CN120529828A

CN120529828A - Plants with increased tolerance to herbicides

Info

Publication number: CN120529828A
Application number: CN202380085210.2A
Authority: CN
Inventors: M·迪巴尔; E·M·戴维斯; H·考尔; E·C·古普顿; P·R·约翰恩; M·T·西赛; S·特雷施; R·坎佩
Original assignee: BASF Agricultural Solutions US LLC
Current assignee: BASF Agricultural Solutions US LLC
Priority date: 2022-12-12
Filing date: 2023-12-04
Publication date: 2025-08-22
Also published as: EP4633362A1; AR131319A1; AU2023398330A1; WO2024126113A1

Abstract

The present invention relates to a plant or plant part comprising a polynucleotide encoding a mutant cellulose synthase (CESA) polypeptide, the expression of which confers tolerance to a CESA-inhibiting herbicide such as azine on the plant or plant part.

Description

Plants with increased tolerance to herbicides

Technical Field

The present invention relates generally to methods for conferring tolerance to agricultural levels of herbicides on plants. In particular, the present invention relates to plants having increased tolerance to herbicides, more particularly to herbicides that inhibit the enzyme cellulose synthase (CESA), also known as Cellulose Biosynthesis Inhibitors (CBI). More particularly, the present invention relates to methods and plants obtained by mutagenesis and cross-breeding and transformation that have increased tolerance to herbicides, particularly CESA-inhibiting herbicides.

Background

Plant cell walls are complex structures composed of high molecular weight polysaccharides, proteins and lignin. Among the parietal polysaccharides, cellulose (hydrogen-bonded ≡1, 4-linked glucan microfibrils) is the main wall component that is subject to loading and a key precursor for industrial applications. Cellulose is synthesized from large multimeric cellulose synthase (CESA) complexes (e.c. 2.4.1.12) and extends along cortical microtubules on plasma membranes. The only known component of these complexes is the cellulose synthase protein. Recent studies have identified experimental interaction partners for CESA and demonstrated that the migration pattern of CESA complexes is dependent on the phosphorylation status (for reviews, see Endler and Persson, molecular Plant, 2011, volume 4, phase 2, pages 199-211, and references contained therein). for example, cotton cellulose synthase genes called CESA1 and CESA2 were identified in a set of Expressed Sequence Tag (EST) sequences based on weak sequence similarity to bacterial derived cellulose synthase genes (Richmond and Somerville. Plant Physiology [ plant Physiology ],2000, vol.124, 495-498; and references contained therein). In addition, these genes were expressed at high levels in cotton fibers at the beginning of secondary wall synthesis, and purified fragments of one of the corresponding proteins were shown to bind to UDP-Glc (a suggested substrate for cellulose biosynthesis). Conclusion that the cotton CESA gene is a cellulose synthase was supported by the results obtained using two cellulose-deficient Arabidopsis (Arabidopsis) mutants rsw1 and irx (Richmond and Somerville. Plant Physiology [ plant Physiology ], vol.124, 2000,495-498; and references contained therein). Genes corresponding to the RSW1 and IRX3 loci exhibit a high degree of sequence similarity to the cotton CESA gene and are considered orthologs. Ten full-length CESA genes from arabidopsis have been sequenced and there are genomic investigation sequences that can indicate one additional family member. Repeated database searches using Arabidopsis Rsw1 (AtCESA 1) and cotton CESA polypeptide sequences as initial query sequences revealed a large superfamily of at least 41 CESA-like genes in Arabidopsis. Based on predicted protein sequences, these genes were grouped into seven distinct families (Richmond and somerville plant Physiology [ plant Physiology ], volume 124, 2000,495-498; and references contained therein) the CESA family (including RSW1 ((AtCESA 1) and IRX3 (AtCESA 7)), and six families of structurally related genes of unknown function, designated as "cellulose synthase-like" genes (CslA, cslB, cslC, cslD, cslE and CslG).

WO 2013/142968 describes arabidopsis cellulose synthase (CESA) alleles identified by mutagenizing plants and screening the plants with Cellulose Biosynthesis Inhibitors (CBI). CBI employed in WO 2013/142968 includes dixynil, oxaden, clomazone, flumioxazin and quinclorac, in particular clomazone or flumioxazin (designated fpx-1 to fxp-3 [ CESA3], fxp-1 to fxp2-3[ CESA1], and ixr1-1 to ixr1-7[ CESA3], ixr2-1 to ixr2-2[ CESA6] mutants of arabidopsis CESA wild-type enzymes). WO 2015/162143 discloses that the mutant CesA enzyme described in WO 2013/142968 confers tolerance to a new class of Cellulose Biosynthesis Inhibitors (CBI), known as azines, when overexpressed in crop plants. WO 2017/068544 describes additional target sites which when substituted with certain amino acids confer tolerance to CBI.

The inventors of the present invention have now surprisingly found that overexpression of the novel mutant sculpin (Glycine max) cellulose synthase form, which is not disclosed in any of WO 2013/142968, WO 2015/162143 or WO 2017/068544, confers tolerance/resistance to a specific class of CESA-inhibiting herbicides (cellulose biosynthesis inhibitors; CBI), respectively, on plants compared to non-transformed and/or non-mutagenized plants or plant cells.

The problem of the present invention can be seen as providing novel traits by identifying target polypeptides, manipulation of which renders plants tolerant to herbicides.

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

Disclosure of Invention

Accordingly, in one aspect, the invention provides a plant or plant part comprising a polynucleotide encoding a mutant CESA polypeptide whose expression confers tolerance to a CESA-inhibiting herbicide on the plant or plant part, wherein the mutant CESA polypeptide is characterised in that the amino acid at the position corresponding to position 1051 of SEQ ID No. 28 is substituted with Leu, met or Thr.

In some aspects, the invention provides seeds capable of germinating into plants comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant.

In one aspect, the present invention provides a plant cell of a plant or a plant cell capable of regenerating into a plant, the plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the plant cell comprises the polynucleotide operably linked to the promoter.

In another aspect, the invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in the cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant.

In other aspects, the invention provides a plant product prepared from a plant or plant part comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant.

In some aspects, the invention provides progeny or offspring plants derived from a plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, wherein the progeny or offspring plant comprises in at least some cells thereof a recombinant polynucleotide operably linked to the promoter, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the progeny or offspring plant.

In other aspects, the invention provides methods for controlling weeds at a locus where plants are growing comprising (a) applying to the locus a herbicide composition comprising a CESA-inhibiting herbicide, and (b) planting a seed at the locus, wherein the seed is capable of producing a plant, the plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in plant cells, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant.

In some aspects, the invention provides methods for controlling weeds at a locus where plants are growing comprising applying to the locus a herbicidal composition comprising a CESA-inhibiting herbicide, wherein the locus is (a) a locus comprising a plant or seed capable of producing the plant, or (b) a locus comprising the plant or seed after said applying, wherein the plant or seed comprises in at least some cells thereof a polynucleotide operably linked to a promoter operable in plant cells, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant.

In one aspect, step (a) occurs before, after, or simultaneously with step (b).

In other aspects, the invention provides methods of producing a plant that is tolerant to a CESA-inhibiting herbicide, the method comprising regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant.

In one aspect, the invention provides a method of producing a progeny plant that is tolerant to a CESA-inhibiting herbicide, the method comprising crossing a first CESA-inhibiting herbicide tolerant plant with a second plant to produce a CESA-inhibiting herbicide tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant.

In addition, the present invention relates to a method for identifying a CESA-inhibiting herbicide by using a mutant CESA of the present invention encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a variant, homologue, paralogue or ortholog thereof.

The method comprises the following steps:

a) Generating a transgenic cell or plant comprising a nucleic acid encoding a mutant CESA of the invention, wherein the mutant CESA of the invention is expressed;

b) Applying a CESA-inhibiting herbicide to the transgenic cells or plants of a) and to control cells or plants of the same variety;

c) Determining the growth or viability of the transgenic cell or plant and the control cell or plant after application of the test compound, and

D) Selecting a test compound that reduces the growth of the control cell or plant as compared to the growth of the transgenic cell or plant.

Another object relates to a method of identifying a nucleotide sequence encoding a mutant CESA that is resistant or tolerant to a CESA-inhibiting herbicide, the method comprising:

a) Generating a library of nucleic acids encoding mutant CESA,

B) Screening a population of the resulting mutant CESA-encoding nucleic acids by expressing each of the nucleic acids in cells or plants and treating the cells or plants with a CESA-inhibiting herbicide,

C) Comparing the level of CESA-inhibitory herbicide tolerance provided by the population of nucleic acids encoding mutant CESA with the level of CESA-inhibitory herbicide tolerance provided by the nucleic acid encoding control CESA,

D) At least one nucleic acid encoding a mutant CESA is selected that provides a significantly increased level of tolerance to a CESA-inhibiting herbicide as compared to the level of tolerance to a CESA-inhibiting herbicide provided by a nucleic acid encoding a control CESA.

In a preferred embodiment, the nucleic acid encoding a mutant CESA selected in step d) provides at least 2-fold resistance to a CESA-inhibiting herbicide as compared to the resistance to a CESA-inhibiting herbicide provided by the nucleic acid encoding a control CESA.

Resistance or tolerance can be determined by generating transgenic plants comprising the nucleic acid sequences of the library of step a) and comparing the transgenic plants to control plants.

Another object relates to a method of identifying a plant or an alga containing a nucleic acid encoding a mutant CESA that is resistant or tolerant to a CESA-inhibiting herbicide, the method comprising:

a) Identifying an effective amount of a CESA-inhibiting herbicide in a culture of plant cells or green algae,

B) Treating the plant cells or green algae with a mutagen,

C) Contacting the mutagenized cell population with an effective amount of a CESA-inhibiting herbicide identified in a),

D) Selecting at least one cell that survives the test conditions,

E) PCR amplification and sequencing of the CESA gene from the cell selected in d) and comparing such sequences with the wild-type CESA gene sequences, respectively.

In a preferred embodiment, the mutagen is ethyl methylsulfonate.

Another object relates to an isolated, recombinantly produced, and/or chemically synthesized nucleic acid encoding a mutant CESA, wherein the mutant CESA polypeptide is characterized by substitution of an amino acid at a position corresponding to position 1051 of SEQ ID NO. 28 with Leu, met, or Thr.

Preferably, the nucleic acid comprises the sequence of SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a variant thereof, as defined below.

In a preferred embodiment, the nucleic acid is identifiable by a method as defined above.

Another object relates to an isolated, recombinantly produced, and/or chemically synthesized mutant CESA polypeptide, wherein the mutant CESA polypeptide is characterized by substitution of an amino acid at a position corresponding to position 1051 of SEQ ID NO. 28 with Leu, met, or Thr.

Preferably, the polypeptide comprises a sequence as shown in SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83, variants, derivatives, orthologs, paralogs or homologues thereof, as defined below.

In still further aspects, the invention provides a plant or plant part comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the plant or plant part further exhibits a second or third herbicide tolerance trait.

In another embodiment, the invention relates to a plant cell transformed with and expressing a mutant CESA nucleic acid according to the invention, or a plant that has been mutated to obtain a plant expressing, preferably overexpressing, a mutant CESA nucleic acid according to the invention, wherein expression of said nucleic acid in the plant cell results in an increased resistance or tolerance to a CESA-inhibiting herbicide compared to a wild-type variety of the plant cell.

In another embodiment, the invention relates to a plant comprising a plant cell according to the invention, wherein expression of a mutant CESA nucleic acid in the plant results in an increase in resistance of the plant to a CESA-inhibiting herbicide compared to a wild-type variety of the plant.

Plants of the invention may be transgenic or non-transgenic.

Preferably, expression of a nucleic acid of the invention in a plant results in an increase in resistance of the plant to a CESA-inhibiting herbicide compared to the wild type variety of the plant. In another embodiment, the invention relates to a seed produced by a transgenic plant comprising a plant cell of the invention, wherein the seed is a true breeding for increased resistance to a CESA-inhibiting herbicide as compared to a wild type variety of the seed.

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

In another embodiment, the invention relates to a method of producing a transgenic plant, the method comprising (a) transforming a plant cell with an expression cassette comprising a polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, and (b) generating from the plant cell a plant having increased resistance to a CESA-inhibiting herbicide.

Preferably, the expression cassette further comprises a transcription initiation regulatory region and a translation initiation regulatory region functional in a plant.

Drawings

FIGS. 1A, 1B, 1C

FIG. 1 shows soybean hairy root assays of missense mutated explants overexpressing CESA3 grown for three weeks on azine selected as (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine. pBas04327 CESA3S1051L, pBas 04328S 1051M, pBas04333 CESA3S 1051T. K599 was used as a non-transgenic control.

1A:1 = K599 on non-selection, 2 = K599 on 2nM (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine, 3 = pBas04327 on 2nM (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine

1B 4 = K599 on non-selection, 5 = K599 on 2nM (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine, 6 = pBas04328 on 2nM K599 on 2nM (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine, 7 = pBas04333 on 2nM K599 on 2nM (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine.

FIG. 2 shows the emergence rate of missense mutated soybean seedlings overexpressing soybean CESA3 with and without azine treatments. pBas04939 CESA 3S 1051L, pBas04940 CESA 3S 1051M, pBas04941 CESA 3S 1051T, SEQ ID NO 28. The upper panel represents the emergence rate of treatment with azines. The lower panel represents the emergence rate of untreated seedlings

Key item of sequence table

TABLE 1

Detailed Description

The articles "a" and "an" as used herein refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one or more elements.

Also as used herein, the term "comprises," "comprising," or a variant thereof, such as "comprises" or "including," is to be interpreted as referring to the elements, integers, or steps, or groups of elements, integers, or steps, but does not exclude the presence of other elements, integers, or steps, or groups of elements, integers, or steps.

The term "controlling undesirable vegetation or weeds" is understood to mean killing the weeds and/or otherwise retarding or inhibiting the normal growth of the weeds. Weeds in the broadest sense are understood to mean all those plants which grow in locations where their emergence is undesirable. Weeds of the invention include, for example, dicotyledonous weeds and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis (Sinapis), lepidium (Lepidium), lagrangia (Galium), palladium (STELLARIA), matricaria (MATRICARIA), matricaria (Anthemis), cyathula (Galinsoga), chenopodium (Chenopodium), urtica (Urtica), senecio (Senecio), amaranthus, portulaca (Portula), xanthium (Xanthium), convolvulus (Convolvulvulus), cyathula (Convolvulus), Sweet potato (Ipomoea), polygonum (Polygonum), sesbania (Sesbania), ragweed (Ambrosia), cirsium (Cirsium), fellium (Carduus), sonchus (Sonchus), solanum (Solanum), roxb (Rorippa), artemisia (Rotala), matricaria (LINDERNIA), indian sesame (Lamium), veronica (Veronica), abutilon (Abutilon), rhus (Emex), Datura (Datura), viola (Viola), weasel (Galeopsis), poppy (Papaver), cornflower (Centaurea), trifolium (Trifolium), ranunculus (Ranunculus) and Taraxacum (Taraxacum). Monocotyledonous weeds include, but are not limited to, weeds of the genera Echinochloa (Echinochloa), setaria (Setaria), panicum (Panicum), crabgrass (DIGITARIA), timothy (Phleum), poa pratensis (Poa), festuca (Festuca), eleusine (Eleusine), brachyotus (Brachiaria), lolium (Lolium), brome (Bromus), avena (Avena), cyperus (Cyperus), sorghum (Sorgum), and combinations thereof, The genera BINGCAO (Agropyron), bermuda (Cynodon), juniperus (Monochoria), fimbristylis (Fimbristyslis), sagittaria (SAGITTARIA), water chestnut (Eleocharis), scirpus (Scirpus), paspalum (Paspalum), duck grass (Ischaemum), pelargonium (Sphenoclea), leptopetalum (Dactyloctenium), agrostis (Agrostis), myrtus (Alopecurus), and Agave (Apera). In addition, weeds of the invention can comprise, for example, crop plants which grow at undesired locations. For example, if a maize plant is undesirable in a field of soybean plants, then the autogenous maize plant in the field containing primarily soybean plants may be considered weeds.

The term "plant" is used in its broadest sense as it relates to organic material and is intended to encompass eukaryotes that are members of the kingdom of plants, examples of which include, but are not limited to, vascular plants, vegetables, grains, flowers, trees, herbs, shrubs, grasses, vines, ferns, mosses, fungi and algae, etc., as well as clones, side shoots (offset) and parts (e.g., cuttings (cutting), tubes (piping), shoots, rhizomes, underground stems, clusters (bounces), crowns, bulbs, rhizomes, plants/tissues produced in tissue culture, etc.) of plants for vegetative propagation. The term "plant" further encompasses whole plants, plant ancestors and progeny and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, florets (floret), fruits, stems (pedicles), inflorescences, stamens, anthers, stigmas, ovaries, petals, sepals, carpels (carpel), root tips, root caps (root caps), root hairs, leaf hairs, seed hairs, pollen grains, microspores, cotyledons, hypocotyls, epicotyls, xylem, phloem, parenchyma, endosperm, companion cells, guard cells, and any other known organ, tissue and organ of a plant, wherein each of the foregoing comprises a gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, calli, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, as well, wherein each of the foregoing comprises the gene/nucleic acid of interest.

In particular, plants useful in the methods of the invention include all plants belonging to the superfamily of green plants (VIRIDIPLANTAE), particularly monocotyledonous and dicotyledonous plants, including forage or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer species (Acer spp.), kiwi species (ACTINIDIA spp.), abelmoschus species (Abelmoschus spp.), sisal (AGAVE SISALANA), umbelliferae species (Agropyron spp.), creeping bentgrass (Agrostis stolonifera), and, Allium spp, amaranthus spp, bindweed Ammophila arenaria, pineapple, annas comosus, annona spp, and the like celery (Apium graveolens), groundnut species (ARACHIS SPP), porcinia species (Artocarpus spp.), asparagus (Asparagus officinalis), oat species (Avena spp.) (e.g., oat (AVENA SATIVA), Wild oat (Avena fatua), praise oat (Avena byzantina), wild oat variety sava (Avena fatua var. Sava), hybrid oat (Avena hybrid), carambola (Averrhoa carambola), trifoliate acanthopanax species (Bambusa sp.), white gourd (Benincasa hispida), brazil chestnut (Bertholletia excelsea), beet (Beta vulgaris), Brassica species (Brassica spp.) (e.g., brassica napus (Brassica napus), guan subspecies (Brassica rapa ssp.) [ canola (canola), brassica napus (oil napus), brassica oleracea (turnip rape) ]), kadaba (Cadaba farinosa), tea (CAMELLIA SINENSIS), canna (CANNA INDICA), cannabis (Cannabis sativa), Capsicum species (Capsicum spp.), sedge (Carex elata), papaya (CARICA PAPAYA), ruscus aculeatus (Carissa macrocarpa), hickory species (Carya spp.), safflower (Carthamus tinctorius), chestnut species (Castanea spp.), gecko (Ceiba pentandra), cultivated chicory (Cichorium endivia), camphor species (Cinnamomum spp), a red flower (p.m.), watermelon (Citrulluslanatus), citrus species (Citrus spp.), coco species (Cocos spp.), caffeia species (cofea spp.), taro (Colocasia esculenta), cola species (Cola spp.), jute species (Corchorus sp.), coriander (Coriandrum sativum), hazelnut species (Corylus spp.), crataegus species (Crataegus spp), Saffron (Crocus sativus), cucurbita species (cuurbita spp.), cucumis species (culumis spp.), cynara species (Cynara spp.), wild carrot (Daucus carota), desmodium species (Desmodium spp.), longan (Dimocarpus longan), dioscorea species (Dioscorea spp.), kaki species (Diospyros spp.), barnyard species, elaeis (Elaeis) (e.g., oil palm africana (Elaeis guineensis), oil palm (35), Oil palm americana (Elaeis oleifera)), finger (Eleusine coracana), teichum (Eragrostis tef), festuca species (Erianthus sp.), loquat (Eriobotrya japonica), eucalyptus species (eucalyptussp), russian (Eugenia uniflora), buckwheat species (Fagopyrum spp.), cyclobalanopsis species (Fagus spp.), festuca (Festuca arundinacea), Fig (Ficus carica), kumquat species (Fortunella spp.), strawberry species (Fragaria spp.), ginkgo (Ginkgo biloba), soybean species (Glycine spp.) (e.g., sculpin, soybean (Soja hispida), or soybean (Soja max)), upland cotton (Gossypium hirsutum), sunflower species (Helianthus spp.) (e.g., sunflower (Helianthus annuus)) Hemerocallis citrina (Hemerocallis fulva), hibiscus spp, hordeum spp (e.g., hordeum vulgare), glycyrrhiza glabra (Ipomoea batatas), juglans spp, lactuca sativa, mucuna Lathyrus spp, lentils (Lens cullaris), linum usitatissimum Linum usitatissimum, Litchi (LITCHI CHINENSIS), baimaigen species (Lotus spp.), luffa acutangula (Luffa acutangula), lupinus species (Lupinus spp.), geotrichum (Luzula sylvatica), lycopersicon spp (e.g., tomato (Lycopersicon esculentum), cherry tomato (Lycopersicon lycopersicum), pear-shaped tomato (Lycopersicon pyriforme)), and combinations thereof, Sclerotium species (Macrotyloma spp.), malus species (Malus spp.), acerola (MALPIGHIA EMARGINATA), malus pumila (MAMMEA AMERICANA), mango (MANGIFERAINDICA), cassava species (Manihot spp.), ginseng fruit (MANILKARA ZAPOTA), alfalfa (Medicago sativa), sweet clover species (Melilotus spp.), peppermint species (Mentha spp.), and combinations thereof, the plant species may include, for example, bastaro (Miscanthus sinensis), momordica species (Momordica spp.), black mulberry (Morus nigra), musa species (Musa spp.), nicotiana species (Nicotiana spp.), olea species (Olea spp.), opuntia species (Opuntia spp.), costus species (Ornithopus spp.), oryza species (Oryza spp.) (e.g., rice (Oryza sativa), broadleaf rice (Oryza latifolia)), Millet (Panicum miliaceum), switchgrass (Panicum virgatum), passion flower (Passiflora edulis), parsnip (PASTINACA SATIVA), pennisetum species (Pennisetum sp.), avocado species (Persea spp.), parsley (Petroselinumcrispum), phalaris (Phalaris arundinacea), phaseolus species (Phaseolus spp.), and combinations thereof, Timothy grass (Phleum pratense), thorn genus species (Phoenix spp.), reed (PHRAGMITES AUSTRALIS), physalis spp.), pinus species (Pinus spp.), pistachio (PISTACIA VERA), pisum species (Pisum spp.), poach species (Poa spp.), populus spp), mesquite species (Prosopis spp), prune species (Prunus spp.), and combinations thereof, Guava species (Psidium spp.), punica granatum (Punica granatum), pyris (Pyrus communis), quercus species (Quercus spp.), radishes (Raphanus sativus), rheum palmatum (Rheum rhabarbarum), ribes species (Ribes spp.), ricinus communis (Ricinus communis), rubus species (Rubus spp.), saccharum species (Saccharum spp.) Willow species (Salix sp.), elder species (Sambucus spp.), rye (SECALE CEREALE), flax species (Sesamum spp.), sinapis species, solanum species (e.g., potato (Solanum tuberosum), red eggplant (Solanumintegrifolium) or tomato (Solanum lycopersicum)), sorghum (Sorghum bicolor), spinach species (Spinacia spp.), the plant species may include, but are not limited to, syzygium species (Syzygium spp.), tagetes spp, acidopsis species (Tamarindusindica), theobroma cacao (Theobroma cacao), trifolium spp, argemone digitata (Tripsacum dactyloides), secale (Triticosecale rimpaui), triticum species (Triticum spp), and (e.g., wheat (Triticum aestivum), Durum wheat (Triticum durum), cone wheat (Triticum turgidum), hybernum wheat (Triticum hybernum), mojia wheat (Triticum macha), float wheat (Triticum sativum), one grain wheat (Triticum monococcum) or common wheat (Triticum vulgare)), trollius chinensis (Tropaeolum minus), trollius chinensis (Tropaeolum majus), and combinations thereof, Vaccinium species (Vaccinium spp.), vicia spp), vicia species (Vigna spp), vigna species (Vigna spp), herba Violae (Viola odorata), vitis species (Vitis spp), semen Maydis (Zea mays), oryza sativa (Zizania palustris), zizania species (Ziziphus spp), herba Amaranthi, globe artichoke, germinatus Phragmitis, broccoli, brussels sprouts, caulis et folium Brassicae Capitatae, canola, radix Dauci Sativae, semen Brassicae Capitatae, and semen Ziziphi Spinosae (Brussels spp.), Broccoli, celery, kale (collard greens), flax, cabbage (kale), lentil (lentil), rape, okra, onion, potato, rice, soybean, strawberry, sugar beet, sugarcane, sunflower, tomato, pumpkin, tea, algae, etc. According to a preferred embodiment of the invention, the plant is a crop plant. Examples of crop plants include, in particular, soybean, sunflower, canola, alfalfa, canola (rapeseed), cotton, tomato, potato or tobacco. Further preferably, the plant is a monocotyledonous plant, such as sugarcane. Further preferred, the plant is a cereal, such as rice, maize, wheat, barley, millet, rye, sorghum or oats.

