Non-gametocide chemical emasculation

Abstract

Provided is an improved method to facilitate breeding hybridization by using chemicals to prevent flower self-pollination in sunflower and other species, bypassing the need for manual emasculation. The high labor cost associated with the production of hybrid seeds in some key crops and some certain plant species is a persistent challenge, due to the laborious and highly specialized nature of the current method of manual emasculation.

Description

[0001]Docket no. PAT-109962-WO-SEC-1 NON-GAMETOCIDE CHEMICAL EMASCULATION FIELD OF THE INVENTION This invention relates to the field of plant breeding, and in particular sunflower breeding, and the production of hybrid plants, especially those varietal plants without a cytoplasmic male sterile system, using a non-gametocide chemical emasculation agent. BACKGROUND Hybridization plays a crucial role in the plant breeding process. However, a significant practical challenge in producing hybrid seeds is the prevention of self-pollination, as it would result in non-hybrid seeds. Currently, manual crossing remains the primary method for creating hybrids. This involves carefully removing anthers from flowers without affecting the female parts and transferring pollen from donor plants to the stigma. However, a major bottleneck in manual crossing is the process of emasculation, which is highly time- consuming and tedious, leading to low efficiency. For instance, developing hybrid sunflower seeds through traditional crossing methods presents significant challenges due to the unique structure and biology of sunflower heads. The composite nature of sunflower inflorescence, with hundreds of individual disc florets arranged in a spiral pattern on a single head, makes manual emasculation and controlled pollination extremely labor-intensive and time- consuming. Each sunflower head contains disc florets at various stages of development, requiring meticulous daily attention to emasculate and pollinate only the mature disc florets. This process typically allows for the treatment of only a single rim of disc florets per day, extending the crossing period over several days or weeks for a single head. The extended flowering period increases the risk of unintended cross-pollination from external sources, potentially compromising the genetic purity of the hybrid seeds. Furthermore, the proximity of numerous disc florets on the head creates a high risk of pollen contamination between adjacent disc florets, demanding extreme precision and care during the emasculation and pollination processes. This tedious and delicate work requires skilled labor and significant time investment, making large-scale hybrid seed production through manual methods economically challenging and logistically complex. Creation of male sterility is a breeding technique that facilitates hybridization by eliminating the need for manual emasculation. Male sterility in plants refers to the inability to produce or release functional pollen due to issues in the formation, development, or release of male Docket no. PAT-109962-WO-SEC-1 gametes. The discovery of male sterility has opened opportunities for developing highly efficient crossing processes in hybrid seed production. Genetic-controlled male sterility (e.g., cytoplasmic male sterility (CMS) and genic male sterility (GMS)) has been successfully employed in breeding systems for a few crop species to produce hybrid seeds. However, for most plant species, CMS or GMS systems are not readily available. Developing such systems is often a time-consuming process that requires identifying and maintaining male sterile and restorer lines, making it impractical for most researchers. Another approach involves inducing non-genetic and non-heritable male sterility using chemicals that render the male part of the plant abortive. Certain chemicals, known as chemical hybridizing agents (CHAs) or gametocides, can disrupt normal pollen development without affecting female functionality. Examples of such chemicals include Ethyl 4- fluorooxanilate, ethrel, 2,4-D, gibberellins, Maleic Hydrazide, Surf Excel, and sodium methyl arsenate. These chemicals have been used as chemical hybridizing agents in specific plant species, and they can have systemic effects on plant development. Moreover, the gametocides are not always 100% effective, resulting in contamination of hybrid seeds by self-pollinated seeds. In addition, these chemicals are often toxic to plants or have negative effects on plant growth and development. Non-systemic chemical-induced flower sterility refers to the application of chemicals directly to the flowers or reproductive structures of the plant, as opposed to applying them to the entire plant or using systemic chemicals that are absorbed and distributed throughout the plant. In the pursuit of producing hybrids, non-systemic chemical-induced flower sterility can serve as a valuable tool for plant breeders. Ideally, the chosen chemicals should selectively affect stamen development to prevent self-crossing, while leaving female fertility and overall plant development unaffected. Additionally, these chemicals should be cost-effective, environmentally friendly, and easy to apply. However, as so far, no such generic non- systemic chemicals have been identified for broad application in plant crossing. The key strategy in the pursuit of producing hybrids is to bypass the manual emasculation processes to increase the efficiency. In the present disclosure, presented is an approach to bypass the manual emasculation process through chemical treatment. Liquid chemicals are directly applied to the flower buds, affecting normal stamen development to prevent self- crossing. These chemicals are non-systemic, selectively preventing self-crossing only in the treated flowers while leaving female fertility and overall plant development unaffected. Docket no. PAT-109962-WO-SEC-1 SUMMARY Production of hybrid seeds is an essential step for traditional