Soybean event cor-23134-4 and methods for detection thereof

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

ATTORNEY DOCKET #: 108747-WO-SEC-1 SOYBEAN EVENT COR-23134-4 AND METHODS FOR DETECTION THEREOF CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 627235, filed on January 31, 2024, the disclosure of which is incorporated herein by reference in its entirety. REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] An XML formatted sequence listing having the file name “108747-WO-SEC- 1_SequenceListing.xml” created on January 14, 2025, and having a size of 1,569,000 bytes is filed in computer readable form concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety. FIELD

[0003] Embodiments disclosed herein relate to the field of plant molecular biology, including to DNA constructs for conferring insect resistance to a plant. Embodiments disclosed herein also include insect resistant soybean plants containing event COR-23134-4 and assays for detecting the presence of event COR-23134-4 in a sample and compositions thereof. BACKGROUND

[0004] Soybean is an important crop and is a primary food source in many areas of the world. Damage caused by insect pests is a major factor in the loss of the world’s soybean crops, despite the use of protective measures such as chemical pesticides. In view of this, insect resistance has been genetically engineered into crops such as soybean in order to control insect damage and to reduce the need for traditional chemical pesticides. One group of genes which have been utilized for the production of transgenic insect resistant crops is the delta-endotoxin group from Bacillus thuringiensis (Bt). Delta-endotoxins have been successfully expressed in crop plants such as cotton, potatoes, rice, sunflower, as well as corn, and in certain circumstances have proven to provide excellent control over insect pests. (Perlak, F.J et al. (1990) Bio / Technology 8:939-943; Perlak, F.J. et al. (1993) Plant Mol. Biol.22:313-321; Fujimoto, H. et al. (1993) Bio / Technology 11:1151-1155; Tu et al. (2000) NatureATTORNEY DOCKET #: 108747-WO-SEC-1 Biotechnology 18:1101-1104; PCT publication WO 01 / 13731; and Bing, J.W. et al. (2000) Efficacy of Cry1F Transgenic Maize, 14th Biennial International Plant Resistance to Insects Workshop, Fort Collins, CO). Soybean is a globally traded commodity produced in both temperate and tropical regions and serves as a key source of vegetable oils and protein. Soybean is grown as a commercial crop in over 35 countries. Brazil, United States, and Argentina together produce about 80% of the world’s soybean. Soybean is grown primarily to produce grain for further processing, has a multitude of uses in the food, feed, and industrial sectors, and represents one of the major sources of edible vegetable oil and of proteins for livestock feed use.

[0005] Certain lepidopteran insects are serious pests of soybean in various geographies. As adoption of Bt soybean has increased, the selection pressure on target insects to develop resistance has become greater. There remains a need for transgenic soybean plants that are resistant to certain insect pests in cultivation areas around the world. SUMMARY

[0006] The embodiments relate to the insect resistant soybean (Glycine max) plant event COR-23134-4, also referred to as “soybean line COR-23134-4,” “soybean event COR-23134- 4,” and “COR-23134-4 soybean,” to the DNA plant expression construct of soybean plant event COR-23134-4, and to methods and compositions for the detection of the transgene construct, flanking, and insertion (the target locus) regions in soybean plant event COR-23134-4 and progeny thereof.

[0007] In one aspect compositions and methods relate to methods for producing and selecting an insect resistant dicot crop plant. Compositions include a DNA construct that when expressed in plant cells and plants confers resistance to insects. In one aspect, a DNA construct, capable of introduction into and replication in a host cell, is provided that when expressed in plant cells and plants confers insect resistance to the plant cells and plants. Soybean event COR-23134-4 was produced by transformation with plasmid PHP90315. As described herein, these events include the cry1B.34.1 gene (polynucleotide SEQ ID NO: 4 and amino acid SEQ ID NO: 5) cassette, cry1B.61.1 gene (polynucleotide SEQ ID NO: 6 and amino acid SEQ ID NO: 7) cassette, and ipd083Cb gene (polynucleotide SEQ ID NO: 8 and amino acid SEQ ID NO: 9) cassette (Table 1), each of which confers resistance to certain lepidopteran plant pests. The insect control components have demonstrated efficacy against lepidopteran insect species.

[0008] Some embodiments relate to specific flanking sequences of COR-23134-4 as described herein, which can be used to develop identification methods for COR-23134-4 inATTORNEY DOCKET #: 108747-WO-SEC-1 biological samples. More particularly, the disclosure relates to 5’ and / or 3’ flanking regions of COR-23134-4, which can be used for the development of specific primers and probes. Further embodiments relate to identification methods for the presence of COR-23134-4 in biological samples based on the use of such specific primers or probes.

[0009] According to some embodiments, methods of detecting the presence of DNA corresponding to the soybean event COR-23134-4 in a sample are provided. Such methods comprise: (a) contacting the sample comprising DNA with a DNA primer set, that when used in a nucleic acid amplification reaction with genomic DNA extracted from soybean comprising event COR-23134-4 produces an amplicon that is diagnostic for soybean event COR-23134-4, respectively; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon. In some aspects, the primer set comprises SEQ ID NOs: 18 and 19, and optionally a probe comprising SEQ ID NO: 20.

[0010] According to some embodiments, methods of detecting the presence of a DNA molecule corresponding to the COR-23134-4 event in a sample comprise: (a) contacting the sample comprising DNA extracted from a soybean plant with a DNA probe molecule that hybridizes under stringent hybridization conditions with DNA extracted from soybean containing event COR-23134-4 and does not hybridize under the stringent hybridization conditions with a control soybean plant DNA; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA extracted from soybean containing event COR-23134-4. More specifically, a method for detecting the presence of a DNA molecule corresponding to the COR-23134-4 event in a sample comprises (a) contacting the sample comprising DNA extracted from a soybean plant with a DNA probe molecule that comprises sequences that are unique to the event, e.g. junction sequences, wherein said DNA probe molecule hybridizes under stringent hybridization conditions with DNA extracted from soybean event COR-23134-4 and does not hybridize under the stringent hybridization conditions with a control soybean plant DNA; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA.

[0011] In addition, a kit and methods for identifying event COR-23134-4 in a biological sample which detects a COR-23134-4 specific region are provided.

[0012] DNA molecules are provided that comprise at least one junction sequence of COR- 23134-4; wherein a junction sequence spans the junction located between heterologous DNA inserted into the genome and the DNA from the soybean cell flanking the insertion site. Detection of the junction sequence can be diagnostic for the COR-23134-4 event.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0013] According to some embodiments, methods of producing an insect resistant soybean plant comprise the steps of: (a) sexually crossing a first parental soybean line comprising the expression cassettes disclosed herein, which confer resistance to insects, and a second parental soybean line that lacks such expression cassettes, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that is insect resistant. Such methods may optionally comprise the further step of back-crossing the progeny plant to the second parental soybean line to produce a true-breeding soybean plant that is insect resistant.

[0014] Some embodiments provide a method of producing a soybean plant that is resistant to insects comprising transforming a soybean cell with the DNA construct PHP90315, growing the transformed soybean cell into a soybean plant, selecting the soybean plant that shows resistance to insects, and further growing the soybean plant into a fertile soybean plant. The fertile soybean plant can be self-pollinated or crossed with compatible soybean varieties to produce insect resistant progeny. In some embodiments, a soybean plant comprises the genotype of the soybean event COR-23134-4, wherein said genotype comprises a nucleotide sequence as set forth in SEQ ID NO: 12 and SEQ ID NO: 15.

[0015] Some embodiments further relate to a DNA detection kit for identifying soybean event COR-23134-4 in biological samples. The kit comprises a first primer which specifically recognizes the 5’ or 3’ flanking region of COR-23134-4, and a second primer which specifically recognizes a sequence within the non-native target locus DNA of COR-23134-4, respectively, or within the flanking DNA, for use in a PCR identification protocol. A further embodiment relates to a kit for identifying event COR-23134-4 in biological samples, which kit comprises a specific probe having a sequence which corresponds or is complementary to, a sequence having between about 80% and 100% sequence identity with a specific region of event COR-23134-4. The sequence of the probe can correspond to a specific region comprising part of the 5’ or 3’ flanking region of event COR-23134-4. In some embodiments, the first or second primer comprises any one of SEQ ID NOs: 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 38, or 39.

[0016] The methods and kits encompassed by the embodiments disclosed herein can be used for different purposes such as, but not limited to the following: to identify event COR- 23134-4 in plants, plant material or in products such as, but not limited to, food or feed products (fresh or processed) comprising, or derived from plant material; additionally or alternatively, the methods and kits can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits can be used to determine the quality of plant material comprising soybeanATTORNEY DOCKET #: 108747-WO-SEC-1 event COR-23134-4. The kits may also contain the reagents and materials necessary for the performance of the detection method.

[0017] A further embodiment relates to the COR-23134-4 soybean plant or its parts, including, but not limited to, pollen, ovules, vegetative cells, the nuclei of pollen cells, and the nuclei of egg cells of the soybean plant COR-23134-4 and the progeny derived thereof. In another embodiment, the DNA primer molecules targeting the soybean plant and seed of COR- 23134-4 provide a specific amplicon product. DESCRIPTION OF THE DRAWINGS

[0018] FIG.1. shows a schematic diagram of plasmid PHP90315 containing the cry1B.34.1, cry1B.61.1, ipd083Cb, and gm-hra_1 gene cassettes. Soybean event COR-23134-4 was created by Agrobacterium-mediated transformation with plasmid PHP90315. The size of plasmid PHP90315 is 35,909 bp. (SEQ ID NO: 1).

[0019] FIG.2. shows a schematic diagram of the PHP90315 T-DNA indicating the cry1B.34.1, cry1B.61.1, ipd083Cb, and gm-hra_1 gene cassettes. The size of the T-DNA is 27,801 bp (SEQ ID NO: 2).

[0020] FIG.3. shows a schematic Diagram of the Transformation and Development of COR-23134-4.

[0021] FIG.4. Illustrates a recombinase-mediated excision.

[0022] FIG.5. Illustrates a DNA DSB-mediated excision.

[0023] FIG.6. Illustrates a type I-mediated sequence removal.

[0024] FIG.7. Illustrates an example of cellular repair of DSB(s) by NHEJ, MMEJ, SSA, or HR can then be used to introduce targeted sequence variation in the promoter(s), intron(s), coding sequence(s) (CDS), terminator(s), and / or flanking sequence of COR-23134-4 to knock- out or disrupt gene expression, up- or down-regulate gene expression, modify the coding content, and / or alter the flanking genomic polynucleotides.

[0025] FIG.8. Illustrates an example of an RE, in which a target (for example but not limited to a loxP site) can be placed (either before or after genomic insertion) in opposing orientations and, then once recognized by the RE, the polynucleotide sequence between the sites inverted. DETAILED DESCRIPTIONATTORNEY DOCKET #: 108747-WO-SEC-1

[0026] As used herein the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the protein" includes reference to one or more proteins and equivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise. Nucleic acid sequences listed in the accompanying sequence listing and referenced herein are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand.

[0027] Compositions of this disclosure include seed, deposited as NCMA Accession No.: 202305013, and plants, plant cells, and seed derived therefrom. Applicant(s) deposited at least 625 seeds of soybean event COR-23134-4 (NCMA Accession No.: 202305013) with the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA), 60 Bigelow Drive, East Boothbay, ME 04544 USA, on May 12, 2023. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. The seeds deposited with the NCMA on May 12, 2023, were taken from the deposit maintained by Pioneer Hi-Bred International, Inc., 7250 NW 62nd Avenue, Johnston, Iowa 50131-1000. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant(s) will make available to the public, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit of at least 625 seeds of hybrid soybean with the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA). This deposit of seed of soybean event COR-23134-4 will be maintained in the NCMA depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant(s) have satisfied all the requirements of 37 C.F.R. §§1.801 - 1.809, including providing an indication of the viability of the sample upon deposit. Applicant(s) have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant(s) do not waive any infringement of their rights granted under this patent or rights applicable to event COR-23134-4 under the Plant Variety Protection Act (7 USC 2321 et seq.). Unauthorized seed multiplication is prohibited. The seed may be regulated.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0028] A first gene cassette (cry1B.34.1 gene cassette) contains the cry1B.34.1 gene, a gene comprised of sequences from a cry1B-class gene and the cry1Ca1 gene, both derived from Bacillus thuringiensis (WO Patent 2016061197 [Izumi Wilcoxon and Yamamoto, 2016]; GenBank accession CAA30396.1, respectively). The expressed Cry1B.34.1 protein confers control of certain susceptible lepidopteran pests. The Cry1B.34.1 protein is 665 amino acids (aa) in length and has a molecular weight of approximately 75 kDa. Expression of the cry1B.34.1 gene is controlled by the promoter region from the maize (Zea mays) histone H2B (zm-H2B) gene (GenBank accession NM_001196058.2; US Patent 6177611 [Rice, 2001]) and the 5' untranslated region (UTR) and intron region of the maize ubiquitin gene 1 (ubiZM1) (Christensen et al., 1992). The terminator for the cry1B.34.1 gene is the terminator region from the rice (Oryza sativa) ubiquitin (os-ubi) gene (WO Patent 2018102131 [Abbitt et al., 2018]). Two additional terminator regions from the sorghum (Sorghum bicolor) ubiquitin (sb-ubi) gene (Phytozome gene ID Sobic.004G049900.1; US Patent 9725731[Abbitt, 2017]) and the sorghum actin (sb-actin) gene (GenBank accession XM_002441128.2; US Patent 9725729 [Abbitt and Jung, 2017]) are present between the first and second gene cassettes. These additional terminators are intended to prevent any potential transcriptional interference. Transcriptional interference is defined as the transcriptional suppression of one gene on another when both are in proximity (Shearwin et al., 2005). The placement of one or multiple transcriptional terminators between gene cassettes has been shown to reduce the occurrence of transcriptional interference (Greger et al., 1998).

[0029] A second gene cassette (cry1B.61.1 gene cassette) contains the cry1B.61.1 gene, a modified cry1B-class gene, derived from Bacillus thuringiensis (WO Patent 2017180715 [Horn et al., 2017]). The expressed Cry1B.61.1 protein confers control of certain susceptible lepidopteran pests. The Cry1B.61.1 protein is 656 aa in length and has a molecular weight of approximately 74 kDa. Expression of the cry1B.61.1 gene is controlled by the promoter and the 5' UTR from the soybean chlorophyll a / b binding protein (gm-cab3) gene (Phytozome gene ID Glyma05g25810; US Patent 20200407742 [Sidorenko et al., 2020]). The os-T28 terminator (US Patent 10059953 [Bhyri et al., 2018]) for the cry1B.61.1 gene is the bidirectional terminator region from the rice NAC domain-containing protein gene (Phytozome gene ID LOC_Os03g60080.1) and the methylenetetrahydrofolate reductase gene (Phytozome gene ID LOC_Os03g60090.1). Two additional terminator regions from the sorghum phosphoenolpyruvic carboxylase (sb-PEPC1) gene (WO Patent 2018102131 [Abbitt et al., 2018]) and the Arabidopsis thaliana ubiquitin 3 (UBQ3) gene (WO Patent 2016149352 [ElsingATTORNEY DOCKET #: 108747-WO-SEC-1 et al., 2016] are present between the second and third gene cassettes to prevent possible transcriptional interference.

[0030] A third gene cassette (ipd083Cb gene cassette) contains the insecticidal protein gene, ipd083Cb, from giant maidenhair fern (Adiantum trapeziforme var. braziliense) (US Patent 10227608 [Barry et al., 2019]). The expressed IPD083Cb protein confers control of certain susceptible lepidopteran pests. The IPD083Cb protein is 853 aa in length and has a molecular weight of approximately 95 kDa. Expression of the ipd083Cb gene is controlled by the promoter region from the common bean (Phaseolus vulgaris) ubiquitin 2 (pv-ubi2) gene including the 5' UTR and intron (Phytozome gene ID Phvul.003G123900.1). The os-T17 terminator (US Patent 10059953 [Bhyri et al., 2018]) for the ipd083Cb gene is the bidirectional terminator region from the rice 5-enolpyruvyl shikimate-3-phosphate synthase (epsps) gene (Phytozome gene ID LOC_Os06g04280.1) and the ribosomal protein S10 gene (Phytozome gene ID LOC_Os06g04290.1).

[0031] A fourth gene cassette (gm-hra_1 gene cassette) contains the gm-hra_1 gene, a modified acetolactate synthase gene, from Glycine max (US Patent 7834242 [Falco and Li, 2010]). The expressed GM-HRA protein in plant tissue serves as a selectable marker during transformation which allows for the growth of tissue in the presence of sulfonylurea herbicides. The GM-HRA protein is 651 aa in length and has a molecular weight of approximately 70 kDa. Expression of the gm-hra_1 gene is controlled by the promoter region from the soybean S- adenosyl-L-methionine synthetase (SAMS) gene including 5’ UTR and intron (US Patent 7834242 [Falco and Li, 2010]). The terminator for the gm-hra_1 gene is the terminator region from the soybean acetolactate synthase (als) gene (Phytozome gene ID Glyma.04G196100; US Patent 7834242 [Falco and Li, 2010]).

[0032] Additional terminator sequences are present adjacent to the Right Border and Left Border within the T-DNA region: the terminator region from the maize globulin-1 (zm-Glb1) gene (WO Patent 2014070646 [Albertsen et al., 2014]) and the terminator region from the maize 19-kDa zein (Z19) gene (GenBank accession KX247647.1; Dong et al., 2016), respectively.

[0033] The PHP90315 T-DNA contains one flippase recombinase target sequence, FRT1 (Proteau et al., 1986), and four attB recombination sites (Hartley et al., 2000; Katzen, 2007; Cheo et al., 2004). The presence of these sites alone does not cause any recombination, since to function, these sites need a specific recombinase enzyme that is not naturally present in plants (Cox, 1988; Dale and Ow, 1990; Thorpe and Smith, 1998).ATTORNEY DOCKET #: 108747-WO-SEC-1

[0034] According to some embodiments, compositions and methods are provided for identifying a novel soybean plant designated COR-23134-4 (NCMA Accession No.: 202305013). The methods are based on primers or probes which specifically recognize 5’ and / or 3’ flanking sequence of COR-23134-4. DNA molecules are provided that comprise primer sequences that when utilized in a PCR reaction will produce amplicons unique to the transgenic event COR-23134-4. In one embodiment, the soybean plant and seed comprising these molecules is contemplated. Further, kits utilizing these primer sequences for the identification of the COR-23134-4 event are provided.

[0035] As used herein, the term “soybean” means Glycine max or soybean and includes all plant varieties that can be bred with soybean, including wild soybean species.

[0036] As used herein, the terms “insect resistant” and “impacting insect pests” refers to effecting changes in insect feeding, growth, and / or behavior at any stage of development, including but not limited to: killing the insect; retarding growth; reducing reproductive capability; inhibiting feeding; and the like.

[0037] As used herein, the terms “pesticidal activity” and “insecticidal activity” are used synonymously to refer to activity of an organism or a substance (such as, for example, a protein) that can be measured by numerous parameters including, but not limited to, pest mortality, pest weight loss, pest attraction, pest repellency, and other behavioral and physical changes of a pest after feeding on and / or exposure to the organism or substance for an appropriate length of time. For example, “pesticidal proteins” are proteins that display pesticidal activity by themselves or in combination with other proteins.

[0038] As used herein, “insert DNA” refers to the heterologous DNA within the expression cassettes used to transform the plant material while “flanking DNA” can refer to either genomic DNA naturally present in an organism such as a plant, or foreign (heterologous) DNA introduced via the transformation process which is extraneous to the original insert DNA molecule, e.g., fragments associated with the transformation event. A “flanking region” or “flanking sequence” as used herein refers to a sequence of at least 10 bp (in some narrower embodiments, at least 20 bp, at least 50 bp, and up to at least 5000 bp), which is located either immediately upstream of and contiguous with and / or immediately downstream of and contiguous with the original non-native insert DNA molecule. Transformation procedures of the foreign DNA may result in transformants containing different flanking regions characteristic and unique for each transformant. When recombinant DNA is introduced into a plant through traditional crossing, its flanking regions will generally not be changed. It may be possible for single nucleotide changes to occur in the flanking regions through generations of plant breedingATTORNEY DOCKET #: 108747-WO-SEC-1 and traditional crossing. Transformants will also contain unique junctions between a piece of heterologous insert DNA and genomic DNA, or two (2) pieces of genomic DNA, or two (2) pieces of heterologous DNA. A "junction" is a point where two (2) specific DNA fragments join. For example, a junction exists where insert DNA joins flanking DNA. A junction point also exists in a transformed organism where two (2) DNA fragments join together in a manner that is modified from that found in the native organism. “Junction DNA” refers to DNA that comprises a junction point. Junction sequences set forth in this disclosure include a junction point located between the soybean genomic DNA and the 5’ end of the insert, which range from at least -5 to +5 nucleotides of the junction point (SEQ ID NO: 12), from at least -10 to +10 nucleotides of the junction point (SEQ ID NO: 13), and from at least -25 to +25 nucleotides of the junction point (SEQ ID NO: 14); and a junction point located between the 3’ end of the insert and soybean genomic DNA, which range from at least -5 to +5 nucleotides of the junction point (SEQ ID NO: 15), from at least -10 to +10 nucleotides of the junction point (SEQ ID NO: 16), and from at least -25 to +25 nucleotides of the junction point (SEQ ID NO: 17). Junction sequences set forth in this disclosure also include a junction point located between the target locus and the 5’ end of the insert. In some embodiments, SEQ ID NOs: 20 or 33 for COR-23134-4 represent the junction point located between the target locus and the 5’ end of the insert. The complete insert with flanking regions is represented in SEQ ID NO: 3.

