J.R. Simplot Company JRS01 Page 1 of 74



## Petition for Determination of Non r egulated S tatus for Innate TM 

## Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise: E vents 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 article s 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 , 201 3

J.R . Simplot Company Petition JRS01

OECD Unique identifier s:
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 CBIJ.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 e xcept 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 Numbe r 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

................................ ................................ 46J.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 Asparagi ne 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 Glycoalkal oids

................................ ................................ .. 50

8.2. Allergenicity of Innate TM 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 Consideratio ns 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 Innate TM Potatoes

................................ . 63

12. Statement of Grounds Unfavorable

................................ ................................ ................................ 65

13. Certification

................................ ................................ ................................ ................................ ..... 66

14. Record of Field Test Reports

................................ ................................ ................................ ........... 6 7

15. Literature Cited

................................ ................................ ................................ ............................... 68J.R. Simplot Company JRS01 Page 6 of 74 List of Tables

Table 1. Innate TM 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 Be tween 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

Fi gure 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 1 0 :

................................ ................................ ................................ .......... Post

- Harvest MonitoringJ.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 Hin d 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

exten d up to the cleavage site in the Left Border or be truncat ed 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

- DNAJ.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

exten d 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

R ight B order 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 o ther forms of vegetative propagation

TPS T rue 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 ValineJ.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 Innate TM technolog ies allow us to transform plants with plant DNA . In the present petition, Innate TM 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 Innate TM 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 Innate TM technolog ies 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 come s 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 p etition, the transformed cultivated potato plants are not parasitic, and our data show no increase in pest potential compared to the controls . Furthe r more, 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 k ey consequence of these beneficial traits is that the harveste d potatoes contain less acrylamide when cooked than untransformed, commercially available potatoes.

Among other things, we have used Innate TM t echnolog ies 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) susceptib ility to enzymatic browning and discoloration, which happens when polyphenol oxidase leaks out fro m 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 glu cose 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 Innate TM technolog ies 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, Innate TM t echnolog ies are particularly attractive because potatoes are notorious for having a high degree of heterozygosity, suffering from inbreeding depression, and limited predominantly to clonal propagat ion . These inherent
factors signif icantly hinder and prevent us from commercially introducing quality, sought

- after traits into this valuable crop. T oday’s traditional breeding techniques simply result in random geno mic rearrangements and trait segregation, and do not allow for the simulta neous addition of multiple
desired traits

.

To illustrate th is 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 t hat 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

.

T his 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 ( Asn 1) and the polyphenol oxidase

- 5 gene ( Ppo5 ), arranged as inverted repeats betw e en the Agp promoter of t he 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 asso ciated 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 Innate TM t echnolog ies , these expression cassettes contain p otato 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 transform ed five different varieties including Ranger Russet, Russet Burbank, Atlantic

, variety G, and variety H with pSIM1278, and identified transformed s hoots using polymerase chain reaction (PCR)
rather than markers

. We also performed n umerous tests and confirm ed that none of the plants
contained vector backbone DNA. Transformed plants were propagated to produce the Innate TM 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 . Innate TM 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 ofJ.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 gene s ( R1 ) and pho sphorylase

- L ( PhL ).

Ultimately, we demonstrate that the Innate TM t ubers exhibited the desi red 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 self

pollinated

, an inability to attract honey bees because they lack nectar

, the pollen transfer range is
limited to about 20 meters , and true seeds would b e unlikely to grow into mature potatoes since potato seeds are not saved and propagated in the typical farming operation. If p otatoes were gro wn from true potato seed, the offspring would be so div ers e 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 concer ns 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 t echniques 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 modification s described in this petition are hi ghly 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 tri als 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 spec ies 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 reque sts 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

. Ra tionale for Potatoes with Low Acrylamide Potential and Reduced Black Spot Bruise

The potato varieties were transformed using Innate TM technolog ies 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 suga rs. Use of Innate TM technolog ies allows for transformation of pot atoes using genetic material that contains only non

- coding regulatory regions

. The desirable traits were simultaneously incorporated into five of the most popular potato processing varietie s . Such trait
addition into existing varieties would not be possible to achieve through traditional breeding because potato is tetraploid, highly heterozygo us and sensitive to inbreeding depression. T he resulting InnateTM potatoes are not plant pests as ver ified by phenotype, Southern blot hybridization, and PCR

.

1 .1

. Basis for Determination of N on r egulated Status

Part 340 Section 7 of the United States F ederal R egister 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 th at 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 event s displayed similar agronomic and phenotyp ic
characteristics compared to their untransformed controls. This submission revi ews 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 event s 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 ( Asn 1) a nd 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 present ed 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

T he Swedish National Food Administration and Stoc kholm 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 201 2 ) . 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 treat ing 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

, a ffecting changes in the color, flavor, functional properties and nutr itional 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
bioch emical 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 prod ucts contribute 35% of the acrylamide exposure through diet in the US. T he 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 201 2 ).

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 introduc tion of InnateTM potatoes with low acrylamide potential would provide potatoes that are largely indistinguishable from existing varieties. The reduction in asparagine and sugars using Innate TM technolog ies and the resu lting 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 add ition, 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 f rench fry and potato chip manufacturers regarding perceived risks from acrylamide

. So me settlements required signs warning consumers

, but in addition, some manufacturers of retail f rench fries and potato chips would be required to reduce acrylamide in their pr oducts or add warning labels. As of December 1, 2011, some potato chip manufactur ers 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 motiva ted to adopt the low acrylamide

, InnateTM po tato products

.