breeding and for modern plant biotechnology. The high labor cost associated with the production of hybrid seeds in some key crops and certain plant species is a persistent challenge, largely due to the laborious and highly specialized nature of the current manual emasculation method. Presented herein is an approach to achieve hybridization without emasculation. The approach has potential to aid in crossing and breeding by serving as an alternative method to facilitate breeding hybridization through using chemicals to prevent flower self-pollination in sunflower and other species, bypassing the need for manual emasculation. Fluorocarbons and their derivatives, including hydrofluoroethers, perfluorooctyl bromides, hydrofluoroalkyl esters, and fluorooctanoyl chlorides, are known for their hydrophobic properties, exceptional biological inertness, low phytotoxicity, low surface tension, and high fluidity. These chemicals are applied directly on flower buds before flower opening and quickly penetrate the floral buds, inducing morphological changes in male reproductive organs such as shortened anthers and filaments. This spatially separates the anthers and stigmas, preventing pollen deposition on the stigmas. Moreover, the treated flowers exhibit improper stamen development, leading to defects in anther dehiscence. This makes the application of liquid fluorocarbons or their derivatives during floral bud treatment an effective way to prevent self-crossing and facilitate the crossing process. It is important to note that the chemical treatment has little to no impact on female fertility. Additionally, these chemicals are non-systemic, selective to prevent self-crossing only in the treated flowers and leave non-treated flowers and overall plant development unaffected. The traditional hybrid agents are commonly known as male gametocides. However, this is the first reported instance of a chemical family that prevents self-crossing without acting as gametocides. These chemicals are biologically inert and do not cause pollen death. Moreover, due to their biological inertness, oxygen solubility, and hydrophobic nature, fluorocarbons and their derivatives have the potential to preserve pollen viability for an extended period of time. Presented herein is a method to emasculate a plant by applying a chemical emasculation agent to the flowers of a plant. The chemical emasculation agent herein is not a gametocide and is applied to the flowers of the plant at a desired developmental stage. In an embodiment, the chemical emasculation agent comprises a compound selected from the group consisting of fluorocarbons, hydrofluoroethers, perfluorooctyl bromides, hydrofluoroalkyl esters, and Docket no. PAT-109962-WO-SEC-1 fluorooctanoyl chlorides. In another embodiment, the chemical emasculation agent comprises a hydrofluoroether compound. The chemical emasculation agent may be HFE7500 or FC-40. The plants of the method may be a dicot selected from the group consisting of sunflower, soybean, tobacco, pepper, and tomato. In one embodiment, the plant is a sunflower. The chemical emasculation agent may be applied to sunflower at the R2 stage, the R3 stage, the R4 stage, both the R3 and R4 stages, or at all R2, R3, and R4 stages. In one embodiment, the sunflower is treated with the emasculation agent at a volume of 0.4 milliliters to 1.5 milliliters. In another embodiment, the dicot is tobacco, and the desired developmental stage is stage 4 – 5. Tobacco may be treated with the emasculation agent at a volume of 5 to 20 microliters. In yet another embodiment, the dicot is pepper, and the desired developmental stage is stage 4- 5. Pepper may be treated with 50 microliters of the emasculation agent. In another embodiment, the dicot is tomato, and the desired developmental stage is stage 4-5. Tomato may be treated with 50 microliters of the emasculation agent. In an embodiment, the emasculation agent of the method is applied in liquid form uniformly across the flower buds. In an embodiment, the emasculation agent is applied using a pipette. In the method, the female organ remains fertile. In another embodiment, the emasculation agent is non-systemic. In yet another embodiment, the emasculation agent application results in at least 90% sterility of the plant. Maintenance of pollen viability is a challenge for hybridization. After pollen is shed, its’ viability can rapidly decline. For instance, corn pollen exposed to hot and dry conditions can lose up to 80% viability within just one hour, becoming completely nonviable within two hours (Johnson R, Herrero MP. Corn pollination under moisture and high temperature stress. H.D. Loden, D. Wilkinson (Eds.), Proceedings of the 36th Annual Corn and Sorghum Industry Research Conference, Chicago, IL, December 9–11 (1981), pp.66-77). Pollen has a longer functional lifespan under relatively low temperatures and high humidity levels, as these factors influence the vapor pressure deficit between the pollen grains and the surrounding air, affecting the moisture content of the pollen. The rapid decrease in pollen viability under high temperature and low humidity conditions can be attributed to the loss of pollen water content. Studies have shown that maize pollen viability is almost lost when the moisture content drops below 30% (Fonseca A, Westgate M. Relationship between desiccation and viability of maize pollen. Field Crops Research.2005 Nov 15; Vol.94:pg 114-125). Therefore, preventing the loss of pollen moisture is crucial for extending its viable Docket no. PAT-109962-WO-SEC-1 period. However, it should be noted that pollen is quickly killed when immersed in water or most other liquids. Fluorocarbons (FC) play a crucial role in maintaining pollen viability by preventing moisture loss. Upon application, they quickly cover the surface of the pollen grains owing to their high fluidity and very low surface tension. As these chemicals are highly hydrophobic, they envelop the pollen grains without