Obtaining haploids via androgenesis

Abstract

Provided herein are methods for obtaining androgen microspores. Methods for producing haploid plants from androgen microspores are also provided. The methods include reducing expression of at least one pollen starch biosynthetic pathway gene. The methods also include androgenic microspores having an increased response as compared to the wild-type microspores. In addition, the methods comprise reducing the expression of at least one pollen starch biosynthetic pathway gene by using gene editing or RNAi knockout or knockdown.

Description

Technical Field

[0001] The present invention generally relates to the production of doubled haploids via androgenesis.

[0002] Sequence Listing

[0003] This application is accompanied by a sequence listing named 82745_ST26.xml created on September 19, 2022, which is approximately 225 kilobytes in size. The sequence listing is incorporated herein by reference in its entirety. The sequence listing is submitted with this application via EFS-Web and complies with 37 C.FR §1.824(a)(2)-(6) and (b). Background Art

[0004] Microspores ("MS") are haploid cells containing the number of gametic chromosomes and can be induced to form embryoids (pseudoembryos). This method is generally referred to as microspore embryogenesis or androgenesis. The haploid embryoids produced by androgenesis can then develop into haploid plants. Haploid plants are usually fragile and infertile. Chromosome doubling caused by spontaneous or chemical induction can double the chromosomes, thereby producing stable, homozygous and fertile diploid or amphidiploid plants. Therefore, microspores can form homozygous double haploid plants (DH) in one generation through androgenesis.

[0005] Can easily separate millions of microspores from a corn ear or several wheat ears in a few minutes with a stirrer.By contrast, the immature haploid embryo of preparing similar number needs more effort, and this needs haploid inducer to be used for haploid generation, then identification, separation and preparation of haploid embryo ("haploid induction") are carried out.Haploid induction produces less than 100 haploid embryos / ears induced usually.Therefore, using microspore as the starting donor to obtain DH plant is simpler and more economically beneficial.Therefore, the male nuclear development from haploid microspore to DH system in one generation is the most effective DH production method.But commercial plant breeding lacks the successful maize male nuclear development method for a wide range of genotypes.

[0006] Previous androgenic schemes required the application of treatment to microspores after isolation. There is a correlation between chemical inducers and the lack of starch formation in microspores and the effect of androgenic induction (Liu et al., [Crop Science] Vol. 42, No. 3, pp. 686-692, 2002). It has been found that several genes are down-regulated in microspores after mannitol treatment to induce barley androgenic development. The down-regulation has also been shown to be similar to the induction of maltase genes and invertase genes. Maltase and invertase are involved in the decomposition of starch and sucrose, respectively (Maraschin et al., [Journal of Experimental Botany], Vol. 56, No. 417, July 2005, pp. 1711-1726). Therefore, genes and their products in the starch accumulation pathway in pollen (Pfister & Zeeman, Cell. Mol. Life Sci. 73, 2781-2807 (2016)) are suitable candidates as possible genes and / or signals that control the triggering of androgenic development.

[0007] Blocking or interfering with the starch accumulation pathway and / or the pollen starch biosynthesis pathway can act as a universal trigger for androgenic development by redirecting microspores from a preprogrammed gametophyte development pathway to a sporophyte development pathway. The present disclosure targets selected genes to block or interfere with the starch accumulation pathway in spores, thereby acting as a universal trigger for androgenic development. Summary of the invention

[0008] Hybrid breeding requires the rapid development of pure inbred lines. Current double haploid (DH) technology and methods are expensive. Through the standard haploid induction process, it is necessary to spend a lot of effort to prepare millions of immature haploid embryos. However, millions of microspores can be easily separated from a corn ear (or several wheat ears) in a few minutes with a stirrer. Therefore, it is simpler and more economically beneficial to use microspores as the starting donor to obtain DH plants. The male nuclear development from haploid microspores to DH system in one generation is the most effective DH production method. At present, commercial plant breeding lacks the successful maize male nuclear development method for a wide range of genotypes. This article provides a method for producing DH system from microspores in one generation.

[0009] The method herein reduces the expression of at least one pollen starch biosynthetic pathway gene selected from ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, glucose phosphomutase 1, glucose phosphomutase 2, and invertase 2. In these methods, at least one pollen starch pathway gene is reduced by knocking out or knocking down using gene editing or RNAi. Gene editing is performed by using a site-directed nuclease. For example, gene editing is performed using a CRISPR nuclease (e.g., Cas12a). In these methods, the androgen response is increased by at least 40% compared to the wild type.

[0010] Brief description of the sequences in the sequence listing

[0011] SEQ ID NO: 1 is the nucleotide sequence of construct 25639.

[0012] SEQ ID NO: 2 is the nucleotide sequence of construct 25635.

[0013] SEQ ID NO: 3 is the nucleotide sequence of construct 25637.

[0014] SEQ ID NO:4 is the nucleotide sequence of construct 25619.

[0015] SEQ ID NO:5 is the nucleotide sequence of construct 25634.

[0016] SEQ ID NO:6 is the nucleotide sequence of construct 25636.

[0017] SEQ ID NO:7 is the nucleotide sequence of construct 25662.

[0018] SEQ ID NO:8 is the nucleotide sequence of construct 25638.

[0019] SEQ ID NO: 9 is the nucleotide sequence of the target gene phosphoglucomutase 1.

[0020] SEQ ID NO: 10 is the nucleotide sequence of the target gene phosphoglucomutase 2.

[0021] SEQ ID NO: 11 is the nucleotide sequence of the target gene ADP glucose pyrophosphorylase II.

