How to Increase Pollen Fertility

Abstract

The present invention relates to a method for improving plant pollen viability using MRN-ATM pathway inhibitors. The improved pollen viability preferably results in improved plant viability. The present invention further relates to MRN-ATM pathway inhibitors for improving plant pollen viability. A preferred MRN-ATM pathway inhibitor for use in the present invention is 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone. The present invention also relates to a method for developing a mature fertile plant scion, comprising contacting an isolated plant part, including an immature flower bud, with a (hazardous or toxic) compound.

Description

[Technical field]

[0001] The present invention relates to the field of plant breeding, more specifically to the field of interspecific hybrid plant breeding. A new method is disclosed for improving plant fertility through the use of pollen treatments and compounds that enhance pollen fertility, which result in improved pollen viability. The pollen can be derived from low-fertility or sterile plants, such as interspecific hybrids. [Background technology]

[0002] The process of plant breeding depends heavily on the availability of genetic variation. This variation is generated by utilizing reverse breeding techniques and CRISPR / Cas9 as well as natural variation found in wild relatives of the target crop. It is desirable to introgress new traits from wild relatives for various reasons, such as to increase pest resistance of cultivated varieties and / or to create new varieties. Furthermore, hybrids often exhibit heterosis or hybrid vigor, which generally enhances biological quality. Therefore, hybrids are of great interest, and it is expected that interspecific hybridization will remain a powerful tool for breeding for the coming decades.

[0003] Depending on the species being crossed, there are several reproductive barriers to the creation and propagation of interspecific hybrids. These postzygotic and prezygotic barriers that prevent the crossing of different plant species range from problems with pollen-stigma interactions, synapsis and recombination between homologous chromosomes during meiosis in F1 hybrids, and pollen tube guidance to embryos or endosperm development. Current methods to circumvent these problems include bud pollination, ovule culture and embryo rescue, but are still far from optimal. So far, the creation of interspecific hybrids has relied heavily on trial and error, which involves extensive breeding efforts that require a lot of time, effort and money. Even more challenging can be the subsequent propagation of these resulting interspecific hybrids. In fact, the majority of interspecific hybrids do not have viable pollen.

[0004] Thus, there is a great need in the art for methods to improve plant fertility, especially for plants with low fertility, such as interspecific hybrids, and more specifically, for increasing pollen viability, for example, in interspecific hybrid breeding and / or production. Summary of the Invention

[0005] The present invention can be summarized in the following embodiments: Embodiment 1.a) providing a first seed plant or part of the plant comprising plant pollen-bearing tissue; b) inhibiting the MRN-ATM pathway in at least a portion of the provided plant or plant part; c) causing said plant or plant part to produce pollen exhibiting improved viability; 16. A method for improving the viability of plant pollen comprising: Embodiment 2. The method of embodiment 1, wherein in step b), the MRN-ATM pathway is inhibited by contacting the plant or plant part with 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone. Embodiment 3. The method of embodiment 1 or 2, wherein said improved viability of plant pollen increases plant fertility. Embodiment 4. The method according to any one of the preceding embodiments, wherein the first plant or part of the plant according to step a) is an interspecific hybrid or part thereof. Embodiment 5. The method of any one of the preceding embodiments, wherein the part of the plant provided in step a) is a cutting, and the cut end of said cutting is contacted with a compound that inhibits the MRN-ATM pathway in step b). Embodiment 6. The method according to embodiment 5, wherein the cutting is a cutting of an angiosperm seed plant and comprises an inflorescence, the inflorescence comprising at least one immature flower bud and preferably no mature flower buds. Embodiment 7. Steps a) to c) are carried out using a part of a plant that is a scion, and the method comprises: d) grafting the scion onto a rootstock. 13. The method of any one of the preceding embodiments, further comprising: Embodiment 8. The method of any one of the preceding embodiments, further comprising allowing said pollen to mature. Embodiment 9 The method of embodiment 8, further comprising isolating mature pollen. Embodiment 10. The method of any one of the preceding embodiments, further comprising self-pollinating said first plant or pollinating a second plant. Embodiment 11. A plant growth medium comprising a compound that inhibits the MRN-ATM pathway. Embodiment 12. Viable pollen of a plant or part of a plant, obtainable by a method according to any one of embodiments 1 to 10. Embodiment 13. a) providing a first seed plant or part of the plant comprising plant pollen-bearing tissue; b) inhibiting the MRN-ATM pathway in at least a portion of the provided plant or plant part; c) causing said plant or plant part to produce pollen exhibiting improved viability; 23. A method for producing a first seed plant having improved pollen viability, comprising: Embodiment 14. A seed plant, preferably an interspecific hybrid, comprising pollen with improved viability obtained by the method according to embodiment 13. Embodiment 15. Use of a compound that inhibits the MRN-ATM pathway, optionally included in a plant growth medium, to improve pollen viability. Embodiment 16. Use of viable pollen according to embodiment 12 or a seed plant according to embodiment 14 for pollinating a second plant, preferably for producing a progeny plant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] definition Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are given their usual meaning in the art to which the present invention pertains unless otherwise indicated. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used to carry out the testing of the present invention, the preferred materials and methods are described herein.

[0007] It will be clear to one of ordinary skill in the art how to carry out the conventional techniques used in the methods of the present invention. Conventional techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well known to those of ordinary skill in the art and are discussed, for example, in the following references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0008] "a," ​​"an," and "the": these singular terms include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0009] As used herein, the term "about" is used to describe and account for small variations. For example, the term can refer to ±10% or less, such as ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, ±0.5% or less, ±0.1% or less, or ±0.05% or less. In addition, amounts, ratios, and other numerical values ​​may be expressed herein in a range format. Such range formats are used for convenience and brevity and should be understood to be flexible to include numerical values ​​explicitly specified as the limits of the range, but all individual numerical values ​​or subranges contained within the range should be understood to be included as if each numerical value and subrange were expressly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly stated limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and subranges such as about 10 to about 50, about 20 to about 100, etc.

[0010] "And / or": The term "and / or" refers to a situation in which one or more of the listed cases may occur alone or in combination with at least one of the listed cases, up to and including all of the listed cases.