penetrating them. Consequently, the pollen grains retain their moisture content. Moreover, these chemicals aid in the survival of pollen by providing oxygen. Oxygen is a basic requirement for metabolic activities in viable pollen grains. Perfluorocarbons (PFC), known for their high oxygen solubility, have been used in liquid ventilation therapy to improve lung function in patients (see Arni S, Necati C, Maeyashiki T, Opitz I, Inci I. Perfluorocarbon-Based Oxygen Carriers and Subnormothermic Lung Machine Perfusion Decrease Production of Pro-Inflammatory Mediators. Cells.2021 Aug 30;10(9):2249. doi: 10.3390 / cells10092249. PMID: 34571898; PMCID: PMC8466246). When pollen grains come in contact with these liquid chemicals, they do not suffocate due to the oxygen-rich nature of fluorocarbons and their derivatives. BRIEF DESCRIPTION OF DRAWINGS FIG.1A shows an untreated R4 stage sunflower head. FIG.1B shows an untreated sunflower head which has produced seeds normally. FIG.1C shows a sunflower head that was treated with HFE7500 at R4 stage and is completely sterile. FIG.2A shows an untreated sunflower head at stage R5 with normal anthers and stigmas. FIG.2B. shows the release of pollen from the anthers of the untreated sunflower head at stage R5 that subsequently attached to the stigma. FIG.2C shows a HFE7500 treated sunflower head at stage R5 displaying only the stigma. FIG.2D shows a close-up of a HFE7500 treated sunflower head at stage R5 with stigmas but no anthers or pollen grains. FIG.3A shows an untreated sunflower disc floret on the left in comparison to a HFE7500 treated disc floret on right exhibiting normal female organs (ovary, style, and stigma), but the anthers are not visible, and no pollen grains are attached to the stigma. Docket no. PAT-109962-WO-SEC-1 FIG.3B shows an untreated sunflower disc floret on the left in comparison to a HFE7500 treated disc floret on right, but without the ovaries. This view shows no visible anthers or pollen grains, providing a closer look at the morphological differences between treated and non-treated disc florets. FIG.4A shows an untreated sunflower disc floret exhibiting longer filaments and a visible anther cylinder emerging from the top of the corolla. Pollen grains have dehisced and are deposited on the stigma. FIG.4B shows a sunflower disc floret that was treated with HFE7500 at stage R4, exhibiting shorter filaments and anthers in comparison to the untreated floret in FIG.4A. FIG.4C shows a sunflower disc floret that was treated with HFE7500 at stage R, where the anthers remain indehiscent with pollen grains inside. FIG.5A shows an entire sunflower head that was treated with HFE7500 at stage R4, where the top half was pollinated and the bottom half of the same sunflower was not pollinated. FIG.5B shows a sunflower head that was treated with HFE7500 at stage R4, where the top half was manually pollinated and the disc florets successfully produced seeds. The bottom half of the same sunflower head was left unpollinated and shows that it remained sterile. FIG.6A shows a sunflower head at R1 stage just prior to chemical treatment. FIG.6B shows normal flowering and anthesis on a sunflower head at R5 stage that was treated with HFE7500 at stage R1. FIG.6C shows normal seed set on a sunflower head after HFE7500 treatment at stage R1. FIG.7A shows a sunflower head at R2 stage just prior to chemical treatment. FIG.7B shows a sunflower head that was treated with HFE7500 at stage R2, where the stamens in the outer rims of disc florets are suppressed and the anthers and pollen are not visible. FIG.7C shows a sunflower head that was treated with HFE7500 at stage R2 where the chemical treatment is ineffective on the inner rims of disc florets. The disc florets develop normal flowering and undergo anthesis, releasing pollen grains around the sunflower head. FIG.7D shows a sunflower head that was treated with HFE7500 at stage R2 which produced seeds. Docket no. PAT-109962-WO-SEC-1 FIG.8A shows a sunflower head at stage R3 just prior to chemical treatment. FIG.8B shows a sunflower head that was treated at stage R3 with HFE7500 where the chemical treatment suppressed the stamens in the outer and middle rims of disc florets. FIG.8C shows a sunflower head that was treated at stage R3 with HFE7500 where the chemical treatment was ineffective on the center disc florets as shown by the normal flowering and pollen release. FIG.8D shows a sunflower head that was treated at stage R3 with HFE7500 where some seed is still produced. FIG.9A shows an untreated sunflower head at R4 stage prior to chemical treatment. FIG.9B shows a sunflower head that was treated at stage R4 with HFE7500 where the chemical treatment has suppressed the stamen development. FIG.9C shows a sunflower head that was treated at stage R4 with HFE7500 where the chemical treatment has induced complete sterility. FIG.9D shows a sunflower head that was treated at stage R4 with HFE7500 that was manually pollinated and produced a substantial number of seeds. FIG.10A shows an untreated sunflower head at stage R5. FIG.10B shows a R5 stage sunflower head that was treated earlier with 0.5 mL HFE7500 at stage R4. FIG.10C shows a R5 stage sunflower head that was treated earlier with 0.8 mL HFE7500 at stage R4. FIG.10D shows a R5 stage sunflower head that was treated earlier with 1.0 mL HFE7500 at stage R4. FIG.11A shows a sunflower head that was treated at stage R4 with HFE7500 where the non- uniform chemical application results in only partial sterility. Pollen was released (indicated by arrows) where no chemical was applied. FIG.11B shows a sunflower head that was treated at stage R4 with HFE7500 where the non- uniform chemical application resulted in partial sterility. Pollen was released (indicated by arrows) where no chemical was applied. Docket no. PAT-109962-WO-SEC-1 FIG.12 shows a sunflower head at stage R5 after treatment with FC-40 at R4 stage where the stamens are not visible during flowering. FIG.13A shows a non-treated