Compositions and methods for efficient targeting of transgenes

Abstract

The present invention provides recombinant DNA molecules and constructs useful for providing efficient transgene subcellular localization in transgenic plants for the production of proteins. Further provided are recombinant DNA molecules and constructs for conferring herbicide tolerance or resistance in plants, as well as plants exhibiting herbicide tolerance and methods of producing or utilizing such plants.

Description

[0001] This application is a divisional application of the invention patent application filed on December 19, 2016, with application number 201680078294.7 and entitled "Composition and Method for Effectively Targeting Transgenic Genes".

[0002] Cross-reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 270,180, filed September 21, 2015, and U.S. Provisional Application No. 62 / 364,715, filed July 20, 2016, which are incorporated herein by reference in their entirety. Invention Field

[0004] This invention generally relates to the fields of agriculture, plant biotechnology, and molecular biology. More specifically, this invention relates to compositions and methods for producing transgenic plants exhibiting herbicide tolerance or resistance.

[0005] Merging of sequence lists

[0006] The sequence list is submitted electronically in a computer-readable form along with this application and is incorporated herein by reference in its entirety. The sequence list is contained in a file named MONS389WO_ST25.txt, which is 122 kilobytes in size (measured in the MS Windows operating system) and was created on December 19, 2016.

[0007] Related technical descriptions

[0008] The generation of novel transgenic plants offers the potential for significantly improved crop plants to exhibit beneficial traits such as improved herbicide tolerance, allowing for enhanced weed control strategies. However, while proteins suitable for producing beneficial traits in crops are known, the efficient subcellular localization (known as targeting) and processing of these recombinant proteins in transgenic plant cells remains a significant hurdle. Therefore, novel transport peptides capable of efficiently localizing recombinant proteins within plant cells are needed.

[0009] summary

[0010] One aspect of the present invention relates to a recombinant DNA molecule comprising a DNA sequence encoding a chloroplast transport peptide (CTP), said DNA sequence being operatively linked to a DNA sequence encoding dicamba monooxygenase (DMO) or protoporphyrinogen oxidase (PPO), wherein the CTP comprises a sequence selected from the group consisting of SEQ ID NO: 1-3. In some embodiments, the DNA sequence encoding CTP comprises a sequence selected from the group consisting of SEQ ID NO: 7-14. In other embodiments, the DMO or PPO comprises a polypeptide selected from the group consisting of SEQ ID NO: 18-27 and 40-59. In one embodiment, the DNA sequence encoding DMO or PPO comprises a sequence selected from the group consisting of SEQ ID NO: 28-37 and 61-102. In a particular embodiment, DMO or PPO is defined as a herbicide tolerance protein capable of conferring herbicide tolerance when expressed in plant cells. In a particular embodiment, the herbicide tolerance protein is a DMO protein and the CTP contains a sequence selected from the group consisting of SEQ ID NO:1-3, or the herbicide tolerance protein is a PPO protein and the CTP contains a sequence selected from the group consisting of SEQ ID NO:1 and 2.

[0011] In another aspect, the present invention provides a DNA construct comprising a recombinant DNA molecule operably linked to a heterologous promoter that functions in plant cells, as described herein.

[0012] In another aspect, the present invention provides a transgenic plant, plant cell, plant part, or seed transformed with the recombinant DNA molecule of the present invention. In a particular embodiment, the plant is a monocotyledonous plant. Monocotyledonous plants that can be used in the context of the present invention include, but are not limited to, corn or wheat plants. In another embodiment, the plant is a dicotyledonous plant. Dicotyledonous plants that can be used in the context of the present invention include, but are not limited to, soybean, cotton, or Brassica plants.

[0013] In another aspect, the present invention provides a recombinant DNA molecule present in non-living plant material. In one example, plant cells containing the recombinant DNA molecule of the present invention are within the scope of the invention. In one embodiment, such plant cells may be regenerative plant cells or non-regenerative plant cells that cannot regenerate.

[0014] In another aspect, the present invention provides a method for producing a commercial product comprising a detectable amount of the recombinant DNA molecule of the present invention, including products generated therefrom. In some embodiments, the commercial product provided by the present invention comprises inactive seeds or portions thereof, dehydrated plant tissue, frozen plant tissue, processed plant tissue, food, powder, flakes, bran, and fiber. The commercial product may be viable or inactive. Inactive commercial products include, but are not limited to, inactive seeds and grains; processed seeds, seed portions, and plant portions; dehydrated plant tissue, frozen plant tissue, and processed plant tissue. The commercial product of the present invention contains a detectable amount of the recombinant DNA molecule as described herein. Methods for detecting the recombinant DNA molecule of the present invention are well known in the art.

[0015] In another aspect, the present invention provides a method for generating herbicide-resistant plants, the method comprising the steps of: a) transforming plant cells with a DNA construct of the present invention, and b) regenerating plants from the transformed plant cells containing the DNA construct. In one embodiment of the method, the regenerated plants are resistant to herbicides selected from the group consisting of dicamba and PPO inhibitors.

[0016] In another aspect, the present invention provides a method for producing herbicide-resistant plants, the method comprising the steps of: a) hybridizing a parent plant containing a recombinant DNA molecule of the present invention with itself or with a second plant to produce one or more progeny plants; and b) selecting the progeny plants containing the DNA molecule. In one embodiment of the method, the progeny plants are resistant to herbicides selected from the group consisting of dicamba and PPO inhibitors.

[0017] In another aspect, the present invention provides a method for expressing PPO or DMO in plant cells, the method comprising introducing the recombinant DNA molecule of the present invention into the plant cells. In one embodiment of the present invention, the introduction of the recombinant DNA molecule comprises transforming plant cells.

[0018] In another aspect, the present invention provides a method for controlling weed growth in a crop growing environment, the method comprising the steps of: a) planting the plants or seeds of the present invention in the crop growing environment; and b) applying to the crop growing environment an amount of dicamba or PPO inhibitor herbicide that can effectively control weed growth. In a particular embodiment, the herbicide is applied pre-emergence or post-emergence. In one embodiment, the amount of herbicide does not harm the plants or seeds. In some embodiments of the method, the plants or seeds are monocotyledonous plants or seeds, such as corn or wheat plants or seeds. In other embodiments, the plants or seeds are dicotyledonous plants or seeds, such as soybean, cotton, or Brassica plants. In other embodiments, the herbicide is dicamba or a PPO inhibitor. Brief description of the attached diagram

[0019] Figure 1 Transgenic F1 maize plants expressing H_N10 (SEQ ID NO:43) operatively linked to APG6 (SEQ ID NO:1) or 12G088600TP (SEQ ID NO:38) were generated after herbicide application treatments of 0.036 lb ai / acre S-3100 at V2, V4 and V8.

[0020] Sequence Summary

[0021] SEQ ID NO:1 is the amino acid sequence of albino and pale green (APG6) CTP from Arabidopsis thaliana.

[0022] SEQ ID NO:2 is the amino acid sequence of an amino-terminal optimized variant of the APG6 CTP of SEQ ID NO:1.

[0023] SEQ ID NO:3 is the amino acid sequence of Arabidopsis thaliana 90kDa heat shock protein (CR88) CTP.

[0024] SEQ ID NO:4 is the amino acid sequence of Ph.ShkG-CTP4 CTP.

[0025] SEQ ID NO:5 is the amino acid sequence of Ps.RbcS-3C CTP.

[0026] SEQ ID NO:6 is the amino acid sequence of Os.Waxy CTP.

[0027] SEQ ID NO:7-11 are nucleic acid sequences encoding APG6 CTP of SEQ ID NO:1, optimized for monocot or dicot expression.

[0028] SEQ ID NO:12 is the nucleic acid sequence encoding APG6 CTP of SEQ ID NO:2.

[0029] SEQ ID NO:13 and 14 are nucleic acid sequences encoding At.CR88 CTP optimized for monocot or dicot expression, respectively.

[0030] SEQ ID NO:15-17 are the nucleic acid sequences encoding SEQ ID NO:4-6, respectively.

[0031] SEQ ID NO:18-27 is the amino acid sequence encoding a variant of dicamba monooxygenase (DMO).

[0032] SEQ ID NO:28-37 are nucleic acid sequences encoding the DMO variants of SEQ ID NO:18-27, respectively.

[0033] SEQ ID NO:38 is the amino acid sequence of the cotton 12G088600TP chloroplast transport peptide optimized for dicotyledonous expression.

[0034] SEQ ID NO:39 is the nucleic acid sequence encoding SEQ ID NO:38.

[0035] SEQ ID NO:40 is the amino acid sequence of H_N90.

[0036] SEQ ID NO:41 is the amino acid sequence of H_N20.

[0037] SEQ ID NO:42 is the amino acid sequence of H_N60.

[0038] SEQ ID NO:43 is the amino acid sequence of H_N10.

[0039] SEQ ID NO:44 is the amino acid sequence of H_N30.

[0040] SEQ ID NO:45 is the amino acid sequence of H_N40.

[0041] SEQ ID NO:46 is the amino acid sequence of H_N50.

[0042] SEQ ID NO:47 is the amino acid sequence of H_N70.

[0043] SEQ ID NO:48 is the amino acid sequence of H_N100.

[0044] SEQ ID NO:49 is the amino acid sequence of H_N110.

[0045] SEQ ID NO:50-56 are the amino acid sequences that lack the initiating methionine, corresponding to SEQ ID NO:40, 41, 43, 44, 45, 46 and 48, respectively.

[0046] SEQ ID NO:57-58 are amino acid variants of SEQ ID NO:50.

[0047] SEQ ID NO:59 is an amino acid variant of SEQ ID NO:56.

[0048] SEQ ID NO:60 is the amino acid sequence of protoporphyrinogen oxidase from Amaranthus tuberculatus (water hemp) (WH_PPO).

[0049] SEQ ID NO:61-70 are nucleotide sequences encoding SEQ ID NO:40-49, which are codons optimized for expression in Escherichia coli (E. coli).

[0050] SEQ ID NO:71-80 are nucleotide sequences encoding SEQ ID NO:40-49, which are optimized for dicotyl expression codons.