In general, the term "herbicide" as used herein means an active ingredient that kills plants, controls or otherwise adversely alters plant growth. The preferred amount or concentration of herbicide is an "effective amount" or "effective concentration". "effective amount" and "effective concentration" refer to amounts and concentrations, respectively, sufficient to kill or inhibit the growth of a similar wild-type plant, plant tissue, plant cell, or host cell, but which do not kill or severely inhibit the growth of the herbicide resistant plant, plant tissue, plant cell, and host cell of the invention. Typically, an effective amount of herbicide is that amount conventionally used in agricultural production systems to kill weeds of interest. Such amounts are known to those of ordinary skill in the art. Herbicides useful in the present invention exhibit herbicidal activity when applied directly to plants or to the locus of plants at any stage of growth or prior to planting or emergence. The observed effect depends on the plant species to be controlled, the stage of growth of the plant, the application parameters-dilution and spray droplet size, the particle size of the solid component, the environmental conditions at the time of use, the particular compound used, the particular adjuvant and carrier used, the type of soil, etc., and the amount of chemical applied. These and other factors can be adjusted as known in the art to promote non-selective or selective herbicidal action. In general, it is preferred to apply the herbicide to relatively immature undesirable vegetation after emergence to achieve maximum control of the weeds.

By "herbicide-tolerance" or "herbicide-resistance" plants is meant plants that are tolerant or resistant to at least one herbicide at a level that would normally kill normal or wild-type plants, or inhibit their growth. Herbicide levels that generally inhibit the growth of non-tolerant plants are known to those skilled in the art and are readily determined. Examples include the amounts recommended by the manufacturer for application. The maximum rate is an example of the amount of herbicide that would normally inhibit the growth of non-tolerant plants. For the purposes of the present invention, the terms "herbicide tolerance" and "herbicide resistance" are used interchangeably and are intended to have equivalent meanings and equivalent scope. Similarly, the terms "herbicide tolerance" and "herbicide resistance" are used interchangeably and are intended to have equivalent meanings and equivalent ranges. Similarly, the terms "tolerance" and "resistance" are used interchangeably and are intended to have an equivalent meaning and range of equivalents. As used herein, with respect to herbicidal compositions useful in various embodiments thereof, terms such as CESA-inhibiting herbicides and the like refer to those agronomically acceptable herbicide active ingredients (a.i.) that are recognized in the art. Similarly, terms such as fungicides, nematicides, pesticides, and the like refer to other agronomically acceptable active ingredients recognized in the art.

When used in reference to a particular mutant enzyme or polypeptide, terms such as herbicide tolerance (herebicide-tolerant and herebicide-tolerance) refer to the ability of such enzyme or polypeptide to exert its physiological activity in the presence of an amount of herbicide a.i. that would normally inactivate or inhibit its activity in the wild-type (non-mutant) form of the enzyme or polypeptide. For example, when specifically used with respect to a CESA enzyme, it specifically refers to the ability to tolerate a CESA inhibitor. By "herbicide-resistant mutant CESA protein" or "herbicide-resistant mutant CESA protein" is meant that such CESA protein exhibits higher CESA activity relative to the CESA activity of the wild-type CESA protein when at least one herbicide known to interfere with CESA activity is present and at a concentration or level known to inhibit CESA activity of the wild-type CESA protein. Furthermore, the CESA activity of such herbicide-tolerant or herbicide-resistant mutant CESA proteins may be referred to herein as "herbicide-tolerant" or "herbicide-resistant" CESA activity.

As used herein, when referring to a nucleic acid or polypeptide, "recombinant" indicates that such material has been altered as a result of human application of recombinant techniques (e.g., by polynucleotide restriction and ligation, by polynucleotide overlap-extension, or by genomic insertion or transformation). A gene sequence open reading frame is recombinant if the nucleotide sequence of that gene sequence open reading frame has been removed from its natural text and cloned into any type of artificial nucleic acid vector. The term recombinant may also refer to organisms having recombinant material, e.g., plants comprising recombinant nucleic acids may be considered recombinant plants.

The term "transgenic plant" refers to a plant comprising a heterologous polynucleotide. Preferably, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is delivered for multiple consecutive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part or plant whose genotype has been altered by the presence of a heterologous nucleic acid, including those transgenic organisms or cells that were originally so altered as well as those produced by the original transgenic organism or cell by hybridization or asexual propagation. In some embodiments, the "recombinant" organism is a "transgenic" organism. As used herein, the term "transgene" is not intended to encompass genomic (chromosomal or extrachromosomal) alterations by conventional plant breeding methods (e.g., crosses) or by naturally occurring events (e.g., such as self-fertilization, random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation).

As used herein, "mutagenized" refers to an organism or DNA thereof that has one or more alterations in the sequence of its natural genetic material compared to the sequence of genetic material of the corresponding wild-type organism or DNA, wherein the one or more alterations in the genetic material are induced and/or selected by human behavior. Examples of human behaviors that can be used to generate mutagenized organisms or DNA include, but are not limited to, tissue culture of plant cells (e.g., callus tissue) and selection thereof with herbicides (e.g., CESA-inhibiting herbicides), treatment of plant cells with chemical mutagens (e.g., EMS) and subsequent selection with one or more herbicides, or selection by treatment of plant cells with x-rays and subsequent selection with one or more herbicides. Any method known in the art may be used to induce the mutation. The method of inducing the mutation may induce a mutation at a random position in the genetic material, or may induce a mutation at a specific position in the genetic material (i.e., may be a directed mutagenesis technique), such as by using a genoplasty (genoplasty) technique.

As used herein, a "Genetically Modified Organism (GMO)" is an organism whose genetic characteristics contain one or more alterations produced by human effort that result in transfection, resulting in transformation of a target organism with genetic material from another organism or "source" organism or synthetic or modified natural genetic material, or for the progeny of retaining inserted genetic material. The source organism may be a different type of organism (e.g., a GMO plant may contain bacterial genetic material), or from the same type of organism (e.g., a GMO plant may contain genetic material from another plant). As used herein, "recombinant," "transgenic," and "GMO" are considered synonymous and indicate the presence of genetic material from different sources with respect to plants and other organisms, in contrast, "mutagenized" is used to refer to plants or other organisms, or DNA thereof, in which such transgenic material is not present, but in which natural genetic material has been mutated to differ from the corresponding wild-type organism or DNA.

As used herein, "wild type" or "corresponding wild type plant" means a typical form of an organism or genetic material thereof that is normally present, as opposed to, for example, mutagenized and/or recombinant forms. Similarly, "control cell" or "similar, wild-type plant, plant tissue, plant cell or host cell" refers to a plant, plant tissue, plant cell or host cell, respectively, that lacks the herbicide resistance characteristics and/or specific polynucleotides of the invention disclosed herein. Thus, the use of the term "wild-type" does not mean that the plant, plant tissue, plant cell, or other host cell lacks recombinant DNA in its genome and/or does not have herbicide resistance characteristics that differ from those disclosed herein.

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

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

The term "seed" includes all types of seeds, such as, for example, real seed, caryopsis, emaciation, fruit, tuber, seedling and the like. In the context of brassica and brassica species, unless otherwise indicated, "seed" refers to one or more solid seeds. For example, the seed may be a seed of a transgenic plant or a plant obtained by conventional breeding methods. Examples of traditional breeding methods may include cross breeding, selfing, backcrossing, embryo rescue, internal cross breeding, outcrossing, inbreeding, selection, asexual reproduction, and other traditional techniques known in the art.

Although exemplified with reference to a particular plant or plant variety and hybrids thereof, in various embodiments, the presently described methods of using CESA-inhibiting herbicides can be used with a variety of commercially valuable plants. CESA-inhibiting herbicide-tolerant plant lines described herein as useful may be used directly or indirectly in weed control methods, i.e., as crops for herbicide treatment or as CESA-inhibiting herbicide-tolerant trait donor lines for development (e.g., by conventional plant breeding), to produce other varieties and/or hybrid crops containing such one or more traits. All such resulting varieties or hybrid crops containing one or more ancestral CESA-inhibitory herbicide-tolerant traits may be referred to herein as progeny or offspring of the one or more ancestral CESA-inhibitory herbicide-tolerant lines. It can be said that such resulting plants retain the "one or more herbicide tolerance characteristics" of the ancestor plants, meaning that they possess and express the ancestor genetic molecular components responsible for the trait.

In one aspect, the invention provides a plant or plant part comprising a polynucleotide encoding a mutant CESA polypeptide whose expression confers tolerance to a CESA-inhibiting herbicide on the plant or plant part, wherein the mutant CESA polypeptide is characterized by substitution of the amino acid at a position corresponding to position 1051 of SEQ ID No. 28 with Leu, met or Thr.

In a preferred embodiment, plants have been previously produced by a method comprising recombinantly producing plants by introducing and overexpressing a mutant CESA transgene according to the invention, as described in more detail below.

In another preferred embodiment, plants have been previously produced by a method comprising in situ mutagenesis of plant cells or seeds to obtain plant cells or plants expressing a mutant CESA.

In another embodiment, the polynucleotide encoding a mutant CESA polypeptide comprises a nucleic acid sequence as set forth in SEQ ID NO. 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98, or a variant or derivative thereof.

In other embodiments, a mutant CESA polypeptide for use according to the invention is a functional variant that has at least about 80%, illustratively at least about 80%, 90%, 95%, 98%, 99% or greater amino acid sequence identity to SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83 over the full length of the variant.

In another embodiment, the mutant CESA polypeptides for use according to the invention are functional fragments of polypeptides having the amino acid sequence shown in SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83.

It will be appreciated that the CESA polynucleotide molecules and CESA polypeptides of the invention encompass polynucleotide molecules and polypeptides comprising a nucleotide sequence set forth in SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a nucleotide or amino acid sequence having sufficient identity to an amino acid sequence set forth in SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83. The term "sufficient identity" is used herein to mean that a first amino acid or nucleotide sequence contains a sufficient or minimum number of amino acid residues or nucleotides that are identical or equivalent (e.g., have similar side chains) to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common domain and/or a common functional activity.

In general, "sequence identity" refers to the degree to which two optimally aligned DNA or amino acid sequences will not change throughout the alignment window of the components (e.g., nucleotides or amino acids). The "identity score" for an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). "percent identity" is the identity score multiplied by 100. The optimal alignment of sequences for the alignment window is well known to those skilled in the art and can be performed by means such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search similarity method of Pearson and Lipman, and preferably by computerized implementation of these algorithms, such as GAP, BESTFIT, FASTA and TFASTA (available as part of the asepsis Le De company (accelrysinc.) of GCG software Package (GCG. Wisconsin Package, berlington, mass.).

Polynucleotides and oligonucleotides

By "isolated polynucleotide" we mean a polynucleotide that is at least partially isolated from a polynucleotide sequence associated with or linked to its native state, including DNA, RNA, or a combination thereof, single-or double-stranded, sense or antisense orientation, or a combination of both. Preferably, the isolated polynucleotides are at least 60% free, preferably at least 75% free, and most preferably at least 90% free of other components with which they are naturally associated. As the skilled artisan will appreciate, an isolated polynucleotide may be an exogenous polynucleotide that is present in, for example, a transgenic organism that does not naturally contain the polynucleotide. Furthermore, the terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", "nucleic acid molecule(s)" are used interchangeably herein and refer to nucleotides of any length (ribonucleotides or deoxyribonucleotides or a combination of both) in polymerized unbranched form.

The term "mutant CESA nucleic acid" refers to a CESA nucleic acid having a sequence that is mutated from a wild-type CESA nucleic acid and confers increased tolerance to CESA-inhibiting herbicides to plants expressing it. Furthermore, the term "mutant cellulose synthase (mutant CESA)" refers to the replacement of the amino acid of the wild-type primary sequence SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83, or a variant, derivative, homologue, ortholog or paralogue thereof, with another amino acid. The expression "mutated amino acid" will be used hereinafter to denote an amino acid that is replaced by another amino acid, thereby indicating a mutation site in the primary sequence of the protein.

In a preferred embodiment, the CESA nucleotide sequence encoding the mutant CESA comprises the sequence of SEQ ID NO:84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a variant or derivative thereof

Furthermore, it will be understood by those skilled in the art that the CESA nucleotide sequence encompasses homologs, paralogs and orthologs of SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 as defined below.

With respect to sequences (e.g., polypeptide or nucleic acid sequences, such as, for example, transcriptional regulatory nucleotide sequences of the present invention), the term "variant" is intended to mean substantially similar sequences. For nucleotide sequences comprising an open reading frame, variants include those sequences that encode the same amino acid sequence of the native protein due to the degeneracy of the genetic code. Naturally occurring allelic variants such as these may be identified by using well known molecular biology techniques, for example, using Polymerase Chain Reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those that encode a native protein comprising the sequence of SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83, as well as those that encode polypeptides having amino acid substitutions relative to the native protein (e.g., mutant CESA according to the invention as disclosed herein), e.g., generated by using site-directed mutagenesis and for open reading frames. Typically, nucleotide sequence variants of the invention will have at least 30%, 40%, 50%, 60%, to 70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%, typically at least 80%, e.g., 81% -84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99% nucleotide "sequence identity" to the nucleotide sequence of SEQ ID NO:84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97%, to 98% or 98. The% identity of the polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program), wherein the GAP creation penalty = 5 and the GAP extension penalty = 0.3. Unless otherwise specified, the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their full length.

Polypeptides

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

The% identity of the polypeptide was determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program), wherein GAP creation penalty = 5 and GAP extension penalty = 0.3. The query sequence is at least 25 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 25 amino acids. More preferably, the query sequence is at least 50 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their full length.

With respect to the defined polypeptides, it is to be understood that% identity numbers higher than the% identity numbers provided above will cover preferred embodiments. Thus, where applicable, it is preferred that the CESA polypeptides of the invention comprise amino acid sequences having 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 to SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82% or 83.

A "variant" polypeptide refers to a polypeptide derived from a protein SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83 by deletion (so-called truncation) or addition of one or more amino acids at the N-and/or C-terminus of the native protein, deletion or addition of one or more amino acids at one or more sites in the native protein, or substitution of one or more amino acids at one or more sites in the native protein. Such variants may be caused, for example, by genetic polymorphisms or by manual manipulation. Methods of such operation are well known in the art.

"Derivatives" of proteins encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activities as the unmodified protein from which they are derived. Thus, functional variants and fragments of CESA polypeptides, as well as nucleic acid molecules encoding them, are also within the scope of the invention, and unless specifically described otherwise, regardless of the source of the polypeptide and whether it occurs naturally. Various assays for the functionality of CESA polypeptides may be employed. For example, functional variants or fragments of a CESA polypeptide may be assayed to determine its ability to confer tolerance to a CESA-inhibiting herbicide. By way of illustration, CESA-inhibiting herbicide tolerance may be defined as an insensitivity to a CESA-inhibiting herbicide sufficient to provide a determinable increase in tolerance to a CESA-inhibiting herbicide in a plant or plant part comprising a recombinant polynucleotide encoding a variant or fragment of a CESA polypeptide, wherein the plant or plant part expresses up to about 0.5%, illustratively about 0.05% to about 0.5%, about 0.1% to about 0.4%, and about 0.2% to about 0.3% of a variant or fragment of a total cellular protein relative to a similarly treated control plant that does not express the variant or fragment.

In preferred embodiments, the mutant CESA polypeptide is a functional variant or fragment of a cellulose synthase having an amino acid sequence set forth in SEQ ID No. 1,3, 11, 16, 20, 23, 28, 35, 45, 51, 55, 66, 69, 74, 75, 79 or 80, wherein the functional variant or fragment has at least about 80% amino acid sequence identity to SEQ ID No. 1,3, 11, 16, 20, 23, 28, 35, 45, 51, 55, 66, 69, 74, 75, 79 or 80.

In other embodiments, the functional variant or fragment further has CESA-inhibiting herbicide tolerance, which is defined as insensitivity to a CESA-inhibiting herbicide sufficient to provide a determinable increase in tolerance to a CESA-inhibiting herbicide in a plant or plant part comprising a recombinant polynucleotide encoding the variant or fragment, wherein the plant or plant part expresses up to about 0.5% of the variant or fragment of the total cellular protein relative to a similarly treated control plant not expressing the variant or fragment.

"Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.

In addition, one of ordinary skill in the art will further appreciate that changes may be introduced into the nucleotide sequences of the present invention by mutation, resulting in changes in the amino acid sequence of the encoded protein without altering the biological activity of the protein. Thus, for example, an isolated polynucleotide molecule encoding a mutant CESA polypeptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83 may be produced by introducing one or more nucleotide substitutions, additions or deletions into the corresponding nucleotide sequence such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. The present invention also encompasses such variant nucleotide sequences. For example, preferably, conservative amino acid substitutions may be made at one or more predicted preferred nonessential amino acid residues. "nonessential" amino acid residues are residues that can be altered from the wild-type sequence of the protein without altering the biological activity, while "essential" amino acid residues are required for the biological activity.

Deletions refer to the removal of one or more amino acids from a protein.

Insertion refers to the introduction of one or more amino acid residues into a predetermined site of a protein. Insertions may comprise N-terminal and/or C-terminal fusions, as well as intra-sequence insertions of single or multiple amino acids. Typically, insertions within the amino acid sequence will be smaller than N-terminal or C-terminal fusions, on the order of about 1 to 10 residues. Examples of N-terminal or C-terminal fusion proteins or peptides include the binding or activation domain of transcriptional activators used in yeast two-hybrid systems, phage coat proteins, (histidine) -6-tag, glutathione S-transferase-tag, protein A, maltose binding protein, dihydrofolate reductase, tag 100 epitope, C-myc epitope,Epitope, lacZ, CMP (calmodulin binding peptide), HA epitope, protein C epitope and VSV epitope.

Substitution refers to the replacement of an amino acid of a protein with other amino acids having similar properties (e.g., similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or disrupt an alpha-helical structure or a beta-sheet structure). Amino acid substitutions are typically single residue substitutions, but may be clustered (depending on the functional limitations imposed on the polypeptide) and may be in the range of 1 to 10 amino acids, with insertions typically being on the order of about 1 to 10 amino acid residues. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions are not made to conserved amino acid residues or to amino acid residues that reside within a conserved motif. Conservative representations are well known in the art (see, e.g., cright on (1984) Proteins [ protein ] W.H.Freeman and Company [ W.H.Frieman Co.) ]).

Amino acid substitutions, deletions and/or insertions can be readily made using peptide synthesis techniques well known in the art (e.g., solid phase peptide synthesis, etc.) or by recombinant DNA procedures. Methods for manipulating DNA sequences to produce substitution, insertion or deletion variants of proteins are well known in the art. For example, techniques for substitution mutagenesis at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB company, cleveland, ohio), quickChange site-directed mutagenesis (Stratagene company, san Diego, calif.), PCR-mediated site-directed mutagenesis, or other site-directed mutagenesis protocols.

"Derivatives" further include peptides, oligopeptides, polypeptides, which may comprise substitution of amino acids with, or addition of non-naturally occurring amino acid residues as compared to the amino acid sequence of a naturally occurring form of the protein (e.g., the protein of interest). "derivatives" of proteins also encompass peptides, oligopeptides, polypeptides, which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulfated, etc.) or non-naturally altered amino acid residues as compared to the amino acid sequence of the naturally occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additives as compared to the amino acid sequence from which it is derived, e.g., a reporter molecule or other ligand that is covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule that is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally occurring protein. In addition, "derivatives" include fusion of naturally occurring forms of the protein with a tag peptide (such as FLAG, HIS6 or thioredoxin) (for reviews of tag peptides, see Terpe, appl. Microbiol. Biotechnol. [ applied microbiology and biotechnology ]60,523-533,2003).

"Orthologs" and "paralogs" encompass evolutionary concepts used to describe the ancestral relationship of genes. Paralogs are genes within the same species that originate from the replication of an ancestral gene, and orthologs are genes from different organisms that originate by speciation and also from a common ancestral gene. Non-limiting lists of examples of such orthologs are shown in table 1. Those skilled in the art will appreciate that the sequences SEQ ID NO:2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83 listed in Table 1 represent orthologues and paralogues of SEQ ID NO. 1.

It is well known in the art that paralogs and orthologs may share different domains carrying suitable amino acid residues at a given site, such as binding pockets for specific substrates or binding motifs for interactions with other proteins.

The term "domain" refers to a group of amino acids that are conserved at specific positions along an alignment of evolutionarily related protein sequences. While amino acids at other positions may vary between homologs, amino acids that are highly conserved at particular positions indicate amino acids that may be necessary in the structure, stability, or function of the protein. "domains" are identified by their high degree of conservation in aligned sequences of a family of protein homologs, which can be used as identifiers to determine whether any polypeptide in question belongs to a previously identified family of polypeptides.

The term "motif" or "consensus sequence" refers to a short conserved region in a sequence of a proteins of interest. Motifs are typically highly conserved parts of a domain, but may also comprise only a part of a domain, or be located outside a conserved domain (if all amino acids of a motif fall outside a defined domain).

Additionally or alternatively, the homologs of the CESA protein have at least 20％、21％、22％、23％、24％、25％、26％、27％、28％、29％、30％、31％、32％、33％、34％、35％、36％、37％、38％、39％、40％、41％、42％、43％、44％、45％、46％、47％、48％、49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％ or 99% overall sequence identity in ascending order of preference to the amino acid represented by SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83. The global alignment algorithm (e.g., NEEDLEMAN WUNSCH algorithm in the GAP program (GCG software package, arser Le De, wi)) is used to determine overall sequence identity, preferably using default parameters and preferably using the sequence of the mature protein (i.e., without consideration of secretion signals or transit peptides).

There are specialized databases for identifying domains such as SMART (Schultz et al (1998) Proc. Natl. Acad. Sci. USA [ national academy of sciences of the United states ]95,5857-5864; leturc et al (2002) Nucleic Acids Res [ Nucleic acids research ]30, 242-244), interPro (Mulder et al, (2003) Nucl. Acids. Res. [ Nucleic acids research ]31, 315-318), prosite (the generalized features of Bucher and Bairoch(1994),A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation.[ for biomolecular sequence motifs and their functions in automated sequence interpretation ] (in) ISMB-94;Proceedings2nd International Conference on Intelligent Systems for Molecular Biology [ International conference of molecular biology, 2. Proceedings ] Altman R., brutlag D., karp P., lathrop R., searls D., editions, pages 53-61, AAAIPRESS [ AAAI ] Portal Park (Menlo Park); hulo et al, nucleic acids Res. Nucleic acids 32: D134-D137, (2004)), or Pfam (Bateman et al, nucleic ACIDS RESEARCH [ Nucleic acids Res. 30 (1): 276-280 (2002)). A set of tools for computer analysis of protein sequences are available on the ExPASy proteomics server (Swiss Bioinformatics institute (Swiss Institute of Bioinformatics)) (Gasteiger et al, exPASy: the proteomics server for in-depth protein knowledge AND ANALYSIS [ ExPASy: proteomics server for in-depth knowledge and analysis of proteins ], nucleic Acids Res. [ nucleic acids research ]31:3784-3788 (2003)). The domains or motifs can also be identified using conventional techniques, for example by sequence alignment.

Methods for aligning sequences for comparison are well known in the art and include GAP, BESTFIT, BLAST, FASTA and tfast a. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol [ journal of molecular biology ] 48:443-453) to find global (i.e., across complete sequences) alignments of two sequences, thereby maximizing the number of matches and minimizing the number of space bits. The BLAST algorithm (Altschul et al (1990) J Mol Biol [ journal of molecular biology ] 215:403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. Software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Centre for Biotechnology Information, NCBI). Homologs can be readily identified using, for example, the ClustalW multisequence alignment algorithm (version 1.83) with default alignment parameters and percentage scoring methods (see fig. 1). Global similarity and percent identity can also be determined using one of the methods available in the MatGAT software package (Campanella et al BMC Bioinformatics [ BMC bioinformatics ] application of 7/10/;4:29.MatGAT:an application that generates similarity/identity matrices using protein or DNA sequences.[MatGAT： to generate a similarity/identity matrix using protein or DNA sequences). It will be apparent to those skilled in the art that a small number of manual edits can be made to optimize the alignment between conserved motifs. In addition, instead of using full length sequences, specific domains can also be used to identify homologs. Sequence identity values can be determined over the entire nucleic acid or amino acid sequence or over a selected domain or one or more conserved motifs using default parameters using the procedure described above. For local alignment, the Smith-Waterman algorithm is particularly useful (Smith TF, waterman MS (1981) J.mol. Biol [ journal of molecular biology ]147 (1); 195-7).

The proteins of the invention may be altered in a variety of ways, including amino acid substitutions, deletions, truncations and insertions. Methods of such operation are well known in the art. For example, amino acid sequence variants may be prepared by mutation in DNA. Methods for mutagenesis and nucleotide sequence alteration are well known in the art. See, e.g., kunkel (1985) PNAS [ Proc. Natl. Acad. Sci. USA ],82:488-492; kunkel et al (1987) Methodsin Enzymol [ methods of enzymology ]154:367-382; U.S. Pat. No. 4,873,192; walker and Gaastra editions (1983) Techniques in Molecular Biology [ techniques of molecular biology ] (Mimi Lun publishing company (MACMILLAN PUBLISHING COMPANY), new York) and references cited therein. Guidance on suitable 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 [ protein sequence and structure atlas ] (national biomedical research foundation (Natl. Biomed. Res. Foundation.), washington Columbia zone), which is incorporated herein by reference. Conservative substitutions (e.g., exchanging one amino acid for another with similar properties) may be preferred.

Alternatively, variant nucleotide sequences may be made by randomly introducing mutations along all or part of the coding sequence (e.g., by saturation mutagenesis), and the resulting mutants may be screened to identify mutants encoding proteins that retain activity. For example, after mutagenesis, the encoded protein may be expressed recombinantly, and the activity of the protein may be determined using standard assay techniques.

