modified promoters of parthenogenic genes

Abstract

The present invention provides a method of generating a mutated gene, wherein the gene comprises a modified promoter and wherein the gene is capable of inducing a parthenocarpic phenotype in a plant. The present invention also provides the mutated gene, an isolated nucleic acid molecule, a construct or vector comprising the same. Furthermore, the present invention provides a method of producing a parthenocarpic plant comprising the mutated gene, and a parthenocarpic plant obtained thereby. The mutated gene is based on the PAR gene of Taraxacum officinale having SEQ ID NO: 5 and orthologs thereof.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and particularly to plant biotechnology, including plant breeding. Specifically, this invention relates to the identification and use of genes associated with, for example, agametosis and haploid induction, and useful in agametosis and haploid induction. This invention particularly relates to mutant promoters of genes associated with parthenogenesis. This invention also relates to methods for inducing parthenogenesis in plants and crops, and to the use of such genes and / or promoters in agametosis, particularly in combinations with agametosis genes, or in the production of haploid plants, wherein chromosomes can be doubled to produce double haploids. Background Technology

[0002] Agametic reproduction (also known as incomplete agametic reproduction) is asexual plant reproduction via seeds. Agametic reproduction has been reported in approximately 400 species of flowering plants (Bicknell and Koltunow, 2004). There are two forms of agametic reproduction in flowering plants:

[0003] (1) Gametophyte agametosis, in which the embryo is produced through parthenogenesis from unreduced, unfertilized egg cells;

[0004] (2) Sporophytes reproduce without gametes, in which the embryo is produced from the somatic cells of the sporophyte.

[0005] Examples of gametophyte agametic reproduction include dandelion (Taraxacum sp.), mountain willow (Hieracium sp.), Kentucky bluegrass (Poa pratensis), and eastern gamagrass (Tripsacum dactyloides). Examples of sporophyte agametic reproduction include citrus (Citrus sp.) and mangosteen (Garcinia mangostana). Gametophyte agametic reproduction involves two developmental processes:

[0006] (1) Avoid meiotic recombination and meiosis (incomplete meiosis); and

[0007] (2) The egg cell develops into an embryo, but is not fertilized (parthenogenesis).

[0008] Seeds produced by agametic reproduction are genetically identical to the parent plants. The usefulness of agametic reproduction in plant breeding has long been recognized (Asker, 1979; Hermsen, 1980; Asker and Jerling, 1990; Vielle-Calzada et al., 1995). The most obvious advantage of introducing agametic reproduction into crops is the true breeding of heterotic F1 hybrids. In most crops, F1 hybrids are the best-performing varieties. However, in sexual crops, F1 hybrids must be reproduced again in each generation through hybridization with homozygous inbred parents because self-fertilization of F1 hybrids leads to the loss of heterosis through recombination in the genomes of F2 offspring. Producing sexually occurring F1 seeds is a repetitive, complex, and expensive process. In contrast, agametic F1 hybrids will reproduce indefinitely. In other words, the genetic fixation of F1 hybrids and the production of homogeneous offspring plants through seeds become possible.

[0009] F1 fixation in agamemation is a special case of the general characteristics of agamemation, namely that any genotype, regardless of its genetic complexity, can be propagated in a single step. This means that agamemation can be used for the immediate fixation of polygenic quantitative traits. It should be noted that most yield traits are polygenic. Agamemation can be used for the superposition (or aggregation) of multiple traits (e.g., various resistances, several transgenes, or multiple quantitative trait loci). Without agamemation, to fix such a set of traits, each trait locus must be prepared individually homozygous and then combined. As the number of loci involved in a trait increases, homozygousing of these trait loci through hybridization becomes time-consuming, logically challenging, and therefore costly. Furthermore, specific epistatic interactions between alleles are lost due to homozygosity. With agamemation, it is possible to repair this non-superpositional genetic variation. Therefore, agamemation, i.e., clonal propagation through seeds, has the potential to lead to a paradigm shift in plant breeding, commercial seed production, and agriculture (van Dijk et al. 2016).

[0010] Besides instantly fixing any genotype, regardless of its complexity, agametosis has other important agricultural applications. Sexual interspecific hybrids and autopolyploids often suffer from sterility due to meiotic problems. Because agametosis skips meiosis, it can solve these problems in interspecific hybrids and autopolyploids. Since agametosis prevents female hybridization, it has been proposed, combined with male sterility, to curb transgenic activity and prevent infiltration of transgenic genes into wild-related genetically modified crops (Daniell, 2002). In insect-pollinated crops (such as Brassica), the seed set rate of agametosis is not limited by insufficient pollination services. This is becoming increasingly important given the growing health problems of pollinating bee populations (Varroa mite infestation, African killer bees, etc.). In tuber-producing crops (such as potatoes), agametosis asexually preserves superior genotypes but reduces or even eliminates the risk of current virus transmission and the associated costs of clean production, containment, and certification. Furthermore, the storage cost of agametic seeds is far lower than that of tubers or other asexually propagated plant parts. In ornamental plants, agametic reproduction can replace labor-intensive and expensive tissue culture propagation. It is believed that, in general, agametic reproduction significantly reduces the cost of variety development and plant propagation.

[0011] Unfortunately, agametosis does not occur in any major crops. Numerous attempts have been made to introduce agametosis into sexual crops. These include, for example, the introgression of agametosis genes, mutations in sexual type species, regeneration of agametosis through hybridization, and cloning of candidate genes. To date, the introgression of agametosis genes from wild agametogens into crop species through extensive hybridization has not been successful (e.g., agametosis from *Frictiona rubra* to maize – Savidan, Y., 2001; Morgan et al., 1998; WO97 / 10704). Regarding mutant sexual type species, WO2007 / 066214 describes the use of an agametosis mutant called Dyad in *Arabidopsis thaliana*. However, Dyad is a recessive mutation with very low penetrance. The use of this mutation in crop species is limited. Reproducing agametosis through hybridization between two sexual ecotypes has not yielded agametocytes of agronomical interest (US2004 / 0168216A1 and US2005 / 0155111A1). The cloning of candidate agametosis genes in maize via transposon markers has been described in US2004 / 0148667. Orthologs of the elongate gene, which are believed to induce agametosis, have been claimed for protection. However, according to Barrell and Grossniklaus (2005), the elongate gene skips meiosis II and therefore does not retain the maternal genotype, which greatly diminishes its usefulness.

[0012] US2006 / 0179498 describes so-called reverse breeding as an alternative to agametosis. However, this is a technically complex in vitro laboratory method, while agametosis is an in vivo method performed by the plant itself. Furthermore, in reverse breeding, hybridization is still required once the parental line is reconstructed (digametic homozygotes).

[0013] Agamemion in naturally occurring agametic organisms usually has a genetic basis (review by Ozias-Akins and VanDijk, 2007). Therefore, another approach might be to isolate agametic genes from naturally occurring agametic species. However, this is not an easy task, as naturally occurring agametic organisms often possess polyploid genomes, and localized cloning in polyploids is extremely difficult. Other complicating factors include recombination repression in agamemion-specific chromosomal regions, repetitive sequences, and segregational aberrations during hybridization.

[0014] As described herein, there is a need for methods to induce agametosis in crops that do not have at least some of the limitations of existing technologies. In particular, there is a need for methods to produce agametogenic plants and agametogenic seeds. There is also a need to provide genes and proteins involved in agametosis, particularly parthenogenesis, that are suitable for introducing agametosis or (di)haploidy into crops and can substantially mimic the agametosis pathway. Summary of the Invention

[0015] The inventors have identified and isolated parthenogenesis loci and genes, alleles associated with parthenogenesis phenotypes (referred to herein as parthenogenesis alleles or Par alleles) and non-parthenogenesis phenotypes (referred herein as sexual alleles of parthenogenesis genes or Par alleles), and their genetic sequences, namely promoter sequences, 5'UTRs, coding sequences, 3'UTR sequences and encoded protein sequences.

[0016] This invention provides a method for modifying or changing a par allele into a Par allele by altering the promoter sequence of the par allele. This invention is particularly applicable to modifying endogenous par alleles into Par alleles, preferably through random or targeted mutagenesis, and optionally through transformation. The resulting mutant allele is capable of transforming a plant and / or its offspring into plants with or with enhanced ability to develop oocytes into embryos without fertilization.

[0017] definition

[0018] As used herein, the term “loci” (plural: loci) refers to one or more specific locations or sites on a chromosome where, for example, a gene or genetic marker is found. For example, a “parthenogenesis locus” refers to the location of a parthenogenesis gene in the genome. Two functional variants of parthenogenesis genes have been identified: the alleles that contribute to the parthenogenesis phenotype, referred herein as the parthenogenesis allele or Par allele, and / or its sexual counterpart, referred herein as the sexual allele or Par allele.

[0019] "Functional in parthenogenesis" genes, alleles, proteins, or nucleic acids should be understood in this text as those that contribute to parthenogenesis phenotypes and / or increase or transform the ability of plants or plant cells to develop egg cells into embryos.

[0020] "Parthenogenesis gene" is a gene associated with parthenogenesis, where "associated" in this document should be understood as indicating a parthenogenesis or non-parthenogenesis (sexual) phenotype. The genetic sequences of the dominant allele (Par allele) conferring parthenogenesis and its two sexual counterparts (par allele or the sexual allele of the parthenogenesis gene) have been first identified in the triploid apomixis isolate A68 of dandelion (Taraxacum officinale), as described in PCT / EP2020 / 064991, which is incorporated herein by reference. Preferably, the Par allele initiates parthenogenesis. The dominant allele conferring parthenogenesis has the genetic sequence of SEQ ID NO: 5 and includes a promoter having the sequence of SEQ ID NO: 2, a coding sequence having the sequence of SEQ ID NO: 3, and a 3' UTR having the sequence of SEQ ID NO: 4. One of the sexual alleles has the genetic sequence of SEQ ID NO: 10 and includes a promoter with the sequence of SEQ ID NO: 7, a coding sequence with the sequence of SEQ ID NO: 8, and a 3'UTR with the sequence of SEQ ID NO: 9. The other sexual allele has the genetic sequence of SEQ ID NO: 15 and includes a promoter with the sequence of SEQ ID NO: 12, a coding sequence with the sequence of SEQ ID NO: 13, and a 3'UTR with the sequence of SEQ ID NO: 14. Based on the characteristics of the proteins encoded by these parthenogenetic genes (denoted as PAR proteins), orthologous genes have been identified in other species. The presence of PAR proteins in oocytes may lead to the inhibition of embryogenesis inhibitors, triggering cell division in the absence of fertilization. These PAR proteins are characterized by containing a zinc finger C2H2-type domain (IPR13087), preferably a zinc finger K2-2-like domain with the concordant sequence C.{2}C.{7}[K / R]A.{2}GH.[R / N].H, which can also be annotated as: CXXCXXXXXXX[K / R]AXXGHX[R / N]XH (SEQ ID NO: 37), where X can be any naturally occurring amino acid, where [K / R] indicates that the amino acid is lysine or arginine, and where [R / N] indicates that the amino acid is arginine or asparagine (see Englbrecht et al., 2004). In addition to the zinc finger C2H2-type domain as defined herein, preferably the zinc finger K2-2-like domain, the protein also includes an EAR motif (an amphiphilic inhibition-associated ethylene-responsive element binding factor) having a shared amino acid sequence DLNXXP (SEQ ID NO: 58) or DLNXP (SEQ ID NO: 59), where X can be any naturally occurring amino acid (see Kagale et al., 2010; and Yang et al.).(al., 2018). Preferably, the EAR motif is located at the C-terminus. Preferably, the zinc finger C2H2-type domain is located at the N-terminus of the EAR motif. Preferably, the PAR protein has a length of up to 400 amino acids, wherein the protein comprises one or two EAR motifs as shown herein and a zinc finger K2-2-like domain as defined herein. Preferably, the protein has a length of up to 400 amino acids, wherein the protein comprises only one or two EAR motifs as shown herein and only one zinc finger K2-2-like domain as defined herein, i.e., no other EAR motifs as defined herein and no other zinc finger K2-2-like domains as defined herein. In addition to a maximum size of 400 amino acids, the presence of only one or two EAR motifs as shown herein, and a single zinc finger K2-2-like domain as defined herein, PAR proteins may contain only one additional zinc finger domain with a zinc finger concordant sequence of C.{2}C.{12}H.{3}H, which may also be annotated as CXXCXXXXXXXXXXXXHXXXH (SEQ ID NO: 38), but more preferably does not contain an additional zinc finger domain with a zinc finger concordant sequence of C.{2}C.{12}H.{3}H (SEQ ID NO: 38). Preferably, the PAR protein has a length of up to 500 amino acids. The PAR protein may be about 50-500, 100-300, or about 150-200 amino acids. The PAR protein may be about 170 amino acids in length. Orthologous parthenogenesis genes can be, but are not limited to, genes encoding any of the following groups of PAR proteins: PAR proteins from pineapple (Ananascomosus) (e.g., UniProtKB: A0A199URK4), PAR proteins from Shenzhen orchid (Apostasia shenzhenica) (e.g., UniProtKB: A0A2I0AZW3), PAR proteins from Arabidopsis thaliana (e.g., UniProtKB: Q8GXP9, A0A178V2S4, O81793, A0A178V1Q3, A0MFC1, O81801), PAR proteins from the Arabidopsis lyrata subsp. Lyrata (e.g., UniProtKB: D7MC52 or D7MCE8), and PAR proteins from peanut (Arachis ipaensis) (e.g., SEQ ID NO: 45 or SEQ ID NO: 45). NO: 49), PAR proteins from Brachypodium distachyon (e.g., UniProtKB: I1J0D9), and from Brassica oleracea.PAR proteins from the oleracea variety (e.g., UniProtKB: A0A0D3A1Q6 or A0A0D3A1Q3), PAR proteins from rapeseed (Brassica campestris) (e.g., UniProtKB: A0A398AHT1), PAR proteins from turnip (Brassica rapa) (e.g., SEQ ID NO: 47), PAR proteins from the turnip subspecies Pekinensis (e.g., UniProtKB: M4D574 or M4D571), PAR proteins from cabbage (e.g., UniProtKB: A0A3P6ESB1 or A0A3P6F726), PAR proteins from rapeseed (e.g., UniProtKB: A0A3P5ZMM3 or A0A3P5Z1M1), PAR proteins from pigeon pea (Cajanus cajan) (e.g., SEQ ID NO: 46), and from Capsella... PAR proteins from rubella (e.g., UniProtKB: R0H2J1 or R0H0C2), PAR proteins from Cephalotus follicularis (e.g., UniProtKB: A0A1Q3CSK1), PAR proteins from Cicerarietinum (e.g., UniProtKB: A0A3Q7YBZ1, A0A1S2YZL9, A0A3Q7Y0Z6 or A0A1S2YZM6; or SEQ ID NO: 55, 56 or 57), PAR proteins from Cichorium endivia (e.g., SEQ ID NO: 39), PAR proteins from Cucumis sativus (e.g., UniProtKB: A0A0A0KGW4 or A0A0A0L0X7), and PAR proteins from Cucumis melon (e.g., Cucumis rubella). PAR proteins from melo (e.g., UniProtKB: A0A1S3BLF2 or A0A1S3B298), cucumber (e.g., UniProtKB: A0A0A0KAW8), squash (Cucurbita amoschata) (e.g., SEQ ID NO: 43), Cuscuta campestris (e.g., UniProtKB: A0A484MGR1), and Dendrobium officinale (Dendrobium)PAR proteins from catenatum (e.g., UniProtKB: A0A2I0V7N9, A0A2I0X2T2, or A0A2I0W0Q8), PAR proteins from Dorcocerashygrometricum (e.g., UniProtKB: A0A2Z7D3Y1), PAR proteins from Eutremasalsugineum (e.g., UniProtKB: V4LSH0; or SEQ ID NO: 44), PAR proteins from Fagus sylvatica (e.g., UniProtKB: A0A2N9E5Y5, A0A2N9HAB9, or A0A2N9H993), PAR proteins from Genlisea aurea (e.g., UniProtKB: S8E1M6), and PAR proteins from soybean (e.g., SEQ ID NO: 44) are included. PAR proteins from various plants, including: NO: 51, 52, 53, or 54; upland cotton (Gossypium hirsutum) (e.g., UniProtKB: A0A1U8LDU9); sunflower (Helianthus annuus) (e.g., SEQ ID NO: 21); Brazilian clover (Hevea brasiliensis) (e.g., SEQ ID NO: 42); Hieracium aurantiacum (Hieracium aurantiacum) (e.g., SEQ ID NO: 40); walnut (Juglansregia) (e.g., UniProtKB: A0A2I4E6B1); lettuce (Lactuca sativa) (e.g., UniProtKB: A0A2J6KZF7; or SEQ ID NO: 22); gourd (Lagenaria siceraria) (e.g., SEQ ID NO: 48); and alfalfa (Medicago esculenta). PAR proteins from various plants include those from *Truncatula* (e.g., UniProtKB: G7K024), *Morus notabilis* (e.g., UniProtKB: W9SMY3 or W9SMQ7), *Mucuna pruriens* (e.g., UniProtKB: A0A371ELJ8), *Nicotiana attenuata* (e.g., UniProtKB: A0A1J6IQI6), and *Nicotiana spp.* (e.g., *Nicotiana spp.*).PAR proteins from various plants include those from *Sylvestris* (e.g., UniProtKB: A0A1U7VXJ0), *Nicotiana tabacum* (e.g., UniProtKB: A0A1S4A651 or A0A1S3YHQ2), *Oryza sativa* subspecies *Japonica* (e.g., UniProtKB: B9FGH8), *Oryza barthii* (e.g., UniProtKB: A0A0D3FWX3), *Panicum miliaceum* (e.g., UniProtKB: A0A3L6Q010 or A0A3L6T1D6), *Parasponia andersonii* (e.g., UniProtKB: A0A2P5BMI5), and *Populus*. PAR proteins from various plants include: *Alba* (e.g., UniProtKB: A0A4U5PSY9), *Populus trichocarpa* (e.g., UniProtKB: B9H661), *Punica granatum* (e.g., UniProtKB: A0A2I0IBB9, A0A218XB85, or A0A218W102), *Senecio cambrensis* (e.g., SEQ ID NO: 41), *Prunus persica* (e.g., SEQ ID NO: 50), *Trema orientale* (e.g., UniProtKB: A0A2P5EB04), *Trifolium pratense* (e.g., UniProtKB: A0A2K3N851), and *Trifolium...* PAR proteins from *Vitis vinifera* (e.g., UniProtKB: A0A2Z6MYD3 or A0A2Z6MDR7), red clover (e.g., UniProtKB: A0A2K3PR44), grape (Vitis vinifera) (e.g., UniProtKB: A0A438C778, A0A438ESC4 or A0A438DBR4), and maize (Zea)PAR proteins (e.g., UniProtKB: A0A1D6HF46, B6UAC5, A0A3L6F4S1, A0A3L6EMC6, A0A3L6EMC6, K7UHQ6, or A0A1D6KHZ4) of the mays. This gene can also encode PAR proteins selected from the following: PAR proteins from *Actinidia chinensis* (e.g., UniProtKB: A0A2R6S2S9), PAR proteins from *Beta vulgaris* (e.g., UniProtKB: XP_010690656.1), PAR proteins from *Solanum tuberosum* (e.g., UniProtKB: XP_015159151.1), PAR proteins from *Solanum lycopersicum* (e.g., UniProtKB: A0A3Q7GXB3, Solyc05g055500, or Solyc06g060480), PAR proteins from *Capsicum baccatum* (e.g., UniProtKB: A0A2G2WJR7), and PAR proteins from *Solanum tuberosum* (e.g., Solanum tuberosum). PAR proteins from various sources include: * *Melongena* (e.g., UniProtKB: AVC18974.1), soybean (Glycine soja) (e.g., GeneBank accession numbers: XP_028201014.1, XP_006596577.1, or UniprotKB: A0A445M3M6), peanut (Arachis hypogaea) (e.g., UniProtKB: A0A444WUX5), common bean (Phaseolus vulgaris) (e.g., UniProtKB: V7CIF6), carrot (Daucus carota) (e.g., GeneBank accession number: XP_017245413.1), and wheat (Triticum...). PAR proteins from *Aestivum* (e.g., UniProtKB: A0A3B6RP64), from the rice subspecies *Indica* (e.g., UniProtKB: A2YH63), from the rice subspecies *Japonica* (e.g., UniProtKB: Q5Z7P5), and from *Theobroma*PAR proteins (e.g., UniProtKB: A0A061DL63) of cacao. Optionally, the ortholog is a gene encoding a PAR protein comprising or composed of the following amino acid sequences, preferably having at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more of identity and / or any of the orthologs provided above when compared over their entire length using, for example, Needleman and Wunsch algorithms (global sequence alignment) with default parameters.

