Soybean transgenic strain GMCSM63770 as well as detection method and application thereof

Abstract

The invention provides a transgenic soybean strain GMCSM63770 and a transgenic soybean strain GMCSM63770. Plants, plant cells, seeds, plant parts, progeny plants and commodity products of the strain GMCSM63770 are included; polynucleotides dedicated to the definition and detection of the line GMCSM63770, as well as plants, plant cells, seeds, plant parts, progeny plants, and commodity products comprising the line GMCSM63770; and methods related to the detection, characterization and selection of the strain GMCSM63770.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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.

[0003] Incorporation of Sequence Listing

[0004] The sequence table is contained in a file named MONS567WO_ST26, which is 341,230 bytes in size (Microsoft Measured in China), created on June 16, 2023, submitted electronically with this application and incorporated by reference. Technical Field

[0005] The present invention relates to recombinant DNA molecules present in and / or isolated from soybean strain GM_CSM63770. The present invention also relates to transgenic soybean plants, plant parts and seeds, cells and agricultural products containing soybean strain GM_CSM63770, as well as methods of using the same and methods of detecting the presence of soybean strain GM_CSM63770. Transgenic soybean plants, plant parts, seeds and cells containing soybean strain GM_CSM63770 DNA exhibit resistance to infestation by lepidopteran insects. Background Art

[0006] Soybean (Glycine max) is an important crop and a major food source in many parts of the world. Biotechnology methods have been applied to soybean to improve the agronomic traits and quality of the product. Insect resistance is one such agronomic trait, which is achieved through the expression of heterologous insect toxins (also called transgenes) inserted into the genome of soybean plants.

[0007] Many different soybean transgenic lines have been described in the art that provide various types of insect resistance, particularly resistance to lepidopteran insects, and include MON87701, MON87751, and DAS81419 (lepidopteran resistance and herbicide tolerance). These transgenic lines have been in commercial use for a long time in many geographic regions around the world, and resistance to toxins expressed by target insect pests in these lines has been observed in many of the geographic regions where these lines have been deployed.

[0008] Therefore, there is a continuing need in the art to provide new transgenic lines of soybean that exhibit resistance to insect infestation, and preferably, the novel transgenic lines will confer resistance to target insects, including those species that have evolved resistance to currently commercially deployed traits, using a mode of action that does not overlap or is similar to the mode of action previously deployed in earlier commercial embodiments. An example of such a novel transgenic line is described herein, which confer resistance to lepidopteran insect infestation, including resistance to lepidopteran insects that have evolved resistance to previously deployed commercial embodiments. Summary of the invention

[0009] In one embodiment, the present invention provides a novel transgenic soybean strain, i.e. GM_CSM63770, which provides insecticidal control of soybean lepidopteran pests. In another embodiment, the present invention also provides transgenic plants, plant cells, seeds, plant parts and commodity products comprising soybean strain GM_CSM63770 DNA. The strain contains the unique and unique novel DNA of this GM_CSM63770 strain, and comprises the transgenic DNA segment inserted and the novel DNA segment adjacent to the DNA segment inserted. These adjacent segments are described as junction sequences in this article, and the sequence corresponds to the DNA sequence extending along the chromosome DNA adjacent to the chromosome breakpoint introduced into the inserted DNA. In another embodiment, the present invention provides soybean strain GM_CSM63770 DNA unique and unique polynucleotides, which can be used for identifying and detecting the presence of soybean strain GM_CSM63770 DNA in biological samples containing soybean tissues, and plants, plant cells, seeds, plant parts, progeny plants and commodity products comprising soybean strain GM_CSM63770 DNA. In yet another embodiment, methods are provided relating to selecting plant cells, plants and seeds comprising soybean line GM_CSM63770 DNA and detecting the presence (or absence) of soybean line GM_CSM63770 DNA in a sample, which methods allow a researcher to confirm whether the line DNA is present in a particular sample subjected to the method or methods relying on the subject nucleotide sequences of the present disclosure.

[0010] Therefore, 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 complementary sequences.

[0011] In one embodiment, the recombinant DNA molecule is derived from a soybean line in a sample containing seeds of soybean line GM_CSM63770, and the soybean line contains a corresponding unique and characteristic DNA segment corresponding to the seeds of soybean line GM_CSM63770, which is deposited under ATCC Accession No. PTA-126048.

[0012] Another aspect of the present invention provides a DNA molecule comprising a polynucleotide segment of sufficient length to serve as a DNA probe, wherein the polynucleotide segment specifically hybridizes with the soybean strain GM_CSM63770 DNA in a sample under stringent hybridization conditions, wherein detection of the hybridization of the DNA probe with the DNA in the sample under the stringent hybridization conditions will diagnose or characterize the presence of the soybean strain GM_CSM63770 DNA in the sample. In certain embodiments, the sample comprises soybean plants, soybean plant cells, soybean seeds, soybean pollen, soybean plant parts, soybean progeny plants, processed soybean seeds, animal feeds containing soybeans, soybean oil, soybean meal, soybean flour, soybean flakes, soybean bran, soybean biomass, and fuel products produced using soybeans and soybean parts, as long as such soybeans and soybean products contain a detectable amount of soybean strain GM_CSM63770 DNA or a detectable amount of novel toxin proteins containing soybean strain GM_CSM63770 produced by soybean plants, cells, etc.

[0013] 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 act as DNA primers when used together in an amplification reaction, the amplification reaction comprising the appropriate reagents required for a DNA amplification procedure for a sample containing a soybean line GM_CSM63770 template DNA to produce an amplicon that diagnoses or characterizes the presence of the soybean line GM_CSM63770 DNA in the sample. The amplicon produced will contain at least 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 complementary sequences.

[0014] Another embodiment of the present invention is a method for detecting the presence of a DNA segment that will be used to confirm the presence of soybean line GM_CSM63770 in a sample. In certain embodiments, the method is performed by contacting the sample with a probe DNA molecule that specifically hybridizes with a DNA uniquely associated with soybean line GM_CSM63770; then subjecting the sample to stringent hybridization conditions with the probe DNA molecule to allow the probe to bind to an appropriate complementary segment of soybean line GM_CSM63770-specific DNA. Detection of hybridization of the probe DNA molecule with the DNA in the sample is definitive, diagnostic, and definite, i.e., the DNA in the sample contains soybean line GM_CSM63770 DNA.

[0015] Yet another embodiment of the present invention is a method for detecting the presence of a DNA segment that diagnoses or characterizes soybean line GM_CSM63770 DNA in a sample containing soybean DNA. In one embodiment, the method comprises the following steps: contacting a biological sample with a pair of DNA molecules that act as thermal amplification primers that are specifically used to amplify a segment of soybean line GM_CSM63770 DNA; performing an amplification reaction sufficient to produce a DNA amplicon; and detecting the presence of a DNA amplicon in the reaction. Detection of the DNA amplicon will diagnose or characterize the presence of a detectable amount of soybean line GM_CSM63770 DNA in the sample, and the amplicon will contain all or a portion of the nucleotide sequence to be amplified between the primer hybridization positions, the portion of the nucleotide sequence to be amplified being 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 complementary sequences.

[0016] Another embodiment of the present invention is a method for detecting the presence of a protein that diagnoses or characterizes soybean line GM_CSM63770 in a sample, the method comprising: (a) contacting the sample with a first monoclonal antibody and a second monoclonal antibody, wherein in an immunoassay, the first monoclonal antibody specifically binds to Cry1A.2 and the second monoclonal antibody specifically binds to Cry1B.2; (2) incubating the immunoassay for a sufficient amount of time to allow the monoclonal antibodies to bind; and (3) detecting the presence of Cry1A.2 and Cry1B.2 proteins in the immunoassay, wherein the detection will diagnose or characterize the presence of the soybean line GM_CSM63770 DNA in the sample. The assay may be selected from the group consisting of: enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and lateral flow immunochromatographic assay.

[0017] Another embodiment of the present invention is a soybean plant, soybean plant part, soybean cell or 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 complete complementary sequences. Each of these SEQ ID NOs is a specific and selective DNA defining soybean line GM_CSM63770. Such soybean plants, soybean plant parts, soybean cells or parts thereof have an insecticidal effect when provided in a diet of lepidopteran insect pests. The target lepidopteran insect pests to be controlled include soybean podworm (Helicoverpa zea), soybean looper (Chrysodeixis includens), velvet bean 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 lesser corn stalk borer (Elasmopalpus lignosellus). In addition, the soybean plant can be further defined as a progeny of any generation of a soybean plant comprising soybean line GM_CSM63770, as long as the progeny also contains the specific and selective DNA defining soybean line GM_CSM63770.

[0018] Yet another embodiment of the present invention is a method for protecting soybean plants from insect infestation, wherein the method comprises providing an insecticidally effective amount of cells or tissues of a soybean plant comprising soybean line GM_CSM63770 in a diet of a lepidopteran insect pest. Contemplated lepidopteran insect pests include soybean pod borer (American bollworm), soybean armyworm (soybean looper), velvet bean caterpillar (Spodoptera exigua), southern cutworm (subtropical armyworm), black cutworm (Spodoptera litura), South American pod borer (Argentine bollworm), sunflower armyworm (Spodoptera exigua), bean stem moth (Spodoptera exigua), green armyworm (Spodoptera lucerna), and small corn stalk borer (Ostrichia annua).

[0019] Another embodiment of the present invention is a method for producing an insect-resistant soybean plant, the method comprising: a) artificially crossing two different soybean plants by conventional breeding to produce progeny, wherein at least one of the two different soybean plants contains soybean line GM_CSM63770 DNA; b) in seeds produced by breeding activities and confirming the presence of DNA segments used to diagnose or characterize soybean line GM_CSM63770 in progeny plants and tissues grown from such seeds; and c) selecting progeny containing soybean line GM_CSM63770 DNA. Such seeds and progeny are soybean plants resistant to Lepidoptera insects.

[0020] Another embodiment of the present invention is a soybean seed, non-living plant material or microorganism, which contains 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 a complete complementary sequence thereof.

[0021] Yet another embodiment is a commodity soy product comprising a detectable amount of a DNA molecule unique to soybean line GM_CSM63770, wherein the molecule 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. Contemplated commodity soy products include, but are not limited to, whole or processed soybean seeds, animal feeds containing soybeans, soybean oil, soybean meal, soybean flour, soy flakes, soybean bran, soybean biomass, and fuel products produced using soybeans and soybean portions.

[0022] Another embodiment of the present invention is a soybean plant, soybean plant part, or soybean seed thereof, comprising DNA that functions as a template when tested in a DNA amplification method, thereby producing an amplicon that is diagnostic or characterizing the presence of soybean line GM_CSM63770 DNA.

[0023] Yet another embodiment of the present invention is a method for determining the zygosity of a soybean plant or soybean seed, the soybean plant or soybean seed comprising a DNA describing soybean line GM_CSM63770. Zygosity is determined by a series of consecutive steps. In the first step, a sample comprising soybean DNA is contacted with a first primer pair, the first primer pair being able to produce an amplicon of a diagnosis or characterization DNA, the DNA describing soybean line GM_CSM63770 and only present in the line. Then, the sample comprising soybean DNA is contacted with a second primer pair, the second primer pair being designed to produce an amplicon of an internal standard known to be single-copy and homozygous in soybean plants. The method further comprises contacting the DNA sample with a probe set, the probe set comprising at least one first probe specifically hybridized with the allele of soybean line GM_CSM63770, and at least one second probe specifically hybridized with an internal standard genomic DNA known to be single-copy and homozygous in soybean plants. The method also includes performing a DNA amplification reaction using real-time PCR and determining a cycle threshold value (Ct value) of an amplicon corresponding to an allele of soybean line GM_CSM63770 and a 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 allele of the soybean line GM_CSM63770 amplicon can be calculated. In one embodiment, zygosity is determined, wherein ΔCt is about zero (0) indicating that the inserted T-DNA of soybean line GM_CSM63770 is homozygous, and ΔCt is about one (1) indicating that the inserted T-DNA of soybean line GM_CSM63770 is heterozygous. In certain embodiments of this method, the primer pair is selected from the group consisting of: a combination of SEQ ID NO: 14 and SEQ ID NO: 15 and a combination of SEQ ID NO: 17 and SEQ ID NO: 18; and wherein the probe is SEQ ID NO: 16 and SEQ ID NO: 19. In another embodiment of the present invention, the ΔCt of about one (1) indicative of heterozygosity for the inserted T-DNA of soybean line MON95275 is in the range of 0.75 to 1.25. In certain embodiments, the ΔCt of about zero (0) may be about 0, 0.05, 0.1, 0.15, 0.2, or 0.25, and in other embodiments, the 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 yet another embodiment, a ΔCt of about one (1) can be in the range of 0.75 to 1.25, 0.8 to 1.25, 0.85 to 1.25, 0.9 to 1.25, 0.95 to 1.25, 1.0 to 1.25, 1.05 to 1.25, 1.1 to 1.25, 1.15 to 1.25, 1.2 to 1.25, 0.75 to 1.2, 0.8 to 1.2, 0.85 to 1.2, 0.9 to 1.2, 0.95 to 1.2, 1.0 to 1.2, 1.05 to 1.2, 1.1 to 1.2, 1.15 to 1.2, 0.75 to 1.15, 0.8 to 1.15, 0.85 to 1.15, 0.9 to 1.15, 0.95 to 1.15, 1.0 to 1.15, 1.05 to 1. 15, 1.1 to 1.15, 0.75 to 1.1, 0.8 to 1.1, 0.85 to 1.1, 0.9 to 1.1, 0.95 to 1.1, 1.0 to 1.1, 1.05 to 1.1, 0.75 to 1.05, 0.8 to 1.05, 0.85 to 1.05, 0.9 to 1.05, 0.95 to 1.05, 1.0 to 1.05, 0.7 From 0.5 to 1.0, 0.8 to 1.0, 0.85 to 1.0, 0.9 to 1.0, 0.95 to 1.0, 0.75 to 0.95, 0.8 to 0.95, 0.85 to 0.95, 0.9 to 0.95, 0.75 to 0.9, 0.75 to 0.85, 0.75 to 0.8, 0.8 to 0.9, 0.8 to 0.85 or 0.85 to 0.9.

[0024] Another embodiment of the present invention is a method for determining the zygosity of a soybean plant, soybean seed, soybean pollen, ovule or cell, wherein each such biological component suspected of containing soybean line GM_CSM63770 DNA 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) wherein a first primer pair consists of a first primer and a second primer different from the first primer, which, when used in an amplification reaction with soybean DNA, is capable of producing a nucleic acid sequence useful for diagnosing or characterizing soybean line GM_CSM63770 in the sample; The method comprises the steps of: (i) detecting a first amplicon for the presence of soybean genomic DNA in the sample, and (ii) a second primer pair consisting of a first primer and a third primer different from the first primer and the second primer, wherein when used in an amplification reaction with soybean DNA, the second primer pair is capable of producing a second amplicon for diagnosing or characterizing natural soybean genomic DNA that does not contain or includes soybean line GM_CSM63770-specific DNA; b) performing a nucleic acid amplification reaction using the sample and the two primer pairs; c) detecting in the nucleic acid amplification reaction the first amplicon for diagnosing or characterizing soybean line GM_CSM63770, or the second amplicon for diagnosing or characterizing natural soybean genomic DNA that does not contain soybean line GM_CSM63770, wherein the presence of only the first amplicon in the sample will be used to diagnose or characterize the homozygous soybean line GM_CSM63770. DNA, and the presence of both the first amplicon and the second amplicon will diagnose or characterize a soybean plant that is heterozygous for the soybean line GM_CSM63770 allele; or b) contacting a sample containing soybean DNA with a probe set, the probe set containing at least one first probe that specifically hybridizes to the soybean line GM_CSM63770 DNA, and at least one second probe that specifically hybridizes to the soybean genomic DNA within a chromosomal DNA segment that is disrupted by the insertion of heterologous DNA of the soybean line GM_CSM63770; i) hybridizing the probe set to the sample under stringent hybridization conditions, wherein detection of only the first probe hybridizing under the hybridization conditions will diagnose or characterize a homozygous allele of the soybean line GM_CSM63770 DNA in the sample, and wherein detection of both the first probe and the second probe hybridizing under the hybridization conditions will diagnose or characterize a heterozygous allele of the soybean line GM_CSM63770 in the sample. In one embodiment of this method, the set of primer pairs comprises a combination of SEQ ID NO: 14 and SEQ ID NO: 15, and a combination of SEQ ID NO: 20 and SEQ ID NO: 15. In another embodiment of this method, the probe set comprises SEQ ID NO: 16 and SEQ ID NO:21.

[0025] Yet another embodiment of the present invention is to provide soybean plants, plant cells, pollen, plant parts and seeds, which contain a recombinant DNA construct integrated into chromosome 19. The recombinant DNA construct causes resistance to lepidopteran insect pest species. The recombinant DNA construct is integrated into the chromosome at a position flanked by at least 50 consecutive nucleotides of SEQ ID NO: 11 or 36 and at least 50 consecutive nucleotides of SEQ ID NO: 12 or 37. The at least 50 consecutive nucleotides of SEQ ID NO: 11 may include one or more nucleotide sequences selected from SEQ ID NO: 118-137. The at least 50 consecutive nucleotides of SEQ ID NO: 36 may include one or more nucleotide sequences selected from SEQ ID NO: 38-117. The at least 50 consecutive nucleotides of SEQ ID NO: 12 may include one or more nucleotide sequences selected from SEQ ID NO: 138-157. The at least 50 consecutive nucleotides of SEQ ID NO: 37 may include one or more nucleotide sequences selected from SEQ ID NO: 158-237.

