EA044296B1 - PLANTS WITH INCREASED RESISTANCE TO HERBICIDES - Google Patents

Description

Field of invention

The invention relates generally to methods for increasing the resistance of agricultural crops to herbicides. In particular, the invention relates to plants with increased resistance to herbicides, more particularly to cellulose biosynthesis inhibitor herbicides, which thereby interfere with cell wall biosynthesis.

Prerequisites for creating an invention

Microorganisms often respond to the introduction of xenobiotics into the environment by developing mechanisms for their use as a source of nutrients and energy for their growth. Because the structure of herbicides, which is based on the s-triazine ring, is different from natural compounds (Esser et al., 1975), microorganisms have slowly evolved enzymes and metabolic pathways capable of degenerating them. The amidohydrolase superfamily includes a striking array of enzymes that catalyze the hydrolysis of a wide variety of substrates containing amide or ester functional groups at carbon and phosphorus centers. In all cases, the nucleophilic water molecule is activated due to complex formation in the mononuclear or binuclear center of the metal. At mononuclear metal sites, the substrate is activated by proton transfer from the active site, and water is activated by the metal ligature and normal base catalysis. Metal centers are deposited on the C-terminal region of the beta-barrel core within the (beta alpha) 8th structural domain. One excellent example is the atrazine chlorohydrase (AtzA)-dependent, Fe(II)-dependent homohexamer ((Seffernick et al., 2002; Wackett et al., 2002a), which catalyzes the hydrolytic dechlorination of atrizine, a herbicide, from which a non-herbicidal product is obtained 2-hydroxyatrazine (de Souza et al., 1996; Seffernick et al., 2002; Sadowsky and Wackett 2000) The closest known related form to AtzA is melamine diamenase (TriA from Pseudomonas sp. strain NRRL B12227; 98% sequence identity) Although highly similar, the sequences of AtzA and TriA differ in their catalytic properties. TriA is a diamenase with dechlorinase activity several orders of magnitude lower than its physiological diamenase activity, whereas AtzA is a dechlorinase with no appreciable diamenase activity. Previous work has shown that three of the nine amino acids that differ between the two proteins (S331C; N328D; and F84I AtzA) are largely responsible for the differences in the occurrence of specific catalytic properties.

The present invention provides new methods for increasing plant resistance to herbicides by introducing bacterial genes encoding target proteins that biodegrade the herbicide, in particular cellulose biosynthesis inhibitors, which are known as azines. Bacterial TriA enzymes were designed in a form to maintain or increase aminohydrolase activity and expand the enzyme repertoire to accept a larger substrate. The present inventors have surprisingly discovered that overexpression of wild type or mutant forms of TriA melamine diamenase confers resistance/tolerance to certain classes of herbicides in plants compared to untransformed and/or unmutated plants or plant cells, respectively. In particular, the present inventors have discovered that expression of TriA confers resistance/resistance to azines.

The object of the present invention can be considered to provide new characteristics by identifying target polypeptides, the manipulation of which confers herbicide resistance in plants.

There are three main strategies for increasing plant resistance to herbicides, namely: (1) detoxification of the herbicide with enzymes that transform the herbicide or its active metabolite into non-toxic products, such as enzymes for increasing resistance to bromoxynil or basta (EP242236, EP337899) ; (2) mutation of the target enzyme into a functional enzyme that will be less sensitive to the herbicide or its active metabolite, for example, enzymes to increase glyphosate resistance (EP293356, Padgette S.R. et al., J. Biol. Chem., 266, 33, 1991 ); or (3) overexpression of a sensitive enzyme to produce sufficient amounts of the target enzyme in the plant relative to the herbicide, in terms of the kinetic constants of that enzyme, to produce sufficient amounts of the functional enzyme despite the presence of its inhibitor.

This problem is solved by the subject matter of the present invention.

Summary of the invention

Accordingly, in one aspect, the present invention provides a plant or plant part comprising a polynucleotide encoding a mutated TriA polypeptide, wherein expression of the polynucleotide confers herbicide resistance to the plants or plant parts.

In accordance with some aspects of the invention, the present invention provides a seed capable of developing into a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts resistance to herbicides to said plant.

In accordance with one aspect, the present invention provides a plant cell or a cell from which a plant can be regenerated that, in at least some of its

- 1 044296 cells contains a polynucleotide operably linked to a promoter functioning in plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of a mutated TriA polypeptide imparts resistance to herbicides to the said plant, wherein said plant cell contains the polynucleotide operably linked to the promoter.

In accordance with yet another aspect, the present invention provides a plant cell containing a polynucleotide operably linked to a promoter operating in the cell, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide confers herbicide resistance to said plant .

In accordance with other aspects of the invention, the present invention provides a plant product obtained from a plant or part of a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter functioning in the plant cells, wherein the promoter is capable of expressing a mutated polypeptide TriA encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide confers herbicide resistance to said plant.

In accordance with some aspects of the invention, the present invention provides a progeny or progeny of a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide , wherein said progeny or descendants of the plant, in at least some of their cells, contain a recombinant polynucleotide operably linked to a promoter, wherein expression of the mutated TriA polypeptide imparts resistance to herbicides to said progeny or descendants of the plant.

In accordance with other aspects of the invention, the present invention provides a method for controlling weeds at a plant site, the method comprising: (a) applying a herbicidal composition containing herbicides to a plant site; and (b) sowing the seed at said location, wherein the seed is capable of developing into a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing the mutated TriA polypeptide, encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts resistance to herbicides to said plant.

In accordance with some aspects of the invention, the present invention provides a method for controlling weeds in a plant habitat, the method comprising: applying a herbicidal composition containing herbicides to a plant site; in this case, in the specified place: (a) a plant grows or a seed is planted that is capable of developing into the specified plant; or (b) a plant will grow or such seed will be planted after application of said herbicidal composition; wherein said plant or seed, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts to said plant herbicide resistance.

In accordance with one aspect, step (a) is performed before step (b), after step (b), or simultaneously with step (b).

In accordance with other aspects, the present invention provides a method for producing a plant with herbicide resistance, the method comprising regenerating the plant from a plant cell transformed using a polynucleotide operably linked to a promoter operating in the cell, wherein the promoter is capable of expressing a mutated TriA polypeptide , encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts resistance to herbicides to said plant.

In accordance with one aspect, the present invention provides a method for producing a progeny plant having herbicide resistance, the method comprising: crossing a first plant having herbicide resistance with a second plant to produce progeny plant having a herbicide resistance, wherein the first plant and progeny plants, in at least some of their cells, contain a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide confers herbicide resistance to said plant .

In addition, the present invention provides a method for detecting a herbicide using wild type or mutated TriA of the present invention encoded by a nucleic acid containing nucleotide sequence No. 1 or a variant, homolog, paralog or orthologue thereof.

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This method includes the following steps:

a) obtaining a transgenic cell or plant with a nucleic acid encoding a mutated TriA, where the mutated TriA of the present invention is expressed;

b) applying the herbicide to the transgenic cell or plant a) and to a control cell or plant of the same species;

c) determining the growth or viability of the transgenic cell or plant and the control cell or plant after application of the specified test compound;

d) selecting test compounds that resulted in reduced growth of the control cell or plant compared to the growth of the transgenic cell or plant.

Another object of the present invention relates to a method for identifying a nucleotide sequence encoding a mutated TriA that is persistent or resistant to a herbicide. The method includes the following steps:

a) obtaining a library of nucleic acids encoding mutated TriA,

b) screening a population of resulting nucleic acids encoding the mutated TriA by expressing each said nucleic acid in a cell or plant and treating the cell or plant with a herbicide,

c) comparing the levels of herbicide resistance that are obtained from a given population of nucleic acids encoding mutated TriA with the levels of herbicide resistance that are obtained from a control nucleic acid encoding TriA,

d) selecting at least one nucleic acid encoding a mutated TriA that exhibits a significantly increased level of herbicide resistance compared to a control nucleic acid encoding TriA.

In a preferred embodiment of the invention, the nucleic acid encoding the mutated TriA and selected in step d) exhibits a herbicide resistance that is at least twofold greater than that of the control nucleic acid encoding TriA.

Persistence or resistance can be determined by obtaining a transgenic plant with the nucleic acid sequence from the library in step a) and by comparing this transgenic plant with a control plant.

Yet another subject relates to isolated, recombinantly produced and/or chemically synthesized nucleic acids encoding wild-type TriA or mutated TriA, which nucleic acid contains the sequences of SEQ ID NO: 1 or a variant thereof, as defined herein.

A preferred embodiment of the invention relates to an isolated and/or recombinantly produced and/or synthetic nucleic acid molecule comprising a nucleic acid molecule encoding a TriA mutated polypeptide selected from the group consisting of:

(a) a nucleic acid molecule encoding a polypeptide with a mutated TriA, and containing the sequence SEQ ID NO: 2 or a variant, paralog, ortholog or homologue thereof;

(b) a nucleic acid molecule containing the sequence SEQ ID NO: 1 or a variant, paralog, ortholog or homologue thereof;

(c) a nucleic acid molecule which, as a result of degeneration of the genetic code, can be transmitted from the polypeptide sequence TriA SEQ ID NO: 2 or a variant, paralog, ortholog or homolog thereof, and conferred increased resistance or resistance to herbicides compared to the corresponding, e.g. untransformed, wild-type plant cell, plant or part thereof;

(d) nucleic acid molecules with an identity of 30% or more, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or more with the nucleic acid molecule sequence of a polynucleotide comprising the nucleic acid molecule of SEQ ID NO: 1 or a variant, paralog, ortholog or homolog thereof, and confers increased herbicide resistance or resistance compared to the corresponding, e.g. untransformed, wild-type plant cell, plant or part thereof;

(e) a nucleic acid molecule encoding a TriA mutated polypeptide with an identity of 30% or more, preferably 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99%, 99.5% or more with the amino acid sequence of the TriA polypeptide of SEQ ID NO: 2 and confers increased herbicide tolerance or resistance compared to a corresponding, for example, untransformed, wild-type plant cell, plant or part thereof;

(f) a nucleic acid molecule that hybridizes with a nucleic acid molecule of (a), (b), (c), (d) or (e) under stringent hybridization conditions and confers increased herbicide resistance or resistance compared to the corresponding , for example, an untransformed, wild-type plant cell, plant or part thereof;

characterized in that the amino acid sequence of the mutated TriA polypeptide differs from the amino acid sequence of the wild type TriA polypeptide in one or more

- 3 044296 positions corresponding to the following sequence positions SEQ ID NO: 2: 69, 70, 71, 74, 82,

84, 85, 87, 88, 89, 91, 92, 93, 96, 126, 128, 129, 130, 131, 155, 157, 160, 167, 170, 174, 180, 182, 216, 217, 219,

220, 246, 247, 248, 249, 250, 251, 298, 301, 302, 304, 328.

Another aspect relates to an expression cassette comprising a nucleic acid molecule of the present invention as well as a promoter operating in plant cells.

Preferably, the promoter is a root promoter or an enhanced root promoter from Glycine max.

Yet another subject relates to an isolated recombinant and/or chemically synthesized TriA mutated polypeptide, the polypeptide comprising the sequence set of SEQ ID NO: 2 or a variant, derivative, ortholog, paralog or homologue thereof as defined herein.

Preferably, the amino acid sequence of the TriA mutated polypeptide differs from the amino acid sequence of the wild type TriA polypeptide at one or more positions corresponding to the following sequence positions SEQ ID NO: 2: 69, 70, 71, 74, 82, 84, 85, 87, 88, 89, 91, 92, 93, 96, 126, 128, 129, 130, 131, 155, 157, 160, 167, 170, 174, 180, 182, 216, 217, 219, 220, 246, 247, 248, 249, 250, 251, 298, 301, 302, 304, 328.

In accordance with other aspects of the invention, the present invention provides a plant or part of a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said plant cell. polynucleotide, wherein expression of the TriA polypeptide with the mutated TriA confers herbicide resistance to said plant, wherein the plant or part of the plant also exhibits a characteristic of resistance to a second or third herbicide.

In yet another embodiment of the invention, the invention relates to a plant cell transformed with or expressing a wild-type TriA nucleic acid or a PPO mutated nucleic acid according to the present invention, or a plant that has been mutated to produce a plant expressing, preferably with overexpressing wild-type or mutated TriA TriA nucleic acid in accordance with the present invention, wherein expression of said nucleic acid in a plant cell results in increased resistance or resistance to the herbicide compared to a wild-type plant cell.

In yet another embodiment of the invention, the invention relates to a plant with a plant cell in accordance with the present invention, in which the expression of a nucleic acid results in increased resistance of the plant to a herbicide compared to a wild-type plant.

Preferably, expression of the nucleic acid of the invention in a plant results in increased plant resistance to the herbicide compared to a wild-type plant.

In another embodiment of the invention, the invention relates to a seed obtained from a transgenic plant with a plant cell of the present invention, in which the seed germinates due to an increase in the plant's herbicide tolerance compared to the wild type seed.

In yet another embodiment of the invention, the invention relates to a method of producing a transgenic plant cell with increased herbicide resistance compared to a wild-type plant cell, including transforming the plant cell with an expression cassette polynucleotide operably linked to a promoter functioning in plant cells, a promoter that is capable of express a mutated TriA polynucleotide-encoded polypeptide.

In yet another embodiment, the invention relates to a method for producing a transgenic plant, comprising: (a) transforming a plant cell with an expression cassette containing a polynucleotide operably linked to a promoter functioning in plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, and (b) obtaining from a plant cell a plant with increased resistance to the herbicide.

Preferably, the expression cassette also includes a transcriptional initiation regulatory site and a translational initiation regulatory region that function in the plant.

Description of the figures

In fig. Figure 1 shows an assessment of the resistance of soybean plants treated with azines before emergence. Development of the phenotype of a wild plant (A) and a transgenic line expressing the TriA mutant of the sequence SEQ ID NO: 2, containing amino acid substitutions in four positions, namely L92A_Y93L_M155T_F157L (B). The indicated values [g/ha] reflect the amount of 6-cyclopentyl-N4(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine. The upper part of each plant shows the affected shoots, and the lower part shows the root damage.

In fig. Figure 2 shows the assessment of pre-emergence resistance of corn plants treated with azines. Development of the phenotype of a wild plant (A) and a transgenic line expressing the TriA mutant of the sequence SEQ ID NO: 2, containing amino acid substitutions in four positions, namely L92A_Y93L_M155T_F157L (B). The indicated values [g/ha] reflect the amount of 6-cyclopentylN4-(2,3,4,5,6-pentafluorophenyl)-1,3,5-triazine-2,4-diamine.

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Detailed Description of the Invention

The indefinite articles used in the present invention refer to one or more (ie, at least one) nouns with which the article is used. For example, the indefinite article with the word element means one or more elements.

As used in the present invention, the word content or variations thereof such as contains or containing implies the inclusion of a specified element, whole number or step or group of elements, whole numbers or steps, but not the exclusion of any other element, whole number or step or groups of elements, integers, or stages.

The term control of unwanted vegetation or weeds implies the destruction of weeds and/or the retardation or inhibition of their normal growth. Weeds, in a broad sense, refer to all plants that grow where they are not wanted. The term weeds in accordance with the present invention includes, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, the following genera:

Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga,

Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Lindernia, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum.

Monocot weeds include, but are not limited to, the following genera:

Echinochloa,

Setaria, Panicum, Digitaria, Phleum, Roa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus and Apera.

In addition, the term weeds in accordance with the present invention may include, for example, crops growing in an undesirable area. For example, corn growing randomly in a field that primarily grows soybeans may be considered a weed if it is undesirable there.

The term plant is used in a broad sense as it refers to organic material and eukaryotic organisms belonging to the Plant Kingdom, examples of which include, but are not limited to, vascular plants, vegetables, cereals, flowers, trees, grasses, bushes, turf, vines , ferns, moss, fungi and algae, etc., as well as clones, shoots and parts of plants used for vegetative propagation (e.g. shoots, stems, shoots, rhizomes, underground roots, clumps, corollas, bulbs, corms, tubers , plants/tissues obtained from tissue culture, etc.). The term plant also means the whole plant, the ancestors and offspring of plants and their parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, inflorescences, fruits, pedicels, peduncles, stamens, anthers, stigmas, ovaries, sepals, carpel, root tip, root cap, root hair, leaf hair, plant fiber, pollen grain, microspores, cotyledon, hypocotyl, epicotyl, xylem, phloem, parenchyma, endosperm, satellite cell, guard cell and other known organs, tissues and cells plants, and tissues and organs, where each of the above contains the gene/nucleic acid of interest. The term plant also includes plant cells, suspension cultures, callus tissue, embryos, meristem regions, gametophytes, sporophytes, pollen and microspores, each of the above containing a gene/nucleic acid of interest.

Plants particularly useful in the methods of the present invention include all plants that belong to the subkingdom Viridiplantae, in particular monocotyledons and dicotyledons, including forage plants or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list, among other things, including

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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., Asparagus officinalis, Avena spp. (e.g. Avena saliva, Avena fatua, Avena byzantina, Avena fatua var. saliva, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, olive turnip]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., 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 (e.g. Elaeis guineensis, Elaeis oleifera ), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., 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. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum , Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, 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, carrots, cauliflower

- 6 044296 empty, celery, collards, flax, cabbage, lentils, oilseed rape, okra, onions, potatoes, rice, soybeans, strawberries, sugar beets, sugar cane, sunflowers, tomatoes, pumpkins, tea and seaweed, in addition other things. According to a preferred embodiment of the present invention, the plant is a crop. Examples of crops include soybeans, sunflowers, canola, alfalfa, canola seeds, cotton, tomatoes, potatoes or tobacco. More preferably, the plant is a monocot such as sugar cane. More preferably, the plant is a grain crop such as rice, corn, wheat, barley, millet, rye, sorghum or oats.

In general, the term herbicide in the present invention refers to an ingredient that kills a plant, controls or in any way interferes with the growth of a plant. The preferred amount or concentration of the herbicide is an effective amount or effective concentration. By effective amount or effective concentration is meant an amount or concentration, respectively, sufficient to kill or inhibit the growth of a similar, wild-type plant, plant tissue, plant cell, or host cell, but the amount does not kill or similarly inhibit the growth of plants. plant tissues, plant cells and host cells resistant to the herbicides described in the present invention. Also, the effective amount of the herbicide is the amount that is typically used in agricultural production systems to kill the weeds of interest. Specialists are well aware of this quantity. The herbicides useful in the present invention are active when applied directly to the plant or to the site of the plant at any stage of growth or before planting or germination. The effect depends on the type of plant, stage of growth, applied solution parameters and droplet size when spraying, particle size of solid components, external conditions during use, specific composition used, auxiliaries and carriers, type of soil, etc., as well as the amount used chemical substances. These and other factors can be adjusted in a manner known to those skilled in the art to cause selective or non-selective herbicide activity. Typically, PPI (pre-plant application), PPSA (post-plant surface application), pre-emergence or post-emergence herbicide treatments can be used. To obtain maximum weed control, post-emergence treatments typically target unwanted plants that have just emerged.

By herbicide-resistant or herbicide-resistant plants, it is meant that the plants are resistant or resistant to at least one herbicide that, in a given amount, would typically kill or inhibit the growth of common or wild plants. One skilled in the art will know the amount of herbicide that typically causes stunting of a fragile plant and can easily determine it. The examples indicate the quantities recommended by manufacturers for use. The maximum level is an example of an amount of herbicide that will stunt the growth of a non-resistant plant. For purposes of the present invention, the terms herbicide-resistant and herbicide-resistant are used interchangeably; they are assumed to have the same meaning and equivalent volume. Likewise, for the purposes of the present invention, the terms herbicide resistance and herbicide resistance are used interchangeably; they are assumed to have the same meaning and equivalent volume. Likewise, for the purposes of the present invention, the terms stable and persistent are used interchangeably; they are assumed to have the same meaning and equivalent volume. As used herein, with respect to the herbicides useful in various embodiments of the present invention, the terms herbicide and the like refer to the agriculturally acceptable active ingredients (A.I.) of the herbicide known in the art. Likewise, terms such as fungicide, nematicide, pesticide and the like refer to other agriculturally acceptable active ingredients commonly used in the art.

When the terms herbicide-tolerant and herbicide-resistant are used in reference to a particular mutant enzyme or polypeptide, they refer to the ability of such enzyme or polypeptide to perform its physiological activity in the presence of that amount of A.I. a herbicide that, under normal conditions, inactivates or inhibits the activity of the wild-type (non-mutant) version of the specified enzyme or polypeptide. On the other hand, when used in particular with respect to the TriA enzyme, the term refers in particular to the ability to metabolize, and thereby inactivate, herbicides that inhibit cellulose biosynthesis, so-called cellulose biosynthesis inhibitors (CBIs). The term herbicide-resistant mutated TriA protein or herbicide-resistant mutated TriA protein means a cellulose protein that exhibits greater metabolic activity than the metabolic activity of wild-type TriA protein in the presence of at least one herbicide that typically affects on cellulose biosynthesis and at the concentration or dose of herbicide application, it usually inhibits cellulose biosynthesis. Moreover, the TriA activity of such wild-type or herbicide-resistant TriA mutated TriA protein may be referred to herein as activity

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TriA, herbicide-resistant or herbicide-resistant.

When used herein, the term recombinant to refer to a nucleic acid or polypeptide indicates that such material has been altered by human recombination technology, such as polynucleotide restriction and ligation, polynucleotide overlapping expansion, or genomic insertion or transformation. An open reading frame of a gene sequence is recombinant if the specific nucleotide sequence has been extracted from its natural context and cloned into any type of artificial nucleic acid vector. The term recombinant may also refer to an organism containing recombinant material, for example, a plant containing a recombinant nucleic acid may be considered a recombinant plant.

The term transgenic plant refers to a plant that contains a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated into the genome, such that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide can be inserted into the genome alone or as part of a recombinant expression cassette. As used herein, the term transgenic refers to any cell, cell line, callus, tissue, plant part, or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including transgenic organisms or cells originally so altered, as well as transgenic organisms or cells created by crossing or asexual reproduction from an original transgenic organism or cell. In some embodiments, a recombinant organism means a transgenic organism. As used herein, the term transgenic does not include genomic changes (chromosomal or extrachromosomal) resulting from conventional plant breeding methods (e.g., crossbreeding) or from naturally occurring phenomena such as, for example, self-fertilization, random cross-pollination, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.

As used herein, the term mutated refers to an organism or its DNA with change(s) in the biomolecular sequence of its native genetic material compared to the sequence of the genetic material of the corresponding organism or wild-type DNA in which the change(s) of genetic material were caused and/or bred by humans. Examples of human actions to produce a mutated organism or DNA include, but are not limited to, treatment with a chemical mutagen such as EMS and subsequent selection using herbicides; or treatment of plant cells with X-rays and subsequent selection using herbicides. Any method known in the art can be used to obtain the mutation. Methods for producing mutations can cause mutations at random positions in the genetic material, or at specific areas of the genetic material (i.e., site-directed mutagenesis techniques can be used), such as the use of geneplasty.

As used herein, a genetically modified organism (GMO) is an organism whose genetic characteristics have been altered by human action resulting in transfection, which results in the transformation of the target organism using genetic material from another organism or source organism or using synthetic or modified natural genetic material or an organism that is its descendant retaining the introduced genetic material. The source organism may be a different type of organism (for example, a GMO plant may contain bacterial genetic material) or the same type of organism (for example, a GMO plant may contain genetic material from another plant). When used throughout this document in relation to plants and other organisms, the terms recombinant, transgenic and GMO are considered synonymous and indicate the presence of genetic material or other source; rather, when used, the term mutated refers to plants or other organisms or the DNA of such plants or organisms that lack such transgenic material, but in which the naturally occurring genetic material has been mutated such that it differs from the material of the corresponding organism or wild-type DNA.

As used herein, the term wild type or corresponding wild type plant refers to the typical form of an organism or its genetic material in its normal form, as distinguished from, for example, mutated and/or recombinant forms. Likewise, by control cell or similar wild-type plant, plant tissue, plant cell, or host cell is meant a plant, plant tissue, plant cell, or host cell, respectively, that lacks herbicide resistance characteristics and/or a specific nucleotide as described in the present invention. The term wild type, therefore, does not imply that the plant, plant tissue, plant cell or other host cell does not have recombinant DNA in the genome, and/or does not have a herbicide tolerance characteristic other than those described herein.

As used throughout this document, the term descendant refers to a plant of any generation. In some embodiments, the offspring is a plant of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth generation.

- 8 044296

As used herein, the term progeny refers to the first generation plant.

The term seed includes seeds of all kinds, such as seeds, caryopsis, achenes, fruits, tubers, seedlings and the like. Unless otherwise noted, in the context of species of the genera Brassica and Sinapis, the term seed refers to the seeds themselves. For example, seeds may refer to seeds of transgenic plants or plants obtained through traditional breeding methods. Examples of traditional breeding methods may include crossbreeding, self-fertilization, backcrossing, embryo rescue, incrossing, outcrossing, inbreeding, selective breeding, asexual propagation and other traditional methods known to those skilled in the art.

Although embodiments of the invention provide examples of application to certain plants or plant species and hybrids thereof, the methods described herein that use herbicides can be applied to a variety of commercially valuable plants. Plant lines with herbicide resistance that can be used in accordance with the present invention as described can be used in methods of controlling weeds directly or indirectly, i.e. as herbicide-treated crops or as herbicide-resistant donor lines for development to produce other varietal and/or hybrid crops having that characteristic or characteristics. All such resulting varieties or hybrid crops containing the herbicide resistance hereditary characteristic or characteristics are referred to herein as the progeny or descendants of the herbicide resistant line(s). It can be said that such resulting plants have retained the herbicide resistance characteristics of the ancestral plant, i.e. this means that they have and express the ancestral genetic molecular components responsible for that characteristic.

In one aspect, the present invention provides a plant or plant part comprising a polynucleotide encoding a mutated TriA polypeptide, wherein expression of the polynucleotide confers herbicide resistance to the plants or plant parts.

In a preferred embodiment of the invention, the plant has previously been produced by a process comprising recombinantly preparing the plant by introducing and overexpressing a wild-type TriA transgene or a mutated TriA transgene in accordance with the present invention, as described in more detail below.

In yet another embodiment of the invention, the polynucleotide encoding the mutated TriA polypeptide contains the nucleotide sequence of SEQ ID NO: 1 or a variant or derivative thereof.

In accordance with other embodiments, the mutated TriA polypeptide of the present invention is a functional variant that has, over the entire length of the variant, at least about 80%, such as at least about 80%, 90%, 95%, 98% , 99% or more amino acid sequence identity to SEQ ID NO: 2.

In yet another embodiment, the mutated TriA polypeptide used in accordance with the present invention is a functional polypeptide fragment that has the amino acid sequence of SEQ ID NO: 2.

The TriA polynucleotide molecules and TriA polypeptides of the invention are intended to include polynucleotide molecules and polypeptides with a nucleotide or amino acid sequence substantially 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 "substantially identical" means, as used herein, a first amino acid or nucleotide sequence that contains a sufficient or minimum number of amino acid radicals or nucleotides identical or equivalent (e.g., with a similar side chain) to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have in common structural domain and/or overall functional activity.

In general, sequence identity refers to the extent to which two optimally aligned DNA or amino acid sequences are consistent across a component alignment window, such as nucleotides or amino acids. The concept of relative identical number for aligned segments of a test or reference sequence means the number of identical components in two aligned sequences divided by the total number of components in a segment of the reference sequence, i.e. the entire reference sequence or a smaller specified portion of the reference sequence. The identical percentage is the relative identical number multiplied by 100. Optimal sequence alignment for comparative region alignment is well known in the art and can be accomplished using tools such as the Smith and Waterman local homology algorithm, the Needleman-Wunsch homology alignment algorithm, and the Pearson similarity search method. -Lipman, and preferably using computerized support for these algorithms, such as GAP, BESTFIT, FASTA and TFASTA, available as part of the GCG software package. Wisconsin Package. (Accelrys Inc. Burlington, Mass.)

- 9 044296

Polynucleotides and oligonucleotides.

The term isolated polynucleotide, including DNA, RNA or a combination thereof, single-stranded or double-stranded, in sense or non-sense orientation or combinations thereof, dsRNA or other variants, should be understood to mean a polynucleotide that is, at least in part, separated from the polynucleotide sequences, with with which he is bound or attached in his natural state. Preferably, the isolated polynucleotide will be at least 60%, more preferably at least 75%, and most preferably 90% free from other components with which it is normally associated in nature. It is known that an isolated polynucleotide can be exogenous and present, for example, in a transgenic organism that does not naturally contain this polynucleotide. In addition, the terms polynucleotide, nucleic acid sequence, nucleotide sequence, nucleic acid, nucleic acid molecule are used interchangeably and refer herein to nucleotides, ribonucleotides and deoxyribonucleotides, or a combination thereof, in polymeric unbranched form of any length.

The term mutated TriA nucleic acid refers to a TriA nucleic acid that has a sequence mutated from a wild-type TriA nucleic acid and that confers increased herbicide resistance to the plant in which it is expressed. In addition, the term mutated melamine diamenase (mutated TriA) implies the replacement of an amino acid of the primary sequence of wild type No: 2 or a variant, derivative, homolog, ortholog or paralogue thereof with another amino acid. The expression mutated amino acid will be used below to define an amino acid that is replaced by another amino acid, thereby indicating the location of the mutation in the primary sequence of the protein.

In a preferred embodiment of the invention, the TriA nucleotide sequence encoding the mutated TriA includes the sequence of SEQ ID NO: 1 or a variant or derivative thereof.

In addition, those skilled in the art will understand that TriA nucleotide sequences include homologs, paralogs, and orthologues of SEQ ID NO: 1, as defined herein.

The term variant with respect to a sequence (eg, a polypeptide sequence or a nucleotide sequence, such as the transcription regulatory nucleotide sequence of the present invention) refers to substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that, due to degeneration of the genetic code, encode an identical amino acid sequence to the native protein. Such natural allelic variants can be identified using known molecular biology methods such as, for example, the polymerase chain reaction (PCR) method and the hybridization method. Variant nucleotide sequences also include nucleotide sequences obtained synthetically, such as sequences generated, for example, by site-directed mutagenesis and for open reading frames encoding the native protein containing the sequence SEQ ID NO: 2, as well as sequences encoding a polypeptide with amino acid substitutions relative to native protein, for example, the mutated TriA of the present invention, as described herein. In general, nucleotide sequence variants according to the invention will have at least 30, 40, 50, 60 and up to 70%, for example preferably 71%, 72%, 73%, 74%, 75%, 76%, 77% , 78% and up to 79%, in general, at least 80%, for example 81%-84%, at least 85%, for example 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, at least 94%, 95%, 96%, 97%, up to 98% and 99% sequence identity to the nucleotide sequence SEQ ID NO: 1. Percentage sequence identity of the polynucleotide is determined by using GAP analysis (Needleman and Wunsch, 1970) (GCG program) with deletion penalty = 5 and deletion penalty = 0.3. Unless otherwise specified, the target sequence is at least 45 nucleotides in length, and GAP analysis aligns two sequences over a region of at least 45 nucleotides in length. Preferably, the search sequence is at least 150 nucleotides in length, and two sequences are aligned using GAP analysis over a region of at least 150 nucleotides in length. More preferably, the target sequence is at least 300 nucleotides in length, and GAP analysis aligns two sequences over a region of at least 300 nucleotides in length. Even more preferably, GAP analysis aligns two sequences along their entire length.

Polypeptides.

By substantially purified polypeptide or purified polypeptide it is meant that it has been separated from one or more lipids, nucleic acids, other polypeptides or other contaminant molecules with which it is naturally associated. Preferably, the substantially purified polypeptide is at least 60% pure, more preferably

- 10 044296 is at least 75%, and most preferably 90% free from other components with which it is normally associated in nature. Those skilled in the art will recognize that the purified polypeptide may be a polypeptide produced by a recombinant process. The terms polypeptide and protein are generally used interchangeably herein and refer to a single chain of polypeptides that may or may not be modified by the addition of non-amino acid groups. It is contemplated that such polypeptide chains may combine with other polypeptides or proteins, or other molecules such as cofactors. The terms proteins and polypeptides as used herein also include variants, mutants, modifications, analogs and/or derivatives of the polypeptides of the invention as described herein.

Percent polypeptide identity is determined using GAP analysis (Needleman and Wunsch, 1970) (GCG program), which has a gap creation gap of 5 and a continuation deletion penalty of 0.3. The search sequence is at least 25 amino acids long, and GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the target sequence is at least 50 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 50 amino acids in length. More preferably, the target sequence is at least 100 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 100 amino acids in length. Even more preferably, the search sequence is at least 250 amino acids in length, and GAP analysis aligns the two sequences over a region of at least 250 amino acids in length. Even more preferably, GAP analysis aligns two sequences along their entire length.

With respect to the described polypeptide, it will be preferred that percentage identity values greater than those indicated above include preferred embodiments of the invention. Thus, where appropriate, given the minimum percentage identity figures, 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% identity with SEQ ID NO: 2.

By variant polypeptide is meant a polypeptide derived from a protein of SEQ ID NO: 2 by deleting (called truncation) or adding one or more amino acids to the N-terminal and/or C-terminal region of the native protein; by deleting (so-called truncation) or adding one or more amino acids at one or more regions of the native protein; or by replacing one or more amino acids at one or more regions of the native protein. Such variants can be obtained, for example, as a result of genetic polymorphism or manipulations performed by humans. Methods for such manipulations are well known to specialists.

Protein derivatives include peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein of interest and having similar biological and functional activity as the unmodified protein from which they are derived. Thus, unless specifically stated otherwise, the present invention includes functional variants and fragments of TriA polypeptides, and the nucleic acid molecules encoding them, regardless of the origin of the polypeptide and regardless of whether it occurs naturally. . Various assays can be used to determine the functionality of the TriA polypeptide. For example, a functional variant or fragment of a TriA polypeptide may be analyzed to determine its ability to impart detoxification to herbicides. For example, the rate of herbicide detoxification may be defined as the rate of catalysis sufficient to provide a measurable increase in herbicide tolerance in a plant or plant part containing a recombinant polynucleotide encoding a TriA polypeptide variant or fragment, wherein in the plant or plant part

- 11 044296 expresses the specified variant or fragment to about 0.5%, as an example: about 0.05-0.5%, about 0.1-0.4% and about 0.2-0.3% of total amount of cellular protein compared to a similarly treated control plant in which the specified variant or fragment is not expressed.

In a preferred embodiment, the mutated TriA polypeptide is a functional variant or fragment of melamine diamenase with the amino acid sequence set forth in SEQ ID NO: 2, wherein the functional variant or fragment has at least about 80% amino acid sequence identity with SEQ ID NO : 2.

In accordance with other embodiments of the invention, said functional variant or fragment has a herbicide detoxification rate, which can be defined as a rate of catalysis sufficient to provide a measurable increase in herbicide resistance in a plant or plant part containing a recombinant polynucleotide encoding said variant or fragment, wherein the plant or part of the plant expresses the specified variant or fragment to about 0.5% of the total amount of cellular protein compared to a similarly treated control plant that does not express the specified variant or fragment.

Protein homologues include peptides, oligopeptides, polypeptides, proteins and enzymes that have amino acid substitutions, deletions and/or insertions relative to the unmodified protein of interest and have similar biological and functional activity as the unmodified protein from which they are derived.

In addition, one skilled in the art will also appreciate that changes may be introduced into the nucleotide sequences of the invention by mutation, thereby altering the amino acid sequence of the encoded proteins without altering the biological activity of the proteins. Thus, for example, an isolated polynucleotide molecule encoding a TriA mutated polynucleotide having an amino acid sequence that differs from that provided in SEQ ID NO: 2 may be created by making one or more nucleotide substitutions, additions or deletions to the corresponding nucleotide sequence, in which one or more amino acid substitutions, additions or deletions will be made in the encoded protein. Mutations can be introduced using standard methods such as site-directed and PCR-mediated mutagenesis. Such variant nucleotide sequences are also included within the scope of the present invention. For example, preferably, conservative amino acid substitutions may be made at one or more predicted nonessential amino acid residues. A nonessential amino acid residue is a residue in a wild-type protein sequence that can be changed without changing biological activity, while an essential amino acid residue is a residue required for biological activity.

The term deletion refers to the removal of one or more amino acids from a protein.

The term insert refers to one or more amino acid residues that are introduced into a specified location in a protein. Insertions may include N- and/or C-terminal fusions, as well as insertions of single or multiple amino acids within a sequence. Typically, insertions within an amino acid sequence will be smaller than N- and C-terminal fusions, on the order of approximately 1-10 residues. Examples of N- and C-terminal fusion proteins or peptides include transcriptional activator binding or activation domain as used in the yeast two-hybrid system, phage coat proteins, histidine-6 marker, glutathione S-transferase marker, protein A, maltose binding protein, dihydrofolate reductase, Tag 100 epitope, c-mic epitope, FLAG® epitope, lacZ, CMP (calmodulin binding protein), HA epitope, protein C epitope and VSV epitope.

The term substitution means the replacement of amino acids in proteins with other amino acids with similar characteristics (such as similar hydrophobicity, hydrophilicity, antigenicity, tendency to form or destroy α-helical or β-sheet structures). Typically, amino acid residues are replaced by single residues, but can be grouped depending on the functional restrictions imposed on the polypeptide, and can vary from 1 to 10 amino acids; insertions typically range from 1 to 10 amino acid residues. By conservative amino acid substitution is meant a substitution in which an amino acid residue is replaced by another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been described in the literature in this field. These families include amino acids with base side chains (e.g., lysine, arginine, histidine), acid side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cystine), non-polar 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 for conserved amino acid residues or for amino acid residues located in a conserved motif. Tables of conservative substitutions are well known to those skilled in the art (for example, see

Creighton (1984) Proteins. W.H. Freeman and Company (Eds).

Amino acid substitutions, deletions and/or insertions can be carried out using peptide synthesis technologies well known in the art, for example, solid phase peptide synthesis, or by manipulation of recombinant DNA. Methods for manipulating DNA sequences to effect substitution, deletion and/or insertion of protein variants are well known in the art. For example, methods for creating mutants by substitution at predetermined DNA sites are known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, ON), site-directed mutagenesis using the QuickChange method (Stratagene, San Diego, CA), PCR mediated site-directed mutagenesis or other site-directed mutagenesis protocols.

Derivatives also include peptides, oligopeptides, polypeptides, which, when compared to the amino acid sequence of a natural form of a protein, such as the protein of interest, may include amino acid substitutions with unnatural amino acid residues, or additions of unnatural amino acid residues. Protein derivatives also include peptides, oligopeptides, polypeptides that include naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulfated, etc.) or unnaturally altered amino acid residues from the amino acid sequence of the natural form of the polypeptide. The derivative may also contain one or more non-amino acid substituents or attachments relative to the amino acid sequence from which it is derived, for example, a reporter molecule or other ligand covalently or non-covalently linked to the amino acid sequence, such as a reporter molecule that is linked to facilitating its detection, and unnaturally formed amino acid residues related to the amino acid sequence of the natural protein. In addition, derivatives also include fusions of the natural form of the protein with marker peptides such as FLAG, HIS6 or thioredoxin (see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).

Orthologs and paralogs include evolutionary concepts that describe the hereditary relationships of genes. Paralogues are genes of the same species, formed as a result of duplication of an ancestral gene; orthologs are genes from different organisms resulting from speciation that also originate from a common ancestral gene.

Variants, orthologs and paralogs of SEQ ID NO: 2 covered by the present invention are illustrated by, but are not limited to, the 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.

Those skilled in the art are well aware that paralogs and orthologs may have common domains containing suitable amino acid moieties at these sites, such as binding pockets for certain substrates or binding motifs for interaction with other proteins.

The term domain refers to a set of amino acids that are conserved at specific positions in the sequence alignment of evolutionarily related proteins. While amino acids at other positions may differ between homologs, amino acids that are highly conserved at specific positions indicate amino acids that are likely to be essential for protein structure, stability, or function. They are identified by a high degree of conservation in aligned sequences of a family of protein homologues and can be used as identifiers to determine whether a given polypeptide belongs to a previously identified family of polypeptides.

The term motif or consensus sequence refers to a short conserved region in the sequence of evolutionarily related proteins. Often, motifs are often highly conserved parts of domains, but they can also include only part of a domain or be located outside a conserved domain (if all the amino acids of the motif are outside this domain).

There are specialized databases for defining domains, for example, 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 sequence motifs and their 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., Ed. ., pp. 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1 ): 276-280 (2002)).

A toolkit for in silico analysis of protein sequences is available on the proteomics server ExPASy (Swedish Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for indepth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788( 2003) Domains or motifs can be determined using standard techniques such as sequence alignment.

