Federal Register / Vol. 88, No. 219 / Wednesday, November 15, 2023 / Notices 78285 

1To view the final rule and supporting 

documents, go to https://www.regulations.gov/ 

docket/APHIS-2018-0034. 

DEPARTMENT OF AGRICULTURE 

Animal and Plant Health Inspection 

Service 

[Docket No. APHIS–2023–0022] 

Movement of Organisms Modified or 

Produced Through Genetic 

Engineering; Notice of Proposed 

Exemptions 

AGENCY: Animal and Plant Health 

Inspection Service, USDA. 

ACTION: Notice. 

SUMMARY: We are advising the public 

that we are proposing to add five new 

types of genetic modifications a plant 

can contain and be exempt from the 

regulations for the movement of 

organisms modified or produced 

through genetic engineering because 

such modifications could otherwise be 

achieved through conventional breeding 

methods. First, we propose any diploid 

or autopolyploid plant with any 

combination of loss of function 

modifications (i.e., a modification that 

eliminates a gene’s function) in one to 

all alleles of a single genetic locus, or 

any allopolyploid plant with any 

combination of loss of function 

modifications in one or both alleles of 

a single genetic locus on up to four pairs 

of homoeologous chromosomes, without 

the insertion of exogenous DNA, would 

qualify for exemption. Second, we 

propose that any diploid or 

autopolyploid plant in which the 

genetic modification is a single 

contiguous deletion of any size, 

resulting from cellular repair of one or 

two targeted DNA breaks on a single 

chromosome or at the same location(s) 

on two or more homologous 

chromosomes, without insertion of 

DNA, or with insertion of DNA in the 

absence of a repair template, would 

qualify for exemption. Third, we 

propose to extend the modifications 

described in certain existing exemptions 

in the regulations to all alleles of a 

genetic locus on the homologous 

chromosomes of an autopolyploid plant. 

Fourth, we propose that plants with up 

to four modifications that individually 

qualify for exemption and are made 

simultaneously or sequentially would 

be exempt from regulation, provided 

that each modification is at a different 

genetic locus. Fifth, we propose that 

plants that have previously completed a 

voluntary review confirming exempt 

status and that have subsequently been 

produced, grown, and observed 

consistent with conventional breeding 

methods appropriate for the plant 

species, could be successively modified 

in accordance with the exemptions. 

This action would reduce the regulatory 

burden for developers of certain plants 

modified using genetic engineering that 

are not expected to pose plant pest risks 

greater than the plant pest risks posed 

by plants modified by conventional 

breeding methods. 

DATES: We will consider all comments 

that we receive on or before December 

15, 2023. 

ADDRESSES: You may submit comments 

by either of the following methods: 

• Federal eRulemaking Portal: Go to 

http://www.regulations.gov. Enter 

APHIS–2023–0022 in the Search field. 

Select the Documents tab, then select 

the Comment button in the list of 

documents. 

• Postal Mail/Commercial Delivery: 

Send your comment to Docket No. 

APHIS–2023–0022, Regulatory Analysis 

and Development, PPD, APHIS, Station 

3A–03.8, 4700 River Road Unit 118, 

Riverdale, MD 20737–1238. 

Supporting documents and any 

comments we receive on this docket 

may be viewed at regulations.gov or in 

our reading room, which is located in 

room 1620 of the USDA South Building, 

14th Street and Independence Avenue 

SW, Washington, DC. Normal reading 

room hours are 8 a.m. to 4:30 p.m., 

Monday through Friday, except 

holidays. To be sure someone is there to 

help you, please call (202) 799–7039 

before coming. 

FOR FURTHER INFORMATION CONTACT: Dr. 

Neil Hoffman, Science Advisor, 

Biotechnology Regulatory Services, 

APHIS, 4700 River Road Unit 98, 

Riverdale, MD 20737–1238; 

Neil.E.Hoffman@usda.gov; (301) 851– 

3947. 

SUPPLEMENTARY INFORMATION: The 

regulations in 7 CFR part 340 govern the 

movement (importation, interstate 

movement, or release into the 

environment) of certain organisms 

modified or produced through genetic 

engineering. The Animal and Plant 

Health Inspection Service (APHIS) first 

issued these regulations in 1987 under 

the authority of the Federal Plant Pest 

Act of 1957 and the Plant Quarantine 

Act of 1912, two acts that were 

subsumed into the Plant Protection Act 

(PPA, 7 U.S.C. 7701 et seq.) in 2000, 

along with other provisions. Since 1987, 

APHIS has amended the regulations 

seven times, in 1988, 1990, 1993, 1994, 

1997, 2005, and 2020. 

On May 18, 2020, we published in the 

Federal Register (85 FR 29790–29838, 

Docket No. APHIS–2018–0034) a final 

rule 1 that marked the first 

comprehensive revision of the 

regulations since they were established 

in 1987. The final rule provided a clear, 

predictable, and efficient regulatory 

pathway for innovators, facilitating the 

development of organisms developed 

using genetic engineering that are 

unlikely to pose plant pest risks. 

The May 2020 final rule included 

regulatory exemptions for certain 

categories of modified plants. 

Specifically, § 340.1(b) exempted plants 

that contain a single modification of one 

of the following types, specified in 

§ 340.1(b)(1) through (3): 

• The genetic modification is a 

change resulting from cellular repair of 

a targeted DNA break in the absence of 

an externally provided repair template; 

or 

• The genetic modification is a 

targeted single base pair substitution; or 

• The genetic modification introduces 

a gene known to occur in the plant’s 

gene pool or makes changes in a 

targeted sequence to correspond to a 

known allele of such a gene or to a 

known structural variation present in 

the gene pool. 

In addition to the modifications listed 

above, § 340.1(b)(4) provides that the 

Administrator may propose to exempt 

plants with additional modifications, 

based on what could be achieved 

through conventional breeding. Such 

proposals may either be APHIS-initiated 

or may be initiated via a request that is 

accompanied by adequate supporting 

information and submitted by another 

party. In either case, APHIS will publish 

a notice in the Federal Register of the 

proposal, along with the supporting 

documentation, and will request public 

comments. After reviewing the 

comments, APHIS will publish a 

subsequent notice in the Federal 

Register announcing its final 

determination. A list specifying 

modifications a plant can contain and 

be exempt pursuant to paragraph (b)(4) 

is available on the APHIS website at 

https://www.aphis.usda.gov/aphis/ 

ourfocus/biotechnology. 