[0026] The foregoing and other aspects of the invention will become apparent from the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1The orientation and alignment of the DNA elements / segments present in the nucleotide sequence of SEQ ID NO: 10 (indicated as

[10] in the figure), which is the sequence of the inserted transgenic DNA and adjacent to the 5' and 3' sequences of the soybean genome present in soybean line GM_CSM63770, are graphically depicted. SEQ ID NO: 1 is indicated as [1] and is a segment of fifty (50) nucleotides that spans twenty-five (25) nucleotides of the inserted DNA and twenty-five (25) nucleotides of any designated 5' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO: 2 is indicated as [2] and is a segment of fifty (50) nucleotides that spans twenty-five (25) nucleotides of the inserted DNA and twenty-five (25) nucleotides of any designated 3' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO:1 is embedded within SEQ ID NO:3 (indicated by [3]), which is a segment of (100) nucleotides that spans fifty (50) nucleotides of the inserted DNA and fifty (50) nucleotides of any specified 5' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO:2 is embedded within SEQ ID NO:4 (indicated by [4]), which is a segment of one hundred (100) nucleotides that spans fifty (50) nucleotides of the inserted DNA and fifty (50) nucleotides of any specified 3' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO:5 is indicated by [5], which is two hundred (200) nucleotides long, contains SEQ ID NO:3 and spans one hundred (100) nucleotides of the inserted DNA and one hundred (100) nucleotides of any specified 5' soybean chromosomal DNA segment adjacent to the inserted DNA. SEQ ID NO:6 is represented by [6], which is two hundred (200) nucleotides long, contains SEQ ID NO:4, and spans one hundred (100) nucleotides of the inserted DNA and one hundred (100) nucleotides of any specified 3' soybean chromosomal DNA segment adjacent to the inserted DNA. [7] represents SEQ ID NO:7, corresponding to one thousand (1,000) consecutive nucleotides of soybean genomic DNA at any specified 5' end of the inserted DNA and one hundred and eighty-one (181) consecutive nucleotides of the adjacent inserted transgenic DNA. [8] represents SEQ ID NO:8, corresponding to one thousand (1,000) consecutive nucleotides of the adjacent soybean genomic DNA at any specified 3' end of the inserted DNA and one hundred and ninety-two (192) consecutive nucleotides at the 3' end of the inserted transgenic DNA. [9] represents SEQ ID NO:9, and is the full length nucleotide segment of the inserted transgenic DNA.The arrows and the label below each arrow indicate the orientation of the transcription and translation directions (if applicable) relative to the applicable expression elements located in the two boxes of the inserted DNA. RB and LB represent the positions of the right and left borders of the Agrobacterium double border mediated transformation vector, the letter P represents the position of the promoter element in the corresponding construct, the letter L represents the position of the leader sequence (5' untranslated region, 5'UTR) in the corresponding construct, the letter I represents the position of the intron sequence in the corresponding construct, and the letter T represents the position of the transcription termination sequence (3' untranslated region, 3'UTR) in the corresponding construct. The elements from P to the immediately following T presented in the graphical depictions of [9] and

[10] represent one construct each. Figure 1 The construct on the left encodes the pest toxin Cry1A.2, while Figure 1The construct on the right encodes Cry1B.2. Cry1A.2 and Cry1B.2 are each distinct toxins that exhibit less than 45% amino acid sequence identity to each other, are both toxic to lepidopteran pests, and each toxin exhibits a different mode of action relative to the other. Table 1 lists the location of each genetic element involved in the expression of these respective toxin proteins in the transgenic insert DNA.

[11] represents SEQ ID NO:11 and indicates the location of the soybean genomic DNA flanking the 5' end of the insert DNA.

[12] represents SEQ ID NO:12 and indicates the location of the soybean genomic DNA flanking the 3' end of the insert DNA.

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

[15] represents SEQ ID NO:15 or primer SQ51400). These are described as being used together as a primer pair that can be used in samples containing line GM_CSM63770 DNA, which when used in a thermal amplification reaction will produce an amplicon of one hundred thirty four (134) nucleotides containing the 3' insert / genomic junction region, with arrows indicating the approximate hybridization position within

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

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

[14] and

[15] together with soybean line GM_CSM63770 DNA as a template to detect the presence of soybean line GM_CSM63770 DNA 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' end of the inserted DNA and, when combined in a thermal amplification reaction with

[15] (SEQ ID NO: 15, primer SQ51400) and conventional soybean DNA lacking or not containing soybean line GM_CSM63770 DNA as a template, will produce an amplicon of one hundred thirty-six (136) nucleotides containing unbroken soybean genomic DNA, and the amplicon is detected to represent a sample that does not contain soybean transgenic line GM_CSM63770 DNA at the chromosomal locus. Probe GM_WT63770PB (SEQ ID NO: 21) represents a probe that binds (or hybridizes) to the amplicon produced using primers

[20] and

[15] to detect an allele lacking or not containing line GM_CSM63770 DNA.Probe GM_WT63770PB hybridizes to the 3' terminal 9 nucleotides of the 5' genomic flanking DNA, 14 nucleotides of the wild-type allele DNA deleted during T-DNA insertion in soybean line GM_CSM63770, and the 5' terminal 2 nucleotides of the 3' genomic DNA flanking the DNA corresponding to the transgenic insert DNA described herein as line GM_CSM63770.

[0029] Sequence Description

[0030] SEQ ID NO:1 is a 50 nucleotide sequence representing the 5' junction of soybean genomic DNA and the integrated transgenic expression cassette (25 nucleotides of soybean genomic DNA at the 5' end of SEQ ID NO:1 and 25 nucleotides of transgenic insert DNA at the 3' end of SEQ ID NO:1) and can be identified at nucleotide positions 976-1,025 in SEQ ID NO:10.

[0031] SEQ ID NO:2 is a 50 nucleotide sequence representing the 3' junction of the integrated transgenic expression cassette and soybean genomic DNA (25 nucleotides of transgenic insert DNA at the 5' end of SEQ ID NO:2 and 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 in SEQ ID NO:10.

[0032] SEQ ID NO:3 is a 100 nucleotide sequence representing the 5' junction of soybean genomic DNA and the integrated transgenic expression cassette (50 nucleotides of soybean genomic DNA at the 5' end of SEQ ID NO:3 and 50 nucleotides of transgenic insert DNA at the 3' end of SEQ ID NO:3) and can be identified at nucleotide positions 951-1,050 in SEQ ID NO:10.

[0033] SEQ ID NO:4 is a 100 nucleotide sequence representing the 3' junction of the integrated transgenic expression cassette and soybean genomic DNA (50 nucleotides of transgenic insert DNA at the 5' end of SEQ ID NO:4 and 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 in SEQ ID NO:10.

[0034] SEQ ID NO:5 is a 200 nucleotide sequence representing the 5' junction 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 and 100 nucleotides of transgenic insert DNA at the 3' end of SEQ ID NO:5) and can be identified at nucleotide positions 901-1,100 in SEQ ID NO:10.

[0035] SEQ ID NO:6 is a 200 nucleotide sequence representing the 3' junction of the integrated transgenic expression cassette and soybean genomic DNA (100 nucleotides of transgenic insert DNA at the 5' end of SEQ ID NO:6 and 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 in SEQ ID NO:10.

[0036] SEQ ID NO:7 is a 1,181 nucleotide sequence representing the 5' junction 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 and 181 nucleotides of transgenic insert DNA at the 3' end of SEQ ID NO:7) and can be identified at nucleotide positions 1-1,181 in SEQ ID NO:10.

[0037] SEQ ID NO:8 is a 1,192 nucleotide sequence representing the 3' junction of the integrated transgenic expression cassette and soybean genomic DNA (192 nucleotides of transgenic insert DNA at the 5' end of SEQ ID NO:6 and 1000 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 in SEQ ID NO:10.

[0038] SEQ ID NO:9 is a 12,240 nucleotide sequence corresponding to the transgenic insertion T-DNA of soybean line GM_CSM63770 and can be identified in SEQ ID NO:10 at nucleotide positions 1,001-13,240.

[0039] SEQ ID NO:10 is a sequence of 14,240 nucleotides corresponding to the overlapping nucleotide sequence of the 5' genomic flanking DNA nucleotide sequence, the T-DNA nucleotide sequence inserted in strain GM_CSM63770, and the 3' genomic flanking DNA nucleotide sequence; and includes SEQ ID NO:11 (nucleotides 1-1,1000), SEQ ID NO:9 (nucleotides 1,001-13,240) and SEQ ID NO:12 (nucleotides 13,241-14,240).

[0040] SEQ ID NO:11 is a 1,000 nucleotide sequence representing the soybean genomic DNA 5' flanking the inserted T-DNA and can be identified in SEQ ID NO:10 at nucleotide positions 1-1,000.

[0041] SEQ ID NO:12 is a 1,000 nucleotide sequence representing the 3' flanking soybean genomic DNA following the inserted T-DNA and can be identified in SEQ ID NO:10 at nucleotide positions 13,241-14,240.

[0042] 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 line GM_CSM63770.

[0043] SEQ ID NO: 14 is a 26-nucleotide sequence corresponding to a thermal amplification primer designated SQ13805 that can be used to identify soybean line GM_CSM63770 DNA in a sample and is identical to the nucleotide sequence corresponding to positions 13,177-13,202 in SEQ ID NO: 10.

[0044] SEQ ID NO:15 is a 31 nucleotide sequence corresponding to a thermal amplification primer designated SQ51400 that can be used to identify soybean line GM_CSM63770 DNA in a sample and is identical to the reverse complement of the nucleotide sequence corresponding to positions 13,280-13,310 in SEQ ID NO:10.

[0045] SEQ ID NO:16 is a 16-nucleotide sequence corresponding to a probe designated PB4832 used to identify soybean line GM_CSM63770 DNA in a sample and is identical to the nucleotide sequence corresponding to positions 13,204-13,219 in SEQ ID NO:10.

[0046] SEQ ID NO: 17 is a 20 nucleotide sequence corresponding to a thermal amplification primer designated SQ549, used as an internal control for line and zygosity assays for soybean line GM_CSM63770, and hybridizes to a region of the soybean genome.

[0047] SEQ ID NO: 18 is a 20 nucleotide sequence corresponding to a thermal amplification primer designated SQ546, used as an internal control for line and zygosity assays for soybean line GM_CSM63770, and hybridizes to a region of the soybean genome.

[0048] SEQ ID NO: 19 is a 28 nucleotide sequence corresponding to a probe designated PB0004, used as an internal control for line and zygosity assays for soybean line GM_CSM63770, and hybridizes to a region of the soybean genome.

[0049] SEQ ID NO:20 is a 25 nucleotide sequence corresponding to a thermal amplification primer called GM_WT63770F used in a zygosity assay for soybean line GM_CSM63770 and hybridizing to 5′ genomic flanking DNA, and is identical to the nucleotide sequence corresponding to positions 949-971 in SEQ ID NO:10.

[0050] SEQ ID NO:21 is a 25 nucleotide sequence corresponding to a probe designated GM_WT63770PB, used in a zygosity assay for soybean line GM_CSM63770, and hybridizes to the last 9 nucleotides of the 5' genomic flanking DNA, 14 nucleotides of the wild-type allele DNA deleted during T-DNA insertion in soybean line GM_CSM63770, and the first 2 nucleotides of the 3' genomic flanking DNA.

[0051] SEQ ID NO: 22 is a 27-nucleotide sequence corresponding to the original guide RNA recognition site (OgRRS), i.e., OgRRS_5-1, which comprises a Cas12a protospacer adjacent motif (PAM) site operably linked to the guide RNA hybridization site.

[0052] SEQ ID NO:23 is a 27-nucleotide sequence corresponding to OgRRS, namely OgRRS_5-2.

[0053] SEQ ID NO:24 is a 27-nucleotide sequence corresponding to OgRRS, namely OgRRS_5-3.

[0054] SEQ ID NO:25 is a 27-nucleotide sequence corresponding to OgRRS, namely OgRRS_3-1.

[0055] SEQ ID NO:26 is a 27-nucleotide sequence corresponding to OgRRS, namely OgRRS_In-1.

[0056] SEQ ID NO:27 is a 27-nucleotide sequence corresponding to OgRRS, namely OgRRS_In-2.

[0057] SEQ ID NO:28 is a 51-nucleotide sequence corresponding to the guide RNA (gRNA), i.e., gRNA_OgRRS_5-1.

[0058] SEQ ID NO:29 is a sequence of 51 nucleotides corresponding to gRNA, i.e., gRNA_OgRRS_5-2.

[0059] SEQ ID NO:30 is a sequence of 51 nucleotides corresponding to gRNA, i.e. gRNA_OgRRS_5-3.

[0060] SEQ ID NO:31 is a sequence of 51 nucleotides corresponding to gRNA, i.e. gRNA_OgRRS_3-1.

[0061] SEQ ID NO:32 is a sequence of 51 nucleotides corresponding to gRNA, i.e. gRNA_OgRRS_In-1.

[0062] SEQ ID NO:33 is a sequence of 51 nucleotides corresponding to gRNA, i.e., gRNA_OgRRS_In-2.

[0063] SEQ ID NO: 34 is designed for expression in plant cells, and the sequence encodes a nuclear-targeted Cas12a CRISPR-associated protein.

[0064] SEQ ID NO: 35 is the amino acid sequence of the nuclear-targeted Cas12a CRISPR-associated protein.

[0065] SEQ ID NO:36 is a 5,000 nucleotide sequence representing soybean genomic DNA flanking the transgenic insert at the 5' end of the insert. Nucleotides 4,001-5,000 of SEQ ID NO:36 are identical to nucleotides 1-1,000 of SEQ ID NO:11.

[0066] SEQ ID NO:37 is a 5,000 nucleotide sequence representing soybean genomic DNA flanking the transgenic insert at the 3' end of the insert. Nucleotides 1-1,000 of SEQ ID NO:99 are identical to nucleotides 1-1000 of SEQ NO: 12. The remaining nucleotides of SEQ ID NO:99 (nucleotides 1,001-5,000) are based on the genomic sequence of the Williams 82 soybean cultivar.

[0067] SEQ ID NOs: 38-117 are additional 50 nucleotide sequences in the 5' flanking genomic sequence of line GM_CSM63770 based on the Williams 82 soybean cultivar genomic sequence.

[0068] SEQ ID NOs: 118-137 are 50 nucleotide sequences in the 5' flanking genomic sequence of strain GM_CSM63770.

[0069] SEQ ID NOs: 138-157 are 50 nucleotide sequences in the 3' flanking genomic sequence of strain GM_CSM63770.

[0070] SEQ ID NOs: 158-237 are additional 50 nucleotide sequences in the 3' flanking genomic sequence of line GM_CSM63770 based on the Williams 82 soybean cultivar genomic sequence. DETAILED DESCRIPTION

[0071] The present invention provides a transgenic soybean line GM_CSM63770, which achieves insecticidal control of certain soybean lepidopteran pest larvae by expressing insecticidal toxins Cry1A.2 and Cry1B.2, which are available in the tissues of soybean plants containing this line and presented to the pest larvae when they consume plant tissues. Specifically, expression of Cry1A.2 and Cry1B.2 insect inhibitory proteins in soybean line GM_CSM63770 will provide resistance to the larval forms of the following lepidopteran insect pests: soybean pod borer (SPW; American bollworm), soybean armyworm (soybean looper), velvet bean caterpillar (Spodoptera exigua), southern cutworm (subtropical armyworm), black cutworm (Spodoptera litura), South American pod borer (Argentine bollworm), sunflower armyworm (Spodoptera exigua), bean stem moth (Spodoptera exigua), green armyworm (Spodoptera lucerne) and small corn stem borer (Ostrichia annua). Line GM_CSM63770 provides an unmet need in the art for the control of these insects in the soybean market because lepidopteran insect species have begun to develop resistance to the pest control proteins used in early forms of transgenic soybean plants expressing such lepidopteran insect control proteins, and chemical insecticides have not provided adequate control of these insects and sometimes require multiple applications of chemicals during the growing season, thereby increasing the input of chemical pesticides into the environment, increasing the carbon footprint at each application, and significantly increasing the production costs of the soybean crop.

[0072] The protection provided by line GM_CSM63770 against infestation by lepidopteran insect species is associated with the expression of DNA segments encoding two different lepidopteran insect-specific insecticidal proteins, which are operably covalently linked in the inserted transgenic DNA, defining soybean line GM_CSM63770 in part. The two insecticidal proteins encoded by the transgenic inserted DNA in soybean line GM_CSM63770 are (i) Cry1A.2 (U.S. Patent No. 10,494,408, wherein the amino acid sequence mentioned is SEQ ID NO:4 and the coding sequence is SEQ ID NO:3); and (ii) Cry1B.2 (U.S. Patent No. 10,669,3017, wherein the amino acid sequence mentioned is SEQ ID NO:10 and the coding sequence is SEQ ID NO:9). These two insecticidal proteins are described herein as being expressed by two different but linked expression cassettes in the inserted transgenic DNA construct, as shown in SEQ ID NO:9 herein and Figure 1 shown.

[0073] The DNA sequence encoding Cry1A.2 protein in soybean strain GM_CSM63770 is operably connected to Arabidopsis thaliana ubiquitin 10 promoter, leader sequence and intron. The DNA sequence encoding Cry1B.2 protein in soybean strain GM_CSM63770 is operably connected to melon (Cucumis melo) chlorophyll ab binding protein 13 gene promoter and leader sequence (U.S. Patent 10,443,066, cited as SEQ ID NO:4-6). The toxin proteins Cry1A.2 and Cry1B.2 are expressed from their respective transgenic boxes (transcribed into mRNA encoding toxin amino acid sequences, and mRNA translated into toxin proteins) in the same direction (head to tail / head to tail) orientation. Figure 1 The relative position of each element is shown: promoter (P), 5'UTR or leader sequence (L), intron (I), toxin coding sequence or ORF (open reading frame) and 3'UTR (T). The ORF of Cry1A.2 and the ORF of Cry1B.2 are contained in SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0074] As described herein, multiple constructs with different use of expression elements, toxin coding sequences, and transcription and translation orientations were evaluated. One hundred and twenty-five (125) constructs containing one or more of twenty-one (21) different insect toxin coding sequences were used to generate lines for testing, thereby selecting soybean line GM_CSM63770. When evaluating the resistance of the corresponding transgenic soybean plants to lepidopteran insect pest infestation, the construct used to generate soybean line GM_CSM63770 (herein provided as SEQ ID NO: 13) provided excellent performance relative to other constructs. In addition, soybean line GM_CSM63770 does not contain markers for selecting transformed plant cells, because the transformation method used ensures that the selective marker and the predetermined trait to be integrated can be isolated from each other by breeding selection. Soybean line GM_CSM63770 was transformed with two T-DNA plant transformation binary vectors, wherein the selection box is contained on a T-DNA different from the T-DNA containing the insect toxin transgenic expression box retained in soybean line GM_CSM63770. After self-pollination of transformed soybean plants of the R0 generation, the R1 progeny are selected based on the presence of the T-DNA containing the insect toxin expression cassette and the absence of the T-DNA used to select the initial transformed line.

[0075] By using plant transformation technology, heterologous DNA (also referred to as transgenic DNA) is randomly inserted into the chromosome of the soybean cell genome to produce genetically engineered cells (also referred to as "transgenic" or "recombination" soybean cells), soybean strain GM_CSM63770 is produced thus. Using this technology, many individual cells are transformed, but because foreign DNA is randomly inserted into the genome, each cell produces a unique "transgenic strain" or "strain". Then, a transgenic plant is regenerated from each transgenic cell. Each cell of the transgenic plant is thus made to contain the uniquely inserted transgenic strain as a stable part of its genome. Then, this transgenic plant can be used to produce seeds, then plant the seeds and grow them into progeny plants, which each contain a unique transgenic strain.

[0076] Soybean strain GM_CSM63770 is produced by agrobacterium-mediated transformation method using binary transformation plasmid construct GM_CSM63770 and dry excised soybean plant explants. This plasmid construct contains two regions, and each region is surrounded by agrobacterium border segments (T-DNA segments). The first T-DNA segment contains two connected plant expression cassettes, an expression cassette encoding selectable marker, and an expression cassette encoding scoreable marker. The second T-DNA segment contains two connected plant expression cassettes, and the plant expression cassette has the required regulatory genetic elements for expressing two different insecticidal proteins Cry1A.2 and Cry1B.2 in soybean plant cells. The T-DNA segment containing the selectable / scorable marker gene is randomly inserted into the soybean genome and at a site different from the integration site of the T-DNA segment containing the Cry1A.2 and Cry1B.2 expression cassettes, thereby allowing the two T-DNA segments to be isolated in the genome of the transformed plant during selfing and / or backcrossing, such as screening transgenic plants of the R1 generation and higher generations. The transformed soybean cells are regenerated into complete soybean plants, and individual plants are selected from the plant population showing the integrity of the second T-DNA segment encoding Cry1A.2 and Cry1B.2 proteins. In the R1 generation and subsequent generations, the selection of the lines is based on the integrity, stability and integrity of the T-DNA segment encoding Cry1A.2 and Cry1B.2 proteins, as well as the absence (i.e., isolation) of the T-DNA segment encoding the selectable / scorable marker cassette and (ii) any plasmid backbone sequence. Further selection was made based on the location of the T-DNA segments encoding the Cry1A.2 and Cry1B.2 proteins within the soybean genome and other characteristics such as efficacy and agronomy. Expression of the Cry1A.2 and Cry1B.2 insecticidal toxic proteins in cells having soybean line GM_CSM63770 conferred resistance to lepidopteran insect pests when soybean cells having line GM_CSM63770 were provided in the diet of insects.

