Method for producing haploid / dihaploid sunflower plants by microspore culture

Abstract

The present invention relates to methods of producing haploid, polyhaploid and / or dihaploid embryos, calluses, seeds and / or plants of Helianthus annus from isolated microspore cultures, and more particularly to methods comprising contacting isolated microspores with a histone deacetylase inhibitor (HDACi) and a complex protein composition. The invention further provides a kit for producing haploid, polyhaploid and / or dihaploid embryos, calluses, seeds and / or plants of a sunflower (Helianthus annus) from at least one isolated microspore, and a method for producing the haploid, polyhaploid and / or dihaploid embryos, calluses, seeds and / or plants of the sunflower (Helianthus annus) from the at least one isolated microspore, and the use of a histone deacetylase inhibitor (HDACi) and complex protein composition for the production of haploid, polyhaploid and / or dihaploid embryos, calluses, seeds and / or plants of Helianthus annus. Finally, the invention also relates to a population of haploid, polyhaploid and / or dihaploid sunflower plants derived directly from at least one sunflower top or lateral head inflorescence.

Description

Technical Field

[0001] The present invention relates to a method for producing haploid, polyhaploid and / or dihaploid embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) from isolated microspore cultures, and more specifically to a method comprising contacting isolated microspores with a histone deacetylase inhibitor (HDACi) and a composite protein composition. The present invention also provides a kit for producing haploid, polyhaploid and / or dihaploid embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) from at least one isolated microspore, and the use of a histone deacetylase inhibitor (HDACi) and a composite protein composition for producing haploid, polyhaploid and / or dihaploid embryos, callus, seeds and / or plants of sunflower (Helianthus annuus). Finally, the present invention also relates to a population of haploid, polyhaploid and / or dihaploid sunflower (Helianthus annuus) plants, which are directly derived from at least one sunflower head or side head, preferably from at least one heart flower of at least one sunflower head or side head. Background Art

[0002] The subspecies of the genus Helianthus, including sunflower from the species Helianthus annuus L., represent a group of large annual non-grape herbaceous plants grown as ornamentals but especially as crops for their high yields of edible oil and edible fruits. It is one of the most important oil crops in Europe (Zhou et al., 2020; Front. Plant Sci., 28 August 2020 | https: / / doi.org / 10.3389 / fpls.2020.01315).

[0003] Sunflower oil is rich in the polyunsaturated fatty acid linoleic acid (C18:2) and is very popular as a cooking oil. In addition, due to its high content of the monounsaturated fatty acid oleic acid (C18:1), it has the potential to be used as a substitute for petroleum-based products in the chemical-technical industry.

[0004] It is well known that the long-term development of inbred parental lines is one of the limiting factors and major problems of competitive F1 hybrid breeding in plant breeding. Double haploid (DH) technology provides a time-saving method to obtain pure lines (Murovec, J., & Bohanec, B. (2011). Haploids and doubled haploids in plant breeding. Plant Breeding, Dr. Ibrokhim Abdurakhmonov (Ed.). -2012.-P, 87-106.). The advantage of double haploid production is the ability to obtain completely homozygous plant genotypes in one generation, and the performance of recessive alleles in haploid plants is usually covered by the heterozygous state of diploid plants, thus promoting the identification, evaluation and selection of plants with proterties of agronomic importance.

[0005] Haploid plants can arise spontaneously, or they can develop as a result of asexual reproduction and chromosome elimination following interspecific or intergeneric hybridization. In addition, haploid plants may be generated following induction of gamete formation in microspore, anther, ovule, or ovary cultures.

[0006] However, in sunflower, even most of these methods have failed or are inefficient so far. Unfortunately, however, for breeding purposes, sunflower has proven to be very resistant to a variety of tissue cultures, including anther culture. In anther culture, for example, the culture response is strongly affected by physical, nutritional, physiological and genetic factors (Gürel et al. (1991) Shoot regeneration from anther culture of sunflower (Helianthus annuus L.) and some interspecific hybrids as affected by genotype and culture procedure. Plant Breeding 106: 68-76), and the regeneration rate is very low. It is possible to successfully generate haploid plants through anther culture, but only for wild species (Nenova et al. (2000) Anther culture regeneration from some wild Helianthus species. Helia (Yugoslavia)) or interspecific hybrids derived from crosses between H. Annuus and, for example, H. tuberosus, H. laetiflorus, H. resinosus (Nurhidayah et al., (1996) High regeneration rates in anther culture of interspecific sunflower hybrids. Plant Cell Reports, 16 (3), 167-173.; Jonard and Mezzarobba, 1990. Sunflower (Helianthus spp.): Anther culture and field studies on haploids. In Legumes and Oilseed Crops I (pp. 485-501) Springer, Berlin, Heidelberg). To date, results for anther culture of cultivated sunflower have been unsatisfactory, and as sunflower becomes an increasingly important crop plant, new culture techniques are urgently needed.

[0007] Coumans and Zhong (1995. Doubled haploid sunflower (Helianthus annuus) plant production by androgenesis: fact or artifact? Part 2. In vitro isolated microspore culture. Plant cell, tissue and organ culture, 41 (3), 203-309) studied the microspore culture of cultivated sunflower to avoid the development of anther wall and other somatic tissues. It was reported that after the addition of the ethylene precursor aminocyclopropanecarboxylic acid, the viability and initial division rate of the microspore response increased, and sustained division and microcallus formation were achieved. The only cell population had a multi-hairy structure that developed into callus. However, the efficient production of doubled haploid plants could not be achieved by this approach at all.

[0008] An alternative method for producing haploids in Helianthus spp. species is parthenogenesis, which allows the rapid generation of double haploid lines in sunflower. Todorova et al. (1997. Doubled haploid production of sunflower (Helianthus annuus L.) through irradiated pollen-induced parthenogenesis. Euphytica, 97 (3), 249-254) used irradiated pollen-induced parthenogenesis to obtain many fertile and agronomically useful DH lines resistant to downy mildew. However, the efficiency of this method is very dependent on the female genotype and pollen donor.

[0009] Therefore, there is currently a great need to establish and provide an efficient sunflower cultivation system allowing the production of haploid and double haploid sunflower (Helianthus annuus) plants, preferably in a genotype-independent manner. Summary of the invention

[0010] As described in the Background section, there is a great need in the art to identify techniques for efficiently producing haploid and doubled haploid sunflower (Helianthus annuus) plants, and to use this understanding to develop new methods and kits for producing such plants. The present invention satisfies this need and other needs.

[0011] In one aspect, the present invention relates to a method for producing haploid, multihaploid and / or dihaploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus), the method comprising:

[0012] i) providing isolated microspores of at least one sunflower (Helianthus annuus) plant,

[0013] ii) culturing at least one microspore from step i) in the presence of the complex protein composition,

[0014] iii) culturing at least one microspore from step ii) in the presence of at least one histone deacetylase inhibitor (HDACi) to induce and thereby obtain callus or embryos,

[0015] iv) culturing the callus or embryos, and

[0016] v) optionally, regenerating and obtaining therefrom at least one plant from said callus or embryo of step iv), and preferably obtaining at least one haploid, multihaploid and / or dihaploid seed thereof.

[0017] In a preferred embodiment, step ii) and step iii) are applied to induce and thereby obtain callus or embryos.

[0018] In one embodiment of the above aspect, the following steps are performed before providing isolated microspores of at least one sunflower (Helianthus annuus) plant according to step i) of the above aspect:

[0019] (a) providing at least one inflorescence head or side inflorescence head of a sunflower (Helianthus annuus) plant,

[0020] (b) harvesting at least one heart flower from at least one top inflorescence or side inflorescence to obtain heart flower material; and

[0021] (c) optionally: sterilizing the surface of the flower material;

[0022] (d) optionally: washing the sterilized heart flower material;

[0023] (e) transferring the optionally sterilized and washed cardamom material to a separation medium containing macro- and micro-salts and sugars and preferably a complex protein composition, and

[0024] (f) homogenizing and optionally sieving the inflorescence material to provide isolated microspores of at least one sunflower (Helianthus annuus) plant.

[0025] In a preferred embodiment, the complex protein composition in step (e) is the same as in step ii).

[0026] In another embodiment of the above aspect, the method further comprises the following steps within step ii):

[0027] (b1) culturing at least one flower of the genus C. or a part thereof in the presence of at least one amino acid, such as glutamine, and / or in the presence of at least one nucleoside, such as uracil and cytosine, at a concentration of about 10 mg / L to about 800 mg / L, preferably about 20 mg / L to about 750 mg / L.

