Soybean transgenic event GM_CSM63770 and methods for its detection and use

Abstract

The present invention provides transgenic soybean event GM_CSM63770, plants, plant cells, seeds, plant parts, progeny plants, commodity products containing event GM_CSM63770, and polynucleotides specific for the definition and detection of plants, plant cells, seeds, plant parts, progeny plants, and commodity products containing event GM_CSM63770, as well as methods related to the detection, characterization, and selection of event GM_CSM63770.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 355,947, filed on June 27, 2022, which is incorporated herein by reference in its entirety.

[0002] Incorporation of Sequence Listing The sequence listing contained in a file named MONS567WO_ST26, which is 341,230 bytes (measured in Microsoft Windows®) in size, was created on June 16, 2023, and is submitted herewith with this specification by electronic submission and is incorporated by reference.

[0003] The present invention relates to recombinant DNA molecules present in and / or isolated from the soybean event GM_CSM63770. The present invention also relates to transgenic soybean plants, plant parts, and seeds, cells, and agricultural products containing the soybean event GM_CSM63770, as well as methods of using them and methods of detecting the presence of the soybean event GM_CSM63770. Transgenic soybean plants, plant parts, seeds, and cells containing the DNA of the soybean event GM_CSM63770 exhibit resistance to invasion by insects of the family Lepidoptera.

Background Art

[0004] Soybean (Glycine max) is an important crop in many regions of the world and a major food supply source. Biotechnology methods have been applied to soybeans for improving the agricultural traits and quality of products. One such agricultural trait is insect resistance, which is achieved through the expression of heterologous insect toxins, also known as transgenes, inserted into the genome of soybean plants.

[0005] Soybeans have several different transgenic events described in the art that confer various types of pest resistance, particularly to Lepidoptera species, including MON87701, MON87751, and DAS81419 (Lepidoptera tolerance and herbicide tolerance). These transgenic events have been used commercially for extended periods in various regions around the world, and resistance to the expressed toxins in these events by target pests has been observed in many of the geographical areas where they are deployed.

[0006] Accordingly, there is a continuing need in the art to provide novel transgenic events in soybeans that exhibit resistance to insect infestation, and preferably, the novel transgenic events use a mode of action that does not overlap and is not similar to the modes of action previously deployed in prior commercial embodiments, to confer resistance to target insects, including varieties that have evolved resistance to existing commercially deployed traits. Described herein are examples of such novel transgenic events that confer resistance to Lepidoptera infestation, including resistance to Lepidoptera that has evolved resistance to prior commercial embodiments. SUMMARY OF THE INVENTION

[0007] In one embodiment, the present invention provides a novel transgenic soybean event, namely GM_CSM63770, which provides insecticidal control against lepidopteran pests of soybean. In further embodiments, the present invention also provides transgenic plants, plant cells, seeds, plant parts, and commodity products comprising the DNA of soybean event GM_CSM63770. The event is specific and unique to this GM_CSM63770 event and comprises novel DNA comprising the inserted transgenic DNA segment and novel DNA segments adjacent to the inserted DNA segment. These adjacent segments are described herein as junction sequences corresponding to DNA sequences extending along chromosomal DNA adjacent to the breakpoint of the chromosome into which the inserted DNA has been introduced. In another embodiment, the present invention provides a polynucleotide that is specific and unique to the DNA of soybean event GM_CSM63770 and can be used to identify and detect the presence of the DNA of soybean event GM_CSM63770 in a biological sample comprising soybean tissue comprising the DNA of soybean event GM_CSM63770, as well as in plants, plant cells, seeds, plant parts, progeny plants, and commodity products. In yet another embodiment, methods are provided relating to the selection of plant cells, plants, and seeds comprising the DNA of soybean event GM_CSM63770, and the detection of the presence (or absence) of the DNA of soybean event GM_CSM63770 in a sample, such methods providing an investigator with confirmation that the DNA of the event is present, or absent, in a particular sample that has been subjected to a method (or methods) that relies on the nucleotide sequences that are the subject of the present disclosure.

[0008] Accordingly, in one aspect, the present invention provides a recombinant DNA molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, and their complete complements.

[0009] In one embodiment, the recombinant DNA molecule is derived from the event in a sample of seeds containing the soybean event GM_CSM63770 and contains a unique and specific DNA segment corresponding to the seeds of the soybean event GM_CSM63770 deposited under ATCC accession number PTA-126048.

[0010] Another aspect of the invention provides a DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that specifically hybridizes with the DNA of the soybean event GM_CSM63770 in a sample under stringent hybridization conditions, wherein detection of the hybridization of the DNA probe to the DNA in the sample under the stringent hybridization conditions is used for or is characteristic of the diagnosis of the presence of the DNA of the soybean event GM_CSM63770 in the sample. In certain embodiments, the sample includes a soybean plant, soybean plant cell, soybean seed, soybean pollen, soybean plant part, soybean progeny plant, processed soybean seed, animal feed containing soybeans, soybean oil, soybean meal, soybean flour, soybean flakes, soybean bran, soybean biomass, and fuel products manufactured using soybeans and soybean parts, provided that such soybeans and soybean products contain a detectable amount of the DNA of the soybean event GM_CSM63770 or contain a detectable amount of a novel toxin protein produced by a soybean plant, cell, etc. and that contains the soybean event GM_CSM63770.

[0011] Yet another aspect of the present invention provides a first DNA molecule and a second DNA molecule different from the first DNA molecule, i.e., a pair of DNA molecules, which, when used in an amplification reaction containing appropriate reagents necessary for performing a DNA amplification procedure together with a sample containing the template DNA of the soybean event GM_CSM63770, function as DNA primers and are used for diagnosing the presence of the DNA of the soybean event GM_CSM63770 in the sample or produce an amplicon characteristic thereof. The produced amplicon comprises a nucleotide sequence selected from the group consisting of at least SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and their perfect complements.

[0012] Another embodiment of the present invention is a method for detecting the presence of a DNA segment used in a diagnosis for confirming the presence or absence of the soybean event GM_CSM63770 in a sample. In certain embodiments, the method comprises contacting the sample with a probe DNA molecule that specifically hybridizes to DNA uniquely associated with the soybean event GM_CSM63770, and then subjecting the sample and the probe DNA molecule to stringent hybridization conditions, which is done by binding the probe to an appropriate complementary segment of the soybean event GM_CSM63770-specific DNA. By detecting the hybridization of the probe DNA molecule to the DNA in the sample, it is conclusive that the DNA in the sample contains the DNA of the soybean event GM_CSM63770 and is used for its diagnosis, as it is limited thereto.

[0013] Yet another embodiment of the present invention is a method for diagnosing the DNA of the soybean event GM_CSM63770 in a sample containing soybean DNA or detecting the presence of a DNA segment characteristic thereof. In one embodiment, the method comprises contacting a biological sample with a pair of DNA molecules that function as thermal amplification primers specific for amplification of a segment of the DNA of the soybean event GM_CSM63770, performing an amplification reaction sufficient to produce an amplicon of the DNA, and detecting the presence of the amplicon of the DNA in the reaction product. Detection of the DNA amplicon is used for or is characteristic of diagnosing the presence of a detectable amount of the DNA of the soybean event GM_CSM63770 in the sample, and the amplicon comprises all or a portion of the nucleotide sequence targeted for amplification that is between the hybridization positions of the primers, and such portion of the nucleotide sequence targeted for amplification is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, and their complete complements.

[0014] Another embodiment of the present invention is a method for diagnosing the soybean event GM_CSM63770 in a sample or detecting the presence of a protein characteristic thereof, the method comprising: (a) contacting the sample with a first and a second monoclonal antibody, wherein the first monoclonal antibody specifically binds to Cry1A.2 and the second monoclonal antibody specifically binds to Cry1B.2 in an immunoassay; (2) incubating the immunoassay for a time sufficient to bind the monoclonal antibodies; and (3) detecting the presence of the Cry1A.2 and Cry1B.2 proteins in the immunoassay, wherein the detection is used for or is characteristic of diagnosing the presence of the DNA of the soybean event GM_CSM63770 in the sample. The assay can be selected from the group consisting of an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, and a lateral flow immunochromatography assay.

[0015] Another embodiment of the present invention is a soybean plant, a soybean plant part, a soybean cell, or a part thereof, comprising a recombinant polynucleotide molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and their perfect complements. These SEQ ID NOs are each specific and selected DNAs that define the soybean event GM_CSM63770. This soybean plant, soybean plant part, soybean cell, or a part thereof is insecticidal when fed to a lepidopteran pest. Target lepidopteran pests intended for control include Helicoverpa zea, Chrysodeixis includens, Anticarsia gemmatalis, Spodoptera eridania, Spodoptera cosmioides, Helicoverpa gelotopoeon, Rachiplusia nu, Crocidosema aporema, Hypena scabra, and Elasmopalpus lignosellus. Further, the soybean plant can be further defined as a progeny of any generation of a soybean plant comprising the soybean event GM_CSM63770, provided that the progeny also contains the specific and selected DNA that defines the soybean event GM_CSM63770.

[0016] Yet another embodiment of the present invention is a method for protecting soybean plants from insect infestation, the method comprising supplying an insecticidally effective amount of cells or tissues of a soybean plant comprising the soybean event GM_CSM63770 to the diet of Lepidopteran pests. Intended Lepidopteran pests include Helicoverpa zea, Chrysodeixis includens, Anticarsia gemmatalis, Spodoptera eridania, Spodoptera cosmioides, Helicoverpa gelotopoeon, Rachiplusia nu, Crocidosema aporema, Hypena scabra, and Elasmopalpus lignosellus.

[0017] Another embodiment of the present invention is a method for producing an insect-resistant soybean plant, comprising: a) manually crossing two different soybean plants by conventional breeding methods to produce offspring, wherein at least one of the two different soybean plants comprises the DNA of the soybean event GM_CSM63770; b) in seeds resulting from the breeding operation, as well as in progeny plants and tissues grown from such seeds, confirming the presence of a DNA segment used for the diagnosis of, or characteristic of, the soybean event GM_CSM63770; and c) selecting progeny comprising the DNA of the soybean event GM_CSM63770. Such seeds and progeny are soybean plants resistant to Lepidoptera.

[0018] A further embodiment of the present invention is a soybean seed, non-living plant material, or microorganism comprising a detectable amount of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or their complete complements.

[0019] Yet another embodiment is a commodity soybean product containing a detectable amount of a DNA molecule specific to the soybean event GM_CSM63770, the molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Commodity soybean products contemplated include, but are not limited to, whole soybean seeds or processed soybean seeds, animal feed containing soybeans, soybean oil, soybean meal, soybean flour, soybean flakes, soybean bran, soybean biomass, and fuel products manufactured using soybeans and soybean parts.

[0020] Another embodiment of the invention is a soybean plant, a soybean plant part, or a soybean seed thereof, containing DNA that functions as a template when tested in a DNA amplification method that is used for diagnosing the presence of DNA of the soybean event GM_CSM63770 or that produces an amplicon characteristic thereof.

[0021] Another embodiment of the present invention is a method for determining the zygosity of a soybean plant or soybean seed containing DNA that describes the soybean event GM_CSM63770. The zygosity is identified in a series of consecutive steps. In the first step, a sample containing soybean DNA is contacted with a first primer pair that is used for diagnosing DNA that describes and is only present in the soybean event GM_CSM63770, or that can produce an amplicon characteristic of it. Next, the sample containing the soybean DNA is contacted with a second primer designed to produce an amplicon of an internal standard that is known to be present in a single copy and be homozygous in the soybean plant. The method further includes contacting the DNA sample with a set of probes that includes at least a first probe that specifically hybridizes to an allele of the soybean event GM_CSM63770, and a second probe that specifically hybridizes to the genomic DNA of an internal standard that is known to be present in a single copy and be homozygous in the soybean plant. The method also includes a DNA amplification reaction, using real-time PCR, to identify the cycle threshold (Ct value) of the amplicon corresponding to the allele of the soybean event GM_CSM63770 and the single-copy homozygous internal standard. After amplification, the difference (ΔCt) between the Ct value of the single-copy homozygous internal standard amplicon and the Ct value of the amplicon of the allele of the soybean event GM_CSM63770 can be calculated. In one embodiment, the zygosity is determined, and a ΔCt of approximately zero (0) indicates the homozygosity of the inserted T-DNA of the soybean event GM_CSM63770, and a ΔCt of approximately one (1) indicates the heterozygosity of the inserted T-DNA of the soybean event GM_CSM63770. In a particular embodiment of the method, the primer pair is selected from the group consisting of the combination of SEQ ID NO: 14 and SEQ ID NO: 15, and the combination of SEQ ID NO: 17 and SEQ ID NO: 18, and the probes are SEQ ID NO: 16 and SEQ ID NO: 19. In yet another embodiment of the present invention, a ΔCt of approximately one (1) indicating the heterozygosity of the inserted T-DNA of the corn event MON95275 is in the range of 0.75 to 1.25.In certain embodiments, ΔCt of about zero (0) may be about 0, 0.05, 0.1, 0.15, 0.2, or 0.25, and in other embodiments, ΔCt of about one (1) may be about 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, or 1.25. In further embodiments, ΔCt of about one (1) may be in the range of 0.75 - 1.25, 0.8 - 1.25, 0.85 - 1.25, 0.9 - 1.25, 0.95 - 1.25, 1.0 - 1.25, 1.05 - 1.25, 1.1 - 1.25, 1.15 - 1.25, 1.2 - 1.25, 0.75 - 1.2, 0.8 - 1.2, 0.85 - 1.2, 0.9 - 1.2, 0.95 - 1.2, 1.0 - 1.2, 1.05 - 1.2, 1.1 - 1.2, 1.15 - 1.2, 0.75 - 1.15, 0.8 - 1.15, 0.85 - 1.15, 0.9 - 1.15, 0.95 - 1.15, 1.0 - 1.15, 1.05 - 1.15, 1.1 - 1.15, 0.75 - 1.1, 0.8 - 1.1, 0.85 - 1.1, 0.9 - 1.1, 0.95 - 1.1, 1.0 - 1.1, 1.05 - 1.1, 0.75 - 1.05, 0.8 - 1.05, 0.85 - 1.05, 0.9 - 1.05, 0.95 - 1.05, 1.0 - 1.05, 0.75 - 1.0, 0.8 - 1.0, 0.85 - 1.0, 0.9 - 1.0, 0.95 - 1.0, 0.75 - 0.95, 0.8 - 0.95, 0.85 - 0.95, 0.9 - 0.95, 0.75 - 0.9, 0.75 - 0.85, 0.75 - 0.8, 0.8 - 0.9, 0.8 - 0.85, or 0.85 - 0.9.

[0022] A further embodiment of the present invention is a method for determining the zygosity of a soybean plant, soybean seed, soybean pollen, egg, or cell, wherein each of such biological components suspected of containing the DNA of the soybean event GM_CSM63770 is subjected to the following method: a) contacting a sample containing DNA obtained from the suspected biological component with at least two different sets of primers, (i) in this case, a first primer pair consisting of a first primer and a second primer different from the first primer is used in an amplification reaction together with soybean DNA, and is used for diagnosing the presence of the DNA of the soybean event GM_CSM63770 in the sample or is capable of producing a first amplicon characteristic thereof, (ii) a second primer pair consisting of the first primer and a third primer different from the first primer and the second primer is used in an amplification reaction together with soybean DNA, and is used for diagnosing natural soybean genomic DNA that does not contain or does not include the soybean event GM_CSM63770-specific DNA or is capable of producing a second amplicon characteristic thereof; b) performing a nucleic acid amplification reaction with the sample and the two primer pairs; c) detecting in the nucleic acid amplification reaction the first amplicon used for diagnosing the soybean event GM_CSM63770 or characteristic thereof, or the second amplicon used for diagnosing natural soybean genomic DNA that does not contain the soybean event GM_CSM63770 or characteristic thereof, in which case the presence of only the first amplicon is used for diagnosing or is characteristic of the DNA of the homozygous soybean event GM_CSM63770 in the sample, and the presence of both the first amplicon and the second amplicon is used for diagnosing or is characteristic of a soybean plant that is heterozygous for the alleles of the soybean event GM_CSM63770; or, b) a sample containing soybean DNA is contacted with at least a first probe that specifically hybridizes to the DNA of the soybean event GM_CSM63770,and contacting a set of probes that specifically hybridizes to soybean genomic DNA over a segment of chromosomal DNA disrupted by the insertion of foreign DNA of soybean event GM_CSM63770 and that includes at least a second probe that does not hybridize to the DNA of soybean event GM_CSM63770, i) hybridizing the set of probes with a sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is used for or is characteristic of the diagnosis of the homozygous allele of the DNA of soybean event GM_CSM63770 in the sample, and detecting hybridization of both the first probe and the second probe under the hybridization conditions is used for or is characteristic of the diagnosis of the heterozygous allele of soybean event GM_CSM63770 in the sample. In one embodiment of the method, the set of primer pairs includes the combination of SEQ ID NO: 14 and SEQ ID NO: 15 and the combination of SEQ ID NO: 20 and SEQ ID NO: 15. In another embodiment of the method, the set of probes includes SEQ ID NO: 16 and SEQ ID NO: 21.,

[0023] Yet another embodiment of the present invention provides soybean plants, plant cells, pollen, plant parts, and seeds, which contain a recombinant DNA construct integrated into chromosome 19. The recombinant DNA construct confers resistance to lepidopteran pest species. The recombinant DNA construct is integrated at a position on the chromosome where at least 50 consecutive nucleotides of SEQ ID NO: 11 or 36 and 50 consecutive nucleotides of SEQ ID NO: 12 or 37 are located side by side. The at least 50 consecutive nucleotides of SEQ ID NO: 11 may include one or more nucleotide sequences selected from SEQ ID NOs: 118 to 137. The at least 50 consecutive nucleotides of SEQ ID NO: 36 may include one or more nucleotide sequences selected from SEQ ID NOs: 38 to 117. The at least 50 consecutive nucleotides of SEQ ID NO: 12 may include one or more nucleotide sequences selected from SEQ ID NOs: 138 to 157. The at least 50 consecutive nucleotides of SEQ ID NO: 37 may include one or more nucleotide sequences selected from SEQ ID NOs: 158 to 237.

[0024] The foregoing and other aspects of the present invention will become more apparent from the following detailed description.

Brief Description of the Drawings

[0025]

Figure 1

[10] in the drawings, where

[10] is the sequence of the inserted transgenic DNA of the soybean genome present within the soybean event GM_CSM63770 as well as the adjacent 5' and 3' sequences. SEQ ID NO: 1 is represented as [1] and is a segment of fifty (50) nucleotides spanning twenty-five (25) nucleotides of the inserted DNA and twenty-five (25) nucleotides of an arbitrarily assigned 5' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO: 2 is represented as [2] and is a segment of fifty (50) nucleotides spanning twenty-five (25) nucleotides of the inserted DNA and twenty-five (25) nucleotides of an arbitrarily assigned 3' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO: 1 is incorporated within SEQ ID NO: 3, represented as [3], which is a segment of one hundred (100) nucleotides spanning fifty (50) nucleotides of the inserted DNA and fifty (50) nucleotides of an arbitrarily assigned 5' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO: 2 is incorporated within SEQ ID NO: 4, represented as [4], which is a segment of one hundred (100) nucleotides spanning fifty (50) nucleotides of the inserted DNA and fifty (50) nucleotides of an arbitrarily assigned 3' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO: 5 is represented as [5], is two hundred (200) nucleotides in length, includes SEQ ID NO: 3, and spans one hundred (100) nucleotides of the inserted DNA and one hundred (100) nucleotides of an arbitrarily assigned 5' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO: 6 is represented as [6], is two hundred (200) nucleotides in length, includes SEQ ID NO: 4, and spans one hundred (100) nucleotides of the inserted DNA and one hundred (100) nucleotides of an arbitrarily assigned 3' soybean chromosomal DNA segment adjacent to the inserted DNA.[7] represents SEQ ID NO: 7 and corresponds to one thousand (1,000) consecutive nucleotides of the soybean genomic DNA at the arbitrarily assigned 5'-end of the inserted DNA and one hundred eighty-one (181) consecutive nucleotides of the adjacent inserted transgenic DNA. [8] represents SEQ ID NO: 8 and corresponds to one thousand (1,000) consecutive nucleotides of the adjacent soybean genomic DNA at the arbitrarily assigned 3'-end of the inserted DNA and one hundred ninety-two (192) consecutive nucleotides at the 3'-end of the inserted transgenic DNA. [9] represents the full-length nucleotide segment of the inserted transgenic DNA. The arrows, and the labels below each arrow, represent the orientation of the direction of transcription and translation, where applicable, from the applicable expression elements located within the two cassettes within the inserted DNA. RB and LB represent the positions of the right and left borders, respectively, of the binary Agrobacterium transformation vector, the letter P represents the position of the promoter element in each construct, the letter L represents the position of the leader sequence (5'-untranslated region, 5'UTR) in each construct, the letter I represents the position of the intron sequence in each construct, and the letter T represents the position of the transcription termination sequence (3'-untranslated region, 3'UTR) in each construct. The elements from P to immediately after T represented in this depiction by [9] and

[10] each represent a construct. The construct on the left side of FIG. 1 encodes the pest toxicity Cry1A.2, and the construct on the right side of FIG. 1 encodes Cry1B.2. Cry1A.2 and Cry1B.2 are each different unique toxins, showing less than 45% amino acid sequence identity to each other, each is toxic to lepidopteran pests, and each toxin shows a different mode of action relative to the other. Table 1 designates the position of each of these genetic elements involved in the expression of each of these toxin proteins within the transgenic inserted DNA.

