Meiotic restitution

Abstract

The present invention relates generally to the use of auxin-type phytohormones to induce diploid (2n) pollen formation in a plant. More particularly the invention relates to a method to modulate meiosis in male germ cells to ectopically induce meiotic restitution and associated formation of diploid pollen. This method provides a straightforward and human controlled way to create polyploid offspring (plant species with more than the usual two sets of chromosomes) with maximum integration of the genetic diversity of the parental line(s).

Description

[0001] M EXOTIC RESTITUTION

[0002] Field of the Invention

[0003] The present invention relates generally to the use of selected phytohormones or mutants in the homeostasis pathway to induce diploid pollen formation in a plant.

[0004] Background of the invention

[0005] Generating diploid pollen is useful primarily related to the process of plant reproduction, crop breeding and the manipulation of plant biodiversity. Control of meiotic cell division, and particularly induction of meiotic restitution, the process where meiosis fails to produce gametes with a reduced chromosome number but instead generates gametes with the somatic chromosome number (so called unreduced or 2n gametes), is of major interest for different crop breeding applications; including restoration of fertility in allohaploids, sexual polyploidization, interploidy hybridization, (near-)reverse breeding, and the production of doubled haploids (DH), as haploid plants undergoing meiotic restitution can often directly form DH seeds without the need to apply mitosis inhibitors to double the chromosome number. Thus, there is a need in the art for induced meiotic restitution.

[0006] Present invention provides a solution to this need by modulating meiosis in germ cells to ectopically induce formation of diploid pollen according to the method described herein.

[0007] Prior art methods to trigger of meiotic restitution, namely heat stress, cold stress and treatments with gibberellic acid (GA), all induce an SDR-type (second division restitution) meiotic restitution. This leads to unbalanced segregation of chromosomes and unbalanced dyads with aneuploid spores.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention solves the problem of SDR type meiotic restitution, as the treatment with auxins lead to FDR with an absence or restoration of imbalanced chromosome segregation in stage MI of the meiosis with a balanced segregation of chromatids for all chromosomes. This advantages are explained in detail in example 13.

[0010] The present invention relates to a method to modulate male germ cells to induce meiotic restitution and to form diploid pollen with the method comprising spraying or dipping or injecting - of at least the microsporangia - of a flowering plant with a treatment medium comprising an aromatic compound of formula D-E, whereby D is a C3-6 carbocyclic ring structure fused with E, E being a C3-6 carbocyclic or aromatic 3-6 membered heterocyclic ring structure with a hetero-atom selected from N, O and S, and whereby E is further substituted on a carbon member with an R group selected from the group consisting of carboxylate group, carboxymethyl group, propanoate group, acetamido group, propionamide group or a stereoisomer, tautomer or salt of such aromatic compound or a mixture thereof. This method provides a human controlled way to induce 2n pollen in plants that can be used to obtain whole genome doubling and to create de novo polyploid offspring (plant species with more than the usual two sets of chromosomes) through sexual polyploidization. This can occur unilateral (leading to 3x progeny) or bilaterial (leading to 4x progeny).

[0011] Several documents are cited throughout the text of this specification. Each of the documents herein (including manufacturer's specifications, instructions, etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the present invention.

[0012] In accordance with the purpose of the invention, as embodied and broadly described herein, one aspect of the invention is broadly drawn to a method for inducing diploid and polyploid spores in flowering plants, the method comprising treating, for instance by spraying or dipping, of -at least the microsporangia of- a flowering plant with a treatment medium comprising an aromatic compound of formula D-E, whereby D is a C3-6 carbocyclic ring structure fused with E, E being a C3-6 carbocyclic or aromatic 3-6 membered heterocyclic ring structure with a hetero-atom selected from N, O and S, and whereby E is further substituted on a carbon member with an R group selected from the groups consisting of a carboxylate group, carboxymethyl group, propanoate group, acetamido group, propionamide group, or a stereo-isomer, tautomer or salt of such aromatic compound or a mixture thereof.

[0013] In one aspect of the invention, the invention concerns a method for inducing di- and polyploid plant spores in flowering plants and therewith to generate polyploid plant progeny, the method comprising the steps of 1) spraying or dipping of -at least the microsporangia of- a flowering plant with or into a treatment medium comprising an aromatic compound of formula D-E, whereby D is a C3-6 carbocyclic ring structure fused with E, E being a C3-6 carbocyclic or aromatic 3-6 membered heterocyclic ring structure with a hetero-atom selected from N, O and S, and whereby E is further substituted on a carbon member with a R group selected from the group consisting of a carboxylate group, carboxymethyl group, propanoate group, acetamido group, propionamide group, or a stereoisomer, tautomer or salt of such aromatic compound or a mixture thereof, and the method further comprising the step 2) of pollination with ectopically generated diploid (2n) or polyploid (>2n) pollen via hybridization crossing to generate polyploid plant progeny.

[0014] In another aspect, the method of present invention provides that therein an aromatic compound is used which is represented by the following formula (I), its salt or solvates thereof, wherein the moietyA is a benzene ring or a pyrrole ring and R represents a carboxylate group, carboxymethyl group, propanoate group, acetamido group or propionamide group.

[0015] In yet another aspect, the method of present invention provides that therein an aromatic compound is used which is a carboxymethyl-fused bicyclic compound of the group consisting of 1-naphthalene acetic acid (NAA) and indole-3-acetic acid (IAA, 3- IAA).

[0016] In yet another aspect, the method of present invention provides that therein an aromatic compound is used which is an aromatic heterocyclic compound that has a benzene ring (D) fused to a pyrrole ring (E).

[0017] In yet another aspect, the method of present invention provides that therein an aromatic compound is used which is an aromatic carbobicyclic compound that has a benzene ring (D) fused to a benzene ring (E).

[0018] The methods described above may be embodied as that the concentration of the compound in the treatment medium is in the range of 200 pM to 2 mM, preferably 250 pM to 1 mM, and more preferably 280 - 320 pM.

[0019] The object of the present invention is also to provide the use of an aromatic compound of formula D-E, whereby D is a C3-6 carbocyclic ring structure fused with E, E being a C3-6 carbocyclic or aromatic 3-6 membered heterocyclic ring structure with a hetero-atom selected from N, O and S, and whereby E is further substituted on a carbon member with an R group selected from the group consisting of acetic acid, propionic acid, acetamide, propionamide or a stereoisomer, tautomer or salt of such aromatic compound or a mixture thereof to induce meiotic restitution in flowering plants.

[0020] The object of the present invention is also to provide the use of an aromatic compound which is represented by the following formula (I), its salt or solvates thereof: wherein the moietyA is a benzene ring or a pyrrole ring and R. represents acetic acid, propionic acid, acetamide, propionamide or a stereoisomer, tautomer or salt of such aromatic compound or a mixture thereof to induce meiotic restitution in the microsporocytes of a flowering plant.

[0021] Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

[0022] The invention is further summarised in the following statements:

[0023] 1) A method for inducing diploid or polyploid spores in flowering plants, the method comprising the steps of: a) applying on at least the microsporangia of a flowering plant an auxin, a stereoisomer, tautomer or salt thereof, or providing a plant with increased auxin levels, and b) identifying from the plant treated with an auxin, or from the plant with increased auxin levels, flowers or flower buds with diploid or polyploid spores.

[0024] 2) The method according statement 1, further comprising step c) of applying di- or polyploid pollen identified in step b) of statement 1 in self-pollination or crosshybridization.

[0025] 3) The method of statement 2, further comprising step d) of generating polyploid progeny, polyploid embryos, polyploid seeds or polyploid plants.

[0026] 4) The method according to any one of statements 1 to 3, comprising the step of applying on at least the microsporangia of a flowering plant an auxin or a stereoisomer, tautomer or salt thereof.

[0027] 5) The method according to any one of statements 1 to 4, wherein the application is performed by spraying, dipping or injecting. 6) The method according to any of statements 1 to 5, wherein the plant is a dicotyl.

[0028] 7) The method according to any of statements 1 to 6, wherein the plant belongs to the systematic class of the Solanaceae, Brassicaceae or Rosaceae.

[0029] 8) The method according to any of statements 1 to 7, wherein the auxin is applied in the first half or in the first quarter of the flowering period of the plant.

[0030] 9) The method according to any one of statement 1 to 8, wherein the auxin is a compound comprising an unsaturated ring and an acidic side chain.

[0031] 10)The method according to statement 10, wherein the unsaturated ring is aromatic ring or a heterocyclic ring.

[0032] 11) The method according to statement 10, wherein the aromatic ring is a benzene or a naphthalene ring.

[0033] 12) The method according to statement 9, wherein the aromatic ring is an indole.

[0034] 13) The method according to statement 9, wherein the acidic side chain is a carboxylic acid.

