NZ735454A - Mutated acetohydroxyacid synthase genes in brassica - Google Patents

Abstract

Disclosed is an isolated nucleic acid encoding a Brassica acetohydroxyacid synthase (AHAS) protein comprising a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1.

Description

MUTATED ACETOHYDROXYACID SYNTHASE GENES IN BRASSICA CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application is a onal application of New Zealand Patent Application No. 717451, which, in turn, is a divisional application of New d Patent Application No. 628537, which, in turn, is a divisional application of New Zealand Patent Application No. 606701, which, in turn, is a divisional application of New Zealand Patent Application No. 584413, which claims the benefit of U.S. Prov. App. No. 60/977,944, filed October 5, 2007.

All of the above applications are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION This invention relates to the field of herbicide ant plants and seeds and more specifically to mutations in the ydroxyacid synthase (AHAS) gene and protein.

BACKGROUND OF THE INVENTION The following description is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art to the invention.

Benefits of herbicide-tolerant plants are known. For example, herbicide-tolerant plants may reduce the need for tillage to control weeds thereby effectively reducing soil erosion.

The introduction of exogenous mutant genes into plants is well documented. For example, U.S. Pat. No. 4,545,060 relates to sing a plant's resistance to glyphosate by introducing into the plant's genome a gene coding for an EPSPS variant having at least one mutation that renders the enzyme more ant to its competitive inhibitor, i.e., glyphosate.

Examples of some of the mutations in AHAS genes are known. See e.g. U.S. Patent No. 7,094,606.

Through chemical mutagenesis, a mutation was found in the lower sed AHAS I gene. This mutation is known as PM- 1 (a mutation at an equivalent position known as 653 based on the acetolactate synthase (ALS) amino acid ce of Arabidopsis, a serine to gine amino acid change, respectively encoded as follows – AGT to AAT). r mutation was found in the higher sed gene AHAS III, known as PM-2 (a mutation at an equivalent position known as 574, a tryptophan to leucine amino acid change, respectively encoded as follows —- TGG to TTG). These two ons, PM—l and PM—Z, are combined in a commercial variety of Canola known as Clearfield Canola (Tan et a[., 2005).

Y OF THE ION The invention relates in part to mutated acetohydroxyacid synthase (AHAS) nucleic acids and the proteins encoded by the mutated nucleic acids. The invention also relates in part to canola plants, cells, and seeds comprising these mutated nucleic acids and proteins.

In one aspect, there is provided an isolated nucleic acid encoding a Brassica acetohydroxyacid synthase protein having a mutation at one or more amino acid positions corresponding to a position ed from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1. In some embodiments, the isolated nucleic acid encodes a protein having one or more mutations selected from the group consisting of: an alanine to valine tution at a position conesponding to position 205 of SEQ ID NO: I, an alanine to ic acid substitution at a position ponding to position 205 of SEQ ID NO: 1, an aspartic acid to glutamic acid substitution at a position corresponding to position 376 of SEQ ID NO: 1, a tryptophan to cysteine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptophan to leucine substitution at a position corresponding to position 574 ofSEQ ID NO: I, a tryptophan to methionine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a phan to serine substitution at a position corresponding to position 574 of SEQ ID NO: 1, an arginine to tryptophan substitution at a position correSponding to on 577 of SEQ ID NO: I, a serine to gine at a position ponding to position 653 of SEQ ID NO: I, and a serine to threonine at a position corresponding to position 653 of SEQ ID NO: 1. In some embodiments, the mutation is a serine to asparagine substitution at a position corresponding to position 653 of SEQ ID NO: I in which the on is not S653N in Brassica AHAS I gene. 4.

In some embodiments, the mutation is a serine to ine substitution at a position corresponding to position 653 of SEQ ID NO: 1. In some embodiments, the mutation is a tryptophan to leucine substitution at a position corresponding to position 574 of SEQ ID NO: 1, in which the mutation is not W5 74L in Brassica AHAS III gene. In some ments, the mutation is an alanine to valine tution at a position corresponding to position 205 of SEQ ID NO: 1. In other embodiments the mutation is an alanine to ic acid substitution at a position corresponding to position 205 of SEQ ID NO: 1. In other embodiments, the isolated nucleic acid encodes a protein having a mutation selected from the mutations shown in Table 2.

In certain ments, the isolated nucleic acid encodes a protein having two or more mutations. In some embodiments, the two or more mutations are selected from Table 2. In some embodiments, the isolated nucleic acid encodes a protein having a mutation at a position corresponding to S653 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a p0sition selected from the group consisting of: A205, D376, W574, and R577 of SEQ ID NO: 1. In other embodiments, the isolated nucleic acid encodes a protein having a mutation at a position corresponding to W574 of SEQ ID NO: 1 and a mutation at a position corresponding to R577 of SEQ ID NO: 1. In some embodiments, the ed nucleic acid s an acetohydroxyacid synthase (AHAS) protein that is resistant to inhibition by an AHAS—inhibiting ide. In some ments, the AHAS-inhibiting herbicide is ed from the group consisting of herbicides of: imidazolinone, sulfonylurea, triazolopyrimidine, pyrimidinylthiobenzoate, sulfonylamino-carbonyltriazolinone, and mixtures thereof. In some embodiments, the herbicide is an olinone herbicide. In some embodiments, the herbicide is a sulfonylurea herbicide. In some embodiments the the isolated nucleic acid encodes an AHAS protein comprising 70% or more identity to one or more of the amino acid sequences in Figure 2. In some embodiments, the isolated c acid encodes a Brassica napus AHAS n. In other embodiments, the isolated nucleic acid encodes a Brassica napus AHAS I protein. In certain embodiments, the isolated nucleic acid encodes a Brassica napus AHAS III protein.

In another aspect, there is provided an expression vector containing an isolated nucleic acid encoding a Brassica acetohydroxyacid synthase protein having a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1. In some embodiments, the expression vector contains an isolated c acid encoding a protein having one or more mutations selected from the group consisting of: an alanine to valine substitution at a on corresponding to position 205 of SEQ ID NO: 1, an alanine to aspartic acid substitution at a position corresponding to position 205 of SEQ ID NO: 1, an aspartic acid to glutamic acid tution at a position corresponding to position 376 of SEQ ID NO: 1, a tryptophan to cysteine substitution at a position corresponding to position 5 74 of SEQ ID NO: 1, a tryptophan to leucine substitution at a position corresponding to position 5 74 of SEQ ID NO: 1, a tryptophan to nine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptophan to serine substitution at a position ponding to position 5 74 of SEQ ID NO: 1, an arginine to tryptophan substitution at a position ponding to position 577 of SEQ ID NO: 1, a serine t0 asparagine at a position corresponding to position 653 of SEQ ID NO: 1, and a serine to threonine at a position corresponding to position 653 of SEQ ID NO: 1. In some embodiments, the mutation is a serine to asparagine substitution at a position corresponding to position 653 of SEQ ID NO: 1 in which the mutation is not S653N in Brassz'ca AHAS I gene. 4. In some ments, the mutation is a serine to ine tution at a position corresponding to position 65 3 of SEQ ID NO: 1. In some embodiments, the mutation is a tryptophan to leucine substitution at a position corresponding to on 574 of SEQ ID NO: 1, in which the on is not W5 74L in Brassica AHAS III gene. In some embodiments, the on is an alanine to valine substitution at a position corresponding to position 205 of SEQ ID NO: 1. In other embodiments the mutation is an alanine to aspartic acid substitution at a position corresponding to position 205 of SEQ ID NO: 1.

In another aspect, there is provided a plant having a Brassica acetohydroxyacid synthase (AHAS) gene, in which the gene encodes a protein having a mutation at one or more amino acid ons corresponding to a position selected from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1. In another aspect, there is provided a plant having a Brassica acetohydroxyacid synthase (AHAS) gene, in which the plant is resistant to an AHAS—inhibiting herbicide, in which the gene encodes a protein having a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1. In some embodiments of the above aspects, the plant has an AHAS gene which encodes a protein having one or more mutations selected from the group consisting of: an alanine to valine substitution at a position corresponding to position 205 of SEQ ID NO: I, an alanine to aspartic acid substitution at a position corresponding to position 205 of SEQ ID NO: I, an aspartic acid to ic acid substitution at a position corresponding to position 376 of SEQ ID NO: I, a tryptophan to cysteine substitution at a position ponding to on 574 of SEQ ID NO: I, a tryptophan to leucine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptOphan to nine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptophan to serine substitution at a position corresponding to position 574 of SEQ ID NO: 1, an arginine to tryptophan substitution at a position corresponding to position 577 of SEQ ID NO: 1, a serine to asparagine at a position corresponding to position 653 of SEQ ID NO: I, and a serine to threonine at a position correSponding to on 653 of SEQ ID NO: I. In some embodiments, the mutation is a serine to asparagine substitution at a position corresponding to position 653 of SEQ ID NO: I in which the mutation is not S653N in Brassz'ca AHAS I gene. 4.

In some embodiments, the mutation is a serine to threonine substitution at a on corresponding to position 653 of SEQ ID NO: I. In some embodiments, the mutation is a tryptophan to e substitution at a position corresponding to on 574 of SEQ ID NO: 1, in which the on is not W574L in Brassica AHAS III gene. In some embodiments, the mutation is an alanine to valine substitution at a on corresponding to position 205 of SEQ ID NO: 1. In other embodiments the mutation is an alanine to ic acid substitution at a position corresponding to position 205 of SEQ ID NO: I. In other embodiments, the mutation selected from the mutations shown in Table 2. In certain embodiments, the plant has an AHAS gene which encodes a protein having two or more mutations. In some embodiments, the two or more mutations are selected from Table 2. In some embodiments, the plant has an AHAS gene which encodes a protein having a mutation at a position corresponding to S653 of SEQ ID NO: I and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: A205, D376, W574, and R577 of SEQ ID NO: 1. In other embodiments, the plant has an AHAS gene which encodes a protein having a mutation at a position corresponding to W574 of SEQ ID NO: 1 and a on at a on corresponding to R577 of SEQ ID NO: 1.