[0021] As used herein, the term "allelic gene" refers to any one or more alternative forms of a gene at a specific locus. In the diploid and / or polyploid cells of an organism, the alleles of a given gene are located at a specific location or locus on a chromosome, with one allele present on each of the set of homologous chromosomes. Diploid and / or polyploid organisms or plant species may contain a large number of different alleles at a given locus.

[0022] As used in this article, the term "dominant allele" refers to the relationship between alleles of a gene, where the effect of one allele (i.e., the dominant allele) masks the effect of the second allele (i.e., the recessive allele) at the same locus. For genes on autosomes (any chromosome other than sex chromosomes), alleles and their associated traits are autosomal dominant or autosomal recessive. Dominance is a key concept in Mendelian and classical genetics. Optionally, the dominant allele encodes a functional protein, while the recessive allele does not. Optionally, the dominant and recessive alleles may encode the same or substantially the same functional protein, but unlike the recessive allele, only the dominant allele is able to express a certain amount of said functional protein in a specific environment and / or in a specific tissue, thereby transforming a specific phenotype, such as parthenogenesis.

[0023] As used in this article, the term "female ovary" refers to the outer shell in which spores form. It can be unicellular or multicellular. All plants, fungi, and many other lineages form an ovary at some point in their life cycle. The ovary produces spores through mitosis or meiosis. Typically, within each ovary, the megasporocyte undergoes meiosis to produce four haploid megaspores. In gymnosperms and angiosperms, only one of these four megaspores is functional at maturity, while the other three are degenerate. The existing megaspore undergoes mitosis and develops into a female gametophyte (macrogametophyte), which eventually produces an egg cell.

[0024] As used in this article, the term "female gamete" refers to a cell in sexually reproducing organisms that fuses with another ("male") cell under normal (sexual) conditions during fertilization (fertilization). In species that produce two morphologically different types of gametes, and in which each individual produces only one type, "female" refers to any individual that produces the larger type of gamete (called an ovule (egg) or ovum). In plants, the female ovule is produced by the ovary of the flower. Upon maturity, the haploid ovule produces the female gamete and is then ready for fertilization. The male cell is the pollen (mostly haploid), which is produced by the anther.

[0025] The term "genetic marker" or "polymorphic marker" refers to a region of genomic DNA that can be used to "mark" a specific location on a chromosome. If a genetic marker is tightly linked to or "on" a gene, it "marks" the DNA where that gene is found, and can therefore be used for (molecular) marker analysis to select for the presence of that gene, for example in marker-assisted breeding / selection (MAS) methods. Examples of genetic markers are AFLP (Amplified Fragment Length Polymorphism, EP534858), microsatellites, RFLP (Restriction Fragment Length Polymorphism), STS (Sequence Marker Site), SNP (Single Nucleotide Polymorphism), SFP (Single Feature Polymorphism; see Borevitz et al., 2003), SCAR (Sequence Feature Amplified Region), CAPS (Cut Amplified Polymorphic Sequence), etc. The farther a marker is from a gene, the greater the likelihood of recombination (hybridization) between the marker and the gene, resulting in loss of linkage (and co-segregation of the marker and gene). The distance between genetic loci is measured based on recombination frequency and expressed in cM (centimoles; 1 cM is the meiotic recombination frequency between two markers of 1%). Due to the large differences in genome size between species, the actual physical distance expressed in 1 cM (i.e., kilobases, kb between two markers) also varies greatly between species.

[0026] It should be understood that when this article refers to “linked” markers, this also includes markers “on” the gene itself.

[0027] “MAS” stands for “Marker-Assisted Selection”, which screens plants for the presence and / or absence of one or more genetic and / or phenotypic markers to accelerate the transfer of DNA regions containing said markers (and optionally lacking flanking regions) into (superior) breeding lines.

[0028] "Molecular marker analysis" (or testing) refers to (DNA-based) analysis that indicates the presence or absence (directly or indirectly) of alleles, such as the Par or par alleles in a plant or plant part. Preferably, it allows one to determine whether a particular allele at a parthenogenetic locus in any single plant is homozygous or heterozygous. For example, in one embodiment, nucleic acids linked to a parthenogenetic locus are amplified using PCR primers, the amplification products are enzymatically digested, and based on the electrophoretic analysis pattern of the amplification products, it can be determined which(s) allele(s) are present in any single plant and the conjugation of alleles at the parthenogenetic locus (i.e., the genotype at each locus). Examples are SCAR markers (Sequence Characteristic Amplified Regions), CAPS markers (Cut Amplified Polymorphic Sequences), and similar marker analyses.

[0029] As used herein, the term "heterozygosity" refers to a genetic condition where two distinct alleles are located at a specific locus but on corresponding homologous chromosome sets in a cell. Conversely, as used herein, the term "homozygosity" refers to a genetic condition where two (or more in the case of polyploidy) identical alleles are located at a specific locus but on corresponding homologous chromosome sets in a cell.

[0030] The term “variety” as used in this paper conforms to UPOV convention and refers to a group of plants in a single botanical taxonomic unit of the lowest known rank that can be defined by the expression of characteristics and can be distinguished from any other group of plants by the expression of at least one of said characteristics and is considered a unit of suitability for its invariant (stable) reproduction.

[0031] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules composed of chains of amino acids, without specifying a particular mode of action, size, three-dimensional structure, or origin. Therefore, a "fragment" or "part" of a protein can still be called a "protein."

[0032] The term "gene" refers to a DNA sequence containing a region (transcribed region) that is transcribed into an RNA molecule (e.g., pre-mRNA processed into mRNA) in the cell, which is operatively linked to a suitable regulatory region (e.g., a promoter). Thus, a gene can contain several operatively linked sequences, such as a promoter, a 5' leader sequence containing, for example, a sequence involved in translation initiation, a (protein) coding region (cDNA or genomic DNA), and a 3' untranslated sequence containing, for example, a transcription termination site.

[0033] A "chimeric gene" (or recombinant gene) is any gene that is not normally found in a species in nature, especially one or more nucleotide sequence portions that are unrelated to each other in nature. For example, a promoter that is not associated with part or all of a transcriptional region or another regulatory region in nature.

[0034] "Natural gene" refers to a sequence that includes a promoter sequence, a coding sequence, and optionally a 3'- Any gene with a sequence, which may also be found in naturally occurring genes. Optionally, the nucleotide sequence of a natural gene is identical to a sequence found in nature. It should be understood herein that a natural gene can be transgenic, and in this embodiment, the natural gene is present in a plant species that does not naturally contain the natural gene.

[0035] This article defines "endogenous genes" as natural genes in the natural environment, that is, those that exist in plant species that naturally contain the gene.

[0036] The “3'UTR” or “3' untranslated sequence” (also often referred to as the 3' untranslated region or 3' end) is a nucleotide sequence found downstream of the gene coding sequence that contains, for example, a transcription termination site and (in most, but not all, eukaryotic mRNAs) a polyadenylation signal (e.g., AAUAAA or its variants). After transcription termination, the mRNA transcript can be cleaved downstream of the polyadenylation signal and a poly(A) tail can be added, which is involved in the transport of mRNA to the cytoplasm (where translation occurs).

[0037] The 5'UTR, also known as the leader sequence or 5' untranslated region, is a region in the mRNA transcript and its corresponding DNA located between the +1 position of mRNA transcription initiation and the translation start codon in the coding region (usually AUG on mRNA or ATG on DNA). The 5'UTR typically contains sites important for translation, mRNA stability and / or turnover, as well as other regulatory elements.

[0038] "Gene expression" refers to the process of transcribing a DNA region operatively linked to a suitable regulatory region, particularly a promoter, into RNA that is biologically active, i.e., capable of being translated into a biologically active protein or peptide (or an active peptide fragment) or inherently active (e.g., in post-transcriptional gene silencing or RNAi). An active protein can be a protein capable of performing its function, which may be, for example, by binding to a regulatory element of the gene's 5'UTR to inhibit the expression of the gene. In some embodiments, an active protein refers to a constitutively active protein. The coding sequence is preferably sense-oriented and encodes the desired biologically active protein or peptide or an active peptide fragment. In gene silencing methods, the DNA sequence is preferably in the form of antisense DNA or inverted repeat DNA, comprising short sequences of the target gene in both sense and antisense orientations.

[0039] “Transcription regulatory sequence” is defined herein as a nucleotide sequence capable of regulating the transcription rate of a (coding) sequence operatively linked to a transcription regulatory sequence. Therefore, the transcription regulatory sequence defined herein will include all sequence elements required to initiate transcription (promoter elements), maintain and regulate transcription, including, for example, attenuators or enhancers. Although primarily referring to upstream (5') transcription regulatory sequences of coding sequences, downstream (3') regulatory sequences of coding sequences have also been found to be included in this definition.

[0040] As used herein, the term "promoter" refers to a nucleic acid segment whose function is to control the transcription of one or more DNA regions located upstream of the transcription start site in the direction of transcription, and is structurally identified by the presence of a DNA-dependent RNA polymerase binding site, a transcription start site, and any other DNA sequence including, but not limited to, transcription factor binding sites, repressor and activator protein binding sites, and any other nucleotide sequence known to those skilled in the art to directly or indirectly regulate the amount of transcription derived from the promoter. Optionally, the term "promoter" herein also includes a 5' UTR region (e.g., a promoter herein may include one or more portions upstream (5') of the translation start codon of a gene, as this region may play a role in regulating transcription and / or translation).

[0041] Constitutive promoters are promoters that are active in most tissues under most physiological and developmental conditions. Inducible promoters are promoters that are physiologically (e.g., through the application of certain compounds) or developmentally regulated. Tissue-specific promoters are active only in specific types of tissues or cells. A promoter that is active in a plant or plant cell refers to the promoter's general ability to drive transcription within a plant or plant cell. It has no effect on the promoter's spatiotemporal activity.

[0042] As used herein, the term “operably ligated” refers to the ligation of polynucleotide elements in a functional relationship. It is “operably ligated” when one nucleic acid is in a functional relationship with another nucleotide sequence. For example, a promoter, or transcriptional regulatory sequence, is operably ligated to a coding sequence if it affects the transcription of that sequence. Operable ligation means that the ligated DNA sequences are typically contiguous and that the promoter sequence can be ligated to a protein-coding sequence or the protein-coding sequence to the 3' UTR if necessary. “Nucleic acid construct” or “vector” is understood herein to refer to an artificial nucleic acid molecule produced using recombinant DNA technology for the delivery of exogenous DNA into a host cell. The vector backbone can be, for example, a binary or superbinary vector (see, for example, US 5591616, US 2002138879, and WO95 / 06722), a co-integration vector, or a T-DNA vector, as known in the art and described elsewhere herein, in which a gene or chimeric gene is integrated, or, if a suitable transcriptional regulatory sequence is already present, the desired nucleotide sequence (e.g., coding sequence, antisense, or inverted repeat sequence) is integrated only downstream of the transcriptional regulatory sequence. Vectors typically contain further genetic elements to facilitate their application in molecular cloning, such as selection markers, multiple cloning sites, etc.

[0043] "Recombinant host cell," "transformed cell," or "transgenic cell" refers to a new single cell (or organism) produced by at least one nucleic acid molecule, particularly containing a transgene and / or chimeric gene that has been introduced into said cell to encode a desired protein, or a nucleotide sequence that, when expressed, produces a specific protein such as PAR protein as defined herein. "Isolated nucleic acid" is used to refer to nucleic acids that are no longer in their natural environment, such as those in vitro or in recombinant bacterial or plant host cells.