sunflower head used as a control demonstrating normal seed set. FIG.13B shows a sunflower head that was treated at stage R4 with 0.2 mL FC-40 and remained fertile for seed set. FIG.13C shows a sunflower head that was treated at stage R4 with 0.8 mL FC-40 and resulted in a high degree of sterility with significant reduction in seed set. FIG.13D shows a sunflower head that was treated at stage R4 with 1.0 mL FC-40 and resulted in total sterility with no resulting seed set. FIG.14 shows a sunflower head that was treated at stage R4 with FC-40, then pollinated with pollen from an untreated flower that resulted in seed set. FIG.15 shows a sunflower head where the treated zone (indicated by arrows) resulted in sterility after contact with FC-40 at stage R4. FIG.16A shows a Nicotiana benthamiana flower at stage 4. FIG 16B shows a Nicotiana benthamiana flower at stage 5. FIG.17A shows a tomato flower bud at stage 5 just prior to HFE7500 treatment. FIG.17B shows a non-developed fruit (left) or very small fruit (right) of a tomato flower bud that was treated with HFE7500 at stage 5. FIG.17C shows a normal size fruit from a non-treated tomato flower bud. FIG.18A shows a flower bud of pepper at stage 4 just prior to HFE7500 treatment. FIG.18B shows two very small fruits that developed from a flower bud of pepper that was treated with HFE7500 at stage 4. FIG.18C shows normal pepper fruit developed from non-treated flower buds. FIG.18D shows the inside of a very small fruit (as seen in FIG.18B) containing very few seeds inside, that developed from a flower bud of pepper that was treated with HFE7500 at stage 4. Docket no. PAT-109962-WO-SEC-1 FIG.19A shows untreated maize pollen with normal pollen tube growth after 8 hours of storage at room temperature. FIG.19B shows HFE7500 treated maize pollen exhibiting longer pollen tube growth compared to its’ respective control after 8 hours of treatment at room temperature. FIG.20A shows the germination of untreated maize pollen stored at 4°C for 48 hours. FIG.20B shows the pollen tube elongation of HFE7500 treated maize pollen which was stored at 4°C for 48 hours. DEFINITIONS 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. The terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. For example, the phrase “a cell” refers to one or more cells, and in some embodiments can refer to a tissue and / or an organ. Similarly, the phrase “at least one”, when employed herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of that entity, including but not limited to all whole number values between 1 and 100 as well as whole numbers greater than 100. 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. As used herein, the term “and / or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, B, C, and / or D” includes A, B, C, and D individually, but also includes any and all combinations and sub-combinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, one of more of the elements to which the “and / or” refers can Docket no. PAT-109962-WO-SEC-1 also individually be present in single or multiple occurrences in the combinations(s) and / or sub-combination(s). 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 of anther development, conversion of anthers into carpels or other organs, blocking filament elongation or anther dehiscence, or any other mechanism. The term “chemical hybridizing agents” or “male gametocides” refers to any agent that inhibits or destroys viable pollen formation. These agents are used to induce male sterility in plants to produce a male sterile line for use in hybridization crosses. The use of “chemical hybridizing agents” or “male gametocides” lead to “chemical-induced male sterility.” “Male gametocides” result in non-viable pollen. The term “chemical-induced male sterility” refers to the non-genetic method of inducing male sterility in plants. The development of the stamen, anthers, or pollen is inhibited or interrupted by the application of a “chemical hybridizing agent” or “gametocide.” 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. The term “cross-pollination” refers to the process of transferring pollen from the anthers of one plant to the pistil of a second plant to bring together the male and female gametes of two same or related species. “Cross-pollination” is used in the production of hybrid plants. It may include the practice of “manual crossing” where the transfer of pollen is done by hand with the use of tools including a brush, cotton swab or delicate instrument to place on the pistil of the second plant for pollination. The term “emasculation” or “manual emasculation” refers to the process of removing the stamens or anthers of a plant by physical removal. “Emasculation” may be done on one flower or floret of a plant to all flowers or florets of a plant. The term “environmentally induced male sterility” refers to the condition of male sterility in a plant that results from exposure to environmental conditions such as temperature, photoperiod, duration and quality of light, as well as other stresses from the soil. Environmental stresses impact normal development of the male reproductive organs of the stamen or pollen. Docket no. PAT-109962-WO-SEC-1 The term “cytoplasmic male sterility” refers to a maternally inherited trait that prevents the production of functional pollen while maintaining female fertility in the same plant. The male sterility cause by interactions of the nuclear and mitochondrial genomes. The term “genic male sterility” refers a trait in plants where male reproductive organs (pollen or stamens) are impaired due to mutations in nuclear genes, leading to male sterility. As used herein, the term “elite line” or “inbred line” refers to any line that has resulted from breeding and selection for superior agronomic performance. An elite line has stable genetics, i.e., it is reasonably or nearly isogenic across its genome. Said another way, an elite line is reasonably or nearly homozygous for all alleles in its