i 

# JR Simplot Company **Petition ( 13**  **- 022**  **- 01p**  **) for Determination of Non**  **- regulated Status**  **of Low**  **Acrylamide Potential and Reduced Black Spot Bruise Potato Events F10, F37, E12, E24, J3, J55,**  **J78, G11, H37, and H50**  **OECD Unique Identifier: SPS**  **- 00F**  **10 -**  **7** , **SPS -**  **00 F37**  **- 7**  , **SPS -**  **00 E12**  **- 8**  , **SPS -**  **00 E24**  **- 2**  , **SPS -**  **000 J3**  **- 4**  , **SPS -**  **00 J55**  **- 2**  , **SPS -**  **00 J78**  **- 7**  , **SPS -**  **00 G11**  **- 9**  , **SPS -**  **00 H37**  **- 9**  , **SPS -**  **00 H50**  **- 4**  

## Plant Pest Risk **Assessment**
**March 2014 Agency Contact**  **Cindy Eck Biotechnology Regulatory Services**  **4700 River Road USDA, APHIS**  **Riverdale, MD 20737 Fax: (301) 734**  **- 8669** 
The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, sex, religion, age, disability, political beliefs, sexual orientation, or marital or family sta tus. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’S TARGET Center at (202) 720

–2600 (voice and TDD). To f ile a complaint of discrimination, write USDA, Director, Office of Civil Rights, Room 326

–W, Whitten Building, 1400 Independence Avenue, SW, Washington, DC 20250

– 9410 or call (202) 720

– 5964 (voice and TDD). USDA is an equal opportunity provider and employer. Mention of companies or commercial products in this report does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Prod

uct names are mentioned solely to report factually on available data and to provide specific information.
This publication reports research involving pesticides. All uses of pesticides must be registered by appropriate State and/or Federal agencies b efore they can be recommended.ii

A. Introduction
................................ ................................ ................................ ................. 1 B. Development of Ten JRSLA Potato Events
................................ ................................ 3 C. Description of Inserted Genetic Material, Its Inheritance and Expression, Gene Products, and Changes to Plant Metabolism
................................ 5 D. Potential Plant Pest and Disease Impacts
................................ ................................ .. 14 E. Potential Impacts on Nontarget Organisms Beneficial to Agriculture
..................... 22 F. Potential for Enhanced Weediness of JRSLA Potatoes
................................ ............ 23 G. Potential Impacts on the Weediness of An y Other Plants with which JRSLA Potatoes Can Interbreed
................................ ................................ .............. 25 H. Potential Changes to Agriculture or Cultivation Practices
................................ ....... 29 I. Potential Impacts from Transfer of Genetic Information to Organisms with which JRSLA Potatoes Cannot Interbreed
................................ ...................... 29 J. Conclusion
................................ ................................ ................................ ................ 31 K. References
................................ ................................ ................................ ................. 331



### A. **Introduction** The JR Simplot Company (JR Simplot) has petitioned the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) for a determination that 10 geneticall y - engineered (GE) potato (Solanum tuberosum L. ) events (F1 0, F37, E12, E24, J3, J55, J78, G11, H37 and H50) with low acrylamide potential and reduced black spot bruise are unlikely to pose a plant pest risk and, therefore, should no longer be regulated articles under the APHIS 7 Code of Federal Regulations (CFR) part 340 ( JR Simplot Company, 2013 ) . This petition was assigned the number 13
- 022
- 01p, and is hereafter referred to as JR Simplot , 2013 . Throughout this document, in cases where the ten potato events are discussed as a group, they will be collectively

referred to as JRSLA potatoes. APHIS administers 7 CFR part 340 under the authority of the plant pest provisions of the Plant Protection Act (PPA) of 2000. 1 This plant pest risk assessment was conducted to determine if JRSLA potatoes are unlikel y to pose a plant pest risk. APHI S regulations in 7 CFR part 340 regulate the introduction (importation, interstate movement, or release into the environment) of certain GE organisms and products. A GE organism is no longer subject to the plant pest provisions of the PPA or to the regulatory requirements of 7 CFR part 340 when APHIS determines that it is unlikely to pose a plant pest risk. A GE organism is considered a regulated arti cle under 7 CFR part 340 if the donor organism, recipient organism, or vector, or vector agent used in engineering the organism belong to an y genera or taxa designated in 7 CFR § 340.2 and meets the definition of plant pest, or is an unclassified organism and/or organism whose classification is unknown, or an y product which contains such an organism, or an y other organism or product altered or produced through genetic engineering which the Administrator determines is a plant pest or has reason to believe is a plant pest. 2 The 10 JRSLA potato events were produced b y Agrobacterium
- mediated transformation and nine of the events include introduced genetic sequences that were designed based on sequences found in plant pest organisms listed in 7 CFR § 340.2 (i.e., border sequences were designed based on border sequences found in Agrobacterium tumefaciens ; Table 5, pp. 29
- 30, JR Simplot, 2013 ). Therefore, the JRSLA potatoes are considered regulated articles under APHI S regulations at 7 CFR part 340. JR Simplot has c onducted introductions of JRSLA potatoes under APHIS
- authorized notifications since 2009 ( JR Simplot, 2013 , Table 13, page 66) in part, to gather information to support that JRSLA potatoes are unlikel y to pose a plant pest risk.

1 Plant Protection Act in 7 U.S.C. 7702 § 403(14) defines plant pest as: “Plant Pest
- The term “plant pest” means any living stage of any

of the following that can directl y or indirectly injure, cause damage to, or cause disease in any plant or plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A fungus. (F) A virus or viroid. (G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding subparagraphs.” 2 Li mited exclusions or exemptions apply for certain engineered microorganisms and for interstate movement of some organisms, as in 7 CFR 340.1 and 340.2.(b).2

Potential impacts to be addressed in this Plant Pest Risk Assessment are those that pertain to plant pest risk associated with the 10 JRSLA potato events and their progeny and their use in the absence of confinement relative to the unmodified recipient cul tivars and/or other appropriate comparators. APHI S used data and information submitted by the applicant, in addition to current literature, to determine if the 10 JRSLA potato events are unlikely to pose a plant pest risk. APHI S regulations in 7 CFR § 340 .6(c) specify the information needed for consideration in a petition for nonregulated status. APHI S will assess information submitted by the applicant about the 10 JRSLA potato events related to: plant pest risk characteristics; expression of the gene pro duct, new enz ymes, or changes to plant metabolism; disease and pest susceptibilities and indirect plant pest effects on other agricultural products; wee diness of the regulated articles; effects of the regulated articles on nontarget organisms; impact on th e weediness of an y other plant with which they

can interbreed; changes to agricultural or cultivation practices that may impa ct diseases and pests of plants; indirect plant pest effects on other agricultural products; and transfer of genetic information to organisms with which they cannot interbreed. APHI S may

also consider information relevant to reviews conducted by other agencies that are part of the ‘Coordinated Framework for the Regulation of Biotechnology ’ ( 51 FR 23302, 1986 ; 57 FR 22984, 1992 )
. Under the Coordinated Framework, the oversight of biotechnology
- derived plants rests with APHI S, the Food and Drug Administration (FDA), and the Office of Pesticide Programs of the U.S. Environmental Protection Agency (EPA). Depending on their characteris tics, certain biotechnology
- derived products are subjected to review b y one or more of these agencies. EPA regulates under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U
. S
. C
. 136 et seq

) the distribution, sale, use and testing of pe sticidal substances produced in plants and microbes, including those pesticides that are produced b y an organism through techniques of modern biotechnology . EPA also sets tolerance limits for residues of pesticides on and in food and animal feed, or estab lishes an exemption from the requirement for a tolerance, under the Federal Food, Drug and Cosmetic Act (FFDCA) (21 U
. S
. C
. Chapter 9 ). Prior to registration for a new use for a new or previously registered pesticide, EPA must determine through testing that the pesticide does not cause unreasonable adverse effects on humans, the environment, and non
- target species when used in accordance with label instructions. EPA must also approve the language used on the pesticide label in accordance with 40 CFR part 15 8. Other applicable EPA regulations include 40 CFR p art 152
- Pesticide Registration and Classification Procedures, p art 174
- Procedures and Requirements for Plant Incor porated Protectants (PIPs) and p art 172
- Experimental Use Permits. The 10 JRSLA potat o events are not engineered to express substances to protect the potatoes against plants pests, and are therefore not subject to EPA review. The FDA under the FFDCA is responsible for ensuring the safety and proper labeling of all plant
- derived foods and feeds, including those developed through modern biotechnology . To help sponsors of foods and feeds derived from genetically engineered crops compl y with their obligations, the FDA encourages them to participate in its3

voluntary earl y food safety evaluation for new non - pesticidal proteins produced b y new plant varieties intended to be used as food ( FDA, 2006 ) and a more comprehensive voluntary consultation p rocess prior to commercial distribution of food or feed ( 57 FR 22984, 1992 )
. JR Simplot stated in their petition that they have initiated the FDA consultation process ( JR Simplot, 2013 , page 15). 

### B. **Development of the Ten JRSLA Potato Events**  Potatoes ( Solanum tuberosum ) belong to the genus Solanum , section petota , subsection potatoe , and series tuberosa, whi ch consists of approximately 54 species, of which onl y S. tuberosum is widely

cultivated for food production ( OECD, 1997 )
. 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 North America and Europe, and the subspecies andigena is also cultivated, but cultivation is largel y restricted to Central and South America ( OECD, 1997 )
. After China, India, Russia, and the Ukraine, the United States is the fifth largest potato producing country ( FAO, 2013 ) , with annual production over the last three years of between 404
- 467 million cwt (centum weight = 100 pounds) , grown on 1.0
- 1.1M acres (USDA
-NASS, 2013 ) . Potatoes are grown across most of the cont inental US, with six States (Colorado, Idaho, Minnesota, North Dakota, Washington and Wisconsin) accounting for approximately 75% of annual production (USDA
-NASS, 2013 ). The average American consumes about 115 lb s of potato annuall y, of which about two thi rds is consumed as processed potato products (USDA
- ERS, 2010 )
. Different potato varieties have been selected for performance in the fresh and processed markets. The russet varieties store well and are the source of most fresh market potatoes and French fries, while the variety Atlantic does not store well and is us ed primarily to produce potato chips ( JR Simplot, 2013 , pp. 25
- 26). The tetraploid nature of commercial potato varieties is a significant impediment to potato breeding ( Hoopes and Plaisted, 1987 ) . Due t o more complex chromosome segregation ratios, poly

ploid crops are inherentl y more difficult to breed. Furthermore, vegetativel y propagated crops like potato are often poor seed producers due to partial or full sterility
. For seed propagated crops, like c orn or so ybean, trait developers often create a single elite event and then backcross that elite event into a wide range of elite germplasm. This is not possible in potato. Each parent variety must be independentl y transformed to achieve the desired phen oty pe in that variety. JR Simplot has used a genetic engineering approach to introduce into the background of commercial potato cultivars two traits that are of interest to potato consumers, producers and processors: reduced acrylamide potential in certain processed or heated potato products and reduced black spot bruise. JR Simplot used the single construct pSIM1278 to transform 5 different commercial parent varieties and created the 10 events described in JR Simplot, 2013 . The objective was to incorpo rate the same new phenoty pes into each of these important varieties, while maintaining all of the desirable characteristics4