Methods for aligning sequences for comparison are well known to those skilled in the art and include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the Needleman-Wunsch algorithm ((1970) J Mol Biol 48: 443-453) to find a global (i.e., sequence-spanning) alignment of two sequences, which maximizes the number of matches and minimizes the number of gaps. Using the BLAST method algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10), it calculates the percentage sequence identity and performs a statistical analysis of the similarity of two sequences. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). Homologues can be easily identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83) with default pairwise alignment parameters and the percentage calculation method (see Figure 1). Global percentage similarity and identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. July 10, 2003; 4:29. MatGAT: an application that generates similarity/identity matrices using sequences proteins or DNA). One skilled in the art will be aware that minor adjustments to the settings can be made to optimize the alignment between conserved motifs. In addition, individual domains can be used instead of full-length sequences when identifying homologues. The sequence identity value can be determined over the entire nucleotide or amino acid sequence, or selected domains, or conserved motif(s), using the programs mentioned above with default parameters. For local alignment, the Smith-Waterman algorithm is particularly useful (Smith T.F., Waterman M.S. (1981) J. Mol. Biol 147(1); 195-7).

The proteins of the invention can be modified in a variety of ways, including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are well known to specialists. For example, amino acid sequence variants can be produced through mutations in DNA. Methods of mutagenesis and changes in nucleotide sequences are well known to specialists; see, for example, Kunkel (1985) PNAS, 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; US Patent No. 4873192; Walker and Gaastra, ed. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.), incorporated herein by reference. Conservative substitutions, such as replacing one amino acid with another amino acid with similar properties, may be preferred.

Alternatively, variant nucleotide sequences can be generated by introducing mutations randomly along all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened to identify mutants encoding proteins that retain activity. For example, after mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined using standard assay methods.

The present inventors have discovered that by replacing one or more key amino acid residues of the TriA enzyme of SEQ ID NO: 2, for example, by using one of the methods described above to mutate the nucleic acids encoding TriA, resistance or resistance to certain herbicides can be significantly increased. Preferred replacements for mutated TriA are those that increase plant resistance to herbicides but do not significantly affect the biological activity of diamenase.

Accordingly, another object of the present invention relates to a mutated TriA polypeptide that contains the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, of key amino acid radicals that are replaced by any other amino acid.

Those skilled in the art will appreciate that amino acids located close to the positions of the following amino acids may also be substituted. Thus, in another embodiment of the invention, a variant of SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof comprises a mutated TriA wherein ±3, ±2 or ±1 amino acid positions from the key amino acid are replaced by any other amino acid.

Using methods well known in the art, a highly characteristic sequence pattern can be generated that can be used to search for other mutated TriA variants with the desired activity.

Searching for other variants of mutated TriA using the appropriate sequence pattern is also covered by the present invention. It will be obvious to a specialist that this

- 14 044296 a sequence pattern is not limited by the exact distance between two adjacent amino acid radicals of that pattern. Each distance between two adjacent radicals in said pattern can, for example, vary independently of each other and be up to ±10, ±5, ±3, ±2 or ±1 amino acid positions without significantly affecting the desired activity.

In addition, by using site-directed mutagenesis techniques, such as saturation mutagenesis (see, for example, Schenk, et al., Biospektrum. 03/2006, pp. 277-279), the inventors of the present invention have identified and created specific amino acid substitutions and combinations thereof which, when introduced into a plant through transformation and expression of a corresponding mutated TriA encoding nucleic acid, confer on said plant increased resistance or resistance to herbicides.

Thus, in a particularly preferred embodiment of the invention, a mutated TriA variant or derivative refers to a TriA polypeptide comprising the sequence of SEQ ID NO: 2, an orthologue, paralog or homolog thereof, wherein the amino acid sequence differs from the amino acid sequence of the wild type TriA polypeptide at one or more positions , corresponding to the following sequence positions SEQ ID NO: 2: 69, 70, 71, 74, 82, 84, 85, 87, 88, 89, 91, 92, 93, 96, 126, 128, 129, 130, 131, 155 , 157, 160, 167, 170, 174, 180, 182, 216, 217, 219, 220, 246, 247, 248, 249, 250, 251, 298, 301, 302, 304, 328.

Examples of differences in specified amino acid positions include, but are not limited to, one or more of the following amino acids:

the amino acid corresponding to position 69 is replaced by any other amino acid;

the amino acid corresponding to position 70 is replaced by any other amino acid;

the amino acid corresponding to position 71 is replaced by any other amino acid; the amino acid corresponding to position 74 is replaced by any other amino acid; the amino acid corresponding to position 82 is replaced by any other amino acid; the amino acid corresponding to position 84 is replaced by any other amino acid; the amino acid corresponding to position 85 is replaced by any other amino acid; the amino acid corresponding to position 87 is replaced by any other amino acid; the amino acid corresponding to position 88 is replaced by any other amino acid; the amino acid corresponding to position 89 is replaced by any other amino acid; the amino acid corresponding to position 91 is replaced by any other amino acid; the amino acid corresponding to position 92 is replaced by any other amino acid; the amino acid corresponding to position 93 is replaced by any other amino acid; the amino acid corresponding to position 96 is replaced by any other amino acid; the amino acid corresponding to position 126 is replaced by any other amino acid; the amino acid corresponding to position 128 is replaced by any other amino acid; the amino acid corresponding to position 129 is replaced by any other amino acid; the amino acid corresponding to position 130 is replaced by any other amino acid; the amino acid corresponding to position 131 is replaced by any other amino acid; the amino acid corresponding to position 155 is replaced by any other amino acid; the amino acid corresponding to position 157 is replaced by any other amino acid; the amino acid corresponding to position 160 is replaced by any other amino acid; the amino acid corresponding to position 167 is replaced by any other amino acid; the amino acid corresponding to position 170 is replaced by any other amino acid; the amino acid corresponding to position 174 is replaced by any other amino acid; the amino acid corresponding to position 180 is replaced by any other amino acid; the amino acid corresponding to position 182 is replaced by any other amino acid; the amino acid corresponding to position 216 is replaced by any other amino acid; the amino acid corresponding to position 217 is replaced by any other amino acid; the amino acid corresponding to position 219 is replaced by any other amino acid; the amino acid corresponding to position 220 is replaced by any other amino acid; the amino acid corresponding to position 246 is replaced by any other amino acid; the amino acid corresponding to position 247 is replaced by any other amino acid; the amino acid corresponding to position 248 is replaced by any other amino acid; the amino acid corresponding to position 249 is replaced by any other amino acid; the amino acid corresponding to position 250 is replaced by any other amino acid; the amino acid corresponding to position 251 is replaced by any other amino acid; the amino acid corresponding to position 298 is replaced by any other amino acid; the amino acid corresponding to position 301 is replaced by any other amino acid; the amino acid corresponding to position 302 is replaced by any other amino acid; the amino acid corresponding to position 304 is replaced by any other amino acid; the amino acid corresponding to position 328 is replaced by any other amino acid;

Examples of differences in specified amino acid positions include, but are not limited to, one

- 15 044296 or more of the following amino acids:

the amino acid corresponding to position 69 is different from Valine; the amino acid corresponding to position 70 is different from Asparagine; the amino acid corresponding to position 71 is different from

Glutamine; the amino acid corresponding to position 74 is different from Leucine; the amino acid corresponding to position 82 is different from Arginine;

the amino acid corresponding to position 84 is different from Leucine; the amino acid corresponding to position 85 is different from Tyrosine; the amino acid corresponding to position 87 is different from Tryptophan; the amino acid corresponding to position 88 is different from Leucine; the amino acid corresponding to position 89 is different from Phenylalanine; the amino acid corresponding to position 91 is different from Valine; the amino acid corresponding to position 92 is different from Leucine; the amino acid corresponding to position 93 is different from Tyrosine; the amino acid corresponding to position 96 is different from Glutamine; the amino acid corresponding to position 126 is different from Asparagine; the amino acid corresponding to position 128 is different from Aspartic acid;

the amino acid corresponding to position 129 is different from Serine; the amino acid corresponding to position 130 is different from Alanine; the amino acid corresponding to position 131 is different from Isoleucine; the amino acid corresponding to position 155 is different from Methionine; the amino acid corresponding to position 157 is different from Phenylalanine; the amino acid corresponding to position 160 is different from Methionine; the amino acid corresponding to position 167 is different from Tyrosine; the amino acid corresponding to position 170 is different from Alanine; the amino acid corresponding to position 174 is different from Lysine; the amino acid corresponding to position 180 is different from Leucine; the amino acid corresponding to position 182 is different from Serine; the amino acid corresponding to position 216 is different from Alanine; the amino acid corresponding to position 217 is different from Isoleucine; the amino acid corresponding to position 219 is different from Proline; the amino acid corresponding to position 220 is different from Alanine; the amino acid corresponding to position 246 is different from Glutamate; the amino acid corresponding to position 247 is different from Serine; the amino acid corresponding to position 248 is different from Aspartate; the amino acid corresponding to position 249 is different from Histidine; the amino acid corresponding to position 250 is different from Aspartate; the amino acid corresponding to position 251 is different from Glutamate; the amino acid corresponding to position 298 is different from Glutamine;

the amino acid corresponding to position 301 is different from Serine; the amino acid corresponding to position 302 is different from Asparagine; the amino acid corresponding to position 304 is different from Tyrosine; the amino acid corresponding to position 328 is different from Aspartate;

In a preferred embodiment of the invention, the mutated TriA contains the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, in which:

the amino acid corresponding to position 70 is Thr, Cys, Gly, Val or Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Thr, Val, Gly, Cys or Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Val, and the amino acid corresponding to position 71 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Val, and the amino acid corresponding to position 71 is Thr.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Thr, and the amino acid corresponding to position 71 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Thr, and the amino acid corresponding to position 71 is Thr.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

- 16 044296 amino acid corresponding to position 155 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 128 is Gly, Pro or Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Val, and the amino acid corresponding to position 155 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Val, and the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 128 is Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 70 is Val, and the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 128 is Thr.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 157 is Thr, Ala, Met or Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 157 is Ala, and the amino acid corresponding to position 70 is Leu, and the amino acid corresponding to position 71 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 157 is Ala, and the amino acid corresponding to position 70 is Ile, and the amino acid corresponding to position 71 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 88 is Val, and the amino acid corresponding to position 92 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 88 is Val, and the amino acid corresponding to position 92 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 157 is Val, and the amino acid corresponding to position 88 is Val, and the amino acid corresponding to position 92 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 157 is Ala, and the amino acid corresponding to position 88 is Val, and the amino acid corresponding to position 92 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 96 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Val, and the amino acid corresponding to position 92 is Val, and the amino acid corresponding to position 155 is Ala, and the amino acid corresponding to position 157 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 128 is Ile, and the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 70 is Leu, and the amino acid corresponding to position 71 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Leu, and the amino acid corresponding to position 91 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Leu, and the amino acid corresponding to position 91 is Ala, and the amino acid corresponding to position 92 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Leu, and the amino acid corresponding to position 91 is Ala, and the amino acid corresponding to position 92 is Val, and the amino acid corresponding to position 88 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 157 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 157 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 69 is Ala, Leu or Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 74 is Val or Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 74 is Ala, and the amino acid corresponding to position 70 is Leu, and the amino acid corresponding to position 71 is Ile.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 82 is Leu, Met or Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 96 is Glu, Asp, Ala, Thr or Asn.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 126 is Ala, Met, Ser or Asp.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 128 is Ser, Ala or Asn.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 155 is Gly, Ala or Glu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 157 is Met or Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 167 is Ile, and the amino acid corresponding to position 88 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 167 is Ile, and the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 84 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 167 is Ile, and the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 84 is Thr.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 216 is Ser or Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 217 is Ala, Ser or Thr.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 219 is Gly, and the amino acid corresponding to position 249 is Asn.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 219 is Gly, and the amino acid corresponding to position 249 is Asn, and the amino acid corresponding to position 217 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 220 is Thr, Ser or Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 220 is Gly, and the amino acid corresponding to position 157 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 246 is Ser, Thr, Gln or Asp.

- 19 044296

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 247 is Ala, Asn, Val, Gly or Pro.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 248 is Ser, Asn or Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 249 is Val, Ile or Asn.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 250 is Glu or Asn.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 251 is Asp.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 251 is Asp, and the amino acid corresponding to position 248 is Glu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 298 is Cys, Asn, Thr or Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 301 is Ala, Thr or Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 302 is Glu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 304 is Lys.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 87 is Thr.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 88 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Phe.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 128 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 128 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 93 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 93 is Phe.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 128 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 88 is Ala, and the amino acid corresponding to position 128 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Phe, and the amino acid corresponding to position 128 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Phe, and the amino acid corresponding to position 128 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 157 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 157 is Val.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 71 is Asn, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 328 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 89 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 89 is Ala, and the amino acid corresponding to position 93 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 89 is Ala, and the amino acid corresponding to position 93 is Ala, and the amino acid corresponding to position 160 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Phe.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 89 is Ala, Val or Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 93 is Ala, Val, Leu or Phe.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 217 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 160 is Gly.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 250 is Ser.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 328 is Gly or Ala.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 129 and/or position 130 has been deleted.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 130 and/or position 131 has been deleted.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 170 and/or position 182 has been deleted.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 174 and/or position 180 has been deleted.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 89 is Ala, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 89 is Leu, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 85 is Leu, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu.

In yet another preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 85 is Leu, and the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Val.

In a particularly preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Val, and the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

In yet another particularly preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, in

- 23 044296 of which:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu, and the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

In yet another particularly preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu, and the amino acid corresponding to position 155 is Val, and the amino acid corresponding to position 157 is Leu.

In yet another particularly preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu, and the amino acid corresponding to position 157 is Leu.

In yet another particularly preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 71 is Asn, the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Phe, and the amino acid corresponding to position 96 is Thr, and the amino acid corresponding to position 128 is itself Gly.

In yet another particularly preferred embodiment of the invention, the mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 71 is Asn, the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu, and the amino acid corresponding to position 96 is Thr, and the amino acid corresponding to position 128 is itself Gly.

Other preferred embodiments of the invention, i.e. combinations of mutations are shown in the following table. 1.

Table 1

Combine tion amine oxide position in SEQ ID NO: 2 preferred substitution amine oxide position in SEQ ID NO: 2 preferred substitution amino acid position in SEQ ID NO: 2 Preferably substitution e amine oxide position in SEQ ID NO: 2 preferred substitution aminok islotnaya position in SEQID NO: 2 preferred substitution amine oxide position in SEQ ID NO: 2 preferred substitution amine oxide position in SEQ ID NO: 2 preferred substitution amine oxide position in SEQ ID NO: 2 preferred substitution amine oxide position in SEQ ID NO: 2 preferred substitution 1 Q71 V L92 V M155 A F157 A N70 V 2 Q71 V L92 V M155 A F157 A N70 I D128 I M155 V N70 L Q71 I F157 L 4 Q71 L V91 A L92 V L88 A F157 L 5 Q71 N L88 A L92 A Y93 L 1217 A 6 Q71 N L88 A L92 A Y93 F F157 L

- 24 044296

7 Q71 Ν L88 Α L92 Α Υ93 F D128 G 8 Q71 Ν L92 Α Υ93 F D128 G Q96 Τ 9 Y85 L F89 Α L92 Α Υ93 L 10 Y85 L L92 Α Υ93 L D250 S eleven Y85 L L92 Α Υ93 L D128 Α D250 S 12 L92 Α Υ93 L D250 S 13 L88 Α L92 Α Υ93 L 14 L88 Α L92 Α Υ93 V 15 Y85 L L88 Α L92 Α Υ93 L 16 Y85 L L88 Α L92 Α Υ93 V 17 Y85 L L88 Α L92 Α Υ93 L D128 G 18 Y85 L L88 Α L92 Α Υ93 V D128 G 19 Y85 L L88 Α L92 Α Υ93 L Q96 Τ D128 G 20 Y85 L L88 Α L92 Α Υ93 V Q96 Τ D128 G 21 Y85 L L88 Α F89 Α L92 Α Υ93 L Q96 Τ D128 G 22 Y85 L L88 Α F89 Α L92 Α Υ93 V Q96 Τ D128 G 23 Q71 Ν L92 Α Υ93 L Q96 Τ D128 G 24 L92 Α Υ93 L Q96 Τ D128 G

-25 044296

25 L92 A Y93 V 26 L92 A Y93 I — 27 L92 A Y93 V M155 T F157 L — 28 L92 A Y93 L M155 T F157 L — 29 L92 A Y93 L F157 L — thirty L88 A L92 A Y93 L F157 L — 31 W87 F L92 A Y93 L — 32 W87 N L92 A Y93 L — 33 W87 A L92 A Y93 L — 34 Y85 L L88 A F89 A L92 A Y93 L Q96 T D128 G M155 V F157 L 35 Y85 L L88 A F89 A L92 A Y93 V Q96 T D128 G M155 V F157 L

Those skilled in the art will be aware of how to identify conserved regions and motifs common to homologs, orthologs, and paralogs encoded by SEQ ID NO: 1. Once such conserved regions have been identified that may constitute suitable binding motifs, amino acids can be selected that can be substituted for any another amino acid, for example, conservative amino acids, preferably the amino acid substitutions described above using SEQ ID NO: 2 as a reference.

Another object of the present invention relates to a method for identifying a nucleotide sequence encoding a mutated TriA that is persistent or resistant to a herbicide. The method includes the following steps:

a) obtaining a library of nucleic acids encoding mutated TriA,

b) screening a population of resulting nucleic acids encoding the mutated TriA by expressing each said nucleic acid in a cell or plant and treating the cell or plant with a herbicide,

c) comparing the levels of herbicide resistance that are obtained from a given population of nucleic acids encoding mutated TriA with the levels of herbicide resistance that are obtained from a control nucleic acid encoding TriA,

d) selecting at least one nucleic acid encoding a mutated TriA that exhibits a significantly increased level of herbicide resistance compared to a control nucleic acid encoding TriA.

Levels of herbicide resistance can also be determined by measuring the rate of detoxification in a cell, tissue or plant.

Detoxification rate is the rate at which a herbicide breaks down over a specified period of time in the tissue concerned. Decomposition and product formation can be determined analytically, for example, by liquid chromatography (LC) coupled with the use of a high resolution (HR) mass spectrometer (MS). The product can be determined by comparison with the original standard and/or by elucidating the structure.

In a preferred embodiment of the invention, the nucleic acid encoding the mutated TriA and selected in step d) exhibits resistance and resistance of the cell or plant to the herbicide that is at least twice that of the control nucleic acid encoding TriA.

In a more preferred embodiment of the invention, the nucleic acid encoding the mutated TriA selected in step d) indicates that the resistance or resistance of the cell or plant to the herbicide is at least 2-fold higher, or at least 2-fold higher. 5 times, at least 10 times, at least 20 times, at least 50 times, at least 100 times, at least 500 times compared to the stability provided by the control nucleic acid encoding TriA.

Persistence or resistance can be determined by obtaining a transgenic plant or host cell, preferably a plant cell, with the nucleic acid sequence from the library in step a), and by comparing this transgenic plant with a control plant or host cell, preferably a plant cell.

Methods are well known to those skilled in the art to obtain suitable nucleic acid variants for identifying the nucleotide sequence encoding the mutated TriA from a variety of different potential parent organisms, including microbes, plants, fungi, algae, mixed crops, etc., as well as from natural sources. DNA, such as soil. These methods include, but are not limited to, the preparation of cDNA or genomic DNA libraries, the use of suitable degenerate oligonucleotide primers, the use of probes based on known sequences or structural complementation analyzes (eg, tyrosine-induced growth), and the use of mutagenesis and mixing to produce recombinant or mixed sequences encoding mutated TriA.

Nucleic acids comprising variant or control sequences encoding TriA may be expressed in yeast, a bacterial host strain, algae, or higher plants such as tobacco or Arabidopsis, and are tested for relative levels of hereditary stability of the sequences encoding TriA based on visual determining the phenotype of a transformed strain or plant in the presence of different concentrations of the selected herbicide. Dose-response data or changes in dose effect associated with such indicator phenotypes (brown leaf color, plant growth inhibition, herbicide exposure, etc.) are conveniently expressed as GR50 values (the concentration at which growth rate plants is reduced by 50%) or MIC (minimum inhibitory concentration), where increasing values correspond to an increase in the innate resistance of the expressed TriA. For example, in a rapid assay system based on the transformation of a bacterium such as E. coli, each sequence encoding the mutated TriA can be expressed, for example, as a DNA sequence under the expression control of a controlled promoter, such as the lacZ promoter, taking into account such indicators (eg , using synthetic DNA) as the frequency of codon usage, to obtain the expression level as similar as possible for different TriA sequences. Such strains expressing nucleic acids including alternative TriA sequence variants can be inoculated at various concentrations of the selected herbicide in addition to tyrosine media, and the relative levels of heritable resistance of the TriA expressed enzymes can be determined based on the extent and minimum inhibitory concentration (MIC). inhibition of the formation of brown ochronotic pigment.

In another embodiment of the invention, candidate amino acids are transformed into plant material to produce a transgenic plant, which is regenerated into morphologically normal fruit-bearing plants, which are then measured for differential resistance to selected herbicides as described in the Examples section above. Many suitable transformation methods are well known to those skilled in the art, using selection markers such as kanamycin, binary vectors such as vectors derived from Agrobacterium and plant regeneration such as tobacco leaf discs. Additionally, a control population of plants is similarly transformed with a nucleic acid expressing the control TriA. The average and distribution of herbicide resistance levels characterizing the results of the primary transformation of the plant or its progeny affected by the herbicides described above are calculated in the usual manner, taking into account plant damage, meristem bleaching symptoms, etc., at various herbicide concentrations. These data can be expressed as, for example, GR50 values obtained from a dose-response curve, where the dose value is marked on the x-axis and the y-axis is the percentage of dead plants, the effect of the herbicide, the number of young shoots, etc. where an increase in GR50 values corresponds to an increase in the level of innate resistance of expressed TriA. Herbicides can be applied before or after plant emergence.

Another aspect of the present invention relates to an isolated, recombinant and/or chemically synthesized nucleic acid encoding a mutated TriA as described above, which nucleic acid may contain the nucleotide sequences of SEQ ID NO: 1 or a variant or derivative thereof.

Examples of differences in specified amino acid positions include, but are not limited to, one or more of the following amino acids:

the amino acid corresponding to position 69 is replaced by any other amino acid;

the amino acid corresponding to position 70 is replaced by any other amino acid;

- 27 044296 the amino acid corresponding to position 71 is replaced by any other amino acid;

the amino acid corresponding to position 74 is replaced by any other amino acid;

the amino acid corresponding to position 82 is replaced by any other amino acid;

the amino acid corresponding to position 84 is replaced by any other amino acid;

the amino acid corresponding to position 85 is replaced by any other amino acid;

the amino acid corresponding to position 87 is replaced by any other amino acid;

the amino acid corresponding to position 88 is replaced by any other amino acid;

the amino acid corresponding to position 89 is replaced by any other amino acid;

the amino acid corresponding to position 91 is replaced by any other amino acid;

the amino acid corresponding to position 92 is replaced by any other amino acid;

the amino acid corresponding to position 93 is replaced by any other amino acid;

the amino acid corresponding to position 96 is replaced by any other amino acid;

the amino acid corresponding to position 126 is replaced by any other amino acid;

the amino acid corresponding to position 128 is replaced by any other amino acid;

the amino acid corresponding to position 129 is replaced by any other amino acid;

the amino acid corresponding to position 130 is replaced by any other amino acid;

the amino acid corresponding to position 131 is replaced by any other amino acid;

the amino acid corresponding to position 155 is replaced by any other amino acid;

the amino acid corresponding to position 157 is replaced by any other amino acid;

the amino acid corresponding to position 160 is replaced by any other amino acid;

the amino acid corresponding to position 167 is replaced by any other amino acid;

the amino acid corresponding to position 170 is replaced by any other amino acid;

the amino acid corresponding to position 174 is replaced by any other amino acid;

the amino acid corresponding to position 180 is replaced by any other amino acid;

the amino acid corresponding to position 182 is replaced by any other amino acid;

the amino acid corresponding to position 216 is replaced by any other amino acid;

the amino acid corresponding to position 217 is replaced by any other amino acid;

the amino acid corresponding to position 219 is replaced by any other amino acid;

the amino acid corresponding to position 220 is replaced by any other amino acid;

the amino acid corresponding to position 246 is replaced by any other amino acid;

the amino acid corresponding to position 247 is replaced by any other amino acid;

the amino acid corresponding to position 248 is replaced by any other amino acid;

the amino acid corresponding to position 249 is replaced by any other amino acid;

the amino acid corresponding to position 250 is replaced by any other amino acid;

the amino acid corresponding to position 251 is replaced by any other amino acid;

the amino acid corresponding to position 298 is replaced by any other amino acid;

the amino acid corresponding to position 301 is replaced by any other amino acid;

the amino acid corresponding to position 302 is replaced by any other amino acid;

the amino acid corresponding to position 304 is replaced by any other amino acid;

the amino acid corresponding to position 328 is replaced by any other amino acid;

In a preferred embodiment of the invention, the encoded mutated TriA is a variant of SEQ ID NO: 2, which includes one or more of the following amino acids:

the amino acid corresponding to position 69 is different from Valine;

the amino acid corresponding to position 70 is different from Asparagine;

the amino acid corresponding to position 71 is different from Glutamine;

the amino acid corresponding to position 74 is different from Leucine;

the amino acid corresponding to position 82 is different from Arginine;

the amino acid corresponding to position 84 is different from Leucine;

the amino acid corresponding to position 85 is different from Tyrosine;

the amino acid corresponding to position 87 is different from Tryptophan;

the amino acid corresponding to position 88 is different from Leucine;

the amino acid corresponding to position 89 is different from Phenylalanine;

the amino acid corresponding to position 91 is different from Valine;

the amino acid corresponding to position 92 is different from Leucine;

the amino acid corresponding to position 93 is different from Tyrosine;

the amino acid corresponding to position 96 is different from Glutamine;

the amino acid corresponding to position 126 is different from Asparagine;

the amino acid corresponding to position 128 is different from Aspartic acid;

the amino acid corresponding to position 129 is different from Serine;

the amino acid corresponding to position 130 is different from Alanine;

the amino acid corresponding to position 131 is different from Isoleucine;

the amino acid corresponding to position 155 is different from Methionine;

- 28 044296 amino acid corresponding to position 157 is different from Phenylalanine;

the amino acid corresponding to position 160 is different from Methionine;

the amino acid corresponding to position 167 is different from Tyrosine;

the amino acid corresponding to position 170 is different from Alanine;

the amino acid corresponding to position 174 is different from Lysine;

the amino acid corresponding to position 180 is different from Leucine;

the amino acid corresponding to position 182 is different from Serine;

the amino acid corresponding to position 216 is different from Alanine;

the amino acid corresponding to position 217 is different from Isoleucine;

the amino acid corresponding to position 219 is different from Proline;

the amino acid corresponding to position 220 is different from Alanine;

the amino acid corresponding to position 246 is different from Glutamate;

the amino acid corresponding to position 247 is different from Serine;

the amino acid corresponding to position 248 is different from Aspartate;

the amino acid corresponding to position 249 is different from Histidine;

the amino acid corresponding to position 250 is different from Aspartate;

the amino acid corresponding to position 251 is different from Glutamate;

the amino acid corresponding to position 298 is different from Glutamine;

the amino acid corresponding to position 301 is different from Serine;

the amino acid corresponding to position 302 is different from Asparagine;

the amino acid corresponding to position 304 is different from Tyrosine;

the amino acid corresponding to position 328 is different from Aspartate.

In a particularly preferred embodiment of the invention, the mutated TriA encoded by the nucleic acid of the present invention contains a variant of SEQ ID NO: 2 in which the amino acids at positions corresponding to positions 92, 93, 155 and 157 of SEQ ID NO: 2 are replaced by any other amino acid .

In a particularly preferred embodiment of the invention, the encoded mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homolog thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Val, and the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

In yet another particularly preferred embodiment of the invention, the encoded mutated TriA comprises the sequence SEQ ID NO: 2, a variant, derivative, ortholog, paralog or homologue thereof, wherein:

the amino acid corresponding to position 92 is Ala, and the amino acid corresponding to position 93 is Leu, and the amino acid corresponding to position 155 is Thr, and the amino acid corresponding to position 157 is Leu.

In accordance with other aspects, the present invention includes progeny or progenies of a plant having resistance to the herbicides of the present invention, as well as seeds and cells that are obtained from plants having resistance to the herbicides of the present invention.

In some embodiments, the present invention provides a progeny or progeny of a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein said progeny or descendants of the plant, in at least some of their cells, contain a recombinant polynucleotide operably linked to a promoter, wherein expression of the mutated TriA polypeptide imparts resistance to herbicides to said progeny or descendants of the plant.

In accordance with one embodiment of the invention, the seeds of the present invention preferably have the herbicide resistance characteristics of said herbicide-resistant plant. In accordance with other embodiments of the invention, a seed is capable of developing into a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, at In this case, the expression of the mutated TriA polypeptide imparts resistance to herbicides to the said plant.

In some embodiments, a plant or part of a plant can be regenerated from plant cells of the present invention. In other embodiments, the plant or plant part cannot be regenerated from the plant cells of the present invention. Examples of cells from which a plant cannot be regenerated include, but are not limited to, endosperm, seed

- 29 044296 shell (testa and pericarp) and root cap.

In yet another embodiment, the present invention provides a plant cell, or a cell from which a plant can be regenerated, that in at least some of its cells contains a polynucleotide operably linked to a promoter functional in plant cells, wherein the promoter is capable of expressing the polypeptide TriA mutated TriA encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts herbicide resistance to said plant, wherein said plant cell contains a recombinant polynucleotide operably linked to a promoter.

In accordance with other embodiments of the invention, the present invention provides a plant cell containing a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide renders said cell resistant to herbicides.

In yet another embodiment of the invention, the invention relates to a plant cell transformed with a nucleic acid encoding a TriA mutated polypeptide in accordance with the present invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or resistance to a herbicide compared to a wild-type plant cell . Preferably, the nucleic acid encoding the TriA mutated polypeptide contains a polynucleotide sequence selected from the group consisting of: a) a polynucleotide as shown in SEQ ID NO: 1, or a variant or derivative thereof; b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO: 2, or a variant or derivative thereof; c) a polynucleotide containing at least 60 contiguous nucleotides of any of a) or b); and d) a polynucleotide adjacent to a polynucleotide of any of items a) to c).

In accordance with certain aspects, the present invention provides a plant product derived from herbicide-tolerant plants. In some embodiments, examples of plant products include, but are not limited to, grains, oils, and flours. In accordance with one embodiment of the invention, the plant product is a plant grain (for example, grain suitable for consumption or processing), vegetable oil (for example, oil suitable for human consumption or for the production of biodiesel fuel) or vegetable flour (for example, flour suitable for human consumption).

In one embodiment, a plant product is provided derived from a plant or part of a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said plant cell. polynucleotide, wherein expression of the mutated TriA polypeptide confers herbicide resistance to said plant or plant part.

In yet another embodiment of the invention, the invention relates to a method for producing a transgenic plant cell with increased herbicide resistance compared to a wild-type plant cell, including transforming the plant cell with an expression cassette polynucleotide operably linked to a promoter functioning in plant cells, a promoter that is capable of express the mutated TriA polynucleotide-encoded polypeptide.

In yet another embodiment, the invention relates to a method for producing a transgenic plant, comprising: (a) transforming a plant cell with an expression cassette containing a polynucleotide operably linked to a promoter functioning in plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, and (b) obtaining from a plant cell a plant with increased resistance to the herbicide.

In accordance with some aspects, the present invention provides a method for producing plants that are resistant to herbicides. In one embodiment, said method comprises regenerating a plant from a plant cell transformed using a polynucleotide operably linked to a promoter operating in the cell, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts to said plant herbicide resistance.

The term expression/expression or expression of genes means the transcription of a specific gene or genes or genetic construct. The term expression or expression of genes, in particular, means the transcription of a specific gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA, followed by translation of the latter into protein or without translation. This process involves transcription of DNA and processing of the resulting mRNA product.

To obtain the desired effect, i.e. plants resistant or resistant to the herbicides described in the present invention, at least one nucleic acid is overexpressed by methods and techniques known to those skilled in the art.

- 30 044296

The term overexpression or overexpression, as used herein, means any form of expression that is added to the level of wild-type expression. Methods for increasing the expression of genes or gene products are described in documents in the art and include, for example, overexpression facilitated by appropriate promoters, transcriptional or translational enhancers. Isolated nucleic acids that serve as promoters or enhancers can be introduced into the appropriate position (usually upstream of transcription) of a non-heterologous form of the polynucleotide to up-regulate the expression of the nucleic acid encoding the target polypeptide. For example, endogenous promoters can be modified in vivo by mutation, deletion and/or substitution (see Kmiec, US 5565350; Zarling et al., WO9322443), or isolated promoters can be introduced into the plant cell in the appropriate direction and at the appropriate distance from the gene of the present invention to control gene expression. If expression of a polypeptide is desired, it is desirable to include a polyadenyl region at the 3' end of the polynucleotide coding region. The polyadenyl region can be obtained from a natural gene, a set of other plant genes, or T-DNA. The added 3'end sequence can be obtained, for example, from the nopaline synthase or octopine synthase genes, or also from another plant gene, or, less preferably, from any other eukaryotic gene. To increase the amount of ready information that accumulates in the cytosol, an intronic sequence can also be added to the 5' untranslated region (UTR) or coding sequence of a partial coding sequence. Incorporation of a binding intron into a transcription element in both plant and animal expression constructs increased gene expression at the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 43954405; Callis et al. (1987) ) Genes Dev 1:1183–1200). This intronic increase in gene expression is usually greatest near the 5' end of the transcription element. The use of corn introns Adhl-S intron 1, 2, and 6, intron Bronze-1 are known to those skilled in the art. For general information, see The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

If necessary, nucleotide sequences can be optimized for increased expression in the transformed plant. For example, coding sequences may be provided that contain plant-preferred codons to enhance expression in the plant; see, for example, Campbell and Gowri (1990) Plant Physiol., 92: 1-11 for a discussion of the use of preferential base codons. Methods for obtaining plant-preferred genes are also known to those skilled in the art; see, for example, US Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.

Therefore, the wild-type TriA/TriA mutated nucleic acids of the invention are presented in expression cassettes for expression in the target plant. The cassette will include regulatory sequences operably linked to the mutated TriA nucleotide sequence of the invention. The term regulatory element as used herein refers to a polynucleotide capable of regulating the transcription of an operably linked polynucleotide. It includes, but is not limited to, promoters, enhancers, introns, 5' UTR and 3' UTR. By operably linked is meant a functional relationship between a promoter and a second sequence, wherein the promoter sequence initiates and promotes transcription of a DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleotide sequences, when linked, are contiguous and, if necessary, combine two protein coding regions adjacent and in the same reading frame. The cassette may also contain at least one additional gene for co-transformation into the body. Alternatively, additional gene(s) may be present in multiple expression cassettes.

Such an expression cassette is provided with multiple restriction sites for insertion of a wild-type mutated TriA/TriA nucleic acid sequence that will be subject to transcriptional regulation by regulatory regions. The expression cassette may also contain a selectable marker gene. The expression cassette of the present invention will include, in the direction of transcription from the 5' to 3' end, a translation or transcription initiation region (i.e., a promoter), a wild-type TriA/TriA mutated encoding a nucleic acid sequence of the present invention, and a transcription termination region or translation (i.e., termination site) that is functional in plants. The promoter may be native or analogous, foreign or heterologous, to the host plant and/or to the wild-type TriA/TriA mutated nucleotide sequence of the invention. Also, the promoter may be a natural sequence or, alternatively, a synthetic sequence. When a promoter is foreign or heterologous to the host plant, it is implied that the promoter is not present in the native plant into which it was introduced. When a promoter is foreign or heterologous to the wild-type TriA/TriA mutated nucleic acid sequence of the present invention, it is understood that the promoter is not

- 31 044296 a native or natural promoter for an operably linked wild-type TriA/TriA mutated nucleic acid sequence of the present invention. As used herein, a chimeric gene contains a coding sequence operably linked to a transcription initiation site that is heterologous to the coding sequence. Since it is preferable to express the nucleic acids of the present invention with wild-type mutated TriA/TriA using heterologous promoters, native promoter sequences can be used. Such constructs will alter protein expression levels with wild-type TriA/TriA mutated in a plant or plant cell. Thus, the phenotype of the plant or its cell changes.

The termination site may be native to the transcription initiation site, native to the operably linked target sequence to the mutated wild-type TriA/TriA, native to the host plant, or may be derived from another source (i.e., may be foreign or heterologous to the promoter, target nucleic acid sequence with mutated TriA/TriA wild type, host plant, or a combination thereof). Suitable termination sites can be obtained from the Ti plasmid of the bacteria A. tumefaciens, such as octopine synthase and nopaline synthase termination sites; see also Guerineau et al. (1991) Mol. 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 et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627–9639. If desired, the gene or genes can be optimized for increased expression in the transformed plant. In other words, genes can be synthesized using plant-preferred codons to improve expression; see, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 discussion of the use of preferential base codons. Methods exist for synthesizing preferably plant genes; see, for example, US Pat. Nos. 5,380,831 and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, incorporated herein by reference.

Thus, the present invention provides an expression cassette comprising a TriA mutated nucleic acid molecule of the present invention as well as a promoter functional in plant cells.

In a preferred embodiment of the invention, the promoter is a root promoter or an enhanced root promoter.

In a particularly preferred embodiment of the invention, the promoter is a root promoter or an enhanced root promoter from Glycine max (eg p-Glyma04g34080, see examples 8 and 9).

Even more preferably, the promoter contains the nucleotide sequence of SEQ ID NO: 32.

Since the polynucleotides of the invention can be used as selectable marker genes for plant transformation, the expression cassettes of the invention can include another selectable marker gene for selecting transformed cells. Selectable marker genes, including the genes of the present invention, are used to select transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO) and hydromycin phosphotransferase (HPT), as well as genes encoding resistance to herbicidal compounds such as ammonium glufosinate, bromoxynil, imidazolinones and 2,4-dichlorophenoxyacetate (2,4-D); look, in general,

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) Mol 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)

- 32 044296

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) Mol Cell Biol 10:33433356; 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 Mol 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, volume 78 (Springer-Verlag, Berlin); Gill et al (1988) Nature 334:721–724.

This information is incorporated herein by reference. The list of marker genes listed above is not exhaustive. Any selectable marker gene can be used in the present invention.

It is also known that additional sequence modifications improve gene expression in the host cell. These include the removal of sequences encoding spurious polyadenylation signals, exon and intron splice site signals, transposon-like repeats, and other similarly defined sequences that may be disruptive to gene expression. The GC content of the sequence can be adjusted to levels average for the specified host cell, which is calculated using known genes expressed in the host cell. Also, if necessary, the sequences can be modified so that they do not contain predicted hairpin secondary mRNA structures. Nucleotide sequences to improve gene expression can also be used in plant expression vectors. These include, for example, the maize Adhl introns, Adhl gene introns 1, 2 and 6 (Callis et al., Genes and Development 1:1183-1200, 1987), and leading sequences, ^-sequence) from tobacco mosaic virus (TMV) , corn chlorospot virus (CMCV) 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 maize shriveled-1 locus shows increased gene expression in chimeric gene constructs. US Patent Nos. 5,424,412 and 5,593,874 describe the use of specific introns in gene expression constructs, and Gallie et al. (Plant Physiol. 106:929-939, 1994) also suggests that introns can be used to regulate gene expression on a tissue-specific basis. To further improve or optimize gene expression, the plant expression vectors of the present invention may also contain DNA sequences with MARs (matrix attachment sites) regions. Plant cells transformed with such modified expression systems may subsequently exhibit overexpression or constitutive expression of the nucleotide sequence of the invention.

The invention also provides an isolated recombinant expression vector comprising a wild-type TriA/TriA mutated nucleic acid expression cassette as described above, wherein expression of the vector in a host cell results in increased herbicide resistance compared to a wild-type host cell. In this context, the term vector refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is associated. One type of vector is a plasmid, which refers to double-stranded DNA loop segments that can be ligated into the viral genome. Another type of vector is a viral vector, where additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial replication origin and episomal vectors related to mammals). Other vectors (eg, non-episomal mammalian vectors) are integrated into the host cell genome upon introduction into the host cell and replicate along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors, in this context, are called expression vectors. Typically, the expression vectors used in recombinant DNA technology take the form of plasmids. As used herein, the terms plasmid and vector are used interchangeably, since plasmid is most often used in vector form. However, the invention also includes

- 33 044296 other forms of expression vectors, such as viral vectors (eg replication-defective retrovirus, adenoviruses and adeno-associated viruses) performing the same functions.

The recombinant expression vectors of the invention include the nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences selected based on the host cells to be used in the expression that functionally linked to the nucleotide sequence for expression. Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and sequences that direct constitutive expression of a nucleotide sequence only in certain host cells or under certain conditions. Those skilled in the art will recognize that the type of expression vector may depend on factors such as the choice of host cell for transformation, the level of expression of the desired polypeptide, etc. Expression vectors of the invention can be introduced into host cells to thereby produce polypeptides or peptides, including polypeptides or fusion peptides encoded by nucleic acids herein (eg, TriA mutated polypeptides, fusion polypeptides, etc.).

