Brassica event MON94100 and its use

Abstract

The present invention provides recombinant DNA molecules specific to Brassica event MON94100, as well as transgenic Brassica plants, Brassica plant parts, Brassica seeds, Brassica cells, and agricultural products containing Brassica event MON94100, and methods of using and detecting Brassica event MON94100. Efficacy: Transgenic Brassica plants containing the Brassica event MON94100 exhibit tolerance to dicamba.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62 / 746,158, filed October 16, 2018. The benefit of prior art is claimed, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Incorporating sequence tables 16,483 bytes (measured on MS-Windows) on September 12, 2019 The sequence table contained in the created file named "MONS450WO_ST25" is , filed herewith by electronic submission and incorporated herein by reference.

[0003] The present invention relates to a recombinant DNA molecule of the Brassica event MON94100. The present invention relates to a transgenic Brassica strain containing the Brassica event MON94100. ssica plants, plant parts, seeds, cells, and agricultural products, as well as methods and methods of using the same and a method for detecting the Brassica event MON94100. Transgenic Brassica plants, plant parts and seeds containing MON94100 The offspring and cells are tolerant to dicamba. [Background technology]

[0004] Brassica is an important crop in many parts of the world and plays a key role in weed control in crop production. The use of herbicides for crop protection is an established tool. Weeds compete for space, nutrients, water, and light. They can easily compete with crops and become contaminated by crops, so weed control has become essential in agriculture. Resistance to certain herbicides is achieved due to the expression of foreign genes, also known as transgenes. To create transgenic Brassica plants with sexual traits, Transgenic herbicide resistance has been used to produce crops without causing phytotoxicity. Allows the use of herbicides in the growing environment, improving weed control and increasing crop yields Transgenesis of glyphosate and glufosinate resistance in Brassica Genetic traits are widely used for weed control in commercial Brassica production. These are examples of herbicide tolerance traits that have been used alone or in combination with other traits. Combinations of herbicide tolerance traits can be used in combination to provide growers with greater flexibility and options. Weed control that allows the use of multiple herbicide modes of action to control a wide range of tough weeds It may be desirable to provide alternatives. Trait combinations are mutually exclusive. This can be achieved by crossing the

[0005] A transgenic trait is a unique DNA sequence at a fixed location on a chromosome. The presence of a transgenic event in the genome. It is created by a one-time random insertion of a cassette into a single specific location in the plant genome. Each event is unique and each event may result in the production of transgenic plants, plant parts, seeds, or Expression of the transgene in the host or cell is determined by the enzyme used in the transgenic expression cassette. The elements, the interactions of those elements, the genomic location of the gene insert, and the gene insert The location of the transgene can be influenced by a number of different factors, including the chromosomal context of the transgene. Efficacy can be affected by these, e.g., the distribution of the transgene among similarly produced events. The overall level of expression or the spatial or temporal pattern of expression of the transgene may vary widely. It has been observed that there may be large variations in the results. It is necessary to develop and test a single event that will ultimately be useful for commercial agricultural applications. The creation and selection of events with the traits required for commercial crop production requires years of plant testing. It is a scientific process that requires molecular characterization, field trials, and data analysis. In addition, transgenic expression cassettes are used for specific transgene expression in specific crop plants. must be designed, tested, and optimized. Then, thousands of unique events are created, Various species are bred over multiple generations of plants to select unique superior events for commercial use. Such events must be analyzed in the field conditions and genetic background of the desired introduced genes. Once identified as having gene expression and molecular characteristics, plant breeding methods can be used to breed other B Introgression of traits into the rassica genetic background to produce native traits, high-yielding germplasm, and disease resistance Sex, traits for hybrid seed production, and other transgenic herbicide tolerance trait(s) Many different Brassica napus strains with novel traits combined with other desirable properties such as It can be used to create the a variety. Summary of the Invention

[0006] The present invention relates to SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO:5, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2, and SEQ ID NO:1. In one embodiment, the recombinant DNA molecule comprises the sequence Bra ssica event MON94100 and derived from a plant, seed, or cell containing said event. A representative sample of seeds containing the plant has been deposited under ATCC accession number PTA-125182. In one embodiment, the recombinant DNA molecule is a plant comprising the Brassica event MON94100. The event is present in an organism, cell, seed, or plant part, and a representative sample of the seed containing the event is registered with the ATCC In another embodiment, the recombinant DNA molecule is , an amplicon diagnostic for the presence of Brassica event MON94100.

[0007] The present invention relates to a Brassica plant, a Brassica seed, or a Brassica stem. It serves as a DNA probe specific for SEQ ID NO:10 in DNA samples derived from cells. A DNA molecule having a sufficient length of consecutive nucleotides of SEQ ID NO: 10 is provided. In an embodiment, the DNA probe comprises SEQ ID NO:13.

[0008] The present invention provides a pair of DNA molecules comprising a first DNA molecule and a second DNA molecule, wherein the first and second DNA molecules each comprise a fragment of SEQ ID NO: 10, and To generate amplicons useful for the diagnosis of the Brassica event MON94100 in Used with DNA containing Brassica event MON94100 in amplification reactions for In one embodiment, the DNA primer includes SEQ ID NO:11 and SEQ ID NO:12.

[0009] The present invention relates to a Brassica plant, a Brassica seed, or a Brassica stem. Detecting the presence of Brassica event MON94100 in DNA samples derived from cells contacting the sample with a DNA probe; subjecting the probe to stringent hybridization conditions, and Detecting hybridization of the DNA probe to DNA molecules in the sample. and wherein said hybridization of said DNA probe to said DNA molecule comprises: The method indicates the presence of Brassica event MON94100 in the DNA sample. to provide.

[0010] The present invention relates to a Brassica plant, a Brassica seed, or a Brassica stem. Detecting the presence of Brassica event MON94100 in DNA samples derived from cells The method comprises contacting the sample with a pair of DNA molecules that function as DNA primers. SEQ ID NO: 10, SEQ ID NO: 9, SEQ ID NO: 8, SEQ ID NO: 7, SEQ ID NO: 6, SEQ ID NO: 5. A sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:2, and SEQ ID NO:1. performing an amplification reaction sufficient to generate a DNA amplicon comprising the sequence; detecting the presence of said DNA amplicon, The method indicates the presence of Brassica event MON94100 in the sample. do.

[0011] The present invention relates to a Brassica plant, a Brassica seed, or a Brassica stem. Method for detecting the presence of Brassica event MON94100 in samples derived from cells - Patents.com said sample being subjected to a step of isolating a protein encoded by Brassica event MON94100; contacting the sample with at least one antibody specific for the protein; detecting binding of said antibody to a protein of interest, The method further comprises: do.

[0012] The present invention relates to a DNA probe specific for SEQ ID NO: 10, Brassica event MON94 A pair of DNA primers that generates 100 diagnostic amplicons, or Bras B. sica event MON94100, including an antibody specific for the protein encoded by the event A kit for detecting the presence of the rassica event MON94100 is provided.

[0013] The present invention relates 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. providing a plant, seed, cell, plant part, or commercial product that contains a DNA molecule that includes the sequence In one embodiment, the plant, seed, cell, or plant part is tolerant to dicamba. .

[0014] The present invention relates to a method for controlling weeds in an area, comprising the steps of: Planting Brassica containing 4100 and harming said Brassica applying an effective amount of dicamba to control said weeds in said area without In one embodiment, the effective amount of dicamba is about 0.1 pounds per growing season. In one embodiment, an effective amount of Dicamba is applied at about 0.5 to 2 pounds of acid equivalents per acre over the growing season. It is a car.

[0015] The present invention relates to native Brassica species, including Brassica event MON94100, in the region. A method for controlling ica comprising the step of: applying a herbicide, wherein the herbicide application is a Brassica event MON94100 In one embodiment, the method comprises the step of: The drugs are 2,4-D (2,4-dichlorophenoxyacetic acid), bromoxynil (3,5-dibromo bromo-4-hydroxybenzonitrile), and MCPA amine (4-chloro-2-methyl phenoxyacetic acid).

[0016] The present invention provides a method for producing a plant that is tolerant to dicamba, the method comprising the steps of: To obtain the Brassica event MON94100, the plant may be cultured either by itself or by a second plant. and identifying progeny seeds containing the Brassica event MON94100. In one embodiment, the method comprises: Identifying progeny seeds containing .00 and growing the progeny seeds to produce progeny plants. treating said progeny plants with an effective amount of dicamba; and In one embodiment, the Brassica event M Identifying progeny seeds containing ON94100 indicates that the progeny seeds are by detecting the presence of Brassica event MON94100. In terms of morphology, identifying progeny seeds containing the Brassica event MON94100 is Coded by Brassica event MON94100 in samples derived from the progeny seeds The method involves detecting the presence of at least one protein that is involved in the detection of the mutation.

[0017] The present invention provides a method for determining plant zygosity for the Brassica event MON94100. The method comprises the steps of: subjecting a sample containing DNA derived from the plant to Brassica event MON94 The first amplicon diagnostic for the presence of 100 and Brassica event MON941 A diagnostic second amplicon for wild-type Brassica genomic DNA that does not contain 00 and carrying out a nucleic acid amplification reaction. and detecting said first amplicon and / or said second amplicon. The presence of both amplicons confirmed that the sample was Brassica event MON94100. and the presence of only the first amplicon indicates that the sample is heterozygous for the first amplicon. The subject is homozygous for the Brassica event MON94100. In one embodiment, the primer set comprises SEQ ID NO:11 and SEQ ID NO:12. include.

[0018] The present invention provides a method for improving tolerance to dicamba in Brassica plants, comprising: (a) Peanut chlorosis streak virus (b) promoter derived from tobacco etch virus (c) ribulose 1,5-bisphosphonate from Pisum sativum (d) chloroplast transit peptide of acid carboxylase; dicamba monooxygenase coding sequence from (e)Me DNA containing an expression cassette containing the 3'UTR from dicago truncatula Obtaining a construct and inserting the DNA construct into the genome of a Brassica cell. regenerating said Brassica cells into Brassica plants; and and selecting a Brassica plant containing the DNA construct. In one embodiment, the selecting comprises treating the Brassica plant with an effective amount of dicamba. The present invention relates to a method for producing dicamba-tolerant plants that are accessible by this method. Provide a Brassica plant, Brassica seed, or Brassica cell. wherein the Brassica plant, Brassica seed, or Brassica In another embodiment, the cells comprise the DNA construct. ca plant, Brassica seed, or Brassica cell comprises SEQ ID NO: 10 . [Brief description of the drawings]

[0019] [Figure 1] 1 shows the sequence of Brassica event MON94100. The horizontal lines correspond to the positions 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:9 relative to SEQ ID NO:10, the horizontal arrows labeled SQ51321 (SEQ ID NO:11) and SQ13805 (SEQ ID NO:12) indicate the approximate location of a pair of primers that may be used to detect Brassica event MON94100, and the horizontal line labeled PB4832 (SEQ ID NO:13) indicates the approximate location of a DNA probe that may be used to detect Brassica event MON94100. [Diagram 2] 1 shows an expression cassette for Brassica event MON94100 with the genetic elements designated as set out in Table 1 for SEQ ID NO:9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] A brief description of the sequence SEQ ID NO:1 represents the 5' junction between the Brassica genomic DNA and the transgene insert. SEQ ID NO:1 is a 30 nucleotide DNA sequence showing the nucleotide sequence of SEQ ID NO:10. This corresponds to Leotid's rankings of 986 to 1015.