[0022] SEQ ID NO: 12 is the nucleotide sequence of the target gene invertase 2.

[0023] SEQ ID NO: 13 is the nucleotide sequence of the target gene hexokinase 5.

[0024] SEQ ID NO: 14 is the nucleotide sequence of the target gene Waxy1.

[0025] SEQ ID NO: 15 is the nucleotide sequence of gRNA target 1 of ADP glucose pyrophosphorylase II.

[0026] SEQ ID NO: 16 is the nucleotide sequence of gRNA target 2 for ADP glucose pyrophosphorylase II.

[0027] SEQ ID NO: 17 is the nucleotide sequence of the RNAi target of ADP glucose pyrophosphorylase II.

[0028] SEQ ID NO: 18 is the nucleotide sequence of gRNA target 1 of hexokinase 5.

[0029] SEQ ID NO: 19 is the nucleotide sequence of gRNA target 2 of hexokinase 5.

[0030] SEQ ID NO:20 is the nucleotide sequence of the RNAi target of hexokinase 5.

[0031] SEQ ID NO:21 is the nucleotide sequence of gRNA target 1 of Waxy1.

[0032] SEQ ID NO:22 is the nucleotide sequence of gRNA target 2 of Waxy1.

[0033] SEQ ID NO:23 is the nucleotide sequence of the RNAi target of Waxy1.

[0034] SEQ ID NO:24 is the nucleotide sequence of gRNA target 1 of invertase 2.

[0035] SEQ ID NO:25 is the nucleotide sequence of gRNA target 2 of invertase 2.

[0036] SEQ ID NO:26 is the nucleotide sequence of the RNAi target of invertase 2.

[0037] SEQ ID NO: 27 is the nucleotide sequence of gRNA target 1 of phosphoglucomutase 1.

[0038] SEQ ID NO: 28 is the nucleotide sequence of gRNA target 1 for phosphoglucomutase 2.

[0039] SEQ ID NO:29 is the primer sequence of TaqMan assay 3682.

[0040] SEQ ID NO:30 is the primer sequence of TaqMan assay 3682.

[0041] SEQ ID NO:31 is the probe sequence of TaqMan assay 3682.

[0042] SEQ ID NO:32 is the primer sequence of TaqMan assay 3683.

[0043] SEQ ID NO:33 is the primer sequence of TaqMan assay 3683.

[0044] SEQ ID NO:34 is the probe sequence of TaqMan assay 3683.

[0045] SEQ ID NO:35 is the primer sequence of TaqMan assay 3684.

[0046] SEQ ID NO:36 is the primer sequence of TaqMan assay 3684.

[0047] SEQ ID NO:37 is the probe sequence of TaqMan assay 3684.

[0048] SEQ ID NO:38 is the primer sequence of TaqMan assay 3685.

[0049] SEQ ID NO:39 is the primer sequence of TaqMan assay 3685.

[0050] SEQ ID NO:40 is the probe sequence of TaqMan assay 3685.

[0051] SEQ ID NO:41 is the primer sequence of TaqMan assay 3686.

[0052] SEQ ID NO:42 is the primer sequence of TaqMan assay 3686.

[0053] SEQ ID NO:43 is the probe sequence of TaqMan assay 3686.

[0054] SEQ ID NO:44 is the primer sequence of TaqMan assay 3687.

[0055] SEQ ID NO:45 is the primer sequence of TaqMan assay 3687.

[0056] SEQ ID NO:46 is the probe sequence of TaqMan assay 3687.

[0057] SEQ ID NO:47 is the primer sequence of TaqMan assay 3688.

[0058] SEQ ID NO:48 is the primer sequence of TaqMan assay 3688.

[0059] SEQ ID NO:49 is the probe sequence of TaqMan assay 3688.

[0060] SEQ ID NO:50 is the primer sequence of TaqMan assay 3689.

[0061] SEQ ID NO:51 is the primer sequence of TaqMan assay 3689.

[0062] SEQ ID NO:52 is the probe sequence of TaqMan assay 3689.

[0063] SEQ ID NO:53 is the primer sequence of TaqMan assay 3690.

[0064] SEQ ID NO:54 is the primer sequence of TaqMan assay 3690.

[0065] SEQ ID NO:55 is the probe sequence of TaqMan assay 3690.

[0066] SEQ ID NO:56 is the primer sequence of TaqMan assay 3691.

[0067] SEQ ID NO:57 is the primer sequence of TaqMan assay 3691.

[0068] SEQ ID NO:58 is the probe sequence of TaqMan assay 3691.

[0069] definition

[0070] Although it is believed that the following terms are well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the subject matter disclosed herein.

[0071] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. Reference to the technology employed herein is intended to refer to the technology commonly understood in the art, including variations of those technologies and / or alternatives to equivalent technologies that are well known to those of ordinary skill in the art. Although it is believed that the following terms can be well understood by those of ordinary skill in the art, the following definitions are stated to facilitate explanation of the subject matter disclosed in this application.

[0072] In accordance with long-standing patent law conventions, the terms "a," "an," and "the" as used in this application, including the claims, mean "one or more." For example, the phrase "a cell" means one or more cells, and in some embodiments may refer to tissues and / or organs. Similarly, the phrase "at least one," when used herein to refer to an entity, means, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more of the entity, including but not limited to all integer values ​​between 1 and 100 and integers greater than 100.