[0011] "Plant": as used herein, should be understood as a plant comprising at least a part of a root system and a part of a shoot system. A plant preferably comprises parts of a plant as defined herein below. Preferably, at least, the plant comprises the tissue that produces plant pollen. Preferably, the plant comprises at least one of a flower bud and an inflorescence (mature or immature). Non-limiting examples of plants include barley, cassava, cotton, groundnut or peanut, maize, millet, oil palm fruit, potato, beans, rapeseed or canola, rice, rye, sorghum, soybean, sugarcane, sugar beet, sunflower, wheat, okra, allium, celery, asparagus, wax gourd, beet, brassicas including cruciferous vegetables, bell peppers, chillies, endives, chicories, melons including watermelons, cucumbers, gherkins, zucchinis, cucumbers ... Crops and cultivated plants include such as cinnamon, pumpkin, artichoke, carrot, rocket, fennel, lettuce, bottle gourd, loofah, bitter melon, parsnip, parsley, beans, ground cherry, tomatillo, radish, eggplant, tomatoes including tomato rootstocks, pepino, spinach, snake gourd, field lettuce, peas including chickpeas, soybeans, fava beans, sweet corn, hemp, hops, berries, dandelions and ornamentals.

[0012] "Part of a plant": This includes at least one of a plant cell, a plant protoplast, a plant cell tissue culture capable of regenerating a plant, a plant callus, a plant mass, a flower bud (mature or immature), an inflorescence and an intact plant cell in a plant or part of a plant, such as an embryo, pollen, an ovule, a seed, a leaf, a flower, a branch, a fruit, a grain, an ear, a cob, a husk, a stem, a root, a root tip, an anther, a grain, etc. Preferably, the part of a plant includes at least a tissue that gives rise to plant pollen. Preferably, the part of a plant is at least one of a flower bud (mature or immature) and an inflorescence.

[0013] As described herein, a "seed plant" refers to a plant or group of plants that produce seeds as the primary mode of sexual reproduction, as opposed to spore-forming plants. This group is also known as spermatophytes and is made up of gymnosperms and angiosperms, which includes all crops associated with agriculture. Non-limiting examples are provided herein under the definition of "plant."

[0014] "Inflorescence" refers to the reproductive unit of a plant. It is a group or cluster of flowers arranged on a stem, preferably consisting of a main branch or an arrangement of branches. It can be a part of the shoot of a seed plant in which flowers are formed. Two specific types of inflorescences can be determined: determinate and indeterminate. Determinate (cyme) inflorescences grow terminal (central) flowers from the terminal bud and stop elongating the central axis. In indeterminate (raceme) inflorescences, the terminal bud continues to grow and forms lateral flowers from the lateral buds. Terminal flowers are never formed. The inflorescence is the means of sexual reproduction of said plant. It therefore includes, but is not limited to, one or more, preferably all developmental stages of carpels, anthers, stigma, style, stamens, petals and sepals.

[0015] An "interspecific hybrid" is a direct progeny ("F1" progeny) from the mating of two species, preferably two individuals within the same genus. The progeny preferably exhibits the traits and characteristics of both parents, but is often sterile, preventing gene flow between the species (Keeton, William T. 1980. Biological science. New York: Norton. ISBN 0-393-95021-2, p. 800). Sterility is often due to differences in the number of chromosomes between the two species. Sterility may be due, at least in part, to the absence of viable pollen.

[0016] The "MRN-ATM pathway" is a pathway involved in the control of DNA damage, more specifically double-stranded (DSB) break repair. The MRN-ATM pathway is highly conserved among plant species (Yoshiyama et al., Biology (Basel). 2013 2(4): 1338-1356; Amiard et al. The Plant Cell, 2010, 22 (9): 3020-3033). The plant MRN complex is a heterotrimer containing three proteins: meiotic recombination 11 (Mre11), DNA repair protein Rad 50, and Nijmegen breakage syndrome 1 (Nbs1). It is known as the key catalytic protein complex in the regulation and sensing of DSBs and initiation of the DNA damage response pathway. The MRN complex senses DSBs and recruits inactive dimeric ATM (ataxia telangiectasia mutated), where it is activated to phosphorylate members of the MRN complex, in addition to various other proteins involved in cell cycle control and DNA repair (Kanaar and Wyman Cell 2008, 135, 14-16.). Activation of the MRN-ATM pathway activates DNA damage repair and allows the cell cycle to continue. During meiosis in plants, subunits of the MRN-ATM pathway are involved in the control of meiosis and pollen formation (Culligan et al., Plant J. 2008 Aug; 55(4): 629-38.; Kurzbauer et al., The Plant Cell 2021; 33(5): 1-24).

[0017] An "MRN-ATM pathway inhibitor" is a compound that downregulates or prevents activation of the MRN-ATM pathway. The compound is preferably a chemical compound.

[0018] As used herein, the terms "pollination" or "pollinating" refer to the process by which pollen is transferred from the anther (male part) to the stigma (female part) of a plant, thereby enabling fertilization and reproduction. Each pollen grain is a male haploid gametophyte that is adapted to be transported to the female gametophyte where it can cause fertilization by producing a male gamete (or multiple gametes) in a process of double fertilization.

[0019] The viable pollen grain (gametophyte) of angiosperms containing the male gametes is transported to the stigma, where it germinates, and the pollen tube grows along the style to the ovary. The two gametes of the pollen grain travel down the tube to where the gametophyte containing the female gametes is held within the carpel. One nucleus fuses with the polar body to produce endosperm tissue, and the other nucleus fuses with the ovule to produce the embryo. Gymnosperms do not produce fruits or flowers. However, like angiosperms, gymnosperms use pollen to promote fertilization.

[0020] Detailed Description The present inventors have discovered that inhibiting the MRN-ATM pathway in plants enables sterile interspecific hybrids to develop fully mature anthers and viable pollen.

[0021] Thus, in a first aspect of the present invention, a) providing a first seed plant or part of the plant comprising plant pollen-bearing tissue; b) inhibiting the MRN-ATM pathway in at least a portion of the provided plant or plant part; c) causing said plant or plant part to produce pollen exhibiting improved viability; The present invention provides a method for improving the viability of plant pollen, comprising:

[0022] The method of the present invention may also be considered a method for producing plant pollen with improved viability.

[0023] Step a) Providing a first seed plant or part of a plant In step a) of the method of the present invention, a first seed plant or a part of a plant is provided. Preferably, the provided seed plant comprises a tissue that produces plant pollen. Also preferably, the provided part of a plant comprises a tissue that produces plant pollen. The provided seed plant is preferably an agriculturally relevant crop plant. In the present specification, a part of a plant is understood to be a part of the first seed plant.

[0024] The term "plant pollen-bearing tissue" is familiar to those skilled in the art. Such tissue is preferably a sporulating tissue, and preferably includes at least one of a sporulating cell, a microspore mother cell (also known as a "pollen mother cell" or a "meiocyte") and a microspore. This tissue may be part of an immature bud or may be contained within an immature bud.

