Methods to enhance the production of haploid plants and chromosome doubling

Abstract

This disclosure provides methods and compositions related to haploid plant production and chromosome doubling of haploids. In one aspect, provided herein are methods for producing haploid plants (e.g., haploid sunflower plants) by limiting fertilization, including limiting double fertilization and / or self-fertilization. Also provided are fertilizer regimes, light regimes, formulations, and methods of delivery thereof for chromosome doubling.

Description

METHODS TO ENHANCE THE PRODUCTION OF HAPLOID PLANTS ANDCHROMOSOME DOUBLINGCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of International Patent Application Serial No. PCT / CN2024 / 090146, filed April 26, 2024, the contents of which are incorporated herein by this reference as if fully set forth herein.FIELD

[0002] This disclosure relates to the field of plant biotechnology.BACKGROUND

[0003] Haploid production and chromosome doubling of haploids are important techniques for production of doubled haploids in plant breeding to accelerate the breeding programs of a range of crops. It is also an effective way to induce polyploids for study of genetics and plant breeding.

[0004] Current haploid production methods are not satisfactory because they require complex procedures and / or often have low efficiency. Haploid induction in sunflower is particularly challenging. In vitro anther culture is generally a time-consuming process, requires skilled experience, and is also highly reliant on genotype. Several reports of using this method in sunflower demonstrated quite a low haploid induction rate. Irradiation-treated pollen is another method used to induce haploid induction in sunflower, but the efficacy is too low to be commercially applied. Androgenesis is also commonly used for haploid generation but is tedious and inconvenient as it requires individual development of a male derived from a haploid embryo from a fertilized egg in the absence of or after elimination of the female nucleus.

[0005] Current methods of chromosome doubling in plants fall into two general categories: (1) application of doubling chemicals during tissue cultures of microspores or immature embryos to induce doubled seedlings; and (2) soaking germinated seedlings in doubling chemical solutions. These methods have several drawbacks, especially when applied to double chromosome number of haploid sunflower plants. The direct seedling-soaking approaches are simple and high throughput, but they require the chemicals to be used at a concentration (for doubling effect) that would pose severe phytotoxicity or lethal effects on plants. The method of targeted meristem application has higher plant survival rate because the plant root systems,stem, and leaves are free from the chemical treatments. However, the doubling efficiency of this method is low by far partly due to the limited chemical penetration into meristem tissues; they usually reach only a few cell layers close to the plant surface.BRIEF SUMMARY

[0006] In one aspect, provided herein are methods for producing haploid plants (e.g., haploid sunflower plants by limiting fertilization, including limiting double fertilization and / or selffertilization).

[0007] In one aspect, provided herein are formulations and method of delivery such for chromosome doubling. The formulation is an emulsion and can be maintained on the plant tissue for a sufficiently long time, for example, at least one cell divisional cycle. This allows the formulation to slowly and continuously supply the doubling agents to meristem cells.

[0008] In some embodiments, provided herein is a method of enhancing haploid production in a sunflower plant, wherein the method comprises interrupting pollen development of pollen producing tissues; or outcrossing the sunflower plant with pollen from a second plant, wherein the sunflower plant is grown under high light intensity and / or long daylength and broadspectrum including UV light. In some embodiments, the high light intensity is provided by sunlight and supplemented with metal halides, incandescent light, light emitting diodes (LEDs) or high-pressure sodium lights (HPS). In some embodiments, the method of enhancing haploid production further comprises the application of a fertilizer two to seven times per week.

[0009] In some embodiments, the method further comprises treating flower heads of the sunflower plant after pollination with one or more of boron, indoleacetic acid (IAA), nicotinamide riboside, sucrose, or 2,4-Dichlorophenoxyacetic acid.

[0010] In some embodiments, the method further comprises recovering seeds from ovaries of the sunflower plant, separating haploid seeds from non-haploid seeds.

[0011] In some embodiments, the method further comprises germinating haploid seeds in soil, germination paper, liquid medium, semi-solid medium, or solid medium. The solid medium comprises one or more of SF-SIM16, SF-SIM18, SF-SIM19, SF-SIM20, SF-SEM2- 1, or SF-SIM21.

[0012] In some embodiments, the method further comprises treating the haploid seedlings with a composition comprising a doubling agent(s) in a semi-solid emulsion.

[0013] In some embodiments, the semi-solid emulsion comprises a fluorocarbon ether polymer of polyhexafluoropropylene oxide, and a hydrofluoroether, wherein the fluorocarbon ether polymer is dissolved in hydrofluoroether droplets contained in the aqueous phase.

[0014] In some embodiments, the sunflower plant of the disclosure is selected from the group consisting of SY48, SY54, and SY58. In another embodiment, the sunflower plant is SY58.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 compares images of diploid and parthenogenic haploid sunflower seeds according to certain aspects of this disclosure.

[0016] FIG. 2A shows a comparison of the phenotypes of a diploid sunflower plant and three haploid sunflower plants according to certain aspects of this disclosure. FIG. 2B shows a comparison of the leaves of hybrid diploid plants in comparison to leaves of three haploid plants. FIG. 2C shows a comparison of the size of stomates between a diploid leaf (on left) and a haploid leaf (on right).

[0017] FIG. 3 shows results of flow cytometry analysis to determine ploidy of leaf tissues according to certain aspects of this disclosure.

[0018] FIGS. 4A-4C show exemplary devices and methods that can be used to separate haploid seeds from diploid seeds according to certain aspects of the disclosure. FIG. 4A shows an exemplary device that can separate haploid seeds from diploid seeds by weight. FIG. 4B shows an exemplary device that can separate haploid seeds from diploid seeds by size. FIG. 4C shows an exemplary method that can separate haploid seeds from diploid seeds by developmental markers.

[0019] FIG. 5 shows the normal shoots / plants from haploid seed produced using one-round and two-round regeneration methods according to certain aspects of the disclosure.

[0020] FIG. 6 shows seedling performance at 13 days after transplanting normal haploid plants generated from haploid seeds in the greenhouse according to certain aspects of this disclosure. Top view and side view of plants P l through P_8 from left to right.

[0021] FIG. 7 shows the seedling performance of doubled sunflower haploid plants as compared to a diploid sunflower plant and a haploid sunflower plant according to certain aspects of this disclosure.

[0022] FIG. 8 shows the effect of acetone on the hydrofluoroether (HFE) phase of formulations according to certain aspects of the disclosure. Tube 1 (“1”) contains a mixture comprising water, a yellow dye (dark top layer in the photo), and HFE7500; Tube 2 (“2”) contains a mixture comprising acetone, a yellow dye, and HFE7500; Tube 3 (“3”) contains a mixture comprising water, a red dye (dark top layer in the photo), and HFE7500; and Tube 4 (“4”) contains a mixture comprising acetone, a red dye, and HFE7500.

[0023] FIG. 9 illustrates the components and formation of an emulsion comprising a doubling agent according to certain aspects of the disclosure. The schematic shows multiple chemicals in the formulation are cross-linked together to form a gel-like network through hydrogen bonds.

[0024] FIG. 10 shows the formation of an emulsion when amounts of components are used at the proper ratios as indicated according to certain aspects of this disclosure.

[0025] FIG. 11A shows the formation of an emulsion according to certain aspects of the disclosure. FIG. 11B shows the sticky formulation after being applied to the plant. FIG. 11C and FIG. 11D show the images of plant leaves 12 hours and 24 hours after contacting the emulsion.

[0026] FIG. 12A shows the light spectrum in the phytotron under normal “control” conditions. FIG. 12B shows the light spectrum in the phytotron under “UV block” conditions according to certain aspects of this disclosure.

[0027] FIG. 13 shows the number of haploid seeds per head across various sunflower germplasms, e.g., SY48, SY54, and SY58.DETAILED DESCRIPTIONI. Terminology

[0028] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques and / or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject.

[0029] As used in herein, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “an antibody” optionally includes a combination of two or more such molecules, and the like.

[0030] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0031] As used herein, the term “comprising” or “comprise” is open-ended. When used in connection with a subject nucleic acid (or amino acid sequence), it refers to a nucleic acid sequence (or an amino acid sequence) that includes the subject sequence as a part or as its entire sequence.

[0032] “Phenotype” is understood within the scope of the present disclosure to refer to a distinguishable characteristic(s) of a genetically controlled trait.

[0033] A “plant” is any plant at any stage of development, particularly a seed plant. In particular, in the context of this disclosure, a plant refers to a sunflower plant.

[0034] A “plant cell” is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant.

[0035] “Plant cell culture” means cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.

[0036] “Plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any group of plant cells organized into structural and / or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0037] The term “plant part” indicates a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which plants can be regenerated. Examples of plant parts include, but are not limited to, single cells andtissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like.

[0038] The terms “variety” or “cultivar” mean a group of similar plants that by structural or genetic features and / or performance can be distinguished from other varieties within the same species.

[0039] The term “haploid induction rate,” or “HIR,” refers to the ratio of haploid seeds among total seeds harvested from a plant that has been subj ected to haploid induction treatment. An enhancement in haploid production can be reflected by an increase in HIR.

[0040] The term “double fertilization” refers to a complex process involving joining of a female gametophyte with two male gametes (sperm). Each pollen grain produces two sperm: one fuses with an egg to form the zygote, and the other fuses with one or more polar nuclei in the female gametophyte (megagametophyte, or also “embryo sac”) to form an endosperm. The term “limit double fertilization” as used herein refers to treatment that reduces the frequency or likelihood of double fertilization. Double fertilization can be limited by abnormal pollen development, abnormal pollen-stigma interaction, abnormal pollen germination and pollen tube development, abnormal embryo sac development and response to pollen tube, abnormal egg cell-sperm cell interaction, abnormal control cell-sperm cell interaction, and / or abnormal embryo or endosperm development. Limited double fertilization can reduce seed setting rate or embryo development rate by at least 0.1%, compared to 100% seed setting rate in normal double fertilization process.

[0041] The term “self-fertilization” refers to a fertilization process in which both the female gametophyte fuses with a male gamete from the same plant.

[0042] The term “fertilization-limited” refers to a plant, or any plant part that has been treated by agents that limits double fertilization or self-fertilization.

[0043] The term “out-cross” refers to a cross between two separate individuals, interspecies or intraspecies, that are not siblings.

[0044] The term “chemical emasculation,” refers to any method that uses chemical treatments of plants that causes the development or production of pollen to fail. This could be through several biological mechanisms which may include pollen abortion, an interruption ofanther development, conversion of anthers into carpels or other organs, blocking filament elongation or anther dehiscence, or any other mechanism.

[0045] The term “fertigation,” refers to a fertilizer treatment mixed with water and provided to the plants during the normal plant watering schedule through the drip irrigation system.

[0046] As used herein, a plant referred to as “haploid” has a single set (genome) of chromosomes and the reduced number of chromosomes (In) in the haploid plant is equal to that of the gamete. As used herein, a plant referred to as “doubled haploid” is developed by doubling the haploid set of chromosomes (from In to 2n). A plant or seed that is obtained from a doubled haploid plant that is selfed to any number of generations may still be identified as a doubled haploid plant. A doubled haploid plant is considered a homozygous plant. A plant is considered to be doubled haploid if it is fertile, even if the entire vegetative part of the plant does not consist of the cells with the doubled set of chromosomes; that is, a plant will be considered doubled haploid if it contains viable gametes, even if it is chimeric.

[0047] As used herein, the term “spontaneous doubling,” “spontaneous chromosome doubling” (“SCD”) or “spontaneous haploid genome doubling” or “haploid male fertility” or “doubled haploid” or “spontaneous genome doubling” are used interchangeably to describe the doubling of haploid genomes without any intervention. SCD allows for the correct meiotic reduction of chromosomes and subsequent formation of mature pollen. In the present disclosure, SCD was calculated by dividing the number of fertile haploid plants / by the total number of plants.

[0048] The term “hybridization” refers to a cross between two plants; it can imply that the cross is between two distinct lines, genetic types, ecotypes, subspecies, or even species.

[0049] The term “wide hybridization” or “wide cross” refers to a hybridization cross between two species or subspecies.

[0050] The term “parthenogenesis” refers to asexual reproduction in which an embryo develops without fertilization of the egg cell. “Parthenogenic” or “parthenogenetic” haploid seeds result when this type of asexual reproduction occurs in plants.

[0051] The term “photoperiod-sensitive genic male sterility (PGMS)” refers to a trait in some plants in which male sterility is triggered through a combination of genetics and environment. That is, in which a certain photoperiod can trigger male sterility in certain genetic backgrounds.

[0052] The term “thermos-sensitive genic male sterility (TGMS)” refers to a trait in some plants in which male sterility is triggered through a combination of genetics and environment. That is, in which a certain temperature can specifically trigger male sterility in certain genetic backgrounds.

[0053] The term “chemical sensitive genic male sterility (CGMS),” refers to a trait in some plants in which male sterility is induced in a certain genotype but can be restored by chemical treatment.

[0054] The term “ploidy” refers to the number of chromosome sets in a cell or an organism. Typically, plants have two sets of chromosomes (one from the mother and father) and are referred to as diploids.

[0055] The term “meristematic tissue” refers to the tissues in the plant shoot apical meristem, at the apex of the shoot, and / or in the axillary meristems.

