Plants with increased herbicide resistance

Abstract

A plant or plant part is provided that contains a polynucleotide encoding a mutant TriA polypeptide, expression of which confers herbicide resistance to the plant or plant part. [Selection diagram] None

Description

[Technical field]

[0001] The present invention relates generally to methods for conferring agricultural levels of tolerance to herbicides to plants. In particular, the present invention relates to plants having increased tolerance to herbicides, and more particularly to herbicides that inhibit cellulose biosynthesis and thus disrupt cell wall biosynthesis. [Background technology]

[0002] Microorganisms often respond to the introduction of xenobiotics into the environment by evolving mechanisms that allow them to utilize these foreign substances as a source of nutrients and energy for growth. Because the structures of s-triazine ring-based herbicides are distinct from naturally occurring compounds (Esser et al. 1975), microorganisms have slowly evolved enzymes and pathways that can degrade them. The amidohydrolase superfamily comprises a remarkable group of enzymes that catalyze the hydrolysis of a wide range of substrates that contain amide or ester functional groups at the carbon and phosphorus centers. In all cases, a nucleophilic water molecule is activated by complexation with a mononuclear or binuclear metal center. In the mononuclear metal center, the substrate is activated by proton transfer from the active site and water is activated by metal ligation and general base catalysis. The metal center is located at the C-terminus of a β-barrel core within a (βα)8 structural domain. One prominent example is atrazine chlorohydrolase (AtzA), an Fe(II)-dependent homohexamer (Seffernick et al. 2002; Wackett et al. 2002a), which catalyzes the hydrolytic dechlorination of the herbicide atrazine to the non-herbicidally active product 2-hydroxyatrazine (de Souza et al. 1996; Seffernick et al. 2002; Sadowsky and Wackett 2000). The closest known relative of AtzA is melamine deaminase (TriA from Pseudomonas sp. NRRL B-12227; 98% sequence identity). Despite their high sequence similarity, AtzA and TriA differ in their catalytic properties; TriA is a deaminase with a dechlorinase activity that is orders of magnitude lower than its physiological deaminase activity, whereas AtzA is a dechlorinase with no detectable deaminase activity. Previous studies have shown that three of the nine amino acids that differ between the two proteins (S331C; N328D; and F84I AtzA) play a major role in the difference in catalytic specificity. Summary of the Invention

[0003] The present invention provides a new method to enhance herbicide tolerance in plants by introducing a bacterial gene encoding a target protein that biodegrades herbicides, specifically cellulose biosynthesis inhibitors named azines. The bacterial enzyme TriA was engineered to retain or increase amidohydrolase activity, as well as to enlarge the enzyme pocket to accommodate larger substrates. The inventors of the present invention surprisingly found that overexpression of wild-type or mutant melamine deaminase TriA confers tolerance / resistance to certain types of herbicides in plants compared to non-transformed and / or non-mutagenized plants or plant cells, respectively. More specifically, the inventors of the present invention found that TriA expression confers tolerance / resistance to azines.

[0004] The objective of the present invention can be seen as the provision of novel traits by identifying target polypeptides whose manipulation renders plants tolerant to herbicides.

[0005] Three main strategies are available to make plants tolerant to herbicides: (1) detoxifying the herbicide with an enzyme that converts the herbicide or its active metabolite into a non-toxic product, e.g., the enzymes for resistance to bromoxynil or basta (EP242236, EP337899); (2) mutating the target enzyme, e.g., the enzymes for resistance to glyphosate, into a functional enzyme that is less sensitive to the herbicide or its active metabolite (EP293356, Padgette SR et al., J. Biol. Chem., 266, 33, 1991); or (3) overexpressing the sensitive enzyme to produce sufficient amounts of the target enzyme for the herbicide in the plant, taking into account the rate constant of this enzyme, so that there is enough functional enzyme available despite the presence of its inhibitor. [Means for solving the problem]

[0006] The above object is achieved by the subject matter of the present invention.

[0007] Summary of the Invention Thus, in one aspect, the invention provides a plant or plant part comprising a polynucleotide encoding a mutant TriA polypeptide, expression of said polynucleotide confers tolerance to a herbicide to the plant or plant part.

[0008] In some embodiments, the present invention provides a seed capable of germinating into a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant.

[0009] In one aspect, the invention provides a plant cell capable of regenerating a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant, wherein the plant cell comprises the polynucleotide operably linked to the promoter.

[0010] In another aspect, the invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in the cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant.

[0011] In another aspect, the invention provides a plant product prepared from a plant or plant part comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant.

[0012] In some aspects, the present invention provides progeny or descendant plants derived from a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least a portion of its cells a recombinant polynucleotide operably linked to the promoter, and expression of the mutant TriA polypeptide confers resistance to a herbicide to the progeny or descendant plant.

[0013] In another aspect, the invention provides a method for controlling weeds at a locus for growing a plant, the method comprising the steps of: (a) applying to the locus a herbicide composition comprising a herbicide; and (b) planting a seed at the locus, wherein the seed is capable of producing a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and wherein expression of the mutant TriA polypeptide confers resistance to the herbicide to the plant.

[0014] In some embodiments, the present invention provides a method for controlling weeds at a locus for the growth of a plant, the method comprising the step of applying to the locus a herbicide composition comprising a herbicide, the locus being (a) a locus containing a plant or a seed capable of producing said plant, or (b) a locus which will contain a plant or seed after said application, wherein the plant or seed comprises in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and wherein expression of the mutant TriA polypeptide confers tolerance to the plant to the herbicide.

[0015] In one embodiment, step (a) occurs before, after, or simultaneously with step (b).

[0016] In another aspect, the invention provides a method for producing a plant having tolerance to a herbicide, the method comprising the step of regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein expression of the mutant TriA polypeptide confers tolerance to the herbicide to the plant.

[0017] In one aspect, the invention provides a method for producing a progeny plant having tolerance to a herbicide, the method comprising crossing a plant tolerant to a first herbicide with a second plant to produce a progeny plant tolerant to the herbicide, wherein the first plant and the progeny plant comprise in at least a portion of their cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to the herbicide to the plant.

[0018] Furthermore, the present invention relates to a method for identifying herbicides by using a wild-type or mutant TriA of the present invention encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO:1, or a mutant, homolog, paralog or ortholog thereof.

[0019] The method comprises: a) producing a transgenic cell or plant which contains a nucleic acid encoding the mutant TriA of the present invention and expresses the mutant TriA of the present invention; b) applying a herbicide to the transgenic cells or plants of a) and to control cells or plants of the same variety; c) determining the growth or viability of the transgenic cells or plants and the control cells or plants following application of the test compound; and d) selecting test compounds that cause reduced growth in control cells or plants compared to the growth of the transgenic cells or plants. Includes.

[0020] Another object relates to a method for identifying a nucleotide sequence encoding a mutant TriA that is resistant or tolerant to a herbicide, the method comprising: a) generating a library of nucleic acids encoding mutant TriAs; b) screening the resulting population of nucleic acids encoding mutant TriAs by expressing each of said nucleic acids in a cell or a plant and treating said cell or plant with a herbicide; c) comparing the level of resistance to the herbicide conferred by the population of nucleic acids encoding mutant TriA with the level of resistance to the herbicide conferred by a control nucleic acid encoding TriA; d) selecting at least one mutant TriA-encoding nucleic acid that confers a significantly increased level of resistance to the herbicide compared to the level conferred by a control TriA-encoding nucleic acid. Includes.

[0021] In a preferred embodiment, the mutant TriA-encoding nucleic acid selected in step d) confers at least 2-fold resistance to an herbicide compared to the resistance conferred by a control TriA-encoding nucleic acid.

[0022] Resistance or tolerance can be determined by generating transgenic plants comprising a nucleic acid sequence from the library of step a) and comparing said transgenic plants to control plants.

[0023] Another object relates to an isolated, recombinant and / or chemically synthesized nucleic acid encoding a mutant TriA, which comprises the sequence of SEQ ID NO: 1, or a variant thereof as defined below.

[0024] The preferred embodiment is: (a) a nucleic acid molecule encoding a mutant TriA polypeptide comprising the sequence of SEQ ID NO:2, or a variant, paralog, ortholog, or homolog thereof; (b) a nucleic acid molecule comprising the sequence of SEQ ID NO:1, or a variant, paralog, ortholog, or homolog thereof; (c) a nucleic acid molecule derivable from the TriA polypeptide sequence of SEQ ID NO:2, or a variant, paralog, ortholog, or homolog thereof, as a result of the degeneracy of the genetic code, which confers increased herbicide tolerance or resistance compared to a corresponding, e.g., untransformed, wild-type plant cell, plant, or plant part; (d) a nucleic acid molecule having 30% or more identity, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity to a nucleic acid molecule sequence of a polynucleotide comprising a nucleic acid molecule of SEQ ID NO:1, or a variant, paralog, ortholog, or homolog thereof, said nucleic acid molecule conferring increased herbicide tolerance or resistance compared to a corresponding, e.g., untransformed, wild-type, plant cell, plant or plant part; (e) a nucleic acid molecule encoding a mutant TriA polypeptide having 30% or more identity, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identity, to the amino acid sequence of the TriA polypeptide sequence of SEQ ID NO:2, which nucleic acid molecule confers increased herbicide tolerance or resistance compared to a corresponding, e.g., untransformed, wild-type plant cell, plant or plant part; (f) a nucleic acid molecule that hybridizes under stringent hybridization conditions to a nucleic acid molecule of (a), (b), (c), (d) or (e), and that confers increased herbicide tolerance or resistance compared to a corresponding, e.g., untransformed, wild-type plant cell, plant or plant part; The present invention relates to an isolated and / or recombinantly produced and / or synthetic nucleic acid molecule comprising a nucleic acid encoding a mutant TriA polypeptide selected from the group consisting of:

[0025] Another object is an expression cassette comprising a nucleic acid molecule of the invention and a promoter operable in a plant cell.

[0026] Preferably, the promoter is a root-specific or root-enhanced promoter from soybean (Glycine max).

[0027] Another object relates to isolated, recombinant, and / or chemically synthesized mutant TriA polypeptides, which include the sequence set forth in SEQ ID NO:2, variants, derivatives, orthologs, paralogs or homologs thereof, as defined below.

[0028] Preferably, the amino acid sequence of the mutant TriA polypeptide differs from the wild-type amino acid sequence of the TriA polypeptide at one or more positions corresponding to the following positions: 92, 93, 155, 157 of SEQ ID NO:2.

[0029] In yet another aspect, the present invention provides a plant or plant part comprising in at least a portion of a plant cell a polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant, and wherein the plant or plant part further exhibits a second or third herbicide-tolerance trait.

[0030] In another embodiment, the present invention relates to a plant cell transformed with and expressing a wild-type or mutant TriA nucleic acid of the invention, or a plant mutated to obtain a plant expressing, preferably overexpressing, a wild-type or mutant TriA nucleic acid of the invention, wherein expression of said nucleic acid in the plant cell confers increased resistance or tolerance to herbicides compared to a wild type variety of the plant cell.

[0031] In another embodiment, the present invention relates to a plant comprising a plant cell of the present invention, wherein expression of the nucleic acid in the plant results in increased resistance of the plant to an herbicide compared to a wild type variety of the plant.

[0032] Preferably, expression of a nucleic acid of the invention in a plant results in increased resistance of the plant to herbicides compared to a wild type variety of the plant.

[0033] In another embodiment, the present invention relates to a seed produced by a transgenic plant comprising a plant cell of the present invention, wherein the seed is true breeding for increased resistance to a herbicide compared to a wild type variety of the seed.

[0034] In another embodiment, the present invention relates to a method for producing a transgenic plant cell having increased resistance to a herbicide compared to a wild-type variety of the plant cell, the method comprising the step of transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide.

[0035] In another embodiment, the present invention relates to a method for producing a transgenic plant, the method comprising: (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide; and (b) producing a plant having increased resistance to a herbicide from the plant cell.

[0036] Preferably, the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region that are functional in plants. [Brief description of the drawings]

[0037] [Figure 1-1] Figure 1 shows the hydroponic tolerance evaluation of soybean plants treated with azines. Phenotypic growth results of wild type (A) and transgenic events expressing the triA mutant variant of SEQ ID NO: 2 containing four amino acid substitutions, namely L92T_Y93L_M155T_F157L (B, C and D). Values ​​shown indicate the amount of 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine in [nM]. [Figure 1-2]Figure 1 shows the hydroponic tolerance evaluation of soybean plants treated with azines. Phenotypic growth results of wild type (A) and transgenic events expressing the triA mutant variant of SEQ ID NO: 2 containing four amino acid substitutions, namely L92T_Y93L_M155T_F157L (B, C and D). Values ​​shown indicate the amount of 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine in [nM]. [Diagram 2] Figure 2 shows the degradation of 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine by the triA mutant polypeptides. The polypeptides were used as is or after incubation with or without the addition of proteases for 72 hours at room temperature. [Diagram 3] Figure 3 shows the degradation of 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine by two triA mutant polypeptides with the mutations L92A_Y93L_M155T_F157L (patent application WO 2018 / 011750) and L92T_Y93L_M155T_F157L. The polypeptides were used as is or after incubation with various concentrations of protease for 20 hours at 30°C.

[0038] Detailed Description As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more elements.

[0039] As used herein, the word "comprising" or variations thereof such as "comprises" or "comprising" indicates the inclusion of a stated element, integer or step, or a group of elements, integers or steps, but is understood to not exclude other elements, integers or steps, or a group of elements, integers or steps.

[0040] The term "control of unwanted vegetation or weeds" is understood to mean killing the weeds and / or otherwise retarding or inhibiting their normal growth. Weeds are understood in the broadest sense to mean any plant that grows where it is not wanted. Weeds in the context of the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, and the like. Weeds in the genera Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum.Monocotyledonous weeds include, but are not limited to, Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, and Brassica napus. Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and Apera. In addition, the weeds of the present invention may include, for example, crop plants that grow in places where they are not wanted. For example, a native corn plant in a field where soybean plants grow mainly can be considered a weed if the corn plant is not wanted in the field of soybean plants.

[0041] The term "plant" is used in its broadest sense as referring to organic material and is intended to include eukaryotic organisms that are members of the Kingdom Plantae, examples of which include, but are not limited to, vascular plants, vegetables, cereals, flowers, trees, grasses, shrubs, turf, vines, ferns, mosses, fungi, and algae, as well as clones, offshoots, and parts of plants used for asexual propagation (e.g., cuttings, piping, shoots, rhizomes, rhizomes, clumps, crowns, bulbs, corms, tubers, rhizomes, plants / tissues produced in tissue culture, etc.). The term "plant" further includes whole plants, ancestors and descendants of plants and plant parts, such as seeds, shoots, stems, leaves, roots (including tubers), flowers, florets, fruits, stalks, pedicels, stamens, anthers, stigmas, styles, ovaries, petals, sepals, carpels, root tips, root caps, root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organs, tissues, and cells, and tissues and organs of a plant, each of the above containing the gene / nucleic acid of interest. The term "plant" also includes plant cells, suspension cultures, callus tissue, embryos, meristematic tissue portions, gametophytes, sporophytes, pollen, and microspores, each of the above containing the gene / nucleic acid of interest.

[0042] Plants that are particularly useful in the methods of the invention include any plant belonging to the superfamily Viridiplantae, particularly Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., Artocarpus spp., and the like. spp.), Asparagus (Asparagus officinalis), Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Star fruit (Averrhoa carambola), Bambusa sp., Wax gourd (Benincasa hispida), Brazil nut (Bertholletia excelsea), Sugar beet (Beta vulgaris), Brassica spp. (e.g. Brassica napus, Brassica rapa var. sativa, ssp.) [canola, rapeseed, oilseed rape], Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp.), Carex elata, Papaya (Carica papaya), Carissa macrocarpa, Pecan (Carya spp.), Safflower (Carthamus tinctorius), Chestnut (Castanea spp.), Kapok (Ceiba pentandra), Endive (Cichorium endivia), Cinnamomum spp., Watermelon (Citrullus lanatus), Citrus spp., Cocos spp., Coffea spp., Taro (Colocasia esculenta), Cola spp., Corchorus sp., Coriander (Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis spp. (e.g. Elaeis guineensis, Elaeis oleracea), Elaeis oleracea (e.g. ... oleifera), finger millet (Eleusine coracana), teff (Eragrostis tef), Erianthus sp., loquat (Eriobotrya japonica), eucalyptus sp., pitanga (Eugenia uniflora), buckwheat (Fagopyrum spp.), beech (Fagus spp.), fescue (Festuca arundinacea), fig (Ficus carica), fortunella spp.), Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), cotton (Gossypium hirsutum), Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), sweet potato (Ipomoea batatas), Juglans spp., lettuce (Lactuca sativa), Lathyrus spp. spp.), Lens (Lens culinaris), Linum (Linum usitatissimum), Litchi (Litchi chinensis), Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Acerola (Malpighia emarginata), Mammea americana, Mango (Mangifera indica), Manihot spp. spp.), Sapodilla (Manilkara zapota), Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp.), Opuntia spp., Ornithopus spp., Oryza spp. (e.g., rice (Oryza sativa, Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), passion fruit (Passiflora edulis), parsnip (Pastinaca sativa), Pennisetum spp., Persea spp., parsley (Petroselinum crispum), reed canary grass (Phalaris arundinacea), Phaseolus spp., Timothy grass (Phleum pratense), Phoenix spp. spp.), common reed (Phragmites australis), nightshade (Physalis spp.), pine (Pinus spp.), pistachio (Pistacia vera), pea (Pisum spp.), Poa spp., poplar (Populus spp.), Prosopis spp., cherry (Prunus spp.), Psidium spp., pomegranate (Punica granatum), pear (Pyrus communis), oak (Quercus spp.), radish (Raphanus sativus), rhubarb (Rheum rhabarbarum), currant (Ribes spp.), castor bean (Ricinus communis, Rubus spp., Saccharum spp., Salix spp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis spp., Solanum spp.) (e.g. potato (Solanum tuberosum), acanthus (Solanum integrifolium) or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach (Spinacia spp.), myrtle (Syzygium spp.), Tagetes spp., tamarind (Tamarindus indica), cocoa (Theobroma cacao), Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, wheat (Triticum spp.) (e.g. bread wheat (Triticum aestivum), durum wheat (Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare, Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp.), amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, rapeseed, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugarcane, sunflower, tomato, pumpkin, tea and algae are included in the list of monocotyledonous and dicotyledonous plants including forage or fodder legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato or tobacco, among others. More preferably, the plant is a monocotyledonous plant such as sugarcane. More preferably, the plant is a cereal such as rice, maize, wheat, barley, millet, rye, sorghum or oats.

[0043] In general, the term "herbicide" is used herein to mean an active ingredient that kills, controls, or otherwise adversely alters the growth of plants. The preferred amount or concentration of the herbicide is an "effective amount" or "effective concentration". "Effective amount" and "effective concentration" mean an amount and concentration sufficient to kill or inhibit the growth of similar wild-type plants, plant tissues, plant cells, or host cells, respectively, but said amount does not kill or seriously inhibit the growth of plants, plant tissues, plant cells, and host cells that are resistant to the herbicide of the present invention. Typically, an effective amount of a herbicide is an amount that is routinely used to kill the target weed in an agricultural production system. Such amounts are known to those skilled in the art. Herbicidal activity is exhibited by herbicides useful in the present invention when the herbicides are applied directly to plants or to the locus of plants at any stage of growth or before planting or emergence. The observed effect depends on the plant species being controlled, the growth stage of the plant, application parameters of dilution and spray droplet size, particle size of the solid component, environmental conditions at the time of use, the specific compound used, the specific adjuvants and carriers used, soil type, etc., as well as the amount of chemical applied. These and other factors can be adjusted as known in the art to enhance non-selective or selective herbicidal action. In general, herbicide treatments can be applied by PPI (pre-plant incorporation), PPSA (post-plant surface application), pre-emergence or post-emergence. Post-emergence treatments are typically applied to relatively immature undesirable vegetation to achieve maximum control of weeds.

[0044] A "herbicide-tolerant" or "herbicide-resistant" plant means a plant that is tolerant or resistant to at least one herbicide at a level that would normally kill or inhibit the growth of a normal or wild-type plant. The level of herbicide that would normally inhibit the growth of a non-tolerant plant is known to those skilled in the art and can be easily determined. An example is the amount that the manufacturer recommends for application. The maximum rate is an example of the amount of herbicide that would normally inhibit the growth of a non-tolerant plant. In the present invention, the terms "herbicide-tolerant" and "herbicide-resistant" are used interchangeably and are intended to have the same meaning and the same scope. Similarly, the terms "herbicide-tolerant" and "herbicide-resistant" are used interchangeably and are intended to have the same meaning and the same scope. Similarly, the terms "tolerant" and "resistant" are used interchangeably and are intended to have the same meaning and the same scope. As used herein, for the herbicide compositions useful in various embodiments thereof, terms such as herbicide, etc. refer to an agriculturally acceptable herbicidal active ingredient (AI) recognized in the art. Similarly, terms such as fungicide, nematicide, insecticide, and the like refer to other agriculturally acceptable active ingredients as recognized in the art.

[0045] When used in relation to a particular mutant enzyme or polypeptide, terms such as herbicide-resistant and herbicide-tolerant relate to the ability of such enzyme or polypeptide to exert its physiological activity in the presence of an amount of herbicide AI that would normally inactivate or inhibit the wild-type (non-mutated) activity of said enzyme or polypeptide. On the other hand, when used specifically in relation to the TriA enzyme, the term specifically refers to the ability to metabolize and thereby inactivate herbicides that inhibit cellulose biosynthesis, so-called cellulose biosynthesis inhibitors (CBIs). By "herbicide-resistant mutant TriA protein" or "herbicide-resistant mutant TriA protein" is meant that such TriA protein exhibits a higher metabolic activity in the presence of at least one herbicide known to interfere with cellulose biosynthesis, at a concentration or level of said herbicide known to inhibit cellulose biosynthesis, compared to the metabolic activity of the wild-type TriA protein. The metabolic activity depends on the intrinsic catalytic activity of the protein and the amount of catalytically active protein present in the plant tissue. For a protein to be catalytically active, it must be correctly folded. Incorrectly folded or partially unfolded proteins are susceptible to degradation by cytoplasmic proteases, which reduces the amount of catalytically active protein in plant tissues. It is therefore desirable for the mutated TriA protein to be resistant to degradation by proteases. The TriA activity of such herbicide-tolerant or herbicide-resistant mutant TriA proteins may also be referred to herein as "herbicide-tolerant" or "herbicide-resistant" TriA activity.

[0046] As used herein, "recombinant" in reference to a nucleic acid or polypeptide indicates that such material has been altered as a result of human application of recombinant techniques, such as by restriction cleavage and ligation of polynucleotides, duplication extension of polynucleotides, or insertion or transformation of a genome. A gene sequence open reading frame is recombinant if its nucleotide sequence has been removed from its natural string and cloned into an artificial nucleic acid vector of any type. The term recombinant can also refer to an organism having a recombinant material, for example, a plant containing a recombinant nucleic acid can be considered a recombinant plant.

[0047] The term "transgenic plant" refers to a plant that contains a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated into the genome so that the polynucleotide is passed on from generation to generation. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part, or plant having a genotype that is so altered by the presence of a heterologous nucleic acid, including the transgenic organism or cell originally so altered, and those created by mating or asexual propagation from the original transgenic organism or cell. In some embodiments, a "recombinant" organism is a "transgenic" organism. The term "transgenic" as used herein is not intended to encompass alterations of the genome (chromosomal or extrachromosomal) by conventional plant breeding methods (e.g., mating) or natural events such as self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0048] As used herein, "mutagenized" refers to an organism or its DNA having an alteration in the sequence of the biomolecules of its native genetic material compared to the sequence of the genetic material of the corresponding wild-type organism or DNA, where the alteration in the genetic material has been induced and / or selected by human action. Examples of human actions that can be used to generate a mutagenized organism or DNA include, but are not limited to, treatment with a chemical mutagen such as EMS followed by selection with a herbicide, or treatment of plant cells with X-rays followed by selection with a herbicide. Any method known in the art can be used to induce mutations. The method of inducing mutations can induce mutations at random positions in the genetic material, or can induce mutations at specific positions in the genetic material (i.e., it can be a directed mutagenesis technique), such as using genoplasty techniques.

[0049] As used herein, a "genetically modified organism" (GMO) is an organism whose genetic characteristics contain alterations produced by human efforts to cause transfection resulting in the transformation of the target organism with genetic material from another or "source" organism or with synthetic or modified natural genetic material, or an organism that descends from it and retains the inserted genetic material. The source organism can be a different type of organism (e.g., a GMO plant can contain bacterial genetic material) or the same type of organism (e.g., a GMO plant can contain genetic material from another plant). When used herein with respect to plants and other organisms, "recombinant," "transgenic," and "GMO" are considered synonymous and indicate the presence of genetic material from different sources. In contrast, "mutagenized" is used to refer to a plant or other organism, or its DNA, in which such transgenic material is present but in which the natural genetic material has been mutated to be different from the corresponding wild-type organism or DNA.

[0050] As used herein, "wild type" or "corresponding wild type plant" refers to the typical form of an organism or its genetic material as it normally exists, e.g., as distinguished from mutagenized and / or recombinant forms. Similarly, "control cell" or "similar wild type plant, plant tissue, plant cell, or host cell" refers to a plant, plant tissue, plant cell, or host cell, respectively, that lacks the inventive herbicide resistance trait and / or specific polynucleotide disclosed herein. Thus, the use of the term "wild type" is not intended to indicate that the plant, plant tissue, plant cell, or other host cell does not lack recombinant DNA in its genome and / or does not possess a different herbicide resistance trait than that disclosed herein.

[0051] As used herein, "progeny" refers to any generation of a plant. In some embodiments, a progeny is a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation plant.

[0052] As used herein, "progeny" refers to a first generation plant.

[0053] The term "seed" includes all kinds of seeds, such as true seeds, caryopsis, fruits, tubers, seedlings, and similar forms. In the context of Brassica and Sinapis species, "seed" refers to true seeds unless otherwise specified. For example, the seed may be the seed of a transgenic plant or a plant obtained by conventional breeding methods. Examples of conventional breeding methods include cross breeding, selfing, backcrossing, embryo rescue, in-crossing, outcrossing, inbreeding, selection, asexual propagation, and other conventional techniques, as known in the art.

[0054] Although exemplified in relation to specific plants or plant varieties and their hybrids, in various embodiments, the herbicide-using methods described herein can be used with commercially valuable plant varieties. The herbicide-tolerant plant lines described as useful in the present invention can be used directly or indirectly in weed control methods, i.e., as crops for herbicide treatment, or as herbicide-tolerant trait donor lines for growth to generate other varieties and / or hybrid crops containing such one or more traits. All such obtained varieties or hybrid crops containing one or more traits of ancestral herbicide resistance can be referred to as descendants or progeny of the ancestral herbicide-tolerant line in the present invention. The plants thus obtained can be referred to as retaining the "herbicide-tolerant trait" of the ancestral plant, meaning that they retain and express the ancestral genetic molecular components that are responsible for the trait.

[0055] In one aspect, the invention provides a plant or plant part comprising a polynucleotide encoding a mutant TriA polypeptide, wherein expression of the polynucleotide confers tolerance to a herbicide to the plant or plant part.

