State University of New York College of 

Environmental Sciences and Forestry Petition (19-309-

01p) for Determination of Nonregulated Status for 

Blight-Tolerant Darling 58 American Chestnut 

OECD Unique Identifier: 

ESF-DAR58-3 

Draft Plant Pest Risk Assessment 

June 2022

Agency Contact 

Cindy Eck

Biotechnology Regulatory Services 

4700 River Road

USDA, APHIS 

Riverdale, MD 20737 

Fax: (301) 734-8669

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information. 

 

 

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TABLE OF CONTENTS PAGE

i

A. INTRODUCTION ...................................................................................................... 1

B. DEVELOPMENT OF BLIGHT-TOLERANT DARLING 58 AMERICAN 

CHESTNUT............................................................................................................... 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......................................... 7

E. POTENTIAL IMPACTS ON NONTARGET ORGANISMS BENEFICIAL 

TO AGRICULTURE................................................................................................. 8

F. POTENTIAL FOR ENHANCED WEEDINESS OF DARLING 58 

AMERICAN CHESTNUT ........................................................................................ 9

G. POTENTIAL IMPACTS ON THE WEEDINESS OF ANY OTHER 

PLANTS WITH WHICH DARLING 58 AMERICAN CHESTNUT CAN 

INTERBREED ........................................................................................................ 10

H. POTENTIAL CHANGES TO SILVICULTURE OR AGRICULTURE 

PRACTICES............................................................................................................ 13

I. POTENTIAL IMPACTS FROM TRANSFER OF GENETIC 

INFORMATION TO ORGANISMS WITH WHICH DARLING 58 

AMERICAN CHESTNUT CANNOT INTERBREED........................................... 17

J. CONCLUSION......................................................................................................... 20

K. REFERENCES ......................................................................................................... 22
1 

A. Introduction

The State University of New York College of Environmental Science and Forestry

(hereafter referred to as ESF) has submitted a petition to Animal and Plant Health 

Inspection Service (APHIS) of the United States Department of Agriculture (USDA) 

seeking a determination of nonregulated status for American chestnut (Castanea dentata) 

event with OECD Unique Identifier “ESF-DAR58-3” (hereafter referred to as Darling 58 

American chestnut) developed using genetic engineering (hereafter referred to as 

modified) for enhanced blight tolerance. The petition presents evidence supporting the 

argument that Darling 58 American chestnut is unlikely to pose a plant pest risk and, 

therefore, should no longer be regulated under the APHIS’ 7 Code of Federal Regulations 

(CFR) part 340. This petition was assigned the number 19-309-01p and is hereafter 

referenced as ESF 2019 (ESF 2019). Under the authority of the plant pest provisions of 

the Plant Protection Act (7 U.S.C. 7701 et seq. 2019), the regulations in 7 CFR part 340, 

"Movement of Organisms Modified or Produced Through Genetic Engineering,” 

regulate, among other things, the importation, interstate movement, and release into the 

environment of organisms modified or produced through genetic engineering that are 

plant pests or pose a plausible plant pest risk. This plant pest risk assessment (PPRA) was 

conducted to determine if the Darling 58 American chestnut is unlikely to pose a plant 

pest risk.

The petition for a determination of nonregulated status described in this PPRA is being 

evaluated under the version of the regulations effective at the time the petition was 

received. APHIS issued a final rule, published in the Federal Register on May 18, 2020 

(85 FR 29790-29838, Docket No. APHIS-2018-0034)1

, revising 7 CFR part 340. Since 

the petition for determination of nonregulated status for Darling 58 American chestnut 

was received on January 21, 2020, before the revisions to the regulations became final, 

this petition request is being evaluated in accordance with the [legacy] regulations at 7 

CFR 340.6 (e) (2020). 

Darling 58 American chestnut was produced by Agrobacterium tumefaciens-mediated 

transformation of somatic embryos from the isogenic line of Darling 58 known as Ellis 

using plasmid p35S-OxO (ESF 2019). Portions of the inserted genetic material were 

derived from plant pest organisms listed in 7 CFR § 340.2 (7 CFR 340 2020) (i.e., a 

promoter sequence from cauliflower mosaic caulimovirus (CaMV) and a terminator 

sequence from A. tumefaciens) (Section 7.1, pp. 86-87, ESF 2019). Therefore, Darling 58 

American chestnut is considered regulated under APHIS regulations at 7 CFR part 340 (7 

CFR 340 2020). ESF has conducted field releases of Darling 58 American chestnut under 

APHIS authorizations since 2014 (ESF 2019), in part, to gather information to support 

that Darling 58 American Chestnut is unlikely to pose a greater plant pest risk than the

unmodified organism from which it was derived.

Potential impacts in this plant pest risk assessment are those that pertain to plant pest risk 

associated with Darling 58 American chestnut and its progeny and their use in the 

absence of confinement relative to the unmodified recipient line and/or other appropriate 

1 To view the final rule, go to www.regulations.gov and enter APHIS-2018-0034 in the Search field.
2

comparators. APHIS utilizes data and information submitted by the applicant, in addition 

to current literature, to determine if Darling 58 American chestnut is unlikely to pose a 

greater plant pest risk than the unmodified organism from which it was derived. APHIS 

regulations in 7 CFR § 340.6(c) (7 CFR 340 2020) specify the information needed for 

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

submitted by the applicant about Darling 58 American chestnut 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; effects of the regulated article on nontarget organisms; weediness 

of the regulated article; impact on the weediness of any other plant with which it can 

interbreed; changes to agricultural or cultivation practices that may impact diseases and 

pests of plants; and transfer of genetic information to organisms with which it 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 its characteristics, certain biotechnology-derived products 

are subjected to review by one or more of these agencies. 

Under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136 et 

seq. 2019 Edition), EPA regulates the distribution, sale, use and testing of pesticidal 

substances produced in plants and microbes, including those pesticides 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. 301 et seq. 2018 Edition.). 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 

nontarget species when used in accordance with label instructions. EPA must also 

approve the language used on the pesticide label in accordance with Data Requirements 

for Pesticides (40 CFR part 158 2019 Edition.). Other applicable EPA regulations include 

Pesticide Registration and Classification Procedures (40 CFR part 152 2019 Edition.),

Procedures and Requirements for Plant Incorporated Protectants (PIPs) (40 CFR part 174 

2019 Edition.), and Experimental Use Permits (40 CFR part 172 2019 Edition.). ESF 

informed APHIS that EPA has deemed the expressed OxO protein in their Darling 58 

American chestnut tree as a PIP and therefore it must be registered as a pesticide under 

Section 3 of FIFRA. However, EPA also advised ESF that Darling 58 American chestnut 

may qualify for an exemption under Section 25(b) of FIFRA, so ESF has decided to go 

through both the Section 3 FIFRA registration and the Section 25(b) FIFRA exemption 

processes simultaneously. After EPA has completed its assessments of ESF’s submissions 

and provided these to APHIS, APHIS will update this PPRA if needed.