Expression vectors may also contain 5' leader sequences in the expression construct. Such leader sequences may improve translation. Translational leader sequences are well known to those skilled in the art and include: picornavirus leader sequences, for example, EMCV leader sequences (non-coding region of the 5' end of encephalomyocarditis) (Elroy-Stein et al. (1989) PNAS, 86:6126-6130); Potyvirus leader sequences, e.g. Tobacco etch virus (TEV) leader (Gallie et al. (1995) Gene 165(2):233-238), Maize dwarf mosaic virus (MDMV) leader sequences (Virology 154:9-20), and immunoglobulin heavy chain binding protein (BiP) (Macejak et al. (1991) Nature 353:90-94); an untranslated leader sequence from the viral protein of alfalfa mosaic virus mRNA (AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); the tobacco mosaic virus (TMV) leader sequence (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York), pp. 237-256); and the leader sequence of maize chlorine mottle virus (MCMV) (Lommel et al. (1991) Virology 81:382-385); see also Della-Cioppa et al. (1987) Plant Physiol. 84:965-968.

Other methods that improve translation, such as introns, etc., can also be used. When preparing an expression vector, various nucleic acid fragments can be modified to produce nucleotide sequences in the desired orientation and, if necessary, reading frame. To this end, adapters or linkers can be used to connect nucleic acid fragments or other manipulations to provide suitable restriction sites, remove excess nucleic acids, remove restriction sites, and the like. For these purposes, in vitro mutagenesis, primer recovery, restriction, renaturation, repeated replacement, for example, transitions and transversions can be used.

In the practice of the invention, several promoters can be used. Promoters can be selected depending on the desired outcome. Nucleic acids can be combined with constitutive, preferably tissue or other promoters for expression in plants.

Constitutive promoters include, for example, the Rsyn7 promoter core promoter and other constitutive promoters described in WO 99/43838 and US Pat. No. 6,072,050; CaMV 35S nuclear 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 (US patent No. 5659026), and similar promoters.

Other constitutive promoters include, for example, those described in US patent No. 5608149; 5608144; 5604121; 5569597; 5466785; 5399680; 5268463; 5608142 and 6177611.

Tissue-specific promoters can be used to target increased expression to a specific plant tissue. Such tissue-specific promoters include, but are not limited to, leaf, root, seed and stem specific promoters. Some examples of tissue-specific 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) VocNatl. Acad. ScL USA 90(20):9586-9590; and GuevaraGarcia et al. (1993) Plant J 4(3):495–505. Promoters can be modified, if necessary, to reduce expression.

In a preferred embodiment of the invention, the promoter is a root-specific or root-enhanced promoter.

In a particularly preferred embodiment of the invention, the promoter contains the nucleic acid sequence SEQ ID NO: 32.

In some embodiments, target nucleic acids can be directed to the chloroplast for expression. Thus, where the target nucleic acid is inserted indirectly into the chloroplast, the expression vector will further comprise a chloroplast targeting sequence containing a nucleotide sequence that encodes a chloroplast transit peptide for targeting the target gene product to the chloroplasts. Such transit peptides are well known to those skilled in the art. With respect to chloroplast-targeting sequences, operably linked means that the nucleotide sequence encoding the transit peptide (i.e., the chloroplast-targeting sequence) is linked to the target coding sequence of the invention such that the two sequences are contiguous and in the same reading frame ; see, for example, 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, any known chloroplast transit peptide can be linked to the amino acid sequence of a TriA polypeptide of the invention by operably linking a chloroplast-targeting sequence to the 5' end of a nucleotide sequence encoding said TriA polypeptide.

Chloroplast-targeting sequences are well known in the art and include the chloroplast subunit ribulose-1,5-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); see also 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.

Methods for transforming chloroplasts are well 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. The method is based on introducing DNA containing a selectable marker using a gene gun and targeting the DNA into the plastid genome by homologous recombination. Plastid transformation can also be accomplished by transactivation of a non-functioning transgene located in the plastid through tissue-specific expression of RNA polymerase encoded in the nucleus and directed to the plastid. Such a system is reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

Chloroplast-targeted nucleic acids can be optimized for expression in the chloroplast to account for differences in codon usage between the plant embryo and that organelle. Thus, target nucleic acids can be synthesized using chloroplast-preferred codons; see, for example, US Patent No. 5,380,831, incorporated herein by reference.

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Various transformation vectors and plant transformation methods are available; see for example

An, G. et al. (1986) Plant PysioL, 81:3O1-3O5; Fry, I, 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.2032\2.203-2\2;

Cousins, et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L.

(1992) Gene.l 1 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. 99:8188; 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, J. A. and Mchughen, A. (1993) Plant ScL 91: 139-148; Franklin, S. I.

and Trieu, T. N. (1993) Plant. Physiol. 102: 167; Golovkin, et al. (1993) Plant ScL 90:4152; Guo Chin ScL Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres

N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 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.

In some embodiments, the method of the invention includes introducing a polynucleotide construct into a plant. The term introduction means the introduction of a polynucleotide construct into a plant such that the construct gains access to the interior of the plant cell. The methods of the invention are independent of the particular method of introducing the polynucleotide construct into a plant, except that the introduction must be carried out in such a way that the polynucleotide construct gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known to those skilled in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The terms introduction and transformation as used herein refer to the transfer of an exogenous polynucleotide into a host cell, regardless of the mode of transfer. Plant tissue capable of further clonal propagation, by organogenesis or embryogenesis, can be transformed to regenerate from it the genetic construct of the present invention and the whole plant. The choice of a particular tissue will depend on the clonal propagation systems available and most suitable for transformation of a particular species. Target tissue samples include leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissues, existing meristem tissue (eg, apical meristem, axillary buds, and root meristems), and induced meristem tissue (eg, cotyledon meristem and hypocotyl meristem). The polynucleotide may be introduced into the host cell temporarily or permanently and may be maintained unchanged, for example, as a plasmid. Or it can be integrated into the main genome. The resulting transformed plant cell can be used to regenerate the transformed plant by methods known to those skilled in the art.

The term stable transformation means that when introduced into a plant, the polynucleotide construct is integrated into the plant genome and is able to be inherited by its descendants. The term transient transformation means that when introduced into a plant, the polynucleotide construct is not integrated into the plant genome.

To transform plants and plant cells, the nucleotide sequences of the invention are inserted using standard methods into any vector known to those skilled in the art that can be used to express the nucleotide sequences in a plant or plant cell. The choice of vector depends on the preferred transformation method and the target plant species being transformed. In one embodiment of the invention, the coding nucleotide sequence is operably linked to a plant promoter, for example, a promoter known to those skilled in the art as a promoter that provides high levels of expression in a plant cell, and the construct is then introduced into a plant cell that is subjected to

- 36 044296 exposure to herbicides, then the transformed plant is regenerated. In some embodiments, the transformed plant is resistant to a level of herbicide exposure that would kill or substantially damage the plant regenerated from a non-transformed cell. The present method can be applied to any plant species or crops.

In general, methodologies for constructing plant expression vectors and introducing foreign nucleic acids into a plant are known to those skilled in the art. For example, foreign DNA can be introduced into plants using tumor-inducing (Ti) plasmid vectors. Other methods used to introduce foreign DNA include the use of PEG-mediated protoplast transformation, electroporation, the use of microneedles for microinjection, and bioballistics or microparticle bombardment for direct DNA uptake. Such methods are known to those skilled in the art.

(US Patent No. 5,405,765 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, ed.) Academic Press, Inc. (1989).

Other suitable methods for introducing a nucleotide sequence into plant cells include, for example, Crossway et al. (1986) Biotechniques 4:320-334, electroporation as described, for example, by Riggs et al. (1986) Proc. Natl. Acad. ScL USA 83:5602-5606, Agrobacterium transformation as described, for example, Townsend et al., US Patent No. 5,563,055, Zhao et al., US Patent No. 5,981,840, direct gene transfer, as described, for example, Paszkowski et al. (1984) EMBO J. 3:2717-2722, ballistic acceleration of particles, as described, for example, in US patent No. 4945050; 5879918; 5886244 and 5932782;

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Tomes et al. (1995)

Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, in 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 see 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 (corn); US Patent Nos. 5,240,855; 5,322,783; and 5,324,646; Tomes et al., (1995) Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (corn);

Klein et al., (1988) Plant Physiol. 91:440-444 (corn); Fromm et al., (1990) Biotechnology 8:833-839 (corn); Hooykaas-Van Slogteren et al., (1984) Nature (London) 31 1:763-764; Bowen et al, US Patent 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 (piercing cells by shaking them in a microneedle suspension); D'Halluin et al., (1992) Plant Cell 4: 14951505 (electroporation); Li et al., (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 on tumor-forming Agrobacterium); each of which is incorporated herein by reference.

Transgenic plants, including transgenic crops, are preferably produced by Agrobacterium-dependent transformation. The preferred method of transformation is in planta transformation. For this purpose, it is possible to allow agrobacteria to act on the plant's seeds or to inoculate the plant meristem with agrobacteria. For the purposes of the present invention, it has been found particularly advantageous for the suspension of transformed Agrobacterium to act on the whole plant or on the flower primordium. The plant then develops to produce the seeds of the treated plant (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for transforming rice by Agrobacterium include known methods for transforming rice, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 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), these documents are incorporated herein by reference in their entirety. In the case of maize transformation, the method described either in Ishida et al. is preferably used. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), these documents are incorporated herein in their entirety by reference. These methods are also described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, volume 1, Engineering and Utilization, ed. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in 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 that is suitable for transformation of Agrobacterium turnefaciens, for example pBin19 pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed with such a vector can then be used in a known manner for

- 38 044296 transformation of plants, for example plants used as a model, for example Arabidopsis (Arabidopsis thaliana is not considered a crop for the purposes of the present invention), or crops, such as, for example, tobacco, by, for example, dipping crushed or chopped leaves into agrobacterial solutions and then cultivating them in a suitable medium. Plant transformation by Agrobacterium tumefaciens is described, for example, by H'''fgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or known, among other things, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Volume 1, Engineering and Utilization, ed. S.D. Kung and R. Wu, Academic Press, 1993, p. 15-38.

One transformation method known to those skilled in the art is to immerse a flowering plant in a solution of Agrobacteria, the Agrobacteria containing the TriA nucleic acid, and then grow the transformed gametes. Plant transformation by Agrobacterium can be achieved using, for example, GV3101(pMP90) (Koncz and Schell, 1986, Mol. Gen. Genet. 204:383-396) or LBA4404 (Clontech), strains of Agrobacterium tumefaciens. Transformation can be achieved using standard transformation and regeneration methods (Deblaere et al., 1994, Nucl. Acids. Res. 13:47774788; 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 cotyledon or hypocotyl transformation (Moloney et al., 1989, Plant Cell Report 8:238-242; De Block et al., 1989, Plant Physiol. 91:694-701). Antibiotic use for Agrobacterium and plant selection depend on the binary vector and Agrobacterium strain used for transformation. When selecting rapeseed seeds, kanamycin is usually used as a selectable plant marker. Gene transfer by Agrobacterium into flax can be accomplished using, for example, the method described by Mlynarova et al., 1994, Plant Cell Report 13:282-285. Additionally, soybean transformation can be achieved using, for example, the method described in European Patent No. 0424047, US Patent No. 5322783, European Patent No. 0397687, US Patent No. 5376543 or US Patent No. 5169770. Corn transformation can be achieved using particle bombardment, absorption DNA via polyethylene glycol or silicon carbide fiber method. (See, for example, Freeling and Walbot The maize handbook Springer Verlag: New York (1993) ISBN 3-540-978267). A specific example of corn transformation is described in US Patent No. 5,990,387, and a specific example of wheat transformation is described in PCT Application No. WO 93/07256.

In some embodiments, the polynucleotides of the present invention can be introduced into plants by contacting the plants with viruses or viral nucleic acids. Typically, such methods involve introducing a polynucleotide construct of the invention into a viral DNA or RNA molecule. It has been found that the polypeptides of the invention can initially be synthesized as part of a viral polyprotein, which can then be processed by proteolysis in vivo or in vitro to obtain the desired recombinant polypeptide. It is also recognized that the promoters of the invention also include promoters used for transcription by viral RNA polymerases. Methods for introducing a polynucleotide construct into a plant and expressing the protein encoded therein using viral DNA and RNA molecules are known to those skilled in the art; see, for example, US Patent Nos. 5889191, 5889190, 5866785, 5589367 and 5316931; incorporated herein by reference. The transformed cells can develop into plants according to conventional methods; see, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. Such plants can then be grown and pollinated using the same transformed line or other lines, after which a hybrid with constitutive expression of the target phenotypic characteristic can be identified. Two or more generations may be grown to ensure that the target phenotypic characteristic is maintained and inherited, and then seeds may be harvested to ensure that expression of the target phenotypic characteristic is achieved.

The present invention can be used to transform any plant species, including, but not limited to, monocots and dicots. Examples of target plant species include, but are not limited to, corn or maize (Zea mays), Brassica species (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species that can be used in oil production from seeds, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet, e.g. Pennisetum glaucum, common millet (Panicum miliaceum), Italian millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annu), safflower (Carthamus tinctorius), wheat (Triticum aestivum, T. Turgidum ssp. durum), soybean (Glycine max), tobacco (Nicotiana tabacum), Potato (Solarium tuberosum), Peanut (Arachis hypogaea), Cotton (Gossypium barbadense, Gossypium hirsutum), Sweet potato (Ipomoea batatus), Cassava (Manihot esculenta), Coffee (Coffea spp.), Coconut (Cocos nucifera), Pineapple (Ananas comosus) ), citrus trees (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

- 39 044296 papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Primus amygdalus), sugar beet (Beta vulgaris), sugar cane (Saccharum spp.), oats, barley, vegetables, ornamental plants and coniferous trees . Preferably, the plants of the present invention are agricultural crops (for example, sunflower, Brassica species, cotton, sugar beets, soybeans, peanuts, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley, triticale, sorghum, millet, etc. .d.).

In addition to the transformation of somatic cells, which are then regenerated into the whole plant, there is the possibility of transformation of plant meristem cells, especially those cells that become gametes. In this case, the development of transformed gametes occurs naturally, forming transgenic plants. For example, Arabidopsis seeds are treated with agrobacteria and seeds are obtained from developing plants, a certain number of which are transformed, i.e. transgenic [Feldman, K.A. and Marks M.D. (1987) Mol Gen Genet 208:274–289; Feldmann K. (1992). In: S. Koncz, N.-H. Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, p. 274-289]. Alternative methods are based on repeated removal of the inflorescences and cultivating the cutting area in the center of the rosette with transformed Agrobacterium, so that transformed seeds can be obtained at a later date (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, the vacuum infiltration method with modifications such as the flower dipping method is especially effective. In the case of vacuum infiltration of Arabidopsis, whole plants are treated under reduced pressure with a suspension of agrobacteria [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], whereas in the case of a protocol called floral-dip, developing floral tissue is incubated using a surfactant-treated Agrobacterium suspension [Clough, S.J. and Bent A.F. (1998) The Plant J. 16, 735-743]. In both cases, a certain number of transgenic seeds are collected; these seeds differ from non-transgenic seeds in that they are grown under the breeding conditions described above. In addition, persistent plastid transformation is preferred because plastids are maternally inherited, hence the risk of transgene flow through pollen is reduced or eliminated for most crops. Transformation of the chloroplast genome is usually achieved through a process schematically described in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. The sequences to be transformed are cloned together with the selectable marker genome between flanking sequences that are homologous to the chloroplast genome. These homologous flanking sequences enter the reservoir through direct specific integration. Plastid transformation has been described for many plant species, an overview of which is given in Bock (2001) Transgenic 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. Further biotechnological development has resulted in marker-free plastid transformants that can be produced by a transient cointegrated marker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229). Genetically modified plant cells can be regenerated using any methods known to those skilled in the art. Suitable methods can be found in the above-listed S.D. publications. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.

Typically, after transformation, plant cells or groups of cells are selected for the presence of one or more markers encoded by plant-expressed genes, which are transferred along with the target gene, after which the transformed material is regenerated into the whole plant. To select transformed plants, the plant material resulting from the transformation is generally subjected to selective conditions such that transformed plants can be distinguished from non-transformed ones. For example, seeds obtained by the method described can be sown and, after an initial growth stage, subjected to a suitable spray selection method. Another possible method is to grow the seeds, if necessary sterilized, in an agar plate using a suitable selective agent so that only the transformed seeds grow in the plants. Alternatively, the transformed plants can be screened for the presence of the selectable marker described above.

Following DNA transfer and regeneration, putatively transformed plants can also be examined using Southern analysis for the presence of the target gene, copy number and/or genomic organization. Alternatively or in addition, expression levels of newly introduced DNA can be examined using Northern analysis and/or Western analysis, both methods are well known in the art.

The generated transformed plants can be propagated in various ways, such as clonal propagation or classical plant propagation. For example, the first generation (or T1) of a transformed plant may be capable of self-fertilization, the transformed cells of the homozygous second generation (or T2) may be selected, and the T2 plants may then be propagated by classical plant breeding methods. The generated transformed organisms can take different forms. For example, these could be hybrids

- 40 044296 transformed and ^transformed cells, clonal transformed cells (for example, all transformed cells contain expression cassettes), grafts of transformed and non-transformed tissues (for example, in plants, a transformed root shoot is transplanted into a ^transformed shoot).

Preferably, expression of the nucleic acid in the plant results in increased resistance of the plant to the herbicide compared to a wild-type plant.

In yet another embodiment of the invention, the invention relates to a plant with a plant cell in accordance with the present invention, in which the expression of a nucleic acid results in increased resistance of the plant to a herbicide compared to a wild-type plant.

The plants described herein may be either transgenic crops or non-transgenic plants.

In addition to the general definition above, the terms transgenic, transgene or recombinant mean, with respect to, for example, a nucleotide sequence, an expression cassette, a gene construct or vector comprising a nucleotide sequence, or an organism transformed with a nucleotide sequence, expression cassettes or vectors according to of the present invention, all of these constructs are obtained by recombinant methods, in which:

(a) nucleotide sequences encoding proteins used in the methods of the present invention, or (b) genetic control sequence(s) that are operably linked to a nucleic acid sequence of the present invention, such as a promoter, or (c) a) and b) are not located in their natural genetic environment or have been altered by recombinant methods, wherein the modification may take the form of, for example, substitution, addition, deletion, inversion or insertion of one or more nucleotide residues to enable expression of the mutated TriA of the present invention.

Natural genetic environment means a natural genomic or chromosomal locus in the original plant or found in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably preserved, at least in part. The medium is adjacent to the nucleic acid sequence on at least one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp . O. and most preferably at least 5000 bp. A natural expression cassette, for example a natural combination of a natural promoter nucleotide sequences with a corresponding nucleotide sequence encoding a polypeptide suitable for the methods of the present invention as defined above, becomes a transgenic expression cassette when the expression cassette is altered by non-natural, synthetic (artificial) methods such as such as mutagenic treatment. Corresponding methods are described, for example, in US 5565350 or WO 00/15815.

Thus, when using the term transgenic plant for the purposes of the present invention, it is meant, as stated above, that the nucleic acids are not located at a natural locus of the genome of the plant, so the nucleic acids can be expressed homologously or heterologously. However, as noted, transgenic also means that, since the nucleic acids of the invention or method used are at a naturally occurring position in the plant genome, the sequence has been altered relative to the naturally occurring sequence and/or the regulatory sequences of the naturally occurring sequences have been altered. The term transgenic preferably means the expression of the nucleic acids according to the invention at a non-natural locus of the genome, i.e. homologous or preferably heterologous expression of nucleic acids occurs. Preferred transgenic plants are indicated herein. Also, the term transgenic 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 firmly integrated into a chromosome or stable extrachromosomal element in order to be transmitted to subsequent generations. For purposes of the present invention, the term recombinant polynucleotide refers to a polynucleotide that has been altered, rearranged, or altered by genetic engineering. Examples include any cloned polynucleotide or polynucleotides joined or joined to heterologous sequences. The term recombinant does not refer to changes in polynucleotides that were produced by natural processes such as spontaneous mutations, or by non-spontaneous mutagenesis and then selection.

Alleles or allelic variants are alternative forms of a particular gene located at the same chromosomal position. Such variants include single nucleotide polymorphism (SNP) and insertion-deletion polymorphism (INDEL). The size of INDEL is usually less than 100 bp. SNPs and INDELs form the largest group of sequence variants in the natural polymorphic sequences of most organisms.

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The term species refers to a group of plants of the same variety, defined by the presence of common characteristics or traits that experts consider sufficient to distinguish one variety or species of plant from another. No concept implies that all plants of any variety or variety will be genetically identical at the whole gene or molecular level or that any plant will be homozygous at all loci. A variety or species is considered to have the ability to transmit a certain trait to its offspring if, upon self-fertilization of the specified variety or species, all offspring contain this trait. The term breeding line or line refers to a group of plants of the same species, defined by the presence of common characteristics or traits that experts consider sufficient to separate one breeding line or line from another. Neither concept implies that all plants of any breeding line or line will be genetically identical at the whole gene or molecular level or that any plant will be homozygous at all loci. A breeding line or line is said to have the ability to pass on a particular trait to its offspring if, when said line or breeding line self-fertilizes, all of the offspring contain that trait. In the present invention, the trait results from a mutation in the TriA gene of the plant or seed.

The herbicide-tolerant plants of the present invention containing polynucleotides encoding polypeptides with mutated TriA are also used in methods of increasing plant resistance to herbicides through standard plant breeding, including sexual propagation. These methods involve crossing a first plant, which is a herbicide-resistant plant of the invention, with a second plant, which may or may not be resistant to the same herbicide or herbicides, or may be resistant to another herbicide or herbicides. from the first plant. The second plant can be any plant capable of producing viable offspring (i.e. seeds) when crossed with the first plant.

Most often, but not necessarily, the first and second plants are of the same species. These methods may optionally include selecting progeny plants containing the TriA mutated polypeptides of the first plant and the herbicide tolerance characteristics of the second plant. The progeny plants produced by the method of the present invention have increased herbicide tolerance compared to the first or second plant or both plants. If the first and second plants are resistant to different herbicides, the progeny plants will have the combined herbicide resistance characteristics of the first and second plants. The methods of the invention may involve one or more generations of backcrossing the progeny of the plants of the first cross with a plant of the same lineage or genotype as the first or second plant. Alternatively, the progeny of the first cross or any subsequent cross may be crossed with a third plant that has a different lineage or genotype than the first or second plant.

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

In another embodiment of the invention, the invention relates to a seed obtained from a transgenic plant with a plant cell of the present invention, in which the seed germinates due to an increase in the plant's herbicide tolerance compared to the wild type seed.

In accordance with other aspects of the invention, plants with herbicide resistance of the present invention can be used as herbicide-resistant donor lines for development, for example, using traditional plant breeding methods to produce other varietal and/or hybrid crops having such characteristic or characteristics. All such resulting varieties or hybrid crops containing the herbicide resistance hereditary characteristic or characteristics are referred to herein as the progeny or descendants of the herbicide resistant line(s).

In accordance with other embodiments, the present invention provides a method for producing plants that are resistant to herbicides. The method comprises: crossing a first plant having herbicide resistance with a second plant to produce a progeny plant having herbicide resistance, wherein the first plant and the progeny plant, in at least some of their cells, contain a polynucleotide operably linked to the promoter, functioning in plant cells, wherein the action of the recombinant polynucleotide in the cells of the first plant leads to the expression of a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts resistance to herbicides to said plant.

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Traditional plant breeding methods can be used to introduce herbicide resistance into the offspring plants resulting from such methods. In one embodiment, the present invention provides a method for creating a herbicide-resistant offspring plant, a method comprising: crossing the parent plant with a herbicide-resistant plant to introduce the herbicide-resistant properties of the herbicide-resistant plant into the germplasm of the offspring plant , characterized in that the descendant plant has increased resistance to herbicides compared to the parent plant. In other embodiments of the invention, the method also includes the step of introducing herbicide resistance properties through the use of traditional plant breeding techniques to produce a progeny plant having herbicide resistance properties.

In other aspects, the plants of the present invention include plants that, in addition to being herbicide resistant, have been further genetically modified by breeding, mutagenesis, or genetic engineering, such as becoming resistant to other specific classes of herbicides, such as AHAS inhibitors, auxin herbicides bleaching herbicides such as hydroxyphenylpyruvate dioxygenase (HPDH) 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); or oxynyl herbicides (ie bromoxynil or ioxynil) as a result of conventional breeding methods or genetic engineering. Thus, the herbicide-tolerant plants of the invention can be made resistant to different classes of herbicides through multiple genetic modifications, such as resistance to glyphosate and both glufosinate, or glyphosate and both a herbicide of another class, such as GPDH inhibitors, AHAS inhibitors or inhibitors. ACCase. These technologies for developing herbicide resistance are described, for example, in Pest Control Science (volume, year, p.): 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 in materials referenced in this document. For example, the herbicide-tolerant plants of the present invention may, in some embodiments, be resistant to ACCase inhibitors such as dims (e.g., cycloxydim, sethoxydim, clethodim, or tepraloxydim), fops (e.g., clodinafop, diclofop, fluazifop, haloxyfop, or chisalofop) and dens (eg, pinoxaden); to auxin herbicides such as dicamba, to EPS inhibitors such as glyphosate; to other cellulose biosynthesis inhibitors, as well as to GS inhibitors such as glufosinate.

In addition to these classes of inhibitors, herbicide-tolerant plants of the present invention may also be resistant to herbicides with other modes of action, such as chlorophyll/carotenoids, cell wall disruptors, photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and their combinations.

Such resistance characteristics may be expressed, for example, as mutant or wild-type GPPD proteins, mutant or wild-type PPO proteins, mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins, or mutant glutamine synthetase proteins; either as a native, inbreed or transgenic aryloxyalkanoate dioxygenase (AAD or DHT), haloarylnitriles (BXN), 2,2-dichloropropionic acid dehalogenase (DEH), glyphosate N-acetyltransferase (GAT), glyphosate decarboxylase (GDC), glyphosate oxidoreductase (GOX), glutathione S-transferase (GST), phosphinothricin acetyltransphase (PAT or bar) or CYP450 proteins having herbicide-reducing activity. The herbicide-tolerant plants of the present invention may also be combined with other traits including, but not limited to, pesticidal traits such as Bt Cry and other proteins with pesticidal activity against Coleoptera, Lepidoptera, nematodes and other pests; nutritional or nutraceutical characteristics such as modified oil content and others known in the art.

Moreover, in other embodiments of the invention, herbicide-resistant plants are also included within the scope of the invention through the use of recombinant DNA technologies and/or selection and/or are otherwise selected for characteristics that are capable of synthesizing one or more insecticidal proteins, especially proteins from a bacterial species Bacillus, in particular Bacillus thuringiensis, such as [delta]endotoxins, for example CryIA(b), CryIA(c), CryIF, CryIF(a2), CryIIA(b), CryIIIA, CryIIIB(b1) or Cry9c; plant insecticidal proteins (VIPs), for example VIP1, VIP2, VIP3 or VIP3A; insecticidal proteins of bacteria-colonizing nematodes, such as Photorhabdus spp. or Xenorhabdus spp.; toxins produced by animals, such as scorpion toxins, arachnid toxins, wasp toxins, or other specific insect neurotoxins; toxins produced by fungi, such as streptomycete toxins; plant lectins such as pea or barley lectins; agglutinin; proteinase inhibitors such as trypsin inhibitors, serine protease inhibitors, patatin, cystatin or papain inhibitors; ribosome inactivating proteins (RIPs), such as ricin, corn-RIP, abrin, luffin, saporin or bryodin; metabolic enzymes

- 43 044296 roids, such as 3-hedroxy-steroid oxidase, ecdysteroid-IDP-glycosyl-transferase, cholesterol oxidase, ecdiozone inhibitors or HMG-CoA reductase; ion channel inhibitors such as sodium or calcium channel inhibitors; juvenile hormone esterase; diuretic hormone receptors (helical kinin receptors); stilbene synthase, bibenzyl synthase, chitinase or glycanase. In the context of the present invention, these insecticidal proteins or toxins should also be understood as pretoxins, fusion proteins, truncated or otherwise modified proteins. Fusion proteins are characterized by new combinations of protein domains (see, for example, WO 02/015701). Examples of such toxins or genetically modified plants capable of synthesizing such toxins are described, for example, in EP-A 374753, WO 93/007278, WO 95/34656, EP-A 427529, EP-A 451878, WO 03/18810 and WO 03 /52073. Methods for producing such genetically modified plants are known to those skilled in the art and are described, for example, in the above-mentioned publications. These insecticidal proteins, which are contained in genetically modified plants, give plants that produce these proteins resistance to pests of all taxonomic groups of arthropods, especially beetles (Coeloptera), dipterans (Diptera), moths (Lepidoptera) and roundworms (Nematoda).

In some embodiments, the expression of one or more protein toxins (eg, insecticidal proteins) in herbicide-resistant plants is an effective means of controlling organisms including, for example, representatives of the classes and orders: Coleoptera, such as the American bean weevil Acanthoscelides obtectus; leaf beetle Agelastica alni; click beetles (Agriotes lineatus, Agriotes obscurus, Agriotes bicolor); oilseed flat beetle Ahasvems advena; June beetle Amphimallon solstitialis; furniture grinder Anobium punctatum; Anthonomus spp. (weevils); beet crumb Atomaria linearis; leather beetles (Anthrenus spp., Attagenus spp.); four-spotted weevil Callosobruchus maculates; hemipteran spangle Carpophilus hemipterus; rape seed moth Ceutorhynchus assimilis; winter rapeseed stalker Ceutorhynchus picitarsis; wireworms Conoderus vespertinus and Conoderus falli; banana elephant Cosmopolites sordidus; New Zealand grass beetle Costelytra zealandica; shiny green beetle Cotinis nitida; sunflower stem stalker Cylindrocopturus adspersus; ham beetle Dermestes lardarius; cereal rootworms Diabrotica virgifera, Diabrotica virgifera virgifera and Diabrotica barberi; Mexican legume Epilachna varivestis; woodcutter Hylotropes bajulus; alfalfa leaf weevil Hypera postica; Gibbium psylloides; tobacco beetle Lasioderma serricorne; Colorado potato beetle Leptinotarsa decemlineata; tree beetle Lyctus spp., rapeseed flower beetle Meligethes aeneus; western May beetle Melolontha melolontha; American grasshopper Mezium americanum; silky fake Niptus hololeucs; bread grinders Oryzaephilus Surinamensis and Oryzaephilus Mercator; Sulcata mower Otiorhynchus sulcatus; horseradish leaf beetle Phaedon cochleariae, cruciferous flea beetle Phyllotreta cruciferae; striped flea beetle Phyllotreta striolata; rapeseed flea beetle Psylliodes chrysocephala; Ptinus spp. (anti-rust); grain borer Rhizopertha dominica; striped weevil Sitona lineatus; rice and granary weevils Sitophilus oryzae and Sitophilus; red sunflower weevil Smicronyx fulvus; grain grinder Stegobium paniceum; large mealworm Tenebrio molitor, small mealworm (Tribolium confusum) Tribolium castaneum and Tribolium confusum; trogoderma (Trogoderma spp.); sunflower cutworm Zygogramma exclamationis; Leatherwings (earwigs), such as the common earwig Forficula auricularia and the coastal earwig Labidura riparia; cockroaches, such as the black cockroach Blatta orientalis; greenhouse centipede Oxidus gracilis; beet fly Pegomyia betae; Swedish fly Oscinella frit; fruit flies (Dacus spp., Drosophila spp.); white ants (termites), including species representing the families of harvester termites, wood-eating termites, Darwinian termites, long-nosed termites, winged termites, Termitidae, Termopsidae; grass bug Lygus lineolaris; beet aphid Aphis fabae; melon and cotton aphid Aphis gossypii; green apple aphid Aphis pomi; citrus whitefly (Dialeurodes citri) Aleurocanthus spinifems; tobacco whitefly Bemesia tabaci; cabbage aphid Brevicoryne brassicae; pear honeydew Cacopsylla pyricola; currant aphid Cryptomyzus ribis; grape leaf phylloxera Daktulosphaira vitifoliae; citrus psyllid Diaphorina citri; potato leafhopper Empoasca fabae; bean planthopper Empoasca Solana; grape leafhopper Empoasca vitis; downy aphid Eriosoma lanigerum; oyster scale Eulecanium corni; pollinated plum aphid Hyalopterus arundinis; dark leafhopper Laodelphax striatellus; potato leaf aphid Macrosiphum euphorbiae; green peach aphid Myzus persicae; green rice leafhopper Nephotettix cinticeps; brown leafhopper Nilaparvata lugens; hop aphid Phorodon humuli; bird cherry aphid Rhopalosiphum padi; large cereal aphid Sitobion avenae; Lepidoptera such as Adoxophyes orana (net leaf roller); Archips podana (omnivorous leaf roller); Bucculatrix pyrivorella (pear miner); Bucculatrix thurberiella (cotton cotton moth); Bupalus piniarius (pine moth); Carpocapsa pomonella (codling moth); Chilo suppressalis (Asian stem moth); Choristoneura fumiferana (spruce budworm); Cochylis hospes (sunflower woolfoot); Diatraea grandiosella (southwestern corn moth); Eupoecilia ambiguella (bunch leaf roller); Helicoverpa armigera (bollworm); Helicoverpa zea (bollworm); Heliothis vires cens (tobacco budworm), Homeosoma

- 44 044296 electellum (sunflower moth); Homona magnanima (western tea bud); Lithocolletis blancardella (fruit leaf miner); Lymantria dispar (gypsy moth); Malacosoma neustria (cocoon moth); Mamestra brassicae (cabbage cutworm); Mamestra configurata (lettuce cutworm); Operophtera brumata (winter moth); Ostrinia nubilalis (corn borer), Panolis flammea (pine armyworm), Phyllocnistis citrella (citrus leaf miner); Pieris brassicae (cabbage whiteweed); Rachiplusia ni (soybean armyworm); Spodoptera exigua (small cutworm); Spodoptera littoralis (cotton caterpillar); Sylepta derogata (cotton budworm); Trichoplusia ni (cabbage cutworm); Orthoptera, such as the common cricket Acheta domesticus, the Egyptian locust (Anacridium spp.), the migratory locust Locusta migratoria, the two-striped grasshopper Melanoplus bivittatus, the distinctive grasshopper Melanoplus differentialis, the red-thighed grasshopper Melanoplus femurrubrum, the migratory grasshopper Melanoplus sanguinipes, the ten-fingered mole cricket Neocurtilla he xadectyla, Nomadacris locust septemfasciata, short-winged mole cricket Scapteriscus abbreviatus, southern mole cricket Scapteriscus borellii, reddish-brown mole cricket Scapteriscus vicinus, and desert locust Schistocerca gregaria; symphylae such as the garden millipede Scutigerella immaculata; Fringedwings, such as tobacco thrips Frankliniella fusca, common thrips Frankliniella intonsa, western flower thrips Frankliniella occidentalism, cotton thrips Frankliniella schultzei, ornamental thrips Hercinothrips femoralis, soybean thrips Neohydatothrips variabilis, citrus thrips Pezothrips kellyanus, avocado thrips Scirt othrips perseae, melon thrips Thrips palmi, and onion thrips Thrips tabaci; and similar organisms, as well as combinations thereof, including one or more of these organisms.

In some embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in herbicide-resistant plants is effective in controlling leaf beetles, that is, members of the leaf beetle subfamily of the leaf beetle family, preferably Phyllotreta spp., such as Phyllotreta cruciferae and/or Phyllotreta triolata. In other embodiments, the expression of one or more protein toxins (eg, insecticidal proteins) in herbicide-resistant plants is effective in controlling rape seed worms, lettuce cutworms, horse flies, or cabbage moths.

Moreover, in one embodiment of the invention, herbicide-tolerant plants are also within the scope of the invention, for example, using recombinant DNA technologies and/or selection and/or otherwise selected for characteristics that are capable of synthesizing one or more proteins to increase resistance or resistance of these plants to bacterial, viral or fungal pathogens. Methods for obtaining such genetically modified plants are known to specialists.

Moreover, in yet another embodiment of the invention, plants that are resistant to herbicides are also within the scope of the invention, for example, using recombinant DNA technologies and/or selection and/or otherwise selected for characteristics that are capable of synthesizing one or more proteins for increasing productivity (i.e. oil content), resistance to drought, salinity or other environmental factors that limit growth, or resistance to pests, as well as bacterial, viral or fungal pathogens of these plants.

Moreover, in other embodiments of the invention, plants that are resistant to herbicides are also included within the scope of the invention, for example, through the use of recombinant DNA technologies and/or selection and/or are otherwise selected for characteristics that are altered such that they contain altered quantity of one or more substances or new substances, for example, to improve human and animal nutrition, for example, oilseeds that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids (for example, Nexera® rape, Dow Agro Sciences, Canada).

Moreover, in some embodiments of the invention, plants that are resistant to herbicides are also included within the scope of the invention, for example, through the use of recombinant DNA technologies and/or selection and/or are otherwise selected for characteristics that are altered such that they contain increased amounts of vitamins and/or minerals and/or have improved nutraceutical compound profiles.

In one embodiment, the plants of the present invention, compared to wild-type plants, contain an increased amount and improved profile of a compound selected from the group consisting of: glucosinolates (e.g., glucoraphanin (4-methylsulfinylbutylglucosinolate), sulforaphane, 3-indolylmethyl-glucosinate (glucobrassicin), 1-methoxy-3indolylmethyl-glucosinate (neoglucobrassicin)); phenolics (e.g. flavonoids (e.g. quercetin, kaempferol), hydroxycinnamoyl derivatives (e.g. 1,2,2'-trizinapoyl gentiobiose, 1,2-diferuloyl gentiobiose, 1,2-disinapoyl-2-feruloyl gentiobiose, 3-O-caffeoyl- quinic (neochlorogenic acid)); and vitamins and minerals (eg, vitamin C, vitamin E, carotene, folic acid, niacin, riboflavin, thiamine, calcium, iron, magnesium, potassium, selenium, and zinc).

In yet another embodiment, plants of the present invention that are herbicide resistant compared to wild type plants contain an increased amount and improved profile of a compound selected from the group consisting of: progoitrin; isothiocyanates; indoles

- 45 044296 (glucosinolate hydrolysis products); glutathione; carotenoids such as beta-carotene, lycopene and xanthophyll; carotenoids such as lutein and zeaxanthin; phenolics containing flavonoids such as flavonols (eg, quercetin, rutin), flavans/tannins (such as coumarin-containing procyanidins, proanthocyanidins, catechins and anthocyanins); flavones; phytoestrogens such as coumestans, lignans, resveratrol, isoflavones such as genistein, daidzein and glycitein; resorcylic acid lactones; organic sulfur compounds; phytosterols; terpenoids such as carnazole, rosmarinic acid, glycyrrhizins and saponins; chlorophyll, chlorophyllin, sugars, anthocyanin and vanilla.

Other embodiments of the invention include plants exhibiting herbicide resistance compared to wild plants that contain a greater amount or improved profile of a compound selected from the group consisting of vincristine, vinblastine, taxanes (e.g., taxol (paclitaxel), baccatin III, 10-deacetyl baccatin III, 10-deacetyl taxol, xylosyl taxol, 7-epitaxol, 7-epibaccatin III, 10-deacetyl cephalomanin, 7-epicephalomanin, taxotere, cephalomanin, xylosyl cephalomanin, taxagifin, 8-benzyloxy taxagifin, 9-acetyloxy tak susin , 9hydroxy taxusin, taiwanxam, taxane 1a, taxane 1b, taxane Ic, taxane Id, GMP paclitaxel, 9-dihydro-13-acetylbaccatin III, 10-deacetyl-7-epitaxol, tetrahydrocannabinol (THC), cannabidiol (CBD), genistein, diadzein , codeine, morphine, quinine, shikonine, aimalazine, serpentine and the like.

In other aspects, a method for treating a plant of the present invention is provided.

In some embodiments of the invention, the method involves applying to the plants a composition that is acceptable from an agricultural point of view. In accordance with one embodiment of the invention, the agriculturally acceptable composition comprises an auxin herbicide A.I.

According to yet another aspect of the invention, the invention provides a method for producing progeny seeds. The method involves planting seeds of a plant of the present invention or seeds from which a plant of the present invention can be obtained. In one embodiment, the method also includes growing a progeny plant from a seed and collecting the progeny seeds of the progeny plant. In other embodiments of the invention, the method also involves applying the herbicidal herbicide composition to the plant.

In yet another embodiment, the present invention relates to the reapable parts of the transgenic plant of the present invention. The harvestable parts preferably contain the TriA nucleic acid or TriA protein of the present invention. The harvested parts may be seeds, roots, leaves and/or flowers containing TriA nucleic acid or TriA protein, or parts thereof. Preferred soy parts are soybeans containing TriA nucleic acid or TriA protein.