[0073] Unless otherwise indicated, all numbers expressing amounts of ingredients, reaction conditions, and the like used in the specification and claims are to be understood as modified in all instances by the term "about". As used herein, the term "about", when referring to a measurable value such as an amount of mass, weight, time, volume, concentration, or percentage, is meant to encompass variations of ±20% from the specified amount in some embodiments, ±10% from the specified amount in some embodiments, ±5% from the specified amount in some embodiments, ±1% from the specified amount in some embodiments, ±0.5% from the specified amount in some embodiments, and ±0.1% from the specified amount in some embodiments, as such variations are suitable for performing the disclosed methods and / or using the disclosed compositions, nucleic acids, polypeptides, and the like. Therefore, unless indicated to the contrary, the numerical parameters set forth in the specification and the appended claims are approximate values ​​that may vary depending upon the desired properties sought to be obtained by the subject matter disclosed herein.

[0074] As used herein, the term "allele" refers to a variant or alternative sequence form at a genetic locus. In a diploid, a single allele at each locus is inherited by the progeny from each parent, respectively. Although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent all the alleles present in the species, the two alleles present in a given locus in a diploid organism occupy corresponding positions on a pair of homologous chromosomes.

[0075] As used herein, the term "amplified" or "amplification" means using at least one nucleic acid molecule as a template to construct multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification systems (TAS), and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., ed., American Society for Microbiology, Washington, DC, (1993). The amplified product is referred to as an "amplicon".

[0076] As used herein, the term "and / or," when used in the context of listing entities, refers to the entities either alone or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, the one or more elements referred to by "and / or" may also be present alone in single or multiple occurrences in one or more combinations and / or one or more subcombinations.

[0077] The term "comprising" is synonymous with "including," "containing," and "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a term that means the specified elements and / or steps are present, but other elements and / or steps may be added and still fall within the scope of the relevant subject matter.

[0078] As used herein, the phrase "consisting of" excludes any elements, steps or ingredients not specifically recited. When the phrase "consisting of" appears in a clause of the body of a claim, rather than directly following the preamble, it limits only the elements set forth in the clause; other elements are not excluded from the claim as a whole.

[0079] With respect to the terms "comprising," "consisting essentially of," and "consisting of," where one of these three terms is used herein, the subject matter disclosed and claimed in the present application may, in some embodiments, include the use of either of the other two terms. For example, if the subject matter, in some embodiments, relates to a nucleic acid encoding a polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or 3. It should be understood that the disclosed subject matter therefore also encompasses nucleic acids encoding polypeptides that, in some embodiments, consist essentially of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or 3, and nucleic acids encoding polypeptides that, in some embodiments, consist of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or 3. Similarly, it should also be understood that in some embodiments, the methods for the disclosed subject matter include the steps disclosed herein, in some embodiments, the methods for the subject matter disclosed in the present application consist essentially of the disclosed steps, and in some embodiments, the methods for the subject matter disclosed in the present application consist of the steps disclosed herein.

[0080] As used herein, the term "de novo haploid induction" refers to triggering haploid induction by introducing a spontaneous haploid induction agent. Such introduction can be achieved by topical spraying, manual pollination, mutagenesis or transgenic methods. Throughout this specification, the terms "de novo haploid induction", "de novo HI" and "haploid induction de novo" are used interchangeably.

[0081] As used herein, the term "superior line" or "inbred line" refers to any line resulting from breeding and selection for superior agronomic performance. A superior line has stable genetics, i.e., it is reasonably or nearly isogenic in its genome. In other words, a superior line is reasonably or nearly homozygous for all alleles in its genome.

[0082] As used herein, "embryoids" or "pseudoembryons" refer to clusters of embryo-like structures formed in vitro by a single microspore cell. These embryoids can form fully grown plants. As used herein, "embryoid-like structures" or "ELS" refer to embryos or calli formed by a single microspore cell and released from the exosporium. "Callus" or "calli" refers to a mass of cells derived from an in vitro culture of microspore cells.

[0083] As used herein, when used with a reference polynucleotide (such as a gene, ORF or part thereof, or transgenic of a plant), the term "expression" refers to a method in which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA or snRNA) by "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase), and is converted into a protein by "translation" of the mRNA in applicable cases (e.g., if the gene encodes a protein). Gene expression can be regulated at many stages of the method. For example, in the case of an antisense construct or a dsRNA construct, expression may refer only to the transcription of the antisense RNA or only to the transcription of the dsRNA. In an embodiment, "expression" refers to the transcription and stable accumulation of a sense (mRNA) or functional RNA. "Expression" may also refer to the production of a protein.

[0084] As used herein, the term "gene" refers to a unit of inheritance comprising a DNA sequence that occupies a specific location on a chromosome and contains the genetic instructions for a specific characteristic or trait in an organism.

[0085] As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. "A group of genetically marked genotypes" of an individual include the specific alleles of one or more genetic marker loci present in the individual. As known in the art, genotypes can relate to a single locus or multiple loci, whether these loci are relevant or unrelated, and / or are linked or non-linked. In certain embodiments, the genotype of an individual relates to one or more relevant genes, because one or more of these genes participate in the expression of a target phenotype (e.g., quantitative traits as defined herein). Therefore, in certain embodiments, genotypes include the summation of one or more alleles present at one or more genetic loci of an individual intrinsic quantitative trait. In certain embodiments, genotypes are represented by haplotypes (defined below).

[0086] As used herein, the term "germplasm" refers to the totality of the genotype of a population or other individual group (e.g., species). The term "germplasm" may also refer to plant material; for example, a group of plants that serve as a repository for various alleles. The phrase "adapted germplasm" refers to plant material that has been confirmed to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original germplasm," and "exotic germplasm" refer to plant material with unknown or unconfirmed genetic value; for example, for a given environment or geographic region; like this, the phrase "unadapted germplasm" refers in some embodiments to plant material that does not belong to an established breeding population and has no known relationship with a member of an established breeding population.