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 
3

biotechnology. To help sponsors of foods and feeds derived from modified crops comply 

with their obligations, the FDA encourages them to participate in its voluntary early food 

safety evaluation for new non-pesticidal proteins produced by new plant varieties 

intended to be used as food (US-FDA 2006) and a more comprehensive voluntary 

consultation process prior to commercial distribution of food or feed (57 FR 22984 

1992). ESF submitted a voluntary consultation with the FDA on October 28, 2021,

concerning food and feed derived from Darling 58 American chestnut and is awaiting

FDA’s response. 

B. Development of Blight-Tolerant Darling 58 American Chestnut

American chestnut (Castanea dentata) was once a dominant forest tree and keystone 

species within its native range that extended across eastern North America, including 

nearly every state east of the Mississippi as well as southern Ontario, Canada (Little 

1977; Shaw et al. 2012; ESF 2019). Prior to the introduction of Cryphonectria parasitica

to the United States, American chestnut seeds were primarily collected from wild trees 

for animal feed or human use by local farmers. Wild trees were also harvested for timber. 

Specimen trees were also planted around farmhouses, along residential streets, and in city 

parks, but due to the abundance of wild trees across forests in the Eastern United States,

the wild long-lived trees provided ample nuts to meet demand for anthropocentric uses.

Once a major component of the Eastern hardwood forest and in some areas comprising 

half of the hardwood tree population, American chestnut is almost extinct in the wild 

now, having succumbed to chestnut blight, a bark fungal disease introduced from Asia

caused by the fungus C. parasitica. The chestnut blight fungus kills the above-ground 

portion of American chestnut trees and as a result, the American chestnut now persists 

mostly in the form of sprouts from old stumps and root systems. Typically, the sprouts 

grow up and possibly flower and fruit for several years before dying back from the blight. 

Before blight introduction, mature trees could reach more than 35 meters tall (>120 feet) 

and 5 meters wide (>17 feet) (Davis 2005). American chestnut is now considered 

functionally extint in modern forests (ESF 2019).

To rescue and ultimately restore American chestnut tree in it native environment, the 

petitioner has developed Darling 58 American chestnut trees with enhanced tolerance to 

chestnut blight. Darling 58 American chestnut was developed from a single immature 

zygotic embryo extracted from a wild-type American chestnut seed. The embryo was first 

subcultured to produce somatic embryo, and then the somatic embryo clumps were

transformed with disarmed A. tumefaciens strain AGL1 containing the plasmid vector 

p35S-OxO (Figure 7.1a, p. 85, ESF 2019). The p35S-OxO Agrobacterium vector 

contains the oxo and nptII expression cassettes that confer tolerance to chestnut blight 

and resistance to aminoglycoside antibiotics, respectively, and plasmid backbone 

sequences necessary for maintenance or selection of the plasmid vector in bacteria but 

which are not expected to be transferred to the American chestnut somatic embryo

(Figure 7.1a., p. 85, ESF 2019).
4

After incubation with the Agrobacterium vector, the American chestnut somatic embryo 

clumps were placed on selection medium with hygromycin to select for transformed 

embryos and with the antibiotic carbenicillin disodium salt to eliminate A. tumefaciens. 

Each clump of surviving somatic embryo tissue on selection medium was considered a 

separate event, presumably originating from a single transformed cell. Putative transgenic 

somatic embryos (events) were propagated and then tested for the presence of oxo gene

via PCR screening. The somatic embryos with the confirmed presence of oxo gene were 

propagated and regenerated. The regenerated shoot was then placed on rooting medium 

to induce the development of roots, resulting in the production of transformed American 

chestnut plantlets (ESF 2019).

The transformed American chestnut plant (Darling 58) expresses the oxo gene encoding

oxalate oxidase (OxO) that neutralizes oxalic acid produced by the chestnut blight fungus

(ESF 2019). Additionally, Darling 58 contains selectable marker gene nptII that 

expresses the neomycin phosphotransferase (NPTII) enzyme to allow successfully 

transformed tissue to survive in the presence of aminoglycoside antibiotics. The NPTII

protein is produced in numerous deregulated commercial plant products, and the safety of 

NPTII proteins present in biotechnology-derived crops has been extensively assessed

(US-FDA 2022; USDA-APHIS 2022).

The isogenic recipient genome of Darling 58 is a wild-type C. dentata tissue culture line 

known as Ellis that was established from a single immature zygotic embryo extracted 

from wild seed of an American chestnut tree (C. dentata (Marsh.) Borkh.) known as Pond 

#1 (ESF 2019). This tree is located near Windsor, NY, on the property of a member of 

The American Chestnut Foundation who reports that in the early 1960s, the property 

contained hundreds of naturalized American chestnuts, including the Pond #1 tree. There 

were no chestnuts from outside sources or other locations planted on this property before 

the establishment of Pond #1. Presently, most of the trees have either died or been 

reduced to stump sprouts.

Control materials used as comparators in the Darling 58 safety assessments include the 

isogenic recipient line Ellis and unmodified full siblings for molecular characterization 

studies (vector insert copy number, insert location, OxO mRNA expression, OxO enzyme 

activity, OxO enzyme quantification). Control materials used as comparators for 

phenotypic characteristics (blight tolerance, growth, respiration and photosynthesis, nut 

nutrition and composition) and environmental interactions (mycorrhizal colonization of 

roots, seed germination in Darling 58 leaf litter, insect herbivory on Darling 58 leaves, 

bumble bee foraging on Darling 58 pollen, and responses to other pests and diseases) 

included wild-type American chestnut, full-siblings, traditionally bred American chestnut 

x Chinese Chestnut hybrids, Chinese chestnut, and European chestnut (ESF 2019).

APHIS is confident that the use of the unmodified isogenic recipient line, unmodified full 

siblings, and the other reference materials used as comparators is sufficient to determine 

that Darling 58 American chestnut does not differ from other American chestnut growing 

wild or in an orchard.
5

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 modified 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, or changes in plant metabolism 

or composition in Darling 58 American chestnut compared to the isogenic and other nontransgenic controls. The assessment encompasses a consideration of the expressed 

oxalate oxidase (OxO) from Triticum aestivum, the expressed neomycyin 

phosphotransferase (NPTII) from Escherichia coli, and any observed or anticipated 

effects on plant metabolism including, for example, any relevant changes in levels of 

metabolites, antinutrients, or nutrients in nuts or leaves derived from Darling 58 

American chestnut compared to those in the isogenic or other non-transgenic controls. 

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 modified chestnut tree event; or for expression of inserted DNA, new proteins or 

enzymes, or changes in metabolism to affect plant pests or diseases, nontarget beneficial 

organisms, weediness, silvicultural and agricultural practices that impact pests or diseases 

or their management, or plant pest risks through horizontal gene flow. 

