitate the flow of goods in commerce and minimize trade disruptions. 

BIO’s Food and Agriculture Section believes that henceforth individual member companies should, prior 

to commercialization meet applicable regulatory requirements in key countries identified in a market 

and trade assessment that have functioning regulatory systems and are likely to import the new 

biotechnology-derived plant products.”

The voluntary guideline was adopted because, according to BIO: “asynchronous authorizations 

combined with importing countries maintaining “zero tolerance” for recombinant-DNA products not yet 

authorized results in the potential for major trade disruptions. The potential occurrences of trade 

disruptions will only increase given the substantial amount of research that will bring many new 

products and combinations of products to market.”

The submission for nonregulated status in the US will be followed by applications submitted in Canada, 

Mexico, and Japan, with the intention of completing approvals before crop introduction.

11. Conclusions: Determination of Nonregulated Status for InnateTM Potatoes

Simplot’s InnateTM technologies allow us to transform plants with potato DNA and only introducing noncoding DNA (DNA not coding for RNA that is translated into protein) into the plant’s genome. We 

transformed five different varieties with pSIM1278, and selected ten of these events for determination 

of nonregulated status: F10 and F37 for Ranger Russet, E12, and E24 for Russet Burbank, J3, J55, and J78 

for Atlantic, G11 for variety G, and H37 and H50 for variety H. As demonstrated in the data presented in 

this Petition, the transformed cultivated potato plants are not parasitic, and our data prove there is no 

reason to believe they are plant pests. Furthermore, the expression of the integrated genetic elements 

results in traits highly sought-after in the potato industry, namely: reduced black spot bruise, low 
J.R. Simplot Company JRS01 Page 64 of 74

asparagine, and lower levels of reducing sugars. Ultimately, we demonstrate that the InnateTM tubers

exhibited the desired traits which can lead to lower acrylamide levels in cooked potatoes and, 

importantly, we showed that the transformed tubers were otherwise substantially equivalent to 

untransformed controls.

To conclude, we have created plants utilizing biotechnology that are not plant pests and which have 

resulted from the addition of well characterized non-coding regions from potato or wild potato that are 

as safe as untransformed potatoes. We therefore seek nonregulated status of potato plants 

transformed using our InnateTM technologies and submit the evidence that these plants should not be 

classified as “Regulated articles” as defined under 7 CFR 340. 
J.R. Simplot Company JRS01 Page 65 of 74

12. Statement of Grounds Unfavorable

JR Simplot is not aware of any information indicating that InnateTM potatoes may pose a greater plant 

pest risk than conventional potatoes. There are no adverse environmental consequences anticipated 

with its introduction based on the data collected to date. The benefits of introduction of commercial 

events are provided in this submitted petition.

J.R. Simplot Company JRS01 Page 67 of 74

14. Record of Field Test Reports

The J.R. Simplot Company has adhered to USDA compliance regulations by submitting the field test 

reports for each notification (Table 13). Volunteer monitoring for 2009, 2010, and 2011 field trials is 

discussed in Appendix 10 (Post Harvest Monitoring).

Table 13. Field release notifications

USDA 

Reference 

Number

Field Trial 

Year

Effective Start and 

Expiration Date

Release Sites (by State) 

Covered by Notification

Field Test 

Report 

Submitted 

(Yes or No)

08-353-103n 2009 1/30/2009 – 1/30/2010 FL Yes

08-353-104n 2009 4/01/2009 – 4/01/2010 MI Yes

09-049-114n 2009 4/13/2009 – 4/13/2010 ID, WI Yes

09-077-112n 2009 4/23/2009 – 4/23/2010 NE Yes

09-336-103n 2010 1/25/2010 – 7/31/2011 FL Yes

10-053-132n 2010 4/05/2010 – 4/05/2011 ID, MI, ND, NE, WA, WI Yes

10-076-103n 2010 4/26/2010 – 4/26/2011 WI Yes

10-326-103n 2011 1/03/2011 – 1/03/2012 FL Yes

11-063-103n 2011 4/04/2011 – 4/03/2012 ID, IN, MI, ND, NE, WA, WI Yes

11-094-106n 2011 5/02/2011 – 5/2/2012 WI Yes

11-150-101n 2011 6/06/2011 – 6/62012 MI Yes

11-083-111n 2011 4/11/2011 – 4/11/2012 ID Yes

11-074-109n 2011 4/04/2011 – 4/04/2012 WA Yes

12-018-110n 2012 2/01/2012 – 2/01/2013 ID No
J.R. Simplot Company JRS01 Page 68 of 74