[11] represents SEQ ID NO: 11 and indicates the position of the soybean genomic DNA adjacent to the 5'-end of the inserted DNA.

[12] represents SEQ ID NO: 12 and indicates the position of the soybean genomic DNA adjacent to the 3'-end of the inserted DNA.

[14] represents SEQ ID NO: 14 or primer SQ13805.

[15] represents SEQ ID NO: 15, or primer SQ51400.These are described for use together as primer pairs applicable to samples containing DNA of event GM_CSM63770, which produces an amplicon of one hundred thirty-four (134) nucleotides containing the 3'-end insert / genomic junction when used in a thermal amplification reaction. The arrows indicate the approximate hybridization positions within

[10] and the direction in which transcription proceeds during the amplification cycle.

[16] represents SEQ ID NO: 16 or probe PB4832 and binds (or hybridizes) to an amplicon produced by using, for example, primers

[14] and

[15] together in an amplification reaction with DNA of soybean event GM_CSM63770 as a template to detect the presence of DNA of soybean event GM_CSM63770 in a sample.

[20] represents SEQ ID NO: 20 or primer GM_WT63770F.

[20] is a primer that binds (or hybridizes) to soybean genomic DNA adjacent to the 5'-side of the inserted DNA and, in a thermal amplification reaction, when combined with

[15] (SEQ ID NO: 15, primer SQ51400) and conventional soybean DNA lacking or missing DNA of soybean event GM_CSM63770 as a template, produces an amplicon of one hundred thirty-six (136) nucleotides containing uninterrupted soybean genomic DNA. Detection of that amplicon represents a sample that does not contain DNA of soybean transgenic event GM_CSM63770 at that chromosomal locus. Probe GM_WT63770PB (SEQ ID NO: 21) represents a probe that binds (or hybridizes) to an amplicon produced by using primers

[20] and

[15] to detect an allele lacking or missing DNA of event GM_CSM63770. Probe GM_WT63770PB hybridizes to 9 nucleotides at the 3'-end of the 5'-genomic flanking DNA, 14 nucleotides of DNA of the wild-type allele deleted during the process of T-DNA insertion in soybean event GM_CSM63770, and 2 nucleotides at the 5'-end of the 3'-genomic DNA adjacent to DNA corresponding to the transgenic inserted DNA described herein as event GM_CSM63770.

[0026] Brief description of the sequences SEQ ID NO: 1 is a 50-nucleotide sequence representing the 5' junction region of the soybean genomic DNA and the integrated transgenic expression cassette (25 nucleotides of soybean genomic DNA at the 5' end of SEQ ID NO: 1, 25 nucleotides of transgenic inserted DNA at the 3' end of SEQ ID NO: 1), and can be identified at nucleotide positions 976-1,025 within SEQ ID NO: 10.

[0027] SEQ ID NO: 2 is a 50-nucleotide sequence representing the 3' junction region of the integrated transgenic expression cassette and the soybean genomic DNA (25 nucleotides of transgenic inserted DNA at the 5' end of SEQ ID NO: 2, 25 nucleotides of soybean genomic DNA at the 3' end of SEQ ID NO: 2), and can be identified at nucleotide positions 13,216-13,265 within SEQ ID NO: 10.

[0028] SEQ ID NO: 3 is a 100-nucleotide sequence representing the 5' junction region of the soybean genomic DNA and the integrated transgenic expression cassette (50 nucleotides of soybean genomic DNA at the 5' end of SEQ ID NO: 3, 50 nucleotides of transgenic inserted DNA at the 3' end of SEQ ID NO: 3), and can be identified at nucleotide positions 951-1,050 within SEQ ID NO: 10.

[0029] SEQ ID NO: 4 is a 100-nucleotide sequence representing the 3' junction region of the integrated transgenic expression cassette and the soybean genomic DNA (50 nucleotides of transgenic inserted DNA at the 5' end of SEQ ID NO: 4, 50 nucleotides of soybean genomic DNA at the 3' end of SEQ ID NO: 4), and can be identified at nucleotide positions 13,191-13,290 within SEQ ID NO: 10.

[0030] SEQ ID NO: 5 is a 200-nucleotide sequence representing the 5'-junction region of soybean genomic DNA and the integrated transgenic expression cassette (100 nucleotides of soybean genomic DNA at the 5'-end of SEQ ID NO: 5, transgenic inserted DNA of 100 nucleotides at the 3'-end of SEQ ID NO: 5), and can be identified at nucleotide positions 901-1,100 within SEQ ID NO: 10.

[0031] SEQ ID NO: 6 is a 200-nucleotide sequence representing the 3'-junction region of the integrated transgenic expression cassette and soybean genomic DNA (100 nucleotides of transgenic inserted DNA at the 5'-end of SEQ ID NO: 6, 100 nucleotides of soybean genomic DNA at the 3'-end of SEQ ID NO: 6), and can be identified at nucleotide positions 13,141-13,340 within SEQ ID NO: 10.

[0032] SEQ ID NO: 7 is a 1,181-nucleotide sequence representing the 5'-junction region of soybean genomic DNA and the integrated transgenic expression cassette (1,000 nucleotides of soybean genomic DNA at the 5'-end of SEQ ID NO: 7, 181 nucleotides of soybean genomic DNA at the 3'-end of SEQ ID NO: 7), and can be identified at nucleotide positions 1-1,181 within SEQ ID NO: 10.

[0033] SEQ ID NO: 8 is a 1,192-nucleotide sequence representing the 3'-junction region of the integrated transgenic expression cassette and soybean genomic DNA (192 nucleotides of transgenic inserted DNA at the 5'-end of SEQ ID NO: 6, 1,000 nucleotides of soybean genomic DNA at the 3'-end of SEQ ID NO: 6), and can be identified at nucleotide positions 13,049-14,240 within SEQ ID NO: 10.

[0034] SEQ ID NO: 9 is a 12,240-nucleotide sequence corresponding to the transgenic insert T-DNA of soybean event GM_CSM63770, and can be identified at nucleotide positions 1,001-13,240 within SEQ ID NO: 10.

[0035] SEQ ID NO: 10 is a 14,240-nucleotide sequence corresponding to the contiguous nucleotide sequence of the 5' genomic flanking DNA nucleotide sequence, the inserted T-DNA nucleotide sequence in event GM_CSM63770, and the 3' genomic flanking DNA nucleotide sequence, and includes SEQ ID NO: 11 (nucleotides 1 to 1,000), SEQ ID NO: 9 (nucleotides 1,001 to 13,240), and SEQ ID NO: 12 (nucleotides 13,241 to 14,240).

[0036] SEQ ID NO: 11 is a 1,000-nucleotide sequence representing the 5' flanking soybean genomic DNA up to the inserted T-DNA and can be identified at nucleotide positions 1 to 1,000 within SEQ ID NO: 10.

[0037] SEQ ID NO: 12 is a 1,000-nucleotide sequence representing the 3' flanking soybean genomic DNA after the inserted T-DNA and can be identified at nucleotide positions 13,241 to 14,240 within SEQ ID NO: 10.

[0038] SEQ ID NO: 13 is a 12,629-nucleotide sequence representing the transgene cassette contained within the binary plasmid transformation vector used to transform soybean to produce soybean event GM_CSM63770.

[0039] SEQ ID NO: 14 is a 26-nucleotide sequence corresponding to the thermal amplification primer called SQ13805 that can be used to identify the DNA of soybean event GM_CSM63770 in a sample and is identical to the nucleotide sequence corresponding to positions 13,177 to 13,202 of SEQ ID NO: 10.

[0040] SEQ ID NO: 15 is a 31-nucleotide sequence corresponding to the thermal amplification primer called SQ51400 that can be used to identify the DNA of soybean event GM_CSM63770 in a sample and is identical to the reverse complementary sequence of the nucleotide sequence corresponding to positions 13,280 to 13,310 of SEQ ID NO: 10.

[0041] SEQ ID NO: 16 is a 16-nucleotide sequence corresponding to a probe called PB4832 that is used to identify the DNA of the soybean event GM_CSM63770 in a sample, and is identical to the nucleotide sequence corresponding to positions 13,204 to 13,219 of SEQ ID NO: 10.

[0042] SEQ ID NO: 17 is a 20-nucleotide sequence corresponding to a thermal amplification primer called SQ549 that is used as an internal control for the event and zygosity assay of the soybean event GM_CSM63770, and hybridizes to the region of the soybean genome.

[0043] SEQ ID NO: 18 is a 20-nucleotide sequence corresponding to a thermal amplification primer called SQ546 that is used as an internal control for the event and zygosity assay of the soybean event GM_CSM63770, and hybridizes to the region of the soybean genome.

[0044] SEQ ID NO: 19 is a 28-nucleotide sequence corresponding to a probe called PB0004 that is used as an internal control for the event and zygosity assay of the soybean event GM_CSM63770, and hybridizes to the region of the soybean genome.

[0045] SEQ ID NO: 20 is a 25-nucleotide sequence corresponding to a thermal amplification primer called GM_WT63770F that is used in the zygosity assay of the soybean event GM_CSM63770, hybridizes to the 5' genomic flanking DNA, and is identical to the nucleotide sequence corresponding to positions 949 to 971 of SEQ ID NO: 10.

[0046] SEQ ID NO: 21 is a 25-nucleotide sequence corresponding to a probe called GM_WT63770PB that is used in the zygosity assay of the soybean event GM_CSM63770, and hybridizes to the last 9 nucleotides of the 5' genomic flanking DNA, 14 nucleotides of the DNA of the wild-type allele deleted during the process of T-DNA insertion in the soybean event GM_CSM63770, and the first 2 nucleotides of the 3' genomic flanking DNA.

[0047] Sequence number 22 is a 27-nucleotide sequence corresponding to OgRRS_5-1, which consists of an originator guide RNA recognition site (OgRRS), i.e., a Cas12a protospacer adjacent motif (PAM) site operably linked to the hybridization site of the guide RNA.

[0048] Sequence number 23 is a 27-nucleotide sequence corresponding to OgRRS, i.e., OgRRS_5-2.

[0049] Sequence number 24 is a 27-nucleotide sequence corresponding to OgRRS, i.e., OgRRS_5-3.

[0050] Sequence number 25 is a 27-nucleotide sequence corresponding to OgRRS, i.e., OgRRS_3-1.

[0051] Sequence number 26 is a 27-nucleotide sequence corresponding to OgRRS, i.e., OgRRS_In-1.

[0052] Sequence number 27 is a 27-nucleotide sequence corresponding to OgRRS, i.e., OgRRS_In-2.

[0053] Sequence number 28 is a 51-nucleotide sequence corresponding to a guide RNA (gRNA), i.e., gRNA_OgRRS_5-1.

[0054] Sequence number 29 is a 51-nucleotide sequence corresponding to gRNA, i.e., gRNA_OgRRS_5-2.

[0055] Sequence number 30 is a 51-nucleotide sequence corresponding to gRNA, i.e., gRNA_OgRRS_5-3.

[0056] Sequence number 31 is a 51-nucleotide sequence corresponding to gRNA, i.e., gRNA_OgRRS_3-1.

[0057] Sequence number 32 is a 51 - nucleotide sequence corresponding to the gRNA, namely gRNA_OgRRS_In - 1.

[0058] Sequence number 33 is a 51 - nucleotide sequence corresponding to the gRNA, namely gRNA_OgRRS_In - 2.

[0059] Sequence number 34 is the sequence of a synthetic DNA coding sequence designed for expression in plant cells encoding a nuclear - targeted Cas12a CRISPR - associated protein.

[0060] Sequence number 35 is the amino - acid sequence of a nuclear - targeted Cas12a CRISPR - associated protein.

[0061] Sequence number 36 is a 5,000 - nucleotide sequence representing the soybean genomic DNA adjacent to the insert at the 5' end of the transgenic insert. Nucleotides 4,001 - 5,000 of sequence number 36 are identical to nucleotides 1 - 1,000 of sequence number 11.

[0062] Sequence number 37 is a 5,000 - nucleotide sequence representing the soybean genomic DNA adjacent to the insert at the 3' end of the transgenic insert. Nucleotides 1 - 1,000 of sequence number 99 are identical to nucleotides 1 - 1000 of sequence number 12. The remaining nucleotides of sequence number 99 (nucleotides 1,001 - 5,000) are based on the genomic sequence of the Williams 82 soybean cultivar.

[0063] Sequence numbers 38 - 117 are sequences of an additional 50 nucleotides in the 5' flanking genomic sequence of event GM_CSM63770 based on the genomic sequence of the Williams 82 soybean cultivar.

[0064] Sequence numbers 118 - 137 are sequences of 50 nucleotides in the 5' flanking genomic sequence of event GM_CSM63770.

[0065] Array numbers 138 to 157 are the sequences of 50 nucleotides in the 3'-flanking genomic sequence of event GM_CSM63770.

[0066] Array numbers 158 to 237 are further sequences of 50 nucleotides in the 3'-flanking genomic sequence of event GM_CSM63770 based on the genomic sequence of the Williams 82 soybean variety.

Mode for Carrying Out the Invention

[0067] The present invention provides a transgenic soybean event GM_CSM63770 that achieves insecticidal control against certain lepidopteran larval pests of soybean by the expression of insecticidal toxins Cry1A.2 and Cry1B.2. The toxins are available in the tissues of soybean plants containing this event and are presented when the larval pests ingest the plant tissues. Specifically, the expression of the insect inhibitory proteins Cry1A.2 and Cry1B.2 in soybean event GM_CSM63770 confers resistance to the larval forms of lepidopteran pests including soybean podworm (SPW, Helicoverpa zea), soybean looper (Chrysodeixis includens), velvetbean caterpillar (Anticarsia gemmatalis), southern armyworm (Spodoptera eridania), black armyworm (Spodoptera cosmioides), South American podworm (Helicoverpa gelotopoeon), sunflower looper (Rachiplusia nu), bean shoot moth (Crocidosema aporema), green cloverworm (Hypena scabra), and sugarcane borer (Elasmopalpus lignosellus). Lepidopteran species are beginning to develop resistance to the pest control proteins previously used in earlier forms of transgenic soybean plants expressing such lepidopteran control proteins, and chemical insecticides have not provided adequate control of these insects, sometimes requiring multiple applications of different chemical properties during the growing season, increasing the input of chemical insecticides into the environment, increasing the carbon footprint per application, and adding significant costs to soybean crop production. Thus, event GM_CSM63770 provides a solution to the unmet needs in the art regarding the control of these insects in the soybean market.

[0068] Protection against infestation by Lepidoptera species provided by event GM_CSM63770 results from the expression of DNA segments encoding two different Lepidoptera-specific insecticidal proteins operably and covalently linked within the inserted transgenic DNA that partially defines soybean event GM_CSM63770. The two insecticidal proteins encoded by the transgenic insert DNA in soybean event GM_CSM63770 are (i) Cry1A.2 (U.S. Patent No. 10,494,408, the amino acid sequence is referenced as SEQ ID NO: 4 and the coding sequence is referenced as SEQ ID NO: 3), and (ii) Cry1B.2 (U.S. Patent No. 10,669,3017, the amino acid sequence is referenced as SEQ ID NO: 10 and the coding sequence is referenced as SEQ ID NO: 9). These two insecticidal proteins are described herein as being expressed from two different but linked expression cassettes within the inserted transgenic DNA construct described in SEQ ID NO: 9 and shown in Figure 1.

[0069] The DNA sequence encoding the Cry1A.2 protein in the soybean event GM_CSM63770 is operably linked to the Arabidopsis thaliana ubiquitin 10 promoter, leader, and intron. The DNA sequence encoding the Cry1B.2 protein in the soybean event GM_CSM63770 is operably linked to the Cucumis melo chlorophyll a-b binding protein 13 gene promoter and leader (referenced as SEQ ID NOs: 4-6 in U.S. Patent No. 10,443,066). Expression of the toxin proteins Cry1A.2 and Cry1B.2 from each transgene cassette (transcription into mRNA encoding the toxin amino acid sequence and translation of the mRNA into the toxin protein) proceeds in the same direction (head-to-tail / head-to-tail). Figure 1 shows the relative positions of each element, namely, the promoter (P), 5’UTR or leader (L), intron (I), toxin coding sequence or ORF (open reading frame), and 3’UTR (T), and the ORFs of Cry1A.2 and Cry1B.2 are contained within SEQ ID NO: 9 and SEQ ID NO: 10, respectively.

[0070] As described herein, a number of different constructs were evaluated with respect to expression elements, toxin coding sequences, and the use of transcriptional and translational orientations. One hundred twenty-five (125) constructs containing one or more of twenty-one (21) different insect toxin coding sequences were used to generate events for assay, leading to the selection of the soybean event GM_CSM63770. The construct used to create the soybean event GM_CSM63770, shown herein as SEQ ID NO: 13, demonstrated superior performance compared to other constructs when evaluated for resistance to lepidopteran pest infestation in the corresponding transgenic soybean plants. Furthermore, the soybean event GM_CSM63770 does not contain a marker used for the selection of transformed plant cells as a result of breeding selection and a transformation method in which a selectable marker and the trait of interest to be incorporated are used to be separable from each other. The soybean event GM_CSM63770 was transformed with two T-DNA plant transformation binary vectors. In this case, the selection cassette was contained on a T-DNA separate from the T-DNA containing the insect toxin transgene expression cassette retained in the soybean event GM_CSM63770. After self-pollination of the R0 generation of transformed soybean plants, the R1 progeny were selected with respect to the presence of the T-DNA containing the insect toxin expression cassette and the absence of the T-DNA used for the selection of the first transformation event.

[0071] The soybean event GM_CSM63770 was created through a plant transformation technology used to produce genetically engineered soybean cells, also called "transgenic" or "recombinant" soybean cells, by randomly inserting foreign DNA (also known as transgenic DNA) into the chromosomes of the genome of soybean cells. Using this technology, many individual cells are transformed, each resulting in a unique "transgenic event" or "event" due to the random insertion of foreign DNA into the genome. Transgenic plants are then regenerated from the individual transgenic cells. As a result, all cells of the transgenic plant contain the transgenic event inserted as a stable part of its genome. The transgenic plant can then be used to produce seeds, which are then planted, and progeny plants each containing the unique transgenic event grow.

[0072] Soybean event GM_CSM63770 was produced by an Agrobacterium-mediated transformation process using the binary transformation plasmid construct GM_CSM63770 and desiccated excised soybean plant explants. This plasmid construct contains two regions, each flanked by the Agrobacterium border segments (T-DNA segments). The first T-DNA segment contains two linked plant expression cassettes, one expression cassette encoding a selectable marker and one expression cassette encoding a scorable marker. The second T-DNA segment contains two linked plant expression cassettes and includes the regulatory genetic elements necessary for the expression of the two different insecticidal proteins, Cry1A.2 and Cry1B.2, in soybean plant cells. The T-DNA segment containing the selectable / scorable marker gene inserted randomly into the soybean genome and at a site distant from the site of integration of the T-DNA segment containing the Cry1A.2 and Cry1B.2 expression cassettes, in turn, allows for the separation of the two T-DNA segments within the genome of the transformed plants during the process of selfing and / or backcrossing, for example, the screening of the R1 and higher generations of transgenic plants. The transformed soybean cells were regenerated into intact soybean plants, and individual plants were selected from a population of plants that demonstrated the integrity of the second T-DNA segment encoding the Cry1A.2 and Cry1B.2 proteins. In the R1 and subsequent generations, the event was selected based on (i) the T-DNA segment encoding the selectable / scorable marker cassette and (ii) the absence (i.e., separation) of any plasmid backbone sequences, based on the integrity, stability, and intactness of the T-DNA segment encoding the Cry1A.2 and Cry1B.2 proteins. Further selection was also performed based on the location of the T-DNA segment encoding the Cry1A.2 and Cry1B.2 proteins within the soybean genome and other characteristics, such as efficacy and agronomics.The expression of the insecticidal toxin proteins Cry1A.2 and Cry1B.2 in the cells of soybean event GM_CSM63770 confers resistance to lepidopteran pests when the soybean cells of event GM_CSM63770 are fed to lepidopteran insects.