[0077] The T-DNA segment encoding Cry1A.2 and Cry1B.2 proteins in the plasmid construct pM63770 is shown in 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. In addition, fourteen (14) consecutive base pairs (bp) of wild-type genomic DNA at the transgenic DNA insertion point were also deleted during the integration process.

[0078] As described in detail herein, soybean line GM_CSM63770 was generated through a complex research and development process in which: (1) more than one hundred plasmid vector constructs were developed, each with a different coding sequence for an insecticidal protein, a different coding sequence for a transcriptional regulatory element, and the number and orientation of expression cassettes within the construct; and the constructs were transformed into soybean cells to generate thousands of lines, which were tested and analyzed to ultimately select the construct used to generate line GM_CSM63770; (2) thousands of soybean cells were transformed with the construct that generated line GM_CSM63770 to generate a population of transgenic plants, each of which contained a unique transgenic line, which was regenerated and tested; and (3) the final line GM_CSM63770 was selected after a rigorous and meticulous line selection process over many years, which involved testing and analyzing molecular characteristics, efficacy, protein expression, and agronomic properties in a variety of soybean genetic backgrounds. Thus, soybean line GM_CSM63770 was generated and selected as a unique superior line that can be used for large-scale agronomic purposes.

[0079] The transgenic DNA inserted into the soybean line GM_CSM63770 genome is characterized by detailed molecular analysis. This analysis includes: the number of inserts (the number of integration sites in the soybean genome), the genome insert position (the specific site where insertion occurs in the soybean genome), the copy number (the copy number of T-DNA in a locus) and the integrity of the transgenic DNA inserted. Detailed molecular analysis shows that the integrated T-DNA containing Cry1A.2 and Cry1B.2 expression cassettes remains intact after integration, while the T-DNA containing selectable / scorable markers does not exist. As used herein, "expression cassette" or "cassette" is a recombinant DNA molecule that comprises a combination of different elements expressed by transformed cells. Table 1 provides a list of elements contained in SEQ ID NO:10 (i.e., corresponding to the DNA sequence dna of soybean line GM_CSM63770).

[0080] Table 1. Description of soybean line GM_CSM63770.

[0081]

[0082]

[0083] The soybean line GM_CSM63770 is characterized by the insertion of a predetermined transgenic DNA into a locus in the soybean genome, resulting in two new loci or junction sequences (e.g., sequences shown 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) between the inserted DNA and the soybean genomic DNA, which are known not to naturally exist in the soybean genome or other transgenic soybean lines, i.e., they are unique to soybean DNA containing line GM_CSM63770. These junction sequences can be used to detect the presence of the line in soybean cells, soybean tissues, soybean seeds, soybean pollen and ovules, and soybean plants or soybean plant products (e.g., soybean commodity products). Described herein are DNA molecular probes and primer pairs developed for identifying the presence of these different junctional segments in biological samples containing or suspected of containing soybean cells, soybean seeds, soybean plant parts, soybean pollen or ovules, or soybean plant tissues containing line GM_CSM63770.

[0084] Sample is intended to refer to a composition of substantially pure soybean DNA or protein, or a composition containing soybean DNA or protein. With regard to a sample containing DNA, the sample is a biological sample, that is, it contains biological material, including but not limited to the DNA directly or indirectly obtained from or derived from the genome of soybean strain GM_CSM63770. "Directly" refers to the ability of technicians to obtain DNA directly from the soybean genome in the following manner: soybean cells are ruptured (or by obtaining a soybean sample containing ruptured soybean cells), and genomic DNA is exposed for detection purposes. "Indirectly" refers to the ability of technicians to obtain target DNA or specific reference DNA (that is, described herein as a novel and unique junction section for diagnosing or characterizing the presence of soybean strain GM_CSM63770 in a specific sample) by certain means, and the means are not directly obtained via rupturing soybean cells or obtaining a soybean sample containing ruptured soybean cells to obtain the DNA. Such indirect means include, but are not limited to: amplifying a DNA segment containing a DNA sequence that is the target of a specific probe designed to specifically bind to the target sequence; or amplifying a DNA segment that can be measured or characterized, i.e., separated from other DNA segments by some high-efficiency matrix such as agarose or acrylamide gel, or characterized by direct sequence analysis of the amplicon, or cloning the amplicon into a vector and directly sequencing the amplicon inserted into the vector.

[0085] Pure soy protein samples or composition samples containing soy protein are biological samples, that is, the samples contain biological materials, including but not limited to proteins obtained from or derived from tissues or cells of soybean strain GM_CSM63770. The sample is contacted with a monoclonal antibody that specifically binds to Cry1A.2 and Cry1B.2 in an immunoassay. Detection of the binding protein will diagnose or characterize the soybean strain GM_CSM63770. The immunoassay method can be, but is not limited to, ELISA (enzyme-linked immunosorbent assay), radioimmunoassay or lateral flow immunochromatography assay. ELISA assays are usually performed in a laboratory using tissue or cell samples obtained from a whole plant or its plant part. Sandwich ELISA assays are often used to detect and quantify proteins in transgenic crops. In order to use sandwich ELISA assays to detect the presence of soybean strain GM_CSM63770 in a sample, a variety of monoclonal antibodies are used, the first monoclonal antibody specifically binds to Cry1A.2, and the second antibody specifically binds to Cry1B.2. A known amount of monoclonal antibodies is bound to a fixed surface. The non-specific sites on the solid surface are blocked by bovine serum albumin, casein or any other neutral solution. The sample containing the protein derived from soybean strain GM_CSM63770 is applied to the plate and captured by the antibody. Unbound antigen is washed off with a washing solution. A secondary antibody is added, which is also conjugated with an enzyme. Unbound antibody is washed off. Substrate is added, enzyme is reacted with the substrate and a product is produced, typically a pigment or photon, which is proportional to the amount of the antigen present. Two toxin proteins in the sample derived from soybean strain GM_CSM63770 are respectively carried out to ELISA assay. Positive ELISA reaction will diagnose or standard soybean strain GM_CSM63770.

[0086] Lateral flow immunochromatographic assays, also known as immunochromatographic test strips (ICS) or test paper tests, can be used in areas where laboratory facilities cannot perform more complex immunoassays. The immunochromatographic test strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane, an absorbent pad, and a backing card. 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 host cell of the organism that is the source of the first monoclonal antibody and the second monoclonal antibody are transferred 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 monoclonal antibody and the second monoclonal antibody labeled with colloidal gold nanoparticles. The imprinted membrane, conjugate pad, sample pad, and absorbent pad are assembled in sequence on a plastic backing plate. The Cry1A.2 and Cry1B.2 proteins present in samples derived from soybean line GM_CSM63770 will combine with their respective gold labeled monoclonal antibodies and then bind to the capture antibodies coated on Test Line 1 and Test Line 2. Visual inspection of Test Line 1 and Test Line 2 will diagnose or characterize soybean line GM_CSM63770.

[0087] Detailed molecular analysis also shows that strain GM_CSM63770 contains a single T-DNA insertion, and one copy each of Cry1A.2 and Cry1B.2 expression cassettes. In strain GM_CSM63770, except for the portion of the left and right border regions of Agrobacterium tumefaciens, no other elements for transferring transgenic DNA from the plant transformation plasmid to the soybean genome were identified from the transformation construct. In addition, thermal amplification was also performed to produce specific amplicons for the presence of strain GM_CSM63770 in the diagnosis or characterization sample, and DNA sequence analysis was performed to determine the arbitrarily specified 5' and 3' insert-plant genome junctions, confirm the organization of each element in the insert, and determine the complete DNA sequence of the inserted transgenic DNA (SEQ ID NO:9). SEQID NO:11 is a sequence of one thousand (1,000) base pairs (bp) adjacent to the arbitrarily specified 5' end of the inserted DNA in the soybean variety A3555 genomic DNA sequence representing the T-DNA sequence inserted with the flanking joint. SEQ ID NO: 12 is a sequence representing one thousand (1,000) bp adjacent to any specified 3' end of the inserted DNA in the soybean variety A3555 genomic DNA sequence flanked by the inserted T-DNA sequence. SEQ ID NO: 7 is SEQ ID NO: 11 plus one hundred and 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 plus one hundred and 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 line GM_CSM63770 and includes a contiguous sequence (contig) comprising 5' A3555 flanking sequences, the transgenic insert of GM_CSM63770 and 3' A3555 flanking sequences, and thus the insert-plant genome junction sequences designated by SEQ ID NOs: 1, 3, 5 and 7 (5' end) and SEQ ID NOs: 2, 4, 6 and 8 (3' end).

[0088] Unless otherwise specified herein, the term should be understood according to the conventional usage of those of ordinary skill in the relevant art. The definition of commonly used terms in molecular biology can be found in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994, and other sources known to those of ordinary skill in the art. As used herein, the term "soybean" refers to species belonging to the genus Glycine, preferably soybean, and includes all plant varieties that can be bred with soybean plants containing strain GM_CSM63770, including wild soybean species, and plants belonging to the genus Glycine and allowing breeding between species.

[0089] The strain GM_CSM63770 was transformed with a DNA construct containing an expression cassette expressing toxic amounts of the insecticidal proteins Cry1A.2 and Cry1B.2. Toxic amounts mean effective amounts, insecticidal amounts, insecticidal effective amounts, target insect inhibition amounts, effective pesticide amounts, insecticidal amounts in insect food for lepidopteran insects, and other similar terms understood by those of ordinary skill in the relevant art according to conventional usage. Soybean plants transformed according to the methods and DNA constructs disclosed herein are resistant to lepidopteran insect pests.

[0090] Transgenic "plants" are produced in the following manner: plant cells are transformed with heterologous DNA (i.e., polynucleic acid constructs including many effective features of interest), plants are regenerated by inserting transgenes into the genome of plant cells, and specific plants are selected, characterized by the number of effective features of transgenic plants inserted into specific genomic positions and regenerated. The term "line" refers to the DNA from the original transformant, comprising the inserted DNA and the flanking genomic sequences of the DNA next to the inserted DNA. This DNA is unique, and it is expected that it will be transferred to the progeny that accepts the inserted DNA (including the transgene of interest), which is produced by sexual hybridization of the parental line (e.g., the progeny produced by the original transformant and self-pollination) including the inserted DNA with the parental line that does not contain the inserted DNA. The present invention also provides the progeny of the original transformant plant and the transformant including heterologous DNA. Such progeny can be produced by sexual hybridization between a plant comprising the line and another plant, wherein the progeny includes heterologous DNA. Even after repeated backcrossing with the recurrent parent, the line is still present at the same chromosome position of the hybrid progeny. The transgenic strain DNA and the genome in which the transgenic strain DNA is detectable in the plant cells, plants, seeds and other plant parts do not pre-exist in nature.

[0091] As used herein, the term "recombinant" refers to non-natural DNA, proteins, or organisms that do not normally occur in nature and are produced by human intervention. A "recombinant DNA molecule" is a DNA molecule that comprises a combination of DNA molecules that do not occur together naturally and are the result of human intervention. For example, a DNA molecule comprises a combination of at least two DNA molecules that are heterologous to each other, such as a DNA molecule comprising a transgene and plant genomic DNA adjacent to the transgene, and is a recombinant DNA molecule.

[0092] The terms "DNA" and "DNA molecule" referred to herein refer to deoxyribonucleic acid (DNA) molecules. DNA molecules can be of genomic or synthetic origin, and are conventionally 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. Conventionally, the disclosed DNA sequences of the present invention and fragments thereof are about only one chain in a double-stranded complementary DNA sequence chain. By implication and intent, the complementary sequences of the sequences provided herein (the sequences of the complementary chains, also referred to as reverse complementary sequences in the art) are within the scope of the present invention and are explicitly intended to be within the scope of the claimed subject matter.

[0093] As used herein, the term "fragment" refers to a smaller portion of a whole. For example, a fragment of SEQ ID NO: 10 includes a sequence that is 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 entire sequence of SEQ ID NO: 10.

[0094] Similarly, a fragment of the 5' flanking sequence (SEQ ID NO: 11 or SEQ ID NO: 36) or the 3' flanking sequence (SEQ ID NO: 12 or SEQ ID NO: 37) of soybean line GM_CSM63770 may comprise SEQ ID NO: 11 or SEQ ID NO: 36; or SEQ ID NO: 12 or SEQ ID NO: 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 contiguous nucleotides of NO:37. In addition, the present disclosure also 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 12 or SEQ ID NO: 36 or 37, or a fragment of any one thereof.

[0095] The "isolated DNA molecule" or equivalent terms or phrases mentioned in the present disclosure are intended to refer to a DNA molecule that exists alone or in combination with other compositions, but is not present in its natural environment. For example, as long as a nucleic acid element naturally present in the DNA of the genome of an organism (such as a coding sequence, an intron sequence, a non-translated leader sequence, a promoter sequence, a transcription termination sequence, etc.) is located within the genome of the organism and at its naturally occurring position within the genome, the element is not considered to be "isolated". However, as long as each of these elements and a sub-portion of these elements is not within the genome of the organism and is not at its naturally occurring position within the genome, the element is "isolated" within the scope of the present disclosure. Similarly, as long as a nucleotide sequence encoding an insecticidal protein or any naturally occurring insecticidal variant of the protein is not in the DNA of a bacterium in which the sequence encoding the protein naturally occurs, the nucleotide sequence is an isolated nucleotide sequence. For the purposes of the present disclosure, a synthetic nucleotide sequence encoding the amino acid sequence of a naturally occurring insecticidal protein will be considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector, will be considered an isolated nucleotide sequence, whether the nucleotide sequence is present in a plasmid or similar structure used to transform cells, in the genome of a plant or bacterium, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacterium. In any case, an isolated DNA molecule is a chemical molecule, whether it is referred to as a nucleic acid, a nucleic acid sequence, a polynucleotide sequence, etc. An "isolated" molecule is a novel inventive molecule that exhibits industrial applicability when present in a plant cell or plant genome, as well as when present outside a plant cell, and therefore exhibits and is expected to exhibit such utility, regardless of where the molecule is located.

[0096] The "isolated protein molecule" or equivalent terms or phrases mentioned in this disclosure are intended to mean that the protein molecule is a protein molecule that exists alone or in combination with other compositions, but does not exist in its natural environment. For example, the insecticidal protein molecule expressed by soybean strain GM_CSM63770 does not naturally exist in natural soybean plant samples. The Cry1A.2 and Cry1B.2 insecticidal proteins of soybean strain GM_CSM63770 are isolated protein molecules as long as the insecticidal protein or its variant is not present in the protein of the bacteria where the protein naturally exists.

[0097] The DNA sequence that spans the region of the phosphodiester linkage that connects one end of the transgenic insert to the flanking soybean genomic DNA is called a "junction point". The junction point is the point of connection that connects the transgenic insert to the flanking DNA as a continuous molecule. One junction point is found at the 5' end of the transgenic insert, and the other is found at the 3' end of the transgenic insert, referred to herein as the 5' junction point and the 3' junction point, respectively. A "junction sequence" refers to a DNA sequence of any length that spans the 5' or 3' junction of a line. The junction sequence of the soybean line GM_CSM63770 using SEQ ID NO: 10 is obvious to those skilled in the art. Examples of the junction sequence of GM_CSM63770 are provided with SEQ ID NO: 1-8. Figure 1 The physical arrangement of the junction sequences arranged 5' to 3' relative to SEQ ID NO: 10 is shown. The junction sequences of GM_CSM63770 may be present as part of the genome of a plant, seed or cell containing soybean line GM_CSM63770. Identification of any one or more of the junction sequences in a sample from a plant, plant part, seed or cell indicates that the DNA was obtained from soybean containing GM_CSM63770 and will be used to diagnose or characterize the presence of soybean line GM_CSM63770.

[0098] The junction sequence of soybean line GM_CSM63770 can be represented by a sequence of 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 sequence provided by 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 sequence provided by 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 sequence provided by 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 sequence provided by SEQ ID NO: 7 (5' junction sequence) and SEQ ID NO: 8 (3' junction sequence). These nucleotide sequences are linked by phosphodiester linkages and are present as part of the recombinant plant cell genome in soybean line GM_CSM63770.

[0099] These junction sequences will be used to diagnose or characterize the presence of soybean line GM_CSM63770 or the construct contained therein. Thus, 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 will be diagnostic that the DNA was obtained from soybean line GM_CSM63770. Thus, 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 DNA segment derived from the transgenic soybean line GM_CSM63770 is within the scope of the present disclosure, the DNA segment being sufficient to include at least one 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. In addition, any polynucleotide comprising a sequence complementary to any of the sequences described in this paragraph is also within the scope of the present invention.

[0100] The invention provides exemplary DNA molecules that can be used as primers or probes to detect the presence of DNA derived from a soybean plant containing line GM_CSM63770 DNA in a sample. Such primers or probes are specific to the target nucleic acid sequence and can therefore be used to identify soybean line GM_CSM63770 nucleic acid sequences by the methods of the invention described herein.

[0101] The use of the term "derived from" is intended to indicate that a particular DNA molecule is in the soybean plant genome or can be detected in soybean plant DNA. "Can be detected" means that a particular DNA segment (i.e., a target DNA segment) can be amplified and characterized or elucidated by DNA sequence analysis for size or sequence, and then the binding of the probe to the target can be detected. The particular DNA segment or target DNA segment of the present invention is present in soybean containing the insertion soybean line GM_CSM63770.

[0102] "Probe" is complementary to a strand of target nucleic acid and can be used for the nucleic acid molecule in hybridization method. The probe can be connected to conventional detectable label or reporter molecule, such as radioisotope, fluorophore, ligand, chemiluminescent agent or enzyme. This probe is complementary to a strand of target nucleic acid, and in the case of the present invention, is complementary to a strand of DNA from GM_CSM63770 (whether from the plant containing GM_CSM63770 or from the sample comprising GM_CSM63770 DNA). Therefore, the probe used herein can include DNA molecules or polynucleotide segments of sufficient length to play a role under stringent hybridization conditions defined herein, thereby combining the specific unique DNA segment present in the sample and used for diagnosis or characterization of strain GM_CSM63770. Such probes can be designed to only combine a single junction or other novel sequences only present in soybean strain GM_CSM63770, or two or more such single junction segments. Probes according to the present invention not only include deoxyribonucleic acid or ribonucleic acid, but also include polyamide and other probe materials that specifically bind to the target DNA sequence and can be used to detect the presence of the target DNA sequence. An exemplary DNA sequence that can be used as a probe to detect soybean line GM_CSM63770 is provided as SEQ ID NO: 16 (PB4832).

