ALS inhibitor herbicide tolerant beet mutants

Abstract

The present invention relates to an ALS inhibitor herbicide tolerant beet plant and parts thereof comprising a mutation of an endogenous acetolactate synthase (ALS) gene wherein the ALS gene encodes an ALS polypeptide containing an amino acid different from tryptophan at its position 569.

Description

[0001] This application is a divisional application of Chinese patent application 201610822031.4, filed on October 13, 2011, entitled "A beet mutant tolerant to ALS inhibitor herbicides". Chinese patent application 201610822031.4 is a divisional application of Chinese patent application 201180049766.3, filed on October 13, 2011, entitled "A beet mutant tolerant to ALS inhibitor herbicides". Technical Field

[0002] This invention relates to sugar beet (Beta vulgaris) plants and parts thereof that are tolerant to ALS inhibitor herbicides, and methods for preparing them. Background Technology

[0003] Cultivated sugar beets (as defined in Ford-Lloyd (2005) Sources of genetic variation, Genus Beta. In: Biancardi E, Campbell LG, Skaraci GN, De Biaggi M (eds.) Genetics and Breeding of Sugar Beet. Science Publishers, Enfield (NH), USA, pp. 25-33) are important crops in temperate and subtropical regions. For example, approximately 20% of global sugar production is based on sugar beets. Because beet seedlings and nascent plants are vulnerable to strong competition from fast-growing weeds during the first 6-8 weeks of their life, which can outcompete the young crop plants, reliable weed control is essential in these crop regions.

[0004] For over 40 years, herbicides have been the preferred tool for weed control in cultivated sugar beets. Products used for this purpose, such as phenmedipham, desmedipham, and metamitron, suppress weed growth in sugar beet fields without harming the crop. However, there is room for improvement in the efficacy of these products under adverse environmental conditions, especially when harmful weeds, such as Chenopodium album, Amaranthus retroflexus, and / or Tripleurospermum inodorata, germinate over extended periods.

[0005] There is a great need for innovative herbicide active ingredients to improve weed control selectivity in sugar beets. Such compounds should be effective against a broad spectrum of weeds (preferably from weed germination to the full development of the weed plant) without affecting the sugar beet crop (regardless of its developmental stage). Through classical herbicide screening pathways, no selective herbicide active ingredient that meets all these stringent properties in an agronomically preferred manner has been found for use in sugar beets in the past few decades.

[0006] Certain chemicals can inhibit the enzyme acetylhydroxy acid synthase (AHAS), also known as acetyllactic acid synthase (ALS [EC 4.1.3.18]). ALS refers to the action sites of five structurally different herbicide families belonging to the ALS inhibitor herbicide class. These herbicide families include, for example, (a) sulfonylurea herbicide (Beyer E.M et al. (1988), Sulfonylureas in Herbicides: Chemistry, Degradation, and Mode of Action; Marcel Dekker, New York, 1988, 117-189), (b) sulfonylaminocarbonyltriazolinone herbicide (Pontzen, R., Pflanz.-Nachrichten Bayer, 2002, 55, 37-52), and (c) imidazolinone herbicide (Shaner, DL, et al., Plant Physiol., 1984, 76, 545-546; Shaner, DL, and O'Connor, SL (Eds.)). (d) triazolopyrimidine herbicide (Kleschick, W.A. et al., Agric. Food Chem., 1992, 40, 1083-1085), and (e) pyrimidinyl(thio)benzoate herbicide (Shimizu, T.J., Pestic. Sci., 1997, 22, 245-256; Shimizu, T. et al., Acetolactate Syntehase Inhibitors in Herbicide Classes in Development, Boger, P., Wakabayashi, K., Hirai, K., (Eds.), Springer Verlag, Berlin, 2002, 1-41).

[0007] ALS is involved in the conversion of two pyruvate molecules into one acetolactate molecule and carbon dioxide. The reaction uses thiamine pyrophosphate to link the two pyruvate molecules. The product of the reaction, acetolactate, is eventually converted into valine, leucine and isoleucine (Singh (1999) "Biosynthesis of valine, leucine and isoleucine", in Plant Amino Acids, Singh, BK, ed., Marcel Dekker Inc. New York, New York, pp. 227-247).

[0008] Inhibitors of ALS can block the biosynthesis of valine, leucine, and isoleucine in plants. As a result, the individual amino acid pools are rapidly depleted, leading to the cessation of protein biosynthesis, which in turn causes plant growth to stop and ultimately results in plant death or at least damage.

[0009] ALS-inhibiting herbicides are widely used in modern agriculture due to their effectiveness at moderate application rates and relative non-toxicity in animals. By inhibiting ALS activity, these herbicide families prevent the further growth and development of susceptible plants, including many weed species. To provide plants with high tolerance to even high concentrations of ALS-inhibiting herbicides required for adequate weed control, additional breeding lines and varieties of ALS-resistant herbicides are needed, as well as methods and compositions for producing and using these breeding lines and varieties.

[0010] A wide variety of ALS-inhibiting herbicides allow farmers to control many weed species (regardless of their growth stage), but these highly effective herbicides cannot be used on sugar beets because conventional sugar beet plants / commercial sugar beet varieties are highly sensitive to these ALS-inhibiting herbicides.

[0011] However, these ALS-inhibiting herbicides have shown excellent herbicidal activity against broadleaf and grassy weeds. The first herbicides with an ALS-inhibiting mechanism of action were developed for agriculture as early as 30 years ago. Now, the active ingredients in these herbicides exhibit strong weed control and are widely used in corn and cereals, as well as dicotyledonous crops, except for sugar beets.

[0012] The only known ALS inhibitor herbicide for post-emergence application in sugar beet cultivation is This herbicide (containing triflusulfuron-methyl as the active ingredient plus specific formulation compounds) can be broken down by sugar beets before inhibiting their endogenous ALS enzymes, but it has a serious gap in weed control in sugar beet growing areas.

[0013] Since the introduction of ALS inhibitor herbicides into agriculture, it has been observed that susceptible plant species (including naturally occurring weeds) occasionally exhibit spontaneous tolerance to these herbicides. Single-base pair substitutions at specific sites in the ALS gene typically result in more or less resistant ALS enzyme variants that show varying degrees of inhibition by ALS inhibitor herbicides.

[0014] Therefore, plants with mutated ALS alleles exhibit varying levels of tolerance to ALS inhibitor herbicides, depending on the chemical structure of the ALS inhibitor herbicide and the point mutation site of the ALS gene.

[0015] For example, Hattori et al. (1995), Mol. Gen. Genet. 246:419-425 documented a single mutation in codon 557 of Trp in a Brassica napus cell line (the position refers to position "574" according to the Arabidopsis sequence number used in the literature to compare all ALS / AHAS mutants), which is equivalent to position 569 of the sugar beet ALS sequence. These authors observed resistance to several members of the ALS inhibitor herbicide subclass (e.g., sulfonylureas, imidazolinones, and triazolopyrimidines).

[0016] A sugar beet mutant with a point mutation in codon Ala 122, resulting in some tolerance to the ALS-inhibiting herbicide imidazolinone subclass (WO 98 / 02526), ​​is described, but this is insufficient for weed control in agricultural application programs. No cross-tolerance to other ALS-inhibiting herbicide classes has been documented with this mutant. Furthermore, sugar beet plants with a second point mutation in codon Pro 197 exhibit moderate tolerance to ALS-inhibiting herbicides belonging to the sulfonylurea herbicide subclass. A double mutant with both mutations is also described (WO 98 / 02527). However, none of these mutants have been used for market introduction into sugar beet varieties because the herbicide tolerance levels to ALS-inhibiting herbicides in these mutants are not high enough for development for agronomic purposes.

[0017] Stougaard et al. (1990), J. Cell Biochem., Suppl. 14E, 310, described the isolation of ALS mutants in tetraploid sugar beet cell cultures. Two distinct ALS genes (ALS I and ALS II) were isolated, differing only at amino acid position 37. Mutant 1 contained two mutations in its ALS I gene, and mutant 2 contained three mutations in its ALS II gene. After the mutations were isolated to determine which mutation conferred resistance to ALS inhibitors, it was found that if the ALS synthesized in recombinant E. coli contained a point mutation at codon 574 of Trp (which, according to the Arabidopsis sequence number used in the literature to compare all ALS / AHAS mutants, is equivalent to position 569 in the sugar beet ALS sequence), then the ALS was herbicide resistant. Stougaard et al. did not demonstrate that any mutation at position 569 in the sugar beet ALS gene was sufficient to achieve acceptable levels of tolerance to ALS inhibitor herbicides. Furthermore, Stougaard et al. did not regenerate or treat sugar beet plants containing mutations, including the Trp->Leu mutation at position 569 of the sugar beet ALS gene.