[0073] The T-DNA segments encoding the Cry1A.2 and Cry1B.2 proteins in plasmid construct pM63770 are shown as SEQ ID NO: 13. During the integration process, two hundred and seventeen (217) and one hundred and sixty-nine (169) consecutive base pairs (bp) were deleted from the right and left borders, respectively. Furthermore, fourteen (14) consecutive base pairs (bp) of wild-type genomic DNA at the insertion point of the transgenic DNA were deleted during the integration process.

[0074] Specifically, as described herein, soybean event GM_CSM63770 was produced by a complex research and development process. In that process, (1) over 100 plasmid vector constructs, i.e., constructs with different coding sequences of insecticidal proteins, coding sequences of transcriptional regulatory elements, and the number and orientation of cassettes within the constructs, were developed, transformed into soybean cells, thousands of events were created, these were tested and analyzed, and the construct used to generate event GM_CSM63770 was selected; (2) thousands of soybean cells were transformed with the construct used to generate event GM_CSM63770 to create a population of transgenic plants each containing a unique transgenic event, which was regenerated and tested; (3) the final event GM_CSM63770 was selected after a rigorous multi-year event selection process including testing and analysis of molecular characteristics, efficacy, protein expression, and agronomic characteristics in various soybean genetic backgrounds. Soybean event GM_CSM63770 was thus produced and selected as a unique and excellent event useful for large-scale agronomic purposes.

[0075] The transgenic DNA inserted into the genome of the soybean event GM_CSM63770 was characterized by detailed molecular analysis. This analysis included the number of insertions (the number of integration sites in the soybean genome), the genomic insertion location (the specific site in the soybean genome where the insertion occurred), the copy number (the number of copies of the T-DNA within a single locus), and the integrity of the transgenic inserted DNA (without any rearrangements). Detailed molecular analysis demonstrated that the integrated T-DNA containing the Cry1A.2 and Cry1B.2 expression cassettes remained intact after integration and that there was no T-DNA containing selectable / scorable markers. As used herein, an "expression cassette" or "cassette" is a recombinant DNA molecule that contains a combination of different elements that are expressed by a transformed cell. Table 1 provides a list of the elements contained in the DNA sequence corresponding to the soybean event GM_CSM63770, SEQ ID NO: 10. [Table 1]

[0076] Soybean event GM_CSM63770 is characterized as the insertion of the transgenic DNA of interest into a single locus in the soybean genome, resulting in two novel locus or junction sequences (e.g., the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8) that are unique to the soybean DNA containing event GM_CSM63770 and that do not naturally occur between the inserted DNA and the soybean genomic DNA or in other transgenic soybean events. These junction sequences are useful for detecting the presence of the event in soybean cells, soybean tissues, soybean seeds, soybean pollen and eggs, and soybean plants or soybean plant products, e.g., soybean commodity products. DNA molecular probes and primer pairs are described herein as being developed for use in identifying the presence of these various junction sequences in biological samples containing, or suspected of containing, soybean cells, soybean seeds, soybean plant parts, soybean pollen or eggs, or soybean plant tissue that contain event GM_CSM63770.

[0077] The sample is intended to refer to a composition that is substantially either pure soybean DNA or protein, or a composition containing soybean DNA or protein. With respect to a sample containing DNA, the sample is a biological sample, i.e., it includes biological materials that are directly or indirectly obtained from, or derived from, the genome of the soybean event GM_CSM63770, including but not limited to. "Directly" refers to the ability of one skilled in the art to directly obtain DNA from the soybean genome by disrupting soybean cells (or obtaining a sample of soybean containing disrupted soybean cells) and exposing the genomic DNA for detection. "Indirectly" refers to the ability of one skilled in the art to obtain a target or specific reference DNA contained in a particular sample, i.e., the novel and unique junction segments described herein that are characteristic of or used in the diagnosis of the presence of the soybean event GM_CSM63770, by means other than directly through disruption of soybean cells or obtaining a sample of soybean containing disrupted soybean cells. Such indirect means include amplification of a DNA segment containing a DNA sequence targeted by a specific probe designed to specifically bind to the target sequence, or measurement and characterization that can be carried out, i.e., measured by separation through some efficient matrix from other DNA segments, such as agarose or acrylamide gel, etc., or direct sequence analysis of the amplicon, or amplification of a DNA segment that can be characterized by cloning the amplicon into a vector and directly sequencing the inserted amplicon present in such vector, including but not limited to these.

[0078] A sample of pure soybean protein or a sample of a composition containing soybean protein is a biological sample, i.e., it contains biological substances including, but not limited to, proteins obtained from or derived from the tissue or cells of the soybean event GM_CSM63770. The sample is subjected to contact with monoclonal antibodies that specifically bind to Cry1A.2 and Cry1B.2 in an immunoassay. Detection of the bound protein is used for or is characteristic of the diagnosis of the soybean event GM_CSM63770. The immunoassay method can be, but is not limited to, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay, or lateral flow immunochromatography assay. The ELISA assay is usually performed in a laboratory using a whole plant or a tissue or cell sample obtained from that plant part. The sandwich ELISA assay is often used to detect and quantify proteins derived from transgenic crops. To detect the presence of the soybean event GM_CSM63770 in a sample using a sandwich ELISA assay, monoclonal antibodies, i.e., a first monoclonal antibody that specifically binds to Cry1A.2 and a second antibody that specifically binds to Cry1B.2, are used. A known amount of the monoclonal antibody is bound to a solid surface. The non-specific sites of the solid surface are blocked with bovine serum albumin, casein, or any other such neutral solution. A sample containing a protein derived from the soybean event GM_CSM63770 is added to the plate and captured by the antibody. Unbound antigen is washed away with a wash solution. A secondary antibody is added, which is also conjugated to an enzyme. Unbound antibody is washed. A substrate is added, and the enzyme reacts with the substrate to produce a product, usually a dye or a photon, proportional to the amount of antigen present. Separate ELISA assays are performed for each of the two toxin proteins in a sample derived from the soybean event GM_CSM63770. A positive ELISA reaction is used for or is characteristic of the diagnosis of the soybean event GM_CSM63770.

[0079] Lateral flow immunoassay chromatography assay, also known as immunoassay chromatography strip (ICS) or dipstick test, can be used in fields where laboratory facilities are not available for more complex immunoassays. The immunoassay chromatography test strip is composed of a sample pad, a conjugate pad, a nitrocellulose membrane, an absorption pad, and a backing card. Transfer the first monoclonal antibody that specifically binds to Cry1A.2, the second monoclonal antibody that specifically binds to Cry1B.2, and the IgG antibody that binds to the antibody derived from the biological host cell from which the first and second monoclonal antibodies are derived to the nitrocellulose membrane to form test line 1 (Cry1A.2), test line 2 (Cry1B.2), and a control line (anti-host IgG antibody). The conjugate pad is coated with the first and second monoclonal antibodies labeled with colloidal gold nanoparticles. Assemble the blotted membrane, conjugate pad, sample pad, and absorption pad continuously onto a plastic backing plate. The Cry1A.2 and Cry1B.2 proteins present in the sample derived from the soybean event GM_CSM63770 bind to their respective gold-labeled monoclonal antibodies and then bind to the capture antibodies coated on test line 1 and test line 2. Visual detection of test line 1 and test line 2 is used for or characteristic of the diagnosis of the soybean event GM_CSM63770.

[0080] Detailed molecular analysis also demonstrated that event GM_CSM6377 contains a single T-DNA insertion with one copy of each of the Cry1A.2 and Cry1B.2 expression cassettes. No additional elements from the transformation construct other than the portions of the left and right border regions of Agrobacterium tumefaciens used for the transfer of transgenic DNA from the plant transformation plasmid into the soybean genome were identified in event GM_CSM63770. Further, thermal amplification and DNA sequence analysis were performed to produce specific amplicons used for the diagnosis of the presence of event GM_CSM63770 in a sample or characteristic thereof, to identify the junctions of the arbitrarily assigned 5' and 3' inserts with the plant genome, to confirm the organization of the elements within the inserts, and to identify the complete DNA sequence (SEQ ID NO:9) of the inserted transgenic DNA. SEQ ID NO:11 represents a sequence of one thousand (1,000) base pairs (bp) adjacent to the arbitrarily assigned 5' end of the DNA inserted into the genomic DNA of soybean variety A3555 adjacent to the inserted T-DNA sequence. SEQ ID NO:12 represents a sequence of one thousand (1,000) bp adjacent to the arbitrarily assigned 3' end of the DNA inserted into the genomic DNA of soybean variety A3555 adjacent to the inserted T-DNA sequence. SEQ ID NO:7 is SEQ ID NO:11 with one hundred eighty-one (181) bp of the 5' end of the inserted T-DNA sequence added to the 3' end of SEQ ID NO:11. SEQ ID NO:8 is SEQ ID NO:12 with one hundred ninety-two (192) bp of the 3' end of the inserted T-DNA sequence added to the 5' end of SEQ ID NO:12. SEQ ID NO:10 corresponds to the DNA sequence defining soybean event GM_CSM63770 and contains a contiguous sequence (contig) including the 5'-flanking A3555 adjacent sequence, the transgene insert of GM_CSM63770, and the 3'-flanking A3555 adjacent sequence, and thus includes both of the junction sequences of the inserts identified as SEQ ID NOs:1, 3, 5, and 7 (5' end) and SEQ ID NOs:2, 4, 6, and 8 (3' end) with the plant genome.

[0081] Unless otherwise noted herein, terms are to be understood as having their ordinary meaning as used by those of ordinary skill in the relevant art in accordance with conventional usage. Definitions of common terms in molecular biology can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5 th edition, Springer-Verlag: New York, 1991, and Lewin, Genes V, Oxford University Press: New York, 1994, as well as other sources of information known to those of ordinary skill in the art. As used herein, the term "soybean" means a species belonging to the genus Glycine, preferably Glycine max, and includes all plant species capable of crossing with a soybean plant containing event GM_CSM63770, including wild soybean species and plants belonging to the genus Glycine that are capable of interspecific crossing.

[0082] Event GM_CSM63770 was transformed with a DNA construct containing an expression cassette that expresses toxic amounts of the insecticidal proteins Cry1A.2 and Cry1B.2. Toxic amount means an effective amount, an insecticidal amount, an insecticidally effective amount, an amount that suppresses target insects, an effective insecticidal amount, an amount of food for lepidopteran insects that is insecticidal, and other similar terms understood by those of ordinary skill in the relevant art in accordance with conventional usage. According to the method, soybean plants transformed with the DNA constructs disclosed herein are resistant to lepidopteran pests.

[0083] A transgenic "plant" is produced by the transformation of a plant cell with a heterologous DNA, i.e., a polynucleotide construct containing effective characteristics for several purposes, the regeneration of a plant obtained from the insertion of the transgene into the genome of the plant cell, and the selection of some effective characteristics of a specific plant and the regenerated transgenic plant characterized by the insertion into a specific genomic location. The term "event" refers to the DNA derived from the original transformant, including the inserted DNA and the flanking genomic sequences immediately adjacent to the inserted DNA. Such DNA is unique and is expected to transfer to the progeny that receive the inserted DNA, including the transgene of interest, as a result of the crossbreeding of a parental line containing the inserted DNA (e.g., the original transformant and the progeny obtained from self-breeding) with a parental line that does not contain the inserted DNA. The present invention also provides the original transformed plant containing the heterologous DNA and the progeny of the transformant. Such progeny may be produced by crossbreeding between a plant containing the event and another plant, and the progeny contains the heterologous DNA. Even after repeated backcrossing to a recurrent parent, the event remains at the same chromosomal location in the progeny of the cross. The DNA of the transgenic event, as well as the genomic DNA within the plant cell, the plant, the seed, and other parts of the plant where the DNA of the transgenic event is detectable, is not naturally occurring in nature.

[0084] As used herein, the term "recombinant" refers to a non-natural DNA, protein, or organism created by human intervention that is not normally found in nature. A "recombinant DNA molecule" is a DNA molecule that contains a combination of DNA molecules that do not naturally occur together and is the result of human intervention. For example, a DNA molecule composed of a combination of at least two DNA molecules that are heterologous to each other, such as a DNA molecule containing a transgene and plant genomic DNA adjacent to the transgene, is a recombinant DNA molecule.

[0085] As used herein, the terms "DNA" and "DNA molecule" refer to deoxyribonucleic acid (DNA) molecules. DNA molecules can be of genomic or synthetic origin and, by convention, run from the 5' (upstream) end to the 3' (downstream) end. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. By convention, the DNA sequences and fragments thereof of the present invention are disclosed by reference to only one of the two complementary DNA sequence strands. Implicitly and intentionally, the complementary sequences (sequences of the complementary strands) of the sequences provided herein, also referred to in the art as reverse complementary sequences, are within the scope of the present invention and are expressly intended to be within the scope of the claimed subject matter.

[0086] As used herein, the term "fragment" refers to a smaller piece of the whole. For example, a fragment of SEQ ID NO: 10 includes sequences that are at least about 12 consecutive nucleotides, at least about 13 consecutive nucleotides, at least about 14 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutive nucleotides, at least about 35 consecutive nucleotides, at least about 40 consecutive nucleotides, at least about 45 consecutive nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, or at least about 100 consecutive nucleotides of the complete sequence of SEQ ID NO: 10.

[0087] Similarly, a fragment of the 5' flank (SEQ ID NO: 11 or SEQ ID NO: 36) or 3' flank (SEQ ID NO: 12 or SEQ ID NO: 37) of the soybean event GM_CSM63770 can contain at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400, or at least about 500 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 36, or SEQ ID NO: 12 or SEQ ID NO: 37. Further, the present disclosure encompasses nucleotide sequences that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% identical to SEQ ID NO: 11 or SEQ ID NO: 12, or SEQ ID NO: 36 or SEQ ID NO: 37, or any fragment thereof.

[0088] References to "isolated DNA molecule" or equivalent terms or expressions in this disclosure are intended to mean that the DNA molecule exists alone or in combination with other compositions, but not in its natural environment. For example, nucleic acid elements naturally found within the DNA of an organism's genome, such as coding sequences, intron sequences, untranslated leader sequences, promoter sequences, transcription termination sequences, etc., are not considered "isolated" as long as the element is within the organism's genome and at the position within the genome where it is naturally found. However, each of these elements, and sub-portions of these elements, are "isolated" within the scope of this disclosure as long as the element is not within the organism's genome and not at the position within the genome where it is naturally found. Similarly, a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of that protein is an isolated nucleotide sequence as long as the nucleotide sequence is not within the DNA of the bacterium in which the sequence encoding the protein is naturally found. A synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein is considered isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA that is inserted into the genome of a plant or bacterial cell or exists within an extrachromosomal vector, is an isolated nucleotide sequence whether it exists within the plasmid or similar construct used for transformation of the cell, within the genome of the plant or bacterium, or is present in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from the plant or bacterium. In any case, the isolated DNA molecule is a chemical molecule regardless of whether it is referred to as a nucleic acid, nucleic acid sequence, polynucleotide sequence, etc. It is a novel, invention-related molecule that exhibits industrial applicability both when present within a plant cell or plant genome and when present outside of the plant cell, and thus, regardless of where the molecule is located, such usefulness is intended to be shown and is shown.

[0089] References to "isolated protein molecules" or equivalent terms or expressions in the present disclosure are intended to mean that the protein molecule exists alone or in combination with other compositions but not in its natural environment. For example, the insecticidal protein molecules expressed by the soybean event GM_CSM63770 are not naturally found in natural soybean plant samples. The insecticidal proteins Cry1A.2 and Cry1B.2 of the soybean event GM_CSM63770 are isolated protein molecules if the insecticidal protein or its variant is not within the proteins of the bacterium in which the protein is naturally found.

[0090] The region of DNA sequence spanning the connection by phosphodiester bonds to adjacent soybean genomic DNA at one end of the transgenic insert is referred to as the "junction". The junction is the point of connection between the transgenic insert as one continuous molecule and the adjacent DNA. One junction is found at the 5' end of the transgenic insert and the other is found at the 3' end of the transgenic insert, and are referred to herein as the 5' and 3' junctions, respectively. "Junction sequence" refers to a DNA sequence of any length spanning the 5' or 3' junction of an event. The junction sequences of the soybean event GM_CSM63770 are apparent to those skilled in the art using SEQ ID NO: 10. Examples of the junction sequences of GM_CSM63770 are provided as SEQ ID NOs: 1-8. Figure 1 shows the physical arrangement of the junction sequences arranged 5' to 3' relative to SEQ ID NO: 10. The junction sequences of GM_CSM63770 may exist as part of the genome of a plant, seed, or cell containing GM_CSM63770. The identification of any one or more of the junction sequences in a sample derived from a plant, plant part, seed, or cell indicates that the DNA was obtained from a soybean containing GM_CSM63770 and is used for the diagnosis of the presence of the soybean event GM_CSM63770 or is characteristic thereof.

[0091] The junction sequence of the soybean event GM_CSM63770 can be represented by a sequence from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. For example, the junction sequence can be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 1 (5' junction sequence) and SEQ ID NO: 2 (3' junction sequence). Alternatively, the junction sequence can be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 3 (5' junction sequence) and SEQ ID NO: 4 (3' junction sequence). Alternatively, the junction sequence can be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 5 (5' junction sequence) and SEQ ID NO: 6 (3' junction sequence). Alternatively, the junction sequence can be arbitrarily represented by the nucleotide sequences provided as SEQ ID NO: 7 (5' junction sequence) and SEQ ID NO: 8 (3' junction sequence). These nucleotide sequences are connected by phosphodiester bonds and, in the soybean event GM_CSM63770, exist as part of the recombinant plant cell genome.

[0092] These junction sequences are used for, or are characteristic of, the diagnosis of the presence of the soybean event GM_CSM63770, or a construct contained therein. Accordingly, the identification of one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10 in a sample derived from a soybean plant, soybean seed, or soybean plant part is used for the diagnosis that the DNA was obtained from the soybean event GM_CSM63770. Accordingly, the present invention provides a DNA molecule comprising at least one of the nucleotide sequences provided as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Any segment of DNA derived from the transgenic soybean event GM_CSM63770 that is sufficient to include at least one of the sequences provided as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10 is within the scope of the present invention. Further, any polynucleotide comprising a sequence complementary to any of the sequences described in this paragraph is within the scope of the present invention.

[0093] The present invention provides exemplary DNA molecules that can be used as either primers or probes for detecting the presence of DNA from a soybean plant containing the DNA of event GM_CSM63770 in a sample. Such primers or probes are specific for the target nucleic acid sequence and are thus useful for identifying the nucleic acid sequence of the soybean event GM_CSM63770 by the methods of the present invention described herein.

[0094] The use of the word "derived" is intended to mean that a particular DNA molecule can be present in the soybean plant genome or detected in soybean plant DNA. "Capable of being detected" refers to the ability of a particular DNA segment to be amplified and its size or sequence, characterized or elucidated by DNA sequence analysis, i.e., the target DNA segment, and the subsequent ability to detect the binding of the probe to that target. The specific DNA segment or target DNA segment of the present invention is present within soybean containing the insertion event GM_CSM63770.

[0095] A "probe" is a nucleic acid molecule that is complementary to a strand of a target nucleic acid and is useful in hybridization methods. The probe can be linked to a conventional detectable label or reporter molecule, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of DNA derived from GM_CSM63770, whether the strand of the target nucleic acid is of plant origin containing GM_CSM63770 or from a sample containing the DNA of GM_CSM63770. Thus, a probe as used herein binds to a specific unique segment of DNA present within the event GM_CSM63770 in a sample and can be used for the diagnosis of that event or can include a DNA molecule or polynucleotide segment of sufficient length to function under stringent hybridization conditions as defined herein that are characteristic of it. Such a probe can be designed to bind to a single junction or other novel sequence present only in the soybean event GM_CSM63770, or to only two or more such single junction segments. Probes according to the present invention include not only deoxyribonucleic acid or ribonucleic acid, but also polyamides and other probe materials that can specifically bind to a target DNA sequence and be used to detect the presence of that target DNA sequence. An exemplary DNA sequence useful as a probe for detecting the soybean event GM_CSM63770 is provided as SEQ ID NO: 16 (PB4832).