[0021] SEQ ID NO:2 is the 3' junction between the Brassica genomic DNA and the transgene insert. SEQ ID NO:2 is a 30 nucleotide DNA sequence showing the nucleotide sequence of SEQ ID NO:10. This corresponds to Leotid's rankings 3899 to 3928.

[0022] SEQ ID NO:3 represents the 5' junction between the Brassica genomic DNA and the transgene insert. SEQ ID NO:3 is a 60 nucleotide DNA sequence showing the nucleotide sequence of SEQ ID NO:10. This corresponds to Leotid's rankings of 971 to 1030.

[0023] SEQ ID NO:4 is the 3' junction between the Brassica genomic DNA and the transgene insert. SEQ ID NO:4 is a 60 nucleotide DNA sequence showing the nucleotide sequence of SEQ ID NO:10. This corresponds to Leotid's rankings 3884 to 3943.

[0024] SEQ ID NO:5 is the 5' junction between the Brassica genomic DNA and the transgene insert. SEQ ID NO:5 is a 100 nucleotide DNA sequence showing the nucleotide sequence of SEQ ID NO:10. This corresponds to nucleotide positions 951 to 1050.

[0025] SEQ ID NO:6 is the 3' junction between the Brassica genomic DNA and the transgene insert. SEQ ID NO:6 is a 100 nucleotide DNA sequence showing the nucleotide sequence of SEQ ID NO:10. This corresponds to nucleotide positions 3864 to 3963.

[0026] SEQ ID NO:7 identifies 1000 nucleotides of the 5' flanking Brassica genomic DNA and the nucleotide sequence A DNA fragment of 1104 nucleotides representing the 5' end of the transgene insert. It is an array.

[0027] SEQ ID NO:8 identifies the 3'-terminal 281 nucleotides and the 3'-flanking Br A DNA sequence of 1281 nucleotides representing 1000 nucleotides of the assica genomic DNA. It is an array.

[0028] SEQ ID NO:9 corresponds to the transgene insert of Brassica event MON94100. It is a DNA sequence of 2913 nucleotides.

[0029] SEQ ID NO:10 is the 4913 nucleotide sequence corresponding to Brassica event MON94100. the 5'-flanking genomic DNA sequence from position 1 to position 1000; Transgenic DNA inserts from positions 1001 to 3913 and 3914 to 491 It contains the 3' flanking genomic DNA sequence of position 3.

[0030] SEQ ID NO: 11 corresponds to a primer designated SQ51321, which identifies the Bra 23 nucleotides used to identify the DNA of ssica event MON94100 This corresponds to positions 3953 to 3931 of SEQ ID NO:10.

[0031] SEQ ID NO: 12 corresponds to a primer designated SQ13805, which identifies the Bra 26 nucleotides used to identify the DNA of ssica event MON94100 This corresponds to positions 3839 to 3864 of SEQ ID NO:10.

[0032] SEQ ID NO: 13 corresponds to a probe designated PB4832, which identifies the Brass The 16-nucleotide D sequence used to identify the DNA of ica event MON94100 This is an NA sequence, which corresponds to positions 3869 to 3881 of SEQ ID NO:10.

[0033] The following definitions and methods are intended to more fully define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms are used according to common practice by those of ordinary skill in the relevant art. It should be understood according to the example usage.

[0034] Plant transformation techniques involve the introduction of foreign DNA (also known as transgenic DNA) into cells. They are randomly inserted into the chromosomes of the genome to create "transgenic" or "recombinant" cells. This technique is used to create genetically engineered cells, also called cells. A large number of individual cells are transformed, each with the random insertion of the foreign DNA into their genome. Each individual produces a unique transgenic event. Transgenic plants are then They are regenerated from another transgenic cell. This is because the unique inserted transgenic Every cell of the transgenic plant contains the check event as a stable part of the genome. The transgenic plants are then produced, each with its own unique transgenic The Brassica event MON may be used to produce progeny plants containing the Brassica event MON. 94100 is a system for (i) transgenic expression cassettes (design of many different expression cassettes); Thousands of Brassica plants were transfected with DNA constructs containing the (ii) transformation of cells into transgenic organisms, each of which contains a unique transgenic event; (iii) regeneration of a variety of plant populations with tens of thousands of plants for thousands of events; Multiplexing with molecular characterization in genetic background, efficacy of herbicide resistance, and agronomic trait testing and analysis. Created and selected through rigorous event selection over several years. Brassica event MO The N94100 was thus created and has unique advantages that make it useful for large-scale commercial agricultural purposes. were selected as events that occurred.

[0035] The act of inserting transgenic DNA into the genome of a Brassica plant is considered a This is accomplished by plant transformation techniques known in the art and is referred to as a "transgenic event" or This creates a new transgenic genomic DNA sequence known as an "event." The elephant DNA sequence is the inserted foreign DNA (called a "transgenic insert" ) and immediately adjacent to the transgenic insert on either side of the insertion site, or The DNA sequence of the event is , which is unique and specific to the event and does not differ from sequences of other events or non-transformed Brassica It can be easily identified when compared to other DNA sequences, such as those of genomic DNA. assica event MON94100 is a novel and unique D NA sequence, which is provided as SEQ ID NO: 9; and the 5' and 3' flanking DNA sequences provided in SEQ ID NO:7 and SEQ ID NO:8, respectively. Therefore, Brassica event MON94100 is a transgenic plant containing the event. DNA molecules that are components of the chromosomes of transgenic Brassica cells and plants. It is therefore static and can be transmitted to progeny cells and plants.

[0036] The present invention relates to the first transformed cells and plants containing the Brassica event MON94100. Such progeny are also provided by cell tissue culture, such as Brassica event MON9. By self-pollination of Brassica plants, including 4100, or by Brassica event M Brassica plants containing ON94100 and the event, with or without It may be produced by sexual outcrossing between another plant, which may be the same or Transgenic plants containing different event(s) or derived from different varieties, etc. The Brassica event MON94100 can be a non-transgenic plant of each It is passed down from the original parent to the next generation.

[0037] As used herein, the term "Brassica" refers to any plant belonging to the genus Brassica. The term "Brassica" refers to a plant and includes all plant varieties that can be crossed with Brassica. Brassica useful for carrying out the method include Brassica napus (commonly known as rapeseed and certain cultivars that may be referred to as canola), Brassica napus sica juncea, Brassica napobrassica, Brassi ca oleracea, Brassica carinata, Brassica n apus, Brassica rapa, and Brassica campestris and any member of the genus Brassica that allows crossing between Brassica species. Other plants include, but are not limited to, Brassica napus, B rassica rapa (formerly Brassica campestris) and Br assica oleracea hybridization and the differences resulting from the retention of both genomes. Brassica including Brassica event MON94100, which is a metatetraploid napus plants were bred to be Brassica event MON94100 and thus It can be used to introduce the birch resistance trait into other members of the genus Brassica. As used herein, the term "rapeseed" or "rapeseed plant" refers to the oil-producing canola plant ( i.e., oil that meets a specific quality specification of containing less than 2% erucic acid Refers to the Brassica plant which can be used for

[0038] The present invention provides Brassica event MON94100, which includes the event Provides tolerance to dicamba in Brassica cells, plants, and seeds. The ica event MON94100 contains an expression cassette for expressing the DMO protein. As used herein, an "expression cassette" or "cassette" refers to a vector that is used to express a gene in a transformed cell. A recombinant DNA molecule that contains a combination of separate elements that are expressed by a cell. provides a listing of the elements contained in SEQ ID NO:9 and illustrated in FIG. TIFF0007636385000001.tif158134

[0039] As used herein, the term "recombinant" refers to a gene that is not normally found in nature and is produced through human intervention. As used herein, the term refers to non-naturally occurring DNA, proteins, or organisms that have been created by introduction. "Recombinant DNA molecules" refer to molecules that are not naturally occurring together and that are not produced without human intervention. DNA molecules that contain combinations of DNA molecules that are the result of, for example, A DNA molecule that is composed of a combination of two DNA molecules, e.g., a transgene and a corresponding A DNA molecule containing plant genomic DNA adjacent to the introduced gene. An example is a DNA molecule comprising at least one sequence selected from SEQ ID NOs: 1-10. As used herein, a "recombinant plant" refers to a plant that does not normally occur in nature and has been modified as a result of human intervention. The result is a plant containing a transgenic DNA molecule. As a result, recombinant plants are novel and distinct from the related wild-type plants. An example of a plant is a Brassica plant containing the Brassica event MON94100. do.

[0040] As used herein, the term "transgene" refers to a gene that has been introduced into a human It refers to a DNA molecule that has been artificially inserted into the genome of an organism as a result of the intervention of another. The offspring may be heterologous to the organism. As used herein, the term "transgenic The insert was then cloned using plant transformation techniques to generate the Brassica event MON94100. This refers to foreign DNA that has been inserted into the Brassica genome by genetic engineering. The sequence of the transgenic insert of MON94100 is provided as SEQ ID NO:9. The term "transgenic" refers to containing a transgene, e.g. "Nicked plant" refers to a plant containing a transgene.

[0041] As used herein, the term "heterologous" refers to a first molecule, which is usually not native to a second molecule. For example, a DNA molecule may be a first molecule that is not related to a molecule or organism. The DNA molecule may originate from one species and be inserted into the genome of a second species. It will be foreign to the microbial community and living organisms.

[0042] As used herein, the term "chimera" refers to a modification of a first DNA molecule to a second DNA molecule. refers to a single DNA molecule created by fusing a first DNA molecule Neither the DNA nor the second DNA molecule is normally found in that configuration fused to the other. A chimeric DNA molecule is a novel DNA molecule not normally found in nature. An example of the A molecule is a DNA molecule comprising at least one sequence selected from SEQ ID NOs: 1 to 10. It is.

[0043] As used herein, the term "DMO" or "dicamba monooxygenase" refers to Dimethylation of the non-herbicidal compound 3,5-dichlorosalicylic acid via O-demethylation Dicamba monooxygenase is a protein that catalyzes the inactivation of dicamba. Stenotrophomonas maltophthora, a microorganism found in the soil rhizosphere. It was first isolated from Ilia. Examples of nucleic acid molecules encoding dicamba monooxygenases Exemplary sequences, and the protein sequences encoded by these nucleic acid molecules, are described in the art. and is described, for example, in U.S. Pat. No. 7,884,262.