[0056] In a preferred embodiment, the plant has previously been produced by a method comprising the step of recombinantly producing the plant by introducing and overexpressing a wild-type or mutant TriA transgene of the invention, as described in more detail below.

[0057] In another embodiment, the polynucleotide encoding the mutant TriA polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 1, or a variant or derivative thereof.

[0058] In other embodiments, the mutant TriA polypeptide used in accordance with the invention is a functional variant having at least about 80%, for example at least about 80%, 90%, 95%, 98%, or 99% or more amino acid sequence identity to SEQ ID NO:2 over the entire length of the variant.

[0059] In another embodiment, the mutant TriA polypeptide used in accordance with the invention is a functional fragment of a polypeptide having the amino acid sequence set forth in SEQ ID NO:2.

[0060] The TriA polynucleotide molecules and TriA polypeptides of the present invention are understood to include polynucleotide molecules and polypeptides that comprise a nucleotide or amino acid sequence that is sufficiently identical to the nucleotide sequence set forth in SEQ ID NO: 1, or the amino acid sequence set forth in SEQ ID NO: 2. The term "sufficiently identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., having similar side chains) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and / or a common functional activity.

[0061] Generally, "sequence identity" refers to the degree to which two optimally aligned DNA or amino acid sequences are invariant within a window of alignment of components, e.g., nucleotides or amino acids. The "percentage of identity" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components within the reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The "percentage of identity" is the percentage of identity x 100. Optimal alignment of sequences to align a comparison window is well known to those skilled in the art and can be performed by means such as the Smith and Waterman local homology algorithm, the Needleman and Wunsch homology alignment algorithm, the Pearson and Lipman similarity search method, preferably by computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, available as part of the GCG. Wisconsin Package (Accelrys Inc. Burlington, Mass.).

[0062] Polynucleotides and Oligonucleotides By "isolated polynucleotide", including DNA, RNA, or combinations thereof, single-stranded or double-stranded, in sense or antisense orientation, or a combination of both, dsRNA, etc., is meant a polynucleotide that is at least partially separated from the polynucleotide sequence with which it is naturally associated or bound. Preferably, an isolated polynucleotide is at least 60%, preferably at least 75%, and most preferably at least 90% free from other components with which it is naturally associated. As will be appreciated by the skilled artisan, an isolated polynucleotide can be, for example, an exogenous polynucleotide present in a transgenic organism that does not naturally contain the polynucleotide. Also, the terms "polynucleotide", "nucleic acid sequence", "nucleotide sequence", "nucleic acid", and "nucleic acid molecule" are used interchangeably herein and refer to nucleotides of any length in a polymeric, unbranched form, either ribonucleotides or deoxyribonucleotides, or a combination of both.

[0063] The term "mutant TriA nucleic acid" refers to a TriA nucleic acid having a sequence that is mutated from a wild-type TriA nucleic acid and confers increased resistance to herbicides to a plant in which it is expressed. The term "mutant melamine deaminase (mutant TriA)" also refers to the replacement of an amino acid in the wild-type primary sequence of SEQ ID NO: 2 or a variant, derivative, homolog, ortholog, or paralog thereof with another amino acid. The expression "mutated amino acid" is used hereinafter to refer to an amino acid that is replaced with another amino acid, and thus refers to the site of the mutation in the primary sequence of the protein.

[0064] In a preferred embodiment, the TriA nucleotide sequence encoding the mutant TriA comprises the sequence of SEQ ID NO: 1, or a variant or derivative thereof.

[0065] As will be appreciated by those of skill in the art, TriA nucleotide sequences also encompass homologues, paralogs and orthologs of SEQ ID NO:1, as defined below.

[0066] The term "variant" with respect to a sequence (e.g., a polypeptide or a nucleic acid sequence, such as, for example, a transcriptional regulatory nucleotide sequence of the invention) is intended to mean a substantially similar sequence. For a nucleotide sequence comprising an open reading frame, variants include sequences that encode the same amino acid sequence as the native protein due to the degeneracy of the genetic code. Naturally occurring allelic variants such as these can be identified using well-known molecular biology techniques, such as, for example, polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include those generated, for example, by using site-directed mutagenesis, that encode a native protein whose open reading frame comprises the sequence of SEQ ID NO:2, and synthetically derived nucleotide sequences, such as those that encode a polypeptide having amino acid substitutions compared to the native protein, such as, for example, a mutant TriA of the invention as disclosed herein. Typically, the nucleotide sequence variants of the invention have at least 30, 40, 50, 60, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, typically at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide "sequence identity" with the nucleotide sequence of SEQ ID NO: 1. Percent polynucleotide identity is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with gap creation penalty = 5 and gap extension penalty = 0.3. Unless otherwise specified, the query sequence is at least 45 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides.Even more preferably, the GAP analysis aligns the two sequences over their entire length.

[0067] Polypeptides By "substantially purified polypeptide" or "purified" is meant a polypeptide that has been separated from one or more lipids, nucleic acids, other polypeptides, or other contaminating molecules with which it is naturally associated. A substantially purified polypeptide is preferably at least 60%, more preferably at least 75%, more preferably at least 90% free from other components with which it is naturally associated. As will be appreciated by the skilled artisan, a purified polypeptide can be a recombinantly produced polypeptide. The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain that may or may not be modified by the addition of non-amino acid groups. It is understood that such a polypeptide chain may be associated with other molecules, such as other polypeptides or proteins or cofactors. The terms "protein" and "polypeptide" as used herein also include variants, mutants, modifications, analogs and / or derivatives of the polypeptides of the invention described herein.

[0068] Percent identity of polypeptides is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 25 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

[0069] It is understood that for a given polypeptide, higher % identity figures than those listed above encompass preferred embodiments. Thus, in terms of the minimum % identity figure, where applicable, it is preferred that the TriA polypeptide of the invention comprises an amino acid sequence that is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to SEQ ID NO:2.

[0070] By "mutant" polypeptide is meant a polypeptide derived from the protein of SEQ ID NO:2 by deletion (so-called truncation) or addition of one or more amino acids to the N-terminus and / or C-terminus of the native protein, deletion or addition of one or more amino acids at one or more sites in the native protein, or substitution of one or more amino acids at one or more sites in the native protein. Such variants may be the result, for example, of genetic polymorphism or human manipulation. Methods for such manipulation are well known in the art.

[0071] A "derivative" of a protein includes peptides, oligopeptides, polypeptides, proteins and enzymes that have amino acid substitutions, deletions and / or insertions compared to the unmodified protein in question and have biological and functional activity similar to the unmodified protein from which they are derived. Thus, functional variants and fragments of TriA polypeptides, as well as the nucleic acid molecules encoding them, are also within the scope of the present invention, regardless of the origin of the polypeptide, and whether naturally occurring or not, unless otherwise specified. Various assays for the functionality of TriA polypeptides can be used. For example, functional variants or fragments of TriA polypeptides can be assayed to determine their ability to confer an antidote effect against herbicides. By way of example, the detoxification rate for a herbicide can be defined as the rate of catalytic reaction sufficient to provide a measurable increase in tolerance to the herbicide in a plant or plant part comprising a recombinant polynucleotide encoding a variant or fragment of a TriA polypeptide, where the plant or plant part expresses the variant or fragment in up to about 0.5% of total cellular protein, e.g., about 0.05 to about 0.5%, about 0.1 to about 0.4%, and about 0.2 to about 0.3%, relative to a similarly treated control plant that does not express the variant or fragment.

[0072] In a preferred embodiment, the mutant TriA polypeptide is a functional variant or fragment of melamine deaminase having the amino acid sequence set forth in SEQ ID NO:2, where the functional variant or fragment has at least about 80% amino acid sequence identity to SEQ ID NO:2.

[0073] In other embodiments, the functional variants or fragments further have a herbicide detoxification rate, which is defined as a catalytic reaction rate sufficient to provide a measurable increase in tolerance to the herbicide in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses the variant or fragment in up to about 0.5% of total cellular protein compared to a similarly treated control plant that does not express the variant or fragment.

[0074] "Homologs" of a protein include peptides, oligopeptides, polypeptides, proteins and enzymes that have amino acid substitutions, deletions and / or insertions compared to the native protein in question and that have the same biological and functional activity as the native protein from which they are derived.

[0075] In addition, as will be further understood by those skilled in the art, by introducing changes into the nucleotide sequences of the invention by mutation, the amino acid sequence of the encoded protein can be altered without altering the biological activity of the protein. Thus, for example, an isolated polynucleotide molecule encoding a mutant TriA polypeptide having an amino acid sequence different from that of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Such mutant nucleotide sequences are also encompassed by the present invention. For example, preferably, conservative amino acid substitutions may be made at one or more predicted, preferably non-essential, amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, and an "essential" amino acid residue is required for biological activity.

[0076] A deletion refers to the removal of one or more amino acids from a protein.

[0077] An insertion refers to the introduction of one or more amino acid residues into a given site in a protein. Insertions can include N- and / or C-terminal fusions as well as intrasequence insertions of single or multiple amino acids. In general, an intrasequence insertion is about 1-10 residues smaller than an N- or C-terminal fusion. Examples of N- or C-terminal fusion proteins or peptides include the binding or activation domain of a transcription activator used in the yeast two-hybrid system, a phage coat protein, a (histidine)-6-tag, a glutathione S-transferase-tag, protein A, maltose binding protein, dihydrofolate reductase, tag 100 epitope, a c-myc epitope, a FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), an HA epitope, a protein C epitope, and a VSV epitope.

[0078] Substitution refers to the replacement of an amino acid in a protein with another amino acid having similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or disrupt α-helical or β-sheet structures). Amino acid substitutions are usually single residues, but may be clusters and range from 1-10 amino acids depending on the functional constraints imposed on the polypeptide, with insertions usually on the order of about 1-10 amino acid residues. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions are not made to conserved amino acid residues or to amino acid residues that lie within a conserved motif. Conservative substitution tables are well known in the art (see, e.g., Creighton (1984) Proteins. WH Freeman and Company (Eds)).

[0079] Amino acid substitutions, deletions and / or insertions can be readily made using peptide synthesis techniques well known in the art, such as solid phase peptide synthesis, or by recombinant DNA manipulation. Methods for manipulating DNA sequences to generate substitution, insertion or deletion mutants of proteins are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those of skill in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange site-directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.

[0080] "Derivatives" further include peptides, oligopeptides, and polypeptides that may contain substitutions of amino acids with non-naturally occurring amino acid residues or additions of non-naturally occurring amino acid residues compared to the amino acid sequence of the naturally occurring form of a protein, such as the subject protein. A "derivative" of a protein also encompasses peptides, oligopeptides, and polypeptides that contain naturally occurring altered (glycosylation, acylation, prenylation, phosphorylation, myristoylation, sulfation, etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of the naturally occurring form of the polypeptide. Derivatives may also contain one or more non-amino acid substitutions or additions compared to the amino acid sequence from which they are derived, such as reporter molecules or other ligands covalently or non-covalently attached to the amino acid sequence, such as reporter molecules attached to facilitate their detection, and non-naturally occurring amino acid residues compared to the amino acid sequence of the naturally occurring protein. "Derivatives" also include fusions of the naturally occurring form of the protein with tag peptides such as FLAG, HIS6, or thioredoxin (for a review of tag peptides see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

[0081] "Ortholog" and "paralog" encompass evolutionary concepts used to describe the ancestral relationship of genes. Paralogs are genes within the same species that arose through duplication of an ancestral gene. Orthologs are genes from different organisms that arose through speciation, also derived from a common ancestral gene.

[0082] Variants, orthologs and paralogs of SEQ ID NO:2 encompassed by the present invention include, but are not limited to, polypeptides comprising SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31.

[0083] As is well known in the art, paralogs and orthologs may share distinct domains with appropriate amino acid residues at given sites, such as binding pockets for specific substrates or binding motifs for interactions with other proteins.

[0084] The term "domain" refers to a set of amino acids that are conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions may vary between homologs, highly conserved amino acids at specific positions indicate amino acids that are likely essential for the structure, stability or function of the protein. Identification of a high degree of conservation in the aligned sequences of a family of protein homologs can be used as a discriminator to determine whether any polypeptide in question belongs to a previously identified polypeptide family.

[0085] The term "motif" or "consensus sequence" refers to a short conserved region in the sequences of evolutionarily related proteins. A motif is most often a highly conserved portion of a domain, but may also include only a portion of a domain or may be located outside a conserved domain (provided all of the amino acids of the motif are outside the defined domain).

[0086] Specialist databases for domain identification, e.g. SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds.. pp53-61, AAAI Press, Menlo Park; There are several tools for computerized analysis of protein sequences, such as the Proteomics Server (Hullo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for computerized analysis of protein sequences is available from the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs can also be identified using routine techniques such as sequence alignment.

[0087] Methods for aligning sequences for comparison are well known in the art, and include GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP uses the Needleman and Wunsch algorithm ((1970) J Mol Biol 48: 443-453) to find a global (i.e., complete sequence) alignment that maximizes the number of matches between two sequences and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologs can be easily identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83) using default pairwise alignment parameters and the percent scoring method (see FIG. 1). Global percentages of similarity and identity may be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul 10;4:29. MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences.). As will be apparent to those skilled in the art, minor manual steps can be used to optimize alignments between conserved motifs. Also, instead of using full-length sequences for the identification of homologs, specific domains can be used. Sequence identity values ​​can be determined over the entire nucleic acid or amino acid sequence, or for selected domains or conserved motifs, using the above-mentioned programs with default parameters.For local alignments the Smith-Waterman algorithm is particularly useful (Smith TF, Waterman MS (1981) J. Mol. Biol 147(1);195-7).

[0088] The proteins of the invention can be altered in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are well known in the art. For example, amino acid sequence variants can be prepared by DNA mutations. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Patent No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the subject protein can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), which is incorporated herein by reference. Conservative substitutions, such as the exchange of one amino acid for another having similar properties, may be preferred.

[0089] Alternatively, mutant nucleotide sequences can be generated by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants can be screened to identify those that encode proteins that retain activity. For example, following mutagenesis, the encoded protein can be recombinantly expressed and the activity of the protein can be measured using standard assay techniques.

[0090] The inventors of the present invention have found that, for example by using one of the methods described above, tolerance or resistance to a particular herbicide can be significantly increased by mutating a nucleic acid encoding TriA by substituting one or more key amino acid residues of the TriA enzyme of SEQ ID NO: 2. Preferred substitutions in the mutant TriA are those that increase the herbicide tolerance of the plant while leaving the biological activity of the deaminase activity substantially unaffected.

[0091] Therefore, another object of the present invention relates to a mutated TriA polypeptide comprising the sequence of SEQ ID NO: 2, or a variant, derivative, ortholog, paralog or homolog thereof, in which key amino acid residues are replaced by any other amino acid.

[0092] As will be appreciated by those skilled in the art, amino acids in positions immediately adjacent to the amino acid positions mentioned below may also be substituted.Thus, in another embodiment, the variant of SEQ ID NO:2, its variants, derivatives, orthologs, paralogs or homologs comprises a mutated TriA, in which the amino acids at positions ±3, ±2 or ±1 amino acids from the critical amino acid are substituted with any other amino acid.

[0093] Based on techniques well known in the art, highly characteristic sequence patterns can be identified that can be used to search for additional candidate mutant TriAs with desired activities.

[0094] The present invention also encompasses the search for further mutant TriA candidates by applying suitable sequence patterns.As will be understood by the skilled reader, the sequence patterns are not limited to the exact interval between two adjacent amino acid residues in the patterns.For example, each of the intervals between two adjacent residues in the above patterns may be changed by ±10, ±5, ±3, ±2 or ±1 amino acid positions independently of each other without substantially affecting the desired activity.

[0095] Also, by applying the method of site-directed mutagenesis, e.g. saturation mutagenesis (see, e.g., Schenk et al., Biospektrum 03 / 2006, pages 277-279), the inventors of the present invention have identified and generated specific amino acid substitutions and combinations thereof, which, when introduced into a plant by transformation and expression, and the nucleic acid encoding the respective mutant TriA, confer on said plant increased herbicide resistance or tolerance to herbicides.

[0096] Thus, in a particularly preferred embodiment, a mutant TriA variant or derivative refers to a TriA polypeptide comprising SEQ ID NO:2, its ortholog, paralog, or homolog, whose amino acid sequence differs from the wild-type amino acid sequence of the TriA polypeptide at one or more positions corresponding to the following positions of SEQ ID NO:2: 92, 93, 155, 157.

[0097] Examples of differences at these amino acid positions include, but are not limited to, one or more of the following: the amino acid corresponding to position 92 is substituted with any other amino acid; the amino acid corresponding to position 93 is substituted with any other amino acid; the amino acid corresponding to position 155 is substituted with any other amino acid; The amino acid corresponding to position 157 is substituted with any other amino acid.

[0098] Examples of differences at these amino acid positions include, but are not limited to, one or more of the following: the amino acid corresponding to position 92 is other than leucine; the amino acid corresponding to position 93 is other than tyrosine; the amino acid corresponding to position 155 is other than methionine; The amino acid corresponding to position 157 is other than phenylalanine.

[0099] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, wherein the amino acid corresponding to position 92 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr.

[0100] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, wherein the amino acid corresponding to position 93 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Leu.

[0101] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, wherein the amino acid corresponding to position 155 is Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr.

[0102] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, wherein the amino acid corresponding to position 157 is Ala, Cys, Asp, Glu, Leu, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr.

[0103] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2 or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, Cys, Asp, Glu, Gly, His, Ile, Met, Asn, Pro, Gln, Ser, Thr, Val, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0104] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0105] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Cys, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0106] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Asp, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0107] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Glu, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0108] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Gly, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0109] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is His, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0110] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ile, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0111] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Met, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0112] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Asn, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0113] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Pro, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0114] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Gln, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0115] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ser, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0116] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Thr, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0117] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Val, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0118] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2 or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0119] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Ala, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0120] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Cys, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0121] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Glu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0122] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Phe, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0123] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Gly, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0124] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is His, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0125] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Ile, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0126] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Lys, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0127] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Met, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0128] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Asn, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0129] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Gln, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0130] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Arg, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0131] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Ser, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0132] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Thr, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0133] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Val, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0134] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Trp, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0135] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2 or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Ala, Cys, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Val, Tyr, and the amino acid corresponding to position 157 is Leu.

[0136] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Ala, and the amino acid corresponding to position 157 is Leu.

[0137] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Cys, and the amino acid corresponding to position 157 is Leu.

[0138] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Gly, and the amino acid corresponding to position 157 is Leu.

[0139] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is His, and the amino acid corresponding to position 157 is Leu.

[0140] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Ile, and the amino acid corresponding to position 157 is Leu.

[0141] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Lys, and the amino acid corresponding to position 157 is Leu.

[0142] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Leu, and the amino acid corresponding to position 157 is Leu.

[0143] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Asn, and the amino acid corresponding to position 157 is Leu.

[0144] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Pro, and the amino acid corresponding to position 157 is Leu.

[0145] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Gln, and the amino acid corresponding to position 157 is Leu.

[0146] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Ser, and the amino acid corresponding to position 157 is Leu.

[0147] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 157 is Leu.

[0148] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Tyr, and the amino acid corresponding to position 157 is Leu.

[0149] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2 or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Ala, Cys, Glu, Gly, His, Ile, Lys, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, Tyr.

[0150] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Ala.

[0151] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Cys.

[0152] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Glu.

[0153] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Gly.

[0154] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is His.

[0155] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Ile.

[0156] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Lys.

[0157] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Met.

[0158] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Asn.

[0159] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Gln.

[0160] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Arg.

[0161] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Ser.

[0162] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Thr.

[0163] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Val.

[0164] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Trp.

[0165] In another preferred embodiment, the mutant TriA comprises the sequence of SEQ ID NO:2, or a variant, derivative, ortholog, paralog, or homolog thereof, in which the amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Tyr.

[0166] It is considered within the knowledge of one of ordinary skill in the art to identify conserved regions and motifs shared among homologs, orthologs and paralogs encoded by SEQ ID NO: 1. Having identified such conserved regions that may represent suitable binding motifs, SEQ ID NO: 2 can be used as a reference to select any other amino acids, e.g., conserved amino acids, preferably amino acid substitutions as described above, to be substituted.

[0167] Another object relates to a method for identifying a nucleotide sequence encoding a mutant TriA that is herbicide resistant or tolerant, the method comprising: a) generating a library of nucleic acids encoding mutant TriAs; b) screening the population of nucleic acids by expressing each of the resulting mutant TriA-encoding nucleic acids in a cell or plant and treating the cell or plant with an herbicide; c) comparing the level of herbicide tolerance conferred by said population of mutant TriA-encoding nucleic acids with the level of herbicide tolerance conferred by a control TriA-encoding nucleic acid; d) selecting at least one mutant TriA-encoding nucleic acid that confers a significantly increased level of resistance to the herbicide over that conferred by the control TriA-encoding nucleic acid.

[0168] The level of herbicide resistance can also be determined by measuring the detoxification rate in cells, tissues, or plants.

[0169] The detoxification rate is the rate of herbicide degradation in each tissue over a given time period. Degradation and product formation can be measured analytically, for example, by liquid chromatography (LC) coupled to a high resolution (HR) mass spectrometer (MS). Products can be measured by comparison to standards and / or by structural elucidation.

[0170] In a preferred embodiment, the mutant TriA-encoding nucleic acid selected in step d) confers at least 2-fold greater resistance or tolerance to a cell or plant against an herbicide than the resistance or tolerance conferred by a control TriA-encoding nucleic acid.

[0171] In further preferred embodiments, the mutant TriA-encoding nucleic acid selected in step d) confers at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 500-fold greater resistance or tolerance to a cell or plant to an herbicide compared to the resistance or tolerance conferred by a control TriA-encoding nucleic acid.

[0172] Resistance or tolerance can be measured by generating a transgenic plant or host cell, preferably a plant cell, containing a nucleic acid sequence of the library of step a) and comparing said transgenic plant to a control plant or host cell, preferably a plant cell.

[0173] Many methods well known to those skilled in the art are available for obtaining suitable candidate nucleic acids for identifying mutant TriA-encoding nucleotide sequences from a variety of potential source organisms, including microorganisms, plants, fungi, algae, mixed cultures, etc., as well as environmental sources of DNA, such as soil. These methods include, inter alia, the preparation of cDNA or genomic DNA libraries, the use of appropriately degenerate oligonucleotide primers, the use of probes based on known sequences or complementation assays (e.g., for growth on tyrosine), and the use of mutagenesis and shuffling to provide recombined or shuffled mutant TriA coding sequences.

[0174] Nucleic acids containing candidate and control TriA coding sequences can be expressed in yeast, bacterial host strains, algae, or higher plants, such as tobacco or Arabidopsis, and the relative levels of resistance conferred by the TriA coding sequences can be examined by visual indicator phenotypes of the transformed strains or plants in the presence of different concentrations of the selected herbicide. Dose responses, as well as relative changes in dose responses related to the indicator phenotypes (brown color production, growth inhibition, herbicidal effect, etc.), are conveniently expressed in terms of GR50 (concentration to reduce growth by 50%) or MIC (minimal inhibitory concentration) values, with an increase in this value corresponding to an increase in resistance conferred by the expressed TriA. In a relatively rapid assay system based on transformation of bacteria such as E. coli, each variant TriA coding sequence can be expressed as a DNA sequence under the expression control of a controllable promoter, such as the lacZ promoter, and the different TriA sequences can be expressed with appropriate consideration of issues such as codon usage, for example by the use of synthetic DNA, to obtain as comparable levels of expression as possible. Such strains expressing nucleic acids containing different candidate TriA sequences can be plated on various concentrations of a selected herbicide, optionally in media supplemented with tyrosine, and the relative levels of resistance provided by the expressed TriA enzymes can be assessed based on the extent of inhibition of browning pigment formation and the MIC, or by measuring the degradation of the herbicide by LC-HRMS (liquid chromatography-high resolution mass spectrometry).

[0175] In another embodiment, the candidate nucleic acid is introduced into plant material by transformation to generate transgenic plants, which are regenerated to morphologically normal fertile plants, and the differences in the resistance of the plants to the selected herbicide are measured as described in the Examples section below. Many suitable methods for transformation are well known in the art, using appropriate selection markers such as kanamycin, binary vectors such as from Agrobacterium, and plant regeneration, such as regeneration from tobacco leaf disks. Optionally, a control population of plants is similarly transformed with a nucleic acid expressing a control TriA. The average and distribution of herbicide resistance levels to the herbicides of the series of original plant transformation events, or their progeny, are evaluated in the usual manner based on plant damage, symptoms of chlorosis of meristems, etc., at various herbicide concentrations. These data are represented, for example, by GR50 values ​​derived from dose / response curves with "dose" plotted on the x-axis and "percent kill", "herbicidal effect", "number of emerging green plants", etc. plotted on the y-axis, with increasing GR50 values ​​corresponding to increasing levels of resistance provided by the expressed TriA. Herbicides can be applied pre- or post-emergence as appropriate.

[0176] Another object of the present invention relates to an isolated, recombinant and / or chemically synthesized nucleic acid encoding said mutant TriA, which comprises the nucleotide sequence of SEQ ID NO: 1, or a variant or derivative thereof.

[0177] Examples of differences at the above amino acid positions include, but are not limited to, one or more of the following: the amino acid corresponding to position 92 is replaced with another amino acid; the amino acid corresponding to position 93 is replaced with another amino acid; the amino acid corresponding to position 155 is replaced with another amino acid; The amino acid corresponding to position 157 is replaced with another amino acid.

[0178] In a preferred embodiment, the encoded mutated TriA is a variant of SEQ ID NO:2, which includes one or more of the following: the amino acid corresponding to position 92 is other than leucine; the amino acid corresponding to position 93 is other than tyrosine; the amino acid corresponding to position 155 is other than methionine; The amino acid corresponding to position 157 is other than phenylalanine.

[0179] In a particularly preferred embodiment, the mutant TriA encoded by the nucleic acid of the invention comprises a variant of SEQ ID NO:2, in which the amino acids at positions corresponding to 92, 93, 155, and 157 of SEQ ID NO:2 have been replaced with other amino acids.

[0180] In a particularly preferred embodiment, the encoded mutant TriA comprises the sequence of SEQ ID NO:2, a variant, derivative, ortholog, paralog, or homolog thereof: The amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Val, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0181] In another particularly preferred embodiment, the encoded mutant TriA comprises the sequence of SEQ ID NO:2, a variant, derivative, ortholog, paralog, or homolog thereof: The amino acid corresponding to position 92 is Thr, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0182] In another particularly preferred embodiment, the encoded mutant TriA comprises the sequence of SEQ ID NO:2, a variant, derivative, ortholog, paralog, or homolog thereof: The amino acid corresponding to position 92 is Ala, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0183] In another particularly preferred embodiment, the encoded mutant TriA comprises the sequence of SEQ ID NO:2, a variant, derivative, ortholog, paralog, or homolog thereof: The amino acid corresponding to position 92 is Ser, the amino acid corresponding to position 93 is Leu, the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

[0184] In other embodiments, the invention includes progeny or descendants of the herbicide-tolerant plants of the invention, as well as seeds derived from the herbicide-tolerant plants of the invention and cells derived from the herbicide-tolerant plants of the invention.