[0051] SEQ ID NO:81-87 are nucleotide sequences encoding SEQ ID NO:50-56, which are optimized for dicotyl expression codons.

[0052] SEQ ID NO:88 and 91 are nucleotide variants of SEQ ID NO:50 and 51, respectively.

[0053] SEQ ID NO:89, 90 and 92 are the nucleotide sequences encoding SEQ ID NO:57-59, respectively.

[0054] SEQ ID NO:93-102 are nucleotide sequences encoding SEQ ID NO:40-49, which are optimized for monocot expression codons. Detailed description

[0055] Chloroplast transport peptides (CTPs) for intracellular localization of herbicide tolerance proteins are known in the art, but the extent of effective subcellular localization and processing of any combination of CTPs and herbicide tolerance proteins is difficult to predict. Localization and processing determine the expression level and function of herbicide tolerance proteins, and thus affect the herbicide tolerance phenotype of transgenic cells, plants, or seeds containing said proteins. Various CTPs have been tested in transgenic plants in the presence of suitable herbicide tolerance proteins, including dicamba monooxygenase (DMO) and protoporphyrinogen oxidase (PPO). However, poor or incomplete processing and localization of the proteins are frequently observed.

[0056] This invention overcomes these obstacles by providing novel recombinant DNA molecules, compositions, and methods of use thereof that enable improved chloroplast localization and processing. The recombinant DNA molecules of this invention comprise a DNA sequence encoding a CTP operably linked to DMO or PPO. The recombinant DNA molecules of this invention provide chloroplast localization of DMO or PPO and improved tolerance to dicamba or PPO herbicides in plants containing the recombinant DNA molecules.

[0057] In some embodiments, the present invention provides a recombinant DNA molecule comprising a DNA sequence encoding a CTP having a sequence selected from the group consisting of SEQ ID NO: 1-3, the DNA sequence being operatively linked to a DNA sequence encoding a herbicide tolerance protein. In some embodiments, the present invention provides a recombinant DNA molecule comprising a DNA sequence encoding a CTP (such as a CTP having a sequence selected from the group consisting of SEQ ID NO: 1-3), the DNA sequence being operatively linked to a DNA sequence encoding a DMO protein (e.g., a DMO protein having a sequence selected from the group consisting of SEQ ID NO: 18-27). In other embodiments, the present invention provides a recombinant DNA molecule comprising a DNA sequence encoding a CTP (such as a CTP having a sequence selected from the group consisting of SEQ ID NO: 1-3), the DNA sequence being operatively linked to a DNA sequence encoding a PPO protein (such as a PPO protein having a sequence selected from the group consisting of SEQ ID NO: 40-60).

[0058] Recombinant DNA molecules

[0059] As used herein, the term "recombinant" refers to non-natural DNA, polypeptides, proteins, cells, seeds, or plants that are the result of genetic engineering and therefore do not normally exist in nature and are formed through human intervention. A "recombinant DNA molecule" is a DNA molecule whose contained DNA sequence is not naturally occurring but is the result of human intervention, such as a DNA molecule comprising a combination of at least two heterologous DNA molecules. An example of a recombinant DNA molecule is a DNA molecule encoding a CTP comprising a sequence selected from the group consisting of SEQ ID NO: 1-3, operatively linked to a DNA sequence encoding a DMO protein comprising a sequence selected from the group consisting of SEQ ID NO: 18-27. Examples of DMO proteins are provided in Table 1 below.

[0060] Table 1. Dicamba monooxygenase (DMO)

[0061]

[0062] Another example of a recombinant DNA molecule is a DNA molecule encoding a CTP comprising a sequence selected from the group consisting of SEQ ID NO: 1-3, operatively linked to a DNA sequence encoding a PPO protein comprising a sequence selected from the group consisting of SEQ ID NO: 40-60. Recombinant cells, seeds, or plants are cells, seeds, or plants containing transgenic DNA, such as transgenic cells, seeds, plants, or plant parts containing the recombinant DNA molecule of the present invention. Examples of PPO proteins are provided in Table 2 below.

[0063] Table 2. Protoporphyrinogen oxidase (PPO)

[0064]

[0065] Table 3 below provides examples of CTP sequences that can be used according to the present invention.

[0066] Table 3. Chloroplast transport peptides (CTPs)

[0067]

[0068]

[0069] As used herein, the term "isolated DNA molecule" means a DNA molecule that exists alone or in combination with other compositions, but not in its natural environment. For example, an isolated DNA molecule is one in which a recombinant DNA molecule containing a protein-coding sequence and a heterologous CTP sequence is present in the genome of a transgenic plant, cell, or seed, because the components of the recombinant DNA molecule are not in its natural environment (i.e., the genome of the organism where each component was first observed). A recombinant DNA molecule present in the genome of a transgenic plant is an isolated DNA molecule, provided that the recombinant DNA molecule is not naturally present in the plant genome and is therefore isolated from its natural environment.

[0070] As used herein, the term "genetic engineering" refers to the artificial creation of DNA, proteins, or organisms that do not normally exist in nature. Genetic engineering can be used to produce DNA, peptides, proteins, cells, seeds, or plants conceived in the laboratory and formed using one or more biotechnologies such as molecular biology, protein biochemistry, bacterial transformation, and plant transformation. For example, genetic engineering can be used to create a chimeric gene comprising a DNA molecule encoding a CTP comprising a sequence selected from the group consisting of SEQ ID NO: 1-3, the DNA molecule being operatively linked to a DMO protein comprising a sequence selected from the group consisting of SEQ ID NO: 18-27; and optionally, a heterologous promoter that may further function in plant cells. In another example, genetic engineering can be used to create a chimeric gene comprising a DNA molecule encoding a CTP comprising a sequence selected from the group consisting of SEQ ID NO: 1-3, the DNA molecule being operatively linked to a PPO protein comprising a sequence selected from the group consisting of SEQ ID NO: 40-60; and optionally, a heterologous promoter that may further function in plant cells. Such chimeric genes can be generated using one or more molecular biology techniques, such as gene cloning, DNA ligation, and DNA synthesis.

[0071] The term "transgenic" refers to a DNA molecule artificially incorporated into the genome of an organism due to human intervention, such as through plant transformation methods. As used herein, the term "transgenic" means containing a transgenic gene; for example, a "transgenic plant" refers to a plant whose genome contains a transgenic gene, and a "transgenic trait" refers to a characteristic or phenotype transmitted or conferred by the presence of a transgenic gene incorporated into the plant's genome. Due to such genomic alterations, transgenic plants are plants that are significantly different from the relevant wild-type plants, and transgenic traits are traits not naturally present in wild-type plants. The transgenic plants of the present invention contain the recombinant DNA molecules provided by the present invention.

[0072] As used herein, the term "heterologous" refers to a relationship between two or more substances that originate from different sources and are therefore not typically related in nature. For example, a DMO protein is heterologous with respect to the CTP it is operatively linked to, if such a combination is not typically found in nature. In another instance, a recombinant DNA molecule encoding a CTP operatively linked to a DMO protein is heterologous with respect to a promoter operatively linked to that function in a plant cell, if such a combination is not typically found in nature. A particular recombinant DNA molecule may also be heterologous with respect to the specific cell, seed, or organism into which it is inserted, if it is not naturally present in that cell, seed, or organism.

[0073] As used herein, the terms "protein-coding DNA molecule" or "peptide-coding DNA molecule" refer to a DNA molecule containing a DNA sequence that encodes a protein or polypeptide, such as proteins or polypeptides used to confer herbicide tolerance or control insects. "Protein-coding sequence" or "peptide-coding sequence" means the DNA sequence that encodes a protein or polypeptide. "Sequence" means the sequential arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence or peptide-coding sequence are typically defined by a 5'-terminal translation start codon and a 3'-terminal translation stop codon. A protein-coding molecule or peptide-coding molecule may contain a DNA sequence that encodes a protein or polypeptide sequence. As used herein, "transgenic expression," "expressing a transgenic," "protein expression," "peptide expression," "expressing a protein," and "expressing a peptide" refer to the production of a protein or polypeptide by transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain that can ultimately fold into a protein. Protein-coding DNA molecules or peptide-coding DNA molecules can be operatively linked to a heterologous promoter in a DNA construct for the expression of a protein or polypeptide in cells transformed with a recombinant DNA molecule. As used herein, "operably linked" means that two DNA molecules are linked in such a way that one DNA molecule can influence the function of the other DNA molecule. Operatically linked DNA molecules can be part of a single, continuous molecule and may or may not be adjacent. For example, a promoter is operably linked to a protein-coding or polypeptide-coding DNA molecule in a DNA construct, wherein the two DNA molecules are arranged such that the promoter can influence the expression of the transgene.

[0074] The recombinant DNA molecule of the present invention comprises a DNA sequence encoding DMO operably linked to a CTP sequence. As used herein, “dicamba monooxygenase” or “DMO” means an oxygenase capable of enzymatically catalyzing the degradation of dicamba (3,6-dichloro-o-methoxybenzoic acid) to 3,6-dichlorosalicylic acid (3,6-DCSA), such as dicamba monooxygenase encoded by the demethylase (dmo) gene from Stenotrophomonas maltophilia. Dicamba monooxygenases are protein sequences known in the art and included in Table 1 as provided and identified in the form of SEQ ID NO:18-27.

[0075] The recombinant DNA molecule of the present invention comprises a DNA sequence encoding PPO operably linked to a CTP sequence. As used herein, “protoporphyrinogen oxidase” or “PPO” means an oxidase capable of enzymatically converting protoporphyrinogen IX to protoporphyrin IX. Protoporphyrinogen oxidases are protein sequences known in the art and included in and identified in Table 2 in the form SEQ ID NO:40-60.