15. Literature Cited

Astwood J, Alibhai M, Lee T, Fuchs R, Sampson H (2000) Identification and characterization of IgE binding 

epitopes of patatin, a major food allergen of potato. Program and abstracts of the 56th annual 

meeting of the American Academy of Allergy, Asthma and Immunology, March 3-8, San Diego, 

Calif. Abstracts 555.

Bamberg JB, del Rio A, Huaman Z, Vega S, Martin M, Salas A, Pavek J, Kiru S, Fernandez C, Spooner D 

(2003) A decade of collecting and research on wild potatoes of the southwest. Am J of Potato 

Res, 80: 159-172.

Bamberg JB, del Rio A (2011a) Use of native potatoes for research and breeding. HortScience, 46: 1444-

1445.

Bamberg JB, del Rio A (2011b) Diversity relationships in tetraploid wild potato native to the USA 

[abstract]. Am J of Potato Res, 88:29. 

http://www.ars.usda.gov/research/publications/publications.htm?seq_no_115=252056

Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA (2008) High-throughput Agrobacteriummediated barley transformation. Plant Methods, 4: 22.

Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach 

to multiple testing. Journal of the Royal Statistical Society, Series B (Methodological), 57: 289–

300.

BIO (2007) US Biotechnology Industry Organization Product Launch Stewardship Policy. 

http://www.bio.org/foodag/stewardship/20070521.asp (Accessed 22 May 2011).

CFIA (1996) Biology Document BIO1996-09: The Biology of Solanum tuberosum (L.) (Potatoes). Canadian 

Food Inspection Agency. http://www.inspection.gc.ca/plants/plants-with-noveltraits/applicants/directive-94-08/biology-documents/solanum-tuberosum-l-

/eng/1330982063974/1330982145930 (Accessed 23 August 2012).

Chawla R, Shakya R, Rommens CM (2012) Tuber specific silencing of asparagine synthetase-1 reduces 

the acrylamide-forming potential of potatoes grown in the field without affecting tuber shape 

and yield. Plant Biotechnol J, 10: 913-924.

Cheng M, Fry JE, Pang SZ, Zhou HP, Hironaka CM, Duncan DR, Conner TW, Wan YC (1997) Genetic 

transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol, 115: 971–980.

Chiou TJ, Aung K, Lin SI, Wu CC, Chiang SF, Su CI (2006) Regulation of phosphate homeostasis by 

microRNA in Arabidopsis. Plant Cell, 18: 412-421.

Conner AJ, Dale PJ (1996) Reconsideration of pollen dispersal data from field trials of transgenic 

potatoes. Theor Appl Genet, 92: 505-508.
J.R. Simplot Company JRS01 Page 69 of 74

Conner AJ, Mlynárová L, Stiekema WJ, Nap JP (1998) Meiotic stability of transgene expression is 

unaffected by flanking matrix-associated regions. Mol Breeding, 4: 47-58.

Courvalin P, Weisblum B, Davies J (1977) Aminoglycoside-modifying enzyme of an antibiotic-producing 

bacterium acts as a determinant of antibiotic resistance in Escherichia coli. P Natl Acad Sci USA,

74: 999-1003.

Daxinger L, Hunter B, Sheikh M, Jauvion V, Gasciolli V, Vaucheret H, Matzke M, Furner, I (2008)

Unexpected silencing effects from T_DNA tags in Arabidopsis. Trends Plant Sci, 13: 4-6.

De Buck S, Jacobs A, Van Montagu M, Depicker A (1999) The DNA sequences of T-DNA junctions suggest 

that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. 

Plant J, 20: 295-304.

De Buck S, Podevin N, Nolf J, Jacobs A, Depicker A (2009) The T-DNA integration pattern in Arabidopsis 

transformants is highly determined by the transformed target cell. Plant J, 60: 134-145.