[0103] "Primer" is usually designed to be used for the DNA molecule in the specific annealing or hybridization method related to thermal amplification. Primer can include the paired different oligonucleotides or polynucleotide segments used in the thermal amplification reaction, and the thermal amplification reaction will amplify the specific DNA target segment. Each primer in the pair is designed to be combined with the specific DNA segment in or near the DNA segment of interest to amplify. The binding mode of these primers makes them act as the local area of ​​nucleic acid sequence polymerization to produce one or more amplicons (target DNA segment of amplification) subsequently. The amplicon produced by this reaction will have a DNA sequence corresponding to the template DNA sequence between the two sites of primer and template hybridization. In certain embodiments, the unique segment designed for combining soybean strain GM_CSM63770 and amplifying the primer of the specific amplicon containing one or more junction sequences described herein, and detecting and / or characterizing such amplicon when the polymerase reaction is completed or terminated, will diagnose or characterize the presence of soybean strain GM_CSM63770 in a specific sample. Those skilled in the art are very familiar with this amplification method, and the specific details of amplification do not need to be elaborated here.

[0104] Primers are typically designed to hybridize with complementary target DNA strands to form a hybrid between the primer and the target DNA strand, and the presence of the primer is a recognition point for the polymerase to begin extension of the primer using the target DNA strand as a template (i.e., polymerizing additional nucleotides into an extended nucleotide molecule). A primer pair refers to a polynucleotide segment between the positions targeted for binding to each member of the primer pair, typically using two primers that bind to opposite strands of a double-stranded nucleotide segment in a thermal amplification reaction or other conventional DNA amplification methods. Exemplary DNA molecules that can be used as primers are provided in SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 20.

[0105] Primer pair SEQ ID NO: 14 and SEQ ID NO: 15 can be used as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both have a sufficient length of consecutive nucleotides in SEQ ID NO: 10 to act as a DNA primer, when it is used in a thermal amplification reaction with a template DNA derived from soybean line GM_CSM63770, it will produce an amplicon that diagnoses or characterizes soybean line GM_CSM63770 DNA in a sample. Primer pair SEQ ID NO: 17 and SEQ ID NO: 18 can be used as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and both have a sufficient length of consecutive nucleotides of a locus within the soybean genome to act as a DNA primer, when it is used in a thermal amplification reaction with a template DNA derived from soybean line GM_CSM63770, it will produce an amplicon that is used as an internal control to diagnose soybean line GM_CSM63770 and determine the zygosity of soybean line GM_CSM63770 DNA in a sample. Primer pair SEQ ID NO:20 and SEQ ID NO:15 can be used as a first DNA molecule and a second DNA molecule different from the first DNA molecule, and each of which has a sufficient length of contiguous nucleotides of a locus within the soybean genome to act as a DNA primer, which when used in a thermal amplification reaction with template DNA derived from soybean line GM_CSM63770 will produce an amplicon that will be diagnostic or characterize the insertion-free wild-type soybean genomic DNA that does not contain line GM_CSM63770. For any particular desired amplification parameters, one skilled in the art will be able to determine which probes and primers are best suited for inclusion in a thermal amplification reaction to detect the presence or absence of the transgenic line DNA of the present invention based on the DNA sequence provided in the insert DNA (SEQ ID NO:9) and the complete DNA segment shown herein as SEQ ID NO:10, which will define the transgenic soybean line GM_CSM63370 of the present invention.

[0106] The length of DNA probes and DNA primers is generally eleven (11) polynucleotides or longer, usually eighteen (18) polynucleotides or longer, twenty-four (24) polynucleotides or longer, or thirty (30) polynucleotides or longer. Such probes and primers are selected to have sufficient length to specifically hybridize to the target sequence under high stringency hybridization conditions. Preferably, the probes and primers according to the present invention have complete sequence similarity to the target sequence, but probes different from the target sequence that retain the ability to hybridize to the target sequence can be designed by conventional methods.

[0107] The nucleic acid probes and primers of the present invention hybridize with the target DNA molecule under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from transgenic plants in a sample. Polynucleic acid molecules are also referred to as nucleic acid segments or fragments thereof and can specifically hybridize with other nucleic acid molecules in certain circumstances.

[0108] As used herein, if two polynucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, the two molecules are considered to be able to specifically hybridize to each other. If a nucleic acid molecule exhibits complete complementarity with another nucleic acid molecule, the nucleic acid molecule is considered to be the "complementary sequence" of the other nucleic acid molecule. As used herein, when each nucleotide of a molecule is complementary to the nucleotide of another molecule, the molecule is considered to exhibit "complete complementarity". If two molecules can hybridize to each other with sufficient stability so that they keep annealing to each other under at least conventional "low stringency" conditions, they are considered to be "minimally complementary". Similarly, if molecules can hybridize to each other with sufficient stability so that they keep annealing to each other under conventional "high stringency" conditions, they are considered to be "complementary". Conventional stringency conditions are described in Sambrook et al., 1989; and Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985). Therefore, deviations from complete complementarity are permissible, as long as these deviations do not completely exclude the ability of molecules to form double-stranded structures. In order for a nucleic acid molecule to function as a primer or probe, it is only necessary that the nucleic acid molecules be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.

[0109] As used herein, substantially homologous sequences are nucleic acid sequences that specifically hybridize to the complementary sequence of the compared nucleic acid sequence under high stringency conditions. Suitable stringency conditions for promoting DNA hybridization are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, such as washing with 6.0 × sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by washing with 2.0 × SSC at 50 ° C. For example, the salt concentration in the washing step can be selected from the low stringency of about 2.0 × SSC at 50 ° C to the high stringency of about 0.2 × SSC at 50 ° C. In addition, the temperature in the washing step can be increased from the low stringency conditions of room temperature (about 22 ° C) to the high stringency conditions of about 65 ° C. Both temperature and salt can be changed, or the temperature or salt concentration can be kept constant while other variables change. In a preferred embodiment, the polynucleic acids of the invention will specifically hybridize to one or more of the nucleic acid molecules shown 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 the complement thereof or a fragment of any of them under moderate stringency conditions (e.g., at about 2.0×SSC and about 65° C.). In a particularly preferred embodiment, the polynucleic acids of the invention will specifically hybridize to one or more of the nucleic acid molecules shown 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 the complement of any of them or a fragment thereof under high stringency conditions. In one aspect of the invention, the preferred marker nucleic acid molecules of the invention have the nucleic acid sequence shown 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 its complementary sequence, or a fragment of any one thereof. The hybridization of the probe to the target DNA molecule can be detected by any method known to those skilled in the art, which methods may include, but are not limited to, fluorescent tags, radioactive tags, antibody-based tags, and chemiluminescent tags.

[0110] For amplifying a target nucleic acid sequence (e.g., by PCR) using a specific amplification primer pair, "stringent conditions" refer to conditions under which the primer pair hybridizes only to a target nucleic acid sequence to which a primer having the corresponding wild-type sequence (or its complementary sequence) will bind and preferably produces a unique amplification product amplicon in a DNA thermal amplification reaction.

[0111] The term "specific for (a target sequence)" indicates that the probe or primer hybridizes only to the target sequence in a sample containing the target sequence under stringent hybridization conditions.

[0112] As used herein, "amplified DNA" or "amplicon" refers to the product of a polynucleic acid amplification method for a target polynucleic acid molecule that is part of a polynucleic acid template. For example, in order to determine whether a soybean plant produced by sexual crosses contains transgenic plant genomic DNA from a soybean plant comprising the strain GM_CSM63770 of the present invention, a primer pair can be used to perform a polynucleic acid amplification method on DNA extracted from a soybean plant tissue sample, wherein the primer pair includes a first primer, which is derived from a genomic DNA sequence in a flanking region of a heterologous inserted DNA of the strain GM_CSM63770, and is extended from 5' to 3' by a polymerase in the direction of the inserted DNA. The second primer is derived from a heterologous inserted DNA molecule and is extended from 5' to 3' by a polymerase along the direction of the flanking genomic DNA as the source of the first primer. The length range of the amplicon can be a primer pair plus one nucleotide base pair, or plus about fifty nucleotide base pairs, or plus about two hundred and fifty nucleotide base pairs, or plus a combined length of about four hundred and fifty or more nucleotide base pairs. Alternatively, the primer pair can be derived from genomic sequences on either side of the inserted heterologous DNA to produce an amplicon that includes the entire inserted polynucleotide sequence (e.g., a forward primer isolated from the genomic portion located at the 5' end of SEQ ID NO: 10 and a reverse primer isolated from the genomic portion located at the 3' end of SEQ ID NO: 10, which amplifies a DNA molecule comprising the inserted DNA sequence (SEQ ID NO: 9) identified herein in the genome of line GM_CSM63770). The members of the primer pair that are derived from plant genomic sequences adjacent to the inserted transgenic DNA are at a distance from the inserted DNA sequence that can range from one nucleotide base pair to as much as about twenty thousand nucleotide base pairs. The use of the term "amplicon" explicitly excludes primer dimers that can be formed in a DNA thermal amplification reaction.

[0113] For practical purposes, primers should be designed to produce amplicons of a limited size range (e.g., between 100 and 1000 bases). In general, amplicons of smaller size (shorter polynucleotide length) are more reliably generated in thermal amplification reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or are suitable for endpoint Sample determination. Smaller amplicons can be produced and detected by methods known in the field of DNA amplicon detection. In addition, the amplicon produced using primers can be cloned into a vector, propagated, separated and sequenced, or can be sequenced directly with the method fully established 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 is an aspect of the present invention, and the primer pair can be used in DNA amplification methods to produce an amplicon for diagnosis or characterization of soybean strain GM_CSM63770 or its progeny. Any single separated DNA polynucleotide primer molecule comprising at least 15 continuous nucleotides of SEQ IDNO:11 or its complementary sequence is an aspect of the present invention, and the primer molecule can be used in DNA amplification methods to produce an amplicon for diagnosis or characterization of soybean strain GM_CSM63770 or its progeny. Any single isolated DNA polynucleotide primer molecule comprising at least 15 consecutive nucleotides of SEQ ID NO: 12 or its complementary sequence is an aspect of the present invention, and the primer molecule can be used in a DNA amplification method to produce an amplicon that diagnoses or characterizes a plant comprising soybean line GM_CSM63770 or its progeny. Any single isolated DNA polynucleotide primer molecule comprising at least 15 consecutive nucleotides of SEQ ID NO: 9 or its complementary sequence is an aspect of the present invention, and the primer molecule can be used in a DNA amplification method to produce an amplicon that diagnoses or characterizes soybean line GM_CSM63770 or its progeny.

[0114] Polynucleic acid amplification can be accomplished by any of various polynucleic acid amplification methods known in the art, including polymerase chain reaction (PCR). Amplification methods are known in the art, and are particularly described in U.S. Patent Nos. 4,683,195 and 4,683,202 and PCR Protocols: A Guide to Methods and Applications, edited by Innis et al., Academic Press, San Diego, 1990. PCR amplification methods have been developed for amplifying genomic DNA up to 22 kb (kilobases) and phage 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 field of DNA amplification can be used to implement the present invention. The sequence of the heterologous DNA insert or flanking genomic DNA sequence from soybean line GM_CSM63770 can be verified (and corrected, if necessary) by amplifying such DNA molecules from soybean seeds containing line GM_CSM63770 DNA or from soybean plants grown from soybean seeds containing line GM_CSM63770 DNA deposited under ATCC Accession No. PTA-126048 using primers derived from the sequences provided herein, followed by standard DNA sequencing of the PCR amplicon or a cloned DNA fragment thereof.

[0115] The diagnostic amplicon produced by these methods can be detected using a variety of techniques. One of the methods is genetic bit analysis (Nikiforov et al., Nucleic Acid Res.22:4167-4175,1994), in which the designed DNA oligonucleotide overlaps with the adjacent lateral genomic DNA sequence and the inserted DNA sequence. The oligonucleotide is fixed in the hole of a microtiter plate. After PCR is performed on the region of interest (using one primer in the inserted sequence and one primer in the adjacent lateral genomic sequence), the single-stranded PCR product can be hybridized with the fixed oligonucleotide and used as a template in the single base extension reaction performed using DNA polymerase and a labeled dideoxynucleotide triphosphate (ddNTP) dedicated to the expected next base. The read segment can be performed based on fluorescence or based on ELISA. The signal indicates the presence of transgenic / genomic sequence due to successful amplification, hybridization and single base extension.

[0116] Another method is the pyrophosphate sequencing technology described by Winge (Innov.Pharma.Tech.00:18-24,2000). In this method, an oligonucleotide overlapping with adjacent genomic DNA and inserted DNA junction sequence is designed. Oligonucleotide is hybridized with a single-stranded PCR product (a primer in the inserted sequence and a primer in the lateral genomic sequence) from the region of interest, and incubated in the presence of DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin. DNTP is added separately, and the enzymatic reaction of luciferase and these reagents and substrate causes the release of photons (light signals), which are then measured or observed. Light signal represents the presence of transgenic / genomic sequence due to successful amplification, hybridization and single base or multi-base extension.

[0117] The fluorescence polarization described by Chen et al. (Genome Res.9:492-498,1999) is a method that can be used to detect the amplicon of the present invention. Using this method, an oligonucleotide overlapping with the genomic flanking sequence and the inserted DNA junction sequence is designed. The oligonucleotide is hybridized with the single-stranded PCR product from the region of interest (one primer in the inserted DNA and one primer in the flanking genomic DNA sequence), and incubated in the presence of DNA polymerase and fluorescently labeled ddNTP. Single base extension leads to the incorporation of ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. The change in polarization indicates the presence of transgenic / genomic sequence due to successful amplification, hybridization and single base extension.

[0118] Real-time polymerase chain reaction (PCR) is able to monitor the progress of PCR as it occurs (i.e., real-time monitoring). Data is collected throughout the PCR process rather than at the end of PCR. In real-time PCR, the reaction is characterized by the time point when the target amplification is first detected during the cycle, rather than the target amount accumulated after a fixed number of cycles. In real-time PCR assays, positive reactions are detected by the accumulation of fluorescent signals. The higher the initial 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 cross the threshold (i.e., exceed 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 greater the amount of target nucleic acid in the sample).

[0119] (PE Applied Biosystems, Foster City, CA) is described as a method for using real-time PCR to detect and quantitative DNA sequence presence, and is fully understood in the instructions provided by the manufacturer. In short, a FRET oligonucleotide probe overlapping with the genomic side joint sequence and the inserted DNA junction sequence is designed. FRET probe and PCR primer (a primer in the inserted DNA sequence and a primer in the side joint genomic sequence) are circulated in the presence of a thermostable polymerase and dNTP. The hybridization of the FRET probe causes the fluorescent part to crack and release from the quenching part on the FRET probe. Fluorescent signal indicates that there is a transgenic / genomic sequence due to successful amplification and hybridization.

[0120] Molecular beacons have been described for sequence detection, as described in Tyangi et al. (Nature Biotec h.14:303-308, 1996). In short, a FRET oligonucleotide probe overlapping the flanking genomic sequence and the inserted DNA junction sequence is designed. The unique structure of the FRET probe contains a secondary structure that allows the fluorescence and quenching parts to remain in close proximity. The FRET probe and PCR primers (one primer in the inserted DNA sequence and one primer in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of the FRET probe with the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescence and quenching parts. A fluorescent signal is generated. The fluorescent signal indicates the presence of the flanking / transgenic insertion sequence due to successful amplification and hybridization.

[0121] The DNA detection kit based on the DNA amplification method contains DNA primer molecules that specifically hybridize with the target DNA and amplify the diagnostic amplicon under appropriate reaction conditions. The kit can provide a detection method based on agarose gel or a variety of methods for detecting diagnostic amplicon known in the art. The DNA detection kit can be developed using the composition disclosed herein, and can be used to identify the soybean strain GM_CSM63770 DNA in the sample, and can be used to cultivate a method for soybean plants containing soybean strain GM_CSM63770 DNA. An object of the present invention is a kit containing DNA primers that are homologous or complementary to any part of the soybean genomic region shown in SEQ ID NO:10 and to any part of the transgenic DNA inserted shown in SEQ ID NO:10. The DNA molecule can be used in a DNA amplification method (PCR) or as a probe in a polynucleic acid hybridization method (i.e. Southern analysis, Northern analysis).

[0122] Although the primers and probes different from the target sequence that retain the ability to hybridize preferentially with the target sequence can be designed by conventional methods, the probe and primer according to the present invention can have complete sequence identity with the target sequence. In order to make nucleic acid molecules used as primers or probes, it is only necessary for the nucleic acid molecules to be fully complementary in sequence to form a stable double-stranded structure under the specific solvent and salt concentration used. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from soybean strain GM_CSM63770 in the sample. The length of the probe and primer is generally at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides or at least about 30 nucleotides or longer. Such probes and primers are specifically hybridized with the target DNA sequence under stringent hybridization conditions. Conventional stringent conditions are described in Sambrook et al., 1989 and Haymes et al., Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).

[0123] Can use the multiple methods well-known to those skilled in the art to separate and manipulate DNA molecules disclosed in the present invention or its fragment, comprise thermal amplification method.Can also obtain DNA molecules or its fragment by other technology, such as by directly synthesizing described fragment with chemical means, as usually implemented using automatic oligonucleotide synthesizer.

[0124] Therefore, the DNA molecules and corresponding nucleotide sequences provided in this article are particularly useful for identifying soybean line GM_CSM63770; detecting the presence of DNA derived from transgenic soybean line GM_CSM63770 in a sample; and monitoring the presence and / or absence of soybean line GM_CSM63770 or plant parts derived from soybean plants containing soybean line GM_CSM63770 in a sample.

[0125] In the case of soybean, soybean plants, soybean plant cells, soybean seeds, soybean pollen and ovules, soybean plant parts, soybean progeny plants, and soybean commodities are all 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 as being used to diagnose or characterize soybean line GM_CSM63770 (e.g., a polynucleotide having at least one 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). The soybean plants, plant cells, seeds, pollen, and or ovules, plant parts, and progeny plants of the present invention may also contain one or more additional transgenes. The one or more additional transgenes may be any nucleotide molecule encoding a protein or RNA molecule that confers a desired trait, including, but not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, and / or increased herbicide tolerance.

[0126] The present invention provides soybean plants, soybean plant cells, soybean seeds, soybean plant parts (such as pollen, ovules, filaments, ears, anthers, cobs, root tissue, stem tissue, leaf tissue), and soybean progeny plants derived from transgenic soybean plants containing GM_CSM63770 DNA. Representative samples of soybean seeds containing soybean line GM_CSM63770 DNA have been deposited with the American Type Culture Collection under the Budapest Treaty. The ATCC repository has assigned patent deposit designation PTA-126048 to seeds containing DNA of soybean line GM_CSM63770.

[0127] The present invention provides soybean plants, plant cells, plant parts and plant seeds, which contain a recombinant DNA construct integrated into chromosome 19, wherein the recombinant DNA construct confers resistance to a lepidopteran insect pest species. The recombinant DNA construct is integrated into the chromosome at a position flanked by at least 50 consecutive nucleotides of SEQ ID NO: 11 or 36 and at least 50 consecutive nucleotides of SEQ ID NO: 12 or 37. The at least 50 consecutive nucleotides of SEQ ID NO: 11 or 36 may contain one or more nucleotide sequences selected from SEQ ID NO: 38-137. At least the consecutive nucleotides of SEQ ID NO: 12 or 37 may contain one or more nucleotide sequences selected from SEQ ID NO: 138-237.