[0185] In some embodiments, the invention provides progeny or descendant plants derived from a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein the progeny or descendant plant comprises in at least a portion of its cells a recombinant polynucleotide operably linked to the promoter, and expression of the mutant TriA polypeptide confers resistance to a herbicide to the progeny or descendant plant.

[0186] In one embodiment, the seed of the present invention preferably comprises the herbicide-tolerant trait of a herbicide-tolerant plant, hi another embodiment, the seed is capable of germinating into a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to a herbicide to a progeny or descendent plant.

[0187] In some embodiments, the plant cells of the present invention are capable of regenerating a plant or plant part. In other embodiments, the plant cells are not capable of regenerating a plant or plant part. Examples of cells that are not capable of regenerating a plant include, but are not limited to, endosperm, seed coats (seed coats and pericarp), and root caps.

[0188] In another embodiment, the invention provides a plant cell capable of regenerating a plant comprising in at least a portion of its cells a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant, wherein the plant cell comprises a recombinant polynucleotide operably linked to the promoter.

[0189] In another embodiment, the invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in a plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, wherein expression of the mutant TriA polypeptide confers tolerance to a herbicide to the cell.

[0190] In another embodiment, the invention relates to a plant cell transformed with a nucleic acid encoding a mutant TriA polypeptide of the invention, wherein expression of the nucleic acid in the plant cell confers increased resistance or tolerance to a herbicide compared to a wild-type variety of the plant cell. Preferably, the nucleic acid encoding the mutant TriA polypeptide comprises a polynucleotide sequence selected from the group consisting of: a) a polynucleotide set forth in SEQ ID NO: 1, or a variant or derivative thereof, b) a polynucleotide encoding the polypeptide set forth in SEQ ID NO: 2, or a variant or derivative thereof, c) a polynucleotide comprising at least 60 contiguous nucleotides of any of a) or b), and d) a polynucleotide complementary to any of the polynucleotides of a) to c).

[0191] In some aspects, the present invention provides plant products prepared from plants tolerant to herbicides of the present invention. In some embodiments, examples of plant products include, but are not limited to, grains, oils, and meals. In one embodiment, the plant product is a plant grain (e.g., grain suitable for use as feed or processing), a plant oil (e.g., oil suitable for use as food or biodiesel), or a plant meal (e.g., meal suitable for use as feed).

[0192] In one embodiment, a plant product is provided prepared from a plant or plant part, the plant or plant part comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to a herbicide to the plant or plant part.

[0193] In another embodiment, the present invention relates to a method for producing a transgenic plant cell having increased resistance to an herbicide compared to a wild-type variety of the plant cell, the method comprising transforming the plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide.

[0194] In another embodiment, the present invention relates to a method for generating a transgenic plant, the method comprising the steps of: (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in the plant cell and capable of expressing a mutant TriA polypeptide encoded by the polynucleotide; and (b) producing a plant having increased resistance to a herbicide from the plant cell.

[0195] In some aspects, the invention provides a method for producing a plant that is tolerant to a herbicide, in one embodiment, the method comprises regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and expression of the mutant TriA polypeptide confers tolerance to the herbicide to the plant.

[0196] The term "expression" or "gene expression" refers to the transcription of one or more specific genes or of a specific genetic construct. The term "expression" or "gene expression" refers in particular to the transcription of one or more genes or genetic constructs into structural RNA (rRNA, tRNA) or mRNA, with or without the subsequent translation of the latter into a protein. The method includes transcription of DNA and processing of the resulting mRNA product.

[0197] It is understood that in order to obtain the desired effect of the present invention, i.e. a plant that is tolerant or resistant to an herbicide that is a derivative of the herbicide, at least one nucleic acid is "overexpressed" by methods and means known to those skilled in the art.

[0198] The term "enhanced expression" or "overexpression" as used herein means any form of expression that is in addition to wild-type expression levels. Methods for increasing expression of genes or gene products are well documented in the art, for example overexpression driven by a suitable promoter, or the use of transcriptional or translational enhancers. An isolated nucleic acid that acts as a promoter or enhancer element may be introduced into a suitable position (usually upstream) of a non-heterologous form of polynucleotide to upregulate expression of a nucleic acid encoding a polypeptide of interest. For example, the endogenous promoter may be modified in vivo by mutation, deletion, and / or substitution (see Kmiec, US 5,565,350; Zarling et al., WO 9322443), or an isolated promoter may be introduced into a plant cell in the correct orientation and spacing from the gene of the invention to control expression of the gene. If expression of a polypeptide is desired, it is generally desirable to include a polyadenylation region at the 3'-end of the polynucleotide coding region. The polyadenylation region can be from the native gene, from a variety of other plant genes, or from T-DNA. The 3' terminal sequence to be added can be, for example, from the nopaline synthase or octopine synthase genes, or from another plant gene, or less preferably from any other eukaryotic gene. Intron sequences can also be added to the 5' untranslated region (UTR) or to the coding sequence of the partial coding sequence to increase the amount of mature message that accumulates in the cytosol. Inclusion of splicable introns in transcription units in both plant and animal expression constructs has been shown to increase gene expression up to 1000-fold at both the mRNA and protein levels (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is usually greatest when placed near the 5' end of the transcription unit. The use of the maize introns Adh1-S introns 1, 2, and 6, the Bronze-1 intron, is known in the art. For more information, see The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, NY (1994).

[0199] Where appropriate, the nucleic acid sequence may be optimized for increased expression in transformed plants. For example, a coding sequence can be provided that contains plant-preferred codons for improved expression in plants. See, for example, Campbell and Gowri (1990) Plant Physiol., 92: 1-11 for a discussion of host-preferred codon usage. Methods for preparing plant-preferred genes are also known in the art. See, for example, U.S. Patent Nos. 5,380,831 and 5,436,391, which are incorporated herein by reference, and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

[0200] Thus, the wild-type / mutant TriA nucleic acid of the present invention is provided in an expression cassette for expression in a plant of interest. The cassette comprises a regulatory sequence operably linked to the wild-type or mutant TriA nucleic acid sequence of the present invention. The term "regulatory element" as used herein refers to a polynucleotide capable of regulating the transcription of an operably linked polynucleotide, including, but not limited to, a promoter, an enhancer, an intron, a 5'UTR, and a 3'UTR. "Operably linked" refers to a functional association between a promoter and a second sequence, where the promoter sequence initiates and mediates the transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous, and where necessary to join two protein coding regions, contiguous and in the same reading frame. The cassette may further contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional genes can be provided on multiple expression cassettes.

[0201] Such expression cassettes are provided with multiple restriction sites for inserting the wild-type / mutant TriA nucleic acid sequence so that it is under the transcriptional control of the regulatory region. The expression cassette may further contain a selectable marker gene. The expression cassette of the present invention comprises, in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter) functional in a plant, a nucleic acid sequence encoding the wild-type / mutant TriA of the present invention, and a transcriptional and translational termination region (i.e., a termination region). The promoter may be native or analogous, or foreign or heterologous, to the plant host and / or the wild-type / mutant TriA nucleic acid sequence of the present invention. Furthermore, the promoter may be a natural sequence or a synthetic sequence. When a promoter is "foreign" or "heterologous" to a plant host, it is intended that the promoter is not found in the native plant into which it is introduced. When a promoter is "foreign" or "heterologous" to the wild-type / mutant TriA nucleic acid sequence of the present invention, it is intended that the promoter is not native or a naturally occurring promoter to the operably linked wild-type / mutant TriA nucleic acid sequence of the present invention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region heterologous to the coding sequence. Although it may be preferred to use a heterologous promoter to express the wild-type / mutant TriA nucleic acid of the present invention, the native promoter sequence may also be used. Such a construct alters the expression level of the wild-type / mutant TriA protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

[0202] The termination region may be native to the transcription initiation region, native to the operably linked wild-type / mutant TriA sequence of interest, native to the plant host, or derived from another source (i.e., foreign or heterologous to the promoter, the wild-type / mutant TriA nucleic acid sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) MoI. Gen. Genet. 262: 141-144, Proudfoot (1991) Cell 64:671-674, Sanfacon et al. (1991) Genes Dev. 5: 141-149, Mogen et al. (1990) Plant Cell 2: 1261-1272, Munroe et al. (1990) Gene 91: 151-158, Ballas t al. (1989) Nucleic Acids Res. 17:7891-7903, and Joshi et al. (1987) Nuclei Acid Res. 15:9627-9639. Where appropriate, genes may be optimized for increased expression in transformed plants. That is, genes may be synthesized with plant-preferred codons for improved expression. For a discussion of host-preferred codon usage, see, e.g., Campbell and Gowri (1990) Plant Physiol. 92: 1-11. Methods for synthesizing plant-preferred genes are available in the art. See, e.g., U.S. Patent Nos. 5,380,831 and 5,436,391, which are incorporated herein by reference, and Murray et al. (1989) Nucleic Acids Res. 17:477-498.

[0203] Thus, the present invention provides an expression cassette comprising a mutant TriA nucleic acid molecule of the present invention and a promoter operable in a plant cell.

[0204] In a preferred embodiment, the promoter is a root-specific or root-enhanced promoter.

[0205] In a particularly preferred embodiment, the promoter is a root-specific or root-enhanced promoter from soybean (Glycine max) (eg, p-Glyma04g34080, see Examples 8 and 9).

[0206] More preferably, the promoter comprises the nucleic acid sequence of SEQ ID NO:32.

[0207] Although the polynucleotides of the present invention can be used as a selectable marker gene for plant transformation, the expression cassettes of the present invention can contain another selectable marker gene for the selection of transformed cells. Selectable marker genes, including those of the present invention, are utilized for the selection of transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), and genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). In general terms, Yarranton (1992) Curr. Opin. Biotech. 3 :506-511, Christophers on et al (1992) Proc. Natl. Acad. ScL USA 89:6314-6318, Yao et al. (1992) Cell 71:63-72, Reznikoff (1992) MoI Microbiol 6:2419-2422, Barkley et al (1980) in The Operon, pp. 177-220, Hu et al (1987) Cell 48:555-566, Brown et al (1987) Cell 49:603-612, Figge et al (1988) Cell 52:713-722, Deuschle et al (1989) Proc. Natl Acad. AcL USA 86:5400-5404, Fuerst et al (1989) Proc. Natl Acad. ScL USA 86:2549-2553, Deuschle et al (1990) Science 248:480-483, Gossen (1993) Ph.D. Thesis, University of Heidelberg, Reines et al (1993) Proc. Natl Acad.ScL USA 90: 1917-1921, Labow et al (1990) MoI Cell Biol 10:3343-3356, Zambretti et al (1992) Proc. Natl Acad. ScL USA 89:3952-3956, Bairn et al (1991) Proc. Natl Acad. ScL USA 88:5072-5076, Wyborski et al (1991) Nucleic Acids Res. 19:4647-4653, Hillenand-Wissman (1989) Topics MoI Struc. Biol 10: 143- 162, Degenkolb et al (1991) Antimicrob. Agents Chemother. 35: 1591-1595, Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104, Bonin (1993) Ph.D. Thesis, University of Heidelberg, Gossen et al (1992) Proc. Natl Acad. ScL USA 89:5547- 5551, Oliva et al (1992) Antimicrob. Agents Chemother. 36:913-919, Hlavka et al (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin), Gill et al (1988) Nature 334:721-724, the disclosures of which are incorporated herein by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene may be used in the present invention.

[0208] Furthermore, additional sequence modifications are known to enhance gene expression in cellular hosts. These include the deletion of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other well-characterized sequences that may be detrimental to gene expression. The GC content of the sequence can be adjusted to an average level for a given cellular host, calculated with reference to known genes expressed in the host cell. Also, if desired, the sequence can be easily modified to avoid predicted hairpin-like secondary mRNA structures. Nucleotide sequences for enhancing gene expression can also be used in plant expression vectors. These include, for example, introns from the maize Adh gene Adh1-S introns 1, 2, and 6 (Callis et al. Genes and Development 1:1183-1200, 1987), as well as leader sequences (W-sequences) from Tobacco Mosaic Virus (TMV), Maize Chlorotic Mottle Virus, and Alfalfa Mosaic Virus (Gallie et al. Nucleic Acid Res. 15:8693-8711, 1987 and Skuzeski et al. Plant Mol. Biol. 15:65-79, 1990). The first intron from the shrunken-1 locus has been shown to increase gene expression in chimeric gene constructs. U.S. Patent Nos. 5,424,412 and 5,593,874 disclose the use of certain introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also show that introns are useful for regulating gene expression in a tissue-specific manner. To further enhance or optimize gene expression, the plant expression vector of the present invention may also contain a DNA sequence containing a matrix attachment region (MAR). Plant cells transformed with such modified expression systems may then exhibit overexpression or constitutive expression of the nucleotide sequence of the present invention.

[0209] The present invention further provides an isolated recombinant expression vector comprising an expression cassette containing a wild-type / mutant TriA nucleic acid as described above, wherein expression of the vector in a host cell results in increased resistance to herbicides compared to a wild-type variant of the host cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. One type of vector is a "plasmid", which is a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into a viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Some vectors are also capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0210] The recombinant expression vector of the invention comprises a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, meaning that the recombinant expression vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed, selected based on the host cell to be used for expression. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of a nucleotide sequence only in certain host cells or under certain conditions. As will be appreciated by those skilled in the art, the design of an expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level of the polypeptide, and the like. The expression vector of the invention can be introduced into a host cell to produce a polypeptide or peptide, including a fusion polypeptide or peptide, encoded by a nucleic acid as described herein (e.g., a wild-type or mutant TriA polypeptide, a fusion polypeptide, and the like).

[0211] The expression vector may further contain a 5' leader sequence in the expression construct. Such a leader sequence can act to enhance translation. Translation leaders are known in the art and include picornavirus leaders such as the EMCV leader (Encephalomyo carditis 5' noncoding region) (Elroy-Stein et al. (1989) PNAS, 86:6126-6130), potyvirus leaders such as the TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154:9-20), and the human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94), the nontranslated leader derived from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 165(2):233-238). 325:622-625), the tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256), and the corn chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 :382-385). See also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

[0212] Other methods known to enhance translation can also be utilized, such as introns. In preparing expression vectors, various nucleic acid fragments can be manipulated to obtain nucleic acid sequences in the correct orientation and, where appropriate, in the correct reading frame. To this end, adapters or linkers can be used to join the nucleic acid fragments, or other manipulations can be used, such as providing convenient restriction sites, removing excess nucleic acid, removing restriction sites, etc. To this end, in vitro mutagenesis, primer repair, restriction cleavage, annealing, resubstitutions, such as transitions and transversions, can be performed.

[0213] Many promoters can be used in the practice of the present invention. The promoter can be selected based on the desired outcome. The nucleic acid can be combined with a constitutive, tissue-selective, or other promoter for expression in plants.

[0214] Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter disclosed in WO99 / 43838 and U.S. Pat. No. 6,072,050 and other constitutive promoters, the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), rice actin (McElroy et al. (1990) Plant Cell 2: 163-171), ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689), pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588), MAS (Velten et al. (1984) EMBO J. 3:2723-2730), ALS promoter (U.S. Pat. No. 5,659,026), etc. Other constitutive promoters are described, for example, in U.S. Pat. Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611.

[0215] Tissue-selected promoters can be utilized to target enhanced expression in specific plants, including, but not limited to, leaf-selected promoters, root-selected promoters, seed-selected promoters, and stem-selected promoters. Some examples of tissue-selective promoters are described, for example, in Yamamoto et al. (1997) Plant J. 12(2):255-265, Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803, Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343, Russell et al. (1997) Transgenic Res. 6(2): 157-168, Rinehart et al. (1996) Plant Physiol. 1 12(3): 1331-1341, Van Camp et al. (1996) Plant Physiol. 112(2):525-535, Canevascini et al. (1996) Plant Physiol. 1 12(2):513- 524, Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778, Lam (1994) Results Probl. Cell Differ. 20:181-196, Orozco ef al. (1993) Plant Mol Biol. 23(6): 1 129-1138, Matsuoka et al. (1993) Voc Natl. Acad. ScL USA 90(20):9586-9590, and Guevara-Garcia et al. (1993) Plant J 4(3):495-505. If necessary, promoters can be modified for weak expression.

[0216] In a preferred embodiment, the promoter is a root-specific or root-enhanced (enhanced) promoter.

[0217] In a particularly preferred embodiment, the promoter comprises the nucleic acid sequence of SEQ ID NO:32.

[0218] In some embodiments, the nucleic acid of interest may be targeted to the chloroplast for expression. In such cases, if the nucleic acid of interest is not directly inserted into the chloroplast, the expression vector further contains a chloroplast targeting sequence, which comprises a nucleotide sequence encoding a chloroplast transit peptide to direct the gene product of interest to the chloroplast. Such transit peptides are known in the art. With respect to the chloroplast targeting sequence, "operably linked" means that the nucleic acid sequence encoding the transit peptide (i.e., the chloroplast targeting sequence) is joined to the desired coding sequence of the present invention such that the two sequences are contiguous and in the same reading frame. See, e.g., Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126, Clark et al. (1989) J Biol. Chem. 264:17544-17550, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968, Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421, and Shah et al. (1986) Science 233:478-481. For example, chloroplast transit peptides known in the art can be fused to the amino acid sequence of a TriA polypeptide of the invention by operably linking a chloroplast targeting sequence to the 5'-end of the nucleotide sequence encoding the TriA polypeptide.

[0219] Chloroplast targeting sequences are known in the art and include those targeted to the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J Biol. Chem. 266(5):3335-3342), EPSPS (Archer et al. (1990) J Bioenerg. Biomemb. 22(6):789-810), tryptophan synthase (Zhao et al. (1995) J Biol. Chem. 270(11):6081-6087), plastocyanin (Lawrence et al. (1997) J Biol. Chem. 272(33):20357-20363), chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457), and light-harvesting chlorophyll a / b-binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263: 14996-14999). Also, Von Heijne et al. (1991) Pant Mol. Biol. Rep. 9: 104- 126, Clark et al. (1989) J Biol. Chem. 264: 17544-17550, Della-Cioppa et al. (1987) Plant Physiol. 84:965-968, Romer et al. (1993) Biochem Biophys. Res. Commun. 196: 1414-1421, and Shah et al. (1986) Science 233:478-481.

[0220] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. ScL USA 87:8526-8530, Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917, Svab and Maliga (1993) EMBO J. 12:601-606. This method utilizes particle gun delivery of DNA containing a selection marker and targeting the DNA to the plastid genome by homologous recombination. Additionally, plastid transformation can be achieved by transactivation of silent plastid-borne transgenes by tissue-selective expression of nuclear-encoded, plastid-directed RNA polymerase. Such a system is reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

[0221] The nucleic acid of interest targeted to chloroplasts can be optimized for expression in chloroplasts to account for the difference in codon usage between the plant nucleus and this organelle. In this way, the nucleic acid of interest can be synthesized using codons that are preferred for chloroplasts. See, for example, U.S. Patent No. 5,380,831, which is incorporated herein by reference.

[0222] I'm not sure if I'm going to be able to do that. An, G. et al. (1986) Plant PysioL、81 :301-305、Fry, J., et al. ( 1987) Plant Cell Rep. 6:321-325、Block, M. (1988) Theor. Appl. Genet .16: 161 -1 1 A、Hinchee, et al. (1990) Stadler. Genet. Symp.20322.203-2 2、Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494、Chee, PP and Slightom, JL (1992) Gene.ll 8:255-260、Christou, et al. (1992) Trends. Biotechnol. 10:239-246、Halluin, et al. (1992) Bio / Technol. 10:309-314、Dhir, et al. (1992) Plant Physiol. 99:81-88、Casas et al. (1993) Proc. Nat. Acad Sd. USA 90: 1 1212-1 1216、Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P.119-124、Davies, et al. (1993) Plant Cell Rep. 12: 180-183、Dong, JA and Mchughen, A. (1993) Plant ScL 91 : 139-148、Franklin, CI and Trieu, TN (1993) Plant. Physiol. 102: 167、Golovkin, et al. (1993) Plant ScL 90:41-52、Guo Chin ScL Bull. 38:2072-2078、Asano, et al. (1994) Plant Cell Rep. 13、Ayeres NM and Park, WD (1994) Crit. Freeze. Plant. Sci.See 13:219-239, Barcelo, et al. (1994) Plant. J. 5:583-592, Becker, et al. (1994) Plant. J. 5:299-307, Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230, Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27, Eapen et al. (1994) Plant Cell Rep. 13:582-586, Hartman, et al. (1994) Bio-Technology 12: 919923, Ritala, et al. (1994) Plant. Mol. Biol. 24:317-325, and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748.

[0223] In some embodiments, the method of the invention includes a step of introducing a polynucleotide construct into a plant. By "introducing" it is meant presenting the construct to the plant so that the polynucleotide construct enters the interior of a cell of the plant. The method of the invention does not depend on a particular method of introducing the polynucleotide construct into the plant, as long as the polynucleotide construct enters the interior of at least one cell of the plant. Methods of introducing a polynucleotide construct into a plant are known in the art, including but not limited to stable transformation methods, transient transformation methods, and virus-mediated methods. As used herein, the term "introducing" or "transforming" further refers to the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for transfer. Plant tissues capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with the genetic constructs of the invention and therefrom regenerated whole plants. The particular tissues selected will vary depending on the clonal propagation systems available and optimal for the particular species being transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristem tissues (e.g., apical meristems, axillary buds, and root meristems), and induced meristem tissues (e.g., cotyledon meristems and hypocotyl meristems). The polynucleotides may be introduced into the host cell either transiently or stably, and may be non-integrated and maintained, for example, as a plasmid. Alternatively, they may be integrated into the host genome. The resulting transformed plant cells may then be used to regenerate transformed plants by methods known to those of skill in the art.

[0224] "Stable transformation" means that a polynucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. "Transient transformation" means that a polynucleotide construct introduced into a plant is not integrated into the genome of the plant.

[0225] For transformation of plants and plant cells, the nucleotide sequence of the present invention is inserted into any vector known in the art suitable for expression of the nucleotide sequence in plants or plant cells using standard techniques. The choice of vector depends on the preferred transformation technique and the target plant species to be transformed. In one embodiment of the present invention, the coding nucleotide sequence is operably linked to a plant promoter, e.g., a promoter known in the art for high level expression in plant cells, and then this construct is introduced into a plant cell sensitive to herbicides and a transformed plant is regenerated. In some embodiments, the transformed plant is tolerant to exposure to levels of herbicide that would kill or severely damage a plant regenerated from a non-transformed cell. This method can be applied to any plant species or crop.

[0226] Methods for constructing plant expression vectors and introducing foreign nucleic acid into plants are widely known in the art. For example, foreign DNA can be introduced into plants using tumor-inducing (Ti) plasmid vectors. Other methods used for delivery of foreign DNA include PEG-mediated protoplast transformation, electroporation, microinjection whiskers, and gene gun or particle bombardment for direct DNA uptake. Such methods are known in the art (see U.S. Patent No. 5,405,765 to Vasil et al; Bilang et al. (1991) Gene 100: 247-250; Scheid et al., (1991) MoL Gen. Genet., 228: 104-1 12; Guerche et al., (1987) Plant Science 52: 1 1 1 -1 16; Neuhause et al., (1987) Theor. Appl Genet. 75: 30-36; Klein et al., (1987) Nature 327: 70-73; Howell et al., (1980) Science 208: 1265; Horsch et al., (1985) Science 227: 1229-1231; DeBlock et al., (1989) Plant Physiology 91 : 694-701, Methods for Plant Molecular Biology (Weissbach and Weissbach, eds.) Academic Press, Inc. (1988) and Methods in Plant Molecular Biology (Schuler and Zielinski, eds.) Academic Press, Inc. (1989).

[0227] Other suitable methods for introducing nucleotide sequences into plant cells include microinjection, as described, for example, in Crossway et al. (1986) Biotechniques 4:320-334; electroporation, as described, for example, in Riggs et al. (1986) Proc. Natl. Acad. ScL USA 83:5602- 5606; Agrobacterium-mediated transformation, as described, for example, in U.S. Pat. No. 5,563,055 to Townsend et al. and U.S. Pat. No. 5,981,840 to Zhao et al.; direct gene transfer, as described, for example, in Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particle acceleration, as described, for example, in U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and 5,932,782 to Tomes et al. (1995) "Direct DNA transfer into intact plant cells by particle bombardment." Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer- Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923-926), and Led transformation (WO 00 / 28058). Also, Weissinger et al., (1988) Ann. Rev. Genet. 22:421-477, Sanford et al, (1987) Particulate Science and Technology 5:27-37 (onion), Christou et al, (1988) Plant Physiol. 87:671-674 (soybean), McCabe et al., (1988) Bio / Technology 6:923-926 (soybean), Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean), Singh et al, (1998) Theor. Appl. Genet.96:319-324 (soybean), Datta et al., (1990) Biotechnology 8:736-740 (rice), Klein et al., (1988) PNAS, 85:4305-4309 (maize), Klein et al., (1988) Biotechnology 6:559-563 (maize), U.S. Patent Nos. 5,240,855, 5,322,783, and 5,324,646, Tomes et al., (1995) "Direct DNA Transfer into Intact Plant Cells by Microprojectile Bombardment," Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer- Verlag, Berlin) (maize), Klein et al., (1988) Plant Physiol. 91 :440-444 (maize), Fromm et al., (1990) Biotechnology 8:833-839 (maize), Hooykaas-Van Slogteren et al., (1984) Nature (London) 31 1 :763-764, Bowen et al, U.S. Pat. No. 5,736,369 (cereals), Bytebier et al, (1987) PNAS 84:5345- 5349 (Liliaceae), De Wet et al., (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al, (Longman, New York), pp. 197-209 (pollen), Kaeppler et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler et al., (1992) Theor. Apph Genet. 84:560-566 (whisker-mediated transformation), D'Halluin et al., (1992) Plant Cell 4: 1495-1505 (electroporation), Li et al.See also, Christou and Ford (1993) Plant Cell Reports 12:250- 255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice), Osjoda et al, (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens), each of which is incorporated herein by reference.