On July 19, 2021, we published a 

notice in the Federal Register (86 FR 

37988–37989, Docket No. APHIS–2020– 

0072) proposing to exempt plants with 

any of the following additional 

modifications: 

• Cellular repair of a targeted DNA 

break in the same location on two 

homologous chromosomes, in the 

absence of a repair template, resulting in 

homozygous or heterozygous biallelic 


ddrumheller on DSK120RN23PROD with NOTICES1
78286 Federal Register / Vol. 88, No. 219 / Wednesday, November 15, 2023 / Notices 

mutations, each of which is a loss of 

function mutation; 

• Contiguous deletion of any size 

resulting from cellular repair of a 

targeted DNA break in the presence of 

an externally supplied repair template; 

or 

• Cellular repair of two targeted DNA 

breaks on a single chromosome or at the 

same location on two homologous 

chromosomes, when the repair results 

in a contiguous deletion of any size in 

the presence or absence of a repair 

template, or in a contiguous deletion of 

any size combined with an insertion of 

DNA in the absence of a repair template. 

We received comments on that notice 

that suggested these exemptions were 

piecemeal and could be replaced with 

an overarching exemption. Furthermore, 

comments included additional 

exemptions beyond those that we 

proposed. 

Based on the comments that we 

received and our own subsequent 

review and analysis of conventional 

breeding techniques that are currently 

employed, we are withdrawing the 

original three proposed exemptions and 

are proposing five new types of 

modifications a plant can contain and 

qualify for exemption from regulation 

pursuant to paragraph (b)(4) of § 340.1. 

First, we propose that a diploid or 

autopolyploid plant with any 

combination of loss of function 

modifications in one to all alleles of a 

single genetic locus, or an allopolyploid 

plant with any combination of loss of 

function modifications in one or both 

alleles of a single genetic locus on up to 

four pairs of homoeologous 

chromosomes, without the insertion of 

exogenous DNA, would qualify for 

exemption (proposed exemption 

340.1(b)(4)(vi)(Additional Modification 

(AM)1)). (Because this exemption would 

be found solely on the internet, and not 

in the regulations themselves, the ‘‘AM’’ 

nomenclature would be used to identify 

the method by which it and the other 

exemptions proposed in this notice 

were added.) This category would apply 

to scenarios that might not be expressly 

described in the exemptions codified in 

the May 2020 final rule (namely, 

paragraphs (b)(1) and (2) of § 340.1) but 

would achieve an end result that can 

also be accomplished by those 

exemptions. In addition, it more broadly 

extends, compared to the 2020 rule, loss 

of function mutations without the 

insertion of exogenous DNA to 

polyploid plants. 

Second, we propose that any diploid 

or autopolyploid plant in which the 

genetic modification is a single 

contiguous deletion of any size, 

resulting from cellular repair of one or 

two targeted DNA breaks on a single 

chromosome or at the same location(s) 

on two or more homologous 

chromosomes, without insertion of 

DNA, or with insertion of DNA in the 

absence of a repair template, would 

qualify for exemption (proposed 

exemption 340.1(b)(4)(vi)(AM2)). As 

proposed, additional modifications to 

homoeologous loci of homoeologous 

chromosomes of allopolyploids would 

not qualify for this exemption. 

Third, we propose to extend the 

modifications described in the 

exemptions found at § 340.1(b)(2) and 

(3) to all alleles of a genetic locus on the 

homologous chromosomes of 

autopolyploids (proposed exemption 

340.1(b)(4)(vi)(AM3)). As proposed, 

additional modifications to 

homoeologous loci of homoeologous 

chromosomes of allopolyploids would 

not qualify for this exemption. 

Fourth, we propose that plants with 

up to four modifications of a certain 

type, made simultaneously or 

sequentially, that individually qualify 

for exemption, and provided each 

modification is at a different genetic 

locus, would be exempt from regulation 

because such modifications are 

achievable through conventional 

breeding methods (proposed exemption 

340.1(b)(4)(vi)(AM4)). Allopolyploid 

plants could contain up to four of the 

proposed loss of function modifications 

described herein or four modifications 

described under § 340.1(b)(2) and (3) or 

a combination thereof, provided each 

modification is introduced into just one 

allele; however, allopolyploid plants 

would not be exempt if they contain a 

modification that is allowable only in 

diploid and autopolyploid plants. 

Fifth, we propose that plants that 

have previously completed voluntary 

reviews confirming the plants’ exempt 

status as described in § 340.1(e), which 

provides the process by which 

developers can request such a 

confirmation of exempt status, and that 

have been produced, grown, and 

observed consistent with conventional 

breeding methods appropriate for the 

plant species, could be successively 

modified in accordance with any 

exemption under § 340.1(b) of the 

regulations (proposed exemption 

340.1(b)(4)(vi)(AM5)). 

We are also making available for 

public review scientific literature that 

we considered prior to initiating this 

notice, which demonstrates that in 

polyploid plants (such as wheat, potato, 

tobacco, and canola), all alleles of a 

single genetic locus can be modified by 

conventional breeding to generate loss 

of function mutations. This notice 

provides scientific literature supporting 

our rationale for why the proposed 

modifications could extend to any 

autopolyploid species and our rationale 

for why some of the proposed 

modifications could extend to any 

allopolyploid species. This notice 

includes examples of conventional 

breeding programs in sterile crops such 

as banana, long cycle crops such as 

forest trees, crops with complex 

genomes such as strawberry and 

sugarcane, and highly heterozygous 

crops such as potato and apple. This 

notice discusses literature describing 

the approach of pyramiding genes (i.e., 

the simultaneous selection for and/or 

introduction of multiple genes during 

plant breeding) to create multiplex edits 

and provide examples in soybean, 

coffee, tobacco, tomato, potato, corn, 

and rice where four to seven traits are 

pyramided by conventional breeding 

methods. We also provide references to 

literature describing how homozygous 

autopolyploids can be created through 

conventional breeding methods in 

autopolyploid plants that are not 

applicable to allopolyploids plants. We 

also explain how the categories for loss 

of function modifications, and 

successive modifications for plants that 

have completed the voluntary 

confirmation process and that have been 

produced, grown, and observed are 

consistent with conventional breeding 

methods for the appropriate plant 

species. This action would reduce the 

regulatory burden for developers of 

certain plants modified using genetic 

engineering that are not expected to 

pose plant pest risks greater than the 

plant pest risks posed by plants 

modified by conventional breeding 

methods and, thus, should not be 

subjected to regulation under part 340. 