The inventors of the present invention have found that by mutating the CESA-encoding nucleic acid by substituting one or more of the key amino acid residues of SEQ ID NO:28 (e.g., by employing one of the methods described above), tolerance or resistance to a particular CESA-inhibiting herbicide (collectively referred to as azines, and described in more detail below) can be significantly increased. Preferred substitutions of mutant CESA are those that increase herbicide tolerance in plants, but do not substantially affect the biological activity of cellulose synthase activity.

Accordingly, in another object of the present invention is a CESA polypeptide comprising a sequence of SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83, a variant, derivative, ortholog, paralog or homologue thereof, the key amino acid residues of which are substituted with any other amino acid.

Those skilled in the art will appreciate that amino acids located immediately adjacent to the amino acid positions described below may also be substituted. Thus, in another embodiment ,SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83, a variant, derivative, ortholog, paralog or homologue thereof, comprises a mutant CESA in which the amino acid at a position + -3, + -2 or + -1 amino acids from the critical amino acid is substituted with any other amino acid.

Based on techniques well known in the art, highly characteristic sequence patterns can be developed by which mutant CESA candidates with the desired activity can be further searched.

The invention also encompasses searching for additional mutant CESA candidates by applying an appropriate sequence pattern. The skilled person will appreciate that the sequence pattern of the invention is not limited by the exact distance between two adjacent amino acid residues of the pattern. Each of the distances between two adjacent amino acids in the above pattern may vary, for example, up to ±10, ±5, ±3, ±2 or ±1 amino acid positions independently of each other without substantially affecting the desired activity.

Furthermore, the inventors of the present invention have identified and generated specific amino acid substitutions and combinations thereof by applying methods of site-directed mutagenesis, particularly saturation mutagenesis (see, e.g., schenk et al, biospektrum [ biological spectrum ]03/2006, pages 277-279), PCR-based site-directed mutagenesis (e.g., the directional mutagenesis kit of Stratagene, california, or the GeneArt mutagenesis service of sammer femto technology, massachusetts, usa) or systematic mutagenesis (the GeneArt system mutagenesis service of sammer femto technology, massachusetts, usa), which confer to plants increased herbicide resistance or tolerance to CESA-inhibiting herbicides when these amino acid substitutions and combinations thereof are introduced into the plants by transforming and expressing the corresponding nucleic acids encoding mutant CESA.

In another embodiment, a variant or derivative of a CESA polypeptide refers to a CESA polypeptide comprising SEQ ID NO. 28, an ortholog, paralog or homolog thereof, wherein the amino acid sequence differs from the wild-type amino acid sequence of the CESA polypeptide at a position corresponding to position 1051 of SEQ ID NO. 28.

In a preferred embodiment, the amino acid at a position corresponding to position 1051 of SEQ ID NO. 28 is Leu, met or Thr.

It will be within the knowledge of one skilled in the art to identify conserved regions and motifs shared between homologs, orthologs and paralogs encoded by SEQ ID NOs 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98, such as those described in Table 1.

a) Generating a library of nucleic acids encoding mutant CESA,

In a preferred embodiment, the nucleic acid encoding a mutant CESA selected in step d) provides at least 2-fold resistance or tolerance of the cell or plant to the CESA-inhibiting herbicide as compared to the resistance or tolerance of the cell or plant provided by the nucleic acid encoding a control CESA to the CESA-inhibiting herbicide.

In a further preferred embodiment, the nucleic acid encoding a mutant CESA selected in step d) provides at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 500-fold resistance or tolerance of the cell or plant to a CESA-inhibiting herbicide as compared to resistance or tolerance of the cell or plant provided by the nucleic acid encoding a control CESA to a CESA-inhibiting herbicide.

Resistance or tolerance can be determined by generating a transgenic plant or host cell, preferably a plant cell, comprising the nucleic acid sequence of the library of step a) and comparing the transgenic plant with a control plant or host cell, preferably a plant cell.

Another object relates to a method of identifying a plant or an alga containing a nucleic acid comprising a nucleotide sequence encoding a mutant CESA that is resistant or tolerant to a CESA-inhibiting herbicide, the method comprising:

a) Identifying in a plant cell or green algae culture an effective amount of a CESA inhibiting herbicide that results in death of the cell.

B) Treating the plant cells or green algae with a mutagen,

D) Selecting at least one cell that survives the test conditions,

In a preferred embodiment, the mutagen is Ethyl Methylsulfonate (EMS).

Many methods well known to those of skill in the art can be used to obtain suitable candidate nucleic acids from a variety of different potential sources including microorganisms, plants, fungi, algae, mixed cultures, etc., as well as environmental sources of DNA such as soil, for identifying nucleotide sequences encoding mutant CESA. These methods include, inter alia, preparation of cDNA or genomic DNA libraries, use of appropriately degenerate oligonucleotide primers, use of probes based on known sequences or complementary assays (e.g., growth on tyrosine), and use of mutagenesis and shuffling to provide recombinant or shuffled sequences encoding mutant CESA.

The nucleic acid comprising sequences encoding the candidate and control CESA may be expressed in yeast, bacterial host strains, algae, or higher plants (e.g., tobacco or arabidopsis), and the relative levels of inherent tolerance of the sequences encoding CESA are screened for a visible indicator phenotype of the transformed strain or plant in the presence of different concentrations of the selected CESA-inhibiting herbicide. The dose response and the relative change in dose response associated with these indicator phenotypes (formation of necrosis, growth inhibition, herbicidal effect, etc.) can conveniently be expressed, for example, as GR50 (concentration of 50% reduction in growth) or MIC (minimum inhibitory concentration) values, where an increase in the value corresponds to an increase in the inherent tolerance of the expressed CESA. For example, in a relatively rapid assay system based on transformed arabidopsis as described in the examples section (example 7), each sequence encoding a mutant CESA may be expressed as a DNA sequence, e.g., under the control of expression of a suitable promoter, and T1 plants may be selected for differential tolerance (as measured by growth) to the CESA-inhibiting herbicide of choice.

In another embodiment, candidate nucleic acids are transformed into plant material to generate transgenic plants, the formally normal fertile plants are regenerated, and then the fertile plants are measured for differential tolerance to a selected CESA-inhibiting herbicide, as described in the examples section below. Many suitable methods for transformation and plant regeneration (e.g. from tobacco leaf discs) using suitable selectable markers (e.g. kanamycin), binary vectors (e.g. from Agrobacterium (Agrobacterium)), are well known in the art. Optionally, the control plant population is also transformed with a nucleic acid that expresses a control CESA. Alternatively, untransformed dicotyledonous plants such as Arabidopsis or tobacco can be used as controls, as such plants express their own endogenous CESA in any event. The average and distribution of herbicide tolerance levels of the above-described series of primary plant transformation events or progeny thereof to CESA-inhibiting herbicides is assessed in a conventional manner based on plant damage, meristem bleaching symptoms, etc. at a range of different herbicide concentrations. These data can be expressed, for example, as GR50 values derived from dose/response curves plotted with "dose" on the x-axis and "percent kill", "herbicidal effect", "number of green plants in the new year", etc., on the y-axis, wherein an increase in GR50 values corresponds to an increase in the inherent tolerance level of the expressed CESA. The herbicide may be applied pre-emergence or post-emergence as appropriate.

Another object of the present invention relates to an isolated and/or recombinantly produced and/or synthetic nucleic acid encoding a mutant CESA as disclosed above, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a variant or derivative thereof.

In one embodiment, the nucleic acid is identifiable by a method as defined above.

For the purposes of the present invention, "recombinant" means that for example nucleic acid sequences, expression cassettes (=genetic constructs, nucleic acid constructs) or vectors containing the nucleic acid sequences according to the invention or organisms transformed with said nucleic acid sequences, expression cassettes or vectors according to the invention, all of those constructs are produced by genetic engineering methods, wherein

(A) A nucleic acid sequence comprising the sequence of SEQ ID NO. 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98, or a homologue thereof, or a derivative or part thereof, or

(B) Genetic control sequences, e.g.3 '-and/or 5' -genetic control sequences, such as promoters or terminators, functionally linked to the nucleic acid sequences described in (a), or

(C) (a) and (b);

Modifications such as substitution, addition, deletion, inversion or insertion of one or more nucleotide residues are not found or have been genetically modified in their natural genetic environment.

"Native genetic environment" means the native genomic or chromosomal locus in the organism of origin or within the host organism, or present in a genomic library. In the case of genomic libraries, the natural genetic environment of the nucleic acid sequence is preferably at least partially preserved. The environment adjoins the nucleic acid sequence at least on one side and has a sequence length of at least 50bp, preferably at least 500bp, particularly preferably at least 1000bp, most particularly preferably at least 5000 bp. When the transgene expression cassette is modified by non-natural synthetic ("artificial") methods such as, for example, mutagenesis, the naturally occurring expression cassette (e.g., a naturally occurring combination of a natural promoter of a nucleic acid sequence according to the invention and a corresponding gene) is converted into a transgene expression cassette. Suitable methods are described by way of example in U.S. Pat. No. 5,565,350 or WO 00/15815.

In a preferred embodiment, the encoded mutant CESA is a variant of SEQ ID NO. 28, wherein the amino acid at the position corresponding to position 1051 of SEQ ID NO. 28 is substituted with Leu, met or Thr.

In other aspects, the invention encompasses progeny or offspring of the CESA-inhibiting herbicide-tolerant plants of the invention, as well as seeds derived from the CESA-inhibiting herbicide-tolerant plants of the invention and cells derived from the CESA-inhibiting herbicide-tolerant plants of the invention.

In some embodiments, the present invention provides progeny or offspring plants derived from a plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, wherein the progeny or offspring plant comprises in at least some cells thereof a recombinant polynucleotide operably linked to the promoter, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the progeny or offspring plant.

In one embodiment, the seed of the invention preferably comprises CESA-inhibiting herbicide tolerance characteristics of a CESA-inhibiting herbicide tolerant plant. In other embodiments, the seed is capable of germinating into a plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, the expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to progeny or offspring plants.

In some embodiments, the plant cells of the invention are capable of regenerating into plants or plant parts. In other embodiments, the plant cell is unable to regenerate a plant or plant part. Examples of cells that cannot regenerate into plants include, but are not limited to, endosperm, seed coat (seed coat and pericarp), and root cap.

In another embodiment, the present invention provides a plant cell of a plant or capable of regenerating into a plant, the plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the plant cell comprises a recombinant polynucleotide operably linked to a promoter.

In other embodiments, the invention provides a plant cell comprising a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the cell.

In another embodiment, the invention relates to a plant cell transformed with a nucleic acid encoding a mutant CESA polypeptide according to the invention, or to a plant cell that has been mutated to obtain a plant expressing a nucleic acid encoding a mutant CESA polypeptide according to the invention, wherein expression of the nucleic acid in the plant cell results in increased resistance or tolerance to a CESA-inhibiting herbicide compared to a wild-type variety of the plant cell. Preferably, the nucleic acid encoding a mutant CESA polypeptide comprises a polynucleotide sequence selected from the group consisting of a) a polynucleotide as shown in SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a variant or derivative thereof, b) a polynucleotide encoding a polypeptide as shown in SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83 or a variant or derivative thereof, c) a polynucleotide comprising at least 60 consecutive nucleotides of any of a) or b), and d) a polynucleotide complementary to a polynucleotide of any of a) to c).

In some aspects, the invention provides plant products prepared from CESA-inhibiting herbicide tolerant plants herein. In some embodiments, examples of plant products include, but are not limited to, kernels, oils, and kibbles. In one embodiment, the plant product is a plant kernel (e.g., kernel suitable for use as a feed or for processing), a plant oil (e.g., oil suitable for use as a food or biodiesel), or a plant meal (e.g., meal suitable for use as a feed).

In one embodiment, a plant product prepared from a plant or plant part is provided, wherein the plant or plant part comprises in at least some cells thereof a polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant or plant part.

In some aspects, the invention provides methods of producing CESA-inhibiting herbicide-tolerant plants. In one embodiment, the method comprises regenerating a plant from a plant cell transformed with a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide on the plant.

The term "expression" or "gene expression" means the transcription of a particular gene(s) or a particular gene construct. The term "expression" or "gene expression" means in particular the transcription of a gene(s) or gene construct into structural RNA (rRNA, tRNA) or mRNA, the latter being subsequently translated into protein or not. This process involves DNA transcription and processing of the resulting mRNA product.

In order to obtain the desired effect, i.e. the plants of the invention which are tolerant or resistant to CESA-inhibiting herbicide derivatives herbicides, it is to be understood that the at least one nucleic acid is "overexpressed" by methods and means known to the person skilled in the art.

As used herein, the term "increased expression" or "overexpression" means any form of expression that is added to the original wild-type expression level. Methods for increasing expression of a gene or gene product are well documented in the art and include, for example, overexpression driven by an appropriate promoter, the use of transcriptional enhancers or translational enhancers. An isolated nucleic acid that acts as a promoter or enhancer element may be introduced into a polynucleotide in a non-heterologous form at a suitable location (typically upstream) so as to up-regulate expression of the nucleic acid encoding the polypeptide of interest. For example, the endogenous promoter may be altered in vivo by mutation, deletion and/or substitution (see Kmiec, U.S. Pat. No. 5,565,350; zarling et al, WO 9322443), or the isolated promoter may be introduced into a plant cell in the appropriate orientation and distance from the gene of the present invention to control expression of the gene. If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3' -end of the polynucleotide coding region. The polyadenylation region may be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3' terminal sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene. Intronic sequences may also be added to the coding sequence of the 5' untranslated region (UTR) or part of the coding sequence to increase the amount of mature message that accumulates in the cytosol. It has been shown that inclusion of a spliceable intron in the transcription unit of plant and animal expression constructs can increase gene expression up to 1000-fold at the mRNA and protein levels (Buchman and Berg (1988) mol. Cell biol. [ molecular cell biology ]8:4395-4405; callis et al (1987) Genes Dev [ Gene & development ] 1:1183-1200). Such intron enhanced gene expression is typically greatest when placed near the 5' end of the transcriptional unit. The use of maize introns (Adh 1-S introns 1, 2 and 6, the bronze-1 intron) is known in the art. For general information see The Maize Handbook [ maize handbook ], chapter 116, freeling and Walbot, editions, springer [ Schpulger Press ], new York (1994).

Where appropriate, the nucleic acid sequences may be optimized to increase expression in transformed plants. For example, a coding sequence comprising plant-preferential codons for improved expression in a plant can be provided. For a discussion of host preference codon usage see, e.g., campbell and Gowri (1990) Plant Physiol [ Plant physiology ],92:1-11. Methods for preparing plant-preferred genes are also known in the art. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al (1989) Nucleic Acids Res [ nucleic acids research ]17:477-498, which are incorporated herein by reference.

Thus, the mutant CESA nucleic acids of the invention are provided in expression cassettes for expression in plants of interest. The cassette will include regulatory sequences operably linked to the mutant CESA nucleic acid sequences of the invention. As used herein, the term "regulatory element" 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 'UTRs and 3' UTRs. "operably linked" refers to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of a DNA sequence corresponding to the second sequence. Typically, operably linked means that the linked nucleic acid sequences are contiguous and, where necessary to join two protein coding regions, the linked nucleic acid sequences are contiguous and in the same reading frame. Alternatively, the cassette may contain at least one additional gene to be co-transformed into the organism. Alternatively, one or more additional genes may be provided on multiple expression cassettes.

Such an expression cassette is provided with a plurality of restriction sites for insertion of the mutant CESA nucleic acid sequence under transcriptional control of the regulatory region. The expression cassette may additionally contain a selectable marker gene. The expression cassettes of the invention will include transcription and translation initiation regions (i.e., promoters) functional in plants, nucleic acid sequences encoding mutant CESA of the invention, and transcription and translation termination regions (i.e., termination regions) in the 5'-3' transcription direction. The promoters may be native or similar, or foreign or heterologous to the plant host and/or the mutant CESA nucleic acid sequences of the invention. Alternatively, the promoter may be a natural sequence or alternatively a synthetic sequence. When a promoter is "foreign" or "heterologous" to a plant host, this means that the promoter is not found in the native plant into which the promoter was introduced. When the promoter is "foreign" or "heterologous" to the mutant CESA nucleic acid sequences of the invention, this refers to a promoter that is not native or naturally occurring to the operably linked mutant CESA nucleic acid sequences of the invention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Although the use of a heterologous promoter to express the mutant CESA nucleic acids of the invention may be preferred, natural promoter sequences may also be used. Such constructs will alter the expression level of the mutant CESA protein in a plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.

The termination region may be native to the transcription initiation region, may be native to the operably linked mutant CESA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the mutant CESA nucleic acid sequence of interest, the plant host, or any combination thereof). Convenient termination regions may be obtained from Ti-plasmids of Agrobacterium tumefaciens (A.tumefaciens), such as octopine synthase and nopaline synthase termination regions. See also Guerineau et al (1991) MoI.Gen.Genet. [ molecular genetics & genomics ]262:141-144, proudfoot (1991) Cell [ Cell ]64:671-674, sanfacon et al (1991) Genes Dev. [ Gene & development ]5:141-149, mogen et al (1990) PLANT CELL [ plant Cell ]2:1261-1272; munroe et al (1990) Gene [ Gene ]91:151-158; ballas et al (1989) Nucleic Acids Res. [ Nucleic acids research ]17:7891-7903; and Joshi et al (1987) Nucleic acids Res. [ Nucleic acids research ]15:9627-9639. Where appropriate, one or more genes may be optimized to increase expression in the transformed plant. That is, plant preference codons can be used to synthesize genes to improve expression. For a discussion of host preference codon usage see, e.g., campbell and Gowri (1990) Plant Physiol [ Plant physiology ]92:1-11. Methods for synthesizing plant-preferential genes are available in the art. See, for example, U.S. Pat. Nos. 5,380,831 and 5,436,391, and Murray et al (1989) Nucleic Acids Res [ nucleic acids research ]17:477-498, which are incorporated herein by reference.

Although the polynucleotides of the invention may be used as selectable marker genes for plant transformation, the expression cassettes of the invention may include another selectable marker gene for selection of transformed cells. Selectable marker genes (including those of the invention) are used to select transformed cells or tissues. Marker genes include, but are not limited to, genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and Hygromycin Phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds such as glufosinate ammonium, bromoxynil, imidazolinones and2, 4-dichlorophenoxyacetate (2, 4-D). See generally Yarranton (1992) curr.Opin.Biotech [ current viewpoint of biotechnology ]3:506-511; christopmers et al (1992) Proc.Natl.Acad.ScL USA [ Proc. Natl Acad.Sci.Sci.USA (Proc. Natl. Acad. Sci.Sci.USA. USA (1992) Cell [ Cell ]71:63-72; reznikoff (1992) MoI Microbiol [ molecular microbiology ]6:2419-2422; barkley et al (1980) in The operator ], pages 177-220; hu et al (1987) Cell [ Cell ]48:555-566; brown et al (1987) Cell [ Cell ]49:603-612; figge et al (1988) Cell [ Cell ]52:713-722; deuschle et al (1989) Proc.Natl Acad.AcL USA [ Proc.Natl Acad.ScL USA [ Sci. USA U.S. Sci ]86:2549-2553; deuschle et al (1990) Science [ Science ]248:480-483; gossen (1993) Ph.D. thesis [ doctor article ], university of Heidelberg [ university of Heidelberg ]; reines et al (1993) Proc.Natl Acad.ScL USA [ Proc. Natl. Acad. Sci. USA ]90:1917-1921; labow et al (1990) MoI Cell Biol [ molecular cell biology ]10:3343-3356; zambretti et al (1992) Proc.Natl Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]89:3952-3956; bairn et al (1991) Proc.Natl Acad. Scl. Sci. U. A. U.S. Sci. No. Sci. 5-5072-5076; wyborski et al (1991) Nucleic Acids Res:4647-4653; hillend-Wissman (1989) Topics MoI. Strul [ thermal molecular structure biology ]10:143-162; degenelb et al (1991) Antimrobotics; university of Heidelberg [ university of Heidelberg ]; gossen et al (1992) Proc.Natl Acad.ScL USA [ Proc. Natl. Acad. Sci. USA ]89:5547-5551; oliva et al (1992) Antimicrob. Agents Chemother. [ antimicrobial and chemotherapy ]36:913-919; hlavka et al (1985) Handbook of Experimental Pharmacology [ handbook of Experimental pharmacology ], volume 78 (Springer-Verlag), berlin; gill et al (1988) Nature [ Nature ]334:721-724. Such disclosure is incorporated herein by reference. The above list of selectable marker genes is not intended to be limiting. Any selectable marker gene may be used in the present invention.

Furthermore, additional sequence modifications are known to enhance gene expression in cellular hosts. These include the elimination of sequences encoding pseudo polyadenylation signals, sequences encoding exon-intron splice site signals, sequences encoding transposon-like repeats, and other such sequences that are well characterized and may be detrimental to gene expression. The G-C content of the sequences can be adjusted to the average level of a given cellular host, as calculated by reference to known genes expressed in the host cell. Furthermore, the sequence can be easily modified to avoid predicted hairpin secondary mRNA structures, if desired. Nucleotide sequences for enhancing gene expression may also be used in plant expression vectors. These include, for example, introns Adh1-S introns 1, 2 and 6 of the maize Adh gene (Callis et al Genes and Development [ Gene & development ]1:1183-1200,1987) and leader sequences (W-sequences) from Tobacco Mosaic Virus (TMV), maize chlorotic mottle virus and alfalfa mosaic virus (Gallie et al Nucleic Acid Res. [ Nucleic acids research ]15:8693-8711,1987 and Skuzeski et al Plant mol. Biol. [ Plant molecular biology ]15:65-79,1990). The first intron from the maize shrunken-1 locus has been shown to increase gene expression in chimeric gene constructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and galie et al (Plant Physiol. [ Plant physiology ]106:929-939,1994) also demonstrate that introns can be used to regulate gene expression on a tissue specific basis. To further enhance or optimize gene expression, the plant expression vectors of the present invention may also comprise DNA sequences comprising Matrix Attachment Regions (MARs). Plant cells transformed with such modified expression systems may then exhibit over-expression or constitutive expression of the nucleotide sequences of the invention.

The invention further provides an isolated recombinant expression vector comprising an expression cassette comprising a mutant CESA nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to a CESA-inhibiting herbicide as compared to a wild-type variety of the host cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". In general, expression vectors useful in recombinant DNA technology are typically in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. The present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

The recombinant expression vectors of the invention comprise the nucleic acids of the invention in a form suitable for expressing the nucleic acids in a host cell, which means that the recombinant expression vector comprises one or more regulatory sequences selected based on the host cell to be used for expression, operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of the nucleotide sequence only in certain host cells or under certain conditions. Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of polypeptide expression, and the like. The expression vectors of the invention can be introduced into host cells to produce polypeptides or peptides, including fusion polypeptides or peptides (e.g., mutant CESA polypeptides, fusion polypeptides, etc.), encoded by nucleic acids as described herein

The expression vector may additionally contain a 5' leader sequence in the expression construct. Such a leader sequence may serve to enhance translation. Translation leader sequences are known in the art and include, for example, picornaviral leader sequences (EMCV leader sequence (encephalomyocarditis 5' non-coding region) (Elroy-Stein et al (1989) PNAS [ Proc. Natl. Acad. Sci. U.S. Sci., 86:6126-6130)), potato viral leader sequences (TEV leader sequence (Tobacco Etch Virus)) (Gallie et al (1995) Gene [ Gene ]165 (2): 233-238), MDMV leader sequences (maize dwarf mosaic virus (Maize Dwarf Mosaic Virus)) (Virology [ Virology ] 154:9-20) and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al (1991) Nature [ Nature ] 353:90-94), non-translated leader sequences (AMV RNA 4) from alfalfa mosaic virus coat protein mRNA (Jobling et al (Nature [ Natl. 325:622-625)), tobacco mosaic virus leader sequences (Gal. V) (Gal. Natl. Gene [ Gene ]165 (2):. 233-238), and human immunoglobulin heavy chain binding protein (BiP) (Macejak et al. [ Nature ] Nature [ Natl. Sci., 3:353-94), human immunoglobulin (3:3) RNA (Lev. Natl. Sci., 3) and human immunoglobulin RNA (3:3.K.K.K.K.K.K.K.K. L.). See also Della-Cioppa et al (1987) Plant Physiol [ Plant physiology ]84:965-968.

Other methods known to enhance translation, such as introns, etc., may also be utilized. In preparing the expression vector, various nucleic acid fragments may be manipulated to provide the nucleic acid sequence in the correct orientation and, where appropriate, in the correct reading frame. To this end, adaptors or linkers may be employed to ligate nucleic acid fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess nucleic acid, remove restriction sites, and the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re-substitution, such as transitions and transversions, may be involved.

Many promoters may be used in the practice of the present invention. Promoters may be selected based on the desired result. The nucleic acid may be combined with a constitutive promoter, a tissue-preferred promoter, or other promoters for expression in the plant.

Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050, the core CaMV 35S promoter (Odell et al (1985) Nature [ Nature ] 313:810-812), rice actin (McElroy et al (1990) PLANT CELL [ Plant cell ] 2:163-171), ubiquitin (Christensen et al (1989) Plant mol. Biol. [ Plant molecular biology ]12:619-632 and Christensen et al (1992) Plant mol. Biol. [ Plant molecular biology ] 18:675-689), pEMU (Last et al (1991) the operator applied. Genet. [ theory and applied genetics ] 81:581-588), MAS (Velten et al (1984) EMJ. [ European molecular biology ] 3:23:279, 026, etc. Other constitutive promoters include, for example, U.S. Pat. nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, and 6,177,611.