[0044] As used herein, the term "isolated" refers to a molecule, typically in its native or natural state. "Isolated" refers to separating a DNA molecule from other molecules with which it is associated. and separated from other DNA molecule(s) with which it is naturally or naturally associated. Such a DNA molecule may refer to a DNA molecule that has been engineered, such as a recombinant DNA molecule. It can exist in a modified form, i.e., removed from its natural state and with different, normally unrelated, A DNA molecule fused to the DNA molecule of interest would be an isolated DNA molecule. The child is entitled to create recombinant DNA, or to introduce a foreign DNA molecule into a cell, plant, or seed. This may be achieved through the use of biotechnological techniques such as chromosomal integration.

[0045] The present invention provides DNA molecules and their corresponding DNA sequences. As used herein, the terms "DNA" and "DNA molecule" refer to a deoxyribonucleic acid (DNA) molecule. The DNA molecule may be of genomic or synthetic origin and, by convention, is endogenous to the 5' (upstream) From the end to the 3' (downstream) end. As used herein, the term "DNA sequence" means refers to the nucleotide sequence of a DNA molecule. The nomenclature used is based on the title of the U.S. Code of Federal Regulations. 37, as required by § 1.822, and the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3. By convention, the DNA sequences of the invention and their A fragment is disclosed with reference to only one strand of two complementary DNA sequences. It is also intended that the complementary sequences of the sequences provided herein (the complementary strand sequences, known in the art) (also referred to in the field as reverse complement sequences) are within the scope of the present invention and claimed subject matter. As used herein, therefore, SEQ ID NOs: 1 to 10 are expressly intended to be within the scope of the present invention. and references to fragments thereof include and refer to the sequence of the complementary strand and fragments thereof.

[0046] As used herein, the term "fragment" refers to a smaller portion of a whole. The fragment of sequence number 10 is a fragment of at least about 10 consecutive nucleotides of the complete sequence of SEQ ID NO:10. nucleotides, at least about 11 contiguous nucleotides, at least about 12 contiguous nucleotides at least about 13 contiguous nucleotides; at least about 14 contiguous nucleotides; At least about 15 contiguous nucleotides, at least about 16 contiguous nucleotides, at least about 17 contiguous nucleotides, at least about 18 contiguous nucleotides, At least about 19 contiguous nucleotides, at least about 20 contiguous nucleotides, at least about 25 contiguous nucleotides, at least about 30 contiguous nucleotides, at least about 35 at least about 40 contiguous nucleotides, at least about 45 contiguous nucleotides contiguous nucleotides, at least about 50 contiguous nucleotides, at least about 60 contiguous nucleotides, at least about 70 contiguous nucleotides, at least about 80 contiguous nucleotides nucleotide, at least about 90 contiguous nucleotides, or at least about 100 contiguous It will contain a sequence that is a nucleotide.

[0047] The DNA sequence of the transgenic insert of Brassica event MON94100 is , provided as SEQ ID NO: 9. Transgenic Inserts and The DNA sequences of Brassica genomic DNA flanking each side of the input fragment are shown in SEQ ID NO: No. 10. Portions of flanking DNA and the 5' end of the transgenic insert are provided as The terminal DNA sequences are provided as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7. The DNA sequence of the 3' end of the transgenic insert and the adjacent DNA fragments are provided as SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8.

[0048] Host markers for Brassica genomic DNA adjacent to one end of the transgenic insert. The DNA sequence of the region spanning the phosphodiester bond linkage is referred to herein as " The junction is the site of the transgenic insert as one continuous molecule. The junction of one end of the transgenic insert is the junction of the other end of the adjacent DNA. one is found at the 3' end of the transgenic insert, and the other is found at the 3' end of the transgenic insert, The "junction sequence" is the 5' junction of the event. or any length of DNA sequence spanning the 3' junction. The junction sequence of N94100 is clear to one skilled in the art using SEQ ID NO: 10. Examples of junction sequences for the ssica event MON94100 are provided as SEQ ID NOs: 1-8. FIG. 1 shows the physical layout of SEQ ID NOs: 1 to 10 arranged 5' to 3'. The junction sequence of the Brassica event MON94100 It may be present as part of the genome of the plant, seed, or cell containing it. Any one of SEQ ID NOs: 1 to 8 or 10 in a sample derived from a child or cell The above identification indicates that the DNA of Brassica monocytes containing the Brassica event MON94100 is ca and was diagnostic of the presence of Brassica event MON94100. This indicates that.

[0049] The plants, seeds, cells, plant parts, and commercial products of the present invention are Brassica event MO The sample may be used to detect DNA or protein molecules that indicate the presence of N94100. Primers for detecting the presence of Brassica event MON94100 in Exemplary DNA molecules that can be used as either probes or plasmids are provided. The immobilizer or probe is specific for the target nucleic acid sequence and thus is a nucleic acid sequence as described herein. The method is useful for identifying the Brassica event MON94100. a Detection of the presence of event MON94100 is by thermal amplification of nucleic acids or by nucleic acid hybridization. Techniques known in the art, such as blotting and Southern analysis, are also known in the art. This can be done by using the method.

[0050] A "primer" is a nucleic acid that is used in the annealing or hybridization method that accompanies an amplification reaction. The amplification reaction is a process in which a DNA molecule is amplified to produce an amplification product. As used herein, an "amplicon" is an in vitro reaction that produces an amplicon. A DNA molecule that has been synthesized using amplification techniques. An amplicon of the present invention is a DNA molecule having the sequence A DNA sequence containing one or more of numbers 1 to 10 or a fragment thereof. The primers are then subjected to an amplification reaction such as polymerase chain reaction (PCR) to generate amplicons. can be used with template DNA, such as a sample of Brassica genomic DNA, in a reaction Here, the amplicon generated is a hybridization sequence of two primers hybridized to the template. The primers generally have a DNA sequence that corresponds to the sequence of the template DNA located between the sites. Usually, it hybridizes with a complementary target DNA strand, forming a hapten between the primer and the target DNA strand. The presence of a primer allows the target DNA strand to act as a template and form a hybrid. This is the position recognized by the polymerase to initiate extension of the primer using The primer pair is a double-stranded nucleic acid sequence for amplifying the nucleotide segment between them. This refers to the use of two primers that bind to opposite strands of a nucleotide segment. Examples of columns are SEQ ID NO:11 (SQ51321) and SEQ ID NO:12 (SQ13805) The primer pair provided as SEQ ID NO: 11 and SEQ ID NO: 12 is a first DNA A molecule and a second DNA molecule, wherein the first DNA molecule is SEQ ID NO: No. 10, wherein the second DNA molecule is a fragment of the transgenic insert DNA sequence of A fragment of the contiguous DNA sequence of SEQ ID NO: 10, which identifies the Brassica event MON Brassica event MON9 to generate amplicons diagnostic for 94100 When used together in an amplification reaction with DNA containing 4100, each In certain embodiments, the present invention provides a method for the preparation of a primer. A primer pair may be defined as comprising a first DNA molecule and a second DNA molecule. wherein the first DNA molecule is a fragment of the Brassica genome portion of SEQ ID NO: 10. the second DNA molecule being a fragment of the transgene portion of SEQ ID NO: 10; To generate amplicons useful for the diagnosis of the Brassica event MON94100 Used together in amplification reactions with DNA containing the Brassica event MON94100 Each is of sufficient length to function as a DNA primer when

[0051] A "probe" is a nucleic acid that is complementary to a strand of a target nucleic acid and is useful in hybridization detection methods. The probe according to the present invention is a nucleic acid molecule which is a deoxyribonucleic acid or a ribonucleic acid alone. but also polyamides and other probe materials that specifically bind to target DNA sequences. Detection of such binding can be useful in detecting the presence or absence of a target DNA sequence. The probe can be labeled with any conventional detectable label, such as a radioisotope, ligand, chemiluminescent agent, or enzyme. or may be attached to a reporter molecule. Exemplary DNA sequences useful as probes for the detection of BRCA1 are SEQ ID NO:13 (PB4832) and and provided.

[0052] Methods for designing and using primers and probes are well known in the art. The DNA molecule containing the fragments of SEQ ID NOs: 1 to 10 is Brassica event MON94100 The present invention provides a method for detecting serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 Such a polypeptide can be readily designed by one skilled in the art using the sequences available.

[0053] Probes and primers according to the invention may have perfect sequence identity with the target sequence, but Primers and primers different from the target sequence that retain the ability to hybridize preferentially to the target sequence The primers and probes can be designed by conventional methods. To function, the sequence of the nucleic acid molecule must be stable under the particular solvent and salt concentration used. They need only be sufficiently complementary to form a double-stranded structure. To identify the presence of transgenic DNA from Brassica event MON94100 Any conventional nucleic acid hybridization or amplification method may be used to detect the presence of the nucleic acid. The probes and primers generally have a length of at least about 11 nucleotides, at least about 18 nucleotides. nucleotide, at least about 24 nucleotides, or at least about 30 nucleotides or more Such probes and primers are suitable for stringent hybridization. It specifically hybridizes to the target DNA sequence under stringent hybridization conditions. Hybridization conditions are known in the art and are described, for example, in MR Gree n and J Sambrook,Molecular cloning: a lab oratory manual,4 th Edition, Cold Spring H arbor Laboratory Press,Cold Spring Harbo As used herein, two nucleic acid molecules are specifically linked to each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. Nucleic acid molecules can hybridize to each other if they show complete complementarity. As used herein, an aligned first nucleic acid molecule is a "complement" of another nucleic acid molecule. If every nucleotide of a molecule is complementary to every nucleotide of a second molecule, The two molecules exhibit "perfect complementarity." The two molecules are at least as complementary as the conventional "low stringency" method. Under "sustaining" conditions, sufficient to allow them to remain annealed to each other. Two molecules are "minimally complementary" if they can hybridize to each other with sufficient stability. Similarly, the molecules may be bonded to one another under conventional "high stringency" conditions. They hybridize to each other with sufficient stability to allow the two to remain in a linked state. Thus, deviations from perfect complementarity are not necessarily required. Deletion is permissible so long as it does not completely eliminate the ability of the molecule to form double-stranded structures.

[0054] Suitable stringency conditions that promote DNA hybridization, e.g., about 6.0× sodium chloride / sodium citrate (SSC) at 45°C, followed by 50°C Washing with 2.0×SSC is known to those of skill in the art or is described in Current Protocols. cols in Molecular Biology,John Wiley & S. Ons, NY (1989), 6.3.1-6.3.6. The salt concentration in the step was low stringency of about 2.0×SSC at 50°C to 50 0.2xSSC at 10°C. The temperature ranges from low stringency at room temperature (about 22°C) to high stringency at about 65°C. Stringent conditions may be increased. Both temperature and salt may be altered, or Either the temperature or the salt concentration is held constant while the other variable is varied.

