Kinase inhibitors for plant cell reprogramming

Abstract

The present invention is directed to methods of chemical reprogramming of a microspore plant cell using tyrosine kinase inhibitors. The tyrosine kinase inhibitors described facilitate microspore embryoid regeneration in maize, including maize varieties previously recalcitrant to microspore embryoid regeneration, and in a variety of other plant species.

Description

[0001] KINASE INHIBITORS FOR PLANT CELL REPROGRAMMING

[0002] FIELD OF THE INVENTION

[0003] [1] The present disclosure relates to the field of plant molecular biology, and more particularly, to kinase inhibitors and methods for use in connection with plant regeneration.

[0004] JOINT RESEARCH AGREEMENT

[0005] [2] Pioneer Hi-Bred International, Inc. and ScreenSYS GmbH are parties to a joint research agreement. Certain subject matter disclosed in WO2023 / 056236 related to the invention claimed herein, and the invention claimed herein, were made by the parties to the joint research agreement, which joint research agreement was in effect before the effective filing date hereof. The invention claimed herein was made as a result of activities undertaken within the scope of the joint research agreement. This statement is made pursuant to 35 U.S.C. 102(b)(2)(C).

[0006] DISCLOSURE STATEMENT

[0007] [3] The disclosure of Apatinib and Dasatinib in WO2023 / 056236 originated from one or more inventors of the present application. This statement is made pursuant to 35 U.S.C. 102(b)(1)(A).

[0008] BACKGROUND

[0009] [4] Plant breeding programs identify new cultivars by screening numerous plants to identify individuals with desirable characteristics. Large numbers of progeny from crosses are typically grown and evaluated, ideally across multiple years and environments, to select the plants with the most desirable characteristics.

[0010] [5] Typical breeding methods cross two parental plants and the filial 1 hybrid (Fl hybrid), is the first filial generation. Hybrid vigor in a commercial Fl hybrid is observed when two parental strains, (typically inbreds), from different heterotic groups are intercrossed. Hybrid vigor, the improved or increased function of any biological quality resulting after combining the genetic contributions of its parents, is important to commercial maize seed production and commercial hybrid performance improvements require continued development of new inbred parental lines. [6] Maize inbred line development methods use maternal (gynogenic) doubled haploid production, in which maternal haploid embryos are selected following the fertilization of the ear of a plant resultant from a first-generation cross that has been fertilized with pollen from a so- called “haploid inducer” line. Pollination of a female flower with pollen of a haploid inducer plant results in elevated levels of ovules that contain only the haploid maternal genome, as opposed to inheriting a copy of both the maternal and paternal genome, thus, creating maternal haploid embryos. Ovules within the female flower are the products of meiosis and each maternal ovule is a unique meiotically recombined haploid genome, thereby allowing immature maternal haploid embryos to be isolated and treated using in vitro tissue culture methods that include chromosome doubling treatments to rapidly enable generating maternal doubled haploid recombinant populations. However, paternal doubled haploids, which are derived solely from a microspore gamete, have potential advantages over maternal doubled haploids, such as an increased number of gametes and doubled haploids generated.

[0011] [7] Many maize inbreds are recalcitrant to microspore in vitro tissue culture and plantlet regeneration methods to create paternal (androgenic) gamete doubled haploids. Thus, there is also a need for a method of producing doubled haploid plants applicable to paternal gamete doubled haploids in maize. Recalcitrance is the inability of a plant cell to respond to tissue culture techniques such as cellular reprogramming, embryogenesis, plant regeneration or totipotency in the absence of specific tissue culture enhancing compounds such as those described herein.

[0012] [8] Plant breeders would thus benefit from methods of developing a population of recombinant inbred lines that do not require extensive pollination control methods or the prolonged time required for propagating self-fertilized lines into isogenic states.

[0013] SUMMARY OF INVENTION

[0014] [9] The kinase inhibitors described herein have been shown to have a beneficial effect in the development of cells into embryos and / or embryo-like structures, which may ultimately be grown into plants, including haploid cell derived plants. The term embryo-like structure as used herein refers to proliferating cell masses derived from stem cells that resemble an embryo, and the term embryoids as used herein refers to a specific type of embryo-like structure that is derived from culturing tetrads or microspore gametes. Combinations of the kinase inhibitors described herein and HD AC inhibitors have also been shown to have a beneficial effect on the development of cells into embryos and / or embryo-like structures, which may ultimately be grown into plants, including haploid cell derived plants.

[0015] DETAILED DESCRIPTION

[0016]

[0010] Embodiments described herein involve the use of kinase inhibitors to facilitate the regeneration of plant cells into embryos and / or embryo-like structures and plants. Such plant cells may haploid cells, specifically tetrad and / or microspore haploid cells that are used to produce paternally derived inbred lines. Plants for which these kinase inhibitors may be utilized include hybrid cereal crops including, but not limited to, wheat, maize (com), rice, barley, oats, rye and sorghum, as well as a broad range of plant species, including dicotyledonous plants and monocotyledonous plants. Other representative examples of plants that can be treated in accordance with the methods disclosed herein include, but are not limited to, cotton, sunflower, safflower, tobacco, Arabidopsis, triticale, millet, flax, sugarcane, banana, cassava, common bean, cowpea, tomato, potato, beet, grape, Eucalyptus, wheat grasses, turf grasses, alfalfa, clover, soybean, peas, peanuts, citrus, papaya, Setaria sp, cacao, cucumber, apple, Capsicum, bamboo, melon, ornamentals including commercial garden and flower bulb species, fruit trees, vegetable species, Brassica species, as well as interspecies hybrids. In a preferred embodiment, the compositions and methods of the disclosure are applied to maize plants.

[0017]

[0011] The disclosure provides efficient and effective methods of producing populations of inbred lines including, but not limited to, methods of initiating embryogenesis in plant cells to enable generating recombinant doubled haploid populations. The inbred lines may comprise a transformed or gene edited cell, which modification may have been introduced in the line from which the gamete cell was produced or during the regeneration process. The disclosure also provides methods of enabling cellular reprogramming and embryogenic growth stimulation in non-transformed cells, and particularly in gametes or haploid cells during the development of the gametes or haploid cells. The present disclosure provides methods of promoting embryo-like regeneration in a cell, tissue or organ of a plant by contacting the cell, tissue or organ with an embryogenesis modulation factor capable of reprogramming the cell, tissue or organ wherein embryogenesis is induced in the cell, tissue or organ, such as, for example, the kinase inhibitors disclosed herein, whether individually or in combination with an embryogenesis inducing exogenous morphogenic developmental gene protein product and / or another secondary embryogenesis inducing compound. In one embodiment, the present disclosure provides methods of promoting microspore derived embryoid formation.

[0018]

[0012] The present disclosure, in one aspect, also includes plants obtained by any of the disclosed methods or compositions herein. In many aspects, the present disclosure also includes seeds from a plant obtained by any of the disclosed methods or compositions herein. As used herein, the term “plant” refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), plant tissues, plant cells, plant parts, seeds, propagules, embryos and progeny of the same. As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated (“multicellular structures”), plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grain and the like. Plant cells include, without limitation, cells from seeds, suspension cultures, explants, immature embryos, embryos, zygotic embryos, somatic embryos, embryogenic callus, meristem, somatic meristems, organogenic callus, protoplasts, meristematic regions, embryos derived from mature ear-derived seed, leaf bases, leaves from mature plants, leaf tips, immature inflorescences, tassel, immature ear, silks, cotyledons, immature cotyledons, embryonic axes, meristematic regions, callus tissue, cells from leaves, cells from stems, cells from roots, cells from shoots, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores. Plant cells can be differentiated or undifferentiated (e.g. callus, undifferentiated callus, immature and mature embryos, immature zygotic embryo, immature cotyledon, embryonic axis, suspension culture cells, protoplasts, leaf, leaf cells, root cells, phloem cells and pollen). Plant parts include differentiated and undifferentiated tissues including, but not limited to, roots, stems, shoots, leaves, pollen, seeds, tumor tissue and various forms of cells in culture (e. g., single cells, protoplasts, embryos, and callus tissue). The plant tissue may be in a plant or in a plant organ, tissue, or cell culture. “Plantlet” refers to regenerable structures of plant cells, such as an embryo with root and shoot, an embryo-like structure, an embryoid (an embryo-like structure or “ELS” regenerated from a tetrad or microspore) or other multi cellular / macroscopic structures, from which plants may be regenerated. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species. Progeny, variants and mutants of the regenerated plants are also included within the scope of the disclosure, provided these progeny, variants and mutants are made using the methods and compositions disclosed herein and / or comprise the introduced polynucleotides.

[0019]

[0013] As used herein, the terms “transformed plant” and “transgenic plant” refer to a plant that comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome of a transgenic or transformed plant such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant DNA construct. It is to be understood that as used herein the term “transgenic” includes any cell, cell line, callus, tissue, plant part or plant the genotype of which has been altered by the presence of a heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. A transgenic plant is defined as a mature, fertile plant that contains a transgene.

[0020]

[0014] As used herein, the term “gene-edited” refers to the use of a gene editing technique such as CRISPR-Cas that may involve modifications such as insertions, deletions or substitutions to the genome of the organism.

[0021]

[0015] Chemical names used herein are inclusive of its salts, isomers, and salts of its isomers, even if such variant or variants would receive a unique CAS registry number.

[0022]

[0016] In some embodiments, the transformation or gene editing is introduced in the haploid cell prior to doubling, which has the benefit of duplicating the transformed event or gene edit upon chromosomal doubling. In some embodiments, the haploid cell is a haploid gamete, which has the advantage of introducing the transformed event or gene edit into all cells of the regenerated plant.

[0017] Methods for harvesting tassels, including sterilization methods, as well as tassel pretreatments, for example, temperature pretreatments, are known in the art and will vary depending on the intended tassel use. Specifically, prior to selecting tassels for microspore culture, microspores must be staged to an appropriate stage typically, between the uninucleate to binucleate stage. Typically, for tassels with anthers and microspores at the appropriate stage, the tassels are detached and each tassel is individually wrapped in for example, aluminum foil. The herein provided methods can comprise a step of pretreatment of anthers (e.g. in case of maize pretreatment of tassels) e.g. at about 10 °C for about for between 1 to 21 days, specifically about 8-16 days, e.g. 8, 9, 10, 11, 12, 13, 14, 15 or 16 days. In one aspect the pretreatment is for about 14 days. Typically, the pretreatment is in darkness.

[0023]

[0018] The method of obtaining a regenerated plantlet from a plant microspore provided herein can comprise a step of isolating a plant microspore. This step of isolating a plant microspore can be optional and may be omitted Thus, the methods can comprise culturing a plant microspore with an embryogenesis inducing tyrosine kinase inhibitor, wherein the plant microspore is (obtained) from a plant, such as maize, rice, sorghum, sunflower, brassica, soybean, wheat, or cotton. The term “plant microspore” and ‘"microspore (obtained) from a plant” can be used interchangeably herein (like “maize microspore’’ and “microspore (obtained) from a plant”) Isolation of microspores typically occurs after a tassel pretreatment in a reduced temperature environment to improve the androgenic response. A commonly used technique is to place foil wrapped tassels at 10° C for between 1 to 21 days. Additionally, preculture of anthers in a mannitol solution, for example 0.3M liquid mannitol plus 50 mg / L ascorbic acid, can be practiced (U.S. Pat. Nos. 5,322,789 and 5,445,961 incorporated herein by reference in their entireties). Prior to use, tassels can be surface-sterilized in a 40% Clorox (8.25% Sodium Hypochlorite diluted v / v) solution plus two drops of Tween 80 for approximately fifteen minutes, with gentle agitation on a reciprocal shaker. The tassels can then be rinsed three or more times in sterile water at room temperature and placed in a large petri dish and typically left uncovered for 1-1.5 hours under aseptic conditions to allow any excess water to evaporate. Another method known in the art includes placing spikelets detached from the tassel into permeable baskets that are then submerged in a 40% Clorox (8.25% Sodium Hypochlorite diluted v / v) solution plus two drops of Tween 80 for fifteen minutes followed by rinsing as described above. The spikelets may then be placed in a large petri dish and typically left uncovered for 1-1.5 hours to allow excess water to evaporate prior to microspore isolation.

[0024]

[0019] A variety of isolation procedures for maize anthers and spikelets are known in the art, including, but not limited to, glass rod maceration methods (Pescitelli, et al., (1990) Plant Cell Rep. 8:628-31), blending methods, razor blade tissue cutting methods (see U.S. Pat. No. 5,445,961 incorporated herein by reference in its entirety), tissue homogenizer methods (Gaillard, et al., (1991) Plant Cell Rep. 10:55-8), and tissue grinder methods (Mandaron et al., (1990) Theor Appl Genet 80: 134-138.

[0025]

[0020] Following isolation of microspores from the surrounding somatic tissue, the microspores are typically separated from any anther debris and placed into a fresh isolation medium. Numerous media compositions are known in the art. A common method of separating microspores from anther debris is to pass a blended microspore anther debris slurry from the isolation procedure through a sieve (Pescitelli (1989) Plant Cell Rep. 7:673-6, Gaillard, et al., (1991), and U.S. Pat. No. 5,445,961 incorporated herein by reference in its entirety). Alternatively, the microspore anther debris slurry is passed through several layers of cheesecloth or a mesh filter (Coumans, (1989) Plant Cell Rep. 7:618-21). Further separation can be performed using a discontinuous density centrifugation method or additional filtration methods including, but not limited to, methods using a sucrose or Percoll gradient (Coumans, (1989), Pescitelli et al., (1990)). Alternatively, selection of cells captured at the 20-30% interface of a Percoll gradient ranging from 20-50% after centrifugation at 225 g for 3 min can be further separated using a final, high sucrose (0.44M) centrifugation method (Gaillard, et al., (1991)). Further variations to separation methods are known in the art (Vergne et al., (1991) In: Negrutiu I. (ed) BioMethods. Birkhauser, Basel, Boston, Bedinger and Edgerton, (1990) Plant Physiol. 92:474-9, Gaillard, et al., (1991)) and can be optimized as needed. Alternatively, in some embodiments, microfluidic methods may be utilized, such as the methods disclosed in US20200238288, W02021212102 and US20210291185, each of which are incorporated herein by reference.

[0021] Specific media used during isolation, for example, typically consists of 6% sucrose, 50 mg / L ascorbic acid, 400 mg / L proline, 0.05 mg / L biotin and 10 mg / L nicotinic acid (see Petolino and Genovesi (1994) The Maize Handbook, Freeling, M., Walbot, V. (eds) Springer-Verlag, New York). Various other media and solutions used for the culturing of maize microspores are similar to those used for other cereal tissue culture procedures and various modifications can be used (see Genovesi and Magill, (1982) Plant Cell Rep. 1 :257-60, Martin and Widholm, (1996) Plant Cell Rep. 15:781-85, Magnard et al., (2000) Plant Mol Biol 44:559-74, Testillano et al., (2002) Int J Dev Biol 46: 1035-47, Testillano et al., (2004) Chromosoma 112:342-9, Shariatpanahi et al., (2006) Plant Cell Rep 25: 1294-9, Shim et al., (2006) Protoplasma 228:79- 86, Soriano et al., (2008) Plant Cell Rep 27:805-11, Cistue et al., (2009) Plant Cell Rep 28:727- 35, Jacquard et al., (2009) Planta 229:393-402, Jacquard et al., (2009) Plant Cell Rep 28: 1329- 39, Shim et al., (2009) Genome 52:166-74, Sanchez-Diaz et al., (2013) Plant Reprod 26: 287- 96). As evidenced in the citations above, common features for maize culture media typically include the use of N6, NLN, or YP basal salt formulations with relatively high sugar concentrations (6-12%) that may have constituents including triiobenzoic acid, various phytohormones, and / or proline.

[0026]

[0022] In an aspect, haploid cells can be contacted with an amount of a chromosome doubling agent to promote chromosome doubling followed by regenerating homozygous diploid plants from the treated haploid cells. The haploid microspore cells can be in contact with the doubling agent before, during, or after initiation of microspore embryoid regeneration or embryo maturation. After chromosome doubling, the doubled haploid cell, or embryo, or plant will contain 2 copies of paternally derived chromosomes. The efficiency of the process for obtaining doubled haploid plants from haploid embryos may be greater than 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, or 90%. The duration of contact between the haploid cells and the chromosomal doubling agent may vary. Contact may be from less than 24 hours, for example 4- 12 hours, to about a week. The duration of contact is generally from about 8 hours to 2 days.

[0027]

[0023] Methods of chromosome doubling are disclosed in Antoine-Michard, S. et al., Plant cell, tissue organ cult., Cordrecht, the Netherlands, Kluwer Academic Publishers, 1997, 48(3) :203 - 207; Kato, A., Maize Genetics Cooperation Newsletter 1997, 36-37; and Wan, Y. et al., TAG, 1989, 77: 889-892. Wan, Y. et al., TAG, 1991, 81 : 205-21 1 . The disclosures of which are incorporated herein by reference. Typical doubling methods involve contacting the cells with colchicine, anti -microtubule agents or anti -microtubule herbicides, pronamide, nitrous oxide, or any mitotic inhibitor to create homozygous doubled haploid cells. The amount of colchicine used in medium is generally 0.01%-0.2% or approximately 0.05% of amiprophos-methyl (APM) (5-225 pM) may be used. The amount of colchicine can range from approximately 400-600 mg / L or approximately 500 mg / L. The amount of pronamide in medium is approximately 0.5-20 pM.

[0028]

[0024] Where colchicine is used for doubling, the concentration in the medium may be generally 0.01%-0.2% or approximately 0.05% or APM (5-225 pM). The range of colchicine concentration may be from about 400-600 mg / L or about 500 mg / L. Where pronamide is used, the medium concentration may be about 0.5-20 pM. Examples of known mitotic inhibitors are listed below. Other agents such as DMSO, adjuvants, or surfactants may be used with the mitotic inhibitors to improve doubling efficiency.

[0029]

[0025] The chromosome doubling agent may be contacted with a haploid embryo at various times. If the embryo is isolated, the doubling agent may come in contact immediately after isolation. The duration of contact between the chromosomal doubling agent may vary. Contact may be from less than 24 hours, for example, 4-12 hours, to about a week. The duration of the contact is generally from about 24 hours to 2 days.

[0030]

[0026] Common or trade names of suitable chromosome doubling agents include colchicine, acetyltrimethylcolchicinic acid derivatives, carbetamide, chloropropham, propham, pronamide / propyzamide tebutam, chlorthal dimethyl (DCPA), Dicamba / dianat / disugran (dicamba-methyl) (BANVEL, CLARITY), benfluralin / benefin / (BALAN), butralin, chloralin, dinitramine, ethalfluralin (Sonalan), fluchloralin, isopropalin, methalpropalin, nitralin, oryzalin (SURFLAN), pendimethalin, (PROWL), prodiamine, profluralin, trifluralin (TREFLAN, TRIFIC, TRILLIN), AMP (Amiprofos methyl); amiprophos-methyl Butamifos, Dithiopyr, and Thiazopyr. Examples of mitotic inhibitors are included in Table 1. Other agents may be used with the mitotic inhibitors to improve doubling efficiency. Such agents include dimethyl sulfoxide (DMSO), adjuvants, surfactants, and the like. Cells, tissue or organs from a maize population with a high frequency of spontaneous haploid genome doubling may also be used, such as disclosed in US11155825, which may reduce or eliminate the amount of chemical doubling agent needed.