originall y selected b y potato breeders. If a single variety were transformed, it would take decades to move these new traits into the other commercial varieties b y conventional breeding, and even then, it would be difficult to reconstitute the desirable characteristics of the original variety. In 2002, Swedish researchers demonstrated that acrylamide forms when starchy foods, such as potatoes and breads, are heated ( Tareke et al., 2002 ) . These scientists were looking for the source of acrylamide
- induced blood adducts in individuals not known to be exposed to acrylamide. They de monstrated that acrylamide forms when starchy foods are heated, however it was not detected in unheated or boiled foods ( Tareke et al., 2002 )
. Therefore, even though dietary exposure to acrylamide is measurable, it is not a natural compositional component of unheated foods derived from plants. The often desired non
- enzy matic browning that occurs when food is cooked is referred to as the Maillard reaction ( Martins et al., 2000 )
. Maillard reaction products, which impact the flavor and texture of the cooked food, are formed by a chemical reaction between an amino acid and a reducing sugar. Stadler et al ( 2002 ) demonstrated that oxidation of the free amino acid asparagine is the main source of acrylamide when starchy foods are baked or fried. Asparagine is a major amino acid in potatoes and cereals ( Mottram et al., 2002 )
. Because acrylamide is a known carcinogen of rodents and a probable carcinogen in people ( WHO
- IARC, 1994 ; NTP, 2011 ) the discovery of acrylamide in cooked potato products raised concerns throughout the potat o processing industry , as well as among consumers. The State of California listed acrylamide as a potential carcinogen under Proposition 65 in 1990 and established a No Significant Risk Level (NSRL) of 0.2 μ g/ day ( CEPA
- OEHHA, 2005 )
. Subsequent to the discovery of acrylamide in cooked foods, this NSRLwas revised to 1.0 μg/day ( CEPA
- OEHHA, 2005 )
. Proposition 65 requires that food manufacturers warn consumers about the dangers of acryl amide in their products. In 2005, the State of California sued Frito
- Lay, Kettle Foods and Lance, Inc. for failing to provide such warnings. In the settl ement, the potato chip manufacturers agreed to reduce the acrylamide in their products to 275 ppb, low enough to avoid the Proposition 65 warning. These three companies also agreed to pay close to $2M in penalties and court costs. The potato processing i ndustry now has a strong financial incentive to reduce the levels of acrylamide in their retail products. Black spot bruising is a post
- harvest ph ys iological disorder primaril y resulting from the handling of potato tubers during harvest, transport and processing. If ph ys ical impact causes cell rupture and/or flesh injury , the injured cells release phenolic co mpounds, normally compartmentalized in the vacuoles, which are converted to o - phenols and o quinones by

the enzy me poly phenol oxidase (PPO). These quinoids auto
- oxidize, forming melanin, leading to blackened tissue which is undesirable in processed potato product ( Hunt et al., 1993 )
.5

The intended purpose of the 10 JRSLA potato lines is to provide the potato processing industry with new varieties with low acrylamide potential and reduced black spot bruise. Both of these changes are intended to benefit potato consumers, producers, and processors. The low acrylamide potential is intended to benefit consumers because of concerns about the health effects of ingesting acrylamide, and to benefit the industry relative to Proposition 65. The reduced black spot bruise is intended to benefit consumers b y providing a higher quality product, to benefit producers b y reducing culls at delivery , and to benefit processors by reducing pick
- outs. 

### C. **Description of Inserted**  **Genetic Material,**  **Its Inheritance and**  **Expression , Gene Products,**  **and Changes to Plant Metabolism** To inform the potential hazards resulting from the genetic modification and potential routes of exposure related to the inserted DNA and its expression products , APHI S assesse d data and information presented in the petition related to : the transformation process ; the source of the inserted genetic material and its function in both the donor organism and the GE crop event ; and the integrity, stability and mode of inheritance of the inserted genetic material through sexual or asexual reproduction based on the location of the insertion ( e.g. nucleus or organelle) and the number of loci inserted. APHI S also assessed data presented in the petition on whether the genetic modification results in expression of new g enes, proteins , or enzy mes , suppression of existing genes and their products, or changes in plant metabolism or composition in JRSLA potatoes relative to the ir nontransgenic counterparts . The assessment encompasses a consideration of the expressed double stranded RNA (dsRNA) and any observed or anticipated effect s on plant metabolism including , e.g. an y relevant changes in levels of metabolite s , antinutrient s , or nutrient s in harvested potatoes derived from the GE crop event compared to those in th e conventional counterpart and other comparators. This information is used later in this risk assess ment to inform whether there is any potential for plant pest vectors or sequences to cause disease or greater plant pest risks in the GE crop event ; or for expression of inserted DNA , new proteins or enzy mes, or changes in metabolism to affect plant pest or disease s, nontarget beneficial organisms, weediness, agricultural practices that impact pest or diseases or their management , or plant pest risks through horizontal gene flow. **Description of the genetic modification and inheritance of inserted DNA**  The 10 JRSLA potato events were produced by using Agrobacterium
- mediated transformation of potato internode explants of 5 different varieties: Ranger Russet, Ru sset Burbank, Atlantic, variety G and variety H ( JR Simplot, 2013 , p. 26
- 29). The binary plasmid vector pSIM1278 (Figure 3 in JR Simplot, 2013), consisting of the vector backbone ( JR Simplot, 2013 , Table 4) and the DNA insert ( JR Simplot, 2013 , Table 5) was used to create all 10 events; only the DNA insert portion was intended to be transferred to the recipient plants. After transformation, the explants were subjected to antibiotic treatment with 150 mg/L timentin, a concentration previously s how to be6

effective to eliminate the A. tumef a ciens vector (Nauerb y et al., 1997 )
. Lack of A. tumefaciens was confirmed by incubating stem fragments on nutrient a gar for 2 weeks at 28 ° C ( JR Simplot, 2013 , p. 25). A marker
- free selection sy stem was used to eliminate plants in which vector backbone inserted into the genome due to inefficient cleavage at the Left Border site ( Gelvin, 2003 ; Richael et al., 2008 ) . To this end, JR S implot inserted the i sopenten yltransferase ( ipt) gene from A. tumefaciens into the vector backbone just outside the L eft Border ( JR Simplot, 2013 , Figure 3 and Table 4). Transgenic plants expressing the ipt gene , which results in the production of the plant hormone cy tokinin, display

a stunted phenoty

pe that can be visuall y identified, allowing the elimination of plants with inserted vector backbone. The remaining plants were then molecularly characterized by

PC R and Southern blots to select those containing the genes of interest , and thus do not contain the ipt gene. The DNA insert in plasmid pSIM1278 is designed to silence four different genes in the potato: asparagine s ynthetase
- 1 ( Asn1 ), pol yphenol oxidase
- 5 ( Ppo5 ), potato phosphorylase L (

PhL ) and the starch
- associated R1 gene ( R1 ). For each gene, the expression cassettes are designed to produce dsRNA that functions through an RNA interference (RNAi) mechanism to degrade transcripts for the genes. They ar e not designed to encode a full open reading frame to produce protein ( for details, see pages 31

– 32 in JR Simplot, 2013 ). The suppression of Asn1 should result in potatoes with reduced free asparagine, and the suppression of PhL and R1 should result in p otatoes with a lower content of reducing sugars. Collectively , the silencing of these 3 genes should result in potato tubers with a reduced acrylamide potential. The suppression of Ppo5 confers the JRSLA potatoes with a non
- browning phenoty pe resulting i n tubers with reduced black spot bruising. The DNA insert of pSIM1278 contains two expression cassettes, the first designed to silence Asn1 and Ppo5 and the second cassette designed to silence PHL and R1. With the exception of the left and right borders, all inserted DNA in the ten JRSLA events was derived from S. tuberosum var. Ranger Russet or from S. verrucosum . Sy

nthetic DNA designed to be similar to and function like Agrobacterium T
- DNA borders was used to generate the left and right borders. The first gene cassette consists of seven genetic elements designed to silence the Asn1 and Ppo5 genes :  pAgp , the ADP glucose py rophosphorylase promoter sequence from S. tuberosum. This promoter directs the silencing construct in the antisense orientation.  fA sn1 , a fragment of the protein coding region of the Asn1 gene from S. tuberosum , arranged in antisense orientation ( Chawla et al., 2012 )
.  tPpo5 , the 3’ untranslated leader sequence of the Ppo5 gene from S. verrucosum , arranged in antisense orientation.  S pacer - 1, a 10 kb fragment derived from S. tuberosum , inserted between the two inverted repeats to create the hairpin which enhances gene silencing.  tPpo5 , the 3’ untranslated leader sequence of the Ppo5 gene from S. verrucosum , arranged in sense orientatio n.7

 fAsn1 , a fragment of the Asn1 gene sequence from S. tuberosum , arranged in sense orientation.  pGbss , the granule
- bound starch s ynthase promoter sequence from S. tuberosum
. This promoter directs the silencing construct in the sense direction, and is oriented convergentl y relative to the first pAgp
. The second gene cassette consists of seven genetic elements designed to silence the PhL and R1 genes:  pAgp , the ADP glucose py rophosphorylase promoter sequence from S. tuberosum. This promoter directs the silencing construct in the antisense orientation.  pPhL , a fragment of the PhL promoter from S. tuberosum , arranged in antisense orientation.  pR1 , a fragment of the R1 promoter sequence from S. tuberosum , arranged in antisense orientation.  Spacer
- 2, a 257 bp fragment derived from S. tuberosum , inserted between the two inverted repeats to create the hairpin which enhances gene silencing.  pR1 , a fragment of the R1 promote r sequence from S. tuberosum , arranged in sense orientation.  pPhL , a fragment of the PhL promoter from S. tuberosum , arranged in sense orientation.  pGbss , the granule
- bound starch s ynthase promoter sequence from S. tuberosum
. This promoter directs the sil encing construct in the sense direction, and is oriented convergentl y relative to the second pAgp
. The first cassette is preceded by the left border and the second cassette is followed by the right border. Table 5 in JR Simplot, 2013 provides a dditional detail for all of these genetic elements. Even though the same pSIM1278 was used to create the 10 JRSLA events, each insertion is unique with some being simple insertions, some double insertions and others more complex (Table 1). More detail for each eve nt can be found in JR Simplot, 2013 , Figure 5. **Table 1.** The parent variety, number of inserts and a brief description of the nature of the inserts in the 10 JRSLA potato events. **Event Parent Variety**  **Insert Copies Comments**  F10 Ranger Russet Single Complete insert F37 Ranger Russet Single Complete insert E12 Russet Burbank Single Complete insert E24 Russet Burbank Single Nearly complete insert with small deletion at the left border J3 Atlantic Single Nearly complete insert with deletion of the left border and a small portion of the Agp promoter of the first cassette, fused to an inverted copy of the first cassette J5 5 Atlantic Single Complete insert fused to an inverted copy of the first cassette)8 J7 8 Atlantic Single Partial insert comprising first cassette plus partial second cassette, truncated after the second R1 fragment (so missing the second PhL fragment and Gbss promoter) G11 G Single Partial insert comprising first cassette only (truncated i n the first PhL fragment of the second cassette) H37 H Complex One complete insert and three unlinked partial inserts, one of which includes a complete second

cassette H50 H Double One complete insert and one unlinked partial insert with the first cassette only (truncated in the first PhL fragment of the second cassette) JR Simplot provided evidence demonstrating that:  The A. tumefaciens strain AGL1 that was used to transform the 5 parental varieties is nonpathogenic, having been disarmed of sequences which lead to tumor formation in plants ( Lazo et al., 1991 )
.  The 10 JRSLA events do not contain vector backbone sequences as determined through Southern blot and Poly merase Chain Reaction assay s ( JR Simplot, 2013 , Appendix 2).  The genomes of the 10 JRSLA pot ato events contain either one or two copies of full or partial inserts between the Left and Right borders in pSIM1278 ( JR Simplot, 2013 , Appendix 1; as summarized in Table 2 above). Events J78 and G11 only contain the first expression cassette and insuff icient portions of the second expression cassette.  The insertions in the 10 JRSLA potato events were stably inherited through at least three generations of vegetative propagation, as confirmed b y DNA gel blot hy bridization (all 10 JRSLA events) and a pheno ty pic assay for the suppression of PPO (all events except J3, J55, and J78) ( JR Simplot, 2013 , Appendix 3). All 10 of the JRSLA potato events contained the first expression cassette containing the genetic elements to silence Asn1 and Ppo5 . Since potatoes are not propagated by seed, stability