First Proposed Exemption 

Commenters to the previous July 2021 

notice suggested that we ‘‘establish a 

single exemption category for indel 

modifications resulting from 

modifications to the alleles of a single 

gene on homologous chromosomes.’’ We 

recognize that as new tools emerge, 

there may be DNA modifications that 

are not expressly covered by the three 

exemptions described in the July 2021 

notice. For example, base editing and 

prime editing involve nicking a single 

strand rather than making double strand 

breaks. In the case of base editing, a 

deaminase further modifies the DNA 

before the changes are resolved by 

natural repair. In prime editing, primeediting guide RNA contains an internal 

template and further uses reverse 

transcriptase to incorporate the edit. 

When base editing is used to introduce 

a loss of function (‘‘LOF’’) mutation to 


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Federal Register / Vol. 88, No. 219 / Wednesday, November 15, 2023 / Notices 78287 

2Li, S., X. Zhong, X. Zhang, M. M. Rahman, J. 

Lan, H. Tang, P. Qi, J. Ma, J. Wang, G. Chen, X. Lan, 

M. Deng, Z. Li, W. Harwood, Z. Lu, Y. Wei, Y. 

Zheng and Q. Jiang (2020). ‘‘Production of waxy 

tetraploid wheat (Triticum turgidum durum L.) by 

EMS mutagenesis.’’ Genetic Resources and Crop 

Evolution 67(2): 433–443). 

3 Krasileva, K. V., H. A. Vasquez-Gross, T. 

Howell, P. Bailey, F. Paraiso, L. Clissold, J. 

Simmonds, R. H. Ramirez-Gonzalez, X. Wang, P. 

Borrill, C. Fosker, S. Ayling, A. L. Phillips, C. Uauy 

and J. Dubcovsky (2017). ‘‘Uncovering hidden 

variation in polyploid wheat.’’ Proc Natl Acad Sci 

U S A 114(6): E913-e921). 

4Pearce, S., L.M. Shaw, H. Lin, J.D. Cotter, C. Li 

and J. Dubcovsky (2017). ‘‘Night-Break Experiments 

Shed Light on the Photoperiod1-Mediated 

Flowering’’ Plant Physiology 174(2): 1139–1150; 

Karunarathna, N.L., H. Wang, H.–J. Harloff, L. Jiang 

and C. Jung (2020). ‘‘Elevating seed oil content in 

a polyploid crop by induced mutations in SEED 

FATTY ACID REDUCER genes.’’ Plant 

Biotechnology Journal 18(11): 2251–2266; Kippes, 

N., Chen, A., Zhang, X., Lukaszewski, A.J., and 

Dubcovsky, J. (2016). Development and 

characterization of a spring hexaploid wheat line 

with no functional VRN2 genes. Theor Appl Genet 

129, 1417–1428. Lewis, R.S., Lopez, H.O., Bowen, 

SW, Andres, K.R., Steede, W.T., and Dewey, R.E. 

(2015). Transgenic and Mutation-Based 

Suppression of a Berberine Bridge Enzyme-Like 

(BBL) Gene Family Reduces Alkaloid Content in 

Field-Grown Tobacco. PLOS ONE 10, e0117273. 

Mccord, P., Zhang, L., and Brown, C. (2012). The 

Incidence and Effect on Total Tuber Carotenoids of 

a Recessive Zeaxanthin Epoxidase Allele (Zep1) in 

Yellow-fleshed Potatoes. American Journal of 

Potato Research 89, 262–268. 

5Udagawa, H., Ichida, H., Takeuchi, T., Abe, T., 

and Takakura, Y. (2021). Highly Efficient and 

Comprehensive Identification of Ethyl 

Methanesulfonate-Induced Mutations in Nicotiana 

tabacum L. by Whole-Genome and Whole-Exome 

Sequencing. Front Plant Sci 12, 671598. 

6 Jenny, C., Tomekpe, K., Bakry, F., and Escalent, 

J.V. (2002). ‘‘Conventional Breeding of Bananas’’, 

in: Mycosphaerella leaf spot diseases of bananas: 

present status and outlook. (eds.) L. Jacome, P. 

Lepoiver, D. Marin, R. Ortiz, R. Romero & J.V. 

Escalent. (San Jose Costa Rica: INIBAP). 

7Harfouche, A., Meilan, R., Kirst, M., Morgante, 

M., Boerjan, W., Sabatti, M., and Scarascia 

Mugnozza, G. (2012). Accelerating the 

domestication of forest trees in a changing world. 

Trends in Plant Science 17, 64–72. 

8Hummer, K.E., and Hancock, J. (2009). 

Strawberry genomics: botanical history, cultivation, 

traditional breeding, and new technologies. 

Genetics and genomics of Rosaceae, 413–435. 

9 Kumar, U., Priyanka, and Kumar, S. (2016). 

‘‘Genetic Improvement of Sugarcane Through 

Conventional and Molecular Approaches’’, 325– 

342. 

10Bonierbale, M.W., Amoros, W.R., Salas, E., and 

De Jong, W. (2020). ‘‘Potato Breeding’’, in The 

Potato Crop: Its Agricultural, Nutritional and Social 

Contribution to Humankind, eds. H. Campos & O. 

Ortiz. (Cham: Springer International Publishing), 

163–217; Bethke, P.C., Halterman, D.A., Francis, 

D.M., Jiang, J., Douches, D.S., Charkowski, A.O., 

and Parsons, J. (2022). Diploid Potatoes as a Catalyst 

for Change in the Potato Industry. American Journal 

of Potato Research 99, 337–357. 

11Sedov, E.N. (2014). Apple breeding programs 

and methods, their development and improvement. 

Russian Journal of Genetics: Applied Research 4, 

43–51. 

12 King, R., Stansfield, W., & Mulligan, P. (2007). 

loss of function mutation. In A Dictionary of 

Genetics. Oxford University Press. Retrieved 6 Jun. 