[0035] Examples of auxins are 2,4- Dichlorophenoxyacetic acid (2,4-D), 2,3,4- Trichlorophenol (Pestanal), 2,4,5-Trichlorophenol (2,4, 5-T), 4- chloroindole-3- acetic acid (4-CI-IAA), indole-3-acetic acid (IAA) or 3-(Carboxymethyl)indole, Benzoic acid, 3,6-dichloro-2-methoxy- (9CI, ACI) (Dicamba), Phenyl acetic acid (PAA), 2- Pyridinecarboxylic acid, 4-amino-3,5,6-trichloro- (9CI, ACI) (Picloram), 1- Naphthaleneacetic acid (NAA), lH-indole-3-butanoic acid (IBA), 5-Chloroindole-3- Acetic Acid (6-CI-IAA), 6-Chloroindole-3-Acetic Acid (6-CI-IAA), 3-Indolepropionic Acid (IPA), Indole-3-Lactic Acid (ILA), Indole-3-Acetyl Alanine (lAAIa), 2,4- Dichlorophenoxybutyric Acid (2,4-DB), 1-Naphthaleneacetamide (NAD or NAAm), 2- (2,4-Dichlorophenoxy)propionic Acid (Dichlorprop), and 2-(4-Chloro-2- methylphenoxy)propionic Acid (Mecoprop)

[0036] 14)The method according to any of statements 1 to 8, wherein the auxin is an aromatic compound with structure D-E, whereby D is a C3-6 carbocyclic ring structure fused with E,

[0037] E being a C3-6 carbocyclic or an aromatic 3-6 membered heterocyclic ring structure with a hetero-atom selected from N, O and S, and whereby E is further substituted on a carbon member with an R group selected from the group consisting of carboxylate, carboxymethyl, propanoate, acetamido, or propionamide group.

[0038] 15) The method according to statement 14, whereby the aromatic compound is represented by the formula (I), its salt or solvates thereof: , wherein the moiety A is a benzene ring or a pyrrole ring and R. represents a carboxylate group, carboxymethyl group, propanoate group, acetamido group or a propionamide group.

[0039] 16)The method according to statement 14 or 15, whereby the aromatic compound is 1-naphthalene acetic acid (NAA) or indole-3-acetic acid (IAA, 3-IAA.

[0040] 17)The method according to statement 15, whereby the aromatic compound is an aromatic heterocycle compound that has a benzene ring (D) fused to a pyrrole ring (E).

[0041] 18)The method according to 15, whereby the aromatic compound is an aromatic carbobicyclic compound that has a benzene ring (D) fused to a benzene ring (E).

[0042] 19)The method according to any of statements 1 to 18, whereby the concentration of the compound in a treatment medium is in a range of 200 pM to 2 mM, preferably of 250 pm to 1 mM, and more preferably 280 to 320 pM.

[0043] 20)The method according to any of statements 1 to 7, comprising the step of providing a plant with increased auxin levels. Apart from mutants these can also be trans- or cis-genic line with an overexpression construct of one or more biosynthesis genes.

[0044] 21)The method according to statement 20, wherein the plant with increased auxin levels is a plant with a mutation in one or more auxin biosynthesis genes and increasing expression of said one or more genes.

[0045] 22)The method according to statement 20, wherein the plant with increased auxin levels is a plant with a mutation in one or more auxin conjugation genes and decreasing expression of said one or more genes.

[0046] 23)The method according to statement 20, wherein the plant with increased auxin levels is a plant with a mutation in one or more auxin degradation genes and decreasing expression of said one or more genes.

[0047] 24)The method according to statement 21, wherein the gene belongs to the family of YUCCA genes or WCCA-like genes.

[0048] 25)The method according to statement 24, wherein the gene is YUCCA6.

[0049] 26)The method according to statement 22, wherein the gene belongs to the family of GRETCHEN HAGEN 3 (GH3) genes or GH3-\ike genes. 27)The method according to statement 26, wherein the gene is an ortholog of the GH3.5 or GH3.9 Arabidopsis genes.

[0050] 28)The method according to statement 20, wherein the plant exhibits enhanced or ectopic auxin signalling due to a mutation in one or more genes encoding for transcriptional repressors of the auxin response and decreasing expression of said gene(s).

[0051] 29)The method according to statement 28, wherein the gene belongs to the family of INDOLEACETIC ACID-INDUCED PROTEINS (IAA) genes or lAA-like genes.

[0052] 30)The method according to statement 29, wherein the gene is IAA8, IAA12 or IAA18.

[0053] 31)The use of an auxin to induce meiotic restitution in male sporogenesis or to induce meiotic restitution in female sporogenesis of a flowering plant.

[0054] 32)The use of a plant with increased auxin levels to induce meiotic restitution in male sporogenesis or to induce meiotic restitution in female sporogenesis of a flowering plant.

[0055] DETAILED DESCRIPTION

[0056] Drawing Description

[0057] BRIEF DESCRIPTION OF THE DRAWINGS

[0058] The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:

[0059] FIG. 1 is a photo that provides an example of a bright field image of Arabidopsis Col- 3 I3bc (+ / -) (CYR / + + + ) qrt (- / -) pollen 8 days after NAA treatment (300 pM). Unaffected pollen are visible as configurated balanced tetrads. The ectopic presence of monads, (un)balanced dyads and (un)balanced triads, as shown by red arrows, indicate defects in male meiotic cell division that result in meiotic restitution and associated formation of diploid spores. Scale bar; 50 pM.

[0060] FIG. 2 is a graphic that shows the quantification of diploid (2n) pollen grain formation in the Arabidopsis Col-3 I3bc (+ / -) qrt (- / -) FTL line 6, 7 and 8 days after exogenous treatment with (in panel A) NAA and (in panel B) IAA (N = 3).

[0061] FIG. 3 is a graphic that provides the percentages of different tetrad-stage meiotic products in the Arabidopsis Col-3 Ilbc (+ / -) qrt (- / -) FTL line 24h after NAA (panels Al & A2) and (in panels Bl & B2) IAA treatments at different concentrations (left graph) with corresponding dose-response curves for observed restituted products given in the right graph (N = 3).

[0062] FIG. 4 is a graphic that provides tetrad analysis data of Arabidopsis Col-3 I3bc (+ / -) qrt (- / -) plants upon NAA (1 mM), IAA (1 mM) and combined treatment (NAA + IAA, 1 mM) (N=3).

[0063] FIG. 5 is a graphic that provides tetrad analysis data of Arabidopsis Col-3 I3bc (+ / -) qrt (- / -) plants (N=3) upon treatment of rosette or inflorescence tissue with 1 mM NAA (N=3).

[0064] FIG.67 is a graphic that demonstrates the comparison between the spraying method and the dipping method for obtaining auxin-induced male meiotic restitution (NAA and IAA, 2 mM) in Arabidopsis Col-3 Ilbc (+ / -) qrt (- / -) plants (N=3).

[0065] FIG. 7 is a graphic (panel A) and a photo (panel B) showing the quantification of male meiotic restitution induced by a 1 mM NAA spray treatment, as compared to mock treatments, in 8 different Arabidopsis ecotypes. Representative pictures of balanced dyads and triads are given below.

[0066] FIG. 8 is a graphic (panel A) and a photo (panel B) that shows the quantification of balanced dyads, triads, and monads in male sporogenesis of yucca6-lD and yucca6- 2D gain-of-function plants (N=3) under room temperature conditions, as compared to Col-3 plants treated with 1 mM NAA (N=3). A representative image of triads and actively dividing meiocytes (indicated in circle) in yucca6-lD and yucca6-2D plants is given below.

[0067] FIG. 9 is a photo that provides visualization of GUS expression in different stages of flower bud development in mock and 1 mM NAA-treated Arabidopsis DR5:GUS plants as a proxy for assaying tissue-specific localization and accumulation of auxin. While mock-treated early stage meiotic flower buds do not show any auxin-triggered GUS signal, NAA-treated buds at similar stages appear to have a high abundance of auxin in all tissue types.

[0068] FIG. 10 is a graphic and showing the quantification of male meiotic restitution induced by a 1 mM NAA spray treatment compared to mock treatments and non-treatment analyses, at time points of 1 day and 2 days after treatments, on Col-3 plants (N=3). FIG. 11 is a graphic that demonstrates the dosage response analysis of the effect of different NAA concentrations on the induction of male meiotic restitution in the Arabidopsis ecotypes Col-0 (panel A), Utrecht (panel B) and Sq-8 (panel C). Conditions with one or two replicates in which less than 50 tetrads were analysed are indicated with one or two asterisks, respectively. N = 3 biologically independent replicates, unless stated otherwise. No statistical differences were detected between different concentrations of NAA treatments.

[0069] FIG.12 is a graphic that shows the developmental stage analysis of the induction of meiotic restitution in the yucca6-2D mutant (panel B) and NAA-treated wild type plants of the Arabidopsis Col-0 (panel A) ecotype background. Tetrad analyses were performed in the first, second and third week of flowering (indicated by week 1, week 2 and week 3, respectively), both for mock and NAA-treated plants (except for yucca6-2D mutant). N = 3 biologically independent replicates, unless stated otherwise. Significant differences are depicted with an asterisk; p < 0.05.

