Method for breeding male sterile line and for seed production

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a method for breeding a male sterile line and for seed production. The method of the present invention is universally applicable and enables the production of a large number of hybrids at a low cost. The heterozygous plant of the present invention can serve, on the one hand, as a male sterile line, and on the other hand, as a maintainer line, for use in the establishment of a hybrid seed production system.

Description

A method for breeding and producing male-sterile lines Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for breeding male-sterile lines and a method for seed production. Background Technology

[0002] Heterosis refers to the phenomenon where the first generation of hybrids outperforms their parents in terms of stress resistance, yield, and quality. Heterosis is a widespread phenomenon observed in various crops. Utilizing heterosis is an important means of achieving high yields in crops. Therefore, improving yield, quality, and stress resistance through hybridization has been widely applied in crops such as corn, rice, watermelon, tomato, and cabbage. For example, corn, as a plant with separate male and female inflorescences, can undergo large-scale and efficient deemasculation using mechanical methods when creating hybrids; rice, as a self-pollinating plant, cannot be efficiently deemasculated mechanically and relies on male-sterile systems.

[0003] Hybrid seed production systems based on male-sterile lines can be divided into two models: the three-line method and the two-line method. The three-line method consists of a male-sterile line, a maintainer line, and a restorer line. The male-sterile line refers to the male-sterile line; the maintainer line is the male-sterile line that produces seeds for hybridization; and the restorer line restores pollen fertility and is used to hybridize with the male-sterile line to produce hybrids with normal fertility. The three-line method can be implemented through screening for natural mutants and transgenic techniques. The three-line system for rice utilizes naturally occurring male-sterile materials, combined with naturally existing maintainer and restorer lines, to achieve large-scale, low-cost production of hybrid rice. The two-line method combines the male-sterile line and maintainer line from the three-line system into a single line, namely a conditional male-sterile line, to simultaneously produce both the male-sterile line and the maintainer line. Two-line methods can be achieved through different technical means. The specific technical routes are as follows: 1) transgenic two-line method, 2) natural mutation two-line method, 3) chemical male-killing two-line method, and 4) mechanical male-killing two-line method.

[0004] Invention Summary

[0005] To address the aforementioned problems in the existing technology, this invention provides a method for breeding male-sterile lines and a method for producing seeds.

[0006] This invention provides a method for breeding male-sterile lines in plants, the steps of which include:

[0007] (a) Obtain heterozygous plants with two different ALS allele mutations, wherein the homozygous plants with the first ALS allele mutation are resistant to herbicide A but not resistant to herbicide B; and the homozygous plants with the second ALS allele mutation are resistant to herbicide B but not resistant to herbicide A.

[0008] (b) Treat the heterozygous plants described in step (a) with both herbicides A and B simultaneously to obtain resistant male-sterile lines; wherein, in step (b), both herbicides A and B are applied simultaneously during the reproductive growth period, or both herbicides A and B are applied simultaneously before the reproductive growth period and the efficacy continues into the reproductive growth period.

[0009] This invention also provides a method for breeding plant conservation lines, the steps of which include:

[0010] (A) Obtain heterozygous plants with two different ALS allele mutations, wherein the homozygous plants with the first ALS allele mutation are resistant to herbicide A but not resistant to herbicide B; and the homozygous plants with the second ALS allele mutation are resistant to herbicide B but not resistant to herbicide A.

[0011] (B) Treat the heterozygous plants described in step (A) with both herbicides A and B simultaneously to obtain resistant maintainer lines; wherein, in step (B), both herbicides A and B are applied simultaneously before the reproductive growth stage and the efficacy does not continue into the reproductive growth stage.

[0012] The present invention also provides a method for isolating heterozygous plants with two different ALS allele mutations in plant progeny, the steps of which include:

[0013] (1) Obtain heterozygous plants with two different ALS allele mutations, wherein the homozygous plants with the first ALS gene mutation are resistant to herbicide A but not resistant to herbicide B; and the homozygous plants with the second ALS gene mutation are resistant to herbicide B but not resistant to herbicide A.

[0014] (2) Use two herbicides, A and B, to treat the offspring produced by self-pollination of the heterozygous plants described in step (1) simultaneously, and isolate heterozygous plants with two different ALS allele mutations.

[0015] In one specific embodiment, when step (2) involves simultaneous treatment with both herbicides A and B before the reproductive growth period and the efficacy does not continue into the reproductive growth period, the heterozygous plants isolated with two different ALS allele mutations are resistant and have normal fertility; when step (2) involves simultaneous treatment with both herbicides A and B during the reproductive growth period, or simultaneous treatment with both herbicides A and B before the reproductive growth period and the efficacy continues into the reproductive growth period, the heterozygous plants isolated with two different ALS allele mutations are resistant and male-sterile.

[0016] In one specific embodiment, the method for obtaining heterozygous plants with two different ALS allele mutations includes hybridization, gene editing, mutagenesis, natural mutation, or transgenic modification.

[0017] In one specific embodiment, the hybridization is performed by crossing homozygous plants containing different ALS gene mutations and having different resistance spectra.

[0018] In one specific embodiment, the alleles are located at corresponding positions on two different homologous chromosomes.

[0019] The present invention also provides a method for producing hybrid seeds, the steps of which include: using plants with normal pollen fertility as male parents and using male-sterile plants obtained by the method as female parents for hybridization to obtain hybrid seeds.

[0020] In one specific embodiment, the paternal parent is a homozygous plant with a third ALS gene mutation, which is resistant to both herbicides A and B, and whose pollen has normal fertility.

[0021] The present invention also provides a method for restoring the fertility of male-sterile plants, the steps of which include: hybridizing the male-sterile plant with a fertile plant that can provide pollen, thereby restoring the fertility of the male-sterile plant.

[0022] The present invention also provides a method for increasing plant yield, the steps of which include: planting maintainer plants obtained by the method, or heterozygous plants with normal fertility and two different ALS allele mutations obtained by the method, or hybrids obtained by the method, in a plant planting site, and spraying herbicides A and / or B in an effective amount, resulting in increased plant yield compared with wild-type plants.

[0023] The present invention also provides a method for improving herbicide resistance in plants, the steps of which include: planting male sterile plants obtained by the method, or maintainer plants obtained by the method, or heterozygous plants with two different ALS allele mutations obtained by the method, or hybrids obtained by the method in a plant planting site, and spraying herbicide A and / or B in an effective amount, after which the plants grow normally.

[0024] In one specific embodiment, the herbicides are all ALS inhibitor herbicides.

[0025] In another specific embodiment, the herbicide is applied by a method selected from top spraying, foliar spraying, soil treatment, irrigation treatment, seed coating treatment, and seed soaking treatment.

[0026] In one specific embodiment, the present invention also provides a plant seed, plant, plant cell, or plant part obtained by the method described.

[0027] In one specific embodiment, the plant includes monocotyledonous plants or dicotyledonous plants.

[0028] The beneficial effects of the present invention are: the method described in the present invention is universal and can obtain a large number of hybrids at low cost. The heterozygous plants described in the present invention can be used as male sterile lines or as maintainer lines for the establishment of hybrid seed production systems.

[0029] Invention Details

[0030] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural indicators. Thus, for example, a reference to “plant” includes a plurality of such plants; a reference to “cell” includes one or more cells and their equivalents known to those skilled in the art.

[0031] In the context of this disclosure, the term "hybrid" or "cross" (or "crossing") refers to the fusion of gametes via pollination to produce offspring (i.e., cells, seeds, or plants). This term encompasses sexual hybridization (one plant being pollinated by another) and self-pollination (self-pollination, i.e., when pollen and ovules (or microspores and megaspores) are from the same plant or plants with identical genes).

[0032] "Expression" usually refers to the production of a functional product. For example, the expression of a nucleic acid fragment can refer to the transcription of the nucleic acid fragment (such as the transcription of mRNA or functional RNA) and / or the translation of mRNA into a precursor or mature protein.

[0033] A "gamete" is a reproductive cell with 1n sets (haploid) of chromosomes, which can fuse with another gamete of the opposite sex during fertilization in an organism undergoing sexual reproduction. As used herein, a gamete in an organism undergoing asexual reproduction refers to a cell with 2n chromosomes (not reduced in number).

[0034] The term "genome" refers to the complete complement of the genetic material (genes and non-coding sequences) present in every cell, virus, or organelle of an organism; and / or the complete set of chromosomes inherited as (haploid) units from a parent.

[0035] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are polymers of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or modified nucleotide bases.

[0036] As used herein, “polynucleotide” includes references to deoxyribonucleotides, ribonucleotides, or their analogues that have the basic properties of natural ribonucleotides, because under strict hybridization conditions they hybridize with nucleotide sequences substantially identical to those of naturally occurring nucleotides and / or allow translation into one or more amino acids identical to one or more naturally occurring nucleotides. Polynucleotides can be natural or heterologous structures or full-length or subsequences of regulatory genes. Unless otherwise specified, the term includes references to the specified sequence and its complementary sequence. Thus, DNA or RNA with a modified backbone for stability or other reasons is a “polynucleotide” as the term implies herein. Furthermore, by just two examples, DNA or RNA containing rare bases (such as inosine) or modified bases (such as triphenylmethylated bases) is a polynucleotide as the term is used herein.

[0037] The terms “polypeptide,” “peptide,” “amino acid sequence,” and “protein” are used interchangeably in this article to refer to polymers of amino acid residues.

[0038] "Offspring" includes any subsequent generations of a plant.

[0039] ALS inhibitors are herbicide-inhibiting enzymes called acetylhydroxy acid synthase (AHAS), also known as acetyllactic acid synthase (ALS [EC 4.1.3.18]). ALS is the site of action of five structurally different herbicide families belonging to the ALS inhibitor herbicide class, such as (a) sulfonylurea herbicides (Beyer EM et al. (1988), Sulfonylureas in Herbicides: Chemistry, Degradation, and Mode of Action; Marcel Dekker, New York, 1988, 117-189), (b) sulfonylaminocarbonyltriazolinone herbicides (Pontzen, R., Pflanz.-Nachrichten Bayer, 2002, 55, 37-52), and (c) imidazolinone herbicides (Shaner, DL, et al., Plant Physiol., 1984, 76, 545-546; Shaner, DL, and O'Connor, SL (Eds.) The Imidazolinone Herbicides, CRC Press, Boca Raton, FL, 1991), (d) triazolopyrimidine herbicides (Kleschick, W.A. et al., Agric. Food Chem., 1992, 40, 1083-1085), and (e) pyrimidinyl (thio)benzoate herbicides (Shimizu, T.J., Pestic. Sci., 1997, 22, 245-256; Shimizu, T. et al., Acetolactate Syntehase Inhibitors in Herbicide Classes in Development, P., Wakabayashi, K., Hirai, K., (Eds.), Springer Verlag, Berlin, 2002, 1-41).

[0040] ALS involves the conversion of two pyruvate molecules into acetolactate and carbon dioxide. The reaction uses thymine pyrophosphate to link the two pyruvate molecules. The resulting product, acetolactate, is ultimately 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). ALS inhibitors disrupt the biosynthesis of valine, leucine, and isoleucine in plants. The result is an immediate depletion of these amino acid pools, leading to the cessation of protein biosynthesis, plant growth arrest, and ultimately plant death or at least damage.

[0041] In one specific implementation, ALS inhibitor herbicides include, but are not limited to:

[0042] (Sulfoamide group (Group (A)) includes:

[0043] The sulfonylurea subgroup (A1) includes:

[0044] Amidesulfuron [CAS RN 120923-37-7] (=A1-1);

[0045] Azimsulfuron [CAS RN 120162-55-2] (=A1-2);

[0046] bensulfuron-methyl [CAS RN 83055-99-6] (=A1-3);

[0047] Chlorimuron-ethyl [CAS RN 90982-32-4] (=A1-4);

[0048] Chlorsulfuron [CAS RN 64902-72-3] (=A1-5);

[0049] Cinosulfuron [CAS RN 94593-91-6] (=A1-6);

[0050] Cyclosulfamuron [CAS RN 136849-15-5] (=A1-7);

[0051] ethametsulfuron-methyl [CAS RN 97780-06-8] (=A1-8);

[0052] Ethoxysulfuron [CAS RN 126801-58-9] (=A1-9);

[0053] Flazasulfuron [CAS RN 104040-78-0] (=A1-10);

[0054] Flucetosulfuron [CAS RN 412928-75-7] (=A1-11);

[0055] Flupyrsulfuron-methyl-sodium [CAS RN 144740-54-5] (=A1-12);

[0056] Foramsulfuron [CAS RN 173159-57-4] (=A1-13);

[0057] Halosulfuron-methyl [CAS RN 100784-20-1] (=A1-14);

[0058] Imazosulfuron [CAS RN 122548-33-8] (=A1-15);

[0059] Sodium iodosulfuron-methyl-sodium [CAS RN 144550-36-7] (=A1-16);

[0060] Mesosulfuron-methyl [CAS RN 208465-21-8] (=A1-17);

[0061] Metsulfuron-methyl [CAS RN 74223-64-6] (=A1-18);

[0062] Monosulfuron [CAS RN 155860-63-2] (=A1-19);

[0063] Nicosulfuron [CAS RN 111991-09-4] (=A1-20);

[0064] Orthosulfamuron [CAS RN 213464-77-8] (=A1-21);

[0065] Oxasulfuron [CAS RN 144651-06-9] (=A1-22);

[0066] primisulfuron-methyl [CAS RN 86209-51-0] (=A1-23);

[0067] Prosulfuron [CAS RN 94125-34-5] (=A1-24);

[0068] Pyrazosulfuron-ethyl [CAS RN 93697-74-6] (=A1-25);

[0069] Rimsulfuron [CAS RN 122931-48-0] (=A1-26);

[0070] Sulfosulfuron-methyl [CAS RN 74222-97-2] (=A1-27);

[0071] Sulfosulfuron [CAS RN 141776-32-1] (=A1-28);

[0072] Thifensulfuron-methyl [CAS RN 79277-27-3] (=A1-29);

[0073] Triasulfuron [CAS RN 82097-50-5] (=A1-30);

[0074] Tribenuron-methyl [CAS RN 101200-48-0] (=A1-31);

[0075] Trifloxysulfuron [CAS RN 145099-21-4] (sodium) (=A1-32);

[0076] Triflusulfuron-methyl [CAS RN 126535-15-7] (=A1-33);

[0077] Tritosulfuron [CAS RN 142469-14-5] (=A1-34);

[0078] NC-330[CAS RN 104770-29-8](=A1-35);

[0079] Metazosulfuron [CAS RN 868680-84-6] (=A1-36);

[0080] Propyrisulfuron [CAS RN 570415-88-2] (=A1-37);

[0081] Monosulfuron-methyl [CAS RN 175076-90-1] (=A1-38);

[0082] 2-Iodo-N-[(4-methoxy-6-methyl-1,3,5-triazinyl)carbamoyl]benzenesulfonamide (=A1-39);

[0083] Compounds of general formula (I)

[0084] Where M+ represents the respective salts of compound (I), namely its lithium salt (=A1-40); its sodium salt (=A1-41); its potassium salt (=A1-42); its magnesium salt (=A1-43); its calcium salt (=A1-44); its ammonium salt (=A1-45); its methylammonium salt (=A1-46); its dimethylammonium salt (=A1-47); its tetramethylammonium salt (=A1-48); its ethylammonium salt (=A1-49); its diethylammonium salt (= A1-50); its tetraethylammonium salt (=A1-51); its propanemonium salt (=A1-52); its tetrapropylammonium salt (=A1-53); its isopropylammonium salt (=A1-54); its diisopropylammonium salt (=A1-55); its butylammonium salt (=A1-56); its tetrabutylammonium salt (=A1-57); its (2-hydroxyethyl-1-yl)ammonium salt (=A1-58); its bis-N,N-(2-hydroxyethyl-1-yl)ammonium Salts (=A1-59); its tri-N,N,N-(2-hydroxyethyl-1-yl)ammonium salt (=A1-60); its 1-phenylethylammonium salt (=A1-61); its 2-phenylethylammonium salt (=A1-62); its trimethylsulfonium salt (=A1-63); its trimethyloxonium salt (=A1-64); its pyridinium salt (=A1-65); its 2-methylpyridinium salt (=A1-66); its 4-methylpyridinium salt (=A1- 67); its 2,4-dimethylpyridinium salt (=A1-68); its 2,6-dimethylpyridinium salt (=A1-69); its piperidineium salt (=A1-70); its imidazole salt (=A1-71); its morpholineium salt (=A1-72); its 1,5-diazabicyclo[4.3.0]non-7-ene salt (=A1-73); its 1,8-diazabicyclo[5.4.0]undec-7-ene salt (=A1-74);

[0085] Or a compound of formula (II) or a salt thereof

[0086] R2 and R3 have the meanings defined in the table below.

