Flavonoid-dependent fertility

Abstract

Flavonoid-dependent fertility provides a safe and convenient method for controlling fertility between plants. Mutations to chalcone synthase, quercetin, kaempferol, and other flavonoids have previously been shown to restore fertility in maize and petunia when applied to pollen or flowers. Here, novel gene targets and their mutations have been discovered in wheat, rice, and maize. By mutating these novel genes in each of the three wheat genomes, the inventors provide flavonoid-dependent fertility in wheat. Also provided are rice FDF genes and an improved FDF system in maize.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to U.S. Patent Application No. 63 / 369,975, filed August 1, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to methods for regulating plant fertility, and more particularly to the creation of flavonoid-dependent fertility in wheat and corn plants by mutating novel genes, and the restoration of fertility in these plants by providing fertility-restoring flavonoids to the plant pollen site.

[0003] Sequence Listing This application is accompanied by a Sequence Listing entitled 82692PCT.xml, created on July 24, 2023, which is approximately 200 kilobytes in size. This Sequence Listing is incorporated herein by reference in its entirety. This Sequence Listing is submitted pursuant to the present application and complies with 37 CFR §§ 1.831-1.835. [Background technology]

[0004] Controlling male fertility in crop plants is a key factor required for maintaining seed purity and controlling commodity costs in hybrid seed production. In corn, male fertility control is often achieved by removing male reproductive tissue (detasseling). In both cereals and corn, male fertility can also be controlled using cytoplasmic male sterility (CMS). Detasseling is a high-cost challenge in hybrid seed production, while CMS systems present challenges with high commodity costs in increasing CMS lines, added complexity in breeding, and unstable male fertility in hybrids. A class of alternative hybrid seed production techniques designed to overcome these challenges associated with detasseling and CMS is called "conditional male sterility." In plants bred for conditional male sterility, growers can control whether pollen is viable or sterile by either selecting specific growing conditions or by exogenous application of chemicals that affect pollen viability.

[0005] One form of conditional male sterility in maize and wheat is flavonoid-dependent fertility (FDF). Flavonoids are a class of secondary metabolites in plants that play diverse biological roles, including tissue pigmentation, acting as signaling compounds, and mediating reactive oxygen species (ROS) homeostasis. Flavonoids are thought to be essential for maize and wheat reproduction because they mediate ROS homeostasis during pollen tube germination, which controls pollen tube rupture. These essential flavonoids accumulate in pollen grains before pollen maturation, ensuring that the compounds are readily available upon pollen shedding and attachment to the receptive stigma. Pollen tube rupture is typically a tightly regulated process that maintains the structural integrity of the pollen tube as it elongates through the receptive stigma. When the pollen tube reaches synergids, it initiates rupture, releasing sperm cells and enabling double fertilization, a necessary part of plant sexual reproduction. In maize and wheat plants whose pollen grains lack these essential flavonoids, pollen tube growth is prematurely lost. The pollen tube either does not successfully extend into the stigma, or the tube prematurely bursts before reaching the stigma. This failure of pollen tube growth completely prevents fertilization, and maize and wheat seed plants whose pollen lacks flavonoids are male sterile.

[0006] The use of flavonols to restore fertility has been previously observed in maize (see, e.g., EH Coe, et al., White pollen in maize, J. Heredity 72:318-320 (1981)). Early examples were the result of random mutations affecting two copies of chalcone synthase. One copy of chalcone synthase (WHITE POLLEN1, WHP1) on maize chromosome 2 is expressed in anthers during the late stages of pollen development. Plants homozygous for the whp1 mutant allele produce and shed pollen that is less yellow than typical pollen (colloquially referred to as "white pollen"). When this white pollen is applied to standard wild-type maize silk, normal pollen tube elongation and fertilization can occur. A second chalcone synthase (COLORLESS2, C2) on maize chromosome 4 is expressed in multiple tissue types, including silk. When both the whp1 and c2 mutations are homozygous in the same plant, the plant produces and sheds pollen that is less yellow and unable to elongate normal pollen tubes during self-pollination or fertilize another maize plant by cross-pollination. It was later discovered that this white pollen is conditionally sterile and can be restored to full fertility by adding exogenous flavonoids at the time of pollen-silk interaction (Y. Mo, et al., Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen, Proc. NAT'L ACAD. Sci. USA 89:7213-7217 (1992)). Application of exogenous flavonoids can be achieved by coating mature white pollen grains with dry powdered flavonoid or by applying the flavonoid directly to the silk before applying the white pollen.

[0007] The discovery of the c2 / whp1 conditional male sterility system prompted patent applications seeking to commercialize the technology in maize through multiple approaches, including the use of GM technology and inducible promoters (see U.S. Patent Nos. 5,432,068 and 5,733,759). While theoretically a promising alternative to detasseling or CMS, this technology has not been commercialized for maize seed production. The most significant drawback is the c2 mutation, which affects both male and female reproductive tissues other than pollen. Loss of this critical chalcone synthase gene inhibits downstream synthesis of flavonoids and anthocyanins in silk, anthers, kernel aleurone, and other tissues. This loss of downstream metabolites could potentially disrupt ROS signaling, inhibit responses to abiotic stress in tissues that affect female receptivity and yield, and alter the plant's visual phenotype in ways that are unattractive to growers. Furthermore, the c2 / whp1 system has not been easily translated into crops other than maize. Summary of the Invention [Means for solving the problem]

[0008] Here, we report the discovery of a novel FDF system in maize and wheat based on mutations in an anther-specific chalcone synthase gene. This novel system, termed flavonoid-dependent fertility ("FDF"), exhibits complete male sterility without the significant drawbacks of c2 / whp1, yet can be restored to male fertility by application of exogenous flavonoids. The FDF mutant allele does not affect downstream synthesis of flavonoids and anthocyanins in tissues other than the anther tapetum cell layer and therefore does not affect the color or stress-response capacity of female reproductive tissues.

[0009] Included within the scope of the present invention is a method of seed production comprising: (a) obtaining a first plant that is a flavonoid-dependent fertile ("FDF") plant having a mutation in the FDF gene; (b) obtaining a second plant that is male fertile; (c) pollinating the FDF plant with pollen from the second plant; and (d) obtaining progeny seeds. The FDF gene can be TaFDF1A, TaFDF1B, TaFDF1D, or a combination thereof (SEQ ID NOS: 1-3), or a homolog thereof, or ZmFDF1, or ZmFDF2, or a combination thereof (SEQ ID NOS: 4-5), or a homolog thereof, such as OsFDF in rice. In one aspect, the first plant and the second plant are monocotyledonous plants. In one aspect, the monocotyledonous plant is selected from the group consisting of wheat, maize, and rice.

[0010] Also included within the scope of the present invention are flavonoid-dependent fertile ("FDF") plants comprising a mutation in the FDF gene. The mutation in the FDF gene may be a knockout mutation, or the plant may be homozygous for the knockout mutation in the FDF gene. The FDF gene may be TaFDF1A, TaFDF1B, TaFDF1D, or a combination thereof (SEQ ID NOS: 1-3), or a homolog thereof, or ZmFDF1, or ZmFDF2, or a combination thereof (SEQ ID NOS: 4-5), or a homolog thereof, such as OsFDF in rice. Additionally, the FDF plant further comprises a mutation in C2 / WHP or a homolog thereof. In one embodiment, the plant is a wheat plant. In an alternative embodiment, the plant is a corn plant.

[0011] Another embodiment of the present invention is a method of propagating an FDF plant, the method comprising: (a) obtaining at least one plant, wherein the plant is a flavonoid-dependent fertile ("FDF") plant comprising a mutation in the FDF gene; (b) applying a composition comprising a flavonoid to the plant of step (a); (c) allowing self-pollination to occur; and (d) obtaining progeny seeds thereof.

[0012] The flavonoid may be quercetin, which may be in a liquid solution (optionally mixed in water, propylene glycol, or other solvent or solution). Quercetin may be mixed in a solvent or solution at a concentration of 1 mg / L to 100 mg / L, 2.5 mg / L to 50 mg / L, or about 5 mg / L, or the liquid solution may be saturated with quercetin. Alternatively, the composition containing quercetin may be a powder, which may be a mixture of quercetin and a carrier compound. The mixture of quercetin and the carrier compound may be in a ratio of 1000:1 to 1:1000. The carrier compound may be crystalline silica, talc, metal powder, or mica mineral. In either a liquid or powder formulation, the flavonoid may be applied to the female organs of the flowers of the female parent plant. The flavonoid composition may be applied topically by painting, misting, spraying, or drenching.

[0013] Another embodiment of the present invention is a method for producing hybrid seed, comprising: (a) obtaining at least one inbred female FDF plant, wherein the inbred female FDF plant contains a mutation in the FDF gene; (b) obtaining pollen from at least one inbred male plant, wherein the inbred male plant produces fertile pollen; (c) pollinating the inbred female FDF plant with the fertile pollen from the inbred male plant; and (d) obtaining hybrid progeny thereof. Another embodiment of this method is a plant produced by the previous method.

