Method for the production of haploid and dihaploid sunflower plants by double-layer system microspore culture

Abstract

The present invention relates to a method for producing haploid, polyploid and / or doubled haploid embryos, calli, seeds and / or plants of the species Helianthus annuus from isolated microspores, more particularly to a method comprising contacting isolated microspores with a histone deacetylase inhibitor (HDACi) and / or a histone methyltransferase inhibitor (HMTi) and a complex protein composition and culturing on a double layer system. The present invention also provides a kit for producing said haploid, polyploid and / or doubled haploid embryos, calli, seeds and / or plants. Finally, the present invention also relates to a population of haploid, polyploid and / or doubled haploid sunflower plants directly derived from at least one sunflower terminal or lateral inflorescence. (Helianthus annuus) ​

Description

Technical Field

[0001] This invention relates to the production of isolated microspore cultures Sunflower (Helianthus annuus) Methods for producing haploid, multi-haploid, and / or double-haploid embryos, calluses, seeds, and / or plants of a species, more specifically, relate to a method comprising contacting isolated microspores with a histone deacetylase inhibitor (HDACi) and / or a histone methyltransferase inhibitor (HMTi) and a complex protein composition, wherein the culture is carried out on a bilayer system. Kits for producing said haploid, multi-haploid, and / or double-haploid embryos, calluses, seeds, and / or plants are also provided. Finally, the invention also relates to haploid, multi-haploid, and / or double-haploid sunflowers directly derived from at least one terminal or lateral capitulum of a sunflower ( Helianthus annuus) The plant population is preferably derived from at least one tubular floret of at least one sunflower terminal or lateral capitulum. Background Technology

[0002] Sunflower genus ( Helianthus Subspecies of ) including those from the species sunflower ( Helianthus annuus L.) Sunflowers, representing a group of large perennial weeds grown as ornamental plants, are also cultivated as crops, particularly due to their high yields of edible oil and fruit. It is one of the most important oilseed 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 (C60-32 ... 18 :2), and it is very popular as an edible oil. Furthermore, oleic acid (C :2), a monounsaturated fatty acid, is... 18 :1) When the content is high, it can be used as a substitute for petroleum-based products in the chemical technology industry.

[0004] It is generally known that long-term development of inbred parent lines is a limiting factor in plant breeding and a major problem in the breeding of competitive F1 hybrids. Double haploid (DH) technology provides a time-saving method for obtaining pure breeding lines (Murovec, J., & Bohanec, B. (2011)). Haploids and doubled haploids in plant breeding. Plant Breeding, Dr. Ibrokhim Abdurakhmonov (Ed.). –2012. –Р(87-106.). The advantage of double haploid production is that it can achieve completely homozygous plant genotypes in one generation and that recessive alleles that are usually masked by the heterozygous state of diploid plants are expressed in haploid plants, thereby promoting the identification, evaluation and selection of plants with important agricultural characteristics.

[0005] Haploid plants can occur spontaneously, or they can be obtained through apomixis and chromosome elimination following interspecific or intergeneric hybridization. Furthermore, haploid plants can be generated after inducing gametogenesis in microspore, anther, ovule, or ovary cultures. Additionally, double haploid (DH) technology provides a time-saving method for obtaining pure breeding lines, reducing the cycle to approximately 3-5 years. The advantages of double haploid production include the ability to achieve completely homozygous plant genotypes in one generation and the manifestation of recessive alleles typically masked by the heterozygous state of diploid plants in haploid plants, thereby facilitating the identification, evaluation, and selection of plants with agriculturally important characteristics.

[0006] However, even these methods are currently unsuccessful or inefficient in sunflowers. Unfortunately, in breeding, sunflowers have proven to be very insensitive to various tissue cultures, including anther culture. In anther culture, for example, the culture response is strongly influenced 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. Successful production of haploid plants can be achieved through anther culture, but only in wild species (Nenova et al. (2000) Anther culture regeneration from some wild Helianthus species). Helia (Yugoslavia) Or made from ordinary sunflowers ( H. annuus ) with, for example, Jerusalem artichoke ( H. tuberosus ), vibrant sunflowers ( H. laetiflorus) Resin sunflower ( H. resinosusInterspecific hybrids produced by crossbreeding (Nurhidayah et al., (1996) High regenerationrates in anther culture of interspecific sunflower hybrids). Plant Cell Reports , 16 (3), 167-173.; Jonard and Mezzarobba, 1990. Sunflower (Helianthusspp.): Anther culture and field studies on haploids. In Legumes and Oilseed Crops I (pp. 485-501) Springer, Berlin, Heidelberg). To date, the results of anther culture of cultivated sunflowers have been unsatisfactory, and new cultivation techniques are urgently needed as sunflowers become an increasingly important crop.

[0007] Coumans and Zhong (1995. Doubled haploid sunflower (Helianthus annuus)plant production by androgenesis: fact or artifact? Part 2. In vitro isolatedmicrospore culture. Plant cell, tissue and organ culture , 41 (3), 203-309) investigated the culture of microspores in cultivated sunflowers to avoid the development of anther walls and other somatic tissues. It was reported that the survival rate and initial division rate of microspores increased upon the addition of aminocyclopropanecarboxylic acid, an ethylene precursor, and that continuous division and microcallus formation were achieved. Only the trichomes of cells developed into callus. However, efficient production of double haploid plants could not be achieved through this pathway.

[0008] An alternative method for haploid production in sunflowers is parthenogenesis, which allows for the rapid production of double haploid lines. Todorova et al. (1997. Doubled haploid production of sunflower (Helianthus annuus L.) through irradiated pollen-induced parthenogenesis.) Euphytica , 97(3), 249-254) Several agriculturally valuable fertile and downy mildew-resistant DH lines were obtained by using radiation-induced parthenogenesis. However, the efficiency of this method is highly dependent on the female genotype and pollen donor.

[0009] Given the specific nature of sunflower microspore development, it is difficult to define suitable culture conditions that provide an optimal environment for the in vitro development of fragile cultured organisms.

[0010] Therefore, there is a need to establish and provide an efficient and stable cultivation system for sunflowers, enabling the production of haploid and double-haploid sunflowers. Helianthus annuus) Plants, preferably in a genotype-independent manner. Summary of the Invention

[0011] As specified in the background section, there are in the art requirements for the efficient production of haploids and double haploids. Towards Sunflower There is a significant demand for plant technology to identify and utilize this understanding to develop novel methods and kits for producing such plants. This invention addresses this and other needs by providing a targeted combination of suitable culture techniques for stabilizing sunflower microspore cultures using a bilayer system, allowing for high yields of callus and embryo regeneration from sensitive microspore cultures, and combining this approach with the provision of specifically optimized culture media and chemicals during the culture steps. This combined approach results in a favorable high ratio of haploid and double-haploid plants, thereby accelerating plant breeding using an increasing number of relevant sunflower plants.

[0012] On one hand, the present invention relates to a method for producing sunflowers ( Helianthus annuus A method for producing haploid, polyhaploid, and / or double haploid cells, embryos, callus, seeds, and / or plants of a species, said method comprising: i) Provide sunflowers ( Helianthus annuus At least one isolated microspore of the plant; ii) Optionally: pre-culture at least one microspore from step i) in the presence of the complex protein composition; iii) In the presence of at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi), and optionally in the presence of a complex protein composition, culture at least one microspore from step i) or ii) in a bilayer system to induce and thus obtain callus or embryo. iv) Culture the callus or embryo; v) Optionally, at least one plant is regenerated and thereby obtained from the callus or embryo of step iv), and preferably at least one haploid, multiple haploid and / or double haploid seed is obtained, preferably wherein activated carbon is added during steps (i), (ii) and / or (iii).

[0013] In a preferred embodiment, steps ii) and iii) are both applied to induce and thus obtain callus or embryo.

[0014] In one embodiment of the above aspects, the following steps are performed before providing at least one isolated microspore of the sunflower plant according to step i) of the above aspects: (a) Provide at least one terminal or lateral capitulum of a sunflower plant; (b) Harvesting at least one tubular floret from at least one terminal or lateral capitulum to obtain tubular floret material; and (c) Optionally: disinfect the surface of the tubular flower material; (d) Optionally: Wash the disinfected tubular flower material; (e) The optionally sterilized and washed tubular flower material is transferred to an isolation medium containing macro- and micro-element salts and sugars, and preferably containing a complex protein composition. (f) Homogenize and optionally sieve the tubular flower material to provide at least one isolated microspore of the sunflower plant.

[0015] In a preferred embodiment, the complex protein composition in step (e) is the same as that in step (ii).

[0016] In another embodiment of the above aspects, the method further includes the following steps in step i) and / or ii): (b1) In the presence of at least one amino acid, such as glutamine, and / or in the presence of at least one nucleoside, such as uridine and cytidine, at a concentration of about 10 mg / L to about 800 mg / L, preferably about 20 mg / L to about 750 mg / L, at a concentration of about 10 mg / L to about 800 mg / L; In yet another embodiment of the above aspects, the method further includes the following step in step iii): (iii.a) Add at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) to a final concentration of 1 nM-10 μM, preferably 100 nM-10 μM, more preferably 1 μM-10 μM; (iii.b) After incubation for at least about 1 hour, preferably at least about 5 hours or more preferably at least about 12 hours and / or incubation for at least up to about 48 hours, preferably at least up to about 50 hours or more preferably at least up to about 60 hours, the at least histone deacetylase inhibitor (HDACi) and / or the at least histone methyltransferase inhibitor (HMTi) are removed, wherein the incubation is optionally carried out in the dark.

[0017] According to one embodiment, an incubation step in the dark may be included, particularly during steps ii)-iv), especially when at least one HDACi and / or at least one HMTi and / or complex protein composition is applied to provide sufficient incubation time. This step may be performed at room temperature or higher, for example, at about 20°C to 30°C, preferably at about 25°C or 26°C. After washing, a new settling and culturing step may be included to adapt to the new culture medium, preferably in the dark and at about 25°C or 26°C.

[0018] In another embodiment of the foregoing, the at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) is selected from: trichostatin A (TSA), hydroxamic acid and hydroxamic acid salts such as vorinostat (SAHA), belistat (PXD101), dacanitia (LAQ824) and palbociclib (LBH589), cyclic tetrapeptides such as tryptophan B and desipeptides such as romidesin (FK228), benzamides such as entenstatin (MS-275), tacidalin (CI994) and moxidinat (MGCD0103), BIX129 4. BIX1338, electrophilic ketones, and aliphatic acid compounds such as phenylbutyric acid and glutaric acid, preferably wherein the histone deacetylase inhibitor (HDACi) and / or the histone methyltransferase inhibitor (HMTi) is trachostatin A (TSA), BIX1294, BIX1338, SAHA, or romidesin (FK228), or a combination thereof, wherein the concentration of at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) in the culture medium used in step iii) is from about 1 nM to about 10 μM, preferably from 100 nM to 10 μM, more preferably from 1 μM to 10 μM. μM, and / or the concentration of the complex protein composition in the culture medium used in step ii) or iii) is 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, and / or the complex protein composition comprises or consists of hydrolyzed or partially hydrolyzed protein substances derived from milk such as casein or whey, animals such as meat or fish, grains such as rice or corn, plants such as soybeans, and combinations 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, The complex protein composition comprises hydrolyzed sodium casein, hydrolyzed calcium casein, hydrolyzed whole milk, partially or completely skimmed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, and combinations thereof, and / or the complex protein composition comprises or consists of protein hydrolysates selected from the group consisting of casein hydrolysates, soy hydrolysates, rice protein hydrolysates, potato protein hydrolysates, fish protein hydrolysates, ovalbumin hydrolysates, lactalbumin hydrolysates, gliadin hydrolysates, 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 complex protein composition is provided together with activated carbon.