[0087] Or the sodium salt of compound (III) (=A1-87), i.e., compound (A1-83).

[0088] Or the compound of formula (IV) (=A1-88), i.e. the sodium salt of compound (A1-82).

[0089] The sulfonylaminocarbonyltriazolinone subgroup (subgroup (A2)) includes:

[0090] Flucarbazone-sodium [CAS RN 181274-17-9] (=A2-1);

[0091] Propoxycarbazone-sodium [CAS RN 181274-15-7] (=A2-2);

[0092] Thiencarbazone-methyl [CAS RN 317815-83-1] (=A2-3);

[0093] The triazolopyrimidine subgroup (subgroup (A3)) includes:

[0094] cloransulam-methyl [147150-35-4] (=A3-1);

[0095] Diclosulam [CAS RN 145701-21-9] (=A3-2);

[0096] Florasulam [CAS RN 145701-23-1] (=A3-3);

[0097] Flumetsulam [CAS RN 98967-40-9] (=A3-4);

[0098] Metosulam [CAS RN 139528-85-1] (=A3-5);

[0099] Penoxsulam [CAS RN 219714-96-2] (=A3-6);

[0100] Pyroxsulam [CAS RN 422556-08-9] (=A3-7);

[0101] The sulfonamide subgroup (subgroup (A4)) includes:

[0102] Compounds selected from the group described by general formula (V) or their salts:

[0103] in

[0104] R1 is a halogen, preferably fluorine or chlorine.

[0105] R2 is hydrogen and R3 is a hydroxyl group, or R2 and R3 together with the carbon atoms they are attached to form a carbonyl group (C=O).

[0106] R4 is either hydrogen or methyl;

[0107] And more particularly compounds having the chemical structures (A4-1) to (A4-8) given below.

[0108] The imidazolinone group (group (B1)) includes:

[0109] Imazamethabenz-methyl [CAS RN 81405-85-8] (=B1-1);

[0110] Methoxymethylene (imazamox) [CAS RN 114311-32-9] (=B1-2);

[0111] Imidazolidinic acid (imazapic) [CAS RN 104098-48-8] (=B1-3);

[0112] Imazapyr [CAS RN 81334-34-1] (=B1-4);

[0113] Imidazolidine (imazaquin) [CAS RN 81335-37-7] (=B1-5);

[0114] Imazethapyr [CAS RN 81335-77-5] (=B1-6);

[0115] SYP-298[CAS RN 557064-77-4](=B1-7);

[0116] SYP-300[CAS RN 374718-10-2](=B1-8);

[0117] The pyrimidinyl (thio)benzoate group (Group (C)) includes:

[0118] The pyrimidinoxybenzoic acid subgroup (subgroup (C1)) includes:

[0119] Bispyribac-sodium [CAS RN 125401-92-5] (=C1-1);

[0120] Pyribenzoxim [CAS RN 168088-61-7] (=C1-2);

[0121] Pyriminobac-methyl [CAS RN 136191-64-5] (=C1-3);

[0122] Pyribambenz-isopropyl [CAS RN 420138-41-6] (=C1-4);

[0123] Pyribambenz-propyl [CAS RN 420138-40-5] (=C1-5);

[0124] The pyrimidinylthiobenzoic acid subgroup (subgroup (C2)) includes:

[0125] Pyriftalid [CAS RN 135186-78-6] (=C2-1);

[0126] Sodium pyrithiobac [CAS RN 123343-16-8] (=C2-2);

[0127] Triafamone [CAS RN 874195-61-6] (=C2-3)

[0128] Pyrimisulfan [CAS RN 221205-90-9] (=C2-4).

[0129] In one specific embodiment, herbicide A is selected from any one or more of sulfonylurea herbicides, triazolidine herbicides, pyrimidinyl (thio)benzoate herbicides, or sulfonamide carbonyl triazolone herbicides, and herbicide B is selected from any one or more imidazolinone herbicides.

[0130] In one specific embodiment, herbicide A is selected from bensulfuron-methyl, mesosulfuron-methyl, nicosulfuron, bensulfuron-methyl, sulfonylsulfuron-methyl, chlorpyrifos, fluazolidone, thiamethoxam, acylsulfuron-methyl, ethersulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, thifensulfuron-methyl, mesosulfuron-methyl, chlorsulfuron-methyl, pyrimisulfuron-methyl, chlorpyrifos, chlorpyrifos-methyl, chlorpyrifos-methyl, fluazinam-sulfuron-methyl, chlorpyrifos-methyl, and / or bispyribac-methyl; herbicide B is selected from imidacloprid, imidacloprid, methoxyimidacloprid, and / or imidacloprid.

[0131] In one specific embodiment, herbicide A is bensulfuron-methyl and herbicide B is methyl benzoate;

[0132] The herbicide A is mesosulfuron-methyl, and B is methyl sulfadiazine;

[0133] The herbicide A is nicosulfuron, and B is methyl benzoate;

[0134] The herbicide A is bensulfuron-methyl, and B is methyl benzoate;

[0135] The herbicide A is sulfadiazine, and B is methyl methazine.

[0136] The herbicide A is chlorpyrifos and B is methyl methazine;

[0137] The herbicide A is flusulfuron-methyl, and B is methyl benzoate;

[0138] The herbicide A is thiamethoxam and B is methyl benzoate;

[0139] The herbicide A is sulfadiazine, and B is methyl methazine;

[0140] The herbicide A is sulfadiazine, and B is methyl methazine;

[0141] The herbicide A is pyrimisulfuron, and B is methyl methazine;

[0142] The herbicide A is pyrimethanil, and B is methyl benzoate;

[0143] The herbicide A is pyrimisulfuron, and B is methyl methazine;

[0144] The herbicide A is thifensulfuron-methyl, and B is methyl benzoate;

[0145] The herbicide A is pyrazosulfuron, and B is imidacloprid;

[0146] The herbicide A is chlorpyrifos-methyl and B is methyl methazine;

[0147] The herbicide A is dichlorvos, and B is methyl methazine;

[0148] The herbicide A is pyrimethanil, and B is methyl methazine;

[0149] The herbicide A is fluroxypyr, and B is methyl methazine;

[0150] The herbicide A is bispyribac-sodium, and B is methyl methazine;

[0151] A is bensulfuron-methyl, and B is imazalil;

[0152] The herbicide A is mesosulfuron-methyl, and B is imazalil;

[0153] The herbicide A is nicosulfuron, and B is imazalil;

[0154] The herbicide A is bensulfuron-methyl, and B is imazalil;

[0155] The herbicide A is sulfadiazine, and B is imazalil;

[0156] The herbicide A is chlorpyrifos and B is imazalil;

[0157] The herbicide A is flusulfuron-methyl, and B is imazalil;

[0158] The herbicide A is thiamethoxam and B is imazalil;

[0159] The herbicide A is sulfadiazine, and B is imazalil;

[0160] The herbicide A is fensulfuron-methyl, and B is imazalil;

[0161] The herbicide A is pyrimisulfuron, and B is imazalil;

[0162] The herbicide A is pyrimethanil, and B is imazalil;

[0163] The herbicide A is pyrimisulfuron, and B is imazalil;

[0164] The herbicide A is thifensulfuron-methyl, and B is imazalil;

[0165] The herbicide A is disulfuron-methyl, and B is imazalil;

[0166] The herbicide A is chlorpyrifos-methyl, and B is imazalil;

[0167] The herbicide A is dichlorvos, and B is imazalil;

[0168] The herbicide A is pyrimethanil, and B is imazalil;

[0169] The herbicide A is fluroxypyr, and B is imazalil;

[0170] The herbicide A is bispyribac-sodium, and B is imazalil;

[0171] A is bensulfuron-methyl, and B is methoxyfenozide.

[0172] The herbicide A is mesosulfuron-methyl, and B is methoxyfenozide.

[0173] The herbicide A is nicosulfuron, and B is methoxyfenozide;

[0174] The herbicide A is bensulfuron-methyl, and B is methoxyfenozide;

[0175] The herbicide A is sulfadiazine, and B is methoxyfenozide;

[0176] The herbicide A is chlorpyrifos and B is methoxyfenozide;

[0177] The herbicide A is flusulfuron-methyl, and B is methoxyfenozide.

[0178] The herbicide A is thiamethoxam and B is methoxyfenozide.

[0179] The herbicide A is sulfadiazine, and B is methoxyfenozide;

[0180] The herbicide A is sulfadiazine, and B is methoxyfenozide;

[0181] The herbicide A is pyrimisulfuron, and B is methoxyfenozide;

[0182] The herbicide A is pyrimethanil, and B is methoxyfenozide.

[0183] The herbicide A is pyrimisulfuron, and B is methoxyfenozide;

[0184] The herbicide A is thifensulfuron-methyl, and B is methoxyfenozide.

[0185] The herbicide A is pyrazosulfuron, and B is methoxyfenozide;

[0186] The herbicide A is chlorpyrifos-methyl, and B is methoxyfenozide.

[0187] The herbicide A is dichlorvos, and B is methoxyfenozide;

[0188] The herbicide A is pyrimethanil, and B is methoxyfenozide;

[0189] The herbicide A is fluroxypyr, and B is methoxyfenozide;

[0190] The herbicide A is bispyribac-sodium, and B is methoxyfenozide.

[0191] A is benzylsulfuron-methyl, and B is imidazoquinoline acid;

[0192] The herbicide A is mesosulfuron-methyl, and B is imidazoquinoline acid;

[0193] The herbicide A is nicosulfuron, and B is imidazoquinoline.

[0194] The herbicide A is bensulfuron-methyl, and B is imidazoquinoline.

[0195] The herbicide A is sulfadiazine, and B is imidazoquinoline.

[0196] The herbicide A is chlorpyrifos and B is imidazoquinoline.

[0197] The herbicide A is flusulfuron-methyl, and B is imidazoquinoline.

[0198] The herbicide A is thiamethoxam and B is imidazoquinoline.

[0199] The herbicide A is sulfadiazine, and B is imidazoquinoline.

[0200] The herbicide A is sulfadiazine, and B is imidazoquinoline.

[0201] The herbicide A is pyrimisulfuron, and B is imidazoquinoline.

[0202] The herbicide A is pyrimethanil, and B is imidazoquinoline.

[0203] The herbicide A is pyrimisulfuron, and B is imidazoquinoline.

[0204] The herbicide A is thifensulfuron-methyl, and B is imidazoquinoline acid;

[0205] The herbicide A is disulfuron-methyl, and B is imidazoquinoline acid;

[0206] The herbicide A is chlorpyrifos-methyl and B is imidazoquinoline.

[0207] The herbicide A is dichlorvos, and B is imidazoquinoline.

[0208] The herbicide A is pyrimethanil, and B is imidazoquinoline.

[0209] The herbicide A is fluroxypyr, and B is imidazoquinoline.

[0210] The herbicide A is bispyribac-sodium, and B is imidazoquinoline.

[0211] The herbicide A is pyrimisulfuron, and B is methyl methazine;

[0212] The herbicide A is methamidosulfuron, and B is methyl benzoate;

[0213] The herbicide A is chlorsulfuron, and B is methyl methazine;

[0214] The herbicide A is pyrimisulfuron, and B is imazalil;

[0215] The herbicide A is methamidosulfuron, and B is imazalil;

[0216] The herbicide A is pyrimisulfuron, and B is imidazoquinoline.

[0217] The herbicide A is methamidosulfuron, and B is imidazoquinoline.

[0218] The herbicide A is chlorsulfuron-methyl, and B is methoxyfenozide.

[0219] The ALS gene is highly conserved in plants and can undergo resistance point mutations. Currently, herbicide-resistant weeds based on ALS gene mutations have emerged in nature. According to information from weedscience.org (Heap, I. The International Herbicide-Resistant Weed Database. Online. Thursday, December 12, 2024. Available www.weedscience.org), using the Arabidopsis thaliana ALS protein sequence as a reference, herbicide-resistant mutation sites appear at G121, A122, V196, P197, R199, M200, Q207, A205, K256, D376, R377, M570, V571, W574, S653, and G654, and different types of mutations exhibit different resistance profiles. For example, *Solanum ptycanthum* with the A122T mutation in the ALS gene (i.e., alanine A at position 122 is mutated to threonine T) is resistant to one tested imidazolinone herbicide but not to one tested sulfonylurea herbicide; *Schoenoplectus juncoides* with the P197S mutation in the ALS gene is not resistant to one tested imidazolinone and pyrimidine benzoate herbicide but is resistant to the tested sulfonylurea herbicide; *Descurainia sophia* with the W574L mutation shows resistance to all five classes of herbicides tested (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinyl (thio)benzoates, and sulfonamide carbonyl triazolinones); and *Amaranthus* with the S653N mutation... The herbicide *Tuberculus tuberculatus* was resistant to one of the tested imidazolinone herbicides but not to the tested sulfonylurea herbicide.

[0220] The ALS gene is prone to resistance mutations, including single-amino acid and multi-amino acid mutations. Various methods exist for inducing mutations in the ALS gene, including traditional chemical mutagens such as EMS and sodium azide, physical mutagens such as radiation, and gene editing tools.In addition, for example, through artificial mutagenesis, it has been found that resistance mutations can occur at more locations in the ALS gene, and the degree of freedom in amino acid changes is very high (Pest Manag Sci 2014; 70:1340–1350); Chen et al. 2017 (Chen Y, Wang Z, Ni H, Xu Y, Chen Q, Jiang L. CRISPR / Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis. Sci China Life Sci. 2017 May; 60(5):520-523. doi:10.1007 / s11427-017-9021-5. Epub 2017 Mar 16.PMID:28303459.) reported the creation of the then-known P197S and P197L resistance sites at the P197 site of the ALS gene in Arabidopsis using gene editing technology, as well as the previously unreported P197F mutant, and demonstrated that the P197F mutant exhibits similar resistance expression to known resistance sites such as P197S; Dong et al. 2020 (Dong H, Wang D, Bai Z, Yuan Y, Yang W, Zhang Y, Ni H, Jiang L. Generation of imidazolinone herbicide resistant trait in Arabidopsis. PLoS One. 2020 May) 22;15(5):e0233503.doi:10.1371 / journal.pone.0233503.PMID:32442184;PMCID:PMC7244175.) reported the creation of a series of resistance mutations near the G654 site of the ALS gene in Arabidopsis thaliana using gene editing technology, including single point mutations such as S653N, G654D, and G655S, and S65 Two-site mutations such as 3N / G654D, S653N / G655S, and G654D / G655S, as well as three-site mutations such as P652P / S653N / G654D, P652P / G654D / G655S, S653N / G654D / G655S, and S653N / G654N / G655S, are newly discovered resistance mutation types, except for S653N and G654D mutations which have been reported before.