In yet another embodiment, the present invention relates to products obtained from a transgenic plant of the present invention, parts thereof or harvested parts of such a plant. The preferred plant product is fodder, cake meal, oil and processed or coated seeds. Preferably, the flour and/or oil contains TriA nucleic acids or TriA proteins.

In yet another embodiment of the invention, the invention relates to a method for producing a product, said method comprising:

a) growing plants according to the invention or plants that can be obtained by methods according to the invention, and

b) the production of the specified product from the plants according to the invention or using the plants according to the invention, or parts of the specified plants, for example, their seeds.

In yet another embodiment of the invention, the method comprises the following steps:

a) growing plants according to the invention,

b) removal of harvestable parts of plants described above, as well as

c) producing said plant product using the harvested parts of the plants according to the invention.

These products may be produced at the place where the plants are grown, or the plants and/or parts thereof may be transferred from the place where the plants are grown to produce the products. Typically, the plant is grown and the required harvestable parts are removed from the plant (if appropriate, through repeated process cycles), after which a product is produced from the harvestable plant parts. When using the methods according to the invention, the plant growing step can be carried out only once, and the product production step can occur in repeated technological cycles, for example, the step of removing plant parts suitable for harvesting according to the invention and, if necessary, a further processing step can be repeated cyclically these plant parts to obtain the product. It is also possible that the stage of growing the plants according to the invention is repeated and the plants or plant parts suitable for harvesting are stored until the stage of production of the product from the accumulated stock of plants or plant parts. Also, the stages of growing plants and producing a product can occur simultaneously, partially or completely. Typically, plants are grown for a certain period of time before the product is produced.

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In one embodiment of the invention, the products obtained by said methods of the invention are plant products, such as, but not limited to, foodstuffs, products used in animal feeding, food additives, feed additives, fibers used in the production of cosmetics and/or pharmaceuticals products. Food includes anything used for nutrition and/or to provide nutrients. In particular, substances and products used for animal nutrition also belong to food products.

In yet another embodiment, the methods of the invention are used to produce agricultural products such as, but not limited to, plant extracts, proteins, amino acids, carbohydrates, fats, oils, polymers, vitamins, and the like.

It is possible that a plant product is largely composed of one or more agricultural products.

Herbicides.

As described above, the present invention provides nucleic acids, polypeptides that confer resistance to compounds/herbicides that inhibit or interfere with cell wall (cellulose) biosynthesis in plants.

Examples of herbicides that can be used in accordance with the present invention, i.e. The herbicides to which the plants of the present invention are resistant/tolerant are compounds known to those skilled in the art, such as azines. Examples of azines that are metabolized by the mutated TriA polypeptides of the present invention are described in detail in the following patent applications listed in the table below. 2.

table 2

Structural formula Application number/internal link; publication number ZT Ζ ^Γ 0ί < PCT/EP2014/065092 PF75365; WO2015/007711 Pages 2-7, line 21 X -АА^ ι· ^н EP 14162309.0 PF76068; WO2015/144881 Page 3, line 4 - page 5, line 3 X •-AA^· ” J· ⁿ EP 14163356.0 PF76069; WO2015/150541 X <AAl ^1st EP 14163742.1 PF76635; WO2015/155129

- 47 044296

X AA/ EP 14163743.9 PF76636; EP2930174 V ^(Ar ' ^Q ^A А ХЧа n^n А (A ⁿ EP 14165565.4 PF76857; WO2015/162166 X An A [fA|— EP 14165624.9 PF76888; WO2015/162169 R R ^a R--- Άα Id 11 RR V / I ^ω /— ^{z TZ} \ EP 14164431.0 PF76890; WO2015/155271 \u-Z—Ш 4./ R ³ --R ⁴ An- ^r2 H EP 14164434.4 PF76930; WO2015/155272 3 R ^{4 R} -°A ⁵ I II 2 ^{(l) a} JA JA A l ^H EP 14164433.6 PF77027; WO2015/155273

Examples of preferred herbicides that can be used in accordance with the present invention are azines having formula (I)

in which A represents phenyl, which is substituted with two to five substituents selected from the group consisting of halogen, CN, NO2, C1-C ₆ -alkyl, C1-Cb-haloalkyl, C ₂ -Cb-alkenyl, C ₂ -C ₆ -haloalkenyl, C ₂ -C ₆ alkynyl, C1-C ₆ -haloalkynyl, OH, C1-C ₆ -alkoxy, C1-C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, ( C1-C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

R ¹ H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy, (Ci-C ₆ alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl, (C1 -C ₆ -alkyl)sulfonyl or phenylsulfonyl, where phenyl is unsubstituted or substituted with one to five substituents selected from the group consisting of halogen, CN, NO2 , C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl and C ₁ -C ₆ -alkoxy;

R ² H, halogen, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C 2 -C ₆ -alkenyl, C ₃ -C ₆ -alkynyl, C _{3 -C 6} -cycloalkyl, C ₃ -C ₆ -cycloalkenyl, OH, C ₁ -C ₆ -alkoxy or C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl;

R ³ H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; R ⁴ H, halogen, CN, C1-C ₆ -alkyl or C ₁ -C ₆ -haloalkyl or

R ³ and R ⁴ together with the carbon atom to which they are attached form a functional group selected from the group consisting of carbonyl, C ₂ -C ₆ -alkenyl, C ₃ -C ₆ -cycloalkyl, C ₃ -C ₆ -cycloalkenyl and three to six membered heterocyclyl, wherein C ₃ -C ₆ -cycloalkyl, C ₃ -C ₆ -cycloalkenyl, or three to six membered heterocyclyl is unsubstituted or substituted with one to three substituents selected from halogen, CN, C ₁ - C ₆ -alkyl and C1-C ₆ -alkoxy; And

R ⁵ H, CN, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl, C1-C ₆ -alkoxy-C1-C ₆ -alkyl, C1-C ₆ -alkoxy, (C1-C6alkyl)carbonyl, (C1 -C ₆ -alkoxy)carbonyl, (C1-C ₆ -alkyl)sulfonyl or phenylsulfonyl, where the phenyl is unsubstituted or is substituted with one to five substituents selected from the group consisting of halogen, CN, NO2, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl and C1-C ₆ -alkoxy; including their agrochemically acceptable salts and N-oxides.

Preferably, the present invention provides azines of formula (I) wherein

A is 2-fluorophenyl, which is substituted with one to four substituents selected from the group consisting of halogen, CN, NO2, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl, OH, C1-C ₆ -alkoxy, C1C ₆ -alkylthio, (C1-C ₆ -alkyl)sulfinyl, (C1-C ₆ -alkyl)sulfonyl, amino, (C1-C ₆ -alkyl)amino, di(C1-C ₆ -alkyl)amino, (C1-C ₆ -alkyl)carbonyl and (C1-C ₆ -alkoxy)carbonyl;

R ¹ H, CN, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl, C1-C ₆ -alkoxy-C1-C ₆ -alkyl, C1-C ₆ -alkoxy, (C1-C ₆ -alkyl)carbonyl , (C1-C ₆ -alkoxy)carbonyl, (C1-C ₆ -alkyl)sulfonyl or phenylsulfonyl, where the phenyl is unsubstituted or substituted with one to five substituents selected from the group consisting of halogen, CN, NO2, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl and C1-C ₆ -alkoxy; R ² H, halogen, CN, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl, C 2 -C ₆ -alkenyl, C ₃ -C ₆ -alkynyl, C _{3 -C 6} -cycloalkyl, C ₃ -C ₆ -cycloalkenyl, OH, C1- _C6 -alkoxy or _{C1C6 -} alkoxy-C1- _C6 -alkyl;

R ³ H, halogen, CN, C1-C6 alkyl, C1-C6 haloalkyl or C1-C6 alkoxy; R ⁴ H, halogen, CN, C1-C6 alkyl or C1-C ₆ haloalkyl; or R ³ and R ⁴ together with the carbon atom to which they are attached form a functional group selected from the group consisting of carbonyl, C ₂ -C ₆ alkenyl, C ₃ -C ₆ cycloalkyl, C ₃ -C ₆ cycloalkenyl and three to six membered heterocyclyl, wherein C ₃ -C ₆ -cycloalkyl, C ₃ -C ₆ -cycloalkenyl or three to six membered heterocyclyl is unsubstituted or substituted with one to three substituents selected from halogen, CN, C1-C ₆ -alkyl and C1-C ₆ -alkoxy; and R ⁵ H, CN, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C1-C ₆ -alkoxy, (C ₁ C ₆ - alkyl)carbonyl, (C1- _C6 -alkoxy)carbonyl, (C1- _C6 -alkyl)sulfonyl or phenylsulfonyl, wherein the phenyl is unsubstituted or is substituted with one to five substituents selected from the group consisting of halogen, CN, NO2, C1 -C ₆ -alkyl, C1-C ₆ -haloalkyl and C1-C ₆ -alkoxy; including their agrochemically acceptable salts and N-oxides.

Also, in the present invention, agrochemical compositions can be used, which include at least one azine of formula (I) and auxiliary substances that are usually used for the preparation of crop protection substances.

The present invention also provides for the use of azines of formula (I) as herbicides, that is, for the purpose of controlling harmful plants.

If azines of formula (I) as defined herein are capable of forming geometric isomers, for example E/Z isomers, both pure isomers and mixtures thereof may be used in the compositions of the invention.

Where azines of formula (I) as described herein have one or more chiral centers and, as a consequence, are present as enantiomers or diastereoisomers, both pure enantiomers and diastereoisomers may be used in the compositions of the invention. mixtures of them.

If the azines of formula (I) as described herein contain ionizable functional groups, they can also be used in the form of their salts, which are acceptable for agricultural purposes. In general, salts of those cations and acid addition salts of those acids are suitable here, whose cations and, accordingly, anions do not adversely affect the activity of the active compounds.

Preferred cations are alkali metal ions, preferably lithium, sodium and potassium, alkaline earth metals, preferably calcium and magnesium, and transition metals, preferably manganese, copper, zinc and iron, more preferably ammonium and substituted ammonium, in which one to four hydrogen atoms are replaced by C1-C ₄ -alkyl, hydroxy-C1C ₄ -alkyl, C ₁ -C ₄ -alkoxy-C1-C ₄ -alkyl, hydroxy-C ₁ -C ₄ -alkoxy-C ₁ -C ₄ -alkyl, phenyl or benzyl, preferably ammonium, methylammonium, isopropylammonium, dimethylammonium, diisopropylammonium, trimethylammonium, heptylammonium, dodecylammonium, tetradecylammonium, tetramethylammonium, tetraethylammonium, tetrabutylammonium, 2-hydroxylammonium (olamine salt), 2-(2-hydroxyeth- 1- oxy)eth-1-ylammonium (diglycolamine salt), di(2-hydroxyeth-1-yl) ammonium (diolamine salt), tris-(2-hydroxyethyl) ammonium (trolamine salt), tris-(2-hydroxypropyl) ammonium, benzyltrimethylammonium, benzyltriethylammonium, K,K,K-trimethylonammonium (choline salt), more preferably phosphonium ions, sulfonium ions, preferably tri(C1-C ₄ -alkyl)sulfonium, such as tri(C1-C ₄ -alkyl)sulfoxonium, and finally salts of polybasic amines such as such as N,N-bis-(3-αminopyl)methylamine and diethylenetriamine.

Anions of suitable acid addition salts include generally chloride, bromide, fluoride, iodide, hydrogen sulfate, methyl sulfate, sulfate, dihydrogen phosphate, hydrogen phosphate, nitrate, bicarbonate, carbonate, hexafluorosilicate, hexafluorophosphate, benzoate, as well as C1- _C4 alkane anions. acids, preferably formate, acetate, propionate and butyrate.

The organic groups indicated in the definition of the variables R1- ^R5 are (such as the term halogen) collective terms for listing the individual members of the group. The term halogen means in each case 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 straight or branched, and the prefix C _n -Cm in each case means the possible number of carbon atoms in the group .

Examples of such values are

C1-C ₄ -alkyl: for example, CH3, C ₂ H ₅ , n-propyl, CH(CH ₃ ) ₂ , n-butyl, CH(CH3)-C ₂ H ₅ , CH ₂ -CH(CH ₃ ) ₂ and C(CHz)z;

C ₁ -C ₆ -alkyl, as well as C ₁ -C ₆ -alkyl groups (C ₁ -C ₆ -alkyl)carbonyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl: C1-C ₄ - alkyl as above, 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 2methylpentyl 3-methylpentyl 4-methylpentyl 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2dimethylbutyl, 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;

C1- _C4 -haloalkyl: a C1- _C4 -alkyl radical as defined above which is partially or completely substituted with fluorine, chlorine, bromine and/or iodine, 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-2fluoroethyl, 2-chloro-2, 2-difluoroethyl, 2,2-dichloro-2-fluoroethyl, 2,2,2-trichloroethyl, pentafluoroethyl, 2-fluoropropyl, 3-fluoropropyl, 2,2-difluoropropyl, 2,3-difluoropropyl, 2-chloropropyl, 3-chloropropyl, 2,3dichloropropyl, 2-bromopropyl, 3-bromopropyl, 3,3,3-trifluoropropyl, 3,3,3-trichloropropyl, 2,2,3,3,3pentafluoropropyl, 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;

C ₁ -C6-haloalkyl: C1-C ₄ -haloalkyl, as above, and also, for example, 5-fluoropentyl, 5-chloropentyl, 5-bromopentyl, 5-iodopentyl undecafluoropentyl, 6-fluorohexyl, 6-chlorohexyl, 6-bromohexyl, 6iodohexyl and dodecafluorohexyl;

C ₃ -C ₆ -cycloalkyl: monocyclic saturated hydrocarbons with 3-6 carbon ring members, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl;

C ₂ -C ₆ -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-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, 2methyl-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, 4methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl- 3-butenyl, 1,2-dimethyl-1-butenyl, 1,2-dimethyl2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-1-butenyl, 1,3-dimethyl-2- butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-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, 2ethyl-1-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2propenyl, 1-ethyl-2-methyl-1-propenyl and 1-ethyl-2-methyl-2-propenyl;

- 50 044296

C ₃ -C ₆ -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, 2,5cyclohexadienyl;

C ₃ -C ₆ -alkynyl: for example, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-methyl-2propynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4- pentynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2methyl-3-butynyl, 3-methyl-1-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 1-hexynyl, 2hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4pentynyl, 2-methyl-3-pentynyl, 2- methyl 4-pentynyl, 3-methyl-1-pentynyl, 3-methyl-4-pentynyl, 4-methyl1-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-3butynyl and 1-ethyl-1-methyl-2-propynyl;

C1-C4 alkoxy: for example methoxy, ethoxy, propoxy, 1-methylethoxy butoxy, 1-methylpropoxy, 2methylpropoxy and 1,1-dimethylethoxy;

C ₁ -C ₆ -alkoxy, as well as C ₁ -C ₆ -alkoxy functional groups (C ₁ -C ₆ -alkoxy)carbonyl, C1-C6alkoxy-C ₁ -C ₆ -alkyl: C1-C ₄ -alkoxy, like as above, as well as, for example, pentoxy, 1-methylbutoxy, 2methylbutoxy, 3-methoxybutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1ethylpropoxy, hexoxy, 1-methylpentoxy, 2-methylpentoxy, 3-methylpentoxy, 4-methylpentoxy, 1,1dimethylbutoxy, 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;

C1- _C4 -alkylthio: for example, methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio, 1methylpropylthio, 2-methylpropylthio and 1,1-dimethylethylthio;

C ₁ -C ₆ -αalkylthio: C1-C ₄ -alkylthio, as above, and also, for example, pentylthio, 1methylbutylthio, 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, 2,2dimethylbutylthio, 2, 3-dimethylbutylthio, 3,3-dimethylbutylthio, 1-ethylbutylthio, 2-ethylbutylthio, 1,1,2trimethylpropylthio, 1,2,2-trimethylpropylthio, 1-ethyl-1-methylpropylthio and 1-ethyl-2-methylpropylthio;

C ₁ -C ₆ -alkylsulfinyl (C ₁ -C ₆ -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, 1,1dimethylpropylsulfinyl, 1,2-dimethylpropylsulfinyl, hexylsulfinyl, 1-methylpentylsulfinyl, 2-methylpentylsulfinyl, 3 -methylpentylsulfinyl, 4-methylpentyl-sulfinyl, 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 1ethyl-2-methylpropylsulfinyl;

C ₁ -C ₆ -alkylsulfonyl (C ₁ -C ₆ -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, 2,2dimethylpropylsulfonyl, 1-ethylpropylsulfonyl, hexylsulfonyl, 1-methylpentylsulfonyl, 2methylpentylsulfonyl, 3 -methylpentylsulfonyl, 4-methylpentylsulfonyl, 1,1-dimethylbutylsulfonyl, 1,2-dimethylbutylsulfonyl, 1,3-dimethylbutylsulfonyl, 2,2-dimethylbutylsulfonyl, 2,3dimethylbutylsulfonyl, 3,3-dimethylbutylsulfonyl, 1-ethylbutylsulfonyl, 2-ethylbutylsulfonyl, 1,1,2-trimethylpropylsulfonyl, 1,2,2-trimethylpropylsulfonyl, 1-ethyl-1-methylpropylsulfonyl and 1ethyl-2-methylpropylsulfonyl;

(C1-C ₄ -alkyl)amino: for example methylamino, ethylamino, propylamino, 1-methylethylamino, butylamino, 1-methylpropylamino, 2-methylpropylamino or 1,1-dimethylethylamino;

(C ₁ -C ₆ -alkyl)amino: (C1-C ₄ -alkylamino), as above, 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, 1,2dimethylbutylamino, 1,3-dimethylbutylamino, 2,2 -dimethylbutylamino, 2,3-dimethylbutylamino, 3,3dimethylbutylamino, 1-ethylbutylamino, 2-ethylbutylamino, 1,1,2-trimethylpropylamino, 1,2,2trimethylpropylamino, 1-ethyl-1-methylpropylamino or 1-ethyl-2- methylpropylamino;

di(C1-C ₄ -alkyl)amino: for example, K,K-dimethylamino, K,K-diethylamino, K,K-di(1-methylethyl)amino, K,K-dipropylamino, K,K-dibutylamino, K ,K-di(1-methylpropyl)amino, K,K-di(2-methylpropyl)amino, K,K-di(1,1-dimethylethyl)amino, K-ethyl-K-methylamino, K-methyl-K -propylamino, K-methyl-K(1-methylethyl)amino, K-butyl-K-methylamino, K-methyl-K-(1-methylpropyl)amino, K-methyl-K-(2

- 51 044296 methylpropyl)amino, K-(1,1-dimethylethyl)-K-methylamino, N-ethyl-K-propylamino, K-ethyl-K-(1methylethyl)amino, N-butyl-K-ethylamino, K- ethyl-K-(1-methylpropyl)amino, K-ethyl-K-(2methylpropyl)amino, K-ethyl-K-(1,1-dimethylethyl)amino, K-(1-methylethyl)-K-propylamino, K -butyl-Npropylamino, K-(1-methylpropyl)-K-propylamino, K-(2-methylpropyl)-K-propylamino, N-(1,1dimethylethyl)-K-propylamino, K-butyl-N-(1 - methylethyl)amino, N-(1-methylethyl)-K-(1-methylpropyl)amino, N-(1-methylethyl)-K-(2-methylpropyl)amino, N-(1,1-dimethylethyl)-K- ( 1 -methylethyl)amino, Nbutyl-K-(1-methylpropyl)amino, K-butyl-K-(2-methylpropyl)amino, K-butyl-K-(1,1-dimethylethyl)amino, N-(1 -methylpropyl)-K-(2-methylpropyl)amino, N-(1,1-dimethylethyl)-K-(1-methylpropyl)amino or N-(1,1dimethylethyl)-K-(2-methylpropyl)amino;

di(C1-C ₆ -alkyl)amino: di(C1-C ₄ -alkyl)amino, as indicated above, and also, for example, K-methyl-Npentylamino, K-methyl-K-(1-methylbutyl)amino, K-methyl-(2-methylbutyl)amino, K-methyl-K-(3methylbutyl)amino, K-methyl-K-(2,2-dimethylpropyl)amino, K-methyl-K-(1-ethylpropyl)amino, K-methyl-Nhexylamino, K-methyl-K-(1,1-dimethylpropyl)amino, K-methyl-K-(1,2-dimethylpropyl)amino, K-methyl-N(1-methylpentyl)amino, K- methyl-K-(2-methylpentyl)amino, K-methyl-K-(3-methylpentyl)amino, N-methylK-(4-methylpentyl)amino, K-methyl-K-(1,1-dimethylbutyl)amino, K-methyl-K-(1,2-dimethylbutyl)amino, Nmethyl-K-(1,3-dimethylbutyl)amino, K-methyl-K-(2,2-dimethylbutyl)amino, K-methyl-K-( 2,3-dimethylbutyl)amino, K-methyl-K-(3,3-dimethylbutyl)amino, K-methyl-K-(1-ethylbutyl)amino, K-methyl-K-(2ethylbutyl)amino, K-methyl -K-(1,1,2-trimethylpropyl)amino, K-methyl-K-(1,2,2-trimethylpropyl)amino, K-methyl-K-(1-ethyl-1-methylpropyl)amino, K- methyl-K-(1-ethyl-2-methylpropyl)amino, K-ethyl-Npentylamino, K-ethyl-K-(1-methylbutyl)amino, K-ethyl-K-(2-methylbutyl)amino, K-ethyl -K-(3methylbutyl)amino, K-ethyl-K-(2,2-dimethylpropyl)amino, K-ethyl-K-(1-ethylpropyl)amino, K-ethyl-Nhexylamino, K-ethyl-N-(1 ,1-dimethylpropyl)amino, K-ethyl-N-(1,2-dimethylpropyl)amino, K-ethyl-N-(1methylpentyl)amino, K-ethyl-K-(2-methylpentyl)amino, K-ethyl- K-(3-methylpentyl)amino, K-ethyl-K-(4methylpentyl)amino, K-ethyl-K-(1,1-dimethylbutyl)amino, K-ethyl-K-(1,2-dimethylbutyl) amino, K-ethyl-N(1,3-dimethylbutyl)amino, K-ethyl-K-(2,2-dimethylbutyl)amino, K-ethyl-K-(2,3-dimethylbutyl)amino, Nethyl- K-(3,3-dimethylbutyl)amino, K-ethyl-K-(1-ethylbutyl)amino, K-ethyl-K-(2-ethylbutyl)amino, N-ethylN-(1,1,2-trimethylpropyl) amino, N-ethyl-N-(1,2,2-trimethylpropyl)amino, K-ethyl-K-(1-ethyl-1-methylpropyl)amino, K-ethyl-K-(1-ethyl-2-methylpropyl) )amino, N-propyl-K-pentylamino, N-butyl-K-pentylamino, N,N-dipentylamino, N-propyl-K-hexylamino, N-butyl-K-hexylamino, N-pentyl-K-hexylamino or N ,N-dihexylamino;

three to six membered heterocyclyl: monocyclic saturated or partially saturated hydrocarbons with 3 to 6 carbon ring members as defined above, which, in addition to carbon atoms, contains one or two heteroatoms selected from O, S and N;

e.g. 2-oxiranyl, 2-oxetanyl, 3-oxetanyl, 2-aziridinyl, 3-thietanyl, 1-azetidinyl, 2azetidinyl, e.g. 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 azolidinyl, 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-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-5yl, 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-2yl, 2,3-dihydropyrazol-3-yl, 2,3-dihydropyrazol-4-yl, 2,3-dihydropyrazol-5-yl, 3,4-dihydropyrazol-3yl, 3,4-dihydropyrazol-4- yl, 3,4-dihydropyrazol-5-yl, 4,5-dihydropyrazol-3-yl, 4,5-dihydropyrazol-4yl, 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-3yl, 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;

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-tetrahydrothiopyranyl, 3tetrahydrothiopyranyl, 4-tetrahydrothiopyranyl, 3-hexahydropyridazinyl, 4-hexahydropyridazinyl, 2-hexahydropyrimidinyl, 4-hexahydropyrimidinyl, 5-hexahydropyrimidinyl, 2-piperazinyl, tetra-52 044296 hydro-1,3-oxazine- 2-yl, tetrahydro-1,3-oxazin-6-yl, 2-morpholinyl, 3-morpholinyl;

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-2Hpyran- 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-2Hpyran-6-yl, 3,4-dihydro-2H-pyran-3-yl, 3,4-dihydro-2H-pyran-4-yl, 3,4-dihydro-2H-pyran-6-yl, 2H-thiopyran-2yl, 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.

The preferred embodiments of the invention set forth below throughout this document are to be understood as being preferred either independently of each other or in combination with each other.

According to a preferred embodiment of the invention, preference is given to azines of formula (I) in which the variables, either independently of each other or in combination with each other, have the following meanings:

Preferred are azines of formula (I), in which

A is phenyl which is substituted with two to five substituents selected from the group consisting of halogen, CN, NO ₂ , C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy , C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably phenyl, which is substituted with two to five substituents selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also, in particular, preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

particularly preferably selected from the group consisting of F, Cl and CN;

particularly preferably phenyl which is substituted with two to four substituents selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C1 -C6alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C1-C ₆ alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

more preferably phenyl which is substituted with two substituents selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ - C ₆ -alkylthio, (C1C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl)amino, ( C1C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

also more preferably phenyl which is substituted with three substituents selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl) )amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy; particularly preferably selected from halogen and CN; also particularly preferably selected from the group consisting of F, Cl, CN and CH3;

more preferably selected from the group consisting of F, Cl and CN;

also more preferably phenyl which is substituted with four substituents selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C1- C6alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C1-C ₆ alkyl)amino, (C1- C ₆ -alkyl)carbonyl, (C1-C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C1-C ₆ -alkyl and C1-C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH3;

more preferably selected from the group consisting of F, Cl and CN.

Also preferred are azines of formula (I) in which A is

- 53 044296

wherein R ^a and R ^e independently of each other represent halogen, CN, NO2, C ₁ -C ₆ -alkyl, C1-C6 haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)-amino, di(C ₁ -C ₆ -alkyl)amino, (C ₁ - C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl; And

R ^b , R ^c and R ^d are independently hydrogen, halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl) )amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably R ^a and R ^e are independently of each other halogen, CN, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -alkoxy; And

R ^b , R ^c and R ^d are independently hydrogen, halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl or C ₁ -C ₆ -alkoxy;

particularly preferably R ^a and R ^e are, independently of each other, halogen or CN; And

R ^b , R ^c and R ^d are independently hydrogen, halogen, CN, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -alkoxy;

more preferably R ^a and R ^e are halogen and

R ^b , R ^c and R ^d are independently hydrogen, halogen or CN;

most preferably R ^a and R ^e are halogen and

R ^b , R ^c and R ^d represent hydrogen;

also most preferably R ^a , R ^b , R ^d and R ^e are halogen and

R ^c represents hydrogen;

also most preferably R ^a , R ^b , R ^c , R ^d and R ^e are halogen.

Also preferred are azines of formula (I) in which A is

wherein R ^a represents halogen or CN;

R ^b and R ^d are H, halogen or CN;

R ^c represents H or halogen;

R ^e represents halogen, CN or C ₁ -C ₆ -alkyl;

in particular, preferably R ^a is halogen;

R ^b , R ^c and R ^d represent H or halogen and

R ^e represents halogen or CN;

particularly preferably R ^a , R ^b , R ^d and R ^e are halogen and

R ^c represents H or halogen;

more preferably R ^a , R ^b , R ^d and R ^e are F and

^Rc is H or F.

Particularly preferred are azines of formula (I), in which

A is selected from the group consisting of (A.1.1), (A.1.2) and (A.1.3);

more preferably selected from the group consisting of (A.1.2) and (A.1.3);

wherein R ^a and R ^e independently of each other represent halogen, CN, NO2, C1-C ₆ -alkyl, C1-C6 haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl; And

R ^b and R ^d independently of each other represent halogen, CN, NO2, C ₁ -C ₆ -alkyl, C1-C6 haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

- 54 044296 in particular, preferably R ^a and R ^e are, independently of each other, halogen, CN,

C1-C ₆ -alkyl or C ₁ -C ₆ -alkoxy; And

R ^b and R ^d are independently halogen, CN, NO2, C ₁ -C ₆ alkyl, Ci-C ₆ haloalkyl or C ₁ -C ₆ alkoxy;

particularly preferably R ^a and R ^e are independently of each other halogen or CN and

R ^b and R ^d are independently of each other halogen, CN, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -alkoxy;

more preferably R ^a and R ^e are halogen and

R ^b and R ^d are independently of each other halogen or CN;

most preferably R ^a , R ^b , R ^d and R ^e are halogen.

Also particularly preferred are azines of formula (I) in which A is

R ^b _R dXJ (Α-ΐ-υ

R®, wherein R ^a , R ^b , R ^d and R ^e have the meanings, in particular the preferred meanings, as defined above.

Also particularly preferred are azines of formula (I) in which A is

^Rb

AM

O (A.1.2)

R ^{e X} , with R ^a , R ^b and R ^e having the meanings, in particular the preferred meanings, as defined above.

Also particularly preferred are azines of formula (I) in which A is

wherein R ^a and R ^e have meanings, particularly preferred meanings, as defined above.

Also preferred are azines of formula (I), in which

A represents 2-fluorophenyl, which is substituted with one four substituents selected from the flu consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy , C1C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ alkyl) amino, (C ₁ -C ₆ -alkyl)carbonyl and (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably 2-fluorophenyl, which is substituted with one to four substituents selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also, in particular, preferably selected from the group consisting of F, Cl, CN and CH3;

particularly preferably selected from the group consisting of F, Cl and CN;

particularly preferably 2-fluorophenyl which is substituted with one to three substituents selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy , C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ C ₆ -alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl and (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy; particularly preferably selected from halogen and CN; also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

more preferably 2-fluorophenyl, which is substituted with one substituent selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ - alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl and (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

also more preferably 2-fluorophenyl which is substituted with two substituents selected

- 55 044296 from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C1-C6 alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ alkyl)amino, (C1-C ₆ -alkyl)carbonyl and (C1-C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C1-C ₆ -alkyl and

C1-C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH3;

more preferably selected from the group consisting of F, Cl and CN;

also more preferably 2-fluorophenyl which is substituted with three substituents selected from the group consisting of halogen, CN, NO2, C1- _C6 alkyl, C1- _C6 haloalkyl, OH, C1- _C6 alkoxy, C1-C6 alkylthio , (C1-C ₆ -alkyl)sulfinyl, (C1-C ₆ -alkyl)sulfonyl, amino, (C1-C ₆ -alkyl)amino, di(C1-C ₆ -alkyl)amino, (C1-C ₆ -alkyl) )carbonyl and (C1-C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C1-C ₆ -alkyl and C1-C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH3;

more preferably selected from the group consisting of F, Cl and CN.

Also preferred are azines of formula (I) in which A is

wherein R ^a represents halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C1-C ₆ alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl , (C ₁ -C ₆ -alkoxy)carbonyl; And

R ^b , R ^c and R ^d are independently hydrogen, halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ -alkyl) )amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably R ^A is halogen, CN, C1-C6 alkyl or C1-C6 alkoxy; and R ^b , R ^c and R ^d are independently hydrogen, halogen, CN, NO2, C1- _C6 alkyl, C1-C6 haloalkyl or _C1 - _C6 alkoxy;

particularly preferably R ^A is halogen or CN; and R ^b , R ^c and R ^d are independently hydrogen, halogen, CN, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -alkoxy;

more preferably R ^A is halogen and

R ^b , R ^c and R ^d are independently hydrogen, halogen or CN;

most preferably R ^A is halogen and

R ^b , R ^c and R ^d represent hydrogen;

also most preferably R ^a , R ^b and R ^d are halogen and

R ^c represents hydrogen;

also most preferably R ^a , R ^b , R ^c and R ^d are halogen.

Also preferred are azines of formula (I) in which A is

wherein R ^a represents halogen, CN or C1-C ₆ -alkyl;

R ^b and R ^d are H, halogen or CN and

R ^c represents H or halogen;

in particular, preferably R ^A is halogen or CN; And

R ^b , R ^c and R ^d represent H or halogen;

particularly preferably R ^a , R ^b and R ^d are halogen; And

R ^c represents H or halogen;

Also particularly preferably, R ^a , R ^b and R ^d are halogen and

R ^c represents H, F, Br or I;

more preferably R ^a , R ^b and R ^d are F and

R ^c represents F, Br or I;

- 56 044296 also more preferably R ^a , R ^b and R ^d are F and

R ^c represents H or F.

Particularly preferred are azines of formula (I) in which A is selected from the group consisting of (A.1a.1), (A.1a.2) and (A.1a.3);

more preferably selected from the group consisting of (A.1.2) and (A.1.3)

wherein R ^a represents halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C1-C ₆ alkyl)amino, (C1-C ₆ -alkyl)carbonyl, (C1-C ₆ -alkoxy)carbonyl; And

R ^b and R ^d independently of each other represent halogen, CN, NO2, C1-C ₆ -alkyl, C ₁ -C ₆ haloalkyl, OH, C1-C ₆ -alkoxy, C1-C ₆ -alkylthio, (C1- C ₆ -alkyl)sulfinyl, (C1-C ₆ -alkyl)sulfonyl, amino, (C1-C ₆ -alkyl)amino, di(C1-C ₆ -alkyl)amino, (C1-C ₆ -alkyl)carbonyl, (C1-C ₆ -alkoxy)carbonyl;

in particular, preferably R ^A is halogen, CN, C1-C6 alkyl or C1-C6 alkoxy; and R ^b and R ^d are independently halogen, CN, NO2, C1-C6 alkyl, C1- _C6 haloalkyl or C1- _C6 alkoxy;

particularly preferably R ^A is halogen or CN; And

R ^b and R ^d are independently halogen, CN, C1- _C6 alkyl or C1- _C6 alkoxy;

more preferably R ^A is halogen; And

R ^b and R ^d are independently of each other halogen or CN;

most preferably R ^a , R ^b and R ^d are halogen.

Also particularly preferred are azines of formula (I) in which A is

wherein R ^a , R ^b and R ^d have meanings, particularly preferred meanings, as defined above. Also particularly preferred are azines of formula (I) in which A is

^Rb

N

Η

(A.1a.2) wherein R ^a and R ^b have values, in particular preferred values, as defined above.

Also particularly preferred are azines of formula (I) in which A is

wherein R ^a has values, in particular preferred values, as defined above.

Also preferred are azines of formula (I), in which

R ¹ represents H, CN, C1- _C6 alkyl, C1- _C6 haloalkyl, _C1 - _C6 alkoxy- _C1 - _C6 alkyl, C1-C6 alkoxy, (C1- _C6 alkyl) carbonyl or (C1-C ₆ -alkyl)sulfonyl;

in particular, preferably H, CN, C1- _C6 -alkyl, _C1 - _C6 -alkoxy-C1 _{- C6} -alkyl, C1- _C6 -alkoxy, ( _C1C6- alkyl)carbonyl or (C1-C ₆ -alkyl)sulfonyl;

particularly preferably H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3;

more preferably hydrogen.

Also preferred are azines of formula (I) in which R ² is H, halogen, C1- _C6 alkyl or C1- _C6 haloalkyl;

- 57 044296 in particular, preferably halogen, C1-C ₆ -alkyl or C1-C ₆ -haloalkyl;

also, in particular, H, F, Cl, CH ₃ or CF ₃ are preferred.

Also preferred are azines of formula (I), in which

R ³ and R ⁴ are, independently of each other, H, halogen, C1- _C6 alkyl or _C1 - _C6 haloalkyl;

or together with the carbon atom to which they are attached, form a functional group selected from the group consisting of C ₃ -C ₆ -cycloalkyl, C ₃ -C ₆ -cycloalkenyl and three to six-membered heterocyclyl, wherein C ₃ -C ₆ -cycloalkyl, C ₃ -C ₆ -cycloalkenyl or three- to six-membered heterocyclyl is unsubstituted or substituted with one to three substituents selected from halogen, CN, C1-C6 alkyl and C1-C ₆ -alkoxy;

independently of each other, in particular, preferably H, halogen, C1-C ₆ -alkyl or Ci-C ₆ haloalkyl; or together with the carbon atom to which they are attached, form a functional group selected from the group consisting of C ₃ -C ₆ -cycloalkyl and C ₃ -C ₆ -cycloalkenyl, wherein C ₃ -C ₆ -cycloalkyl or C ₃ -C ₆ -cycloalkenyl is unsubstituted or substituted with one to three substituents selected from halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

Independently of each other, H, halogen, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -haloalkyl are particularly preferred;

independently of each other, more preferably H, halogen or C ₁ -C ₆ -alkyl.

Also preferred are azines of formula (I), in which

R ² represents H, halogen, C ₁ -C ₆ -alkyl; And

R ³ and R ⁴ independently represent H, halogen, C ₁ -C ₆ -alkyl, or together with the carbon atom to which they are attached form C ₃ -C ₆ -cycloalkyl;

in particular, preferably R ² represents H, halogen or C ₁ -C ₆ -alkyl;

R ³ represents C ₁ -C ₆ -alkyl;

R ⁴ represents H, halogen or C ₁ -C ₆ -alkyl;

R ³ and R ⁴ together with the carbon atom to which they are attached form C ₃ -C ₆ -cycloalkyl;

particularly preferably R ² represents halogen or C ₁ -C ₆ -alkyl;

R ³ represents C ₁ -C ₆ -alkyl;

R ⁴ represents H or C ₁ -C ₆ -alkyl;

more preferably R ² is halogen; And

R ³ and R ⁴ represent C ₁ -C ₆ -alkyl.

Also preferred are azines of formula (I), in which

R ⁵ represents H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C1-C ₆ alkoxy, (C ₁ - C ₆ -alkyl)carbonyl or (C ₁ -C ₆ -alkyl)sulfonyl;

in particular, preferably H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy, (C1C ₆ -alkyl)carbonyl or (C ₁ -C ₆ -alkyl)sulfonyl;

particularly preferably H, Cn, CH ₃ , CH ₂ OCH ₃ , OCH ₃ , COCH ₃ or SO ₂ CH ₃ ;

more preferably hydrogen.

Also preferred are azines of formula (I), in which

A represents phenyl, which is substituted with two to five substituents selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also, in particular, preferably selected from the group consisting of F, Cl, CN and CH3;

particularly preferably selected from the group consisting of F, Cl and CN;

in particular, preferably phenyl which is substituted with two to four substituents selected from the group consisting of halogen, CN, NO ₂ , C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ - alkoxy, C ₁ -C ₆ -alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C1C ₆ - alkyl)amino, (C1-C ₆ -alkyl)carbonyl, (C1-C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C1-C ₆ -alkyl and C1-C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

particularly preferably phenyl which is substituted with two substituents selected from the group consisting of halogen, CN, NO ₂ , C 1 -C ₆ -alkyl, C 1 -C ₆ -haloalkyl, OH, C 1 -C ₆ -alkoxy, C 1 -C ₆ - alkylthio, (C1-C ₆ -alkyl)sulfinyl, (C1-C ₆ -alkyl)sulfonyl, amino, (C1-C ₆ -alkyl)amino, di(C1-C ₆ -alkyl)amino, (C1-C ₆ -alkyl)carbonyl, (C1-C ₆ -alkoxy)carbonyl; in particular, preferably selected from the group consisting of halogen, CN, C1-C ₆ -alkyl and C1-C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

also particularly preferred is phenyl which is substituted with three substituents selected from

- 58 044296 group consisting of halogen, CN, NO2, C1-C ₆ -alkyl, C1-C ₆ -haloalkyl, OH, C1-C ₆ -alkoxy, C ₁ -C ₆ alkylthio, (C ₁ -C ₆ - alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and

C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH3;

more preferably selected from the group consisting of F, Cl and CN;

also particularly preferably phenyl, which is substituted with four substituents selected from the group consisting of halogen, CN, NO2, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, OH, C ₁ -C ₆ -alkoxy, Q- C6alkylthio, (C ₁ -C ₆ -alkyl)sulfinyl, (C ₁ -C ₆ -alkyl)sulfonyl, amino, (C ₁ -C ₆ -alkyl)amino, di(C ₁ -C ₆ alkyl)amino, (C ₁ -C ₆ -alkyl)carbonyl, (C ₁ -C ₆ -alkoxy)carbonyl;

in particular, preferably selected from the group consisting of halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

particularly preferably selected from halogen and CN;

also particularly preferably selected from the group consisting of F, Cl, CN and CH ₃ ;

more preferably selected from the group consisting of F, Cl and CN;

R ¹ represents H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C ₁ -C ₆ alkoxy, (C ₁ -C ₆ -alkyl)carbonyl or (C ₁ -C ₆ -alkyl)sulfonyl;

in particular, preferably H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy, (C ₁ -C ₆ -alkyl)carbonyl or (C ₁ -C ₆ -alkyl)sulfonyl;

particularly preferably H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3; more preferably hydrogen.