[0028] In yet another embodiment of the above aspect, the method further comprises the following steps in step iii):

[0029] (iiia) adding at least one histone deacetylase inhibitor (HDACi) to a final concentration of 1 nM to 10 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM; and

[0030] (iiib) removing the at least one histone deacetylase inhibitor (HDACi) after incubation for at least about 1 h, preferably at least about 5 h or more preferably at least about 12 h and / or at least up to about 48 h, preferably at least up to about 50 h or more preferably at least up to about 60 h, wherein the incubation is optionally carried out in the dark.

[0031] According to one embodiment, an incubation step in the dark may be included, particularly during steps ii) to iv), particularly when at least one HDACi and / or complex protein composition is applied to give sufficient incubation time. This step may be performed at room temperature or above, for example at about 20°C to 30°C, preferably at about 25°C or 26°C. After washing and allowing to settle in new culture medium, a new standing and incubation step may be included, preferably in the dark and at about 25°C or 26°C.

[0032] In another embodiment of the above aspects, at least one histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), dagnostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228) and / or the composite protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk such as casein or whey, animals such as meat or fish, cereals such as rice or corn, plants such as soy, or combinations thereof, and / or the composite protein composition comprises or consists of: a hydrolyzed milk protein isolate; , hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or completely skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof, and / or the composite protein composition comprises or consists of a protein hydrolyzate selected from the group consisting of casein hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and a combination thereof, preferably with a degree of hydrolysis ranging from about 20 to about 80%, preferably from about 30 to about 80%, particularly preferably from about 40 to about 60%, optionally wherein the composite protein composition is provided together with activated carbon.

[0033] In yet another embodiment of the above aspects, at least one histone deacetylase inhibitor (HDACi) is present in a concentration of about 1 nM to about 10 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM in the culture medium used in step ii), and / or wherein the complex protein composition is present in a concentration of about 100 to about 20,000 mg / L, preferably about 500 to about 15,000 mg / L, particularly preferably about 2,000 to about 10,000 mg / L in the culture medium used in step ii) and / or iii), and / or wherein the complex protein composition is present in a concentration of about 100 to about 20,000 mg / L, preferably about 500 to about 15,000 mg / L, particularly preferably about 2,000 to about 10,000 mg / L in the separation medium used in step (e).

[0034] In one embodiment of the above described aspects, in step iii) and / or iv) and / or v), in step iii), at least one microspore during callus induction, or a callus or embryo producing at least one microspore, is contacted with one or more plant growth regulators selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins, and abscisic acid and mixtures thereof.

[0035] In another embodiment of the above described aspects, one or more chromosome doubling agents such as colchicine, amisulalin and / or trifluralin are added during step iii) and / or step iv) and / or step v).

[0036] A second aspect relates to a kit for producing haploid, multihaploid and / or dihaploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) from at least one isolated microspore, comprising:

[0037] (a) at least one histone deacetylase inhibitor (HDACi); and

[0038] (b) a complex protein composition; and

[0039] (c) optional additional ingredients, including at least one plant growth regulator:

[0040] Wherein the at least one histone deacetylase inhibitor (HDACi) and the complex protein composition are contained in the same container or in two or more separate containers.

[0041] In one embodiment of the kit, at least one histone deacetylase inhibitor (HDACi) is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), dagnostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228) and / or the composite protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk such as casein or whey, animal For example, meat or fish, cereals such as rice or corn, plants such as soybeans, or combinations thereof, and / or the complex protein composition comprises or consists of: hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolyzate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or completely skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or combinations thereof, and / or the complex protein composition comprises or consists of a protein hydrolyzate selected from: casein White hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and combinations thereof, preferably with a hydrolysis degree ranging from about 20 to about 80%, preferably from about 30 to about 80%, particularly preferably from about 40 to about 60%; and, in certain embodiments, wherein the kit further comprises one or more plant growth regulators, the plant growth regulators being selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxy Acetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins, and mixtures thereof, and / or the kit further comprises one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin.

[0042] Yet another aspect relates to the use of a histone deacetylase inhibitor (HDACi) and a complex protein composition, preferably as defined in the first aspect, or the use of a kit as defined in the second aspect above, preferably for use in a method according to the first aspect for producing haploid, multihaploid and / or double haploid embryos, callus and / or plants or seeds of sunflower (Helianthus annuus).

[0043] The final aspect relates to a population of haploid, polyhaploid and / or dihaploid plants of sunflower (Helianthus annuus), directly derived from at least one sunflower head or side head, preferably derived from at least one heart flower of at least one sunflower head or side head, more preferably derived from a single heart flower, preferably obtained or obtainable by a method according to the first aspect above, preferably wherein the population comprises at least 10 individuals.

[0044] definition

[0045] Androgenesis is defined as the process of generating individuals whose genetic background is derived entirely from nuclei of male origin. In other words, androgenesis refers to the generation of plants entirely from male haploid gamete precursors (gametophytes).

[0046] Haploidy is a property of cells or plants or plant parts that contain only one copy (n) of each chromosome in their nucleus.

[0047] Diploidy is a property applicable to cells or plants or plant parts whose nuclei contain two copies (2n) of each chromosome.

[0048] Doubled haploidy is a property applicable to cells, or plants or plant parts comprising such cells, whose chromosome stock is artificially multiplied, most often by chemical treatment, for example with a chromosome doubling agent, including in particular antimitotic agents such as colchicine, amosulfuron and / or trifluralin, or by spontaneous doubling. Such doubling of the chromosome stock makes it possible to obtain cells, plants or plant parts having two copies (2n) of each chromosome in their nucleus, wherein the cells, plants or plant parts are completely homozygous or substantially homozygous.

[0049] Polyhaploidy is a property applicable to cells, or plants or plant parts comprising such cells, which were originally haploid and whose chromosome stocks spontaneously increased by three or more times. Said cells, plants or plant parts have at least three copies (3n or 4n etc.) of each chromosome in their nucleus, wherein said cells, plants or plant parts are completely homozygous or substantially homozygous.

[0050] The term microspore is used herein to refer to the immature male gametophyte of a plant at all stages of its in vitro growth, including its multicellular form derived from the division of the sporophyte of a single-celled isolated microspore and still enclosed in the original outer cell wall (this multicellular form is also referred to herein as a multicellular structure). In the uninucleate development stage of the microspore, the nucleus has not yet divided. Tetrad refers to microspores arranged in groups of four after a diploid cell undergoes meiosis to form four haploid microspores.

[0051] In the context of the present disclosure, a complex protein composition refers to a mixture of proteins derived from milk, meat, fish, cereals or soy. Preferably, the complex protein composition comprises or consists of at least partially hydrolyzed proteins. In particular, the complex protein composition can be a protein hydrolyzate, i.e. a mixture of proteins obtained by enzymatic hydrolysis catalyzed by a protease. The degree of hydrolysis is the percentage of cleaved peptide bonds relative to the total number of bonds available for protein hydrolysis. Examples of complex protein compositions are given in the detailed description.

[0052] Histone deacetylase is an enzyme that removes acetyl from acetyl lysine at the N-terminus of histones to increase the affinity of histones to DNA. Therefore, histone deacetylase plays a role in the regulation of DNA expression. Inhibitors of histone deacetylase (HDACi) inhibit histone deacetylase and therefore prevent the removal of acetyl from histones. Histone deacetylase inhibitors are used to treat several diseases.

[0053] The MS can be selected from ammonium nitrate (NH 4 NO 3 ), calcium chloride (CaCl 2× 2H 2 O), magnesium sulfate (MgSO 4 ×7H 2 O), monopotassium phosphate (KH 2 PO 4 ), dipotassium phosphate (K 2 HPO 4 ), potassium nitrate (KNO 3 ), calcium nitrate (Ca(NO 3 ) 2 ×4H 2 O); the micro salt can be selected from boric acid (H 3 BO 3 ), cobalt chloride (CoCl 2 ×6H 2 O), ferrous sulfate (FeSO 4 ×7H 2 O), manganese(II) sulfate (MnSO 4 ×4H 2 O), potassium iodide (KI), sodium molybdate (Na2 MoO 4 ×2H 2 O), zinc sulfate (ZnSO 4× 7H 2 O), sodium ferric ethylenediaminetetraacetate (FeNaEDTA), copper sulfate (CuSO 4 ×5H 2 O), see, for example, Murashige T. and Skoog F., Physiol. Plant, 15, 473 (1962).

