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-00F10-7, SPS-00F37-7, SPS-00E12-8, 

SPS-00E24-2, SPS-000J3-4, SPS-00J55-2, SPS-00J78-7, SPS-00G11-

9, SPS-00H37-9, SPS-00H50-4

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 status. 

(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 file 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. Product 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 before 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 Any 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................................................................................................................. 33
1

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 genetically-engineered (GE) potato (Solanum tuberosum L.) 

events (F10, 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 unlikely to 

pose a plant pest risk.

APHIS 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 article under 7 CFR part 340 if the 

donor organism, recipient organism, or vector, or vector agent used in engineering the 

organism belong to any 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 any product which contains such an organism, or any 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.2The 10 

JRSLA potato events were produced by 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 APHIS regulations at 7 CFR part 340. JR Simplot has conducted 

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 unlikely 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 directly 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

Limited 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 cultivars and/or 

other appropriate comparators. APHIS 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. APHIS regulations in 7 CFR §340.6(c) specify the 

information needed for consideration in a petition for nonregulated status. APHIS will 

assess information submitted by the applicant about the 10 JRSLA potato events related 

to: plant pest risk characteristics; expression of the gene product, new enzymes, or 

changes to plant metabolism; disease and pest susceptibilities and indirect plant pest 

effects on other agricultural products; weediness of the regulated articles; effects of the 

regulated articles on nontarget organisms; impact on the weediness of any other plant 

with which they can interbreed; changes to agricultural or cultivation practices that may

impact 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.

APHIS 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 APHIS, the Food and Drug Administration 

(FDA), and the Office of Pesticide Programs of the U.S. Environmental Protection 

Agency (EPA). Depending on their characteristics, certain biotechnology-derived 

products are subjected to review by 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 pesticidal substances produced 

in plants and microbes, including those pesticides that are produced by an organism 

through techniques of modern biotechnology. EPA also sets tolerance limits for residues 

of pesticides on and in food and animal feed, or establishes 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 158. Other applicable EPA regulations 

include 40 CFR part 152 - Pesticide Registration and Classification Procedures, part 174 -

Procedures and Requirements for Plant Incorporated Protectants (PIPs) and part 172 -

Experimental Use Permits. The 10 JRSLA potato 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 comply with their obligations, the FDA encourages them to participate in its 
3

voluntary early food safety evaluation for new non-pesticidal proteins produced by new 

plant varieties intended to be used as food (FDA, 2006) and a more comprehensive 

voluntary consultation process 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, 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, and the 

subspecies andigena is also cultivated, but cultivation is largely 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 continental 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 lbs of potato annually, of which about two thirds 

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 used 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 to more complex chromosome segregation 

ratios, polyploid crops are inherently more difficult to breed. Furthermore, vegetatively 

propagated crops like potato are often poor seed producers due to partial or full sterility. 

For seed propagated crops, like corn or soybean, 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 independently transformed to 

achieve the desired phenotype 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 incorporate the same new phenotypes into 

each of these important varieties, while maintaining all of the desirable characteristics 
4

originally selected by potato breeders. If a single variety were transformed, it would take 

decades to move these new traits into the other commercial varieties by 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 demonstrated 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-enzymatic 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 potato 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 

μg/day(CEPA-OEHHA, 2005). Subsequent to the discovery of acrylamide in cooked 

foods, this NSRL was revised to 1.0 μg/day (CEPA-OEHHA, 2005).

Proposition 65 requires that food manufacturers warn consumers about the dangers of 

acrylamide 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 settlement, 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 industry now has a 

strong financial incentive to reduce the levels of acrylamide in their retail products.

Black spot bruising is a post-harvest physiological disorder primarily resulting from the 

handling of potato tubers during harvest, transport and processing. If physical impact 

causes cell rupture and/or flesh injury, the injured cells release phenolic compounds, 

normally compartmentalized in the vacuoles, which are converted to o-phenols and oquinones by the enzyme polyphenol 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 by providing a higher quality product, to benefit producers by 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, APHIS 

assessed 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. 

APHIS also assessed data presented in the petition on whether the genetic modification 

results in expression of new genes, proteins, or enzymes, suppression of existing genes 

and their products, or changes in plant metabolism or composition in JRSLA potatoes

relative to their nontransgenic counterparts. The assessment encompasses a consideration 

of the expressed double stranded RNA (dsRNA) and any observed or anticipated effects

on plant metabolism including, e.g. any relevant changes in levels of metabolites, 

antinutrients, or nutrients in harvested potatoes derived from the GE crop event compared

to those in the conventional counterpart and other comparators.