R ² represents H, halogen, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -haloalkyl; in particular, preferably halogen, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -haloalkyl; also particularly preferably H, F, CH3 or CF3;

R3 and ^R4 are, independently of each other, H, halogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ haloalkyl; or together with the carbon atom to which they are attached, form a functional group selected from the group consisting of C ₃ -C ₆ -cycloalkyl, C ₃ -C ₆ -cycloalkenyl and three- to six-membered heterocyclyl, wherein C3-C6-cycloalkyl , C3-C6-cycloalkenyl or three to six-membered heterocyclyl is unsubstituted or substituted with one to three substituents selected from halogen, CN, C ₁ -C6 alkyl and C1C ₆ -alkoxy;

independently of each other, particularly preferably H, halogen, C ₁ -C ₆ alkyl or C ₁ -C ₆ haloalkyl; or together with the carbon atom to which they are attached, form a functional group selected from the group consisting of C ₃ -C ₆ -cycloalkyl and C ₃ -C ₆ -cycloalkenyl, wherein C ₃ -C ₆ -cycloalkyl or C ₃ -C ₆ -cycloalkenyl is unsubstituted or substituted with one to three substituents selected from halogen, CN, C ₁ -C ₆ -alkyl and C ₁ -C ₆ -alkoxy;

Independently of each other, H, halogen, C ₁ -C ₆ -alkyl or C ₁ -C ₆ -haloalkyl are particularly preferred;

independently of each other, more preferably H, halogen or C ₁ -C ₆ -alkyl; And

R ⁵ represents H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -haloalkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C1-C6 alkoxy, (C ₁ -C ₆ -alkyl)carbonyl or (C ₁ -C ₆ -alkyl)sulfonyl;

in particular, preferably H, CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy-C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy, (C _{1 -C 6} -alkyl)carbonyl or (C ₁ -C ₆ -alkyl)sulfonyl;

particularly preferably H, CN, CH3, CH2OCH3, OCH3, COCH3 or SO2CH3; more preferably hydrogen.

It is particularly preferred to use azines of formula (Ia) which correspond to azines of formula (I), characterized in that A is (A.1) and R ¹ and R ⁵ are H

characterized in that the variables R ^a , R ^b , R ^c , R ^d , R ^e , R ² , R ³ and R ⁴ have values, in particular preferred values, as defined above;

Particular preference is given to azines of the formulas (Ial)-(Ia 1406), given in table. A, where the definitions of the variables R ^a , R ^b , R ^c , R ^d , R ^e , R ² , R ³ and R ⁴ are of particular importance for the compounds of this invention, not only in combination with each other, but also in each case, separately.

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Table A

No. R ^{a Rb} R ^{c Rd} R ^e R ² R ³ R ⁴ I.a.l F H H H F СНз N n I.a.2 Cl H H H F СНз N n I.a.3 Br H H H F СНз N n I.a.4 CN H H H F СНз N n I.a.5 СНз H H H F СНз N n I.a.6 F H H F F СНз N n I.a.7 Cl H H F F СНз N n I.a.8 F H H Cl F СНз N n I.a.9 Cl H H F F СНз N n I.a.lO CN H H F F СНз N n La. eleven F H H CN F СНз N n La. 12 CN H H F F СНз N n I.a.13 F H F H F СНз N n La. 14 Cl H F H F СНз n n I.a.15 CN H F H F СНз n n I.a.16 F F F H F СНз n n I.a.17 Cl F F H F СНз n n I.a.18 F Cl F H F СНз n n I.a.19 Cl F F H F СНз n n I.a.20 CN F F H F СНз n n I.a.21 F CN F H F СНз n n I.a.22 CN F F H F СНз n n I.a.23 F F H F F СНз n n I.a.24 Cl F H F F СНз n n I.a.25 F Cl H F F СНз n n I.a.26 CN F H F F СНз n n I.a.27 F CN H F F СНз n n I.a.28 F F F F F СНз n n I.a.29 Cl F F F F СНз n n I.a.3O F Cl F F F СНз n n

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I.a.31 CN F F F F СНз N N I.a.32 F CN F F F СНз N N I.a.33 H F F F F СНз N N I.a.34 F F Br F F СНз N N I.a.35 F F C=CH F F СНз N N I.a.36 CF ₃ Cl H H F СНз N N I.a.37 F F I F F СНз N N I.a.38 F H H H F СНз СНз N I.a.39 Cl H H H F СНз СНз N La.40 Br H H H F СНз СНз N La.41 CN H H H F СНз СНз n I.a.42 СНз H H H F СНз СНз n I.a.43 F H H F F СНз СНз n I.a.44 Cl H H F F СНз СНз n I.a.45 F H H Cl F СНз СНз n I.a.46 Cl H H F F СНз СНз n I.a.47 CN H H F F СНз СНз n I.a.48 F H H CN F СНз СНз n I.a.49 CN H H F F СНз СНз n La.5O F H F H F СНз СНз n La.51 Cl H F H F СНз СНз n I.a.52 CN H F H F СНз СНз n I.a.53 F F F H F СНз СНз n I.a.54 Cl F F H F СНз СНз n I.a.55 F Cl F H F СНз СНз n I.a.56 Cl F F H F СНз СНз n I.a.57 CN F F H F СНз СНз n I.a.58 F CN F H F СНз СНз n I.a.59 CN F F H F СНз СНз n La. 60 F F H F F СНз СНз n I.a.61 Cl F H F F СНз СНз n I.a.62 F Cl H F F СНз СНз n

-61 044296

La.63 CN F H F F СНз СНз N La. 64 F CN H F F СНз СНз N I.a.65 F F F F F СНз СНз N La. 66 Cl F F F F СНз СНз N I.a.67 F Cl F F F СНз СНз N I.a.68 CN F F F F СНз СНз N I.a.69 F CN F F F СНз СНз N I.a.70 H F F F F СНз СНз N I.a.71 F F Br F F СНз СНз N I.a.72 F F C=CH F F СНз СНз N La. 73 CF ₃ Cl H H F СНз СНз n I.a.74 F F I F F СНз СНз n I.a.75 F H H H F СНз СНз СНз I.a.76 Cl H H H F СНз СНз СНз La. 77 Br H H H F СНз СНз СНз I.a.78 CN H H H F СНз СНз СНз I.a.79 СНз H H H F СНз СНз СНз I.a.8O F H H F F СНз СНз СНз I.a.81 Cl H H F F СНз СНз СНз I.a.82 F H H Cl F СНз СНз СНз I.a.83 Cl H H F F СНз СНз СНз La. 84 CN H H F F СНз СНз СНз I.a.85 F H H CN F СНз СНз СНз I.a.86 CN H H F F СНз СНз СНз I.a.87 F H F H F СНз СНз СНз I.a.88 Cl H F H F СНз СНз СНз I.a.89 CN H F H F СНз СНз СНз I.a.90 F F F H F СНз СНз СНз I.a.91 Cl F F H F СНз СНз СНз I.a.92 F Cl F H F СНз СНз СНз I.a.93 Cl F F H F СНз СНз СНз I.a.94 CN F F H F СНз СНз СНз

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I.a.95 F CN F H F СНз СНз СНз I.a.96 CN F F H F СНз СНз СНз I.a.97 F F H F F СНз СНз СНз I.a.98 Cl F H F F СНз СНз СНз I.a.99 F Cl H F F СНз СНз СНз La. 100 CN F H F F СНз СНз СНз I.a.101 F CN H F F СНз СНз СНз La. 102 F F F F F СНз СНз СНз La. 103 Cl F F F F СНз СНз СНз La. 104 F Cl F F F СНз СНз СНз I.a.105 CN F F F F СНз СНз СНз La. 106 F CN F F F СНз СНз СНз La. 107 H F F F F СНз СНз СНз I.a.108 F F Br F F СНз СНз СНз La. 109 F F C^CH F F СНз СНз СНз I.a.110 CF ₃ Cl H H F СНз СНз СНз I.a.lll F F I F F СНз СНз СНз La. 112 F H H H F F F F I.a.l 13 Cl H H H F F F F La. 114 Br H H H F F F F Lal 15 CN H H H F F F F La. 116 СНз H H H F F F F I.a.117 F H H F F F F F Lal 18 Cl H H F F F F F I.a.119 F H H Cl F F F F La. 120 Cl H H F F F F F I.a.121 CN H H F F F F F La. 122 F H H CN F F F F La. 123 CN H H F F F F F La. 124 F H F H F F F F La. 125 Cl H F H F F F F La. 126 CN H F H F F F F

- 63 044296

La. 127 F F F H F F F F La. 128 Cl F F H F F F F La. 129 F Cl F H F F F F I.a.130 Cl F F H F F F F I.a.131 CN F F H F F F F I.a.132 F CN F H F F F F I.a.133 CN F F H F F F F I.a.134 F F H F F F F F I.a.135 Cl F H F F F F F I.a.136 F Cl H F F F F F I.a.137 CN F H F F F F F I.a.138 F CN H F F F F F I.a.139 F F F F F F F F La. 140 Cl F F F F F F F I.a.141 F Cl F F F F F F La. 142 CN F F F F F F F La. 143 F CN F F F F F F La. 144 H F F F F F F F La. 145 F F Br F F F F F La. 146 F F C=CH F F F F F La. 147 CF ₃ Cl H H F F F F La. 148 F F I F F F F F La. 149 F H H H F F CF ₃ F I.a.150 Cl H H H F F CF ₃ F I.a.151 Br H H H F F CF ₃ F I.a.152 CN H H H F F CF ₃ F I.a.153 CH ₃ H H H F F CF ₃ F I.a.154 F H H F F F CF ₃ F I.a.155 Cl H H F F F CF ₃ F I.a.156 F H H Cl F F CF ₃ F I.a.157 Cl H H F F F CF ₃ F I.a.158 CN H H F F F CF ₃ F

- 64 044296

I.a.159 F H H CN F F CF ₃ F La. 160 CN H H F F F CF ₃ F I.a.161 F H F H F F CF ₃ F La. 162 Cl H F H F F CF ₃ F La. 163 CN H F H F F CF ₃ F La. 164 F F F H F F CF ₃ F I.a.165 Cl F F H F F CF ₃ F La. 166 F Cl F H F F CF ₃ F La. 167 Cl F F H F F CF ₃ F I.a.168 CN F F H F F CF ₃ F La. 169 F CN F H F F CF ₃ F La. 170 CN F F H F F CF ₃ F I.a.171 F F H F F F CF ₃ F La. 172 Cl F H F F F CF ₃ F La. 173 F Cl H F F F CF ₃ F La. 174 CN F H F F F CF ₃ F I.a.175 F CN H F F F CF ₃ F La. 176 F F F F F F CF ₃ F La. 177 Cl F F F F F CF ₃ F I.a.178 F Cl F F F F CF ₃ F La. 179 CN F F F F F CF ₃ F I.a.180 F CN F F F F CF ₃ F I.a.181 H F F F F F CF ₃ F I.a.182 F F Br F F F CF ₃ F I.a.183 F F C=CH F F F CF ₃ F I.a.184 CF ₃ Cl H H F F CF ₃ F I.a.185 F F I F F F CF ₃ F I.a.186 F H H H F F CH ₃ F I.a.187 Cl H H H F F CH ₃ F I.a.188 Br H H H F F CH ₃ F I.a.189 CN H H H F F CH ₃ F La. 190 СНз H H H F F CH ₃ F

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I.a.191 F H H F F F СНз F La. 192 Cl H H F F F СНз F La. 193 F H H Cl F F СНз F La. 194 Cl H H F F F СНз F I.a.195 CN H H F F F СНз F La. 196 F H H CN F F СНз F La. 197 CN H H F F F СНз F I.a.198 F H F H F F СНз F La. 199 Cl H F H F F СНз F I.a.200 CN H F H F F СНз F I.a.201 F F F H F F СНз F I.a.202 Cl F F H F F СНз F I.a.203 F Cl F H F F СНз F I.a.204 Cl F F H F F СНз F I.a.205 CN F F H F F СНз F I.a.206 F CN F H F F СНз F I.a.207 CN F F H F F СНз F I.a.208 F F H F F F СНз F I.a.209 Cl F H F F F СНз F I.a.210 F Cl H F F F СНз F I.a.211 CN F H F F F СНз F I.a.212 F CN H F F F СНз F I.a.213 F F F F F F СНз F I.a.214 Cl F F F F F СНз F I.a.215 F Cl F F F F СНз F I.a.216 CN F F F F F СНз F I.a.217 F CN F F F F СНз F I.a.218 H F F F F F СНз F I.a.219 F F Br F F F СНз F I.a.220 F F C=CH F F F СНз F I.a.221 CF ₃ Cl H H F F СНз F I.a.222 F F I F F F СНз F

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I.a.223 F H H H F F СНз N La.224 Cl H H H F F СНз N La.225 Br H H H F F СНз N I.a.226 CN H H H F F СНз N I.a.227 СНз H H H F F СНз N I.a.228 F H H F F F СНз N I.a.229 Cl H H F F F СНз N La.230 F H H Cl F F СНз N La.231 Cl H H F F F СНз N I.a.232 CN H H F F F СНз N I.a.233 F H H CN F F СНз n La.234 CN H H F F F СНз n La.235 F H F H F F СНз n La.236 Cl H F H F F СНз n I.a.237 CN H F H F F СНз n La.238 F F F H F F СНз n La.239 Cl F F H F F СНз n La.240 F Cl F H F F СНз n La.241 Cl F F H F F СНз n I.a.242 CN F F H F F СНз n I.a.243 F CN F H F F СНз n I.a.244 CN F F H F F СНз n I.a.245 F F H F F F СНз n I.a.246 Cl F H F F F СНз n I.a.247 F Cl H F F F СНз n I.a.248 CN F H F F F СНз n I.a.249 F CN H F F F СНз n La.250 F F F F F F СНз n La.251 Cl F F F F F СНз n I.a.252 F Cl F F F F СНз n La.253 CN F F F F F СНз n I.a.254 F CN F F F F СНз n

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I.a.255 H F F F F F СНз N I.a.256 F F Br F F F СНз N I.a.257 F F C=CH F F F СНз N I.a.258 CF ₃ Cl H H F F СНз N I.a.259 F F I F F F СНз N La.260 F H H H F F СНз СНз La.261 Cl H H H F F СНз СНз I.a.262 Br H H H F F СНз СНз I.a.263 CN H H H F F СНз СНз I.a.264 СНз H H H F F СНз СНз I.a.265 F H H F F F СНз СНз I.a.266 Cl H H F F F СНз СНз I.a.267 F H H Cl F F СНз СНз I.a.268 Cl H H F F F СНз СНз I.a.269 CN H H F F F СНз СНз La.270 F H H CN F F СНз СНз La.271 CN H H F F F СНз СНз I.a.272 F H F H F F СНз СНз I.a.273 Cl H F H F F СНз СНз I.a.274 CN H F H F F СНз СНз I.a.275 F F F H F F СНз СНз I.a.276 Cl F F H F F СНз СНз I.a.277 F Cl F H F F СНз СНз I.a.278 Cl F F H F F СНз СНз I.a.279 CN F F H F F СНз СНз La.280 F CN F H F F СНз СНз La.281 CN F F H F F СНз СНз I.a.282 F F H F F F СНз СНз La.283 Cl F H F F F СНз СНз I.a.284 F Cl H F F F СНз СНз I.a.285 CN F H F F F СНз СНз I.a.286 F CN H F F F СНз СНз

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I.a.287 F F F F F F СНз СНз I.a.288 Cl F F F F F СНз СНз I.a.289 F Cl F F F F СНз СНз La.290 CN F F F F F СНз СНз La.291 F CN F F F F СНз СНз I.a.292 H F F F F F СНз СНз La.293 F F Br F F F СНз СНз I.a.294 F F C=CH F F F СНз СНз I.a.295 CF ₃ Cl H H F F СНз СНз I.a.296 F F I F F F СНз СНз I.a.297 F H H H F Cl СНз СНз I.a.298 Cl H H H F Cl СНз СНз I.a.299 Br H H H F Cl СНз СНз La.3OO CN H H H F Cl СНз СНз La.301 СНз H H H F Cl СНз СНз La.302 F H H F F Cl СНз СНз La.3O3 Cl H H F F Cl СНз СНз La.304 F H H Cl F Cl СНз СНз La.3O5 Cl H H F F Cl СНз СНз La.306 CN H H F F Cl СНз СНз La.307 F H H CN F Cl СНз СНз La.3O8 CN H H F F Cl СНз СНз La.309 F H F H F Cl СНз СНз La.31O Cl H F H F Cl СНз СНз La.311 CN H F H F Cl СНз СНз La.312 F F F H F Cl СНз СНз I.a.313 Cl F F H F Cl СНз СНз La.314 F Cl F H F Cl СНз СНз La.315 Cl F F H F Cl СНз СНз La.316 CN F F H F Cl СНз СНз La.317 F CN F H F Cl СНз СНз La.318 CN F F H F Cl СНз СНз

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I.a.319 F F H F F Cl CH ₃ CH ₃ La.320 Cl F H F F Cl CH ₃ CH ₃ La.321 F Cl H F F Cl CH ₃ CH ₃ La.322 CN F H F F Cl CH ₃ CH ₃ I.a.323 F CN H F F Cl CH ₃ CH ₃ I.a.324 F F F F F Cl CH ₃ CH ₃ I.a.325 Cl F F F F Cl CH ₃ CH ₃ I.a.326 F Cl F F F Cl CH ₃ CH ₃ I.a.327 CN F F F F Cl CH ₃ CH ₃ I.a.328 F CN F F F Cl CH ₃ CH ₃ I.a.329 H F F F F Cl CH ₃ CH ₃ I.a.330 F F Br F F Cl CH ₃ CH ₃ La.331 F F C=CH F F Cl CH ₃ CH ₃ I.a.332 CF ₃ Cl H H F Cl CH ₃ CH ₃ I.a.333 F F I F F Cl CH ₃ CH ₃ I.a.334 F H H H F F _{C2H5 _} CH ₃ I.a.335 Cl H H H F F _{C2H5 _} СНз I.a.336 Br H H H F F _{C2H5 _} СНз I.a.337 CN H H H F F _{C2H5 _} СНз La.338 CH ₃ H H H F F _{C2H5 _} СНз I.a.339 F H H F F F _{C2H5 _} СНз La.340 Cl H H F F F _{C2H5 _} СНз La.341 F H H Cl F F _{C2H5 _} СНз I.a.342 Cl H H F F F _{C2H5 _} СНз I.a.343 CN H H F F F _{C2H5 _} СНз I.a.344 F H H CN F F _{C2H5 _} СНз I.a.345 CN H H F F F _{C2H5 _} СНз I.a.346 F H F H F F _{C2H5 _} СНз I.a.347 Cl H F H F F _{C2H5 _} СНз I.a.348 CN H F H F F _{C2H5 _} СНз I.a.349 F F F H F F _{C2H5 _} СНз La.35O Cl F F H F F _{C2H5 _} СНз

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I.a.351 F Cl F H F F _{C2H5 _} СНз I.a.352 Cl F F H F F _{C2H5 _} СНз La.353 CN F F H F F _{C2H5 _} СНз La.354 F CN F H F F _{C2H5 _} СНз I.a.355 CN F F H F F _{C2H5 _} СНз I.a.356 F F H F F F _{C2H5 _} СНз I.a.357 Cl F H F F F _{C2H5 _} СНз I.a.358 F Cl H F F F _{C2H5 _} СНз I.a.359 CN F H F F F _{C2H5 _} СНз La.36O F CN H F F F _{C2H5 _} СНз La.361 F F F F F F _{C2H5 _} СНз I.a.362 Cl F F F F F _{C2H5 _} СНз La.363 F Cl F F F F _{C2H5 _} СНз I.a.364 CN F F F F F _{C2H5 _} СНз La.365 F CN F F F F _{C2H5 _} СНз I.a.366 H F F F F F _{C2H5 _} СНз I.a.367 F F Br F F F _{C2H5 _} СНз La.368 F F C=CH F F F _{C2H5 _} СНз I.a.369 CF ₃ Cl H H F F _{C2H5 _} СНз La.37O F F I F F F _{C2H5 _} СНз La.371 F H H H F F _{C2H5 _} C ₂ H ₅ I. a. 372 Cl H H H F F _{C2H5 _} C ₂ H ₅ La.373 Br H H H F F _{C2H5 _} C ₂ H ₅ I.a.374 CN H H H F F _{C2H5 _} C ₂ H ₅ I.a.375 СНз H H H F F _{C2H5 _} C ₂ H ₅ I.a.376 F H H F F F _{C2H5 _} C ₂ H ₅ I.a.377 Cl H H F F F _{C2H5 _} C ₂ H ₅ La.378 F H H Cl F F _{C2H5 _} C ₂ H ₅ I.a.379 Cl H H F F F _{C2H5 _} C ₂ H ₅ La.38O CN H H F F F _{C2H5 _} C ₂ H ₅ La.381 F H H CN F F _{C2H5 _} C ₂ H ₅ I.a.382 CN H H F F F _{C2H5 _} C ₂ H ₅

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I.a.383 F H F H F F _{C2H5 _ C2H5 _} I.a.384 Cl H F H F F _{C2H5 _ C2H5 _} I.a.385 CN H F H F F C2H5 _{C2H5 _} I.a.386 F F F H F F _{C2H5 _ C2H5 _} I.a.387 Cl F F H F F _{C2H5 _ C2H5 _} I.a.388 F Cl F H F F _{C2H5 _ C2H5 _} I.a.389 Cl F F H F F _{C2H5 _ C2H5 _} La.390 CN F F H F F _{C2H5 _ C2H5 _} La.391 F CN F H F F _{C2H5 _ C2H5 _} I.a.392 CN F F H F F _{C2H5 _ C2H5 _} I.a.393 F F H F F F _{C2H5 _ C2H5 _} I.a.394 Cl F H F F F _{C2H5 _ C2H5 _} I.a.395 F Cl H F F F _{C2H5 _ C2H5 _} I.a.396 CN F H F F F _{C2H5 _ C2H5 _} I.a.397 F CN H F F F _{C2H5 _ C2H5 _} I.a.398 F F F F F F _{C2H5 _ C2H5 _} I.a.399 Cl F F F F F _{C2H5 _ C2H5 _} La.400 F Cl F F F F _{C2H5 _ C2H5 _} La.401 CN F F F F F _{C2H5 _ C2H5 _} I.a.402 F CN F F F F _{C2H5 _ C2H5 _} La.403 H F F F F F _{C2H5 _ C2H5 _} La.404 F F Br F F F _{C2H5 _ C2H5 _} I.a.405 F F C=CH F F F _{C2H5 _ C2H5 _} La.406 CF ₃ Cl H H F F _{C2H5 _ C2H5 _} La.407 F F I F F F _{C2H5 _ C2H5 _} I.a.408 F H H H F H -(CH ₂ ) ₂ - La.409 Cl H H H F H -(CH ₂ ) ₂ - La.410 Br H H H F H -(CH ₂ ) ₂ - La.411 CN H H H F H -(CH ₂ ) ₂ - La.412 CH ₃ H H H F H -(CH ₂ ) ₂ - La.413 F H H F F H -(CH ₂ ) ₂ - La.414 Cl H H F F H -(CH ₂ ) ₂ -

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La.415 F H H Cl F H -(CH ₂ ) ₂ - La.416 Cl H H F F H -(CH ₂ ) ₂ - I.a.417 CN H H F F H -(CH ₂ ) ₂ - La.418 F H H CN F H -(CH ₂ ) ₂ - La.419 CN H H F F H -(CH ₂ ) ₂ - La.420 F H F H F H -(CH ₂ ) ₂ - La.421 Cl H F H F H -(CH ₂ ) ₂ - I.a.422 CN H F H F H -(CH ₂ ) ₂ - I.a.423 F F F H F H -(CH ₂ ) ₂ - I.a.424 Cl F F H F H -(CH ₂ ) ₂ - I.a.425 F Cl F H F H -(CH ₂ ) ₂ - I.a.426 Cl F F H F H -(CH ₂ ) ₂ - I.a.427 CN F F H F H -(CH ₂ ) ₂ - I.a.428 F CN F H F H -(CH ₂ ) ₂ - I.a.429 CN F F H F H -(CH ₂ ) ₂ - La.430 F F H F F H -(CH ₂ ) ₂ - La.431 Cl F H F F H -(CH ₂ ) ₂ - I.a.432 F Cl H F F H -(CH ₂ ) ₂ - I.a.433 CN F H F F H -(CH ₂ ) ₂ - La.434 F CN H F F H -(CH ₂ ) ₂ - I.a.435 F F F F F H -(CH ₂ ) ₂ - I.a.436 Cl F F F F H -(CH ₂ ) ₂ - I.a.437 F Cl F F F H -(CH ₂ ) ₂ - I.a.438 CN F F F F H -(CH ₂ ) ₂ - I.a.439 F CN F F F H -(CH ₂ ) ₂ - I.a.440 H F F F F H -(CH ₂ ) ₂ - La.441 F F Br F F H -(CH ₂ ) ₂ - I.a.442 F F C=CH F F H -(CH ₂ ) ₂ - I.a.443 CF ₃ Cl H H F H -(CH ₂ ) ₂ - I.a.444 F F I F F H -(CH ₂ ) ₂ - I.a.445 F H H H F H -(CH ₂ )s- I.a.446 Cl H H H F H -(CH ₂ )s-

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I.a.447 Br H H H F H -(CH ₂ )s- I.a.448 CN H H H F H -(CH ₂ )s- I.a.449 СНз H H H F H -(CH ₂ )s- La.450 F H H F F H -(CH ₂ ) ₃ - La.451 Cl H H F F H -(CH ₂ ) ₃ - I.a.452 F H H Cl F H -(CH ₂ ) ₃ - I.a.453 Cl H H F F H -(CH ₂ ) ₃ - I.a.454 CN H H F F H -(CH ₂ ) ₃ - I.a.455 F H H CN F H -(CH ₂ ) ₃ - I.a.456 CN H H F F H -(CH ₂ ) ₃ - I.a.457 F H F H F H -(CH ₂ ) ₃ - La.458 Cl H F H F H -(CH ₂ ) ₃ - I.a.459 CN H F H F H -(CH ₂ ) ₃ - La.460 F F F H F H -(CH ₂ ) ₃ - La.461 Cl F F H F H -(CH ₂ )s- I.a.462 F Cl F H F H -(CH ₂ ) ₃ - I.a.463 Cl F F H F H -(CH ₂ ) ₃ - I.a.464 CN F F H F H -(CH ₂ ) ₃ - I.a.465 F CN F H F H -(CH ₂ ) ₃ - I.a.466 CN F F H F H -(CH ₂ )s- I.a.467 F F H F F H -(CH ₂ ) ₃ - La.468 Cl F H F F H -(CH ₂ ) ₃ - I.a.469 F Cl H F F H -(CH ₂ ) ₃ - I.a.470 CN F H F F H -(CH ₂ ) ₃ - La.471 F CN H F F H -(CH ₂ ) ₃ - I.a.472 F F F F F H -(CH ₂ ) ₃ - I.a.473 Cl F F F F H -(CH ₂ ) ₃ - I.a.474 F Cl F F F H -(CH ₂ ) ₃ - I.a.475 CN F F F F H -(CH ₂ ) ₃ - I.a.476 F CN F F F H -(CH ₂ ) ₃ - I.a.477 H F F F F H -(CH ₂ ) ₃ - I.a.478 F F Br F F H -(CH ₂ )s-

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I.a.479 F F C=CH F F H -(CH ₂ )s- La.480 CF ₃ Cl H H F H -(CH ₂ )s- La.481 F F I F F H -(CH ₂ )s- I.a.482 F H H H F H -(CH ₂ ) ₄ - I.a.483 Cl H H H F H -(CH ₂ ) ₄ - I.a.484 Br H H H F H -(CH ₂ ) ₄ - La.485 CN H H H F H -(CH ₂ ) ₄ - La.486 СНз H H H F H -(CH ₂ ) ₄ - I.a.487 F H H F F H -(CH ₂ ) ₄ - La.488 Cl H H F F H -(CH ₂ ) ₄ - I.a.489 F H H Cl F H -(CH ₂ ) ₄ - La.490 Cl H H F F H -(CH ₂ ) ₄ - La.491 CN H H F F H -(CH ₂ ) ₄ - I.a.492 F H H CN F H -(CH ₂ ) ₄ - I.a.493 CN H H F F H -(CH ₂ ) ₄ - I.a.494 F H F H F H -(CH ₂ ) ₄ - I.a.495 Cl H F H F H -(CH ₂ ) ₄ - I.a.496 CN H F H F H -(CH ₂ ) ₄ - I.a.497 F F F H F H -(CH ₂ ) ₄ - I.a.498 Cl F F H F H -(CH ₂ ) ₄ - I.a.499 F Cl F H F H -(CH ₂ ) ₄ - La.5OO Cl F F H F H -(CH ₂ ) ₄ - La.501 CN F F H F H -(CH ₂ ) ₄ - La.502 F CN F H F H -(CH ₂ ) ₄ - La.5O3 CN F F H F H -(CH ₂ ) ₄ - La.504 F F H F F H -(CH ₂ ) ₄ - La.5O5 Cl F H F F H -(CH ₂ ) ₄ - La.506 F Cl H F F H -(CH ₂ ) ₄ - La.507 CN F H F F H -(CH ₂ ) ₄ - La.5O8 F CN H F F H -(CH ₂ ) ₄ - La.509 F F F F F H -(CH ₂ ) ₄ - La.51O Cl F F F F H -(CH ₂ ) ₄ -

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I.a.511 F Cl F F F H -(CH ₂ )4- La.512 CN F F F F H -(CH ₂ )4- I.a.513 F CN F F F H -(CH ₂ ) ₄ - La.514 H F F F F H -(CH ₂ ) ₄ - La.515 F F Br F F H -(CH ₂ ) ₄ - La.516 F F C=CH F F H -(CH ₂ ) ₄ - La.517 CF ₃ Cl H H F H -(CH ₂ )4- La.518 F F I F F H -(CH ₂ ) ₄ - La.519 F H H H F H -(CH ₂ )5- La.520 Cl H H H F H -(CH ₂ )5- La.521 Br H H H F H -(CH ₂ )5- I.a.522 CN H H H F H -(CH ₂ ) ₅ - I.a.523 CH ₃ H H H F H -(CH ₂ ) ₅ - I.a.524 F H H F F H -(CH ₂ ) ₅ - I.a.525 Cl H H F F H -(CH ₂ ) ₅ - I.a.526 F H H Cl F H -(CH ₂ ) ₅ - I.a.527 Cl H H F F H -(CH ₂ ) ₅ - I.a.528 CN H H F F H -(CH ₂ ) ₅ - I.a.529 F H H CN F H -(CH ₂ ) ₅ - La.53O CN H H F F H -(CH ₂ )5- La.531 F H F H F H -(CH ₂ ) ₅ - I.a.532 Cl H F H F H -(CH ₂ ) ₅ - I.a.533 CN H F H F H -(CH ₂ ) ₅ - I.a.534 F F F H F H -(CH ₂ ) ₅ - I.a.535 Cl F F H F H -(CH ₂ ) ₅ - I.a.536 F Cl F H F H -(CH ₂ )5- I.a.537 Cl F F H F H -(CH ₂ ) ₅ - La.538 CN F F H F H -(CH ₂ )5- I.a.539 F CN F H F H -(CH ₂ ) ₅ - La.540 CN F F H F H -(CH ₂ ) ₅ - I.a.541 F F H F F H -(CH ₂ ) ₅ - I.a.542 Cl F H F F H -(CH ₂ ) ₅ -

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I.a.543 F Cl H F F H -(CH ₂ ) ₅ - I.a.544 CN F H F F H -(CH ₂ ) ₅ - La.545 F CN H F F H -(CH ₂ ) ₅ - La.546 F F F F F H -(CH ₂ ) ₅ - La.547 Cl F F F F H -(CH ₂ ) ₅ - La.548 F Cl F F F H -(CH ₂ ) ₅ - I.a.549 CN F F F F H -(CH ₂ ) ₅ - La.55O F CN F F F H -(CH ₂ ) ₅ - La.551 H F F F F H -(CH ₂ ) ₅ - La.552 F F Br F F H -(CH ₂ ) ₅ - La.553 F F C=CH F F H -(CH ₂ ) ₅ - I.a.554 CF ₃ Cl H H F H -(CH ₂ ) ₅ - La.555 F F I F F H -(CH ₂ ) ₅ - La.556 F H H H F СНз -(CH ₂ ) ₂ - La.557 Cl H H H F СНз -(CH ₂ ) ₂ - La.558 Br H H H F СНз -(CH ₂ ) ₂ - La.559 CN H H H F СНз -(CH ₂ ) ₂ - La.56O СНз H H H F СНз -(CH ₂ ) ₂ - La.561 F H H F F СНз -(CH ₂ ) ₂ - La.562 Cl H H F F СНз -(CH ₂ ) ₂ - La.563 F H H Cl F СНз -(CH ₂ ) ₂ - La.564 Cl H H F F СНз -(CH ₂ ) ₂ - La.565 CN H H F F СНз -(CH ₂ ) ₂ - La.566 F H H CN F СНз -(CH ₂ ) ₂ - La.567 CN H H F F СНз -(CH ₂ ) ₂ - La.568 F H F H F СНз -(CH ₂ ) ₂ - I.a.569 Cl H F H F СНз -(CH ₂ ) ₂ - La.57O CN H F H F СНз -(CH ₂ ) ₂ - La.571 F F F H F СНз -(CH ₂ ) ₂ - La.572 Cl F F H F СНз -(CH ₂ ) ₂ - La.573 F Cl F H F СНз -(CH ₂ ) ₂ - La.574 Cl F F H F СНз -(CH ₂ ) ₂ -

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I.a.575 CN F F H F CH ₃ -(CH ₂ ) ₂ - I.a.576 F CN F H F CH ₃ -(CH ₂ ) ₂ - I.a.577 CN F F H F CH ₃ -(CH ₂ ) ₂ - La.578 F F H F F CH ₃ -(CH ₂ ) ₂ - I.a.579 Cl F H F F CH ₃ -(CH ₂ ) ₂ - La.58O F Cl H F F CH ₃ -(CH ₂ ) ₂ - I.a.581 CN F H F F CH ₃ -(CH ₂ ) ₂ - I.a.582 F CN H F F CH ₃ -(CH ₂ ) ₂ - I.a.583 F F F F F CH ₃ -(CH ₂ ) ₂ - I.a.584 Cl F F F F СНз -(CH ₂ ) ₂ - I.a.585 F Cl F F F СНз -(CH ₂ ) ₂ - I.a.586 CN F F F F СНз -(CH ₂ ) ₂ - I.a.587 F CN F F F СНз -(CH ₂ ) ₂ - La.588 H F F F F СНз -(CH ₂ ) ₂ - I.a.589 F F Br F F СНз -(CH ₂ ) ₂ - La.59O F F C=CH F F СНз -(CH ₂ ) ₂ - La.591 CF ₃ Cl H H F СНз -(CH ₂ ) ₂ - I.a.592 F F I F F СНз -(CH ₂ ) ₂ - I.a.593 F H H H F СНз -(CH ₂ ) ₃ - I.a.594 Cl H H H F СНз -(CH ₂ ) ₃ - I.a.595 Br H H H F СНз -(CH ₂ ) ₃ - I.a.596 CN H H H F СНз -(CH ₂ ) ₃ - I.a.597 CH ₃ H H H F СНз -(CH ₂ )s- La.598 F H H F F СНз -(CH ₂ ) ₃ - I.a.599 Cl H H F F СНз -(CH ₂ ) ₃ - La. 600 F H H Cl F СНз -(CH ₂ ) ₃ - La. 601 Cl H H F F СНз -(CH ₂ ) ₃ - La. 602 CN H H F F СНз -(CH ₂ ) ₃ - La. 603 F H H CN F СНз -(CH ₂ ) ₃ - La. 604 CN H H F F СНз -(CH ₂ ) ₃ - La. 605 F H F H F СНз -(CH ₂ ) ₃ - La. 606 Cl H F H F СНз -(CH ₂ ) ₃ -

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La. 607 CN H F H F СНз -(CH ₂ )s- La. 608 F F F H F СНз -(CH ₂ )s- La. 609 Cl F F H F СНз -(CH ₂ ) ₃ - I.a.610 F Cl F H F СНз -(CH ₂ ) ₃ - I.a.611 Cl F F H F СНз -(CH ₂ ) ₃ - I.a.612 CN F F H F СНз -(CH ₂ ) ₃ - I.a.613 F CN F H F СНз -(CH ₂ ) ₃ - I.a.614 CN F F H F СНз -(CH ₂ ) ₃ - I.a.615 F F H F F СНз -(CH ₂ ) ₃ - I.a.616 Cl F H F F СНз -(CH ₂ ) ₃ - I.a.617 F Cl H F F СНз -(CH ₂ ) ₃ - I.a.618 CN F H F F СНз -(CH ₂ ) ₃ - I.a.619 F CN H F F СНз -(CH ₂ ) ₃ - I.a.620 F F F F F СНз -(CH ₂ ) ₃ - I.a.621 Cl F F F F СНз -(CH ₂ )s- I. a. 622 F Cl F F F СНз -(CH ₂ ) ₃ - I.a.623 CN F F F F СНз -(CH ₂ ) ₃ - I. a. 624 F CN F F F СНз -(CH ₂ ) ₃ - I.a.625 H F F F F СНз -(CH ₂ ) ₃ - I.a.626 F F Br F F СНз -(CH ₂ )s- I.a.627 F F C^CH F F СНз -(CH ₂ ) ₃ - I.a.628 CF ₃ Cl H H F СНз -(CH ₂ ) ₃ - I.a.629 F F I F F СНз -(CH ₂ ) ₃ - I.a.630 F H H H F СНз -(CH ₂ ) ₄ - I.a.631 Cl H H H F СНз -(CH ₂ ) ₄ - I.a.632 Br H H H F СНз -(CH ₂ ) ₄ - I.a.633 CN H H H F СНз -(CH ₂ ) ₄ - I.a.634 СНз H H H F СНз -(CH ₂ ) ₄ - I.a.635 F H H F F СНз -(CH ₂ ) ₄ - I.a.636 Cl H H F F СНз -(CH ₂ ) ₄ - I.a.637 F H H Cl F СНз -(CH ₂ ) ₄ - I.a.638 Cl H H F F СНз -(CH ₂ ) ₄ -

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La.639 CN H H F F CH ₃ -(CH ₂ ) ₄ - La. 640 F H H CN F СНз -(CH ₂ ) ₄ - La. 641 CN H H F F СНз -(CH ₂ ) ₄ - I. a. 642 F H F H F СНз -(CH ₂ ) ₄ - La. 643 Cl H F H F СНз -(CH ₂ ) ₄ - I. a. 644 CN H F H F СНз -(CH ₂ ) ₄ - La. 645 F F F H F СНз -(CH ₂ ) ₄ - La. 646 Cl F F H F СНз -(CH ₂ ) ₄ - La. 647 F Cl F H F СНз -(CH ₂ ) ₄ - La. 648 Cl F F H F СНз -(CH ₂ ) ₄ - La. 649 CN F F H F СНз -(CH ₂ ) ₄ - I.a.650 F CN F H F СНз -(CH ₂ ) ₄ - I.a.651 CN F F H F СНз -(CH ₂ ) ₄ - I.a.652 F F H F F СНз -(CH ₂ ) ₄ - I.a.653 Cl F H F F СНз -(CH ₂ ) ₄ - I.a.654 F Cl H F F СНз -(CH ₂ ) ₄ - I.a.655 CN F H F F СНз -(CH ₂ ) ₄ - I.a.656 F CN H F F СНз -(CH ₂ ) ₄ - I.a.657 F F F F F СНз -(CH ₂ ) ₄ - I.a.658 Cl F F F F СНз -(CH ₂ ) ₄ - I.a.659 F Cl F F F СНз -(CH ₂ ) ₄ - La. 660 CN F F F F СНз -(CH ₂ ) ₄ - La. 661 F CN F F F СНз -(CH ₂ ) ₄ - La. 662 H F F F F СНз -(CH ₂ ) ₄ - La. 663 F F Br F F СНз -(CH ₂ ) ₄ - La. 664 F F C=CH F F СНз -(CH ₂ ) ₄ - La. 665 CF ₃ Cl H H F СНз -(CH ₂ ) ₄ - I.a.666 F F I F F СНз -(CH ₂ ) ₄ - La. 667 F H H H F СНз -(CH ₂ ) ₅ - La. 668 Cl H H H F СНз -(CH ₂ ) ₅ - La. 669 Br H H H F СНз -(CH ₂ ) ₅ - I.a.670 CN H H H F СНз -(CH ₂ ) ₅ -