[0018] Knowing this, Stougaard et al. constructed plant transformation vectors containing different ALS genes for plant transformation. However, for more than 20 years since then, these or other authors have not published further data on genetically engineered or mutant plants—especially not on the effects of applying ALS inhibitor herbicides to sugar beet plants, including those with the mutation, and / or agricultural areas.

[0019] WO 99 / 57965 broadly describes sulfonylurea-resistant sugar beet plants and the method for obtaining them via EMS (ethyl methanesulfonate) mutagenesis. However, aside from the research required to obtain such mutants, the publication provides neither the plant itself nor describes any specific location in the ALS gene that might be associated with obtaining ALS-inhibitor herbicide-resistant mutants, nor discloses any related agronomic uses. Furthermore, there is a strong possibility that the use of such strong mutagenic compounds (e.g., EMS) could generate multiple additional mutations elsewhere in the genome, potentially leading to defective plants resulting in infertility and / or stunted growth. Additionally, EMS application may be insufficient to induce specific mutations (e.g., converting the triplet TGG to TTG) due to its chemical interaction with DNA, as EMS always converts guanosine to adenosine.

[0020] In certain weed species, such as the genus *Amaranthus*, the Trp 574Leu mutation has been detected in plant populations repeatedly exposed to ALS-inhibiting herbicides. These Trp 574Leu mutants exhibit high levels of tolerance to several chemical classes of ALS-inhibiting herbicides, such as those selected from sulfonylureas and sulfonamide carbonyl triazolinones.

[0021] WO 2008 / 124495 discloses double and triple mutants of ALS. Specific mutations in the ALS gene are provided according to WO 2009 / 046334. However, to date, no agronomically usable herbicide-tolerant sugar beet mutant containing these mutations from WO 2009 / 046334 has been obtained.

[0022] Furthermore, considering that sugar beets account for approximately 20% of global sugar production, there is a strong need to obtain sugar beet plants that exhibit a growth advantage over vigorous weeds. Therefore, there is a significant need to obtain non-GMO sugar beet plants (including sugar beet plants) that are tolerant of ALS-inhibiting herbicides, relative to the ALS gene. Thus, there is a need for such non-GMO sugar beet plants, especially those tolerant of ALS-inhibiting herbicides at agronomically approved levels.

[0023] Therefore, the technical problem described is to meet this need. Summary of the Invention

[0024] The present invention addresses this need and, therefore, provides a solution to the aforementioned technical problem by providing a sugar beet plant and a portion thereof that is tolerant to ALS inhibitor herbicides and contains a mutation in the endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing an amino acid different from tryptophan at position 569.

[0025] The seeds of this invention were deposited on March 12, 2010, at the Food Industry and Marine Bacteria Culture Collection in Aberdeen, UK (NCIMB, Aberdeen, UK) with accession number NCIMB 41705. Detailed Implementation

[0026] By applying a variety of breeding methods, the initially obtained mutant plants can then be used to develop high-yielding commercial varieties with strong ALS inhibitor herbicide tolerance that are highly competitive in all specific markets.

[0027] It is important to note that, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein include plural referents. Thus, for example, reference to “reagent” includes one or more such different reagents, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art that can be modified or substituted for the methods described herein.

[0028] All publications and patents cited in this disclosure are incorporated herein by reference in their entirety. If any content incorporated herein by reference contradicts or is inconsistent with this specification, this specification shall supersede any such content.

[0029] Unless otherwise stated, the term "at least" used before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to identify many equivalent embodiments of the invention described herein using only conventional experimentation. The invention is intended to include such equivalents.

[0030] Unless the context otherwise requires, in this specification and the following claims, the term "comprise" and its variations, such as "comprises" and "comprising," should be understood to mean including the stated whole or step, or whole or group of steps, without excluding any other whole or step, or whole or group of steps. On the one hand, the term "comprise" and its variations, and on the other hand, "contain" and its similar variations, may be used interchangeably without preference for any one of them.

[0031] In this invention, a sugar beet plant was obtained comprising an altered endogenous ALS gene (also known as the “AHAS” gene) with a point mutation at codon 569 of Trp (relative to the sugar beet ALS amino acid reference sequence shown in SEQ ID NO:2; this is equivalent to position 574 of the reference Arabidopsis sequence shown in SEQ ID NO:6), said point mutation being obtained through several rounds of selection for a specifically selected ALS inhibitor herbicide.

[0032] Since the sugar beet plants of the present invention are obtained by isolating spontaneous mutant plant cells, these spontaneous mutant plant cells can be directly regenerated into fully fertile sugar beet plants with the point mutations described in detail herein. With respect to the ALS gene involved, these plants are non-transgenic.

[0033] Furthermore, the plants of the present invention and their progeny are fertile and therefore can be used for breeding purposes without any other manipulations that could lead to stress-induced alterations to the genetic background. Plants obtained according to the selection steps described herein can be used directly to produce sugar beet varieties and / or hybrids with agronomically usable levels of ALS-inhibiting herbicide tolerance, thus allowing for new weed control measures in sugar beet growing areas.

[0034] When used herein, the term “transgenic” means the introduction of a gene (which may be of the same or different species) into a plant by means of a suitable biological vector (e.g., Agrobacterium tumefaciens) or by any other physical means (e.g., protoplast transformation or ion bombardment), and the gene is capable of being expressed in a new host environment (i.e., a genetically modified organism (GMO)).

[0035] According to the aforementioned definition, the term "non-GMO" means exactly the opposite: that the individual genes have not been introduced through a suitable biological carrier or any other physical means. However, mutated genes can be transferred through pollination (naturally or through breeding methods) to produce another non-GMO plant containing that specific gene.

[0036] "Endogenous" genes refer to genes in plants that were not introduced into the plants through genetic engineering techniques.

[0037] When used in this article, the term “sequence” refers to nucleotide sequences, polynucleotides, nucleic acid sequences, nucleic acids, nucleic acid molecules, peptides, polypeptides, and proteins, depending on the context in which the term is used.

[0038] The terms “nucleotide sequence,” “polynucleotide,” “nucleic acid sequence,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, ribonucleotides, or deoxyribonucleotides of any length in a polymeric, unbranched form, or a combination thereof. Nucleic acid sequences include DNA, cDNA, genomic DNA, and RNA, including synthetic forms and mixed polymers, including sense and antisense strands, or may contain non-natural or derived nucleotide bases, as will be readily understood by those skilled in the art.

[0039] When used herein, the terms “polypeptide” or “protein” (these two terms are used interchangeably herein) mean a peptide, protein, or polypeptide comprising an amino acid chain of a given length, wherein the amino acid residues are linked by covalent peptide bonds. However, the invention also includes peptide analogs of said proteins / polypeptides (where the amino acids and / or peptide bonds have been replaced by functional analogs), as well as amino acids other than the 20 gene-encoded amino acids, such as selenocysteine. Peptides, oligopeptides, and proteins may be referred to as polypeptides. The term polypeptide also refers to (not excluding) modifications of polypeptides, such as glycosylation, acetylation, phosphorylation, etc. Such modifications are well documented in basic literature and described in more detail in monographs and research literature. Preferred polypeptides (or proteins) herein refer to beet ALS polypeptides (or ALS proteins) [SEQ ID NO:2].

[0040] Amino acid substitution involves changes in amino acids, where an amino acid is replaced by a different, naturally occurring amino acid residue. This substitution can be classified as "conserved," where amino acid residues present in wild-type ALS proteins are replaced by other naturally occurring amino acids with similar properties, such as… or The substitutions included in this invention may also be “non-conservative,” wherein amino acid residues present in wild-type ALS proteins are replaced by amino acids with different properties, such as naturally occurring amino acids from different groups (e.g., replacing charged or hydrophobic amino acids with alanine). As used herein, “similar amino acids” refers to amino acids having similar amino acid side chains, i.e., amino acids with polar, nonpolar, or near-neutral side chains. As used herein, “dissimilar amino acids” refers to amino acids with different amino acid side chains; for example, amino acids with polar side chains are dissimilar to those with nonpolar side chains. Polar side chains generally tend to be located on the surface of proteins, where they can interact with the aqueous environment present in the cell (“hydrophilic” amino acids). On the other hand, “nonpolar” amino acids tend to be located in the center of the protein, where they can interact with similar nonpolar neighboring molecules (“hydrophobic” amino acids). Examples of amino acids with polar side chains are arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, serine, and threonine (all hydrophilic amino acids, except for cysteine, which is hydrophobic). Examples of amino acids with nonpolar side chains include alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan (all of which are hydrophobic, except for glycine, which is neutral).

[0041] Generally, those skilled in the art can recognize, based on their common sense and the context of the use of the terms ALS, ALSL, AHAS, or AHASL, as referring to the nucleotide sequence or nucleic acid, or the amino acid sequence or polypeptide, respectively.