[0087] As used herein, a plant referred to as a "haploid" has a single chromosome set (genome), and the number of chromosomes (1n) reduced in a haploid plant is equal to the number of chromosomes in a gamete. As used herein, a plant referred to as a "double haploid" is developed by doubling the haploid chromosome set (from 1n to 2n). Plants or seeds obtained from selfing to any number of generations of double haploid plants can still be identified as double haploid plants. Double haploid plants are considered to be homozygous plants. If a plant is fertile, even if the entire vegetative part of the plant is not composed of cells with a doubled chromosome set, the plant is considered to be a double haploid; that is, if a plant contains surviving gametes, even if it is mosaic, it will also be considered to be a double haploid.

[0088] As used herein, the term "heterologous" when used in reference to a gene or nucleic acid means that the gene encoding a factor is not in its natural environment (i.e., has been artificially altered). For example, a heterologous gene may include a gene introduced from one species into another species. A heterologous gene may also include a gene that is native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.). A heterologous gene may further include a plant gene polynucleotide that includes a cDNA form of a plant gene; the cDNA may be expressed in a sense direction (to produce mRNA) or an antisense direction (to produce an antisense RNA transcript complementary to an mRNA transcript). In one aspect of the invention, heterologous genes are distinguished from endogenous plant genes in that the heterologous gene polynucleotides are typically joined to polynucleotides containing regulatory elements such as promoters, these polynucleotides are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene polynucleotides in chromosomes, or these polynucleotides are associated with parts of chromosomes not found in nature (e.g., genes expressed in loci that do not normally express the gene). In addition, in embodiments, a "heterologous" polynucleotide is a polynucleotide that is not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.

[0089] As used herein, the term "heterozygous" refers to a genetic condition that exists when different alleles are located at corresponding loci on homologous chromosomes.

[0090] As used herein, the term "homozygous" refers to a genetic condition that exists when identical alleles are located at corresponding loci on homologous chromosomes.

[0091] As used herein, when used in the context of nucleic acid molecules of the present invention or polynucleotides, the term "isolated" refers to polynucleotides that are identified and separated / separated in the context of chromosome polynucleotides in the corresponding source organism. If the isolated nucleic acid or polynucleotides do have naturally occurring counterparts, the isolated nucleic acid or polynucleotides are not nucleic acids that exist in their natural environment. In contrast, non-isolated nucleic acids are nucleic acids (such as DNA and RNA), which are found under the state of nature. For example, given polynucleotides (such as genes) are found on the host cell chromosome near adjacent genes. Isolated nucleic acid molecules can exist in single-stranded or double-stranded forms. Alternatively, it can include sense strands and antisense strands (that is, nucleic acid molecules can be double-stranded). In a preferred embodiment, nucleic acid molecules of the present invention are interpreted as isolated.

[0092] As used herein, the term "locus" refers to a location on a chromosome in a given species (eg, the location of a gene, genetic marker, etc.).

[0093] As used herein, the term "human-induced mutation" refers to any mutation that occurs due to direct or indirect human action. This term includes, but is not limited to, mutations obtained by any targeted mutagenesis method.

[0094] As used herein, the term "hybrid" refers to the offspring produced by crossing two genetically different parent plants. The resulting progeny of this cross is the "biparental" population.

[0095] As used herein, the terms "label probe" and "probe" refer to nucleotide sequences or nucleic acid molecules that can be used to detect the presence or absence of a sequence within a larger sequence, for example, a nucleic acid probe that is complementary to all or part of a marker or marker locus by nucleic acid hybridization. Label probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides can be used for nucleic acid hybridization.

[0096] As used herein, when identifying the presence / absence of SCD or other loci of interest, the term "molecular marker" can be used to refer to a genetic marker as defined above, or a coded product (e.g., protein) thereof used as a reference point. Molecular markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., derived from RNA, cDNA, etc.). The term also refers to a nucleotide sequence complementary to a marker sequence or to a nucleotide sequence flanking it, such as a nucleotide sequence used as a probe and / or primer capable of amplifying a marker sequence. When nucleotide sequences specifically hybridize in a solution, these nucleotide sequences are "complementary" (e.g., according to Watson-Crick base pairing principles). This term also refers to a genetic marker that indicates a trait by the absence of a nucleotide sequence complementary to a marker sequence or to a nucleotide sequence flanking it (e.g., a nucleotide sequence used as a probe and / or primer capable of amplifying a marker sequence).

[0097] As used herein, the terms "nucleotide sequence", "polynucleotide", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid fragment" refer to polymers of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, and / or altered nucleotide bases. "Nucleotide" is a monomeric unit from which DNA or RNA polymers are constructed and consists of a purine or pyrimidine base, a pentose sugar and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenylic acid or deoxyadenylic acid (for RNA or DNA, respectively), "C" represents cytidylic acid or deoxycytidylic acid, "G" represents guanylic acid or deoxyguanylic acid, "U" represents uridylic acid, "T" represents deoxythymidylic acid, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0098] The terms "identity" or "identical" in the context of two nucleic acid or amino acid sequences refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of a reference ("query") sequence (or its complementary strand) when the two sequences are fully aligned, such as compared to a test ("subject") sequence. Unless otherwise indicated, sequence identity as used herein refers to the value obtained by using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) implemented in the EMBOSS Needle alignment tool, using the default matrix file EBLOSUM62 (for proteins) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5) or DNAfull (for nucleic acids) and default parameters (gap open=10, gap extension=0.5, terminal gap penalty=false, terminal gap open=10, terminal gap extension=0.5); or any equivalent program thereof. EMBOSS Needle is available, for example, from EMBL-EBI, for example at the following website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: "The EMBL-EBI search and sequence analysis tools APIs in 2019." Madeira et al. Nucleic Acids Research, June 2019, 47(W1): W636-W641. As used herein, the term "equivalent program" refers to any sequence comparison program that, for any two sequences in question, generates an alignment with identical nucleotide or amino acid residue matches and identical percentages of sequence identity when compared to the corresponding alignment generated by EMBOSS Needle. In some embodiments, substantially identical nucleic acid or amino acid sequences can perform substantially the same function.