Description of the genetic modification and inheritance of inserted DNA

The inserted DNA in Darling58 American chestnut, with both the oxo and nptII gene 

expression cassettes from T-DNA for production of OxO and NPTII proteins, is 

described as containing the following genetic elements (Figure 7.1a, p85, ESF 2019):

• B-Right Border Region: A specific DNA region from A. tumefaciens containing 

the 71 base pair right border sequence used for transfer of the T-DNA.

• CaMV: Promoter Sequence of the constitutive cauliflower mosaic virus (CaMV) 

35S promoter (Guilley et al. 1982).

• Ta-oxo: Coding Sequence, 672 base pairs, for oxalate oxidase enzyme (OxO) 

protein from Triticum aestivum that confers tolerance to blight in American 

chestnut.

• T-actII: Terminator Sequence, actin 2 terminator from Arabidopsis thaliana.

• P-UBQ10: Promoter Sequence, the constitutive promoter of the Ubiquitin 10 

gene from Arabidopsis thaliana.
6

• CS-nptII: Coding Sequence, for neomycyin phosphotransferase (NPTII) from 

Escherichia coli. 

• Tnos: Terminator Sequence, 3’ UTR from nopaline synthase gene, nos, of A. 

tumefaciens.

• B-Left Border Region: A specific DNA region from A. tumefaciens containing 

the left border sequence used for transfer of the T-DNA.

ESF confirmed the insertion of the genetic elements listed above by conducting a detailed 

molecular characterization of the inserted DNA sequences in Darling 58 American 

chestnut tree. ESF used a combination of PCR and Sequence Analysis to demonstrate the 

insertion of transgenes (oxo and nptII genes) but without causing any extraneous insertion 

of vector DNA sequences or deletions of existing DNA compared to the Ellis isogenic 

line. APHIS reviewed the molecular characterization data and methods provided in 

Section 7 of the petition (ESF 2019):

• The T-DNA inserted into the Darling 58 American chestnut genome is present at 

a single locus on chromosome 7, about 10.9 and 5.5 kilobases away from the 

nearest upstream and downstream chestnut genes, respectively.

• Darling 58 does not contain any sequence from the plasmid p35S-OxO backbone 

or from T-DNA.

• The T-DNA sequence in Darling 58 has an inversion of about 600 base pairs of 

genomic DNA just outside the left T-DNA border. However, the inversion is not 

near any known genes.

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

Blight tolerance in Darling 58 American chestnut is conferred by an oxalate oxidase gene 

(oxo) from T. aestivum. The mechanism of the blight tolerance mediated by oxalate 

oxidase enzyme is to detoxify oxalic acid secreted by the fungal pathogen, preventing the 

oxalate acid from killing the chestnut’s tissues. Oxalate oxidase is widely present in all 

grains of crops and many wild plants and microbes, and it is well understood with a 

history of over 100 years’ studies. Wheat oxalate oxidase enzyme has been well 

characterized, can effectively detoxify oxalate, and has no known safety concern for 

consumption by human and livestock. 

The Darling 58 oxalate oxidase enzyme was shown to be expressed in all the tested leaf, 

stem, root, and nut tissues of the chestnut tree, but in a decremental sequential order. 

The petitioner carried out nut nutrition and composition analysis of Darling 58 and its 

non-transgenic American chestnuts to assess whether levels of key nutrients and 

compositions in Darling 58 American chestnut were equivalent to levels in the nontransgenic control. It showed that Darling 58 and non-transgenic nuts are nutritionally 

almost identical when rounded to FDA guidelines for food labels. While difference does 

exist in American chestnut nutritional measurements between source trees, and between 

similar trees over different years, the values of transgenic chestnuts are within the ranges 
7

found in non-transgenic chestnuts. This supports the conclusion that Darling 58 American 

chestnut is both nutritionally and compositionally similar to its related non-transgenic

controls.

Based on all the above noted considerations, APHIS concludes that the inserted genetic 

material, its inheritance and expression are stable, and the resulting proteins do not alter 

Americann chestnut plant metabolisms except the intended blight tolerance trait 

compared to non-transgenic controls.

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 Darling 58 American chestnut 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 whether Darling 58 American 

chestnut is likely to have significantly increased disease and pest susceptibility based on 

data and observations from field trials 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 modified 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.

American chestnut itself is not considered a plant pest in the U.S. (7 CFR § 340.2 (7 CFR 

340 2020)). The plant pest derived vector DNA and the plant pest vector used to insert 

the DNA do not pose a plant pest risk to Darling 58 American chestnut. The binary 

plasmid vector p35S-OxO proved to be disarmed; the T-DNA inserted into Darling 58 

American chestnut contained only the intended sequences, along with the typical 

insertion site mutations, and lacked sequences from Tumor-inducing (Ti) plasmids 

normally responsible for the formation of crown gall tumors upon A. tumefaciens

infection (Hoekema et al. 1983; Hellens et al. 2000). The sequences derived from plant 

pathogens retained in Darling 58 American chestnut (i.e., promoter sequence from 

caulimovirus and T-DNA border sequences from A. tumefaciens) are non-coding 

sequences that do not cause plant disease. 

Darling 58 American chestnut was grown within confined field trials in the U.S. since

2011 across seven states covering a diverse range of environmental conditions 

representative of where American chestnut is currently grown and bred, and where 

Darling 58 American chestnut is expected to be grown. In addition to the observational 

data that ESF annually reported to USDA-APHIS from these product development trials, 
8

which would have included reports of unusual pest and/or disease incidence, ESF also 

assessed phenotypic and environmental interaction characteristics for Darling 58 

American chestnut compared with the non-transgenic controls.

Data from these tests showed that the most substantial difference in Darling 58 American 

chestnuts compared to the non-transgenic controls is its oxalic acid tolerance that allows 

the tree to coexist with the blight fungus. However, Darling 58 American chestnuts are 

not associated with increased susceptibility or tolerance to specific abiotic stressors, 

arthropods, or diseases compared to controls. The results support the conclusion that the 

blight tolerance trait in Darling 58 American chestnut is not expected to alter the response 

of Darling 58 to diseases or arthropod pests under natural levels of these stressors, nor 

cause pest arthropods to be more abundant around Darling 58 plots, compared to 

conventional non-transgenic controls. 

In summary, the introduced genes did not significantly alter the observed insect pest 

infestation and disease occurrence or resulting damage on Darling 58 compared to the 

control and other reference lines. As presented later in this document, the blight tolerance

trait did not significantly alter the observed agronomic and phenotypic traits and did not 

reveal any significant changes that would indirectly indicate that Darling 58 is or could 

be more susceptible to pests and diseases compared to the control lines. Thus, Darling 58

is unlikely to be more susceptible to plant pathogens and insect pests than conventional 

American chestnut and existing commercial hybrids, and it is unlikely to differ from 

conventional American chestnut 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

APHIS assessed whether exposure or consumption of Darling 58 American chestnut

would have a direct or indirect adverse impact 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 

data and information on Darling 58 American chestnut compared to the conventional 

control and other comparators used as a reference range for 1) any biologically relevant 

changes in the phenotype or substances produced that may be novel or expressed at 

significantly altered amounts and are associated with impacts on organisms beneficial to 

agriculture, and/or 2) any observations of beneficial organisms associated with the plants. 