Following the discovery of acrylamide in foods and i n 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 tr aditional 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 sugar s . In a crop like potatoes, which is highly heterozy gous 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 char acteristics of the original parent.J.R. Simplot Company JRS01 Page 14 of 74

Potato t ransformation 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 sugar s (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 acr ylamide.

1 .3

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

The event s 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 r ecent toxicology studies (NTP 201 2 )

.

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 fl avors associated with additives such as aspara ginase or citrate. Typically, fry manuf acturers add dextrose during processing of f rench fries to develop the desired golden brown color, but dextrose also increases the formation of acrylamide through the Mai llard reaction. Significant reductions in acrylamide occur by merely omitting dextrose from th e process ; however, the signature golden brown colors must then be developed some other way ( such as though the addition of co lors like

annatto )

. The use of alt ernate colors, results in an absence of the typical flavors that develop through
those brown ing 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, t he Innate TM potato es 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 affect s quality and recovery in processing f rench fries and chips. Potatoes that have been damaged and show b lack spot bruise must be trimmed or could
be rejected before processing , resulting in quality challenges or economic loss

. M anagement 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 Innate TM technolog ies exhibit signi ficantly less black spot bruising and lower
redu cing sugar content, which will likely have economic benefits for grower s and processors.J.R. Simplot Company JRS01 Page 15 of 74

1

. 4

. Submissions to Other Regulatory Agencies

Simplot has initiate d and will complete a consultation process with the Food and Drug Administration (FDA) prior to commercial distribution of tubers or products from the 1 0 event s.

An assessment of the safety of these events will also be submitted to Health Canada and the Canadian Food Inspection Agency (CFIA) in 201 3 . F urthermore, submissions in support of approval to import products from the event s 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, the re is an important need to improve the quality and reduce levels of acrylamid e in fried
and baked potatoes

. Because i t was not feasible t o reduce acrylamide formation and black spot bruise in existing varieties through traditional breeding , we accomplished this goal by applying Innate TM
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 s eek nonregulated status for

10 events based on the weight of evidence demonstrating their safety. W e have transformed plants
with the addition of genomic DNA from potato and wild potato where the integrated genetic material contains only well characterize d 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 Eco nomic Co

- Operation and Development’s (OECD) series on harmonization of regulatory oversight in biotechnology no. 8, Consensus document on the biology of Solanum tuberosum, 1 9 97.

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

Innate TM potatoes. All commercial potato varieties must be propagated throu gh 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 C hile (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 fiel d 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 theJ.R. Simplot Company JRS01 Page 16 of 74

crop a lso 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 20 11 , the United States harvested 2 1. 4 million tons of potatoes, enough to make it the world's fourth biggest producer (NPC 201 2 ) . Potatoes in the United States are grown in

many state s , 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 2010 b )

. 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), c hi ps, dehydrated potato an d 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 F eed in the USA

Harvested potatoe s 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 201 2 ).

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

- processed raw potato products) and processed discards (french fry, hash br own, 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 G enus 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 thousa nd 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 S olanum 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, Nort h America and Europe. The subspecies andigena is also a cultivated species, but cultivation is restricted to Central a nd South America ( Hanneman 1994).

2

. 4

. Genetics of P otato

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

. tuberosum can be diploids (2n= 2x= 24) or tetraploids ( 2 n= 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 c ultivated 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, t he 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 whe n receiving plants were separated by more than 20 met er s 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 fert ility. E vents of three
of five varieties in this petition produce abundant flowers and pollen. However, Russet Burbank and the

H variet ies 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) S prouts 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 enlarg e

. 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 cyc le begins with c ertified 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. T ubers 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 . W hen 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

. T he combination of these processes results in the swellin g of the subapical part of
the stolon. D uring enlargement

, tubers become the largest nutrient sink of the potato plant storing large amounts of carbohydrates (mainly starch) and also significant amounts of protein

. F urthermore , potato
tubers decrease their general metabolic activity and as such behave as typical storage sinks. P otato
tubers of varieties such as Ranger Russet, Russet Burbank, Atlantic, G, and H are harvested from 90 toJ.R. Simplot Company JRS01 Page 18 of 74

160 days after planting , which may vary with production area and marketin g conditions

. Typical potatoes are about 20

- 24% dry matter, of which approximately 60

- 80% consists of starch. A fter potato
vines die back

, the tuber skin s thicken and harden which provides greater protection to tubers during harvest and blocks entry of pa thogen s . Although dormancy is defined as the absence of visible growth,
dormant tuber meristems are metabolically active . I n general, rates of many cellular processes such as respiration, transcription, and translation are suppressed during dormancy and no n

- 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 seed s 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 sufficie nt amounts of tuber seed needed for commercialization. A t ypical
seed bulk

- up program would progress as follows. In the initial year, propagation in tissue culture would produce about 100 plants , and then t hese plants are gr own 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 in crease 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 SchematicJ.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 US 1