[0054] Plant growth regulators are compounds that affect plant growth and development, for example by promoting or inhibiting growth. Natural plant growth regulators are plant hormones produced by the plants themselves. On the other hand, synthetic plant growth regulators do not occur naturally in plants. Examples of plant growth regulators are given in the detailed description.

[0055] Haploid plants may undergo spontaneous chromosome doubling or chromosome doubling may be enhanced or promoted by a chromosome doubling agent that blocks the function of spindle fibers, for example, during meiosis or mitosis. The most commonly used chromosome doubling agent is colchicine or another anti-mitotic agent such as amosulfuron or trifluralin.

[0056] The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Furthermore, in describing exemplary embodiments, specific terminology will be employed for the sake of clarity.

[0057] It must also be noted that the singular forms "a", "an", and "the" used in the specification and the appended claims include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended to include compositions of multiple components. Reference to a composition containing "an" ingredient is intended to include other ingredients in addition to the specified ingredient. In other words, the terms "a", "an", and "the" do not indicate a limitation of quantity, but rather indicate the presence of "at least one" of the referenced item.

[0058] As used herein, the term "and / or" may mean "and," "or," "exclusive-or," "one," "some but not all," "neither," and / or "both." The term "or" is intended to mean an inclusive "or."

[0059] To facilitate understanding of the principles and features of various embodiments of the present invention, various illustrative embodiments are explained below. Although exemplary embodiments of the present invention are explained in detail, it should be understood that other embodiments are contemplated. Therefore, it is not intended that the scope of the present invention be limited to the details of the subject matter set forth in the following description or examples and the arrangement of the components.

[0060] In addition, when describing exemplary embodiments, terms will be used for clarity. Each term is intended to cover its broadest meaning understood by those skilled in the art and includes all technical equivalents that operate in a similar manner to achieve similar purposes. It should be understood that the embodiments of the disclosed technology can be practiced without these specific details. In other examples, well-known methods, structures and techniques are not shown in detail so as not to obscure the understanding of this specification. References to "one embodiment", "embodiment", "exemplary embodiment", "some embodiments", "certain embodiments", "multiple embodiments" and the like indicate that the embodiments of the disclosed technology described in this way may include specific features, structures or characteristics, but not every embodiment must include the specific features, structures or characteristics. In addition, the repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may refer to the same embodiment.

[0061] As used herein, the term "about" should be interpreted as referring to two numbers designated as the endpoints of any range. Any reference to a range should be deemed to provide support for any subset within the range. The range can be expressed herein as from "about" or "approximately" or "substantially" a specific value and / or to "about", "approximately" or "substantially" another specific value. When expressing such a range, other exemplary embodiments include from a specific value and / or to another specific value. In addition, the term "about" means within the acceptable error range of a specific value determined by one of ordinary technicians in the art, which will depend in part on how to measure or determine the value, i.e., the limitation of the measurement system. For example, according to the practice of the art, "about" can mean within an acceptable standard deviation. Alternatively, "about" can mean a range of up to ±20% of a given value, preferably up to ±10%, more preferably up to ±5%, and also more preferably up to ±1%. Alternatively, particularly for biological systems or processes, the term can be expressed within an order of magnitude of a numerical value, preferably within 2 times. Where particular values ​​are described in the application and claims, unless otherwise stated, the term "about" is implicit and means herein within an acceptable error range for the particular value.

[0062] Throughout this disclosure, aspects of the present invention can be presented in the form of ranges. It should be understood that the description of range form is only for convenience and simplicity and should not be interpreted as an inflexible limitation to the scope of the present invention. Therefore, the description of the scope should be considered to specifically disclose all possible sub-ranges and the single numerical values ​​in the range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the single numerals in the range are such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. It is applicable regardless of the width of the scope.

[0063] “Comprising” or “containing” or “including” means that at least the specified compound, element, particle or method step is present in the composition, article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if other such compounds, materials, particles, method steps have the same function as specified.

[0064] Throughout the specification, various components with specific values ​​or parameters may be identified, however, these items are provided as exemplary embodiments. In fact, exemplary embodiments do not limit the various aspects and concepts of the present invention, as many comparable parameters, sizes, ranges and / or values ​​may be achieved. The terms "first", "second", etc., "primary", "secondary", etc. do not indicate any order, quantity or importance, but are used to distinguish one element from another.

[0065] It should be noted that terms such as "particularly," "preferably," "typically," "generally," and "frequently" are not used herein to limit the scope of the claimed invention or to imply that certain features are critical, necessary, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention. It should also be noted that terms such as "substantially" and "about" are utilized herein to represent the inherent uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation.

[0066] The dimensions and values ​​disclosed herein should not be understood as being strictly limited to the exact values ​​recited. Instead, unless otherwise specified, each such dimension is intended to represent the recited value and a functionally equivalent range around that value. For example, a dimension disclosed as "50 mm" means "about 50 mm".

[0067] It is also to be understood that the reference to one or more process steps does not exclude the presence of additional process steps or intermediate process steps between those steps specifically identified. Similarly, it is also to be understood that the reference to one or more components in a composition does not exclude the presence of additional components other than those specifically identified.

[0068] The materials that constitute the various elements of the present invention described below are intended to be illustrative and not restrictive. It is intended that many suitable materials that perform the same or similar functions as the materials described herein are included within the scope of the present invention. Such other materials not described herein may include, but are not limited to, materials developed after the time of developing the present invention, for example. Any dimensions listed in multiple drawings are only for illustrative purposes and are not intended to be limiting. Other dimensions and ratios are also considered and intended to be included within the scope of the present invention.

[0069] In accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art may be employed. Such techniques are fully explained in the literature. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (this article "Sambrook et al., 1989"); DNA Cloning: APractical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed.1984); Nucleic Acid Hybridization (BDHames&S.J.Higginseds.(1985); Transcription and Translation (BDHames&S.J.Higgins,eds.(1984)); Animal Cell Culture (RIFreshney,ed.(1986); Immobilized Cells and Enzymes (IRLPress, (1986)); B.Perbal, A Practical Guide To Molecular Cloning (1984); FMAusubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 Plant material for harvesting is shown: A. Head morphology of sunflower (Helianthus annuus); B. Heart flower appearance and size; C. Isolated heart flower.

[0071] Figure 2 The development from sunflower microspores to callus structures is shown, from microspore nuclear divisions (A) to multinucleate cells (B) and callus structures (C).

[0072] Figure 3 The development from sunflower microspores to callus structures is shown.

[0073] Figure 4 (A, B, and C) show shoot induction (arrows) from sunflower microspore-derived callus.

[0074] Figure 5 Shown are: A. a microspore-derived sunflower plant after in vitro development; B. a microspore-derived sunflower plant at the flowering stage after previous in vitro development as described herein.

[0075] Figure 6 Shown are flow cytometry bar graph results from diploid control donor material (see Example 6).

[0076] Figure 7 Shown are flow cytometry bar graph results from plant material derived from microspores after spontaneous doubling, ie, doubled haploid (DH) lines generated based on the methods disclosed herein (see Example 6 for details).

[0077] Figure 8 (A and B) shows the effect of certain 2,4D-analogs on callus number ( Fig. 8A ) and regeneration ( Figure 8B )’s impact.

[0078] Fig. 9 Microspore division and callus formation are shown, where several 2,4-D concentrations on the 007 genotype were tested as detailed in Example 4.3. Top row: Arrows indicate where the embryos are located (magnification). Bottom row: corresponding petri dishes.

[0079] Fig.10 Shown is a microspore-derived sunflower with root formation after prior in vitro development as described herein, in particular as detailed in Example 5.2 below.

[0080] FIG. 11 shows a doubled haploid sunflower plant obtained using the method disclosed herein ( Fig.11A Plants were fertile and pollen was readily observed on anthers and pistils ( Fig. 11B and Example 7). DETAILED DESCRIPTION

[0081] As described in the Background section above, there is an urgent need in the art to identify techniques for effectively producing haploid and dihaploid sunflower (H. annuus) plants, in order to use these techniques to develop new methods and kits for producing such plants in a more economical, versatile and faster manner. The present invention meets this need and other needs. Embodiments of the present invention generally relate to methods and kits for producing haploid, polyhaploid and / or dihaploid sunflower (Helianthus annuus) plants, particularly in a genotype-independent manner, by isolated microspore cultures, and more specifically to methods and kits for producing such sunflower (Helianthus annuus) plants comprising contacting isolated microspores with an inhibitor of histone deacetylase (HDACi) and a composite protein composition.