[0056] The term “photosynthetic photon flux density” (PPFD) refers to the amount of photosynthetically active radiation (PAR) light (generally considered 400-700nm in wavelength) is received by a unit area, such as a leaf surface, over a given time. The units are micromols of PAR per meter squared per second (pmol / m2 / s).II. Promoting haploid production by limiting fertilization

[0057] Disclosed herein are methods of producing haploid plants by limiting fertilization. As disclosed herein, the term “inducing haploid production” refers to causing the production of one or more haploid seeds from a plant. These haploid seeds can be doubled to generate homozygous diploid or polyploid plants having desired genes and traits. Various exemplary methods of limiting fertilization to produce haploids are disclosed below. The methods disclosed herein can be performed on sunflower plants to induce haploid production. In some embodiments, the sunflower plant is from the SY15 variety. In some embodiments, the sunflower plant is from the SY54 variety. In some embodiments, the sunflower plant is from other sunflower varieties. a. Chemical-based method

[0058] In some embodiments, this disclosure provides methods to limit double fertilization for enhancement in haploid production. Limited double fertilization can reduce seed setting rate or embryo development rate by at least 0.1%, for example, at least 0.2%, at least 0.3%, atleast 0.4%, at least 0.5%, at least 0.6%, or at least 0.8% as compared to the seed setting rate in normal double fertilization process (which can be set as a rate of 100%). In some embodiments, limited double fertilization can reduce seed setting rate or embryo development rate by about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, or about 0.8%. Double fertilization is known to be beneficial for increasing seed production in sunflower plants. In some embodiments, limiting double fertilization may promote haploid seed production. In some embodiments, limiting double fertilization in the sunflower plant can be achieved by contacting pollen of the pollen-donor plant (also referred to herein as the “pollen donor”) with a Formula-A composition (Table 3) or Formula-B composition (Table 4). A Formula-A composition in this disclosure comprises at least one of oleic acid ethyl ester (OAEE), methyl a-linolenyl fluorophosphonate (MLnFP), and / or manoalide. In some embodiments, Formula-A further comprises one or more of dimethyl lactamide (DML), phosphate buffered saline (PBS), Blend 91, and Blend 92. The compositions of Blend 91 and Blend 92 are set forth in Table 1 and Table 2, respectively. Unless otherwise noted, all percentages reported in this application are volume percentages.Table 1. Blend 91 ingredientsTable 2. Blend 92 ingredients

[0059] In some embodiments, the OAEE is present in Formula-A in an amount of 0.40% to 3.00%, for example, 1.00% to 2.10 %, or from 1.60% to 2.10%. In some embodiments, the MLnFP is present in an amount of 0.20% to 5.00%, for example, 0.40% to 3.00%, or 1.00% to 2.10%. In some embodiments, the manoalide is present in an amount of 0.20% to 3%, for example, 0.40% to 2.10%, or 0.40%. In some embodiments, the dimethyl lactamide (DML) ispresent in the amount of 0.5% to 2%, e.g., 1%. In some embodiments, Blend 91 is present in an amount of 2.6% to 13.9%. In some embodiments, Blend 92 is present in an amount of 2.7% to 14.5%. Table 3 shows the ingredients in an exemplary Formula-A composition.

[0060] In some embodiments, a method of using a Formula-A composition to limit double fertilization for enhancing haploid production comprises spraying a Formula-A composition onto pollen collected from a pollen donor plant, self-pollination, or out-cross pollination. Any device that can be used to spray a liquid is suitable for use to spray Formula-A composition onto the pollen. One exemplary device is a nebulizer. The nebulizer could be, but not limited to, Omron C101 Basic Nebulizer, Omron Compressor Nebulizer NE-C28 (https: / / www.omronhealthcare-ap.com / ap / category / 9-respiratory-therapy) , or PARI Boy SX Nebulizer (https: / / www.pari.com / int / products / inhalation-devices-for-the-lungs / pari-boy-sx- int / ).

[0061] In some embodiments, the spray is performed in the open-air, and the spray may last 1-10 minutes, e.g., 2-8 minutes, or about 3 minutes. In some embodiments, the spray is performed in a sealed container that houses the pollen, e.g., a sealed plastic bag. In some embodiments, the spray is performed in the closed container and lasts 1-10 minutes, e.g., 2-8 minutes, or about 2 minutes.

[0062] In some embodiments, limiting double fertilization in the sunflower plant can be achieved by contacting pollen obtained from the pollen-donor plant with a Formula-B composition. A Formula-B composition disclosed herein comprises (i) at least one of oleic acid ethyl ester (OAEE), methyl a-linolenyl fluorophosphonate (MLnFP), and / or manoalide, and (ii) oil. Various types of oils, such as cooking oil and lubricants, can be used in Formula-B compositions. Non-limiting examples of oils include com oil, sunflower oil, mineral oil, and the like. In some embodiments, OAEE is present in Formula B in an amount of 1% to 3%. In some embodiments, the MLnFP is present in an amount of 1% to 3%. In some embodiments, manoalide is present in an amount of 1% to 3%. As shown in Table 5, the compositions disclosed herein have resulted in haploid induction rates in a range from 0.34% to 66.67%. In some cases, plants having a high haploid induction rate produced less seeds.

[0063] In some embodiments, a method of using Formula-B composition to limit double fertilization comprises treating pollen by soaking them in a Formula-B composition for a time period. The time period may last 5 minutes to 60 minutes, e.g., 10 minutes to 40 minutes, orabout 20 minutes. In some cases, the pollen is soaked in a Formula-B composition with a gentle shaking, e.g., at 200 rpm, to facilitate the mixing of the composition with pollen.

[0064] Pollen having been treated by a Formula A composition or a Formula B composition as described above are then collected and applied to the head of a pollen-recipient sunflower plant for fertilization to produce seeds. In some embodiments, the recipient sunflower plant is a cytoplasmic male sterility (CMS) plant. CMS plants do not produce workable pollen and therefore do not self-fertilize. As shown in Table 5, a percentage of the seeds so produced were haploid seeds, with haploid induction rates (ratio of haploid seeds among total seeds harvested) varying from 0.34% to 66.67%. b. Wide-cross method

[0065] In some embodiments, the enhancement of haploid production via limiting double fertilization can also be achieved by distant hybridization (interspecific distant hybridization and intergeneric distant hybridization), also referred to as a wide cross. In some embodiments, the sunflower plant is emasculated and then crossed with pollen from a plant species that is reproductively isolated from sunflower. A reproductively isolated species from sunflower refers to a species that does not produce viable progeny plants when crossing with sunflower. In some embodiments, the plant from the reproductively isolated species is a monocot plant, for example, corn, wheat, sugarcane, or rice. In some embodiments, the plant from the reproductively isolated species is a dicot plant, for example, tomato, soybean, spinach, pepper, broccoli, cauliflower, carrot, or potato. In some embodiments, the plant is a corn plant from ZM01, ZM02, ZM03, or ZM04 variety. As shown in Table 6, maize pollen enhanced the production of sunflower haploid seed compared to the control. For example, a distant hybridization between sunflower SY15 plant and maize ZM01 plant produced 12 haploid seeds per head of the sunflower. c. Plant hormone-based method

[0066] Double fertilization of a sunflower plant can also be limited by contacting the sunflower plant (e.g., flower buds of the sunflower plant) with plant hormones. Useful hormones that can be used for this purpose include, but not limited to, gibberellic acid (GA) or jasmonic acid (J A) or ethylene antagonists. In some embodiments, a solution comprising one or more of these hormones (or antagonists) is applied to the flower buds of a sunflower plant. In some embodiments, the solution comprises GAs in an amount of 25 ppm to 300 ppm, for example 50 ppm to 200 ppm, or about 125 ppm. In some embodiments, a solution comprisingknown inhibitors of hormone pathway factors is applied to the flower buds of a sunflower plant. In some embodiments, these inhibitors, such as Jarin-1, COR-MO, or NOPh, either block perception or synthesis of jasmonic acid (JA). In some embodiments, these inhibitors either block the perception or synthesis of ethylene, including 1 -Aminocyclopropane- 1 -carboxylic acid (ACC), aminoethoxyvinyl glycine (AVG), cobalt ions, or silver thiosulphate (STS). In some embodiments, the solution comprises GA, JA and / or ethylene antagonists in any amount described above. In some embodiments, the solution comprising the more hormones is applied to flower buds at a frequency of at least every two days, for example, at least every three days, at least every four days, or at least every five days. In some embodiments, the solution is applied at least once before fertilization.

[0067] In some embodiments, the solution comprising the one or more hormones are applied to flower buds that are at the R1 stage or later, e.g., R2, R3, R4, R5, R6, R7, R8, R9. A plant is at R1 stage when the terminal bud forms a miniature floral head, and the immature bracts form a many-pointed starlike appearance when viewed from directly above. See, e.g., Schneiter, A.A., and Miller, J.F. 1981. Description of Sunflower Growth Stages. Crop Science. Vol. 21(6): 901-903. In one exemplary study described herein, GAs were applied to flower buds at R1 stage and induced greater than 1% male sterility (Tables 7A-C, 8, and 9). The treatment also resulted in production of a significant number of haploid seeds, with 22-23 haploid seeds per head in average. d. Emasculation and Cytoplasmic Male Sterility (CMS) method

[0068] In some embodiments, haploid production via limiting double fertilization can be achieved by performing manual emasculation on the sunflower plant or by using a sunflower plant from a CMS line. In some embodiments, manual emasculation is performed on the plant when the anther of a plant flower is emerged and pollen is still absent (i.e., before the pollen falls off the plant). Manual emasculation includes removing the anthers from the plant. Manual emasculation is typically performed in the morning of a day when the florets are open. In some embodiments, the flowers are washed before and after emasculation to remove pollen. As shown in FIG. 1, FIG. 2, and Table 10, emasculation was successful in generating haploid seeds from emasculated plants.

[0069] In some embodiments, the method of enhancing haploid production comprises growing a CMS plant and isolating haploid seeds from the flowers of the CMS plant. CMS lines do not produce functional pollen so can naturally limit self-fertilization based doublefertilization. The term “functional pollen” refers to the pollen that is able to result in fertilization when contacting the stigma of a plant. As such they can produce a higher number of haploid seed as compared to a normally cross-pollinated CMS line. In some embodiments, sunflower CMS is from petiolaris (PET 1) male sterile source (Nandini, C., et al. 2016. Evaluation of hybrids developed from diversified CMS lines for resistance to powdery mildew in sunflower (Helianthus annuus L.). Electronic Journal of Plant Breeding, Vol. 7(4): 947-952.) or other sources. In some embodiments, collecting haploid seeds from a CMS line sunflower plant comprises bagging the CMS sunflower heads before flowering, harvesting the heads after flowering, drying the heads, and isolating haploid seed from the heads. In some embodiments, harvesting sunflower seeds occurs 30-40 days after flowering. As shown in Table 10 the CMS line sunflower plants produced multiple haploid seeds per flower.

[0070] In some embodiments, a male sterile line is generated by modifying genes that are responsible for sterility, for example, the male-sterile 9 (ms9) gene (Chen, J., et al. 2006. Molecular mapping of a nuclear male-sterility gene in sunflower (Heliantus annuus L.) using TRAP and S SR markers. Theor Appl Genet. Vol. 113(1): 122-7). In some embodiments, a male sterile line is generated by modifying genes that are responsible for the thermo-sensitive genic male sterility (TGMS), for example, TMS5 (Barman, H.N., et al. 2019. Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR / Cas9 system. BMC Plant Biology . Vol. 19: 109). In some embodiments, a male sterile line is generated by modifying genes that are responsible for the photoperiod-sensitive genic male sterility (PGMS) gene, for example, carbon starved anther (CSA) gene (Zhang, H., et al. 2013. Mutation in CSA creates a new photoperiod-sensitive genic male sterile line applicable for hybrid rice seed production. Proc Natl Acad Sci U S A. 110(l):76-81). In some embodiments, a male sterile line is generated by modifying genes that are responsible for the chemical-sensitive genic male sterility (CGMS) gene, for example, oxophytodienoic acid reductase 7 (OsOPR7) gene (Pak, H., et al. 2021. Creation of male-sterile lines that can be restored to fertility by exogenous methyl jasmonate for the establishment of a two-line system for the hybrid production of rice (Oryza sativa L.) Plant Biotechnology Journal Vol. 19: 365- 374). e. Determination of ploidy

[0071] The ploidy status of the haploid seeds produced from plants treated as disclosed above can be verified by using methods well known in the art. In some embodiments, ploidy isdetermined using flow cytometry, for example, as disclosed in Bohanec, B. 2003. Ploidy determination using flow cytometry. In: Maluszynski, M., Kasha, K. J., Forster, B.P., Szarejko, I. (eds) Doubled Haploid Production in Crop Plants. Springer, Dordrecht. An illustrative example of using flow cytometry to determine ploidy status and the results are shown in FIG. 3. In general, a haploid showed a lower value in the FL2-A channel as compared to a diploid; thus, the value of FL2-A can be used to determine the ploidy status of the seeds. In some embodiments, ploidy is determined via plant size, usually haploid is smaller compared to diploid or doubled haploid seeds (Yao, L., et al. 2018. OsMATL mutation induces haploid seed formation in indica rice. Nat Plants. Vol. 4(8): 530-533). In some embodiments, ploidy is determined via plant organ development, for example, stomata develop smaller in size in haploid compared to diploid (see FIG. 2C) (Liu, H., et al. 2020. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Hgro zctera / m-mediated CRISPR system, Journal of Experimental Botany, Vol. 71(4): 1337-1349) or no trichome in haploid (Kelliher, T., et al. 2019. One-step genome editing of elite crop germplasm during haploid induction. Nature Biotechnology. Vol. 37(3): 287-292). In some embodiments, ploidy is determined via immature seed / mature seed / seedling color, for example Rl-nj anthocyanin marker (Kelliher, T., et al. 2019. One-step genome editing of elite crop germplasm during haploid induction. Nature Biotechnology. Vol. 37(3): 287-292). In some embodiments, ploidy is determined via male sterility or female sterility (Zhang, W ., et al. 2014. Production and identification of haploid dwarf male sterile wheat plants induced by com inducer. Botanical Studies. Vol. 55:26).III. Increasing haploid seed setting

[0072] In some embodiments, the method for enhancing haploid production in a sunflower plant further comprises growing parent sunflower plants under high light intensity and / or long daylength with a broad spectrum including ultraviolet (UV). In some embodiments, the plants are grown with sunlight and supplemental light including metal halide (MH) bulbs, incandescent light, light emitting diodes (LEDs), or high-pressure sodium (HPS) lights. In some embodiments, the method comprises growing the parent sunflower plants under a light intensity between 500 and 1400 pmol / m2 / s photosynthetic photon flux density (PPFD), optimally between 1100-1300 pmol / m2 / s PPFD. In some embodiments, the method comprises providing this light intensity for 12 to 18 hours, optimally for a 16-hour daylength, for a total daily light integral (DLI) of 65-75 mol / m2 / d. An optimal supplemental lighting source, to beused alone or in combination with sunlight or incandescent light, is to primarily use metal halide bulbs which emit intensely in both the visible and UV spectrum.