In some embodiments, the plant has an AHAS gene which encodes a protein that is resistant to inhibition by an AHAS-inhibiting herbicide. In some embodiments the plant has an AHAS gene which encodes a protein sing 70% or more identity to one or more of the amino acid sequences in Figure 2. In some embodiments, the plant is resistant to the application of at least one AHAS—inhibiting ide. In some embodiments, the AHAS—inhibiting herbicide is selected from the group ting of herbicides of: imidazolinone, sulfonylurea, triazolopyrimidine, dinylthiobenzoate, sulfonylamino-carbonyltriazolinone, and es thereof. In some embodiments, the herbicide is an imidazolinone herbicide. In some embodiments, the herbicide is a sulfonylurea ide. In some embodiments, the plant is a Brassica plant produced by growing a seed of a line selected from the lines listed in Table 2. In some embodiments, the plant is a Brassz'ca species. In other embodiments, the plant is Brassica napus. In some embodiments, the plant is selected from Spring Oilseed Rape and Winter Oilseed Rape. In some embodiments, the plant has an AHAS gene which encodes a Brassica napus AHAS protein. In other embodiments, the plant has an AHAS gene which s a Brassica napus AHAS I protein. In n embodiments, the plant has an AHAS gene which encodes a Brassz'ca napus AHAS III protein. In some embodiments, the plant is non—transgenic.

In one aspect there is provided a seed having a Brassz'ca acetohydroxyacid synthase (AHAS) gene encoding a protein having a mutation at one or more amino acid positions corresponding to a position selected fi‘om the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1. In some embodiments, the seed has an AHAS gene which encodes a protein having one or more mutations selected from the group consisting of: an alanine to valine substitution at a position ponding to position 205 of SEQ ID NO: 1, an alanine to aspartic acid substitution at a position corresponding to position 205 of SEQ ID NO: I, an aspartic acid to glutamic acid substitution at a position corresponding to position 376 of SEQ ID NO: 1, a phan to cysteine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptophan to leucine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptophan to methionine substitution at a position corresponding to position 574 of SEQ ID NO: 1, a tryptophan to serine substitution at a position corresponding to position 574 of SEQ ID NO: 1, an arginine to phan substitution at a position corresponding to position 577 of SEQ ID NO: 1, a serine to asparagine at a position corresponding to position 653 of SEQ ID NO: 1, and a serine to threonine at a position corresponding to on 653 of SEQ ID NO: 1. In some embodiments, the mutation is a serine to asparagine substitution at a position corresponding to position 653 of SEQ ID NO: 1 in which the mutation is not S653N in Brassica AHAS I gene. 4. In some embodiments, the mutation is a serine to ine substitution at a position corresponding to position 653 of SEQ ID NO: 1. In some embodiments, the mutation is a tryptophan to leucine substitution at a position corresponding to position 574 of SEQ ID NO: 1, in which the mutation is not W574L in Brassica AHAS III gene. In some embodiments, the mutation is an alanine to valine substitution at a position corresponding to position 205 of SEQ ID NO: 1. In other embodiments the mutation is an alanine to aspartic acid substitution at a position corresponding to position 205 of SEQ ID NO: 1. In other embodiments, the mutation selected from the mutations shown in Table 2. In certain embodiments, the seed has an AHAS gene which encodes a protein having two or more mutations. In some embodiments, the Mo or more mutations are selected from Table 2. In some embodiments, the seed has an AHAS gene which encodes a protein having a mutation at a on corresponding to S653 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: A205, D376, W574, and R577 of SEQ ID NO: 1. In other embodiments, the seed has an AHAS gene which s a protein having a mutation at a position corresponding to W574 of SEQ ID NO: 1 and a mutation at a position corresponding to R577 of SEQ ID NO: I.

In some embodiments, the seed has an AHAS gene which encodes a protein that is ant to irdiibition by an AHAS—inhibiting ide. In some embodiments the seed has an AHAS gene which encodes a protein sing 70% or more ty to one or more of the amino acid sequences in Figure 2. In some embodiments, the seed is resistant to the application of at least one AHAS—inhibiting herbicide. In some ments, the nhibiting herbicide is selected from the group consisting of herbicides of: olinone, sulfonylurea, triazolopyrimidine, pyrimidinylthiobenzoate, su1fonylamino—carbonyltriazolinone, and mixtures thereof. In some embodiments, the herbicide is an imidazolinone herbicide. In some embodiments, the herbicide is a sulfonylurea herbicide. In some embodiments, the seed is a Brassz'ca seed. In some embodiments, the seed has an AHAS gene which encodes a Brassica napus AHAS protein. In other embodiments, the seed has an AHAS gene which encodes a Brassica napus AHAS I protein. In certain embodiments, the seed has an AHAS gene which encodes a Brassica napus AHAS III protein. In some embodiments, the seed is a seed of a Brassica plant line in which the line is selected from the lines listed in Table 2. In some ments, the seed is non—transgenic. In some ments, there is provided a seed produced by a plant of the methods sed herein. In other embodiments, the seed is a canola seed.

In another , there is provided a method for producing an herbicide—resistant plant by introducing into a plant cell a gene repair oligonucleobase (GRON) with a targeted mutation in an ydroxyacid synthase (AHAS) gene to produce a plant cell with an AHAS gene that expresses an AHAS protein having a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: A205, D376, W574, and S653 of SEQ ID NO: 1; and identifying a plant cell having substantially normal growth and catalytic activity as compared to a corresponding wild—type plant cell in the presence of an AHASw inhibiting herbicide; and rating a non—transgenic herbicide—resistant plant having a d AHAS gene from said plant cell. In another aspect, there is provided a method for increasing the herbicide—resistance of a plant by: (a) crossing a first Brassica plant to a second Brassica plant, in which the first plant comprises a Brassica acetohydroxyacid synthase (AHAS) gene, in which the gene encodes a n having a mutation at one or more amino acid positions corresponding to a on selected from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1; (b) screening a population resulting from the cross for increased AHAS ide— resistance; (c) selecting a member resulting from the cross having increased AHAS herbicide- resistance; and (d) producing seeds resulting from the cross. In some embodiments, a hybrid seed is produced by any of the above methods. In some embodiments, plants are grown from seeds produced by any of the above methods. In another aspect, there is provided a method of controlling weeds in a field containing plants by applying an effective amount of at least one AHAS~inhibiting herbicide to a field containing said weeds and plants, the plant having a ca acetohydroxyacid synthase (AHAS) gene, in which the gene encodes a protein having a on at one or more amino acid positions corresponding to a on selected from the group consisting of: A205, D376, W574, R577, and S653 of SEQ ID NO: 1. 134. In some embodiments, the AHAS~inhibiting herbicide is selected from the group ting of herbicides of: imidazolinone, sulfonylurea, triazolopyrimidine, pyrimidinylthiobenzoate, sulfonylamino- carbonyltriazolinone, and mixtures thereof. In other embodiments, the AHAS—inhibiting ide is an imidazolinone herbicide. In other embodiments, the AHAS-inhibiting herbicide is a sulfonylurea herbicide.

The term “nucleic acid” or "nucleic acid sequence" refers to an oligonucleotide, nucleotide 0r cleotide, and fragments or portions thereof, which may be single or double ed, and represent the sense or antisense . A nucleic acid may include DNA or RNA, and may be of natural or synthetic origin. For example, a nucleic acid may include mRNA or cDNA. c acid may include nucleic acid that has been amplified (e.g. , using polymerase chain reaction). The convention “Nth###NTmut” is used to indicate a mutation that results in the wild~type nucleotide Nth at position ### in the nucleic acid being replaced with mutant NTmut. The single letter code for nucleotides is as described in the U.S. Patent Office Manual of Patent Examining Procedure, n 2422, table 1. In this regard, the nucleotide designation “R” means purine such as guanine or adenine, “Y” means pyrimidine such as cytosine or th ine uracil if RNA ‘ “M” means adenine or c osine; “K” means uanine or th me and 3 “W” means adenine or thymine.

A "gene" refers to a DNA sequence that comprises control and coding ces necessary for the production of an RNA, which may have a non~coding function (e. g., a ribosomal or transfer RNA) or which may include a polypeptide or a polypeptide precursor. The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained. As used herein, the term “AHAS Gene” refers to a gene that has homology to a ca AHAS gene. In n embodiments, the AHAS gene has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a specific Brassica AHAS gene; for example the Brassica napus AHAS gene I or the Brassica napus AHAS gene III. In certain embodiments, the AHAS gene has 60%; 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% ty to a sequence selected from the sequences in Figure 3. In certain embodiments, the AHAS gene is modified with at least one on. In other embodiments, the AHAS gene is modified with at least two mutations. In some embodiments, the AHAS gene is modified with at least one mutation selected from the mutations shown in Table 2. In certain embodiments; the AHAS gene is modified with at least two ons shown in Table 2. In certain embodiments; the mutation is a conserved mutation.

By “coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof; that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a nt thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti~sense strand is the complement of such a nucleic acid; and the encoding sequence can be d rom.

By “non-coding sequence” is meant a sequence of a nucleic acid or its complement, or a part thereof; that is not ribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts; and gene-associated sequences such as promoters; enhancers, ers, etc.

A nucleobase is a base, which in n preferred embodiments is a purine; pyrimidine; or a derivative or analog thereof. Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2’-deoxyriboside. Nucleosides can be linked by one of several linkage moieties; which may or may not contain phosphorus. Nucleosides that are linked by unsubstituted phosphodiester linkages are termed nucleotides. The term “nucleobase” as used herein includes peptide nucleobases, the subunits of e nucleic acids, and morpholine nucleobases as well as nucleosides and nucleotides.

An oligonucleobase is a polymer comprising nucleobases; preferably at least a portion of which can hybridize by -Crick base pairing to a DNA having the complementary sequence. An oligonucleobase chain may have a single 5' and 3’ terminus, which are the ultimate nucleobases of the r. A particular oligonucleobase chain can contain nucleobases of all types. An oligonucleobase compound is a nd comprising one or more ucleobase chains that may be complementary and hybridized by Watson—Crick base pairing. Ribo-type nucleobases e pentosefuranosyl containing nucleobases wherein the 2' carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases are nucleobases other than ribo—type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.

In certain ments, an ucleobase strand may e both oligonucleobase chains and segments or regions of oligonucleobase chains. An oligonucleobase strand may have a 3' end and a 5’ end, and when an oligonucleobase strand is coextensive with a chain, the 3' and ' ends of the strand are also 3' and 5' termini of the chain.