through seed generations is not relevant to the present anal ys is. **Expression of inserted DNA, changes in gene expression , new proteins**  **or metabolism**  APHI S assessed whether changes in plant metabolism or composition of JRSLA potatoes are likel y to alter plant pest risk relative to the untransformed control. The assessment encompasses (1) a consideration of the specific effects on plant metabolism due to silencing Asn1 , Ppo5 , PhL and R1 ; and (2) evaluation of whether the nutrient and antinutrient levels in harvested tubers of the 10 JRSLA events are comparable to those in the respective parental varieties and other reference potato cultivars. Host plant quality (including such components as carbon, nitrogen, amino acid sources, trace elements, and9

defensive metabolites) is known to affect herbivore performance and fecundity ; and higher
- trophic level interactions, such as the performance of predators and p arasitoids, may also be affected (reviewd by Awmack and Leather, 2002 ) . Similarly a vast array of secondary metabolites in plants is known to provide defense against microbes ( Dixon, 2001 ) . Thus changes in host plant quali ty and composition have the potential to affect the performance of JRSLA potatoes against pest s and disease s and to affect the suitability of JRSLA tubers as a food source for non
- target organisms
. To as sess the efficacy

of the gene silencing constructs, JR Simplot examined Asn1 , Ppo5 , PhL and R1 transcript levels in tubers, stolons, roots, stems, leaves, and flowers in the 10 JRSLA events. In addition, to assess the efficacy of gene silencing and determi ne whether there are an y unintended compositional changes to JRSLA potatoes, JR Simplot compared the tuber composition of the 10 JRSLA events with the tuber composition of their respective parent varieties. The analyzed tubers were sampled from 23 differe nt trials conducted in 2009, 2010 and 2011 in 8 different states; however, not all varieties were included in every

trial. JR Simplot selected locations to represent the major potato production areas in the US; trials were conducted in Id aho, Washington, North Dakota, and Wisconsin , states that account for more than half of US potato production (USDA
NASS, 2013 )
. The Ranger Russet, Russet Burbank and Atlantic events were sampled all three ye ars and the data presented as combined analy ses across locations and years. For the G and H varieties the compositional analy ses are all based on 2010 samples ( JR Simplot, 2013 , Appendix 9). The exact function of PPO in plants remains elusive ( Bachem et al., 1994 ; Mayer, 2006 )
. The potential role of PPO in plant defense against pests and pathogens is discussed in the next section below. In potato, PPO is involved in black spot bruise formation, which reduces the quality

of harvested tubers ( Bachem et al., 1994 ; Cors ini et al., 1999 ; Partington et al., 1999 ) . PPO oxidizes monophenols and o - diphenols to o - quinones, which are then further oxidized non
- enzy maticall y to poly

phenols. These dark
pigmented pol yphenols, also referred to as melanins, result in the darkening of potato tissue following mechanical bruising. Black spot bruise can lead to economic losses as high as 20% ( Partington et al., 1999 ) ; the potato industry

therefore has a vested interest in minimizing these losses. Bachem et al 1994 demonstrated that black spot bruise can be reduced b y silencing Ppo genes in potatoes, and JR Simplot, 2013 has further developed this concept in the design of JRSLA potatoes. PPO is not substrate specific and is capable of oxidizing a wide variety of monophenols and o - diphenols. Chlorogenic acid is the major phenolic component in potato tubers, making up 90% of the total phenolic content ( Friedman, 1997 ) , and is therefore the primary PPO substrate in potato tubers. There is a correlation between PPO activity and the rate of browning in different p otato varieties, but browning is not correlated with content of chlorogenic acid. Internal blackspot in potatoes is caused by internal enzy matic browning type reactions initiated b y PPO catal ys is with ty rosine as the primary substrate ( Friedman, 1997 )
.10

In potato, PPO comprises a multigene family with at l east six genes differing in temporal and tissue
- specific expression ( Thygesen et al., 1995 )
. Thygesen and colleagues reported that one member of the PPO gene famil y, Ppo5 (called POT32 in Thygesen et al 1995) is the primary

form found in potato tubers, and is the primary message detected in older tubers. While also expressed in roots, no expression was detected in photosy nthetic tissues. NOR333 (also called P2 ), another member of the potato PPO gene family , was detected in young leaves and tissue near the tuber skin, but was highl y expressed in flowers ( Th ygesen et al., 1995 ) . Other PP O genes are expressed in tubers in lower amounts: POT33 is expressed mainly in the tissue near the skin and POT72 is expressed in developing tubers; expression patterns for POT41 and P1 , which are 95% and 97% homologous with Ppo5 and NOR333 respectivel y, were not reported ( Th yg esen e t al., 1995 ) . JRSLA potatoes are engineered to specifically reduce expression of Ppo5
. JR Simplot provides evidence demonstrating that:  Based on qualitative RNA (northern) blot anal ys es of various tissues of the 10 JRSLA events and nontransgenic co ntrol varieties, Ppo5 transcripts were down
regulated in tubers of all 10 events, in stems of nine events (all except G11), in roots of five events (F10, F37, E12, H37, and H50) and in stolons of three events (E24, G11, and H50) ( JR Simplot, 2013 , Table 6, p. 40, and Appendix 5). No transcripts h ybridizing to the Ppo5 probe were detected in leaves of nontransgenic control varieties or in leaves of an y of the 10 JRSLA events. Similarly , transcripts hy bridizing to the Ppo5 probe were not detected in flowers of the eight JRSLA events assayed that produce flowers or of their nontransgenic control varieties. From the data provided, it is not possible to definitely determine whether the results reflect down
- regulation of Ppo5 transcripts alone, or whether the re sults also or instead reflect down
- regulation of some of the other Ppo genes, particularl y POT41 , since its expression pattern has not been characterized nor its DNA sequence made available. However, it is unlikely that NOR333 transcripts were down
- regulat ed via RNAi since there is no homology between the tPpo5 sequence introduced into the JRSLA potatoes and the NOR333 gene.  Using a catechol assay , there was a lack of functional PPO activity in tubers of seven of the 10 events (see Table 2; JR Simplot, 20 13 , Appendix 3). The three Atlantic events (J3, J55 and J78) were not tested with the catechol assay because the Atlantic parent variety is known to have some resistance to black spot bruising ( JR Simplot, 2013 , p.26) and did not react with catechol ( JR S implot, 2013 , p. 47). In contrast, Russet Burbank and particularl y, Ranger Russet, are more susceptible to black spot bruise ( Potato Association of America, 2009 )
. JRSLA potatoes were designed to have reduced levels of free asparagine by silencing Asn1 as well as reduced levels of reducing sugars by silencing PhL and R1 . Together, the silencing of these three genes was expected to reduce the acrylamide content in cooked potato products. JR S implot qualitatively

examined the Asn1 , R1 , and PhL transcrip t levels in tubers from field and greenhouse grown plants and in stolons, roots, stems, leaves, and flowers of11

greenhouse grown plants ( JR Simplot, 2013 , Appendix 5). These data are summarized below and in Table 6, page 40, of JR Simplot, 2013. Free glut amine, free asparagine, and reducing sugar levels in tubers were also examined (see Table 2), and tubers were processed into French fries and potato chips in order to anal yz e acrylamide content ( JR Simplot, 2013 , Appendix 9). JR S implot provides evidence d emonstrating that:

 Asn1 transcripts were down
- regulated in tubers of all 10 events, in stolons of nine events (all but H37) , in flowers of all eight flowering events (all except H37 and H50) , in roots of six events (F10, F37, E12, J3, J55, and H50) , in st ems of four events (F37, E24, J3, and H50) , and in leaves of one event (F10)
.  Seven of the 10 JRSLA events (F10, F37, J3, J55, G11, H37, and H50) displayed a statistically

significant reduced content of free asparagine while three events (E12, E24 and J78) had only numerically

reduced levels.  Five of the 10 JRSLA events (F10, F37, G11, H37, and H50) displayed a statistically

significant increased content of free glutamine while the other five events (E12, E24, J3, J55 and J78) had only numerically

increased levels. In creased content of free glutamine is an expected consequence of reducing the content of free asparagine. This is because the enzy me asparagine s ynthetase also functions to deaminate glutamine to glutamate ( JR Simplot, 2013 , Figure 6).  R1 tran scripts were down
- regulated in greenhouse grown tubers of eight events (all except J78 and G11) and in field grown tubers of some of these events ( JR Simplot, 2013 , Appendix 5, Figure 4). R1 transcripts were also down
- regulated in stolons of the same eight events , roots of three eve nt s (F10, F37 and H50) , stems of one event (G11) and leaves of two or three event s (G11 and H50, and possibly H37) ( JR Simplot, 2013 , Table 6, and see Appendix 5, Figure 12 for R1 in leaf tissue )
. R1 transcripts were not down
- regulated in G11 tubers due to a truncation of the inserted DNA which removed the pR1 fragments ( JR Simplot, 2013 , Figure 5 ). R1 transcripts were also not down
- regulated in greenhouse grown tubers of J78, but were down
- regulated in field grown tubers of that line. R1 transcripts were unexpectedly

down
- regulated in stems and leaves of event G11, which does not contain the PhL/R1 silencing cassette. The reason for this apparent down
regulation, which was not observed in tubers, stolons, or roots, is unkn own.  PhL transcripts were down
- regulated in tubers of six events (E12, E24, J3, J55, H37, and H50) and in stolons of seven events (F10, F37, E12, E24, J55, H37, and H50) (see also JR Simplot, 2013 , Appendix 5, Figure 7). PhL transcripts appear to be slightly

down
- regulated in leaves of three events (F10, J55 and J78) (see also JR Simplot, 2013 , Appendix 5, Figure 11), but were not down
- regulated in roots or stems of an y event . PhL transcripts were not down
- regulated in J78 or G11 tubers due to a truncatio n of the inserted DNA which removed some or all of the PhL/R1 silencing cassette ( JR Simplot 2013, Figure 5 ).  Six events (F10, F37, E12, E24, J3 and J55) had significant decrease in tuber reducing sugar (fructose + glucose) content after 1 month in storage ( JR Simplot, 2013 , Appendix 9). However, these differences were not detected in fresh tubers (except for J3 and J55) or after longer periods of storage. A decrease in reducing sugars was not tightl y correlated with the presence of the complete silencing cassette for the R1 or PhL genes. In particular, there is no significant decrease in12 reducing sugars in fresh tubers in events F10, F37, E12, E24, H37 and H50, all of which contain this cassette. An unexpected decrease in reducing sugars was observed in f

resh J78 tubers which do not contain the full silencing cassette for the R1 or PhL genes.  Cooked potato products from fresh tubers of all 10 events, as well from tubers after up to 2 or 3 months in storage, had statistically significant reductions in acryl amide content. For events F10, F37, E1 2 and E24, these reductions were statistically

significant through 7 months of storage ( JR Simplot, 2013 , Appendix 9). Cooked potato products of events J78 and G11, which lack the second cassette of pSIM1278, had levels of acryl ami de similar to those in the other 8 JRSLA events . Consistent (although not alway s statistically significant) reductions in free asparagine and increases in free glutamine across all 10 JRSLA events suggest that silencing Asn1 was suff icient to achieve the desired low acrylamide phenoty pe, and reductions in reducing sugar content may

not be required. Considering the variable and often insignificant differences in reducing sugar content in the JRSLA tubers relative to their respective p arent varieties ( JR Simplot, 2013 , Appendix 9), the efficacy of silencing PhL and R1 conferred b y pS IM1278 appears limited. pSIM1278 did not confer the desired phenotype of lower levels of reducing sugars in JRSLA tubers. **Table 2.** Tuber composition pheno ty pes of JRSLA Potatoes **Event PPO** 