This information is used later in this risk assessment 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 enzymes, or 

changes in metabolism to affect plant pest or diseases, 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, Russet 

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 show to be 
6

effective to eliminate the A. tumefaciens vector (Nauerby et al., 1997). Lack of A. 

tumefaciens was confirmed by incubating stem fragments on nutrient agar for 2 weeks at 

28° C (JR Simplot, 2013, p. 25).

A marker-free selection system 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 Simplot inserted the isopentenyl transferase (ipt) 

gene from A. tumefaciens into the vector backbone just outside the Left Border (JR 

Simplot, 2013, Figure 3 and Table 4). Transgenic plants expressing the ipt gene, which 

results in the production of the plant hormone cytokinin, display a stunted phenotype that 

can be visually identified, allowing the elimination of plants with inserted vector 

backbone. The remaining plants were then molecularly characterized by PCR 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 synthetase-1 (Asn1), polyphenol 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 are 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 potatoes 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 phenotype resulting in 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. Synthetic 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 pyrophosphorylase promoter sequence from S. tuberosum. 

This promoter directs the silencing construct in the antisense orientation.

 fAsn1, 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.

 Spacer-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 orientation.
7

 fAsn1, a fragment of the Asn1 gene sequence from S. tuberosum, arranged in 

sense orientation.

 pGbss, the granule-bound starch synthase promoter sequence from S. tuberosum. 

This promoter directs the silencing construct in the sense direction, and is oriented 

convergently 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 pyrophosphorylase 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 promoter 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 synthase promoter sequence from S. tuberosum. 

This promoter directs the silencing construct in the sense direction, and is oriented 

convergently 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 additional 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 event 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

J55 Atlantic Single Complete insert fused to an 

inverted copy of the first cassette)
8

J78 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 in 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 Polymerase Chain Reaction assays (JR Simplot, 2013, 

Appendix 2).

 The genomes of the 10 JRSLA potato 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 insufficient 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 by DNA gel blot 

hybridization (all 10 JRSLA events) and a phenotypic 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 analysis.

Expression of inserted DNA, changes in gene expression, new proteins or metabolism

APHIS assessed whether changes in plant metabolism or composition of JRSLA potatoes 

are likely 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, and 
9

defensive metabolites) is known to affect herbivore performance and fecundity; and 

higher-trophic level interactions, such as the performance of predators and parasitoids, 

may also be affected (reviewd byAwmack 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 quality and composition have the potential to affect 

the performance of JRSLA potatoes against pests and diseases and to affect the suitability

of JRSLA tubers as a food source for non-target organisms.

To assess 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 determine 

whether there are any 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 different 

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 Idaho, Washington, North Dakota, 

and Wisconsin, states that account for more than half of US potato production (USDANASS, 2013). The Ranger Russet, Russet Burbank and Atlantic events were sampled all 

three years and the data presented as combined analyses across locations and years. For 

the G and H varieties the compositional analyses 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; Corsini et al., 1999; 

Partington et al., 1999). PPO oxidizes monophenols and o-diphenols to o-quinones, 

which are then further oxidized non-enzymatically to polyphenols. These darkpigmented polyphenols, 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 by 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 potato varieties, but browning is not correlated with 

content of chlorogenic acid. Internal blackspot in potatoes is caused by internal 

enzymatic browning type reactions initiated by PPO catalysis with tyrosine as the 

primary substrate (Friedman, 1997).
10

In potato, PPO comprises a multigene family with at least six genes differing in temporal 

and tissue-specific expression (Thygesen et al., 1995). Thygesen and colleagues reported 

that one member of the PPO gene family, 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 photosynthetic 

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 highly expressed in 

flowers (Thygesen et al., 1995). Other PPO 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 respectively, were not reported (Thygesen et al., 

1995). JRSLA potatoes are engineered to specifically reduce expression of Ppo5. 