[0031]

[0027] TABLE 1

[0032]

[0028] Protein kinases constitute a large family of structurally related enzymes that are responsible for the control of a variety of signal transduction processes within cells (see, e.g., Hardie and Hanks, The Protein Kinase Facts Book, I and II, Academic Press, San Diego, Calif, 1995). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function. Almost all kinases contain a similar 250- 300 amino acid catalytic domain. The kinases can be categorized into families by the substrates they phosphorylate (e.g., protein-tyrosine, protein-serine / threonine, lipids, etc ). Sequence motifs have been identified that generally correspond to each of these families (see, e.g., Hanks & Hunter, (1995), FASEB J. 9:576-596; Knighton et al., (1991), Science 253:407-414; Hiles et al., (1992), Cell 70:419-429; Kunz et al., (1993), Cell 73:585-596; Garcia-Bustos et al., (1994), EMBO J. 13:2352-2361).

[0033]

[0029] Kinase inhibitors that have been identified and newly described herein for use in regenerating multicellular structures or plantlets, optionally in conjunction with doubled haploid production, include the Vascular Endothelial Growth Factor Receptor (VEGFR) tyrosine kinase inhibitor Apatinib and the BCR / ABL and SRC tyrosine kinase inhibitor Dasatinib, as well as the analog derivatives of each as shown below, including chemicals listed in Tables 2 and 3 below, and modifications thereof. After tassel pretreatment at 10 degrees C for about 14 days, microspores were isolated, and Apatinib (CAS811803-05-1) and Dasatinib (CAS302692-49-8) were tested by continuous treatment during cell culture. Apatinib and Apatinib derivates are described in US20040259916, and Dasatinib and Dasatinib derivatives are described in W02000 / 62778, each of which are incorporated by reference herein.

[0034]

[0030] The effectiveness of each of the tyrosine kinase inhibitors, Apatinib and Dasatinib, has been confirmed in both ATCC germplasm 40520, known to be conducive to microspore embryo regeneration, as well as elite germplasm varieties otherwise recalcitrant to a microspore embryogenic response, and thus, lacks a resulting embryo regeneration capability.

[0031] It was predicted that, having identified the success of Apatinib and Dasatinib in increasing the number of embryo-like / macroscopic structures recovered from ATCC germplasm 40520 as well as recalcitrant inbred lines, that additional analogs and derivatives of each will perform as well or better than Apatinib and Dasatinib. Such prediction has been confirmed as described herein. Such compounds that have been identified include compounds that fall within the following groups:

[0035]

[0032] Apatinib

[0036]

[0033] Apatinib (CAS 811803-05-1) and Apatanib analogs and derivatives that are members of the Markush group(s) identified in US20040259916, and pharmaceutically and agriculturally acceptable salts thereof.

[0037]

[0034] Formula Al (or Formula (I))

[0038] Formula (I)

[0039] Wherein, in the compounds of Formula Al,

[0040] X is O or S, preferably O;

[0041] Y is — N(R4) — , preferably — NH — ; Zi, Z2, Z3, Z4 are independently CRs or N; preferably Zi, Z2, Z3 are C and Z4is C or N as a phenyl or a pyridyl ring which is optional substituted up to three times independently by R5;

[0042] A is selected from direct bond, lower alkylenyl and lower alkenlenyl; preferably direct bond or lower alkylenyl;

[0043] B is selected from direct bond, lower alkylenyl, lower alkenlenyl, — O — , — N(Rn) — , — C(O)N(R4)— , -OC(O)N(R4)-, -N(R4)C(O)-, -N(R4)C(O)O-, -N(R4)C(0)N(R4)-, — C(O)— , — S(O)— , -S(O)2-, -S(O)N(R4)-, -S(O)2N(R4)-, -N(R4)S(O)-, -N(R4)S(O)2-, - N(R4)S(O)N(R4)-, — N(Rt)S(O)2N(R4) — ; preferably direct bond or lower alkylenyl;

[0044] Ri is selected from cycloalkyl, cycloalkenyl, aryl and heterocyclyl; preferably phenyl which is optionally substituted by hydrogen, halogen or R2;

[0045] Cy is selected from cycloalkyl, cycloalkenyl and heterocyclyl; preferably selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 4 to 7 membered lactam and lactone;

[0046] R2 is selected from halogen-lower alkyl, lower alkyl, lower alkenyl, lower alkynyl, Co- C6Cyano, Co-C6hydroxy, Co-C6alkoxy, Co-C6alkoxyalkoxyl, Co-C6amino, Co- C6alkoxyamino, Co- C6Carboxy, Co-C6carboxyalkyl, Co-C6Carbonylamino, Co-C6Carbonylalkyl, Co-C6oxycarbonylalkyl, Co-C6Oxycarbonylamino, Co- C6aminocarbonylalkyl, Co-C6aminocarbonyloxyalkyl, Co-C6aminocarbonylamino, Co- C6aminosulfonylalkyl, Co-C6cycloalkyl, Co-C6Cycloalkenyl, Co-C6aryl, Co-C6Oxyaryl, Co-C6alkoxyaryl, Co-C6aminoaryl, Co-C6aminoalkylaryl, Co-C6heterocyclyl, Co- C6oxyheterocyclyl, Co-C6alkoxyheterocyclyl, Co-C6aminoheterocyclyl and Co- C6aminoalkylheterocyclyl; wherein any above C1-C6groups and amino groups can be optionally unsubstituted, mono-substituted or possibly disubstituted by lower alkyl; preferably R2 is selected from cyano, methyleneoxomethyl, methylenehydroxy, methyleneamino, methylene N,N-dim- ethylamino, methyleneazetidine, methylenepyrrolidine, methylenepiperidine, methylenemorpholine, methylenepip- erazine, N-methyl-methylenepiperazine, carbonyl-N,N- dimethylamino and carbonyl N- methyl-piperazine;

[0047] Ri and R2 are combined together as a fused spiro ring G comprising C, N, O or S, wherein ring G is selected from cycloalkyl, cycloalkenyl, aryl and heterocyclyl, which can be saturated or partially saturated and unsubstituted, mono or polysubstituted; preferably G is selected from 5 to 7 membered saturated or partial saturated heterocyclyl ring which can be unsubstituted or mono or polysubstituted independently by halogen or R2;

[0048] V is C, N or SO2; preferably C;

[0049] W and W are independently of each other hydrogen, halogen or lower alkyl; or together with the carbon atom to which they are attached as a cycloalkyl, a cycloalkenyl, or a heterocyclyl ring; preferably, W and W are independently hydrogen or fluoro; n is an integer from 0 to 6; preferably 0, 1, 2 or 3;

[0050] Rs is a heterocyclyl or an aryl; preferably selected from pyridyl, pyrimidinyl, quinolinyl, quinazolinyl, inda- zole, indolinone and phenyl;

[0051] Rt is H or a lower alkyl; preferably H;

[0052] Rs is H, halogen or lower alkyl; preferably H or fluoro; or of a N-oxide or a possible tautomer thereof;

[0053]

[0035] Compounds within Formula Al include compounds of the following Formula A2 and pharmaceutically or agriculturally effective salts thereof:

[0054] Formula A2 where A, B, Cy, Ri, Ra, Ra, n, V, W, W’, and Z4 are as described as above for Formula Al.

[0055]

[0036] Compounds within Formula A2 include compounds of the following Formula A3 (in other words, Markush group A3 is a subset of the compounds within Markush group A2) and pharmaceutically or agriculturally effective salts thereof:

[0056] Formula A3

[0057] O l2

[0058] N H

[0059] MH where Cy, Ra, and Z4 are as described as above for Formula Al and A2.

[0060]

[0037] In some embodiments, the invention comprises the use of one or more of the specific Apatinib analog compounds described in Table 2.

[0038] TABLE 2 - Specific Apatinib analog compounds.

[0061] Number Name CAS number if known. _

[0062] 1 N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4- 811802-99-0 ylmethyl)amino)benzamide _

[0063] 2 N-(4-( 1 -cyanocyclopropyl)phenyl)-2-((pyridin-4- 811803-00-6 ylmethyl)amino)benzamide _

[0064] 3 N-(4-( 1 -cyanocy clopentyl)phenyl)-2-((pyridin-4- 811803-01-7 ylmethyl)amino)benzamide _

[0065] 4 N-(4-( 1 -cyanocy clohexyl)phenyl)-2-((pyridin-4- 811803-02-8 ylmethyl)amino)benzamide _

[0066] 5 N-(4-( 1 -cyanocyclobutyl)phenyl)-2-((pyridin-4- 811803-03-9 ylmethyl)amino)nicotinamide _

[0067] 6 N-(4-( 1 -cyanocyclopropyl)phenyl)-2-((pyridin-4- 811803-04-0 ylmethyl)amino)nicotinamide _

[0068] 7 N-phenyl-2-((pyridin-4-ylmethyl)amino)mcotinamide _ Not known

[0069] 8 N-(4-( 1 -cyanocyclohexyl)phenyl)-2-((pyridin-4- 811803-06-2 ylmethyl)amino)nicotinamide _

[0070] 9 N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- 811803-07-3 ylmethyl)amino)benzamide _

[0071] 10 N-(4-( 1 -(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- 811803-08-4 ylmethyl)amino)nicotinamide _

[0072] 11 N-(4-( 1 -(hydroxymethyl)cy clobutyl)phenyl)-2-((pyridin-4- 811803-09-5 ylmethyl)amino)benzamide _

[0073] 12 N-(4-( 1 -(hydroxymethyl )cyclobutyl)phenyl)-2-((pyridin-4- 811803-10-8 ylmethyl)amino)nicotinamide _

[0074] 13 N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- 811803-11-9 ylmethyl)amino)nicotinamide _

[0075] 14 N-(4-( 1 -(methoxymethyl)cyclohexyl)phenyl)-2-((pyridin-4- 811803-12-0 ylmethyl)amino)nicotinamide _

[0076] 15 N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- 811803-13-1 ylmethyl)amino)benzamide _ _

[0077] 16 N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- 811803-14-2 ylmethyl)amino)nicotinamide _ _

[0078] 17 N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- 811803-15-3 ylmethyl)amino)benzamide _

[0079] 18 N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- 811803-16-4 ylmethyl)amino)ni cotinamide _ _

[0080] 19 2-(( lH-indazol-6-yl)amino)-N-(4-( 1 - 811803-17-5 cyanocyclopentyl)phenyl)nicotinamide _

[0081] 20 2-(( lH-indazol-6-yl)amino)-N-(4-( 1 - 811803-18-6 cyanocyclobutyl)phenyl)nicotinamide _

[0082] 21 2-((lH-indazol-6-yl)amino)-N-(4-( 1 -cyanocyclopropy 811803-19-7 l)phenyl)nicotinamide

[0039] The structure of select Apatinib analog compounds, from Table 2, rows 1-6, row 8, row 11 and row 13, are provided below:

[0083]

[0084] 1 N-(4-(1-cyanocydobutyl)phenyl)-2-((pyrldln-4-ylmethyl)amlno)benzamlde

[0085] 2 N-(4-(1-cyanocydopropyi)phenyi)-2-((pyridin-4-y1methyi)amino)benzamide

[0086] 3 N-(4-(1-cyanocydopentyl)phenyl)-2-((pyridln-4-ylmethyl)amlno)benzamlde

[0087]

[0040] 4 N-(4-(1-cyanocydohexyl)phenyl)-2-((pyr1dln-4-ylmethyl)amlno)benzamlde

[0088]

[0089] 5 N-(4-(1-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide

[0090] 6 N-(4-(1-cyanocyclopropyl)phenyl)-2-((pyi1din-4-ylmethyl)amino)nicotinamide

[0091] 8 N-(4-(1-cyanocydohexyi)phenyl)-2-((pyridin-4-ylmethyi)amino)nicx)tinamide

[0092] 11 N-(4-(1-(hydroxymethylXyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amlno)benzamide

[0093]

[0094] 13 N-(4-(1-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide

[0095] Dasatinib

[0096] Dasatinib (CAS 302962-49-8) and Dasatinib analogs and derivatives include members of the Markush group identified in WO 00 / 62778, which is incorporated by reference herein, and pharmaceutically and agriculturally effective salts thereof. where

[0097] Q is:

[0098] (1) a 5-membered heteroaryl ring;

[0099] (2) a 6-membered heteroaryl ring; or

[0100] (3) an aryl ring; optionally substituted with one or more groups Ri;

[0101] Z is:

[0102] (1) a single bond;

[0103] (2) -RI5C=CH-; or

[0104] (3) -(CH2)m-, where m is 1 to 2; Xi and X2 are each hydrogen, or together form =0 or =S;

[0105] Ri is:

[0106] ( 1 ) hydrogen or R6 , where Re is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, heterocyclo, or heterocycloalkyl, each of which is unsubstituted or substituted with Zi, Z2 and one or more (preferably, one or two) groups Z3;

[0107] (2) -OH or -ORe;

[0108] (3) -SH or -SRe;

[0109] (4) -C(0)2H, -C(O)qRe, or -O-C(O)qRe, where q is 1 or 2;

[0110] (5) -SO3H or -S(O)qR6;

[0111] (6) halo;

[0112] (7) cyano;

[0113] (8) nitro;

[0114] (9) -Z4-NR7R8;

[0115] (10) -Z4-N(R9)-Z6-NRIORII;

[0116] (11) -Z4-N(Ri2)-Z5-Re;

[0117] (12) -P(O)(ORe)2;

[0118] R2 and R3 are each independently:

[0119] (1) hydrogen or R6;

[0120] (2) -Z4-R6; or

[0121] (3) -Z13-NR7R8;

[0122] R4 and Rs:

[0123] (1) are each independently hydrogen or R6;

[0124] (2) -Z4-N(R9)-Z5-NRIORII;

[0125] (3) -N(R9)Z4Re; or

[0126] (4) together with the nitrogen atom to which they are attached complete a 3- to 8- membered saturated or unsaturated heterocyclic ring which is unsubstituted or substituted with Zi, Z2 and Z3, which heterocyclic ring may optionally have fused to it a benzene ring itself unsubstituted or substituted with Zi, Z2 and Z3;

[0127] R7, R8, R9, RIO, R11 and R12:

[0128] (1) are each independently hydrogen or R6; (2) R.7 and Rs may together be alkylene, alkenylene or heteroalkyl, completing a 3- to 8- membered saturated or unsaturated ring with the nitrogen atom to which they are attached, which ring is unsubstituted or substituted with Zi, Z2 and Z3; or

[0129] (3) any two of R9, Rio and R11 may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Zi, Z2 and Z3;

[0130] R13 is:

[0131] (1) cyano;

[0132] (2) nitro;

[0133] (3) -NH2;

[0134] (4) -NHOalkyl;

[0135] (5) -OH;

[0136] (6) -NHOaryl;

[0137] (7) -NHCOOalkyl;

[0138] (8) -NHCOOaiyl;

[0139] (9) -NHSOalkyl;

[0140] (10) -NHSO2aryl;

[0141] (11) aryl;

[0142] (12) heteroaryl;

[0143] (13) -Oalkyl; or

[0144] (14) -Oaryl;

[0145] R14 is:

[0146] (1) -NO2;

[0147] (2) -COOalkyl; or

[0148] (3) -COOaryl;

[0149] R15 is:

[0150] (1) hydrogen;

[0151] (2) alkyl;

[0152] (3) aryl;

[0153] (4) aryl alkyl; or

[0154] (5) cycloalkyl; Zi, Z2 and Z3 are each independently:

[0155] (1) hydrogen or Ze, where Ze is (i) alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl, aryl, aralkyl, alkylaryl, cycloalkylaryl, heterocyclo, or heterocycloalkyl; (ii) a group (i) which is itself substituted by one or more of the same or different groups (i); or (iii) a group (i) or (ii) which is substituted by one or more of the following groups (2) to (16) of the definition of Zl, Z2 and Z3;

[0156] (2) -OH or -OZ6;

[0157] (3) -SH or -SZ6;

[0158] (4) -C(O)qH, -C(O)qZ6, or -O-C(O)qZ6;

[0159] (5) -SO3H, -S(O)qZ6; or S(O)qN(Z9)Ze;

[0160] (6) halo;

[0161] (7) cyano;

[0162] (8) nitro;

[0163] (9) -Z4-NZ7Z8;

[0164] (10) -Z4-N(Z9)-Z5-NZ7Z8;

[0165] (11) -Z4-N(ZIO)-Z6-Z6;

[0166] (12) -Z4-N(ZIO)-Z5-H;

[0167] (13) oxo;

[0168] (14) -O-C(O)-Z6;

[0169] (15) any two of Zi, Z2, and Z3 may together be alkylene or alkenylene completing a 3- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached; or

[0170] (16) any two of Zi, Z2, and Z3 may together be -O-(CH2)r-O- .where r is 1 to 5, completing a 4- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached;

[0171] Z4 and Z5 are each independently:

[0172] (1) a single bond;

[0173] (2) -Z11-S(O)q-Zi2-;

[0174] (3) -Z11-C(O)-Zi2-;

[0175] (4) -Zu C(S)-Zi2-;

[0176] (5) -Z11-O-Z12-; (6) -Z11-S-Z12-;

[0177] (7) -Z11-0-C(0)-Zi2-; or

[0178] (8) -Z11-C(0)-0-Zi2-; Z7, Z8, Z9and Z10:

[0179] (1) are each independently hydrogen or Z6;

[0180] (2) Z7 and Zs, or Ze and Z10, may together be alkylene or alkenylene, completing a 3- to 8-membered saturated or unsaturated ring together with the atoms to which they are attached, which ring is unsubstituted or substituted with Zi, Z2 and Z3; or

[0181] (3) Z7 or Zs, together with Z9, may be alkylene or alkenylene completing a 3- to 8- membered saturated or unsaturated ring together with the nitrogen atoms to which they are attached, which ring is unsubstituted or substituted with Zi, Z2 and Z3;

[0182] Z11 and Z12 are each independently:

[0183] (1) a single bond;

[0184] (2) alkylene;

[0185] (3) alkenylene; or

[0186] (4) alkynylene; and

[0187] Z13 is:

[0188] (1) a single bond;

[0189] (2) -Z11-S(0)q-Zi2-;

[0190] (3) -Z11-C(0)-Zi2-;

[0191] (4) -Z11-C(S)-Zi2-;

[0192] (5) -Z11-O-Z12-;

[0193] (6) -Z11-S-Z12-;

[0194] (7) -Z11-0-C(0)-Zi2-;

[0195] (8) -Z11-C(0)-0-Zi2-;

[0196] (9) -C(NRI3)-;

[0197] (10) -C(CHRI4)-; or

[0198] (11) -C(C(Ri4)2)-; and include pharmaceutically or agriculturally acceptable salts thereof.

[0199] Formula D2

[0200] where n is 1 or 2

[0201] A is selected from carbon and nitrogen;

[0202] B is selected from nitrogen, oxygen and sulfur;

[0203] X3 is oxygen or sulfur; and

[0204] Ri, R2, R3, R4 and R5 are as described above.

[0205] Compounds within formula D2 include compounds of the following formula D3 and pharmaceutically or agriculturally effective salts thereof:

[0206] Formula D3 where Ri, R2, R3, R4 and Rs are as described above for Formula’s DI and D2.

[0207]

[0045] Compounds within formula D3 include compounds of the following formula D4 (in other words, Markush group D4 is a subset of the compounds within Markush group D3) and pharmaceutically or agriculturally effective salts thereof: Formula D4 where Ri, R2, R3 and R4 are described above for Formula’s DI, D2 and D3

[0208]

[0046] In some embodiments, the invention comprises the use of one or more of the specific Dasatinib analog compounds listed in Table 3.