[0055] To generate antibodies to detect the presence of Brassica event MON94100 in samples. Such antibodies are provided in the form of proteins that can be used to generate Brassica spp. The protein is specific for one or more of the proteins encoded by the mutant MON94100. The DNA sequence encoding such a protein is provided in SEQ ID NO: 10, and the start of the coding sequence is The DNA sequences encoding each protein and the stop positions are shown in Table 1. The encoded protein can be isolated by the methods described herein using the Brassica event MO It is useful for generating antibodies to detect the presence of N94100. Detection of the presence of event MON94100 can be achieved by any protein assay known in the art. Detection techniques, e.g., Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) A) a detectable label or reporter molecule (e.g., a radioisotope, a ligand, a Uses attachment of an antibody to a chemiluminescent agent, or enzyme, or the action of an enzyme on a reporter molecule One method is to clone the sample with Brassica event MON94100. contacting the DMO protein with an antibody that binds to the DMO protein to be encoded, and then detecting the presence or absence of antibody binding. The binding of such antibodies provides a means for detecting the presence or absence of Brassica events. It is diagnostic for the presence of one or more proteins encoded by MON94100.

[0056] Protein and Nucleic Acid Tests to Detect the Presence of Brassica Event MON94100 A variation of such a kit is also provided that includes the compositions disclosed herein. and methods, as well as methods well known in the art of protein and nucleic acid detection. The protein and nucleic acid detection kit can be used to detect Brassica event MON94100. Such a kit can be applied to a method for crossing with a plant containing SEQ ID NO: 1 to 1. 0 fragment or the Brassica event MON941 00 and one or more reaction reagents, etc. Other components (e.g., nucleotides, polymerase, buffers) may be included. It may also include a gender control, a negative control, and a protocol for use.

[0057] An example of a detection kit is a kit for detecting the presence or absence of Brassica event MON94100 in a sample. is a sequence number of sufficient length to serve as a DNA probe useful for detecting the absence It contains at least one DNA molecule of 10 consecutive nucleotides. An exemplary DNA molecule sufficient for use in the present invention is one that includes the sequence provided as SEQ ID NO:13. Other probes can be readily designed by one of skill in the art. Another example of a detection kit includes a test Useful for detecting the presence or absence of Brassica event MON94100 in samples The method includes at least one primer pair useful for generating an amplicon of interest. The method may also include sequencing the amplicon or a fragment thereof. Exemplary DNA molecules sufficient for use in the present invention are provided as SEQ ID NO:11 and SEQ ID NO:12, respectively. Other primer pairs can be readily designed by one of skill in the art. The kits of the invention optionally include a reagent for carrying out the detection or diagnostic reactions described herein. Another example of a detection kit may include reagents for detecting Brassica event MON9410. At least one antibody specific to at least one protein encoded by For example, such a kit may include a lateral flow strip, The lateral flow strip contains a reagent that is activated when the end comes into contact with an aqueous solution. An exemplary protein sufficient for use in generating antibodies is provided as SEQ ID NO:10. or any fragment thereof.

[0058] The present invention relates to a Brassica plant containing the Brassica event MON94100, The Company provides the progeny, seeds, cells, and plant parts, as well as the commercial products produced therefrom. The plants, progeny, seeds, cells, plant parts, and commercial products of the present invention are provided as set forth in SEQ ID NOs: 1 to 4. 8 and SEQ ID NO: 10. Contains DNA.

[0059] The plants, progeny, seeds, cells, and plant parts of the present invention may be modified to express one or more additional desired traits (eg, Such desired traits may include transgenic traits, natural traits, or It may be a trait created by other methods, such as genome editing or other conventional mutagenesis methods. The desired trait is, for example, Brassica monatum, which contains the Brassica event MON94100. Crossing a Brassica plant with another Brassica plant containing the additional trait(s). Such traits can be combined with the Brassica event MON94100. improved insect resistance, improved hulling or shredding, improved oil quality, improved water use efficiency, improved yield Improved yield, improved drought tolerance, improved seed quality, improved nutritional quality, hybrid seed production, and / or or improved herbicide tolerance, where the trait is such a trait. The transgenic trait is measured relative to Brassica plants lacking the transgenic trait.

[0060] The plants of the present invention are selected to produce progeny containing the Brassica event MON94100. As used herein, "progeny" includes SEQ ID NOs: 1 to 8 and SEQ ID NO: No. 10, wherein the plant comprises a DNA molecule having at least one sequence selected from the group consisting of Any plant containing the Brassica event MON94100 inherited from an ancestral plant that The plants, seeds, and cells are Brassica event MON. The progeny plants may be homozygous or heterozygous for 94100. From seeds produced by Brassica plants containing the ica event MON94100 or Brassica event MON94100. It can be grown from seeds produced by the ica plant.

[0061] As used herein, the "plant part" of the present invention refers to Brassica event MON94. 100. Plant parts include tissue samples, pollen, and the like. The term "organic plant" includes, but is not limited to, the whole or parts of the plant, ovule, pod, seed, flower, root, stem, fiber, and leaf. Plant parts may be viable or non-viable, but are not limited to these.

[0062] The present invention relates to commercial products produced from plants containing the Brassica event MON94100. The commercial product of the present invention is selected from the group consisting of SEQ ID NOs: 1 to 10. As used herein, "commercially available" refers to a substance that contains a detectable amount of DNA that contains a DNA sequence. "Substance" refers to a plant, seed, cell, or plant part containing Brassica event MON94100. It refers to any composition or product made from materials derived from natural products. , processed seeds, grains, plant parts, meals, and oils. No. Commodity products are non-living plant matter, i.e. non-living Brassica a Substance derived from a plant, seed, cell, or plant part containing event MON94100 The commercial product of the present invention is a detectable strain corresponding to Brassica event MON94100. The detection of one or more of these DNA species in a sample indicates that the product is commercially available. The detection methods disclosed herein can be used to determine the contents or source of an object. Any standard method for detection of DNA molecules can be used, including methods.

[0063] As used herein, dicamba refers to 3,6-dichloro-2-methoxybenzoic acid. Herbicidal active ingredients having the chemical name, as well as dicamba-Na salt, dicambabutotyl, dicamba dicamba dimethylamine salt, dicamba dimethylammonium salt , Diethanolammonium Dicanba, N,N-Bis-(aminopropyl)methylamine Salts, dicambaisopropylammonium, dicamba potassium, dicamba sodium, and Any salt or ester of dicamba, including but not limited to dicamba trolamine Dicamba may be used in formulations with one or more additional herbicide(s). do.

[0064] As used herein, "tolerant to herbicides" or "herbicide tolerance" or "Tolerance" refers to the ability of a plant to be completely or partially immune to the presence or application of one or more herbicides. ability to be partially insulated, e.g. to resist the toxic effects of herbicides when applied The cells, seeds, or plants are grown at least in the presence of one or more herbicides. If the plant is able to maintain some degree of normal growth or phenotype, it is considered to be "tolerant to the herbicide." A trait is one whose presence is indicative of a trait that is "indicative of" or has "improved resistance" to wild-type or control cells. Improved resistance of the cell, plant, or seed to a herbicide compared to the cell, plant, or seed. If the herbicide is capable of conferring resistance, it is a herbicide tolerance trait. Crops containing herbicide tolerance traits are , can continue to grow and is minimally affected by the presence of herbicides. The expression of the protein is increased in cells, plants, or seeds compared to wild-type or control cells, plants, or seeds. If a compound can confer improved resistance to a herbicide to a plant, plant, or seed, it is called a "herbicide." An example of a herbicide resistance protein is dicamba monooxygenase. Herbicide resistance can be complete or partial insensitivity to a particular herbicide and can be It may be expressed as percent (%) resistance or insensitivity to the herbicide.

[0065] As used herein, "chemical injury" or "injury" refers to damage to a plant resulting from the application of a herbicide. "Injury rate" or "percent injury" refers to the amount of injury caused by a herbicide. Refers to visual assessment of damage. In the case of a plant, the plant shows reduced injury after application of dicamba. Dicamba is an indole-3 -It can affect plant growth in the same way as natural auxins such as acetic acid, but is metabolized by the plant. Dicamba is a synthetic auxin that is not regulated. As a result, plant tissues treated with dicamba are The leaf epiphytic growth continues even if it has a negative effect on the plant. Visual assessment of injury is a noticeable downward bending or curvature of the leaves as a result of growth disturbance from the plant. This is one effect of dicamba that makes it useful.

[0066] As used herein, a "weed" is any undesirable plant. In addition, they may be considered undesirable for agricultural or horticultural purposes (e.g., Amar anthus species), or may be considered undesirable in certain circumstances (e.g. Weeds are generally plants that grow in the soil and are planted in the soil. It is well known in the art and varies with geography, season, growing conditions, and time of year. Lists of weed species are available from agricultural associations and societies (e.g., the Weed Science Society). of America and Canadian Weed Science Society ety), government agencies (e.g., United States Department of f Agriculture), as well as industrial and farmer associations (e.g. Canola Council of Canada.

[0067] The present invention is a method for controlling weeds in Brassica growing areas by applying dicamba. The present invention provides a method for controlling Br before, during, or after application of a herbicide. Seeds or plants containing the assica event MON94100 were planted in the area and herbicides Application will prevent or inhibit the growth of weeds and will not harm Brassica plants. The method of the present invention may or may not contain weed seeds or plants at the time of herbicide application. Dicamba may be used alone or in combination with one or more herbicides during the growing season. The herbicide(s) used in the method of the present invention may be applied in combination with One or more herbicides may be applied in a timed manner (e.g., as a tank mix or sequentially) in space (e.g., different growth periods during the growing season, including before and after planting of Brassica seeds) For example, to control weeds, planting seeds containing Brassica event MON94100 in the area; and and the Brassica event MON94100. A herbicidally effective amount of dicamba, alone or in combination with another herbicide, is administered to control grasses. and applying the same to the plant over the growing season. Pre-planting (for killing weeds, i.e., for application to weeds that have appeared or existed before the seeds are planted) Any prior to planting of seeds containing the Brassica event MON94100, time point), pre-emergence (after the seeds containing Brassica event MON94100 were planted) and at any time before the emergence of plants containing the Brassica event MON94100 ), or post-emergence (after the plants containing Brassica event MON94100 emerge Multiple applications of dicamba or a combination of dicamba and one or more other pesticides may be used. Multiple applications of a combination of herbicides (multiple applications) either simultaneously or separately, e.g. two applications (e.g., pre-planting and post-emergence applications, or pre-emergence and post-emergence applications) or three or more applications (e.g., a preplant application and two postemergence applications) may improve growth. It can be used for a long period of time.

[0068] Herbicide applications in practicing the method of the present invention may be at commercially recommended rates or any fraction thereof. The amount of herbicide may be a combination or multiple of the amount of herbicide used, for example, twice the amount commercially recommended. Depending on the agent and formulation, the acid equivalents per pound per acre (lb.acid equivalent / acre) may be calculated. car) or pound of active ingredient per acre (lbs. active ingredient / acre) The herbicidally effective amount of dicamba used in an area to control weeds can be expressed as: From about 0.1 lb. acid equivalent / acre to about 16 lb. acid equivalent / acre over the growing season. (e.g., dicamba should range from about 0.5 lb. acid equivalent / acre to about (May be applied at a rate of 2.0 pounds acid equivalent per acre).