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 P 2 O 5 – 330 lb K 2 O per

acre For 600 cwt/A yields and optimum soil test
levels:
250 lb N

– 100 lb P 2 O 5 – 330 lb K 2 O 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 seed s 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 Innate TM po tatoes 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 f ew 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.) thatJ.R. Simplot Company JRS01 Page 21 of 74

typically don’t travel much more than 3 kilometers, rather than wind pollinat ion (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 rate s w ere zero whe n receiving plants were separated by more than 20 meters from genetically modified plants (Conner and
Dale 1996). Additionally, p otato pollen grains are extremely temperature sensiti ve

. 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 publ ished by the American Society of Plant Taxonomists entitled Wild Potatoes ( Solanum section P etota; Solanaceae ) of North and Central America (Spooner et al. 2004). The USDA maintains the US Potato Genebank and participates in the Association of Potato Inte r

- 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 s pecimens exist in gene banks, include the tetraploid species S. fendleri (recently reclassified as S. stoloniferu m ; 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 tho ught 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. james i i

, 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 (Ba mberg 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 possi bility 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 organ ic soil of alluvial valleys, sandy fallow fields, grasslands, juniper

- pin yo n 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. 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) indic ate 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 p otency 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 s tates with w ild p otato p opulations (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 (Lo ve 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 hybridizati on 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

p loidy 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 ba rriers to hybridization, it is unlikely that inter

- species pollination would occur with the Innate TM 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 fol lowing 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 chal lenges 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, P ythium leak, Verticillium wilt, and powdery scab (Hopkins et al. 2003).

P otatoes 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 coloni zer of unmanaged ecosystems. I n 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 non

target organisms) making pest potential negligible.

Although al most 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 disadvan tage 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 establis h 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 p otato 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 diversi ty 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 andJ.R. Simplot Company JRS01 Page 24 of 74

geographically isolated from commercial producti on areas, further reducing concerns about cross

pollination 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 Innate TM 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: Descriptio n of Marker

- Free DNA Transformation

3 .1

. Introduction

Simplot’s Innate TM technolog ies comprise many aspects of plant biology all working together to produce traits of interest in transformed plants

. S pecifically

, the potatoes were modified to r educ e asparagine and black spot bruise

. Along with the potato derived expression cassettes , are promoters and spacers, also from the potato genome, all creat ing the gene silencing system. The targeted genes are then combined with the backbone elements of pSIM1 278, 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 po tatoes, 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). T ransformed 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, n o antibiotics remain, and the result is that genomic DNA from the potato or sexually compatible species
ha ve 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 Agrobacteriu m AGL1 strain carrying pSIM1278, and
transferred to tissue culture medi a 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 timenti n 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 plas mid 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 sta rted expressing
th e isopentenyltransferase ( ipt ) gene result ing 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 medi um lack ing any synthetic
hormones and incubated in a Percival growth chamber under a 16 hr photoperiod at 24 o C where they started to form shoots. Many shoots expressed the ipt gene and displayed a cytokinin overproduction phenotype ; t hese 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 p ublished by
Richael et al. ( 2008 )

.

The process of eliminating Agrobacterium start ed 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

. P roof was o btained by incubating stem fragment s of transformed event s on nutrient
broth

- yeast extract ( NBY medium ) for 2 weeks at 28 O C (repeated twice) . In accordance with 7 CFR Part 340, transformed plants were transported and planted in the field only when free of live Agrobacterium

. T he potato events were confirmed to be free of Agrobacterium

- derived vector backbone DNA by the following t hree methods : 1) If plants had the negative selectable isopentenyl isomerase ( ipt ) marker gene in the vector backbone , they wer e discarded

. Plants with ipt gene expression would have stunted phenotypes, abnormal leaves, or the inability to root due to the cytokinin overproduction caused by i pt expression ; 2) A bsence of the backbone DNA was confirmed with Southern blot hybridizati on ; 3) PCR
was used to confirm no fragments of the backbone DNA were present

.

3 .2

. Parental Varieties

Russe t 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 Vertic illium 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. Jelly

end 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 f rench 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 pro duce 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 f rench fries. Tubers are susceptible to common scab and black
spot bruise. Ranger Russet matures earlier than Russet Burbank and would be considered a medium

length stora ge variety

. The variety is fertile and mainly grown in the Northwest, especially for the production of f rench 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 t ertiary leaflets. Flowers are profuseJ.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 i nches ) 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 an d 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 g ene 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 (= 9,51 2

- 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 inse rt 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 10,14 8

- bp.J.R. Simplot Company JRS01 Page 28 of 74

3 .4

. Backbone P ortion of pSIM1278

The purpose of the backbone portion of pSIM1278 is to maintain the DNA ins ert 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

P olyubiquitin 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

- 19,21 8

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

199 4 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

r eplication 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 ) site 1 Synthetic Site for secondary cleavage to release single

- stranded DNA insert from pSIM1278 AY566555 2
(bases 1

- 25) 19,469

–

19,493 van Haaren

et al. 1989

2. DNA flanking the LB sequence S. tuberosum var. Ranger

Russet Su pports secondary cleavage at LB AY566555 2
(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 ( 1 st 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. 20 12 3

6. 3’

- untranslated sequence of the polyphenol oxidase

- 5 gene ( Ppo5 ) ( 1 st 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 2,81 7

