Method for producing haploid, and doubled haploid pepper plants by isolated microspore culture

Abstract

The present invention relates to a method for the production of haploid, polyhaploid and / or doubled haploid embryos, calli, seeds and / or plants of the species Capsicum annuum from isolated microspore cultures, especially in a non-genotype dependent way, more specifically to a method comprising contacting the isolated microspores with a histone deacetylase inhibitor (HDACi) in a double layer system.

Description

[0001] Method for producing haploid, and doubled haploid pepper plants by isolated microspore culture

[0002] Technical Field

[0003] The present invention relates to a method for the production of haploid, polyhaploid and / or doubled haploid embryos, calli, seeds and / or plants of the species Capsicum annuum from isolated microspore cultures, especially in a non-genotype dependent way, more specifically to a method comprising contacting the isolated microspores with a histone deacetylase inhibitor (HDACi) in a double layer system.

[0004] Background

[0005] The long-term development of parental lines by inbreeding is one of the limiting factors and a major problem in competitive F1 hybrids breeding. Doubled haploids (DH) technologies offer a time-saving approach to obtain pure breeding lines by reducing the period to approximately 3-5 years. Advantages of the doubled haploid production are the ability to achieve fully homozygous plant genotypes in one generation as well as the manifestation of recessive alleles in haploid plants usually masked by the heterozygous state of a diploid plant, whereby the identification, assessment, and selection of plants with traits of agronomic importance is facilitated.

[0006] Haploid plants can occur spontaneously or develop as the result of apomixis and chromosome elimination after interspecific or intergeneric hybridization. Additionally haploid plants may be generated after induction of gametogenesis in microspore, anther, ovule or ovary culture.

[0007] In pepper, however, even at date most of these methods are inefficient and / or laborious. Established protocols for the production of haploid, doubled haploid and / or polyploid cells in pepper start with laborious isolation of anthers from flower buds with subsequent induction and isolation of microspores, induction of embryos and, in some cases, the regeneration of plants (Supena and Custers, 2011 ; Lantos et al, 2012; Cheng et al, 2013 or Ari et al, 2016). The authors show production of normal looking doubled haploid embryos, however there is still a need to provide methods that are less laborious and have a high efficiency for production of haploid, polyploid and / or doubled haploid Capsicum annuum plants.

[0008] Summary of the Invention

[0009] In one aspect, the present invention relates to method for producing a haploid, polyhaploid and / or doubled haploid cell, embryo, callus, seed and / or plant of the species Capsicum annuum, the method comprising: i) providing at least one isolated microspore of a Capsicum annuum plant, and ii) culturing the at least one microspore from step i) in the presence of at least one histone deacetylase inhibitor (HDACi) in a double layer system to induce and thus obtain a callus and / or embryo, and iii) cultivating the callus and / or embryo, preferably on a solid medium, and iv) optionally, regenerating and thereby obtaining at least one plant from the embryo of step iii), and preferably obtaining at least one haploid, polyhaploid and / or doubled haploid seed thereof.

[0010] The at least one histone deacetylase inhibitor (HDACi) that may be used in any method of the invention described herein may be selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably the HDACi is trichostatin A (TSA) or romidepsin (FK228). Each HDACi may be used alone or in combination with other HDACis disclosed herein.

[0011] The step of regeneration and thereby obtaining at least one plant from the embryo and / or callus of step iii) is preferably performed on a membrane separating the embryo from the medium. The membrane may for example consist of filter paper, cellulose, nitro cellulose, gaze or other material suitable for avoiding direct contact between plant material and medium while allowing transfer of chemicals comprised in the medium. In one embodiment of the invention as defined above, the microspore, callus and / or embryo in step ii) and / or iii) is contacted with 2,4-D, Phenoxyacetic acid (PHAA) and / or p-Chlorophenoxyacetic acid (4-CIPA), wherein the concentration of the auxins is between about 0,5 and 5 pM, preferably between about 1 and 4 pM, more preferably between about 2 and 3 pM.

[0012] The term “double layer system” when used herein is referring to a media system comprising a solid medium as basis with a liquid medium containing the microspores on top. The skilled person is aware of various media that may be useful in such double layer system. For example, the liquid medium may be a sucrose starvation medium, for example using polyols such as mannitol as described in Kim et al., 2008, and / or sorbitol and / or amino acids such as proline whereas the solid medium may be based on for example MS, Gamborg-B5, Nitsch, NLN or Chu N6 basal medium, preferably Chu N6 basal medium including vitamins with a sugar source such as for example sucrose, and / or fructose, and / or glucose, and / or maltose, preferably maltose and / or sucrose, most preferably sucrose, in a range of for example 10 to 200 g / l, preferably 50 to 150 g / l and preferably comprises activated charcoal in a range of 1 to 15 g / l, more preferably 2 to 14 g / l, more preferably 3 to 13 g / l, even more preferably 4 to 12 g / l, even more preferably 5 to 11 g / l, even more preferably 6 to 10 g / l, even more preferably 7 to 9 g / l.

[0013] A further embodiment of the invention is the method as defined above, wherein the provision of at least one isolated microspore of a Capsicum annuum plant, according to step i) preceded by the following steps:

[0014] (a1) providing whole flower buds comprising late uninucleate to early binucleate state microspores,

[0015] (a2) pre-treatment said whole flower buds for about 12h to 4 days, preferably for about 24h to 3 days, more preferably for about 2 days, at about 1 to 10°C, preferably about 3 to 7°C, more preferably about 4°C,

[0016] (a3) optionally disinfection said whole flower buds, wherein disinfection means disinfecting the surface of said whole flower buds,

[0017] (a3) homogenizing said whole flower buds, preferably, wherein the homogenizing is performed in a complex protein composition and wherein the homogenizing is preferably performed using a masticator paddle tissue homogenizer (MPH), (a4) purifying the microspores comprised in the homogenate, for example by filtration, wherein purifying means separation of microspores from debris of the crushed whole flower buds, and optionally,

[0018] (a5) enriching the microspores for example by pelleting the microspores or separation microspores of specific stage using density gradient centrifugation.