[0073] In some embodiments, the method further comprises inducing or enhancing the formation of haploid seed by growing the parent sunflower plants with a fertilizer regimen. In some embodiments, the method includes providing the sunflower plants with fertilizer every day in the irrigation water (‘fertigation’). In other embodiments, the method optimally includes providing the plants with fertilizer two times per week, restricting the total amount of fertilizer nutrients delivered to the plant roots. Without wishing to be bound by theory, the combination of high light intensity and reduced fertilizer induces a stress in the plant which may trigger high rates of haploid seed formation under conditions where male-sterile conditions have been met - i.e., in genetically sterile plants or in plants where anthers were removed or chemically aborted.

[0074] In some embodiments, the method further comprises applying one or more haploid seed setting reagents to the plant. These haploid seed setting reagents enhance haploid seed production by facilitating fertilization limitation and / or enhancing haploid seed development. Nonlimiting examples of haploid seed setting reagents include micronutrients (e.g., boron), cytokinins (e.g., ribosides, such as nicotinamide riboside), and plant hormones (e.g., indoleacetic acid, jasmonates, ethylene). In some embodiments, sucrose or 2,4- Dichlorophenoxyacetic acid can also serve as haploid seed setting reagents. These haploid seed setting reagents can be applied to the heads of sunflower plants that have been treated as described above in Section II. These plants can be, for example, a CMS sunflower plant, a sunflower plant that has undergone manual emasculation, or a plant that has been treated by the plant hormone to limit fertilization, or a plant that has been contacted with pollens that have been treated by fertilization-limiting chemicals as described above. In some embodiments, the haploid seed setting reagents are injected into the sunflower heads.IV. Separating haploid seeds from non-haploid seeds

[0075] Haploid seeds produced using the methods and the compositions disclosed herein can be separated from non-haploid seeds using methods well known in the art. In some embodiments, the separation can be performed manually based on the difference in shape and color between the haploid seeds and non-haploid (e.g., diploid) seeds (See FIG. 1). In some embodiments, the separation can be performed using a seed blower based on that the haploid seeds are different from the non-developed seeds in weight (See FIG. 4A). In someembodiments, the separation can be performed using grid based on that haploid seeds are smaller than non-haploid seeds (See FIG. 4B). In some embodiments, the separation can be performed using imaging methods on the basis that the structure of haploid seed is different from the structure of non-haploid seed. In some embodiments, the separation is performed by detecting oil signal in haploid seeds that is distinguishable from non-haploid seeds. In some embodiments, the separation can be performed by using markers specifically associated with haploid seeds, for example markers in hull, seed coat, cotyledon of haploid seeds. In some embodiments, the separation is performed based on developmental markers that are specific to either haploid seeds or the non-haploid seeds, for example, the anthocyanin markers (See FIG. 4C).V. Regenerating / germinating haploid seedlings from haploid seeds

[0076] In some embodiments, the haploid seeds are sowed in soil directly and grown into haploid plants. In some embodiments, the haploid seeds are germinated on a germination paper (such as, https: / / www.ssfilters.com / pdf / seed-germination-paper.pdf), a liquid medium, semisolid medium, or solid medium. Non-limiting examples of solid media that are suitable for use to grow the haploid seeds include SF-SIM16, SF-SIM18, SF-SIM19, SF-SIM20, SF-SEM2-1, SF-SIM21, as shown in Table 17, below.

[0077] In some embodiments, haploid seeds collected from the sunflower plant are first soaked in sterile water for 2 to 5 hours (e.g., 3 hours). Optionally, seed pericarps are then removed from the embryos and embryos are sterilized before being placed in one of the media described above. In one embodiment, the sterilization solution comprises sodium hypochlorite and Tween 20.

[0078] In some embodiments, the method further comprises a one-round regeneration process. In some embodiments, the method further comprises a two-round regeneration process. In some embodiments, the method further comprises a multiple-round regeneration process. Others have discussed regeneration from sunflowers, for example, as in Chraibi, Khalid M.B., et al. 1992. A genotype-independent system of regeneration from cotyledons of sunflower (Helianthus annuus L.), The role of ethylene. Plant Science, Vol. 86(2): 215-221.

[0079] The one-round regeneration process typically comprises (i) culturing the embryos on petri dishes (e.g., 90 x 24 mm) at ambient temperature (e.g., 25°C) in the dark for 5 days, (ii) then transferring the culture to the environment at ambient temperature, under 16 hour (light) / 8 hour (dark) photoperiod with light intensity of approx. 7500 Lux for approximately 4 to 7 days,(iii) transferring the embryos into a larger volume of medium (e.g., 100 ml) and growing the embryos under the same conditions for up to 4 weeks, with subcultures of the original culture being prepared every two weeks to accommodate the rapid growth (FIG. 5). Subcultures of the plant can be prepared using methods well known in the art, for example, by taking parts of the plant of the original culture and transferring it to fresh culture medium. Exemplary procedures are described, for example, in Volk, G.M., Denoma, J., Hummer, K.E., Chen, K. 2021. Introduction of clean plants into tissue culture: Temperate crops. In: Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Volk, G.M., ed. Colorado State University (Fort Collins, Colorado), available at colostate. pressbooks. pub / clonalcryopreservation / chapter / introduction-of-plants-into-tissue-culture. Shoots that were approximately >1.5 cm in length were transferred to a rooting medium for 2 to 3 weeks, then transferred to the greenhouse after rooting. (FIG. 5 and 6) .

[0080] The two-round regeneration process comprises performing an additional round of regeneration on the shoots or plantlets obtained from the one-round regeneration method as described above. Specifically, the additional round of regeneration comprises cutting nodal sections (0.5cm in length) above and below the junctions of leaves and main stem from the shoots or plantlets and placing them in a medium described above in 88 x 76mm clear plastic culture boxes. Cultures comprising these nodal sections are then placed in a growth room at ambient temperature (e.g., 25 °C), under 16-hour (light) / 8-hour (dark) photoperiod with light intensity of approx. 7500 Lux for 2 to 3 weeks. Shoots that have grown to approx. > 1.5 cm in length are then transferred to a medium to produce roots for approximately 2 to 3 weeks (FIG. 5).

[0081] As shown in Table 18, one-round regeneration method produced 91% of normal haploid plants and / or shoots from SY54 haploid seeds grown in medium SF-SIM19 (FIGs. 5 and 6). Two-round regeneration method produced 200% of normal plants and / or shoots from SF54 haploid seeds using medium SF-SIM19 (FIGs. 5 and 6, Table 17). In some embodiments, normal haploid plant, as used herein, refers to a plant that has shoots, two cotyledons, one apical meristem, and roots. A normal haploid plant is able to grow into reproductive stage. Conversely, an abnormal haploid plant refers to a haploid plant that lacks one or more of the features above and cannot grow into reproductive stage. In some embodiments, a normal haploid plant is plant that can be doubled into sunflower seedlings, further which can grow diploid seeds. In some embodiments, 200% means 2 normal plants developed from 1 haploidseed. After transplanting the normal plants into the greenhouse, 88.9% of the generated plants survived in greenhouse conditions and 87.5% of the plants were haploids (FIG. 6).VI. Chromosome doubling

[0082] Chemicals capable of inducing chromosome doubling in a plant are referred to as doubling agents. As used herein, a doubling composition or doubling formulation refers to a mixture comprising doubling agents and organic or inorganic solvents used to dissolve the doubling agents. In some embodiments, the doubling composition comprises one or more additional components such as, dimethyl sulfoxide (DMSO) or glycerol.

[0083] In general, chromosome doubling can be artificially induced through application of doubling agents to block cell division by preventing the microtubules formation. There are multiple strategies for chromosome doubling in plants: (1) application of doubling agents during tissue cultures of microspores or immature embryos to induce doubled seedlings; (2) soaking the germinated seedlings into chemical solutions; and (3) targeted delivery of doubling agents on the shoot apical meristematic region in the plant, which is further described below in section VI.

[0084] In some embodiments, one or more doubling agents may be used in the methods disclosed herein comprise colchicine, herbicide, or a combination thereof. Exemplary herbicides include Oryzalin (4-(dipropylamino)-3,5-dinitrobenzenesulfonamide) and Trifluralin (2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline. In some embodiments, the doubling agent is colchicine. For example, colchicine can be present in the solution in a concentration ranging from 50 pM-200 pM. In some embodiments, the doubling agent is Oryzalin. For example, the Oryzalin can be present in a concentration ranging from 20 pmol / L to 100 pmol / L, from 30 pmol / L to 80 pmol / L, or from 35 pmol / L to 70 pmol / L. In some embodiments, the doubling agent is Trifluralin. For example, the Trifluralin can be present at a concentration of 20 pmol / L to 150 pmol / L, 30 pmol / L to 100 pmol / L, 48 pmol / L to 95 pmol / L.

[0085] Delivery of the doubling composition to target areas (e.g., the meristem tissues) can be achieved via dripping doubling agent(s) directly onto / into meristem tissues, e.g., shoot apical meristem and / or axillary meristem tissues. Dripping can be achieved by pipette, pipet, needle, sprayer, nebulizer, et al.

[0086] One exemplary method of chromosome doubling (including doubling a haploid plant) includes the steps of (i) germinating sunflower seeds (e.g., sunflower haploid seeds) to form sunflower seedling (e.g., sunflower haploid seedlings) and growing the sunflower seedling until VE stage (a stage where two cotyledons are fully expanded, but the first true leaf is not elongated yet), (ii) merging the whole seedling or whole apex and axillary meristems into the chemical solution for a time period (5 seconds - 48 hours, e.g., 5 minutes to 24 hours, 5 hours to 12 hours or 1 hour to 8 hours) or dropping the chemical solution on seedlings between two cotyledons to cover the region of apex and axillary meristems; (iii) removing residual formulation from seedlings, for example, by washing the seedlings. In some embodiments, the seedlings that have been treated with the formulation are covered (e.g., with domes) to decrease formulation evaporation. The ploidy level in newly extruded tissues, such as leaves, may be verified by methods known in the art, e.g., by flow cytometry.VII. Formulation and targeted delivery

[0087] Haploids produced using the methods and compositions disclosed herein can be treated to increase the chromosome number to produce the doubled haploids. Disclosed in this section specifically is a formulation-based targeted delivery of doubling agents into plant seedling meristem regions for chromosome doubling. The composition is a sticky emulsion that can be applied to cover meristem regions and allows continuous release of doubling agents to meristems. As used herein, sticky emulsion refers to a viscous, adherent emulsion that remains substantially in the location to which it is applied. The chromosome-doubling effect of the composition lasts for 1-2 cell division cycles. An additional advantage of the doubling composition disclosed in this section is that it does not contain any carbon sources (e.g., carbohydrates; carbon hydrate). Thus, the provided compositions are substantially free from fungal and other microbial contamination issues. The methods and compositions therefore provide simple and efficient approaches for chromosome doubling and producing doubled haploids.(1) Formulations components

[0088] The formulation comprises or consists of one or more of the following components: 1) one or more doubling agents; 2) water or a buffer; 3) a fluorocarbon ether polymer of polyhexafluoropropylene oxide (e.g., Krytox™ fluorinated oils); 4) a hydrofluoroether. The formulation exists as a semi-solid emulsion of an aqueous phase, a hydrofluoroether, and an oil comprising fluorocarbon ether polymer of poly hexafluoropropylene oxide (comprising,e.g., Krytox™ fluorinated oils). This is an oil in water emulsion. The fluorocarbon ether polymer is dissolved in hydrofluoroether droplets contained in the aqueous phase. The term “semi-solid emulsion” refers to an emulsion that is highly viscous and is able to maintain its location and consistency when placed on a solid surface (e.g., a flat or relatively flat surface such as a leaf) for at least 0.5 hour, at least 1 hour, at least 2 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 30 hours, or at least 36 hours. Generally, the formulation is an emulsion having a homogeneous distribution of hydrofluoroether droplets contained in the aqueous phase. Generally, the formulation has a uniform consistency.