[0039] In one embodiment, seeds, plants, and plant parts comprising soybean event COR- 23134-4 are provided, wherein said seeds, plants, and plant parts comprise a DNA sequence chosen from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, or a DNA sequence chosen from a sequence having at least 95% sequence identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, wherein a representative sample of the soybean event COR- 23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013. In another embodiment, seeds, plants, and plant parts comprising soybean event COR-23134-4 are provided, wherein said seeds, plants, and plant parts comprise SEQ ID NO: 3 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3, wherein a representative sample of the soybean event COR-23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0040] As used herein, “heterologous” in reference to a nucleic acid sequence is a nucleic acid sequence that originates from a different non-sexually compatible species, or, if from the same species, is substantially modified from its native form in composition and / or genomicATTORNEY DOCKET #: 108747-WO-SEC-1 locus by deliberate human intervention. For example, a promoter operably linked to a heterologous nucleotide sequence can be from a species different from that from which the nucleotide sequence was derived, or, if from the same species, the promoter is not naturally found operably linked to the nucleotide sequence. A heterologous protein may originate from a foreign species, or, if from the same species, is substantially modified from its original form by deliberate human intervention.

[0041] The term “regulatory element” refers to a nucleic acid molecule having gene regulatory activity, i.e., one that has the ability to affect the transcriptional and / or translational expression pattern of an operably linked transcribable polynucleotide. The term “gene regulatory activity” thus refers to the ability to affect the expression of an operably linked transcribable polynucleotide molecule by affecting the transcription and / or translation of that operably linked transcribable polynucleotide molecule. Gene regulatory activity may be positive and / or negative and the effect may be characterized by its temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemically responsive qualities as well as by quantitative or qualitative indications.

[0042] “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence comprises proximal and more distal upstream elements, the latter elements are often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different regulatory elements may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical or similar promoter activity.

[0043] The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translationATTORNEY DOCKET #: 108747-WO-SEC-1 leader sequence may affect numerous parameters including, processing of the primary transcript to mRNA, mRNA stability and / or translation efficiency.

[0044] The “3’ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3’ end of the mRNA precursor.

[0045] A DNA construct is an assembly of DNA molecules linked together that provide one or more expression cassettes. The DNA construct may be a plasmid that is enabled for self- replication in a bacterial cell and contains various endonuclease enzyme restriction sites that are useful for introducing DNA molecules that provide functional genetic elements, i.e., promoters, introns, leaders, coding sequences, 3’ termination regions, among others; or a DNA construct may be a linear assembly of DNA molecules, such as an expression cassette. The expression cassette contained within a DNA construct comprises the necessary genetic elements to provide transcription of a messenger RNA. The expression cassette can be designed to express in prokaryotic cells or eukaryotic cells. Expression cassettes of the embodiments are designed to express in plant cells.

[0046] The DNA molecules disclosed herein are provided in expression cassettes for expression in an organism of interest. The cassette includes 5’ and 3’ regulatory sequences operably linked to a coding sequence. “Operably linked” means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. Operably linked is intended to indicate a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes or multiple DNA constructs.

[0047] The expression cassette may include in the 5’ to 3’ direction of transcription: a transcriptional and translational initiation region, a coding region, and a transcriptional and translational termination region functional in the organism serving as a host. The transcriptional initiation region (e.g., the promoter) may be native or analogous, or foreign or heterologous to the host organism. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. The expression cassettes may additionally contain 5’ leader sequences in the expression cassette construct. Such leader sequences can act to enhance translation.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0048] It is to be understood that as used herein the term “transgenic” generally includes any cell, cell line, callus, tissue, plant part, or plant, the genotype of which has been altered by the presence of a heterologous nucleic acid including those initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic and retains such heterologous nucleic acids.

[0049] A transgenic “event” is produced by transformation of plant cells with a heterologous DNA construct(s), including a nucleic acid expression cassette that comprises a transgene of interest, the regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location. An event is characterized phenotypically by the expression of the transgene. At the genetic level, an event is part of the genetic makeup of a plant. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another variety, wherein the progeny includes the heterologous DNA. After back-crossing to a recurrent parent, the inserted DNA and the linked flanking genomic DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. A progeny plant may contain sequence changes to the insert arising as a result of conventional breeding techniques. The term “event” also refers to DNA from the original transformant comprising the inserted DNA and flanking sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.

[0050] An insect resistant COR-23134-4 soybean plant may be bred by first sexually crossing a first parental soybean plant having the transgenic COR-23134-4 event plant and progeny thereof derived from transformation with the expression cassettes of the embodiments that confers insect resistance, and a second parental soybean plant that lacks such expression cassettes, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is resistant to insects; and selfing the first progeny plant, thereby producing a plurality of second progeny plants; and then selecting from the second progeny plants an insect resistant plant. These steps can further include the back-crossing of the first insect resistant progeny plant or the second insect resistant progeny plant to the second parental soybean plant or a third parental soybean plant, thereby producing a soybean plant that is resistant to insects. The term “selfing” refers to self-pollination, including the union of gametes and / or nuclei from the same organism.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0051] As used herein, the term "plant" includes reference to whole plants, parts of plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same. In some embodiments, parts of transgenic plants comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, and roots originating in transgenic plants or their progeny previously transformed with a DNA molecule disclosed herein, and therefore consisting at least in part of transgenic cells.

[0052] As used herein, the term "plant cell" includes, without limitation, seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. The class of plants that may be used is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.

[0053] “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host plants containing the transformed nucleic acid fragments are referred to as “transgenic” plants.

[0054] As used herein, the term "progeny," in the context of event COR-23134-4, denotes an offspring of any generation of a parent plant which comprises soybean event COR-23134-4.

[0055] Isolated polynucleotides disclosed herein may be incorporated into recombinant constructs, typically DNA constructs, which are capable of introduction into and replication in a host cell. Such a construct may be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. A number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al., (1985; Supp.1987) Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach (1989) Methods for Plant Molecular Biology, (Academic Press, New York); and Flevin et al., (1990) Plant Molecular Biology Manual, (Kluwer Academic Publishers). Typically, plant expression vectors include, for example, one or more cloned genes under the transcriptional control of 5’ and 3’ regulatory sequences and a dominant selectable marker. Such plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and / or a polyadenylation signal.

[0056] During the process of introducing an insert into the genome of plant cells, it is not uncommon for some deletions or other alterations of the insert and / or genomic flanking sequences to occur. Thus, the relevant segment of the plasmid sequence provided herein mightATTORNEY DOCKET #: 108747-WO-SEC-1 comprise some minor variations, including truncations. The same is possible for the flanking sequences and junction sequences provided herein. Thus, a plant comprising a polynucleotide having some range of identity with the subject flanking and / or insert sequences is within the scope of the subject disclosure. Identity to the sequence of the present disclosure may be a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified or described herein. Hybridization and hybridization conditions as provided herein can also be used to define such plants and polynucleotide sequences of the subject disclosure. A sequence comprising the flanking sequences plus the full insert sequence can be confirmed with reference to the deposited seed.

[0057] In some embodiments, two different transgenic plants can also be crossed to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation.

[0058] A “probe” is an isolated nucleic acid to which is attached a conventional, synthetic detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid, for example, to a strand of isolated DNA from soybean event COR-23134-4 whether from a soybean plant or from a sample that includes DNA from the event. Probes may include not only deoxyribonucleic or ribonucleic acids but also polyamides and other modified nucleotides that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.

[0059] “Primers” are isolated nucleic acids that anneal to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, then extended along the target DNA strand by a polymerase, e.g., a DNA polymerase. Primer pairs refer to their use for amplification of a target nucleic acid sequence, e.g., by PCR or other conventional nucleic-acid amplification methods. “PCR” or “polymerase chain reaction” is a technique used for the amplification of specific DNA segments (see, U.S. Patent Nos.4,683,195 and 4,800,159; herein incorporated by reference).

[0060] Probes and primers are of sufficient nucleotide length to bind to the target DNA sequence specifically in the hybridization conditions or reaction conditions determined by the operator. This length may be of any length that is of sufficient length to be useful in a detectionATTORNEY DOCKET #: 108747-WO-SEC-1 method of choice. Generally, 11 nucleotides or more in length, 18 nucleotides or more, and 22 nucleotides or more, are used. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Probes and primers according to embodiments may have complete DNA sequence similarity of contiguous nucleotides with the target sequence, although probes differing from the target DNA sequence and that retain the ability to hybridize to target DNA sequences may be designed by conventional methods. Probes can be used as primers, but are generally designed to bind to the target DNA or RNA and are not used in an amplification process.

[0061] Specific primers may be used to amplify an integration fragment to produce an amplicon that can be used as a “specific probe” for identifying event COR-23134-4 in biological samples. When the probe is hybridized with the nucleic acids of a biological sample under conditions which allow for the binding of the probe to the sample, this binding can be detected and thus allow for an indication of the presence of event COR-23134-4 in the biological sample. In an embodiment of the disclosure, the specific probe is a sequence which, under appropriate conditions, hybridizes specifically to a region within the 5’ or 3’ flanking region of the event and also comprises a part of the foreign DNA contiguous therewith. The specific probe may comprise a sequence of at least 80%, from 80 and 85%, from 85 and 90%, from 90 and 95%, and from 95 and 100% identical (or complementary) to a specific region of the event.

[0062] Methods for preparing and using probes and primers are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.1989 (hereinafter, “Sambrook et al., 1989”); Ausubel et al. eds., Current Protocols in Molecular Biology, , Greene Publishing and Wiley-Interscience, New York, 1995 (with periodic updates) (hereinafter, “Ausubel et al., 1995”); and Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990. PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as the PCR primer analysis tool in Vector NTI version 6 (Informax Inc., Bethesda MD); PrimerSelect (DNASTAR Inc., Madison, WI); and Primer (Version 0.5©, 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.). Additionally, the sequence can be visually scanned and primers manually identified using guidelines known to one of skill in the art.

[0063] A “kit” as used herein refers to a set of reagents, and optionally instructions, for the purpose of performing method embodiments of the disclosure, more particularly, the identification of event COR-23134-4 in biological samples. A kit may be used, and itsATTORNEY DOCKET #: 108747-WO-SEC-1 components can be specifically adjusted, for purposes of quality control (e.g., purity of seed lots), detection of event COR-23134-4 in plant material, or material comprising or derived from plant material, such as but not limited to food or feed products. “Plant material” as used herein refers to material which is obtained or derived from a plant.

[0064] Primers and probes based on the flanking DNA and insert sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such sequences. The nucleic acid probes and primers hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method may be used to identify the presence of DNA from a transgenic event in a sample.

[0065] A nucleic acid molecule is said to be the “complement” of another nucleic acid molecule if they exhibit complete complementarity or minimal complementarity. As used herein, molecules are said to exhibit “complete complementarity” when every nucleotide of one of the molecules is complementary to a nucleotide of the other. Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, D.C. (1985), departures from complete complementarity are therefore permissible, as long as such departures do not completely preclude the capacity of the molecules to form a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.

[0066] In hybridization reactions, specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. The thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem.138:267-284: Tm = 81.5 °C + 16.6 (log M) + 0.41 (%GC) - 0.61 (% form) - 500 / L; where M is the molarity of monovalent cations, %GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about 1ATTORNEY DOCKET #: 108747-WO-SEC-1 °C for each 1% of mismatching; thus, Tm, hybridization, and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10 °C. Generally, stringent conditions are selected to be about 5 °C lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, in some embodiments, other stringency conditions can be applied, including severely stringent conditions can utilize a hybridization and / or wash at 1, 2, 3, or 4 °C lower than the Tm; moderately stringent conditions can utilize a hybridization and / or wash at 6, 7, 8, 9, or 10 °C lower than the Tm; low stringency conditions can utilize a hybridization and / or wash at 11, 12, 13, 14, 15, or 20 °C lower than the Tm.

[0067] Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and / or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45 °C (aqueous solution) or 32 °C (formamide solution), a user may choose to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) and Sambrook et al. (1989).

[0068] In some embodiments, a complementary sequence has the same length as the nucleic acid molecule to which it hybridizes. In some embodiments, the complementary sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, the complementary sequence is 1%, 2%, 3%, 4%, or 5% longer or shorter than the nucleic acid molecule to which it hybridizes. In some embodiments, a complementary sequence is complementary on a nucleotide-for-nucleotide basis, meaning that there are no mismatched nucleotides (each A pairs with a T and each G pairs with a C). In some embodiments, a complementary sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or less mismatches. In some embodiments, the complementary sequence comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% or less mismatches.

[0069] "Percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, forATTORNEY DOCKET #: 108747-WO-SEC-1 instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence ×100).

[0070] Regarding the amplification of a target nucleic acid sequence (e.g., by PCR) using a particular amplification primer pair, stringent conditions permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and optionally to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.

[0071] As used herein, “amplified DNA” or “amplicon” refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, to determine whether a soybean plant resulting from a sexual cross contains transgenic event genomic DNA from the soybean plant disclosed herein, DNA extracted from a tissue sample of a soybean plant may be subjected to a nucleic acid amplification method using a DNA primer pair that includes a first primer derived from flanking sequence adjacent to the insertion site of inserted heterologous DNA, and a second primer derived from the inserted heterologous DNA to produce an amplicon that is diagnostic for the presence of the event DNA. Alternatively, the second primer may be derived from the flanking sequence. The amplicon is of a length and has a sequence that is also diagnostic for the event. The amplicon may range in length from the combined length of the primer pairs plus one nucleotide base pair to any length of amplicon producible by a DNA amplification protocol. Alternatively, primer pairs can be derived from flanking sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucleotide sequence of the PHP90315 expression construct as well as a portion of the sequence flanking the transgenic insert. A member of a primer pair derived from the flanking sequence may be located a distance from the inserted DNA sequence, this distance can range from one nucleotide base pair up to the limits of the amplification reaction. The use of the term “amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.

[0072] Nucleic acid amplification can be accomplished by any of the various nucleic acid amplification methods known in the art, including PCR. A variety of amplification methods are known in the art and are described, inter alia, in U.S. Pat. Nos.4,683,195 and 4,683,202 and inATTORNEY DOCKET #: 108747-WO-SEC-1 Innis et al., (1990) supra. PCR amplification methods have been developed to amplify up to 22 Kb of genomic DNA and up to 42 Kb of bacteriophage DNA (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the embodiments of the present disclosure. It is understood that a number of parameters in a specific PCR protocol may need to be adjusted to specific laboratory conditions and may be slightly modified and yet allow for the collection of similar results. These adjustments will be apparent to a person skilled in the art.

[0073] The amplicon produced by these methods may be detected by a plurality of techniques, including, but not limited to, Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res.22:4167-4175, 1994) where a DNA oligonucleotide is designed which overlaps both the adjacent flanking DNA sequence and the inserted DNA sequence. The oligonucleotide is immobilized in wells of a microwell plate. Following PCR of the region of interest (for example, using one primer in the inserted sequence and one in the adjacent flanking sequence) a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labeled ddNTPs specific for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert / flanking sequence due to successful amplification, hybridization, and single base extension.

[0074] Another detection method is the pyrosequencing technique as described by Winge (2000) Innov. Pharma. Tech.00:18-24. In this method an oligonucleotide is designed that overlaps the adjacent DNA and insert DNA junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (for example, one primer in the inserted sequence and one in the flanking sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5’ phosphosulfate and luciferin. dNTPs are added individually and the incorporation results in a light signal which is measured. A light signal indicates the presence of the transgene insert / flanking sequence due to successful amplification, hybridization, and single or multi-base extension.

[0075] Fluorescence polarization as described by Chen et al., (1999) Genome Res.9:492- 498 is also a method that can be used to detect an amplicon. Using this method an oligonucleotide is designed which overlaps the flanking and inserted DNA junction. The oligonucleotide is hybridized to a single-stranded PCR product from the region of interest (for example, one primer in the inserted DNA and one in the flanking DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the ddNTP. Incorporation can be measured as a change inATTORNEY DOCKET #: 108747-WO-SEC-1 polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert / flanking sequence due to successful amplification, hybridization, and single base extension.

[0076] Quantitative PCR (qPCR) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by commercially available manufacturers. Briefly, in one such qPCR method, a FRET oligonucleotide probe is designed which overlaps the flanking and insert DNA junction. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization.

[0077] Molecular beacons have been described for use in sequence detection as described in Tyangi et al. (1996) Nature Biotech.14:303-308. Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (for example, one primer in the insert DNA sequence and one in the flanking sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization.

[0078] A hybridization reaction using a probe specific to a sequence found within the amplicon is yet another method used to detect the amplicon produced by a PCR reaction.

[0079] Insect pests include insects selected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera, Mallophaga, Homoptera, Hemiptera, Orthoptera, Thysanoptera, Dermaptera, Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularly Lepidoptera.

[0080] Of interest are larvae and adults of the order Lepidoptera including, but not limited to, armyworms, cutworms, loopers and heliothines in the family Noctuidae, Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus (cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogonia Morrison (western cutworm); A. subterranea Fabricius (granulate cutworm); AlabamaATTORNEY DOCKET #: 108747-WO-SEC-1 argillacea Hübner (cotton leaf worm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindara Barnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris (darksided cutworm); Earias insulana Boisduval (spiny bollworm); E. vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialis Grote (citrus cutworm); borers, casebearers, webworms, coneworms, and skeletonizers from the family Pyralidae Ostrinia nubilalis Hübner (European corn borer); Amyelois transitella Walker (naval orangeworm); Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautella Walker (almond moth); Chilo suppressalis Walker (rice stem borer); C. partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth); Crambus caliginosellus Clemens (corn root webworm); C. teterrellus Zincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leaf roller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinata Linnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraea grandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius (surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestia elutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus (greater wax moth); Herpetogramma licarsisalis Walker (sod webworm); Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellus Zeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser wax moth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalis Walker (tea tree web moth); Maruca testulalis Geyer (bean pod borer); Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulas Walker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); and leafrollers, budworms, seed worms and fruit worms in the family Tortricidae Acleris gloverana Walsingham (Western blackheaded budworm); A. variana Fernald (Eastern blackheaded budworm); Archips argyrospila Walker (fruit tree leaf roller); A. rosana Linnaeus (European leaf roller); and other Archips species, Adoxophyes orana Fischer von Rösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham (banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C. pomonella Linnaeus (coding moth); Platynota flavedana Clemens (variegated leafroller); P. stultana Walsingham (omnivorous leafroller); Lobesia botrana Denis & Schiffermüller (European grape vine moth); Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth); Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguella Hübner (vine moth); Bonagota salubricola MeyrickATTORNEY DOCKET #: 108747-WO-SEC-1 (Brazilian apple leafroller); Grapholita molesta Busck (oriental fruit moth); Suleima helianthana Riley (sunflower bud moth); Argyrotaenia spp.; and Choristoneura spp..

[0081] In one embodiment, of interest are larvae and adults of the order Lepidoptera including, but not limited to, Spodoptera frugiperda JE Smith (fall armyworm); S. exigua Hübner (beet armyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar); Agrotis ipsilon Hufnagel (black cutworm); Trichoplusia ni Hübner (cabbage looper); Pseudoplusia includens Walker (soybean looper); Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabra Fabricius (green cloverworm); Heliothis virescens Fabricius (tobacco budworm); Helicoverpa armigera Hübner (American bollworm); H. zea Boddie (corn earworm or cotton bollworm); and Pyralidae Ostrinia nubilalis Hübner (European corn borer).

[0082] In another embodiment, of interest are larvae and adults of the order Lepidoptera including, but not limited to, Spodoptera cosmioides Walker (garden armyworm); Spodoptera albula Walker (gray-streaked armyworm); Spodoptera eridania Stoll (Southern armyworm); Chrysodeixis includens Walker; Rachiplusia nu Guenée (sunflower looper); Helicoverpa gelotopoeon Dyar (South American bollworm); Chloridea virescens Fabricius (this is current approved scientific name for tobacco budworm); Chrysodeixis acuta Walker (Tunbridge Wells Gem); and Thysanoplusia orichalcea Fabricius (slender burnished brass).

[0083] Selected other agronomic pests in the order Lepidoptera include, but are not limited to, Alsophila pometaria Harris (fall cankerworm); Anarsia lineatella Zeller (peach twig borer); Anisota senatoria J.E. Smith (orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese Oak Tussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiella Busck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfa caterpillar); Datana integerrima Grote & Robinson (walnut caterpillar); Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomos subsignaria Hübner (elm spanworm); Erannis tiliaria Harris (linden looper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisina americana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviae Cockrell (range caterpillar); Hyphantria cunea Drury (fall webworm); Keiferia lycopersicella Walsingham (tomato pinworm); Lambdina fiscellaria Hulst (Eastern hemlock looper); L. fiscellaria lugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus (satin moth); Lymantria dispar Linnaeus (gypsy moth); Manduca quinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M. sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumata Linnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm); Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidia californica Packard (California oakworm); Phyllocnistis citrella Stainton (citrus leafminer); Phyllonorycter blancardella Fabricius (spottedATTORNEY DOCKET #: 108747-WO-SEC-1 tentiform leafminer); Pieris brassicae Linnaeus (large white butterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus (green veined white butterfly); Platyptilia carduidactyla Riley (artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth); Pectinophora gossypiella Saunders (pink bollworm); Pontia protodice Boisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée (omnivorous looper); Schizura concinna J.E. Smith (red humped caterpillar); Sitotroga cerealella Olivier (Angoumois grain moth); Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar); Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick (tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothis subflexa Guenée; Malacosoma spp. and Orgyia spp.