[0019] The whole flower buds harvested for isolation of microspores in the invention at hand are about 2 to 10 mm in size, preferably about 4 to 6 mm in size, most preferably about 5 mm in size. They are harvested preferably while the anthers are green with slightly purple tips as described for example in Supena and Custers 2011 , Supena 2021 , Dumas de Vaulx et al. 1981.

[0020] The homogenizing of said whole flower buds may be performed using a MPH, a Waring blender, a GentleMACS™ Octo Dissicator, or may be performed manually. Preferably an MPH is used. In one experiment about 1 to about 300, preferably about 50 to about 150, more preferably about 100 flower buds were immersed in 10 to 150 ml medium, preferably 40 to 100 ml, most preferably 60 ml medium in sealed bags, and processed two times for about 30 s to about 180 s, preferably 60 s to 150 s, more preferably for about 90 s.

[0021] The complex protein composition used in the method of the invention is preferably a protein hydosylate and may comprise or consist of hydrolysed or partially hydrolysed protein matter derived from milk, such as casein or whey, animals, such as meat or fish, cereal, such as rice or corn, plants, such as soybean or combinations thereof and / or the complex protein composition comprises or consists of hydrolysed milk protein isolates, hydrolysed lactoprotein concentrate, hydrolysed casein isolates, casein hydrolysates, hydrolysed lactalbumin, hydrolysed casein sodium, hydrolysed calcium caseinate, hydrolysed full cow's milk, partially or completely skimmed milk, hydrolysed soy protein isolate, hydrolysed soybean concentrate or combinations thereof and / or the complex protein composition comprises or consists of a proteolysate selected from the group consisting of casein hydrolysate, soybean hydrolysate, rice proteolysate, potato protein hydrolysate, fish protein hydrolysate, ovalbumin hydrolysate, lactalbumin hydrolysate, gluten hydrolysate, animal and plant proteolysate and a combination thereof, preferably the hydrolysis degree is in a range from about 20 to about 80%, preferably from about 30 to about 80%, particularly preferably from about 40 to about 60%.

[0022] In a further embodiment, the method of the invention as defined above is further comprising within step ii) the following steps:

[0023] (iia) adding in the liquid medium the at least one histone deacetylase inhibitor (HDACi) to a final concentration of 100 nM to about 10 pM, preferably 1 pM to 10 pM, more preferably 1.25 pM to 5 pM, most preferably 1.25 pM, 2.5 pM or 5 pM to the microspore suspension in a double layer system, and

[0024] (iib) removing the at least one histone deacetylase inhibitor (HDACi) after incubation for about 1 h to 72 h, preferably for about 8 h to 60 h, more preferably for about 12 h to 48 h, most preferably for about 48h; wherein incubation optionally takes place in the dark.

[0025] A further embodiment of the invention is the method as described above wherein the steps are performed at a temperature of 20 to 34 °C, preferably at 24 to 30 °C, more preferably 26 to 29 °C most preferably at about 28 °C. The method as described above is to be performed without applying a heat shock at temperatures at or above 34 °C.

[0026] In a further embodiment of the method of the invention as described above the one or more chromosome doubling agent(s) is / are added during step ii) and / or step iii) and / or step iv), preferably during step ii). Chromosome doubling agents are preferably selected from the list consisting of colchicine, trifluralin, amiprophos- methyl, oryzalin, pronamide, podophyllin and nitrous oxide. Preferably, colchicine is used.

[0027] In a further embodiment of the invention the at least one histone deacetylase inhibitor (HDACi) is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0028] A further embodiment of the invention is the use of a histone deacetylase inhibitor (HDACi) for producing a haploid, polyhaploid and / or doubled haploid embryo, callus and / or plant or seed of the species Capsicum annuum in a method as described above.

[0029] The HDACi in the above mentioned embodiment is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0030] A further embodiment of the invention is a population of haploid, polyhaploid and / or doubled haploid plants of the species Capsicum annuum, directly derived from one or more isolated microspore, obtained by a method as defined above.

[0031] A further embodiment of the invention is said population of haploid, polyhaploid and / or doubled haploid plant of the species Capsicum annuum, wherein the population comprises at least 10 individuals.

[0032] In another embodiment of the method according to any of the embodiments described above, the step ii) of the method further comprises one or more of the following steps:

[0033] (i) optionally, cultivating the microspore suspension in the dark in a double layer system,

[0034] (j) adding at least one histone deacetylase inhibitor (HDACi) to a final concentration of 100 nM to about 10 pM, preferably 1 pM to 10 pM, more preferably 1 .25 pM to 5 pM, most preferably 1 .25 pM, 2.5 pM or 5 pM to the microspore suspension in a double layer system, and (k) removing the at least one histone deacetylase inhibitor (HDACi) from the microspore culture of step ii).

[0035] In one embodiment of the method described above, step ii) comprises all steps (i)- (k). In a further embodiment of the method according to any of the embodiments described above, the at least one histone deacetylase inhibitor (HDACi) is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably wherein the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0036] In one embodiment of the method according to any of the embodiments described above, the at least one histone deacetylase inhibitor (HDACi) is present at a concentration from about 100 nM to about 10 pM, preferably 1 pM to 10 pM, more preferably 1 .25 pM to 5 pM, most preferably 1 .25 pM, 2.5 pM or 5 pM in the culture medium used in step ii).

