Title of Invention

NUCLEIC ACIDS ENCODING IMIDIZOLINONE - TOLERANT AHAS ENZYMES

Abstract The present invention is directed to nucleic acids encoding polypeptides that confer upon a plant tolerance to an imidazolinone and/or other acetohydroxyacid synthase (AHAS) inhibiting herbicide when expressed in the plant. The present invention also provides plants having increased tolerance to an imidazolinone and/or other AHAS-inhibiting herbicide. More particularly, the present invention includes plants containing at least one IMI nucleic acid. The present invention also includes seeds produced by these plants and methods of controlling weeds in the vicinity of these wheat plants.
Full Text

NOVEL MUTATION INVOLVED IN INCREASED TOLERANCE TO IMIDAZOLINONE HERBICIDES IN PLANTS
FIELD OF THE INVENTION
[0001] The present invention relates in general to plants having an increased
tolerance to imidazolinone herbicides. More specifically, the present invention relates to plants obtained by mutagenesis, cross-breeding, and transformation that have an increased tolerance to imidazolinone herbicides.
BACKGROUND OF THE INVENTION
[0002] Acetohydroxyacid synthase (AHAS; EC 4.1.3.18; acetolactate synthase
(ALS)), encoded by the Als nucleic acid, is the first enzyme that catalyzes the biochemical synthesis of the branched chain amino acids valine, leucine, and isoleucine (Singh B. K., 1999, Biosynthesis of valine, leucine and isoleucine in: Singh B. K. (Ed) Plant amino acids. Marcel Dekker Inc. New York, New York. Pg 227-247). AHAS is the site of action of four structurally diverse herbicide families including the sulfonylureas (LaRossa RA and Falco SC, 1984, Trends Biotechnol. 2:158-161), the imidazolinones (Shaner et aL, 1984, Plant Physiol. 76:545-546), the triazolopyrimidines (Subramanian and Gerwick, 1989, Inhibition of acetolactate synthase by triazolopyrimidines in (ed) Whitaker JR, Sonnet PE Biocatalysis in agricultural biotechnology. ACS Symposium Series, American Chemical Society. Washington, D.C. Pg 277-288), and the pyrimidyloxybenzoates (Subramanian et aL, 1990, Plant Physiol. 94: 239-244.). Imidazolinone and sulfonylurea herbicides are widely used in modern agriculture due to their effectiveness at very low application rates and relative non-toxicity in animals. By inhibiting AHAS activity, these families of herbicides prevent further growth and development of susceptible plants including many weed species. Several examples of commercially available imidazolinone herbicides are PURSUIT® (imazethapyr), SCEPTER® (imazaquin) and ARSENAL® (imazapyr). Examples of sulfonylurea herbicides are chlorsulfiiron, metsulfirron methyl, sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl, nicosulfcron, ethametsulfuron methyl, rimsulfuron, triflusulforon methyl, triasulfuron, primisulfuron methyl, cinosulfuron, amidosulfuron, fluzasulforon, imazosulforon, pyrazosulfuron ethyl, and halosulfuron.

[0003] Due to their high effectiveness and low toxicity, imidazolinone
herbicides are favored for application by spraying over the top of a wide area of vegetation. The ability to spray an herbicide over the top of a wide range of vegetation decreases the costs associated with plantation establishment and maintenance, and decreases the need for site preparation prior to use of such chemicals. Spraying over the top of a desired tolerant species also results in the ability to achieve Tnqxiimnn yield potential of the desired species due to the absence of competitive species. However, the ability to use such spray-over techniques is dependent upon the presence of imidazolinone tolerant species of the desired vegetation in the spray over area.
[0004] Among the major agricultural crops, some leguminous species such as
soybean are naturally tolerant to imidazolinone herbicides due to their ability to
rapidly metabolize the herbicide compounds (Shaner and Robson, 1985, Weed Sci.
33:469-471). Other crops such as com (Newhouse et ai, 1992, Plant Physiol.
100:882-886) and rice (Barrett et al.,1989, Crop Safeners for Herbicides, Academic
Press New York, pp. 195-220) are susceptible to imidazolinone herbicides. The
differential sensitivity to the imidazolinone herbicides is dependent on the chemical
nature of the particular herbicide and differential metabolism of the compound from a
toxic to a non-toxic form in each plant (Shaner et aL, 1984, Plant Physiol. 76:545-
546; Brown et aL, 1987, Pestic. Biochem. Physiol. 27:24-29). Other plant
physiological differences such as absorption and translocation also play an important
role in sensitivity (Shaner and Robson, 1985, Weed Sci. 33:469-471).
[0005] Crop cultivars tolerant to imidazolinones, sulfonylureas, and
triazolopyrimidines have been successfully produced using seed, microspore, pollen, and callus mutagenesis in Zea mays, Brassica napus, Glycine max, and Nicotiana tabacum (Sebastian et al., 1989, Crop Sci. 29:1403-1408; Swanson et aL, 1989, Theor. Appl. Genet. 78:525-530; Newhouse et al., 1991, Theor. Appl. Genet. 83:65-70; Sathasivan et al., 1991, Plant Physiol. 97:1044-1050; Mourand et al., 1993, J. Heredity 84:91-96). In all cases, a single, partially dominant nuclear gene conferred tolerance. Four imidazolinone tolerant wheat plants were also previously isolated following seed mutagenesis of Triticum aestivum L. cv Fidel (Newhouse et al., 1992, Plant Physiol. 100:882-886). Inheritance studies confirmed that a single, partially dominant gene conferred tolerance. Based on allelic studies, the authors concluded that the mutations in the four identified lines were located at the same locus. One of

the Fidel cultivar tolerance genes was designated FS-4 (Newhouse et al., 1992, Plant Physiol. 100:882-886).
[0006] Computer-based modeling of the three dimensional conformation of
the AHAS-inhibitor complex predicts several amino acids in the proposed inhibitor binding pocket as sites where induced mutations would likely confer selective tolerance to imidazolinones (Ott et al., 1996, J. MoL Biol. 263:359-368). Tobacco plants produced with some of these rationally designed mutations in the proposed binding sites of die AHAS enzyme have in feet exhibited specific tolerance to a single class of herbicides (Ott et al., 1996, J. MoL Biol. 263:359-368). [0007] ant tolerance to imidazolinone herbicides has also been reported in a number of patents. U.S. Patent Nos. 4,761,373, 5,331,107, 5,304,732, 6,211,438, 6,211,439, and 6,222,100 generally describe the use of an altered Als nucleic acid to elicit herbicide tolerance in plants, and specifically disclose certain imidazolinone tolerant com lines. U.S. Patent No. 5,013,659 discloses plants exhibiting herbicide tolerance possessing mutations in at least one amino acid in one or more conserved regions. The mutations described therein encode either cross-tolerance for imidazolinones and sulfonylureas or sulfonylurea-specific tolerance, but imidazolinone-specific tolerance is not described. Additionally, U.S. Patent No. 5,731,180 and U.S. Patent No. 5,767,361 discuss an isolated gene having a single amino acid substitution in a wild-type monocot AHAS amino acid sequence that results in imidazolinone-specific tolerance.
[0008] To date, the prior art has not described mutations in the Als J gene that
confer increased tolerance to an imidazolinone herbicide other than the mutation in die FS-4 imidazolinone tolerant line. Nor has the prior art described imidazolinone tolerant wheat or triticale plants comprising at least one altered Als nucleic acid from a Triticum aestivum Shiloh cultivar. Therefore, what is needed in the art is the identification of additional mutations that confer tolerance to imidazolinone herbicides. What are also needed in the art are wheat plants and triticale plants having increased tolerance to herbicides such as imidazolinone and containing at least one altered Als nucleic acid. Also needed are methods for controlling weed growth in the vicinity of such wheat plants and triticale plants. These compositions and methods would allow for the use of spray over techniques when applying herbicides to areas containing wheat plants and triticale plants.

SUMMARY OF THE INVENTION
[0009] The present invention provides wheat plants comprising IMI nucleic
acids, wherein the wheat plant has increased tolerance to an imidazolinone herbicide
as compared to a wild-type plant. The wheat plants can contain one, two, three, or
more IMI alleles. In one embodiment, the wheat plant comprises at least one IMI
nucleic acid. In another embodiment, the at least one IMI nucleic acid is an Imil
nucleic acid. In another embodiment, the at least one IMI nucleic acid comprises a
Triticum aestivum IMI nucleic acid. In another embodiment, the at least one IMI
nucleic acid comprises a Shiloh cultivar Dvll nucleic acid, hi yet another embodiment,
the whe^ptai1T5bmprises multiple MI nucleic acids located on different genomes.
In another embodiment, the multiple IMI nucleic acids comprise a Triticum aestivum
Shiloh cultivar Imil nucleic acid. Preferably, the Shiloh cultivar Imil nucleic acid
encodes a protein comprising a mutation in a conserved amino acid sequence selected
from the group consisting of a Domain A, a Domain B, a Domain C, a Domain D, and
a Domain E. More preferably, the mutation is in a conserved Domain C. Also
provided are plant parts and plant seeds derived from the wheat plants described
herein.
[0010] The present invention also provides triticale plants comprising IMI
nucleic acids, wherein the triticale plant has increased tolerance to an imidazolinone
herbicide as compared to a wild-type triticale plant In one embodiment, the triticale
plant comprises at least one IMI nucleic acid. In another embodiment, the at least one
IMI nucleic acid is an Imil nucleic acid. In another embodiment, the at least one IMI
nucleic acid comprises a Triticum aestivum Shiloh cultivar IMI nucleic acid. In
another embodiment, the wheat plant comprises multiple IMI nucleic acids located on
different genomes. In yet another embodiment, the multiple IMI nucleic acids
comprise a Shiloh cultivar Imil nucleic acid. In another embodiment, the IMI nucleic
acids encode proteins comprising a mutation in a conserved amino acid sequence
selected from the group consisting of a Domain A, a Domain B, a Domain C, a
Domain D, and a Domain E. More preferably, the mutation is in a conserved Domain
C. Even more preferably, the mutation encodes a polypeptide having an alanine to
threonine substitution at position 96 of the ALS polypeptide. Also provided are plant
parts and plant seeds derived from the triticale plants described herein.
[0011] The IMI nucleic acids of the present invention can comprise a
polynucleotide sequence selected from the group consisting of: a polynucleotide as

defined in SEQ ID N0:1; a polynucleotide sequence encoding a polypeptide as
defined in SEQ ID NO:2; a polynucleotide comprising at least 60 consecutive
nucleotides of any of the aforementioned polynucleotides; and a polynucleotide
complementary to any of the aforementioned polynucleotides.
[0012] The plants of the present invention can be transgenic or non-
transgenic. An example of a non-transgenic wheat plant having increased tolerance to an imidazolinone herbicide is the wheat plant having an ATCC Patent Deposit Designation Number PTA-5625; or a mutant, recombinant, or genetically engineered derivative of the plant with ATCC Patent Deposit Designation Number PTA-5625; or any prdph^trf1E& plant with ATCC Patent Deposit Designation Number PTA-5625; or a plant that is a progeny of any of these plants.
[0013] In addition to the compositions of the present invention, several
methods are provided- Described herein are methods of modifying a plant's tolerance to an imidazolinone herbicide comprising modifying the expression of an IMI nucleic acid in the plant. Also described are methods of producing a transgenic plant having increased tolerance to an imidazolinone herbicide comprising, transforming a plant cell with an expression vector comprising one or more IMI nucleic acids and generating the plant from the plant cell. The invention further includes a method of controlling weeds within the vicinity of a wheat or triticale plant, comprising applying an imidazolinone herbicide to the weeds and to the wheat or triticale plant, wherein the wheat or triticale plant has increased tolerance to the imidazolinone herbicide as compared to a wild type wheat or triticale plant and wherein the plant comprises one or more IMI nucleic acids. In some preferred embodiments of these methods, the plants comprise multiple IMI nucleic acids that are located on different wheat genomes.
[0014] Also provided are expression cassettes, transformation vectors,
transformed non-human host cells, and transformed plants, plant cells, plant parts, and seeds that comprise one or more the IMI nucleic acids of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 shows an alignment of the cDNA sequence of the Shiloh-8
hnil nucleic acid (SEQ ID NO:l), the wild type Alsl nucleic acid (SEQ ID NO:3), and a consensus nucleic acid sequence (SEQ ID NO:5). The base pair substituted in the Imil sequence is indicated in bold.

[0016] Figure 2 shows an alignment of the deduced amino acid sequence of
the Shiloh-8IMIIl polypeptide (SEQ ID NO:2), a wild type ALS1 polypeptide (SEQ ID NO:4)? and a consensus amino acid sequence (SEQ ID NO:6). The amino acid substituted in the IMI1 sequence is indicated in bold.
[0017] Figure 3 is a schematic representation of the conserved amino acid
sequences in the AHAS genes implicated in tolerance to various AHAS inhibitors. The specific amino acid site responsible for tolerance is indicated by an underline. (Modified from Devine, M. D. and Eberlein, C. V., 1997, Physiological, biochemical, and molecular aspects of herbicide tolerance based on altered target sites in Herbicide Activi Toxicity Biochemistry, and Molecular Biology, IOS Press Amersterdam, p. 159-185).
DETAILED DESCRIPTION
[0018] The present invention is directed to isolated nucleic acids encoding
polypeptides that confer increased tolerance to an imidazolinone herbicide when
expressed in a plant. The present invention is also directed to wheat or triticale plants,
wheat or triticale plant parts, and wheat or triticale plant cells having increased
tolerance to imidazolinone herbicides. The present invention also includes seeds
produced by the wheat or triticale plants described herein and methods for controlling
weeds in the vicinity of the wheat or triticale plants described herein.
[0019] As used herein, the term "wheat plant" refers to a plant that is a
member of the Triticum genus. The wheat plants of the present invention can be members of a Triticum genus including, but not limited to, T. aestivum, T. turgidum, T. timopheevii, T monococcum, T. zhukovskyi, and T urartu> and hybrids thereof. Examples of T aestivum subspecies included within the present invention are aestivum (common wheat), compaction (club wheat), macha (macha wheat), vavilovi (vavilovi wheat), spelta and sphaecrococcum (shot wheat). Examples of T turgidum subspecies included within the present invention are turgidum, carthlicum, dicoccom, durum, paleocolchicum, polonicum, turanicum, and dicoccoides. Examples of T. monococcum subspecies included within the present invention are monococcum (einkorn) and aegilopoides. In one embodiment of the present invention, the wheat plant is a member of the Triticum aestivum L. species, and more particularly, a Shiloh cultivar.

[0020] The term "wheat plant" is intended to encompass wheat plants at any
stage of maturity or development, as well as any tissues or organs (plant parts) taken or derived from any such plant unless otherwise clearly indicated by context. Plant parts include, but are not limited to, stems, roots, flowers, ovules, stamens, leaves, embryos, meristematic regions, callus tissue, anther cultures, gametophytes, sporophytes, pollen, microspores, protoplasts, and the like. The present invention also includes seeds produced by the wheat plants of the present invention. In one embodiment, the seeds are true breeding for an increased tolerance to an imidazolinone herbicide as compared to a wild type wheat plant seed. [0021]— %- ^Hie present invention also encompasses triticale plants, triticale plant parts, and triticale plant cells having increased tolerance to imidazolinone herbicides. As used herein, a 'triticale plant" refers to a plant that is created by crossing a rye plant (Secale cereale) with either a tetraploid wheat plant (e.g. Triticum turgidum) or a hexaploid wheat plant (e.g. Triticum aestivum). The present invention also includes seeds produced by the triticale plants described herein and methods for controlling weeds in the vicinity of the triticale plants described herein.
[0022] The present invention describes a wheat plant comprising at least one
IMI nucleic acid, wherein the wheat plant has increased tolerance to an imidazolinone herbicide as compared to a wild-type plant It is possible for the wheat plants of the present invention to have multiple IMI nucleic acids from different genomes since these plants can contain more than one genome. For example, a Triticum aestivum wheat plant contains three genomes referred to as the A, B, and D genomes. Because ABAS is a required metabolic enzyme, it is assumed that each genome has at least one gene coding for the AHAS enzyme (Le. at least one Als gene), commonly seen with other metabolic enzymes in hexaploid wheat that have been mapped. As used herein, the term "Als gene locus" refers to the position of an Als gene on a genome, and the terms "Als gene" and "Als nucleic acid" refer to a nucleic acid encoding the AHAS enzyme. The Als nucleic acid on each genome differs in its nucleotide sequence from an Als nucleic acid on another genome. One of skill in the art can determine the genome of origin of each Als nucleic acid through genetic crossing and/or either sequencing methods or exonuclease digestion methods known to those of skill in the art. As used herein, the terms "Alsl nucleic acid," "Als2 nucleic acid," and "Als3 nucleic acid" refer to Als nucleic acids located on three different genomes. For the purposes of this invention, the Als3 gene locus is located on the A genome,