The term "gene repair oligonucleobase" as used herein denotes oligonucleobases, including mixed duplex oligonucleotides, non—nucleotide containing molecules, single stranded oligodeoxynucleotides and other gene repair molecules.

By “isolated”, when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) is meant a nucleic acid that is apart from a ntial portion of the genome in which it lly occurs and/or is substantially separated from other cellular components which naturally accompany such nucleic acid. For example, any nucleic acid that has been produced synthetically (e.g., by serial base condensation) is considered to be isolated.

Likewise, nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise d from a genome are also considered to be isolated.

An "amino acid sequence" refers to a polypeptide or protein sequence. The convention “AAwt###AAmut” is used to indicate a on that results in the wild—type amino acid AAwt at position ### in the polypeptide being replaced with mutant AAmut.

By “complement” is meant the complementary sequence to a nucleic acid according to standard Watson/Crick pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complement sequence, and can also be a cDNA.

By "substantially complementary” is meant that tWo sequences hybridize under stringent ization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length.

As used herein the term “codOn” refers to a sequence of three adjacent nucleotides (either RNA or DNA) constituting the genetic code that determines the ion of a c amino acid in a polypeptide chain during protein synthesis or the signal to stop protein synthesis.

The term “codon” is also used to refer to the corresponding (and complementary) sequences of three nucleotides in the messenger RNA into which the al DNA is transcribed.

As used , the term “AHAS Protein” refers to a protein that has homology to a Brassica AHAS protein. In certain embodiments, the AHAS protein has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a specific Brassz'ca AHAS protein, such as for example, the Brassica napus AHAS protein I or the Brassica napus AHAS n III. In certain embodiments, the AHAS protein has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a sequence selected from the sequences in Figure 2. In certain embodiments, the AHAS protein is d with at least one mutation. In other embodiments, the AHAS protein is modified with at least two mutations. In some ments, the AHAS protein is modified with at least one mutation ed from the mutations shown in Table 2. In certain embodiments, the AHAS protein is d with at least two mutations shown in Table 2. In certain embodiments, the mutation is a conserved mutation.

The term "wild-type" refers to a gene or a gene t that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild—type gene is that which is most frequently ed in a population and is thus arbitrarily designated the "normal" or type" form of the gene. “Wild-type” may also refer to the sequence at a specific nucleotide position or positions, or the sequence at a particular codon position or positions, or the sequence at a particular amino acid position or positions.

As used herein, "mutant,” or "modified" refers to a nucleic acid or protein which displays ations in sequence and or functional properties (126., altered characteristics) when ed to the wild-type gene or gene product. "Mutant," or "modified" also refers to the sequence at a specific nucleotide position or positions, or the sequence at a ular codon position or positions, or the sequence at a particular amino acid position or positions which displays modifications in sequence and or onal properties (126., altered characteristics) when compared to the wild—type gene or gene product.

A “mutation” is meant to encompass at least a single nucleotide variation in a nucleic acid sequence or a single amino acid variation in a polypeptide relative to the normal ce or wild—type sequence. A mutation may include a substitution, a deletion, an ion or an insertion.

As used herein, the term “homology” refers to sequence similarity among proteins and DNA. The term “homology” or “homologous” refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that has less than 100% sequence identity when compared to another sequence.

“Heterozygous” refers to having different s at one or more genetic loci in homologous chromosome segments. As used herein “heterozygous” may also refer to a sample, a cell, a cell tion or an organism in which different alleles at one or more genetic loci may be detected. Heterozygous samples may also be determined via methods known in the art such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows two peaks at a single locus and both peaks are roughly the same size, the sample may be characterized as heterozygous. Or, if one peak is smaller than another, but is at least about 25% the size of the larger peak, the sample may be characterized as heterozygous. In some embodiments, the smaller peak is at least about 15% of the larger peak. In other embodiments, the smaller peak is at least about 10% of the larger peak. In other embodiments, the smaller peak is at least about 5% of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.

As used herein, “homozygous” refers to having identical alleles at one or more genetic loci in homologous chromosome segments. “Homozygous” may also refer to a , a cell, a cell population or an organism in which the same alleles at one or more genetic loci may be ed. gous samples may be determined via s known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing opherogram shows a single peak at a particular locus, the sample may be termed “homozygous” with respect to that locus.

The term “hemizygous” refers to a gene or gene segment being present only once in the genotype of a cell or an organism because the second allele is deleted. As used herein “hemizygous” may also refer to a sample, a cell, a cell tion or an organism in which an allele at one or more genetic loci may be detected only once in the genotype.

The term “zygosity status” as used herein refers to a sample, a cell population, or an sm as ing heterozygous, homozygous, 0r hemizygous as determined by testing methods known in the art and described herein. The term “zygosity status of a nucleic acid” means ining whether the source of nucleic acid appears heterozygous, homozygous, or hemizygous. The “zygosity status” may refer to differences in a single nucleotide in a sequence.

In some methods, the zygosity status of a sample with respect to a single mutation may be categorized as homozygous wild-type, zygous (118., one wild-type allele and one mutant allele), homozygous mutant, or hemizygous (116., a single copy of either the wild-type or mutant allele).

As used herein, the term “RTDS” refers to The Rapid Trail pment SystemTM (RTDS) developed by Cibus. RTDS is a site-specific gene modification system that is effective at making precise changes in a gene sequence without the incorporation of foreign genes or control sequences.

The term “about” as used herein means in quantitative terms plus or minus 10%. For example, “about 3%” would encompass 2.7-3.3% and “about 10%” would encompass 9-1 1%.

BRIEF DESCRIPTION OF THE FIGURES FIGURE 1 shows an amino acid alignment of Arabidopsis acetohydroxyacid synthase AHAS (SEQ ID NO: 1), ca napus AHAS I (SEQ ID NO: 2), and Brassica napus AHAS III (SEQ ID NOs: 3 and 4). SEQ ID NO: 1 is the amino acid sequence ofthe Arabidopsis AHAS At3 g48 560 based on the annotated genomic DNA sequence Genbank accession number NC003074. SEQ ID NO: 2 is the amino acid sequence of ca napus AHAS I from Cibus elite lines BN-2 and BN—l 1. This sequence is identical to the translated product of Genbank accession 21 1524. SEQ ID NO: 3 is the amino acid sequence of Brassica napus AHAS III from Cibus elite line BN—2. This sequence is identical to the translated product of Genbank accession Z11526, excepting a D325E substitution at amino acid 325. SEQ ID NO: 4 is the amino acid sequence sica napus AHAS 111 from Cibus elite line BN-l 1. This sequence is identical to the translated product of SEQ ID NO: 3, ing an E343 at amino acid 343 as in SEQ ID NO: Figure 2 shows amino acid sequences of translated genes referred to in Table 2. Amino acids indicated in bold represent the mutation.

Figure 3 shows nucleotide sequences ed to in Table 2. Nucleotides indicated in bold represent the mutation.

Figure 4 shows results of the spray trial described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION Provided are compositions and methods d in part to the successful targeting of ydroxyacid synthase (AHAS) genes in Brassica using, for example, the Rapid Trait Development System (RTDSTM) technology developed by Cibus. In combination or alone, plants containing any of the mutations disclosed herein can form the basis of new herbicide- resistant products. Also ed are seeds ed from the mutated plants in which the AHAS genes are either homozygous or heterozygous for the mutations. The mutations disclosed herein can be in combination with any other mutation known or with mutations discovered in the RTDS is based on altering a targeted gene by utilizing the cell’s own gene repair system to specifically modify the gene sequence in situ and not insert foreign DNA and gene expression control sequences. This procedure effects a precise change in the genetic sequence while the rest of the genome is left unaltered. In contrast to conventional enic GMOs, there is no integration of foreign genetic material, nor is any foreign genetic material left in the plant. The changes in the genetic sequence introduced by RTDS are not randomly inserted. Since affected genes remain in their native location, no random, uncontrolled or adverse pattern of expression OCCUIS .

The RTDS that effects this change is a ally synthesized oligonucleotide which may be composed ofboth DNA and modified RNA bases as well as other al moieties, and is designed to hybridize at the targeted gene location to create a mismatched base~pair(s). This mismatched base—pair acts as a signal to attract the cell’s own natural gene repair system to that site and correct (replace, insert or delete) the designated nucleotide(s) within the gene. Once the correction process is complete the RTDS le is degraded and the now-modified or repaired gene is expressed under that gene’s normal endogenous control isms.

The subject mutations in the AHAS I and III genes were described using the Brassica napus AHAS genes and proteins (see SEQ ID N03: 2, 3 and 4). The compositions and methods also encompass mutant AHAS genes of other s (paralogs). r, due to variations in the AHAS genes of different species, the number of the amino acid residue to be changed in one species may be different in another species. heless, the analogous position is readily identified by one of skill in the art by sequence homology. For e, FIGURE 1 shows the aligned amino acid sequences of the Arabz'dopsis AHAS (SEQ ID NO:1) and Brassz'ca napus AHAS I (SEQ ID NO:2) and AHAS III (SEQ ID NO:3 and SEQ ID NO: 4) paralogs. Thus, analogous positions in these and other paralogs can be identified and mutated.

The compositions and methods relate in part to mutations in an AHAS gene that render a plant resistant or tolerant to an herbicide of the AHAS~inhibiting or ALS~inhibiting family of herbicides. The compositions and methods also relate to the use of a gene repair oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for an AHAS n. The mutated protein, which ntially ins the catalytic activity of the wild-type protein, allows for increased resistance or nce of the plant to an herbicide of the AHAS~inhibiting family, and allows for the substantially normal growth or development of the plant, its organs, tissues, or cells as compared to the wild-type plant irrespective of the presence or absence of the herbicide. The compositions and methods also relate to a non-transgenic plant cell in which an AHAS gene has been mutated, a non-transgenic plant regenerated rom, as well as a plant resulting from a cross using a rated non— transgenic plant to a plant having a mutation in a ent AHAS gene or to a plant having a mutated EPSPS gene, for example.

Imidazolinones are among the five chemical families of AHAS-inhibiting herbicides.

The other four families are sulfonylureas, triazolopyrimidines, pyrimidinylthiobenzoates and sulfonylamino—carbonyltriazolinones (Tan et al., 2005).