- 2,973

8. 3’

- untranslated sequence of the polyphenol oxidase

- 5 gene ( Ppo5 ) ( 2 nd 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 ( 2 nd 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. 20 12 3

10. Promoter for the granule

- bound starch synthase ( pGbss ) gene ( 1 st copy, convergent orientation relative
to the 1 st 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 3,5 30

- 4,215

11. pAgp, 2 nd 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 4,2 32

- 6,491

12. Fragment of promoter for the potato phosphorylase

- L ( pPhL ) gene ( 1 st 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 ) ( 1 st 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

1 4

. Spacer

- 2 S. tuberosum var. Ranger

Russet Sequence between the 2nd inverted

repeat HM363756 7,53 9

- 7,796J.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 RB

Like site AY566555 2
(bases 231

- 391) 9,77

–

9,931

20. Right Border (RB) sequence 1 Synthetic Site for primary cleavage to release single stranded DNA insert from pSIM1278 AY566555 2
(bases 392

- 416) 9,932

-

9,956 van

Haaren et
al. 1989

1 The LB and RB s equences (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 B order 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 A ssociated w ith the DNA Insert

Open reading frames (ORFs) are long sequence s 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 10,1 4 8

- bp DNA insert , from the c leavage site in the Left Border element to the cleavage site in the Righ t Border element contain s fragments associated with an ORF of the Asn1 gene and the trailer
associated with an ORF of the Ppo5 gene. These inverted repeat s produce a pool of variably

- sized RNAs that trigger gene silencing and are not translated into prote in when transcribed

.

3

. 7

. Conclusions of Method s of Development: Description of Marker

- Free DNA Transformation

A description of Innate TM technolog ies outlines the plant biolog ical sy s tems 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

. T he Innate TM 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 T ransferred DNA and Gene Regulation

The potato events all produce potatoes that are silenced for specific genes. The silencing methods are part of Simplot’s Innate TM technolog ies 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 m RNA s that would normally code for an enzyme, like asparagine synthetase.
This results in much reduced leve ls 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 in formation of the junctions provided the basis for developing event

- specific PCR tests for all events.

4 .1

. Description and H istory of the M odification

Mechanism for Gene Silencing The silencing approach applied here to down

- regulate the transcript level s of several genes is illustrated

in Figure 4

. T wo 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 s ilencing 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 promoter s 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 evolve d naturally as a consequence of the duplication of genes and regulatory elements

. One example relates to a 27

- kb region of theJ.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 inhi bition of a metabolic pathway, which functions specifically in one tissue while allowing e xpression in other parts of the plant.

Expression of Neighboring Genes Previous studies on convergent transcription ha ve shown that transcripts are of variabl e size but smaller than the distance between the two promoters , both in yeast (Prescott and Proud foot 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 w as 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 s tranded transcripts are ineffective in triggering gene silencing (Yan et al. 2006). Furthermore, studies on the expression of

a n antibiotic selectable marker gene next to a convergent transcription

- based silencing construct
demonstrated that this antibioti c gene was not affected by expression of the silencing construct (Yan et
al. 2006). In the unlikely event that read

- through occurred, and lower ed the expression of an adjacent gene in any of the 10 events, it would have been evident through change in plant phenotype. After

e xtensive evaluations from multiple field trials over t hree years, no unexpected agronomic , phenotypic,
or biochemical differences were observed with these 10 events when compared with control varieties.

4

. 2

. Verification of I nsert I ntegrity and N umber of I nserted S equences

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 o f Inserted DNA

. T wo 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)

.

Th e comprehensive molecular analys e s confirm ed 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 remain ing event H37 contain s 1 cop y with 3 additional fragments . This finding indicate d that the number of copies of the DNA insert is similar to that reported
elsewhere for T

- DNA s in potato (Wolters et al. 1998) and other crops such as tobacco ( D e Neve
et al. 1997; D e Buck et al. 1999), Arabidopsis ( D e Buck et al. 2009) , rice (Sallaud et al. 2003 ; Yang
et al. 2005 ) , wheat (Cheng et a l. 1997), and barley (Bartlett et al. 2008)

. Although h igh 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 activit ies between higher

- copy

eve nt s and the events with only one cop y

. (2) Event J55 contain ed two linked DNA insert s positioned as an inverted repeat

. Lechtenberg et al. (2003) showed with bacterial T

- DNA that the presence of a second gene copy either in tandemJ.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 repeat s 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 e xample of an event with such sequences
include s the 49

- bp sequence between the two DNA insert s 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 insert s are shorter than the full distance between the Left a nd 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 F 10 ( a 38

- bp deletion at the Right
Border ) and F 37 (a 12

- bp deletion at the Left Border ). L arge deletions at the Right Border side
that resulted in loss of the PhL/R1 promoter silencing casset te 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 R egions of I nserted S equences

Extensive resear ch 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 method s

.

Among the evidence supporting safety r elative to the insertion site are the following : (1) most insertions
do not disru pt 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 b e unlikely to result in an observable phenotype (in contrast to most other crops, transformed potatoes are not self

- fertilized (backcrossed) to develop event s that are homozygous for the insert); andJ.R. Simplot Company JRS01 Page 38 of 74

(3) if a n unusual and undesirable phenotype occurred , th at event would be selected only if equivalent or superior to the untransformed potato variety.