[0069] The present invention relates to a method for the preparation of a medicament for use in the manufacture of a medicament for the treatment of a herbicide comprising administering to said patient a herbicidally effective amount of another herbicide, such as 2,4-D (2,4-dichlorophenoxy) bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), and MCPA amine (4-chloro-2-methylphenoxyacetic acid). Brassica in areas for crop cultivation by applying synthetic auxins A method for controlling native Brassica plants containing event MON94100 is provided, wherein the Application of the herbicide inhibits the growth of Brassica including Brassica event MON94100. 2. Prevent the growth of wild Brassica. 4-D herbicide is effective at delivering from about 0.1 lb. acid equivalent / acre to about 16 lb. acid equivalent / acre over the growing season. equivalents / acre (e.g., 2,4-D can be applied at about 0.1 mg / acre over the growing season). (It may be applied at a rate of .5 lbs. acid equivalent / acre to about 2.0 lbs. acid equivalent / acre). Herbicidally effective rates of bromoxynil for use in areas to control native Brassica. The herbicide may be applied at a rate of about 0.1 lb. acid equivalent / acre to about 16 lb. acid equivalent / acre over the growing season. (e.g., bromoxynil can be applied at about 0.5 ppm over the growing season. (It can be applied at a rate of about 2.0 lb. acid equivalents / acre to about 2.0 lb. acid equivalents / acre). A herbicidally effective amount of MCPA amine herbicide should be used in the area to control C. rassica. From about 0.1 lb. acid equivalent / acre to about 16 lb. acid equivalent / acre over the growing season. (e.g., MCPA amine may be applied at about 0.5 pounds of acid over the growing season. equivalents / acre to about 2.0 lbs. acid equivalents / acre).

[0070] Methods for producing plants and seeds containing Brassica event MON94100 are provided. Plants can be cultured by any method known in the art, for example, as described in WR Fehr, n Breeding Methods for Cultivar Developm ent,Wilcox J.ed.,American Society of Agr. Commonly used breeding materials can be found in The American Pest Control Society, Madison WI (1987). Plants can be cross-pollinated using the methods described in Methods. Can be pollinated (also known as "crossed"). Contains Brassica event MON94100 The plants were a pure lineage of plants homozygous for the Brassica event MON94100. Inbred lines can be produced by self-pollination, which produces progeny known as "inbreds." It can be used to produce inbred lines that are genetically uniform. Plants containing event MON94100 may be cross-flowered to produce varieties or hybrid seeds. The plant may be crossed with another plant, either transgenic or non-transgenic. The seeds and progeny plants produced by the methods of the present invention are Brassica event MON941 00. Brassica event MON94100 contains one or more of the species to which it confers resistance. Herbicide applications were used to select for progeny containing Brassica event MON94100. Alternatively, the progeny may be a plant containing the Brassica event MON94100. The progeny may be analyzed using diagnostic methods to select for a variety or seed. The plant may be of the species Brassica MON94100. From seeds produced or from plants containing the Brassica event MON94100 may be grown from seeds produced by plants fertilized with pollen of Brassica One may be homozygous or heterozygous for the event MON94100.

[0071] The plants, progeny, seeds, cells, and plant parts of the present invention may be derived from one or more additional Brassica a trait(s) or transgenic event(s). The trait(s) or transgenic event(s) may include, for example, improved insect resistance; Improved water use efficiency, improved yield, improved drought tolerance, improved seed quality, improved nutritional quality, These include, but are not limited to, hybrid seed production, male sterility, and herbicide tolerance. The traits are measured relative to Brassica plants lacking the transgenic trait. Transgenic events in Brassica are known to those skilled in the art and can be found, for example, in A list of the traits that can be used is provided by the United States Department of Agriculture. iculture (USDA) Animal and Plant Health I Each is provided by the American Pest Control Inspection Service (APHIS). Crossing the two parent plants containing the transgenic event(s) and collecting progeny seeds. By selecting progeny seeds or plants containing two or more transgenic events. Therefore, two or more transgenic events may be combined in a progeny seed or plant. These steps then result in the desired combination of transgenic events in progeny. This may be repeated until a match is achieved. Outcrossing with clone plants is also contemplated, as well as vegetative propagation.

[0072] A representative sample of seeds containing the Brassica event MON94100 was collected according to the Budapest Treaty. Therefore, 10801 University Boulevard, Manassas, American Type with address in Virginia 20110 (USA) Culture Collection (ATCC®) Patent Department The seed AT containing the Brassica event MON94100 was deposited in the ository. The CC patent deposit name (accession number) is PTA-125182, and the deposit date is August 2018. The deposit will be valid for 30 years, or 5 years from the date of the last claim, or the term of the patent. The documents will be maintained in the depository for the period of one year or two years, whichever is longer.

[0073] As used herein, the term "including" means "including, but not limited to." Taste. EXAMPLES

[0074] The following examples are included to more fully illustrate the invention. Six different expression constructs required for the creation and final selection of MON94100 Construction and testing of the construct, generation of 2,775 unique transformation events, and rigorous molecular testing The analysis of millions of individual plants over five years from agronomic and field trials was summarized. do.

[0075] Numerous modifications can be made in the specific examples disclosed and still obtain similar results. It will be understood by those skilled in the art that the present invention relates to a compound that exhibits both chemical and physiological relevance. A given agent may be substituted for the agents described herein so long as the agent achieves the same or similar results. All such substitutions and modifications apparent to those skilled in the art are within the scope of the present invention. It is considered that there is.

[0076] Example 1: Design of expression constructs, production of events, and testing of R0 and R1 plants This example describes the design of six different expression constructs for dicamba tolerance, Generation of thousands of unique Brassica events using plant vectors containing Testing of the resulting transgenic Brassica plants over two generations (R0 and R1) The experiments and analyses are described.

[0077] Six expression constructs were designed and cloned into plant transformation vectors. The expression elements and DMO transgenes linked to the Brain marker gene are uniquely combined. Three different promoters, three different CTPs, and two different Four single expression cassette constructs (D T-1, DT-2, DT-3, and DT-4) were designed. Each was operably linked The same CP4-EPS with a unique combination of expression elements and DMO transgenes Two different promoters, two different CTPs, and and two dual expression vectors allowing for testing of two different DMO variant transgenes. Cassette constructs (DT-5 and DT-6) were designed. The constructs are shown in Table 2. Seed expression constructs were cloned into plant transformation vectors. TIFF0007636385000002.tif51133

[0078] Six plant transformation vectors were restored to fertility using methods known in the art. The lineage, Brassica napus cultivar 65037 (Canadian Plant Breeder's Rights Application No. 0 6-5517) for Agrobacterium-mediated transformation, 2,775 unique transformation events were generated. Each transformation event contained the same transgene insert fragment as the It was generated by random insertion at a unique position into the Brassica genome. R0 plants were regenerated from transgenic cells and rooted with normal phenotypic characteristics. Plants were transferred to soil for growth and further evaluation.

[0079] 2,775 R0 plants were transfected with intact single-copy transgenic inserts. This initial molecular analysis demonstrated that the vector contains genomic DNA and is free of vector backbone sequences. Through this, 201 unique events were identified as the highest quality events to move forward.

[0080] Plants representing the DT-1, DT-2, and DT-3 constructs were selected and subjected to R0 phenotype testing in the greenhouse. For efficacy (dicamba tolerance) testing, dicamba was applied at 1.0 lb acid equivalent / acre. Clarity® herbicide (2x dose) or 2.0 lb acid equivalent / acre C Dicamba was applied at the V3 growth stage at either 4x the dose of either dicamba or dicamba. The damage caused by this to plants is characterized as epiphytic growth (curving or twisting of leaves), reduced growth, The plant was visually assessed based on the assessment of chlorosis, necrosis, and chlorosis. Plants showing more than 20% damage were discarded. For the DT-1 and DT-3 constructs, the R0 plants were resistant to the double dose in the greenhouse. Although the DT-2 construct showed resistance (less than 20% injury), all R0 plants were resistant to the 2x dose Analysis of this data showed that the DT-2 construct was able to inhibit dicamba-induced morphological changes in the DT-2 mutant. It was decided not to proceed with any events generated using

[0081] The first 2,775 unique transformation events generated using six transformation vectors Combining molecular analysis data and R0 dicamba resistance testing, 206 unique events were identified. were selected for forwarding. The R0 plants of the selected events were homozygous for R1 testing. They were allowed to self-pollinate to produce sexual seeds.

[0082] A greenhouse test of dicamba tolerance was carried out using R1 plants of 169 unique events. The ba was sprayed with Clarity herbicide (double rate) at a rate of 1120 g acid equivalent / hectare in V3. The application was performed at the growth stage. For the 2010-2013 crop, dicamba was added as a tank mix of glyphosate at 3600 g acid equivalent / g. The dicamba was applied at twice the rate of Roundup® per hectare. The plant injury induced by dicamba was evaluated as described above. We prioritized the plants that we could move forward with.

[0083] R1 plants were 7.48% with the DT-1 construct, 72.66% with the DT-3 construct, and 72.66% with the DT-5 construct. The mean injury rates were 20.7% for the DT-6 construct and 33.4% for the DT-7 construct. However, individual events with less than 20% damage will be moved forward unless the damage is epigenetic. This resulted in nine events from the DT-1 construct in addition to one event from the DT-4 construct. events, 17 events from the DT-5 construct, and 9 events from the DT-6 construct were selected. .

[0084] Initial molecular analysis (copy number, intact insert, and absence of vector backbone) Based on the analysis of data from the R0 and R1 herbicide resistance evaluations in the greenhouse, 36 Unique events were selected for progression to F1 field trials, and the relevant data are summarized in Table 3. The R1 plants of the selected events were cross-pollinated with conventional plants to produce seeds. TIFF0007636385000003.tif51132

[0085] Example 2: First season field trials This example illustrates the first step of cloning plants containing each of the 36 unique events proceeding from the R1 analysis. Describe the first season field trials. For each unique event, several thousand plants were tested in the first season field trials. Plants were tested at 8-13 different locations over a 2-year period for trait availability (dicamba tolerance), agronomic performance, and These data were analyzed to determine the overall yield of all plants and all sites. The performance of each event was compared in field conditions across a number of locations. Then, first season field trials were conducted. The data was used to select superior events for progression into second season field trials.

[0086] Brassica napus female parent lines were cross-flowered with R1 or R2 plants of selected events. The field trials were carried out by pollinating the plants and producing hybrid F1 seeds (hemizygous for the event). Hybrid F1 plants were produced for the first season of the two-year field trials. Different Brassica napus female parental lines were cross-pollinated with R1 plants of selected events. The female parent line was then crossed with the 100-kDa hybrid line to produce F1 hybrid seeds. The female parent line was also crossed with the 100-kDa hybrid line to produce F1 hybrid seeds. The plants were cross-pollinated with non-transgenic plants of the same genetic background. The use of appropriate controls for testing and comparison of events in each dam line was ensured.

[0087] In the first season of testing, 36 unique events were identified from the first 2,775 events in the field trials. These 36 events were selected for testing. The results showed: 9 events for construct DT-1, 1 event for construct DT-4, 17 events for structure DT-5 and 9 events for structure DT-6. The field tests were conducted over a two-year period, with the first season agronomic performance field tests for each event. The trials were conducted simultaneously (during the same season) with the first season trait effectiveness field trials. The field trials used randomized complete block designs and were conducted at 8-13 different locations in North America. Ta.