[0044] "Host cell" is the original cell that has been transgenic into a recombinant host cell. The host cell is preferably a plant cell or a bacterial cell. The recombinant host cell may contain a nucleic acid construct as an extrachromosomal (free) replication molecule, or more preferably, a gene or chimeric gene integrated into the nuclear or plasmonic genome of the host cell.

[0045] A “recombinant plant” or “recombinant plant part” or “transgenic plant” is a plant or plant part (e.g., seed, fruit, or leaf) that contains a recombinant gene or chimeric gene or transgene, even if the gene may not be expressed in all cells or in all cells.

[0046] An "elite event" is a recombinant plant in which a selected location in the genome contains a recombinant gene or transgene, resulting in favorable phenotypes and / or agronomic traits. The flanking DNA of the integration site can be sequenced to characterize the integration site and distinguish the event from other transgenic plants containing the same recombinant gene at other locations in the genome.

[0047] The term "selection marker" is a term familiar to those skilled in the art and is used herein to describe any genetic entity that, when expressed, can be used to select one or more cells containing a selection marker. The product of a selection marker gene confers, for example, antibiotic resistance, or more preferably herbicide resistance, or another selectable trait, such as a phenotypic trait (e.g., a change in pigmentation) or nutritional requirement. The term "reporter" is primarily used to refer to visible markers such as green fluorescent protein (GFP), eGFP, luciferase, GUS, etc.

[0048] The term "ortholog" in this article refers to a homologous gene or protein found in another species that has the same function as the gene or protein, but (typically) diverged sequentially from the point in time when the species carrying the gene began to diverge (i.e., the gene evolved from a common ancestor through speciation). Therefore, orthologs of the Taxaracum parthenogenetic gene can be identified in other plant species based on sequence comparisons (e.g., based on the percentage of sequence similarity across the entire sequence or specific domains) and functional analysis.

[0049] The terms “homologous” and “heterologous” refer to the relationship between a nucleic acid or amino acid sequence and its host cell or organism, especially in the context of transgenic organisms. Thus, a homologous sequence is naturally found in the host species (e.g., lettuce plants transformed with a lettuce gene), while a heterologous sequence is not naturally found in the host cell (e.g., lettuce plants transformed with a sequence from a potato plant). Depending on the context, the terms “homologous” or “homogeneous” may alternatively refer to sequences that are descendants of a common ancestral sequence (e.g., they can be orthologous).

[0050] “Strong hybridization conditions” are used to identify nucleotide sequences that are substantially identical to a given nucleotide sequence. Strict conditions depend on the sequence and will vary under different conditions. Typically, at specified ionic strengths and pH, strict conditions are chosen to be approximately 5°C lower than the thermal melting point (Tm) of the specific sequence. Tm is the temperature at which 50% of the target sequence hybridizes with a perfectly matched probe (at specified ionic strengths and pH). Strict conditions are typically chosen where the salt concentration is approximately 0.02 mol at pH 7 and the temperature is at least 60°C. Decreasing the salt concentration and / or increasing the temperature increases strictness. Strict conditions for RNA-DNA hybridization (using, for example, a 100 nt probe using Northern blotting) include, for example, washing at 63°C in 0.2× SSC for at least 20 minutes, or equivalent conditions. Strict conditions for DNA-DNA hybridization (using, for example, a 100 nt probe using Southern blotting) include, for example, washing at at least once (usually twice) at a temperature of at least 50°C, typically approximately 55°C, in 0.2× SSC for 20 minutes, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

[0051] For example, "highly stringent" conditions can be provided by hybridization in an aqueous solution at 65°C containing 6x SSC (20x SSC contains 3.0M NaCl, 0.3M sodium citrate, pH 7.0), 5x Denhardt's (100x Denhardt's contains 2% Ficoll, 2% polyvinylpyrrolidone, 2% bovine serum albumin), 0.5% sodium dodecyl sulfate (SDS), and 20 μg / ml denatured vector DNA (single-stranded fish sperm DNA, with an average length of 120-3000 nucleotides) as non-specific competitors. Following hybridization, highly stringent washing can be performed in several steps, with the final wash (approximately 30 minutes) at the hybridization temperature in 0.2-0.1× SSC and 0.1% SDS.

[0052] "Medium stringency" refers to conditions comparable to hybridization in the above solutions, but at approximately 60-62°C. In this case, the final wash is performed at the hybridization temperature in 1x SSC and 0.1% SDS.

[0053] “Low stringency” refers to conditions equivalent to hybridization at approximately 50–52 °C in the above solutions. In this case, the final wash is performed at the hybridization temperature in 2x SSC and 0.1% SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

[0054] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or two nucleotide sequences using a global or local alignment algorithm (depending on the length of the two sequences). Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch), which optimally aligns sequences across their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Then, sequences can be described as "substantially identical" or "substantially similar" when they share at least a minimum percentage of sequence identity (as defined herein) when optimally aligned using, for example, the GAP or BESTFIT program with default parameters. The percentage of sequence identity is preferably determined using the "BESTFIT" or "GAP" program of Sequence Analysis Software Package™ (version 10; GeneticsComputer Group, Inc., Madison, Wis.). GAP uses the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, Journal of Molecular Biology 48:443-453, 1970) to align two sequences across their entire length (full length), maximizing the number of matches and minimizing the number of gaps. Global alignment is suitable for determining sequence identity when two sequences are of similar length. Typically, using GAP's default parameters, the gap generation penalty is 50 (nucleotides) / 8 (proteins) and the gap extension penalty is 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and percentage sequence identity scores can be determined using computer programs, such as the GCG Wisconsin package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or open-source software, such as the programs "needle" (using the global Needleman-Wunsch algorithm) or "water" (using the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as the GAP above, or using the default settings (for "needle" and "water," and for protein and DNA alignments, the default GAP open penalty is 10.0, and the default gap extension penalty is 0.5; for proteins, the default score matrix is ​​Blossum62, and for DNA, the default score matrix is ​​DNAFull). “BESTFIT” uses Smith and Waterman’s local homology algorithm to perform optimal alignment of the best segments of similarity between two sequences and inserts gaps to maximize the number of matches (Smith and Waterman, Advances in Applied Mathematics, 2:482-489, 1981; Smith et al., Nucleic Acids Research 11:2205-2220, 1983). When sequences have substantially different total lengths, local alignment, such as that using Smith and Waterman’s algorithm, is preferred.

[0055] As used herein, “sequence identity” refers to the degree to which two best-aligned polynucleotide or peptide sequences remain unchanged throughout the alignment window of their components, such as nucleotides or amino acids. The “identity score” of the aligned fragments of the test and reference sequences is the number of common components shared by the two aligned sequences divided by the total number of components in the reference sequence fragment (i.e., the entire reference sequence or a smaller defined portion of the reference sequence). The “identity percentage” is the identity score multiplied by 100.

[0056] Useful methods for determining sequence identity are also disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994 and Carillo, H., and Lipton, D., Applied Math (1988) 48:1073. More specifically, preferred computer programs for determining sequence identity include the Basic Local Alignment Search Tool (BLAST) program, which is publicly available from the National Center for Biotechnology Information (NCBI) of the National Institutes of Health, National Library of Medicine, Bethesda, Md. 20894; see BLAST Manual, Altschul et al., J. Mol. Biol. 215:403-410 (1990); BLAST program version 2.0 or higher allows the introduction of gaps (deletions and insertions) in the alignment; for peptide sequences, BLASTX can be used to determine sequence identity; and for polynucleotide sequences, BLASTN can be used to determine sequence identity.

[0057] Optionally, the percentage similarity or identity can be determined by searching public databases using algorithms such as FASTA and BLAST. Therefore, the nucleic acid and protein sequences of this invention can be further used as "query sequences" to search public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program with a score of 100 and a word length of 12 to obtain nucleotide sequences homologous to the oxidoreductase nucleic acid molecule of this invention. BLAST protein searches can be performed using the BLASTx program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecule of this invention. To obtain gap alignments for comparative purposes, Gapped BLAST (Gapped BLAST) as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402 can be used. When using the BLAST and Gapped BLAST procedures, the default parameters of the corresponding procedures (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information: http: / / www.ncbi.nlm.nih.gov / .

[0058] As used in this article, “sexual plant reproduction” refers to a developmental pathway in which a somatic cell called a “megasporocyte” (e.g., diploid) undergoes meiosis to produce four meiotic megaspores. One of these megaspores undergoes mitosis to form a macrogametophyte (also called an embryo sac), which contains a meiotic egg cell (i.e., a cell with a reduced number of chromosomes compared to the maternal parent) and two meiotic polar nuclei. One sperm cell in the pollen grain fertilizes the egg cell to produce a (e.g., diploid) embryo, while a second sperm cell fertilizes the two polar nuclei to produce a (e.g., triploid) endosperm (a process called double fertilization).

[0059] As used in this article, the term "megasporocyte mother cell" or "megasporonocyte" refers to the cell that produces megaspores through meiosis (usually meiosis) to generate four haploid megaspores that will develop into female gametophytes. In angiosperms (also known as flowering plants), megasporocyte mother cells produce megaspores, which develop into macrogametophytes through two distinct processes: megasporogenesis (formation of megaspores or megasporangia within the nucellus) and macrogametophyte formation (development of megaspores into macrogametophytes).

[0060] The term "asexual reproduction" as used in this article refers to the process by which plants reproduce without fertilization and gamete fusion. Asexual reproduction produces new individuals that are genetically identical to and similar to the parent plant, except through mutation or somatic cell recombination. There are two main types of asexual reproduction in plants: vegetative propagation (i.e., budding, tillering, etc., of the vegetative parts of the original plant) and agametosis.

[0061] As used herein, the term "agametic reproduction" refers to the formation of seeds through an asexual process. One form of agametic reproduction is characterized by: 1) incomplete meiosis, which refers to the formation of unreduced embryo sacs in the ovary, and 2) parthenogenesis, which refers to the development of unreduced eggs into embryos. Hundreds of wild plant species are characterized by agametic and asexual reproduction. Incomplete meiosis is a process that produces unreduced egg cells with the same chromosome number and genotype as the parent plant's somatic cell tissue. Unreduced egg cells can originate from unreduced megaspores (plosporogenesis) or primary somatic cells (asporogenesis). In the case of plosporogenesis, megasporogenesis is replaced by mitosis or modified meiosis. Modified meiosis is preferably a first-division restorer without recombination. Alternatively, modified meiosis can be a second-division restorer. In a preferred embodiment, incomplete meiosis is disporogenic incomplete meiosis affecting the first meiotic division. Agametic reproduction is known to occur in different forms, including at least two, known as gametophytic agametic reproduction and sporophytic agametic reproduction (also called adventitious embryos). Examples of plants that undergo gametophytic agametic reproduction include dandelion (Taraxacum), wild willow (Lactuca indica), Kentucky bluegrass (Poa tigrinosa), and eastern gama grass (Poa rubescens). Examples of plants that undergo sporophytic agametic reproduction include citrus (Citrus) and mangosteen (Mangosteen).

[0062] The term "plosporogenesis" as used in this article refers to the derivation of unmeiotic embryo sacs directly from megasporocytes via mitosis or terminated meiotic events. Three main types of plosporogenesis have been reported, named after the plants in which they occur: dandelion, *Ixeris*, and *Antennaria* types. In the dandelion type, promeiosis begins but is subsequently terminated, resulting in two unmeiotic didiglia, one of which produces an embryo sac via mitosis. In the *Ixeris* type, two further mitotic divisions of the nucleus following the isomerization in promeiosis produce an octenuclear embryo sac. The dandelion and *Ixeris* types are referred to as meiotic plosporogenesis because they involve modifications of meiosis. Conversely, in the *Antennaria* type, called mitotic plosporogenesis, the megasporocyte does not initiate meiosis and directly divides three times to produce an unmeiotic embryo sac. In gametophytic agametosis via plosporogenesis, the unreduced gametophyte is produced from unreduced megaspores. These unreduced megaspores arise from mitotic-like division (mitosis) or modified meiosis (meiotic disporogenesis). In both gametophytic agametosis via asporogenesis and gametophytic agametosis via plosporogenesis, the unreduced ovum develops asexually into an embryo. Agametosis in dandelions is disporogenic, meaning that the first female meiosis (meiosis I) is skipped, producing two unreduced megaspores with the same genotype as the mother plant. One of these megaspores degenerates, while the other surviving unreduced megaspore produces an unreduced megasporophyte (or embryo sac) containing an unreduced ovum. This unreduced ovum develops into an embryo with the same genotype as the mother plant without fertilization. Seeds produced by gametophytic agametosis are called agametogenic seeds.

[0063] The term "ploidysporogenesis function" refers to the ability of a plant, preferably in the female ovary, and more particularly in the megasporocyte and / or female gamete, to induce ploidysporogenesis. Therefore, a plant with ploidysporogenesis function is capable of undergoing the ploidysporogenesis process, i.e., resuming the production of unmeiotic gametes through meiosis I.

[0064] As used herein, the term "agametic seed" refers to a seed obtained from an agametic plant species or from a plant or crop that undergoes agametosis, particularly through the formation of gametophytes from ploid spores. Agametic seeds are characterized by being clones and genetically identical to their parent plants, and germinating into plants capable of true reproduction. In this invention, "agametic seed" also refers to "cloned agametic seed."

[0065] As used herein, the term "agametic plant" refers to a plant that reproduces itself asexually without fertilization. An agametic plant can be a sexual plant modified to reproduce agametically, such as a sexual plant that has been genetically modified, for example, with one or more parthenogenetic genes taught herein to obtain an agametic plant, or a plant that is the offspring of an agametic plant. In this case, the offspring produced by agametosis are genetically identical to the parent plant.

[0066] A "clone" of a cell, plant, plant part, or seed is characterized by being genetically identical to its sibling and the parent plant from which it originated. Individual clones have nearly identical genomic DNA sequences; however, mutations can cause minute differences.

[0067] As used in this article, the term "true reproduction" or "true reproduction organism" (also known as a purebred organism) refers to an organism that always passes on a certain phenotypic trait to its offspring with no change or almost no change. An organism is referred to as a true reproducer for each trait to which it applies, and the term "true reproduction" is also used to describe an individual's heritable traits.

[0068] As used herein, the term "F1 hybrid" (or F1 hybrid) refers to the first offspring of children with significantly different parental types. Parental types may or may not be inbred lines. F1 hybrids are used in genetics and selective breeding, where they may appear as F1 hybrids. Offspring with significantly different parental types produce new, uniform phenotypes that combine traits from both parents. F1 hybrids have significant advantages, such as heterosis, and are therefore very popular in agricultural practice. In one embodiment of the invention, the methods, genes, proteins, variants, or fragments thereof taught herein can be used to fix the genotype of an F1 hybrid, regardless of its genetic complexity, and allow for the production of reproductive organisms in a single step.

[0069] As used in this article, "pollination" or "pollination of..." refers to the process by which pollen is transferred from the anther (male part) of a plant to the stigma (female part), enabling fertilization and reproduction. It is unique to flowering angiosperms. Each pollen grain is a male haploid gametophyte adapted for transport to the female gametophyte, where fertilization can occur during double fertilization by producing male gametes (or multiple gametes). A successful angiosperm pollen grain (gametophyte) containing male gametes is transported to the stigma, where it germinates and its pollen tube grows along the style to the ovary. Its two gametes travel along the tube to the location where the gametophyte containing the female gamete is preserved within the carpel. One nucleus fuses with the polar body to form the endosperm tissue, and the other nucleus fuses with the ovule to form the embryo.

[0070] As used herein, the term "parthenogenesis" refers to a form of asexual reproduction in which the embryo grows and develops without fertilization. The genes and proteins of this invention can, preferably, combine with dispore factors, such as genetic or chemical factors, to produce gamete-infected offspring.