[0019] In another embodiment of the foregoing aspects, the concentration of at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) in the culture medium used in step ii) is from about 1 nM to about 10 μM, preferably from 100 nM to 10 μM, more preferably from 1 μM to 10 μM, and / or wherein the concentration of the complex protein composition in the culture medium used in steps ii) and / or iii) is from about 100 to about 20,000 mg / L, preferably from about 500 to about 15,000 mg / L, particularly preferably from about 2,000 to about 10,000 mg / L, and / or wherein the concentration of the complex protein composition in the separation culture medium used in step (e) is from about 100 to about 20,000 mg / L, preferably from about 500 to about 15,000 mg / L, particularly preferably from about 2,000 to about 10,000 mg / L.

[0020] In one embodiment of the above aspects, in step iii), the at least one microspore during callus induction, or the callus or embryo obtained from the 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-dichlorophthalic acid (dicyclopentadiene), naphthaleneacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA); cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylacrylamido)purine (2iP), and thidiazuron (TDZ); gibberellins; and abscisic acid; and mixtures thereof, in steps iii) and / or iv) and / or v).

[0021] In another embodiment of the above aspects, at least one HDACi and / or at least one HMTi and at least one plant growth regulator are used together in the culture medium used in step iii), wherein the at least one HDACi and / or at least one HMTi is preferably provided together with activated carbon.

[0022] In another embodiment of the above aspects, one or more chromosome doubling agents, such as colchicine, oryzalin, and / or trifluralin, are added during steps iii) and / or iv) and / or v).

[0023] The second aspect relates to a kit for producing haploid, polyhaploid, and / or double haploid cells, embryos, callus, seeds, and / or plants of the species sunflower (Helianthus annuus) from at least one isolated microspore, comprising: (a) at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi), and optionally comprising a complex protein composition; and (b) A means for providing at least two culture media in a bilayer system, the bilayer system comprising a liquid culture medium and a solid culture medium; and (c) Optional further components, comprising at least one plant growth regulator: The at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi), as well as the complex protein composition, if present, are contained in the same container or in two or more separate containers.

[0024] In one kit-related embodiment, the at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) is selected from: trichostatin A (TSA), isohydroxamic acid and isohydroxamic acid salts (e.g., vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), pabilistat (LBH589)); cyclic tetrapeptides (e.g., aprotinin B); cyclic peptides (e.g., romidesin (FK228)); and benzamides (e.g., entenostatin (MS-275), tazloride (CI994)). Moxistat (MGCD0103); BIX1294, BIX1338; electrophilic ketones; and fatty acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) and / or histone methyltransferase inhibitor (HMTi) is trachostatin A (TSA), BIX1294, BIX1338, vorinostat (SAHA), or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228), and / or the complex protein. The composition comprises or is composed of hydrolyzed or partially hydrolyzed protein substances derived from milk (e.g., casein or whey), animal (e.g., meat or fish), grain (e.g., rice or corn), plant (e.g., soybean), or combinations thereof, and / or the complex protein composition comprises or is composed 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 combinations thereof, and / or the complex protein composition. The kit comprises or consists of protein hydrolysates selected from: casein hydrolysate, soybean hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant complex protein hydrolysates, and combinations thereof, preferably with a degree of hydrolysis of 20 to 80%, more preferably 30 to 80%, particularly preferably 40 to 60%; and in some embodiments, 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-dichlorobenzoic acid (dicamba), naphthaleneacetic acid (NAA), indoleacetic acid (IAA) and indole-3-butyric acid (IBA), cytokinins including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP) and thiamethoxam (TDZ), gibberellins, mixtures thereof, and / or the kit further comprising one or more chromosome doubling agents, such as colchicine, asulfonamide and / or trifluralin.

[0025] Another aspect relates to the use of histone deacetylase inhibitors (HDACi) and / or histone methyltransferase inhibitors (HMTi), preferably used with a bilayer system comprising a solid layer and a liquid layer as defined in the first aspect, and optionally to the use of complex protein compositions with histone deacetylase inhibitors (HDACi) and / or with histone methyltransferase inhibitors (HMTi), preferably with as defined in the first aspect and / or with a bilayer system comprising a solid layer and a liquid layer and / or with activated charcoal, or to the use of kits for producing haploid, multi-haploid, and / or double-haploid embryos, callus tissue, and / or plants or seeds of the species sunflower, preferably according to the method of the first aspect.

[0026] The final aspect of the invention relates to a species of sunflower directly derived from at least one terminal or lateral capitulum, preferably derived from at least one tubular floret from at least one terminal or lateral capitulum, and more preferably from a single tubular floret. Helianthus annuus) A population of haploid, multiple haploid, and / or double haploid plants, preferably obtained by the method described in the first aspect above or can be obtained by the method described in the first aspect above, preferably wherein the population contains at least 10 individuals.

[0027] definition Androgenesis is defined as the process by which an individual's genetic background originates solely from a male-derived cell nucleus. In other words, androgenesis refers to the production of plants solely from male haploid gametophyte precursors (gametophytes).

[0028] Haploidy is a property that applies to cells, plants, or plant parts in which each chromosome contained in its nucleus has only one copy (n).

[0029] Diploidy refers to a property applicable to cells, plants, or parts of plants in which the nucleus contains two copies (2n) of chromosomes.

[0030] Double haploidy is a property applicable to cells or plants or plant parts containing said cells, whose chromosome library is artificially doubled, most often through chemical treatment, such as with chromosome doubling agents, including but not limited to mitosis inhibitors such as colchicine, asulfonamide, and / or trifluralin, or through spontaneous doubling. This doubling of the chromosome library makes it possible to obtain cells, plants, or plant parts having two copies (2n) of each chromosome in their nuclei, wherein said cells, plants, or plant parts are completely homozygous or substantially homozygous.

[0031] Multiple haploidy is a property applicable to cells or plants or plant parts containing said cells, which are originally haploid and whose chromosome library spontaneously triploids or polyploidizes. A cell, plant, or plant part having at least three copies (3n or 4n, etc.) of each chromosome in its nucleus, wherein said cell, plant, or plant part is entirely homozygous or substantially homozygous.

[0032] In this text, the term microspore is used to refer to the immature male gametophyte of a plant, encompassing all stages of its in vitro growth, including the multicellular form (also referred to herein as a multicellular structure) derived from the division of the unicellular sporophyte from the isolated microspore and still surrounded by the primitive exowall. During the mononuclear development stage of the microspore, the nucleus has not yet divided. A tetrad refers to a group of four microspores formed after diploid cells undergo meiosis to create four haploid microspores.

[0033] The complex protein compositions disclosed herein refer to mixtures of proteins derived from milk, meat, fish, grains, or soybeans. Preferably, the complex protein composition comprises or consists of at least partially hydrolyzed proteins. Specifically, the complex protein composition can be a protein hydrolysate, i.e., a mixture of proteins obtained by enzymatic hydrolysis catalyzed by a protease. The degree of hydrolysis refers to the percentage of peptide bonds cleaved relative to the total number of bonds available for protease hydrolysis. Examples of complex protein compositions are given in detail in the specification.

[0034] Histone deacetylases are enzymes that increase the affinity of histones for DNA by removing the acetyl group from the N-terminal acetyl-lysine residue of histones. Histone deacetylases thus play a role in the regulation of DNA expression. Histone deacetylase inhibitors (HDACi) inhibit histone deacetylases and therefore prevent the removal of the acetyl group from histones. Histone deacetylase inhibitors are used to treat a variety of diseases.

[0035] Histone methyltransferase inhibitors (HMTi) are known epigenetic drugs that can write or delete epigenetic information by blocking histone methyltransferases. Therefore, HMTis are used, for example, in cancer treatment.

[0036] The macro-element salts (MS) can be selected from ammonium nitrate (NH4NO3), calcium chloride (CaCl2x 2H2O), magnesium sulfate (MgSO4x7H2O), dipotassium hydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), potassium nitrate (KNO3), and calcium nitrate (Ca(NO3)2x 4H2O); the micro-element salts can be selected from boric acid (H3BO3), cobalt chloride (CoCl2x 6H2O), ferrous sulfate (FeSO4x 7H2O), manganese(II) sulfate (MnSO4x 4H2O), potassium iodide (KI), sodium molybdate (Na2MoO4x 2H2O), zinc sulfate (ZnSO4x 7H2O), ferric sodium ethylenediaminetetraacetate (FeNaEDTA), and copper sulfate (CuSO4x 5H2O), see, for example, Murashige T. and Skoog F., Physiol. Plant, 15, 473 (1962).

[0037] Plant growth regulators are chemical compounds that affect plant growth and development, for example, by promoting or inhibiting growth. Natural plant growth regulators are plant hormones, which are produced by the plant itself. Synthetic plant growth regulators, on the other hand, do not exist naturally in plants. Examples of plant growth regulators are given in detail in the specification.

[0038] Haploid plants can undergo spontaneous chromosome doubling, or chromosome doubling can be enhanced or promoted by chromosome doubling agents, such as those that block the function of spindle fibers during meiosis or mitosis. The most commonly used chromosome doubling agents are colchicine or other antimitotic agents, such as asulfanilamide or trifluralin.

[0039] This invention can have other embodiments and can be implemented or carried out in various ways. Furthermore, specific terminology will be used for clarity in describing exemplary embodiments.

[0040] It should also be noted that the singular forms “a,” “an,” and “the” used in the specification and appended claims include plural references unless the context clearly indicates otherwise. For example, references to components are also intended to include compositions of multiple components. References to compositions containing “one component” are intended to include other components besides the one specified. In other words, the terms “a,” “an,” and “the” do not indicate a limitation of quantity, but rather the presence of “at least one” of the referenced item.

[0041] As used herein, the term “and / or” can mean “and”, “or”, “exclusive”, “one”, “some but not all”, and / or “both”. The term “or” implies inclusive “or”.

[0042] To facilitate understanding of the principles and features of the various embodiments of the present invention, illustrative embodiments are explained below. Although exemplary embodiments of the present invention have been explained in detail, it is understood that other embodiments are also conceivable. Therefore, it is not intended that the scope of the present invention be limited to the subject matter and component arrangement details set forth in the following description or embodiments.