Tissue-preferred promoters may be used to target enhanced expression within specific plant tissues. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Some examples of tissue-preferred promoters are described, for example, by Yamamoto et al (1997) Plant J. [ Plant J. ]12 (2): 255-265; kawamata et al (1997) PLANT CELL Plant physiology ]38 (7): 792-803; hansen et al (1997) Mol. Genet. [ molecular Genet. ]254 (3): 337-343; russell et al (1997) TRANSGENIC RES. [ transgenic research ]6 (2): 157-168; rinehart et al (1996) Plant Physiol. [ Plant physiology ]1 (3): 1331-1341; van cam et al (1996) Plant Physiol. [ Plant physiology ]112 (2): 525-535; canascini et al (1996) Plant Physiol. ]1 (2): 513-524; russel et al (1997) 35 (1998): 35-35) Plant physiology ]35 (1996) and by Programm 35 (1993) [ Plant Physiol ]35 (35) Plant Physiol ]1 (3): 1331-1341; buddha et al (1996) Plant Physiol, and by Proc 35 (1996) Plant Physiol et al, 35 (1996) Plant Physiol. If desired, the promoter may be modified for weak expression.

In some embodiments, the nucleic acid of interest can be targeted to chloroplasts for expression. In this way, when the nucleic acid of interest is not inserted directly into a chloroplast, the expression vector will additionally contain a chloroplast targeting sequence comprising a nucleotide sequence encoding a chloroplast transit peptide to direct the gene product of interest to the chloroplast. Such transit peptides are known in the art. By "operably linked" with respect to a chloroplast targeting sequence is meant that the nucleic acid sequence encoding the transit peptide (i.e., the chloroplast targeting sequence) is linked to the desired coding sequence of the invention such that the two sequences are contiguous and in the same reading frame. See, e.g., von Heijne et al (1991) Plant mol. Biol. Rep. [ Plant molecular biology guide ]9:104-126; clark et al (1989) J biol. Chem. [ journal of biochemistry ]264:17544-17550; della-Ciopa et al (1987) Plant Physiol. [ Plant physiology ]84:965-968; romer et al (1993) biochem. Res. Commun. [ Biochem. Biol. Commun. [ Biochem. Comm. ]196:1414-1421; and Shah et al (1986) Science [ Science ] 233:478-481). For example, a chloroplast transit peptide known in the art may be fused to the amino acid sequence of a CESA polypeptide of the invention by operably linking a chloroplast targeting sequence to the 5' -end of the nucleotide sequence encoding the CESA polypeptide.

Chloroplast targeting sequences are known in the art and include the small chloroplast subunit of ribulose-l, 5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al (1996) Plant mol. Biol. [ Plant molecular biology ]30:769-780; schnell et al (1991) J biol. Chem. [ J biochem. ]266 (5) 3335-3342), EPSPS (Archer et al (1990) J bioeng. Biomembrane. [ bioenergy and biofilm journal ]22 (6): 789-810)), tryptophan synthase (Zhao et al (1995) J biol. Chem. [ biochem. [ 270 (11): 6081-6087), plastocyanin (Lawrence et al (1997) J biol. Chem. [ J biol. Chem. ] 33): 57-20363), chorismate synthase (Schdt et al (1993) J biol. 263): (1493) and chlorophyll binding proteins (hander et al) (1493) J biol. Chem. J biol. 37 (1996) and (19864). See also Von Heijne et al (1991) Plant mol. Biol. Rep. [ guide for Plant molecular biology ]9:104-126; clark et al (1989) J biol. Chem. [ journal of biochemistry ]264:17544-17550; della-Ciopa et al (1987) Plant Physiol. [ Plant physiology ]84:965-968; romer et al (1993) Biochem Biophys.Res. Commun. [ communication for biochemistry and biophysics ]196:1414-1421; and Shah et al (1986) Science [ Science ] 233:478-481).

Methods for transforming chloroplasts are known in the art. See, for example, svab et al (1990) Proc. Natl. Acad. ScL USA [ journal of the national academy of sciences USA ]87:8526-8530; svab and Malega (1993) Proc. Natl. Acad. Sci. USA [ journal of the national academy of sciences USA ]90:913-917; svab and Malga (1993) EMBO J. ]12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker, and targeting of the DNA into the plastid genome by homologous recombination. Alternatively, plastid transformation may be accomplished by transactivating silent plastid-carried transgenes through tissue-preferential expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al (1994) Proc.Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA, U.S. national academy of sciences ] 91:7301-7305.

The nucleic acid of interest to be targeted to the chloroplast can be optimized for expression in the chloroplast to account for the difference in codon usage between the plant nucleus and the organelle. In this way, chloroplast-preferred codons can be used to synthesize the nucleic acid of interest. See, for example, U.S. Pat. No. 5,380,831, which is incorporated herein by reference.

A number of plant transformation vectors and methods for transforming plants are available. See, e.g., an, G.et al (1986) Pv. [ Plant physiology ],81:301-305; fry, J.et al (1987) PLANT CELL REP ] [ Plant cell report ]6:321-325; block, M. (1988) Theor. Appl. Genet. [ theory & applied genetics ]16:161-11A; hinchee et al (1990) Stadler. Genet. Symp. [ Stadler genetics monograph ] 2032/2; coussins et al (1991) Aust. J.plant Physiol. [ Aust. J.plant physiology ]18:481-494; chee, P.P.and Slightom; J.L. (1992) Gene ] 8:255-260; chustone et al (1992) Trends. Biotechnology [ biological 239 ] 10:246 ] 6:Symp. (1992) and [ View ] 3:57:57:3:90 ] Plant Physics [ View ] [ 1991) Physics ] [ View ];29P.119-124; davies et al (1993) PLANT CELL REP [ Plant cell report ]12:180-183; dong, J.A. and Mchughen, A. (1993) Plant ScL [ Plant science ]91:139-148; franklin, C.I. and Trieu, T.N. (1993) Plant Physiol [ Plant physiology ]102:167; golovkin et al (1993) Plant ScL [ Plant science ]90:41-52;Guo Chin ScL Bull [ science report ]38:2072-2078; asano et al (1994) PLANT CELL REP ]13; ayeres N.M. and Park, (1994) crit.rev.plant.sci. [ Plant science review ]13:219-239; barcelo et al (1994) plant.J. [ Plant journal ]5:583-592; becker et al (1994) plant.J. [ Plant journal ]5:299-307; borkowska et al (1994) acta.physiol Plant. [ Plant Physiol. Report ]16:225-230; christou, P. (1994) Agro.food.Ind.Hi Tech. [ agricultural food industry high Tech ]5:17-27; epen et al (1994) PLANT CELL REP. [ Plant cell report ]13: -586; hartman et al (1994) Bio-Technology [ biotechnology ] 12:919; ria et al (1994) Plant. Mol. Plant. Biol. 24:325; warstal et al (1994) biological molecular weight [ 24:325; leur.:.37.c.p.) [ agricultural food industry high Tech ] 5:582; epen et al (1994) biological Technology ] 12:919:9.

In some embodiments, the methods of the invention involve introducing the polynucleotide construct into a plant. "introduced" is intended to present the polynucleotide construct to a plant in such a way that the construct enters the interior of a plant cell. The methods of the invention do not depend on the particular method used to introduce the polynucleotide construct into the plant, but only on the entry of the polynucleotide construct into the interior of at least one cell of the plant. Methods for introducing polynucleotide constructs into plants are known in the art and include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. The term "introducing" or "transforming" as referred to herein further means transferring an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue that can be subsequently propagated by cloning, either by organogenesis or embryogenesis, can be transformed with the gene construct of the present invention and regenerated therefrom into whole plants. The particular tissue selected will vary depending on the clonal propagation system available and best suited to the particular species being transformed. Exemplary tissue targets include leaf discs (leaf discs), pollen, embryos, cotyledons, hypocotyls, female gametophytes, callus, existing meristems (e.g., apical meristem, axillary buds, and root meristems), and induced meristems (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into the host cell and may remain non-integrated, for example as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cells may then be used to regenerate transformed plants in a manner known to those skilled in the art.

"Stable transformation" refers to the integration of a polynucleotide construct in a plant into the genome of the plant and the ability to be inherited by its progeny. "transient transformation" refers to the integration of a polynucleotide construct into a plant into the genome of the plant.

For transformation of plants and plant cells, the nucleotide sequences of the invention are inserted into any vector known in the art suitable for expressing the nucleotide sequences in plants or plant cells using standard techniques. The choice of vector depends on the preferred transformation technique and the target plant species to be transformed. In embodiments of the invention, the coding nucleotide sequence is operably linked to a plant promoter (e.g., a promoter known in the art for high level expression in plant cells) and then the construct is introduced into a plant cell susceptible to a CESA-inhibiting herbicide, and regenerated into a transformed plant. In some embodiments, the transformed plant is resistant to exposure to a level of CESA-inhibiting herbicide that would kill or significantly damage plants regenerated from untransformed cells. The method may be applied to any plant species or crop.

Methods for constructing plant expression vectors and introducing foreign nucleic acids into plants are well known in the art. For example, a tumor-inducing (Ti) plasmid vector may be used to introduce foreign DNA into plants. Other methods for foreign DNA delivery involve the use of PEG-mediated protoplast transformation, electroporation, microinjection of whiskers, biolistics or microprojectile bombardment for direct uptake of DNA. Such methods are known in the art (Vasil et al U.S. Pat. No. 5,405,765; bilang et al (1991) Gene [ Gene ]100:247-250; scheid et al, (1991) MoL Gen.Genet. Genet., moL. Science and genomics, 228:104-112; guerche et al, (1987) PLANT SCIENCE [ Plant Science ]52:111-116; neuhuse et al, (1987) Theor. Appl Gene. [ theory and applied genetics ]75:30-36; klein et al, (1987) Nature [ Nature ]327:70-73; howell et al, (1980) Science [ Science ]208:1265; howell et al, (1985) Science [ 227:1221 ]; deock et al, (1989) Plant Physiology [ 34694-34 ] (Schusch) MoL. Biol.37:34 (1989) and (J.S.Chemie., and (J.P.M.25, J.P.) (J.M.25, J.Chem.) (J.37, J.25, J.P.) (J.L.25, J.M.)) (1987) J..

Other suitable methods of introducing nucleotide sequences into plant cells include microinjection described by, for example, crossway et al (1986) Biotechniques [ Biotechnology ]4:320-334, electroporation described by, for example, riggs et al (1986) Proc.Natl. Acad.ScL USA [ Proc. Natl Acad. Sci. USA U.S. Pat. No. 5,563,055 of Townsend et al, agrobacterium mediated transformation described by, for example, U.S. Pat. No. 5,981,840 of Zhao et al, direct gene transfer described by, for example, paszkowski et al (1984) EMBO J. [ European molecular biology J ]3:2717-2722, and particle acceleration by, for example, described below, in U.S. Pat. No. 4,945,050, 5,879,918, 5,886,244, and 5,932,782; tomes et al (1995) "DIRECT DNA TRANSFER into INTACT PLANT CELLS VIA Microprojectile Bombardment [ direct transfer of DNA into intact plant cells via microprojectile bombardment ]," in PLANT CELL, tissue, and Organ Culture: fundamental Methods [ plant cells ], Tissue and organ culture, basic methods, editing Gamborg and Phillips (Springer-Verlag, berlin), mcCabe et al (1988) Biotechnology [ Biotechnology ] 6:923-926), and Led transformation (WO 00/28058). See also WEISSINGER et al, (1988) Ann.Rev.Genet. [ genealogy "22:421-477; sanford et al, (1987) particle SCIENCE AND Technology [ particle science and Technology ]5:27-37 (onion); christou et al, (1988) Plant Physiol. [ Plant Physiol. ]87:671-674 (soybean); mcCabe et al, (1988) Bio/Technology [ Biotechnology ]6:923-926 (soybean); finer and McMullen (1991) InVitro Cell Dev.biol. [ In Vitro Cell and developmental biology ]27P:175-182 (soybean); singh et al, (1998) Theor.Appl.Genet al. [ theory and applied genealogy ]96:319-324 (soybean); datta et al, (1990) Biotechnology [ biology ]8:736 (1998); kbook) 6:3-926 (soybean); U.S. 1) In Vitro Cell Dev.Biol.; finder.S. and McMullen (1991) In.; in.Vitro Cell Dev.27:175-182 (soybean); singh et al, (1998) applied science and applied Gene.; kbook 6:, 5,322,783, and 5,324,646; tomes et al, (1995), "DIRECT DNA TRANSFER into INTACT PLANT CELLS VIA Microprojectile Bombardment [ direct transfer of DNA into intact plant cells via microprojectile bombardment ]," in PLANT CELL, tissue, and Organ Culture: fundamental Methods [ plant cells ], tissue and organ culture, basic methods ], editing Gamborg (Springer-Verlag, berlin) (maize); klein et al, (1988) Plant Physiol [ Plant physiology ]91:440-444 (maize); from Wet et al, (1990) Biotechnology [ Biotechnology ]8:833-839 (maize); hooykaas-Van Slogteren et al, (1984) Nature [ Nature ] (London) 31:763-764; bowen et al U.S. Pat. No. 5,736,369 (cereal); bytebier et al, (1987) PNAS [ Proc. Natl. Acad. Sci. USA ]84:5345-5349 (Liliaceae); deWet et al, (1985) in The Experimental Manipulation of Ovule Tissues [ experiment operations of ovule tissue ], chapman et al, (Lantern publishing (Longman), new York), pages 197-209 (pollen); kaeppler et al, (1990) PLANT CELL Reports [ Plant cell ]9:415-418 and Kaeppler et al, (1992) The et al) applied to Plant cells [ applied to the same ] Plant society [ society ]84:5345-5349 (maize); de Wet et al, (1985) and (1995) Plant science) applied to Plant cell theory [ 35:25:35:25-25, and (1995) Plant whisker (1995) applied to Plant society [ society ] of electrical laboratory operations [ 35:5345-5349 (Phasexual, human being (1995), (1996) Nature Biotechnology [ Nature Biotechnology ]14:745-750 (via Agrobacterium tumefaciens maize); each of which is incorporated herein by reference.

Transgenic plants (including transgenic crop plants) are preferably produced via agrobacterium-mediated transformation. An advantageous transformation method is plant in situ transformation. For this purpose, it is possible, for example, to have the genus Agrobacterium act on plant seeds or to inoculate plant meristems with the genus Agrobacterium. It has proven to be particularly advantageous according to the invention to have the transformed Agrobacterium suspension act on the whole plant or at least on the floral primordia. Plants were then grown until seeds of the treated plants were obtained (Clough and Bent, plant J. [ J.Phytocet ] (1998) 16,735-743). Methods for Agrobacterium-mediated transformation of rice include well-known methods for rice transformation, as described in any of European patent applications EP 1198985 A1,Aldemita and Hodges (Planta [ botanic ]199:612-617,1996), chan et al (Plant Mol Biol [ Plant molecular biology ]22 (3): 491-506, 1993), hiei et al (Plant J [ Plant J ]6 (2): 271-282, 1994), the disclosures of which are incorporated herein by reference as if fully set forth. In the case of maize transformation, the preferred methods are described in Ishida et al (Nat. Biotechnol. Nature Biotechnology ]14 (6): 745-50, 1996) or Frame et al (Plant Physiol [ Plant Physiol ]129 (1): 13-22,2002), the disclosures of which are incorporated herein by reference as if fully set forth. The method is further described by way of example in B.Jenes et al, techniques for GENE TRANSFER [ gene transfer technology ], in TRANSGENIC PLANTS [ transgenic plants ], volume 1, ENGINEERING AND Utilization [ engineering and Utilization ], editors S.D.Kung and R.Wu, academic Press [ academic Press ],1993,128-143, and in Potrykus Annu.Rev.plant Physiol.plant molecular biology [ annual. Biol ]42 (1991) 205-225. The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transformation of Agrobacterium tumefaciens, such as pBin19 (Bevan et al, nucleic acids Res. [ nucleic acids Res. ]12 (1984) 8711). Plants of the genus Agrobacterium transformed with such vectors can then be used in a known manner, such as plants used as models, like Arabidopsis plants (Arabidopsis thaliana (Arabidopsis thaliana) is within the scope of the present invention, not considered crop plants), or crop plants such as, for example, tobacco plants, for example by immersing scratched or chopped leaves in an Agrobacterium solution, and then culturing them in a suitable medium. For example, transformation of plants by Agrobacterium tumefaciens from, for exampleAnd Willmitzer, described in nucleic acid Res (1988) 16,9877, or known inter alia from F.F.white, vectors for GENE TRANSFER IN HIGHER PLANTS [ Vectors for higher plant gene transfer ]; in TRANSGENIC PLANTS [ transgenic plants ], vol.1, ENGINEERING AND hybridization [ engineering and Utilization ], editors S.D.Kung and R.Wu, academic Press [ academic Press ],1993, pages 15-38.

One transformation method known to those skilled in the art is to immerse the flowering plant in a solution of agrobacterium, wherein the agrobacterium contains CESA nucleic acid, followed by breeding the transformed gametes. Agrobacterium-mediated plant transformation can be performed using, for example, the Agrobacterium tumefaciens strain GV3101 (pMP 90) (Koncz and Schell,1986, mol. Gen. Genet. [ molecular genetics & genomics ] 204:383-396) or LBA4404 (Clontech). Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al, 1994, nucl. Acids. Res. [ nucleic acids research ]13:4777-4788;Gelvin,Stanton B and Schilperort, robert A, plant Molecular Biology Manual [ handbook of plant molecular biology ], 2 nd edition-Dordrecht: kluwer Academic Publ [ Duode Reich Tex Kluyverz publishing, 1995 ] -in section Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; glick, bernard R. And Thompson, john E., methods in Plant Molecular Biology and Biotechnology [ methods of plant molecular biology and biotechnology ], boca Raton: CRC Press [ Bokapton: CRC publishing ],1993 360S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et al, 1989,Plant Cell Report [ plant cell report ]8:238-242; de Block et al, 1989,Plant Physiol. [ plant physiology ] 91:694-701). The use of antibiotics for agrobacterium and plant selection depends on the binary vector and agrobacterium strain used for transformation. Rapeseed selection is typically performed using kanamycin as a selectable plant marker. Agrobacterium-mediated gene transfer of flax can be performed using techniques such as those described by Mlynarova et al 1994,Plant Cell Report [ plant cell report ] 13:282-285. alternatively, the conversion of soybeans may be performed using techniques described in, for example, european patent No. 0424 047, U.S. Pat. No. 5,322,783, european patent No. 0397 687, U.S. Pat. No. 5,376,543, or U.S. Pat. No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via silicon carbide fiber technology. (see, e.g., freeling and Walbot, "The maize handbook [ maize handbook ]" SPRINGER VERLAG [ Schpulin Greek Press ]: ISBN 3-540-97826-7, new York (1993)). Specific examples of maize transformation are found in U.S. Pat. No. 5,990,387, while specific examples of wheat transformation can be found in PCT application No. WO 93/07256.

In some embodiments, the polynucleotides of the invention may be introduced into a plant by contacting the plant with a virus or viral nucleic acid. Typically, such methods involve incorporating the polynucleotide constructs of the invention into viral DNA or RNA molecules. It will be appreciated that the polypeptides of the invention may be initially synthesized as part of a viral polyprotein which may then be processed by in vivo or in vitro proteolysis to produce the desired recombinant polypeptide. Furthermore, it should be appreciated that the promoters of the present invention also encompass promoters for transcription by viral RNA polymerase. Methods for introducing polynucleotide constructs into plants and expressing the proteins encoded therein (involving viral DNA or RNA molecules) are known in the art. See, for example, U.S. patent nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, and 5,316,931, which are incorporated herein by reference. The cells that have been transformed can be grown into plants according to conventional methods. See, e.g., mcCormick et al (1986) PLANT CELL Reports [ plant cell Reports ]5:81-84. These plants can then be grown and pollinated with the same transformed strain or a different strain and the resulting hybrids of constitutive expression with the desired phenotypic characteristics identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited, and then seeds harvested to ensure that expression of the desired phenotypic characteristic has been achieved.

The invention may be used to transform any plant species including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, maize or maize (Zea mays), brassica species (e.g., brassica napus, guan root, brassica juncea), particularly those used as a source of seed oil, alfalfa (Medicago sativa), rice (rice, oryza sativa), rye (rye, SECALE CEREALE), sorghum (Sorghum, sorghum bicolor, sorghum vulgare), millet, e.g., pearl millet (Pennisetum glaucum)), and, Millet (Panicum miliaceum)), millet (foxtailmillet, setariaitalica), finger millet (Eleusine coracana), sunflower (sun flower, helianthus annuus), safflower (saffiower, carthamus tinctorius), wheat (common wheat (Triticum aestivum), durum wheat (T.Turgium ssp. Durum)), soybean (Glycine max)), tobacco (tobacco, Nicotiana tabacum), potato (potto, solarium tuberosum), peanut (Arachis hypogaea)), cotton (gossypium barbadense (Gossypium barbadense), gossypium hirsutum (Gossypium hirsutum)), sweet potato (sweet potto, ipomoea batatus), tapioca (cassava, manihot esculenta), coffee (coffee species (coffire spp)), coconut (coconut), Cocos nucifera), pineapple (Ananas comosus), citrus tree (Citrus species (Citrus spp.)), cocoa (cocoa, theobroma cacao), tea (wild tea tree (CAMELLIA SINENSIS)), banana (Musa species (Musa spp.)), avocado (avocado, PERSEA AMERICANA), fig (fig, ficus casica), guava (guava, psidium guajava), mango (mango), MANGIFERAINDICA), olives (Oleaceae), papaya (CARICA PAPAYA), cashew (cashew, anacardium occidentale), macadamia nuts (macadamia, MACADAMIA INTEGRIFOLIA), almonds (almond), beets (sugar beets, beta vulgares), sugarcane (Saccharum (Saccharum) species), oats, barley, vegetables, ornamental plants and conifers. Preferably, the plant of the invention is a crop plant (e.g., sunflower, brassica species, cotton, sugar, beet, soybean, peanut, alfalfa, safflower, tobacco, corn, rice, wheat, rye, barley, triticale, sorghum, millet, etc.).

In addition to transformant cells, which then have to be regenerated into whole plants, it is also possible to transform cells of plant meristems, in particular those which develop into gametes. In this case, the transformed gametes follow natural plant development, resulting in transgenic plants. Thus, for example, arabidopsis seeds are treated with Agrobacterium and seeds are obtained from developing plants, wherein a proportion of the plants are transformed and thus transgenic [ Feldman, KA and Marks MD (1987) [ Mol Gen Genet [ molecular genetics & genomics ]208:274-289; feldmann K (1992): C Koncz, N-H Chua and J Shell editions, methodsin Arabidopsis Research. [ Arabidopsis research methods ] Word Scientific [ world science ], singapore, pages 274-289 ]. An alternative method is based on repeated removal of inflorescences and incubation of the excised sites in the center of rosettes with transformed Agrobacterium, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. [ J. Plant ]5:551-558; katavic (1994). Mol Genet [ molecular genetics and genomics ], 245:363-370). However, particularly effective methods are vacuum infiltration methods and modifications thereof, such as the "floral dip" method. In The case of vacuum infiltration of Arabidopsis, whole plants under reduced pressure were treated with Agrobacterium suspensions [ Bechthold, N (1993). C RACAD SCI PARIS LIFE SCI [ Proc. Natl. Acad. Sci. Paris, 316:1194-1199], whereas in The case of The "flower dipping" method, developing flower tissue was incubated briefly with surfactant-treated Agrobacterium suspensions [ Clough, SJ and Bent AF (1998) The Plant J. [ J. Plant J. ]16,735-743]. In both cases a proportion of transgenic seeds were harvested and by growing under the selection conditions described above, these seeds were distinguishable from non-transgenic seeds. In addition, stable transformation of plastids is advantageous because plastids are maternally inherited in most crops, thereby reducing or eliminating the risk of transgene loss through pollen. Transformation of the chloroplast genome is generally achieved by the method schematically shown in Klaus et al 2004[Nature Biotechnology [ Nature Biotechnology ]22 (2), 225-229 ]. Briefly, the sequence to be transformed is cloned together with the selectable marker gene between flanking sequences homologous to the chloroplast genome. these homologous flanking sequences direct site-specific integration into the plastid genome. Plastid transformation of many different plant species has been described and an overview is given in Bock (2001) TRANSGENIC PLASTIDS IN basic RESEARCH AND PLANT biotechnology [ transgenic plastids in basic research and plant biotechnology ] J Mol Biol [ journal of molecular biology ] month 9, 21, 312 (3): 425-38 or Malega, P (2003) Progress towards commercialization of plastid transformation technology ] [ progress of plastid transformation technology commercialization ] Trends Biotechnol ] [ biotechnological trend ]21,20-28. More recently, additional biotechnology advances have been reported in the form of marker-free plastid transformants, which can be generated by transient co-integration of the marker gene (Klaus et al 2004,Nature Biotechnology [ Nature Biotechnology ]22 (2), 225-229). The genetically modified plant cells can be regenerated via all methods familiar to the skilled worker. Suitable methods can be found in S.D.Kung and R.Wu, potrykus orAnd Willmitzer, among the aforementioned publications.

Typically, after transformation, plant cells or cell populations are selected for the presence of one or more markers encoded by plant-expressible genes co-transferred with the gene of interest, and the transformed material is subsequently regenerated into whole plants. In order to select for transformed plants, the plant material obtained in the transformation is generally subjected to selection conditions so that the transformed plants can be distinguished from untransformed plants. For example, seeds obtained in the manner described above may be planted and, after the initial growth phase, subjected to a suitable selection by spraying. Another possibility is that, if appropriate, after sterilization, the seeds are grown on agar plates with a suitable selection agent, so that only transformed seeds can grow into plants. Alternatively, transformed plants are screened for the presence of a selectable marker (such as the selectable markers described above).