[0096] A "primer" is typically a DNA molecule designed to be used in a specific annealing or hybridization method involving thermal amplification. A primer can include pairs of different oligonucleotide or polynucleotide segments that are used in a thermal amplification reaction to amplify a specific target segment of DNA. Each primer in the pair is designed to bind to a fairly specific segment of DNA within or near the segment of DNA to be amplified. The primers bind such that they then act as sites of localization for polymerization of the nucleic acid sequence, resulting in the production of one or more amplicons (the amplified target segments of DNA). The amplicons produced from such a reaction have a DNA sequence corresponding to the sequence of the template DNA located between the two sites to which the primer hybridized to the template. In certain embodiments, the use of a primer designed to bind to a unique segment of the hybridization event GM_CSM63770 and to amplify a specific amplicon containing one or more of the junction sequences described herein, as well as the detection and / or characterization of such an amplicon upon completion or termination of the polymerase reaction, is used to diagnose or is characteristic of the presence of the hybridization event GM_CSM63770 in a particular sample. Those skilled in the art are well versed in this amplification method, and the details of amplification need not be enumerated here.

[0097] A primer is typically designed to hybridize to a complementary target DNA strand and form a hybrid between the primer and the target DNA strand, and the presence of the primer is a recognition point by a polymerase for initiating extension of the primer (i.e., polymerization of additional nucleotide molecules using the target DNA strand as a template). A primer pair typically refers to the use of two primers that bind to opposite strands of a double-stranded nucleotide segment for the purpose of linearly amplifying the polynucleotide segment between positions targeted for binding by each of the primer pair in a thermal amplification reaction or other conventional nucleic acid amplification method. Exemplary DNA molecules useful as primers are provided as SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:20.

[0098] The primer pair of SEQ ID NO: 14 and SEQ ID NO: 15 are useful as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both function as DNA primers when used in a thermal amplification reaction together with a template DNA derived from the soybean event GM_CSM63770, and are used for the diagnosis of the DNA of the soybean event GM_CSM63770 in a sample, or are of a sufficient length of consecutive nucleotides of SEQ ID NO: 10 to produce an amplicon characteristic thereof. The primer pair of SEQ ID NO: 17 and SEQ ID NO: 18 are useful as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both function as DNA primers when used in a thermal amplification reaction together with a template DNA derived from the soybean event GM_CSM63770, and are of a sufficient length of consecutive nucleotides of a locus in the soybean genome to produce an amplicon that functions as an internal control for both the diagnosis of the soybean event GM_CSM63770 and the zygosity of the DNA of the soybean event GM_CSM63770 in a sample. The primer pair of SEQ ID NO: 20 and SEQ ID NO: 15 are useful as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both function as DNA primers when used in a thermal amplification reaction together with a template DNA derived from the soybean event GM_CSM63770, and are used for the diagnosis of non-inserted wild-type soybean genomic DNA that does not contain the event GM_CSM63770, or are of a sufficient length of consecutive nucleotides of a locus in the soybean genome to produce an amplicon characteristic thereof. For any particular desired amplification parameter, based on the DNA sequence provided in the inserted DNA (SEQ ID NO: 9) and all segments of the DNA described herein as SEQ ID NO: 10 that define the transgenic soybean event GM_CSM63370 of the present invention, it is within the skill of one of ordinary skill in the art to identify which probes and primers are optimal for inclusion in a thermal amplification reaction to detect the presence or absence of the DNA of the transgenic event of the present invention.

[0099] DNA probes and DNA primers generally have a polynucleotide length of eleven (11) or more nucleotides, and often have a polynucleotide length of eighteen (18) or more nucleotides, twenty-four (24) or more nucleotides, or thirty (30) or more nucleotides. Such probes and primers are selected to be of sufficient length to hybridize specifically to the target sequence under high-stringency hybridization conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity with the target sequence, but different from the target sequence, and probes having the ability to hybridize to the target sequence may be designed by conventional methods.

[0100] The nucleic acid probes and primers of the present invention hybridize to target DNA molecules under stringent conditions. The presence of transgenic plant-derived DNA in a sample can be identified using any conventional nucleic acid hybridization or amplification method. A polynucleotide molecule, also referred to as a nucleic acid segment or a fragment thereof, can specifically hybridize to other nucleic acid molecules under certain circumstances.

[0101] As used herein, two polynucleic acid molecules are said to be capable of specifically hybridizing to each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. Nucleic acid molecules are said to be "complements" of each other if they exhibit complete complementarity. As used herein, molecules are said to exhibit "complete complementarity" if all of the nucleotides of one of the molecules are complementary to the other nucleotides. Two molecules are said to be "minimally complementary" if they can hybridize to each other with sufficient stability such that they remain annealed to each other, at least under conventional "low stringency" conditions. Similarly, the molecules are said to be "complementary" if they can hybridize to each other with sufficient stability such that they remain annealed to each other under conventional "high stringency" conditions. Conventional stringency conditions are described in Sambrook et al., 1989, and Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, D.C. (1985). Deviations from complete complementarity are thus tolerated as long as such deviations do not completely preclude the ability of the molecules to form a double-stranded structure. For a nucleic acid molecule to function as a primer or probe, it is only required that the sequence be sufficiently complementary to enable it to form a stable double-stranded structure under the particular solvent and salt concentrations used.

[0102] As used herein, a substantially identical sequence is a nucleic acid sequence that specifically hybridizes to a complement of a nucleic acid sequence being compared under high stringency conditions. Appropriate stringency conditions that promote DNA hybridization, for example, 6.0× sodium chloride / sodium citrate (SSC) at about 45°C, followed by a wash in 2.0× SSC at 50°C, are known to those of skill in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), Sections 6.3.1-6.3.6. For example, the salt concentration in the wash step can be selected from about 2.0× SSC at 50°C for low stringency to about 0.2× SSC at 50°C for high stringency. Additionally, the temperature of the wash step can be increased from low stringency conditions at room temperature, i.e., about 22°C, to high stringency conditions at about 65°C. Both temperature and salt can be varied, or one of the temperature or salt concentration can be held constant while the other variable is changed. In a preferred embodiment, the polynucleic acid of the present invention specifically hybridizes under moderately stringent conditions, such as about 2.0× SSC and about 65°C, to one or more of the nucleic acid molecules set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or a complement or any fragment thereof. In a particularly preferred embodiment, the nucleic acid of the present invention specifically hybridizes under high stringency conditions to one or more of the nucleic acid molecules set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, or a complement or any fragment thereof. In one aspect of the present invention, a preferred marker nucleic acid molecule of the present invention has a nucleic acid sequence set forth in SEQ ID NO:1, or SEQ ID NO:2, or SEQ ID NO:3, or SEQ ID NO:4, or SEQ ID NO:5, or SEQ ID NO:6, or SEQ ID NO:7, or SEQ ID NO:8, or SEQ ID NO:9, or SEQ ID NO:10, or a complement or any fragment thereof.Hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, including but not limited to fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

[0103] With respect to the amplification of a target nucleic acid sequence using a specific amplification primer pair (e.g., by PCR), "stringent conditions" mean that in a DNA thermal amplification reaction, the primer pair hybridizes only to the target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complement) binds, and preferably enables the production of a unique amplification product, i.e., an amplicon.

[0104] The term "specific (for the target sequence)" indicates that a probe or primer hybridizes only to the target sequence in a sample containing the target sequence under stringent hybridization conditions.

[0105] As used herein, "amplified DNA" or "amplicon" refers to the product of a nucleic acid amplification method targeted at a target nucleic acid molecule that is part of a polynucleotide template. For example, to determine whether a soybean plant obtained from a cross between male and female contains genomic DNA of a transgenic plant derived from a soybean plant containing the event GM_CSM63770 of the present invention, the DNA extracted from a soybean plant tissue sample may be subjected to a nucleic acid amplification method using a primer pair that includes a first primer derived from a genomic DNA sequence in a region adjacent to the heterologous inserted DNA of event GM_CSM63770 and that extends by polymerase in the 5' to 3' direction of the inserted DNA. The second primer is derived from the heterologous inserted DNA molecule and extends by polymerase in the 5' to 3' direction of the adjacent genomic DNA from which the first primer is derived. The amplicon can have a total length that extends to and obtains a length that is the primer pair plus one nucleotide base pair, or plus about 50 nucleotide base pairs, or plus about 250 nucleotide base pairs, or plus about 450 nucleotide base pairs or a combination thereof or more. As another method, the primer pair can be derived from genomic sequences on both sides of the inserted heterologous DNA, and an amplicon containing the entire inserted polynucleotide sequence is produced (for example, a forward primer isolated from the genomic portion at the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion at the 3' end of SEQ ID NO: 10, which amplifies a DNA molecule containing the inserted DNA sequence (SEQ ID NO: 9) identified herein in the genome of event GM_CSM63770). The members of the primer pair derived from the plant genomic sequence adjacent to the inserted transgenic DNA are located away from the inserted DNA sequence, and this distance can range from 1 nucleotide base pair to about 20,000 nucleotide base pairs. The use of the term "amplicon" clearly excludes primer dimers that may be formed during the thermal amplification reaction of the DNA.

[0106] In practice, primers should be designed to produce amplicons within a limited size range, for example, amplicons of 100 to 1000 bases. Generally, smaller-sized (shorter polynucleotide length) amplicons are more reliably produced in thermal amplification reactions, allow for shorter cycle times, can be easily separated and visualized on an agarose gel, or can be adapted for use in endpoint TAQMAN®-like assays. Smaller amplicons can be produced and detected by DNA amplicon detection methods known in the art. Additionally, amplicons produced using the primer pair may be cloned into a vector, propagated, isolated, and sequenced in some cases, or directly sequenced by methods well-known in the art in other cases. Any primer pair derived from the combination of SEQ ID NO: 11 and SEQ ID NO: 9, or the combination of SEQ ID NO: 12 and SEQ ID NO: 9, which is useful for a DNA amplification method for producing an amplicon characteristic of or used in the diagnosis of the soybean event GM_CSM63770 or its progeny, is an aspect of the present invention. Any single isolated DNA polynucleotide primer molecule comprising at least 15 consecutive nucleotides of SEQ ID NO: 11, or its complement, which is useful for a DNA amplification method for producing an amplicon characteristic of or used in the diagnosis of the soybean event GM_CSM63770 or its progeny, is an aspect of the present invention. Any single isolated DNA polynucleotide primer molecule comprising at least 15 consecutive nucleotides of SEQ ID NO: 12, or its complement, which is useful for a DNA amplification method for producing an amplicon characteristic of or used in the diagnosis of a plant comprising the soybean event GM_CSM63770 or its progeny, is an aspect of the present invention. Any single isolated DNA polynucleotide primer molecule comprising at least 15 consecutive nucleotides of SEQ ID NO: 9, or its complement, which is useful for a DNA amplification method for producing an amplicon characteristic of or used in the diagnosis of the soybean event GM_CSM63770 or its progeny, is an aspect of the present invention.

[0107] Amplification of the polynucleic acid can be achieved by any of a variety of polynucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). The 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 PCR Protocols: A Guide to Methods and Applications, ed. Innis et al., Academic Press, San Diego, 1990. The PCR amplification method was developed to amplify genomic DNA up to 22 kb (kilobases) and bacteriophage DNA up to 42 kb (Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699, 1994). These methods and other methods known in the art of DNA amplification can be used in the practice of the present invention. The sequence of the heterologous DNA insert or the adjacent genomic DNA sequence from the soybean event GM_CSM63770 can be verified (and corrected if necessary) by standard DNA sequencing of the PCR amplicon or the cloned DNA fragment thereof, after amplifying such a DNA molecule from a soybean seed containing the DNA of the event GM_CSM63770 deposited with the ATCC having accession number PTA-126048 or a soybean plant grown from a soybean seed containing the DNA of the event GM_CSM63770 using primers derived from the sequences provided herein.

[0108] The diagnostic amplicons produced by these methods can be detected by a plurality of techniques. One such method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994), where DNA oligonucleotides that overlap both the adjacent genomic DNA sequence and the inserted DNA sequence are designed. The oligonucleotides are immobilized in the wells of a microtiter plate. Following PCR of the region of interest (using one primer in the inserted sequence and one in the adjacent genomic sequence), the single-stranded PCR product can hybridize to the immobilized oligonucleotide and function as a template for a single-base extension reaction using DNA polymerase and a labeled dideoxynucleotide triphosphate (ddNTP) specific for the expected next base. The readout can be by fluorescence or ELISA-based. The signal indicates the presence of the transgene / genomic sequence by virtue of the achievement of amplification, hybridization, and single-base extension.

[0109] Another method is pyrosequencing technology, as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method, oligonucleotides that overlap the adjacent genomic DNA and the inserted DNA junction are designed. The oligonucleotides are hybridized to a single-stranded PCR product from the region of interest (one primer in the inserted sequence and one in the adjacent genomic sequence) and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5’ phosphosulfate, and luciferin. The dNTPs are added individually, and the enzymatic reaction of luciferase with those reagents and substrates results in the release of photons (a light signal), which is then measured or observed. The light signal indicates the presence of the transgene / genomic sequence by virtue of the achievement of amplification, hybridization, and single- or multi-base extension.

[0110] The fluorescence polarization described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method that can be used to detect the amplicons of the present invention. Using this method, oligonucleotides that overlap with genomic flanks and the inserted DNA junctions are designed. The oligonucleotides are hybridized to single-stranded PCR products from the region of interest (one primer is in the inserted DNA and one is in the adjacent genomic DNA sequence) and incubated in the presence of DNA polymerase and fluorescently labeled ddNTPs. Incorporation of the ddNTPs occurs by single-base extension. Incorporation can be measured as a change in polarization using a fluorometer. The change in polarization indicates the presence of the transgene / genomic sequence by the achievement of amplification, hybridization, and single-base extension.

[0111] Real-time polymerase chain reaction (PCR) is the ability to observe the progress of PCR as it occurs (i.e., in real time). Data are collected throughout the PCR process, not at the end of PCR. In real-time PCR, the reaction is characterized by the point in the cycle at which target amplification is first detected, rather than the amount of target accumulated after a fixed number of cycles. In a real-time PCR assay, a positive reaction is detected by the accumulation of a fluorescent signal. The higher the starting copy number of the nucleic acid target, the earlier a significant increase in fluorescence is observed. The cycle threshold (Ct value) is defined as the number of cycles required for the fluorescent signal to exceed a threshold (i.e., above the background level). The Ct level is inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct value, the higher the amount of target nucleic acid in the sample).

[0112] Taqman® (PE Applied Biosystems, Foster City, CA) is a method for detecting and quantifying the presence of DNA sequences using real-time PCR and is well understood in the manufacturer's instructions. Briefly, FRET oligonucleotide probes that overlap genomic flanks and the inserted DNA junction are designed. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one in the adjacent 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 of the FRET probe. The fluorescent signal indicates the presence of the transgene / genomic sequence by achieving amplification and hybridization.

[0113] Molecular beacons are described for use in sequence detection and are described in Tyangi, et al. (Nature Biotech. 14:303-308, 1996). Briefly, FRET oligonucleotide probes that overlap adjacent genomic and inserted DNA junctions are designed. The unique structure of the FRET probe results in a secondary structure that maintains the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one in the adjacent genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following achievement of PCR amplification, hybridization of the FRET probe to the target sequence results in removal of the probe's secondary structure and spatial separation of the fluorescent and quenching moieties. A fluorescent signal results. The fluorescent signal indicates the presence of the adjacent / transgene insertion sequence by achieving amplification and hybridization.

[0114] A DNA detection kit based on a DNA amplification method contains DNA primer molecules that specifically hybridize to a target DNA and amplify diagnostic amplicons under appropriate reaction conditions. The kit may provide an agarose gel-based detection method or various methods for detecting diagnostic amplicons known in the art. The DNA detection kit can be developed using the compositions disclosed herein, is useful for identifying the DNA of the hybridization event GM_CSM63770 in a sample, and can be applied to methods for breeding soybean plants containing the DNA of the soybean event GM_CSM63770. Kits containing DNA primers that are homologous or complementary to any portion of the soybean genomic region shown in SEQ ID NO: 10 and any portion of the inserted transgenic DNA shown in SEQ ID NO: 10 are the subject of the present invention. The DNA molecules can be used as primers in DNA amplification methods (PCR) or in nucleic acid hybridization methods, i.e., as probes in Southern analysis and Northern analysis.

[0115] The probes and primers according to the present invention may have perfect sequence identity with the target sequence, but different from the target sequence, primers and probes that retain the ability to preferentially hybridize to the target sequence may be designed by conventional methods. For a nucleic acid molecule to function as a primer or probe, it is only necessary that the sequences are sufficiently complementary to allow the formation of a stable double-stranded structure under the particular solvent and salt concentrations used. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA derived from the hybridization event GM_CSM63770 in the sample. Probes and primers generally have a length of at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides or more. Such probes and primers specifically hybridize to the target DNA sequence under stringent hybridization conditions. Conventional stringency conditions are described in Sambrook et al., 1989, and Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0116] The DNA molecules, or fragments thereof, disclosed in the present invention can be isolated and manipulated using any method well known to those skilled in the art, including thermal amplification methods. The DNA molecules, or fragments thereof, can also be obtained by direct synthesis of the fragments by chemical means, which is generally carried out by using other techniques, such as an automated oligonucleotide synthesizer.

[0117] Accordingly, the DNA molecules and corresponding nucleotide sequences provided herein are useful, inter alia, for identifying the soybean event GM_CSM63770, detecting the presence of DNA derived from the transgenic soybean event GM_CSM63770 in a sample, and observing a sample for the presence and / or absence of the soybean event GM_CSM63770 or a plant part derived from a soybean plant containing the soybean event GM_CSM63770.

[0118] By reference to soybean, soybean plants, soybean plant cells, soybean seeds, soybean pollen and eggs, soybean plant parts, soybean progeny plants, and soybean commercial products are intended to be within the scope of the present invention as long as each embodiment contains a detectable amount of DNA corresponding to any one, two, or more of the segments described herein (e.g., polynucleotides having at least one of the sequences provided as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10) that are used in or are characteristic of the diagnosis of the presence of the soybean event GM_CSM63770. The soybean plants, plant cells, seeds, pollen and / or eggs, plant parts, and progeny plants of the present invention may contain one or more additional transgenes. Such additional transgene(s) can be any nucleotide sequence encoding a protein or RNA molecule that confers a desired trait including, but not limited to, improved pest resistance, improved water use efficiency, improved yield, improved drought tolerance, improved seed quality, and / or improved herbicide tolerance.

[0119] The present invention provides a soybean plant, a soybean plant cell, a soybean seed, a soybean plant part (e.g., pollen, ovule, hair, spike, anther, rachis, root tissue, stem tissue, leaf tissue), and a soybean progeny plant derived from a transgenic soybean plant containing the DNA of GM_CSM63770. A representative sample of a soybean seed containing the DNA of the soybean event GM_CSM63770 has been deposited in accordance with the Budapest Treaty with the American Type Culture Collection (ATCC®). The ATCC repository has assigned the patent deposit designation PTA-126048 to the seed containing the DNA of the soybean event GM_CSM63770.

[0120] The present invention provides a soybean plant, plant cell, plant part, and plant seed containing a recombinant DNA construct integrated into chromosome 19, the recombinant DNA construct conferring resistance to lepidopteran pest species. The recombinant DNA construct is integrated at a position on the chromosome where at least 50 consecutive nucleotides of SEQ ID NO: 11 or 36 and 50 consecutive nucleotides of SEQ ID NO: 12 or 37 are positioned side by side. The at least 50 consecutive nucleotides of SEQ ID NO: 11 or 36 may include one or more nucleotide sequences selected from SEQ ID NOs: 38 to 137. The at least nucleotides of SEQ ID NO: 12 or 37 may include one or more nucleotide sequences selected from SEQ ID NOs: 138 to 237.

[0121] The present invention provides a microorganism containing a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 in its genome. An example of such a microorganism is a transgenic plant cell.