[0042] When used herein, the term "gene" refers to a polymer of nucleotides (ribonucleotides or deoxyribonucleotides) of any length. The term includes double-stranded and single-stranded DNA and RNA. It also includes modifications of known types, such as methylation, "capping," and substitution of one or more naturally occurring nucleotides with analogs. Preferably, a gene comprises a coding sequence encoding a polypeptide as defined herein. A "coding sequence" is a nucleotide sequence that, when placed or under the control of a suitable regulatory sequence, can be transcribed into mRNA and / or translated into a polypeptide. The boundaries of the coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. Coding sequences may include, but are not limited to, mRNA, cDNA, recombinant nucleic acid sequences, or genomic DNA, but in some cases, introns may also be present.

[0043] When used herein, the term “Beta vulgaris” may be abbreviated as “B. vulgaris”. Additionally, the term “beet” is used herein. These three terms are used interchangeably and should be understood to fully encompass cultivated forms of sugar beet as defined in Ford-Lloyd (2005) Sources of genetic variation, Genus Beta. In: Biancardi E, Campbell LG, Skaraci GN, De Biaggi M (eds) Genetics and Breeding of Sugar Beet. Science Publishers, Enfield (NH), USA, pp. 25-33. Similarly, for example, the term “Arabidopsis thaliana” may be abbreviated as “A. thaliana”. These two terms are used interchangeably herein.

[0044] When used in this invention, the term "position" means the location of an amino acid in the amino acid sequence described herein or the location of a nucleotide in the nucleotide sequence described herein. The term "corresponding" as used herein also includes positions not only determined by the aforementioned nucleotide / amino acid number.

[0045] The position of a given nucleotide that can be substituted in this invention can vary due to the deletion or insertion of nucleotides at other locations in the ALS 5' untranslated region (UTR) (including the promoter and / or any other regulatory sequence) or in the gene (including exons and introns). Similarly, the position of a given amino acid that can be substituted in this invention can vary due to the deletion or insertion of amino acids at other locations in the ALS polypeptide.

[0046] Therefore, in this invention, "corresponding position" should be understood as meaning that the nucleotides / amino acids at the indicated number may be different, but may still have similar adjacent nucleotides / amino acids. The interchangeable, deleted, or inserted nucleotides / amino acids are also included in the term "corresponding position".

[0047] To determine whether a nucleotide residue or amino acid residue in a given ALS nucleotide / amino acid sequence corresponds to a certain position in the nucleotide sequence SEQ ID NO:1 or the amino acid sequence SEQ ID NO:2, those skilled in the art may use tools and methods known in the art, such as manual or by using computer programs for alignment, such as BLAST (Altschul et al. (1990), Journal of Molecular Biology, 215, 403-410) (which represents a basic local alignment search tool) or ClustalW (Thompson et al. (1994), Nucleic Acid Res., 22, 4673-4680) or any other suitable program for generating sequence alignments.

[0048] SEQ ID NO:1 is the nucleotide sequence encoding wild-type ALS in beet. SEQ ID NO:2 is the beet amino acid sequence derived from SEQ ID NO:1. Therefore, the codon at positions 1705-1707 of the nucleotide sequence SEQ ID NO:1 encodes the amino acid at position 569 of SEQ ID NO:2, which is either the three-letter code for the amino acid "Trp" or the single-letter code for "W".

[0049] In the alternative schemes for determining whether a nucleotide residue or amino acid residue in a given ALS nucleotide / amino acid sequence corresponds to certain positions in the nucleotide sequence SEQ ID NO:1, the nucleotide sequence encoding the Arabidopsis wild-type ALS shown in SEQ ID NO:5 can be used. SEQ ID NO:6 is the Arabidopsis amino acid sequence derived from SEQ ID NO:5.

[0050] Therefore, the codon at positions 1720-1722 of SEQ ID NO:5 encodes the amino acid at position 574 of SEQ ID NO:6 (i.e., the amino acid "Trp" in the three-letter code or "W" in the single-letter code).

[0051] If the Arabidopsis wild-type ALS nucleotide sequence shown in SEQ ID NO:5 is used as a reference sequence (because most relevant literature does so, so this sequence is used to make comparison with such known sequences easier), then the codon encoding an amino acid different from tryptophan is at position 1720-1722 corresponding to the Arabidopsis ALS gene nucleotide sequence shown in SEQ ID NO:5.

[0052] However, in all subsequent disclosures, SEQ ID NO:1 is preferred as the reference nucleotide sequence and SEQ ID NO:2 is preferred as the reference amino acid sequence.

[0053] The table below provides an overview of the reference sequences used in this article, at which point the location of point mutations in the nucleotide sequence or the location of substitutions in the amino acid sequence has been determined:

[0054] SEQ ID NO: sequence type Species 1 nucleotide sequence beet 2 amino acid sequence beet 3 Nucleotide sequence (mutated) beet 4 Amino acid sequence (mutated) beet 5 nucleotide sequence Arabidopsis 6 amino acid sequence Arabidopsis

[0055] Therefore, in any case, equivalent positions can still be determined by comparison with reference sequences such as SEQ ID NO:1 or 5 (nucleotide sequence) or SEQ ID NO:2 or 6 (amino acid sequence).

[0056] Considering the differences between the wild-type ALS gene in sugar beets and the ALS gene contained in the sugar beet plant of the present invention, the ALS gene (or polynucleotide or nucleotide sequence) contained in the sugar beet plant of the present invention may also be referred to as a "mutated ALS gene," "mutated ALS allele," "mutated ALS polynucleotide," etc. Therefore, throughout this specification, the terms "mutated allele," "mutated ALS allele," "mutated ALS gene," or "mutated ALS polynucleotide" may be used interchangeably.

[0057] Unless otherwise stated herein, these terms refer to a nucleotide sequence containing a codon encoding an amino acid other than tryptophan at positions 1705-1707 of the beet ALS gene nucleotide sequence shown in SEQ ID NO:1. When set according to the Arabidopsis reference sequence shown in SEQ ID NO:5, the codon positions are 1720-1722.

[0058] Similarly, these terms refer to the nucleotide sequence encoding an ALS protein having an amino acid different from tryptophan at position 569, corresponding to the amino acid sequence of the beet ALS protein shown in SEQ ID NO:2. When set according to the Arabidopsis reference sequence shown in SEQ ID NO:6, the position is 574.

[0059] "Amino acids other than tryptophan" (represented as "Trp" in the three-letter code or equivalently as the single-letter code "W") includes any naturally occurring amino acid other than tryptophan. These naturally occurring amino acids include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tyrosine (Y), or valine (V).

[0060] However, preferably, the amino acid different from tryptophan (which belongs to the neutral-polar amino acid group) is an amino acid with physicochemical properties different from tryptophan, i.e., any amino acid exhibiting neutral-nonpolar, acidic, or basic properties. More preferably, the amino acid different from tryptophan is selected from alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, valine, and arginine. Even more preferably, the amino acid is a neutral-nonpolar amino acid, such as alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, or valine. Particularly preferably, the amino acid is alanine, glycine, isoleucine, leucine, or valine. Even more preferably, it is glycine and leucine. More preferably, it is leucine.

[0061] In contrast, unless otherwise stated, the terms "wild-type allele," "wild-type ALS allele," "wild-type ALS gene," or "wild-type ALS polynucleotide" refer to the nucleotide sequence encoding an ALS protein without the W569 substitution of SEQ ID NO:2 (or the W574 substitution of SEQ ID NO:6). These terms also refer to the nucleotide sequence containing a codon encoding an amino acid different from tryptophan at positions 1705-1707 corresponding to the nucleotide sequence of the beet ALS gene shown in SEQ ID NO:1.

[0062] Such “wild-type alleles”, “wild-type ALS alleles”, “wild-type ALS genes” or “wild-type ALS polynucleotides” may or may not contain mutations other than those that could lead to the W569 substitution.

[0063] Essentially, the only difference between the wild-type beet plant and the beet plant of the present invention regarding the ALS gene is preferably (and explicitly) that, at the location specifically described herein (specifically at positions 1705-1707 corresponding to the nucleotide sequence of the beet ALS gene shown in SEQ ID NO:1), the beet plant of the present invention contains a codon encoding an amino acid other than tryptophan, preferably the codon encoding an amino acid listed elsewhere herein. However, as stated above, other differences, such as additional mutations, may exist between the mutated ALS alleles listed herein and the wild-type ALS alleles. However, these additional differences are insignificant as long as the differences previously explained exist.

[0064] Therefore, the W569 substitution (or, when using the Arabidopsis ALS amino acid sequence of SEQ ID NO:6 as a reference, the W574 substitution) is due to a codon change at positions corresponding to nucleotide sequence positions 1705-1707 shown in SEQ ID NO:1 (or positions corresponding to nucleotide sequence positions 1720-1722 shown in SEQ ID NO:5).