the Als2 gene locus is located on the B genome, and the Alsl gene locus is located on the D genome. Also for the purposes of this invention, 1MI nucleic acids derived from the A, B, or D genomes are distinguished and designated as Imi3, Imi2, or Imil nucleic acids, respectively.
[0023] As used herein, the term "IMI nucleic acid" refers to an Als nucleic
acid having a sequence that is mutated from a wild type Als nucleic acid and that
confers increased imidazolinone tolerance to a plant in which it is expressed. As used
herein, the terms "Imil nucleic acid," "Imi2 nucleic acid," and "hni3 nucleic acid" are
IMI nucleic acids that refer to the imidazolinone tolerance alleles of the Alsl, Als2,
and Afe5*gto^Ti^spectively. Because wheat plants have two copies of each genome,
a wheat plant contains two copies of each particular Als nucleic acid. For example, a
Triticum aestivum wheat plant comprises two copies each of the A, B, and D
genomes, and therefore, two copies each of the Als3, Als2, and Alsl genes. As used
herein, the term "IMI allele" refers to a single copy of a particular IMI nucleic acid.
Accordingly, for the purposes of the present invention, a wheat plant may have two
Imil alleles, one on each of two copies of the D genome.
[0024] hi another embodiment, the wheat plant comprises multiple IMI
nucleic acids. As used herein, when describing a plant that comprises "multiple IMI nucleic acids," the phrase "multiple IMI nucleic acids" refers to the presence of different IMI nucleic acids in the plant and not to whether the plant is homozygous or heterozygous at a particular Als locus. For example, a plant comprising multiple IMI nucleic acids may comprise an Imil and an hni2 nucleic acid, as opposed to having two copies of an Imil nucleic acid
[0025] The Imil class of nucleic acids includes the FS-4 gene as described by
Newhouse et al. (1992 Plant Physiol. 100:882-886) and the Shiloh-8 gene described in more detail below. Each class can include members from different wheat species. Therefore, each Imi class includes IMI nucleic acids that differ in their nucleotide sequence but that are nevertheless designated as originating from, or being located on, the same wheat genome using inheritance studies as known to those of ordinary skill in the art.
[0026] Accordingly, the present invention includes a wheat plant comprising
at least one IMI nucleic acid, wherein the wheat plant has increased tolerance to an imidazolinone herbicide as compared to a wild-type plant and wherein the at least one IMI nucleic acid is an Imil nucleic acid. In a preferred embodiment, the Imil nucleic

acid comprises the polynucleotide sequence shown in SEQ ID NO: 1. In another
preferred embodiment, the wheat plant comprises multiple IMI nucleic acids.
[0027] The present invention also encompasses an imidazolinone tolerant
triticale plant. As used herein, a t4triticale plant" refers to a plant that is created by
crossing a rye plant (Secale cereale) with either a tetraploid wheat plant (e.g. Triticum
turgidum) or a hexaploid wheat plant (e.g. Triticum aestivuni). For the purposes of
the present invention, an inridazolinone tolerant triticale plant comprises at least one
IMI nucleic acid, wherein the triticale plant has increased tolerance to an
imidazolinone herbicide as compared to a wild-type plant and wherein the at least one
IMI nucleic acid is an Imil nucleic acid. In a preferred embodiment, the Imil nucleic
acid comprises the polynucleotide sequence of SEQ ID NO:l. In another preferred
embodiment, the triticale plant comprises multiple IMI nucleic acids.
[0028] As used herein with regard to nucleic acids, the term "from" refers to a
nucleic acid "located on" or "derived from" a particular genome. The term "located on" refers to a nucleic acid contained within that particular genome. As also used herein with regard to a genome, the term "derived from" refers to a nucleic acid that has been removed or isolated from that genome. The term "isolated" is defined in more detail below.
[0029] The present invention includes wheat plants comprising one, two,
three, or more IMI alleles, wherein the wheat plant has increased tolerance to an
imidazolinone herbicide as compared to a wild-type plant. The IMI alleles can
comprise a nucleotide sequence selected from the group consisting of a
polynucleotide as defined in SEQ ID NO:l; a polynucleotide encoding a polypeptide
as defined in SEQ ID NO:2; a polynucleotide comprising at least 60 consecutive
nucleotides of any of the aforementioned polynucleotides; and a polynucleotide
complementary to any of the aforementioned polynucleotides.
[0030] The present invention also includes triticale plants comprising one,
two, three, or more IMI alleles, wherein the triticale plant has increased tolerance to an imidazolinone herbicide as compared to a wild-type plant. The IMI alleles can comprise a polynucleotide sequence selected from the group consisting of a polynucleotide as defined in SEQ ID NO:l; a polynucleotide encoding a polypeptide as defined in SEQ ID NO:2; a polynucleotide comprising at least 60 consecutive nucleotides of any of the aforementioned polynucleotides; and a polynucleotide complementary to any of the aforementioned polynucleotides.

[0031] In one embodiment, the wheat plant or triticale plant comprises two
different IMI nucleic acids. Preferably, at least one of the two nucleic acids is an Imil nucleic acid. More preferably, at least one of the two IMI nucleic acids comprises the polynucleotide sequence of SEQ ID NO:l. In another embodiment, the wheat plant or triticale plant comprises one IMI nucleic acid, wherein the nucleic acid comprises the polynucleotide sequence of SEQ ID NO:L In yet another embodiment, the wheat plant comprises greater than two IMI nucleic acids wherein each IMI nucleic acid is from a different genome. Preferably, at least one of the IMI nucleic acids comprises a polynucleotide sequence encoding the polypeptide sequence of SEQ ID NO:2, or the
polynucleoltde^s&fuence of SEQ ID NO: 1.
[0032] In a preferred embodiment of the present invention, the isolated IMI
nucleic acid encodes an amino acid sequence comprising a mutation in a domain that
is conserved among several AHAS proteins. These conserved domains are referred to
herein as Domain A, Domain B, Domain C, Domain D, and Domain E. Figure 3
shows the general location of each domain in an AHAS protein. Domain A contains
the amino acid sequence ATTGQVPRRMIGT (SEQ ID NO:7). Domain B contains
the amino acid sequence QWED (SEQ ID NO:8). Domain C contains the amino acid
sequence WAYPGGASMEIHQALTRS (SEQ ID NO:9). Domain D contains the
amino acid sequence AFQETP (SEQ ID NO.10). Domain E contains the amino acid
sequence IPSGG (SEQ ID NO: 11). The present invention also contemplates that
there may be slight variations in the conserved domains, for example, in cockleber
plants, the serine residue in Domain E is replaced by an alanine residue.
[0033] Accordingly, the present invention includes a wheat plant or triticale
plant comprising an IMI nucleic acid that encodes an amino acid sequence having a mutation in a conserved domain selected from the group consisting of a Domain A, a Domain B, a Domain C, a Domain D, and a Domain E. In one embodiment, the wheat plant or triticale plant comprises an IMI nucleic acid that encodes an amino acid sequence having a mutation in a Domain E. In further preferred embodiments, the mutations in the conserved domains occur at the locations indicated by the following underlining: ATTGQVPRRMIGT (SEQ ID NO:7); QWED (SEQ ID NO:8); VFAYPGGASMEIHQALTRS (SEQ ID NO:9); AFQETP (SEQIDNO:10), and IPSGG (SEQ ID NO:l 1). One preferred substitution is an alanine to threonine in Domain C. Even more preferably, the substitution is an alanine to threonine substitution at position 96 of the ALS polypeptide.

[0034] The present invention provides methods for enhancing the tolerance or
resistance of a plant, plant tissue, plant cell, or other host cell to at least one herbicide
that interferes with the activity of the AHAS enzyme. The present invention further
provides plants, plant cells, plant parts, plant organs, plant tissues, seeds, and host
cells with tolerance to at least one herbicide, particularly an AHAS-inhibiting
herbicide. Preferably, such an AHAS-inhibiting herbicide is an imidazolinone
herbicide, a sulfonylurea herbicide, a triazolapyrimidine herbicide, a
pyrimidinyloxybenzoate herbicide, a sulfon^damino-carbonyltriazolinone herbicide, or
mixture thereof. More preferably, such a herbicide is an imidazolinone herbicide or
mixture^of^ro^'more imidazolinone herbicides. For the present invention, the
imidazolinone herbicides include, but are not limited to, PURSUIT® (imazethapyr),
CADRE® (imazapic), RAPTOR® (imazamox), SCEPTER® (imazaquin),
ASSERT® (imazethabenz), ARSENAL® (imazapyr), a derivative of any of the
aforementioned herbicides, and a mixture of two or more of the aforementioned
herbicides, for example, imazapyr/imazamox (ODYSSEY®). More specifically, the
imidazolinone herbicide can be selected from, but is not limited to, 2- (4-isopropyl-4-
methyl-5-oxo-2«imidiazolin-2-yl) -nicotinic acid, [2- (4~isopropyl)-4-] [methyl-5-oxo-
2-imidazolin-2-yl)-3-quinolinecarboxyhc] acid, [5-ethyl-2- (4-isopropyl-] 4-methyl-5-
oxo-2-imidazolin-2-yl) -nicotinic acid, 2- (4-isopropyl-4-methyl-5-oxo«2- imidazolin-
2-yl)-5- (methoxymethyl)-nicotrnic acid, [2- (4-isopropyl-4-methyl-5-oxo-2-]
imidazolin-2-yl)-5»methylmcotimc acid, and a mixture of methyl [6- (4-isopropyl-4-j
methyl-5-oxo-2-imidazolin-2-yl) -m-toluate and methyl [2- (4-isopropyl-4-methyl-5-]
oxo-2-imidazolin-2-yl) -p-toluate. The use of 5-ethyl-2- (4-isopropyl-4-methyl-5-
oxo- 2-imidazolin-2-yl) -mcotimc acid and [2- (4-isopropyl~4-methyl~5-oxo-2-
imidazolin-2-] yl)-5- (methoxymethyl)-nicotinic acid is preferred. The use of [2- (4-
isopropyl-4-] methyl«5-oxo-2-imidazolin-2-yi)-5- (methoxymethyl)-nicotinic acid is
particularly preferred.
[0035] For the present invention, the sulfonylurea herbicides include, but are
not limited to, chlorsulfuron, metsulfuron methyl, sulfometaron methyl, chlorimuron
ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl, nicosulfuron,
ethametsulfuron methyl, rimsulferon, triflusulfuron methyl, triasul&ron,
primisulfuron methyl, cinosulfuron, amidosulfiuon, fluzasulfuron, imazosulfuron,
pyrazosulfixron ethyl, halosulfuron, azimsulfuron, cyclosulfuron, ethoxysulforon,
flazasulfuron, flupyrsulfuron methyl, foramsulfuron, iodosulfuron, oxasulfuron,


of any of the aforementioned herbicides, and a mixture of two or more of the
aforementioned herbicides. The triazolopyrimidine herbicides of the invention
include, but are not limited to, cloransulam, diclosulam, florasulam, flumetsulam,
metosulam, and penoxsulam. The pyrimidinyloxybenzoate herbicides of the
invention include, but are not limited to, bispyribac, pyrithiobac, pyriminobac,
pyribenzoxim and pyriftalid. The sulfonylamino-carbonyltriazolinone herbicides
include, but are not limited to, flucarbazone and propoxycarbazone.
[0036] It is recognized that pyrimidinyloxybenzoate herbicides are closely
relatedlo"ttep^rnidinylthiobenzoate herbicides and are generalized under the heading of the latter name by the Weed Science Society of America, Accordingly, the herbicides of the present invention further include pyrimidinylthiobenzoate herbicides, including, but not limited to, the pyrimidinyloxybenzoate herbicides described above.
[0037] The wheat plants described herein can be either transgenic wheat
plants or non-transgenic wheat plants. Similarly, the triticale plants described herein can be either transgenic triticale plants or non-transgenic triticale plants. As used herein, 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 stable extra-chromosomal element, so that it is passed on to successive generations. For the purposes of the invention, the term "recombinant polynucleotide" refers to a polynucleotide that has been altered, rearranged or modified by genetic engineering. Examples include any cloned polynucleotide, or polynucleotides, that are linked or joined to heterologous sequences. The term 'recombinant" does not refer to alterations of polynucleotides that result from naturally occurring events, such as spontaneous mutations, or from non-spontaneous mutagenesis followed by selective breeding. Plants containing mutations arising 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 wherein the wheat plant or triticale plant is transgenic and comprises multiple IMI nucleic acids, the nucleic acids can be derived from different genomes or from the same genome. Alternatively, in embodiments wherein the wheat plant or triticale plant is

non-transgenic and comprises multiple IMI nucleic acids, the nucleic acids are located on different genomes or on the same genome.
[0038] An example of a non-transgenic wheat plant cultivar comprising one
IMI nucleic acid is the plant cultivar deposited with the ATCC under Patent Deposit
Designation Number PTA-5625 and designated herein as the Shiloh-8 line. The
Shiloh-8 line contains an Imil nucleic acid. The partial-length nucleotide sequence
corresponding to the Shiloh-8 gene is shown in SEQ ID NO:l.
[0039] A deposit of 2500 seeds of the Shiloh-8 line (designated i4417-8) was
made with the American Type Culture Collection, Manassas, Virginia on October 30,
2003. TEestrdeposits were made in accordance with the tarns and provisions of the
Budapest Treaty relating to the deposit of microorganisms. The deposits were made
for a term of at least thirty years and at least five years after the most recent request
for the furnishing of a sample of the deposit is received by the ATCC. The deposited
seeds were accorded Patent Deposit Designation Number PTA-5625.
[0040] The present invention includes the wheat plant having a Patent Deposit
Designation Number PTA-5625; a mutant, recombinant, or genetically engineered
derivative of the plant with Patent Deposit Designation Number PTA-5625; any
progeny of the plant with Patent Deposit Designation Number PTA-5625; and a plant
that is the progeny of any of these plants. In a preferred embodiment, the wheat plant
of the present invention additionally has the herbicide tolerance characteristics of the
plant with Patent Deposit Designation Number PTA-5625.
[0041] Also included in the present invention are hybrids of the Shiloh-8
wheat plants described herein and hybrids of the Shiloh-8 with another wheat plant. The other wheat plant includes, but is not limited to, T. aestivum L. cv Fidel and any wheat plant harboring a mutant gene FS-1, FS-2, FS-3 or FS-4. (See U.S. Patent No. 6,339,184 and U.S. Patent Application No. 08/474,832).
[0042] The terms "cultivaf* and "variety" refer to a group of plants within a
species defined by the sharing of a conraion set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one cultivar or variety from another cultivar or variety. There is no implication in either term that all plants of any given cultivar or variety will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A cultivar or variety is considered 'true breeding" for a particular trait if, when the true-breeding cultivar or variety is self-pollinated, all of the progeny contain the trait The terms "breeding

line" or "line" refer to a group of plants within a cultivar defined by the sharing of a common set of characteristics or traits accepted by those skilled in the art as sufficient to distinguish one breeding line or line from another breeding line or line. There is no implication in either tenn that all plants of any given breeding line or line will be genetically identical at either the whole gene or molecular level or that any given plant will be homozygous at all loci. A breeding line or line is considered "true breeding" for a particular trait if, when the true-breeding line or breeding line is self-pollinated, all of the progeny contain the trait. In the present invention, the trait arises from a mutation in an Als gene of the wheat or triticale plant or seed.
[0043] *~ T%thermore, the use of the terns "cultivaf * and "variety" herein is not intended to limit the plants of the present invention to one or more plant varieties. While the present invention encompasses plant varieties, the plants of the present invention include any plants that comprise the heibicide-tolerance characteristics of the plant of ATCC Patent Deposit Number 5625 and/or one or more of the IMI nucleic acids of the present invention.
[0044] It is to be understood that the wheat or triticale plant of the present
invention can comprise a wild type Als nucleic acid in addition to an IMI nucleic acid. As described in Example 1, it is contemplated that the Shiloh-8 line contains a mutation in only one of multiple AHAS isoenzymes. Therefore, the present invention includes a wheat plant or triticale plant comprising at least one IMI nucleic acid in addition to one or more wild type Als nucleic acids.
[0045] In addition to wheat and triticale plants, the present invention
encompasses isolated IMt proteins and nucleic acids. .The nucleic acids comprise a polynucleotide selected from the group consisting of a polynucleotide as defined in SEQ ID NO: 1; a polynucleotide encoding of a polypeptide as defined in SEQ ID NO:2; a polynucleotide comprising at least 60 consecutive nucleotides of any of the aforementioned polynucleotides; and a polynucleotide complementary to any of the aforementioned polynucleotides. In a preferred embodiment, the IMI nucleic acid comprises a polynucleotide sequence of SEQ ID NO:l.
[0046] The terms "AHAS protein," "AHAS polypeptide," "ALS protein," and
"ALS polypeptide" refer to an acetohydroxyacid synthase protein, and the terms "IMI protein" or "IMI polypeptide" refer to any AHAS protein that is mutated from a wild type AHAS protein and that confers increased imidazolinone tolerance to a plant, plant cell, plant part, plant seed, or plant tissue when it is expressed therein. In a

