[Federal Register Volume 88, Number 219 (Wednesday, November 15, 2023)]
[Notices]
[Pages 78285-78291]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 2023-25122]



[[Page 78285]]

-----------------------------------------------------------------------

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; [email protected]; (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.
---------------------------------------------------------------------------

    \1\ To view the final rule and supporting documents, go to 
https://www.regulations.gov/docket/APHIS-2018-0034.
---------------------------------------------------------------------------

    The May 2020 final rule included regulatory exemptions for certain 
categories of modified plants. Specifically, Sec.  340.1(b) exempted 
plants that contain a single modification of one of the following 
types, specified in Sec.  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, Sec.  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

[[Page 78286]]

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 Sec.  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 Sec.  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 Sec.  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 Sec.  
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 
Sec.  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 Sec.  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, prime-editing 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

[[Page 78287]]

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 Sec.  
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 Sec.  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 exome-sequencing 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.
---------------------------------------------------------------------------

    \2\ Li, 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).
    \4\ Pearce, 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.
    \5\ Udagawa, 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).
    \7\ Harfouche, 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.
    \8\ Hummer, 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.
    \10\ Bonierbale, 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.
    \11\ Sedov, E.N. (2014). Apple breeding programs and methods, 
their development and improvement. Russian Journal of Genetics: 
Applied Research 4, 43-51.
---------------------------------------------------------------------------

    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 
Sec.  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

[[Page 78288]]

usually dominant, semidominant, or codominant. In some cases, a 
mutation can render a protein to be non-functioning 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 Sec.  
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 exemptions-confirmations website, there are some GOF 
mutations that could meet the criteria for exemptions at Sec.  
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 Sec.  
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 
Sec.  340.1(b)(1) exemption. In any plant, GOF modifications from 
faulty DNA repair qualify under exemptions Sec.  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.
---------------------------------------------------------------------------

    \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/acref-9780195307610-e-3651.
    \13\ Lackie, 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/acref-9780191829116-e-3735.
    \14\ Rodr[iacute]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.
    R[ouml]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.
    \15\ Patzoldt, 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.
---------------------------------------------------------------------------

    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.
---------------------------------------------------------------------------

    \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., Mej[iacute]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[oslash]nderk[aelig]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 doubling-induced the 
enrichment of H3K27ac and potentially enhanced resistance to cold-
induced sweetening in tubers. Horticulture Research 10.
    \18\ Bourke, 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.
---------------------------------------------------------------------------

Third Proposed Exemption

    We propose to extend the modifications described in Sec.  
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:

[[Page 78289]]

     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 
Sec.  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.
---------------------------------------------------------------------------

    \19\ Majhi, 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.
    \20\ Lewis, 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.
    \21\ Singh, Y., Shrivastava, M., and Banerjee, J. (2021). 
``Chapter -3 Gene Pyramiding in Soybean.'').
    \22\ Rogozina, 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.
    \23\ Saavedra, L.M., Caixeta, E.T., Barka, G.D., Bor[eacute]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.
    \24\ Zambrano, 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.
    \25\ Hanson, 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 
marker-assisted selection and pyramiding of genes for multiple 
disease resistance in tomato. Scientia Horticulturae 201, 346-354.
    \26\ Ramalingam, 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.
---------------------------------------------------------------------------

    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:
     Sec.  340.1(b)(2)--a genetic modification is a targeted 
single base pair substitution; and
     Sec.  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

[[Page 78290]]

the possibility for sequential modification (i.e., subsequent or 
further modification to an exempt plant) in a future notice using the 
process described in Sec.  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 Sec.  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
--------------------------------------------------------------------------------------------------------------------------------------------------------
                                 Sec.   340.1(b)(1)  The genetic                          1 pair of homologous chromosomes             Yes.
                                                      modification is a
                                                      change resulting from
                                                      cellular repair of a
                                                      targeted DNA break in
                                                      the absence of an
                                                      externally provided
                                                      repair template.
                                                                             ---------------------------------------------------------
                                 Sec.   340.1(b)(2)  The genetic                          1 pair of homologous chromosomes             Yes.
                                                      modification is a
                                                      targeted single base
                                                      pair substitution.
                                                                             ---------------------------------------------------------
                                 Sec.   340.1(b)(3)  The genetic                          1 pair of homologous chromosomes             Yes.
                                                      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.
                                                                             ---------------------------------------------------------
Overarching LOF exemption......  340.1(b)(4)(vi)(AM  Any diploid or           All alleles of a   All alleles of a   Any combination    No.
                                  1) on the           autopolyploid plant      single genetic     single genetic     of loss of
                                  exemptions-         that contains any        locus on homo-     locus on homo-     function
                                  confirmations       combination of loss of   logous             logous             modifications in
                                  website.            function modifications   chromosomes.       chromosomes.       one or both
                                                      in one to all alleles                                          alleles of a
                                                      of a single genetic                                            single genetic
                                                      locus, or any                                                  locus on up to
                                                      allopolyploid plant                                            four pairs of
                                                      with any combination                                           homoeologous
                                                      or loss of function                                            chromosomes.
                                                      modification in one or
                                                      both alleles of a
                                                      single genetic locus
                                                      on up to four pairs of
                                                      homoeol-ogous chromo-
                                                      somes, without the
                                                      insertion of exogenous
                                                      DNA.
Deletion of any size; one or     340.1(b)(4)(vi)(AM  A single contiguous      Applicable.......  Applicable.......  Does not apply...  Yes.
 two cuts; external repair        2) as listed in     deletion of any size,
 template for deletion diploids   the exemptions-     resulting from
 and autopoly-ploids.             confirmations       cellular repair of one
                                  website.            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.

[[Page 78291]]

 
Expand Sec.   340.1(b)(2) and    340.1(b)(4)(vi)(AM  The genetic              Not relevant.....  Applicable.......  Does not apply...  Yes.
 (3) to auto-polyploids.          3) as listed in     modification is a
                                  the exemptions-     targeted single base
                                  confirmations       pair substitution or
                                  website.            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.
Allow up to 4 multiplex or       340.1(b)(4)(vi)(AM  Any combination of up    Applicable.......  Applicable.......  Applicable.......  For allopoly-
 sequential modi-fications.       4) as listed in     to 4 multiplexed or                                                               ploids, multiple
                                  the exemptions-     sequentially made                                                                 hetero-zygous
                                  confirmations       modifications provided                                                            modifications
                                  website.            that each edit is at a                                                            are Applicable.
                                                      different genetic
                                                      locus and would
                                                      individually qualify
                                                      for an existing
                                                      exemption.
Process for further              340.1(b)(4)(vi)(AM  Plants that have         Applicable.......  Applicable.......  Applicable.......  For allopoly-
 modification of exempt plants.   5) in the           previously completed                                                              ploids, multiple
                                  exemptions-         voluntary confirmation                                                            hetero-zygous
                                  confirmations       process and have been                                                             modifications
                                  website.            produced, grown, and                                                              are applicable.
                                                      observed consistent
                                                      with conventional
                                                      breeding methods for
                                                      the appropriate plant
                                                      species, could be
                                                      further modified in
                                                      accordance with the
                                                      exemptions.
--------------------------------------------------------------------------------------------------------------------------------------------------------

    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