[0065] Preferably, the substitution at position 569 is a W→L substitution, wherein "L" is encoded by any one of the codons "CTT", "CTC", "CTA", "CTG", "TTA" or "TTG".

[0066] More preferably, the substitution at position 569 is a W→L substitution, due to the transposition of the "G" nucleotide to a "T" nucleotide at position 1706 corresponding to the nucleotide sequence shown in SEQ ID NO:1 (or at position 1721 corresponding to the nucleotide sequence shown in SEQ ID NO:5). Therefore, the codon at positions 1705-1707 corresponding to the nucleotide sequence shown in SEQ ID NO:1 (or at positions 1720-1722 corresponding to the nucleotide sequence shown in SEQ ID NO:5) is changed from "TGG" to "TTG". However, codon "TGG" encodes tryptophan, and codon "TTG" encodes leucine.

[0067] Therefore, in the most preferred embodiment, the present invention provides a sugar beet plant in which the nucleotide sequence of the endogenous ALS gene of the sugar beet plant contains the codon TTG (encoding leucine) at positions 1705-1707 corresponding to the nucleotide sequence positions of the sugar beet ALS mutant gene shown in SEQ ID NO:1, and the nucleotide sequence contains SEQ ID NO:3 (or is preferably composed of therein).

[0068] The sugar beet plant encoding the ALS polypeptide (which has an amino acid different from tryptophan at position 569 of the sugar beet ALS protein amino acid sequence shown in SEQ ID NO:2) preferably contains a codon encoding an amino acid different from tryptophan at positions 1705-1707 of the nucleotide sequence of its endogenous ALS gene, corresponding to positions 1705-1707 of the sugar beet ALS gene nucleotide sequence shown in SEQ ID NO:1.

[0069] The term beet "ALS" or "AHAS" gene also includes beet nucleotide sequences having at least 90%, 95%, 97%, 98%, or 99% identity with the beet ALS nucleotide sequence of SEQ ID NO:1 or 3, wherein these nucleotide sequences having 60%, 70%, 80%, 90%, 95%, 97%, 98%, or 99% identity contain a codon encoding an amino acid other than tryptophan at positions 1705-1707 corresponding to the nucleotide sequence of SEQ ID NO:1.

[0070] Similarly, these nucleotide sequences having at least 90%, 95%, 97%, 98%, or 99% identity encode ALS polypeptides containing an amino acid different from tryptophan at position 569 corresponding to SEQ ID NO:2. The identity nucleotide sequences encode ALS proteins that retain the activities described herein, and more preferably, the encoded ALS polypeptides are resistant to one or more ALS inhibitor herbicides described herein. This term also includes allelic variants and homologues encoding ALS polypeptides, which are preferably resistant to one or more ALS inhibitor herbicides described herein.

[0071] To determine whether a nucleic acid sequence is identical to the nucleotide sequence of the present invention to some degree, those skilled in the art may use tools and methods known in the art, such as comparison (manually or by using a computer program), for example those mentioned below related to the definition of the term "hybridization" and the degree of homology.

[0072] For example, BLAST, which stands for Basic Local Alignment Search Tool (Altschul, Nucl. Acids Res. 25 (1997), 3389-3402; Altschul, J. Mol. Evol. 36 (1993), 290-300; Altschul, J. Mol. Biol. 215 (1990), 403-410), can be used to search for local sequence alignments. BLAST produces alignments of nucleotide and amino acid sequences to determine sequence similarity. Due to the local nature of these alignments, BLAST is particularly useful in determining exact matches or identifying similar sequences. The basic unit output by the BLAST algorithm is the High-scoring Segment Pair (HSP). An HSP consists of two arbitrary but equal-length sequence fragments whose alignment is the local maximum and whose alignment score reaches or exceeds a user-defined threshold or critical score. The BLAST method searches for Highly Significant Spectrum Pairs (HSPs) between the query sequence and database sequences to assess the statistical significance of any matches found, and only reports those matches that meet the user-selected significance threshold. The parameter E establishes the threshold used to report statistical significance for database sequence matches. E is interpreted as an upper bound on the expected frequency of occurrence of an HSP (or set of HSPs) across the entire database search. Any database sequence whose matches satisfy E is reported in the program output.

[0073] Similar computer techniques to BLAST (Altschul (1997), loc.cit.; Altschul (1993), loc.cit.; Altschul (1990), loc.cit.) are used to search for identical or related molecules in nucleotide databases (e.g., GenBank or EMBL). This analysis is much faster than multiple membrane-based hybridization. Furthermore, the sensitivity of the computer search can be modified to determine whether any specific match is classified as exact or similar. The search is based on the multiplicative integral value, which is defined as:

[0074]

[0075] It takes into account the degree of similarity between two sequences and the length of the sequence match. For example, a multiplier value of 40 indicates a precise match within a 1-2% error; a multiplier value of 70 indicates a precise match. Similar molecules are typically identified by selecting those exhibiting multiplier values ​​between 15 and 40, but lower scores may identify related molecules.

[0076] The term "beet sugar ALS" or "AHAS" polypeptide also includes an amino acid sequence having at least 90%, 95%, 97%, 98%, or 99% identity with the ALS amino acid sequence of SEQ ID NO:2 or 4, wherein these amino acid sequences having at least 90%, 95%, 97%, 98%, or 99% identity contain an amino acid different from tryptophan at position 569 corresponding to SEQ ID NO:2. The identical amino acid sequence maintains the activity of the ALS described herein, and more preferably, the ALS polypeptide is resistant to the ALS inhibitor herbicides described herein.

[0077] If necessary, ALS activity can be measured according to the assay described in Singh (1991), Proc. Natl. Acad. Sci. 88:4572-4576.

[0078] However, the ALS nucleotide sequence encoding the ALS polypeptide mentioned herein preferably confers tolerance to one or more ALS inhibitor herbicides described herein (or, conversely, lower sensitivity to ALS inhibitor herbicides). This is due to the point mutations that result in amino acid substitutions described herein.

[0079] Therefore, tolerance to ALS inhibitor herbicides (or, conversely, lower sensitivity to ALS inhibitor herbicides) can be measured by obtaining ALS from cell extracts of plants containing mutant ALS sequences and plants without mutant ALS sequences in the presence of ALS inhibitor herbicides and comparing their activity, for example, by the method described in Singh et al (1988) [J. Chromatogr., 444, 251-261].

[0080] However, a preferred activity assay for the ALS polypeptide encoded by a nucleotide sequence containing a codon encoding an amino acid different from tryptophan at positions 1705-1707 of the beet ALS gene nucleotide sequence shown in SEQ ID NO:1 can be performed as follows:

[0081] According to the manufacturer's instructions, the coding sequences of wild-type and mutant beet plants are cloned into a vector such as Novagen pET-32a(+), and the vector is transformed into, for example, *E. coli* AD494. Preferably, the bacteria are cultured at 37°C in a medium under selective pressure (e.g., LB medium containing 100 mg / L carbenicillin and 25 mg / L kanamycin) at OD... 600Preferably, induction is performed at approximately 0.6 μL using, for example, 1 mM isopropyl-β-D-thiogalactopyranoside, and the culture is preferably carried out at 18°C ​​for approximately 16 hours, followed by harvesting by centrifugation. The bacterial pellet is resuspended at a concentration of 1 g wet weight / 25 ml buffer in 100 mM pH 7.0 sodium phosphate buffer (containing 0.1 mM thiamine pyrophosphate, 1 mM MgCl2, and 1 μM FAD) and the bacteria are lysed by sonication. The crude protein extract obtained after centrifugation is used for ALS activity measurement.

[0082] ALS was then determined using a modified procedure described in Ray (1984), Plant Physiol., 75, 827-831, for example, in a 96-well microtiter plate. The reaction mixture preferably contained 20 mM potassium phosphate buffer (pH 7.0), 20 mM sodium pyruvate, 0.45 mM thiamine pyrophosphate, 0.45 mM MgCl2, 9 μM FAD, ALS enzyme, and various concentrations of ALS inhibitors, with a final volume of approximately 90 μl.

[0083] The assay was initiated by adding the enzyme, preferably after incubation at 30°C for 75 min, and terminated by adding 40 μl of 0.5 M H₂SO₄. After approximately 60 min at room temperature, approximately 80 μl of a solution of 1.4% α-naphthol and 0.14% creatine in 0.7 M NaOH was added, and the assay was incubated for another approximately 45 min at room temperature before measuring the absorbance at 540 nm. The pI₅₀ value of ALS inhibition was determined using the XLFit Excel plugin version 4.3.1 curve fitting program from Guildford ID Business Solutions Limited, UK, as described by Ray (1984) (Plant Physiol., 75, 827-831).