[0221] Novel resistance mutation sites, including single-amino acid and multi-amino acid mutations, can also be discovered in the ALS gene using gene editing technology in other crops. For example, Azameti et al (MK, Dauda WP. Base Editing in Plants: Applications, Challenges, and Future Prospects. Front Plant Sci. 2021 Jul 27; 12:664997. doi:10.3389 / fpls.2021.664997.PMID:34386023; PMCID:PMC8353127.) reviewed the creation of a large number of novel single-site and multi-site resistance mutations in the ALS gene of rice, wheat, tomato, watermelon, rapeseed, and maize using gene editing technology. Ouyang et al (Ouyang,C.,Jin,X.,Zhao,H.et al.Generating Broad-Spectrum Resistance to ALS-Inhibiting Herbicides in Rice by CRISPR / Cas9-Mediated NHEJ.Rice 18,86(2025).https: / / doi.org / 10.1186 / s12284-025-00845-w) reported the creation of a triple-mutant resistant material P171T / R172G / M174L in rice using gene editing technology, and reviewed the creation of a variety of resistance mutations in the ALS gene through various pathways (including conventional mutagenesis and precise mutagenesis).

[0222] As used herein, herbicide “tolerance” or “resistance” refers to the ability to be completely or partially unaffected by the presence or application of one or more herbicides, such as the ability to resist the toxic effects of herbicides when applied. A cell or organism is herbicide-resistant if it can maintain at least some normal growth or phenotype in the presence of one or more herbicides. A trait is herbicide-tolerant if its presence, compared to wild-type or control cells, plants, or seeds, confers increased tolerance to herbicides. A target enzyme is herbicide-resistant if it exhibits increased enzyme activity relative to wild-type or control enzymes in the presence of herbicides. Herbicide tolerance can be complete or partial insensitivity to a particular herbicide and can be expressed as a percentage (%) of tolerance or insensitivity to a particular herbicide.

[0223] The two different ALS allele mutations described in this invention are selected from amino acid sequences that have undergone arbitrary mutations at sites 121, 196, 197, 199, 205, 570, 571, 653 and / or 654 corresponding to the Arabidopsis amino acid sequence SEQ ID NO: 1, as long as they possess the herbicide resistance.

[0224] In one specific implementation, the first ALS mutation includes:

[0225] At position 197 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, proline is mutated to the amino acid sequence of serine, leucine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, or valine.

[0226] At position 121 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, glycine is mutated to proline, serine, leucine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, or valine.

[0227] At position 196 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, valine is mutated to proline, serine, leucine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, or glycine.

[0228] At position 205 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, alanine is mutated to proline, serine, leucine, valine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, or glycine.

[0229] At position 570 of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1, methionine is mutated to proline, serine, leucine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, lysine, valine, phenylalanine, threonine, tryptophan, tyrosine, or glycine; and / or

[0230] At position 571 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, valine is mutated to proline, serine, leucine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, or glycine.

[0231] The second type of ALS mutation includes:

[0232] At position 653 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, serine is mutated to the amino acid sequence of asparagine, isoleucine, alanine, arginine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine.

[0233] At position 199 of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1, arginine is mutated to an amino acid sequence of asparagine, isoleucine, alanine, serine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine; and / or

[0234] At position 654 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, glycine is mutated to an amino acid sequence of asparagine, isoleucine, alanine, serine, aspartic acid, cysteine, glutamine, glutamic acid, arginine, histidine, leucine, lysine, methionine, phenylalanine, proline, threonine, tryptophan, tyrosine, or valine.

[0235] In one specific embodiment, the first ALS gene mutation is selected from the following positions at positions 121, 196, 197, 205, 570, and / or 571 corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1: G121A, G121D, G121N, G121S, V196M, P197A, P197R, P197C, P197Q, P197M, P197N, P197E, P197G, P197H, P197S, P197W, P197Y, P197V, P197L ...R, P197C, P197Q, P197M, P197N, P197E, P197G, P197H, P197S, P197W, P197Y, P197V, P197L, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P197R, P1 The first type of ALS gene mutation is selected from mutations in 7F, A205V, A205R, A205N, A205D, A205C, A205E, A205T, A205W, A205Y, M570A, M570N, M570C, V571A, V571N, V571C, V571Q, V571I, V571S and / or V571W; the second type of ALS gene mutation is selected from mutations at positions 199, 653 and / or 654 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, namely R199A, R199E, S653N, S653T, S653I, S653F, G654E and / or G654D.

[0236] In one specific embodiment, the first type of ALS mutation is a change from proline to serine or leucine at position 197 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1; the second type of ALS mutation is a change from serine to asparagine or isoleucine at position 653 of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1.

[0237] In one specific embodiment, the first type of ALS mutation is a change from proline to serine or leucine at position 197 of the amino acid sequence of the rapeseed C genome ALS (NP_001412453.1); the second type of ALS mutation is a change from serine to asparagine or isoleucine at position 653 of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1.

[0238] In one specific embodiment, the first type of ALS mutation is to change proline to serine or leucine at position 171 of the amino acid sequence of rice ALS (NP_001403551.1); the second type of ALS mutation is to change serine to asparagine or isoleucine at position 627 of the amino acid sequence of Arabidopsis ALS SEQ ID NO: 1.

[0239] In one specific embodiment, the first type of ALS mutation is a change from proline to serine or leucine at position 171 of the amino acid sequence of wheat D genome ALS (XP_044420692.1); the second type of ALS mutation is a change from serine to asparagine or isoleucine at position 627 of the amino acid sequence of Arabidopsis ALS SEQ ID NO: 1.

[0240] In one specific embodiment, the first type of ALS mutation is to change proline to serine or leucine at position 165 in the amino acid sequence of maize ALS108 (NP_001151761.2); the second type of ALS mutation is to change serine to asparagine or isoleucine at position 621 in the amino acid sequence of Arabidopsis ALS SEQ ID NO: 1.

[0241] In another specific embodiment, the first type of ALS mutation is to change proline to serine or leucine at position 179 in the amino acid sequence of pepper ALS (NP_001311628.1); the second type of ALS mutation is to change serine to asparagine or isoleucine at position 636 in the amino acid sequence of Arabidopsis ALS SEQ ID NO: 1.

[0242] In one specific embodiment, the third ALS gene mutation is selected from mutations that have herbicide resistance at positions 122, 206, 256, 351, 376, 377 and / or 574 corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1.

[0243] In another specific embodiment, the third ALS gene mutation is selected from the following positions at positions 122, 206, 256, 351, 376, 377 and / or 574 corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1: A122R, A122N, A122D, A122C, A122E, A122Q, A122H, A122I, A122L, A122K, A122M, A122F, A122P, A122S, A122T, A122W, A122Y, A122V, F206A, F206H, F206W, F206Y, K256D, K256N, K256G, K256P, K256T, M351C, M351, and M351, respectively. Mutations in Q, M351G, M351K, M351P, M351Y, M351V, D376E, D376A, D376N, D376C, D376G, D376P, D376S, D376W, D376V, R377H, R377K, W574R, W574G, W574M, W574A, W574D, W574N, W574C, W574Q, W574E, W574H, W574I, W574L, W574K, W574F, W574S, W574T, W574Y and / or W574V.

[0244] In one specific embodiment, the third ALS mutation is a change from tryptophan to leucine or methionine at position 574 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1.

[0245] In one specific embodiment, the third ALS mutation is a change from tryptophan to leucine or methionine at position 574 of the amino acid sequence of ALS (NP_001412453.1) in the rapeseed C genome.

[0246] In one specific embodiment, the third ALS mutation is a change from tryptophan to leucine or methionine at position 548 of the amino acid sequence of rice ALS (NP_001403551.1).

[0247] In one specific embodiment, the third ALS mutation is a change from tryptophan to leucine or methionine at position 548 of the amino acid sequence of ALS (XP_044420692.1) in the wheat D genome.

[0248] In one specific embodiment, the third ALS mutation is a change from tryptophan to leucine or methionine at position 542 of the maize ALS108 (NP_001151761.2) amino acid sequence.

[0249] In another specific embodiment, the third ALS mutation is a change from tryptophan to leucine or methionine at position 557 of the amino acid sequence of chili ALS (NP_001311628.1).

[0250] The term "mutation" refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the normal sequence, wild-type sequence, or reference sequence. In some embodiments, a mutation refers to a single amino acid variation in a polypeptide and / or at least a single nucleotide variation in a nucleic acid sequence relative to the nucleotide or amino acid sequence of a non-herbicide-resistant Arabidopsis ALS protein. In some embodiments, a mutation refers to one or more mutations at amino acid positions in the Arabidopsis ALS amino acid sequence as shown in any of SEQ ID NO:1 or at homologous positions in homologous genes of different species. In some embodiments, a mutation may include substitution, deletion, inversion, or insertion. In some embodiments, substitution, deletion, insertion, or inversion may include variations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides. In some implementations, substitutions, deletions, insertions, or inversions may include variations at positions 1, 2, 3, 4, 5, 6, 7, or 8 amino acids.

[0251] For the terminology related to amino acid substitutions used in this specification, the first letter represents a naturally occurring amino acid at a specific position in a particular sequence, the following number represents the position relative to a reference sequence (such as SEQ ID NO:1), and the second letter represents the different amino acid that replaces that natural amino acid. For example, P197S indicates that, relative to the amino acid sequence of SEQ ID NO:1, proline at position 197 is replaced by serine. For amino acid substitutions where the first letter is absent, it refers to the substitution of a natural amino acid at the position corresponding to the reference sequence (such as SEQ ID NO:1) relative to the amino acid sequence of its wild-type protein, by the amino acid represented by the letter following the number. For double or multiple mutations, the mutations are separated by " / " or "+". For example, P197S+S653N or P197S / S653N indicates that, relative to the amino acid sequence of SEQ ID NO:1, proline at position 197 is replaced by serine, and serine at position 653 is replaced by asparagine, with both mutations present in the specific ALS protein.

[0252] The terms "corresponding to" and "relative to" are used interchangeably. In this invention, the specific amino acid position (number) within the protein is determined using standard sequence alignment tools by comparing the amino acid sequence of the target protein with SEQ ID NO:1, etc. For example, the Smith-Waterman algorithm or the CLUSTALW2 algorithm can be used to align two sequences, where the sequence is considered aligned when the alignment score is the highest. The alignment score can be calculated according to the method described in Wilbur, WJ and Lipman, DJ (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm, the default parameters are preferably used: protein gap opening penalty = 10.0; protein gap extension penalty = 0.2; protein matrix = Gonnet; protein / DNA end gap = -1; protein / DNA GAPDIST = 4.

[0253] The AlignX program (part of the vectorNTI group) is preferably used with default parameters suitable for multiple alignments (gap opening penalty: 10; gap extension penalty: 0.05) to determine the position of specific amino acids in the protein of the present invention by comparing the amino acid sequence of the protein with SEQ ID NO:1.

[0254] Those skilled in the art will also understand that the structure of a protein can be altered without adversely affecting its activity and function. For example, one or more conserved amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional conformation of the protein molecule. Examples and implementations of conserved amino acid substitutions are familiar to those skilled in the art. Specifically, an amino acid residue can be substituted with another amino acid residue belonging to the same group as the site to be substituted, i.e., a nonpolar amino acid residue can replace another nonpolar amino acid residue, a polar uncharged amino acid residue can replace another polar uncharged amino acid residue, a basic amino acid residue can replace another basic amino acid residue, and an acidic amino acid residue can replace another acidic amino acid residue. Conservative substitutions in which an amino acid is replaced by another amino acid belonging to the same group fall within the scope of this invention, provided that the substitution does not impair the biological activity of the protein.

[0255] Therefore, in addition to the mutations described above, the ALS protein of the present invention may also contain one or more other mutations, such as conserved substitutions, in its amino acid sequence. Furthermore, the present invention also covers ALS proteins containing one or more other non-conserved substitutions, provided that such non-conserved substitutions do not significantly affect the desired function and biological activity of the protein of the present invention.

[0256] As is well known in the art, one or more amino acid residues can be deleted from the N and / or C-terminus of a protein while retaining its functional activity. Therefore, in another aspect, the present invention also relates to fragments of ALS proteins that have one or more amino acid residues deleted from their N and / or C-terminus while retaining their desired functional activity; these are also within the scope of the present invention and are referred to as bioactive fragments. In the present invention, a "bioactive fragment" refers to a portion of the ALS protein of the present invention that retains the biological activity of the ALS protein of the present invention. For example, a bioactive fragment of an ALS protein may be a portion of the protein in which one or more (e.g., 1-50, 1-25, 1-10, or 1-5, e.g., 1, 2, 3, 4, or 5) amino acid residues are deleted from the N and / or C-terminus, but which still retains the biological activity of the full-length protein.

[0257] In one specific embodiment, the homozygous plant with the first ALS gene mutation is not limited to the aforementioned mutation, but may also include any mutation at other positions (such as L183M, L189I, V187I, I163V, K160F, S135T, L183M+S135T, L183M+S135T+L189I, L183M+S135T+L189I, L183M+S135T+L189I+V187I corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1), as long as it does not affect the resistance to the corresponding herbicide.

[0258] In another specific embodiment, the homozygous plant with the second ALS gene mutation is not limited to the aforementioned mutation, but may also include any mutation at other positions (such as N658K, D620E, A619E, T656A, C641V, N658K+D620E, N658K+A619E, D620E+A619E, N658K+D620E+A619E, N658K+D620E+A619E, N658K+D620E+A619E, N658K+D620E+A619E+T656A corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1), as long as it does not affect the resistance to the corresponding herbicide.

[0259] In another specific embodiment, the homozygous plant with the third ALS gene mutation is not limited to the aforementioned mutation, but may also include any mutation at other positions (such as F587Y, V560I, Q593R, D595N, M572V, F587Y+V560I, F587Y+Q593R, F587Y+V560I+Q593R, F587Y+V560I+Q593R, F587Y+V560I+Q593R+D595N corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1), as long as it does not affect the resistance to the corresponding herbicide and the pollen has normal fertility.

[0260] The term "plant" means the whole plant, plant organs, plant tissues, seeds, plant cells, and the offspring of a plant. Plant cells include, but are not limited to, cells derived from: seeds, suspension cultures, embryos, meristematic regions, callus, leaves, roots, buds, gametophytes, sporophytes, pollen, and microspores. Plant parts include differentiated and undifferentiated tissues, including but not limited to roots, stems, branches, leaves, pollen, seeds, tumor tissue, and various forms of cells and cultures (e.g., single cells, protoplasts, embryos, and callus). Plant tissues can be in the plant or in plant organs, tissues, or cell cultures. The term "plant organ" refers to plant tissue or a group of tissues that constitute different morphological and functional parts of a plant.