**activity ASN/GLN** 

**level Reducing sugars** 

**level Acrylamide level**  F10 absent ↓/ ↑ ↓at 1 month ↓, through 7 months F37 absent ↓/ ↑ ↓at 1 month ↓, through 7 months E12 absent */* ↓at 1 month ↓, through 7 months E24 absent */* ↓at 1 month ↓, through 7 months J3 not tested ↓/* ↓at 1 month ↓, through 2 months J55 not tested ↓/* ↓at 1 month ↓, through 2 months J78 not tested */* no change ↓, through 2 months G11 absent ↓/ ↑ no change ↓, through 2 months H37 absent ↓/ ↑ no change ↓, through 2 months H50 absent ↓/ ↑ no change ↓, through 2 months *Reduction in Asn and/or increase in Gln occurred but was not statistically significant No functional enzy

mes or proteins are expressed by the inserted DNA. The ipt gene used for negative selection resides in the vector backbone and is therefore not expressed in the 10 JRSLA events, all of which are free of vector backbone ( JR Simplot, 2013 , Appendix 2). JR Simplot also analy

zed tubers of the 10 JRSLA events and the ir respective parents for the following constituents: protein, fat, ash, crude fiber, carbohydrates, calories, moisture, vitamin B 3 , vitamin B 6 , vitamin C, Cu, Mg, K, amino acids, sugars, and gl ycoalkaloids13

( JR Simplot, 2013 , Appendix 9). This composition al anal ys is of tubers confirmed the following:

 For key

proximates, vitamins, and minerals, there were no statisticall y significant differences between an y of the 10 events and their respective parent varieties for content of protein, fat, ash, crude fiber , carboh ydrates, calories, copper, magnesium or potassium. However, events F10 and F37 had statisticall y significant increases in niacin (vitamin B3 ) and vitamin C content, and events J3 and J55 had statistically

significant decreases in pyridoxine (vitam in B6) content ( JR Simplot, 2013 , Appendix 9).  For amino acids, event F37 had statistically significant increases in free aspartate and free arginine content, event G11 had statistically significant increases in free ly sine and free proline content while e vent H37 had a statistically significant decrease in free l ys ine content, and events J3 and J55 had statistically significant decreases in free valine content ( JR Simplot, 2013 , Appendix 9). Consistent with the decreased content of free asparagine and inc reased content of free glutamine in all events as mentioned above (see Table 2 above), as expected most of the events (F10, F37, E12, E24, G11, H37, and H50) also had corresponding significantl y decreased levels of total aspartate and asparagine and increa sed levels of total glutamate and glutamine.  Events E12 and E24 had statistically significant decreases, and event H37 had a statistically

significant increase, in sucrose content.  In all cases, these differences were within the 99% tolerance intervals g enerated from nine non - GE commercial varieties grown concurrentl y at the same field sites ( JR Simplot, 2013 , Appendix 9).  There were no significant differences between any of the 10 events and their respective parents for mean gl yc oalkaloid toxin content ( JR Simplot, 2013 Appendix 9). Although the upper end of the range of gl yco alkaloid content was higher in event H50 and much higher in event G11 than the limit of 20 mg per 100 g of potato that is generall y accepted as safe, the upper ends of the ranges fo r the control varieties were also high. The mean values for H50 and its control were below 20 mg per 100 g of potato, while the mean values for G11 and its control were very slightly above and below 20 mg per 100 g of potato, respectively , and did not diff

er statistically. A third event, E24, also exhibited elevated levels of gl yco alkaloids at the upper end of the range while its control did not. However, the mean gl ycoalkaloid value for event E24 was well below 20 mg per 100 g potato and did not differ statistically from its control. JR Simplot notes that handling conditions can result in higher gl yco alkaloid levels ( JR Simplot, 2013 , Appendix 9, p. 67). Based on the compositional anal ys es presented in JR Simplot, 2013 (Appendix 9), APHI S concludes that a lthough raw tubers of some of the JRSLA potato events had some statistically

significant differences in nutritional components (vitamins C, B3 and B6; certain amino acids, sucrose and reducing sugars) compared to their respective parent varieties, they

are nutritionally

equivalent to other commercial potato varieties. No new proteins are expected to be produced based on the inserted genetic elements and the14

genetic modification. For the few cases when significant differences were detected between the JRSLA events and their respective parents, the mean values for the JRSLA events all fell within the tolerance interval and in most cases, also fell within the combined literature range for potatoes. The primary Ppo gene expressed in roots, tubers, and stolons is suppressed in the JRSLA events, and reduction in PPO activity in JRSLA event tubers has been demonstrated. As discussed in the next section, the JRSLA potatoes were not observed to consistently exhibit any

increase in susceptibility to plant pathogens or pests as a result of the reduction in PPO activity. APHI S therefore concludes that the JRSLA potatoes pose no more of a plant pest risk from new gene products, changes to plant metabolism or composition than their respective parent varieties or other conventional potato varieties. 

### D. **Potential Plant Pest and Disease Impacts**  APHI S assessed whether potential plant pest or disease impacts are likel y t o result from the transformation process, from DNA sequences from plant pests, or from any other expression products, new enzy mes, proteins or changes in plant metabolism or composition in the JRSLA potatoes that are known or anticipated to cause disease sy mptoms, or to affect plant pests or diseases o r plant defense responses (as identified from the previous section). APHI S also assessed or whether the JRSLA potatoes are likely to have significantly

increased disease and pest susceptibility based on d ata and observations from field trials and laborato ry experiments on specific pest and disease damage or incidence and any agronomic data that might relate to such damage. Impacts or ch anges are assessed to determine if they would (1) affect the new GE crop and/or result in significant introduction or spread of a damaging pest or disease to other plants; (2) result in the introduction, spread, and/or creation of a new disease ; and/ or (3) result in a significant exacerbation of a pest or disease for which APHI S has a control program. Any increase in pest or disease susceptibility is evaluated with respect to the context of currentl y cultivated varieties, the ability to manage the pest or disease, and the potential impact on agriculture. Plant Protection and Quarantine (PPQ) is an APHIS program that safegu ards agriculture and natural resources from the entry , establishment, and spread of animal and plant pests and noxious weeds into the United States of America; and supports trade and exports of U.S. agricultural products. PPQ responds to many new introduc tions of plant pests to eradicate, suppress, or contain them through various programs in cooperation with state departments of agriculture and other government agencies. These may be emergency

or longer term domestic programs that target a specific pest. A variet y of insect, plant disease, mollusk, nematode or weed programs exist (USDA
- APHIS, 2013a )
. Currently , PPQ has active pest management programs for the golden nematode ( Globodera rostochiensis ) and the pale cy st nematode ( G lobodera pallida ), both of which can be very

destructive pests of potato (USDA
- APHI S, 2013a)
. Golden nematode is a major potato pest in Europe and is currently limited to L ong Island and six counties in western New York. Pale cy st nematode is widel y distributed throughout the world, and15

was first detected in the United States in I daho in 2006. It is currentl y limited to a five mile radius within two counties in I daho. PPQ also has an active pest management program for potato tuber necrotic ringspot disease (PTNRD) caused by necrotic strains of Potato Virus Y (PVYN) (USDA
- APHIS 2013 b ). PVYN is common in Europe and found in Canada and was first reported in the U.S. Pacific Northwest in 2002. In the earl y 1990's, Canada and the U.S. implemented a Management Plan with the objective of controlling its spread. Two additional potato pathogens, Synchytrium endobioticum (the cause of potato wart) and Ralstonia s olanacearum race 3 biovar 2 (the cause of brown rot) are APHI S quarantine pests and are listed as Select Agents in the U.S. ( US
- FSAP, 2013 )
. S. endobioticum is the most important world
- wide quarantine pest of potato. In 2007, USDA established a quarantine plan for S. endobioticum, to be used in the event of its detection in the U.S. (USDA, 2007 )
. R. solanacearum race 3 biovar 2 is found worldwide except the U.S. and Canada. There have been sporadic introductions into the U.S. via imports of geranium plants. These introductions have been limited to greenhouses. In 2008, USDA
- APHI S established New Pest Response Guidelines for this pathogen, to be used in the event of its detection in the U.S. (USDA
- APHIS, 2008b )
. Additional information on all of these programs can be found on the PPQ website (http://www.aphis.usda.gov/plant_health/plant_pest_info/index.shtml ) The potato crop is intensively managed with integrated pest management (IPM) to control a variety

of in sect and disease ( Johnson, 2007 ) . In partic ular, infestations of Colorado potato beetle (

Leptinotarsa decemlineata ) and the late blight fungus ( Phytophthora infestans ) are often countered b y spraying insecticides and fungicides, respectivel y. Other economically important potato insect and disease pests managed in the IPM programs include: the green peach aphid ( Myzus persicae ); wireworms ( Limonius californicus, L. canus, Ctenicera pruinina ), which damage tubers and shoots; the potato leafhopper (

Emposasca fabae ); and the earl y blight fungus ( Altern aria solani ). Potatoes can also become infected with a number of viruses (e.g. potato leafroll virus, and potato viruses A, M, X and Y); however, IP M options for virus pests are limited to insecticidal control of their insect vectors and planting of resis tant varieties ( Arif et al., 2012 )
. JR Simplot presented pest and disease response data for the 10 JRSLA events from two distinct sources: (1) They took observ ational data on any insects or diseases present in the regulated field trials conducted in 2009, 2010 and 2011 ( JR Simplot, 2013 , Appendix 6); and (2) they

conducted laboratory

and field disease assay s to generate response data to two important diseases of potato: late blight caused by P. infestans and bacterial soft rot caused b y Erwinia carotovora ( JR Simplot, 2013 , Appendix 8). In 18 field trials conducted in 2009, 2010 and 2011, JR Simplot collected data on insect, disease and abiotic stressors. Fi eld trial sites were selected to represent the major production areas for each variety. The Ranger Russet and Russet Burbank types were primarily field tested in Idaho, while the bulk of the field trials for the G and H ty pes were conducted in Wisconsin, F lorida and Michigan. The Atlantic types were field tested in 8 states ( JR Simplot, 2013 , Appendix 6, Table 1). The agronomic practices and pest control measures employed were location
- specific, recommended b y experts, and ty pical16

for potato cultivation. The events and untransformed varieties received identical inputs and treatments within each site. Stressor data were collected earl y, mid, and late season on insect populations, disease sy mptoms or pressure, and abiotic stressor s ymptoms. A list of common potato disease and insect pests is presented ( JR Simplot, 2013 , Appendix 6, Table 26). Not all insects or diseases were evaluated or found at each site. Rather, data were collected on those insect and disease pests which were observed or were specificall y looked for in each field trial. Stressors were rated on a 1
- 5 scale (1=high, 3=average, 5=low ), and the ranges of the observed data for each JRSLA event were compared to those of its respective parent variet y. If a stressor was specificall y looked for b ut not found, then a rating of 0 was recorded. These data are summarized below. “Incidental” stressor data were also recorded for diseases at mid and late season and for insects at mid
- season and observations were recorded as present or absent for the JR SLA event and its respective parent variety. However, these data were recorded simply as incidental insect, Verticillium, virus, and “other” and rarely was the presence of the stressor encountered, except for Verticillium. For the insect stressors, JR Simplot collected 630 observations, of which nine were reported with minor differences ( JR Simplot, 2013 , Appendix 6) (see Table 3 below). In seven of these nine cases, the JRSLA events had more insect stress than the controls: event F37 had greater stre ss from seed corn maggots during one observation in Adams County , Washington in 2011; event G11 had greater stress from Colorado potato beetles and potato leafhopper during one of eight observations, and from grasshoppers during two of eight observations i n Oneida County , Wisconsin in 2011; event H50 had greater stress from blister beetles and grasshoppers during one of eight observations each in Oneida County , Wisconsin in 2011 ( JR Simplot, 2013 , Appendix 6). **Table 3.** Field Trial Insect and Disease Stressor Observations for Ranger Russet Events F10 and F37 in 2010
- 2011, Russet Burbank events E
- 12 and E
- 24 in 2009 and 2011, Atlantic Events J3, J55, and J78, G variety Event G11, and H variet y Events H37 and H50 in 2011 (a dapted from JR Simplot, 2013 , Appendix 6). **Stressor Total # Observations for each Event (number of observations where the** 