[0082] In a first aspect, the present invention provides a method for producing haploid, multihaploid and / or dihaploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus), wherein the method may comprise:

[0083] i) providing isolated microspores of at least one sunflower (Helianthus annuus) plant,

[0084] ii) culturing at least one microspore from step i) in the presence of the complex protein composition,

[0085] iii) culturing at least one microspore from step ii) in the presence of at least one histone deacetylase inhibitor (HDACi) to induce and thereby obtain callus or embryos including microcallus,

[0086] iv) culturing the callus or embryos, and

[0087] v) optionally, regenerating and obtaining therefrom at least one plant from said callus or embryo of step iv), and preferably obtaining at least one haploid, multihaploid and / or double haploid seed thereof.

[0088] The method according to the invention provides the advantage that hundreds of haploid or double haploid plants can be obtained from one heart or inflorescence of at least one sunflower head or side head, and using tissue culture, at most several haploid plants can be obtained. Another advantage is that haploid plants obtained by microspore culture appear to have a much higher spontaneous doubling rate than plants obtained from ovule culture. Therefore, most of the plants obtained by the method according to the invention can be used directly for breeding without the need to apply chromosome doubling agents.

[0089] As used herein, the term "callus" may also refer to microcallus or structures, or portions thereof.

[0090] Therefore, the method provided herein starts from the initial step of isolating living plant material, preferably isolating living plant material from the top or side head of sunflower (Helianthus) as starting material. Then, the first critical step is to prepare and especially cultivate the material under suitable reaction conditions and in a suitable medium supplemented with certain ingredients to allow microspore propagation to obtain callus or embryonic structure that can be further cultivated.

[0091] In a preferred embodiment, step ii) and step iii) are applied to induce and thereby obtain callus or embryos.

[0092] In one embodiment of the above aspect, the following steps may be performed before providing isolated microspores of at least one sunflower (Helianthus annuus) plant according to step i) of the above aspect:

[0093] (a) providing at least one inflorescence head or side inflorescence head of a sunflower (Helianthus annuus) plant,

[0094] (b) harvesting at least one heartflower or part thereof from the at least one top inflorescence or side inflorescence to obtain heartflower material; and

[0095] (c) optionally: sterilizing the surface of the flower material;

[0096] (d) optionally: washing the sterilized heart flower material;

[0097] (e) transferring the optionally sterilized and washed cardamom material to a separation medium containing macro- and micro-salts and sugars and preferably a complex protein composition, and

[0098] (f) homogenizing and optionally sieving the inflorescence material to provide isolated microspores of at least one sunflower (Helianthus annuus) plant.

[0099] In a preferred embodiment, the complex protein composition in step (e) is the same as in step ii).

[0100] In some embodiments, the heart flower material can be directly stored for further processing before or after disinfection and / or washing, preferably after harvesting. In some embodiments, this may be beneficial to give the material a period of recovery to increase the yield of viable microspores. The storage of the heart flower material can be completed at room temperature (meaning a temperature between about 18°C ​​to about 25°C), at a temperature above 28°C, or at a chilling temperature or freezing temperature. In a preferred embodiment, the heart flower material, including the heart flower or part thereof, is removed from a sunflower (Helianthus annuus) plant. The removed heart flower or part thereof can be stored at a moderate freezing temperature (e.g., at about 2°C to about 10°C, or at about 4°C to 8°C) or a lower freezing temperature (e.g., below 2°C). The formation of ice crystals should be avoided to ensure the integrity of the cell structure. Freezing temperature can be applied during all steps before step (f).

[0101] In a preferred embodiment of the method described above, an initial cell suspension is obtained at any stage after harvesting in step (b) and during step (b) and before step (e), and the cell suspension obtained by applying a separation buffer or separation medium (or a solubilization step during the previous step (b) and optional steps (c) and (d)) can be purified or the cells can be further separated by passing through a filter during step (b) or (e), followed by passing through a cell filter having a pore size ranging from about 10 to 200 μm, preferably 20 to 100 μm, such as a nylon mesh, and through a cell filter having a pore size of about 20 to 80 μm, preferably 25 to 50 μm. Preferably, passing through each filter with a different pore size is performed at least 1 to 3 times or exactly 1 to 3 times. Alternatively, the cell suspension can be purified by passing through a filter in step (b) or (e) repeatedly through a cell filter having a pore size of about 25 to 100 μm, preferably at least 1 to 6 times or exactly 1 to 6 times.

[0102] In one embodiment, after harvesting in step (b) above, in step (e), the early propagation material is transferred to a suitable separation buffer or separation medium, wherein the separation medium may include one or more amino acids such as glutamine and / or nucleosides such as uracil and cytosine. Each of these additional components may be present at a concentration of about 20 to 750 mg / L.

[0103] In certain embodiments, in step (e), the sugar is a monosaccharide such as glucose monohydrate (dextrose), fructose, or galactose, which may be present in a concentration of 20 to 750 mg / L.

[0104] In one embodiment, a method may therefore be provided, the method further comprising the following steps between steps (b) and (f):

[0105] (b1) culturing at least one flower of the genus C. or a part thereof in the presence of at least one amino acid, such as glutamine, and / or in the presence of at least one nucleoside, such as uracil and cytosine, at a concentration of about 10 mg / L to about 800 mg / L, preferably about 20 mg / L to about 750 mg / L.

[0106] In some embodiments of the methods described above, prior to regeneration and towards the end of callus / embryo induction during step iv), the structure of the callus or embryo may be disrupted by applying physical force, for example the callus or embryo may be broken into pieces, crushed or pounded.

[0107] In yet another embodiment of the above aspect, the method may further comprise the following steps within step iii):

[0108] (iiia) adding at least one histone deacetylase inhibitor (HDACi) to a final concentration of 1 nM to 10 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM; and

[0109] (iiib) removing the at least one histone deacetylase inhibitor (HDACi) after incubation for at least about 1 h, preferably at least about 5 h or more preferably at least about 12 h and / or at least up to about 48 h, preferably at least up to about 50 or more preferably at least up to about 60 h, wherein the incubation is optionally carried out in the dark.

[0110] In another embodiment of the above aspects, at least one histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), danositol (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0111] In yet another embodiment, a complex protein composition can be used, which comprises or consists of a hydrolyzed or partially hydrolyzed protein material, the protein material being derived from milk, such as casein or whey, animals, such as meat or fish, cereals, such as rice or corn, plants, such as soy, or a combination thereof, and / or the complex protein composition comprises or consists of: hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolyzate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or completely skimmed milk , hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof, and / or the composite protein composition comprises or consists of a protein hydrolyzate, the protein hydrolyzate being selected from: casein hydrolyzate, soy hydrolyzate, rice hydrolyzate, potato protein hydrolyzate, fish protein hydrolyzate, egg white protein hydrolyzate, lactalbumin hydrolyzate, gluten hydrolyzate, animal and plant protein hydrolyzate and a combination thereof, preferably with a degree of hydrolysis ranging from about 20 to about 80%, preferably from about 30 to about 80%, particularly preferably from about 40 to about 60%, optionally, wherein the composite protein composition is provided together with activated carbon.

[0112] During the isolation / early culture step ii) and / or additionally or optionally during step b), when the microspores are cultured in an induction medium, at least in the first culture period, the complex protein composition may be used for callus or embryo induction in step iii). In certain embodiments, the complex protein composition will only be a complex protein composition that can be used during the isolation / early culture step and in an early induction medium for microspore differentiation and development (e.g., induction medium I), but not in a late differentiation step toward callus and / or embryonic tissue (e.g., induction medium II).

[0113] In certain embodiments, and depending in particular on the length of tissue culture and the tissue culture system, at least one antibiotic such as carbenicillin, penicillin or timentin may be added to incubation medium I and / or incubation medium II during steps ii) and / or iii) and / or iv).

[0114] The complex protein composition may be obtained from any protein source such as milk, meat, fish, cereals or other plants. In particular, good results were obtained with a protein composition derived from milk and subjected to at least partial hydrolysis.

[0115] In any embodiment of the method described above, the complex protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk, such as casein or whey, animals, such as meat or fish, cereals, such as rice or corn, plants, such as soy, or a combination thereof.

[0116] Preferably, in any embodiment of the method described above, the complex protein composition comprises or consists of: hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or fully skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof.