[0088] First season trait efficacy trials evaluate F1 hybrid plants for tolerance to dicamba Two types of dicamba applications were tested for all events: Treatment 1 (TRT1) , 2.4 kg acid equivalent / hectare (twice the commercial amount) of BANVEL® II Preemergence treatment with 1.2 kg acid equivalent / ha (twice the commercial amount) of BANVEL( Treatment 2 (TRT2) included a postemergence treatment at the 3-leaf growth stage (V3) with 100 mg / kg of 10 ... ) is BANVEL® I at 2.4 kg acid equivalent / hectare (twice the commercial amount). Preemergence treatment with BANVEL I at 1.2 kg acid equivalent / ha (twice the commercial amount) Postemergence treatment with V3 with 1.2 kg acid equivalent / hectare (commercial The treatments included postemergence treatments at the first flower stage with BANVEL® II (twice the targeted dose). Percentage plant injury induced by herbicides 7 days after each application (V3 or first flower, respectively) The rate was based on the assessment of vegetative growth (curving or twisting of leaves), reduced growth, chlorosis, and necrosis. Agronomic evaluations were collected over the course of the field testing season. Instead, yields were determined as pounds per acre (lb / ac).

[0089] Trait validity data from first season field trials was compiled. For each unique event, Metadata of aggregated data across all locations and all individual plants in the trait efficacy field trial. An analysis was performed to compare the hybrid injury ratings. Table 4 shows the results for the two dicamba treatment regimes. Provides the average injury rating for each event across all locations (NA is for treatments). (Indicates that the data is unavailable.) A meta-analysis of trait effectiveness field trials across seasons was Generally, plants in all events had mild injury from dicamba, but DT-1 and DT-4 The construct events had lower injury ratings, indicating very good performance. TIFF0007636385000004.tif188135 TIFF0007636385000005.tif19134

[0090] Yield data from first season trait effectiveness field trials was compiled. Yields aggregated across all locations and all individual plants in a one-season trait availability field trial. A meta-analysis of the abundance data was performed for comparison. Table 5 shows the results for the two dicamba treatment regimes. Provides the average yield as pounds per acre (lb / ac) for each event across all locations. (NA indicates that data for a treatment is not available). A meta-analysis of yields from trait efficacy field trials generally showed that plants in all events were higher in the unsprayed versus untreated control. The results showed that the plants had the same yield as the control plants. Meta-analysis of the combined data showed that plants containing events with the DT-5 construct had significantly lower mean yields than plants containing events with the DT-5 construct. Had the quantity. TIFF0007636385000006.tif187134 TIFF0007636385000007.tif164135

[0091] In the first season of agronomic testing, the F1 hybrid plants were evaluated for agronomic performance and yield. The test plots were maintained completely weed-free and were treated with the test herbicides (dicamba or dicamba and glyphosate). No fertilizers (including fertilizers) were applied during the growing season. The following agronomic evaluations were collected for all events: : Early vigor, emergence uniformity, first flowering date, end of flowering date, plant height, maturity date, actual Harvested kernel weight, percent kernel moisture, harvest date, and, where applicable, Stan dability, dehulling, Sclerotinia infestation, and disease and pests Insect pressure. Agronomic evaluations were collected over the field trial season. At the end of the season, pound Agronomic yields were determined as lb / acre (ac).

[0092] Yield data from the first season agronomic performance field trials was compiled. For each unique event, Meta-analysis of aggregated data across all sites and all individual plants in a seasonal agricultural field trial were performed for comparison. Table 6 shows the lbs / acre across all locations for each event. The average yield in lb / ac is given. Yields from agricultural field trials over each season are Meta-analysis of the amount of DT-4 constructs showed that event 68403 and DT-4 constructs had significantly lower yields. Generally, all events were plant-specific, except for events 43166 and 43237, which were produced using the DT-5 construct. The results showed that the DT-1 construct produced a yield comparable to that of unsprayed control plants. Four events had significantly lower agronomic yields than the other five events with the DT-1 construct. TIFF0007636385000008.tif188134 TIFF0007636385000009.tif166136

[0093] (1) evaluate trait effectiveness for tolerance to commercial amounts of dicamba, and (2) evaluate agronomic performance. Data collected from field trials of hybrid plants were analyzed using constructs DT-1, DT-4, DT-5, and D The 36 events tested for T-6 were analyzed. This analysis for each event is shown in Example 1. Combined with the results of thorough molecular characterization described in 3, the product will proceed to a second season field trial. We selected events that could be considered as

[0094] Example 3: Molecular characterization This example describes the detailed molecular characterization of selected events that was conducted in parallel with field trials. Molecular characterization of each event determines whether an event should be selected for further development. was used to determine.

[0095] DNA and RNA analysis of the events are carried out using a variety of techniques known in the art. Southern blot analysis was performed on genomic DNA to identify the transgenic plants. contains a single copy of the entire transgene insert without any vector backbone DNA amplification and sequencing were used to confirm the transgenic insert for each event. It was confirmed that the structure and sequence of the inserted fragment were unchanged. The DNA adjacent to each end (5' and 3' ends) of the insert was sequenced and each junction Northern analysis was performed to determine the dmo in transgenic plants of each event. The mRNA transcripts of the genes and the cp4 gene (where applicable) were detected and measured.

[0096] Protein analysis of the plants containing each event was carried out using techniques known in the art. The N of DMO protein purified from transgenic plants containing each event was Terminal protein sequencing was performed to confirm the recombinant protein sequence. Protein analysis was performed on protein extracts from plants containing each event to identify DMO proteins. Using enzyme-linked immunosorbent assay (ELISA), the researchers confirmed that proteins had been produced. The protein levels of DMO proteins in plant leaves, seeds, roots, and pollen were examined. It was decided that

[0097] The insertion site of each event in the genome was analyzed. The flanking sequences were compared to the binomial location of the event on the chromosome. The insertion sites of each event were identified using the publicly available Brass ica napus genome (Boulos Chalhoub, et al. “Earl y allopolyploid evolution in the post-Ne olithic Brassica napus oilseed genome”,S science,Vol 345 Issue 6199(22 August 2014 The DNA amplification across the wild-type allele in the genome was then mapped to the neighbors of each event. The wild-type insertion site sequence was used to perform the analysis. The reference genome of Brassica napus was used to identify unique transgene integration sites for elephants. was mapped to.

[0098] Detailed molecular characterization of each event was performed on first season field samples for each event. Combined with trait efficacy and agronomic performance data from the study. This combined information was used ,To advance 13 unique events from 36 tested in the first season field trial, After analysis, none of the nine events in construct DT-6 were selected for further development. ,10 of 17 events in DT-5 did not proceed, 5 events in construct DT-1, construct Thirteen unique events were reported, including one event in construct DT-4 and seven events in construct DT-5. was selected to proceed.

[0099] Example 4: Second season field trials This example illustrates the development of a plant containing each of the 13 unique events that were developed from the first season field trial. For each specific event, describe the second season field trials. Thousands of plants were tested for trait efficacy (tolerance to dicamba and glyphosate) over a two-year period in multiple locations. The data were analyzed to determine the variability, agronomic performance, and yield of all plants. The performance of each event in field conditions across all plants and locations was then compared. Using data from the first season of field trials, we will identify outstanding cases to advance to third season field trials. Selected.

[0100] Brassica napus female parental lines were cross-pollinated with R2 plants of selected events. Second season field trial by producing hybrid F1 seeds (hemizygous for the event) Hybrid F1 plants were produced for the study. The female parent line carrying the DT-1 and DT-4 constructs was used to cross with the R2 plants containing the DT-1 and DT-4 constructs. This produced F1 plants that were tolerant to both dicamba and glyphosate. In each of the two years in which the second season field trials were conducted, one to three different Brassica The napus female parental lines were cross-pollinated with R2 plants of the selected events to produce F1 hybrid seeds. This female parent line was also produced in the same genetic background as the R2 line for use as a control. This strategy allowed for the identification of the different female parent lines. The use of appropriate controls for elephant testing and comparison was ensured.

[0101] In the second season of testing, the 13 unique events selected for testing were five of construct DT-1. 1 event for construct DT-4, and 7 events for construct DT-5. The seasonal tests were conducted over a two-year period, with the second season agricultural performance test for each event. The field trial was conducted simultaneously (during the same season) with the second season trait effectiveness field trial. All field trials used randomized complete block designs and were conducted at 4-9 different locations across North America. did.

[0102] In the second season trait efficacy trials, the F1 hybrid plants were exposed to dicamba and glyphosate. The plants were evaluated for their tolerance to 1.2 kg acid equivalent / ha (commercially available) at the V3 stage. dicamba (BANVEL II) at 1.8 kg acid equivalents / hectare (twice the commercial dose) Glyphosate (Roundup) followed by 1.2 kg acid equivalent / ha at first flowering stage. (twice the commercial amount) of dicamba (BANVEL II) and 1.8 kg acid equivalents / hectare glyphosate (Roundup) postemergence (as a tank mix). Seven days after each application (V3 or first flower, respectively), herbicide-induced plant injury was measured. The percentages were used to determine vegetative growth (curving or twisting of leaves), reduced growth, chlorosis, and necrosis. Agronomic evaluations were collected over the course of the field trial season. Yields were determined as pounds per acre (lb / ac) at the end of each period.

[0103] Trait validity data from second season field trials was compiled. For each unique event, trait validity was calculated. A meta-analysis of aggregated data across all sites and all individual plants in efficacy field trials was performed. A comparison of injury assessments was performed. Table 7 shows the results for each treatment plan across all locations. Meta-analyses of trait effectiveness field trials across seasons generally In contrast, plants with the DT-1 and DT-4 constructs had lower injury ratings and performed very well. It showed that it was good. TIFF0007636385000010.tif78134

[0104] Yield data from the second season trait effectiveness field trials were compiled. Yields aggregated across all locations and all individual plants in a two-season trait efficacy field trial. A meta-analysis of the abundance data was performed for comparison. Table 8 shows the abundance of each event across all locations. The pounds per acre (lbs / acre) of treated and untreated plants (control) in the treatment regimen b / ac) Yields of trait effectiveness field trials over each season are provided. The meta-analysis generally found that plants containing the Brassica event MON94100 were more likely to be chronologically related to the The highest yields were achieved when plants were sprayed with methadone and glyphosate compared to 12 other plants. It showed that it had. TIFF0007636385000011.tif137133

[0105] In the second season agronomic performance test, the F1 hybrid plants were grown in agronomic conditions as described in Example 2. Performance and yield were evaluated at the end of the season in pounds per acre (lb / ac) and The yield data from the second season of agronomic performance field trials were compiled. The occurrence of any of the above events was examined across all locations and all individual plants in the second season agricultural field trials. A meta-analysis of the aggregated yield data was performed for comparison. Table 9 shows the results for each event. The average yield as pounds per acre (lb / ac) across all locations is provided. A meta-analysis of yields from agricultural field trials across seasons showed that DT-5 construct event 40819 In general, plants in all events had yields comparable to the control plants, except for the significantly lower yields. It showed that it had. TIFF0007636385000012.tif172134

[0106] (1) Molecular analysis and phenotypic expression of dicamba and glyphosate-resistant com- mercial doses (2) Data collected from field trials with hybrid plants to evaluate their agronomic performance were used to validate the construct. Thirteen events tested in DT-1, DT-4, and DT-5 were analyzed. Conduct analysis of trait effectiveness, agronomic performance, and yield data from second season field trials on Combined with the results of thorough molecular characterization described in Example 3, the results of the third season field trials Four unique events were selected for testing. The four unique events were the two events of construct DT-1. There were two events, one for construct DT-1 and one for construct DT-5.