[0228] Preferably, transgenic plants, including transgenic crop plants, are produced by Agrobacterium-mediated transformation. An advantageous transformation method is transformation in planta. For this, it is possible to apply Agrobacterium to plant seeds or to inoculate plant meristems with Agrobacterium. It has proven particularly advantageous to apply a suspension of Agrobacterium transformed according to the invention to intact plants or at least to flower primordia. The plants are then grown until treated plant seeds are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well-known rice transformation methods, such as those described in any of the following: European Patent Application Publication No. 1198985, Aldemita and Hodges (Plant 199: 612-617, 1996), Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), the disclosures of which are incorporated herein by reference as if fully set forth in their entireties. For maize transformation, preferred methods are described in Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), the disclosures of which are incorporated herein by reference as if fully set forth in their entireties. The method is further described, for example, in B. Jenes et al., Techniques for Gene Transfer, Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SD Kung and R. Wu, Academic Press (1993) 128-143 and Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225).The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transforming Agrobacterium tumefaciens, such as pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacterium transformed with such a vector can then be used as known for the transformation of plants such as plants used as models, such as Arabidopsis (Arabidopsis thaliana is not considered a crop plant within the scope of the present invention), or crop plants, such as tobacco plants for example, by immersing wounded or chopped leaves in a solution of Agrobacterium followed by culturing in a suitable medium. Transformation of plants by Agrobacterium tumefaciens is described, for example, by Hoefgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from FF White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SD Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0229] One transformation method known to those skilled in the art is to soak flowering plants in a solution of Agrobacterium containing TriA nucleic acid, followed by breeding of the transformed gametophytes. Agrobacterium-mediated plant transformation can be carried out, for example, using Agrobacterium tumefaciens strains GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech). Transformation can be carried out by standard transformation and regeneration techniques (Deblaere et al., 1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed. - Dordrecht: Kluwer Academic Publ., 1995. - in Sect., Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R. and Thompson, John E., Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed by transformation of cotyledons or hypocotyls (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). The use of Agrobacterium and antibiotics for plant selection depends on the binary vector and Agrobacterium strain used for transformation. Selection in rapeseed is usually performed using kanamycin as a selectable plant marker. Agrobacterium-mediated gene transfer into flax can be performed using the technique described, for example, in Mlynarova et al., 1994, Plant Cell Report 13:282-285.Additionally, soybean transformation can be carried out using techniques described, for example, in EP 0424 047, U.S. Pat. No. 5,322,783, EP 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. Maize transformation can be achieved by particle bombardment, polyethylene glycol-mediated DNA uptake, or silicon carbide fiber methods (e.g., Freeling and Walbot, "The Maize Handbook," Springer Verlag: New York (1993) ISBN 3-540-97826-7). Specific examples of maize transformation can be found in U.S. Pat. No. 5,990,387, and wheat transformation can be found in WO 93 / 07256.

[0230] In some embodiments, the polynucleotide of the invention can be introduced into a plant by contacting the plant with a virus or viral nucleic acid. In general, such methods include incorporating a polynucleotide construct of the invention into a viral DNA or RNA molecule. It is recognized that the polypeptide of the invention may be initially synthesized as part of a viral polyprotein, which may then be processed by in vivo or in vitro proteolysis to generate the desired recombinant polypeptide. It is further recognized that the promoter of the invention also encompasses promoters utilized for transcription by viral RNA polymerase. Methods of introducing polynucleotide constructs into plants and expressing proteins encoded therein involving viral DNA or RNA molecules are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931, which are incorporated herein by reference. The transformed cells can be grown into plants according to conventional methods. See, e.g., McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be grown and pollinated with the same transformed line or a different line, and the resulting hybrids that constitutively express the desired phenotypic trait can be identified. Two or more generations can be grown to ensure that expression of the desired phenotypic trait is stably maintained and inherited, after which seeds can be harvested to confirm whether expression of the desired phenotypic trait has been achieved.

[0231] The present invention can be used for the transformation of any plant species, including but not limited to monocotyledonous and dicotyledonous plants.Examples of plant species of interest include, but are not limited to, corn or maize (Zea mays), Brassica species (e.g., B. napus, B. rapa, B. juncea), particularly those useful as a source of seed oils, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Seedum cepa), sorghum (Sorghum bicolor, Sorghum vulgare), millet, e.g., Pennisetum glaucum, Panicum miliaceum, Setaria italica, Eleusine coracana), sunflower (Helinthus annuus), safflower (Carthamus tinctorius), and the like. tinctorius), wheat (T. Turgidum ssp.), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solarium tuberosum), peanut (Arachis hypogaea), cotton (Gossypium barbadense), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus tree (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia nut (Macadamia integrifolia), almond (Prunus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.Preferably, the plants of the present invention are crop plants (e.g., sunflower, Brassica spp., cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum, millet, etc.).

[0232] In addition to transforming somatic cells that will be regenerated into complete plants, it is also possible to transform cells of the plant meristem, in particular those that will become gametophytes. In this case, the transformed gametophytes will develop like natural plants and become transgenic plants. Thus, for example, Arabidopsis seeds are treated with Agrobacterium and seeds are obtained from the growing plants, a certain proportion of which are transformed and therefore transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, NH Chua and J Shell, Eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. An alternative method is based on repeated removal of the inflorescence and incubation of the excised portion with transformed Agrobacterium in the center of the rosette, whereby transformed seeds can also be obtained at a later time point (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, a particularly effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, whole plants are treated with a suspension of Agrobacterium under reduced pressure [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1194-1199], whereas in the "floral dip" method, growing floral tissue is briefly incubated with a suspension of detergent-treated Agrobacterium [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743]. In both cases, a certain percentage of transgenic seeds are harvested, which can be differentiated from non-transgenic seeds by growing them under the selective conditions mentioned above.Furthermore, stable transformation of plastids is advantageous since plastids are maternally inherited in most crops, reducing or eliminating the risk of transgene shedding through pollen. Transformation of the chloroplast genome is generally achieved by the method outlined in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly, the sequence to be transformed is cloned together with a selectable marker gene between the chloroplast genome and homologous flanking sequences. These homologous flanking sequences direct site-specific integration into the plastome. Plastid transformation has already been described for many different plant species, and a general overview can be found in Bock (2001) Transgeneic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Recently, further biotechnological advances have been reported in the form of marker-free plastid transformants, which can be generated by transiently cointegrated marker genes (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). The genetically modified plant cells can be regenerated by all methods familiar to the skilled worker. Suitable methods can be found in the abovementioned publications by SD Kung and R. Wu, Potrykus or Hoefgen and Willmitzer.

[0233] Generally, after transformation, the plant cell or cells are selected for the presence of one or more markers encoded by genes expressible in the plant that have been co-transferred with the gene of interest, and the transformed material is then regenerated into whole plants. To select transformed plants, the plant material obtained by transformation is generally subjected to selection conditions, which allows the transformed plants to be distinguished from non-transformed plants. For example, the seeds obtained as described above can be sown and, after the first growing season, subjected to an appropriate selection by spraying. A further possibility is to grow the seeds, after appropriate sterilization, on agar plates with an appropriate selection agent such that only transformed seeds can develop into plants. Alternatively, the transformed plants are screened for the presence of a selection marker such as those described above.

[0234] Following DNA transfer and regeneration, putatively transformed plants may also be assessed for the presence, copy number and / or genomic organization of the gene of interest using, for example, Southern analysis. Alternatively, or in addition, expression levels of the newly introduced DNA may be monitored using Northern and / or Western analysis, both techniques well known to those of skill in the art.

[0235] The transformed plants produced can be propagated by various means, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants can be self-pollinated and homozygous second generation (or T2) transformants can be selected, and the T2 plants can then be further propagated by classical breeding techniques. The transformed organisms produced can take various forms, such as chimeras of transformed and untransformed cells, clonal transformants (e.g., all cells transformed to contain the expression cassette), and grafts of transformed and untransformed tissues (e.g., a plant comprising a transformed rootstock grafted onto an untransformed scion).

[0236] Preferably, expression of the nucleic acid in the plant results in an increased resistance of the plant to the herbicide compared to a wild type variety of the plant.

[0237] In another embodiment, the present invention relates to a plant comprising a plant cell according to the present invention, wherein expression of the nucleic acid in the plant confers increased resistance of the plant to herbicides compared to a wild type variety of the plant.

[0238] The plants described herein can be either transgenic crop plants or non-transgenic plants.

[0239] In addition to the general definitions above, "transgenic," "transgenic," or "recombinant" means, for example, with respect to a nucleic acid sequence, an expression cassette, genetic construct, or vector comprising the nucleic acid sequence, or an organism transformed with a nucleic acid sequence, expression cassette, or vector of the invention, all of which constructions are carried out by recombinant methods, where (a) a nucleic acid sequence encoding a protein useful in the methods of the invention, or (b) a gene control sequence, e.g., a promoter, operably linked to the nucleic acid sequence of the invention; or (c) a) and b) Either of the above is not present in its natural genetic environment or has been modified by recombinant methods to allow expression of the mutant TriA of the invention, which modification can take the form of, for example, substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. Natural genetic environment is understood to mean the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, preferably the natural genetic environment of the nucleic acid sequence is at least partially retained. The environment is located at least on one side of the nucleic acid sequence and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette, such as the naturally occurring combination of the natural promoter of a nucleic acid sequence and the corresponding nucleic acid sequence encoding a polypeptide useful for the method of the invention as defined above, becomes a transgenic expression cassette when this expression cassette is modified by non-natural synthetic ("artificial") methods, such as, for example, mutagenic treatment. Suitable methods are described, for example, in US Pat. No. 5,565,350 or WO 00 / 15815.

[0240] Thus, for the purposes of the present invention, transgenic plants are understood to mean that the nucleic acids of the invention are not in their natural locus in the genome of said plant, as described above, and these nucleic acids can be expressed homologously or heterologously. However, as described above, transgenic also means that the nucleic acids according to the invention or used in the methods of the invention are in their natural location in the genome of the plant, but their sequence is modified with respect to the natural sequence and / or the regulatory sequences of the natural sequence are modified. Transgenic is preferably understood to mean that expression of the nucleic acids of the invention, which are in a non-natural locus in the genome, occurs, i.e. that a homologous, preferably heterologous, expression of the nucleic acid occurs. Preferred transgenic plants are described herein. The term "transgenic" also refers to any plant, plant cell, callus, plant tissue or plant part that contains all or part of at least one recombinant polynucleotide. In many cases, all or part of the recombinant polynucleotide is stably integrated into a chromosome or a stable extrachromosomal element, so that it can be passed on to the next generation. For purposes of the present invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged, or modified by genetic engineering. Examples include any cloned polynucleotide, or a polynucleotide that has been combined or spliced ​​with a heterologous sequence. The term "recombinant" does not refer to alterations of a polynucleotide that result from natural events, such as spontaneous mutations, or from non-natural mutagenesis followed by selective breeding.

[0241] An "allele" or "allelic variant" is an alternative form of a given gene at the same chromosomal location. Allelic variants include single nucleotide polymorphisms (SNPs) and small insertion / deletion polymorphisms (INDELs). INDELs are typically less than 100 bp in size. SNPs and INDELs form the largest set of sequence variants in the naturally occurring polymorphic populations of most organisms.

[0242] The term "variety" refers to a group of plants within a species defined by the sharing of a common set of characteristics or traits that are accepted by those of skill in the art as sufficient to distinguish one cultivar or variety from another. There is no implication in either term that all plants of any given cultivar or variety are genetically identical at the all-gene or molecular level, or that a given plant is homozygous at all loci. A cultivar or variety is considered to be "true breeding" for a particular trait if, when a true breeding cultivar or variety is self-pollinated, all of the progeny contain the trait. The term "breeding line" or "line" refers to a group of plants within a cultivar defined by the sharing of a common set of characteristics or traits that are accepted by those of skill in the art as sufficient to distinguish one breeding line or line from another. There is no implication in either term that all plants of any given breeding line or line are genetically identical at the every gene or molecular level, or that a given plant is homozygous at every genetic locus. A breeding line or line is considered to be "true breeding" for a particular trait if, when the true breeding line or breeding line is self-pollinated, all of the progeny contain the trait. In the present invention, the trait results from a mutation in the TriA gene of the plant or seed.

[0243] The herbicide-resistant plant of the present invention comprising a polynucleotide encoding a mutant TriA polypeptide can also be used in a method of increasing herbicide resistance of a plant by conventional plant breeding involving sexual reproduction. The method includes crossing a first plant, which is a herbicide-resistant plant of the present invention, with a second plant, which may or may not be resistant to one or more herbicides the same as the first plant, or may be resistant to one or more herbicides different from the first plant. The second plant can be any plant that can produce a viable progeny plant (i.e., seed) when crossed with the first plant. Typically, but not necessarily, the first and second plants are of the same species. The method can optionally include selecting progeny plants that comprise the mutant TriA polypeptide of the first plant and the herbicide-resistant trait of the second plant. The progeny plants produced by this method of the present invention have increased herbicide resistance when compared to either or both of the first or second plants. When the first and second plants are resistant to different herbicides, the progeny plants have a combination of the herbicide-tolerant properties of the first and second plants. The method of the present invention can further include one or more generations of backcrossing the progeny plants of the first cross with a plant of the same line or genotype as either the first or second plant. Alternatively, the progeny of the first cross or any subsequent cross can be crossed with a third plant of a different line or genotype than the first or second plant.

[0244] The present invention also provides plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells transformed with at least one polynucleotide molecule, expression cassette, or transformation vector of the present invention. Such transformed plants, plant organs, plant tissues, plant cells, seeds, and non-human host cells have increased tolerance or resistance to at least one herbicide at levels of the herbicide that kill or inhibit the growth of non-transformed plants, plant tissues, plant cells, or non-human host cells, respectively. Preferably, the transformed plants, plant tissues, plant cells, and seeds of the present invention are Arabidopsis thaliana and crop plants.

[0245] In another embodiment, the present invention relates to a seed produced by a transgenic plant comprising a plant cell of the present invention, the seed being a pure breeding line for increased resistance to herbicides compared to a wild type variety of the seed.

[0246] In other embodiments, the herbicide-tolerant plants of the present invention can be used as herbicide-tolerant trait donor lines for development, such as by conventional plant breeding, to produce other variety and / or hybrid crops containing such one or more traits. All of the resulting variety or hybrid crops containing such ancestral herbicide-tolerant one or more traits may be referred to herein as progeny or descendants of the ancestral herbicide-tolerant line.

[0247] In another embodiment, the present invention provides a method of producing a plant that is tolerant to a herbicide, the method comprising crossing a plant tolerant to a first herbicide with a second plant to produce a progeny plant tolerant to the herbicide, wherein the first plant and the progeny plant comprise in at least some of their cells a polynucleotide operably linked to a promoter operable in a plant cell, the recombinant polynucleotide being effective to express in the cells of the first plant a mutant TriA polypeptide encoded by the polynucleotide, and wherein expression of the mutant TriA polypeptide confers tolerance to the herbicide to the plant.

[0248] Conventional plant breeding may be used to introduce herbicide-tolerant traits into progeny plants obtained therefrom. In one embodiment, the present invention provides a method for producing herbicide-tolerant progeny plants. The method includes crossing a parent plant with a herbicide-tolerant plant to introduce the herbicide-tolerant trait of the herbicide-tolerant plant into the germplasm of the progeny plant, where the progeny plant has increased tolerance to the herbicide compared to the parent plant. In another embodiment, the method further includes introgressing the herbicide-tolerant trait by conventional plant breeding techniques to obtain progeny plants having the herbicide-tolerant trait.

[0249] In another embodiment, the plants of the present invention, in addition to being tolerant to herbicides that inhibit cellulose biosynthesis, include plants that have been subjected to further genetic modification by breeding, mutagenesis or genetic engineering, for example, plants that have been made tolerant to the application of AHAS inhibitors, auxin-containing herbicides, chlorosis herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or phytoene desaturase (PDS) inhibitors, EPSPS inhibitors such as glyphosate, glutamine synthetase (GS) inhibitors such as glufosinate, lipid biosynthesis inhibitors such as acetyl-CoA carboxylase (ACCase) inhibitors, or certain other classes of herbicides such as oxynil (i.e. bromoxynil or ioxynil) herbicides as a result of conventional methods of breeding or genetic engineering. Thus, the herbicide-tolerant plants of the present invention can be made tolerant to multiple types of herbicides, such as resistance to both glyphosate and glufosinate, or to both glyphosate and another type of herbicide such as HPPD inhibitors, AHAS inhibitors, or ACCase inhibitors, by multiple genetic modifications. These herbicide resistance technologies are described, for example, in Pest Management Science (in order of volume, year, and page): 61, 2005, 246; 61,2005, 258; 61, 2005, 277; 61, 2005, 269; 61, 2005, 286; 64, 2008, 326; 64, 2008, 332; Weed Science 57, 2009, 108; Australian Journal of Agricultural Research 58, 2007, 708; Science 316, 2007, 1185, and references cited therein.For example, herbicide-tolerant plants of the present invention may, in some embodiments, be tolerant to ACCase inhibitors such as the "dims" (e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), the "hops" (e.g., clodinafop, diclofop, fluazifop, haloxyfop, or quizalofop), and the "dens" (e.g., pinoxaden), auxin-containing herbicides such as dicamba, EPSPS inhibitors such as glyphosate, other cellulose biosynthesis inhibitors, and GS inhibitors such as glufosinate.

[0250] In addition to these classes of inhibitors, plants that are tolerant to the herbicides of the present invention may also be tolerant to herbicides with other modes of action, such as chlorophyll / carotenoid pigment inhibitors, cell membrane disruptors, photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and combinations thereof.

[0251] Such resistance traits can be expressed, for example, as mutant or wild-type HPPD proteins, as mutant or wild-type PPO proteins, as mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins, or as mutant native, inbred, or transgenic aryloxyalkanoate dioxygenase (AAD or DHT), haloarylnitrilase (BXN), 2,2-dichloropropionic acid dehalogenase (DEH), glyphosate-N-acetyltransferase (GAT), glyphosate decarboxylase (GDC), glyphosate oxidoreductase (GOX), glutathione-S-transferase (GST), phosphinothricin acetyltransferase (PAT or bar), or CYP450 proteins having herbicide degradation activity. The herbicide-tolerant plants of the present invention can also be stacked with other traits including, but not limited to, insecticidal traits such as Bt Cry and other proteins with insecticidal activity against beetles, lepidopterans, nematodes, or other pests, nutritional or nutraceutical traits such as modified oil content or oil profile traits, high protein or high amino acid concentration traits, and other trait types known in the art.

[0252] Furthermore, in other embodiments, the use of recombinant DNA techniques and / or breeding and / or selection for other such properties may result in the production of one or more insecticidal proteins, particularly those known from the bacteria Bacillus genus, in particular Bacillus thuringiensis, such as [delta]-endotoxins, such as CrylA(b), CrylA(c), CrylF, CryIF(a2), CryllA(b), CrylllA, CrylllB(bl) or Cry9c; plant insecticidal proteins (VIPs), such as VIP1, VIP2, VIP3 or VIP3A; nematode-colonizing bacteria, such as Photorhabdus spp. or Xenorhabdus spp. spp.); toxins produced by animals, such as scorpion venom, arachnid venom, wasp venom, or other insect-specific neurotoxins; toxins produced by fungi, such as actinomycete venoms; Also covered are herbicide-resistant plants which are enabled to synthesize plant lectins, such as pea or barley lectins; agglutinins; proteinase inhibitors, such as trypsin inhibitors, serine protease inhibitors, patatins, cystatins or papain inhibitors; ribosome-inactivating proteins (RIPs), such as ricin, maize-RIPs, abrin, luffin, saporin or bryodin; steroid metabolic enzymes, such as 3-hydroxysteroid oxidase, ecdysteroid-IDP-glycosyl-transferase, cholesterol oxidase, ecdysone inhibitors or HMG-CoA-reductase; ion channel blockers, such as blockers of sodium or calcium channels; juvenile hormone esterase; diuretic hormone receptors (helicokinin receptors); stilbene synthase, bibenzyl synthase, chitinase or glucanase. In the context of the present invention, these insecticidal proteins or toxins are also expressly to be understood as pretoxins, hybrid proteins, truncated or otherwise modified proteins. Hybrid proteins are characterized by novel combinations of protein domains (see, for example, WO02 / 015701).Further examples of such toxins or genetically modified plants capable of synthesizing such toxins are disclosed, for example, in EP 374 753, WO 93 / 007278, WO 95 / 34656, EP 427 529, EP 451 878, WO 03 / 18810 and WO 03 / 52073. Methods for producing such genetically modified plants are widely known to those skilled in the art and are described, for example, in the above-mentioned publications. These insecticidal proteins contained in the genetically modified plants confer resistance to pests of all taxonomic groups of arthropods, especially beetles (Coeloptera), two-winged insects (Diptera), and moths (Lepidoptera) and nematodes (Nematoda).

[0253] In some embodiments, expression of one or more protein toxins (eg, insecticidal proteins) in plants that are tolerant to herbicides is effective in controlling organisms including, for example, those of the following classes and orders: Coleoptera, e.g. Bean weevil (Acanthoscelides obtectus), Leaf beetle (Agelastica alni), Click beetle (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor), Grain beetle (Ahasverus advena), Summer chafer (Amphimallon solstitialis), Furniture beetle (Anobium punctatum), Anthonomus spp. (Weevils), Pygmy mangold beetle (Atomaria linearis). linearis), carpet beetles (Anthrenus spp., Attagenus spp.), cowpea weevil (Callosobruchus maculates), chestnut leaf beetle (Carpophilus hemipterus), cabbage pod weevil (Ceutorhynchus assimilis), rapeseed stalk weevil (Ceutorhynchus picitarsis), wireworms (Conoderus vespertinus and Conoderus falli), banana weevil (Cosmopolites sordidus), sordidus), New Zealand grass beetle (Costelytra zealandica), cockroach beetle (Cotinisnitida), sunflower stem weevil (Cylindrocopturus adspersus), striped grass beetle (Dermestes lardarius), root cut beetle (Diabrotica virgifera, Diabrotica virgifera virgifera, and Diabrotica barberi), ladybird beetle (Epilachna varivestis), house longhorn beetle (Hylotropes bajulus), alfalfa weevil (Hypera postica), grasshopper beetle (Gibbium psiloides), psylloides), cigarette beetle (Lasioderma serricorne), Colorado potato beetle (Leptinotarsa ​​decemlineata), flat-headed bark beetle (Lyctus spp.), pollen beetle (Meligethes aeneus), European cockroach beetle (Melolontha melolontha), American leaf beetle (Mezium americanum), golden leaf beetle (Niptus hololeucus), grain beetle (Oryzaephilus surinamensis), surinamensis and Oryzaephilus Mercator), Chinese vine weevil (Otiorhynchus sulcatus), mustard beetle (Phaedon cochleariae), oilseed leaf beetle (Phyllotreta cruciferae), striped flea beetle (Phyllotreta striolata),striolata), cabbage stem flea beetle (Psylliodes chrysocephala), Ptinus spp. (leopard beetles), grain beetle (Rhizopertha dominica), red-legged weevil (Sitona lineatus), rice weevils (Sitophilus oryzae and Sitophilus granaries), red sunflower seed weevil (Smicronyx fulvus), orange beetle (Stegobium paniceum), paniceum), Brown mealworm (Tenebrio molitor), War beetles (Tribolium castaneum and Tribolium confusum), Spotted cutworms and cabinet beetles (Trogoderma spp.), Sunflower beetle (Zygogramma exclamationis), Dermoptera (Earwigs) such as European earwig (Forficula auricularia) and Asian earwig (Labidura riparia), Dictyoptera such as Oriental cockroach (Blatta orientalis), orientalis), burnt millipede (Oxidus gracilis), beet root fly (Pegomyia betae), fruit fly (Oscinella frit), fruit flies (Dacus spp. and Drosophila spp.),spp.), Isoptera (Termites), e.g. species of the families Hodotermitidae, Kalotermitidae, Mastotermitidae, Rhinotermitidae, Serritermitidae, Termitidae, Termopsidae, the Japanese cricket bug (Lygus lineolaris), the black bean aphid (Aphis fabae), the cotton aphid (Aphis gossypii), the apple aphid (Aphis pomi), Asian citrus spiny whitefly (Aleurocanthus spiniferus), Tobacco whitefly (Bemesia tabaci), Radish aphid (Brevicoryne brassicae), European pear psylla (Cacopsylla pyricola), Currant aphid (Cryptomyzus ribis), Grape phyll aphid (Daktulosphaira vitifoliae), Asian citrus psylla (Diaphorina citri), Potato leafhopper (Empoasca fabae), Bean leafhopper (Empoasca solana), Grape leaf leafhopper (Empoasca vitis), Apple aphid (Eriosoma lanigerum), European scale insect (Eulecanium corni), Peach butterbur aphid (Hyalopterus arundinis), Small brown planthopper (Laodelphax striatellus), Tulip long-horned aphid (Macrosiphum euphorbia)euphorbiae), green peach aphid (Myzus persicae), green rice leafhopper (Nephotettix cinticeps), brown planthopper (Nilaparvata lugens), hop wart aphid (Phorodon humuli), wheat curl aphid (Rhopalosiphum padi), wheat long-horn aphid (Sitobion avenae), Lepidoptera, e.g. Adoxophyes orana (apple tortrix), Archips podana (Fruit Tree Tortricker), Bucculatrix pyrivorella (Pear Leaf Moth), Bucculatrix thurberiella (Cotton Leaf Perforator), Bupalus piniarius (Pine Looper), Carpocapsa pomonella (Codling Moth), Chilo suppressalis (Rice Stem Borer), Choristoneura fumiferana (Eastern Spruce Beetle), Cochylis hospes (Banded Sunflower Moth), Diatraea grandiosella (Southwestern Corn Borer), Eupoecilia ambiguella ambiguella (Grape vine moth), Helicoverpa armigera (Tobacco budworm), Helicoverpa zea (Tobacco budworm), Heliothis virescens (Tobacco budworm), Homeosoma electellum (Sunflower moss), Homona magnanima (Homonamagnanima (Tea Tortrix Moth), Lithocolletis blancardella (Apple Bark Moth), Lymantria dispar (Gypsy Moth), Malacosoma neustria (Tent Caterpillar), Mamestra brassicae (Cuttle Moth), Mamestra configurata (Bertha Armyworm), Operophtera brumata (Winter Geometrid), Ostrinia nubilalis (European Corn Borer), Panolis flammea (Pine Tree Moth), Phyllocnistis citrella (Citrella leafminer), Pieris brassicae (Cabbage white butterfly), Rachiplusia ni (Soybean looper), Spodoptera exigua (Beet armyworm), Spodoptera littoralis (Cotton leafworm), Sylepta derogata (Cotton stem borer), Trichoplusia ni (Nettle looper), Orthoptera such as house cricket (Acheta domesticus), tree locust (Anacridium spp.), migratory grasshopper (Locusta migratoria), migratoria), Two-striped grasshopper (Melanoplus bivittatus), Differential grasshopper (Melanoplus differentialis), Red-legged grasshopper (Melanoplus femurrubrum), Spring grasshopper (Melanoplus sanguinipes), Northern mole cricket (Neocurtillahexadectyla, red grasshopper (Nomadacris septemfasciata), short-winged mole cricket (Scapteriscus abbreviatus), southern mole cricket (Scapteriscus borellii), Tawny mole cricket (Scapteriscus vicinus, and Egyptian ground locust (Schistocerca gregaria), synoptera such as garden symphylans (Scutigerella immaculata), Thysanoptera such as tobacco thrips (Frankliniella fusca), plain-leaf thrips (Frankliniella intonsa), western flower thrips (Frankliniella occidentalis), cottonbud thrips (Frankliniella schultzei), banded greenhouse thrips (Hercinothrips femoralis), soybean thrips (Neohydatothrips variabilis), Kelly's citrus thrips (Pezothrips kellyanus), avocado thrips (Scirtothrips perseae), southern melon thrips (Thrips palmi), and onion thrips (Thrips tabaci). tabaci), and the like, as well as combinations comprising one or more of the foregoing organisms.

[0254] In some embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in herbicide-resistant plants is effective to control flea beetles, i.e., flea beetles of the family Chrysomelidae, preferably members of the genus Phyllotreta pp., such as Phyllotreta cruciferae and / or Phyllotreta triolata. In other embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in herbicide-resistant plants is effective to control cabbage weevils, Bertha armyworm, Lygus bugs, or diamondback moths.