JR Simplot provides evidence demonstrating that:

 Based on qualitative RNA (northern) blot analyses of various tissues of the 10 

JRSLA events and nontransgenic control varieties, Ppo5 transcripts were downregulated 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 hybridizing to the Ppo5 probe were detected in leaves of nontransgenic 

control varieties or in leaves of any of the 10 JRSLA events. Similarly, transcripts 

hybridizing 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 results 

also or instead reflect down-regulation of some of the other Ppo genes, 

particularly POT41, since its expression pattern has not been characterized nor its 

DNA sequence made available. However, it is unlikely that NOR333 transcripts 

were down-regulated 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, 2013, 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 Simplot, 2013, p. 

47). In contrast, Russet Burbank and particularly, 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 Simplot qualitatively examined the Asn1, R1, and PhL transcript levels in tubers from 

field and greenhouse grown plants and in stolons, roots, stems, leaves, and flowers of 
11

greenhouse grown plants (JR Simplot, 2013, Appendix 5). These data are summarized 

below and in Table 6, page 40, of JR Simplot, 2013. Free glutamine, 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 analyze acrylamide content (JR 

Simplot, 2013, Appendix 9). JR Simplot provides evidence demonstrating 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 stems 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. 

Increased content of free glutamine is an expected consequence of reducing the 

content of free asparagine. This is because the enzyme asparagine synthetase also 

functions to deaminate glutamine to glutamate (JR Simplot, 2013, Figure 6). 

 R1 transcripts 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 events (F10, F37 and H50), stems 

of one event (G11) and leaves of two or three events (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 downregulation, which was not observed in tubers, stolons, or roots, is unknown. 

 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 any event. PhL transcripts were not down-regulated in J78 or G11 

tubers due to a truncation 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 tightly correlated with the presence of the complete silencing

cassette for the R1 or PhL genes. In particular, there is no significant decrease in 
12

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 fresh 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 

acrylamide content. For events F10, F37, E12 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 acrylamide similar to those in the other 8 JRSLA events. 

Consistent (although not always statistically significant) reductions in free asparagine and 

increases in free glutamine across all 10 JRSLA events suggest that silencing Asn1 was 

sufficient to achieve the desired low acrylamide phenotype, 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 

parent varieties (JR Simplot, 2013, Appendix 9), the efficacy of silencing PhL and R1

conferred by pSIM1278 appears limited. pSIM1278 did not confer the desired phenotype 

of lower levels of reducing sugars in JRSLA tubers.

Table 2. Tuber composition phenotypes 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 enzymes 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 analyzed tubers of the 10 JRSLA events and their respective parents for 

the following constituents: protein, fat, ash, crude fiber, carbohydrates, calories, moisture, 

vitamin B3, vitamin B6, vitamin C, Cu, Mg, K, amino acids, sugars, and glycoalkaloids 
13

(JR Simplot, 2013, Appendix 9). This compositional analysis of tubers confirmed the 

following:

 For key proximates, vitamins, and minerals, there were no statistically significant 

differences between any of the 10 events and their respective parent varieties for 

content of protein, fat, ash, crude fiber, carbohydrates, calories, copper, 

magnesium or potassium. However, events F10 and F37 had statistically 

significant increases in niacin (vitamin B3) and vitamin C content, and events J3 

and J55 had statistically significant decreases in pyridoxine (vitamin 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 

lysine and free proline content while event H37 had a statistically significant 

decrease in free lysine 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 increased 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 

significantly decreased levels of total aspartate and asparagine and increased 

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 generated 

from nine non-GE commercial varieties grown concurrently 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 glycoalkaloid toxin content (JR Simplot, 2013 

Appendix 9). Although the upper end of the range of glycoalkaloid 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 generally accepted as safe, the upper ends of the ranges for 

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 differ statistically. A third event, E24, also exhibited elevated levels of 

glycoalkaloids at the upper end of the range while its control did not. However, 

the mean glycoalkaloid 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 glycoalkaloid levels (JR Simplot, 2013, 

Appendix 9, p. 67).

Based on the compositional analyses presented in JR Simplot, 2013 (Appendix 9), 

APHIS concludes that although 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 the 
14

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. 

APHIS 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

APHIS assessed whether potential plant pest or disease impacts are likely to result from 

the transformation process, from DNA sequences from plant pests, or from any other 

expression products, new enzymes, proteins or changes in plant metabolism or 

composition in the JRSLA potatoes that are known or anticipated to cause disease 

symptoms, or to affect plant pests or diseases or plant defense responses (as identified 

from the previous section). APHIS also assessed or whether the JRSLA potatoes are

likely to have significantly increased disease and pest susceptibility based on data and 

observations from field trials and laboratory experiments on specific pest and disease 

damage or incidence and any agronomic data that might relate to such damage. Impacts 

or changes 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 APHIS has a control program. 