[0261] Plants particularly useful in the method of this invention include all plants belonging to the superfamily of the Kingdom Viridiplantae, especially monocots and dicots, including legumes used for fodder or feed, ornamental plants, food crops, trees or shrubs, wherein said plants are selected from a list containing the following species: Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp., and Artocarpus spp. spp.), Asparagus officinalis, Avena spp. (e.g., Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Star fruit (Averrhoacarambola), Bambusa sp., Benincasa hispida, Brazil chestnut (Bertholletia excelsea), Beetroot (Beta vulgaris), Brassica spp. (e.g., Brassicanapus, Brassica rapa ssp.), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, papaya (Caricapapaya), large-fruited false tiger thorn (Carissa macrocarpa), species of the genus Carya (Carya spp.), red flower (Carthamus tinctorius), species of the genus Castanea (Castanea spp.).), American kapok (Ceiba pentandra), chicory (Cichorium endivia), Cinnamomum species (Cinnamomum spp.), watermelon (Citrullus lanatus), citrus species (Citrus spp.), coconut species (Cocos spp.), coffee species (Coffea spp.), taro (Colocasia esculenta), African sycamore species (Cola spp.), jute (Corchorus sp.), coriander (Coriandrum sativum), hazel species (Corylus spp.), hawthorn species (Crataegus spp.), saffron (Crocus sativus), squash species (Cucurbita spp.), cantaloupe species (Cucumis spp.), artichoke species (Cynara spp.), carrot (Daucus carota), grasshopper species (Desmodium spp.), longan (Dimocarpus) species of the genera *Dioscorea*, *Diospyros*, *Echinochloa*, *Elaeis* (e.g., *Elaeis guineensis*, *Elaeis oleifera*), *Eleusine coracana*, *Eragrostis tef*, *Erianthus* sp., *Eriobotrya japonica*, *Eucalyptus* sp., *Eugenia uniflora*, *Fagopyrum* sp., *Fagus* sp., *Festuca arundinacea*, *Ficus carica*, *Fortunella* sp., *Fragaria* sp., and *Ginkgo*. genus *Bibona*, genus *Glycine* (e.g., *Glycine max*, *Soja hispida*, or *Soja max*), upland cotton (*Gossypiumhirstum*), and species of the genus *Helianthus* (*Helianthus* spp.).(e.g., sunflower (Helianthus annuus)), daylily (Hemerocallis fulva), hibiscus species (Hibiscus spp.), barley (Hordeum spp.) (e.g., barley (Hordeum vulgare)), sweet potato (Ipomoea batatas), walnut species (Juglans spp.), lettuce (Lactuca sativa), pea species (Lathyrus spp.), lentil (Lens culinari), flax (Linumus itatisimum), litchi (Litchi chinensis), lotus species (Lotus spp.), loofah (Luffaacutangula), lupinus species (Lupinus spp.), Luzula sylvatica, tomato species (Lycopersicon spp.) (e.g., tomato (Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme), species of the genera *Macrotyloma*, *Malus*, *Malpighia emarginata*, *Mammea americana*, *Mangifera indica*, species of the genera *Manihot*, *Manilkara zapota*, *Medicago sativa*, species of the genera *Melilotus*, *Mentha*, *Miscanthus sinensis*, species of the genera *Momordica*, *Morus nigra*, species of the genera *Musa*, species of the genera *Nicotiana*, species of the genera *Olea*, species of the genera *Opuntia*, species of the genera *Ornithopus*, and species of the genera *Oryza*. (e.g., rice (Oryza sativa), broadleaf rice (Oryza latifolia)), millet (Panicum miliaceum), switchgrass (Panicum virgatum), passion fruit (Passiflora edulis), European parsnip (Pastinaca sativa), species of the genus Pennisetum (Pennisetum sp.)), avocado species (Persea spp.), parsley (Petroselinum crispum), purslane (Phalaris arundinacea), common bean species (Phaseolus spp.), cat's tail grass (Phleum pratense), prickly ash species (Phoenix spp.), southern reed (Phragmites australis), groundcherry species (Physalis spp.), pine species (Pinus spp.), pistachio species (Pistacia vera), pea species (Pisum spp.), Kentucky bluegrass species (Poa spp.), poplar species (Populus spp.), prosopis species (Prosopis spp.), plum species (Prunus spp.), guava species (Psidium spp.), pomegranate (Punica granatum), European pear (Pyrus communis), oak species (Quercus spp.) Radish (Raphanus sativus), Rheum rhabarbarum, Ribes spp., Castor bean (Ricinus communis), Rubus spp., Saccharum spp., Salix sp., Sambucus spp., rye (Secale cereale), Sesamum spp., Sinapis sp., Solanum spp. (e.g., potato (Solanum tuberosum), red eggplant (Solanum integrifolium), or tomato), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarind (Tamarindus) (Indica), cacao (Theobroma cacao), Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp.(For example, common wheat (Triticum aestivum), durum wheat (Triticum durum), cylindrical wheat (Triticum turgidum), Triticum hybernum, macha wheat (Triticum macha), common wheat (Triticum sativum), emmer wheat (Triticum monococcum), or common wheat (Triticum vulgare)), tarope (Tropaeolumminus), tarope (Tropaeolum majus), species of the genus *Vaccinium* (Vaccinium spp.), species of the genus *Vicias* (Vicias spp.), species of the genus *Vigna* (Vigna spp.), violet (Viola odorata), species of the genus *Vitis* (Vitis spp.), maize (Zea mays), *Zizania palustris*, species of the genus *Ziziphus* This includes, but is not limited to, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrots, cauliflower, celery, kale, flax, kale, lentils, rapeseed, okra, onions, potatoes, rice, soybeans, strawberries, sugar beets, sugarcane, sunflowers, tomatoes, squash, tea, and algae, along with others. According to a specific embodiment of the invention, the plants are crop plants. Examples of crop plants particularly include Arabidopsis thaliana, rapeseed, rice, wheat, peppers, tomatoes, eggplants, Chinese cabbage, cabbage, mustard greens, sesame, cotton, sunflowers, watermelons, lettuce, sugar beets, potatoes, sweet potatoes, barley, sorghum, millet, corn, soybeans, squash, oats, buckwheat, flax, or lentils.

[0262] As used in this article, "male-sterile line" or "male-sterile plant" refers to a plant that does not produce viable male gametes or is otherwise capable of fertilization and is female-fertile.

[0263] A "sterile line" refers to a specific plant strain that possesses stable hereditary male sterility, with pollen that is sterile or nonfunctional, preventing self-pollination and seed production. However, its pistil develops normally and it can accept foreign pollen to produce seeds. This strain serves as the basic maternal line for hybrid seed production.

[0264] A "maintainer line" refers to a specific plant line that has a genetic background highly similar to a particular sterile line, but whose male reproductive organs are normally developed, it can self-pollinate and produce fruit, and when used as a male parent to cross with the sterile line, it can stably transmit male sterility traits to its offspring. Maintainer lines are used to propagate and maintain the population of the sterile line.

[0265] The "reproductive growth period" of plants begins with the differentiation of flower buds or young spikelets and continues until the fruits and seeds mature. This period is mainly characterized by the formation of reproductive organs such as flowers, fruits, and seeds.

[0266] As used herein, the terms “heterozygous plant,” “heterozygous mutant,” or “heterozygote” include plants with two distinct ALS allele mutations and offspring with the same genotype produced in different ways. A “heterozygous plant,” “heterozygous mutant,” or “heterozygote” as used herein is a plant with two distinct ALS allele mutations, for example, one allele mutation changing proline to serine or leucine at position 197 of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1, and the other changing serine to asparagine or isoleucine at position 653 of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1.

[0267] "Effective dosage" refers to the effective application amount selected at a specific time to achieve a specific technical objective, based on the specific application scenario, target, and expected effect. This dosage is not a fixed value and needs to be determined through routine screening based on actual conditions. Its specific value will be affected by a variety of factors, such as the target (e.g., different crop categories), the nature of the drug or active ingredient, the application environment, and the target control effect, and therefore there will be corresponding differences. For example, as described herein, the “effective amount” for making the hybrid ALS-P197S / S653N Arabidopsis thaliana resistant but fertile (male-killing) is the application of bensulfuron-methyl (e.g., between 1 g ai / ha and 500 g ai / ha, preferably between 1 g ai / ha and 200 g ai / ha, particularly between 1 g ai / ha and 50 g ai / ha) + methyl nicotinic acid (e.g., between 1 g ai / ha and 2400 g ai / ha, preferably between 5 g ai / ha and 1200 g ai / ha, more preferably between 5 g ai / ha and 600 g ai / ha, particularly between 10 g ai / ha and 100 g ai / ha) herbicide during the reproductive growth stage (budding stage). Attached Figure Description

[0268] Figure 1. Growth status of wild-type (WT), homozygous ALS-P197S, homozygous ALS-S653N, homozygous ALS-W574L, and heterozygous ALS-P197S / S653N Arabidopsis thaliana on MS, bensulfuron-methyl, mefenoxamic acid, and bensulfuron-methyl + mefenoxamic acid media; the concentrations were 2.5 mg / L bensulfuron-methyl, 0.24 mg / L mefenoxamic acid, and 2.5 mg / L bensulfuron-methyl + 0.24 mg / L mefenoxamic acid, respectively.

[0269] Figure 2. Pollen viability analysis of heterozygous ALS-P197S / S653N Arabidopsis thaliana plants; the top left panel shows a treatment dose of 0 g ai / ha, the top right panel shows a treatment dose of 1.875 g ai / ha bensulfuron-methyl, the bottom left panel shows a treatment dose of 9 g ai / ha mefenoxamic acid, and the bottom right panel shows a treatment dose of 1.875 g ai / ha bensulfuron-methyl + 9 g ai / ha mefenoxamic acid.

[0270] Figure 3. Pollen viability analysis of homozygous ALS-W574L Arabidopsis thaliana plants; the top left panel shows a treatment dose of 0 g ai / ha, the top right panel shows a treatment dose of 1.875 g ai / ha bensulfuron-methyl, the bottom left panel shows a treatment dose of 9 g ai / ha mefenoxamic acid, and the bottom right panel shows a treatment dose of 1.875 g ai / ha bensulfuron-methyl + 9 g ai / ha mefenoxamic acid.

[0271] Figure 4. Pod condition of heterozygous P197S / S653N (double-drug treatment) × homozygous W574L (double-drug treatment).

[0272] Figure 5. Fruit set of heterozygous P197S / S653N (double-drug treatment) × homozygous W574L (double-drug treatment).

[0273] Figure 6. Growth status of Arabidopsis thaliana in wild type (WT), homozygous ALS-W574L, heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous ALS-W574L (double-drug treatment), heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous ALS-W574L (untreated), and heterozygous ALS-P197S / S653N (double-drug treatment) × WT (untreated) on MS, bensulfuron-methyl, metronidazole, and bensulfuron-methyl + metronidazole media; the dosages were 2.5 mg / L bensulfuron-methyl, 0.24 mg / L metronidazole, and 2.5 mg / L bensulfuron-methyl + 0.24 mg / L metronidazole, respectively.

[0274] Figure 7. Growth status of F1 progeny of heterozygous ALS-P197S / S653N (dual-drug treatment) × homozygous W574L (dual-drug treatment) 14 days after treatment with bensulfuron-methyl (left), metronidazole (middle), and bensulfuron-methyl + metronidazole (right). The bensulfuron dosage from left to right is 0 g ai / ha, 0.234 g ai / ha, 0.469 g ai / ha, 0.938 g ai / ha, 1.875 g ai / ha, 3.75 g ai / ha, 7.5 g ai / ha, and 15 g ai / ha; the metronidazole dosage from left to right is 0 g ai / ha, 1.125 g ai / ha, 2.25 g ai / ha, 4.5 g ai / ha, 9 g ai / ha, 18 g ai / ha, 36 g ai / ha, and 72 g ai / ha. The dosages of bensulfuron-methyl + metronidazole nicotinic acid from left to right are as follows: 0 g ai / ha, 0.234 g ai / ha bensulfuron-methyl + 1.125 g ai / ha metronidazole nicotinic acid, 0.469 g ai / ha bensulfuron-methyl + 2.25 g ai / ha metronidazole nicotinic acid, 0.938 g ai / ha bensulfuron-methyl + 4.5 g ai / ha metronidazole nicotinic acid, 1.875 g ai / ha bensulfuron-methyl + 9 g ai / ha metronidazole nicotinic acid, 3.75 g ai / ha bensulfuron-methyl + 18 g ai / ha metronidazole nicotinic acid, 7.5 g ai / ha bensulfuron-methyl + 36 g ai / ha metronidazole nicotinic acid, and 15 g ai / ha bensulfuron-methyl + 72 g ai / ha metronidazole nicotinic acid.

[0275] Figure 8. The effect of nicosulfuron + methoxyfenozide on the male-killing effect of heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana; the upper figure shows the pollen activity of heterozygous ALS-P197S / S653N Arabidopsis thaliana treated with 0.9375 g ai / ha nicosulfuron + 3.75 g ai / ha methoxyfenozide, and the lower figure shows the pollen activity of homozygous ALS-W574L Arabidopsis thaliana treated with 0.9375 g ai / ha nicosulfuron + 3.75 g ai / ha methoxyfenozide.

[0276] Figure 9. Fruit set of heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana after treatment with nicosulfuron + methoxyfenozide. The top left image shows the fruit set of homozygous ALS-W574L Arabidopsis thaliana under the blank treatment, and the top right image shows the fruit set of homozygous ALS-W574L Arabidopsis thaliana under the treatment with 0.9375 g ai / mu nicosulfuron + 3.75 g ai / mu methoxyfenozide. The bottom left image shows the fruit set of heterozygous ALS-P197S / S653N Arabidopsis thaliana under the blank treatment, and the bottom right image shows the fruit set of heterozygous ALS-P197S / S653N Arabidopsis thaliana under the treatment with 0.9375 g ai / ha nicosulfuron + 3.75 g ai / ha methoxyfenozide.

[0277] Figure 10. The effect of mesosulfuron-methyl + imidazoline acid on the male-killing effect of heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana; the upper figure shows the pollen activity of heterozygous ALS-P197S / S653N Arabidopsis thaliana treated with 0.9375 g ai / ha mesosulfuron-methyl + 3.75 g ai / ha imidazoline acid, and the lower figure shows the pollen activity of homozygous ALS-W574L Arabidopsis thaliana treated with 0.9375 g ai / ha mesosulfuron-methyl + 3.75 g ai / ha imidazoline acid.

[0278] Figure 11. Fruit set of heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana after treatment with mesosulfuron-methyl and imidazoline acid; the upper left image shows the fruit set of homozygous ALS-W574L Arabidopsis thaliana under the blank treatment, and the upper right image shows the fruit set of homozygous ALS-W574L Arabidopsis thaliana under the treatment with 0.9375 g ai / ha mesosulfuron-methyl + 3.75 g ai / ha imidazoline acid; the lower left image shows the fruit set of heterozygous ALS-P197S / S653N Arabidopsis thaliana under the blank treatment, and the lower right image shows the fruit set of heterozygous ALS-P197S / S653N Arabidopsis thaliana under the treatment with 0.9375 g ai / ha mesosulfuron-methyl + 3.75 g ai / ha imidazoline acid.

[0279] Figure 12. The effect of chlorpyrifos + methoxyfenozide on the male-killing effect of heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana; the upper figure shows the pollen activity of heterozygous ALS-P197S / S653N Arabidopsis thaliana treated with 0.234 g ai / ha chlorpyrifos + 1.875 g ai / ha methoxyfenozide, and the lower figure shows the pollen activity of homozygous ALS-W574L Arabidopsis thaliana treated with 0.234 g ai / ha chlorpyrifos + 1.875 g ai / ha methoxyfenozide.