genome. The term “flowers,” “florets,” or “flower buds” refers to the reproductive structure of a plant that naturally contains both male and female reproductive structures including stamens and pistil. “Flowers,” “florets,” or “flower buds” may refer to the floral structure at various stages of development and include non-reproductive tissues of petals and sepals in addition to the stamens and pistil. The term “disc florets” refers to the small, tubular flowers found in the center of the sunflower head referred to as the disc. These “disc florets” contain both male and female reproductive structures and can produce seed when fertilized. The term “gene” refers to a hereditary unit including a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or train in an organism. The term “genotype” and variants thereof refers to the genetic composition of an organism, including, for example, whether a diploid organism is heterozygous (i.e., has two different alleles for a given gene or QTL) or homozygous (i.e., has the same allele for a given gene or QTL) for one or more genes or loci (e.g., a SNP, a haplotype, a gene mutation, an insertion, or a deletion). As used herein, the term “at least heterozygous” for a particular allele indicates that at least one copy of the allele is present. For example, a maize plant that is at least heterozygous for a HI allele of a gene has either one or two copies (i.e., is either heterozygous or homozygous) of the HI allele. The term “germplasm” refers to the totality of the genotypes of a population or other group of individuals (e.g., a species or plant line). The phrase “adapted germplasm” refers to plant materials of proven genetic superiority; e.g., for a given environment or geo-graphical area, while the phrases “non-adapted germplasm”, “raw germplasm”, and “exotic germplasm” Docket no. PAT-109962-WO-SEC-1 refer to plant materials of unknown or unproven genetic value; e.g., for a given environment or geographical area; as such, the phrase “non-adapted germplasm” refers in some embodiments to plant materials that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population. The terms “hybrid”, “hybrid plant”, and “hybrid progeny” in the context of plant breeding refer to a plant that is the offspring of genetically dissimilar parents produced by crossing plants of different lines or breeds or species, including but not limited to the cross between two inbred lines (e.g., a genetically heterozygous or mostly heterozygous individual). The phrase “single cross FI hybrid” refers to an FI hybrid produced from a cross between two inbred lines. 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. The phrase “inbred line” refers to a genetically homozygous or nearly homozygous population. An inbred line, for example, can be derived through several cycles of brother / sister breedings or of selfing. In some embodiments, inbred lines breed true for one or more phenotypic traits of interest. An “inbred,” “inbred individual,” or “inbred progeny” is an individual sampled from an inbred line. The term “inbred” means a substantially homozygous individual or line. An inbred line may also be referred to as a “parent line” when used in a breeding program. The term “male sterility” refers to the failure of plants to produce functional anthers, pollen, or male gametes, which is due to the malformation of male flowers or anthers or defective anther dehiscence of pollen. Plants that are male sterile can be used as a “male sterile line” in hybrid seed production. The term “non-systemic chemical induced flower sterility” refers to sterility in the flower of a plant that results from direct contact with a chemical agent. The term “offspring” plant refers to any plant resulting as progeny from a vegetative or sexual reproduction from one or more parent plants or descendants thereof. For instance, an offspring plant may be obtained by cloning or selfing of a parent plant or by crossing two parent plants and includes selfings as well as the F1 or F2 or still further generations. An F1 is a first- generation offspring produced from parents at least one of which is used for the first time as donor of a trait, while offsprings of second generation (F2) or subsequent generations (F3, F4, etc.) are specimens produced from selfings of F1s, F2s etc. An F1 may thus be a hybrid Docket no. PAT-109962-WO-SEC-1 resulting from a cross between two true breeding parents, while an F2 may be an offspring resulting from self-pollination of said F1 hybrids. “Phenotype” is understood within the scope of the present disclosure to refer to a distinguishable characteristic(s) of a genetically controlled trait. The phrase “phenotypic trait” refers to the appearance or other detectable characteristic of an individual, resulting from the interaction of its genome with the environment. A “plant” is any plant at any stage of development, particularly a seed plant. In the context of this disclosure, a plant refers to a sunflower, tomato, tobacco, or pepper plant. 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. “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. “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. 