[0209]

[0047] TABLE 3 - Specific Dasatinib analog compounds

[0210]

[0048] The structure of select Dasatinib analog compounds, from Table 3, rows 1 and rows 20- 23, are provided below: 2-((6-(4-(2-hydroxyethyl)piperazin-1-yl)-2-methylpyrimidin-4- yl)amino)-N-mesitylthiazole-5-carboxamide

[0211] N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin-1- yl)pyrimidin-4-yl)amino)thiazole-5-carboxamide

[0212] N-(2-chloro-6-methylphenyl)-2-((2-methyl-6- morpholinopyrimidin-4-yl)amino)thiazole-5-carboxamide

[0213] N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-

[0214] 1-yl)pyridin-2-yl)amino)thiazole-5-carboxamide

[0215] 23 N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-1- yl)pyridin-2-yl)amino)thiazole-5-carboxamide

[0216]

[0049] In various embodiments, the invention comprises methods of obtaining a regenerated plantlet from a plant microspore comprising isolating a plant microspore, culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor selected from: a. Apatinib (CAS811803-05-1), b. Dasatinib (CAS302692-49-8), c. A combination of Apatinib (CAS811803-05-1) and Dasatinib (CAS302692-49-8), d. Any one or more members of the Markush group for Apatinib provided in formula Al, A2, or A3, e. Any one or more members of the Markush group for Dasatinib provided in formula DI, D2, D3 or D4, f. Any one or more combinations of the foregoing a.- e., and regenerating a plantlet from the cultured microspore.

[0217]

[0050] While specific dosage ranges are provided below, generally, Apatinib, Dasatinib,

[0218] Apatinib analogs and / or Dasatinib analogs, optionally in combination with a HD AC inhibitor, are used herein at a dosage sufficient for embryogenesis induction. Herein exemplary dosages are described that are shown to be sufficient, and in some cases optimal, for embryogenesis induction. For example, the method exemplified in Example 1 and / or 8 herein can be used to test or further adjust for sufficient and / or optimal dosage. As explained in those examples, the culture plates were incubated and screened for embryo-like structures (including embryoids) after e.g. 2 weeks of culture, and / or after 45-60 days of culture. The number of embryo-like structures (ELS) and other macroscopic structures were determined and the percentage of ELS and / or macroscopic structures compared to the initial number of microspores was calculated. Thus, the percentage of embryogenesis induction ((formation of) embryo-like structures (ELS) and / or other macroscopic structures) was calculated and / or the relative increase of embryogenesis induction ((formation of) embryo-like structures (ELS) and / or other macroscopic structures) relative to control was determined (control is e.g. culturing the plant microspore in the absence of an embryogenesis inducing agent, in particular in the absence of Apatinib, Dasatinib, Apatinib analogs and / or Dasatinib analogs, optionally in combination with a HDAC inhibitor). As is exemplified in the examples, the tyrosine kinase inhibitor(s) and / or HDAC inhibitors to be used herein preferably are used at a dosage sufficient to induce embryogenesis and / or are capable of inducing embryogenesis, in particular to a greater extent when compared to the control, for example resulting in greater than 10 %, 15 %, 20 %, 25 % or 30% in embryo-like structure formation when compared to the control. Ideally, tyrosine kinase inhibitor(s) and / or HDAC inhibitors are used at the lowest maximum effective dosage or less.

[0219]

[0051] Apatinib (CAS811803-05-1) may be used herein at a dosage between 5-60 pM, e.g between 5-50 pM, 5-40 pM, 10-50 pM, or 10 to 40 pM or 10 to 30 pM, or as it has been observed for optimal effect, at a dosage between 10-20pM. The term “between” as used herein generally includes the endpoints. For example a dosage between 5-60 pM includes any dosage of from 5 up to 60 pM, including 5 pM and 60 pM, e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 pM. In other words, a dosage between e.g. 5-60 pM means that any concrete dosage in that range may be used, e.g. 5, 6, 7, 8 pM and so on. In a preferred aspect, Apatinib may be used at a dosage between 10-20pM. Preferred Apatinib analogs described herein are analogs #1, 2, 3, 4, 5, 6, 8, 11 and 13 from Table 12. Apatinib analogs may generally be used at the same dosage as Apatinib. Particularly preferred are CAS 811802-99-0 (#1), CAS 811803-00-6 (#2), and CAS 811803-01-7 (#3). CAS 811802-99-0 (#1) is preferably used at a dosage between 5 pM and 30 pM (e.g. especially preferred at 10 pM or 20 pM or in between 10 pM and 20 pM). CAS 811803-00-6 (#2) is preferably used at a dosage between 4 pM and 40 pM (e.g. especially preferred at about 20 pM). CAS 811803-01-7 (#3) is preferably used at a dosage of about 30 pM.

[0052] Dasatinib (CAS302692-49-8) may be used at a dosage between 1-50 pM, 2.5-50 pM, or 5-50 pM, e.g between 10-50 pM, or 10 to 40 pM or, has been observed in some experiments, for optimal effect betweenlO to 30 pM. For example, a dosage between 1-50 pM includes any dosage of from 1 up to 50 pM, including 1 pM and 50 pM, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pM. In a preferred aspect, Dasatinib may be used at a dosage between 10-30pM. Dasatinib analogs may generally be used at the same dosage as Dasatinib. Herein preferred Dasatinib analogs are analogs #1, 20, 21, 22 and 23 from Table 13. #1 from Table 13 is preferably used at a range between 20-40 pM (e.g. most preferably at about 40pM). #20 from Table 13 (CAS1335054-68-6) is preferably used at a range between 5-40 pM (e.g. most preferably at about 5-10 pM based on the results shown in each of Tables 13 and 14). #21 from Table 13 (CAS 302962-43-2) is preferably used at a range between 5-40pM (e.g. most preferable at about 5-15 pM based on the results shown in each of Tables 13 and 14). #22 from Table 13 is preferably used at a range between 5-40 pM (e.g. most preferable at a dosage of about 30 pM). #23 from Table 13 (CAS 302961-72-4) is preferably used at a range between 5-40 pM (e.g. most preferably at a dosage of about 40pM).

[0220]

[0053] When used in combination each of Apatinib (CAS811803-05-1) and Dasatinib (CAS302692-49-8) may be used at a dosage between 5-50 pM, e.g between 10-50 pM, or 10 to 40 pM or 10 to 30 pM. In a preferred aspect, Apatinib may be used at a dosage between 10- 20pM and Dasatinib may be used at a dosage between 10-30 pM.

[0221]

[0054] The Apatinib analogs and Dasatinib analogs may be used at the dosages indicated for Apatinib and Dasatinib, respectively, and has been observed herein, effective doses have been generally observed between 5-40 pM. Thus, Apatinib analogs and / or Dasatinib analogs may each be used at a dosage between 5-40 pM.

[0222]

[0055] The term “dosage” as used herein can refer to the concentration of the compound in a medium.

[0056] In some embodiments, the members of the Apatinib Markush group are one or more of:

[0223] 1) N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide

[0224] (CAS#811802-99-0),

[0225] 2) N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide

[0226] (CAS#811803-00-6),

[0227] 3) N-(4-(l-cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide

[0228] (CAS#811803-01-7),

[0229] 4) N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide

[0230] (CAS#811803-02-8),

[0231] 5) N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide

[0232] (CAS#811803-03-9),

[0233] 6) N-(4-( 1 -cyanocy cl opropyl)phenyl)-2-((pyri din-4- ylmethyl)amino)nicotinamide (CAS#811803-04-0),

[0234] 7) N-phenyl-2-((pyridin-4-ylmethyl)amino)ni cotinamide,

[0235] 8) N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)ni cotinamide (CAS#811803-06-2),

[0236] 9) N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-07-3),

[0237] 10) N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-08-4),

[0238] 11) N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-09-5),

[0239] 12) N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-10-8),

[0240] 13) N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0241] (CAS#811803-11-9),

[0242] 14) N-(4-(l-(methoxymethyl)cyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)ni cotinamide (CAS#811803-12-0), 15) N-(2'-oxospiro[cyclopentane-l ,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0243] (CAS#811803-13-1),

[0244] 16) N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0245] (CAS#811803-14-2),

[0246] 17) N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0247] (CAS#811803-15-3),

[0248] 18) N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0249] (CAS#811803-16-4),

[0250] 19) 2-(( lH-indazol-6-yl)amino)-N-(4-( 1 -cyanocyclopentyl)phenyl)nicotinamide (CAS#811803-17-5),

[0251] 20) 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclobutyl)phenyl)nicotinamide (CAS#811803-18-6), and

[0252] 21 ) 2-(( lH-indazol-6-yl)amino)-N-(4-( 1 -cyanocyclopropy l)phenyl)nicotinamide (CAS#811803-19-7).

[0253]

[0057] In some embodiments, the members of the dasatinib markush group are one or more of:

[0254] 1) 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- mesitylthiazole-5-carboxamide

[0255] 2) N-(2-fluoro-5-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide

[0256] 3) N-(4-bromo-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide

[0257] 4) 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N-(naphthalen- 2-yl)thiazole-5-carboxamide

[0258] 5) 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N-(5-isopropyl- 2-methylphenyl)thiazole-5-carboxamide 6) N-(2,6-dimethoxyphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l -yl)-2-methylpyrimidin- 4-yl)amino)thiazole-5-carboxamide

[0259] 7) tert-butyl (5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)carbamate (CAS#302964-06-3),

[0260] 8) 2-(3-butylureido)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide

[0261] 9) N-(2-chloro-6-methylphenyl)-2-(3-cyclohexylureido)thiazole-5-carboxamide

[0262] 10) N-(2-chloro-6-methylphenyl)-2-(3-(3-methoxypropyl)ureido)thiazole-5-carboxamide

[0263] 11) N-(2-chloro-6-methylphenyl)-2-(3-(3-fluoropropyl)ureido)thiazole-5-carboxamide

[0264] 12) N-(2-chloro-6-methylphenyl)-2-(cyclopropanecarboxamido)thiazole-5-carboxamide (CAS#302961-12-2),

[0265] 13) N-(2-chloro-6-methylphenyl)-2-(cyclohexanecarboxamido)thiazole-5-carboxamide (CAS#302961-45-l),

[0266] 14) N-(2-chloro-6-methylphenyl)-2-(2-methylpentanamido)thiazole-5-carboxamide (CAS#302961-58-6),

[0267] 15) 2-butyramido-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (CAS#302961-34-8),

[0268] 16) N-(2-chloro-6-methylphenyl)-2-(nicotinamido)thiazole-5-carboxamide (CAS# 302961-51-9),

[0269] 17) N-(2-chloro-6-methylphenyl)-2-(picolinamido)thiazole-5-carboxamide (CAS#302961-50-8),

[0270] 18) N-(2-chloro-6-methylphenyl)-2-((2-methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (CAS#823206-99-l),

[0271] 19) N-(2-chloro-6-methylphenyl)-2-((6-(dimethylamino)-2-methylpyrimidin-4- yl)amino)thiazole-5-carboxamide,

[0272] 20) N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin-l-yl)pyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#1335054-68-6),

[0273] 21 ) N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-morpholinopyrimidin-4-yl)amino)thiazole-5- carboxamide (CAS#302962-43 -2),

[0274] 22) N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin- 1 -yl)pyridin-2- yl)amino)thiazole-5-carboxamide,

[0275] 23) N-(2-chloro-6-rnethylphenyl)-2-((4-methylpyridin-2-yl)amino)thiazole-5-carboxamide (CAS#302961-72-4), 24) N-(2-chloro-6-methylphenyl)-2-(pyridin-4-ylamino)thiazole-5-carboxamide (CAS#302961 - 88-2),

[0276] 25) N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin- 4-yl)amino)-4-methylthiazole-5-carboxamide,

[0277] 26) N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin- 1 -yl)-2-methylpyrimidin- 4-yl)amino)-4-(trifluoromethyl)thiazole-5-carboxamide, and

[0278] 27) N-(2-chloro-6-rnethylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin- 4-yl)amino)-4-phenylthiazole-5-carboxamide.

[0279]

[0058] The methods provided herein comprise a step of culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor as defined herein.

[0280]

[0059] “Culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor” generally means that the plant microspore is cultured under conditions sufficient to allow regeneration of a plantlet. The regeneration of a plantlet can comprise formation of an embryo, embryo-like structure (inclusive of an embryoid structure) and / or multicellular / macroscopic structures. The method comprises the regeneration of a plant from such a plantlet (e.g. from an embryo, embryo-like structure and / or multicellular / macroscopic structures). In one embodiment, the method comprises regeneration of a plant from an embryoid structure.

[0281]

[0060] The “culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor” generally involves contacting the plant microspore with an embryogenesis inducing tyrosine kinase. For example, the microspore may be contacted with the tyrosine kinase inhibitor in that the tyrosine kinase inhibitor is present in the microspore culture media used for culturing the plant microspore, or is added a later culturing stage.

[0282]

[0061] An exemplary culture medium for culturing the plant microspore is a basic induction media (liquid induction medium ), that may, for example, be prepared as follows (or consist of the respective components): 1.77g NLN Basal medium (Cat# N479, PhytoTechnology Laboratories), 100g sucrose, 0.00001g (or 100 pl of 0.1 mg / mLstock) Kinetin, KOH (1.0 and 0.1M) to adjust pH to 5.8, water adjusted to 1000 mL. the tyrosine kinase inhibitors to be used herein are added to the culture medium to result in the desired dosages as defined herein above.

[0283]

[0062] As mentioned, “dosage” refers particularly in this context to the concentration of the tyrosine kinase inhibitors in the culture medium.

[0284]

[0063] The microspores may be cultured in appropriate vessels, for example in petri dishes. The number of microspores per vessel is chosen appropriate and may range, for example, between 1 and 100,000, e.g. 1,000, 5,000, 10,000, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000 or 90,000 microspores may be used per vessel. The volume of the culture medium is chosen appropriately, such as for example, culturing in 1.5 ml liquid induction medium with approximately 40,000-60,000 microspores.

[0285]

[0064] The microspores are cultured for a sufficient time allowing the formation of plantlets (and regeneration thereof), e.g. of from 2 to 8 weeks, e.g. 2, 3, 4, 5, 6, 7, 8 weeks or longer if needed. The temperature may range from 25 to 32 °C and typically is about 28 °C, but can be varied as appropriate for the species or variety. Culturing is typically performed in darkness or low light conditions until photosynthesis begins. Nutrients, such as sugars or other energy sources, may be applied to the culture.

[0286]

[0065] In some aspects, the tyrosine kinase inhibitor is added to the microspore culture at day 0. In other aspects, the tyrosine kinase inhibitor may not be added to the microspore culture until at least 3 days after culture initiation. In other words, in a first step of the culturing step, the plant microspores may be cultured in the absence of a tyrosine kinase inhibitor for a given time, e.g. for 1, 2, 3, 4 and tip to 5 days. In one preferred aspect, culturing is performed for 3 days.

[0287] This may be conveniently done by culturing the plant microspore in a culture medium as described above, with the exception that the tyrosine kinase inhibitor is not present in the culture medium. For example, if the culture is initiated on a Monday (day 0) and the culture is performed for 3 days in the absence of the tyrosine kinase inhibitor, the microspores are cultured until Thursday (day 3), at which time the tyrosine kinase inhibitor may be added.

[0066] The tyrosine kinase inhibitor can subsequently be added by adding the inhibitor to the culture medium to achieve the desired dosage. Alternatively, the culture medium where no inhibitor is present may be replaced / exchanged with a culture medium where the inhibitor is present at the indicated dosage.

[0288]

[0067] Any inhibitor described herein may be used in this context. In a preferred aspect, the inhibitor is Dasalinib. Such a first step of culturing the microspore in the absence of a tyrosine kinase inhibitor is then followed by a subsequent, step of culturing the microspore in the presence of a tyrosine kinase inhibitor as described further above, e.g. for about an additional 2 to 8 weeks.

[0289]

[0068] The plant microspore may be a maize microspore or a microspore from a different plant species.

[0290]

[0069] In some aspect, the plant microspore is a maize microspore from a maize plant or maize variety that is conducive to microspore plantlet formation and / or regeneration, such as a formation and / or regeneration into an embryo with root and shoot formation, and / or an embryolike structure formation or a multicellular / macroscopic structure formation. In some aspects the plant microspore is from sunflower, soybean or another plant species (such as a rice, sorghum, brassica, wheat, or cotton microspore), where the plant or plant variety is conducive to microspore plantlet formation and / or regeneration.

[0291]

[0070] In some embodiments, the plant microspore is a maize microspore from a maize plant or maize variety that is recalcitrant to microspore-derived plantlet formation and / or regeneration, such as a formation and / or regeneration into an embryo with root and shoot formation, and / or an embryo-like structure formation or a multicellular / macroscopic structure formation. For example, a maize microspore from a maize variety that is recalcitrant to microspore plantlet regeneration may be defined as a microspore from a maize variety that does not regenerate into an embryo-like structure. It is understood that a maize microspore from a maize variety that is recalcitrant to microspore plantlet regeneration is, in each case, recalcitrant without the addition of a compound such as any one of the Apatinib, Apatinib analog, Dasatinib, or Dasatinib analog compounds described herein. Likewise, the plant microspore may be a variety from a plant species other than maize (such as sunflower, soybean, rice, sorghum, brassica, wheat, or cotton microspore), where that plant or plant variety is particularly recalcitrant to microspore plantlet formation and / or regeneration.

[0292]

[0071] “Recalcitrant” microspore / plant / plant variety means that no or essentially no plantlets are formed and / or regenerated in the absence of an enhancer of plantlet formation and / or regeneration, such as the tyrosine kinase inhibitors described herein, such that formation and / or regeneration of that variety (or even all varieties of a species) is not efficient enough to be feasible for plant variety development.

[0293]

[0072] The microspore may be either haploid or diploid. For haploid microspores, the haploid microspore may be placed in contact with a chromosome doubling agent for a period sufficient to generate a doubled haploid cell, plant embryo, plant embryoid, or plant. The contact with the chromosome doubling agent may occur prior to, contemporaneous with or following contact with the tyrosine kinase inhibitor. The microspore cells used as starting material may be a mixed population of uninucleate and binucleate microspores, a purified population of uninucleate microspores, or a purified population of binucleate microspores. The kinase inhibitor may be present in the microspore culture media, the germination media, and / or the tissue culture media. Optionally, the kinase inhibitor is not added to the microspore culture media until at least 3 days after culture initiation. The microspore may be derived from sunflower, rice, sorghum, brassica, soybean, wheat, or cotton. An optional heat shock step and the optional addition of Trichostatin A (TSA) or another HD AC inhibitor (such as SAHA, Apicidin or Fimepinostat) or any combination thereof may be utilized as described herein and / or in the examples.

[0294]

[0073] The herein disclosed tyrosine kinase inhibitors have been found to be useful for culturing conducive or recalcitrant microspores (or likewise microspores from conducive or recalcitrant plants / plant species). The term “microspores from plants / plant species / plant variety” can be interchangeably used herein with the terms “microspores obtained from plants / plant species / plant variety” or “plant / plant species / plant variety microspore”. For example, “microspore from maize”, “microspore obtained from maize” and “maize microspore” can be used interchangeably herein.

[0295]

[0074] To further enhance formation and / or regeneration of plantlets, the herein disclosed tyrosine kinase inhibitors may be used in combination with one or more HDAC inhibitors. The herein disclosed tyrosine kinase inhibitors may be used in combination with one or more HDAC inhibitors for culturing conducive or recalcitrant microspores (or likewise microspores from conducive or recalcitrant plants / plant species or varieties). The additional use of HDAC inhibitors may be particularly useful for culturing recalcitrant microspores.