[0084] When using plants, ALS activity is preferably determined in wild-type cell or leaf extracts and in beet cell or leaf extracts of the obtained mutant in the presence of multiple concentrations of ALS inhibitor herbicides (preferably sulfonylurea herbicides or sulfonamide-carbonyl triazolinone herbicides), more preferably in the presence of multiple concentrations of the ALS inhibitor herbicide "formamide sulfuron". Therefore, ALS is preferably extracted from sugar beet leaves or sugar beet tissue cultures as described in Ray (1984) in Plant Physiol 75:827-831.

[0085] Preferably, the beet plants of the present invention have lower sensitivity to ALS inhibitors, more preferably at least 100 times lower, more preferably 500 times lower, even more preferably 1000 times lower, and most preferably 2000 times lower. When used herein, lower sensitivity, and vice versa, can be considered as "higher tolerance" or "higher resistance." Similarly, "higher tolerance" or "higher resistance," and vice versa, can be considered as "lower sensitivity."

[0086] For example, the beet plants of the present invention (especially the beet plants described in the accompanying examples) are at least 2,000 times less sensitive to the ALS inhibitor herbicide formamide sulfonyl (a member of the ALS inhibitor subclass "sulfonylurea herbicides") than the wild-type enzyme.

[0087] Preferably, the sugar beet plants of the present invention are less sensitive to members of a variety of ALS-inhibiting herbicides (e.g., sulfonylurea herbicides, sulfonamide-carbonyltriazolone herbicides, and imidazolinone herbicides). Preferably, sulfonylurea and sulfonamide-carbonyltriazolone herbicides to which the plants are less sensitive are selected. In a particularly preferred embodiment, the sugar beet plants of the present invention are less sensitive to the ALS-inhibiting herbicide formasulfuron (a sulfonylurea herbicide) alone, or to its combination with one or more other ALS-inhibiting herbicides (from a sulfonylurea herbicide subclass or any other ALS-inhibiting herbicide subclass).

[0088] Therefore, the beet plants of the present invention that are less sensitive to ALS inhibitor herbicides can also be characterized as having “higher tolerance” to ALS inhibitors (i.e., plants that are tolerant to ALS inhibitors).

[0089] Therefore, "ALS-tolerant plants" refers to plants, especially sugar beets, which exhibit higher tolerance to at least one ALS-inhibiting herbicide at levels that typically inhibit the growth of normal or wild-type plants, preferably those herbicides that control normal or wild-type plants. In the nucleotide sequence of any allele of the endogenous ALS gene in the normal or wild-type plant, positions 1705-1707 corresponding to the sugar beet ALS gene nucleotide sequence shown in SEQ ID NO:1 do not contain a codon encoding an amino acid other than tryptophan.

[0090] The nucleotide sequence can also typically be characterized as a nucleotide sequence "tolerant to ALS inhibitor herbicides". "A nucleotide sequence tolerant to ALS inhibitor herbicides" means a nucleic acid molecule that, compared to an ALS protein that does not contain an amino acid different from tryptophan at position 569 of the beet ALS protein amino acid sequence shown in SEQ ID NO:2, contains at least a nucleotide sequence that produces a mutation encoding an amino acid different from tryptophan, wherein the at least one mutation results in the expression of an ALS protein with lower sensitivity to ALS inhibitor herbicides.

[0091] "Herbicide-tolerant ALS protein" means an ALS protein that exhibits higher ALS activity compared to wild-type ALS protein in the presence of at least one known ALS inhibitor herbicide that interferes with ALS activity and said herbicide is at a concentration or level known to inhibit the ALS activity of wild-type ALS protein.

[0092] Similarly, the terms "ALS inhibitor herbicide" or the simplified "ALS inhibitor" are used interchangeably. When used herein, "ALS inhibitor herbicide" or "ALS inhibitor" is not intended to be limited to a single herbicide that interferes with ALS enzyme activity. Therefore, unless otherwise stated or apparent from the context, "ALS inhibitor herbicide" or "ALS inhibitor" can be one herbicide known in the art or a mixture of two, three, four, or more herbicides, preferably those listed herein, all of which interfere with ALS enzyme activity.

[0093] Surprisingly, it has been found that even a single point mutation of the present invention can confer agronomically useful and stable levels of tolerance to ALS-inhibiting herbicides in sugar beets and their progeny, especially when homozygous mutations are established. Compared to herbicide-tolerant sugar beets with the same genetic background (where such mutations exist only in heterozygous form), sugar beets with herbicide-tolerant mutations in homozygous form exhibit higher agronomical levels of tolerance to ALS-inhibiting herbicides.

[0094] Therefore, the present invention relates to sugar beets tolerant to ALS inhibitor herbicides by having mutations in the endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing an amino acid different from tryptophan at position 569. The mutations may be heterozygous and are preferably the only mutation in the ALS gene. More preferably, the mutations may be homozygous; most preferably, the mutation is homozygous as the only mutation in the endogenous ALS gene.

[0095] It is also unrealistic to expect that a single mutation in the ALS gene in sugar beets would be sufficient, as, for example, WO 2010 / 037061 teaches that two or three mutations in the ALS gene are necessary to confer agronomically useful tolerance to ALS inhibitor herbicides.

[0096] Therefore, heterozygous beet plants and portions thereof for the aforementioned mutation are subpreferably included, but are still covered by the present invention and may be sufficient for certain applications and / or certain environmental conditions. The present invention also covers plants in which at least one allele of the endogenous ALS gene contains a codon encoding an amino acid (preferably leucine) different from tryptophan at position 1705-1707 of the beet ALS gene nucleotide sequence shown in SEQ ID NO:1, and the endogenous ALS gene contains one (when diploid) or more (when polyploid) other alleles with one or more other mutations.

[0097] Therefore, when used herein, the terms “heterozygous” or “heterozygous” mean plants of the present invention that have different alleles at specific loci, particularly at the ALS locus.

[0098] "Homozygous" or "homozygous" refers to the plant of the present invention having two copies of the same allele in different DNA strands, especially at the ALS gene locus.

[0099] When used in this article, unless otherwise expressly stated, the term "plant" means any plant at any stage of development.

[0100] Preferably, the beet plant of the present invention is orthoploid or odd-ploid. Orthoploid plants are preferably haploid, diploid, tetraploid, hexaploid, octoploid, decaploid, or dodecoploid, and odd-ploid plants are preferably triploid or pentaploid.

[0101] A part of a plant can be attached to the whole plant or can be separated from the whole plant. Such parts of a plant include, but are not limited to, plant organs, tissues and cells, preferably seeds.

[0102] Therefore, the sugar beet plants of the present invention are non-transgenic in terms of endogenous ALS genes. Of course, exogenous genes can be transferred into said plants through genetic engineering or conventional methods such as hybridization. These genes may be genes that confer herbicide tolerance (preferably genes that confer herbicide tolerance different from that of ALS inhibitor herbicides), genes that increase yield, genes that improve resistance to the organism, and / or genes involving changes in contents.

[0103] In another aspect, the present invention relates to a method for preparing sugar beet plants and their parts, comprising the following steps:

[0104] (a) Expose the callus (preferably from sugar beet) to approximately 10 -7 M-10 -9 M's ALS inhibitor herbicide (preferably formamide sulfonamide);

[0105] (b) The option can be available in up to 3×10 -6 Cell clones grown under the influence of M's ALS inhibitor herbicide (preferably formamide sulfonamide, [CAS RN173159-57-4]);

[0106] (c) Regeneration of shoot tips in the presence of an ALS inhibitor herbicide (preferably formamide sulfonamide);

[0107] (d) Select regenerated plantlets with an ALS inhibitor herbicide (preferably formamide sulfur, iodosulfuron-methyl-sodium [CAS RN 144550-36-7] and / or a mixture of both), wherein the dosage of formamide sulfur is preferably equivalent to 7-70 g ai / ha and the dosage of iodosulfuron-methyl-sodium is preferably equivalent to 1-10 g ai / ha.

[0108] On the other hand, the regenerated plantlets obtained according to the above processes (a) to (d) can be used for further preparation of sugar beet plants by applying the following steps (e) to (m):

[0109] (e) The individual plantlets from step (d) were propagated vegetatively by establishing cell lines (clones) of each plantlet tolerant to ALS inhibitor herbicides to obtain different positive variants.

[0110] (f) Long-term storage of each established clone in a vegetative state;

[0111] (g) Transfer the cloned plants of each clone that have been stored for a long time to a greenhouse;

[0112] (h) Vernalization and adaptation in a vernalization chamber to induce flowering;

[0113] (i) Transfer vernalized plants to a growing room (controlled temperature and light);

[0114] (j) Select the best pollen-producing plants of the best flowering clone for hybridization with emasculated plants of superior varieties but sensitive to ALS inhibitor herbicides to overcome the negative impact of somatic asexual variation on the fertility (male and female) of the plants in step (d).

[0115] (k) Backcross to improved strains until fertility is restored, and finally allow the heterozygous plants to self-pollinate to achieve a homozygous state.