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 and tissues 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. The term “progeny” refers to the descendant(s) of a particular cross. Typically, progeny result from breeding of two individuals, although some species (particularly some plants and hermaphroditic animals) can be selfed (i.e., the same plant acts as the donor of both male and female gametes). The descendant(s) can be, for example, of the F1, the F2, or any subsequent generation. Docket no. PAT-109962-WO-SEC-1 The term “restorer line” refers to an inbred line used in plant breeding used to produce a hybrid which is male fertile. The “restorer line” carries one or more “restorer factor,” or “restorer allele” genes that restore male fertility in the progeny of a cross. The term “self-fertilization” refers to a fertilization process in which both the female gametophyte fuses with a male gamete from the same plant. The phrases “sexually crossed” and “sexual reproduction” in the context of the present disclosure refer to the fusion of gametes to produce progeny (e.g., by fertilization, such as to produce seed by pollination in plants). In some embodiments, a “sexual cross” or “cross- fertilization” is fertilization of one individual by another (e.g., cross-pollination in plants). In some embodiments the term “selfing” refers to the production of seed by self-fertilization or self-pollination; i.e., pollen and ovule are from the same plant. The terms “solvent” and / or “solution” refer to a liquid in which the flavonoid can be mixed. Examples include water, alcohol, propylene glycol, DMSO, Tris buffer, and other liquids available to a person skilled in the art. The flavonoid need not be dissolved in the liquid; a mixture or emulsion can be sufficient. 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. EXAMPLES Example 1: Fluorinated oils prevent self-pollination in sunflower Environmental stress, particularly water deficiency, can induce male sterility in plants while often preserving female fertility. This phenomenon, known as environmentally induced male sterility, occurs due to the heightened sensitivity of stamen and pollen development to stress compared to female organs. The method herein exploits this vulnerability by applying fluorinated oils to flower buds and creating localized abiotic stress. These hydrophobic compounds rapidly penetrate the buds, expelling water from delicate tissues and inducing short-term water stress coupled with physical impact. The high fluidity of fluorocarbons ensures the stress is transient, allowing female organs to recover while disrupting stamen development, thereby achieving male sterility. In the present experiment, two specific fluorinated oils: HFE7500 (for example, 3M™ Novec™ 7500 Engineered Fluid, herein referred to as HFE7500) and 3M™ Fluorinert™ FC- Docket no. PAT-109962-WO-SEC-1 40, herein referred to as FC-40, were selected for evaluation. These compounds are typically colorless, odorless, tasteless, low in toxicity, have low viscosity, and remain liquid at room temperature. They have been used in various industrial applications; however, they have not been used for inducing male sterility in biological systems. Our selection criteria prioritized environmental safety and cost-effectiveness. Both HFE7500 and FC-40 have zero ozone depletion potential (ODP) and are exempt from U.S. EPA and most state definitions of volatile organic compounds (VOCs) due to their negligible photochemical reactivity, thus not contributing to ground-level smog formation (See 3M™ Novec™ 7500 Engineered Fluid Product Information. September 2009 and 3M™ Fluorinert™ Electronic Liquid FC-40 Technical Data. September 2019). They are biocompatible and pose minimal risk for human use. Additionally, these chemicals offer a cost-efficient solution, making them suitable candidates for potential large-scale applications in inducing plant male sterility. (1) Induction of flower sterility in sunflower using HFE7500 To evaluate the ability of HFE7500 to induce flower sterility in plants, SYN-SF1 was cultivated in a controlled greenhouse environment. HFE7500 was administered at the R4 stage, during which the sunflower inflorescence begins to open (See FIG.1A). The treatment applied 1 mL of HFE7500 to the middle of the developing sunflower head with the use of a pipette. To confirm the chemical-induced sterility, precautions were taken by bagging the sunflower heads to prevent cross-pollination. At the maturation stage, the untreated plants exhibited complete fertility and produced self-pollinated seeds (See FIG.1B). In contrast, the sunflower heads treated with HFE 7500 at the R4 stage were entirely sterile (See FIG.1C). These results clearly demonstrated that the treatment could induce complete sterility for a sunflower head. (2) Chemical treatment resulted in stamen abnormality leading to flower sterility After applying the treatment of HFE7500 at R4 stage, we observed the treated and untreated disc florets during the blossoming R5 stage. We could not detect any significant morphological differences in the female organs between the treated and untreated disc florets. However, abnormalities were detected in the anthers of the treated disc florets. In the untreated disc florets, we clearly observed the release of pollen from the anthers that subsequently attached to the stigma (See FIG.2A and FIG.2B). However, the treated disc Docket no. PAT-109962-WO-SEC-1 florets displayed only the stigma (See FIG.2C and FIG.2D). Neither the anther nor the pollen grains were visible in the treated disc florets (See also FIG.3A and 3B). Disc florets were examined under a dissection microscope to determine why the pollen was not visible in the treated disc florets. Sunflowers have a unique reproductive structure where the stamens are arranged in a circular pattern (anther cylinder) around the central female reproductive structures. The normal sunflower anthesis process begins with rapid elongation of anther filaments, causing the anther tube to protrude above the corolla. Once extended, pollen is released within the anther tube. Subsequently, the style starts to elongate rapidly, pushing through the pollen-filled anther tube. As the style continues its growth, it pushes the stigma beyond the anther tube. The style's surface is covered with tiny sweeping hairs (also known as collecting hairs) that rake pollen out of the anther tube as it grows through. At the tip of the style, the stigma emerges from the anther tube. Initially, the stigma's two lobes remain closed, but once fully exposed, they open outward, becoming receptive to pollen for pollination. Chemical treatment of sunflower disc florets significantly alters the anthesis and pollination processes. Primarily, the treatment suppresses