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La.671 СНз H H H F СНз -(CH ₂ ) ₅ - I.a.672 F H H F F СНз -(CH ₂ ) ₅ - La.673 Cl H H F F СНз -(CH ₂ ) ₅ - I.a.674 F H H Cl F СНз -(CH ₂ ) ₅ - La.675 Cl H H F F СНз -(CH ₂ ) ₅ - La.676 CN H H F F СНз -(CH ₂ ) ₅ - I.a.677 F H H CN F СНз -(CH ₂ ) ₅ - La.678 CN H H F F СНз -(CH ₂ ) ₅ - La.679 F H F H F СНз -(CH ₂ ) ₅ - La.680 Cl H F H F СНз -(CH ₂ ) ₅ - La.681 CN H F H F СНз -(CH ₂ ) ₅ - La.682 F F F H F СНз -(CH ₂ ) ₅ - La.683 Cl F F H F СНз -(CH ₂ ) ₅ - La.684 F Cl F H F СНз -(CH ₂ ) ₅ - I.a.685 Cl F F H F СНз -(CH ₂ ) ₅ - La.686 CN F F H F СНз -(CH ₂ ) ₅ - La.687 F CN F H F СНз -(CH ₂ ) ₅ - La.688 CN F F H F СНз -(CH ₂ ) ₅ - La.689 F F H F F СНз -(CH ₂ ) ₅ - I. a. 690 Cl F H F F СНз -(CH ₂ ) ₅ - I. a. 691 F Cl H F F СНз -(CH ₂ ) ₅ - I.a.692 CN F H F F СНз -(CH ₂ ) ₅ - La.693 F CN H F F СНз -(CH ₂ ) ₅ - I.a.694 F F F F F СНз -(CH ₂ ) ₅ - I. a. 695 Cl F F F F СНз -(CH ₂ ) ₅ - La.696 F Cl F F F СНз -(CH ₂ ) ₅ - I. a. 697 CN F F F F СНз -(CH ₂ ) ₅ - I. a. 698 F CN F F F СНз -(CH ₂ ) ₅ - I. a. 699 H F F F F СНз -(CH ₂ ) ₅ - La.700 F F Br F F СНз -(CH ₂ ) ₅ - La.701 F F C=CH F F СНз -(CH ₂ ) ₅ - La.702 CF ₃ Cl H H F СНз -(CH ₂ ) ₅ -

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I.a.703 F F I F F СНз -(CH ₂ ) ₅ - I.a.704 F H H H F F -(CH ₂ ) ₂ - La.705 Cl H H H F F -(CH ₂ ) ₂ - I.a.706 Br H H H F F -(CH ₂ ) ₂ - I.a.707 CN H H H F F -(CH ₂ ) ₂ - La.708 СНз H H H F F -(CH ₂ ) ₂ - I.a.709 F H H F F F -(CH ₂ ) ₂ - La.710 Cl H H F F F -(CH ₂ ) ₂ - I.a.711 F H H Cl F F -(CH ₂ ) ₂ - I.a.712 Cl H H F F F -(CH ₂ ) ₂ - I.a.713 CN H H F F F -(CH ₂ ) ₂ - I.a.714 F H H CN F F -(CH ₂ ) ₂ - La.715 CN H H F F F -(CH ₂ ) ₂ - La.716 F H F H F F -(CH ₂ ) ₂ - I.a.717 Cl H F H F F -(CH ₂ ) ₂ - La.718 CN H F H F F -(CH ₂ ) ₂ - I.a.719 F F F H F F -(CH ₂ ) ₂ - La.720 Cl F F H F F -(CH ₂ ) ₂ - I.a.721 F Cl F H F F -(CH ₂ ) ₂ - I. a. 722 Cl F F H F F -(CH ₂ ) ₂ - I.a.723 CN F F H F F -(CH ₂ ) ₂ - I. a. 724 F CN F H F F -(CH ₂ ) ₂ - I.a.725 CN F F H F F -(CH ₂ ) ₂ - I.a.726 F F H F F F -(CH ₂ ) ₂ - I.a.727 Cl F H F F F -(CH ₂ ) ₂ - I.a.728 F Cl H F F F -(CH ₂ ) ₂ - I.a.729 CN F H F F F -(CH ₂ ) ₂ - La. 730 F CN H F F F -(CH ₂ ) ₂ - La. 731 F F F F F F -(CH ₂ ) ₂ - La. 732 Cl F F F F F -(CH ₂ ) ₂ - La. 733 F Cl F F F F -(CH ₂ ) ₂ - La. 734 CN F F F F F -(CH ₂ ) ₂ -

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La. 735 F CN F F F F -(CH ₂ ) ₂ - La. 736 H F F F F F -(CH ₂ ) ₂ - I. a. 73 7 F F Br F F F -(CH ₂ ) ₂ - La. 738 F F C=CH F F F -(CH ₂ ) ₂ - La. 739 CF ₃ Cl H H F F -(CH ₂ ) ₂ - I.a.740 F F I F F F -(CH ₂ ) ₂ - I.a.741 F H H H F F -(CH ₂ ) ₃ - I. a. 742 Cl H H H F F -(CH ₂ ) ₃ - I.a.743 Br H H H F F -(CH ₂ ) ₃ - I. a. 744 CN H H H F F -(CH ₂ ) ₃ - I.a.745 CH ₃ H H H F F -(CH ₂ ) ₃ - I.a.746 F H H F F F -(CH ₂ ) ₃ - I.a.747 Cl H H F F F -(CH ₂ ) ₃ - I.a.748 F H H Cl F F -(CH ₂ ) ₃ - I.a.749 Cl H H F F F -(CH ₂ ) ₃ - I.a.750 CN H H F F F -(CH ₂ ) ₃ - I.a.751 F H H CN F F -(CH ₂ ) ₃ - I.a.752 CN H H F F F -(CH ₂ ) ₃ - I.a.753 F H F H F F -(CH ₂ ) ₃ - I.a.754 Cl H F H F F -(CH ₂ ) ₃ - I.a.755 CN H F H F F -(CH ₂ ) ₃ - I.a.756 F F F H F F -(CH ₂ ) ₃ - I.a.757 Cl F F H F F -(CH ₂ ) ₃ - I.a.758 F Cl F H F F -(CH ₂ ) ₃ - I.a.759 Cl F F H F F -(CH ₂ ) ₃ - I. a. 760 CN F F H F F -(CH ₂ ) ₃ - I.a.761 F CN F H F F -(CH ₂ ) ₃ - I.a.762 CN F F H F F -(CH ₂ ) ₃ - I.a.763 F F H F F F -(CH ₂ ) ₃ - I.a.764 Cl F H F F F -(CH ₂ ) ₃ - I.a.765 F Cl H F F F -(CH ₂ ) ₃ - I.a.766 CN F H F F F -(CH ₂ ) ₃ -

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I.a.767 F CN H F F F -(CH ₂ ) ₃ - I.a.768 F F F F F F -(CH ₂ ) ₃ - I.a.769 Cl F F F F F -(CH ₂ ) ₃ - La. 770 F Cl F F F F -(CH ₂ ) ₃ - La. 771 CN F F F F F -(CH ₂ ) ₃ - La. 772 F CN F F F F -(CH ₂ ) ₃ - La. 773 H F F F F F -(CH ₂ ) ₃ - La. 774 F F Br F F F -(CH ₂ ) ₃ - La. 775 F F C=CH F F F -(CH ₂ ) ₃ - La. 776 CF ₃ Cl H H F F -(CH ₂ ) ₃ - La. 777 F F I F F F -(CH ₂ ) ₃ - La. 778 F H H H F F -(CH ₂ ) ₄ - La. 779 Cl H H H F F -(CH ₂ ) ₄ - I.a.780 Br H H H F F -(CH ₂ ) ₄ - I.a.781 CN H H H F F -(CH ₂ ) ₄ - I.a.782 CH ₃ H H H F F -(CH ₂ ) ₄ - I.a.783 F H H F F F -(CH ₂ ) ₄ - I.a.784 Cl H H F F F -(CH ₂ ) ₄ - I.a.785 F H H Cl F F -(CH ₂ ) ₄ - I.a.786 Cl H H F F F -(CH ₂ ) ₄ - I.a.787 CN H H F F F -(CH ₂ ) ₄ - I.a.788 F H H CN F F -(CH ₂ ) ₄ - I.a.789 CN H H F F F -(CH ₂ ) ₄ - I.a.790 F H F H F F -(CH ₂ ) ₄ - I.a.791 Cl H F H F F -(CH ₂ ) ₄ - I.a.792 CN H F H F F -(CH ₂ ) ₄ - I.a.793 F F F H F F -(CH ₂ ) ₄ - I.a.794 Cl F F H F F -(CH ₂ ) ₄ - I.a.795 F Cl F H F F -(CH ₂ ) ₄ - I.a.796 Cl F F H F F -(CH ₂ ) ₄ - I.a.797 CN F F H F F -(CH ₂ ) ₄ - I.a.798 F CN F H F F -(CH ₂ ) ₄ -

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I.a.799 CN F F H F F -(CH ₂ ) ₄ - La.8OO F F H F F F -(CH ₂ ) ₄ - La.8Ol Cl F H F F F -(CH ₂ ) ₄ - La.802 F Cl H F F F -(CH ₂ ) ₄ - La.8O3 CN F H F F F -(CH ₂ ) ₄ - La.804 F CN H F F F -(CH ₂ ) ₄ - La.8O5 F F F F F F -(CH ₂ ) ₄ - La.806 Cl F F F F F -(CH ₂ ) ₄ - La.807 F Cl F F F F -(CH ₂ ) ₄ - La.808 CN F F F F F -(CH ₂ ) ₄ - La.809 F CN F F F F -(CH ₂ ) ₄ - La.81O H F F F F F -(CH ₂ ) ₄ - La.811 F F Br F F F -(CH ₂ ) ₄ - La.812 F F C=CH F F F -(CH ₂ ) ₄ - I.a.813 CF ₃ Cl H H F F -(CH ₂ ) ₄ - La.814 F F I F F F -(CH ₂ ) ₄ - La.815 F H H H F F -(CH ₂ ) ₅ - La.816 Cl H H H F F -(CH ₂ ) ₅ - La.817 Br H H H F F -(CH ₂ ) ₅ - La.818 CN H H H F F -(CH ₂ ) ₅ - La.819 CH ₃ H H H F F -(CH ₂ ) ₅ - La.820 F H H F F F -(CH ₂ ) ₅ - La.821 Cl H H F F F -(CH ₂ ) ₅ - I.a.822 F H H Cl F F -(CH ₂ ) ₅ - I.a.823 Cl H H F F F -(CH ₂ ) ₅ - I.a.824 CN H H F F F -(CH ₂ ) ₅ - La.825 F H H CN F F -(CH ₂ ) ₅ - I.a.826 CN H H F F F -(CH ₂ ) ₅ - I.a.827 F H F H F F -(CH ₂ ) ₅ - La.828 Cl H F H F F -(CH ₂ ) ₅ - I.a.829 CN H F H F F -(CH ₂ ) ₅ - La.83O F F F H F F -(CH ₂ ) ₅ -

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I.a.831 Cl F F H F F -(CH ₂ ) ₅ - I.a.832 F Cl F H F F -(CH ₂ ) ₅ - I.a.833 Cl F F H F F -(CH ₂ ) ₅ - I.a.834 CN F F H F F -(CH ₂ ) ₅ - I.a.835 F CN F H F F -(CH ₂ ) ₅ - I.a.836 CN F F H F F -(CH ₂ ) ₅ - I.a.837 F F H F F F -(CH ₂ ) ₅ - I.a.838 Cl F H F F F -(CH ₂ ) ₅ - I.a.839 F Cl H F F F -(CH ₂ ) ₅ - La. 840 CN F H F F F -(CH ₂ ) ₅ - La. 841 F CN H F F F -(CH ₂ ) ₅ - La. 842 F F F F F F -(CH ₂ ) ₅ - La. 843 Cl F F F F F -(CH ₂ ) ₅ - La. 844 F Cl F F F F -(CH ₂ ) ₅ - La. 845 CN F F F F F -(CH ₂ ) ₅ - La. 846 F CN F F F F -(CH ₂ ) ₅ - La. 847 H F F F F F -(CH ₂ ) ₅ - La. 848 F F Br F F F -(CH ₂ ) ₅ - La. 849 F F C=CH F F F -(CH ₂ ) ₅ - I.a.85O CF ₃ Cl H H F F -(CH ₂ ) ₅ - I.a.851 F F I F F F -(CH ₂ ) ₅ - I.a.852 F H H H F Cl -(CH ₂ ) ₂ - I.a.853 Cl H H H F Cl -(CH ₂ ) ₂ - I.a.854 Br H H H F Cl -(CH ₂ ) ₂ - I.a.855 CN H H H F Cl -(CH ₂ ) ₂ - I.a.856 СНз H H H F Cl -(CH ₂ ) ₂ - I.a.857 F H H F F Cl -(CH ₂ ) ₂ - I.a.858 Cl H H F F Cl -(CH ₂ ) ₂ - I.a.859 F H H Cl F Cl -(CH ₂ ) ₂ - I.a.860 Cl H H F F Cl -(CH ₂ ) ₂ - I.a.861 CN H H F F Cl -(CH ₂ ) ₂ - I.a.862 F H H CN F Cl -(CH ₂ ) ₂ -

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I.a.863 CN H H F F Cl -(CH ₂ ) ₂ - I.a.864 F H F H F Cl -(CH ₂ )2- I.a.865 Cl H F H F Cl -(CH ₂ ) ₂ - I.a.866 CN H F H F Cl -(CH ₂ ) ₂ - I.a.867 F F F H F Cl -(CH ₂ ) ₂ - I.a.868 Cl F F H F Cl -(CH ₂ ) ₂ - I.a.869 F Cl F H F Cl -(CH ₂ ) ₂ - La.870 Cl F F H F Cl -(CH ₂ ) ₂ - La.871 CN F F H F Cl -(CH ₂ ) ₂ - I.a.872 F CN F H F Cl -(CH ₂ ) ₂ - I.a.873 CN F F H F Cl -(CH ₂ ) ₂ - I.a.874 F F H F F Cl -(CH ₂ ) ₂ - I.a.875 Cl F H F F Cl -(CH ₂ ) ₂ - I.a.876 F Cl H F F Cl -(CH ₂ ) ₂ - I.a.877 CN F H F F Cl -(CH ₂ ) ₂ - I.a.878 F CN H F F Cl -(CH ₂ ) ₂ - I.a.879 F F F F F Cl -(CH ₂ ) ₂ - La.88O Cl F F F F Cl -(CH ₂ ) ₂ - La.881 F Cl F F F Cl -(CH ₂ ) ₂ - I.a.882 CN F F F F Cl -(CH ₂ ) ₂ - I.a.883 F CN F F F Cl -(CH ₂ ) ₂ - I.a.884 H F F F F Cl -(CH ₂ ) ₂ - I.a.885 F F Br F F Cl -(CH ₂ ) ₂ - I.a.886 F F C=CH F F Cl -(CH ₂ ) ₂ - I.a.887 CF ₃ Cl H H F Cl -(CH ₂ ) ₂ - I.a.888 F F I F F Cl -(CH ₂ ) ₂ - I.a.889 F H H H F Cl -(CH ₂ ) ₃ - La.890 Cl H H H F Cl -(CH ₂ ) ₃ - La.891 Br H H H F Cl -(CH ₂ ) ₃ - I.a.892 CN H H H F Cl -(CH ₂ ) ₃ - I.a.893 CH ₃ H H H F Cl -(CH ₂ ) ₃ - I.a.894 F H H F F Cl -(CH ₂ ) ₃ -

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I.a.895 Cl H H F F Cl -(CH ₂ ) ₃ - I.a.896 F H H Cl F Cl -(CH ₂ ) ₃ - I.a.897 Cl H H F F Cl -(CH ₂ ) ₃ - La.898 CN H H F F Cl -(CH ₂ ) ₃ - I.a.899 F H H CN F Cl -(CH ₂ ) ₃ - I. a. 900 CN H H F F Cl -(CH ₂ ) ₃ - I. a. 901 F H F H F Cl -(CH ₂ ) ₃ - I. a. 902 Cl H F H F Cl -(CH ₂ ) ₃ - I. a. 903 CN H F H F Cl -(CH ₂ ) ₃ - I. a. 904 F F F H F Cl -(CH ₂ ) ₃ - I. a. 905 Cl F F H F Cl -(CH ₂ ) ₃ - I. a. 906 F Cl F H F Cl -(CH ₂ ) ₃ - I. a. 907 Cl F F H F Cl -(CH ₂ ) ₃ - I. a. 908 CN F F H F Cl -(CH ₂ ) ₃ - I. a. 909 F CN F H F Cl -(CH ₂ ) ₃ - La.910 CN F F H F Cl -(CH ₂ ) ₃ - La.911 F F H F F Cl -(CH ₂ ) ₃ - La.912 Cl F H F F Cl -(CH ₂ ) ₃ - La.913 F Cl H F F Cl -(CH ₂ ) ₃ - La.914 CN F H F F Cl -(CH ₂ ) ₃ - La.915 F CN H F F Cl -(CH ₂ ) ₃ - La.916 F F F F F Cl -(CH ₂ ) ₃ - La.917 Cl F F F F Cl -(CH ₂ ) ₃ - La.918 F Cl F F F Cl -(CH ₂ ) ₃ - La.919 CN F F F F Cl -(CH ₂ ) ₃ - I. a. 920 F CN F F F Cl -(CH ₂ ) ₃ - I. a. 921 H F F F F Cl -(CH ₂ ) ₃ - I. a. 922 F F Br F F Cl -(CH ₂ ) ₃ - I.a.923 F F C=CH F F Cl -(CH ₂ ) ₃ - I. a. 924 CF ₃ Cl H H F Cl -(CH ₂ ) ₃ - I. a. 925 F F I F F Cl -(CH ₂ ) ₃ - I.a.926 F H H H F Cl -(CH ₂ ) ₄ -

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I.a.927 Cl H H H F Cl -(CH ₂ )4- I. a. 928 Br H H H F Cl -(CH ₂ ) ₄ - I.a.929 CN H H H F Cl -(CH ₂ ) ₄ - La.93O СНз H H H F Cl -(CH ₂ ) ₄ - La.931 F H H F F Cl -(CH ₂ ) ₄ - I.a.932 Cl H H F F Cl -(CH ₂ ) ₄ - La.933 F H H Cl F Cl -(CH ₂ ) ₄ - La.934 Cl H H F F Cl -(CH ₂ ) ₄ - La.935 CN H H F F Cl -(CH ₂ ) ₄ - I.a.936 F H H CN F Cl -(CH ₂ ) ₄ - I.a.937 CN H H F F Cl -(CH ₂ ) ₄ - La.938 F H F H F Cl -(CH ₂ ) ₄ - I.a.939 Cl H F H F Cl -(CH ₂ ) ₄ - I. a. 940 CN H F H F Cl -(CH ₂ ) ₄ - I. a. 941 F F F H F Cl -(CH ₂ ) ₄ - I. a. 942 Cl F F H F Cl -(CH ₂ ) ₄ - I.a.943 F Cl F H F Cl -(CH ₂ ) ₄ - I. a. 944 Cl F F H F Cl -(CH ₂ ) ₄ - I. a. 945 CN F F H F Cl -(CH ₂ ) ₄ - I.a.946 F CN F H F Cl -(CH ₂ ) ₄ - I.a.947 CN F F H F Cl -(CH ₂ ) ₄ - I. a. 948 F F H F F Cl -(CH ₂ ) ₄ - I.a.949 Cl F H F F Cl -(CH ₂ ) ₄ - La.95O F Cl H F F Cl -(CH ₂ ) ₄ - La.951 CN F H F F Cl -(CH ₂ ) ₄ - I.a.952 F CN H F F Cl -(CH ₂ ) ₄ - La.953 F F F F F Cl -(CH ₂ ) ₄ - I.a.954 Cl F F F F Cl -(CH ₂ ) ₄ - I.a.955 F Cl F F F Cl -(CH ₂ ) ₄ - I.a.956 CN F F F F Cl -(CH ₂ ) ₄ - I.a.957 F CN F F F Cl -(CH ₂ ) ₄ - I.a.958 H F F F F Cl -(CH ₂ ) ₄ -

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I.a.959 F F Br F F Cl -(CH ₂ )4- I. a. 960 F F C=CH F F Cl -(CH ₂ ) ₄ - I. a. 961 CF ₃ Cl H H F Cl -(CH ₂ ) ₄ - I. a. 962 F F I F F Cl -(CH ₂ ) ₄ - I. a. 963 F H H H F Cl -(CH ₂ ) ₅ - I. a. 964 Cl H H H F Cl -(CH ₂ ) ₅ - I. a. 965 Br H H H F Cl -(CH ₂ ) ₅ - I. a. 966 CN H H H F Cl -(CH ₂ ) ₅ - I. a. 967 CH ₃ H H H F Cl -(CH ₂ ) ₅ - I. a. 968 F H H F F Cl -(CH ₂ ) ₅ - I. a. 969 Cl H H F F Cl -(CH ₂ ) ₅ - I. a. 970 F H H Cl F Cl -(CH ₂ ) ₅ - I. a. 971 Cl H H F F Cl -(CH ₂ ) ₅ - I.a.972 CN H H F F Cl -(CH ₂ ) ₅ - I. a. 973 F H H CN F Cl -(CH ₂ ) ₅ - I.a.974 CN H H F F Cl -(CH ₂ ) ₅ - I. a. 975 F H F H F Cl -(CH ₂ ) ₅ - I. a. 976 Cl H F H F Cl -(CH ₂ ) ₅ - I.a.977 CN H F H F Cl -(CH ₂ ) ₅ - I. a. 978 F F F H F Cl -(CH ₂ ) ₅ - I. a. 979 Cl F F H F Cl -(CH ₂ ) ₅ - La.980 F Cl F H F Cl -(CH ₂ ) ₅ - La.981 Cl F F H F Cl -(CH ₂ ) ₅ - I.a.982 CN F F H F Cl -(CH ₂ ) ₅ - I.a.983 F CN F H F Cl -(CH ₂ ) ₅ - I.a.984 CN F F H F Cl -(CH ₂ ) ₅ - I.a.985 F F H F F Cl -(CH ₂ ) ₅ - I.a.986 Cl F H F F Cl -(CH ₂ ) ₅ - I.a.987 F Cl H F F Cl -(CH ₂ ) ₅ - I.a.988 CN F H F F Cl -(CH ₂ ) ₅ - I.a.989 F CN H F F Cl -(CH ₂ ) ₅ - I. a. 990 F F F F F Cl -(CH ₂ ) ₅ -

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I. a. 991 Cl F F F F Cl -(CH ₂ ) ₅ - I.a.992 F Cl F F F Cl -(CH ₂ ) ₅ - I.a.993 CN F F F F Cl -(CH ₂ ) ₅ - I.a.994 F CN F F F Cl -(CH ₂ ) ₅ - I.a.995 H F F F F Cl -(CH ₂ ) ₅ - I.a.996 F F Br F F Cl -(CH ₂ ) ₅ - I.a.997 F F C=CH F F Cl -(CH ₂ ) ₅ - I.a.998 CF ₃ Cl H H F Cl -(CH ₂ ) ₅ - I.a.999 F F I F F Cl -(CH ₂ ) ₅ - La. 1000 F H H H F _{C2H5 _} CH ₃ n I.a.1001 Cl H H H F _{C2H5 _} CH ₃ n La. 1002 Br H H H F _{C2H5 _} CH ₃ n La. 1003 CN H H H F _{C2H5 _} CH ₃ n La. 1004 CH ₃ H H H F _{C2H5 _} CH ₃ n La. 1005 F H H F F _{C2H5 _} CH ₃ n La. 1006 Cl H H F F _{C2H5 _} CH ₃ n La. 1007 F H H Cl F _{C2H5 _} CH ₃ n La. 1008 Cl H H F F _{C2H5 _} CH ₃ n La. 1009 CN H H F F _{C2H5 _} CH ₃ n I.a.1010 F H H CN F _{C2H5 _} CH ₃ n I.a.1011 CN H H F F _{C2H5 _} CH ₃ n I.a.1012 F H F H F _{C2H5 _} CH ₃ n I.a.1013 Cl H F H F _{C2H5 _} CH ₃ n I.a.1014 CN H F H F _{C2H5 _} СНз n I.a.1015 F F F H F _{C2H5 _} СНз n I.a.1016 Cl F F H F _{C2H5 _} СНз n I.a.1017 F Cl F H F _{C2H5 _} СНз n I.a.1018 Cl F F H F _{C2H5 _} СНз n I.a.1019 CN F F H F _{C2H5 _} СНз n La. 1020 F CN F H F _{C2H5 _} СНз n I.a.1021 CN F F H F _{C2H5 _} СНз n La. 1022 F F H F F _{C2H5 _} СНз n

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La. 1023 Cl F H F F _{C2H5 _} СНз n La. 1024 F Cl H F F _{C2H5 _} СНз n La. 1025 CN F H F F _{C2H5 _} СНз n La. 1026 F CN H F F _{C2H5 _} СНз n La. 1027 F F F F F _{C2H5 _} СНз n La. 1028 Cl F F F F _{C2H5 _} СНз n La. 1029 F Cl F F F _{C2H5 _} СНз n I.a.1030 CN F F F F _{C2H5 _} СНз n La. 1031 F CN F F F _{C2H5 _} СНз n La. 1032 H F F F F _{C2H5 _} СНз n I.a.1033 F F Br F F _{C2H5 _} СНз n La. 1034 F F C=CH F F _{C2H5 _} СНз n I.a.1035 CF ₃ Cl H H F _{C2H5 _} СНз n I.a.1036 F F I F F _{C2H5 _} СНз n I.a.1037 F H H H F _{C2H5 _} from ₂ to ₅ n I.a.1038 Cl H H H F _{C2H5 _} from ₂ to ₅ n I.a.1039 Br H H H F _{C2H5 _} from ₂ to ₅ n La. 1040 CN H H H F _{C2H5 _} from ₂ to ₅ n I.a.1041 CH ₃ H H H F _{C2H5 _} from ₂ to ₅ n La. 1042 F H H F F _{C2H5 _} from ₂ to ₅ n La. 1043 Cl H H F F _{C2H5 _} from ₂ to ₅ n La. 1044 F H H Cl F _{C2H5 _} from ₂ to ₅ n La. 1045 Cl H H F F _{C2H5 _} from ₂ to ₅ n La. 1046 CN H H F F _{C2H5 _} from ₂ to ₅ n La. 1047 F H H CN F _{C2H5 _} from ₂ to ₅ n La. 1048 CN H H F F _{C2H5 _} from ₂ to ₅ n La. 1049 F H F H F _{C2H5 _} from ₂ to ₅ n I.a.1050 Cl H F H F _{C2H5 _} from ₂ to ₅ n I.a.1051 CN H F H F _{C2H5 _} from ₂ to ₅ n La. 1052 F F F H F _{C2H5 _} from ₂ to ₅ n I.a.1053 Cl F F H F _{C2H5 _} from ₂ to ₅ n La. 1054 F Cl F H F _{C2H5 _} from ₂ to ₅ n

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I.a.1055 Cl F F H F _{C2H5 _ C2H5 _} H La.1056 CN F F H F _{C2H5 _ C2H5 _} H La. 1057 F CN F H F _{C2H5 _ C2H5 _} H La.1058 CN F F H F _{C2H5 _ C2H5 _} H La.1059 F F H F F _{C2H5 _ C2H5 _} H La. 1060 Cl F H F F _{C2H5 _ C2H5 _} H La.1061 F Cl H F F _{C2H5 _ C2H5 _} H La. 1062 CN F H F F _{C2H5 _ C2H5 _} H La. 1063 F CN H F F _{C2H5 _ C2H5 _} H La. 1064 F F F F F _{C2H5 _ C2H5 _} H La. 1065 Cl F F F F _{C2H5 _ C2H5 _} H La. 1066 F Cl F F F _{C2H5 _ C2H5 _} H La. 1067 CN F F F F _{C2H5 _ C2H5 _} H La. 1068 F CN F F F _{C2H5 _ C2H5 _} H La. 1069 H F F F F _{C2H5 _ C2H5 _} H La. 1070 F F Br F F _{C2H5 _ C2H5 _} H La. 1071 F F C=CH F F _{C2H5 _ C2H5 _} H La. 1072 CF ₃ Cl H H F _{C2H5 _ C2H5 _} H La. 1073 F F I F F _{C2H5 _ C2H5 _} H La. 1074 F H H H F _{C2H5 _ C2H5 _} CH ₃ La. 1075 Cl H H H F _{C2H5 _ C2H5 _} СНз La. 1076 Br H H H F _{C2H5 _ C2H5 _} СНз La. 1077 CN H H H F _{C2H5 _ C2H5 _} СНз La. 1078 CH ₃ H H H F _{C2H5 _ C2H5 _} СНз La. 1079 F H H F F _{C2H5 _ C2H5 _} СНз La. 1080 Cl H H F F _{C2H5 _ C2H5 _} СНз La.1081 F H H Cl F _{C2H5 _ C2H5 _} СНз La. 1082 Cl H H F F _{C2H5 _ C2H5 _} СНз La. 1083 CN H H F F _{C2H5 _ C2H5 _} СНз La. 1084 F H H CN F _{C2H5 _ C2H5 _} СНз La.1085 CN H H F F _{C2H5 _ C2H5 _} СНз La. 1086 F H F H F _{C2H5 _ C2H5 _} СНз

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La. 1087 Cl H F H F _{C2H5 _ C2H5 _} CH ₃ I.a.1088 CN H F H F _{C2H5 _ C2H5 _} CH ₃ La. 1089 F F F H F _{C2H5 _ C2H5 _} CH ₃ La. 1090 Cl F F H F _{C2H5 _ C2H5 _} CH ₃ I.a.1091 F Cl F H F _{C2H5 _ C2H5 _} CH ₃ La. 1092 Cl F F H F _{C2H5 _ C2H5 _} CH ₃ La. 1093 CN F F H F _{C2H5 _ C2H5 _} CH ₃ La. 1094 F CN F H F _{C2H5 _ C2H5 _} CH ₃ La. 1095 CN F F H F _{C2H5 _ C2H5 _} CH ₃ La. 1096 F F H F F _{C2H5 _ C2H5 _} CH ₃ La. 1097 Cl F H F F _{C2H5 _ C2H5 _} CH ₃ La. 1098 F Cl H F F _{C2H5 _ C2H5 _} CH ₃ La. 1099 CN F H F F _{C2H5 _ C2H5 _} CH ₃ La. 1100 F CN H F F _{C2H5 _ C2H5 _} CH ₃ I.a.1101 F F F F F _{C2H5 _ C2H5 _} CH ₃ La. 1102 Cl F F F F _{C2H5 _ C2H5 _} CH ₃ La. 1103 F Cl F F F _{C2H5 _ C2H5 _} CH ₃ La. 1104 CN F F F F _{C2H5 _ C2H5 _} CH ₃ I.a.1105 F CN F F F _{C2H5 _ C2H5 _} CH ₃ La. 1106 H F F F F _{C2H5 _ C2H5 _} CH ₃ La. 1107 F F Br F F _{C2H5 _ C2H5 _} CH ₃ La. 1108 F F C=CH F F _{C2H5 _ C2H5 _} CH ₃ La. 1109 CF ₃ Cl H H F _{C2H5 _ C2H5 _} CH ₃ Lal 110 F F I F F _{C2H5 _ C2H5 _} CH ₃ I.a.l 111 F H H H F _{C2H5 _} CH ₃ CH ₃ La. 1112 Cl H H H F _{C2H5 _} CH ₃ CH ₃ Lal 113 Br H H H F _{C2H5 _} CH ₃ CH ₃ La. 1114 CN H H H F _{C2H5 _} CH ₃ CH ₃ Lal 115 CH ₃ H H H F _{C2H5 _} CH ₃ CH ₃ Lal 116 F H H F F _{C2H5 _} CH ₃ CH ₃ Lal 117 Cl H H F F _{C2H5 _} CH ₃ CH ₃ Lal 118 F H H Cl F _{C2H5 _} CH ₃ CH ₃

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I.a.l 119 Cl H H F F _{C2H5 _} CH ₃ CH ₃ La. 1120 CN H H F F _{C2H5 _} CH ₃ CH ₃ Lal 121 F H H CN F _{C2H5 _} CH ₃ CH ₃ La. 1122 CN H H F F _{C2H5 _} CH ₃ CH ₃ La. 1123 F H F H F _{C2H5 _} CH ₃ CH ₃ La. 1124 Cl H F H F _{C2H5 _} CH ₃ CH ₃ La. 1125 CN H F H F _{C2H5 _} CH ₃ CH ₃ La. 1126 F F F H F _{C2H5 _} CH ₃ CH ₃ La. 1127 Cl F F H F _{C2H5 _} CH ₃ CH ₃ La. 1128 F Cl F H F _{C2H5 _} CH ₃ CH ₃ La. 1129 Cl F F H F _{C2H5 _} CH ₃ CH ₃ I.a.1130 CN F F H F _{C2H5 _} CH ₃ CH ₃ Lal 131 F CN F H F _{C2H5 _} CH ₃ CH ₃ I.a.1132 CN F F H F _{C2H5 _} CH ₃ CH ₃ Lal 133 F F H F F _{C2H5 _} CH ₃ CH ₃ I.a.1134 Cl F H F F _{C2H5 _} CH ₃ CH ₃ Lal 135 F Cl H F F _{C2H5 _} CH ₃ CH ₃ I.a.1136 CN F H F F _{C2H5 _} CH ₃ CH ₃ I.a.1137 F CN H F F _{C2H5 _} CH ₃ CH ₃ I.a.1138 F F F F F _{C2H5 _} CH ₃ CH ₃ I.a.1139 Cl F F F F _{C2H5 _} CH ₃ CH ₃ La. 1140 F Cl F F F _{C2H5 _} CH ₃ CH ₃ Lal 141 CN F F F F _{C2H5 _} CH ₃ CH ₃ La. 1142 F CN F F F _{C2H5 _} CH ₃ CH ₃ La. 1143 H F F F F _{C2H5 _} CH ₃ CH ₃ La. 1144 F F Br F F _{C2H5 _} CH ₃ CH ₃ La. 1145 F F C=CH F F _{C2H5 _} CH ₃ CH ₃ La. 1146 CF ₃ Cl H H F _{C2H5 _} CH ₃ CH ₃ La. 1147 F F I F F _{C2H5 _} CH ₃ CH ₃ La. 1148 F H H H F Cl CH ₃ H La. 1149 Cl H H H F Cl CH ₃ H I.a.1150 Br H H H F Cl CH ₃ H

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I.a.l 151 CN H H H F Cl СНз N La.1152 СНз H H H F Cl СНз N I.a.l 153 F H H F F Cl СНз N La.1154 Cl H H F F Cl СНз N La.1155 F H H Cl F Cl СНз N La.1156 Cl H H F F Cl СНз N La.1157 CN H H F F Cl СНз N I.a.l 158 F H H CN F Cl СНз N I.a.1159 CN H H F F Cl СНз N I.a.l 160 F H F H F Cl СНз n I.a.l 161 Cl H F H F Cl СНз n I.a.l 162 CN H F H F Cl СНз n I.a.l 163 F F F H F Cl СНз n I.a.l 164 Cl F F H F Cl СНз n I.a.1165 F Cl F H F Cl СНз n I.a.l 166 Cl F F H F Cl СНз n I.a.l 167 CN F F H F Cl СНз n I.a.1168 F CN F H F Cl СНз n I.a.l 169 CN F F H F Cl СНз n I.a.l 170 F F H F F Cl СНз n I.a.l 171 Cl F H F F Cl СНз n I.a.l 172 F Cl H F F Cl СНз n I.a.l 173 CN F H F F Cl СНз n I.a.l 174 F CN H F F Cl СНз n I.a.1175 F F F F F Cl СНз n I.a.l 176 Cl F F F F Cl СНз n I.a.l 177 F Cl F F F Cl СНз n I.a.1178 CN F F F F Cl СНз n I.a.l 179 F CN F F F Cl СНз n I.a.1180 H F F F F Cl СНз n I.a.l 181 F F Br F F Cl СНз n I.a.1182 F F C=CH F F Cl СНз n

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I.a.l 183 CF ₃ Cl H H F Cl CH ₃ H I.a.1184 F F I F F Cl CH ₃ H I.a.l 185 F H H H F CH ₂ C1 Cl CH ₃ I.a.1186 Cl H H H F CH2C1 Cl CH ₃ I.a.1187 Br H H H F CH2C1 Cl CH ₃ I.a.l 188 CN H H H F CH2C1 Cl CH ₃ I.a.1189 CH ₃ H H H F CH2C1 Cl СНз I.a.l 190 F H H F F CH2C1 Cl СНз I.a.l 191 Cl H H F F CH2C1 Cl СНз I.a.l 192 F H H Cl F CH2C1 Cl СНз I.a.l 193 Cl H H F F CH2C1 Cl СНз I.a.l 194 CN H H F F CH2C1 Cl СНз I.a.1195 F H H CN F CH2C1 Cl СНз I.a.l 196 CN H H F F CH2C1 Cl СНз I.a.l 197 F H F H F CH2C1 Cl СНз I.a.1198 Cl H F H F CH2C1 Cl СНз I.a.l 199 CN H F H F CH2C1 Cl СНз La. 1200 F F F H F CH2C1 Cl СНз I.a.1201 Cl F F H F CH2C1 Cl СНз La. 1202 F Cl F H F CH2C1 Cl СНз La. 1203 Cl F F H F CH2C1 Cl СНз La. 1204 CN F F H F CH2C1 Cl СНз La. 1205 F CN F H F CH2C1 Cl СНз La. 1206 CN F F H F CH2C1 Cl СНз La. 1207 F F H F F CH2C1 Cl СНз La. 1208 Cl F H F F CH2C1 Cl СНз La. 1209 F Cl H F F CH2C1 Cl СНз I.a.1210 CN F H F F CH2C1 Cl СНз I.a.1211 F CN H F F CH2C1 Cl СНз I.a.1212 F F F F F CH2C1 Cl СНз I.a.1213 Cl F F F F CH2C1 Cl СНз I.a.1214 F Cl F F F CH2C1 Cl СНз

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I.a.1215 CN F F F F CH ₂ C1 Cl CH ₃ La.1216 F CN F F F CH2CI Cl CH ₃ La.1217 H F F F F CH2CI Cl CH ₃ La.1218 F F Br F F CH2CI Cl CH ₃ La.1219 F F C=CH F F CH2CI Cl CH ₃ La. 1220 CF ₃ Cl H H F CH2CI Cl CH ₃ La. 1221 F F I F F CH2CI Cl СНз La. 1222 F H H H F CN CH ₃ СНз La. 1223 Cl H H H F CN CH ₃ СНз La. 1224 Br H H H F CN CH ₃ СНз La. 1225 CN H H H F CN CH ₃ СНз La. 1226 CH ₃ H H H F CN CH ₃ СНз La. 1227 F H H F F CN CH ₃ СНз La. 1228 Cl H H F F CN CH ₃ СНз La. 1229 F H H Cl F CN CH ₃ СНз La. 1230 Cl H H F F CN CH ₃ СНз La. 1231 CN H H F F CN CH ₃ СНз La. 1232 F H H CN F CN CH ₃ СНз La. 1233 CN H H F F CN CH ₃ СНз La. 1234 F H F H F CN CH ₃ СНз La.1235 Cl H F H F CN CH ₃ СНз La. 1236 CN H F H F CN CH ₃ СНз La. 1237 F F F H F CN CH ₃ СНз La.1238 Cl F F H F CN CH ₃ СНз La. 1239 F Cl F H F CN CH ₃ СНз La. 1240 Cl F F H F CN CH ₃ СНз La. 1241 CN F F H F CN CH ₃ СНз La. 1242 F CN F H F CN CH ₃ СНз La. 1243 CN F F H F CN CH ₃ СНз La. 1244 F F H F F CN CH ₃ СНз La. 1245 Cl F H F F CN CH ₃ СНз La. 1246 F Cl H F F CN CH ₃ СНз

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La. 1247 CN F H F F CN CH ₃ СНз La. 1248 F CN H F F CN CH ₃ СНз La. 1249 F F F F F CN CH ₃ СНз La. 1250 Cl F F F F CN CH ₃ СНз I.a.1251 F Cl F F F CN CH ₃ СНз La. 1252 CN F F F F CN CH ₃ СНз La. 1253 F CN F F F CN СНз СНз La. 1254 H F F F F CN СНз СНз I.a.1255 F F Br F F CN СНз СНз La. 1256 F F C=CH F F CN СНз СНз La. 1257 CF ₃ Cl H H F CN СНз СНз I.a.1258 F F I F F CN СНз СНз La. 1259 F H H H F OCH ₃ n n La. 1260 Cl H H H F OCH ₃ n n I.a.1261 Br H H H F OCH ₃ n n La. 1262 CN H H H F OCH ₃ n n La. 1263 CH ₃ H H H F OCH ₃ n n La. 1264 F H H F F OCH ₃ n n La. 1265 Cl H H F F OCH ₃ n n La. 1266 F H H Cl F OCH ₃ n n La. 1267 Cl H H F F OCH ₃ n n La. 1268 CN H H F F OCH ₃ n n La. 1269 F H H CN F OCH ₃ n n La. 1270 CN H H F F OCH ₃ n n I.a.1271 F H F H F OCH ₃ n n La. 1272 Cl H F H F OCH ₃ n n La. 1273 CN H F H F OCH ₃ n n La. 1274 F F F H F OCH ₃ n n La. 1275 Cl F F H F OCH ₃ n n La. 1276 F Cl F H F OCH ₃ n n La. 1277 Cl F F H F OCH ₃ n n La. 1278 CN F F H F OCH ₃ n n