[0071] As used in this article, the term "parthenogenesis phenotype" refers to the ability of a plant and / or its offspring to grow and develop an embryo from an egg cell without fertilization.

[0072] As used herein, the term "pyramiding" or "stacking" of genes refers to the process of synthesizing related or unrelated genomes from different parental lines into a single plant, which are the basis for desired or advantageous traits (such as disease resistance, color, drought resistance, insect resistance, etc.). Gene pyramiding or stacking can be performed using conventional breeding methods, or it can be accelerated by using molecular markers to identify and retain plants containing the desired allele combination while discarding those that do not. In one embodiment of the invention, the parthenogenetic genes taught herein can be advantageously used in gene pyramiding or stacking procedures to produce agametogenic plants or to introduce agametosis into sexual crops.

[0073] In this document and its claims, the verb "comprising" and its variations are used in a non-limiting sense to indicate that the item following the word is included, but not to exclude items not specifically mentioned. Furthermore, the use of the indefinite article "a" or "an" to refer to an element does not preclude the possibility that more than one element exists, unless the context explicitly requires the presence of exactly one element. Therefore, the indefinite article "a" or "an" generally means "at least one". It should also be understood that when "sequence" is referred to herein, it generally refers to an actual physical molecule having a specific subunit sequence (e.g., amino acids).

[0074] As used herein, the term "plant" includes plant cells, plant tissues or organs, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant cell masses, and intact plant cells in plants or plant parts such as embryos, pollen, ovules, fruits, flowers, leaves (e.g., harvested lettuce crops), seeds, roots, root tips, etc. Detailed Implementation

[0075] This invention provides a method for generating a mutant gene, wherein the mutant gene is functional in parthenogenesis. Preferably, the method includes the step of mutating the promoter sequence of a sexual variant of a parthenogenesis gene, represented herein as the par allele, to increase the expression of the encoded PAR protein. The resulting mutant gene can be considered a Par allele because it can induce a parthenogenesis phenotype in plants. In cases where the par allele is an endogenous allele in plants or plant cells lacking a dominant Par allele, the method of this invention, by modifying the promoter of said par allele, transforms plants or plant cells that do not exhibit parthenogenesis into plants or plant cells that exhibit parthenogenesis. This invention provides a method for converting a sexual par allele into a parthenogenesis Par allele by modifying the promoter sequence of the par allele. This invention provides a method for generating a mutant gene functional in parthenogenesis, comprising the following steps:

[0076] (a) Providing a gene containing a sequence encoding a PAR protein operatively linked to a promoter; and

[0077] (b) Modifying the promoter by modifying the sequence upstream of the sequence encoding the PAR protein to increase the expression of the encoded PAR protein, preferably in mature female gametophytes.

[0078] More specifically, the present invention provides a method for generating a mutated gene that functions in parthenogenesis, comprising the following steps:

[0079] (a) Provides a gene comprising a sequence encoding a PAR protein operably linked to a promoter containing one or more transcription factor MYB binding sites; and

[0080] (b) Modifying the promoter by modifying the sequence upstream of the MYB binding site of one or more transcription factors to increase the expression of the encoded PAR protein, preferably in mature female gametophytes.

[0081] The sequence upstream of the PAR protein encoding sequence, preferably the sequence upstream of one or more transcription factor MYB binding sites, can be modified by introducing an enhancer sequence that enhances PAR protein expression and / or by removing a repressor sequence that inhibits PAR protein expression. Preferably, an enhancer sequence is inserted, preferably a female gametophyte-specific enhancer sequence. The insert may be a MITE sequence as defined herein. Alternatively or additionally, the promoter sequence upstream of the PAR protein encoding sequence, preferably the promoter sequence upstream of one or more MYB binding sites, can be altered by (random) mutagenesis resulting in one or more nucleotide substitutions, insertions, and / or deletions to introduce one or more enhancer sequences and / or increase the expression of the encoded PAR protein.

[0082] Alternatively or concurrently, the present invention provides a method for generating a mutant gene that functions in parthenogenesis, comprising the following steps:

[0083] (a) Provides a gene comprising a sequence encoding a PAR protein operably linked to a promoter containing one or more transcription factor MYB binding sites; and

[0084] (b) Modify the promoter by modifying at least one of the MYB binding sites of the one or more transcription factors to increase the expression of the encoded PAR protein.

[0085] Preferably, when present in plants, the modified promoter of the mutant gene of the present invention results in increased expression of the PAR protein encoded by a coding sequence operatively linked to the promoter, compared to the unmodified counterpart, i.e., the (endogenous or natural) promoter from which the promoter of the present invention is derived as defined herein. Preferably, the increased expression is at least in the egg cells of plants containing the modified promoter of the present invention and / or the mutant gene. Preferably, the increase in expression compared to the original non-mutant gene (i.e., the gene of step (a) of the method of the present invention) is an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

[0086] In one embodiment, the gene encoding the PAR protein in step a) is not derived from Arabidopsis thaliana. In one embodiment, the mutant gene of the present invention does not contain a mutant promoter derived from Arabidopsis thaliana DAZ3 with mutations at one or more MYB binding sites.

[0087] The gene provided in step (a) is a parthenogenesis gene, preferably a sexual variant of the gene, i.e., the par allele. Since the obtained mutant gene is functional in parthenogenesis, the method of the present invention can also be considered as a method for converting the sexual allele (par allele) of a parthenogenesis gene into an allele (Par allele) that is functional in parthenogenesis.

[0088] The transcription factor MYB binding site (also referred to as the "MYB binding site" in this paper) is a sequence within the promoter that is recognized and bound by the transcription factor MYB. MYB proteins are a family of DNA-binding proteins containing varying numbers of MYB domain repeats, which endow them with the ability to bind DNA at the MYB binding site, thereby regulating transcription.

[0089] The inventors identified one or more MYB binding sites in a region approximately 50-150 bp upstream of the start codon of the par allele. The MYB binding site is defined herein as a preferred 7-nucleotide sequence, preferably having the nucleotide sequence NACCNNN, preferably AACCNNN, more preferably AACCGNN, even more preferably AACCG[C / T]N, even more preferably AACCG[C / T]C, and can be AACCGCC, AACCGTC, or [T / A]AACCGCC (Borg et al., 2011). Preferably, one or more MYB binding sites are located approximately 60-140 bp, 70-130 bp, 80-120 bp, or 90-110 bp upstream of the start codon in the sequence encoding the PAR protein, preferably up to approximately 200, 190, 180, 170, 160, 150, 140, 130, 120, or 110 nucleotides upstream of the start codon. More preferably, the 3' end of one or more, and preferably two, MYB binding sites is located 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 nucleotides upstream of the start codon in the sequence encoding the PAR protein. In some cases, two MYB binding sites are located in the aforementioned region. For example, in lettuce (Lactuca sativa), the first MYB binding site is located 117-110 nucleotides upstream of the ATG start codon, and the second MYB binding site is located 104-98 nucleotides upstream of the ATG start codon.

[0090] The MYB binding site is preferably a binding site for the transcription factor MYB, wherein the MYB transcription factor is an R2R3 transcription factor or an R2R3-MYB transcription factor, and wherein the transcription factor MYB can be DUO1 (UniProtKB accession number A0A178VEK7), or a variant, homology, or ortholog of DUO1. The transcription factor preferably has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID NO: 62.

[0091] Alternatively, the present invention can also be considered as a method for generating mutant genes capable of inducing a parthenogenetic phenotype in plants, wherein the method includes the following steps:

[0092] (a) Providing a gene comprising a sequence encoding a PAR protein, wherein said sequence is operatively linked to a promoter, wherein said promoter optionally comprises one or more transcription factor MYB binding sites; and

[0093] (b) Modifying the promoter to increase the expression of the encoded PAR protein, preferably by at least one of the following:

[0094] i) Introducing an insert upstream of the start codon of a sequence encoding a PAR protein as defined herein, or even more preferably upstream of or directly upstream of one or more MYB binding sites as defined herein, to increase the expression of the encoded PAR protein, preferably by introducing an enhancer sequence or removing a repressor sequence.

[0095] ii) Introducing substitutions and / or deletions in a promoter sequence upstream of the start codon of a sequence encoding a PAR protein as defined herein, even more preferably upstream or directly upstream of one or more MYB binding sites as defined herein, wherein the substitutions and / or deletions preferably increase the expression of the encoded PAR protein by introducing an enhancer sequence or removing a repressor sequence; and

[0096] Combinations of (iii), (i), and (ii).

[0097] In the methods of this invention, promoter modification can be performed using any conventional method known in the art, such as, but not limited to, preferably by random or targeted mutagenesis, and optionally by homologous recombination, to introduce an insertion or deletion in a promoter directly upstream of one or more MYB binding sites as defined herein.

[0098] Optionally, insertions, substitutions, or deletions in the promoter may modify or remove one or more MYB binding sites as defined herein.

[0099] Therefore, the present invention can also be considered a method for generating mutant genes capable of inducing parthenogenesis phenotypes in plants, wherein the method includes the following steps:

[0100] (a) Provides a gene comprising a sequence encoding a PAR protein, wherein said sequence is operatively linked to a promoter comprising one or more transcription factor MYB binding sites; and

[0101] (b) Modifying the promoter by modifying the sequence upstream of the MYB binding site of the one or more transcription factors to increase the expression of the encoded PAR protein by introducing an insertion or deletion in the promoter directly upstream of the one or more MYB binding sites of the promoter, as defined herein.

[0102] Alternatively, the method of the present invention may include modifying the promoter by inducing, modifying, or removing one or more MYB binding sites as defined herein.

[0103] In cases where the promoter contains one or more, preferably two, MYB binding sites as defined herein, these one or more, preferably two, MYB binding sites may be optionally modified or removed to reduce the binding of the transcription factor MYB and / or to introduce insertions or deletions upstream of these one or more MYB binding sites. The modification of the MYB binding site in the method of the present invention may be a modification of 1, 2, 3, 4, 5, 6, or 7 nucleotides, preferably 1, 2, or 3 nucleotides, or even more preferably 1 nucleotide, such that the sequence is no longer a MYB binding site. Preferably, the modification is at least one mutation (nucleotide exchange, insertion, or deletion) at the first, second, third, fourth, or fifth position of the 7-nucleotide-long MYB binding motif as described above, i.e., modification of at least one of A, A, C, C, and G in the motif AACCGNN. Preferably, the modification is at least one mutation (nucleotide exchange, insertion, or deletion) at the first, second, third, or fourth position of the 7-nucleotide-long MYB binding motif, i.e., modification of at least one of A, A, C, and C of the motif AACCGNN, or optionally, modification of two, three, or all four nucleotides at these positions.

[0104] Preferably, the modification results in a reduction or elimination of the binding of the transcription factor MYB to the MYB binding site. Preferably, when tested under suitable experimental conditions, such as as described above by Kelemen et al., the binding affinity is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. Optionally, the modification of the MYB binding site can be the deletion of the MYB binding site. The modification can be carried out by random mutagenesis (e.g., by chemical or radiation mutagenesis) or targeted mutagenesis (e.g., CRISPR-mediated mutagenesis). Optionally, multiple (two, three, or more) MYB binding sites present in the gene are modified as defined in the method of the present invention. Optionally, the mutant gene comprises a modified promoter of lettuce, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 17. Optionally, the mutant gene includes the promoter operatively linked to a sequence encoding a lettuce PAR protein, preferably the coding sequence comprising or consisting of the sequence of SEQ ID NO: 33. Optionally, the mutant gene includes or consists of the sequence of SEQ ID NO: 35. The invention also includes plants or plant cells comprising the mutant gene and / or construct, preferably lettuce plants or plant cells.

[0105] In one embodiment, the promoter of a sexual gene, preferably a par gene, can be modified by introducing an insert upstream of one or more MYB binding sites. The insert can be introduced near or directly adjacent to one or more MYB binding sites. The distance between the insert and the MYB binding site (or, in the case of multiple MYB binding sites, the most upstream MYB binding site in the coding sequence) is preferably a maximum of 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 nucleotides. Preferably, the insert is introduced directly upstream of one or more MYB binding sites. In the method of the present invention, the insert introduced into the gene promoter preferably includes a nucleic acid insert, preferably a double-stranded DNA insert, wherein the length of the insert is 50 to 2000 bp, 100 to 1900 bp, 200 to 1800 bp, 300 to 1700 bp, 400 to 1600 bp, 500 to 1500 bp, 600 to 1400 bp, 1000 to 1400 bp, 1200 to 1400 bp, or 1300 to 1400 bp. Even more preferably, the insert has a length of about 1300 bp. Alternatively or additionally, the insert is between about 1-50 bp, about 5-30 bp, or about 10-20 bp. Preferably, the insert is introduced into the promoter upstream (5') of the MYB binding site as defined herein, preferably such that the distance between the MYB binding site and the insert (preferably the 3'-terminus of the insert) is between 0 and 200 bp, preferably at most 0, 10, 20, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 bp. Preferably, the insert is positioned such that the 3'-terminal nucleotide of the insert is homologous to nucleotide 1798 of SEQ ID NO: 2 and / or nucleotide 1798 of SEQ ID NO: 5. Preferably, the insert has no open reading frame.

[0106] The insert may be a non-autonomous transposable element, preferably a non-autonomous transposable sub-element derived from hAT. The insert may contain enhancer sub-elements, preferably female gametophyte-specific enhancer sub-elements. Even more preferably, the insert is a miniature inverted repeat transposable element (MITE) or a MITE-like sequence, wherein the MITE or MITE-like sequence is a non-autonomous element, preferably a non-autonomous transposable element, characterized by containing an inner sequence without an open reading frame, flanked by terminal inverted repeats (TIRs) flanked by small direct repeats (target site repeats, TSDs). The TIR may have the sequence CAGGGCCGG and / or CCGGCCCTG. The TSD may have the ACTGCTAC sequence. For a further description of the MITE, TIR and sequence, see Guo et al., Scientific Reports. 2017 Jun 1; 7(1):2634, which is incorporated herein by reference. The insert, preferably the MITE or MITE-like sequence, may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more identity with SEQ ID NO: 60. The insert may be introduced by recombination and / or targeted genome editing.

[0107] Optionally, the mutant gene comprises a modified lettuce promoter, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 18. Optionally, the mutant gene comprises the promoter operatively linked to a sequence encoding a lettuce PAR protein, preferably, the coding sequence comprises or consists of the sequence of SEQ ID NO: 33. Optionally, the mutant gene comprises or consists of the sequence of SEQ ID NO: 36. Optionally, the mutant gene comprises a modified lettuce promoter, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 18 and the promoter is operatively linked to a sequence encoding a dandelion PAR protein, preferably, the coding sequence comprises or consists of the sequence of SEQ ID NO: 3. Optionally, the mutant gene comprises or consists of the sequence of SEQ ID NO: 64. The present invention also includes plants or plant cells containing the mutant gene and / or construct, preferably lettuce plants or plant cells.

[0108] Optionally, the mutant gene comprises a dandelion Par promoter, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 2. Optionally, the mutant gene comprises the promoter operatively linked to a sequence encoding a PAR protein, wherein the PAR protein is not a (natural) dandelion PAR protein. Optionally, the mutant gene comprises the promoter operatively linked to a sequence encoding an orthologous PAR protein as defined above. Optionally, the mutant gene comprises a dandelion Par promoter, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 2, and wherein the promoter is operatively linked to a sequence encoding a lettuce PAR protein, preferably the encoding sequence comprising or consisting of the sequence of SEQ ID NO: 33. Optionally, the mutant gene comprises or consists of the sequence of SEQ ID NO: 65.