2023, from https://www.oxfordreference.com/view/ 

10.1093/acref/9780195307610.001.0001/acref9780195307610-e-3651. 

13Lackie, J. gain-of-function mutation. In Nation, 

B. (Ed.), A Dictionary of Biomedicine.: Oxford 

University Press. Retrieved 6 Jun. 2023, from 

https://www.oxfordreference.com/view/10.1093/ 

acref/9780191829116.001.0001/acref9780191829116-e-3735. 

a single genetic locus, multiple changes 

may occur within the single genetic 

locus. The fact that multiple changes 

occur is irrelevant if one or more of the 

changes leads to a loss of function. Both 

base-editing and prime-editing can be 

used to make modifications that 

conform to the spirit of the 

modifications codified in § 340.1(b)(1) 

that are exempt from regulation, but 

they are not expressly described in the 

modifications. Creating a category for 

any DNA modification that leads to LOF 

of a single gene on homologous 

chromosomes would cover scenarios we 

did not specifically describe that are 

nevertheless consistent with our intent 

for modifications that would qualify for 

exemption in § 340.1(b)(1) because they 

are achievable through conventional 

breeding methods. 

Accordingly, in this notice, we 

propose that diploid or autopolyploid 

plants with any combination of loss of 

function modifications in one to all 

alleles of a single genetic locus, or 

allopolyploid plants with any 

combination of loss of function 

modifications in one or both alleles of 

a single genetic locus on up to four 

homoeologous chromosomes, without 

the insertion of exogenous DNA, would 

be exempt from regulation. In the 

comment period for the previous notice, 

several papers were brought to our 

attention describing the successful 

breeding of tetraploid (AABB genomes) 

and hexaploid (AABBDD genomes) 

wheat lines with loss of function alleles 

for all four or six homoeologous alleles, 

respectively. In one case,2 homologous 

null mutations in starch synthase from 

both the A and B genomes were isolated 

from the M2 generation of ethyl 

methansesulfonate (EMS) mutagenized 

tetraploid wheat lines. Both null 

mutants were crossed to generate the 

null lacking all 4-functioning starch 

synthase alleles. In a second case,3 the 

exomes of 2735 EMS mutagenized lines 

were sequenced, and more than 10 

million mutations were identified 

covering about 90 percent of the three 

wheat genomes. The authors explained 

how loss of function homozygous 

mutants could be successfully isolated 

from both genomes in the third 

generation of tetraploid wheat and 

homozygous mutants across all three 

genomes in the fourth generation of a 

hexaploid wheat. The literature contains 

several additional cases of double and 

triple null mutants successfully created 

by conventional breeding (naturally 

occurring transposon induced mutation/ 

ems mutagenesis, tilling, and marker 

assisted breeding) in the polyploids, 

wheat, tobacco, potato and canola.4 The 

combination of mutagenesis and exomesequencing described by Krasileva et al. 

2017, has also been applied in tetraploid 

tobacco.5 Based on these examples, it 

appears this methodology can be used to 

create the modifications captured by the 

exemption in any species that can be 

bred conventionally. Breeding programs 

exist for crops that are challenging to 

breed, such as the largely sterile triploid 

bananas,6 forest trees with long 

generation times,7 and crops with 

complex genomes such as strawberry 8

and sugarcane,9 or highly heterozygous 

genomes such as potato 10 or apple.11

We propose that any diploid or 

autopolyploid plant that contains any 

combination of loss of function 

modifications in one to all alleles of a 

single genetic locus without the 

insertion of exogenous DNA, or any 

allopolyploid plant with any 

combination of loss of function 

modifications in one or both alleles of 

a single genetic locus on up to four 

homoeologous chromosomes, would 

qualify for exemption because such 

modifications are achievable through 

conventional breeding methods. The 

limitation to four homoeologous 

chromosomes in polyploid plants is 

explained further below. 

Modifications resulting from 

insertions of exogenous DNA do not 

currently qualify for exemption and, 

likewise, LOF mutations created 

through insertion of exogenous DNA 

such as T–DNA (the transferred DNA of 

the (Ti) plasmid of Agrobacterium used 

in the transformation of plant cells) or 

transposons (DNA sequences that can 

move and integrate to different locations 

within the genome), would not qualify 

for exemption as proposed. LOF 

mutations could qualify for more than 

one exemption. For example, LOF 

mutations may still qualify for 

exemption § 340.1(b)(3), if they are 

already known to occur in the gene pool 

of the plant species. 

By loss of function, we mean a 

mutation in which the altered gene 

product prevents the normal gene 

product from being produced or renders 

it inactive.12 By gain of function (GOF) 

mutation, we mean a mutation that 

alters the properties of the protein 

product so that it has novel properties 

or has greater activity because a 

regulatory site has been lost 13 and is 


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14Rodrı ́guez-Leal, D., Lemmon, Z.H., Man, J., 

Bartlett, M.E., and Lippman, Z.B. (2017). 

Engineering Quantitative Trait Variation for Crop 

Improvement by Genome Editing. Cell 171, 470– 

480.e478. 

Ro ̈nspies, M., Schindele, P., and Puchta, H. 

(2021). CRISPR/Cas-mediated chromosome 

engineering: opening up a new avenue for plant 

breeding. J Exp Bot 72, 177–183. Xu, C., Liberatore, 

K.L., Macalister, C.A., Huang, Z., Chu, Y.-H., Jiang, 

K., Brooks, C., Ogawa-Ohnishi, M., Xiong, G., Pauly, 

M., Van Eck, J., Matsubayashi, Y., Van Der Knaap, 

E., and Lippman, Z.B. (2015). A cascade of 

arabinosyltransferases controls shoot meristem size 

in tomato. Nature Genetics 47, 784–792. 

15Patzoldt, W.L., Hager, A.G., McCormick, J.S., 

and Tranel, P.J. (2006). A codon deletion confers 

resistance to herbicides inhibiting 

protoporphyrinogen oxidase. Proceedings of the 

National Academy of Sciences 103, 12329–12334. 