Also ed is a transgenic or non-transgenic plant or plant cell having one or more mutations in the AHAS gene, for example, such as disclosed herein. In certain embodiments, the plant or plant cell having one or more mutations in the AHAS gene has increased resistance or tolerance to a member of the AHAS—inhibiting. In certain embodiments, the plant or plant cell having one or more mutations in the AHAS gene may exhibit substantially normal growth or development of the plant, its organs, tissues or cells, as ed to the corresponding wild-type plant or cell. In particular aspects and embodiments provided are ansgenic plants having a mutation in an AHAS gene, for example, such as disclosed herein, which in certain embodiments has sed resistance or tolerance to a member of the AHAS-inhibiting herbicide family and may exhibit substantially normal growth or development of the plant, its organs, tissues or cells, as compared to the corresponding wild-type plant or cell, i.e., in the presence of one or more herbicide such as for example, an imidazolinone and/or sulfonyl urea, the mutated AHAS protein has substantially the same catalytic activity as compared to the wild—type AHAS protein.

Further provided are s for producing a plant having a mutated AHAS gene, for example, having one or more mutations as described herein; preferably the plant substantially maintains the catalytic activity of the wild—type protein irrespective of the presence or absence of a relevant herbicide. In certain embodiments, the methods include introducing into a plant cell a gene repair oligonucleobase with one or more targeted mutations in the AHAS gene (for example, such as sed herein) and fying a cell, seed, or plant having a mutated AHAS gene.

In various embodiments, plants as disclosed herein can be of any species of dicotyledonous, monocotyledonous or gymnospennous plant, including any woody plant species that grows as a tree or shrub, any eous species, or any species that es edible fruits, seeds or vegetables, or any species that produces colorful or aromatic flowers. For example, the plant may be selected from a species of plant from the group consisting of canola, sunflower, tobacco, sugar beet, cotton, maize, wheat, barley, rice, sorghum, , mango, peach, apple, pear, strawberry, banana, melon, potato, carrot, e, onion, soya spp, sugar cane, pea, field beans, , grape, , alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, cucumber, morning glory, , pepper, eggplant, ld, lotus, cabbage, daisy, carnation, tulip, iris, lily, and nut producing plants insofar as they are not already specifically mentioned.

The gene repair oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers (biolistic delivery), microfibers, polyethylene glycol (PEG)—mediated uptake, electroporation, and microinjection.

Also ed are methods and compositions related to the e of cells mutated according to methods as disclosed herein in order to obtain a plant that produces seeds, henceforth a "fertile plant", and the production of seeds and additional plants from such a fertile plant.

Also provided are s of selectively controlling weeds in a field, the field comprising plants with the disclosed AHAS gene alterations and weeds, the method comprising application to the field of an herbicide to which the plants have been rendered resistant.

Also provided are ons in the AHAS gene that confer resistance or tolerance to a member of the nt herbicide to a plant or n the mutated AHAS gene has substantially the same enzymatic ty as compared to wild-type AHAS.

Gene Repair Oligonucleobases [0055) The methods and compositions disclosed herein can be practiced or made with “gene repair Oligonucleobases” having the conformations and chemistries as described in detail below.

The “gene repair Oligonucleobases” as contemplated herein have also been described in published scientific and patent literature using other names including “recombinagenic oligonucleobases;” “RNA/DNA chimeric oligonucleotides;” “chimeric oligonucleotides;” “mixed duplex oligonucleotides” (MDONs); “RNA DNA oligonucleotides (RDOs);” “gene targeting oligonucleotides; 3, “genoplasts;” e stranded modified ucleotidesg” “Single stranded oligodeoxynucleotide mutational vectors” s); x mutational vectors;” and “heteroduplex mutational vectors.” [0056) Oligonucleobases having the conformations and chemistries described in US. Pat. No. ,565,350 by Kmiec (Kmiec I) and US. Pat. No. 5,731,181 by Kmiec (Kmiec II), hereby incorporated by reference, are suitable for use as “gene repair Oligonucleobases” of the invention. The gene repair Oligonucleobases in Kmiec I and/or Kmiec 11 contain two complementary strands, one of which contains at least one segment of RNA-type nucleotides (an "RNA segment") that are base paired to DNA-type nucleotides of the other strand.

Kmiec II discloses that purine and dine base-containing non—nucleotides can be substituted for nucleotides. Additional gene repair molecules that can be used for the present invention are described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 983; 5,795,972; ,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No.

OO/23457; and in International Patent Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; W0 99/58702; and WO 99/40789, which are each hereby incorporated in their entirety.

In one embodiment, the gene repair oligonucleobase is a mixed duplex oligonucleotides (MDON) in which the RNA—type tides of the mixed duplex oligonucleotide are made RNase resistant by replacing the roxyl with a fluoro, chloro or bromo functionality or by g a substituent on the 2'—O. Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents include the substituents taught by U.S. Pat. No. 71 l (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications), which are hereby incorporated by reference. As used herein, a 2'—fluoro, chloro or bromo tive of a cleotide or a ribonucleotide having a 2'- OH substituted with a substituent described in the Martin Applications or Sproat is termed a "2'— Substituted Ribonucleotide." As used herein the term "RNA—type nucleotide" means a 2'— hydroxyl or 2'—Substituted Nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non—natural linkages taught by Kmiec I or Kmiec II. As used herein the term "deoxyribo—type nucleotide" means a nucleotide having a 2’—H, which can be linked to other nucleotides of a gene repair oligonucleobase by an tituted phosphodiester linkage or any of the non—natural linkages taught by Kmiec I or Kmiec II.

In a particular embodiment of the present invention, the gene repair oligonucleobase is a mixed duplex oligonucleotides (MDON) that is linked solely by unsubstituted phosphodiester bonds. In alternative embodiments, the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus—based linkages as taught by Kmiec II. In yet another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2'—Substituted Nucleotide. Particular preferred embodiments of stituted Ribonucleotides are 2'-flu0ro, 2'- methoxy, 2'~propyloxy, 2'-allyloxy, 2'—hydroxylethyloxy, 2'-methoxyethyloxy, 2'- fluoropropyloxy and 2'-trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2'—Substituted Ribonucleotides are 2'-fluoro, 2'—methoxy, 2'-methoxyethyloxy, and 2'~allyloxy substituted nucleotides. In another embodiment the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds. gh mixed duplex oligonucleotides (MDONs) having only a single type of 2'- substituted RNA~type nucleotide are more conveniently synthesized, the methods of the invention can be practiced with mixed duplex ucleotides having two or more types of RNA-type nucleotides. The on of an RNA segment may not be affected by an interruption caused by the introduction of a deoxynucleotide between two pe trinucleotides, accordingly, the term RNA segment encompasses terms such as "interrupted RNA segment.“ An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment an RNA t can contain alternating RNase-resistant and unsubstituted 2'-OH nucleotides. The mixed duplex oligonucleotides preferably have fewer than 100 tides and more preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands are Watson-Crick base . In one embodiment the strands of the mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3' and a single 5' end. The 3' and 5' ends can be protected by the addition ofa "hairpin cap" whereby the 3' and 5' terminal nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second s distant from the 3' and 5' ends, so that the Watson-Crick pairing between the first and second strands is ized.

The first and second strands contain two regions that are homologous with two fragments of the target gene, i.e., have the same sequence as the target gene. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of ting DNA segment and may also contain pe tides that are not within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a "heterologous region. " The logous region can contain one, two or three mismatched nucleotides. The mismatched nucleotides can be contigmous or alternatively can be separated by one or two nucleotides that are homologous with the target gene. Alternatively, the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene only by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide. The length and position of the heterologous region is, in this case, deemed to be the length of the deletion, even though no tides of the mixed duplex oligonucleotide are within the heterologous region. The distance between the fragments of the target gene that are complementary to the two homologous regions is identical to the length of the heterologous region where a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.

The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous , i.e., a region that is identical in sequence to a fragment of the target gene, which ts er preferably contain at least 13 RNA—type tides and preferably from 16 to 25 RNA—type nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one embodiment, RNA segments of the homology regions are separated by and adjacent to, i.e., "connected by" an intervening DNA segment. In one embodiment, each nucleotide of the logous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a or segment." In another embodiment of the present invention, the gene repair oligonucleobase (GRON) is a single stranded oligodeoxynucleotide mutational vector (SSOMV), which is sed in International Patent Application 00/23457, US. Pat. Nos. 360, 6,479,292, and 7,060,500 which is incorporated by reference in its entirety. The sequence of the SSOMV is based on the same principles as the mutational s bed in US. Pat. Nos. ,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 02; and WO 99/40789. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired c tion termed the mutator region. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region can cause a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such an SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene but separated by one, two, or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of the target gene.

The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3’ terminal and/or 5’ terminal intemucleotide e or alternatively the two 3’ terminal and/or 5' terminal intemucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an intemucleotide linkage is the linkage n nucleotides of the SSOMV and does not include the linkage n the 3' end nucleotide or 5’ end nucleotide and a blocking tuent. In a specific embodiment the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.

The SSOMV can be designed to be complementary to either the coding or the non- coding strand of the target gene. When the d mutation is a substitution of a single base, it is preferred that both the mutator nucleotide and the targeted nucleotide be a pyrimidine. To the extent that is consistent with achieving the desired functional result, it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines.

Particularly preferred are SSOMVs that encode transversion mutations, i.e., a C or T mutator nucleotide is ched, tively, with a C or T nucleotide in the complementary strand.

In addition to the oligodeoxynucleotide, the SSOMV can contain a 5' blocking substituent that is attached to the 5' terminal carbons through a linker. The try of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible. A variety of non-toxic substituents such as biotin, terol or other steroids or a non-intercalating cationic fluorescent dye can be used. Particularly preferred reagents to make SSOMVs are the reagents sold as Cy?)TM and Cy5TM by Glen Research, Sterling Va. (now GE Healthcare), which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3',3'«tetramethyl sopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is particularly preferred.

When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5' terminal of the oligodeoxynucleotide as a phosphodiester with a 5‘ terminal phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience. When the cially available Cy3 phosphoramidite is used as ed, the resulting 5' modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N'—phosphatidylpropyl 3,3,3',3'—tetramethyl indomonocarbocyanine.