[0014] Another embodiment of the present invention is a method for restoring fertility to an FDF plant by applying a composition containing a flavonoid to the FDF plant. The FDF plant can be corn, wheat, or rice. In the case of wheat, the FDF wheat plant contains a mutation in a gene selected from the group consisting of SEQ ID NOS: 1-3, and combinations thereof. In the case of corn, the FDF corn plant contains a mutation in a gene selected from the group consisting of SEQ ID NOS: 4-5, and combinations thereof, and optionally further contains a mutation in C2 (SEQ ID NOS: 26) and / or WHP (SEQ ID NOS: 28). When the FDF plant is rice, the FDF rice plant contains a mutation in SEQ ID NOS: 57. The flavonoid can include quercetin, which can be mixed in a liquid such as water, propylene glycol, or other solvent or solution. Alternatively, the flavonoid can be mixed as a powder with a carrier compound such as crystalline silica, talc, metal powder, and mica minerals. The composition can be applied to plant parts such as anthers, silks, stigmas, florets, spikes, leaf whorls, leaf canopy, and / or roots by painting, misting, spraying, and root drenching.

[0015] A brief description of the sequences in the sequence listing SEQ ID NO:1 is TraesCS1A02G160300 (also called TaFDF1A), a gene on chromosome 1A of Triticum aestivum. This gene encodes a functional copy of chalcone synthase that is expressed in developing anthers. Loss of function of this gene is required for FDF in wheat.

[0016] SEQ ID NO:2 is TraesCS1B02G176300 (also called TaFDF1B), a gene on chromosome 1B of Triticum aestivum. This gene encodes a functional copy of chalcone synthase that is expressed in developing anthers. Loss of function of this gene is required for FDF in wheat.

[0017] SEQ ID NO: 3 is TraesCS1D02G157500 (also called TaFDF1D), a gene on chromosome 1D of Triticum aestivum. This gene encodes a functional copy of chalcone synthase that is expressed in developing anthers. Loss of function of this gene is required for FDF in wheat.

[0018] SEQ ID NO: 4 is Zm00001d032662 (also known as ZmFDF1, ZmCHS_Chr1, chalcone synthase 2 (chls2), or GRMZM2G380650), a gene on Zea mays chromosome 1. This sequence was downloaded from maizeGDB.org and refers to the Zm-B73-REFERENCE-GRAMENE-4.0 genome version. This gene encodes a functional copy of chalcone synthase, which is primarily expressed in meiotic tassels and developing anthers, with low or no expression in other tissues. Loss of function of this gene is likely required for FDF in maize.

[0019] SEQ ID NO: 5 is Zm00001d013991 (also known as ZmFDF2, ZmCHS_Chr5, chalcone synthase 11 (chls11), or GRMZM2G477683), a gene on Zea mays chromosome 5. This sequence was downloaded from maizeGDB.org and refers to the Zm-B73-REFERENCE-GRAMENE-4.0 genome version. This gene encodes a functional copy of chalcone synthase that is primarily expressed in meiotic tassels and developing anthers, with low or no expression in other tissues. Loss of function of this gene is likely required for FDF in maize.

[0020] SEQ ID NO:6 is the edited TraesCS1A02G160300. It contains a 1 base pair deletion relative to SEQ ID NO:1.

[0021] SEQ ID NO:7 is the edited TraesCS1B02G176300. It contains a 2 base pair deletion relative to SEQ ID NO:2.

[0022] SEQ ID NO:8 is the edited TraesCS1D02G157500. It contains a 1 base pair deletion relative to SEQ ID NO:3.

[0023] SEQ ID NO:9 is a DNA sequence encoding a guide RNA sequence targeting SEQ ID NO:1.

[0024] SEQ ID NO: 10 is a DNA sequence encoding a guide RNA sequence targeting SEQ ID NO: 2. We found no evidence of genome editing with this guide.

[0025] SEQ ID NO:11 is a repeat of SEQ ID NO:10.

[0026] SEQ ID NO: 12 is construct 25206.

[0027] SEQ ID NO: 13 is TraesCS2A03G12234, the wheat chromosome A ortholog of maize c2 / whp1.

[0028] SEQ ID NO: 14 is TraesCS2B03G140060, the wheat chromosome B ortholog of maize c2 / whp1.

[0029] SEQ ID NO: 15 is TraesCS2D03G11827, the wheat chromosome D ortholog of maize c2 / whp1.

[0030] SEQ ID NO: 16 is construct 27726.

[0031] SEQ ID NO: 17 is construct 27738.

[0032] SEQ ID NO: 18 is construct 27769.

[0033] SEQ ID NOs: 19 to 24 are primers LZ311, LZ312, LZ313, LZ314, LZ315, and LZ316, respectively; see Table 5.

[0034] SEQ ID NO:25 is GRMZM2G380650, which is a repeat of SEQ ID NO:4.

[0035] SEQ ID NO: 26 is the reference sequence for C2 in maize.

[0036] SEQ ID NO: 27 is the reference sequence for c2 in maize.

[0037] SEQ ID NO: 28 is the reference sequence of Whp1 in maize.

[0038] SEQ ID NO: 29 is a DNA sequence encoding a guide RNA sequence designated ZmFDF1\target37.

[0039] SEQ ID NO: 30 is a DNA sequence encoding a guide RNA sequence designated ZmFDF1\target74.

[0040] SEQ ID NO: 31 is a DNA sequence encoding a guide RNA sequence designated ZmFDF2\target72.

[0041] SEQ ID NO: 32 is a DNA sequence encoding a guide RNA sequence designated ZmFDF2\target74.

[0042] SEQ ID NO: 33 is a DNA sequence encoding a guide RNA sequence designated ZmFDF2\target2.

[0043] SEQ ID NO: 34 is a DNA sequence encoding a guide RNA sequence designated ZmFDF2\target16.

[0044] SEQ ID NOs: 35 to 52 are primers and probes listed in Table 9.

[0045] SEQ ID NO: 53 is the nucleotide sequence of Os05g0212900 presented in reverse orientation (3'-5').

[0046] SEQ ID NO: 54 is the nucleotide sequence of Os07g0214900 presented in reverse orientation (3'-5').

[0047] SEQ ID NO: 55 is the nucleotide sequence of Os07g0501100 presented in reverse orientation (3'-5').

[0048] SEQ ID NO: 56 is the nucleotide sequence of Os11g0530600 presented in reverse orientation (3'-5').

[0049] SEQ ID NO: 57 is the nucleotide sequence of OsFDF (also referred to as Os10g0484800) presented in the 5'-3' direction

[0050] definition All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined below. References to technology used herein are intended to refer to technology as commonly understood in the art, including variations of those technologies and / or equivalent technology alternatives that would be apparent to those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, definitions are provided below to facilitate description of the subject matter of the present disclosure.

[0051] The terms "a," "an," and "the," when used in this application, including the claims, refer to "one or more." For example, the phrase "a cell" refers to one or more cells, and in certain embodiments, may refer to a tissue and / or an organ. Similarly, the phrase "at least one," when used herein to refer to an entity, refers to, 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 relevant entity, including, but not limited to, all integer values between 1 and 100, as well as integer values greater than 100.

[0052] The term "about," as used herein, refers to the normal range of error for the respective value, readily known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5% within the intended meaning of the recited value.

[0053] The term "allele" refers to any of one or more alternative forms of a gene, all of which relate to at least one trait or characteristic. In diploid cells, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. In some instances (e.g., for QTLs), it is more accurate to refer to a "haplotype" (i.e., an allele of a chromosome segment) instead of an "allele," but in those instances, the term "allele" should be understood to include the term "haplotype." When two individuals (e.g., two plants) have the same allele at a particular locus, the alleles are said to be "homoeologous" if they are inherited from a common ancestor (i.e., the alleles are copies of the same parental allele). Otherwise, the alleles are "identical by state" (i.e., the alleles appear identical but are derived from two different copies of the allele). Homoeologous information is useful for linkage analysis; both homoeologous and homoeologous information can be used in association studies, but homoeologous information can be particularly useful.

[0054] As used herein, the term "and / or," when used in reference to a list of entities, refers to those entities present either singly or in combination. Thus, for example, the phrase "A, B, C and / or D" includes not only A, B, C, and D individually, but also 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, one or more of the elements referred to by "and / or" may also be present individually in single or multiple occurrences in combinations and / or subcombinations.

[0055] An "anther-specific gene" is defined as a gene that has high expression in developing anthers, including developing microspores, and low or no expression in all other tissues tested, including female reproductive tissues.

[0056] The term "backcrossing" is understood within the scope of this disclosure to refer to a process in which hybrid progeny are repeatedly crossed back to one of the parents.

[0057] The term "carrier" as used herein means a compound, preferably in powder form, that acts as an agent to accompany the collected pollen. Suitable carrier compounds can be, but are not limited to, talc powder, silica powder, etc.

[0058] As used herein, the terms "comprising" or "comprise" are open-ended. When used in the context of a subject nucleic acid (or amino acid sequence), it refers to a nucleic acid sequence (or amino acid sequence) that includes the subject sequence as a portion or in its entirety.

[0059] As used herein, the term "elite line" or "inbred line" refers to any line resulting from breeding and selection for superior agronomic performance. An elite line has genetic stability, i.e., it is reasonably or nearly isogenic across its genome. Stated differently, an elite line is reasonably or nearly homozygous for all alleles in its genome.

[0060] The term "FDF gene" (and also used interchangeably throughout "FDF allele") refers to a gene that, when mutated or otherwise defective or inhibited, renders a plant sterile due to a flavonoid deficiency, and further, the deficiency can be corrected by application of a flavonol and / or flavonoid. For example, wheat FDF genes include TraesCS1A02G160300 (SEQ ID NO: 1; also referred to as TaFDF1A), TraesCS1B02G176300 (SEQ ID NO: 2; also referred to as TaFDF1B), and TraesCS1D02G157500 (SEQ ID NO: 3; also referred to as TaFDF1D). For example, maize FDF genes include Zm00001d032662 (SEQ ID NO: 4; also referred to as ZmFDF1 or ZmCHS_Chr1) and Zm00001d013991 (SEQ ID NO: 5; also referred to as ZmFDF2 or ZmCHS_Chr5).