[0107] Example 5: Third season field trials This example illustrates the use of plants containing each of the four unique events that were developed from a second season field trial. For each specific event, the results of the third season field trials are listed. Thousands of plants were tested in multiple locations over two years for trait availability (dicamba tolerance), agronomic performance, and These data were analyzed to determine the relative yield and yield of all plants and all locations. The performance of each event was compared under field conditions over a period of three seasons. The data was used to select leading events for commercialization.

[0108] Brassica napus female parent lines were cross-flowered with R2 or R3 plants of the selected events. The third seed was produced by pollinating the plants and producing hybrid F1 seeds (hemizygous for the event). Hybrid F1 plants were produced for field trials. In each year, two to three different Brassica napus female parent lines were selected. The R1 plants were cross-pollinated to produce F1 hybrid seeds. This female parent line was then cross-cultured with other non-transgenic plants of the same genetic background as the R2 or R3 line. This strategy provided a suitable platform for testing and comparing events in different female parental lines. The use of controls was ensured.

[0109] The third season test was conducted over a two-year period, with the third season agricultural Performance field trials are conducted simultaneously (during the same season) with third season trait effectiveness field trials. All field trials used randomized complete block designs and were conducted in 5-10 different locations across North America. It was carried out at the following location.

[0110] In the third season trait efficacy trials, the F1 hybrid plants were grown using XtendiMax (registered trademark). The target herbicide was used to evaluate tolerance to dicamba as described in Example 2. Yields were determined at the end of the season as pounds per acre (lb / ac). The trait validity data from the field trials was compiled. For each unique event, the trait validity field trials were A meta-analysis of aggregated data across all sites and all individual plants in the study was performed for the hybrid injury assessment. For comparison, Table 10 shows the averages for each event across all locations for the treatment plan. Meta-analyses of trait effectiveness field trials across seasons generally provide an assessment of DT The results showed that plants with the -1 construct had lower injury ratings and performed very well. did. TIFF0007636385000013.tif46136

[0111] Yield data from the third season trait effectiveness field trials were compiled. Yields aggregated across all locations and all individual plants in a three-season trait availability field trial. A meta-analysis of the abundance data was performed for comparison. Table 11 shows the results for the two dicamba treatment regimes. The average yield was provided as pounds per acre (lb / ac) for each event across all locations. (NA indicates that data is not available for the treatment). A meta-analysis of yields from the trials generally showed that plants from all events had yields comparable to unsprayed controls. It was shown that the amount TIFF0007636385000014.tif45134

[0112] In the third season agronomic performance test, the F1 hybrid plants were grown in agronomic conditions as described in Example 2. Performance and yield were evaluated at the end of the season in pounds per acre (lb / ac). Agronomic yields were determined by the above. Yield data from the third season of agronomic performance field trials were compiled. The phenomenon was examined across all locations and all individual plants in the third season agricultural field trials. A meta-analysis of the aggregated yield data was performed for comparison. Table 12 shows the results for each event. The average yield as pounds per acre (lb / ac) across all locations in the Meta-analyses of yields from commercial field trials generally showed that plants in all events showed higher yields than untreated controls. It was shown that they had comparable yields and there was no statistically significant difference. TIFF0007636385000015.tif53136

[0113] Data collected from molecular analyses and third season field trials were analyzed for four events: Trait validity, agronomic performance, and yield data from the third season field trials for each event were collected. The analysis was combined with the results of thorough molecular characterization described in Example 3 to evaluate the events. In addition, a dicamba tolerance trait that is not molecularly linked to another herbicide tolerance trait is preferred. The construction of a single dicamba resistance expression cassette was considered to be effective for self-control, weed control, and Allowing farmers greater freedom and choice in their farm management and crop rotation Two events representing the DT-1 construct were analyzed using combined data from three seasons of field trials. We then proceeded to data analysis.

[0114] Example 6: Meta-analysis of combined field trial data This example describes a meta-analysis of all field trial data for the final two events. This is the result of hybridization under field conditions over many years, many locations, and hundreds of thousands of plants. Allowing for larger scale comparisons of plant trait performance and yield effects.

[0115] A meta-analysis of trait validity for the two selected events by construct DT-1 was performed on all This was done by aggregating data from the first and third seasons across locations. This allowed for a larger comparison of the hybrid injury ratings. Table 13 shows the results for each event for each treatment. The mean injury rating is given. A meta-analysis of trait efficacy field trials showed that both The events showed that both treatments had very low injury ratings. TIFF0007636385000016.tif29117

[0116] All results of the trait validity field trials for the two selected events with construct DT-1. A meta-analysis of abundance data was performed by aggregating data from seasons 1 and 3 across all locations. Table 14 shows the pounds per acre (l) of each event for each treatment. b / ac) (for treated plants and treated plants containing the same event) (Calculated as the difference in yield in pounds per acre between plants that were treated with and without the treatment). A meta-analysis of the trial results showed that MON94100 performed very well, with both treatments showing better efficacy. The data showed that event 170060 produced a higher average yield than event 170060. It is important to select the best commercial events and to evaluate the Br under dicamba application in field conditions. The results demonstrated the remarkable performance of plants containing the M. assica event MON94100. TIFF0007636385000017.tif31119

[0117] All agricultural field tests on two selected events and control plants with construct DT-1 A meta-analysis of all yield data was performed by aggregating data from all three seasons across all locations. This was done by comparing the yield data with and without dicamba. Table 15 shows the average yield in pounds per acre for each event for each treatment. A meta-analysis of yield data from agricultural field trials showed that MON94 was significantly lower in unsprayed conditions than 100 and 170060 provided similar yields to the control plants, and when compared to the control plants, There was no statistically significant difference in yield from plants containing either of these events. TIFF0007636385000018.tif40121

[0118] Analysis of cumulative data showed Brassica event MON941 compared with event 170060 00 herbicide application conditions and demonstrated advantages in dicamba tolerance and crop yield. The elephant was selected as a superior event useful for commercial purposes.

[0119] Example 7: Detection of Brassica event MON94100 This example describes the detection of Brassica event MON94100. Detection of the Brassica event MON94100 was performed using DNA, RNA, or protein Detection techniques can be used. Exemplary detection methods and materials are provided below. Br The DNA sequence information of the assica event MON94100 is set forth herein as SEQ ID NOs: 1 to 10. The transgenic insertion of Brassica event MON94100 is provided in the literature. The fragment contains the elements described in Table 1.

[0120] Detection determines the presence or absence of Brassica event MON94100 in a sample. This can be used to identify the Brassica event MON94 in a sample of genomic DNA. 100 genome copy number (i.e., hemizygous, homozygous, or heterozygous) Event-specific Applied Biosystems™ TAQMAN Thermo Fisher Scientific c) was developed to identify the Brassica event MON94100 in the sample. The DNA primers and probes used in the endpoint assay were primer S Q51321 (SEQ ID NO: 11), SQ13805 (SEQ ID NO: 12), and 6-FAM ( The labeled probe is PB4832 (SEQ ID NO: 13). Fluorescein is a fluorophore attached to a DNA probe (Applied Biosystems) (Foster City, CA) is a fluorescent dye product. ) probe, the 5' exonuclease activity of Taq DNA polymerase was detected by fluoro The probe between the endophore and the quencher is cleaved from the 5' end. When hybridized, the quencher and fluorophore are sufficiently close together that a fluorescent signal is produced. These reaction methods and PB4832 are well separated, which causes the emission of fluorescence. When used together, SQ51321 and SQ13805 inhibit the Brassica event MO This analysis generates a DNA amplicon useful for diagnosing N94100. A positive control, non-transgenic Bras assica event MON94100, was included. A negative control derived from sica and a negative control containing no template DNA should be included. Additionally, the PCR reaction controls preferably include a single copy number of the nucleotide sequence in the Brassica genome. Gene-specific internal control primers and probes should be included. The assay is run using the Applied Biosystems Genome eAmp® PCR System 9700 (Thermo Fisher Scientific) The device is optimized for use with a 3D scanner, but other devices may be used.

[0121] Used with the TAQMAN method to detect the Brassica event MON94100 Examples of conditions useful for this purpose are as follows: Step 1: Adjust to a final volume of 5 μl. Step 2: Add 2.28 μl of 2× Univ. ersal Master Mix (dNTPs, enzymes, buffer). Step 3: 0.9μ 0.05 μl of event primer 1 (SQ51321) and event primer 2 (SQ51322) to a final concentration of M Primer 2 (SQ13805) (100 uM concentration for each primer in 18 megaohm water) Step 4: 0.01 μl of 6-FAM for a final concentration of 0.2 μM MGB event probe PB4832 (resuspended in 18 megaohm water to a concentration of 100 μM) Step 5: 0.05 μl of internal control primer 1 and internal control primer 2 for a final concentration of 0.9 μM. Mix 2 control primers (100 μM for each primer in 18 megaohm water) Step 6: 0.01 μl of internal control V for a final concentration of 0.2 μM. IC™ probe (resuspended in 18 megaohm water to a concentration of 100 μM). 7: 2.5 μl of extracted DNA (template) of each sample with one of the following: (a) Analysis (b) negative control (non-transgenic DNA); (c) negative water control (template) (d) a positive pair containing DNA of the Brassica event MON94100 Illumination: Brassica. Step 8: Thermocycler conditions are as follows: 95°C. 1 cycle at 95°C for 20 seconds, 3 cycles at 95°C for 3 seconds, then 40 cycles at 60°C for 20 seconds, and a final cycle of 10°C.