[0025] Preferably, the first seed plant or part of the plant provided in step a) comprises an immature shoot. Preferably, the part of the plant is a cutting. Thus, preferably, said cutting comprises an immature shoot.

[0026] Preferably, the plant pollen-bearing tissue may be part of or may be included within an inflorescence. Preferably, the plant part provided in step a) is or includes an inflorescence. Preferably, the cutting is or includes an inflorescence. The inflorescence preferably includes at least one immature flower bud. The inflorescence may include at least 1, 2, 3, 4, 5, 5, 6, 7, 8, 9, 10 or more immature flower buds. Preferably, the inflorescence includes less than 10, 9, 8, 7, 6, 5, 4, 3, 2 or less than 1 mature flower bud. Preferably, the plant part for use in the method of the present invention is an inflorescence that includes at least one immature flower bud and does not include any mature flower bud. Optionally, any mature flower buds have been removed from the plant or plant part before step a) of the method of the present invention.

[0027] Preferably, the first seed plant or part thereof provided is an angiosperm seed plant. Preferably, the cutting is an angiosperm seed plant cutting.

[0028] Preferably, the first seed plant has a reduced ability to produce viable pollen.Preferably, at least about 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the pollen produced by the first seed plant is non-viable.Preferably, less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or less than about 1% of the pollen produced by the first seed plant is viable. Preferably, the first seed plant cannot produce viable pollen, and the first seed plant may not produce any viable pollen. The reduced ability of the first seed plant to produce viable pollen preferably results in the plant being a plant with limited ability or unable to produce offspring by pollination.

[0029] The viability of plant pollen can be easily determined using any conventional method known to those skilled in the art.As a non-limiting example, viability can be determined using conventional MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium or MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, for example, using the method steps described in Example 1 below.

[0030] Alternatively or additionally, preferably, in the first seed plant provided, at least about 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the anthers remain closed upon pollen maturation. Preferably, less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or less than about 1% of the anthers open upon pollen maturation. Anther opening can be assessed by visual inspection, optionally using a microscope to magnify the image.

[0031] The first seed plant used in the method of the present invention can be a plant with low or non-viable pollen viability and / or a plant with closed anthers and / or a plant with low fertility. Optionally, the plant is sterile or partially sterile. The first seed plant can be an interspecific hybrid. A first seed plant is understood herein to be a plant that produces seeds as the main form of sexual reproduction, and is a member of the seed plant classification, including but not limited to all major crop plants. The first seed plant provided in step a) can be an angiosperm or gymnosperm, preferably an angiosperm. Preferably, in step a), a part of an angiosperm plant, preferably an inflorescence from an angiosperm, is provided. The plant may belong to, but is not limited to, the families Solanaceae, Cucurbitaceae, Brassicaceae, Asteraceae, Malvaceae, Fabaceae, Poaceae, Amaranthaceae, Leguminosae, Musaceae, Gramineae, Rosaceae, Euphorbiaceae, Apiaceae, and Rubiaceae.Plants include the following: Solanum (including Lycopersicum), Allium, Beta, Brassica, Nicotiana, Capsicum, Petunia, Triticum, Oryza, Glycine, Cucumis, Cichorium, Chrysanthemum, Pennisetum, Secale, Lactuca, Hordeum, Helianthus, Cannabis, Avena, and Corn. The plant may belong to, but is not limited to, the genera Sorghum, Gossypium, Medicago, Phaseolus, Sorghum, Saccharum, Hordeum, Taraxacum, Pisum, Zea, Medicago, Phaseolus, Rosa, Lilium, Coffea, Linum, Mannihot, Daucus, Cucurbita, Citrullus and Musa. Preferably the plant is a plant of the Brassicaceae family, preferably a plant of the genus Brassica.

[0032] The first seed plant that is prepared can be a monoecious plant or a dioecious plant.Preferably, the first seed plant that is prepared is a plant with reduced or low fertility or a sterile plant.Preferably, the first seed plant is a plant with reduced or low pollen viability or a plant with non-viable pollen.Preferably, the first seed plant has limited ability or cannot produce offspring through pollination.

[0033] The first seed plant can be an interspecific hybrid.Interspecific hybrid can be generated from any combination of species that can produce viable offspring.In other words, interspecific hybrid can be the hybrid of any two parent plants of different species that produce viable plants.Preferably, interspecific hybrid is the hybrid between two species of the same genus.This interspecific hybrid can be characterized by having hybrid vigor for parent plants and / or a mixture of chromosomes from each parent.

[0034] Preferably, interspecific hybrid is a hybrid between two parent plants that differ in chromosome number and / or ploidy.It is understood that the present invention is not limited to any particular interspecific hybrid, but is generally applicable.The method of the present invention can be applied to any interspecific hybrid, i.e., any cross between two species, preferably of the same genus, resulting in a partially or completely sterile interspecific hybrid, and / or a plant that can benefit from a method of increasing pollen viability, opening anthers and / or increasing fertility.

[0035] Optionally, the interspecific hybrid is an interspecific hybrid between two species of the genus Brassica, preferably with different chromosome numbers between the parents. The interspecific hybrid may be a hybrid of one parent plant of a species selected from the group consisting of B.rapa (AA 2n=20), B.nigra (BB 2n=16) and B.oleracea (CC 2n=18) with another parent plant of another species of the group. The interspecific hybrid may be an interspecific hybrid of a cross between Brassica oleraceae and Brassica rapa. Optionally, the interspecific hybrid is a hybrid between two species of the genus Lactuca. The interspecific hybrid may be an interspecific hybrid of a cross between Lactuca sativa and Lactuca virosa.

[0036] Optionally, the first seed plant is the progeny of interspecific hybrid, preferably the progeny with reduced pollen viability.The progeny can be the progeny of interspecific hybrid backcross, self-pollination or combination thereof.Optionally, when interspecific hybrid is considered as "first generation plant", the progeny is the second generation, third generation, fourth generation, fifth generation, sixth generation, seventh generation, eighth generation, ninth generation or tenth generation.