[0296]

[0075] For example, when a HDAC inhibitor (such as TSA, Apicidin or Fimepinostat) is used, the following protocol may be followed. The microspore is cultured in the presence of a HDAC inhibitor. “Presence” can mean culturing the microspores in a medium in which the HDAC inhibitor is included or has been added. In a preferred aspect, the culture is initiated prior to the addition of the tyrosine kinase inhibitor. Suitable media and culture conditions have been described above in context of culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor These explanations apply, rautatis mutandis, here.

[0297] [76 j The microspores are pre-cultured or cultured in the presence of a HDAC inhibitor for a given time, e.g. for 15 minutes, 30 minutes, 1 hour, 2 hours, between 3 to 23 hours, 1 day, 2 days, 3, 4 and up to 5 days. In one aspect, culturing is performed for 3 days.

[0298]

[0077] Particularly for preculture in the presence of a HDAC inhibitor the temperature may range from 8 to 12 °C and typically is about 10 °C. Culturing is typically performed in darkness or low light conditions. The temperature may range from 8 to 32 °C and typically is about 28 °C. Culturing is typically performed in darkness or low light conditions. An optional heat shock process may be used, either with or without HDAC inhibitor pre-culture. The heat shock process involves a period of incubation of the microspore culture at a higher temperature. One heat shock protocol (in this case, including an optional HDAC inhibitor pretreatment) is to incubate isolated microspore cells with an HDAC inhibitor, such as lOOnM TSA, in isolation medium for 30 minutes. Following a washing step with isolation medium, culture microspore cells are cultured and immobilized in plates, and liquid induction medium either comprising or supplemented with the tyrosine kinase inhibitor. The heat shock process comprises culturing the plate at a temperature of approximately 32°C (or at any one or more points in the range of 29°C to 40°C for the first 1-6 days of culture and then transferring to a cooler culturing temperature (such as 28°C or less) for the remaining culture period until embryogenesis has progressed to the desired point. One specific protocol determined to be effective is an initial culture at 32°C for 3-5 days, followed by the remaining culture at 28°C.

[0299]

[0078] In one protocol found to be effective, the tyrosine kinase inhibitor is added to the microspore culture at day (). In another aspect, the tyrosine kinase inhibitor is not added to the microspore culture until at least 3 days after culture initiation. In other words, in a first step of the culturing step, the plant microspores are cultured in the absence of a tyrosine kinase inhibitor for a given time, e.g. for 10 minutes, 30 minutes, 1 hour, 2 hours, between 3 to 23 hours, 1 day, 2 days, 3, 4 and up to 5 days. In one preferred aspect, culturing was successfully performed for 3 days prior to the addition of the tyrosine kinase inhibitor. In such case, the HD AC inhibitor was TSA. In another aspect, culturing was successfully performed with culturing with TSA and SAHA for 30 minutes, followed by 5 days with Apicidm.

[0300]

[0079] The subsequent culturing of the microspores in the presence of the tyrosine kinase inhibitor may then be performed as described above. In a preferred aspect, the further culturing is performed in the absence of the HDAC inhibitor. This may conveniently be achieved by replacing / exchanging the culture medium in which the HDAC inhibitor is present with a culture medium in which the HDAC inhibitor is absent, and the tyrosine kinase inhibitor is present (at the indicated dosages).

[0301]

[0080] In one aspect observed to be effective, the HDAC inhibitor to be used herein is Trichostatin A (at a dosage between 1 nM to 1 pM, e.g. between 10 nM to 900 nM 20 nM to 800 nM, or 30 nM to 700 nM, or 40 nM to 600 nM. In a preferred aspect the dosage is between 50nM to 100 nM, e.g. 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM. TSA may be used either alone (i.e. as sole HDAC inhibitor) or in combination with one or more further HDAC inhibitor, e g SAHA, Fimepinostat and / or Apicidan.

[0081] In one aspect, the HD AC inhibitor to be used herein is Fimepinostat (CAS 1339928-25-4) (at a dosage between 1 nM to 1 pM, e.g. between 10 nM to 900 nM 20 nM to 800 nM, or 30 nM to 700 nM, or 40 nM to 600 nM. In a preferred aspect the dosage is between 50nM to 300 nM, e.g. 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM 120 nM. 130 nM,140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, or 200 nM. 210 nM 220 nM, 230 nM, 240 nM, 250 nM, 260 nM, 270 nM, 280 nM, 290 nM, or 300 nM. In a preferred aspect the dosage is 200nM. Fimepinostat may be used either alone (i.e. as sole I ID AC inhibitor) or in combination with one or more further HD AC inhibitor, e.g. SAHA, TSA and / or Apicidin.

[0302]

[0082] In one aspect, the HDAC inhibitor to be used herein is SAHA (at a dosage between 1 pM to 100 pM, e.g. between 2 pM to 90 pM, 3 pM to 80 pM, 4 pM to 70 pM, 5 pM to 60 pM, 7 pM to 50 pM, 8 pM to 40 pM or 9 pM to 30 pM. The upper limit may be 25 pM, 20 pM, 15 pM or less, e.g. 14, 13, 12 or 11 pM. In a preferred aspect the dosage is 10 pM.

[0303]

[0083] In one aspect, the HDAC inhibitor to be used herein is Apicidin (at a dosage between 1 nM to 100 nMI, e.g. between 5 nM to 90 nM, between 10 nM to 80 nM, between 15 nM to 70 nM, between 20 nM to 60 nM, between 25 nM to 50 nM, between 30 nM to 40 nM, e.g. 31 , 32, 33, 34, 35. 36, 37, 38 or 39 nM,. In a preferred aspect the dosage is 35 nM.

[0304]

[0084] Exemplary histone deacetylase inhibitor (HDACi) to be used herein may be Trichostatin A (ISA), hydroxamic acids and hydroxamates, such as vorinostat (SAHA), belinosiat (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 aci d compounds such as phenyl butyrate and valproic acid, Fimepinostat (CAS 133992.8-25-4) and Apicidin (CAS 183506-66-3) either alone or in combination

[0305]

[0085] Another protocol for using a HDAC inhibitor is as follows (specifically, when the HDAC inhibitor is Fimepinostat, TSA, SAHA and / or Apicidin or a combination of two or more of Fimepinostat, TSA, SAHA and / or Apicidin). In one aspect, a combination of TSA and Apicidin is used. In one aspect, a combination of SAHA and Apicidin may be used.

[0306]

[0086] The microspore is initially cultured in the presence of a HD AC inhibitor, such as Fimepinostat, TSA or SAHA, or in the absence of a tyrosine kinase inhibitor. As described above, “presence” can mean culturing the microspores in a medium in which the HD AC inhibitor is included or has been added. Suitable media and culture conditions have been described above in context of culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor. These explanations apply, mutatis mutandis, here. The microspores may be cultured in the presence of a HD AC inhibitor for a given time, e.g. for 10 minutes, 15 minutes, 2.0 minutes, 25 minutes 30 minutes, 1 hour, 2 hours, between 3 to 23 hours, 1 day, 2 days, 3, 4 and up to 5 days In one aspect, culturing is performed for between 10 minutes to one hour, such as between 15 minutes and 50 minutes, or between 20 minutes and 40 minutes. In one embodiment, culturing is performed 15 minutes or for 30 minutes

[0307]

[0087] Suitable media for the initial culture in the presence of a HD AC inhibitor are known in the art. Exemplary microspore isolation media to be used in accordance with the invention, e.g. for sunflower, soybean or maize, are described in the examples and references cited herein.

[0308]

[0088] The temperature for the HD AC inhibitor pre-culture may range from 8 to 12 °C and typically is about 10 °C, although other culture conditions could be used, such within the range of 8 to 34 °C, including use of the culture temperature conditions described herein. Culturing is typically performed in darkness or low light.

[0309]

[0089] In one aspect, the tyrosine kinase inhibitor may be added to the microspore culture after the HD AC inhibitors (specifically TSA and / or SAHA and / or Fimepinostat) have been removed, e.g. by replacing the culture medium in which the HD AC inhibitors (specifically TSA and / or SAHA and / or Fimepinostat) are present by a culture medium in which the HD AC inhibitors (specifically TSA and / or SAHA and / or Fimepinostat) is absent, while the tyrosine kinase inhibitor is present. Suitable media for the culture in the absence of a HD AC inhibitor and presence of tyrosine kinase inhibitor are known in the art. Exemplary media to be used in accordance with the invention e.g. for maize, sunflower and soybean are described in the examples and references cited herein.

[0310]

[0090] The subsequent culturing of the microspores in the presence of the tyrosine kinase inhibitor may then be performed as described above, with the exception that one or more different HDAC inhibitors may, optionally, be present (in the culture medium), such as the HDAC inhibitor Apicidin. In a preferred aspect, the further culturing is performed in the absence of the HDAC inhibitor used in the preceding step (specifically TSA and / or SAHA). This may conveniently be achieved by replacing / exchanging the culture medium in which the initially used HDAC inhibitor (specifically TSA and / or SAHA) is present with a culture medium in which the initial HDAC inhibitor is absent, and the tyrosine kinase inhibitor and different HDAC inhibitor (such as Apicidin) is present (at the indicated dosages).

[0311]

[0091] The microspores are cultured in the presence of the second (different) HDAC inhibitor, and tyrosine kinase inhibitor for a given time, e.g. 1 day, 2. days, 3, 4, 5, 6 and up to 7 days. In one preferred aspect, culturing is performed for 5 days. The temperature may range from 25 to 33 °C and typically is about 32 °C. Culturing is typically performed in darkness.

[0312]

[0092] The microspores can further be cultured (in a subsequent culturing step) in the presence of the second (different) HDAC inhibitor and tyrosine kinase inhibitor for a given time, e.g. 7 days, 10 days, 12, 14, 16, 18 and up to 20 days. . In one preferred aspect, further culturing is performed for 16 days. The temperature may range from 25 to 33 °C and typically is about 28 °C. Culturing is typically performed in darkness.

[0313]

[0093] Any inhibitor described herein may be used in this context. In a preferred aspect, die inhibitor is Apatinib, specifically when used in combination with SAHA and Apicidin.

[0314]

[0094] The following combinations may be used: tyrosine kinase inhibitor (such as Apatinib) in combination with SAHA and Apicidin; tyrosine kinase inhibitor (such as Apatinib) in combination with I SA and Apicidin.

[0095] Alternatively, the subsequent culturing of the microspores in the presence of the tyrosine kinase inhibitor may then be performed as described above. In one aspect, the subsequent culturing of the microspores in the presence of a tyrosine kinase inhibitor is performed in the absence of HDAC inhibitors as disclosed e.g in the absence of Fimepinostat, SAHA, TSA and / or Apicidin.

[0315]

[0096] This may conveniently be achieved by replacing / exchanging the culture medium in which the initially used HDAC inhibitor (specifically Fimepinostat, TSA and / or SAHA) is present with a culture medium in which the initial HDAC inhibitor is absent, and the tyrosine kinase inhibitor is present (at the indicated dosages). This described aspect may be particularly advantageous for culturing microspores of dicotyledonous plants such as sunflower or soybean.

[0316]

[0097] Further, the subsequent culturing of the microspores in the presence of the tyrosine kinase inhibitor may additionally comprise culturing in the presence of one or more cytokine, such as BAP or TDZ, e.g. the cytokine may be used at a dosage of 0.1 pM to 0.5 pM, e.g. 0.1 pM, 0.2 pM, 0.3 pM, 04 pM or 0 5 pM. In a preferred aspect, the cytokine may be used at a dosage of 0.2 pM.

[0317]

[0098] The microspores are cultured in the presence of the tyrosine kinase inhibitor for a given time, e g. 1 day, 2 days, 3, 4, 5, 6 and up to 7 days. In one preferred aspect, culturing is performed for 5 days. The temperature may range from 25 to 33 °C and typically is about 32 °C. Culturing is typically performed in darkness.

[0318]

[0099] Any inhibitor described herein may be used in this context. In a preferred aspect, the inhibitor is Apatinib, specifically when used in combination with Fimepinostat.

[0319]

[0100] Dasatinib may in the alternative be used, specifically when used in combination with TSA. Any suitable dosage of Dasatinib may be used, e.g. at a dosage of between 1-50 pM, 2.5-50 pM, or 5-50 pM, e.g between 10-50 pM, or 10 to 40 pM or 10 to 30 pM. For example a dosage between 1-50 pM includes any dosage of from 1 up to 50 pM, including 1 pM and 50 pM, e g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 3738, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 pM. Specifically, for microspores from dicotyledonous plants, such as sunflower or soybean, Dasatinib may be used at a dosage between 1-30 pM, 2.5-30 pM, or 5-30 pM.

[0320]

[0101] The microspores may subsequently be cultured in the absence of the tyrosine kinase inhibitor (and / or HDAC inhibitor and / or cytokine) for a given time, e.g 2 to 6 weeks, e.g.

[0321] 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more. In one preferred aspect, culturing is performed for 3 to 5 w'eeks. The temperature may range from 25 to 33 °C and typically is about 28 °C. Culturing is typically performed in darkness. This may conveniently be achieved by replacing / exchanging the culture medium in which the tyrosine kinase inhibitor (and / or HDAC inhibitor and / or cytokine) is present with a culture medium in which the tyrosine kinase inhibitor (and / or HDAC inhibitor and / or cytokine) is absent. This described aspect may be particularly advantageous for culturing microspores of dicotyledonous plants such as sunflower or soybean.

[0322]

[0102] In some aspects, the present embodiments may be defined as follows: a. A method of obtaining a plantlet from a plant microspore comprising.

[0323] Isolating a plant microspore,

[0324] Culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor selected from any one of: i. Apatinib (CAS811803-05-1) at a dosage between 10-20pM, ii. Dasatinib (CAS302692-49-8) at a dosage between 10-30pM, or iii. A combination of Apatinib (CAS811803-05-1) at a dosage between 10-20pM and Dasatinib (CAS302692-49-8) at a dosage between 10-30pM, iv. Any Apatinib analog listed in Table 2, v. Any Dasatinib analog listed in Table 3, vi. Any Apatinib and Dasatinib combination from iv and v above; vii. Any Apatinib analog within the Markush group described in Formula Al, A2 or A3, viii. Any Dasatinib analog within the Markush group described in Formula DI, D2, D3 or

[0325] D4, ix. Any Apatinib and Dasatinib combination from vii and viii above, x. Any combination from one or more of i, ii, iv, v, vii and viii above, and generating a plantlet from the cultured microspore. b. The method of a, wherein the plant microspore is a maize microspore. c. The method of a or b, wherein the plant microspore is a maize microspore from a plant variety that is recalcitrant to the formation of embryo-like structures or multi cellular / microscopic structures. d. The method of any one of a-c, wherein the microspore is haploid. e. The method of d, further comprising contacting the haploid microspore or one or more cells derived from the haploid microspore with a chromosome doubling agent for a period sufficient to generate a doubled haploid cell, plant embryoid or plant. f. The method of any one of a-e, wherein the microspore is treated with a doubling agent. g- The method of any one of a-f, wherein the microspore is a mixed population of uninucleate and binucleate microspores, or a purified population of either uninucleate or binucleate microspores. h. The method of any one of a-g, wherein the embryogenesis inducing kinase inhibitor is present in the microspore culture media. i. The method of any one of a-h, wherein the kinase inhibitor, such as Dasatinib, is not added to the microspore culture until at least 3 days after culture initiation. j- The method of any one of a and c to i, wherein the plant microspore is obtained from sunflower. k. The method of any one of a and c to j wherein the plant microspore is obtained from rice, sorghum, brassica, soybean, wheat, or cotton.

[0326] 1. The method of a to k, wherein an HD AC inhibitor, such as Trichostatin A, Fimepinostat, or Apicidin, is used in combination with the tyrosine kinase inhibitor. Further, the invention relates to uses of a tyrosine kinase inhibitor, and optionally, one or more HD AC inhibitor, for regenerating a plantlet from a plant microspore, and wherein the tyrosine kinase inhibitor may be Apatinib in the dosage indicated herein or an apatinib analog, Dasatinib in the dosage indicated herein or a Dasatinib analog, or a combination of Apatinib or an Apatinib analog or Dasatinib or a Dasatinib analog in the dosages described herein, as well as a kit comprising the same. The definition and explanations given herein in context of a method for regenerating a plantlet from a plant microspore apply, mutatis mutandis, in context of these uses and kits, specifically in relation to the HDAC inhibitor, plantlet, plant, plant microspore.

[0327]

[0103] The terms ‘embryogenesis inducing tyrosine kinase inhibitor” and “tyrosine kinase inhibitor” can be used interchangeably herein. The term “embryogenesis inducing” is meant to specify that the “tyrosine kinase inhibitor” is capable of inducing embryogenesis and / or is suitable to be used for regenerating a plantlet from a plant microspore. The terms ‘contacting’ or ‘treating' with a chemical compound may refer to such contact or treatment occurring as part of a further culturing step. The terms “treatment’ ’ / “treating”, “further cultured’7“further culturing” or “contacting” can be used interchangeably herein.

[0328]

[0104] In one aspect, the invention relates to the use of Apatinib, Dasatinib or a combination of Apatinib and Dasatinib, and optionally, one or more HDAC inhibitor, for regenerating a plantlet from a plant microspore,

[0329] (i) Apatinib (CAS811803-05-1) is used at a dosage between 10-30pM,

[0330] (ii) Dasatinib (CAS302692-49-8) is used at a dosage between 10-50pM, or

[0331] (iii) a combination of Apatinib (CAS811803-05-1) is used at a dosage between 10-30pM and Dasatinib (CAS302692-49-8) at a dosage between 10-50pM.

[0105] In one aspect, the invention relates to the use of an embryogenesis inducing tyrosine kinase inhibitor, and optionally, one or more HD AC inhibitor, for regenerating a plantlet from a plant microspore, wherein the tyrosine kinase inhibitor is an Apatinib analog, a Dasatinib analog, or a combination of an Apatinib analog and a Dasatinib analog, and wherein the Apatinib analog is selected from any one or more of: i. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811802-99-0), ii. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-00-6), iii. N-(4-(l-cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-01-7), iv. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-02-8), v. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-03-9), vi. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide (CAS#811803-04-0), vii. N-phenyl-2-((pyridin-4-ylmethyl)amino)nicotinamide, viii. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-06-2), ix. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0332] (CAS#811803-07-3), x. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0333] (CAS#811803-08-4), xi. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0334] (CAS#811803-09-5), xii. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide (CAS#81 1803-10-8), xiii. N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0335] (CAS#811803-11-9), xiv. N-(4-(l-(methoxymethyl)cyclohexyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0336] (CAS#811803-12-0), xv. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0337] (CAS#811803-13-1), xvi. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0338] (CAS#811803-14-2), xvii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0339] (CAS#811803-15-3), xviii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0340] (CAS#811803-16-4), xix. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopentyl)phenyl)ni cotinamide (CAS#811803-17-5), xx. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclobutyl)phenyl)nicotinamide (CAS#811803-18-6), xxi. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopropy l)phenyl)nicotinamide

[0341] (CAS#811803-19-7), and / or

[0342] B. wherein the Dasatinib analog is selected from any one or more of: i. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- mesitylthiazole-5-carboxamide ii. N-(2-fluoro-5-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l -yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iii. N-(4-bromo-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iv. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- (naphthal en-2-y 1 )thi azol e- 5 -carb oxami de v. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-rnethylpyrirnidin-4-yl)amino)-N-(5- isopropyl-2-methylphenyl)thiazole-5-carboxamide vi. N-(2,6-dimethoxyphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide vii. tert-butyl (5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)carbamate (CAS#302964-06-3) viii. 2-(3-butylureido)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide ix. N-(2-chloro-6-methylphenyl)-2-(3-cyclohexylureido)thiazole-5-carboxamide x. N-(2-chloro-6-methylphenyl)-2-(3-(3-methoxypropyl)ureido)thiazole-5-carboxamide xi. N-(2-chloro-6-methylphenyl)-2-(3-(3-fluoropropyl)ureido)thiazole-5-carboxamide xii. N-(2-chloro-6-methylphenyl)-2-(cyclopropanecarboxamido)thiazole-5-carboxamide (CAS#302961-12-2) xiii. N-(2-chloro-6-methylphenyl)-2-(cyclohexanecarboxamido)thiazole-5-carboxamide (CAS#302961-45-1) xiv. N-(2-chloro-6-methylphenyl)-2-(2-methylpentanamido)thiazole-5-carboxamide (CAS#302961-58-6) xv. 2-butyramido-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (CAS#302961- 34-8) xvi. N-(2-chloro-6-methylphenyl)-2-(nicotinamido)thiazole-5-carboxamide (CAS#302961-51-9) xvii. N-(2-chloro-6-methylphenyl)-2-(picolinamido)thiazole-5-carboxamide (CAS#302961-50-8) xviii. N-(2-chloro-6-rnethylphenyl)-2-((2-methylpyrimidin-4-yl)amino)thiazole-5- carboxamide (CAS#823206-99-l) xix. N-(2-chloro-6-methylphenyl)-2-((6-(dimethylamino)-2-methylpyrimidin-4- yl)amino)thiazole-5-carboxamide xx. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin- 1 -yl)pyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#1335054-68-6) xxi. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-morpholinopyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#302962-43 -2) xxii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)pyridin-2- yl)amino)thiazole-5-carboxamide xxiii. N-(2-chloro-6-methylphenyl)-2-((4-methylpyri din-2 -yl)amino)thiazole-5- carboxamide (CAS#302961-72-4) xxiv. N-(2-chloro-6-methylphenyl)-2-(pyridin-4-ylamino)thiazole-5-carboxamide (CAS#302961-88-2) xxv. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-methylthiazole-5-carboxamide xxvi. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-(trifluoromethyl)thiazole-5-carboxamide xxvii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-phenylthiazole-5-carboxamide.