[0280] Figure 13. Fruit set of heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana after treatment with clopyralid + methoxyfenozide. The upper left image shows the fruit set of homozygous ALS-W574L Arabidopsis thaliana under the blank treatment, and the upper right image shows the fruit set of homozygous ALS-W574L Arabidopsis thaliana under the treatment with 0.234 g ai / ha clopyralid + 1.875 g ai / ha methoxyfenozide. The lower left image shows the fruit set of heterozygous ALS-P197S / S653N Arabidopsis thaliana under the blank treatment, and the lower right image shows the fruit set of heterozygous ALS-P197S / S653N Arabidopsis thaliana under the treatment with 0.234 g ai / ha clopyralid + 1.875 g ai / ha methoxyfenozide.

[0281] Figure 14. Effect of two applications of 0.6 g ai / ha bensulfuron-methyl + 2.88 g ai / ha mepiquat nicotinic acid on male killing in ALS-P197S / S653N hybrid rapeseed.

[0282] Figure 15. Pollen activity detection of heterozygous OsALS-P171S / S627N and homozygous OsALS-W548L rice after spraying with 120g ai / ha bensulfuron-methyl + 300g ai / ha nicotinic acid during the second booting stage.

[0283] Figure 16. Schematic diagram of the selection and breeding of sterile lines and maintainer lines.

[0284] Figure 17. Schematic diagram of hybrid breeding and restoration of fertility of sterile lines.

[0285] Figure 18. Schematic diagram of screening heterozygotes (sterile lines or maintainer lines). Detailed Implementation

[0286] Various features and implementations of this disclosure are exemplified in the following representative embodiments, which are intended to be exemplary and not to be limiting.

[0287] Example 1: Analysis of conserved amino acid sites in ALS genes in different plants

[0288] By performing conservation analysis on the amino acid sequences of acetolactate synthase (ALS) from different plants, highly conserved sites were obtained.

[0289] Mutations in the Arabidopsis AtALS gene at sites 121, 122, 196, 197, 199, 200, 207, 205, 256, 376, 377, 570, 571, 574, 653, and 654 produce resistance to acetolactate synthase inhibitors (ALS inhibitors) herbicides. Bioinformatics analysis revealed that the relevant ALS amino acid sequences in other crops showed higher conservation at the sites corresponding to the Arabidopsis ALS amino acid sequence (SEQ ID NO: 1) compared to Arabidopsis. Some information is shown in Table 1.

[0290] Table 1. Analysis of conserved amino acid sites in ALS genes in different plants. Note: / represents missing.

[0291] Example 2: Arabidopsis thaliana mutant plants and seed production methods

[0292] 1. Resistance profile analysis of wild-type and mutant Arabidopsis thaliana to ALS inhibitor herbicides

[0293] Wild-type and ALS mutant Arabidopsis thaliana plants at the budding stage were treated with different types of ALS inhibitor herbicides at varying concentrations. Resistance levels were investigated and analyzed after 30 days. Partial resistance profiles are shown in Table 2. The mutations mentioned in the table correspond to the ALS amino acid sequence SEQ ID NO: 1 in Arabidopsis thaliana.

[0294] Table 2 Resistance profiles of different ALS mutants to different types of ALS inhibitor herbicides Note: √ indicates resistance, × indicates no resistance, / indicates no data.

[0295] The following detailed descriptions use ALS-P197S, ALS-S653N, and ALS-W574L as examples; other ALS mutants will not be discussed further.

[0296] Benzsulfuron-methyl and mefenoxamic acid were selected for plate tests of resistance in wild-type Arabidopsis thaliana and its mutants. The specific steps are as follows: Wild-type, homozygous ALS-P197S, homozygous ALS-S653N, homozygous ALS-W574L, and heterozygous ALS-P197S / S653N Arabidopsis thaliana seeds were obtained and spread on the same MS medium containing 2.5 mg / L benzsulfuron-methyl and 0.24 mg / L mefenoxamic acid or 2.5 mg / L benzsulfuron-methyl + 0.24 mg / L mefenoxamic acid. The plate resistance differences were analyzed. As shown in Figure 1, it was verified that wild-type Arabidopsis thaliana showed no resistance to bensulfuron-methyl and / or methyl nicotinic acid; homozygous ALS-P197S Arabidopsis thaliana was resistant to bensulfuron-methyl but not to methyl nicotinic acid; homozygous ALS-S653N Arabidopsis thaliana was resistant to methyl nicotinic acid but not to bensulfuron-methyl; heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana were resistant to bensulfuron-methyl and / or methyl nicotinic acid, and could grow and bear fruit normally when transplanted into the soil. This indicates that when treated with effective amounts of methyl nicotinic acid and bensulfuron-methyl during non-reproductive growth periods (such as the seed germination period of vegetative growth), the ALS-P197S / S653N heterozygous plants showed resistance and fertility.

[0297] Spray wild-type, homozygous ALS-P197S, homozygous ALS-S653N, homozygous ALS-W574L, and heterozygous ALS-P197S / S653N Arabidopsis thaliana at the budding stage with bensulfuron-methyl, mesosulfuron-methyl, nicosulfuron-methyl, bensulfuron-methyl, pyrimisulfuron-methyl, sulfadiazine, chlorpyrifos, fluazolidone, thiamethoxam, penoxsulam, diflubenzuron, pyrimisulfuron, and chlorpyrifos. Twenty-nine ALS herbicides, including chlorpyrifos, dichlorvos, pyrazosulfuron, sulfadiazine, fensulfuron, pyrimisulfuron, pyrimisulfuron, thifensulfuron, flufensulfuron, bispyribac-sodium, pyrazosulfuron, pyrimisulfuron-methyl, imazalil, metribuzin, methoxyfenozide, and imazalil-quinolinic acid, were investigated for herbicide phytotoxicity after 19 days. Among them, the homozygous ALS-P197S herbicide showed the best phytotoxicity in Arabidopsis thaliana. Sulfonylurea herbicides such as bensulfuron-methyl, mesosulfuron-methyl, nicosulfuron-methyl, bensulfuron-methyl, sulfadiazine, chlorpyrifos-methyl, fluazolidone, thiamethoxam, sulfadiazine, ethersulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, thifensulfuron and other sulfonylurea herbicides, and triazolopyrimidine ALS inhibitor herbicides such as pyrazosulfuron-methyl, chlorpyrifos-methyl, cyhalothrin, pyrazosulfuron-methyl, fluazinam, and bispyribac-methyl have good efficacy. Resistance was observed, but the homozygous ALS-S653N Arabidopsis thaliana showed no resistance to the aforementioned herbicides; the homozygous ALS-S653N Arabidopsis thaliana exhibited strong resistance to four imidazolinone herbicides: methyl methoxyfenozide, imidacloprid, methoxyfenozide, and imidazoline, but the homozygous ALS-P197S Arabidopsis thaliana showed no resistance to them; the wild type was not resistant to any herbicides; the homozygous ALS-W574L was resistant to all herbicides.

[0298] 2. Analysis of resistance and fertility of wild-type and mutant Arabidopsis thaliana

[0299] Wild-type, homozygous ALS-P197S, homozygous ALS-W574L, homozygous ALS-S653N, and heterozygous ALS-P197S / S653N Arabidopsis thaliana at the budding stage were treated with stems and leaves at different concentrations of bensulfuron-methyl, imidazolic acid, or combinations thereof. Ten concentration gradients were set for bensulfuron-methyl: 0 g ai / ha, 1.875 g ai / ha, 3.75 g ai / ha, 7.5 g ai / ha, 15 g ai / ha, 30 g ai / ha, 60 g ai / ha, 120 g ai / ha, 240 g ai / ha, and 480 g ai / ha. Ten concentration gradients were set for imidazolic acid: 0 g ai / ha, 9 g ai / ha, 18 g ai / ha, 36 g ai / ha, 72 g ai / ha, 144 g ai / ha, and 288 g ai / ha. Ten concentration gradients were established: ai / ha, 576g ai / ha, 1152g ai / ha, and 2304g ai / ha. The dual-drug combination concentrations were any combination of these gradients. After 30 days, resistance levels, fruit set, and pollen viability were investigated and analyzed. Resistance and fertility analyses at representative drug concentrations are shown in Tables 3-4, and representative pollen viability analyses are shown in Figures 2-3. Pollen from ALS-P197S / S653N heterozygous plants treated with both bensulfuron-methyl and mefenoxamic acid showed no viability; pollen from ALS-P197S / S653N heterozygous plants treated with either bensulfuron-methyl or mefenoxamic acid alone showed viability. Pollen from ALS-W574L homozygous plants treated with bensulfuron-methyl and / or mefenoxamic acid showed viability and normal fruit set.

[0300] Table 3 Resistance analysis under single treatments with bensulfuron-methyl and mefenoxamic acid

[0301] Table 4. Analysis of resistance and fertility under the combined treatment of bensulfuron-methyl and mefenoxamic acid.

[0302] Artificial pollination was performed on hybrid ALS-P197S / S653N sterile plants (maternal parent) treated with bensulfuron-methyl and metronidazole as the male parent, using pollen from wild-type Arabidopsis thaliana as the male parent or pollen from homozygous ALS-W574L Arabidopsis thaliana treated with bensulfuron-methyl and metronidazole as the male parent. The hybridization results with homozygous ALS-W574L as the male parent are shown in Figures 4 and 5, indicating fertility and normal fruit set.

[0303] In summary, when benzsulfuron-methyl and mefenoxamic acid were applied during the budding stage of reproductive growth, the ALS-P197S / S653N heterozygous plants, although exhibiting normal resistance, showed self-sterility, and pollen viability testing showed male sterility. However, artificial pollination with pollen from wild-type or homozygous ALS-W574L plants resulted in fertility.

[0304] 2. Genetic analysis of Arabidopsis ALS-P197S / S653N heterozygous plants

[0305] F2 generation seeds of fertile ALS-P197S / S653N self-pollination were sterilized and sown on MS medium. The medium was then cultured in a light incubator (16 hours light, 8 hours dark). After one week, the seedlings were transplanted into soil. After two weeks of growth, 40 F2 generation plants were randomly selected for DNA extraction.

[0306] PCR amplification of the P197 and S653 loci was performed, and first-generation sequencing was used to detect the P197 and S653 loci. The detection results were ALS-P197S homozygous: ALS-P197S / S653N heterozygous: ALS-S653N homozygous = 7:20:13, which basically conforms to the theoretical prediction value of Mendel's law of segregation, ALS-P197S / P197S homozygous: ALS-P197S / S653N heterozygous: ALS-S653N / S653N homozygous = 1:2:1.

[0307] 3. Analysis of resistance differences and fertility in the offspring of hybridization between Arabidopsis thaliana ALS-P197S / S653N heterozygous plants and ALS-W574L homozygous plants.

[0308] Seeds of wild-type, homozygous ALS-W574L, heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous ALS-W574L (double-drug treatment), heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous ALS-W574L (untreated), and heterozygous ALS-P197S / S653N (double-drug treatment) × wild-type F1 progeny were sown on MS, 2.5 mg / L bensulfuron-methyl, 0.24 mg / L methylimidazole, and 2.5 mg / L bensulfuron-methyl + 0.24 mg / L methylimidazole, respectively, and incubated at 22℃. Results were observed after 8 days. The double-drug treatment refers to spraying with 3.75 g ai / ha bensulfuron-methyl + 18 g ai / ha methylimidazole at the Arabidopsis thaliana budding stage.

[0309] The results showed that the seeds of the F1 progeny of heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous W574L (double-drug treatment) and the F1 progeny of heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous W574L (untreated) were resistant to both bensulfuron-methyl and mefenoxamic acid; the seeds of the F1 progeny of heterozygous ALS-P197S / S653N (double-drug treatment) × wild type (WT) were resistant to either bensulfuron-methyl or mefenoxamic acid alone, but not both simultaneously (Figure 6).

[0310] The F1 progeny of heterozygous ALS-P197S / S653N (double-drug treatment) × homozygous W574L (double-drug treatment) showed normal plant growth (Figure 7) and normal pollen development after being sprayed with 3.75 g ai / ha bensulfuron-methyl, 18 g ai / ha imidacloprid, or both simultaneously. The length of the pods and the number of seeds per pod were not different from those of the parent plant.

[0311] 4. Analysis of the male-killing effect and seed setting rate of different ALS herbicide combinations on heterozygous ALS-P197S / S653N and homozygous ALS-W574L Arabidopsis thaliana.

[0312] Five herbicides resistant to homozygous ALS-P197S Arabidopsis thaliana (including bensulfuron-methyl, nicosulfuron-methyl, sulfadiazine, mesosulfuron-methyl, and chlorpyrifos) and four herbicides resistant to homozygous ALS-S653N Arabidopsis thaliana (including imidacloprid, imidacloprid, methoxyfenozide, and imidacloprid) were selected. Nineteen other different combinations of the above nine herbicides were used to test the male-killing effects on heterozygous ALS-P197S / S653N Arabidopsis thaliana.

[0313] During the budding stage, wild-type, homozygous ALS-P197S, homozygous ALS-S653N, homozygous ALS-W574L, and heterozygous ALS-P197S / S653N Arabidopsis thaliana were sprayed with bensulfuron-methyl + imidacloprid, bensulfuron-methyl + methoxyimidacloprid, bensulfuron-methyl + imidacloprid, nicosulfuron-methyl + imidacloprid, nicosulfuron-methyl + methoxyimidacloprid, nicosulfuron-methyl + imidacloprid, sulfonyl ... The following combinations of pesticides were used: pyrisulfuron + imazalil, sulfonylsulfuron + methoxyimazalil, sulfonylsulfuron + imazaloline acid, mesosulfuron + imazaloline acid, mesosulfuron + imazalil, mesosulfuron + methoxyimazalil, mesosulfuron + imazaloline acid, chlorpyrisulfuron + imazaloline acid, chlorpyrisulfuron + imazalil, chlorpyrisulfuron + methoxyimazalil, or chlorpyrisulfuron + imazaloline acid. After 14 days, pollen activity and plant resistance were observed, and the seed setting rate was investigated in the later stages.

[0314] The results showed that the drug combination had excellent male-killing effect on heterozygous ALS-P197S / S653N Arabidopsis thaliana without killing pollen of homozygous ALS-W574L Arabidopsis thaliana. It can be used for male-sterile line selection and seed production. Representative results are shown in Figures 8-13. Under the treatment of three groups of drugs, namely 0.9375g ai / ha nicosulfuron + 3.75g ai / ha methoxyfenozide, 0.9375g ai / ha mesosulfuron + 3.75g ai / ha imidazoline, and 0.234g ai / ha clopyralid + 1.875g ai / ha methoxyfenozide, the heterozygous ALS-P197S / S653N Arabidopsis thaliana grew normally, but the pollen was inactive and did not produce seeds. The homozygous ALS-W574L Arabidopsis thaliana grew normally, had normal pollen activity, and produced seeds normally after being treated with two drugs.

[0315] In addition, using ALS inhibitors such as bensulfuron-methyl, fluazolidone, thiamethoxam, acesulfame, ethersulfuron, pyrimisulfuron, pyrimisulfuron, pyrimisulfuron, thifensulfuron, pyrimisulfuron, pyrimisulfuron, thifensulfuron, chlorpyrifos, chlorpyrifos, pyrimisulfuron, fluazinam, and bispyribac-sodium, which are resistant to ALS-P197S Arabidopsis thaliana but not to ALS-S653N Arabidopsis thaliana, to replace bensulfuron-methyl, or using ALS inhibitors such as imazalil, methoxyimazalil, and imazalil-quinolinic acid, which are resistant to ALS-S653N Arabidopsis thaliana but not to ALS-P197S Arabidopsis thaliana, to perform dual-drug treatment, can achieve the same technical effect as bensulfuron-methyl + imazalil.