[0061] The term "gene" refers to a hereditary unit comprising a sequence of DNA that occupies a specific location on a chromosome and contains the genetic instructions for a particular characteristic or trait in an organism.

[0062] "Gene editing" generally refers to the use of site-specific nucleases (including, but not limited to, CRISPR / Cas, zinc fingers, meganucleases, etc.) to cleave a nucleotide sequence at a desired location. This may result in an insertion / deletion ("indel") mutation (i.e., "SDN1"), base editing (i.e., "SDN2"), or allele insertion or replacement (i.e., "SDN3"). SDN2 or SDN3 gene editing may involve providing one or more recombination templates (e.g., in vectors) containing the gene sequence of interest that can be used for homology-directed repair (HDR) in a plant (i.e., to be introduced into the plant genome).

[0063] Breaks in the plant genome may be introduced within, upstream of, and / or downstream of the target sequence. In some embodiments, double-stranded DNA breaks are made within or near the target sequence locus. In some embodiments, breaks are made upstream and downstream of the target sequence locus, which may lead to its excision from the genome. In some embodiments, one or more single-stranded DNA breaks (nicks) are made within, upstream of, and / or downstream of the target sequence (e.g., using a nickase Cas9 mutant). Such breaks may be repaired through the process of non-homologous end joining (NHEJ), which can result in the generation of small insertions or deletions (indels) at the repair site. Such indels may lead to frameshift mutations or other types of loss-of-function mutations that cause premature stop codons in the targeted gene.

[0064] In some embodiments, gene editing may involve transient, inducible, or constitutive expression of the gene editing component or system in the target plant. Gene editing may also involve genomic integration or episomal presence of the gene editing component or system in the target plant.

[0065] In certain embodiments, nucleic acid modification or mutation is carried out by (modified) zinc finger nuclease (ZFN) system. ZFN system uses artificial restriction enzymes, which are produced by fusing zinc finger DNA binding domains with DNA cleavage domains, which can be engineered to target desired DNA sequences. Exemplary methods of genome editing using ZFN can be found in, for example, U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; and 6,979,539.

[0066] In certain embodiments, nucleic acid modification is accomplished by (modified) meganucleases, which are endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methods for using meganucleases can be found in U.S. Patent Nos. 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134.

[0067] In certain embodiments, the nucleic acid modification is accomplished by a (modified) CRISPR / Cas complex or system. In certain embodiments, the CRISPR / Cas system or complex is a Class 2 CRISPR / Cas system. In certain embodiments, the CRISPR / Cas system or complex is a Type II, Type V, or Type VI CRISPR / Cas system or complex. CRISPR / Cas systems do not require the production of specialized proteins to target specific sequences; instead, a single Cas protein can be programmed by an RNA guide (gRNA) to recognize a specific nucleic acid target; in other words, the Cas enzyme protein can be recruited to a specific nucleic acid target locus of interest (which may comprise or consist of RNA and / or DNA) using the short RNA guide.

[0068] In general, CRISPR / Cas or CRISPR system, as used herein in the aforementioned documents, refers collectively to the transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, including sequences encoding Cas genes and one or more tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr mate sequences (including "direct repeats" and tracrRNA-processing partial direct repeats in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems) or "RNAs" as that term is used herein (e.g., RNAs that guide Cas, such as Cas9, e.g., CRISPR RNAs and, if applicable, trans-activating (tracr) RNAs or single guide RNAs (sgRNAs) (chimeric RNAs)), or other sequences and transcripts from a CRISPR locus. Generally, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of a target sequence (also referred to as protospacers in the context of endogenous CRISPR systems). In the context of CRISPR complex formation, "target sequence" refers to a sequence that a guide sequence is designed to be complementary to, and hybridization between the target sequence and the guide sequence promotes the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide.

[0069] In certain embodiments, the gRNA is a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, the gRNA comprises a guide sequence and a tracr mate sequence (or direct repeat). In certain embodiments, the gRNA comprises a guide sequence, a tracr mate sequence (or direct repeat), and a tracr sequence. In certain embodiments, the CRISPR / Cas systems or complexes described herein do not include and / or are not dependent on the presence of a tracr sequence (e.g., when the Cas protein is Cas12a).

[0070] The Cas proteins referred to herein, including but not limited to Cas9, Cas12a (formerly called Cpf1), Cas12b (formerly called C2c1), Cas13a (formerly called C2c2), C2c3, and Cas13b proteins, may originate from any suitable source and thus include various orthologs originating from various (prokaryotic) organisms, as is well described in the art. In certain embodiments, the Cas protein is a (modified) Cas9, preferably a (modified) Staphylococcus aureus Cas9 (SaCas9) or a (modified) Streptococcus pyogenes Cas9 (SpCas9). In certain embodiments, the Cas protein is Cas12a, optionally Acidaminococcus sp., e.g., Acidaminococcus sp. BV3L6 Cpf1 (AsCas12a), or Lachnospiraceae bacterium Cas12a, e.g., Lachnospiraceae bacterium MA2020 or Lachnospiraceae bacterium MD2006 (LBCas12a). See U.S. Patent No. 10,669,540. Alternatively, the Cas12a protein may be from Moraxella bovoculi AAX08_00205 [Mb2Cas12a] or Moraxella bovoculi AAX11_00205 [Mb3Cas12a]. See WO 2017 / 189308. In certain embodiments, the Cas protein is a (modified) C2c2, preferably Leptotrichia wadei C2c2 (LwC2c2) or Listeria newyorkensis FSL M6-0635 C2c2 (LbFSLC2c2). In certain embodiments, the (modified) Cas protein is C2c1. In certain embodiments, the (modified) Cas protein is C2c3.In certain embodiments, the (modified) Cas protein is Cas13b. Other Cas enzymes are available to those skilled in the art.

[0071] The term "genotype" and variations thereof refer to the genetic composition of an organism, including, for example, whether a diploid organism is heterozygous (i.e., has two different alleles for a given gene or QTL) or homozygous (i.e., has the same allele for a given gene or QTL) for one or more genes or loci (e.g., SNPs, haplotypes, genetic mutations, insertions, or deletions). As used herein, the term "at least heterozygous" for a particular allele indicates that at least one copy of the allele is present. For example, a corn plant that is at least heterozygous for the HI allele of a gene has one or two copies of the HI allele (i.e., is heterozygous or homozygous).

[0072] The term "germplasm" refers to the genotypic totality of a population or other group of individuals (e.g., a species or plant line). The phrase "adapted germplasm" refers to plant material with proven genetic superiority, e.g., for a given environment or geographic area, while the phrases "unadapted germplasm," "natural germplasm," and "exotic germplasm" refer to plant material with unknown or unproven genetic value, e.g., for a given environment or geographic area; thus, the phrase "unadapted germplasm," in some embodiments, refers to plant material that is not part of an established breeding population and has no known relationship to members of an established breeding population.

[0073] The term "haplotype" can refer to a set of alleles inherited by an individual from one parent. A diploid individual therefore has two haplotypes. The term "haplotype" can also be used in a more limited sense to refer to physically linked and / or unlinked genetic markers (e.g., sequence polymorphisms) associated with a phenotypic trait. The phrase "haplotype block" (sometimes simply referred to in the literature as a haplotype) refers to a group of two or more genetic markers that are physically linked on a single chromosome (or a portion thereof). Typically, each block has a small number of common haplotypes, and a subset of genetic markers (i.e., a "haplotype tag") can be selected that uniquely identifies each of these haplotypes.

[0074] The term "heterosis" refers to hybrid vigor, i.e., the improvement or enhancement of the performance of any biological trait (e.g., size, growth rate, fertility, yield, etc.) in the offspring of a hybrid compared to its parents. For example, the offspring of a cross between inbred plant lines from different heterosis groups may exhibit more heterosis than their parent lines, as described above. The first generation offspring of such a cross generally exhibit the desired characteristics of both parents to a greater extent. When first generation hybrids are crossed with each other, this heterosis may decrease in subsequent generations.

[0075] The terms "heterosis group" and "heterosis pool" are used interchangeably and refer to a group of genotypes or inbred lines that exhibit similar heterosis responses when crossed with genotypes or inbred lines from other genetically distinct germplasm groups. When comparing between heterosis groups, lines that are more closely related are contained within the heterosis group relative to lines that are more distantly related. Generally, hybrids of two inbred lines crossed with each other within the same heterosis group exhibit much less heterosis than hybrids in which an inbred line from one heterosis group is crossed with an inbred line from a different heterosis group. A particular heterosis group can contain a large number of lines with widely varying genetic properties.

[0076] The terms "hybrid," "hybrid plant," and "hybrid progeny," in relation to plant breeding, refer to plants that are the offspring of genetically dissimilar parents (e.g., genetically heterozygous or mostly heterozygous individuals) produced by crossing plants of different lines or cultivars or species, including, but not limited to, crosses between two inbred lines. The phrase "single-cross FI hybrid" refers to an FI hybrid produced from a cross between two inbred lines.

[0077] The phrase "inbred line" refers to a genetically homozygous or near-homozygous population. Inbred lines can be derived, for example, through several cycles of brother / sister breeding or selfing. In some embodiments, inbred lines breed purebreds for one or more phenotypic traits of interest. An "inbred line," "inbred individual," or "inbred progeny" is an individual sampled from an inbred line. The term "inbred line" refers to a substantially homozygous individual or line. Inbred lines may also be referred to as "parent lines" when used in a breeding program.