[0043] Furthermore, terminology will be used for clarity in the description of exemplary embodiments. The terms used in this invention are meant in their broadest sense as understood by those skilled in the art and include all technical equivalents that operate in a similar manner to achieve similar purposes. It should be understood that various embodiments of the disclosed technology may be implemented without using these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail so as not to obscure the understanding of this specification. References to “one embodiment,” “an embodiment,” “exemplary embodiment,” “some embodiments,” “specific embodiment,” “various embodiments,” etc., indicate that embodiments of the described disclosed technology may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes that particular feature, structure, or characteristic. Furthermore, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

[0044] The term “about” as used herein should be interpreted as referring to two numerical values ​​specified as endpoints of any range. Any reference to a range should be considered as support for any subset of that range. A range herein can be expressed as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When expressed with such a range, other exemplary embodiments include from one particular value and / or to another particular value. Furthermore, the term “about” refers to a range of acceptable error for a particular value as determined by one of ordinary skill in the art, depending on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation according to practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and even more preferably up to ±1% of a given value. Or, particularly for biological systems or processes, the term can mean within the order of magnitude of a value, preferably within twice the order of magnitude. Unless otherwise stated, where a particular value is described in the application and claims, the term “approximately” implicitly indicates, and in this context means, that the value is within an acceptable range of error for that particular value.

[0045] Throughout this disclosure, various aspects of the invention may be presented in a scope format. It should be understood that the scope format is for convenience and brevity purposes only and should not be construed as an inflexible limitation on the scope of the invention. Therefore, a scope description should be considered to specifically disclose all possible sub-ranges within that range, as well as individual numerical values. For example, a scope description such as from 1 to 6 should be considered to specifically disclose sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the scope.

[0046] The use of "comprising," "containing," or "including" means that at least the stated 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, or method steps, even if such other compounds, materials, particles, or method steps have the same function as the named one.

[0047] Throughout this specification, various components with specific values ​​or parameters may be identified; however, these items are provided as exemplary embodiments. In fact, the exemplary embodiments do not limit the aspects and concepts of the invention, as many comparable parameters, dimensions, ranges, and / or values ​​can be implemented. The terms “first,” “second,” etc., “main,” “subsidiary,” etc., do not indicate any order, quantity, or importance, but are used to distinguish one element from another.

[0048] Note that terms such as “specifically,” “preferably,” “typically,” “generally,” and “usually” used herein are not intended to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are intended only to highlight alternative or additional features that may or may not be employed in specific embodiments of the invention. It should also be noted that terms such as “substantially” and “approximately” used herein are attributable to the inherent degree of uncertainty in any quantitative comparison, value, measurement, or other representation.

[0049] The dimensions and values ​​disclosed herein should not be construed as strictly limited to the exact numerical values ​​listed. Rather, unless otherwise stated, each such dimension refers to the stated value and the range of functionally equivalent values ​​around that value. For example, a dimension disclosed as “50 mm” means “approximately 50 mm”.

[0050] It should also be understood that mentioning one or more method steps does not preclude the presence of additional method steps or intermediate method steps between those expressly identified steps. Similarly, it should be understood that mentioning one or more components in a composition does not preclude the presence of other components besides those expressly identified.

[0051] The materials used as various elements of the invention as described below are intended to be illustrative and not limiting. Many materials suitable for performing the same or similar functions as those described herein are intended to be included within the scope of the invention. Other materials not described herein may include, but are not limited to, materials developed after the invention. The dimensions listed in the figures are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and expected to be included within the scope of the invention.

[0052] According to the present invention, conventional molecular biology, microbiology, and recombinant DNA technologies can be employed to the extent known to those skilled in the art. These technologies are well explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (BD Hames&S.J. Higgins, eds. (1985); Transcriptionand Translation (BD Hames&S.J. Higgins, eds. (1984)); Animal Cell Culture (RI Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); FM Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley&Sons, Inc. (1994); etc. Attached Figure Description

[0053] Figure 1 The plant material used for harvesting is shown: A. Head morphology of sunflower; B. Appearance and size of tubular florets; C. Separate tubular florets.

[0054] Figure 2 The development from sunflower microspores to callus structures is shown. From microspore nuclear division (A) to multinucleated cells (B) and callus structures (C).

[0055] Figure 3The development from sunflower microspores to callus tissue structures is shown.

[0056] Figure 4 (A, B, and C) show the bud induction of sunflower microspore-derived callus (arrows).

[0057] Figure 5 Display: A. Sunflower plants derived from microspores after the preceding in vitro development; B. Sunflower plants derived from microspores during the flowering stage after the preceding in vitro development described in this article.

[0058] Figure 6 The flow cytometry histogram results of the diploid control donor material are shown (see Example 6).

[0059] Figure 7 The flow cytometry histogram results of the spontaneous doubling from microspore-derived plant material are shown, i.e., double haploid (DH) lines generated based on the methods disclosed herein (see details of Example 6).

[0060] Figures 8 (A and B) show the effects of certain 2,4-dichlorophenoxyacetic acid analogues on callus count ( Figure 8A ) and regeneration ( Figure 8B The effect of ) compared to the control (Ctrl) is as described in Example 4.2.

[0061] Figure 9 The microspore division and callus formation at several 2,4-D concentrations tested on the 007 genotype are shown, as described in detail in Example 4.3. Top row: Arrows indicate the location of the embryo (magnification). Bottom row: Corresponding culture dish.

[0062] Figure 10 The image shows a sunflower derived from microspores, which forms a root after undergoing in vitro development as described herein, particularly in the detailed description of Example 5.2 below.

[0063] Figure 11 shows double haploid sunflower plants obtained using the methods disclosed herein. Figure 11A The plant is fertile, and pollen can be easily observed on both the stamens and pistils. Figure 11B (and Example 7).

[0064] Figure 12 shows the effect of charcoal on callus induction in sunflower plants. Figure 12A The role of carbon in the presence and absence of double layer (DL) for two genotypes during the microspore culture stage. Figure 12B The effect of activated carbon (AC) during the microspore culture stage of five different sunflower genotypes in a two-layer system.

[0065] Figure 13As shown in Example 10, the effect of the lactalbumin hydrolysate (Ctrl = control) as a complex protein composition favorably influences microspore division and callus growth. Detailed Implementation

[0066] As described in the background section, there is an urgent need in the art to identify methods for the efficient production of haploid and double haploid sunflowers (…). H. annuus) Plant technologies are used to develop novel methods and kits for producing such plants more economically, versatilely, and rapidly. This invention addresses these and other needs. Embodiments of the invention generally relate to methods and kits for producing, particularly in a genotype-independent manner, haploid, polyhaploid, and / or double haploid sunflower (Helianthus annuus) plants from isolated microspore cultures, and more specifically to methods and kits for producing such sunflower plants, comprising contacting isolated microspores with histone deacetylase inhibitors (HDACi) and / or HMTi, as well as a complex protein composition.

[0067] In a first aspect, the present invention provides a method for generating species. sunflower Methods for the production of haploid, polyhaploid, and / or diploid cells, embryos, callus, seeds, and / or plants of Helianthus annuus, wherein the methods may include: i) Provide sunflowers ( Helianthus annuus At least one isolated microspore of the plant; ii) Optionally: culture at least one microspore from step i) in a bilayer system in the presence of the complex protein composition; iii) In the presence of at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi), and optionally in the presence of a complex protein composition, culture at least one microspore from step i) or ii) to induce and thus obtain callus, including microcallus, and / or embryos. iv) Culture the callus or embryo; v) Optionally, at least one plant is regenerated and thereby obtained from the callus or embryo of step iv), and preferably at least one haploid, multiple haploid and / or double haploid seed is obtained, preferably wherein activated carbon is added during steps (i), (ii) and / or (iii).

[0068] The method of this invention offers the advantage of obtaining hundreds of haploid or double haploid plants from a single tubular flower or inflorescence of at least one sunflower terminal or lateral capitulum in a highly efficient manner by culturing sensitive microspores in an optimal manner using a two-layer system in combination with HDACi and / or HMTi; whereas tissue culture yields at most a few plants, and if any, only a minority of them are haploid. Another advantage is that haploid plants obtained through microspore culture appear to have a higher natural doubling rate than those obtained from ovule culture. Therefore, most of the plants obtained according to the method of this invention can be used directly for breeding without the need for chromosome doubling agents.

[0069] The term "callus" as used in this article may also refer to microcallus or structures, or a portion thereof.

[0070] In some preferred embodiments, activated charcoal is added to the liquid or solid medium of the DL system during steps (i), (ii), or (iii), or, for selected sunflower genotypes, the solid medium is used instead of the liquid induction medium for microspore activation. The addition of charcoal promotes callus development in certain sunflower genotypes. Preferably, activated charcoal is added to the solid medium that forms the solid layer of the DL system.

[0071] In some embodiments, and depending on the genotype of the microspores derived from sunflower, activated charcoal may be used in a liquid system or in a DL system. In one embodiment using a DL system, activated charcoal may be present in two layers. In another embodiment, activated charcoal is added only to the liquid or solid medium of the DL system during the microspore culture step, thereby further promoting callus regeneration and advantageously increasing the haploid induction rate.

[0072] In some embodiments, at the concentrations disclosed herein, at least one HDACi and / or at least one HMTi will be used in the liquid and / or solid phases of the DL system.

[0073] In some implementations, specific combinations of more than one histone deacetylase inhibitor (HDACi) and / or more than one histone methyltransferase inhibitor (HMTi) were tested individually or in combination, and even better results could be obtained when they were added during microspore culture.

[0074] In one embodiment, the combination of at least two HDACi and / or at least two histone methyltransferase inhibitors (HMTi) is selected from, but not limited to: SAHA and TSA, romedixin and TSA, BIX1294 (G9a histone methyltransferase inhibitor) and TSA, or BIX1338 and TSA.

[0075] The use of HDACi and / or HMTi in the DL system unexpectedly significantly improved microspore-initiated callus formation and the production of haploid, multi-haploid, and / or double-haploid cells and final seeds.

[0076] In some implementations, and depending on the sunflower genotype, the addition of activated charcoal during the culture step has shown an enhancing effect on haploid, multi-haploid, and / or double-haploid cells, as well as the final seed.

[0077] Therefore, the method presented herein begins with the initial step of isolating living plant material as the starting material, preferably from... sunflower Living plant material is isolated from terminal or lateral capitulum inflorescences. The first key step is to prepare and, in particular, culture the material under suitable reaction conditions and in a suitable culture medium supplemented with certain components to allow for the propagation of microspores, thereby obtaining callus or embryonic structures that can be further cultured.

[0078] In a preferred embodiment, steps ii) and iii) are both used to induce and thus obtain callus or embryo.

[0079] In one embodiment of the foregoing, the following steps may be performed before providing at least one isolated microspore of the sunflower plant according to step i) of the foregoing: (a) Provide at least one terminal or lateral capitulum of a sunflower plant; (b) Harvesting at least one tubular floret or a portion thereof from at least one terminal or lateral capitulum to obtain tubular floret material; and (c) Optionally: disinfect the surface of the tubular flower material; (d) Optionally: Wash the disinfected tubular flower material; (e) The optionally sterilized and washed tubular flower material is transferred to an isolation medium containing macro- and micro-element salts and sugars, and preferably containing a complex protein composition. (f) Homogenize and optionally sieve the tubular flower material to provide at least one isolated microspore of the sunflower plant.