preferred embodiment, the 1MI protein comprises a polypeptide encoded by a
polynucleotide sequence comprising SEQ ID NO: 1. Such IMI proteins comprise
herbicide-tolerant AHAS activity, particularly imidazolinone-tolerant AHAS activity.
Such herbicide-tolerant AHAS activity can be evaluated by AHAS activity assays.
See, for example, Singh et al. (1988) Anal Biochem. 171:173-179, herein
incorporated by reference.
[0047] In another preferred embodiment, the IMI protein comprises a
polypeptide comprising SEQ ID NO:2. As also used herein, the terms "nucleic acid"
and "polynucleotide" refer to RNA or DNA that is linear or branched, single or
double"sEraiMe3rcfr a hybrid thereof. The term also encompasses RNA/DNA hybrids.
These terms also encompass untranslated sequence located at both the 3' and 5' ends
of the coding region of the gene: at least about 1000 nucleotides of sequence upstream
from the 5' end of the coding region and at least about 200 nucleotides of sequence
downstream from the 3' end of the coding region of the gene. Less common bases,
such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also
be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides
that contain C-5 propyne analogues of uridine and cytidine have been shown to bind
RNA with high affinity and to be potent antisense inhibitors of gene expression.
Other modifications, such as modification to the phosphodiester backbone, or the 2'-
hydroxy in the ribose sugar group of the RNA can also be made. The antisense
polynucleotides and ribozymes can consist entirely of ribonucleotides, or can contain
mixed ribonucleotides and deoxyribonucleotides. The polynucleotides of the
invention may be produced by any means, including genomic preparations, cDNA
preparations, in vitro synthesis, RT-PCR, and in vitro ox in vivo transcription.
[0048] An "isolated" nucleic acid molecule is one that is substantially
separated from other nucleic acid molecules, which are present in the natural source
of the nucleic acid (i.e., sequences encoding other polypeptides). Preferably, an
"isolated" nucleic acid is free of some of the sequences that naturally flank the nucleic
acid (i.e., sequences located at the 5' and 3* ends of the nucleic acid) in its naturally
occurring replicon. For example, a cloned nucleic acid is considered isolated. In
various embodiments, the isolated IMI nucleic acid molecule can contain less than
about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived (e.g., a Triticum aestivum cell). A nucleic acid is also

considered isolated if it has been altered by human intervention, or placed in a locus or location that is not its natural site, or if it is introduced into a cell by agroinfection, biolistics, or any other method of plant transformation. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be free from some of the other cellular material with which it is naturally associated, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized.
[0049] Specifically excluded from the definition of "isolated nucleic acids"
are: naturally^-occurring chromosomes (such as chromosome spreads), artificial
chromosom^K^ries, genomic libraries, and cDNA libraries that exist either as an in
vitro nucleic acid preparation or as a transfected/transfonned host cell preparation,
wherein the host cells are either an in vitro heterogeneous preparation or plated as a
heterogeneous population of single colonies. Also specifically excluded are the above
libraries wherein a specified nucleic acid makes up less than 5% of the number of
nucleic acid inserts in the vector molecules. Further specifically excluded are whole
cell genomic DNA or whole cell RNA preparations (including whole cell preparations
that are mechanically sheared or enzymatically digested). Even further specifically
excluded are the whole cell preparations found as either an in vitro preparation or as a
heterogeneous mixture separated by electrophoresis wherein the nucleic acid of the
invention has not further been separated from the heterologous nucleic acids in the
electrophoresis medium (e.g., further separating by excising a single band from a
heterogeneous band population in an agarose gel or nylon blot).
[0050] A nucleic acid molecule of the present invention, e,g., a nucleic acid
molecule containing a nucleotide sequence of SEQ ID NO: 1 or a portion thereof^ can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a T. aestivum IMI cDNA can be isolated from a T. aestivum library using all or a portion of the sequence of SEQ ID NO: 1. Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:l can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon this sequence. For example, mRNA can be isolated from plant cells (e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et aL, 1979, Biochemistry 18:5294-5299), and cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Petersburg, FL).

Synthetic oligonucleotide primers for polymerase chain reaction amplification can be
designed based upon the nucleotide sequence shown in SEQ ID NO:l. A nucleic acid
molecule of the invention can be amplified using cDNA or, alternatively, genomic
DNA, as a template and appropriate oligonucleotide primers according to standard
PCR amplification techniques. The nucleic acid molecule so amplified can be cloned
into an appropriate vector and characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to an IMI nucleotide sequence can be prepared by
standard synthetic techniques, e.g., using an automated DNA synthesizer.
[0051] The MI nucleic acids of the present invention can comprise sequences
encoding^ifH^fptotein (i.e., "coding regions"), as well as 5' untranslated sequences and 3' untranslated sequences. Alternatively, the nucleic acid molecules of the present invention can comprise only the coding regions of an IMI gene, or can contain whole genomic fragments isolated from genomic DNA. A coding region of these sequences is indicated as an "QRF position." Moreover, the nucleic acid molecule of the invention can comprise a portion of a coding region of an IMI gene, for example, a fragment that can be used as a probe or primer. The nucleotide sequences determined from the cloning of the IMI genes from T. aestivwn allow for the generation of probes and primers designed for use in identifying and/or cloning IMI homologs in other cell types and organisms, as well as IMI homologs from other wheat plants and related species. The portion of the coding region can also encode a biologically active fragment of an IMI protein.
[0052] As used herein, the term '"biologically active portion of an IMI protein
is intended to include a portion, e.g., a domain/moti£ of an IMI protein that, when produced in a plant increases the plant's tolerance to an imidazolinone herbicide as compared to a wild-type plant. Methods for quantitating increased tolerance to imidazolinone herbicides are provided in the Examples below. Biologically active portions of an IMI protein include peptides derived from SEQ ID NO:2 which include fewer amino acids than a full length IMI protem and impart increased tolerance to an imidazolinone herbicide upon expression in a plant. Typically, biologically active portions (e.g., peptides which are, for example, 5,10,15,20, 30, 35,36,37,38,39, 40, 50,100, or more amino acids in length) comprise a domain or motif with at least one activity of an IMI protein. Moreover, other biologically active portions in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the activities described herein.

Preferably, the biologically active portions of an IMI protein include one or more conserved domains selected from the group consisting of a Domain A, a Domain B, a Domain C, a Domain D and a Domain E, wherein the conserved domain contains a mutation.
[0053] The invention also provides IMI chimeric or fusion polypeptides. As
used herein, an IMI "chimeric polypeptide" or "fusion polypeptide" comprises an IMI polypeptide operatively linked to a non-IMI polypeptide. A "non-IMI polypeptide" refers to a polypeptide having an amino acid sequence that is not substantially identical to an IMI polypeptide, e.g., a polypeptide that is not an IMI isoenzyme, which p^tJfre^ffoxms a different function than an IMI polypeptide. As used herein with respect to the fusion polypeptide, the term "operatively linked" is intended to indicate that the IMI polypeptide and the non-IMI polypeptide are fused to each other so that both sequences fulfill the proposed function attributed to the sequence used. The non-IMI polypeptide can be fused to the N-terminus or C-terminus of the IMI polypeptide. For example, in one embodiment, the fusion polypeptide is a GST-IMI fusion polypeptide in which the IMI sequence is fused to the C-terminus of the GST sequence. Such fusion polypeptides can facilitate the purification of recombinant IMI polypeptides. In another embodiment, the fusion polypeptide is an IMI polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or seCTetion of an IMI polypeptide can be increased through use of a heterologous signal sequence.
[0054] An isolated nucleic acid molecule encoding an IMI polypeptide having
a certain percent sequence identity to a polypeptide of SEQ ID NO:2 can be created by introducing one or more nucleotide substitutions, additions, or deletions into a nucleotide sequence of SEQ ID NO:l such that one or more amino acid substitutions, additions, or deletions are introduced into the encoded polypeptide. Mutations can be introduced into a sequence of SEQ ID NO:l by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
[0055] A "conservative amino acid substitution" is one in which the amino
acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine,

histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g., threonine, valine,
isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in an IMI polypeptide is
preferably replaced with another amino acid residue from the same side chain family.
Alternatively, in another embodiment, mutations can be introduced randomly along
all or part of an IMI coding sequence, such as by saturation mutagenesis, and the
resultantrmmtafltS -can be screened for an IMI activity described herein to identify
mutants that retain IMI activity. Following mutagenesis of the sequence of SEQ ID
NO: 1, the encoded polypeptide can be expressed recombinantly and the activity of the
polypeptide can be determined by analyzing the imidazolinone tolerance of a plant
expressing the polypeptide as described in the Examples below.
[0056] To determine the percent sequence identity of two amino acid
sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one polypeptide for optimal alignment with the other polypeptide). The amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence is occupied by the same amino acid residue as the corresponding position in the other sequence, then the molecules are identical at that position. The same type of comparison can be made between two nucleic acid sequences. The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent sequence identity = numbers of identical positions/total numbers of positions x 100). For the purposes of the invention, the percent sequence identity between two nucleic acid or polypeptide sequences is determined using the Vector NTT 6.0 (PC) software package (InforMax, 7600 Wisconsin Ave., Bethesda, MD 20814). A gap opening penalty of 15 and a gap extension penalty of 6.66 are used for determining the percent identity of two nucleic acids. A gap opening penalty of 10 and a gap extension penalty of 0.1 are used for determining the percent identity of two polypeptides. All other parameters are set at the default settings.
[0057] It is to be understood that for the purposes of determining sequence
identity, when comparing a DNA sequence to an RNA sequence, a thymidine nucleotide is equivalent to a uracil nucleotide. Preferably, the isolated IMI nucleic

acids of the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75% 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to the entire polynucleotide sequence shown in SEQ ID NO:l. In another embodiment, the isolated IMI nucleic acids included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to an entire polynucleotide sequence shown in SEQ ID
NO:l.
[0058] *~ Tifeferably, the isolated IMI polypeptides of the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to an entire amino acid sequence shown in SEQ ID NO:2. In another embodiment, the isolated IMI polypeptides included in the present invention are at least about 50-60%, preferably at least about 60-70%, and more preferably at least about 70-75%, 75-80%, 80-85%, 85-90%, or 90-95%, and most preferably at least about 96%, 97%, 98%, 99%, or more identical to an entire amino acid sequence shown in SEQ ID NO:2.
[0059] Additionally, optimized IMI nucleic acids can be created. Preferably,
an optimized IMI nucleic acid encodes an IMI polypeptide that modulates a plant's tolerance to imidazolinone herbicides, and more preferably increases a plant's tolerance to an imidazolinone herbicide upon its overexpression in the plant As used herein, "optimized" refers to a nucleic acid that is genetically engineered to increase its expression in a given plant or animal. To provide plant optimized IMI nucleic acids, the DNA sequence of the gene can be modified to 1) comprise codons prefeired by highly expressed plant genes; 2) comprise an A+T content in nucleotide base composition to that substantially found in plants; 3) form a plant initiation sequence, 4) eliminate sequences that cause destabilization, inappropriate polyadenylation, degradation and teratination of RNA, or that form secondary structure hairpins or RNA splice sites. Increased expression of IMI nucleic acids in plants can be achieved by utilizing the distribution frequency of codon usage in plants in general or a particular plant. Methods for optimizing nucleic acid expression in plants can be found in EPA 0359472; EPA 0385962; PCT Application No. WO 91/16432; U.S. Patent No. 5,380,831; U.S. Patent No. 5,436,391; PerlacketaL, 1991, Proc. Natl

Acad. Sci. USA 88:3324-3328; and Murray et aL, 1989, Nucleic Acids Res. 17:477-498.
[0060] As used herein, "frequency of preferred codon usage" refers to the
preference exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. To determine the frequency of usage of a particular codon in a gene, the number of occurrences of that codon in the gene is divided by the total number of occurrences of all codons specifying the same amino acid in the gene. Similarly, the frequency of preferred codon usage exhibited by a host cell can be calculated by averaging frequency of preferred codon usage in a large number of genes ~exprfcsse?Tby the host cell. It is preferable that this analysis be limited to genes that are highly expressed by the host cell. The percent deviation of the frequency of preferred codon usage for a synthetic gene from that employed by a host cell is calculated first by determining the percent deviation of the frequency of usage of a single codon from that of the host cell followed by obtaining the average deviation over all codons. As defined herein, this calculation includes unique codons (i.e., ATG and TGG). In general terms, the overall average deviation of the codon usage of an optimized gene from that of a host cell is calculated using the equation 1A = n = 1 Z Xn - Yn XQ times 100 Z where Xn - frequency of usage for codon n in the host cell; Yn = frequency of usage for codon n in the synthetic gene, n represents an individual codon that specifies an amino acid and the total number of codons is Z. The overall deviation of the frequency of codon usage, A, for all amino acids should preferably be
less than about 25%, and more preferably less than about 10%.
[0061] Hence, an IMI nucleic acid can be optimized such that its distribution
frequency of codon usage deviates, preferably, no more than 25% from that of highly
expressed plant genes and, more preferably, no more than about 10%. In addition,
consideration is given to the percentage G+C content of the degenerate third base
(monocotyledons appear to favor G+C in this position, whereas dicotyledons do not).
It is also recognized that the XCG (where X is A, T, C, or G) nucleotide is the least
preferred codon in dicots whereas the XTA codon is avoided in both monocots and
dicots. Optimized IMI nucleic acids of this invention also preferably have CG and
TA doublet avoidance indices closely approximating those of the chosen host plant
(i.e., Triticum aestivum). More preferably these indices deviate from that of the host
by no more than about 10-15%.

[0062] In addition to the nucleic acid molecules encoding the IMI
polypeptides described above, another aspect of the invention pertains to isolated nucleic acid molecules that are antisense thereto. Antisense polynucleotides are thought to inhibit gene expression of a target polynucleotide by specifically binding the target polynucleotide and interfering with transcription, splicing, transport, translation and/or stability of the target polynucleotide. Methods are described in the prior art for targeting the antisense polynucleotide to the chromosomal DNA, to a primary RNA transcript or to a processed mRNA. Preferably, the target regions include splice sites, translation initiation codons, translation termination codons, and other se^Sta^Within the open reading frame.
[0063] The term "antisense," for the purposes of the invention, refers to a
nucleic acid comprising a polynucleotide that is sufficiently complementary to all or a
portion of a gene, primary transcript, or processed mRNA, so as to interfere with
expression of the endogenous gene. "Complementary" polynucleotides are those that
are capable of base pairing according to the standard Watson-Crick complementarity
rules. Specifically, purines will base pair with pyrimidines to form a combination of
guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in
the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is
understood that two polynucleotides may hybridize to each other even if they are not
completely complementary to each other, provided that each has at least one region
that is substantially complementary to the other. The tern "antisense nucleic acid"
includes single stranded RNA as well as double-stranded DNA expression cassettes
that can be transcribed to produce an antisense RNA. "Active" antisense nucleic
acids are antisense RNA molecules that are capable of selectively hybridizing with a
primary transcript or mRNA encoding a polypeptide having at least 80% sequence
identity with the polypeptide sequence of SEQ ID NO:2.
[0064] In addition to the IMI nucleic acids and polypeptides described above,
the present invention encompasses these nucleic acids and polypeptides attached to a moiety. These moieties include, but are not limited to, detection moieties, hybridization moieties, purification moieties, delivery moieties, reaction moieties, binding moieties, and the like. A typical group of nucleic acids having moieties attached are probes and primers. Probes and primers typically comprise a substantially isolated oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes undo- stringent conditions to at least

about 12, preferably about 25, more preferably about 40, 50, or 75 consecutive nucleotides of a sense strand of the sequence set forth in SEQ ED NO:l, an anti-sense sequence of the sequence set forth in SEQ ID NO:l, or naturally occurring mutants thereof. Primers based on a nucleotide sequence of SEQ ID NO:l can be used in PCR reactions to clone IMI homologs. Probes based on the MI nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous polypeptides. In preferred embodiments, the probe further comprises a label group attached thereto, e.g. the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a genomic markertesHdrfcr identifying cells which express an IMI polypeptide, such as by measuring a level of an IMI-encoding nucleic acid, in a sample of cells, e.g., detecting IMI mRNA levels or determining whether a genomic IMI gene has been mutated or deleted.
[0065] The invention further provides an isolated recombinant expression
vector comprising an IMI nucleic acid as described above, wherein expression of the vector in a host cell results in increased tolerance to an imidazolinone herbicide as compared to a wild type host cell. As used herein, the tenn "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been 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, wherein 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. Moreover, 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 of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0066] The recombinant expression vectors of the invention comprise a
nucleic acid of the invention in a form suitable for expression of the nucleic acid in a
host cell, which means that the recombinant expression vectors include one or more
regulatory sequences, selected on the basis of the host cells to be used for expression,
which is operatively linked to the nucleic acid sequence to be expressed. With respect
to a recombinant expression vector, "operatively linked" is intended to mean that the
nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner
which allows for expression of the nucleotide sequence (e.g., in an in vitro
transcription/ translation system OT in a host cell when the vector is introduced into the
host cell). *ffieTerm "regulatory sequence" is intended to include promoters,
enhancers, and other expression control elements (e.g., polyadenylation signals).
Such regulatory sequences are described, for example, in Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990)
and Gruber and Crosby, in: Methods in Plant Molecular Biology and Biotechnology,
eds. Glick and Thompson, Chapter 7, 89-108, CRC Press: Boca Raton, Florida,
including the references therein. 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. It will be appreciated by those skilled in the art that the design of
the expression vector can depend on such factors as the choice of the host cell to be
transformed, the level of expression of polypeptide desired, etc. The expression
vectors of the invention can be introduced into host cells to thereby produce
polypeptides or peptides, including fusion polypeptides or peptides, encoded by
nucleic acids as described herein (e.g., 1MI polypeptides, fusion polypeptides, etc.).
[0067] In a preferred embodiment of the present invention, the IMI
polypeptides are expressed in plants and plants cells such as unicellular plant cells
(such as algae) (See Falciatore et al., 1999, Marine Biotechnology 1(3):239-251 and
references therein) and plant cells from higher plants (e.g., the spermatophytes, such
as crop plants). An IMI polynucleotide may be "introduced" into a plant cell by any
means, including txansfection, transformation or transduction, electroporation, particle
bombardment, agroinfection, biolistics, and the like.
[0068] Suitable methods for transforming or transfecting host cells including
plant cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory
Manual. 2nd, ed., Cold Spring Haibor Laboratory, Cold Spring Harbor Laboratory

Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals such as Methods in Molecular Biology, 1995, Vol. 44; Agrobacterium protocols, ed: Gartland and Davey, Humana Press, Totowa, New Jersey. As increased tolerance to imidazolinone herbicides is a general trait wished to be inherited into a wide number of plants like maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed and canola, manihot, pepper, sunflower and tagetes, solanaceous plants like potato, tobacco, eggplant, and tomato, Vicia species, pea, alfalfa, bushy plants (coffee, cacao, tea), Salix species, trees (oil palm, coconut), perennial grasses, and forage crops, these plants are also preferred target plants for a genetic engineering as one further embodimefttdTthe present invention. In a preferred embodiment, the plant is a wheat plant. Forage crops include, but arc not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildrye Grass, Bluegrass, Grchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover.
[0069] In one embodiment of the present invention, transfection of an IMI
polynucleotide into a plant is achieved by Agrobacterium mediated gene transfer. One transformation method known to those of skill in the art is the dipping of a flowering plant into an Agrobacteria solution, wherein the Agrobacteria contains the IMI nucleic acid, followed by breeding of the transformed gametes. Agrobacterium mediated plant transformation can be performed using for example the GV3101(pMP90) (Koncz and Schell, 1986, MoL Geru Genet 204:383-396) or LBA4404 (Clontech) Agrobacterium tumefaciens strain. Transformation can be performed by standard transformation and regeneration techniques (Deblaere et al.,
1994, Nucl. Acids. Res. 13:4777-4788; Gelvin, Stanton B. and Schilperoort, Robert A, Plant Molecular Biology Manual, 2nd Ed. - Dordrecht: Kluwer Academic PubL,
1995, - in Sect, Ringbuc Zentrale Signatur: BT11-P ISBN 0-7923-2731-4; Glick, Bernard R. and Thompson, John R, Methods in Plant Molecular Biology and Biotechnology, Boca Raton: CRC Press, 1993 360 S., ISBN 0-8493-5164-2). For example, rapeseed can be transformed via cotyledon or hypocotyl transformation (Moloney et aL, 1989, Plant Cell Report 8:238-242; De Block et al, 1989, Plant Physiol. 91:694-701). Use of antibiotics for Agrobacterium and plant selection depends on the binary vector and the Agrobacterium strain used for transformation. Rapeseed selection is normally performed using kanamycin as selectable plant marker. Agrobacterium mediated gene transfer to flax can be performed using, for example, a technique described by Mlynarova et al., 1994, Plant Cell Report 13:282-

285. Additionally, transformation of soybean can be performed using for example a technique described in European Patent No. 0424 047, U.S. Patent No. 5,322,783, European Patent No. 0397 687, U.S. Patent No. 5,376,543, or U.S. Patent No. 5,169,770. Transformation of maize can be achieved by particle bombardment, polyethylene glycol mediated DNA uptake, or via the silicon carbide fiber technique. (See, for example, Freeling and Walbot 'The maize handbook" Springer Verlag: New York (1993) ISBN 3-540-97826-7). A specific example of maize transformation is found in U.S. Patent No. 5.990,387, and a specific example of wheat transformation can be found in PCT Application No. WO 93/07256.
[0070] *"~ "According to the presort invention, the introduced IMI polynucleotide may be maintained in the plant cell stably if it is incoiporated into a non-chromosomal autonomous replicon or integrated into the plant chromosomes. Alternatively, the introduced IMI polynucleotide may be present on an extra-chromosomal non-replicating vector and be transiently expressed or transiently active. In one embodiment, a homologous recombinant microorganism can be created wherein the IMI polynucleotide is integrated into a chromosome, a vector is prepared which contains at least a portion of an AHAS gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the endogenous AHAS gene and to create an IMI gene. To create a point mutation via homologous recombination, DNA-RNA hybrids can be used in a technique known as chimeraplasty (Cole-Strauss et a!., 1999, Nucleic Acids Research 27(5): 1323-1330 and Rmiec, 1999, Gene therapy American Scientist 87(3):240-247). Other homologous recombination procedures in Triticum species are also well known in the art and are contemplated for use herein.
[0071] In the homologous recombination vector, the IMI gene can be flanked
at its 5' and 3' ends by an additional nucleic acid molecule of the AHAS gene to allow for homologous recombination to occur between the exogenous IMI gene carried by the vector and an endogenous AHAS gene, in a microorganism or plant. The additional flanking AHAS nucleic acid molecule is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several hundreds of base pairs up to kilobases of flanking DNA (both at the 5' and 3' ends) are included in the vector (See, e.g., Thomas, EL R-, and Capecchi, M. R., 1987, Cell 51:503 for a description of homologous recombination vectors or Strepp et al., 1998, PNAS, 95(8):4368-4373 for cDNA based recombination in Physcomitretta patens).

However, since the IMI gene normally differs from the AHAS gene at very few amino
acids, a flanking sequence is not always necessary. The homologous recombination
vector is introduced into a microorganism or plant cell (e.g., via polyethylene glycol
mediated DNA), and cells in which the introduced IMI gene has homologously
recombined with the endogenous AHAS gene are selected using art-known
techniques.
[0072] In another embodiment, recombinant microorganisms can be produced
that contain selected systems that allow for regulated expression of the introduced
gene. For example, inclusion of an IMI gene on a vector placing it under control of
the lac^efenpeamits expression of the IMI gene only in the presence of IPTG. Such
regulatory systems are well known in the art.
[0073] Whether present in an extra-chromosomal non-replicating vector or a
vector that is integrated into a chromosome, the IMI polynucleotide preferably resides
in a plant expression cassette. A plant expression cassette preferably contains
regulatory sequences capable of driving gene expression in plant cells that are
operatively linked so that each sequence can fulfill its function, for example,
termination of transcription by polyadenylation signals. Preferred polyadenylation
signals are those originating from Agrobacterium tumefaeiens t-DNA such as the gene
3 known as octopine synthase of the Ti-plasmid pTiACH5 (Gielen et aL, 1984,
EMBO J. 3:835) or functional equivalents thereof but also all other terminators
functionally active in plants are suitable. As plant gene expression is very often not
limited on transcriptional levels, a plant expression cassette preferably contains other
operatively linked sequences like translational enhancers such as the overdrive-
sequence containing the 5 '-untranslated leader sequence from tobacco mosaic virus
enhancing the polypeptide per RNA ratio (Gallie et aL, 1987, NucL Acids Research
15:8693-8711). Examples of plant expression vectors include those detailed in:
Becker, D. et aL, 1992, New plant binary vectors with selectable markers located
proximal to the left border, Plant Mol. Biol. 20:1195-1197; Bevan, M.W., 1984,
Binary Agrobacterium vectors for plant transformation, NucL Acid. Res. 12:8711-
8721; and Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, Vol. 1,
Engineering and Utilization, eds.: Rung and R. Wu, Academic Press, 1993, S. 15-38.
[0074] Plant gene expression should be operatively linked to an appropriate
promoter conferring gene expression in a timely, cell type-preferred, or tissue-preferred manner. Promoters useful in the expression cassettes of the invention

include any promoter that is capable of initiating transcription in a plant cell. Such promoters include, but are not limited to those that can be obtained from plants, plant viruses and bacteria that contain genes that are expressed in plants, such as Agrobacteriwn mdRhizobium.
[0075] The promoter may be constitutive, inducible, developmental stage-
preferred, cell type-preferred, tissue-preferred or organ-preferred. Constitutive
promoters are active under most conditions. Examples of constitutive promoters
include the CaMV 19S and 35S promoters (Odell et al., 1985, Nature 313:810-812),
the sX CaMV 35S promoter (Kay et al., 1987, Science 236:1299-1302) the Sepl
promoter, ffie'nce actin promoter (McElroy et al., 1990, Plant Cell 2:163-171), the
Arabidopsis actin promoter, the ubiquitin promoter (Christensen ei at, 1989, Plant
Molec. Biol. 18:675-689); pEnm (Last et al., 1991, Theor. AppL Genet 81:581-588),
the figwort mosaic virus 35S promoter, the Smas promoter (Velten et al., 1984,
EMBO J. 3:2723-2730), the GRP1-8 promoter, the cinnamyl alcohol dehydrogenase
promoter (U.S. Patent No. 5,683,439), promoters from the T-DNA of Agrobacterium,
such as mannopine synthase, nopaline synthase, and octopine synthase, the small
, subunit of ribulose biphosphate caiboxylase (ssuRUBISCO) promoter, and the like.
[0076] Inducible promoters are active under certain environmental conditions,
such as the presence or absence of a nutrient or metabolite, heat or cold, light,
pathogen attack, anaerobic conditions, and the like. For example, the hsp80 promoter
from Brassica is induced by heat shock; the PPDK promoter is induced by light; the
PR-1 promoter from tobacco, Arabidopsis, and maize are inducible by infection with
a pathogen; and the Adhl promoter is induced by hypoxia and cold stress. Plant gene
expression can also be facilitated via an inducible promoter (For review, see Gatz,
1997, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:89-108). Chemically inducible
promoters are especially suitable if time-specific gene expression is desired.
Examples of such promoters are a salicylic acid inducible promoter (PCT Application
No. WO 95/19443), a tetracycline inducible promoter (Gatz et al., 1992, Plant J.
2:397-404) and an ethanol inducible promoter (PCT Application No. WO 93/21334).
[0077] Developmental stage-preferred promoters are preferentially expressed
at certain stages of development Tissue and organ preferred promoters include those that are preferentially expressed in certain tissues or organs, such as leaves, roots, seeds, or xylem. Examples of tissue preferred and organ preferred promoters include, but are not limited to fruit-preferred, ovule-preferred, male tissue-preferred, seed-

preferred, integument-preferred, tuber-preferred, stalk-prefeired, pericarp-preferred, and leaf-preferred, stigma-preferred, pollen-preferred, anther-preferred, a petal-preferred, sepal-preferred, pedicel-preferred, silique-preferred, stem-preferred, root-preferred promoters, and the like. Seed preferred promoters are preferentially expressed during seed development and/or germination. For example, seed preferred promoters can he embryo-preferred, endosperm preferred, and seed coat-preferred. See Thompson et al., 1989, BioEssays 10:108. Examples of seed preferred promoters include, but are not limited to cellulose synthase (celA), Ciml, gamma-zein, globulin-1, maize 19 kD zein (cZ19Bl), and the like.
[0078] Other suitable tissue-preferred or organ-preferred promoters include
the napin-gene promoter from rapeseed (U.S. Patent No. 5,608,152), the USP-promoter from Viciafaba (Baeumlein et al., 1991, Mol Gen Genet 225(3):459-67), the oleosin-promoter from Arabidopsis (PCT Application No. WO 98/45461), the phaseolin-promoter fzomPhaseolus vulgaris (U.S. Patent No. 5,504,200), the Bce4-promoter from Brassica (PCT Apphcation No. WO 91/13980), or the legumin B4 promoter (LeB4; Baeumlein et al, 1992, Plant Journal, 2(2):233-9), as well as promoters conferring seed specific expression in monocot plants like maize, barley, wheat, rye, rice, etc. Suitable promoters to note axe the lpt2 or Iptl-gene promoter from barley (PCT Apphcation No. WO 95/15389 and PCT Apphcation No. WO 95/23230) or those described in PCT Application No. WO 99/16890 (promoters from the barley hordein-gene, rice glutelin gene, rice oryzin gene, rice prolamin gene, wheat gliadin gene, wheat glutelin gene, oat glutelin gene, Sorghum kasirin-gene, and
rye secalin gene).
[0079] Other promoters useful in the expression cassettes of the invention
include, but are not limited to, the major chlorophyll a/b binding protein promoter, histone promoters, the Ap3 promoter, the j5-conglycin promoter, the napin promoter, the soybean lectin promoter, the maize 15kD zein promoter, the 22kD zein promoter, the 27kD zein promoter, the g-zein promoter, the waxy, shrunken 1, shrunken 2, and bronze promoters, the Zml3 promoter (U.S. Patent No. 5,086,169), the maize polygalacturonase promoters (PG) (U.S. Patent Nos. 5,412,085 and 5,545,546), and the SGB6 promoter (U.S. Patent No. 5,470,359), as well as synthetic or other natural promoters.
[0080] Additional flexibility in controlling heterologous gene expression in
plants may be obtained by using DNA binding domains and response elements from

heterologous sources (i.e., DNA binding domains from non-plant sources). An example of such a heterologous DNA binding domain is the LexA DNA binding domain (Brent and Ptashne, 1985, Cell 43:729-736).
[0081] The present invention provides expression cassettes for expressing the
polynucleotide molecules of the invention in plants, plant cells, and other, non-human
host cells. The expression cassettes comprise a promoter expressible in the plant,
plant cell, or other host cells of interest operably linked to an IMI nucleic acid. If
necessary for targeting expression to the chloroplast, the expression cassette can also
comprise an operably linked chloroplast-targeting sequence that encodes of a
chlonSpIasftramSit peptide to direct an expressed IMI protein to the chloroplast
[0082] In one embodiment, the IMI nucleic acids are targeted to the
chloroplast for expression. In this manner, where the IMI nucleic acid is not directly inserted into the chloroplast, the expression cassette will additionally contain a chloroplast-targeting sequence comprising a nucleotide sequence that encodes a chloroplast transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art With respect to chloroplast-targeting * sequences, "operably linked" means that the nucleic acid sequence encoding a transit peptide {i.e., the chloroplast-targeting sequence) is linked to the IMI nucleic acid of the invention such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et ah (1991) Plant Mot Biol. Rep. 9:104-126; Clark et ah (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et ah (1987) Plant Physiol 84:965-968; Romeref ah (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481. While the IMI proteins of the invention can include a native chloroplast transit peptide, any chloroplast transit peptide known in art can be fused to the amino acid sequence of a mature IMI protein of the invention by operably linking a chloroplast-targetmg sequence to the 5'-end of a
nucleotide sequence encoding a mature IMI protein of the invention.
[0083] Chloroplast targeting sequences are known in the art and include the
chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de
Castro Silva Filho et ah (1996) Plant Moh Biol. 30:769-780; Schnell et ah (1991) J.
Bioh Chem. 266(5):3335-3342); 5-(enolpyravyl)shikimate-3-phosphate synthase
(EPSPS) (Archer et ah (1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan
synthase (Zhao et ah (1995) /. Biol. Chem. 270(11):6081-6087); plastocyanin
(Lawrence et ah (1997) J. Biol Chem. 272(33):20357-20363); chorismate synthase

(Schmidt et aL (1993) J. Biol. Chem. 268(36):27447-27457); and the light harvesting
chlorophyll a/b binding protein (LHBP) (Lamppa et aL (1988) J. Biol. Chem.
263:14996-14999). See also Von Heijne et aL (1991) Plant Mol. Biol Rep. 9:104-
126; Clark et aL (1989) J. Biol Chem. 264:17544-17550; Della-Cioppa et aL (1987)
Plant Physiol 84:965-968; Romer et aL (1993) Biochem. Biophys. Res. Commun.
196:1414-1421; and Shah et aL (1986) Science 233:478^81.
[0084] The IMI nucleic acids or expression cassettes comprising the IMI
nucleic acids can also be introduced into the chloroplast for expression therein.
Methods for transformation of chloroplasts are known in the art See, for example,
Svab efal^9$JProa Natl Acad ScL USA 87:8526-8530; Svab andMaliga (1993)
Proa Natl Acad ScL USA 90:913-917; Svab and MaHga (1993) EMBO J. 12:601-
606. The method relies on particle gun delivery of DNA containing a selectable
marker and targeting of the DNA to the plastid genome through homologous
recombination. Additionally, plastid transformation can be accomplished by
transactivation of a silent plastid-borne transgene by tissue-preferred expression of a
nuclear-encoded and plastid-directed RNA polymerase. Such a system has been
reported in McBride et aL (1994) Proa Natl Acad. Set USA 91:7301-7305.
[0085J Tke Dvll nucleic acids to be targeted to the chloroplast may be
optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Patent No. 5,380,831, herein incorporated by reference. If necessary for chloroplast expression, the expression cassette can further comprise a chloroplast promoter operably linked to the IMI nucleic acid. Such chloroplast promoters are known in the art.
[0086] Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but they also ^pply to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herdn. A host cell can be any prokaryotic or eukaryotic cell. For example, an IMI polynucleotide can be expressed in bacterial cells such as