[0037] In another embodiment of the method according to any of the embodiments described above, the callus or embryo is contacted with one or more plant growth regulator(s) selected from auxins, cytokinins, gibberellins, abscisic acid and mixtures thereof in step iv). Preferably the callus and / or embryo in step iv) is contacted with BAP and / or kinetin, preferably in a concentration of 0.01 to 0.1 mg / l BAP, more preferably 0.025 to 0.075 mg / l BAP, most preferably 0.05 mg / l BAP or 0.05 to 0.2 mg / l kinetin, preferably 0.075 to 0.15 mg / l kinetin, most preferably 0.1 mg / l kinetin. Preferably the growth regulator(s) are comprised in a % MS medium, most preferably in % MS basal salts comprising full MS vitamins as described for example in Supena et al. 2006.

[0038] In a further embodiment of the method according to any of the embodiments described above, one or more chromosome doubling agent(s), such as trifluralin, amiprophos-methyl, oryzalin, pronamide, podophyllin and nitrous oxide, preferably, colchicine, is / are added during step ii) and / or step iii) and / or step iv). In yet another aspect, the present invention relates to a population of haploid, polyhaploid and / or doubled haploid Capsicum annuum plants directly derived from one or more whole flower buds, preferably obtained by a method according to any of the embodiments described above.

[0039] In one embodiment of the population described above, the population comprises at least 10 individuals.

[0040] Definitions

[0041] Androgenesis is defined as the process of generation of an individual whose genetic background is derived exclusively from a nucleus of male origin. That is, androgenesis is the generation of a plant exclusively from a male, haploid gamete precursor (gametophyte).

[0042] Haploid is an attribute applicable to cells or to plants or parts of plants, of which the chromosomes contained in their nucleus are each in only one copy (n).

[0043] Diploid is an attribute applicable to cells or to plants or parts of plants, of which the chromosomes contained in their nucleus are each in two copies (2n).

[0044] Doubled haploid is an attribute applicable to cells or to plants or parts of plants comprising said cells, the chromosome stock of which was multiplied artificially, most often by chemical treatment, such as with colchicine, trifluralin, amiprophos- methyl, oryzalin, pronamide, podophyllin and nitrous oxide, preferably, colchicine or by spontaneous doubling. This doubling of the chromosome stock makes it possible to obtain a cell, plant or plant part that has two copies of each chromosome in its nucleus (2n), wherein said cell, plant or plant part is entirely homozygous or essentially homozygous.

[0045] Polyhaploid is an attribute applicable to cells or to plants or parts of plants comprising said cells, these cells being haploid initially, and their chromosome stock having tripled or more spontaneously. The cell, plant or plant part that has at least three copies of each chromosome in its nucleus (3n or 4n, etc.), wherein said cell, plant or plant part is entirely homozygous or essentially homozygous. The term microspore is herein used to designate an immature male gametophyte of a plant at all stages of its in vitro growth, including its multicellular form derived from the sporophytic divisions of a single cell isolated microspore, and still enclosed within the original exine wall (this multicellular form is herein also referred to as a multicellular structure). At the uninucleate development stage of microspores, the nucleus is not divided yet and located in the centre of the cell. At late uninucleate stage the nucleus has migrated to the periphery of the cell, and the microspore is considered polarized. At early binucleate stage the nucleus already divided into a vegetative nucleus located in the centre and a generative nucleus located at the periphery.

[0046] A plant of the species Capsicum annuum is, for example, a bell pepper, blocky pepper, cayenne pepper, sweet pepper, hot pepper, serrano or chili pepper or an ornamental pepper.

[0047] Histone deacetylases are enzymes, which remove acetyl groups from acetyl lysine at the N-terminus of histones thereby increasing the affinity of the histone to DNA. Histone deacetylases therefore play a role in the regulation of DNA expression. Inhibitors of histone deacetylases (HDACi) inhibit histone deacetylases and therefore prevent the removal of acetyl groups from the histones. Histone deacetylase inhibitors are used in the treatment of several diseases.

[0048] A flower bud refers to a reproductive structure containing the reproductive organs, developing from meristems located either at the leaf nodes or at the tip of a stem.

[0049] Macro salts may be selected from the group consisting of ammonium nitrate (NH4NO3), calcium chloride (CaCI2x 2 H2O), magnesium sulfate (MgSO4x 7 H2O), monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), potassium nitrate (KNO3), calcium nitrate (Ca(NO3)2x4 H2O); micro salts may be selected from boric acid (H3BO3), cobalt chloride (CoCI2x 6 H2O), ferrous sulfate (FeSO4x 7 H2O), manganese(ll) sulfate (MnSO4x 4 H2O), potassium iodide (KI), sodium molybdate (Na2Mo04x 2 H2O), zinc sulfate (ZnSO4x 7 H2O), ethylenediaminetetraacetic acid ferric sodium (FeNaEDTA), copper sulfate (CUSO4X 5 H2O). Plant growth regulators are chemical compounds, which affect the growth and development of plants, e.g. by promoting or inhibiting growth. Natural plant growth regulators are plant hormones, which plants produce themselves. Synthetic plant growth regulators, on the other hand, do not naturally occur in plants. Examples of plant growth regulators are given in the detailed description.

[0050] Haploid plants can undergo spontaneous chromosome doubling or chromosome doubling can be enhanced or facilitated by a chromosome doubling agent, which e.g. blocks the function of the spindle fibers during meiosis or mitosis. Exemplary chromosome doubling agents are colchicine, trifluralin, amiprophos-methyl, oryzalin, pronamide, podophyllin and nitrous oxide. The most commonly used chromosome doubling agent is colchicine.