[0080] In a preferred embodiment, the complex protein composition in step (e) is the same as that in step (ii).

[0081] In some embodiments, the tubular flower material can be stored for further processing before or after sterilization and / or washing, preferably directly after harvest. In some embodiments, this may be beneficial to give the material some time to recover to increase the yield of surviving microspores. The tubular flower material can be stored at room temperature (between about 18°C ​​and about 25°C), above 28°C, or at cooling or freezing temperatures. In a preferred embodiment, the tubular flower material, comprising tubular flowers or portions thereof, is removed from the sunflower plant. The removed tubular flowers or portions thereof can be stored at intermediate cooling temperatures, such as about 2°C to about 10°C, or about 4°C to 8°C, or below freezing temperatures, such as below 2°C. Ice crystal formation should be avoided to ensure the integrity of the cell structure. Freezing temperatures may be applied throughout step (f).

[0082] In some embodiments, physical factors such as heat shock and cold shock treatments, combined with the use alone or in combination of 2,4-D or 2,4-D analogs and different HDACi and / or HMTi, can further promote sunflower microspore development in the DL system of this disclosure, thereby providing haploid, multi-haploid and double-haploid sunflower cells.

[0083] In some implementations, a cold shock pulse treatment at 2-6 degrees Celsius for 2-7 days may be applied during sunflower microspore culture in steps (i)-(iii).

[0084] In other embodiments, a heat shock pulse treatment at 29-35 degrees Celsius for 2-7 days may be applied during sunflower microspore culture in steps (i)-(iii).

[0085] In another embodiment, the physical factors used in the sunflower microspore culture process in steps (i)-(iii), such as light intensity and exposure time in various spectra, can vary from low to high and differ during the culture steps, thereby further optimizing the microspore culture. The light intensity used in this paper is 50 μmol / m². 2 / s can be considered as low light intensity, while 200μmol / m 2 / s can be considered as the intensity of the light.

[0086] In a preferred embodiment of the above method, an initial cell suspension is obtained in step (b) after harvesting, and at any stage during step (b) and before step (e), the cell suspension is obtained by applying a separation buffer or separation medium (or before the dissolution step during steps (b) and optionally steps (c) and (d). The cell suspension can be purified or cells further separated during step (b) or (e) by passing it through a sieve, for example, through a cell sieve with a pore size in the range of about 10 to 200 μm, preferably 20 to 100 μm, and through a cell sieve with a pore size in the range of about 20 to 80 μm, preferably 25 to 50 μm. Preferably, the cell suspension is passed through each sieve with a different pore size at least 1 to 3 times, or exactly 1 to 3 times. Alternatively, the cell suspension can be purified using a sieve in step (b) or (e) by repeatedly, preferably at least 1 to 6 times, or exactly 1 to 6 times, passing the cell suspension through a cell sieve with a pore size range of about 25 to 100 μm.

[0087] In one embodiment, following the harvest in step (b) above, in step (e), the early proliferating material is transferred to a suitable separation buffer or one or more separation media, wherein the separation media may contain one or more amino acids such as glutamine and / or nucleosides such as uridine and cytidine. Each of these additional components may be present at a concentration of about 20 to 750 mg / L.

[0088] In a preferred embodiment, microspores are cultured on top of the DL system. Relevant supplements can then be provided to the microspores via diffusion from the liquid and / or solid culture medium of the DL system, thereby efficiently providing an excellent environment for microspore culture and development, and generating haploid cells, multiple haploid cells, and / or double haploid cells in the desired direction.

[0089] In some embodiments, in step (e), the sugar is a monosaccharide such as glucose monohydrate (dextrose), fructose, or galactose, which may be present at a concentration of 20 to 750 mg / L.

[0090] In one embodiment, a method may be provided that further includes the following steps between steps (b) and (f): (b1) In the presence of at least one amino acid, such as glutamine, and / or in the presence of at least one nucleoside, such as uridine and cytidine, at a concentration of about 10 mg / L to about 800 mg / L, preferably about 20 mg / L to about 750 mg / L, at a concentration of about 10 mg / L to about 800 mg / L; In some embodiments of the above method, before regeneration and during the period near the end of callus / embryo induction, in step iv), the structure of the callus or embryo can be destroyed by applying physical force, for example, the callus or embryo can be divided into blocks, crushed or pulverized.

[0091] In yet another implementation of the above aspects, the method may further include the following steps within step iii): (iii.a) Add at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) to a final concentration of 1 nM-10 μM, preferably 100 nM-10 μM, more preferably 1 μM-10 μM; (iii.b) After incubation for at least about 1 hour, preferably at least about 5 hours or more preferably at least about 12 hours and / or incubation for at least up to about 48 hours, preferably at least up to about 50 hours or more preferably at least up to about 60 hours, the at least histone deacetylase inhibitor (HDACi) and / or the at least histone methyltransferase inhibitor (HMTi) are removed, wherein the incubation is optionally carried out in the dark.

[0092] In another embodiment of the foregoing, the at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) are selected from: trichostatin A (TSA); isohydroxamic acid and isohydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and pabisostat (LBH589); cyclic tetrapeptides, such as aprotinin B; cyclic condensates, such as romidesin (FK228); and benzamides, such as entenoxetine (MS-275) and talcum powder. The histone deacetylase inhibitor (HDACi) and / or HMTi are preferably trachomatis A (TSA), BIX1294, BIX1338, vorinostat (SAHA), or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228).

[0093] In another embodiment, a complex protein composition may be used, comprising or consisting of hydrolyzed or partially hydrolyzed protein substances derived from milk (e.g., casein, whey), animal sources (e.g., meat, fish), grains (e.g., rice, corn), plants (e.g., soybeans), or combinations of the above sources; 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 casein sodium, hydrolyzed casein calcium, hydrolyzed whole milk, semi-skimmed / fully skimmed hydrolyzed milk, Hydrolyzed soy protein isolate, hydrolyzed soy protein concentrate, or a combination of the above substances; and / or, the complex protein composition comprises or is selected from the following protein hydrolysates: casein hydrolysate, soy hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant complex protein hydrolysate, and combinations of the above hydrolysates; preferably, its degree of hydrolysis is about 20% to 80%, more preferably about 30% to 80%, particularly preferably about 40% to 60%; optionally, the complex protein composition is provided together with activated carbon.

[0094] In a preferred embodiment, the composite protein composition or protein hydrolysate comprises or consists of hydrolyzed lactalbumin. Lactalbumin surprisingly enhances microspore division and callus growth properties (see Example 10 and...). Figure 13 ).

[0095] The complex protein composition may be used during the isolation / early culture step ii) and / or additionally or alternatively during step b) of culturing microspores in induction medium, at least during the first culture cycle for callus or embryo induction in step iii). In some embodiments, the complex protein composition may be used during the isolation / early culture step and in the early induction medium (e.g., induction medium I) for microspore differentiation and development, but not in the late differentiation step toward callus and / or embryo (e.g., induction medium II).

[0096] In some embodiments, and particularly depending on the tissue culture length 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).

[0097] This complex protein composition can be obtained from any protein source, such as milk, meat, fish, grains, or other plants. In particular, good results are obtained by using a protein composition derived from milk and subjected to at least partial hydrolysis.

[0098] In any embodiment of the above method, the complex protein composition comprises or consists of hydrolyzed or partially hydrolyzed protein substances derived from milk such as casein or whey, animals such as meat or fish, grains such as rice or corn, plants such as soybeans, or combinations thereof.

[0099] Preferably, in any embodiment of the above method, the composite protein composition comprises or consists of hydrolyzed whey 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, preferably hydrolyzed lactalbumin or a combination thereof.

[0100] Particularly preferably, in any embodiment of the above method, the complex protein composition comprises or consists of proteolytic hydrolysates selected from casein hydrolysates, soybean hydrolysates, rice hydrolysates, potato hydrolysates, fish hydrolysates, ovalbumin hydrolysates, whey protein hydrolysates, gluten hydrolysates, animal and plant protein hydrolysates, preferably whey protein hydrolysates and combinations thereof.

[0101] More preferably, in the complex protein composition, the degree of hydrolysis is in the range of about 20-80%, preferably in the range of about 30-80%, and particularly preferably in the range of about 40-60%.

[0102] More preferably, the composite protein composition is applied together with activated charcoal to a culture medium or buffer to stabilize and activate the mixture, and to support callus or embryo induction and / or plant, plant tissue or seed regeneration from callus or embryo.

[0103] In one embodiment of the method according to any of the above embodiments, the concentration of at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) in the culture medium used in step iii) is from about 1 nM to about 10 μM, preferably from 100 nM to 10 μM, more preferably from 1 μM to 10 μM.

[0104] In another embodiment of the method according to any of the above embodiments, the concentration of the complex protein composition in the culture medium used in steps ii) and / or iii) is 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, and / or wherein the concentration of the complex protein composition in the separation culture medium used in step (e) is 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.

[0105] In another embodiment of the method according to any of the above embodiments, if the complex protein composition is a lactalbumin hydrolysate or contains a lactalbumin hydrolysate, its concentration in the culture medium used in steps ii) and / or iii) is about 100 to about 20,000 mg / L, preferably about 200 to about 15,000 mg / L, particularly preferably about 300 to about 10,000 mg / L, more preferably about 400 to about 7,500 mg / L, and even more preferably about 500 to about 5,000 mg / L, and / or the concentration of the complex protein composition in the separation culture medium used in step (e) is about 100 to about 20,000 mg / L, preferably about 200 to about 15,000 mg / L, particularly preferably about 300 to about 10,000 mg / L, more preferably about 400 to about 7,500 mg / L, and even more preferably about 500 to about 5,000 mg / L.

[0106] In any of the above embodiments, in step iii), at least one microspore or callus or embryo generated from said at least one microspore during callus induction 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), p-chlorophenoxyacetic acid (4-CPA or 4CIPA), 3,6-dichlorophthalic acid (dicyclopentadiene), naphthaleneacetic acid (NAA), indoleacetic acid (IAA) and indole-3-butyric acid (IBA), cytokinins including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylacrylamido)purine (2iP) and thidiazuron (TDZ), gibberellins, and abscisic acid and mixtures thereof, in steps iii) and / or iv) and / or v), wherein in some embodiments, said plant growth regulators and mixtures thereof are applied in steps iii) and / or iv). In some embodiments, the concentration of 2,4-dichlorophenoxyacetic acid or any auxin or any synthetic auxin analogue and / or BAP or any other cytokinin may be from about 0.01 mg / mL to 10 mg / mL, preferably from about 0.05 mg / mL to about 3 mg / mL.