[0084] In some embodiments the COR-23134-4 soybean event may further comprise a stack of additional traits. Plants comprising stacks of polynucleotide sequences can be obtained by either or both of traditional breeding methods or through genetic engineering methods. These methods include, but are not limited to, breeding individual lines each comprising a polynucleotide of interest, transforming a transgenic plant comprising a gene disclosed herein with a subsequent gene and co- transformation of genes into a single plant cell. As used herein, the term “stacked” includes having the multiple traits present in the same plant (i.e., both traits are incorporated into the nuclear genome, one trait is incorporated into the nuclear genome and one trait is incorporated into the genome of a plastid or both traits are incorporated into the genome of a plastid).

[0085] In some embodiments the COR-23134-4 soybean event disclosed herein, alone or stacked with one or more additional insect resistance traits can be stacked with one or more additional input traits (e.g., herbicide resistance, fungal resistance, virus resistance, stress tolerance, disease resistance, male sterility, stalk strength, and the like) or output traits (e.g., increased yield, modified starches, improved oil profile, balanced amino acids, high lysine or methionine, increased digestibility, improved fiber quality, drought resistance, and the like). Thus, the embodiments can be used to provide a complete agronomic package of improved crop quality with the ability to flexibly and cost effectively control any number of agronomic pests.

[0086] In a further embodiment, the COR-23134-4 soybean event may be stacked with one or more additional insecticidal toxins, including, but not limited to, DAS814.19 (Conkesta soybean) (US Patent Number 8,680,363), MON87701 (US Patent Number 8,455,198); MON87751 (US Patent Number 9,719,145); In a further embodiment, the COR-23134-4 soybean event may be stacked with one or more additional transgenic events containing herbicide events, including, but not limited to, MON04032 (Roundup ready soybean);ATTORNEY DOCKET #: 108747-WO-SEC-1 MON89788 (US Patent Number 7,632,985); MON87708 (U.S. Patent No.9,447,428); A5547- 127; LL2704; BPS-CV127-9 (U.S. Patent No.9,024,114); MST-FG072-3; DP305423 (US Patent Number 9,816,098); DAS44406 (US Patent No: 9,540,655). Other such transgenic alleles conferring desirable traits may include herbicide tolerance: GTS 40-3-2, A2704-12, A2704-21, A5547-35, A5547-127, BPS-CV127-9 (U.S. Patent No.9,024,114), DP356043 (U.S. Patent No.7,951,995), GU262, W62, W98, DAS-68416-4 (US Patent No.9,944,944), FG72, BPS-CV127-9, SYHT04R (U.S. Patent No.10,006,044), SYHT0H2, 3560.4.3.5, EE- GM3, pDAB4472-1606, pDAB4468-0416, pDAB8291.45.36, 127, increased enhanced oil composition: DP-305423 (US Patent Number 8,609,935), G94-1, G94-19, G168, OT96-15, MON87705 (U.S. Patent No.8,329,989), MON87769 (U.S. Patent No.8,692,076); increased yield: MON 87712 (U.S. Patent No.9,493,786), or nitrogen fixation traits, traits modulating the use of water, resistance to fungal infestation, resistance to nematode infestation, and the like. A non-transgenic property (e.g., QTL or maturity group) may also confer a desirable trait and one with skill in the art would know how to breed soybean to contain such non-transgenic trait and event COR-23134-4.

[0087] In a further embodiment, the COR-23134-4 soybean event may be stacked with one, two, three or more additional herbicidal tolerance traits enabling the selective use of multiple herbicide modes of action alone or in combinations. Glyphosate herbicide acts by inhibiting the EPSPS enzyme (5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involved in the biosynthesis of aromatic amino acids that are essential for growth and development of plants. A variety of glyphosate-insensitive EPSPS or glyphosate-metabolism enzymes are known. Genes that encode such enzymes can be operably linked to the gene regulatory elements of the subject disclosure. In an embodiment, such genes may include, but are not limited to, genes encoding glyphosate tolerance genes, which include: glyphosate- insensitive EPSPS genes such as 2mEPSPS, cp4 EPSPS, mEPSPS, dgt-28; epsps grg23ace5; and aroA genes; and as well as glyphosate-degradation genes such as glyphosate acetyl transferase genes (gat), and glyphosate oxidase genes (gox), for example. These traits may be found in products currently marketed as EnlistE3®, GT27, Gly-TolTM, Optimum® GAT®, Genuity Roundup Ready2 Yield®, and Roundup Ready®. Tolerance genes for glufosinate and / or bialaphos compounds include dsm-2, mat, hpat, bar and pat genes. The pat gene is in the trait currently marketed as LibertyLink®, LibertyLink GT27, EnlistE3®, ConkestaE3®, and Genuity Roundup Ready2 XtendFlex Soybean. Also included are tolerance genes that provide tolerance to 2,4-D (and other phenoxy auxin herbicides) such as aad-1, rdpA, and ft-t genes (it should be noted that these genes have further activity on aryloxyphenoxypropionate (aka ‘fop’)ATTORNEY DOCKET #: 108747-WO-SEC-1 herbicides) and aad-12, spdA, and tfdA genes (it should be noted that aad-12 genes have further activity on pyridyloxyacetate synthetic auxins like fluroxypyr and triclopyr). AAD-12 traits are marketed as Enlist® crop protection technology. Resistance genes for ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) are known in the art. These resistance genes most commonly result from point mutations to the ALS encoding gene sequence. Other ALS inhibitor resistance genes include hra genes, the csr1-2 genes, surA genes, and surB genes. Some of the traits are marketed under the tradename Cultivance, STS, Bolt, or Optimum GAT. Herbicides that inhibit HPPD include the pyrazolones such as pyrazoxyfen, benzofenap, and topramezone; triketones such as mesotrione, sulcotrione, tembotrione, benzobicyclon; and diketonitriles such as isoxaflutole. These exemplary HPPD herbicides can be tolerated by known traits. Examples of HPPD inhibitors include hppdPF_W336 , hppdPf-4Pa , tdo / his1 / hsl1, avhppd-03 genes (for resistance to isoxaflutole, meostrione, or other HPPD- inhibitor herbicides). An example of oxynil herbicide tolerant traits include the bxn gene, which has been showed to impart resistance to the herbicide / antibiotic bromoxynil. Resistance genes for dicamba include the dicamba monooxygenase gene (dmo) as disclosed in International PCT Publication No. WO 2008 / 105890. Resistance genes for PPO or PROTOX inhibitor type herbicides (e.g., acifluorfen, butafenacil, flupropazil, pentoxazone, carfentrazone, fluazolate, pyraflufen, aclonifen, azafenidin, flumioxazin, flumiclorac, bifenox, oxyfluorfen, lactofen, fomesafen, fluoroglycofen, epyrfenacil, saflufenacil, trifludimoxazin, tiafenacil, and sulfentrazone) are known in the art. Exemplary genes conferring resistance to PPO include herbicide insensitive forms of the PPO target enzyme from plants (US10100329, US10041087, US7671254, US6288306). Further exemplary genes conferring resistance to PPO include over expression of a wild-type Arabidopsis thaliana PPO enzyme (Lermontova I and Grimm B, (2000) Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl- ether herbicide acifluorfen. Plant Physiol 122:75–83.), the B. subtilis PPO gene (Li, X. and Nicholl D.2005. Development of PPO inhibitor-resistant cultures and crops. Pest Manag. Sci. 61:277-285 and Choi KW, Han O, Lee HJ, Yun YC, Moon YH, Kim MK, Kuk YI, Han SU and Guh JO, (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. Biosci Biotechnol Biochem 62:558–560.) Resistance genes for ayrloxyphenoxypropionate(AOPP) and cyclohexone (DIM) include the herbicide insensitive forms of ACCase (AcetylCoA Carboxylase) genes (e.g., Acc1-S1, Acc1-S2 and Acc1-S3). Exemplary genes conferring resistance to cyclohexanediones and / or aryloxyphenoxypropanoic acid includeATTORNEY DOCKET #: 108747-WO-SEC-1 sethoxydim, clethodim, cycloxydim, haloxyfop, diclofop, fenoxyprop, fluazifop, and quizalofop, among others. AOPP herbicide tolerance is also a second herbicide class enabled by genes such as aad-1, rdpA, and ft_t. Finally, herbicides can inhibit photosynthesis, including triazine or benzonitrile are provided tolerance by psbA genes (tolerance to triazine), 1s+ genes (tolerance to triazine), and nitrilase genes (tolerance to benzonitrile). The above list of herbicide tolerance genes is not meant to be limiting. Any herbicide tolerance genes are encompassed by the present disclosure.

[0088] In some embodiments, the disclosed compositions can be introduced into the genome of a plant using genome editing technologies, or previously introduced polynucleotides in the genome of a plant may be edited using genome editing technologies. For example, the disclosed polynucleotides can be introduced into a desired location in the genome of a plant through the use of genome editing systems such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, the disclosed polynucleotides can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion. The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with existing trait(s) of interest. Existing trait(s) of interest could be either endogenous traits or previously introduced traits.

[0089] In some embodiments, where the disclosed polynucleotide has previously been introduced into a genome, genome editing or genome engineering technologies may be used to alter or modify the introduced polynucleotide sequence, including flanking chromosomal genomic sequences. Site specific modifications that can be introduced into the disclosed compositions include those produced using any method for introducing site specific modification, including, but not limited to, through the use of sequence repair oligonucleotides, alone, or through the use of site-directed genome modification tools such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like, with or without donor DNA. Site-specific modifications to the disclosed polynucleotides (including genomic flanking and junction sequences) may include, but are not limited to, changes to codon usage, changes to regulatory elements such as promoters, introns, terminators, enhancers, 5’ or 3’ untranslated regions (UTRs), or other noncoding sequences, and other regions of the polynucleotide, where the modifications do not adversely affect the phenotypic characteristics of the resulting soybean plant. COR-23134-4 event plants containing modified polynucleotide sequences are also contemplated herein.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0090] Cas polypeptides suitable for introducing site-specific modifications include, for example, Cas9, Cas12f (Cas-alpha, Cas14), Cas12l (Cas-beta), Cas12a (Cpf1), Cas12b (a C2c1 protein), Cas13 (a C2c2 protein), Cas12c (a C2c3 protein), Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas12j, Cas12k, Cas3, Cas3-HD, Cas 5, Cas6, Cas7, Cas8, Cas10, or combinations or complexes of these. In some aspects, transposon-associated TnpB, a programmable RNA- guided DNA endonuclease can be used.

[0091] In some aspects, a genome editing system comprises a Cas-alpha (e.g., Cas12f) endonuclease and one or more guide polynucleotides that introduce one or more site-specific modifications in a target polynucleotide sequence, resulting in a modified target sequence. As used herein, “altered target site”, “altered target sequence”, “modified target site,” and “modified target sequence” are used interchangeably and refer to a target sequence as disclosed herein that comprises at least one alteration or modification when compared to a non-altered target sequence. Such alterations or modifications include, for example: (i) replacement or substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).

[0092] In some aspects, a genome editing system comprises a Cas-alpha endonuclease, one or more guide polynucleotides, and optionally a donor DNA. Some exemplary Cas-alpha endonucleases are described, for example, in WO2020123887.

[0093] In some aspects, a genome editing system comprises a Cas polypeptide, one or more guide polynucleotides, and optionally donor DNA, and editing a target polynucleotide sequence comprises nonhomologous end-joining (NHEJ) or homologous recombination (HR) following a Cas polypeptide-mediated double-strand break. Once a double-strand break is induced in the DNA, the cell's DNA repair mechanism is activated to repair the break. The most common repair mechanism to bring the broken ends together is the nonhomologous end-joining pathway (Bleuyard et al., (2006) DNA Repair 5:1-12). As a result, deletions, insertions, or other rearrangements are possible (Siebert and Puchta, (2002) Plant Cell 14:1121-31; Pacher et al., (2007) Genetics 175:21-9). Alternatively, the double-strand break can be repaired by homologous recombination between homologous DNA sequences. Once the sequence around the double-strand break is altered, for example, by exonuclease activities involved in the maturation of double-strand breaks, gene conversion pathways can restore the original structure if a homologous sequence is available, such as a homologous chromosome in non-dividing somatic cells, or a sister chromatid after DNA replication (Molinier et al., (2004) Plant Cell 16:342-52). Ectopic and / or epigenic DNA sequences may also serve as a DNA repair template for homologous recombination (Puchta, (1999) Genetics 152:1173-81).ATTORNEY DOCKET #: 108747-WO-SEC-1

[0094] In some aspects, the genome editing system comprises a Cas polypeptide, one or more guide polynucleotides, and a donor DNA. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of interest to be inserted into the genomic target site of a Cas polypeptide. Once a double-strand break is introduced in the target site by the endonuclease, the first and second regions of homology of the donor DNA can undergo homologous recombination with their corresponding genomic regions of homology resulting in exchange of DNA between the donor DNA and the target genomic region. As such, the provided methods result in the integration of the polynucleotide of interest of the donor DNA into the double-strand break in the target site in the plant genome, thereby altering the original target site and producing an altered genomic target site.

[0095] In some aspects, a genome editing system comprises a base editing agent and a plurality of guide polynucleotides and editing a target polynucleotide sequence comprises introducing a plurality of nucleobase edits in the target polynucleotide sequence resulting in a variant nucleotide sequence. Other aspects include modified COR-23134-4 event plants produced using a genome editing system.

[0096] One or more nucleobases of a target genomic sequence can be chemically altered, in some cases to change the base from one type to another, for example from a Cytosine to a Thymine, or an Adenine to a Guanine. In some aspects, a plurality of bases, for example 2 or more, 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more 90 or more, 100 or more, or even greater than 100, 200 or more, up to thousands of bases may be modified or altered, to produce a plant with a plurality of modified bases.

[0097] Any base editing complex, such as a base editing agent associated with an RNA- guided polypeptide (such as e.g., dCas associated with a deaminase), may be used to target and bind to a desired locus in the genome of an organism and chemically modify one or more nucleotides of a target genomic sequence.

[0098] Site-specific nucleotide base conversions can be achieved to engineer one or more nucleotide changes to create one or more edits into the genome. These include for example, a site-specific base edit mediated by a C•G to T•A or an A•T to G•C base editing deaminase enzymes (Gaudelli et al., Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage." Nature (2017); Nishida et al. “Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.” Science 353 (6305) (2016); Komor et al. “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.” Nature 533 (7603) (2016):420-4. A catalytically “dead” or inactive Cas (dCas)ATTORNEY DOCKET #: 108747-WO-SEC-1 polypeptide, for example an inactive Cas9 (dCas9), Cas12f (dCas12f), or another Cas polypeptide disclosed herein, fused to a cytidine deaminase or an adenine deaminase protein becomes a specific base editor that can alter DNA bases without inducing a DNA break. Base editors convert C->T (or G->A on the opposite strand) or an adenine base editor that would convert adenine to inosine, resulting in an A->G change within an editing window specified by the guide polynucleotides. Any molecule that effects a change in a nucleobase is a “base editing agent”. The dCas forms a functional complex with a guide polynucleotide that shares homology with a genomic sequence at the target site, and is further complexed with the deaminase molecule. The guided Cas polypeptide recognizes and binds to a target sequence, opening the double-strand to expose individual bases. In the case of a cytidine deaminase, the deaminase deaminates the cytosine base and creates a uracil. Uracil glycosylase inhibitor (UGI) is provided to prevent the conversion of U back to C. DNA replication or repair mechanisms then convert the Uracil to a thymine (U to T), and subsequent repair of the opposing base (formerly G in the original G-C pair) to an Adenine, creating a T-A pair.

[0099] One or more nucleotides of the inserted event and / or the flanking genomic DNA can be modified using a prime editing technology. See e.g., Anzalone et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019). A prime editing complex includes a prime editing protein that contains an RNA-guided DNA- nicking domain, such as a Cas nickase (e.g., Cas9 nickase, Casd12f1 nickase), fused to a reverse transcriptase domain and complexed with a pegRNA. The PE–pegRNA complex is able to introduce targeted DNA edits at desired locations in the genome, by binding the target DNA and nicking the PAM-containing strand. The resulting 3′ end hybridizes to a chosen primer binding site and then primes reverse transcription of new DNA sequence containing the desired edit using the reverse transcriptase template of the pegRNA. The resulting regulatory expression elements of the disclosed recombinant expression cassette(s) may be truncated or may include a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a regulatory element sequence exemplified or described herein.

[0100] Other modifications may include modifications to other portions of the DNA of the COR-23134-4 event. In some embodiments, genome engineering technologies can be used to relocate one or more expression cassettes described herein to one or more different locations of the same chromosome, or different chromosomes of soybean or a different crop. In such embodiment, polynucleotides comprising one or more of the junction sequences describedATTORNEY DOCKET #: 108747-WO-SEC-1 herein (e.g., SEQ ID NOs: 12 and / or 15) may be retained with the expression cassette(s), either partially or fully, or may be removed. Furthermore, genomic flanking sequence(s) described herein may also be retained with the expression cassette(s), either partially or fully, or may be removed.

[0101] In another embodiment, genome engineering technologies may be used to co-locate one or more transgene(s) or expression cassette(s) in physical proximity to the 5’ or 3’ junction sequence(s) described herein. With regard to physical position on a chromosome, co-located transgenes and / or expression cassettes can be separated from the 5’ or 3’ junction sequence(s), e.g., by about 1 megabase (MB; 1 million nucleotides), about 500 kilobases (Kb; 1000 nucleotides), about 400 Kb, about 300 Kb, about 200 Kb, about 100 Kb, about 50 Kb, about 25 Kb, about 10 Kb, about 5 Kb, about 4 Kb, about 3 Kb, about 2 Kb, about 1 Kb, about 500 nucleotides, about 250 nucleotides, or less. With regard to genetic distance on a chromosome, co-located transgenes and / or expression cassettes can be separated from the 5’ or 3’ junction sequence(s), e.g., by about 10 cM, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25, or 0.1 cM. For example, one or more of the expression cassette(s) obtained from one or more of the additional transgenic events described above may be co-located in physical proximity to the 5’ or 3’ junction sequence(s) described herein.

[0102] In another embodiment, polynucleotides comprising one of the junction sequences (e.g., SEQ ID NOs: 12 or 15) may be introduced at either or both ends of the inserted heterologous DNA. For example, a polynucleotide comprising the 5’ junction sequence may be deleted and replaced with a polynucleotide comprising the 3’ junction sequence, or vice versa.

[0103] In another embodiment, genome editing technologies may be used to modify the previously introduced polynucleotide(s) by inverting at least one of the polynucleotide(s) of the inserted DNA of the COR-23134-4 event. Such genome editing technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion and / or substitution of one or more nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, but are not limited to, additional expression elements, such as enhancer and promoter sequences. Sequences that may be deleted include, but are not limited to, regulatory elements or portions thereof that when deleted do not adversely affect function. Modifications to modulate expression patterns (e.g., reducing the expression level of the insecticidal polypeptide in certain tissue) is also contemplated by site-directed modification to the introduced expression cassette.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0104] In another embodiment, genome engineering technologies may be used to delete or modify all or part of one or more expression cassette(s) of the COR-23134-4 event as deposited with the NCMA on May 12, 2023, having NCMA Accession No.: 202305013. In this embodiment, the resulting soybean plant derived from the COR-23134-4 event as deposited with the NCMA on May 12, 2023, having NCMA Accession No.: 202305013 may comprise a portion of the expression cassette(s) described herein, none of the expression cassette(s) described herein, or modifications of the expression cassette(s) described herein.

[0105] In another embodiment, targeted DSB technologies may be used to position additional insecticidally-active proteins in close proximity to the disclosed compositions disclosed herein within the genome of a plant, in order to generate molecular stacks of insecticidally-active proteins.

[0106] In another embodiment, the polynucleotide sequences disclosed herein are used in a method comprising designing guide polynucleotides, such as guide RNAs (gRNAs), that recognize said polynucleotide sequences, synthesizing or obtaining said guide polynucleotides, and introducing said guide polynucleotides as part of genome engineering compositions to modify the DNA of the COR-23134-4 event as deposited with the NCMA on May 12, 2023, having NCMA Accession No.: 202305013. Such resulting modifications may include a polynucleotide sequence having at least 65% sequence identity, at least 70% sequence identity, at least 75% sequence identity at least 80% identity, or at least 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with a sequence exemplified or described herein.

[0107] Embodiments include modified COR-23134-4 event plants produced using genome engineering technologies described herein.

[0108] One embodiment includes a soybean plant comprising the genotype of the soybean event COR-23134-4, wherein said genotype comprises a nucleotide sequence as set forth in SEQ ID NO: 12, 13, or 14, or SEQ ID NO: 15, 16, or 17 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12 or SEQ ID NO: 15.

[0109] Another embodiment includes a soybean plant comprising the genotype of the soybean event COR-23134-4, wherein said genotype comprises a nucleotide sequence as set forth in SEQ ID NO: 12, 13, or 14, and SEQ ID NO: 15, 16, or 17 or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 12, 13, or 14, and SEQ ID NO: 15, 16, or 17. Still another embodiment includes a soybean plant comprising the genotype of the soybean event COR-23134-4 or any priorATTORNEY DOCKET #: 108747-WO-SEC-1 embodiment, wherein said genotype comprises a nucleotide sequence having 1, 2, 3, 4, or 5 nucleotide changes in one or more of SEQ ID NO: 12-17 or SEQ ID NO: 3.