[0122] Microorganisms, such as the plant cells of the present invention, are useful in many industrial applications including, but not limited to: (i) use as research tools for scientific or industrial research, (ii) use in culture to produce endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products, or small molecules that can subsequently be used for scientific research or as industrial products, and (iii) use with modern plant tissue culture techniques to produce transgenic plants or plant tissue cultures that can subsequently be used in agricultural research or production. The production and use of microorganisms, such as transgenic plant cells, utilizes modern microbiological techniques and human intervention to produce unique artificial microorganisms. In this process, recombinant DNA is inserted into the genome of a plant cell, creating a unique transgenic plant cell distinct from the naturally occurring plant cell. This transgenic plant cell can then be cultured, using modern microbiological techniques, to closely resemble bacterial and yeast cells and can exist in an undifferentiated single cell state. The new genetic constitution and phenotype of the transgenic plant cell are the technical effects created by integrating the heterologous DNA into the genome of the cell. Another aspect of the present invention is a method of using the microorganisms of the present invention. Methods of using the microorganisms of the present invention, such as transgenic plant cells, include: (i) a method of generating transgenic cells by integrating recombinant DNA into the genome of the cell and then using this cell to induce further cells having the same heterologous DNA, (ii) a method of culturing cells containing recombinant DNA using modern microbiological techniques, (iii) a method of producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid, or protein products from the cultured cells, and (iv) a method of using modern plant tissue culture techniques with transgenic plant cells to produce transgenic plants or transgenic plant tissue cultures.

[0123] The plants of the present invention can transmit the DNA of soybean event GM_CSM63770, including the transgene inserted into soybean event GM_CSM63770, to their progeny, usually through conventional breeding and selection. As used herein, "progeny" includes any plant, plant cell, seed, and / or regenerable plant part that contains the DNA of soybean event GM_CSM63770 derived from an ancestral plant and / or contains a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. The plants, progeny, and seeds may be homozygous or heterozygous for the transgene of soybean event GM_CSM63770. The progeny may be grown from seeds produced by a plant containing soybean event GM_CSM63770 and / or from seeds produced by a plant pollinated with pollen from a plant containing soybean event GM_CSM63770.

[0124] Progeny plants may be self-pollinated (also known as "selfing") to produce plants of a pure breeding line, i.e., plants that are homozygous for the transgene. By appropriate selfing of the progeny, plants that are homozygous for both of the added foreign genes can be produced.

[0125] Alternatively, progeny plants may be crossbred, e.g., with another unrelated plant, to produce variant or hybrid seeds or plants. The other unrelated plant may be transgenic or non-transgenic. The variant or hybrid seeds or plants of the invention may thus be obtained by cross-fertilizing a first parent lacking the specific and unique DNA of the soybean event GM_CSM63770 with a second parent containing the soybean event GM_CSM63770 to yield a hybrid containing the specific and unique DNA of the soybean event GM_CSM63770. Each parent may be a hybrid or an inbred / variant plant as long as the crossbreeding or breeding results in seeds having at least one allele containing the DNA of the plant or seed of the invention, i.e., the soybean event GM_CSM63770, and / or a DNA molecule having at least one sequence selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Thus, two different transgenic plants may be crossbred to produce progeny of hybrids containing two independently segregating, additional, foreign genes. For example, the soybean event GM_CSM63770 containing Cry1A.2 and Cry1B.2 conferring insect pest resistance to soybeans may be crossbred with other transgenic soybean plants to produce plants having the characteristics of both transgenic parents. An example of this is to crossbreed the soybean event GM_CSM63770 containing Cry1A.2 and Cry1B.2 conferring lepidopteran resistance to soybeans with a plant having one or more additional traits, e.g., herbicide tolerance, insect pest resistance, or drought tolerance, to yield progeny plants or seeds having resistance to lepidopteran pests and at least one or more additional traits. Backcrossing to the parent plant and outcrossing with non-transgenic plants are also contemplated, as is vegetative propagation. Descriptions of other breeding methods commonly used for different traits and crops can be found in one of several references, e.g., Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison WI (1987).

[0126] The plants, progeny, seeds, pollen and eggs, cells, and plant parts of the present invention may also include a soybean plant containing one or more additional soybean trait(s) or transgenic event(s), particularly the soybean event GM_CSM63770, which may be introduced by crossing with another soybean event containing the additional trait(s) or transgenic event(s). Such trait(s) or transgenic event(s) include, but are not limited to, improved insect pest resistance, herbicide tolerance, improved water use efficiency, improved yield, improved drought tolerance, improved seed quality, improved nutritional value, hybrid seed production, or disease or fungal resistance. Transgenic events in soybeans are known to those skilled in the art. For example, a list of such traits is provided by the United States Department of Agriculture (USDA) Animal and Plant Health Inspection Service (APHIS) and can be found on the World Wide Web at the website www.aphis.usda.gov. Thus, two or more transgenic events may be combined in progeny seeds or plants by crossing two parent plants, each containing one or more transgenic events, collecting the progeny seeds, and selecting progeny seeds or plants containing two or more transgenic events. These steps may be repeated until the desired combination of transgenic events in the progeny is achieved. Backcrossing to the parent plant and outcrossing to non-transgenic plants are also contemplated, as is vegetative propagation.

[0127] Plant parts derived from soybean plants containing the soybean event GM_CSM63770 are also provided. As used herein, "plant part" refers to any part of a plant composed of material derived from a soybean plant containing the event GM_CSM63770. Plant parts include, but are not limited to, pollen, ovules, sheaths, flowers, root or stem tissue, fibers, and leaves. The plant parts may be capable of growing, non-growing, renewable, and / or non-renewable.

[0128] Further provided is a commercial product derived from a soybean plant comprising the soybean event GM_CSM63770 and containing a detectable amount of a nucleic acid specific to the soybean event GM_CSM63770. As used herein, "commercial product" refers to any composition or product composed of a soybean plant containing the DNA of the soybean event GM_CSM63770, whole soybean seeds or processed soybean seeds, or materials derived from one or more plant cells and / or plant parts. Non-growing commercial products include, but are not limited to, non-growing seeds, whole soybean seeds or processed soybean seeds, seed parts, and plant parts, animal feed containing soybeans, soybean oil, soybean protein, soybean meal, soybean flour, soybean flakes, soybean bran, soy milk, soybean cheese, soybean wine, paper containing soybeans, cream containing soybeans, soybean biomass, and fuel products manufactured using soybean plants and soybean parts. Growing commercial products include, but are not limited to, seeds, plants, and plant cells. Thus, a soybean plant comprising the soybean event GM_CSM63770 can be used to produce any commercial product normally obtained from soybeans. Any such commercial product derived from a soybean plant comprising the soybean event GM_CSM63770 may contain at least a detectable amount of the specific and unique DNA corresponding to the soybean event GM_CSM63770, and in particular, may contain a detectable amount of a polynucleotide comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Any standard detection method for nucleotide molecules can be used, including the detection methods disclosed herein. A commercial product is within the scope of the present invention if it contains in the commercial product any detectable amount of a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0129] The soybean plants, soybean plant cells, soybean seeds, soybean plant parts (e.g., pollen, ovules, hairs, spikes, anthers, rachis, root tissue, stem tissue, leaf tissue), soybean progeny plants, and commodity products of the present invention are thus useful, inter alia, for agricultural purposes, for growing plants for the purpose of producing seeds and / or plant parts containing the soybean event GM_CSM63770, for breeding and research purposes of plants to produce progeny containing the soybean event GM_CSM63770, for use in microbiological techniques for industrial and research applications, and for sale to consumers.

[0130] Provided are methods for producing pest-resistant soybean plants comprising a DNA sequence specific and unique to the event GM_CSM63770 of the present invention. The transgenic plants used in these methods may be homozygous or heterozygous for the transgene. Progeny plants produced by these methods may be variant or hybrid plants and may be grown from seeds produced by plants containing the soybean event GM_CSM63770 and / or from seeds of plants pollinated with pollen from plants containing the soybean event GM_CSM63770, and may be homozygous or heterozygous for the transgene. The progeny plants may then be self-pollinated to produce plants of a pure breeding line, i.e., plants that are homozygous for the transgene, or alternatively, outcrossed, for example, to another unrelated plant, to produce variant or hybrid seeds or plants.

[0131] Provided is a method for detecting the presence of DNA derived from soybean cells, soybean tissues, soybean seeds, or soybean plants containing the soybean event GM_CSM63770 in a sample. One method includes (i) extracting a DNA sample from at least one soybean cell, soybean tissue, soybean seed, or soybean plant, (ii) contacting the DNA sample with at least one primer capable of producing a DNA sequence specific to the DNA of event GM_CSM63770 under conditions appropriate for DNA sequencing, (iii) performing a DNA sequencing reaction, and then (iv) confirming that the nucleotide sequence contains a nucleotide sequence specific to event GM_CSM63770 of the construct contained therein, for example, one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Another method includes (i) extracting a DNA sample from at least one soybean cell, soybean tissue, soybean seed, or soybean plant, (ii) contacting the DNA sample with a primer pair capable of producing an amplicon from the DNA of event GM_CSM63770 under conditions appropriate for DNA amplification, (iii) performing a DNA amplification reaction, and then (iv) detecting the amplicon molecule and / or confirming that the nucleotide sequence of the amplicon contains a nucleotide sequence specific to event GM_CSM63770, for example, one selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. The amplicon should be an amplicon containing a sequence specific to event GM_CSM63770, for example, SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6. Detection of the nucleotide sequence specific to soybean event GM_CSM63770 in the amplicon is used for and / or is characteristic of determining and / or diagnosing the presence of soybean event GM_CSM63770-specific DNA in the sample.Examples of primer pairs capable of producing amplicons from the DNA of event GM_CSM63770 under conditions appropriate for DNA amplification are provided as SEQ ID NO: 14 and SEQ ID NO: 15. Other primer pairs may be readily designed by those skilled in the art to produce amplicons that include SEQ ID NO: 1, or SEQ ID NO: 2, or SEQ ID NO: 3, or SEQ ID NO: 4, or SEQ ID NO: 5, or SEQ ID NO: 6. Such primer pairs include at least one primer within a genomic region adjacent to the insert and a second primer within the insert. Another method for detecting the presence of DNA derived from soybean cells, soybean tissue, soybean seeds, or soybean plants containing the soybean event GM_CSM63770 in a sample includes (i) extracting a DNA sample from at least one soybean cell, soybean tissue, soybean seed, or soybean plant, (ii) contacting the DNA sample with a DNA probe specific for the DNA of event GM_CSM63770, (iii) hybridizing the probe to the DNA sample under stringent hybridization conditions, and then (iv) detecting hybridization between the probe and the target DNA sample. An example of the sequence of a DNA probe specific for event GM_CSM63770 is provided as SEQ ID NO: 16. Other probes may be readily designed by those skilled in the art and include at least one fragment of genomic DNA adjacent to the insert and at least one fragment of the inserted DNA, e.g., the sequences provided in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 10. Detection of hybridization of the probe to the DNA sample is used to diagnose or is characteristic of the presence of GM_CSM63770-specific DNA in the sample. The absence of hybridization is used to diagnose or is characteristic of an alternative absence of GM_CSM63770-specific DNA in the sample.

[0132] Provided is a DNA detection kit that is useful for identifying the DNA of the soybean event GM_CSM63770 in a sample and can also be applied to a method for breeding soybean plants containing the DNA of an appropriate event. Such a kit contains DNA primers and / or probes containing the fragments of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. An example of such a kit contains at least one DNA molecule of a continuous nucleotide of SEQ ID NO: 10 that is long enough to function as a DNA probe useful for detecting the presence and / or absence of DNA derived from a transgenic soybean plant containing the event GM_CSM63770 in a sample. The DNA derived from a transgenic soybean plant containing the event GM_CSM63770 contains a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10. Provided is a DNA molecule sufficient for use as a DNA probe that is useful for determining, detecting, or diagnosing the presence and / or absence of the DNA of the soybean event GM_CSM63770 in a sample provided as SEQ ID NO: 16. Other probes may be easily designed by those skilled in the art and should contain at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 consecutive nucleotides of SEQ ID NO: 10 and should be sufficiently specific for the DNA of the soybean event GM_CSM63770 to identify the DNA derived from the event.

[0133] Another type of kit includes primer pairs useful for producing amplicons useful for detecting the presence and / or absence of DNA derived from the transgenic soybean event GM_CSM63770 in a sample. Such a kit contacts a target DNA sample with the primer pairs described herein and then performs a nucleic acid amplification reaction sufficient to produce an amplicon comprising a DNA molecule having at least one sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and then detects the presence and / or absence of the amplicon. Such a method may also include sequencing the amplicon or a fragment thereof, which determines the presence of GM_CSM63770-specific DNA in the target DNA sample, i.e., is used for its diagnosis or is characteristic thereof. Other primer pairs may be readily designed by those skilled in the art and include, but are not limited to, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the sequences provided in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 and should be sufficiently unique to the DNA of soybean event GM_CSM63770 to identify DNA derived from the event.

[0134] The kits and detection methods of the present invention are useful, inter alia, for identifying soybean event GM_CSM63770, selecting plant species or hybrids comprising soybean event GM_CSM63770, detecting the presence of DNA derived from transgenic soybean plants comprising event GM_CSM63770 in a sample, and observing a sample or a plant part derived from a soybean plant comprising event GM_CSM63770 for the presence and / or absence of soybean plants comprising event GM_CSM63770.

[0135] The sequence, junction sequence, or flanking sequence of the heterologous DNA insert from the soybean event GM_CSM63770 can be verified (and corrected as necessary) by standard DNA sequencing of the amplicon or cloned DNA after amplifying such a sequence from the event using primers derived from the sequences provided herein.

[0136] Methods are provided for detecting the zygosity of a transgene allele in DNA derived from a soybean cell, soybean tissue, soybean seed, or soybean plant comprising the soybean event GM_CSM63770 in a sample. One method comprises (i) extracting a DNA sample from at least one soybean cell, soybean tissue, soybean seed, or soybean plant, (ii) contacting the DNA sample with a primer pair capable of producing a first amplicon diagnostic of or characteristic of soybean event GM_CSM63770, (iii) contacting the DNA sample with a primer pair capable of producing a second amplicon diagnostic of or characteristic of native soybean genomic DNA not containing soybean event GM_CSM63770, (iv) performing a DNA amplification reaction, and then (v) detecting the amplicon, wherein the presence of only the first amplicon is diagnostic of homozygous soybean event GM_CSM63770 DNA in the sample and the presence of both the first amplicon and the second amplicon is diagnostic of a soybean plant heterozygous for an allele of soybean event GM_CSM63770. Exemplary sets of primer pairs are shown as SEQ ID NO: 14 and SEQ ID NO: 15, which produce amplicons for or characteristic of the event GM_CSM63770, and SEQ ID NO: 20 and SEQ ID NO: 15, which produce amplicons for or characteristic of wild-type soybean genomic DNA that does not contain the soybean event GM_CSM63770. A set of probes can also be incorporated into such an amplification method that is used in real-time PCR format using the above-mentioned sets of primer pairs. Exemplary sets of probes are shown as SEQ ID NO: 16 (used to diagnose or characteristic of the amplicons of soybean event GM_CSM63770) and SEQ ID NO: 21 (used to diagnose or characteristic of the amplicons of wild-type soybean genomic DNA that does not contain the soybean event GM_CSM63770).

[0137] Another method for determining zygosity comprises: (i) extracting a DNA sample from at least one soybean cell, soybean tissue, soybean seed, or soybean plant; (ii) contacting the DNA sample with a set of probes comprising at least a first probe that specifically hybridizes to the DNA of event GM_CSM63770 and at least a second probe that specifically hybridizes to soybean genomic DNA disrupted by the insertion of the foreign DNA of soybean event GM_CSM63770 and does not hybridize to the DNA of soybean event GM_CSM63770; (iii) hybridizing the set of probes with the sample under stringent hybridization conditions, wherein detecting hybridization of only the first probe under the hybridization conditions is used for or is characteristic of the diagnosis of homozygous alleles of the DNA of soybean event GM_CSM63770 in the sample, and detecting hybridization of both the first probe and the second probe under the hybridization conditions is used for or is characteristic of the diagnosis of heterozygous alleles of soybean event GM_CSM63770 in the DNA sample.

[0138] Yet another method for determining zygosity involves: (i) extracting a sample containing DNA from at least one soybean cell, soybean tissue, soybean seed, or soybean plant; (ii) contacting the DNA sample with a primer pair capable of producing an amplicon that is used for, or is characteristic of, the diagnosis of the alleles of the soybean event GM_CSM63770; (iii) contacting the DNA sample with a primer pair capable of producing an amplicon of an internal standard known to be present in single copy and homozygous in the soybean plant; (iv) contacting the DNA sample with a set of probes comprising at least a first probe that specifically hybridizes to the alleles of event GM_CSM63770 and at least a second probe that specifically hybridizes to the genomic DNA of an internal standard known to be present in single copy and homozygous in the soybean plant; (v) performing a DNA amplification reaction using real-time PCR to determine the cycle threshold (Ct value) of the amplicon corresponding to the sequence encoding the toxin and the single-copy homozygous internal standard; (vi) calculating the difference (ΔCt) between the Ct value of the amplicon of the single-copy homozygous internal standard and the Ct value of the amplicon of the sequence encoding the toxin; and (vii) determining zygosity, where ΔCt of approximately zero (0) indicates homozygosity of the inserted T-DNA and ΔCt of approximately one (1) indicates heterozygosity of the inserted T-DNA. Heterozygous and homozygous events are discriminated by ΔCt value units of approximately one (1). Considering the normal variations observed in real-time PCR due to multiple factors such as amplification efficiency and optimal annealing temperature, the range of “approximately one (1)” is defined as ΔCt 0.75 to 1.25. The primer pairs and probes for the above method for determining zygosity are capable of amplifying and detecting amplicons from the alleles of event GM_CSM63770 and the internal standard. Exemplary primer pairs for the detection of amplicons corresponding to the alleles of event GM_CSM63770 and the internal standard are shown as the combination of SEQ ID NO: 14 and SEQ ID NO: 15 (alleles of event GM_CSM63770) and the combination of SEQ ID NO: 17 and SEQ ID NO: 18 (internal standard).Exemplary probes associated therewith are shown as SEQ ID NO: 16 (allele of event GM_CSM63770) and SEQ ID NO: 19 (internal standard).

[0139] Deposit Information The deposit of a representative sample of soybean seeds containing event GM_CSM63770 was made on August 21, 2019, with the American Type Culture Collection (ATCC), located at 10801 University Boulevard, Manassas, Virginia 20110, USA, in accordance with the Budapest Treaty, and ATCC Accession No. PTA-126048 was assigned. Access to the deposit is available during the pendency of the application to the Commissioner of Patents and Trademarks and to those determined by the Commissioner to have a right to access upon request. When the patent issues, all restrictions on availability to the public are irrevocably removed. The deposit will be maintained by the depository institution for a period of thirty (30) years, or five (5) years from the last request, or for the life of the patent, whichever is longer, and will be replaced as necessary during that period.

Examples

[0140] Examples are given below to explain the present invention in more detail. In summary, it involves the creation and selection of soybean event GM_CSM63770, including the structure and testing of 125 constructs, the generation of 3,343 events, and the analysis of hundreds of individual plants out of thousands over six years through rigorous molecular, agronomic, and field tests.

[0141] Those skilled in the art will understand that, in light of the present disclosure, many changes can be made in the specific embodiments disclosed, but still, similar or analogous results can be obtained without departing from the spirit and scope of the present invention.

[0142] Example 1 Expression cassette test, construct design, construct selection, molecular characterization, efficacy test, field test, and event selection Often, it is necessary to create and screen multiple gene expression constructs and transformation events to identify constructs that, while showing optimal expression of the introduced gene of interest, also identify events that do not produce agronomic or phenotypic abnormalities.

[0143] For these reasons, large-scale research, development, and analysis were required for the development of transgenic soybean plants containing insecticidal proteins active against Lepidoptera without adversely affecting agronomics, yield, or stacking survival rates. Specifically, approximately 3,343 proof-of-concepts over six years and commercial transgenic events derived from 125 different plasmid vector constructs were developed, tested, and analyzed.

[0144] In this example, the design and testing of 125 different constructs in soybean plants will be described to identify constructs favorable for event creation. Each construct differed with respect to the coding sequences of the insecticidal protein and transcriptional regulatory elements, and these were tested to select constructs favorable for use in the expression of the insecticidal protein in plants. Each construct had a unique composition that differed by the composition (both the insecticidal protein and expression elements) of the expression cassette, orientation, and whether the protein targeted insertion into the chloroplast.