[0128] The present invention provides a microorganism comprising a DNA molecule present in its genome, the DNA molecule having at least one 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. An example of such a microorganism is a transgenic plant cell.

[0129] Microorganisms, such as plant cells of the present invention, can be used in many industrial applications, including but not limited to: (i) as a research tool for scientific inquiry or industrial research; (ii) used in culture to produce endogenous or recombinant carbohydrates, lipids, nucleic acids or protein products or small molecules that can be used for subsequent scientific research or as industrial products; and (iii) used with modern plant tissue culture techniques to produce transgenic plants or plant tissue cultures, which can then be used for agricultural research or production. The production and use of microorganisms (such as transgenic plant cells) will utilize modern microbiological technology and human intervention to produce artificial unique microorganisms. In this process, recombinant DNA is inserted into the genome of plant cells to produce transgenic plant cells that are different and unique from naturally occurring plant cells. Then, this transgenic plant cell can be cultured using modern microbiological techniques like bacterial and yeast cells, and can exist in an undifferentiated single cell state. The new genetic composition and phenotype of the transgenic plant cell are technical effects produced by integrating heterologous DNA into the cell genome. Another aspect of the present invention is a method of using the microorganism of the present invention. Methods for using the microorganisms (e.g., transgenic plant cells) of the present invention include: (i) methods for producing transgenic cells by integrating recombinant DNA into the genome of the cell and then using such cells to obtain additional cells with the same heterologous DNA; (ii) methods for culturing cells containing recombinant DNA using modern microbiological techniques; (iii) methods for producing and purifying endogenous or recombinant carbohydrate, lipid, nucleic acid or protein products from cultured cells; and (iv) methods for producing transgenic plants or transgenic plant tissue cultures using modern plant tissue culture techniques with transgenic plant cells.

[0130] Plants of the present invention can pass soybean line GM_CSM63770 DNA (including transgenes inserted into soybean line GM_CSM63770) to subsequent generations, which is usually carried out through conventional breeding and selection. As used herein, "progeny" includes any plant, plant cell, seed and / or regenerable plant part that contains line GM_CSM63770 DNA derived from an ancestral plant and / or contains a DNA molecule having at least one sequence selected from the following: 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. Plants, progeny and seeds can be homozygous or heterozygous for the transgene of line GM_CSM63770. Progeny can be grown from seeds produced by plants containing soybean line GM_CSM63770 and / or from seeds produced by plants fertilized with pollen from plants containing soybean line GM_CSM63770.

[0131] Progeny plants can be self-pollinated (also called "selfing") to produce true plant breeding lines, ie, plants homozygous for the transgene. Selfing of appropriate progeny can produce plants that are homozygous for both added exogenous genes.

[0132] Alternatively, progeny plants can be outcrossed, for example bred together with another unrelated plant, to produce variants or hybrid seeds or plants. Other unrelated plants can be transgenic or non-transgenic. Therefore, it is possible to produce hybrids comprising the unique and unique DNA of soybean line GM_CSM63770 by sexually hybridizing the first parent lacking the unique and unique DNA of soybean line GM_CSM63770 with the second parent comprising soybean line GM_CSM63770, thus obtaining variants or hybrid seeds or plants of the present invention. Each parent can be a hybrid or inbred line / variant, as long as hybridization or breeding produces plants or seeds of the present invention, that is, having at least one allele containing soybean line GM_CSM63770 DNA and / or having at least one seed of a DNA molecule selected from the following sequence: 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 SEQID NO:10. Therefore, two different transgenic plants can be hybridized to produce hybrid offspring containing two independently isolated, added foreign genes. For example, Cry1A.2 and Cry1B.2 containing soybean strain GM_CSM63770 and conferring soybean insect resistance can be hybridized with other transgenic soybean plants to produce plants with the characteristics of two transgenic parents. One of them is a plant hybridization containing soybean strain GM_CSM63770 and Cry1A.2 and Cry1B.2 that make soybean have lepidopteran insect resistance and one or more additional traits (such as herbicide tolerance, insect resistance or drought tolerance), thereby producing a progeny plant or seed that is resistant to lepidopteran pests and has at least one or more additional traits. As vegetative propagation, backcrossing with parental plants and outcrossing with non-transgenic plants are also covered. Descriptions of other breeding methods commonly used for different traits and crops can be found in one of several references, such as Fehr, Breeding Methods for Cultivar Development, Wilcox J., ed., American Society of Agronomy, Madison WI (1987).

[0133] Plant of the present invention, progeny, seed, pollen and ovule, cell and plant part also can contain one or more extra soybean traits or transgenic strains, particularly by making the soybean plant containing soybean strain GM_CSM63770 and another plant hybridization containing described one or more extra traits or transgenic strains and introduce those.These one or more traits or transgenic strains include but are not limited to the insect resistance, herbicide tolerance, the water use efficiency of improvement, the yield performance of improvement, the drought resistance of improvement, the seed quality of improvement, the nutritional quality of improvement, the hybrid seed yield or the disease or fungus resistance of increase.Soybean transgenic strains are well known to those skilled in the art.For example, the United States Department of Agriculture (United States Department of Agriculture's, USDA) Animal and Plant Health Inspection Service (Animal and Plant Health Inspection Service, APHIS) provides the list of such traits, and the list can be found in its website www.aphis.usda.gov on the World Wide Web. Therefore, two or more transgenic lines can be combined in the progeny seed or plant by hybridizing two parent plants that each contain one or more transgenic lines, collecting progeny seeds and selecting progeny seeds or plants containing the two or more transgenic lines. These steps can be repeated until the desired transgenic line combination is obtained in the progeny. Backcrossing with the parent plant and outcrossing with non-transgenic plants are also encompassed, and are vegetative propagation.

[0134] Also provided are plant parts derived from soybean plants comprising soybean line GM_CSM63770. As used herein, "plant part" refers to any part of a plant comprising material derived from a soybean plant comprising line GM_CSM63770. Plant parts include, but are not limited to, pollen, ovules, pods, flowers, root or stem tissue, fibers, and leaves. Plant parts can be vigorous, inactive, regenerable, and / or non-regenerable.

[0135] A commercial product is also provided, the commercial product is derived from a soybean plant comprising soybean strain GM_CSM63770, and contains a detectable amount of nucleic acid unique to soybean strain GM_CSM63770. As used herein, "commercial product" refers to any composition or product comprising a soybean plant, complete or processed soybean seeds, or one or more plant cells and / or plant parts containing soybean strain GM_CSM63770 DNA. Non-viable commercial products include, but are not limited to, inactive seeds, complete or processed seeds, seed parts, and plant parts; animal feed, soybean oil, soybean protein, soybean meal, soybean meal, soybean flakes, soybean bran, soy milk, soy cheese, soy wine, paper comprising soybeans, cream comprising soybeans, soybean biomass, and fuel products produced using soybean plants and soybean parts. Vigorous commercial products include, but are not limited to, seeds, plants, and plant cells. Therefore, soybean plants comprising soybean strain GM_CSM63770 can be used to make any commercial products commonly obtained from soybeans. Any such commodity product derived from a soybean plant comprising soybean line GM_CSM63770 may contain at least a detectable amount of a unique and distinctive DNA corresponding to soybean line GM_CSM63770, and in particular, may contain a detectable amount of a polynucleotide comprising a DNA molecule having at least one 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. Any standard method for detecting nucleotide molecules may be used, including the detection methods disclosed herein. If any detectable amount of a DNA molecule having at least one 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 is present in the commodity product, the commodity product is within the scope of the present invention.

[0136] Therefore, the soybean plants, soybean plant cells, soybean seeds, soybean plant parts (e.g., pollen, ovules, silks, ears, anthers, cobs, root tissue, stem tissue, leaf tissue), soybean progeny plants and commodity products of the present invention are particularly useful for growing plants to produce seeds and / or plant parts comprising soybean line GM_CSM63770 for agricultural purposes, producing progeny comprising soybean line GM_CSM63770 for plant breeding and research purposes, for use with microbial technology in industrial and research applications, and for sale to consumers.

[0137] Provided are methods for producing insect-resistant soybean plants, the soybean plants comprising the unique and distinctive DNA sequences of the strain GM_CSM63770 of the present invention. The transgenic plants used in these methods can be homozygous or heterozygous for the transgene. The progeny plants produced by these methods can be variants or hybrid plants; they can be grown from seeds produced by plants containing the soybean strain GM_CSM63770 and / or from seeds produced by plants fertilized with pollen from plants containing the soybean strain GM_CSM63770; and they can be homozygous or heterozygous for the transgene. The progeny plants can then be self-pollinated to produce true plant breeding lines, i.e., plants that are homozygous for the transgene; or alternatively, outcrossing can be performed, such as breeding with another unrelated plant, to produce varieties or hybrid seeds or plants.

[0138] Provided are methods for detecting the presence of DNA derived from a soybean plant, soybean tissue, soybean seed or soybean plant comprising soybean line 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 at least one primer capable of producing a DNA sequence specific to line GM_CSM63770 DNA under conditions suitable for DNA sequencing; (iii) performing a DNA sequencing reaction; and then (iv) confirming that the nucleotide sequence comprises a nucleotide sequence specific to line GM_CSM63770 in a construct contained therein, such as 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 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 an amplicon from the strain GM_CSM63770 DNA under conditions suitable 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 comprises a nucleotide sequence specific to the strain GM_CSM63770, such as 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 amplicon specific to the strain GM_CSM63770, such as an amplicon comprising 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 a nucleotide sequence specific to soybean line GM_CSM63770 in the amplicon will determine and / or diagnose or characterize the presence of DNA specific to soybean line GM_CSM63770 in the sample. An example of a primer pair capable of generating an amplicon from line GM_CSM63770 DNA under conditions suitable for DNA amplification is provided as SEQ ID NO: 14 and SEQ ID NO: 15.Other primer pairs can be easily designed by one skilled in the art, and these primer pairs will produce an amplicon comprising 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, wherein such primer pairs comprise at least one primer within the genomic region flanking the insert and a second primer within the insert. Another method for detecting the presence of DNA derived from a soybean cell, soybean tissue, soybean seed, or soybean plant comprising soybean line GM_CSM63770 in a sample 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 DNA probe specific for line GM_CSM63770 DNA; (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 a sequence of a DNA probe specific for line GM_CSM63770 is provided as SEQ ID NO: 16. Other probes can be easily designed by one skilled in the art, and these probes will contain at least one fragment of the genomic DNA flanking the insert and at least one fragment of the insert DNA, such as 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 will diagnose or characterize the presence of soybean line GM_CSM63770 specific DNA in the sample. Alternatively, the absence of hybridization will diagnose or characterize the absence of soybean line GM_CSM63770 specific DNA in the sample.

[0139] The DNA detection kits provided can be used to identify soybean line GM_CSM63770 DNA in a sample, and can also be applied to methods for breeding soybean plants containing appropriate line DNA. These kits contain DNA primers and / or probes comprising 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 comprises at least one DNA molecule having a sufficient length of continuous nucleotides of SEQ ID NO:10, acting as a DNA probe, which can be used to detect the presence and / or absence of DNA derived from a transgenic soybean plant comprising line GM_CSM63770 in a sample. DNA derived from a transgenic soybean plant comprising line GM_CSM63770 will comprise a DNA molecule having at least one 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. A DNA molecule sufficient for use as a DNA probe that can be used to determine, detect, or diagnose the presence and / or absence of soybean line GM_CSM63770 DNA in a sample is provided as SEQ ID NO: 16. Other probes can be readily designed by those skilled in the art and should comprise 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 be sufficiently unique to soybean Line GM_CSM63770 DNA to identify DNA derived from said line.

[0140] Another type of kit comprises a primer pair that can be used to generate an amplicon that can be used to detect the presence and / or absence of DNA derived from transgenic soybean line GM_CSM63770 in a sample. This kit will use a method that includes contacting a target DNA sample with a primer pair as described herein, then performing a nucleic acid amplification reaction sufficient to generate an amplicon, the 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 detecting the presence and / or absence of the amplicon. This method can also include sequencing the amplicon or a fragment thereof, which can determine, i.e., diagnose or characterize the presence of soybean line GM_CSM63770-specific DNA in the target DNA sample. Other primer pairs can be readily designed by those skilled in the art, and these primer pairs should comprise 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 sequence provided in, but not limited to, 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 be sufficiently unique to the soybean line GM_CSM63770 DNA to identify DNA derived from said line.

[0141] The kits and detection methods of the present invention are particularly useful for identifying soybean line GM_CSM 63770; selecting plant varieties or hybrids comprising soybean line GM_CSM63770; detecting the presence of DNA derived from a transgenic soybean plant comprising line GM_CSM63770 in a sample; and monitoring the presence and / or absence of a soybean plant comprising line GM_CSM63770 or a plant part derived from a soybean plant comprising line GM_CSM63770 in a sample.

[0142] The sequence of the heterologous DNA insert, junction sequence, or flanking sequence from soybean line GM_CSM63770 can be verified (and corrected, if necessary) by amplifying such sequences from the line using primers derived from the sequences provided herein, followed by standard DNA sequencing of the amplicon or cloned DNA.

[0143] Provided are methods for detecting the zygosity of transgenic alleles of DNA derived from a soybean plant, soybean tissue, soybean seed or soybean plant containing soybean line GM_CSM63770 in a sample. A 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 that is diagnostic or characterizing line GM_CSM63770; (iii) contacting the DNA sample with a primer pair capable of producing a second amplicon that is diagnostic or characterizing native soybean genomic DNA that does not contain soybean line GM_CSM63770; (iv) performing a DNA amplification reaction; and then (v) detecting the amplicons, wherein the presence of only the first amplicon will diagnose homozygous soybean line GM_CSM 63770 DNA in the sample, and the presence of both the first amplicon and the second amplicon will diagnose a soybean plant that is heterozygous for the soybean line GM_CSM63770 allele. An exemplary set of primer pairs is as follows: SEQ ID NO: 14 and SEQ ID NO: 15, which primer pairs produce an amplicon that diagnoses or characterizes the line GM_CSM63770; and SEQ ID NO: 20 and SEQ ID NO: 15, which primer pairs produce an amplicon that diagnoses or characterizes wild-type soybean genomic DNA without soybean line GM_CSM63770. A set of probes can also be incorporated into this amplification method for use in a real-time PCR format using the primer pair sets described above. An exemplary set of probes is as follows: SEQ ID NO: 16 (an amplicon that diagnoses or characterizes soybean line GM_CSM63770) and SEQ ID NO: 21 (an amplicon that diagnoses or characterizes wild-type soybean genomic DNA without soybean line GM_CSM63770).

[0144] 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 probe set, the probe set comprising at least one first probe that specifically hybridizes to DNA of line GM_CSM63770 and at least one second probe that specifically hybridizes to soybean genomic DNA that is interrupted by insertion of heterologous DNA of soybean line GM_CSM63770 and does not hybridize to DNA of soybean line GM_CSM63770; (iii) hybridizing the probe set to the sample under stringent hybridization conditions, wherein detection of hybridization of only the first probe under the hybridization conditions is diagnostic or characterizing homozygous alleles of soybean line GM_CSM63770 DNA in the sample; and wherein detection of hybridization of both the first probe and the second probe under the hybridization conditions is diagnostic or characterizing heterozygous alleles of soybean line GM_CSM63770 in the DNA sample.

[0145] Yet 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 primer pair capable of producing an amplicon that is diagnostic or characterizing an allele of line GM_CSM63770; (iii) contacting the DNA sample with a primer pair capable of producing an amplicon of an internal standard that is known to be single copy and homozygous in soybean plants; (iv) contacting the DNA sample with a probe set comprising at least one first probe that specifically hybridizes to an allele of line GM_CSM63770 and at least one probe that specifically hybridizes to an internal standard. (v) performing a DNA amplification reaction using real-time PCR and determining a cycle threshold (Ct value) for the amplicon corresponding to the toxin encoding sequence and the single copy homozygous internal standard; (vi) calculating the difference (ΔCt) between the Ct value of the single copy homozygous internal standard amplicon and the Ct value of the toxin encoding sequence amplicon; and (vii) determining zygosity, wherein a ΔCt of about zero (0) indicates that the inserted T-DNA is homozygous, and a ΔCt of about one (1) indicates that the inserted T-DNA is heterozygous. Heterozygous lines are distinguished from homozygous lines by a ΔCt value of about one (1). Taking into account the normal variability observed in real-time PCR due to factors such as amplification efficiency and ideal annealing temperature, the range of "about one (1)" is defined as a ΔCt of 0.75 to 1.25. The primer pairs and probes used in the above-mentioned method of determining zygosity can amplify and detect amplicons from alleles of strain GM_CSM63770 and internal standards. Exemplary primer pairs for detecting amplicons corresponding to alleles of strain GM_CSM63770 and internal standards are presented as a combination of SEQ ID NO: 14 and SEQ ID NO: 15 (alleles of strain GM_CSM63770) and a combination of SEQ ID NO: 17 and SEQ ID NO: 18 (internal standard). Accompanying exemplary probes are presented as SEQ ID NO: 16 (alleles of strain GM_CSM63770) and SEQ ID NO: 19 (internal standard).

[0146] Preservation Information

[0147] On August 21, 2019, a representative sample of soybean seed containing line GM_CSM63770 was deposited with the American Type Culture Collection (ATCC) at 10801 University Boulevard, Manassas, Virginia USA, 20110, under the Budapest Treaty, and assigned ATCC Accession No. PTA-126048. During the pendency of this application, the deposit may be accessed upon request by the Commissioner of Patents and Trademarks and persons qualified by the Commissioner. Upon issuance of a patent, all restrictions on public availability will be irrevocably lifted. The deposit will be maintained at the depository for a period of thirty (30) years, or five (5) years after the last request, or for the life of the patent, whichever is longer.

[0148] Example

[0149] The following examples are included to more fully describe the invention.The rigorous molecular, agronomic, and field testing required to generate and select soybean line GM_CSM63770 summarized the construction and testing of 125 constructs, the generation of 3,343 lines, and the analysis of hundreds of thousands of individual plants over more than 6 years.

[0150] Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0151] Example 1

[0152] Expression cassette testing, construct design, construct selection, molecular characterization, efficacy testing, field trials, and line selection

[0153] It is often necessary to generate and screen large numbers of gene expression constructs and transformed lines in order to identify constructs and then lines that exhibit optimal expression of the introduced gene of interest without producing agronomic or phenotypic off-types.

[0154] For these reasons, extensive research, development, and analysis were required to develop a transgenic soybean plant that contained insecticidal proteins effective against lepidopteran insects without any negative impact on agronomics, yield, or stacking viability. Specifically, approximately 3,343 proof-of-concept and commercial transgenic lines derived from 125 different plasmid vector constructs were developed, tested, and analyzed over a 6-year period.

[0155] This example describes the design of 125 different constructs and testing in soybean plants to identify preferred constructs for producing strains. Each construct differs in the coding sequence of the insecticidal protein and the transcriptional regulatory elements, and these constructs are tested to select preferred constructs for expressing the insecticidal protein in plants. Each construct has a unique configuration that varies depending on the expression cassette composition (insecticidal protein and expression element), orientation, and whether the protein is inserted into the chloroplast.