[0122] Plants containing the Brassica event MON94100 were cultured for either the event or the wild-type allele. Zygosity assays have been developed to determine heterozygosity or homozygosity. Amplification reaction assays can be designed using the sequence information provided herein. For example, such a PCR assay includes at least three primers: primer 1; The design of primers includes primer 1, primer 2, and primer 3, where primer 1 is Brassica The 3' flanking DNA of event MON94100 is specific for Brassica genomic DNA. , primer 2 is for the transgenic insert of Brassica event MON94100 Primer 1 is specific for the wild-type allele, and primer 2 is specific for the wild-type allele. When used as a pair, primer 1, together with primer 2, detects Brassica events. The PCR reaction will generate a PCR amplicon specific for MON94100. When used as a primer pair, primer 1, together with primer 3, detects the wild-type allele. This will generate PCR amplicons specific to the Brassica genome. In the PCR reaction performed, each PCR amplicon generated by primer 1 + primer 2 Each PCR amplicon generated by primer 1 + primer 3 was The sequences and sizes of the precons will differ. Three primers were used to identify the Brassica event PCR reactions with DNA extracted from plants homozygous for MON94100 If the primer is 1+primer 2, the amplicon (Brassica event MON Three primers were used to generate the Bras P in DNA extracted from plants heterozygous for the sica event MON94100 When included in the CR reaction, the amplicon with primer 1 + primer 2 (Brassii ca event MON94100 insert specific) and primer 1 + primer 3 amplification Plicon (specific for wild-type allele or Brassica event MON94100 insert fragment Three primers were used to generate both the Brassica a Extracted from plants that are null (i.e., wild type) for event MON94100. When mixed in a PCR reaction with DNA containing primer 1 + primer 3, Only amplicons (specific for the wild-type allele) will be generated. The generated amplicons may be identified or analyzed using any method known in the art. can be distinguished.

[0123] Another conjugation assay for Brassica event MON94100 was performed using TAQMAN thermal amplification In this type of assay, in addition to the primers mentioned above, the assay also contains two The probes are fluorophore-labeled with two fluorescent probes. Probe 1 is the Brassica event MON94100 and probe 2 is specific for Brassica event MON94100 (wild type). The two probes are specific for null (wild type) Brassica plants, contains a different fluorescent label, e.g., a 6-FAM label or a VIC™ label. When used in a QMAN reaction, primer 1 + primer 2 + probe 1 is Br The first fluorescent signal was generated specific for the assica event MON94100, using primer 1 + primer 3 + probe 2 is a second fluorescent signal specific for wild-type Brassica Three primers and two probes will generate the Brassica event MO TAQMAN reactions using DNA extracted from plants homozygous for N94100 If the first fluorescent signal (specific to primer 1 + primer 2 + probe 1) is included in Three primers were used to identify the Brassica event MON941. Included in a TAQMAN reaction using DNA extracted from plants heterozygous for 00 If the first fluorescent signal (specific for primer 1 + primer 2 + probe 1) and Both the first and second fluorescent signals (specific for primer 1 + primer 3 + probe 2) were generated. Three primers will be used for the Brassica event MON94100. Together in a TAQMAN reaction with DNA extracted from null (wild type) plants When the two are mixed together, the second fluorescent signal (specific to primer 1 + primer 3 + probe 2) Only the abnormal (non-normal) will be generated.

[0124] Another method to detect the presence of Brassica event MON94100 in plant samples Southern Analysis. Those skilled in the art can perform Southern analysis specific for Brassica event MON94100. Hybridization probe(s) and Brassica event MON94100 A second Southern hybrid specific for (wild-type) Brassica plants that are null for In Southern analysis, the first Signals detected only from the hybridization probe are Brassica events. The first Southern hybridization analysis showed homozygous plants for MON94100. The signal detected by both the chromosome probe and the second Southern hybridization probe was Null indicates a plant heterozygous for the Brassica event MON94100, and the second The signal detected only from the Southern hybridization probe of Brassic a DNA was extracted from plants that were null (wild type) for event MON94100. would show.

[0125] Another example of a detection kit is a kit for detecting the minor genotype encoded by Brassica event MON94100. For example, such a kit may include at least one antibody specific for at least one protein. The present invention relates to a lateral flow strip, which, when the tip of the strip comes into contact with an aqueous solution, The lateral flow strips containing the activated reagents can be used for antibody production. An exemplary protein sufficient for the synthesis of the ribozyme is encoded by the sequence provided as SEQ ID NO:10. or any fragment thereof.

[0126] The sample is a plant, seed, cell, or plant part containing Brassica event MON94100. Protein detection methods will be developed to determine whether the protein is derived from Brassi. A small number of genes specific for at least one protein encoded by the ca event MON94100 At least one antibody was identified in the sample that was causative of the Brassica event MON94100. Used to detect proteins that are transduced in the Brassica event MON9410 A detection kit comprising one or more antibodies specific for one or more proteins encoded by .alpha.-.0. The lateral flow strip is activated when the tip of the strip comes into contact with an aqueous solution. The lateral flow strips containing the reagents to be activated can be used. a Tissue sample is ground and dissolved in water or an aqueous buffer (e.g., detergent and bovine serum albumin). Proteins can be extracted for analysis using phosphate buffered saline (PBS) containing 100% ethanol. After centrifugation, the sandwich former is placed on a lateral flow strip with an absorbent pad. The aqueous supernatant is analyzed using the ELISA method of the present invention. Detection is initiated by dipping the tip of the strip into a solution. The aqueous solution wicks through the strip by capillary action. The gold-labeled antibody is then transported to the top of the trip and solubilizes the gold-labeled antibody on the strip. Assica event MON94100 is specific for at least one protein encoded by the The antibody is specific to the epitope of the protein in the sample and forms an antibody-antigen complex. The gold-labeled antibody-antigen complex is then transported up the strip to the nitrocellulose membrane. The membrane is regulated by a protein encoded by the Brassica event MON94100. A test line of immobilized antibody that binds to a second distinct epitope is included, If the protein encoded by mutant MON94100 is present in the sample, the test strain This will cause a visible line to appear across the top of the screen.

[0127] Example 8: Self-control This example describes a method for controlling plants containing Brassica event MON94100. For the purpose of self-control, the Brassica event MON94100 is included. Any herbicide to which the plant has high sensitivity is useful.

[0128] Exemplary herbicides for purposes of native control include synthetic auxin herbicides other than dicamba. Dicamba on plants containing Brassica event MON94100 Sensitivity to synthetic auxin herbicides other than dicamba makes it undesirable for farmers. Brassica (which includes the Brassica event MON94100) This provides the ability to remove the plant from the environment, which may be contaminated by other desirable crops or Brassica napus. a May or may not include Brassica that does not contain event MON94100.

[0129] Plants containing the Brassica event MON94100 and non-transgenic plants of the same germplasm as controls. Transgenic plants were grown in a greenhouse using a randomized complete block design. Four synthetic auxin herbicides: dicamba (XtendiMax®) )), 2,4-D Amine 4 (dimethylamine salt of 2,4-dichlorophenoxyacetic acid), Lomoxynil (Buctril®, 3,5-dibromo-4-hydroxybenzoate) nitrile), and MCPA amine (4-chloro-2-methylphenoxyacetic acid) The plant injury rate was measured 9 days after treatment.

[0130] After spraying commercial amounts (1x) of dicamba, Brassica event MON94100 The plants containing 10.1% glycerol had no injury and the control plants had 21.3% injury. However, the other three 1x synthetic auxin herbicides (2,4-D amine, bromoxynil, and MCPA) or twice as much, the plant and plant containing Brassica event MON94100 were Both the illuminated and unilluminated plants showed injury rates of 70% to 90%. Plants containing ON94100 and control plants responded similarly to the three auxin herbicides The data are shown in Table 16. TIFF0007636385000019.tif110133

[0131] A representative sample of seeds containing the Brassica event MON94100 was collected according to the Budapest Treaty. Therefore, 10801 University Boulevard, Manassas, American Type with address in Virginia 20110 (USA) Culture Collection (ATCC®) Patent Department The seed AT containing the Brassica event MON94100 was deposited in the ository. The CC patent deposit name (accession number) is PTA-125182, and the date of deposit is 201 The deposit was made on August 21, 1998. The deposit will be valid for 30 years, or 5 years from the date of the last claim, or until the date of the patent application. The documents will be maintained in the depository for the period of validity of either the original or the original documents, whichever is longer.

Claims

1. A recombinant DNA molecule comprising the sequence of SEQ ID NO:

9.

2. 2. The recombinant DNA molecule of claim 1, wherein said recombinant DNA molecule is derived from a plant, seed, or cell containing Brassica event MON94100, and a representative sample of seeds containing said event has been deposited under ATCC Accession No. PTA-125182.

3. 2. The recombinant DNA molecule of claim 1, wherein the recombinant DNA molecule is present in a plant, cell, seed, or plant part comprising Brassica event MON94100, and a representative sample of a seed comprising the event has been deposited under ATCC Accession No. PTA-125182.

4. An amplicon comprising the recombinant DNA molecule of claim 1.

5. A pair of DNA primers comprising a first DNA primer and a second DNA primer, wherein the first and second DNA primers each comprise a fragment of SEQ ID NO:10 and function when used with DNA containing Brassica event MON94100 in an amplification reaction to generate an amplicon diagnostic of Brassica event MON94100 in a sample, the amplicon comprising a 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 and SEQ ID NO:

10.

6. 6. The pair of DNA primers according to claim 5, wherein the first DNA primer comprises SEQ ID NO:11 and the second primer comprises SEQ ID NO:

12.

7. A kit for detecting the presence of Brassica event MON94100, comprising a pair of DNA primers according to claim 5.

8. A Brassica plant, seed, cell, plant part, or commercial product comprising a DNA molecule comprising the sequence of SEQ ID NO:

9.

9. 9. The Brassica plant, seed, cell, or plant part of claim 8, wherein the plant, seed, cell, or plant part exhibits tolerance to dicamba.

10. 13. A method for controlling weeds in an area comprising planting Brassica containing the recombinant DNA molecule of claim 1 and applying an effective amount of dicamba to control said weeds in said area without harming said Brassica.

11. 11. The method of claim 10, wherein the effective amount of dicamba is from 0.5 pounds acid equivalents per acre to 2 pounds acid equivalents per acre over a growing season.

12. 13. A method for controlling volunteer Brassica containing the recombinant DNA molecule of claim 1 in an area comprising applying a herbicidally effective amount of at least one herbicide other than dicamba, said herbicide application preventing the growth of Brassica containing said recombinant DNA molecule.

13. 13. The method of claim 12, wherein the herbicide other than dicamba is selected from the group consisting of 2,4-D (2,4-dichlorophenoxyacetic acid), bromoxynil (3,5-dibromo-4-hydroxybenzonitrile), and MCPA amine (4-chloro-2-methylphenoxyacetic acid).

14. 1. A method for producing a Brassica plant that is tolerant to dicamba, comprising: a) crossing a plant containing the recombinant DNA molecule of claim 1 with itself or with a second plant to produce seeds; b) identifying progeny seeds which contain said recombinant DNA molecule.

15. identifying progeny seeds containing the recombinant DNA molecule; i. growing the progeny seed to produce a progeny plant; ii. treating the progeny plant with an effective amount of dicamba; iii. selecting progeny plants that exhibit tolerance to dicamba.

16. 15. The method of claim 14, wherein identifying a progeny seed containing the recombinant DNA molecule is by detecting the presence of SEQ ID NO:9 in a sample derived from the progeny seed.

17. 15. The method of claim 14, wherein identifying progeny seeds containing the recombinant DNA molecule is by detecting the presence of at least one protein encoded by SEQ ID NO:9 in a sample derived from the progeny seeds.

Images