[0109] In another embodiment, the promoter of the sexually transmitted gene, preferably the par gene, can be modified by introducing a deletion upstream of one or more MYB binding sites. The deletion can be introduced near or directly adjacent to one or more MYB binding sites. The distance between the deletion and the MYB binding site (or, in the case of multiple MYB binding sites, the MYB binding site located upstream of the coding sequence) is preferably a maximum of 200, 150, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide. Preferably, the deletion is introduced directly upstream of one or more MYB binding sites. Deletions that can be introduced into the gene promoter in the method of the invention preferably include nucleic acid deletions, preferably double-stranded DNA deletions, wherein the length of the deletion is 10 to 1000 bp, 50 to 900 bp, 100 to 800 bp, 200 to 700 bp, 350 to 600 bp, preferably about 400 bp. Preferably, the deletion has no open reading frame. The deletion can be introduced by recombination and / or targeted or random genome editing. Optionally, the mutant gene comprises a lettuce-modified promoter, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 20. Optionally, the mutant gene comprises the promoter operatively linked to a sequence encoding a lettuce PAR protein, preferably, the coding sequence comprises or consists of the sequence of SEQ ID NO: 33. Optionally, the mutant gene comprises or consists of the sequence of SEQ ID NO: 61. The invention also includes plants or plant cells comprising the mutant gene and / or construct, preferably lettuce plants or plant cells.

[0110] Preferably, the gene in step a) of the method of the present invention and the mutant gene obtainable by the method of the present invention are nucleic acid molecules, preferably DNA molecules, even more preferably genomic DNA molecules, or a portion thereof. Optionally, the genomic DNA molecule is in a plant cell, preferably a plant protoplast. Compared with a control plant, modifying the promoter of a sexual gene by the method of the present invention, when located in a plant cell, can result in a significant increase in the parthenogenetic phenotype of the plant derived from the plant cell, wherein preferably, the control plant does not exhibit a parthenogenetic phenotype, while the plant containing the mutant gene of the present invention exhibits a parthenogenetic phenotype. The control plant preferably differs from the plant derived from the plant cell only in that the promoter of the par allele is not modified as defined herein. Preferably, the control plant or control plant cell differs from the plant cell or plant of the present invention only in that the control plant or control plant cell does not contain the genetic modification defined herein.

[0111] Preferably, the gene in step (a) is a par allele and the promoter of the par allele is modified as defined herein, resulting in a plant exhibiting a parthenogenetic phenotype when located in a plant cell that cannot reproduce asexually. Since the mutant gene obtainable by the method of the present invention is functional in parthenogenesis, the mutant gene can be considered a par allele, and the method of the present invention can also be considered a method for converting a par allele into a par allele.

[0112] Optionally, the modification of the promoter by the method of the present invention affects the binding of the transcription factor MYB to the promoter, resulting in increased expression of the PAR protein encoded by the gene containing the promoter. Optionally, the modification results in a decrease or elimination of the binding of the transcription factor MYB. The binding of the transcription factor MYB to the promoter can be assessed by any suitable assay known to those skilled in the art, such as, but not limited to, the in vivo yeast one-hybrid system (see, for example, Kelemen et al., PLoSOne. 2015; 10(10):e0141044).

[0113] Random mutagenesis can be, but is not limited to, chemical mutagenesis, gamma radiation, X-rays, or fast neutron radiation. Non-limiting examples of chemical mutagenesis include, but are not limited to, EMS (ethyl methanesulfonate), MMS (methyl methanesulfonate), NaN3 (sodium azide)D, ENU (N-ethyl-N-nitrosourea), AzaC (azytidine), and NQO (4-nitroquinoline 1-oxide). Optionally, mutagenesis systems such as TILLING (Targeting Induced Local Lesions IN Genomics; McCallum et al., 2000, NatBiotech 18:455 and McCallum et al. 2000, Plant Physiol. 123, 439-442, both incorporated herein by reference) can be used to generate plant lines with genes having modifications as defined herein. TILLING uses conventional chemical mutagenesis (e.g., EMS mutagenesis) followed by high-throughput screening for mutations. Therefore, plants, seeds, and tissues containing genes with one or more desired mutations can be obtained using TILLING. Targeted mutagenesis is mutagenesis that can be programmed to alter specific nucleotide or nucleic acid sequences. Targeted mutagenesis can be selected from, but is not limited to, techniques selected from: oligotargeted mutagenesis, RNA-guided endonucleases (e.g., CRISPR technology), TALENs or zinc finger techniques, and combinations thereof.

[0114] Optionally, the gene in step (a) of the method of the present invention is a natural sequence. The gene in step (a) of the method of the present invention preferably comprises or consists of a promoter operatively linked to a sequence encoding a PAR protein, subsequently optionally followed by a 3'UTR sequence. The gene provided in step (a) may be part of a plant cell, preferably a plant protoplast, which does not contain a parthenogenetic allele of a parthenogenetic gene. Preferably, the plant cell or protoplast is non-parthenogenetic, i.e., the plant regenerated from the cell or protoplast does not exhibit a parthenogenetic phenotype. In other words, the gene provided in step (a) is preferably contained in a plant cell, preferably a plant protoplast, wherein a mutation, as defined herein, leads to the induction or increase of parthenogenesis in the plant cell, i.e., the plant regenerated from the mutated cell or protoplast exhibits a parthenogenetic phenotype.

[0115] The gene provided in step (a) of the method of the present invention can be a natural gene, preferably naturally occurring in plant cells. A natural sequence is a sequence found in nature, also referred to herein as "wild-type" or "natural". Thus, in this embodiment, the promoter, coding sequence, and optional 3'UTR are derived from a single plant species.

[0116] Alternatively, the gene provided in step (a) of the method of the present invention is a non-natural and / or synthetic gene, also referred to herein as a chimeric gene. Optionally, in the chimeric gene, a promoter is operatively linked to a coding sequence encoding a PAR protein and / or a 3'UTR, wherein the coding sequence and / or 3'UTR is heterologous to the promoter. As a non-limiting example, the promoter may be from another plant species, such as the coding sequence and / or 3'UTR. Optionally, the promoter and 3'UTR are from a single plant species, while the coding sequence is from another plant species.

[0117] Preferably, the gene provided in step (a) of the method of the present invention is present in plant cells or protoplasts. Therefore, the method of the present invention may include a step of providing a protoplast or plant cell containing the gene of step (a) prior to the step of modifying the gene promoter. Preferably, the gene is an endogenous gene present in the genome of the protoplast or plant cell. Preferably, the protoplast or plant cell is isolated from a plant, preferably a non-parthenogenetic plant. Preferably, the plant does not possess the Par allele in its genome. Preferably, the promoter of the gene in the protoplast or plant cell provided in step (a) is modified as defined herein by targeted or random mutagenesis, preferably targeted mutagenesis.

[0118] In a specific embodiment of the method of the present invention, the plant cell containing the gene of step (a) may be located in a plant seed. Preferably, the seed does not have the Par allele in its genome. Preferably, in the method of the present invention, the promoter of the gene within the seed is modified by targeted or random mutagenesis, preferably random mutagenesis.

[0119] Following the step of modifying the promoter of the gene, the method of the present invention may include the step of regenerating a plant from the protoplast or the step of growing a plant from the seed.

[0120] Furthermore, the method of the present invention may include screening and / or genotyping steps. Genotyping can be performed by sequencing at least a portion of the promoter after the modification step (optionally preceded by PCR amplification of genomic DNA and / or a target sequence containing the promoter of interest) or by any genomic variation analysis method or molecular marker analysis known in the art, such as, but not limited to, sequence-based genotyping (SBG). SNPSelect analysis is performed. Event-specific PCR diagnostic methods can also be developed, where PCR primers are based on plant DNA flanking the modification (see US6563026). Similarly, event-specific AFLP fingerprints or RFLP fingerprints can be developed to identify transgenic plants or any plant, seed, tissue, or cell from which they originate. Genotyping can be performed directly after the modification step or after callus, tissue, or plant tissue has grown from protoplasts or seeds.

[0121] Screening for parthenogenesis can be directly assessed by comparing the ability of plants containing the mutant gene obtained by the method of the present invention (referred to herein as test plants) to grow and develop embryos from unfertilized egg cells. Preferably, this ability is compared with that of a control plant, which preferably differs from the test plant only in that it does not contain the mutant gene obtained by the method of the present invention. Preferably, the control plant is a plant that does not contain the Par allele.

[0122] Alternatively or additionally, the function of the mutant gene obtained by the method of the present invention in parthenogenesis can be assessed by supplementing the construct containing the mutant gene of the present invention with a plant having agametogenesis deletion. Such agametogenesis-deprived plant can be dandelion isolate A68 that has been modified to lose the agametogenesis phenotype by modifying a functional Par allele (e.g., by deletion or knockout). Such agametogenesis-deprived plant can be dandelion isolate A68 containing the Par allele, wherein SEQ ID NO: 23 (encoding the PAR protein of SEQ ID NO: 32) as defined herein has been modified to any one of SEQ ID NO: 24-27 to encode the proteins of SEQ ID NO: 28-31 respectively (see Table 1). This loss of agametogenesis in dandelion isolate A68 can be achieved by targeted genome editing using a CRISPR-Cas9 / guide RNA complex, wherein the guide RNA (also referred to herein as gRNA) contains the target-specific sequence of SEQ ID NO: 19, as exemplified herein. The deletion of the Par allele in dandelion isolate A68 leads to the loss of parthenogenesis, and consequently, the loss of agametogenesis. The mutant gene obtained by the method of this invention has the ability to induce parthenogenesis, and the agametogenic phenotype is restored (or rescued) upon introduction or transfection of a construct or vector containing the mutant gene. For dandelion isolate A68, a high seed set rate in the absence of cross-pollination is a clear indicator of agametogenesis. Self-pollination in this isolate can be excluded as an alternative explanation because sexually produced oocytes and pollen grains would have very low fertility due to unbalanced triploid male and female meiosis. Preferably, in the above complementation analysis, the mutant gene of this invention, in contrast to the original non-mutant gene, is able to restore agametogenesis in loss-of-function plants. Preferably, the capability means the restoration of agametosis in at least 1 out of 200 plants transformed with the mutant gene, 1 out of 100 plants, 10 out of 100 plants, 20 out of 100 plants, 30 out of 100 plants, 40 out of 100 plants, 50 out of 100 plants, 60 out of 100 plants, 70 out of 100 plants, 80 out of 100 plants, 90 out of 100 plants, or all plants that have lost function, preferably compared to 1 out of 500 plants, 1 out of 600 plants, 1 out of 700 plants, 1 out of 800 plants, 1 out of 900 plants, or 1 out of 1000 plants transformed with the original non-mutant gene (i.e., the gene provided in step a of the method of the present invention).

[0123] This invention also provides mutant genes obtained by or achievable through the methods of this invention. Preferably, the mutant gene differs from an endogenous or natural gene (only) in that it includes an insertion or deletion and / or one or more modified or removed MYB binding sites in a promoter as defined herein. Preferably, the mutant gene of this invention containing a promoter with modifications as defined herein is capable of inducing a parthenogenetic phenotype in plants. In other words, preferably, the mutant gene containing a promoter with modifications as defined herein is functional in parthenogenesis.

[0124] Optionally, the gene in step (a) of the method of the present invention and / or the mutant gene obtained by the method of the present invention is an isolated nucleic acid molecule or nucleic acid construct or (expression) vector, or a portion thereof. The present invention also provides such an isolated nucleic acid molecule, construct, or (expression) vector comprising the mutant gene, wherein the construct or vector is capable of transforming the mutant gene into the plant when the plant is transfected with the vector construct. The nucleic acid molecule may be, but is not limited to, DNA, and may be genomic DNA or may be derived from genomic DNA. The present invention also provides the use of the mutant gene of the present invention and / or the isolated nucleic acid, construct, or vector comprising the mutant gene in the purpose of increasing or inducing a parthenogenetic phenotype of protoplasts, plant cells, or plants.

[0125] The mutant gene of the present invention may be a chimeric gene as defined herein, optionally being part of a genetic construct or nucleic acid vector. The mutant gene of the present invention may optionally be contained in isolated nucleic acids, constructs, or vectors. In one embodiment of the invention, a nucleic acid containing or composed of the mutant gene of the present invention may be used to prepare a construct and / or vector containing the nucleic acid for transfer to a host cell and for generating a functional (preferably inducible parthenogenetic) protein encoded by said nucleic acid in the host cell.

[0126] Vectors suitable for introducing the mutant gene of the present invention into plant cells are referred to herein as “expression vectors”. The host cell is preferably a plant cell. The construction of mutant genes, constructs, and / or vectors for optionally transiently but preferably stably introducing the mutant gene sequence into the host cell genome is well known in the art.

[0127] The present invention also provides plant cells, plant protoplasts, plant tissues, seeds or plants containing mutant genes obtained by or obtainable by the methods of the present invention and / or containing said nucleic acid molecules or vectors.

[0128] The present invention also provides plant cells, plant protoplasts, plant tissues, seeds, or plants containing mutant genes, nucleic acid molecules, constructs, or vectors containing mutant genes as defined herein. Optionally, the plant cells, plant protoplasts, plant tissues, seeds, or plants are capable of incomplete meiosis, preferably agametosis.

[0129] Preferably, the nucleic acid of the present invention is an isolated nucleic acid. In one embodiment, the nucleic acid of the present invention may be derived from the dandelion family (e.g., broadly defined medicinal dandelion (Taraxacum officinale sensu lato)) or from other plant species. In one embodiment, the nucleic acid of the present invention is derived from a source other than that of the genus Taraxacum or broadly defined medicinal dandelion.

[0130] Optionally, nucleic acids or nucleic acid constructs of the present invention comprising or derived from the mutant gene obtained by the methods of the present invention can be stably inserted into the nuclear genome of a single plant cell, and the plant cells thus transformed can be used to produce transformed plants with an altered phenotype, i.e., a parthenogenetic phenotype. In a non-limiting example, a T-DNA vector from *Agrobacterium tumefaciens* containing the mutant gene taught herein can be used to transform plant cells, from which transformed plants can then be regenerated using, for example, the methods described in EP0116718, EP0270822, PCT publication WO84 / 02913, and published European patent application EP0242246, as well as Gould et al. (1991). The construction of T-DNA vectors for *Agrobacterium*-mediated plant transformation is well known in the art. The T-DNA vector can be a binary vector as described in EP0120561 and EP0120515 or a co-integration vector as described in EP0116718 that can be integrated into the Agrobacterium Ti plasmid via homologous recombination. For example, Michelmore et al. (1987) and Chupeau et al. (1989) have described lettuce transformation protocols.

[0131] Gielen et al. (1984) described the boundary sequence. Of course, other types of vectors can be used to transform plant cells, using methods such as direct gene transfer (as described in EP0223247), pollen-mediated transformation (as described in EP0270356 and WO85 / 01856), protoplast transformation (as described in US4,684,611), plant RNA virus-mediated transformation (as described in EP0067553 and US4,407,956), liposome-mediated transformation (as described in US4,536,475), and others.

[0132] In another embodiment, the mutant gene of the present invention can be introduced via somatic cell hybridization. Somatic cell hybridization can be accomplished via protoplast fusion (see, for example, Holmes, 2018).

[0133] The mutant gene of the present invention can also be integrated into the genome, for example, by introducing double-strand breaks at appropriate sites in the genome using one or more specific endonucleases (such as CRISPR-endonuclease / guide RNA complexes) and by integrating it into the genome using a donor construct containing the mutant gene of the present invention. Those skilled in the art know how to design such CRISPR-endonuclease / guide RNA complexes for introducing double-strand breaks and donor constructs suitable for integration (see Bortesi and Fischer, 2015 for a review).

[0134] Similarly, the selection and regeneration of transformed plants from transformed cells is well known in the art. Clearly, this method is particularly suitable for high-frequency regeneration of transformants, regardless of the species or even different varieties or cultivars of a single species. The invention also includes offspring of transformed plants exhibiting parthenogenesis and containing the mutant gene of the present invention.

[0135] In addition to nuclear genome transformation, plastid genome transformation, preferably chloroplast genome transformation, is also included in this invention. One advantage of plastid genome transformation is that it can reduce the risk of transgenic transmission. Plastid genome transformation can be performed as is known in the art, see, for example, Sidorov et al. (1999) or Lutz et al. (2004).

