EA044838B1 - CORN TRANSFORMANT MON87429 AND WAYS TO USE IT - Google Patents

Description

Cross references to related applications

This application claims benefit from U.S. Provisional Patent Application No. 62/625537, filed February 2, 2018, which is incorporated herein by reference in its entirety.

Enabling sequence listing

The sequence listing, contained in a 44.2 KB file entitled MONS430WO-seq.txt (MS-Windows), created on January 17, 2019, is filed electronically with this document and is incorporated herein by reference.

Field of technology

The invention relates to recombinant DNA molecules of maize transformant MON87429. The invention also relates to transgenic corn plants, plant parts, seeds, cells and agricultural products containing the corn transformant MON87429, as well as methods for using transgenic corn plants, plant parts, seeds, cells and agricultural products containing the corn transformant MON87429, and methods for detecting maize transformant MON87429. Transgenic maize plants, plant parts, seeds and cells containing the maize transformant MON87429 exhibit tolerance to acetyl-CoAcarboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, such as chisalofop and haloxyfop; synthetic auxins such as dicamba and 2,4-D; glutamine synthetase inhibitors such as glufosinate; as well as 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors such as glyphosate.

Prerequisites for creating the invention

Corn (Zea mays) is an important crop in many parts of the world, and the use of herbicides in crop production to control weed growth is an established practice. Biotechnological methods are used to produce transgenic corn that is tolerant to a particular herbicide through the expression of a heterologous gene, also known as a transgene. The herbicide tolerance trait may be used alone or in combination with other traits, such as tolerance to another herbicide or resistance to pests or pathogens. Combinations of herbicide tolerance traits are desirable to provide weed control options that increase grower flexibility and allow the use of different herbicides to control problem weeds. A combination of traits can be obtained by crossing each individual trait with each other. Crossbreeding individual traits in maize and maintaining that combination through crosses with a diverse pool of elite germplasm is a labor-intensive and expensive process. A combination of traits can also be obtained by combining multiple traits at one location, or locus, in the genome, thereby simplifying the process of crossing. One way to achieve this is through a single transgene insert containing multiple transgenes. The combination of multiple herbicide tolerance traits at a single locus in corn would provide a useful weed control tool that would be much easier and less expensive to maintain through crossbreeding with a diverse pool of elite germplasm.

The expression of a transgene in a transgenic plant, plant part, seed or cell, and thus its effectiveness, can be influenced by many different factors, such as the elements used in the transgene expression cassette and the interactions of such elements. This is further complicated in the case of a transgene insert containing two or more expression cassettes, where each expression cassette contains a transgene that provides a separate trait and is also called a multigene transgene transformant. A commercially available multigene transgenic transformant assumes that each of the transgenes in the transgene insert will be expressed as required for each trait. To achieve this, individual expression cassettes are first developed and tested in plants, and the best expression cassettes are selected for each trait. The selected expression cassettes for one trait are then combined with the selected expression cassettes for the other trait(s) into one construct, and the construct is tested to ensure that all expression cassettes function well together and that each transgene is expressed properly. The selected combination of expression cassettes is then used as a single transgene insert to produce hundreds of multigene transgenic transformants, each of which is the result of random insertion of foreign DNA at different genomic locations.

Each transgenic transformant is unique. The unique transformants are then analyzed across multiple plant generations to select the best transformant for commercial use. The properties of the transformant in the transgenic plant, plant part, seed or cell, and thus its effectiveness, can be influenced by the genomic location of the transgenic insertion. Transformants may have the same transgene insertion and yet have different levels of transgene expression and different properties in different tissues, at different stages of development, in different germplasm or under particular growth conditions. Development of a multigene transformant for commercial use requires extensive molecular characterization, greenhouse testing and field trials over many years, in different locations and under different conditions, so that extensive agronomic, phenotypic and molecular data can be collected. The data obtained must then be analyzed by scientists and agronomists so that a transformant suitable for commercial use can be selected. The commercial multigene transformant can then be introgressed as a single locus with multiple herbicide tolerance traits into a new germplasm using plant crossing techniques.

Summary of the invention

The invention provides recombinant DNA molecules containing a sequence selected from the group consisting of 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 is obtained from a plant, seed or cell containing the maize transformant MON87429, wherein a representative sample of the seed , containing the transformant, was deposited under the accession number ATCC RTA-124635. In another embodiment, the recombinant DNA molecule is in a plant, cell, seed, or plant part containing the maize transformant MON87429, wherein a representative sample of seed containing the transformant is deposited as ATCC PTA-124635. In another embodiment, the recombinant DNA molecule is an amplicon capable of diagnosing the presence of the maize transformant MON87429.

The invention proposes a DNA construct containing four expression cassettes, the first expression cassette containing in functional connection (I) the promoter, leader and intron of ubiquitin from Erianthus ravennae, (II) a sequence encoding phosphinothricin N-acetyltransferase, and (III) 3'- HTO of fructose bisphosphate aldolase from Setaria italica; the second expression cassette contains in a functional connection (I) the promoter, leader and ubiquitin intron from Coix lacryma-jobi, (II) the sequence encoding the albino and pale green 6 chloroplast transit peptide from Arabidopsis thaliana, (III) the sequence encoding dicamba- monooxygenase, and (IV) 3'-UTR of metallothionein-like protein from Oryza sativa; the third expression cassette contains in functional connection (I) the promoter, leader and intron of ubiquitin from Arundo donax, (II) the sequence encoding the chloroplast transit peptide of malate dehydrogenase from Arabidopsis thaliana, (III) the sequence encoding the FT_T protein, and (IV) 3' -UTR of non-apical meristem protein from Oryza sativa; and the fourth expression cassette contains in functional connection (I) the promoter and leader of CaMV 35S, (II) the leader of chlorophyll a/b-binding protein from Triticum aestivum, (III) actin 1 intron from Oryza sativa, (IV) a sequence encoding a transit peptide chloroplast ShkG, from Arabidopsis thaliana, (V) sequence encoding glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase, from Agrobacterium sp strain CP4, (VI) target miRNA specific for male plant tissues, from Zea mays, and ( VII) 3'-UTR of glycine-rich RNA-binding protein from Oryza sativa.

The invention provides a DNA molecule having a sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as a DNA probe specific for SEQ ID NO: 10 in a DNA sample obtained from a corn plant, corn seed or corn cell. In one embodiment, the DNA probe contains SEQ ID NO: 13.

The invention provides a pair of DNA molecules comprising a first DNA molecule and a second DNA molecule, wherein the first and second DNA molecules contain the fragment SEQ ID NO: 10 and function as DNA primers when used together in an amplification reaction with DNA containing the maize transformant MON87429 , to obtain an amplicon that allows diagnosing the maize transformant MON87429 in the sample. In one embodiment, the at least one DNA primer contains a fragment of a sequence selected from the group consisting of SEQ ID NO: 7 and SEQ ID NO: 8. In another embodiment, the first DNA molecule contains SEQ ID NO: 11, and the second DNA molecule contains SEQ ID NO: 12.

The invention provides a method for detecting the presence of the corn transformant MON87429 in a DNA sample obtained from a corn plant, corn seed or corn cell, the method comprising: contacting the sample with a DNA probe; subjecting the sample and DNA probe to stringent hybridization conditions and detecting hybridization of the DNA probe with a DNA molecule in the sample, wherein hybridization of the DNA probe with the DNA molecule indicates the presence of the maize transformant MON87429 in the DNA sample.

The invention provides a method for detecting the presence of the corn transformant MON87429 in a DNA sample obtained from a corn plant, a corn seed or a corn cell, the method comprising: contacting the sample with a pair of DNA molecules that function as DNA primers; performing an amplification reaction sufficient to produce a DNA amplicon containing a sequence selected from the group consisting of 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; and detecting the presence of the DNA amplicon, wherein the presence of the DNA amplicon indicates the presence of the maize transformant MON87429 in the sample.

The invention provides a method for detecting the presence of the maize transformant MON87429 in a sample obtained from a maize plant, a maize seed or a maize cell, the method

- 2 044838 includes: contacting the sample with at least one antibody specific for at least one or more proteins encoded by the maize transformant MON87429; and detecting binding of the antibody to a protein in the sample, wherein binding of the antibody to the protein indicates the presence of the maize transformant MON87429 in the sample. In another embodiment, the method includes further contacting the sample with at least a second antibody specific for a second protein encoded by the maize transformant MON87429. In another embodiment, the method includes further contacting the sample with at least a second antibody and a third antibody specific for a second protein and a third protein, respectively, encoded by the maize transformant MON87429. In another embodiment, the method includes further contacting the sample with at least a second antibody, a third antibody, and a fourth antibody specific for a second protein, a third protein, and a fourth protein, respectively, encoded by the maize transformant MON87429.

The invention provides a kit for detecting the presence of the maize transformant MON87429, containing a DNA probe specific for SEQ ID NO: 10, a pair of DNA primers that produce an amplicon allowing the diagnosis of the maize transformant MON87429, or at least one antibody specific for at least one protein encoded by the maize transformant MON87429.

The invention provides a plant, seed, cell, plant part or commodity containing a DNA molecule containing 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, SEQ ID NO: 9, and SEQ ID NO: 10. In one embodiment, the plant, seed, cell, or plant part is tolerant to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, or any combination of them. In another embodiment, the plant, seed, cell or plant part is tolerant to chisalofop, haloxyfop, dicamba, 2,4-D and glufosinate.

The invention provides a method for controlling weed growth in a crop growing area, comprising planting corn containing the corn transformant MON87429 in the crop growing area and applying to the crop growing area, or any portion thereof, an effective amount of at least one herbicide, selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, or any combination thereof, to control weed growth in this area without harming the corn. In one embodiment, the method includes the use of at least two or more herbicides selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors, and 5enolpyruvylshikimate-3 inhibitors -phosphate synthase (EPSPS), for the harvest season. In another embodiment, the method includes using a herbicide selected from the group consisting of chisalofop, haloxyfop, dicamba, 2,4-D, glufosinate and glyphosate, or any combination thereof. In one embodiment, the effective amount of dicamba is from about 0.1 lb ae/acre to about 16 lb ae/acre per crop season. In one embodiment, the effective amount of dicamba is from about 0.5 lb ae/acre to about 2 lb ae/acre per crop season. In one embodiment, the effective amount of glufosinate is from about 0.1 lb ae/acre to about 16 lb ae/acre per crop season. In one embodiment, the effective amount of glufosinate is from about 0.4 lb ae/acre to about 1.59 lb ae/acre per crop season. In one embodiment, the effective amount of 2,4-D is from about 0.1 lb ae/acre to about 10 lb ae/acre per crop season. In one embodiment, the effective amount of 2,4-D is from about 0.75 lb ae/acre to about 1.0 lb ae/acre per crop season. In one embodiment, the effective amount of FOP herbicide is from about 0.01 lb ai/acre to about 1.0 lb ai/acre per crop season. In one embodiment, the effective amount of the FOP herbicide is from about 0.034 lbs ai/acre to about 0.083 lbs ai/acre of chisalofop per crop season. In one embodiment, the effective amount of the FOP herbicide is from about 0.018 lb ai/acre to about 0.07 lb ai/acre haloxyfop per crop season.

The invention provides a method of controlling self-seeded corn containing the maize transformant MON87429 in a field, comprising applying a herbicidal effective amount of at least one cyclohexanedione (DIM) herbicide, wherein the use of the herbicide prevents the growth of corn containing the corn transformant MON87429. In one embodiment, the cyclohexanedione (DIM) herbicide is selected from the group consisting of clethodim, sethoxydim and tralkoxydim.

The invention provides a method for producing a plant that is tolerant to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase inhibitors

- 3 044838 (ACC) in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors or any combination thereof, the method comprising: crossing a plant containing the maize transformant MON87429 , with itself or with a second plant to produce a seed and identifying the progeny seed that contains the maize transformant MON87429. In one embodiment, identification of a progeny seed that contains the maize transformant MON87429 occurs by germinating the progeny seed to produce progeny plants; treating the progeny plants with an effective amount of at least one herbicide selected from the group consisting of chisalofop, haloxyfop, dicamba, 2,4-D, glufosinate, glyphosate, or any combination thereof; and selecting a progeny plant that is tolerant to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5enolpyruvyl shikimate-3-phosphate inhibitors synthase (EPSPS). In one embodiment, identification of a progeny seed that contains the maize transformant MON87429 occurs by detecting the presence of the maize transformant MON87429 in a sample obtained from the progeny seed. In one embodiment, identification of a progeny seed that contains the maize transformant MON87429 occurs by detecting the presence of at least one protein encoded by the maize transformant MON87429 in a sample obtained from the progeny seed.

The invention provides a method for producing a hybrid seed, comprising: growing a plant containing SEQ ID NO: 10; applying an effective amount of glyphosate before or during the development of male reproductive organ tissue of the plant, thereby causing male sterility in the plant; fertilizing the plant with pollen from a second plant and collecting the hybrid seed from the plant. In one embodiment, glyphosate is applied before or during development at an effective amount of about 0.25 lb ae/acre to about 11.0 lb ae/acre. In one embodiment, glyphosate is applied before or during development in an effective amount of from about 0.5 lb ae/acre to about 2.5 lb ae/acre in the aggregate of one or more applications. In one embodiment, an effective amount of glyphosate is applied at a developmental stage selected from the group consisting of stages V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 of the corn plant development. The invention provides a hybrid seed containing SEQ ID NO: 10 and obtained by a method for producing a hybrid seed, comprising: growing a plant containing SEQ ID NO: 10; applying an effective amount of glyphosate before or during the development of male reproductive organ tissue of the plant, thereby causing male sterility in the plant; fertilizing the plant with pollen from a second plant and collecting the hybrid seed from the plant.