[0156] During the initial proof-of-concept and development phase, approximately 992 transformed lines were generated using 121 constructs containing different combinations of 29 different promoters, 1 enhancer, 16 different introns, and 21 different insect toxin coding sequences and 10 different 3'UTRs. These lines were evaluated for phenotype or agronomic heteromorphism, expression levels of insect toxin proteins, and efficacy against selected lepidopteran insect pest species. The resulting efficacy and protein expression data, as well as any information on phenotype and agronomic heteromorphism, were used to eliminate ineffective proteins, expression elements, and combinations, and these were used to design a smaller number of binary commercial transformation plasmid constructs for the next phase of development.

[0157] In the development of the next stage, 4 new commercial constructs are produced again. These constructs include a combination of 2 to 3 insect toxin transgenic expression cassettes in different orientations (convergence or divergence). According to the study of the mode of action of an insect toxin protein expressed in the construct, a construct is eliminated before transformation. The remaining 3 constructs, i.e. pM63770, construct-2 and construct-3 are used to produce transformation strains (also referred to as "transformants"). Table 2 below shows the strain selection and elimination process of all three commercial construct transformation strains.

[0158] Table 2. Line selection and elimination of stably transformed soybean lines, from which GM_CSM63770 was selected.

[0159]

[0160] After buds formed in culture, a subset of transformed lines were selected based on visual characteristics and early molecular analysis. After transformation, 12,036 transformants were transferred to culture plates containing selective medium (also known as "plugged"). After preliminary molecular characterization, 9,685 transformants were eliminated. Of the remaining 2,351 lines, 1,877 lines were eliminated based on observations of plant health and survival. The remaining 474 R0 lines were transplanted into pots and grown in a greenhouse (GH) for further testing. The molecular characteristics and efficacy of the 474 lines were evaluated, and 327 lines were eliminated based on these studies.

[0161] The remaining 147 R0 lines were self-pollinated to produce R1 seeds. The R1 lines were further molecularly characterized and assessed for zygosity, performance, and maturity. Based on this analysis, 116 lines were eliminated. At this selection point, it was also observed that the lines derived from construct-3 did not meet the criteria for further study, and this construct and its related lines were excluded from further testing and analysis. The remaining 9 lines derived from construct pM63770 and 14 lines derived from construct-2 were selected for further molecular characterization and field study evaluation.

[0162] In the R2 generation, the molecular characteristics and performance of each strain are further evaluated.During the Argentine growing season from 2017 to 2018, 23 strains (9 strains are derived from construct pM63770, and 14 strains are derived from construct-2) are evaluated for effect in net house and field trials and agronomy in field trials.According to the R2 standard (molecular characteristics and effect), 1 strain derived from construct pM63770 is eliminated, and 7 strains derived from construct-2 are also eliminated.According to yield measurement, 1 strain derived from each of the two constructs is eliminated.Another strain derived from construct pM63770 is eliminated because it cannot be crossbred with superior germplasm material.

[0163] Evaluate the effect of remaining 20 strains (wherein 7 are derived from construct pM63770 and 13 are derived from construct-2) in net house and field test and field agronomy test.In addition, further molecular research has also been carried out, comprises the T-DNA of identifying each strain and inserts gene seat, adjacent gene and the repetitive sequence near the insertion gene seat.After having commented on effect and agronomy data and more meticulous molecular characterization, eliminated 5 strains that are derived from construct pM63770 and 12 strains that are derived from construct-2, only leave 2 events that are derived from construct pM63770 and 1 event that is derived from construct-2.After having commented on the side joint sequence details that electronic version Southern (eSouthern) data provides, eliminated 1 event that is derived from construct pM63770. Based on the final comparison of yield, efficacy and molecular characteristics of each of the remaining lines derived from construct pM63770 and construct-2, line GM_CSM63770 derived from construct pM63770 was selected as the commercial product.

[0164] Thus, after multiple rounds of testing and comparison of various constructs, it was found that lines generated with the transgenic cassette provided by SEQ ID NO: 13 (construct pM63770) exhibited superior efficacy against the following lepidopteran pest species: soybean pod borer (SPW; American bollworm), soybean armyworm (Soybean looper), velvet bean caterpillar (Spodoptera: lilii), southern cutworm (Subtropical armyworm), black cutworm (Spodoptera: litura), South American pod borer (Argentine bollworm), sunflower armyworm (Spodoptera: bicolor), bean stem moth (Spodoptera: citrinopilidae), green armyworm (Spodoptera: alfalfa looper), and small corn stalk borer (Ostrinia nubilalis) when compared to lines generated with all other constructs evaluated. Soybean line GM_CSM63770 was selected from a large number of lines generated using this pM63770 construct because it had superior efficacy and agronomic performance characteristics compared to other lines generated using this construct.

[0165] Example 2

[0166] The soybean line GM_CSM63770 exhibits resistance to the lepidopteran insect pests soybean pod borer, soybean armyworm, velvet bean caterpillar, southern cutworm, black cutworm, South American pod borer, sunflower armyworm, bean stem moth, green armyworm and small corn stalk borer.

[0167] This example describes the activity of soybean line GM_CSM63770 against several different lepidopteran insect pests of soybean. The insect toxin proteins Cry1A.2 and Cry1B.2, when expressed together in soybean line GM_CSM63770, provide resistance to soybean pod borer (SPW; American bollworm), soybean armyworm (SBL; soybean looper), velvet bean caterpillar (VBC; lily bean armyworm), southern cutworm (SAW; subtropical armyworm), black cutworm (BLAW; Spodoptera litura), South American pod borer (SAPW; Argentinian bollworm), sunflower armyworm (SFL; two-color bearded moth), bean stem moth (BSM; night squirrel), green armyworm (GCW; green clover armyworm) and small corn stalk borer (LCSB; small corn borer).

[0168] A nethouse trial was conducted at three locations to evaluate resistance to the southern armyworm (SAW; subtropical armyworm) as well as the velvet bean caterpillar (VBC; Spodoptera exigua) and soybean pod borer (SPW; American bollworm). Soybean line GM_CSM63770 and lines transformed with constructs pM63770, construct-2, and construct-3 were evaluated using a randomized complete block design. Each line plot was planted with a row spacing of 6 feet, with approximately 8 seeds per foot. There were 3 replicates (reps) for each line; therefore, each line was represented in the nethouse by 3 independent plots that were randomly positioned within the nethouse. The untransformed line was used as a negative control, and its plots were also randomly assigned to locations within the nethouse.

[0169] Adult moths were used to achieve infestation of SPW and VBC. These insects were reared to pupation in insect adult hatching cages and placed in a climate-controlled incubator. These insects were released into the mesh house. About 1,200 to 2,000 adults were released into the mesh house at one time. For SPW, adults were released into the mesh house weekly from the R1 to R2 stage of soybean development. For VBC, adults were released into the mesh house every two weeks between the V4 to R3 stages of development. About 1,200 to 2,000 adults were released into the mesh house at one time. Adult moths require constant exposure to a 10% sucrose solution to maintain normal lifespan and fecundity. Fill a plastic food container with cotton wool and pour the sugar solution into the container until the cotton wool is completely soaked. Replenish the sugar solution daily until adult activity subsides, which is usually about 2 weeks after the final release of the adults.

[0170] Since SAW do not lay eggs preferentially or evenly on soybeans, infestations were made directly using eggs of this insect. Infestations were made every two weeks from the R1 to R3 developmental stages, using approximately 250,000 to 320,000 eggs per infestation. Paper sheets containing equal amounts of SAW eggs were folded and placed over sturdy petioles in the upper part of the canopy and attached to the plants by staples firmly together. One sheet was placed on the plants within 1 foot of the front of the plot, the second sheet was placed on the plants in the middle of the plot, and the third sheet was placed on the plants within 1 foot of the back of the plot.

[0171] After each infestation, the defoliation caused by all three insects was evaluated twice: once about 2 weeks after infestation and the other about four weeks after infestation. Plant growth stage and defoliation percentage were recorded after each evaluation. In order to determine the defoliation percentage, the canopy of each plot was divided into 5 districts. From each district, a leaf with three leaves was selected, and the damage presented by the leaf represented the damage of the selected district. The defoliation percentage was determined using the damage chart provided to each assessor. The average defoliation percentage was calculated by the average value of the single leaf with three leaves selected from the 5 districts. The pod yield and damage in the presence of SPW were determined when the soybean plant matured. Five plants were randomly selected from each plot, and the total number of pods and the number of damaged pods of each plant were recorded. By sampling 5 plants, the average number of pods and the average percentage of damaged pods were determined. Table 3 below shows the average defoliation percentage of soybean line GM_CSM63770 and negative control evaluated in the presence of all three insects SAW, VBC and SPW. Table 4 below shows the average number of pods and the average percentage of pod damage caused by SPW.

[0172] Table 3. Average percent leaf drop for soybean line GM_CSM63770 infested with SAW, VBC, and SPW and negative controls in the 2017 US nethouse trial.

[0173]

[0174]

[0175] Table 4. Average pod number and average percentage of damaged pods for soybean line GM_CSM63770 and negative controls infested with SPW in 2017 US nethouse trials.

[0176] strain Average number of pods Average damage rate% Pod damage reduction % Negative 25 8％ - GM_CSM63770 47 5％ 36％

[0177] As can be seen from Table 3 above, soybean line GM_CSM63770 provides superior resistance to SAW, VBC and SPW compared to the negative control. In addition, as can be seen from Table 4, the percentage of damaged pods in soybean line GM_CSM63770 is reduced relative to the negative control.

[0178] The net house and field efficacy test were conducted. Each plot in the net house contained 42 seeds in one row with a row spacing of 2 meters. Each strain was represented by 3 replicates randomly arranged in the net house. The net house test was conducted for the lepidopteran insect pests Velvet Bean Caterpillar (VBC; Spodoptera litura), Black Spodoptera litura (BLAW; Spodoptera litura), South American Podworm (SAPW; Argentine Bollworm) and Sunflower Moth (SFL; Spodoptera litura). During each infestation, up to about 500 adult moths were released into the net house. Two infestations were conducted, the first at the V3 development stage and the second at the R2 development stage. Adult moths were raised as described above using 10% sugar solution soaked in absorbent cotton in a container. The percentage of defoliation was determined according to the above method. Table 5 below shows the average percentage of defoliation for the determined soybean line GM_CSM63770 and the negative control.

[0179] Table 5. Average percent leaf drop of soybean line GM_CSM63770 and negative control infected with BLAW, SAPW, SBL and SFL.

[0180]

[0181]

[0182] As can be seen from Table 5 above, soybean line GM_CSM63770 exhibited excellent resistance to BLAW, SAPW, SBL and SFL when compared to the negative control.

[0183] Field efficacy trials were also conducted at six different locations (AF). Each plot contained 4 rows with a row spacing of 10 meters, 25 seeds per meter, and approximately 1 meter apart. Three replicate plots were randomly arranged within the field for each line. Sampling was carried out in the field to determine the types of Lepidoptera species and the corresponding percentages of these insects in each field. The Lepidoptera species found in the field were: Velvet Bean Caterpillar (VBC; Spodoptera velutipes); Geometrids (SBL / SFL), namely soybean armyworm (SBL; soybean looper) and sunflower armyworm (SFL; bicolor bearded moth); Spodoptera spp. (SPO); and South American pod borer (SAPW; Argentine bollworm). Insect monitoring was initiated in the non-transgenic border rows at the beginning of the V3 stage, with at least 10 samples evenly distributed around the border. Sampling was carried out every 7 to 10 days in the border rows. Monitoring continued until the R6 stage, when pod filling was complete. Defoliation was determined in a similar manner as in the net house trial. Pod damage was also carried out in a similar manner to the net house experiment. In addition, the number of larvae per meter of planting row was also recorded. When compared with the negative control plants, there were fewer Lepidoptera larvae on the transformed strains, which indicated resistance because the resistant plants killed the larvae and left very few larvae on the plants. Table 6 below shows the average percentage of maximum leaf fall, cumulative larvae per meter of row, and pod damage. In addition, the percentage of each Lepidoptera species at each location was also provided.

[0184] As can be seen in Table 6 below, soybean line GM_CSM63770 showed resistance to lepidopteran insects in naturally infested fields when compared to the negative control. Most lepidopteran pest species in most fields were VBC and SBL / SFL. One location had a high percentage of SAPW and almost no VBC. As can be seen from the results below, GM_CSM63770 was effective against VBC, SBL / SFL, and SAPW, as shown by a reduction in the percentage of defoliation, a reduction in the cumulative number of larvae per meter of row, and a reduction in pod damage.

[0185]

[0186]

[0187] Plants at both sites were also examined for damage caused by bean moth (BSM; Crocidosema aporema). Ten plants were randomly selected from each of the three plots for each line. The average percent damage of the controls was 6% and 4.7% in Site 1 and Site 2, respectively. Soybean line GM_CSM63770 did not show any damage at Site 1 and only 0.3% damage at Site 2.

[0188] The net house efficacy trial was conducted at multiple locations against the lepidopteran insect pest species southern cutworm (SAW, subtropical armyworm), soybean armyworm (SBL; soybean looper), soybean pod borer (SPW; American bollworm) and velvet bean caterpillar (VBC; Velvet bean armyworm). SBL, SPW and VBC infestations were performed as described above using adult moths. SAW infestations were performed as described above by egg infestations. Leaf drop was determined as described above. Plot size and number of replicates were also as described above. Table 7 below shows the infestation frequency, stage of infestation, maximum percentage of leaf drop and percentage reduction of leaf drop corresponding to line GM_CSM63770 compared to the negative control.

[0189] Table 7. Infection frequency and stage as well as maximum leaf drop and percentage reduction of soybean line GM_CSM63770 and negative control infected with SAW, SBL, SPW and VBC.

[0190]

[0191] As can be seen from Table 7 above, soybean line GM_CSM63770 is effective against SAW, SBL, SPW and VBC as shown by lower maximum percentage of leaf drop and percentage reduction of leaf drop compared to the negative control. In these studies, soybean line GM_CSM63770 provided resistance to southern armyworm (SAW; subtropical armyworm), soybean armyworm (SBL; soybean looper), soybean pod borer (SPW; American bollworm) and velvet bean caterpillar (VBC; Velvet bean worm).

[0192] Field efficacy trials were conducted against natural lepidopteran insect pressure at five different locations (JN). Each plot contained approximately 4 rows of 25 feet each with approximately 8 seeds per foot. Each line was represented by 4 plots randomly arranged in the field. Defoliation measurements were made in a similar manner to those described above and were performed approximately every 10 days from the R1 to R6 developmental stages. Pod injury was assessed after the maximum injury occurred in the negative control. Five plants were randomly selected for each line to assess pod injury. Lepidopteran species population assessments were performed at the R1 to R6 developmental stages. One or more fields were found to be infested with velvet bean caterpillar (VBC; Spodoptera velutipes), soybean armyworm (SBL; soybean looper), soybean podworm (SPW; American bollworm), green armyworm (GCW; alfalfa green armyworm), yellowstriped armyworm (YAW; yellowstriped armyworm (Spodoptera ornithogalli)) and beet armyworm (BAW; Spodoptera exigua). The cumulative number of larvae per meter of row was also determined for each line. Table 8 below shows the percentage of Lepidoptera species, maximum percentage of defoliation and percentage reduction for each location, as well as the cumulative number of larvae per meter of row and percentage reduction of larvae.

[0193] Table 8. Lepidopteran species, maximum percent defoliation and percent defoliation reduction, cumulative numbers, and percent larval reduction for soybean line GM_CSM63770 versus negative controls in naturally infested fields.

[0194]

[0195]

[0196] As can be seen from Table 8 above, based on the percentage of the more prominent Lepidoptera species in each field, soybean line GM_CSM63770 was effective against velvet bean caterpillar (VBC; Spodoptera velvetifolia), soybean armyworm (SBL; soybean looper), and green armyworm (GCW; lucerne armyworm), as evidenced by the percentage reduction in defoliation and larvae for soybean line GM_CSM63770 when compared to the negative control. For example, in site D, the most dominant insect pest species were GCW and SBL, and accounted for ninety-four percent (94%) of the total species observed in the field. No larvae were observed in GM_CSM63770, confirming that this line was highly resistant to these two species, as no live larvae were found in the line records, while for the negative control, more than 21 larvae were observed per meter of row.

[0197] Pod damage was assessed in site M, where the major pest species was SBL. Table 9 shows the average percentage of pod damage and the percentage reduction in pod damage for soybean line GM_CSM63770.

[0198] Table 9. Average percent and reduction in seed damage for soybean line GM_CSM63770 and negative control.

[0199]

[0200] As can be seen in Table 9 above, while soybean line GM_CSM63770 and the negative control had similar numbers of pods per plant, line GM_CSM63770 showed 80% reduction in pod damage in a field where the majority of the lepidopteran insects were SBLs.Soybean line GM_CSM63770 exhibited resistance to SBLs.

[0201] Soybean line GM_CSM63770 is effective against the lepidopteran insect pests Velvet Bean Caterpillar (VBC; Spodoptera: Soybean Armyworm), Spodoptera: Soybean Looper (SBL; Soybean Looper), and Green Armyworm (GCW; Green Armyworm) based on measurements of defoliation, larval numbers, and pod damage in naturally infested fields.

[0202] The net house and field efficacy studies were conducted at two locations (OP). The net house trials were conducted in Acevedo and Fontezuela, Buenos Aires Province, and were conducted against the lepidopteran insect pests Velvet Bean Caterpillar (VBC; Spodoptera litura), Black Spodoptera litura (BLAW; Spodoptera litura), South American Podworm (SAPW; Argentine Bollworm), Soybean Looper (SBL; Soybean Looper) and Sunflower Looper (SFL; Two-color Bearded Looper). Three plots similar to those described above for the 2017-2018 Argentina net house trials were randomly set up in the net house. Each strain was represented by three plots. Each net house was infested with adult moths, and the moths were maintained on a sugar diet as described above. Defoliation measurements were performed as described above. Table 10 below shows the plant growth stage at which infestation occurred, the maximum defoliation percentage for each insect at the two locations, and the percentage reduction.

[0203] Table 10. Maximum percent leaf drop and percent reduction in leaf drop for soybean line GM_CSM63770 and negative control.

[0204]

[0205]

[0206] As can be seen from Table 10 above, soybean line GM_CSM63770 is effective against velvet bean caterpillar (VBC; Spodoptera litura), black cutworm (BLAW; Spodoptera litura), South American pod borer (SAPW; Argentine bollworm), soybean armyworm (SBL; soybean looper) and sunflower armyworm (SFL; Spodoptera litura).

[0207] Field trials were conducted at five locations (QU). Sampling was performed in the field to determine the types of lepidopteran species and the corresponding percentages of these insects in each field. The lepidopteran species found in the field were: velvet bean caterpillar (VBC; Spodoptera velutipes); loopers (SBL / SFL), namely soybean armyworm (SBL; soybean looper) and sunflower armyworm (SFL; two-color bearded armyworm); Spodoptera spp. (SPO); and South American pod borer (SAPW; Argentine bollworm). Each plot contained 4 rows, each row was 8 meters long, and each row had 25 seeds. Each strain was represented by 3 replicate plots randomly arranged in the field. Defoliation, cumulative larval number, and pod damage were measured as described above. Table 11 shows the maximum percentage of defoliation and the percentage reduction of defoliation for GM_CSM63770 and the negative control; the cumulative number of larvae per meter of row and the percentage reduction of larvae; the average percentage of pod damage and the percentage reduction of pod damage.