[0107] In a preferred embodiment, more than one plant growth regulator is used, wherein the mixture may include at least 2,4-D, and at least one of 3,5ME, 4-CPA / 4CIPA, and / or BAP. Mixtures containing 2,4-D exhibit excellent performance if used in combination with expensive auxins (analytes) and if it allows for the substitution of a portion of the expensive auxin (analyte) with inexpensive and readily available 2,4-D.

[0108] In some embodiments, particularly for the rooting and rooting medium, PAA and / or IBA 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, and even more preferably about 0.05 to about 0.1 mg / mL.

[0109] In a preferred embodiment, during callus induction and culture in steps iii) and iv) of the method according to the first aspect disclosed herein, at least one of 4CIPA and / or 3.5ME and / or PHAA is used as a plant growth regulator in the method disclosed herein, because the inventors have surprisingly found that these compounds can advantageously increase callus formation and regeneration when used during callus induction.

[0110] In another embodiment, in step iv) of the method of the first aspect, preferably at least one auxin, synthetic auxin, or auxin analogue such as PAA and / or IBA is used as a plant growth regulator to enhance rooting and thus regenerate the plant. Given the fairly high survival rate of callus induced using the methods described herein, it is not necessary to add a growth regulator as a rooting promoter in the regeneration step, or to add the growth regulator before that step to obtain effective rooting (see Example 5.2 below).

[0111] According to the regeneration protocol, buffers and culture media without growth regulators may be beneficial for allowing efficient and natural plant regeneration.

[0112] In another embodiment of the above aspects, one or more chromosome doubling agents, such as colchicine, sulfadiazine, and / or trifluralin, are added during step iii) and / or steps iv) and / or v). The chromosome doubling agent may be administered before, with, or subsequently to the HDACi and / or HMTi of interest.

[0113] In another implementation, spontaneous chromosome doubling can be induced.

[0114] In an embodiment of the above method, in step v), a regenerated plant population is formed, having a spontaneous chromosome doubling 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.

[0115] In certain embodiments of the method disclosed herein, the method is used to increase the spontaneous chromosome doubling rate according to any of the above embodiments, wherein callus or embryo 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-dichlorobenzoic acid (dicamba), naphthaleneacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA); cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP), and thidazuron (TDZ); gibberellins; abscisic acid; and mixtures thereof, in steps iii) and / or iv) and / or v). This may be significant when spontaneous chromosome doubling is important. In some embodiments, in steps iii) and / or iv), but not in step v) of regeneration, or not throughout step v), the callus or embryo is contacted with one or more plant growth regulators selected from the group consisting of: auxins, synthetic auxins or auxin analogues, including 2,4-dichlorophenoxyacetic acid, 3,5-dimethylphenoxyacetic acid (3,5ME), phenoxyacetic acid (PHAA), phenylacetic acid (PAA), p-chlorophenoxyacetic acid (4-CPA), 3,6-dichlorobenzoic acid (dicamba herbicide), naphthaleneacetic acid (NAA), indoleacetic acid (IAA) and indole-3-butyric acid (IBA), cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylacrylamido)purine (2iP) and thidiazuron (TDZ), gibberellins and abscisic acid, and mixtures thereof.

[0116] In a preferred embodiment, one or more plant regulators are used, wherein the mixture may contain at least 2,4-D, which is combined with at least one of 3,5ME, 4-CPA / 4CIPA and / or BAP.

[0117] The double haploid plants obtained by the above method are fertile and can be directly used as maternal or paternal parents for further breeding with other plants, preferably for the development of DH lines / DH populations or for hybridization.

[0118] The techniques used in these chromosome doubling steps are known in the art, thereby producing double haploid material from haploid plants, cells, tissues, organs or materials or seeds obtained according to the method of the invention, wherein the material can be used to accelerate plant breeding.

[0119] Another aspect involves the production of the sunflower species from at least one isolated microspore. Helianthus annuus) Kits for haploid, polyhaploid, and / or double haploid cells, embryos, callus, seeds, and / or plants, including: (a) at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi); and (b) a complex protein composition; and (c) Optional further components, including at least one plant growth regulator and / or one or more chromosome doubling agents, such as colchicine, sulfadiazine and / or trifluralin; The at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) and the complex protein composition are contained in the same container or in two or more separate containers.

[0120] In one kit-related embodiment, the at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) is selected from: trichostatin A (TSA), isohydroxamic acid and isohydroxamic acid salts (e.g., vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), pabilistat (LBH589)); cyclic tetrapeptides (e.g., aprotinin B); cyclic peptides (e.g., romidesin (FK228)); and benzamides (e.g., entenostatin (MS-275), tazloride (CI994)). Moxistat (MGCD0103); BIX1294, BIX1338; electrophilic ketones; and fatty acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) and / or histone methyltransferase inhibitor (HMTi) is trachostatin A (TSA), BIX1294, BIX1338, vorinostat (SAHA), or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228), and / or the complex protein. The composition comprises or is composed of hydrolyzed or partially hydrolyzed protein substances derived from milk (e.g., casein or whey), animal (e.g., meat or fish), grain (e.g., rice or corn), plant (e.g., soybean), or combinations thereof, and / or the complex protein composition comprises or is composed 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 combinations thereof, and / or the complex protein composition. The kit comprises or consists of protein hydrolysates selected from: casein hydrolysate, soybean hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant complex protein hydrolysates, and combinations thereof, preferably with a degree of hydrolysis of 20 to 80%, more preferably 30 to 80%, particularly preferably 40 to 60%; and in some embodiments, 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-dichlorobenzoic acid (dicamba), naphthaleneacetic acid (NAA), indoleacetic acid (IAA) and indole-3-butyric acid (IBA), cytokinins including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP) and thiamethoxam (TDZ), gibberellins, mixtures thereof, and / or the kit further comprising one or more chromosome doubling agents, such as colchicine, asulfonamide and / or trifluralin.

[0121] In a preferred embodiment, more than one plant regulator is used in the kit, wherein the mixture may include at least 2,4-D, in combination with at least one of 3,5ME, 4-CPA / 4CIPA and / or BAP.

[0122] In one embodiment, the kit comprises a first composition containing HDACi and / or HMTi; and a second composition containing a complex 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 containing HDACi and / or HMTi and a complex protein composition. In one embodiment, the composition is a culture medium for plant cell culture. The kit may include a set of instructions for using HDACi and / or HMTi and / or the complex protein composition. One or both of HDACi and / or HMTi and the complex protein composition may be in concentrated form and require dilution before use.

[0123] In one embodiment of the kit according to any of the above embodiments, the at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) is selected from: trichostatin A (TSA); hydroxamic acid and hydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and pabilistat (LBH589); cyclic tetrapeptides, such as aprotinin B; cyclic condensates, such as romidesin (FK228); and benzamides, such as entenoxetine (MS-275). Tazoline (CI994), moxistat (MGCD0103); BIX1294, BIX1338; electrophilic ketones; and fatty acid compounds such as phenylbutyrate and valproic acid, preferably, the histone deacetylase inhibitor (HDACi) and / or HMTi is trichostatin A (TSA), BIX1294, BIX1338, vorinostat (SAHA) or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228).

[0124] Removing HDACi and / or HMTi before culturing to obtain callus results in particularly effective callus formation. Removal of HDACi and / or HMTi can be performed by centrifugation, washing of microspores treated with HDACi and / or HMTi, or by replacing the medium with the same medium or a different medium without HDACi and / or HMTi, or any combination of these steps. Such steps for removing HDACi and / or HMTi can be applied once, repeatedly, or multiple times before and / or during callus induction. The same steps apply to removing at least one plant growth medium from one step (containing) to another (excluding plant growth regulators).

[0125] Those skilled in the art are aware of various different HDACi and / or HMTi. Good results are particularly obtained when TSA or romidesin (FK228) is used as one of the combination partners. In some embodiments, TSA and romidesin can be used. In other embodiments, either TSA or romidesin is used in combination with an HDACi and / or HMTi preferably selected from BIX1294, BIX1338, and SAHA.

[0126] Another aspect relates to the use of histone deacetylase inhibitors (HDACi) and / or at least one HMTi (preferably as defined in the first aspect), and the use of complex protein compositions (preferably as defined in the first aspect), or the use of kits as defined in the second aspect above, for producing haploid, multi-haploid and / or double-haploid embryos, callus and / or plants or seeds of sunflower species, preferably according to the method of the first aspect.

[0127] In the plants obtained according to the present invention, chromosome doubling can occur spontaneously or can be promoted, aided, or enhanced by chromosome doubling agents.

[0128] In one embodiment of the method for preparing cultivated sunflower microspores and / or cultivating haploid, multi-haploid, double-haploid cells, embryos, callus, seeds, and plants of the species, and / or in the kits described in any of the foregoing technical solutions and embodiments, the at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) is selected from the following substances: trichostatin A (TSA); isohydroxamic acid and isohydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and parbistat (LBH589); cyclic tetrapeptides, such as aprotinin B; and cyclic peptides, such as romidesin (F... K228); benzamides, such as entenostat (MS-275), tazloridine (CI994), moxistat (MGCD0103); BIX1294, BIX1338; electrophilic ketones; and fatty acid compounds, such as phenylbutyrate, valproic acid. Preferably, the histone deacetylase inhibitor (HDACi) and / or histone methyltransferase inhibitor (HMTi) is trichostatin A (TSA), BIX1294, BIX1338, vorinostat (SAHA), or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228).

[0129] In another embodiment of the kit according to any of the above embodiments, the complex protein composition comprises or consists of hydrolyzed or partially hydrolyzed protein substances derived from milk such as casein or whey, animals such as meat or fish, grains such as rice or corn, plants such as soybeans, or combinations thereof.

[0130] Preferably, the complex protein composition comprises or consists of hydrolyzed whey 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, preferably hydrolyzed lactalbumin or a combination thereof.

[0131] Particularly preferably, the complex protein composition comprises or consists of proteolytic hydrolysates selected from casein hydrolysates, soybean hydrolysates, rice hydrolysates, potato hydrolysates, fish hydrolysates, ovalbumin hydrolysates, whey protein hydrolysates, gluten hydrolysates, animal and plant protein hydrolysates, preferably whey protein hydrolysates and combinations thereof.

[0132] In one implementation, activated carbon or activated carbon may be provided with at least one container of the kit, or used as a separate container with another container of the kit.

[0133] In one embodiment, the degree of hydrolysis is in the range of 20-80%, preferably 30-80%, and particularly preferably 40-60%.

[0134] In another embodiment of the kit according to any of the above embodiments, 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-dichlorobenzoic acid (dicamba), naphthaleneacetic acid (NAA), indoleacetic acid (IAA) and indole-3-butyric acid (IBA); cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylallylamino)purine (2iP) and thidazuron (TDZ); gibberellins; abscisic acid; and mixtures thereof.

[0135] One or more plant growth regulators may be present in the same container or in separate containers with HDACi and / or HMTi and / or complex protein compositions.

[0136] In addition, kits for generating solid culture media for the DL system may include, for example, agarose, gelrite, or agar.