APHIS reviewed information ESF provided justifying the safety of Darling 58, as well as 

additional literature.

As indicated earlier in this plant pest risk assessment, the mechanism of the blight 

defense in Darling 58 American chestnut is mediated by wheat oxalate oxidase enzyme to 

detoxify oxalic acid secreted by the fungal pathogen, which allows the tree to tolerate and 

coexist with the blight fungus. Also, oxalate oxidase is widely present in a variety of

plants and microbes, has been well characterized, and has no known safety concern for 

consumption by human and livestock. The petitioner’s characterization of Darling 58
9

showed nutrient composition levels in nuts were comparable to the conventional control, 

and that the Darling 58 OxO expression level is low, so there is unlikely to be nontarget 

effects resulting from changes in composition or from consumption of Darling 58 OxO. 

Also, the study on environmental interactions found that there were no changes in 

beneficial arthropod abundance in field plots of Darling 58. 

Therefore, based on the above analysis of ESF’s studies on Darling 58 nut nutrient and 

composition, levels of OxO in tissues, and environmental interactions with beneficial 

arthropods, APHIS concludes exposure to and/or consumption of Darling 58 are unlikely 

to have any adverse impacts on organisms beneficial to agriculture.

F. Potential for Enhanced Weediness of Darling 58 American Chestnut

APHIS assessed whether Darling 58 is likely to become more weedy (i.e., more 

prevalent, competitive, damaging or difficult-to-control in situations where it is not 

wanted) than the non-transgenic controls. The assessment considers the basic biology of 

the plant, the situations in which volunteers or feral populations are considered weeds, 

and an evaluation of Darling 58 compared to its progenitor chestnuts under field 

conditions characteristic for the regions of the U.S. where American chestnut is grown 

(and/or evaluated under laboratory or greenhouse conditions) 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. The assessment 

also considers whether the engineered trait affects methods of control for the plant in 

situations where it is managed as a weed or volunteer in subsequent crops or in feral 

populations. 

American chestnut is a generalist species with competitive growth rate, it exhibits

intermediate shade tolerance, and ability to regenerate vegetatively from the root collar

(Jacobs et al. 2013). These traits allow American chestnut to establish and colonize areas 

where soil and climatic conditions are suitable, which includes much of the eastern 

United States and southeastern Canada. Though American chestnut grows considerably 

faster than other hardwoods on the same site (McEwan et al. 2006), American chestnut 

spreads at an average rate of 100 m/yr., which is much slower than oaks which spread at 

an average rate of 350 m/yr., and American beech at an average rate of 200 m/yr. (Davis 

1981; Davis 1983; McEwan et al. 2006). This slow-spread of American chestnut trees 

may be due to the production of less than one viable offspring per year until they were 

greater than 17 years old, and even trees > 70 years old would produce less than 5 

offspring per year (Rogstad and Pelikan 2014; ESF 2019).

The natural spread of chestnut species to a new location can occur only by seed because

chestnut sprouts new shoots only from the immediate location of the former tree (ESF 

2019). However, American chestnut takes at least several years to produce seeds, and a 

good number of seeds would be produced only after 20th year (Zon 1904; Cook and 

Forest 1979). American chestnut seeds show little dormancy because mature seeds are 

released from burs through fall frost and then can sprout the following spring (ESF 
10

2019). Furthermore, actual seed germination and the subsequent seedling establishment

of chestnut is rare due to the chestnut seeds’ high predation rate by wildlife and insects. 

Even after being established, chestnut seedlings are also subject to herbivory and damage 

by a variety of mammals. Therefore, American chestnut trees are not able to spread 

quickly without human assistance.

American chestnut is better adapted to fire and intensive logging than its competitors due 

to its ability to sprout after fire or from the cut stumps (Abrams 2003; Nowacki and 

Abrams 2008; Faison and Foster 2014). It was also empirically shown that American 

chestnut has the ecological capacity to achieve canopy dominance on favorable sites

(Paillet and Rutter 1989). One of the most reliable predictors of whether a species would 

be invasive was whether the plant was known to be invasive elsewhere in the world

(Reichard and Hamilton 1997). Chestnuts have been introduced throughout the United 

States, as well as in Argentina, Australia, and New Zealand (Jaynes and DePalma 1984). 

However, American chestnut has become naturalized in only a few places outside its 

native range (Section 2.1, ESF 2019). American chestnuts have been planted on the west 

coast of the United States for well over a century and have not established any

naturalized populations (Section 2.1.1, ESF 2019). These data indicate that Darling 58

American chestnut is unlikely to display the highly competitive and fast spreading

behavior associated with weedy and/or invasive species.

In the United States, American chestnut is not listed as a weed in any of the major weed 

databases, nor is it designated as a noxious weed or invasive species by the United States

federal government or any other countries. American chestnut exhibited historically slow 

spread rates, low propagule pressure, and need for disturbance to provide sufficient light 

for fast growth, the rate of increase would be very slow without human assistance, 

requiring centuries before chestnut becomes a significant presence in the landscape. 

Considering that there are no differences in the seedling establishment, growth, 

reproduction and dispersal of Darling 58 and wild-type American chestnut, it is implied 

that Darling 58 American chestnut trees may also take a century or more to become 

dominant after the first pioneer trees become established in a given area and it is unlikely 

that Darling 58 American chestnut populations establish in non-native habitats.

G. Potential Impacts on the Weediness of Any Other Plants with which 

Darling 58 American Chestnut 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; 

Grant 1994; 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; Rieseberg 1997; Preston 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 
11

relatives, as observed in rice, sorghum, sunflower and a few other crops (see Table 1 in 

Ellstrand et al. (1999)). By providing fitness-related traits such as resistance to insects, 

diseases, herbicides or harsh growing conditions, gene flow from crops to their wild 

relatives could allow the hybrids to compete better, produce more seeds, and become 

more abundant (Snow 2002). Besides weediness, other concerns are the loss of herbicide 

resistance as a tool to protect crops from closely related weeds (Gepts and Papa 2003). 

This topic is covered in two sections: 1) the potential for gene flow, hybridization and 

introgression from Darling 58 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 following introgression, based on the 

phenotypic changes that have been observed in the engineered plants. 

Potential for gene flow, hybridization and gene introgression

The American chestnut (Castanea dentata (Marsh.) Borkh) is a deciduous tree with a 

widespread historical range in the eastern United States and southeastern Canada before 

the introduced blight killed the chestnut trees in the early 20th century (Emily 1987). 