[0255] Also included in one embodiment are herbicide-tolerant plants that have been rendered capable, for example, by use of recombinant DNA technology and / or by breeding and / or selection for other such traits, of synthesizing one or more proteins to increase the plant's resistance or tolerance to bacterial, viral or fungal pathogens. Methods for producing such genetically modified plants are well known to those of skill in the art.

[0256] Also included in other embodiments are herbicide-tolerant plants that have been rendered, for example by the use of recombinant DNA technology and / or breeding and / or selection for other such traits, capable of synthesizing one or more proteins to increase productivity (e.g., oil content), resistance to drought, salinity or other growth-limiting environmental factors, or resistance to plant pests and fungal, bacterial or viral pathogens.

[0257] Also included in other embodiments are herbicide-tolerant plants that have been modified, e.g., by the use of recombinant DNA technology and / or breeding and / or selection for other such traits, to contain altered amounts of one or more substances or new substances, e.g., oil crops that produce health-promoting long chain omega-3 fatty acids or unsaturated omega-9 fatty acids (e.g., Nexera® canola, Dow Agro Sciences, Canada), e.g., to improve human or animal nutrition.

[0258] Also included in some embodiments are herbicide-tolerant plants that have been modified to contain increased amounts of vitamins and / or minerals, and / or an improved profile of nutraceutical compounds, for example, by the use of recombinant DNA technology and / or breeding and / or selection for other such traits.

[0259] In one embodiment, herbicide-tolerant plants of the invention exhibit increased amounts or improved profiles of the following as compared to wild-type plants: glucosinolates (e.g., glucoraphanin (4-methylsulfinylbutyl-glucosinolate), sulforaphane, 3-indolylmethyl-glucosinolate (glycobrassicin), 1-methoxy-3-indolylmethyl-glucosinolate (neoglucobrassicin)), phenolics (e.g., flavonoids (e.g., quercetin, kaempferol), hydroxycinnamoyl derivatives (e.g., 1,2,2′-tricinapoyl ... The composition comprises a compound selected from the group consisting of: 1,2-diferuloylgentiobiose, 1,2'-diferuloylgentiobiose, 1,2'-disinapoyl-2-feruloylgentiobiose, 3-0-caffeoyl-quinic acid (neochlorogenic acid), and vitamins and minerals (e.g., vitamin C, vitamin E, carotene, folic acid, niacin, riboflavin, thiamine, calcium, iron, magnesium, potassium, selenium, and zinc).

[0260] In another embodiment, the herbicide-tolerant plants of the present invention have increased amounts or improved profiles of the following compared to wild-type plants: progoitrin, isothiocyanates, indoles (products of glucosinolate hydrolysis), glutathione, carotenoids such as beta-carotene, lycopene, and xanthophyll carotenoids such as lutein and zeaxanthin, flavonoids such as flavonols (e.g., quercetin, rutin), flavans / tannins (e.g., coumarins, proanthocyanidins, arginines, and the like), and / or soluble amines such as glyceryl stearate, ... The active ingredients include compounds selected from the group consisting of phenols including procyanidins including dicine, catechin, and anthocyanins, flavones, phytoestrogens such as coumestin, lignans, resveratrol, isoflavones such as genistein, daidzein, and glycitein, resorcylic acid lactones, organic sulfur compounds, phytosterols, terpenoids such as carnosol, rosmarinic acid, glycyrrhizin, and saponins, chlorophyll, chlorophyllin, sugars, anthocyanins, and vanilla.

[0261] In another embodiment, the herbicide-tolerant plants of the present invention exhibit increased amounts or improved profiles of the following herbicides as compared to wild-type plants: vincristine, vinblastine, taxanes (e.g., taxol (paclitaxel), baccatin III, 10-desacetylbaccatin III, 10-desacetyltaxol, xylosyltaxol, 7-epitaxol, 7-epibaccatin III, 10-desacetylcephalomannine, 7-epicephalomannine, taxotere, cephalomannine, xylosylcephalomannine, taxaggifin, ifine), 8-benzoyloxytaxine, 9-acetyloxytaxine, 9-hydroxytaxine, taiwanxam, taxane la, taxane lb, taxane Ic, taxane Id, GMP paclitaxel, 9-dihydro-13-acetylbaccatin III, 10-desacetyl-7-epitaxol, tetrahydrocannabinol (THC), cannabidiol (CBD), genistein, daidzein, codeine, morphine, quinine, shikonin, ajmalicine, serpentine, and the like.

[0262] In another aspect, a method of treating a plant of the present invention is provided.

[0263] In some embodiments, the method includes contacting the plant with an agriculturally acceptable composition. In one embodiment, the agriculturally acceptable composition includes an auxinic herbicide AI.

[0264] In another aspect, the present invention provides a method for producing progeny seeds. The method includes planting a seed capable of producing a plant of the present invention. In one embodiment, the method further includes growing a progeny plant from the seed and harvesting a progeny seed from the progeny plant. In another embodiment, the method further includes applying a herbicide composition of a herbicide to the progeny plant.

[0265] In another embodiment, the present invention relates to a harvestable part of a transgenic plant of the present invention. Preferably, the harvestable part comprises a TriA nucleic acid or a TriA protein of the present invention. The harvestable part may be a seed, root, leaf and / or flower or part thereof comprising a TriA nucleic acid or a TriA protein. A preferred part of a soybean plant is a soybean comprising a TriA nucleic acid or a TriA protein.

[0266] In another embodiment, the present invention relates to a product derived from a transgenic plant of the present invention, a part thereof or a harvestable portion thereof. Preferred plant products are feed, seed meal, oil, or seed treatment coated seeds. Preferably, the meal and / or oil comprises a TriA nucleic acid or a TriA protein.

[0267] In another embodiment, the present invention relates to a method for producing an article of manufacture, the method comprising: a) growing a plant of the invention or obtainable by a method of the invention, and b) Producing said product from or by plants of the invention and / or parts of these plants, such as seeds. Includes.

[0268] In a further embodiment, the method comprises the steps: a) growing a plant of the invention; b) harvesting from the plant a harvestable part as defined above; c) Producing said product from or with the harvestable part of the invention. Includes.

[0269] The product is produced at the site where the plant is grown, and the plant and / or parts thereof are removed from the site where the plant is grown to produce the product. Typically, the plant is grown and the desired harvestable parts are removed from the plant, possibly in a repeated cycle, and the product is made from the harvestable parts of the plant. The step of growing the plant may be performed only once each time the method of the invention is performed, while the step of producing the product may be repeated, for example by repeatedly removing harvestable parts of the plant of the invention and further processing these parts into the product if necessary. It is also possible to repeat the step of growing the plant of the invention, to accumulate plants or harvestable parts, and then perform a single production of the product on the accumulated plants or plant parts. Also, the steps of growing the plant and producing the product may overlap in time, be performed approximately simultaneously, or be performed sequentially. Typically, the product is produced after the plant has been grown for a period of time.

[0270] In one embodiment, the product produced by the method of the invention is a plant product, such as, but not limited to, a food product, a feed, a food supplement, a feed supplement, a fiber, a cosmetic and / or a pharmaceutical. A food product refers to a composition used for nutrition and / or to supplement nutrition. In particular, animal feed and animal feed supplements are considered food products.

[0271] In another embodiment, the process of the present invention is used to produce agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.

[0272] A plant product can generally consist of one or more agricultural products.

[0273] Herbicides As noted above, the present invention provides nucleic acids, polypeptides that confer resistance in plants to compounds / herbicides that disrupt or inhibit cell wall (cellulose) biosynthesis.

[0274] Examples of herbicides that can be used according to the invention, i.e. to which the plants of the invention are tolerant / resistant, are compounds known to those skilled in the art as azines. Examples of azines metabolized by the mutant TriA polypeptides of the invention are described in detail in the patent applications listed in Table 2 below.

[0275] TIFF0007619806000001.tif200154TIFF0007619806000002.tif219153TIFF0007619806000003.tif73155

[0276] Examples of preferred herbicides that can be used in accordance with the present invention are the azines (including their agriculturally acceptable salts or N-oxides) having the formula (I): [ka] (In the formula, A is phenyl substituted by 2 to 5 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, C2-C6-alkenyl, C2-C6-haloalkenyl, C2-C6-alkynyl, C1-C6-haloalkynyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, and (C1-C6-alkoxy)carbonyl; R 1 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, (C1-C6-alkyl)sulfonyl or phenylsulfonyl, where phenyl is unsubstituted or substituted by 1 to 5 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl and C1-C6-alkoxy, R 2is H, halogen, CN, C1-C6-alkyl, C1-C6-haloalkyl, C2-C6-alkenyl, C3-C6-alkynyl, C3-C6-cycloalkyl, C3-C6-cycloalkenyl, OH, C1-C6-alkoxy or C1-C6-alkoxy-C1-C6-alkyl, R 3 is H, halogen, CN, C1-C6-alkyl, C1-C6-haloalkyl or C1-C6-alkoxy, R 4 is H, halogen, CN, C1-C6-alkyl or C1-C6-haloalkyl, or R 3 and R 4 together with the carbon atom to which they are attached form a moiety selected from the group consisting of carbonyl, C2-C6-alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkenyl and 3-6-membered heterocyclyl, wherein the C3-C6-cycloalkyl, C3-C6-cycloalkenyl or 3-6-membered heterocyclyl is unsubstituted or substituted by 1-3 substituents selected from halogen, CN, C1-C6-alkyl and C1-C6-alkoxy; R 5 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, (C1-C6-alkyl)sulfonyl or phenylsulfonyl, where the phenyl is unsubstituted or substituted by 1 to 5 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl and C1-C6-alkoxy.

[0277] Preferably, the present invention relates to azines of formula (I) A is 2-fluoro-phenyl substituted by 1 to 4 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl and (C1-C6-alkoxy)carbonyl; R 1 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, (C1-C6-alkyl)sulfonyl or phenylsulfonyl, where the phenyl is unsubstituted or substituted by 1 to 5 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl and C1-C6-alkoxy, R 2 is H, halogen, CN, C1-C6-alkyl, C1-C6-haloalkyl, C2-C6-alkenyl, C3-C6-alkynyl, C3-C6-cycloalkyl, C3-C6-cycloalkenyl, OH, C1-C6-alkoxy or C1-C6-alkoxy-C1-C6-alkyl, R 3 is H, halogen, CN, C1-C6-alkyl, C1-C6-haloalkyl or C1-C6-alkoxy, R 4 is H, halogen, CN, C1-C6-alkyl or C1-C6-haloalkyl, or R 3 and R 4together with the carbon atom to which they are attached form a moiety selected from the group consisting of carbonyl, C2-C6-alkenyl, C3-C6-cycloalkyl, C3-C6-cycloalkenyl and 3-6-membered heterocyclyl, wherein the C3-C6-cycloalkyl, C3-C6-cycloalkenyl or 3-6-membered heterocyclyl is unsubstituted or substituted by 1-3 substituents selected from halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, R 5 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, (C1-C6-alkyl)sulfonyl or phenylsulfonyl, wherein the phenyl is unsubstituted or substituted by 1 to 5 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl and C1-C6-alkoxy (including agriculturally acceptable salts or N-oxides thereof).

[0278] Also useful in the present invention are agricultural compositions comprising at least one azine of formula (I) and auxiliaries customary for formulating crop protection agents.

[0279] The invention also provides the use of the azines of formula (I) as herbicides, i.e. for controlling harmful plants.

[0280] Where the azines of formula (I) described herein can form geometric isomers, such as E / Z isomers, it is possible to use both the pure isomers and mixtures thereof in the compositions of the present invention.

[0281] Where the azines of formula (I) described herein possess one or more chiral centers and, as a result, exist as enantiomers or diastereomers, it is possible for both the pure enantiomers and diastereomers and mixtures thereof to be used in the compositions of the present invention.

[0282] When the azines of formula (I) described herein have an ionizable functional group, they can also be used in the form of their agriculturally acceptable salts. In general, salts of cations and acid addition salts of acids whose cations and anions, respectively, do not have a deleterious effect on the activity of the active compound are suitable.

[0283] Preferred cations are ions of alkali metals, preferably lithium, sodium and potassium, alkaline earth metals, preferably calcium and magnesium, and transition metals, preferably manganese, copper, zinc and iron, as well as ammonium and substituted ammonium in which one to four hydrogen atoms are replaced by C1-C4-alkyl, hydroxy-C1-C4-alkyl, C1-C4-alkoxy-C1-C4-alkyl, hydroxy-C1-C4-alkoxy-C1-C4-alkyl, phenyl or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium, tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2-methyl ... ammonium (diolamine salt), tris(2-hydroxyethyl)ammonium (trolamine salt), tris(2-hydroxypropyl)ammonium, benzyltrimethylammonium, benzyltriethylammonium, N,N,N-trimethylethanolammonium (choline salt), also phosphonium ions, sulfonium ions, preferably tri(C1-C4-alkyl)sulfonium, for example trimethylsulfonium, and sulfoxonium ions, preferably tri(C1-C4-alkyl)sulfoxonium, and finally the salts of polybasic amines, for example N,N-bis-(3-aminopropyl)methylamine and diethylenetriamine.

[0284] Useful anions of acid addition salts are primarily chloride, bromide, fluoride, iodide, hydrogensulfate, methylsulfate, sulfate, dihydrogenphosphate, hydrogenphosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, and also anions of C1-C4-alkanoic acids, preferably formate, acetate, propionate and butyrate.

[0285] Variable groups, e.g. R 1 ~R 5 The organic moieties mentioned in the definition of are generic to the members of the individual groups listed, as is the term halogen. The term halogen in each case means fluorine, chlorine, bromine or iodine. All hydrocarbon chains, i.e. alkyl, haloalkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, (alkyl)amino, di(alkyl)amino chains, can be linear or branched and are denoted by the prefix C. n ~C m means in each case the possible number of carbon atoms in the group.

[0286] Examples of such meanings are:

[0287] C1-C4-Alkyl: for example CH3, C2H5, n-propyl, CH(CH3)2, n-butyl, CH(CH3)-C2H5, CH2-CH(CH3)2 and C(CH3)3;

[0288] C1-C6-alkyl and (C1-C6-alkyl)carbonyl, C1-C6-alkyl moiety of C1-C6-alkyloxy-C1-C6-alkyl: the above-mentioned C1-C4-alkyl, and also, for example, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, ethyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methylpropyl or 1-ethyl-2-methylpropyl, preferably methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1,1-dimethylethyl, n-pentyl or n-hexyl,

[0289] C1-C4-Haloalkyl: the abovementioned C1-C4-alkyl groups which are partially or completely substituted by fluorine, chlorine, bromine and / or iodine, such as, for example, chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, chlorofluoromethyl, dichlorofluoromethyl, chlorodifluoromethyl, bromomethyl, iodomethyl, 2-fluoroethyl, 2-chloroethyl, 2-bromoethyl, 2-iodoethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-chloro-2-fluoroethyl, 2-chloro-2,2-difluoroethyl, 2,2-dichloro-2-fluoroethyl, 2,2,2-trichloroethyl, pentafluoroethyl, 2-fluoroethyl,

[0036] Oropropyl, 3-fluoropropyl, 2,2-difluoropropyl, 2,3-difluoropropyl, 2-chloropropyl, 3-chloropropyl, 2,3-dichloropropyl, 2-bromopropyl, 3-bromopropyl, 3,3,3-trifluoropropyl, 3,3,3-trichloropropyl, 2,2,3,3,3-pentafluoropropyl, heptafluoropropyl, 1-(fluoromethyl)-2-fluoroethyl, 1-(chloromethyl)-2-chloroethyl, 1-(bromomethyl)-2-bromoethyl, 4-fluorobutyl, 4-chlorobutyl, 4-bromobutyl, nonafluorobutyl, 1,1,2,2-tetrafluoroethyl and 1-trifluoromethyl-1,2,2,2-tetrafluoroethyl.

[0290] C1-C6-Haloalkyl: the abovementioned C1-C4-haloalkyl, and also, for example, 5-fluoropentyl, 5-chloropentyl, 5-bromopentyl, 5-iodopentyl, undecafluoropentyl, 6-fluorohexyl, 6-chlorohexyl, 6-bromohexyl, 6-iodohexyl and dodecafluorohexyl,

[0291] C3-C6-Cycloalkyl: monocyclic saturated hydrocarbons having 3 to 6 ring members, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl;

[0292] C2-C6-alkenyl: for example, ethenyl, 1-propenyl, 2-propenyl, 1-methylethenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-methyl-1-propenyl, 2-methyl-1-propenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-1-butenyl, 2-methyl-1-butenyl, 3-methyl-1-butenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl ethyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-1-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-1-propenyl, 1-ethyl-2-propenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-1-pentenyl, 2-methyl-1-pentenyl, 3-methyl-1-pentenyl, 4-methyl-1-pentenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethy methyl-3-butenyl, 2,3-dimethyl-1-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-1-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-1-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1-ethyl-2-methyl-1-propenyl and 1-ethyl-2-methyl-2-propenyl,

[0293] - C3-C6-cycloalkenyl: 1-cyclopropenyl, 2-cyclopropenyl, 1-cyclobutenyl, 2-cyclobutenyl, 1-cyclopentenyl, 2-cyclopentenyl, 1,3-cyclopentadienyl, 1,4-cyclopentadienyl, 2,4-cyclopentadienyl, 1-cyclohexenyl, 2-cyclohexenyl, 3-cyclohexenyl, 1,3-cyclohexadienyl, 1,4-cyclohexadienyl, 2,5-cyclohexadienyl,

[0294] C3-C6-alkynyl: for example, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 3-methyl-1-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-3- pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-1-pentynyl, 3-methyl-4-pentynyl, 4-methyl-1-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 3,3-dimethyl-1-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl,

[0295] C1-C4-alkoxy: for example methoxy, ethoxy, propoxy, 1-methylethoxybutoxy, 1-methylpropoxy, 2-methylpropoxy and 1,1-dimethylethoxy;

[0296] C1-C6-alkoxy and also (C1-C6-alkoxy)carbonyl, the C1-C6-alkoxy moiety of C1-C6-alkoxy-C1-C6-alkyl: the above-mentioned C1-C4-alkoxy, and also, for example, pentoxy, 1-methylbutoxy, 2-methylbutoxy, 3-methoxybutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, hexoxy, 1-methylpentoxy, , 2-methylpentoxy, 3-methylpentoxy, 4-methylpentoxy, 1,1-dimethylbutoxy, 1,2-dimethylbutoxy, 1,3-dimethylbutoxy, 2,2-dimethylbutoxy, 2,3-dimethylbutoxy, 3,3-dimethylbutoxy, 1-ethylbutoxy, 2-ethylbutoxy, 1,1,2-trimethylpropoxy, 1,2,2-trimethylpropoxy, 1-ethyl-1-methylpropoxy and 1-ethyl-2-methylpropoxy,

[0297] C1-C4-Alkylthio: for example methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio and 1,1-dimethylethylthio;

[0298] C1-C6-Alkylthio: the abovementioned C1-C4-alkylthio, and also, for example, pentylthio, 1-methylbutylthio, 2-methylbutylthio, 3-methylbutylthio, 2,2-dimethylpropylthio, 1-ethylpropylthio, hexylthio, 1,1-dimethylpropylthio, 1,2-dimethylpropylthio, 1-methylpentylthio, 2-methylpentylthio, 3-methylpentylthio, 4-methylpentylthio, 1,1-dimethylbutylthio, 1,2-dimethylbutylthio, 1,3-dimethylbutylthio, 2,2-dimethylbutylthio, 2,3-dimethylbutylthio, 3,3-dimethylbutylthio, 1-ethylbutylthio, 2-ethylbutylthio, 1,1,2-trimethylpropylthio, 1,2,2-trimethylpropylthio, 1-ethyl-1-methylpropylthio and 1-ethyl-2-methylpropylthio,

[0299] C1-C6-Alkylsulfinyl (C1-C6-alkyl-S(=O)-): for example, methylsulfinyl, ethylsulfinyl, propylsulfinyl, 1-methylethylsulfinyl, butylsulfinyl, 1-methylpropylsulfinyl, 2-methylpropylsulfinyl, 1,1-dimethylethylsulfinyl, pentylsulfinyl, 1-methylbutylsulfinyl, 2-methylbutylsulfinyl, 3-methylbutylsulfinyl, 2,2-dimethylpropylsulfinyl, 1-ethylpropylsulfinyl, 1,1-dimethylpropylsulfinyl, 1,2-dimethylpropylsulfinyl, hexylsulfinyl, 1-methylethylsulfinyl, 2-methylpropylsulfinyl, 2-methylpropylsulfinyl, 1,1-dimethylpropylsulfinyl, 1,2-dimethylpropylsulfinyl, hexylsulfinyl, 1-methylethylsulfinyl, 2-methylpropylsulfinyl, 2-methylpropylsulfinyl, 1,1-dimethylpropylsulfinyl, 1,2-dimethylpropylsulfinyl, 2-methylpropylsulfinyl, 1-methylpropylsulfinyl, 2 ... pentylsulfinyl, 2-methylpentylsulfinyl, 3-methylpentylsulfinyl, 4-methylpentylsulfinyl, 1,1-dimethylbutylsulfinyl, 1,2-dimethylbutylsulfinyl, 1,3-dimethylbutylsulfinyl, 2,2-dimethylbutylsulfinyl, 2,3-dimethylbutylsulfinyl, 3,3-dimethylbutylsulfinyl, 1-ethylbutylsulfinyl, 2-ethylbutylsulfinyl, 1,1,2-trimethylpropylsulfinyl, 1,2,2-trimethylpropylsulfinyl, 1-ethyl-1-methylpropylsulfinyl and 1-ethyl-2-methylpropylsulfinyl.

[0300] C1-C6-Alkylsulfonyl (C1-C6-alkyl-S(O)2-): for example, methylsulfonyl, ethylsulfonyl, propylsulfonyl, 1-methylethylsulfonyl, butylsulfonyl, 1-methylpropylsulfonyl, 2-methylpropylsulfonyl, 1,1-dimethylethylsulfonyl, pentylsulfonyl, 1-methylbutylsulfonyl, 2-methylbutylsulfonyl, 3-methylbutylsulfonyl, 1,1-dimethylpropylsulfonyl, 1,2-dimethylpropylsulfonyl, 2,2-dimethylpropylsulfonyl, 1-ethylpropylsulfonyl, hexylsulfonyl, 1-methylpropylsulfonyl, pentylsulfonyl, 2-methylpentylsulfonyl, 3-methylpentylsulfonyl, 4-methylpentylsulfonyl, 1,1-dimethylbutylsulfonyl, 1,2-dimethylbutylsulfonyl, 1,3-dimethylbutylsulfonyl, 2,2-dimethylbutylsulfonyl, 2,3-dimethylbutylsulfonyl, 3,3-dimethylbutylsulfonyl, 1-ethylbutylsulfonyl, 2-ethylbutylsulfonyl, 1,1,2-trimethylpropylsulfonyl, 1,2,2-trimethylpropylsulfonyl, 1-ethyl-1-methylpropylsulfonyl and 1-ethyl-2-methylpropylsulfonyl,

[0301] (C1-C4-alkyl)amino: for example methylamino, ethylamino, propylamino, 1-methylethylamino, butylamino, 1-methylpropylamino, 2-methylpropylamino or 1,1-dimethylethylamino;

[0302] (C1-C6-alkyl)amino: the abovementioned (C1-C4-alkylamino), and also, for example, pentylamino, 1-methylbutylamino, 2-methylbutylamino, 3-methylbutylamino, 2,2-dimethylpropylamino, 1-ethylpropylamino, hexylamino, 1,1-dimethylpropylamino, 1,2-dimethylpropylamino, 1-methylpentylamino, 2-methylpentylamino, 3-methylpentylamino, 4-methylpentylamino, 1,1-dimethylbutylamino, 1,2-dimethylbutylamino, 1,3-dimethylbutylamino, 2,2-dimethylbutylamino, 2,3-dimethylbutyl-amino 3,3-dimethylbutylamino, 1-ethylbutylamino, 2-ethylbutylamino, 1,1,2-trimethylpropylamino, 1,2,2-trimethyl-propylamino, 1-ethyl-1-methylpropylamino or 1-ethyl-2-methylpropylamino,

[0303] Di(C1-C4-alkyl)amino: for example, N,N-dimethylamino, N,N-diethylamino, N,N-di(1-methylethyl)amino, N,N-dipropylamino, N,N-dibutylamino, N,N-di(1-methylpropyl)amino, N,N-di(2-methylpropyl)amino, N,N-di(1,1-dimethylethyl)amino, N-ethyl-N-methylamino, N-methyl-N-propylamino, N-methyl-N-(1-methylethyl)amino , N-butyl-N-methylamino, N-methyl-N-(1-methylpropyl)amino, N-methyl-N-(2-methylpropyl)amino, N-(1,1-dimethylethyl)-N-methylamino, N-ethyl-N-propylamino, N-ethyl-N-(1-methylethyl)amino, N-butyl-N-ethylamino, N-ethyl-N-(1-methylpropyl)amino, N-ethyl-N-(2-methylpropyl)amino, N-ethyl-N-(1,1-dimethylethyl)amino n-(1-methylethyl)amino, N-(1-methylethyl)-N-propylamino, N-butyl-N-propylamino, N-(1-methylpropyl)-N-propylamino, N-(2-methylpropyl)-N-propylamino, N-(1,1-dimethylethyl)-N-propylamino, N-butyl-N-(1-methylethyl)amino, N-(1-methylethyl)-N-(1-methylpropyl)amino, N-(1-methylethyl)-N-(2-methylpropyl)amino N-(1,1-dimethylethyl)-N-(1-methylethyl)amino, N-butyl-N-(1-methylpropyl)amino, N-butyl-N-(2-methylpropyl)amino, N-butyl-N-(1,1-dimethylethyl)amino, N-(1-methylpropyl)-N-(2-methylpropyl)amino, N-(1,1-dimethylethyl)-N-(1-methylpropyl)amino or N-(1,1-dimethylethyl)-N-(2-methylpropyl)amino,