[0117] Particularly preferably, in any embodiment of the method described above, the complex protein composition comprises or consists of a protein hydrolysate selected from the group consisting of casein hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and combinations thereof.

[0118] Further preferably, in the complex protein composition, the degree of hydrolysis is in the range of about 20 to about 80%, preferably about 30 to about 80%, particularly preferably about 40 to about 60%.

[0119] Further preferably, the complex protein composition is applied together with activated charcoal in the culture medium or buffer to stabilize and activate the mixture and support callus or embryo induction and / or regeneration of plants or plant tissues or seeds from callus or embryos.

[0120] In one embodiment of the method according to any of the embodiments described above, the at least one histone deacetylase inhibitor (HDACi) is present in the culture medium used in step iii) at a concentration of about 1 nM to about 10 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM.

[0121] In another embodiment of the method according to any of the embodiments described above, the complex protein composition is present in a concentration of about 100 to about 20,000 mg / L, preferably about 500 to about 15,000 mg / L, particularly preferably about 2,000 to about 10,000 mg / L in the culture medium used in step ii) and / or iii), and / or wherein the complex protein composition is present in a concentration of about 100 to about 20,000 mg / L, preferably about 500 to about 15,000 mg / L, particularly preferably about 2,000 to about 10,000 mg / L in the separation medium used in step (e).

[0122] The method according to any one of the preceding claims, wherein in step iii) and / or iv) and / or v), in step iii), the at least one microspore during callus induction or the callus or embryo producing the at least one microspore is contacted with one or more plant growth regulators selected from the group consisting of auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), parachlorophenoxyacetic acid (PAA), phenoxyacetic acid (PHAA ... Phenoxyacetic acid (4-CPA or 4CIPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins, and abscisic acid, and mixtures thereof, wherein in certain embodiments, plant growth regulators and mixtures thereof are applied in steps iii) and / or iv). In certain embodiments, the concentration of 2,4-D or any auxin or any synthetic auxin analog and / or BAP or any other cytokinin used may be about 0.01 mg / mL to 10 mg / mL, preferably about 0.05 mg / mL to about 3 mg / mL.

[0123] In certain embodiments, particularly for rooting and rooting media, PAA and / or IBA, for example, are typically applied at a concentration of about 0.001 to 10 mg / L, preferably about 0.01 to about 3 mg / mL, more preferably about 0.05 to about 2 mg / mL, even more preferably about 0.05 to about 0.1 mg / mL.

[0124] In a preferred embodiment, at least one of 4CIPA and / or 3.5ME and / or PHAA is used as a plant growth regulator in the method disclosed herein during callus induction and cultivation according to steps iii) and iv) of the method according to the first aspect disclosed herein, because the inventors surprisingly found that these compounds can advantageously increase callus formation and regeneration when used during callus induction.

[0125] In yet another embodiment, at least one auxin, synthetic auxin or auxin analogue such as PAA and / or IBA is used as a plant growth regulator in step iv), preferably step (v) of the method of the first aspect to enhance rooting of the plant, thereby promoting plant regeneration. In view of the fact that callus induced using the methods described herein is quite viable, rooting will also be effective when no plant growth regulator is added as a rooting enhancer during or before the regeneration step (see Example 5.2 below).

[0126] Depending on the regeneration protocol, buffers and media without growth regulators may be advantageous to allow efficient and natural plant regeneration.

[0127] In another embodiment of the above described aspects, one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin are added during step iii) and / or step iv) and / or step v). The chromosome doubling agent can be applied before, together with or after the HDACi of interest.

[0128] In another embodiment, spontaneous chromosome doubling can be induced.

[0129] In one embodiment of the method described above, the plant population regenerates in step v) undergoes spontaneous chromosome doubling at a rate of at least 40%, at least 50%, at least 60% or at least 70%. In this context, the population comprises at least 10 individuals.

[0130] In certain embodiments of the methods disclosed herein, the method is used to increase the rate of spontaneous chromosome doubling according to any of the embodiments described above, wherein the callus or embryo is contacted with one or more plant growth regulators in step iii) and / or iv) and / or v), the plant growth regulator being selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins, and abscisic acid, and mixtures thereof. This may be of interest when spontaneous chromosome doubling is of interest. In certain embodiments, the callus or embryo is contacted with one or more plant growth regulators during steps iii) and / or iv), but not during them or not during the entire step v) of regeneration, selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins, and abscisic acid and mixtures thereof.

[0131] The doubled haploid plants obtained by the method described above are fertile and can be used directly for further breeding with other plants as maternal or paternal crossing partners, preferably for developing DH lines / DH groups or for hybrid crossing.

[0132] These chromosome doubling steps using techniques known in the art produce doubled haploid material from the haploid plants, cells, tissues, organs or materials or seeds obtained according to the method of the invention, wherein this material is useful for accelerating plant breeding.

[0133] Another aspect relates to a kit for producing haploid, multihaploid and / or dihaploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) from at least one isolated microspore, comprising:

[0134] (a) at least one histone deacetylase inhibitor (HDACi); and

[0135] (b) a complex protein composition; and

[0136] (c) optionally further components, including at least one plant growth regulator and / or one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin;

[0137] Wherein the at least one histone deacetylase inhibitor (HDACi) and the complex protein composition are contained in the same container or in two or more separate containers.

[0138] In one embodiment of the kit, the at least one histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), dagnostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228) and / or the composite protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk such as casein or whey, animal For example, meat or fish, cereals such as rice or corn, plants such as soybeans, or combinations thereof, and / or the complex protein composition comprises or consists of: hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolyzate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or completely skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or combinations thereof, and / or the complex protein composition comprises or consists of a protein hydrolyzate selected from: casein White hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and combinations thereof, preferably with a degree of hydrolysis ranging from about 20 to about 80%, preferably from about 30 to about 80%, particularly preferably from about 40 to about 60%; optionally wherein the kit further comprises one or more plant growth regulators, the plant growth regulators being selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME ), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins and abscisic acid and mixtures thereof, and / or wherein the kit further comprises one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin.

[0139] In one embodiment, the kit comprises a first composition and a second composition, wherein the first composition comprises an HDACi and the second composition comprises a composite protein composition. In one embodiment, the first and second compositions are culture media for plant cell culture. In one embodiment, the first composition is in a first container and the second composition is in a second container. In another embodiment, the kit comprises a composition comprising an HDACi and a composite protein composition. In one embodiment, the composition is a culture media for plant cell culture. The kit may comprise a set of instructions for using the HDACi and / or the composite protein composition. Either or both of the HDACi and the composite protein composition may be in concentrated form and require dilution before use.

[0140] In one embodiment of the kit according to any of the embodiments described above, the at least one histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), danositol (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0141] Removing the HDACi before culturing to obtain callus can result in particularly efficient callus formation. Removing the HDACi can be performed by the following steps: centrifugation, washing the microspores treated with the HDACi, or replacing the culture medium with the same culture medium without the HDACi or a different culture medium without the HDACi, or any combination of these steps. Such steps of removing the HDACi can be applied once, repeatedly or several times before and / or during callus induction. The same steps are applicable to removing at least one plant growth regulator from one step (with plant growth regulator) to another step (without plant growth regulator).

[0142] Various HDACi are known to the person skilled in the art. In particular good results have been obtained with TSA or romidepsin.

[0143] Yet another aspect relates to the use of a histone deacetylase inhibitor (HDACi), preferably as defined in the first aspect, and a complex protein composition, preferably as defined in the first aspect, or the use of a kit as defined in the above second aspect, preferably in a method according to the first aspect, for producing haploid, multihaploid and / or double haploid embryos, callus and / or plants or seeds of sunflower (Helianthus annuus).

[0144] In the plants obtained according to the present invention, chromosome doubling may occur spontaneously, or may be promoted, enhanced or strengthened by a chromosome doubling agent.

[0145] In one embodiment of the method for preparing microspores and / or for producing haploid, multihaploid and / or double haploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) and / or as part of a kit according to any aspect and embodiment described above, the at least one histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), dagnostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0146] In another embodiment of the kit according to any of the embodiments described above, the complex protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk, such as casein or whey, animals, such as meat or fish, cereals, such as rice or corn, plants, such as soy, or a combination thereof.

[0147] Preferably, the complex protein composition comprises or consists of hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or fully skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof.