[0089] The aqueous phase of the formulation is composed of water or a buffer. In the context of this disclosure, a buffer is an aqueous solution that maintains a constant pH over a given range by neutralizing the effects of hydrogen ions. A buffer is a solution containing either a weak acid and its conjugate base or a weak base and its conjugate acid. The buffer contains an ionizable salt such as phosphate, acetate, citrate, bicarbonate, Tris, Tris-Acetate, and the like, with a counter-ion. Exemplary buffers include phosphate buffered saline (PBS), 2-[(2-amino- 2-oxoethyl)amino] ethanesulfonic acid (ACES), sodium; 2-[bis(2- hydroxyethyl)amino]ethanesulfonate (BES), N-(2-Hydroxyethyl)piperazine-N’-2- ethanesulfonic acid (HEPES), 2-morpholin-4-ylethanesulfonic acid;hydrate (MES), 3- morpholin-4-ylpropane-l -sulfonic acid (MOPS), 3-(N-morpholino)-2-hydroxy-l- propanesulfonic acid (MOPSP), 1,4-Piperazinedi ethanesulfonic acid (PIPES), etc.

[0090] Any of the above-referenced doubling agents can be used in the formulation disclosed herein. (See Section V.) Certain doubling agents (for example, colchicine and herbicides) may be toxic to plants when used at high concentrations. The formulation disclosed herein has been optimized such that it comprises doubling agents at concentrations that are effective for chromosome doubling but are also safe enough for plants to allow the development of meristem tissues into mature plants. In some embodiments, colchicine is present in a concentration in a range from 0.5 pM - 200 pM, 0.5 pM - 100 pM, 25 pM - 150 pM, or 25 pM - 75 pM, e.g., 30 pM - 36 pM.

[0091] The doubling agent may be dissolved in organic or inorganic solvents. Organic solvents are carbon-based solvents. Nonlimiting examples of organic solvents that can be used include alcohols (e.g., methanol, ethanol, isopropanol), ethers, esters, ethyl acetate, ketones, and so on. Inorganic solvents are solvents other than water that do not have organic componentsin them. In some embodiments, the solvent is acetone or dimethyl sulfoxide (DMSO). Nonlimiting examples of inorganic solvents that can be used include ammonia, sulfuric acid, and fluoride sulfuryl chloride.

[0092] Hydrofluoroethers is a class of organic solvents. Hydrofluoroethers are hydrophobic and immiscible, and they are soluble to Krytox™ fluorinated oil.

[0093] One example of fluorocarbon ether polymer of poly hexafluoropropylene oxide is the Krytox™ fluorinated oils. They are generated by polymerization of hexafluoropropylene oxide. Fluorocarbon ether polymers are not water-soluble, but they are soluble in hydrofluoroethers. Krytox™ fluorinated oil molecules contain water soluble groups, which can trap water molecules through strong hydrogen bonds and contribute to the semi-solid property of the formulation.

[0094] Optionally, the formulation comprises other components that can be used to facilitate penetration, stabilize the formulation, and enhance doubling efficiency, such as DMSO, acetone, glycerol, or non-ionic surfactants (e.g., Silwet® L-77).

[0095] In some embodiments, the formulation comprises acetone. Acetone is an organic compound and is miscible with water and serves as an important organic solvent. As shown in FIG. 8, acetone can be used in the formulation to stabilize hydrofluoroether in semi-solid emulsion. The semi-solid emulsion, when applied on a plant tissue, e.g., a leaf of a haploid plant, as shown in FIG. 11, substantially retains its consistency and remains in the location in which it has been placed so as to continuously deliver the doubling agent to the plant tissue over a period of time. In some embodiments, the length of the time is greater than the time period required for 2 cell cycles of the plant, but less than the time period required for 3 cell cycles. In some embodiments, the length of the time is between 12 hours and 36 hours, between 18 hours and 30 hours, between 22 hours and 26 hours, or about 24 hours.

[0096] In some embodiments, the formulation comprises non-ionic surfactant, such as Silwet ®L-77. Silwet® L-77 is a nonionic organosilicone surfactant co-polymer that has enhanced wetting and spreading characteristics. Silwet® L-77 can help doubling chemicals penetrate meristem to increase doubling efficiency. As shown in Table 24, 0.05% Silwet® L-77 significantly increased doubling rate from 0% to 18%.(2) Formulation properties

[0097] The formulation disclosed herein in this Section VII is produced by mixing the components as described above at appropriate concentrations. The fluorocarbon ether polymer of the formulation is dissolved in hydrofluoroether droplets that are contained (suspended) in the aqueous phase, as shown in FIG. 9. The hydrofluoroether soluble groups of the fluorocarbon ether polymer (e.g., fluorinated oil) associate with the hydrofluoroether component, and the water-soluble head groups of the fluorocarbon ether face outwards into the aqueous phase containing the doubling agents, thereby forming an emulsion of fluorocarbon ether polymer-encapsulated hydrofluoroether droplets in the aqueous phase. Without being held to any particular theory, the droplets can be cross-linked to one another through hydrogen bonds in the aqueous phase (e.g., via water molecules), forming an emulsion.

[0098] In some embodiments, the composition comprises 1-3% (w / v) of fluorocarbon ether polymer of polyhexafluoropropylene oxide and 11-39%, e.g., 15-35%, e.g., 20-30% hydrofluoroether (v / v). As shown in FIG. 10, a semi-solid emulsion formed successfully from Hydrofluoroether oil (HFE7500 oil. 20-30% v / v), Krytox™ 157 FSH (1-3% w / v), and water (70-80% v / v) when they were used in concentrations in the referenced ranges.

[0099] In some embodiments, the formulation confers a chromosome doubling rate of greater than 30% in cells of new tissue growth when applied on haploid sunflower seedlings (See Table 20).

[0100] The formulation temporarily remains as a semi-solid emulsion upon application to the tissue. However, the doubling agents are continuously released from the formations into the tissue for 24 hours or more after application. The superspreading surfactants and hydrofluoroethers in the formulation facilitates the doubling agents to penetrate through the plant tissues.(3) Formulation applications

[0101] In some embodiments, the method comprises applying the formulation as disclosed above on a seedling center of a sunflower plant such that the formulation covers a portion of meristematic tissues (e.g., the apex meristem and / or and axillary meristem region). The method further comprises maintaining the haploid sunflower seedling with the formulation thereon in a high humidity condition (60-100%) for sufficient time for new tissue growth in the haploid sunflower seedling. In some embodiments, the plants are kept in a high humidity condition for2-3 days (1-2 cell division cycles for sunflower) to avoid formulation evaporation and allows for 1-2 cell division cycles in tissue of the haploid sunflower seedling.(4) Plant care after treatments:

[0102] After the treatment, seedlings are washed with water showering to remove residual formulations to avoid multiple times of chromosome doubling. The seedlings are moved to the suitable growth conditions for seed production or for ploidy testing.EXEMPLARY EMBODIMENTS

[0103] This disclosure provides the following nonlimiting embodiments.

[0104] Embodiment 1 is a method of enhancing haploid production in a sunflower plant, wherein the method comprises interrupting pollen development of pollen producing tissues ; or outcrossing the sunflower plant with pollen from a second plant, wherein the sunflower plant is grown under high light intensity and / or long daylength and a broad-spectrum including UV light.

[0105] Embodiment 2 is the method of embodiment 1, wherein the interrupting pollen development comprises treating a flower bud of the sunflower plant with a plant hormone.

[0106] Embodiment 3 is the method of embodiment 2, wherein the plant hormone is gibberellic acid (GA), or jasmonate, or ethylene, or related antagonist(s).

[0107] Embodiment 4 is the method of embodiment 3, wherein gibberellic acid (GA) is at a concentration of approximately 125 ppm.

[0108] Embodiment 5 is the method of embodiment 4, wherein gibberellic acid (GA) is applied to sunflower plants at development stage R1 or later.

[0109] Embodiment 6 is the method of any one of embodiments 1-5, wherein the second plant is a monocot plant or a dicot plant.

[0110] Embodiment 7 is the method of any one of embodiment 6, wherein the monocot plant is corn, wheat, sugarcane, or rice.[OHl] Embodiment 8 is the method of embodiment 6, wherein the dicot plant is tomato, soybean, spinach, pepper, broccoli, cauliflower, carrot, or potato.

[0112] Embodiment 9 is the method of any one of embodiments 1-8, further comprising treating flower heads of the sunflower plant after pollination with one or more of boron, indoleacetic acid (IAA), nicotinamide riboside, sucrose, or 2,4-Dichlorophenoxyacetic acid.

[0113] Embodiment 10 is the method of any one of embodiments 1-9, wherein the method further comprises emasculating flowers of the sunflower plant by removing anthers therein before pollen emission.

[0114] Embodiment 11 is the method of any one of embodiments 1-10, wherein the method further comprises recovering seeds from ovaries of the sunflower plant, separating haploid seeds from non-haploid seeds.

[0115] Embodiment 12 is the method of embodiment 11, wherein the method further comprises developing haploid seeds to generate haploid seedlings and further doubling to generate diploid seedlings / progenies.

[0116] Embodiment 13 is the method of embodiment 11, wherein the haploid sunflower seedlings are derived from a cytoplasmic male sterility plant line or a genetic male sterility plant line.

[0117] Embodiment 14 is the method of embodiment 11, wherein separating of haploid seeds from non-haploid seeds is based on seed weight, seed size, seed color, or seed structure.

[0118] Embodiment 15 is the method of embodiment 11, wherein separating of haploid seeds from non-haploid seeds is based on seedling color.

[0119] Embodiment 16 is the method of embodiment 11, wherein separating of haploid seeds from non-haploid seeds further comprising using seed blower, seed grid, photographing, and / or manual isolation.

[0120] Embodiment 17 is the method of embodiment 11, wherein the method further comprises germinating haploid seeds in soil, germination paper, liquid medium, semi-solid medium, or solid medium.

[0121] Embodiment 18 is the method embodiment 17, wherein the solid medium comprises one or more of SF-SIM16, SF-SIM18, SF-SIM19, SF-SIM20, SF-SEM2-1, or SF-SIM21.

[0122] Embodiment 19 is the method of embodiment 18, wherein the method further comprises at least one round of regeneration process in the liquid medium, semi-solid medium, or solid medium.

[0123] Embodiment 20 is the method of embodiment 12, wherein the method further comprises treating the haploid seedlings with at least one doubling agent to produce a diploid sunflower plant.

[0124] Embodiment 21 is the method of embodiment 20, wherein the doubling agent is colchicine, an herbicide or combination of colchicine and an herbicide.

[0125] Embodiment 22 is the method of embodiment 21, wherein the herbicide is Oryzalin (4-(dipropylamino)-3,5-dinitrobenzenesulfonamide), Trifluralin (2,6-dinitro-N,N-dipropyl-4- (trifluoromethyl)aniline.

[0126] Embodiment 23 is the method of any one of embodiments 20-22 wherein treating the haploid seedlings comprises dripping at least one doubling agent onto the shoot apical meristem of haploid seedling.

[0127] Embodiment 24 is the method of embodiment 11, wherein the method further comprises treating the haploid seedlings with a composition comprising a doubling agent(s) in a semi-solid emulsion.

[0128] Embodiment 25 is the composition of embodiment 24, wherein the semi-solid emulsion comprises a fluorocarbon ether polymer of polyhexafluoropropylene oxide, and a hydrofluoroether, wherein the fluorocarbon ether polymer is dissolved in hydrofluoroether droplets contained in the aqueous phase.

[0129] Embodiment 26 is the composition of embodiment 25, wherein the composition comprises 1-3% (w / v) of fluorocarbon ether polymer of polyhexafluoropropylene oxide and 30% hydrofluoroether (v / v).

[0130] Embodiment 27 is the composition of any one of embodiments 24-26, wherein the composition is free of carbon hydrate.

[0131] Embodiment 28 is the composition of any one of embodiments 23-27, wherein the doubling agent is colchicine or an herbicide or a combination thereof.

[0132] Embodiment 29 is the composition of embodiments 28, wherein the herbicide is Trifluralin or Oryzalin.

[0133] Embodiment 30 is the composition of embodiments 29, wherein colchicine is present in the composition in a concentration ranging from 0.5 pM - 100 pM inclusive.

[0134] Embodiment 31 is the composition of embodiments 28 wherein the colchicine is present in the composition in a concentration ranging from 30 pM - 36 pM.

[0135] Embodiment 32 is the composition of any one of embodiments 25-31, wherein the composition further comprises one or more additional chemicals for facilitating penetration and / or stabilizing the composition.

[0136] Embodiment 33 is the composition of embodiment 32, wherein the one or more additional chemicals are one or more of dimethyl sulfoxide (DMSO), acetone, glycerol, or nonionic surfactants.

[0137] Embodiment 34 is the composition of any one of embodiments 25-33, wherein semisolid emulsion confers a chromosome doubling rate of greater than 30% in cells of new tissue growth when applied on haploid sunflower seedlings.