[0116] (I) Test crosses were performed with ligands sensitive to ALS inhibitor herbicides, and self-pollinated seeds of each backcross line were generated for field evaluation.

[0117] (m) Apply different ALS inhibitor herbicides with relevant dosage rates in agronomy to select the best-performing strains, preferably homozygous ones.

[0118] The lines obtained according to steps (a) to (m) above form the basis for developing commercial varieties according to steps known to the Breeding Association, which are supported by molecular breeding techniques (e.g., marker-assisted breeding or marker-assisted selection) to accelerate the process and ensure the correct selection of plants to obtain the homozygous form of the mutation, or to correctly select heterozygous plants that actually contain the W569 mutation of the present invention in at least one allele when one or more mutations are present at multiple locations of the endogenous gene encoding ALS (for the review, see Bertrand CY et al. (2008), Phil. Trans. R. Soc. B., 363, 557-572).

[0119] Callus tissue can be obtained using tools and methods well known in the art (such as those described in the accompanying embodiments).

[0120] The seeds obtained from the above step (m) have been deposited at the Food Industry and Marine Bacteria Culture Collection in Aberdeen, UK (NCIMB, Aberdeen, UK) with accession number NCIMB 41705.

[0121] On the other hand, the present invention relates to a method for producing herbicide-tolerant sugar beets and portions thereof, the mechanical parts of the sugar beet comprising (i) a mutation in an endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing an amino acid different from tryptophan at position 569, and (ii) an additional mutation in the endogenous ALS gene, the method comprising the steps of:

[0122] (a) Producing sugar beets that are tolerant to ALS inhibitor herbicides and contain mutations in the endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide (parent A) containing an amino acid different from tryptophan at position 569.

[0123] (b) Cross parent A with a sugar beet plant (parent B) that contains one or more other mutations at a position different from amino acid position 569 in the endogenous ALS gene;

[0124] (c) Obtain beet offspring that are heterozygous for the ALS gene mutation at amino acid position 569 and for one or more other ALS gene mutations encoded by parent B.

[0125] (d) The breeding process described herein is controlled by the following two

[0126] (i) Application of marker-assisted breeding and / or microsequencing techniques, and / or

[0127] (ii) Applying an agronomically appropriate dose of one or more ALS inhibitor herbicides, said ALS inhibitor herbicides being tolerated by the offspring produced in step (c).

[0128] Therefore, it is conceivable that the present invention also relates to beet plants obtainable by the aforementioned preparation method.

[0129] In one non-limiting example, the sugar beet plant of the present invention is obtained by performing the following non-limiting procedure. Not limited by the theory, the same procedure can be used to obtain beet plants other than sugar beets.

[0130] Sugar beet cell cultures were initiated from diploid sugar beet seedlings of genotype 7T9044 (e.g., described by Alexander Dovzhenko, PhD Thesis, Title: "Towards plastid transformation in rapeseed (Brassica napus L.) and sugarbeet (Beta vulgaris L.)", Ludwig-Maximilians-Universitat Munich, Germany, 2001). Sugar beet seeds were immersed in 70% ethanol for 60 seconds, then washed twice in sterile water with 0.01% detergent, and incubated in 1% NaOCl bleach for 1–4 hours. The seeds were then washed three times with sterile H2O and stored overnight in sterile water at 4°C. The embryos were then separated using forceps and a scalpel.

[0131] Freshly prepared seedlings were immersed in 0.5% NaOCl for 30 minutes, then washed three times with sterile water. After the final wash, they were placed on hormone-free MS agar medium (Murashige and Skoog (1962), Physiol. Plantarum, 15, 473-497). Those seedlings that developed into sterile seedlings were used as the initiation medium for regenerative sugar beet cell cultures.

[0132] Cotyledons and hypocotyls were cut into 2-5 mm segments and cultured on MS medium (0.8%) fixed with agar containing 1 mg / L purine benzoate (BAP) or 0.25 mg / L phenylthiazolyl (TDZ). After 4 weeks, the developing shoot tip cultures were transferred to fresh agar medium of the same composition and then subcultured monthly. The cultures were kept in dark at 25°C with a 12h / 12h light / dark cycle.

[0133] Following 7-10 days of subculture, shoot tip cultures grown on media containing phenylthiazolyl hydrochloride formed distinct callus morphologies that were rapidly growing, soft, and brittle. These callus morphologies ranged in color from pale yellow to pale green. Some of these brittle callus morphologies consistently produced chlorophyll-containing shoot tip primordia from the embryoid structure. These rapidly growing, regenerating callus morphologies were used to select sugar beet mutants resistant to ALS-inhibiting herbicides.

[0134] When this type of callus is exposed to 10 -9 When M-sulfonylurea formamide sulfonamide (CAS RN 173159-57-4) was used, the cells survived, but produced less than 50% of the biomass of their lineage cells produced on a medium without the inhibitor. At a concentration of 3 × 10⁻⁶... -8 No growth was detected on the culture medium containing M-formamidesulfuron. For large-scale mutant selection experiments, 10 mutants were selected. -7 M formamide sulfonamide. Count surviving and growing cell clones and transfer them to cells containing 3 × 10⁻⁶ cells after 4–6 weeks. -7 On fresh culture medium containing the inhibitor described in M. One of these cell clones was not only able to grow at this concentration of the inhibitor, but even at 3 × 10⁻⁶. -6 Shoot tips were grown in the presence of M-formamide-sulfuron-methyl. From this clone (SB574TL), shoot tips were regenerated in the presence of an ALS inhibitor herbicide, and then transferred to MS medium containing 0.05 mg / L naphthaleneacetic acid (NAA).

[0135] Within 4–12 weeks, the shoot tips develop roots, and they are then transferred to sterile plant containers filled with moist, sterilized perlite and watered with half-strength MS inorganic components. Alternatively, the plantlets are transferred directly from agar-solidified medium to a soil mixture containing perlite in a greenhouse. For the first 10–15 days after transfer to the soil-containing substrate, the plants are kept in a high-humidity environment. During and after the gradual transition to normal greenhouse humidity, the plants are kept in the greenhouse under artificial light (12 h) and day / night temperatures of 20 ± 3 °C / 15 ± 2 °C.

[0136] Three to five weeks later, regenerated plants from the formamide-sulfuron-tolerant cell culture (SB574TL) obtained above, as well as from wild-type cell cultures, were treated with formamide-sulfuron, sodium iodide-methyl (CAS RN 144550-3-7), and a mixture of the two active ingredients. The tested herbicide doses were equivalent to 7-70 g ai / ha of formamide-sulfuron and 1-10 g ai / ha of sodium iodide-methyl. Regenerated plants from this tolerant cell line tolerated even the highest herbicide doses (formamide-sulfuron, sodium iodide-methyl, and a 7:1 mixture), while even the lowest doses killed wild-type plants.

[0137] Test the offspring as follows (in a non-restrictive manner):

[0138] Based on SB574TL, F2 and F3 seeds of experimental hybrids containing heterozygous resistance alleles, and F4-F6 seeds containing homozygous mutant alleles, were sown in the field. When the plants developed 3-5 rosette leaves, they were treated with formamide-sulfuron, sodium iodide-sulfuron, and a mixture of these two ALS-inhibiting herbicides. The homozygous seedlings tolerated a mixture of 35 g formamide-sulfuron / ha + 7 g sodium iodide-sulfuron / ha without stunted growth or any visible damage. In several cases, at these ratios, heterozygous lines showed signs of stunted growth and some leaf chlorosis, but they recovered within 3-5 weeks; however, conventional sugar beet seedlings were killed by the ALS-inhibiting herbicides.

[0139] The ALS mutants were characterized as follows:

[0140] The extraction and nucleic acid sequence analysis of the obtained mutants were performed by LGC Genomics GmbH in Berlin, Germany, according to a modified standard protocol.

[0141] The nucleic acid sequence obtained from the sugar beet mutant SB574TL is shown in SEQ ID NO:3. SEQ ID NO:4 represents the corresponding amino acid sequence; however, SEQ ID NO:1 was obtained by sequencing wild-type sugar beet. SEQ ID NO:2 represents the corresponding amino acid sequence of the wild-type sugar beet.

[0142] Comparison of all these sequences showed that the mutation was only present at position 574 of the endogenous ALS gene, and no other changes were observed in any other part.