[0051] Detailed Description

[0052] As specified in the Background Section, there is a great need in the art to identify technologies for efficient production of haploid and double haploid Capsicum annuum plants and use this understanding to develop novel methods for producing such plants. The present invention satisfies this and other needs. Embodiments of the present invention relate generally to methods for the production of haploid, polyhaploid and / or doubled haploid Capsicum annuum plants by isolated microspore cultures, especially in a genotype independent way, and more specifically to methods for producing such Capsicum annuum plants comprising contacting the isolated microspores with an inhibitor of histone deacetylase (HDACi) in a double layer system.

[0053] To facilitate an understanding of the principles and features of the various embodiments of the invention, various illustrative embodiments are explained below. Although exemplary embodiments of the invention are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the invention is limited in its scope to the details of construction and arrangement of components set forth in the following description or examples.

[0054] The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the exemplary embodiments, specific terminology will be resorted to for the sake of clarity. It must also be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a composition containing “a” constituent is intended to include other constituents in addition to the one named. In other words, the terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.

[0055] As used herein, the term “and / or” may mean “and,” it may mean “or”, it may mean “one,” it may mean “some, but not all,” and / or it may mean “both.” The term “or” is intended to mean an inclusive “or.”

[0056] Also, in describing the exemplary embodiments, terminology will be resorted to for the sake of clarity. It is intended that each term contemplates its broadest meaning as understood by those skilled in the art and includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. It is to be understood that embodiments of the disclosed technology may be practiced without these specific details. In other instances, well-known methods, structures, and techniques have not been shown in detail in order not to obscure an understanding of this description. References to “one embodiment,” “an embodiment,” “example embodiment,” “some embodiments,” “certain embodiments,” “various embodiments,” etc., indicate that the embodiment(s) of the disclosed technology so described may include a particular feature, structure, or characteristic, but not every embodiment necessarily includes the particular feature, structure, or characteristic. Further, repeated use of the phrase “in one embodiment” does not necessarily refer to the same embodiment, although it may.

[0057] As used herein, the term “about” should be construed to refer to both of the numbers specified as the endpoint (s) of any range. Any reference to a range should be considered as providing support for any subset within that range. Ranges may be expressed herein as from “about” or “approximately” or “substantially” one particular value and / or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplary embodiments include from the one particular value and / or to the other particular value. Further, the term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part 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, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, preferably up to ±10%, more preferably up to ±5%, and more preferably still up to ±1% of a given value.

[0058] Throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges 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, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0059] Similarly, as used herein, “substantially free” of something, or “substantially pure”, and like characterizations, can include both being “at least substantially free” of something, or “at least substantially pure”, and being “completely free” of something, or “completely pure”.

[0060] By “comprising” or “containing” or “including” is meant that at least the named compound, element, particle, or method step is present in the composition or article or method, but does not exclude the presence of other compounds, materials, particles, method steps, even if the other such compounds, material, particles, method steps have the same function as what is named.

[0061] Throughout this description, various components may be identified having specific values or parameters, however, these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present invention as many comparable parameters, sizes, ranges, and / or values may be implemented. The terms “first,” “second,” and the like, “primary,” “secondary,” and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. It is noted that terms like “specifically,” “preferably,” “typically,” “generally,” and “often” are not utilized herein 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 merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the present invention. It is also noted that terms like “substantially” and “about” are utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation.

[0062] It is also to be understood that the mention of one or more method steps does not preclude the presence of additional method steps or intervening method steps between those steps expressly identified. Similarly, it is also to be understood that the mention of one or more components in a composition does not preclude the presence of additional components than those expressly identified.

[0063] The materials described hereinafter as making up the various elements of the present invention are intended to be illustrative and not restrictive. Many suitable materials that would perform the same or a similar function as the materials described herein are intended to be embraced within the scope of the invention. Such other materials not described herein can include, but are not limited to, materials that are developed after the time of the development of the invention, for example. Any dimensions listed in the various drawings are for illustrative purposes only and are not intended to be limiting. Other dimensions and proportions are contemplated and intended to be included within the scope of the invention.

[0064] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully 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 (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.(1985); Transcription and Translation (B.D. Hames & S.J. Higgins, eds. (1984); Animal Cell Culture (R.l. Freshney, ed. (1986); Immobilized Cells and Enzymes (IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); Plant propagation by Tissue Culture 3rdEdition, Volume 1 , The Background; George et al. 2008, among others.

[0065] Methods of the Invention

[0066] In this context, one of the aims of the present invention is to provide a method for the production of haploid, polyhaploid and / or doubled haploid Capsicum annuum plants by or from isolated microspore cultures, especially in a non-genotype dependent (i.e., genotype independent) way.

[0067] Another aim of the invention is to provide calli, embryos, plantlets and plants regenerated from the embryo, progeny of such plants and seed from such plants, usable in or obtained by a method for the production of haploid, polyhaploid and / or doubled haploid Capsicum annuum plants by or from isolated microspore cultures, especially in a non-genotype dependent way.

[0068] Such aims are achieved by the present invention, which relates, in one aspect, to a method for producing a haploid, polyhaploid and / or doubled haploid embryo, callus, seed and / or plant of the species Capsicum annuum comprising: i) providing at least one isolated microspore of a Capsicum annuum plant, ii) culturing the at least one microspore from step i) in the presence of at least one histone deacetylase inhibitor (HDACi) in a double layer system to induce and thus obtain a callus and / or embryo, iii) cultivating the callus and / or embryo, and iv) optionally, regenerating and thereby obtaining at least one plant from the embryo and / or callus of step iii), and preferably obtaining at least one haploid, polyhaploid and / or doubled haploid seed thereof.