[0136] The resulting transformed plants can be used in conventional plant breeding programs to produce more transformed plants containing the described mutant gene. Methods such as Southern blot analysis or PCR-based methods can be used. Technical analysis (ThirdWave Technologies, Inc.) is used to select single-copy transformants. Transformed cells and plants can be easily distinguished from non-transformed cells and plants by the presence of the mutant gene of this invention, characterized by a promoter containing modifications as defined herein. Plant DNA sequences flanking the mutant gene insertion site can also be sequenced, thereby enabling the development of "event-specific" detection methods for routine use. See, for example, WO0141558, which describes, for example, excellent event detection kits (e.g., PCR detection kits) based on integrated sequences and flanking (genomic) sequences.

[0137] In one embodiment, the present invention comprises a mutant gene derived from the par allele, which originates from a plant that is inherently non-parthenogenic (and non-asexual), and modified by the methods of the present invention. This plant may be a wild or cultivated plant. The mutant gene is preferably obtained by modifying a promoter using the methods of the present invention, characterized in that it includes insertions or deletions in the promoter and / or one or more modified or removed MYB binding sites in the promoter as defined herein.

[0138] In one embodiment, the mutant gene of the present invention or the nucleic acid, vector or construct containing the mutant gene has a dominant function (genetically), preferably provided by (over)expression of a functional protein having the amino acid sequence SEQ ID NO: 1 or a variant or fragment thereof, such as an ortholog or fragment thereof found in another plant (i.e., other than the genus Taraxacum or Taraxacum in the broad sense).

[0139] Preferably, the mutant gene of the present invention, or the nucleic acid, vector, or construct containing the mutant gene, encodes a protein or a functional fragment thereof, which, when produced in a plant, is functional and induces and / or enhances parthenogenesis.

[0140] Preferably, the modified promoter, mutant gene, nucleic acid, vector and / or construct does not exist naturally, i.e., it does not exist in nature.

[0141] The gene provided in step (a) of the method of the present invention can be an endogenous gene present in the genome of a plant cell or protoplast. Preferably, the plant cell or protoplast is a portion of or isolated from a plant that does not have a parthenogenetic phenotype (hereinafter referred to as the originating plant). The plant cell or protoplast containing the mutant gene obtained by the method of the present invention can be a portion of or a regeneration of a plant that has a parthenogenetic phenotype or shows a significant increase in the parthenogenetic phenotype compared to the original plant. Therefore, the present invention also provides a method for converting a plant to a parthenogenetic phenotype or for increasing a plant to a parthenogenetic phenotype. In other words, the method of the present invention provides a method for producing parthenogenetic plants.

[0142] Therefore, the present invention provides a method for producing parthenogenetic plants, comprising the following steps:

[0143] (A) Regeneration and / or growth of plant tissues or plants from plant cells or protoplasts, said plant cells or protoplasts containing mutant genes obtainable by methods for generating mutant genes as defined herein; and

[0144] (B) Optionally, the plant tissues or plants obtained in step (A) are screened and / or genotyped.

[0145] Therefore, the present invention also provides a method for producing parthenogenetic plants, wherein the method includes the following steps:

[0146] a) Provide one or more plants, plant protoplasts, plant cells, plant tissues or plant seeds containing a gene encoding a PAR protein operatively linked to a promoter, wherein the promoter preferably contains one or more transcription factor MYB binding sites;

[0147] b) Modify the promoter by modifying at least one of the MYB binding sites of the one or more transcription factors and / or the sequence upstream of the MYB binding sites of the one or more transcription factors to increase the expression of the encoded PAR protein;

[0148] c) Optionally, one or more plants are grown from the modified plant protoplasts, plant cells, plant tissues, or seeds obtained in step b); and

[0149] d) Optionally, the plant protoplasts, plant cells, plant tissues or seeds obtained in step b) or the plants obtained in step b) or c) are screened and / or genotyped.

[0150] Alternatively or additionally, the present invention provides a method for producing parthenogenetic plants, wherein the method comprises the following steps:

[0151] a) Provide one or more plants, plant protoplasts, plant cells, plant tissues or plant seeds containing a gene encoding a PAR protein, which is operatively linked to a promoter that optionally contains one or more transcription factor MYB binding sites;

[0152] (b) Modifying the promoter to increase the expression of the encoded PAR protein, preferably by at least one of the following:

[0153] i) An insertion is introduced upstream of the start codon of a sequence encoding a PAR protein as defined herein, preferably upstream of or directly upstream of a promoter sequence at one or more transcription factor MYB binding sites, wherein the insertion preferably introduces an enhancer sequence or removes a repressor sequence.

[0154] ii) Introducing substitutions and / or deletions in the promoter sequence upstream of the start codon of the sequence encoding the PAR protein as defined herein, preferably upstream of or directly upstream of one or more transcription factor MYB binding sites, wherein the substitutions and / or deletions preferably introduce enhancer sequences or remove repressor sequences; and

[0155] Combinations of (iii), (i), and (ii).

[0156] c) Optionally, one or more plants are grown from the modified plant protoplasts, plant cells, plant tissues, or seeds obtained in step b); and

[0157] d) Optionally, the plant protoplasts, plant cells, plant tissues or seeds obtained in step b) or the plants obtained in step b) or c) are screened and / or genotyped.

[0158] Parthenogenetic plants produced by the method of the present invention can be plants with normal meiotic function, i.e., plants that do not exhibit incomplete meiosis, preferably not dispores and / or do not exhibit ploid spore formation. Preferably, the gametophyte of the plant can have a reduced ploidy compared to its somatic cells. In the case of diploid plants (i.e., plants with diploid somatic cells), the reduced ploidy can be a monohaploid. After inducing parthenogenesis in plants by the method of the present invention, the gametophyte of the plant can develop in a plant with reduced ploidy, preferably a haploid plant. Therefore, the method of the present invention can be a method for producing plants with reduced ploidy, preferably a method for producing haploid plants. The method preferably includes the step of providing parthenogenetic plants obtainable by the above method, and a subsequent step of allowing the plant to produce seeds without fertilization, allowing one or more of the seeds to germinate and regenerate into plants with reduced ploidy, preferably haploid plants. The method may include the step of inducing parthenogenesis as defined herein, subsequently allowing the plant to produce seeds without fertilization, and allowing one or more of the seeds to germinate and regenerate into plants with reduced ploidy, preferably haploid plants (plants with haploid somatic cells).

[0159] A genome with reduced ploidy, preferably a haploid genome, can spontaneously or inducedly double, preferably through chemical treatment. Preferred chemical treatments are described, for example, in Touchell DH et al, Front Plant Sci. 2020 Jun 3; 11:722, which is incorporated herein by reference. The chemical treatment can be a treatment with at least one of colchicine, oryzalin, trifluralin, and nitrous oxide. Chemical treatment of plants preferably produces plants with a double haploid genome.

[0160] Doubled monohaploid plants are plants that achieve homozygosity at all loci and can be obtained through whole-genome replication of the monohaploid genome, preferably using the methods described herein. Such perfectly homozygous plants have significant commercial value as parent plants in the production of F1 hybrid seeds. Therefore, the present invention also provides a method for producing doubled monohaploid plants, the method comprising the steps of producing parthenogenetic plants as defined herein, optionally including the steps of chemically induced genome replication, selecting doubled monohaploid seeds, and optionally allowing said seeds to germinate and regenerate into doubled haploid plants.

[0161] The method of this invention is not limited to the production of doubled haploid plants. The method described herein is also applicable to the production of other doubled haploid plants, such as, but not limited to, doubled diploids, doubled triploids, doubled tetraploids, doubled pentaploids, and doubled hexaploids.

[0162] As a non-limiting example, in polyploid crops (e.g., tetraploid potatoes), the method of the present invention can be used to produce bihaploid offspring. These bihaploids will have a much lower heterozygosity than their polyploid parents. Therefore, selection at the bihaploid level will be far more efficient than selection at the tetraploid level. The method of the present invention can be further used to produce unit haploids from these perfectly homozygous bihaploids. Bihaploids can facilitate the construction of genetic maps and enable the assembly of staged whole-genome sequences. Valuable traits of interest in wild diploid species can be introgressed using bihaploids derived from cultivars produced by the method of the present invention. The resulting bihaploids with valuable introgressed traits can, for example, be made into doubled bihaploids using the chemical treatments described above, thereby allowing the traits of interest to introgress into tetraploid cultivars. Those skilled in the art will readily understand that similar methods can be used to produce diploid, triploid, pentaploid, hexaploid, heptaploid, and other cultivars with introgressed wild-type traits of interest.

[0163] Preferably, the gene encoding the PAR protein in step a) is the par allele. Preferably, the promoter of the gene is modified in step b) as defined herein, thereby converting the par allele into the Par allele. Since the Par allele may be dominant, changing a single par allele into the Par allele of a plant or plant cell, or introducing the mutant gene into a plant or plant cell, for example, by transfecting the plant or plant cell with a vector containing the mutant gene of the present invention, may be sufficient to convert the plant from a sexual phenotype to a parthenogenetic phenotype, i.e., producing a plant and / or its offspring capable of growing and developing an embryo from an egg cell without fertilization. Therefore, preferably, a single sexual gene present in the plant cell, preferably an endogenous gene, is modified by the method of the present invention. Optionally, multiple genes present in the plant cell, preferably endogenous genes, are modified by the method of the present invention.

[0164] In cases where the modified promoter is present in plant cells or protoplasts, which may be part of a plant or regenerated into a plant, the plant is transformed from having a non-parthenogenesis phenotype to having a parthenogenesis phenotype. Therefore, the present invention also provides a method for conferring a parthenogenesis phenotype on a plant that does not exhibit a parthenogenesis phenotype by modifying the promoter of the par allele. In other words, the method of the present invention is a method for transforming a plant that does not exhibit parthenogenesis into a plant that exhibits parthenogenesis. Preferably, one or more plants, plant protoplasts, plant cells, plant tissues, or plant seeds in step a) lack the par allele and / or do not exhibit a parthenogenesis phenotype. Similarly, the method of the present invention is a method for transforming a plant that will exhibit limited parthenogenesis into a plant with increased parthenogenesis. An increase or induction of parthenogenesis preferably means that at least 1 in 200, at least 1 in 100, at least 10 in 100, at least 20 in 100, at least 30 in 100, at least 40 in 100, at least 50 in 100, at least 60 in 100, at least 70 in 100, at least 80 in 100, at least 90 in 100, or all plants transformed with the mutant gene of the present invention exhibit parthenogenesis, preferably compared to less than 1 in 500, 1 in 600, 1 in 700, 1 in 800, 1 in 900, or 1 in 1000 plants transformed with the original non-mutant gene (i.e., the gene provided in step a of the method of the present invention).

[0165] Preferably, the gene in step a) of the method of the present invention is a natural gene. Optionally, the gene modified by the method of the present invention is the par allele of dandelion or any orthologous parthenogenetic gene as defined herein. Preferably, the par allele is present in plants, plants, plant protoplasts, plant cells, plant tissues, or plant seeds that lack the par allele and therefore do not have a parthenogenetic phenotype. By conferring the par allele onto the par allele, the method of the present invention preferably results in the transformation of plants without a parthenogenetic phenotype into plants with a parthenogenetic phenotype.

[0166] Optionally, in step a), a variety of plants, plant protoplasts, plant cells, plant tissues, or plant seeds are provided, and after step b), one or more plants, plant protoplasts, plant cells, plant tissues, or plant seeds are selected that contain the modifications of the present invention, i.e., insertion or deletion and / or modification or removal of one or more MYB binding sites, preferably determined by genotyping and / or screening as defined herein. Therefore, the screening in step b) can be screening for parthenogenetic phenotypes.

[0167] In addition to genetically modifying the naturally occurring par allele in plant cells (which may be plant tissues, plant seeds, or a part of a whole plant) or plant protoplasts to confer the par allele to the Par allele, and growing and / or developing the cells or protoplasts into parthenogenetic plants, parthenogenetic plants can also be obtained by transforming plants or plant cells with nucleic acids, constructs, or vectors containing the mutant gene of the present invention, i.e., the mutant gene contains a promoter operatively linked to a modified coding sequence encoding a PAR protein as defined herein, optionally linked to a 3'UTR sequence.

[0168] The mutant gene obtained by the method of this invention can be introduced into one or more plant cells through transformation, introgression, somatic cell hybridization, and / or protoplast fusion. This mutant gene can be located on a foreign nucleic acid, i.e., a nucleic acid that does not exist in the plant cells in nature.

[0169] Therefore, the present invention also provides a method for producing parthenogenetic plants, wherein the method includes the following steps:

[0170] a. Provide one or more plants, plant protoplasts, plant cells, plant tissues, or plant seeds;

[0171] b. Transform one or more plants, plant protoplasts, plant cells, plant tissues, or plant seeds with a nucleic acid construct containing the mutant gene of the present invention;

[0172] c. Optionally, one or more plants are grown from the plant protoplasts, plant cells, plant tissues, or seeds transformed in step b); and

[0173] d. Optionally, the plant protoplasts, plant cells, plant tissues or seeds obtained in step b), or the plants obtained in step b) or c), are screened and / or genotyped.

[0174] Preferably, one or more plants in step a lack the Par allele and / or do not exhibit a parthenogenetic phenotype.

[0175] On the other hand, the present invention relates to plants (including, for example, plant cells, organs, seeds, and plant parts) obtained by any of the methods defined above. Preferably, these are parthenogenetic plants, or exhibit increased parthenogenesis compared to natural or unmodified plants. Preferably, the plants of the present invention are obtained by technical means, more preferably by the methods described herein. Such technical means are well known to those skilled in the art and include genetic modifications, such as at least one of random mutagenesis, targeted mutagenesis, and nucleic acid insertion or deletion.

[0176] Preferably, the plants of the present invention are not obtained by substantially biological methods. Preferably, the plants of the present invention are not obtained solely by substantially biological methods. Preferably, the plants of the present invention are not obtained by any substantially biological method of introducing parthenogenesis into the plant, and preferably are not obtained directly. Preferably, the plants of the present invention are not obtained solely by any substantially biological method of introducing parthenogenesis into the plant. Preferably, the plants of the present invention are not naturally occurring plants, i.e., plants that do not exist in nature.

[0177] In one embodiment, the promoter of the gene in the method of the present invention is an upstream transcriptional regulatory region, for example, within about 2000 bp upstream of the translation start codon and / or transcription start site of the gene, and can be isolated from agametogenic plants and / or other plants using known methods such as TAIL-PCR (Liu et al., 1995; Liu et al., 2005), adapter-PCR, or inverse PCR (IPCR). The chimeric gene as defined herein can be generated by ligating a promoter to a coding sequence taught herein, preferably having the amino acid sequence of SEQ ID NO: 1 or a functional variant and / or fragment thereof, which optionally is subsequently ligated upstream (i.e., 5') of a suitable 3' terminal untranslated region (“3' end” or 3'UTR). Suitable 3' ends include the 3' ends of the CaMV 35S gene (“3'35S”), the carmine synthase gene (“3'nos”) (Depicker et al., 1982), the octopus alkaloid synthase gene (“3'ocs”) (Gielen et al., 1984), and the T-DNA gene 7 (“3'gene 7”) (Velten and Schell, 1985), which serve as 3'-untranslated DNA sequences in transformed plant cells, etc. In one embodiment, the 3'UTR of a natural parthenogenetic gene, or a 3'UTR derived therefrom, is used. For example, any 3'UTR derived from SEQ ID NO: 4, or a variant or fragment thereof, can be used. The 3'UTR may have the nucleotide sequence of SEQ ID NO: 4.

[0178] Introducing T-DNA vectors into Agrobacterium can be done using known methods, such as electroporation or triparental hybridization.

[0179] The mutant gene taught in this paper can optionally be inserted into the plant genome as a hybrid gene sequence linked within a frame to a gene encoding an optional or quantifiable marker (US5,254,799; Vaeck et al., 1987), such as the neo (or nptII) gene (EP0242236) encoding kanamycin resistance, making plants containing this nucleic acid readily detectable.

[0180] Optionally, the mutant gene of the present invention can be modified to possibly (further) modify the transcription factor binding site, preferably modifying the binding site of a transcription factor that represses gene transcription.