Following DNA transfer and regeneration, the putative transformed plants may also be assessed for the presence of the gene of interest, copy number and/or genomic tissue, for example using southern blot analysis. Alternatively or additionally, northern blot and/or western blot analysis may be used to monitor the expression level of newly introduced DNA, both techniques being well known to those of ordinary skill in the art.

The resulting transformed plants may be propagated in a variety of ways, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and a homozygous second generation (or T2) transformant selected, and the T2 plant may then be further propagated by classical breeding techniques. The resulting transformed organisms may take a variety of forms. For example, they may be chimeras of transformed and non-transformed cells, clonal transformants (e.g., transformed into all cells containing the expression cassette), grafts of transformed and non-transformed tissue (e.g., in plants, the transformed stock is grafted onto the non-transformed scion).

Preferably, expression of the nucleic acid in the plant results in an increase in tolerance of the plant to the CESA-inhibiting herbicide as compared to the wild type variety of the plant.

In another embodiment, the invention relates to a plant comprising a plant cell according to the invention, wherein expression of the nucleic acid in the plant results in an increase in resistance of the plant to a CESA-inhibiting herbicide compared to a wild-type variety of the plant.

The plants described herein may be transgenic crop plants or non-transgenic plants.

In addition to the general definitions given above, "transgenic" or "recombinant" means that for example nucleic acid sequences, expression cassettes comprising nucleic acid sequences, genetic constructs or vectors or organisms transformed with nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructs are obtained by recombinant methods, wherein

(A) Nucleic acid sequences encoding proteins useful in the methods of the invention, or

(B) One or more genetic control sequences operably linked to a nucleic acid sequence according to the invention, for example promoters, or

(C) a) and b)

Not located in its natural genetic environment or has been modified by recombinant means, which modification may take the form of, for example, substitution, addition, deletion, inversion or insertion of one or more nucleotide residues, to allow expression of the mutant CESA of the invention. Natural genetic environment is understood to mean the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of genomic libraries, the natural genetic environment of the nucleic acid sequence is preferably at least partially preserved. The environment is flanked on at least one side by nucleic acid sequences and has a sequence length of at least 50bp, preferably at least 500bp, particularly preferably at least 1000bp, most preferably at least 5000 bp. Naturally occurring expression cassettes, e.g. naturally occurring promoters of nucleic acid sequences, are transformed into transgenic expression cassettes when modified by non-natural, synthetic ("artificial") methods, e.g. mutagenesis, as defined above, in combination with naturally occurring nucleic acid sequences encoding the corresponding polypeptides useful in the methods of the invention. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.

Thus, transgenic plants for the purposes of the present invention are understood to mean that, as mentioned above, the nucleic acids of the invention are not at their natural locus in the genome of the plant, and these nucleic acids may be expressed homologously or heterologously. However, as described above, transgenic also means that, although the nucleic acids according to the invention or used in the method of the invention are in their natural position in the plant genome, the sequences have been modified with respect to the natural sequences and/or the regulatory sequences of the natural sequences have been modified. Transgenic is preferably understood to mean the expression of a nucleic acid according to the invention at a non-natural locus in the genome, i.e. homologous or preferably heterologous expression of the nucleic acid takes place. Preferred transgenic plants are mentioned herein. Furthermore, 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 stably integrated into a chromosome or into a stable extrachromosomal element, thereby delivering it to successive offspring. For the purposes of the present invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged or modified by genetic engineering. Examples include polynucleotides linked or joined to any one or more clones of heterologous sequences. The term "recombinant" does not refer to a change in a polynucleotide caused by a naturally occurring event (e.g., spontaneous mutation) or a change in a polynucleotide caused by non-spontaneous mutagenesis followed by selective breeding.

Plants containing mutations due to non-spontaneous mutagenesis and selective breeding are referred to herein as non-transgenic plants and are included in the present invention. In embodiments in which the plant is transgenic and comprises multiple mutant CESA nucleic acids, the nucleic acids may be derived from different genomes or from the same genome. Alternatively, in embodiments in which the plant is non-transgenic and comprises multiple mutant CESA nucleic acids, the nucleic acids are located on different genomes or on the same genome.

In certain embodiments, the invention relates to herbicide resistant plants produced by mutation breeding. Such plants comprise a polynucleotide encoding a mutant CESA and are tolerant to one or more CESA-inhibiting herbicides. Such methods may involve, for example, exposing the plant or seed to a mutagen, particularly a chemical mutagen such as Ethyl Methanesulfonate (EMS), and selecting plants having enhanced tolerance to at least one or more CESA-inhibiting herbicides [ see example 1].

However, the present invention is not limited to herbicide tolerant plants produced by mutagenesis methods involving chemical mutagens EMS. Any mutagenesis method known in the art may be used to produce the herbicide resistant plants of the invention. Such mutagenesis methods may involve, for example, the use of any one or more of the group of mutagens such as X-rays, gamma rays (e.g., cobalt 60 or cesium 137), neutrons (e.g., nuclear fission products of uranium 235 in an atomic reactor), beta radiation (e.g., radiation emitted by a radioisotope such as phosphorus 32 or carbon 14), and ultraviolet radiation (preferably 250 to 290 nm), as well as chemical mutagens such as base analogues (e.g., 5-bromo-uracil), related compounds (e.g., 8-ethoxycaffeine), antibiotics (e.g., streptavidin), alkylating agents (e.g., sulfur mustard, nitrogen mustard, epoxides, vinylamines, sulfates, sulfonates, sulfones, lactones), azides, hydroxylamines, nitrous acid, or acridines. Herbicide resistant plants can also be produced by selecting plant cells comprising the herbicide resistant mutation using a tissue culture method, and then regenerating herbicide resistant plants therefrom. See, for example, U.S. patent nos. 5,773,702 and 5,859,348, both of which are incorporated herein by reference in their entirety. Additional details of mutation breeding can be found in "PRINCIPALS OF CULTIVAR DEVELOPMENT [ principles of cultivar development ]" Fehr,1993 mimilan publishing company, the disclosure of which is incorporated herein by reference

Alternatively, herbicide resistant plants according to the invention can also be produced by selecting plant cells comprising the herbicide resistant mutation using a genome editing method, and then regenerating herbicide resistant plants therefrom. "genome editing" refers to a type of genetic engineering in which an engineered nuclease is used to insert, delete, or replace DNA in the genome of an organism. Those skilled in the art know that these nucleases produce a site-specific double strand break at a desired location in the genome. The induced double strand breaks are repaired by non-homologous end joining or homologous recombination, resulting in targeted mutations. Four families of engineered nucleases, meganucleases, zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) based and CRISPR-Cas systems are currently known in the art for the purposes of the present invention. For reference, see, e.g., esvelt, KM. and Wang, HH. (2013) "Genome-SCALE ENGINEERING for SYSTEMS AND SYNTHETIC biology [ Genome-scale engineering of systems and synthetic biology ]", mol Syst Biol.[ molecular systems biology ]9 (1): 641; tan, WS. Et al, (2012) "Precision editing of LARGE ANIMAL genomes [ precision editing of large animal genomes ]", adv Genet @ genetic progress ]80:37-97; puchta, H. And Fauser, F. (2013) "GENE TARGETING IN PLANTS:25years later [ plant genes targeting after 25years ]", int.J.Dev. Biol. [ J. J. International journal of developmental biology ]57:629-637; boglioli, elsy and Richard, magali "REWRITING THE book oflife: A NEW ERAIN precision Genome editing [ new era of precise Genome editing ]", boston Consulting Group [ Boston consultant group ], search for 30 months; method of the Year 2011:20116 (2011) Nature 20111, method 2011.

The plants of the invention comprise at least one mutant CESA nucleic acid or overexpressed wild-type CESA nucleic acid and have increased tolerance to a CESA-inhibiting herbicide as compared to the wild-type variety of the plant. Plants of the invention may have multiple mutant CESA nucleic acids from different genomes, as these plants may contain more than one genome. For example, plants contain two genomes, commonly referred to as the a and B genomes. Since CESA is an essential metabolic enzyme, it is assumed that each genome has at least one gene encoding a CESA enzyme (i.e., at least one CESA gene). As used herein, the term "CESA locus" refers to the location of the CESA gene on the genome, and the terms "CESA gene" and "CESA nucleic acid" refer to nucleic acids encoding a CESA enzyme. The nucleotide sequence of the CESA nucleic acid on each genome is different from the nucleotide sequence of the CESA nucleic acid on the other genome. The genome from which each CESA nucleic acid originates can be determined by genetic hybridization and/or sequencing methods or exonuclease digestion methods known to those skilled in the art.

The present invention includes plants comprising one, two, three or more mutant CESA alleles, wherein the plant has increased tolerance to a CESA-inhibiting herbicide as compared to a wild type variety of the plant. The mutant CESA allele may comprise a nucleotide sequence selected from the group consisting of a polynucleotide defined in SEQ ID No. 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98, or a variant or derivative thereof, a polynucleotide encoding a polypeptide defined in SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83, or a variant or derivative, homolog, ortholog, paralog thereof, a polynucleotide comprising at least 60 consecutive nucleotides of any of the polynucleotides, and a polynucleotide complementary to any of the polynucleotides.

An "allele" or "allelic variant" is an alternative form of a given gene located at the same chromosomal location. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs) and small insertion/deletion polymorphisms (INDELs). INDEL is typically less than 100bp in size. SNPs and INDELs form the largest set of sequence variants in most naturally occurring polymorphic strains of organisms.

The term "variety" refers to a group of plants within a species defined by sharing a common set of characteristics or traits that are acceptable to one skilled in the art to distinguish one cultivar or variety from another cultivar or variety. No term means that all plants of any given cultivar or variety are genetically identical at the entire gene or molecular level, or that any given plant is homozygous at all loci. A true breeding cultivar or variety is considered to be "true breeding" for a particular trait if all progeny contain that trait when the cultivar or variety is self-pollinated. The term "breeding line" or "line" refers to a group of plants within a cultivar defined by sharing a set of common features or traits that are acceptable to one of skill in the art to distinguish one breeding line or line from another. No term means that all plants of any given breeding line or line are genetically identical at the whole genetic or molecular level, or that any given plant is homozygous at all loci. A true breeding line or line is considered to be "true breeding" with a particular trait if all progeny contain that trait when the true breeding line or line is self-pollinated. In the present invention, the trait is derived from mutation of CESA gene of a plant or seed.

The herbicide resistant plants of the invention comprising polynucleotides encoding mutant CESA polypeptides are also useful in methods of increasing herbicide resistance of plants by conventional plant breeding involving sexual propagation. These methods comprise 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 as the first plant, or may be resistant to a different herbicide or herbicides than the first plant. The second plant may be any plant that is capable of producing viable progeny plants (i.e., seeds) when crossed with the first plant. Typically, but not necessarily, the first plant and the second plant are of the same species. These methods may optionally involve selecting a progeny plant comprising the mutant CESA polypeptide of the first plant and the herbicide resistance characteristics of the second plant. The progeny plants produced by the method of the invention have increased resistance to herbicides when compared to the first plant or the second plant or both. When the first and second plants are resistant to different herbicides, the progeny plants will have a combination of the herbicide tolerance characteristics of the first and second plants. The methods of the invention may further involve backcrossing the progeny plant of the first cross with a plant of the same line or genotype as the first or second plant for one or more generations. Alternatively, the progeny of the first cross or any subsequent cross may be crossed to a third plant that is of a different line or genotype than the first or second plants. The invention also provides plants, plant organs, plant tissues, plant cells, seeds and non-human host cells transformed with at least one polynucleotide molecule, expression cassette or transformation vector of the invention. Such transformed plants, plant organs, plant tissues, plant cells, seeds and non-human host cells have enhanced tolerance or resistance to at least one herbicide at herbicide levels that kill or inhibit growth of the non-transformed plants, plant tissues, plant cells or non-human host cells, respectively. Preferably, the transformed plants, plant tissues, plant cells and seeds of the invention are arabidopsis and crop plants.

It will be appreciated that in addition to the mutant CESA nucleic acid, the plants of the invention may comprise a wild-type CESA nucleic acid. It is contemplated that CESA-inhibiting herbicide-tolerant lines may contain mutations in only one of the CESA isozymes. Thus, the present invention includes plants comprising one or more mutant CESA nucleic acids in addition to one or more wild-type CESA nucleic acids.

In another embodiment, the invention relates to a seed produced by a transgenic plant comprising a plant cell of the invention, wherein the seed is a true breeding for increased resistance to a CESA-inhibiting herbicide as compared to a wild type variety of the seed.

In other aspects, the CESA-inhibiting herbicide-tolerant plants of the invention can be used as CESA-inhibiting herbicide-tolerant trait donor lines for development (e.g., by conventional plant breeding) to produce other varieties and/or hybrid crops containing such one or more traits. All such resulting varieties or hybrid crops containing one or more ancestral CESA-inhibitory herbicide-tolerant traits may be referred to herein as progeny or offspring of the one or more ancestral CESA-inhibitory herbicide-tolerant lines.

In other embodiments, the invention provides methods of producing CESA-inhibiting herbicide-tolerant plants. The method comprises crossing a first CESA-inhibiting herbicide tolerant plant with a second plant to produce a CESA-inhibiting herbicide tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the recombinant polynucleotide being effective to express in the cells of the first plant a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant.

In some embodiments, traditional plant breeding is employed whereby CESA-inhibiting herbicide tolerance traits are introduced into the progeny plants produced therefrom. In one embodiment, the invention provides a method for producing a CESA-inhibiting herbicide-tolerant progeny plant, the method comprising crossing a parent plant with a CESA-inhibiting herbicide-tolerant plant to introduce a CESA-inhibiting herbicide tolerance characteristic of the CESA-inhibiting herbicide-tolerant plant into the germplasm of the progeny plant, wherein the progeny plant has increased tolerance to the CESA-inhibiting herbicide relative to the parent plant. In other embodiments, the method further comprises the step of introgressing the CESA-inhibitory herbicide tolerance trait by conventional plant breeding techniques to obtain a progeny plant having the CESA-inhibitory herbicide tolerance trait.

In other aspects, the plants of the invention include those plants that have undergone additional genetic modification by breeding, mutagenesis or genetic engineering in addition to tolerance to a CESA-inhibiting herbicide, for example, tolerance to application of certain other classes of herbicides, such as AHAS inhibitors, auxin herbicides, bleaching herbicides such as hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors or Phytoene Desaturase (PDS) inhibitors, EPSPS inhibitors such as glyphosate, glutamine Synthetase (GS) inhibitors such as glufosinate, lipid biosynthesis inhibitors such as acetyl CoA carboxylase (ACCase) inhibitors, or cyanohydrin (i.e. bromoxynil or ioxynil) herbicides, as a result of conventional breeding or genetic engineering methods. Thus, CESA-inhibiting herbicide-tolerant plants of the invention may be made resistant to multiple classes of herbicides, such as resistance to both glyphosate and glufosinate, or resistance to both glyphosate and herbicides from another class (such as HPPD inhibitors, AHAS inhibitors, or ACCase inhibitors), by a variety of genetic modifications. These herbicide resistance techniques are described, for example, in PEST MANAGEMENT SCIENCE [ pest management science ] (volume, year, page): 61,2005,246;61,2005,258;61,2005,277;61,2005,269;61,2005,286;64,2008,326;64,2008,332;Weed Science [ weed science ]57,2009,108;Australian Journal of Agricultural Research [ journal of australian agricultural research ]58,2007,708; science [ science ]316,2007,1185; and references cited therein. For example, in some embodiments, CESA-inhibiting herbicide-tolerant plants of the invention may be tolerant to ACCase inhibitors such as "dims" { e.g., buprofezin, sethoxydim, clethodim, or pyrone), "fops" { e.g., clodinafop-propargyl, norzafop, fluazifop-p, or quizalofop) and "dens" (e.g., pinoxaden); to auxin herbicides such as dicamba; to EPSPS inhibitors such as glyphosate; to other CESA inhibitors, and to GS inhibitors such as glufosinate.

In addition to these classes of inhibitors, the CESA-inhibiting herbicide-tolerant plants of the present invention may also be tolerant to herbicides having other modes of action, such as chlorophyll/carotenoid pigment inhibitors, cell membrane disrupters (disrupter), photosynthesis inhibitors, cell division inhibitors, root inhibitors, shoot inhibitors, and combinations thereof.

Such tolerance traits may be expressed, for example, as mutant or wild-type HPPD proteins, mutant AHASL proteins, mutant ACCase proteins, mutant EPSPS proteins or mutant glutamine synthetase proteins, or mutant natural, inbred or transgenic aryloxyalkanoate dioxygenases (AAD or DHT), haloarylnitrilases (BXN), 2-dichloropropionic Dehalogenases (DEH), glyphosate-N-acetyltransferases (GAT), glyphosate Decarboxylases (GDC), glyphosate Oxidoreductase (GOX), glutathione-S-transferases (GST), phosphinothricin acetyltransferases (PAT or bar) or CYP450S proteins with herbicide degrading activity. CESA-inhibiting herbicide tolerant plants herein can also be overlaid with other traits, including, but not limited to, pesticidal traits, such as Bt Cry and other proteins having pesticidal activity against coleopteran (coleopteran), lepidopteran (lepidopteran), nematodes or other pests, nutraceutical or nutraceutical traits, such as modified oil content or oil characterization traits, high protein or high amino acid concentration traits, and other trait types known in the art.

In addition, in other embodiments, CESA-inhibiting herbicide tolerant plants are also covered, which plants, by using recombinant DNA techniques and/or by breeding and/or otherwise selecting for features that enable the synthesis of one or more insecticidal proteins, especially those known to be from the genus Bacillus (Bacillus), particularly from Bacillus thuringiensis (Bacillus thuringiensis), such as [ delta ] -endotoxins, e.g., crylA (b), crylA (c), crylF, cryIF (a 2), and, CrllA (b), crylllA, crylllB (bl) or Cry9c; asexual insecticidal proteins (VIP), for example VIP1, VIP2, VIP3 or VIP3A; insecticidal proteins of bacteria colonizing nematodes, for example, light-emitting Bacillus species (Photorhabdus spp.) or Xenorhabdus species (Xenorhabdus spp.), toxins produced by animals, such as scorpions, spider toxins, wasp toxins or other insect-specific neurotoxins, toxins produced by fungi, such as streptomyces toxins, phytolectins (lecins), such as pea or barley lectins, lectins (agglutanin), protease inhibitors, such as trypsin inhibitors, trypsin inhibitors, Serine protease inhibitors, potato glycoprotein, cysteine protease inhibitors or papain inhibitors, ribosome Inactivating Proteins (RIPs) such as ricin, maize-RIP, abrin, luffa seed protein, saporin or other diarrhea toxin proteins, steroid metabolizing enzymes such as 3-hydroxy-steroid oxidase, ecdysteroid-IDP-glycosyl-transferase, cholesterol oxidase, ecdysone inhibitors or HMG-CoA reductase, ion channel blockers such as sodium or calcium channel blockers, juvenile hormone esterase, diuretic hormone receptor (Helicokinin receptor), stilbene synthase, Bibenzyl synthase, chitinase or glucanase. In the context of the present invention, these insecticidal proteins or toxins are also to be understood in particular as being protoxins, hybrid proteins, truncated or otherwise modified proteins. Hybrid proteins are characterized by a novel combination of protein domains (see, e.g., WO 02/015701). Further examples of such toxins or genetically modified plants capable of synthesizing such toxins are disclosed, for example, in EP-A374 753, WO 93/007578, WO 95/34656, EP-A427 529, EP-A451 878, WO 03/18810 and WO 03/52073. Methods for producing such genetically modified plants are generally known to the person skilled in the art and are described, for example, in the publications mentioned above. These insecticidal proteins contained in the genetically modified plants confer tolerance to harmful pests from all arthropod taxa to plants producing these proteins, in particular beetles (coleoptera), diptera (Diptera) and moths (lepidoptera) and nematodes (Nematoda).

In some embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in CESA-inhibiting herbicide-tolerant plants is effective to control organisms including, for example, members of the order coleoptera, such as the us bean image (Acanthoscelides obtectus)), the leaf beetles (the red bean image (AGELASTICA ALNI)), the tap beetles (the straight tap beetles (Agriotes lineatus), the dark tap beetles (Agriotes obscurus), the plant species, and the members of the order coleoptera, such as the red bean image (Acanthoscelides obtectus)) The plant species comprise, inter alia, a two-color click beetle (Agriotes bicolor)), a flat valley beetle (milpa oryzae (Ahasverus advena)), potato gills (summer schafer, amphimallon solstitialis), a furniture beetle (furniture beetle, anobium punctatum), a flower species of the genus Philippica (Anthonius spp.) (elephantom (weevil)), a dwarf's jujube (Pygmy mangold beetle), beet cryptoshi beetle (Atomaria linearis), a red bark beetle (carpet beetles) (a bark beetle species (Anthrenus spp.) the species of the codling genus (Attagenus spp.)), cowpea weevil (four-grain weevil (Callosobruchus maculates)), dried fruit beetles (fried fruit beetle), yellow tail beetles (Carpophilus hemipterus), cabbage weevils (cabbage hale weevil (Ceutorhynchus assimilis)), winter rape stem weevil (RAPE WINTER STEM WEEVIL, ceutorhynchus picitarsis), nematodes, tobacco wireworms (Conoderus vespertinus) and platythorn (Conoderus falli), banana weevil (banana weevil, cosmopolites sordidus), new Zealand grassland grubs (New Zealand grass golden turtle (Costelytra zealandica)), juniper (green juniper (Cotinis nitida)), sunflower stem weevil (dense point weevil (Cylindrocopturus adspersus)), ham bark beetles (larder beetle, DERMESTES LARDARIUS), corn rootworm (corn root weevil (Diabrotica virgifera)), corn rootworm (Diabrotica virgifera), diabrotica and Diabrotica (); the plant species may be selected from the group consisting of ladybird (,)), hornworm (,), alfalfa leaf beetle (,), gymnosperm (,), tobacco beetle (,), corridomile (potato leaf beetle ()), silverfish beetle ({ pinus species (spp.), pollen beetle (rape beetle ()), gill-horn beetle (), american spider beetle (), flat-valley beetle (saw-valley beetle (), and large-eye saw-valley beetle ()), grape blackberry beetle (black, mustard beetle (horseradish beetle ()), brassicaceae plant strip flea (vegetable yellow strip flea ()), yellow leaf flea beetle (,); cabbage flea (rape flea beetle ()), spider species (p.)), rhizopertha dominica), pea and bean (Rhizobium striped root-weevil (Sitona lineatus)), rice and valley (rice (Sitophilus oryzae) and valley (Sitophilus granaries)), red melon (yellow brown petiole (Smicronyx fulvus))), beetle (medicinal material A (Stegobium paniceum)), yellow meal worm (Tenebrio molitor)), yellow meal worm (Tribolium castaneum) and weevil (Triboliumconfusum)), warehouse and cabinet beetles (warehouse and cabinet beetle) { Pityrospermum species (Trogoderma spp.)), beetle (sunflower beetle, zygogramma exclamationis), leather wing (DERMAPTERA) (earwig (earwig)), such as earwig (ordinary earwig (Forficula auricularia)) and earwig (STRIPED EARWIG, labidura riparia), net wing (Dictyoptera), such as Blatta orientalis (oriental cockroach, blatta orientalis), qian (greenhouse millipede, oxidus gracilis), beet leaf fly (beet fly, pegomyia betae), leaf fly (Oscinella frit) and fruit stalk (Dahousefly (35) Drosophila species (Drosophila spp.)), isoptera (Isoptera) (termites (termite)), including species from the families Amolonchidae (Hodotermitidae), hylothectorite (Kalotermitidae), australian termite (Mastotermitidae), rhapontiidae (Rhinotermitidae), mao Baiyi (SERRITERMITIDAE), termite (TERMITIDAE), The plant growth promoting agent is selected from the group consisting of Proteidae ()/Aphis pratensis (bug,)/Aphis gossypii or Aphis melo (Aphis gossypii))/Aphis malabarica (Aphis gossypii); aleurites citri (Aleurites citri), aleurites alfa (Bemisia tabaci))/Aleuro (Bemisia tabaci)), brassica oleracea (,), pythia (pear psy); aleuro (black currant cryptotaza aphid ()/Pythia (grape root nodule aphid (,); aleuro citri (Diaphorina citri), aleuro citri (potato leafhopper (); aleuro vitis (grape leafhopper ()), aleuro (Aleuro mali (Aphis mali) Ulmori, ulmoschus fasciatus (Bemisia tabaci); pimenta (myzu) and Pythium graciliatum, and Pythium gracile (). Fion, and the plant growth promoting agent is able to be a. Sitobion avena e), lepidoptera, such as Philippine flea-borer (Adoxophyes orana, summer fruit tortrix moth), fruit Huang Juane (fruit tree tip moth (fruit tree tortrix moth)), plutella xylostella (Bucculatrix pyrivorella, PEARLEAFMINER), philippine flea-borer (Bucculatrix thurberiella, cotton leaf perforator), pine looper (Bupalus piniarius, pine looper), codling moth (Carpocapsa pomonella, codling moth), chilo suppressalis (Chilo suppressalis, STRIPED RICE borer), spruce leaf moth (Choristoneura fumiferana, eastern spruce budworm), stripe sunflower moth (banded sunflower moth)), southwestern corn borer (Diatraea grandiosella) (southwestern corn borer), ring needle moth (Eupoecilia ambiguella, european grape berry moth), cotton bollworm (Helicoverpa armigera, cotton bollworm), fall webworm (Helicoverpa zea) (cotton bollworm), tobacco bud moth (Heliosis VIRES CENS) (noctuid (tobacco budworm)) The plant species include Heliothis armyworms (Homeosoma electellum) (Helicoverpa armyworms), helicoverpa armyworms (Homona magnanima, oriental tea tree tortrix moth), leaf-spot-curtain leaf-miners (Lithocolletis blancardella, spotted tentiform leafminer), gypsy moths (LYMANTRIA DISPAR, gypsy moth), yellow-brown-day-curtain caterpillars (Malacosoma neustria) (Monday-curtain caterpillars), cabbage loopers (Mamestra brassicae, cabbage armyworm), spodoptera frugiperda (Mamestra configurata, bertha armyworm), winter geometrid moths (Operophtera brumata, winter moths), european corn borers (Ostrinia nubilalis, european corn borer), Noctuid (Panolis flammea), pine moth (Pine beauty moth), Citrus leaf miners (Phyllocnistis citrella, citrus leafminer), cabbage butterflies (Pieris brassicae) (cabbage butterflies), peppermint spodoptera (rachiplus ni) (soybean loopers), beet armyworms (Spodoptera exigua, beet armywonn), sea-dust spodoptera (Spodoptera littoralis, cotton leafworm), cotton leaf rollers (Sylepta derogata, cotton leaf roller), armyworms (Trichoplusia ni) (cabbage loopers), orthoptera (Orthoptera), such as cricket (common cricket, acheta domesticus), and cotton bollworms (cotton bollworms), Locust (tree locust) (species of genus Ulva (Anacridium spp.)), migratory locust (migratory locust, locusta migratoria), double-banded black locust (twostriped grasshopper, melanoplus bivittatus), heteroblack locust (DIFFERENTIAL GRASSHOPPER, melanoplus DIFFER ENTIALIS), red-legged black locust (REDLEGGED GRASSHOPPER, melanoplus femurrubrum), Locusts (Melanoplus sanguinipes), northern mole cricket (Neocurtilla hexadectyla), red wing locusts (red locust, nomadacris septemfasciata), mole cricket (shortwinged mole cricket, scapteriscus abbreviatus), southern mole cricket (southern mole cricket, scapteriscus borellii), gryllotalpa (tawny mole cricket, scapteriscus vicinus) and grasshopper (desert locust, schistocerca gregaria), complex class (Symphyla) such as white pine beetles (GARDEN SYMPHYLAN, scutigerellaimmaculata), thysanoptera (Thysanoptera) such as thrips tabaci (tobacco thrip, FRANKLINIELLA FUSCA), Flower thrips (flower thrip, frankliniellaintonsa), western flower thrips (western flower thrip, FRANKLINIELLA OCCIDENTALISM), cotton bud thrips (cotton bud thrip, FRANKLINIELLA SCHULTZEI), ribbon-shaped greenhouse thrips (banded greenhouse thrip, hercinothrips femoralis), soybean thrips (soybean thrip, neohydatothrips variabilis), Ponkan Thrips (Pezothrips kellyanus), avocado Thrips (avocado thrip, scirtothrips perseae), melon Thrips (THRIPS PALMI), and onion Thrips (Thrips tabaci)), and the like, as well as combinations comprising one or more of the foregoing organisms.