16 Xu, X., Pan, S., Cheng, S., Zhang, B., Mu, D., 

Ni, P., Zhang, G., Yang, S., Li, R., Wang, J., Orjeda, 

G., Guzman, F., Torres, M., Lozano, R., Ponce, O., 

Martinez, D., De La Cruz, G., Chakrabarti, S.K., 

Patil, V.U., Skryabin, K.G., Kuznetsov, B.B., Ravin, 

N.V., Kolganova, T.V., Beletsky, A.V., Mardanov, 

A.V., Di Genova, A., Bolser, D.M., Martin, D.M.A., 

Li, G., Yang, Y., Kuang, H., Hu, Q., Xiong, X., 

Bishop, G.J., Sagredo, B., Mejı ́a, N., Zagorski, W., 

Gromadka, R., Gawor, J., Szczesny, P., Huang, S., 

Zhang, Z., Liang, C., He, J., Li, Y., He, Y., Xu, J., 

Zhang, Y., Xie, B., Du, Y., Qu, D., Bonierbale, M., 

Ghislain, M., Del Rosario Herrera, M., Giuliano, G., 

Pietrella, M., Perrotta, G., Facella, P., O’brien, K., 

Feingold, SE, Barreiro, L.E., Massa, G.A., Diambra, 

L., Whitty, B.R., Vaillancourt, B., Lin, H., Massa, 

A.N., Geoffroy, M., Lundback, S., Dellapenna, D., 

Robin Buell, C., Sharma, S.K., Marshall, D.F., 

Waugh, R., Bryan, G.J., Destefanis, M., Nagy, I., 

Milbourne, D., Thomson, S.J., Fiers, M., Jacobs, 

J.M.E., Nielsen, K.L., S<nderk#r, M., Iovene, M., 

Torres, G.A., Jiang, J., Veilleux, R.E., Bachem, 

C.W.B., De Boer, J., Borm, T., Kloosterman, B., Van 

Eck, H., Datema, E., Te Lintel Hekkert, B., Goverse, 

A., Van Ham, R.C.H.J., Visser, R.G.F., The Potato 

Genome Sequencing, C., The Potato Genome, C., 

Shenzhen, B.G.I., et al. (2011). Genome sequence 

and analysis of the tuber crop potato. Nature 475, 

189–195. 

17 Guo, H., Zhou, M., Zhang, G., He, L., Yan, C., 

Wan, M., Hu, J., He, W., Zeng, D., Zhu, B., and 

Zeng, Z. (2023). Development of homozygous 

tetraploid potato and whole genome doublinginduced the enrichment of H3K27ac and potentially 

enhanced resistance to cold-induced sweetening in 

tubers. Horticulture Research 10. 

18Bourke, P.M., Voorrips, R.E., Visser, R.G., and 

Maliepaard, C. (2015). The Double-Reduction 

Landscape in Tetraploid Potato as Revealed by a 

High-Density Linkage Map. Genetics 201, 853–863. 

usually dominant, semidominant, or 

codominant. In some cases, a mutation 

can render a protein to be nonfunctioning but lead to a new 

phenotype. For example, mutations that 

knockout the repressor protein CLV3 

(CLAVATA 3) result in larger sized 

fruit.14 These mutations are a LOF 

modification that would qualify for 

exemption. In cases where a deletion or 

frameshift mutation leads to a new 

molecular function or increased 

expression of the altered gene product, 

the modification would not qualify for 

the new exemption. For example, a 

codon deletion in protoporphyrinogen 

oxidase conferred resistance to PPO 

type herbicide inhibitors.15 This 

deletion results in a protein with a new 

molecular function, is dominant, and 

does not lack the molecular function of 

the wild type (it is still able to convert 

protoporphyrinogen IX to 

protoporphyrin IX). This particular 

example is a naturally occurring 

mutation described in Amaranthus 

tuberculatus. If genome editing were 

used to confer herbicide tolerance to a 

crop plant by deleting the 

corresponding codon by DNA break and 

repair, the modified plant would likely 

qualify for the exemption found at 

§ 340.1(b)(1). Thus, although GOF 

mutations will not qualify for the 

proposed exemption 

340.1(b)(4)(vi)(AM4) as listed in the 

above-mentioned exemptionsconfirmations website, there are some 

GOF mutations that could meet the 

criteria for exemptions at § 340.1(b)(1) 

through (3). For example, promoter 

deletions can result in either LOF or 

GOF. If a promoter deletion eliminates 

or greatly decreases expression of the 

downstream gene, that would be a LOF 

modification and would qualify for this 

exemption or the § 340.1(b)(1) 

exemption. If the promoter deletion 

results in an increase of expression of 

the downstream gene, that would be a 

GOF modification and it would not 

qualify for this exemption but would 

qualify for the § 340.1(b)(1) exemption. 

In any plant, GOF modifications from 

faulty DNA repair qualify under 

exemptions § 340.1(b)(1) for a DNA 

break on a single chromosome or at the 

same location on two homologous 

chromosomes. In addition, GOF 

modifications from faulty repair could 

qualify for exemption under 

340.1(b)(4)(vi) AM2 for one or two DNA 

breaks to the same location in the 

absence of an external template on all 

homologous chromosomes in 

autopolyploids (see below). In short, our 

proposal does not extend to all 

modifications that involve the insertion 

or deletion of bases (‘‘indel’’) because 

GOF modifications are statistically less 

common than LOF mutations and the 

same GOF mutation would not be 

expected to occur across multiple alleles 

in allopolyploids by conventional 

breeding. 

We welcome comments from the 

public on the scope of this proposed 

exemption. 

Second Proposed Exemption 

In the published notice of July 2021, 

we proposed that plants with a 

modification that results in a single 

contiguous deletion of any size using an 

external repair template or using two 

targeted DNA breaks on a single 

chromosome would be exempt from 

regulation because they are achievable 

through conventional breeding methods. 

This type of modification allows 

deletions to contain more than one 

genetic locus. Based on the comments 

and information we received in 

response to the July 2021 notice, we are 

clarifying how the contiguous deletion 

of any size would apply to polyploids. 