The invention provides a method for determining the zygosity of a plant for the maize transformant MON87429, which includes: contacting a sample containing DNA obtained from the plant with a set of primers capable of producing a first amplicon that allows diagnosing the presence of the maize transformant MON87429, and a second amplicon that allows the diagnosis wild-type maize genomic DNA not containing the maize transformant MON87429; carrying out a nucleic acid amplification reaction; detection of the first amplicon and the second amplicon, the presence of both amplicons indicating that the sample is heterozygous for the maize transformant MON87429, and the presence of only the first amplicon indicating that the sample is homozygous for the maize transformant MON87429. In one embodiment, the primer set comprises SEQ ID NO: 11 and SEQ ID NO: 12.

The invention provides a method for increasing tolerance to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvyl shikimate-3-phosphate inhibitors synthase (EPSPS), or any combination thereof, in a maize plant, comprising: (a) obtaining a DNA construct containing four expression cassettes, wherein the first expression cassette contains in functional linkage (I) a promoter, a leader and a ubiquitin intron from Erianthus ravennae, (II) the sequence encoding phosphinothricin N acetyltransferase, and (III) the 3'-UTR of fructose bisphosphate aldolase from Setaria italica; the second expression cassette contains in a functional connection (I) the promoter, leader and ubiquitin intron from Coix lacryma-jobi, (II) the sequence encoding the albino and pale green 6 chloroplast transit peptide from Arabidopsis thaliana, (III) the sequence encoding dicamba- monooxygenase, and (IV) 3'UTR of metallothionein-like protein from Oryza sativa; the third expression cassette contains in functional linkage (I) the ubiquitin promoter, leader and intron from Arundo donax, (II) the sequence encoding the chloroplast transit peptide of malate dehydrogenase from Arabidopsis thaliana, (III) the sequence encoding the FTT protein, and (IV) 3' -UTR of non-apical meristem protein from Oryza sativa; and the fourth expression cassette contains in functional connection (I) the promoter and leader of CaMV 35S, (II) the leader of chlorophyll a/b-binding protein from Triticum aestivum, (III) actin 1 intron from Oryza sativa, (IV) a sequence encoding a transit peptide chloroplast ShkG, from Arabidopsis thaliana, (V) sequence encoding glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase, from

- 4 044838 strain CP4 of Agrobacterium sp, (VI) siRNA target specific to male plant tissues from Zea mays, and (VII) 3'-UTR of a glycine-rich RNA-binding protein from Oryza sativa; (b) inserting a DNA construct into the genome of a maize cell; (c) regenerating a corn cell into a corn plant; and (d) selecting a corn plant containing the DNA construct. In one embodiment, selection occurs by treating the corn plant with an effective amount of at least one herbicide selected from the group consisting of chisalofop, haloxyfop, dicamba, 2,4-D, glufosinate, or glyphosate. In one embodiment, the invention provides a corn plant, corn seed or corn cell tolerant to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, or any combination thereof, and obtained by a method in which a corn plant, corn seed or corn cell contains a DNA construct. In a further embodiment, a corn plant, corn seed, or corn cell obtained by this method comprises SEQ ID NO: 10.

Brief description of graphic materials

In fig. Figure 1 shows the sequence of the maize transformant MON87429. 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 SQ51062 (SEQ ID NO: 11) and SQ51053 (SEQ ID NO: 12) indicate the approximate position of a pair of primers that can be used to detect the maize transformant MON87429; and the horizontal line designated PB50370 (SEQ ID NO: 13) shows the approximate position of a DNA probe that can be used to detect the maize transformant MON87429.

In fig. 2 shows four expression cassettes of the maize transformant MON87429 relative to SEQ ID NO: 9 with their corresponding genetic elements designated as described in table. 1.

In fig. 3 shows the construction, testing, characterization and selection of the MON87429 transformant as described herein. All times are approximate.

Brief description of sequences

SEQ ID NO: 1 is a thirty nucleotide DNA sequence representing the 5' junction of maize genomic DNA and the transgenic insert. SEQ ID NO: 1 corresponds to nucleotide positions 1015 to 1044 in SEQ ID NO: 10.

SEQ ID NO: 2 is a thirty nucleotide DNA sequence representing the 3' junction of maize genomic DNA and the transgenic insert. SEQ ID NO: 2 corresponds to nucleotide positions 15023 to 15052 in SEQ ID NO: 10.

SEQ ID NO: 3 is a sixty nucleotide DNA sequence representing the 5' junction of maize genomic DNA and the transgenic insert. SEQ ID NO: 3 corresponds to nucleotide positions 1000 to 1059 in SEQ ID NO: 10.

SEQ ID NO: 4 is a sixty nucleotide DNA sequence representing the 3' junction of maize genomic DNA and the transgenic insert. SEQ ID NO: 4 corresponds to nucleotide positions 15008 to 15067 in SEQ ID NO: 10.

SEQ ID NO: 5 is a one hundred nucleotide DNA sequence representing the 5' junction of maize genomic DNA and the transgenic insert. SEQ ID NO: 5 corresponds to nucleotide positions 980 to 1079 in SEQ ID NO: 10.

SEQ ID NO: 6 is a one hundred nucleotide DNA sequence representing the 3' junction of maize genomic DNA and the transgenic insert. SEQ ID NO: 6 corresponds to nucleotide positions 14988 to 15087 in SEQ ID NO: 10.

SEQ ID NO: 7 is a 1350 nucleotide DNA sequence representing 1029 nucleotides of the 5' flanking maize genomic DNA and 321 nucleotides at the 5' end of the transgenic insert.

SEQ ID NO: 8 is a 1069 nucleotide DNA sequence representing 38 nucleotides at the 3' end of the transgenic insert and 1031 nucleotides 3' flanking maize genomic DNA.

SEQ ID NO: 9 - DNA sequence of 14008 nucleotides corresponding to the transgenic insert of the maize transformant MON87429.

SEQ ID NO: 10 - DNA sequence of 16068 nucleotides corresponding to the maize transformant MON87429; the sequence contains a 5' flanking genomic DNA sequence at positions 1 to 1029, a transgenic DNA insert at positions 1030 to 15037, and a 3' flanking genomic DNA sequence at positions 15038 to 16068.

SEQ ID NO: 11 is the 29 nucleotide DNA sequence corresponding to the primer referred to as SQ51062 and used to identify the DNA of the maize transformant MON87429 in the sample; it corresponds to positions 15038 to 15066 in SEQ ID NO: 10.

SEQ ID NO: 12 is the 17 nucleotide DNA sequence corresponding to the primer designated SQ51053 and used to identify the DNA of the maize transformant MON87429 in the sample; it corresponds to positions 14987 to 15003 in SEQ ID NO: 10.

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SEQ ID NO: 13 is a 16 nucleotide DNA sequence corresponding to a probe called PB50370 used to identify the DNA of the maize transformant MON87429 in the sample;

it corresponds to positions 15009 to 15024 in SEQ ID NO: 10.

Detailed description of the invention

The following definitions and methods are presented to better describe the invention and to instruct those skilled in the art in putting the present invention into practice. Unless otherwise indicated, those skilled in the art should understand the terms in accordance with common usage.

Plant transformation techniques are used to insert foreign DNA (also known as transgenic DNA) randomly into the chromosome of a cell's genome to produce a genetically engineered cell, also called a transgenic or recombinant cell. Using this technique, many individual cells are transformed and each transformation results in a unique transgenic transformant due to the random insertion of foreign DNA into the genome. The transgenic plant is then regenerated from each individual transgenic cell. This results in each transgenic plant cell containing a uniquely inserted transgenic transformant as a stable part of its genome. This transgenic plant can then be used to produce progeny plants, each of which will contain a unique transgenic transformant. The maize transformant MON87429 was generated by: (i) transforming thousands of maize cells with a DNA construct that contains four expression cassettes (each expression cassette was selected by individual testing followed by testing in combination with the other three expression cassettes), (ii) population regeneration transgenic plants, each containing a unique transgenic transformant, and (iii) rigorous multi-year selection of transformants, including testing and analysis of the molecular properties, herbicide tolerance efficiency and agronomic properties of various background genotypes of thousands of transformants among tens of thousands of plants. Thus, maize transformant MON87429 was obtained and selected as a unique superior transformant suitable for large-scale commercial agronomic applications.

As used herein, a transgenic transformant or transformant is a DNA molecule created by inserting a transgenic DNA molecule into the genomic DNA of a plant cell using plant transformation methods known in the art. Such an insertion creates a new transgenic genomic DNA sequence that consists of the inserted foreign DNA (called the transgenic insert) and the genomic DNA located immediately next to the transgenic insert (or flanking the transgenic insert) on either side of the insertion site (called the flanking DNA). The DNA sequence of a transformant is unique and specific to that transformant and can be readily identified by comparison with other DNA sequences, such as those of other transformants or untransformed maize genomic DNA. The maize transformant MON87429 has a novel and unique DNA sequence shown as SEQ ID NO: 10, which contains the transgenic insert sequence shown as SEQ ID NO: 9, and 5' and 3' flanking DNA sequences shown as SEQ ID NO: : 7 and SEQ ID NO: 8, respectively. Thus, the maize transformant MON87429 is a DNA molecule that is an integral part of the chromosome of transgenic maize cells and plants containing this transformant, and is therefore static and can be transmitted to progeny cells and plants.

The present invention also provides progeny of the original transformed cell and plant that contain the maize transformant MON87429. Such progeny can be obtained from tissue culture cells, by self-fertilization of a corn plant containing the corn transformant MON87429, or by sexually crossing a corn plant containing the corn transformant MON87429 with another plant that may or may not contain this transformant. Such other plant may be a transgenic plant containing similar or different transformant(s), or a non-transgenic plant, such as a plant of a different species. The maize transformant MON87429 is passed on to the offspring from the original parent every generation.

As used herein, the term corn refers to Zea mays (also called maize) and covers all plant species that can be crossed with Zea mays.

The invention provides a maize transformant MON87429, which confers tolerance to acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, such as chisalofop and haloxyfop, to the cells, plants and seeds of maize that contain this transformant; synthetic auxins such as dicamba and 2,4-D; glutamine synthetase inhibitors such as glufosinate; and the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor glyphosate. The maize transformant MON87429 contains four expression cassettes. As used herein, an expression cassette or cassette is a recombinant DNA molecule containing a combination of individual elements to be expressed by the transformed cell. In table 1 provides a list of elements contained in SEQ ID NO:

10, as shown in FIG. 2.

Table 1

Description of maize transformant MON87429

Element Position in SEQ ID NO: 10 Description 5' flanking DNA 1-1029 DNA sequence flanking the 5' end of the transgene insert Left area borders 1030-1288 Region of DNA from Agrobacterium tumefaciens containing the left border sequence Intermediate sequence 1289-1359 Sequence used when DNA cloning V-Ea.ubq 1360-3541 Sequences of the promoter, 5'-UTR and intron of the ubiquitin gene from Erianthus ravennae Intermediate sequence 3542-3546 Sequence used when DNA cloning CS-Sv.pat 3547-4098 Coding sequence for phosphinothricin N-acetyltransferase (PAT) Ύ-Si.fba 4099-4475 Sequence of the 3'-UTR gene Fructose bisphosphate aldolase from Setaria italica Intermediate sequence 4476-4537 Sequence used when DNA cloning V-Clj.ubq 4538-6463 Sequences of the promoter, 5'-UTR and intron of the ubiquitin gene from Coix lacryma-jobi Intermediate sequence 6464-6473 Sequence used when DNA cloning

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TS-At.apg6 6474-6677 Codon-optimized targeting albino and pale green 6 gene sequence from Arabidopsis thaliana CS-Sm.dmo 6678-7700 Coding sequence for dicamba monooxygenase (DMO) protein Intermediate sequence 7701-7708 Sequence used when DNA cloning VOs.mt 7709-8008 Sequence 3'-UTR metal othionein- similar protein from Oryza sativa Intermediate sequence 8009-8016 Sequence used when DNA cloning P-Ad.ubq 8017-9973 Sequences of the promoter, 5'-UTR and intron of the ubiquitin gene from Arundo donax Intermediate sequence 9974-9986 Sequence used when DNA cloning TS-At.mdh 9987-10229 Sequence of the transit peptide of the malate dehydrogenase gene from Arabidopsis thaliana CS-Sh.ftt 10230-11117 Coding sequence for the FT T protein Intermediate sequence 11118-11132 Sequence used when DNA cloning Ί-Os.pat 11133-11649 Sequence of the 3'-UTR of the non-apical meristem protein from Oryza sativa Intermediate sequence 11650-11655 Sequence used when DNA cloning P-CaMF.35S 11656-11979 Promoter and leader from cauliflower mosaic virus 35S RNA Intermediate sequence 11980-12001 Sequence used when DNA cloning

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E-Ta.cab 12002-12062 Leader sequence of 5'-UTR from chlorophyll a/b-binding protein from Triticum aestivum Intermediate sequence 12063-12078 Sequence used when DNA cloning 1-0 s. gact 1 12079-12558 Intron and UTR sequence of actin 1 protein from Oryza sativa Intermediate sequence 12559-12567 Sequence used when DNA cloning TS-At.CTP2 12568-12795 Sequence of the transit peptide of the ShkG gene from Arabidopsis thaliana CS-cp4epsps 12796-14163 Coding sequence for the protein 5enolpyruvylshikimate-3-phosphate synthase (CP4EPSPS) Intermediate sequence 14164-14169 Sequence used when DNA cloning mts-miRNA 14170-14370 Sequence of target miRNA specific to male plant tissues Intermediate sequence 14371-14378 Sequence used when DNA cloning Ί-Os.grpS 14379-14989 Sequence of the 3'-UTR of the glycine-rich RNA-binding protein (Grp3) gene from Oryza sativa Intermediate sequence 14990-15030 Sequence used when DNA cloning Right border area 15031-15037 Region of DNA from Agrobacterium tumefaciens containing the right border sequence 3' flanking 15038-16068 Flanking DNA DNA

As used herein, the term recombinant refers to non-natural DNA, protein or organism that is not normally found in nature and has been created through human intervention. As used herein, a recombinant DNA molecule is a DNA molecule containing a combination of DNA molecules that do not naturally occur together and is the result of human intervention, for example, a DNA molecule that contains a combination of at least two DNA molecules that are heterologous with respect to to each other, such as the DNA molecule that contains the transgene and the plant genomic DNA adjacent to the transgene. An example of a recombinant DNA molecule is a DNA molecule containing at least one sequence selected from SEQ ID NO: 1-10. As used herein, a recombinant plant is a plant that does not normally exist in nature, that is the result of human intervention, and that contains a transgenic DNA molecule. As a result of this genomic change, the recombinant plant is something new and clearly different from its wild-type relative. An example of a recombinant plant is a maize plant containing the maize transformant MON87429.