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154] However, generally preferably, the sugar beet plants and portions thereof of the present invention are agronomically usable. "Agronomically usable" means that the sugar beet plants and portions thereof can be used for agronomic purposes. For example, the sugar beet plants should be usable for purposes of sugar production, biofuel production (e.g., biogas, biobutanol), ethanol production, betaine and / or uridine production. The term "agronomically usable" as used herein also includes: the sugar beet plants of the present invention preferably have lower sensitivity to ALS inhibitor herbicides, more preferably at least 100 times lower, more preferably 500 times lower, even more preferably 1000 times lower, and most preferably less than 2000 times lower. The ALS inhibitor herbicide is one or more described herein, preferably it is formamide sulfonamide alone or a combination of formamide sulfonamide with one or more other ALS inhibitor herbicides from the sulfonylurea herbicide subclass or any other subclass of ALS inhibitor herbicides, most preferably it is a combination of formamide sulfonamide with other ALS inhibitors from the sulfonylurea herbicide and / or sulfonamide carbonyl triazoline herbicide subclass.

[0155] Preferably, the agronomically usable sugar beet plants of the present invention (most preferably, sugar beet plants) are fully fertile, and more preferably have wild-type fertility. Fertility is of paramount importance for the sugar beet plants of the present invention in order to be agronomically usable.

[0156] One example of an agronomically usable sugar beet plant is sugar beet. When cultivated in an area of ​​1 hectare of field, the sugar beet plant of the present invention (about 80,000 to 90,000 sugar beet plants) should preferably be used to produce at least 4 tons of sugar.

[0157] Alternatively, the sugar beet plant of the present invention should preferably contain 15-20% (preferably at least 17%) sugar content in order to be agronomically usable. Therefore, a sugar beet plant having a sugar content of 15-20% (preferably at least 17%) is a preferred embodiment of the present invention.

[0158] The plants of this invention can be identified using any genotyping method. Genotyping of the plants includes the use of techniques such as isoenzyme electrophoresis, restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), random-initiated polymerase chain reaction (AP-PCR), allele-specific PCR (AS-PCR), DNA amplified fingerprint (DAF), sequence characteristic amplified region (SCAR), amplified fragment length polymorphism (AFLP), and simple repeat sequence (SSR) (also known as “microsatellites”). Additional compositions and methods for analyzing the genotypes of the plants provided herein include those disclosed in U.S. Publication Nos. 2004 / 0171027, 2005 / 02080506, and 2005 / 0283858.

[0159] Another aspect of the invention is the use of the sugar beet plants described herein and / or the harvestable parts or propagation materials described herein for the preparation / breeding of sugar beet plants. Methods for preparing / breeding sugar beet plants are described elsewhere herein. Such preparation / breeding methods can be used to produce sugar beet plants of the invention that also include new plant traits, such as stress resistance, for example, but not limited to, drought stress, heat stress, cold stress, or salt stress.

[0160] In another aspect, the present invention covers the use of herbicide-tolerant sugar beets and / or harvestable portions or propagation materials derived therefrom in screening methods for selecting ALS inhibitor herbicides.

[0161] The invention and its many advantages can be better understood from the following embodiments, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0162] Example 1: Mutant Isolation

[0163] Sugar beet cell cultures were initiated from diploid sugar beet genotype 7T9044 (e.g., described by Alexander Dovzhenko, PhD Thesis, Title: "Towards plastid transformation in rapeseed (Brassica napus L.) and sugarbeet (Beta vulgaris L.)", Ludwig-Maximilians-Universitat Munchen, Germany, 2001).

[0164] Sugar beet seeds were immersed in 70% ethanol for 60 seconds, then washed twice in sterile water containing 0.01% detergent, and then incubated in 1% NaOCl bleach for 1–4 hours. The seeds were then washed three times with sterile H2O and stored overnight at 4°C in sterile H2O. The embryo was then separated using forceps and a scalpel.

[0165] Freshly prepared seedlings were immersed in 0.5% NaOCl for 30 minutes, then washed three times with sterile H2O. After the final wash, they were placed on hormone-free MS agar medium (Murashige and Skoog (1962), Physiol. Plantarum, 15, 473-497). Those seedlings that developed into sterile seedlings were used to initiate regenerative sugar beet cell cultures.

[0166] Cotyledons and hypocotyls were cut into 2-5 mm segments and cultured on MS agar medium (0.8%) fixed with 1 mg / L purine benzoate (BAP) or 0.25 mg / L phenylthiazolyl (TDZ). After 4 weeks, the developing shoot tip cultures were transferred to fresh MS agar medium with the same composition and then subcultured monthly. The cultures were kept in dark at 25°C with a 12h / 12h light / dark cycle.

[0167] After 7-10 days, subcultures grown on media containing phenylthiazolyl hydrochloride formed distinct callus morphologies in the shoot tips, which were rapidly growing, soft, and brittle. These callus morphologies ranged in color from pale yellow to pale green. Some of these brittle callus morphologies consistently produced chlorophyll-containing shoot tip primordia from the embryoid structure. These rapidly growing, regenerating callus morphologies were used to select sugar beet mutants tolerant to ALS-inhibiting herbicides.

[0168] When this type of callus is exposed to 10 -9 When the ALS inhibitor herbicide formamidesulfuron (belonging to the sulfonylurea subclass, see above) is used, the cells survive, but produce less than 50% of the biomass of their lineage cells on a medium without the inhibitor. At a concentration of 3 × 10⁻⁶... -8 No growth was detected on the culture medium containing M-formamidesulfuron. For large-scale mutant selection experiments, 10 mutants were selected. -7 M formamide sulfonamide. Count surviving and growing cell clones and transfer them to cells containing 3 × 10⁻⁶ cells after 4–6 weeks. -7 On fresh culture medium containing the inhibitor described in M. One of these cell clones was not only able to grow at this concentration of the inhibitor, but even at 3 × 10⁻⁶. -6It grows in the presence of M-formamide sulfonamide.

[0169] From this clone (SB574TL), shoot tips were regenerated in the presence of an ALS inhibitor herbicide, and then the shoot tips were transferred to MS medium containing 0.05 mg / L naphthaleneacetic acid (NAA).

[0170] Within 4–12 weeks, the shoot tips develop roots, and they are then transferred to sterile plant containers filled with moist, sterilized perlite and watered with a half-concentration MS inorganic component. Alternatively, the plantlets are transferred directly from agar-solidified medium to a soil mixture containing perlite in a greenhouse. For the first 10–15 days after transfer to the soil-containing substrate, the plants are kept in a high-humidity environment. During and after the gradual transition to normal greenhouse humidity, the plants are kept in the greenhouse under artificial light (12 h) and day / night temperatures of 20 ± 3 °C / 15 ± 2 °C.

[0171] Three to five weeks later, regenerated plants from the formamide-sulfuron-tolerant cell culture (SB574TL) obtained above, as well as from wild-type cell cultures, were treated with formamide-sulfuron, sodium iodide-methyl (CAS RN 144550-3-7), and a mixture of the two active ingredients. The tested herbicide doses were equivalent to 7-70 g ai / ha of formamide-sulfuron and 1-10 g ai / ha of sodium iodide-methyl. Regenerated plants from this tolerant cell line tolerated even the highest herbicide doses (formamide-sulfuron, sodium iodide-methyl, and a 7:1 mixture), while even the lowest doses killed wild-type plants.

[0172] Example 2: Testing of Offspring

[0173] Based on SB574TL, F2 and F3 seeds of experimental hybrids containing heterozygous resistance alleles, and F4-F6 seeds containing homozygous mutant alleles, were sown in the field. When the plants developed 3-5 rosette leaves, they were treated with a mixture of formamide-sulfuron, sodium iodide-sulfuron, and these two ALS inhibitor herbicides. The homozygous seedlings tolerated a mixture of 35 g formamide-sulfuron / ha + 7 g sodium iodide-sulfuron / ha without stunted growth or any visible damage. At these ratios, the heterozygous lines showed signs of stunted growth and some leaf chlorosis, but they recovered within 3-5 weeks; however, conventional sugar beet seedlings were killed by the ALS inhibitor herbicides.

[0174] Example 3: Molecular characterization of the obtained sugar beet mutant (SB574TL)

[0175] The extraction and nucleic acid sequence analysis of the obtained mutants were performed by LGC Genomics GmbH in Berlin, Germany, according to a modified standard protocol.

[0176] The nucleic acid sequence obtained from the sugar beet mutant SB574TL is shown in SEQ ID NO:3, SEQ ID NO:4 represents the corresponding amino acid sequence, and SEQ ID NO:1 is obtained by sequencing wild-type sugar beet. SEQ ID NO:2 represents the corresponding amino acid sequence of the wild-type sugar beet.

[0177] A comparison of all these sequences clearly shows that there is a mutation only at position 569 of the endogenous ALS gene in this sugar beet plant material, and no other changes have occurred in any other part.