C. glutamicurn, insect cells, fungal cells, or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells), algae, ciliates, plant cells, fungi, or other
microorganisms like C glutamicum. Other suitable host cells are known to those
skilled in the art.
[0087] A host cell of the invention, such as aprokaryotic or eukaryotic host
cell in culture, can be used to produce (i.e., express) an IMI polynucleotide.
Accordingly, the invention further provides methods for producing IMI polypeptides
using the host cells of the invention. In one embodiment, the method comprises
culturing the host cell of invention (into which a recombinant expression vector
encoding afrlRfi polypeptide has been introduced, or into which genome has been
introduced a gene encoding a wild-type or IMI polypeptide) in a suitable medium until
IMI polypeptide is produced In another embodiment, the method further comprises
isolating IMI polypeptides from the medium or the host cell. Another aspect of the
invention pertains to isolated IMI polypeptides, and biologically active portions
thereof. An "isolated" or "purified" polypeptide or biologically active portion thereof
is free of some of the cellular material when produced by recombinant DNA
techniques, or chemical precursors or other chemicals when chemically synthesized.
The language "substantially free of cellular material" includes preparations of IMI
polypeptide in which the polypeptide is separated from some of the cellular
components of the cells in which it is naturally or recombinantly produced. In one
embodiment, the language "substantially free of cellular material" includes
preparations of an IMI polypeptide having less than about 30% (by dry weight) of
non-IMI material (also referred to herein as a "contaminating polypeptide'), more
preferably less than about 20% of non-IMI material, still more preferably less than
about 10% of non-IMI material, and most preferably less than about 5% non-IMI
material.
[0088] When the IMI polypeptide, or biologically active portion thereof, is
recombinantly produced, it is also preferably substantially free of culture medium, i.e.,
culture medium represents less than about 20%, more preferably less than about 10%,
and most preferably less than about 5% of the volume of the polypeptide preparation.
The language "substantially free of chemical precursors or other chemicals" includes
preparations of IMI polypeptide in which the polypeptide is separated from chemical
precursors or other chemicals that are involved in the synthesis of the polypeptide. In
one embodiment, the language "substantially free of chemical precursors or other

chemicals" includes preparations of an IMI polypeptide having less than about 30% (by dry weight) of chemical precursors or chemicals, more preferably less than about 20% chemical precursors or chemicals, still more preferably less than about 10% chemical precursors or chemicals, and most preferably less than about 5% chemical precursors or chemicals. In preferred embodiments, isolated polypeptides, or biologically active portions thereof, lack contaminating polypeptides from the same organism from which the IMI polypeptide is derived. Typically, such polypeptides are produced by recombinant expression of, for example, a Triticum aestivum IMI polypeptide in plants other than Triticum aestivum or microorganisms such as C. glutarnicuiltrcXfiStes, algae, or fungi.
[0089] The IMI polynucleotide and polypeptide sequences of the invention
have a variety of uses. The nucleic acid and amino acid sequences of the present
invention can be used to transform plants, thereby modulating the plant's tolerance to
imidazolinone herbicides. Accordingly, the invention provides a method of producing
a transgenic plant having increased tolerance to an imidazolinone herbicide
comprising, (a) transforming a plant cell with one or more expression vectors
comprising one or more IMI nucleic acids, and (b) generating from the plant cell a
transgenic plant with an increased tolerance to an imidazolinone herbicide as
compared to a wild type plant. In one embodiment, the multiple IMI nucleic acids are
derived from different genomes. Also included in the present invention are methods
of producing a transgenic plant having increased tolerance to an imidazolinone
herbicide comprising, (a) transforming a plant cell with an expression vector
comprising an IMI nucleic acid, wherein the nucleic acid is a non-Imil nucleic acid
and (b) generating from the plant cell a transgenic plant with an increased tolerance to
an imidazolinone herbicide as compared to a wild type plant
[0090] The present invention includes methods of modifying a plant's
tolerance to an imidazolinone herbicide comprising modifying the expression of one or more IMI nucleic acids. The plant's tolerance to the imidazolinone herbicide can be increased or decreased as achieved by increasing or decreasing the expression of an IMI polynucleotide, respectively. Preferably, the plant's tolerance to the imidazolinone herbicide is increased by increasing expression of an IMI polynucleotide. Expression of an IMI polynucleotide can be modified by any method known to those of skill in the art The methods of increasing expression of IMI polynucleotides can be used wherein the plant is either transgenic or not transgenic.

In cases when the plant is transgenic, the plant can be transformed with a vector containing any of the above described IMI coding nucleic acids, or the plant can be transformed with a promoter that directs expression of endogenous IMI polynucleotides in the plant, for example. The invention provides that such a promoter can be tissue specific or developmentally regulated Alternatively, non-transgenic plants can have endogenous IMI polynucleotide expression modified by inducing a native promoter. The expression of polynucleotides comprising a polynucleotide sequence as defined in SEQ ID NO:l in target plants can be accomplished by, but is not limited to, one of the following examples: (a) constitutive promoter, ^b) chemical-induced promoter, and (c) engineered promoter over-expression with for example zinc-finger derived transcription factors (Greisman and Pabo, 1997, Science 275:657).
[0091] In a preferred embodiment, transcription of the IMI polynucleotide is
modulated using zinc-finger derived transcription factors (ZFPs) as described in Greisman and Pabo, 1997, Science 275:657 and manufactured by Sangamo Biosciences, hie. These ZFPs comprise both a DNA recognition domain and a functional domain that causes activation or repression of a target nucleic acid such as an IMI nucleic acid. Therefore, activating and repressing ZFPs can be created that specifically recognize the IMI polynucleotide promoters described above and used to increase or decrease IMI polynucleotide expression in a plant, thereby modulating the herbicide tolerance of the plant
[0092] As described in more detail above, the plants produced by the methods
of the present invention can be monocots or dicots. The plants can be selected from maize, wheat, rye, oat, triticale, rice, barley, soybean, peanut, cotton, rapeseed, canola, manihot, pepper, sunflower, tagetes, solanaceous plants, potato, tobacco, eggplant, tomato, Vicia species, pea, alfalfa, coffee, cacao, tea, Salix species, oil palm, coconut, perennial grass, and forage crops, for example. Forage crops include, but are not limited to, Wheatgrass, Canarygrass, Bromegrass, Wildiye Grass, Bluegrass, Orchardgrass, Alfalfa, Salfoin, Birdsfoot Trefoil, Alsike Clover, Red Clover, and Sweet Clover, hi a preferred embodiment, the plant is a wheat plant or triticale plant. In each of the methods described above, the plant cell includes, but is not limited to, a protoplast, gamete producing cell, and a cell that regenerates into a whole plant As used herein, 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 stable extra-chromosomal element, so that it is passed on to successive generations.
[0093] As described above, the present invention teaches compositions and
methods for increasing the imidazolinone tolerance of a plants or seed as compared to a wild-type plant or seed. In a preferred embodiment, the imidazolinone tolerance of a wheat plant or seed is increased such that the plant or seed can withstand an imidazolinone herbicide application of preferably approximately 10-300 g ai ha"1, more preferably 20-160 g ai ha"1, and most preferably 40-80 g ai ha'1. As used herein, to "witEsteiSa" an imidazolinone herbicide application means that fee plant is either not killed or not injured by such application.
[0094] The present invention provides plants, plant parts, plant organs, plant
tissues, plant cells, seed, and host cells with increased tolerance to at least one imidazolinone herbicide when compared to a wild-type, plant, plant part, plant organ, plant tissue, plant cell, seed, or host cell, respectively. By such a "wild-type plant, plant part, plant organ, plant tissue, plant cell, seed, or host cell" is intended that the plant, plant part, plant organ, plant tissue; plant cell, seed, or host cell, respectively, is wild-type with respect to the herbicide-tolerance characteristics of the plant of ATCC Patent Deposit Number 5625 and/or the IMI nucleic acids of the present invention. That is, such a wild-type plant, plant part, plant organ, plant tissue, plant cell, seed, or host cell does not comprise the herbicide-resistance characteristics of the plant of ATCC Patent Deposit Number 5625 and/or does not comprise the IMI nucleic acids of the present invention. The use of the term "wild-type" is not, therefore, intended to imply that a plant, plant part, plant organ, plant tissue, plant cell, seed, or host cell lacks recombinant DNA in its genome, and/or does not comprise herbicide tolerance characteristics and/or IMI nucleic acids that are different from those herbicide
tolerance characteristics and IMI nucleic acids of the present invention.
[0095] Additionally provided herein is a method of controlling weeds within
the vicinity of a wheat or triticale plant, comprising applying an imidazolinone
herbicide to the weeds and to the wheat or triticale plant, wherein the wheat or
triticale plant has increased tolerance to the imidazolinone herbicide as compared to a
wild type wheat or triticale plant, and wherein the imidazolinone tolerant wheat or
triticale plant comprises at least one IMI nucleic acid, hi one embodiment, the plant
comprises multiple IMI nucleic acids. In another embodiment, the plant comprises an

Imil nucleic acid. By providing for wheat and triticale plants having increased tolerance to imidazolinone, a wide variety of formulations can be employed for protecting wheat and triticale plants from weeds, so as to enhance plant growth and reduce competition for nutrients. An imidazolinone herbicide can be used by itself for pre-emergence, post-emergence, pre-planting, and at-planting control of weeds in areas surrounding the wheat plants described herein or an imidazolinone herbicide formulation can be used that contains other additives. The imidazolinone herbicide can also be used as a seed treatment. Additives found in an imidazolinone herbicide formulation include other herbicides, detergents, adjuvants, spreading agents, sticking agentsTitaBHiSSg agents, or the like. The imidazolinone herbicide formulation can be a wet or dry preparation and can include, but is not limited to, flowable powders, emulsifiable concentrates, and liquid concentrates. The imidazolinone herbicide and herbicide formulations can be applied in accordance with conventional methods, for example, by spraying, irrigation, dusting, or the like.
[0096] The present invention further provides transformation vectors
comprising a selectable marker gene of the invention. The selectable marker gene comprises a promoter that drives expression in a host cell operably linked to a IMI nucleic acid of the invention. The transformation vector can additionally comprise a gene of interest to be expressed in the host cell and can also, if desired, include a chloroplast-targeting sequence that is operably linked to fee polynucleotide of the invention.
[0097] The present invention further provides methods for using the
transformation vectors of the invention to select for cells transformed with the gene of
interest. Such methods involve the transformation of a host cell with the
transformation vector, exposing the cell to a level of an imidazolinone or sulfonylurea
herbicide that would kill or inhibit the growth of a non-4ransformed host cell, and
identifying the transformed host cell by its ability to grow in the presence of the
herbicide, hi one embodiment of the invention, the host cell is a plant cell and the
selectable marker gene comprises a promoter that drives expression in a plant cell.
[0098] The transfonnation vectors of the invention can be used to produce
plants transformed with a gene of interest The transformation vector will comprise a selectable marker gene of the invention and a gene of interest to be introduced and typically expressed in the transformed plant Such a selectable marker gene comprises an IMI nucleic acid of the invention operably linked to a promoter that

drives expression in "a host cell. The DvH nucleic acid comprises the polynucleotide sequence set forth in SEQ ID NO:l, a polynucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO:25 and functional fragments and variants of either of these polynucleotide sequences, wherein the fragment or variant encodes a polypeptide that comprises an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide. For use in plants and plant cells, the transformation vector comprises a selectable marker gene comprising an IMI nucleic acid of the invention operably linked to a promoter that drives expression in a plant cell.
[0099] *^ The invention also relates to a plant expression vector comprising a
promoter that drives expression in a plant operably linked to an IMI nucleic acid of
the invention. The IMI nucleic acid comprises the polynucleotide sequence set forth
in SEQ ID NO:l, a polynucleotide sequence encoding the amino acid sequence set
forth in SEQ ID NO:2, and functional fragments and variants of either of these
polynucleotide sequences, wherein the fragment or variant encodes a polypeptide that
comprises an alanine to threonine substitution at position 96 corresponding to a wild
type AHAS polypeptide. The plant expression vector of the invention does not
depend on a particular promoter, only that such a promoter is capable of driving gene
expression in a plant cell. Preferred promoters include constitutive promoters and
tissue-preferred promoters.
[00100] The genes of interest of the invention vary depending on the desired
outcome. For example, various changes in phenotype can be of interest including modifying the fatty acid composition in a plant, altering the amino acid content of a plant, altering a plant's insect and/or pathogen defense mechanisms, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in plants. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the plant These changes result in a change in phenotype of the transformed plant
[00101] In one embodiment of the invention, the genes of interest include
insect resistance genes such as, for example, Bacillus thuringiensis toxin protein genes (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et ah (1986) Gene 48:109).

[00102] The IMI proteins or polypeptides of the invention can be purified
from, for example, canola plants and can be used in compositions. Also, an isolated IMI nucleic acid encoding an IMI protein of the invention can be used to express an IMI protein of the invention in a microbe such as E, coli or a yeast The expressed IMI protein can be purified from extracts of E. coli or yeast by any method known to those or ordinary skill in the art.
[00103] In certain embodiments of the invention, the methods involve the use
of herbicide-tolerant or herbicide-resistant plants. By an "herbicide-tolerant" or
"herbicide-resistant" plant, it is intended that a plant that is tolerant or resistant to at
least oneli&bittfre at a level that would normally kill, or inhibit the growth ot, a
normal or wild-type plant. In one embodiment of the invention, the herbicide-tolerant
plants of the invention comprise an IMI nucleic acid that encodes an IMI protein.
[00104] For the present invention, the terms "herbicide-tolerant" and
"herbicide-resistant" are used interchangeable and are intended to have an equivalent meaning and an equivalent scope. Similarly, the terms "herbicides-tolerance" and "herbicide-resistance" are used interchangeable and are intended to have an equivalent meaning and an equivalent scope. Likewise, the terms "imidazolinone-resistant" and "imidazolinone-resistance" are used interchangeable and are intended to be of an equivalent meaning and an equivalent scope as the terms "inridazolinone-tolerant" and "imidazolinone-tolerance", respectively.
[00105] The present invention provides plants, plant tissues, plant cells, and
host cells with increased resistance or tolerance to at least one herbicide, particularly a herbicide that interferes with the activity of the AHAS enzyme, more particularly an irnidazolinone or sulfonylurea herbicide. The preferred amount or concentration of the herbicide is an "effective amount" or "effective concentration." By "effective amount" and "effective concentration" is intended an amount and concentration, respectively, that is sufficient to kill or inhibit the growth of a similar, wild-type, plant, plant tissue, plant cell, microspore, or host cell, but that said amount does not kill or inhibit as severely the growth of the herbicide-resistant plants, plant tissues, plant cells, microspores, and host cells of the presort invention. Typically, the effective amount of a herbicide is an amount that is routinely used in agricultural production systems to kill weeds of interest Such an amount is known to those of ordinary skill in the art, or can be easily determined using methods known in the art. Furthermore, it is recognized that the effective amount of a herbicide in an agricultural

production system might be substantially different than an effective amount of a
herbicide in an in vitro plant culture system.
[00106] The IMI nucleic acids of the present invention may be used for
transformation of any plant species, including, but not limited to, monocots and
dicots. Examples of plant species of interest include, but are not limited to, corn or
maize (Zea mays), Brassica sp. (e.g., B. napus, B. rapas B.jimcea), particularly those
Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza
sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet
(e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail
millet JSetdHaiidlica), finger millet (Eleusine coracand)), sunflower (Helianthus
annuus), safflower (Carihamns tinctorius), wheat (Triticwn aestivum, T. Turgidum
ssp. durum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum
tuberosum), peanuts (Arackis Irypogaea), cotton (Gossypium barbadense, Gossypium
hirsutism), sweet potato (Ipomoea batatas), cassava (Mamhot esculenta), coffee
(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees
(Citrus spp.), cocoa (Tfieobroma cacao), tea (Camellia sinensis), banana (Musa spp.),
avocado (Persea ameiicana), fig (Ficus casicd), guava (Psidium guajava), mango
(Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew
(Anacardium occidentale), macadamia (Macadamia integrifolid), almond (Prunus
amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,
vegetables, ornamentals, and conifers. Preferably, plants of fee present invention are
crop plants (for example, sunflower, Brassica sp.t cotton, sugar, beet, soybean,
peanut, alfalfa, safQower, tobacco, corn, rice, wheat, rye, barley triticale, sorghum,
millet, etc.).
[00107] The herbicide resistant plants of the invention find use in methods for
controlling weeds. Thus, the present invention further provides a method for
controlling weeds in the vicinity of a herbicide-resistant plant of the invention. The
method comprises applying an effective amount of a herincide to the weeds and to the
herbicide-resistant plant, wherein the plant has increased resistance to at least one
herbicide, particularly an imidazolinone or sulfonylurea herbicide, when compared to
a wild-type plant. In such a method for controlling weeds, the herbicide-resistant
plants of the invention are preferably crop plants, including, but not limited to,
sunflower, alfalfa, Brassica sp., soybean, cotton, safflower, peanut, tobacco, tomato,
potato, wheat, rice, maize, sorghum, barley, rye, millet, and sorghum.