[0304] Di(C1-C6-alkyl)amino: the abovementioned di(C1-C4-alkyl)amino, and also, for example, N-methyl-N-pentylamino, N-methyl-N-(1-methylbutyl)amino, N-methyl-N-(2-methylbutyl)amino, N-methyl-N-(3-methylbutyl)amino, N-methyl-N-(2,2-dimethylpropyl)amino, N-methyl-N-(1-ethylpropyl)amino, N-methyl-N-hexylamino, N-methyl-N-(1,1-dimethylpropyl)amino, N-methyl-N-(1,2-dimethylpropyl)amino, N-methyl N-methyl-N-(1-methylpentyl)amino, N-methyl-N-(2-methylpentyl)amino, N-methyl-N-(3-methylpentyl)amino, N-methyl-N-(4-methylpentyl)amino, N-methyl-N-(1,1-dimethylbutyl)amino, N-methyl-N-(1,2-dimethylbutyl)amino, N-methyl-N-(1,3-dimethylbutyl)amino, N-methyl-N-(2,2-dimethylbutyl)amino, N-methyl-N-(2,3-dimethylbutyl)amino, N-methyl-N-(3,3-dimethylbutyl)amino, N-methyl-N-(1-ethyl-N-ethyl)amino, N-methyl-N-(2-ethyl-N-ethyl)amino, N-methyl-N-(2-ethyl-N-ethyl)amino, N-methyl-N-(3-ethyl-N-ethyl)amino, N-methyl-N-(1 ...1-ethyl-N-ethyl)amino, N-methyl-N-(1-ethyl-N-ethyl)amino, N-methyl-N-(1-ethyl-N-ethyl)amino, N-methyl-N-(1-ethyl-N-ethyl)amino, N-methyl-N-(1-ethyl-N-ethyl) N-ethyl-N-(2-ethylbutyl)amino, N-methyl-N-(1,1,2-trimethylpropyl)amino, N-methyl-N-(1,2,2-trimethylpropyl)amino, N-methyl-N-(1-ethyl-1-methylpropyl)amino, N-methyl-N-(1-ethyl-2-methylpropyl)amino, N-ethyl-N-pentylamino, N-ethyl-N-(1-methylbutyl)amino, N-ethyl-N-(2-methylbutyl)amino, N-ethyl-N-(3-methylbutyl)amino, N-ethyl-N-(2,2-dimethylpropyl)amino n-ethyl-N-(1-ethylpropyl)amino, N-ethyl-N-hexylamino, N-ethyl-N-(1,1-dimethylpropyl)amino, N-ethyl-N-(1,2-dimethylpropyl)amino, N-ethyl-N-(1-methylpentyl)amino, N-ethyl-N-(2-methylpentyl)amino, N-ethyl-N-(3-methylpentyl)amino, N-ethyl-N-(4-methylpentyl)amino, N-ethyl-N-(1,1-dimethylbutyl)amino, N-ethyl-N-(1,2-dimethylbutyl)amino, N-ethyl-N-(1,3-dimethylbutyl)amino, N-ethyl-N-(2,2-dimethylbutyl)amino, N-ethyl-N-(2,3-dimethylbutyl)amino, N-ethyl-N-(3,3-dimethylbutyl)amino, N-ethyl-N-(1-ethylbutyl)amino, N-ethyl-N-(2-ethylbutyl)amino, N-ethyl-N-(1,1,2-trimethylpropyl)amino, N-ethyl-N-(1,2,2-trimethylpropyl)amino, N-ethyl-N-(1-ethyl-1-methylpropyl)amino, N-ethyl-N-(1-ethyl-2-methylpropyl)amino, N-propyl-N-pentylamino, N-butyl-N-pentylamino, N,N-dipentylamino, N-propyl-N-hexylamino, N-butyl-N-hexylamino, N-pentyl-N-hexylamino or N,N-dihexylamino,

[0305] 3-6 membered heterocyclyl: a monocyclic saturated or partially unsaturated hydrocarbon having 3-6 ring members as described above, containing, in addition to carbon atoms, 1 or 2 heteroatoms selected from O, S and N; For example, 2-oxiranyl, 2-oxetanyl, 3-oxetanyl, 2-aziridinyl, 3-thietanyl, 1-azetidinyl, 2-azetidinyl,

[0306] For example, 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothienyl, 3-tetrahydrothienyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 3-isoxazolidinyl, 4-isoxazolidinyl, 5-isoxazolidinyl, 3-isothiazolidinyl, 4-isothiazolidinyl, 5-isothiazolidinyl, 3-pyrazolidinyl, 4-pyrazolidinyl, 5-pyrazolidinyl, 2-oxazolidinyl, 4-oxazolidinyl, 5-oxazolidinyl, 2-thiazolidinyl, 4-thiazolidinyl, 5-thiazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl, For example, 2,3-dihydrofur-2-yl, 2,3-dihydrofur-3-yl, 2,4-dihydrofur-2-yl, 2,4-dihydrofur-3-yl, 2,3-dihydrothien-2-yl, 2,3-dihydrothien-3-yl, 2,4-dihydrothien-2-yl, 2,4-dihydrothien-3-yl, 4,5-dihydropyrrol-2-yl, 4,5-dihydropyrrol-3-yl, 2,5-dihydropyrrol 2,5-dihydropyrrol-2-yl, 2,5-dihydropyrrol-3-yl, 4,5-dihydroisoxazol-3-yl, 2,5-dihydroisoxazol-3-yl, 2,3-dihydroisoxazol-3-yl, 4,5-dihydroisoxazol-4-yl, 2,5-dihydroisoxazol-4-yl, 2,3-dihydroisoxazol-4-yl, 4,5-dihydroisoxazol-5-yl, 2,5 -dihydroisoxazol-5-yl, 2,3-dihydroisoxazol-5-yl, 4,5-dihydroisothiazol-3-yl, 2,5-dihydroisothiazol-3-yl, 2,3-dihydroisothiazol-3-yl, 4,5-dihydroisothiazol-4-yl, 2,5-dihydroisothiazol-4-yl, 2,3-dihydroisothiazol-4-yl, 4,5-dihydroisothiazol-5-yl, 2,5-dihydroisothiazol-5-yl, 2,3-dihydroisothiazol-5-yl, 2,3-dihydropyrazol-2-yl, 2,3-dihydropyrazol-3-yl, 2,3-dihydropyrazol-4-yl, 2,3-dihydropyrazol-5-yl, 3,4-dihydropyrazol-3-yl, 3,4-dihydropyrazol-4-yl, 3,4-dihydropyrazol-5-yl, 4,5-Dihydropyrazol-3-yl, 4,5-dihydropyrazol-4-yl, 4,5-dihydropyrazol-5-yl, 2,3-dihydroimidazol-2-yl, 2,3-dihydroimidazol-3-yl, 2,3-dihydroimidazol-4-yl, 2,3-dihydroimidazol-5-yl, 4,5-dihydroimidazol-2-yl, 4,5-dihydroimidazol-4-yl, 4,5-dihydroimidazol-5-yl, 2,5-dihydroimidazol-2-yl, 2,5-dihydroimidazol-4-yl, 2,5-dihydroimidazol-5-yl, 2,3-dihydrooxazol-3-yl 2,3-dihydrooxazol-4-yl, 2,3-dihydrooxazol-5-yl, 3,4-dihydrooxazol-3-yl, 3,4-dihydrooxazol-4-yl, 3,4-dihydrooxazol-5-yl, 2,3-dihydrothiazol-3-yl, 2,3-dihydrothiazol-4-yl, 2,3-dihydrothiazol-5-yl, 3,4-dihydrothiazol-3-yl, 3,4-dihydrothiazol-4-yl, 3,4-dihydrothiazol-5-yl, 3,4-dihydrothiazol-2-yl, 3,4-dihydrothiazol-3-yl, 3,4-dihydrothiazol-4-yl,

[0307] For example, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 1,3-dioxan-2-yl, 1,3-dioxan-4-yl, 1,3-dioxan-5-yl, 1,4-dioxan-2-yl, 1,3-dithian-2-yl, 1,3-dithian-4-yl, 1,4-dithian-2-yl, 1,3-dithian-5-yl, 2-tetrahydropyranyl, 3-tetrahydropyranyl, 4-tetrahydropyranyl, 2-tetrahydropyranyl, tetrahydrothiopyranyl, 3-tetrahydrothiopyranyl, 4-tetrahydro-thiopyranyl, 3-hexahydropyridazinyl, 4-hexahydropyridazinyl, 2-hexahydropyrimidinyl, 4-hexahydropyrimidinyl, 5-hexahydropyrimidinyl, 2-piperazinyl, tetrahydro-1,3-oxazin-2-yl, tetrahydro-1,3-oxazin-6-yl, 2-morpholinyl, 3-morpholinyl,

[0308] For example, 2H-pyran-2-yl, 2H-pyran-3-yl, 2H-pyran-4-yl, 2H-pyran-5-yl, 2H-pyran-6-yl, 3,6-dihydro-2H-pyran-2-yl, 3,6-dihydro-2H-pyran-3-yl, 3,6-dihydro-2H-pyran-4-yl, 3,6-dihydro-2H-pyran-5-yl, 3,6-dihydro-2H-pyran-6-yl, -yl, 3,4-dihydro-2H-pyran-3-yl, 3,4-dihydro-2H-pyran-4-yl, 3,4-dihydro-2H-pyran-6-yl, 2H-thiopyran-2-yl, 2H-thiopyran-3-yl, 2H-thiopyran-4-yl, 2H-thiopyran-5-yl, 2H-thiopyran-6-yl, 5,6-dihydro-4H-1,3-oxazin-2-yl.

[0309] It is to be understood that the preferred embodiments of the invention described hereinafter may be preferred independently of one another or in combination with one another.

[0310] According to a preferred embodiment of the invention, azines of formula (I) in which the variables have, independently of one another or in combination with one another, the following meanings are preferred:

[0311] Preferred azines of formula (I) are those in which A is, phenyl substituted by 2 to 5 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, and (C1-C6-alkoxy)carbonyl; Particularly preferably, Selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy. Particularly preferably selected from halogen and CN, Particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, Particularly preferably, it is selected from the group consisting of F, Cl and CN. phenyl substituted with 2 to 5 substituents; Particularly preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. phenyl substituted with 2 to 4 substituents; More preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. is a phenyl substituted by two substituents; And more preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. is a phenyl substituted with three substituents And more preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. It is a phenyl substituted with four substituents.

[0312] Likewise preferred azines of formula (I) are those in which A, [ka] and During the ceremony, R a and R e are independently of each other halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, R b , R c and R d are, independently of one another, hydrogen, halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, Particularly preferably, R a and R e are, independently of one another, halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, R b , R c and R d are independently of one another hydrogen, halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl or C1-C6-alkoxy, Particularly preferably, R a and R e are each independently halogen or CN, R b , R c and R d are, independently of one another, hydrogen, halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, More preferably, R a and R e is a halogen, R b , R c and Rd are each independently hydrogen, halogen or CN, Most preferably R a and R e is a halogen, R b , R c and R d is hydrogen, Also, most preferably R a , R b , R d and R e is a halogen, R c is hydrogen, Also, most preferably R a , R b , R c , R d and R e is a halogen.

[0313] Likewise preferred azines of formula (I) are those in which A, [ka] and During the ceremony, R a is halogen or CN, R b and R d is H, halogen or CN, R c is H or a halogen, R e is halogen, CN or C1-C6-alkyl, Particularly preferably, R a is a halogen, R b , R c and R d is H or a halogen, R e is halogen or CN, Particularly preferably, R a , R b , R d and R e is a halogen, Rc is H or a halogen, More preferably, R a , R b , R d and R e is F, R c is H or F.

[0314] Particularly preferred azines of formula (I) are those in which A is (A.1.1), (A.1.2) and (A.1.3): [ka] (In the formula, R a and R e are independently of each other halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, R b and R d are independently of each other halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, Particularly preferably, R a and R e are, independently of one another, halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, R b and R d are independently of one another halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl or C1-C6-alkoxy, Particularly preferably, R aand R e are each independently halogen or CN, R b and R d are, independently of one another, halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, More preferably, R a and R e is a halogen, R b and R d are each independently halogen or CN, Most preferably R a , R b , R d and R e is a halogen) and more preferably selected from the group consisting of (A.1.2) and (A.1.3).

[0315] Likewise particularly preferred are azines of the formula (I), in which A, [ka] where R a , R b , R d and R e has the meanings, particularly preferred meanings, as defined above.

[0316] Likewise particularly preferred are azines of the formula (I), in which A, [ka] where R a , R b and R e has the meanings, particularly preferred meanings, as defined above.

[0317] Likewise particularly preferred are azines of the formula (I), in which A, [ka] where R a and R e has the meanings, particularly preferred meanings, as defined above.

[0318] Also preferred azines of formula (I) are those in which A, 2-fluoro-phenyl substituted by 1 to 4 substituents selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl and (C1-C6-alkoxy)carbonyl; Particularly preferably, selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy; Particularly preferably selected from halogen and CN, Particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, Particularly preferably, it is selected from the group consisting of F, Cl and CN. 2-fluoro-phenyl substituted by 1 to 4 substituents; Particularly preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl and (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. 2-fluoro-phenyl substituted by 1 to 3 substituents; More preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl and (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. 2-fluoro-phenyl substituted by one substituent; And more preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl and (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. 2-fluoro-phenyl substituted by two substituents; And more preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl and (C1-C6-alkoxy)carbonyl; Particularly preferably selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably selected from halogen and CN, Also particularly preferably selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. It is 2-fluoro-phenyl substituted by three substituents.

[0319] Likewise preferred azines of formula (I) are those in which A, [ka] and During the ceremony, R a is halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, R b , R c and R dare, independently of one another, hydrogen, halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, Particularly preferably, R a is halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, R b , R c and R d are independently of one another hydrogen, halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl or C1-C6-alkoxy, Particularly preferably, R a is halogen or CN, R b , R c and R d are, independently of one another, hydrogen, halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, More preferably, R a is a halogen, R b , R c and R d are each independently hydrogen, halogen or CN, Most preferably R a is a halogen, R b , R c and R d is hydrogen, Also, most preferably R a , R b and R d is a halogen, R c is hydrogen, Also, most preferably R a , R b , R c and R d is a halogen.

[0320] Likewise preferred azines of formula (I) are those in which A, [ka] and In the formula, R a is halogen, CN or C1-C6-alkyl, R b and R d is H, halogen or CN, R c is H or a halogen, Particularly preferably, R a is halogen or CN, R b , R c and R d is H or a halogen, Particularly preferably, R a , R b and R d is a halogen, R c is H or a halogen, Also particularly preferably R a , R b and R d is a halogen, R c is H, F, Br or I, More preferably, R a , R b and R d is F, R cは F, Br or I; More preferably, R a , R b and R d is F, R c is H or F.

[0321] Particularly preferred azines of formula (I) are those in which A is (A.1a.1), (A.1a.2) and (A.1a.3): [ka] (In the formula, R a is halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, R b and R d are independently of each other halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl, Particularly preferably, R a is halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, R b and R d are independently of one another halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl or C1-C6-alkoxy, Particularly preferably, R a is halogen or CN, R b and R d are, independently of one another, halogen, CN, C1-C6-alkyl or C1-C6-alkoxy, More preferably, R a is a halogen, R b and R d are each independently halogen or CN, Most preferably R a , R b and R d is a halogen.) and more preferably selected from the group consisting of (A.1.2) and (A.1.3).

[0322] Likewise particularly preferred are azines of the formula (I), in which A, [ka] where R a , R b and R d has the meanings, particularly preferred meanings, as defined above.

[0323] Likewise particularly preferred are azines of the formula (I), in which A, [ka] where R a and R b has the meanings, particularly preferred meanings, as defined above.

[0324] Likewise particularly preferred azines of the formula (I) are those in which A, [ka] where R a has the meanings, particularly preferred meanings, as defined above.

[0325] Likewise preferred azines of formula (I) are those in which R 1 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, C1-C6-alkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3, Hydrogen is more preferred.

[0326] Likewise preferred azines of formula (I) are those in which R 2 is H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, Particularly preferred is halogen, C1-C6-alkyl or C1-C6-haloalkyl. Also particularly preferred is H, F, Cl, CH3 or CF3.

[0327] Likewise preferred azines of formula (I) are those in which R 3 and R 4 teeth, are independently H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, or together with the carbon atom to which they are attached form a moiety selected from the group consisting of C3-C6-cycloalkyl, C3-C6-cycloalkenyl and 3- to 6-membered heterocyclyl, wherein C3-C6-cycloalkyl, C3-C6-cycloalkenyl or 3- to 6-membered heterocyclyl is unsubstituted or substituted by 1 to 3 substituents selected from halogen, CN, C1-C6-alkyl and C1-C6-alkoxy; Independently of one another, particularly preferably H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, or together with the carbon atom to which they are attached form a moiety selected from the group consisting of C3-C6-cycloalkyl and C3-C6-cycloalkenyl, wherein C3-C6-cycloalkyl or C3-C6-cycloalkenyl is unsubstituted or substituted by 1-3 substituents selected from halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Independently of each other, particularly preferably H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, More preferably, each independently is H, halogen or C1-C6-alkyl.

[0328] Likewise preferred azines of formula (I) are those in which R 2 is H, halogen, C1-C6-alkyl, R 3 and R 4 are independently of one another H, halogen, C1-C6-alkyl or, together with the carbon atom to which they are attached, form a C3-C6-cycloalkyl, Particularly preferably, R 2 is H, halogen or C1-C6-alkyl, R 3 is C1-C6-alkyl, R 4 is H, halogen or C1-C6-alkyl, R 3 and R 4 together with the carbon atom to which they are attached form a C3-C6-cycloalkyl; Particularly preferably, R 2 is halogen or C1-C6-alkyl, R 3 is C1-C6-alkyl, R 4 is H or C1-C6-alkyl, More preferably, R 2 is a halogen, R 3 and R 4 is C1-C6-alkyl.

[0329] Likewise preferred azines of formula (I) are those in which R 5 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, C1-C6-alkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3, Hydrogen is more preferred.

[0330] Likewise preferred azines of formula (I) are those in which A is selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, particularly preferably selected from halogen and CN, Also particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, Particularly preferably, it is selected from the group consisting of F, Cl and CN. phenyl substituted with 2 to 5 substituents; Particularly preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably, it is selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably, it is selected from halogen and CN, Also particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. phenyl substituted with 2 to 4 substituents; Particularly preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably, it is selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably, it is selected from halogen and CN, Also particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. is a phenyl substituted by two substituents; Also particularly preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably, it is selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably, it is selected from halogen and CN, Also particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. is a phenyl substituted by three substituents; Also particularly preferably, selected from the group consisting of halogen, CN, NO2, C1-C6-alkyl, C1-C6-haloalkyl, OH, C1-C6-alkoxy, C1-C6-alkylthio, (C1-C6-alkyl)sulfinyl, (C1-C6-alkyl)sulfonyl, amino, (C1-C6-alkyl)amino, di(C1-C6-alkyl)amino, (C1-C6-alkyl)carbonyl, (C1-C6-alkoxy)carbonyl; Particularly preferably, it is selected from the group consisting of halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Particularly preferably, it is selected from halogen and CN, Also particularly preferably, it is selected from the group consisting of F, Cl, CN and CH3, More preferably, it is selected from the group consisting of F, Cl and CN. is substituted with four substituents and is phenyl; R 1 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, C1-C6-alkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3, More preferably, it is hydrogen. R 2 is H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, Particularly preferred is halogen, C1-C6-alkyl or C1-C6-haloalkyl. Particularly preferred is H, F, CH3 or CF3, R 3 and R 4 are, independently of one another, H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, or together with the carbon atom to which they are attached form a moiety selected from the group consisting of C3-C6-cycloalkyl, C3-C6-cycloalkenyl and 3-6-membered heterocyclyl, where C3-C6-cycloalkyl, C3-C6-cycloalkenyl or 3-6-membered heterocyclyl is unsubstituted or substituted by 1-3 substituents selected from halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Independently of one another, particularly preferably H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, or together with the carbon atom to which they are attached form a moiety selected from the group consisting of C3-C6-cycloalkyl and C3-C6-cycloalkenyl, wherein C3-C6-cycloalkyl or C3-C6-cycloalkenyl is unsubstituted or substituted by 1-3 substituents selected from halogen, CN, C1-C6-alkyl and C1-C6-alkoxy, Independently of each other, particularly preferably H, halogen, C1-C6-alkyl or C1-C6-haloalkyl, More preferably, independently of each other, H, halogen or C1-C6-alkyl; R 5 is H, CN, C1-C6-alkyl, C1-C6-haloalkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, C1-C6-alkyl, C1-C6-alkoxy-C1-C6-alkyl, C1-C6-alkoxy, (C1-C6-alkyl)carbonyl or (C1-C6-alkyl)sulfonyl, Particularly preferred is H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3, Hydrogen is more preferred.

[0331] A is (A.1) and R 1 and R5 Particularly preferred are azines of formula (Ia) which correspond to the azines of formula (I) in which [ka] [Wherein, the variable group R a , R b , R c , R d , R e , R 2 , R 3 and R 4 have the above defined meanings, especially the preferred meanings]; Particularly preferred are the azines of formulae (Ia1) to (Ia1406) of Table A, in which the variable group R a , R b , R c , R d , R e , R 2 , R 3 and R 4 The definitions of are particularly important not only in combination with each other but also individually: Table A TIFF0007619806000018.tif206157TIFF0007619806000019.tif248147TIFF0007619806000020.tif248147TIFF0007619806000021.tif248147TIFF0007619806000022.tif249148TIFF0007619806000023.tif249148TIFF0007619806000024.tif248148TIFF0007619806000025.tif248148TIFF0007619806000026.tif248148TIFF0007619806000027.tif248148TIFF0007619806000028.tif248148TIFF0007619806000029.tif248148TIFF0007619806000030.tif248148TIFF0007619806000031.tif248148TIFF0007619806000032.tif248148TIFF0007619806000033.tif248148TIFF0007619806000034.tif248148TIFF0007619806000035.tif248148TIFF0007619806000036.tif248148TIFF0007619806000037.tif249147TIFF0007619806000038.tif248147TIFF0007619806000039.tif249147TIFF0007619806000040.tif248148TIFF0007619806000041.tif248148TIFF0007619806000042.tif248148TIFF0007619806000043.tif248148TIFF0007619806000044.tif248147TIFF0007619806000045.tif248147TIFF0007619806000046.tif248147TIFF0007619806000047.tif248148TIFF0007619806000048.tif248148TIFF0007619806000049.tif248148TIFF0007619806000050.tif248148TIFF0007619806000051.tif249148TIFF0007619806000052.tif249148TIFF0007619806000053.tif101147.

[0332] The herbicidal compounds useful in the present invention can also be used in conjunction with additional herbicides to which crop plants are naturally resistant, or to which they have been made resistant by mutagenesis as described above, or to which they have been made resistant by the expression of one or more additional transgenes as described above.The herbicides useful in the present invention are often best applied in conjunction with one or more other herbicides to provide broader control of undesirable vegetation.When used in conjunction with other herbicides (hereinafter referred to as compound B), the claimed compounds of the present invention can be formulated with one or more other herbicides, tank mixed with one or more other herbicides, or applied sequentially with one or more other herbicides.

[0333] The further herbicide compound B (component B) is in particular selected from the herbicides of classes b1) to b15) (as well as the agriculturally acceptable salts or derivatives thereof, such as ethers, esters or amides). b1) lipid biosynthesis inhibitors, b2) acetolactate synthase inhibitors (ALS inhibitors), b3) photosynthesis inhibitors, b4) protoporphyrinogen IX oxidase inhibitors, b5) bleaching herbicides; b6) Enolpyruvylshikimate 3-phosphate synthase inhibitors (EPSP inhibitors), b7) glutamine synthetase inhibitors, b8) 7,8-dihydropteroate synthase inhibitors (DHP inhibitors), b9) mitotic inhibitors; b10) Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors), b11) cellulose biosynthesis inhibitors, b12) decoupler herbicides; b13) auxin herbicides, b14) auxin transport inhibitors, and b15) Bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumylron, dalapon, dazomet, difenzoquat, difenzoquat-methyl sulfate, dimethipine, DSMA, dymron, endothal and its salts, etobenzanide, flamprot, flamprot-isopropyl, flamprot-methyl, flamprot-M-isopropyl, flamprot-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methiozoline (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam, tridiphane and other herbicides selected from the group consisting of 6-chloro-3-(2-cyclopropyl-6-methylphenoxy)-4-pyridazinol (CAS 499223-49-3) and its salts and esters.

[0334] Preference is given to compositions according to the invention which comprise at least one herbicide B selected from the herbicides of classes b1, b6, b9, b10 and b11.

[0335] Examples of herbicides B which can be used in combination with the compounds of formula (I) of the present invention are given below.

[0336] b1) From the group of lipid biosynthesis inhibitors: ACC-herbicides, for example, alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-P, fenoxaprop-P-ethyl, fluazifop, fluazifop-butyl, fluazifop-P, fluazifop-P-butyl, haloxyfop, haloxyfop-methyl ... Xyfop-P, Haloxyfop-P-methyl, Metamifop, Pinoxaden, Profoxydim, Propaquizafop, Quizalofop, Quizalofop-ethyl, Quizalofop-tefuryl, Quizalofop-P, Quizalofop-P-ethyl, Quizalofop-P-tefuryl, Sethoxydim, Tepraloxydim, Tralkoxydim, 4-(4'-chloro-4-cyclopropyl-2'-fluoro[1,1'-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-72-6;4-(2',4'-dichloro-4-cyclopropyl[1,1'-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1312337-45-3);4-(4'-chloro-4-ethyl-2'-fluoro[1,1'-biphenyl]-3-yl)-5-hydroxy-2,2,6,6-tetramethyl-2H-pyran-3(6H)-one (CAS 1033757-93-5);4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-2,2,6,6-tetramethyl-2H-pyran-3,5(4H,6H)-dione (CAS 1312340-84-3;5-(acetyloxy)-4-(4'-chloro-4-cyclopropyl-2'-fluoro[1,1'-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312337-48-6);5-(acetyloxy)-4-(2',4'-dichloro-4-cyclopropyl-[1,1'-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one;5-(Acetyloxy)-4-(4'-chloro-4-ethyl-2'-fluoro[1,1'-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1); 5-(Acetyloxy)-4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6-tetramethyl-2H-pyran-3-one (CAS 1033760-55-2); 4-(4'-chloro-4-cyclopropyl-2'-fluoro[1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-yl methyl carbonate (CAS 1312337-51-1;4-(2',4'-dichloro-4-cyclopropyl-[1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonic acid methyl ester;4-(4'-chloro-4-ethyl-2'-fluoro[1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonic acid methyl ester (CAS 1312340-83-2);4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonic acid methyl ester (CAS 1033760-58-5), and non-ACC herbicides such as benfuresate, butyrate, cycloate, dalapon, dimepyrate, EPTC, esprocarb, ethofumesate, flupropanate, molinate, orbencarb, pebulate, prosulfocarb, TCA, thiobencarb, thiocarbazyl, triallate and vernolate;

[0337] b2) From the group of ALS inhibitors: Sulfonylureas, such as amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlorimuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupyrsulfuron, flupyrsulfuron-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron ... sulphon-sodium, mesosulfuron, metazosulfuron, metsulfuron, metsulfuron-methyl, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, primisulfuron-methyl, propyrisulfuron, prosulfuron, pyrazosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron, sulfometuron-methyl, sulfosulfuron, thifensulfuron, thifensulfuron-methyl, triasulfuron, tribenuron, tribenuron-methyl, trifloxysulfuron, triflusulfuron, triflusulfuron-methyl and tritosulfuron, Imidazolinones such as Imazamethabenz, Imazamethabenz-methyl, Imazamox, Imazapic, Imazapyr, Imazaquin and Imazethapyr, triazolopyrimidine herbicides and sulfonanilides such as Cloransulam, Cloransulam-methyl, Diclosulam, Flumetsulam, Florasulam, Metosulam, Penoxsulam, Pyrimisulfan and Pyroxsulam, Pyrimidinyl benzoates, for example bispyribac, bispyribac-sodium, pyribenzoxim, pyriftalid, pyriminobac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium, 4-[[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid-1-methylethyl ester (CAS 420138-41-6), 4-[[[2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]phenyl]methyl]amino]-benzoic acid propyl ester (CAS 420138-40-5), N-(4-bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenemethanamine (CAS 420138-01-8); Sulfonylaminocarbonyl-triazolinone herbicides, such as flucarbazone, flucarbazone-sodium, propoxycarbazone, propoxycarbazone-sodium, thiencarbazone and thiencarbazone-methyl; and triafamone, Among these, preferred embodiments of the present invention relate to compositions comprising at least one imidazolinone herbicide.