[0099] As used herein, the terms "introgression," "introgressed," and "introgressing" refer to natural and artificial processes in which a genomic region of one species, variety, or cultivar is moved to the genome of another species, variety, or cultivar by crossing the other species, variety, or cultivar. The process can optionally be accomplished by backcrossing to the recurrent parent.

[0100] The term "open reading frame" (ORF) refers to a nucleic acid sequence encoding a polypeptide. In some embodiments, an ORF comprises a translation initiation codon, a translation termination (termination) (i.e., stop) codon, and a nucleic acid sequence encoding amino acids present in the polypeptide therebetween. The terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides (i.e., codons) in a coding sequence, which correspondingly indicate the start and chain termination of protein synthesis (mRNA translation).

[0101] As used herein, the term "phenotype", "phenotypic trait" or "trait" refers to one or more traits of a plant or plant cell. The phenotype is observable by the naked eye or by any other evaluation method known in the art (e.g., microscopy, biochemical analysis, or electromechanical determination). In some cases, the phenotype is directly controlled by a single gene or genetic locus (i.e., corresponding to a "single gene trait"). For example, in the case of a haploid plant for evaluating SCD, the phenotype may refer to fertile pollen shed and / or seeds obtained by pollination with the pollen. In other cases, the phenotype is the result of the interaction between several genes, which in some embodiments is also caused by the interaction of a plant and / or a plant cell with its environment.

[0102] As used herein, the term "plant" may refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, unless otherwise indicated, the term "plant" may refer to any of the following: a whole plant, a plant component or organ (e.g., leaves, stems, roots, etc.), a plant tissue, a seed, and / or a plant cell.

[0103] Plant cells are plant cells obtained from plants, or plant cells derived from cells taken from plants by culture. Thus, the term "plant cell" includes, but is not limited to, cells within seeds, suspension cultures, embryos, meristems, callus, leaves, buds, gametophytes, sporophytes, pollen, and microspores. The phrase "plant part" refers to a part of a plant, including single cells and cell tissues (such as intact plant cells in plants), cell clumps, and tissue cultures that can regenerate plants. Examples of plant parts include, but are not limited to, single cells and tissues from: pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, and seeds; as well as scions, rhizomes, protoplasts, callus, and the like.

[0104] As used herein, the term "population" means a genetically heterogeneous collection of plants sharing a common genetic derivation.

[0105] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to a nucleic acid target (in some embodiments, specifically annealing to a nucleic acid target) when placed under conditions that induce synthesis of a primer extension product (e.g., in the presence of nucleotides and an agent for polymerization (e.g., a DNA polymerase) and at a suitable temperature and pH) to allow a DNA polymerase and / or reverse transcriptase to attach thereto, thereby serving as a starting point for DNA synthesis. In some embodiments, one or more primers are employed to amplify plant nucleic acids (e.g., using a polymerase chain reaction; PCR).

[0106] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a double-stranded or higher-order nucleic acid chain, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, the probe has a sufficient length to form a stable and sequence-specific duplex molecule with its complementary sequence, and this can be used in some embodiments to detect a target sequence present in multiple nucleic acids.

[0107] As used herein, the terms "offspring" and "offspring plant" refer to plants produced by asexual or sexual reproduction from one or more parent plants. In haploid induction, the seed on the maternal parent is haploid, and therefore is not an offspring of an induced haploid line. The offspring of haploid seeds is not the only offspring desired. There are also subsequent plants and seed offspring of HI seeds and haploid induced plants. Both haploid seeds and HI seeds can be offspring. Progeny plants can be obtained by cloning a single parent plant or selfing a single parent plant or by hybridizing two or more parent plants. For example, progeny plants can be obtained by cloning or selfing a parent plant or by hybridizing two parent plants, and include selfing bodies and F1 or F2 or even further generations. F1 is the first generation of progeny generated from two parents (at least one of the two parents is used as a donor for the trait for the first time), while the progeny of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples generated from self-crossing, reciprocal crosses, backcrosses and / or other crosses of F1, F2, etc. Therefore, F1 can be (and in some embodiments is) a hybrid produced from a cross between two purebred parents (i.e., the purebred parents are each homozygous for the trait of interest or its alleles), and F2 can be (and in some embodiments is) the progeny produced from self-pollination of the F1 hybrid.

[0108] As used herein, the term "regeneration" and grammatical variations thereof refer to the generation of plants from tissue culture.

[0109] As used herein, "spontaneously doubled haploid plant" refers to a plant whose florets have undergone spontaneous doubling. Other tissues of the spontaneously doubled haploid plant may retain their haploid state (e.g., roots, leaves, stems).

[0110] As used herein, the term "trait" refers to a phenotype of interest, a gene that contributes to a phenotype of interest, and a nucleic acid sequence associated with a gene that contributes to a phenotype of interest.