**range of ratings for the event was outside the range of its parental control variety)**  **Insects F10**  **F37 E12**  **E24 J3**  **J55 J78**  **G11 H37**  **H50** Aphids 18 12 18 18 21 21 21 8 8 8 Blister Beetle 8 8 8 (1) Colorado Potato Beetle 18 12 18 18 65 65 65 8 (1) 8 8 Flea Beetle 8 8 8 8 8 8 Grasshopper 8 (2) 8 8 (1) Looper 3 3 3 3 3 3 3 Potato Leafhopper 6 6 6 8 (1) 8 8 Seed corn ma ggot 3 3 (1) 3 (1) 3 (1) 3 3 317

**Total observations**  42 30 48 48 106 106 106 48 48 48 **Diseases F10**  **F37 E12**  **E24 J3**  **J55 J78**  **G11 H37**  **H50 Bacterial**  Black leg 3 3 3 3 3 3 3 **Fungal** Botrytis 12 6 (1) 12 12 24 24 24 Early Blight 15 (1) 9 (2) 15 (1) 15 (1) 51 (1) 51 (1) 51 Late Blight 3 3 3 Rhizoctonia 3 3 3 3 6 6 6 Verticillium 3 (1) 3 3 3 3 (1) 3 (1) 3 (1) 4 4 4 White Mold 9 9 9 9 (1) 6 6 6 **Virus** 3 3 3 3 3 3 3 **Total observations**  48 36 48 48 99 99 99 4 4 4 For the disease stressors, JR Simplot collected 489 observations, of which 13 were reported with differences (see Table 3 above adapted from JR Simplot, 2013 , Appendix 6). In two of these 13 cases, the JRSLA events had less disease stress than the controls, in four cases the range of disease stress observed was slightly broader than the control and skewed towards less disease, and in one case the range of disease stress observed was slightly broader than control in both directions. The JRSLA event had more disease stress than the control in the other six cases: F10
- earl y blight ( A. solani ), Grand Forks Co, ND in 2011; E24
- white mold or Sclerotinia stalk rot (Sclerotinia sclerotiorum ), Bingham Co, ID in 20 11; F10, J3, J55, and J78
- Verticillium spp., Bingham Co, ID in 2011). However, with the exception of earl y blight in event F10 at Grand Forks, ND in 2011, these observed differences were small, and 14 other observations demonstrated no difference betwe en F10 and controls in stress due to earl y blight. Among important diseases affecting potato tubers, there were many observations collected for black leg (soft rot in tubers; E. carotovora ) and Rhizoctonia ( R. solani ), but few observations for late blight (

P. infestans ), and no observations were sy stematically collected for Fusarium dry rot ( Fusarium spp.), ring rot ( C. michiganensis spp. sepedonicus ), and water rot/Py thium leak (

P. ultimum ). However, there were no significant differences between events J3 , J55, J78, H37, and H50 and their controls in the number of tubers discarded (pick
- outs) at harvest due to quality issues, i.e. rot or mold, while the number of tubers discarded for event G11 was significantly lower than its control (JR Simplot, 2013, App endix 6). Pick
- outs for Events F10, F37, E12 and E24 were not recorded. During the cold storage period for trials in three locations in either 2009 or 2010, pink rot ( Phytophthora erythroseptica ) and soft rot and P ythium leak diseases were occasionall y ob served on both untransformed and unspecified JRSLA potato events, but no data were provided, and the disease incidence was attributed to environmental soil conditions that favored infection ( JR Simplot, 2013 , pg. 46). While a number of viruses also affect18

tubers (e.g., potato leaf roll virus), these also have above ground s ymptoms and such sy mptoms were not observed during the field trials. JR Simplot also recorded data on abiotic stressors (frost, hail, heat, herbicide, and wind) for 7 of the 10 JRSLA events ( JR Simplot, 2013 , Appendix 6). For events F10, F37, E12, E24, J3, J55 and J78, JR Simplot recorded 341 stressor observations and in no case did the JRSLA potatoes fall outside the range of their respective parental varieties. No abiotic stressor data was presented for the G11, H37 and H50 events. JR Simplot evaluated resistance to late blight (P. infestans ) in both laboratory and field experiments and to soft rot ( E. carotovora ) in laboratory experiments. Two separate laboratory

assay s were con ducted in 2009 and one in 2011. All 10 JRSLA events were not included in every

test for late blight and soft rot; Table 4 summarizes which events were included in each test and their disease response/level of susceptibility relative to their parental control. In 2009, the Ranger Russet and Russet Burbank ty pes (E12, E24 and F10) were tested in one assay and the G and H ty

pes (G11, H37, H50) were tested in a second assay . The 2011 laboratory assay included the Ranger Russet, Russet Burbank and Atlantic ty

pes, but not the G and H t ypes. The 2011 field experiment included the E12, E24, F10, J3, J5 and J78. **Table 4.** Summary

of disease ratings for late blight ( P. infestans ) and soft rot ( E. carotovora subsp. carotovora ) lab and field studies for JRSLA events compared to their parental controls conducted by JR Simplot ( JR Simplot, 2013 , Appendix 8). (< = less susceptible, > = more susceptible, nsd = no significant difference). Disease Assay Pathogen

Strain Year F10 F37 E12 E24 J3 J5 5 J7 8 G11 H37 H50 Late Blight Lab Tubers US8 2009 < > nsd < < > Late Blight Lab Tubers US10 2009 nsd nsd nsd Late Blight Lab Tubers US22 2009 < > > Late Blight Lab Tubers US8 2011 nsd nsd < nsd > nsd nsd Late Blight Field

Foliar US22 2011 < nsd nsd nsd nsd nsd Soft Rot Lab Tubers 2009 nsd nsd nsd nsd nsd Soft Rot Lab Tubers 2011 nsd nsd nsd nsd < < nsd nsd nsd nsd Total assays 4 3 5 5 3 3 3 5 5 4 The data from all laboratory late blight assay s was highl y variable, and in some cases, large differences were not found to be significant ( JR Simplot, 2013 , Appendix 8, Tables 1, 2 and 3), and no trends were apparent (see Table 4 above). E12 had significantly more19

disease than the controls in 2009, but had significantly l ess disease than the controls in 2011, using the same US8 strain of P. infestans in both years. In the 2009 assay that included the G and H typ es, three different late blight strains were used (US8, US10 and US22) and the results were inconsistent across strains and between the events G11, H37 and H50, except that all three events were similar in having no significant difference in their response to strain US10 relative to their respective controls. The most robust data for late blight reactions comes from the 2011 field trial conducted in Michigan ( JR Simplot, 2013 , Appendix 8, Table 4). This trial received a foliar inoculation of P. infestans strain US22, a relatively new genotype that has been prevalent in the Midwestern US ( Gevens et al., 2011 ) , and percent foliar infection was recorded at several points in time. Event F10 has significantly less foliar infection, and there were no differences for the other five events tested (see Table 4 above). These data provide no indication that the six JRSLA events tested have increased susceptibility to late blight. Events F37, G11, H37 and H50 were not included in this test, however there appears to be no strong correlation with presence of one or both expression cassettes and an increase in susceptibility . In addition to the challenge experiments, there were no differences observed in late blight in the field in the absence of challenge inoculum for J3, J55 and J78 compared to the Atlantic control (see Table 3). JR Simplot eva luated resistance to bacterial soft rot by percent weight loss of inoculated tubers. In the 2009 assay, only 5 of the 10 JRSLA events were tested. No significant differences were detected; however, this data was only minimally

informative due to the larg e standard errors ( JR Simplot, 2013 , Appendix 8, Table 5). All 10 JRSLA events were included in the 2011 soft rot assay s. The Atlantic events J3 and J55 had significantl y less weight loss than their parental varieties, and six other events had numericall y less weight loss ( JR Simplot, 2013 , Appendix 8, Table 6). These data (see Table 4 above) provide no indication that any of the 10 JRSLA events have increased susceptibility

to bacterial soft rot. The biological function of PPO in plants remains elusive ( Mayer, 2006 ) . The oxidation products of PPO appear to play a role in general plant defense mechanisms against pathogens and pests; Mayer 200 6 has reviewed these interactions in potato. Much of this literature is focused on specific phenolic compounds, rather than on PPO activity per se . Phenolics can inhibit the growth of specific pathogens and can also inhibit enzy mes involved in pathogenes is ( Lyon, 1989 ) . Additional factors that complicate the anal ys is of PPO relative to pest and pathogen resistance include: (1) both the substrates and the products of PPO have been implicated in resistance to plant pathogens; and (2) PPO can exist in latent forms such that no Ppo transcription is required to initiate the PPO activity ( Partington et al., 1999 )
. Lyo n 1989 reviewed the biochemical basis for resistance of potato to bacterial soft rot caused by Erwinia spp. Because it is the dominant monophenol, chlorogenic acid has been a focus in man y of these studies. Chlorogenic acid did not inhibit the in vitro growth of Erwinia spp. or P. infestans (the causal agent of late blight), and there remains20

n o proof that phenols are important in the interaction between potato and Erwinia spp. (Lyon 1989). Kroner et al 2012evaluated the role of specific phenolics in quantitative resistance to the elicitors of two pathogens, P. infestans , the causal agent of late blight, and Pectobacterium atroseptiucm (s ynon ym : E. carotovora subsp. atroseptica ), the causal agent of bacterial soft rot. Increasing concentrations of total phenolics tended toward a positive correlation with quantity of s ymptoms due to the late bli ght pathogen, but were negativel y correlated with increased tuber rot severity due to the soft rot pathogen. Because chlorogenic acid accumulates in response to soft rot elicitors, these authors suggest that chlorogenic acid could be used as a marker for resistance to soft rot ( Kroner et al., 2012 ) . Since chlorogenic acid is a PPO substrate, silencing of PPO would not be expected to reduce the le vel of chlorogenic acid in potato tubers. All of the JRSLA potato events contain the gene cassette for silencing the Ppo5 gene in tubers and Ppo5 transcripts were down
- regulated in tubers of all 10 events, and reduced levels of PPO activity

were confirm ed for all but the Atlantic events J5, J55, and J78. Taking this into consideration, the weight of evidence from field observations, challenge experiments with the late blight pathogen and tuber soft rot, and the level of tuber discards (pick
- outs) due to quality issues including rots, suggests that JRSLA potatoes are unlikely to be more susceptible than their control variety to either late blight or tuber rots due to silencing of the Ppo5 gene in tubers. The relationship between PPO and resistance to he rbivores has also been studied. PPO activity increases when potato leaves are wounded and at higher rates in response to regurgitant from the pest Colorado potato beetle ( Kruzmane et al., 2002 ) . In other plants the increase of PPO activity is a dir ect induced defense against insect pests that decreases nutrient availability ( Baldwin and Preston, 1999 ) . In addition, PPO in glandular trichomes of wild potatoes (and other plants) is involved in resistance to insects. However, the trichomes of cultivated potatoes contain low amounts of PPO which is not thought to be involved resistance to pests ( Friedman, 1997 )
. In the JRSLA potatoes, Ppo5 has been sil enced in tubers by use of a tuber
- specific promoter. JR Simplot provided expression data showing that PPO transcripts are down
regulated in tubers of all 10 events, stems of 9 events, roots of 5 events and s tolons of 3 events (JR Simplot, 2013, Table 6, p age 43). Transcripts hy bridizing to the Ppo5 probe were not detected in greenhouse grown leaves or flowers of the JRSLA events or their controls. Another Ppo gene transcript ( NOR333 ) has been shown to be more highl y expressed in young leaves and flowers of field
- grown potatoes. APHI S has determined that NOR333 shares no homology with the tPpo5 sequence used to silence the Ppo5 gene in the JRSLA events and thus should not be down
- regulated via RNAi in JRSLA potatoes. This suggests that the JRSLA potatoes are expected to be unchanged relative to PPO gene expression and PPO levels in leaves and therefore unchanged in any potential interactions between potato foliar PPO and foliar pathogens or pests. No consistent differences were observed in foliar pest and pathogens on JRSLA potatoes compared to their control varieties. Nematode damage of JRSLA potatoes was not assessed. The21