[0316] 5. Screening of ALS-P197S / S653N heterozygous plants

[0317] Taking bensulfuron-methyl and mefenoxamic acid as examples, at the 5-leaf stage of F2 progeny Arabidopsis thaliana of wild type, homozygous ALS-P197S, homozygous ALS-S653N and heterozygous ALS-P197S / S653N, 15g ai / ha bensulfuron-methyl and 36g ai / ha mefenoxamic acid were sprayed simultaneously, and the resistance ratio of plants was observed and counted after 14 days.

[0318] The results showed that wild-type, homozygous ALS-P197S, and homozygous ALS-S653N Arabidopsis thaliana all died after being sprayed with 15 g ai / ha bensulfuron-methyl and 36 g ai / ha mefenazate. 51.6% of the F2 progeny of heterozygous ALS-P197S / S653N Arabidopsis thaliana survived and grew normally, indicating that ALS-P197S / S653N heterozygous plants can be efficiently screened from mixed plants.

[0319] Alternatively, replacing W574L with ALS-W574M can also efficiently screen for ALS-P197S / S653N heterozygous plants in mixed plants.

[0320] In summary, it has been verified that when treated with effective amounts of methyl methacrylate and bensulfuron-methyl during non-reproductive growth stages (such as the seed germination stage of vegetative growth), the ALS-P197S / S653N heterozygous plants exhibited resistance and normal fertility. When sprayed with effective amounts of bensulfuron-methyl + methyl methacrylate during the reproductive growth stage (such as the budding stage), the ALS-P197S / S653N heterozygous plants exhibited resistance and male sterility (see Figure 16). Furthermore, fertility was restored after hybridization with pollen from wild-type plants or fertile plants resistant to both drugs (such as the homozygous ALS-W574L plant) (see Figure 17).

[0321] Therefore, by using the ALS-P197S / S653N heterozygous male-sterile line (achieving male sterility through spraying with two drugs) as the female parent and the ALS-W574L homozygous plant (or other plants resistant to the two drugs) as the male parent for hybridization, hybrid varieties resistant to the two drugs can be produced in large quantities, as shown in Figure 17. By self-pollinating fertile ALS-P197S / S653N heterozygous plants or by crossing ALS-P197S homozygous plants with ALS-S653N homozygous plants, and then treating the self-pollinated and hybrid offspring with an effective amount of the two drugs at a specific time (such as before the reproductive growth period), fertile ALS-P197S / S653N heterozygous maintainer lines can be selected. These heterozygous plants, when sprayed with an effective amount of the two drugs during the reproductive growth period, can produce a large number of ALS-P197S / S653N male-sterile lines, as shown in Figure 18.

[0322] This seed production system is highly operable, and the ALS-P197S / S653N hybrid plants can be propagated in large quantities. They can be used as maintainer lines or male sterile lines as needed, which solves the problem of low efficiency in existing breeding technologies and allows for the low-cost acquisition of a large number of hybrids.

[0323] In summary, the above experimental results are sufficient to verify that, for heterozygous plants with two different ALS allele mutations (e.g., the first and second ALS mutations in Table 2) as described in this application, simultaneous treatment with both herbicides A and B during the reproductive growth period, or simultaneous treatment with both herbicides A and B before the reproductive growth period with the efficacy lasting into the reproductive growth period, can yield resistant male-sterile lines; simultaneous treatment with both herbicides A and B before the reproductive growth period without the efficacy lasting into the reproductive growth period can yield resistant maintainer lines; and hybridization can be performed by using the male-sterile plants as the female parent and plants with normal pollen fertility as the male parent (e.g., homozygous plants with the third ALS mutation in Table 2). Herbicides A and B can be screened according to the following principles: homozygous plants with the first type of ALS gene mutation are resistant to herbicide A but not to herbicide B; homozygous plants with the second type of ALS gene mutation are resistant to herbicide B but not to herbicide A. Therefore, this invention can not only achieve the breeding of male-sterile lines and maintainer lines, but also realize a closed-loop hybrid seed production system, producing a large number of hybrids resistant to both herbicides.

[0324] Example 3: Rapeseed mutant plants and seed production methods

[0325] 1. Resistance spectrum and fertility analysis of rapeseed mutant plants to ALS-type herbicides

[0326] Homozygous C-genome ALS-P197S (westar) plants were crossed with homozygous C-genome ALS-S653N plants (Zhongshuang 11) to obtain ALS-P197S / S653N heterozygous plants. After harvesting, these plants were simultaneously sown with wild-type, homozygous ALS-P197S, homozygous ALS-S653N, and homozygous ALS-W574L seedlings. When the materials reached one pair of true leaves, they were treated with ALS-based herbicides. Herbicide tolerance was assessed 28 days after treatment. The results showed that homozygous ALS-P197S oil... The rapeseed plants exhibited resistance to herbicides such as bensulfuron-methyl, mesosulfuron-methyl, nicosulfuron-methyl, bensulfuron-methyl, chlorpyrifos-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrazosulfuron-methyl, and pyrimethanil-methyl; however, the homozygous ALS-S653N rapeseed plant showed no resistance to these herbicides. The homozygous ALS-S653N rapeseed plant also showed resistance to nicotinic acid, imidacloprid, and methoxyfenozide, but the homozygous ALS-P197S rapeseed plant showed no resistance. The wild-type rapeseed was not resistant to any ALS inhibitor herbicides; the homozygous ALS-W574L rapeseed was resistant to all ALS inhibitor herbicides. A combination of bensulfuron-methyl and nicotinic acid was selected for further testing.

[0327] Wild-type, homozygous ALS-P197S, homozygous ALS-S653N, homozygous ALS-W574L, and heterozygous ALS-P197S / S653N plants were sown and divided into two groups for treatment. In the first group, when the plants reached two pairs of true leaves, they were treated with 15 g ai / ha bensulfuron-methyl + 72 g ai / ha imidacloprid. Results showed that wild-type, homozygous ALS-P197S, and homozygous ALS-S653N plants died, while homozygous ALS-W574L survived normally and exhibited normal fertility. Although the heterozygous ALS-P197S / S653N plants experienced phytotoxicity, they recovered later and exhibited normal fertility. In the second group, when the plants were in the budding stage, they were sprayed with 0.6g ai / ha bensulfuron-methyl + 2.88g ai / ha mefenazate. The results showed that the growth of wild-type, homozygous ALS-P197S and homozygous ALS-S653N plants was inhibited and they gradually died. Multiple applications were possible. Homozygous ALS-W574L and heterozygous ALS-P197S / S653N plants survived. After the initial flowering of the heterozygous ALS-P197S / S653N plants, pollen in the anthers was examined. As shown in Figure 14, no plants with pollen or micro-powder were found. Furthermore, after the application, ALS-P197S / S653N plants failed to produce seeds through self-pollination. This indicates that ALS-P197S / S653N heterozygous plants exhibit resistance and normal fertility when sprayed with bensulfuron-methyl + mefenoxamic acid during the non-reproductive growth period; and that treatment with bensulfuron-methyl + mefenoxamic acid during the budding stage can achieve the purpose of chemically killing males.

[0328] The fertility of ALS-P197S / S653N heterozygous plants treated simultaneously with bensulfuron-methyl and mefenoxamic acid was analyzed.

[0329] (1) Pollen activity detection

[0330] ALS-P197S / S653N hybrid plants were sown and divided into two groups. Group 1 was treated with 0.6 g ai / ha bensulfuron-methyl + 2.88 g ai / ha methomyl nicotinic acid herbicide at the budding stage. Group 2, the same batch of control plants, was sprayed with an equal amount of water. After the rapeseed began to flower, the pollen in the anthers was examined. No pollen or micropollen was found in Group 1. Pollen was found in Group 2. A small amount of pollen was taken for pollen viability testing using the acetic acid-carmine staining method. The results showed that the pollen was viable.

[0331] (2) Condition of the fruit

[0332] ALS-P197S / S653N heterozygous plants were sown and divided into three groups. Groups 1 and 2 were treated with a herbicide of 0.6 g ai / ha bensulfuron-methyl + 2.88 g ai / ha methomyl nicotinic acid at the budding stage. Group 3, the same batch of control plants, was sprayed with an equal amount of water. All plants in Group 1 were individually bagged during flowering to prevent external pollination. Plants in Group 2 served as the female parent for hybridization with homozygous ALS-W574L plants or wild-type plants. During the fruit set period, no effective siliques were found in Group 1, with a silique and fruit set rate of 0%, and a sterility induction rate of 100%. The fruit set of the hybrid plants in Group 2 was good compared to the silique and fruit set rates of the naturally pollinated plants in Group 3, with no significant differences between treatments.

[0333] The results showed that ALS-P197S / S653N heterozygous plants treated with bensulfuron-methyl and mefenoxamic acid during the reproductive growth period (such as the budding stage) were male-sterile, and their fertility could be restored after hybridization with pollen from fertile plants.

[0334] 2. Genetic analysis of rapeseed ALS-P197S / S653N heterozygous plants

[0335] Fertility-producing ALS-P197S / S653N heterozygous plants were self-pollinated, bagged, and the self-pollinated seeds were harvested. After sowing, 300 seedlings were obtained (three parallel experiments were set up). Leaves were collected from these plants, and genomic DNA was extracted as templates for PCR amplification.

[0336] PCR amplification products from different lines were sequenced, and the sequencing results were compared with the ALS gene of wild-type rapeseed (Wester). The results showed that the segregation ratios of ALS-P197S / P197S homozygous: ALS-P197S / S653N heterozygous: ALS-S653N / S653N homozygous were 23:52:25, 28:51:21, and 24:48:28, respectively, basically conforming to the 1:2:1 ratio, which is consistent with Mendelian inheritance laws.

[0337] 3. Screening of rapeseed ALS-P197S / S653N heterozygous plants

[0338] Heterozygous ALS-P197S / S653N plants were selected post-emergence. Homozygous ALS-P197S, homozygous ALS-S653N, and heterozygous ALS-P197S / S653N plants were sown, and treated with 0.942 g ai / ha bensulfuron-methyl + 4.5 g ai / ha imidacloprid at the cotyledon stage. Homozygous ALS-P197S and homozygous ALS-S653N plants died, while heterozygous ALS-P197S / S653N plants grew normally.

[0339] Heterozygous ALS-P197S / S653N plants were selected by seed coating. Seeds from homozygous ALS-P197S, homozygous ALS-S653N, and heterozygous ALS-P197S / S653N plants were coated with a herbicide consisting of 0.03125 g ai / kg bensulfuron-methyl and 0.15 g ai / kg methyl imidacloprid. After sowing, homozygous ALS-P197S and homozygous ALS-S653N seeds showed severe herbicide damage and significantly inhibited growth; heterozygous ALS-P197S / S653N seeds germinated normally, showing no significant difference from the water treatment.

[0340] Heterozygous ALS-P197S / S653N plants were selected by soaking seeds. Seeds from homozygous ALS-P197S, homozygous ALS-S653N, and heterozygous ALS-P197S / S653N plants were soaked in a herbicide solution of 10 mg ai / L bensulfuron-methyl + 15 mg ai / L methomyl nicotinic acid for 24 hours at a herbicide-to-seed ratio of 2:1. After sowing, seeds from homozygous ALS-P197S and homozygous ALS-S653N plants showed severe herbicide damage after emergence. In contrast, seeds from heterozygous ALS-P197S / S653N plants emerged normally without herbicide damage, showing no significant difference from those treated with water.

[0341] The results showed that multiple screening methods could achieve the goal of screening heterozygous plants by soaking seeds.

[0342] In addition, we also used drugs that are resistant to bensulfuron-methyl (ALS-P197S) but not to bensulfuron-methyl (ALS-S653N), such as mesosulfuron-methyl, nicosulfuron-methyl, bensulfuron-methyl, chlorpyrifos, pyrimisulfuron-methyl, pyrimisulfuron-methyl, or pyrimethanil-methyl, to replace bensulfuron-methyl, or drugs that are resistant to bensulfuron-methyl (ALS-S653N) but not to bensulfuron-methyl (ALS-P197S), such as imidacloprid and methoxyfenozide, to replace nicotinic acid in a two-drug treatment, achieving the same technical effect as bensulfuron-methyl + nicotinic acid. In summary, it has been verified that similar effects to Arabidopsis thaliana can be achieved in rapeseed, and a simple and feasible new seed production method can be provided by referring to Arabidopsis thaliana.

[0343] Example 4: Rice mutant plants and seed production methods

[0344] 1. Resistance analysis of rice mutant plants to ALS-type herbicides

[0345] Rice plants with homozygous mutants of ALS protein P171S, W548M, W548L, and S627N were obtained. The corresponding mutation types of P171S, W548M, W548L, and S627N in Arabidopsis thaliana plants are P197S, W574M, W574L, and S653N, respectively. The obtained OsALS-P171S and OsALS-S627N rice mutants were crossed to obtain F1 generation plants. After genotyping, multiple OsALS-P171S / S627N heterozygous rice plants were obtained.

[0346] Wild-type, homozygous OsALS-P171S, homozygous OsALS-W548L, OsALS-W548M, and homozygous OsALS-S627N rice plants were simultaneously sown. When the plants reached the three-leaf stage, ALS-based herbicides were sprayed. Herbicide tolerance was assessed 25 days after treatment. Results showed that homozygous OsALS-P171S rice tolerated benzsulfuron-methyl, mesosulfuron-methyl, pyrimisulfuron-methyl, sulfadiazon-methyl, fluazolidone, thiamethoxam, diflubenzuron, pyrimisulfuron-methyl, and chlorpyrifos. The rice variety exhibited resistance to bensulfuron-methyl, dichlorvos, pyrazosulfuron-methyl, thifensulfuron-methyl, pyrimisulfuron-methyl, and metsulfuron-methyl, but homozygous OsALS-S627N rice showed no resistance to these herbicides. Homozygous OsALS-S627N rice was resistant to methylphenidate and methoxyfenozide, but homozygous OsALS-P171S rice showed no resistance. The wild-type variety was not resistant to any ALS inhibitor herbicides. Homozygous OsALS-W548L and OsALS-W548M were resistant to all ALS inhibitor herbicides. A combination of bensulfuron-methyl and methylphenidate was selected for further testing.

[0347] The results showed that wild-type and homozygous OsALS-S627N rice exhibited inhibited growth and severe dwarfing under treatment with 60 g ai / ha of bensulfuron-methyl. Treatment with 1920 g ai / ha of bensulfuron-methyl resulted in severe phytotoxicity and leaf tip dieback. In contrast, homozygous OsALS-P171S and homozygous OsALS-W548L rice showed no significant phytotoxicity. Wild-type and homozygous OsALS-P171S rice were essentially or completely killed under treatment with 300 g ai / ha of imidacloprid. In contrast, homozygous OsALS-S627N and homozygous OsALS-W548L showed normal growth.

[0348] After testing, fluzosulfonamide, thiazosulfonamide, and bensulfonamide showed consistent effects. Therefore, dual-drug combinations of fluzosulfonamide with metronidazole and thiazosulfonamide with metronidazole can also be used for subsequent androgen extermination tests.

[0349] 2. Genetic analysis of OsALS-P171S / S627N heterozygous rice plants

[0350] Fertility-producing OsALS-P171S / S627N plants were self-crossed, and the OsALS genotype of 1000 self-crossed progeny plants was identified. The results showed that 239 plants had both alleles of P171S, 254 plants had both alleles of S627N, and 507 plants had one allele of P171S and the other of S627N, consistent with Mendel's laws of inheritance.