[0138] Embodiment 35 is a method for doubling chromosomes in cells in a haploid sunflower seedling, wherein the method comprises: contacting a seedling center of the haploid sunflower seedling with the composition of any one of embodiments 25-34; and maintaining the haploid sunflower seedling with the composition thereon in a high humidity condition for sufficient time for new tissue growth in the haploid sunflower seedling, the new tissue growth comprising diploid cells.

[0139] Embodiment 36 is the method of embodiment 35, wherein the sufficient time is a period of time that allows for 1-2 cell division cycles in tissue of the haploid sunflower seedling.

[0140] Embodiment 37 is the method of embodiment 35, wherein the seedling center comprises an apex region and / or an axillary meristem region of the haploid sunflower seedling.

[0141] Embodiment 38 is the method of any one of embodiments 35-37, wherein the haploid sunflower seedling is at seedling emerge (VE) stage.

[0142] Embodiment 39 is the method of any one of embodiments 35-38, wherein the haploid sunflower seedling is obtained from the method of embodiment 15.

[0143] Embodiment 40 is the method of any one of embodiments 35-38, wherein the haploid sunflower seedling is derived from a Cytoplasmic male sterility plant line, a TGMS line, a thermo-sensitive genic male sterility (TGMS) line, a photoperiod-sensitive genic male sterility(PGMS) line, a chemical-sensitive genic male sterility (CGMS) line, a manually- emasculated line, or double-fertilization limited line.

[0144] Embodiment 41 is the method of embodiment 40, wherein the Cytoplasmic male sterility line is of variety SY15 or SY54.

[0145] Embodiment 42 is the method of any one of embodiments 35-41, wherein the method further comprises: washing the haploid sunflower seedling to remove residual composition from the seedling; and / or assessing ploidy levels in a newly formed tissue from the seedling.

[0146] Embodiment 43 is the method of embodiment 12, further comprising growing the haploid seedling that has been contacted with the composition of any one of embodiments 28- 37 to form a plant, thereby producing a diploid sunflower plant.

[0147] Embodiment 44 is the method of embodiment 1 , wherein the high light intensity is provided by sunlight and supplemented with metal halides, incandescent light, light emitting diodes (LEDs), or high-pressure sodium lights (HPS).

[0148] Embodiment 45 is the method of embodiment 44, wherein the high light intensity is provided by sunlight and supplemented with metal halide bulbs.

[0149] Embodiment 46 is the method of any one of embodiments 44-46, wherein the high light intensity is between 500 and 1400 pmol / m2 / s photosynthetic photon flux density (PPFD).

[0150] Embodiment 47 is the method of any one of embodiments 44-46, wherein the high light intensity is provided for 12 to 18 hours resulting in a total daily light integral (DLI) of 65 to 75 mol / m2 / d.

[0151] Embodiment 48 is the method of embodiment 1, wherein the method further comprises growing the sunflower plants with fertilizer application selected from Osmocote or Jack’s fertilizer.

[0152] Embodiment 49 is the method of embodiment 48, wherein the fertilizer is applied between two and seven times per week.

[0153] Embodiment 50 is the method of embodiment 1, wherein the sunflower plant is selected from the group consisting of SY48, SY54, and SY58.

[0154] Embodiment 51 is the method of embodiment 50, wherein the sunflower plant isSY58EXAMPLESExample 1: Limiting fertilization by treating sunflower plants with chemical compounds

[0155] Maize haploid induction gene ZmMATL, encoding a pollen-specific phospholipase A2, releases fatty acids from the second carbon group of glycerol. Haploid induction in maize and rice was achieved by loss-of-function of MATL. In this example, we tested the hypothesis that chemicals limiting fertilization can also induce haploid seed development in Sunflower. Three chemicals were selected and tested, methyl a-linolenyl fluorophosphonate (MLnFP), oleic acid ethyl ester (OAEE) and manoalide (Table 3). The CMS line of the SY15 variety was used as the female to avoid manual emasculation and contamination from selfing. Pollen from the SY83 variety, a restorer line, was treated with chemicals following the methods below to limit pollen fertilization capability.

[0156] Method 1 comprises spraying chemical Formula-A (Table 3) onto pollen in open air for 3 minutes with a nebulizer. Method 2 comprises spraying chemical Formula-A (Table 3) onto pollen in sealed plastic bag for 2 minutes with a nebulizer. Method 3 comprises collecting pollen and soaking them in chemical Formula-B (Table 4) for 20 minutes at 25°C at 200 rpm. In Table 3: DML, dimethyl lactamide; PBS, Phosphate buffer solution, pH 6.1, IX. Components of Blend 91 and Blend 92 are described in Tables 1 and 2 above.Table 3. Detailed recipe of Formula-A used for spray.

[0157] Corn oil and sunflower oil, as cooking oil, were purchased from the market. Mineral oil, a lubricant commonly used in manufacturing, can also be used in the Formula-B, see below.Table 4. Detailed recipe of Formula-B used for pollen soaking.

[0158] Treated pollen was transferred with ink brush to fertilize CMS heads. A SNP marker is used to pick haploids from diploid Fl and accompanied with ploidy analysis using flow cytometry. With all 3 chemicals, we obtained sunflower haploid seeds (Table 5) with haploid induction rates (HIR) varying from 0.34% to 66.67 (ratio of haploid seeds among total seeds harvested). Oil treatments led to higher HIR but with less seeds set. Among 23 haploids, we obtained 5 putatively spontaneously doubled haploids (pSDH) . The mechanism of spontaneous doubling is not well known in sunflower or in other crops where it has been identified, such as in wheat and maize.Table 5. Haploid seeds induced by fertilization-limited pollen after different treatments.Example 2: Limiting fertilization by using pollen in a wide cross

[0159] Wide cross usually means outcross between two reproductive isolated species or subspecies, which can block fertilization. Corn is a typical monocot crop and sunflower is a dicot crop. Corn pollen was collected and used to fertilize CMS lines of sunflower variety SY15 and SY54. One field corn (ZM01, inbred) and 3 sweet corn varieties (ZM02-04, hybrid) were used as pollen donors to block fertilization in sunflower. As illustrated in Table 6, maize pollen can trigger sunflower haploid seed development (4-145 haploid seeds per head depending on treatment). Boron (B) is a micronutrient critical to the growth and health of all crops as a component of plant cell walls and reproductive structures. Boron spray changes sunflower haploid seed setting either via assisting fertilization blocking / limiting or enhancing haploid seed development. Cytokinin controls development of flower buds and seeds at different stages. Riboside (ZR), one type of cytokinin, was injected into sunflower heads after pollination with maize pollen, which can also affect sunflower haploid seeds development. Another key plant hormone, indoleacetic acid (IAA), had similar observations (Table 6). One may apply both hormones and micronutrients in combination to further increase haploid seed set.Table 6. Haploid seeds induced by maize pollen with different treatments.Example 3: Limiting fertilization by plant hormones

[0160] Gibberellins (GAs) and jasmonates are plant hormones with diverse roles in plant growth and development. Male sterility is one of several GA responses. We dissolved 0.1 grams 90% GA3 powder in 1 tsp. (~5 mL) of 70% or greater ethyl alcohol and diluted in 720 mL of DI water to prepare 125 ppm GA3. We applied GA3 solution directly to flower buds at the R1 stage with 3-4 spray pumps (~2 mL solution) from a standard spray bottle. The application was repeated more than 1 time, dependent on the sunflower line being treated, witha 4-day interval between each application. Heads were bagged after all applications were complete and prior to flowering to prevent any cross-pollination. Sunflower seeds were harvested once they reached physiological maturity. Hand sorting or a seed blower was used to separate haploid seeds from diploid seeds. Varying application methods, timings, and concentrations of GA3 were tested on 13 different fertile sunflower lines in a total of 8 experiments. The GA3 emasculation protocol developed through this testing achieves > 99% male sterility for the two parthenogenesis (PRG) sunflower lines tested (SY54 and SY58) and > 93% male sterility for all 13 lines tested (Tables 7A and 7B). Some varieties, like SY10, need to be sprayed multiple times. Instead of spray, dripping GA3 with a pipette onto R1 buds also can introduce chemical-induced male sterility in sunflower. GA3 induced male sterility in SY54 and SY58 triggered haploid seed development, with 22-23 haploid seeds per head on average. Percent (%) Sterile, as reported in Tables 7A, 7B, and 7C was calculated as: 100 - [(Average number of Diploid Seeds for treatment / Average number of seeds for control) * 100],Table 7 A. GA3 induced male sterility in greenhouse conditions.Table 7B. GA3 inducec male sterility in greenhouse conditions.Table 7C. GA induced male sterility in greenhouse conditions.

[0161] A study was conducted to mimic field conditions in Southern France (a predominant sunflower breeding area) at the time of flowering to determine the influence of high temperatures on the effectiveness of GA3 sterilization. The greenhouse was set on a schedule to directly mimic field temperatures during the 2019 growing season in Toulouse, France. Temperature data at the time of GA3 application and flowering are presented in Table 8. The increased temperatures during application and flowering proved to have no effect on the sterility of the GA3 application in this small study across 8 sunflower lines (Table 9).Table 8. Field conditions when applying GA3 induced male sterility treatment.Table 9. GA3 induced male sterility in greenhouse conditions mimicking field conditions.Example 4. Haploid production by manual emasculation and cytoplasmic male sterility (CMS).

[0162] Firstly, manual emasculation was applied. Sunflower will flower in spiral with one to two “rows” of flowers opening every day: early in the morning the anthers will come out, during the morning the anther will break and release pollen, and later the stigma will emerge through the anthers. Pollen is viable as it is released and remains viable for several hours. Pollen can also be kept in the refrigerator for several months as long as it’s stored in low humidity. The stigma may be receptive some hours after emerging and will be receptive for several days. Once the stigma is pollinated, it will retract (if not pollinated, it will simply dry out).Emasculation can be performed by using tweezers to remove the anthers, usually between 8:00 - 10:00 am (it depends on the genotype and the environment). The anther must be fully emerged in order not to take the stigma away with it and must be completed before pollen is shed. Usually, we “flush” the flowers with water before and after emasculating to eliminate any visible pollen. With manual emasculation treatment, varieties SY15 and SY54 were tested with multiple heads, and there were 13-24 seeds per head developed on average (Table 10, FIG. 1). Those seeds were germinated and sampled to determine ploidy level via flow cytometry to identify true haploids (FIG. 2, FIG. 3).Table 10. Haploid seeds developed in sunflower with manual emasculation and in cytoplasmic male sterility (CMS) sunflower.

[0163] Via ploidy analysis (see generally Bohanec B. (2003) Ploidy determination using flow cytometry. In: Maluszynski M., Kasha K.J., Forster B.P., Szarejko I. (eds) Doubled Haploid Production in Crop Plants. Springer, Dordrecht, doi.org / 10.1007 / 978-94-017-1293- 4_52), most plants were haploids. Meanwhile, we found a triploid plant from those seeds collected from manually emasculated sunflower heads. The ratio of triploid is around 5-10%, varying in different treatments and germplasms.

[0164] Further CMS (cytoplasmic male sterility) lines of varieties SY15 and SY54 were tested, which do not produce pollen and naturally block self-fertilization. CMS heads were bagged at R1 stage and without fertilization treatment. Haploid seeds were observed in those CMS heads of both varieties (Table 10), with similar average haploid seeds setting per head of non-CMS lines. Modifying nuclear male-sterility, such as ms9 in sunflower, can also lead to haploid seed development. Similar cases exist in thermo-sensitive genic male sterility (TGMS) gene modification and the photoperiod-sensitive genic male sterility (PGMS) gene modification.The following growth conditions and protocols were used in the emasculation and CMS experiments:

[0165] Sunflower seeds were sown in 32-well germination trays with a well size of 7 cm X 7 cm. The tray was filled with a soil mixture of Turf 876 (https: / / klasmann-deilmann.com / wp- content / uploads / 8982_KD_Aktualisierung_Easy _Growing_EN.pdf) and SN peat moss at a volume ratio of 4: 1 and contained 10g Osmocote slow-release fertilizer per tray. Sunflower seeds were sown in the mixture 1 cm below the surface and then covered with Turf 876. Trays were placed in a growth chamber at the following conditions (Table 11).Table 11. Sunflower Germination Growth Conditions.

[0166] Seedlings emerged about 1 week after sowing and were transferred to larger 10-liter pots in the greenhouse. The larger pots were filled with Turf 876, Turf SN peat moss, and perlite at a ratio of 4:4: 1. After transplanting, 45 g Osmocote slow-release fertilizer (nitrogen, phosphate, potash ratio “N:P:K” of 15:9: 12) was added in each pot. CMS plants were planted in pollen-free greenhouse room (no male-fertile plants in the room). Irrigation was managed through a drip irrigation system. Pests, including thrips, were controlled by a biweekly pesticide spray, with the following growing conditions (Table 12).Table 12. Sunflower Plant Growth Conditions.