In a red embodiment the indocarbocyanine dye is tetra substituted at the 3 and 3' positions of the indole rings. Without tions as to theory these substitutions t the dye from being an intercalating dye. The identity of the substituents at these positions is not critical.

The SSOMV can in addition have a 3' blocking substituent. Again the chemistry of the 3‘ blocking substituent is not critical.

The mutations herein described might also be obtained by mutagenesis (random, somatic or directed) and other DNA g or ination technologies including, but not limited to, gene targeting using pecific homologous recombination by zinc finger nucleases.

Delivery‘of Gene Repair Oligonucleobases into Plant Cells Any commonly known method used to transform a plant cell can be used for delivering the gene repair ucleobases. rative methods are listed below.

Microcarriers and Microfibers The use of metallic microcarriers spheres) for introducing large fragments of DNA into plant cells having cellulose cell walls by projectile penetration is well known to those skilled in the relevant art (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for projecting them.

Specific conditions for using microcarriers in the methods of the present invention are described in International Publication WO 99/07865. In an illustrative technique, ice cold microcarriers (60 mgmL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M C8C12 and 0.1 M spermidine are added in that order; the mixture gently agitated, e. g., by vortexing, for 10 minutes and then left at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, fuged and ended in 100% l. Good results can be obtained with a concentration in the adhering solution of 8-10 uguL microcarriers, 14—1 7 ugmL mixed duplex oligonucleotide, 1.1-1.4 M C3C12 and 18-22 mM dine. Optimal results were observed under the conditions of 8 uguL microcarriers, 16.5 ug/mL mixed duplex oligonucleotide, 1.3 M CaClg and 21 mM spermidine.

Gene repair oligonucleobases can also be introduced into plant cells for the practice of the present invention using micro fibers to penetrate the cell wall and cell membrane. U.S. Fat.

No. 5,302,523 to Coffee er al. describes the use of es.0.5 um and 10.times.0.3 um silicon carbide fibers to facilitate transformation of suspension maize cultures of Black Mexican Sweet.

Any mechanical technique that can be used to introduce DNA for transformation of a plant cell using microfibers can be used to deliver gene repair oligonucleobases for transmutation.

An illustrative technique for microfiber delivery of a gene repair oligonucleobase is as follows: Sterile microfibers (2 ug) are suspended in 150 uL of plant culture medium containing about 10 pg of a mixed duplex ucleotide. A sion e is allowed to settle and equal volumes of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 h as is appropriate for the particular trait.

Protoplast Electroporation In an alternative embodiment, the gene repair ucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part. The protoplasts are formed by enzymatic treatment of a plant part, particularly a leaf, according to techniques well known to those skilled in the art. See, e.g., Gallois er £11., 1996, in s in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp er £11., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, NJ. The lasts need not be cultured in growth media prior to electroporation. Illustrative conditions for electroporation are s.10.sup.5 protoplasts in a total volume of 0.3 mL with a concentration of gene repair oligonucleobase of between 0.64 ug/mL.

Protoplast PEG—mediated DNA uptake In an alternative embodiment, nucleic acids are taken up by plant protoplasts in the presence of the membrane~modifying agent polyethylene glycol, according to ques well known to those skilled in the art (see, e.g., Gharti-Chhetri er al., 1992; Datta er al., 1992).

Microinjection In an alternative embodiment, the gene repair oligonucleobases can be delivered by injecting it with a microcapillary into plant cells or into protoplasts (see, e.g., Miki er al., 1989; Schnorfera1., 1991).

Selection of Herbicide Resistant Plants and Application of Herbicide Plants and plant cells can be tested for resistance or tolerance to an herbicide using commonly known methods in the art, e.g., by growing the plant or plant cell in the presence of an herbicide and measuring the rate of growth as compared to the growth rate in the absence of the As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or rate of cell division of the plant, plant organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild—type AHAS protein.

As used herein, substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as the occurrence of one or more development events in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild-type AHAS protein.

In certain embodiments plant organs provided herein include, but are not limited to, leaves, stems, roots, tive buds, floral buds, meristems, embryos, cotyledons, endosperm, , petals, pistils, s, stamens, s, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or sections, slices or discs taken therefrom. Plant tissues include, but are not limited to, callus tissues, ground s, ar tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, gall tissues, plant tumor tissues, and reproductive tissues.

Plant cells include, but are not limited to, ed cells with cell walls, variously sized aggregates thereof, and protoplasts.

Plants are substantially ant" to a relevant herbicide when they are subjected to it and provide a dose/response curve which is shifted to the right when compared with that provided by similarly ted lerant like plant. Such esponse curves have "dose" plotted on the X—axis and "percentage kill", "herbicidal effect", etc., plotted on the y—axis.

Tolerant plants will require more herbicide than non—tolerant like plants in order to produce a given herbicidal effect. Plants that are substantially "resistant" to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions, when subjected to herbicide at concentrations and rates which are typically ed by the emical community to kill weeds in the field.

Plants which are resistant to an herbicide are also tolerant of the herbicide.

Following are examples, which illustrate procedures for practicing the ion.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

Example 1: Preparation of Herbicide—Resistant Brassica s Unless otherwise specified, as used herein, numbering of the gene(s) is based on the amino acid sequence of the Arabidopsis acetolactate synthase (ALS) or acetohydroxyacid synthase (AHAS) At3g48560 (SEQ ID NO: I). In laboratory notebook references prior to October 2005, the S653 position (based on the Arabz‘dopsz‘s amino acid sequence) is referred to as S621 based on the corn amino acid sequences ZmAHAS108 and ZmAHAS109 (Fang et (11., 1992).

One goal was to make the imazethapyr (Imi) resistant amino acid substitution S653N in either or both BnAHAS I and III of spring Canola (Brassica napus, Spring d Rape ~— designated BN—2) and Winter Oilseed Rape (WOSR, also Brassz'ca napus - designated BN—l l).

In order to amplify the target regions of BnAHAS I and III from Brassz’ca napus (initially elite Canola line BN—Z), the oligo pair BnALSl and BnALS2 was designed (SEQ ID NOs: 9 and 10). Since BnAHAS I and III do not contain introns, BnALSl and BnALS2 amplify the target regions of 284 bp from both genomic DNA and cDNA flanking the S653 site. This primer pair was also designed to enable amplification of the ALS target region from Arabidopsis.

The s were received and resuspended in sterile water.

Initially the BnALSl/BnALS2 primer pair was used to PCR amplify the BnAHAS I and III C—terrninal target regions from BN—2. Later, the ALS target regions were ed from additional BN—2 samples and cloned into pGEM—T easy. Cloned inserts from 12 colonies from each of 3 genomic DNA and cDNA samples grown in ght cultures were plasmid prepped and sequenced, ing the target sequence. Glycerol stocks were prepared for BN-2 ALS 2c—21 as the BnAHAS I representative and BN—2 ALS YB10—2g-l 4 as the BnAHAS III entative for these 284 bp target regions.

The genomic and cDNA target region BnALS sequences from line BN-2 were analyzed and PCR errors recorded. Based on the sequence of BnAHAS I and III from BN—2 a single GRON (Gene Repair Oligonucleotide) BnALSl 621/C/4l/5’Cy3/3’idC (SEQ ID NO: 5) was designed to make a serine to asparagine amino acid substitution (AGT -> AAT) at position 653 (See Table l). The initial syntheses were resuspended and their concentrations determined. The ended oligos were stored frozen at v700C. The non—coding version of this GRON was called BnALS1621/NC/4l/5’Cy3/3’idC (SEQ ID NO: 6). At a later date, the BnALS1621/NC was compared with all Genbank sequences Where the only sequences with >16 tides of 41 hybridizing were BnAHAS I and III and where the oligo had the single mismatch G->A designed to introduce the S653N amino acid substitution.

TABLE 1. GRON SEQUENCES GRON Sequence BnALSl 621/C/41/5’Cy3/3 ’idC VTGTGTTACCGATGATCCCAAATGGTGGCACTTTC AAAGATGH (SEQ ID NO: 5) BnALS 1 621/NC/41/5’Cy3/3 ’idC VCATCTTTGAAAGTGCCACCATTTGGGATCATCGG TAACACAH (SEQ ID NO: 6) BnALS1574/C/4l/5 ’Cy3/3 ’idC GATGGTCATGCAATTGGAAGATCGGTTCT ACAAAGCH (SEQ ID NO: 7) BnALSlS74/NQ’41/5’Cy3/3’idC VGCTTTGTAGAACCGATCTTCCAATTGCATGACCA TCCCAAGH (SEQ ID NO: 8) The converting base is shown in bold. V=CY3; H=3‘DMT dC CPG BnALS target regions, from a Cibus elite Winter Oil Seed Rape (WOSR) line BN-l 1, were amplified from genomic DNA and cDNA of single plants known to represent line BN-l 1.

The BN-ll BnALS sequences were analyzed and PCR errors recorded.

In addition to characterizing the BnALS target s from BN-2 and BN—l 1, the same target regions were also characterized in the commercial variety eld Canola (BN-15). The Clearfield BnAHAS sequences were analyzed and PCR errors recorded showing the expected amino acid changes, S653N (AGT -> AAT) in BnAHAS I and W574L (TGG —> TTG) in BmAHAS III. Ultimately full coding sequences for BnAHAS I and III were amplified using BnALS3/BnALSOR2 (SEQ ID NOs: 11 and 12) and BnALS3/BnALSOR3 (SEQ ID NOs: 11 and 13) respectively, cloned and sequenced from lines BN-2, BN—l 1 and BN-15 to serve as reference sequences for comparative es Imi resistant BN-2 (Canola) callus s BnALS1621N-44 to 53, arising from various treatments, were extracted using the Edwards at al. (1991) method. Genomic DNA from this material was screened using an allele specific PCR (AS-PCR) mix (MM#23 composed of oligos BnALSOFI, BrLALSORZ, BnALSOR3 and BnALSIRIA) (SEQ ID NOs: 14, 12, 13, and , tively) to specifically detect the S653N change and allele specific PCR mix (MM#29 composed of oligos BnALSOFZ, BnALSOR4 and BnALSIFlT) (SEQ ID NOS: 16, 17, and 18, respectively) to specifically detect the W574L change in either BnAHAS I or 111. These initial AS-PCR results were inconclusive, therefore, additional samples BnALS1621N-54 to 58 for most of samples BnALS1621N-44 to 53 were ed, and extracted using the s et al. (1991) method with additional purification steps to obtain more pure genomic DNA.