[0137] This invention also relates to a method or kit according to the invention for producing species from isolated microspores via androgenesis. sunflowerUses of haploid, polyhaploid, and / or dihaploid plants of *Helianthus annuus*. Prior art solutions, such as those described in the background section, are presented. supra It is incorporated into this article by reference.

[0138] The present invention also relates to the use of HDACi and / or HMTi and protein complex compositions for producing haploid, polyhaploid, and / or double haploid embryos and / or plants of the species sunflower (Helianthus annuus) from isolated microspores via androgenesis.

[0139] In one embodiment, the present invention relates to the use of histone deacetylase inhibitors (HDACi) and / or HMTi, and complex protein compositions, or the use of kits as defined in any of the foregoing embodiments, for generating haploid, multiple haploid, and / or double haploid species. sunflower Embryo, callus and / or plant or seed of (Helianthus annuus).

[0140] Preferably, histone deacetylase inhibitors (HDACi) and / or HMTI, as well as complex protein compositions or kits, are used at least during step iii) in the method according to any of the above embodiments.

[0141] In one embodiment of the above-described use, the histone deacetylase inhibitor (HDACi) and / or the histone methyltransferase inhibitor (HMTi) are selected from: trichostatin A (TSA); isohydroxamic acid and isohydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and pabisostat (LBH589); cyclic tetrapeptides, such as aprotinin B; cyclic peptides, such as romidesin (FK228); and benzamides, such as entenoxetine (MS-275) and tazloridine (CI). 994), moxistat (MGCD0103); BIX1294, BIX1338; electrophilic ketones; and fatty acid compounds such as phenylbutyrate and valproic acid, preferably, the histone deacetylase inhibitor (HDACi) and / or HMTi is trichostatin A (TSA), BIX1294, BIX1338, vorinostat (SAHA) or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228).

[0142] In a preferred embodiment of the above-described use, the degree of hydrolysis of the complex protein composition is 20-80%, preferably 30-80%, and particularly preferably 40-60%.

[0143] One final aspect involves species derived directly from a single tubular flower. sunflower A population of haploid, multi-haploid, and / or double-haploid plants, preferably obtained or obtainable by any of the methods described in the first aspect above, preferably wherein the population comprises at least 10 individuals.

[0144] Advantageously, the method of the present invention allows for the production of a large number of haploid, polyhaploid, and / or double haploid sunflower plants from a single tubular flower or a single capitulum, while using ovule culture, up to four plants can be obtained from a single flower.

[0145] In one implementation of the aforementioned group, the group comprises at least 10 individuals, preferably at least 50 individuals, and more preferably at least 100 individuals.

[0146] Another advantage is that plants obtained by the method of this invention show a spontaneous chromosome doubling rate of over 50%, while plants obtained by ovule culture show a maximum spontaneous doubling rate of only 5%. Therefore, most of the obtained plants can be used directly for breeding purposes without the use of chromosome doubling agents.

[0147] According to any of the above embodiments, in another embodiment, the population comprises at least 40%, at least 50%, at least 60%, or at least 70% of double haploid plants.

[0148] The present invention also relates to the use of histone deacetylase inhibitors (HDACi) and / or HMTi and complex protein compositions, or to the use of kits as defined in any of the embodiments described above, for increasing the spontaneous chromosome doubling rate in sunflower populations derived from single flowers or single capitulums and obtained by microspore culture.

[0149] In a preferred embodiment of the above-described uses, the species prepared according to any of the above embodiments... sunflower In methods for producing haploid, polyhaploid, and / or diploid embryos, callus, seeds, and / or plants of Helianthus annuus, histone deacetylase inhibitors (HDACi) and / or HMTi, as well as the complex protein composition or kit described above, are used.

[0150] In one embodiment of the use, the histone deacetylase inhibitor (HDACi) and / or the histone methyltransferase inhibitor (HMTi) are selected from: trichostatin A (TSA); isohydroxamic acid and isohydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and pabisostat (LBH589); cyclic tetrapeptides, such as aprotinin B; cyclic peptides, such as romidesin (FK228); and benzamides, such as entenoxetine (MS-275) and tazloridine (CI9). 94) Moxistat (MGCD0103); BIX1294, BIX1338; electrophilic ketones; and fatty acid compounds such as phenylbutyrate and valproic acid, preferably, the histone deacetylase inhibitor (HDACi) and / or HMTi is trichostatin A (TSA), BIX1294, BIX1338, vorinostat (SAHA) or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228).

[0151] In some embodiments, activated carbon may be provided together with the complex protein composition used herein.

[0152] In another embodiment of the use according to any of the above embodiments, the complex protein composition comprises or consists of hydrolyzed or partially hydrolyzed protein substances derived from milk such as casein or whey, animals such as meat or fish, grains such as rice or corn, plants such as soybeans, or combinations thereof.

[0153] Preferably, the complex protein composition comprises or consists of hydrolyzed whey 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, preferably hydrolyzed lactalbumin or a combination thereof.

[0154] Particularly preferably, the complex protein composition comprises or consists of proteolytic hydrolysates selected from casein hydrolysates, soybean hydrolysates, rice hydrolysates, potato hydrolysates, fish hydrolysates, ovalbumin hydrolysates, whey protein hydrolysates, gluten hydrolysates, animal and plant protein hydrolysates, preferably whey protein hydrolysates and combinations thereof.

[0155] In a preferred embodiment of the above-described use, the degree of hydrolysis of the complex protein composition is 20-80%, preferably 30-80%, and particularly preferably 40-60%.

[0156] The invention will now be further illustrated by the following non-limiting embodiments.

[0157] Example: Example 1: Harvesting and Preparation of Plant Material As an initial harvesting step, the sunflower's ray florets turn from green to yellow and begin to grow above the disc florets. Figure 1 When A), remove the terminal capitulum. The intact capitulum is retained in the germination box, with moistened filter paper at the bottom and the top covered. The harvested material is stored at 4°C until further processing, not exceeding 14 days.

[0158] Remove the ray florets and collect the tubular florets with a diameter between 4 mm and 6 mm using a scalpel. Figure 1 (B and 1C). The tubular flowers are assembled in a plastic cup for further processing. Throughout the process, the material is kept under suitable temperature conditions.

[0159] Example 2: Surface disinfection of plant materials Surface sterilization was performed under a clean bench. 96% EtOH was added to each plastic cup containing tubular flowers for a few seconds, followed by rinsing with sterile water. 3% NaOCl was added for approximately half an hour, followed by several rinsings with sterile water. The plant material was transferred to plastic bags, a suitable separation buffer was added (see, for example, Table 1), and the bags were sealed. The bags were stored under suitable conditions until further processing.

[0160] Table 1

[0161] The separation buffer serves as the basic liquid culture medium in the DL system further described herein. Further supplements, particularly at least one HDACi and / or at least one histone methyltransferase inhibitor (HMTi), or preferably a combination thereof, are added to the DL system (liquid and / or solid culture medium) during microspore culture.

[0162] Example 3: Microspore isolation Then homogenize the tubular flowers in the bag. After further homogenization, add additional separation medium directly to the bag. Pour the resulting material through a nylon sieve (approximately 25 μm to 100 μm pore size) and wash with more separation medium. Load the solution containing microspores into tubes. Centrifuge the tubes at 200 g for 10 minutes.

[0163] The supernatant was decanted, and sufficient separation medium was added to agitate the precipitate. The solutions from all tubes were combined and decanted multiple times through a nylon sieve (20 μm to 100 μm). Any remaining microspores on the sieve were washed off with separation buffer.

[0164] To further stabilize microspores and improve supplement availability, optimization of post-isolation culture was also tested. For this purpose, a bilayer system was established in all media used during sunflower microspore culture and callus induction, as well as during desired haploid induction. At least a portion of the media was solidified using agarose, agar, or gellan gum (another suitable solidifying agent currently being tested). This creates a physically optimal environment that allows for the stabilization of microspore development during culture. A portion of the components is rapidly available from the liquid layer, while other supplements and components placed on top of the solid layer of the bilayer system for microspores diffuse only slowly. Therefore, the relevant supplements can always be used during development, which is more efficient at producing haploid cells than simple culture in the medium alone.

[0165] Example 4: Callus Induction Example 4.1: TSA and colchicine Centrifuge a solution of 80,000 microspores per milliliter at 200 g for 10 minutes and discard the supernatant. Add induction medium I (see, for example, Table 2) to the remaining microspores and aliquot into plastic tubes. Histone deacetylase inhibitor (HDACi), or optionally or alternatively HMTi, and colchicine are added to each tube, and the microspore solution is stored at 26°C for two days. If HDACi (triazolin A) is used, it is dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1 nM–10 μM, preferably 100 nM–10 μM, more preferably 1 μM–10 μM, depending on the genotype and plant donor source. If colchicine is used, it is dissolved in water to a final concentration of 1 mg–500 mg, preferably 10 mg–200 mg, more preferably 50 mg–100 mg, depending on the genotype and plant donor source. The microspores were removed by centrifugation at 200g for 10 minutes, removing HDACi and / or optionally at least one HMTi (e.g., the BIX-type HMTi mentioned herein) as well as the chromosome doubling agent. The supernatant was decanted, and the appropriate amount of induction medium I was added to resuspend the microspores. The tubes were incubated in the dark at 26°C for several days.

[0166] Table 2

[0167] The microspore solution was then centrifuged at 200g for 10 minutes, and the supernatant was discarded. The appropriate amount of induction medium II (see, for example, Table 3.1) was added, and the microspore solution was plated in petri dishes and placed at 26°C for callus induction.

[0168] Table 3.1

[0169] Depending on the tissue culture length and the tissue culture system, at least one antibiotic, such as ampicillin, penicillin, or temonidine, can be added to incubation medium II. The first microspore nuclear division (DAPI staining) can be observed one to two weeks after isolation. Figure 2 Callus tissue structures became visible to the naked eye approximately three weeks after separation. Figure 3 ).

[0170] Example 4.2: Callus induction - auxin analogue Centrifuge a solution of 80,000 microspores per milliliter at 200 g for 10 minutes and discard the supernatant. Add induction medium I (see Table 3.2, e.g.) to the remaining microspores and aliquot into plastic tubes. Histone deacetylase inhibitor (HDACi), and / or optionally histone methyltransferase inhibitor (HMTi), and colchicine are added to each tube, and the microspore solution is stored at 26°C for 2 days. If using HDACi trichostatin A (TSA), dissolve it in dimethyl sulfoxide (DMSO) to a final concentration of 1 nM–10 μM, preferably 100 nM–10 μM, more preferably 1 μM–10 μM, depending on genotype and plant donor source. If using colchicine, dissolve it in water to a final concentration of 1 mg–300 mg, preferably 10 mg–200 mg, more preferably 50 mg–100 mg, depending on genotype and plant donor source. Another antimitotic agent, such as asulfanilamide and / or trifluralin, may also be used. HDACi and / or optionally at least one HMTi and chromosome doubling agent are removed by centrifugation for 10 minutes at 200g. The supernatant is discarded, and the appropriate amount of induction medium I is added to resuspend the microspores. Typically, auxin or its (synthetic) analogues are applied at a concentration of approximately 0.05 to approximately 3 mg / L. The tubes are incubated in the dark at 26°C for several days.