[0208]

[0209] As can be seen from Table 11 above, the largest proportion of lepidopteran insect pests in the fields of Site Q and Site U were VBC and SBL / SFL, while VBC was the main insect pest in the fields of Site S. From the above results, it can be seen that soybean line GM_CSM63770 is effective against VBC and SBL / SFL, which is reflected in the reduction of percentage of leaf fall, cumulative number of larvae per meter of row, and pod damage.

[0210] The effectiveness of soybean line GM_CSM63770 against small corn stem borer (LCSB; Ostrinia nubilalis) was determined using the leaf disc assay. Eight (8) plants of soybean line GM_CSM63770 and a negative control were grown in a greenhouse. Sixteen leaves were collected from each plant and assayed with first-instar larvae. The mortality rate and number of first-instar larvae (mortality + L1) were measured. Table 12 below shows the average mortality + L1 percentages for GM_CSM63770 and the negative control.

[0211] Table 12. Average mortality +L1 percentage of soybean line GM_CSM63770 infected with LSCB.

[0212] strain Mortality + L1 Negative 12.5％ GM_CSM63770 100.0％

[0213] As can be seen from Table 12 above, soybean line GM_CSM63770 is very effective against small corn stalk borer (LCSB; Little Corn Borer).

[0214] From the data presented above, it can be seen that soybean variety GM_CSM63770 provides resistance to the following Lepidoptera insect pest species: soybean pod borer (American bollworm), soybean armyworm (Soybean looper), velvet bean caterpillar (Spodoptera: Soybean moth), southern cutworm (Subtropical armyworm), black cutworm (Spodoptera: Spodoptera litura), South American pod borer (Argentine bollworm), sunflower armyworm (Spodoptera: Bicolor), bean stem moth (Spodoptera: Soybean looper), green armyworm (Spodoptera: Alfalfa looper) and small corn stalk borer (Ostrichia: Corn borer).

[0215] Example 3

[0216] Soybean line GM_CSM63770 provides similar consistent yield and agronomic performance to untransformed A3555 soybean plants

[0217] This example demonstrates that the transgenic soybean line GM_CSM63770 provides similar consistent yield and agronomic performance in the field as untransformed A3555 soybean plants.

[0218] Field trials were conducted in four different growing seasons to evaluate the agronomic traits and yield of soybean line GM_CSM63770 and compared with the parental transformation background under field conditions. Agronomic data for each plot were evaluated, such as emergence, date of first flowering, plant height, days to maturity, and yield (yield per acre corrected for moisture).

[0219] When compared to the transformed background A3555, soybean line GM_CSM63770 did not show any significant differences in agronomic traits such as emergence, flowering date and maturity except plant height; soybean line GM_CSM63770 was about 1 inch taller than the wild-type A3555 control, which was not biologically significant. When compared to the non-transgenic control, the expression of insecticidal proteins Cry1A.2 and Cry1B.2 in soybean line GM_CSM63770 did not negatively affect the agronomic and yield performance characteristics of soybean line GM_CSM63770.

[0220] Example 4

[0221] Soybean line GM_CSM63770 line-specific endpoint Determination

[0222] The following example describes a method that can be used to identify the presence of GM_CSM63770 in soybean samples. A pair of PCR primers and a probe were designed to identify GM_CSM63770 at the line-specific endpoint. The unique junction sequence formed between soybean genomic DNA and the inserted GM_CSM63770 DNA in PCR. Tables 13 and 14 describe the unique junction sequences used for line-specific endpoints. Example of conditions for identifying the presence of GM_CSM63770 in soybean samples in PCR.

[0223] The sequence of oligonucleotide forward primer SQ13805 (SEQ ID NO: 14) is identical to the nucleotide sequence corresponding to positions 13,177-13,202 of SEQ ID NO: 10. The sequence of oligonucleotide reverse primer SQ51400 (SEQ ID NO: 15) is identical to the reverse complementary sequence of the nucleotide sequence corresponding to positions 13,280-13,310 of SEQ ID NO: 10. The sequence of oligonucleotide probe PB4832 (SEQ ID NO: 16) is identical to the nucleotide sequence corresponding to positions 13,204-13,219 of SEQ ID NO: 10. Primers SQ13805 (SEQ ID NO: 14) and SQ51400 (SEQ ID NO: 15) and probe PB4832 (SEQ ID NO: 16) can be used for endpoint In a PCR assay to identify the presence of DNA derived from GM_CSM63770 in a sample, the probe may be fluorescently labeled (e.g., 6-FAM TM Fluorescent labeling).

[0224] In addition to SQ13805 (SEQ ID NO: 14), SQ51400 (SEQ ID NO: 15) and PB4832 (SEQ ID NO: 16), it should be apparent to those skilled in the art that other primers and / or probes can be designed to amplify or hybridize sequences within SEQ ID NO: 10 that are unique and can be used to detect the presence of DNA derived from GM_CSM63770 in a sample.

[0225] Following standard molecular biology laboratory practice, a PCR assay for strain identification was developed to detect GM_CSM63770 in samples. Each set of primer pairs and probes (e.g., probes with fluorescent labels such as 6-FAM TM Labeled probes) to optimize standard PCR assays or Parameters for PCR assays were used to detect the presence of DNA derived from GM_CSM63770 in samples. Controls for the PCR reactions included internal control primers and an internal control probe specific for a region within the soybean genome that served as an internal control (e.g. markers), which are primers SQ549 (SEQ ID NO: 17), SQ546 (SEQ ID NO: 18) and The probe PB0004 (SEQ ID NO: 19) was labeled.

[0226] In general, the parameters optimized for detecting GM_CSM63770 in a sample include primer and probe concentrations, template DNA amount, and PCR amplification cycle parameters. Controls for this analysis include a positive control from soybeans containing GM_CSM63770, a negative control from non-GM soybeans, and a negative control without template DNA.

[0227] Table 13. GM_CSM63770 strain-specific endpoints PCR reaction components.

[0228]

[0229]

[0230] Table 14. Endpoints Thermal cycler conditions

[0231]

[0232] Example 5

[0233] use Determination of zygosity of soybean line GM_CSM63770

[0234] The following example describes a method that can be used to identify the zygosity of line GM_CSM63770. PCR primer pairs and probes were designed to identify the specific characteristics of the T-DNA insertion positive allele that produced line GM_CSM63770, and to serve as an internal control probe specific to a region within the soybean genome that serves as an internal control and is represented as homozygous in the soybean genome.

[0235] In a real-time PCR reaction, such as the PCR reaction described in Example 6 above, a PCR primer pair and probe specific for the GM_CSM63770 transgenic allele described in Example 4, i.e., PCR primers SQ13805 (SEQ ID NO: 14), SQ51400 (SEQ ID NO: 15) and 6-FAM TM The labeled probe PB4832 (SEQ ID NO: 16), and a PCR primer pair and probe specific for the internal control, namely primers SQ549 (SEQ ID NO: 17), SQ546 (SEQ ID NO: 18) and Labeled probe PB0004 (SEQ ID NO: 19).

[0236] After amplification, the cycle threshold (Ct value) of the amplicon corresponding to the GM_CSM63770 insertion allele and the single copy homozygosity internal standard is determined. The difference (ΔCt) between the Ct value of the single copy homozygosity internal standard amplicon and the Ct value of the GM_CSM63770 insertion allele amplicon is determined. With respect to zygosity, ΔCt is about zero (0) indicating that the inserted GM_CSM63770 T-DNA is homozygous, while ΔCt is about one (1) indicating that the inserted GM_CSM63770 T-DNA is heterozygous. The lack of an amplicon corresponding to the GM_CSM63770 insertion allele indicates that there is no inserted GM_CSM63770 T-DNA in the sample. The Ct value in thermal amplification methods will vary somewhat depending on a variety of factors such as amplification efficiency and ideal annealing temperature. Therefore, the range of "about one (1)" is defined as a ΔCt of 0.75 to 1.25.

[0237] Example 6

[0238] use Determination of zygosity of soybean line GM_CSM63770

[0239] The following example describes a method that can be used to identify the zygosity of line GM_CSM63770 in a soybean sample.

[0240] PCR primer pairs and probes were designed to identify the specific characteristics of the T-DNA insertion positive and negative alleles that produced line GM_CSM63770. Tables 15 and 16 provide the strain-specific zygosity Example of conditions for PCR. To perform this assay, four primers and two probes were mixed with the sample. The DNA primer pairs used in the zygosity assay were primers 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 probe used in the zygosity assay was 6FAM TM Labeled probe PB4832 (SEQ ID NO: 16) and Labeled probes GM_WTA3555PB (SEQ ID NO: 21). SQ13805 (SEQ ID NO: 14) and SQ51400 (SEQ ID NO: 15) and 6FAM TMThe labeled probe PB4832 (SEQ ID NO: 16) will diagnose or characterize soybean line GM_CSM63770 DNA. When there are no copies of soybean line GM_CSM63770, primers GM_WTA3555F (SEQ ID NO: 20) and SQ51400 (SEQ ID NO: 15) and The labeled probes GM_WTA3555PB (SEQ ID NO: 21) are diagnostic; that is, they will diagnose or characterize the wild-type allele.

[0241] When three primers and two probes were mixed in a PCR reaction with DNA extracted from heterozygous plants of soybean line GM_CSM63770, 6FAM TM Labeled probe PB4832 (SEQ ID NO: 16) and The labeled probe GM_WTA3555PB (SEQ ID NO: 21) will release a fluorescent signal, thereby indicating and diagnosing or characterizing a soybean line GM_CSM63770 heterozygous plant. When the three primers and two probes are mixed with DNA extracted from a GM_CSM63770 homozygous plant in a PCR reaction, only the presence of 6FAM TM The fluorescent signal of the labeled probe PB4832 (SEQ ID NO: 16) was not The fluorescent signal of the labeled probe GM_WTA3555PB (SEQ ID NO: 21). When these three primers and two probes were mixed with DNA extracted from plants without soybean line GM_CSM63770 (i.e., wild type) in a PCR reaction, only The labeled probe GM_WTA3555PB (SEQ ID NO: 21) will generate a fluorescent signal. The template DNA samples and controls used for this analysis were positive controls from soybeans containing GM_CSM63770 DNA (from known homozygous samples and known heterozygous samples), negative controls from non-transgenic soybeans, and negative controls without template DNA.

[0242] Table 15. GM_CSM63770 zygosity PCR.

[0243]

[0244]

[0245] Table 16. Zygosity Thermal cycler conditions

[0246]

[0247] Example 7

[0248] Identification of soybean line GM_CSM63770 in any GM_CSM63770 breeding line

[0249] The following examples describe how to identify soybean line GM_CSM63770 in the progeny of any breeding campaign using soybean line GM_CSM63770. For example, soybean line GM_CSM63770 can be stacked with other lines known in the art to control lepidopteran pests through breeding or site-directed gene introgression, such as any of the following soybean lines, including but not limited to MON87701, MON87751, DAS81419 (lepidopteran resistance and herbicide tolerance). The soybean line GM_CSM63770 can also be stacked with other transgenic soybean lines known in the art through breeding or site-directed gene introgression to provide resistance to herbicides, including 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, WO201102248). 11022471), DAS44406-6 (US Patent Application Publication No. US2014041083), DAS68416-4 (US Patent Application Publication No. US20130296170), DAS81419-2 (US Patent Application Publication No. US2013338006), DP356043-5 (US Patent Application Publication No. US20100184079), FG72 (US Patent Application Publication No. US2011162098), GMB151 (US Patent Application Publication No. US2019345512), GTS 40-3-2, MON87708 (U.S. Patent Application Publication No. US, MON87751 (U.S. Patent Application Publication No. US20140373191), MON89788, SYHT0H2 (U.S. Patent Application Publication No. US20140201860) and SYHT04R (U.S. Patent Application Publication No. US20140310835). The soybean line GM_CSM63770 can also be stacked with other transgenic soybean lines known in the art through breeding or site-directed gene introgression to provide protection against the addition of Resistance to herbicides and modified oils, or enhanced photosynthesis, or drought tolerance, including 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).

[0250] The DNA primer pairs are used to generate an amplicon that is diagnostic or characterizing soybean line GM_CSM63770. The amplicon that is diagnostic or characterizing soybean line GM_CSM63770 comprises at least one junction sequence. The junction sequences of soybean line 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 ( Figure 1 , [1], [2], [3], [4], [5] and [6] in SEQ ID NO:10, respectively). 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. SEQ ID NO:1 is located at nucleotides 976-1,025 of SEQ ID NO:10. SEQ ID NO:2 is a 50 nucleotide sequence representing the 3' junction region of the soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO:2 is located at nucleotides 13,216-13,265 of SEQ ID NO:10. 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. SEQ ID NO:3 is located at nucleotides 951-1,050 of SEQ ID NO:10. SEQ ID NO:4 is a 100 nucleotide sequence representing the 3' junction region of the soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO:4 is located at nucleotides 13,191-13,290 of SEQ ID NO:10. SEQ ID NO:5 is a 200 nucleotide sequence representing the 5' junction region of the soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO:5 is located at nucleotides 901-1,100 of SEQ ID NO:10. SEQ ID NO:6 is a 200 nucleotide sequence representing the 3' junction region of the soybean genomic DNA and the integrated transgenic expression cassette. SEQ ID NO:6 is located at nucleotides 13,141-13,340 of SEQ ID NO:10.

[0251] The primer pairs that generate the amplicon that diagnoses or characterizes the line GM_CSM63770 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). In order to obtain a diagnostic amplicon that finds SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, it is necessary to design a forward primer molecule based on the 5' flanking soybean genomic DNA (SEQ ID NO: 11) from bases 1-1,000, and a reverse primer molecule based on the inserted T-DNA (SEQ ID NO: 9) from positions 1,001-13,240, wherein the primer molecules have a sufficient length of consecutive nucleotides to specifically hybridize with SEQ ID NO: 11 and SEQ ID NO: 9. In order to obtain a diagnostic amplicon for finding SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6, it is necessary to design a forward primer molecule based on the inserted T-DNA from positions 1,001-13,240 (SEQ ID NO:9) and a reverse primer molecule based on the 3' flanking soybean genomic DNA from positions 13,241-14,240 (SEQ ID NO:12), wherein the primer molecules have a sufficient length of consecutive nucleotides to specifically hybridize to SEQ ID NO:9 and SEQ ID NO:12.

[0252] For practical purposes, primers should be designed to produce amplicons of a limited size range (preferably between 200 and 1000 bases). In general, smaller amplicons are more reliably produced in thermal amplification reactions, allow for shorter cycle times, and can be easily separated and visualized on agarose gels or suitable for endpoint In a sample assay. In addition, the amplicons generated using the primer pairs can be cloned into a vector, propagated, isolated and sequenced, or can be directly sequenced using methods established 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 is an aspect of the present disclosure, and the primers can be used in a DNA amplification method to produce an amplicon that diagnoses or characterizes soybean line GM_CSM63770 or its progeny. Any single isolated DNA polynucleotide primer molecule comprising at least eleven (11) consecutive nucleotides of SEQ ID NO: 11, SEQ ID NO: 9 or SEQ ID NO: 12 or its complementary sequence is an aspect of the present disclosure, and the primer molecule can be used in a DNA amplification method to produce an amplicon that diagnoses or characterizes soybean line GM_CSM63770 or its progeny.

[0253] Examples of amplification conditions used for this analysis are shown in Tables 14 and 15. Any modification of these methods or the use of DNA primers homologous or complementary to SEQ ID NO: 11 or SEQ ID NO: 12, or the DNA sequence of the genetic elements contained in the transgenic insert of GM_CSM63770 (SEQ ID NO: 9) are within the art, which will produce an amplicon that is diagnostic or characterizes soybean line GM_CSM63770. Diagnostic amplicons contain DNA molecules that are homologous or complementary to at least one transgenic / genomic binding DNA or a substantial portion thereof.

[0254] Analysis of soybean line GM_CSM63770 plant tissue samples 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 pairs will amplify endogenous soybean DNA molecules and serve as internal controls for DNA amplification conditions. Those skilled in the art of DNA amplification methods can select additional primer sequences from SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 9. The conditions for producing amplicons selected by the methods shown in Table 13 and Table 14 may be different, but will all produce amplicons that diagnose or characterize soybean line GM_CSM63770 DNA. The use of DNA primer sequences within the methods of Table 13 and Table 14 or modifications to the methods are 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 is an aspect of the present invention, which amplicon will diagnose or characterize soybean line GM_CSM63770.

[0255] A DNA test kit is one aspect of the invention, the kit containing at least one DNA primer of sufficient length of contiguous nucleotides derived from SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO: 9, which, when used in a DNA amplification method, produces a diagnostic amplicon for GM_CSM63770 or its progeny. A soybean plant or seed is one aspect of the invention, wherein the genome of the soybean plant or seed, when tested in a DNA amplification method, will produce an amplicon that is diagnostic or characterizes soybean line GM_CSM63770. Applied Biosystems GeneAmp TM PCR System 9700, Stratagene The soybean line GM_CSM63770 amplicons can be assayed using a Gradient thermal cycler or any other amplification system, which can be used to generate the amplicons shown in Table 15 that are diagnostic or characterizing of the soybean line GM_CSM63770.

[0256] Example 8

[0257] Insertion of sequences into soybean line GM_CSM63770 using single guide RNA to facilitate removal of transgenic insertions

[0258] The following example describes how to use genome editing technology to remove the transgenic insertion present in soybean line GM_CSM63770. Sequences that can be used to remove the transgenic insertion or expression cassette of soybean line GM_CSM63770 in SEQ ID NO: 10 can be introduced by using a variety of methods, in particular by using a clustered regularly interspaced short palindromic repeat (CRISPR) editing system for genome editing. 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, 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. CRISPR-associated proteins and one or more guide RNAs (gRNAs) can be introduced into plant cells corresponding to soybean line GM_CSM63770 to target specific sequences within the transgenic insertion locus through double-strand break repair pathways, 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 a combination thereof at the genomic target site. One or more sequences can be inserted into the soybean line GM_CSM63770 transgenic insertion locus, thereby allowing the excision of the transgenic insert from a specific expression cassette within soybean line GM_CSM63770 or SEQ ID NO: 10.

[0259] Scan the sequences corresponding to the 5' and 3' genomic flanking sequences of soybean line GM_CSM63770 (shown in SEQ ID NO: 11 and 12), 5' and 3' junction regions (represented by SEQ ID NO: 1-6), and inserted T-DNA to find potential original guide RNA recognition sites (OgRRS), which include protospacer adjacent motifs (PAM) sites operably connected to guide RNA hybridization sites. OgRRS can be located in the 5' or 3' genomic sequence of the side joint, or in the 5' or 3' junction region, or in the inserted T-DNA. OgRRS will be determined according to the selected special CRISPR editing system. For example, Cas9 recognizes the protospacer adjacent motifs (PAM) rich in G at the 3' end of its guide RNA binding site, while the Cas12a system recognizes the protospacer adjacent motifs (PAM) rich in T at the 5' end of its guide RNA binding site.