[0037] Step b) Inhibiting the MRN-ATM pathway in a prepared plant or plant part. In step b) of the method of the present invention, the MRN-ATM pathway can be inhibited in at least a part of the plant.Preferably, the MRN-ATM pathway is inhibited at least in the tissue that produces plant pollen.The inhibition of the MRN-ATM pathway can include inhibiting the activity of a protein that is a member of the pathway, where preferably the protein is selected from the group consisting of Mre11, Rad50, Nbs1 (together the MRN complex) and ATM protein.Preferably, the inhibition of the MRN-ATM pathway can include inhibiting the MRN complex, preferably inhibiting the activity of a protein that is a member of the complex, for example inhibiting the activity of at least one of Mre11, Rad50 and Nbs1.

[0038] The reduction in activity of Mre11 is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in activity. The reduction in activity of Rad50 is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in activity. The reduction in activity of Nbs1 is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in activity. The reduction in activity of ATM is preferably at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in activity. A 100% reduction in activity is understood herein as a complete lack of activity.

[0039] The inhibition of MRN-ATM pathway can be partial or complete inhibition. The inhibition of MRN-ATM pathway is preferably carried out by exposure to a compound. The inhibition of MRN-ATM pathway in the tissue that produces plant pollen of the first seed plant or a part thereof in step b) is also considered to be the treatment of the tissue that produces plant pollen of the first seed plant or a part thereof with an MRN-ATM inhibitor. The tissue that produces plant pollen is preferably part of an inflorescence or is contained within an inflorescence. The inhibition of MRN-ATM pathway in the inflorescence of the first seed plant or a part thereof in step b) is also considered to be the treatment of the inflorescence of the first seed plant or a part thereof with an MRN-ATM inhibitor.

[0040] Additionally or alternatively, the MRN-ATM pathway can be inhibited or blocked by downregulating or eliminating the expression of one or more proteins of the pathway, for example by suppressing the expression of one or more proteins, for example by using RNAi, and / or by disrupting the gene encoding the one or more proteins. The mutation of the gene encoding the one or more proteins can result in reduced or eliminated expression of functional protein. The mutation can be in a promoter sequence, which can result in reduced and / or eliminated expression, and / or the mutation can be in a coding sequence, which can result in a dysfunctional and / or truncated protein and / or a reduced function protein. Optionally, the inhibition of the MRN-ATM pathway is carried out by RNAi, where the expressed siRNA / miRNA is under the control of a promoter, preferably a tissue-specific promoter. The tissue-specific promoter is preferably active in the tissue that produces plant pollen, and optionally only in the tissue that produces plant pollen.

[0041] Preferably, the MRN-ATM pathway is inhibited by exposure to a compound. Thus, step b) of the method of the invention may also be a step of contacting at least a portion of the provided plant or plant part with a compound that inhibits the MRN-ATM pathway. Preferably, at least the plant pollen-bearing tissue is contacted with said compound. Preferably, said compound is a chemical compound, said compound preferably inhibiting the 3'→5' exonuclease activity associated with Mre11. Said compound has the molecular formula C 10 H8N2O2S, and may be 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone, also known as mirin (PubChem ID 1206243, CAS number 299953-00-7). Optionally, the compound for use in the method of the present invention is a derivative or analog of mirin. Preferred derivatives of mirin are those described in Shibata et al., Mol Cell (2014) 53:7-18, which is incorporated herein by reference.

[0042] Other potentially useful inhibitory compounds may be, for example, but are not limited to, any one of the following inhibitors of ATM kinase activity: - Molecular formula C 30 H 33 2-((2S,6R)-2,6-dimethylmorpholino)-N-(5-(6-morpholino-4-oxo-4H-pyran-2-yl)-9H-thioxanthen-2-yl)acetamide, having N3O5S and also known as KU-60019 (PubChem ID 15953870, CAS number 925701-49-1); - Molecular formula C 21 H 17 2-(4-morpholinyl)-6-(1-thianthrenyl)-4H-pyran-4-one, also known as KU-55933, having NO3S2 (PubChem ID 5278396, CAS number, 587871-26-9); and - Molecular formula C 17 H 15N7O2, 1-(6,7-dimethoxyquinazolin-4-yl)-3-(pyridin-2-yl)-1H-1,2,4-triazol-5-amine, also known as CP-466722 (PubChem ID 44551660, CAS number 1080622-86-1).

[0043] Inhibiting the MRN-ATM pathway is preferably carried out by inhibiting Mre11. Preferably, the 3' to 5' exonuclease activity associated with Mre11 is inhibited. Preferably, said Mre11 inhibitor is mirin.

[0044] The whole prepared plant can be contacted with a compound that inhibits the MRN-ATM pathway, also referred to herein as "inhibitory compound". Optionally, only a part of the plant is contacted with the inhibitory compound. As a non-limiting example, only (a part of) the root system or shoot system is contacted with the inhibitory compound. Optionally, only a part of the plant is contacted with the inhibitory compound, but the compound can then move through the plant tissue and reach the tissue that produces pollen and / or the cells of immature flower buds.

[0045] The MRN-ATM pathway may be inhibited in all cells of the prepared plant or plant part. Alternatively, the MRN-ATM pathway is inhibited in only some cells. For example, the MRN-ATM pathway may be inhibited in at least about 0.5%, 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% of the plant cells of the prepared plant or plant part. The MRN-ATM pathway may be inhibited in cells of the root system or cells of the shoot system, preferably cells of the shoot system. The MRN-ATM pathway is preferably inhibited at least in tissues that give rise to plant pollen. The MRN-ATM pathway may be inhibited in at least one or more immature flower buds. The MRN-ATM pathway may be inhibited at least in the florescense of the prepared plant or plant part.

[0046] Preferably, at least a part of the plant is contacted with the inhibitory compound. The MRN-ATM pathway can be inhibited in step b) by contacting at least the tissue that produces plant pollen with the inhibitory compound. Preferably, the compound is contacted only with the tissue that produces plant pollen, only with the immature flower buds, only with the inflorescence, or only with the part of the plant provided in step a). Preferably, at least the tissue that produces plant pollen is contacted with the compound that inhibits the MRN-ATM pathway. Preferably, at least one or more immature flower buds of the provided plant or part of the plant are contacted with the inhibitory compound. Preferably, at least the inflorescence of the provided plant or part of the plant is contacted with the inhibitory compound.

[0047] The chemical compounds can be added by any suitable means known in the art, including, but not limited to, spraying the flower buds and / or foliage with a composition containing the chemical compound, immersing the flower buds in a medium containing the chemical compound, or mixing the compound into the soil in which the plant is growing so that the compound reaches the flower buds via the roots.

[0048] The MRN-ATM inhibitor may be a compound that is toxic to humans at the concentrations required for the method of the present invention. Therefore, the compound is preferably not used in the form of an aerosol, and preferably the amount of chemical compound used is minimized.