[0070] FIG. 13 is a graphic that provides meiotic restitution frequencies observed after treatments with auxin analogues in Arabidopsis, given together with the molecular structures of each compound. A comparison of restitution frequencies after 2 mM treatments of each analogue is given (panel A). Structures of IAA and NAA are shown to enable comparison with other analogues (panel B). Comparison of restitution frequencies through dosage analyses are provided for each analogue: picloram (panel

[0071] C), 2,4, 5-T (panel D), PAA (panel E), 4-CI-IAA (panel F), 2,4-D (panel G) and dicamba (panel H). N = 3 biologically independent replicates. Significant differences are depicted with letters in panel A and with asterisk (*) in panel C, p < 0.05.

[0072] FIG. 14 is a graphic that shows gene structure of the T-DNA insertion mutants that were investigated for meiotic restitution (panel A). White boxes represent 5' or 3' UTRs, black boxes represent exons and horizontal lines upstream of a 5' UTR represent the upstream parts of the promoter. Triangles indicate the T-DNA insertion sites. Observed meiotic restitution frequencies in auxin mutants are given in panel B. N = 3 biologically independent replicates, unless stated otherwise. No significant differences were found.

[0073] FIG. 15 is a photo (panels A, B, C, D, E, F, G, I, J, K, L, M, N, O, P, Q) and a graphic (panels H and R) that demonstrates the frequency of meiotic end products observed in a haploid (n) yucca6-2D plant. Representative pictures of balanced dyads (panel A), unbalanced dyads (panel B), unbalanced triads (panel C), balanced triads (panel

[0074] D), balanced tetrads (panel E), unbalanced tetrads (panel F) and polyads (panel G) are given. Meiotic end products observed in haploid Col-0 and yucca6-2D (H), N = l. FDA-based viability staining of diploid Col-0 (panels I-K), haploid Col-0 (panels L-N) and haploid yucca6-2D (panels O-Q), as well as overlay (panels I, L and O), bright- field (panels J, M and P) and fluorescence images (panels K, N and Q) are shown. Comparative quantification of FDA-stained pollen observed in diploid Col-0, haploid Col-0 and haploid yucca6-2D are given (panel R), N=4. Scale bars represent 10 pm for A-G and 100 pm for I-Q.

[0075] FIG. 16 is a graphic that provides meiotic restitution frequencies in mock- and NAA- treated plants for Solarium iycopersicum 'MicroTom' (panels A and B) and Brassica rapa (panel C). Plants have been treated by dipping buds in the mock and auxin solutions (panels A and C), or by injecting the solutions into the buds (panel B). N = 3 biologically independent replicates, unless stated otherwise.

[0076] The following detailed description of the invention refers to the accompanying drawings. The same reference numbers in different drawings identify the same or similar elements. Also, the following detailed description does not limit the invention. Instead, the scope of the invention is defined by the appended claims and equivalents thereof.

[0077] The term "auxin" is herein employed in the sense first suggested by Kogi (1932) Forsch und Fortschr. 8, 409-410, and refers to those chemical substances which bring about such growth reaction in higher plants as is conveniently measurable by the curvature of Avena coleoptiles. In the physiological sense the term "auxin" includes some "growth hormones" but is not inclusive of plant stimulant materials which serve solely in a nutrient capacity or of materials such as ammonia or ethylene.

[0078] Auxin A, auxin B, heteroauxin (also known as indole-3-acetic acid (IAA)), and mixtures thereof, as well as numerous auxin-like materials, such as naphthalene acetic acid, indol-3-butyric acid, naphthalene propionic acid, phenoxy acetic acid, their salts and esters, naphthalene acetamide, etc., are representative of this class of materials. All have been found to initiate and promote rooting action, gravitropism and light-regulated growth responses (e.g., shading avoidance) in growing plants. The auxin family comprises the synthetically generated naphthalene acetic acid (NAA) and the natural indole-3-acetic acid (IAA, 3-IAA) and indole-3-butyric acid (IBA) or synthesised structurally similar compounds, the most common naturally occurring plant hormones of the auxin class, together with several other naturally occurring auxins that have been described to date: IBA, Phenylacetic acid (PAA) and 4-Chloroindole-3-acetic acid (4-CI-IAA). Naturally occurring auxins are found in plants as free acid or in conjugated forms or they can be synthesised structurally similar compounds.

[0079] The term "Meiosis" means a type of cell division that results in four daughter cells, each with half the number of chromosomes of the parent cell. It is a type of cell division that occurs in sexually reproducing organisms, leading to the formation of spores that contain or bring along the gametes (sperm and egg cell). This process is essential in the production of plant spores and the development of enclosed gametes. The primary purpose of meiosis is to reduce the chromosome number by half, ensuring that when two gametes fuse during fertilization, the resulting zygote has the correct somatic (2n) number of chromosomes. Meiosis involves one DNA replication phase (S-phase) followed by two consecutive divisions, called meiosis I and meiosis II, each consisting of prophase, metaphase, anaphase, and telophase stages. The end result of meiosis is the production of four genetically non-identical haploid daughter cells (spores), each with half the number of chromosomes as the original diploid progenitor cell. Organisms that reproduce sexually undergo a special type of cell division called meiosis. Meiosis occurs only in the reproductive organs (i.e., flowers in angiosperms, More specifically in the stamen (male reproductive tissue) and in the pistil (female reproductive structure) during the process of sporogenesis and produces daughter cells with halved ploidy level (the number of homologous chromosome sets within the nucleus). The meiotic process involves multiple consecutive cytological steps, including DSB induction, homolog pairing, synapsis and cross-over formation, that are tightly controlled at the molecular and cellular level to ensure the formation of haploid gametes.

[0080] The term "meiotic restitution" means a process in which meiosis fails to produce gametes with a reduced chromosome number, for instance as a result of an error in one of the two subsequent nuclear divisions that occur during meiosis, and the consequent occurrence or formation of diploid or unreduced gametes ("2n gametes"). The reduction in chromosome number, which is a characteristic of meiosis, is abolished in the process of meiotic restitution. Instead of the chromosome number being halved, resulting in the formation of haploid cells (gametes), the cells retain the diploid number of chromosomes. This failure in chromosome reduction during meiosis leads to the production of daughter cells and spores with an identical chromosome number as the original parent cell. In the kingdom of plants, diploid spores that are the product of spontaneous meiotic restitution have been observed, albeit at low frequency (<0,l%). In the context of the present invention Induced polyploidy after auxin treatments can be detected through several cytological analysis. Diploid pollen grains are generally larger in size (approximately 1.2 times) compared to normal haploid (n) pollen. By collecting pollen from treated and control (untreated) plants and measuring their diameters under a light microscope (through either an automated quantifier or manual counting), the presence and ratio of enlarged pollen can be assessed. Additionally, dyes such as DAPI (4,6-diamidino-2-phenylindole) can be used to stain the nuclei of pollen grains, which enables their visualization. Diploid pollen exhibit larger nuclei due to their higher DNA content. These stains can also reveal abnormalities in pollen development. Lastly, for more accurate and high-throughput confirmation, flow cytometry can be used to measures the DNA content of individual pollen grains, allowing for the clear distinction between haploid and diploid pollen based on fluorescence intensity. Thus flow cytometry can provide a more precise method for ploidy assessment of the pollen, but it requires specialized equipment and some level of optimization of protocols.

[0081] The term "Microsporangia" means the sporangia or sporocyte cells that generate microspores that in both flowering plants (angiosperms) and cone-bearing plants (gymnosperms) give rise to male gametophytes, i.e., the mature pollen grains, that contain the male gametic or sperm cells. Microsporangia occur in all vascular plants that have a heterosporous life cycle and are situated in the reproductive organs. Phytohormones are chemical compounds (natural or chemically synthesised) that occur in small amounts or concentrations in the plant and regulate and govern a variety of physiological processes in plants, including cell division, elongation, differentiation, and responses to environmental stimuli, even at extremely low concentrations. Different phytohormones have distinct functions, and they work in concert to coordinate the plant's growth and development. Examples of known categories of phytohormones are for instance of the group consisting of cytokinins, auxins, gibberellins, abscisic acid (ABA), ethylene, strigolactones, brassinosteroids, jasmonates, salicylic acid (SA) and oligosaccharides. Auxins and cytokinins are generally crucial regulators for the initiation and maintenance of cell division as a whole and can act both synergistically and antagonistically.