[0167] CMS sunflower heads were bagged before flowering, and the heads were harvested 30 to 40 days after flowering. Harvested heads were dried for 2 days, then all seeds were threshed from the heads. Developed seeds were picked out manually or by using a seed blower.Example 5. Enhancing the formation of haploid seeds

[0168] We observed that haploid seed set tended to drop in the winter and rise in the summer. To test the effects of photoperiod and light spectrum on haploid seed set, we grew the SY58 CMS plants under different lighting regimes and photoperiods, including light emitting diodes (LEDs) and high-pressure sodium (HPS) lights. In the first set of treatments, which used a 16- hour photoperiod and moderate light intensity (150-250 pmol / m2 / s PPFD) resulting in a 12-17 Daily Light Integral (DLI) (moles / m2 / day), we observed a dramatic drop in the rate of haploid formation in the SY58 CMS line. Most heads had zero haploids, and all experiments averagedless than 5 haploids per head for all treatments. A second round of experiments were performed using higher intensity LED lights with a 1 :3 Blue to Red ratio, at 400-600 PPFD at a 14-hour photoperiod (a DLI of about 25 mol / m2 / d). In this test, the haploid rate remained low, averaging around 10 to 15 haploid seeds per head. In contrast, plants grown in a tent house in the spring and early summer, which has abundant sunlight plus HPS supplemental light, were able to produce well over 60 haploids per head.

[0169] We grew SY58 CMS in Phytotron growth chambers that yielded a record 130 haploids per head. The light intensity was 800 PPFD with a 16-hour photoperiod using a mixture of incandescent and metal halide lights. Fafard Sunshine® Mix #1 (https: / / www.sungro.com / en- ca / professional-product / sunshine-mix-1 / ) was the soil substrate and JR Peter’s Jack’s Professional® LX fertilizer “Jack’s” (https: / / www.jrpeters.com / 15-5-15-ca-mg-lx) was provided twice a week with watering (diluted to 200 parts per million (ppm) nitrogen). Compared to high pressure sodium (HPS) and light emitting diodes (LED) lights, metal halides come closest to mimicking sunlight. After seeing these surprisingly high numbers of haploid seed per head, we noted that all prior trials of SY58 to induce haploids either used LED or HPS supplemental lights. We also noted that prior trials in spring (when there is extra sunlight) had more haploids than trials in the winter. We also observed higher levels of light were not sufficient to enhance haploid seed rate on its own. When we matched the light intensity in a separate experiment grown in a greenhouse (800 PPFD at a shorter, 14-hour photoperiod), the yield from SY58 averaged only 30 haploid seeds per head. This is more than 4x lower than what was observed in the growth chambers. One difference between the two trials was the supplemental light: HPS lights were used in the greenhouse compared to metal halides and incandescent light in the chambers. HPS lights generally lack the shorter wavelengths (blues and UV) of the light spectrum. However, the soil type and fertilizer regime were also different between the two experiments, so the level of importance of the light source was unclear without further experiments.

[0170] From this information, it appeared that both the intensity and spectrum of light were important to high rates of haploid seed set. To understand this further, SY58 CMS plants were grown in a LED chamber with typical plant settings of a 3: 1 ratio of red to blue, which is purportedly most optimal for photosynthesis since it reflects the relative use of light during photosynthesis by the plant’s photoreceptors. We compared this typical LED chamber to a spectrum that is more like sunlight and what is achieved with metal halides (i.e., a broader and more even spectrum with a balance closer to 1 :2 red to blue. To ensure enough light wasprovided, we matched the 800 PPFD intensity and 16-hour photoperiod to what had been delivered at the Phytotron growth chambers for a total 46 DLI, in both the typical LED treatment and the “blue-enhanced” LED treatment.

[0171] Against our expectation, the broader spectrum including ‘enhanced’ blue light yielded fewer haploids per head (24) than the typical LED spectrum (37). Before starting this experiment, we sought to evaluate other variables between the Phytotron growth chambers and the greenhouse, including soil utilized in the Phytotron growth chambers (Fafard Sunshine® mix #1 [SS# 1 ]), daily hand-watering, and fertilizing two times per week with Jack’s fertilizer at a final dilution of 200ppm nitrogen, rather than daily fertigation. We also maintained the same 60% relative humidity. Despite efforts to copy the Phytotron growth chamber’s process and mimicking the broader, metal halide-like spectrum, the overall numbers of haploids remained 4x lower than expected in both light treatments. Therefore, while anecdotal data was pointing towards a broad light spectrum being important, we did not see that hypothesis validated in this particular experiment. We also concluded that higher blue light, Fafard SS# 1 , and reduced use of Jack’s fertilizer were likely not the critical elements driving the higher rate of haploid seed set in the Phytotron growth chambers.

[0172] To further explore these variables, we used a greenhouse room with high light conditions (800 PPFD, again with HPS + sunlight), and set up a 2 x 2 matrix to compare soil and fertilizer. We tested two soil types: Fafard SS#1 versus Fafard 3B (MM830) (https: / / www.sungro.com / professional-product / metro-mix-830 / ). We also compared hand fertilizing 2x-per week using Jack’s versus daily fertigation using General Hydroponics Floraseries® “Flora” (https: / / generalhydroponics.com / products / floraseries / ). The overall composition of these fertilizers was similar. The primary difference between the fertilization regimes is that the “Flora” method used fertigation - that is, the fertilizer was delivered daily in every watering rather than only twice per week (Table 13). The haploid yield in this trial was again disappointing, with all treatments resulting in fewer than 40 haploid seed per head. However, we did observe a clear benefit of using the reduced fertilizer regime protocol compared to the daily fertigation. Soil type did not make an impact on haploid yield, but switching to a reduced fertilizer more than doubled the haploid yield under both soil types. Interestingly, the plants with a reduced fertilizer regime did not have any height difference, but had lower biomass overall, with much smaller leaf area, resulting in less shading.Table 13. Testing the impact of soil and fertilizer regime on sunflower haploid seed yield.

[0173] All treatments in Table 13 were still far below what had been obtained in the Phytotron growth chambers. To understand why, we returned to the light spectrum, photoperiod, and intensity. We first sought to determine the optimal light intensity for haploid yield in the Phytotron growth chambers. As shown in Table 14, the haploid yield peaked when the SY58 CMS plants were grown under a very high intensity of light (1200 pmol / m2 / s), a striking 199 haploid seeds per head. The haploid yield dropped off at 850 and 1400 pmol / m2 / s PPFD. Generally, 1200 pmol / m2 / s of light is considered more than the plant can use for photosynthesis, so this may be considered a high light stress treatment.

[0174] The trial for Table 12 used HPS lights which have a narrower spectrum compared to metal halides, but we previously ruled out the importance of blue light during the wide spectrum LED experiment. One key component of the metal halide spectrum that is missing from HPS and LED lights is UV. UV from the sun is also significantly blocked by the glass in the greenhouse room (we measured 4000 uW / cm2UV lighting power density on a sunny day outside the greenhouse and only 300 uW / cm2under the glass). To measure the impact of UV on haploid seed set, we ran a UV-block experiment in the Phytotron growth chambers using plastic acrylite OP3 (https: / / www.acrylite.co / products / our-brands / acrylite-gallery / uv- filtering). The acrylite OP3 removed 96% of the UV light inside the growth chamber from reaching plant level when set up beneath the overhead lights. We measured 2800 uW / cm2UV lighting power density is received by plants under the normal full spectrum at 1200 PPFD (FIG. 12A), whereas just 100 uW / cm2UV is received under the acrylite OP3 (FIG. 12B). This block reduced the level of visible light by about 10%. Sufficient gaps in the plastic ensured airflow in the chamber, and environmental readings did not change after installation. The UV block reduced haploid yield by approximately 50%, from 199 to 96.2 haploid seed / head (Table 14).Table 14. Testing the impact of light intensity on sunflower haploid seed yield.

[0175] A trial was run to replicate the most favorable lighting conditions in the greenhouse at a different location. This included the addition of ten Philips metal halide 400 UNV bulbs and modification of photoperiod to 16-hour daylength. The results demonstrated that due to facility constraints, the most favorable lighting conditions were unable to be met.

[0176] The following growth conditions and protocols were used in the Phytotron experiments:

[0177] Seeds were sown in 24 cell trays. Each cell is 8x6 cm at the surface and 5.5 cm deep. Unless otherwise requested as part of an experiment, all soil used was Sunshine mix 1 (SS#1) (https: / / www.sungro.com / professional-product / sunshine-mix-l / ). Sunflower seeds were sown in the soil 1 cm below the surface. Trays were placed in a phytotron room at the following conditions (Table 15).Table 15. Sunflower Germination and Growth Conditions.

[0178] Seedlings emerged about 1 week after sowing. Between 10-14 days after sowing, seedlings were transferred to larger 7.5 -liter pots in the same phytotron. The larger pots were filled with SS#1 soil. The pots have a top diameter of 8 inches, bottom diameter of 7 inches, and a height of 93 / 4 inches. Irrigation was managed through hand watering with “Jack’s” 15-5- 15 cal-mag fertilizer provided in the water at a final concentration of 200ppm two days per week and watered with RO water the other days of the week. No pests were present, so no controls were applied to the plants or soil. Ants in the growth chambers were treated with Terro ant bait placed on the floor.

[0179] The following growth conditions and protocols were used in the greenhouse experiments:

[0180] Sunflower seeds were sown in 32-well germination trays with a well size of 6.35 cm X 6.35 cm. The tray was filled with Fafard 3B soil. Sunflower seeds were sown in the soil 1 cm below the surface. Trays were placed in a glasshouse room at the following conditions (Table 16).Table 16: Sunflower germination and plant growth conditions in the greenhouse.

[0181] Seedlings emerged about 1 week after sowing and were transferred to larger 1 ,2-liter pots in the greenhouse approximately 21 days after sowing. The larger pots were filled with Fafard 3B soil. Plants continued to be grown using the same conditions shown in Table 16. Irrigation was managed through a drip irrigation system with fertilizer provided in the water (Fertigation), using daily “Flora” fertilizer. Pests, including thrips, were controlled by a pesticide spray, as needed.Example 6. Separating haploid seeds from non-haploid seeds.

[0182] Blocking and / or limiting fertilization usually leads to seed abortion, but haploid seeds can develop. Intact haploid seed can be picked by hand, but this is time-consuming and labor intensive. We developed several ways to select haploid seeds in a more efficient manner based on the physical differences between haploid seeds and non-developed seeds (e.g., weight, buoyancy, size, and appearance). For each method, all seeds were shelled from the flower head. Based on the observation that small haploid seeds are heavier than non-developed seeds, a seed blower (CFY-II Electric Seed Blower by Zhejiang TOP Cloud-Agri Technology Co. Ltd.) was used to collect haploid seeds in a high throughput way (FIG. 4A). Similar seed blowers can also be used, such as South Dakota Seed Blower (https: / seedburo.com / collections / seed- blower / products / 3430) or Oregon Seed Blower (https: / / www.hoffmanmfg.com / products / oregon-seed-blower-en). Similarly, differences in buoyancy in liquid can be used to pick haploid seeds, which will sink. Based on the smaller size of haploid seeds, they can also be selected by grid, e.g., with a 2 mm size filter (FIG. 4B). Haploid seeds can also be selected via micro-computed tomography (micro-CT), by which clear difference in seed structure can be observed. Diploid seeds have well-developed embryo, while haploid seeds have more than 1 distorted meristems. Nuclear Magnetic Resonance Spectrometry (NMR) can also be used to assess oil signal of intact haploid seeds and pick out from non-developed seeds. Lastly, markersin hull, seed coat, cotyledon can be used to pick haploid seeds, such as RFP, CFP, GFP, anthocyanin marker, developmental markers (See FIG. 4C).Example 7. Regeneration of haploid seeds.

[0183] Haploid seeds obtained from above-described methods can be sowed in soil directly and grow into normal haploid plants with low frequency of l%-30%. The normal plant means seedling goes to normal reproductive stage. Large numbers of haploid seeds produced from above-described methods were abnormal without double fertilization, including haploid seeds containing only axillary bud meristems, or meristematic cells or tissues without apical and root meristems, which cannot be developed into normal haploid plants via germination in soil and / or germination medium. During in vitro culture, two methods, a single-cycle regeneration method and a two-cycle regeneration method, can be used to rescue abnormal haploid seeds such that normal haploid plants can be obtained from those seeds. These methods induce redifferentiation via organogenesis and regenerate buds / shoots from meristematic cells / tissues of abnormal haploid seeds using plant growth regulators under in-vitro culture conditions. The methods are further described below.

[0184] For mature haploid embryo extraction and sterilization, haploids seeds were rinsed 2 times with sterile water and soaked in sterile water for 3 hours to facilitate mature embryo extraction. Seed pericarps were removed by hand and the isolated mature embryos were dried in a petri dish overnight. Mature embryos were rinsed with 75% ethanol, with shaking by hand for 30 seconds. Mature embryos were soaked in sterilizing solution (10% (v / v) sodium hypochlorite solution, Sigma catalog # 239305 500ml; Chlorine 4 - 4.99%) with 1 drop of Tween 20 per 50 ml sterilizing solution for 15 minutes with shaking at approx. 110 rpm. Mature embryos were rinsed with sterile water 5 to 6 times to remove residual sterilizing solution. The sterilized embryos were placed in a plate with 3-5 sheets of sterile papers to dry the excess water.