[0178] Example 4: Enzyme Activity Measurement

[0179] According to the manufacturer's instructions, the coding sequences of the ALS gene from wild-type beet and the W574L- mutant (SB574TL) were cloned into the Novagen pET-32a(+) vector, which was then used to transform *E. coli* AD494. The bacteria were cultured at 37°C in LB medium (Luria-Broth medium) containing 100 mg / L carbenicillin and 25 mg / L kanamycin, and the results were analyzed at OD... 600 At a concentration of 0.6, the bacteria were induced with 1 mM isopropyl-β-D-thiogalactopyranoside and cultured at 18°C ​​for 16 hours, followed by harvesting by centrifugation. The bacterial pellet was resuspended at a concentration of 1 g wet weight / 25 ml buffer in 100 mM pH 7.0 sodium phosphate buffer (containing 0.1 mM thiamine pyrophosphate, 1 mM MgCl2, and 1 μM FAD) and lysed by sonication. The crude protein extract obtained after centrifugation was used for ALS activity measurement.

[0180] ALS assays were performed in 96-well microtiter plates using a modified protocol based on the steps described by Ray (1984). The reaction mixture contained 20 mM potassium phosphate buffer (pH 7.0), 20 mM sodium pyruvate, 0.45 mM thiamine pyrophosphate, 0.45 mM MgCl2, 9 μM FAD, ALS enzyme, and various concentrations of ALS inhibitors, to a final volume of 90 μL. The assay was initiated by adding the enzyme and terminated by adding 40 μL of 0.5 M H2SO4 after incubation at 30 °C for 75 min. After 60 min at room temperature, 80 μL of a solution of 1.4% α-naphthol and 0.14% creatine in 0.7 M NaOH was added, followed by incubation at room temperature for another 45 min, and absorbance was measured at 540 nm. The pI50 values ​​for ALS inhibition were determined using the XLFitExcel plugin version 4.3.1 curve fitting program from ID Business Solutions Limited, as described by Ray (1984).

[0181] In total, compared with the wild-type enzyme, the mutant enzyme is at least 2000 times less sensitive to the ALS inhibitor formamide sulfonamide.

[0182] Example 5: Enzyme activity measurement (from plants)

[0183] ALS was extracted from sugar beet leaves or sugar beet tissue cultures as described by Ray (1984), Plant Physiol 75:827-831.

[0184] As described in Example 4, ALS activity was determined in leaf extracts of wild-type and sugar beet, as well as in the obtained SB574TL leaf extract, in the presence of various concentrations of formamide sulfonamide.

[0185] In total, compared with the wild-type enzyme, the mutant enzyme is at least 2000 times less sensitive to the ALS inhibitor formamide sulfonamide.

[0186] Example 6: Field trial using homozygous sugar beet plants tolerant of ALS inhibitor herbicides

[0187] Based on SB574TL, F4-F6 seeds containing mutant alleles of the endogenous ALS gene in a homozygous state were used for further testing.

[0188] Seeds of homozygous SB574TL mutant plants and seeds of the traditional variety KLARINA (commonly used sugar beet varieties with ALS inhibitor sensitivity references, which do not have a corresponding mutation at position 569 in their ALS protein) were sown in the field and cultured to various growth stages according to BBCH standards (as defined by monographie, Entwicklungsstadien mono- und dikotyler Pflanzen, 2nd edition, 2001, ed. Uwe Meier, Biologische Bundesanstalt fur Landund Forstwirtschaft).

[0189] The plants were then treated with the various ALS inhibitor herbicides listed in Table 1 below, the same herbicides used in the selection step.

[0190] The amount of water used in various applications is equal to 200 l / ha.

[0191] Damage (phytotoxicity / plant) on different sugar beet plants was scored from 0% to 100% on days 8, 14, and 28 following application of the various ALS inhibitor herbicides (DAA) (as shown in Table 1). In this context, "0%" means "no phytotoxicity / plant" and "100%" means the plant was completely killed.

[0192] Table 1

[0193]

Claims

1. A sugar beet plant and a portion thereof that are tolerant to an ALS inhibitor herbicide, comprising a non-transgenic mutation of its endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing an amino acid different from tryptophan at position 569.

2. The beet plant and its portion according to claim 1, wherein the ALS polypeptide at position 569 is replaced by the naturally encoded amino acid tryptophan with the amino acids alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, valine or arginine.

3. The beet plant and its parts according to claim 1, wherein the ALS gene encodes an ALS polypeptide, and the ALS polypeptide contains an amino acid different from tryptophan at position 569 of the ALS polypeptide sequence as shown in SEQ ID NO:

2.

4. The beet plant and its parts according to claim 1, wherein the amino acid is leucine, and wherein the endogenous ALS gene is identical to the nucleotide sequence identified in SEQ ID NO:

3.

5. The sugar beet plant and its parts according to claim 1, which are tolerant to one or more ALS inhibitor herbicides, said ALS inhibitor herbicides being selected from sulfonylurea herbicides, sulfonamide carbonyl triazolinone herbicides, imidazolinone herbicides, triazolopyrimidine herbicides, and pyrimidinyl (thio)benzoate (ester) herbicides.

6. The sugar beet plant and its parts according to claim 1, wherein the non-transgenic mutation is homozygous.

7. A portion of a sugar beet plant according to claim 1, wherein the portion is an organ, tissue, or cell of the plant, and preferably a seed.

8. The use of a non-GMO sugar beet plant, its parts, its harvestable parts or its propagation material that is tolerant to an ALS inhibitor herbicide for the preparation / breeding of sugar beets, sugar production, biofuel production, ethanol production, betaine production or uridine production, wherein the sugar beet plant, its parts, its harvestable parts or its propagation material contains a mutation in the endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing leucine replacing the naturally encoded amino acid tryptophan at position 569, and wherein the mutation is present as a unique mutation in a homozygous form.

9. The use according to claim 8, wherein the ALS gene encodes an ALS polypeptide, the ALS polypeptide containing leucine at position 569 of the ALS polypeptide sequence as shown in SEQ ID NO:2, in place of the naturally encoded amino acid tryptophan.

10. The use according to claim 8, wherein the amino acid is leucine, and wherein the endogenous ALS gene is identical to the nucleotide sequence identified in SEQ ID NO:

3.

11. The use according to claim 8, wherein the non-GMO sugar beet plant, its parts, its harvestable parts or its propagation material that are tolerant to one or more ALS inhibitor herbicides, said ALS inhibitor herbicide being selected from sulfonylurea herbicides, sulfonamide carbonyl triazolinone herbicides, imidazolinone herbicides, triazolopyrimidine herbicides and pyrimidinyl (thio)benzoate (ester) herbicides.

12. The use according to claim 8, wherein said part is a plant organ, tissue, or cell, and preferably a seed.

13. A non-GMO sugar beet plant portion or harvestable portion tolerant to an ALS inhibitor herbicide, comprising a mutation in the endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing leucine replacing the naturally encoded amino acid tryptophan at position 569, wherein the mutation is present as a unique mutation in a homozygous form, and wherein the sugar beet plant portion or harvestable portion is non-reproductive.

14. A portion or harvestable part of the sugar beet plant of claim 13, wherein the ALS gene encodes an ALS polypeptide, the ALS polypeptide containing leucine at position 569 of the ALS polypeptide sequence as shown in SEQ ID NO:2, in place of the naturally encoded amino acid tryptophan.

15. The sugar beet plant portion or harvestable portion of claim 13, wherein the amino acid is leucine, and wherein the endogenous ALS gene is identical to the nucleotide sequence identified in SEQ ID NO:

3.

16. A portion or harvestable part of the sugar beet plant of claim 13, which is tolerant to one or more ALS-inhibiting herbicides, said ALS-inhibiting herbicide being selected from sulfonylurea herbicides, sulfonamide carbonyl triazolinone herbicides, imidazolinone herbicides, triazolopyrimidine herbicides, and pyrimidinyl (sulfur) benzoate (ester) herbicides.

17. A cell or leaf extract of a non-GMO beet plant and a portion thereof that is tolerant to an ALS inhibitor herbicide, comprising a mutation in the endogenous acetolactate synthase (ALS) gene, wherein the ALS gene encodes an ALS polypeptide containing leucine replacing the naturally encoded amino acid tryptophan at position 569, and wherein the mutation is present as the only mutation in a homozygous form.

18. The cell extract or leaf extract of claim 17, wherein the ALS gene encodes an ALS polypeptide, wherein the ALS polypeptide contains leucine at position 569 of the ALS polypeptide sequence as shown in SEQ ID NO:2, replacing the naturally encoded amino acid tryptophan.

19. The cell extract or leaf extract of claim 17, wherein the amino acid is leucine, and wherein the endogenous ALS gene is identical to the nucleotide sequence identified in SEQ ID NO:

3.

20. Use of a sugar beet plant or its harvestable portion that is tolerant to an ALS inhibitor herbicide in a screening method for selecting an ALS inhibitor herbicide, said sugar beet plant or its harvestable portion containing a mutation in the endogenous acetolactate synthase (ALS) gene, said ALS gene encoding an ALS polypeptide containing leucine replacing the naturally encoded amino acid tryptophan at position 569, and said mutation being present as a unique mutation in a homozygous form.

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