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As used herein, the term transgene refers to a DNA molecule artificially introduced into the genome of an organism as a result of human intervention, for example, through plant transformation techniques. The transgene may be heterologous to the organism. The term transgenic insertion as used herein refers to foreign DNA inserted through plant transformation techniques into the maize genome to produce the maize transformant MON87429. The sequence for the transgenic insert of the maize transformant MON87429 is provided as SEQ ID NO: 9. The term transgenic refers to anything containing a transgene, for example, the term transgenic plant refers to a plant containing a transgene.

As used herein, the term heterologous refers to a first molecule that is not typically associated with a second molecule or organism in nature. For example, a DNA molecule may come from a first species and be inserted into the genome of a second species. The DNA molecule will thus be heterologous with respect to the genome and the organism.

As used herein, the term chimeric refers to a single DNA molecule produced by fusing a first DNA molecule with a second DNA molecule, wherein neither the first nor the second DNA molecule typically occurs in a similar configuration when fused to another molecule. A chimeric DNA molecule is therefore a new DNA molecule that is not normally found in nature. An example of a chimeric DNA molecule is a DNA molecule containing at least one sequence selected from SEQ ID NO: 1-10.

As used herein, the term isolated refers to the separation of a molecule from other molecules that are normally associated with it in its native or natural state. Thus, the term isolated may refer to a DNA molecule that has been separated from the other DNA molecule(s) with which it is associated in its native or natural state. Such a DNA molecule may be present in a recombinant state, for example, as a recombinant DNA molecule. Thus, a DNA molecule removed from its natural state and fused with another DNA molecule with which it is not normally associated would be an isolated DNA molecule. Such an isolated DNA molecule can be obtained by using biotechnological techniques such as the creation of recombinant DNA or the integration of foreign DNA into the chromosome of a cell, plant or seed.

The invention provides DNA molecules and their corresponding DNA sequences. As used herein, the terms DNA and DNA molecule refer to the deoxyribonucleic acid (DNA) molecule. The DNA molecule can be genomic or synthetic in origin and typically runs from the 5' (upper) end to the 3' (lower) end. As used herein, the term DNA sequence refers to the nucleotide sequence of a DNA molecule. The nomenclature used complies with the requirements of 37 CFR § 1.822 and is set out in the tables of WIPO standard ST.25 (1998), appendix 2, table. 1 and 3. By convention, the DNA sequences of the invention and fragments thereof are described with reference to only one strand of two complementary strands of the DNA sequence. It is the spirit or intent of complementary sequences of the sequences provided herein (complementary strand sequences), also referred to in the art as reverse complementary sequences, that are within the scope of the present invention and are expressly intended to be encompassed within the scope of the claimed subject matter. Thus, as used herein, references to SEQ ID NO: 1-10 and fragments thereof include and refer to the complementary strand sequence and fragments thereof.

As used herein, the term fragment refers to a smaller piece of a whole. For example, fragments of SEQ ID NO: 10 will include sequences that are at least about 10 contiguous 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 contiguous nucleotides, at least about 40 contiguous nucleotides, at least about 45 contiguous nucleotides, at least about 50 consecutive nucleotides, at least about 60 consecutive nucleotides, at least about 70 consecutive nucleotides, at least about 80 consecutive nucleotides, at least about 90 consecutive nucleotides, or at least about 100 consecutive nucleotides from the complete sequence SEQ ID NO: 10 .

The DNA sequence for the maize transformant transgenic insert MON87429 is provided as SEQ ID NO: 9. DNA sequence of the transgenic insert and flanking maize genomic DNA

- 10 044838 each side of the transgenic insert, presented as SEQ ID NO: 10. The DNA sequences of the part of the flanking DNA and the 5' end of the transgenic insert are presented as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID

NO: 5 and SEQ ID NO: 7. The DNA sequences of the portion of the flanking DNA and the 3' end of the transgene insert are shown as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8.

The DNA sequence of the region attributable to the phosphodiester linkage of one end of the transgenic insert to the flanking maize genomic DNA is referred to herein as a junction. The junction is the point where the transgene insert binds to the flanking DNA into one continuous molecule. One junction is at the 5' end of the transgenic insert and the other junction is at the 3' end of the transgene insert, referred to herein as the 5' and 3' junctions, respectively. A junction sequence refers to a DNA sequence of any length that spans the 5' or 3' junction of a transformant. The junction sequences of the maize transformant MON87429 will be apparent to one skilled in the art using SEQ ID NO: 10. Examples of the junction sequences of the corn transformant MON87429 are provided as SEQ ID NO: 1-8. In fig. 1 illustrates the physical arrangement of SEQ ID NO: 1-10 located 5' to 3'. The maize transformant MON87429 junction sequences may be present as part of the genome of the plant, seed, or cell containing the maize transformant MON87429. Identification of any one or more of SEQ ID NO: 1-8 or 10 in a sample obtained from a plant, plant part, seed or cell indicates that the DNA was obtained from corn containing the corn transformant MON87429 and allows diagnosis of the presence of the transformant corn MON87429.

The plants, seeds, cells, plant parts and products of the invention can be used to detect DNA or protein molecules indicative of the presence of the maize transformant MON87429. Examples of DNA molecules are provided that can be used as primers or probes to detect the presence of the maize transformant MON87429 in a sample. Such primers or probes are specific for the target nucleic acid sequence and are thus useful for identifying the maize transformant MON87429 by the methods described herein. Detection of the presence of the maize transformant MON87429 can be accomplished by methods known in the art, such as thermal nucleic acid amplification or nucleic acid hybridization techniques (such as Northern blotting and Southern blotting).

A primer is a DNA molecule designed for use in annealing or hybridization methods that involve an amplification reaction. An amplification reaction is an in vitro reaction that amplifies a DNA template to produce an amplicon. As used herein, an amplicon is a DNA molecule that has been synthesized using amplification techniques. The amplicons of the invention contain a DNA sequence containing one or more of SEQ ID NOs: 1-10 or fragments thereof. A pair of primers can be used with a DNA template, such as a corn genomic DNA sample, in an amplification reaction, such as a polymerase chain reaction (PCR), to produce an amplicon, wherein the resulting amplicon will have a DNA sequence corresponding to the sequence of the DNA template located between the two sites. where the primers hybridized to the template. The primer is typically designed to hybridize to a target complementary DNA strand to form a hybrid between the primer and the target DNA strand. The presence of a primer is the recognition point for the polymerase to begin extending the primer using the target DNA strand as a template. Primer pairing refers to the use of two primers linking opposite strands of a double-stranded nucleotide segment to amplify the nucleotide segment between them. Examples of primer sequences are provided as SEQ ID NO: 11 (SQ51062) and SEQ ID NO: 12 (SQ51053). The primer pairs shown as SEQ ID NO: 11 and SEQ ID NO: 12 are used as a first DNA molecule and a second DNA molecule, wherein the first DNA molecule is a fragment of the DNA sequence SEQ ID NO: 10 of the transgenic insert, and the second DNA molecule is a fragment of the flanking DNA sequence of SEQ ID NO: 10, and both are of sufficient length to function as DNA primers when used together in an amplification reaction with DNA containing the maize transformant MON87429 to produce an amplicon capable of diagnosing the maize transformant MON87429 in the sample. The primer pairs of the present invention may, in certain embodiments, be defined as those that comprise a first and a second DNA molecule, wherein the first DNA molecule is a fragment of the maize genomic portion of SEQ ID NO: 10 and the second DNA molecule is a fragment of the transgenic portion of SEQ ID NO: 10 : 10, and both are of sufficient length to function as DNA primers when used together in an amplification reaction with DNA containing the maize transformant MON87429 to produce an amplicon capable of diagnosing the maize transformant MON87429 in a sample.

A probe is a nucleic acid molecule that is complementary to a target nucleic acid strand and is used in hybridization detection methods. Probes in accordance with the invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that specifically bind to a target DNA sequence, and detection of such binding may be useful in detecting the presence or absence of a target DNA sequence. The probe may be attached to a conventional detectable label or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent agent or enzyme. An example of a DNA sequence useful as a probe for the detection of the maize transformant MON87429 is provided as SEQ ID NO: 13 (PB50370).

Methods for designing and using primers and probes are well known in the art. DNA molecules containing full length or fragments of SEQ ID NO: 1-10 are useful as primers and probes for the detection of the maize transformant MON87429 and can be easily designed by one of ordinary skill in the art using the sequences provided herein.

The probes and primers of the invention may have complete sequence identity to the target sequence, although primers and probes different from the target sequence that retain the ability to hybridize preferentially to the target sequences can be designed using conventional techniques. For a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to be able to form a stable double-stranded structure at the particular solvent and salt concentrations used. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of transgenic DNA from the maize transformant MON87429 in a sample. Probes and primers are typically at least about 11 nucleotides, at least about 18 nucleotides, at least about 24 nucleotides, or at least about 30 nucleotides in length. Such probes and primers specifically hybridize to the target DNA sequence under stringent hybridization conditions. Typical stringency conditions are described in MR Green and J Sambrook, Molecular cloning: a laboratory manual, ^4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012). As used herein, two nucleic acid molecules are capable of specifically hybridizing with each other if the two molecules are capable of forming an antiparallel double-stranded nucleic acid structure. A nucleic acid molecule is the complement of another nucleic acid molecule if they exhibit complete complementarity. As used herein, two molecules exhibit complete complementarity if, in an alignment, every nucleotide of the first molecule is complementary to every nucleotide of the second molecule. Two molecules are minimally complementary if they can hybridize to each other sufficiently stably to allow them to remain annulated to each other at least under normal low stringency conditions. Likewise, molecules are complementary if they can hybridize to each other sufficiently stably to allow them to remain annulated to each other under normal high stringency conditions. Deviations from complete complementarity are therefore acceptable as long as such deviations do not completely prevent the molecules from forming a double-stranded structure.

Appropriate stringency conditions that promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C followed by a wash in 2.0 x SSC at 50°C, are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. For example, the salt concentration in the washing step can be selected ranging from a low hardness of about 2.0 x SSC at 50°C to a high hardness of about 0.2 x SSC at 50°C. In addition, the temperature of the washing step can be increased ranging from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. You can vary both the temperature and the salt concentration, or you can hold one of the temperature or the salt concentration constant and change the other value.

Proteins are proposed that can be used to generate antibodies to detect the presence of the maize transformant MON87429 in a sample. Such antibodies are specific for one or more proteins that are encoded by the maize transformant MON87429. The DNA sequence encoding such proteins is presented as SEQ ID NO: 10, and the starting positions and ending positions of the coding sequence are indicated in table. 1. The DNA sequence encoding each protein and the protein encoded by the sequence are used to generate antibodies to detect the presence of the maize transformant MON87429 by the methods described herein. Detection of the presence of the maize transformant MON87429 can be accomplished using any protein detection techniques known in the art, such as Western blotting, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), coupling of an antibody to a detectable label or reporter molecule (such as a radioactive isotope, ligand, chemiluminescence). 12 044838 cent agent or enzyme) or enzymatic activity on a reporter molecule. One method involves contacting a sample with an antibody that binds to the DMO, PAT, FT_T, or CP4-EPSPS protein encoded by the maize transformant MON87429, and then detecting the presence or absence of antibody binding. The binding of such an antibody allows the presence of one or more proteins encoded by the maize transformant MON87429 to be diagnosed.

Protein and nucleic acid detection kits have been proposed to detect the presence of the maize transformant MON87429. Variations of such kits can also be developed using the compositions and methods described herein, as well as methods well known in the field of protein and nucleic acid detection. Protein and nucleic acid detection kits can be used in crossbreeding methods with plants containing the maize transformant MON87429. Such kits contain primers or probes containing fragments of SEQ ID NO: 1-10 or antibodies specific for the protein encoded by the maize transformant MON87429.

One example of a detection kit includes at least one DNA molecule having a sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as a DNA probe useful for detecting the presence or absence of the maize transformant MON87429 in a sample. An example of a DNA molecule that can be used as a probe is one containing the sequence shown as SEQ ID NO: 13. Other probes can be easily designed by one skilled in the art. Another example of a detection kit includes at least one pair of primers used to generate an amplicon useful for detecting the presence or absence of the maize transformant MON87429 in a sample. Such a method may also include sequencing the amplicon or a fragment thereof. Examples of DNA molecules that can be used as a primer pair are those containing the sequences shown as SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Other primer pairs can be easily designed by one skilled in the art. The kits of the invention may also optionally contain reagents to perform the detection or diagnostic reactions described herein. Another example of a detection kit includes at least one antibody specific for at least one protein encoded by the maize transformant MON87429. For example, such a kit may use a lateral diffusion test strip containing reagents that are activated when the tip of the strip comes into contact with an aqueous solution. Examples of proteins that can be used to produce antibodies are those encoded by the sequence shown in SEQ ID NO: 10, or any fragment thereof.