[0148] Particularly preferably, the complex protein composition comprises or consists of a protein hydrolysate selected from the group consisting of casein hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and combinations thereof.

[0149] In one embodiment, activated carbon may be provided with at least one container of the kit, or as a separate container for use with another container of the kit.

[0150] In one embodiment, the degree of hydrolysis ranges from 20 to 80%, preferably from 30 to 80%, particularly preferably from 40 to 60%.

[0151] In another embodiment of the kit according to any of the embodiments described above, the kit further comprises one or more plant growth regulators selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins, and abscisic acid, and mixtures thereof.

[0152] The one or more plant growth regulators may be contained in the same container as the HDACi and / or the complex protein composition or in a separate container.

[0153] The invention also relates to the use of a method or kit according to the invention for producing haploid, polyhaploid and / or dihaploid plants of sunflower (Helianthus annuus) from isolated microspores by androgenesis. Schemes from the prior art are described in the background section above, e.g. Coumans and Zhong (1995) supra, incorporated herein by reference.

[0154] The present invention also relates to the use of HDACi and complex protein compositions for producing haploid, multihaploid and / or double haploid embryos and / or plants of sunflower (Helianthus annuus) from isolated microspores through androgenesis.

[0155] In one embodiment, the present invention relates to the use of a histone deacetylase inhibitor (HDACi) and a composite protein composition or kit as defined in any of the embodiments described above for producing haploid, multihaploid and / or double haploid embryos, callus and / or plants or seeds of sunflower (Helianthusannuus).

[0156] Preferably, the histone deacetylase inhibitor (HDACi) and the complex protein composition or kit is used in the method according to any one of the embodiments described above at least during step iii).

[0157] In one embodiment of the use described above, the histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), danositol (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0158] In a preferred embodiment of the use described above, the degree of hydrolysis of the complex protein composition ranges from 20 to 80%, preferably from 30 to 80%, particularly preferably from 40 to 60%.

[0159] A final aspect relates to a population of haploid, polyhaploid and / or dihaploid plants of sunflower (Helianthus annuus) directly derived from a monocarpic flower, preferably obtained or obtainable by a method according to the first aspect above, preferably wherein the population comprises at least 10 individuals.

[0160] Advantageously, the method of the invention allows obtaining a large number of haploid, polyhaploid and / or dihaploid sunflower (Helianthus annuus) plants from a monocarpal flower or a single inflorescence, whereas using the ovule culture method, up to four plants can be obtained from one flower.

[0161] In one embodiment of the population described above, the population comprises at least 10 individuals, preferably at least 50 individuals, more preferably at least 100 individuals.

[0162] Another advantage is that the plants obtained by the method of the present invention show a spontaneous chromosome doubling rate of more than 50%, while the plants obtained by ovule culture only show a maximum of 5% spontaneous doubling. Therefore, most of the plants obtained can be used directly for breeding purposes without applying chromosome doubling agents.

[0163] In another embodiment of the population according to any of the above embodiments, the population comprises at least 40%, at least 50%, at least 60% or at least 70% doubled haploid plants.

[0164] The present invention also relates to the use of a histone deacetylase inhibitor (HDACi) and a complex protein composition or a kit as defined in any of the embodiments described above for increasing the spontaneous chromosome doubling rate in a population of sunflower (Helianthus annuus) plants derived from a single flower or a single inflorescence and obtained by microspore culture.

[0165] In a preferred embodiment of the uses described above, the histone deacetylase inhibitor (HDACi) and the complex protein composition or kit defined in any of the embodiments described above are used in a method for producing haploid, multi-haploid and / or double haploid embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) according to any of the embodiments described above.

[0166] In one embodiment of the use, the histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamic esters such as vorinostat (SAHA), belinostat (PXD101), danositol (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0167] In certain embodiments, activated carbon may be provided with the composite protein compositions used herein.

[0168] In another embodiment of the use according to any of the embodiments described above, the complex protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk, such as casein or whey, animals, such as meat or fish, cereals, such as rice or corn, plants, such as soy, or a combination thereof.

[0169] Preferably, the complex protein composition comprises or consists of hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or fully skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof.

[0170] Particularly preferably, the complex protein composition comprises or consists of a protein hydrolysate selected from the group consisting of casein hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and combinations thereof.

[0171] In a preferred embodiment of the use described above, the degree of hydrolysis of the complex protein composition ranges from 20 to 80%, preferably from 30 to 80%, particularly preferably from 40 to 60%.

[0172] The invention will now be further illustrated by the following non-limiting examples.

[0173] Example

[0174] Example 1: Harvesting and Plant Material Preparation

[0175] As an initial harvesting step, remove the sunflower head ( Figure 1 A). Keep the intact flower heads in a germination box with a moistened filter paper at the bottom and covered on top. Store the harvested material at 4°C for no longer than 14 days until further processing.

[0176] Remove the side flowers and use a scalpel to collect the central flowers between 4 mm and 6 mm (see Figure 1 B and 1C). The heart flowers are combined in a plastic cup for further processing. During the whole process, the material is kept under suitable temperature conditions.

[0177] Example 2: Surface disinfection of plant material

[0178] Surface sterilization was performed under a clean bench. 96% ethanol was added to each plastic cup containing the heart flower for a few seconds, then washed with sterile water. 3% NaOCl was added for about half an hour, then washed several times with sterile water. The plant material was transferred to a plastic bag, a suitable isolation buffer (see, e.g., Table 1) was added, and the bag was sealed. The bag was kept under suitable conditions until further processing.

[0179] Table 1

[0180]

[0181] Example 3: Microspore Isolation

[0182] The heart flowers in the bag are then homogenized. After a further homogenization step, additional separation medium is added directly to the bag. The obtained material is poured through a nylon sieve (pore size of about 25 μm to 100 μm) and washed with more separation medium. The solution containing the microspores is filled into a tube. The tube is centrifuged at 200g for 10 min.

[0183] The supernatant was decanted and enough separation medium was added to disturb the precipitation. The solutions from all tubes were combined and poured through a nylon mesh (20 μm to 100 μm) several times. The microspores retained on the mesh were washed off the mesh with separation buffer.

[0184] Example 4: Callus induction

[0185] Example 4.1: TSA and colchicine

[0186] The solution of 80,000 microspores / ml with microspore density was centrifuged at 200g for 10min, and the supernatant was poured out. Inducing medium I (see, for example, Table 2) is added to the remaining microspores and distributed in a plastic tube. Histone deacetylase inhibitor (HDACi) and colchicine are added to each tube, and the microspore solution is stored at 26°C for 2 days. If HDACi trichostatin A (TSA) is used, it is dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1nM to 10μM, preferably 100nM to 10μM, more preferably 1μM to 10μM, depending on genotype and plant donor source. If colchicine is used, it is dissolved in water to a final concentration of 1mg to 500mg, preferably 10mg to 200mg, more preferably 50mg to 100mg, depending on genotype and plant donor source. HDACi and chromosome doubling agent are removed by centrifugation at 200g for 10 minutes. Pour off the supernatant, add the corresponding amount of induction medium I, and resuspend the microspores. Incubate the tube at 26 °C in the dark for several days.

[0187] Table 2

[0188]

[0189]

[0190] Thereafter, the microspore solution was centrifuged at 200 g for 10 min, and the supernatant was decanted. A corresponding amount of induction medium II (see, for example, Table 3.1) was added, and the microspore solution was inoculated into a petri dish and placed at 26° C. for callus induction.

[0191] Table 3.1

[0192]

[0193] Depending on the length of tissue culture and the tissue culture system, at least one antibiotic such as carbenicillin, penicillin or timentin may be added to medium II. One to two weeks after isolation, the first microspore nuclear divisions can be observed (DAPI staining) ( Figure 2 About three weeks after separation, the callus structure can be seen with the naked eye ( Figure 3 ).

[0194] Example 4.2: Callus Induction - Auxin Analogues

[0195] The solution with a microspore density of 80,000 microspores / ml was centrifuged at 200g for 10min, and the supernatant was poured out. Induction medium I (see, for example, Table 3.2) was added to the remaining microspores and distributed in a plastic tube. Histone deacetylase inhibitor (HDACi) and colchicine were added to each tube, and the microspore solution was stored at 26°C for 2 days. If HDACi trichostatin A (TSA) is used, it is dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1nM to 10μM, preferably 100nM to 10μM, more preferably 1μM to 10μM, depending on genotype and plant donor source. If colchicine is used, it is dissolved in water to a final concentration of 1mg to 300mg, preferably 10mg to 200mg, more preferably 50mg to 100mg, depending on genotype and plant donor source. Another antimitotic agent may also be used, such as amosulfuron and / or trifluralin, or any other chromosome doubling agent. HDACi and chromosome doubling agents are removed by centrifugation at 200 g for 10 min. The supernatant is poured out, a corresponding amount of induction medium I is added, and the microspores are resuspended. Typically, auxin or its (synthetic) analog is typically applied at a concentration of about 0.05 to about 3 mg / L. The tube is incubated in the dark at 26 ° C for several days.