[0185] For embryo culture, embryos were placed onto 90 x 25 mm petri dishes with 50 ml SF-SIM19 medium per plate (Table 17), and the radicle end of mature embryos were placed into medium and cultured at 25°C in the dark for 5 days. Five days after culture, the mature embryo culture was moved to a growth room at 25°C, under 16 hours (light) / 8 hours (dark) photoperiod with light intensity of approximately 7500 Lux for approximately 4 to 7 days. Then, embryos were transferred to 88 mm diameter x 76 mm high clear plastic culture boxes (4 per box) with 100 ml of SF-SIM19 medium (Table 17) and cultures were placed in a growthroom at 25°C, under 16 hours (light) / 8 hours (dark) photoperiod with light intensity of approximately 7500 Lux for up to 4 weeks. Every two weeks sub-culturing was needed due to sunflower’s rapid growth. In the meantime, some normal plants or shoots were generated. As soon as normal plants were generated, they were transplanted into the greenhouse. Normal shoots were transferred to a rooting media, SF-R4 medium (Table 17), to produce roots (4 shoots per box) for approximately 2 to 3 weeks.

[0186] The two-cycle regeneration method has an additional cycle that is shoot proliferation, following by rooting, as compared with the single-cycle method (FIG. 5). Each nodal section (0.5 cm in length) was cut above and below junction of leaf and main stem from normal shoots or normal plantlets obtained from above single-cycle method, and the nodal sections were placed on 88 mm diameter x 76 mm high clear plastic culture boxes with 100 ml of SF-SIM19 medium. Cultures were placed in a growth room at 25°C under 16 hours (light) / 8 hours (dark) photoperiod with light intensity of approximately 7500 Lux for 2 to 3 weeks. Once normal shoots were approximately > 1.5 cm in length, they were transferred to SF-R4 rooting medium to produce roots (4 shoots per box) for approximately 2 to 3 weeks.Table 17. Medium compositions.

[0187] With the single-cycle method, 91% of normal plants and / or shoots were obtained from SY54 haploid seeds derived using medium SF-SIM19 (FIG. 6, Table 18). With the two- cycle method, 200% of normal plants and / or shoots were obtained from SF54 haploid seeds using medium SF-SIM19 (FIG. 6, Table 18). The percentage is determined by dividing the number of normal plants obtained by the number of haploid seeds that were initially grown in the rooting medium to obtain these normal plants. In theory, two normal haploid seedlings regenerated from one haploid seed. After transplanting the normal plants into the greenhouse, 88.9% of the generated plants survived and 87.5% of the plants that were haploids; P l, P_2, P_3, P_4, P_5, P_6, P_7, and P_8 are exemplary haploid plants (FIG. 6).Table 18. Effect of mediums (plant growth hormones, concentration, and nutrients) on percentage of normal shoot and plant generation from SY54 haploid seeds derived using singlecycle and two-cycle methods.Example 8. Chromosome doubling in generalA. Haploid doubling process in general

[0188] Haploids produced using the methods disclosed herein can be treated to increase the chromosome number to produce the doubled haploids. This process is referred to herein as a doubling process (“chromosome doubling” or “doubling”).

[0189] One exemplary method of chromosome doubling includes the steps of (i) germinating sunflower seeds to form sunflower seedling and growing the sunflower seedling until VE stage (a stage where two cotyledons are fully expanded, but the first true leaf is not elongated yet), (ii) merging the whole seedling or whole apex and axillary meristems into the chemical solution for several minutes (5 seconds - 48 hours depending on sunflower genotype) or dripping the chemical solution on seedlings between two cotyledons to cover the region of apex and axillary meristems; (iii) removing residual formulation from seedlings with water wash, if needed;covering seedlings with domes to decrease formulation evaporation; and (iv) checking ploidy level in newly extruded tissues, such as leaves.B. Formulation induced chromosome doubling in sunflower using colchicine

[0190] Colchicine is a commonly used doubling agent. In this experiment, SY58 diploid seedlings were utilized. All concentrations (50 pM-200 pM) tested are functional in doubling (Table 19). The colchicine concentrations can extend from IpM to lOmM depending on sunflower germplasm. High concentrations of colchicine tend to be toxic to plant development and survival rates.Table 19. Testing the doubling effect of colchicine in different concentrations.C. Formulation induced chromosome doubling in sunflower using Oryzalin and Trijluralin

[0191] Some herbicides can affect normal mitosis via different mechanisms, including dinitroanilines (oryzalin and trifluralin), phosphorothioamidates (aminoprophos-methyl or APM), benzamides (pronamide), carbamates (chlorpropham and isopropyl N-3 -chlorophenyl carbamate) and others. Oryzalin and APM can bind to tubulin, inhibiting microtubule polymerization and promoting depolymerization of the anaphase spindle. Trifluralin disrupts the spindle microtubules in a similar manner to colchicine. Trifluralin, oryzalin, APM and other similar herbicides have potential to double ploidy level in sunflower.

[0192] In one experiment, a doubling agent comprising Oryzalin and / or Trifluralin was dripped onto shoot apical meristem (SAM) or the whole SAM was soaked in the doubling agent of VE stage sunflowers. The composition of the doubling agent solution is shown in Table 20.Table 20. Compositions of doubling agent solutions

[0193] Table 21 shows the results of using Oryzalin and Trifluralin to induce chromosome doubling in haploid and diploid sunflower seedlings. The double haploids were confirmed by flow cytometry and phenotyping (FIG. 7). Meanwhile, some haploid sunflower seedlings after treatment were triploids and tetrapioids.Table 21. Oryzalin and Trifluralin as doubling agents in sunflowerExample 9. Formulation and delivery of the doubling agents.A. Targeted delivery of doubling chemicals onto / into meristem

[0194] Targeted delivery can be achieved via dripping doubling agent(s) directly onto / into the shoot apical meristem (SAM). Dripping can be achieved by pipette, pipet, needle, sprayer, nebulizer, et al. Meristem includes shoot apical meristem and axillary meristem.

[0195] Beyond dripping, placing a doubling agent in the semi-solid emulsion was developed as another targeted delivery method.

[0196] The semi-solid emulsion consists of the following components: one or more doubling agents dissolved in an organic or inorganic solvent, an aqueous phase comprising water or buffer, Krytox™ fluorinated oils, hydrofluoroether, and other components for enhancedpenetration, stability, and / or doubling efficiency (for example, DMSO, acetone, glycerol, Sil wet® L-77, and the like).

[0197] The semi-solid emulsion contains hydrofluoroethers, Krytox™ fluorinated oil, and an aqueous phase. Hydrofluoroethers are hydrophobic and immiscible. Krytox™ fluorinated oil is not water-soluble, but the moles contain a water-soluble group (-COOH), which can trap water moles through strong hydro-bonds. Krytox™ fluorinated oil also contains groups soluble to hydrofluoroether oil.

[0198] If the mixture of three components is in proper ratio (the ratio varies in different Krytox™ fluorinated oil and hydrofluoroethers - one example is illustrated in Table 22), the moles of these components will cross-link to each other. The system exhibits a semi-solid property, combining the behaviors of liquids and solids in a soft matter. The aqueous phase, including the doubling chemicals, are entrapped into the network mesh. If HFE7500 is used as hydrofluoroether oil, and Krytox™ 157 FSH is used as Krytox™ fluorinated oil, the semisolid matter will be formed in the following ratio: 70% volume of water, 30% volume of HFE7500, and 1-3% (w / v) Krytox™ 157 FSH.Table 22. Chemical components in the doubling formulation and controls

[0199] The semi-solid emulsion disclosed herein is relatively stable, but the stability is only temporary. The liquid with doubling agents is continuously released from the formations. The superspreading surfactants and hydrofluoroethers in the formulation assist doubling agents topenetrate in plant tissues. The emulsion does not contain carbon sources, so there are no contamination issues from fungi and other microbial infection.

[0200] In some embodiments, a targeted delivery via a semi-solid emulsion was performed as follows: the formulation was applied onto the seedling center (seedling at cotyledon opening stage for sunflower) to cover the apex and axillary meristem regions. The treated seedlings were then kept in a high humidity condition for 2-3 days to avoid formulation evaporation (the formulations are sticky and temporarily stay on the meristem areas). The formulation enables continuous releases of doubling chemicals in meristem tissues. After treatment, the seedlings were washed with water showering to remove residual formulations. The seedlings were then transferred to the suitable growth conditions for seed production or for ploidy testing.

[0201] FIG. 11 A and FIG. 1 IB show the formulation and application of the formulation on the seedling. As shown in FIG. 11C and FIG. 11D, the formulation was able to stick on the plant tissues after application for more than 24 hours, which is the time of one cell division cycle, and penetration into the plant tissue was visibly discernable. The stickiness allowed the formulation to slowly penetrate into plant tissues. There was no obvious plant damage after application of the formulation.

[0202] Compared to the delivery method by directly dripping the doubling agent to the plant tissues, targeted delivery of the doubling agent in the semi-solid emulsion significantly increased doubling rate to 50% with 36 pM colchicine using SY54B diploid seedlings (Table 23). The semi-solid emulsion method also increased the rate of haploid doubling using 30 pM colchicine from 33% to 50% compared to control which was formulated with 1.5% DMSO only (Table 23). The semi-solid emulsion method also enhanced the doubling of haploids that were treated with colchicine plus trifluralin (Table 23). Trifluralin as the additive for colchicine can enhance doubling efficiency (Table 23).Table 23. Doubling results of different target delivery methods using SY54 drip method onto the meristem.B. Chemicals to facilitate penetration, stabilize the formulation and enhance doubling efficiency

[0203] Chemicals in doubling recipes can facilitate penetration, stabilize the formulation, and enhance doubling efficiency as helpers, which include, but are not limited to, DMSO, acetone, Silwet® L-77, glycerol, and Tween® 80.

[0204] Acetone is an organic compound, the simplest and smallest ketone. Acetone is miscible with water and serves as an important organic solvent. Acetone is used to dissolve chromosome doubling reagents, such as Trifluralin, colchicine, Oryzalin and amiprophos methyl. Hydrofluoroether is an excellent component to penetrate plant tissues. However, hydrofluoroether is a hydrophobic liquid and it is incapable to carry water and other chemicals. We found acetone is miscible to hydrofluoroether, so we believed acetone may be able to emulsify chemicals into hydrofluoroether liquid to form a stable three-phase emulsion. We assessed the impact of acetone on the stability of the hydrofluoroether phase in the formulation by testing four formulations: (1) a mixture comprising water, a yellow dye, and HFE7500; (2) a mixture comprising acetone, a yellow dye, and HFE7500; (3) a mixture comprising water, a red dye, and HFE7500; and (4) a mixture comprising acetone, a red dye, and HFE7500. We chose dyes because they are visible in the solution and easy to evaluate the solubility. We mixed the components, including HFE7500, water, and fluorescein together. Fluorescein dissolved into water, but the water and dye were insoluble in HFE7500. HFE7500 stayed in the bottom while the water / fluorescein biphase floated on the top. However, when we replacedwater with acetone, three phases mixed well and formed a uniformed emulsion. To verify the stability of the emulsion was not chemical-specific, we replaced fluorescein with another chemical dye called Allura Red AC (red color) and got very similar results. Allura Red AC with water could not mix with HFE7500, but Allura Red AC, acetone, and HFE7500 formed a uniform emulsion. Acetone can thus stabilize hydrofluoroether phase in an emulsion (FIG. 8).

[0205] Silwet® L-77 is a nonionic organosilicone surfactant co-polymer that has enhanced wetting and spreading characteristics. Silwet® L-77 can help doubling chemicals penetrate meristem tissue to increase doubling efficiency. As shown in Table 24, 0.05% Silwet® L-77 significantly increased the doubling rate to 18%.Table 24. Doubling rate with and without Silwet® L-77D: Production of sunflower doubled haploids through application of formulation on haploid- sunflower meristem regions(1) The process• Formulation components are listed in the Table 23. In this experiment, we used 30uM colchicine as the doubling agent.• Haploid sunflower seedlings: sunflower haploid seeds were sown into small soil pots in flat trays. After germination and two cotyledons were fully expanded, we applied the formulation before the first true leaf was elongated.• Formulation application: dropped 60 pl formulation on the meristem region and covered the apex as well as axillary meristem region.• Two days after the treatment, washed the seedling with water showering to remove residual chemicals.• Transplanted into big pots for seed production. Did not remove branches. Kept the primary stem and branches for seed production. The primary stems and the branches have the same chance to produce doubled haploid seeds.(2) The results: 9 haploid plants were used for the testing.• Three plants only had main head and they were fertile.• Four plants had both main head and the secondary heads. The seeds were primarily produced from secondary heads.• One plant did not produce seeds, indicating the plant was not doubled.• One plant died from treatment.• Overall, the doubling was successful with a rate of 7 / 9 (78%)Results are shown in Table 25 below.Table 25. Sunflower doubled haploids through application of formulation on haploid- sunflower meristem regions.• Primary heads doubling rate (>0 seeds): 5 / 9 =55%• Primary heads doubling rate (>50 seeds): 3 / 9 =33%• Secondary heads doubling rate (>50 seeds): 4 / 9 = 44%• Overall success rate (>50 seeds): 7 / 9 = 78%

[0206] In yet another experiment, nine (9) haploid plants were tested by applying the semisolid emulsion disclosed in FIG. 10, comprised of -80% water, 20% HFE7500 with Krytox™ 157 FSH 1-3% (Weightvolume). The results (Table 26) showed that eight (8) plants doubled in chromosome numbers, and seven (7) plants (shown in the first seven rows in Table 26) produced more than 50 seeds, all of which had been doubled (based on seed morphology).Table 26. Effect of formulations in producing DH seeds