[0171] Table 3.2

[0172] Then, centrifuge the microspore solution at 200g for 10 minutes and decant the supernatant. Add the appropriate amount of induction medium II (see, for example, Table 3.3) according to the treatment method, place the microspore solution in a petri dish, and incubate 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.

[0173] Table 3.3

[0174] Depending on the tissue culture length and the tissue culture system, at least one antibiotic can be added to incubation medium II, such as carbenicillin, penicillin, or timentin. First microspore nuclear division (DAPI staining) can be observed 1 to 2 weeks after isolation. Callus structures are visible to the naked eye approximately three weeks after isolation.

[0175] Auxin analogues were titrated at the different concentrations described above.

[0176] This experiment surprisingly showed that different auxins, synthetic auxins, and auxin analogs could induce microspore division and callus formation from different genotypes. Different treatments had no negative impact on callus formation, and the hormones 4CIPA / 4-CPA showed a trend towards increasing callus number over a wider concentration range. Figure 8A ), where exemplarily shown Figure 8A The data were generated at a concentration of 1 mg / L. It was also confirmed that the use of hormones in callus induction and embryo induction steps has an impact on further plant regeneration. Figure 8B Analysis of the number of shoots regenerated per unit of callus tissue showed that auxin analogs 3.5ME and 4CIPA / 4-CPA exhibited a trend of increasing regeneration. Figure 8B ).

[0177] Example 4.3: Callus induction by different concentrations of 2,4-D A microspore density solution of 80,000 microspores per milliliter was centrifuged at 200 g for 10 minutes, and the supernatant was discarded. Induction medium I (see, for example, Table 3.4) was added to the remaining microspores and aliquoted into plastic tubes. Histone deacetylase inhibitor (HDACi) and / or optionally at least one HMTi and colchicine were added to each tube, and the microspore solution was stored at 26°C for two days. If HDACi trichostatin A (TSA) was used, it was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1 nM–10 μM, preferably 100 nM–10 μM, more preferably 1 μM–10 μM, depending on the genotype and plant donor source. If colchicine was used, it was dissolved in water to a final concentration of 1 mg–500 mg, preferably 10 mg–200 mg, more preferably 50 mg–100 mg, depending on the genotype and plant donor source. HDACi and / or optionally at least one HMTi and chromosome doubling agent were removed by centrifugation for 10 minutes at 200g. The supernatant was discarded, and the appropriate amount of induction medium I was added to resuspend the microspores. The tubes were incubated in the dark at 26°C for several days.

[0178] Table 3.4

[0179] Subsequently, the microspore solution was centrifuged at 200g for 10 minutes, and the supernatant was discarded. Depending on the treatment method, different concentrations of 2,4-D induction medium II were added in appropriate amounts (see, for example, Table 3.5), and the microspore solution was spread in petri dishes and placed at 26°C for callus induction.

[0180] Table 3.5

[0181] Depending on the tissue culture length and the tissue culture system, at least one antibiotic, such as carbenicillin, penicillin, or temenine, can be added to induction medium II. The first microspore nuclear division (DAPI staining) can be observed 1 to 2 weeks after isolation. The callus structure can be seen with the naked eye approximately three weeks after isolation.

[0182] This experiment shows that we were able to induce microsporogenesis and callus formation using several concentrations of 2,4-D. Different treatments neither reduced nor enhanced callus formation, and the production of microsporocyte-derived embryos was observed in all treatments and the control. Figure 9 ).

[0183] Example 5: Plant Regeneration Example 5.1: First step of regeneration Callus tissue was removed from liquid medium and transferred to regeneration medium containing 1 mg / L BAP (see, for example, Table 4) and grown at 26°C under light until plant regeneration was observed, approximately 2 weeks after transfer. Figure 4 ) Table 4:

[0184] For example, by presenting as Figure 5 A and Figure 5 As shown in the photograph of B. Here, microspores cultured in vitro according to the present invention produce viable sunflower plants, which can be further analyzed and cultured.

[0185] Example 5.2. Plant rooting Transfer the seedlings to a growth / rooting medium (see Table 5, for example), with or without a auxin (PAA (phenylacetic acid) or IBA is used in this example), and culture under light at 24°C until root formation is observed, approximately 2 weeks after transfer. Figure 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 obvious root formation. Interestingly, in the hormone-free medium, 56% of the plants were able to form roots.

[0186] Table 5:

[0187] Example 6: Plant ploidy examination by flow cytometry To examine the ploidy of plant materials obtained, for example, in the experiments described in detail in Example 5.1 above, a ploidy examination based on flow cytometry was performed. The procedure used was the same as that described by Noga et al. for oats ( Avenasativa The procedure is equivalent to (Noga, A). et al .Conversion of oat( Avenasativa )haploidembryos into plants in relation to embryo developmental stage and regeneration media. In Vitro Cell. Dev.Biol. -Plant 52,590–597 (2016). Flow cytometry for sunflower analysis was performed as follows: Leaf samples were manually removed with a scalpel and cut into small pieces. The sliced ​​material was added to tubes containing combined extraction and staining solutions, namely Cystain UV Ploidy (Sysmex). The solution was then filtered and placed in a 96-well plate for measurement. MACSQuant was then used. TM Analyzer 10 measures DAPI emission under 40s excitation at a maximum of 461nm.

[0188] Exemplary results Figure 6 and Figure 7 The diagram shows a comparison of the scattering patterns of control diploid donor plants with the results of microspore-derived plants of spontaneous double haploid (DH) lines produced according to the present invention. Both patterns shown are comparable and represent the same ploidy rank. The results thus confirm that the method of the present invention is perfectly suited for the convenient and reliable production of DH sunflower lines via the microspore pathway.

[0189] Example 7: Plant transfer to greenhouse and seed production Plants with good external root systems were transferred to soil and placed in a fog chamber with a relative humidity of 90%, a day length of 16-18 hours, and a temperature of 25°C. After 21 days, the humidity was reduced to 60%. After 7 days, the plants were finally grown in a chamber with a relative humidity of 50%, a day length of 16-18 hours, and a temperature of 25°C. The sunflower double haploid plants were fertile, and the anthers and stigmas were easily observed in the pollen. Figure 11A(and B). Before flowering, cover the sunflower inflorescences with plastic bags to ensure self-pollination. Seeds can be harvested 6–10 weeks after the plants are moved to the greenhouse. These experiments demonstrate that the newly established method can quickly and conveniently produce viable sunflowers.

[0190] While several possible embodiments have been disclosed above, the embodiments of the present invention are not limited thereto. These exemplary embodiments are not intended to be exhaustive or unnecessarily limiting of the scope of the invention, but rather have been chosen and described to explain the principles of the invention so that those skilled in the art can practice it. In fact, many modifications of the invention will be apparent to those skilled in the art from the foregoing description, in addition to those described herein. These modifications are intended to fall within the scope of the appended claims. Furthermore, the terminology used herein is for the purpose of describing exemplary embodiments only, and is not intended to be limiting, as the scope of the various embodiments of the invention is defined only by the appended claims and their equivalents. Therefore, the scope of the invention is indicated by the following claims, rather than by the foregoing description and the above embodiments, and is intended to cover all changes that fall within their meaning and equivalents.

[0191] All patents, applications, publications, test methods, documents and other materials cited herein are incorporated herein by reference in their entirety as if they were physically present in this specification.

[0192] Example 8: Optimization of microspore culture using DL and optional activated carbon The study also specifically tested whether using a two-layer system instead of liquid culture medium alone showed better results. Experiments conducted in a liquid culture medium system confirmed this (see [link to study].) Figure 12A (No other data is displayed).

[0193] In addition to using a bilayer culture system during microspore culture, further attempts were made to improve callus induction rate and haploid yield. Activated charcoal was added to the bilayer for this purpose.

[0194] for Figure 12A The experimental results shown confirm the following data: Table 6

[0195] For what is shown Figure 12B Based on the results of the experiment, the following data were used to confirm the various genotypes tested: Table 7

[0196] Surprisingly, certain genotypes using the DL system and, advantageously, additionally using activated charcoal (alone or with the DL system) produced more callus per milliliter of initial sunflower microspores, such as Figure 12A and 12B As shown. For certain genotypes, it was confirmed that using the DL system instead of the conventional medium resulted in particularly good microspore growth and development. Activated charcoal showed an improving effect on most of the genotypes tested. For certain genotypes (data not shown), activated charcoal alone in the liquid medium system (using the aforementioned beneficial supplements, particularly at least one HDACI and / or at least one HMTi) also increased the number of callus units formed per milliliter of initial microspore culture.

[0197] Example 9: Callus induction-auxin mixture Centrifuge the solution containing 80,000 microspores per milliliter at 200 g for 10 minutes and discard the supernatant. Add induction medium I (see, for example, Table 8) to the remaining microspores and divide into plastic tubes. Histone deacetylase inhibitor (HDACi) and colchicine are added to each tube, and the microspore solution is stored at 26°C for two days. If using HDACi trichostatin A (TSA), dissolve it in dimethyl sulfoxide (DMSO) to a final concentration of 1 nM–10 μM, preferably 100 nM–10 μM, more preferably 1 μM–10 μM, depending on the genotype and plant donor source.

[0198] If colchicine is used, it is dissolved in water to a final concentration of 1 mg-300 mg, preferably 10 mg-200 mg, more preferably 50 mg-100 mg, depending on the genotype and plant donor source. Other antimitotic agents such as azoxystrobin and / or trifluralin may also be used.

[0199] Table 8

[0200] Then, centrifuge the microspore solution at 200g for 10 minutes and discard the supernatant. Add the appropriate amount of induction medium II (see, for example, Table 9) according to the treatment method for different auxin combinations, and plate the microspore solution in petri dishes and place at 26°C for callus induction. Abbreviations used in Table 9: 3,5ME = 3,5-dimethylphenoxyacetic acid; 4-CPA or 4CIPA = p-chlorophenoxyacetic acid.

[0201] Table 9

[0202] Depending on the tissue culture length and the tissue culture system, at least one antibiotic, such as carbenicillin, penicillin, or temontin, may be added to incubation medium II. First microspore nuclear division (DAPI staining) can be observed within one to two weeks after isolation. Callus structures are visible to the naked eye approximately three weeks after isolation.

[0203] This experiment demonstrates that mixtures of different types of auxins and auxin analogs can induce microspore division and callus formation from different genotypes. In particular, the experiment confirmed the use of balanced auxin mixtures, with a significant component being 2,4-D, which is well-known and relatively inexpensive when used in appropriate and balanced combinations with other auxins or auxin analogs. Different treatments had no negative impact on callus formation and were consistent with test results for different genotypes in the control group (results in Table 10).