[0110] Another embodiment includes the soybean plant comprising the genotype of the soybean event COR-23134-4 of any prior embodiment, wherein said genotype comprises the nucleotide sequence set forth in SEQ ID NO: 13 and SEQ ID NO: 16, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 13 and SEQ ID NO: 16.

[0111] Another embodiment includes the soybean plant comprising the genotype of the soybean event COR-23134-4 of any prior embodiment, wherein said genotype comprises the nucleotide sequence set forth in SEQ ID NO: 14 and SEQ ID NO: 17, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 14 and SEQ ID NO: 17.

[0112] One embodiment includes a DNA construct comprising an operably linked first, second, and third expression cassettes.

[0113] In a further embodiment, the first expression cassette comprises: 1) a zm-H2B Promoter; 2) an ubiZM1 Intron; 3) an cry1B.34.1; and 4) an os-ubi Terminator.

[0114] In a further embodiment, the second expression cassette comprises: a) a gm-cab3 Promoter; b) a cry1B.61.1; and c) an os-T28 Terminator.

[0115] In yet another embodiment, the third expression cassette comprises: i) a pv-ubi2 Promoter; ii) a pv-ubi2 Intron; iii) an ipd083C; and iv) an os-T17 Terminator.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0116] Another embodiment includes a plant comprising the DNA construct comprising at least two operably linked expression cassette of any prior embodiment.

[0117] A further embodiment includes a plant comprising the DNA construct comprising at least two operably linked expression cassettes of any prior embodiment, wherein said plant is a soybean plant.

[0118] One embodiment includes a plant comprising the sequence set forth in SEQ ID NO: 33, or a sequence having at least 95% sequence identity to SEQ ID NO: 33.

[0119] One embodiment includes a soybean event COR-23134-4, wherein a representative sample of seed of said soybean event has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0120] Other embodiments include plant parts of the soybean event COR-23134-4 of any prior embodiments, wherein a representative sample of seed of said soybean event has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0121] One embodiment includes seed comprising soybean event COR-23134-4, wherein said seed comprises a DNA molecule chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein a representative sample of the soybean event COR-23134-4 seed of has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0122] Another embodiment includes a soybean plant, or part thereof, grown from the seed comprising soybean event COR-23134-4 of any prior embodiment, wherein said seed comprises a DNA molecule chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein a representative sample of the soybean event COR-23134-4 seed of has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0123] A further embodiment includes a transgenic seed produced from the soybean plant comprising soybean event COR-23134-4 of any prior embodiment, wherein a representative sample of the soybean event COR-23134-4 seed of has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0124] Other embodiments include a transgenic soybean plant, or part thereof, grown from the seed soybean produced from the soybean plant of soybean event COR-23134-4 of any prior embodiment, wherein a representative sample of the soybean event COR-23134-4 seed of hasATTORNEY DOCKET #: 108747-WO-SEC-1 been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0125] One embodiment includes an isolated nucleic acid molecule comprising a nucleotide sequence chosen from SEQ ID Nos: 12-17, 33-37, and 41, and full length complements thereof.

[0126] One embodiment includes an amplicon comprising the nucleic acid sequence chosen from SEQ ID NOs: 33-37, or 41 and full length complements thereof.

[0127] One embodiment includes a biological sample or extract derived from soybean event COR-23134-4 plant, tissue, or seed, wherein said sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013.

[0128] Another embodiment includes the biological sample or extract derived from soybean event COR-23134-4 plant, tissue, or seed, wherein said sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013 of any prior embodiment, wherein said biological sample comprises plant, plant tissue, or seed of transgenic soybean event COR-23134-4.

[0129] Another embodiment includes the biological sample or extract derived from soybean event COR-23134-4 plant, tissue, or seed, wherein said biological sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013 of any prior embodiment, wherein said biological sample or extract is a DNA sample extracted from the transgenic soybean plant event COR-23134-4, and wherein said DNA sample comprises one or more of the nucleotide sequences chosen from SEQ ID NOs: 12-17, 33-37, and 41, and the complement thereof.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0130] Another embodiment includes the biological sample or extract derived from soybean event COR-23134-4 plant, tissue, or seed, wherein said biological sample or extract comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, wherein said nucleotide sequence is detectable in said sample or extract using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013 of any prior embodiment, wherein said biological sample or extract is chosen from soybean flour, soybean meal, and soybean oil manufactured in whole or in part to contain soybean by- products.

[0131] One embodiment includes a method of producing hybrid soybean seeds comprising: a) sexually crossing a first inbred soybean line comprising a nucleotide chosen from SEQ ID NOs: 12-17, 33-37, and 41 and a second inbred line having a different genotype; b) growing progeny from said crossing; and c) harvesting the hybrid seed produced thereby.

[0132] Another embodiment includes the method of producing hybrid soybean seeds of any prior embodiment, wherein the first inbred soybean line is a female or a male parent.

[0133] One embodiment includes a method for producing a soybean plant resistant to lepidopteran pests comprising: a) sexually crossing a first parent soybean plant with a second parent soybean plant, wherein said first or second parent soybean plant comprises event COR-23134-4 thereby producing a plurality of first-generation progeny plants; b) selfing the first-generation progeny plant, thereby producing a plurality of second-generation progeny plants; and c) selecting from the second-generation progeny plants that comprise the event COR-23134-4 and are resistant to a lepidopteran pest.

[0134] Another embodiment includes a method of producing hybrid soybean seeds comprising: a) sexually crossing a first inbred soybean line comprising the DNA construct of claim 1 with a second inbred line not comprising the DNA construct of claim 1; and b) harvesting the hybrid seed produced thereby.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0135] Another embodiment includes the method of producing a soybean plant resistant to lepidopteran pests of any prior embodiment, further comprising the step of backcrossing a second-generation progeny plant that comprises soybean event COR-23134-4 to the parent plant that lacks the soybean event COR-23134-4 DNA, thereby producing a backcross progeny plant that is resistant to a lepidopteran pest.

[0136] One embodiment includes a method of determining zygosity of a soybean plant comprising event COR-23134-4 in a biological sample comprising: a) contacting said sample with a first pair of DNA molecules and a second distinct pair of DNA molecules such that: 1) when used in a nucleic acid amplification reaction comprising soybean event COR-23134-4 DNA, produces a first amplicon that is diagnostic for event COR- 23134-4, and 2) when used in a nucleic acid amplification reaction comprising soybean genomic DNA other than COR-23134-4 DNA, produces a second amplicon that is diagnostic for soybean genomic DNA other than COR-23134-4 DNA; b) performing a nucleic acid amplification reaction; and c) detecting the amplicons so produced, wherein detection of the presence of both amplicons indicates that said sample is heterozygous for soybean event COR-23134- 4 DNA, wherein detection of only the first amplicon indicates that said sample is homozygous for soybean event COR-23134-4 DNA.

[0137] Another embodiment includes the method of determining zygosity of a soybean plant comprising event COR-23134-4 in a biological sample in any prior embodiment, wherein the first pair of DNA molecules comprises primer pair SEQ ID NOs: 18 and 19.

[0138] A further embodiment includes the method of determining zygosity of a soybean plant comprising event COR-23134-4 in a biological sample in any prior embodiment, wherein the first and second pair of DNA molecules comprise a detectable label.

[0139] Another embodiment includes the method of determining zygosity of a soybean plant comprising event COR-23134-4 in a biological sample in any prior embodiment, wherein the detectable label is a fluorescent label.

[0140] A further embodiment includes the method of determining zygosity of a soybean plant comprising event COR-23134-4 in a biological sample in any prior embodiment, wherein the detectable label is covalently associated with one or more of the primer molecules.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0141] One embodiment includes a method of detecting the presence of a nucleic acid molecule that is unique to event COR-23134-4 in a sample comprising soybean nucleic acids, the method comprising: a) contacting the sample with a pair of primers that, when used in a nucleic-acid amplification reaction with genomic DNA from event COR-23134-4 produces an amplicon that is diagnostic for event COR-23134-4; b) performing a nucleic acid amplification reaction, thereby producing the amplicon that is diagnostic for event COR-23134-4; and c) detecting the amplicon that is diagnostic for event COR-23134-4.

[0142] Another embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event COR-23134-4 in a sample comprising soybean nucleic acids of any prior embodiment, wherein the nucleic acid molecule that is diagnostic for event COR- 23134-4 is an amplicon produced by the nucleic acid amplification chain reaction.

[0143] Another embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event COR-23134-4 in a sample comprising soybean nucleic acids of any prior embodiment, wherein the method further comprises contacting the sample with a probe.

[0144] A further embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event COR-23134-4 in a sample comprising soybean nucleic acids, further comprises contacting the sample with a probe of any prior embodiment, wherein the probe comprises a detectable label.

[0145] A further embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event COR-23134-4 in a sample comprising soybean nucleic acids further comprising contacting the sample with a probe, wherein the probe comprises a detectable label of any prior embodiment, wherein the detectable label is a fluorescent label.

[0146] A further embodiment includes the method of detecting the presence of a nucleic acid molecule that is unique to event COR-23134-4 in a sample comprising soybean nucleic acids further comprising contacting the sample with a probe, wherein the probe comprises a

[0147] detectable label of any prior embodiment, wherein the detectable label is covalently associated with the probe.

[0148] One embodiment includes a plurality of polynucleotide primers comprising one or more polynucleotides which target event COR-23134-4 DNA template in a sample to produce an amplicon diagnostic for event COR-23134-4 as a result of a polymerase chain reaction method.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0149] Another embodiment includes a plurality of polynucleotide primers according to any prior embodiment, wherein a) a first polynucleotide primer comprises a nucleotide sequence as set forth in SEQ ID NO: 18, and the complements thereof; and b) a second polynucleotide primer comprises a nucleotide sequence as set forth in SEQ ID NO: 19, and the complements thereof.

[0150] Another embodiment includes the primers of any prior embodiment, wherein said first primer and said second primer are at least 19 nucleotides.

[0151] One embodiment includes a method of detecting the presence of DNA corresponding to event COR-23134-4 in a sample, the method comprising: a) contacting the sample comprising soybean DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from soybean event COR-23134-4 and does not hybridize under said stringent hybridization conditions with a non- COR-23134-4 soybean plant DNA; b) subjecting the sample and probe to stringent hybridization conditions; and c) detecting hybridization of the probe to the DNA; wherein detection of hybridization indicates the presence of event COR-23134-4.

[0152] One embodiment includes a kit for detecting nucleic acids that are unique to event COR-23134-4 comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event COR-23134-4 in the sample.

[0153] Another embodiment includes the kit for detecting nucleic acids that are unique to event COR-23134-4 comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event COR-23134-4 in the sample of any prior embodiment, wherein the nucleic acid molecule comprises a nucleotide sequence from SEQ ID NO: 12-41.

[0154] Another embodiment includes the kit for detecting nucleic acids that are unique to event COR-23134-4 comprising at least one nucleic acid molecule of sufficient length ofATTORNEY DOCKET #: 108747-WO-SEC-1 contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event COR-23134-4 in the sample of any prior embodiment, wherein the nucleic acid molecule is a primer chosen from SEQ ID NOs: 12-41, and the complements thereof.

[0155] Another embodiment includes the soybean plant comprising the genotype of the soybean event COR-23134-4 of any prior embodiment, wherein the genotype comprises a nucleotide sequence having 1, 2, 3, 4, or 5 nucleotide changes in one or more of SEQ ID Nos: 12-17, or SEQ ID NO:3.

[0156] Another embodiment includes the corn plant comprising the genotype of the soybean event COR-23134-4 of any prior embodiment, further comprising the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence of SEQ ID NO: 3. In yet another embodiment, the corn plant comprising the genotype of the soybean event COR-23134-4 of any prior embodiment, further comprising the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes to the nucleotide sequence of SEQ ID NO: 3.

[0157] One embodiment includes a method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event.

[0158] Another embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event of any prior embodiment, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 3. In yet another embodiment, the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean eventATTORNEY DOCKET #: 108747-WO-SEC-1 of any prior embodiment, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes to the nucleotide sequence of SEQ ID NO: 3.

[0159] Another embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event of any prior embodiment, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having all or a portion of SEQ ID NO: 12 or SEQ ID NO: 15 duplicated in said modified DNA sequence.

[0160] Another embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event of any prior embodiment, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3.

[0161] A further embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, wherein said excision comprises an excision from one or more regulatory elements of SEQ ID NO: 3 that does not substantially affect the activity of said one or more regulatory elements.

[0162] A further embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, comprising modifying the DNA of said COR-23134-4 soybean event to produce aATTORNEY DOCKET #: 108747-WO-SEC-1 modified DNA sequence having all or a portion of SEQ ID NO: 12 or SEQ ID NO: 15 excised from said modified DNA sequence.

[0163] Another embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having at least 30% of SEQ ID NO: 3 excised from said modified DNA sequence.

[0164] A further embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, wherein at least 80% of SEQ ID NO: 3 is excised from said modified DNA sequence.

[0165] Another embodiment includes the method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO: 3 of any prior embodiment, wherein all of SEQ ID NO: 3 is excised from said modified DNA sequence.

[0166] One embodiment includes a method of generating guide polynucleotides for use with a COR-23134-4 soybean event genome editing system comprising designing one or more guide polynucleotides that recognize at least a portion of SEQ ID NO: 3 and synthesizing said guide polynucleotides.

[0167] Another embodiment includes a method of modifying the DNA of the COR-23134-4 event having NCMA Accession No.: 202305013 comprising introducing said one or more guide polynucleotides for use with a COR-23134-4 soybean event genome editing system ofATTORNEY DOCKET #: 108747-WO-SEC-1 any prior embodiment as part of a genome engineering composition to a DNA of the COR- 23134-4 event to modify the DNA of the COR-23134-4 event.

[0168] One embodiment includes a COR-23134-4 soybean event genome editing system comprising a CAS polypeptide, one or more guide polynucleotides, and COR-23134-4 soybean event donor DNA.

[0169] One embodiment includes a method of modifying at least one expression cassette of the COR-23134-4 event as deposited with the NCMA having NCMA Accession No.: 202305013, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting soybean plant derived from the COR- 23134-4 event comprises at least one modified cassette.

[0170] Another embodiment includes the method of modifying at least one expression cassette of the COR-23134-4 event as deposited with the NCMA having NCMA Accession No.: 202305013, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting soybean plant derived from the COR- 23134-4 event comprises at least one modified cassette of any prior embodiment, wherein the method comprises altering expression of cry1B.34.1.

[0171] Another embodiment includes the method of modifying at least one expression cassette of the COR-23134-4 event as deposited with the NCMA having NCMA Accession No.: 202305013, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting soybean plant derived from the COR- 23134-4 event comprises at least one modified cassette of any prior embodiment, wherein the method comprises altering expression of cry1B.61.1.

[0172] Another embodiment includes the method of modifying at least one expression cassette of the COR-23134-4 event as deposited with the NCMA having NCMA Accession No.: 202305013, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting soybean plant derived from the COR- 23134-4 event comprises at least one modified cassette of any prior embodiment, wherein the method comprises altering expression of IPD083Cb.

[0173] One embodiment includes a method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR-23134-4.

[0174] Another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4 of any prior embodiment, wherein the Lepidopteran insect is Fall Armyworm.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0175] Another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4 of any prior embodiment, wherein the Lepidopteran insect is Soybean Looper.

[0176] Another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4 of any prior embodiment, wherein the Lepidopteran insect is Velvetbean Caterpillar.

[0177] Another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4 of any prior embodiment, wherein the damage from the Lepidopteran insect is controlled for soybean event COR-23134-4.

[0178] Another embodiment includes a method of producing a commodity plant product comprising processing grain produced from a soybean event COR-23134-4 plant comprising a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) with NCMA Accession No.: 202305013, wherein said grain is processed into a commodity plant product chosen from soybean flour, soybean meal, and soybean oil manufactured in whole or in part to contain soybean by-products, wherein said composition / commodity plant product comprises a detectable amount of said nucleotide sequence.

[0179] One embodiment includes a method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR-23134-4.

[0180] Another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4, wherein the Lepidopteran insect is Fall Armyworm (Spodoptera frugiperda).

[0181] Yet another embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4, wherein the Lepidopteran insect is Fall Armyworm (Spodoptera frugiperda), Soybean Looper (Chrysodeixis includens), or Velvetbean Caterpillar (Anticarsia gemmatalis).

[0182] A further embodiment includes the method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR- 23134-4, wherein damage from the Lepidopteran insect is controlled for soybean pods or seeds from event COR-23134-4.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0183] In some embodiments, a soybean plant comprising a COR-23134-4 event may be treated with a seed treatment. In some embodiments, the seed treatment may be a fungicide, an insecticide, or a herbicide.

[0184] The following examples are offered by way of illustration and not by way of limitation.ATTORNEY DOCKET #: 108747-WO-SEC-1 EXAMPLES

[0185] The following Examples are included to more fully describe embodiments of the development of soy event COR-23134-4, which resulted from the construction of 19 different construct designs having IPD083Cb, Cry1B.61.1, and Cry1B.34.1. About 4,750 T0 events were generated, of which 69 events were tested in T1 for homozygous plants, of which 57 events were tested in field trials. Eighteen events were tested in up to four different genotypes. Of these, 8 events were tested in seed field trials and soy event COR- 23134-4 was subsequently selected. These field tests were conducted over the course of 5 years. Example 1. Cassette Design -

[0186] Soybean (Glycine max [L.] Merr) was created by Agrobacterium-mediated transformation with plasmid PHP90315 (Figure 1). The T-DNA region is represented schematically in Figure 2. Summary of the genetic elements and their positions on the T-DNA are provided in Table 1.

[0187] The T-DNA of plasmid PHP90315 contains four gene cassettes. The first gene cassette (cry1B.34.1 gene cassette) contains the cry1B.34.1 gene, a gene composed of sequences from a cry1B-class gene and the cry1Ca1 gene, both derived from Bacillus thuringiensis (WO Patent 2016061197 [Izumi Wilcoxon and Yamamoto, 2016]; GenBank accession CAA30396.1, respectively). The expressed Cry1B.34.1 protein confers control of certain susceptible lepidopteran pests. The Cry1B.34.1 protein is 665 amino acids (aa) in length and has a molecular weight of approximately 75 kDa. Expression of the cry1B.34.1 gene is controlled by the promoter region from the maize (Zea mays) histone H2B (zm- H2B) gene (GenBank accession NM_001196058.2; US Patent 6177611 [Rice, 2001]) and the 5' untranslated region (UTR) and intron region of the maize ubiquitin gene 1 (ubiZM1) (Christensen et al., 1992). The terminator for the cry1B.34.1 gene is the terminator region from the rice (Oryza sativa) ubiquitin (os-ubi) gene (WO Patent 2018102131 [Abbitt et al., 2018]). Two additional terminator regions from the sorghum (Sorghum bicolor) ubiquitin (sb-ubi) gene (Phytozome gene ID Sobic.004G049900.1; US Patent 9725731[Abbitt, 2017]) and the sorghum actin (sb-actin) gene (GenBank accession XM_002441128.2; US Patent 9725729 [Abbitt and Jung, 2017]) are present between the first and second gene cassettes. These additional terminators are intended to prevent any potential transcriptional interference. Transcriptional interference is defined as the transcriptional suppression of oneATTORNEY DOCKET #: 108747-WO-SEC-1 gene on another when both are in proximity (Shearwin et al., 2005). The placement of one or multiple transcriptional terminators between gene cassettes has been shown to reduce the occurrence of transcriptional interference (Greger et al., 1998).

[0188] The second gene cassette (cry1B.61.1 gene cassette) contains the cry1B.61.1 gene, a modified cry1B-class gene, derived from Bacillus thuringiensis (WO Patent 2017180715 [Horn et al., 2017]). The expressed Cry1B.61.1 protein confers control of certain susceptible lepidopteran pests. The Cry1B.61.1 protein is 656 aa in length and has a molecular weight of approximately 74 kDa. Expression of the cry1B.61.1 gene is controlled by the promoter and the 5' UTR from the soybean chlorophyll a / b binding protein (gm- cab3) gene (Phytozome gene ID Glyma05g25810; US Patent 20200407742 [Sidorenko et al., 2020]). The os-T28 terminator (US Patent 10059953 [Bhyri et al., 2018]) for the cry1B.61.1 gene is the bidirectional terminator region from the rice NAC domain- containing protein gene (Phytozome gene ID LOC_Os03g60080.1) and the methylenetetrahydrofolate reductase gene (Phytozome gene ID LOC_Os03g60090.1). Two additional terminator regions from the sorghum phosphoenolpyruvic carboxylase (sb- PEPC1) gene (WO Patent 2018102131 [Abbitt et al., 2018]) and the Arabidopsis thaliana ubiquitin 3 (UBQ3) gene (WO Patent 2016149352 [Elsing et al., 2016] are present between the second and third gene cassettes to prevent possible transcriptional interference.

[0189] The third gene cassette (ipd083Cb gene cassette) contains the insecticidal protein gene, ipd083Cb, from giant maidenhair fern (Adiantum trapeziforme var. braziliense) (US Patent 10227608 [Barry et al., 2019]). The expressed IPD083Cb protein confers control of certain susceptible lepidopteran pests. The IPD083Cb protein is 853 aa in length and has a molecular weight of approximately 95 kDa. Expression of the ipd083Cb gene is controlled by the promoter region from the common bean (Phaseolus vulgaris) ubiquitin 2 (pv-ubi2) gene including the 5' UTR and intron (Phytozome gene ID Phvul.003G123900.1). The os- T17 terminator (US Patent 10059953 [Bhyri et al., 2018]) for the ipd083Cb gene is the bidirectional terminator region from the rice 5-enolpyruvyl shikimate-3-phosphate synthase (epsps) gene (Phytozome gene ID LOC_Os06g04280.1) and the ribosomal protein S10 gene (Phytozome gene ID LOC_Os06g04290.1).