Fossils of Castanea that were detected in Greenland and throughout western North 

America and Asia can be dated back to more than 85-60 million years ago (mya) and 

indicate that the species once had a much wider distribution than today (Dane 2022). 

There are at least 26 Castanea species known to occur in North America (GBIF 2022) of 

which the occurance of some Castanea species (C. dentata, C. crenata, C. mill, C x 

neglecta, C. pumila, C. sativa) were also mapped by USDA and found substantial overlap 

in their range with American chestnut (USDA-NRCS 2022). Also, figure 2.1.1a of this 

petition describes overlap of distribution range for North American Castanea species: C. 

dentata, C. pumila, and C. ozarkensis. However, none of the Castanea species are listed 

as weeds or invasive species (CABI 2022; WSSA 2022).

C. dentata (American chestnut) is substantially outcrossing and self-incompatible. A 

recent whole genome analysis of 384 putative American chestnut individuals showed that 

340 had >99% ancestry assigned to the C. dentata populations. Whereas ten individuals 

showed a significant level of ancestry (>10%) from other Castanea species. Of these four 

samples were identified as C. sativa x C. dentata hybrids, and three were C. mollissima x 

C. dentata hybrids. Overall, the American chestnut samples sequenced revealed some 

degree of introgression with other Castanea species (Sandercock et al. 2022). It was also 

described in the petition that the American chestnut can outcross to other chestnut 

species, including Chinese chestnut (C. mollissima), Japanese chestnut (C. crenata), 

European chestnut (C. sativa), and chinquapin (C. pumila) (Jaynes and DePalma 1984) to 

form hybrids (Section 2.1.2). However, many such crosses may result in problems such 

as abnormal nut development called Internal Kernel Breakdown (Fulbright et al. 2012), a 

semi-lethal condition called “cracked bark” in young seedlings (Jaynes and DePalma 

1984), and male sterility in hybrid offspring (Anagnostakis 2012; Sisco et al. 2012). 

The widespread chestnut blight fungus, which colonizes wounded cambium and forms a 

canker that eventually results in death of the aboveground stem, has led to the decline of 

sexual reproduction of American chestnut in the North American range to the point of 

being very rare. The blight does not attack roots, so the current remaining wild American 
12

chestnut population is characterized by an asexual cycle of root-collar sprouting from the 

surviving trunks, eventual infection by blight before the tree can reach the forest canopy 

and form flowers, and stem dieback. In the U.S., 431 ± 30.2 million American chestnut 

trunks remain with the vast majority of these trunks (360 ± 22 million, 84%) having a 

diameter at breast height (dbh) of <2.5 cm. (Dalgleish et al. 2015). The pollen-flow from 

Darling 58 to these American chestnut trunks and to the sprouts emerging from them, 

will not result in the successful production of hybrid offspring.

There are several reports of effective pollination distance for American chestnut from 

reviewed and non-reviewed sources. These reports would include both wind and insect 

pollination because they examine viable nut production success, regardless of how the 

pollen was vectored. Jacobs et al. (2016) report that trees need to be within 100 meters (~ 

328 ft) for successful pollination. Rutter (1990) states that trees only 100 feet apart have

reduced pollination success, and trees 1000 ft apart are essentially reproductively isolated 

from one another. To prevent Internal Kernel Breakdown (IKB) caused by crosses 

between Chinese chestnut and the Colossal hybrid, the Midwest Nut Producers Council 

recommends keeping these trees at least 1000 ft from each other to prevent pollination 

(Long 2012).

Pollen-mediated gene-flow from events derived from Darling 58 to the wild relatives 

would be possible because Castaenea species can hybridize freely (Lang et al. 2006).

Also, there are reports of natural occurance of interspecific hybrids such as C. dentata x 

C. mollissima and Castenea x neglecta (a natural hybrid between C. dentata x C. pumila)

with C. dentata as one of the parental species (Binkley 2008; Perkins et al. 2019; 

Sandercock et al. 2022). These analyses suggest that there is a low degree of possibility 

for gene flow via introgression between sympatric Darling 58 American chestnut and 

other Castanea species.

Potential for enhanced weediness of recipients after hybridization and/or introgression

Darling 58 American chestnut, described in the current petition, was transformed with 

oxo gene to produce oxalate oxidase (OxO) enzyme that can detoxify the oxalate 

produced by the blight-causing fungus, Cryphonectria parasitica. The OxO enzyme 

detoxify the oxalate by breaking it down to carbon dioxide and hydrogen peroxide. The 

hydrogen peroxide byproduct might have a second function of strengthening the lignin in 

the barrier produced by the chestnut in an effort to wall off the fungal infection. Though 

there is some evidence that the OxO enzyme can enhance lignin formation (Welch et al. 

2007), this is not to modify any other traits related to weediness such as competitiveness, 

responses to other pests, interactions with other organisms in the environment, or survival 

(besides blight tolerance).

If genetic introgression of the transgene in Darling 58 American chestnut to a sexually 

compatible relative were to occur, the offspring would be expected to gain the ability to 

synthesize oxalate oxidase and tolerance to blight fungus. However, similarly, to 

modified chestnut, the OxO production is not expected to influence the weediness of 

sexually compatible relatives of chestnut that acquire the transgene. American chestnut 
13

and its wild relative are not listed as weeds or invasive species by any US states or other 

countries.

Based on the information presented in the petition and in relevant literature, APHIS has 

reached the following conclusions. Gene flow, hybridization and/or introgression of 

genes from American chestnut to its sexually compatible relatives, including wild, feral 

or cultivated species in the U.S. and its territories is likely to occur if the plants are within 

the pollen dispersal distance, and depending on their potential for crossing with C. 

dentata, as discussed above. The genetic modification in Darling 58 American chestnut is 

not expected to increase the potential for hybridization, gene flow and and/or 

introgression to occur to sexually compatible taxa compared to the non-transgenic 

recipient or other varieties of chestnut that are commonly growing in the forests and in 

the sylviculture settings. The genetic modification in Darling 58 American chestnut is 

unlikely to confer novel weedy properties to chestnut or its wild relatives. Therefore, 

APHIS has determined that Darling 58 American chestnut is not expected to increase the 

weed risk potential of other species with which it can interbreed in the U.S. and its 

territories.

H. Potential Changes to Silviculture or Agriculture Practices

APHIS assessed whether significant changes to silvicultural or agricultural practices from 

adoption of Darling 58 are likely to impact American chestnut’s silviculture or 

agriculture practices. This includes consideration of any changes in phenotypic 

characteristics and environmental interactions.