[0082] Arabidopsis thaliana is a model plant species, with a diploid genome comprising a total of ten chromosomes (2n=2x=10). Under normal conditions, a haploid gamete of A. thaliana has five chromosomes, as opposed to the ten chromosomes seen in a diploid somatic cell. However, in case of meiotic restitution, diploid gametes are formed that have the same ploidy as somatic cells. The fusing of 2n gametes after fertilization, either with a haploid gamete or with another 2n gamete, results in polyploid progeny, respectively a triploid (3x) or a tetrapioid (4x). This sexual polyploid ization process can take place spontaneously in nature, and is thought to be the main pathway for natural polyploidization in the plant kingdom (De Storme et al. (2012) Plant Physiol. 160, 1808-1826). Polyploids are divided into two categories: allopolyploids and autopolyploids, respectively. Autopolyploid organisms are the consequence of a genome doubling event occurring within a single species, while allopolyploid organisms result from the hybridization and genome merger of two different biological species (De Storme & Geelen (2013) New Phytol. 198(3), 670- 684).

[0083] EXAMPLES

[0084] Example 1

[0085] Example la Screen

[0086] For the present invention different classes of (bio-)molecules have been screened for their effect on meiotic processes. Molecules in solid form were dissolved and applied onto flowering Arabidopsis plants by spraying. The screen on affected meiotic processes was carried out using the Arabidopsis fluorescent tagged lines (FTL) system. FTLs contain linked reporter transgenes that are introduced in the genome by random insertion mediated b Agrobacterium tumefaciens transfer-DNA (T-DNA). These reporter transgenes are under the control of the post-meiotic pollen-specific LAT52 promotor and either express dsRed (R), eCFP (C) or eYFP (Y) fluorescent proteins in mature pollen grains.

[0087] The investigation of male meiotic processes using the engineered FTL system in Arabidopsis can be done by the linked fluorescent tags in the hemizygous state, but also by the maintained physical connection of the male meiotically related products in a so-called tetrad configuration. This is mediated by a loss-of-function in the QUARTET gene. In Arabidopsis, QUARTET QRT) genes (e.g. QRTl') encode for pectin methylesterase (PME) enzymes that are essential for the proper physical separation of the four microspores that are formed by a single meiotic event during floral development. Generally, the developing pollen mother cell (PMC) is surrounded by a primary and a secondary cell wall composed of (hemi)cellulose and pectin, and callose, respectively. As a meiotic event results in the division of the diploid PMC into four haploid microspores, four individual microspores are released in the locules of the anther that, -upon gametogenesis development-, are released as mature, single pollen grains. This physical separation of the four microspores at the end of male meiosis requires an active degradation of the meiotic cell wall, and this is mediated by the activity of multiple enzymes, including PME. As a consequence, loss-of- function mutations in QRT1 (or QRT2 or QRT3) block the process of PMC cell wall degradation, and thereby disrupt proper separation of the microspores originating from a single meiotic event, leading to the development and eventual release of male meiotically related pollen as tetrahedrally configured tetrads [Francis et al. (2006) Plant Physiol. 142, 1004-1013],

[0088] Example lb Effects of externally applied auxins on stability of male meiotic cell division, and meiotic restitution in Arabidopsis thaliana.

[0089] The first three types of auxin molecules (indole and naphthalene class molecules) that were tested included Indole-3-butyric acid (lH-indole-3-butanoic acid, IBA) and Indole-3-acetic acid (IAA, 3-IAA), which are naturally occurring auxins, and 1-Naphthalene acetic acid (NAA) which is a synthetically synthesized auxin. These all concern heterocyclic aromatic compounds or heteroaromatics with the carboxylic acid group as differentiator, either acetic acid (with its two carbon atoms) or butyric acid (four carbons). When NAA and IAA were externally applied to flowering Arabidopsis plants at concentrations greater than 200 or 300 pM, they caused formation of diploid pollen through ectopic induction of meiotic restitution in Arabidopsis male sporogenesis. Treatment of diploid qrtl(- / -) Arabidopsis plants in the Col-0 background with NAA or IAA resulted in a mixture of haploid and diploid pollen, occurring either as single pollen grains or in their meiotic 'tetrad' configuration (Figure 1 & Figure 2).

[0090] IBA (indole-3-butanoic acid), which is a naturally occurring auxin, can also act as a precursor that is converted into the commonly found auxin IAA through the process of beta-oxidation. When Arabidopsis plants were treated with different concentrations of IBA (300 pM and 1 mM), very low percentages of diploid pollen (<0.01%) were occasionally observed. These findings indicate that IBA also induces meiotic restitution in A. thaliana, albeit at lower frequencies compared to NAA and IAA.

[0091] This lower efficacy of IBA (indole-3-butanoic acid) in inducing male meiotic restitution in Arabidopsis could be due to structural differences, different transport mechanisms, or other factors within the plant that cause IBA not reach the floral buds as efficiently as IAA (indole-3-acetic acid) and NAA (1-naphthaleneacetic acid) after spraying. This is supported by the absence of other auxin-related phenotypic effects, such as single qrtl(- / -) pollen grains and delayed floral opening, in exogenous IBA treatments (Figure 1).

[0092] Example 2 Auxin dosage curve analysis in Arabidopsis for determining the optimal auxin concentration for maximizing induction of male meiotic restitution

[0093] In order to understand the relationship between the dosage of externally applied auxin and the induction of male meiotic restitution in Arabidopsis, as well as to determine the optimal concentration of NAA and IAA that results in the highest percentage of meiotic restitution (for optimization of polyploidization induction in the progeny), dosage curves were created for NAA and IAA. Arabidopsis Col-3 qrtl^- / -) plants were spray-treated with increasing concentrations of NAA or IAA ranging from 0 mM to 2 mM. One day after auxin treatment, the male meiotic end products were visually examined using bright-field microscopy and the relative amount of restituted meiocytes (i.e., dyads and triads) were quantified. These data were used to generate detailed dose-response curves both for NAA and IAA (Figure 3).

[0094] From these curves, it can be inferred that with spray delivery both auxins induce restitution from about 300 pM onwards) to, and as the auxin concentration gradually increases, there is a gradual increase in the mean percentage of restitution. The induced restitution products primarily concern a combination of triads and balanced dyads, with a lesser occurrence of unbalanced dyads. At similar concentrations (<1 mM), NAA resulted in slightly higher percentages of diploid spores compared to IAA. However, this distinction becomes less significant at extreme high concentrations ( / .e., 2 mM), where both NAA and IAA lead to similar percentages of meiotic restitution. This could be due to saturation of the treated plant tissue with exogenous auxins, hindering further diffusion or physiological interference.

[0095] Example 3 Investigating putative effects between NAA and IAA on inducing meiotic restitution

[0096] To investigate whether the restitution-inducing auxins NAA and IAA have an additive, multiplicative or other interfering effect, combined treatments with NAA and IAA were conducted alongside individual treatments. Arabidopsis Col-3 plants were spray- treated with a mock solution, 1 mM NAA, 1 mM IAA, or a combination of 1 mM NAA and 1 mM IAA. One day after the treatment, the male meiotic products were analysed using tetrad analysis (Figure 4). In all auxin treatments, restitution of male meiosis was observed with a primarily production of triads, followed by low percentages of balanced and unbalanced dyads. The fraction of restituted meiotic products after treatment with 1 mM NAA is significantly higher compared to the fraction of restituted products after treatment with 1 mM IAA. However, there is no significant difference between the percentage of restituted meiocytes between plants treated with 1 mM NAA alone and plants treated with a combination of 1 mM NAA and 1 mM IAA. These findings indicate that combined treatments with NAA and IAA do not have an additive effective on the induction of meiotic restitution, suggesting they function through the same molecular pathway within the plant cell.

[0097] Example 4 Characterization of direct or indirect effects of auxin treatments on meiotic restitution

[0098] In the previous examples, auxin solutions have been applied to Arabidopsis plants through spraying. During spraying, most of the mock or hormone solution is directly targeted at the inflorescences. However, small amounts of the hormone solution also reach the rosette leaves, roots, and other parts of the plant. This implies that the observed meiotic restitution following auxin spraying could be a direct result of auxin penetrating the local flower bud tissue where it then directly interferes with the integrity of the meiotic cell division program. Alternatively, auxins applied to other parts of the plant can act in a more systemic way or indirectly interfere with meiotic cell division, for example by altering the overall photosynthetic capacity and energy availability in the plant or by triggering specific signalling cascades that can eventually lead to meiotic restitution. To investigate whether NAA and IAA directly or indirectly cause male meiotic restitution, separate auxin spray treatments were conducted on Arabidopsis inflorescences (along with the upper part of the stems) and rosette leaves using NAA (1 mM), after which tetrad analysis was conducted (Figure 5).

[0099] The results revealed that treating only the rosette leaves did not lead to aberrantly configured tetrads, indicating absence of meiotic restitution. However, treating only the inflorescences did lead to restituted meiotic products with similar percentages and ratios of dyads and triads as previous NAA treatments involving spraying of whole plant. These findings suggest that the meiotic restitution induced by external spray application of NAA or IAA (>300 pM) to the plants is likely due to a direct effect of auxin penetrating the floral bud tissue, though alternatively can still be attributed to an indirect effect of auxin penetrating nearby tissues adjacent to the inflorescences.