[0190] The fourth gene cassette (gm-hra_1 gene cassette) contains the gm-hra_1 gene, a modified acetolactate synthase gene, from Glycine max (US Patent 7834242 [Falco and Li, 2010]). The expressed GM-HRA protein in plant tissue serves as a selectable marker during transformation which allows for the growth of tissue in the presence of sulfonylurea herbicides. The GM-HRA protein is 651 aa in length and has a molecular weight ofATTORNEY DOCKET #: 108747-WO-SEC-1 approximately 70 kDa. Expression of the gm-hra_1 gene is controlled by the promoter region from the soybean S-adenosyl-L-methionine synthetase (SAMS) gene including 5’ UTR and intron (US Patent 7834242 [Falco and Li, 2010]). The terminator for the gm- hra_1 gene is the terminator region from the soybean acetolactate synthase (als) gene (Phytozome gene ID Glyma.04G196100; US Patent 7834242 [Falco and Li, 2010]).

[0191] Additional terminator sequences are present adjacent to the Right Border and Left Border within the T-DNA region: the terminator region from the maize globulin-1 (zm- Glb1) gene (WO Patent 2014070646 [Albertsen et al., 2014]) and the terminator region from the maize 19-kDa zein (Z19) gene (GenBank accession KX247647.1; Dong et al., 2016), respectively.

[0192] The PHP90315 T-DNA contains one flippase recombinase target sequence, FRT1 (Proteau et al., 1986), and four attB recombination sites (Hartley et al., 2000; Katzen, 2007; Cheo et al., 2004). The presence of these sites alone does not cause any recombination, since to function, these sites need a specific recombinase enzyme that is not naturally present in plants (Cox, 1988; Dale and Ow, 1990; Thorpe and Smith, 1998). Table 1: Description of Genetic Elements in the T-DNA Region from Plasmid PHP90315ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1Example 2. Transformation of Soybean by Agrobacterium transformation and Regeneration of Transgenic Plants

[0193] COR23134 soybean was created by Agrobacterium-mediated transformation of soybean variety 93Y21 with plasmid PHP90315. Immature soybean cotyledons were inoculated Agrobacterium tumefaciens strain AGL1 containing plasmid PHP90315. Agrobacterium tumefaciens strain AGL1 is a disarmed strain that contains the vir genes and enables efficient transfer of the transfer DNA (T-DNA) region of the transformationATTORNEY DOCKET #: 108747-WO-SEC-1 plasmid to the inoculated host plant tissue. Healthy green callus was transferred to a solid maturation medium and incubated, followed by desiccation of the resulting embryos. Embryos were then transferred to a solid germination medium to initiate shoot and root development. Once shoots and roots were established, healthy plants were selected, and PCR was used to confirm the presence of the PHP90315 T-DNA insert. Plants that were regenerated from transformation and tissue culture (designated T0 plants) were selected for further characterization. Example 3. Identification of Soybean Events COR-23134-4

[0194] Soybean (Glycine max (L.) Merr.) was modified by the insertion of the gm-hra, cry1B.34, cry1B.61, and ipd083Cb genes via insertion of T-DNA (Figure 2) from plasmid PHP90315 (Figure 1) to create event COR-23134-4 (also referred to as COR23134 soybean).

[0195] Polymerase chain reaction (PCR) amplification of unique regions within the introduced genetic elements can distinguish the test plants from their non-genetically modified counterparts and can be used to screen for the presence of the inserted T-DNA region of plasmid PHP90315.

[0196] Real-time PCR analyses of COR23134 soybean using event-specific and construct-specific assays confirmed the COR23134 soybean plants from two segregating generations (T2 and T3) contained the inserted T-DNA regions of plasmid PHP90315, as demonstrated by the CT values produced in all test plants analyzed and confirmed the absence of event COR-23134-4 in the negative segregant plants within each generation. These results also indicate stable integration and segregation of a single copy of the inserted genes with transfer to subsequent generations. The data were highly reproducible across technical replicates of the plants tested. The soybean endogenous reference gene assay for detection of the Lectin (Le1) gene amplified, as expected, for all the plants analyzed.

[0197] The sensitivity of the construct-specific PCR detection methods in COR23134 soybean, under the conditions performed in 5 ng of soybean genomic DNA, has demonstrated ability to detect to approximately 5 pg of the gm-hra gene, 20 pg of the cry1B.34 gene, 10 pg of the cry1B.61 gene, 5 pg of the ipd083Cb gene, and 10 pg of the COR-23134-4 event. These concentrations are equivalent to 0.1%, 0.4%, 0.2%, 0.1%, and 0.2% of the COR23134 soybean genomic DNA, respectively.

[0198] For detection of the gm-hra, cry1B.34, cry1B.61, and ipd083Cb genes contained within COR23134 soybean, regions spanning between 57 bp and 109 bp were amplifiedATTORNEY DOCKET #: 108747-WO-SEC-1 using primers and Taqman®probes specific for each unique sequence. Additionally, a 66-bp region of an endogenous reference gene, Lectin (Le1; GenBank accession number K00821.1), was validated to be used in duplex with each assay for both qualitative and quantitative assessment of each assay and to demonstrate the presence of sufficient quality and quantity of DNA within the PCR reaction (Kuribara et al., 2002). CT data from Le1 was used in calculations regarding scoring with regard to the event or gene tested. Data were compared to the performance of either the validated positive or copy number calibrator as well as negative genomic controls as described below.

[0199] The real-time PCR reaction exploited the 5’ nuclease activity of the heat-activated DNA polymerase. Two primers and one probe annealed to the target DNA with the probe, which contained a 5’ fluorescent reporter dye and a 3’ quencher dye. With each PCR cycle, the reporter dye was cleaved from the annealed probe by the polymerase, emitting a fluorescent signal that intensified with each subsequent cycle. The cycle at which the emission intensity of the sample amplicon rose above the detection threshold was referred to as the CT value. When no amplification occurred, there was no CT calculated by the instrument and was assigned a CT value of 40.00.

[0200] Samples tested qualitatively were determined to be positive or negative for a specific gene of interest using the following criteria: • Positive: o Endogenous gene CT < 35 o Gene of interest (GOI) CT< 35 o ΔCT (Endogenous CT – GOI CT) > -5 • Negative: o Endogenous gene CT < 35 o Gene of interest (GOI) CT > 35 o ΔCT (Endogenous CT – GOI CT) < or > -5

[0201] If copy number of the test samples was to be determined to determine a quantitative result, copy number calibrators (samples known to contain defined copies of the gene of interest, e.g., 1 or 2 copies) were used as controls for both the endogenous gene and gene of interest. Fold differences were used to apply a copy number for each test sample. FoldATTORNEY DOCKET #: 108747-WO-SEC-1 difference, or fold change, is calculated using the formula of 2-ΔCT. The ΔCT was calculated for the test samples and copy number calibrators as described above. A copy number of 1 was applied to the sample population producing a fold change between 0 and 0.7 with a maximum range of 0.75 when compared to the 2-copy calibrators. Likewise, a copy number of 2 was applied to a sample population producing a fold change ranging between 1.5 and 2.2 with a maximum range of 0.91 when compared to the single copy calibrators; and a copy number of 3 was applied to a sample population producing a fold change ranging between 1.3 and 1.5 with a maximum range of 0.35 when compared to the 2-copy calibrators.

[0202] Genomic DNA was isolated from COR23134 soybean leaf tissue for 58 and 81 plants from each of the T2 and T3 generations, respectively. The DNA samples were extracted using a high alkaline buffer comprised of sodium hydroxide, ethylenediaminetetraacetic acid disodium salt dihydrate (Na2-EDTA), and Tris hydrochloride. Approximately 3 ng of template DNA were used per reaction.

[0203] Each assay supporting event COR-23134-4, as well as the transgenes contained within event COR-23134-4 soybean, were multiplexed with the Le1 endogenous reference assay. Reaction mixes were prepared, each comprised of all components to support both the gene of interest and the endogenous gene for the PCR reaction. The base master mix, Taqman SureAmp™Lo-ROX master mix, was used. Individual concentrations of primer varied per assay between 300 nM and 900 nM, dependent on the optimal concentration established for the specific target and the endogenous reference during analysis validation. Individual concentrations of probe per assay were either 80 nM or 120 nM. Assay controls included no template controls (NTC) which consisted of molecular biology grade water or Tris-EDTA (TE) buffer (10 mM Tris pH 8.0, 1mM EDTA) as well as copy number calibrator and negative controls, all of which were validated for each assay performed. Annealing temperatures and number of cycles used during the PCR analyses are provided in Tables 2 and 3. The primer and probes used for each PCR analysis are provided in Tables 4 and 5. Master mix formulations for each PCR analysis are provided in Tables 6-10. PCR Parameters

[0204] The PCR parameters used during PCR analysis are listed below:ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 2. Annealing Temperatures and Cycles used During the PCR Reaction (COR- 23134-4 and ipd083Cb)Table 3. Annealing Temperatures and Cycles used During the PCR Reaction (gm-hra, cry1B.34, and cry1B.61)Primers and Probes

[0205] The primers and probe used for each transgene are listed in Table 4. The primers and probe used for the Le1 endogenous reference gene are listed in Table 5. Table 4. Primers and Probe for PCR Analysis of the COR-23134-4 Soybean Event and gm-hra, cry1B.34, cry1B.61, and ipd083Cb GenesATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1Table 5. Primers and Probe for PCR Analysis of the Le1 Endogenous Reference GenePreparation of Master Mix

[0206] The components and concentrations supporting each master mix are listed below: Table 6. Master Mix Supporting Multiplex Assay: COR-23134-4 and Le1a Final volume of each reaction was 6 µL comprised of 5.0 µL of Master Mix and 1.0 µL of genomic DNA template. b N_A is equivalent to Not Applicable.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 7. Master Mix Supporting Multiplex Assay: gm-hra and Le1aFinal volume of each reaction was 6 µL comprised of 5.0 µL of Master Mix and 1.0 µL of genomic DNA template.bN_A is equivalent to Not Applicable. Table 8. Master Mix Supporting Multiplex Assay: cry1B.34 and Le1aFinal volume of each reaction was 6 µL comprised of 5.0 µL of Master Mix and 1.0 µL of genomic DNA template.bN_A is equivalent to Not Applicable.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 9. Master Mix Supporting Multiplex Assay: cry1B.61 and Le1a Final volume of each reaction was 6 µL comprised of 5.0 µL of Master Mix and 1.0 µL of genomic DNA template. b N_A is equivalent to Not Applicable. Table 10. Master Mix Supporting Multiplex Assay: ipd083Cb and Le1a Final volume of each reaction was 6 µL comprised of 5.0 µL of Master Mix and 1.0 µL of genomic DNA template. b N_A is equivalent to Not Applicable. PCR Analysis

[0207] Genomic DNA samples isolated from collected leaf samples of 151 COR23134 soybean plants (582plants from the T2 and 813plants from the T3 generations) along with copy number calibrator, negative, and NTC controls, were subjected to qPCR amplification using TaqMan SureAmp™Lo-ROX master mix (ThermoFisher; California, USA) in theATTORNEY DOCKET #: 108747-WO-SEC-1 presence of primer pair and probes specific for genes gm-hra, cry1B.34, cry1B.61, and ipd083Cb as well as the COR-23134-4 event which allowed for the unique identification of the PHP90315 T-DNA insertion in COR23134 soybean. For assay and DNA quality monitoring, soybean Le1 was included in duplex with each reaction as an endogenous control. Each qPCR reaction was set up in a total volume of 6 µL with approximately 3 ng (1.0 µL of volume) of the isolated genomic DNA. Copy number results for the T2 and T3 generations tested are provided in Table 11. Table 11. Copy Number Determination of Event COR-23134-4 and the gm-hra, cry1B.34, cry1B.61 and ipd083Cb Genes in Two Generations of COR23134 Soybean y ber l l3l l l l l3l llATTORNEY DOCKET #: 108747-WO-SEC-1 1 6 plants were omitted from the T2 generational data due to ambiguous copy number calls. 2 6 plants were omitted from the T3 generational data due to ambiguous copy number calls. 3 Although some late and non-linear amplification was detected in the Null scoring samples, the calculated fold change using both the Calibrator 1 and Calibrator 2 genomic controls was outside of the maximum range for a 1 or 2 copy number assignment resulting in Null final scores. Event-Specific and Construct-Specific PCR Analyses for COR23134 Soybean

[0208] The results of the qPCR copy number analyses indicate stable integration and segregation of a single copy of the genes within the T-DNA of plasmid PHP90315, with demonstrated transfer to subsequent generations.

[0209] PCR products ranging in size between 57 bp to 109 bp, representing event COR- 23134-4 as well as the genes within the T-DNA from plasmid PHP90315, were amplified and observed in leaf samples of COR23134 soybean as well as in eight copy number 1 calibrator genomic controls and eight copy number 2 genomic controls but were absent in each of the four negative genomic controls and four NTC controls.

[0210] Using the soybean endogenous reference gene Le1, a PCR product of 66 bp was amplified and observed in leaf samples from COR23134 soybean as well as in eight copy number 1 calibrator, eight copy number 2 calibrator, and four negative genomic controls. Amplification of the endogenous gene was not observed in the four NTC controls tested with no generation of CT values.

[0211] For each sample, all assays were performed in duplex (specific target with endogenous reference gene), analyzing for the insertion site and all genes. For each sample, CT values, ΔCT values, and copy numbers (if applicable) were calculated. Sensitivity of Construct-Specific PCR Analyses for COR23134 Soybean

[0212] To assess the sensitivity of the construct-specific PCR assays, COR23134 soybean DNA was diluted in control soybean genomic DNA, resulting in test samples containing various amounts of COR23134 soybean (5 ng, 1 ng, 500 pg, 250 pg, 100 pg, 50 pg, 20 pg, 10 pg, 5 pg, and 0 pg) in a total of 5 ng soybean DNA. These various amounts of COR23134 soybean DNA correspond to 100%, 20%, 10%, 5%, 2%, 1%, 0.4%, 0.2%, 0.1%, and 0% of COR23134 soybean DNA in total soybean genomic DNA, respectively. TheATTORNEY DOCKET #: 108747-WO-SEC-1 various amounts of COR23134 soybean DNA were subjected to real-time PCR amplification for gm-hra, cry1B.34, cry1B.61, and ipd083Cb genes and the COR-23134-4 soybean event. Based on these analyses, the limit of detection (LOD) in 5 ng of total DNA for COR23134 soybean was determined to be approximately 5 pg for gm-hra (0.1%), 20 pg for cry1B.34 (0.4%), 10 pg for cry1B.61 (0.2%), 5 pg for ipd083Cb (0.1%), and 10 pg for COR23134 soybean (0.2%). The determined sensitivity of each assay described is sufficient for many screening applications. Each concentration was tested a total of five times. At the point where amplification of the target tested was not detected in each replicate, the preceding concentration was determined to be the limit of sensitivity.

[0213] Real-time PCR analyses of COR23134 soybean DNA using event-specific and construct-specific assays confirm the stable integration and segregation of a single copy of the T-DNA of plasmid PHP90315 in leaf samples tested, as demonstrated by the quantified detection of event COR-23134-4 and the gm-hra, cry1B.34, cry1B.61, and ipd083Cb genes in COR23134 soybean plants. These results were reproducible among all the replicate qPCR analyses conducted. The soybean endogenous reference gene assay for detection of Le1 amplified as expected in all the test samples and negative controls and was not detected in the NTC samples. The sensitivity of each assay under the conditions described ranged between 5 pg to 20 pg DNA, all sufficient for many screening applications by PCR. Example 4. Southern-by-Sequencing (SbS) Analysis of COR-23134-4 soybean for the Insertion Organization and Copy Number

[0214] An application of Next-generation sequencing (NGS) called Southern‑by‑Sequencing (SbS) analysis was conducted on the T0 generation of COR- 23134-4 soybean to demonstrate that a single insertion has occurred in soybean event COR- 23134-4..

[0215] Genomic DNA was extracted from leaf tissue of the T0 generation of COR-23134-4 soybean. SbS utilizes probe-based sequence capture, Next Generation Sequencing (NGS) techniques, and bioinformatics procedures to isolate, sequence, and identify inserted DNA within the soybean genome. By compiling a large number of unique sequencing reads and comparing them to the transformation plasmid sequence and the soybean genome, unique junctions resulting from inserted DNA are identified in the bioinformatics analysis and can be used to determine the number of insertions within the plant genome. A single insertion will have two genomic-insertion junctions, one at each end of the insertion. The T0 plant ofATTORNEY DOCKET #: 108747-WO-SEC-1 COR-23134-4 soybean was analyzed by SbS to determine the copy number and integrity of the insertion.

[0216] A series of unique sequences encompassing the PHP90315 plasmid sequence was used to design overlapping biotinylated oligonucleotides as capture probes. The capture probes were designed and synthesized by Roche NimbleGen, Inc. Sequences in the COR- 23134-4 genome that match the PHP90315 transformation plasmid, either from the T-DNA insertion or endogenous soybean sequences similar to elements in PHP90315, would be enriched by hybridization to the probes during the capture process. The probes were compared to the soybean genome to determine the level of soybean genomic sequence that would be captured and sequenced simultaneously with sequences derived from PHP90315.

[0217] Separate NGS libraries were constructed for COR-23134-4 soybean and control soybean. SbS was performed essentially as described in Zastrow-Hayes, et al (2015). The sequencing libraries were hybridized to the capture probes through two rounds of hybridization to enrich the targeted sequences. Following NGS (Illumina NextSeq), the sequencing reads entered the bioinformatics pipeline for trimming and quality assurance. Reads were aligned against the soybean genome and the T-DNA region from plasmid PHP90315 to determine the copy number and insertion organization derived from the T- DNA. Reads were also aligned to the full sequence of PHP90315 to determine if any additional plasmid sequences were incorporated into the genome. Reads that contained both genomic and plasmid sequence and reads that contained noncontiguous plasmid sequence were identified as junction reads.

[0218] To identify putative junctions that were due to endogenous soybean sequences, a control soybean genomic DNA library was separately captured and sequenced in the same manner as the T0 COR-23134-4 soybean plant. This library was sequenced to approximately the same average depth as the COR-23134-4 soybean plant sample. This increased the probability that the endogenous junctions captured by the probes would be detected in the control sample, so that they could be identified and removed in the COR- 23134-4 soybean sample.

[0219] Copy number of the DNA insertion in COR-23134-4 soybean, derived from plasmid PHP90315 (Figure 1), was determined. A schematic map of the PHP90315 T-DNA is provided in Figure 2.

[0220] SbS was conducted on the T0 plant of COR-23134-4 soybean to determine the copy number and the insertion organization in the genome. Alignments of the SbS sequencing reads to the T-DNA from PHP90315 resulted in two unique plasmid-genome junctionsATTORNEY DOCKET #: 108747-WO-SEC-1 between the flanking genomic sequence and the inserted DNA, indicating the presence of a single insertion in COR-23134-4 soybean. A single plasmid-plasmid junction was identified within the insertion, indicating that there is a 21-bp deletion in the pv-ubi2 promoter of the ipd083Cb gene cassette that differs from the PHP90315 T-DNA sequence. There were no additional junctions between the PHP90315 sequence and the soybean genome detected in the T0 plant, indicating that there are no additional plasmid-derived insertions present in COR-23134-4 soybean. Furthermore, there were no junctions between soybean genome sequences and the backbone sequence of PHP90315 in the T0 plant, demonstrating that no plasmid backbone sequences were incorporated into COR-23134-4 soybean. Example 5. Insect efficacy of soybean events COR-23134-4

[0221] Soybean (Glycine max [L.] Merr.) event COR-23134-4 (referred to as COR23134 soybean) expresses the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins for control of certain susceptible lepidopteran pests, and the GM-HRA protein that was used as a selectable marker.

[0222] Greenhouse experiments were conducted to evaluate efficacy of event COR-23134- 4 (containing insecticidal proteins Cry1B.34.1, Cry1B.61.1, and IPD083Cb) in Toledo, Brazil over 2 years. A separate experiment was conducted for each of the three Lepidopteran target species: velvetbean caterpillar (Anticarsia gemmatalis), soybean looper (Chrysodeixis includens), and fall armyworm (Spodoptera frugiperda).

[0223] The two treatments were COR23134 and wild type soybean, both in soybean variety 93Y21. The experiments were arranged in a randomized complete block design with three replications. Each plot consisted of 16 plants.

[0224] For all species, insects were manually infested at uniform rates to each plot when soybean plants reached the R2 growth stage. Visual ratings for percent defoliation (0-100%) were assessed on each plot approximately 14 days after initial feeding was observed. Statistical analysis was conducted using linear mixed models to evaluate percent defoliation results for COR23134 soybean and wild type soybean. Table 12. Lepidopteran Target SpeciesATTORNEY DOCKET #: 108747-WO-SEC-1Statistical Analysis

[0225] A linear mixed model was applied to model percent defoliation for each experiment separately. Data for percent defoliation (Yipmns) of replication (R)i, protein (P)p, construct (C)m, event (E)n and plot s, were modeled as a function of an overall mean μ, factors for replication, construct, event, construct by event by and a residual εipmns. The model can be specified as: Yipmns = μ + Ri + Pp + (C × E)mn + εipmns where protein was treated as fixed effect, and all the other effects except the residual were treated as independent normally distributed random variables with means of zero.