De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA integration patterns in cotransformed plant cells suggest that T-DNA repeats originate from ligation of separate T-DNAs. 

Plant J, 11: 15–29.

De Swert LF, Cadot P, Ceuppens JL (2002) Allergy to cooked white potatoes in infants and young 

children: A cause of severe, chronic allergic disease. J Allergy Clin Immun, 110: 524-535.

De Swert LF, Cadot P, Ceuppens JL (2007) Diagnosis and natural course of allergy to cooked potatoes in 

children. Allergy, 62: 750-757.

Dwelle, RB (2003) Potato growth and development. In: Potato Production Systems (Eds. Stark JC and 

Love S) University of Idaho Agricultural Communications, Moscow, pp. 9-19.

Federal Register (2009) “Acrylamide in Food, Request for Comments and for Scientific Data and 

Information.” Federal Register 74 (26 August 2009): 43134-43138. Print.

Federal Register (2011) ”Pesticides; Data Requirements for Plant-incorporated Protectants (PIPs) and 

Certain Exemptions for PIPs; Notification to the Secretaries of Agriculture and Health and 

Human Services, Proposed Rule.” Federal Register 76 (16 March 2011): 14358-14359. Print.

Garbarino JE, Belknap WR (1994) Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and 

expression of its promoter in transgenic plants. Plant Mol Biol, 24: 119-127.

Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the "genejockeying" tool. Microbiol. Mol Biol R, 67: 16-37.

Gheysen G, Villarroel R, Van Montagu M (1991) Illegitimate recombination in plants: a model for T-DNA 

integration. Gene Dev, 5: 287-297.

Gutiérrez RA, Stokes TL, Thum K, Xu X, Obertello M, Katari MS, Tanurdzic M, Dean A, Nero DC, McClung 

CR, Coruzzi GM (2008) Systems approach identifies an organic nitrogen-responsive gene 
J.R. Simplot Company JRS01 Page 70 of 74

network that is regulated by the master clock control gene CCA1. P Natl Acad Sci, 105: 4939-

4944.

Hagio M, Sakurai I, Sato S, Kato T, Tabata S, Wada H (2002) Phosphatidyglycerol is essential for the 

development of thylakoid membranes in Arabidopsis thaliana. Plant Cell Physiol, 43: 1456-1464.

Hakimi SM, Krohn BM, Stark DM: Monsanto Technology LLC (2006) United States Patent: Method of 

imparting disease resistance to plants by reducing polyphenol oxidase activities. Patent No.: US 

7,122,719 B2.

Hall KD, Holloway RL, Smith DT (2000) Texas Crop Profile: Potatoes. Texas Agricultural Extension Service, 

Texas A & M University, TX.

Hanneman RE (1994) The testing and release of transgenic potatoes in the North American Center of 

diversity. In: Biosafety of Sustainable Agriculture: Sharing Biotechnology Regulatory Experiences 

of the Western Hemisphere (Eds. Krattiger AF and Rosemarin A). ISAAA, Ithaca and SEI, 

Stockholm, pp. 47-67.

Hijmans RJ, Spooner DM (2001) Geographic distribution of wild potato species. Am J of Botany, 88: 

2101-2112.

Hokanson K, Heron D, Gupta S, Koehler S, Roseland C, Shantharam S, Turner J, White J, Schechtman M, 

McCammon S, Bech R (1999) The concept of familiarity and pest resistant plants. Proceedings of 

a workshop on: Ecological Effects of pest resistance genes in managed ecosystems. January 31-

February 3, 1999, Bethesda, MD. USDA-APHIS Plant Protection and Quarantine, Scientific 

Services.

Hopkins BG, Stark J, Hafez SL, Hutchinson PJS, Patterson P, Miller J, Thornton M, and Alvarez, J (2003) 

Field Selection, Crop Rotations, and Soil Management. In: Potato Production Systems (Eds. Stark 

JC and Love S) University of Idaho Agriculutral Communications, Moscow, pp. 1-7.