[0049] In a preferred embodiment, a part of a plant containing the tissue that produces plant pollen may be cut from the rest of the plant. The part of a plant may be a cutting. Preferably, the part of a plant may be placed at its cut end in a growth medium containing the above chemical compound, preferably in a minimal amount of growth medium. To prevent the compound from evaporating into the environment, the growth medium containing the compound may be covered or sealed, for example, with parafilm. Optionally, the compound may be applied as shown in FIG. 1.

[0050] Preferably, the MRN-ATM pathway inhibitor is used only on a specific part of the plant. Preferably, the MRN-ATM pathway is inhibited in a tissue of said plant part that gives rise to plant pollen. The tissue is preferably comprised in or on a part of a flower bud. Thus, the plant part preferably comprises at least one flower bud. Preferably, said flower bud is immature and the MRN-ATM pathway is inhibited throughout the bud's maturation period until flowering. Preferably, said flower bud is immature and the plant or plant part is exposed to a compound that inhibits the MRN-ATM pathway throughout the bud's maturation period until flowering. Preferably, said MRN-ATM pathway is inhibited temporarily from the moment of development of the immature flower bud to the moment the bud matures and flowers. Preferably, the plant or plant part is exposed temporarily to a compound that inhibits the MRN-ATM pathway from the moment of development of the immature flower bud to the moment the bud matures and flowers. Preferably, in step a) of the method of the present invention, an inflorescence is provided that comprises at least one immature flower bud. Preferably, said inflorescence does not include mature flower buds. Preferably, said immature flower buds have not yet formed pollen. Said inflorescence is obtained by removing mature flower buds and keeping only one or more immature flower buds.

[0051] Preferably, before contacting the first seed plant or part of the plant with an MRN-ATM pathway inhibitor, a part of the plant comprising at least one immature flower bud is cut from the first seed plant. Preferably, in step b), an inflorescence comprising at least one immature flower bud is cut from the plant. Preferably, the part of the plant comprising the immature flower bud is then placed at its cut end in a medium comprising an MRN-ATM pathway inhibitor at a concentration effective to inhibit the MRN-ATM pathway, preferably the MRN complex in said pathway, and more preferably, said concentration is effective to inhibit Mre11 in tissues and / or cells that give rise to plant pollen. Preferably, said medium is a medium for maturing immature shoots. Such a medium may be a vitamin-containing Murashige-Skoog medium as described in Murashige T. and Skoog F., Physiol. Plant, 15, 473 (1962).

[0052] The concentration of the MRN-ATM pathway inhibitor in step b) is present at an effective concentration, which in the present specification should be understood as the concentration of inhibitor sufficient to elicit the desired effect, i.e., to increase pollen viability and / or result in anther opening. When the MRN-ATM pathway inhibitor is mirin, the concentration can be up to about 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 mM, 70 nM, 60 nM, 50 nM, 40 nM or 30 nM, and the concentration can be at least about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM or 20 nM. Preferably, the concentration is in the range of about 1 nM to 5 μM, about 1 nM to 1 μM, about 5 nM to 500 nM, about 5 nM to 100 nM, about 10 nM to 100 nM, about 5 nM to 50 nM, about 10 nM to 50 nM, or preferably about 20 nM to 30 nM.

[0053] Preferably, the immature flower bud of the first seed plant or part of the plant provided in step a) of the method of the present invention is treated in step b) with an MRN-ATM pathway inhibitor during the maturation period of said flower bud, preferably in a medium containing said inhibitor at a concentration as indicated herein. The treatment is effective on immature flower buds before pollen formation, and may be terminated when pollen is formed. Preferably, a flower bud is considered mature when it opens. Preferably, the flower bud is treated with an MRN-ATM pathway inhibitor by placing an inflorescence containing said flower bud in a medium containing an MRN-ATM pathway inhibitor at an effective concentration as indicated herein during the maturation of said flower bud, i.e., from when the flower bud is immature to when the flower bud opens.

[0054] Plant pollen producing tissue, preferably immature flower buds, are treated with an MRN-ATM pathway inhibitor, preferably for at least about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 16 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. Preferably, the flower buds are treated for up to about 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. Plant pollen producing tissue, preferably immature flower buds, are treated for a period of preferably about 1 hour to 20 days, about 1 day to 20 days, about 1 day to 10 days, about 5 days to 15 days, or about 8 days to 12 days.

[0055] The treatment period may depend on the duration of the maturation period of flower buds. The maturation period may be known or may be determined by those skilled in the art. In some cases, immature flower buds are treated only during a part of the maturation period. The maturation period of flower buds preferably includes at least the pre-meiotic stage, the first meiotic division, and microsporogenesis, which may include the second meiotic division. Therefore, the period of treating immature flower buds with the compound of the method of the present invention is preferably during the microsporogenesis, which may include at least the pre-meiotic stage and the first meiotic division, and may also include the second meiotic division.

[0056] Treatment may be for the entire period of complete maturation of the flower buds, e.g., 100% of that period, or for a portion of that period, e.g., about 20%, 40%, 60%, 80%, 90%, 95%, 96%, 97%, 98%, 99% of the period required for maturation of immature flower buds.

[0057] Step c) Producing pollen with improved viability In the present specification, improved pollen viability is to be understood as increased pollen viability of a first seed plant or part thereof treated by the method of the present invention in comparison with the pollen of a control plant or part thereof.

[0058] Preferably, improved pollen viability can be determined by comparing the pollen viability of the plant pollen-bearing tissue treated by the method of the present invention with the plant pollen-bearing tissue not treated by the method of the present invention, also referred to herein as the control tissue. Preferably, the treated tissue and the control tissue are of the same species, preferably of the same plant. Preferably, the treated tissue and the control tissue have a similar or the same genetic background.

[0059] Preferably, improved pollen viability may be determined by comparing the pollen viability of flower buds treated by the method of the present invention with flower buds of a control plant or part of that plant, also referred to herein as control flower buds.