[0343]

[0106] In one aspect the invention comprises a kit for regenerating a plantlet from a plant microspore, the kit comprising a embryogenesis inducing tyrosine kinase inhibitor, and optionally, one or more HDAC inhibitor, wherein the tyrosine kinase inhibitor is Apatinib or an Apatinib analog, Dasatinib or a Dasatinib analog, or a combination of Apatinib or an Apatinib analog and Dasatinib or a Dasatinib analog,

[0344] A. wherein the Apatinib analog is selected from any one or more of: i. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811802-99-0), ii. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-00-6), iii. N-(4-(l-cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide

[0345] (CAS#811803-01-7), iv. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-02-8), v. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-03-9), vi. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide (CAS#811803-04-0), vii. N-phenyl-2-((pyridin-4-ylmethyl)amino)nicotinamide, viii. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-06-2), ix. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0346] (CAS#811803-07-3), x. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0347] (CAS#811803-08-4), xi. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0348] (CAS#811803-09-5), xii. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0349] (CAS#811803-10-8), xiii. N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0350] (CAS#811803-11-9), xiv. N-(4-(l-(methoxymethyl)cyclohexyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0351] (CAS#811803-12-0), xv. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0352] (CAS#811803-13-1), xvi. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6l-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0353] (CAS#811803-14-2), xvii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0354] (CAS#811803-15-3), xviii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0355] (CAS#811803-16-4), xix. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopentyl)phenyl)nicotinamide (CAS#811803-17-5), xx. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclobutyl)phenyl)nicotinamide (CAS#811803-18-6), xxi. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopropy l)phenyl)nicotinamide (CAS#811803-19-7), and / or

[0356] B. wherein the Dasatinib analog is selected from any one or more of: i. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- mesitylthi azol e- 5 -carb oxami de ii. N-(2-fluoro-5-methylphenyl)-2-((6-(4-(2-hydroxy ethyl )piperazin- 1 -yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iii. N-(4-bromo-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iv. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- (naphthal en-2-y l)thiazol e- 5 -carb oxami de v. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N-(5- isopropyl-2-methylphenyl)thiazole-5-carboxamide vi. N-(2,6-dimethoxyphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide vii. tert-butyl (5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)carbamate (CAS#302964-06-3) viii. 2-(3-butylureido)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide ix. N-(2-chloro-6-methylphenyl)-2-(3-cyclohexylureido)thiazole-5-carboxamide x. N-(2-chloro-6-methylphenyl)-2-(3-(3-methoxypropyl)ureido)thiazole-5-carboxamide xi. N-(2-chloro-6-methylphenyl)-2-(3-(3-fluoropropyl)ureido)thiazole-5-carboxamide xii. N-(2-chloro-6-methylphenyl)-2-(cyclopropanecarboxamido)thiazole-5-carboxamide (CAS#302961-12-2) xiii. N-(2-chloro-6-methylphenyl)-2-(cyclohexanecarboxamido)thiazole-5-carboxamide (CAS#302961-45-1) xiv. N-(2-chloro-6-methylphenyl)-2-(2-methylpentanamido)thiazole-5-carboxamide (CAS#302961-58-6) xv. 2-butyramido-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (CAS#302961- 34-8) xvi. N-(2-chloro-6-methylphenyl)-2-(nicotinamido)thiazole-5-carboxamide (CAS#302961-51-9) xvii. N-(2-chloro-6-methylphenyl)-2-(picolinamido)thiazole-5-carboxamide (CAS#302961-50-8) xviii. N-(2-chloro-6-methylphenyl)-2-((2-methylpyrimidin-4-yl)amino)thiazole-5- carboxamide (CAS#823206-99-l) xix. N-(2-chloro-6-methylphenyl)-2-((6-(dimethylamino)-2-methylpyrimidin-4- yl)amino)thiazole-5-carboxamide xx. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin-l-yl)pyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#1335054-68-6) xxi. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-morpholinopyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#302962-43 -2) xxii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)pyridin-2- yl)amino)thiazole-5-carboxamide xxiii. N-(2-chloro-6-methylphenyl)-2-((4-methylpyridin-2-yl)amino)thiazole-5- carboxamide (CAS#302961-72-4) xxiv. N-(2-chloro-6-methylphenyl)-2-(pyridin-4-ylamino)thiazole-5-carboxamide (CAS#302961-88-2) xxv. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-methylthiazole-5-carboxamide xxvi. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-(trifluoromethyl)thiazole-5-carboxamide xxvii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-phenylthiazole-5-carboxamide.

[0357]

[0107] The invention relates, inter alia, to the following aspects:

[0358]

[0108] XI . A method of obtaining a regenerated plantlet from a plant microspore comprising:

[0359] Isolating a plant microspore,

[0360] Culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor selected from: i. Apatinib (CAS811803-05-1) at a dosage between 5-40 pM, preferably 10-30pM, or an Apatinib analog, preferably represented by the structure of Formula A3, ii. Dasatinib (CAS302692-49-8) at a dosage between 2.5-50pM or a Dasatinib analog, preferably represented by the structure of Formula D4, or iii. A combination of Apatinib (CAS811803-05-1) at a dosage between 5-40 pM, preferably 10-30pM, and Dasatinib (CAS302692-49-8) at a dosage between 2.5- 50pM, iv. A combination of Apatinib (CAS811803-05-1) and a Dasatinib analog , preferably represented by the structure of Formula D4, v. A combination of an Apatinib analog, preferably represented by the structure of Formula A3 and Dasatinib (CAS302692-49-8), vi. A combination of an Apatinib analog, preferably represented by the structure of Formula A3 and a Dasatinib analog, preferably represented by the structure of Formula D4; and

[0361] Regenerating a plantlet from the cultured microspore.

[0109] X2. The method of XI, wherein the plant microspore is a maize microspore.

[0362]

[0110] X3. The method of item X2, wherein the Apatinib is at a dosage between 5-40 pM, preferably 10-30pM, preferably at a dosage between 10-20 pM, the Dasatinib is at a dosage between 2.5-50pM, preferably a dosage between 10-30 pM, or the combination of Apatinib and Dasatinib is between 10-20 pM for Apatinib and between 10-30 pM for Dasatinib, or wherein the Apatinib analog is at a dosage between 5-40pM, or wherein the Dasatinib analog is at a dosage between 5-40pM.

[0363] [H l] X4. The method of any one of items X1-X3, wherein the microspore is further cultured or treated with a histone deacetylase inhibitor (HD AC inhibitor) in addition to the tyrosine kinase inhibitor, optionally wherein the microspore is pretreated with an HD AC inhibitor prior to treatment with the tyrosine kinase inhibitor.

[0364]

[0112] X5. The method of any one of items X1-X4, wherein the microspore is haploid.

[0365]

[0113] X6. The method of any one of items XI to X5, further comprising contacting or treating the haploid microspore or microspore derived cells with a chromosome doubling agent for a period of time sufficient to generate a doubled haploid cell, plant embryo, plant embryoid or plant, optionally wherein said contacting or treating follows contacting or treating with the HD AC inhibitor.

[0366]

[0114] X7. The method of any one of items X1-X4, wherein the embryogenesis inducing tyrosine kinase inhibitor is present in the microspore culture media.

[0367]

[0115] X8. The method of item X7, wherein the tyrosine kinase inhibitor is not added to the microspore culture until at least 3 days after culture initiation.

[0368]

[0116] X9. The method of item X8, wherein the tyrosine kinase inhibitor is Dasatinib or a Dasatinib analog.

[0369]

[0117] XI 0. The method of item XI or X4, wherein the plant microspore is obtained from rice, sorghum, sunflower, brassica, soybean, wheat, or cotton or is a rice, sorghum, sunflower, brassica, soybean, wheat, or cotton microspore.

[0370]

[0118] Xl l. A method of obtaining a regenerated plantlet from a plant microspore comprising:

[0371] Isolating a plant microspore,

[0372] Culturing the plant microspore with an HD AC inhibitor in combination with an embryogenesis inducing tyrosine kinase inhibitor selected from: i. Apatinib (CAS811803-05-1) or an Apatinib analog, preferably represented by the structure of Formula A3, ii. Dasatinib (CAS302692-49-8) or a Dasatinib analog, preferably represented by the structure of Formula D4, or iii. A combination of Apatinib (CAS811803-05-1) and Dasatinib (CAS302692-49-8), iv. A combination of Apatinib (CAS811803-05-1) and a Dasatinib analog, preferably represented by the structure of Formula D4, v. A combination of an Apatinib analog, preferably represented by the structure of Formula A3 and Dasatinib (CAS302692-49-8), vi. A combination of an Apatinib analog, preferably represented by the structure of Formula A3 and a Dasatinib analog, preferably represented by the structure of Formula D4, and regenerating a plantlet from the cultured microspore.

[0373]

[0119] X12. The method of item XI 1, wherein the plant microspore is from a plant or plant variety that is recalcitrant to microspore regeneration.

[0374]

[0120] X13. The method of any one of items X4, XI 1 or X12, wherein the HDAC inhibitor is Trichostatin A, Fimepinostat, or Apicidin.

[0375]

[0121] X14. The method of any one of items XI 1- X13, wherein the microspore is maize.

[0376]

[0122] X15. The method of any one of items XI 1- X14, wherein heat stress is applied to the plant microspore during culture with the tyrosine kinase inhibitor and the HDAC inhibitor.

[0377]

[0123] X16. A method of obtaining a regenerated plantlet from a plant microspore comprising:

[0378] Isolating a plant microspore,

[0379] Culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor selected from any one or more of:

[0380] A. An Apatinib analog, selected from any one or more of: i. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811802-99-0), ii. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-00-6), iii. N-(4-(l-cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-01-7), iv. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-02-8), v. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-03-9), vi. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide (CAS#811803-04-0), vii. N-phenyl-2-((pyridin-4-ylmethyl)amino)nicotinamide, viii. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-06-2), ix. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0381] (CAS#811803-07-3), x. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0382] (CAS#811803-08-4), xi. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0383] (CAS#811803-09-5), xii. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0384] (CAS#811803-10-8), xiii. N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0385] (CAS#811803-11-9), xiv. N-(4-(l-(methoxymethyl)cyclohexyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0386] (CAS#811803-12-0), xv. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide (CAS#811803-13-1), xvi. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0387] (CAS#811803-14-2), xvii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0388] (CAS#811803-15-3), xviii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0389] (CAS#811803-16-4), xix. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopentyl)phenyl)nicotinamide

[0390] (CAS#811803-17-5), xx. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclobutyl)phenyl)nicotinamide (CAS#811803-18-6), xxi. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopropy l)phenyl)nicotinamide (CAS#811803-19-7), and / or

[0391] B. A Dasatinib analog, selected from any one or more of: i. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- mesitylthiazole-5-carboxamide ii. N-(2-fluoro-5-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iii. N-(4-bromo-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iv. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- (naphthal en-2-y l)thi azol e- 5 -carb oxami de v. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N-(5- isopropyl-2-methylphenyl)thiazole-5-carboxamide vi. N-(2,6-dimethoxyphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide vii. tert-butyl (5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)carbamate (CAS#302964-06-3) viii. 2-(3-butylureido)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide ix. N-(2-chloro-6-methylphenyl)-2-(3-cyclohexylureido)thiazole-5-carboxamide x. N-(2-chloro-6-methylphenyl)-2-(3-(3-methoxypropyl)ureido)thiazole-5-carboxamide xi. N-(2-chloro-6-methylphenyl)-2-(3-(3-fluoropropyl)ureido)thiazole-5-carboxamide xii. N-(2-chloro-6-methylphenyl)-2-(cyclopropanecarboxamido)thiazole-5-carboxamide (CAS#302961-12-2) xiii. N-(2-chloro-6-methylphenyl)-2-(cyclohexanecarboxamido)thiazole-5-carboxamide (CAS#302961-45-1) xiv. N-(2-chloro-6-methylphenyl)-2-(2-methylpentanamido)thiazole-5-carboxamide (CAS#302961-58-6) xv. 2-butyramido-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (CAS#302961- 34-8) xvi. N-(2-chloro-6-methylphenyl)-2-(nicotinamido)thiazole-5-carboxamide (CAS#302961-51-9) xvii. N-(2-chloro-6-methylphenyl)-2-(picolinamido)thiazole-5-carboxamide (CAS#302961-50-8) xviii. N-(2-chloro-6-methylphenyl)-2-((2-methylpyrimidin-4-yl)amino)thiazole-5- carboxamide (CAS#823206-99-l) xix. N-(2-chloro-6-methylphenyl)-2-((6-(dimethylamino)-2-methylpyrimidin-4- yl)amino)thiazole-5-carboxamide xx. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin-l-yl)pyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#1335054-68-6) xxi. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-morpholinopyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#302962-43 -2) xxii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)pyridin-2- yl)amino)thiazole-5-carboxamide xxiii. N-(2-chloro-6-methylphenyl)-2-((4-methylpyridin-2-yl)amino)thiazole-5- carboxamide (CAS#302961-72-4) xxiv. N-(2-chloro-6-methylphenyl)-2-(pyridin-4-ylamino)thiazole-5-carboxamide (CAS#302961-88-2) xxv. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-methylthiazole-5-carboxamide xxvi. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-(trifluoromethyl)thiazole-5-carboxamide xxvii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-phenylthiazole-5-carboxamide, and regenerating a plantlet from the cultured microspore.

[0392]

[0124] X17. The method of item X16, wherein the plant microspore is a maize microspore.

[0393]

[0125] X18. The method of any one of items X16 - X17, wherein the microspore is from a plant or plant variety that is recalcitrant to microspore regeneration.

[0394]

[0126] X19. The method of any one of items X16 - X18, wherein the microspore is further cultured or treated with a histone deacetylase inhibitor (HD AC inhibitor) in addition to the tyrosine kinase inhibitor.

[0395]

[0127] X20. The method of any one of items X16 - X19, wherein the microspore is haploid.

[0396]

[0128] X21. The method of any one of items X16 to X20, further comprising contacting or treating the haploid microspore or microspore derived cells with a chromosome doubling agent for a period of time sufficient to generate a doubled haploid cell, plant embryo, plant embryoid or plant, optionally wherein said contacting or treating follows contacting or treating with the HD AC inhibitor.

[0397]

[0129] X22. The method of any one of items X16 - X21, wherein the embryogenesis inducing kinase inhibitor is present in the microspore culture media.

[0398]

[0130] X23. The method of any one of items X16 - X22, wherein heat stress is applied to the plant microspore during culture with the tyrosine kinase inhibitor and, optionally, the HD AC inhibitor.

[0399]

[0131] X24. The method of any one of items XI 6 - X23, wherein the tyrosine kinase inhibitor is not added to the microspore culture until at least 3 days after culture initiation.

[0132] X25. The method of item X24, wherein the tyrosine kinase inhibitor is Dasatinib or a Dasatinib analog.

[0400]

[0133] X26. The method of item X16, wherein the plant microspore is obtained from rice, sunflower, sorghum, brassica, soybean, wheat, or cotton or is a rice, sunflower, sorghum, brassica, soybean, wheat, or cotton microspore.

[0401]

[0134] X27. The method of any one of items XI to X26, further comprising regenerating a plant from the plantlet, wherein the plant is capable of generating seed.

[0402]

[0135] X28. A plantlet, a plant or population of plants obtained or obtainable by the method of any one of items XI to X27.

[0403]

[0136] X29. Use of Apatinib, Dasatinib or a combination of Apatinib and Dasatinib, an Apatinib analog, preferably represented by the structure of Formula A3, a Dasatinib analog, preferably represented by the structure of Formula D4, a combination of Apatinib and a Dasatinib analog preferably represented by the structure of Formula D4, or a combination of an Apatinib analog preferably represented by the structure of Formula A3 and Dasatinib, or a combination of an Apatinib analog preferably represented by the structure of Formula A3 and a Dasatinib analog preferably represented by the structure of Formula D4, and optionally, one or more HDAC inhibitor, for regenerating a plantlet from a plant microspore, wherein

[0404] (i) Apatinib (CAS811803-05-1) is used at a dosage between 5-40 pM, preferably 10- 30pM,

[0405] (ii) Dasatinib (CAS302692-49-8) is used at a dosage between 10-50pM,

[0406] (iii) a combination of Apatinib (CAS811803-05-1) is used at a dosage between 5-40 pM, preferably 10-30pM and Dasatinib (CAS302692-49-8) at a dosage between 10-50pM;

[0407] (iv) wherein the Apatinib analog is at a dosage between 5-40pM, and / or

[0408] (v) wherein the Dasatinib analog is at a dosage between 5-40pM.