[0076] The recombinant DNA molecule of the present invention comprises a DNA sequence encoding a CTP sequence operably linked to a protein-coding DNA molecule provided by the present invention, whereby the CTP facilitates the intracellular localization of the recombinant protein molecule. CTP is also referred to in the art as a signal sequence, targeting sequence, targeting peptide, and localization sequence. Chloroplasts are also referred to in the art as plastids. By promoting intracellular protein localization, CTP ensures that proteins are localized to chloroplasts to obtain optimal enzymatic activity and can increase the accumulation of recombinant proteins and prevent proteolytic degradation. Upon translocation to chloroplasts, CTP typically cleaves from the protein, a process also referred to as processing. CTP processing can be complete (meaning that complete CTP is cleaved from the amino terminus of the protein), incomplete (meaning that one or more amino acids of the CTP remain at the amino terminus of the protein), or result in the removal of one or more amino acids from the amino terminus of the protein. Complete processing of CTP from DMO proteins increases the level of protein accumulation, thereby increasing dicamba tolerance and reducing damage levels in transgenic cells, seeds, or organisms after herbicide application. CTPs are provided and identified in Table 3 in the form of SEQ ID NOs: 1-6 and 38. DNA sequences encoding each CTP, optimized for expression in both dicotyledons and monocotyledons, are provided in the form of SEQ ID NO:7-17 and 39.

[0077] The recombinant DNA molecules of this disclosure can be synthesized and modified, either wholly or partially, by methods known in the art, particularly when it is desirable to provide sequences suitable for DNA manipulation (such as restriction enzyme recognition sites or recombination-based cloning sites), plant-preferred sequences (such as plant codon usage or Kozak common sequences), or sequences suitable for DNA construct design (such as spacer or adapter sequences). The recombinant DNA molecules of this disclosure comprise degenerate DNA sequences encoding amino acid sequences identical to those provided herein. Degenerate DNA sequences can be prepared using methods and DNA codon tables known in the art. This invention includes recombinant DNA molecules and proteins having at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, and at least 99% sequence identity with any of the recombinant DNA molecules or polypeptide sequences provided herein. For example, the recombinant DNA molecule of the present invention may comprise a DNA sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 7-14 or with a sequence selected from the group consisting of SEQ ID NO: 28-37 and 61-102. The recombinant DNA molecule of the present invention may encode a protein sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-3 or with a sequence selected from the group consisting of SEQ ID NO: 18-27 and 40-59.

[0078] As used herein, the terms "sequence identity percentage" or "sequence identity %" refer to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of the reference ("query") sequence (or its complement) compared to the test ("subject") sequence (or its complement) when two sequences are optimally aligned (with less than 20% of appropriate nucleotide or amino acid insertions, deletions, or vacancies in total within the comparison window). Optimal sequence alignment for the comparison window is well known to those skilled in the art and can be implemented using tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, and Pearson and Lipman's similarity search method, and is implemented by computerized implementations of these algorithms, such as using default parameters. Wisconsin GAP, BESTFIT, FASTA, and TFASTA are available from Accelrys Inc. (San Diego, CA), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, WI 53715), and MUSCLE (version 3.6) (Edgar, Nucleic Acids Research 32(5):1792-7, 2004). The “identity score” of the aligned fragments of the test and reference sequences is calculated by dividing the number of common components shared by the two aligned sequences by the total number of components in the reference sequence fragment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The sequence identity percentage is expressed as an identity score multiplied by 100. Comparisons of one or more sequences can be made for the full-length sequence or a portion thereof, or for a longer sequence.

[0079] As used herein, a “DNA construct” is a recombinant DNA molecule containing two or more heterologous DNA sequences. DNA constructs are suitable for transgenic expression and can be contained in vectors and plasmids. DNA constructs can be used in vectors for transformation purposes (i.e., the introduction of heterologous DNA into host cells) to produce transgenic plants and cells, and therefore can also be contained in plasmid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts. As used herein, “vector” means any recombinant DNA molecule that can be used for plant transformation purposes. For example, a recombinant DNA molecule as described in the sequence listing can be inserted as part of a vector having a recombinant DNA molecule operatively linked to a gene expression element that functions in the plant to affect the expression of a protein encoded by the recombinant DNA molecule. Methods for constructing DNA constructs and vectors are well known in the art. Components of a DNA construct or a vector containing a DNA construct typically include one or more gene expression elements operatively linked to a transcribed DNA sequence, such as: a promoter for expressing the operatively linked DNA, an operatively linked protein-coding DNA molecule, and a 3' untranslated region. Gene expression elements suitable for practicing this invention include, but are not limited to, one or more of the following types of elements: promoter, 5' untranslated region, enhancer, leader sequence, cis-acting element, intron, 3' untranslated region, and one or more optional marker transgenes.

[0080] The DNA constructs of the present invention may include a promoter operatively linked to a protein-coding DNA molecule provided by the present invention, thereby driving the expression of a recombinant protein molecule. Promoters suitable for practicing the present invention include those that function in cells to express operatively linked polynucleotides, such as cellular or plant promoters. Plant promoters are diverse and well known in the art, and include inducible, viral, synthetic, constitutive, time-regulated, space-regulated, and / or spatiotemporally regulated plant promoters.

[0081] As used in this article, "negative control" and "positive control" refer to experimental controls designed for comparative purposes. For example, a negative or positive control in transgenic plant analysis may be a plant of the same type as the experimental plant (i.e., the plant to be tested) but without the transgenic insert, recombinant DNA molecule, or DNA construct of the experimental plant. Examples of plants suitable for comparison with transgenic maize plants include non-transgenic LH244 maize (US Patent No. 6,252,148) or non-transgenic 01DKD2 maize (US Patent No. 7,166,779); non-transgenic A3555 soybean (US Patent No. 7,700,846) or non-transgenic A3244 soybean (US Patent No. 5,659,114, PVP 9600246) suitable for comparison with transgenic soybean plants; non-transgenic Brassica napus restorer line 65037 suitable for comparison with transgenic canola or Brassica napus plants; non-transgenic wheat variety Samson germplasm (PVP 1994) suitable for comparison with transgenic wheat plants; and non-transgenic DP393 (US Patent No. 6,930,228, PVP 200400266) suitable for comparison with transgenic cotton plants.

[0082] Genetically modified plants

[0083] One aspect of the present invention includes transgenic plant cells, transgenic plant tissues, transgenic plants, and transgenic seeds comprising the recombinant DNA molecules provided by the present invention. These cells, tissues, plants, and seeds comprising the recombinant DNA molecules exhibit tolerance to herbicides.

[0084] The insertion of transgenic DNA (referred to as a “transgenic”) into the genome of a plant can be achieved through plant transformation and induces the formation of a new transgenic genomic molecular sequence, referred to as an “event.” Each event is unique, and the DNA sequence of an event is event-specific. Suitable methods for transforming host plant cells for use in this invention include virtually any method that introduces DNA into cells (e.g., in which a recombinant DNA construct is stably integrated into the plant chromosome) and are well known in the art. Exemplary methods for introducing a recombinant DNA construct into a plant include the *Agrobacterium* transformation system and DNA particle bombardment, both of which are well known to those skilled in the art. Another exemplary method for introducing a recombinant DNA construct into a plant is by site-directed integration, inserting the recombinant DNA construct into the plant genome at a predetermined site. Site-directed integration can be accomplished by any method known in the art, such as using zinc finger nucleases, engineered or naturally occurring broad-spectrum nucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., the CRISPR / Cas9 system). The transgenic plant can then be regenerated from the transformed plant cells by plant cell culture. A homozygous transgenic plant (i.e., a plant with two copies of the transgenic allele) can be obtained by self-pollinating (self-pollinating) a transgenic plant containing a single transgenic allele with itself (e.g., an R0 plant) to produce R1 seeds. One-quarter of the resulting R1 seeds will be homozygous regarding the transgene. The conjugability of plants grown from germinating R1 seeds is typically tested using SNP analysis, DNA sequencing, or thermoamplification analysis that allows differentiation between heterozygotes and homozygotes; this is called conjugability analysis.

[0085] The plants, seeds, plant parts, plant tissues, and cells provided by this invention exhibit tolerance to the herbicide dicamba. Dicamba can be applied to plant-growing areas containing the plants and seeds provided by this invention for weed control, including methods of preventing weed growth. The plants and seeds provided by this invention contain herbicide tolerance traits and are therefore tolerant to dicamba application. Herbicide application can be at the recommended commercial rate (1X) or any fraction or multiple thereof, such as twice the recommended commercial rate (2X). The dicamba application rate can be expressed as per pound acid equivalent per acre (lb ae / acre) or per gram acid equivalent per hectare (g ae / ha). When applying the herbicide, the plant-growing area may or may not include weed plants. The herbicidally effective dose of dicamba for controlling weeds in an area should consist of a range of about 0.1X to about 30X label ratios over the growing season. The 1X label ratio for dicamba is 0.5 lb ae / acre. The herbicide ratio can be converted between imperial and metric units as follows: (lb ai / ac)*1.12=(kg ai / ha), and (kg ai / ha)*0.89=(lb ai / ac).

[0086] Plants, seeds, plant parts, plant tissues, and cells may exhibit tolerance to one or more PPO inhibitors, known as PPO herbicides. One or more PPO herbicides may be applied to plant growing areas containing the plants and seeds provided by this invention for weed control, including methods of preventing weed growth. The plants and seeds provided by this invention contain herbicide tolerance traits and are therefore tolerant to the application of one or more PPO herbicides. Herbicide application may be at the recommended commercial ratio (1X) or any fraction or multiple thereof, such as twice the recommended commercial ratio (2X). The plant growing area may or may not include weed plants when the herbicide is applied. The herbicidally effective dose of the PPO herbicide used to control weeds in an area should consist of a range from about 0.1X to about 30X of the labeled ratio during the growing season. PPO herbicides are well known in the art and are commercially available. Examples of PPO herbicides include, but are not limited to, diphenyl ethers (such as acifluorfen, its salts and esters, aclonifen, bifenox, its salts and esters, ethoxyfen, its salts and esters, fluoronitrofen, furyloxyfen, halosafen, chlomethoxyfen, fluoroglycofen, its salts and esters, lactoferrin, its salts and esters, oxyfluorfen, and fomesafen, its salts and esters); and thiadiazoles (such as fluthiacet-methyl and thidiazimin). ; pyrimidinidones or phenyluracils (such as benzfendizone, butafenacil, ethyl[3-2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridinoxy]ethyl acetate (CAS registration number 353292-31-6 and referred to herein as S-3100), flupropacil, saflufenacil, and tiafenacil); phenylpyrazoles (such as fluazolate, pyraflufen, and pyraflufen-ethyl); oxadiazoles (such as oxadiargyl and oxadiazon);Triazoline ketones (such as azafenidin, bencarbazone, carfentrazone, their salts and esters, and sulfentrazone); oxazolidinones (such as pentoxazone); N-phenylphthalimides (such as cinidon-ethyl, flumiclorac, and flumiclorac-pentyl) And flumioxazin; benzoxazinone derivatives (such as 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3,4-dihydro-3-oxo-4-prop-2-ynyl-2H-1,4-benzoxazin-6-yl)-1,3,5-triazin-2,4-dione); flufenpyr and flufenpyr-ethyl; pyraclonil; and profluazol.