At this point, samples BnALS1621N—54 to 58 were amplified with a BnALSOF2, BnALSORZ and BnALSOR3 primer combination, and a small amount of amplified product was obtained. These products were cloned into pGEM-T easy and 12 White colonies per line setup as overnight cultures, plasmid prepped (using the Qiagen plasmid miniprep kit), and sequenced using BigDye 3.1 sequencing try.

Preliminary sequence analysis determined that BnALS1621N—55~13 had an S653N mutation in BnAHAS III (AGT -> AAT) and two other clones from this line, BnALSl621N—55- and 19, were wildtype in BnAHAS 111. Full sequence is was recorded. To confirm that this single positive did not result from a PCR error, an additional 38 es from this line were ed by AS—PCR. The strongest 8 positives from this AS-PCR screen BnALS1621N-55~1 to 8 were setup as overnight cultures, and plasmid DNA was isolated and sequenced. Seven of the 8 positive colonies, BnALS1621N-55—2 to 8 by AS-PCR were positive by sequencing for the S653N mutation in BnAHAS III; the other BnALS1621N—55‘1 was a pe BnAHAS I sequence. Together these results indicated that line BnALS1621N-55 is heterozygous for the S653N mutation in BnAHAS III.

Sample BnALS1621N-55 was a parallel callus sample from the same initial Imi resistant callus as BnALS1621Nv49. Genomic DNA from these samples was amplified With the BnALSOF2, BnALSOR2 and BnALSOR3 primer combination, and the fragments cloned into pGEM-T easy and transformed. MM#23 was used to screen 38 White colonies for the S653N mutation, With 4 positive colonies being identified (BnALS1621N9 to 12). These colonies were sequenced. Three of the four colonies 1621N—49—9, 10 and 12) had the S653N on in BnAHAS III.

A similar method of target region cloning followed by AS—PCR with sequence ation was used to identify other lines with the S653N polymorphism. Among these was line BnALS—97. BnALS—97 was regenerated into a plant and allowed to set seed. Since this line was a prototype (as a plant), the full coding sequences of both BnAHAS I and III were cloned and ced. In this line, the only rphism compared to the BN—2 wildtype sequence was the ACT '> AAT codon change which s in the 8653N amino acid substitution in BnAHAS III.

Preliminary sequence analysis of random cloned F2i‘BnALSOR2 and BnALSOF2/BnALSOR3 amplicons from line BnALSl62lN—57 indicated that both clones BnALSl621N43 and 46 had the W574L mutation (TGG —> TTG) in BnAHAS 1. Full sequence analysis was recorded, and indicated line 57 is heterozygous, since clones 38 and 41 were W574. Therefore, the BnALSOF2, BnALSOR2 and BnALSOR3 primer combination was used to amplify this fragment from BnALSl62lN-57 and BnALSl62lN—45, a parallel callus sample from the same initial lmi resistant callus as BnALSl621N-57, ligated into pGEM-T easy, ormed and plated with hite selection.

MM#29 was used to screen 19 white colonies for the W574L from each of the plates for BnALSl621N—57 and BnALS 1621N—45, with 4 positive colonies for each (BnALSl62lN-45~l, 2, 9 and 18, and BnALSl62lNl, 7, l3 and 16) being plasmid prepped and sequenced.

Sequence analysis showed that all 8 es had the W574L mutation in BnAHAS I. The armealing temperature for MM#29 was optimized, and an additional 19 colonies screened using these new conditions. Three positive colonies (BnALSl621N—57—1 7, 18 and 19) were plasmid prepped and sequenced.

Additional Imi resistant Canola samples BnALS-68 to 91 were received and genomic DNA extracted using the Edwards er a1. (1991) method. From each sample, the BnALSOF2, BnALSOR2 and BnALSOR3 primer combinations were used to amplify fragments of BnAHAS I and III and clone them into pGEM-T easy.

Using MM#23 in AS-PCR reactions to detect the S653N mutation, 12 colonies per line were screened, and results showed 2 positives from line Sl —81-203 and 208) and 4 from BnALS—76 —76-153, 154, 156 and 162) to contain the S653N mutation in BnAHAS III by sequencing. Full sequence analysis was recorded. By AS—PCR screening with MM#23, both plified target regions or of bacterial colonies with single cloned inserts, line BnALS—159 ular biology sample 102; colony 655) was identified as having the S653N mutation in BnAHAS I, Cibus’ initial S653N event in this gene.

Finally, in line BnALS—83 (molecular biology sample 91), wildtype (colony 408) and mutant (colonies 403, 405 and 406) were fied to indicate this line was heterozygous for the S653T mutation (AGT -> ACT) in BnAHAS I. As plants, line BnALS-83 was confirmed to be heterozygous in BnAHAS I for the S653T on. Further screening with MM#23 (S653N) and MM#29 (W574L) of new Imi resistance BN-2 Canola lines BnALS-76 and BnALS—123 was performed. Since the aforementioned ed mutations were not found in these samples, 12 F2/OR2/3 Were cloned and sequenced. All 7 clones of BnAHAS III for BnALS—123 (Colonies 5, 17-19, 21, 22, 26 and 28) Were positive for the S653T mutation. However, seed from the R1 plants was ped as heterozygous.

Additionally, N-terminal PCR fragments of each of BnAHAS I and III were cloned and sequenced for BnALS-58, 68 and 69 using BnALS3/BnALS8 (SEQ ID NOs: 11 and 19 respectively) and BnALSB/BnALS9 (SEQ ID NOs: 1 1 and 20, respectively) oligo combinations respectively. A new tissue sample for BnALS— 96 was obtained and N—terminal PCR fragments of BnAHAS I and III were cloned and sequenced to show line BnALS-96 had an A205V mutation (GCC -> GTC) in BnAHAS I. All four clones of the full length coding sequence BnALS3/OR2 amplicon from plant material derived from callus line BnALS-68 had an A205V change (GCG -> GTG) in BnAHAS III, identical to the change in BnALS-69.

Lines determined to have a mutation, the experiment they were derived from and the treatment provided are summarized in Table 2. Line BnALS—159 died as a callus line and did not regenerate shoots. Table 3 shows exemplary oligos used in the ments disclosed herein.

TABLE 2. MUTATIONS IN IMI RESISTANT TISSUE SAMPLES OF BN CANOLA.

Line (CS#) Mutation SNP Gene i Line BnALS-96 A205V GCC —> GTC I I BN2 BmALs-68 A205V GCG a GTG 111 BN2 BmALs-69 A205V GCG a GTG 111 BN2 ' fl r ’ ’ 58 A205D GCC _, GAC I BN2 BmALs-63 w574c TGG _, TGT 111 BN2 BmALs-57 W574L TGG _, TTG 1 BN2 i"\11/574L r BMLS—67 TGG _, TTG 1 BN2 BN02—204—A01 W574L;R577W TGG a TTG; CGG a TGG 111 BN2 BN02C01 W574L (HET) TGG a TTG 111 LBN2 A L l BN02—224—B01 W574M (HET) TGG a ATG 111 BN2 BnALS—76 *"w574s TGG —> TCG ”111 BN2 J’ 7 BmALs-159* S653N AGT —> AAT 1 BN2 BnALS—SS S653N AGT —> AAT 111 BN2 ‘ ’ BnALS—97 S653N AGT —> AAT 111 BN2 J l él S653N AGT _, AAT 111 BN2 BmALs—84 S653N "AGT _, AAT 111 '"BN2 ” w‘ ‘ BmALs-83 S653T AGT _, ACT 1 BN2 “ ‘ BmALs—123 S653T AGT _, ACT L111 BN2 ‘ “ BN02—139—E07 W574C(het);S653N TGG —> TGC; AGT 6 AAT 111 BN2 BN02—139—D05 A205V;S653N GCG —> T —> AAT 111 FBN2 ‘ 1 BN02—139-F08 A205V;S653N GCG a GTG;AGT —> AAT 111 BN2 BN02—139-C03 A205V(het);S653N GCC —> GTC;AGT —> AAT 1;111 BN2 ’ ’ BN02D06 W574C;S653N TGG —» T —» AAT 1;111 BN2 BNO2E10 W574L;S653N TGG —» TTG;AGT —» AAT 1;111 BN2 BN02A13 FW574L(het);S653N * ‘ TGG —> T —> AAT 111 BN2 ‘ A “ BNO2A12 +D376E;S653N GAC —» GAG;AGT —» AAT 111 BN2 BN02F09 A122V;S653N GCT—>GTT;AGT —» AAT 1;111 BN2 BN02A01 A2051), S653N GCG —» GAC;AGT —» AAT F1;111 BN2 - . 1 BNO2D-04 ws74c; S653N TGG —» TGC;AGT —» AAT 1;111 BN2 J 1 BN02B11 W574C; S653N TGG —» T —» AAT 1;111 BN2 TABLE 3: EXEMPLARY OLIGOS Name Length Oligo Sequence 7311ALSI (SEQ ID 1 "’ 24 ATGCAATGGGAAGATCGGTTCTAC NO: 9) BnALS2 (SEQ ID 29 CCATCYCCTTCKGTTATKACATCKTTGAA NO: 10) BnALS3 (SEQ ID 2o CTAACCATGGCGGCGGCAAC NO: 11) BnALSOR2 (SEQ ID 28 AGTCTGGGAACAAACCAAAAGCAGTACA NO: 12) BnALSOR3 (SEQ ID 28 CGTCTGGGAACAACCAAAAGTAGTACAA NO: 13) BnALSOFl (SEQ ID 28 AGTGACGAAGAAAGAAGAACTCCGAGAA NO: 14) BnALSIRlA (SEQ 30 LCTGTTATTACATCTTTGAAAGTGCCACAAT ID NO: 15) BnALSOFz (SEQ ID 28 bTGACGGTGATGGAAGCTTCATAATGAAC NO: 16) BnALSOR4 (SEQ ID 28 i—GTCCWGGTGTATCCAGCATTGTCTGAAT NO: 17) L—BnALSIFlT (SEQ ID H26 hCAGCATCTTGGGATGGTCATGCAGTT NO: 18) BnALSS (SEQ ID 71 CCCATCAAAGTACTCGCACCG NO: 19) BnALS9 (SEQ ID T21 AACGTACTCGCACCA NO: 20) As shoots become available, s are being made in all permutations and ations. Line BnALS—97 is the reference S653N in BnAHAS 111. Line BnALS-57 is the reference W574L in BnAHAS I. Both BnALS-68 and 69 are being advanced as reference lines for A205V in BnAHAS Ill. Line BnALS-83 is the first line that was heterozygous for S653T in BnAHAS I as callus to also be zygous as a plant.