Based on examples and methods 

described above, we propose that any 

diploid or autopolyploid plant with a 

genetic modification that is a single 

contiguous deletion of any size, 

resulting from cellular repair of one or 

two targeted DNA breaks on a single 

chromosome or at the corresponding 

location(s) on two or more homologous 

chromosomes, without insertion of 

DNA, or with the insertion of DNA in 

the absence of a repair template, would 

be exempt because these modifications 

are achievable through conventional 

breeding methods. This proposed 

modification allows for multiple 

modifications in autopolyploids, but not 

allopolyploids. This is because the 

literature indicates this type of 

modification can be achieved through 

conventional breeding in 

autopolyploids to produce the same 

deletion throughout the genome. For 

example, though potato is highly 

heterozygous, a highly homozygous line 

was established from a doubled 

monoploid derived by another culture 

of a heterozygous diploid 16 and this 

line in turn was used to create 

homozygous tetraploid lines by another 

round of whole genome doubling.17 In 

this way, conventional breeding was 

used to produce homozygous 

autopolyploids from allele variants in 

the haploid genome. Additionally, 

through random assortment of 

homologous chromosomes in 

autopolyploids, it is possible to achieve 

homozygosity of a modification across 

all chromosomes, while maintaining a 

high degree of heterozygosity across a 

genome, particularly when double 

reduction progeny are selected.18 Based 

on our review of the literature, we 

believe that this type of modification is 

not possible through conventional 

breeding methods for allopolyploids, 

which is why the proposed modification 

applies only to autopolyploids. 

Third Proposed Exemption 

We propose to extend the 

modifications described in § 340.1(b)(2) 

and (3) to all alleles of a genetic locus 

on the homologous chromosomes of 

autopolyploids. This would allow the 

following modifications to all alleles of 

a single gene on all homologous 

chromosomes in autopolyploids: 


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Federal Register / Vol. 88, No. 219 / Wednesday, November 15, 2023 / Notices 78289 

19Majhi, P. (2020). ‘‘GENE PYRAMIDING.’’), 3– 

16; Chapagain, S., Pruthi, R., and Subudhi, P.K. 

(2023). Pyramiding QTLs using multiparental 

advanced generation introgression lines enhances 

salinity tolerance in rice. Acta Physiologiae 

Plantarum 45, 59.; Dormatey, R., Sun, C., Ali, K., 

Coulter, J.A., Bi, Z., and Bai, J. (2020). Gene 

Pyramiding for Sustainable Crop Improvement 

against Biotic and Abiotic Stresses. Agronomy 10, 

1255.; Malav, A.K., Indu, and Chandrawat, K.S. 

(2016). Gene Pyramiding: An Overview. 

International Journal of Current Research in 

Biosciences and Plant Biology 3, 22–28; 

Muthurajan, R., and Balasubramanian, P. (2009). 

‘‘Pyramiding Genes for Enhancing Tolerance to 

Abiotic and Biotic Stresses,’’ in Molecular 

Techniques in Crop Improvement: 2nd Edition, eds. 

S.M. Jain & D.S. Brar. (Dordrecht: Springer 

Netherlands), 163–184; Servin, B., Martin, O., 

Mezard, M., and Hospital, F. (2004). Toward a 

Theory of Marker-Assisted Gene Pyramiding. 

Genetics 168, 513–523. 

20Lewis, R.S., Dewey, R.E., and Tamburrino, J.S. 

(2023). US Patent Application for GENETIC 

APPROACH FOR ACHIEVING ULTRA LOW 

NICOTINE CONTENT IN TOBACCO Patent 

Application (Application #20230029171 issued 

January 26, 2023)—Justia Patents Search. 

21Singh, Y., Shrivastava, M., and Banerjee, J. 

(2021). ‘‘Chapter –3 Gene Pyramiding in 

Soybean.’’). 

22Rogozina, E.V., Beketova, M.P., Muratova, O.A., 

Kuznetsova, M.A., and Khavkin, E.E. (2021). 

Stacking Resistance Genes in Multiparental 

Interspecific Potato Hybrids to Anticipate Late 

Blight Outbreaks. Agronomy 11, 115. 

23Saavedra, L.M., Caixeta, E.T., Barka, G.D., 

Bore ́m, A., Zambolim, L., Nascimento, M., Cruz, 

C.D., Oliveira, A.C.B.D., and Pereira, A.A. (2023). 

Marker-Assisted Recurrent Selection for Pyramiding 

Leaf Rust and Coffee Berry Disease Resistance 

Alleles in Coffea arabica L. Genes 14, 189. 

24Zambrano, J.L., Jones, M.W., Brenner, E., 

Francis, D.M., Tomas, A., and Redinbaugh, M.G. 

(2014). Genetic analysis of resistance to six virus 

diseases in a multiple virus-resistant maize inbred 

line. Theoretical and Applied Genetics 127, 867– 

880. 

25Hanson, P., Lu, S.-F., Wang, J.-F., Chen, W., 

Kenyon, L., Tan, C.-W., Tee, K.L., Wang, Y.-Y., Hsu, 

Y.-C., Schafleitner, R., Ledesma, D., and Yang, R.- 

Y. (2016). Conventional and molecular markerassisted selection and pyramiding of genes for 

multiple disease resistance in tomato. Scientia 

Horticulturae 201, 346–354. 

26Ramalingam, J., Raveendra, C., Savitha, P., 

Vidya, V., Chaithra, T.L., Velprabakaran, S., 

Saraswathi, R., Ramanathan, A., Arumugam Pillai, 

M.P., Arumugachamy, S., and Vanniarajan, C. 

(2020). Gene Pyramiding for Achieving Enhanced 

Resistance to Bacterial Blight, Blast, and Sheath 

Blight Diseases in Rice. Frontiers in Plant Science 

11. 

• a targeted single base pair 

substitution, or 

• introduction of a gene known to 

occur in the plant’s gene pool or make 

changes in a targeted sequence to 

correspond to a known allele of such a 

gene or to a known structural variation 

present in the gene pool. 

For the reasons discussed above, the 

modifications described in § 340.1(b)(2) 

and (3) would only extend to all loci on 

the homologous chromosomes in 

autopolyploids plants and not to all 

homoeologous loci in allopolyploids 

plants. 