4

. 5

. Stability of I nserted 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 vegetativ e ly propagated crop and reproduction does not involve meiosis , DNA in sertions were expected to be stable

. Nevertheless, the stability of integrated DNA insert s 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 n umber 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 i nstability 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 t esting will

monitor phenoty pic 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, event

specific PCR was developed for all the events submitted for nonregulated status

. The methods developed will be used to monitor plants and tubers in fie ld 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 cop y of the DNA insert from pSIM1278 and, in the case of H37, t hree additional fragments of pSIM1278 . Although the inserts were not always full

length, therefore, each event contained at least one functional Asn1 / Ppo5 gene silencing cassette. This
means there are three main differences between the se events and their untransformed counterparts : 1)

Innate TM tubers contained less asparagine , 2) more glutamine and 3) at least some degree of black spot bruise tolerance. The one exception being that conven tional Atlantic al ready 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

conta ined 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 enhan cer 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 interrupt ed the exp ression 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 ge netic material from a donor organism where the material is well characterized and contains only non

- coding regulatory regions of DNA

.

5

. Express ion of the Gene Products

The DNA insert used to transform these ten potato events contain s two expression cassettes designed to lower the transcript levels of endogenous genes. The first cassette contain s two fragments of the asparagine synthetase

- 1 ( Asn1 ) gene (404

- bp) and the polyphenol oxidase

- 5 ( Ppo5 ) gene (143

- bp)

, inserted as inverted repe at s 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.

T ranscript 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 H 50

.

It is possible that these intended changes could trigge r uninten ded 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.

Pre vious 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 eve nts contain only

non

- coding regulatory sequences

. M ultiple 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

. E fficacy 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 . T ra nscript 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 Nort hern 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 an d tissue.

Innate TM technolog ies ha ve 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 strong er 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 Tubers 1 Stolons 1 Roots 1 Stems 1 Leaves 1 Flowers 1, 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

H37 3 A P L R L R P P R

H50 3 A P L R A P L R A P R A P R

1 A = Asn1 , P = Ppo 5, 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. Sin ce ASN and GLN are similar non

- essential

a mino 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 f rom 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 e vents, 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 stolo ns than in the roots, stems, leaves, or flowers ( Table 6 ).J.R. Simplot Company JRS01 Page 41 of 74

E vent 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 integratio n 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
ch aracterization that showed all events contained the Asn1 and Ppo5 genes, but two events, J78 and

G1 1 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

Innate TM 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 end ogenous 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 i s illustrated in the a gronomy and composition studies

. I t 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 In nate TM potat o es 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

. Agronom ic Performance , Disease, and Efficacy of Traits

The purpose of the agronomic trials was to confirm that the Innate TM potatoes had equivalent phenotypes compared with the ir untransformed controls when grown at multiple locations representing the major areas for potato production in the US. Observations th roughout the growing season allowed for a thorough assessment of growth, disease and pest susceptibility, and measurements of tuber yield and quality. The field assessments ai ded in the selection of the events that contained the intentionally incorporated new traits and maintained all benefits of the conventional parent varieties . The se

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 charac teristics

.

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, t he potatoes were tested for acrylamide. A brief summary of the acrylamide results will be presented here with the supporting detail s available in Appendix 9. Compositional Analyses

.J.R. Simplot Company JRS01 Page 42 of 74

7 .1

. Agronomy and Phenotypic Evaluation

Thi s summary includes agronomic evaluation s of 1 0 events , representing 5 varieties , at geographical ly
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 Evaluation s )

. All agronomic trials were conducted under Biotechnology Quality Management System (BQMS) standards and USDA

- APHIS compliance (For a complete listing of trials, see Table 1 3 in Section 14. Record of Field Test Reports )

. Planting was carried out m echan ically to facilitate subsequent harvests and to ensure that Innate TM 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 T able 7 . Summary of Agronomic Observations

Characteristic Number of Site Years 1

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 e very site.

The agronomic and ecological data that are presented in Appendix 6 ( Fiel d Performance and Tuber
Evaluations ) show that over multi

- year and multi

- site field trials no variety specific differences lead ing to increased weed or pest potential wer e 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 Innate TM varieties and appropriate controls. We use d a step

- wise approach to interpret any differences that were observed (both statistically significant and non

- statisticallyJ.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, statist ical 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 in dicated statistical significance or the data did not support
calculation o f 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 th e 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 o r 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 r ange 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 combine d 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 t he events, and these attributes will be investigated further in commercial trials. Thus, we concluded that n o biologically meaningful differences that would contribute to increased weediness or pest potential were observed for any of the agronomic characte ristics, 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 P erformance and Tuber Evaluations )

. There were no differences in phenotype and tuber composition ( Appendix 9. Compositional Analyses ) or disease susceptibility ( Appendix 8. Disease Susceptibility ). I t can be concluded that the event s are not different
from the ir untransformed controls in terms of agronomy, tuber yield, and tuber composition. Therefore,

t he transformation of potato with pSIM1278 does not introduce characteristics that will encourage or
enhance weediness or pest potential.