results of Osman et al 2012, suggest that PPO might be involved in resistance to some plant parasitic nematodes, but do not provide dat a demonstrating such a role or address the species that affect potatoes ( Osman et al., 2012 ) . Conversel y, lower PPO levels might increase the resistance of JRSLA potatoes to some nematodes. Other researchers found that tubers of potato c ultivars resistant to G. pallida have lower levels of phenols and discolored less than tubers of susceptible cultivars ( Mondy et al., 1985 )
. They

suggest that PPO
- mediated tanning of nematode cysts enables eggs to remain viable in soil for a longer time. Itis not possible to draw a conclusion regarding the susceptibility of JRSLA potatoes to G. rostochiensis or G. pallida based on PPO silenci ng. Changes in amino acid composition have also been implicated in plant pest interactions or defenses. L
- glutamic acid and its derivatives  and  - aminobut yric acids and L
gl ycine are chemoattractants for G. rostochiensis and G. pallida ( Riga et al., 1997 ; Rasmann et al., 2012 ) . However, there are no sign ificant changes in glutamic acid or gl ycine levels in tubers of the JRSLA events ( JR Simplot, 2013 , Appendix 9). In another instance, researchers demonstrated that changes in the amino acid profile in potato phloem sap as the plants mature may affect aphid s ( Karley

et al., 2002 ) . Aphids performed relativel y poorly on more mature plants in the tuber
- filling phase; these mature plants had reduced levels of glutamine and asparagine and increased levels of glutamate in leaf phloem. JRSLA potato tubers have reduced levels of aspar agine; however they have increased levels of glutamine ( JR Simplot, 2013 , Appendix 9). No leaf compositional data was presented b y JR Simplot, 2013 . It is difficult to accuratel y predict from the tuber compositional data whether there would be changes in amino acid composition in JRSLA tissues that aphids feed upon that would enhance aphid performance. However, no increase in aphid infestation or damage was reported in the field observations for all 10 JRSLA potato events ( JR Simplot, 2013 , Appendix 6). Similarly , although aphids are vectors for viruses such as potato virus Y (USDA
- APHI S, 2013b ) , no increase in viral disease was reported in field observations for seven of the events ( JR Simplot, 2013 , Appendix 6). There are no indications that JRSLA potatoes would have an adverse impact on the implementation of APHIS PPQ pest management programs for nematode pests of potato. These pests are of limited distribution and there are ongoing efforts to detect them and prevent their spread. Even if JRSLA potato varieties were to be more susceptibl e to nematodes than their parent varieties, this should not prevent the successful implementation of APHIS PPQ best management practices and control efforts, which include cultural practices, crop rotations including resistant varieties and non
- crops, planting certified nematode free seed potatoes, and preventing movement of infected soil, equipment and plant material (USDA
- APHIS, 2008a ; 2009 ) The introduced genes did not significantl y alter the observed insect pest infestation and disease occurrence or resulting damage in JRSLA potatoes relative to the control line under the t ypical recommended pest management conditions. However, the only obse rvations reported for insect pests that affect potato tubers or seed potatoes were for flea beetle and seed corn maggot (no observations were reported for nematodes), and the only field observations for pathogens recorded for events G11, H50 and H37 were f or22

Verticillium. Nonetheless, the tubers discarded due to tuber rot and inoculated disease assay s did not provide any indication that the JRSLA potatoes have increased incidence of pathogens or disease sy mptoms (with the possible exception of increased sus ceptibility to late blight strain US8 in events H50 and J3 events or to strain US22 in events H50 and H37). As discussed earlier there were no significant changes in the composition of JRSLA potatoes that would render them more susceptible to pests and dis eases over their control or reference potato varieties. As presented later in this document, the observed agronomic traits also did not reveal any significant changes that would indirectly indicate that JRSLA potatoes are or could be relativel y more susceptible to pests and diseases than control or reference potato varieties. Thus JRSLA potatoes are unlikel y to be more susceptible to plant pathogens and insect pests than existing commercial varieties of conventional potatoes and are not expected to adverse ly impact APHIS PPQ pest management programs for potato pests. For these reasons, JRSLA potatoes are unlikel y to differ from conventional potatoes in its ability to harbor or transmit plant pathogens or pests and cause indirect plant pest effects on other agricultural products. 

### E. **Potential Impacts on Nontarget Organisms Beneficial to Agricultur e**  JRSLA potatoes are not engineered for pest resistance , thus there are no “target” species and thus no “no ntarget” species either. APHIS assessed wh ether exposure to or consumption of JRSLA potato plants would have a direct or indirect adverse effect on species beneficial to agriculture. Organisms considered were representatives of the species associated with production of the regulated crop in the agricultural environment. The assessment include s an analy sis of the JRSLA potatoes compared to the non
- GE counterpart (or reference varieties) for an y substances (proteins, RNAi, nutrients, antinutrients, metabolites, etc.) produced b y the GE plant that may be novel or expressed at significantly altered amounts , changes in the phenoty pe, and/or an y reported impacts on organisms beneficial to agriculture
. As discussed above, JRSLA potato tubers are nutritionally and compositionally

similar to their respecti ve parental varieties and/or other commercial potato varieties, with the exception of the intentional changes conferred b y the introduced genes. These intentional changes fall into three broad categories: (1) reduction of PPO enzy me levels in tubers; (2) alteration of the levels of asparagine and glutamine in the pool of free amino acids in tubers; and (3) reduction in the levels of the reducing sugars glucose and fructose in tubers. The altered composition of JRSLA tubers is unlikely to alter the suitabil ity

of potato tubers as a foodstuff for beneficial organisms. Furthermore, any organism that feeds on potato tubers is likely to be considered a plant pest. Although the composition of other plant parts was not assessed in JRSLA potatoes, as noted above, JR Simplot collected 630 observations of insect stressors during field trials of the JRSLA potatoes, of which onl y 9 were reported with minor differences from the parental varieties. In onl y two cases did the JRSLA potatoes exhibit less insect stress than controls, indicating that JRSLA potatoes do not cause harm to insect pests that feed on potato leaves. By

extension, JRSLA potatoes are unlikely to adversel y affect other insects23

that may feed on potato leaves. None of the insect stressor observations in cluded insects that damage or feed on flowers. Beneficial organisms associated with potatoes in the field include pollinators, particularl y bumblebees ( OECD, 1997 ) . ASN1 transcripts were down
- regulated in JRSLA flowers of the eight varieties that produce flowers ( JR Simplot, 2013 , Appendix 5), suggesting that asparagine levels might be reduced in the flowers of these events. However, asparagine is not an essential amino acid for honey bees, which are in the same famil y as bumblebees ( Cook et al., 2003 ) , or man y other insects ( Boudko, 2012 ) . Thus, it is unlikely that there will be a negative effect on bumblebees as a result of potential alter ed composition of JRSLA flowers. Moreover, since potatoes are propagated vegetativel y from seed potato tuber pieces, pollination by bumblebees is not important to potato tuber production. In addition to observations on insect stressors, there were no tren ds observed with respect to significant differences in JRSLA potato responses to disease stressors in the 489 observations made, including bacterial and fungal pathogens, so by extension, JRSLA potatoes are unlikel y to adversel y impact soil or plant
- associ ated beneficial fungi or bacteria. RNAi mediated gene suppression generally requires sequence homology of at least 90% between the silencing construct and the target sequence to be successful and even higher degrees of homology

over 21
- 23 nucleotide stre tches ( Sharp, 2001 ) . It is not likely that the genetic cons truct components responsible for gene silencing in the JRSLA events would contribute to silencing of genes in other non
- target organisms through direct consumption of pollen by pollinators or through secondary exposure of beneficial predator or parasitic arthropods or other potential biological control agents for potato pests ( Lacey

et al., 2001 ) since sequences from arthropods, bacteria, fungi and viruses are expected to be highl y divergent fro m the sequences used to silence genes in the JRSLA potatoes. Furthermore, indirect exposure scenarios are unlikel y to lead to impacts to non
target predators and parasitic arthropods since 1) they may not receive effective doses, 2) intracellular amplific ation of siRNA, the active gene silencing component derived from dsRNA, is not widely

found in insects, 3) environmental and phy siological conditions in the gut may

destroy the RNA, 4) and they

may not have the appropriate receptors to allow transmembrane movement of dsRNA or the appropriate enzy me to direct RNAi (e.g. Dicer, Argonaute, RdRP, RNA and DNA helicases) ( Lundgren and Duan, 2013 )
. Therefore, based on the compositional similarity of JRSLA potato tubers to the parent varieties, the observed interactions of JRSLA potatoes with insects and pathogens, and the unlikely

impacts of nontarget effects due to RNA i, APHI S concludes that exposure to and/or consumption of JRSLA tubers or other plant parts is unlikely to have adverse impacts on organisms beneficial to agriculture. 

### F. **Potential for Enhanced Weediness**  **of JRSLA Potatoes** APHI S assessed whether JRSLA potat oes are likely to become more weedy

(i.e. more prevalent, competitive, damaging or difficult
- to - control in situations where it is not wanted) than the nontransgenic progenitors from which they were derived, or other varieties of the crop currently under cu ltivation. The assessment considers the basic biology of the crop and the situations in which crop volunteers or feral populations are24

considered weeds. The assessment also compares the GE crop events to the nontrangsenic progenitors, when cultivated unde r field conditions characteristic for the regions of the US where the GE crop is intended to be grown, for characteristics related to establishment, competiveness, reproduction, survival, persistence and/or spread that could influence weediness and the abi lity to manage the crop as a weed. For this cro p, such characteristics include tuber sprouting (emergence), vigor, vine size, tuber set (as measured b y yi eld), and tolerance to biotic and abiotic stress. Potatoes are not known to be weedy or persistent; they are incapable of survival outside of cultivation ( Holm et al., 1979 ; Muenscher, 1980 ; Lo ve, 1994 ; OECD, 1997 ) . Potato tubers have a fairl y low frost tolerance; shallow tubers and those exposed to the surface are often destro yed b y frost, but in temperate climates up to 20% of tubers left in the soil show no dormancy

and will sprout the next season ( Andersson and de Vicente, 2010 )
. Volunteer potatoes, growing from overwintered tubers, can be a weed problem in the following crop but are easily controlled with cultivation and herbicides and do not persist as weeds for more than one or a few years ( Andersson and de Vicente, 2010 ) . Since tubers are a source of volunteers, tuber yi eld is directly related to volunteer potential. Another potential source of volunteers is true potato seed (TPS). While many potato cultivars are partia lly

or fully sterile, rarel y producing fruits, some cultivars are capable of prolific fruit and seed production. Lawson reports that TPS produced by the cv. Desiree can survive in the soil for up to 7 years in a potato rotation in Scotland ( Lawson, 1983 ) . Of the five parent varieties used to develop the 1 0 JRSLA events , Russet Burbank and “H” are fully sterile, precluding an y possibility of TPS production. However, the JRSLA ev ents derived from Ranger Russet, Atlantic and “G” are fertile and may produce TPS. Regardless, plants produced from TPS are no weedier than volunteer plants produced from over
- wintered tubers and are relatively eas y to control in rotational crops. JR Simplot evaluated phenoty pic and agronomic characteristics of JRSLA potatoes relative to their parent varieties at several field locations over 2
- 3 years at geographically distinct sites that represent most of the main production areas for potatoes in the USA ( JR Simplot, 2013 , Appendix 6). The data presented indicated:

 Relative to their respective parent varieties, there was no consistent trend among the 10 events for significant increases in early emergence, final emergence, plant vigor or total yi eld ( JR Simplot, 2013 , Appendix 6). o Five events (F37, E12, E24, G11 and H37) had statistically significant increase s in plant vigor, but the relative difference was small (on the 1 to 5 scale, the means differed by less than 1 from the control), and no statistical ly significant differences were detected for this characteristic for the other events. o Five events (F10, F37, E24, G11 and H37) had statistically significant reductions in total yield and no statistically significant differences were detected for this characteristic for the other events. There was no obvious correlation between decreased yi eld and increased disease or pest stress25 except possibly

for event G11, since greater insect stress was observed than in the parental control at four of 48 insect observ ations. o Event J55 had a statistically significant reduc tion in earl y emergence.  Relative to their respective parent varieties, events G11, H37, and H50 showed no difference in vine size ( JR Simplot, 2013 , Appendix 6). Vine size was not assessed for the oth er seven events.  Relative to their respective parent varieties, most JRSLA potatoes did not display any differences in toleranc e to abiotic stresses ( JR Simplot, 2013 , Appendix 6). Abiotic stress data was not presented for events G11, H37 and H55.  As disc ussed above (see Potential Impacts on Disease and Pest Susceptibilities ), JRSLA potatoes did not display notable differences in tolerance to biotic stresses. The data presented b y J R Simplot demonstrate that the JRSLA potatoes are for the most part pheno ty

picall y and agronomically similar to the respective parent varieties and do not exhibit meaningful changes in characteristics that would make them weedier or more persistent than their respective parent varieties. Because of the reductions in emergence and yield, some of the JRSLA potatoes appear to have a reduced potential for weediness. Furthermore, JR Simplot did not observe an y differences during the completed post
harvesting volunteer monitoring of the JRSLA potato field test sites from 3 years of f ield testing that would lead them to believe that these potatoes have properties that would increase their survivability compared to conventional potatoes ( JR Simplot, 2013 , Section 9.3, and Appendix 10). Volunteers were rarel y observed, were easil y controlled, and are not engineered for resistance to herbicides. Based on the agronomic field data and literature survey concerning weediness potential of the crop, the JRSLA potatoes are unlikel y to persist as a troublesome weed or to have an impact on curr ent weed management practices. Furthermore, post
- harvest monitoring of field trial plots planted with the GE crop event under USDA
- APHI S notifications or permits (JR Simplot, 2013, Table 13, page 67) did not reveal an y differences in survivability

or persi stence relative to other varieties of the same crop currently being grown . These data suggest that the JRSLA potatoes are no more likel y to become weeds than conventional varieties of the crop. 

### G. **Potential Impact**  **s on the Weediness of**  **Any Other Plants with which**  **JRSLA Potatoes Can Interbreed**  Gene flow is a natural biological process with significant evolutionary importance. A number of angiosperm taxa are believed to be derived from hy bridization or introgression between closely

related taxa ( Grant, 1981 ; Rieseberg and Wendel, 1993 ; Soltis et al., 1993 ; Hegde et al., 2006 ) , and even in the existing floras, the occurrence of hy bridization or introgression is reported to be widespread ( Stace, 1987 ; Rieseberg and Wendel, 1993 ; Peterson et al., 2002 ) . It has been a common practice by plant breeders to artificially introgress traits from wild relatives into crop plants to develop new cultivar s ( Kh oury et al., 2013 ) . However, gene flow from crops to wild relatives is also thought of as having a potential to enhance the weediness of wild relatives, as observed in rice, sorghum,26

sunflower and a few other crops (see Table 1 in ( 1999 )
. This topic is covered in two sections: 1) the potential for gene flow , hy bridization and introgression from the GE crop event to sexually

compatible relatives , including wild, weedy, feral or cultivated species in the United S tates and its territories , and 2) if so, the risk potential with res pect to weediness of those taxa based on the phenotyp ic changes that have been observed in the engineered plants. As described previousl y, potatoes (Solanum tuberosum ) belong to the genus Solanum , section p etota , subsection potatoe , and series tuberosa, which consists of approximately 54 species, of which only S. tuberosum is widely cultivated for food production (OECD 1997). 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 North America and Europe. The subspecies andigena is also cultivated, but cultivation is largel y restricted to Central and South America (OECD 1997). The center of diversity for wild tuber
- bearing potatoes (sect. petota ) and the center of origin for domesticated potatoes is in L atin America ( Andersson and de Vicente, 2010 ) .The genus Solanum includes over 1000 species, however, cultivated potato is only sexually
- compatible with some of the other tuber bearing species in the section petota and rarel y with the non
tuber
- bearing species in the section etuberosum ; there are very strong barriers to hy bridization to other Solanum species ( Jackson and Hanneman Jr., 1999 ; Andersson and de Vicente, 2010 ) . Numerous Solanum spp. occur in the US, including other cultivated species, weeds, and three Federal noxious weeds (7 CFR part 360, subpart 200)
- S. tampicense , S. torvum , and S. viarum ; however cultivated potato is not sexually compatible with these species. Among native Solanum spp. in the US, cultivated potato is potentially

sexually
- compatible only with the two tuber
- bearing species, S. jamesii and S. stoloniferum (previously S. fendleri ( Spooner et al., 2004 ) ). Neither of these species is listed on US or State weed lists (USDA
-NRCS, 2013a)
. These two species are found onl y in Texas, New Mexico, and Arizona, and S. jamesii is further found in Colorado and Utah. In some cases these species are found in counties with commercial potato production (pages 2 1
- 22 in JR Simplot Company , 2013 ; USDA
-NRCS, 2013c ; 2013b )
. The distribution map for S. jamesii in the USDA Plants Database includes Iowa ; however this is not correct ( Bamberg, 2012 ) and this map should be updated in the near future. As stated above, the five parent varieties of the 10 JRSLA events vary in fertility . Since Russet Burbank produces few flowers and is male sterile and events derived from variety “H” are sterile (do not produce flowers) ( JR Simplot, 2013 , pp. 17 and 40), pollen
mediated gene
- flow from events derived from these varieties would not be possible. The events derived from Ranger Russet, At lantic, and “G” are likel y to produce fertile pollen. The new traits engineered into JRSLA events are not expected to expand the range of environments or locations in which cultivated potatoes are grown or affect their outcrossing capacity
. Numerous biolo gical and geographic obstacles make gene flow from these cultivated potato varieties to the two wild relatives a highl y unlikely occurrence , and there have been no reports that such crosses have ever occurred naturall y ( Love, 1994 ; US
- EPA,27

2011 )
. Ploidy

level and endosperm balance numbers (EBN) are genetic barriers which reduce the likelihood of gene flow from cultivated potato varieties into S. jamesii and S. stoloniferum . The concept of EBN was developed to explain endosperm level post
zygotic barriers that prevent seed development after fertilization in crosses among various Solanum spp. ( Carputo et al., 1999 )
. The se EBNs are h ypothetical genetic factors, independent of ploidy ( Spooner et al., 2004 ) . The EBN represents the effective ploidy , and must be in a 2:1 maternal to paternal ratio in the hy brid endosperm for normal endosperm deve lopment ( Carputo et al., 1999 ) . In general, crosses between species with the same EBN are successful, while crosses between species of differing EBN are not ( Spooner et al., 2004 )
. Modern potato varieties are tetraploid with an EBN of 4. S. jamesii is diploid with an EBN of 1, while S. stoloniferum is tetraploid with an EBN of 2 ( Andersson and de Vicente, 2010 )
. Since the EBNs of the two wild species differ from cultivated potato, these crosses would be expected to fail. Jackson and Hanneman evaluated the crossability

of 200 wild relatives with tetraploid pot ato (S. tuberosum ssp. tuberosum ) cultivars ( Jackson and Hanneman Jr., 1999 ) . For c rosses involving S. jamesii , no fruits formed after 109 and 24 pollinations with S. jamesii as the male and female parent, respectivel y. These data support the conclusion that crosses between cultivated potato and S. jamesii are expected to fail and thus that gene flow from cultivated potato to S. jamesii is unlikely

to occur. Unreduced gametes, which are not uncommon in Solanum spp., provide an exception to the general rule the crosses between species with differing EBN are not successful. Unreduced game tes of S. stoloniferum have an EBN of 4 and thus could potentially lead to successful h ybridization with tetraploid potato cultivars. In the experiments of Jackson and Hanneman, when S. stoloniferum (including plants categorized as S. fendleri and S. papita ( Spooner et al., 2004 ) ) was used as the male parent, 14 fruits containing 180 seeds formed after 852 pollinations, for a crossing efficiency of 0.0 15 3 When S. stoloniferum (including plants categorized as S. fendleri and S. papita ) was used as the female, 18 fruits containing 43 seeds formed after 205 pollinations, for a crossing efficiency of 0.012. Seed germination rates in these latter crosses we re approximately 50%. In comparison, when S. tuberosum ssp. tuberosum females were crossed to S. tuberosum ssp. tuberosum males, 14 fruits containing 252 seeds formed after 110 pollinations, for an overall crossing efficiency of 0.16 ( Jackson and Hanneman Jr., 1999 ) . These results demonstrate that gene flow from cultivated potato to wild S. stoloniferum is scientifically plausible, but that hy

bridization occurs at roughly 10% of the efficiency

with which it occurs in crosses when both parents are cultivated potatoes. Andersson and de Vicente conclude that although cultivated potato is not expected to cross easil y with S. stoloniferum in the environment, hy bridization between the two species is theoreticall y

3 Crossing efficiency

= # seeds per fruit/# pollinations. Crossing efficiency “takes out some of the bias intro duced by getting a large number of seeds from a few frui t, but requiring a great number of pollinations to get one or more fruit .” (Jackson and Hanneman, 1999).28

possible if they

are growing in close proximity to each other ( Andersson and de Vicente, 2010 )
. As noted above, S. stoloniferum (sy n. S. fendleri ) is found in several counties in Arizona ( Apache , Cochise , Coconino, Gila , Graham , Greenlee , Pima , Pinal, Santa Cruz ), New Mexico ( Catron , Dona Ana , Grant , Lincoln, Otera , Sierra, Socorro ) , and Texas (Brewster, Jeff Davis , Presidio) ( Bamberg et al., 2003 ; USDA
-NRCS, 2013b ; USGS, 2 013 )
. Of these, the 2007 Census of Agriculture found that potatoes were harvested on a total of 59 acres distributed among 82 farms in Apache, Chochise, Coconino, Gila, Dona Ana, and Socorro Counties, plus an unknown number of a cres distributed among 7 farms in Brewster, Grant, Pinal, and Sierra Counties (USDA
-NASS, 2009a ; 2009b ; 2009c )
. Since cultivated potatoes and S. stolo niferum co - occur in several counties in Arizona and New Mexico, and in one in Texas, and hy bridization between the two species is scientifically

plausible, this risk analy sis further considers the potential impact on the weediness of S. stoloniferum if gen e introgression from JRSLA potatoes were to occur. To be clear, gene introgression per se is not a risk, rather it is an exposure pathway . The risk to be evaluated is the nature of the altered phenoty pe should transgenes become stably introgressed into p opulations of the wild potatoes. As discussed above, the genetic material and resulting novel phenotypes (i.e. low acryl amide potential and reduced black spot bruise) incorporated into the JRSLA potatoes did not impart any significant and consistently obs erved changes to the agronomic properties or responses to biotic or abiotic stresses of cultivated potatoes under recommended management practices that would cause them to be more weedy, and they are not engineered for resistance to herbicides or to plant pests or insects. As also discussed above, S. stoloniferum is not listed on US or State weed lists (USDA
-NRCS, 2013a)
. Solanum stoloniferum exhibits a fairly wide range of genotypic and phenoty pic variability

in the environment ( Bamberg et al., 2003 ; Spooner et al., 2004 ) , suggesting that introgression of genetic material from cultivated JRSLA potatoes may not substantially

change its ability

to act as a weed. Based on the information presented in the petition and in relevant literature, APHI S has reached the following conclusions. T he genetic modification in the JRSLA potatoes is not expected to increase the potential for gene flow, hy bridization and/ or introgression to occur to sexually

compatible taxa compared to the nontransgenic recipient or other varieties of the crop commonly grown. Gene flow, h yb ridization and/or introgressio n of the introduced genetic material from JRSLA potatoes to the wild relatives S. stoloniferum and S. jamesii are unlikely to occur. In the case of S. stoloniferum , where there is a remote possibility of gene introgression, APHI S concludes that even if suc h introgression were to occur, this species is not considered a weed, and the gene silencing cassettes originating from JRSLA potatoes are unlikely to impact the weediness of this wild species since the JRSLA potatoes do not exhibit characteristics that ca use them to be an y weedier than other cultivated potatoes
.29

Therefore, the JRSLA potatoes are not expected to increase the weed risk potential of other species with which they can interbreed in the U.S
. and its territories. 