Fourth Proposed Exemption 

We have received several comments 

that multiplexing genome edits that 

individually qualify for exemption 

should qualify for exemption when 

achieved simultaneously or sequentially 

because conventional breeding allows 

the combination of multiple desired 

traits. In the 2020 preamble, APHIS 

noted, ‘‘[i]nitially, the exemptions will 

apply only to plants containing a single 

targeted modification in one of the 

categories listed. APHIS anticipates 

scientific information and/or experience 

may, over time, allow APHIS to list 

additional modifications that plants can 

contain and still be exempted from the 

regulations so that the regulatory system 

stays up to date and keeps pace with 

advances in scientific knowledge, 

evidence, and experience. This may 

include multiple simultaneous genomic 

changes.’’ 85 FR 29790, 29794. We have 

verified that there is literature on this 

topic, including literature describing 

gene pyramiding.19 One commenter 

provided us with a patent for a tobacco 

plant made homozygous in five separate 

loci through conventional breeding.20

Additionally, we observed cases where 

four to seven traits were combined in 

soybean,21 potato,22 coffee,23 corn,24

tomato,25 and rice 26 suggesting that 

pyramiding genes is becoming a 

standard practice in conventional 

breeding and four traits are 

conservatively within the norm. The 

examples provided include four 

different diploid species, an 

autopolyploid species (potato), an 

allopolyploid species (coffee), which is 

also a tree, suggesting that gene 

pyramiding is widely applicable to crop 

plants. When discussing the first 

proposed exemption, we noted new 

techniques that created DNA 

modifications using chemical 

mutagenesis while characterizing the 

genome using molecular analysis both 

of which are applicable to any species. 

We also provide examples of crops that 

have active breeding programs even 

though they are challenging to breed. 

Based on feedback during the comment 

period of the 2021 notice and our own 

review of the literature, it is our current 

view that a single targeted modification 

is more conservative than what can be 

achieved by conventional breeding in 

all species. 

Accordingly, we propose that plants 

with up to four modifications of a 

certain type that individually qualify for 

exemption and that are made 

simultaneously or sequentially would 

be exempt from regulation, provided 

that that each modification is at a 

different genetic locus. This is because 

such modifications are achievable 

through conventional breeding methods. 

For the reasons discussed above, 

allopolyploid plants could contain up to 

four of the proposed loss of function 

modifications described herein. 

Allopolyploid plants would also qualify 

for exemption with the following 

changes to a single pair of homologous 

chromosomes: 

• § 340.1(b)(2)—a genetic 

modification is a targeted single base 

pair substitution; and 

• § 340.1(b)(3)—the introduction of a 

gene known to occur in the plant’s gene 

pool or makes changes in a targeted 

sequence to correspond to a known 

allele of such a gene or to a known 

structural variation present in the gene 

pool. 

We propose that up to four such 

modifications would qualify for 

exemption in allopolyploids provided 

that each change is heterozygous. We 

note that the introduction of multiple 

dominant resistance traits has been 

accomplished by conventional breeding 

in both allopolyploid coffee (see 

footnote 23) and autopolyploid potato 

(see footnote 22). However, we are not 

aware of multiple homologous traits 

pyramided in allopolyploids. 

Modifications would be counted 

based on loci modified. For an 

autopolyploid, such as potato, which 

has four alleles of the same genetic 

locus, a change to make four 

homozygous copies of an allele would 

count as one multiplex modification. 

However, in an allopolyploid, such as 

canola, which has two pairs of 

homoeologous chromosomes, LOF edits 

to all alleles (two loci and four alleles) 

would count as two multiplex 

modifications. We welcome comments 

from the public on the number of 

individual modifications that are 

achievable simultaneously or 

sequentially in plants based on 

conventional breeding methods, and 

comments on the reasons for or against 

allowing for simultaneous or sequential 

modifications in all plants. We 

emphasize that multiplexed or 

sequential modifications must be made 

to distinct loci; multiple modifications 

to a single gene would not qualify for 

exemption except in the cases where the 

gene is known to occur in the plant’s 

gene pool. 

Fifth Proposed Exemption 

We have also received questions on 

whether a modified plant that meets the 

criteria for exemption from the 

regulations at part 340, may undergo 

successive or further modification. In 

the preamble that accompanied the final 

rule, we noted that we would address 


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78290 Federal Register / Vol. 88, No. 219 / Wednesday, November 15, 2023 / Notices 

the possibility for sequential 

modification (i.e., subsequent or further 

modification to an exempt plant) in a 

future notice using the process 

described in § 340.1(b)(4). In 

conventional breeding, it is standard 

practice to introduce new traits through 

successive crosses. Conventional 

breeding affords the opportunity to 

evaluate and select the progeny of a 

cross that will be advanced in the 

breeding program. Along these lines, we 

propose that plants that have previously 

completed the voluntary confirmation 

process (also called the ‘‘CR’’ process) 

found at § 340.1(e) and that have been 

produced, grown, and observed 

consistent with conventional breeding 

methods for the appropriate plant 

species, may be successively modified 

in accordance with the exemptions 

because allowing for such successive 

modification is consistent with plant 

development in conventional breeding 

programs. Plants that are merely 

hypothetical in nature would not be 

eligible for subsequent hypothetical 

modifications because they have not yet 

been produced, grown, and observed 

consistent with conventional breeding 

methods for the appropriate plant 

species. 

The following table summarizes the 

proposed exemptions and their 

applicability to polyploids: 

TABLE 1—SUMMARY OF PROPOSED EXEMPTION CHANGES AND APPLICABILITY TO POLYPLOIDS. 

Notes Designation Exemption Diploids Autoploids Alloploids GOF 

§ 340.1(b)(1) ............... The genetic modification is a 

change resulting from cellular repair of a targeted 

DNA break in the absence 

of an externally provided repair template.

1 pair of homologous chromosomes Yes. 

§ 340.1(b)(2) ............... The genetic modification is a 

targeted single base pair 

substitution.

1 pair of homologous chromosomes Yes. 

§ 340.1(b)(3) ............... The genetic modification introduces a gene known to 

occur in the plant’s gene 

pool or makes changes in a 

targeted sequence to correspond to a known allele of 

such a gene or to a known 

structural variation present 

in the gene pool.

1 pair of homologous chromosomes Yes. 

Overarching 

LOF exemption.

340.1(b)(4)(vi)(AM1) 

on the exemptionsconfirmations 

website.

Any diploid or autopolyploid 

plant that contains any combination of loss of function 

modifications in one to all 

alleles of a single genetic 

locus, or any allopolyploid 

plant with any combination 

or loss of function modification in one or both alleles of 

a single genetic locus on up 

to four pairs of homoeologous chromo-somes, without the insertion of exogenous DNA.

All alleles of a 

single genetic locus 

on homologous chromosomes.