[0111] As used herein, the term "targeted mutagenesis" or "mutagenesis strategy" refers to any mutagenesis method that results in the intentional mutagenesis of a selected gene. Targeted mutagenesis includes CRISPR, TILLING, TALEN methods, and other methods that have not yet been discovered but can be used to achieve the same results.

[0112] As used herein, haploid induction rate ("HIR") means the number of haploid kernels that survive pollination of an ear with pollen from a haploid induction line divided by the total number of kernels. DETAILED DESCRIPTION

[0113] A method for obtaining androgenic microspores is described herein. The method includes reducing the expression of at least one pollen starch biosynthetic pathway gene, wherein the androgenic microspore has an increased androgen response compared to wild-type microspores. The method further includes reducing the expression of at least one pollen starch biosynthetic pathway gene by knocking out or knocking down. In an embodiment, the pollen starch biosynthetic pathway gene is selected from the group consisting of: ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase 1 and phosphoglucomutase 2, and invertase 2. In another embodiment, ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase 1 and phosphoglucomutase 2, and invertase 2 genes respectively include SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 12.

[0114] The knockout of pollen starch biosynthesis pathway genes can be achieved by gene editing, and knocking down can be achieved by RNAi. In an embodiment, gene editing is performed using a site-directed nuclease selected from the group consisting of CRISPR nuclease, meganuclease, zinc finger nuclease, and transcription activator-like effector nuclease. In one embodiment, the site-directed nuclease is a CRISPR nuclease, and in another embodiment, the CRISPR nuclease is selected from the group consisting of: Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and its nickase or inactivated form. In another embodiment, the CRISPR nuclease is Cas12a. In one embodiment, knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NO: 15 and 16. In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 18 and 19. In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 21 and 22. In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 24 and 25. In one embodiment, the knockout includes the use of multiple gRNAs, wherein the gRNAs include SEQ ID NOs: 15, 16, 18, 19, 21, 22, 27 and 28. In one embodiment, the increased androgen response is increased by at least 40% compared to the wild type. In another embodiment, the microspores obtained are selected from the group consisting of maize, rice and wheat. The microspores obtained can be derived from maize.

[0115] Also described herein are methods for producing haploid plants. These methods include first obtaining androgenic microspores, wherein the androgenic microspores have an increased androgenic response compared to the wild type. The obtained microspores are cultured, and haploid plants are regenerated from the microspores. In an embodiment, the increased androgenic response is achieved by knocking out or knocking down at least one pollen starch biosynthetic pathway gene. The pollen starch biosynthetic pathway gene is selected from the group consisting of: ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, glucose phosphomutase 1 and glucose phosphomutase 2, and invertase 2, and may include SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

[0116] In one embodiment, knockout can be achieved by gene editing, and knockdown can be achieved by RNAi. Gene editing can be performed using a site-directed nuclease selected from the group consisting of CRISPR nucleases, meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases. In one embodiment, the site-directed nuclease is a CRISPR nuclease, and the CRISPR nuclease can be selected from the group consisting of: Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and nickases or inactivated forms thereof. In one embodiment, the CRISPR nuclease can be Cas12a.

[0117] In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 15 and 16. In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 18 and 19. In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 21 and 22. In one embodiment, the knockout is a single gene knockout including the use of two gRNAs, and the two gRNAs include SEQ ID NOs: 24 and 25. In one embodiment, the knockout includes the use of multiple gRNAs, wherein the gRNAs include SEQ ID NOs: 15, 16, 18, 19, 21, 22, 27 and 28. In one embodiment, the increased androgen response is increased by at least 40% compared to the wild type. In another embodiment, the microspores obtained are selected from the group consisting of maize, rice and wheat. The microspores obtained can be derived from maize.

[0118] In another embodiment, these methods include doubling the chromosome of a haploid plant to produce a double haploid plant. Chromosome doubling can be achieved via chemical induction or spontaneous doubling. In an embodiment, chromosome doubling is achieved via chemical induction, and by selecting the chemical of the group consisting of colchicine, trifluralin, natrium, dithiopyr, nitrous oxide and amoxicillin to achieve. In an embodiment, the method for producing a double haploid plant by microspores occurs in one generation. Plants produced by the method are also provided herein.

[0119] Examples

[0120] 1. Androgenesis Protocol Used to Test the Hypothesis of This Article

[0121] Use improved androgeny scheme (referring to Zheng et al., 2003) to compare the potential androgeny (i.e. the number of embryoid-like structures in 100 spikelets) of GE-modified microspores and wild-type microspores. Harvest spikes with mid-late uninucleate stage microspores, wrap in aluminum foil, and store in the dark at 6°C-8°C for 14 days. Then, use a stirrer to separate the microspores and purify them using maltose gradient centrifugation. Use two kinds of fresh wheat ovaries / mL culture medium from wheat variety AC Nanda to cultivate isolated microspores at 28°C in the dark to produce callus or embryoids. This scheme is used as a baseline for comparing the androgeny potential between GE-modified microspores and non-modified microspores.

[0122] 2. Transformable genotypes, candidate gene selection, vector construction, and event selection

[0123] genotype

[0124] Four genotypes (3 inbreds and 1 hybrid (F1)) were tested to select options for subsequent transformation to produce transgenic microspores. Based on transformability data and preferences (Table 1), hybrid 1 and inbred 1 were selected for transformation to carry the designed constructs. Inbreds were selected only for the FIREWORKS editing method, while hybrids were selected for all eight constructs in Table 2 below.

[0125] Table 1. List of genotypes tested for transformability to carry constructs.