[0100] Example 5 Comparative analysis of administration methods on auxin- induced meiotic restitution in Arabidopsis plants; stem cutting, spraying, and dipping methods

[0101] As the method of auxin administration to the inflorescence may influence the efficacy of induction of male meiotic restitution, three different NAA application methods were tested and compared. In the stem cutting method, the stems of flowering Arabidopsis Col-3 plants were cut at the base around 20-25 cm starting from the inflorescence. The cutting was performed under water to prevent the formation of air bubbles which may block the stem vasculature. The cut stems were then placed in individual falcon tubes filled with either mock solution or NAA solution at different concentrations (500 pM, 1 mM, and 2 mM). In parallel, negative control groups placed in mock solution were included in this experiment to account for potential stress responses resulting from the stem wounding that could putatively activate meiotic restitution. In all the treatments, stems started to wilt at 24 hours after treatment initiation, with the most pronounced effect observed at the highest NAA concentration, after which the wilting cumulatively proceeded in the following days. No restituted meiocytes were observed one day after treatment with 500 pM, 1 mM, and 2 mM NAA, nor was this observed in the mock treatment. These findings suggest that the auxin solution does not adequately reach the inflorescences within the time between administration and analysis, or alternatively that this specific way of NAA administration does not have the capacity to induce meiotic restitution at all. To address this issue and differentiate between these two explanations, the experiment was repeated with shorter stems, ~4 cm in length starting from the top of the inflorescence.

[0102] In addition to spraying and the stem cutting method, we have also tested the efficacy of an auxin dipping method for inducing male meiotic restitution. In this method, the inflorescences of intact Arabidopsis Col-3 plants were directly dipped in a 2 mM NAA or IAA solution for 30-60 seconds, or parallelly in the corresponding mock solution, and then returned to standard growth conditions. One day later, male meiotic products at the tetrad stage were analysed under a microscope. The results showed that dipping the inflorescences in NAA or IAA effectively induces meiotic restitution and in general leads to a higher level of male restituted produces as compared to the conventional spraying method (Figure 6). More specifically, dipping with NAA (2 mM) resulted in a significant increase in the mean percentage of restitution compared to the spraying method. However, for IAA, although restitution levels showed a higher trend upon dipping, no significant differences were observed with the spray method. Previous experiments have shown that NAA leads to a significantly higher percentage of restituted products as compared to IAA, however this difference was not observed when auxins were applied via the dipping approach. This could simply reflect the natural biological variability in induction of meiotic restitution, or could be caused by variations in environmental conditions, such as temperature or humidity, and / or subjective quantification of tetrads. Interestingly, for both auxins (i.e., NAA and IAA), the dipping method led to a relatively higher fraction of balanced dyads as compared to the spray method, suggesting that the application methodology not only affects the level of restitution but also may influence the underlying cellular mechanism, or at least the extent to which it interferes with meiotic cell division, most likely through variability in endogenous auxin levels. It is worth noting that when using the dipping method even the mock-treated plants showed a low percentage of restituted products (<0.5%), which could be attributed to the physical stress resulting from stem bending or from the short period of hypoxic conditions during the treatment.

[0103] Altogether, these results show that exogenous application of NAA or IAA via the dipping method is more effective than the spraying method for inducing male meiotic restitution both in terms of efficiency and homogeneity. Example 6 External application of NAA consistently induces male meiotic restitution in different Arabidopsis ecotypes

[0104] An ecotype is defined as a distinct form or variant of a species that has evolved to be adapted to a specific ecological condition or niche. As a consequence, different plant ecotypes often have broad genetic differences that have been accumulated over long periods of time to allow them to better adapt to specific soil types and climatic weather conditions of a particular ecosystem. The Arabidopsis 1001 Genomes Project has created a large database of Arabidopsis thaliana ecotypes, including phenotypic characterization and genotyping of more than 1135 variants collected from all around the world, providing a valuable research platform to study (natural) genetic variability on a large scale for any phenotype-of-interest. Due to high level of genetic variance (i.e., mainly SNPs) between ecotypes, this natural germplasm collection shows strong variability in many phenotypic attributes, such as plant habitus, flowering time, and leaf morphology. If the Arabidopsis ecotypes would show drastic differences in either the occurrence or level of auxin-induced meiotic restitution, this would indicate a genetic basis for the sensitivity of male meiosis to auxin, with major implications for application in crops, as the efficacy and efficiency of the method would largely depend on the genotypic background of the line / cultivar treated. In order to investigate this, an assessment of phenotypic variance between Arabidopsis ecotypes was conducted by analyzing the extent of NAA-induced male meiotic restitution in a confined set of 20 randomly selected ecotypes via meiotic tetrad analysis (Figure 7).

[0105] The results indicate that exogenous application of NAA consistently induces meiotic restitution in all Arabidopsis thaliana ecotypes that have been tested, and that the overall percentage of restitution products is highly similar, or at least within the same range (2-5%), in most lines (Figure 7). Parallel to balanced triads, which is the most commonly observed restitution product in all backgrounds, ecotypes like Col-3 and Altai-5 also show a significant fraction of balanced dyads, whereas in Ler-0, Chat-1 and Alst-1, also monads could be found. This variability in both the frequency and aspect of meiotic restitution across ecotypes, albeit minor, indicates the existence of some level of natural genetic variance in the sensitivity of the meiotic cell division program to alterations in hormone homeostasis. However, as all Arabidopsis ecotypes tested show a certain level of meiotic restitution upon application of NAA, these data show that this genetic effect is rather minor, implying that this method for induction of meiotic restitution is broadly applicable and effective, independently of the genetic background of the treated plant / line . Example 7 Increased auxin levels in yucca6 gain-of-function lines also lead to meiotic restitution

[0106] The YUCCA gene family, encoding for flavin monooxygenases, plays unique roles in plant development and growth, particularly the YUCCA6 enzyme that is essential in the tryptophan-dependent pathway for auxin biosynthesis. Overexpression of the YUCCA6 gene, via the 35S promoter, leads to increased enzyme activity and auxin concentrations, resulting in higher expression of lAA-inducible genes, like Aux / IAA. In previous research specific gain-of-function mutants of Arabidopsis YUCCA6 have been uncovered, i.e., the yucca6-lD and yucca6-2D, that both show a significant increase in endogenous IAA levels. It was hereby further specified that late-stage inflorescences of these yucca6 mutants contain 32% more free IAA as compared to wild-type plants. As such, both the yucca6-lD and -2D gain-of-function Arabidopsis mutants establish a valuable genetic tool to validate and characterize the effects of endogenous IAA modulation on the stability of meiotic cell division and its capacity to induce male meiotic restitution. In order to monitor for putative alterations in male meiotic cell division, yucca6-lD and -2D plants were subjected to tetrad analysis. The results revealed that both yucca6-lD and -2D plants, besides a large amount of balanced tetrads, also generate a significant fraction of dyads and triads (~5-10%), indicating occurrence of meiotic restitution (Figure 8). As such, these data confirm that an endogenous increase of auxin levels in early stage flower buds leads to ectopic induction of meiotic restitution in Arabidopsis male sporogenesis.

[0107] Example 8 Auxin localization in NAA-treated meiotic buds using the DR5::GUS reporter system

[0108] The DR5::GUS reporter system, including the auxin responsive promoter DR5 that promotes the expression of the p-glucuronidase (GUS) enzyme in tissues that contain auxin, is a widely used tool to monitor auxin localization in Arabidopsis thaliana. This reporter gene system relies on the exogenous application of 5-bromo-4-chloro-3- indolyl glucuronide (X-Gluc), that serves as a precursor for GUS enzyme activity and thereby is converted into a stable coloured product that can be easily visualized using bright field microscopy. As such, accumulation of auxin (maxima) in fixated plant tissues can be easily assessed using this approach. The DR.5-GUS system is beneficial for our research to determine tissue-specific shifts in endogenous auxin accumulation upon exogenous IAA or NAA treatments (particularly in the reproductive organs), but also for conducting auxin localization studies in non-treated Arabidopsis plants to determine the normal spatio-temporal auxin distribution in reproductive organs. By taking advantage of the DR5::GUS reporter line, localization of auxin in developing floral buds has been characterized after X-Gluc application, both under control and IAA treatment conditions. Under regular conditions (mock treatment), Arabidopsis early stage flower buds up till stage 9 do not show presence of auxin, neither in the anthers nor anywhere else in the bud, indicating complete absence of auxin during male sporogenesis. However, in later stages of flower development, i.e., from stage 9 onwards, flower buds show a gradual increase in DR.5-GUS expression, indicating progressive accumulation of endogenous auxin (Figure 9). In contrast, when inflorescences were exogenously treated with 1 mM NAA, a strong GUS signal was observed in all tissues in all stages of flower bud development, indicating overall auxin abundance in the anthers. Auxin is known to act as a primary growth regulator in plants and its specific localization pattern plays a critical role in many developmental processes, in particular for driving cell division in meristematic or proliferating tissues. However, despite its central role in promoting cell division, the complete absence of auxin in early stage floral buds suggests lack of functionality or even an antagonistic role of this hormone in meiotic cell division, and thereby also hints for the presence of molecular mechanisms that prevent local auxin biosynthesis, accumulation, and / or signalling in early stage anthers / flowers to safeguard stability of male meiosis.