Example 2: Materials and Methods Cell Culture Work Description. Shoots derived both from seeds and from microspore-derived embryos were propagated under sterile conditions in vitro. Cuttings were subcultured every 2 - 4 weeks and cultured in petri dishes (25 mm X 90 mm) in a volume of 40 - 45 mL RS medium (Dovzhenko, 2001). The dishes were sealed with Micropore tape (3M Company). Young leaves were used for protoplast ion.

Protoplast isolation and purification. About 600 mg of leaf tissue of 2 - 3 week-old in vitro shoots were cut into small strips with a l in a petri dish with 5 mL medium B (Pelletier et al., 1983), pH adjusted to 5.8. After approximately 1 h, medium B was replaced with enzyme solution, ting of medium B in which 0.5% (w/v) Cellulase YC and 0.75% (w/v) Macerozyrne R10 (both from Karlan Research Products, Cottonwood, Arizona), 1 g/L bovine serum albumin, and 1 g/L 2~morpholinoethanesulfonic acid were dissolved. The enzyme solution was vacuum—infiltrated into the leaf tissue, and the dish with leaf pieces in enzyme solution was incubated for at 25°C in darkness. last purification was performed using an iodixanol density gradient (adapted from Optiprep Application Sheet C18; Purification of Intact , Plant Protoplasts; Axis«Shie1d USA, 10 Commerce Way, Norton, MA 02776). After the density gradient centrifugation, the band with purified protoplasts was removed er with about 5 mL W5 medium (Frigerio et a[., 1998). The protoplast yield was determined with a hemocytometer, and the lasts were stored for 2 h at 4°C.

Gene Repair Oligonucleotide GRON introduction. The protoplast suspension was mixed with an equal volume ofW5 medium, transferred to a 50 mL centrifuge tube, and centrifuged for 5 min at the lowest setting of a clinical fuge (about 50 x g). The supernatant was d and replaced with TM medium (Klaus, 2001), adjusting the protoplast density to 5 x 106/mL. Aliquots of 100 nL containing 5 x 106 protoplasts each were distributed into 12 mL round bottom centrifuge tubes. GRONS targeted at a mutation in one or both AHAS genes were then introduced into the lasts using a PEG treatment. To introduce the GRONs into the protoplasts, 12.5 pg GRON dissolved in 25 uL purified water and 125 uL of a polyethylene glycol solution (5 g PEG MW 1500, 638 mg mannitol, 207 mg CaN03 x 4H20 and 8.75 mL purified water; pH adjusted to about 9.0) was added. After a 30 min incubation on ice, the protoplast-PEG sion was washed with W5 medium and resuspended in medium B.

The suspension was kept overnight in a refrigerator at about 4°C.

Embedding of protoplasts in calcium alginatc. One day after the GRON introduction, protoplasts were embedded in calcium alginate. The embedding of protOplasts in gel substrates (e. g., agarose, alginate) has been shown to enhance protoplast survival and to se division frequencies of last—derived cells. The method applied was based on that bed in Dovzhenko .

Protoplast culture and selection of imazcthapyr~resistant calli. The selection of imazethapyr—resistant calli was carried out using sequential subcultures of the alginates in media according to Pelletier et al. (1983). Selection was started one week after the PEG/GRON ent at a concentration of 0.5 uM imazethapyr. The herbicide did not have an immediate effect. Initially, all microcolonies that had formed during the early culture phase without hapyr continued to grow, but slower than controls without added herbicide. One to two weeks after the onset of selection, the colonies slowed down in growth or stopped g.

Before the end of the selection phase in liquid medium, cells and colonies were released from the alginate by treating them for 30 — 45 min with culture medium containing 50 mM sodium citrate. At the moment of transferring released colonies from liquid to solid medium, the majority of colonies were either dead, or consisted of a greenish center, covered with outer layers of dead cells. On the solidified selection medium E the majority of microcalli that still ned living cells stopped g and turned sh. Limited growth of individual calli continued occasionally, but all non—resistant calli eventually turned brown and died. Two to three weeks after the transfer to solidified selection medium (occasionally earlier), actively growing calli ed among a background of brownish cells and microcalli.

Regeneration of plants from protoplast~derived, herbicide—tolerant calli with a ed mutation in an AHAS gene. Imi—tolerant calli that had developed on solidified selection medium and whose DNA upon analysis had shown the presence of a mutation were transferred to herbicide—free medium E (Pelletier et a[., 1983) to accelerate development.

Individual callus lines varied in their growth rates and morphologies. In general, the development towards shoot regeneration followed these steps: Undifferentiated, green callus ~> callus with dark green areas -> development of roots -> development of shoot initials ~> development of stunted shoots with hyperhydric (Vitrified) leaves.

The development of individual callus line was variable, but through continuous subculture and lication on medium E or modifications of medium E with lower concentrations of alpha—naphthalene acetic acid (NAA) eventually many callus lines produced shoots.

Once shoots with three to four leaves had formed on medium E, they were transferred to RS medium (Dovzhenko, 2001). On this medium, over time shoot and leaf tissue developed that was logically l’ (i.e., non-hyperhydric). After in vitro plantlets had produced roots, standard protocols were used for the adaptation to greenhouse conditions.

Example 4: Herbicide Spray Data B. napus plants at the 5—6 leaf stage were sprayed with various AHAS inhibiting herbicides. B. napus plants sprayed ed the parental line BN02 (or BN1 1 as required), BN15 (Clearfield, a commercial two gene check) and mutants as ed below. Herbicides were sprayed in the presence of 0.25% AU391 surfactant at the following rates: ox (BeyondTM) 0, 2, 4, 6, 8, 12, 16, 32 and 48 oz active ingredient/Acre (ai/A) Thifensulfuron 0.028, 0.056, 0.112, 0.168 lb ai/A Tribenuron 0.015, 0.03, 0.06, 0.12, 0.18 lb ai/A Nicosulfiiron lb 0.06, 0.120, 0.24, 0.36 ai/A Rimsulfuron 0.015, 0.03, 0.06, 0.12, 0.18 lb ai/A 2:1 weightzweight Thifensulfuron/Tribenuron 0.056, 0.1 12, 0.224, 0.336 lb ai/A 2.22:1 ThifensulfiJrorMNicosulfuron 0.058, 0.116, 0.232 lb ai/A Primisulfuron 0.035, 0.070, 0.140 lb ai/A Flumetsulam0.040, 0.080, 0.16 lb ai/A Cloransulam0.039, 0.078, 0.156 lb ai/A Herbicides were applied by foliar spray with control plants being lefi unsprayed.

Excepting lmazamox, all AHAS inhibiting herbicide trials were evaluated 14 days post spraying using a damage scale of l—10 with 1 being dead, and 10 being the undamaged unsprayed controls. dual plant lines were scored at each spray rate compared to the performance of the controls at that particular rate. Results of the Spray trial are ted in Figure 4.

Chemistry tested: Genotype (# of seedlings for all rates) Thifensulfuron: BN2 (6), BN15 (6), BN15xBnALS—57 (18) Tribenuron: BN2 (6), BN15 (6), BN15xBnALS-57 (18), 63 (9) Nicosulfuron: BN2 (6), BN15 (6), BN15xBnALS—57 (18) THI / TRI: BN2 (6), BN15 (6), BN15xBnALS—57 (18) Rimsulfuron: BN2 (6), BN15 (6), nALS—57 (18), 63 (9) THI / Nic: BN2 (18), 63 (9), BN15xBnALS—57 (36), BN15 (18) ulfuron: BN2 (9), 63 (9), BN15 (9), BN15xBnALS—57 (18) Flumetsulam: BN2 (9), 63 (9), BN15 (9), BN15xBnALS~57 (18) Cloransulam: BN2 (9), 63 (9), BN15 (9), BN15xBnALS—57 (18) Trials with Imazamox were evaluated as follows: 10d score for: 8, 16, 32, and 48 oz/A oanALS—83xBnALS—123, BnALS—96xBnALS—123, BnALS—97xBnALS—57, BN15xBnALS-57, BN2, BN15, BnALS—97xBN15 17d score for: 4 and 12 oz/A —97xBnALS—57, BN15xBnALS—97, BN2 28d score for: 2, 4, 6, 8 oz/A for all the single factor mutations. lmazamox: 2 oz/A: BN2 (18), BnALS-123 (9), BnALS-96 (9), BnALS-97 (18), BnALS-83 (6), BnALS-76 (18), BnALS-58 (9), BnALS—57 (12) 4 oz/A: BN2 (18), BnALS-123 (9), BnALS-96 (9), BnALS-97 (18), 83 (9), BnALS~76 ( 18), BnALS—58 (9), BnALS-57 (12), BnALS-97xBN15 (15), BnALS-97xBnALS-57 (14) 6 oz/A: BN2 (9), BnALS-123 (9), 96 (12), BnALS-97 (9), BnALS—83 (12), BnALS-76 (9), BnALS-57 (12) 8 oz/A: BnALS-123 (9), BnALS—96 (12), BnALS-83 (12), BnALS—83xBnALS—123 (18), BnALS-96xBnALS-123 (18), BN2 (18), BN15 (18), nALS-57 (15), BnALS- 97xBnALS-57 (18), BN15xBnALS—97 (18) 12 oz/A: BnALS-97xBN15 (15), BnALS-97xBnALS—57 (14), BN2 (12) 16 oz/A: BnALS—83xBnALS—123 (18), BnALS—96xBnALS-123 (18), BN2 (15), BN15 (18), BN15xBnALS—57 (18), BnALS-97xBnALS—57 (18), nALS-97 (18) 32 oz/A: BnALS-83xBnALS-123 (18), BnALS—96xBnALS-123 (18), BN2 (13), BN15 (18), BN15xBnALS—57 (18), BnALS—97xBnALS—57 (18), nALS—97 ( 18) 48 oz/A: BnALS-83xBnALS—123 (18), BnALS—96xBnALS—123 (18), BN2 (6), BN15 (18), nALS—57 (18), BnALS—97xBnALS—57 (18), BN15xBnALS—97 (18) REFERENCES Datta SK, Datta K, Soltanifar N, Donn G, Potrykus l (1992) Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts. Plant Molec.