[0226] For the residual, instead of assuming independence among plots, 2-dimensional separable first-order autoregressive correlation (AR1 X AR1) structure was applied to capture plot-to-plot correlations in both row and column directions of the field, besides the plot-to-plot variation. T-tests using standard errors from the model were conducted to compare treatment effects. A difference was considered statistically significant if the P- value of the difference was less than 0.05. All data analysis and comparisons were made in ASReml 4.0 (VSN International, Hemel Hempstead, UK, 2009). RESULTS

[0227] Mean percent defoliation injury results are summarized below for velvetbean caterpillar (Table 13), soybean looper (Table 14), and fall armyworm (Table 15) as means, standard errors, and P-values. In all experiments, percent defoliation injury was significantly higher in the wild type soybean with ≥ 70% defoliation compared to the COR23134 soybean which was ≤ 4.7%. Results from these studies demonstrate that eventATTORNEY DOCKET #: 108747-WO-SEC-1 COR-23134-4 provides protection from velvetbean caterpillar, soybean looper, and fall armyworm. Table 13. Insect Efficacy Results for COR23134 Soybean: Percent Defoliation Injury from Velvetbean Caterpillar* A statistically significant difference (P-value < 0.05) was observed. Table 14. Insect Efficacy Results for COR23134 Soybean: Percent Defoliation Injury from Soybean Looper* A statistically significant difference (P-value < 0.05) was observed. Table 15. Insect Efficacy Results for COR23134 Soybean: Percent Defoliation Injury from Fall ArmywormNote: Means were estimated from the linear mixed model. * A statistically significant difference (P-value < 0.05) was observed.ATTORNEY DOCKET #: 108747-WO-SEC-1 Example 6. Agronomic and yield field evaluations of soybean events COR-23134-4 Agronomic Characteristics of a Soybean Line Containing Event COR-23134-4

[0228] Soybean (Glycine max [L.] Merr.) event COR-23134-4 (referred to as COR23134 soybean) expresses the Cry1B.34.1, Cry1B.61.1, and IPD083Cb proteins for control of certain lepidopteran pests and the GM-HRA protein that was used as a selectable marker.

[0229] The objective of this study phase was to evaluate agronomic characteristics of COR23134 soybean - both for untreated COR23134 soybean or for COR23134 soybean treated with diclosulam (referred to as diclosulam-treated COR23134 soybean).

[0230] The field portion of this study was conducted during the 2022 growing season at 12 sites in commercial soybean-growing regions of the United States (two sites in each of Iowa, Illinois, and Indiana, and one site in each of Missouri, Nebraska, Pennsylvania, and Wisconsin) and Canada (two sites in Ontario). A randomized complete block design with four blocks was utilized at each site. Each block included untreated COR23134 soybean, diclosulam-treated COR23134 soybean, non-genetically modified (non-GM) near-isoline control soybean (referred to as control soybean), and four out of eighteen non-GM commercial soybean lines (referred to as reference soybean).

[0231] Data were collected for the following agronomic endpoints: early stand count, days to flowering, plant height, days to maturity, lodging, shattering, final stand count, pod count, yield, harvest seed moisture, and 100-seed weight. Statistical analyses were conducted to evaluate and compare agronomic results derived from COR23134 soybean to the control soybean.

[0232] The results obtained in this study phase demonstrated that agronomic characteristics of COR23134 soybean were comparable to those of conventional soybean represented by non-GM near-isoline control soybean and non-GM commercial soybean. For untreated COR23134 soybean, a statistically significant difference was identified in pod count, with 46 of 48 observations within the reference range. For diclosulam-treated COR23134 soybean, a statistically significant difference was identified in early stand count, with 47 of 48 observations within the reference range. MATERIALS

[0233] The test system in this study was soybean (Glycine max [L.] Merr.). The test substance consisted of event COR-23134-4 contained within soybean seed. The study included a non-genetically modified (non-GM) near-isoline soybean line (referred to as control soybean), which did not contain event COR-23134-4. Additionally, a total of 18ATTORNEY DOCKET #: 108747-WO-SEC-1 non-GM commercial soybean lines (referred to as reference soybean) were included in the study. METHODS Experimental Design

[0234] The field portion of this study was conducted during the 2022 growing season at 12 sites in commercial soybean-growing regions of the United States (two sites in each of Iowa, Illinois, and Indiana, and one site in each of Missouri, Nebraska, Pennsylvania, and Wisconsin) and Canada (two sites in Ontario). A randomized complete block design with four blocks was utilized at each site. Each block included untreated COR23134 soybean, diclosulam-treated COR23134 soybean, non-genetically modified (non-GM) near-isoline control soybean (referred to as control soybean), and four of the following non-GM commercial soybean lines (referred to as reference soybean): 92M35, 92B63, 92M72, BK291, P29T50, BK310, BK317, BK331N, P33T60, BK340, 93Y41, P34A50, P35A41, BK360, BK361, 93M62, BK370, and 93B82 soybean.

[0235] Bias in the generation of agronomic data in this study was controlled by randomization of the entries within each block and uniform maintenance treatments across all plots at each site. Field Trial Planting

[0236] Each block contained COR23134 soybean, control soybean, and four reference soybean lines planted in 4-row plots at a rate of approximately 168 seeds per row. Each row was approximately 7.6 m (25 ft) in length and approximately 76 cm (30 in.) in width at all sites, with the exception of site IN3, where row length was 6.1 m (20 ft). Each block was separated by an alley of at least 0.9 m (36 in.) in width, and each plot was bordered on either side by one row of soybeans. Maintenance Product Applications

[0237] At a given site, maintenance products were uniformly applied, as needed, to all plots to minimize weed, insect, and disease pressure. Glyphosate and glufosinate herbicides were not used post emergence as maintenance applications in this study. Diclosulam, quizalofop, and fomesafen herbicides and insecticides containing Bacillus thuringiensis (Bt) were not used as maintenance applications in this study.ATTORNEY DOCKET #: 108747-WO-SEC-1 Herbicide Treatment Untreated COR23134 soybean:

[0238] This COR23134 soybean entry is referred to as “untreated” because it was only treated with herbicides labelled for use in conventional soybean and was not treated with diclosulam. The untreated COR23134 soybean plots, as well as all control soybean and reference soybean plots in each block at all field sites were treated with the herbicides quizalofop and fomesafen at the V3-V4 growth stage. Herbicide applications were applied at the target rates at all sites, with the exception of site IL5, where fomesafen was applied at 115% of the target rate.

[0239] A visual evaluation of the plants was completed 12-17 days after each treatment to confirm that no unexpected herbicide injury was observed. As expected, minor foliar leaf burn from the fomesafen application was observed at some sites resulting in no negative impact to plant growth and development. Diclosulam-treated soybean study:

[0240] While a quizalofop and fomesafen herbicide treatment was applied to the untreated COR23134 soybean plot and all control and reference soybean plots at the V3-V4 growth stage, the herbicide diclosulam was applied at the V3-V4 growth stage to the diclosulam- treated plot of COR23134 soybean in each block. Herbicide applications were applied at the target rates at all sites, with the exception of site IL5, where fomesafen was applied at 115% of the target rate.

[0241] A visual evaluation of the plants was completed 12-17 days after each treatment to confirm that no unexpected herbicide injury was observed. As expected, minor foliar leaf burn from the fomesafen application was observed at six of the 12 sites resulting in no negative impact to plant growth and development. Very minor herbicide injury (leaf cupping or chlorosis) from the diclosulam application was observed in plots of herbicide- treated COR23134 soybean at two of the 12 sites resulting in no negative impact to plant growth and development. Agronomic Characteristics Data Collection

[0242] Agronomic characteristics were evaluated at given soybean growth stages for each plot. The following characteristics were evaluated: Early Stand CountATTORNEY DOCKET #: 108747-WO-SEC-1

[0243] The total number of plants emerged in each of three marked sections (each one row by one meter) in Rows 1-2 was determined between the VC and V2 growth stages. Additional calculations are provided in the statistical analysis section below. Days to Flowering

[0244] The date when at least one flower was open for at least 50% of the plants in Rows 1- 2 was recorded. These dates were used in subsequent statistical analysis to calculate days to flowering in the statistical analysis section below. Plant Height

[0245] Plant height was measured in centimeters from the soil surface to the uppermost node on the main stem at the R8 growth stage for five individual plants in Rows 1-2. Additional calculations are provided in the statistical analysis section below. Days to Maturity

[0246] The date when 95% of the pods in Rows 1-2 were at the physiological mature color (R8 growth stage) was recorded. These dates were used in subsequent statistical analysis to calculate days to maturity in the statistical analysis section below. Lodging

[0247] Lodging was recorded as the percentage of plants in Rows 1-2, to the nearest 10%, inclined more than 45° from vertical at the R8 growth stage. Pod Count Untreated COR23134 soybean:

[0248] The number of pods (containing seed) per plant from five plants in Rows 1-2 was recorded at the R8 growth stage. Additional calculations are provided in the statistical analysis section below. The same five plants were utilized for pod count and shattering data collection. Diclosulam-treated soybean:

[0249] The number of pods (containing seed) per plant from five plants in Rows 1-2 was recorded at the R8 growth stage. Due to an error in data collection, pod count data was not collected for one plot of diclosulam-treated COR23134 soybean at site MO2. Additional calculations are provided in the statistical analysis section below. The same five plants were utilized for pod count and shattering data collection. ShatteringATTORNEY DOCKET #: 108747-WO-SEC-1

[0250] The number of shattered pods per plant from five plants in Rows 1-2 was recorded at the R8 growth stage. Additional calculations are provided in the statistical analysis section below. Final Stand Count

[0251] The total number of plants emerged in each of three marked sections (each one row by one meter) in Rows 1-2 was determined at the R8 growth stage. Each section was previously used to evaluate early stand count. Additional calculations are provided in the statistical analysis section below. Yield Untreated COR23134 soybean:

[0252] The seed from all plants in Rows 1 and 2 of each plot was harvested at the R8 growth stage. The weight of the seed was recorded in kilograms. Using the harvest seed moisture, seed weight values from all sites were adjusted to a standardized moisture content and used to calculate yield during subsequent statistical analysis. Rows 1 and 2 of one plot of COR23134 soybean at site ON3 were adjusted to a length of 5.6 m (18 ft) for yield calculations due to missing plants from a fertilizer spill. Diclosulam-treated soybean:

[0253] The seed from all plants in Rows 1 and 2 of each plot was harvested at the R8 growth stage. The weight of the seed was recorded in kilograms. Using the harvest seed moisture, seed weight values from all sites were adjusted to a standardized moisture content and used to calculate yield during subsequent statistical analysis. Harvest Seed Moisture

[0254] The moisture content (%) of harvested seed from Rows 1 and 2 at the R8 growth stage was recorded. 100-Seed Weight

[0255] The weight (g) of 100 seeds from the seed harvested from Rows 1 and 2 at the R8 growth stage was recorded. 100-seed weight data were collected on the same day as harvest seed moisture data collection at all sites, with the exception of site WI1, where 100-seed weight data were collected one day after harvest seed moisture. 100-seed weight values were adjusted to a standardized moisture content (see statistical analysis section below). Statistical AnalysisATTORNEY DOCKET #: 108747-WO-SEC-1

[0256] Statistical analyses were conducted to evaluate and compare agronomic characteristics of untreated COR23134 soybean and diclosulam-treated COR23134 soybean to the control soybean. Processing of Data Early Stand Count and Final Stand Count

[0257] For early stand count and final stand count, the recorded count values for three separate one meter row lengths were each divided by the count area to calculate the number of plants per square meter. The calculated values were then used to calculate the plot average. Days to Flowering and Days to Maturity

[0258] For days to flowering data, the number of days was calculated from the recorded planting date to the recorded flowering date. For days to maturity data, the number of days was calculated from the recorded planting date to the recorded maturity date. Plant Height, Pod Count, and Shattering

[0259] For plant height, pod count, and shattering data, the recorded values for five individual plants were used to calculate the plot average. Yield

[0260] Yield was determined based on the weight of seed collected at typical harvest maturity as follows: Seed weight was adjusted to 0% moisture content (seed dry weight): Seed dry weight (lb) = Seed fresh weight (lb) ^ [(100 - % actual moisture) / 100] Seed dry weight was then adjusted to 13% moisture content: Seed weight at 13% moisture (lb) = Seed dry weight (lb) / ((100 – 13% moisture) / 100) Seed weight at 13% moisture was then converted to a yield in bushels per acre (bu / A): (Seed weight (lb) at 13% moisture) ^ (43,560 Yield (bu / A at 13% = ft2 / A) moisture) (plot area (ft2)) ^ (60 lb / bu)

[0261] If applicable, plot dimensions recorded in meters were converted to feet and the weight unit of seed was converted to pounds prior to yield calculations.ATTORNEY DOCKET #: 108747-WO-SEC-1 100-Seed Weight

[0262] 100-seed weight for each plot was determined as follows: Weight of 100 seeds was adjusted to 0% moisture content (100-seed dry weight): 100-seed dry weight (g) = 100-seed fresh weight (g) ^ [(100 - % actual moisture) / 100] 100-seed dry weight was then adjusted to 13% moisture content: 100-seed weight at 13% moisture (g) = 100-seed dry weight (g) / ((100 – 13% moisture) / 100) Selection of Statistical Method

[0263] The following rules were implemented for each agronomic characteristic: If < 50% of sites had uniform data values for either COR23134 soybean or the control soybean, and < 50% of all data across sites for each entry were at a uniform value, then an across-site mixed model analysis would be conducted. In addition, if both soybean lines had at least two data points at a given site that were not at a uniform value, then an individual-site mixed model analysis would be conducted. If ≥ 50% of sites had uniform data values across both soybean lines, then statistical analyses would not be performed.

[0264] If the criteria described above were not met, then an across-site analysis using the generalized Cochran-Mantel-Haenszel (CMH) test would be conducted. Individual-site analyses would not be performed. Across-Site Analysis Mixed Model Analysis

[0265] For a given agronomic characteristic, data were analyzed using the following linear mixed model: Model 1 yijk= μi+ ℓj+ rk(j)+ (μℓ)ij+ εijkℓj ~ iid N(0, σ2Site), rk(j) ~ iid N(0, σ2Rep), (μℓ)ij ~ iid N(0, σ2Ent×Site), and εijk ~ iid N(0, σ2Error), where μi denotes the mean of the ithentry (fixed effect), ℓj denotes the effect of the jthsite (random effect), rk(j) denotes the effect of the kthblock within the jthsite (random effect),ATTORNEY DOCKET #: 108747-WO-SEC-1 (μℓ)ij denotes the interaction between the entries and sites (random effect), and εijk denotes the effect of the plot assigned the ithentry in the kthblock of the jthsite (random effect or residual). Notation ~ iid N(0, σ2a) indicates random variables that are identically independently distributed (iid) as normal with zero mean and variance σ2a. Subscript a represents the corresponding source of variation.

[0266] The residual maximum likelihood estimation procedure was utilized to generate estimates of variance components and entry means across sites. The estimated means are known as empirical best linear unbiased estimators (hereafter referred to as LS-Means). The statistical comparison was conducted by testing for a difference in LS-Means between COR23134 soybean and the control soybean. The approximated degrees of freedom for the statistical test were derived using the Kenward-Roger method (Kenward and Roger, 2009). A significant difference was identified if a P-value was < 0.05.

[0267] For each agronomic characteristic, goodness-of-fit of the model was assessed in terms of meeting distributional assumptions of normally, independently distributed errors with homogeneous variance. Deviations from assumptions were addressed using an appropriate transformation or allowing for heterogeneous error variance among sites. Generalized CMH Test

[0268] The generalized CMH test is more appropriate in the instance where the normality assumption of mixed model analysis cannot be achieved for discrete data. The test was developed specifically for stratified nominal-by-ordinal contingency tables (Agresti, 2002; Koch et al., 1990). It compares entries (a nominal variable) based on their values (recorded on an ordinal scale) while controlling for location (the stratifying variable). Due to the data values being used as the scores in the generalized CMH test, the test’s P-value can be directly interpreted as testing for the difference between the arithmetic means of two entries. A significant difference was identified if a P-value was < 0.05. Individual-Site Analyses

[0269] For a given agronomic characteristic, individual sites were analyzed separately using the following linear mixed model: yik = μi + rk + εik Model 2ATTORNEY DOCKET #: 108747-WO-SEC-1 rk ~ iid N(0,σ2Rep) and εik ~ iid N(0, σ2Error), where μi denotes the mean of the ithentry (fixed effect), rk denotes the effect of the kthblock (random effect), and εik denotes the residual for the observation obtained from the plot assigned to the ithentry in the kthblock.

[0270] The residual maximum likelihood estimation procedure was used to generate estimates of variance components and entry means (LS-Means). The statistical comparison was conducted by testing for difference in LS-Means between diclosulam-treated or untreated COR23134 soybean and the control soybean. The approximated degrees of freedom for the statistical test were derived using the Kenward-Roger method. False Discovery Rate Adjustment

[0271] The false discovery rate (FDR) method (Benjamini and Hochberg, 1995; Westfall et al., 1999) was used to control for false positive outcomes across all agronomic characteristics analyzed using linear mixed models or generalized CMH tests. A false positive outcome occurs if the difference in means between two entries is declared significant, when in fact the two means are not different. Since the introduction of the FDR approach in the mid-1990s, it has been widely employed across a number of scientific disciplines, including genomics, ecology, medicine, plant breeding, epidemiology, dairy science, and signal / image processing (e.g., Pawitan et al., 2005; Spelman and Bovenhuis, 1998). In the FDR method, the false discovery rate is held at 5% across comparisons of multiple agronomic characteristics via an adjustment to the P-value and is not inflated by the number of agronomic characteristics in the comparison. The FDR adjustment of raw P- values was conducted separately for the across-site analysis and each of the individual-site analyses. Statistical Software and Procedures

[0272] Statistical analyses were conducted using SAS software, Version 9.4 (SAS Institute Inc.). SAS PROC MIXED was utilized to fit Models 1 and 2, and to provide LS-Means, 95% confidence intervals, and statistical comparisons. SAS PROC FREQ was used to perform the generalized CMH test. SAS PROC MULTTEST was utilized to provide FDR adjusted P-values. All other data processing was conducted in Base SAS.ATTORNEY DOCKET #: 108747-WO-SEC-1 Interpretation of Statistical Results

[0273] For a given agronomic characteristic, when a statistically significant difference (P- value < 0.05) was identified in the across-site analysis, the respective range of individual values from diclosulam-treated or untreated COR23134 soybean was compared to the in- study reference range comprised of all individual values across-sites from all non-GM reference soybean lines included in this study. In cases when a raw P-value indicated a significant difference but the FDR adjusted P-value was > 0.05, it was concluded that the difference was likely a false positive. In addition, for agronomic characteristics exhibiting a statistically significant difference (P-value < 0.05) in the across-site analysis, the results for individual sites were evaluated. Reported Statistics

[0274] For agronomic characteristics examined using mixed model analysis, the following statistical results were reported: LS-Means, ranges, 95% confidence intervals, FDR- adjusted P-values, and non-adjusted P-values. For agronomic characteristics examined using CMH test, the following statistical results were reported: arithmetic means, ranges, FDR-adjusted P-values, and non-adjusted P-values. For agronomic characteristics which were not statistically analyzed, arithmetic means and ranges were reported. Additionally, the in-study reference range was provided for all agronomic characteristics.

[0275] Note: The lower and / or upper confidence limits might occasionally fall outside the measurement scale (e.g., 1-9) for an agronomic characteristic. In this case, the lower and / or upper limit of the measurement scale was used as the corresponding confidence limit for reporting purposes. Results of Agronomic Characteristic Evaluation untreated COR23134 soybean study:

[0276] A total of 11 agronomic endpoints were included in the assessment: nine were evaluated using mixed model analysis and one was evaluated using the generalized CMH test. The remaining agronomic endpoint (shattering) did not meet criteria for minimum levels of non-uniformity and was therefore not subjected to comparative analyses.

[0277] No statistically significant differences were identified between COR23134 soybean and the control soybean for six agronomic endpoints that went through across-site analysis.ATTORNEY DOCKET #: 108747-WO-SEC-1

[0278] A statistically significant difference, before FDR-adjustment, between COR23134 soybean and the control soybean was observed in the across-site analysis for harvest seed moisture. The non-significant FDR-adjusted P-value indicates that this difference was likely a false positive. For harvest seed moisture, 47 of 48 values (one value below the lower reference range) for COR23134 soybean were within the reference data range. No statistically significant difference was observed in the individual-site analyses.

[0279] A statistically significant difference, before and after FDR-adjustment, between COR23134 soybean and the control soybean was observed in the across-site analysis for plant height, 100-seed weight, and pod count. For plant height and 100-seed weight, all values for COR23134 soybean were within the reference data range. Additionally, a statistically significant difference was observed in the individual-site analyses at seven and six sites, respectively. For pod count, 46 of 48 values (one value below the lower reference range and one value above the upper reference range) for COR23134 soybean were within the reference data range. Additionally, a statistically significant difference was observed in the individual-site analyses at four sites. Diclosulam-treated soybean:

[0280] A total of 11 agronomic endpoints were included in the assessment: eight were evaluated using mixed model analysis and two were evaluated using the generalized CMH test. The remaining agronomic endpoint (shattering) did not meet criteria for minimum levels of non-uniformity and was therefore not subjected to comparative analyses.