The American chestnut was once one of the most abundant trees within its native range in 

the eastern United States. It was a fast-growing and long-lived canopy tree that produced 

a consistent crop of nuts, could be harvested for lumber, and was considered a keystone 

species for wildlife in the past. However, American chestnuts are now considered 

functionally extinct due to a fungal canker disease caused by an invasive fungal pathogen 

C. parasitica.

ESF intends to use Darling 58 blight-tolerant American chestnut for restoration and forest 

health and to establish and colonize much of the eastern United States where stunted trees 

persist. The long-term goal is for the American chestnut to become a self-sustaining 

forest tree species within its native range. Initial distribution will consist of long-term 

research plots and relatively small-scale public horticultural plantings to inform 

subsequent larger-scale distributions. The American Chestnut Foundation (TACF) and 

ESF plan to cross Darling 58 with a diverse set of surviving American chestnuts over 

multiple generations, which should result in a diverse and resilient population suitable for 

potential large-scale restoration efforts. 

As previously described, tolerance to the invasive, exotic fungal pathogen, C. parasitica, 

in Darling 58 American chestnuts was enhanced by inserting the wheat oxo gene for the 

oxalate oxidase (OxO) enzyme. This enzyme has no direct fungicidal properties, but 
14

rather detoxifies oxalic acid (oxalate) produced by the fungus, preventing the acid from 

killing the chestnut’s tissues which can lead to lethal cankers on the tree. And thus, the 

introduced oxalic acid tolerance conferred by oxo gene allows coexistence of American 

chestnut tree and the fungus. This means that modified American chestnut trees will not 

require forest management intervention practices used to maintain a plant defense 

mechanism.

Control materials used as comparators for phenotypic characteristics (blight tolerance, 

growth, respiration and photosynthesis) and environmental interactions (mycorrhizal 

colonization of roots, seed germination in Darling 58 leaf litter, insect herbivory on 

Darling 58 leaves, bumble bee foraging on Darling 58 pollen, and responses to other 

pests and diseases) included wild-type American chestnut, full-siblings, traditionally bred 

American chestnut x Chinese Chestnut hybrids, Chinese chestnut, and European chestnut

(ESF 2019).

Phenotypic observations including growth and photosynthesis (Section 8) and including 

growth and photosynthesis and environmental interaction experiments on Darling 58 

have been conducted under laboratories, greenhouses, and field conditions under APHIS 

BRS authorizations. The purpose of the authorizations includes Phytophthora screenings, 

outdoor plantings to evaluate growth, controlled pollinations, ecological interactions, and 

blight tolerance. No unusual occurrences, non-target interactions, or deleterious effects 

have been observed which demonstrate that Darling 58 is not significantly different from 

unmodified chestnut controls (Appendix I, ESF 2019).

Phenotypic characteristics

ESF evaluated phenotypic characteristics as compared to unmodified and traditionally 

bred controls in field, laboratory, or greenhouse experiments. Additionally, data from 

legacy modified event, Darling 4, provide further evidence to support that the presence of 

oxo gene or the transformation process does not significantly affect growth or 

performance of modified American chestnuts. Modified American chestnut trees had 

similar growth and photosynthetic and respiratory rates compared to unmodified control 

trees.

Furthermore, the data support the conclusion that the Darling 58 American chestnut is no 

more likely to pose a plant pest risk than comparators such as the Ellis isogenic line, fullsibling controls, or traditionally bred American chestnuts. The most substantial difference 

is oxalic acid/blight tolerance trait that allows chestnut trees to coexist with the blight 

fungus.

Environmental Interactions

Mycorrhizal colonization of Darling 58 roots

There were no significant differences in colonization by ectomycorrhizal fungi in roots 

compared to unmodified controls. These results suggest that presence or expression of 

OxO in Darling 58 does not pose novel pest risks to native soil fungi that are ecologically 
15

important for American chestnuts and other trees (ESF 2019). The introduction of 

Darling 58 back into eastern forest is unlikely to have negative impacts on soil quality.

Responses of plants found in chestnut habitats: germination of seeds in Darling 58 

leaf litter

A greenhouse bioassay was performed to evaluate interactions between leaf litter from 

modified American chestnut trees and seeds from other species that are native or 

naturalized in American chestnut habitats (Newhouse et al. 2018). No significant 

differences in seed germination in the presence of modified vs. unmodified American 

chestnut leaf litter, in terms of numbers of seeds germinated or total dry biomass of 

germinated seedlings was observed. These studies reinforce that modified American 

chestnuts are not substantially different than unmodified American chestnuts (apart from 

their enhanced blight tolerance), and do not pose a novel pest risk to several types of 

representative native plants.

Insect herbivory on chestnut leaves

Interactions between chestnuts and herbivorous insects tested to date are not significantly 

affected by OxO. Plant/herbivore/biocontrol interaction tests to evaluate gypsy moth 

(Lymantria dispar) caterpillars feeding on various modified chestnut lines confirmed that 

insect herbivory on OxO-expressing modified American chestnuts is not significantly 

different than that on unmodified controls. Darling 58 modified leaves do not 

differentially affect biopesticides or parasitoids used to control insect pests.

Bumble bees and pollen with OxO

As previously mentioned, oxalate oxidase itself does not have known harmful effects 

against living organisms, but the hydrogen peroxide byproduct can have antimicrobial 

properties (Baldry 1983) or sub-lethal effects on insects (Ramputh et al. 2002).

Chestnut pollen has been shown to be especially nutritious to bumble bees (Bombus 

terrestris) (Tasei and Aupinel 2008). However, a realistic concentration of oxalate 

oxidase in chestnut pollen does not present novel risks to bumble bees (Section 9.1.4, 

ESF 2019).

Responses to other pests and environmental stresses

Apart from chestnut blight tolerance, no differences between modified and unmodified 

trees have been observed in response to plant-disease, plant-arthropod, or plantenvironment interactions. Modified and unmodified American chestnuts in greenhouse 

conditions do not appear differentially susceptible to damage from common greenhouse 

pests such as mealybugs, spider mites, and powdery mildew.

Phytophthora cinnamomi is especially relevant to American chestnuts as it is a major pest 

in the southern part of the chestnut’s natural range (Wang et al. 2013). Studies by ECF 

confirm that presence or expression of OxO in Darling 58 offspring does not affect 

susceptibility to Phytophthora, further confirming a lack of plant-pest interactions outside 

of blight tolerance.
16

Field-grown chestnuts have also been observed to sustain incidental insect herbivory, 

powdery mildew infections, and rodent chewing, regardless of transgene presence. There 

is no evidence that planting of Darling 58 American chestnuts would affect incidence of 

other pests or pathogens any more than planting traditionally bred chestnuts.