[0338] b3) From the group of photosynthetic inhibitors: Amicarbazone, inhibitors of photosystem II, for example triazine herbicides such as chlorotriazine, triazinones, triazinediones, methylthiotriazines and pyridazinones, for example ametryn, atrazine, chloridazon, cyanazine, desmetryn, dimethametryn, hexazinone, metribuzin, prometon, prometryn, propazine, simazine, simetryn, terbumeton, terbuthylazine, terbutryn and trietazine, aryl ureas such as chlorobromuron, chlorotoluron, chloroxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, methabenzthiazuron, methobenzuron , metoxuron, monolinuron, nebulon, siduron, tebuthiuron and thiadiazuron, phenylcarbamates such as desmedipham, carbutilate, phenmedipham, phenmedipham-ethyl, nitrile herbicides such as bromofenoxime, bromoxynil and its salts and esters, ioxynil and its salts and esters, uracils such as bromacil, lenacil and terbacil, as well as bentazone and bentazone-sodium, pyridate, pyridahol, pentanochlor and propanil, and inhibitors of photosystem I such as diquat, diquat-dibromide, paraquat, paraquat-dichloride and paraquat-dimethylsulfate.

[0339] Of these, preferred embodiments of the invention relate to compositions comprising at least one aryl urea herbicide. Of these, preferred embodiments of the invention relate to compositions comprising at least one triazine herbicide. Of these, preferred embodiments of the invention relate to compositions comprising at least one nitrile herbicide.

[0340] b4) From the group of the protoporphyrinogen IX oxidase inhibitors: Acifluorfen, Acifluorfen-sodium, Azafenidin, Bencarbazone, Benzphendizone, Bifenox, Butafenacil, Carfentrazone, Carfentrazone-ethyl, Chlomethoxyfen, Cinidon-ethyl, Fluazolate, Flufenpyr, Flufenpyr-ethyl, Flumiclorac, Flumiclorac-pentyl, Flumioxazin, Fluoroglycofen, Fluoroglycofen-ethyl, Fluthiacet, Fluthiacet-methyl, Fomesafen , halosafen, lactofen, oxadiargyl, oxadiazon, oxyfluorfen, pentoxazone, profluazole, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiadiamine, thiaphenacyl, ethyl [3-[2-chloro-4-fluoro-5-(1-methyl-6-trifluoromethyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidin-3-yl)phenoxy]-2-pyridyloxy]acetate (CAS 353292-31-6;S-3100), N-ethyl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3-[7-fluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[1,3,5]triazinane-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-ynyl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinane-2,4-dione (CAS 1258836-72-4), 2-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-4,5,6,7-tetrahydro-isoindole-1,3-dione, 1-methyl-6-trifluoromethyl-3-(2,2,7-trifluoro-3-oxo-4-prop-2-ynyl-3,4-dihydro-2H-benzo[1,4]oxazin-6-yl)-1H-pyrimidine-2,4-dione, methyl(E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4-fluoro-phenoxy]-3-methoxy-but-2-enoate [CAS 948893-00-3], and 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1H-benzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)-1H-pyrimidine-2,4-dione (CAS 212754-02-4);

[0341] b5) From the group of the bleaching herbicides: PDS inhibitors such as beflubutamide, diflufenican, fluridone, flurochloridone, flurtamone, norflurazone, picolinafen, and 4-(3-trifluoromethylphenoxy)-2-(4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), HPPD inhibitors such as benzobicyclon, benzofenap, clomazone, fenquinotrion, isoxaflutole, mesotrione, pyrasulfotole, pyrazolinate, pyrazoxyfen, sulcotrione, tefuryltrione, tembotrione, topramezone, and bicyclopyrone, whitening agents, unknown targets such as aclonifen, amitrole, and fluometuron,

[0342] b6) From the group of EPSP synthase inhibitors: Glyphosate, glyphosate-isopropylammonium, glyphosate-potassium and glyphosate-trimesium (sulfonates),

[0343] b7) From the group of glutamine synthetase inhibitors: Biranaphos (Bialaphos), Biranaphos-sodium, Glufosinate, Glufosinate-P and Glufosinate-ammonium,

[0344] b8) From the group of the DHP synthase inhibitors: Ashram,

[0345] b9) From the group of the mitotic inhibitors: Compounds of group K1, for example dinitroanilines, such as benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramidates, such as amiprophos, amiprophos-methyl and butamiphos, benzoic acid herbicides, such as chlorthal, chlorthal-dimethyl, pyridines, such as dithiopyr and thiazopyr, benzamides, such as propyzamide and tebutam, compounds of group K2, such as chlorpropham, propham and carbetamide, of which compounds of group K1, especially the dinitroanilines, are preferred.

[0346] b10) From the group of VLCFA inhibitors: Chloroacetamides such as acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, dimethenamid-P, metazochlor, metolachlor, metolachlor-S, petoxamide, pretilachlor, propachlor, propisochlor and thenylchlor, oxyacetanilides such as flufenacet and mefenacet, acetanilides such as diphenamide, naproanilide, napropamide and napropamide-M, tetrazolinones such as fentrazamide, and other herbicides such as anilofos, cafenstrole, fenoxasulfone, ipfencarbazone, piperophos, pyroxasulfone, and isoxazoline compounds of the formulae II.1, II.2, II.3, II.4, II.5, II.6, II.7, II.8 and II.9. [ka]

[0347] Isoxazoline compounds of formula (I) are known in the art, for example from WO2006 / 024820, WO2006 / 037945, WO2007 / 071900 and WO2007 / 096576. Among the VLCFA inhibitors, chloroacetamides and oxyacetamides are preferred.

[0348] b11) From the group of the cellulose biosynthesis inhibitors: Chlorthiamid, dichlobenil, flupoxam, isoxaben and 1-cyclohexyl-5-pentafluorophenyloxy-1 4 -[1,2,4,6]thiatriazin-3-ylamine,

[0349] b12) From the group of the decoupler herbicides: Dinoseb, dinoterb and DNOC and its salts,

[0350] b13) From the group of the auxin herbicides: 2,4-D and its salts and esters, such as clacyfos, 2,4-DB and its salts and esters, aminocyclopyrachlor and its salts and esters, aminopyralid and its salts, such as aminopyralid-dimethylammonium, aminopyralid-tris(2-hydroxypropyl)ammonium and its esters, benazolin, benazolin-ethyl, chloramben and its salts and esters, clomeprop, clopyralid and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichlorprop-P and its salts and esters, fluroxypyr, fluroxypyr-butometyl, fluroxypyr-meptyl, halauxifen and its salts and esters (CAS 943832-60-8), MCPA and its salts and esters, MCPA-thioethyl, MCPB and its salts and esters, mecoprop and its salts and esters, mecoprop-P and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac, TBA(2,3,6) and its salts and esters, and triclopyr and its salts and esters,

[0351] b14) From the group of auxin transport inhibitors: Diflufenzopyr, diflufenzopyr-sodium, naptalam and naptalam-sodium,

[0352] b15) From the group of other herbicides: Bromobutide, chlorflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, cyclopyrimorate (CAS 499223-49-3) and its salts and esters, dalapon, dazomet, difenzoquat, difenzoquat-methyl sulfate, dimethipine, DSMA, dymron, endothal and its salts, etobenzanide, flamprot, flamprot-isopropyl, flamprot-methyl, flamprot-M-isopropyl, flamprot-M-methyl, flurenol, flurenol-butyl, flurprimidol, fosamine, fosamine-ammonium, indanofan, indaziflam, maleic hydrazide, mefluidide, metam, methiozoline (CAS 403640-27-7), methyl azide, methyl bromide, methyl-dymron, methyl iodide, MSMA, oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam and tridiphane.

[0353] The active compounds B and C having a carboxyl group can be used in the compositions of the invention in the form of the acid, in the form of an agriculturally suitable salt as described above, or in the form of an agriculturally acceptable derivative.

[0354] In the case of dicamba, suitable salts include those in which the counterion is an agriculturally acceptable cation. For example, suitable salts of dicamba are dicamba-sodium, dicamba-potassium, dicamba-methylammonium, dicamba-dimethylammonium, dicamba-isopropylammonium, dicamba-diglycolamine, dicamba-olamine, dicamba-diolamine, dicamba-trolamine, dicamba-N,N-bis-(3-aminopropyl)methylamine, and dicamba-diethylenetriamine. Examples of suitable esters are dicamba-methyl and dicamba-butotyl.

[0355] Suitable salts of 2,4-D are 2,4-D-ammonium, 2,4-D-dimethylammonium, 2,4-D-diethylammonium, 2,4-D-diethanolammonium (2,4-D-diolamine), 2,4-D-triethanolammonium, 2,4-D-isopropylammonium, 2,4-D-triisopropanolammonium, 2,4-D-heptylammonium, 2,4-D-dodecylammonium, 2,4-D-tetradecylammonium, 2,4-D-triethylammonium, 2,4-D-tris(2-hydroxypropyl)ammonium, 2,4-D-tris(isopropyl)ammonium, 2,4-D-trolamine, 2,4-D-lithium, 2,4-D-sodium. Examples of suitable esters of 2,4-D are 2,4-D-butotyl, 2,4-D-2-butoxypropyl, 2,4-D-3-butoxypropyl, 2,4-D-butyl, 2,4-D-ethyl, 2,4-D-ethylhexyl, 2,4-D-isobutyl, 2,4-D-isooctyl, 2,4-D-isopropyl, 2,4-D-meptyl, 2,4-D-methyl, 2,4-D-octyl, 2,4-D-pentyl, 2,4-D-propyl, 2,4-D-tefuryl and clasifos.

[0356] Suitable salts of 2,4-DB are, for example, 2,4-DB-sodium, 2,4-DB-potassium and 2,4-DB-dimethylammonium. Suitable esters of 2,4-DB are, for example, 2,4-DB-butyl and 2,4-DB-isooctyl.

[0357] Suitable salts of dichlorprop are, for example, dichlorprop-sodium, dichlorprop-potassium and dichlorprop-dimethylammonium. Examples of suitable esters of dichlorprop are dichlorprop-butotyl and dichlorprop-isooctyl.

[0358] Suitable salts and esters of MCPA include MCPA-butotyl, MCPA-butyl, MCPA-dimethylammonium, MCPA-diolamine, MCPA-ethyl, MCPA-thioethyl, MCPA-2-ethylhexyl, MCPA-isobutyl, MCPA-isooctyl, MCPA-isopropyl, MCPA-isopropylammonium, MCPA-methyl, MCPA-olamine, MCPA-potassium, MCPA-sodium, and MCPA-trolamine.

[0359] A preferred salt of MCPB is sodium MCPB. A preferred ester of MCPB is MCPB-ethyl.

[0360] Suitable salts of clopyralid are clopyralid-potassium, clopyralid-olamine and clopyralid-tris-(2-hydroxypropyl)ammonium. An example of a suitable ester of clopyralid is clopyralid-methyl.

[0361] Examples of suitable esters of fluroxypyr are fluroxypyr-meptyl and fluroxypyr-2-butoxy-1-methylethyl, with fluroxypyr-meptyl being preferred.

[0362] Preferred salts of picloram are picloram-dimethylammonium, picloram-potassium, picloram-triisopropanolammonium, picloram-triisopropylammonium and picloram-trolamine. A preferred ester of picloram is picloram-isooctyl.

[0363] A suitable salt of triclopyr is triclopyr-triethylammonium. Suitable esters of triclopyr are, for example, triclopyr-ethyl and triclopyr-butotyl.

[0364] Suitable salts and esters of chloramben include chloramben-ammonium, chloramben-diolamine, chloramben-methyl, chloramben-methylammonium and chloramben-sodium. Suitable salts and esters of 2,3,6-TBA include 2,3,6-TBA-dimethylammonium, 2,3,6-TBA-lithium, 2,3,6-TBA-potassium and 2,3,6-TBA-sodium.

[0365] Suitable salts and esters of aminopyralid include aminopyralid-potassium, aminopyralid-dimethylammonium, and aminopyralid-tris(2-hydroxypropyl)ammonium.

[0366] Suitable salts of glyphosate are, for example, glyphosate-ammonium, glyphosate-diammonium, glyphosate-dimethylammonium, glyphosate-isopropylammonium, glyphosate-potassium, glyphosate-sodium, glyphosate-trimesium as well as the ethanolamine and diethanolamine salts, preferably glyphosate-diammonium, glyphosate-isopropylammonium and glyphosate-trimesium (sulfonates).

[0367] A suitable salt of glufosinate is, for example, glufosinate-ammonium.

[0368] A suitable salt of glufosinate-P is, for example, glufosinate-P-ammonium.

[0369] Suitable salts and esters of bromoxynil are, for example, bromoxynil-butyrate, bromoxynil-heptanoate, bromoxynil-octanoate, bromoxynil-potassium and bromoxynil-sodium.

[0370] Suitable salts and esters of ioxynil are for example ioxynil-octanoate, ioxynil-potassium and ioxynil-sodium.

[0371] Suitable salts and esters of mecoprop include mecoprop-butotyl, mecoprop-dimethylammonium, mecoprop-diolamine, mecoprop-ethadyl, mecoprop-2-ethylhexyl, mecoprop-isooctyl, mecoprop-methyl, mecoprop-potassium, mecoprop-sodium and mecoprop-trolamine.

[0372] Suitable salts of mecoprop-P are, for example, mecoprop-P-butotyl, mecoprop-P-dimethylammonium, mecoprop-P-2-ethylhexyl, mecoprop-P-isobutyl, mecoprop-P-potassium and mecoprop-P-sodium.

[0373] A suitable salt of diflufenzopyr is, for example, diflufenzopyr-sodium.

[0374] A suitable salt of naptalam is for example naptalam-sodium.

[0375] Suitable salts and esters of aminocyclopyrachlor are for example aminocyclopyrachlor-dimethylammonium, aminocyclopyrachlor-methyl, aminocyclopyrachlor-triisopropanolammonium, aminocyclopyrachlor-sodium and aminocyclopyrachlor-potassium.

[0376] A suitable salt of quinclorac is, for example, quinclorac-dimethylammonium.

[0377] A suitable salt of quinclorac is, for example, quinclorac-dimethylammonium.

[0378] A suitable salt of imazamox is, for example, imazamox-ammonium.

[0379] Suitable salts of imazapic are, for example, imazapic-ammonium and imazapic-isopropylammonium.

[0380] Suitable salts of imazapyr are, for example, imazapyr-ammonium and imazapyr-isopropylammonium.

[0381] A suitable salt of imazaquin is, for example, imazaquin-ammonium.

[0382] Suitable salts of imazethapyr are, for example, imazethapyr-ammonium and imazethapyr-isopropylammonium.

[0383] A suitable salt of topramezone is, for example, topramezone sodium.

[0384] Particularly preferred herbicide compounds B are the herbicides B as defined above, in particular the herbicides B.1 to B.189 listed in Table B below. Table B JPEG0007619806000055.jpg86152JPEG0007619806000056.jpg237152JPEG0007619806000057.jpg234152JPEG0007619806000058.jpg98151

[0385] It may also be useful to apply the compound of formula (I) in combination with a safener and optionally one or more additional herbicides.Safeners are chemical compounds that prevent or reduce damage to useful plants without significantly affecting the herbicidal action of the compound of formula (I) on undesirable plants.They can be applied before sowing the seeds of useful plants (for example, seed treatment, shoots or seedlings) or in pre-emergence or post-emergence applications.Safeners and the compound of formula (I) and optionally herbicide B can be applied simultaneously or successively.

[0386] Suitable safeners are, for example, (quinoline-8-oxy)acetic acid, 1-phenyl-5-haloalkyl-1H-1,2,4-triazole-3-carboxylic acid, 1-phenyl-4,5-dihydro-5-alkyl-1H-pyrazole-3,5-dicarboxylic acid, 4,5-dihydro-5,5-diaryl-3-isoxazolecarboxylic acid, dichloroacetamide, alpha-oximinophenylacetonitrile, acetophenone oxime, 4,6-dihalo-2-phenylpyrimidine, N-[[4-(aminocarbonyl)phenyl]sulfonyl]-2-benzoic acid amide, 1,8-naphthalic anhydride, 2-halo-4-(haloalkyl)-5-thiazolecarboxylic acid, phosphorothioates and N-alkyl-O-phenylcarbamates and their agriculturally acceptable salts and agriculturally acceptable derivatives, such as amides, esters, and thioesters, provided that they contain an acidic group.

[0387] Examples of preferred safeners C are benoxacor, cloquintocet, cyometrinil, cyprosulfamide, dichlormid, dicyclonone, dietholate, fenchlorazole, fenclorim, flurazole, fluxofenim, furilazole, isoxadifen, mefenpyr, mephenate, naphthalic anhydride, oxabetrinil, 4-(dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane (MON4660, CAS 71526-07-3), 2,2,5-trimethyl-3-(dichloroacetyl)-1,3-oxazolidine (R-29148, CAS 52836-31-4) and N-(2-methoxybenzoyl)-4-[(methylaminocarbonyl)amino]benzenesulfonamide (CAS 129531-12-0).

[0388] Particularly preferred safeners C are the following compounds C.1 to C.17.

[0389] JPEG0007619806000059.jpg68157

[0390] The active compound B and the safener compound C of group b1) to b15) are known herbicides and safeners.See, for example, The Compendium of Pesticide Common Names (http: / / www.alanwood.net / pesticides / ); Farm Chemicals Handbook 2000 volume 86, Meister Publishing Company, 2000; B. Hock, C. Fedtke, RR Schmidt, Herbizide [Herbicides], Georg Thieme Verlag, Stuttgart 1995; WH Ahrens, Herbicide Handbook, 7th edition, Weed Science Society of America, 1994, and KK Hatzios, Herbicide Handbook, Supplement for the 7th edition, Weed Science Society of America, 1998. 2,2,5-Trimethyl-3-(dichloroacetyl)-1,3-oxazolidine [CAS No. 52836-31-4] is also known as R-29148. 4-(Dichloroacetyl)-1-oxa-4-azaspiro[4.5]decane [CAS No. 71526-07-3] is also known as AD-67 and MON 4660.

[0391] The allocation of the active compounds to each mechanism of action is based on current knowledge. If several mechanisms of action apply to one active compound, this substance was assigned to only one mechanism of action.

[0392] It is generally preferred to use the compounds of the present invention in combination with herbicides that are selective to the crops being treated and that complement the range of weeds controlled by these compounds at the rates used.Furthermore, it is generally preferred to apply the compounds of the present invention and other complementary herbicides simultaneously as a combination formulation or tank mix.

[0393] In another embodiment, the present invention relates to a method for identifying a herbicide by using a mutant TriA encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1, or a mutant or derivative thereof.

[0394] The method comprises: a) generating a transgenic cell or plant that contains a nucleic acid encoding a wild-type or mutant TriA and in which the mutant TriA is expressed; b) applying a herbicide to the transgenic cells or plants of a) and to control cells or plants of the same variety; c) determining the growth or viability of the transgenic cells or plants and the control cells or plants after application of the herbicide; and d) selecting a "herbicide" that confers reduced growth to the control cells or plants compared to the growth of the transgenic cells or plants. Includes.

[0395] As mentioned above, the present invention teaches compositions and methods for increasing the resistance of a crop plant or seed compared to a wild type variety of the plant or seed. In a preferred embodiment, the resistance of the crop plant or seed is increased when the plant or seed is preferably at about 1-1000 g ai ha -1 , more preferably 1-200g ai ha -1 , and even more preferably 5-150g ai ha -1 , most preferably 10-100g ai ha -1 The plant's ability to withstand the application of a herbicide of a high activity is increased. As used herein, "withstands" the application of a herbicide means that the plant is not killed or only moderately damaged by such application. Those skilled in the art will understand that the application rate may vary depending on environmental conditions such as temperature or humidity, and depending on the type of herbicide (active ingredient ai) selected.

[0396] Postemergence weed control methods useful in various embodiments of the present invention utilize about >0.3x application rate of herbicide, which in some embodiments can be, for example, about >0.3x, >0.4x, >0.5x, >0.6x, >0.7x, >0.8x, >0.9x, or >1x herbicide. In one embodiment, the herbicide-tolerant plants of the present invention are tolerant to postemergence application of herbicide in an amount of about 25 to about 200 g ai / ha. In some embodiments, when the herbicide-tolerant plants are dicotyledons (e.g., soybean, cotton), the postemergence application of herbicide is in an amount of about 50 g ai / ha. In another embodiment, when the herbicide-tolerant plants are monocotyledons (e.g., maize, rice, sorghum), the postemergence application of herbicide is in an amount of about 200 g ai / ha. In another embodiment, where the herbicide tolerant plant is a Brassica (e.g., canola), the post-emergence application of the herbicide is at a rate of about 25 g ai / ha. In some embodiments, the post-emergence weed control methods of the invention can utilize an application rate of herbicide at about 7-10 days after emergence. In another embodiment, the application rate can be greater than 1x herbicide, and in some embodiments, the rate can be up to 4x herbicide, but is more typically about 2.5x or less, or about 2x or less, or about 1x or less.

[0397] The present invention also provides a method comprising the use of at least one herbicide, optionally in combination with one or more herbicide compounds B and optionally a safener C, as described in detail above.

[0398] In these methods, the herbicide can be applied by any method known in the art, including but not limited to seed treatment, soil treatment, and foliar treatment.Before application, the herbicide can be converted into a conventional formulation, such as a solution, emulsion, suspension, dust, powder, paste, and granule.The form of use depends on the specific intended purpose.In any case, it should ensure a fine and uniform distribution of the compound of the present invention.

[0399] Various formulations can be used to protect plants from weeds to enhance plant growth and reduce competition for nutrients by providing plants with increased tolerance to herbicides. Herbicides can be used pre-emergence, post-emergence, pre-planting, and at planting to control weeds in the area around the crop plants described herein, or herbicide formulations containing other additives can be used. Herbicides can also be used as seed treatments. Additives found in herbicide formulations include other herbicides, surfactants, adjuvants, spreading agents, sticking agents, stabilizers, and the like. Herbicide formulations can be wet or dry preparations, including, but not limited to, flowable powders, emulsifiable concentrates, and concentrates. Herbicides and herbicide formulations can be applied by conventional methods, such as spraying, irrigation, dusting, and the like.

[0400] Suitable formulations are described in detail in PCT / EP2009 / 063387 and PCT / EP2009 / 063386, which are incorporated herein by reference.

[0401] As disclosed herein, the TriA nucleic acid of the present invention is used to enhance CBI herbicide tolerance in plants that contain a gene in their genome that encodes a wild-type or mutant TriA protein that is herbicide resistant. As described above, such a gene may be an endogenous gene or a transgene. Furthermore, in certain embodiments, the nucleic acid of the present invention is stacked with any combination of polynucleotide sequences of interest to create a plant with a desired phenotype. For example, the nucleic acids of the invention can be used to encode proteins encoding, for example, Bacillus thuringiensis toxin proteins (described in U.S. Patent Nos. 5,366,892, 5,747,450, 5,737,514, 5,723,756, 5,593,881, and Geiser et al. (1986) Gene 48: 109), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate acetyltransferase (GAT), cytochrome P450 monooxygenase, phosphinothricin acetyltransferase (PAT), acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also referred to as acetolactate synthase or ALS), hydroxyphenylpyruvate pyruvate dioxygenase (HPPD), phytoene desaturase (PD), protoporphyrinogen oxidase (PPO) and the dicamba degrading enzymes disclosed in WO02 / 068607, or the phenoxyacetic acid- and phenoxypropionic acid-derivative degrading enzymes disclosed in WO2008141154 or WO2005107437. The combinations made can also include multiple copies of any of the polynucleotides of interest.

[0402] Thus, the herbicide-tolerant plants of the present invention can be used in combination with the herbicide to which they are tolerant. The herbicide can be applied to the plants of the present invention using any technique known to those skilled in the art. The herbicide can be applied at any time during the plant cultivation process. For example, the herbicide can be applied before planting, at planting, before emergence, after emergence, or a combination thereof. The herbicide can be applied to the seed and dried to form a layer on the seed.

[0403] In some embodiments, the seeds are treated with a safener and then a herbicide is applied post-emergence. In one embodiment, the post-emergence application of the herbicide is about 7-10 days after planting the safener-treated seeds. In some embodiments, the safener is cloquintocet, dichlormid, fluxofenim, or combinations thereof.

[0404] Method for controlling weeds or unwanted vegetation In another aspect, the invention provides a method of controlling weeds at a locus for the growth of a plant or plant part thereof, the method comprising the step of applying to the locus a composition comprising a herbicide.

[0405] In some aspects, the invention provides methods for controlling weeds at a locus for growing a plant, the methods comprising the step of applying to the locus a herbicide composition comprising a herbicide, the locus being (a) a locus containing a plant or a seed capable of producing said plant, or (b) a locus which will contain a plant or seed after said application, wherein the plant or seed comprises in at least a portion of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide encoded by the polynucleotide, and wherein expression of the mutant TriA polypeptide confers resistance to the plant to the herbicide.

[0406] The herbicidal compositions of the present invention can be applied, for example, as a foliar treatment, a soil treatment, a seed treatment, or a soil drench. Application can be made, for example, by spraying, dusting, dusting, or by any other useful method known in the art.

[0407] In one embodiment, the herbicide can be used to control the growth of weeds that may be found growing adjacent to the herbicide-resistant plants of the present invention. In such an embodiment, the herbicide can be applied to the land where the herbicide-resistant plants of the present invention grow adjacent to the weeds. In this case, the herbicide to which the herbicide-resistant plants of the present invention are resistant can be applied to the land at a concentration sufficient to kill or inhibit the growth of the weeds. Concentrations of herbicide sufficient to kill or inhibit the growth of weeds are known in the art and are disclosed above.

[0408] In another embodiment, the present invention provides a method for controlling weeds adjacent to a plant tolerant to the herbicide of the present invention. The method includes applying an effective amount of a herbicide to a weed and a plant tolerant to an auxinic herbicide, wherein the plant has increased tolerance to the auxinic herbicide when compared to a wild-type plant. In some embodiments, the plant tolerant to the herbicide of the present invention is preferably a crop plant, such as, but not limited to, sunflower, alfalfa, Brassica spp., soybean, cotton, safflower, peanut, tobacco, tomato, potato, wheat, rice, corn, sorghum, barley, rye, millet, and sorghum.

[0409] In other aspects, the herbicide (e.g., multiple herbicides) can also be used as a seed treatment. In some embodiments, an effective concentration or amount of the herbicide, or a composition containing an effective concentration or amount of the herbicide, can be applied directly to the seed before or during sowing of the seed. The seed treatment formulation can further include a binder and optionally a colorant.