[0069] In a preferred embodiment of the method described above, a population of plants is regenerated in step iv), which undergoes spontaneous chromosome doubling at a rate of at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70%. In this context, a population comprises at least 10 individuals. In one embodiment of the method described above, step i) is preceded by one or more of the following steps:

[0070] (a1) providing at least one whole flower bud comprising uninucleate to early binucleate, preferably late uninucleate to early binucleate state microspores,

[0071] (a2) pre-treat said at least one whole flower bud for about 12 h to 4 days, preferably for about 24 h to 3 days, more preferably for about 2 days at 1 to 10 °C, preferably at 4 °C,

[0072] (a3) optionally disinfection said at least one whole flower bud,

[0073] (a3) homogenizing the at least one whole flower bud from step (a2) or (a3),

[0074] (a4) purify the microspores comprised in the homogenate.

[0075] In a preferred embodiment, in step (a1) the whole flower buds are removed from a Capsicum annuum plant; whole flower buds may preferably be removed before the flower opens. The removed whole flower buds may be stored intermediately at room temperature, preferably at chilling temperatures, for example 1 to 10 °C, preferably 2 to 8 °C, more preferably 4 °C.

[0076] In a preferred embodiment of the method described above, the cell suspension is purified in step (a4) by means of a strainer by subsequently passing it through a cell strainer with a pore size of about 50 pm, a strainer with a pore size of about 40pm, a strainer with a pore size of about 30 pm and a strainer with a pore size of about 20 pm. Preferably, passing through each of the strainers with different pore sizes is performed at least 1 to 3 times or exactly 1 to 3 times. Alternatively, the cell suspension may be purified by means of a strainer in step (a4) by passing it through a cell strainer with a pore size in a range from about 25 to 100 pm repeatedly, preferably at least 1 to 6 times or exactly 1 to 6 times.

[0077] In one embodiment of the method according to any of the embodiments described above, the step ii) of the method further comprises one or more of the following steps:

[0078] (i) optionally, cultivating the microspore suspension in the dark in a double layer system, and (j) adding at least one histone deacetylase inhibitor (HDACi) to a final concentration of 100 nM to about 10 pM, preferably 1 pM to 10 pM, more preferably 1.25 pM to 5 pM, most preferably 1.25 pM, 2.5 pM or 5 pM to the microspore suspension in a double layer system and

[0079] (k) removing the at least one histone deacetylase inhibitor (HDACi) from the microspore culture of step ii).

[0080] In one embodiment of the method described above, step ii) comprises all steps (i)-(k).

[0081] Removing the HDACi prior to the culturing to obtain an embryo and / or a callus leads to particularly efficient callus formation. Removing of HDACi may be conducted by a step of centrifugation, washing of the microspores treated with HDACi, or replacing of the cultivation medium by the same medium without the HDACi or a different medium without the HDACi, or any combination of these steps. Such steps for removing the HDACi may be applied once, repeated or several times, prior to and / or during callus induction.

[0082] In any of the embodiments of the methods described above, the at least one histone deacetylase inhibitor (HDACi) is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0083] A range of different HDACis are known to the skilled person. Good results are in particular obtained with TSA.

[0084] In one embodiment of the method according to any of the embodiments described above, the at least one histone deacetylase inhibitor (HDACi) is present at a concentration from about 100 nM to about 10 pM, preferably 1 pM to 10 pM, more preferably 1 .25 pM to 5 pM, most preferably 1 .25 pM, 2.5 pM or 5 pM in the culture medium used in step ii). Regenerating a plant in step iv) may involve the use of one or more plant growth regulators.

[0085] In one embodiment of the method according to any of the embodiments described above, the callus or embryo is contacted with one or more plant growth regulator(s) selected from auxins, cytokinins, gibberellins, abscisic acid and mixtures thereof in step iii) and / or iv).

[0086] The cytokinins include kinetin, 6-benzyl amino purine (BAP), 6-(gamma,gamma- Dimethylallylamino)purine (2iP) and thidiazuron (TDZ).

[0087] In one embodiment of the method described above, in step iii) and or iv), the callus or embryo resulting from the at least one microspore is contacted with one or more plant growth regulator(s) selected from auxins, synthetic auxins or auxin analogs, including 2,4-Dichlorophenoxyacetic acid (2,4-D), 3,5-dimethylphenoxyacetic acid (3.5ME), Phenoxyacetic acid (PHAA), Phenylacetic acid (PAA), p- Chlorophenoxyacetic acid (4-CPA), 3,6-dichloro-o-anisic acid (dicamba), naphthaleneacetic acid (NAA), indole acetic acid (IAA), and indole-3-butyric acid (IBA), preferably 2,4-D, 3.5ME, PHAA or 4-CPA, more preferably PHAA or 4-CPA.

[0088] The auxin analog is present in a culture medium in a concentration between 1 and 5 pM, preferably between 1 ,5 and 4 pM, more preferably between 2 and 3 pM, more preferably between 2,1 and 2,5 pM.

[0089] In the plants obtained according to the present invention, chromosome doubling can occur either spontaneously or it can be promoted, facilitated or enhanced by a chromosome doubling agent.

[0090] In one embodiment of the method according to any of the embodiments described above, one or more chromosome doubling agent(s) is / are added during step iii) and / or step iv). Chromosome doubling agents are preferably selected from the list consisting of colchicine, trifluralin, amiprophos-methyl, oryzalin, pronamide, podophyllin and nitrous oxide. In particular, colchicine may be added during step iii) and / or step iv). The present invention also relates to the use of a HDACi and a double layer system for producing haploid, polyhaploid and / or doubled haploid embryos and / or plants of the species Capsicum annuum by androgenesis from isolated microspores.