[0281] No statistically significant differences were identified between diclosulam-treated COR23134 soybean and the control soybean for five agronomic endpoints that went through across-site analysis.

[0282] A statistically significant difference, before FDR-adjustment, between diclosulam-treated COR23134 soybean and the control soybean was observed in the across- site analysis for final stand count. The non-significant FDR-adjusted P-value indicates that this difference was likely a false positive. For final stand count, 47 of 48 values (one value above the upper reference range) for diclosulam-treated COR23134 soybean were within the reference data range. No statistically significant difference was observed in the individual-site analyses.

[0283] A statistically significant difference, before and after FDR-adjustment, between diclosulam-treated COR23134 soybean and the control soybean was observed in the across- site analysis for early stand count, plant height, pod count, and 100-seed weight. For earlyATTORNEY DOCKET #: 108747-WO-SEC-1 stand count, 47 of 48 values (one value above the upper reference range) for diclosulam-treated COR23134 soybean were within the reference data range. Additionally, a statistically significant difference was observed in the individual-site analyses at one site. For plant height, pod count, and 100-seed weight, all values for diclosulam-treated COR23134 soybean were within the reference data range. Additionally, a statistically significant difference was observed in the individual-site analyses at five sites for each endpoint. Across-Site Agronomic Characteristics Results

[0284] The results for the across-site analysis of agronomic characteristics are provided in Tables 16 and 17. CONCLUSION

[0285] The results obtained in this study demonstrated that agronomic characteristics of diclosulam-treated or untreated COR23134 soybean were comparable to those of conventional soybean represented by non-GM near-isoline control soybean and non-GM commercial soybean. For untreated COR23134 soybean, a statistically significant difference was identified in pod count, with 46 of 48 observations within the reference range. For diclosulam-treated soybean, a statistically significant difference was identified in early stand count, with 47 of 48 observations within the reference range.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 16. Across-Site Analysis of Agronomic Characteristics Results Agronomic Reported Control Untreated teristic Statistics Soybean COR231 Reference Data Charac 34 Soybean Range Mean 27.9 27.3 Range 21.0 - 42.0 21.4 - 42.9 Early Stand Count Confidence 25.2 - 30.6 24.6 - 30.0 2(count / m ) Interval Adjusted P-Value -- 0.294 P-Value -- 0.176 Mean 96.4 90.1 Range 60.6 - 113.4 59.6 - 103.0 Plant Height (cm) Confidence 89.4 - 103.3 83.1 - 97.0 Interval 44.8 - 122.8 Adjusted P-Value -- 0.000432†P-Value -- <0.0001*Mean 44.1 52.6 Range 22 - 66 20 - 113 Pod Count (count) Confidence 36.3 - 52.0 44.7 - 60.4 Interval 24 - 99 Adjusted P-Value -- 0.0107†P-Value -- 0.00321*Mean 13.3 13.1 Range 0.0 - 100.0 0.0 - 100.0 Lodging (%) Confidence NA NA Interval 0.0 - 90.0 Adjusted P-Value -- 0.959 P-Value -- 0.955 Mean 0.3 0.2 Range 0 - 4 0 - 2 Shattering (count) Confidence NA NA Interval 0 - 3 Adjusted P-Value -- NA P-Value -- NA Mean 49.0 49.3 Range 39 - 60 39 - 62 Days to Flowering Confidence 44.7 - 53.3 45.0 - 53.6 ys) Interval 37(da - Adjusted P-Value -- 0.567 P-Value -- 0.397 Days to Maturity Mean 126.3 126.3 (days) Range 110 - 145 110 - 145ATTORNEY DOCKET #: 108747-WO-SEC-1 Agronomic Reported Control Untreated Soybean COR2 Reference Data Characteristic Statistics 3134 Soybean Range Confidence 119.4 - 133.1 119.4 - 133.1 Interval Adjusted P-Value -- 0.959 P-Value -- 0.949 Mean 26.0 25.9 Range 19.2 - 35.0 17.1 - 37.6 Final Stand Count Confidence 23.4 - 28.6 23.3 - 28.5 2(count / m ) Interval Adjusted P-Value -- 0.958 P-Value -- 0.958 Mean 10.5 10.3 Range 7.2 - 13.6 6.8 - 13.8 Harvest Seed Confidence 9.2 - 11.7 9.1 - 11.6Moisture (%) Interval Adjusted P-Value -- 0.0983 P-Value -- 0.0393*Mean 59.7 61.9 Range 26 - 92 30 - 87 Yield (bu / A) Confidence 50.2 - 69.2 52.4 - 71.4 Interval 17 - 99 Adjusted P-Value -- 0.228 P-Value -- 0.126 Mean 18.4 17.0 Range 11.4 - 22.5 10.9 - 21.8 100-Seed Weight (g) Confidence 17.1 - 19.8 15.6 - 18.3 Interval 9.5 - 24.2 Adjusted P-Value -- 0.000419†P-Value -- <0.0001*Note: Not Applicable (NA); mixed model analysis was not performed. * A statistically significant difference (P-value < 0.05) was observed. † Adjusted P-value < 0.05 was observed.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 17. Across-Site Analysis of Agronomic Characteristics Results Diclosulam- Agronomic Reported Control Treated Reference Data Characteristic Statistics Soybean COR23134 Range Soybean Mean 27.9 26.6 Range 21.0 - 42.0 18.9 - 40.2Mean 44.1 52.8 Range 22 - 66 32 - 88 Pod Count (count) Confidence 36.3 - 52.0 44.9 - 60.6 Interval 24 - 99 Adjusted P-Value -- 0.00665†P-Value -- 0.00266*Mean 13.3 13.4 Range 0.0 - 100.0 0.0 - 100.0 Lodging (%) Confidence NA NA Interval 0.0 - 90.0 Adjusted P-Value -- 0.976 P-Value -- 0.976 Mean 0.3 0.3 Range 0 - 4 0 - 1 Shattering (count) Confidence NA NA Interval 0 - 3 Adjusted P-Value -- NA P-Value -- NA Mean 49.0 48.9 Range 39 - 60 38 - 62 Days to Flowering Confidence 44.7 - 53.3 44.6 - 53.2(days) Interval Adjusted P-Value -- 0.950 P-Value -- 0.855 Mean 126.3 126.2 114 - 146ATTORNEY DOCKET #: 108747-WO-SEC-1 Diclosulam- Agronomic Reported Control Treated Reference Data Characteristic Statistics Soybean COR23134 Range Soybean Range 110 - 145 111 - 145 Days to Maturity Confidence 119.4 - 133.1 NA (days) Interval Adjusted P-Value -- 0.934 P-Value -- 0.747 Mean 26.0 25.1 Range 19.0 - 35.0 18.0 - 38.0 Final Stand Count Confidence 23.4 - 28.6 22.5 - 27.7 2(count / m ) Interval Adjusted P-Value -- 0.0623 P-Value -- 0.0311*Mean 26.0 25.1 Range 19.2 - 35.0 17.9 - 38.5 Final Stand Count Confidence 23.4 - 28.6 22.5 - 27.7 2(count / m ) Interval Adjusted P-Value -- 0.0606 P-Value -- 0.0303*Mean 10.5 10.4 Range 7.2 - 13.6 6.9 - 13.6 Harvest Seed Confidence 9.2 - 11.7 9.1 - 11.6Moisture (%) Interval Adjusted P-Value -- 0.254 P-Value -- 0.178 Mean 59.7 61.9 Range 26 - 92 29 - 84 Yield (bu / A) Confidence 50.2 - 69.2 52.4 - 71.4 Interval 17 - 99 Adjusted P-Value -- 0.210 P-Value -- 0.126 Mean 18.4 17.0 Range 11.4 - 22.5 11.2 - 22.3 100-Seed Weight (g) Confidence 17.1 - 19.8 15.7 - 18.4 Interval 9.5 - 24.2 Adjusted P-Value -- 0.000627†P-Value -- <0.0001*Note: Herbicide-treated refers to treatment with diclosulam. Not applicable (NA); mixed model analysis was not performed. * A statistically significant difference (P-value < 0.05) was observed. † Adjusted P-value < 0.05 was observed.ATTORNEY DOCKET #: 108747-WO-SEC-1 Example 7. Protein Expression and Concentration

[0286] COR-23134-4 soybean plants were grown during the 2022 growing season at six sites in commercial soybean-growing regions of the United States (one site in Iowa, Illinois, Indiana, Nebraska, and Pennsylvania) and Canada (one site in Ontario). A randomized complete block design with four blocks was utilized at each site. Each block included COR- 23134-4 soybean and herbicide-treated COR-23134-4 soybean.

[0287] The following samples were collected and processed for each entry: leaf (V5, R1, and R3 growth stages), flowers (R1-R2 growth stage), root (R3 growth stage), forage (R3 growth stage), and seed (R8 growth stage). Growth stage descriptions are provided in Table 18. Samples were analyzed for Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA protein concentrations using quantitative enzyme-linked immunosorbent assay (ELISA) methods.

[0288] Control of bias for expressed trait protein analysis was achieved through the use of replicate testing, appropriate assay controls, and pre-set data acceptability criteria. Sample Collection and Processing

[0289] Leaf (V5, R1, and R3 growth stages), flowers (R1-R2 growth stage), root (R3 growth stage), forage (R3 growth stage), and seed (R8 growth stage) samples were collected, lyophilized, homogenized, and stored frozen. Protein Concentration Determination

[0290] The concentrations of Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA proteins were determined using quantitative ELISA methods that have been internally validated to demonstrate method suitability. The number of samples analyzed is provided in Table 19. Protein Extraction

[0291] Processed tissue sub-samples were weighed at the following target weights: 5 mg for flowers, 10 mg for leaf and seed, and 20 mg for root and forage. Sub-samples were stored in a -20 ºC freezer unit until analysis.

[0292] Each sample analyzed for Cry1B.34.1, Cry1B.61.1, and IPD083Cb was extracted with 0.60 ml of chilled phosphate-buffered saline containing 0.05% polysorbate 20 (PBST). Each leaf, root, forage, and seed sample analyzed for GM-HRA was extracted with 0.60 ml of chilled buffer which was comprised of PBST with 0.2% CHAPS. Flower samplesATTORNEY DOCKET #: 108747-WO-SEC-1 analyzed for GM-HRA were extracted with 0.60 ml of chilled buffer comprised of PBST with 0.2% CHAPS and 5% StabilZyme Select. Extracted samples were centrifuged, and then supernatants were removed and prepared for analysis. Cry1B.34.1 Protein ELISA Method

[0293] Prior to analysis, samples were diluted as applicable in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with a monoclonal antibody. Following incubation, unbound substances were washed from the plate. A different monoclonal antibody conjugated to the enzyme horseradish peroxidase (HRP) was added to the plate and incubated. Unbound substances were washed from the plate. Detection of the bound Cry1B.34.1-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the optical density (OD) of each well was determined using a plate reader. Cry1B.61.1 Protein ELISA Method

[0294] Prior to analysis, samples were diluted as applicable in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with a monoclonal antibody. Following incubation, unbound substances were washed from the plate. A different monoclonal antibody conjugated to the enzyme HRP was added to the plate and incubated. Unbound substances were washed from the plate. Detection of the bound Cry1B.61.1-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader. IPD083Cb Protein ELISA Method

[0295] Prior to analysis, samples were diluted as applicable in PBST. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with a monoclonal antibody. Following incubation, unbound substances were washed from the plate. A different monoclonal antibody conjugated to the enzyme HRP was added to the plate and incubated. Unbound substances were washed from the plate. Detection of the bound IPD083Cb-antibody complex was accomplished by theATTORNEY DOCKET #: 108747-WO-SEC-1 addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader. GM-HRA Protein ELISA Method

[0296] Prior to analysis, samples were diluted as applicable in PBST containing 0.2% CHAPS and 5% StabilZyme Select. Standards (typically analyzed in triplicate wells) and diluted samples (typically analyzed in duplicate wells) were incubated in a plate pre-coated with a polyclonal antibody. Following incubation, unbound substances were washed from the plate and the bound GM-HRA protein was incubated with a monoclonal antibody, conjugated to the enzyme HRP. Unbound substances were washed from the plate. Detection of the bound GM-HRA-antibody complex was accomplished by the addition of substrate, which generated a colored product in the presence of HRP. The reaction was stopped with an acid solution and the OD of each well was determined using a plate reader. Calculations for Determining Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA Protein Concentrations

[0297] SoftMax Pro GxP Version 7.0.3 (Molecular Devices) microplate data software was used to perform the calculations required to convert the OD values obtained for each set of sample wells to a protein concentration value.

[0298] A standard curve was included on each ELISA plate. The equation for the standard curve was derived by the software, which used a quadratic fit to relate the OD values obtained for each set of standard wells to the respective standard concentration (ng / ml).

[0299] The sample concentration values were adjusted for a dilution factor expressed as 1:N by multiplying the interpolated concentration by N. Adjusted Concentration = Interpolated Sample Concentration x Dilution Factor

[0300] Adjusted sample concentration values obtained from SoftMax Pro GxP software were converted from ng / ml to ng / mg sample weight as follows: Sample Concentration Sample Extraction Buffer Volume (ng protein / mg sample = Concentration x (ml) weight) (ng / ml) Sample Target Weight (mg)ATTORNEY DOCKET #: 108747-WO-SEC-1

[0301] The reportable assay lower limit of quantification (LLOQ) in ng / ml was calculated as follows: Reportable Assay LLOQ (ng / ml) = (lowest standard concentration - 10%) x minimum dilution The LLOQ, in ng / mg sample weight, was calculated as follows: Extraction Buffer Volume LLOQ = Reportable Assay LLOQ x (ml)Sample Target Weight (mg) Trait Confirmation

[0302] To confirm sample identity, event-specific polymerase chain reaction (PCR) analyses were performed for samples with unexpected ELISA results. If a given test sample was confirmed as not containing the event of interest, the protein results were excluded from reporting. Statistical Analysis

[0303] Statistical analysis of the protein concentration results consisted of the calculations of means, ranges, and standard deviations. Individual sample results below the LLOQ were assigned a value equal to the LLOQ for calculation purposes. Results

[0304] The concentration results for the Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM- HRA proteins are provided in Tables 20, 21, 22, and 23, respectively. CONCLUSION

[0305] Cry1B.34.1, Cry1B.61.1, IPD083Cb, and GM-HRA protein concentration results for COR-23134-4 soybean and herbicide-treated COR-23134-4 soybean are summarized across sites as means, ranges, and standard deviations. Table 18. Soybean Growth Stage DescriptionsATTORNEY DOCKET #: 108747-WO-SEC-1Note: Growth stages (Pedersen, 2004). Table 19. Summary of Soybean Samples AnalyzedATTORNEY DOCKET #: 108747-WO-SEC-1Note: Growth stages (Pedersen, 2004). Herbicide-treated refers to treatment with diclosulam.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 20. Across-Site Summary of Expressed Trait Cry1B.34.1 Protein ConcentrationsNote: Growth stages (Pedersen, 2004). Herbicide-treated refers to treatment with diclosulam.aLower limit of quantification (LLOQ) in ng / mg tissue dry weight.bOne forage sample was confirmed negative for the event of interest by polymerase chain reaction (PCR) analysis.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 21. Across-Site Summary of Expressed Trait Cry1B.61.1 Protein ConcentrationsNote: Growth stages (Pedersen, 2004). Herbicide-treated refers to treatment with diclosulam. a Lower limit of quantification (LLOQ) in ng / mg tissue dry weight.bSome, but not all, sample results were below the LLOQ. A value equal to the LLOQ value was assigned to those samples to calculate the mean and standard deviation. c One forage sample was confirmed negative for the event of interest by polymerase chain reaction (PCR) analysis.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 22. Across-Site Summary of Expressed Trait IPD083Cb Protein ConcentrationsNote: Growth stages (Pedersen, 2004). Herbicide-treated refers to treatment with diclosulam.aLower limit of quantification (LLOQ) in ng / mg tissue dry weight.bOne forage sample was confirmed negative for the event of interest by polymerase chain reaction (PCR) analysis.ATTORNEY DOCKET #: 108747-WO-SEC-1 Table 23. Across-Site Summary of Expressed Trait GM-HRA Protein ConcentrationsNote: Growth stages (Pedersen, 2004). Herbicide-treated refers to treatment with diclosulam. a Lower limit of quantification (LLOQ) in ng / mg tissue dry weight.bSome, but not all, sample results were below the LLOQ. A value equal to the LLOQ value was assigned to those samples to calculate the mean and standard deviation. c One forage sample was confirmed negative for the event of interest by polymerase chain reaction (PCR) analysis.ATTORNEY DOCKET #: 108747-WO-SEC-1 Example 8. Sequence Characterization of Insert and Flanking Genomic Regions of COR-23134-4 Soybean

[0306] Sequence characterization analysis was performed to determine the DNA sequence of the COR23134 insert and flanking genomic regions.

[0307] The sequence of the insert and its flanking genomic regions was determined to confirm the integrity of the inserted DNA in COR23134 soybean. The gDNA extracted from COR23134 soybean plants was used for PCR amplification. Seven overlapping PCR fragments, spanning the insert and flanking genomic regions, were each amplified in two independent reactions. PCR products at the expected size were amplified only from the gDNA of COR23134 soybean but not from the non-GM control soybean or the no-template control. The PCR products were cloned, and at least six plasmids (three from each of the two independent PCR reactions) for each PCR fragment were sequenced in both forward and reverse directions using Sanger sequencing to cover every nucleotide, and the resulting sequencing reads were used to determine the consensus sequence for each PCR fragment. The consensus sequences from all seven overlapping PCR fragments were combined to determine the sequence for COR23134 soybean. The total length of sequence determined for COR23134 soybean is 30,019 base pairs (bp), composed of 1,024 bp of 5' flanking genomic sequence, 1,425 bp of 3' flanking genomic sequence, and 27,570 bp of inserted DNA. In comparison with the sequence of the PHP90315 T-DNA, the COR23134 insert is composed of bps 26 to 27,616 of the PHP90315 T-DNA, except for a 21-bp deletion in the pv-ubi2 promoter at bps 21,719-21,739 relative to the PHP90315 T-DNA sequence. All remaining sequence is intact and identical to the PHP90315 T-DNA sequence.

[0308] The junction sequences of Soybean Event COR-23134-4 are listed in Table 24 below: Table 24. Junction SequencesATTORNEY DOCKET #: 108747-WO-SEC-1Example 9: Modification of COR-23134-4 event using genome editing

[0001] In this Example, methods to modify the polynucleotide sequence of COR-23134- 4 and / or surrounding genomic polynucleotide sequences (SEQ ID NO:3) using genome editing are described.

[0002] In one method, one or more genome editing reagents (for example but not limited to a recombinase (RE) (Wang et al. (2010) Plant Cell Rep.30:267-285 and Van Duyne (2015) Microbiol Spectr. 3:10.1128 / microbiolspec.mdna3-0014-2014), zinc finger nuclease (ZFN) (Urnov et al. (2010) Nat Rev Genet. 11:636-646), transcription activator-like effector nuclease (TALEN) (Joung and Sander (2013) Nat Rev Mol Cell Biol. 14:49-55), homing endonuclease (HE) (Belfort and Bonocora (2014) Methods Mol Biol.1123:1-26 and Stoddard (2014) Mobile DNA.5:7), clustered regularly interspaced short palindromic repeat (CRISPR) associated (Cas) effector nuclease (Cong et al. (2013) Science. 339:819-823, Zetsche et al. (2015) Cell.163:759-771, Yan et al. (2018) Science.363:88-91, Pausch et al. (2020) Science. 369:333-337, Karvelis et al. (2020) Nucleic Acids Res.48:5016-5023, Yoshimi and Mashimo (2022) Gen and Genome Editing. 3-4:100013, and Urbaitis et al. (2022) EMBO Rep. 23:e55481), transposase associated B (TnpB) nuclease (Karvelis et al. (2021) Nature. 599:692-696 and Altae-Tran et al. (2021) Science. 374:57-65), Fanzor (Saito et al. (2023) Nature. 620:660-668 and Jiang et al. (2023) Sci. Adv. 9:eadk0171), and / or hydrolytic endonucleolytic ribozyme (HYER) (Liu et al. (2024) Science. 383:eadh4859)) are used to excise all or a portion of the polynucleotide sequence of COR-23134-4. In the case of a RE, a target site can be placed (either before or after the transgene or cisgene is inserted into the genome) flanking the polynucleotide region to be excised (FIG. 4). If the target site is included before insertion into the genome, it can be incorporated into the trait DNA sequence by first synthesizing a DNA fragment containing the site (Integrated DNA Technologies, GenScript, and / or Twist Bioscience) and then inserted using a NEBuilder HiFi DNA Assembly kit per the manufacturer’s instruction (New England Biolabs) although other vector construction methods can be used (for example but not limited to restriction enzyme, Golden Gate, and Gateway cloning). If the target site is incorporated following genomic insertion, it can be introduced using standard genome editing methodologies which include prime editingATTORNEY DOCKET #: 108747-WO-SEC-1 (Anzalone et al. (2019) Nature. 576:149-157) or non-homologous end-joining (NHEJ), micro-homology mediated end-joining (MMEJ), and / or homologous recombination (HR) repair of a targeted DNA nick or double-strand break (DSB) in the presence of a DNA repair template containing the target site of interest (Yao et al. (2017) Cell Research.27:801-814). Once introduced, the recombinase can be delivered (for example but not limited to particle bombardment (Ozyigit and Kurtoglu (2020) Mol Biol Rep.47:9831-9847) or Agrobacterium transformation (Sardesai and Subramanyam (2018) In: Gelvin SB (ed) Agrobacterium Biology: From Basic Science to Biotechnology. Cham: Springer International Publishing, 463-488)) either transiently or from a DNA expression cassette in a constitutive or tissue- specific manner. Once present in the cell, the RE can recognize its target site(s) and through strand exchange loop-out the intervening sequence (FIG.4) (Dale and Ow (1991) Proc Natl Acad Sci USA.88:10558-10562 and Russell et al. (1992) Mol Gen Genet.234:49-59).