Biol. 20:619—629 Dovzhenko A (2001) Towards plastid transformation in rapeseed (Brassica napus L.) and sugarbeet (Beta vulgaris L.). PhD Dissertation, LMU Munich, y of Biology Edwards K, Johnstone C, Thompson C (1991) A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Res. 1921349.

Frigerio L, Vitale A, Lord JM, Ceriotti A, Roberts LM (1998) Free ricin A chain, proricin, and native toxin have different cellular fates when expressed in tobacco protoplasts. J Biol Chem 273:14194—14199 Fang LY, Gross PR, Chen CH, Lillis M (1992) Sequence of two acetohydroxyacid synthase genes from Zea mays. Plant Mol Biol. 18(6):]185-7 Gharti~Chhetri GB, Cherdshewasart W, Dewulf J, Jacobs M, Negrutiu I (1992) Polyethylene glycol~mediated direct gene transfer in Nicotiana spp. Physiol. Plant. 85 :345-35 1 Klaus S (2003) Markerfreie transplastome flanzen (Marker—free transplastomic tobacco ). PhD Dissertation, LMU Munich, Faculty of y Miki B, Huang B, Bird S, Kemble R, Simmonds D, Keller W (1989) A procedure for the njection of plant cells and protoplasts. Meth. Cell Science 12: 1 39—144 Pelletier G, Primard C, Vedel F, Chetrit P, Remy R, Rouselle P, Renard M (1983) Intergeneric cytoplasm ization in Cruciferae by protoplast fusion. Mol. Gen. Genet. 191: 244-250 Schnorf M, Neuhaus-Url G, Galli A, Iida S, Potiykus I, Neuhaus G (1991) An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis.

Transgen. Res. 1:23— 30 Tan S, Evans RR, Dahmer ML, Singh BK, Shaner DL (2005) Imidazolinone~tolerant crops: y, current status and future. Pest Manag Sci. 61(3):246-57.

Unless otherwise defined, all cal and scientific terms used herein have the same g as commonly understood by one of ordinary skill in the art to which this invention belongs.

The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms ising,” “including,” ining,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of tion, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or ns thereof.

It is recognized that various modifications are possible Within the scope of the invention claimed.

Thus, it should be understood that although the t invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement, and variation of the ions disclosed may be resorted to by those skilled in the art, and that such modifications, ements and variations are considered to be within the scope of this ion. The materials, methods, and examples provided here are representative of red embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative tion removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the h group.

All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, inducing definitions, will control.

Definitions of specific embodiments of the invention as claimed herein follow.

According to a first embodiment of the invention, there is provided a non-transgenic herbicide-resistant Brassica plant sing: an acetohydroxyacid synthase (AHAS) I gene, wherein said gene encodes an AHAS I protein comprising a tryptophan to leucine substitution at a on corresponding to position 559 of SEQ ID NO: 2, and an AHAS III gene, wherein said gene encodes an AHAS III protein comprising a phan to leucine substitution at a position ponding to position 556 of SEQ ID NO: 3.

According to a second embodiment of the invention, there is ed a seed produced by the plant according to the first embodiment, wherein the genome of the seed comprises: the AHAS I gene, wherein said gene encodes the AHAS I protein comprising the tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and the AHAS III gene, wherein said gene encodes the AHAS III n comprising the tryptophan to leucine substitution at a position corresponding to position 556 of SEQ ID NO: 3.

According to a third embodiment of the invention, there is provided a plant grown from the seed of the second embodiment. ing to a fourth embodiment of the invention, there is provided a plant element from the plant of the third embodiment, wherein the plant element is ed from the group consisting of pollen, protoplast, ovule, and cell.

According to a fifth embodiment of the ion, there is provided a seed produced by a plant of the first embodiment.

According to a sixth embodiment of the invention, there is provided a seed produced by a plant of the first embodiment, wherein said seed is a seed of a Brassica plant line wherein said line is selected from the lines listed in Table 2.

According to a seventh embodiment of the invention, there is provided a seed produced by a plant of the first embodiment, wherein said seed is non-transgenic.

According to an eighth embodiment of the invention, there is provided a seed of an AHAS-inhibiting herbicide-resistant plant capable of ing a plant having AHAS herbicide resistance, wherein said seed is produced by a plant of the first embodiment. ing to a ninth embodiment of the ion, there is provided a cell from a non-transgenic herbicide-resistant Brassica plant according to the first embodiment, wherein the genome of the cell comprises: the AHAS I gene, wherein said gene encodes the AHAS I protein comprising the tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and the AHAS III gene, wherein said gene encodes the AHAS III protein sing the tryptophan to leucine substitution at a position corresponding to position 556 of SEQ ID NO: 3. ing to a tenth embodiment of the invention, there is ed a method for producing a non-transgenic herbicide-resistant Brassica plant, said method sing: (a) introducing into a plant cell a gene repair ucleobase (GRON) with a targeted mutation in an AHAS I gene and with a targeted mutation in an AHAS III gene to e a plant cell with: (i) said AHAS I gene that expresses an AHAS I protein comprising a tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and (ii) said AHAS III gene that expresses an AHAS III protein comprising a tryptophan to leucine substitution at a position ponding to position 556 of SEQ ID NO: 3; (b) identifying a plant cell having substantially normal growth and catalytic activity as compared to a corresponding wild-type plant cell in the presence of an AHAS- inhibiting herbicide; and (c) regenerating a non-transgenic herbicide-resistant plant having a mutated AHAS I gene and an AHAS III gene from said plant cell.

According to an eleventh embodiment of the invention, there is ed a method for increasing the herbicide-resistance of a plant comprising: crossing a first ansgenic Brassica plant to a second non-transgenic second Brassica plant, wherein said first plant comprises an AHAS I gene and said second plant ses an AHAS III gene, said AHAS I gene encodes an AHAS I protein comprising a tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and said AHAS III gene encodes an AHAS III protein comprising a tryptophan to leucine substitution at a on corresponding to position 556 of SEQ ID NO: 3; screening a population resulting from the cross for increased AHAS herbicideresistance selecting a member resulting from the cross having increased AHAS herbicideresistance ; and ing seeds resulting from the cross.

According to a h embodiment of the invention, there is provided a hybrid non- transgenic Brassica seed produced by the method of the eleventh embodiment.

According to a enth embodiment of the invention, there is provided a method of controlling weeds in a field containing non-transgenic herbicide-resistant Brassica plants, said method comprising applying an ive amount of at least one AHAS-inhibiting herbicide to said field containing said weeds and plants, said plants having an AHAS I gene and an AHAS III gene, wherein said AHAS I gene encodes an AHAS I protein comprising a tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and wherein said AHAS III gene s an AHAS III protein comprising a tryptophan to leucine substitution at a position corresponding to position 556 of SEQ ID NO: 3.

According to a fourteenth embodiment of the invention, there is ed a plant according to the first embodiment, a seed according to the second embodiment, or a method according to the tenth embodiment, the eleventh embodiment or the thirteenth embodiment, substantially as hereinbefore described with reference to the accompanying Examples and Figures.

According to a fifteenth embodiment of the invention, there is provided a plant when produced by the method according to the tenth embodiment or the fourteenth ment.

Other embodiments are set forth within the following claims.

We

Claims (15)

Claim:

1. A non-transgenic herbicide-resistant Brassica plant comprising: an acetohydroxyacid synthase (AHAS) I gene, wherein said gene encodes an AHAS I protein sing a tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and an AHAS III gene, wherein said gene encodes an AHAS III protein comprising a tryptophan to leucine substitution at a position ponding to position 556 of SEQ ID NO: 3.

2. The plant of claim 1, n said Brassica plant is resistant to inhibition by an AHAS-inhibiting herbicide.

3. The plant of claim 2, wherein said AHAS-inhibiting herbicide is selected from the group consisting of herbicides of: imidazolinone, sulfonylurea, triazolopyrimidine, pyrimidinylthiobenzoate, sulfonylamino-carbonyltriazolinone, and mixtures thereof.

4. The plant of claim 3, wherein said AHAS-inhibiting herbicide is an imidazolinone herbicide.

5. The plant of claim 3, wherein said AHAS-inhibiting herbicide is a ylurea

6. The plant of any one of claims 1-5, wherein said plant is resistant to the application of at least one AHAS-inhibiting herbicide.

7. The plant of any one of claims 1-6, n said plant is resistant to the application of at least one AHAS-inhibiting herbicide selected from the group consisting of herbicides of: imidazolinone, sulfonylurea, triazolopyrimidine, pyrimidinylthiobenzoate, sulfonylaminocarbonyltriazolinone , and mixtures f.

8. The plant of any one of claims 1-7, wherein said plant is produced by growing a seed of a line selected from the lines listed in Table 2.

9. The plant of any one of claims 1 to 8, wherein said plant is a ca napus plant.

10. A seed produced by the plant according to any one of claims 1-9, wherein the genome of the seed comprises: the AHAS I gene, wherein said gene encodes the AHAS I protein comprising the tryptophan to leucine substitution at a position corresponding to position 559 of SEQ ID NO: 2, and the AHAS III gene, wherein said gene encodes the AHAS III protein comprising the tryptophan to leucine substitution at a position corresponding to position 556 of SEQ ID NO: 3.

11. The seed of claim 10, n said AHAS I and AHAS III genes encode proteins that are resistant to inhibition by an nhibiting herbicide.

12. The seed of claim 11, wherein said AHAS-inhibiting herbicide is selected from the group consisting of herbicides of: imidazolinone, sulfonylurea, triazolopyrimidine, pyrimidinylthiobenzoate, ylamino-carbonyltriazolinone, and mixtures thereof.

13. The seed of claim 12, wherein said AHAS-inhibiting herbicide is an imidazolinone herbicide.

14. The seed of claim 12, wherein said AHAS-inhibiting herbicide is a sulfonylurea herbicide.

15. The seed of any one of claims 10-14, wherein said AHAS I gene encodes a protein comprising 70% or more ty to one or more of the amino acid sequences in