[0087] Herbicide application can be carried out sequentially or mixed with one, two, or a combination of several herbicides or any other compatible herbicide. During the growing season, one herbicide or a combination of two or more herbicides, or applied individually and multiple times, can be used on areas containing the transgenic plants of the present invention to control broad-spectrum dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (such as pre-planting and post-emergence or pre-emergence and post-emergence) or three applications (such as pre-planting, pre-emergence, and post-emergence or pre-emergence and two post-emergence applications).

[0088] As used herein, “tolerance” and “herbicide tolerance” refer to the ability of a plant, seed, or cell to resist the toxic effects of herbicide application. Herbicide tolerance of a plant, seed, plant tissue, plant part, or cell can be measured by comparing the plant, seed, plant tissue, plant part, or cell to a suitable experimental control. For example, herbicide tolerance can be measured or assessed by applying a herbicide to a plant containing a recombinant DNA molecule encoding a protein that confers herbicide tolerance (test plant) and to the same species of plant not containing a recombinant DNA molecule encoding a protein that confers herbicide tolerance (negative control plant), and then comparing the plant damage to the two plants, wherein the herbicide tolerance of the test plant is indicated by a reduction in the damage level compared to the damage level of the negative control plant. Herbicide-tolerant plants, seeds, plant tissues, plant parts, or cells exhibit a reduced response to the toxic effects of the herbicide compared to negative control plants, seeds, plant tissues, plant parts, or cells. As used in this article, "herbicide tolerance trait" refers to a transgenic trait that confers improved herbicide tolerance to plants compared to negative control plants.

[0089] The transgenic plants, offspring, seeds, plant cells, and plant parts of the present invention may also contain one or more additional transgenic traits. Additional transgenic traits can be introduced by hybridizing a plant containing a transgenic gene comprising a recombinant DNA molecule provided by the present invention with another plant containing the additional transgenic trait. As used herein, “hybridization” means breeding two individual plants to produce offspring plants. Thus, two transgenic plants can be hybridized to produce offspring containing the transgenic trait. As used herein, “offspring” means any generation of the parent plant, and the transgenic offspring contain a DNA construct provided by the present invention and inherited from at least one parent plant. Alternatively, the additional transgenic trait can be obtained by co-transforming a DNA construct comprising a recombinant DNA molecule provided by the present invention (e.g., where all DNA constructs are part of the same vector used for plant transformation) or by inserting the additional trait into a transgenic plant containing a DNA construct provided by the present invention, or vice versa (e.g., by using any method of plant transformation of a transgenic plant or plant cell). Such additional transgenic traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield performance, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, wherein the trait is measured relative to the wild-type plant. Such additional transgenic traits are known to those skilled in the art; for example, a list of such traits is provided by the Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA).

[0090] Transgenic plants and progeny containing the transgenic traits provided by this invention can be used with any breeding method generally known in the art. In plant lines containing two or more transgenic traits, the transgenic traits can be independently segregated, linked, or a combination of both in plant lines containing three or more transgenic traits. Backcrossing with parental plants and heteromorphic hybridization with non-transgenic plants, as well as asexual reproduction, are also considered. Descriptions of breeding methods commonly used for different traits and crops are well known to those skilled in the art. To confirm the presence of transgenes in a particular plant or seed, various analyses can be performed. Such analyses include, for example, molecular biological analyses such as Southern blotting and Northern blotting, PCR, and DNA sequencing; biochemical analyses such as detecting the presence of protein products by, for example, immunological means (ELISA and Western blots) or by enzyme function; plant part analyses such as leaf or root analyses; and analyses of the phenotype of the whole plant. To analyze CTP processing in specific transgenic plants or seeds, recombinant DMO or PPO proteins obtained from transgenic cells, plants, or seeds can be analyzed using methods such as Edman degradation sequencing or mass spectrometry, and the resulting sequence data can be compared with those of DMO or PPO proteins.

[0091] As a result of backcrossing transformation, transgenic traits are introduced into plant genotypes. Plant genotypes with introduced transgenic traits can be called backcrossed genotypes, lines, inbreds, or hybrids. Similarly, plant genotypes lacking the desired transgenic trait can be called untransformed genotypes, lines, inbreds, or hybrids.

[0092] As used in this article, the term "comprising" means "including but not limited to". Example

[0093] The following embodiments are included to illustrate embodiments of the invention. Based on this disclosure, those skilled in the art will recognize that many changes can be made to the specific embodiments provided, and the same or similar results can still be obtained without departing from the scope and concept of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant reagents can be substituted with reagents described herein that yield the same or similar results. All such substitutions and modifications that are apparent to those skilled in the art are considered to be within the scope and concept of the invention.

[0094] Example 1: Expression and localization of CTP-DMO in soybean protoplasts

[0095] Soybean protoplast analysis was used to assess the relative chloroplast targeting efficiency of recombinant proteins containing one of five CTPs operatively linked to a DMO sequence (SEQ ID NO:27). To monitor the cytosolic and chloroplast distribution of the recombinant proteins, a sequence encoding green fluorescent protein was added to a cassette encoding the recombinant CTP and DMO combination (referred to herein as CTP-DMO), such that the green fluorescent protein was fused to the C-terminus of the DMO.

[0096] Protoplasts were prepared from soybean cotyledons (germplasm A3244). Immature soybean seed pods were harvested and seeds (4-6 mm long) were removed using aseptic techniques. The cotyledons of each seed were manually removed, transversely cut into 1 mm fragments, and incubated in CPW buffer (pH 5.8) in the presence of 0.7 M mannitol at 24-26°C in the dark for 1 hour with shaking at 40 RPM. Then remove the buffer and replace it with enzyme buffer (4% cellulase 'onozuka' R-10; 2% hemicellulase; 0.3% cleavage enzyme R-10; in CPW buffer (pH 5.8; containing 0.49M mannitol). Incubate the cotyledon tissue on a rotary shaker at 50 rpm at 24–26°C for 2 hours. At the end of this incubation, release soybean protoplasts from the cotyledon tissue by manually rotating the plate and filtering the suspension through a double-layered 60 μm nylon mesh into a 50 mL conical tube. Gently wash the protoplasts once with resuspension and centrifugation. Resuspend the final globules in buffer (4 mM MES, pH 5.7; 150 mM NaCl; 5 mM CaCl2; 0.5 M mannitol) and incubate on ice for 1 hour. Then centrifuge the protoplasts and resuspend the globules in transformation buffer (0.4 M mannitol; 15 mM MgCl2; 4 mM MES, pH 5.7). In step 5.7), adjust the volume to allow 1 x 10,000,000 protoplasts per mL. Transformation is accomplished by mixing 12.5 μg DNA for each construct. Gently mix the DNA with 1.5 x 1,000,000 protoplasts, followed by the addition of an equal volume of PEG buffer. Incubate for 5 minutes, then slowly dilute with 300 μl of W5 buffer (154 mM NaCl; 125 mM CaCl2; 5 mM KCl; 2 mM MES, pH 5.7). Incubate for 5–10 minutes, then slowly add 900 μl of W5 buffer. Allow the protoplasts to aggregate and resuspend them in WI buffer (0.5 M mannitol; 4 mM MES (pH 5.7); 20 mM PEG buffer). Incubation was performed in KCl at 24–26 °C in the dark. Microscopic analysis was performed using a Zeiss LSM510 META laser scanning microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY) equipped with a krypton-argon ion (458, 488 nm) laser, a green (543 nm) helium-neon laser, and a combination of FITC and Texas red filters. Image acquisition and analysis were performed using a ZEN 2012v.8.1 (Carl Zeiss MicroImaging, Inc., Thornwood, NY) and a 40X immersion 1.2 numerical aperture objective.The excitation wavelengths used were 488 nm (GFP) and 543 nm (chloroplast autofluorescence), and the emission filters were 500–530 nm (GFP) and 630–700 nm (chloroplast autofluorescence). For each construct, construct localization was scored for at least 50 individual cells: cytosol, plastid, or both. Results were recorded as the percentage of cells with proteins localized in cytosol or plastid (or both) out of the total number of cells analyzed, and are provided in Table 4.

[0097] Table 4. Targeting analysis of soybean protoplasts

[0098]

[0099] Of the five CTP-DMO combinations analyzed, only APG6 CTP (SEQ ID NO:1) resulted in 100% of cells showing protein localization only in plastids. At.CR88 CTP (SEQ ID NO:3) resulted in 94% of cells showing protein localization only in plastids, and 6% of cells showing protein localization in both cytosol and plastids. 'A' CTP resulted in 79% of cells showing protein localization only in plastids, and 21% of cells showing localization in both cytosol and plastids. 'B' CTP resulted in 9% of cells showing protein localization only in plastids, and 91% of cells showing localization in both plastids and cytosol. 'C' CTP resulted in 18% of cells showing protein localization only in plastids, and 82% of cells showing localization in both plastids and cytosol. Without CTP, the protein was only present in the cytosol. These results indicate that APG6 CTP is 100% effective in targeting CTP-DMO to plasmids, and At.CR88 CTP is 94% effective in targeting CTP-DMO to plasmids.