[0126]

[0127] *Select only for FIREWORKS builds

[0128] **Selected for all 8 constructs

[0129] ***Not enough ears are produced to obtain adequate immature embryos

[0130] Gene selection

[0131] Five genes in the maize pollen starch biosynthesis pathway were selected to demonstrate that disruption of the starch biosynthesis pathway results in enhanced androgenesis: ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase 1 and 2, and invertase 2. We generated T0 events using two strategies: knockout (loss of function) via CRISPR gene editing and knockdown (reduced expression) via RNAi.

[0132] Vector construction

[0133] We created three types of constructs for the above two strategies. First, we created a single gene knockout construct using two gRNA / genes. Second, we created a single gene knockdown construct using RNAi. Finally, we used the FIREWORKS method (see WO 2020 / 176412) to multiplex gRNAs for multiple genes to produce microspores with multiple gene knockouts. Table 2 shows the multiple constructs generated and tested. Table 2. Table 2 shows a list of multiple constructs created and tested with pollen starch biosynthesis pathway gene targets.

[0134]

[0135] KD = knockdown; KO = knockout

[0136] Event Selection

[0137] We used the following method to screen T0 events and subsequent T1 plants for CRISPR / Cas12a-induced gene editing and RNAi-mediated gene silencing. T0 plants were generated by Agrobacterium-mediated transformation according to established procedures (Zhong et al., 2018.). Genomic DNA and total RNA were isolated from T0 plants and molecular analysis was performed. TaqMan copy number assays combined with targeted next-generation sequencing (NGS) or TaqMan copy number assays combined with qRT-PCR were used to detect knockout (KO) and knockdown (KD) events, respectively. T0 transgenic plants carrying monoallelic or biallelic mutations and single-copy transgenes without vector backbones were selected as T0 KO plants. Transgenic plants showing reduced gene expression by qRT-PCR were selected as T0 KD events. The selected T0 events were grown in the greenhouse and crossed with DH inbred line 1 (as females) to produce T1 plants (T0×inbred line 1). Androgen responses were evaluated on T0 and T1 plants. Non-transgenic plants were used as controls.

[0138] 3. Assessment of androgenesis in microspores

[0139] In our evaluation, we observed three examples of increased androgen response in GE-microspores of KO or KD genes in the T0 generation and the subsequent T1 generation (generated by the first backcross). First, the KD T0 event with the best response occurred in the hybrid line 1 genotype from RNAi construct 25639. Compared with unedited microspores (wild type), this event showed that the expression of Waxy1 was suppressed, resulting in an increase of more than 300% in androgen response (based on the production of embryoid-like structures (ELS) in 100 spikelets). Subsequent T1 microspores also had an increase in androgen response of more than 300%. T2 microspores had an average increase of 155% in androgen response (based on the production of ELS from 200,000 microspores).

[0140] Secondly, the KO construct (25635) targeting the hexokinase 5 gene in hybrid line 1 resulted in a 70% increase in androgen response (measured as ELS from cultured T0 microspores of 100 spikelets) compared to unedited microspores (wild type). Subsequent T1 generation microspores had an increase in androgen response of 338% to 602% (measured as ELS from 200,000 microspores). This increase in the T1 generation may be due to less stress and shorter plants compared to the T0 generation, which also occurs in T0. Compared to unedited microspores, T2 offspring from three different ancestral T1 plants showed an increase in androgen response of 575% to 1406% ​​(measured as ELS from 200,000 microspores).

[0141] Finally, the FIREWORK construct (25619) that simultaneously targeted several genes (ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, and phosphoglucomutase) in inbred line 1 resulted in an increase of more than 400% in androgen response (measured in ELS from cultured TO microspores of 100 spikelets) compared to unedited microspores (wild type). However, T1 seeds were not produced from the best responding TO event.

[0142] Table 3. The following table shows the androgenic results for all constructs tested in the T0 and T1 generations. Three specific examples of increased responses discussed above in Example 3 are shown in bold.

[0143]

[0144]

[0145] KD = knockdown, KO = knockout, NS = not significant

[0146] Table 4. The following table shows the androgenic results in the T2 generation of the best responding plants from the T1 generation of hybrid line 1 (the first two rows in Table 3 above).

[0147]

[0148] References

[0149] 1. Zheng, M.Y., Weng, Y., Sahibzada, R., Konzak, C.F. (2003). Isolated microspore culture in maize (Zea mays L.), production of doubled-haploids via induced androgenesis. In: Maluszynski, M., Kasha, K.J., Forster, B.P., Szarejko, I. (eds) Doubled Haploid Production in Crop Plants. Springer, Dordrecht. https: / / doi.org / 10.1007 / 978-94-017-1293-4_15

[0150] 2. Weiguo Liu, Ming Y. Zheng, Enrique A. Polle, Calvin F. Konzak. Highly Efficient Doubled-Haploid Production in Wheat (Triticum aestivum L.) via Induced Microspore Embryogenesis. 2002. Crop Science Volume 42, Issue 3. Pp. 686 - 692. https: / / doi.org / 10.2135 / cropsci2002.6860

[0151] 3. S.F. Maraschin, W. de Priester, H.P. Spaink, M. Wang, Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. 2005. Journal of Experimental Botany, Volume 56, Issue 417, July 2005, Pages 1711 - 1726, https: / / doi.org / 10.1093 / jxb / eri190.

[0152] 4.Pfister,B.,Zeeman,S.C.Formation of starch in plantcells.Cell.Mol.Life Sci.73,2781-2807(2016).https: / / doi.org / 10.1007 / s00018-016-2250-x.