[0260] Then, the homologous guide RNA recognition site (CgRRS) is defined using the OgRRS sequence, and the site is inserted into the transgenic insertion locus of strain GM_CSM63770 using the CRISPR editing system. CgRRS includes the gRNA target sequence identical to the selected OgRRS. CgRRS is inserted into the transgenic insertion locus of strain GM_CSM63770 relative to OgRRS in the region on the opposite side of the transgenic insertion, and its insertion mode will allow the use of a single gRNA to excise the DNA fragment corresponding to the entire transgenic insertion of strain GM_CSM63770, or the fragment in the transgenic insertion of soybean strain GM_CSM63770, such as the genetic element in the expression cassette or transgenic cassette. For example, if OgRRS is located in 3' genome flanking sequence or 3' junction region, CgRRS will be inserted in 5' genome flanking sequence or 5' junction region, or in the transgenic insertion, such as between the genetic elements in the expression cassette or expression cassette. Insertion of a CgRRS on the opposite side of the transgenic insert or in the region between expression cassettes relative to the OgRRS will allow for excision of the transgenic insert or a specific expression cassette to be excised using a single gRNA. The OgRRS located between the expression cassettes of soybean line GM_CSM63770 can be used to design a CgRRS that can be inserted into either the 5' or 3' genomic flanking sequence to allow for excision of one or the other expression cassette using a single gRNA.

[0261] Table 17 below shows exemplary OgRRS sequences located within the 5' and 3' genomic flanking sequences and between its two expression cassettes of soybean line GM_CSM63770, which can be used in a CRISPR editing system using Cas12a (a type V CRISPR-associated protein; coding sequence represented by SEQ ID NO: 34; protein sequence represented by SEQ ID NO: 35).

[0262] Table 17. Exemplary OgRRS sequences within soybean line GM_CSM63770.

[0263]

[0264] Table 18 below shows gRNAs comprising a poly-T transcription termination region that can be used to target Cas12a nucleases to cut within OgRRS and CgRRS sequences.

[0265] Table 18. gRNAs that can be used to target Cas12a nuclease.

[0266]

[0267]

[0268] Any OgRRS sequence shown in Table 17 can be used alternatively as a site for inserting CgRRS, and the CgRRS is designed using different OgRRS. For example, CgRRS can be inserted into the side sequence to allow the entire transgenic insert of strain GM_CSM63770 to be excised. To illustrate this method, OgRRS_3-1 is selected as the OgRRS for designing the DNA fragment containing the corresponding CgRRS, and OgRRS_5-3 is selected as the target site for inserting the DNA fragment containing CgRRS. Using the Cas12a editing system, gRNA (i.e., gRNA_OgRRS_5-3 shown in Table 18) is used to target the OgRRS_5-3 site to cut in the OgRRS_5-3 site. Then, the DNA fragment containing CgRRS and comprising the OgRRS_3-1 target site is inserted into the cleavage site, and the cleavage site is introduced into the OgRRS_5-3 sequence. After selecting a transgenic line containing the introduced CgRRS site, the line can be cultivated into another germplasm. When necessary, the transgenic insert of soybean line GM_CSM63770 can be excised from the plant using the Cas12a editing system and gRNA (i.e., gRNA_OgRRS_3-1 shown in Table 18).

[0269] Any OgRRS sequence in the 5' and 3' genomic flanking sequences of soybean line GM_CSM63770 shown in Table 17 can be used as a site for inserting a DNA fragment containing CgRRS, and the fragment is included in the OgRRS sequence between the expression cassettes, to allow the use of a single gRNA to excise a specific expression cassette. To illustrate this method, OgRRS_In-1 is selected as the OgRRS for designing a DNA fragment containing corresponding CgRRS, and OgRRS_5-2 is selected as the target site for inserting a DNA fragment containing CgRRS. Using the Cas12a editing system, gRNA (i.e., gRNA_OgRRS_5-2 shown in Table 18) is used to target the OgRRS_5-2 site to cut in the OgRRS_5-2 site. Then, the DNA fragment containing CgRRS and comprising the OgRRS_In-1 target site is inserted into the cleavage site, and the cleavage site is introduced into the OgRRS_5-2 sequence. After selecting a transgenic line containing the introduced CgRRS site, the line can be cultivated into another germplasm. If necessary, the first expression cassette expressing the Cry1A.2 toxin protein can be excised from the plant using the Cas12a editing system and gRNA (ie, gRNA_OgRRS_In-1 shown in Table 18).

[0270] CgRRS can be introduced into the transgenic insertion locus using the CRISPR system by a variety of methods. For example, the CRISPR system can be used to target the insertion of a flat-ended double-stranded DNA fragment into a genomic target site of interest, such as OgRRS, in a 5' insertion manner, which is not the OgRRS selected for designing CgRRS. The endonuclease activity mediated by CRISPR can introduce double-strand breaks (DSBs) in the selected genomic target site, and DNA repair, such as non-homologous end-joining DNA repair driven by microhomology, allows the flat-ended double-stranded DNA fragment to be inserted into the DSB. The flat-ended double-stranded DNA fragment can be designed to have a microhomology of 1-10bp on the 5' and 3' ends of the DNA fragment, which corresponds to the 5' and 3' flanking sequences at the cleavage site of the original spacer in the genomic target site.

[0271] The CRISPR system can be introduced into strain GM_CSM63770 by several methods. One or more expression cassettes are transiently introduced into cells, and the expression cassette encodes the gRNA and / or CRISPR-associated protein components of type I, type II, type III, type IV, type V or type VI CRISPR-Cas systems. The one or more expression cassettes encoding gRNA and / or CRISPR-associated proteins introduced and the DNA fragments comprising the CgRRS are provided in an amount sufficient to modify the cell, but do not continue after the expected time period or after one or more cell divisions. In such an embodiment, no additional steps are required to remove or separate one or more expression cassettes encoding gRNA and / or CRISPR-associated proteins from the modified cells.

[0272] Alternatively, an expression construct comprising one or more expression cassettes for expressing gRNA and an expression construct encoding type I, type II, type III, type IV, type V or type VI CRISPR associated proteins is stably transformed into strain GM_CSM63770 to introduce CgRRS into the desired target locus. gRNA will instruct nuclease to cut in the target locus, and the target locus may be an OgRRS different from the selected OgRRS. The expression construct will also include a CgRRSDNA fragment, the 5' and 3' flanking desired locus (i.e., an OgRRS different from the selected OgRRS) PAM / gRNA sequence of the fragment, which will allow excision of the CgRRSDNA fragment, which can then be introduced into the target locus by a double-strand break repair approach.

[0273] 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 outcome 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 where they can be found. The content shown in bold has been previously listed in Table 17.

[0274] Table 19. Potential OgRRS sequences and elements within SEQ ID NO: 10.

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286] Example 9

[0287] Genome editing technology using two guide RNAs to improve soybean lines

[0288] Modification of GM_CSM63770 This example describes the excision of all or any portion of the transgenic insert DNA or expression cassettes within the transgenic insert DNA defined and present in soybean line GM_CSM63770 by genome editing methods using a CRISPR editing system comprising two guide RNAs. Various methods can be used to excise the transgenic insert of line GM_CSM63770 or the expression cassettes within SEQ ID NO: 9 or SEQ ID NO: 10 by genome editing. In one embodiment, a clustered regularly interspaced short palindromic repeat (CRISPR) editing system comprising a 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 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, such as but not limited to Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas Csx1, Csx2, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, and Mad7. CRISPR-associated proteins and two guide RNAs (gRNAs) can be introduced into plant cells comprising soybean line GM_CSM63770 to target specific sequences within the transgenic insertion locus. In one embodiment, the CRISPR nuclease system cuts at two different guide RNA hybridization sites, thereby allowing the excision of intervening sequences. After DNA cleavage, the genomic sequence can be repaired by double-strand break repair pathways, 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 a combination thereof at the genomic target site.

[0289] The guide RNA shown in Table 18 of Example 8 is used to excise the entire transgenic box, or to remove one of the two expression boxes in soybean line GM_CSM63770. For example, the gRNA selected from the group consisting of SEQ ID NO:28-30 and the gRNA as shown in SEQ ID NO:31 are used to guide the Cas12a nuclease to cut in the region of the 5' and 3' genomic flanking sequences of soybean line GM_CSM63770, thereby causing the excision of the entire transgenic insert. Alternatively, in order to excise the Cry1A.2 expression cassette from soybean line GM_CSM63770, the gRNA selected from the group consisting of SEQ ID NO:28-30 and the gRNA selected from the group consisting of SEQ ID NO:32 and 33 are used to guide the Cas12a nuclease to cut in the region of the 5' genomic flanking sequence and in the region between the two transgenic boxes, thereby causing the excision of the Cry1A.2 expression cassette. Similarly, to excise the Cry1B.2 expression cassette from soybean line GM_CSM63770, a gRNA selected from the group consisting of SEQ ID NOs: 32 and 33 and a gRNA as shown in SEQ ID NO: 31 were used to guide the Cas12a nuclease to cut in the region between the two transgenic cassettes and in the region of the 3' flanking genomic sequence, thereby causing excision of the Cry1B.2 expression cassette.

[0290] All publications and published patent documents cited in this specification and which are material to the present invention are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0291] Having shown and described the principles of the present invention, it will be apparent to those skilled in the art that the present invention may be modified in arrangement and detail without departing from such principles. We claim all modifications that come within the spirit and scope of the following claims.

Claims

1. 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 the complete complementary sequence of any one of the foregoing.

2. The recombinant DNA molecule of claim 1, wherein the molecule is derived from soybean line GM_CSM63770, a representative sample of seeds comprising the line having been deposited under ATCC Accession No. PTA-126048.

3. A DNA molecule comprising a polynucleotide segment of sufficient length to serve as a DNA probe, wherein the polynucleotide segment specifically hybridizes with soybean line GM_CSM63770 DNA in a sample under stringent hybridization conditions, wherein detection of hybridization of the DNA molecule under the stringent hybridization conditions is diagnostic of the presence of soybean line GM_CSM63770 in the sample.

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

5. A DNA molecule pair, comprising a first DNA molecule and a second DNA molecule different from the first DNA molecule, wherein the DNA molecule pair acts as a DNA primer when used in an amplification reaction together with a sample containing soybean line GM_CSM63770 template DNA to produce an amplicon that diagnoses the presence of the soybean line GM_CSM63770 DNA in the sample, wherein the amplicon comprises a nucleotide sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID 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.

6. A method for detecting the presence of a DNA segment diagnostic for soybean line GM_CSM63770 DNA in a sample, the method comprising: a. contacting the sample with the DNA molecule according to claim 3; b. subjecting the sample and the DNA molecule to stringent hybridization conditions; as well as c. detecting hybridization of the DNA molecule with the DNA in the sample, Wherein said detection will diagnose the presence of said soybean line GM_CSM63770 DNA in said sample.

7. A method for detecting the presence of a DNA segment diagnostic for soybean line GM_CSM63770 DNA in a sample, the method comprising: a. contacting the sample with the DNA molecule pair according to claim 5; b. performing an amplification reaction sufficient to produce DNA amplicons; as well as c. detecting the presence of said DNA amplicon in said reaction, 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 diagnostic for soybean line GM_CSM63770 in a sample, the method comprising: a. contacting the sample with a first monoclonal antibody 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; b. incubating the protein binding assay for a sufficient amount of time to allow the monoclonal antibody to bind; and c. detecting the presence of said Cry1A.2 protein and said Cry1B.2 protein in said assay, Wherein said detection will diagnose the presence of said soybean line GM_CSM63770 DNA in said sample.

9. The method of claim 8, wherein the assay is performed by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay or lateral flow immunochromatographic assay.

10. A soybean plant, soybean plant part, soybean cell or part thereof comprising soybean line GM_CSM63770 DNA, characterized in that The presence of a recombinant polynucleotide molecule can be detected, wherein the recombinant polynucleotide molecule 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; wherein the soybean plant, plant part, cell or part thereof exhibits insecticidal activity when provided in a diet of a lepidopteran insect pest.

11. The soybean plant, soybean plant part, soybean cell or part thereof of claim 10, wherein the lepidopteran insect pest is selected from the group consisting of soybean pod borer (American bollworm), soybean armyworm (Soybean looper), velvet bean caterpillar (Spodoptera: Cottonworm), southern cutworm (Subtropical armyworm), black cutworm (Spodoptera: Litura), South American pod borer (Argentine bollworm), sunflower armyworm (Spodoptera: Bicolor), bean stem moth (Spodoptera: Corythuja), green armyworm (Spodoptera: Alfalfa looper) and small corn stalk borer (Ostrichia: Corn borer).

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

13. A method for protecting a soybean plant from insect infestation, wherein the method comprises providing an insecticidally effective amount of cells or tissues of a soybean plant comprising soybean line GM_CSM63770 in a diet of a lepidopteran insect pest.

14. The method of claim 13, wherein the lepidopteran insect pest is selected from the group consisting of soybean pod borer (American bollworm), soybean armyworm (Soybean looper), velvet bean caterpillar (Spodoptera: Cottonworm), southern cutworm (Subtropical armyworm), black cutworm (Spodoptera: Spodoptera litura), South American pod borer (Argentine bollworm), sunflower armyworm (Spodoptera: Bicolor), bean stem moth (Spodoptera: Corythuja), green armyworm (Spodoptera: Alfalfa looper), and small corn stalk borer (Ostrichia: Corn borer).

15. A method for producing a soybean plant resistant to lepidopteran insects, comprising: a. cultivating two different soybean plants to produce progeny, wherein at least one of the two different soybean plants comprises soybean line GM_CSM63770 DNA; b. confirming the presence of a DNA segment diagnostic of soybean line GM_CSM63770 DNA in said progeny; and c. selecting the progeny comprising soybean line GM_CSM63770 DNA; The progeny of step (c) is resistant to lepidopteran insects.

16. A soybean seed 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 the complete complementary sequence thereof.

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

18. A microorganism comprising a detectable amount of the DNA molecule of claim 1.

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

20. A commercial product comprising a detectable amount of a DNA molecule unique to soybean line GM_CSM63770, wherein the molecule comprises the DNA molecule of claim 1.

21. The commodity product of claim 20, further selected from the group consisting of whole or processed soybean seed, animal feed comprising soybeans, soybean oil, soybean meal, soybean flour, soy flakes, soy bran, soy biomass, and fuel products produced using soybeans and soybean fractions.

22. A soybean plant, soybean plant part, or soybean seed thereof, comprising DNA that functions as a template in a DNA amplification method that produces an amplicon diagnostic for soybean line GM_CSM63770 DNA.

23. A method for determining the zygosity of a soybean plant or soybean seed comprising soybean line GM_CSM63770, comprising: a. contacting a sample containing soybean DNA with a primer pair capable of producing an amplicon diagnostic of an allele corresponding to soybean line GM_CSM63770 DNA; b. contacting the sample with a second primer pair capable of generating an amplicon of an internal standard soybean genomic DNA known to be single copy and homozygous in the soybean plant using a thermal amplification reaction; c. contacting the sample with a probe set comprising at least one first probe that specifically hybridizes to an allele DNA of soybean line GM_CSM63770 and at least one second probe that specifically hybridizes to an internal standard soybean genomic DNA that is known to be single copy and homozygous in the soybean plant; d. performing a DNA amplification reaction using real-time PCR and determining the cycle threshold (Ct value) of the amplicon corresponding to the allele DNA of soybean line GM_CSM63770 and the single copy homozygous internal standard; e. calculating 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 soybean line GM_CSM63770 sequence amplicon; and f. Determine zygosity, where a ΔCt of about zero (0) indicates that the inserted T-DNA of line GM_CSM63770 is homozygous, and a ΔCt of about one (1) indicates that the inserted T-DNA of soybean line GM_CSM63770 is heterozygous.

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

19.

25. The method of claim 23, wherein the ΔCt of about one (1) indicating that the inserted T-DNA of GM_CSM63770 is heterozygous is in the range of 0.75 to 1.

25.

26. A method for determining the zygosity of a soybean plant or soybean seed comprising soybean line GM_CSM63770, comprising: a. contacting a sample comprising soybean DNA with a set of primer pairs, the primer pairs comprising at least two different primer pairs, the primer pairs being capable of producing a first amplicon that is diagnostic of soybean line GM_CS M63770 and a second amplicon that is diagnostic of natural soybean genomic DNA that does not contain soybean line GM_CSM63770; b. performing a nucleic acid amplification reaction using the sample and the set of primers; and c. detecting in the nucleic acid amplification reaction the first amplicon diagnostic for soybean line GM_CSM63770 DNA or the second amplicon diagnostic for native soybean genomic DNA that does not contain soybean line GM_CSM63770, wherein the presence of only the first amplicon will diagnose a soybean plant or soybean seed homozygous for soybean line GM_CSM63770 DNA, and the presence of both the first amplicon and the second amplicon will diagnose a soybean plant or soybean seed heterozygous for soybean line GM_CSM63770 DNA; or d. contacting a sample comprising soybean DNA with a probe set, the probe set comprising at least one first probe that specifically hybridizes to soybean line GM_CSM63770 DNA and at least one second probe that specifically hybridizes to soybean genomic DNA and does not hybridize to line GM_CSM63770 DNA, the soybean genomic DNA being disrupted by the insertion of heterologous DNA of soybean line GM_CSM63770; e. hybridizing the probe set to the sample under stringent hybridization conditions, wherein detection of hybridization of only the first probe under the hybridization conditions will diagnose a homozygous allele of the soybean line GM_CSM63770 DNA, and wherein detection of hybridization of both the first probe and the second probe under the hybridization conditions will diagnose a heterozygous soybean plant or soybean seed of the soybean line GM_CSM63770 in the sample.

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

15.

28. The method of claim 27, wherein the probe set comprises SEQ ID NO: 16 and SEQ ID NO:

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% identical to the full length of SEQ ID NO:9; and wherein the DNA construct comprises at the 5' end or the 3' end of the construct: (i) at least 50 consecutive nucleotides of SEQ ID NO:11 or SEQ ID NO:36; or (ii) at least 50 consecutive nucleotides of SEQ ID NO:12 or SEQ ID NO:

37.

30. DNA construct as claimed in claim 29, wherein said DNA construct comprises at least 50 consecutive nucleotides of SEQ ID NO:11 or SEQ ID NO:36 at the 5' end of said construct and comprises at least 50 consecutive nucleotides of SEQ ID NO:12 or SEQ ID NO:37 at the 3' end of said construct.

31. A DNA construct as described in any one of claims 29-30, wherein the construct comprises one or more nucleotide sequences selected from SEQ ID NO:38-137 at the 5' end of the construct.

32. A DNA construct as described in any one of claims 29-31, wherein the construct comprises one or more nucleotide sequences selected from SEQ ID NOs: 138-237 at the 3' end of the construct.

33. A soybean plant, plant cell, plant part or plant seed comprising the DNA construct of 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 a lepidopteran insect pest species, and wherein the recombinant DNA construct is integrated in the chromosome at a position flanked by 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.

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

36. The soybean plant, plant cell, plant part or plant seed of claim 35, wherein the at least 50 consecutive nucleotides of SEQ ID NO: 12 or SEQ ID NO: 37 comprise one or more nucleotide sequences selected from SEQ ID NOs: 138-237.

Images