[0351] When F2 seedlings were treated with bensulfuron-methyl and mefenoxamic acid, neither homozygous P171S nor homozygous S627N individuals survived, while OsALS-P171S / S627N heterozygous plants grew normally and produced fertile offspring at a ratio of 1:2:1.

[0352] 3. Analysis of fertility and seed setting rate of rice OsALS-P171S / S627N heterozygous plants and OsALS-W548L homozygous plants after spraying with bensulfuron-methyl and mefenoxamic acid during the booting stage.

[0353] Rice materials OsALS-P171S / S627N and OsALS-W548L were sown and transplanted in plots of 36 seedlings each. At the second booting stage, bensulfuron-methyl and methyl nicotinic acid were applied in gradients at dosages of 30 g ai / ha bensulfuron-methyl + 75 g ai / ha methyl nicotinic acid, 60 g ai / ha bensulfuron-methyl + 150 g ai / ha methyl nicotinic acid, and 120 g ai / ha bensulfuron-methyl + 300 g ai / ha methyl nicotinic acid. Water was sprayed simultaneously as a control. Each treatment was replicated four times. Pollen was collected during the flowering period and its activity was observed by staining with potassium iodide, as shown in Figure 15. Under the treatment of 120g ai / ha bensulfuron-methyl + 300g ai / ha imidazonic acid, the pollen of heterozygous OsALS-P171S / S627N rice lost its activity, while the pollen of homozygous OsALS-W548L rice was almost entirely active.

[0354] Pollen from heterozygous OsALS-P171S / S627N rice plants treated with 120g ai / ha bensulfuron-methyl + 300g ai / ha imidacloprid was used as the male parent, and wild-type or homozygous OsALS-W548L rice plants were used as the female parent. Hybridization experiments were conducted, and the seed yield of the hybrid plants was investigated. The results showed that none of the hybrid plants in this combination could yield seeds. Pollen from wild-type or homozygous OsALS-W548L rice plants was used as the male parent, and OsALS-P171S / S627N heterozygous rice plants simultaneously sprayed with bensulfuron-methyl and imidacloprid during the second booting stage were used as the female parent. Hybridization experiments were conducted, and the seed yield of the hybrid plants was investigated. The results showed that all hybrid plants in this combination could yield seeds. Genotyping of the harvested seeds revealed that all were single plants carrying either OsALS-P171S or OsALS-S627N. The results showed that the pollen of the hybrid OsALS-P171S / S627N rice plants, which were sprayed with an effective dose of bensulfuron-methyl and mefenoxamic acid simultaneously during the second booting stage, was sterile, but fertility could be restored after hybridization with pollen from fertile plants.

[0355] In addition, we tested the use of herbicides resistant to OsALS-P171S but not to OsALS-S627N, such as mesosulfuron-methyl, pyrimisulfuron-methyl, sulfadiazine, fluazolidone, thiamethoxam, diflubenzuron, chlorpyrifos, dichlorvos, pyrimisulfuron-methyl, thifensulfuron-methyl, pyrimisulfuron-methyl, and mesosulfuron-methyl, to replace bensulfuron-methyl, or herbicides resistant to OsALS-S627N but not to OsALS-P171S, such as imazalil and imidacloprid, to replace mesosulfuron-methyl in dual-treatment. These methods achieved the same technical effects as bensulfuron-methyl + mesosulfuron-methyl. The results showed similar effects in rice and Arabidopsis thaliana, suggesting the potential for similar hybrid seed production systems.

[0356] Example 5: Wheat mutant plants and their seed production methods

[0357] 1. Obtaining wheat TaALS-P171S / S627N heterozygous plants

[0358] Wheat plants with homozygous mutants of ALS proteins P171S, W548L, and S627N were obtained from the D genome of wheat. The corresponding mutation types of P171S, W548L, and S627N in Arabidopsis plants are homozygous TaALS-P197S, TaALS-W574L, and TaALS-S653N, respectively. The homozygous TaALS-P171S and TaALS-S627N wheat mutants obtained above were crossed to obtain F1 generation plants. After genotyping, multiple heterozygous wheat plants with TaALS-S627N and TaALS-P171S aggregation were obtained and named wheat TaALS-P171S / S627N heterozygous mutant.

[0359] 2. Resistance analysis of wheat mutants to the combination of nicosulfuron and / or mepiquat nicotinic acid

[0360] To examine the differences in resistance to combinations of nicosulfuron and / or metronidazole nicotinic acid among different mutants, effective doses of nicosulfuron and / or metronidazole nicotinic acid were applied to wild-type, homozygous TaALS-P179S, homozygous TaALS-S636N, homozygous TaALS-W557L, and heterozygous TaALS-P179S / S636N mutant plants at the 3-leaf-1-heart stage. The results showed that the growth of wild-type and S636N mutants was inhibited and their plant height was severely stunted under nicosulfuron treatment. In contrast, P179S, W557L and ALS-P179S / S636N heterozygous mutants showed no obvious phytotoxicity. Wild-type and P179S mutants died almost completely or almost completely under nicosulfuron treatment. In contrast, S636N, W557L and ALS-P179S / S636N heterozygous mutants showed no obvious phytotoxicity.

[0361] 3. Genetic analysis of wheat TaALS-P171S / S627N heterozygous plants

[0362] The TaALS genotype was identified in individual plants from the self-crossed F1 generation of 950 TaALS-P171S / S627N. The results showed that 225 plants had both TaALS alleles at P171S, 238 plants had both TaALS alleles at S627N, and 487 plants had one allele at P171S and the other at S627N, consistent with Mendelian inheritance laws.

[0363] When F2 seedlings were treated with a combination of nicosulfuron and metronidazole, neither homozygous P171S nor homozygous S627N individuals survived, while TaALS-P171S / S627N heterozygous plants grew normally and produced fertile offspring at a ratio of 1:2:1.

[0364] 4. Fertility analysis of wheat TaALS-P171S / S627N heterozygous plants

[0365] Pollen from TaALS-P171S / S627N heterozygous wheat plants that were simultaneously sprayed with nicosulfuron and mefenoxam during the young spike stage was used as the male parent, and wild-type wheat plants were used as the female parent. Hybridization experiments were conducted, and the seed yield of the hybrid plants was investigated. The results showed that none of the hybrid plants in this combination could yield seeds. Conversely, pollen from wild-type wheat plants was used as the male parent, and TaALS-P171S / S627N heterozygous wheat plants that were simultaneously sprayed with nicosulfuron and mefenoxam during the young spike stage were used as the female parent. Hybridization experiments were conducted, and the seed yield of the hybrid plants was investigated. The results showed that all hybrid plants in this combination could yield seeds. Genotyping of the harvested seeds revealed that all were single plants carrying either TaALS-P171S or TaALS-S627N. The results showed that pollen from TaALS-P171S / S627N heterozygous wheat plants that were sprayed with nicosulfuron and mefenoxam at the young spike stage was sterile, and fertility could be restored after hybridization with pollen from fertile plants.

[0366] We also tested ALS allele mutations in wheat genomes A and B, achieving similar technical effects to those in genome D. Replacing nicotinic acid with imidacloprid or imidazoline acid in a dual-treatment regimen yielded the same technical effects as nicosulfuron + nicotinic acid. A similar hybrid seed production system could also be developed, referencing Arabidopsis thaliana.

[0367] Example 6: Maize mutant plants and their seed production methods

[0368] 1. Obtain homozygous maize plants of ZmALS108-P165S / W542L / S621N.

[0369] Maize plants with homozygous mutants of ALS protein P165S, W542L, and S621N were obtained by mutagenesis. The corresponding mutation types of P165S, W542L, and S621N in Arabidopsis plants are P197S, W574L, and S653N, respectively. The homozygous maize mutants ZmALS108-P165S and ZmALS108-S621N obtained above were crossed to obtain F1 generation plants. After genotyping, multiple ZmALS108-P165S / S621N heterozygous maize plants were obtained.

[0370] 2. Resistance analysis of maize mutants to the combination of bensulfuron-methyl and / or methyl nicotinic acid

[0371] To examine the differences in resistance to combinations of bensulfuron-methyl and / or nicotinic acid among different mutants, effective doses of bensulfuron-methyl and / or nicotinic acid were applied to wild-type, homozygous ZmALS108-P165S, homozygous ZmALS108-W542L, homozygous ZmALS108-S621N, and heterozygous ZmALS108-P165S / S621N maize plants at the three-leaf-one-heart stage. The results showed that wild-type and homozygous ZmALS108-S621N maize exhibited inhibited growth and severe dwarfing under treatment with 15 g ai / ha bensulfuron-methyl, while homozygous ZmALS108-P165S and homozygous ZmALS108-W542L maize showed no obvious phytotoxicity. Wild-type and homozygous ZmALS108-P165S maize were almost or completely killed under treatment with 150 g ai / ha imidacloprid, while homozygous ZmALS108-S621N and homozygous ZmALS108-W542L maize showed normal growth.

[0372] 3. Genetic analysis of maize ZmALS108-P165S / S621N heterozygous plants

[0373] Genotyping of ZmALS108 was performed on individual plants from 1000 self-crossed progeny of ZmALS108-P165S / S621N. The results showed that 243 plants had both alleles of P165S, 251 plants had both alleles of S621N, and 506 plants had one allele of P165S and the other of S621N, consistent with Mendelian inheritance laws.

[0374] When F2 seedlings were treated with bensulfuron-methyl and mefenoxamic acid, neither homozygous P165S nor homozygous S621N individuals survived, while the ZmALS108-P165S / S621N heterozygous plants grew normally and produced fertile offspring in a ratio of 1:2:1.

[0375] 4. Fertility analysis of maize ZmALS108-P165S / S621N heterozygous plants and ZmALS108-W542L homozygous plants sprayed with bensulfuron-methyl and mefenoxamic acid during the tasseling stage.

[0376] Maize materials ZmALS108-P165S / S621N and ZmALS108-W542L were sown and sprayed with effective doses of bensulfuron-methyl and mefenoxamic acid at the V8 / V10 stage. Water was sprayed simultaneously as a control. Each treatment was repeated in triplicate. Pollen was collected during pollen shedding and stained with potassium iodide to observe pollen viability. Pollen samples from heterozygous ZmALS108-P165S / S621N maize showed no activity, while pollen samples from homozygous ZmALS108-W542L maize showed almost complete activity.

[0377] Pollen from heterozygous maize plants of ZmALS108-P165S / S621N, which were simultaneously sprayed with bensulfuron-methyl and methyl nicotinic acid during the V8 / V10 stage, was used as the male parent. Wild-type or homozygous ZmALS108-W542L maize plants after pesticide application were used as the female parent. A hybridization experiment was conducted to investigate the seed yield of the hybrid plants. The results showed that none of the hybrid plants in this combination yielded seeds.

[0378] Pollen from wild-type or herbicide-treated homozygous ZmALS108-W542L maize plants was used as the male parent, and heterozygous ZmALS108-P165S / S621N maize plants simultaneously sprayed with bensulfuron-methyl and methyl nicotinic acid at the V8 / V10 stage were used as the female parent. A hybridization experiment was conducted, and the seed yield of the hybrid plants was investigated. The results showed that all hybrid plants treated with this herbicide combination could produce seeds. Genotyping of the harvested seeds revealed that all were single plants carrying either ZmALS108-P165S or ZmALS108-S621N. The results indicate that the pollen from ZmALS108-P165S / S621N heterozygous maize plants simultaneously sprayed with bensulfuron-methyl and methyl nicotinic acid at the V8 / V10 stage is sterile, and fertility can be restored after hybridization with pollen from fertile plants.

[0379] We also tested the use of pyrimisulfuron, metsulfuron-methyl, pyrimethanil, mesosulfuron-methyl, fluoxetine, chlorsulfuron, and pyrazosulfuron to replace bensulfuron-methyl, or the use of methoxyfenozide to replace nicotinic acid, in dual-drug treatments, achieving the same technical effects as bensulfuron-methyl + nicotinic acid. Based on the above results, a similar hybrid seed production system can be developed, referencing Arabidopsis thaliana.

[0380] Example 7: Pepper mutant plants and their seed production methods

[0381] 1. Obtain the pepper ALS-P179S / W557L / S636N mutant.

[0382] Pepper mutants with homozygous mutations of ALS protein P179S, W557L, and S636N were obtained by mutagenesis. The corresponding mutation types of P179S, W557L, and S636N in Arabidopsis plants were P197S, W574L, and S653N, respectively. The obtained P179S and S636N mutants were crossed to obtain F1 generation plants. After genotyping, multiple heterozygous peppers with P179S and S636N aggregation were obtained and named pepper ALS-P179S / S636N heterozygous mutants.

[0383] 2. Resistance analysis of pepper mutants to the combination of nicosulfuron and / or mepiquat nicotinic acid

[0384] The study investigated the differences in resistance to the combination of nicosulfuron and / or methyl nicotinic acid in different mutants. Effective doses of nicosulfuron and / or methyl nicotinic acid were applied to wild-type, homozygous ALSP179S, homozygous ALSS636N, homozygous ALSW557L, and ALS-P179S / S636N heterozygous mutants at the two-true-leaf stage. Results showed that wild-type and S636N mutants exhibited inhibited growth and severe dwarfing under nicosulfuron treatment, while P179S, W557L, and ALS-P179S / S636N heterozygous mutants showed no significant phytotoxicity. Wild-type and P179S mutants died almost entirely or completely under methyl nicotinic acid treatment, while S636N, W557L, and ALS-P179S / S636N heterozygous mutants showed no significant phytotoxicity.

[0385] 3. Genetic analysis of the ALS-P179S / S636N heterozygous mutant in chili pepper

[0386] Genotyping was performed on individual plants from 1000 self-crossed progeny of the ALS-P179S / S636N heterozygous mutant of pepper. The results showed that 246 plants had both alleles of P179S, 253 plants had both alleles of S636N, and 501 plants had one allele of P179S and the other of S636N, consistent with Mendel's laws of inheritance.

[0387] When F2 seedlings were treated with a combination of nicosulfuron and metronidazole, neither homozygous P179S nor homozygous S636N individuals survived, while ALS-P179S / S636N heterozygous plants grew normally and produced fertile offspring at a ratio of 1:2:1.

[0388] 4. Fertility analysis of chili pepper ALS-P179S / S636N heterozygous and ALS-W557L mutant plants sprayed with nicosulfuron and mefenazate.

[0389] ALS-P179S / S636N heterozygous and W557L mutant materials were sown and sprayed with effective doses of nicosulfuron and mefenoxam at the budding stage. Water was sprayed simultaneously as a control, with three replicates for each treatment. Pollen was collected at the pollen shedding stage and its viability was observed using potassium iodide staining. Pollen samples from the ALS-P179S / S636N heterozygous mutant pepper showed no viability, while almost all pollen samples from the W557L mutant pepper showed viability.

[0390] Pollen from ALS-P179S / S636N heterozygous mutant pepper plants that were simultaneously sprayed with nicosulfuron and mefenoxam at the budding stage was used as the male parent, and wild-type or pesticide-treated W557L mutant pepper plants were used as the female parent. Hybridization experiments were conducted to investigate the seed yield of the hybrid plants. The results showed that none of the hybrid plants in this combination yielded seeds.