[0091] In one embodiment, the present invention relates to the use of a histone deacetylase inhibitor (HDACi) as defined in any of the embodiments described above for producing a haploid, polyhaploid and / or doubled haploid embryo, callus and / or plant or seed of the species Capsicum annuum.

[0092] In one embodiment of the use described above, the histone deacetylase inhibitor (HDACi) is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0093] In another embodiment of the use according to any of the embodiments described above, the complex protein composition comprises or consists of hydrolysed or partially hydrolysed protein matter derived from milk, such as casein or whey, animals, such as meat or fish, cereal, such as rice or corn, plants, such as soybean or combinations thereof.

[0094] Preferably, the complex protein composition comprises or consists of hydrolysed milk protein isolates, hydrolysed lactoprotein concentrate, hydrolysed casein isolates, casein hydrolysates, hydrolysed lactalbumin, hydrolysed casein sodium, hydrolysed calcium caseinate, hydrolysed full cow's milk, partially or completely skimmed milk, hydrolysed soy protein isolate, hydrolysed soybean concentrate or combinations thereof.

[0095] Particularly preferably, the complex protein composition comprises or consists of a proteolysate selected from the group consisting of casein hydrolysate, soybean hydrolysate, rice proteolysate, potato protein hydrolysate, fish protein hydrolysate, ovalbumin hydrolysate, lactalbumin hydrolysate, glutin hydrolysate, animal and plant proteolysate and a combination thereof.

[0096] In a preferred embodiment of the use described above, the hydrolysis degree of the complex protein composition is in a range from 20 to 80%, preferably 30 to 80%, particularly preferably 40 to 60%.

[0097] In yet another aspect, the present invention relates to a population of haploid, polyhaploid and / or doubled haploid Capsicum annuum plants directly derived from at least one flower bud, preferably obtained by a method according to any of the embodiments described above.

[0098] In another embodiment of the population according to any of the embodiments described above, the population comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% doubled haploid plants.

[0099] In one embodiment of the use, the histone deacetylase inhibitor (HDACi) is selected from the group consisting of trichostatin A (TSA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinostat (PXD101), dacinostat (LAQ824), and panobinostat (LBH589), cyclic tetrapeptides, such as trapoxin B and depsipeptides, such as romidepsin (FK228), benzamides such as entinostat (MS-275), tacedinaline (CI994), and mocetinostat (MGCD0103), electrophilic ketones, and aliphatic acid compounds such as phenylbutyrate and valproic acid, preferably the histone deacetylase inhibitor (HDACi) is trichostatin A (TSA) or romidepsin (FK228).

[0100] Preferably, in step i) microspores from at least one flower bud of Capsicum annuum have been provided in a density of at least 10,000 microspores per ml, more preferred a density of at least 25,000 microspores per ml or at least 50,000 microspores per ml, most preferred a density of at least 100,000 microspores per ml.

[0101] Preferably, more than one plant is obtained in step iv). More preferably, a population of plants is obtained in step iv), wherein the population of plants regenerated in step iv) comprises at least 10 individuals, at least 50 individuals or at least 100 individuals. In a preferred embodiment of the method described above, the population regenerated in step iv) undergoes spontaneous chromosome doubling at a rate of at least 20%, at least 30%, 40%, at least 50%, at least 60% or at least 70%.

[0102] The present invention therefore also provides a method for producing a hybrid Capsicum annuum plant comprising the crossing of a doubled haploid plant obtained or obtainable by microspore culture with another plant of the same species with a different genotype, and optionally obtaining, identifying or selecting seeds grown on the maternal parent of the cross or obtaining, identifying or selecting progeny plants from said cross.

[0103] Brief Description of the Drawings

[0104] Figure 1

[0105] Fig. 1 shows the development of pepper microspores after 14- 19d.

[0106] Figure 2

[0107] Fig. 2 shows the development of pepper microspores to globulary embryos.

[0108] Figure 3

[0109] Fig. 3 (A) Embryos in different developmental stages after 41 d in culture, (B) Mature embryos after 52d in culture.

[0110] Figure 4

[0111] Fig. 4 shows embryo to plant conversion from microspore-derived pepper embryos after 14d (A) and 28d (B).

[0112] Figure 5

[0113] Fig. 5 shows a comparison between using and not using TSA. Each column represents a different genotype. Each row shows a different treatment (agent indicated on the left before each row). Time point, duration and medium used were always identical for all treatments in the different rows. The different rows thus only differ from each other in the kind of concentration of the inhibitor used. Induction of microspore embryogenesis is highly effective using TSA (1 ,25 pM and 2,5 pM) not depending on the genotypes used and results showed that without TSA only very few embryos or no embryo regeneration at all occurred.

[0114] Figure 6 Fig. 6 shows the difference in pepper embryo to plant conversion using a germination medium without activated charcoal (A / B) or with activated charcoal (C / D). As well, conversion without membrane (A / C) and with membrane (B / D) was tested. Using a membrane for embryo to plant conversion clearly improves conversion rate and developing proper shoots.

[0115] Examples

[0116] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.

[0117] 1. Donor Plant Cultivation

[0118] Using a fully air-conditioned greenhouse chamber, Capsicum annuum seeds were sown in 77-celled multipot trays in Hawita Pikiersubatrat ®. Four weeks after sowing, the young plants were potted in 5 I pots in Hawita Topferde ®.

[0119] Cultivation parameters

[0120] Temperature: fully air-conditioned greenhouse chamber

[0121] •Day Heating: < 22 °C / Ventilation: > 25 °C

[0122] •Night Heating: < 18 °C / Ventilation: > 20°C

[0123] Air humidity:65 -70 %

[0124] Lighting: 18 h day length; min. 150 pmol / m2 / s; HPS 400 W.