The invention provides corn plants, its progeny, seeds, cells and plant parts containing the corn transformant MON87429, as well as products that are produced using them. The plants, progeny, seeds, cells, plant parts and products of the invention contain a detectable amount of DNA containing at least one of the sequences set forth in SEQ ID NO: 1-8 and SEQ ID NO: 10.

The plants, progeny, seeds, cells and plant parts of the invention may also contain one or more additional transgenic traits, particularly those introduced by crossing a maize plant containing the maize transformant MON87429 with another plant containing the additional transgenic trait(s). Such traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield, increased drought tolerance, increased seed quality, increased nutritional value, hybrid seed production, and/or increased herbicide tolerance, where the trait is evaluated in relation to the corn plant , which does not have such a transgenic trait.

Plants of the invention can be used to produce progeny that contain the maize transformant MON87429. As used herein, progeny includes any plant, seed, and cell containing a maize transformant MON87429 inherited from a progenitor plant, as indicated by a plant containing a DNA molecule having at least one sequence selected from SEQ ID NO: 1-8 and SE ID NO: 10. Plants, seeds and cells may be homozygous or heterozygous for the maize transformant MON87429. Progeny plants can be grown from seeds obtained from a corn plant containing the corn transformant MON87429, or from seeds obtained from a corn plant fertilized with pollen containing the corn transformant MON87429.

As used herein, a plant part of the invention is any part of a plant containing the maize transformant MON87429. Plant parts include, but are not limited to, tissue samples, pollen, ovule, pod, flower, roots, stems, fibers, and leaves in whole or in parts. Plant parts may be viable or non-viable.

The invention provides a product that is produced from plants containing the maize transformant MON87429. The products of the invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NOs: 1-10. As used herein, commodity refers to any composition or product that consists of material derived from a plant, seed, cell or plant part containing the maize transformant MON87429. Products include, but are not limited to, processed seeds, grains, plant parts and flour. The product of the invention will contain a detectable amount of DNA corresponding to the maize transformant MON87429. The detection of one or more of these DNAs in a sample can be used to determine the composition or origin of a product. Any standard method for detecting DNA molecules can be used, including the detection methods described herein.

The maize transformant MON87429 contains four expression cassettes that together confer tolerance to acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group; synthetic auxins; glutamine synthetase inhibitors and 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors.

As used herein, acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group (referred to as OPP herbicides) include, but are not limited to, clodinafop, clodinafop-ethyl, clodinafop-propargyl, cyhalofop, cyhalofop-butyl, diclofop , diclofop-methyl, diclofop-R, diclofop-R-methyl, fenoxaprop, fenoxaprop-R, fenoxaprop-R-ethyl, fentiaprop, fluazifop, fluazifop-butyl, fluazifop-R, fluazifop-R-butyl, fluroxypyr, haloxyfop, haloxyfop -ethyl, haloxyfop-methyl, haloxyfop-R, haloxyfop-R-methyl, isoxapyrifop, metamifop, propaquizafop, chisalofop, chisalofop-ethyl, chisalofop-R, chisalofop-R-ethyl, chisalofop-R-tefuryl and trifop.

As used herein, synthetic auxins include, but are not limited to, benzoic acid herbicides, phenoxy acid herbicides, aryl picolinate herbicides, and pyridinyloxy acid herbicides. Examples of benzoic acid herbicides include, but are not limited to, dicamba (3,6-dichloro-2-methoxybenzoic acid), dicamba salts, dicamba butothyl, dicamba diglycolamine salt, dicamba dimethyl ammonium, dicamba diethanolammonium, dicamba isopropylammonium, dicamba potassium, dicamba sodium and dicambatrolamine. Examples of phenoxy acid herbicides include 2,4-D (2,4dichlorophenoxyacetic acid), 2,4-O-butothyl, 2,4-O-butyl, 2,4-D-choline, 2,4-D-dimethylammonium, 2,4D-diolamine, 2,4-O-ethyl, 2,4-O-2-ethylhexyl, 2,4-O-isobutyl, 2,4-D-isoctyl, 2,4-D-isopropyl, 2, 4-Diisopropylammonium, 2,4-O-potassium, 2,4-O-sodium, 2,4-D-triisopropanolammonium, 2,4-O-trolamine, clomeprop, dichlorprop, phenoprop, MCPA, MCPA-butotyl, MCPA- dimethylammonium, MCPA-2-ethylhexyl, MCPA-isopropylammonium, MCPA-potassium, MCPA-sodium, MCPA-thioethyl, 2,4-DB, MCPB, MCPB-methyl, MCPA-ethyl-sodium and mecoprop. Examples of aryl picolinate herbicides are halauxifen, halauxifen-methyl and florpyrauxifen-benzyl. Examples of pyridinyloxy acid herbicides are triclopyr, fluroxypyr, aminopyralid, clopyralid and picloram.

As used herein, glutamine synthetase inhibitors include, but are not limited to, phosphinothricin, glufosinate, glufosinate salts, glufosinate ammonium, glufosinate sodium, glufosinate P, L-glufosinate ammonium, and L-glufosinate sodium.

As used herein, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors include, but are not limited to, glyphosate, glyphosate salts, glyphosate-isopropylammonium, glyphosate-ammonium, glyphosate-dimethylammonium, glyphosate-trimesium (=sulfosate), glyphosate- diammonium, glyphosate-potassium and glyphosate-sodium.

As used herein, herbicide tolerant, or herbicide tolerance, or tolerance means the ability to be completely or partially unaffected by the presence or use of one or more herbicides, for example, to resist the toxic effect of the herbicide when applied. A cell, seed, or plant is tolerant or enhanced tolerant to a herbicide if it is capable of maintaining at least certain normal growth or phenotype in the presence of one or more herbicides. A trait is indicative of herbicide tolerance if its presence is capable of conferring increased tolerance to the herbicide to a cell, plant or seed compared to a wild-type cell, plant or seed or a control cell, plant or seed. Crops that have a herbicide tolerance trait can continue to grow in the presence of the herbicide and such crops will be minimally affected by the presence of the herbicide. A protein will confer herbicide tolerance if expression of the protein can confer increased herbicide tolerance to a cell, plant or seed compared to a wild-type cell, plant or seed or a control cell, plant or seed. Examples of herbicide tolerance proteins include phosphinothricin N acetyltransferase, the sequence encoding dicamba monooxygenase, the FT_T protein, and the sequence encoding glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthase from strain CP4 of Agrobacterium sp. Herbicide tolerance may be complete or partial immunity to a particular herbicide and may be expressed as a percentage (%) of tolerance or immunity to a particular herbicide.

As used herein, a weed is any undesirable phenomenon. The plant may be considered undesirable in general for agriculture or crop production

- 14 044838 (eg Amaranthus spp.) or may be considered undesirable in a particular situation (eg crop of one species in a field with plants of another species, also known as self-sowing plant). Weeds are widely known in the art and vary depending on geographic area, season, growing conditions and time. Lists of weed species are available from agricultural and scientific societies and initiatives (such as the American Weed Science Society, the Canadian Weed Science Society, the Brazilian Weed Science Society, the International Weed Science Society, and the International Weed Resistance Research Society). weed killer), government agencies (such as the US Department of Agriculture and the Australian Department of Environment and Energy), and industry and farming associations (such as the National Corn Growers Association).

The invention provides methods for controlling the growth of weeds in a corn crop by using at least one herbicide selected from the group consisting of (i) acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, such as chisalofop and haloxyfop ; (ii) synthetic auxins such as dicamba and 2,4-D; (iii) glutamine synthetase inhibitors such as glufosinate; and (iv) a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor glyphosate, wherein seeds or plants containing the corn transformant MON87429 are cultivated on the site before, during or after application of the herbicide, and application of the herbicide prevents or inhibits the growth of weeds without harming corn plants. The growing area may or may not have weeds at the time the herbicide is applied. The herbicide(s) used in the methods of the invention may be used alone or in combination with one or more herbicides per crop season. The herbicide(s) used in the methods of the invention may be applied in combination with one or more herbicides in a temporal aspect (for example, as a tank mixture or by sequential application), in a spatial aspect (for example, at different points in time during the crop season , including the time before and after planting corn seeds) or both. For example, a method of controlling weed growth is provided that includes planting a seed containing the corn transformant MON87429 on a plot and applying a herbicidally effective amount of one or more of dicamba, glufosinate, glyphosate, 2,4D or FOP herbicide per crop season, alone or in any combination with another herbicide to control the growth of weeds on the site without harming plants containing the corn transformant MON87429. Such application of the herbicide(s) may occur preplanting (any time before planting seed containing the corn transformant MON87429, including for the purpose of complete eradication, i.e. application to emerging or already growing weeds before planting), preemergence (any time after planting the seed containing the maize transformant MON87429 and before the plants containing the maize transformant emerge) or after germination (any time after the plants containing the maize transformant MON87429 emerge). Several applications of one or more herbicides or a combination of herbicides together or separately are possible per crop season, for example, two applications (for example, a pre-planting application and a post-emergence application, or a pre-emergence application and a post-emergence application) or three or more applications (for example, application before planting and two applications after germination).

Application of the herbicide in the methods of the invention may be at the recommended commercial rate or any part or multiple thereof, for example, twice the recommended commercial rate. Herbicide application rates may be expressed as acid equivalent per pound per acre (lb e/ac) or active ingredient per pound per acre (lb ai/ac), depending on the herbicide and its formulation. The herbicide may be applied at the recommended commercial application rate or any part or multiple thereof. Use of acres at herbicide application rates as suggested herein is for informational purposes only; Herbicide application rates equivalent to any of the application rates proposed herein may be used on plots larger or smaller in area than an acre. The use of herbicides includes at least one herbicide selected from the group consisting of (i) acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, such as chisalofop and haloxyfop; (ii) synthetic auxins such as dicamba and 2,4-D; (iii) glutamine synthetase inhibitors such as glufosinate; and (iv) the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor glyphosate. The growing area may or may not have weeds at the time the herbicide is applied. The herbicidal effective amount of FOP herbicides used on a site to control weed growth should range from about 0.01 lb ai/acre to about 1.0 lb ai/acre per crop season (for example, chisalofop can be applied at a rate application rates from about 0.034 lb ai/acre to about 0.083 lb ai/acre, and haloxyfop can be applied at application rates from about 0.018 lb ai/acre to about 0.07 lb ai/acre). The herbicidally effective amount of synthetic auxins (phenoxy acids) that is used on site for weed control should range from about 0.1 lb ae/acre to about 10 lb ae/acre per crop season (e.g. 2,4-D can be applied at application rates from about 0.75 lb ae/acre to about 1.0 lb ae/acre). Herbicidal effective

- 15 044838 The amount of synthetic auxins (pyridinyloxy acids) used on site for weed control should range from about 0.05 lb ae/acre to about 5.0 lb ae/acre per crop season (e.g. fluroxypyr can be applied at application rates from about 0.14 lb ae/acre to about 0.49 lb ae/acre). The herbicidally effective amount of synthetic auxin (benzoic acid) that is applied to a site for weed control should range from about 0.1 lb ae/acre up to about 16 lb ae/acre per crop season (for example, dicamba can be used with application rates ranging from about 0.5 lb ae/acre to about 2.0 lb ae/acre). The herbicidal effective amount of glutamine synthetase inhibitors that are used on site to control weed growth should range from about 0.1 lb ae/acre to about 10 lb ae/acre per crop season (for example, glufosinate can be applied at a rate of about 0.4 lbs ai/acre to about 1.59 lbs ai/acre). The herbicidally effective amount of EPSPS inhibitors that are used on a site to control weed growth should range from about 0.5 lb ae/acre to about 12 lb ae/acre per crop season (for example, glyphosate can be applied at an application rate of about 0.75 lb ae/acre to about 2.25 lb ae/acre).

The invention provides methods for controlling self-sown corn containing the maize transformant MON87429 in a cultivation area by applying a herbicidal effective amount of at least one cyclohexanedione (DIM) herbicide, such as clethodim, sethoxydim and tralkoxydim, wherein the use of the herbicide prevents the growth of corn containing the corn transformant MON87429 . The herbicidally effective amount of DIM herbicide that is used on a site to control self-planted corn should range from about 0.03 lb ai/acre to about 2.75 lb ai/acre per crop season (for example, clethodim can be applied at a rate application rates from about 0.0625 lb ai/acre to about 0.125 lb ai/acre, and sethoxydim can be applied at application rates from about 0.188 lb ai/acre to about 0.281 lb ai/acre).

Methods have been proposed for obtaining plants and seeds containing the maize transformant MON87429. The plants can be bred by any method known in the art, for example, a description of propagation methods that are widely used can be found in W.R. Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison WI (1987). Plants can be self-pollinated (also called self-fertilized) or cross-pollinated (also called crossed). Plants containing the maize transformant MON87429 can self-pollinate to produce a pure line cross of plants that are homozygous for the maize transformant MON87429. Self-fertilization produces offspring called inbreds and can be used to produce inbred lines that are genetically the same. Alternatively, plants containing the maize transformant MON87429 can be cross-pollinated (crossed with another plant that is transgenic or non-transgenic) to produce varietal or hybrid seed. Seeds and progeny plants obtained by the methods of the invention contain the maize transformant MON87429. One or more herbicides tolerant to the corn transformant MON87429 can be used to select progeny that contain the corn transformant MON87429. Alternatively, the progeny can be analyzed using diagnostic methods to select plants or seeds containing the maize transformant MON87429. The offspring may be varietal or hybrid plants; can be grown from seeds obtained from a plant containing the maize transformant MON87429, or from seeds obtained from a plant fertilized with pollen from a plant containing the maize transformant MON87429; and may also be homozygous or heterozygous for the maize transformant MON87429.