[0100] Example 2: CTP-DMO processing in genetically modified wheat

[0101] Protein expression and CTP processing were assessed using transgenic wheat plants transformed with DNA constructs containing a recombinant DNA molecule encoding one of four independent CTPs operatively linked to DMO.

[0102] Four different plant transformation vectors were used to produce transgenic wheat plants, each containing a DNA construct of one of four different CTPs operably linked to a DMO operably linked to a promoter. Pre-cultured Samson germplasm (PVP 1994) wheat immature embryos were transformed with *Agrobacterium tumefaciens* using methods known to those skilled in the art to produce transgenic seedlings. Leaf samples were taken for molecular analysis to confirm the transgenic copy number in the genome for each unique event, and R0 plants with one transgenic copy were self-pollinated and R1 seeds were collected.

[0103] Grind the seeds (50g) into powder, then add it to 250ml of extraction buffer (1xTBE (89mM Tris-borate, 2mM EDTA, pH 8.4), 200mM NaCl, 10% glycerol, 1mM benzyl sulfonyl fluoride (PMSF), 5mM benzalkonium chloride, 2mM dithiothreitol (DTT), cOmplete TM In protease inhibitors (Roche Diagnostics Corporation, Indianopolis, IN), and used... Homogenize (VWR, Radnor, PA) for approximately 20 seconds, then incubate at 4°C with shaking for 1 to 2 hours. Centrifuge the mixture at 9,000 rpm for 25 min at 4°C and precipitate the supernatant sequentially with 10% and 55% saturated ammonium sulfate (AS), each precipitation step centrifuged at 18,000 rpm for 20 min. Discard the pellets from the 10% AS precipitate.

[0104] Mix pellets from the 10-55% fraction with one piece of COmplete TM The protease inhibitor was dissolved together with PBS (0.1M sodium phosphate, 0.15M NaCl) in 30 ml. The dissolved pellets were centrifuged, and the supernatant was filtered through a 0.22 μm membrane. Goat polyclonal antibody serum against DMO was then mixed with Pierce... TM A 1:1 suspension of protein A / G agarose resin (ThermoFischer Scientific, Grand Island, NY) was mixed. After 1.5 hours, the protein A / G agarose resin loaded with anti-DMO Ab was washed three times with PBS and added to approximately 30 ml of 10%–55% AS filter fraction. After incubation, the resin was rotated and washed three times with PBS, then resuspended in 1 ml of PBS and transferred to a microcentrifuge tube to agglomerate again.

[0105] The final spheres were resuspended in 2X Laemmli buffer, boiled for 5 minutes, and the samples were run on 10% SDS-PAGE gels in Tris-glycine buffer at 185V (constant). Proteins from the SDS-PAGE gels were transferred to PVDF membranes using CAPS transfer buffer and incubated at 4°C and 100V for 30 minutes. Proteins bound to the PVDF membrane were stained with Coomassie blue for approximately 30 seconds, and bands corresponding to each DMO protein in the 10%–55% AS fraction were excised from the PVDF ink dots and used for N-terminal protein sequencing. N-terminal protein sequencing was performed using automated Edman degradation chemistry, with 15 cycles per analysis. The Applied Biosystems 494 system was used, featuring a 140C microgradient pump and a Perkin Elmer Series 200UV / Vis detector. The sequencing system was used for analysis, controlled using Procise Control (version 2.1) software (ThermoFischer Scientific, Grand Island, NY). Version 2.1 of the protein sequencing analysis software collects chromatographic data. If at least eight amino acids are observed to match the predicted sequence of the expected protein, the identity of each protein is determined. Table 5 presents the results of N-terminal sequencing.

[0106] Table 5. N-terminal sequencing of recombinant proteins

[0107]

[0108] The names DMO, DMO+1, DMO+10, and DMO+12 were used to indicate that protein sequencing showed 0, 1, 10, or 12 amino acids, respectively, retained at the N-terminus of the DMO in the post-processed CTP. The name DMO-1 was used to indicate that the first methionine in the DMO was removed after processing. Two unique events were tested for APG6 CTP (SEQ ID NO:18) operably linked to DMO (SEQ ID NO:18). Both samples showed one amino acid retained at the N-terminus of the DMO in the post-processed CTP (DMO+1). Three unique events were tested for At.CR88 CTP (SEQ ID NO:3) operably linked to DMO (SEQ ID NO:18). All three samples showed zero or one amino acid retained at the N-terminus of the DMO in the post-processed CTP (DMO and DMO+1). The events tested from CTP4 (SEQ ID NO:4) operably linked to DMO (SEQ ID NO:19) showed that twelve amino acids of the processed CTP were retained at the N-terminus of the DMO (DMO+12). Two unique events were tested for Os. Waxy CTP (SEQ ID NO:6) operably linked to DMO (SEQ ID NO:18). One sample showed that ten amino acids of the processed CTP were retained at the N-terminus of the DMO (DMO+10), and another showed that the first methionine of the DMO was removed after processing (DMO-1). These results indicate that APG6 CTP and At.CR88CTP are efficiently processed from DMO when expressed in transgenic plants.

[0109] Example 3: CTP-DMO expression in transgenic Brassica napus

[0110] The use of transgenic Brassica napus plants was used to evaluate the ability of DNA constructs containing recombinant DNA molecules encoding one of three independent CTPs operablely linked to DMO to provide dicamba tolerance.

[0111] Transgenic Brassica napus plants were produced using three different plant transformation vectors, each containing a DNA construct with one of three different CTPs operably linked to a DMO operably linked to a promoter. Agrobacterium-mediated transformation was performed using the Brassica napus cultivar 65037 restorer line, and R0 plants were grown in a greenhouse. Screening was conducted for unique events based on transgenic copy number. R0 plants with one transgenic copy were self-pollinated, and R1 seeds were collected.

[0112] Dicamba tolerance was assessed using R0 plants with one transgenic copy plus a vector backbone or two transgenic copies. Dicamba tolerance was defined as 20% or less dicamba damage under greenhouse conditions. R0 events in pods were divided into three groups, and dicamba was applied at one of the following three ratios. (1) No dicamba, (2) 1 lb ae / acre dicamba (2X ratio), or (3) 2 lb ae / acre dicamba (4X ratio). Transgenic plants were sprayed and damage ratings were recorded after 21 days. Plants containing the “A” CTP operably linked to DMO (SEQ ID NO:21) showed no dicamba tolerance events. Plants containing the RbcS CTP (SEQ ID NO:5) operably linked to DMO (SEQ ID NO:21) showed tolerance to dicamba at the 2X ratio in 8 out of 9 events and to the 4X ratio in 7 out of 7 events. Plants containing the APG6 CTP (SEQ ID NO:1) operably linked to DMO (SEQ ID NO:20) showed tolerance to dicamba at the 2X ratio in 7 out of 14 events and to the 4X ratio in 6 out of 18 events. Results are presented in Table 6.

[0113] Table 6. Dicamba tolerance in R0 Brassica napus

[0114]

[0115] Tolerance to dicamba was assessed in R0 plants with one transgenic copy. Plants were sprayed with dicamba (Clarity) at a rate of 1 lb ae / acre (2X ratio) in a greenhouse, and tolerance was determined after 14 to 21 days. Plants containing the APG6 CTP operably linked to DMO (SEQ ID NO:20) showed tolerance to dicamba in 13 out of 31 events. Plants containing the RbcS CTP operably linked to DMO (SEQ ID NO:21) showed tolerance to dicamba in 13 out of 17 events. Plants containing the “A” CTP operably linked to DMO (SEQ ID NO:21) showed tolerance to dicamba in 7 out of 18 events. Results are presented in Table 7.

[0116] Table 7. Dicamba tolerance in R0 Brassica napus

[0117] APG6 (SEQ ID NO:1) SEQ ID NO:20 13 / 31 RbcS (SEQ ID NO:5) SEQ ID NO:21 13 / 17 A (Constructor 7) SEQ ID NO:21 7 / 18

[0118] Ten seeds from each of 28 R1 plants containing APG6 CTP (APG6+DMO) operably linked to DMO (SEQ ID NO:20) and ten seeds from each of 17 R1 plants containing RbcS CTP (RbcS+DMO) operably linked to DMO (SEQ ID NO:21) were grown in a greenhouse. Plants were sprayed with 2 lb ae / acre dicamba (4X) on the day of planting, followed by spraying with 1 lb ae / acre dicamba (2X) at stage V3, and again at initial flowering (defined as >90% of plants having bolls and approximately 25% having at least one open flower). Damage was rated 7 days after each spray and expressed as a percentage of damage compared to the sprayed control. For plants containing APG6+DMO, a total of 9 progeny from 2 events had a dicamba damage rating of ≤20% in each of the three rating phases. For plants containing RbcS+DMO, 77 plants from 16 events had a dicamba tolerance of less than 20% in each of the three rating phases.

[0119] Protein characterization was performed using leaves harvested from the R0 event. Leaf tissue was milled in liquid nitrogen and extracted with two volumes of 2X Limri buffer (BioRad, Hercules, CA) containing 10% 2-mercaptoethanol and 5 mM DTT. The samples were boiled and 10 μl was loaded into 4–20% Criterion. TM The protein was loaded onto a pre-prepared gel (BioRad, Hercules, CA) and run at 250 V for 45 minutes in Tris / glycine / SDS buffer. The protein in the gel was then transferred to a PVDF membrane at 400 mA in Tris / glycine buffer containing 20% ​​methanol for 30 minutes. DMO protein was detected using polyclonal rabbit anti-DMO antiserum and an HRP-conjugated anti-rabbit secondary antibody. SuperSignal was used. TMThe signal was detected using a West Pico chemiluminescence assay kit (ThermoFischer Scientific, Grand Island, NY). For each of the three events containing APG6-DMO, a single band of approximately 38 kDa was observed, which is the expected size of fully processed DMO protein. For each of the six events containing RbcS-DMO, two bands of approximately 38 kDa and approximately 41 kDa were observed. The 41 kDa band is consistent with DMO+27 and has been previously documented in soybeans containing RbcS-DMO (US Patent No. 7,838,729). In all events containing “A”CTP-DMO, DMO protein expression was very low, and the signals detected after prolonged exposure were bands of approximately 50 kDa and approximately 39 kDa. The 50 kDa band approximates the expected size of unprocessed “A”CTP-DMO. These results indicate that APG6-DMO produces a single band of the expected size consistent with fully processed DMO.