[0409]

[0137] X30. Use of an embryogenesis inducing tyrosine kinase inhibitor, and optionally, one or more HDAC inhibitor, for regenerating a plantlet from a plant microspore, wherein the tyrosine kinase inhibitor is an Apatinib analog, a Dasatinib analog, or a combination of an Apatinib analog and a Dasatinib analog. A. wherein the Apatinib analog is selected from any one or more of: i. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811802-99-0), ii. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-00-6), iii. N-(4-(l-cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-01-7), iv. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-02-8), v. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-03-9), vi. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide (CAS#811803-04-0), vii. N-phenyl-2-((pyridin-4-ylmethyl)amino)nicotinamide, viii. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-06-2), ix. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0410] (CAS#811803-07-3), x. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0411] (CAS#811803-08-4), xi. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0412] (CAS#811803-09-5), xii. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0413] (CAS#811803-10-8), xiii. N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0414] (CAS#811803-11-9), xiv. N-(4-(l -(methoxymethyl )cy cl ohexyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0415] (CAS#811803-12-0), xv. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0416] (CAS#811803-13-1), xvi. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0417] (CAS#811803-14-2), xvii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0418] (CAS#811803-15-3), xviii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0419] (CAS#811803-16-4), xix. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopentyl)phenyl)nicotinamide (CAS#811803-17-5), xx. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclobutyl)phenyl)nicotinamide (CAS#811803-18-6), xxi. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopropy l)phenyl)nicotinamide (CAS#811803-19-7), and / or

[0420] B. wherein the Dasatinib analog is selected from any one or more of: i. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- mesitylthiazole-5-carboxamide ii. N-(2-fluoro-5-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iii. N-(4-bromo-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iv. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- (naphthalen-2-yl)thiazole-5-carboxamide v. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N-(5- isopropyl-2-methylphenyl)thiazole-5-carboxamide vi. N-(2,6-dimethoxyphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide vii. tert-butyl (5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)carbamate (CAS#302964-06-3) viii. 2-(3-butylureido)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide ix. N-(2-chloro-6-methylphenyl)-2-(3-cyclohexylureido)thiazole-5-carboxamide x. N-(2-chloro-6-methylphenyl)-2-(3-(3-methoxypropyl)ureido)thiazole-5-carboxamide xi. N-(2-chloro-6-methylphenyl)-2-(3-(3-fluoropropyl)ureido)thiazole-5-carboxamide xii. N-(2-chloro-6-methylphenyl)-2-(cyclopropanecarboxamido)thiazole-5-carboxamide (CAS#302961-12-2) xiii. N-(2-chloro-6-methylphenyl)-2-(cyclohexanecarboxamido)thiazole-5-carboxamide (CAS#302961-45-l) xiv. N-(2-chloro-6-methylphenyl)-2-(2-methylpentanamido)thiazole-5-carboxamide (CAS#302961-58-6) xv. 2-butyramido-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (CAS#302961- 34-8) xvi. N-(2-chloro-6-methylphenyl)-2-(nicotinamido)thiazole-5-carboxamide (CAS#302961-51-9) xvii. N-(2-chloro-6-methylphenyl)-2-(picolinamido)thiazole-5-carboxamide (CAS#302961-50-8) xviii. N-(2-chloro-6-methylphenyl)-2-((2-methylpyrimidin-4-yl)amino)thiazole-5- carboxamide (CAS#823206-99-l) xix. N-(2-chloro-6-methylphenyl)-2-((6-(dimethylamino)-2-methylpyrimidin-4- yl)amino)thiazole-5-carboxamide xx. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin-l-yl)pyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#1335054-68-6) xxi. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-morpholinopyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#302962-43 -2) xxii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)pyridin-2- yl)amino)thiazole-5-carboxamide xxiii. N-(2-chloro-6-methylphenyl)-2-((4-methylpyridin-2-yl)amino)thiazole-5- carboxamide (CAS#302961-72-4) xxiv. N-(2-chloro-6-methylphenyl)-2-(pyridin-4-ylamino)thiazole-5-carboxamide (CAS#302961-88-2) xxv. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-methylthiazole-5-carboxamide xxvi. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-(trifluoromethyl)thiazole-5-carboxamide xxvii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-phenylthiazole-5-carboxamide.

[0421]

[0138] X31. A kit for regenerating a plantlet from a plant microspore, the kit comprising an embryogenesis inducing tyrosine kinase inhibitor, and optionally, one or more HDAC inhibitor, wherein the tyrosine kinase inhibitor is Apatinib or an Apatinib analog, Dasatinib or a Dasatinib analog, or a combination of Apatinib or an Apatinib analog and Dasatinib or a Dasatinib analog, optionally wherein the Apatinib analog is represented by the structure of Formula A3, further optionally wherein the Dasatinib analog is represented by the structure of Formula D4, optionally,

[0422] A. wherein the Apatinib analog is selected from any one or more of: i. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811802-99-0), ii. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-00-6), iii. N-(4-(l-cyanocyclopentyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-01-7), iv. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (CAS#811803-02-8), v. N-(4-(l-cyanocyclobutyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-03-9), vi. N-(4-(l-cyanocyclopropyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide (CAS#811803-04-0), vii. N-phenyl-2-((pyridin-4-ylmethyl)amino)nicotinamide, viii. N-(4-(l-cyanocyclohexyl)phenyl)-2-((pyridin-4-ylmethyl)amino)nicotinamide (CAS#811803-06-2), ix. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0423] (CAS#811803-07-3), x. N-(4-(l-(methoxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0424] (CAS#811803-08-4), xi. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0425] (CAS#811803-09-5), xii. N-(4-(l-(hydroxymethyl)cyclobutyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0426] (CAS#811803-10-8), xiii. N-(4-(l-(methoxymethyl)cyclopentyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0427] (CAS#811803-11-9), xiv. N-(4-(l-(methoxyrnethyl)cyclohexyl)phenyl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0428] (CAS#811803-12-0), xv. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide

[0429] (CAS#811803-13-1), xvi. N-(2'-oxospiro[cyclopentane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0430] (CAS#811803-14-2), xvii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)benzamide (CAS#81 1803-15-3), xviii. N-(2'-oxospiro[cyclopropane-l,3'-indolin]-6'-yl)-2-((pyridin-4- ylmethyl)amino)nicotinamide

[0431] (CAS#811803-16-4), xix. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopentyl)phenyl)nicotinamide (CAS#811803-17-5), xx. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclobutyl)phenyl)nicotinamide (CAS#811803-18-6), xxi. 2-((lH-indazol-6-yl)amino)-N-(4-(l-cyanocyclopropy l)phenyl)nicotinamide (CAS#811803-19-7), and / or

[0432] B. wherein the Dasatinib analog is selected from any one or more of: i. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- mesitylthiazole-5-carboxamide ii. N-(2-fluoro-5-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iii. N-(4-bromo-2-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide iv. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N- (naphthalen-2-yl)thiazole-5-carboxamide v. 2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2-methylpyrimidin-4-yl)amino)-N-(5- isopropyl-2-methylphenyl)thiazole-5-carboxamide vi. N-(2,6-dimethoxyphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)thiazole-5-carboxamide vii. tert-butyl (5-((2-chloro-6-methylphenyl)carbamoyl)thiazol-2-yl)carbamate (CAS#302964-06-3) viii. 2-(3-butylureido)-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide ix. N-(2-chloro-6-methylphenyl)-2-(3-cyclohexylureido)thiazole-5-carboxamide x. N-(2-chloro-6-methylphenyl)-2-(3-(3-methoxypropyl)ureido)thiazole-5-carboxamide xi. N-(2-chloro-6-methylphenyl)-2-(3-(3-fluoropropyl)ureido)thiazole-5-carboxamide xii. N-(2-chloro-6-methylphenyl)-2-(cyclopropanecarboxamido)thiazole-5-carboxamide (CAS#302961-12-2) xiii. N-(2-chloro-6-methylphenyl)-2-(cyclohexanecarboxamido)thiazole-5-carboxamide (CAS#302961-45-1) xiv. N-(2-chloro-6-methylphenyl)-2-(2-methylpentanamido)thiazole-5-carboxamide (CAS#302961-58-6) xv. 2-butyramido-N-(2-chloro-6-methylphenyl)thiazole-5-carboxamide (CAS#302961- 34-8) xvi. N-(2-chloro-6-methylphenyl)-2-(nicotinamido)thiazole-5-carboxamide (CAS#302961-51-9) xvii. N-(2-chloro-6-methylphenyl)-2-(picolinamido)thiazole-5-carboxamide (CAS#302961-50-8) xviii. N-(2-chloro-6-methylphenyl)-2-((2-methylpyrimidin-4-yl)amino)thiazole-5- carboxamide (CAS#823206-99-1) xix. N-(2-chloro-6-methylphenyl)-2-((6-(dimethylamino)-2-methylpyrimidin-4- yl)amino)thiazole-5-carboxamide xx. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-methylpiperazin- 1 -yl)pyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#1335054-68-6) xxi. N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-morpholinopyrimidin-4- yl)amino)thiazole-5-carboxamide (CAS#302962-43 -2) xxii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)pyridin-2- yl)amino)thiazole-5-carboxamide xxiii. N-(2-chloro-6-methylphenyl)-2-((4-methylpyri din-2 -yl)amino)thiazole-5- carboxamide (CAS#302961-72-4) xxiv. N-(2-chloro-6-methylphenyl)-2-(pyridin-4-ylamino)thiazole-5-carboxamide (CAS#302961-88-2) xxv. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-methylthiazole-5-carboxamide xxvi. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-(trifluoromethyl)thiazole-5-carboxamide xxvii. N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-hydroxyethyl)piperazin-l-yl)-2- methylpyrimidin-4-yl)amino)-4-phenylthiazole-5-carboxamide.

[0433]

[0139] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention. For instance, while particular examples may illustrate the methods and embodiments described herein using com, the principles in these examples may be applied to any plant. Therefore, it will be appreciated that the scope of this invention is encompassed by the embodiments recited herein.

[0434]

[0140] It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions belong. In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined herein.

[0435]

[0141] For chemical terms in connection with Formulas Al, A2 and A3, the term “lower alkylenyl”, unless otherwise indicated, includes 1 to 6 saturated — CH2 — radicals. The term “lower alkenlenyl”, as used herein, unless otherwise indicated, includes lower alkylenyl groups, as defined above, having at least one carbon-carbon double bond, such as — CH2 — CH=CH — . The term “halogen”, as used herein, unless otherwise indicated, includes fluoro, chloro, bromo or iodo, such as fluoro and chloro. The term “halogen-lower alkyl”, as used herein, unless otherwise indicated, includes 1 to 6 halogen substituted alkyl, such as trifluoromethyl. The term “lower alkyl”, as used herein, unless otherwise indicated, includes 1 to 6 saturated monovalent hydrocarbon radicals having straight or branched moieties, including, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, and the like. The term “lower alkenyl”, as used herein, unless otherwise indicated, includes lower alkyl groups, as defined above, having at least one carbon-carbon double bond, such as -CH2-CH=CH2. The term “lower alkynyl”, as used herein, unless otherwise indicated, includes lower alkyl groups, as defined above, having at least one carbon-carbon triple bond, such as — CH2 — acetylene. The term “alkoxy”, as used herein, unless otherwise indicated, includes — O-lower alkyl groups wherein lower alkyl is as defined above, such as methoxy and ethoxy. The term “alkoxyalkoxy”, as used herein, unless otherwise indicated, includes — O-lower alkyl-O-lower alkyl groups wherein lower alkyl is as defined above, such as -OCH2CH2OCH3. The term “C0-C6”, as used herein, unless otherwise indicated, includes zero carbon and lower alkyl wherein lower alkyl is as defined above. The term “amino”, as used herein, unless otherwise indicated, includes — NH2 group, — NH-lower alkyl group, or — N(lower alkyl)2 group wherein lower alkyl is as defined above, such as methylamine and dimethylamine. The term “alkoxyamino”, as used herein, unless otherwise indicated, includes — O-lower alkyl-NH2 group, — O-lower alkyl-NH-lower alkyl group, or — O-lower alkyl-N(lower alkyl)2 group wherein lower alkyl is as defined above, such as — OCH2CH2NHCH3 . The term “carboxyalky”, as used herein, unless otherwise indicated, includes — C(O)O-lower alkyl as an ester group wherein lower alkyl is as defined above, such as -C(O)OCH3. The term “carbonylalkyl”, as used herein, unless otherwise indicated, includes — C(O)-lower alkyl as a ketone group wherein lower alkyl is as defined above, such as -C(O)CH3. The term “oxycarbonylalkyl”, as used herein, unless otherwise indicated, includes — OC(O)- lower alkyl as an ester group wherein lower alkyl is as defined above, such as — OC(O)CH3. The term “carboxy”, as used herein, unless otherwise indicated, includes — C(O)OH. The term “carbonylamino”, as used herein, unless otherwise indicated, includes — C(0)NH2 group, — C(O)NH-lower alkyl group, or — C(O)N(lower alkyl)2 as a amide group wherein lower alkyl is as defined above. The term “oxycarbonylamino”, as used herein, unless otherwise indicated, includes — 0C(0)NH2, — OC(O)NH-lower alkyl or — OC(O)N(lower alkyl)2 as a carbamate group wherein lower alkyl is as defined above. The term “aminocarbonylalkyl”, as used herein, unless otherwise indicated, includes — NHC(O) — or — N(lower alkyl)-C(O)-lower alkyl as an amide group wherein lower alkyl is as defined above. The term “aminocarbonyloxyalkyl”, as used herein, unless otherwise indicated, includes — NHC(O)O- lower alkyl or — N(lower alkyl)- C(O)O-lower alkyl as a carbamate group wherein lower alkyl is as defined above. The term “aminocarbonylamino”, as used herein, unless otherwise indicated, includes — NHC(0)NH2, — N(lower alkyl)C(O)NH2, — NHC(O)NH(lower alkyl), — NHC(O)N(lower alkyl)2, — N (lower alkyl)C(0)NH(lower alkyl), — N (lower alkyl)C(O)N(lower alkyl)2, as an urea wherein lower alkyl is as defined above. The term “aminosulfonylalkyl”, as used herein, unless otherwise indicated, includes — NHS(O)2-lower alkyl group wherein lower alkyl is as defined above. The term “aryl”, as used herein, unless otherwise indicated, includes an organic radical derived from an aromatic hydrocarbon by removal of one hydrogen, such as phenyl or naphthyl, and is unsubstituted or substituted by one, two or three substituents, selected from halogen, halo- gen- lower alkyl, lower alkyl, lower alkenyl, lower alkynyl, C0-C6cyano, C0-C6hydroxy, C0- C6alkoxy, C0-C6alkoxyalkoxyl, C0-C6amino, C0-C6alkoxyamino, C0-C6carboxy, C0- C6Carboxyalkyl, C0-C6Carbonylamino, C0-C6Carbonylalkyl, C0-C6 oxycarbonylalkyl, C0- C6oxycarbonylamino, C0-C6aminocarbonylalkyl, C0-C6aminocarbonyloxyalkyl, C0- C6aminocarbonylamino, C0-C6aminosulfonylalkyl, C0-C6cycloalkyl, C0-C6Cycloalkenyl, C0- C6aryl, CO-C60xyaryl, C0-C6alkoxyaryl, C0-C6aminoaryl, C0-C6aminoalkyaryl, C0- C6heterocyclyl, C0-6oxyheterocyclyl, C0-C6alkoxyheterocyclyl, C0-C6aminoheterocyclyl, C0- C6aminoalkyheterocyclyl, C0-C6phenyl, C0-C6phenoxy, C0-C6phenylthio, C0-C6phenyl lower alkylthio, C0-C6Sulfinyl, C0-C6phenylC0-C6sulfmyl, C0-C6Sulfonyl, C0-C6phenylC0- C6sulfonyl, and C0-C6heterocyclyl; wherein any above C1-C6 groups and amino groups can be optionally unsubstituted, mono-substituted or maybe disubstituted by lower alkyl; aryl includes one aromatic ring fused with an aliphatic ring, such as a saturated or partially saturated ring, such as tetrahydronaphthyl. The term “oxyaryl”, as used herein, unless otherwise indicated, includes — O-aryl group wherein aryl is as defined above. The term “alkoxyaryl”, as used herein, unless otherwise indicated, includes — O-lower alkyl-aryl group wherein lower alkyl and aryl are as defined above. The term “aminoaryl”, as used herein, unless otherwise indicated, includes amino-aryl group wherein amino and aryl are as defined above. The term “aminoalkylaryl”, as used herein, unless otherwise indicated, includes amino-lower alkyl-aryl group wherein amino, lower alkyl and aryl are as defined above. The term “cycloalkyl”, as used herein, unless otherwise indicated, includes cyclic radicals having from three to eight ring carbon atoms, including, but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. The cycloalkyl groups may be optionally substituted one or more times, substituents selected from the group defined above as substituents for aryl, preferably halogen, lower alkyl. The term “cycloalkenyl”, as used herein, unless otherwise indicated, includes cycloalkyl groups, as defined above, having at least one carbon-carbon double bond. The term “heterocyclyl”, as used herein, unless otherwise indicated, includes non-aromatic, single and fused rings suitably containing up to four heteroatoms in each ring, each of which independently selected from O, N and S, and which rings, may be unsubstituted or substituted independently by, for example, up to three substituents. Each heterocyclic ring suitably has from 4 to 7, preferably 5 or 6, ring atoms. A fused heterocyclic ring system may include carbocyclic rings and need include only one heterocyclic ring which may be partially saturated or saturated. The heterocyclyl includes mono, bicyclic and tricyclic heteroaro- matic ring systems comprising up to four, preferably 1 or 2, heteroatoms each selected from O, N and S. Each ring may have from 4 to 7, preferably 5 or 6, ring atoms. A bicyclic or tricyclic ring system may include a carbocyclic ring. Carbocyclic ring includes cycloalkyl, cycloalkenyl or aryl ring. Examples of heterocyclyl groups include pyrrolidine, pyrrolidione, piperidine, piperidinone, piperazine, morpho- line, imidazolidine, pyrazolidine, hydantoin, oxetane, tet- rahydrofuran, tetrahydropyran, pyrrole, indole, pyrazole, indazole, trizole, benzotrizole, imidazole, benzoimdazole, thiophene, benzothiophene, thiozole, benzothiozole, furan, benzofuran, oxazole, benzoxazole, isoxazole, tetrazole, pyridine, pyrimidine, trizine, quinoline, isoquinoline, quinazoline, indoline, indolinone, benzotetrahydrofuran, tet- rahydroquinoline, tetrahydroisoquinoline and methylenedioxyphenyl. The heterocyclic rings may be optionally substituted and substituents selected from the group defined above as substituents for aryl. The term “oxyheterocyclyl”, as used herein, unless otherwise indicated, includes — O-heterocyclyl group wherein heterocyclyl is as defined above. The term “alkoxyheterocyclyl”, as used herein, unless otherwise indicated, includes — O- lower alkyl-het- erocyclyl group wherein lower alkyl and heterocyclyl are as defined above. The term “aminoheterocyclyl”, as used herein, unless otherwise indicated, includes amino- heterocyclyl group wherein amino and heterocyclyl are as defined above. The term aminoalkylheterocyclyl”, as used herein, unless otherwise indicated, includes amino-lower alkyl- heterocyclyl group wherein amino, lower alkyl and heterocyclyl are as defined above.