Methods have been proposed for producing a hybrid seed using the maize transformant MON87429. Plants containing the maize transformant MON87429 exhibit expression of the glyphosate-tolerant protein CP4-EPSPS in all tissues except that of the male reproductive organs. This leads to the development of tolerance to glyphosate in vegetative tissues and tissues of the female reproductive organs, as well as sensitivity to glyphosate in the tissues of the male reproductive organs. Such sensitivity to glyphosate can be used to induce male sterility by appropriate application of glyphosate. Glyphosate is a systemic herbicide that translocates from its source into plant rhizome tissue. Based on the rate of glyphosate metabolism in corn, its application to plants containing the corn transformant MON87429 before male organ tissue has developed may interfere with pollen production, pollen shedding, or anther release. Such glyphosate-induced male sterility can be used to increase the efficiency of hybrid seed production, for example, by eliminating or reducing the need to physically castrate the corn plant used as the female of a given cross in the process of producing hybrid seeds.

The invention provides a method for producing a hybrid seed, comprising: (a) growing a plant containing the maize transformant MON87429, (b) applying to the plant an effective amount of glyphosate to induce male sterility, wherein the use of a herbicide

- 16 044838 occurs before or during the development of tissues of the male reproductive organs of the plant, thus causing male sterility in the plant; (c) fertilizing the plant with pollen from a second plant; and (d) collecting hybrid seeds from the plant. In one embodiment, glyphosate is applied before or during development in an effective amount of from about 0.25 lb ae/acre to about 11.0 lb ae/acre in the aggregate of one or more applications. In another embodiment, the fertilization step may occur by passive fertilization (eg, wind pollination) or by other methods such as mechanical pollination, hand pollination, or combinations thereof. Application of the herbicide may occur one or more times before or during the development of male organ tissue, for example, at a stage selected from the group consisting of stages V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 of corn plant development, and may inhibit at least pollen production, pollen shedding, or anther release. Male sterility can be partial or complete. In one embodiment, the effective amount of glyphosate will be from about 0.5 lb ae/acre to about 2.5 lb ae/acre in the aggregate (in one application or divided into two or more applications) and applied at stages V4 through V8 or up to 100 growing degree units (GDU) before flowering.

The plants, progeny, seeds, cells and plant parts of the invention may also contain one or more additional maize traits or transgenic transformants, particularly those introduced by crossing a maize plant containing the maize transformant MON87429 with another maize plant containing the additional traits or transgenic transformants . Such traits or transgenic transformants include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield, increased drought tolerance, increased seed quality, increased nutritional value, hybrid seed production, and herbicide tolerance, where the trait is evaluated in relation to the corn plant , which does not have such a transgenic trait. Transgenic maize transformants are known to one skilled in the art; for example, a list of such traits is offered by the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) and can be found on their website at www. aphis.usda.gov. Thus, two or more transgenic transformants can be combined in a progeny plant or seed by crossing two parent plants each containing one or more transgenic transformants, collecting the progeny seeds, and selecting the progeny seeds or plants that contain two or more transgenic transformants; these steps can then be repeated until the desired combination of transgenic transformants is obtained in the progeny. Backcrossing to the parent plant and outcrossing to a non-transgenic plant are also suggested, as is vegetative propagation.

A representative sample of seed containing the maize transformant MON87429 has been deposited under the Treaty of Budapest with the American Type Crop Collection (ATCC®) at 10801 University Boulevard, Manassas, Virginia, USA, Zip Code 20110. ATCC Patent Deposit Indication Number (Accession No. ) for seeds containing the maize transformant MON87429, PTA-124635, and the deposit date is January 12, 2018. The deposit will be kept in depository for 30 years, or 5 years from the date of last request, or for the duration of the patent, depending on what will last longer.

As used herein, the term comprising means including without limitation.

Examples

The following examples are included in order to more fully describe the invention. The creation and testing of thirty-five expression constructs, the production of over fifteen thousand unique transformants, and the analysis of hundreds of thousands of individual plants over seven years through the rigorous molecular, agronomic, and field testing required to create and ultimately select the maize transformant MON87429 are summarized.

Those skilled in the art will appreciate that many modifications can be made to the specific examples disclosed and still achieve a similar result. Certain agents that are both chemically and physiologically related may be substituted for the agents described herein while still achieving the same or similar results. All such substitutions and modifications apparent to those skilled in the art are considered to be within the scope of the invention.

Example 1: Testing of expression cassettes, development of constructs and testing of R0 plants.

This case study describes the development and testing of thirty-five different constructs in maize plants. Each construct contained four expression cassettes, each expression cassette being used to express a different transgene. This testing was conducted to select the best construct to use to express all four transgenes in maize. Each construct had a unique configuration, differing in orientation and expression elements of the expression cassettes.

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Various individual expression cassettes with different combinations of expression elements and transgenes were designed, cloned into plant transformation vectors, and tested for trait performance in maize plants to create a pool of the best individual expression cassettes. Using this pool of individual expression cassettes, 35 different constructs were created such that each one contained four expression cassettes (each expression cassette containing a transgene for PAT, DMO, CP4-EPSPS or FT) and could be used to test a four-way combination of different expression cassettes. cassettes. Expression cassette combinations differed in expression elements, protein-coding sequence, and orientation. This resulted in testing two PAT expression cassettes, 6 DMO expression cassettes, 5 FT expression cassettes, 18 CP4-EPSPS expression cassettes.

constructs containing four expression cassettes were cloned into plant transformation vectors, and these vectors were used for Agrobacterium-mediated transformation of LH244 immature maize embryos using methods known in the art, yielding 15,326 unique transformation transformants, each of which was generated by random introduction of a transgenic insert into the maize genome. R0 plants were then regenerated from the transgenic cells and rooted plants with normal phenotypic characteristics were transferred to soil for growth and subsequent evaluation.

15,326 R0 plants were analyzed for the presence of one copy of the transgenic insert and the absence of the vector backbone sequence. Plants with one copy of the insert were moved on to test the effectiveness of herbicide tolerance. Single-copy R0 plants were assessed in the greenhouse for tolerance to chisalofop (0.16 lb ae/acre Assure II herbicide) and then to a tank mix of glufosinate (0.98 lb ae/acre Ignite 280 herbicide), dicamba (2.0 lb ae/acre of Clarity herbicide) or 2,4-D (2.0 lb ae/acre of 2,4-D Amine 4 herbicide) (or any combination thereof) sprayed at the V1/V2 growth stage. Plants that were >30% damaged were discarded.

From an initial 15,326 unique transformation transformants generated by 35 transformation vectors, 1,945 unique transformants were selected after analysis of copy number and herbicide spray data. The data is presented in table. 2. R0 plants for selected transformants were selfed to produce R1 seed or crossed to produce F1 seed, which proceeded to the first season field trial.

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table 2

Example 2. Field tests of the first season.

Field trials were conducted over many years with transformants that progressed beyond the R0 analysis for each of the 35 constructs, and then the properties of each plant for each construct were analyzed during the first season of field trials in aggregate. Thus, each design was represented by many unique transformants. This allowed more designs to be tested in the first season's field trials, with only the designs with the best properties progressing beyond the first season's trials. Separate

- 19 044838 first season field trials were carried out on R2 plants (homozygous for each transformant) regarding (1) the effectiveness of tolerance to glufosinate + dicamba, chisalofop and 2,4-D in inbred plants, (2) the effectiveness of the Roundup Hybridization system System RHS) and (3) increasing herbicide rate testing for tolerance to higher application rates of the herbicides used: chisalofop, 2,4-D, glufosinate, and dicamba.

The effectiveness of inbred plants was screened to assess tolerance to the herbicides glufosinate + dicamba, chisalofop and 2,4-D. Herbicide treatments included: a tank mix of glufosinate at a rate of 0.8 lb ae/acre and dicamba at a rate of 2.0 lb ae/acre; chisalofop at 0.16 lb ai/acre or 2,4-D at 2.0 lb ai/acre. Plots were visually assessed for crop damage 10-14 days after herbicide treatment on a scale of 0-100, where 0 meant no damage to the crop and 100 meant complete destruction of the crop. The following indicators were also assessed: plant height (PHT), ear height (ENT), days until the appearance of 50% silk threads (S50D), days until the appearance of 50% pollen (P50D), husk weight (SHW), natural weight (TWT), moisture content (MST) and grain yield (YLD). All data were subjected to analysis of variance and separation of mean values at p<0.05. Overall average values for multiple plants containing the same transformant were used, and inbred plant performance was summarized for glufosinate+dicamba, chisalofop and 2,4-D as excellent (4), good (3), fair (2), poor ( 1) or not applicable (n/a). The data is presented in table. 3.

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Table 3

Efficiency of inbred plants after field trials of the first season

Design G l ufosinate/d icamba Hysalofop 2,4-D NT4-1 4 4 4 NT4-2 4 4 4 NT4-3 4 4 4 NT4-4 4 4 4 NT4-5 4 4 4 NT4-6 4 4 4 NT4-7 4 4 4 NT4-8 4 4 4 NT4-9 4 4 4 NT4-10 4 4 4 NT4-11 4 4 4 NT4-12 4 4 4 NT4-13 4 4 4 NT4-14 4 4 4 NT4-15 4 4 4 NT4-16 4 4 4 NT4-17 4 4 4 NT4-18 4 4 3 NT4-19 4 4 4 NT4-20 4 4 4 NT4-21 4 4 4 NT4-25 4 4 4 NT4-26 n/a n/a n/a NT4-27 n/a n/a n/a NT4-29 4 4 4 NT4-30 4 4 4 NT4-31 4 4 4 NT4-32 4 4 4 NT4-34 4 4 4 NT4-36 4 4 4 NT4-37 4 4 4 NT4-38 4 4 4 NT4-39 4 4 4 NT4-50 4 4 4 NT4-51 4 4 4

Screening for RHS effectiveness in plants was conducted to determine differences in glyphosate tolerance and glyphosate-induced panicle sterility in inbred material. The screening used a single herbicide treatment that included glyphosate at a rate of 1.5 lb ae/acre applied at stage V2, followed by a rate of 0.75 lb ae/acre until approximately stage V8 (875 days of growth rate), and then to approximately stage V10 (1025 days of growth rate). Plots were visually assessed for % crop damage (CIPV2, CIPV8, CIPV10 and CIPVT) 10-14 days after herbicide application on a scale of 0 to 100 and a final damage assessment was made at the VT stage (after panicle emergence). Sites were also visually scored for % silk filament emergence [(SES9A (S90) and SES9C (S90 plus 4 days) SES9E (S90 plus 8 days)] and % anther ejection [(AES9A (S90), AES9C (S90 plus 4 days) and AES9E (S90 plus 8 days)] on a similar scale from 0 to 100, where 0 means no appearance of silk threads or

- 21 044838 ejection of anthers, and 100 - complete appearance of silk threads or ejection of anthers. Other agronomic parameters were also assessed, as in the case of screening the effectiveness of inbred plants. Overall average values for multiple plants containing the same transformant were used, and glyphosate tolerance, panicle sterility, and yield scores were summarized as excellent (4), good (3), fair (2), poor (1), or not applicable ( n/a). The data is presented in table. 4.