[0078] The terms "introgression," "introgressed," and "introgressing" refer to both natural and artificial processes in which a genomic region of one species, variety, or cultivar is transferred to the genome of another species, variety, or cultivar by crossing the species. The process may optionally be completed by backcrossing with the recurrent parent.

[0079] The term "loss-of-function mutation" refers to a change in the DNA sequence of a gene (i.e., a "mutation") that results in a mutated gene product lacking the molecular function of the wild-type gene. Four major genetic alterations can lead to loss-of-function mutations: 1) mutations resulting in a premature stop codon, producing a truncated protein sequence; 2) mutations occurring at canonical splice sites that affect splicing (resulting in the inclusion of an intron or the elimination of an exon in the mRNA transcript); 3) insertion or deletion variants with a non-integer multiple of three located in the gene coding region, causing a frameshift by disrupting the complete transcript; and 4) mutations resulting in the loss of a start codon (e.g., ATG), preventing gene transcription if no alternative start codon is present near the mutation. Additionally, mutations in the promoter or untranslated region (UTR) of a gene can reduce or eliminate gene expression, resulting in loss of function. The term "knockout mutation" is used interchangeably.

[0080] The terms "nucleic acid" and "polynucleotide" are used interchangeably and, as used herein, refer to both sense and antisense strands of RNA, cDNA, genomic DNA, mitochondrial DNA, and synthetic forms, as well as mixed polymers of the above. In certain embodiments, nucleotide refers to ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotide, and combinations thereof. The term also includes, but is not limited to, single- and double-stranded forms of DNA and / or RNA. In addition, polynucleotides disclosed herein, such as circular DNA templates and nucleic acid concatemers disclosed herein, may contain either or both naturally occurring and modified nucleotides linked to each other by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications, such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to encompass any conformation of any topology, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlock conformations. A reference to a nucleic acid sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand with its complementary sequence. Nucleotide sequences are "complementary" if they hybridize specifically in solution (e.g., according to Watson-Crick base-pairing rules).The term also includes codon-optimized nucleic acids that encode the same polypeptide sequence. It is also understood that the nucleic acid may be crude, purified, or attached to synthetic materials such as beads or column matrices.

[0081] As used herein, the terms "nucleotide sequence," "polynucleotide," "nucleic acid sequence," "nucleic acid molecule," and "nucleic acid fragment" refer to a polymer of RNA or DNA that is single- or double-stranded and optionally contains synthetic, non-natural, and / or altered nucleotide bases. A "nucleotide" is the monomeric unit from which DNA or RNA polymers are built, and consists of a purine or pyrimidine base, a pentose, and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their one-letter abbreviation as follows: "A" stands for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" stands for cytidylate or deoxycytidylate, "G" stands for guanylate or deoxyguanylate, "U" stands for uridylate, "T" stands for deoxythymidylate, "R" stands for purine (A or G), "Y" stands for pyrimidine (C or T), "K" stands for G or T, "H" stands for A or C or T, "I" stands for inosine, and "N" stands for any nucleotide.

[0082] The term "offspring" plant refers to any plant resulting as a descendant from one or more parent plants or their progeny by vegetative or sexual reproduction. For example, offspring plants may be obtained by cloning or selfing a parent plant or by crossing two parent plants, including selfed and F1 or F2 or further generations. F1 is the first generation offspring produced from parents, at least one of which is used for the first time as a trait donor, while second generation (F2) or subsequent generation (F3, F4, etc.) offspring are specimens produced from selfing F1s, F2s, etc. Thus, F1 can be a hybrid resulting from a cross between two true breeding parents, while F2 can be offspring resulting from self-pollination of the F1 hybrid.

[0083] The term "PCR (polymerase chain reaction)" is understood within the scope of the present invention to refer to a method for producing relatively large amounts of specific regions of DNA, thereby allowing various analyses based on those regions.

[0084] "Phenotype" is understood within the scope of this disclosure to refer to the distinguishable characteristics of genetically controlled traits. The phrase "phenotypic traits" refers to the appearance or other detectable characteristics of an individual that result from the interaction of the environment and its genome.

[0085] A "plant" is any plant at any stage of development, particularly a seed plant. In particular, in the context of this disclosure, plant means a corn plant.

[0086] A "plant cell" is the structural and physiological unit of a plant, including the protoplast and cell wall. Plant cells may be in the form of isolated single cells or cultured cells, or as part of a more highly organized unit, such as a plant tissue, a plant organ, or a whole plant.

[0087] The term "population" refers to a genetically heterogeneous collection of plants that share a common genetic origin.

[0088] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to (and in some embodiments, specifically annealing to) a nucleic acid target, allowing a DNA polymerase and / or reverse transcriptase to attach to it, thereby serving as a point of initiation for DNA synthesis when placed under conditions that induce the synthesis of a primer extension product (e.g., in the presence of nucleotides and an agent for polymerization, such as a DNA polymerase, and at a suitable temperature and pH). In some embodiments, one or more primers are used to amplify plant nucleic acids (e.g., using the polymerase chain reaction, PCR).

[0089] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a strand of a double-stranded or higher-order nucleic acid, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, a probe is of sufficient length to form a stable and sequence-specific duplex molecule with its complement, and thus, in certain embodiments, can be used to detect a sequence of interest present in multiple nucleic acids.

[0090] The term "progeny" refers to the offspring of a particular cross. Typically, progeny result from the breeding of two individuals, although some species (particularly some plants and hermaphroditic animals) are capable of selfing (i.e., the same plant serves as the donor of both male and female gametes). The progeny may be, for example, an F1, F2, or any subsequent generation.

[0091] The term "random mutagenesis" refers to an alternative method of mutation. In certain embodiments, nucleic acid modification is performed by random mutagenesis. Cells or organisms can be exposed to mutagens such as UV radiation or mutagenic chemicals (e.g., ethyl methanesulfonate (EMS)), and then mutants with desired properties are selected. Mutants can be identified, for example, by TILLING (targeted induced local lesions in the genome). This method combines mutagenesis using chemical mutagens such as ethyl methanesulfonate (EMS) with highly sensitive DNA screening techniques to identify single-base / point mutations in target genes. The TILLING method relies on the formation of DNA heteroduplexes, which are formed when multiple alleles are amplified by PCR, then heated and slowly cooled. A "bubble" forms at the mismatch between the two DNA strands, which is then cleaved by a single-stranded nuclease. The products are then separated by size, for example, by HPLC. See McCallum, et al., Targeted screening for induced mutations, Nat. Biotechnol. 18(4):455-57 (2000) and McCallum, et al., Targeting induced local lesions IN genomes (TILLING) for plant functional genomics, Plant Physiol. 123(2):439-42 (2000).

[0092] The phrases "sexually mated" and "sexual reproduction" in the context of this disclosure refer to the fusion of gametes to produce offspring (e.g., by fertilization, such as to produce seeds by pollination in a plant). In some embodiments, "sexual mating" or "cross-fertilization" is the fertilization of one individual by another (e.g., cross-pollination in a plant). In some embodiments, the term "selfing" refers to self-fertilization or the production of seeds by self-pollination; i.e., the pollen and ovules are from the same plant.

[0093] The terms "solvent" and / or "solution" refer to a liquid in which the flavonoid can be mixed. Examples include water, alcohol, propylene glycol, DMSO, Tris buffer, and other liquids available to those skilled in the art. The flavonoid does not need to be dissolved in a liquid; a mixture or emulsion may suffice.

[0094] "Transformable," "transformable," and the like refer to a plant, plant line, or plant cell (such as callus tissue or protoplast) that is more readily able to accept foreign DNA and stably integrate the foreign DNA into its genome.

[0095] The term "variety" or "cultivar" means a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or performance. DETAILED DESCRIPTION OF THE INVENTION

[0096] An embodiment of the present invention is a seed production method comprising: (a) obtaining at least one first plant, wherein the first plant is a flavonoid-dependent sterile ("FDF") plant comprising a mutation in the FDF gene; (b) obtaining at least one second plant, wherein the second plant is male fertile; (c) allowing the at least one second plant to pollinate the at least one first plant; and (d) obtaining progeny seeds thereof. In one aspect, the first plant and the second plant are monocotyledonous plants. In a further aspect, the monocotyledonous plant is selected from the group consisting of wheat, maize, and rice. In another aspect, the FDF gene is selected from the group consisting of SEQ ID NOs: 1-5 and 57.

[0097] Another embodiment of the present invention is a flavonoid-dependent fertile ("FDF") plant comprising a mutation in the FDF gene. In one aspect, the mutation in the FDF gene is a knockout mutation. In another aspect, the plant is homozygous for the knockout mutation in the FDF gene. In one aspect, the FDF plant is a wheat plant, and the FDF gene is selected from TaFDF1A (SEQ ID NO: 1), TaFDF1B (SEQ ID NO: 2), TaFDF1D (SEQ ID NO: 3), and combinations thereof. In another aspect, the FDF plant is a corn plant, and the FDF gene is ZmFDF1 (SEQ ID NO: 4) or ZmFDF2 (SEQ ID NO: 5), or both. In a further aspect, the FDF plant comprises a mutation in C2 / WHP. In another aspect, the FDF plant is a rice plant, and the FDF gene is OsFDF (SEQ ID NO: 57).