[0436]

[0142] For chemical terms used in connection with Formulas DI, D2, D3 and D4, the terms "alk" or "alkyl" refer to straight or branched chain hydrocarbon groups having 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms. The expression "lower alkyl" refers to alkyl groups of 1 to 4 carbon atoms. The term "alkenyl" refers to straight or branched chain hydrocarbon groups of 2 to 10, preferably 2 to 4, carbon atoms having at least one double bond. Where an alkenyl group is bonded to a nitrogen atom, it is preferred that such group not be bonded directly through a carbon bearing a double bond. The term "alkynyl" refers to straight or branched chain hydrocarbon groups of 2 to 10, preferably 2 to 4, carbon atoms having at least one triple bond. Where an alkynyl group is bonded to a nitrogen atom, it is preferred that such group not be bonded directly through a carbon bearing a triple bond. The term "alkylene" refers to a straight chain bridge of 1 to 5 carbon atoms connected by single bonds (e.g., -(CH2)x- wherein x is 1 to 5), which may be substituted with 1 to 3 lower alkyl groups. The term "alkenylene" refers to a straight chain bridge of 2 to 5 carbon atoms having one or two double bonds that is connected by single bonds and may be substituted with 1 to 3 lower alkyl groups. Exemplary alkenylene groups are -CH=CH-CH=CH-, -CH2-CH=CH-, -CH2-CH=CH-CH2-, - C(CH3)2CH=CH- and -CH(C2H5)-CH=CH-. The term "alkynylene" refers to a straight chain bridge of 2 to 5 carbon atoms that has a triple bond therein, is connected by single bonds, and may be substituted with 1 to 3 lower alkyl groups. Exemplary alkynylene groups are -C= C-, - CH2-C= C-, -CH(CH5)-C= C- and -C= C-CH(C2H5)CH2-. The terms “ar” or "aryl" refer to aromatic cyclic groups (for example 6 membered monocyclic, 10 membered bicyclic or 14 membered tricyclic ring systems) which contain 6 to 14 carbon atoms. Exemplary aryl groups include phenyl, naphthyl, biphenyl and anthracene. The terms "cycloalkyl" and "cycloalkenyl" refer to cyclic hydrocarbon groups of 3 to 12 carbon atoms. The terms "halogen" and "halo" refer to fluorine, chlorine, bromine and iodine. The term “unsaturated ring” includes partially unsaturated and aromatic rings. The terms "heterocycle", "heterocyclic" or "heterocyclo" refer to fully saturated or unsaturated, including aromatic (i.e. “heteroaryl”) cyclic groups, for example, 4 to 7 membered monocyclic, 7 to 11 membered bicyclic, or 10 to 15 membered tricyclic ring systems, which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and / or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system. Exemplary monocyclic heterocyclic groups include pyrrolidinyl, pyrrolyl, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, 4-piperidonyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, tetrahydropyranyl, morpholinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, 1,3-dioxolane and 25 tetrahydro-1, 1- dioxothienyl, triazolyl, triazinyl, and the like. Exemplary bicyclic heterocyclic groups include indolyl, benzothiazolyl, benzoxazolyl, benzodioxolyl, benzothienyl, quinuclidinyl, quinolinyl, tetra-hydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuryl, chromonyl, coumarinyl, benzopyranyl, cinnolinyl, quinoxalinyl, indazolyl, pyrrolopyridyl, furopyridinyl (such as furo[2,3-c]pyridinyl, furo[3,2-b]pyridinyl] or furo[2,3-b]pyridinyl), dihydroisoindolyl, dihydroquinazolinyl (such as 3,4-dihydro-4-oxo-quinazolinyl), tetrahydroquinolinyl and the like. Exemplary tricyclic heterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl, acridinyl, phenanthridinyl, xanthenyl and the like. The term "heteroaryl” refers to aromatic heterocyclic groups. Exemplary heteroaryl groups include pyrrolyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furyl, thienyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazolyl, triazinyl, and the like. Where q is 1 or 2, “-C(O)qH” denotes -C(O)-H or -C(O)-OH; “-C(O)qR6” or “-C(O)qZ6” denote, respectively, -C(0)-R6 or -C(0)-0R6, or -C(O)-Z6 or - C(O)-OZ6; “-O-C(O)qR6” or O-C(O)qZ6” denote, respectively, -0-C(0)-R6 or - 0-C(0)-0R6, or -O-C(O)-Z6 or -O-C(O)- OZ6; and “-S(O)qR6” or “-S(O)qZ6” denote, respectively, -SO-R6 or -SO2-R6, or -SO-Z6 or - SO2-Z6.

[0437]

[0143] “Pharmaceutically and agriculturally acceptable salts” of compounds will be apparent to those skilled in the art and include those described in J. Pharm. Sci., 1977, 66, 1-19, such as acid addition salts formed with inorganic acid e.g. hydrochloric, hydrobromic, sulphuric, nitric or phosphoric acid; and organic acids e.g. succinic, maleic, acetic, fumaric, citric, tartaric, benzoic, p-toluenesulfonic, methanesulfonic or naphthalenesulfonic acid. Other salts may be used, for example in the isolation or purification of compounds of formulas described herein and are included within the scope of this invention. Non-toxic salts are preferred, although other salts are useful, for example, in isolation or purification steps which may be employed during preparation. Salts of the compounds herein may be formed, for example, by reacting a compound with an amount of acid or base, such as an equivalent amount, in a medium such as one in which the salt precipitates or in an aqueous medium followed by lyophilization. Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acid, for example, trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates, benzenesulfonates, bisulfates, borates, butyrates, citrates, camphorates, camphorsulfonates, cyclopentanepropionates, digluconates, dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates, glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides, hydrobromides, hydroiodides, 2-hydroxy ethanesulfonates, lactates, maleates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oxalates, pectinates, persulfates, 3 -phenylpropionates, phosphates, picrates, pivalates, propionates, salicylates, succinates, sulfates (such as those formed with sulfuric acid), sulfonates (such as those mentioned herein), tartrates, thiocyanates, toluenesulfonates, undecanoates, and the like. Exemplary basic salts (formed, for example, where the R substituents comprise an acidic moiety such as a carboxyl group) include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, salts with organic bases (for example, organic amines) such as benzathines, dicyclohexylamines, hydrabamines, N-methyl-D-glucamines, N-methyl-D-glucamides, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. The basic nitrogen-containing groups may be quaternized with agents such as lower alkyl halides (e.g. methyl, ethyl, propyl, and butyl chlorides, bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl, dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides), aralkyl halides (e.g. benzyl and phenethyl bromides), and others.

[0438]

[0144] The compounds of this invention may be in crystalline or non-crystalline form, and, if crystalline, may optionally be hydrated or solvated. This invention includes within its scope stoichiometric hydrates as well as compounds containing variable amounts of water.

[0439]

[0145] The invention extends to all isomeric forms including stereoisomers and geometric isomers of the compounds of the formulas described herein including enantiomers and mixtures thereof e.g. racemates. The different isomeric forms may be separated or resolved one from the other by conventional methods, or any given isomer may be obtained by conventional synthetic methods or by stereospecific or asymmetric syntheses.

[0440]

[0146] As used herein the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the protein” includes reference to one or more proteins and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0441]

[0147] As used herein, the term “about” indicates and encompasses an indicated value and a range above and below that value. For example, the term “about” may indicate the designated value ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ±4% unless otherwise indicated.

[0442]

[0148] As used herein, the terms “comprising”, “including”, ’’having” or grammatical variants thereof are to be taken as specifying the stated features, integers, steps or components but do not preclude the addition of one or more additional features, integers, steps, components or groups thereof.

[0443]

[0149] All patents, publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this disclosure pertains. All patents, publications and patent applications are herein incorporated by reference in the entirety to the same extent as if each individual patent, publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0444]

[0150] Examples

[0445]

[0151] Example 1 : Improved Maize Embryogenic Response from Maize Microspores of ATCC germplasm 40520, with use of Apatinib and Dasatinib.

[0446]

[0152] ATCC germplasm 40520 is known to be conducive to microspore embryo regeneration. Com microspores were cultured in the presence of the tyrosine kinase inhibitors Dasatinib and Apatinib. Dasatinib and Apatinib stock solutions were prepared in DMSO at the concentration of lOmM. Tassels were maintained at 10 deg. C for 14 days prior to microspore isolation, and isolated essentially in the manner described in Pescitelli et. al. High frequency androgenesis from isolated microspores of maize. Plant Cell Reports 7, 673-676 (1989), Gaillard, et. al. Optimization of maize microspore isolation and culture conditions for reliable plant regeneration Plant Cell Reports 10, 55-58 (1991), and US2020 / 0263189, by blending, filtering, centrifuging and washing the microspore pellet. After tassel pretreatment and isolation, Apatinib (CAS811803-05-1), Dasatinib (CAS302692-49-8), were each added to basic induction media (liquid induction medium ) consisting of 1.77g NLN Basal medium (Cat# N479, PhytoTechnology Laboratories, essentially as described in Nitsch J.P. & Nitsch C., Science, (1969), 163, 85 - 87), 100g sucrose, 0.00001g (or 100 pl of 0.1 mg / mLstock) Kinetin, KOH (1.0 and 0.1M) to adjust pH to 5.8, water adjusted to 1000 mL media and maintained for the duration of cell culture. Three chemical concentrations were used for both compounds (cone 1=1 OpM, cone 2=20pM, cone 3= 30pM), where the concentrations were the final concentrations in the cell culture media. Each petri dish contained the same number of microspores: 40k in 1.5 ml liquid induction medium. The microspores were cultured at 28°C in darkness. After 3 weeks, the macroscopic structures were counted. At all tested doses, the number of macroscopic embryolike structures were greater in the Apatinib and Dasatinib treated cultures as compared to the control cultures (with no Apatinib or Dasatinib present).

[0447]

[0153] TABLE 4 (number of macroscopic embryo-like structures per 40,000 microspores)

[0448]

[0154] Example 2A: Dasatinib and Apatinib dose optimization,

[0449]

[0155] Using the protocol described in Example 1 above, Dasatinib was tested across a wider range of concentrations (cone l=10pM, cone 2=20pM, cone 3= 30pM, conc4=40pM, conc5=50pM) to optimize the effective amount. It was determined that the effect of Dasatinib on the development of macroscopic embryo-like structures was dose dependent, with effect in the 10-50 pM range and optimal effect in the 10-30pM range. After 19 days, macroscopic structures formation was evaluated Dasatinib at a rate of 50pM or higher had a negative effect on embryo size. Embryos derived from corn microspore cultured with the supplementation of 50 pM Dasatinib were significantly smaller than the control culture.

[0450]

[0156] TABLE 5 - Optimal Dose of Apatinib and Dasatinib based on Examples 1 and 2A.

[0451]

[0157] Example 2B - Additional Dose Optimization Study

[0452]

[0158] Further studies on dose optimization were conducted for both Apatinib and Dasatinib. Maize micropore cells were cultured and immobilized in multiwell format as described in US patent 10,301,635B2 and in the corresponding PCT application WO 2012 / 066147. The microspore cells were immobilized in a gel at the bottom of the plate and liquid induction medium, as previously described in Example 1, was added on top of the gel. Dasatinib or Apatinib were added to the liquid induction medium. Dasatinib was tested across a concentration range of 20pM, 40pM, 60pM, 80pM, lOOpM. Apatinib was tested across a concentration range of 5pM, lOpM, 20pM, 30pM, 40pM. After approximately 21 days of culture at 28°C, the number of embryo-like structures (ELS) per each well were counted and normalized to the number of cells / well (%ELS). Average number of cells / well was 3,174. The percentage of ELS across eight wells were averaged and compared with the % ELS in control wells where no kinase inhibitor was added to the culture medium. At all tested doses the treatment with Apatinib resulted in a statistically significant (p-value 0.05) increase in embryolike structure formation when compared to the control. Dasatinib treatment resulted in a significant increase in %ELS at the dose of 20pM. A strong inhibition of ELS formation was observed at 60pM, 80pM and lOOpM with the extent of inhibition increasing at the higher doses.

[0453]

[0159] TABLE 6 - Average % of embryo-like structures measured per Apatinib tested across a range of concentrations (5pM, lOpM, 20pM, 30pM, 40pM). Embryogenic activity: a plus sign (+) indicates treatment showing a statistically significant (p-value 0.05) increase greater than 30% improvement in the number of the ELS when compared to the corresponding control. * indicates the most effective embryogenic dose.

[0454]

[0160] TABLE 7 - Average % of embryo-like structures measured per Dasatinib tested across a range of concentrations (20pM, 40pM, 60pM, 80pM, lOOpM). Embryogenic activity: a plus sign (+) indicates treatment showing a statistically significant (p-value 0.05) increase greater than 30% improvement in the number of ELS when compared to the corresponding control; * indicates the most effective embryogenic dose observed. A minus sign (-) indicates a statistically significant decrease in % ELS when compared to the control (< 70% of the respective control).

[0161] Example 3 - Dasatinib timing.

[0455]

[0162] Maize micropore cells were cultured and immobilized in multiwell as described in example 2B. 15pM of Dasatinib was added to the liquid induction medium either immediately after plate preparation or added to the existing media 3 days after culture initiation to achieve the desired concentration. After approximately 19 days of culture, an increased number of embryolike structures was observed for both conditions as compared to the control sample (without Dasatinib). The Dasatinib added on top of the gel was effective, albeit less so then when it was added directly to the liquid culture media as in Example 1. A very large increase in macroscopic structures was observed when Dasatinib was applied 3 days after culture initiation.

[0456]

[0163] Table 8 - Count of macroscopic structures (number macroscopic structures / 3000 microspores.

[0457]

[0164] Example 4 - Dasatinib plantlet regeneration

[0458]

[0165] Embryos derived with Dasatinib in the liquid induction media were cultured following standard procedure for com microspore regeneration as described in Example 1. After about 3 weeks, embryos were transferred from liquid culture to standard solid maturation media where they further developed and formed roots and shoots using typical plant regeneration conditions. Upon further transfer to germination medium, the shoots successfully developed into plantlets.

[0166] Example 5 - Dasatinib in combination with HDACi chemical pretreatment induced embryogenesis in recalcitrant line PHP38,

[0459]

[0167] Tassels were cold pretreated at 10 degrees Celsius for between 9-11 days, anthers were collected in a petri dish containing a pre-culture of 50nM HD AC inhibitor Trichostatin A (and control sample without Trichostatin A) and further incubated for 3 days at 10°C in darkness. Prior testing had determined 50nM to 100 nM of Trichostatin A to be the optimal amount after testing in the low nM to low pM range, and the lower end of the range was used because of the long incubation period. Following anther-pretreatment, microspores were isolated and further cultured in the liquid induction medium described in Example 1 in the presence or absence of Dasatinib 30pM. Multicellular structures, some embryo-like, were observed after 5 weeks of culture only when anther pretreatment with TSA was combined with Dasatinib.

[0460]

[0168] Example 6 - Maize Microspores derived from Recalcitrant Maize Inbred Line PHP24E, using Apatinib in combination with HDAC inhibitors.

[0461]

[0169] Tassels were cold pretreated at 10 degrees Celsius for between 7-10 days prior to microspore isolation. Isolated microspores were incubated for about 30 min. with either lOOnM HDAC inhibitor TSA or alternatively lOpM HDAC inhibitor SAHA or 7mM DMSO (as a control). After removal of the HDAC inhibitors / DMSO by washing with culture media as in Example 1, microspores were incubated with Apatinib ( 15uM) in induction media supplemented with 35nM HDAC inhibitor Apicidan for 5 days at 32°C, followed by incubation in culture media without Apatinib for 16 days at 28°C in the darkness. After 21 days, multicellular structures were counted and evaluated utilizing the ScreenSYS Al imaging system described in WO2021 / 127110, which is hereby incorporated by reference. The combination of the HDAC inhibitor Api cidin and the kinase inhibitor Apatinib resulted in the formation of multicellular structures. Further improvement of multicellular structure formation (macroscopic / embryo-like structures) was observed when this treatment was combined with additional pretreatment with HDAC inhibitor SAHA, but not TSA.

[0170] Table 9 - Count of macroscopic / embryo-like structures using Apatinib and

[0462] HD AC inhibitors

[0463]

[0171] Example 7 - Viability Maintenance or Improvement for Maize Microspores from

[0464] Recalcitrant Maize Inbred Line PHH5G with use of Apatinib and Dasatinib.

[0465] In an additional experiment, microspores from inbred genotype PHH5G that is known to be recalcitrant to microspore culture and subsequent embryo regeneration were tested for successful embryogenesis activity with Apatinib and Dasatinib, as well as a combination of the two. PHH5G tassels were maintained at about 10°C for 7-8 days prior to microspore isolation, and isolated microspores were added to liquid induction medium as in Example 1, to which either a control with no Apatinib or Dasatinib, 10 pM of Apatinib, 20 pM of Dasatinib, or a combination of 10 pM of Apatinib and 20 pM of Dasatinib had been added in order to test for adverse effects of Apatinib and Dasatinib on microspore viability. Isolated microspores were tested for percent viability using impedance flow cytometry after both 3 and 10 days of treatment at 28°C under dark conditions. Microspores in the control group maintained a 10% viability rate after 3 days in the media, which viability percentage dropped to about 1% after 10 days in the media. Error bars ranged from 9% to 11% viability rates for day 3 testing. Microspores treated with 10 pM of Apatinib also maintained a 10% viability rate after 3 days in the media, which viability percentage dropped to about 1.75% after 10 days in the media, indicating that Apatinib did not adversely affect microspore viability in comparison to the control group. Error bars ranged from 8% to 12% viability rates for day 3 testing.

[0466] Surprisingly, PHH5G microspores treated with 20 pM of Dasatinib maintained a 16% viability rate after 3 days in the media, which viability percentage dropped to about 3.5% after 10 days in the media, indicating that Dasatinib did not have an adverse effect on microspore viability, and to the contrary, exhibited a positive effect. Error bars ranged from 14% to just over 18% viability rates for day 3 testing of this sample. Similarly, microspores treated with a combination of lOpM of Apatinib and 20pM of Dasatinib maintained a 16% viability rate after 3 days in the media, which viability percentage dropped to about 3.25% after 10 days in the media. Error bars ranged from 15% to 17% viability rates for the combined sample. Such data indicate a distinct and unexpectedly positive effect of Dasatinib on microspore viability rates, whether Dasatinib is utilized alone or in combination with Apatinib.

[0467]

[0172] Example 8 -Additional Verification of Embryogenic Effect of the Tyrosine Kinase Inhibitors Apatinib and Dasatinib on Recalcitrant Fl Germplasm.

[0468]

[0173] Two different hybrid varieties derived from two recalcitrant maize elite Fl donor genotypes (designated hybrid 1 and hybrid 2 herein) were grown to provide tassels that were maintained at 10 deg. C for 14 days prior to microspore isolation as described in Example 1. After isolation and centrifugation, the microspore pellet was resuspended in a 20% maltose gradient and centrifuged to separate the live microspores, essentially as described in Goralski et. al. Influence of Sugars on Isolated Microspore Development in Maize, Acta Biologica Cracoviensia 44: 203-212 (2002). Live microspores were collected and plated on induction media with treatments of I OpM Apatinib, 25 pM Dasatinib, and a control sample with only induction media, using the method previously described in Example 1. Four or five replications were conducted for each of hybrid 1 and hybrid 2. The culture plates were incubated and screened for embryo-like structures and other multicellular structures after 2 weeks of culture, and for macroscopic structures after 45-60 days of culture.

[0469]

[0174] Table 10 - Average numbers of embryo-like and other multicellular macroscopic / structures, in each case, per 60,000 microspores using Apatinib and Dasatinib.

[0470]

[0175] The formation of embryo-like structures and macroscopic structures generally is lower in recalcitrant germplasm compared to germplasm conducive to microspore embryo regeneration (such as ATCC germplasm 40520).

[0471]

[0176] As data in Table 10 indicates, for both breeding crosses from two recalcitrant elite Fl donor genotypes, when Apatinib and Dasatinib were present in induction media, the numbers of embryo-like and other macroscopic structures per 60K microspores were present and greater in comparison to the media without chemicals (liquid induction medium). More importantly, when Dasatinib was added to basic liquid induction medium, there were embryo-like and other multicellular / macroscopic structures on both genotypes, and when Apatinib was added to basic liquid induction medium, there were embryo-like and other multicellular / macroscopic structures on one genotype. By contrast no multicellular / macroscopic structures were found in the control group’s induction medium media in which no Apatinib or Dasatinib was added.

[0472]

[0177] Example 9, Sunflower microspore pretreatment with Apatinib in combination with an HD AC inhibitor increases formation of multicellular structures.