IPD (2011) Inter-genebank Potato Database. Wild Potato Collection. http://research.cip.cgiar.org/IPD/ 

(Accessed 10 November 2011).

Kim SI, Veena, Gelvin SB (2007) Genome-wide analysis of Agrobacterium T-DNA integration sites in the 

Arabidopsis genome generated under non-selective conditions. Plant J, 51: 779-791.

Kononov ME, Bassuner B, Gelvin SB (1997) Integration of T-DNA binary vector ‘backbone’ sequences 

into the tobacco genome: evidence for multiple complex patterns of integration. Plant J, 11:

945-957.

Kozukue N, Yoon KS, Byun GI, Misoo S, Levin CE, Friedman M (2008) Distribution of glycoalkaloids in 

potato tubers of 59 accessions of two wild and five cultivated Solanum species. J Agric Food 

Chem, 56: 11920-11928.

Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in 

Agrobacterium. Biotechnology, 9: 963-967.
J.R. Simplot Company JRS01 Page 71 of 74

Lechtenberg B, Schubert D, Forsbach A, Gils M, Schmidt R (2003) Neither inverted repeat T-DNA 

configurations nor arrangements of tandemly repeated transgenes are sufficient to trigger 

transgene silencing. Plant J, 34: 507-517.

Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in 

enhanced bacterial disease resistance. Planta, 215: 239-247.

Llorente B, Alonso GD, Bravo-Almonacid F, Rodríguez V, López MG, Carrari F, Torres HN, Flawiá MM 

(2011) Safety assessment of nonbrowning potatoes: opening the discussion about the relevance 

of substantial equivalence on next generation biotech crops. Plant Biotechnol J, 9: 136-150.

Lorberth R, Ritte G, Willmitzer L, Kossmann J (1998) Inhibition of a starch-granule-bound protein leads to 

modified starch and repression of cold sweetening. Nat Biotechnol, 16: 473-477.

Love S (1994) Ecological risk of growing transgenic potatoes in the United States and Canada. Am Potato 

J, 71: 647-658.

Lusk JL, Rozan A (2006) Consumer acceptance of ingenic foods. Biotechnol J, 1: 1433-1434.

Lusk JL, Sullivan P (2002) Consumer acceptance of genetically modified foods. Food Technology, 56: 32-

37.

Masclaux-Daubresse C, Reisdorf-Cren M, Pageau K, Lelandais M, Grandjean O, Kronenberger J, Valadier 

MH, Feraud M, Jouglet T, Suzuki A (2006) Glutamine synthetase- glutamate synthase pathway 

and glutamate dehydrogenase play distinct roles in the sink-source nitrogen cycle in tobacco. 

Plant Physiol, 140: 444-456.

Mayer AM (2006) Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67: 

2318-2331.

McGrath RB, Coruzzi GM (1991) A gene network controlling glutamine and asparagines biosynthesis in 

plants. The Plant J, 1: 275-280.

Mignery GA, Pikaard CS, Park WD (1988) Molecular characterization of the patatin multigene family of 

potato. Gene, 62: 27-44.

Müller AJ, Mendel RR, Schiemann J, Simoens C, Inzé D (1987) High meiotic stability of a foreign gene 

introduced into tobacco by Agrobacterium-mediated transformation. Mol Gen Genet, 207: 171-

175.

Nakata PA, Anderson JM, Okita TW (1994) Structure and expression of the potato ADP-glucose 

pyrophosphorylase small subunit. J Bio Chem 269: 30798-30807.

National Atlas (2004a) “Colorado.” The National Atlas of the United States of America. 2004. 

http://www.nationalatlas.gov (Accessed 11 November 2011).

National Atlas (2004b) “Texas.” The National Atlas of the United States of America. 2004. 

http://www.nationalatlas.gov (Accessed 12 November 2011).
J.R. Simplot Company JRS01 Page 72 of 74

NPC (2012) National Potato Council, 2012 Potato Statistical Yearbook.

NTP (2012) Technical Report on the Toxicology and Carcinogenesis Studies of Acrylamide in F344/N Rats 

and B6C3F1 Mice. National Toxicology Program. National Institutes of Health. Public Health 

Service. U.S. Dept. of Health and Human Services. 