[00108) By providing plants having increased resistance to herbicides,
particularly imidazolinone and sulfonylurea herbicides, a wide variety of formulations
can be employed for protecting plants from weeds, so as to enhance plant growth and
reduce competition for nutrients. A herbicide can be used by itself for pre-emergence,
post-emergence, pre-planting and at planting control of weeds in areas surrounding the
plants described herein or an imidazolinone herbicide formulation can be used that
contains other additives. The herbicide can also be used as a seed treatment That is
an effective concentration or an effective amount of the herbicide, or a composition
comprising an effective concentration or an effective amount of the herbicide can be
appliedrtirfcetPTto the seeds prior to or during the sowing of the seeds. Additives
found in an imidazolinone or sulfonylurea herbicide formulation or composition
include other herbicides, detergents, adjuvants, spreading agents, sticking agents,
stabilizing agents, or the like. The herbicide formulation can be a wet or dry
preparation and can include, but is not limited to, flowable powders, emulsifiable
concentrates and liquid concentrates. The herbicide and herbicide formulations can be
applied in accordance with conventional methods, for example, by spraying,
irrigation, dusting, coating, and the like.
[00109] The present invention provides non-transgenic and transgenic seeds
with increased tolerance to at least one herbicide, particularly an AHAS-inhibiting herbicide, more particularly an imidazolinone herbicide. Such seeds include, for example, non-transgenic wheat seeds comprising the herbicide-tolerance characteristics of the plant with ATCC Patent Deposit Number 5625, and transgenic seeds comprising an IMI nucleic acid molecule of the invention that encodes a IMI protein.
[00110] The present invention provides methods for producing a herbicide-
resistant plant, particularly a herbicide-resistant wheat or triticale plant, through conventional plant breeding involving sexual reproduction. The methods comprise crossing a first plant that is resistant to a herbicide to a second plant that is not resistant to the herbicide. The first plant can be any of the herbicide resistant plants of the present invention including, for example, transgenic plants comprising at least one of the polynucleotides of the present invention that encode a herbicide resistant IMI protein and non-transgenic wheat plants that comprise the herbicide-tolerance characteristics of the wheat plant with ATCC Patent Deposit Number 5625. The second plant can 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 and
second plants are of the same species. The methods of the invention can further
involve one or more generations of backcrossing the progeny plants of the first cross
to a plant of the same line or genotype as either the first or second plant.
Alternatively, the progeny of the first cross or any subsequent cross can be crossed to
a third plant that is of a different line or genotype than either the first or second plant.
The methods of the invention can additionally involve selecting plants that comprise
the herbicide tolerance characteristics of the first plant
[00111] The present invention further provides methods for increasing the
herbicide-rfeis^ice of a plant, particularly a herbicide-resistant wheat plant, through
conventional plant breeding involving sexual reproduction. The methods comprise
crossing a first plant that is resistant to a herbicide to a second plant that may or may
not be resistant to the herbicide or may be resistant to different herbicide or herbicides
than the first plant. The first plant can be any of the herbicide resistant plants of the
present invention including, for example, transgenic plants comprising at least one of
the IMI nucleic acids of the present invention that encode IMI protein and non-
transgenic wheat and triticale plants that comprise the herbicide-tolerance
characteristics of the wheat plant with ATCC Patent Deposit Number 5625. The
second plant can 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 and
second plants are of the same species. The progeny plants produced by this method of
the present invention have increased resistance to a herbicide when compared to either
the first or second plant or both. When the first and second plants are resistant to
different herbicides, the progeny plants will have the combined heibicide tolerance
characteristics of the first and second plants. The methods of the invention can further
involve one or more generations of backcrossing the progeny plants of the first cross
to a plant of the same line or genotype as either the first or second plant
Alternatively, the progeny of the first cross or any subsequent cross can be crossed to
a third plant that is of a different line or genotype than either the first or second plant
The methods of the invention can additionally involve selecting plants that comprise
the herbicide tolerance characteristics of the first plant, the second plant, or both the
first and the second plant
[00112] The plants of the present invention can be transgenic or non-transgenic.
An example of a non-transgenic wheat plant having increased resistance to

imidazolinone is the wheat plant (Shiloh-8) having ATCC Patent Deposit No. 5625; or mutant, recombinant, or a genetically engineered derivative of the plant having ATCC Patent Deposit No. 5625; or of any progeny of the plant having ATCC Patent Deposit No. 5625; or a plant that is a progeny of any of these plants; or a plant that comprises the herbicide tolerance characteristics of the plant having ATCC Patent Deposit No. 5625.
[00113] The present invention also provides plants, plant organs, plant tissues,
plant cells, seeds, and non-human host cells that are transformed with the at least one polynucleotide molecule, expression cassette, or transfonnation vector of the invention. *SucB 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 levels of the herbicide that kill or inhibit the growth of an untransformed plant, plant tissue, plant cell, or non-human host cell, respectively. Preferably, the transformed plants, plant tissues, plant cells, and seeds of the invention are Arabidopsis thaliana and crop plants.
[00114] The present invention provides methods that involve the use of at least
one AHAS-inhibiting herbicide selected from the group consisting of imidazolinone herbicides, sulfonylurea herbicides, triazolopyrimidine heibicides, pyrimidinyloxybenzoate herbicides, sulfonylamino-carbonyltriazolinone heibicides, and mixtures thereof. In these methods, the AHAS-inhibiting herbicide can be applied by any method known in the art including, but not limited to, seed treatment, soil treatment, and foliar treatment.
[00115] Prior to application, the AHAS-inhibiting heriricide can be converted
into the customary formulations, for example solutions, emulsions, suspensions, dusts, powders, pastes and granules. The use form depends on the particular intended purpose; in each case, it should ensure a fine and even distribution of the compound according to the invention.
[00116] The formulations are prepared in a known manner (see e.g. for review
US 3,060,084, EP-A 707 445 (for liquid concentrates), Browning, "Agglomeration", Chemical Engineering, Dec. 4,1967,147-48, Perry's Chemical Engineer's Handbook, 4th Ed., McGraw-Hill, New York, 1963, pages 8-57 and et seq. WO 91/13546, US 4,172,714, US 4,144,050, US 3,920,442, US 5,180,587, US 5,232,701, US 5,208,030, GB 2,095,558, US 3,299,566, Klingman, Weed Control as a Science, John Wiley and Sons, Inc., New York, 1961, Hance et al., Weed Control Handbook, 8th Ed.,

Blackwell Scientific Publications, Oxford, 1989 andMollet, H., Grubemann, A.,
Formulation technology, Wiley VCH Verlag GmbH, Weinheim (Germany), 2001, 2.
D. A. Knowles, Chemistry and Technology of Agrochemical Formulations, Kluwer
Academic Publishers, Dordrecht, 1998 (ISBN 0-7514-0443-8), for example by
extending the active compound with auxiliaries suitable for the formulation of
agrochemicals, such as solvents and/or carriers, if desired emulsifiers, surfactants and
dispersants, preservatives, antifoaming agents, anti-freezing agents, for seed treatment
formulation also optionally colorants and/or binders and/or gelling agents.
[00117] Examples of suitable solvents are water, aromatic solvents (for
examplFSflfreSSb products, xylene), paraffins (for example mineral oil fractions), alcohols (for example methanol, butanol, pentanol, benzyl alcohol), ketones (for example cyclohexanone, gamma-butyrolactone), pyrrolidones (NMP, NOP), acetates (glycol diacetate), glycols, fatty acid dimethylamides, fatty acids and fatty acid esters. In principle, solvent mixtures may also be used.
[00118] Examples of suitable carriers are ground natural minerals (for example
kaolins, clays, talc, chalk) and ground synthetic minerals (for example highly disperse silica, silicates).
[00119] Suitable emulsifiers are nonionic and anionic emulsifiers (for example
polyoxyethylene fatty alcohol ethers, alkylsulfonates and aiyisulfonates).
[00120] Examples of dispersants are Ugnin-sulfite waste liquors and
methylcellulose.
[00121] Suitable surfactants used are alkali metal, alkaline earth metal and
ammonium salts of lignosulfonic acid, naphthalenesulfonic acid, phenolsulfonic acid, dibutylnaphthalenesulfonic acid, alkylarylsulfonates, alkyi sulfates, alkylsulfonates, fatty alcohol sulfates, fatty acids and sulfated fatty alcohol glycol ethers, furthermore condensates of sulfonated naphthalene and naphthalene derivatives with formaldehyde, condensates of naphthalene or of naphthalenesulfonic acid with phenol and formaldehyde, polyoxyethylene octylphenol ether, ethoxyiated isooctylphenol, octylphenol, nonylphenol, alkylphenol polyglycol ethers, tributylphenyl polyglycol ether, tristearylphenyl polyglycol ether, alkylaryl polye&er alcohols, alcohol and fatty alcohol ethylene oxide condensates, ethoxyiated castor oil, polyoxyethylene alkyi ethers, ethoxyiated polyoxypropylene, lauryl alcohol polyglycol ether acetal, sorbitol esters, lignosulfite waste liquors and methylcellulose.

[00122] Substances which are suitable for the preparation of directly sprayable
solutions, emulsions, pastes or oil dispersions are mineral oil fractions of medium to
high boiling point, such as kerosene or diesel oil, furthermore coal tar oils and oils of
vegetable or animal origin, aliphatic, cyclic and aromatic hydrocarbons, for example
toluene, xylene, paraffin, tetrahydronaphthalene, alkylated naphthalenes or their
derivatives, methanol, ethanol, propanol, butanol, cyclohexanol, cyclohexanone,
isophorone, highly polar solvents, for example dimethyl sulfoxide, N-
methylpyrrolidone or water.
[00123] Also anti-freezing agents such as glycerin, ethylene glycol, propylene
glycorand*Bacteficides such as can be added to the formulation-
[00124] Suitable antifoaming agents are for example antifoaming agents based
on silicon or magnesium stearate.
[00125] Suitable preservatives are for example Dichlorophen und
enzylalkoholhemiformaL
[00126] Seed Treatment formulations may additionally comprise binders and
optionally colorants.
[00127] Binders can be added to improve the adhesion of the active materials
on the seeds after treatment. Suitable binders are block copolymers EO/PO surfactants
but also polyvinylalcoholsl, polyvinylpyrrolidones, polyacrylates, polymethacrylates,
polybutenes, polyisobutylenes, polystyrene, polyetitiyleneammes, polyethyieneamides,
polyethyleneimines (Lupasol®, Polymin®), polyethers, polyurethans,
polyvinylacetate, tylose and copolymers derived from these polymers.
[00128] Optionally, also colorants can be included in the formulation. Suitable
colorants or dyes for seed treatment formulations are Rhodamin B, C J. Pigment Red
112, C.L 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.
[00129] An examples of a suitable gelling agent is carrageen (Satiagel^.
[00130] Powders, materials for spreading, and dustable products can be
prepared by mixing or concomitantly grinding the active substances with a solid
carrier.

[00131] Granules, for example coated granules, impregnated granules and
homogeneous granules, can be prepared by binding the active compounds to solid
carriers. Examples of solid carriers are mineral earths such as silica gels, silicates,
talc, kaolin, attaclay, limestone, hme, chalk, bole, loess, clay, dolomite, diatomaceous
earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic
materials, fertilizers, such as, for example, ammonium sulfate, ammonium phosphate,
ammonium nitrate, ureas, and products of vegetable origin, such as cereal meal, tree
bark meal, wood meal and nutshell meal, cellulose powders and other solid carriers.
[00132] In general, the formulations comprise from 0.01 to 95% by weight,
preferaBTy frbnTO.l to 90% by weight, of the AHAS-inhibiting herbicide. In this case, the AHAS-inhibiting herbicides are employed in a purity of from 90% to 100% by weight, preferably 95% to 100% by weight (according to NMR spectrum). For seed treatment purposes, respective formulations can be diluted 2-10 fold leading to concentrations in the ready to use preparations of 0.01 to 60% by weight active compound by weight, preferably 0.1 to 40% by weight
[00133] The AHAS-inhibiting herbicide can be used as such, in the form of
their formulations or the use forms prepared therefrom, for example in the form of
directly sprayable solutions, powders, suspensions or dispersions, emulsions, oil
dispersions, pastes, dustable products, materials for spreading, or granules, by means
of spraying, atomizing, dusting, spreading or pouring. The use forms depend entirely
on the intended purposes; they are intended to ensure in each case the finest possible
distribution of the AHAS-inhibiting herbicide according to the invention.
[00134] Aqueous use forms can be prepared from emulsion concentrates, pastes
or wettable powders (sprayable powders, oil dispersions) by adding water. To prepare emulsions, pastes or oil dispersions, the substances, as such or dissolved in an oil or solvent, can be homogenized in water by means of a wetter, tadrifier, dispersant or emulsifier. However, it is also possible to prepare concentrates composed of active substance, wetter, tackifier, dispersant or emulsifier and, if appropriate, solvent or oil, and such concentrates are suitable for dilution with water.
[00135] The active compound concentrations in the ready-to-use preparations
can be varied within relatively wide ranges. In general, they are from 0.0001 to 10%, preferably from 0.01 to 1% per weight.
[00136] The AHAS-inhibiting herbicide may also be used successfully in the
ultra-low-volume process (ULV), it being possible to apply formulations comprising

over 95% by weight of active compound, or even to apply the active compound
without additives.
[00137] The following are examples of formulations:
[00138] 1. Products for dilution with water for foliar applications. For
seed treatment purposes, such products may be applied to the seed diluted or undiluted.

[00139] [00140]
[00141] [00142]
[00143] [00144]
[00145] [00146]

A) Water-soluble concentrates (SL, LS)
Ten parts by weight of the AHAS-inhibiting heibicide are dissolved in 90 parts by weight of water or a water-sohible solvent- As an alternative, welters or other auxiliaries are added The AHAS-inhibiting heibicide dissolves upon dilution with water, whereby a formulation with 10 % (w/w) of AHAS-inhibiting herbicide is obtained,
B) Dispersible concentrates (DC)
Twenty parts by weight of the AHAS-inhibiting herbicide are dissolved in 70 parts by weight of cyclohexanone with addition of 10 parts by weight of a dispersant, for example polyvinylpyrrolidone. Dilution with water gives a dispersion, whereby a formulation with 20% (w/w) of AHAS-inhibiting herbicide is obtained.
C) Emulsifiable concentrates (EC)
Fifteen parts by weight of the AHAS-inhibiting herbicide are dissolved in 7 parts by weight of xylene with addition of calcium dodecylbenzenesulfonate and castor oil ethoxylate (in each case 5 parts by weight). Dilution with water gives an emulsion, whereby a formulation with 15% (w/w) of AHAS-inhibiting herbicide is obtained.
D) Emulsions (EW, EO, ES)
Twenty-five parts by weight of the AHAS-inhibiting herbicide are dissolved in 35 parts by weight of xylene with addition of calcium dodecylbenzenesulfonate and castor oil ethoxylate (in

each case 5 parts by weight). This mixture is introduced into 30 parts by weight of water by means of an emulsifier machine (e.g. Ultratuirax) and made into a homogeneous emulsion. Dilution with water gives an emulsion, whereby a formulation with 25% (w/w) of AHAS-inhibiting herbicide is obtained.
[00147] E) Suspensions (SC, OD, FS)
[00148] In an agitated ball mill, 20 parts by weight of the AHAS-
inhibiting herbicide are conrarintrted with addition of 10 parts by weight of dispersants, welters and 70 parts by weight of water or of an organic solvent to give a fine AHAS-inhibiting herbicide suspension. Dilation with water gives a stable suspension of the AHAS-inhibiting hearbicide, whereby a formulation with 20% (w/w) of AHAS-inhibiting herbicide is obtained.
[00149] F) Water-dispersible granules and water-soluble granules
(WG, SG)
[00150] Fifty parts by weight of the AHAS-inhibiting herbicide are
ground finely with addition of 50 parts by weight of dispersants and wetters and made as water-dispersible or water-soluble granules by means of technical appliances (for example extrusion, spray tower, fluidized bed). Dilution with water gives a stable dispersion or solution of the AHAS-inhibiting herbicide, whereby a formulation with 50% (w/w) of AHAS-inhibiting herbicide is obtained.
[00151] G) Water-dispersible powders and water-soluble powders
(WP, SP, SS, WS)
[00152] Seventy-five parts by weight of the AHAS-inhibiting herbicide
are ground in a rotor-stator mill wife addition of 25 parts by weight of dispersants, wetters and silica gel. Dilution with water gives a stable dispersion or solution of the AHAS-inhibiting herbicide, whereby a formulation with 75% (w/w) of AHAS-inhibiting herbicide is obtained.