[0125] After approx. 7-8 weeks, the first whole flower buds comprising microspores at the uninucleate to early binucleate development stage can be harvested.

[0126] 2. Harvest and plant material preparation

[0127] Flower buds, appr. 5 mm in size with mainly late uninucleate microspores were removed from the donor plants. Anthers with appropriate microspores show purple tips as described in Supena and Custers 2011. These were kept in plastic boxes with a moistened filter paper on the base. The harvested buds were stored at 4°C for 2d until further processing.

[0128] 3. Surface disinfection of plant material

[0129] All steps were carried out under a clean bench.

[0130] The buds were transferred immersed in 70% EtOH and incubated for 1 min, followed by washing with sterile water. 4 % NaOCI (+20 drops / L of Tween20) was added and incubated for 5 min on a shaker. Then material was washed 10x for 2 min with sterile water. Buds were transferred to plastic bags (maximum of appr. 100 buds per bag) and 60 ml Isolation Buffer medium was added. Bags were sealed and kept in the fridge until further processing.

[0131] 4. Microspore Isolation

[0132] Bags were processed 2x in a masticator paddle tissue homogenizer (MPH) for 90 seconds.

[0133] Additional Isolation bufferwas added directly into the bags following homogenization and material was poured through a 50 pm nylon sieve. The crushed plant material was disturbed using a spatula and the material in the sieve was washed with more Isolation buffer. The solution containing the microspores was poured into 50 ml centrifugation tubes. This process was continued until the final solution was clear and completely diluted. Tubes were centrifuged for 5 min at 200 rpm at 4°C with acceleration level 6 and deceleration level 6.

[0134] Supernatant in the tubes was decanted and Isolation buffer was added enough to disturb the pellet. Solution from all the tubes was combined in one or two tubes and poured through a 40 pm nylon sieve and the sieve was washed thoroughly with Isolation buffer afterwards.

[0135] The solution that has passed through was poured through a 30 pm sieve and the sieve was washed thoroughly with Isolation buffer.

[0136] The solution that has passed through was poured through a 20 pm sieve and the microspores remain on the sieve and do not pass through. The sieve was reversed and placed on a 200 ml container, and 100 ml Isolation buffer was used to wash microspores off the sieve. The solution was transferred to two 50 ml centrifugation tubes.

[0137] Tubes were centrifuged for 5 min at 200 rpm at 4°C with acceleration level 6 and deceleration level 6.

[0138] Supernatant in the tubes was decanted and 20 ml Isolation buffer was added to each centrifugation tube to disturb the pellet. Solution from both tubes was combined in one centrifugation tube.

[0139] 15 pl of the solution was taken for estimation of cell density using a hemicytometer and density was adjusted to 100 000 mic / ml.

[0140] Tube with microspore solution was centrifuged for 5 min at 200 rpm at 4°C with acceleration level 6 and deceleration level 6. The supernatant was decanted, and Induction medium I was added to the remaining microspores to adjust a final density of 100 000 mic / ml. The microspore solution was divided into vessels already containing a solid layer of induction medium II. TSA was added to each vessel and vessels were stored at 28°C in the dark for 2 days to a final concentration of 1 .25 pM to 2.5 pM.

[0141] After 2-day treatment, the TSA was removed. Solutions were transferred to 50 ml centrifugation tubes and centrifuged for 5 min at 200 rpm at RT with acceleration level 6 and deceleration level 6. Supernatant was decanted and the corresponding amount of culture medium I was added. Microspore pellet was resuspended and either 10 ml of the microspore solution was plated in 9 cm petri dishes, already containing 10 ml of a solid layer of culture medium II or 5 ml of the microspore solution was plated in 6 cm petri dishes, already containing 2.5 ml of a solid layer of culture medium II. Plates were placed in the dark at 26°C. Using 6 cm petri dishes for cultivation, microspore solution was transferred to 9 cm petri dishes already containing 10 ml of a solid layer of culture medium II and 5 ml of culture medium I was added additionally after aprox. 28d. Using 9 cm petri dishes for cultivation from the beginning, after 28 d 5 ml of culture medium I was added. | Gelrite | 10000

[0142] 5. Embryo Regeneration

[0143] Approximately 14-19 d after isolation, the first cell divisions and embryo like structures were observed (Fig. 1)

[0144] First globularly embryos were seen after 20-28 d in culture (Fig. 2)

[0145] Torpedo embryos with approx. 2-3 mm in size with visible cotyledon like structures (cotyledonary stage) occurred approx. 40-50 d after transfer to petri dishes for cultivation with culture media I and II (Fig. 3). These were then transferred to germination medium.

[0146] 6. Embryo Germination

[0147] Torpedo embryos were transferred to 9 cm petri dishes containing 30 ml of solid germination medium and a filter paper (080 mm, 65g / m2, grade 3 hw) on top (Fig. 4). The germination process was performed at 26°C with a photoperiod of 16h light, using approx. 40 pmol / m2s. | Gelrite | 5000

[0148] 7. Shoot formation

[0149] After four weeks on embryo germination medium, the developed shoots were transferred to 250 ml containers with 60 ml R2 medium from Dumas de Vaulx et al. 1981 for further maintenance.

[0150] 8. Effect of TSA in different concentrations

[0151] The aim of this experiment was to test the necessity of Trichostatin A (TSA, chemical regeneration booster) for embryo regeneration. Therefore, three different variants were carried out with different concentrations of TSA. Variant one using 2,5 pM TSA for 2d and variant two using 1 ,25 pM TSA for 2d, whereas variant 3 was carried out without any use of TSA as control.