All alleles of a 

single genetic locus 

on homologous chromosomes.

Any combination of loss 

of function 

modifications 

in one or 

both alleles 

of a single 

genetic locus 

on up to four 

pairs of 

homoeologous chromosomes.

No. 

Deletion of any 

size; one or 

two cuts; external repair 

template for 

deletion 

diploids and 

autopolyploids.

340.1(b)(4)(vi)(AM2) 

as listed in the exemptions-confirmations website.

A single contiguous deletion of 

any size, resulting from cellular repair of one or two 

targeted DNA breaks on a 

single chromosome or at 

the same location(s) on two 

or more homologous chromosomes, without insertion 

of DNA, or with insertion of 

DNA in the absence of a repair template.

Applicable ....... Applicable ....... Does not apply Yes. 


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Federal Register / Vol. 88, No. 219 / Wednesday, November 15, 2023 / Notices 78291 

TABLE 1—SUMMARY OF PROPOSED EXEMPTION CHANGES AND APPLICABILITY TO POLYPLOIDS.—Continued 

Notes Designation Exemption Diploids Autoploids Alloploids GOF 

Expand 

§ 340.1(b)(2) 

and (3) to 

autopolyploids.

340.1(b)(4)(vi)(AM3) 

as listed in the exemptions-confirmations website.

The genetic modification is a 

targeted single base pair 

substitution or the genetic 

modification introduces a 

gene known to occur in the 

plant’s gene pool or makes 

changes in a targeted sequence to correspond to a 

known allele of such a gene 

or to a known structural variation present in the gene 

pool.

Not relevant .... Applicable ....... Does not apply Yes. 

Allow up to 4 

multiplex or 

sequential 

modifications.

340.1(b)(4)(vi)(AM4) 

as listed in the exemptions-confirmations website.

Any combination of up to 4 

multiplexed or sequentially 

made modifications provided that each edit is at a 

different genetic locus and 

would individually qualify for 

an existing exemption.

Applicable ....... Applicable ....... Applicable ....... For allopolyploids, multiple heterozygous 

modifications 

are Applicable. 

Process for further modification of 

exempt 

plants.

340.1(b)(4)(vi)(AM5) in 

the exemptions-confirmations website.

Plants that have previously 

completed voluntary confirmation process and have 

been produced, grown, and 

observed consistent with 

conventional breeding methods for the appropriate plant 

species, could be further 

modified in accordance with 

the exemptions.

Applicable ....... Applicable ....... Applicable ....... For allopolyploids, multiple heterozygous 

modifications 

are applicable. 

After reviewing any comments we 

receive, we will announce in a future 

notice our decision regarding any 

modifications that plants can contain 

and qualify for exemption. 

Authority: 7 U.S.C. 7701–7772 and 

7781–7786; 31 U.S.C. 9701; 7 CFR 2.22, 

2.80, and 371.3. 

Done in Washington, DC, this 7th day of 

November 2023. 

Michael Watson, 

Acting Administrator, Animal and Plant 

Health Inspection Service. 

[FR Doc. 2023–25122 Filed 11–14–23; 8:45 am] 

BILLING CODE 3410–34–P 

DEPARTMENT OF AGRICULTURE 

Forest Service 

Black Hills National Forest Advisory 

Board 

AGENCY: Forest Service, Agriculture 

USDA. 

ACTION: Notice of meeting. 

SUMMARY: The Black Hills National 

Forest Advisory Board will hold a 

public meeting according to the details 

shown below. The Board is authorized 

under the Forest and Rangeland 

Renewable Resources Planning Act of 

1974, the National Forest Management 

Act of 1976, the Federal Lands 

Recreation Enhancement Act, and 

operates in compliance with the Federal 

Advisory Committee Act (FACA). The 

purpose of the Board is to provide 

advice and recommendations on a broad 

range of forest issues such as forest plan 

revisions or amendments, forest health 

including fire, insect and disease, travel 

management, forest monitoring and 

evaluation, recreation fees, and sitespecific projects having forest-wide 

implications. 

DATES: An in-person meeting will be 

held on December 6, 2023, 1 p.m.–4:30 

p.m. mountain standard time (MST). 

Written and Oral Comments: Anyone 

wishing to provide in-person oral 

comments must pre-register by 11:59 

p.m. MST on December 1, 2023. Written 

public comments will be accepted up to 

11:59 p.m. MST on December 1, 2023. 

Comments submitted after this date will 

be provided to the Forest Service, but 

the Committee may not have adequate 

time to consider those comments prior 

to the meeting. 

All board meetings are subject to 

cancellation. For status of the meeting 

prior to attendance, please contact the 

person listed under FOR FURTHER

INFORMATION CONTACT. 

ADDRESSES: This meeting will be held in 

person at the U.S. Forest Service Mystic 

Ranger District Office, 8221 Mount 

Rushmore Road, Rapid City, South 

Dakota 57702. Board information and 

meeting details can be found at the 

following website: https:// 

www.fs.usda.gov/main/blackhills/ 

workingtogether/advisorycommittees or 

by contacting the person listed under 

FOR FURTHER INFORMATION CONTACT. 

Written Comments: Written comments 

must be sent by email to 

scott.j.jacobson@usda.gov or via mail 

(i.e., postmarked) to Scott Jacobson, 

8221 Mount Rushmore Road, Rapid 

City, South Dakota 57702. The Forest 

Service strongly prefers comments be 

submitted electronically. 

Oral Comments: Persons or 

organizations wishing to make oral 

comments must pre-register by 11:59 

p.m. MST, December 1, 2023, and 

speakers can only register for one 

speaking slot. Oral comments must be 

sent by email to scott.j.jacobson@

usda.gov or via mail (i.e., postmarked) 

to Scott Jacobson, 8221 Mount 

Rushmore Road, Rapid City, South 

Dakota 57702. 

FOR FURTHER INFORMATION CONTACT: Ivan 

Green, Designated Federal Officer 

(DFO), by phone at 605–673–9201, or 

email at ivan.green@usda.gov or Scott 

Jacobson, Committee Coordinator, at 

605–440–1409 or email at 

scott.j.jacobson@usda.gov. 

SUPPLEMENTARY INFORMATION: The 

meeting agenda will include: 

1. Off-Highway Vehicle (OHV) 

discussion; 

2. Mitchell Lake discussion; 


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