In some embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in CESA-inhibiting herbicide tolerant plants is effective to control flea beetles, i.e., members of the phyllotoxin family (Chrysomelidae) flea beetles, preferably against species of phyllotoxin genus (Phyllotreta spp.) such as cruciferous species phyllotoxin and/or phyllotreta striolata (Phyllotreta striolata). In other embodiments, expression of one or more protein toxins (e.g., insecticidal proteins) in CESA-inhibiting herbicide tolerant plants is effective to control cabbage trunk weevil, spodoptera frugiperda, lygus bug (Lygus bug), or diamond back moth (diamondback moth).

In addition, in one embodiment, CESA-inhibiting herbicide tolerant plants are also covered, which plants are enabled to synthesize one or more proteins to increase the resistance or tolerance of those plants to bacterial, viral or fungal pathogens, for example, by using recombinant DNA techniques and/or by breeding and/or otherwise selecting such traits. Methods for producing such genetically modified plants are generally known to those skilled in the art.

In addition, in another embodiment, CESA-inhibiting herbicide tolerant plants are also covered, which plants are enabled to synthesize one or more proteins to increase productivity (e.g., oil content) of those plants, tolerance to drought, salinity, or other growth-limiting environmental factors, or tolerance to pests and fungal, bacterial, or viral pathogens, for example, by using recombinant DNA technology and/or by breeding and/or otherwise selecting such traits.

In addition, in other embodiments, CESA-inhibiting herbicide tolerant plants are also covered, which plants are altered, e.g., by using recombinant DNA techniques and/or by breeding and/or otherwise selecting such traits, to contain a modified amount of one or more substances or novel substances, e.g., to improve human or animal nutrition, e.g., oil crops (e.g., nexera (R) canola, dow's agricultural science and technology company (Dow Agro Sciences, canada)) that produce health-promoting long-chain omega-3 fatty acids or unsaturated omega-9 fatty acids.

In addition, in some embodiments CESA-inhibiting herbicide tolerant plants are also covered, which plants are altered to contain increased amounts of vitamins and/or minerals, and/or nutraceutical compounds that improve characteristics, for example, by using recombinant DNA techniques and/or by breeding and/or otherwise selecting for such traits.

In one embodiment, the CESA-inhibiting herbicide tolerant plants of the invention comprise increased or improved amounts or characteristics of a compound selected from the group consisting of glucosinolates (e.g., glucoraphanin (4-methylsulfinylbutyl-glucosinolate), glucoraphanin, 3-indolylmethyl-glucosinolate (brassica glucoside), l-methoxy-3-indolylmethyl-glucosinolate (neobrassica glucoside)) relative to wild-type plants, phenols (e.g., flavonoids (e.g., quercetin, kaempferol), hydroxycinnamoyl derivatives (e.g., 1,2 '-triseruption gentiobiose, 1, 2-diferuption gentiobiose, l,2' -diseruption-2-feruloyl gentiobiose, 3-0-caffeoyl-quinic acid (neochlorogenic acid)), and vitamins and minerals (e.g., vitamin C, vitamin E, folic acid, niacin, riboflavin, amin, calcium, iron, magnesium, potassium, and zinc).

In another embodiment, the CESA-inhibiting herbicide tolerant plants of the invention comprise an increased amount or improved profile of a compound selected from the group consisting of pro-goitrons (progoitrin), isothiocyanates, indoles (glucosinolate hydrolysis products), glutathione, carotenoids such as beta-carotene, lycopene and lutein carotenoids such as lutein (lutein) and zeaxanthin, flavonoids containing phenols such as flavonols (e.g., quercetin, rutin), flavans/tannins (e.g., procyanidins containing coumarin, proanthocyanidins, catechin and anthocyanin), flavones, phytoestrogens such as coumestrol (coumestan), lignans, resveratrol, isoflavones, e.g., genistein, daidzein and glycitein, dihydroxybenzoic acid (resorcyclic acid) lactones, organosulfur compounds, phytosterols, terpenes such as carnosol (carnosol), rosmarinic acid, glycyrrhizin and saponins, chlorophyll, chlorophyllin (chlorophyllin), sugar, anthocyanin and vanilla.

In other embodiments, CESA-inhibiting herbicide tolerant plants of the invention comprise increased amounts or improved characteristics of a compound selected from the group consisting of vincristine, vinblastine, a taxane (e.g., paclitaxel (taxol, paclitaxel), baccatin III, 10-deacetylbaccatin III, 10-deacetyl taxol, xylosyl taxol, 7-epitaxol, 7-epibaccatin III, 10-deacetylcephalomannine, 7-epicephalomannine, taxotere, cephalomannine, xylosyl cephalomannine, taxus Ji Fen, 8-benzoyloxy taxane Ji Fen, 9-acetoxytaxol, 9-hydroxy taxol, taiwanxam, taxane la, taxane lb, taxane Ic, taxane Id, taxol, 9-dihydro 13-acetyl baccatin III, 10-deacetyl-7-epitaxol, tetrahydrocannabinol (THC), cannabinol (CBD), xanthene (CBD), emodin, diacetylflavin, alexin (37), radices (37), and the like, relative to wild type plants.

In other aspects, methods for treating plants of the invention are provided.

In some embodiments, the method comprises contacting the plant with an agronomically acceptable composition. In one embodiment, the agronomically acceptable composition comprises a CESA-inhibiting herbicide a.i, such as azine as described herein.

In another aspect, the invention provides a method for preparing offspring seed. The method comprises growing seeds of the plant of the invention or seeds capable of producing the plant of the invention. In one embodiment, the method further comprises growing a progeny plant from the seed, and harvesting the progeny seed from the progeny plant. In other embodiments, the method further comprises applying to the progeny plant a CESA-inhibiting herbicide herbicidal composition.

In another embodiment, the invention relates to harvestable parts of a plant according to the invention. Preferably, the harvestable parts comprise a CESA nucleic acid or a CESA protein of the invention. The harvestable parts may be seeds, roots, leaves and/or flowers comprising CESA nucleic acids or CESA proteins or parts thereof. The preferred part of the soybean plant is soybean comprising CESA nucleic acid or CESA protein.

In another embodiment, the invention relates to a product derived from a plant according to the invention, a part thereof or a harvestable part thereof. Preferred plant products are feed, seed meal, oil, or seed-coated seeds (seed-treated seed). Preferably, the meal and/or oil comprises CESA nucleic acid or CESA protein.

In another embodiment, the invention relates to a method for producing a product, the method comprising

A) Growing the plant of the invention or obtainable by the method of the invention, and

B) The products are produced from or by the plants of the invention and/or parts of such plants (e.g., seeds).

In a further embodiment, the method comprises the steps of:

a) The plants of the present invention are allowed to grow,

B) Removing harvestable parts as defined above from plants, and

C) The product is produced from or by the harvestable parts of the invention.

The product may be produced at a site where the plant has grown, and the plant and/or parts thereof may be removed from the site where the plant has grown to produce the product. Typically, plants are grown, and if feasible, the desired harvestable parts are removed from the plants in repeated cycles, and the product is made from the harvestable parts of the plants. The step of growing the plant may be carried out only once, each time carrying out the method of the invention, while allowing the step of producing the product to be repeated a number of times, for example by repeatedly removing harvestable parts of the plant of the invention and if necessary further processing these parts to obtain the product. The step of growing the plant of the invention may also be repeated and the plant or harvestable parts stored until the product is then produced once to accumulate the plant or plant parts. Furthermore, the steps of growing the plants and producing the product may overlap in time, even largely simultaneously or sequentially. Typically, plants are grown for a period of time prior to production of the product.

In one embodiment, the product produced by the methods of the present invention is a plant product such as, but not limited to, food, feed, food supplement, feed supplement, fiber, cosmetic, and/or pharmaceutical. Food products are considered to be compositions for nutrition and/or for supplementing nutrition. In particular, animal feeds and animal feed supplements are considered to be food products.

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

Plant products may consist to a large extent of one or more agricultural products.

Herbicide

As described above, the present invention provides nucleic acids, polypeptides that confer upon plants tolerance to compounds/herbicides that interfere with or inhibit cell wall (cellulose) biosynthesis ("CESA-inhibiting herbicides") by interfering with the activity of cellulose synthase, which compounds/herbicides are also known to those skilled in the art as Cellulose Biosynthesis Inhibitors (CBI).

Examples of herbicides that can be used according to the invention (i.e. to which the plants according to the invention are tolerant/resistant) are compounds known to the person skilled in the art as azines. Examples of azines ("compound a") are described in detail in the following patent applications described in table 1 below, which are incorporated by reference in their entirety.

TABLE 1

Examples of preferred CESA-inhibiting herbicides from the group of the named azines which can be used according to the invention are compounds of formula (I), which are known to the person skilled in the art as azines.

The herbicidal compounds (component a) useful in the present invention (as disclosed above as e.g. numbered 1-16) may be further used in combination with further herbicides which are naturally tolerant to crop plants, or which have been tolerant by mutagenesis as described above, or to which resistance is provided via expression of one or more further transgenes as described above. The CESA-inhibiting herbicides useful in the present invention are typically preferably applied in combination with one or more other herbicides to control more undesirable vegetation. When used in combination with other herbicides (hereinafter referred to as compound B), the presently claimed compounds may be formulated with the other herbicide(s), mixed with the other herbicide(s) in a bucket, or applied sequentially with the other herbicide(s).

The further herbicidal compounds B (component B) are in particular selected from the classes B1) to B15) herbicides:

b1 Lipid biosynthesis inhibitors;

b2 Acetolactate synthase inhibitors (ALS inhibitors);

b3 A photosynthesis inhibitor;

b4 A protoporphyrinogen-IX oxidase inhibitor,

B5 A bleach herbicide;

b6 Enolpyruvylshikimate 3-phosphate synthase inhibitor (EPSP inhibitor);

b7 Glutamine synthetase inhibitors;

b8 7, 8-dihydropteroic acid synthase inhibitor (DHP inhibitor);

b9 Mitotic inhibitors;

b10 Very long chain fatty acid synthesis inhibitors (VLCFA inhibitors);

b11 Cellulose biosynthesis inhibitors;

b12 A decoupling herbicide (decoupler herbicide);

b13 Auxin herbicide;

b14 Auxin transport inhibitor, and

B15 Other herbicides selected from the group consisting of bromobutamide, plastic alcohol methyl ester, cycloheptyl methyl ether, bensulfuron (cumyluron), coumoxaprop, dazomet, delphinidin-methylsulfonate, thiamethoxam, DSMA, diuron (dymron), endothal (endothal) and salts thereof, ethofenpyr, wheat straw fluoro (flamprop), wheat straw fluoro-isopropyl ester (flamprop-isopropyl), wheat straw fluoro-methyl ester (flamprop-methyl), wheat straw fluoro-isopropyl ester (flamprop-M-isopropyl), wheat straw fluoro-methyl ester (flamprop-M-methyl), imazalil (flurenol), imazethapyr-butyl (flurenol-butyl), furazanol, carbostyril, dime, dimefon-ammonium, indenone, indenofloxamide, maleic hydrazide, fluorosulfonamide, carbosulfan (CAS) 403640-27-methyl, methyl oleate, methyl quinone, methyl-6-propyl, 6-methyl-propyl, 6-propyl, and chlorpyribac-methyl;

Including agriculturally acceptable salts or derivatives thereof, such as ethers, esters or amides.

Preferred are those compositions according to the invention which comprise at least one herbicide B selected from the classes B1, B6, B9, B10 and B11.

Examples of herbicides B which can be used in combination with the compounds of formula (I) according to the invention are:

b1 From the group of lipid biosynthesis inhibitors:

ACC-herbicides such as molinate, molinate-sodium, benalachlor, clethodim, clodinafop-propargyl clodinafop-propargyl, thioxanthone, cyhalofop-butyl, halofop-butyl, graminezic acid, halofop-butyl, and halofop-butyl clodinafop-propargyl, thioxanthone, cyhalofop-butyl acid cyhalofop-butyl, gramineralic acid, halofop-butyl, haloxyfop-methyl, high-efficiency haloxyfop-methyl acid, haloxyfop-methyl high-efficiency haloxyfop-methyl, oxazachlor, pinoxaden, fenpropion high-efficiency haloxyfop-methyl, oxazoxamide, and preparation method thereof pinoxaden, fenpropiophenone,

4- (4 '-Chloro-4-cyclopropyl-2' -fluoro [1,1 '-biphenyl ] -3-yl) -5-hydroxy-2, 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, 6-tetramethyl-2H-pyran-3 (6H) -one (CAS 1312337-45-3), 4- (4 '-chloro-4-ethyl-2' -fluoro [1,1 '-biphenyl ] -3-yl) -5-hydroxy-2, 6-tetramethyl-2H-pyran-3 (6H) -one (CAS 1033757-93-5), 4- (2', 4 '-dichloro-4-ethyl [1,1' -biphenyl ] -3-yl) -2, 6-tetramethyl-2H-pyran-3, 5 (4H, 6H) -dione (CAS 1312340-84-3); 5- (acetoxy) -4- (4 '-chloro-4-cyclopropyl-2' -fluoro [1,1 '-biphenyl ] -3-yl) -3, 6-dihydro-2, 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, 6-tetramethyl-2H-pyran-3-one, 5- (Acetyloxy) -4- (4 '-chloro-4-ethyl-2' -fluoro [1,1 '-biphenyl ] -3-yl) -3, 6-dihydro-2, 6-tetramethyl-2H-pyran-3-one (CAS 1312340-82-1), 5- (Acetyloxy) -4- (2', 4 '-dichloro-4-ethyl [1,1' -biphenyl ] -3-yl) -3, 6-dihydro-2, 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, 6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonate (CAS 1312337-51-1); 4- (2', 4 '-dichloro-4-cyclopropyl- [1,1' -biphenyl ] -3-yl) -5, 6-dihydro-2, 6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonate, 4- (4 '-chloro-4-ethyl-2' -fluoro [1,1 '-biphenyl ] -3-yl) -5, 6-dihydro-2, 6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonate (CAS 1312340-83-2), 4- (2', 4 '-dichloro-4-ethyl [1,1' -biphenyl ] -3-yl) -5, 6-dihydro-2, 6-tetramethyl-5-oxo-2H-pyran-3-ylcarbonate (CAS 1033760-58-5), and non-ACC herbicides such as furben (benfurate), Ding Caodi (butylate), cycloxaprid (cycloate), coumoxystrobin, pethidine, EPTC, penoxsulam, ethofumesate, tetrafluoropropionic acid, molinate (molinate), triclopyr (orbencarb), molinate (pebulate), prosulfocarb, TCA, graminine, secondary chlorpyrifos, wild mevalonate and molinate (ernolate);

b2 Group from ALS inhibitors:

Sulfonylureas of the general formula (i) are described, such as amidosulfuron, tetrazole-sulfuron, bensulfuron-methyl, chlorimuron-ethyl, cinosulfuron, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron, flazasulfuron-methyl, flazasulfuron-ethyl flupyrazosulfuron, fluflazasulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl, iodosulfuron-methyl-sodium, iodosulfuron-sodium, disulfonic acid, halosulfuron-methyl-sodium, halosulfuron-methyl-sodium, halosulfuron-flupyrsulfuron-methyl, fluflazasulfuron-methyl-sodium, foramsulfuron, halosulfuron-methyl pyrazosulfuron-ethyl, iodosulfuron-methyl-sodium, iodosulfuron-methyl, iodosulfuron-sodium, disulfonic acid, sodium-methyl-sodium, sodium-methyl-ethyl, sodium-methyl-ethyl-sodium-methyl-sodium-methyl-ethyl, sodium-methyl-ethyl-sodium-methyl-sodium-methyl-sodium,

An imidazolidinone compound, which is selected from the group consisting of, such as imazethapyr, imazethapyr-methyl, imazethapyr, imazaquin and imazethapyr, triazolopyrimidine herbicides, and a sulfonamide group, the sulfonamide group being a sulfonamide group, such as cloransulam-methyl, cloransulam-methyl sulfenamide, flumetsulam, florasulam sulfenamide, flumetsulam florasulam, a kind of compound,

Pyrimidinyl benzoates, such as bispyribac-sodium, pyribenzoxim, pyriftalid acid (pyriminobac), pyriftalid-methyl, pyriftalid-sodium, 4- [ [ [2- [ (4, 6-dimethoxy-2-pyrimidinyl) oxy ] phenyl ] methyl ] amino ] -benzoic acid-1-methylethyl (CAS 420138-41-6), 4- [ [ [2- [ (4, 6-dimethoxy-2-pyrimidinyl) oxy ] phenyl ] methyl ] amino ] -benzoic acid propyl (CAS 420138-40-5), N- (4-bromophenyl) -2- [ (4, 6-dimethoxy-2-pyrimidinyl) oxy ] benzyl amine (CAS 420138-01-8),

Sulfonylaminocarbonyl-triazolinone herbicides such as flucarbazone-sodium, bensulfuron-methyl, bensulfuron-sodium, thidiazuron-acid and thidiazuron-Long Suan methyl ester; fluoroketosulfenamide;

Among other things, preferred embodiments of the present invention relate to those compositions comprising at least one imidazolinone herbicide;

b3 Group from photosynthesis inhibitors:

Amicarbazone, inhibitors of photosystem II, for example triazine herbicides, including chlorotriazine, triazinone, triazindione, thiotriazinone and pyridazinones, such as ametryn, atrazine, chloroxamine, cyanoacenitrile, diquat, isozin, hexazinone, metribuzin, prometryn, simazine, simetryn, terbutazone, terbutryn and dydrone (trietazin), aryl ureas, such as tribenuron, chlormeuron, cumuron, oxazomet, diuron, fuzouron, isoproturon, isoxauron, linuron, benzinone, metsulfuron, pyranone, methosulfuron, lufenuron, cyclouron, tebuthiuron and thidiazuron, phenyl carbamates, such as betalain, triamcinolone (karbutilat), betanin-ethyl, nitrile herbicides, such as bromfenacet, bromoxynil and its salts and esters, ioxynil and its salts and esters, uracils, such as triclopyr, cyprodinil and terfenadine, and thioflat-sodium, pyridate, metaflumorph (pyridafon), triclosan and propanil, and inhibitors of photosystem I, such as diquat, diquat-dibromo, paraquat-dichloride and paraquat-dimethyl sulfate. Among other things, preferred embodiments of the present invention relate to those compositions comprising at least one aryl urea herbicide. Among other things, preferred embodiments of the present invention are also directed to those compositions comprising at least one triazine herbicide. Among other things, preferred embodiments of the present invention are also directed to those compositions comprising at least one nitrile herbicide;

b4 From the group of protoporphyrinogen-IX oxidase inhibitors:

Acifluorfen, acifluorfen sodium, carfentrazone-ethyl, bensulfuron methyl, and bupirimate, carboxin, flumetsulam, triadimefon, and the like dipyr, carboxin, weeding ether flumetsulam, triadimefon, and fluoroglycofen, fluoroglycofen-ethyl, oxaziclomefone, methyl oxazin, fomesafen, flusulfamide, lactofen, oxadiargyl, oxadiazon, and the like oxyfluorfen, cyclopentaoxadiazon, flumetsulam, bispyraclonil pyraclonil acid, pyraclonil, saflufenacil, sulfenacil, Thioxazin, flumetsulam, [3- [ 2-chloro-4-fluoro-5- (1-methyl-6-trifluoromethyl-2, 4-dioxo-1, 2,3, 4-tetrahydropyrimidin-3-yl) phenoxy ] -2-pyridyloxy ] acetic acid ethyl ester (CAS 353292-31-6;S-3100), N-ethyl-3- (2, 6-dichloro-4-trifluoromethylphenoxy) -5-methyl-1H-pyrazole-1-carboxamide (CAS 452098-92-9), N-tetrahydrofurfuryl-3- (2, 6-dichloro-4-trifluoromethylphenoxy) -5-methyl-1H-pyrazole-1-carboxamide (CAS 915396-43-9), N-ethyl-3- (2-chloro-6-fluoro-4-trifluoromethylphenoxy) -5-methyl-1H-pyrazole-1-carboxamide (CAS 452099-05-7), N-tetrahydrofurfuryl-3- (2-chloro-6-fluoro-4-trifluoromethylphenoxy) -5-methyl-1H-pyrazole-1-carboxamide (CAS 452100-03-7), 3- [ 7-fluoro-3-oxo-4- (prop-2-ynyl) -3, 4-dihydro-2H-benzo [1,4] oxazin-6-yl ] -1, 5-dimethyl-6-thioxo- [1,3,5] triazin-2, 4-dione, 1, 5-dimethyl-6-thioxo-3- (2, 7-trifluoro-3-oxo-4- (prop-2-ynyl) -3, 4-dihydro-2H-benzo [ b ] [1,4] oxazin-6-yl) -1,3, 5-triazin-e-2, 4-dione (CAS 1258836-72-4), 2- (2, 7-trifluoro-3-oxo-4-prop-2-ynyl-3, 4-dihydro-2H-benzo [1,4] oxazin-6-yl) -4,5,6, 7-tetrahydro-isoindole-1, 3-dione, 1-methyl-6-trifluoromethyl-3- (2, 7-trifluoro-3-oxo-4-prop-2-ynyl-3, 4-dihydro-2H-benzo [1,4] oxazin-6-yl) -1H-pyrimidine-2, 4-dione, (E) -4- [ 2-chloro-5- [ 4-chloro-5- (difluoromethoxy) -1H-methyl-pyrazol-3-yl ] -4-fluoro-phenoxy ] -3-methoxy-but-2-enoic acid methyl ester [ CAS 948893-00-3], and 3- [ 7-chloro-5-fluoro-2- (trifluoromethyl) -1H-benzoimidazol-4-yl ] -1-methyl-6- (trifluoromethyl) -1H-pyrimidine-2, 4-dione (CAS 212754-02-4);

b5 Group from bleach herbicides:

PDS inhibitors, which are fluobutamide, diflufenican, fluazinam, flufloxapyrone (flurochloridone), furbenone, flubenoxaden, flupirfenidone, and 4- (3-trifluoromethylphenoxy) -2- (4-trifluoromethylphenyl) pyrimidine (CAS 180608-33-7), HPPD inhibitors, which are bicyclozin (benzobicycloton), pyriftalid (benzofenap), clomazone (clomazone), fenquizalofop, isoxaflutole (isoxaflutole), mesotrione, sulfonylgrass pyrazole, pyrazolote (pyrazolynate), benoxadiazon (pyrazoxyfen), sulfentrazone, furfurfuryl-trione, cyclosulfentrazone, topramezone, bleach, unknown targets, which are benoxadiazon, clomazone, and flucarbazone (flumeturon);

b6 Group from EPSP synthase inhibitors:

glyphosate, glyphosate-isopropylammonium, glyphosate-potassium, and glyphosate-trimethylsulfur (glyphosate);

b7 Group from glutamine synthase inhibitors:

bialaphos @ bialaphos), a bialaphos-sodium glufosinate, spermate, and glufosinate-ammonium;

b8 Group from DHP synthase inhibitors:

sulbenazolin (asulam);

b9 Group from mitotic inhibitors:

The compounds of group K1, dinitroanilines such as flumetsulam, butralin, dichlormid, butachlor (ethalfluralin), chloroethyl (fluchloralin), pendimethalin (oryzalin), pendimethalin (PENDIMETHALIN), amisulfydryl (prodiamine) and trifluralin (trifluralin), phosphoramides such as amifos, amifos-methyl and imazalil (butamiphos), benzoic acid herbicides such as dichlormid (chlorthal), diquat-dimethyl, pyridines such as dithiopyr and thiabendazole, benzamides such as propyzamide (propyzamide) and flumetsulam (tebutam), the compounds of group K2, chlorpropyzamide, propyzamide and carpronium, among which the compounds of group K1, in particular dinitroaniline, are preferred;

b10 Group from VLCFA inhibitors:

Chloroacetamides, such as acetochlor (acetochlor), alachlor (alachlor), butachlor, dimethenamine (dimethachlor), dimethenamine (DIMETHENAMID), dimethenamine, mefenacet, iprovalicarb (metolachlor), dimethenamine (metolachlor-S), dimethenamine (pethoxamid), pretilachlor (pretilachlor), verapamil, iprovalicarb (propisochlor) and thenalachlor (thenylchlor), oxacetamides, such as flufenacet and mefenacet, acetamides, such as benazelamide (diphenamid), napropylamine, dichlormid and dichlormid-M, tetrazolinones, such as tebufenpyr (fentrazamide), and other herbicides, such as anilofos (anilofos), flumorph (cafenstrole), benoxazamate (fenoxasulfone), triazoxamine (ipfencarbazone), pirfenpyr, paraquat (pyroxasulfone) and isoxaprop-ethyl having the formula II, 3.4.5.7.9.8, II,

Isoxazoline compounds having formula (I) are known in the art, for example from WO 2006/024820, WO 2006/037945, WO 2007/071900 and WO 2007/096576;

among the VLCFA inhibitors, chloroacetamides and oxyacetamides are preferred;

b11 From the group of cellulose biosynthesis inhibitors:

Oxaden, diuron, flumetsulam, isoxaflutole and 1-cyclohexyl-5-pentafluorophenyloxy-1 ⁴ - [1,2,4,6] thiatriazin-3-ylamine;

b12 Group from decoupling herbicides:

delrin, terlaol (dinoterb), and DNOC and salts thereof;

b13 Group from auxin herbicides:

2,4-D and salts and esters thereof, such as clopyralid (clacyfos), 2,4-DB and salts and esters thereof, aminopyralid (aminocyclopyrachlor) and salts and esters thereof, aminopyralid (aminopyralid) and salts thereof, such as aminopyralid-dimethylammonium, aminopyralid-tris (2-hydroxypropyl) ammonium and esters thereof, benazolidinate (benazolin), benazolidinate-ethyl, leguminous (chloramben) and salts and esters thereof, chloroformyl oxamine (clomeprop), clopyralid (clopyrad) and salts and esters thereof, dicamba and salts and esters thereof, 2, 4-dipropionic acid (dichlorprop) and salts and esters thereof, fine 2, 4-aminopropionic acid and salts and esters thereof, fluroxypyr (fluroxypyr), fluroxypyr Ding Yangyi propyl, fluroxypyr isooctyl, fluroxypyr (halauxifen) and salts and esters thereof (CAS 943832-60-8), MCPA and salts and esters thereof, MCPA-thio-ethyl, MCPB and salts and esters thereof, triclopyr and esters and salts and esters thereof, triclopyr and salts and esters thereof, and triclopyr and esters and salts and esters thereof, and triclopyr and salts and esters thereof;

b14 A group from auxin transport inhibitors, diflufenzopyr (diflufenzopyr), diflufenzopyr-sodium, naproxen (naptalam) and naproxen-sodium;

b15 Brombutamide, plastic alcohol methyl ester, cycloheptyl methyl ether, benuron, acibenzolar-s-methyl (cyclopyrimorate) (CAS 499223-49-3) and salts and esters thereof, coumox, dazomet, difenoconazole-methyl sulfonate, thiamethoxam, DSMA, chloruron, adoxole and salts thereof, ethofenpyr, dicamba-isopropyl, dicamba-methyl ester, smart dicamba-isopropyl, smart dicamba-methyl ester, imazalil, butyl, furazone, mulin, imazapine-ammonium, indenone, indenofloxacin, maleic hydrazide, fluorosulfonamide, valicarb, methimazone (CAS 403640-27-7), methyl azide, methyl bromide, methyl-chlor, methyl iodide, MSMA, oleic acid, oxazinone, pelargonic acid, barnacle, chlor, chloranil, chlorpyrifos, triazophos, and trifluo.

The active compounds B and C having carboxyl groups can be used in the compositions according to the invention in the form of acids, in the form of agriculturally suitable salts as mentioned above or in the form of agriculturally acceptable derivatives.

In the case of dicamba, suitable salts include salts in which the counter ion is an agriculturally acceptable cation. For example, suitable salts of dicamba are dicamba-sodium, dicamba-potassium, dicamba-methyl ammonium, dicamba-dimethyl ammonium, dicamba-isopropyl ammonium, dicamba-diglycolamine, dicamba-ethanolamine, dicamba-diethanolamine, dicamba-triethanolamine, dicamba-N, N-bis- (3-aminopropyl) methyl amine, and dicamba-diethylenetriamine. Examples of suitable esters are dicamba-methyl ester and dicamba-butoxyethyl ester (dicamba-butotyl).

Suitable salts of 2,4-D are 2, 4-D-ammonium, 2, 4-D-dimethylammonium, 2, 4-D-diethylammonium, 2,4-D-diethanolammonium (2, 4-D-diethanolammonium, 2, 4-D-diolamine), 2, 4-D-triethanolammonium, 2, 4-D-isopropylammonium, 2, 4-D-triisopropanolammonium, 2, 4-D-heptylammonium, 2, 4-D-dodecylammonium, 2, 4-D-tetradecylammonium, 2, 4-D-triethylammonium, 2, 4-D-tris (2-hydroxypropyl) ammonium, 2, 4-D-tris (isopropyl) ammonium, 2, 4-D-triethanolamine, 2, 4-D-lithium, 2, 4-D-sodium. Examples of suitable esters of 2,4-D are 2, 4-D-butoxyethyl ester, 2, 4-D-2-butoxypropyl ester, 2, 4-D-3-butoxypropyl ester, 2, 4-D-butyl ester, 2, 4-D-ethyl ester, 2, 4-D-ethylhexyl ester, 2, 4-D-isobutyl ester, 2, 4-D-isooctyl ester, 2, 4-D-isopropyl ester, 2, 4-D-1-methylheptyl ester (2, 4-D-meptyl), 2, 4-D-methyl ester, 2, 4-D-octyl ester, 2, 4-D-pentyl ester, 2, 4-D-propyl ester, 2, 4-D-tetrahydrofurfuryl ester and chloroglyphosate.

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

Suitable salts of 2, 4-D propionic acid are, for example, sodium 2, 4-D propionate, potassium 2, 4-D propionate and dimethyl ammonium 2, 4-D propionate. Examples of suitable esters of 2, 4-D propionic acid are 2, 4-D propionic acid-butoxyethyl ester and 2, 4-D propionic acid-isooctyl ester.

Suitable salts and esters of MCPA include MCPA-butoxyethyl ester, MCPA-butyl ester, MCPA-dimethylammonium, MCPA-diethanolamine, MCPA-ethyl ester, MCPA-thioethyl ester, MCPA-2-ethylhexyl ester, MCPA-isobutyl ester, MCPA-isooctyl ester, MCPA-isopropyl ester, MCPA-isopropylammonium, MCPA-methyl ester, MCPA-ethanolamine, MCPA-potassium, MCPA-sodium, and MCPA-triethanolamine.

A suitable salt of MCPB is sodium MCPB. A suitable ester of MCPB is MCPB-ethyl ester.

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

Examples of suitable esters of fluroxypyr are fluroxypyr-meptyl and fluroxypyr-2-butoxy-1-methylethyl ester, of which fluroxypyr-meptyl is preferred.

Suitable salts of picloram are picloram-dimethylammonium, picloram-potassium, picloram-triisopropylammonium and picloram-triethanolamine. A suitable ester of picloram is picloram-isooctyl ester.

A suitable salt of triclopyr is triclopyr-triethylammonium. Suitable esters of triclopyr are, for example, triclopyr-ethyl ester and triclopyr-butoxyethyl ester.

Suitable salts and esters of leguminous wei include leguminous wei-ammonium, leguminous wei-diethanolamine, leguminous wei-methyl esters, leguminous wei-methyl ammonium and leguminous wei-sodium. Suitable salts and esters of 2,3,6-TBA include 2,3, 6-TBA-dimethylammonium, 2,3, 6-TBA-lithium, 2,3, 6-TBA-potassium and 2,3, 6-TBA-sodium.

Suitable salts and esters of aminopyralid include aminopyralid-potassium, aminopyralid-dimethyl ammonium and aminopyralid-tris (2-hydroxypropyl) ammonium.

Suitable salts of glyphosate are, for example, glyphosate-ammonium, glyphosate-diammonium, glyphosate-dimethylammonium, glyphosate-isopropylammonium, glyphosate-potassium, glyphosate-sodium, glyphosate-trimethylthio and ethanolamine and diethanolamine salts, preferably glyphosate-diammonium, glyphosate-isopropylammonium and glyphosate-trimethylthio (glyphosate).

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

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

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

Suitable salts and esters of dimethyltetrachloropropionic acid include dimethyltetrachloropropionic acid-butoxyethyl ester, dimethyltetrachloropropionic acid-dimethylammonium, dimethyltetrachloropropionic acid-diethanolamine, dimethyltetrachloropropionic acid-ethyleneglycol (ethadyl), dimethyltetrachloropropionic acid-2-ethylhexyl ester, dimethyltetrachloropropionic acid-isooctyl ester, dimethyltetrachloropropionic acid-methyl ester, dimethyltetrachloropropionic acid-potassium, dimethyltetrachloropropionic acid-sodium, and dimethyltetrachloropropionic acid-triethanolamine.

Suitable salts of fine dimethyltetrachloropropionic acid are, for example, fine dimethyltetrachloropropionic acid-butoxyethyl, fine dimethyltetrachloropropionic acid-dimethylammonium, fine dimethyltetrachloropropionic acid-2-ethylhexyl, fine dimethyltetrachloropropionic acid-isobutyl, fine dimethyltetrachloropropionic acid-potassium and fine dimethyltetrachloropropionic acid-sodium.

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

A suitable salt of naproxen is, for example, naproxen-sodium.

Suitable salts and esters of aminopyrimidic acid are, for example, aminopyrimidic acid-dimethylammonium, aminopyrimidic acid-methyl ester, aminopyrimidic acid-triisopropanolamine, aminopyrimidic acid-sodium and aminopyrimidic acid-potassium.

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

A suitable salt of cloquintocet-mexyl is, for example, quinclorac-dimethylammonium.

A suitable salt of imazethapyr is, for example, imazethapyr-ammonium.

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

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

A suitable salt of topramezone is, for example, topramezone-sodium.

Particularly preferred herbicidal compounds B are herbicides B as defined above, in particular herbicides B.1 to B.189 as listed in Table B below:

Furthermore, it may be useful to apply the compounds of formula (I) in combination with safeners and optionally with one or more further herbicides. Safeners are chemical compounds which prevent or reduce damage to useful plants without having a significant effect on the herbicidal action of the compounds of formula (I) against unwanted plants. They may be applied prior to sowing of the useful plants (e.g. on seed treatments, shoots or seedlings) or applied pre-emergence or post-emergence. The safener and the compound of the formula (I) and optionally the herbicide B can be applied simultaneously or sequentially.

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

Examples of preferred safeners C are clomazone, oxalic acid, clofenamate, cyclopropanesulfonamide, dichlorpropenamine (dichlormid), dicyclopyrrolidone (dicycloonon), synergistic phosphorus (dietholate), clomazone (fenchlorazole), clomazone (fenclorim), clomazone (flurazole), fluroxypyr (fluxofenim), clomazone (furilazole), bisbenzoxazole acid (isoxadifen), pyrazole oxalic acid (mefenpyr), mefenamate (mefenamate), naphthalene dicarboxylic anhydride, clomazone, 4- (dichloroacetyl) -1-oxa-4-azaspiro [4.5] decane (MON 4660, CAS 71526-07-3), 2, 5-trimethyl-3- (dichloroacetyl) -1, 3-oxazolidine (R-29148, CAS 836-31-4) and N- (2-methoxybenzoyl) -4- [ (methylaminocarbonyl) amino ] benzenesulfonamide (CAS 129531-12-0).

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

B1 Active compounds B and safener compounds C of groups B15) are known herbicides and safeners, see, for example, the Compendium of Pesticide Common Names [ pesticide general name schema ] (http:// www.alanwood.net/pesticides /); FARM CHEMICALS Handbook [ Handbook of agrochemicals ]2000, volume 86, meister Publishing Company [ Michelt release company ],2000, B.hock, C.Fedtke, R.R.Schmidt, herbizide [ herbicide ], georg THIEME VERLAG [ Georg Algerd Sitty, stuttgart) 1995;W.H.Ahrens,Herbicide Handbook [ herbicide Handbook ], 7 th edition, WEED SCIENCE Society of America [ society of weed science ],1994; K.K.Hatzios, herbicide Handbook [ herbicide Handbook ], 7 th edition journal of increasing, WEED SCIENCE Society of America [ society of weed science ],1998.2, 5-trimethyl-3- (dichloroacetyl) -1, 3-oxazolidine [ CAS number 52836-31-4] is also known as R-29148.4- (dichloroacetyl) -1-oxa-4-azaspiro [4.5] decane [ CAS number 71526-07-3] was also known as AD-67 and MON 4660.

The partitioning of the active compounds into the corresponding mechanisms of action is based on current knowledge. If several mechanisms of action apply to an active compound, this substance is assigned to only one mechanism of action.

It is generally preferred to use the compounds of the present invention in combination with herbicides which are selective for the treated crop and which complement the weed spectrum controlled by the compounds at the application rates employed. It is generally further preferred to apply the compounds of the invention and other complementary herbicides simultaneously, either as a combined formulation or as a tank mix.

In another embodiment, the invention relates to a method for identifying a CESA-inhibiting herbicide by using a mutant CESA encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 or 98 or a variant or derivative thereof.

The method comprises the following steps:

a) Generating a transgenic cell or plant comprising a nucleic acid encoding a mutant CESA, wherein the mutant CESA is expressed;

c) Determining the growth or viability of the transgenic cell or plant and the control cell or plant after application of the CESA-inhibiting herbicide, and

D) Selecting a "CESA-inhibiting herbicide" that reduces the growth of the control cell or plant as compared to the growth of the transgenic cell or plant.

As described above, the present invention teaches compositions and methods for increasing CESA inhibitory tolerance of a crop plant or seed as compared to a wild type variety of the plant or seed. In a preferred embodiment, the CESA-inhibiting tolerance of the crop plant or seed is increased such that the plant or seed is capable of withstanding CESA-inhibiting herbicide application of preferably about 1-1000g aiha ^-1, more preferably 1-200g aiha ^-1, even more preferably 5-150g ai ha ^-1, and most preferably 10-100g ai ha ^-1. As used herein, "withstand" CESA-inhibiting herbicide application means that the plant is not killed by such application or is only moderately damaged by such application. Those skilled in the art will appreciate that the rate of application may vary, depending on environmental conditions, such as temperature or humidity, and on the type of herbicide selected (active ingredient ai).

The methods of pre-emergence and/or post-emergence weed control useful in the various embodiments herein utilize CESA-inhibiting herbicides at an application rate of about >0.3x, which in some embodiments may be, for example, about >0.3x, >0.4x, >0.5x, >0.6x, >0.7x, >0.8x, >0.9x, or >1x CESA-inhibiting herbicides. In one embodiment, the CESA-inhibiting herbicide-tolerant plants of the invention are tolerant to CESA-inhibiting herbicides in amounts of about 25 to about 500g ai/ha applied pre-emergence and/or post-emergence. In some embodiments, wherein the CESA-inhibiting herbicide tolerant plant is a dicot (e.g., soybean, cotton), the amount of CESA-inhibiting herbicide applied pre-emergence and/or post-emergence is about 25-250g ai/ha. In another embodiment, wherein the CESA-inhibiting herbicide tolerant plant is a monocot (e.g., maize, rice, sorghum), the amount of CESA-inhibiting herbicide applied pre-emergence and/or post-emergence is about 50-500g ai/ha. In other embodiments, wherein the CESA-inhibiting herbicide tolerant plant is brassica (e.g., canola), the amount of CESA-inhibiting herbicide applied pre-emergence and/or post-emergence is about 25-200g ai/ha. In the pre-emergence and/or post-emergence weed control methods herein, in some embodiments, the methods can use CESA-inhibiting herbicide application rates from about 7 to 10 days pre-emergence and/or post-emergence. In another embodiment, the application rate may exceed 8x CESA-inhibiting herbicide, in some embodiments, the application rate may be as high as 4x CESA-inhibiting herbicide, although more typically the application rate will be about 2.5x or less, or about 2x or less, or about 1x or less.

Furthermore, the present invention provides methods involving the use of at least one CESA-inhibiting herbicide, optionally in combination with one or more herbicidal compounds B and optionally safeners C as described in detail above.

In these methods, the CESA inhibiting herbicide may be applied by any method known in the art, including, but not limited to, seed treatment, soil treatment, and foliar treatment. Prior to application, the CESA-inhibiting herbicide may be converted into conventional formulations, such as solutions, emulsions, suspensions, powders, pastes, and granules. The form of use depends on the particular intended purpose, in each case a fine and uniform distribution of the compounds according to the invention should be ensured.

By providing plants with increased tolerance to CESA-inhibiting herbicides, a variety of formulations can be employed to protect plants from weeds, thereby enhancing plant growth and reducing competition for nutrients. CESA-inhibiting herbicides themselves may be used for weed control in the peri-crop plant areas described herein, pre-emergence, post-emergence, pre-planting and at-planting, or CESA-inhibiting herbicide formulations containing other additives may be used. CESA-inhibiting herbicides can also be used as seed treatments. Additives found in CESA-inhibiting herbicide formulations include other herbicides, detergents, adjuvants, spreaders (SPREADING AGENT), adhesives, stabilizers, and the like. CESA-inhibiting herbicide formulations may be wet or dry formulations and may include, but are not limited to, flowable powders, emulsifiable concentrates, and liquid concentrates. CESA-inhibiting herbicides and herbicide formulations can be applied in conventional manner, for example, by spraying, irrigation, dusting, and the like.

Suitable formulations are described in detail in PCT/EP 2009/063287 and PCT/EP 2009/063286, which are incorporated herein by reference.

As disclosed herein, CESA nucleic acids of the invention are useful for enhancing CBI herbicide tolerance in plants that comprise in their genome a gene encoding a herbicide tolerance mutant CESA protein. Such genes may be endogenous genes or transgenes, as described above. Additionally, in certain embodiments, the nucleic acids of the invention can be stacked with any combination of polynucleotide sequences of interest to produce plants having a desired phenotype. For example, the nucleic acids of the invention may be overlaid with any other polynucleotide encoding a polypeptide having pesticidal and/or insecticidal activity, such as a Bacillus thuringiensis toxin protein (described in U.S. Pat. Nos. 5,366,892, 5,747,450, 5,737,514, 5,723,756, 5,593,881; and Geiser et al (1986) Gene [ Gene ] 48:109), a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), a Glyphosate Acetyltransferase (GAT), a cytochrome P450 monooxygenase, a Phosphinothricin Acetyltransferase (PAT), an acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), a hydroxyphenylpyruvate dioxygenase (HPPD), a Phytoene Desaturase (PD), a protoporphyrinogen oxidase (PPO) and a dicamba degrading enzyme, or a phenoxyacetic acid derivative and a phenoxypropionic acid derivative degrading enzyme as disclosed in WO 2008141154 or WO 2005107437. The resulting combination may also include multiple copies of any of the polynucleotides of interest.

Thus, the herbicide tolerant plants of the invention may be used in combination with herbicides to which they are tolerant. The herbicide may be applied to the plants of the invention using any technique known to those skilled in the art. The herbicide may be applied at any point during the plant cultivation process. For example, the herbicide may be applied prior to planting, at planting, prior to emergence, after emergence, or a combination thereof. The herbicide may be applied to the seeds and dried to form a layer on the seeds.

In some embodiments, the seed is treated with a safener, followed by application of the CESA-inhibiting herbicide after emergence. In one embodiment, the post-emergence application of the CESA inhibiting herbicide is about 7 to 10 days after planting the safener treated seed. In some embodiments, the safener is oxalic acid, dichloropropylamine, fluroxypyr, or a combination thereof.

Method for controlling weeds or undesirable vegetation

In other aspects, the invention provides methods for controlling weeds at a locus of growth of a plant or plant part thereof, the methods comprising applying to the locus a composition comprising a CESA-inhibiting herbicide.

In some aspects, the invention provides methods for controlling weeds at a locus where plants are growing comprising applying to the locus a herbicide composition comprising a CESA-inhibiting herbicide, wherein the locus is (a) a locus comprising a plant or seed capable of producing the plant, or (b) a locus comprising the plant or seed after said applying, wherein the plant or seed comprises in at least some cells thereof a polynucleotide operably linked to a promoter operable in plant cells, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant.

The herbicide compositions herein may be applied, for example, as leaf treatments, soil treatments, seed treatments, or soil drenches. The application may be performed, for example, by spraying, dusting, broadcasting, or any other pattern known to be useful in the art.

In one embodiment, the herbicide may be used to control the growth of weeds that may be found to grow in the vicinity of the herbicide tolerant plants of the invention. In this type of embodiment, the herbicide may be applied to plots where the herbicide tolerant plants of the invention are grown near weeds. The herbicide tolerated by the herbicide tolerant plants of the invention can then be applied to the plot at a concentration sufficient to kill or inhibit weed growth. Herbicide concentrations sufficient to kill or inhibit weed growth are known in the art and are disclosed hereinabove.

In other embodiments, the invention provides methods for controlling weeds in the vicinity of a CESA-inhibiting herbicide-tolerant plant of the invention. The method comprises applying an effective amount of a CESA-inhibiting herbicide to the weeds and to herbicide-tolerant plants, wherein the plants have increased tolerance to the CESA-inhibiting herbicide when compared to wild type plants. In some embodiments, the CESA-inhibiting herbicide-tolerant plants of the present invention are preferably crop plants, including but not limited to sunflower, alfalfa, brassica species, soybean, cotton, safflower, peanut, tobacco, tomato, potato, wheat, rice, maize, sorghum, barley, rye, millet, and sorghum.

In other aspects, one or more herbicides (e.g., CESA-inhibiting herbicides) can also be used as a seed treatment. In some embodiments, an effective concentration or effective amount of one or more herbicides, or a composition comprising an effective concentration or effective amount of one or more herbicides, may be applied directly to the seed prior to sowing the seed or during sowing. The seed treatment formulation may additionally comprise a binder and optionally a colorant.

A binder may be added to improve the adhesion of the treated active material to the seed. In one embodiment, suitable binders are block copolymer EO/PO surfactants, but also polyvinyl alcohol, polyvinylpyrrolidone, polyacrylate, polymethacrylate, polybutene, polyisobutylene, polystyrene, polyvinylamine, polyvinylamide, polyethylenimine (Lupasol (R), polymin (R)), polyethers, polyurethanes, polyvinyl acetate, tylose, and copolymers derived from these polymers. Optionally, colorants may also be included in the formulation. Suitable colorants or dyes for the seed treatment formulation are rhodamine B (c.i.), pigment red 112 (c.i.), solvent red 1, pigment blue 15:4, pigment blue 15:3, pigment blue 15:2, pigment blue 15:1, pigment blue 80, pigment yellow 1, pigment yellow 13, pigment red 112, pigment red 48:2, pigment red 48:1, pigment red 57:1, pigment red 53:1, pigment orange 43, pigment orange 34, pigment orange 5, pigment green 36, pigment green 7, pigment white 6, pigment brown 25, basic violet 10, basic violet 49, acid red 51, acid red 52, acid red 14, acid blue 9, acid yellow 23, basic red 10, basic red 108.

The term seed treatment includes all suitable seed treatment techniques known in the art, such as seed dressing, seed coating, seed dusting, seed soaking and seed pelleting. In one embodiment, the present invention provides a method of treating soil by applying a granular formulation (e.g., a granular formulation) containing a CESA-inhibiting herbicide, as a composition/formulation (e.g., a granular formulation) with optionally one or more solid or liquid, an agriculturally acceptable carrier, and/or optionally with one or more agriculturally acceptable surfactants, particularly into a planter. The method is advantageously used in seedbeds of cereals, maize, cotton and sunflower, for example.