[0060] A control flower bud is a flower bud equivalent or identical to the flower bud of the method of the present invention, differing only in that it has not been subjected to the method of the present invention. When the method of the present invention involves treating a part of a plant containing an immature flower bud, a part of a similar plant can serve as a control. The part of the similar plant can be of the same or similar plant, and preferably the part of the similar plant has been subjected to the same treatment as the part of the plant treated by the method of the present invention, except that the part of the similar plant has not been contacted with an MRN-ATM pathway inhibitor as described herein. Preferably, the control plant or part of the plant containing the (control) flower bud at the same developmental stage is placed in the same medium and for the same period of time as the plant or part of the plant of the method of the present invention, but without an MRN-ATM pathway inhibitor, i.e., with a zero concentration of the MRN-ATM pathway inhibitor. The similar plant or part of the plant can be a plant or part of the plant of the same species or a descendant of the same parent plant. Preferably, the part of the plant is an inflorescence. When the part of the plant in step a) is an inflorescence of a plant, a similar plant or an equivalent inflorescence of the same plant can be used as a control. Enhanced pollen viability is preferably determined as an increase in viable pollen, which may be established using an MTT assay as exemplified herein.

[0061] Preferably, the increase in viable pollen is an increase in the percentage of viable pollen, i.e., the amount of viable pollen relative to the total amount of pollen produced by the flower bud. Preferably, the increase in the percentage of viable pollen is an increase compared to the percentage of viable pollen produced by a control flower bud as defined herein. Preferably, the improved viability is at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% increase.

[0062] Additionally or alternatively, the flower buds treated by the method of the present invention preferably have an increased number of open anthers compared to a control flower bud as defined herein. The increased number of open anthers can be an increased proportion of open anthers, i.e., the number of open anthers relative to the total number of anthers produced by the flower bud is increased. Preferably, the increased proportion of open anthers is an increase compared to the proportion of open anthers produced by a control flower bud as defined herein. Preferably, the increase in the number of open anthers is at least about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.

[0063] Alternatively, or in addition, after exposure to an MRN-ATM pathway inhibitor as defined herein, preferably at least about 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the pollen produced by the plant pollen-bearing tissue is viable. Preferably, less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or less than about 1% of the pollen produced by the plant pollen-bearing tissue is non-viable.

[0064] This enhanced pollen viability can be determined by MTT assay, which indicates the metabolic activity of the pollen. Preferably, at least 0.001%, 0.01%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30% or 40% of the total amount of pollen from plant pollen-bearing tissue treated by the methods of the invention is viable, as opposed to only 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.01%, 0.01%, 0.001% or 0.0001% of the total amount of pollen from plant pollen-bearing tissue not treated by the methods of the invention.

[0065] Alternatively, or in addition, after exposure of plant pollen-bearing tissue to an MRN-ATM pathway inhibitor as defined herein, preferably at least about 2%, 4%, 6%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the formed anthers are open anthers. Preferably, less than about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 4%, 2% or less than about 1% of the formed anthers remain closed.

[0066] Preferably, the increased amount of viable pollen and / or the increased number of open anthers increases the fertility of said plant. Thus, the method of the present invention is also considered to be a method of increasing the fertility of a plant.

[0067] After or simultaneously with treating the plant or plant part, preferably the plant pollen-bearing tissue, preferably the immature flower bud, preferably the inflorescence, of step a) of the method of the invention with an MRN-ATM pathway inhibitor of the method of the invention, the method can further comprise the step of allowing said plant or plant part to develop mature pollen, such mature or ripe pollen can then be effectively used for pollination.

[0068] When the plant part treated with the MRN-ATM pathway inhibitor in the method of the present invention is separated from the rest of the plant, the treated plant part can then be grafted onto another plant part to further mature and grow into a whole plant.Preferably, the plant part treated by the method of the present invention is an inflorescence that is then grafted onto a rootstock as a scion.Preferably, the plant part is a scion.

[0069] In a preferred embodiment, the rootstock and the MRN-ATM inhibitor-treated scion are derived from the same or similar plant, preferably from the same genus and possibly from the same species. Preferably, both the rootstock and the scion are derived from the progeny plant of the same parental cross. In another embodiment, the MRN-ATM inhibitor-treated scion is derived from a different species than the plant that provides the rootstock, but the scion and the rootstock are such that they can form a viable graft. The graft can then be further developed and pollen can be collected and / or matured to a stage that can be used for subsequent pollination, preferably cross-pollination or self-pollination.

[0070] Treating a part of a plant containing immature flower buds with a compound, as exemplified herein with mirin, followed by grafting the treated part as a scion onto a rootstock, preferably a rootstock of the same genus, and then allowing the flower buds to successfully mature fully as part of the grafted plant, can be considered as a method beyond the method of the present invention for improving pollen viability. Those skilled in the art will appreciate that such a method is suitable for use in treating such a part of a plant with any compound that affects processes during the early development of the flower buds, preferably during the early emergence of pollen, and is preferably used in small amounts, for example because it is dangerous and / or harmful, or expensive or rare. In particular, (i) isolating a part of a plant containing an immature flower bud, preferably an inflorescence; (ii) contacting a part of the plant with a compound that affects a process during early flower bud development, preferably during early pollen development; (iii) grafting a portion of the plant onto a scion to form a viable scion; (iv) growing the scion into a fertile plant, preferably containing fertile pollen; The present invention provides a method for developing a fertile plant scion, comprising:

[0071] The compound is preferably a compound that affects the meiotic process in the development of the gametes of the plant, preferably in the development of pollen. Preferably, the plant is contacted with said compound during the early maturation of the flower bud, preferably during microsporogenesis including at least the pre-meiotic stage and the first meiotic division, and optionally also during the second meiotic division.

[0072] The method of the present invention may further comprise a step of isolating mature pollen. Preferably, the anthers containing mature viable pollen are open. Alternatively, closed anthers may be opened to expose the pollen. Viable pollen can be selected through various methods, including but not limited to: sieving, centrifugation or fluorescence activated cell sorting (FACS). Preferably, the method of collecting viable pollen comprises a step of discarding a fraction of non-viable pollen. Preferably, the method of collecting viable pollen comprises a step of selecting a fraction of pollen of the same or equal size as viable pollen, while discarding pollen smaller and / or larger than viable pollen, which may be non-viable.

[0073] In a further embodiment, viable mature pollen is used to pollinate the first seed plant and (self-pollination) / or the second plant (cross-pollination), and optionally isolated viable mature pollen is used. Pollination preferably results in the development of seeds. The second plant is preferably a second seed plant. The second plant can be a plant of the same species as one of the parents of the first plant. Optionally, the second plant is a parent plant of the first seed plant, i.e., a mother plant or a father plant. The second plant can be of the same species as the first plant. Pollination can be backcrossing. Optionally, the second plant is the same plant as the first plant. Pollination can be self-pollination. If the first plant is an interspecific hybrid, the second plant can be a similar interspecific hybrid, i.e., has a parent plant of the same species as the parent plant of the first plant, and optionally has the same parent plant. The pollination step using viable pollen obtained by the method of the present invention can be, but is not limited to, crossing, backcrossing and self-pollination.