[0109] Example 9 Investigating the concentration of NAA that is the most suitable for inducing meiotic restitution at the highest level

[0110] To investigate the most effective concentration of NAA for inducing maximal levels of meiotic restitution, a dosage analysis was performed on Arabidopsis ecotype Col-0 plants and two other randomly selected ecotypes (Utrecht and Sq-8). Hereby, plants were treated with four different concentrations of NAA, namely 100 pM, 500 pM, ImM and 2 mM. This dosage analysis was performed on three Arabidopsis ecotypes to examine whether there is a genetic basis for differences in the frequency of meiotic restitution induced by NAA.

[0111] For each of the three ecotypes tested, no significant differences in total meiotic restitution frequency were identified between the different NAA concentrations (Figure 11). In the Col-0 ecotype, the restitution frequencies were observed to be 0.8%, 3.9%, 3.1% and 5.6% for 100 pM, 500 pM, 1 mM and 2 mM NAA, respectively, although no significant increase was detected due to high variance between biological replicates (Figure 11A). However, the large standard deviation at 1 and 2 mM NAA treatments indicate high variability in these observations, indicating that 500 pM appears to be the concentration leading to the most consistent induction of meiotic restitution. In the Utrecht ecotype, 500 pM NAA induced the highest frequency of total meiotic restitution (2.8%), although the difference with 1 mM and 2 mM NAA was not significant (Figure 11B). By contrast, in Sq-8, meiotic restitution was observed only at 1 mM and 2 mM, but not at 500 pM NAA (Figure 11C). This provides evidence that there is genetic variability in the sensitivity of male meiosis in response to NAA treatment. However, this is only apparent at lower concentrations, as at higher concentrations NAA induces meiotic restitution in all genotypes tested.

[0112] Example 10 Assessment of whether the developmental age of the plant influences the frequency of meiotic restitution induced by NAA

[0113] To test whether the frequency of meiotic restitution that is induced by NAA differs depending on the age of the treated plant, a developmental stage analysis was conducted. Hereby, NAA dipping treatments were performed on Arabidopsis Col-0 plants during their first, second and third week of flowering (approximately four, five and six weeks old plants, respectively), after which immediate tetrad analysis was performed. In addition, a similar 'developmental stage analysis' was conducted on the IAA overproduction mutant yucca6-2D to also investigate a putative agedependent effect on the extent of meiotic restitution induction in the case of endogenous auxin production.

[0114] Overall, for both the NAA treatments as well as the yucca6-2D mutant, a decreasing trend in total meiotic restitution frequency was observed as plants got older, although only in yucca6-2D the difference between the first and third week of flowering was significant (Figure 12). In Col-0 plants subjected to NAA treatment, the total restitution frequency decreased from 3.0% during week 1 to 1.5% during week 3 (Figure 12A), while in yucca6-2D (untreated) this frequency decreased from 4.3% in week 1 to 0.9% in week 3 (Figure 12B). It should be noted that 0.4% meiotic restitution was observed in mock-treated plants in the second week of flowering of Col-0, which is significantly lower (p<0.05) than the restitution levels observed with NAA treatments on the same week. (Figure 12A). Thus, this percentage likely results from one triad that was possibly formed through unexpected minor stress events, like a heat or a cold shock. Example 11 Testing analogous auxin compounds for their ability to induce male meiotic restitution

[0115] The natural auxin IAA and the synthetic auxin analogue NAA have been shown to induce meiotic restitution. However, several other natural and synthetic auxin analogues exist, including picloram (4-amino-3,5,6-trichloorpyridine-2-carbonic acid), 2,4,5-Trichlorophenoxyacetic acid (2,4, 5-T), phenylacetic acid (PAA), 4- chloroindole-3-acetic acid (4-CI-IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and dicamba (3,6-Dichloro-2-methoxybenzoic acid) (Table 1). To test whether these analogues also induce meiotic restitution, Arabidopsis Col-0 plants were treated by dipping their inflorescences into a solution with 2 mM of each analogue, after which tetrad analyses were performed on the next day. The applied concentration of 2 mM is considered to be very high for auxin analogues, however, this was deliberately chosen since it was assumed that if an analogue has the ability to induce meiotic restitution, this would be detectable at 2 mM. Similar as IAA and NAA (Figure 3), all tested analogues were found to induce male meiotic restitution at a concentration of 2 mM (Figure 13A). The highest total meiotic restitution frequencies were detected upon application of picloram and 2,4, 5-T, amounting to 4.3% and 4.2%, respectively. PAA, 4-CI-IAA and 2,4-D were found to induce intermediate frequencies of restitution between 2.0% and 2.5%, while dicamba induced the lowest frequency of approximately 0.8%. The extent of meiotic restitution induction was significant between picloram and dicamba and between 2,4, 5-T and dicamba.

[0116] Since all tested analogues induce meiotic restitution at 2 mM, for each analogue a dosage analysis was performed to determine which concentration leads to the highest restitution frequency. Plants were treated via dipping with concentrations of 100 pM, 500 pM, 1 mM and 2 mM, followed by tetrad analyses. For most analogues, including picloram, 2,4, 5-T, PAA and 4-CI-IAA, total restitution frequencies showed a linearly increasing trend with increasing concentrations (Figure 13, panels C-F). For these compounds, the highest total restitution frequency was found at 2 mM. However, the difference between 100 pM and 2 mM was only found to be significant for picloram. Furthermore, for some of these analogues, for example 2,4, 5-T and 4-CI-IAA, a plateau seemed to be reached at 500 pM, whereby higher concentrations did not result in substantially higher restitution frequencies. In contrast, for 2,4-D and dicamba, the highest restitution frequency was observed at 1 mM (Figure 13, panels G and H). This frequency amounted to approximately 1.1% for both analogues and thus appeared relatively low. Interestingly, for all auxin analogues tested, the majority of induced restitution products consisted of triads, although picloram and 4- CI-IAA also induced a substantial fraction of balanced dyads (Figure 13, panels A, C and F). For dicamba, the type of restitution product was less consistent, with only balanced dyads identified during the initial screen (2 mM concentration), whereas only triads were observed in the dosage analysis (Figure 13, panels A and H).

[0117] Differences in the type and frequency of male meiotic restitution induced by the different auxin analogues could potentially be related to differences in their molecular structures. Each analogue has at least one aromatic ring with a side chain that contains a carboxyl group (Figure 13, panels B-H). Moreover, except for PAA, each of the tested analogues contains one, two, or three chloride groups. Relative to the side chain with the carboxyl group, the chloride groups can have an ortho, meta or para positioning. Picloram and 2,4, 5-T, the auxin analogues that induce the highest frequency of meiotic restitution, both have chloride groups at each of these three positions. Furthermore, 2,4-D and dicamba both have two chloride groups, i.e., at the ortho and para and the ortho and meta position, respectively. Thus, among the tested auxin analogues, picloram and 2,4, 5-T have a unique combination of chloride groups at the meta and para position. This suggests that the combination of chloride groups at those two specific positions could contribute to the relatively high restitution frequency of 2,4, 5-T and picloram. However, it should be noted that NAA, while inducing a relatively high meiotic restitution rate of 3.9% (see Figure 12A), does not contain any chloride group.

[0118] Example 12 Several auxin-related mutants show induction of meiotic restitution

[0119] The presence of auxin in cells can be sensed via receptors and this presence can trigger the up- or downregulation of genes through a short signaling pathway. These auxin-inducible genes contain auxin response elements (AREs) in their promoter, which are bound by dimeric Auxin Response Factors (ARFs) [Leyser (2018) Plant Physio. I 176, 465-479; Taele (2006) Nature Rev Mol Cell Biol 7, 847-859], These ARFs regulate the expression of the gene, for example by recruiting chromatin remodelers. In the absence of auxin, Auxin / Indole-3-Acetic Acid (Aux / IAA) repressors dimerize with the ARFs and inhibit their function, typically leading to the inhibition of gene expression . Hereby, Aux / IAA repressors can form complexes and recruit Topless (TPL), a chromatin remodeler that induces gene silencing . However, in the presence of auxin, auxin functions as a molecular glue between the Aux / IAA repressors and F-box proteins from the Transport Inhibitor Response 1 / Auxin Signaling F-Box (TIR1 / AFB) family . These F-box proteins are part of an SCF-type E3 ubiquitin ligase complex, which ubiquitinates proteins, targeting them for degradation by the 26S proteasome. Thus, when auxin is present, the Aux / IAA repressors interact with TIR1 / AFB proteins, leading to their ubiquitination and degradation. As a result, the ARTs are no longer inhibited and the auxin-inducible genes are transcribed. In A. thaliana, 23 different ARFs, 29 Aux / IAA repressors and 6 TIRl / AFBs are currently identified . This plethora of signaling proteins results in diverse and specific proteinprotein and protein-DNA interactions, auxin sensitivities and cellular outputs. As a result, the effect of auxin is determined by the tissue and developmental stage of a cell, as these factors determine the proteins that are expressed and their relative expression levels.