Obrien J, Morrissey PA (1989) Nutritional and toxicological aspects of the Maillard browning reaction in 

foods. Crit Rev Food Sci, 38: 211-248.

OECD (1997) Organization for Economic Co-Operation and Development, Consensus document on the 

biology of Solanum tuberosum subsp. tuberosum (potato), 38 pages.

OEHHA (2011a) Acrylamide: frequently asked questions. Office of Environmental Health Hazard 

Assessment. http://www.oehha.ca.gov/prop65/acrylamideqaall.pdf (Accessed 24 May 2011).

OEHHA (2011b) Proposition 65: Acrylamide (2011). Office of Environmental Health Hazard Assessment. 

http://www.oehha.ca.gov/prop65/acrylamide.html (Accessed 24 May 2011).

Oltmanns H, Frame B, Lee LY, Johnson S, Li B, Wang K, Gelvin SB (2010) Generation of backbone-free, 

low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. Plant 

Physiol, 152: 1158-1166.

Pallais N, Mulcahy D, Fong N, Falcon R, Schmiediche P (1988) The relationship between potato pollen 

and true seed: effects of high temperature and pollen size. In Sexual Reproduction in Higher 

Plants. Spinger-Verlag, Berlin Heidelberg.

Percival G (1999) Light-induced glycoalkaloid accumulation of potato tubers (Solanum tuberosum L). J Sci 

Food Agric, 79: 1305-1310.

Plaisted RL (1980) Potato. In: Hybridization of crop plants (Eds. Fehr, W.R. and Hadley, H.H.). American 

Society of Agronomy and Crop Science Society of America, publishers, Madison, Wisconsin, 483-

494.

Prescott EM, Proudfoot NJ (2002) Transcriptional collision between convergent genes in budding yeast. 

P Natl Acad Sci USA, 99: 8796-8801.

Ramon M, Rolland F, Sheen J (2008) Sugar Sensing and Signaling. The Arabidopsis Book (TAB), ISSN: 

1543-8120 http://www.aspb.org/publications/arabidopsis/pages 1-22.

Richael CM, Kalyaeva M, Chretien RC, Yan H, Adimulam S, Stivison A, Weeks JT, Rommens CM (2008) 

Cytokinin vectors mediate marker-free and backbone-free plant transformation. Transgenic Res,

17: 905-917. 

Sallaud C, Meynard D, van Boxtel J, Gay C, Bès M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix 

M, Ouwerkerk PB, Rueb S, Delseny M, Guiderdoni E (2003) Highly efficient production and 

characterization of T-DNA plants for rice ( Oryza sativa L.) functional genomics. Theor Appl 

Genet, 106: 1396-1408.

Sinden SL (1987) Potato glycoalkaloids. Acta Hortic., 207: 41-48.
J.R. Simplot Company JRS01 Page 73 of 74

Skirvin RM, Norton M, McPheeters KD (1993) Somaclonal variation: has it proved useful for plant 

improvement? Acta Horticulturae, 336: 333-340.

Smigocki AC, Owens LD (1988) Cytokinin gene fused with a strong promoter enhances shoot 

organogenesis and zeatin levels in transformed plant cells. P Natl Acad Sci USA, 85: 5131-5135.

Spooner DM, van den Berg RG, Rodriguez A, Bamberg JB, Hijmans RJ, Cabrera SIL (2004) Wild potatoes 

(Solanum section Petota: Solanaceae) of North and Central America. Syst Bot Monogr, 68: 1-

209.

Spooner DM, McLean K, Ramsay G, Waugh R, Bryan GJ (2005) A single domestication for potato based 

on multilocus amplified fragment length polymorphism genotyping. P Natl Acad Sci USA, 102: 

14694-14699.

Stadler RH, Blank I, Varga N, Robert F, Hau J, Guy PA, Robert MC, Riediker S (2002) Acrylamide from 

Maillard reaction products. Nature, 419: 449-50.

Steffens JC, Harel E, Hunt MD (1994) Polyphenol Oxidase. In: Genetic Engineering of Plant Secondary 

Metabolism (Ed. Ellis BE) Plenum Press, New York, pp. 275-312.