[0074] The second plant preferably provides ovules for germination of a cross between the first plant and the second plant using viable pollen of the first plant obtained by the method of the invention. Preferably, the second plant is the same plant as the first plant of the method of the invention or a sibling thereof.

[0075] The method may further include the steps of seed generation and optionally the production of progeny plants.

[0076] Optionally, the method of the present invention does not include essentially biological steps for the production of a plant.

[0077] In a further aspect, the present invention provides the use of an MRN-ATM pathway inhibitor as defined herein for improving pollen viability.Preferably, said pollen is derived from an interspecific hybrid as defined herein.Preferably, said MRN-ATM pathway inhibitor is an MRN complex inhibitor, more preferably an Mre11 inhibitor, even more preferably 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone or a derivative thereof.

[0078] In a further aspect, the present invention provides viable pollen obtained by the method of the present invention provided herein. Preferably, the pollen has improved viability. Preferably, the pollen is mature pollen. Optionally, the pollen is isolated pollen, preferably isolated mature pollen. Preferably, the pollen is pollen of an interspecific hybrid. Preferably, the pollen is of an interspecific hybrid, and at least 10% of the pollen of the plant (before carrying out the method of the present invention) is non-viable. Preferably, the pollen is of an interspecific hybrid that is otherwise sterile. In other words, preferably, the pollen of the present invention is characterized by being viable or having improved viability, in contrast to the pollen of a plant, preferably an interspecific hybrid or part thereof, that has not been treated according to the method of the present invention.

[0079] Optionally, the pollen of the present invention has one or more genomic changes resulting from or associated with an MRN-ATM pathway inhibitor, preferably resulting from or associated with treatment with an MRN complex inhibitor, more preferably resulting from or associated with treatment with an Mre11 inhibitor. Preferably, the viable pollen has one or more genomic changes resulting from or associated with treatment with 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone or a derivative thereof. Optionally, the viable pollen may be characterized as having an inhibited MRN-ATM pathway, an inhibited MRN complex, and / or an inhibited Mre11 activity. Optionally, the viable pollen of the present invention is characterized as having 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone or a trace amount of 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone.

[0080] In one aspect, the invention relates to at least one of a plant, plant part, flower bud and inflorescence comprising pollen having improved viability as defined herein.

[0081] In one aspect, the present invention relates to a method for producing a first seed plant with improved pollen viability using the method steps defined herein. Preferably, the method comprises: a) providing a first seed plant or part of a plant comprising plant pollen-bearing tissue; b) inhibiting the MRN-ATM pathway in at least a portion of the plant or part thereof of step a); c) allowing the contacted tissue to produce pollen exhibiting improved viability; Includes.

[0082] When a plant part is provided in step a), the method may further comprise the step of grafting said plant part onto a rootstock as defined herein. Preferably, the plant part provided is a scion. Preferably, the plant pollen-bearing tissue is part of an immature flower bud. Preferably, the immature flower bud is part of an inflorescence. Thus, preferably, the plant part in step a) is an inflorescence.

[0083] In one aspect, the present invention relates to a seed plant comprising pollen with improved viability, wherein the seed plant is obtainable by the method of the present invention as defined herein.The seed plant can be an interspecific hybrid and / or a graft.Optionally, the seed plant of the present invention is identical to the first seed plant as defined herein, except that the pollen has improved viability.

[0084] Preferably, the seed plant comprising pollen with improved viability is not obtained essentially or exclusively by a biological process. Preferably, the seed plant of the present invention is obtained by a method comprising technical steps. Preferably, the seed plant comprising pollen with improved viability is artificial.

[0085] In one aspect, the present invention relates to seeds and / or progeny produced using pollen with improved viability, wherein the pollen is obtainable by the method of the present invention as defined herein. The pollen is used as a father. The mother used for seed production is preferably at least one of the first and second plants as defined herein.

[0086] In a further aspect, there is provided a use of the improved viability pollen of the present invention in producing progeny by pollination, preferably at least one of self-pollination and cross-pollination.

[0087] In a further aspect, the present invention relates to an agricultural carrier comprising a compound that inhibits the MRN-ATM pathway, preferably the compound is 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone or a derivative thereof. Preferably, the concentration of the compound in the agricultural carrier is about 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 900 nM, 800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 300 nM, 200 nM, 100 nM, 90 nM, 80 mM, 70 nM, 60 nM, 50 nM, 40 nM or 30 nM, and can be at least about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM or 20 nM. Preferably, the concentration is in the range of about 1 nM to 5 μM, about 1 nM to 1 μM, about 5 nM to 500 nM, about 5 nM to 100 nM, about 10 nM to 100 nM, about 5 nM to 50 nM, about 10 nM to 50 nM, or preferably about 20 nM to 30 nM. Preferred agricultural carriers are selected from the group consisting of plant growth medium, soil, fertilizer, plant-based oil and humectant. Preferably, the agricultural carrier is a plant growth medium.

[0088] The plant growth medium or plant tissue culture growth medium preferably supports plant viability and / or promotes plant growth. The plant growth medium preferably promotes plant growth, preferably promoting the development of at least one of the plant roots and the plant shoots. The plant growth medium preferably comprises at least (macro)nutrients. The plant growth medium may comprise at least one of nitrates, agar, sugars, vitamins and growth regulators. Preferred growth regulators are auxins, such as but not limited to IAA, and cytokinins, such as but not limited to kinetin. A preferred growth medium is Murashige and Skoog, as described in Murashige T. and Skoog F., supra. [Brief description of the drawings]

[0089] [Figure 1]FIG. 1 shows an outline of the experimental setup in which anthers of prepared AC hybrids were placed in glass tubes containing 0.5MS medium and appropriate concentrations of mirin. [Diagram 2] FIG. 1 shows an example of viable pollen (purple) mixed with non-viable pollen (brown). [Diagram 3] Figure 1 shows a range of different concentrations of mirin applied to immature flower buds of a B. rapa x B. oleracea hybrid. Viable pollen grains were visualized using a 2,5-diphenylmonotetrazolium bromide (MTT) assay, which stains viable pollen purple. Mirin at low doses of 20 nM resulted in a higher number of viable pollen grains. Higher concentrations resulted in larger but nonviable pollen. [Figure 4] Figure 1 shows open anthers of a B. rapa x B. oleracea cross after application of 20 nM mirin. Normally, the F1 of this cross does not form open anthers. EXAMPLES

[0090] [Example 1] Mirin application improves fertility in interspecific hybrids. We have found that treatment of inflorescences of AC interspecific hybrids from a cross between Brassica oleracea and Brassica rapa with mirin results in a higher frequency of viable pollen grains and larger viable pollen grains, which is surprising since interspecific AC hybrids between B. oleracea and B. rapa are known to be sterile.