[0145] In the initial proof-of-concept and development stages, 121 constructs containing different combinations of 29 distinct promoters, 1 enhancer, 16 distinct introns, and 21 distinct insect toxin coding sequences, as well as 10 distinct 3'UTRs, were used to generate approximately 992 transformed events. These events were evaluated for phenotypic or agronomic abnormalities, expression levels of the insect toxin protein, and efficacy against selected Lepidoptera pest species. The resulting efficacy and protein expression data, as well as any information regarding phenotypic and agronomic abnormalities, were used to eliminate ineffective proteins, expression elements, and combinations, and these were used to design a smaller number of commercial binary transformation plasmid constructs for use in the next development stage.

[0146] In the next development stage, four new commercial constructs were created. These constructs contained combinations of two to three insect toxin transgene expression cassettes in different orientations (convergent or divergent). One of these constructs was excluded prior to transformation based on the study of the mode of action of one of the insect toxin proteins expressed in the construct. The remaining three, namely pM63770, Construct 2, and Construct 3, were used to generate transformation events (also referred to as "transformants"). Table 2 below shows the process of event selection and exclusion for all three of the transformation events of the commercial constructs.

Table 2

[0147] After shoot formation in culture, a subset of the transformation events was selected based on visual characteristics and initial molecular analysis. After transformation, 12,036 transformants were transferred to culture plates containing selection medium (also referred to as "plugged"). After the first molecular characterization assessment, 9,685 were excluded. Of the remaining 2,351 events, 1,877 were excluded based on observations of plant health and survival. The remaining 474 R0 events were transplanted into pots and grown in a greenhouse (GH) for further assays. These 474 events were evaluated for molecular characteristics and efficacy, and 327 events were excluded based on these tests.

[0148] The remaining 147 R0 events were self-pollinated to produce R1 seeds. These R1 events were further molecularly characterized and evaluated for zygosity, productivity, and maturity. From this analysis, 116 events were excluded. At this point of selection, it was also recognized that the events derived from Construct 3 did not meet the criteria for further advancement, and this construct and the related events were excluded from further tests and analysis. The remaining 9 events derived from Construct pM63770 and 14 events from Construct 2 were selected for further molecular characterization and evaluation in field trials.

[0149] During the process of R2 generation, these events were further evaluated in terms of molecular characteristics and productivity. During the growing seasons of Argentina in 2017 - 2018, 23 events (9 events derived from construct pM63770 and 14 events from construct 2) were evaluated for effectiveness in greenhouse and field trials, as well as for agronomics in field trials. Based on the R2 criteria (molecular characteristics and effectiveness), 1 event derived from construct pM63770 was excluded, and 7 events derived from construct 2 were also excluded. 1 event derived from each of these two constructs was excluded based on yield measurements. Another event derived from construct pM63770 was excluded after failure of cross - breeding with elite germplasm materials.

[0150] The remaining 20 events, namely 7 events derived from construct pM63770 and 13 events derived from construct 2, were evaluated for effectiveness in greenhouse and field trials as well as in field agronomic trials. Additionally, further molecular tests were conducted, including the identification of the T - DNA insertion locus, adjacent genes, and repetitive sequences near the insertion locus for each event. After considering effectiveness and agronomic data, as well as more detailed molecular characteristic evaluations, 5 events derived from construct pM63770 and 12 events derived from construct 2 were excluded, leaving only 2 events derived from construct pM63770 and 1 event derived from construct 2. 1 event derived from construct pM63770 was excluded after examining the details of the adjacent sequences provided by electronic Southern (eSouthern) data. The event GM_CSM63770 derived from construct pM63770 was selected as a product based on the final comparison of yield, effectiveness, and molecular characteristics between each single remaining event derived from construct pM63770 and construct 2.

[0151] Therefore, through multiple tests and comparisons of various constructs, construct pM63770, which is the transgene cassette provided as SEQ ID NO: 13, produced events that showed excellent efficacy against soybean podworms (SPW, Helicoverpa zea), soybean loopers (Chrysodeixis includens), velvetbean caterpillars (Anticarsia gemmatalis), southern armyworms (Spodoptera eridania), black armyworms (Spodoptera cosmioides), South American podworms (Helicoverpa gelotopoeon), sunflower loopers (Rachiplusia nu), bean shoot moths (Crocidosema aporema), green cloverworms (Hypena scabra), and sorghum webworms (Elasmopalpus lignosellus), which are lepidopteran pest species, compared to the events produced by all other constructs evaluated. Soybean event GM_CSM63770 was selected from a large group of events generated using this construct based on its excellent properties regarding efficacy and agricultural productivity compared to other events generated using this pM63770 construct.

[0152] Example 2 Soybean event GM_CSM63770 shows resistance to soybean podworms, soybean loopers, velvetbean caterpillars, southern armyworms, black armyworms, South American podworms, sunflower loopers, bean shoot moths, green cloverworms, and sorghum webworms, which are lepidopteran pests. In this example, the activity of soybean event GM_CSM63770 against several different lepidopteran pests of soybeans is described. The insecticidal toxin proteins Cry1A.2 and Cry1B.2, when expressed together in soybean event GM_CSM63770, confer resistance against soybean podworm (SPW, Helicoverpa zea), soybean looper (SBL, Chrysodeixis includens), velvetbean caterpillar (VBC, Anticarsia gemmatalis), southern armyworm (SAW, Spodoptera eridania), black armyworm (BLAW, Spodoptera cosmioides), South American podworm (SAPW, Helicoverpa gelotopoeon), sunflower looper (SFL, Rachiplusia nu), bean shoot moth (BSM, Crocidosema aporema), green cloverworm (GCW, Hypena scabra), and sorghum webworm (LCSB, Elasmopalpus lignosellus).

[0153] Screenhouse trials were conducted at three locations to evaluate resistance against southern armyworm (SAW, Spodoptera eridania), velvetbean caterpillar (VBC, Anticarsia gemmatalis), and soybean podworm (SPW, Helicoverpa zea). Soybean event GM_CSM63770 was evaluated using the complete randomized block design along with events derived from transformation from constructs, namely pM63770, construct 2, and construct 3. For each event plot, approximately 8 seeds per foot were sown in 6-foot rows. Each event had 3 replicates and thus each event was represented in the screenhouse by 3 separate plots randomly placed within the screenhouse. The non-transformed event functioned as a negative control and its plots were also randomly assigned locations within the screenhouse.

[0154] Infestation of SPW and VBC was achieved using adult moths. Insects were raised to pupation in insect emergence cages maintained in temperature- and humidity-controlled incubators. Insects were released into screenhouses. Approximately 1,200–2,000 adults were used for each release in the screenhouse. For SPW, adults were released into the screenhouse weekly until the R1–R2 stages of soybean development. For VBC, adults were released into the screenhouse once every two weeks between the V4 and R3 developmental stages. Approximately 1,200–2,000 adults were released into the screenhouse each time. Adult moths required continuous access to a 10 percent sucrose solution for normal life span and fecundity. Cotton wool was placed in a plastic food container, and then the sugar solution was poured into the container and allowed to completely soak into the cotton. The sugar solution was replenished daily until adult activity decreased, which was usually about two weeks after the final release of adults.

[0155] Direct egg infestation was used for SAW because this insect does not oviposit preferentially or uniformly in soybean. Approximately 250,000-320,000 eggs were used per infestation applied once every 2 weeks during developmental stages R1-R3. Pieces of paper containing equal numbers of SAW eggs were attached by wrapping the paper around stout leaf stalks in the upper canopy and stapling the paper securely. One piece of paper was placed on a plant within 1 foot of the front edge of the plot, a second piece of paper was placed on a plant in the middle of the plot, and a third piece of paper was placed on a plant within 1 foot of the rear edge of the plot.

[0156] The litter for all three insect species was evaluated twice after each infestation, i.e., once at approximately two weeks after infestation and again at approximately four weeks after infestation. After each evaluation, the plant growth stage and the percentage of litter were recorded. To measure the percentage of litter, the grass canopy of each plot was divided into five zones. Trifoliate leaves were selected from each zone that showed damage representative of the zone it was selected from. The percentage of litter was measured using the diagrams of the damage scale provided to each evaluator. The average percentage of litter was calculated from the average of a single trifoliate leaf selected from each of the five zones. The production and damage of sheaths related to SPW were judged at the maturity of the soybean plants. Five plants were randomly selected from each plot, and the total number of sheaths and the number of damaged sheaths of each plant were recorded. The average number of sheaths and the average percentage of damaged sheaths were identified from the extracted samples of the five plants. Table 3 below shows the average percentage of litter evaluated for all three insect species, SAW, VBC, and SPW, for the soybean event GM_CSM63770 and the negative control. Table 4 below shows the average number of sheaths and the average percentage of sheath damage caused by SPW.

Table 3

Table 4

[0157] As can be seen from Table 3 above, the soybean event GM_CSM63770 provided excellent resistance against SAW, VBC, and SPW when compared to the negative control. Furthermore, as can be seen from Table 4, the soybean event GM_CSM63770 showed a decrease in the percentage of damaged sheaths compared to the negative control.

[0158] Screenhouse and field efficacy trials were conducted. Each plot in the screenhouse contained 42 seed rows in 2-meter rows. Each event was represented by three replicates randomly placed within the screenhouse. The screenhouse trials were conducted against the lepidopteran pests velvetbean caterpillar (VBC, Anticarsia gemmatalis), black armyworm (BLAW, Spodoptera cosmioides), South American armyworm (SAPW, Helicoverpa gelotopoeon), and soybean looper (SFL, Rachiplusia nu). Approximately 500 adult moths were released into the screenhouse per infestation. Two infestations were conducted, the first at the V3 developmental stage and the second at the R2 developmental stage. Adult moths were maintained as described above by saturating cotton wool in the containers with a 10% sugar solution. Defoliation percentage was measured as described above. Table 5 below shows the mean defoliation percentages measured for the soybean event GM_CSM63770 and the negative control.

Table 5

[0159] As can be seen from Table 5 above, the soybean event GM_CSM63770 showed very good resistance against BLAW, SAPW, SBL, and SFL when compared to the negative control.

[0160] The field efficacy trials were also conducted at six different locations (A - F). Each plot consisted of four rows of 10 meters and contained 25 seeds per meter at approximately 1 - meter intervals. Each event had three replicate plots randomly placed within the field. Sample extraction in the field was performed to measure the type of Lepidoptera species in each field and their respective percentages. The prominent Lepidoptera species in the fields were Velvetbean Caterpillar (VBC, Anticarsia gemmatalis), Soybean Looper species (SBL / SFL) which included Soybean Looper (SBL, Chrysodeixis includens) and Sunflower Looper (SFL, Rachiplusia nu), Spodoptera species (SPO), and South American Armyworm (SAPW, Helicoverpa gelotopoeon). Insect observations were started at the beginning of the V3 stage in the non - transgenic border rows, and at least 10 samples were evenly distributed around the border. Sample extraction was done in the border rows every 7 - 10 days. Observations were continued until the R6 stage when pod filling was complete. Defoliation was measured in the same way as in the screenhouse trials. Pod damage was also conducted in the same way as in the screenhouse experiments. Additionally, the number of larvae per meter of the planted row was recorded. Since the resistant plants kill young larvae and leave few on the plant, a lower presence of Lepidoptera larvae in the transgenic events compared to the negative control plants is an indicator of resistance. Table 6 below shows the maximum defoliation, cumulative larvae per meter of row, and mean percentage of damaged pods. Additionally, the percentage of each Lepidoptera species is provided for each location.

[0161] As can be seen from Table 6 below, the soybean event GM_CSM63770 demonstrated Lepidoptera resistance under natural infestation in the field compared to the negative control. The major Lepidoptera pest species in most of the fields were VBC and SBL / SFL. In one location, the percentage of SAPW was high and VBC was hardly or not present at all. From the following results, GM_CSM63770 was effective against VBC, SBL / SFL, and SAPW, as demonstrated by the percentage reduction in defoliation, reduction in cumulative larval counts per meter of row, and reduction in pod damage.

Table 6

[0162] For the plants at two of these locations, damage by bean shoot moth (BSM, Crocidosema aporema) was also investigated. Ten plants were randomly selected from each of three plots for each event. The mean percent damage for the controls at Location 1 and Location 2 was 6 percent and 4.7 percent, respectively. The soybean event GM_CSM63770 showed no damage at Location 1 and only 0.3 percent damage at Location 2.

[0163] Screenhouse efficacy trials were conducted at multiple locations against the lepidopteran pest species southern armyworm (SAW, Spodoptera eridania), soybean looper (SBL, Chrysodeixis includens), soybean podworm (SPW, Helicoverpa zea), and velvetbean caterpillar (VBC, Anticarsia gemmatalis). Infestations of SBL, SPW, and VBC were carried out using adult moths as described above. Infestation of SAW was carried out by egg infestation as described previously. Defoliation was measured as described previously. Plot size and replication were also as described above. Table 7 below shows the infestation frequency, stage at which infestation occurred, maximum percent defoliation, and percent reduction in defoliation corresponding to event GM_CSM63770 compared to the negative control.

Table 7

[0164] As can be seen from Table 7 above, the soybean event GM_CSM63770 was effective against SAW, SBL, SPW, and VBC, as demonstrated by a lower maximum percent defoliation and percent reduction in defoliation compared to the negative control. In these tests, the soybean event GM_CSM63770 conferred resistance against the southern armyworm (SAW, Spodoptera eridania), soybean looper (SBL, Chrysodeixis includens), soybean podworm (SPW, Helicoverpa zea), and velvetbean caterpillar (VBC, Anticarsia gemmatalis).

[0165] Field efficacy trials were conducted at five different locations (J - N) against natural lepidopteran damage. Each plot consisted of approximately 4 rows of 25 feet containing approximately 8 seeds per foot. Each event was represented by 4 plots randomly placed within the field. Defoliation was measured in the same manner as above and performed approximately every 10 days at developmental stages R1 - R6. Sheath damage was evaluated after maximum damage occurred to the negative control. Five plants were randomly selected per event to evaluate sheath damage. Population evaluations of lepidopteran species were conducted at developmental stages R1 - R6. In one or more fields, it was found to be invaded by the velvetbean caterpillar (VBC, Anticarsia gemmatalis), soybean looper (SBL, Chrysodeixis includens), soybean podworm (SPW, Helicoverpa zea), green cloverworm (GCW, Hypena scabra), yellow-striped armyworm (YAW, Spodoptera ornithogalli), and beet armyworm (BAW, Spodoptera exigua). The cumulative number of larvae per meter of row was also measured per event. Table 8 below shows the percent of lepidopteran species per location, the maximum and percent reduction in defoliation, as well as the cumulative number and percent reduction in larvae per meter of row.

Table 8

[0166] As can be seen from Table 8 above, based on the percentage of more prominent Lepidoptera species in each field, the soybean event GM_CSM63770 is effective against the velvetbean caterpillar (VBC, Anticarsia gemmatalis), soybean looper (SBL, Chrysodeixis includens), and green cloverworm (GCW, Hypena scabra), as demonstrated by the percentage reduction in defoliation and the percentage reduction in larvae for the soybean event GM_CSM63770 when compared to the negative control. For example, at location D, the most dominant pest species were GCW and SBL, representing 94% of all species observed in that field. While no surviving larvae were seen in the event entry, more than 21 larvae per linear meter were observed in the negative control, thus the absence of larvae in GM_CSM63770 supports that this event is highly resistant to these two species.

[0167] Sheath damage was evaluated at location M where the dominant pest species was SBL. Table 9 shows the average percentage of damaged sheaths and the percentage reduction in sheath damage for the soybean event GM_CSM63770.

Table 9

[0168] As can be seen from Table 9 above, both the soybean event GM_CSM63770 and the negative control had similar numbers of sheaths per plant, but the event GM_CSM63770 showed an 80% reduction in sheath damage in the field where most Lepidoptera insects were SBL. The soybean event GM_CSM63770 demonstrated resistance to SBL.

[0169] Based on the measurement of fallen leaves, larval numbers, and pod damage in fields invaded naturally, the soybean event GM_CSM63770 was effective against the velvetbean caterpillar (VBC, Anticarsia gemmatalis), soybean looper (SBL, Chrysodeixis includens), and green cloverworm (GCW, Hypena scabra), which are Lepidoptera pests.

[0170] Screenhouse and field efficacy trials were conducted at two locations (O - P). The screenhouse trials were planted in Acevedo and Fontes Eira, Buenos Aires Province, and were conducted against the velvetbean caterpillar (VBC, Anticarsia gemmatalis), black armyworm (BLAW, Spodoptera cosmioides), South American corn earworm (SAPW, Helicoverpa gelotopoeon), soybean looper (SBL, Chrysodeixis includens), and sunflower looper (SFL, Rachiplusia nu), which are Lepidoptera pests. Three plots similar to those described above for the 2017 - 2018 Argentine screenhouse trials were randomly placed within the screenhouse. Each event was represented by three plots. Each screenhouse was invaded with adult moths and maintained on a sugar diet as described previously. The measurement of fallen leaves was conducted as described previously. Table 10 below shows the growth stage of the plants where invasion occurred, the maximum percentage and the percentage decrease of fallen leaves, for each insect species from these two locations. [Table 10]

[0171] As can be seen from Table 10 above, the soybean event GM_CSM63770 was effective against velvet bean caterpillar (VBC, Anticarsia gemmatalis), black armyworm (BLAW, Spodoptera cosmioides), South American bollworm (SAPW, Helicoverpa gelotopoeon), soybean looper (SBL, Chrysodeixis includens), and sunflower looper (SFL, Rachiplusia nu).

[0172] Field trials were conducted at five locations (Q - U). Field samples were taken to measure the type of lepidopteran species and their respective percentages in each field. The prominent lepidopteran species in the field were velvet bean caterpillar (VBC, Anticarsia gemmatalis), soybean looper species (SBL / SFL) soybean looper (SBL, Chrysodeixis includens) and sunflower looper (SFL, Rachiplusia nu), Spodoptera species (SPO), and South American bollworm (SAPW, Helicoverpa gelotopoeon). Each plot consisted of 4 rows of 8 meters and contained 25 seeds per row. Each event was represented by 3 replicated plots randomly placed within the field. Measurements of defoliation, cumulative larvae, and pod damage were as described previously. Table 11 shows the maximum percent defoliation and percent reduction in defoliation, cumulative larvae per linear meter of row and percent reduction in larvae, average percentage of damaged pods and percent reduction in pod damage for GM_CSM63770 and the negative control.

Table 11

[0173] As can be seen from Table 11 above, the maximum percentages of Lepidoptera pests in the fields of locations Q and U were VBC and SBL / SFL, while in the field of location S, VBC was the dominant pest. From the above results, the soybean event GM_CSM63770 was effective against VBC and SBL / SFL, as demonstrated by the percentage reduction in defoliation, the reduction in the cumulative larval amount per 1-meter row, and the reduction in pod damage.

[0174] The soybean event GM_CSM63770 was assayed against the lesser cornstalk borer (LCSB, Elasmopalpus lignosellus) using a leaf disk assay. Eight (8) plants from the soybean event GM_CSM63770 and the negative control were grown in a greenhouse. Sixteen leaf disks were collected from each plant and used for the assay with first-instar larvae. Measurements of the number of dead and first-instar larvae (number of dead + L1) were obtained. Table 12 below shows the mean number of dead + L1 percentages for GM_CSM63770 and the negative control.

Table 12

[0175] As can be seen from Table 12 above, the soybean event GM_CSM63770 is very effective against the lesser cornstalk borer (Elasmopalpus lignosellus).

[0176] From the above data, the soybean event GM_CSM63770 confers resistance to the lepidopteran pest species, soybean pod borer (Helicoverpa zea), soybean looper (Chrysodeixis includens), velvetbean caterpillar (Anticarsia gemmatalis), southern armyworm (Spodoptera eridania), black armyworm (Spodoptera cosmioides), South American pod borer (Helicoverpa gelotopoeon), sunflower looper (Rachiplusia nu), bean shoot moth (Crocidosema aporema), green cloverworm (Hypena scabra), and sorghum webworm (Elasmopalpus lignosellus).

[0177] Example 3 The soybean event GM_CSM63770 gives consistent yields and agronomics similar to those of untransformed A3555 soybean plants This example demonstrates that the transgenic soybean event GM_CSM63770 gives consistent yields and agronomics similar to those of untransformed A3555 soybean plants in the field.