filament elongation, causing the anther cylinder to remain within the floral tube (See FIGs.4A, 4B, and 4C). The stamens, particularly the filaments, are notably shorter compared to untreated disc florets. The ovaries, styles, and stigmas develop normally in morphology and function. Rapid style elongation typically occurs during anthesis, allowing the stigma to push through the anther tube and emerge beyond the corolla top. The stigma's two lobes then open outward for pollen reception. Importantly, no pollen grains are released from the anther tubes, leaving the stigmas pollen-free. Microscopic observation reveals that anther dehiscence fails to occur as the stigma pushes through the anther tubes. The pollen grains remain within the anthers. Further dissection of treated disc florets shows that some anthers can dehisce a small number of pollen grains, but this occurs significantly later, after the stigma has already emerged from the anther tubes. Pollen germination tests on a medium plate confirm that these later-dehisced pollen grains retain their ability to germinate. However, trapped within the anther tubes, these viable pollen grains have no opportunity for pollination. In summary, chemical treatment effectively arrests stamen elongation and suppresses anther dehiscence, creating physical barriers that prevent pollination in treated sunflower disc florets. (3) The chemical treatment does not affect female organ fertility, allowing crossing without emasculation. Docket no. PAT-109962-WO-SEC-1 To ascertain that the observed sterility in the plants could be attributed solely to the male organs, the top half of sunflower heads were manually pollinated with healthy pollen obtained from untreated plants while the other halves of the sunflower heads remained unpollinated (See FIG.5A). Remarkably, the pollinated disc florets produced seeds while their unpollinated counterparts in the same heads remained sterile (See FIG.5B). These results unequivocally demonstrate that the implemented chemical treatments entirely inhibit self-fertilization while the female organs remain operative. (4) Chemical treatment induces disc floret sterility only during the R2-R4 stages and is effective only in the disc florets specifically from post-stamen formation through to near maturation. Sunflowers transition from vegetative to reproductive growth, progressing through stages R1 to R4 before the flower head opens. Disc floret development varies depending on the head's developmental stage and the disc floret's location within the same head. To evaluate the effectiveness of chemical induced male sterility at different development stages, chemical treatments were applied during sunflower stages R1 to R4. To assess whether the chemical treatments are effective on more than one sunflower germplasm, another sunflower line (SYN-SF2) was selected for evaluation. HFE7500 was applied in the amount of 1 mL using a pipette to the center of each sunflower head at developmental stages R1 through R4. (a) The chemical is ineffective on disc florets at R1 stage. At the R1 stage (See FIG.6A), the reproductive phase begins with the formation of a miniature floral head in the terminal bud, not yet visible from above. Disc florets are in early developmental stages, only observable through dissection. Disc florets in the outer rims show initial stamen primordia differentiation and anther sporogenous tissue development, while inner rim floret primordia remain undifferentiated. The chemical application resulted in no visible morphological or developmental changes, with floret flowering, anthesis, and seed production proceeding normally, comparable to untreated flowering heads. (See FIGs. 6B-6C.) (b) The chemical is effective only on disc florets at the outer rims of the sunflower head during the R2 stage. At this stage, the immature bud is visible from above the nearest leaf, and disc florets are developing, but not yet mature. In the outer rim disc florets, stamens are differentiating with distinct filaments and anthers, and microspore mother cells are forming. The chemical agent suppresses anther development in these outer disc florets. However, the treatment is ineffective on the inner rim disc florets, which are at an earlier Docket no. PAT-109962-WO-SEC-1 developmental stage with primordial disc florets forming, stamen primordia beginning to differentiate, and sporogenous tissue developing in anthers. Consequently, these inner disc florets undergo normal anthesis, releasing pollen grains around the sunflower head to produce seeds (See FIGs.7A-7C). (c) At the R3 stage, the chemical treatment is effective on disc florets at the outer and middle rims of the sunflower head, but ineffective on disc florets in the center of the head. In the outer rims, disc florets begin to mature, with elongating stamens and distinct, but immature anthers. Microspore mother cells in these disc florets are undergoing meiosis, with early microspores forming. The chemical treatment suppresses further anther development in these outer and middle rim disc florets. In contrast, the inner whorls contain more defined, but still immature disc florets, with differentiating stamens and microspore mother cells being formed. These inner disc florets, unaffected by the treatment, undergo normal anthesis, releasing pollen grains around the sunflower head and producing some seeds (See FIGs.8A-8D). (d) The chemical treatment is effective on all sunflower disc florets at the R4 stage, when the inflorescence begins to open, and the first ray florets become visible. At this stage, disc florets in the outer rims are mature, with anthers nearly full length and beginning to mature. Meiosis has completed in these disc florets, with microspores developing into immature pollen grains. In contrast, inner whorl disc florets are well-defined, but not yet mature, with elongating stamens and microspore mother cells undergoing meiosis or early microspore formation. Chemical treatment of SYN-SF2 at