[0091] Interspecific hybrids (AC) between Brassica oleracea var. albograbra (C genome) as the father and Brassica rapa var. albograbra (A genome) as the mother were prepared by bud pollination and embryo rescue. B. oleracea and B. rapa parental lines were grown in a greenhouse at 20-22 °C with a 12-h photoperiod. After the plants flowered, B. rapa was emasculated and artificially pollinated by B. oleracea.

[0092] Five to nine days after cross-pollination, siliques that had developed to the globular or heart-shaped embryo stage were dehisced and placed in in vitro conditions for embryo rescue. The resulting embryos were cultured in 1 mg mL -1 of 6-benzylaminopurine and 0.01 mg mL -1 The embryos were then transferred to controlled conditions with a 12-hour photoperiod and a temperature cycle of 15°C day / 10°C night. The best-developed plants were transferred to a greenhouse and grown for further experiments until inflorescences developed.

[0093] A range of concentrations of mirin was prepared in 30 ml of 0.5 Murashige and Skoog (MS) medium from a final concentration of 20 nM to 1 μM. This was added to a flat-bottom glass tube and sealed with parafilm. A small hole was made in the parafilm through which one inflorescence of the AC interspecific hybrid was placed. The inflorescence was prepared by removing all siliques, open flowers and mature flower buds, leaving only very immature buds. An overview of the setup can be seen in Figure 1. The resulting setup was placed under controlled conditions of 23 °C / 21 °C (day / night), 16H / 8H (light / dark) for 10 days. Pollen from open flowers was collected and analyzed to determine the effect of mirin on pollen viability.

[0094] Pollen viability was measured using a standardized 2,5-diphenylmonotetrazolium bromide (MTT) assay. This assay is based on mitochondrial activity, in which live cells turn a deep purple color, while non-viable cells remain unstained. Briefly, the assay uses a solution of 1% MTT in 5% sucrose in demineralized H2O (demi H2O), which is filtered to remove precipitates. This solution should be stored at 4 °C in the dark. 50 µL of MTT solution is dropped onto a microslide and a small amount of pollen, collected by opening an anther with a needle, is dispersed evenly in the MTT droplet. A glass cover slide is applied and incubated at room temperature for 5-10 min, after which staining should be visible (Figure 2). Viable pollen can be scored using a microscope.

[0095] Observation of pollen at different mirin concentrations showed that low concentrations of mirin (optimally around 20 nM) resulted in an increase in viable pollen (Figure 3). In addition, this concentration of mirin was found to induce anther opening (Figure 4). Concentrations above 0.5 μM were found to result in larger but non-viable pollen.

[0096] [Example 2] Chemically treated inflorescences are grafted and grown to full plant maturity in the greenhouse. Brassica rapa rootstock cuttings were prepared by removing all the leaves and axillary buds of the rootstock, leaving only the dominant shoot. The chemically treated young immature inflorescences obtained in Example 1 were grafted onto these rootstock cuttings. For grafting, a rootstock with a similar thickness to the scion tip was selected. Before grafting, the shoot tip or inflorescence of the rootstock was cut off using a scalpel blade (Swann Morton #10). Before grafting, a vertical incision was made in the rootstock by moving the scalpel upwards to the cut end. A silicon clip large enough to hold the rootstock was used for grafting. A wedge or T was made in the scion, with the ends removed with the scalpel blade, leaving the central area of ​​the scion intact. The wedge / T-shaped scion was placed into the vertical incision of the rootstock, and the silicon clip was moved gently but firmly to cover the grafting joint. The grafted cuttings / plants were covered with a plastic cover to ensure that the humidity was approximately 100% for at least the first few days. After one week (7-9 days), the grafts were healed. The silicone clips were removed from the graft joints. The plastic cover was removed and the chemically treated inflorescences were allowed to grow normally. This method allowed the flower buds to fully mature.

Claims

1. a) providing a first seed plant or part of the plant comprising plant pollen-producing tissue; b) inhibiting the MRN-ATM pathway in at least a portion of the provided plant or plant part; and c) causing said plant or plant part to produce pollen exhibiting improved viability.

2. 2. The method of claim 1, wherein in step b), the MRN-ATM pathway is inhibited by contacting the plant or plant part with 2-amino-5-[(4-hydroxyphenyl)methylene]-4(5H)-thiazolone, or a derivative or analog thereof.

3. 10. The method of claim 1, wherein said improved viability of plant pollen increases plant fertility.

4. 2. The method of claim 1, wherein the plant or plant part of step a) is an interspecific hybrid or part thereof.

5. 2. The method of claim 1, wherein the plant part provided in step a) is a cutting, and the cut end of the cutting is contacted with a compound that inhibits the MRN-ATM pathway in step b).

6. 6. The method of claim 5, wherein the cutting is a cutting of an angiosperm seed plant and comprises an inflorescence, the inflorescence comprising at least one immature flower bud and preferably no mature flower buds.

7. Steps a) to c) are carried out using a part of a plant that is a scion, said method comprising: d) grafting the scion onto a rootstock. The method of claim 1 further comprising:

8. The method of claim 1 further comprising the step of allowing the pollen to mature.

9. 9. The method of claim 8, further comprising isolating mature pollen.

10. 10. The method of claim 1, further comprising the step of self-pollinating the first plant or pollinating a second plant.

11. A plant growth medium containing a compound that inhibits the MRN-ATM pathway.

12. Viable pollen of a plant or plant part obtained by the method of any one of claims 1 to 10.

13. a) providing a first seed plant or part of the plant comprising plant pollen-producing tissue; b) inhibiting the MRN-ATM pathway in at least a portion of the provided plant or plant part; and c) causing the plant or part of the plant to produce pollen that exhibits improved viability.

14. 14. A seed plant, preferably an interspecific hybrid, comprising pollen with improved viability obtainable by the method according to claim 13.

15. Use of a compound that inhibits the MRN-ATM pathway, optionally included in a plant growth medium, to improve pollen viability.

16. 15. Use of viable pollen according to claim 12 or a seed plant according to claim 14 for the pollination of a second plant, preferably for the production of a progeny plant.

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