Any increase in pest or disease susceptibility is evaluated with respect to the context of 

currently 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 safeguards 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 introductions 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 variety 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 cyst nematode (Globodera pallida), both of which 

can be very destructive pests of potato (USDA-APHIS, 2013a) . Golden nematode is a 

major potato pest in Europe and is currently limited to Long Island and six counties in 

western New York. Pale cyst nematode is widely distributed throughout the world, and 
15

was first detected in the United States in Idaho in 2006. It is currently limited to a five 

mile radius within two counties in Idaho. 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 2013b). PVYN is common in Europe and found 

in Canada and was first reported in the U.S. Pacific Northwest in 2002. In the early 

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 solanacearum race 3 biovar 2 (the cause of brown 

rot) are APHIS 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-APHIS 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 insect and disease (Johnson, 2007). In particular, infestations of 

Colorado potato beetle (Leptinotarsa decemlineata) and the late blight fungus 

(Phytophthora infestans) are often countered by spraying insecticides and fungicides, 

respectively. 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 early blight fungus (Alternaria 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, IPM options for virus pests are limited to 

insecticidal control of their insect vectors and planting of resistant 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 observational 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 assays to generate response data to 

two important diseases of potato: late blight caused by P. infestans and bacterial soft rot 

caused by 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. Field 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 types 

were conducted in Wisconsin, Florida 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 by experts, and typical 
16

for potato cultivation. The events and untransformed varieties received identical inputs 

and treatments within each site.

Stressor data were collected early, mid, and late season on insect populations, disease 

symptoms or pressure, and abiotic stressor symptoms. 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 specifically 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 

variety. If a stressor was specifically looked for but 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 JRSLA 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 stress 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 in 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 variety Events H37 and 

H50 in 2011 (adapted 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 

maggot

3 3 (1) 3 (1) 3 (1) 3 3 3
17

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 - early blight (A. solani), Grand Forks Co, ND 

in 2011; E24 - white mold or Sclerotinia stalk rot (Sclerotinia sclerotiorum), Bingham 

Co, ID in 2011; F10, J3, J55, and J78 - Verticillium spp., Bingham Co, ID in 2011). 

However, with the exception of early blight in event F10 at Grand Forks, ND in 2011, 

these observed differences were small, and 14 other observations demonstrated no 

difference between F10 and controls in stress due to early 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 systematically collected for Fusarium 

dry rot (Fusarium spp.), ring rot (C. michiganensis spp. sepedonicus), and water 

rot/Pythium 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, 

Appendix 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 Pythium leak diseases were occasionally 

observed 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 affect
18

tubers (e.g., potato leaf roll virus), these also have above ground symptoms and such 

symptoms 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 assays were conducted 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 types (E12, E24 

and F10) were tested in one assay and the G and H types (G11, H37, H50) were tested in 

a second assay. The 2011 laboratory assay included the Ranger Russet, Russet Burbank 

and Atlantic types, but not the G and H types. 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 J55 J78 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 assays was highly 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 more 
19

disease than the controls in 2009, but had significantly less 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 types, 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 evaluated 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 

large standard errors (JR Simplot, 2013, Appendix 8, Table 5). All 10 JRSLA events 

were included in the 2011 soft rot assays. The Atlantic events J3 and J55 had 

significantly less weight loss than their parental varieties, and six other events had 

numerically 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 2006 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 enzymes 

involved in pathogenesis (Lyon, 1989). Additional factors that complicate the analysis 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).

Lyon 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 many 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 remains 
20

no 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 (synonym: E. carotovora subsp. atroseptica), the causal 

agent of bacterial soft rot. Increasing concentrations of total phenolics tended toward a 

positive correlation with quantity of symptoms due to the late blight pathogen, but were 

negatively 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 level 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 confirmed 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 herbivores 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 direct 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 silenced in tubers by use of a tuber-specific 

promoter. JR Simplot provided expression data showing that PPO transcripts are downregulated in tubers of all 10 events, stems of 9 events, roots of 5 events and stolons of 3 

events (JR Simplot, 2013, Table 6, page 43). Transcripts hybridizing 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 highly 

expressed in young leaves and flowers of field-grown potatoes. APHIS 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. The 
21

results of Osman et al 2012, suggest that PPO might be involved in resistance to some 

plant parasitic nematodes, but do not provide data demonstrating such a role or address 

the species that affect potatoes (Osman et al., 2012). Conversely, lower PPO levels might 

increase the resistance of JRSLA potatoes to some nematodes. Other researchers found 

that tubers of potato cultivars 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. It is not possible to draw a conclusion regarding the susceptibility of JRSLA 

potatoes to G. rostochiensis or G. pallida based on PPO silencing. 