[0207] The main heads and the secondary heads had similar fertility of about 30% - 55%. However, the overall success rate was greater with the combination of both primary and secondary heads. The doubled shoots showed more robust and dominant growth than haploid shoots, so doubled branches could produce decent heads to increase doubling efficiency.REFERENCES1. M. Todorova, et al., Doubled haploid production of sunflower (Helianthus annuus L.) through irradiated pollen-induced parthenogenesis. Euphytica Vol. 97: 249-254 (1997).2. Patil N.D. and Bhalerao S.R, In vitro haploid production in sunflower genotypes and homozygosity assessment using microsatellite marker. Research Journal of Biotechnology, Vol. 16 (6) June (2021).3. Coumans, M. & Zhong, D., Doubled haploid sunflower (Helianthus annuus L.) plant production by androgenesis: fact or artifact? Part 2. In vitro isolated microspore culture. Plant Cell, Tissue and Organ Culture, Vol. 41 : 203-309 (1995).4. Schneiter, A.A., and Miller, J.F. 1981. Description of Sunflower Growth Stages. Crop Science. Vol. 21(6): 901-903.5. Nandini, C., Shadakshari, Y.G., Karuna, K., et al. 2016. Evaluation of hybrids developed from diversified CMS lines for resistance to powdery mildew in sunflower (Helianthus annuus L.). Electronic Journal of Plant Breeding, Vol. 7(4): 947-952.6. Chen J., Jinguo, H., Brady, A.V., Jan, C.C. 2006. Molecular mapping of a nuclear malesterility gene in sunflower (Heliantus annuus L.) using TRAP and SSR markers. Theoritical Applied Genetics, Vol. 113(1): 122-7.Barman, H.N., Sheng, Z., Fiaz, S. et al. 2019. Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of 7MS’5 gene through CRISPR / Cas9 system. BMC Plant Biology, Vol.19: 109. Zhang, H., Xu, C., He, Y., Zong, J., Yang, X., Si, H., Sun, Z., Hu, J., Liang, W ., Zhang, D. 2013. Mutation in CSA creates a new photoperiod-sensitive genic male sterile line applicable for hybrid rice seed production. Proc Natl Acad Sci USA. Vol. 110(1): 76- 81. Pak, H., Wang, H., Kim, Y., Song et al. 2021. Creation of male-sterile lines that can be restored to fertility by exogenous methyl j asm onate for the establishment of a two-line system for the hybrid production of rice (Oryza sativa L.) Plant Biotechnology Journal Vol. 19: 365-374. Chraibi, Khalid M.B., Castelle, J.C., Latche, A., Roustan, J-P., Fallot, J. 1992. A genotype-independent system of regeneration from cotyledons of sunflower (Helianthus annuus L.), The role of ethylene. Plant Science, Vol. 86(2): 215-221. Bohanec, B. 2003. Ploidy determination using flow cytometry. In: Maluszynski, M., Kasha, K.J., Forster, B.P., Szarejko, I. (eds.) Doubled Haploid Production in Crop Plants. Springer, Dordrecht. Yao, L., Zhang, Y., Liu, C., Liu, Y., Wang, Y., Liang, D., Liu, J., Sahoo, G., Kelliher T. 2018. OsMATL mutation induces haploid seed formation in indica rice. Nat Plants. Vol. 4(8): 530-533. Liu, H., Wang, K., Jia, Z., Gong, Q., Lin, Z., Du, L., Pei, X., Ye, X. 2020. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Hgrotocterzwm-mediated CRISPR system, Journal of Experimental Botany, Vol. 71(4): 1337-1349. Kelliher, T., Starr, D., Su, X., Tang, G., Chen, Z., Carter, J., Wittich, P.E., Dong, S., Green, J., Burch, E., et al. 2019. One-step genome editing of elite crop germplasm during haploid induction. Nature Biotechnology. Vol. 37(3): 287-292. Zhang, W ., Wang, K., Lin, Z. S., Du, L.P., Ma, H.L., Xiao, L.L., Ye, X.G. 2014. Production and identification of haploid dwarf male sterile wheat plants induced by corn inducer. Botanical Studies. Vol. 55:26. Volk, G.M., Denoma, J., Hummer, K.E., Chen, K. 2021. Introduction of clean plants into tissue culture: Temperate crops. In: Volk. G.M. (ed.) Training in Plant GeneticResources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University.

[0208] It is to be understood that the figures and descriptions of the disclosure have been simplified to illustrate elements that are relevant for a clear understanding of the disclosure. It should be appreciated that the figures are presented for illustrative purposes and not as construction drawings. Omitted details and modifications or alternative embodiments are within the purview of persons of ordinary skill in the art.

[0209] It can be appreciated that, in certain aspects of the disclosure, a single component may be replaced by multiple components, and multiple components may be replaced by a single component, to provide an element or structure or to perform a given function or functions. Except where such substitution would not be operative to practice certain embodiments of the disclosure, such substitution is considered within the scope of the disclosure.

[0210] The examples presented herein are intended to illustrate potential and specific implementations of the disclosure. It can be appreciated that the examples are intended primarily for purposes of illustration of the disclosure for those skilled in the art. There may be variations to these diagrams, or the operations described herein without departing from the spirit of the disclosure. For instance, in certain cases, method steps or operations may be performed or executed in differing order, or operations may be added, deleted or modified.

[0211] Where a range of values is provided, it is understood that each intervening value, to the smallest fraction of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Any narrower range between any stated values or unstated intervening values in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of those smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the technology, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0212] In the foregoing description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the invention described in this disclosure may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known tothose skilled in the art have not been described in order to avoid obscuring the invention. Embodiments of the disclosure have been described for illustrative and not restrictive purposes. Although the present invention is described primarily with reference to specific embodiments, it is also envisioned that other embodiments will become apparent to those skilled in the art upon reading the present disclosure, and it is intended that such embodiments be contained within the present inventive methods. Accordingly, the present disclosure is not limited to the embodiments described above or depicted in the drawings, and various embodiments and modifications can be made without departing from the scope of the claims below.

Claims

WHAT IS CLAIMED IS:

1. A method of enhancing haploid production in a sunflower plant, wherein the method comprises: interrupting pollen development of pollen producing tissues; or outcrossing the sunflower plant with pollen from a second plant wherein the sunflower plant is grown under high light intensity and / or long daylength and a broad-spectrum including UV light; or outcrossing the sunflower plant with pollen from a second plant wherein the sunflower plant is grown under high light intensity and / or long daylength and a broad-spectrum including UV light.

2. The method of claim 1, wherein interrupting pollen development comprises treating a flower bud of the sunflower plant with a plant hormone.

3. The method of claim 2, wherein the plant hormone is gibberellic acid (GA) orjasmonate or ethylene or related antagonist(s).

4. The method of claim 3, wherein gibberellic acid (GA) is at a concentration of approximately 125 ppm.

5. The method of claim 4, wherein gibberellic acid (GA) is applied to sunflower plants at development stage R1 or later.

6. The method of claim 1, wherein the second plant is a monocot plant or a dicot plant.

7. The method of claim 6, wherein the monocot plant is corn, wheat, sugarcane, or rice.

8. The method of claim 6, wherein the dicot plant is tomato, soybean, spinach, pepper, broccoli, cauliflower, carrot, or potato.

9. The method of claim 1, further comprising treating flower heads of the sunflower plant after pollination with one or more of boron, indoleacetic acid (IAA), nicotinamide riboside, sucrose, or 2,4-Dichlorophenoxyacetic acid.

10. The method claim 1 , wherein the method further comprises emasculating flowers of the sunflower plant by removing anthers therein before pollen emission.

11. The method of any one of claims 1-10, wherein the method further comprises recovering seeds from ovaries of the sunflower plant, separating haploid seeds from non- haploid seeds.

12. The method of claim 11, wherein the method further comprises developing haploid seeds to generate haploid seedlings.

13. The method of claim 12, wherein the haploid sunflower seedling is derived from a cytoplasmic male sterility plant line or a genetic male sterility plant line.

14. The method of claim 11, wherein separating of haploid seeds from non-haploid seeds is based on seed weight, seed size, seed color, or seed structure.

15. The method of claim 11, wherein separating of haploid seeds from non-haploid seeds is based on seedling color.

16. The method of claim 11, wherein separating of haploid seeds from non-haploid seeds further comprising using seed blower, seed grid, photographing, and manual isolation.

17. The method of claim 11, wherein the method further comprises germinating haploid seeds in soil, germination paper, liquid medium, semi-solid medium, or solid medium.

18. The method claim 17, wherein the solid medium comprises one or more of SF-SIM16, SF-SIM18, SF-SIM19, SF-SIM20, SF-SEM2-1, or SF-SIM21.

19. The method of claim 18, wherein the method further comprises at least one round of regeneration process in the liquid medium, semi-solid medium, or solid medium.

20. The method of claim 12, wherein the method further comprises treating the haploid seedlings with at least one doubling agent to produce a diploid sunflower plant.

21. The method of claim 20, wherein the doubling agent is colchicine, an herbicide, or combination of colchicine and an herbicide.

22. The method of claim 21, wherein the herbicide is Oryzalin (4-(dipropylamino)-3,5- dinitrobenzenesulfonamide), Trifluralin (2,6-dinitro-N,N-dipropyl-4-(trifluoromethyl)aniline.

23. The method of any one of claims 20-22 wherein treating the haploid seedlings comprises dripping at least one doubling agent onto the shoot apical meristem of the haploid seedling.

24. The method of claim 11, wherein the method further comprises treating the haploid seedlings with a composition comprising a doubling agent(s) in a semi-solid emulsion.

25. The method of claim 24, wherein the semi-solid emulsion comprises a fluorocarbon ether polymer of polyhexafluoropropylene oxide, and a hydrofluoroether, wherein the fluorocarbon ether polymer is dissolved in hydrofluoroether droplets contained in the aqueous phase.

26. The method of claim 25, wherein the composition comprises 1-3% (w / v) of fluorocarbon ether polymer of polyhexafluoropropylene oxide and 30% hydrofluoroether (v / v).

27. The method of any one of claims 24-26, wherein the composition is free of carbon hydrate.

28. The method of any one of claims 23-27, wherein the doubling agent is colchicine or an herbicide or a combination thereof.

29. The method of claim 28, wherein the herbicide is Trifluralin or Oryzalin.

30. The method of claim 28, wherein colchicine is present in the composition in a concentration ranging from 0.5 pM - 200 pM inclusive.

31. The method of claim 28, wherein the colchicine is present in the composition in a concentration ranging from 30 pM - 36 pM.

32. The method of any one of claims 25-31, wherein the composition further comprises one or more additional chemicals for facilitating penetration and / or stabilizing the composition.

33. The method of claim 32, wherein the one or more additional chemicals are one or more of dimethyl sulfoxide (DMSO), acetone, glycerol, or non-ionic surfactants.

34. The method of any one of claims 24-33, wherein semi-solid emulsion confers a chromosome doubling rate of greater than 30% in cells of new tissue growth when applied on haploid sunflower seedlings.

35. A method for doubling chromosomes in cells in a haploid sunflower seedling, wherein the method comprises: contacting a seedling center of the haploid sunflower seedling with the composition of any one of claims 25-34; and maintaining the haploid sunflower seedling with the composition thereon in a high humidity condition for sufficient time for new tissue growth in the haploid sunflower seedling, the new tissue growth comprising diploid cells.

36. The method of claim 35, wherein the sufficient time is a period of time that allows for 1-2 cell division cycles in tissue of the haploid sunflower seedling.

37. The method of claim 35, wherein the seedling center comprises an apex region and / or an axillary meristem region of the haploid sunflower seedling.

38. The method of any one of claims 35-37, wherein the haploid sunflower seedling is at seedling emerge (VE) stage.

39. The method of any one of claims 35-38, wherein the haploid sunflower seedling is obtained from the method of claim 12.

40. The method of any one of claims 35-38, wherein the haploid sunflower seedling is derived from a cytoplasmic male sterility plant line, a thermo-sensitive genic male sterility (TGMS) line, a photoperiod-sensitive genic male sterility (PGMS) line, a chemical-sensitive genic male sterility (CGMS) line, a manually emasculated line, or double-fertilization limited line.

41. The method of claim 40, wherein the cytoplasmic male sterility plant line is of variety SY15 or SY54.

42. The method of any one of claims 35-41, wherein the method further comprises: washing the haploid sunflower seedling to remove residual composition from the seedling; and / or assessing ploidy levels in a newly formed tissue from the seedling.

43. The method of claim 13, further comprising growing the haploid seedling that has been contacted with the composition of any one of claims 24-33 to form a plant, thereby producing a diploid sunflower plant.

44. The method of claim 1, wherein the high light intensity is provided by sunlight and supplemented with metal halides, incandescent light, light emitting diodes (LEDs), or high- pressure sodium lights (HPS).

45. The method of claim 44, wherein the high light intensity is provided by sunlight and supplemented with metal halide bulbs.

46. The method of claim 45, wherein the high light intensity is between 500 and 1400 pmol / m2 / s photosynthetic photo flux density (PPFD).

47. The method of claim 46, wherein the high light intensity is provided for 12 to 18 hours resulting in a total daily light integral (DLI) of 65 to 75 mol / m2 / d.

48. The method of claim 1, wherein the method further comprises applying a fertilizer to the sunflower plant selected from the group consisting of Osmocote and Jack’s.

49. The method of claim 48, wherein the fertilizer is applied between two and seven times per week.

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