[0204] Table 10

[0205] Example 10: Callus induction - lactalbumin A solution of 80,000 microspores per milliliter was centrifuged for 10 minutes at 200 g, and the supernatant was discarded. The remaining microspores were added to induction medium I with varying concentrations of lactalbumin (see, for example, Table 11) and aliquoted into plastic tubes according to the treatment. Histone deacetylase inhibitor (HDACi) and colchicine were added to each tube, and the microspore solution was stored at 26°C for two days. If HDACi trichostatin A (TSA) was used, it was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 1 nM–10 μM, preferably 100 nM–10 μM, more preferably 1 μM–10 μM, depending on the genotype and plant donor source. If colchicine was used, it was dissolved in water to a final concentration between 1 mg and 300 mg, preferably 10 mg to 200 mg, more preferably 50 mg to 100 mg, depending on the genotype and plant donor source. Another antimitotic agent, such as sulfadiazine and / or trifluralin, can also be used.

[0206] Table 11

[0207] Then, the microspore solution was centrifuged at 200g for 10 minutes, and the supernatant was discarded. The appropriate amount of induction medium II (see Table 12) was added, and the microspore solution was spread in a petri dish and placed at 26°C for callus induction.

[0208] Table 12

[0209] Depending on the tissue culture length and the tissue culture system, at least one antibiotic can be added to incubation medium II, such as carbenicillin, penicillin, or timentin. First microspore nuclear division (DAPI staining) can be observed 1 to 2 weeks after isolation. Callus structures can be observed with the naked eye approximately three weeks after isolation.

[0210] Surprisingly, the experiment showed that lactalbumin hydrolysate affected microspore division and callus growth. The presence of lactalbumin hydrolysate increased the amount of callus, and even in some genotypes, its availability was crucial for callus production. Figure 13 ).

Claims

1. A method used to produce the sunflower species (Helianthus annuus) A method for processing haploid, polyhaploid, and / or diploid cells, embryos, callus tissue, seeds, and / or plants, said method comprising: i) Provide at least one isolated microspore of a sunflower plant; ii) Optionally: pre-culture at least one microspore from step i) in the presence of the complex protein composition; iii) In the presence of at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi), and optionally in the presence of a complex protein composition, at least one microspore from step i) or ii) is cultured in a bilayer system to induce and thus obtain callus and / or embryos. iv) Culture the said callus and / or embryo; and v) Optionally, regeneration is performed from the callus or embryo of step iv) to obtain at least one plant, and preferably at least one haploid, multiple haploid and / or double haploid seed. Preferably, activated carbon is added in steps (i), (ii) and / or (iii).

2. The method of claim 1, wherein prior to step i) of claim 1, providing at least one isolated microspore of a sunflower plant, the following steps are performed: (a) Provide at least one terminal or lateral capitulum of a sunflower plant; (b) Harvesting at least one tubular floret or a portion thereof from at least one terminal or lateral capitulum to obtain tubular floret material; and (c) Optionally: disinfect the surface of the tubular flower material; (d) Optionally: Wash the disinfected tubular flower material; (e) The optionally sterilized and washed tubular flower material is transferred to an isolation medium containing a macro- and micro-element salts, sugars, and a complex protein composition; and (f) Homogenize and optionally sieve the tubular flower material to provide at least one isolated microspore of the sunflower plant.

3. The method according to claim 1 or 2, further comprising the following steps in step i) and / or ii): (b1) In the presence of at least one amino acid, such as glutamine, and / or in the presence of at least one nucleoside, such as uridine and cytidine, at a concentration of about 10 mg / L to about 800 mg / L, preferably about 20 mg / L to about 750 mg / L, at a concentration of about 10 mg / L to about 800 mg / L.

4. The method according to any of the preceding claims, further comprising the following step in step iii): (iii.a) Add at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) to a final concentration of 1 nM-10 μM, preferably 100 nM-10 μM, more preferably 1 μM-10 μM; (iiib) After incubation for about 1 hour to 60 hours, preferably from 5 hours to 50 hours, more preferably from 12 hours to 48 hours, the at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) are removed, wherein the incubation is optionally carried out in the dark.

5. The method according to any one of the preceding claims, wherein the at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) is selected from: trichostatin A (TSA); hydroxamic acid and hydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and pabistat (LBH589); cyclic tetrapeptides, such as aprotinin B, and cyclic condensates, such as romidesin (FK228); benzamides, such as entenostat (MS-275), tazloride (CI994), and moxistat (MGCD0103); BIX1294 and BIX1338; electrophilic ketones; and fatty acid compounds, such as phenylbutyrate and valproic acid, preferably, the histone deacetylase inhibitor (HDACi) i) and / or at least one histone methyltransferase inhibitor (HMTi) is trachomatis A (TSA), BIX1294, BIX1338, SAHA, or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228); preferably, in the culture medium used in step iii), the concentration of the at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi) is about 1 nM to about 10 µM, preferably 100 nM to 10 µM, more preferably 1 µM to 10 µM; and / or, in the culture medium used in step ii) or step iii), the concentration of the complex protein composition is about 100 mg / L to about 20000 mg / L, preferably about 500 mg / L. The concentration is approximately 15,000 mg / L to about 15,000 mg / L, particularly preferably about 2,000 mg / L to about 10,000 mg / L.

6. The method according to any one of the preceding claims, wherein the complex protein composition comprises or is composed of hydrolyzed or partially hydrolyzed protein substances derived from milk (e.g., casein, whey), animal sources (e.g., meat, fish), grains (e.g., rice, corn), plants (e.g., soybeans), or combinations of the above sources; and / or, the complex protein composition comprises or is composed of: hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium casein, hydrolyzed calcium casein, hydrolyzed whole milk, semi-skimmed / fully skimmed hydrolyzed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, preferably hydrolyzed lactalbumin, or combinations of the above substances; and / or, the complex protein composition comprises or is composed of protein hydrolysates selected from the following: Casein hydrolysate, soybean hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant protein hydrolysates, preferably lactalbumin hydrolysate, and combinations of the above hydrolysates; preferably, the degree of hydrolysis is about 20% to about 80%, more preferably about 30% to about 80%, particularly preferably about 40% to about 60%; optionally, the complex protein composition is provided together with activated carbon.

7. The method according to any preceding claim, wherein in step iii), the at least one microspore during callus induction, or the callus or embryo obtained from the 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-anisolic acid (dicamba), naphthaleneacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA); cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylacrylamido)purine (2iP), and thidiazuron (TDZ); gibberellins; and abscisic acid; and mixtures thereof, in steps iii) and / or iv) and / or v).

8. The method according to any of the preceding claims, wherein at least one HDACi and / or at least one HMTi is used together with at least one plant growth regulator in the culture medium used in step iii), preferably together with activated carbon.

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

10. A kit for producing haploid, multi-haploid, and / or double-haploid cells, embryos, callus, seeds, and / or plants of the species sunflower from at least one isolated microspore, comprising: (a) at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi), and optionally a complex protein composition; as well as (b) A means for providing at least two culture media in a bilayer system, the bilayer system comprising a liquid culture medium and a solid culture medium; and (c) Optional further components, including at least one plant growth regulator and / or one or more chromosome doubling agents, such as colchicine, sulfadiazine and / or trifluralin; The at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi), as well as the complex protein composition, if present, are contained in the same container or in two or more separate containers.

11. The method according to claim 10, wherein the at least one histone deacetylase inhibitor (HDACi) and / or the at least one histone methyltransferase inhibitor (HMTi) is selected from: trichostatin A (TSA); hydroxamic acid and hydroxamic acid salts, such as vorinostat (SAHA), belistat (PXD101), dacelostat (LAQ824), and pabistat (LBH589); cyclic tetrapeptides, such as aprotinin B, and cyclic peptides, such as romidesin (FK228); and benzamides, such as entenoxetine (MS-275). The histone deacetylase inhibitors (HDACi) and / or HMTi are preferably trachomatis A (TSA), BIX1294, BIX1338, SAHA, or romidesin (FK228), or a combination of at least two of TSA, BIX1294, BIX1338, SAHA, and romidesin (FK228).

12. The method according to claim 10 or 11, wherein the complex protein composition comprises or is composed of hydrolyzed or partially hydrolyzed protein substances derived from milk (e.g., casein, whey), animal sources (e.g., meat, fish), grains (e.g., rice, corn), plants (e.g., soybeans), or combinations of the above sources; and / or, the complex protein composition comprises or is composed of: hydrolyzed milk protein isolate, hydrolyzed milk protein concentrate, hydrolyzed casein isolate, casein hydrolysate, hydrolyzed lactalbumin, hydrolyzed sodium casein, hydrolyzed calcium casein, hydrolyzed whole milk, semi-skimmed / fully skimmed hydrolyzed milk, hydrolyzed soy protein isolate, hydrolyzed soy concentrate, preferably hydrolyzed lactalbumin, or combinations of the above substances; and / or, the complex protein composition comprises or is composed of protein hydrolysates selected from the following: Casein hydrolysate, soybean hydrolysate, rice protein hydrolysate, potato protein hydrolysate, fish protein hydrolysate, egg white protein hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, and animal-plant complex protein hydrolysate, preferably hydrolyzed lactalbumin, and combinations of the above hydrolysates; preferably, the degree of hydrolysis is 20%–80%, more preferably 30%–80%, and particularly preferably 40%–60%; Optionally, the kit further comprises one or more plant growth regulators selected from auxins, synthetic auxins, or auxin analogues, 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-anisolic acid (dicamba), naphthaleneacetic acid (NAA), indoleacetic acid (IAA), and indole-3-butyric acid (IBA); cytokinins, including 6-benzylaminopurine (BAP), 6-(γ,γ-dimethylacrylaldehyde amino)purine (2iP), and thidiazuron (TDZ); gibberellins; and abscisic acid; and mixtures thereof, preferably mixtures comprising at least 2,4-D and at least one of 3.5ME, 4-CPA, and / or BAP, and / or the kit further comprises one or more chromosome doubling agents such as colchicine, asulfanilamide, and / or trifluralin.

13. Use of at least one histone deacetylase inhibitor (HDACi) and / or at least one histone methyltransferase inhibitor (HMTi), preferably as defined in claim 5 and in conjunction with a bilayer system comprising a liquid layer and a solid layer, and optionally: use of a complex protein composition, preferably as defined in claim 6 and in conjunction with a bilayer system comprising a liquid layer and a solid layer and / or in conjunction with a histone deacetylase inhibitor (HDACi) and / or a histone methyltransferase inhibitor (HMTi), preferably in conjunction with activated charcoal, or use of a kit as defined in any one of claims 10 to 12, for producing haploid, multi-haploid, and / or double-haploid embryos, callus, and / or plants or seeds of the species sunflower, preferably by the method of any one of claims 1 to 9.

14. A species of sunflower ( Helianthus annuus A population of haploid, polyhaploid, and / or dihaploid plants, which are directly derived from a single tubular flower, preferably obtained by the method according to any one of claims 1-9 or by the method according to any one of claims 1-9.

15. A population of haploid, polyhaploid, and / or double haploid plants of the species sunflower according to claim 14, wherein the population comprises at least 10 individuals.

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