Table 4

RHS performance after first season field trials

Design Glyphosate tolerance Panicle sterility Exit NT4-1 4 2 n/a NT4-2 4 2 n/a NT4-3 2 2 n/a NT4-4 4 2 n/a NT4-5 2 2 n/a NT4-6 2 2 n/a NT4-7 4 2 n/a NT4-8 4 2 n/a NT4-9 2 2 n/a NT4-10 2 2 n/a NT4-11 2 2 n/a NT4-12 4 2 n/a NT4-13 4 2 n/a NT4-14 4 4 4 NT4-15 4 2 n/a NT4-16 3/2 4 1 NT4-17 4 2 n/a NT4-18 2 2 n/a NT4-19 1 n/a n/a NT4-20 1 n/a n/a NT4-21 4 2 n/a NT4-25 2 2 n/a NT4-26 n/a n/a n/a NT4-27 n/a n/a n/a NT4-29 1 n/a n/a NT4-30 4 2 n/a NT4-31 2 2 n/a NT4-32 4 4 4 NT4-34 4 4 4 NT4-36 4 2 n/a NT4-37 4 2 n/a NT4-38 4 2 n/a NT4-39 4 2 n/a NT4-50 2 2 n/a NT4-51 2 2 n/a

A herbicide rate increase test was conducted to evaluate crop tolerance at the construct level to higher rates of the herbicides applied: chisalofop, 2,4-D, glufosinate and dicamba as follows. Chisalofop treatments included: 1) chisalofop at an application rate of 0.32 lb ai/acre (4X) plus 0.25% v/v. nonionic surfactant

-22044838 (NIS), used at stages VE-V2, and then at stage V4, and then at stage V8; 2) chisalofop at an application rate of 0.64 lb ai/acre (8X) plus 0.25% v/v. NIS applied at stages VE-V2, and then at stage V4, and then at stage V8; or 3) chisalofop at an application rate of 1.28 lb ai/acre (16X) plus 0.25% v/v. NIS applied at stages VE-V2, and then at stage V4, and then at stage V8. The 2,4-D treatments included: 1) 2,4-D amine at an application rate of 2 lb. ai/acre plus 0.25% v/v. non-ionic surfactant (NIS) applied at stages VE-V2, and then at stage V4, and then at stage V8; 2) 2,4-D amine at an application rate of 4 lbs. ai/acre plus 0.25% v/v. NIS applied at stages VE-V2, and then at stage V4, and then at stage V8; 3) 2,4-D amine at an application rate of 8 lb. a.i./acre plus 0.25% v/v. NIS applied at stages VE-V2, and then at stage V4, and then at stage V8; or 4) 2,4-D amine at an application rate of 16 lbs. a.i./acre plus 0.25% v/v. NIS applied at stages VE-V2, and then at stage V4, and then at stage V8. Glufosinate treatments included: 1) glufosinate at 1.0 lb ai/acre applied at the V2 stage, then at the V4 stage, and then at the V8 stage; 2) glufosinate at 2.0 lb ai/acre applied at the V2 stage, then at the V4 stage, and then at the V8 stage; 3) glufosinate at 4.0 lb ai/acre applied at V2, then V4, and then V8; or 4) glufosinate at an application rate of 8.0 lb ai/acre applied at the V2 stage, then at the V4 stage, and then at the V8 stage. Dicamba treatments included: 1) dicamba at a rate of 2.0 lb applied at the V2 stage, then at the V4 stage, and then at the V8 stage; 2) dicamba at an application rate of 4.0 lb applied at the V2 stage, then at the V4 stage, and then at the V8 stage; 3) dicamba at an application rate of 8.0 lb applied at the V2 stage, then at the V4 stage, and then at the V8 stage; 4) Dicamba at 16 lb. applied at V2 stage, then V4 stage, then V8 stage. Plots were visually assessed for crop damage and agronomic parameters obtained, as in the case of inbred plant performance screening. Overall average values for multiple plants containing the same transformant were used, and herbicide tolerance performance was summarized for each of the four herbicides as excellent (4), good (3), fair (2), poor (1), or not applicable ( n/a). The data is presented in table. 5.

Table 5

Test by increasing the herbicide application rate as part of field trials of the first season

Design Glufosinate Dicamba Hysalofop 2,4-D NT4-1 4 4 3 3 NT4-2 4 4 4 4 NT4-3 4 4 4 4 NT4-4 4 4 4 4 NT4-5 4 4 4 4 NT4-6 4 4 4 4 NT4-7 4 4 4 4 NT4-8 4 4 4 4 NT4-9 4 4 4 4 NT4-10 4 4 4 4 NT4-11 4 4 4 4 NT4-12 4 4 4 4 NT4-13 4 4 4 4 NT4-14 4 4 4 4 NT4-15 4 4 4 4

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NT4-16 4 4 4 4 NT4-17 4 4 4 4 NT4-18 4 4 4 4 NT4-19 4 4 4 4 NT4-20 4 4 4 4 NT4-21 4 4 4 4 NT4-25 4 4 4 4 NT4-26 n/a n/a n/a n/a NT4-27 n/a n/a n/a n/a NT4-29 4 4 4 4 NT4-30 4 4 4 4 NT4-31 4 4 4 4 NT4-32 4 4 4 4 NT4-34 4 4 4 4 NT4-36 4 4 4 4 NT4-37 4 4 4 4 NT4-38 4 4 4 4 NT4-39 4 4 4 4 NT4-50 4 4 4 4 NT4-51 4 4 4 4

Overall R2 plant trait data from each of the 35 constructs were compiled and analyzed in relation to (1) inbred plant performance testing for tolerance to glufosinate+dicamba, chisalofop and 2,4-D, (2) performance testing of RHS for tolerance to glyphosate, panicle sterility and yield, and (3) escalation trials for tolerance to higher application rates of the herbicides used, chisalofop, 2,4-D, glufosinate, and dicamba. Using these data, from the 35 designs tested, 3 designs (NT4-14, NT4-32 and NT4-34) were selected for further study. The transformants for these 3 designs then moved on to field testing for the second season.

Example 3: Molecular analysis.

Molecular analysis was carried out simultaneously with field trials for transformants that progressed further. DNA amplification and sequencing were used to confirm the sequence of the insert, the copy number of the insert, and the absence of a backbone in the insert. The insertion site for each transformant was mapped in the maize genome. Northern blot analysis was performed to detect and measure mRNA transcripts of the pat, dmo,ft_t and cp4-epsps genes. N-terminal sequencing of PAT, DMO, FT_T, and CP4EPSPS proteins purified from transgenic plants was performed to confirm the sequence of the recombinant protein. Western blot analysis to detect PAT, DMO, FT_T and CP4-EPSPS proteins was performed on transgenic plant samples. In-depth Southern blot analysis was performed on genomic DNA obtained from R1 plants to confirm copy number and backbone absence.

Example 4: Subsequent field tests.

Subsequent field trials (field trials of the second season and subsequent seasons) were carried out over many years for transformants that progressed beyond the first season of field trials of designs HT4-14, HT4-32 and HT4-34. The properties of many individual plants for each transformant in each field trial were analyzed together. Thus, each transformant was represented by many unique plants. This made it possible to analyze the properties of each transformant under many conditions, in different locations and geographical areas, and in relation to different characteristics.

Field trials were conducted on inbred plants (homozygous for the transformant) and hybrid plants (hemizygous for the transformant) to evaluate (1) the effectiveness of tolerance traits to commercial rates of glufosinate, dicamba, chisalofop and 2,4-D, (2) agronomic properties, (3) the effectiveness of the Roundup Hybridization System (RHS) on glyphosate tolerance, and (4) a herbicide rate increase test on tolerance to higher application rates of the herbicides used, hisalofop, 2,4-D, glufosinate, and dicamba.

During the Season 1 field trials, 30 transformants were tested for the HT4-14 construct, 41 transformants were tested for the HT4-32 construct, and 21 transformants were tested for the const.

- 24 044838 ruktsi NT4-34. Using the pooled data from these trials, transformants were selected for further study. During the Season 2 field trials, 15 transformants were tested for the HT4-14 construct, 38 transformants were tested for the HT4-32 construct, and 38 transformants were tested for the HT4-34 construct (this figure included some transformants that were not tested as part of the field trials 1 season due to a shortage of seeds this season). Transformants for the HT4-14 construct were characterized by molecular analysis as described in Example 3, and these data were also used for transformant selection. The summary data from these tests and in-depth molecular characterization of the transformants for the HT4-14 construct were used to select 3 transformants for further study for the HT4-14 construct. The summary data from these tests was used to select 24 transformants for further study for the HT4-32 construct, and to decide whether to stop further testing for any transformants for the HT4-34 construct.

During the field trials of the 3rd season, 3 transformants for the HT4-14 design and 24 transformants for the HT4-34 design were tested. The summary data from these tests was used to select 2 transformants for further study for the HT4-14 construct, and to decide whether to stop further testing for any transformants for the HT4-32 construct. Season 4 field trials were used to compare the final two transformants in a large number of locations, under different conditions, and in hybrid and inbred germplasm to provide the data needed to select the best transformant. In table Figure 6 shows the number of unique transformants tested for each design during field trials that were carried out in each season.

Table 6

Summary of Subsequent Field Tests

Stage NT4-14 NT4-32 NT4-34 Field season 1 thirty 41 21 Field season 2 15 38 38 Field season 3 3 24 0 Field Season 4 2 0 0 Selection of final transformants 1 0 0

Field trials for agronomic traits were conducted in the same season as field trials for trait performance. All field trials used a completely randomized block design and were conducted in different locations. Field tests were carried out in locations in North America and South America. For both the efficiency field trials and the agronomic field trials, an agronomic score was given throughout the field trial season and yield (effective yield or agronomic yield) was determined at the end of the season. Field efficacy trials were conducted to assess crop damage within 10-14 days of herbicide application. The target crop damage score for further transformant research was less than 10%. For field trials of agronomic properties, plots were kept weed-free and none of the herbicides tested were applied during the crop season. Field tests of the agronomic properties of hybrid plants included controls in the form of a comparable hybrid plant (hybrid control), obtained using the same maize parental lines that were used to produce the transgenic hybrid cross, but did not contain the transgenic transformant. Inbred controls were inbred plants comparable to the transgenic inbred lines.

To compare field trial data, a meta-analysis was performed using a pool of field trial data from all plants over several seasons, in several locations. As an example, in table. Figure 7 illustrates the number of replications over several seasons for which the observation was repeated for the two selected transformants HT4-14, as well as the total number of individual plants tested in each trial for each transformant.

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Table 7

Field trial replications

Stage Transformant Total replications Total plants Field season 1 TRANSFORMANT 2 235 16 450 Field season 1 MON87429 235 16 450 Field Season 2 TRANSFORMANT 2 100 7,000 Field Season 2 MON87429 100 7,000 Field Season 3 TRANSFORMANT 2 529 37 030 Field season 3 MON87429 513 35 910 Field season 4 TRANSFORMANT 2 228 15 960 Field Season 4 MON87429 228 15 960 Field Season 4 TRANSFORMANT 2 56 3 920 Field Season 4 MON87429 2465 172 550

A meta-analysis of several field trials of hybrid plant effectiveness was performed to compare damage rates of hybrid plants. As an example, in table. Figure 8 presents a multi-season damage assessment for two selected HT4-14 transformants obtained at the V8 stage (with the V8 stage analysis covering cumulative damage after herbicide application at stages V2, V4, V6 and V8), with the statistically smallest significant difference observed at 95% confidence level (NCR at p<0.05). Plants containing maize transformant MON87429, as well as plants containing transformant 2, performed well in these tests.

Table 8

Meta-analysis of damage assessment in field trials of hybrid plant effectiveness.

Stage Transformant V8 damage NZR (P<0.05) Field season 1 MON87429 1.3 2.1 Field season 1 TRANSFORMANT 2 1.1 2.1 Field Season 2 MON87429 1.7 3.1 Field Season 2 TRANSFORMANT 2 2.6 3.1 Field Season 3 MON87429 0.08 3.4 Field Season 3 TRANSFORMANT 0.13 3.4 2

A meta-analysis of several field trials of hybrid plant performance was performed to compare bushel/acre yields. As an example, in table. Figure 9 shows the yield for hybrid plants over several seasons for two selected transformants HT4-14, and the statistically smallest significant difference is observed at the 95% confidence level (NZR at p<0.05). Plants containing maize transformant MON87429, as well as plants containing transformant 2, performed well in these tests.

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Table 9

Meta-analysis of yield in field trials of hybrid plant efficacy

Stage Transformant Yield (bushels/acre) NZR (p<0.05) Field season 1 MON87429 222 12 Field season 1 TRANSFORM NT 2 222 12 Field Season 2 MON87429 197 15.4 Field season 2 TRANSFORM NT 2 197 15.4 Field Season 3 MON87429 210.9 9.1 Field Season 3 TRANSFORM NT 2 211.5 9.1

Field trials with increasing application rates of herbicides applied at higher application rates than commercial ones were carried out on hybrid and inbred plants containing one transgenic transformant. Herbicides glufosinate (range 1.6 to 6.4 lb ai/acre), dicamba (range 2.0 to 16 lb ai/acre), chisalofop (range 0.32 to 1.28 lb ai/acre) acre), 2,4-D (ranging from 2.0 to 8.0 lb ae/acre) and glyphosate (at an application rate of 3.0 lb ae/acre) were applied in field trials to test increasing rate of trait effectiveness tolerance to herbicides. At the end of the season, hybrid plants from the increasing herbicide field test were harvested and yield (bushels/acre) was determined. As an example, in table. Figure 10 presents data on the yield of hybrid and inbred plants in different tests for two selected transformants HT4-14, and the statistically smallest significant difference is observed at the 95% confidence level (NZR at p<0.05). Plants containing the maize transformant MON87429, as well as plants containing transformant 2, performed well in hybrid and inbred yield tests for all herbicide treatments. The results indicate that further field trials determined the superiority of maize transformant MON87429 over transformant 2 in terms of inbred plant yield.

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Table 10

Efficiency obtained during field trials by increasing the application rate of herbicides for hybrid/inbred plants

Herbicide Plant Transformant Yield (bushels/acre) NZR (p<0.05) Glufosinate Hybrid plant MON87429 256 28.4 Glufosinate Hybrid plant TRANSFORMANT 2 240 28.4 Dicamba Hybrid plant MON87429 264 39.2 Dicamba Hybrid plant TRANSFORMANT 2 256 39.2 Hysalofop Hybrid plant MON87429 251 48.5 Hysalofop Hybrid plant TRANSFORMANT 2 257 48.5 2,4-D Hybrid plant MON87429 261 38.4 2,4-D Hybrid plant TRANSFORMANT 2 254 38.4 Glyphosate Inbred plant MON87429 90.1 46 Glyphosate Inbred plant TRANSFORMANT 2 67.2 46

Field tests of the agronomic properties of hybrid plants were carried out, agronomic measurements were carried out during the season and agronomic yield was determined at the end of the season. A meta-analysis among field trials of the agronomic properties of hybrid plants over several seasons and in several locations was used to compare the yield of hybrid control and hybrid plants. As an example, in table. Yield data (bushels/acre) for the two selected transformants HT4-14 are presented, with the statistically least significant difference observed at the 95% confidence level (LSI at p<0.05). Plants containing the maize transformant MON87429, as well as plants containing transformant 2, performed well in these tests; no statistical difference in hybrid yield was detected for plants containing either transformant when compared with control plants.