[0120] The response to auxin in plants is determined by both absolute and relative auxin concentrations. Auxin concentrations exhibit spatial and temporal variation and are determined by feedback loops within and between cells, tissues and organs. The higher the auxin concentration within a cell, the more Aux / IAA inhibitors are degraded and the higher the expression level of an auxin-inducible gene . Hence, these auxin-inducible genes are not simply switched on and off, but display gradients in expression levels . The concentration of auxin in a certain cell or tissue is determined by several processes. First, auxin levels are increased by its biosynthesis. In A. thaliana, the main precursor for auxin is the amino acid tryptophan (T rp), which is converted into auxin through the activity of two enzymes. Tryptophan Aminotransferase Of Arabidopsis 1 (TAA1) converts Trp into indole-3-pyruvic acid (IPA), which is subsequently converted into IAA by members of the YUCCA (YUC) flavin monooxygenase family [Mashiguchi (2011) Proc Natl. Acad. Sci. USA 108, 18512-18517]. The conversion of Trp to IAA by these enzymes encompasses the main auxin biosynthetic pathway, although alternative pathways are suggested to exist. Secondly, the concentration of active auxin is determined by the conjugation of auxin to other small molecules, such as amino acids . These conjugates are considered inactive storage forms of auxins. The synthesis of these conjugates is established by Gretchen Hagen 3 (GH3) amidosynthases [Guo (2022) Bloch Biophys Res Comm 589, 16-22]. To release free auxin from these storage forms, the conjugates can be hydrolyzed by lAA-Leu-Resistant 1 / ILRl-Like (ILR1 / ILL) enzymes. Thirdly, the concentration of auxin can be decreased by its degradation. Auxin degradation is initiated by the oxidation of auxin conjugates by Dioxygenase For Auxin Oxidation (DAO) enzymes [Hayashi (2021) Nature Comm. 12, 6752]. Subsequently, these oxidized conjugates are hydrolyzed by ILR1 / ILL enzymes, leading to the release of inactive 2-oxindole-3-acetic acid (oxIAA) (Hayashi et al. (2021) cited above). This oxidized auxin form does not interact with TIRl / AFBs and consequently does not activate auxin signaling mechanisms. Auxin oxidation is irreversible and leads to its further degradation . Finally, auxin levels in a specific cell or tissue are determined by its transport. Auxin can be rapidly transported between tissues via the phloem, but a more precise transport between cells is mediated by the cell-to-cell polar auxin transport. In this polar auxin transport, Pin-Formed (PIN) auxin efflux carriers play a crucial role by localizing auxin efflux to one side of the cell and thus determining the direction of auxin transport.

[0121] To obtain insights into the mechanistic basis of auxin-induced meiotic restitution, seven publicly available auxin-related Arabidopsis transgenic lines were assessed for putative defects in meiotic cell division, namely 35S:ARF2, iaa8-l, iaal2-l, iaal8-3, gh3.5_l, gh3.9_2 and daol-1. As mentioned earlier, AR.Fs are positive regulators of auxin-inducible gene expression and their overexpression leads to constitutive auxin signaling. By contrast, Aux / IAAs inhibit the activity of ARFs and are thus negative regulators of auxin signaling. Hence, knock-out mutants of Aux / IAAs typically show an increased activity of ARFs, thereby leading to enhanced auxin signaling. TAA1 and YUCCA proteins catalyze the biosynthesis of auxin, and correspondingly their overexpression leads to an increased level of endogenous auxin in plant inflorescence tissue , as for example shown for the dominant negative yucca6-2D mutant line [Kim (2007) Plant Physiol. 145, 722-735]. At the other side, auxin is inactivated via conjugation to amino acids and this is specifically mediated by proteins of the GH3 family, so a knock-out of these GH3s also leads to higher level of endogenously active auxin. Finally, conjugated auxin can be oxidized by DAO proteins, leading to its degradation. Thus, a knock-out of DAOs result in less auxin degradation and increased auxin levels. For each of these mutant or transgenic lines, homozygous plants were identified and subsequently tested for stability of male meiotic cell division using tetrad analysis. All mutants analyzed are SALK lines and either have a T-DNA insert in their promoter, which is the case for gh3.5_l and gh3.9_2, or in an exon, which is the case for Iaa8-1, iaal2-l, iaal8-3 and daol-1 (Figurel4A).

[0122] In all three tested IAA mutants, more specifically iaa8-l, iaal2-l and iaal8-3, meiotic restitution was observed at low frequencies varying between 0.2% and 0.6% (Figure 14B). Since no restitution was detected in the wild type control lines, this effect -although minor compared to external treatments- is still significant. Meiotic restitution was also detected in both gh3.5_l and gh3.9_2, at frequencies of 2.1% and 0.5%, respectively (Figure 14B). In contrast, male meiotic restitution was not observed in the 35S:ARF2 and mutant daol-1 lines. Together with the observation that the auxin overproduction mutant yucca6-2D exhibits male meiotic restitution, these results further demonstrate that endogenous alterations of auxin homeostasis, i.e., either by increasing the level of free auxin (gh3 mutants) or ectopically activating the downstream signaling pathway ( aa mutants), leads to a substantial induction of meiotic restitution. Although no significant differences in total meiotic restitution frequency were observed between the different lines, mutations in the pathways that regulate free auxin levels seem to be most efficient at inducing meiotic restitution, with the highest frequency so far observed in the yucca6-2D line (4.3% -+1.4%).

[0123] Example 13 Auxin induces FDR-type restitution in Arabidopsis male meiosis To determine the type of meiotic restitution induced by auxin, i.e., first division restitution (FDR), second division restitution (SDR) or intermediate-type of restitution (IMR), the ability of auxin to overcome male reproductive sterility in haploid plants was assessed. In brief, this principle is based on the fact that haploid plants typically produce inviable spores and pollen because of gametophytic hypoploidy (i.e., less than the basic set of five chromosomes) caused by the double reductional separation of one single haploid (n = 5) chromosome set, and that this can be restored by FDR- type restitution (i.e., absence or restoration of imbalanced chromosome segregation in MI with a balanced segregation of chromatids for all five chromosomes leading to balanced dyads with two haploid spores) but not by SDR-type restitution (i.e., the unbalanced segregation of chromosomes in MI remains and thus unbalanced dyads with aneuploid spores are formed). For this analysis, haploid (n) Arabidopsis Col-0 and yucca6-2D plants were generated by crossing homozygous diploid plants with the haploid inducer line CENH3-GFP-tailswap. In the resulting progeny pool haploid plants were selected based upon DNA flow cytometry combined with phenotypic plant analysis (i.e., small inflorescences and underdeveloped siliques). Next, the effect of auxin overproduction on male meiotic cell division, and particularly the type of restitution induced (balanced vs unbalanced restituted products), in haploid yucca6- 2D plants was investigated, using haploid Col-0 plants as a reference control.

[0124] The occurrence and frequency of male meiotic end products for both wild type Col-0 and yucca6-2D plants was determined by tetrad counting. In Col-0 (n) haploids, only unbalanced tetrads were observed. By contrast, in yucca6-2D haploids (n), besides a significant fraction of unbalanced tetrads (69.5%), a plethora of other meiotic end products were observed, including balanced tetrads (15.7%), dyads (10.9%), triads (2.8%), and polyads (1.1%) (Figure 15, panels A-H). Out of these meiotic products, the frequency of dyads were higher than expected. In Col-0 haploids (n), balanced dyads are expected to occur in 1 / 32 of the meiotic products, corresponding to 3.125%. This frequency is calculated based upon the probability that all five chromosomes migrate to the same pole during MI (which amounts to 1 / 25), as this is the only instance by which Arabidopsis wild type haploid meiocytes can spontaneously undergo restitution and generate 'dyad' end products. Nevertheless, in our analysis, no dyads were observed in Col-0 haploids (n), most likely due to the small sample size. In yucca6-2D haploids (n), however, a total number of 10.9% dyads were observed (Figure 15H). Indeed, apart from the expected 'baseline' frequency of 3.1% balanced dyads, the additional formation of balanced dyads in haploids can only result from a lack or disruption of chromosome segregation in MI (or even a rejoining of the chromosomes in Mil), leading to a single mitotic-like division with segregation of the sister chromatids of all five chromosomes towards two opposite poles in MIL The ~4-fold higher ratio of balanced to unbalanced dyads (8.7% balanced compared to 2.2% unbalanced dyads) observed in yucca6-2D haploid (n) meiosis indicates that yucca6-2D mutation (and thus excess of auxin) predominantly induces an FDR-type of meiotic restitution.