[0098] Another embodiment of the present invention is a method for propagating FDF plants, comprising: (a) obtaining at least one plant, wherein the plant is a flavonoid-dependent fertile ("FDF") plant containing a mutation in the FDF gene; (b) applying a composition comprising a flavonoid to the plant of step (a); (c) allowing self-pollination to occur; and (d) obtaining progeny seeds thereof. In one aspect, the flavonoid is quercetin. In another aspect, the composition comprising quercetin is a liquid solution. In another aspect, the liquid solution comprises quercetin mixed in water, propylene glycol, or other solvent or solution. In yet another aspect, the liquid solution comprises quercetin suspended in a solvent or solution at a concentration of 1 mg / L to 100 mg / L, 2.5 mg / L to 50 mg / L, or approximately 5 mg / L. In yet another aspect, the liquid solution comprising quercetin is saturated. In an alternative aspect, the composition comprising quercetin is a powder. In one aspect, the powder comprises a mixture of quercetin and a carrier compound. In one aspect, the mixture of quercetin and the carrier compound is in a ratio of 1000:1 to 1:1000. In another aspect, the carrier compound is selected from the group consisting of crystalline silica, talc, metal powder, and mica mineral. In yet another aspect, a composition comprising a flavonoid is applied to the female organ of a flower on at least one plant designated as a female plant. In yet another aspect, the composition is applied by a method selected from the group consisting of painting, misting, spraying, and root drenching. In one aspect, the FDF gene is a wheat gene selected from the group consisting of SEQ ID NOs: 1-3 and combinations thereof. In another aspect, the FDF gene is a maize gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof. In yet another aspect, the FGF gene is a rice gene comprising SEQ ID NO: 57. One embodiment of the present invention is a plant produced by the method described above.

[0099] Another embodiment of the present invention is a method for hybrid seed production, comprising: (a) obtaining at least one inbred female FDF plant, wherein the inbred female FDF plant contains a mutation in the FDF gene; (b) obtaining pollen from at least one inbred male plant, wherein the inbred male plant produces fertile pollen; (c) pollinating the inbred female FDF plant with the fertile pollen from the inbred male plant; and (d) obtaining hybrid progeny thereof. Another embodiment of this method is a plant produced by the previous method. In one aspect, the inbred female FDF plant is a corn plant, a wheat plant, or a rice plant. In another aspect, the FDF wheat plant contains a mutation in a gene selected from the group consisting of SEQ ID NOs: 1-3, and combinations thereof. In yet another aspect, the FDF corn plant contains a mutation in a gene selected from the group consisting of SEQ ID NOs: 4-5, and combinations thereof. In yet another aspect, the FDF rice plant contains a mutation in SEQ ID NO: 57. An embodiment of the present invention is a plant produced by the method described above.

[0100] Another embodiment of the present invention is a method for restoring fertility to an FDF plant containing a mutation in the FDF gene, the method comprising applying a composition comprising a flavonoid to the FDF plant. In one aspect, the FDF plant is a corn plant, a wheat plant, or a rice plant. In another aspect, the FDF wheat plant contains a mutation in a gene selected from the group consisting of SEQ ID NOS: 1-3, and combinations thereof. In yet another aspect, the FDF corn plant contains a mutation in a gene selected from the group consisting of SEQ ID NOS: 4-5, and combinations thereof. In yet another aspect, the FDF corn plant further contains a mutation in a chalcone synthase gene. In yet another aspect, the FDF rice plant contains a mutation in SEQ ID NOS: 57. In one aspect, the flavonoid comprises quercetin. In another aspect, the quercetin is mixed in water, propylene glycol, or other solvent or solution. In an alternative aspect, the composition comprising a flavonoid further comprises a carrier compound. In an aspect, the carrier compound is selected from the group consisting of crystalline silica, talc, metal powder, and mica minerals. In one aspect, the flavonoid-containing composition is applied to anthers, silks, stigmas, florets, spikes, leaf whorls, canopy, or roots. In an aspect, the composition is applied by a method selected from the group consisting of painting, misting, spraying, and root drenching. [Example]

[0101] 1. Identification of the FDF gene in maize. The anther-specific chalcone synthase gene was first identified in maize during studies of the causal mutations underlying the publicly available colorless2 (c2) and whitepollen1 (whp1) alleles in the maize mutant line 224H. In the 224H line, whitepollen1 is homozygous for the recessive whp1 mutant allele and segregates for the mutant colorless2 allele, c2. A male sterility phenotype is observed in individual plants homozygous for c2, while plants heterozygous or homozygous for C2 are male fertile. In the 224H line, colorless2 zygosity is visually monitored by the presence or absence of purple aleurone. Embryos homozygous or heterozygous for wild-type C2 are able to synthesize anthocyanins in the grain aleurone, resulting in visually purple kernels. Embryos homozygous for the c2 mutant are unable to synthesize anthocyanins in the kernel aleurone, resulting in a colorless aleurone that clearly exhibits a colorless (white) endosperm underneath (in the 224H line, the endosperm is always white; endosperm color is controlled by an independent, unrelated gene, Y1). While these mutant alleles are known to the maize research community, we were unable to identify a published source for the causative mutations underlying these mutant alleles. To identify the causative mutations underlying colorless2, we planted a population of 224H seeds segregating for c2 (mutant) and C2 (wild type). Tissue samples were collected, and the entire colorless2 gene was amplified using a set of PCR primers. PCR products were sequenced by Sanger sequencing. SNP haplotypes distinct from C2 in the B73, NP2222, and W22 reference genomes (Table 1) were identified as segregating 1:2:1 in the 224H plant population. Multiple seedlings derived from white kernels were found to be homozygous for the SNP haplotype. The whp1 allele was also amplified with PCR primers, and the PCR products were sequenced by Sanger sequencing for comparison with the B73, AX5707, and W22 reference genomes.No putative causative mutation in the whp1 mutant allele was identified.

[0102] [Table 1]

[0103] Further investigation of the c2 SNP haplotype focused on the C1973T SNP, which resulted in an R72C amino acid substitution. This amino acid substitution is not part of the chalcone synthase catalytic triad. Without wishing to be bound by theory, the radical amino acid substitution of a positively charged side chain on arginine to a thiol side chain on cysteine likely has a deleterious effect on protein function. The first evidence supporting the causal role of the C1973T SNP was found in publications detailing that the arginine at amino acid position 72 in C2 is part of a conserved motif required for coenzyme A binding in chalcone synthases (see, e.g., J.L. Ferrer, et al., Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis, Nature Structural Biol. 6(8):775-784 (1999) and M.B. Austin & J.P. Noel, The chalcone synthase superfamily of type III polyketide synthases, Nat. Prod. Rep. 20:79-110 (2003)). Further evidence was identified by comparing the mutant c2 amino acid sequence containing the R72C substitution with the wild-type C2 sequence using PROVEAN software (Y. Choi & A.P. Chan, PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels, Bioinformatics 31(16):2745-2747 (2015).). The software returned a score of -6.055 for the R72C amino acid substitution, predicting that this mutation is deleterious to protein function.Finally, the project team confirmed that a similar amino acid substitution had been identified in a Matthiola incana mutant line (V. Hemleben, et al., Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae), Plant Mol. Biol. 55:455-465 (2004)). Hemleben et al. characterized the R72S amino acid substitution, which eliminates chalcone synthase protein activity and results in a colorless flower phenotype in Matthiola incana, and further emphasized that the substitution at the R72 position results in a loss of function and subsequent inability to synthesize flavonoids required for pigment synthesis.

[0104] Based on the identification of the mutation responsible for the c2 mutant allele in maize, the inventors began searching for orthologs of c2 in wheat that could be targeted using gene editing, including, but not limited to, CRISPR-based editing. During this search in wheat, additional chalcone synthase genes in maize were identified, including chalcone synthase 2 (chls2) GRMZM2G380650 (SEQ ID NO: 4). By including chls2 in the analysis, the project team identified additional chalcone synthase genes with anther-specific expression patterns (TraesCS1A02G160300 (SEQ ID NO: 1), TraesCS1B02G176300 (SEQ ID NO: 2), and TraesCS1D02G157500 (SEQ ID NO: 3)). These additional chalcone synthase genes were found to have anther-specific expression patterns and were included as candidate genome editing targets for generating FDS in wheat. According to the Kyoto Enzyme Gene and Genomes (KEGG) database (www.genome.jp / kegg / annotation / enzyme.html), maize has six chalcone synthases that catalyze the same enzymatic step in the flavonoid biosynthesis pathway (this step is designated EC 2.3.1.74). These six chalcone synthases include C2, WHP1, and CHLS2. To determine whether orthologs other than CHLS2 should be included as candidates for editing to create an FDF system in maize, expression patterns across cellular tissues were analyzed using RNA-seq data from various developmental stages in two datasets: publicly hosted RNA expression at MaizeGDB.org and an internal RNA-seq atlas. The qualitative results for the six maize genes catalyzing EC step 2.3.1.74 are summarized in Table 2. Four of these genes (C2, WHP1, CHLS2, and CHLS11) all had very high expression in anther tissue. A BLAST search showed that CHLS11 was found to be the closest hit to CHLS2, with 92% sequence identity at the nucleotide level, even higher when comparing only the coding sequences.Due to its expression pattern, enzyme profile, and sequence similarity, CHLS11 was also included as a promising editing candidate for maize FDF.