Table 11

Meta-analysis of yield in field trials of agronomic properties of hybrid plants

Stage Transformant Yield (bushels/acre) NZR (p<0.05) Field season 1 Control - absent 222 9 Field season 1 MON87429 220 9 Field season 1 TRANSFORMANT 2 224 9 Field season 2 Control - absent 224.6 13.7 Field season 2 MON87429 211 13.7 Field season 2 TRANSFORMANT 2 215 13.7 Field season 3 Control - absent 213.6 9.7 Field Season 3 MON87429 213.3 9.7 Field season 3 TRANSFORMANT 2 212.4 9.7

Field performance tests of inbred plants were conducted on glyphosate tolerance and the Roundup Hybridization System (RHS) and yield was determined at the end of the season. Glyphosate was applied at a rate of 1.5 lb ae/acre to control weed growth, followed by two applications of glyphosate to achieve sterility at a rate of 0.75 lb ae/acre at approximately V8, and then at a rate of 0.75 lb ae/acre at approximately stage V10. A meta-analysis among field trials of inbred plant performance across multiple seasons and locations was used to compare plant yields. As an example, in table. Figure 12 shows yield data (bushels/acre) for two selected transformants HT4-14, with the smallest statistically significant difference observed at the 95% confidence level (LSI at p<0.05). Field season 2 and field season 3 trials showed a statistically significant reduction in yield for plants containing transformant 2 compared to plants containing transformant MON87429. These data indicate superior yield performance testing of inbred plants containing the maize transformant MON87429.

Table 12

Meta-analysis of yield in field trials of the effectiveness of inbred plants treated with glyphosate

Stage Transformant Yield (bushels/acre) NZR (p<0.05) Field season 1 MON87429 89.9 14.3 Field season 1 TRANSFORMANT 2 87.5 14.3 Field season 2 MON87429 119.6 18.0 Field season 2 TRANSFORMANT 2 99.0 18.0 Field Season 3 MON87429 105.6 10.5 Field Season 3 TRANSFORMANT 2 90.13 10.5

Field tests of the agronomic properties of inbred plants were carried out and the yield for untreated plants was determined at the end of the season. The trials included inbred line controls comparable to transgenic inbred lines. A meta-analysis among field trials of agronomic properties of inbred plants across multiple seasons and locations was conducted to compare yields for mated controls and transgenic inbred plants. As an example, in table. Figure 13 shows yield data (bushels/acre) for two selected transformants HT4-14, with the smallest statistically significant difference observed at the 95% confidence level (LCI at p < 0.05). There was no statistical difference in inbred agronomic yield between control plants and plants containing the maize transformant MON87429. In contrast, in field season 3 trials, there was a statistically significant reduction in yield for plants containing transformant 2 compared to control plants and plants containing maize transformant MON87429. These data indicate superior performance in agronomic inbred yield trials for plants containing the maize transformant MON87429.

Table 13 Meta-analysis of yield in field trials of agronomic properties of inbred plants

Stage Transformant Total (bushels/acre) NZR (p<0.05) Field season 1 No control 105.2 8.9 Field season 1 MON87429 104.1 8.9 Field season 1 TRANSFORMANT 2 102.5 8.9 Field season 2 No control 103.7 15.3 Field Season 2 MON87429 93.4 15.3 Field season 2 TRANSFORMANT 2 91.4 15.3 Field season 3 No control 116.6 6.2 Field season 3 MON87429 112.8 6.2 Field season 3 TRANSFORMANT 2 106.1 6.2

Hybrid plant efficacy trials were conducted at four locations in Argentina to evaluate plant tolerance to glufosinate, dicamba, chisalofop, haloxyfop, 2,4-D and glyphosate. Plants containing MON87429 were crossed with plants containing both the maize transformant MON88017 and the maize transformant MON89034 to produce progeny containing all three transformants (MON87429 x MON88017 x MON89034). Herbicide treatments included: 1)

- 29 044838 raw control; 2) glufosinate with an application rate of 0.448 kg ai/ha, applied at the V2 stage, and then with a similar application rate at the V6 stage; 3) glufosinate with an application rate of 0.896 kg ai/ha, applied at the V2 stage, and then with a similar application rate at the V6 stage; 4) dicamba at a rate of 0.56 lb ae/acre applied at V2 and then at the same rate at V6; 5) dicamba at a rate of 1.12 lb ae/acre applied at V2 and then at the same rate at V6; 6) chisalofop with an application rate of 0.09 kg ae/ha, applied at the V2 stage, and then with a similar application rate at the V6 stage; 7) chisalofop with an application rate of 0.18 kg ae/ha, applied at the V2 stage, and then with a similar application rate at the V6 stage; 8) haloxyfop with an application rate of 0.1 kg ae/ha, applied at stage V2, and then with a similar application rate at stage V6; 9) haloxyfop with an application rate of 0.2 kg ae/ha, applied at stage V2, and then with a similar application rate at stage V6; 10) 2,4-D at a rate of 1.12 lbs. ai/acre applied at V2 and then at the same rate at V6; 11) 2,4-D at a rate of 2.24 lb ae/acre applied at V2 and then at the same rate at V6; or 12) glyphosate at an application rate of 2.24 lb ae/acre applied at stage V2 and then at a similar rate at stage V6. Data obtained included crop damage 10-14 days after herbicide application at stages V2 and V6, as well as final assessment at stage VT, number of days until 50% pollen appeared, number of days until 50% silk appeared, plant height, height ears, husk weight, natural weight, moisture content and grain yield. All data were subjected to analysis of variance and separation of mean values at p<0.05.

Herbicide tolerance of plants containing MON87429 x MON88017 x MON89034 was excellent (<10% crop damage) at all application rates of glyphosate, glufosinate, dicamba, chisalofop, haloxyop and 2,4-D tested. The application rates of herbicides for plant treatment did not affect the visual assessment of crop damage. Head height in any of the treatments was not significantly different compared to the standard herbicide treatment 12 (glyphosate at 2.24 lb ae/acre). There was no significant reduction in plant height or body weight, increase in grain moisture, delay in maturation (measured as an increase in the number of days until 50% pollen or silk filaments appeared), or decrease in grain yield in plants containing MON87429 x MON88017 x MON89034 in any of the treatments compared to untreated plants. Hybrid plants produced by crossing a plant containing MON87429 with a plant containing a transformant that provides glyphosate tolerance in male tissues (such as the commercially available maize transformants MON88017 or NK603) have excellent vegetative tolerance to glyphosate, glufosinate, dicamba, chisalofop, gal oxyfop and 2,4-D when applied at commercially stated application rates.

A three-year field trial was conducted to control plants containing the maize transformant MON87429 using clethodim. These trials evaluated methods for controlling spontaneous growth with the herbicide DIM applied to plants containing the maize transformant MON87429. Plants were treated with clethodim at commercially stated application rates and complete control of plants containing the maize transformant MON87429 was observed.

Data obtained from molecular analysis and field trials of inbred and hybrid plants to evaluate (1) the effectiveness of tolerance traits to commercial rates of glufosinate, dicamba, chisalofop and 2,4-D, (2) agronomic properties, (3) the effectiveness of the hybridization system Roundup (RHS) and glyphosate tolerance, and (4) herbicide ramp-up trials for tolerance to higher rates of the herbicides used, chisalofop, 2,4-D, glufosinate, and dicamba, were analyzed for all transformants tested for the HT4- constructs. 14, NT4-32 and NT4-34. Analysis of the aggregate data demonstrated overall superior performance of the maize transformant MON87429 compared to other transformants and led to the selection of this transformant for commercial use.

Example 5. Molecular characterization of the maize transformant MON87429.

The maize transformant MON87429 was subjected to extensive molecular characterization following selection for commercial use. The transgenic insert of the maize transformant MON87429 contains the elements and sequences described in table. 1.

The DNA sequences of the maize transformant MON87429 were analyzed. Southern blot analysis was performed to confirm that plants containing the maize transformant MON87429 contained one intact copy of the entire transgene insert without the transformation vector backbone. Flanking DNA was sequenced at the 5′ and 3′ ends of the insert, and corresponding junctions were determined using sequence capture, enrichment, sequencing, inverse PCR, and genome walking techniques. The flanking DNA sequences for the maize transformant MON87429 were aligned with the known physical assembly of the maize genome. Sequence information about the insertion site was used for bioinformatic analysis of the chromosomal location of the transformant. The integrity of the insertion site was determined by SLR among the wild-type allele using primers specific for the flanking regions of the maize transformant MON87429. The wild-type insertion site was used to map the unique site of the transgenic

- 30 044838 integration for the maize transformant MON87429 with the maize reference genome. To ensure that no changes or mutations were introduced into any of the transgene regions during transformation, the entire transgenic insert of maize transformant MON87429 was isolated from the plant and sequenced. Sequence information for the 5' junction, 3' junction and transgene insert is provided herein as SEQ ID NO: 1-10.

RNA analysis was carried out on plants containing the maize transformant construct MON87429. Northern blot analysis was performed on total RNA isolated from grains of plants containing the maize transformant MON87429. He confirmed the transcript size and the number of pat, dmo, ft_t and cp4-epsps mRNA products. RNA expression levels for CP4-EPSPS were also measured by real-time PNR using samples obtained from plant tissue containing the maize transformant MON87429. Rapid amplification of cDNA ends (RACE) was used to identify CP4-EPSPS cleavage products to confirm that cleavage of the CP4-EPSPS transcript occurs only in panicles of plants containing the maize transformant MON87429 and that it is caused by male endogenous small interfering RNA (siRNA)-specific sequence way. Small molecule Northern blot analysis was performed to demonstrate that CP4-EPSPS siRNAs, which may reduce glyphosate tolerance, were absent in tissue other than panicle tissue.

Protein analysis was carried out on plants containing the maize transformant construct MON87429. Using immunoaffinity purified protein extracts from grains, N-terminal sequencing of the expressed PAT, DMO, FT_T and CP4-EPSPS proteins was performed to confirm the true N-terminal amino acid sequence. Western blot analysis was performed on protein extracts from kernels containing the maize transformant MON87429 to confirm that a single protein of the expected size was produced for PAT, DMO, FT_T, and CP4-EPSPS, respectively. ELISA was used to determine the content of PAT, DMO, FT_T and CP4-EPSPS proteins in leaves, seeds, roots and pollen of plants.

Example 6: Detection of the maize transformant MON87429.

Detection of maize transformant MON87429 in a sample can be accomplished using DNA, RNA, or protein detection techniques. Examples of detection methods and materials are presented below. Detection methods can determine the presence or absence of the maize transformant MON87429 in a sample. The detection may indicate the number of genomic copies of the maize transformant MON87429 (ie, hemizygosity, homozygosity, or heterozygosity) in the genomic DNA sample.

A transformant-specific Applied Biosystems™ TAQMAN endpoint thermal amplification method (Thermo Fisher Scientific) was developed to identify the maize transformant MON87429 in a sample. The DNA primers and probes used in the endpoint assay are primers SQ51062 (SEQ ID NO: 11), SQ51053 (SEQ ID NO: 12) and the PB50370 6-FAM labeled probe (SEQ ID NO: 13). 6-FAM is a fluorescent dye from Applied Biosystems (Foster City, CA) attached to a DNA probe. In the case of the TAQMAN MGB 5' probes, the exonuclease activity of Taq DNA polymerase cleaves the probe at the 5' end between the fluorophore and the quencher. When hybridized to the target DNA strand, the quencher and fluorophore are separated enough to generate a fluorescent signal, thereby releasing fluorescence. SQ51062 and SQ51O53, when used in similar reaction methods and together with PB50370, produce a DNA amplicon that allows diagnosis of the maize transformant MON87429. Controls in this assay should include a positive control containing the maize transformant MON87429, a negative control derived from non-transgenic corn, and a negative control that does not contain the DNA template. In addition, the control in the PNR reaction should optimally include internal control primers and an internal control probe specific for a single copy gene in the maize genome. These assays are optimized for use with the Applied Biosystems GeneAmp PCR System 9700 (Thermo Fisher Scientific) at maximum speed, but other equipment may be used.

An example of the conditions used in the TAQMAN methods for detecting the maize transformant MON87429 is as follows. Step 1: 18 MΩ water, corrected to a final volume of 5 µL. Step 2. 2.28 µl of 2X universal master mix (dNTP, enzyme, buffer) to 1X final concentration. Step 3: 0.05 µl of object primer-1 (SQ51062) and object primer-2 (SQ51053) (resuspended in 18 MΩ water to a concentration of 100 µM for each primer) to a final concentration of 0.9 µM. Step 4: 0.01 µl of PB50370 6-FAM MGB object probe (resuspended in 18 MΩ water to a concentration of 100 µM) to a final concentration of 0.2 µM. Step 5. 0.05 µl of a mixture of internal control primer-1 and internal control primer-2 (resuspended in 18 MΩ water to a concentration of 100 µM for each primer) to a final concentration of 0.9 µM. Step 6: 0.01 µl of VIC internal control probe (resuspended in 18 MΩ water to a concentration of 100 mM) to a final concentration of 0.2 µM. Step 7. 2.5 µl of extracted DNA (template) for each sample with one of the following: (a) leaf samples to be analyzed; (b) negative control (non-transgenic DNA); (c) negative control water (no template) and (d) positive control corn DNA containing the corn transformant

MON87429. Step 8: Thermal cycler conditions: one cycle at 95°C for 20 s; forty cycles of 95°C for 3 s, then 60°C for 20 s, and a final cycle of 10°C.