7.2

. Disease susce ptibility and polyphenol oxidase

Polyphenol oxidase enzymes (P po ) are found in most organisms including animals, plants, fungi and
bacteria

. Although much is known about the molecular biology of P po and its role in enzymatic browning, little is understood about the function of P po

- mediated browning in plant physiology. Some have hypothesized that P po s represent part of the plant’s defense response against insects and pathogens. Considering that the present submission involves silencing of P po in tubers, we reviewed relevant literature and conducted studies relating to the disease response of the events.

P po s are copper metalloenzymes which oxidize mono

- and o

- diphenols to o

- diquinones by utilizing molecular oxygen (Thipyapong et al . 2004). Typically, P po 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, P po enzyme is released and reacts along with oxygen molecules to produce quinonesJ.R. Simplot Company JRS01 Page 45 of 74

(Thipyapong et al

. 2004) . The production of black and brown quinone s 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 P po have been identified, tomato ( Lycopersicon
esculentum ) possesses 7 PPO genes, and banana ( Musa acuminata ) is known to contain a P po family of
at least 4 genes (Thipyapon g et al. 2007 ; Mayer 2006 ). A number of researchers evaluated induced P po 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 hi gh levels of P po in leaf tissue and increased resistance to pathogens and insect pests. In one study, three lines with higher P po expression were tested against the bacterial pathogen Pseudomonas syringae pv

. T omato , 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 P po , the mechanism for disease resistance r emains unknown (Li and Steffens 2002).

In potato, impacts sustained during harvest and postharvest activities induce the release of P po from cell plastids, facilitating oxidation of phenols to quinones, and resulting in negative effects on quality
and r ecovery in processing french fries and chips, as well as the marketability of fresh potatoes. A family of 6 genes encoding P po exists in potato, with one gene ( Ppo5 ) being tuber

- specific and the remaining 5 genes responsible for P po expression in other tissues. The tuber

- specific Ppo5 gene was down

regulated, resulting in reduced susceptibility to black spot bruise , as shown in the current petition for nonregulated status.

Researchers found that by down

- regulating P po activity in p otato 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 certa in 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 occur s in the fruit of the apple ( Mal us 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 inv estigated in Golden Delicious apples . A significant increase in decay was observed following the treatment of peeled apple s with a P po substrate which had induc ed high er P po activity levels. T he P po enzyme, and particularly the browning process induced by treatment
with P po substrate, may have contributed to increase decay and indicates that an overexpression of P po

may lead to higher level of sensitivity towards the p athogen (Valentines et al. 2005 ).

S ome researchers have proposed that enhanced P po may increase resistance to disease, while others
claim that reduced P po could also increase resistance . Con sidering that some evidence exist s for a relationship between P po and diseases, we chose to test the events for several important potato
diseases an d 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 Innate TM potatoes from the Canyon County, ID, and Bingham County , ID, field trial sites. In addition, P ythium leak disease was observed on some untransformed and Innate TM potatoes from Adams County , WA

, and Bingham County , ID, for the 2010 cold storage period. These observed
disease incidences we re attri buted to environmental soil conditions that favored infectio n of tubers and the relatively high presence of disease within the specific field trial sites.

7

. 4

. Disease Incidence : La te Blight and Bacterial Soft Rot

Disease incidence was evaluated by intentionally infecting the events and their untransformed cont rols with the causal agents of late blight ( Phytop h thora 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 mu ltiple 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 deli berately 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 Innate TM 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 e vents 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 s ilencing 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 event s show ed slightly higher susceptibility to disease , it would not enhanc e the weediness or result in the creation of plant
pest s

.

7

. 5

. Evidence of Trait Efficacy: Reducing Sugars

Tubers contain large amounts of phosphorylated starch, some of which is degraded d uring storage to produce glucose and fructose

. T hese reducing sugars react with amino acids to form Maillard productsJ.R. Simplot Company JRS01 Page 47 of 74

including acrylamide when heated at temperatures above 120 O C

. Two enzymes involved in starch phosphorylation are water dikinase R1 and ph osphorylase

- L ( R1 and PhL ) . The partial o r complete silencing of the associated genes in tubers can li mit 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 t wo events, J78 and G11,
that did not contain the R1/PhL promoter silencing cassette

. Tu bers of the event s G11 , H37, and H50 contain the same amount of reducing sugars as tubers of their untransformed counterpart s

. The inability of the silencing construct to limit glucose/fructose formation in H37 and H50 may be due to the
fact that the H var iety 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 through out 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 displa y 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 combine s with reducing sugars in the Maillard reaction to form acrylamide

, results in average reduction s of 58 to 7 2 % acrylamide in fries and chips ( Table 8 )

. A long 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 Asparagi ne and Acrylamide Between Controls and Events Variety Free Asparagine (ppm) Percent Reduction
from Control Acrylamide (ppb) 1 Percent 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% 8 42.7 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% 5 31.2 0%

H37 877.5 69% 2 23.0 58 %

H50 672.7 76% 1 74.7 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 c rucial 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 o rganic 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 syn thetase 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 amid e nitrogen group, and converting glutamine to glutamate . The asparagine biosy n thesis pathway is represented in Figure 6

. Using I nnate TM technologies, the Asn1 gene is silenced in potato tubers, thereby allowing glutamine to
retain its a mide group, and to remain as glutamine in the system. This explains the reason lower asparagine levels in I nnate TM 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 p rocessors 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 th e year following.
We h ave 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

. I f 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
potat oes, 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 contribut ed 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 enhanceJ.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 n o biologically meanin gful 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

interacti ons, and plant interactions with abiotic stressors) of the events compared to their
untransformed controls

. I t can be concluded that event s are not different from the ir untransformed controls in terms of agronomy, tuber yield, and tuber composition. Therefore, t he 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 Innate TM potatoes to 1) show equivalence to the untransformed
controls , 2) compare the Innate TM potatoes to literature ranges, where applicable; and 3 ) show that
there were no change s in potenti al toxins. Ultimately, results of the composition studies showed food safety equivalence between the events and the untransformed controls.