[0105] [Table 2]

[0106] 1A. Identification of candidate genes for FDF in rice After identifying Zmchls2 and Zmchls11 as targets for FDF, we sought to identify chalcone synthase genes in rice that could be targeted for the creation of an FDF system. In rice (Oryza sativa), there is only a single ortholog for ZmC2 and ZmWHP: OsCHS1 (Os11g0530600). Mutations in OsCHS1 have previously been reported to cause male sterility (Wang, Lanxiang, Pui Ying Lam, Andy CW Lui, Fu-Yuan Zhu, Mo-Xian Chen, Hongjia Liu, Jianhua Zhang, and Clive Lo. 2020. “Flavonoids Are Indispensable for Complete Male Fertility in Rice.” Journal of Experimental Botany 71(16):4715–28. doi.org / 10.1093 / jxb / eraa204). However, no reference to orthologs of ZmCHLS2 and ZmCHLS11 has been reported to date. Using BLAST searches to compare the genomic DNA sequences, spliced RNA sequences, and protein amino acid sequences between maize ZmCHLS2 and ZmCHLS11 and the rice genome (NCBI, genome assembly IRGSP-1.0), the best match was the rice gene OsFDF, which has 85% sequence similarity with its maize ortholog (Table 3).

[0107] [Table 3]

[0108] The rice gene OsFDF has not been assigned to any metabolic step in the KEGG database, so it is unclear whether it catalyzes KEGG enzyme step 2.3.1.74, the step that catalyzes flavonoid biosynthesis by the maize genes ZmC2, ZmWHP1, ZmCHLS2, and ZmCHLS11. However, it is predicted by Uniprot to be a "chalcone and stilbene synthase" based on protein domain and structure (www.uniprot.org / uniprotkb / Q7X795 / entry), the same protein class to which the maize FDF gene is assigned. There are four other rice genes predicted to catalyze step 2.3.1.74 (Table 4), but when their expression patterns were compared to OsFDF in an internal RNA-seq dataset, only one of these four genes (Os07g0411300) showed an expression pattern enriched in the highly male reproductive tissues required for FDF. OsFDF is most strongly expressed in male tissues (Table 4). The combination of its sequence similarity, the presence of a chalconestilbene synthase protein domain, and the required male-enriched gene expression pattern makes OsFDF an ideal candidate for mutation to establish the FDF system in rice.

[0109] [Table 4]

[0110] 2. Wheat transformation. We designed a binary vector that would generate loss-of-function alleles in the TraesCS1A02G160300, TraesCS1B02G176300, and TraesCS1D02G157500 genome editing targets. A wheat-codon-optimized version of the Cas9 enzyme from Streptococcus pyogenes was selected to generate double-strand breaks in the targeted genes. This version of Cas9 contained the L1181V and L1196V amino acid substitutions at both the N- and C-termini of the amino acid sequence, as well as a nuclear localization sequence. Cas9 transcription was driven by the constitutive sugarcane ubiquitin 4 promoter from Saccharum officinarum. This promoter was enhanced by three elements: the nopaline synthase enhancer from Agrobacterium tumefaciens, the figwort mosaic virus enhancer from figwort mosaic caulimovirus, and the cauliflower mosaic virus 35S enhancer region from cauliflower mosaic caulimovirus. A terminator sequence based on the maize metallothionine-like gene was included after Cas9 to terminate transcription.

[0111] Two separate dual guide RNAs targeting the first exons in TraesCS1A02G160300, TraesCS1B02G176300, and TraesCS1D02G157500 were designed based on consensus sequences from Chinese spring wheat. One guide RNA targeted the fourth codon in each of the three chalcone synthase gene targets and was designated TaGHSGTarget1. The second guide RNA targeted the 51st codon in each of the three chalcone synthase gene targets and was designated TaGHSGTarget2. Guide RNA transcription was driven by the constitutive sugarcane ubiquitin 4 promoter from Saccharum officinarum, which does not contain an enhancer element. For efficient guide RNA processing, TaGHSGTarget1 and TaGHSGTarget2 were separated into HammerHead RNA sequences from the transactivating crRNA sequence derived from Streptococcus pyogenes. After transcription, these units form combined guide RNA and crRNA sequences for each of the two guide RNAs (referred to herein as a "dual-guide" system). These combined guide RNA and crRNA sequences were designated rCrRNATaCHSG-01 and rCrRNATaCHSG-02. An additional sequence for the transactivating crRNA was included in the design and designated rTracrRNA. To further promote efficient guide RNA processing, the design included a self-cleaving hammerhead RNA derived from tobacco etch potyvirus, designated rHH, and multiple copies of a hammerhead element with pairing sequences specific for each guide RNA and crRNA complex, designated rHHTaCHSG-01 and rHHTaCHSG-02, respectively. The multiple copies of the self-cleavable ribozyme derived from hepatitis delta virus were designated rHDV.These elements were ligated into a contiguous sequence in the following order: rHH, rTracrRNA, HDV, rHH, rHH, rTracrRNA, rHV, rHH, rHHTaCHSG-01, rCrRNATaCHSG-01, rHDV, rHHTaCHSG-02, rCrRNATaCHSG-02, rHDV. Transcription of this guide RNA and processing element complex was terminated by the nopaline synthase terminator from Agrobacterium tumefaciens.

[0112] Phosphomannose isomerase (PMI; see U.S. Patent No. 5,767,378) was chosen as the selectable marker for stable plant transformation. PMI transcription was driven by a ubiquitin promoter, and transcription was terminated by a ubiquitin terminator, both derived from Zea mays. The Cas9 expression cassette, guide RNA expression and processing cassette, and PMI selectable marker cassette were ligated into a standard binary vector containing left and right border elements and backbone elements derived from Agrobacterium tumefaciens. The backbone elements included a copy of the aminoglycoside 3' adenyltransferase gene from Escherichia coli, which confers resistance to spectinomycin and streptomycin, for vector maintenance in E. coli and Agrobacterium. The backbone also contained bacterial expression cassettes for two Agrobacterium tumefaciens genes driven by the VirG promoter, including the VirG transcriptional activator virulence factor and the RepA plasmid partition protein. The backbone also contained the oVS1 origin of replication element from Pseudomonas sp. for plasmid replication in Agrobacterium tumefaciens and the ColE1 origin of replication element for plastic replication in Escherichia coli. All of the above elements were assembled into a single binary vector in September and October 2019, verified by sequencing, and designated Syngenta construct 25206.

[0113] Syngenta construct 25206 was stably transformed into Fielder wheat cultivar using Agrobacterium-mediated plant transformation. Thirty-four T0 wheat plants were generated from the transformation experiment. All T0 plants were self-pollinated to generate T1 seeds. T1 seed lots from the low-copy-number T0 parent were planted and screened for editing activity at the TraesCS1A02G160300, TraesCS1B02G176300, and TraesCS1D02G157500 target sites. Screening was accomplished by using PCR to amplify regions of each gene surrounding the rCrRNATaCHSG-01 and rCrRNATaCHSG-02 cleavage sites and sequencing the resulting PCR products. The primers used for PCR amplification are listed in Table 5. PCR conditions for all primers included annealing at 66°C, an extension time of 60 seconds, and 40 cycles of amplification. This PCR screening identified editing at the rCrRNATaCHSG-01 cleavage site in separate T1 plants, TraesCS1A02G160300 and TraesCS1B02G176300. All genome editing in this study occurred at the rCrRNATaCHSG-01 cleavage site, and no evidence of editing was found at the rCrRNATaCHSG-02 cleavage site throughout the study. Pollen was collected from a T1 plant containing edits at the TraesCS1B02G176300 target and used to pollinate two T1 plants with edits at the TraesCS1A02G160300 target. T2 seeds generated by this cross between sibling T1 plants were planted and screened for edits at all targets using the same method. This screen confirmed that the edits at TraesCS1A02G160300 and TraesCS1B02G176300 were combined into one T2 plant, and that another T2 sibling plant had both an edit at the TraesCS1A02G160300 target and a de novo edit at the rCrRNATaCHSG-01 cleavage site in TraesCS1D02G157500.T2 plants with genome editing for the TraesCS1A02G160300 and TraesCS1B02G176300 editing targets were self-pollinated for phenotyping in the T3 generation. T3 progeny of these plants were selected for individuals homozygous for the genome editing in TraesCS1A02G160300 and TraesCS1B02G176300, grown to maturity, and observed for male fertility. No evidence of male sterility or changes to plant phenotype was observed in individuals homozygous for the genome editing in TraesCS1A02G160300 and TraesCS1B02G176300. It was later demonstrated that the functional wild-type allele of TraesCS1D02G157500 was sufficient to maintain full male fertility.

[0114] [Table 5]

[0115] Pollen from a T2 segregant with a genome edit at TraesCS1B02G176300 was used to pollinate individuals with edits at the TraesCS1A02G160300 and TraesCS1D02G157500 targets. The T3 progeny of this cross were planted and screened by PCR and sequencing to select individuals with genome edits at the rCrRNATaCHSG-01 cleavage site in all three targets. The selected T3 progeny were naturally selfed to generate a T4 generation segregating for individuals with homozygous edits at all three targets. The T4 generation was planted and screened by sequencing of PCR products. Segregation distortion was observed in the T4 generation due to poor pollen transmission with edits to all three targets (Table 6). One individual T4 plant was identified as having homozygous loss-of-function edits at all three target sites (Table 6). Additional T4 plants with knockout-edited alleles at five of the six possible target copies (six representing the total number of copies from the three diploid A, B, and D wheat genomes) were selected for phenotyping and used as pollen donors. The additional T4 plants included individuals with one functional copy of TraesCS1B02G176300 and one functional copy of TraesCS1D02G157500. The knockout-edited allele at the TraesCS1A02G160300 target was allowed to fix to homozygosity in this line by the T4 generation. All edits sequenced from T4 generation individuals were listed in Table 6.