[00153] I) Gel-Formulation (GF)
[00154] In an agitated ball mill, 20 parts by weight of the AHAS-
inhibiting herbicide are comminuted with addition of 10 parts by weight of dispersants, 1 part by weight of a gelling agent wetters and 70 parts by weight of water or of an organic solvent to give a fine AHAS-inhibiting herbicide suspension. Dilution with water gives a stable suspension of the AHAS-inhibiting herbicide, whereby a formulation with 20% (w/w) of AHAS-inhibiting herbicide is obtained. This gel formulation is suitable for us as a seed treatment
[01)155] z. Products to be applied undiluted for foliar applications. For
seed treatment purposes, such products may be applied to the seed diluted.
[00156] A) Dustable powders (DP, DS)
[00157] Five parts by weight of the AHAS-inhibiting herbicide are
ground finely and mixed intimately with 95 parts by weight of finely divided kaolin. This gives a dustable product having 5% (w/w) of AHAS-inhibiting herbicide.
[00158] B) Granules (GR, FG, GG, MG)
[001591 One-half part by weight of the AHAS-inhibiting herbicide is
ground finely and associated with 95.5 parts by weight of carriers, whereby a formulation with 0.5% (w/w) of AHAS-ihhibiting herbicide is obtained. Current methods are extrusion, spray-drying or the fluidized bed. This gives granules to be applied undiluted for foliar use.
[00160] Conventional seed treatment formulations include for example
flowable concentrates FS, solutions LS, powders for dry treatment DS, water dispersible powders for slurry treatment WS, water-soluble powders SS and emulsion ES and EC and gel formulation GF. These formulations can be applied to the seed diluted or undiluted. Application to the seeds is carried out before sowing, either directly on the seeds.
[001611 In a preferred embodiment a FS formulation is used for seed treatment
Typcially, a FS formulation may comprise 1-800 g/1 of active ingredient, 1-200 gA

Surfactant, 0 to 200 g/1 antifreezing agent, 0 to 400 g/1 of binder, 0 to 200 g/1 of a pigment and up to 1 liter of a solvent, preferably water.
[00162] The present invention non-transgenic and transgenic seeds of the
herbicide-resistant plants of the present invention. Such seeds include, for example,
non-transgenic wheat seeds comprising the herbicide-tolerance characteristics of the
plant with ATCC Patent Deposit Number 5625, and transgenic seeds comprising a
polynucleotide molecule of the invention that encodes an IMI protein.
[00163] For seed treatment, seeds of the herbicide resistant plants according of
the present invention are treated with herbicides, preferably herbicides selected from
the group dShsistmg of AHAS-inhibiting herbicides such as anridosulfuron,
azimsulfuron, bensulfuron, chlorimuron, cMorsulfuron, ciiK>sulfim>n,
cyclosulfamuron, ethametsulfuron, elhoxysulfuron, flazasulforon, flupyrsulfuron,
foramsulfuron, halosulfuron, imazosulfuron, iodosulfuron, mesosulfuron,
metsulfuron, nicosulforon, oxasulfuron, primisulfuron, prosul&ron, pyrazosulfuron,
rimsulfuron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfuron, tribenuron,
trifloxysulfuron, triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic,
rmazapyr, imazaquin, imazethapyr, cloransulam, diclosulam, florasulam, flumetsulam,
metosulam, penoxsulam, bispyribac, pyriminobac, propoxycarbazone, flucafbazone,
pyribenzoxim, pyriftalid, pyrithiobac, and mixtures thereof or with a formulation
comprising a AHAS-inhibiting herbicide.
[00164] The term seed treatment comprises all suitable seed treatment
techniques known in the art, such as seed dressing, seed coating, seed dusting, seed soaking, and seed pelleting.
[00165] In accordance with one variant of the present invention, a further
subject of the invention is a method of treating soil by the application, in particular
into the seed drill: either of a granular formulation containing the AHAS-inhibiting
herbicide as a composition/formulation (e.g .a granular formulation, with optionally
one or more solid or liquid, agriculturally acceptable carriers and/or optionally with
one or more agriculturally acceptable surfactants. This method is advantageously
employed, for example, in seedbeds of cereals, maize, cotton, and sunflower.
[00166] The present invention also comprises seeds coated with or containing
with a seed treatment formulation comprising at least one AHAS-inhibiting herbicide selected from the group consisting of amidosulfiiron, azimsulfuron, bensulfuron, chlorimuron, chlorsulfuron, cinosulfuron, cyclosulfamuron, ethametsulfiiron,

ethoxysulfuron, flazasulfuron, flupyrsulfuron, foramsulfijron, halosulforon, imazosrulfuron, iodosulforon, mesosulfuron, metsul&ron, nicosuliuron, oxasulfiiron, primisulftrron, prosulfuron, pyrazosuLforon, rimsul&ron, sulfometuron, sulfosulfuron, thifensulfuron, triasulfbron, tribenuron, trifloxysulforon, triflusulfuron, tritosulfuron, imazamethabenz, imazamox, imazapic, imazapyr, imazaquin, imazethapyr, cloransulam, diclosulam, florasulam, flumetsulam, metosulam, penoxsulam, bispyribac, pyriminobac, propoxycarbazone, flucaibazone, pyribenzoxim, pyriftaHd and pyrithiobac.
[00167] The term seed embraces seeds and plant propagules of all kinds
including ffut not limited to true seeds, seed pieces, suckers, conns, bulbs, fruit, tubers, grains, cuttings, cut shoots and the like and means in a preferred embodiment true seeds.
[00168] The term "coated with and/or containing" generally signifies that the
active ingredient is for the most part on the surface of the propagation product at the
time of application, although a greater or lesser part of the ingredient may penetrate
into the propagation product, depending on the method of application. When the said
propagation product is (re)planted, it may absorb the active ingredient.
[00169] The seed treatment application with the AHAS-inhibiting herbicide or
with a formulation comprising the AHAS-inhibiting herbicide is carried out by spraying or dusting the seeds before sowing of the plants and before emergence of the plants.
[00170] In the treatment of seeds, the corresponding formulations are applied
by treating the seeds with an effective amount of the AHAS-inhibiting herbicide or a formulation comprising the AHAS-inhibiting herbicide. Herein, the application rates are generally from 0.1 g to 10 kg of the ai. (or of the mixture of a.L or of the formulation) per 100 kg of seed, preferably from 1 g to 5 kg per 100 kg of seed, in particular from 1 g to 2.5 kg per 100 kg of seed. For specific crops such as lettuce the rate can be higher.
[00171] The present invention provides a method for combating undesired
vegetation or controlling weeds comprising contacting the seeds of the resistant plants according to the present invention before sowing and/or after pregermination with an AHAS-inhibiting herbicide. The method can further comprise sowing the seeds, for example, in soil in a field or in a potting medium in greenhouse. The method finds

particular use in combating undesired vegetation or controlling weeds in the immediate vicinity of the seed.
[00172] The control of undesired vegetation is understood as meaning the
killing of weeds and/or otherwise retarding or inhibiting the normal growth of the
weeds. Weeds, in the broadest sense, are understood as meaning all those plants
which grow in locations where they are undesired.
[00173] The weeds of the present invention include, for example,
dicotyledonous and monocotyledonous weeds. Dicotyledonous weeds include, but are not limited to, weeds of the genera: Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, ftalinsbga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Ciisium, Carduus, Sonchus, Solanum, Rorippa, Rotala, Iindeoria, Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, and Taraxacum. Monocotyledonous weeds include, but axe not limited to, weeds of of the genera: Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristyslis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, and
Apera.
[00174] In addition, the weeds of the present invention can include, for
example, crop plants that are growing in an undesired location. For example, a
volunteer maize plant that is in a field that predominantly comprises soybean plants
can be considered a weed, if the maize plant is undesired in the field of soybean
plants.
[00175] The articles "a" and "an" are used herein to refer to one or more than
one (i.e., to at least one) of the grammatical object of the article. By way of example,
"an element" means one or more elements.
[00176] As used herein, the word "comprising," or variations such as
"comprises" or "comprising," will be understood to imply the inclusion of a stated
element, integer or step, or group of elements, integers or steps, but not the exclusion
of any other element, integer or step, or group of elements, integers or steps.
[00177] The following examples are offered by way of illustration and not by
way of limitation.

EXAMPLE 1
Mutagenesis and Selection of Tolerant Wheat Lines
[00178] Samples of 1,500 Shiloh variety seeds were each placed in a 1,000 ml
beaker and covered with deiomzed water to at least 1 inch above the seed level. The beakers were then placed in a refrigerator at 4°C for 15-20 hours. The seed samples were removed from the refrigerator and brought up to room temperature over an approximately 3 hour period by placing the beaker at room temperature. In some cases, the wanning process was accelerated by adding deiomzed water to the beakers. [00179J~ *~ ^fhe deionized water was drained off die seeds, and the beaker was filled with a sodium azide solution to at least 1 inch above the seed leveL The sodium azide solution was prepared by adding 27.218 g KH2PO4 to 1,500 ml deionized water, bringing the solution to pH 3 with concentrated H3PO4, and bringing the final solution to 2 L volume with deionized water. Just prior to use, 0.2604 g NaN3 was added, and the solution was kept in the dark. After addition of the sodium azide solution to the seeds, the beakers were incubated in a dark area at room temperature for 2 hours, with occasional stirring.
[00180] The sodium azide treatment solution was decanted, and the seed
samples were rinsed twice with deionized water. Then the seed samples were covered with deionized water to at least 1 inch above seed level and soaked at room temperature for 1 hour, with occasional stirring. The deionized water was decanted, and the seeds were spread evenly on paper towels to dry. The seeds were planted in the field near Berthoud, Colorado in six 5 feet by 40 feet plots. Approximately 15 pounds of M2 seed were harvested, and approximately 466,000 seeds wore planted near Platteville, Colorado. Hie fields were sprayed with lx (40 g ai ha"1 (imazamox)) or 2x (80 g ai ha"1 (imazamox)).
[00181] Plants tolerant to the herbicide were identified and transplanted into 1
gallon pots, and put into vernalization for 4 weeks at 45 °F. Fourteen single plant selections were made out of the 2x rate area. The tolerant M2 plants were taken out of vernalization, grown out in a Berthoud, Colorado greenhouse, and M3 plants ware planted in approximately 4 feet by 5 feet plots near Berthoud, Colorado. The plots were sprayed with 80 g ai ha-1 (imazamox) when the plants were at the three-leaf stage, and the results of the fourteen progenies were rated as shown in Table 1.



gene. This mutant Alsl polynucleotide encodes a mutant AHAS polypeptide that has an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide and that confers upon the plant tolerance to imidazolinone herbicides. The Shiloh-8 line did not harbor a mutation in thcAls2 or Als3 genes.
EXAMPLE 3 Characterization of the Imidazolinone Tolerant Trait of the Shiloh-8 Line
[00186] Agronomic and comparative imidazolinone herbicide imazamox
tolerance were then evaluated under field conditions. Table 4 summarizes the yield and agronomic evaluations comparing Shiloh-8 and wild type Shiloh plants. These trials were typical cereal evaluation experiments using incomplete randomized block three replication design. Plots were 1.54 m x 4.62 m at harvest Table 5 summarizes multiple field imidazolinone herbicide imazamox tolerance comparisons between Shiloh-8 and the standard tolerant control 9804. These plots ranged from a single row 1 m to larger 1.54 m x 4.62 m plots.



[00187] All publications and patent applications mentioned in the specification
are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[00188] Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.










THAT WHICH IS CLAIMED:
1. A wheat plant comprising at least one IMI nucleic acid selected from
the group consisting of:
(a) a Triticum aestivum Imil nucleic acid encoding an IMI protein comprising a mutation in Domain C that results in a alanine to threonine substitution in the IMI protein as compared to a wild-type AHAS protein,
(b) an IMI nucleic acid comprising the polynucleotide sequence set forth in SEQIDNO:l,and
(c) an IMI nucleic acid encoding a proton comprising the amino acid sequence set forth in SEQ ID NO:2;
wherein the IMI nucleic acid confers upon the plant increased tolerance to an imidazolinone herbicide as compared to a wild-type wheat plant
2. The wheat plant of claim 1, wherein the plant comprises the Triticum aestivum Imil nucleic acid of (a).
3. The wheat plant of claim 1, wherein the plant comprises the IMI nucleic acid of (b).
4. The wheat plant of claim 1, wherein the plant comprises the IMI nucleic acid of (c).
5. The wheat plant of claim 1 or 2, wherein the Triticum aestivum Imil nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
(i) the polynucleotide sequence set forth in SEQ ID NO:l;
(ii) a polynucleotide sequence encoding the polypeptide set forth in
SEQ ID NO:2; (iii) a polynucleotide sequence comprising at least 95% sequence
identity to the complement of the polynucleotide sequence set
forth in SEQ ID NO: 1;

(iv) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and
(v) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (i) or (ii).
6. The wheat plant of any one of claims 1-5, wherein the alanine to threonine substitution is at position 96 corresponding to a wild type AHAS polypeptide.
7. The wheat plant of any one of claims 1-6, wherein the plant is not
transgenic.
8. The wheat plant of any one of claims 1-6, wherein the plant is transgenic.
9. The wheat plant of any one of claims 1-8, wherein the imidazolinone herbicide is selected from the group consisting of [2- (4-isopropyl-4-methyl-5-oxo-2-] imidiazolin-2-yl)-nicotinic acid, 2- (4-isopropyl)-4-methyl-5-oxo-2- imidazolin-2-yl) -3-quinolinecarboxyHc acid, [5-ethyl-2- (4-isopropyl~4-methyl-] 5-oxo-2-imidazolin-2-yl) -nicotinic acid, 2- (4-isopropyl-4-methyl-5-oxo-2- imidazolin-2-yI)-5-(methoxymethyl)-nicotinic acid, 2- (4-isopropyl-4-methyi- 5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid, and a mixture of methyl 6- (4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl) -m-toluate, methyl [2- (4-j isopropyl^methyl-5K>xo-2-imidazolin-2-yl)-p-toluate, and mixtures thereof.
10. The wheat plant of any one of claims 1-9, wherein the imidazolinone herbicide is [5-ethyl-2- (4-isopropyl-4-methyl-] 5-oxo-2-imidazolin-2-yl) -nicotinic acid.
11. The wheat plant of any one of claims 1-9, wherein the imidazolinone herbicide is 2- (4-isopropyl-4-methyl-5-oxo-2- imidazolin-2-yl)-5- (methoxymethyl)-nicotinic acid.

12. A plant part of the wheat plant of any one of claims 1-11.
13. A plant cell of the wheat plant of any one of claims 1-11.
14. A seed produced by the wheat plant of any one of claims 1-11, wherein the seed comprises at least one of the IMI nucleic acids.
15. A wheat plant comprising the heibicide tolerance characteristics of the plant with American Type Culture Collection (ATCQ Patent Deposit Designation Numb^pfA^5.
16. The wheat plant of claim 15, wherein:

(a) the wheat plant has ATCC Patent Deposit Designation Number PTA-5625;
(b) the wheat plant is a recombinant or genetically engineered derivative of the plant with ATCC Patent Deposit Designation Number PTA-5625;
(c) the wheat plant is any progeny of the plant with ATCC Patent Deposit Designation Number PTA-5625; or
(d) the wheat plant is a progeny of any of the plants of (a) through (c).

17. The wheat plant of claim 15 or 16, wherein the wheat plant is a Triticum aestivum wheat plant.
18. The wheat plant of any one of claims 15-17, wherein the plant has increased tolerance to an imidazolinone heibicide as compared to a wild-type wheat plant.
19. The wheat plant of claim 18, wherein the imidazolinone herbicide is selected from the group consisting of [2- (4-isopropyl-4-methyl-5-oxo-2-] imidiazolin-2-yl)~nicotinic acid, 2- (4-isopropyI)-4-methyl-5-oxo-2~ imidazolin-2-yi) -3-quinolinecarboxylic acid, [5-ethyI-2- (4-isopropyl-4-methyi-] 5-oxo-2-imidazolin-2-yl) -nicotinic acid, 2- (4-isopropyl-4-methyl-5-oxo-2- imidazolin-2-yI)-5-(methoxymethyl)-nicotinic acid, 2- (4-isopropyl-4-methyl- 5-oxo-2-imidazolin-2-yl)-5-methylnicotinic acid, and a mixture of methyl 6- (4-isopropyl-4-methyl-5-oxo-2-

imidazolin-2-yl) -m-toluate, methyl [2- (4-j isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)-p-toluate, and mixtures thereof.
20. A plant part of the wheat plant of any one of claims 15-19.
21. A plant cell of the wheat plant of any one of claims 15-19.
22. A seed produced by the wheat plant of any one of claims 15-19, wherein the seed comprises the herbicide tolerance characteristics of the plant with ATCCl?atent Deposit Designation Number PTA-5625.
23. A triticale plant comprising at least one IMI nucleic acid selected from the group consisting of:

(a) a Triticum aestivum hnil nucleic acid encoding an IMI protein comprising a mutation in Domain C that results in a alanine to threonine substitution in the IMI protein as compared to a wild-type AHAS protein,
(b) an IMI nucleic acid comprising the polynucleotide sequence set forth in SEQIDNO:l,and
(c) an IMI nucleic acid encoding a protein comprising the amino acid sequence set forth in SEQ ID NO:2;
wherein the IMI nucleic acid confers upon the plant increased tolerance to an imidazolinone herbicide as compared to a wild-type triticale plant
24. The triticale plant of claim 23, wherein the plant comprises the Triticum aestivum hnil nucleic acid of (a).
25. The triticale plant of claim 23, wherein the plant comprises the IMI nucleic acid of (b).
26. The triticale plant of claim 23, wherein the plant comprises the IMI nucleic acid of (c).