[0391] Wild-type or herbicide-treated W557L mutant pepper plants were used as the male parent, and pollen from ALS-P179S / S636N heterozygous mutant pepper plants simultaneously sprayed with nicosulfuron and mefenoxamic acid at the budding stage was used as the female parent. Hybridization experiments were conducted to investigate the seed yield of the hybrid plants. Results showed that all hybrid plants treated with this herbicide combination could yield seeds. Genotyping of the harvested seeds revealed that all were single plants carrying either P179S or S636N. The results indicate that the pollen from ALS-P179S / S636N heterozygous mutant pepper plants simultaneously sprayed with nicosulfuron and mefenoxamic acid at the budding stage is sterile, and fertility can be restored after hybridization with pollen from fertile plants.

[0392] We also tested other ALS herbicides and achieved the same technical results as nicosulfuron + mefenoxam. Based on these results, a similar hybrid seed production system can be developed, referencing Arabidopsis thaliana.

[0393] In summary, extensive experiments have verified that the methods described in this invention for obtaining male-sterile lines, maintainer lines, and hybrids are universally applicable to various plants (such as tomatoes, barley, sorghum, millet, soybeans, watermelons, pumpkins, eggplants, lettuce, shepherd's purse, sugar beets, and cotton), and have broad application value.

[0394] While the invention is accomplished through many different embodiments, it should be understood that this disclosure is to be considered as an example of the principles of the invention and not intended to limit the invention to the specific embodiments described herein, as detailed in conjunction with preferred embodiments of the invention.

Claims

1. A method for breeding male-sterile lines in plants, comprising the following steps: (a) Obtain heterozygous plants with two different ALS allele mutations, wherein the homozygous plants with the first ALS allele mutation are resistant to herbicide A but not resistant to herbicide B; and the homozygous plants with the second ALS allele mutation are resistant to herbicide B but not resistant to herbicide A. (b) Treat the heterozygous plants described in step (a) with both herbicides A and B simultaneously to obtain resistant male-sterile lines; wherein, in step (b), both herbicides A and B are applied simultaneously during the reproductive growth period, or both herbicides A and B are applied simultaneously before the reproductive growth period and the efficacy continues into the reproductive growth period.

2. A method for breeding plant maintainer lines, comprising the following steps: (A) Obtain heterozygous plants with two different ALS allele mutations, wherein the homozygous plants with the first ALS allele mutation are resistant to herbicide A but not resistant to herbicide B; and the homozygous plants with the second ALS allele mutation are resistant to herbicide B but not resistant to herbicide A. (B) Treat the heterozygous plants described in step (A) with both herbicides A and B simultaneously to obtain resistant maintainer lines; wherein, in step (B), both herbicides A and B are applied simultaneously before the reproductive growth stage and the efficacy does not continue into the reproductive growth stage.

3. A method for isolating heterozygous plants with two different ALS allele mutations in plant progeny, comprising the steps of: (1) Obtain heterozygous plants with two different ALS allele mutations, wherein the homozygous plants with the first ALS gene mutation are resistant to herbicide A but not resistant to herbicide B; and the homozygous plants with the second ALS gene mutation are resistant to herbicide B but not resistant to herbicide A. (2) Use two herbicides, A and B, to treat the offspring produced by self-pollination of the heterozygous plants described in step (1) simultaneously, and isolate heterozygous plants with two different ALS allele mutations.

4. According to the method of claim 3, when step (2) involves simultaneous treatment with both herbicides A and B before the reproductive growth period and the efficacy does not continue into the reproductive growth period, the heterozygous plants with two different ALS allele mutations obtained are resistant and have normal fertility; when step (2) involves simultaneous treatment with both herbicides A and B during the reproductive growth period, or simultaneous treatment with both herbicides A and B before the reproductive growth period and the efficacy continues into the reproductive growth period, the heterozygous plants with two different ALS allele mutations obtained are resistant and male-sterile.

5. The method according to any one of claims 1-4, wherein the method for obtaining heterozygous plants with two different ALS allele mutations includes hybridization, gene editing, mutagenesis, natural mutation, or transgenic modification; preferably, the hybridization is performed by hybridizing homozygous plants containing different ALS gene mutations and having different resistance spectra.

6. The method according to any one of claims 1-5, wherein the alleles are located at corresponding positions on two different homologous chromosomes.

7. The method according to any one of claims 1-6, wherein the two different ALS allele mutations are selected from mutations that have herbicide resistance at positions 121, 196, 197, 199, 205, 570, 571, 653 and / or 654 corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1; Preferably, the first ALS gene mutation is selected from the following positions at SEQ ID NO: 121, 196, 197, 205, 570 and / or 571 corresponding to the Arabidopsis ALS amino acid sequence: G121A, G121D, G121N, G121S, V196M, P197A, P197R, P197C, P197Q, P197M, P197N, P197E, P197G, P197H, P197S, P197W, P197Y, respectively. Mutations in P197V, P197L, P197F, A205V, A205R, A205N, A205D, A205C, A205E, A205T, A205W, A205Y, M570A, M570N, M570C, V571A, V571N, V571C, V571Q, V571I, V571S and / or V571W; The second type of ALS gene mutation is selected from mutations at positions 199, 653, and / or 654 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1, namely R199A, R199E, S653N, S653T, S653I, S653F, G654E, and / or G654D, respectively. More preferably, the first type of ALS mutation is a change from proline to serine or leucine at position 197 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO: 1; the second type of ALS mutation is a change from serine to asparagine or isoleucine at position 653 of the corresponding Arabidopsis ALS amino acid sequence SEQ ID NO:

1.

8. A hybrid seed production method, the steps of which comprise: Using plants with normal pollen fertility as the male parent and male-sterile plants obtained by the method described in claim 1 or any one of claims 3-7 as the female parent, hybrids are obtained. Preferably, the paternal parent is a homozygous plant with a third ALS gene mutation, which is resistant to both herbicides A and B, and whose pollen has normal fertility.

9. The method according to claim 8, wherein the third ALS gene mutation is selected from mutations that have herbicide resistance at positions 122, 206, 256, 351, 376, 377 and / or 574 corresponding to the Arabidopsis ALS amino acid sequence SEQ ID NO: 1; Preferably, the third ALS gene mutation is selected from the following positions corresponding to the 122, 206, 256, 351, 376, 377 and / or 574 positions of the Arabidopsis ALS amino acid sequence SEQ ID NO: 1: A122R, A122N, A122D, A122C, A122E, A122Q, A122H, A122I, A122L, A122K, A122M, A122F, A122P, A122S, A122T, A122W, A122Y, A122V, F206A, F206H, F206W, F206Y, K256D, K256N, K256G, K256P, K256T, M351C, M351N, and M351N, respectively. Mutations of Q, M351G, M351K, M351P, M351Y, M351V, D376E, D376A, D376N, D376C, D376G, D376P, D376S, D376W, D376V, R377H, R377K, W574R, W574G, W574M, W574L, W574A, W574D, W574N, W574C, W574Q, W574E, W574H, W574I, W574K, W574F, W574S, W574T, W574Y and / or W574V; More preferably, the third ALS mutation is a change from tryptophan to leucine or methionine at position 574 of the Arabidopsis ALS amino acid sequence SEQ ID NO:

1.

10. A method of restoring fertility to a male sterile plant, the steps of which comprise: The male-sterile plant described in any one of claims 1 or 3-7 is hybridized with a fertile plant that can provide pollen, thereby restoring the fertility of the male-sterile plant.

11. A method of increasing yield in a plant, the steps of which comprise: The maintainer plants obtained by the method of claim 2, or the heterozygous plants with normal fertility and two different ALS allele mutations obtained by the method of any one of claims 3-7, or the hybrids obtained by the method of claim 8 or 9, are planted in a plant cultivation site and sprayed with an effective amount of herbicide A and / or B. Compared with wild-type plants, they have increased plant yield.

12. A method of increasing herbicide resistance in a plant, the steps of which comprise: The male-sterile plant obtained by the method of claim 1, or the maintainer plant obtained by the method of claim 2, or the heterozygous plant with two different ALS allele mutations obtained by the method of any one of claims 3-7, or the hybrid obtained by the method of claim 8 or 9, is planted in a plant cultivation site, and after spraying with an effective amount of herbicide A and / or B, the plant grows normally.

13. The method according to any one of claims 1-12, wherein the herbicide is an ALS inhibitor herbicide; Preferably, herbicide A is selected from any one or more of sulfonylurea herbicides, triazolidine herbicides, pyrimidinyl (thio)benzoate herbicides, or sulfonamide carbonyl triazolone herbicides, and herbicide B is selected from any one or more imidazolinone herbicides. More preferably, herbicide A is selected from bensulfuron-methyl, mesosulfuron-methyl, nicosulfuron, bensulfuron-methyl, sulfadiazine, chlorpyrifos, fluazolidone, thiamethoxam, acylsulfuron-methyl, ethersulfuron, pyrimisulfuron, pyrimisulfuron, thifensulfuron, mesosulfuron-methyl, chlorsulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, pyrimisulfuron-methyl, and / or bispyribac-methyl; herbicide B is selected from imidacloprid, imidacloprid, methoxyimidacloprid, and / or imidacloprid. More preferably, the herbicide A is bensulfuron-methyl and B is methyl benzoate; The herbicide A is mesosulfuron-methyl, and B is methyl sulfadiazine; The herbicide A is nicosulfuron, and B is methyl benzoate; The herbicide A is bensulfuron-methyl, and B is methyl benzoate; The herbicide A is sulfadiazine, and B is methyl methazine. The herbicide A is chlorpyrifos and B is methyl methazine; The herbicide A is flusulfuron-methyl, and B is methyl benzoate; The herbicide A is thiamethoxam and B is methyl benzoate; The herbicide A is sulfadiazine, and B is methyl methazine; The herbicide A is sulfadiazine, and B is methyl methazine; The herbicide A is pyrimisulfuron, and B is methyl methazine; The herbicide A is pyrimethanil, and B is methyl benzoate; The herbicide A is pyrimisulfuron, and B is methyl methazine; The herbicide A is thifensulfuron-methyl, and B is methyl benzoate; The herbicide A is pyrazosulfuron, and B is imidacloprid; The herbicide A is chlorpyrifos-methyl and B is methyl methazine; The herbicide A is dichlorvos, and B is methyl methazine; The herbicide A is pyrimethanil, and B is methyl methazine; The herbicide A is fluroxypyr, and B is methyl methazine; The herbicide A is bispyribac-sodium, and B is methyl methazine; A is bensulfuron-methyl, and B is imazalil; The herbicide A is mesosulfuron-methyl, and B is imazalil; The herbicide A is nicosulfuron, and B is imazalil; The herbicide A is bensulfuron-methyl, and B is imazalil; The herbicide A is sulfadiazine, and B is imazalil; The herbicide A is chlorpyrifos and B is imazalil; The herbicide A is flusulfuron-methyl, and B is imazalil; The herbicide A is thiamethoxam and B is imazalil; The herbicide A is sulfadiazine, and B is imazalil; The herbicide A is fensulfuron-methyl, and B is imazalil; The herbicide A is pyrimisulfuron, and B is imazalil; The herbicide A is pyrimethanil, and B is imazalil; The herbicide A is pyrimisulfuron, and B is imazalil; The herbicide A is thifensulfuron-methyl, and B is imazalil; The herbicide A is disulfuron-methyl, and B is imazalil; The herbicide A is chlorpyrifos-methyl, and B is imazalil; The herbicide A is dichlorvos, and B is imazalil; The herbicide A is pyrimethanil, and B is imazalil; The herbicide A is fluroxypyr, and B is imazalil; The herbicide A is bispyribac-sodium, and B is imazalil; A is bensulfuron-methyl, and B is methoxyfenozide. The herbicide A is mesosulfuron-methyl, and B is methoxyfenozide. The herbicide A is nicosulfuron, and B is methoxyfenozide; The herbicide A is bensulfuron-methyl, and B is methoxyfenozide; The herbicide A is sulfadiazine, and B is methoxyfenozide; The herbicide A is chlorpyrifos and B is methoxyfenozide. The herbicide A is flusulfuron-methyl, and B is methoxyfenozide. The herbicide A is thiamethoxam and B is methoxyfenozide. The herbicide A is sulfadiazine, and B is methoxyfenozide; The herbicide A is sulfadiazine, and B is methoxyfenozide; The herbicide A is pyrimisulfuron, and B is methoxyfenozide; The herbicide A is pyrimethanil, and B is methoxyfenozide. The herbicide A is pyrimisulfuron, and B is methoxyfenozide; The herbicide A is thifensulfuron-methyl, and B is methoxyfenozide. The herbicide A is pyrazosulfuron, and B is methoxyfenozide; The herbicide A is chlorpyrifos-methyl, and B is methoxyfenozide. The herbicide A is dichlorvos, and B is methoxyfenozide; The herbicide A is pyrimethanil, and B is methoxyfenozide; The herbicide A is fluroxypyr, and B is methoxyfenozide; The herbicide A is bispyribac-sodium, and B is methoxyfenozide. A is benzylsulfuron-methyl, and B is imidazoquinoline acid; The herbicide A is mesosulfuron-methyl, and B is imidazoquinoline acid; The herbicide A is nicosulfuron, and B is imidazoquinoline. The herbicide A is bensulfuron-methyl, and B is imidazoquinoline. The herbicide A is sulfadiazine, and B is imidazoquinoline. The herbicide A is chlorpyrifos and B is imidazoquinoline. The herbicide A is flusulfuron-methyl, and B is imidazoquinoline. The herbicide A is thiamethoxam and B is imidazoquinoline. The herbicide A is sulfadiazine, and B is imidazoquinoline. The herbicide A is sulfadiazine, and B is imidazoquinoline. The herbicide A is pyrimisulfuron, and B is imidazoquinoline. The herbicide A is pyrimethanil, and B is imidazoquinoline. The herbicide A is pyrimisulfuron, and B is imidazoquinoline. The herbicide A is thifensulfuron-methyl, and B is imidazoquinoline acid; The herbicide A is disulfuron-methyl, and B is imidazoquinoline acid; The herbicide A is chlorpyrifos-methyl and B is imidazoquinoline. The herbicide A is dichlorvos, and B is imidazoquinoline. The herbicide A is pyrimethanil, and B is imidazoquinoline. The herbicide A is fluroxypyr, and B is imidazoquinoline. The herbicide A is bispyribac-sodium, and B is imidazoquinoline. The herbicide A is pyrimisulfuron, and B is methyl methazine; The herbicide A is methamidosulfuron, and B is methyl benzoate; The herbicide A is chlorsulfuron, and B is methyl methazine; The herbicide A is pyrimisulfuron, and B is imazalil; The herbicide A is methamidosulfuron, and B is imazalil; The herbicide A is pyrimisulfuron, and B is imidazoquinoline. The herbicide A is methamidosulfuron, and B is imidazoquinoline. The herbicide A is chlorsulfuron-methyl, and B is methoxyfenozide.

14. The method according to any one of claims 1-13, wherein the herbicide is applied by a method selected from top spraying, foliar spraying, soil treatment, irrigation treatment, seed coating treatment, and seed soaking treatment.

15. A plant seed, plant, plant cell, or plant part obtained by the method as described in any one of claims 1-14.

16. The method according to any one of claims 1-14 or the plant seed, plant, plant cell or plant part according to claim 15, wherein the plant includes monocotyledonous plants or dicotyledonous plants; Preferably, the plants include Arabidopsis thaliana, rapeseed, rice, wheat, chili pepper, tomato, eggplant, Chinese cabbage, kale, mustard greens, sesame, cotton, sunflower, watermelon, lettuce, beet, potato, sweet potato, barley, sorghum, millet, corn, soybean, pumpkin, oats, buckwheat, flax, or lentils.

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