[0196] Table 3.2

[0197]

[0198]

[0199] Thereafter, the microspore solution was centrifuged at 200 g for 10 min and the supernatant was decanted. According to the treatment, the corresponding amount of induction medium II with different auxins and auxin analogs (see, for example, Table 3.3) was added, and the microspore solution was plated in a Petri dish and placed at 26°C for callus induction. Abbreviations used in Table 3.3: 3.5ME = 3,5-dimethylphenoxyacetic acid; PHAA = phenoxyacetic acid; 4-CPA or 4CIPA = p-chlorophenoxyacetic acid.

[0200] Table 3.3

[0201]

[0202]

[0203] Depending on the length of tissue culture and the tissue culture system, at least one antibiotic such as carbenicillin, penicillin or timentin may be added to medium II. One to two weeks after separation, the first microspore nuclear division can be observed (DAPI staining). After about three weeks of separation, callus structures can be seen with the naked eye.

[0204] Auxin analogs were titrated at different concentrations as described above.

[0205] This experiment surprisingly showed that the use of different auxins, synthetic auxins and auxin analogs can induce microspore division and callus formation from different genotypes. The different treatments had no negative impact on callus formation, and the hormone 4CIPA / 4-CPA showed a trend to increase callus number over a wider range of concentrations ( Fig. 8A ),in Fig. 8A The data shown exemplarily in the Figures were generated at a concentration of 1 mg / L. It was also demonstrated that the use of hormones in the callus and embryo induction steps had an effect on further plant regeneration ( Figure 8B ). Analysis of the number of shoots regenerated per callus unit showed that the auxin analogs 3.5ME and 4CIPA / 4-CPA showed a tendency to increase regeneration ( Figure 8B ).

[0206] Example 4.3: Callus Induction - Different 2,4-D Concentrations

[0207] The solution with a microspore density of 80,000 microspores / ml was centrifuged at 200g for 10min, and the supernatant was poured out. Induction medium I (see, for example, Table 3.4) was added to the remaining microspores and distributed in a plastic tube. Histone deacetylase inhibitor (HDACi) and colchicine were added to each tube, and the microspore solution was stored at 26°C for 2 days. If HDACi trichostatin A (TSA) is used, it is dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1nM to 10μM, preferably 100nM to 10μM, more preferably 1μM to 10μM, depending on genotype and plant donor source. If colchicine is used, it is dissolved in water to a final concentration of 1mg to 500mg, preferably 10mg to 200mg, more preferably 50mg to 100mg, depending on genotype and plant donor source. HDACi and chromosome doubling agent are removed by centrifugation at 200g for 10min. Pour off the supernatant, add the corresponding amount of induction medium I, and resuspend the microspores. Incubate the tube at 26 °C in the dark for several days.

[0208] Table 3.4

[0209]

[0210]

[0211] Thereafter, the microspore solution was centrifuged at 200 g for 10 min, and the supernatant was decanted. According to the treatment, the corresponding amount of induction medium II with different 2,4-D concentrations (see, for example, Table 3.5) was added, and the microspore solution was inoculated in a petri dish and placed at 26° C. for callus induction.

[0212] Table 3.5

[0213]

[0214] Depending on the length of tissue culture and the tissue culture system, at least one antibiotic such as carbenicillin, penicillin or timentin may be added to medium II. One to two weeks after separation, the first microspore nuclear division can be observed (DAPI staining). After about three weeks of separation, callus structures can be seen with the naked eye.

[0215] This experiment showed that we were able to induce microspore division and callus formation at several 2,4-D concentrations. Callus formation was not reduced or enhanced by the different treatments, and the generation of microspore-derived embryos was also observed in all treatments as well as in the control ( Fig. 9 ).

[0216] Example 5: Plant Regeneration

[0217] Example 5.1: First step of regeneration

[0218] Calli were removed from liquid culture and transferred to regeneration medium containing 1 mg / L BAP (see, e.g., Table 4) and grown at 26°C in the light until plant regeneration was observed approximately 2 weeks after transfer ( Figure 4 ).

[0219] Table 4:

[0220]

[0221] like Figure 5 A and Figure 5 This is documented by the photograph shown in B. Here, the in vitro cultured microspores according to the present invention give rise to living sunflower plants which can be further analyzed and cultivated.

[0222] Example 5.2: Plant rooting

[0223] Shoots were transferred to growth / rooting medium (see, e.g., Table 5) with or without an auxin (PAA (phenylacetic acid) or IBA was used in the examples) and grown at 24°C in the light until plant regeneration was observed approximately 2 weeks after transfer ( Fig.10 ). In the control medium, 35% of the cultivated plants showed root formation, while 68% of the plants grown in 0.5 IBA medium showed root formation. Interestingly, 56% of the plants were able to form roots in the hormone-free medium.

[0224] Table 5:

[0225] Rooting medium (MS) mg / L Comparison 0.5IBA No hormones <![CDATA[KNO 3 ]]> 800 800 800 <![CDATA[MgSO 4 ]]> 90 90 90 <![CDATA[NH 4 NO 3 ]]> 85 85 85 <![CDATA[KH 2 AFTER 4 ]]> 42.5 42.5 42.5 <![CDATA[H 3 BO 3 ]]> 3.1 3.1 3.1 <![CDATA[MnSO 4 ×7H 2 The]]> 8.45 8.45 8.45 <![CDATA[Na 2 MoO 4 ×2H 2 O]]> 0.125 0.125 0.125 <![CDATA[CoCl 2 ×6H 2 The]]> 0.0125 0.0125 0.0125 <![CDATA[CuSO 4 ×5H 2 The]]> 0.0125 0.0125 0.0125 <![CDATA[ZnSO 4 ×2H 2 The]]> 4.3 4.3 4.3 KI 0.415 0.415 0.415 FaNaEDTA 18.35 18.35 18.35 Myo-inositol 50 50 50 <![CDATA[CaCl 2 ×2H 2 The]]> 220 220 220 Thiamine Hydrochloride 0.05 0.05 0.05 niacin 0.25 0.25 0.25 Pyridoxine Hydrochloride 0.25 0.25 0.25 Glycine 1 1 1 PAA 0.5 - - IBA - 0.5 - sucrose 30000 30000 30000 Agar 12000 12000 12000

[0226] Example 6: Plant ploidy testing via flow cytometry

[0227] To check the degree of ploidy of plant materials obtained in the experiments detailed in Example 5.1 above, a flow cytometry-based ploidy check was performed. The protocol used was similar to that of Noga et al. (Noga, A., et al. Conversion of oat (Avena sativa L.) haploid embryos into plants in relation to embryodevelopmental stage and regeneration media. In Vitro Cell. Dev. Biol. -Plant 52, 590-597 (2016). https: / / doi.org / 10.1007 / s11627-016-9788-z ) is comparable to the protocol described for oats (Avena sativa L.).

[0228] Flow cytometry for sunflower analysis was performed as follows: Leaf samples were manually cut with a scalpel and cut into small pieces. The cut material was added to a tube containing a combined extraction and staining solution, Cystain UV ploidy (Sysmex). The solution was then filtered and placed in a 96-well plate for measurement. The analyzer 10 measures DAPI emission under 40 s excitation at a maximum of 461 nm.

[0229] Figure 6 and Figure 7 Exemplary results are provided showing the dispersal pattern of a control diploid donor plant compared to plants produced according to the invention as a source of microspores for a spontaneously doubled haploid (DH) line. Figure 6 and Figure 7 The two patterns shown in are comparable and demonstrate the same ploidy hierarchy. Thus, the results confirm that the method of the present invention is fully suitable for generating DH sunflower lines via the microspore route in a convenient and reliable manner.