Changes in amino acid composition have also been implicated in plant pest interactions 

or defenses. L-glutamic acid and its derivatives  and -aminobutyric acids and Lglycine are chemoattractants for G. rostochiensis and G. pallida (Riga et al., 1997; 

Rasmann et al., 2012). However, there are no significant changes in glutamic acid or 

glycine 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 aphids (Karley et al., 2002). Aphids 

performed relatively 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 asparagine; however they

have increased levels of glutamine (JR Simplot, 2013, Appendix 9). No leaf 

compositional data was presented by JR Simplot, 2013. It is difficult to accurately 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-APHIS, 

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 susceptible 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 significantly alter the observed insect pest infestation and 

disease occurrence or resulting damage in JRSLA potatoes relative to the control line 

under the typical recommended pest management conditions. However, the only 

observations 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 for 
22

Verticillium. Nonetheless, the tubers discarded due to tuber rot and inoculated disease 

assays did not provide any indication that the JRSLA potatoes have increased incidence 

of pathogens or disease symptoms (with the possible exception of increased susceptibility

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 diseases 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 relatively more susceptible to pests and diseases than 

control or reference potato varieties. Thus JRSLA potatoes are unlikely to be more 

susceptible to plant pathogens and insect pests than existing commercial varieties of 

conventional potatoes and are not expected to adversely impact APHIS PPQ pest 

management programs for potato pests. For these reasons, JRSLA potatoes are unlikely 

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 Agriculture

JRSLA potatoes are not engineered for pest resistance, thus there are no “target” species 

and thus no “nontarget” species either. APHIS assessed whether 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 includes an analysis of the JRSLA potatoes compared to the non-GE 

counterpart (or reference varieties) for any substances (proteins, RNAi, nutrients, 

antinutrients, metabolites, etc.) produced by the GE plant that may be novel or expressed 

at significantly altered amounts, changes in the phenotype, and/or any reported impacts 

on organisms beneficial to agriculture.

As discussed above, JRSLA potato tubers are nutritionally and compositionally similar to 

their respective parental varieties and/or other commercial potato varieties, with the 

exception of the intentional changes conferred by the introduced genes. These intentional 

changes fall into three broad categories: (1) reduction of PPO enzyme 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 suitability 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 only 9 were reported with minor differences from the 

parental varieties. In only 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 adversely affect other insects 
23

that may feed on potato leaves. None of the insect stressor observations included insects 

that damage or feed on flowers. Beneficial organisms associated with potatoes in the 

field include pollinators, particularly 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 family as bumblebees (Cook et al., 2003), or many other insects(Boudko, 

2012). Thus, it is unlikely that there will be a negative effect on bumblebees as a result of 

potential altered composition of JRSLA flowers. Moreover, since potatoes are propagated 

vegetatively 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 

trends 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 unlikely to adversely impact soil or plant-associated 

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 stretches (Sharp, 2001). It is not likely that 

the genetic construct 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 highly divergent from the sequences used to silence genes in the JRSLA 

potatoes. Furthermore, indirect exposure scenarios are unlikely to lead to impacts to nontarget predators and parasitic arthropods since 1) they may not receive effective doses, 2) 

intracellular amplification of siRNA, the active gene silencing component derived from 

dsRNA, is not widely found in insects, 3) environmental and physiological 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 enzyme 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 RNAi, APHIS 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

APHIS assessed whether JRSLA potatoes 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 cultivation. The assessment considers the basic 

biology of the crop and the situations in which crop volunteers or feral populations are 
24

considered weeds. The assessment also compares the GE crop events to the 

nontrangsenic progenitors, when cultivated under 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 ability to manage the crop as a weed. For this crop, 

such characteristics include tuber sprouting (emergence), vigor, vine size, tuber set (as 

measured by yield), 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; Love, 1994; OECD, 1997). Potato 

tubers have a fairly low frost tolerance; shallow tubers and those exposed to the surface 

are often destroyed by 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 yield is directly related to volunteer potential.