[0153] 5.Zhong,H.et al.(2018).Advances in Agrobacterium-mediated MaizeTransformation.In:Lagrimini,L.(eds)Maize.Methods in Molecular Biology,vol1676.Humana Press,New York,NY.https: / / doi.org / 10.1007 / 978-1-4939-7315-6_3.

Claims

1. A method for obtaining androgenic microspores, the method comprising reducing the expression of at least one pollen starch biosynthetic pathway gene, wherein the androgenic microspores have an increased androgen response compared to wild-type microspores.

2. The method of claim 1, wherein the expression of the at least one pollen starch biosynthesis pathway gene is reduced by knockout or knockdown.

3. The method of claim 2, wherein the knockout is achieved by gene editing.

4. The method of claim 2, wherein the knockdown is achieved by RNAi.

5. The method of claim 2, wherein the gene editing is performed using a site-directed nuclease selected from the group consisting of a CRISPR nuclease, a meganuclease, a zinc finger nuclease, and a transcription activator-like effector nuclease.

6. The method of claim 5, wherein the site-directed nuclease is a CRISPR nuclease.

7. The method of claim 6, wherein the CRISPR nuclease is selected from the group consisting of Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and nickases or inactivated forms thereof.

8. The method of claim 7, wherein the CRISPR nuclease is Cas12a.

9. The method of claim 3, wherein the knockout is a single gene knockout comprising the use of two gRNAs.

10. The method of claim 9, wherein the two gRNAs comprise SEQ ID NOs: 15 and 16.

11. The method of claim 9, wherein the two gRNAs comprise SEQ ID NOs: 18 and 19.

12. The method of claim 9, wherein the two gRNAs comprise SEQ ID NOs: 21 and 22.

13. The method of claim 9, wherein the two gRNAs comprise SEQ ID NOs: 24 and 25.

14. The method of claim 3, wherein the knockout comprises using multiple gRNAs.

15. The method of claim 14, wherein the gRNA comprises SEQ ID NOs: 15, 16, 18, 19, 21, 22, 27, and 28.

16. The method of claim 2, wherein the pollen starch biosynthesis pathway genes are selected from the group consisting of ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase 1, phosphoglucomutase 2, and invertase 2.

17. The method of claim 16, wherein the ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase, and invertase 2 genes comprise SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

18. The method of claim 1, wherein the increased androgen response is increased by at least 40% compared to the wild type.

19. The method of claim 1, wherein the obtained microspores are selected from the group consisting of maize, rice and wheat.

20. The method of claim 19, wherein the microspores obtained are derived from maize.

21. A method for producing a haploid plant, the method comprising a) obtaining androgenic microspores, wherein the androgenic microspores have an increased androgen response compared to the wild type, b) culturing the microspores, and c) regenerating a haploid plant from the microspores of b).

22. The method of claim 21, wherein the increased androgen response is achieved by knocking out or knocking down at least one pollen starch biosynthesis pathway gene.

23. The method of claim 22, wherein the knockout is achieved by gene editing.

24. The method of claim 22, wherein the knockdown is achieved by RNAi.

25. The method of claim 23, wherein the gene editing is performed using a site-directed nuclease selected from the group consisting of a CRISPR nuclease, a meganuclease, a zinc finger nuclease, and a transcription activator-like effector nuclease.

26. The method of claim 25, wherein the site-directed nuclease is a CRISPR nuclease.

27. The method of claim 26, wherein the CRISPR nuclease is selected from the group consisting of Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and nickases or inactivated forms thereof.

28. The method of claim 27, wherein the CRISPR nuclease is Cas12a.

29. The method of claim 23, wherein the knockout is a single gene knockout comprising the use of two gRNAs.

30. The method of claim 29, wherein the two gRNAs comprise SEQ ID NOs: 15 and 16.

31. The method of claim 29, wherein the two gRNAs comprise SEQ ID NOs: 18 and 19.

32. The method of claim 29, wherein the two gRNAs comprise SEQ ID NOs: 21 and 22.

33. The method of claim 29, wherein the two gRNAs comprise SEQ ID NOs: 24 and 25.

34. The method of claim 23, wherein the knockout comprises using multiple gRNAs.

35. The method of claim 34, wherein the gRNA comprises SEQ ID NOs: 15, 16, 18, 19, 21, 22, 27, and 28.

36. The method of claim 21, wherein the pollen starch biosynthetic pathway genes are selected from the group consisting of ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase, and invertase 2.

37. The method of claim 36, wherein the ADP glucose pyrophosphorylase, Waxy1, hexokinase 5, phosphoglucomutase 1, phosphoglucomutase 2, and invertase 2 genes comprise SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.

38. The method of claim 21, wherein the androgen response is increased by at least 40% compared to the wild type.

39. The method of claim 21, wherein the chromosomes of the haploid plant are doubled to produce a doubled haploid plant.

40. The method of claim 39, wherein the chromosome doubling is achieved via chemical induction or spontaneous doubling.

41. The method of claim 40, wherein the chromosome doubling is achieved via chemical induction.

42. The method of claim 41, wherein the chromosome doubling is achieved by a chemical selected from the group consisting of colchicine, trifluralin, natriuretic, dithiopyr, nitrous oxide and amosulfuron.

43. The method of claim 39, wherein said process of producing doubled haploid plants from microspores occurs in one generation.

44. The method of claim 21, wherein the microspores obtained are selected from the group consisting of maize, rice and wheat.

45. The method of claim 44, wherein the microspores obtained are derived from maize.

46. ​​A plant produced by the method of claim 21.

47. A plant produced by the method of claim 39.

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