[0181] In one embodiment, the PAR protein encoded by the nucleic acid of the present invention taught herein is co-expressed with other proteins that control, preferably enhance or induce parthenogenesis, incomplete meiosis or agametosis in a single host, optionally under the control of different promoters. Such other genes may be genes for conferring incomplete meiosis, such as ploidyspore formation, as described in WO2017 / 039452A1, which is incorporated herein by reference.

[0182] In another embodiment, the mutant gene of the present invention is infiltrated into germplasm that preferably contains other genes of interest, such as genes for conferring incomplete meiosis (e.g., genes for ploid spore formation). Hybrids are produced through hybridization and selection, wherein several genes of interest may be superimposed.

[0183] Optionally, the plant mutated or transformed by the method of the present invention is a plant capable of incomplete meiosis. Preferably, the plant capable of incomplete meiosis is modified to include a functional mutant gene in parthenogenesis as defined herein. Such mutation or modification will result in parthenogenetic plants or plant cells. In this case, the screening in step B) of the method for producing parthenogenetic plants as defined herein may be targeted at the parthenogenetic phenotype. Plant cells capable of incomplete meiosis can be obtained by introducing nucleic acids capable of conferring incomplete meiosis. Optionally, the nucleic acid is introduced into the plant cell before, together with, or after the introduction of the mutant gene of the present invention.

[0184] The present invention also provides a method for producing parthenogenetic hybrid seeds, comprising the following steps:

[0185] (1) To fertilize the pollen of the first sexually reproduced plant with that of the second plant to produce F1 hybrid seeds; and

[0186] (2) Optionally, select from the F1 seeds a seed containing a parthenogenetic phenotype;

[0187] The first and / or second plant is capable of incomplete meiosis, and the second plant is a parthenogenetic plant obtained or available by the method of the present invention, wherein the selection is preferably made by genotyping. Optionally, the method further includes the step of selecting seeds containing a parthenogenetic phenotype from the F1, preferably by genotyping, and optionally, growing at least one F1 plant from the F1 hybrid seeds.

[0188] Preferably, for selection purposes and for weed control, the transgenic plants of the present invention are also transformed with DNA encoding proteins that confer herbicide resistance, such as broad-spectrum herbicides, such as herbicides based on glufosinate as the active ingredient (e.g., ...). Or BASTA; resistance is conferred by the PAT or bar gene; see EP 0 242 236 and EP 0 242 246) or glyphosate (e.g. Resistance can be conferred by EPSPS genes (see, for example, EP0 508 909 and EP0 507 698). Using herbicide resistance genes (or other genes that confer the desired phenotype) as selective markers also has the advantage of avoiding the introduction of antibiotic resistance genes.

[0189] Alternatively or additionally, other selective marker genes, such as antibiotic resistance genes, can be used. Since retention of antibiotic resistance genes in transformed host plants is generally unacceptable, these genes can be removed again after selection of the transformants. Different techniques exist for removing transgenes. One method to achieve removal is to place a lox site on the flanking side of the transgene and, after selection, hybridize the transformed plant with a plant expressing CRE recombinase (see, for example, EP506763B1). Site-specific recombination results in the excision of the marker gene. Another site-specific recombination system is the FLP / FRT system described in EP686191 and US5527695. Site-specific recombination systems such as CRE / LOX and FLP / FRT can also be used for gene stacking purposes. Furthermore, single-component excision systems have been described (see, for example, WO9737012 or WO9500555).

[0190] Preferably, the mutant gene of the present invention is used to produce transgenic plant cells, plants, plant seeds, etc., and any derivatives / offspring thereof, which have an enhanced parthenogenetic phenotype. Preferably, the transgenic plants of the present invention contain enhanced parthenogenesis compared to unmodified control plants. Thus, transgenic lettuce plants containing, for example, enhanced parthenogenesis are provided. Therefore, plants containing said mutant gene show a significant increase in parthenogenesis compared to the same plants that do not contain the mutant gene of the present invention. The enhanced parthenogenetic phenotype can be fine-tuned by expressing appropriate amounts of proteins encoded by the mutant gene of the present invention capable of inducing parthenogenesis at appropriate times and / or locations. This fine-tuning can be accomplished by determining the most suitable promoter modification and / or by selecting transgenic "events" that display the desired expression level.

[0191] Transformers, hybrids, or inbred lines expressing the desired level of the protein encoded by the mutant gene of this invention are selected by, for example, analyzing copy number (Southern blot analysis), mRNA transcription levels (e.g., RT-PCR using primer pairs or flanking primers capable of amplifying the protein encoded by the mutant gene of this invention), or by analyzing the presence and levels of parthenogenetic proteins in various tissues (e.g., SDS-PAGE; ELISA assays, etc.). For example, for regulatory reasons, single-copy transformants may be selected, and the sequences flanking the insertion site of the mutant gene may be analyzed, preferably sequenced to characterize the "event." Transgenic events leading to high or moderate expression of the protein encoded by the mutant gene of this invention are selected for further development until a high-performance, superior event with stable transgenes is obtained.

[0192] Transformers containing the mutant genes of this invention may also contain (other) transgenes, such as genes conferring disease resistance or tolerance to other biotic and / or abiotic stresses, or genes conferring ploidyspore formation. To obtain such plants with "overlay" transgenes, other transgenes may be introduced into the transformants, or the transformants may be subsequently transformed with one or more other genes, or alternatively, several chimeric genes may be used to transform plant lines or varieties. For example, several transgenes may be present on a single vector or may be present on different vectors co-transformed.

[0193] In one embodiment, the following genes are combined with the mutant genes of the present invention: known disease resistance genes, particularly genes conferring enhanced resistance to necrotic pathogens, viral resistance genes, insect resistance genes, abiotic stress resistance genes (e.g., drought tolerance, salt tolerance, heat or cold tolerance, etc.), herbicide resistance genes, etc. Therefore, the superimposed transformants can possess even broader tolerance to biotic and / or abiotic stresses, including resistance to pathogens, insects, nematodes, salinity, cold stress, heat stress, water stress, etc.

[0194] The whole plant, plant parts (e.g., seeds, cells, tissues), and plant products (if fruits) and progeny of any plant described herein are included and can be identified by the presence of mutated genes, for example by using total genomic DNA as a template and PCR analysis using PCR primer pairs specific to the mutated genes of this invention and / or by using genomic variation analysis such as, but not limited to, sequence-based genotyping (SBG) or SNPSelect analysis. Event-specific PCR diagnostic methods can also be developed, where PCR primers are based on inserted modified or transgenic flanking plant DNA, see US6563026. Similarly, event-specific AFLP fingerprints or RFLP fingerprints can be developed to identify transgenic or mutant plants of the present invention, or any plant, seed, tissue, or cell derived therefrom.

[0195] It should be understood that the transgenic or mutant plants according to the present invention preferably do not exhibit undesirable phenotypes, such as reduced yield, increased susceptibility to diseases (especially necrosis factor), or undesirable structural changes (dwarfing, deformity), etc. Furthermore, if such phenotypes are observed in the primary transformant, they can be removed by conventional methods. Any transgenic or mutant plant described herein may be heterozygous, homozygous, or hemizygous for the mutant gene.

[0196] This invention also relates to plants, seeds, plant parts (e.g., plant cells), and plant products obtained or obtainable by the methods detailed herein, preferably comprising the mutant gene of this invention, the nucleic acid of this invention, and / or the construct of this invention. Preferably, the mutant gene, nucleic acid, and / or construct is capable of inducing parthenogenesis and / or is functional in parthenogenesis, as detailed herein. The plants of this invention are preferably species listed herein as suitable host plants. Methods for obtaining plants of this invention include, but are not limited to, random or targeted mutagenesis, introgression of the mutant gene of this invention into offspring from plants, and / or transformation of plant cells by the mutant gene of this invention, and subsequent regeneration of plants from said plant cells.

[0197] Preferably, the plant, plant parts, and / or plant products are not broadly defined dandelion species containing the mutant gene of the present invention. Preferably, the plant, plant parts, and / or plant products are dicotyledonous plants. The plant or plant cells are preferably species listed herein as suitable host plants, preferably selected from the Brassicaceae, Cucurbitaceae, Fabaceae, Poaceae, Solanaceae, and Asteraceae families.

[0198] Preferably, the plant, plant part, and / or plant product containing the mutant gene of the present invention is obtained through genetic modification or introgression, wherein preferably, the mutant gene is located in its genome. Preferably, the plant, plant part, and / or plant product is capable of parthenogenesis and / or exhibits parthenogenesis. Even more preferably, the plant, plant part, and / or plant product is also capable of incomplete meiosis. The present invention provides seeds, plant parts, or plant products of the plant or plant cells of the present invention.

[0199] This invention also relates to plant parts and plant products derived from the plants of this invention, wherein the plant parts and / or plant products contain, as defined herein, the mutant genes, nucleic acids, and / or constructs of this invention, which may be fragments as defined herein, allowing for the assessment of the presence of such proteins, mutant genes, nucleic acids, or constructs in the plant from which the plant parts of the plant products are derived. Such parts and / or products may be seeds or fruits and / or products derived therefrom (e.g., sugars or proteins). Such parts, products, and / or products derived therefrom may be non-reproductive material.

[0200] Any plant can be a suitable host, but the most preferred host plant species should be those that benefit from enhanced parthenogenesis. Suitable hosts include any plant species. In particular, cultivars or breeding lines with other favorable agronomic characteristics are preferred. Those skilled in the art know how to test whether the mutant genes and / or variants or fragments taught herein can confer the desired increase or decrease in parthenogenesis in a host plant by producing transgenic plants and evaluating parthenogenesis, along with suitable control plants.

[0201] Suitable host plants include, for example, those belonging to the Brassicaceae, Cucurbitaceae, Fabaceae, Gramineae, Solanaceae, Asteraceae, Rosaceae, or Poaceae families.

[0202] In a preferred embodiment, the host plant may be a plant species selected from the genera *Taraxacum*, *Lactuca*, *Vaccinium*, *Capsicum*, *Solanum*, *Cucumis*, *Zea*, *Cotton*, *Soybean*, *Tryticum*, *Oryza*, and *Sorghum*.

[0203] In a preferred embodiment, the plants, plant parts, plant cells, or seeds taught herein are selected from the genera *Taraxacum*, *Lactuca*, *Vitis*, *Capsicum*, *Solanum*, *Cucumis*, *Zea*, *Cotton*, *Soybean*, *Wheat*, *Oryza*, *Allium*, *Brassica*, *Sunflower*, *Helianthus*, *Citrus*, *Chicory*, *Chrysanthemum*, *Pennisetum*, *Rye*, *Barley*, *Alfalfa*, *Phaseolus*, *Rosa*, *Lilium*, *Coffee*, *Flaxum*, *Cannabis*, *Cassava*, *Carrot*, *Cucumis*, *Watermelon*, and *Sorghum*.

[0204] Suitable host plants include, for example, maize / corn (maize species), wheat (wheat species), barley (e.g., Hordeum vulgare), oats (e.g., Avena sativa), sorghum (Sorghum bicolor), rye (Secalecereale), soybean (soybean species, e.g., soybean max), cotton (cotton species, e.g., upland cotton (G. hirsutum), sea island cotton (G. barbadense)), brassica species (e.g., rapeseed (B. napus), broadleaf mustard (B. juncea), kale (B. oleracea), turnip, etc.), sunflower (Helianthus annus), safflower, yam, cassava, alfalfa (Medicago sativa)), and rice (Oryza species, e.g., O. sativa indica cultivar or japonica). Cultivar group), forage grasses, pearl millet (foxtail grass species, such as white-haired foxtail grass (P. glaucum)), tree species (pine, poplar, fir, plantain, etc.), tea, coffee, oil palm, coconut, vegetable varieties such as peas, zucchini, legumes (such as common bean varieties), peppers, cucumbers, artichokes, asparagus, eggplant, cauliflower, garlic, leeks, lettuce, onions, radishes, turnips, tomatoes, potatoes, Brussels sprouts, carrots, cauliflower, chicory, celery, spinach, endive, fennel Fragrant, beet, succulent fruit trees (grapes, peaches, plums, strawberries, mangoes, apples, prunes, cherries, apricots, bananas, blackberries, blueberries, citrus fruits, kiwifruit, figs, lemons, limes, nectarines, raspberries, watermelons, oranges, grapefruits, etc.), ornamental plants (such as roses, petunias, chrysanthemums, lilies, gerberas), herbaceous plants (mint, parsley, basil, thyme, etc.), woody trees (such as poplars, willows, oaks, eucalyptus), fiber species such as flax (cultivated flax (Linum usitatissimum)) and hemp (Cannabis sativa).

[0205] The mutant gene obtained or obtainable by the method of the present invention, or the nucleic acid of the present invention containing the mutant gene, can be used to confer parthenogenesis, to confer agametic reproduction for increasing ploidy, and / or to produce diploids. Preferably, the use is in plant biotechnology and / or breeding, i.e., in plants or plant cells.

[0206] Parthenogenesis is an element of agametosis. Genes for parthenogenesis can be combined with genes for incomplete meiosis (e.g., ploidysporogenesis) to produce agametosis, preferably for the applications listed herein. These genes can be introduced into sexual crops by transformation, introgression, or modification of suitable endogenous genes to convert them into genes for incomplete meiosis (or ploidysporogenesis). Knowledge of the structure and function of agametosis genes can also be used to modify endogenous sexual reproduction genes to make them agametosis genes. A preferred use is to place agametosis genes under an inducible promoter so that agametosis can be turned off when sexual reproduction produces new genotypes, and turned on when agametosis is needed to propagate a superior genotype.

[0207] The mutant gene of this invention can be used as a component of agametosis. Functional gametophyte agametosis requires incomplete meiosis and parthenogenesis. Incomplete meiosis can be achieved through a combination of mutations affecting meiosis (Crismani et al., 2013), resulting in no reduction of chromosomes in the megaspore, i.e., mitosis instead of meiosis. Somatic cells that present gametophyte fate through epigenetic alterations (Grimanelli, 2012) also produce unmeiotic spore-like cells, which may produce unmeiotic gametes (egg cells). In another embodiment, incomplete meiosis is achieved through transgenic or non-transgenic expression of a naturally occurring incomplete meiosis gene. Regardless of the manner in which unmeiotic egg cells are formed, appropriate temporal and spatial expression of the mutant gene of this invention can induce the egg cells to exhibit zygote characteristics and divide without fertilization.

[0208] The mutant genes of this invention can be used in entirely new ways, for example, not directly as a tool for agametosis. For example, in agametosis, parthenogenesis and incomplete meiosis are both combined in a single plant, while using incomplete meiosis in one generation and parthenogenesis in the next generation would link the sexual gene pool of the crop at the diploid and polyploid levels, increasing the ploidy level through incomplete meiosis and decreasing it through parthenogenesis. This is very useful because polyploid populations may be more suitable for mutation induction because they can tolerate more mutations. Polyploid plants may also be more vigorous. However, diploid populations are more suitable for selection, and diploid hybridization is more suitable for genetic mapping, BAC library construction, etc. Parthenogenesis in polyploids can produce haploids that can hybridize with diploids. Diploid ploid spore formation produces unmeiotic 2n egg cells, which can be fertilized by polyploid pollen to produce polyploid offspring. Therefore, the alternation of incomplete meiosis and parthenogenesis in different reproductive generations links the diploid and polyploid gene pools.

[0209] Another use of the mutant gene in this invention is to produce haploid offspring without utilizing incomplete meiosis. It can be used to produce haploids and to double the genome through doubling haploids (DHs) (e.g., spontaneous genome doubling, colchicine, sodium azide, or other chemicals). Doubling haploids can be used as parents to produce sexual F1 hybrids. Doubling haploids are the fastest way to make plants homozygous. With doubling haploids, plants can be made homozygous, whereas the second fastest method, self-pollination, requires 5-7 generations to reach a sufficiently high level of homozygosity in diploid plants. Several methods can produce doubling haploids. In some plant species, haploids can be produced through microspore culture. Other methods involve producing haploid embryos (gynogenesis) through pollination with irradiated pollen (melon) or with specific pollinating populations (corn, potato). These methods have limitations, such as cost, difficulty in genotypic adaptation, and high labor intensity. In some crops, there is no method for haploid production (e.g., tomato). By utilizing the dominant alleles of parthenogenesis genes, the frequency of gynogenesis can be significantly increased, and the cost of haploid production can be reduced.