### H. **Potential Chan ges to Agriculture or**  **Cultivation Practices** APHI S could not identify any significant changes to agricultural or cultivation practices (e.g. pesticide applications, tillage, irrigation, harvesting, etc .) from adoption of the GE crop ; therefore, no impact on plant diseases or pests or their management is likely to occur. 

### I. **Potential Impacts from Transfer of Genetic Information to Organism**  **s with which**  **JRSLA potatoes Cannot Interbreed**  APHI S examined the potential for the new genetic material inserted into JRSLA potatoes to be horizontally

transferred without sexual reproduction to other organisms and whether such an event could lead directly or indirectl y to disease, damage, injury or harm to plants, including the creation of new or more virulent p ests, pathogens , or parasitic plants
. The horizontal gene transfer between unrelated organisms is one of the most intensively

studied fields in the biosciences since 1940, and the issue gained extra attention with the release of transgenic plants into the environment ( Dröge et al., 1998 )
. Potent ial risks from stable horizontal gene transfer (HGT) from genetically engineered organisms to another organism without reproduction or human intervention were recently reviewed ( Keese, 2008 )
. Mechanisms of HGT include conjugation, transformation and transduction, and other diverse mechanisms of DNA an d RNA uptake and recombination and rearrangement, most notably through viruses and mobile genetic elements. HGT has been a major contributor to the spread of antibiotic resistance amongst pathogenic bacteria ; emergence of increased virulence in bacteria, eukaryotes and viruses ; and , in the long run , to major transitions in evolution. **Potential for horizontal gene transfer to bacteria , fungi, or invertebrates**  Horizontal gene transfer and expression of DNA from a plant species to bacterial , fungal or invertebrate species is unlikely to occur based on the following observations. Although there are man y opportunities for plants to directl y interact with fungi and bacteria (e.g. as commensals, sy

mbionts, parasites, pathogens, decomposers, or i n the guts of herbivores) and with invertebrates as plant pests , there are almost no evolutionary examples of HGT from eukaryotes to bacteria or from plants to fungi or invertebrates ( Keese, 2008 )
. Examples of HGT between eukaryotes and fungi primarily involve gene acquisition or transfer b y fungi to o r from other distantly related fungi or bacteria ( Keeling and Palmer, 2008 ; Keese, 2008 ) and HGT between plants and fungi is e xtremel y rare ( Richards et al., 2009 ) . Examples of HGT between plants and invertebrates are also extremely rare, and most examples of HGT in insects involve acquisition of gen es from their pathogens or endosy mbionts ( Keese, 2008 ; Zhu et al., 2011 ; Acuna et al., 2012 )
.30

Horizontal transfer from and expression in bacteria of the foreign DNA inserted into the nuclear genome of the GE plant is unlikely to occur. Man y genomes (or parts thereof ) have been sequenced from bacteria that are closely associated with plants including Agrobacterium and Rhizobium ( Wood et al., 2001 ; Kaneko et al., 2002 )
. There is no evidence that these organisms contain genes de rived from plants. HGT from plants to bacteria is a very

low frequency

event, primaril y because functional and selective barriers to HGT increase with genetic distance ( Keese, 2008 )
. In cases where review of sequence data implied that horizontal gene transfer occurred, these events are inferred to occur on an evolutionary

time scale on the order of millions of years ( Brown, 2003 ; EFSA, 2009 ; Koonin et al., 2011 )
. In addition, the inserted gene cassettes in JRSLA potatoes are derived onl y from DNA from Solanum speci es, and are designed to be expressed and silence specific genes in potato. The sequences are optimized for plant expression, not prokaryotic bacterial expression. Thus even if horizontal gene transfer occurred, proteins corresponding to the transgenes are not likely to be produced. APHI S therefore concludes that the likelihood of HGT from JRSLA potatoes to bacteria or fungi is extremely low, and would not be expected to lead to an increased plant pest risk. **Potential for horizontal gene transfer to viru ses**  APHI S also considered whether horizontal transfer of DNA from the GE plant to plant viruses was likel y to occur and would lead to the creation or selection of plant viruses that are more virulent or have a broader host range. This issue has been consi dered before by other science review panels and government regulatory bodies ( EPA
- FIFRA
- SAP, 2006 ; Keese, 2008 )
. HGT is not unusual among plant viruses; however this is generall y limited to ex change between viruses present in the same host organism in mixed infections, and most commonly involves homologous recombination, rel yi ng on sequence similarity at the point of crossover ( Keese, 2008 )
. HGT from virus sequences engineered into plants has been demonstrated with infecting or challenge v iruses, including both DNA viruses (e.g. geminiviruses which replicate in the nucleus) ( Frischmuth and Stanley , 1998 ) and RNA viruses (which ty pically replicate in the cy

toplasm); however most have been under conditions that favor recombination to restore a defective virus ( Fuchs and Gonsalves, 2007 ; Keese, 2008 ; Thompson and Tepfer, 2010 )
. JRSLA potatoes contain no introduced DNA sequences derived from plant viruses ( JR Simplot, 2013 , Table 5). Therefore, the likelihood of HGT from JRSLA potatoes to plant viruses is expected to be very low and of no consequence
. **Potential for horizontal gene transfer to parasitic plants** Evidence for HGT from plants to other plants is limi ted to two specific scenarios: (1) exchange of genes between a parasitic plant and its host; and (2) exchange of genes between cells of two plants living in close proximity , such as in a graft junction. In both cases, this ty

pe of HGT requires phy sical co ntacts between the two plants. Most cases of HGT in plants involve transfer of mitochondrial genomes , which are primarily maternall y inherited in plants ( Barr et al., 2005 ) , to other mitochondria genomes, and mostly involve parasitic plants and their hosts ( Richardson and Palmer, 2007 )
. Recentl y, Yoshida an colleagues ( 2010 ) , through a comparative genomics anal ys is , implicated HGT for the31

incorporation of a specific genetic sequence in the parasitic plant purple witchweed (Striga hermonthica ) from its monocot host plant. According to this study, the incorporation of the specific genetic sequence (with an unknown function) occurred between sorghum and purple witchweed. However, this HGT occurred before speciation of purple witchweed and related cowpea witchweed (S. gesnerioides ) from their commo n ancestor. More recent studies demonstrated that in a few parasitic species of the Rafflesiaceae famil y, out of several genetic sequences examined, about 2.1% of nuclear (Xi et al., 2012 ) and 24

–41% of mitochondrial (Xi et al., 2013 ) gene transcripts appeared to be acquired from their obligate host species. However, all the above
- mentioned instances of HGT between parasitic plants and their hosts were repor ted to be of ancient origins, on an evolutionary time scale spanning thousand s to millions of years ago. Some non
- native species of Dodder ( Cuscuta spp.) (non
- native) are parasitic for potato ( Asigh and Marquez, 2010 )
. If JRSLA potatoes were infected by a parasitic plant or were naturally grafted to another plant in a potato field, there is a very low probability

that HGT could result in the other plant acquiring DNA from JRSLA potatoes. However, in both scenarios this newl y introduced DNA would likely reside in somatic cells, and with little chance of reaching the germ cells, this introduced DNA could not persist in subsequent generations unless the recipient plant reproduced asexually from the affected cells. Dodder reproduces sexually ( Asigh and Marquez, 2010 ) . APHI S therefore concludes that the likelihood of HGT from JRSLA potat oes to another plant including parasitic plants, is extremely low. **Overall conclusion for impacts from HGT** : Based on the above anal ys is APHI S therefore concludes that HGT of the new genetic material inserted into the JRSLA potatoes to other organisms is highly unlikel y, and is not expected to lead directly or indirectly to disease, damage, injury or harm to plants, including the creation of new or more virulent p ests, pathogens , or parasitic plants
. 

### J. **Conclusion** APHI S has reviewed the information submitte d in the petition, supporting documents, public comments in response to the Federal Register notice concerning this petition, and other relevant information to assess the plant pest risk of the 10 JRSLA potato events co mpared to the unmodified varieties from which they were derived. APHIS concludes that the 10 JRSLA potato events are unlikely to pose a plant pest risk based on the following findings.  No plant pest risk was identified from the transformation process or the insertion of new genetic mate rial in the 10 JRSLA potato events because the A. tumef a ciens vector was eliminated using antibiotics, no plant pest sequences were used other than Agrobacterium border sequences which do not confer plant pest risk, and no significant differen ce in disease and pest incidence were observed in the JRSLA potatoes compared to their nontransgenic counterparts or other comparators.  No increase in plant pest risk was identified in the 10 JRSLA potato events from expression of the inserted genetic material or changes in metabolism or composition32

because d isease and pest incidence and/or damage were not observed to be significantl y increased or atypical in the 10 JRSLA potato events compared to the ir nontransgenic counterpart s or other comparators in field trial s conducted in growing regions representative of where the JRSLA potatoes are expected to be grown and in laboratory , greenhouse studies. Observed agronomic traits also did not reveal an y significant differences that would indirectly indicate that the GE crop event is more susc eptible to pests or diseases. Therefore no plant pest effects are expected on these or other agricultural products and no impacts are expected to APHI S pest control programs.  Exposure to and/or consumption of JRSLA potatoes are unlikel y to have any adverse impacts on organisms beneficial to agriculture b ased on the compositional similarity of JRSLA potato tubers to the parent varieties, the observed interactions of JRSLA potatoes with insects and pathogens, and the unlikely impacts of nontarget effects due to RNAi
.  The 10 JRSLA potato events are no more likely to become weeds than conventional varieties of the crop based on their observed agronomic characteristics, weediness potential of the crop and current management pract ices available to control JRSLA potatoes as weeds.  The 10 JRSLA potato events are not expected to increase the weed risk potential of other species with which they can interbreed in the U.S. or its territories. G ene flow, hy bridization and/or introgressi on of inserted genes from JRSLA potatoes to other sexually compatible relatives with which they can interbreed is not likel y to occur. These compatible relatives are not considered weedy or invasive. The new phenoty pe(s) conferred by

genetic engineering are not likely to increase the weediness of these compatible relatives or affect the current ability to control these relatives in situations where they are considered weedy or invasive.  S ignificant changes to agricultural or cultivation practices (e.g. p esticide applications, tillage, irrigation, harvesting, etc.) from adoption of the 10 JRSLA potatoes were not identified.  H orizontal gene transfer of the new genetic material inserted into the 10 JRSLA potato events to other organisms is highly unlikel y, and is not expected to lead directly or indirectl y to disease, damage, injury or harm to plants, including the creation of new or more virulent pests, pathogens, or parasitic plants.33



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