27. The triticale plant of claim 23 or 24, wherein the Triticwn aestivum
Imil nucleic acid comprises a polynucleotide sequence selected from the group
consisting of:
(i) the polynucleotide sequence set forth in SEQ ID NO; 1 ;
(ii) a polynucleotide sequence encoding the polypeptide set forth in
SEQ ID NO:2; (iii) a polynucleotide sequence comprising at least 95% sequence
identity to the complement of the polynucleotide sequence set
forth in SEQ ID NO:l; **~ ""X*V) a polynucleotide sequence encoding an amino acid sequence
comprising at least 95% sequence identity to the amino acid
sequence set forth in SEQ ID NO:2; and (v) a polynucleotide sequence comprising at least 60 consecutive
nucleotides of (i) or (ii).
28. The triticale plant of any one of claims 23-27, wherein the alanine to threonine substitution is at position 96 corresponding to a wild type AHAS polypeptide.
29. The triticale plant of any one of claims 23-28, wherein the plant is transgenic or non-transgenic
30. A plant part of the triticale plant of any one of claims 23-29.
31. A plant cell of the triticale plant of any one of claims 23-29.
32. A seed produced by the triticale plant of any one of claims 23-29, wherein the seed comprises at least one of the IMI nucleic acids.
33. A triticale plant comprising the herbicide tolerance characteristics of the plant with ATCG Patent Deposit Designation Number PTA-5625.
34. The triticale plant of claim 33, wherein:

(a) the triticale plant is a recombinant or genetically engineered derivative of the plant with ATCC Patent Deposit Designation Number PTA-5625;
(b) the triticale plant is any progeny of the plant with ATCC Patent Deposit Designation Number PTA-5625; or
(c) the triticale plant is a progeny of any of the plants of (a) through (b).

35. The triticale plant of claim 33 or 34, wherein the plant has increased tolerance to an imidazolinone herbicide as compared to a wild-type triticale plant
36. A plant part of the triticale plant of any one of claims 33-35.
37. A plant cell of the triticale plant of any one of claims 33-35.
38. A seed produced by the triticale plant of any one of claims 33-35, wherein the seed comprises the herbicide tolerance characteristics of the plant with
. ATCC Patent Deposit Designation Number PTA-5625.
39. An isolated IMI nucleic acid comprising a polynucleotide sequence
selected from the group consisting of:
(a) the polynucleotide sequence set forth in SEQ ID NO:l;
(b) a polynucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2;
(c) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID NO:l, wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;

(e) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (a) or (b), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(f) a polynucleotide sequence that is coinplementary to any one of (a), (b), (c), (d), or (e), wherein the complement of the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide.
40. *~ An expression cassette comprising a promoter operably linked to the
IMI nucleic acid of claim 39.
41. The expression cassette of claim 40, wherein the promoter is capable of driving gene expression in a bacterium, a fungal cell, an animal cell, or a plant cell.
42. A non-human host cell transformed with the expression cassette of claim 40 or 41.
43. The host cell of claim 42, wherein the host cell is selected from the group consisting of a bacterium, a fungal cell, an animal cell, and a plant cell.
44. A transformation vector comprising a gene of interest and a selectable marker gene, the selectable marker gene comprising a promoter operably linked to the IMI nucleic acid of claim 39, wherein the promoter drives expression in a host cell.
45. The transformation vector of claim 44, wherein the host cell is selected from the group consisting of a bacterium, a fungal cell, an animal cell, and a plant cell.
46. A transformed plant comprising stably incorporated in its genome a polynucleotide molecule comprising an IMI nucleic acid operably linked to a promoter that drives expression in a plant cell, wherein the IMI nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
(a) the polynucleotide sequence set forth in SEQ ID NO:l;

(b) a polynucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2;
(c) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID NO:l, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(e) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (a) or (b), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(f) a polynucleotide sequence that is complementary to any one of (a), (b), (c), (d), or (e); wherein the complement of the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
wherein the IMI nucleic acid confers upon the plant increased tolerance to an imidazolinone herbicide as compared to an untransfonned plant
47. The transformed plant of claim 46, wherein the promoter is selected from the group consisting of constitutive promoters and tissue-preferred promoters.
48. The transformed plant of claim 46 or 47, wherein the IMI nucleic acid further comprises an operably linked chloroplast-targeting sequence.
. 49. The transformed plant of any one of claims 46-48, wherein the AHAS activity of the transformed plant is increased relative to an untransfonned plant.

50. The transformed plant of claim 46, wherein the imidazolrnone
herbicide is selected from the group consisting of: [2- (4-isopropyl-4-methyl-5-oxo-2-
] imidiazolin-2-yl)-nicotinic acid, 2- (4-isopropyl)-4-methyl-5-oxo-2- imidazolin-2-yl)
-3-quinolinecarboxylic acid, [5-ethyl-2- (4-isopropyl-4-methyl-J 5-oxo-2-imidazolin-
2-yl) -nicotinic acid, 2- (4-isopropyl-4-methyl-5-oxo-2- imidazolin-2-yl)-5-
(methoxymethyl)-nicotinic acid, 2- (4-isopropyl-4-metiiyl- 5-oxo-2-imidazolin-2-yl)-
5-methylnicotinic acid, and a mixture of methyl 6- (4-isopiopyl-4-methyi-5-oxo-2-
imidazolin-2-yl) -m-toluate, methyl [2- (4-] isopropyi^methyl-5H>xo-2-imidazolin-2-
yI)-p-toluate, and mixtures thereof
—■ %^ ***-,
51. The transformed plant of any one of claims 46-50, wherein the transformed plant is a dicot or a monocot
52. The transformed plant of claim 51, wherein the dicot is selected from the group consisting of sunflower, soybean, cotton, Brassica spp., Arabidopsis thalianay tobacco, tomato, potato, sugar beet, alfalfa, safflower, and peanut
53. The transformed plant of claim 51, wherein the monocot is selected from the group consisting of wheat, rice, maize, barley, rye, oafs, triticale, millet, and sorghum.
54. A seed of the transformed plant of any one of claims 46*53, wherein the seed comprises the IMI nucleic acid
55. A transformed plant cell comprising stably incorporated in its genome a polynucleotide molecule comprising an IMI nucleic acid operably linked to a promoter that drives expression in a plant cell, wherein the IMI nucleic acid comprises a polynucleotide sequence selected from the group consisting of:

(a) the polynucleotide sequence set forth in SEQ ID NO: 1;
(b) a polynucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2;
(c) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID

N0:1, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(e) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (a) or (b), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(f) a polynucleotide sequence that is complementary to any one of (a), (b), (c), (d), or (e); wherein the complement of the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
wherein the IMI nucleic acid confers upon the plant cell increased tolerance to an imidazolinone herbicide as compared to an untransformed plant cell
56. A method of controlling weeds in the vicinity of a plant, comprising applying an effective amount of an imidazolinone herbicide to the weeds and the plant, wherein the plant has increased tolerance to the imidazolinone herbicide as compared to a wild type plant, and wherein the plant comprises at least erne IMI nucleic acid selected from the group consisting of:
(a) a Triticum aestivum hnil nucleic acid encoding an IMI protein comprising a mutation in Domain C that results in a alanine to threonine substitution in the IMI protein as compared to a wild-type AHAS protein,
(b) an IMI nucleic acid comprising the polynucleotide sequence set forth in SEQK>NO:l,and
(c) an IMI nucleic acid encoding a protein comprising the amino acid sequence set forth in SEQ ID NO:2.

57. The method of claim 56, wherein the Triticwn aestivum Imil nucleic
acid comprises a polynucleotide sequence selected from the group consisting of;
(i) the polynucleotide sequence set forth in SEQ ID NO: 1;
(ii) a polynucleotide sequence encoding the polypeptide set forth ir
SEQ ID NO:2; (iii) a polynucleotide sequence comprising at least 95% sequence
identity to the complement of the polynucleotide sequence set
forth in SEQ ID NO: 1; (iv) a polynucleotide sequence encoding an amino acid sequence
comprising at least 95% sequence identity to the amino acid
sequence set forth in SEQ ID NOJ2; and (v) a polynucleotide sequence comprising at least 60 consecutive
nucleotides of (i) or (ii).
58. A method of controlling weeds in the vicinity of a plant, comprising applying an effective amount of an imidazolinone herbicide to the weeds and the plant, wherein the plant has increased tolerance to the imidazolinone herbicide as compared to a wild type plant, wherein the plant is a wheat or triticale plant, and wherein the plant comprises the herbicide tolerance characteristics of the plant with ATCC Patent Deposit Designation Number PTA-5625.
59. A method of producing a transformed plant having increased tolerance to an imidazolinone herbicide comprising:

(a) transforming a plant cell with a polynucleotide molecule comprising an IMI nucleic acid operably linked to a promoter that drives expression in a plant cell; and
(b) regenerating from the plant cell a transgenic plant with an increased tolerance to an imidazolinone herbicide as compared to a wild type plant;
wherein the IMI nucleic acid is selected from the group consisting of:
(i) the polynucleotide sequence set forth in SEQ ID NO: 1; (ii) a polynucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2;

(iii) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID NO:l? wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(iv) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2? wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(v) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (i) or (ii), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide.
60. The method of claim 59, wherein the promoter is selected from the group consisting of constitutive promoters and tissue-preferred promotes.
61. The method of claim 59 or 60, wherein the polynucleotide molecule further comprises an operably linked chloroplast-targeting sequence.
62. The method of any one of claims 59-61, wherein the AHAS activity of the transformed plant is increased relative to an untransformed plant
63. The method of any one of claims 59-62, wherein the herbicide is an imidazolinone herbicide.
64. The method of any one of claims 59-63, wherein the plant cell comprises resistance to at least one herbicide, prior to the transformation step.

65. The method of any one of claims 59-64, wherein the plant cell comprises at least one IMI nucleic acid, prior to the transformation step.
66. A method for increasing AHAS activity in a plant comprising transforming a plant cell with a polynucleotide molecule comprising an IMI nucleic acid operably linked to a promoter that drives expression in a plant cell and regenerating a transformed plant from the transformed plant cell, wherein the IMI nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
~~(a)*~ ^Ife polynucleotide sequence set forth in SEQ ID NO:l;
(b) a polynucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2;
(c) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID NO:l» wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(e) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (a) or (b), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
wherein AHAS activity is increased in the transformed plant or at least one part thereof when compared to an untransformed plant
67. A method for selecting for a transformed plant cell comprising the
steps of,
transforming a plant cell with the plant transformation vector, exposing the transformed plant cell to at least one herbicide at a concentration tibat inhibits the growth of an untransformed plant cell, and

identifying the transformed plant cell by its ability to grow in the presence of the herbicide;
wherein the plant transformation vector comprises a selectable marker gene comprising a promoter that drives expression in a plant cell and an operably linked IMI nucleic acid, wherein the IMI nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
(a) the polynucleotide sequence set forth in SEQ ID NO:l;
(b) a polynucleotide sequence encoding the polypeptide set forth in SEQ ID NO:2;
(c) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID NO:l, wherein the polynucleotide sequence encodes an IME protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type ABAS polypeptide; and
(e) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (a) or (b), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide.

68. The method of claim 67, wherein the plant transformation vector further comprises at least one gene of interest
69. The method of claim 67 or 68, further comprising the step of regenerating a transformed plant from the transformed plant cell.
70. A method of controlling weeds in the vicinity of a transformed plant, the method comprising applying an effective amount of an imidazolinone herbicide to the weeds and to the transformed plant, wherein the transformed plant has increased resistance to the herbicide as compared to an untransformed plant and the

transformed plant comprises in its genome at least one expression cassette comprising an IMI nucleic acid operably linked to a promoter that drives gene expression in a plant cell, wherein the IMI nucleic acid comprises a polynucleotide sequence selected from the group consisting of:
(a) the polynucleotide sequence set forth in SEQ ID NO:l;
(b) a polynucleotide sequence encoding the polypeptide set forth in SEQ IDNO:2;
(c) a polynucleotide sequence comprising at least 95% sequence identity to the complement of the polynucleotide sequence set forth in SEQ ID NO:l, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) a polynucleotide sequence encoding an amino acid sequence comprising at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, wherein the polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(e) a polynucleotide sequence comprising at least 60 consecutive nucleotides of (a) or (b), wherein polynucleotide sequence encodes an IMI protein having an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide.
7L An isolated polypeptide comprising an amino acid sequence selected from the group consisting of:
(a) the amino acid sequence set forth in SEQ ID NO:2;
(b) the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO:l;
(c) an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO;2, wherein the polypeptide comprises herbicide-resistant AHAS activity and has an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide;
(d) an amino acid sequence encoded by a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO:l, wherein the polypeptide comprises herbicide-resistant AHAS activity and has

an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide; and
(f) an amino acid sequence encoded by a nucleotide sequence having at least 95% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, wherein the polypeptide comprises herbicide-resistant AHAS activity and has an alanine to threonine substitution at position 96 corresponding to a wild type AHAS polypeptide.
72. A method for producing a herbicide-resistant plant comprising crossing a first plant that is resistant to a herbicide to a second plant that is not resistant to the herbicide, wherein the first plant is the plant of any one of claims 1-13, 15-19,23-29, 33-35, and 46-53.
73. The method of claim 72 further comprising selecting for a progeny plant that is resistant to the herbicide.
74. A herbicide-resistant plant produced by the method of claim 72 or 73.
75. A seed of the plant of claim 74, wherein the seed comprises the herbicide resistant characteristics of the first plant
76. A method for increasing the herbicide-resistance of a plant comprising crossing a first plant to a second plant, wherein the first plant is the plant of arty one of claims 1-13,15-19,23-29,33-35,46-53, and 74.
77. The method of claim 76 further comprising selecting for a progeny plant that comprises increased herbicide resistance when compared to the herbicide resistance of the second plant
78. A plant produced by the method of claim 76 or 77.
79. A seed of the plant of claim 78, wherein the seed comprises the increased herbicide resistance.

80. A seed of the plant of any one of claims 1-13, 15-19, 23-29, 33-35, 46-
53, 74, and 78 wherein the seed is treated with an AHAS-inhibiting herbicide.
81. A method for combating undesired vegetation comprising contacting a
seed of the plant of any one of claims 1-13,15-19, 23-29, 33-35, 46-53, 74, and 78
before sowing and/or after pregennination with an AHAS-inhibiting herbicide.


Documents:

2937-chenp-2007 amended pages of specification 22-07-2011.pdf

2937-CHENP-2007 AMENDED PAGES OF SPECIFICATION 26-05-2011.pdf

2937-CHENP-2007 AMENDED CLAIMS 26-05-2011.pdf

2937-chenp-2007 amended claims 22-07-2011.pdf

2937-chenp-2007 form-1 26-05-2011.pdf

2937-chenp-2007 form-3 26-05-2011.pdf

2937-CHENP-2007 FORM-6 26-05-2011.pdf

2937-CHENP-2007 OTHER PATENT DOCUMENT 26-05-2011.pdf

2937-CHENP-2007 POWER OF ATTORNEY 26-05-2011.pdf

2937-CHENP-2007 EXAMINATION REPORT REPLY RECEIVED 26-05-2011.pdf

2937-chenp-2007 form-1 22-07-2011.pdf

2937-CHENP-2007 CORRESPONDENCE OTHERS 22-07-2011.pdf

2937-CHENP-2007 CORRESPONDENCE OTHERS 30-09-2010.pdf

2937-CHENP-2007 CORRESPONDENCE OTHERS. 23-08-2011.pdf

2937-chenp-2007-abstract.pdf

2937-chenp-2007-assignement.pdf

2937-chenp-2007-claims.pdf

2937-chenp-2007-correspondnece-others.pdf

2937-chenp-2007-description(complete).pdf

2937-chenp-2007-drawings.pdf

2937-chenp-2007-form 1.pdf

2937-chenp-2007-form 3.pdf

2937-chenp-2007-form 5.pdf

2937-chenp-2007-pct.pdf


Patent Number 248553
Indian Patent Application Number 2937/CHENP/2007
PG Journal Number 30/2011
Publication Date 29-Jul-2011
Grant Date 25-Jul-2011
Date of Filing 02-Jul-2007
Name of Patentee BASF AGROCHEMICAL PRODUCTS, B.V
Applicant Address Groningensingel 1, NL-6835 EA Arnhem
Inventors:
# Inventor's Name Inventor's Address
1 MOFFATT, John 29128 South Wells Road, Cheney, WA 99004
2 BRUNS, Rob 300 Ecr 30, Fort Collins, CO 80525
3 BIRK, Iwona 2608 Sunnystone Way, Raleigh, NC 27613
4 SINGH, Bijay 301 Connemara Drive, Cary, NC 27519
PCT International Classification Number C12N 15/10
PCT International Application Number PCT/US05/43577
PCT International Filing date 2005-12-01
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/632,376 2004-12-01 U.S.A.