[0120] Recombinant proteins were purified from leaf tissues of R0 plants containing either APG6-DMO or RbcS-DMO. N-terminal sequence analysis was performed using Edman degradation chemistry as described. N-terminal sequence analysis confirmed the presence of the DMO N-terminal sequences DMO+27 and DMO-1 in plants containing RbcS-DMO, consistent with the size of the DMO band observed on western ink spots. N-terminal sequence analysis confirmed the presence of only the DMO N-terminal sequence DMO+1 in plants containing APG6-DMO, consistent with the size of the DMO band observed on western ink spots. This result confirms the complete processing of operatively linked DMO in plants using APG6CTP.

[0121] Example 4: CTP-DMO expression in transgenic maize

[0122] The expression of DNA constructs containing a recombinant DNA molecule encoding one of two independent CTPs operablely linked to DMO was analyzed in transgenic maize cells and plants.

[0123] Relative DMO expression of the two CTP-DMO combinations was evaluated using transient transformation of maize mesophyll protoplasts. The DNA constructs were identical, except that the CTP operably linked to DMO (SEQ ID NO:18) was either APG6 (SEQ ID NO:1) or CTP4 (SEQ ID NO:4). Protoplasts were prepared essentially as described in Example 1. After transformation, cells were harvested and DMO protein levels were determined using enzyme-linked immunosorbent assay (ELISA). For each CTP-DMO combination, protein from the four transformed protoplast samples was measured in nanograms (ng) of DMO per milligram (mg) of total protein. Protoplasts transformed with APG6-DMO showed approximately 4-fold higher DMO levels compared to protoplasts transformed with CTP4-DMO. Data are provided in Table 8.

[0124] Transgenic maize plants were generated using DNA constructs, and R0 plants were grown. Leaf samples were collected from R0 plants representing eight unique single-copy events and used for quantitative ELISA to measure DMO levels. DMO expression in R0 leaf tissues was approximately 4-fold higher in events containing APG6-DMO compared to events containing CTP4-DMO. Data are presented in Table 8.

[0125] N-terminal sequencing was performed on DMO expressed in transgenic maize plants. Essentially as described in Example 2, proteins were purified from transgenic maize plants expressing CTP4-DMO or APG6-DMO and prepared for Edman degradation sequencing. N-terminal sequence analysis confirmed the presence of DMO+6, DMO+7, and DMO+12 N-terminal sequences in plants containing CTP4-DMO. N-terminal sequence analysis confirmed the presence of DMO and DMO+1 N-terminal sequences in plants containing APG6-DMO. These results indicate that CTP processing is more complete in the APG6 case, as evidenced by fewer CTP amino acids remaining at the N-terminus of DMO compared to CTP4. Data are provided in Table 8.

[0126] Table 8. DMO protein expression in maize

[0127]

[0128] Transgenic maize was produced by transformation mediated by *Agrobacterium* using DNA constructs containing recombinant DNA molecules encoding APG6-DMO or CTP4-DMO, employing methods known to those skilled in the art. Tolerance to dicamba was assessed in a field trial of the transgenic F1 hybrids. The field trial consisted of four treatments administered at two sites, each treatment repeated twice. The four treatments were: (1) application of dicamba at 2 lb ae / acre (4X) sequentially at V2, V4, and V8. (2) Dicamba was applied sequentially at 4 lb at / acre (8X) in V2, V4, and V8; (3) dicamba was applied sequentially at 8 lb at / acre (16X) in V2, V4, and V8; and (4) dicamba was applied sequentially at 16 lb at / acre (32X) in V2, V4, and V8. Crop damage was rated at 10 days post-treatment and measured as a percentage of crop damage for each V-stage (CIPV2, CIPV4, or CIPV8). Grains were harvested at the end of the season and yield was measured in bushels / acre. For CIPV ratings versus yield, the least significant difference (LSD) was calculated at a probability of 5% (p = 0.05). The highest dicamba ratios (16X and 32X) applied to F1 hybrids containing APG6-DMO showed slightly less nutrient damage and higher grain yields than those containing CTP4-DMO. Data are provided in Table 9.

[0129] Table 9. Field Trial Results of MCPA Damage and Yield in F1 Hybrids

[0130]

[0131] Example 5: CTP-DMO expression in transgenic cotton and soybean

[0132] APG6 CTP was optimized to improve protein translation efficiency (protein synthesis) and increase protein accumulation. The optimized APG6 CTP (SEQ ID NO:2) has an amino acid change from threonine (T) to serine (S) at positions 3 and 4 of the APG6 CTP (SEQ ID NO:1). DNA constructs were prepared to compare two CTPs, each operatively ligated to DMO from soybean.

[0133] Transgenic soybean plants were generated using two identical DNA constructs, except for the APG6 CTP. The first DNA construct had APG6 (SEQ ID NO:1) operably ligated to DMO (SEQ ID NO:18). The second DNA construct had an optimized APG6 (SEQ ID NO:2) operably ligated to DMO (SEQ ID NO:18). A3555 soybeans were transformed using each DNA construct via an Agrobacterium-mediated transformation method. Following transformation, R0 transgenic plants containing a single transgenic copy were identified by PCR analysis. The single-copy R0 plants were grown in a greenhouse, and R1 seeds were harvested. Ten R1 seeds from each of the four events generated using each of the two DNA constructs, along with AG3555 seeds, were planted to assess crop tolerance to post-emergence dicamba treatment under standard greenhouse growing conditions. Dicamba (Clarity) was applied at 1120 g ai / ha in stage V4. Crop damage was rated 10 days post-treatment. Leaf samples from dicamba-tolerant soybean plants were taken for recombinant protein level measurements and N-terminal sequence analysis. For single-copy dicamba-tolerant R1 transgenic soybean plants with an APG6 CTP (SEQ ID NO:1) operably linked to DMO (SEQ ID NO:18), the DMO protein level was 13.35 ± 2.7 ng / mg, detected by ELISA. For single-copy dicamba-tolerant R1 transgenic soybean plants with an optimized APG6 CTP (SEQ ID NO:2), the DMO protein level was 18.55 ± 3.1 ng / mg, detected by ELISA. DMO protein was not detected in the leaf tissue of the negative control A3555 soybean. The dicamba damage rating of single-copy R1 transgenic soybean plants with an APG6 CTP (SEQ ID NO:1) operably linked to DMO (SEQ ID NO:18) was 3.6%. The dicamba damage rating of single-copy R1 transgenic soybean plants with an optimized APG6 CTP (SEQ ID NO:2) operably linked to DMO (SEQ ID NO:18) was 2.7%. The negative control A3555 soybean showed a dicamba damage rating of 99.8%. Leaf samples from single-copy dicamba-tolerant R1 transgenic soybean plants were subjected to N-terminal sequencing (as described in Examples 2 and 4). N-terminal sequence analysis confirmed that processing of APG6-DMO and optimized APG6-DMO resulted in complete processing of CTPs from the N-terminus of the DMO protein. DMO levels, dicamba damage, and APG6-DMO processing indicate that both APG6 and optimized APG6, when operably linked to DMO, provide tolerance to dicamba, and that both CTPs are fully processed in the plants. Data are provided in Table 10.

[0134] Table 10. R1 Soybean Greenhouse Test

[0135]

[0136] Transgenic cotton plants were produced using two identical DNA constructs, except for the APG6 CTP. The first DNA construct had APG6 (SEQ ID NO:1) operably linked to DMO (SEQ ID NO:18). The second DNA construct had an optimized APG6 CTP (SEQ ID NO:2) operably linked to DMO (SEQ ID NO:18). Each DNA construct was transformed into cotton via Agrobacterium-mediated transformation using methods known to those skilled in the art. After transformation, R0 transgenic cotton plants containing a single transgenic copy were identified by PCR analysis, grown in a greenhouse, and R1 seeds were harvested. Ten R1 seeds from each of the ten events of each construct and seeds from DP393 cotton were planted to assess crop tolerance to post-emergence dicamba application. Dicamba (Clarity) was applied at 1120 g ai / ha in stage V4. Crop damage percentage rating was performed 9 days post-treatment. Leaf samples from tolerant cotton plants were used for protein level measurements and N-terminal sequence analysis of APG6-DMO or optimized APG6-DMO. For single-copy dicamba-tolerant R1 transgenic cotton plants with an APG6 CTP (SEQ ID NO:1) operably linked to DMO (SEQ ID NO:18), the DMO protein level detected by ELISA was 176.2 ± 103 ng / mg. For single-copy dicamba-tolerant R1 transgenic cotton plants with an optimized APG6 CTP (SEQ ID NO:2), the DMO protein level detected by ELISA was 136.5 ± 58.6 ng / mg. DMO protein was not detected in the leaf tissue of the negative control DP393 cotton. The dicamba damage in single-copy R1 transgenic cotton plants with an APG6 CTP (SEQ ID NO:1) operably linked to DMO (SEQ ID NO:18) was 2.6%. Single-copy R1 transgenic plants with optimized APG6 CTP (SEQ ID NO:2) operably linked to DMO (SEQ ID NO:18) showed 2.2% dicamba damage. The negative control DP393 cotton showed 85% damage. Leaf samples from single-copy dicamba-tolerant R1 plants were subjected to N-terminal sequencing (as described in Examples 2 and 4). N-terminal sequence analysis confirmed that processing of APG6-DMO and optimized APG6-DMO resulted in complete processing of the CTP from the N-terminus of the DMO protein. DMO protein expression levels, dicamba damage, and N-terminal processing of APG6-DMO and optimized APG6-DMO indicated that both APG6 and optimized APG6, when operably linked to DMO, provide tolerance to dicamba, and that both CTPs are fully processed in the plants.The data is provided in Table 11.