[0178] Field trials were conducted at four separate growth stages to evaluate the agronomic traits and yields of the soybean event GM_CSM63770 compared to the parental transformation background under field conditions. Agronomic data, such as germination, first flowering date, plant height, days to maturity, and yield (moisture - corrected yield per acre) were evaluated plot by plot.

[0179] When compared to its transformation background A3555, soybean event GM_CSM63770 showed no significant differences in other agronomic traits, namely, the number of days to germination, flowering, and maturity. With the exception of plant height, soybean event GM_CSM63770 was approximately 1 inch taller than the wild-type A3555 control, but this was considered not biologically significant. The expression of the insecticidal proteins Cry1A.2 and Cry1B.2 in soybean event GM_CSM63770 had no adverse effect on the agricultural productivity and yield of soybean event GM_CSM63770 when compared to non-transgenic controls.

[0180] Example 4 Soybean event GM_CSM63770 event-specific endpoint TAQMAN® assay In the following examples, methods useful for identifying the presence of GM_CSM63770 in soybean samples are described. In event-specific endpoint TAQMAN® PCR, primer pairs and probes were designed for the purpose of identifying unique junctions formed between soybean genomic DNA and the inserted GM_CSM63770 DNA. Examples of conditions used to identify the presence of GM_CSM63770 in soybean samples in event-specific endpoint TAQMAN® PCR are described in Tables 13 and 14.

[0181] The sequence of the oligonucleotide forward primer SQ13805 (SEQ ID NO: 14) is identical to the nucleotide sequence corresponding to positions 13,177 to 13,202 of SEQ ID NO: 10. The sequence of the oligonucleotide reverse primer SQ51400 (SEQ ID NO: 15) is identical to the reverse complement of the nucleotide sequence corresponding to positions 13,280 to 13,310 of SEQ ID NO: 10. The sequence of the oligonucleotide probe PB4832 (SEQ ID NO: 16) is identical to the nucleotide sequence corresponding to positions 13,204 to 13,219 of SEQ ID NO: 10. Primers SQ13805 (SEQ ID NO: 14) and SQ51400 (SEQ ID NO: 15), together with probe PB4832 (SEQ ID NO: 16) that can be fluorescently labeled (e.g., 6-FAM (trademark) fluorescent label), can be used in an endpoint TAQMAN (registered trademark) PCR assay to identify the presence of DNA derived from GM_CSM63770 in a sample.

[0182] It should be apparent to those skilled in the art that, in addition to SQ13805 (SEQ ID NO: 14), SQ51400 (SEQ ID NO: 15), and PB4832 (SEQ ID NO: 16), other primers and / or probes can be designed to amplify or hybridize to sequences within SEQ ID NO: 10 that are unique and useful for detecting the presence of DNA derived from GM_CSM63770 in a sample.

[0183] Following standard molecular biology laboratory practices, a PCR assay for the identification of events was developed for the detection of GM_CSM63770 in samples. The parameters of either a standard PCR assay or a TAQMAN® PCR assay were optimized for each set of primer pairs and probes (e.g., probes labeled with a fluorescent tag such as 6-FAM™) used to detect the presence of DNA derived from GM_CSM63770 in the sample. Controls for the PCR reaction included an internal control primer and an internal control probe specific for a region within the soybean genome used as an internal control (e.g., labeled with VIC®), the primers SQ549 (SEQ ID NO: 17), SQ546 (SEQ ID NO: 18), and the probe PB0004 (SEQ ID NO: 19) labeled with VIC®.

[0184] Generally, the parameters optimized for the detection of GM_CSM63770 in samples included the concentrations of the primers and probes, the amount of template DNA, and the PCR amplification cycle parameters. Controls for this analysis included a positive control from soybean containing GM_CSM63770, a negative control from non-transgenic soybean, and a negative control without template DNA.

Table 13

Table 14

[0185] Example 5 Assay for Determining the Zygosity of the Soybean Event GM_CSM63770 Using TAQMAN® The following examples describe a method useful for identifying the zygosity of event GM_CSM63770. For the purpose of identifying specific characteristics of the positive allele for the T-DNA insertion that gave rise to event GM_CSM63770, pairs of PCR primers and probes were designed, and pairs of PCR primers and a probe specific for a region within the soybean genome that is used as an internal control represented within the soybean genome as a homozygous form were designed as an internal control probe.

[0186] Pairs of PCR primers and probes specific for the GM_CSM63770 transgenic allele described in Example 4, namely, PCR primers SQ13805 (SEQ ID NO: 14), SQ51400 (SEQ ID NO: 15), and 6-FAM™ labeled probe PB4832 (SEQ ID NO: 16), and pairs of PCR primers and probes specific for the internal control, namely, primers SQ549 (SEQ ID NO: 17), SQ546 (SEQ ID NO: 18), and VIC® labeled probe PB0004 (SEQ ID NO: 19), are used in a real-time PCR reaction as described in Example 6 above.

[0187] After amplification, the cycle threshold (Ct value) of the amplicon corresponding to the inserted allele of GM_CSM63770 and the single-copy homozygous internal standard is determined. The difference (ΔCt) between the Ct value of the single-copy homozygous internal standard amplicon and the Ct value of the amplicon of the inserted allele of GM_CSM63770 is determined. With respect to zygosity, a ΔCt of approximately zero (0) indicates homozygosity of the inserted T-DNA of GM_CSM63770, and a ΔCt of approximately one (1) indicates heterozygosity of the inserted T-DNA of GM_CSM63770. The absence of an amplicon corresponding to the inserted allele of GM_CSM63770 indicates that the sample is null for the inserted T-DNA of GM_CSM63770. The Ct value in the TAQMAN® thermal amplification method has some variation due to multiple factors such as amplification efficiency and the ideal annealing temperature. Therefore, the range of "about one (1)" is defined as ΔCt 0.75 to 1.25.

[0188] Example 6 Assay for Determining the Zygosity of the Soybean Event GM_CSM63770 Using TaqMan® In the following example, a method useful for identifying the zygosity of event GM_CSM63770 in soybean samples is described.

[0189] For the purpose of identifying the specific characteristics of the positive and negative alleles for the T-DNA insertion that gave rise to event GM_CSM63770, pairs of PCR primers and probes are designed. Examples of conditions that can be used in event-specific zygosity TaqMan® PCR are shown in Tables 15 and 16. In this assay, four primers and two probes are mixed together with the sample. The DNA primer pairs used in the zygosity assay are primer SQ13805 (SEQ ID NO: 14) and SQ51400 (SEQ ID NO: 15), and GM_WTA3555F (SEQ ID NO: 20) and SQ51400 (SEQ ID NO: 15). The probes used in the zygosity assay are the 6FAM™-labeled probe PB4832 (SEQ ID NO: 16) and the VIC®-labeled probe GM_WTA3555PB (SEQ ID NO: 21). SQ13805 (SEQ ID NO: 14), SQ51400 (SEQ ID NO: 15), and the 6FAM™-labeled probe PB4832 (SEQ ID NO: 16) are used for or are characteristic of the diagnosis of the DNA of soybean event GM_CSM63770. The primers GM_WTA3555F (SEQ ID NO: 20) and SQ51400 (SEQ ID NO: 15) and the VIC®-labeled probe GM_WTA3555PB (SEQ ID NO: 21) are used for the diagnosis in the case where there is no copy of soybean event GM_CSM63770. That is, they are used for or are characteristic of the diagnosis of the wild-type allele.

[0190] When these three primers and two probes are mixed together with DNA extracted from a plant that is heterozygous for the soybean event GM_CSM63770 in a PCR reaction, fluorescence signals from both the 6FAM™-labeled probe PB4832 (SEQ ID NO: 16) and the VIC®-labeled probe GM_WTA3555PB (SEQ ID NO: 21) are present, which indicates a plant that is heterozygous for the soybean event GM_CSM63770 and is used for or is characteristic of its diagnosis. When these three primers and two probes are mixed together with DNA extracted from a plant that is homozygous for GM_CSM63770 in a PCR reaction, a fluorescence signal from only the 6FAM™-labeled probe PB4832 (SEQ ID NO: 16) is present, and no fluorescence signal from the VIC®-labeled probe GM_WTA3555PB (SEQ ID NO: 21) is present. When these three primers and two probes are mixed together with DNA extracted from a plant that is null (i.e., wild type) for the soybean event GM_CSM63770 in a PCR reaction, a fluorescence signal from only the VIC®-labeled probe GM_WTA3555PB (SEQ ID NO: 21) is present. The template DNA samples and controls for this analysis are a positive control derived from soybean containing the DNA of GM_CSM63770 (both known homozygous and known heterozygous samples), a negative control derived from non-transgenic soybean, and a negative control without template DNA.

Table 15

Table 16

[0191] Example 7 Identification of the Soybean Event GM_CSM63770 in Any GM_CSM63770 Breeding Event In the following examples, a method for identifying the soybean event GM_CSM63770 within the progeny of any breeding operation using the soybean event GM_CSM63770 will be described. For example, the soybean event GM_CSM63770 can be stacked by breeding with any of the soybean events known in the art for controlling Lepidoptera pests, such as, but not limited to, MON87701, MON87751, DAS81419 (Lepidoptera resistance and herbicide resistance), or by site-specific gene transfer. The soybean event GM_CSM63770 can also be stacked by breeding with other transgenic soybean events known in the art for providing herbicide resistance, namely, but not limited to, A2704-12 (U.S. Patent Application Publication No. US20080320616), A5547-127 (U.S. Patent Application Publication No. US2008196127), CV127 (International Patent Application Publication No. WO2010080829), DS40278-9 (International Patent Application Publication Nos. WO2011022469, WO2011022470, WO2011022471), DAS44406-6 (U.S. Patent Application Publication No. US2014041083), DAS68416-4 (U.S. Patent Application Publication No. US20130296170), DAS81419-2 (U.S. Patent Application Publication No. US2013338006), DP356043-5 (U.S. Patent Application Publication No. US20100184079), FG72 (U.S. Patent Application Publication No. US2011162098), GMB151 (U.S. Patent Application Publication No. US2019345512), GTS 40-3-2, MON87708 (U.S. Patent Application Publication US, MON87751 (U.S. Patent Application No. US20140373191), MON89788, SYHT0H2 (U.S. Patent Application Publication No. US20140201860), and SYHT04R (U.S. Patent Application Publication No. US20140310835), etc., or by site-specific gene transfer.The soybean event GM_CSM63770 can also be stacked by breeding with other transgenic soybean events known in the art for providing resistance to herbicides and modified oils, or improvement in photosynthesis, or tolerance to drought, i.e., but not limited to, DP305423-1 (U.S. Patent Application Publication No. US20080312082), MON87705 (U.S. Patent Application Publication No. US2014373191), MON87712 (U.S. Patent Application Publication No. US2014373191), MON87769 (U.S. Patent Application Publication No. US2011067141), and HB4 (U.S. Patent Application Publication No. US2022090114), etc., or by site-specific gene transfer.

[0192] A DNA primer pair is used to produce an amplicon that is used for the diagnosis of the soybean event GM_CSM63770 or is characteristic of it. The amplicon used for the diagnosis of the soybean event GM_CSM63770 or characteristic of it contains at least one junction sequence. The junction sequences of the soybean event GM_CSM63770 are SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (in Figure 1, [1], [2], [3], [4], [5], and [6], respectively). SEQ ID NO: 1 is a 50-nucleotide sequence representing the 5' junction region of soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 1 is located at nucleotide positions 976 to 1,025 in SEQ ID NO: 10. SEQ ID NO: 2 is a 50-nucleotide sequence representing the 3' junction region of soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 2 is located at nucleotide positions 13,216 to 13,265 in SEQ ID NO: 10. SEQ ID NO: 3 is a 100-nucleotide sequence representing the 5' junction region of soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 3 is located at nucleotide positions 951 to 1,050 in SEQ ID NO: 10. SEQ ID NO: 4 is a 100-nucleotide sequence representing the 3' junction region of soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 4 is located at nucleotide positions 13,191 to 13,290 in SEQ ID NO: 10. SEQ ID NO: 5 is a 200-nucleotide sequence representing the 5' junction region of soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 5 is located at nucleotide positions 901 to 1,100 in SEQ ID NO: 10. SEQ ID NO: 6 is a 200-nucleotide sequence representing the 3' junction region of soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO: 6 is located at nucleotide positions 13,141 to 13,340 in SEQ ID NO: 10.

[0193] Examples of primer pairs that are used for the diagnosis of event GM_CSM63770 or that produce amplicons characteristic thereof include primer pairs based on the flanking sequences (SEQ ID NO: 11 and SEQ ID NO: 12) and the inserted T-DNA (SEQ ID NO: 9). To obtain a diagnostic amplicon in which SEQ ID NO: 1, or SEQ ID NO: 3, or SEQ ID NO: 6 is found, a forward primer molecule is designed based on the 5'-flanking soybean genomic DNA (SEQ ID NO: 11) from bases 1 to 1,000, and a reverse primer molecule is designed based on the inserted T-DNA (SEQ ID NO: 9) from positions 1,001 to 13,240. These primer molecules consist of a continuous nucleotide sequence of sufficient length to specifically hybridize to SEQ ID NO: 11 and SEQ ID NO: 9. To obtain a diagnostic amplicon in which SEQ ID NO: 2, or SEQ ID NO: 4, or SEQ ID NO: 6 is found, a forward primer molecule is designed based on the inserted T-DNA (SEQ ID NO: 9) from positions 1,001 to 13,240, and a reverse primer molecule is designed based on the 3'-flanking soybean genomic DNA (SEQ ID NO: 12) from positions 13,241 to 14,240. These primer molecules consist of a continuous nucleotide sequence of sufficient length to specifically hybridize to SEQ ID NO: 9 and SEQ ID NO: 12.

[0194] In practice, primers should be designed to produce amplicons within a limited size range, preferably 200 to 1000 bases. Generally, smaller-sized amplicons are more reliably produced in PCR reactions, allow for shorter cycle times, can be easily separated and visualized on agarose gels, or can be adapted for use in endpoint TAQMAN®-like assays. Additionally, the amplicons produced using the primer pair may be cloned into a vector, propagated, isolated, and sequenced, or may be directly sequenced by methods well known in the art. Any primer pair derived from the combination of SEQ ID NO: 11 and SEQ ID NO: 9, or the combination of SEQ ID NO: 12 and SEQ ID NO: 9, which is useful for the diagnosis of the soybean event GM_CSM63770 or its progeny, or for a DNA amplification method for producing an amplicon characteristic thereof, is an aspect of the present invention. Any single isolated DNA polynucleotide primer molecule containing at least eleven (11) contiguous nucleotides of SEQ ID NO: 11, SEQ ID NO: 9, or SEQ ID NO: 12, or their complements, which is useful for the diagnosis of the soybean event GM_CSM63770 or its progeny, or for a DNA amplification method for producing an amplicon characteristic thereof, is an aspect of the present invention.

[0195] Examples of amplification conditions for this analysis are shown in Tables 14 and 15. The use of these methods or DNA primers homologous or complementary to SEQ ID NO: 11 or SEQ ID NO: 12, or any modification of the DNA sequence of the genetic elements contained in the transgene insert (SEQ ID NO: 9) of GM_CSM63770, which is used for the diagnosis of the soybean event GM_CSM63770 or for producing an amplicon characteristic thereof, is within the scope of the art. Diagnostic amplicons contain DNA molecules homologous or complementary to at least one transgene / genome junction DNA or a substantial portion thereof.

[0196] Analysis of plant tissue samples of soybean GM_CSM63770 should include a positive tissue control from a plant containing GM_CSM63770, a negative control from a soybean plant (e.g., A3555) that does not contain GM_CSM63770, and a negative control that does not contain soybean genomic DNA. The primer pair amplifies endogenous soybean DNA molecules and serves as an internal control for DNA amplification conditions. Additional primer sequences can be selected from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 by those skilled in the art of DNA amplification methods. The conditions selected for the production of amplicons by the methods shown in Tables 13 and 14 may vary, but will result in amplicons that are diagnostic of or characteristic of the DNA of soybean event GM_CSM63770. The use of DNA primer sequences within or with modifications to the methods of Tables 13 and 14 is within the scope of the present invention. An amplicon produced by at least one DNA primer sequence derived from SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:9 that is diagnostic of or characteristic of the soybean event GM_CSM63770 is an embodiment of the present invention.

[0197] A DNA detection kit comprising at least one DNA primer of sufficient length derived from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9 that produces a diagnostic amplicon for GM_CSM63770 or its progeny when used in a DNA amplification method is an embodiment of the present invention. A soybean plant or seed whose genome produces an amplicon that is used for or is characteristic of the diagnosis of the soybean event GM_CSM63770 when tested by a DNA amplification method is an embodiment of the present invention. An assay for the amplicon of the soybean event GM_CSM63770 can be performed using an Applied Biosystems GeneAmp™ PCR System 9700, Stratagene Robocycler®, Eppendorf®, Mastercycler® Gradient thermocycler, or any other amplification system that can be used to produce an amplicon that is used for or is characteristic of the diagnosis of the soybean event GM_CSM63770 as shown in Table 15.

[0198] Example 8 Insertion of a sequence into the soybean event GM_CSM63770 to facilitate removal of a transgene insertion using a single guide RNA The following examples illustrate how the transgene insertion in soybean event GM_CSM63770 can be excised using genome editing techniques. Sequences useful for excision of the soybean event GM_CSM63770 transgene insertion or expression cassette within SEQ ID NO: 10 can be introduced via genome editing using various methods, in particular, using a clustered regularly interspaced short palindromic repeat (CRISPR) editing system. The CRISPR-associated protein can be selected from a type I CRISPR-associated protein, a type II CRISPR-associated protein, a type III CRISPR-associated protein, a type IV CRISPR-associated protein, a type V CRISPR-associated protein, or a type VI CRISPR-associated protein, for example, but not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a (also known as Cpf1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, and Mad7. The CRISPR-associated protein and one or more guide RNAs (gRNAs) may be introduced into plant cells corresponding to soybean event GM_CSM63770 at the genomic target site via a double-strand break repair pathway to target a specific sequence within the transgene insertion locus, and examples of this pathway include, but are not limited to, non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or combinations thereof. Since one or more sequences can be inserted into the soybean event GM_CSM63770 transgene insertion locus, excision of the transgene insertion from soybean event GM_CSM63770 or a specific expression cassette within SEQ ID NO: 10 may be possible.

[0199] Sequences corresponding to the 5' and 3' genomic flanking sequences of the soybean event GM_CSM63770 (shown as SEQ ID NOs: 11 and 12), sequences corresponding to the 5' and 3' junction regions (shown as SEQ ID NOs: 1-6), and the inserted T-DNA were scanned for potential originator guide RNA recognition sites (OgRRS) containing a protospacer adjacent motif (PAM) site operably linked to a guide RNA hybridization site. The OgRRS can be located within the adjacent 5' or 3' genomic sequences, or within the 5' or 3' junction regions, or within the inserted T-DNA. The OgRRS is determined based on the specific CRISPR editing system selected. For example, Cas9 recognizes a G-rich protospacer adjacent motif (PAM) on the 3' side of its guide RNA binding site, whereas the Cas12a system recognizes a T-rich protospacer adjacent motif (PAM) on the 5' side of its guide RNA binding site.

[0200] Next, the OgRRS sequence is used to define a cognate guide RNA recognition site (CgRRS), which is inserted into the transgene insertion locus of event GM_CSM63770 using a CRISPR editing system. The CgRRS contains the same gRNA target sequence as the selected OgRRS. The CgRRS is inserted into a region within the transgene insertion locus of event GM_CSM63770, on the opposite side of the transgene insertion from the OgRRS, either the entire transgene insertion of soybean event GM_CSM63770 or a DNA fragment corresponding to a fragment within the transgene insertion of soybean event GM_CSM63770, e.g., in a manner that enables cleavage of an expression cassette or genetic element within the transgene cassette using a single-stranded gRNA. For example, as long as the OgRRS is located within the 3' genomic flanking sequence or 3' junction region, the CgRRS is inserted into the 5' genomic flanking sequence, or within the 5' junction region, or within the transgene insert, e.g., between expression cassettes or between genetic elements within an expression cassette. Insertion of the CgRRS on the opposite side of the transgene insertion from the OgRRS, or within the region between expression cassettes, enables cleavage of the transgene insertion or a specific expression cassette to be cleaved using a single-stranded gRNA. Using an OgRRS located between the expression cassettes of soybean event GM_CSM63770, a CgRRS can be designed that can be inserted into either the 5' or 3' genomic flanking sequence, enabling excision of either one of the expression cassettes using a single-stranded gRNA.

