Notice2023-25122
Movement of Organisms Modified or Produced Through Genetic Engineering; Notice of Proposed Exemptions
Primary source
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Published
November 15, 2023
Issuing agencies
Agriculture DepartmentAnimal and Plant Health Inspection Service
Abstract
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.
Full Text
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[Federal Register Volume 88, Number 219 (Wednesday, November 15, 2023)]
[Notices]
[Pages 78285-78291]
From the Federal Register Online via the Government Publishing Office [<a href="http://www.gpo.gov">www.gpo.gov</a>]
[FR Doc No: 2023-25122]
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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.
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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:
<bullet> Federal eRulemaking Portal: Go to <a href="http://www.regulations.gov">http://www.regulations.gov</a>. Enter APHIS-2023-0022 in the Search field. Select
the Documents tab, then select the Comment button in the list of
documents.
<bullet> 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 <a href="http://regulations.gov">regulations.gov</a> 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; <a href="/cdn-cgi/l/email-protection#256b404c490b600b6d4a434348444b65505641440b424a53"><span class="__cf_email__" data-cfemail="f8b69d9194d6bdd6b0979e9e959996b88d8b9c99d69f978e">[email protected]</span></a>; (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.
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\1\ To view the final rule and supporting documents, go to
<a href="https://www.regulations.gov/docket/APHIS-2018-0034">https://www.regulations.gov/docket/APHIS-2018-0034</a>.
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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):
<bullet> 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
<bullet> The genetic modification is a targeted single base pair
substitution; or
<bullet> 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
<a href="https://www.aphis.usda.gov/aphis/ourfocus/biotechnology">https://www.aphis.usda.gov/aphis/ourfocus/biotechnology</a>.
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:
<bullet> 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;
<bullet> Contiguous deletion of any size resulting from cellular
repair of a targeted DNA break in the presence of an externally
supplied repair template; or
<bullet> 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.
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\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.
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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.
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\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 <a href="https://www.oxfordreference.com/view/10.1093/acref/9780195307610.001.0001/acref-9780195307610-e-3651">https://www.oxfordreference.com/view/10.1093/acref/9780195307610.001.0001/acref-9780195307610-e-3651</a>.
\13\ Lackie, J. gain-of-function mutation. In Nation, B. (Ed.),
A Dictionary of Biomedicine.: Oxford University Press. Retrieved 6
Jun. 2023, from <a href="https://www.oxfordreference.com/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-3735">https://www.oxfordreference.com/view/10.1093/acref/9780191829116.001.0001/acref-9780191829116-e-3735</a>.
\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.
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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]]
<bullet> a targeted single base pair substitution, or
<bullet> 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:
<bullet> Sec. 340.1(b)(2)--a genetic modification is a targeted
single base pair substitution; and
<bullet> 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
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