[0137] Table 11. R1 Cotton Greenhouse Test

[0138]

[0139] Example 6: CTP-PPO expression in transgenic maize

[0140] A herbicide-resistant bacterial screening system was used to identify novel PPOs resistant to PPO herbicides. This screening system used growth analysis of knockout E. coli strains in LB broth containing PPO herbicides to identify PPOs insensitive to PPO herbicides.

[0141] The knockout *E. coli* strain was transformed with a bacterial expression vector containing defined PPO activity and cultured in LB broth. Purified crystalline forms of one of five different PPO herbicides representing three different PPO chemical subclasses (trifluralin (1 mM), propyzamide (0.5 mM), quizalofop-P-ethyl (0.5 mM), flufenoxuron (1 mM), and S-3100 (100 μM)) were added to the medium. Recombinant protein expression was performed, and *E. coli* growth rates were measured. Growth curves (OD600) of different variants were measured at selected time points from 0 to 24 hours in the presence and absence of PPO herbicides. The growth of the transformed knockout *E. coli* strain in LB broth in the presence of PPO herbicides indicated that the gene used for transformation of *E. coli* encodes herbicide-insensitive protoporphyrinogen oxidase (iPPO).

[0142] The study found that all ten PPOs provided in SEQ ID NO:40-49 induced normal growth rates in knockout E. coli strains in LB medium in the presence of PPO herbicides, indicating that these proteins are herbicide-insensitive protoporphyrinogen oxidases (iPPO). The knockout E. coli strain expressing WH_PPO (SEQ ID NO:60) was sensitive to all five PPO herbicides, confirming that the analysis could distinguish between sensitive and insensitive PPOs for each herbicide.

[0143] Four plant transformation vectors were developed for in situ expression of PPO H_N10 (SEQ ID NO:43) in plants. Transformation constructs 1 and 11 have the same promoter plus leader sequence plus intron combination, the same 3'UTR sequence, and the same PPO H_N10 (SEQ ID NO:43), but different CTP sequences, and were used in soybean transformation. Transformation constructs 6 and 16 have the same promoter plus leader sequence plus intron combination, the same 3'UTR sequence, and the same PPO H_N10 (SEQ ID NO:43), but different CTP sequences, and were used in maize transformation. Table 12 provides the configurations of the PPO H_N10 plant transformation constructs.

[0144] Table 12. Configurations of constructs containing PPO H_N10

[0145]

[0146] PPO enzymes were expressed in transgenic maize plants, and the PPO herbicide tolerance of the transgenic plants was analyzed. Plant transformation vectors were constructed comprising a recombinant DNA molecule encoding one of the PPO enzymes provided in SEQ ID NO:40-59. The DNA sequence encoding the PPO enzyme may include a codon about methionine at the 5' end, commonly referred to as the start codon, or this codon may be omitted to facilitate the operative attachment of the chloroplast transport peptide sequence to the 5' end of the coding sequence. Examples of PPO enzyme protein sequences containing methionine at the N-terminus are provided in SEQ ID NO:40-49. Examples of PPO enzyme protein sequences without methionine at the N-terminus are provided in SEQ ID NO:50-59. For plant transformation, the nucleotide sequence encoding the putative PPO enzyme was optimized for dicotyledonous or monocotyledonous expression. Table 2 provides the SEQ ID NOs corresponding to the protein and nucleotide sequences of the PPO enzymes in the transformation vector.

[0147] For in situ testing of maize plants, maize (LH244) was transformed using *Agrobacterium tumefaciens* and standard methods known in the art. Herbicide tolerance was tested in a greenhouse on transgenic F1 plants produced by heteromorphic hybridization of single-copy R0 plants expressing H_N10 (SEQ ID NO:43) in one of two construct configurations. Plants were treated with 40 g / ha S-3100 at the V3 growth stage, and damage was rated seven days post-treatment. Transgenic maize plants expressing H_N10 (SEQ ID NO:43) in construct configuration 6 (APG6 (SEQ ID NO:1) operably linked to PPO H_N10 (SEQ ID NO:43)) resulted in highly tolerant plants (10% or less damage) in 13 out of 18 events, but construct configuration 16 (12G088600TP (SEQ ID NO:38) operably linked to PPO H_N10 (SEQ ID NO:43)) did not produce highly tolerant plants.

[0148] Herbicide tolerance was tested in the field on transgenic F1 plants produced by heteromorphic hybridization of single-copy R0 plants expressing H_N10 (SEQ ID NO:43) in one of two constructs (constructs 6 and 16). This F1 population was segregated (50% hemizygotes and 50% naked) and no transgenic plants were selected prior to damage rating. Due to the difficulty in distinguishing transgenic from non-transgenic plants, the overall mean damage rating of this population was predicted to be higher than that of the homogeneous transgenic population. The trials were conducted at two sites, each repeated twice, with three treatments per construct. Non-transgenic maize plants were used as negative controls. Herbicide application treatments were as follows: Treatment 1 was 0.036 lb ai / acre S-3100 applied sequentially to V2, V4, and V8; Treatment 2 was 0.072 lb ai / acre S-3100 applied sequentially to V2, V4, and V8; Treatment 3 was 0.144 lb ai / acre S-3100 applied sequentially to V2, V4, and V8. The percentage rating of crop damage was assessed 5 to 7 days after treatment at both the V2 growth stage (CIPV2) and the V4 growth stage (CIPV4) (errors of half the least significant difference (LSD) for V2 and V4). The crop damage ratings from both sites were combined. For each of the three treatments, all non-GMO plants and plants in the event case induced using construct 16 (12G088600TP (SEQ ID NO:38) operably linked to PPO H_N10 (SEQ ID NO:43)) showed damage between 94.6% and 99.5% after herbicide application for both V2 and V4. Plants in the event case induced using construct 6 (APG6 (SEQ ID NO:1) operably linked to PPO H_N10 (SEQ ID NO:43)) showed damage only 30% to 50% after herbicide application for V2 and no damage after herbicide application for V4. Data are provided in Table 13.

[0149] Table 13. Field trials of the efficacy of F1 maize containing PPO H_N10 (SEQ ID NO:43).

[0150]

[0151]

[0152] Greenhouse and field data for F1 transgenic maize demonstrated that expression of APG6 (SEQ ID NO:1) operatively linked to PPO H_N10 (SEQ ID NO:43) in transgenic plants produced a reduced level of damage compared to the damage level observed when 12G088600TP (SEQ ID NO:38) operatively linked to PPO H_N10 (SEQ ID NO:43) was expressed in transgenic plants. See also Figure 1 .

[0153] Plant transformation vectors were developed for in situ expression of PPO H_N40 (SEQ ID NO:54) or PPO H_N90 (SEQ ID NO:50) operatively ligated to APG6 (SEQ ID NO:1), CTP D, or CTP E. Maize (01DKD2) was transformed using *Agrobacterium tumefaciens* and standard methods known in the art. Leaf samples taken from the resulting R0 plants were analyzed by PCR to determine the copy number of the transgenic insert. At approximately the V5 growth stage, R0 plants each containing a unique transformation event were sprayed with 40 g ai / ha or 80 g ai / ha of S-3100, and damage was rated 4–7 days post-treatment. The number of plants with ≤10% damage (highly tolerant) or ≤20% damage (tolerant) out of the total number of sprayed plants was recorded. Plants identified as having a single copy event and with ≤20% damage after spraying were further used for self-pollination and cross-pollination. Data are presented in Table 14.

[0154] Table 14. Assessment of CTP-PPO herbicide tolerance in genetically modified maize

[0155]

[0156] The results showed that when operably linked to H_N40 (SEQ ID NO:54) or H_N90 (SEQ ID NO:50), APG6 (SEQ ID NO:1) consistently produced higher herbicide tolerance compared to plants transformed with CTP D or CTP E. For S-3100 at 80 g ai / ha, operably linked APG6 to H_N40 produced 21.4% to 40.2% highly tolerant transgenic plants and 41.1% to 58.9% tolerant transgenic plants. For S-3100 at 40 g ai / ha, operably linked APG6 to H_N90 produced 49.1% highly tolerant transgenic plants and 56.3% tolerant transgenic plants. For S-3100 at 80 g ai / ha, operably linked CTP D to H_N40 produced 0% highly tolerant and 2.2% tolerant transgenic plants. For S-3100 with a lower herbicide ratio of 40 g ai / ha, when CTP E is operably linked to H_N40, it produces 0% to 8% highly tolerant and 12.9% to 32.1% tolerant transgenic plants.

[0157] Tolerances of transgenic F1 hybrid maize expressing APG6 operablely linked to PPO H_N10 to seven different PPO herbicides were evaluated: S-3100, flumetsulam, trifluralin, quizalofop-P-ethyl, propyzamide, metsulfuron-methyl, and pyrimisulfuron. Seeds representing five distinct events were planted in pots in a greenhouse along with hybrid maize seeds as a negative control.

[0158] To test pre-emergence herbicide tolerance, PPO herbicides were individually applied at one of two ratios, with six replicates for each treatment: S-3100 (80 or 160 g ai / ha), flufenacet (… 840 or 1680 g ai / ha), propyzinamide ( SX, 210 or 420g ai / ha), mesotrione ( 4L, 840 or 1680g ai / ha) and bensulfuron-methyl ( (200 or 400 gai / ha). The percentage of crop damage to plants was rated 20 days after treatment, including maize seeds as a negative control. Transgenic plants containing APG6 operably linked to PPO H_N10 had damage ratings ranging from 0% to 5.8% for different pre-emergence PPO herbicides, indicating that APG6 operably linked to PPO H_N10 provides excellent pre-emergence tolerance to all five PPO herbicides for maize at two-herbicide ratios. Damage ratings for negative control maize plants ranged from 17.5% to 94.2%, except for sulfadiazine, which was predicted because this herbicide was marketed for use in conventional maize plants. Data are presented in Table 15, where standard errors are indicated as + / -.