8 .1

. P roximates, V itamins, M inerals, and G lycoalkaloids

The se analyses were conducted to confirm that compositio n 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 assessment s determined
the amounts of 1) moisture, protein, total fat, ash, crude fiber, carbohydrate, and calories ; 2) vitaminsJ.R. Simplot Company JRS01 Page 51 of 74

B6, B3, and C ; 3) minerals copper, magnesium, and potassium ; 4) glycoalkaloids ; 5) free amino ac ids ; 6)
total amino acids ; and 7) reducing sugars for tubers collecte d from events grown in potato

- growing
areas of the United States. For all the nutrients listed above, the goal was to demonstrat e that event s contain ed equivalent amounts compared with the untransformed controls

. Along with agronomic data, the compositiona l analyses help ed 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

va lues between each of the events and their untransformed co ntrols

. 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 tolera nce intervals: Snowden, Chieftain, Red Norland, Ida Rose

, Russet Burbank, Ranger Russet, Atlantic, G, and H.
The tolerance interval s w ere 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 int erval 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 1 0 for Ranger
Russet and Russet Burbank and in Table 1 1 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, E 24), and increased sucrose (H37). Both the intended changes and these observed differences fell wi thin the tolerance intervals or combined literature values, and therefore were considered to be withi n normal ranges for potatoes.

It’s important to note that for events E12 and E24, the free ASN levels w ere not statistically different
from controls at the adjusted p < .05 level . However, these events showed a 7 4 to 76% reduction in free

ASN

. Such larg e 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 sig nificance 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 pu rpose 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 change s between InnateTM tubers and their untransformed controls relates to
reduction of the amino acid asparagine (ASN) and reducing sugars

. Reduced amounts of free ASN andJ.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 Table s 10 and 11 as well as Appendix 9. Compositional Analyses )

.

Table 10 . Statistical Differences in C omposition 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

De creased 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

Within TI and CLR Reduced Acrylamide Yes

Decreased Sucrose Reduced Acrylamide No

Yes Within TI and CLR

TI = tolerance interval CLR = combined literature rangeJ.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 (B 6 ) 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 Pyr idox ine (B 6 ) 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 afte r 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 promotersJ.R. Simplot Company JRS01 Page 54 of 74

associat ed with the PhL/R1 genes effectively lowered reducing sugars near the time of harvest but these
differences were not su stained 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, acr ylamide 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 modificat ions from insertion of pS IM 1278 resulted in dramatic reductions in acrylamide that persisted throughout the typical storage periods.

This research also confirm ed that the events are as safe as controls and did not contain increased levels of natural glyc oalkaloids, which are toxins commonly found in Solanaceous crops

, including potato

. The princip al glycoalkaloids in potatoes are 

- solanine and 

- chaconine. For food safety purposes, an upper limit for glycoalkaloid content of 20mg per 100g of potato is ge nerally accepted

.

The InnateTM potato events F10, F37, E 12, E24, J3, J55, J78 , H37, H50 and their corresponding untransformed controls had mean glyc oalkaloid ranges from 5.7 to 11.3 mg/ 100g ( Table 1 2 ) ; all below the 20mg/ 100g safety limit described by S inden (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 me an glycoalkaloids of 20.3 mg/100g and untransformed contr ols
contained 19.8 mg/100g ( Table 1 2 ) . The higher mean level s 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 differ ences between the

event s and control s , 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 p otato, 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 CLR 2

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 2.2 0

- 154.2 0

G11 20.32 5.81

- 55.3 0.9153

H Control 10.65 5. 96

- 21.07

1.47 0

- 303.5 2.2 0

- 154.2 H37 10.76 5.41

- 17.25 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 sequence s 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 promoter s . 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 pro teins. 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 ( Asn 1, PPO) and prevent translation to form active gene products such as a protein. There fore no changes to amino acid sequences or proteins are found with InnateTM potatoes.

8

. 2

. A llergen icity of Innate TM 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 glyco protein that displays lipase activity and makes up about 40% of the s oluble protein in tubers (Mignery et al. 1988). Allergic reactions to processed potatoes are considered to beJ.R. Simplot Company JRS01 Page 56 of 74

very uncommon and have been reported for children only (De Swert et al. 2002; De Swe rt 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 the m and changes to patatin levels would not alter that behavior.