[0116] [Table 6]

[0117] [Table 7]

[0118] 3. Phenotyping of homozygous edited wheat plants. The opening phenotype of a single T4 plant homozygous for the edited allele, knocking out all six copies of the editing target, shared similarities with that commonly observed in Timopheevii CMS wheat. See, for example, J. Song, "A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat," Plant Mol. Biol. 26:535-539 (1994). The outer florets of individual spikes released yellow anthers during opening. These anthers did not dehisce along the lodicules and did not shed pollen, as observed in fertile wheat. A few days after the onset of anther release, secondary dehiscence was observed, during which all individual florets opened, allowing external access of pollen to the stigma. In contrast to timopheevii CMS wheat, all directly observed florets were expected to have three fully formed anthers per floret, and the anthers themselves were similar in size to male-fertile Fielder plants. No instances of underdeveloped or improperly shaped anthers, commonly observed in timopheevii CMS wheat, were recorded. Six spikes showing secondary dehiscence were pollinated using pollen from sibling segregant plants carrying one intact copy of an anther-specific chalcone synthase (TraesCS1B02G176300 or TraesCS1D02G157500). All individuals with one intact copy of the anther-specific chalcone synthase exhibited a normal phenotype throughout their life cycle and set normal seeds at harvest. Furthermore, all individuals containing one copy of a functional anther-specific chalcone synthase were observed to shed normal amounts of fertile yellow pollen during pollen collection. No differences were observed between individuals with a single intact copy of TraesCS1B02G176300 and individuals with a single intact copy of TraesCS1D02G157500.

[0119] [Table 8]

[0120] 4. Fertility restoration in wheat Application of exogenous flavonoids to developing wheat anthers is hypothesized to restore male fertility in full knockout plants (aabbdd genotype). Male fertility restoration treatments can be applied to individual spikes of plants homozygous for all three targeted knockout-edited alleles. The treatment can consist of quercetin applied exogenously at one or more microspore developmental stages. Quercetin can be applied exogenously as a dry powder, dissolved in a solvent or solution, or suspended in a formulation. Liquid solutions and suspensions of quercetin dihydrate can include inert ingredients that enhance the penetration of quercetin into plant tissues. Liquid and dry powder treatments can be applied by immersion or drenching to the stigma within individual florets of a wheat spike, to the entire wheat spike, to the entire tiller from which the spike originates, or to the entire plant.

[0121] 5. Maize Transformation Six guide RNA sequences (gRNAs) targeting the second exon in ZmFDF1 and ZmFDF2 for gene editing were designed based on the genome sequence from strain NP2222v5. The design and validation of these gRNAs was performed by Syngenta from September to November 2021, prior to vector design and assembly. These gRNAs were designed by identifying all possible PAM recognition sites (TTTN in the case of LbCas12a) in the gene coding sequence and then selecting the guide closest to the transcription start site of the target gene that was least likely to cause any off-target editing (i.e., fewest potential sequence matches elsewhere in the genome, especially in other chalcone synthase genes) and most likely to disrupt the conserved catalytic triad in exon 2, which is known to be essential for chalcone synthase function across several species (M.B. Austin & J.P. Noel, The chalcone synthase superfamily of type III polyketide synthases, Nat. Prod. Rep. 20:79-110 (2003)). This list was then further prioritized by screening for optimal gRNA parameters, including elimination of TTTT PAM sequences, elimination of gRNAs not predicted to generate the desired hairpin structure, and screening for gRNAs with high GC content (>50%) or long runs of Ts in the sequence. The editing efficiency of 36 potential gRNAs was tested in transient protoplast assays by delivering ribonucleoprotein (RNP) complexes (synthetic gRNAs (purchased from IDT) pre-complexed with purified LbCas12a protein) into maize leaf protoplasts using a polyethylene glycol transfection protocol. After RNP delivery, protoplast DNA was extracted, and the target edited region was amplified from it by polymerase chain reaction (PCR). To confirm the presence and frequency of editing at the target site, the products of the PCR reactions were subsequently analyzed for evidence of editing using both a T7E1-based enzyme assay and next-gen (Illumina sequencing), and the gRNAs were ranked according to their efficiency.From these data, six final gRNAs were selected to go into the vector for stable transformation: two targeting ZmFDF1, two targeting ZmFDF2, and two targeting both ZmFDF1 and ZmFDF2 (Table 9).

[0122] [Table 9]

[0123] [Table 10]

[0124] A series of binary vectors that would generate loss-of-function alleles for ZmFDF1, ZmFDF2, or both ZmFDF1 and ZmFDF2 genome editing targets were designed by Syngenta between January and March 2022. A maize codon-optimized version of the Cas12a enzyme from the Lachnospiraceae bacterium was selected to generate double-strand breaks in the target genes. This Cas12a version contained the C965S amino acid substitution, an intron from the Arabidopsis thaliana BAF60 gene, which encodes a protein belonging to the chromodomain remodeling complex, and nuclear localization sequences from Simian Virus 40 at both the N- and C-termini of the amino acid sequence. Cas12a transcription was driven by the constitutive sugarcane ubiquitin 4 promoter from Saccharum officinarum. Cas12a transcription was terminated by the nopaline synthase terminator from Agrobacterium tumefaciens. In all vectors, guide RNA transcription was driven by the Oryza sativa U3 promoter for Pol III-dependent transcription of noncoding RNA. For efficient guide RNA processing, all six guides were individually ligated to a transactivating crRNA sequence derived from Streptococcus pyogenes to form a combined guide RNA and crRNA sequence.

[0125] For all three vectors, phosphomannose isomerase (PMI) was chosen as the selectable marker for stable plant transformation. PMI transcription was driven by a ubiquitin promoter, and transcription was terminated by a ubiquitin terminator, both derived from Zea mays. The Cas9 expression cassette, guide RNA expression and processing cassette, and PMI selectable marker cassette were ligated into a standard binary vector containing left and right border elements and backbone elements derived from Agrobacterium tumefaciens. The backbone elements included a copy of the aminoglycoside 3' adenyltransferase gene from Escherichia coli, which confers resistance to spectinomycin and streptomycin, for vector maintenance in E. coli and Agrobacterium. The backbone also contained bacterial expression cassettes for two Agrobacterium tumefaciens genes driven by the VirG promoter, including the VirG transcriptional activator virulence factor and the RepA plasmid partition protein. The backbone also contained the oVS1 origin of replication element from Pseudomonas sp. for plasmid replication in Agrobacterium tumefaciens and the ColE1 origin of replication element for plastic replication in Escherichia coli. All of the above elements were assembled into three binary vectors in April 2022, verified by sequencing, and designated Syngenta constructs 27726, 27738, and 27769.

[0126] Constructs 27726, 27738, and 27769 were separately transformed into the maize inbred line NP2222 via Agrobacterium-mediated transformation. Transformants found to stably integrate the selectable marker were regenerated into plantlets and grown to maturity under greenhouse conditions. The presence of edits in the target was confirmed first via a TaqMan assay designed to detect edits at the cleavage site of each gRNA, and a second assay designed to detect the wild-type (uncleaved allele). In individual TO plants where the presence of the cleavage allele was detected by TaqMan, follow-up analysis involved next-generation sequencing (Illumina) to characterize the exact indels present and select TO plants with the highest likelihood of chalcone synthase loss-of-function. In this analysis, any indels that resulted in a shift in the reading frame within an exon were interpreted as loss-of-function alleles.

[0127] 6. Phenotyping of edited maize plants. T0 maize plants with various edits were observed for phenotypes associated with sequenced loss-of-function alleles in ZmFDF1 and ZmFDF2. Generally, plant male reproductive phenotypes were associated with the total number of loss-of-function alleles for ZmFDF1 and ZmFDF2 out of a possible total of four loss-of-function alleles. No abnormal phenotypes were observed in T0 plants with one or two loss-of-function alleles. Interestingly, complete loss of function of one of the edited targets due to frameshift alleles in both copies of ZmFDF1 and ZmFDF2 did not result in deviation from the normal phenotype unless both copies of the other respective gene contained loss-of-function alleles. This suggests that ZmFDF1 and ZmFDF2 are functional duplicates. Some T0 plants exhibited abnormal anthers that were reduced in size upon release compared to most plants. These abnormally small anthers shed less pollen compared to most plants. Abnormal pollen shedding was observed at approximately 10 microliters per tassel per day, compared to the typical 500–1000 microliters per tassel per day for maize. This low level of pollen was nonetheless the standard yellow color commonly observed in maize. The T0 plants with abnormal pollen and anthers were plants with frameshift indels resulting in loss-of-function alleles for three of the four possible gene editing targets. T0 plants with abnormal anthers shedding low levels of pollen were hand-selfed and self-fertile. No T0 individuals with the four loss-of-function alleles (frameshift indels for both copies of ZmFDF1 and both copies of ZmFDF2) were present in the T0 generation. Common phenotypes associated with c2 / whp1 maize (colorless aleurone, colorless silks, white pollen, and premature cessation of pollen tube growth) were not observed in this population.