[0159] Table 15. Pre-emergence damage rating of PPO herbicides in maize

[0160]

[0161]

[0162] To test post-emergence (V3 to V4) herbicide tolerance, PPO herbicides were individually applied at one of three ratios, with six replicates for each treatment: S-3100 (40, 80, or 160 g ai / ha), flufenacet (… 420, 840 or 1680 g ai / ha), trifluralin (Ultra) 420, 840 or 1680g ai / ha), fluoridation quizalofop-p-ethyl ( 220, 440 or 880g ai / ha), propyzinamide ( SX, 105, 210 or 420g ai / ha), mesotrione ( 4L, 420, 840 or 1680g ai / ha) and bensulfuron-methyl ( (100, 200, or 400 g ai / ha). The percentage of crop damage to the plants was rated 14 days after treatment, and conventional hybrid maize seeds were included as a negative control. Transgenic plants containing APG6 operably linked to PPO H_N10 had damage ratings ranging from 0.5% to 5.8% for different post-emergence PPO herbicides, except for flufenacet at 1680 g ai / ha, where the damage rating was 13.8%, indicating that APG6 operably linked to PPO H_N10 provides excellent post-emergence tolerance to all seven PPO herbicides for maize at all herbicide ratios. The damage ratings of the negative control maize plants ranged from 36.7% to 100%. Data are presented in Table 16, where standard errors are indicated as + / -.

[0163] Table 16. Post-emergence damage rating of PPO herbicides in maize

[0164]

[0165]

[0166] Example 7: CTP-PPO expression in transgenic soybeans

[0167] PPO enzymes operablely linked to different CTPs were expressed in transgenic soybean plants, and the PPO herbicide tolerance of transgenic plants was analyzed.

[0168] Plant transformation vectors were formed for in situ plant expression of 12G088600TP (SEQ ID NO:38) operably linked to PPO H_N10 (SEQ ID NO:43) or APG6 (SEQ ID NO:1) operably linked to PPO H_N10 (SEQ ID NO:43). These plant transformation vectors and *Agrobacterium tumefaciens* were used to transform soybean A3555 using standard methods known in the art. Regenerated R0 transgenic seedlings were grown in a greenhouse, self-pollinated, and R1 seeds were collected. Transgenic R1 plants were sprayed in a greenhouse with one of three herbicide treatments applied in V4 and R1: (1) 5 g ai / ha S-3100, (2) 10 g ai / ha S-3100, or (3) 30 g ai / ha S-3100. Crop damage ratings were assessed ten days after treatment. At ratios of 5, 10, and 30 g ai / ha, the damage ratings of transgenic plants expressing APG6 (SEQ ID NO:1) operably linked to PPO H_N10 (SEQ ID NO:43) ranged from 4.2%, 7.8%, and 9.4% in stage V4, and from 3%, 6.5%, to 15.7% in stage R1. At ratios of 5, 10, and 30 g ai / ha, the mean damage ratings of transgenic plants expressing 12G088600TP (SEQ ID NO:38) operably linked to PPO H_N10 (SEQ ID NO:43) ranged from 82.7%, 92.7%, to 98.2%, and none survived in stage R1 for rating. The negative control plants had similar mean damage ratings of 89%, 98%, and 100% at ratios of 5, 10, and 30 g ai / ha, and none survived in stage R1 for rating. Data are provided in Table 17.

[0169] Table 17: PPO herbicide test results for R1 soybeans

[0170]

[0171] Plant transformation vectors were developed to express in situ the PPO H_N90 (SEQ ID NO:47) operatively linked to one of three different CTPs: APG6 (SEQ ID NO:1), CTP F, and CTP H. These plant transformation vectors and *Agrobacterium tumefaciens* were used to transform soybean A3555 using standard methods known in the art. Regenerated R0 transgenic seedlings were grown in a greenhouse, and leaf samples taken from the resulting R0 plants were analyzed by PCR to identify plants containing a single event copy. The transgenic single-copy R0 plants, each representing a unique event, were sprayed in a greenhouse with a herbicide treatment of approximately 20 g ai / ha S-3100 at stage V3. Damage ratings, in numerical form, were obtained 14 days post-treatment and were considered either highly tolerant (≤10% damage) or tolerant (≤20% damage). Transgenic plants expressing APG6 (SEQ ID NO:1) operably linked to PPO H_N90 (SEQ ID NO:47) produced 21.4% of highly tolerant and 57.1% of tolerant unique events. Transgenic plants expressing CTP F operably linked to PPO H_N90 (SEQ ID NO:47) produced 11.7% of highly tolerant and 41.1% of tolerant unique events. Transgenic plants expressing CTP H operably linked to PPO H_N90 (SEQ ID NO:47) did not produce highly tolerant or tolerant unique events. Data are presented in Table 18.

[0172] Table 18. Efficacy evaluation of S-3100 in R0 soybeans

[0173] APG6 H_N90 3 / 14(21.4％) 8 / 14(57.1％) F H_N90 2 / 17(11.7％) 7 / 17(41.1％) H H_N90 0 / 22(0％) 0 / 22(0％)

[0174] These data demonstrate that specific CTPs operablely linked to PPO enzymes are crucial for achieving herbicide tolerance, thus highlighting the importance of CTP selection and the unexpected superiority of the APG6 CTP over other CTPs in producing herbicide-tolerant transgenic plants.

[0175] Example 8: CTP-PPO expression in transgenic cotton

[0176] Plant transformation vectors were developed to express APG6 (SEQ ID NO:1) operably linked to PPO H_N10 (SEQ ID NO:43) in transgenic cotton plants, and PPO herbicide tolerance in transgenic plants was analyzed. Cotton DP393 was transformed using the plant transformation vector and *Agrobacterium tumefaciens* using standard methods known in the art. Regenerated R0 transgenic seedlings were grown in a greenhouse, and leaf samples taken from the resulting R0 plants were analyzed by PCR to identify plants containing a single event copy. Single-copy R0 plants, each representing a unique event, were sprayed in the greenhouse with a herbicide treatment of 20 g ai / ha S-3100 at stage V2. Additionally, multi-copy transgenic plants (≥2 copies / plant) were sprayed in the greenhouse with a herbicide treatment of 20 g ai / ha S-3100 at stage V2. Crop damage was assessed three days post-treatment.

[0177] The negative control cotton DP393 showed 100% damage three days after treatment with a 20 g ai / ha S-3100 herbicide. In contrast, the average damage of the 21 single-copy R0 plants was 26.7%. The damage distribution among these 21 single-copy R0 plants was as follows: 3 plants with no damage; 3 plants with 10% damage; 3 plants with 15% damage; 2 plants with 20% damage; 7 plants with 30% damage; and 3 plants with 40% damage. For multi-copy R0 plants, 14 plants received herbicide treatment and showed an average damage of 10.4%. The damage distribution among these 14 multi-copy plants was as follows: 5 plants with no damage; 3 plants with 5% damage; 1 plant with 10% damage; 2 plants with 15% damage; 1 plant with 20% damage; 1 plant with 30% damage; and 1 plant with 40% damage. These data indicate that R0 transgenic cotton expressing APG6 (SEQ ID NO:1) operatively linked to PPO H_N10 (SEQ ID NO:43) is tolerant to application of herbicide S-3100 at 20 g ai / ha during the V2 stage.

Claims

1. A recombinant DNA molecule comprising a DNA sequence encoding a chloroplast transport peptide (CTP) as shown in SEQ ID NO:2 or SEQ ID NO:3, said DNA sequence being operatively linked to a DNA sequence encoding a dicamba monooxygenase (DMO) as shown in SEQ ID NO:18 or 27.

2. The recombinant DNA molecule of claim 1, wherein the DNA sequence encoding the CTP comprises a sequence selected from the group consisting of SEQ ID NO:12-14.

3. The recombinant DNA molecule of claim 1, wherein the DNA sequence encoding DMO comprises a sequence selected from SEQ ID NO: 28 or 37.

4. A DNA construct comprising the DNA molecule of claim 1, said DNA molecule being operatively linked to a heterologous promoter that functions in plant cells.

5. A transgenic plant, plant cell, plant part or seed comprising the DNA molecule as described in claim 1.

6. The transgenic plant, plant cell, plant part or seed as described in claim 5, wherein the plant is a monocotyledonous plant.

7. The transgenic plant, plant cell, plant part or seed as described in claim 6, wherein the plant is a corn or wheat plant.

8. The transgenic plant, plant cell, plant part or seed as described in claim 5, wherein the plant is a dicotyledonous plant.

9. The transgenic plant, plant cell, plant part or seed as described in claim 8, wherein the plant is soybean, cotton or Brassica.

10. A method for producing dicamba-tolerant plants, the method comprising the steps of: a) Transforming plant cells using the DNA construct as described in claim 4; and b) Regenerate a plant from the transformed plant cells containing the DNA construct.

11. The method of claim 10, wherein the regenerated plants are tolerant to dicamba.

12. A method for producing dicamba-tolerant plants, the method comprising the steps of: a) Hybridizing a parent plant containing the DNA molecule as described in claim 1 with itself or with a second plant to produce one or more offspring plants; and b) Select progeny plants containing the DNA molecule.

13. The method of claim 12, wherein the offspring plants are tolerant to dichotoma.

14. A method for expressing dicamba monooxygenase (DMO), the method comprising introducing a DNA molecule as described in claim 1 into a plant cell.

15. The method of claim 14, wherein the introduction includes transforming the plant cells.

16. A method for controlling weed growth in a crop growing environment, the method comprising the following steps: a) Planting plants or seeds containing the recombinant DNA molecule as described in claim 1 in a crop growth environment; as well as b) Apply an amount of dicamba to the crop growing environment that can effectively control weed growth.

17. The method of claim 16, wherein the dicamba does not harm the plant or seeds.

18. The method of claim 16, wherein the plant or seed is a monocotyledonous plant or seed.

19. The method of claim 18, wherein the plant is a corn or wheat plant.

20. The method of claim 16, wherein the plant or seed is a dicotyledonous plant or seed.

21. The method of claim 20, wherein the plant is soybean, cotton or Brassica.

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