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La. 1279 F CN F H F OCH ₃ H H La. 1280 CN F F H F OCH ₃ H H La.1281 F F H F F OCH ₃ H H La. 1282 Cl F H F F OCH ₃ H H La. 1283 F Cl H F F OCH ₃ H H La. 1284 CN F H F F OCH ₃ H H La. 1285 F CN H F F OCH ₃ H H La. 1286 F F F F F OCH ₃ H H La. 1287 Cl F F F F OCH ₃ H H La. 1288 F Cl F F F OCH ₃ H H La. 1289 CN F F F F OCH ₃ H H La. 1290 F CN F F F OCH ₃ H H La.1291 H F F F F OCH ₃ H H La. 1292 F F Br F F OCH ₃ H H La. 1293 F F C=CH F F OCH ₃ H H La. 1294 CF ₃ Cl H H F OCH ₃ H H La. 1295 F F I F F OCH ₃ H H La. 1296 F H H H F OCH ₃ CH ₃ H La. 1297 Cl H H H F OCH ₃ CH ₃ H La. 1298 Br H H H F OCH ₃ CH ₃ H La. 1299 CN H H H F OCH ₃ CH ₃ H La.1300 CH ₃ H H H F OCH ₃ CH ₃ H La.1301 F H H F F OCH ₃ CH ₃ H La. 1302 Cl H H F F OCH ₃ CH ₃ H La.1303 F H H Cl F OCH ₃ CH ₃ H La. 1304 Cl H H F F OCH ₃ CH ₃ H La.1305 CN H H F F OCH ₃ CH ₃ H La.1306 F H H CN F OCH ₃ CH ₃ H La.1307 CN H H F F OCH ₃ CH ₃ H La.1308 F H F H F OCH ₃ CH ₃ H La.1309 Cl H F H F OCH ₃ CH ₃ H I.a.1310 CN H F H F OCH ₃ CH ₃ H

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I.a.1311 F F F H F OSNZ СНз N La.1312 Cl F F H F OSNZ СНз N I.a.1313 F Cl F H F OSNZ СНз N La.1314 Cl F F H F OSNZ СНз N I.a.1315 CN F F H F OSNZ СНз N La.1316 F CN F H F OSNZ СНз N La.1317 CN F F H F OSNZ СНз N I.a.1318 F F H F F OSNZ СНз N La.1319 Cl F H F F OSNZ СНз N La. 1320 F Cl H F F OSNZ СНз N I.a.1321 CN F H F F OSNZ СНз n La. 1322 F CN H F F OSNZ СНз n La. 1323 F F F F F OSNZ СНз n La. 1324 Cl F F F F OSNZ СНз n I.a.1325 F Cl F F F OSNZ СНз n La. 1326 CN F F F F OSNZ СНз n La. 1327 F CN F F F OSNZ СНз n I.a.1328 H F F F F OSNZ СНз n La. 1329 F F Br F F OSNZ СНз n I.a.1330 F F C=CH F F OSNZ СНз n I.a.1331 CF ₃ Cl H H F OSNZ СНз n I.a.1332 F F I F F OSNZ СНз n I.a.1333 F H H H F OSNZ СНз СНз I.a.1334 Cl H H H F OSNZ СНз СНз I.a.1335 Br H H H F OSNZ СНз СНз I.a.1336 CN H H H F OSNZ СНз СНз I.a.1337 СНз H H H F OSNZ СНз СНз I.a.1338 F H H F F OSNZ СНз СНз I.a.1339 Cl H H F F OSNZ СНз СНз La. 1340 F H H Cl F OSNZ СНз СНз I.a.1341 Cl H H F F OSNZ СНз СНз La. 1342 CN H H F F OSNZ СНз СНз

- 101 044296

La. 1343 F H H CN F OCH ₃ CH ₃ CH ₃ La. 1344 CN H H F F OCH ₃ CH ₃ CH ₃ I.a.1345 F H F H F OCH ₃ CH ₃ CH ₃ La. 1346 Cl H F H F OCH ₃ CH ₃ CH ₃ La. 1347 CN H F H F OCH ₃ CH ₃ CH ₃ I.a.1348 F F F H F OCH ₃ CH ₃ CH ₃ La. 1349 Cl F F H F OCH ₃ CH ₃ CH ₃ I.a.1350 F Cl F H F OCH ₃ CH ₃ CH ₃ I.a.1351 Cl F F H F OCH ₃ CH ₃ CH ₃ I.a.1352 CN F F H F OCH ₃ CH ₃ CH ₃ I.a.1353 F CN F H F OCH ₃ CH ₃ CH ₃ I.a.1354 CN F F H F OCH ₃ CH ₃ CH ₃ I.a.1355 F F H F F OCH ₃ CH ₃ CH ₃ I.a.1356 Cl F H F F OCH ₃ CH ₃ CH ₃ I.a.1357 F Cl H F F OCH ₃ CH ₃ CH ₃ I.a.1358 CN F H F F OCH ₃ CH ₃ CH ₃ I.a.1359 F CN H F F OCH ₃ CH ₃ CH ₃ I.a.1360 F F F F F OCH ₃ CH ₃ CH ₃ I.a.1361 Cl F F F F OCH ₃ CH ₃ CH ₃ La. 1362 F Cl F F F OCH ₃ CH ₃ CH ₃ I.a.1363 CN F F F F OCH ₃ CH ₃ CH ₃ La. 1364 F CN F F F OCH ₃ CH ₃ CH ₃ I.a.1365 H F F F F OCH ₃ CH ₃ CH ₃ I.a.1366 F F Br F F OCH ₃ CH ₃ CH ₃ I.a.1367 F F C=CH F F OCH ₃ CH ₃ CH ₃ I.a.1368 CF ₃ Cl H H F OCH ₃ CH ₃ CH ₃ I.a.1369 F F I F F OCH ₃ CH ₃ CH ₃ I.a.1370 F H H H F H -O(CH ₂ ) ₃ - I.a.1371 Cl H H H F H -O(CH ₂ ) ₃ - La. 1372 Br H H H F H -O(CH ₂ ) ₃ - I.a.1373 CN H H H F H -O(CH ₂ ) ₃ - La. 1374 CH ₃ H H H F H -O(CH ₂ ) ₃ -

- 102 044296

1.a.1375 F H H F F H -O(CH ₂ ) ₃ - 1.a.1376 Cl H H F F H -O(CH ₂ ) ₃ - 1.a.1377 F H H Cl F H -O(CH ₂ ) ₃ - 1.a.1378 Cl H H F F H -O(CH ₂ ) ₃ - 1.a.1379 CN H H F F H -O(CH ₂ ) ₃ - 1.a.1380 F H H CN F H -O(CH ₂ ) ₃ - La. 1381 CN H H F F H -O(CH ₂ ) ₃ - 1.a.1382 F H F H F H -O(CH ₂ ) ₃ - I.a.1383 Cl H F H F H -O(CH ₂ ) ₃ - I.a.1384 CN H F H F H -O(CH ₂ ) ₃ - I.a.1385 F F F H F H -O(CH ₂ ) ₃ - I.a.1386 Cl F F H F H -O(CH ₂ ) ₃ - I.a.1387 F Cl F H F H -O(CH ₂ ) ₃ - I.a.1388 Cl F F H F H -O(CH ₂ ) ₃ - I.a.1389 CN F F H F H -O(CH ₂ ) ₃ - I.a.1390 F CN F H F H -O(CH ₂ ) ₃ - I.a.1391 CN F F H F H -O(CH ₂ ) ₃ - La. 1392 F F H F F H -O(CH ₂ ) ₃ - I.a.1393 Cl F H F F H -O(CH ₂ ) ₃ - La. 1394 F Cl H F F H -O(CH ₂ ) ₃ - I.a.1395 CN F H F F H -O(CH ₂ ) ₃ - I.a.1396 F CN H F F H -O(CH ₂ ) ₃ - I.a.1397 F F F F F H -O(CH ₂ ) ₃ - I.a.1398 Cl F F F F H -O(CH ₂ ) ₃ - I.a.1399 F Cl F F F H -O(CH ₂ ) ₃ - La. 1400 CN F F F F H -O(CH ₂ ) ₃ - I.a.1401 F CN F F F H -O(CH ₂ ) ₃ - La. 1402 H F F F F H -O(CH ₂ ) ₃ - La. 1403 F F Br F F H -O(CH ₂ ) ₃ - La. 1404 F F C^CH F F H -O(CH ₂ ) ₃ - La. 1405 CF ₃ Cl H H F H -O(CH ₂ ) ₃ - La. 1406 F F I F F H -O(CH ₂ ) ₃ -

The herbicidal compounds useful in the present invention may also be used in combination with additional herbicides to which the cereal crop is generally resistant or has become resistant through mutagenesis, as described above, or has become resistant through expression of one or more additional transgenes, as described above. The herbicides useful in the present invention are generally best used in combination with one or more other herbicides to control a wide variety of unwanted vegetation species. When used in combination with other herbicides (hereinafter referred to as compound B), the compounds claimed in the present invention can be used in the same formulation with

- 103 044296 with another herbicide or herbicides, in the form of a tank mixture with another herbicide or herbicides, or used sequentially with another herbicide or herbicides.

In particular, the additional herbicidal compound B is selected from herbicides of class b1) to b15):

b1) lipid biosynthesis inhibitors;

b2) acetolactate synthase inhibitors (ALS inhibitors);

b3) photosynthesis inhibitors;

b4) inhibitors of protoporphyrinogen-IX oxidase,

b5) bleaching herbicides;

b6) enolpyruvylshikimate 3-phosphate synthase inhibitors (EPSP inhibitors);

b7) glutamine synthetase inhibitors;

b8) 7,8-dihydropteroate synthase inhibitors (DHP inhibitors);

b9) mitosis inhibitors;

b10) inhibitors of the synthesis of very long chain fatty acids (VLCFA inhibitors);

b11) cellulose biosynthesis inhibitors;

b12) dissociating herbicides;

b13) auxin herbicides;

b14) auxin transport inhibitors and b15) other herbicides selected from the group consisting of bromobutide, chloroflurenol, chlorflurenol-methyl, cinmethylin, cumyluron, dalapon, dazomet, difenzoquat, difenzoquat-methyl sulfate, dimethipine, DSMA (disodium methyl arsenate), dimron , endothal and its salts, etobenzanide, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-M-isopropyl, flamprop-K-methyl, flurenol, flurenol-butyl, flurprimidol, fosamin, fosamin-ammonium, indanofan, indaziflam, maleic hydrazide acids, mefluidide, metam, methiozolin (CAS 403640-27-7) methyl azide, methyl bromide, methyl dimron, methyl iodide, MSMA (MMHA), oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam, tridifane, and 6-chloro-3 -(2-cyclopropyl-6-methylphenoxy)-4pyridazinol (CAS 499223-49-3), as well as its salts and esters;

including their agronomically acceptable salts or derivatives such as ethers, esters or amides.

Preferred are compositions in accordance with the present invention containing at least one herbicide B selected from herbicide classes bl, b6, b9, b10 and b11.

The following are examples of herbicides B that can be used in combination with the compounds of formula (I) in accordance with the present invention:

b1) from the group of lipid biosynthesis inhibitors:

ACC herbicides, such as alloxydim, alloxydim-sodium, butroxydim, clethodim, clodinafop, clodinafop-propargyl, cycloxydim, cyhalofop, cyhalofop-butyl, diclofop, diclofop-methyl, fenoxaprop, fenoxaprop-ethyl, fenoxaprop-R, fenoxaprop-R- ethyl, fluazifop, fluazifop-butyl, fluazifop-R, fluazifop-R-butyl, haloxyfop, haloxyfop-methyl, haloxyfop-R, haloxyfop-R-methyl, metamifop, pinoxaden, profoxydim, propaquizafop, quizalofop, quizalofop-ethyl, quizalofop- tefuryl, quizalofop-R, quizalofop-R-ethyl, quizalofop-R-tefuryl, sethoxydim, tepraloxydim and 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-tetramethyl2n-pyran-3(6H)-one (CAS 1312337-45-3); 4-(4'-chloro-4-ethyl-2'-fluoro[1,1'-biphenyl]-3-yl)-5-hydroxy-2,2,6,6tetramethyl-2H-pyran-3(6H) -on (CAS 1033757-93-5); 4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-2,2,6,6tetramethyl-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-2Hpyran -3-on; 5-(acetyloxy)-4-(4'-chloro-4-ethyl-2'-fluoro[1,1'-biphenyl]-3-yl)-3,6-dihydro-2,2,6,6- tetramethyl2H-pyran-3-one (CAS 1312340-82-1); 5-(acetyloxy)-4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-3,6dihydro-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 ester (CAS 1312337-51-1); 4-(2',4'-dichloro-4-cyclopropyl-[1,1-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6-tetramethyl5-oxo-2H-pyran- 3-yl methyl carbonic acid ester; 4-(4'-chloro-4-ethyl-2'-fluoro[1,1'-biphenyl]-3-yl)-5,6dihydro-2,2,6,6-tetramethyl-5-oxo-2H- pyran-3-yl methyl carbonate 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-yl methyl carbonic acid ester (CAS 1312340-83-2); 4-(2',4'-dichloro-4-ethyl[1,1'-biphenyl]-3-yl)-5,6-dihydro-2,2,6,6tetramethyl-5-oxo-2H-pyran- 3-yl methyl carbonic acid ester (CAS 1033760-58-5); and non-ACC herbicides such as benfuresate, butylate, cycloate, dalapon, dimepiperate, EPTC, esprocarb, ethophumesate, flupropanate, molinate, orbencarb, pebulate, prosulfocarb, tricarboxylic acid, thiobencarb, thiocarbazyl, trillate and vernolate;

b2) from the group of ALS inhibitors:

sulfonylurea derivatives such as amidosulfuron, azimsulfuron, bensulfuron, bensulfuron-methyl, chlorimuron, chlorimuron-ethyl, chlorsulfuron, cinosulfuron, cyclosulfuron,

- 104 044296 etametsulfuron, etametsulfuron-methyl, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupirsulfuron, flupirsulfuron-methyl-sodium, foramsulfuron, halosulfuron, halosulfuron-methyl, imazosulfuron, iodosulfuron, iodosulfuron-methyl-sodium, iofensulfuron, and ofensulfuron-sodium, mesosulfuron, metazosulfuron, metsulfuron , metsulfuron-methyl, nicosulfuron, orthosulfamuron, oxasulfuron, primisulfuron, primisulfuron-methyl, propyrisulfuron, procylfuron, pyrazosulfuron, pyrazosulfuron-ethyl, rimsulfuron, sulfometuron, sulfometuronmethyl, sulfosulfuron, thifensulfuron, thifensulfuron-methyl, triacylfu ron, tribenuron, tribenuron-methyl, torifloxysulfuron , triflusulfuron, triflusulfuron-methyl and tritosulfuron, imidazolinones such as imazamethabenz, imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin and imazethapyr, triazolopyrimidine herbicides and sulfonanilides such as chloransulam, chloransulam-methyl, diclosulam, flumetsulam, florasulam, methosulam , penoxulam, pyrimisulfan and pyroxulam, pyrimidinyl benzoates such as bispiribac, bispiribac-sodium, pyribenzoxime, pyrifthalide, pyriminobac, pyriminobac-methyl, pyrithiobac, pyrithiobac-sodium, 4-[[[2-[(4,6-dimethoxy-2pyrimidinyl) 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), K-(4-bromophenyl)-2-[(4,6-dimethoxy-2-pyrimidinyl)oxy]benzenemethanamine (CAS 420138-01-8) , herbicides of the sulfonylaminocarbonyl-triazolinone group, such as flucarbazone, flucarbazone sodium, propoxycarbazone, propoxycarbazone sodium, thiencarbazone and thiencarbazone-methyl; and also Triathamon;

including, a preferred embodiment of the invention relates to such compositions containing at least one herbicide from the group of imidazolinones;

b3) from the group of photosynthesis inhibitors:

amicarbazone, photosystem II inhibitors, for example, triazine herbicides, including chlorotriazine, triazinones, triazinediones, methylthiothriazines and pyridazinones, such as ametrine, atrazine, chloridazone, cyanazine, desmethrin, dimethamethrin, hexazinone, metribuzin, promethone, prometrin, propazine, simazine, symetrine , terbumetone, terbutylazine, terbutryn and triethazine, arylureas such as chlorbromuron, chlortoluron, chlorxuron, dimefuron, diuron, fluometuron, isoproturon, isouron, linuron, metamitron, metabenzthiazuron, metobenzuron, methoxuron, monolinuron, neburon, siduron, tebuthiuron and thiadiazuron, phenylcarbamates , such as desmedipham, carbutylate, phenmedipham, phenmedipham-ethyl, nitrile herbicides such as bromphenoxime, bromoxynil and its salts and esters, ioxynil and its salts and esters, uracils such as bromacil, lenacyl and terbacil, and bentazone and bentazone -sodium, pyridate, pyridafol, pentanochlor and propanil and photosystem I inhibitors such as diquat, diquat dibromide, paraquat, paraquat dichloride and paraquat dimethyl sulfate. In particular, a preferred embodiment of the invention relates to such compositions containing at least one herbicide, an aryl urea derivative; In the same way, a preferred embodiment of the invention relates to such compositions containing at least one triazine herbicide. In the same way, a preferred embodiment of the invention relates to such compositions containing at least one nitrile herbicide;

b4) from the group of protoporphyrinogen-IX oxidase inhibitors:

acifluorfen, acifluorfen-sodium, azaphenidine, bencarbazone, benzfendizone, bifenox, butafenacil, carfentrazone, carfentrazone-ethyl, chlomethoxifen, cynidone-ethyl, fluazolate, flufenpyr, flufenpyr-ethyl, flumiclorac, flumiclorac-pentyl, flumioxazin, fluoroglycophene , fluoroglycophene-ethyl, flutiacet, flutiacetmethyl, fomesafen, halosaphen, lactofen, oxadiargyl, oxadiazone, oxyfluorfen, pentoxazone, profluazole, pyraclonil, pyraflufen, pyraflufen-ethyl, saflufenacil, sulfentrazone, thidiazimine, 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-pyrazol-1carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3-(2,6-dichloro-4 -trifluoromethylphenoxy)-5-methyl-1Hpyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3-(2-chloro-6-fluoro-4-trifluoromethylphenoxy)-5-methyl-1Hpyrazole-1- carboxamide (CAS 452099-05-7), K-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-2Hbenzo[1,4]oxazin-6-yl]-1,5-dimethyl-6-thioxo-[ 1,3,5]triazinan-2,4-dione, 1,5-dimethyl-6-thioxo-3-(2,2,7-trifluoro3-oxo-4-(prop-2-inyl)-3,4 -dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1,3,5-triazinan-2,4-dione (CAS 1258836-724), 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-tetrahydroisoindole1,3-dione, 1 -methyl-6-trifluoromethyl-3 -(2,2,7-trifluoro-3-oxo-4-prop-2-inyl-3,4-dihydro-2H-benzo[1,4]oxazin-6yl)-1H -pyrimidine-2,4-dione, methyl (E)-4-[2-chloro-5-[4-chloro-5-(difluoromethoxy)-1H-methyl-pyrazol-3-yl]-4fluorophenoxy]-3- methoxy-but-2-enoate [CAS 948893-00-3] and 3-[7-chloro-5-fluoro-2-(trifluoromethyl)-1Hbenzimidazol-4-yl]-1-methyl-6-(trifluoromethyl)- 1 H-pyrimidine-2,4-dione (CAS 212754-02-4);

b5) from the group of whitening herbicides:

phytoene desaturase (PDS) inhibitors: beflubutamide, diflufenican, fluridone, fluorochloridone, flurtamone, norflurazon, picolinafen and 4-(3-trifluoromethylphenoxy)-2-(4-trifluoromethylphenyl)pyrimidine (CAS 180608-33-7), HPPD inhibitors: benzobicyclone , benzophenap, clomazone, isoxaflutole, mesotrione, pyrasulfo- 105 044296 tol, pyrazolinate, pyrazoxifene, sulcotrione, tefuryltrione, tembotrion, topramesone and bicyclopyrone, herbicide-bleach, target enzyme unknown: aclonifen, amitrol and flumeturon;

b6) from the group of EPSP synthase inhibitors:

glyphosate, glyphosate-isopropylammonium, glyphosate-potassium and Glyphosate-trimesium (sulfosate);

b7) from the group of glutamine synthase inhibitors:

bilanaphos (bialaphos), bilanaphos sodium, glufosinate, glufosinate-P and glufosinate ammonium;

b8) from the group of DHP synthase inhibitors:

asulam;

b9) from the group of mitosis inhibitors:

compounds of group K1: dinitroanilines such as benfluralin, butralin, dinitramine, ethalfluralin, fluchloralin, oryzalin, pendimethalin, prodiamine and trifluralin, phosphoramids such as amiprofos, amiprofos-methyl and butamiphos, herbicides, benzoic acid derivatives such as chlorthal, chlorthal- dimethyl, pyridines such as dithiopyr and thiazopyr, benzamides such as propyzamide and tebutam; compounds of group K2: chlorpropham, profame and carbetamide, including compounds of group K1, in particular dinitroanilines are preferred;

b10) from the group of inhibitors of the synthesis of very long chain fatty acids (VLCFA inhibitors):

chloroacetamides such as acetochlor, alachlor, butachlor, dimethachlor, dimethenamid, dimethenamid-P, metazachlor, metolachlor, metolachlor-S, pentoxamide, pretilachlor, propachlor, propisochlor and tenichlor, oxyacetanilides such as flufenacet and mefenacet, acetanilides such as diphenamide , naproanilide, naproamide and naproamide-M, tetrazolines such as fentrazamide and other herbicides such as anilofos, cafenstrol, phenoxasulfone, ipfencarbazone, piperophos, pyroxasulfone compounds of formulas II.1, II.2, II.3, II.4, II .5, II.6, II.7, II.8 and II.9

isoxazoline compounds of formula (I) are known to those skilled in the art, for example from WO 2006/024820, WO 2006/037945, WO 2007/071900 and WO 2007/096576;

among VLCFA inhibitors, chloroacetamides and hydroxyacetamides are preferred;

b11) from the group of cellulose biosynthesis inhibitors:

chlorothiamide, dichlobelin, flupoxam, isoxaben and 1-cyclohexyl-5-pentafluorophenyloxy-1 ⁴ [1,2,4,6]thiatriazin-3-ylamine;

b12) from the group of uncoupling herbicides:

dinoseb, dinoterb and dinitro-o-cresol (DNOC) and its salts;

- 106 044296 b13) from the group of auxin herbicides:

2,4-D and its salts and esters such as claciphos, 2,4-DB and its salts and esters, aminocyclopyrachlor and its resins and esters, aminopyralid and its salts such as aminopyraliddimethylammonium, aminopyralid-tris-( 2-hydroxypropyl)ammonium and its esters, benazoline, benazoline-ethyl, chloramben and its salts and esters, clomeprop, clopyralide and its salts and esters, dicamba and its salts and esters, dichlorprop and its salts and esters, dichloroprop-P and its salts and esters, fluroxypyr, fluroxypyr-butomethyl, fluroxypyr-meptyl, halauxifene and its salts and esters (CAS 943832-60-8); MCPA (2-methyl-4-chlorophenoxyacetic acid) and its salts and esters, MCPA-thioethyl, MCPA (2-methyl-4-chlorophenoxyisobutanoic acid) and its salts and esters, mecoprop and its salts and esters, mecoprop- R and its salts and esters, picloram and its salts and esters, quinclorac, quinmerac, tributylamine (TBA) (2,3,6) and its salts and esters and triclopyr and its salts and esters;

b14) from the group of auxin transport inhibitors: diflufenzopyr, diflufenzopyr-sodium, naptalam and naptalam-sodium;

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 (DNAM , disodium methyl arsenate), dimron, endothal and its salts, etobenzanide, flamprop, flamprop-isopropyl, flamprop-methyl, flamprop-Nisopropyl, flamprop-N-methyl, flurenol, flurenol-butyl, flurprimidol, fosamin, fosamin-ammonium, indanofan , indaziflam, maleic acid hydrazide, mefluidide, metam, methiozoline (CAS 403640-277) methyl azide, methyl bromide, methyl dimron, methyl iodide, MSMA (MMHA), oleic acid, oxaziclomefone, pelargonic acid, pyributicarb, quinoclamine, triaziflam, tridi fan.

The active compounds of type B and C, which contain a carboxyl group, can be used in the form of an acid, in the form of an agriculturally suitable salt as described above, or in the form of an agronomically acceptable derivative compound in the compositions of the invention.

In the case of dicamba, suitable salts include those in which the counterion is an agronomically tolerant cation. For example, suitable dicamba salts include dicamba sodium, dicamba potassium, dicamba methyl ammonium, dicamba dimethyl ammonium, dicamba isopropylammonium, dicambadiglycolamine, dicamba olamine, dicamba diolamine, dicamba trolamine, dicamba-N,N-bis(3aminopropyl )methylamine and dicamba-diethylenetriamine. Examples of suitable esters include dicamba methyl and dicamba butothyl.

Suitable 2,4-D salts include the following: 2,4-S-ammonium, 2,4-D-dimethylalammonium, 2,4-Sdiethylammonium, 2,4-S-diethanolammonium (2,4-D-diolamine), 2 ,4-S-tritanolammonium, 2,4-Sisopropylammonium, 2,4-D-triisopropylammonium, 2,4-D-heptylammonium, 2,4-O-dodecylammonium, 2,4-Dtetradecylammonium, 2,410-triethylammonium, 2 ,4Iu-tris-(2-hydroxypropyl)ammonium, 2,4Iu-tris(isopropyl)ammonium, 2,4-D-trolamine, 2,4-D-lithium, 2,4-O-sodium. Examples of suitable 2,4-D esters are 2,4-D-butyl, 2,4-S-2-butoxypropyl, 2,4-S-3-butoxypropyl, 2,4-S-butyl, 2,4D- ethyl, 2,4-S-ethylhexyl, 2,4-S-isobutyl, 2,4-S-isooctyl, 2,4-S-isopropyl, 2,4-O-meptyl, 2,4-S-methyl, 2,4-S-octyl, 2,4-S-pentyl, 2,4-S-propyl, 2,4-S-tefuryl and clacifos.

Suitable 2,4-DB salts are, for example, 2,4-DB-sodium, 2,4-DB-potassium and 2,4-DB-dimethylammonium. Suitable 2,4-DB esters are, for example, 2,4-OB-6util and 2,4-OB-isoctyl.

Suitable dichlorprop salts are, for example, sodium dichlorprop, potassium dichlorprop and dichlorprop-dimethylammonium. Examples of suitable dichlorprop esters include dichlorprop-butotyl and dichlorprop-isoctyl.

Suitable MCFC salts and esters include MCFC-butotyl, MCFC-butyl, MCFC-dimethyl, MCFC-diolamine, MCFC-ethyl, MCFC-thioethyl, MCFC-2-ethylhexyl, MCFC-isobutyl, MCFC-isoctyl, MCFC-isopropyl, MCFC-isopropylammonium , MCFC-methyl, MCFC-olamine, MCFC-potassium, MCFC-sodium and MCFC-trolamine.

A suitable MCPB salt is MCPB sodium. A suitable MSPB ester is MSPB-ethyl.

Suitable salts of clopyralid are clopyralid potassium, clopyralid olamine and clopyralid tris-(2hydroxypropyl)ammonium. Examples of suitable clopyralid esters include clopyralid-methyl.

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

Suitable salts of picloram are picloram-dimethylammonium, picloram-potassium, picloram-triisopropanolammonium, picloram-triisopropylammonium and picloram-trolamine. A suitable picloram ester is picloram-isoctyl.

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

Suitable salts and esters of chloramben include chloramben ammonium, chlorambendiolamine, chloramben methyl, chloramben methyl ammonium 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 ,6TBA-sodium.

Suitable aminopyralide salts and esters include aminopyralide-potassium aminopyralide-dimethylammonium, and aminopyralide-tris-(2-hydroxypropyl)ammonium.

Suitable glyphosate salts include, for example, glyphosate ammonium, glyphosate diammonium, glyphosate dimethyl ammonium, glyphosate isopropylammonium, glyphosate potassium, glyphosate sodium, glyphosate trimesium, as well as ethanolamine and diethanolamine salts, preferably glyphosate diammonium, glyphosate isopropylammonium and glyphosate - trimesium (sulfosate).

A suitable salt of glufosinate is, for example, glufosinate ammonium.

A suitable glufosinate-P salt is, for example, glufosinate-P-ammonium.

Suitable bromoxynyl salts and esters include, for example, bromoxynyl butyrate, bromoxynyl heptanoate, bromoxynyl octanoate, bromoxynyl potassium and bromoxynyl sodium.

Suitable ioxynyl salts and esters include, for example, ioxynyl octanoate, ioxynyl potassium and ioxynyl sodium.

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

Suitable mecoprop-R salts include, for example, mecoprop-R-butotyl, mecoprop-Rdimethylammonium, mecoprop-R-2-ethylhexyl, mecoprop-R-isobutyl, mecoprop-R-potassium and mecoprop-R sodium.

A suitable salt of diflufenzopyr is, for example, diflufenzopyr sodium.

A suitable napthalam salt is, for example, sodium napthalam.

Suitable aminocyclopyrachlor salts and esters include, for example, aminocyclopyrachlor-dimethylammonium, aminocyclopyrachlor-methyl, aminocyclopyrachlor-triisopropanolammonium, aminocyclopyrachlor-sodium and aminocyclopyrachlor-potassium.

A suitable salt of quinclorac is, for example, quinclorac-dimethyl ammonium.

A suitable quinmerac salt is, for example, quinmerac-dimethyl ammonium.

A suitable imazamox salt is, for example, imazamox ammonium.

Suitable imazapic salts are, for example, imazapic ammonium and imazapic isopropylammonium.

Suitable imazapic salts are, for example, imazapic ammonium and imazapic isopropylammonium.

A suitable imasaquin salt is, for example, imasaquin ammonium.

Suitable salts of imazethapyr are, for example, imazethapyr ammonium and imazethapyrisopropylammonium.

A suitable salt of topramozone is, for example, topramozone sodium.

In particular, preferred herbicidal compounds B are herbicides B as defined above; in particular, herbicides B.1-B.189, the list of which is given below in table. IN.

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Herbicide B IN 1 clethodim AT 2 clodinafop-propargyl V.Z cycloxdim AT 4 cyhalofop-butyl AT 5 fenoxaprop-ethyl AT 6 fenoxaprop-R-ethyl AT 7 metamifop AT 8 pinoxaden AT 9 profoxidim AT 10 methoxydim in and tepraloxydim AT 12 tralkoxydim B.13 esprocarb B.14 this is a fumesate

Table B

Herbicide B B.15 molinate V.16 prosulfocarb B.17 thiobencarb V.18 triallate V.19 bensulfuron-methyl IN 20 bispiribac sodium AT 21 chloransulam-methyl V.22 chlorsulfuron V.23 chlorimuron V.24 cyclosulfamuron V.25 diclosulam V.26 florasulam V.27 flumetsulam V.28 flupirsulfuron-methylsodium

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B.29 foramsulfuron B.30 imazamox B.31 imazamox-ammonium V.32 imazapic V.33 imazapic-ammonium V.34 imazapic-isopropylammonium V.35 imazapyr V.36 imazapyr-ammonium V.37 imazapyr-isopropylammonium V.38 imazaquin V.39 imasaquin ammonium B.40 imazethapyr V.41 imazapyr-ammonium V.42 imazapyr-isopropylammonium V.43 imazosulfuron V.44 iodosulfuron-methyl-sodium V.45 iofensulfuron V.46 iofensulfuron sodium V.47 mesosulfuron-methyl V.48 metazosulfuron V.49 bensulfuron-methyl V.50 methosulam V.51 nicosulfuron V.52 penoxulam V.53 propoxycarbazone sodium V.54 pyrazosulfuron-ethyl V.55 pyribenzoxime V.56 pyrifthalide V.57 pyroxulam V.58 propyrisulfuron V.59 rimsulfuron B.60 sulfosulfuron

V.61 thiencarbazone-methyl V.62 tripensulfuron-methyl V.63 tribenuron-methyl V.64 tritosulfuron V.65 triathamon V.66 ametrine V.67 atrazine V.68 bentazone V.69 bromoxynyl V.70 bromoxynyl octanoate V.71 bromoxynyl heptanoate V.72 bromoxynyl potassium V.73 Diuron V.74 fluometuron V.75 hexazinone V.76 isoproturon V.77 linuron V.78 metamitron V.79 metribuzin V.80 propanil V.81 simazine V.82 terbutylazine V.83 terbutryn V.84 paraquat dichloride V.85 acifluorfen V.86 butafenacil V.87 carfentrazone-ethyl V.88 flumioxazin V.89 Fomesafen V.90 oxadiargyl V.91 oxyfluorphene V.92 saflufenacil

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V.93 sulfentrazone V.94 ethyl [3-[2-chloro-4-fluoro-5-(1methyl-6-trifluoromethyl-2,4diocho-1,2,3,4- tetrahydropyrimidin-3 yl)phenoxy]-2- pyr idyl oxy] acetate (C AS 353292-31-6) V.95 1,5-d imethyl-6-thioxo-3 (2,2,7-trifluoro-3-oxo-4(prop-2-ynyl)-3,4-dihydro2H-benzo[b] [1,4]oxazine -6yl)-1,3,5-triazinan-2,4-dione (CAS 1258836-72-4) V.96 benzobicyclops V.97 clomazone V.98 diflufenican V.99 flurochloridone V.100 isoxaflutole V.101 mesotrione V.102 norflurazon V.103 picolinafen V.104 sulcotrione V.105 tefuryltrione V.106 tembotrione V.107 topramesone V.108 topramesone sodium V.109 bicyclopyrone V.110 amitrol V.111 fluometuron V.112 phenquintrione V.IZ glyphosate

V.114 glyph osat-ammonium V.115 glyph osat-d imethylammonium V.116 glyphosate isopropylammonium V.117 glyphosate-trimesium (sulfosate) V.118 glyph osat-kaliy V.119 glufosinate V.120 glufosinate ammonium V.121 glufosinate-R V.122 glufosinate-P-ammonium V.123 pendimethalin V.124 trifluralin V.125 acetochlor V.126 butachlor V.127 cafenstrol V.128 dimethenamid-R V.129 fentr azamide V.130 flufenacet V.131 mefenacet V.132 metazachlor V.133 metolachlor V.134 S-metolachlor V.135 pretilachlor V.136 phenoxasulfone V.137 isoxaben V.138 ipfencarbazone V.139 pyroxasulfone V.140 2,4-D V.141 2,4-O-isobutyl V.142 2,4-O-dimethylammonium V.143 2,4-D-N,N,N- trimethylethanolammonium

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V.144 aminopyralide V.145 aminopyralid-methyl V.146 aminopyralide- dimethylammonium V.147 aminopyralide-tris(2hydroxypropyl)ammonium V.148 clopyralid V.149 clopyralid-methyl V.150 clopyralide-olamine V.151 dicamba V.152 dicamba-butothyl V.153 dicamba-diglycoamine V.154 dicamba-dimethylammonium V.155 dicamba diolamine V.156 dicamba isopropylammonium V.157 dicamba potassium V.158 dicamba sodium V.159 dicamba-trolamine V.160 dicamba-X,X-bis-(3 aminopropyl)methylamine V.161 dicamba-diethylenetriamine V.162 fluroxypyr V.163 floxypyr-meptyl V.164 MHFC V.165 MCFC-2-ethylhexyl V.166 MCFC-dimethylammonium V.167 quinclorac V.168 quinclorac-dimethylammonium V.169 hinmerak V.170 quinmerac-dimethylammonium V.171 aminocyclopyrachlor V.172 aminocyclopyrachlor-potassium

V.173 aminocyclopyrachlor-methyl V.174 diflufenzopyr V.175 diflufenzopyr sodium V.176 dimron V.177 indanofan V.178 indaziflam V.179 oxaziclomefone V.180 triaziflam V.181 II. 1 V.182 P.2 V.183 II. 3 V.184 P.4 V.185 II. 5 V.186 II. 6 V.187 II. 7 V.188 II. 8 V.189 II. 9

Moreover, it is advisable to use the compounds of formula (I) in combination with antidotes and, if necessary, with one or more other herbicides. Antidotes are chemical compounds that prevent or reduce damage to beneficial plants without significantly affecting the herbicidal action of the compounds of formula (I) on undesirable plants. They can be applied either before sowing (for example, when treating seeds, shoots or seedlings) or during the pre-emergence or post-emergence period of the respective plants. Antidotes and compounds of formula (I) and, if necessary, herbicides B can be used simultaneously or in succession.

Suitable antidotes are, for example, (quinoline-8-hydroxy)acetic acids, 1-phenyl-5-haloalkyl-1H-1,2,4-triazole-3-carboxylic acids, 1-phenyl-4,5-dihydro-5-alkyl -1H-pyrazole-3,5dicarboxylic acids, 4,5-dihydro-5,5-diaryl-3-isoxazole carboxylic acids, dichloroacetamides, alpha-hydroxyminophenylacetonitriles, acetophenone oximes, 4,6-dihalo-2-phenylpyrimidines, N-[[ 4- 112 044296 (aminocarbonyl)phenyl]sulfonyl]-2-benzoic amides, 1,8-naphthoic anhydride, 2-halo-4(haloalkyl)-5-thiazolecarboxylic acids, phosphothioates and N-alkyl-O-phenylcarbamates and their agronomic permissible salts, as well as their agronomically permissible derivatives, such as amides, esters and thioesters in the presence of an acid group.

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

In particular, the following compounds C.1 to C.17 are preferred C antidotes.

C.1 benoxacor s.z cloquintocet-mexyl C.5 dichlormide P.7 fenchlorazole-ethyl P.9 furilazole S.I isoxadifen-ethyl P.13 mefenpyr-diethyl P.15 4-(dichloroacetyl)-1-oxa-4azaspiro[4.5]decane P.17 H-(2-methoxybenzoyl)-4[(methylaminocarbonyl)amino]b enzylsulfonamide C.2 cloquintocet C.4 cyprosulfamide C.6 fenchlorazole P.8 fenklorim P.10 isoxadifene P.12 mefenpir P.14 naphthoic anhydride P.16 2,2,5-trimethyl-3 (dichloroacetyl)-1,3- oxazolidine

Active compounds B of groups b1)-b15) and safe compounds C 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, R.R. Schmidt, Herbizide [Herbicides], Georg Thieme Verlag, Stuttgart 1995; W.H. Ahrens, Herbicide Handbook, 7th ed., Weed Science Society of America, 1994; and K.K. Hatzios, Herbicide Handbook, Supplement to 7th ed., Weed Science Society of America, 1998. 2,2,5-trimethyl-3(dichloroacetyl)-1,3-oxazolidine [CAS No. 52836-31-4] also known as R-29148. 4-(Dichloroacetyl)-1oxa-4-azaspiro[4.5]decane [CAS No. 71526-07-3], also known as AD-67 and M0N 4660.

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The explanation of the relevant mechanisms of action of the active compounds is based on the current state of knowledge. When multiple mechanisms of action were applicable to a single active compound, the substance was assigned to only one mechanism of action.

In general, it is preferable to use the compounds of the invention in combination with herbicides that are selective for the crops of interest and that complement the spectrum of weeds that the compounds control at a given application rate. It is also generally preferred to apply the compounds of the invention and other additional herbicides simultaneously as a combination formulation or tank mix.

In yet another embodiment, the present invention relates to a method for detecting a herbicide using a TriA mutated type encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 1 or a variant or derivative thereof.

This method includes the following steps:

a) obtaining a transgenic cell or plant with a nucleic acid encoding a mutated TriA, characterized in that the mutated TriA is expressed;

b) applying the herbicide to the transgenic cell or plant a) and to a control cell or plant of the same species;

c) determining the growth or viability of the transgenic and control cell or plant after application of said herbicide, and

d) selecting herbicides that resulted in reduced growth of the control cell or plant compared to the growth of the transgenic cell or plant.

As described above, the present invention provides information about compositions and methods for increasing the resistance of a crop plant or seed compared to a wild type plant or seed. In a preferred embodiment of the invention, the resistance of the crop plant or seed is increased such that the plant or seed can withstand the application of a herbicide in an amount of preferably 1-1000 g ai/ha^-1, more preferably 1-200 g ai/ha 1, even more preferably 5-150 g ai/ha^-1 and most preferably 10-100 g ai/ha^-1. As used herein, herbicide resistance means that the plant is either not killed or is only partially damaged by such application. One skilled in the art will recognize that application rates may vary depending on environmental conditions such as temperature and humidity, and also depending on the type of herbicide (active ingredient, ai) selected.