[0128] [Table 11]

[0129] To further characterize the association between the loss-of-function genotype and plant reproductive phenotype, T1 seeds obtained by self-pollinating T0 plants were planted. Genotyping selected progeny homozygous for the loss-of-function allele for ZmFDF1 or ZmFDF2, progeny with a complete loss-of-function for either ZmFDF1 or ZmFDF2 and one functional copy of the other respective gene, and progeny homozygous for the loss-of-function allele for both ZmFDF1 and ZmFDF2. As part of the pollen viability screening, T1 plants were self-pollinated to generate T2 seeds. T2 seeds were also planted in the greenhouse, and the same panel of genotypes was selected for phenotyping. Similar associations between edited allele genotypes and plant phenotypes were observed for the screened T1 and T2 plants. Complete loss of function of one of the editing targets due to a frameshift allele in both copies of ZmFDF1 or ZmFDF2 did not result in deviation from the normal phenotype unless both copies of the other respective gene carried the loss-of-function editing allele. T1 and T2 plants potentially exhibiting three of the four loss-of-function frameshift alleles continued to exhibit abnormal pollen and anther phenotypes. In these individuals, anthers were consistently smaller than the lineage norm, and these smaller anthers shed less pollen. Abnormal pollen shedding was observed to range from approximately 10 microliters per tassel per day to 100 microliters per tassel per day, compared to the 500–1000 microliters per tassel per day typical for maize. Across the entire range of daily pollen shedding, the pollen was the standard yellow color observed in maize. Pollen samples were self-fertile in all T1 and T2 plants tested.

[0130] T1 and T2 plants carrying homozygous frameshift indels resulting in complete loss of function in both ZmFDF1 and ZmFDF2 were observed to exhibit a variety of phenotypes. Progeny selection showed reduced anther size and pollen shedding phenotypes similar to those of siblings with one intact copy of ZmFDF1 or ZmFDF2. Pollen from these progeny was self-fertile. Progeny from other siblings showed a loss of normal anther development, with anthers not fully maturing, not being released from the florets, and not containing any viable pollen grains. These individuals were all functionally male sterile due to the complete absence of pollen. Further selection for specific indels in ZmFDF1 and ZmFDF2 is required to demonstrate whether the complete loss-of-function phenotype is characterized by a range in anther and pollen phenotypes or a consistent anther-free, pollen-free phenotype. Selected indels that result in frameshifts in the coding sequence may cause a complete loss of function to the gene target, while other indels that result in frameshifts may not completely eliminate function based on their exact location in the target coding sequence. Further phenotyping may demonstrate that a range in anther and pollen phenotypes is influenced by environmental factors independent of the specific indels in ZmFDF1 and ZmFDF2.

[0131] It is hypothesized that application of exogenous flavonoids to developing anthers of antherless, pollenless genotypes restores male fertility. Male fertility restoration treatments can be applied to developing tassels by delivering the treatment to the leaf whorls and injecting it through the leaf whorls into the air pockets surrounding the developing tassels, or by injecting the treatment directly into the developing tassels emerging from the leaf whorls. Male fertility restoration treatments can also be applied to the leaf whorls, as a foliar treatment to the canopy, or as a drench. The treatment can consist of quercetin exogenously applied at one or more microspore developmental stages. Quercetin can be applied exogenously as a dry powder, dissolved in a solvent or solution, or suspended in a formulation. Liquid solutions and suspensions of quercetin dihydrate can include inactive ingredients that enhance the penetration of quercetin into plant tissues.

[0132] [Table 12]

[0133] 7. Construct Information

[0134] [Table 13-1]

[0135] [Table 13-2]

[0136] [Table 14]

[0137] [Table 15]

[0138] [Table 16]

Claims

1. 1. A method of seed production comprising: a. Obtaining at least one first plant, wherein said first plant is a flavonoid-dependent fertility plant comprising a mutation in a flavonoid-dependent fertility ("FDF") gene; b. Obtaining at least one second plant, wherein the second plant is male fertile; and c. allowing said at least one second plant to pollinate said at least one first plant; d. Obtaining the progeny seeds thereof; A method comprising:

2. 10. The method of claim 1, wherein the first plant and the second plant are monocotyledonous plants.

3. 2. The method of claim 1, wherein the monocotyledonous plant is selected from the group consisting of wheat, corn, and rice.

4. A flavonoid-dependent fertile ("FDF") plant containing a mutation in the FDF gene.

5. The FDF plant of claim 4 , wherein the mutation in the FDF gene is a knockout mutation.

6. 6. The FDF plant of claim 5, wherein the plant is homozygous for the knockout mutation in the FDF gene.

7. The FDF plant of claim 3 , wherein the FDF plant is a wheat plant.

8. The FDF plant of claim 6, wherein the FDF gene is selected from TaFDF1A (SEQ ID NO: 1), TaFDF1B (SEQ ID NO: 2), TaFDF1D (SEQ ID NO: 3), and combinations thereof.

9. The FDF plant of claim 3 , wherein the FDF plant is a corn plant.

10. The FDF plant of claim 9, wherein the FDF gene is ZmFDF1 (SEQ ID NO: 4), ZmFDF2 (SEQ ID NO: 5), or a combination thereof.

11. The FDF plant of claim 10, wherein the FDF plant further comprises a mutation in C2 (SEQ ID NO: 26) and / or WHP (SEQ ID NO: 28).

12. The FDF plant according to claim 3 , wherein the FDF plant is a rice plant.

13. The FDF plant of claim 12, wherein the FDF plant gene is OsFDF (SEQ ID NO: 57).

14. 1. A method for propagating FDF plants, comprising: a. Obtaining at least one plant, wherein said plant is a flavonoid-dependent fertility ("FDF") plant comprising a mutation in an FDF gene; b. applying a composition comprising a flavonoid to the plant of step a.; c. Allowing self-pollination to occur; d. Obtaining the progeny seeds thereof; A method comprising:

15. 15. The method of claim 14, wherein the flavonoid is quercetin.

16. 16. The method of claim 15, wherein the composition comprising quercetin is a liquid solution.

17. 17. The method of claim 16, wherein the liquid solution comprises quercetin mixed in water, propylene glycol, or other solvent or solution.

18. 18. The method of claim 17, wherein the liquid solution comprises quercetin suspended in a solvent or solution at a concentration of 1 mg / L to 100 mg / L, 2.5 mg / L to 50 mg / L, or about 5 mg / L.

19. 18. The method of claim 17, wherein the liquid solution containing quercetin is saturated.

20. 16. The method of claim 15, wherein the liquid solution containing quercetin is a powder.

21. 21. The method of claim 20, wherein the powder comprises a mixture of quercetin and a carrier compound.

22. 22. The method of claim 21, wherein the mixture of quercetin and carrier compound is in a ratio of 1000:1 to 1:1000.

23. 22. The method of claim 21, wherein the support compound is selected from the group consisting of crystalline silica, talc, metal powders, and mica minerals.

24. 15. The method of claim 14, wherein the composition comprising a flavonoid is applied to the female organs of flowers on the at least one plant designated as a female plant.

25. 25. The method of claim 24, wherein the composition is applied by a method selected from the group consisting of painting, misting, spraying, and root drenching.

26. 15. The method of claim 14, wherein the FDF gene is a wheat gene selected from the group consisting of SEQ ID NOs: 1 to 3 and combinations thereof.

27. 15. The method of claim 14, wherein the FDF gene is a maize gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof.

28. The method of claim 14, wherein the FGF gene is a rice gene comprising SEQ ID NO:

57.

29. 15. A plant produced by the method of claim 14.

30. 1. A method for hybrid seed production comprising: a. Obtaining at least one inbred female FDF plant, wherein said inbred female FDF plant contains a mutation in the FDF gene; b. Obtaining pollen from at least one inbred male plant, wherein said inbred male plant produces fertile pollen; c. pollinating the inbred female FDF plants with the fertile pollen from the inbred male plants; d. Obtaining the hybrid offspring thereof; A method comprising:

31. 31. The method of claim 30, wherein the inbred female FDF plant is a corn plant, a wheat plant, or a rice plant.

32. 32. The method of claim 31, wherein the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 1-3, and combinations thereof.

33. 32. The method of claim 31, wherein the FDF corn plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof.

34. 32. The method of claim 31 , wherein the FDF rice plant comprises a mutation in SEQ ID NO:

57.

35. 31. A plant produced by the method of claim 30.

36. 1. A method for restoring fertility to an FDF plant containing a mutation in the FDF gene, said method comprising applying to said FDF plant a composition comprising a flavonoid.

37. 37. The method of claim 36, wherein the FDF plant is a corn plant, a wheat plant, or a rice plant.

38. 38. The method of claim 37, wherein the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 1-3, and combinations thereof.

39. 38. The method of claim 37, wherein the FDF corn plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof.

40. 40. The method of claim 39, wherein the FDF corn plant further comprises a mutation in C2 (SEQ ID NO: 26) and / or WHP (SEQ ID NO: 28).

41. 38. The method of claim 37, wherein the FDF rice plant comprises a mutation in SEQ ID NO:

57.

42. 37. The method of claim 36, wherein the flavonoid comprises quercetin.

43. 43. The method of claim 42, wherein the quercetin is mixed in water, propylene glycol, or other solvent or solution.

44. 37. The method of claim 36, wherein the composition comprising a flavonoid further comprises a carrier compound.

45. 45. The method of claim 44, wherein the support compound is selected from the group consisting of crystalline silica, talc, metal powders, and mica minerals.

46. 37. The method of claim 36, wherein the composition comprising a flavonoid is applied to anthers, silks, stigmas, florets, spikes, leaf whorls, canopy, or roots.

47. 47. The method of claim 46, wherein the composition is applied by a method selected from the group consisting of painting, misting, spraying, and root drenching.

Images