[0410] Binders can be added to improve the adhesion of the active substances on the seeds after treatment. In one embodiment, suitable binders are block copolymer EO / PO surfactants, but also polyvinyl alcohol, polyvinylpyrrolidone, polyacrylates, polymethacrylates, polybutenes, polyisobutylenes, polystyrenes, polyethyleneamines, polyethyleneamides, polyethyleneimines (Lupasol®, Polymin®), polyethers, polyurethanes, polyvinyl acetates, tyloses and copolymers derived from these polymers. Optionally, colorants can also be included in the formulation. Suitable colorants or dyes for the seed treatment formulations are Rhodamin B, CI Pigment Red 112, CI Solvent Red 1, pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment blue 15:1, pigment blue 80, pigment yellow 1, pigment yellow 13, pigment red 1 12, pigment red 48:2, pigment red 48:1, pigment red 57:1, pigment red 53:1, pigment orange 43, pigment orange 34, pigment orange 5, pigment green 36, pigment green 7, piment white 6, pigment brown 25, basic violet 10, basic violet 49, acid red 51, acid red 52, avid red 14, acid blue 9, acid yellow 23, basic red 10, and basic red 108.

[0411] The term seed treatment includes all suitable seed treatment techniques known in the art, such as seed dressing, seed coating, seed dusting, soaking, and seed pelleting. In one embodiment, the present invention provides a method for treating soil by applying a granular formulation containing a herbicide as a composition / formulation (e.g., a granular formulation), optionally together with one or more solid or liquid agriculturally acceptable carriers and / or optionally one or more agriculturally acceptable surfactants, in particular in a seed drill. This method is advantageously used, for example, in cereal, maize, cotton, and sunflower seedbeds.

[0412] The present invention also relates to herbicides and antibacterial agents, such as amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfuron, ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, and / or a seed that is coated with or contains a seed treatment formulation comprising at least one other herbicide such as an AHAS-inhibitor selected from the group consisting of sucrose, trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, cloransulam, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac, pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxim, pyriftalid and pyrithiobac.

[0413] The term "coated and / or comprising" generally means that the majority of the active ingredient is on the surface of the propagation product when applied, but more or less some part of the ingredient may penetrate into the propagation product depending on the application method. When said propagation product is (re)planted, the active ingredient may be absorbed.

[0414] In some embodiments, seed treatment applications with herbicides or with formulations containing herbicides are carried out by spraying or dusting the seeds before sowing of the plants and before emergence of the plants.

[0415] In another embodiment, in the treatment of seeds, the corresponding formulations are applied by treating the seeds with an effective amount of the herbicide or a formulation containing the herbicide.

[0416] In another aspect, the present invention provides a method for combating unwanted vegetation or controlling weeds, comprising contacting the seeds of plants resistant to the herbicides of the present invention with the herbicide before sowing and / or after germination. The method can further comprise sowing the seeds in, for example, field soil or in potting medium in a greenhouse. The method is particularly used in combating unwanted vegetation or controlling weeds in the immediate vicinity of the seeds. Controlling unwanted vegetation is understood to mean killing the weeds and / or otherwise retarding or inhibiting the normal growth of the weeds. Weeds are understood in the broadest sense to mean any plant that grows in an undesired place.

[0417] Weeds according to the present invention include, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis, Lepiclium, Galium, Chickweed, Matricaria, Anthemis, Bryum, Chenopodium, Urtica, Amaranth, Portulaca, Xanthium, Convolvulus, Sweet potato, Polygonum, Sesbania, Ambrosia, Cirsium, Cornus, Cornus oleracea, Sowberry, Solarium, Carpinus, Rhizome, Lindera, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaverum, Centaurea, Cinnamon, Ranunculaceae, and Dandelion. Monocotyledonous weeds include, but are not limited to, weeds of the genera Echinochloa, Setaria, Millet, Digitaria, Timothys, Pose, Fescue, Goosegrass, Brachiaria, Solanum, Bromeliad, Avena, Cyperus, Sorghum, Triticum, Cyperus, Monochoria, Tentella, Ardisia, Artemisia, Bulbul, Paspalum, Platypus, Rhus, Scutellaria, Scutellaria, Scutellaria, and Apella.

[0418] In addition, weeds of the present invention can include, for example, crop plants growing where they are not wanted, for example, corn plants growing in a field containing primarily soybean plants can be considered weeds if the corn plants are not wanted in the field of soybean plants.

[0419] In another embodiment, in the treatment of seeds, the corresponding formulations are applied by treating the seeds with an effective amount of the herbicide or a formulation containing the herbicide.

[0420] In yet another aspect, the treatment of the locus, plant, plant part, or seed of the present invention comprises application of an agriculturally acceptable composition that does not contain an AI. In one embodiment, the treatment comprises application of an agriculturally acceptable composition that does not contain a herbicide AI. In some embodiments, the treatment comprises application of an agriculturally acceptable composition that does not contain a herbicide AI, where the composition comprises one or more agriculturally acceptable carriers, diluents, excipients, plant growth regulators, etc. In other embodiments, the treatment comprises application of an agriculturally acceptable composition that does not contain a herbicide AI, where the composition comprises an adjuvant. In one embodiment, the adjuvant is a surfactant, a spreading agent, a sticking agent, a penetrating agent, a drift control agent, a crop oil, an emulsifier, a compatibilizer, or a combination thereof.

[0421] It should also be understood that the above relates to preferred embodiments of the present invention, and that numerous modifications can be made without departing from the scope of the present invention. The present invention is further illustrated in the following examples, which are not intended to limit the scope of the present invention in any way. On the contrary, those skilled in the art who have read the description herein will clearly understand that various other embodiments, modifications and equivalents that may be suggested can be used without departing from the spirit and / or scope of the present invention. (Example) Example 1

[0422] Bacterial strains. Chemically competent Escherichia coli TOP10 (Life Technologies; US) and BL21(DE3) Gold (Agilent Technologies; Germany) were used as recipients for transformation experiments. Transformation was performed as described in Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Habor Laboratory Press, Cold Spring Habor; NY (1982). Agrobacterium tumefaciens can be used to introduce T-DNA regions into Arabidopsis, maize, and soybean.

[0423] Bacterial cultures were routinely grown in LB medium or on LB mixed with agar (15% w / v) at 37°C. When necessary, the medium was supplemented with the antibiotics kanamycin and / or chloramphenicol. Plasmid DNA was prepared using the GeneJet Plasmid Miniprep kit (Thermo Scientific, US). TriA and its variants were produced by gene synthesis (Eurofins, Germany). The synthesized genes carried XhoI and NcoI restriction sites and were cloned into the pET24d N-HIS vector with kanamycin resistance. The chaperone plasmid pGro7 (chaperones groEL and groES) with chloramphenicol resistance was obtained from TaKaRa (Japan). Example 2

[0424] Gene synthesis, restriction enzyme digestion and cloning. Gene synthesis with appropriate cloning into pMK-RQ vector was performed by Eurofins (Germany). Restriction enzymes were purchased from New England. Restriction enzymes were used according to the manufacturer's instructions. Example 3

[0425] Protein purification. TriA and its variants were produced in E. coli BL21(DE3) Gold (Agilent Technologies, Germany). Therefore, E. coli was transformed with the appropriate pET24d N-HIS tag expression vector and the chaperone plasmid pGro7 (chaperones groEL and groES). Bacterial strains were grown in 100 mL LB at 30°C for 20 h and protein expression was induced with 0.1 mM IPTG at 25°C for 20 h. Cells were harvested by centrifugation at 3000 rpm for 20 min at 4°C and resuspended in Bug Buster protein extraction reagent (Novagen, Germany) according to the manufacturer's instructions. Lysates were clarified by centrifugation. Bovine serum albumin (5, 10, and 20 g) samples were loaded onto the gel, respectively, and analyzed by densitometry to provide an internal standard. Protein quantification was verified using Coomassie protein assay dye according to the manufacturer's instructions (Thermo Scientific; USA). HIS-tagged enzymes were purified by metal ion affinity chromatography using the Ni-IDA 1000 kit (Macherey-Nagel, Germany) according to the manufacturer's instructions. Protein purity was assessed by SDS-PAGE using NuPAGE Novex 4-12% Bis-Tris precast gels (Life Technologies; USA) stained with Coomassie brilliant blue (Serva, Germany). Protein concentration was estimated by measuring absorbance at 280 nm using a Lambda Bio+ (Perkin Elmer, USA). Example 4

[0426] Enzyme kinetics. Resting cell suspensions containing the TriA gene were incubated with various azines, melamine, and atrazine, and the culture filtrates were analyzed by UPLC-HR-MS. Substrates were purchased from Sigma-Aldrich or synthesized in-house. Synthetic standards and enzyme reaction products were analyzed by UPLC-HR-MS (Thermo / Dionex UPLC UltiMate3000 interfaced with a QExactive high-resolution mass spectrometer). A Waters Acquity HSS T3 column (2.1 mm; 100 mm; 1.8 μM) was used with a flow rate of 0.6 μl min -1 The mobile phase was water / acetonitrile (0.1% formic acid). The enzyme was dissolved in 25 mM sodium phosphate buffer (pH 7.2) at 30°C, and the substrate concentration ranged from 1 nM to 10 nM. Over time, the original azine peak disappeared, whereas the OH-metabolites (reaction products) produced increased. The products were identified by the determination of the correct formula and by the analysis of the correct MS-MS fragments. In addition, some of the products were co-eluted with standards. The degradation in % was calculated against cells carrying the empty vector as a control. The results are shown in Table 3. TIFF0007619806000060.tif21877TIFF0007619806000061.tif22477TIFF0007619806000062.tif253114TIFF0007619806000063.tif181114 Example 5

[0427] Directed evolution of amidohydrolases. Azines were docked into the active site of the TriA model (based on the trzN and AtzA crystal structures) by superimposing the molecule on the melamine. On this basis, the residues forming the active site and the substrate binding pocket were identified. The main regions involved in the coordination of metal ions in the active site; residues known to be essential for amidohydrolase activity; residues forming the hydrophobic "base" of the active site or essential for hydrolase activity interactions with aromatic rings of the substrate were left unchanged. However, amino acids were modified to enlarge the enzyme pocket. The model was used, on the one hand, to predict amino acid targets away from the active site that could affect the acceptance of triazines in general, and, on the other hand, to identify space-requiring amino acids in the enzyme pocket that could be changed to smaller amino acids with similar hydrophobicity to achieve the accommodation of larger azines without changing the enzyme activity. In addition, full saturation mutagenesis was performed at favorable positions, including positions 92, 93, 155 and 157. Amino acid changes at some positions significantly increased the amidohydrolase activity towards larger azines.

[0428] To study the enzyme stability and protease resistance, solutions containing TriA protein in 25 mM phosphate buffer (pH 7.2) were mixed with various concentrations of protease or left untreated. The protease used was subtilisin A from Bacillus licheniformis obtained from Sigma Aldrich. After the incubation period, the above solutions were incubated with various azines, melamine, or atrazine, and the filtrates were analyzed as described in Example 4. The results are shown in Figures 2 and 3. Example 6

[0429] Generation of herbicide-resistant model plants. Generation of azine-resistant Arabidopsis plants with mutant amidohydrolase sequences. For transformation of Arabidopsis thaliana, wild-type or amidohydrolase sequences based on sequence 1, encoding sequence 2, are cloned into a binary vector by standard cloning techniques as described in Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press), which contains a resistance marker gene cassette (AHAS) and mutant amidohydrolase sequences (denoted GOI) between a ubiquitin promoter (PcUbi) and a nopaline synthase terminator (NOS) sequence. The binary plasmid is introduced into Agrobacterium tumefaciens for plant transformation. The mutant amidohydrolase sequences are transformed into Arabidopsis by the floral dip method as described in McElver and Singh (WO 2008 / 124495). Arabidopsis transgenic plants are subjected to TaqMan analysis to analyze the number of integration sites. Example 7

[0430] Testing of herbicide-tolerant model plants. Expression of triA and its variants is used to select azine-tolerant Arabidopsis thaliana plants. Selected Arabidopsis lines were assayed for improved resistance to azines such as 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine in 48-well plates. Thus, T2 seeds are surface-sterilized by agitating for 5 min in ethanol + water (70 + 30 volume ratio), rinsing once with ethanol + water (70 + 30 volume ratio) and rinsing twice with sterile deionized water. Seeds were resuspended in 0.1% agar (w / v) in water. Four or five seeds per well are plated on solid nutrient medium consisting of half-diluted Murashige and Skoog nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia 40 Plantarum 15: 473-497). Compounds are dissolved in dimethyl sulfoxide (DMSO) and added to the medium before solidification (final DMSO concentration 0.1%). Multiwell plates are grown in a growth chamber at 22°C and 75% relative humidity, with a photon intensity of 110 μmol m -2 s -1 The plants are incubated at 4 °C for 12 h and a 14:10 h light:dark photoperiod. Growth inhibition is assessed 7-10 days after sowing in comparison to wild-type plants. Resistance factors are calculated based on the IC50 values ​​of growth inhibition of transformants over non-transformed Arabidopsis plants. In addition, transgenic T2 or T3 Arabidopsis plants are tested in greenhouse experiments for improved tolerance to herbicides that inhibit cellulose biosynthesis. Example 8

[0431] Creation and testing of herbicide-tolerant crops (soybean and corn). Binary vectors are created as described in Example 9. Soybean cultivar Jake is transformed as described in Siminszky et al., Phytochem Rev. 5:445-458 (2006). After regeneration, transformants are transplanted into soil in small pots and placed in a growth box (16 h day / 8 h night; 25° C. day / 23° C. night; 65% relative humidity; 130-150 μE m -2 s -1 ), and then examined for the presence of T-DNA by TaqMan analysis. After several weeks, healthy, transgenic positive single copy events are transferred to larger pots and grown in growth boxes. Plants are then transferred to the greenhouse for T1 seed production and harvest.

[0432] The best shoots to take are 3-4 inches tall and have at least two nodes. Cuttings are taken from each original transformant (mother plant) and placed in rooting hormone powder (indole-3-butyric acid, IBA). The cuttings are then placed in oasis wedges inside the biodome. The mother plants are allowed to reach maturity in the greenhouse and harvested for seed. Wild-type cuttings are also taken to serve as negative controls. The cuttings are maintained in the biodome for 5-7 days. 7-10 days after transfer to the oasis wedge, the roots are treated with herbicide via the nutrient solution. 3-4 days after treatment, the plants are evaluated for typical phytotoxin symptoms, such as clubbed roots. Transgenic plants with no or less damage compared to wild-type plants are considered herbicide-resistant.

[0433] For pre-emergence treatment, the growing boxes used can be plastic flower pots containing a ferrous sandy soil with about 3.0% humus as a growth medium. Seeds of the test plants are sown separately for each species / case. The active ingredients used are suspended or emulsified in water and applied directly after sowing using fine spray nozzles. To promote germination and growth, the growing boxes are gently watered and then covered with a transparent plastic hood until the plants are rooted. This cover causes uniform germination of the test plants, except in cases where the active ingredients have impaired germination.

[0434] Immature corn embryos are transformed according to the procedures outlined in Peng et al. (WO2006 / 136596). Plants are tested for the presence of the T-DNA by TaqMan analysis, which targets the nos terminator present in all constructs. Healthy looking plants are placed in the greenhouse for hardening and subsequent spray testing. Plants are individually repotted in MetroMix 360 potting soil in 4" pots. Plants are grown for 14 days in a greenhouse (27°C / 21°C day / night cycle, 14-hour photoperiod maintained with 600W high pressure sodium lamps). Maize transgenic plants are grown to T1 seeds for herbicide resistance testing. 14 days after repotting, the roots are treated with herbicides via nutrient solution. 3-4 days after treatment, typical phytotoxic symptoms such as clubbed roots are evaluated. Transgenic plants with no or less damage compared to wild type plants are considered herbicide resistant.

[0435] For pre-emergence treatment, the growing boxes used are plastic pots containing a composted sandy soil with approximately 3.0% humus as a substrate. Seeds of the test plants are sown separately for each species / event. The active ingredients used are suspended or emulsified in water and applied directly after sowing using fine spray nozzles. To promote germination and growth, the growing boxes are gently watered and then covered with a transparent plastic hood until the plants are rooted. This cover causes uniform germination of the test plants, except in cases where the active ingredients have impaired germination.

[0436] For post-emergence treatment, the test plants are first grown to a height of 3-15 cm depending on the early stage and only then treated with the herbicide. For this purpose, the test plants are either directly sown and grown in the same box or first grown separately and then transferred to the test growing box a few days before treatment. The damage caused by the herbicide is assessed 2 and 3 weeks after treatment. The damage to the plants is assessed on a scale from 0% to 100%, 0% being no damage and 100% being complete death. 100 means that the plants do not emerge or that at least the above-ground parts are completely destroyed, 0 means that there is no damage or that the growth process is normal. A moderate herbicidal activity is given at a value of at least 60, a good herbicidal activity at least 70 and a very good herbicidal activity at least 85.

[0437] T1 seeds were sown in open seedling flats with holes in the bottom to allow drainage. The flats were filled halfway to the top with Palmetto Medium C-3 Vermiculite. 60 seeds were evenly scattered on top of this layer and then covered with a new layer of vermiculite. The trays were watered and moved to a greenhouse under standard conditions and kept well watered with a water / fertilizer solution for 4 days. After 4 days, the germinated seeds were removed from the vermiculite, gently scraped off the roots to remove the medium, and placed into 15 mL glass tubes containing the appropriate concentration of 6-cyclopentyl-N4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine dissolved in deionized water. A small hole was made in the cap to allow the roots to just penetrate. The tubes were placed in black boxes with holes in the top to hold the 15 mL tubes neatly and grown under standard soybean conditions in a growth box for 5 days before imaging. The plants were digitally imaged and assessed for resistance to herbicide treatments. Resistance was assessed by comparing shoots and / or roots to non-transformed (wild type) controls. The results are shown in Figure 1. Example 9

[0438] Construction of binary vectors. Cloning methods, such as the use of restriction endonucleases to cleave double-stranded DNA at specific positions, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, ligation of DNA fragments, transformation of E. coli cells, and bacterial culture were performed as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). Polymerase chain reaction was performed using Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions. In general, primers used for PCR were designed such that at least 20 nucleotides at the 3' end of the primer perfectly annealed with the template to be amplified. Restriction enzyme sites were added to the 5' end of the primer by appending nucleotides corresponding to the restriction enzyme site. Fusion PCR, e.g. as described in K. Heckman and LR Pease, Nature Protocols (2207) 2, 924-932, was alternatively used to link two fragments of interest, e.g. to link a promoter to a gene or a gene to a terminator. Gene synthesis was performed at Life Technologies using the GeneArt® service, e.g. as described in Czar et al. (Trends in Biotechnology, 2009, 27(2): 63-72).

[0439] Genes were evaluated for codon usage and the presence of restriction sites that could hinder cloning efforts. If necessary, genes were codon-optimized using standard protocols for maximum expression in crops (see, for example, Puigbo et al. 2007 and Gasper et al. 2012) and, of course, undesirable restriction sites were removed. Genes were either synthesized by GeneArt (Regensburg) or PCR-amplified from cDNA using Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions. In both cases, NcoI and / or AscI restriction sites were introduced at the 5' end and an SbfI restriction site at the 3' end to allow cloning of the genes between functional elements such as promoters and terminators using these restriction sites. Promoter-terminator or promoter-intron-terminator modules were generated by nucleotide synthesis. Terminator sequences were synthetically linked to the promoter or promoter-intron sequences, and restriction endonuclease recognition sequences were added to both sides of the modules, and recognition sites for the restriction endonucleases NcoI, AscI and SbfI were introduced between the promoter and terminator or between the intron and terminator. To obtain the final expression modules, PCR-amplified genes were cloned between the promoter and terminator or between the intron and terminator using the AscI and / or SbfI restriction enzyme recognition sites. The completed binary vectors were used to transform E. coli as described in Sambrook et al. 1989.

[0440] Alternatively, gene synthesis can be performed using the GeneArt® service at Life Technologies, e.g., as described in Czar et al. (Trends in Biotechnology, 2009, 27(2): 63-72). Standard methods such as cloning, restriction, molecular analysis, transformation of E. coli cells, and bacterial culture can be performed as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). Polymerase chain reaction can be performed using Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions. Fusion PCR can be performed as described in K. Heckman and LR Pease, Nature Protocols (2207) 2, 924-932. In both cases, an NcoI and / or AscI restriction site can be introduced at the 5' end and a PacI restriction site at the 3' end, allowing the cloning of these genes between the functional elements. Promoter-terminator or promoter-intron-terminator modules were produced by total synthesis by GeneArt (Regensburg) or by linking the corresponding expression elements using fusion PCR and cloning the PCR product into the TOPO vector pCR2.1 (Invitrogen) according to the manufacturer's instructions. Either by synthesis of the entire cassette or by fusion PCR, the terminator sequence can be linked to the promoter or promoter-intron sequence while at the same time recognition sequences for restriction endonucleases can be added at both ends of the module, and recognition sites for the restriction endonucleases NcoI, AscI and PacI can be introduced between the promoter and terminator or between the intron and terminator.To obtain the final expression module, the PCR amplified genes can be cloned between the promoter and terminator or between the intron and terminator via NcoI and / or Pac I restriction enzyme sites. The genes of interest can be codon-optimized using standard protocols (see, for example, Puigbo et al. 2007 and Gasper et al. 2012) to obtain maximum expression in the crop as well as to remove undesirable restriction enzyme sites, and can be synthesized by GeneArt (Regensburg, Germany).

[0441] References Esser HO, Dupuis G, Ebert E, Marco GJ, Vogel C (1975) s-Triazines. In: Kearney PC, Kaufman DJ (eds) Herbicides, chemistry, degradation and mode of action. Marcel Dekker, New York, pp 129-208 Seffernick JL, McTavish H, Osborne JP, de Souza ML, Sadowsky MJ, Wackett LP (2002) Atrazine chlorohydrolase from Pseudomonas sp. strain ADP is a metalloenzyme. Biochemistry 41: 14430-14437 Wackett et al.; Biodegradation of atrazine and related s-triazine compounds: from enzymes to field studies, Applied Microbiology and Biotechnology; 58 (1), 39-45, 2002 de Souza ML, Sadowsky MJ, Wackett LP (1996) Atrazine chlorohydrolase from Pseudomonas sp strain ADP: Gene sequence, enzyme purification, and protein characterization. Journal of Bacteriology 178: 4894-4900. Sadowski et al.; US 6369299, Transgenic plants expressing bacterial atrazine degrading gene AtzA Padgette S. R. et al., Site directed mutagenesis of a conserved region of the 5-Enolpyruvylshikimate-3-phosphate synthase actives-site.; J.Biol. Chem., 266, 33, 1991 Maniatis et al. Molecular Cloning: A Laboratory Manual, Cold Spring Habor Laboratory Press, Cold Spring Habor; N.Y. (1982) Gasper P., Oliveira J-L., Frommlet J., Santos M.A.S., Moura G. (2012) EuGene: maximizing synthetic gene design for heterologous expression. Bioinformatics 28(20), 2683-2684. Murashige and Skoog 1962 Physiologia 40 Plantarum 15: 473-497, Molecular cloning Cold Spring Harbor Laboratory Press (2001) Komori T., Imayama T., Kato N., Ishida Y., Ueiki J., Komari T. (2007) Current Status of Binary Vectors and Sub-binary Vectors. Plant Physiology 145, 1155-1160.Puigbo P., Guzman E., Romeu A., Garcia-Valve A. (2007) OPTIMIZER: A Web Server for Optimizing the Codon Usage of DNA Sequences. Nucleic Acids Research 35 web server edition. W126-W131. Siminszky B., Plant cytochrome P450-mediated herbicide metabolism, Phytochem Rev. 5:445-458, 2006 The present disclosure includes the following embodiments. [1] A plant or plant part comprising a polynucleotide encoding a mutant TriA polypeptide, wherein expression of the polynucleotide confers tolerance to a herbicide on the plant or plant part. [2] The plant or plant part of embodiment 1, wherein the herbicide comprises a compound that inhibits cellulose biosynthesis. [3] The plant or plant part of embodiment 1 or 2, wherein the polynucleotide encoding the mutant TriA polypeptide comprises the nucleic acid sequence set forth in SEQ ID NO: 1, or a homologue,...

Claims

1. 2. A plant or plant part comprising a polynucleotide encoding a mutant TriA polypeptide, the mutant TriA polypeptide having at least 90% amino acid sequence identity to SEQ ID NO:2 over its entire length and having melamine deaminase activity, wherein the amino acid corresponding to position 92 in SEQ ID NO:2 is substituted with Thr, and wherein in the mutant TriA polypeptide, the amino acid corresponding to position 93 in SEQ ID NO:2 is substituted with Leu, the amino acid corresponding to position 155 in SEQ ID NO:2 is substituted with Thr, and the amino acid corresponding to position 157 in SEQ ID NO:2 is substituted with Leu, and wherein expression of the polynucleotide confers tolerance to a herbicide to the plant or plant part.

2. 2. The plant or plant part of claim 1, wherein the herbicide comprises a compound that inhibits cellulose biosynthesis.

3. 3. The plant or plant part of claim 1 or 2, wherein the polynucleotide encoding the mutant TriA polypeptide comprises a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO:

1.

4. 1. A method for controlling weeds in a locus for growing a plant, comprising: (a) applying to the locus a herbicide composition comprising a herbicide; and (b) planting a seed in the locus, wherein the seed is capable of producing a plant comprising in at least some of its cells a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant TriA polypeptide described in claim 1, and wherein expression of the mutant TriA polypeptide confers resistance to the herbicide to the plant.

5. 5. The method of claim 4, wherein the herbicidal composition is applied to weeds and to plants produced by seeds.

6. 6. The method of claim 4 or 5, wherein the herbicide comprises a compound that inhibits cellulose biosynthesis.

7. 1. A method for producing a plant having resistance to a herbicide, comprising the step of regenerating a plant from a plant cell transformed with a recombinant polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant TriA polypeptide described in claim 1, wherein expression of the mutant TriA polypeptide confers resistance to the herbicide to the plant.

8. (a) a nucleic acid molecule encoding a mutant TriA polypeptide comprising a variant of the sequence of SEQ ID NO:2, the nucleic acid molecule having at least 90% sequence identity to SEQ ID NO:2, the nucleic acid molecule encoding a polypeptide that has melamine deaminase activity and confers resistance to a herbicide to a plant or plant part; (b) a nucleic acid molecule comprising a variant of the sequence of SEQ ID NO:1, wherein the variant has at least 90% sequence identity to SEQ ID NO:1 and encodes a polypeptide that has melamine deaminase activity and confers resistance to herbicides to a plant or plant part; An isolated and / or recombinantly produced and / or synthetic nucleic acid molecule comprising a nucleic acid encoding a mutant TriA polypeptide selected from the group consisting of: A nucleic acid molecule comprising, in a mutant TriA polypeptide, the amino acid corresponding to position 92 of SEQ ID NO:2 is substituted with Thr, the amino acid corresponding to position 93 of SEQ ID NO:2 is substituted with Leu, the amino acid corresponding to position 155 of SEQ ID NO:2 is substituted with Thr, and the amino acid corresponding to position 157 of SEQ ID NO:2 is substituted with Leu.

9. 9. An expression cassette comprising the nucleic acid molecule of claim 8 and a promoter operable in a plant cell.

10. 10. The expression cassette of claim 9, wherein the promoter is a root-specific promoter.

11. 11. A vector containing a nucleic acid molecule according to claim 8, or an expression cassette according to claim 9 or 10.

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