Post-emergence weed control methods that can be used in various embodiments of the invention use application rates of >0.3 x herbicide; in some embodiments, such dosages may be, for example, about >0.3x, >0.4x, >0.5x, >0.6x, >0.7x, >0.8x, >0.9x, or >1x herbicide . In accordance with one embodiment of the invention, the herbicide-tolerant plants of the present invention are resistant to post-emergence herbicide applications in amounts of approximately 25-200 g ai/ha. In some embodiments, when the herbicide-tolerant plant is a dicotyledon (eg, soybean, cotton), herbicides are applied post-emergence in amounts of approximately 50 g ai/ha. In other embodiments, when the herbicide-tolerant plant is a monocot (eg, corn, rice, sorghum), herbicides are applied post-emergence in amounts of approximately 200 g ai/ha. In other embodiments, when the herbicide-tolerant plant is Brassica (eg, canola), herbicides are applied post-emergence in amounts of approximately 25 g ai/ha. When using the post-emergence weed control methods of the present invention, in some embodiments, herbicide dosages may be applied approximately 7-10 post-emergence. According to another embodiment, the herbicide application rate may be greater than 1x; in some embodiments, the herbicide application rate may be 4x, however, typically the application rate will be about 2.5x or less, or about 2x or less, or about 1x or less.

In addition, the present invention provides methods that include the use of at least one herbicide, in some cases with one or more herbicidal compounds B, and in some cases an antidote C as defined above.

In these methods, the herbicide may be applied by any method known to those skilled in the art, including, but not limited to, seed, soil, and foliar applications. Before use, the herbicide can be converted into standard formulations, such as solutions, emulsions, suspensions, dusts, powders, pastes and granules. The choice of form of use depends on the specific purpose; in any case, it must ensure high-quality and uniform distribution of the compound in accordance with the invention.

Once plants have increased herbicide resistance, a wide variety of plant protection compounds can be used to protect plants from weeds, thereby improving plant growth and reducing competition for nutrients. The herbicide may be used to control weeds pre-emergence, post-emergence, pre-planting, and during the growing of the crops described herein in their growing areas, or formulations containing the herbicide and other additives may be used. Herbicides can also be used to treat seeds. Herbicide additives include other herbicides, detergents, adjuvants, lyophilizing agents, adhesives, stabilizing agents and the like. Herbicide formulations may be wet or dry formulations and include, but are not limited to, bulk powders, emulsion concentrates, and liquid concentrates. The herbicide and herbicide preparations can be applied in accordance with standard methods, for example, by spraying, irrigation, pollination or other similar methods.

Suitable formulations are described in detail in PCT/EP2OO9/O63387 and PCT/EP2OO9/O63386, which are incorporated herein by reference.

As described herein, the TriA nucleic acids of the present invention can be used in increasing herbicide resistance in plants that include in their genomes a gene encoding a wild-type or mutated TriA herbicide-resistant PPO protein. This gene may be endogenous or transgenic as described above. Also, in some embodiments, the nucleic acids of the present invention can be grouped using any combination of polynucleotide sequences of interest to create plants with the desired phenotype. For example, the nucleic acids of the present invention can be grouped with any other polynucleotides encoding polypeptides with pesticidal and/or insecticidal activity, such as, for example, the toxin proteins of Bacillus thuringiensis (described in US patent No. 5366892; 5747450; 5737514; 5723756; 5593881; and Geiseret al. (1986) Gene 48: 109), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), glyphosate acetyltransphase (GAT), cytochrome P450 monooxygenase, phosphinothricin acetyltransferase (PAT), AHAS (AHAS; EC 4.1. 3.18, also known as acetolactate synthase or ALS), hydroxyphenylpyruvate dioxygenase (HPPD), phytoene desaturase (PD), protoporphyrinogen oxidase (PPO) and dicamba-degrading enzymes as described in WO 02/068607 or phenoxyacetic and phenoxypropionic acid derivative-degrading enzymes in as described in WO 2008141154 or WO 2005107437. The resulting combinations may also include multiple copies of any of the polynucleotides of interest.

Therefore, the herbicide-resistant plants of the present invention can be used in combination with a herbicide to which they are resistant. The plants of the present invention can be treated with herbicides using any methods known to those skilled in the art. Herbicides can be used at any stage of plant cultivation. For example, herbicides can be applied at the pre-planting, planting, pre-emergence, post-emergence stages or several of them. Herbicides can be applied to the seeds and dried to form a layer on the seeds.

In some embodiments, the seeds are treated with an antidote, followed by a post-emergence application of herbicides. In accordance with one embodiment of the invention, the application of post-emergence herbicides is carried out approximately 7-10 days after sowing the antidote-treated seeds. In some embodiments, the antidote is cloquintocet, dichlormide, fluxofenim, or combinations thereof.

Ways to control weeds or unwanted vegetation.

In accordance with other aspects, the present invention provides a method for controlling weeds at the site of a plant or part thereof, the method comprising: applying a composition containing a herbicide to the site.

In accordance with some aspects of the invention, the present invention provides a method for controlling weeds in a plant habitat, the method comprising: applying a herbicidal composition containing herbicides to a plant site; in this case, at the specified place: (a) a plant grows or a seed is planted that is capable of developing into the specified plant; or (b) a plant will grow or such seed will be planted after application of said herbicidal composition; wherein said plant or seed, in at least some of its cells, contains a polynucleotide operably linked to a promoter operating in the plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide imparts to said plant herbicide resistance.

Herbicidal compositions can also be used, for example, as a foliar treatment, soil treatment, seed treatment or soil drench. Application may be by spraying, spraying, broadcasting, or any other effective method known in the art.

In one embodiment of the invention, herbicides can be used to control the growth of weeds that may grow in close proximity to the herbicide-resistant plants of the present invention. In such embodiments, the herbicide may be applied to an area in which herbicide-resistant plants of the invention grow in immediate

- 115 044296 proximity to weeds. The herbicide to which the herbicide-resistant plant of the present invention is resistant may be applied to the site in a concentration sufficient to kill or inhibit the growth of the weed. Those skilled in the art will know the above concentrations of herbicide that are sufficient to kill or inhibit the growth of the weed.

In accordance with other embodiments, the present invention provides a method for controlling weeds growing near a plant that is resistant to the herbicides of the invention. The method involves applying an effective amount of a herbicide to auxinic weeds and herbicide-resistant plants, wherein the plant has increased resistance to the auxinic herbicide compared to a wild-type plant. In some embodiments, plants that are resistant to the herbicides of the invention are preferably crops including, but not limited to, sunflower, alfalfa, Brassica spp., soybean, cotton, safflower, peanut, tobacco, tomato, potato, wheat, rice, maize, sorghum , barley, rye, millet and sorghum.

In accordance with other aspects, the herbicide(s) (eg, herbicides) may also be used as a seed treatment. In some embodiments, an effective concentration or an effective amount of a herbicide or herbicides, or a composition comprising an effective concentration or an effective amount of a herbicide or herbicides, can be applied directly to the seeds before or during planting of the seeds. Seed treatment formulations may additionally contain binders and, in some cases, dyes.

Binders may be added to improve adhesion of the active materials to the seeds after treatment. In other embodiments, suitable binders include surfactants based on block copolymers of ethylene oxide and propylene oxide, as well as polyvinyl alcohols, polyvinyl pyrrolidones, polyacrylates, polymethacrylates, polybutenes, polyisobutylenes, polystyrene, polyethylene amines, polyethylene amides, polyethylene imines (Lupasol(R), Polymin(R) )), polyesters, polyurethanes, polyvinyl acetate, tylose and copolymers derived from these polymers. In some cases, the formulation may also include dyes. Suitable dyes or pigments that can be used in seed treatment preparations are: Rhodamine B, C.I. red pigment 112, C.I. solvent red 1, blue pigment 15:4, blue pigment 15:3, blue pigment 15:2, blue pigment 15:1, blue pigment 80, yellow pigment 1, yellow pigment 13, red pigment 1 12, red pigment 48:2 , red pigment 48:1, red pigment 57:1, red pigment 57:1, red pigment 53:1, orange pigment 43, orange pigment 34, orange pigment 5, green pigment 36, green pigment 7, white pigment 6, brown pigment 25, base violet 10, base violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, base red 10, base red 108.

The term seed treatment includes all appropriate seed treatment methods known to those skilled in the art, such as seed dressing, seed coating, seed dusting, seed soaking and seed granulation. In accordance with one embodiment of the invention, the present invention provides a method of treating soil by applying, in particular in a seed furrow: a granular preparation containing herbicides as a composition/preparation (for example, a granular preparation), in some cases, together with one or several solid or liquid agronomically acceptable carriers and/or, in some cases, together with one or more agronomically acceptable surfactants. This method is primarily used for cultivating soil prepared for planting, such as grain crops, maize, cotton and sunflowers.

The present invention also includes seeds coated with seed treatment preparations containing such preparations that contain herbicides and at least one more herbicide, such as an AHAS inhibitor selected from the group consisting of amidosulfuron, azimsulfuron, bensulfuron, chlorimuron, chlorsufuron, cinosulfuron , cyclosulfamuron, etamethsulfuron, ethoxysulfuron, flazasulfuron, flucetosulfuron, flupirsulfuron, foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron, metsulfuron, nicosulfuron, oxasulfuron, primisulfuron, prosulfuron, pyrazosulfuron, rimsulfuron, sulf ometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron, trifloxysulfuron, triflusulfuron , tritosulfuron, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, chlorasulam, dclosulam, florasulam, flumetsulam, methosulam, penoxulam, bispiribac, pyriminobac, propoxycarbazone, flucarbazone, pyribenzoxime, pyrifthalide and pyrithiobac.

The term coated and/or containing generally means that the active ingredient is present primarily on the surface of the grow product during use, however, more or less of the ingredient may penetrate into the grow product depending on the method applications. When planting (replanting) a product to grow, the active ingredient may be absorbed by it.

In some embodiments, treating the seeds with herbicides or a herbicide-containing preparation is done by spraying or coating the seeds, seedlings, or soil before or after planting or before or after plant emergence.

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In accordance with other embodiments of the invention, when treating seeds, appropriate preparations are applied by treating the seeds with an effective amount of herbicides or a preparation containing herbicides.

In accordance with other aspects, the present invention provides a method for controlling unwanted vegetation or weeds, comprising contacting seeds of herbicide-resistant plants of the present invention with a herbicide before sowing and/or after pre-germination. The method may also include sowing the seeds, for example, into soil in a field or into potting medium in a greenhouse. This method is especially applicable when controlling unwanted vegetation or weeds in the immediate vicinity of the plant. The term pest control refers to the destruction of weeds and/or the retardation or inhibition of their normal growth. Weeds, in a broad sense, refer to all plants that grow where they are not wanted.

The term weeds in accordance with the present invention includes, for example, dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, the following genera:

Sinapis, Lepiclium, Galium, Stellaria,

Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solarium, Rorippa, Rotala, Lindemia, Lamium, Veronica, Abutilon, Em ex ,

Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus and Taraxacum.

Monocot weeds include, but are not limited to, the following genera:

Echinochloa, Setaria, Panicum, Digitaria, Phleum, Roa, Festuca, Eleusine, Brachiaria,

Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus and Apera.

In addition, the term weeds in accordance with the present invention may include, for example, crops growing in an undesirable area. For example, corn growing randomly in a field that primarily grows soybeans may be considered a weed if it is undesirable there.

In accordance with other embodiments of the invention, when treating seeds, appropriate preparations are applied by treating the seeds with an effective amount of herbicides or a preparation containing herbicides.

In accordance with other aspects, treatment of growing sites, plants, plant parts or seeds of the present invention includes the use of an agronomically acceptable composition that does not contain A.I. In accordance with one embodiment of the invention, such treatment includes the use of an agronomically acceptable composition containing no herbicides as the A.I. In accordance with some embodiments of the invention, such treatment includes the use of an agronomically acceptable composition containing no herbicide as the A.I., said composition containing one or more agronomically acceptable carriers, diluents, auxiliaries, growth regulators, and the like. In accordance with other embodiments of the invention, the treatment comprises the use of an agronomically acceptable composition containing no herbicide as the A.I., said composition comprising an excipient. According to one embodiment of the invention, said excipient is a surfactant, an A.I. spreading agent, an adhesive agent, a penetrating agent, an anti-bleed agent, an oily concentrate, an emulsifier, a compatibilizer, or combinations thereof.

It should be noted that the following information relates to embodiments of the present invention, and without departing from the purpose of the invention, numerous changes may be made herein. The invention is further illustrated by examples, which should not be construed as limiting the purpose of the invention. Otherwise, it should be understood that it is possible to resort to various other embodiments, modifications and equivalents thereof which, upon reading, those skilled in the art may wish to employ without departing from the general spirit of the present invention and/or the intent of the appended claims.

Examples

Example 1. Bacterial strains.

Chemically competent Escherichia coli samples TOP10 (Life Technologies; USA) and BL21(DE3) Gold (Agilent Technologies; Germany) were used as recipients for transformation experiments. Transformation was performed as described in Maniatis et al., Molecular

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Cloning: A Laboratory Manual, Cold Spring Habor Laboratory Press, Cold Spring Habor; N.Y. (1982). Agrobacterium tumefaciens has been used to introduce the T-DNA region into Arabidopsis, corn and soybean.

Bacterial cultures were routinely grown in Luria broth (LB) or at 37°C in LB mixed with agar (15% weight-to-volume ratio). LB was also supplemented with the antibiotic kanamycin and/or chloramphenicol when necessary. Plasmid DNA was prepared using the GeneJet Plasmid Miniprep kit (Thermo Scientific, USA). TriA and its variants were obtained by gene synthesis (Eurofins, Germany). The synthesized genes containing XhoI and NcoI restriction sites were cloned into the pET24d N-HIS vector, which is resistant to kanamycin. The chaperone plasmid pGro7 (chaperones groEL and groES), which is resistant to chloamphenicol, was obtained from TaKaRa (Japan).

Example 2: Gene synthesis, restriction enzyme digestion and cloning.

Gene synthesis and necessary cloning into the pMK-RQ vector were performed by Eurofins (Germany). Restriction enzymes were purchased from New Zealand. They were used in accordance with the manufacturer's instructions.

Example 3: Protein purification.

TriA and its variants were produced in E. coli BL21(DE3) Gold (Agilent Technologies, Germany). Therefore, E. coli was transformed using the appropriate tag expression vector pET24d N-HIS and chaperone plasmid pGro7 (chaperones groEL and groES). Bacterial strains were grown at 30°C in 100 ml LB for 20 hours, and protein expression was induced using 0.1 mmol IPTG at 25°C for 20 hours. Cells were collected by centrifugation at 3000 rpm in current 20 min at 4°C, after which they were re-placed in the Bug Buster protein extraction reagent in accordance with the manufacturer's instructions. Lysates were cleared by centrifugation. Samples of bovine serum albumin (5, 10 and 20 g) were loaded onto each of the test gels using densitometry to determine the internal standard. The found protein parameters were checked using a protein assay dye in accordance with the manufacturer's instructions (Thermo Scientific; USA). HIS-tagged enzymes were purified by metal affinity chromatography using a 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% pre-cast gel (Life Technologies; CIF) stained with Coomassie Brilliant Blue (Serva, Germany). Protein concentration was assessed by measuring absorbance at 280 nm using Lambda Bio+ (Perkin Elmer, USA).

Example 4. Enzyme kinetics.

A quiescent cell suspension containing the TriA gene was incubated in various azines, melamine, and atrazine, and the culture filtrate was analyzed by high-resolution UHPLC mass spectrometry. Substrates were obtained from Sigma-Aldrich or synthesized in-house. Synthetic standards and enzyme reaction products were analyzed by high resolution UHPLC mass spectrometry (Thermo/Dionex UPLC UltiMate3000 coupled with a QExactive high resolution mass spectrometer). A Waters Acquity HSS T3 column (2.1 mm; 100 mm; 1.8 µmol) with a mobile phase of water/acetonitrile (0.1% formic acid solution) at a flow rate of 0.6 µl min ^-1 was used. We used enzymes dissolved in 25 mmol of sodium phosphate buffer (pH 7.2) with substrate concentrations ranging from 1 μmol to 10 μmol at 30°C. Over time, the initial azine peak disappeared, while the amount of OH metabolite (reaction product) formed increased. The product was discovered through determination of the exact formula and MS/MS analysis of the exact fragments. Moreover, some of the resulting products were co-eluted according to the original standard. The degree of degradation in % was calculated from cells that contained empty vector as a control group. The results are presented in table. 3.

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Table 3

Mutation Melamine 6-cyclopentylN4-(2,3,4,5,6pentafluorophenyl)-1,3,5-triazine-2,4diamine N70T 0 0 N70C 0 0 N70G 0 0 N70V 0 4 Q71T 0 0 Q71V 0 0 Q71G 19 0 Q71C 5 0 N70VQ71V 0 0 N70VQ71T 0 0 N70TQ71V 0 3 M155L 4 0 D128G 0 0 D128P 0 0

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D128V 0 2 N70VM155V 0 0 N70VD128SM155V 0 0 N70VD128TM155V 4 1 F157T 60 0 N70LQ71LF157A 2 0 N70IQ71IF157A 0 3 L88VL92I 8 0 L88VL92V 0 0 L88VL92IF157V 5 0 L88VL92VF157A 0 0 Q96V 4 0 Q71L92VM155AF157A 3 1 N70 VQ71L92 VM15 5 AF15 7 A 0 1 N70IQ71L92VM155AF157A 0 1 N70LQ71ID128IM155V 9 0 N70LQ71ID128IM155VF157L 3 0 Q71LV91A 0 0 Q71LV91AL92V 7 3 Q71LL88AV91AL92V 0 1 Q71LL88AV91AL92VF157L 4 1 L88AF157I 0 0 L88AF157L 2 0 V69A 5 0 V69L 0 0 V69S 5 3 L74V 3 0 L74A 0 1 N70LQ71IL74A 2 0 R82L 1 1 R82M 2 2 R82G 0 0 Q96E 0 0

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Q96D 0 4 Q96A 0 0 Q96N 2 2 N126A 2 4 N126M 3 0 N126S 2 1 N126D 0 0 D128S 0 0 D128A 3 5 D128N 0 2 M155G 4 0 M155A 0 4 M155E 0 0 F157M 17 0 F157A 19 0 L88AY167I 2 0 L84VL88AY167I 7 3 L84TL88AY167I 9 4 A216S 45 1 A216G 64 0 I217A 100 4 I217S 100 3 I217T 100 3 P219GH249N 2 0 I217VP219GH249N 8 0 A220T 6 1 A220S 0 3 A220G 0 0

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F157AA220G 0 0 E246S 0 1 E246T 0 3 E246Q 4 0 E246D 0 5 S247A 1 0 S247N 0 2 S247V 3 2 S247G 2 2 S247P 3 0 D248S 20 3 D248N eleven 4 D248G 7 0 H249V 100 0 H249I 100 1 H249N 1 0 D250E 3 2 D250N 3 1 E251D 100 35 D248EE251D 1 2 Q298C 100 23 Q298N 5 0 Q298T 63 0 Q298S 83 0 S301A 0 6 S301T 0 1 S3O1V 0 0 N302E 8 1 Y304K 2 0 AS129_A130 0 3 ΔΑ130_Ι131 3 0 AA170_S182 0 2 AR174_L180 1 0

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N70S 0 0 Q71S 43 0 F157S 10 0 Q87T 0 0 Q71NL92A 87 99 Q71NL88AL92A 40 64 Q71NL92AY93L 99 100 Q71NL92AY93F 78 100 Q71NL92AD128G 0 61 Q71NL92AD128A 0 98 Q71NL88AL92AY93L 4 99 Q71NL88AL92AY93F 5 92 Q71NL88 AL92 AY93LI217 A 12 99 Q71NL88AL92AY93FF157L 1 91 Q71NL88AL92AD128A 0 4 Q71NL88AL92AD128G 0 31 Q71NL92 AY93FD128 A 0 98 Q71NL92 AY93FD128G 0 74 Q71NL92 AY93FD128GQ96T 1 99 Q71NL92AM155TF157L 44 98 Q71NL92AM155TF157V 52 99 Q71NL92AM155 VF157L 67 100 Q71NL92AD328G 0 6 L92AF89A 100 100 L92AF89AY93A 63 100 L92AY93A 1 56 L92AY93L 100 99 L92AY93F 100 100

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F89A 100 26 Y93A 100 66 Y93V 100 98 Y93L 100 82 Y93F 98 33 I217G 100 9 I217G 99 16 M160G 100 29 D250S 0 16 D328G 0 19 D328A 5 95 F89AL92AY93L 7 99 F89LL92AY93L 0 35 Y85LL92AY93V 0 24 Y85LF89AL92AY93L 0 15 Y85LL92AY93LD250S 0 36 Y85LL92AY93LD128AD250S 0 9 L92AY93LD250S 0 99 L88AL92AY93L 0 35 L88AL92AY93V 0 25 Y85LL88AL92AY93L 0 13 Y85LL88AL92AY93V 0 13 Y85LL88AL92AY93LD128G 0 17 Y85LL88AL92AY93VD128G 0 AND Y85LL88AL92AY93LQ96TD128G 0 12 Y85LL88AL92AY93VQ96TD128G 0 14 Y85LL88AF89AL92AY93LQ96TD128G 0 9 Y85LL88AF89AL92AY93VQ96TD128G 0 13 Q71NL92 AY93LQ96TD128G 0 85 L92AY93LQ96TD128G 0 94 L92AY93V 0 58 L92AY93I 0 75 L92AY93 VM155TF157L 0 99

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L92AY93LM155TF157L 41 99 L92AY93LF157L 43 97 L88AL92AY93LF157L 1 99 W87FL92AY93LF157L 1 99 W87HL92AY93LF157L 0 18 W87AL92AY93LF157L 3 12 Y85LL88AF89AL92AY93LQ96TD128GM155VF15 7L 1 13 Y85LL88AF89AL92AY93VQ96TD128GM155VF15 7L 6 13

Example 5. Directed evolution of aminohydrolase.

Azines were placed in the active region of the TriA model (based on the crystal structure of TrzN and AtzA), placing the molecules on top of melamine. Thus, radicals were found that form the active region and binding pocket for substrates. The main regions responsible for coordinating the metal ion in the active region; radicals reported to be important for amidohydrolase activity; radicals that form the hydrophobic base of the active region or are important for hydrolase activity during interactions with the aromatic ring of the substrate were not subject to changes. However, amino acids were modified to expand the set of enzymes. This model was used, on the one hand, to predict amino acid targets from outside the active region that may overall affect the ability to accept triazines, and, on the other hand, the model was used to find amino acids that occupy space in the enzyme pocket, and which can be replaced with smaller amino acids of similar hydrophobicity to provide room for more large azines without changing enzyme activity.

Example 6. Creation of model plants with herbicide resistance.

Construction of Arabidopsis plants resistant to azine herbicides with mutated amidohydrolase sequences. When transforming Arabidopsis thaliana, wild-type or mutated amidohydrolase sequences based on one SEQ ID NO: 1 encoding SEQ ID NO: 2 are cloned using standard cloning techniques as described by Sambrook et al. (Molecular cloning (2001) Cold Spring Harbor Laboratory Press) into a binary vector containing a resistance marker gene cassette (AHAS) and a mutated amidohydrolase sequence (labeled GOI) between the ubiquitin promoter (PcUbi) and the nopaline synthase (NOS) terminator sequence. To transform the plant, binary plasmids are introduced into Agrobacterium tumefaciens. Arabidopsis thaliana is transformed with mutated amidohydrolase sequences by the floral dip method as described by McElver and Singh (WO 2008/124495). Transgenic Arabidopsis plants were subjected to TaqMan analysis to determine the number of integration loci.

Example 7. Testing of model plants with herbicide resistance.

Expression of TriA and its variants is used to select Arabidopsis thaliana plants that are resistant to azines. Selected Arabidopsis thaliana lines were analyzed for improved resistance to azains 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 sterilized on the surface by stirring for 5 minutes in an ethanol-water solution (70+30 by volume), washed once with an ethanol-water solution (70+30 by volume) and twice with sterilized deionized water. The seeds were re-placed in 0.1% agar dissolved in water (w/v). Four to five seeds are placed in a solid nutrient medium consisting of Murashige and Skoog's semi-concentrated nutrient solution, pH 5.8 (Murashige and Skoog (1962) Physiologia 40 Plantarum 15: 473-497). The compounds were dissolved in dimethyl sulfoxide (DMSO) and added to the medium until solidified (final DMSO concentration 0.1%). Incubation on multiwell plates is carried out in a growth chamber at a temperature of 22°C, relative humidity 75% and 110 µmol Phot * m-2 * s-1 with a 14: 10 hour dark/light photoperiod. Growth retardation is assessed seven to ten days after sowing compared to wild-type plants. Resistance factors are calculated based on _IC50 values for growth inhibition of transformed versus transformed Arabidopsis plants. In addition, transgenic T2 or T3 Arabidopsis plants are tested for increased resistance to cellulose biosynthesis inhibitor herbicides in greenhouse studies.

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Example 8. Creation and testing of crops with herbicide resistance.

Binary vectors were prepared as described in Example 9. Jake soybean was transformed as described in Siminszky et al., Phytochem Rev. 5:445-458 (2006). After regeneration, the transformants were transplanted into soil in small pots, placed in growth chambers (16 hours day/8 hours night; 25°С day/23°С night; 65% relative humidity; 130-150 mE m ^-2 s ^-1 ) and further tested for the presence of T-DNA using the Taqman assay. After a few weeks, healthy, single-copy transgenic positive lines are transplanted into larger pots and grown in a growth chamber. The optimal shoot length for cutting is about 3-4 inches, where at least two nodes should be present. Each cutting is taken from the original transformant (mother plant) and dipped in powder containing a root-promoting hormone (indole-3-butyric acid, IBA). The scion is then placed in Oasis Wedges media inside the dome. The mother plant developed to maturity and was used to produce seeds. Wild-type shoots were also taken at the same time and served as negative controls. The shoots are stored in the biodome for 5-7 days. 7-10 days after transfer to Oasis Wedges media, the roots are treated with herbicide using a nutrient solution. 3-4 days after treatment, typical phytotoxic symptoms such as club root are assessed. Transgenic plants with less or no damage compared to wild-type plants are considered herbicide-resistant plants.

For pre-emergence treatment, plastic flower pots filled with sandy loam containing approximately 3.0% humus were used as planting containers as the substrate. Seeds of test plants were sown separately for each species/event. The used active ingredients, which have been suspended or emulsified in water, are applied directly after sowing using fine spray nozzles. The containers were irrigated gently to promote germination and growth and then covered with clear plastic covers until roots emerged. The canopy ensured uniform germination of the test plants until the active ingredients were used.

The results are presented in table. 4 and fig. 1.

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Table 4

Amino acids involved in metabolism Line Tolerance factor Q71NL92 AY93FQ96TD128G RAHEKH ++ Q71NL92 AY93FQ96TD128G RAHEGE +++ Q71NL92 AY93FQ96TD128G RAHDYQ + Q71NL92 AY93FQ96TD128G RAHDZA - Q71NL92 AY93FQ96TD128G RAHDYT + Q71NL92 AY93FQ96TD128G RAHDZD + Q71NL92 AY93FQ96TD128G RAHDYS + Q71NL92 AY93FQ96TD128G RAHEGW + Q71NL92 AY93FQ96TD128G RAHEBZ - Q71NL92 AY93FQ96TD128G RAHDYV - L92AY93L RAHEGO ++ L92AY93L RAHEEP + L92AY93L RAHEFH + L92AY93L RAHDYF - L92AY93L RAHDYD - L92AY93L RAHEEZ ++ L92AY93L RAHEAB - L92AY93L RAHEBR + L92AY93L RAHEEN - L92AY93L RAHEGN - L92AY93F RAHEJE ++ L92AY93F RAHEFX +

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L92AY93F RAHEFY + L92AY93F RAHEJZ - L92AY93F RAHEJD - L92AY93F RAHEJC - L92AY93F RAHEBE - L92AY93F RAHEFQ - L92AY93F RAHEFS ++ L92AY93F RAHEJH + Q71N_L92A_Y93L RAHCUU + Q71N_L92A_Y93L RAHCUT - Q71N_L92A_Y93L RAHDZM - Q71N_L92A_Y93L RAHDRT - Q71N_L92A_Y93L RAHCUS - Q71N_L92A_Y93L RAHDRS - Q71N_L92A_Y93L RAHDRQ + Q71N_L92A_Y93L RAHCUV + Q71N_L92A_Y93L RAHDRY ++ Q71N_L92A_Y93L RAHDXD + L92AY93LM155 V_F 157L RAHIEH +++ L92AY93LM155 V_F 157L RAHIEO + L92AY93LM155 V_F 157L RAHIEQ - L92AY93LM155 V_F 157L RAHILU ++ L92AY93LM155 V_F 157L RAHIDY ++ L92AY93LM155 V_F 157L RAHILT + L92AY93LM155 V_F 157L RAHIAT - L92A_Y93L_M155 V_F 157L RAHIDW - L92AY93LM155 V_F 157L RAHIAV + L92AY93LM155 V_F 157L RAHIEN + L92AY93LF157L RAHJCN - L92AY93LF157L RAHIMK - L92AY93LF157L RAHJCJ - L92AY93LF157L RAHHZW + L92AY93LF157L RAHJCP ++ L92AY93LF157L RAHHZU ++ L92AY93LF157L RAHHZV + L92AY93LF157L RAHJCT - L92AY93LF157L RAHIDK - L92AY93LF157L RAHIDJ -

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Immature maize embryos were transformed according to the procedure described in Peng et al. (WO2006/136596). Plants were tested for the presence of T-DNA by performing a Taqman assay, which targets the nos terminator present in all constructs. Plants without signs of damage were sent to the greenhouse for hardening and spray testing. Plants were individually potted into MetroMix 360 soil in 4-inch pots. After being placed in a greenhouse (day/night cycle at 27°C/21°C, day length 14 hours, which was ensured by using 600 W high-pressure sodium lamps), they germinated for 14 days. Transgenic corn plants were grown to produce T1 seeds for testing herbicide resistance. 7-10 days after transfer, the roots are treated with a herbicide using a nutrient solution. 3-4 days after treatment, typical phytotoxic symptoms such as club root are assessed. Transgenic plants with less or no damage compared to wild-type plants are considered herbicide-resistant plants.

For pre-emergence treatment, plastic flower pots filled with sandy loam containing approximately 3.0% humus were used as planting containers as the substrate. Seeds of test plants were sown separately for each species/event. The used active ingredients, which have been suspended or emulsified in water, are applied directly after sowing using fine spray nozzles. The containers were gently irrigated to promote germination and growth and then covered with clear plastic covers until roots emerged. The canopy ensured uniform germination of the test plants until the active ingredients were used.

For post-emergence treatments, test plants are first grown to a height of 3-15 cm, depending on the nature of the plant, and only then treated with herbicides. For this purpose, test plants are either sown directly into containers and grown there, or they are first grown separately and transplanted into test containers a few days before treatment. Herbicide damage assessments are carried out 2 and 3 weeks after treatment. Damage to the plant is assessed on a scale from 0% to 100%, where 0 means no harm and 100% means complete destruction. 100 means no germination of plants or complete destruction of at least the fragments located on the surface, and 0 means complete absence of damage or normal growth. Moderate herbicidal activity is represented by values of at least 60, good herbicidal activity is represented by values of at least 70, and very good herbicidal activity is represented by values of at least 85.

The results are presented in table. 5 and fig. 2.

Table 5

Phyto

Construct Event 0g AI/Ha 32g AI/Ha 64g AI/Ha 128g AI/Ha 256g AI/Ha Root The escape Root The escape Root The escape Root The escape Root The escape Wild type Wild type 0 0 3(> 5 50 0 (>0 15 85 45 L92A_Y93L_M155T_F157L RAHLMX 0 0 15 15 35 25 ()5 15 80 45 L92A_Y93L_M155T_F157L RAHLMY 0 0 5 45 0 60 5 i—AI 10 L92A_Y93L_M155T_F157L RAHLMV 0 5 15 0 50 10 55 5 (.0 10 L92A_Y93L_M155T_F157L RAHLMW 0 0 35 5 45 1o 7(1 2o 85 45 L92A_Y93L_M155T_F157L RAHLMU 0 0 thirty 0 40 5 <>0 5 55 15

Example 9. Construction of a binary vector.

Cloning methods, such as the use of restriction endonuclease to cut double-stranded DNA in specified areas, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nyctocellulose and nylon membranes, joining of DNA fragments, transformation of E. coli cells and bacterial cultures were carried out in accordance with described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). The polymerase chain reaction was performed using Phusion™ High Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions. In general, primers that have been used in PCR have been designed such that at least 20 nucleotides of the 3rd end of the primer ideally anneal to the extension template. Restriction sites have been added to attach the corresponding recognition site nucleotides to the 5th end of the primer. Polymer chain reaction synthesis, for example, as described in K. Heckman and L.R. Pease, Nature Protocols (2207) 2, 924-932, has been used as an alternative method for joining two target fragments, such as a promoter to a gene or a gene to a terminator. Gene synthesis, as for example described in Czar et al. (Trends in Biotechnology, 2009, 27(2): 63-72), was performed by Life Technologies using their Geneart® service.

Genes were assessed for codon usage and the presence of restriction sites that might interfere with cloning. If necessary, codons in genes were optimized

- 129 044296 using standard protocols to maximize expression in the crop plant (for example, see Puigbo et al., 2007 and Gasper et al., 2012), as well as to remove unwanted restriction sites. Genes were synthesized by GeneArt (Regensburg) or amplified by chain reaction synthesis using Phusion™ High Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions from cDNA. In both cases, NcoI and/or AscI restriction sites were introduced at the 5th end, as well as a PasI restriction site at the 3rd end, allowing cloning of these genes between functional elements such as promoters and terminators using these restriction sites. Promoter-terminator modules or promoter-intron-terminator modules were generated by total synthesis by GeneArt (Regensburg) or by coupling appropriate expression elements using PNR synthesis and cloning the PNR product into the TOPO vector pCR2.1 (Invitrogen) according to the manufacturer's instructions. During the attachment of terminator sequences to promoter sequences or promoter-intron sequences by synthesizing the entire cassette or using PCR synthesis, restriction endonuclease recognition sequences were added to either side of the module, restriction endonuclease recognition sites NcoI, AscI and PacI were introduced between the promoter and terminator or between the intron and the terminator. To obtain final expression modules, PCR-enhanced genes were cloned between a promoter and a terminator or between an intron and a terminator through NcoI and/or PacI restriction sites.

Alternatively, gene synthesis, such as described in Czar et al. (Trends in Biotechnology, 2009, 27(2): 63-72), can be performed by Life Technologies using their Geneart® service. Standard methods such as cloning, restriction digestion, molecular analysis, transformation of E. coli cells and bacterial cultures can be carried out as described in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87965-309-6). The polymerase chain reaction can be performed using Phusion™ High Fidelity DNA Polymerase (NEB, Frankfurt, Germany) according to the manufacturer's instructions. Polymer chain reaction synthesis can be performed as described by K. Heckman and L.R. Pease, Nature Protocols (2207) 2, 924-932. In both cases, NcoI and/or AscI restriction sites can be introduced at the 5th end, as well as a PasI restriction site at the 3rd end, which ensures cloning of these genes between functional elements. Promoter-terminator modules or promoter-intron-terminator modules were generated by total synthesis by GeneArt (Regensburg) or by coupling appropriate expression elements using PCR synthesis and cloning the PCR product into the TOPO vector pCR2.1 (Invitrogen) according to the manufacturer's instructions. During the attachment of terminator sequences to promoter sequences or promoter-intron sequences by whole cassette synthesis or using PCR synthesis, restriction endonuclease recognition sequences can be added to either side of the module, restriction endonuclease recognition sites NcoI, AscI and PacI can be introduced between the promoter and terminator, or between the intron and the terminator. To obtain final expression modules, PCR-enhanced genes can be cloned between a promoter and a terminator or between a nitrone and a terminator through NcoI and/or PacI restriction sites. Codons in target genes can be optimized using standard protocols to maximize expression in the crop plant (e.g., see Puigbo et al., 2007 and Gasper et al., 2012), as well as to remove unwanted restriction sites, which may be synthesized by GeneArt (Regensburg, Germany).

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Links

Esser HO, Dupuis G, Ebert E, Marco GJ, Vogel C (1975) s-triazines. In: Kearney PC, Kaufman DJ (ed) Herbicides, chemistry, degradation and modes of action. Marcel Dekker, New York, pp. 129--208

Seffernick JL, McTavish H, Osborne JP, de Souza ML, Sadowsky MJ, Wackett LP (2002) Atrazine chlorhydrolase 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 xnophydrolase 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 5Enolpyruvylshikimate-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

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Claims (8)

CLAIM 1. A plant or part of a plant containing a polynucleotide encoding a mutated melamine deaminase (TriA) polypeptide that has, over the entire length of the variant, at least about 80, 90, 95, 98, 99% or more amino acid sequence identity with SEQ ID NO: 2, wherein the encoded TriA polypeptide has melamine deaminase activity and the amino acid sequence differs from the wild-type amino acid sequence in that the amino acid at position corresponding to position 93 of SEQ ID NO: 2 is replaced by Leu, Phe, Ala or Val, wherein expression of said polynucleotide imparts or plant parts are resistant to triazine herbicides. 2. The plant or part of a plant according to claim 1, characterized in that the polynucleotide encoding the mutated TriA polypeptide contains a nucleotide sequence having at least 80% identity with the sequence of SEQ ID NO: 1. 3. A seed capable of developing into a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter functioning in plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide as defined in claim 1, encoded by the said polynucleotide, wherein the expression of the mutated TriA polypeptide gives the said plant resistance to herbicides of the triazine group. 4. A plant cell that contains a polynucleotide operably linked to a promoter operating in the cell, wherein the promoter is capable of expressing a mutated TriA polypeptide as defined in claim 1, encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide confers herbicide resistance to said plant triazine groups. 5. A method of improving plant growth by controlling weeds at the site where the plant grows, wherein said method includes: (a) applying a herbicidal composition containing triazine herbicides to the site where the plant grows and (b) sowing a seed at said location, wherein the seed is capable of developing into a plant that, in at least some of its cells, contains a polynucleotide operably linked to a promoter functioning in plant cells, wherein the promoter is capable of expressing a mutated TriA polypeptide as defined in claim 1, encoded by said polynucleotide, wherein expression of the mutated TriA polypeptide gives the said plant resistance to herbicides of the triazine group. 6. The method according to claim 5, characterized in that the weeds and the plant obtained from said seed are treated with the herbicidal composition. 7. A method for producing a plant with resistance to triazine herbicides, wherein the method includes regenerating the plant from a plant cell transformed using a recombinant polynucleotide operably linked to a promoter operating in the cell, wherein the promoter is capable of expressing a mutated TriA polypeptide, as defined in claim 1, encoded by the specified polynucleotide, wherein the expression of the mutated TriA polypeptide gives the specified plant resistance to herbicides of the triazine group. 8. An isolated and/or recombinantly produced and/or synthetic nucleic acid molecule containing a nucleic acid molecule encoding a mutated TriA polypeptide having melamine deaminase activity selected from the group consisting of: (a) a nucleic acid molecule encoding a mutated TriA polypeptide and containing the sequence SEQ ID NO: 2; (b) a nucleic acid molecule containing the sequence SEQ ID NO: 1; (c) a nucleic acid molecule which, as a result of degeneration of the genetic code, can be transferred from the sequence of the TriA polypeptide SEQ ID NO: 2 and confers increased resistance or tolerance to herbicides compared to a corresponding, for example untransformed, wild-type plant cell, plant or part thereof; (d) a nucleic acid molecule having 80, 85, 90, 95, 96, 97, 98, 99, 99.5% or more identity with the nucleic acid molecule sequence of the polynucleotide containing the nucleic acid molecule of SEQ ID NO: 1, confers increased stability or resistance to herbicides compared to a corresponding wild-type plant cell, plant or part thereof; (e) a nucleic acid molecule encoding a mutated TriA polypeptide having 80, 85, 90, 95, 96, 97, 98, 99, 99.5% or more identity with the amino acid sequence of the TriA polypeptide of SEQ ID NO: 2 and conveying an increased resistance or resistance to herbicides compared to a corresponding wild-type plant cell, plant or part thereof; (f) a nucleic acid molecule that hybridizes with a nucleic acid molecule according to claims (a) to (d) or (e) under stringent hybridization conditions and confers increased herbicide resistance or resistance compared to a corresponding wild-type plant cell, plant or part of it; characterized in that the amino acid sequence of the mutated TriA polypeptide differs from the amino acid sequence of the wild type TriA polypeptide in that the amino acid at the position corresponding to position 93 of SEQ ID NO: 2 is replaced by Leu, Phe, Ala or Val. - 132 -