The zygosity test is designed to determine whether a plant containing the maize transformant MON87429 is heterozygous or homozygous for the transformant or the wild-type allele. Using the sequence information provided herein, an amplification reaction assay can be developed. For example, such a PCR assay would include a design of at least three primers: Primer-1, Primer-2, and Primer-3, wherein Primer-1 is specific for maize genomic DNA on the 3' flank of the maize transformant MON87429; primer-2 is specific for the transgenic insert of the maize transformant MON87429; and primer-3 is specific for the wild-type allele. When used as a pair of primers in an amplification reaction, primer-1 and primer-2 produce a PCR amplicon specific for the maize transformant MON87429. When used as a primer pair in an amplification reaction, primer-1 and primer-3 produce a PCR amplicon that is specific for the wild-type allele. In a PCR reaction performed on maize genomic DNA, the corresponding PCR amplicons obtained from Primer-1 and Primer-2, and from Primer-1 and Primer-3, will differ in sequence and amplicon size. When the three primers are included in a PCR reaction with DNA extracted from a plant homozygous for the maize transformant MON87429, only the amplicon of primer-1 and primer-2 (specific for the maize MON87429 insertion) will be obtained. When the three primers are included in a PCR reaction with DNA extracted from a plant heterozygous for the maize transformant MON87429, both the primer-1 and primer-2 amplicon (specific for the maize MON87429 insertion) and the primer1 and primer-3 amplicon ( specific for the wild-type or absence allele of maize MON87429). When the three primers are mixed together in a PCR reaction with DNA extracted from a maize transformant null plant MON87429 (i.e., wild type), only the amplicon of primer-1 and primer-3 (specific for the wild-type allele) will be obtained. The amplicons obtained by the PCR reaction can be identified or determined by any method known in the art.

Another test for zygosity for the maize transformant MON87429 is the TAQMAN heat amplification reaction. For this type, in addition to the primers as described above, the assay will include two fluorescently labeled probes. Probe-1 will be specific for the maize transformant MON87429, and probe-2 will be specific for the maize plant that is null for the maize transformant MON87429 (wild type), and the two probes contain different fluorescent tags, for example, 6-FAM- tag or VIC tag. When used in the TAQMAN reaction, primer-1+primer-2+probe-1 will generate the first fluorescent signal specific for the maize transformant MON87429, primer-1+primer-3+probe-2 will generate the second fluorescent signal specific for wild-type corn . When three primers and two probes are included in a TAQMAN reaction with DNA extracted from a plant homozygous for the maize transformant MON87429, only the first fluorescent signal (specific to primer-1+primer-2+probe-1) will be generated. When the three primers are included in a TAQMAN reaction with DNA extracted from a plant heterozygous for the maize transformant MON87429, both a first fluorescent signal (specific for primer-1+primer2+probe-1) and a second fluorescent signal (specific for primer-1+primer3+probe-2). When the three primers are mixed together in a TAQMAN reaction with DNA extracted from a maize transformant null plant MON87429 (wild type), only a second fluorescent signal (specific to primer-1 + primer-3 + probe-2) will be generated.

Another way to detect the presence of the maize transformant MON87429 in a plant sample is by Southern blot analysis. One skilled in the art will appreciate how to design a Southern blot hybridization probe(s) specific for the maize transformant MON87429 and a second Southern blot hybridization probe(s) specific for a maize plant null for the maize transformant MON87429 ( wild type). In Southern blot analysis, the signal detected from only the first Southern blot hybridization probe will indicate a plant homozygous for the maize transformant MON87429; the signal detected from the first Southern blot hybridization probe and the second Southern blot hybridization probe will indicate a plant heterozygous for the maize transformant MON87429; and a signal detected from only the second Southern blot hybridization probe would indicate that the DNA was extracted from a plant null for the maize transformant MON87429 (wild type).

Another example of a detection kit includes at least one antibody specific for at least one protein encoded by the maize transformant MON87429. For example, such a kit may use a test strip with lateral

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Claims (32)

diffusion, containing reagents that are activated when the tip of the strip comes into contact with an aqueous solution. Examples of proteins that can be used to produce antibodies are those encoded by the sequence shown in SEQ ID NO: 10, or any fragment thereof. The protein detection method is designed to determine whether the sample came from a plant, seed, cell or plant part containing the maize transformant MON87429. At least one antibody specific for at least one protein encoded by the maize transformant MON87429 is used to detect the protein encoded by the maize transformant MON87429 in the sample. A detection kit containing one or more antibodies specific for one or more proteins encoded by the maize transformant MON87429 may use a lateral diffusion technology test strip containing reagents that are activated when the tip of the strip comes into contact with an aqueous solution. Corn tissue samples can be ground and protein extracted for analysis using water or an aqueous buffer (eg, phosphate-buffered saline containing detergent and bovine serum albumin). After centrifugation, the aqueous supernatant is analyzed using a sandwich ELISA on a lateral diffusion test strip containing an absorbent pad. Detection is activated by immersing the tip of the test strip in an aqueous solution containing the sample to be tested. The aqueous solution rises up the test strip by capillary action and solubilizes the gold-labeled antibodies on the test strip. The gold-labeled antibodies are specific for at least one protein encoded by the maize transformant MON87429 and will bind to an epitope on the protein in the sample to form an antibody-antigen complex. The gold-labeled antibody-antigen complex then moves up the test strip to the nitrocellulose membrane. The membrane contains a test line of immobilized antibodies that bind to a second distinct epitope on the protein encoded by the maize transformant MON87429, resulting in a visible line appearing on the test strip if the protein encoded by the maize transformant MON87429 is present in the sample. CLAIM 1. A recombinant DNA molecule containing a sequence selected from the group consisting of SEQ ID NO: 10, 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, wherein the presence of said DNA molecule in a DNA sample obtained from a corn plant, corn seed or corn cell indicates the presence of the corn transformant MON87429 and tolerance of at least to one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, or any combination thereof. 2. The recombinant DNA molecule according to claim 1, characterized in that the recombinant DNA molecule is obtained from a plant, seed or cell containing the maize transformant MON87429, and a representative sample of the seed containing the transformant is deposited under ATCC accession number PTA-124635, where said transformant corn MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 3. The recombinant DNA molecule according to claim 1, characterized in that the recombinant DNA molecule is located in a plant, cell, seed or part of a plant containing the maize transformant MON87429, and a representative sample of the seed containing the transformant is deposited under ATCC accession number PTA-124635, wherein said maize transformant MON87429 contains a sequence selected from the group consisting of SEQ iD NO: 10, 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. 4. The recombinant DNA molecule according to claim 1, wherein the recombinant DNA molecule is an amplicon that allows the presence of a maize transformant MON87429 to be diagnosed, wherein said maize transformant MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 5. A DNA molecule having a sufficient length of contiguous nucleotides of SEQ ID NO: 10 to function as a DNA probe specific to SEQ ID NO: 10 in a DNA sample obtained from a corn plant, corn seed or corn cell, wherein the DNA -probe contains SEQ ID NO: 1 or SEQ ID NO: 2. 6. The DNA molecule according to claim 5, characterized in that the DNA probe contains SEQ ID NO: 13. 7. A pair of DNA molecules containing a first DNA molecule and a second DNA molecule, wherein the first DNA molecule contains a sufficient length of contiguous nucleotides within nucleotides 1030-15037 SEQ ID NO: 10 to function as a DNA primer, wherein the second DNA molecule contains sufficient length of contiguous nucleotides within nucleotides 1-1029 or within nucleotides - 33 044838 15038-16068 SEQ ID NO: 10 to function as a DNA primer, and wherein the DNA molecules, when used together in a DNA amplification reaction, comprise the maize transformant MON87429 to produce an amplicon allowing the diagnosis of the maize transformant MON87429 in the sample, wherein said amplicon contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 8. A pair of DNA molecules according to claim 7, wherein the first DNA molecule contains a sufficient length of contiguous nucleotides within nucleotides 1030-1350 or within nucleotides 1499915037 SEQ ID NO: 10 to function as a DNA primer. 9. A pair of DNA molecules according to claim 7, characterized in that the first DNA molecule contains SEQ ID NO: 11, and the second DNA molecule contains SEQ ID NO: 12. 10. A method for detecting the presence of the corn transformant MON87429 in a DNA sample obtained from a corn plant, a corn seed or a corn cell, the method comprising: a) bringing the sample into contact with the DNA probe according to claim 5; b) subjecting the sample and DNA probe to stringent hybridization conditions, and c) detecting hybridization of the DNA probe to a DNA molecule in the sample, wherein hybridization of the DNA probe to the DNA molecule indicates the presence of the maize transformant MON87429 in the DNA sample, and wherein said maize transformant MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 11. A method for detecting the presence of the corn transformant MON87429 in a DNA sample obtained from a corn plant, a corn seed or a corn cell, the method comprising: a) bringing the sample into contact with a pair of DNA molecules according to claim 7; b) performing an amplification reaction sufficient to produce a DNA amplicon containing a sequence selected from the group consisting of SEQ ID NO: 10, 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; And c) detecting the presence of a DNA amplicon, wherein the presence of a DNA amplicon indicates the presence of a maize transformant MON87429 in the sample, and wherein said maize transformant MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 12. A kit for detecting the presence of the maize transformant MON87429, containing a DNA probe in accordance with paragraph 5, or a pair of DNA molecules in accordance with paragraph 7. 13. A plant or part of a plant containing a DNA molecule containing 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, wherein the presence of said DNA molecule indicates the presence of the corn transformant MON87429 and tolerance to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, or any combination thereof. 14. The plant or plant part of claim 13, wherein the plant or plant part is tolerant to chisalofop, haloxyfop, dicamba, 2,4-D, glufosinate and glyphosate. 15. A method for controlling weed growth in a crop growing area, including: a) planting corn containing the corn transformant MON87429 in a crop growing area where said corn transformant MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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; And b) applying to the crop growing area, or any part thereof, an effective amount of at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and inhibitors 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), or any combination thereof, to control weed growth in the area without harming the corn. 16. The method of claim 15, wherein the use of an effective amount of at least one herbicide includes the use of at least two or more herbicides selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (FOP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors, for the harvest season. 17. The method of claim 15, wherein the use of an effective amount of at least one herbicide includes the use of a herbicide selected from the group consisting of chisalofop, haloxyfop, dicamba, 2,4-D, glufosinate and glyphosate, or any of them combinations. - 34 044838 18. The method of claim 17, wherein the effective amount of dicamba is from about 0.56 kg acid equivalent (ae)/ha to about 2.24 kg ae/ha per crop season. 19. The method of claim 17, wherein the effective amount of glufosinate is from about 0.45 kg ai/ha to about 1.78 kg ai/ha per crop season. 20. The method of claim 17, wherein the effective amount of 2,4-D is from about 0.84 to about 1.12 kg ae/ha per crop season. 21. The method of claim 17, wherein the effective amount of chisalofop is from about 0.038 to about 0.093 kg ai/ha per crop season. 22. The method of claim 17, wherein the effective amount of haloxyfop is from about 0.020 to about 0.078 kg ai/ha per crop season. 23. A method of controlling self-seeded corn containing the corn transformant MON87429 on a site, comprising using a herbicidally effective amount of at least one cyclohexanedione (DIM) herbicide, wherein the use of the herbicide prevents the growth of corn containing the corn transformant MON87429, wherein said corn transformant MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 24. The method of claim 23, wherein the use of a herbicidally effective amount of at least one cyclohexanedione (DIM) herbicide includes the use of a cyclohexanedione (DIM) herbicide selected from the group consisting of clethodim, sethoxydim and tralkoxydim. 25. A method of producing a plant that is tolerant to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvyl shikimate inhibitors. 3-phosphate synthase (EPSPS) or any combination thereof, the method comprising: a) crossing a plant containing the maize transformant MON87429 with itself or with a second plant to produce a seed, and b) identifying the progeny seed that contains the maize transformant MON87429; wherein said maize transformant MON87429 contains a sequence selected from the group consisting of SEQ ID NO: 10, 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. 26. The method according to claim 25, characterized in that identifying the progeny seed that contains the maize transformant MON87429 includes: a) germinating the progeny seed to produce progeny plants; b) treating the progeny plants with an effective amount of at least one herbicide selected from the group consisting of chisalofop, haloxyfop, dicamba, 2,4-D, glufosinate and glyphosate, or any combination thereof; And c) selecting a progeny plant that is tolerant to at least one herbicide selected from the group consisting of acetyl-CoA carboxylase (ACC) inhibitors in the aryloxyphenoxy propionate (OP) group, synthetic auxins, glutamine synthetase inhibitors and 5-enolpyruvyl shikimate inhibitors. 3-phosphate synthase (EPSPS) or any combination thereof. 27. The method of claim 25, wherein identifying a progeny seed that contains the maize transformant MON87429 includes detecting the presence of the maize transformant MON87429 in a sample obtained from the progeny seed. 28. The method of claim 25, wherein identifying a progeny seed that contains the maize transformant MON87429 includes detecting the presence of at least one protein encoded by the maize transformant MON87429 in a sample obtained from the progeny seed. 29. A method for producing a hybrid seed, including: a) growing a plant containing SEQ ID NO: 10; b) using an effective amount of glyphosate before or during the development of the male reproductive organ tissues of the plant, thereby causing male sterility in the plant; c) fertilization of the plant with pollen from a second plant, and d) collecting hybrid seeds from the plant. 30. The method of claim 29, wherein using an effective amount of glyphosate includes using glyphosate before or during development at an effective amount of from about 0.56 to about 2.80 kg ae/ha. 31. The method of claim 29, wherein using an effective amount of glyphosate includes using glyphosate at a developmental stage selected from the group consisting of stage V4, V5, V6, V7, V8, V9, V10, V11, V12, V13 and V14 of maize plant development. 32. Hybrid seed obtained by the method according to claim 29, characterized in that the hybrid seed contains SEQ ID NO: 10. -