[0473]

[0178] The optimal sunflower inflorescences containing microspores at mid- to late mononucleate stage of several different varieties were selected and harvested as described by Garkusha et al., 2017, followed by a pretreatment at 10°C for up to 10 days (7 to 10 days typically). After this pretreatment all the outer leaves and the corollas were removed. The surface sterilization of floral buds and microspore isolation was carried out according to Todorova et al., 1993, with a following modification: 30 sec incubation in 70% ethanol was introduced before treatments with the sterilization solution consisting of 3 % NaOCl and 0.02% Tween-20 and subsequent four times washing with sterile ultrapure water. Microspores were collected from the white to yellow florets by utilizing an IKA multidrive blender containing microspore isolation medium consisting of the macro and micro elements from Gamborg B5 medium (Gamborg, et al, 1968), the organics (vitamins & amino acids) from NLN medium (Nitsch and Nitsch, 1967) and 13% sucrose.

[0474]

[0179] After two blendings at 6000 rpm for 20 sec each per bud, the suspension was filtered through 227 pm and 63 pm stainless steel mesh filters respectively, each followed by centrifugation at 200 g for 2 min. The pellet was washed twice with 50 ml isolation medium per bud, each followed by centrifugation at 200g for 2 min and afterwards resuspended in 10 ml isolation medium. 2 ml of suspension containing sunflower microspores in isolation medium was overlayed on 4 ml of 40% (w / v) maltose solution and centrifuged at 20 g for 4 min. Cells from the interphase were collected in 50 ml centrifugation tubes, resuspended in 50 ml sunflower isolation medium, and pelleted at 200 g for 2 min. The microspore density was adjusted with sunflower isolation medium to 70,000 cells per ml.

[0475]

[0180] Isolated sunflower microspores were treated with the Fimepinostat (CAS 1339928-25-4) at 200 nM concentration for 30 minutes in the microspore isolation medium (consisting of the macro and micro elements from Gamborg B5 medium (Gamborg, et al, 1968), the organics (vitamins & amino acids) from NLN medium (Nitsch and Nitsch, 1967) and 13% sucrose). The medium was then replaced with liquid sunflower culture medium 1 (modified Medium B (Touraev A and Heberle-Bors E, 1999) containing the organics (vitamins & amino acids) from NLN medium (Nitsch and Nitsch, 1967), 12% mannitol, 500 mg / 1 casein hydrolysate and 0.5 mg / 1 BAP), which removed the Fimepinostat.

[0476]

[0181] Cell density was adjusted to 70,000 cells per ml. Apatinib (CAS811803-05-1) was added to achieve 10 pM concentration and cells were cultured at 32°C in 2ml Eppendorf tubes in the darkness for 5 days. After 5 days, the sunflower culture medium 1 with added Apatinib was replaced with 1 ml sunflower culture medium 2 (sunflower isolation medium supplemented with 500 mg / 1 casein hydrolysate, 500 mg / 1 proline and 0.5 mg / 1 BAP).

[0477]

[0182] 300 pl per well of microspore suspension was distributed in multiwell plates (IBIDI 96-well plates) and cultured at 28°C in the darkness for up to 5 weeks. For 6 cm petri dishes, 7 ml of cell suspension per plate were distributed.

[0478]

[0183] The combination of pretreatment Fimepinostat and the Apatinib treatment at 32°C resulted in an increased number of cell divisions and improved multicellular structure formation, with 7 multicellular structures per 23,000 microspores, in comparison to samples with no Fimepinostat or Apatinib treatment, each of which did not result in any multicellular structure formation. The sample treatment with only Fimepinostat resulted in only a single multicellular structure. In addition to the increase in multicellular structure formation, multicellular structures were also observed to develop faster in treatments with the combination of Fimepinostat and Apatinib, and plantlets were able to be successfully regenerated from these multicellular structures.

[0479]

[0184] Five weeks after isolation, the microspore derived multicellular structures were transferred to regeneration medium 1 (MS medium (Murashige and Skoog, 1962), supplemented with 6% sucrose, 500 mg / 1 casein hydrolysate, lmg / 1 6-Benzylaminopurine (BAP) and 0.5mg / l

[0480] 1 -Naphthaleneacetic acid (NAA), solidified with 0.3% Gelrite) and cultured in 48 or 96-well multi-well plates at 28°C in the dark with 2-week subculture intervals. After reaching a minimum size of 0.5 cm, multicellular structures were then transferred to a second regeneration medium 2 supplemented with 2 mg / 1 BAP and 0.5 mg / 1 NAA and cultured at 26°C in 16 / 8h light / dark regime at 60% humidity with 2-week subculture intervals. Shoot initiation and elongation were observed 6-8 weeks after transfer to the light / dark regime. Shoots of 0.5 to 1 cm in length were transferred to rooting medium (1 / 2 MS medium (Murashige and Skoog, 1962) supplemented with 3% sucrose and 1 mg / 1 IBA and solidified with 0.3% Gelrite) and cultured under the same conditions as during shoot initiation and elongation. Root formation was observed approximately 2 weeks after transfer.

[0185] Example 10. Soybean microspore pretreatment with Apatinib in combination with an HD AC inhibitor increases formation of multicellular structures.

[0481]

[0186] The optimal Soybean (variety Adessa) flower buds of 2.5-3.6 mm bud size, containing microspores at mid- to late mononucleate stage, were selected and harvested as described by Sumarmi et al. (2015), followed by a pretreatment at 10 degrees C for up to 5 days. Buds were surface sterilized according to Rodrigues et al., 2006 with increased concentration of NaOCl up to 3%. After two blendings at 7000 rpm for 20 sec each, in microspore isolation medium (consisting of the macro and micro elements from Gamborg B5 medium (Gamborg, et al, 1968), the organics (vitamins & amino acids) from NLN medium (Nitsch and Nitsch, 1967) and 13% sucrose), the suspension was subsequently filtered through 100 pm and 40 pm stainless steel mesh filters respectively, each followed by centrifugation at 160 g for 5 min and resuspension in isolation medium. 2 ml of suspension containing soybean microspores in isolation medium was overlayed on 4 ml of 40% (w / v) maltose solution and centrifuged at 20 g for 4 min. Cells from the interphase were collected, transferred to 50 ml centrifugation tubes, resuspended in 50 ml of microspore isolation medium and pelleted at 200 g for 2 min.

[0482] Microspore density was adjusted with microspore isolation medium to 70,000 cells per ml.

[0483]

[0187] Microspores were then pretreated with 100 nM TSA for 15 minutes in microspore isolation medium (abbr. mim, in Table 11 below). After the pretreatment, microspores were washed, resuspended in an initial soybean culture medium (abbr. ICM, of modified Medium B (Touraev A and Heberle-Bors E, 1999) containing the organics (vitamins & amino acids) from NLN medium (Nitsch and Nitsch, 1967) and 12% mannitol) supplemented with either 0 (Control) or 0.2pM cytokinins 6-Benzylaminopurine (BAP) or Thidiazuron (TDZ) in the presence or absence of 10 pM Apatinib (see Table 11 for tested combinations). Microspore cultures were incubated in multiwell plates at 32°C for 5 days in the darkness. Afterwards, microspores were washed and resuspended in a soybean culture medium (abbr. GCM, with macro and micro elements from Gamborg B5 medium (Gamborg, et al. 1968), the organics (vitamins & amino acids) from NLN medium (Nitsch and Nitsch, 1967), 13% sucrose, 100 mg / 1 L-serine, 400 mg / 1 glutamate, 10 mg / 1 coconut water and 0.02 mg / 1 BAP) to promote growth culture and were cultured for 3 weeks at 28°C in the darkness (See Table 1 1 for tested combinations with Apatinib and TDZ or BAP added). Microspores-derived ELSs were counted after 3 weeks of culture. The results indicated that the presence of Apatinib was mandatory for initiation of microspores, with the several fold increase when Apatinib and cytokinins were present in both culture conditions. No microspore formation was observed in absence of Apatinib or in the presence of only cytokinins, whether BAP or TDZ.

[0484]

[0188] Table 11 - Results of soybean microspore pretreatment with Apatinib.

[0485]

[0189] Additional studies were conducted on soybean microspores with Dasatinib using the protocol described above, but after pretreatment with 100 nM TSA for 15 minutes in mim medium, the microspores were washed and resuspended in 1ml fresh initial culture medium and incubated at 32°C in multiwell plates for 5 days in the darkness. Afterwards, the microspores were washed and resuspended in growth culture medium supplemented with 0 (control) or 2.5 pM Dasatinib and cultured for 5 weeks at 28°C. Formation of multiple multicellular structures were observed only in the samples where the growth culture medium contained the addition of 2.5pM Dasatinib (70 multicellular structures were observed in 3 wells, which was 0.1% of the total cell number or 23.3 multicellular structures per well on average) with no multicellular structures formed in the absence of Dasatinib.

[0190] Example 1 1 : Improved Maize Embryogenic Response from Maize Microspores of

[0486] ATCC germplasm 40520, with use of Apatinib analogs.

[0487]

[0191] Apatinib analogs were tested across a range of concentrations (5pM, lOpM,

[0488] 20pM, 30pM, 40pM) to optimize the effective amount. The tested analogs were added to the liquid induction medium after plate preparation. After approximately 21 days of culture at 28°C, the number of embryo-like structures (ELS) per each well were counted and normalized to the number of cells / well (%ELS). The percentage of ELS across four wells were averaged and compared with the % ELS in control wells where no analogue was added to the culture medium. All tested Apatinib analogs shown in Table 12 below resulted in a significant increase (p-value 0.05) greater than 30% in embryo-like structure formation when compared to the control, with all such successfully tested Apatinib analogs falling within the structure represented by Formula A3 above. Among the most effective analogs tested were, with reference to the Table 2 list of Apatinib analogs herein, the analogs in rows number 1 (CAS 811802-99-0), number 2 (CAS 811803-00-6), and number 3 (CAS 811803-01-7), with an embryogenic efficiency of 0.62%- 0.71% at the optimal dose. The optimal dose varied from lOpM to 40pM for the different analogs.

[0489]

[0192] Table 12 - Average % of embryo-like structures measured per Apatinib analogs tested across a range of concentrations (5pM, lOpM, 20pM, 30pM, 40pM). Embryogenic activity: a plus sign (+) indicates = treatment showing a statistically significant (p-value 0.05) increase greater than 30% improvement in the number of the embryo-like structures when compared to the corresponding control run with that sample. * indicates the most effective embryogenic dose per analogue observed. The abbreviation N / D means not tested.

[0490]

[0193] Example 12: Improved Maize Embryogenic Response from Maize Microspores of

[0491] ATCC germplasm 40520, with use of Dasatinib analogs.

[0492]

[0194] Using the protocol described in Example 3 above, Dasatinib analogs were tested across a range of concentrations (5pM, lOpM, 20pM, 30pM, 40pM) to optimize the effective amount. The tested analogs were added to the liquid induction medium after plate preparation.

[0493] After approximately 21 days of culture at 28°C, the number of embryo-like structures (ELS) per each well were counted and normalized to the number of cells / well (%ELS). The percentage of ELS across four wells were averaged and compared to the % ELS in control wells where no analogue was added to the culture medium. All tested Dasatinib analogs shown in Table 13 resulted in a significant increase: a greater than 30% increase in embryo-like structure formation when compared to the control, with all such successfully tested Dasatinib analogs falling within the structure represented by Formula D4 above. The optimal dose, the dose with the strongest embryogenic effect, varied from analogue to analogue, from 5pM for the analog show in row number 21 of Table 3 above (CAS 302962-43-2), to 40pM for the analog shown in row number 1 [2-((6-(4-(2-hydroxyethyl)piperazin- 1 -yl)-2-methylpyrimidin-4-yl)amino)-N-mesitylthiazole- 5-carboxamide] and row number 23 (CAS 302961-72-4).

[0494]

[0195] Table 13 - Average % of embryo-like structures measured per Dasatinib analogs tested across a range of concentrations (5pM, 10pM, 20pM, 30pM, 40pM). Embryogenic activity: a plus sign (+) indicates the treatment showing a statistically significant (p-value 0.05) increase, greater than 30%, in the number of the embryo-like structures when compared to the corresponding control run with that sample. * indicates the most effective embryogenic dose per analogue observed.

[0196] Example 13: Additional Verification of Embryogenic Effect of Dasatinib analogs in combination with HDACi chemical pretreatment induced embryogenesis in recalcitrant line PHP38,

[0495]

[0197] In an additional experiment, microspores from inbred genotype PHP38 that is known to be recalcitrant to microspore culture and subsequent embryo regeneration were tested for successful embryogenesis activity with Dasatinib analogs. PHP38 tassels were maintained at about 10°C for 7-10 days prior to microspore isolation. Micropore pretreatment and culture with HDACi SAHA and Api cidin was performed in the manner described in Example 6. Either one of the three Dasatinib analogs was added to the culture medium at a dose ranging from 5pM to 40pM as indicated in Table 14. After approximately 21 days of culture at 28°C, the number of embryo-like structures (ELS) per well were counted and normalized to the total number of cells (%ELS). The percentage of ELS across eight wells were averaged and compared to the % ELS in control wells where no Dasatinib analogue was added to the culture medium. When microspores were cultured in the presence of Dasatinib analogue (the analog from row number 20 of Table 3 (CAS 1335054-68-6) at the dose of 5pM or the Dasatinib analogue from row number 21 of Table 3 (CAS 302962-43-2) at a dose of 15pM, a statistically significant increase (p-value 0.05) in number of ELS was observed for both analogs.

[0496]

[0198] Table 14 - Average % of ELS measured per Dasatinib analogs tested across a range of concentrations (5pM, lOpM, 15pM, 20pM, 30pM, 40pM). Embryogenic activity: a plus sign (+) indicates the treatment showing a statistically significant (p-value 0.05) increase, greater than 30%, in the number of the embryo-like structures when compared to the control; * indicates the most effective dose per analogue observed. The abbreviation N / D means not tested.

[0497]

[0199] In all of the examples described above, microspores used were mixed populations of uninucleate and binucleate stage of development, although purified population of either uninucleate or binucleate microspores may be used. Microspores were cultured in a petri dish with approximately 1.5ml in volume or in a 96-well plate with approximately 200pL in volume per well. Microspore density was typically adjusted to approximately 30,000-40,000 microspores per ml. With each method, pulse treatment and / or heat stress treatment of microspores and / or derived cells may optionally be utilized, as needed, so long as adverse effects on cellular health are not observed.

[0498]

[0200] Full references for citations in the examples are as follows:

[0499]

[0201] Garkusha S., Savenko E., Glazyrina V., et al. (2017) Development of methodological procedures for culturing sunflower anthers in vitro. Journal of Biotech Research [ISSN: 1944-3285], 8: 138-150

[0202] Todorova M., Dahlhoff M. and Friedt W. (1993) Microspore Culture in Sunflower (Helianthus Annuus L.), Biotechnology & Biotechnological Equipment, 7:4, 83-90, doi: 10.1080 / 13102818.1993.10818712

[0500]

[0203] Murashige, T and Skoog, F. 1962. A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15 (3): 473-497. doi: 10.1111 / j.1399-3054.1962.tb08052.x

[0501]

[0204] Nitsch, C and Nitsch J P. 1967. The induction of flowering in vitro in stem segments of Plumbago indica L. Planta, 72(4): 355-370. doi: 10.1007 / BF00390146.

[0502]

[0205] Gamborg O.L, Miller R.A and Ojima K. 1968. Nutrient requirement of suspensions cultures of soybean root cells. Exp. Cell Res., 50(1 ): 151 -158. doi: 10.1016 / 0014- 4827(68)90403-5

[0503]

[0206] Touraev A and Heberle-Bors E. 1999. Microspore embryogenesis and in vitro pollen maturation in tobacco.

[0504]

[0207] Sumarmi S., Daryono B. S., Rachmawati D., Indrianto A. (2015) Determination of soybean (Glycine max L. [Merrill]) microspores development stage based on the length of flower buds. Journal of Biological Researches: 20 (6-11). doi: 10.23869 / bphjbr.20.1.20142

[0505]

[0208] In: Hall, R.D. (eds) Plant Cell Culture Protocols. Methods In Molecular Biology™, 1999. vol 111: 281-291. Humana Press, doi: 10.1385 / 1-59259-583-9:281.

[0506]

[0209] Rodrigues LR, de Camargo Forte B and Bodanese-Zanettini M. 2006. Isolation and culture of soybean (Glycine max L. Merrill) microspores and pollen grains. Braz Arch Biol and Tech 49 (4); 537-545 doi: 10.1590 / S1516-89132006000500002.

Claims

We claim:

1. A method of obtaining a regenerated plantlet from a plant microspore comprising: Isolating a plant microspore,Culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor selected from: i. Apatinib (CAS811803-05-1) at a dosage between 5-40pM, or ii. An Apatinib analog represented by the structure of Formula A3, and Regenerating a plantlet from the cultured microspore.

2. The method of claim 1, wherein the Apatinib or Apatanib analog is used in combination with Dasatinib or a Dasatinib analog represented by the structure of Formula D4.

3. The method of any one of claims 1 or 2, wherein the microspore is treated with a histone deacetylase inhibitor (HD AC inhibitor) in addition to the tyrosine kinase inhibitor.

4. The method of claim 3, wherein the microspore is pretreaied with an HDAC inhibitor prior to treatment with the tyrosine kinase inhibitor.

5. The method of claim 3 or 4, wherein the HDAC inhibitor is Trichostatin A, Fimepinostat, SAHA, or Apicidin.

6. The method of any one of claim 3 - 5, wherein the microspore derived cells are treated with a chromosome doubling agent for a period of time sufficient to generate a doubled haploid cell, plant embryoid or plant.

7. The method of any one of claims 1- 6, wherein the Apatinib analog is represented by the structure of Formula A3 is provided at a dosage between 5-40pM.

8. The method of any one of claims 1 - 7, wherein the plant microspore is a maize microspore.

9. The method of any one of claim 1 - 7, wherein the plant microspore is a rice, sorghum, sunflower, brassica, soybean, wheat, or cotton microspore.

10. The method of any one of claims 1-9, wherein Apatinib (CAS811803-05-1) is at a dosage between 10-30pM.

11. A method of obtaining a regenerated plantlet from a plant microspore comprising: Isolating a plant microspore,Culturing the plant microspore with an embryogenesis inducing tyrosine kinase inhibitor selected from: i. Dasatinib (CAS302692-49-8) at a dosage between 2.5-50pM, or ii. A Dasatinib analog represented by the structure of Formula D4, and Regenerating a plantlet from the cultured microspore.

12. The method of claim 11, wherein the Dasatinib or a Dasatinib analog is used in combination an Apatinib or an Apatanib analog that is represented by the structure of Formula A3.

13. The method of any one of claims 11 or 12, wherein the microspore is treated with a histone deacetylase inhibitor (HD AC inhibitor) in addition to the tyrosine kinase inhibitor.

14. The method of any one of claims 11 - 13, wherein the microspore is pretreated with an HD AC inhibitor prior to treatment with the tyrosine kinase inhibitor.

15. The method of claim 13 or 14, wherein the HDAC inhibitor is Trichostatin A, Fimepinostat, SAHA or Apicidin.

16. The method of any one of claims 13 - 15, wherein the microspore derived cells are treated with a chromosome doubling agent for a period of time sufficient to generate a doubled haploid cell, plant embryoid or plant.

17. The method of any one of claims 11 - 16, wherein the Dasatinib analog represented by the structure of Formula D4 is provided at a dosage between 5-40pM.

18. The method of any one of claims 11 - 17, wherein the plant microspore is a maize microspore.

19. The method of any one of claims 11 - 17, wherein the plant microspore is a rice, sorghum, sunflower, brassica, soybean, wheat, or cotton microspore.

20. The method of any one of claims 1 - 6, 8-10 or 12 - 19, wherein the Apatinib analog represented by the structure of Formula A3 is provided at a dosage between 5 - 40pM.

21. The method of any one of claims 2 - 16 or 18-19, wherein the Dasatinib analog represented by the structure of Formula D4 is provided at a dosage between 5 - 40pM.

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