[0125] Secondly, fluorescein diacetate (FDA) staining was performed to evaluate the number and viability of produced pollen in Col 0 (2n), Col-0 (n) and yucca6-2D (n). The FDA- stained anthers were visualized with fluorescence microscopy and the signal indicates that the stained cells have an active metabolism, and thus are viable. As expected, anthers of Col-0 (2n) were full of viable pollen and many pollen could be also observed around the anthers (Figure 15, panels I-K). The anthers of Col-0 (n) were remarkably smaller and contained very few number of viable pollen per anther (0.75 -+ 0.41), which was expected and indicates that these haploid plants were not fertile (Figure 15, panels L-N and R). However, in yucca6-2D (n), the anthers contained in average 12.25 -+4 viable pollen per anther (Figure 15, panels O-R), fewer than Col- 0 (2n), which contained 112 -+ 18 per anther, but clearly more compared to Col-0 (n). This result indicates that male reproductive fertility in yucca6-2D haploid (n) plants was partially restored, suggesting that the pollen grains contain one set of chromosomes, and thus are haploid, further corroborating with the finding that yucca6-2D haploids undergo single mitotic-like division in male meiosis. Consistently with the observations during tetrad analysis, this indicates the occurrence of FDR- type restitution in yucca6-2D. Interestingly, the three previously known exogenous triggers of meiotic restitution, namely heat stress, cold stress and treatments with gibberellic acid (GA), all induce an SDR-type meiotic restitution in A. thaliana through organizational defects of the radial microtubule arrays (RMAs) [De Storme (2012) Plant Physiol. 160, 1808-1826; De Storme & Geelen (2020) Comm Biol 3, 187; Leyser (2018) Plant Physio! 176, 465-479]. Hence, this finding mechanistically differentiates the restitution induced by auxin (FDR-type) from that induced by GA or temperature stress (SDR-type), and thus for the first time provides a means to induce FDR-type restitution via an exogenous treatment.

[0126] Example 14 Auxin induces male meiotic restitution in Solatium lycopersicum 'MicroTom' and Brassica rapa

[0127] Finally, it was investigated whether auxin can also induce meiotic restitution in economically relevant crops. Therefore, NAA treatments and subsequent tetrad analyses were performed on the tomato variety MicroTom and on B. rapa. Since it was not known when the effect of NAA would be detectable, tetrad analysis was performed on three different time points, namely the day of the treatment, the day after treatment and two days after treatment. On MicroTom, treatments were initially performed by dipping the inflorescences in the NAA and mock solutions, similar to the treatments on A. thaliana. However, no meiotic restitution was detected on the day of the treatment and only 0.1% restitution was observed on the day after the treatment (Figure 16A). A possible reason for this low restitution frequency is the hydrophobicity of the outer layer of the tomato buds, which could impair the entry of the NAA solution inside the buds and the anthers. To overcome this hydrophobic layer, MicroTom plants were treated by injecting the NAA or mock solution directly into the buds, which was possible due to the larger sizes of tomato buds in comparison to A. thaliana and B. rapa. On the day of the treatment by injection, 0.3% meiotic restitution could be observed (Figure 16B). On the day after the treatment this frequency increased to 0.5%, while two days after the treatment no meiotic restitution was observed. On B. rapa, all treatments were performed by dipping the inflorescences in the NAA or mock solutions. Similar to the treatments by injection in MicroTom, in B. rapa 0.3% meiotic restitution was observed the day of the treatment and 0.6% the day after the treatment (Figure 16C). Two days after the treatment, no meiotic restitution was detected.

[0128] These results indicate that auxin does not only induce meiotic restitution in A. thaliana, but also in economically relevant crops. Both in MicroTom and B. rapa the highest restitution frequencies were observed the day after the treatment, similarly to A. thaliana.

[0129] Table of the application

[0130]

[0131]

[0132]

[0133] Table 1.

[0134] An extensive list of known auxin analogues, including the compounds that were tested for their effect on male meiotic stability in Arabidopsis thaliana and for putative induction of meiotic restitution. The table indicates whether the auxin analog is naturally occurring 5 in the plant kingdom, or synthetically synthesized, as well as at which concentration it has been applied.

Claims

CLAIMS1) A method for inducing diploid or polyploid spores in flowering plants, the method comprising the steps of: a) applying on at least the microsporangia of a flowering plant an auxin, a stereoisomer, tautomer or salt thereof, or providing a plant with increased auxin levels, and b) identifying from the plant treated with an auxin, or from the plant with increased auxin levels, flowers or flower buds with diploid or polyploid spores.2) The method according claim 1, further comprising step c) of applying di- or polyploid pollen identified in step b) of claim 1 in self-pollination or crosshybridization.3) The method of claim 2, further comprising step d) of generating polyploid progeny, polyploid embryos, polyploid seeds or polyploid plants.4) The method according to any one of claims 1 to 3, comprising the step of applying on at least the microsporangia of a flowering plant an auxin or a stereoisomer, tautomer or salt thereof.5) The method according to any one of claims 1 to 4, wherein the application is performed by spraying, dipping or injecting.6) The method according to any of claims 1 to 5, wherein the plant is a dicotyl.7) The method according to any of claims 1 to 6, wherein the plant belongs to the systematic class of the Solanaceae, Brassicaceae or Rosaceae.8) The method according to any of claims 1 to 7, wherein the auxin is applied in the first half or in the first quarter of the flowering period of the plant.9) The method according to any one of claim 1 to 8, wherein the auxin is a compound comprising an unsaturated ring and an acidic side chain.10) The method according to claim 10, wherein the unsaturated ring is aromatic ring or a heterocyclic ring.11) The method according to claim 10, wherein the aromatic ring is a benzene or a naphthalene ring.12) The method according to claim 9, wherein the aromatic ring is an indole.13) The method according to claim 9, wherein the acidic side chain is a carboxylic acid.14) The method according to any of claims 1 to 8, wherein the auxin is an aromatic compound with structure D-E, whereby D is a C3-6 carbocyclic ring structure fused with E,E being a C3-6 carbocyclic or an aromatic 3-6 membered heterocyclic ring structure with a hetero-atom selected from N, O and S, and whereby E is further substituted on a carbon member with an R group selected from the group consisting of carboxylate, carboxymethyl, propanoate, acetamido, or propionamide group.15) The method according to claim 14, whereby the aromatic compound is represented by the formula (I), its salt or solvates thereof:wherein the moiety A is a benzene ring or a pyrrole ring and R represents a carboxylate group, carboxymethyl group, propanoate group, acetamido group or a propionamide group.16) The method according to claim 14 or 15, whereby the aromatic compound is 1-naphthalene acetic acid (NAA) or indole-3-acetic acid (IAA, 3-IAA).17) The method according to claim 15, whereby the aromatic compound is an aromatic heterocycle compound that has a benzene ring (D) fused to a pyrrole ring (E).18) The method according to 15, whereby the aromatic compound is an aromatic carbobicyclic compound that has a benzene ring (D) fused to a benzene ring (E).19) The method according to any of claims 1 to 18, whereby the concentration of the compound in a treatment medium is in a range of 200 pM to 2 mM, preferably of 250 pm to 1 mM, and more preferably 280 to 320 pM.20) The method according to any of claims 1 to 7, comprising the step of providing a plant with increased auxin levels.21) The method according to claim 20, wherein the plant with increased auxin levels is a plant with a mutation in one or more auxin biosynthesis genes and increasing expression of said one or more genes.22) The method according to claim 20, wherein the plant with increased auxin levels is a plant with a mutation in one or more auxin conjugation genes and decreasing expression of said one or more genes.23) The method according to claim 20, wherein the plant with increased auxin levels is a plant with a mutation in one or more auxin degradation genes and decreasing expression of said one or more genes.24) The method according to claim 21, wherein the gene belongs to the family of YUCCA genes or WCGA-like genes.25) The method according to claim 24, wherein the gene is YUCCA6.26) The method according to claim 22, wherein the gene belongs to the family of GRETCHEN HAGEN 3 (GH3) genes or GH3-\ike genes.27) The method according to claim 26, wherein the gene is an ortholog of the GH3.5 or GH3.9 Arabidopsis genes.28) The method according to claim 20, wherein the plant exhibits enhanced or ectopic auxin signalling due to a mutation in one or more genes encoding fortranscriptional repressors of the auxin response and decreasing expression of said gene(s).29) The method according to claim 28, wherein the gene belongs to the family of INDOLEACETIC ACID-INDUCED PROTEINS (lAA) genes or IAA- li ke genes.30) The method according to claim 29, wherein the gene is IAA8, IAA12 or IAA18.31) The use of an auxin to induce meiotic restitution in male sporogenesis or to induce meiotic restitution in female sporogenesis of a flowering plant.32) The use of a plant with increased auxin levels to induce meiotic restitution in male sporogenesis or to induce meiotic restitution in female sporogenesis of a flowering plant.

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