[0003] Since ZFNs, TALENs, Cas endonucleases, TnpBs, Fanzors, and / or HYER nucleases can be programmed to recognize new targets, one or more target sequences in COR- 23134-4 or its surrounding sequence can be selected and the respective endonuclease(s) engineered to cleave the new site(s). Next, the reprogrammed nuclease can be delivered using particle bombardment or Agrobacterium as described above. Then upon delivery, the DNA editing reagent can be directed to recognize and cut its DNA target(s). The sequence between recognition site(s) can then be removed by leveraging cellular repair of the resulting DNA double-strand breaks (DSBs) (for example but not limited to non-homologous end-joining (NHEJ), micro-mediated end-joining (MMEJ), single-strand annealing (SSA), or homologous recombination (HR) (Gao et al. (2020) Nat Biotech. 38:579-581 and Sfeir and Symington (2015) Trends Biochem Sci.40:701-714)) (FIG.5). In the instance of Type I Cas effector complexes, one or more targets can be selected in the vicinity of COR-23134-4 and then once delivered transiently or expressed in a tissue-specific or constitutive manner used to processively remove sequences adjacent to the target site(s) optionally in combination with an orthogonal nuclease inactive enzyme (FIG.6) (Dolan et al. (2019) Mol Cell.74:936-950 and Li et al. (2024) Sci Adv.10:eadk8052).

[0004] In a second method, one or more genome editing reagents (for example but not limited to a RE, ZFN, TALEN, HE, Cas effector nuclease, TnpB nuclease, Fanzor, and / or HYER nuclease) are used to introduce polynucleotide sequence variation in or in the vicinity of COR-23134-4. For this, one or more target sites are first selected and then a reprogrammed RE, ZFN, TALEN, HE, Cas effector nuclease, TnpB nuclease, Fanzor, and / or HYER nuclease can be used to generate one or more DSBs. Cellular repair of the resulting DSB(s)ATTORNEY DOCKET #: 108747-WO-SEC-1 by NHEJ, MMEJ, SSA, or HR can then be used to introduce targeted sequence variation in the promoter(s), intron(s), coding sequence(s) (CDS), terminator(s), and / or flanking sequence of COR-23134-4 to knock-out or disrupt gene expression, up- or down-regulate gene expression, modify the coding content, and / or alter the flanking genomic polynucleotides (FIG.7). Additionally, an adenine or cytosine deaminase and / or glycosylase can be recruited to the Cas, TnpB, and / or Fanzor gRNA complex and used to introduce cytosine to thymine, adenine to guanine, and other transition or inversion single polynucleotide polymorphisms (SNPs) in COR-23134-4 (Komor et al. (2016) Nature. 533:420-424, Nishida et al. (2016) Science. 353: aaf8729, Gaudelli et al. (2017) Nature. 551:464-471, Tong et al. (2023) National Science Review. 10: nwad143, and Yi et al. (2024) Nat Commun. 15:6397). Moreover, prime editing can be utilized to introduce SNPs, insertions, deletions, and combinations thereof into Event COR-23134-4 (Chen and Liu (2022) Nat Rev Genet.24:161– 177). Similar to that described above, this can be used to disrupt or knock-out gene expression, up- or down-regulate gene expression, and / or generate variation in the coding content of one or more genes encoded in COR-23134-4 (FIG. 7). For a RE, a target (for example but not limited to a loxP site) can be placed (either before or after genomic insertion) in opposing orientations and, then once recognized by the RE, the polynucleotide sequence between the sites inverted (FIG.8). This can be used to modulate gene expression turning it either on or off or used to switch between constitutive and tissue-specific expression or vice versa. Moreover, a RE can be used to perform recombinase mediated cassette exchange (RMCE) to swap-out one or more genetic elements (for examples but not limited to promoter(s), intron(s), CDS(s), and terminator(s)) with different ones (Gao et al. (2020) Front Plant Sci.11:535).

[0005] Table 25 lists polynucleotide target sites (comprised of a guide RNA recognition site (gRNArs) and 3’ NGG target or protospacer adjacent motif (TAM or PAM) where N can be either a cytosine, adenine, thymine, or guanine polynucleotide) for a Cas9 (SEQ ID NO: 45) that can be used to excise all or a portion of COR-23134-4 or introduce targeted polynucleotide sequence variation in COR-23134-4. Target sites were initially selected by first identifying an appropriate PAM in the insert and flanking regions on either DNA strand of COR-23134-4 (SEQ ID NO:3). Next, the 16-30 nts immediately 5’ of the PAM were selected and off-target sites predicted using Cas-OFFinder (Bae et al. (2014) Bioinformatics. 30:1473-1475) against the Williams 82 soybean genomic reference sequence (Garg et al. (2023) Plant Genome.16:e20382). Unique targets, defined as having at least two nucleotide differences from the closest sequence in the Williams 82 genome and less than or equal to 3ATTORNEY DOCKET #: 108747-WO-SEC-1 consecutive adenine, thymine, guanine, and cytosine nts in the gRNArs, can then be prioritized for COR-23134-4 editing. To modulate the expression of genes within COR- 23134-4, targets can be further selected within the regulatory regions of the respective gene (for example but not limited to a TATA box, initiator element, downstream promoter element, TFIIB recognition element, downstream core element, motif ten element, X core promoter element, intron, 5’ untranslated region (UTR), and / or 3’ UTR). In some instances, this can be completed using a neural network trained for prediction of gene expression in plants (Peleke et al. (2024) Nat Commun. 15:3488). To improve the fitness and / or efficacy of proteins encoded within COR-23134-4 (for example but not limited to Cry1B.34.1 (SEQ ID NO:5), Cry1B.61.1 (SEQ ID NO:7), and IPD083 Cb (SEQ ID NO:9)), in silico evolution can be performed and various protein models (for example but not limited to data on efficacy and / or toxicity and / or ProteinMPNN (Dauparas et al. (2022) Science. 378:49-56)) used to rank variants. Next, depending on how many in silico changes were introduced, an editing strategy can be devised (for example but not limited to homologous recombination, prime editing, and / or base editing) and targets selected. Table 25. Cas9 target sites to facilitate the removal of all or a portion of COR-23134-4 or introduce targeted polynucleotide sequence variation in elements of COR-23134-4.ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1ATTORNEY DOCKET #: 108747-WO-SEC-1

[0309] The above description of various illustrated embodiments of the disclosure is not intended to be exhaustive or to limit the scope to the precise form disclosed. While specific embodiments of and examples are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. The teachings provided herein can be applied to other purposes, other than the examples described above. Numerous modifications and variations are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

[0310] These and other changes may be made in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the scope to the specific embodiments disclosed in the specification and the claims.

[0311] The entire disclosure of each document cited (including patents, patent applications, journal articles, abstracts, manuals, books or other disclosures) in the Background, Detailed Description, and Examples is herein incorporated by reference in their entireties.

[0312] Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight; temperature is in degrees celsius; and pressure is at or near atmospheric.

Claims

ATTORNEY DOCKET #: 108747-WO-SEC-1 WHAT IS CLAIMED IS:

1. A soybean plant comprising the genotype of the soybean event COR-23134-4, wherein said genotype comprises a nucleotide sequence as set forth in SEQ ID NO: 12 or SEQ ID NO: 15, or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 12 or SEQ ID NO:

15.

2. The soybean plant of claim 1, wherein said genotype comprises the nucleotide sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 16, or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 13 or SEQ ID NO:

16.

3. The soybean plant of claim 1, wherein said genotype comprises the nucleotide sequence set forth in SEQ ID NO: 14 or SEQ ID NO: 17, or a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 14 or SEQ ID NO:

17.

4. A DNA construct comprising operably linked first, second, and third expression cassettes, wherein the first expression cassettes comprises: 1) a zm-H2B Promoter; 2) an ubiZM1 Intron; 3) an cry1B.34.1; and 4) an os-ubi Terminator; wherein the second expression cassette comprises: a) a gm-cab3 Promoter; b) a cry1B.61.1; and c) an os-T28 Terminator; and wherein the third expression cassette comprises: i) a pv-ubi2 Promoter; ii) a pv-ubi2 Intron; iii) an ipd083C; and iv) an os-T17 Terminator.

5. A plant comprising the DNA construct of claim 4.

6. The plant of claim 5, wherein said plant is a soybean plant.ATTORNEY DOCKET #: 108747-WO-SEC-1 7. A plant comprising the sequence set forth in SEQ ID NO: 33, or a sequence having at least 95% sequence identity to SEQ ID NO:

33.

8. A soybean event COR-23134-4, wherein a representative sample of seed of said soybean event has been deposited with NCMA with NCMA Accession No.: 202305013.

9. Plant parts of the soybean event of claim 8.

10. Seed comprising soybean event COR-23134-4, wherein said seed comprises a DNA molecule chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein a representative sample of the soybean event COR-23134-4 seed of has been deposited with NCMA with NCMA Accession No.: 202305013.

11. A soybean plant, or part thereof, grown from the seed of claim 10.

12. A transgenic seed produced from the soybean plant of claim 8.

13. A transgenic soybean plant, or part thereof, grown from the seed of claim 12.

14. An isolated nucleic acid molecule comprising a nucleotide sequence chosen from SEQ ID NOs: 12-17, 33-37, and 41, and full length complements thereof.

15. An amplicon comprising the nucleic acid sequence chosen from SEQ ID NOs: 33-37, or 41, and full length complements thereof.

16. A biological sample derived from soybean event COR-23134-4 plant, tissue, or seed, wherein said sample comprises a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein said nucleotide sequence is detectable in said sample using a nucleic acid amplification or nucleic acid hybridization method, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with NCMA with NCMA Accession No.: 202305013.

17. The biological sample of claim 16, wherein said biological sample comprises plant, plant tissue, or seed of transgenic soybean event COR-23134-4.

18. The biological sample of claim 17, wherein said biological sample is a DNA sample extracted from the transgenic soybean plant event COR-23134-4, and wherein said DNA sample comprises one or more of the nucleotide sequences chosen from SEQ ID NOs: 12- 17, 33-37, and 41, and the complement thereof.

19. The biological sample of claim 16, wherein said biological sample is chosen from soybean flour, soybean meal, and soybean oil manufactured in whole or in part to contain soybean by-products.ATTORNEY DOCKET #: 108747-WO-SEC-1 20. An extract derived from soybean event COR-23134-4 plant, tissue, or seed and comprising a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein a representative sample of said soybean event COR- 23134-4 seed has been deposited with NCMA with NCMA Accession No.: 202305013.

21. The extract of claim 20, wherein said nucleotide sequence is detectable in said extract using a nucleic acid amplification or nucleic acid hybridization method.

22. The extract of claim 21, wherein said extract comprises plant, plant tissue, or seed of transgenic soybean plant event COR-23134-4.

23. The extract of claim 22, wherein the extract is a composition chosen from soybean flour, soybean meal, and soybean oil manufactured in whole or in part to contain soybean by- products, wherein said composition comprises a detectable amount of said nucleotide sequence.

24. A method of producing hybrid soybean seeds comprising: a sexually crossing a first inbred soybean line comprising a nucleotide chosen from SEQ ID NOs: 12-17, 33-37, and 41 and a second inbred line having a different genotype; b growing progeny from said crossing; and c harvesting the hybrid seed produced thereby. 25 The method according to claim 24, wherein the first inbred soybean line is a female parent or a male parent. 26 A method for producing a soybean plant resistant to lepidopteran pests comprising: a sexually crossing a first parent soybean plant with a second parent soybean plant, wherein said first or second parent soybean plant comprises event COR-23134-4 thereby producing a plurality of first-generation progeny plants; b selfing the first-generation progeny plant, thereby producing a plurality of second- generation progeny plants; and c selecting from the second-generation progeny plants that comprise the event COR-23134-4 and are resistant to a lepidopteran pest. 27 A method of producing hybrid soybean seeds comprising: a sexually crossing a first inbred soybean line comprising the DNA construct of claim 4 with a second inbred line not comprising the DNA construct of claim 4; andATTORNEY DOCKET #: 108747-WO-SEC-1 b. harvesting the hybrid seed produced thereby.

28. The method of claim 26, further comprising the step of backcrossing a second-generation progeny plant that comprises soybean event COR-23134-4 to the parent plant that lacks the soybean event COR-23134-4 DNA, thereby producing a backcross progeny plant that is resistant to a lepidopteran pest.

29. A method for producing a soybean plant resistant to Lepidopteran pest, said method comprising: a crossing a first parent soybean plant with a second parent soybean plant, wherein said first or second parent soybean plant comprises event COR-23134-4, thereby producing a plurality of first-generation progeny plants; b selecting a first-generation progeny plant that comprises the event COR-23134-4; c backcrossing the first-generation progeny plant of step (b) with a parent plant that lacks the soybean event COR-23134-4 DNA, thereby producing a plurality of backcross progeny plants; and d selecting from the backcross progeny plants, a plant that comprises the event COR-23134-4; wherein the selected backcross progeny plant of step (d) comprises SEQ ID NO: 12-17, 33- 37, or 41. 30 The method according to claim 29, wherein the plants of the first parent soybean line are the female parents or male parents. 31 Hybrid seed produced by the method of claim 29. 32 A method of determining zygosity of a soybean plant comprising event COR-23134-4 in a biological sample comprising: a contacting said sample with a first pair of DNA molecules and a second distinct pair of DNA molecules such that: when used in a nucleic acid amplification reaction comprising soybean event COR-23134-4 DNA, produces a first amplicon that is diagnostic for event COR-23134-4, and when used in a nucleic acid amplification reaction comprising soybean genomic DNA other than COR-23134-4 DNA, produces a second amplicon that is diagnostic for soybean genomic DNA other than COR-23134-4 DNA; b performing a nucleic acid amplification reaction; andATTORNEY DOCKET #: 108747-WO-SEC-1 c. detecting the amplicons so produced, wherein detection of the presence of both amplicons indicates that said sample is heterozygous for soybean event COR-23134-4 DNA, wherein detection of only the first amplicon indicates that said sample is homozygous for soybean event COR-23134-4 DNA.

33. The method of claim 32, wherein the first pair of DNA molecules comprises primer pair SEQ ID NOs: 18 and 19.

34. The method of claim 32, wherein the first and second pair of DNA molecules comprise a detectable label.

35. The method of claim 34, wherein the detectable label is a fluorescent label.

36. The method of claim 34, wherein the detectable label is covalently associated with one or more of the primer molecules.

37. A method of detecting the presence of a nucleic acid molecule that is unique to event COR- 23134-4 in a sample comprising soybean nucleic acids, the method comprising: a contacting the sample with a pair of primers that, when used in a nucleic-acid amplification reaction with genomic DNA from event COR-23134-4 produces an amplicon that is diagnostic for event COR-23134-4; b performing a nucleic acid amplification reaction, thereby producing the amplicon that is diagnostic for event COR-23134-4; and c detecting the amplicon that is diagnostic for event COR-23134-4. 38 The method of claim 37 wherein the nucleic acid molecule that is diagnostic for event COR-23134-4 is an amplicon produced by the nucleic acid amplification chain reaction. 39 The method of claim 37, wherein the method further comprises contacting the sample with a probe. 40 The method of claim 39, wherein the probe comprises a detectable label. 41 The method of claim 40, wherein the detectable label is covalently associated with the probe. 42 A plurality of polynucleotide primers comprising one or more polynucleotides which target event COR-23134-4 DNA template in a sample to produce an amplicon diagnostic for event COR-23134-4 as a result of a polymerase chain reaction method. 43 The plurality of polynucleotide primers according to claim 42, whereinATTORNEY DOCKET #: 108747-WO-SEC-1 a. a first polynucleotide primer comprises a nucleotide sequence as set forth in SEQ ID NO: 18, and the complements thereof; and b. a second polynucleotide primer comprises a nucleotide sequence as set forth in SEQ ID NO: 19, and the complements thereof.

44. The primers of claim 43, wherein said first primer and said second primer are at least 19 nucleotides.

45. A method of detecting the presence of DNA corresponding to event COR-23134-4 in a sample, the method comprising: a contacting the sample comprising soybean DNA with a polynucleotide probe that hybridizes under stringent hybridization conditions with DNA from soybean event COR- 23134-4 and does not hybridize under said stringent hybridization conditions with a non- COR-23134-4 soybean plant DNA; b subjecting the sample and probe to stringent hybridization conditions; and c detecting hybridization of the probe to the DNA; wherein detection of hybridization indicates the presence of event COR-23134-4. 46 A kit for detecting nucleic acids that are unique to event COR-23134-4 comprising at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event COR-23134-4 in the sample. 47 The kit according to claim 46, wherein the nucleic acid molecule comprises a nucleotide sequence from SEQ ID NO: 12-41. 48 The kit according to claim 46, wherein the nucleic acid molecule is a primer chosen from SEQ ID NOs: 12-41, and the complements thereof. 49 The soybean plant of claim 3, wherein the genotype comprises a nucleotide sequence having 1, 2, 3, 4, or 5 nucleotide changes in one of SEQ ID NO: 14 or SEQ ID NO:

17. 50 The soybean plant of claim 1, further comprising the nucleotide sequence set forth in SEQ ID NO: 3, or a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 3.ATTORNEY DOCKET #: 108747-WO-SEC-1 51. A method of modifying the COR-23134-4 soybean event, wherein a representative sample of seed of said soybean event was deposited with the NCMA with NCMA Accession No.: 202305013, comprising applying genome engineering technology to a DNA sequence of said COR-23134-4 soybean event to modify the DNA of said soybean event.

52. The method of claim 51, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having at least 90% sequence identity to SEQ ID NO:

3.

53. The method of claim 51, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having all or a portion of SEQ ID NO: 12 or SEQ ID NO: 15 duplicated in said modified DNA sequence.

54. The method of claim 51, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence comprising an excision from SEQ ID NO:

3.

55. The method of claim 54, wherein said excision comprises an excision from one or more regulatory elements of SEQ ID NO: 3 that does not substantially affect the activity of said one or more regulatory elements.

56. The method of claim 54, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having all or a portion of SEQ ID NO: 12 or SEQ ID NO: 15 excised from said modified DNA sequence.

57. The method of claim 54, comprising modifying the DNA of said COR-23134-4 soybean event to produce a modified DNA sequence having at least 30% of SEQ ID NO: 3 excised from said modified DNA sequence.

58. The method of claim 57, wherein at least 80% of SEQ ID NO: 3 is excised from said modified DNA sequence.

59. The method of claim 57, wherein all of SEQ ID NO: 3 is excised from said modified DNA sequence.

60. A method of generating guide polynucleotides for use with a COR-23134-4 soybean event genome editing system comprising designing one or more guide polynucleotides that recognize at least a portion of SEQ ID NO: 3 and synthesizing said guide polynucleotides.

61. A method of modifying the DNA of the COR-23134-4 event having accession No.: 202305013 comprising introducing said one or more guide polynucleotides of claim 60 asATTORNEY DOCKET #: 108747-WO-SEC-1 part of a genome engineering composition to a DNA of the COR-23134-4 event to modify the DNA of the COR-23134-4 event.

62. A COR-23134-4 soybean event genome editing system comprising a CAS polypeptide, one or more guide polynucleotides, and COR-23134-4 soybean event donor DNA.

63. A method of modifying at least one expression cassette of the COR-23134-4 event as deposited with the NCMA having accession number NCMA ACCESSION NO.: 202305013, wherein the method comprises using genome editing technologies to modify at least one expression cassette, wherein the resulting soybean plant derived from the COR- 23134-4 event comprises at least one modified cassette.

64. The method of claim 63, wherein the method comprises altering expression of at least one of the polynucleotide coding sequences selected from the group consisting of: cry1B.34.1, cry1B.61.1, and ipd083Cb.

65. A method of producing a commodity plant product comprising processing grain produced from a soybean event COR-23134-4 plant comprising a nucleotide sequence which is or is complementary to a sequence chosen from SEQ ID NO: 12 and SEQ ID NO: 15, wherein a representative sample of said soybean event COR-23134-4 seed has been deposited with NCMA with NCMA Accession No.: 202305013, wherein said grain is processed into a commodity plant product chosen from soybean flour, soybean meal, and soybean oil manufactured in whole or in part to contain soybean by-products, wherein said composition / commodity plant product comprises a detectable amount of said nucleotide sequence.

66. A method of controlling Lepidopteran insects, comprising exposing the Lepidopteran insects to insect resistant soybean plants of event COR-23134-4.

67. The method of claim 66, wherein the Lepidopteran insect is Fall Armyworm (Spodoptera frugiperda).

68. The method of claim 66, wherein the Lepidopteran insect is Soybean Looper (Chrysodeixis includens).

69. The method of claim 66, wherein the Lepidopteran insect is Velvetbean Caterpillar (Anticarsia gemmatalis).

70. The method of claim 66, wherein damage from the Lepidopteran insect is controlled for soybean from event COR-23134-4.