Another potential source of volunteers is true potato seed (TPS). While many potato 

cultivars are partially or fully sterile, rarely 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 10 JRSLA events, Russet Burbank 

and “H” are fully sterile, precluding any possibility of TPS production. However, the 

JRSLA events 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 easy to control in rotational crops.

JR Simplot evaluated phenotypic 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 yield (JR Simplot, 2013, Appendix 6).

o Five events (F37, E12, E24, G11 and H37) had statistically significant 

increases 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 

statistically 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 yield and increased disease or pest stress 
25

except possibly for event G11, since greater insect stress was observed 

than in the parental control at four of 48 insect observations.

o Event J55 had a statistically significant reduction in early 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 other seven events.

 Relative to their respective parent varieties, most JRSLA potatoes did not display

any differences in tolerance to abiotic stresses (JR Simplot, 2013, Appendix 6). 

Abiotic stress data was not presented for events G11, H37 and H55.

 As discussed above (see Potential Impacts on Disease and Pest Susceptibilities), 

JRSLA potatoes did not display notable differences in tolerance to biotic stresses. 

The data presented by JR Simplot demonstrate that the JRSLA potatoes are for the most 

part phenotypically 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 any differences during the completed postharvesting volunteer monitoring of the JRSLA potato field test sites from 3 years of field 

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 rarely observed, were easily 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 unlikely to persist as a troublesome weed or to have 

an impact on current weed management practices. Furthermore, post-harvest monitoring 

of field trial plots planted with the GE crop event under USDA-APHIS notifications or 

permits (JR Simplot, 2013, Table 13, page 67) did not reveal any differences in 

survivability or persistence relative to other varieties of the same crop currently being 

grown. These data suggest that the JRSLA potatoes are no more likely to become weeds 

than conventional varieties of the crop.

G. Potential Impacts 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 hybridization 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 hybridization 

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 cultivars (Khoury 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, hybridization and introgression from the GE crop 

event to sexually compatible relatives, including wild, weedy, feral or cultivated species 

in the United States and its territories, and 2) if so, the risk potential with respect to 

weediness of those taxa based on the phenotypic changes that have been observed in the 

engineered plants. 

As described previously, potatoes (Solanum tuberosum) belong to the genus Solanum, 

section petota, 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 

largely 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 Latin 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 rarely with the nontuber-bearing species in the section etuberosum; there are very strong barriers to 

hybridization 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 only 

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 21-22 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), pollenmediated gene-flow from events derived from these varieties would not be possible. The 

events derived from Ranger Russet, Atlantic, and “G” are likely 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 biological and geographic obstacles make gene flow from these cultivated 

potato varieties to the two wild relatives a highly unlikely occurrence , and there have 

been no reports that such crosses have ever occurred naturally (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 postzygotic barriers that prevent seed development after fertilization in crosses among 

various Solanum spp. (Carputo et al., 1999). These EBNs are hypothetical 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 hybrid endosperm for normal 

endosperm development (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 potato (S. tuberosum ssp. tuberosum) 

cultivars (Jackson and Hanneman Jr., 1999). For crosses involving S. jamesii, no fruits 

formed after 109 and 24 pollinations with S. jamesii as the male and female parent, 

respectively. 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 gametes of S. stoloniferum have an EBN of 4 and thus could potentially lead 

to successful hybridization 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 0.0153 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 were 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 hybridization 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 easily with S. 

stoloniferum in the environment, hybridization between the two species is theoretically 

 

3 Crossing efficiency = # seeds per fruit/# pollinations. Crossing efficiency “takes out 

some of the bias introduced by getting a large number of seeds from a few fruit, 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 (syn. 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, 

2013). 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 acres distributed among 7 farms 

in Brewster, Grant, Pinal, and Sierra Counties (USDA-NASS, 2009a; 2009b; 2009c).

Since cultivated potatoes and S. stoloniferum co-occur in several counties in Arizona and 

New Mexico, and in one in Texas, and hybridization between the two species is 

scientifically plausible, this risk analysis further considers the potential impact on the 

weediness of S. stoloniferum if gene 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 phenotype should transgenes become 

stably introgressed into populations of the wild potatoes.