[0210] The following non-limiting examples illustrate different embodiments of the invention. Unless otherwise stated in the examples, all recombinant DNA techniques were performed according to the standard protocols described in Sambrook et al. (1989) and Sambrook and Russell (2001); and Ausubel et al. (1994), Volumes 1 and 2. Standard materials and methods for plant molecular work are described in RDCroy's Plant Molecular Biology Labfax (1993), a joint publication of BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications.

[0211] Table 1. Overview of SEQ ID NO used in this article.

[0212]

[0213]

[0214] Attached Figure Description

[0215] Figure 1. Complementation and transformation experiments of dandelion CRISPR / Cas9 parthenogenetic deletion mutants and sexual lettuce. A) Different promoter-gene constructs used for complementation of dandelion LOP mutants and the number of lines successfully complemented. Showing the ToPar promoter with the dandelion Par gene and the sex homolog (Lspar) from lettuce (Lspar) and the Arabidopsis oocyte (EC1.1) promoter. B) Similarity of dandelion ToPar gene transformation into lettuce driven by the Arabidopsis oocyte EC1.1 promoter. C), D), E) Embryo-like structures in lettuce decapitated inflorescences transformed with the pEC1.1::Par construct. C) Embryo sac from control untransformed lettuce 75 hours after decapitation. Unfertilized oocyte (ec) and central cell (cc) nucleus are visible. D) Embryo sac with developing embryo-like structures 75 hours after embryo decapitation. E) Embryo sac with multiple embryo-like structures. An asterisk indicates a single embryo-like structure. F) Flow cytometry analysis of embryo sacs from control non-transformed lettuce 5 days after self-pollination. G) Flow cytometry analysis of embryo sacs from transgenic lettuce carrying the pEC1.1::Par construct 5 days after decapitation.

[0216] Figure 2 Polymorphism of the Par / par promoter in the genus *Taraxacum*. ClustalW alignment of the 350 bp upstream region of the ATG start codon (underlined) of the Par allele and three sexual alleles: par-1 and par-2 of the sexual alleles of *Taraxacum koksaghyz* (Tkpar). A 1335 bp MITE insertion has been removed from the ToPar promoter. The 6 bp direct repeat sequence, i.e., the MITE insertion site, is underlined. Of the 13 SNPs, 3 are located between the PAR promoter and the sexual promoter (bold and underlined); ten occur between sexual promoters.

[0217] Example

[0218] Example 1

[0219] Induction of parthenogenesis via Par promoter

[0220] To test whether the *Taraxacum* Par promoter plays a role in the genetic control of parthenogenesis, we tested whether it could combine with a Par-coding sequence homolog from a sexual species to induce parthenogenesis. The *Taraxacum* Par promoter drives the expression of a homologous gene (Lspar) from lettuce, a related species in the Asteraceae family and an important vegetable crop. This construct was transformed into a self-incompatible tetraploid *Taraxacum* CRISPR / Cas9 parthenogenesis deletion (LOP) mutant derived from a hybridization of a 3x PAR CRISPR mutant from the A68 line with pollen from the diploid plant FCH72, and failed to produce viable seeds. Since the Par allele is dominant, testing was performed on primary transformants (T0). Notably, the Par::Lspar construct resulted in seed formation and tetraploid (due to the presence of a dominant ploid sporulation gene) progeny in four independent transformants (Table 2). This indicates that the *Taraxacum* Par promoter can induce parthenogenesis in lettuce. No genetic polymorphism specific to the ToPar coding sequence was found in the lettuce gene (when compared with the sexual alleles par1 and par2), ruling out the possibility that coding sequence polymorphism is the cause of parthenogenesis. Moving upstream from the Par ATG initiation site, when compared with the three sexual alleles from dandelion (par1, par2, and par...), TKS , Figure 2 In contrast, the MITE insertion exhibits the first genetic polymorphism specific to the asexual reproduction allele. Only 13 SNPs were found among the four dandelion promoters 350 bp upstream of the ATG (when MITE was excised from the PAR allele), of which only 3 were Par-allele specific. Taken together, this provides strong evidence that the functional differences among the dandelion Par alleles are promoter-driven, rather than coding sequence-driven. As previously mentioned, different constructs of the Par gene expressed under the oocyte-specific Arabidopsis EC1 (pEC1::Par) promoter can also lead to complementation of CRISPR / Cas9 LOP mutants, consistent with the hypothesis that oocyte expression of PAR can induce parthenogenesis (see Example 2 and Table 3 of PCT / EP2020 / 064991).

[0221] This experiment demonstrates that the dandelion Par promoter driving sexual lettuce gene expression rescues the parthenogenetic phenotype in dandelion plants that have lost parthenogenesis. In other words, the dandelion Par promoter driving sexual lettuce gene expression can induce parthenogenesis.

[0222] Table 2. Gamete reproductive complementation between the 4x CRISPR / Cas9 PAR deletion mutant of dandelion and the ToPar promoter fused with the lettuce Lspar gene.

[0223] Primary transformants were grown in a greenhouse, seeds were collected, and germination tests were conducted on up to three seed spikes (SH; 30 seeds per spike). All tested progeny plants contained a PCR marker of the dominant DIP gene, indicating that recombination and meiosis did not occur during female meiosis. Ploidy levels in the progeny were determined by flow cytometry (FCM). Parthenogenesis produced tetraploid seedlings; hexaploid (6x) seedlings were derived from self-fertilization (tetraploid ploid spores forming oocytes fertilized by meiotic diploid pollen grains). Four of the eight lines produced parthenogenetic tetraploid progeny. Although *Taraxacum officinale* is self-incompatible, the SI system is known to be potentially leaky (Morita et al. 1990; and Tas and Van Dijk, 1999). Hexaploid progeny were observed intermittently in control plants transformed with the 35S::GUS construct, suggesting that self-fertilization can (rarely) occur in this genetic context. This could explain the single hexaploid offspring of the non-complementary line yellow 12b.

[0224]

[0225] Table 3. Gamete reproductive complementation of the ToPAR gene under the Arabidopsis EC1.1 promoter to the dandelion 4x CRISPR / Cas9 PAR deletion mutant. See Table 2 for explanation.

[0226] In plants transformed using complementary structures, one plant produced four 6x seedlings through self-pollination.

[0227]

[0228] Example 2

[0229] plant materials

[0230] For this experiment, wild-type lettuce was used: Iceberg, Legacy, Takii Japan, Red Romaine, and Baker Creek Heirloom Seeds.

[0231] DNA construct

[0232] A binary vector was constructed using the T-DNA region containing the construct shown in SEQ ID NO: 34. The construct consists of the following sequential elements: a lettuce LSAT_8X112340 promoter (SEQ ID NO: 16) driving the expression of the lettuce LSAT_8X112340 CDS sequence (SEQ ID NO: 33), followed by the first 1000 bases of the 3'UTR of the dandelion Par allele (SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20001.

[0233] A second binary vector was constructed using the T-DNA region containing the construct shown in SEQ ID NO: 35. This construct consists of the following sequential elements: a lettuce LSAT_8X112340 promoter modified to remove two MYB binding sites, containing the sequences AACCGCCA and AACCGTC (SEQ ID NO: 17) driving the expression of the lettuce LSAT_8X112340 CDS sequence (SEQ ID NO: 33), followed by the first 1000 bases of the 3'UTR of the dandelion Par allele (the first 1000 bases of SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20002.

[0234] A third binary vector was constructed using the T-DNA region containing the construct shown in SEQ ID NO: 36. This construct consists of the following sequential elements: a lettuce LSAT_8X112340 promoter containing the dandelion MITE promoter element (SEQ ID NO: 18) Par allele driving the expression of the lettuce LSAT_8X112340 CDS sequence (SEQ ID NO: 33), followed by the first 1000 bases of the 3'UTR of the dandelion Par allele (the first 1000 bases of SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20003.

[0235] A fourth binary vector was constructed using the T-DNA region of the construct shown in SEQ ID NO: 61, the construct consisting of the following sequential elements: a lettuce LSAT_8X112340 promoter with a deletion upstream of two MYB binding sites (SEQ ID NO: 20) driving the expression of the lettuce LSAT_8X112340 CDS sequence (SEQ ID NO: 33), followed by the first 1000 bases of the 3' UTR of the dandelion Par allele (the first 1000 bases of SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20003.

[0236] Plant transformation methods

[0237] Agrobacterium transformation is carried out using genotype-independent transformation of lettuce using Agrobacterium tumefaciens. This method is well known in the art and is taught, for example, in Curtis et al. (1994). Any other method suitable for genetic transformation of lettuce can be used to produce plants containing the desired T-DNA, as described in Michelmore et al. (1987) or Chupeau et al. (1989).

[0238] result

[0239] As described in the “DNA Constructs” section above, parthenogenesis was assessed in plants that tested positive for the presence of transgenes. Since the trait is dominant, testing was performed on primary transformed plants (T0). Parthenogenetic oocytes develop into embryos without hybridization or self-fertilization. To prevent fertilization of any transgenic plants, the plants were grown in a greenhouse, and all flowers were artificially emasculated before microscopic observation. Mastamina was performed by pruning the involucre before corolla growth. Parthenogenesis in non-agametic plants with ovules removed was detected under a microscope using a Nomarski differential interference microscope (DIC). In this study, the removal method using chloral hydrate was applied; a method commonly used to remove plant ovules for microscopic imaging (see, for example, Franks RG, 2016). Flower buds were harvested 75 hours after emasculation, and ovules were removed using chloral hydrate. In the transgenic lines pKG20002 and pKG20003, multiple embryos were observed in these removed ovules. Flow cytometry of the embryo sac pool revealed that these embryos were haploid. In non-transformed control plants and the evaluated pKG20001 transgenic line, emasculation and imaging in the same manner did not reveal any embryos.

[0240] These results indicate that both inserting the MITE promoter element into the Par allele of dandelion and removing the MYB binding site from the LSAT_8X112340 promoter of lettuce are sufficient to alter the expression of the LSAT_8X112340 gene, enabling it to induce haploid embryo formation in lettuce.

[0241] Example 3

[0242] plant materials

[0243] For this experiment, wild-type lettuce, Red Romaine type, Baker Creek Heirloom Seeds, was used.

[0244] DNA construct

[0245] A binary vector was constructed using the T-DNA region of the construct shown in SEQ ID NO: 63, the construct consisting of the following sequential elements: a lettuce LSAT_8X112340 promoter (SEQ ID NO: 16) driving the expression of the dandelion Par CDS sequence (SEQ ID NO: 3), followed by the first 1000 bases of the 3'UTR of the dandelion Par allele (the first 1000 bases of SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20004.

[0246] A second binary vector was constructed using the T-DNA region of the construct shown in SEQ ID NO: 64, the construct consisting of the following sequential elements: a lettuce LSAT_8X112340 promoter with a Par allele insertion containing the dandelion MITE promoter element (SEQ ID NO: 18) driving the expression of the dandelion Par CDS sequence (SEQ ID NO: 3), followed by the first 1000 bases of the 3' UTR of the dandelion Par allele (the first 1000 bases of SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20006.

[0247] A third vector was constructed using the T-DNA region containing the construct shown in SEQ ID NO: 65, the construct consisting of the following sequential elements: a promoter for the dandelion Par allele driving the expression of the LSAT_8X112340CDS sequence (SEQ ID NO: 33) of lettuce (SEQ ID NO: 2), followed by the first 1000 bases of the 3'UTR of the dandelion Par allele (SEQ ID NO: 4), followed by a 35S terminator and a neomycin phosphotransferase gene (nptII) for selection. A suitable technique for generating this binary vector is... Golden Gate or Gibson (See, for example, Ma et al., 2015). Transgenic lines containing this T-DNA are numbered with the code pKG20008.

[0248] Plant transformation methods

[0249] Agrobacterium transformation is performed by genotype-independent transformation of lettuce using Agrobacterium tumefaciens. This method is well known in the art and is taught, for example, by Curtis et al. (1994). Any other method applicable to genetic transformation of lettuce can be used to produce plants containing the desired T-DNA, as described by Michelmore et al. (1987) or Chupeau et al. (1989).

[0250] result

[0251] As described in the “DNA Constructs” section above, parthenogenesis was assessed in plants testing positive for the transgene. Since the trait is dominant, testing was performed on primary transformed plants (T0). Parthenogenetic oocytes develop into embryos without hybridization or self-fertilization. To prevent fertilization of any transgenic plants, the plants were grown in a greenhouse, and all flowers were artificially emasculated before microscopic observation. Mastamina was performed by pruning the involucre before corolla growth. Parthenogenesis in non-agametic plants with ovules removed was examined under a Nomarski differential interference microscope (DIC). In this study, the removal method using chloral hydrate was applied; a method commonly used to remove plant ovules for microscopic imaging (see, for example, Franks RG. 2016). Flower buds were harvested 75 hours after emasculation, and ovules were removed using chloral hydrate. In the transgenic lines pKG20005, pKG20006, pKG20007, and pKG20008, multiple embryos were observed in these demasked ovules (see Table 3). Flow cytometry of the embryo sac pools showed that these embryos were haploid. In control plants transformed with the standard GUS construct and in the evaluated pKG20004 transgenic line, no embryos were observed after emasculation and imaging in the same manner.

[0252] These results demonstrate that the MITE promoter element of the dandelion Par allele is sufficient to alter expression, allowing the LSAT_8X112340 gene to induce haploid embryo formation in lettuce. This is a clear example of inducing parthenogenesis in lettuce through promoter modification of the lettuce LSAT_8X112340 gene, since the oocyte develops into an embryo without hybridization or autofertilization.

[0253] Table 3: Embryo observation in flower buds of transgenic lettuce lines 75 hours after emasculation. The indicator "yes" means that there is at least one embryo in all buds.

[0254]

[0255]

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Claims

1. A mutant or chimeric gene capable of inducing a parthenogenic phenotype, comprising: - a sequence encoding a PAR protein as set forth in SEQ ID NO: 1 operably linked to a promoter having the sequence of any one of SEQ ID NOs: 17, 18 or 20, or - a sequence encoding a PAR protein as set forth in SEQ ID NO: 22 operably linked to a promoter having the sequence of any one of SEQ ID NOs: 2, 17, 18 or 20.

2. The mutant or chimeric gene of claim 1, wherein the gene has the sequence of any one of SEQ ID NOs: 35, 36, 61, 64 or 65.

3. A nucleic acid molecule, construct or vector comprising the mutant or chimeric gene of claim 1 or 2.

4. A method of producing a parthenogenic plant, comprising the steps of: (a) regenerating and / or growing plant tissue or a plant from a plant cell comprising the mutant or chimeric gene of claim 1 or 2 and / or the nucleic acid molecule, construct or vector of claim 3, wherein the plant cell is of a species of the Asteraceae; and (b) optionally, screening and / or genotyping the plant tissue or plant obtained in step (a).

5. A method of producing an apomeiotic plant, comprising the steps of claim 4, wherein the plant cell of step (a) is capable of apomeiosis.

6. A method of producing apomeiotic F1 hybrid seed, comprising the steps of: (I) crossing a sexually reproducing first plant with pollen of a second plant to fertilize to produce F1 hybrid seed, wherein the second plant comprises the mutant or chimeric gene of claim 1 or 2 and / or the nucleic acid molecule, construct or vector of claim 3, and wherein the first and / or second plant is capable of apomeiosis; and (II) optionally, selecting from the F1 hybrid seed seed comprising an apomeiotic phenotype, wherein the seed is of a species of the Asteraceae.

7. The method of claim 6, wherein the selection of step (II) is by genotyping.

8. Use of the mutant or chimeric gene of claim 1 or 2, or the nucleic acid molecule, construct or vector of claim 3, for inducing a parthenogenic phenotype in a protoplast, plant cell or plant, wherein the protoplast, plant cell or plant is of a species of the Asteraceae.

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