8

. 3

. Conclusions of C ompositional A ssessment

T he intentional changes in ASN and reducing sugars accomplished through t ransformation of various potato varieties with the DNA insert of pSIM1 278 resulted in lower acrylamide levels in f rench 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) a ny impact on nutritional quality would
be minimal from the observed differences in the levels of ASN, ASP, GLU and GLN because as non

essential amino acids they can be synthesized by the body. Unintentional changes in the amounts of specific amino acids, v itamin 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 diffe rent from untransformed controls and were within the literature ranges reported for potato varieties

. Therefore, i t can be conclude d 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

Innate TM potatoes, the evidence submitted demonstrates that the Innate TM technolog ies 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 Innate TM potat oes 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 represen ting 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). Ad ditionally

, 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 introd uced 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 plantsJ.R. Simplot Company JRS01 Page 57 of 74

into the environment and compare genetica lly engineered plants to their non

- engineered counterparts.
In the case of Innate TM 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 consump tion.

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 pota toes 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 g row 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 a re 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 pot ato 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 a void 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 o f the U nited States (Bamberg et al. 2003; Spooner et al. 2004 )

. T hese 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 extr emely 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 a nd 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 loca tions 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 majorJ.R. Simplot Company JRS01 Page 58 of 74

potato production a rea, lies between the mountainous regions w h ere 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 growi ng near potato fields ( Love

1994 ). Spooner et al. (2004) describe S. jamesii habitat in the U.S. as a mong boulders on hillsides, sandy alluvial stream bottoms, in gravel along trails o r roadways, rich organic soil of alluvial valleys, sandy
fallow fields, grasslands, juniper

- piny on 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 laborat ory techniques (Love 1994 ). Love concluded that, based on the barriers that exist, including geographic isolation (as described above), endosperm imbalances, and multiple ploidy le vels, natural hybridization is hig hly unlikely, and introgression ( cross hybridizat ion over multiple generations) impossibl e 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 c ultivate d 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 aborti on, 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. Weedine ss 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 provi de 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 potatoesJ.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 o n 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 petiti on 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 si milar 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 t hree different crop yea rs 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 Innate TM potatoes would mean that all insects would be considered non

target. There are a large number of insects that feed on potato leaves and other insects that feed on pests. Many of these insects, in cluding 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 t hree 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 ofJ.R. Simplot Company JRS01 Page 60 of 74

acres. The modifi cation 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, o r 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 pes ticides 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

s usceptibility, 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. U se of Innate TM technolog ies 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 t he 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 saf e 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.

1 0

. Crop Introduction and the Potato Industry

1 0 .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 l argely indistinguishable from existing varieties. The reduction in asparagine and sugars using Innate TM technolog ies 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 re duce 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 t he 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 blac k spot bruise in Ranger Russet may increase the popularity of that variety.J.R. Simplot Company JRS01 Page 61 of 74

1 0 .2. Potato uses and exports

Potatoes are grown commercially in 36 of the 50 states in the US. Total US production in 20 10 was 4 0

. 4 billion lbs, with total value of $3. 72 billion, planted on 1.0 3 million acres (NPC 201 2 ). The commercial
uses include 3 7 % as frozen, 2 7 % sold fresh, 1 4 % chips, 8 % dehydrated, 5% seed potatoes, and 1% canned (NPC 201 2 ). Annual per capita consumption in the US was expected to be approximately 11 2 lbs per person in 201 2 (NPC 201 2 ).

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 2010 b ). 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 E RS 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 20 10 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 weigh t production (USDA ERS 2010).

1 0 .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.

1 0 .4. Impact on the Organ ic 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
f arming, 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 org anic 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

1 0

. 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 co untries, 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 th e 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 2010 a ) 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 2010 a ). These two crops provide significant quantities of GM

- derived ingredients in widely marketed pro cessed 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 acr ylamide 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 an d the products derived from them.

1 0

. 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
pot atoes. 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 r ecord 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 p rior 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 actionsJ.R. Simplot Company JRS01 Page 63 of 74

to avoid com pliance 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 s lowly 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 indust ry 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 s tewardship 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 th e 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 importin g 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 w ill 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 introduct ion.

11. Conclusions : Determination of Non r egulated Status for Innate TM Potatoes

Simplot’s Innate TM technologies allow us to transform plants with p otato DNA and only introducing non

coding 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 expr ession of the integrated genetic elements results in traits highly sought

- after in the potato industry, namely: reduced black spot bruise, lowJ.R. Simplot Company JRS01 Page 64 of 74

asparagine, and lower levels of reducing sugars. Ultimately, we demonstrate that the Innate TM t ubers
exhibited t he 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

untrans formed control s

.

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
transfo rmed using our Innate TM technologies and submit the evidence that these plants should not be classified as “Regulated artic les” as defined under 7 CFR 340.J.R. Simplot Company JRS01 Page 65 of 74

1 2

. Statement of G rounds U nfavorable

JR Simplot is not aware of any information indicating that Innate TM 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

1 4

. 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 1 3 ). Volunteer monitoring for 2009, 2010, and 2011 field trials is
discussed in Appendix 10 ( Post Harvest Monitoring ).

Table 13 . Field re lease 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 20 10 1/25/20 10

– 7/31/201 1 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 5/0 2 /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/ 0 4/2011

– 4/04/2012 WA Yes

12

- 018

- 110n 2012 2/01/2012

– 2/01/2013 ID NoJ.R. Simplot Company JRS01 Page 68 of 74

1 5

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