Potential spread of OxO to other Castanea species

There is no data on natural pollination rates for Darling 58 trees yet. However, there is no 

reason to expect that pollen viability, fertilization rates, or any other aspect of sexual 

reproduction would differ between Darling 58 American chestnut and unmodified 

American chestnut

Persistence of OxO activity in Darling 58 leaves

To address a potential concern over persistence of OxO activity in modified American 

chestnut leaves, ESF tested and monitored the OxO activity in mature leaves of lateseason Darling 58 plants. It was shown that OxO activity in abscised leaves was

detectable for over five days but was not detectable when a leaf dried enough to begin to

curl (Section 9, ESF 2019). Furthermore, the similarity of results between modified lines 

with different expression levels suggests that overall OxO transcript levels in active 

plants do not substantially affect the persistence of OxO activity in desiccating/inactive 

tissues.

Potential effects of blight-tolerant chestnuts on C. parasitica

Darling 58 blight-tolerant trees can survive and serve as a host of C. parasitica

indefinitely, allowing the fungus to persist, while blight-susceptible trees eventually die 

back.

Furthermore, reproductively mature Darling 58 trees will continue to produce 

unmodified, blight-susceptible offspring through crossing with other American chestnut 

trees for the following reasons. American chestnut normally needs two trees to produce 

viable nuts because it is monoecious and predominantly self-infertile. Darling 58 is a 

hemizygous event with only one copy of the inserted oxo gene present in the genome. 

Thus, a cross with such hemizygous transgenic tree as a parent will always result in the 

offspring without transgene. Even when two Darling 58 trees are crossed, about 25% of 

their offspring would be non-transgenic and blight-susceptible (Section 6.4, ESF 2019).

Thus, it is likely that potential chestnut restoration scenarios including blight-tolerant host 

trees would not be detrimental to the blight fungus, allowing it to persist by increasing the 

longevity and/or prevalence of host trees.

Taken all together, Darling 58 American chestnut is similar to unmodified controls in its 

agronomic, phenotypic, environmental response and compositional characteristics and 

has levels of pests and diseases or their damage levels comparable to unmodified

American chestnuts. Furthermore, Darling 58 trees will be reintroduced as a selfsustaining tree species in its native range and these trees will not require additional forest 

management interventions. Also, because the progeny of Darling 58 trees will include 

both plants with transgene and plants without transgene, the original wild-type American 

chestnut will be conserved in the future (ESF 2019). In conclusion, APHIS could not 
17

identify any significant changes to silvicultural or agricultural or practices from adoption 

of the modified chestnut.

I. Potential Impacts from Transfer of Genetic Information to 

Organisms with which Darling 58 American Chestnut Cannot 

Interbreed

APHIS examined the potential for the new genetic material inserted into Darling 58 

American chestnut to be horizontally transferred to other organisms without sexual 

reproduction 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 (HGT) between unrelated organisms is one of the most 

intensively studied fields in the biosciences since the 1940s (Soucy et al. 2015), and the 

issue gained extra attention with the release of modified plants into the environment

(Dröge et al. 1998). Potential risks from stable HGT from genetically engineered 

organisms to another organism without reproduction or human intervention have been

reviewed (Keese 2008b). 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 (Keese 

2008b; Soucy et al. 2015). 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 (Brown 

2003b; Keeling and Palmer 2008; Keese 2008b).

Potential for horizontal gene transfer to bacteria, fungi, or invertebrates

Darling 58 American chestnut contains protein coding regions derived from the oxo gene 

from the wheat plant (Triticum aestivum) and the nptII gene from Escherichia coli. It also 

contains non-protein-coding regions from A. tumefaciens, A. thaliana, and the cauliflower 

mosaic virus (CaMV). 

One example of HGT involves a class of enzymes similar to mono-oxygenase gene 

(DMO). Chakraborty et al. (2012) propose that HGT contributed to the distribution of 

ring-hydroxylating oxygenase (rho) genes among prokaryotic phyla (proteobacteria, 

actinobacteria, cyanobacteria, and archaea), and note that homologues of rho genes are 

found in plants (in strains of Arabidopsis, Zea mays, Oryza sativa, Physcomitrella patens 

and Amaranthus tricolor). Ring-hydroxylating oxygenases (RHO) catalyze the addition 

of hydroxyl groups to aromatic ring compounds, initiating one of the major pathways for 

oxidative degradation of both natural and synthetic aromatic compounds in the 

environment (Peng et al. 2010a, b). Dicamba mono-oxygenase is a unique type of RHO 

that initiates the degradation of dicamba by oxygenating the exocyclic methyl group, 

rather than the more conventional oxygenation of the aromatic ring of the substrate seen 

in most other RHOs (Dumitru et al. 2009). Chakraborty et al. (2012) suggest that 
18

distribution and diversification of rho genes can be explained by the mechanisms of gene 

duplication, transposition events and DNA rearrangements in most cases.

In other cases, HGT is assumed to be the primary mechanism where occurrence of the 

genes was found to be limited to just one or two organisms within phyla (such as rho

genes in some cyanobacteria, firmicutes and crenarchaeota), since the possibility of being 

remnants of a partially deleted rho operon is ruled out due to the absence of similar genes 

in any other member of these genera. Although it is widely accepted that HGT has 

generated novel degradation capabilities and increased metabolic diversity among 

bacterial communities exposed to an ever-evolving array of polycyclic aromatic 

compounds, such degradative capabilities are mostly indicative of divergent evolution 

from a common ancestor, not HGT (Peng et al. 2010a).

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 2008b). 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 2008b) 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 2008a; Zhu et al. 2011; Acuna et al. 

2012).

Horizontal transfer from and expression in bacteria of the foreign DNA inserted into the 

nuclear genome of the GE plant is unlikely to occur. First, many genomes (or parts 

thereof) have been sequenced from bacteria that are closely associated with plants 

including Agrobacterium sp. and Rhizobium sp. (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 2008b). Second, 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 (Koonin et al. 2001; Brown 2003a; EFSA 2009a). Third, transgene DNA promoters 

and coding 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. Fourth, both the FDA (US-FDA 1998) and the

European Food Safety Authority (EFSA 2009b) have evaluated horizontal gene transfer 

from the use of antibiotic resistance marker genes and concluded that the likelihood of 

transfer of antibiotic resistance genes from plant genomes to microorganisms in the 

gastrointestinal tract of humans or animals, or in the environment, is very rare or remote.
19

Potential for horizontal gene transfer to viruses 

APHIS also considered whether horizontal transfer of DNA from the modified chestnut 

tree 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

(Keese 2008b). 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 2008b). HGT from virus sequences engineered 

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

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

1998) and RNA viruses (that typically replicate in the cytoplasm); however most have 

been under conditions that favor recombination to restore a defective virus (Thompson 

and Tepfer 2010). Populations of recombinants between virus transgenes expressed in 

transgenic plants infected with related viruses are similar to recombinants found in mixed 

infections of the same viruses in non-transgenic plants, indicating that there was no novel 

recombination mechanism in the transgenic plants and no increased risk is expected over 

what is expected from mixed infections (Keese 2008b; Turturo et al. 2008).