[0201] Table 17 below shows exemplary OgRRS sequences, which are located within the 5' and 3' genomic flanking sequences of soybean event GM_CSM63770 and between two expression cassettes and can be used in a CRISPR editing system employing Cas12a, i.e., a type V CRISPR-associated protein (the coding sequence is shown in SEQ ID NO: 34 and the protein sequence is shown in SEQ ID NO: 35).

Table 17

[0202] Table 18 below shows gRNAs that can be used to target the Cas12a nuclease for cleavage within both the OgRRS and CgRRS sequences and contain a polyT transcription termination region. [Table 18]

[0203] Any of the OgRRS sequences shown in Table 17 above can alternatively be used as a site for inserting a CgRRS designed using a different OgRRS. For example, to enable excision of the entire transgene insertion of event GM_CSM63770, the CgRRS can be inserted into the flanking sequence. To illustrate this approach, OgRRS_3-1 is selected as the OgRRS used to design the corresponding CgRRS-containing DNA fragment, and OgRRS_5-3 is selected as the target site into which the CgRRS-containing DNA fragment is inserted. Using the Cas12a editing system, the OgRRS_5-3 site is targeted using a gRNA, namely gRNA_OgRRS_5-3 shown in Table 18, and cleaved within the OgRRS_5-3 site. Next, the CgRRS-containing DNA fragment containing the OgRRS_3-1 target site is inserted into the cleavage site introduced into the OgRRS_5-3 sequence. After selection of the transgenic event containing the introduced CgRRS site, the event can be bred into another germplasm. If desired, the transgene insertion of soybean event GM_CSM63770 can be excised from the plant using the Cas12a editing system and a gRNA, namely gRNA_OgRRS_3-1 shown in Table 18.

[0204] Any of the OgRRS sequences shown in Table 17 within the 5' or 3' genomic flanking sequences of the soybean event GM_CSM63770 can be used as a site for inserting the CgRRS-containing DNA fragment, contains the OgRRS sequence present between the expression cassettes, and enables excision of a specific expression cassette using single-stranded gRNA. To illustrate this approach, OgRRS_In-1 is selected as the OgRRS used to design the corresponding CgRRS-containing DNA fragment, and OgRRS_5-2 is selected as the target site where the CgRRS-containing DNA fragment is inserted. Using the Cas12a editing system, the OgRRS_5-2 site is targeted and cleaved within the OgRRS_5-2 site using a gRNA, namely gRNA_OgRRS_5-2 shown in Table 18. Next, the CgRRS-containing DNA fragment containing the OgRRS_In-1 target site is inserted into the cleavage site introduced into the OgRRS_5-2 sequence. After selection of the transgenic event containing the introduced CgRRS site, the event can be bred into another germplasm. If desired, the first expression cassette expressing the Cry1A.2 toxin protein can be excised from the plant using the Cas12a editing system and a gRNA, namely gRNA_OgRRS_In-1 shown in Table 18.

[0205] CgRRS can be introduced into the transgene insertion locus via multiple methods using the CRISPR system. For example, the CRISPR system can be used to target the 5' insertion of a blunt-ended double-stranded DNA fragment to a genomic target site of interest, such as an OgRRS other than the OgRRS selected for the design of CgRRS. The endonuclease activity mediated by CRISPR can introduce a double-stranded break (DSB) into the selected genomic target site, and the blunt-ended double-stranded DNA fragment is inserted into the DSB by DNA repair, such as microhomology-driven non-homologous end joining DNA repair. The blunt-ended double-stranded DNA fragment can be designed with 1-10 bp of microhomology at both the 5' and 3' ends of the DNA fragment corresponding to the 5' and 3' flanking sequences at the cleavage site of the protospacer at the genomic target site.

[0206] The CRISPR system can be introduced into event GM_CSM63770 by several methods. One or more expression cassettes encoding the gRNA and / or the CRISPR-associated protein components of the type I, type II, type III, type IV, type V, or type VI CRISPR-Cas system are transiently introduced into the cell. The DNA fragment containing the CgRRS, together with the introduced one or more expression cassettes encoding the gRNA and / or the CRISPR-associated protein, is provided in an amount sufficient to modify the cell but does not persist after the intended period has elapsed or after one or more cell divisions. In such embodiments, no further steps are required to remove or separate the one or more expression cassettes encoding the gRNA and / or the CRISPR-associated protein from the modified cell.

[0207] Alternatively, an expression construct containing one or more expression cassettes for the expression of the gRNA, and an expression construct encoding a type I, type II, type III, type IV, type V, or type VI CRISPR-associated protein are stably transformed into event GM_CSM63770, and the CgRRS is introduced into the desired target locus. The gRNA instructs the nuclease to cleave within the target locus, which can be a different OgRRS from the selected OgRRS. This expression construct also contains the DNA fragment of the CgRRS flanking the PAM / gRNA sequence of the desired locus on the 5' and 3' sides (i.e., a different OgRRS from the selected OgRRS), which allows for the excision of the DNA fragment of the CgRRS, which can then be introduced into the target locus via the double-strand break repair pathway.

[0208] Other Cas12a PAM / gRNA sites can be found within SEQ ID NO: 10, which can be used as potential OgRRS sequences depending on the desired result after genome editing. Table 19 below shows the coordinates of each of the 418 potential OgRRS sequences within SEQ ID NO: 10 and the elements in which they can be present. Those shown in bold are those shown previously in Table 17.

Table 19-1

Table 19-2

Table 19-3

Table 19-4

Table 19-5

Table 19-6

Table 19-7

Table 19-8

Table 19-9

Table 19-10

Table 19-11

[0209] Example 9 Modification of soybean event GM_CSM63770 to facilitate genome editing technology using two guide RNAs In this example, a CRISPR editing system containing two guide RNAs was used by a genome editing method to define the soybean event GM_CSM63770 and to illustrate the excision of all or any part of the transgenic inserted DNA or the expression cassette within the transgenic inserted DNA present therein. Excision of the transgenic insert or the expression cassette within SEQ ID NO: 9 or SEQ ID NO: 10 of event GM_CSM63770 can be performed by genome editing using various methods. In one embodiment, a CRISPR editing system comprising a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein and two cognate guide RNAs can be used for targeted excision. The CRISPR-associated protein is an RNA-guided nuclease and can be selected from type I CRISPR-associated proteins, type II CRISPR-associated proteins, type III CRISPR-associated proteins, type IV CRISPR-associated proteins, type V CRISPR-associated proteins, or type VI CRISPR-associated proteins, such as, but not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a (also known as Cpf1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, and Mad7. The CRISPR-associated protein and the two guide RNAs (gRNAs) can be introduced into plant cells containing the soybean event GM_CSM63770 to target specific sequences within the transgene insertion locus. In one embodiment, the CRISPR nuclease system cleaves at two different guide RNA hybridization sites, thereby enabling the excision of the intervening sequence.After DNA cleavage, the genomic sequence can be repaired at the genomic target site via a double-strand break repair pathway, which can include, for example, non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination, synthesis-dependent strand annealing (SDSA), single-strand annealing (SSA), or combinations thereof.

[0210] The guide RNAs shown in Table 18 of Example 8 are used to excise the entire transgene cassette or, alternatively, to remove one of the two expression cassettes of the soybean event GM_CSM63770. For example, using a gRNA selected from the group consisting of SEQ ID NOs: 28 - 30 and the gRNA shown as SEQ ID NO: 31, the Cas12a nuclease is guided to cleave within the regions of the 5' and 3' genomic flanking sequences of the soybean event GM_CSM63770, and the entire transgene insert is excised. Alternatively, to excise the Cry1A.2 expression cassette from the soybean event GM_CSM63770, a gRNA is selected from the group consisting of SEQ ID NOs: 28 - 30, a gRNA is selected from the group consisting of SEQ ID NOs: 32 and 33, and is used to guide the Cas12a nuclease to cleave within the region of the 5' genomic flanking sequence and the region between the two transgene cassettes, and the Cry1A.2 expression cassette is excised. Similarly, to excise the Cry1B.2 expression cassette from the soybean event GM_CSM63770, a gRNA is selected from the group consisting of SEQ ID NOs: 32 and 33, and the gRNA shown as SEQ ID NO: 31 is used to guide the Cas12a nuclease to cleave within the region between the two transgene cassettes and the region of the 3' flanking genomic sequence, and the Cry1B.2 expression cassette is excised.

[0211] All publications and patent documents cited herein, to the extent each such publication or patent application is specifically and individually indicated to be incorporated by reference, are hereby incorporated by reference into this specification to the same extent.

[0212] Although the principles of the present invention have been illustrated and described, it should be apparent to those skilled in the art that the composition and details of the present invention can be changed without departing from such principles. The inventors claim all modifications that come within the spirit and scope of the appended claims.

Claims

1. A recombinant DNA molecule containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, and any of the aforementioned complete complements.

2. The recombinant DNA molecule according to claim 1, which originates from soybean event GM_CSM63770, and a representative sample of seeds containing the event is deposited as ATCC accession number PTA-126048.

3. A DNA molecule comprising a polynucleotide segment of sufficient length to function as a DNA probe that specifically hybridizes with the DNA of soy event GM_CSM63770 in a sample under stringent hybridization conditions, wherein the detection of the hybridization of the DNA molecule under stringent hybridization conditions is used to diagnose the presence of the DNA of soy event GM_CSM63770 in the sample.

4. The DNA molecule according to claim 3, wherein the sample comprises soybean plants, soybean plant cells, soybean seeds, soybean plant parts, soybean progeny plants, processed soybean seeds, animal feed containing soybeans, soybean oil, soybean meal, soybean flour, soybean flakes, soybean bran, soybean biomass, and fuel products manufactured using soybeans and soybean parts.

5. A pair of DNA molecules comprising a first DNA molecule and a second DNA molecule different from the first DNA molecule, which functions as a DNA primer when used in an amplification reaction with a sample containing template DNA of soy event GM_CSM63770, and produces an amplicon used to diagnose the presence of the soy event GM_CSM63770 DNA in the sample, wherein the amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, SEQ ID NOs: 2, SEQ ID NOs: 3, SEQ ID NOs: 4, SEQ ID NOs: 5, SEQ ID NOs: 6, SEQ ID NOs: 7, SEQ ID NOs: 8, SEQ ID NOs: 9, and SEQ ID NOs:

10.

6. A method for detecting the presence of a DNA segment used for diagnosing the DNA of soybean event GM_CSM63770 in a sample, a. Contacting the sample with the DNA molecule described in claim 3, b. Subjecting the sample and the DNA molecule to stringent hybridization conditions, and c. Detecting the hybridization of the DNA molecule with respect to the DNA in the sample. The method includes the above, The method wherein the detection is used to diagnose the presence of DNA of the soybean event GM_CSM63770 in the sample.

7. A method for detecting the presence of a DNA segment used for diagnosing the DNA of soybean event GM_CSM63770 in a sample, a. The sample is brought into contact with the DNA molecule pair described in claim 5. b. Performing an amplification reaction sufficient to produce DNA amplicons, and c. Detecting the presence of the DNA amplicon in the reaction. The method includes the above, The method wherein the DNA amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO:

10.

8. A method for detecting the presence of a protein used in the diagnosis of soybean event GM_CSM63770 in a sample, a. The sample is brought into contact with the first and second monoclonal antibodies, in which case the first monoclonal antibody specifically binds to Cry1A.2 and the second monoclonal antibody specifically binds to Cry1B.

2. b. Incubating the protein binding assay for a sufficient time to bind the monoclonal antibody, and c. The assay to detect the presence of the Cry1A.2 and Cry1B.2 proteins. The method includes the above, The method wherein the detection is used to diagnose the presence of DNA of the soybean event GM_CSM63770 in the sample.

9. The method according to claim 8, wherein the assay is an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay, or a lateral flow immunochromatography assay.

10. A soybean plant, plant part, cell, or part thereof containing DNA of soybean event GM_CSM63770, characterized by the detectable presence of a recombinant polynucleotide molecule containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, wherein the soybean plant, plant part, cell, or part thereof exhibits insecticidal activity when supplied as food to lepidopteran pests.

11. The aforementioned lepidopteran pests include the soybean pod worm (Helicoverpa zea), soybean inchworm (Chrysodeixis includedes), velvet bean caterpillar (Anticarsia gemmatalis), southern army worm (Spodoptera eridania), black army worm (Spodoptera cosmioides), South American pod worm (Helicoverpa gelotopoeon), sunflower inchworm (Rachiplusia nu), bean shoot moss (Crocidosema aporema), green clover worm (Hypena scabra), and sorghum leaf moth (Elasmopalpus) A soybean plant, soybean plant part, soybean cell, or part thereof, selected from the group consisting of lignosellus, according to claim 10.

12. The soybean plant, soybean plant part, soybean cell, or part thereof according to claim 10, wherein the soybean plant is further defined as a progeny of any generation of the soybean plant containing the soybean event GM_CSM63770.

13. A method for protecting soybean plants from insect invasion, comprising supplying an insecticidal amount of soybean plant cells or tissues containing soybean event GM_CSM63770 to the food of lepidopteran pests.

14. The aforementioned lepidopteran pests include the soybean pod worm (Helicoverpa zea), soybean inchworm (Chrysodeixis includedes), velvet bean caterpillar (Anticarsia gemmatalis), southern army worm (Spodoptera eridania), black army worm (Spodoptera cosmioides), South American pod worm (Helicoverpa gelotopoeon), sunflower inchworm (Rachiplusia nu), bean shoot moss (Crocidosema aporema), green clover worm (Hypena scabra), and sorghum leaf moth (Elasmopalpus) The method according to claim 13, selected from the group consisting of lignosellus.

15. A method for producing lepidopteran-resistant soybean plants, a. Crossing two different soybean plants, at least one of which contains the DNA of soybean event GM_CSM63770, to produce offspring; b. Confirming the presence of a DNA segment used for diagnosing the DNA of soybean event GM_CSM63770 in the offspring; and c. The method comprising selecting the progeny containing the DNA of the soybean event GM_CSM63770, The method wherein the progeny of step (c) is lepidopteran-resistant.

16. Soybean seeds containing a detectable amount of a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, or their complete complements.

17. A non-living soybean plant material containing a detectable amount of the DNA molecule described in claim 1.

18. A microorganism containing a detectable amount of the DNA molecule described in claim 1.

19. The microorganism according to claim 18, selected from the group consisting of bacterial cells and plant cells.

20. A commercial product containing a detectable amount of a DNA molecule specific to soybean event GM_CSM63770, wherein the molecule includes the DNA molecule described in claim 1.

21. Furthermore, the product according to claim 20, selected from the group consisting of whole soybean seeds or processed soybean seeds, animal feed containing soybeans, soybean oil, soybean meal, soybean flour, soybean flakes, soybean bran, soybean biomass, and fuel products manufactured using soybeans and soybean parts.

22. A soybean plant, a part of the soybean plant, or a soybean seed containing DNA that functions as a template in a DNA amplification method that produces an amplicon used for the DNA diagnosis of soybean event GM_CSM63770.

23. A method for determining the conjugation status of soybean plants or soybean seeds containing soybean event GM_CSM63770, a. Contacting a sample containing soybean DNA with a primer pair capable of producing an amplicon used for diagnosing the allele corresponding to the DNA of the soybean event GM_CSM63770, b. Contacting the sample with a second primer pair capable of producing an amplicon of an internal standard soybean genomic DNA known to be a single copy and homozygous in the soybean plant using a thermal amplification reaction. c. Contacting the sample with a set of probes comprising at least a first probe that specifically hybridizes with (or to) the DNA of the allele of soybean event GM_CSM63770, and a second probe that specifically hybridizes with an internal standard soybean genomic DNA known to be a single copy and homozygous in the soybean plant. d. Perform DNA amplification reactions using real-time PCR to identify the DNA of the allele of the soybean event GM_CSM63770 and the cycle threshold (Ct value) of the amplicon corresponding to the single-copy homozygous internal standard. e. Calculate the difference (ΔCt) between the Ct value of the single copy homozygous internal standard amplicon and the Ct value of the amplicon corresponding to the allele DNA of the amplicon sequence of the soybean event GM_CSM63770, and f. Determining the compatibility of the materials. The method comprising, wherein ΔCt approximately zero (0) indicates homozygosity of the T-DNA of the inserted event GM_CSM63770, and ΔCt approximately one (1) indicates heterozygosity of the T-DNA of the inserted soybean event GM_CSM63770.

24. The method according to claim 23, wherein the primer pair is selected from the group consisting of the combination of SEQ ID NO: 14 and SEQ ID NO: 15 and the combination of SEQ ID NO: 17 and SEQ ID NO: 18, and the probe is SEQ ID NO: 16 and SEQ ID NO:

19.

25. The method according to claim 23, wherein the ΔCt approximately -(1) indicating heterozygosity of the inserted GM_CSM63770 T-DNA is in the range of 0.75 to 1.

25.

26. A method for determining the conjugation status of soybean plants or soybean seeds containing soybean event GM_CSM63770, a. Contacting a sample containing soybean DNA with a set of primer pairs comprising at least two different primer pairs capable of producing a first amplicon used for diagnosing soybean event GM_CSM63770 and a second amplicon used for diagnosing natural soybean genomic DNA that does not contain soybean event GM_CSM63770; b. Performing a nucleic acid amplification reaction with the sample and the set of primer pairs; c. In the nucleic acid amplification reaction, the first amplicon used for diagnosing the DNA of soybean event GM_CSM63770, or the second amplicon used for diagnosing natural soybean genomic DNA that does not contain soybean event GM_CSM63770, in which case the presence of only the first amplicon is used to diagnose soybean plants or soybean seeds homozygous for the DNA of soybean event GM_CSM63770, and the presence of both the first and second amplicons is used to diagnose soybean plants or soybean seeds heterozygous for the DNA of soybean event GM_CSM63770, or d. Contacting a sample containing soybean DNA with a set of probes comprising at least a first probe that specifically hybridizes with the DNA of soybean event GM_CSM63770, and at least a second probe that specifically hybridizes with soybean genomic DNA disrupted by the insertion of heterologous DNA of soybean event GM_CSM63770, but does not hybridize with the DNA of event GM_CSM63770. e. Hybridizing the probe set with the sample under stringent conditions. The method includes the above, The method wherein, in the sample, detecting hybridization of only the first probe under the hybridization conditions is used to diagnose homozygous alleles of the DNA of soybean event GM_CSM63770, and detecting hybridization of both the first probe and the second probe under the hybridization conditions is used to diagnose soybean plants or soybean seeds heterozygous for soybean event GM_CSM63770.

27. The method according to claim 26, wherein the set of primer pairs includes the combination of SEQ ID NO: 14 and SEQ ID NO: 15, and the combination of SEQ ID NO: 20 and SEQ ID NO:

15.

28. The method according to claim 27, wherein the set of probes includes sequence number 16 and sequence number 21.

29. A DNA construct comprising a polynucleotide having a sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, wherein the 5' or 3' end of the construct comprises (i) at least 50 consecutive nucleotides of sequence number 11 or sequence number 36, or (ii) at least 50 consecutive nucleotides of sequence number 12 or sequence number 37.

30. The DNA construct according to claim 29, wherein the 5' end of the construct comprises at least 50 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 36, and the 3' end of the construct comprises at least 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO:

37.

31. The DNA construct according to any one of claims 29 to 30, wherein the 5' end of the construct contains one or more nucleotide sequences selected from SEQ ID NOs: 38 to 137.

32. The DNA construct according to any one of claims 29 to 30, wherein the 3' end of the construct contains one or more nucleotide sequences selected from SEQ ID NOs: 138 to 237.

33. A soybean plant, plant cell, plant part, or plant seed comprising the DNA construct described in claim 30.

34. A soybean plant, plant cell, plant part, or plant seed comprising a recombinant DNA construct integrated into chromosome 19, wherein the recombinant DNA construct confers resistance to lepidopteran insect pest species, and the recombinant DNA construct is integrated into the chromosome at a position where at least 50 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 36 and 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 37 are located laterally.

35. The soybean plant, plant cell, plant part, or plant seed according to claim 34, wherein at least 50 consecutive nucleotides of SEQ ID NO: 11 or SEQ ID NO: 36 include one or more nucleotide sequences selected from SEQ ID NOs: 38 to 137.

36. The soybean plant, plant cell, plant part, or plant seed according to any one of claims 35, wherein at least 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 37 include one or more nucleotide sequences selected from SEQ ID NOs: 138 to 237.

Images