The invention also comprises seeds coated with or containing a seed treatment formulation comprising a CESA-inhibiting herbicide and at least one other herbicide, such as, for example, an AHAS inhibitor selected from the group consisting of: amidosulfuron, tetrazole-sulfuron, bensulfuron-methyl, chlorimuron-ethyl, cinosulfuron-methyl, cyclosulfamuron, ethametsulfuron-methyl, ethoxysulfuron-methyl, flazasulfuron, fluflazasulfuron, formamidosulfuron halosulfuron-methyl, iodosulfuron-methyl, disulfonic acid, methylsulfuron-methyl, nicosulfuron, epoxy-azosulfuron-methyl, fluosulfuron-methyl, pyrazosulfuron-ethyl, rimsulfuron-methyl, methylsulfuron-methyl, sulfonyl sulfuron-methyl halosulfuron-methyl, iodosulfuron-methyl, disulfonic acid, metsulfuron-methyl, nicosulfuron, cyclosulfuron-methyl Fluosulfuron, pyrazosulfuron, rimsulfuron, sulfosulfuron, sulfosulfuron.

The term "coated with and/or containing" generally means that in most cases the active ingredient is on the surface of the propagation product at the time of application, although depending on the method of application, more or less of the ingredient may penetrate into the propagation product. When the propagation product is (re) planted, it may absorb the active ingredient.

In some embodiments, seed treatment application with a CESA-inhibiting herbicide or with a formulation comprising a CESA-inhibiting herbicide is performed by spraying or dusting the seeds prior to sowing the plants and prior to emergence of the plants.

In other embodiments, in seed treatment, the corresponding formulation is applied by treating the seed with an effective amount of a CESA-inhibiting herbicide or formulation comprising a CESA-inhibiting herbicide.

In other aspects, the invention provides methods for combating undesired vegetation or controlling weeds, comprising contacting seeds of a CESA-inhibiting herbicide-tolerant plant of the invention with a CESA-inhibiting herbicide prior to sowing and/or after pre-emergence. The method may further comprise sowing the seeds in soil, for example in a field, or in potting medium within a greenhouse. The method is particularly useful for combating undesirable vegetation or controlling weeds in close proximity to seeds. Controlling undesirable vegetation is understood to be killing weeds and/or otherwise retarding or inhibiting the normal growth of weeds. Weeds in the broadest sense are understood to mean all those plants which grow in locations where their emergence is undesirable.

Weeds of the invention include, for example, dicotyledonous weeds and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera Sinapis, lepidium (Lepidium), lala, chickweed, matricaria, achyranthes, chenopodium, urtica, senecio, amaranthus, portulaca, xanthium, inulae, ipomoea, polygonum, sesbania, ragweed, cirsium, fagopyrum, solanum, robinia, artemisia, matricaria, veronica, abutilon, horseradish, datura, viola, clematis, papaver, cornflower, axis, ranunculus, taraxacum. Monocotyledonous weeds include, but are not limited to, barnyard grass (Echinochloa), setaria (Setaria), broomcorn (Panicum), crabgrass (DIGITARIA), timothy (Phleum), bluegrass (Poa), festuca (Festuca), eleusine (Eleusine), brachyo (Brachiaria), lolium (Lolium), brome (Bromus), avena (Avena), sedge (Cyperus), sorghum (Sorghum), icy (Agropyron), bermuda (Cynodon), sedum (Monochoria), wampee (Fimbristyslis), arrowhead (SAGITTARIA), chufa (Eleocharis), scirpus (Scirpus), spartina (Paspalum), duckbill (Ischaemum), pointed california (Sphenoclea), festuca (Dactyloctenium), prunella (agrestis), seen (Alopecurus) and apricots (Apera).

In addition, weeds of the invention can comprise, for example, crop plants which grow at undesired locations. For example, if a maize plant is undesirable in a field of soybean plants, then the autogenous maize plant in the field containing primarily soybean plants may be considered weeds.

In still further aspects, the treatment of a locus, plant part or seed of the invention comprises applying an agronomically acceptable composition that does not contain a.i. In one embodiment, the treatment comprises applying an agronomically acceptable composition that does not contain CESA inhibiting herbicide a.i. In some embodiments, the treatment comprises applying an agronomically acceptable composition that does not contain CESA inhibiting herbicide a.l, wherein the composition comprises one or more of an agronomically acceptable carrier, diluent, excipient, plant growth regulator, and the like. In other embodiments, the treatment comprises applying an agronomically acceptable composition that does not contain a CESA inhibiting herbicide a.i., wherein the composition comprises an adjuvant. In one embodiment, the adjuvant is a surfactant, a spreading agent, an adhesive, a penetrant, a drift control agent, a crop oil, an emulsifier, a compatibilizer, or a combination thereof.

It should also be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The following examples further illustrate the invention and should not be construed as limiting its scope in any way. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAMPLE 1 identification of cellulose biosynthesis inhibitor (CBI; CESA inhibitor) resistant plants

To select cellulose biosynthesis inhibitor resistant plants, EMS mutagenized seed populations of Arabidopsis or scull plants are used. EMS-mutagenized seed populations were purchased from LEHLE SEEDS (Chamber D (1102South Industrial Blvd.Suite D,Round Rock,Texas USA) of southern Industrial Co., calif. of Texas) or generated as described elsewhere (Heim DR et al (1989) Plant Physiol [ Plant Physiol ] 90:146-150). Pathogenic mutations in CESA03 wild-type sequences (e.g., SEQ ID NO:3 or 28) are identified as described by Scheible WR et al (2001,Proc.Natl.Acad.of Sci. [ Proc. Natl. Acad. Sci. USA ] 98:10079-10084), mcCourt et al (WO 2013/142968), or using next generation sequencing methods as described by Austin RS et al (2011) Plant Journal [ J. Plant ]67:715-725 et al.

The selected arabidopsis lines were assayed for improved resistance to azines like the following in 48 well plates:

-6-cyclopentyl-N4- (2, 3,4,5, 6-pentafluorophenyl) -1,3, 5-triazine-2, 4-diamine;

-6- (1-fluoro-1-methyl-ethyl) -N4- (2, 3,4,5, 6-pentafluorophenyl) -1,3, 5-triazine-2, 4-diamine;

-6-cyclohexyl-N2- (2, 3,4,5, 6-pentafluorophenyl) -1,3, 5-triazine-2, 4-diamine;

-6- (2, 6-difluorophenyl) -N2- (2, 3,5, 6-tetrafluorophenyl) -1,3, 5-triazine-2, 4-diamine;

-N2- (4-chloro-3, 5, 6-trifluoro-2-pyridinyl) -6- (1-fluoro-1-methyl-ethyl) -1,3, 5-triazine-2, 4-diamine

- (1-Fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine.

Thus, M2 or M3 seeds were surface sterilized by stirring in ethanol+water (70+30 by volume) for 5min, rinsing once with ethanol+water (70+30 by volume), and rinsing twice with sterile deionized water. Seeds were resuspended in 0.1% agar (w/v) in water. Four to five seeds per well were plated on solid nutrient medium consisting of half strength Murashige Skoog nutrient solution (pH 5.8) (Murashige and Skoog (1962) Physiologia Plantarum [ plant physiology ] 15:473-497). The compound was dissolved in dimethyl sulfoxide (DMSO) and added to the medium before solidification (final DMSO concentration 0.1%). The multi-well plates were incubated in the growth chamber at 22 ℃, 75% relative humidity and 110 μmolPhot x m ^-2*s^-1 with 14:10h light for a dark photoperiod. Growth inhibition was assessed seven to ten days after sowing compared to wild type plants. Tolerance factors were calculated based on the IC50 values of growth inhibition of transformed (compared to untransformed or unmutated) arabidopsis plants.

In addition, in greenhouse studies, M3 Arabidopsis seeds were tested for improved tolerance to CESA-inhibiting herbicides with azine compounds like, for example, (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine.

Example 2 engineering azine tolerant Arabidopsis plants with wild-type or mutant cellulose synthase sequences.

For transformation of Arabidopsis thaliana, the wild-type or mutant cellulose synthase sequence encoding one of the following protein sequences SEQ ID NO:1 or 3 was cloned into a binary vector containing a resistance marker gene cassette (AHAS) and a mutant cellulose synthase sequence (labeled GOI) between the ubiquitin promoter (PcUbi) and nopaline synthase terminator (NOS) sequences using standard cloning techniques as described in Sambrook et al (Molecular cloning [ molecular cloning ] (2001) Cold Spring Harbor Laboratory Press [ Cold spring harbor laboratory Press ]). The binary plasmid was introduced into agrobacterium tumefaciens for plant transformation. Arabidopsis thaliana was transformed with wild-type or mutant cellulose synthase sequences by the floral dip method as described in McElver and Singh (WO 2008/124495). TaqMan assays were performed on transgenic Arabidopsis plants to analyze the number of integration loci.

The tolerance phenotype of transgenic T1 arabidopsis plants germinated on sand using azine herbicides was assessed 14-21 days post-germination. Improved tolerance of transgenic arabidopsis plants (T2) to azine herbicides like for example (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine was determined in 48-well plates. Thus, T2 seeds were surface sterilized by stirring in ethanol+water (70+30 by volume) for 5min, rinsing once with ethanol+water (70+30 by volume), and rinsing twice with sterile deionized water. Seeds were resuspended in 0.1% agar (w/v) in water. Four to five seeds per well were plated on solid nutrient medium consisting of half strength Murashige Skoog nutrient solution (pH 5.8) (Murashige and Skoog (1962) Physiologia Plantarum [ plant physiology ] 15:473-497). The compound was dissolved in dimethyl sulfoxide (DMSO) and added to the medium before solidification (final DMSO concentration 0.1%). The multi-well plates were incubated in the growth chamber at 22 ℃, 75% relative humidity and 110 μmol Phot x m ^-2*s^-1 with 14:10h light for a dark photoperiod. Growth inhibition was assessed seven to ten days after sowing compared to wild type plants. Tolerance factors were calculated based on the IC50 values of growth inhibition of transformed (compared to untransformed) arabidopsis plants.

Relative tolerance of transgenic arabidopsis plants compared to non-transgenic arabidopsis plants (non-transgenic = 1.0) with treatment with various cellulose biosynthesis inhibitors. Growth inhibition was assessed seven to ten days after sowing compared to wild type plants.

In the Arabidopsis germination assay, resistance to azine was also observed in stable Arabidopsis transformants over-expressing AtCESA S1037L/SEQ ID NO:3 (corresponding to S1051L in SEQ ID NO: 28; scull bean) (Table X)

Table X. Tolerance factors of Arabidopsis mutants of AtCESA S1037L (corresponding to S1051L in SEQ ID NO: 28; scull bean) were overexpressed in germination assays in the presence of both azines.

Example 3 identification of homologous cellulose synthase isoforms in crop plants

To identify homologous cellulose synthase genes from soybean, maize and rice, BLAST searches were performed using protein sequences of Arabidopsis cellulose synthase isoforms (Altschul et al (1990) J Mol Biol [ journal of molecular biology ] 215:403-10). Phylogenetic relationship of genes encoding cellulose synthase proteins from maize, soybean, rice, brassica napus and sunflower was analyzed by R software library phangorn (Schliep KP. (2011) Bioinformatics [ Bioinformatics ] 27:592-593). Bootstrap analysis was calculated for statistical validation of the monoclinic group. In addition, genes were classified according to their expression levels and expression patterns (Hruz T et al (2008) adv. In Bioinformatics [ Bioinformatics progression ] 2008:1-5) (fig. 1-3) and selected for plant transformation. For plant species not available in Geninvestigator, the genes were selected as follows. cDNA libraries, for example, of brassica napus seedlings were sequenced with one lane of Winner Corp (Illumina) Hiseq2000 using TruSeq SBS kit v3 (Illumina Inc. san Diego, calif. CA) FC-401-3001 at a 2X 100bp paired end run. Sequencing raw data was analyzed with FASTQC quality checker (Babraham Bioinformatics 2014), trimmed using EA-Utils fastq-mcf (https:// code. Google. Com/p/EA-Utils /), and further analyzed using CutAdapt codes (http:// code. Google. Com/p/cutadapt /) to remove any sequence of adaptors from the company. For analysis of the relative expression of the cellulose synthase gene in, for example, brassica napus, the readings were plotted using TopHat2 (Kim et al 2013) and counted using HTseq counts (Anders et al 2015).

Example 4 tissue culture conditions.

In vitro tissue culture mutagenesis assays were developed to isolate and characterize plant tissues (e.g., corn, rice) that are tolerant to cellulose synthase-inhibiting herbicides (e.g., (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine, and diuron, a photosynthesis inhibitor, as a negative control). The assay exploits the clonal variation of somatic cells found in vitro tissue culture. Spontaneous mutations derived from the clonal variation of the somatic cells can be enhanced by chemical mutagenesis (e.g., ethyl methylsulfonate, ethyl ethylsulfonate, N-nitroso-N-ethylurea, ethylnitrosourea, nitrous acid, bromouracil, 2-aminopurine, 5-fluorodeoxyuridine, hydroxylamine, N-methyl-N' -nitro-N-nitrosoguanidine) and subsequent selection in a stepwise manner based on increasing concentrations of herbicide.

The present invention provides tissue culture conditions that promote the growth of regenerable friable embryogenic maize or rice calli. Callus was initiated from 4 different maize or rice cultivars, covering maize as well as Japonica (Japonica) (north of the platform 309, nipponbare, polished rice (Koshihikari)) and Indica (Indica 1) varieties, respectively. The seeds were surface sterilized in 70% ethanol for about 1min, followed by 20 min with 20% commercial Clorox bleach. The seeds were rinsed with sterile water and spread on callus induction medium. Various callus induction media were tested. Table E4-1 lists the ingredients of the media tested.

Table E4-1

R001M callus induction medium was selected after testing for multiple changes. Cultures were kept in the dark at 30 ℃. Embryogenic callus was subcultured to fresh medium after 10-14 days.

Example 5 selection of azine-herbicide tolerant calli.

Once the tissue culture conditions are determined, a further establishment of selection conditions is established by analyzing the tissue viability in the killing curves of azine herbicides such as, for example, (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine and the photosynthesis inhibitor diuron as a negative control. Careful consideration is given to the accumulation of herbicide in the tissue and its persistence and stability in cells and culture media. Through these experiments, a sub-lethal dose for the initial selection of mutant material was established. After establishing an initial dose of azine herbicides like for example (1-fluoro-1-methyl-ethyl) -N4- (2, 3, 5-trifluoro-6-methoxy-phenyl) -1,3, 5-triazine-2, 4-diamine and the photosynthesis inhibitor diuron as negative control, the tissue is selected in a stepwise manner by increasing the concentration of the cellulose synthase inhibitor at each transfer until recovering cells that grew in the presence of toxic doses. The resulting calli were further sub-cultured every 3-4 weeks to R001M containing selection agent. Selection of over 26,000 calli was performed for 4-5 subcultures until the selection pressure was above the toxicity level determined by the kill curve and the observation of continuous culture. Alternatively, liquid culture was started from calli in MS711R, slowly shaken, and subcultured weekly. Once liquid culture was established, the selection agent was added directly to the flask at each subculture. After 2-4 rounds of liquid selection, the cultures were transferred to a filter on solid R001M medium for further growth.

Example 6 regeneration of plants.

Resistant tissues are regenerated and molecularly characterized for cellulose synthase gene sequence mutations. In addition, genes that are directly and/or indirectly involved in cell wall biosynthesis and/or metabolic pathways are also sequenced to characterize the mutations. Finally, enzymes that alter fate (e.g., metabolism, translocation, transport) are also sequenced to characterize mutations. Following herbicide selection, calli were regenerated using a medium protocol of R025M for 10-14 days, R026M for approximately 2 weeks, R327M until well formed shoots appeared, and R008S until shoots rooted well for transfer to the greenhouse. Regeneration is performed under illumination. No selection agent is included during regeneration. Once strong roots are established, M0 regenerated adults are transplanted into square or round pots (pot) in the greenhouse. The grafts were kept under transparent plastic cups until they were adapted to greenhouse conditions. The day/night cycle of the greenhouse was set to 27/21 (80/70) with 600W high pressure sodium lamp supplemental lighting to maintain a 14 hour day length. Depending on the weather, plants are watered as needed and fertilized daily.

Example 7 sequence analysis.

Leaf tissue was collected from clonal plants isolated for transplantation and analyzed as individuals. As directed by the manufacturer, useGenomic DNA was extracted from 96 magnetic DNA plant systems kit (Promega, U.S. Pat. Nos. 6,027,945 and 6,368,800). The isolated DNA was PCR amplified using appropriate forward and reverse primers.

PCR amplification was performed using Hotstar Taq DNA polymerase (Qiagen), using a drop-down thermal cycling program (touchdown thermocycling program) for 15min at 96℃followed by 35 cycles (96℃30 seconds; 58℃to 0.2℃per cycle, 30 seconds; 72℃for 3min and 30 seconds) at 72℃for 10min. The concentration of the PCR products and the fragment size were verified by agarose gel electrophoresis. The dephosphorylated PCR products were analyzed by direct sequencing using PCR primers (DNA LANDMARKS, inc.). Mutations of the chromatogram trace file (.scf) relative to the wild-type gene were analyzed using Vector NTI ADVANCE, ^TM (Invitrogen). Based on the sequence information, mutations were identified in several individuals. Sequence analysis was performed on the representative chromatograms, corresponding AlignX alignment was performed at default settings and edited to invoke secondary peaks.

Example 8 soybean conversion and cellulose biosynthesis inhibitor tolerance test.

Binary vectors were generated as described in example 2. Soybean cultivars Jake (cv Jake) were transformed as previously described by Siminszky et al, phytochem Rev [ phytochemical review ]5:445-458 (2006). After regeneration, the transformants were transplanted into soil of a small pot, placed in a growth chamber (16 hours day/8 hours night; 25 ℃ day/23 ℃ night; 65% relative humidity; 130-150microE m-2 s-1), and then tested for the presence of T-DNA via Taqman analysis. After a few weeks, healthy, transgenic positive, single copy events were transplanted into larger pots and allowed to grow in the growth chamber. The optimal shoot height for cutting is about 3-4 inches, with at least two knots. Each cuttings was taken from the original transformant (female parent plant) and immersed in rooting hormone powder (indole-3-butyric acid, IBA). The cuttings are then placed in the oasis wedge within the biological dome. Mature female parent plants were harvested in the greenhouse and seeds harvested. Wild-type cuttings were also taken as negative controls. The cuttings are kept in the biological dome for 5-7 days. 3-4 days after transferring to the flower mud wedge, the buds are treated with herbicide through nutrient solution. Typical phytotoxic symptoms (like club roots) were evaluated 3-4 days after treatment. Less or no damage to the transgenic plant compared to the wild type plant is interpreted as herbicide tolerance.

Example 9 engineering cellulose biosynthesis inhibitor tolerant maize or rice plants with mutant cellulose synthase sequences.

Immature embryos can be transformed according to the procedure outlined in Peng et al (WO 2006/136596). Plants were tested for the presence of T-DNA by Taqman analysis, where the target was the nos terminator present in all constructs. The seemingly healthy plants were sent to a greenhouse for hardening and subsequent spray testing. Plants were individually transplanted into 4 pot metamix 360 soil. Once in the greenhouse (day/night period of 27/21 ℃ with day length of 14 hours supported by 600W high pressure sodium lamps), they were allowed to grow for 14 days. Transformation of rice (Oryza sativa, rice) was performed by protoplast transformation as described in Peng et al (U.S. 6653529). Transgenic maize and rice plants were grown to T1 seeds for herbicide tolerance testing.

EXAMPLE 11 demonstration of herbicide tolerance

In greenhouse studies and mini-plot studies using azine herbicides, soybean, maize, rice, sunflower and brassica napus T0 or T1 transgenic plants containing a cellulose synthase sequence or mutant gene variants thereof were tested for improved tolerance to herbicides. For pre-emergence treatments, the herbicide is applied directly after sowing through finely distributed nozzles. The containers were irrigated gently to promote germination and growth and then covered with a clear plastic cover until the plants rooted. This coverage resulted in uniform germination of the test plants unless this was damaged by the herbicide. For post-emergence treatment, the test plants were first grown to a height of 3 to 15cm, depending on the plant habit, and were treated with herbicide only at this point. For this purpose, the test plants are either directly sown and grown in the same containers, or they are first grown separately and transplanted into the test containers a few days before treatment.

Herbicide damage assessment was performed 2 weeks and 3 weeks after treatment. Plant injury ratings were 0% to 100%,0% indicating no injury, and 100% indicating complete death.

Example 12 Soy hairy root assay

Explants were prepared from five-day old seedlings and transformed with agrobacterium containing constructs with the desired transgene and selectable markers. Five days after co-cultivation, explants were selected on Arsenal to select for successfully transformed explants. Successfully transformed seedlings will produce hairy roots when grown on media with selection. Root cuttings were then transferred to azine selection for 10-14 days, and root growth was observed as a measure of azine tolerance. Roots showing significant growth were considered tolerant to azines, while untransformed WT seedlings did not show any additional root growth upon azine selection.

In Arabidopsis, the missense mutation S1037F in the CESA3 gene (At 5G 05170) confers tolerance to CBI flumetsulam (Shim et al 2018). To test whether a similar position in soybean CESA3 (Glyma.12G237000; S1051) confers resistance to internal azine chemistry, eighteen alternative amino acid residues were tested at this position in the soybean hairy root assay. When grown on azine selection, transgenic root cuttings over-expressing one of the three missense mutations in CESA3 (S1051L, S1051M and S1051T) showed significant root growth (error | inability to find the reference source).

Stable soybean transformants overexpressing three missense mutations in CESA3 were generated and then tested for azine tolerance in a greenhouse assay. Ten events were generated for each of the three missense mutations, and tolerance to both azine compounds was screened at the T1 generation. The results show a pronounced resistance to at least one azine chemistry in some events compared to the wild type (error | failure to find the reference source).

Claims

1. A plant or plant part comprising a polynucleotide encoding a mutant cellulose synthase (CESA) polypeptide whose expression confers tolerance to a CESA-inhibiting herbicide on the plant or plant part, wherein the mutant CESA polypeptide is characterized by substitution of an amino acid at a position corresponding to position 1051 of SEQ ID No. 28 with Leu, met or Thr.

2. The plant or plant part of any one of claims 1 to 2, wherein the mutant CESA polypeptide is a functional variant having at least about 60%, illustratively at least about 80%, 90%, 95%, 98%, 99% or greater amino acid sequence identity to SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82 or 83 over the full length of the variant.

3. A seed capable of germinating into a plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the mutant CESA polypeptide is characterized by substitution of the amino acid at a position corresponding to position 1051 of SEQ ID No. 28 with Leu, met or Thr.

4. A plant cell of a plant or a plant cell capable of regenerating into a plant, the plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in the plant cell, the promoter capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, the expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the plant cell comprises a polynucleotide operably linked to a promoter, and wherein the mutant CESA polypeptide is characterized in that the amino acid at the position corresponding to position 1051 of SEQ ID No. 28 is substituted with Leu, met or Thr.

5. A plant cell comprising a polynucleotide operably linked to a promoter operable in the cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the mutant CESA polypeptide is characterized by substitution of an amino acid at a position corresponding to position 1051 of SEQ ID No. 28 with Leu, met or Thr.

6. A progeny or descendent plant derived from a plant comprising in at least some cells thereof a polynucleotide operably linked to a promoter operable in a plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, wherein the progeny or descendent plant comprises in at least some cells thereof a polynucleotide operably linked to the promoter, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the progeny or descendent plant, wherein the mutant CESA polypeptide is characterized by substitution of an amino acid at a position corresponding to position 1051 of SEQ ID NO:28 with Leu, met or Thr.

7. A method for controlling weeds at a locus where plants are growing comprising (a) applying to the locus a herbicide composition comprising a CESA-inhibiting herbicide, and (b) planting a seed at the locus, wherein the seed is capable of producing a plant which comprises in at least some cells thereof a polynucleotide operably linked to a promoter operable in plant cells, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant, wherein the mutant CESA polypeptide is characterized in that the amino acid at the position corresponding to position 1051 of SEQ ID NO 28 is substituted with Leu, met or Thr.

8. The method of claim 7, wherein a herbicide composition is applied to the weeds and to the plants produced from the seed.

9. A method of producing a plant that is tolerant to a CESA-inhibiting herbicide, the method comprising regenerating a plant from a plant cell transformed with a recombinant polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to a CESA-inhibiting herbicide to the plant, wherein the mutant CESA polypeptide is characterized by substitution of an amino acid at a position corresponding to position 1051 of SEQ ID NO:28 with Leu, met or Thr.

10. A method of producing a progeny plant that is tolerant to a CESA-inhibiting herbicide, the method comprising crossing a first CESA-inhibiting herbicide tolerant plant with a second plant to produce a CESA-inhibiting herbicide tolerant progeny plant, wherein the first plant and the progeny plant comprise in at least some cells thereof a polynucleotide operably linked to a promoter operable in the plant cell, the promoter being capable of expressing a mutant CESA polypeptide encoded by the polynucleotide, expression of the mutant CESA polypeptide conferring tolerance to the CESA-inhibiting herbicide to the plant.

11. A method for producing a plant product from the plant of claim 1, the method comprising processing the plant or plant part thereof to obtain the plant product.

12. The method of claim 11, wherein the plant product is feed, seed meal, oil, or seed coated with a seed treatment.

13. A plant product obtained from the plant of claim 1, wherein the plant or plant part further exhibits a second or third herbicide tolerance trait.

14. The plant product of claim 13, wherein the product is a feed, a seed meal, an oil, or a seed coated with a seed treatment.