Nonhomologous recombination in HGT among viruses or between virus transgenes and 

infecting viruses can occur, but frequently results in gene deletions that can result in 

nonviable viruses (Morroni et al. 2013). Depending on the particular virus and sequences 

involved, various hot-spots for recombination have been found in both coding and 

noncoding regions, and strategies implemented in design of transgenes to avoid 

recombination have been suggested. No recombinant or undesirable viruses with new 

properties have been detected for over at least 10 years in field tests or during 

commercial growth of deregulated virus resistant plum, squash, or papaya engineered 

with genes from viruses that have been deregulated in the U.S. (Fuchs and Gonsalves 

2007).

The only virus sequence inserted in Darling 58 American chestnut is the 35S promoter 

for the cauliflower mosaic virus (CaMV) involved in regulating gene expression. CaMV 

is the type species of the genus Caulimovirus in the family Caulimoviridae – a group of 

double-stranded DNA viruses known as pararetroviruses, in which replication occurs in 

the cytoplasm via reverse transcription of an RNA intermediate. CaMV is a plant virus 

transmitted by sap inoculation, and in nature by aphids such as Brevicoryne 

brassicae, Myzus persicae, and at least 25 other species in a semi-persistent manner. 

Caulimoviruses generally have a narrow host range limited to plants of the family 

Brassicaceae, but some isolates are able to infect plants of the family Solanaceae 

experimentally (Hansen and Heslop-Harrison 2004). American chestnut is not known to 

be susceptible to CaMV or any Caulimoviruses.

Recombination in Caulimoviruses occurs predominantly, if not exclusively, in the 

cytoplasm by template switching between RNA transcripts during the replication process, 

although a low level of recombination involving viral DNA may occur in the nucleus 

(Froissart et al. 2005). Since the Caulimovirus promoter sequences are not transcribed in 

transgenic plants, there is little or no opportunity for them to recombine with any related 
20

Caulimoviruses that may infect American chestnut. Based on the foregoing, horizontal 

transfer of DNA from Darling 58 American chestnut to plant viruses is unlikely to occur 

or is unlikely to lead to the creation or selection of plant viruses that are more virulent or 

have a broader host range. 

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. 2005a, b), to other mitochondria genomes, and mostly 

involve parasitic plants and their hosts (Richardson and Palmer 2007). Recently, a 

comparative genomics analysis implicated HGT for the incorporation of a specific 

genetic sequence in the parasitic plant purple witchweed (Striga hermonthica) from its 

monocot host plant (Yoshida et al. 2010). 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. 2012a) (Xi et al. 

2012b)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. Furthermore, in the 

GE crop, the DNA sequences were inserted into the nuclear genome, not the

mitochondrial genome.

If the GE plant becomes infected by a parasitic plant or is naturally grafted to another 

plant, there is a very low probability that HGT could result in the other plant acquiring 

DNA from the GE plant. 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. 

Based on the above analysis, APHIS therefore concludes that HGT of the new genetic 

material inserted into Darling 58 American chestnut to other organisms with which it 

cannot interbreed 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 
21

other relevant information to assess the plant pest risk of blight-tolerant Darling 58 

American chestnut compared to the unmodified wild-type parent tree from which it was 

derived and other unmodified American chestnut controls. APHIS concludes that Darling 

58 American chestnut is unlikely to pose a greater plant pest risk than its unmodified 

parent based on the following findings: 

• No plant pest risk was identified from the transformation process or the insertion of 

new genetic material in Darling 58 American chestnut. The Agrobacterium

transformation vector was disarmed and therefore lacked sequences from Tumorinducing (Ti) plasmids normally responsible for the formation of galls in host plants

of A. tumefaciens; transformed material was treated to kill the bacterium; a 

terminator sequence donated from A. tumefaciens and a promoter sequence donated 

from Cauliflower mosaic virus do not code for or express any gene products, and 

those inserted plant pest sequences do not cause disease or create infectious agents. 

• No increase in plant pest risk was identified from expression of the inserted genetic 

material, the new Darling 58 OxO or NPTII proteins, or changes in metabolism or 

composition. The composition of Darling 58 American chestnut tissues were 

determined to be substantially equivalent to the wild-type parent tree from which 

Darling 58 was derived and other unmodified American chestnut controls. The mode 

of action and specificity of Darling 58 OxO or NPTII proteins raise no plant pest 

concerns.

• Disease and pest incidence and/or damage, other than reductions in chestnut blight 

from the target organism Cryphonectria parasitica, were not observed to be 

significantly different or atypical in Darling 58 American chestnut compared to the 

unmodified American chestnut controls in field trials conducted in growing regions 

representative of where Darling 58 American chestnut is expected to be grown. The 

oxalate oxidase blight tolerance and the neomycin phosphotransferase selectable 

marker traits did not significantly alter the response of Darling 58 to diseases or 

arthropod pests under natural levels of these stressors, and pest arthropods were not 

more abundant around Darling 58 plots compared to the unmodified controls. 

Observed agronomic traits also did not reveal any significant differences that would 

indirectly indicate that Darling 58 is more susceptible to pests or diseases. Therefore,

no plant pest effects are expected on these or other forest or agricultural products and 

no impacts are expected to APHIS pest control programs. 

• Exposure to and/or consumption of Darling 58 American chestnut is unlikely to have 

any adverse impacts on organisms beneficial to agriculture based on APHIS’ analysis 

of studies on Darling 58 American chestnut food and feed safety, nutrient and antinutrient composition, levels of OxO and NPTII in tissues, environmental interactions 

with beneficial arthropods, and pollen characteristics. 

• Darling 58 American chestnut is unlikely to become more of a weed or volunteer 

problem than wild-type American chestnut based on its observed agronomic 

characteristics, the low weediness potential of American chestnut, and current 

management practices available to control Darling 58 as a weed. Darling 58 

American chestnut volunteers can be controlled with all currently available weed 

control methods, should a landowner wish to remove Darling 58 American chestnut 

from their property.
22 

• Darling 58 American chestnut is not expected to increase the weed risk potential of 

other American chestnut, and other Castanea species with which it can interbreed. 

The genetic modification in Darling 58 American chestnut is not expected to increase 

its potential for gene flow, hybridization, and/or introgression to sexually compatible 

taxa, nor is it likely to increase their weediness potential in the event that such species 

were to be introduced. Introgression of the oxalate oxidase blight tolerance and 

neomycin phosphotransferase selectable marker traits into other American chestnut or 

related species will likely make them resistant to chestnut blight.

• Changes to silvicultural and orchard management practices (e.g., planting, pesticide 

applications, irrigation, nut harvesting, coppicing, timber harvesting, etc.) from 

adoption of Darling 58 American chestnut are only related to blight management 

practices and this is unlikely to increase pests or diseases or adversely impact their 

management, nor will they impact APHIS pest control programs. 

• Horizontal gene transfer of the new genetic material inserted into Darling 58 

American chestnut 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.

 

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