Thipyapong P, Hunt MD, Steffens JC (2004) Antisense downregulation of polyphenol oxidase results in 

enhanced disease susceptibility. Planta, 220: 105-117.

Thipyapong P, Stout MJ, Attajarusit J (2007) Review: Functional analysis of polyphenol oxidases by 

antisense/sense technology. Molecules, 12: 1569-1595.

Thornton M (2008) The rise and fall of NewLeafTM potatoes. 

http://nabc.cals.cornell.edu/pubs/nabc_15/chapters/Thornton.pdf (Accessed 31 May 2011).

Tuteja JH, Zabala G, Varala K, Hudson M, Vodkin LO (2009) Endogenous, tissue-specific short interfering 

RNAs silence the chalcone synthase gene family in glycine max seed coats. Plant Cell, 21: 3063-

3077.

Urquhart AA, Joy KW (1981) Use of phloem exudates technique in the study of amino acid transport in 

pea plants. Plant Physiol, 68: 750-754.

US EPA (2011) United States Environmental Protection Agency. Introduction to Biotechnology 

Regulation for Pesticides. http://epa.gov/oppbppd1/biopesticides/regtools/biotech-regprod.htm (Accessed 27 January 2012).

USDA ERS (2010) Vegetables and Melons Outlook. United States Department of Agriculture Economic 

Research Service. http://www.ers.usda.gov/publications/vgs (Accessed 2 April 2012).

USDA ERS (2011) Organic Production. United States Department of Agriculture Economic Research 

Service. http://www.ers.usda.gov/Data/Organic (Accessed 15 June 2011).

USDA-NASS (2010a) United States Department of Agriculture, National Agricultural Statistics Service. 

Acreage from June 2000-June 2010 (Accessed 24 May 2011).
J.R. Simplot Company JRS01 Page 74 of 74

USDA-NASS (2010b) United States Department of Agriculture, National Agricultural Statistics Service. 

Potatoes 2009 Summary ISSN: 1949-1514 (Accessed 24 May 2011).

Valentines MC, Vilaplana R, Torres R, Usall J, Larrigaudiere C (2005) Specific roles of enzymatic browning 

and lignifications in apple disease resistance. Postharvest Biotech and Tech, 36: 227-234.

Van Haaren MJJ, Sedee NJA, de Boer HA, Schilperoort RA, Hooykaas PJJ (1989) Mutational analysis of the 

conserved domains of a T-region border repeat of Agrobacterium tumefaciens. Plant Mol Bio, 

13: 523-531.

Visser RGF, Stolte A, Jacobsen E (1991) Expression of a chimeric granule-bound starch synthase-GUS 

gene in transgenic potato plants. Pant Mol Bio, 691-699.

Webb RE, Wilson DR, Shumaker JR, Graves B, Henninger NR, Watts J, Frank JA, Murphy HJ (1978) 

Atlantic: a new potato variety with high solids, good processing quality, and resistance to pests. 

Am Potato J, 55: 141-145.

Windels P, DeBuck S, VanBockstaele E, DeLoose M, Depicker A (2003) T-DNA Integration in Arabidopsis 

Chromosomes. Presence and Origin of Filler DNA Sequences. Plant Physiol, 133: 2061-2068.

Wolters AA, Trindade LM, Jacobsen E, Visser RGF (1998) Fluorescence in situ hybridization on extended 

DNA fibres as a tool to analyse complex T-DNA loci in potato. Plant J, 13: 837-847.

Yan H, Chretien R, Ye J, Rommens CM (2006) New construct approaches for efficient gene silencing in 

plants. Plant Physiol, 141: 1508-1518.

Yang L, Ding J, Zhang C, Jia J, Weng H, Liu W, Zhang D (2005) Estimating the copy number of transgenes 

in transformed rice by real-time quantitative PCR. Plant Cell Rep, 23: 759-763.

Zhu W, Ouyang S, Iovene M, O'Brien K, Vuong H, Jiang J, Buell CR (2008) Analysis of 90 Mb of the potato 

genome reveals conservation of gene structures and order with tomato but divergence in 

repetitive sequence composition. BMC Genomics, 9:286.