[0230] Example 7: Transfer of plants to greenhouse and seed production

[0231] Plants that were fully eradicated in vitro were transferred to soil and transferred to a mist chamber at 90% relative air humidity, 16-18 h day length, and 25° C. After 21 days, the humidity was reduced to 60%. After 7 days, the plants were finally grown in a chamber at 50% relative air humidity, 16-18 h day length, and 25° C. Doubled haploid plants of sunflower were fertile, and pollen could be easily observed on anthers and pistils ( Fig.11A and B). Before flowering, the sunflower head was covered with a plastic bag to ensure self-pollination. The seeds were harvested 6-10 weeks after the plants were transferred to the greenhouse. These experiments confirm that the new method established produces live sunflowers in a fast and convenient manner.

[0232] Although several possible embodiments are disclosed above, embodiments of the present invention are not limited thereto. These exemplary embodiments are not intended to be exhaustive or to limit the scope of the present invention unnecessarily, but are selected and described to explain the principles of the present invention so that other technical personnel in the art can practice the present invention. In fact, according to the foregoing description, various modifications of the present invention other than those described herein will become apparent to those skilled in the art. Such modifications are intended to fall within the scope of the appended claims. In addition, the terms used herein are only used for the purpose of describing exemplary embodiments, and the terms are not intended to be limited, because the scope of the various embodiments of the present invention will be limited only by the appended claims and their equivalents. Therefore, the scope of the present invention is indicated by the appended claims rather than the foregoing description and the embodiments discussed above, and all changes falling within the meaning and scope of its equivalents are intended to be included therein.

[0233] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present in this specification.

Claims

1. A method for producing haploid, polyhaploid and / or dihaploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus), the method comprising: i) providing isolated microspores of at least one sunflower (Helianthus annuus) plant, ii) culturing the at least one microspore from step i) in the presence of the complex protein composition, iii) culturing the at least one microspore from step ii) in the presence of at least one histone deacetylase inhibitor (HDACi) to induce and thereby obtain callus or embryos, iv) culturing the callus and / or embryos, and v) optionally, regenerating and obtaining therefrom at least one plant from said callus or embryo of step iv), and preferably obtaining at least one haploid, multihaploid and / or double haploid seed thereof.

2. The method of claim 1, wherein the steps of: (a) providing at least one inflorescence head or side inflorescence head of a sunflower (Helianthus annuus) plant, (b) harvesting at least one heartflower or part thereof from the at least one top inflorescence or side inflorescence to obtain heartflower material; as well as (c) optionally: sterilizing the surface of the flower material; (d) optionally: washing the sterilized heart flower material; (e) transferring the optionally sterilized and washed radish material to a separation medium containing macro- and micro-salts, sugars and a complex protein composition, and (f) homogenizing and optionally sieving the sunflower material to provide isolated microspores of at least one sunflower (Helianthus annuus) plant.

3. The method of claim 1 or 2, further comprising the following steps in step ii): (b1) culturing at least one flower of the genus Pinnatifida or a part thereof in the presence of at least one amino acid, such as glutamine, and / or in the presence of at least one nucleoside, such as uracil and cytosine, at a concentration of about 10 mg / L to about 800 mg / L, preferably about 20 mg / L to about 750 mg / L.

4. The method according to any one of the preceding claims, further comprising the following steps within step iii): (iiia) adding at least one histone deacetylase inhibitor (HDACi) to a final concentration of 1 nM to 10 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM; and (iiib) removing the at least one histone deacetylase inhibitor (HDACi) after incubation for about 1 h to 60 h, preferably 5 h to 50 h, more preferably 12 h to 48 h, wherein the incubation is optionally performed in the dark.

5. The method of any of the preceding claims, wherein the at least one histone deacetylase inhibitor (HDACi) is selected from: trichostatin A (TSA), hydroxamic acids and hydroxamates such as vorinostat (SAHA), belinostat (PXD101), danositol (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994) and mosetinol (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

6. The method according to any one of the preceding claims, wherein the complex protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk, such as casein or whey, animals, such as meat or fish, cereals, such as rice or corn, plants, such as soy, or a combination thereof, and / or the complex protein composition comprises or consists of: Hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or completely skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof, and / or the composite protein composition comprises or consists of a protein hydrolyzate selected from the group consisting of casein hydrolysate, soy hydrolysate, rice hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates and combinations thereof, preferably with a degree of hydrolysis ranging from about 20 to about 80%, preferably from about 30 to about 80%, particularly preferably from about 40 to about 60%, optionally wherein the composite protein composition is provided together with activated carbon.

7. The method according to any of the preceding claims, wherein the at least one histone deacetylase inhibitor (HDACi) is present in a concentration of about 1 nM to about 10 μM, preferably 100 nM to 10 μM, more preferably 1 μM to 10 μM in the culture medium used in step iii) and / or wherein the complex protein composition is present in a concentration of about 100 to about 20,000 mg / L, preferably about 500 to about 15,000 mg / L, particularly preferably about 2,000 to about 10,000 mg / L in the culture medium used in step ii) or iii).

8. A method according to any one of the preceding claims, wherein In step iii) and / or iv) and / or v), in step iii), the at least one microspore during callus induction or the callus or embryo producing the at least one microspore is contacted with one or more plant growth regulators, the plant growth regulators being selected from: auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA); cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidiazuron (TDZ), gibberellins and abscisic acid, and mixtures thereof.

9. The method according to any of the preceding claims, wherein one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin are added during step iii) and / or step iv) and / or step v).

10. A kit for producing haploid, multihaploid and / or dihaploid cells, embryos, callus, seeds and / or plants of sunflower (Helianthus annuus) from at least one isolated microspore, comprising: (a) at least one histone deacetylase inhibitor (HDACi); and (b) a complex protein composition; and (c) optionally further components, including at least one plant growth regulator and / or one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin; Wherein the at least one histone deacetylase inhibitor (HDACi) and the complex protein composition are contained in the same container or in two or more separate containers.

11. The kit of claim 10, wherein the at least one histone deacetylase inhibitor (HDACi) is selected from the group consisting of: trichostatin A (TSA), hydroxamic acids and hydroxamates such as vorinostat (SAHA), belinostat (PXD101), danositol (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides such as trapoxin B and depsipeptides such as romidepsin (FK228), benzamides such as entinostat (MS-275), acetyldinaline (CI994), and mosetinot (MGCD0103), electrophilic ketones and fatty acid compounds such as phenylbutyric acid and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

12. The kit of claim 10 or 11, wherein the complex protein composition comprises or consists of a hydrolyzed or partially hydrolyzed protein material derived from milk, such as casein or whey, animals, such as meat or fish, cereals, such as rice or corn, plants, such as soy, or a combination thereof, and / or the complex protein composition comprises or consists of: Hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolyzate, hydrolyzed lactalbumin, hydrolyzed sodium caseinate, hydrolyzed calcium caseinate, hydrolyzed whole milk, partially or completely skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, or a combination thereof, and / or the composite protein composition comprises or consists of a protein hydrolyzate selected from the group consisting of casein hydrolyzate, soy hydrolyzate, rice hydrolyzate, potato protein hydrolyzate, fish protein hydrolyzate, egg white protein hydrolyzate, lactalbumin hydrolyzate, gluten hydrolyzate, animal and plant protein hydrolyzate and a combination thereof, preferably with a degree of hydrolysis ranging from 20 to 80%, preferably from 30 to 80%, particularly preferably from 40 to 60%; Optionally, the kit further comprises one or more plant growth regulators selected from the group consisting of auxins, synthetic auxins or auxin analogs, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3.5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid, naphthylacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP) and thiadiazolam (TDZ), gibberellins and abscisic acid and mixtures thereof and / or the kit further comprises one or more chromosome doubling agents such as colchicine, amosulfuron and / or trifluralin.

13. Use of a histone deacetylase inhibitor (HDACi) and a complex protein composition, or a kit as defined in any one of claims 10 to 12, for producing haploid, multihaploid and / or double haploid embryos, callus and / or plants or seeds of sunflower (Helianthus annuus), preferably in a method according to any one of claims 1 to 9, wherein the histone deacetylase inhibitor (HDACi) is preferably a histone deacetylase inhibitor (HDACi) as defined in claim 5, and the complex protein composition is preferably a complex protein composition as defined in claim 6.

14. A population of haploid, polyhaploid and / or dihaploid plants of sunflower (Helianthus annuus) directly derived from monocarpic flowers preferably obtained or obtainable by a method according to any one of claims 1 to 9.

15. A population of haploid, multihaploid and / or double haploid plants of sunflower (Helianthus annuus) according to claim 14, wherein said population comprises at least 10 individuals.

Images