[0152] All variants were carried out using a double layer tube system with Induction medium 1 as liquid medium for microspores and Induction medium II as solid layer for the induction phase. After two days, the liquid medium was replaced by Culture medium I and plated in petri dishes as well using a double layer system with Culture medium II as solid medium.

[0153] Results showed that without TSA only very few embryos or no embryo regeneration at all could be observed, whereas embryo regeneration occurred using TSA (fig. 5). Regarding TSA concentration (2,5 pM or 1 ,25 pM) no huge differences could be observed.

[0154] 9. Test of double layer system or liquid system for embryo induction

[0155] The aim of this experiment was to test the effect of a double layer media system with or without Activated Charcoal (AC) during the 2d induction phase on embryo regeneration comparing to liquid medium only. Three different treatments were compared: variant one with a double layer system with AC used in solid medium, variant two with a double layer system without AC in solid medium and variant three only using liquid medium. For all three variants 2,5 pM TSA was used.

[0156] Results showed that using a double layer system with AC led to embryo regeneration and a double layer without AC to embryo and / or callus development (depending on the genotype), whereas using a liquid culture clearly led to callus development instead. Although TSA was used (referring to the previous experiment 8, fig. 5), no embryo development could be observed using liquid medium only. Using a double layer system with AC and TSA during induction phase can influence the developmental pathway of the microspores towards embryo induction / development instead of callus.

[0157] 10. Embryo to plant conversion

[0158] For embryo to plant conversion, two different ways were tested. Germination medium was used either with activated charcoal (fig. 6 C / D) or without activated charcoal (fig. 6 A / B) in 9 cm petri dishes and the embryos were either placed directly on top of the medium (fig. 6 A / C) or with a cellulose membrane in between (fig. 6 B / D).

[0159] Results showed, that using a cellulose membrane tremendously improve embryo to plant conversion with providing good quality shoots. Not using a cellulose membrane leads to growth of callus and shoot-like structures.

[0160] 11. Effect of 2,4-D analogs

[0161] The aim of this experiment was to test the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) analogs on embryo regeneration and embryo to plant conversion compared to 2,4-D itself. Therefore, the same culture medium I with different corresponding amounts of auxin and auxin analogs were used for microspore culture.

[0162] All variants were carried out using a double layer media system with induction medium I as liquid medium for microspores and induction medium II as solid layer for the induction phase. After two days, induction medium I was replaced by liquid culture medium I either containing 2,26 pM 2,4-D as auxin or Phenoxyacetic acid (PHAA) or p-Chlorophenoxyacetic acid (4-CIPA) as 2,4-D analogs. In addition, for all variants, TSA was added as HDACi.

[0163] Results are shown in Table 1. Compared to the use of 2,4-D both PHAA and 4- CIPA increased the number of plants regenerated per bud (Plant / bud) and the number of double haploid plants regenerated per bud (DH / bud) in the tested germplasms PP008-F2, PP009-F2 and PP010-F2.

[0164] Table 1

Claims

CLAIMS1. A method for producing a haploid, polyhaploid and / or doubled haploid cell, embryo, callus, seed and / or plant of the species Capsicum annuum, the method comprising: i) providing at least one isolated microspore of a Capsicum annuum plant, ii) culturing the at least one microspore from step i) in the presence of at least one histone deacetylase inhibitor (HDACi) in a double layer system to induce and thus obtain a callus and / or embryo, iii) cultivating the callus and / or embryo, and iv) optionally, regenerating and thereby obtaining at least one plant from the embryo of step iii), and preferably obtaining at least one haploid, polyhaploid and / or doubled haploid seed thereof.

2. The method of claim 1 , wherein the microspore, callus and / or embryo in step ii) and / or iii) is contacted with 2,4-D, PHAA or 4-CIPA.

3. The method of claim 1 or 2, wherein in step iv) the embryo is incubated on a germination medium for obtaining at least one plant wherein a membrane is separating the embryo from said gemination medium.

4. The method of any of claims 1 to 3, wherein the provision of at least one isolated microspore of a Capsicum annuum plant, according to step i) of claim 1 is preceded by the following steps:(a1) providing at least one whole flower bud comprising uninucleate to early binucleate state microspores,(a2) pre-treat said at least one whole flower bud for 12h to 4 days at 1 to 10 °C,(a3) optionally disinfection said at least one whole flower bud,(a3) homogenizing the at least one whole flower bud,(a4) purify the microspores comprised in the homogenate.

5. The method of any of the preceding claims, further comprising within step ii) the following steps:(iia) adding the at least one histone deacetylase inhibitor (HDACi) to a final concentration of 100 nM to 10 pM and(iib) removing the at least one histone deacetylase inhibitor (HDACi) after incubation for about 1 h to 72 h; wherein incubation optionally takes place in the dark.

6. The method of any of the preceding claims wherein the steps are performed at a temperature at 20 to 34 °C.

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

8. The method according to any of the preceding claims, wherein one or more chromosome doubling agent(s), such as colchicine, is / are added during step ii) and / or step iii) and / or step iv).

9. Use of a histone deacetylase inhibitor (HDACi) for producing a haploid, polyhaploid and / or doubled haploid embryo, callus and / or plant or seed of the species Capsicum annuum in a method according to any of claims 1 to 8.

10. A population of haploid, polyhaploid and / or doubled haploid plant of the species Capsicum annuum, directly derived from isolated microspores, preferably obtained by a method according to any of claims 1 to 8.

11. A population of haploid, polyhaploid and / or doubled haploid plant of the species Capsicum annuum, according to claim 8, wherein the population comprises at least 10 individuals.

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