As discussed above, the genetic material and resulting novel phenotypes (i.e. low 

acrylamide potential and reduced black spot bruise) incorporated into the JRSLA 

potatoes did not impart any significant and consistently observed 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 phenotypic 

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, APHIS has 

reached the following conclusions. The genetic modification in the JRSLA potatoes is not

expected to increase the potential for gene flow, hybridization and/or introgression to 

occur to sexually compatible taxa compared to the nontransgenic recipient or other 

varieties of the crop commonly grown. 

Gene flow, hybridization and/or introgression 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, 

APHIS concludes that even if such 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 cause them to be any 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 Changes to Agriculture or Cultivation Practices

APHIS 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 

Organisms with which JRSLA potatoes Cannot Interbreed

APHIS 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 indirectly to disease, damage, injury or harm to 

plants, including the creation of new or more virulent pests, 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). 

Potential 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 and 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 many opportunities for plants to directly interact with fungi and bacteria (e.g. as 

commensals, symbionts, parasites, pathogens, decomposers, or in 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 by fungi to or from other distantly related fungi or bacteria (Keeling and Palmer, 

2008; Keese, 2008) and HGT between plants and fungi is extremely 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 genes from their pathogens or 

endosymbionts (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. Many 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 derived from plants. HGT from plants to 

bacteria is a very low frequency event, primarily 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 only from DNA from Solanum species, 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. APHIS 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 viruses 

APHIS also considered whether horizontal transfer of DNA from the GE plant to plant 

viruses was likely 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 considered 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 generally 

limited to exchange between viruses present in the same host organism in mixed 

infections, and most commonly involves homologous recombination, relying on sequence 

similarity at the point of crossover (Keese, 2008). HGT from virus sequences engineered 

into plants has been demonstrated with infecting or challenge viruses, including both 

DNA viruses (e.g. geminiviruses which replicate in the nucleus)(Frischmuth and Stanley, 

1998) and RNA viruses (which typically replicate in the cytoplasm); 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 limited 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 type of HGT requires physical contacts between the two plants. Most cases of 

HGT in plants involve transfer of mitochondrial genomes, which are primarily maternally 

inherited in plants (Barr et al., 2005), to other mitochondria genomes, and mostly involve 

parasitic plants and their hosts (Richardson and Palmer, 2007). Recently, Yoshida an 

colleagues (2010), through a comparative genomics analysis, implicated HGT for the 
31

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 common 

ancestor. More recent studies demonstrated that in a few parasitic species of the 

Rafflesiaceae family, 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 reported to be of ancient 

origins, on an evolutionary time scale spanning thousands 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 newly 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). APHIS therefore 

concludes that the likelihood of HGT from JRSLA potatoes to another plant including 

parasitic plants, is extremely low.

Overall conclusion for impacts from HGT: Based on the above analysis APHIS 

therefore concludes that HGT of the new genetic material inserted into the JRSLA 

potatoes to other organisms is highly unlikely, 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 pests, pathogens, or parasitic plants.

J. Conclusion

APHIS has reviewed the information submitted 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

compared 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 material in the 10 JRSLA potato events because the A. tumefaciens

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 difference 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 composition
32

because disease and pest incidence and/or damage were not observed to be 

significantly increased or atypical in the 10 JRSLA potato events compared to their

nontransgenic counterparts or other comparators in field trials 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 any 

significant differences that would indirectly indicate that the GE crop event is more 

susceptible to pests or diseases. Therefore no plant pest effects are expected on these 

or other agricultural products and no impacts are expected to APHIS pest control 

programs.

 Exposure to and/or consumption of JRSLA potatoes are unlikely to have any adverse 

impacts on organisms beneficial to agriculture 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 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 practices 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. Gene flow, 

hybridization and/or introgression of inserted genes from JRSLA potatoes to other 

sexually compatible relatives with which they can interbreed is not likely to occur. 

These compatible relatives are not considered weedy or invasive. The new 

phenotype(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.

 Significant changes to agricultural or cultivation practices (e.g. pesticide applications, 

tillage, irrigation, harvesting, etc.) from adoption of the 10 JRSLA potatoes were not 

identified. 

 Horizontal gene transfer of the new genetic material inserted into the 10 JRSLA 

potato events to other organisms is highly unlikely, 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 pests, pathogens, or parasitic plants.
33

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