the R4 stage achieved complete sterility, matching the effect observed in SYN-SF1 (See FIGs.9A-9C). Manual pollination of treated sunflowers produced a substantial seed yield, demonstrating again that the chemical does not affect female organ function (See FIG.9D). (e) Correlation of chemical treatment efficacy with stamen development stages In summary, the HFE7500 treatment's effectiveness is contingent on stamen formation in sunflowers. At the R4 stage, all disc florets have developed distinct stamens, allowing the treatment to induce complete sterility. Conversely, at the R1 stage, stamens have not yet developed in all disc florets, rendering the treatment ineffective. During the R1-R3 stages, at least the inner rims of disc florets lack distinct stamens, resulting in only partial sterility when treated. The stamen developmental stage and the effectiveness in chemical treatment is summarized in Table 1 below. Table 1: Stamen development stage and HFE7500 treatment efficacy for sterility induction in sunflower Docket no. PAT-109962-WO-SEC-1 Stamen developmental stages Floret locations Effective to induce Sterility Treatment with HFE7500 at the R4 stage alone can induce complete sterility, but may fail if the chemical does not contact all disc florets. To ensure complete sterility and mitigate potential application issues, a two-stage treatment approach is recommended: applying the chemical at the R4 stage, combined with an earlier application at either the R2 or R3 stage. This method has proven effective in consistently inducing complete sterility. (5) Dosage and application uniformity affects efficacy The amount of chemical used impacts how well the treatment works. HFE7500 was applied onto sunflower heads at R4 developmental stage, in volumes of 0.5 mL, 0.8 mL, and 1 mL using a pipette. At a lower volume, some stamens extended out of the corolla and were visible. With higher volumes of applied HFE7500, almost all stamens failed to extend and were not visible. These results demonstrate that chemical volume is critical for achieving complete flower sterility in sunflowers. The explanation is straightforward: insufficient chemical volume fails to cover the entire sunflower head, leading to incomplete sterility. To ensure complete sterility, the applied chemical volume must be adequate to cover the whole head. Larger sunflower heads, which contain thousands of disc florets, would require an increased chemical volume to ensure contact with every single disc floret in the head (See FIGs.10A-10D). Docket no. PAT-109962-WO-SEC-1 The uniformity of chemical application is crucial for inducing complete sterility in sunflower heads. HFE7500, being a non-systemic chemical, only affects the disc florets it directly contacts, with no impact on untreated disc florets. To achieve complete sterility, the chemical must be applied to every single disc floret within the head. Any non-uniform application that misses disc florets can result in pollen production and release from these untreated areas, leading to pollen contamination during the crossing process (See FIGs.11A-11B). This contamination compromises the integrity of the intended cross and reduces the efficiency of hybrid seed production. (6) Another fluorinated oil, FC-40, has similar effects to HFE7500 3M™ Fluorinert™ FC-40 was evaluated to determine if it could produce effects like HFE7500. When 1 mL of FC-40 was applied using a pipette to a single sunflower head at stage R4, the results were consistent with those observed using HFE7500: the stamens failed to extend beyond the corolla and remained concealed throughout the flowering period (See FIG.12). Various quantities of FC-40 (0.2 mL, 0.8 mL, and 1 mL) were administered to sunflower heads. The chemical liquid was evenly dispersed using a pipette over the disc florets of the head in each treatment during stage R4. The treatments with lower doses of 0.2 or 0.8 mL applied either did not induce sterility or resulted in partial sterility. However, complete sterility was achieved in the sunflower heads when the higher 1 mL dose of FC-40 was administered (See FIG.13D). When treated with 0.8 mL, the sunflower heads exhibited a high level of sterility but still produced a few seeds (See FIG.13C). Conversely, when the volume was decreased to 0.2 mL, the heads remained entirely fertile (See FIG.13B). Fluorinated oils comprise high fluidity. Even when applied in small volumes, such as 0.2 mL, these oils can quickly spread and become highly diluted. This rapid dispersion and dilution may result in insufficient concentration to effectively impact stamen development. These findings underscore the dose-dependent nature of chemical-induced male sterility, with more significant degrees of infertility occurring as the dose increased. To investigate whether FC-40 affected female organ fertility, 1 mL of FC-40 was administered by pipette to a sunflower head at stage R4 to induce flower sterility followed by collection of pollen grains from another untreated sunflower head. The collected pollen grains were sprinkled onto the treated sunflower head. The cross-pollinated sunflower head produced seeds, indicating that FC-40 had a similar effect as HFE7500 by inducing male Docket no. PAT-109962-WO-SEC-1 sterility while having no adverse effect on female organ fertility (See FIG.14). In summary, FC-40 had a comparable function to HFE7500. The induction of sterility by the chemical FC-40 is not systemic. To confirm, 300 uL of FC- 40 was applied by pipette at stage R4 to a single spot on the sunflower head. Although the chemical liquid spread slightly from the applied spot, it did not cover the entire sunflower head due to the limited volume applied. Consequently, some disc florets came into contact with the chemical while others did not. The results showed that sterility occurred only in the area where the chemical spread, while the non-treated area within the same head remained fertile, producing seeds by self-pollination. These findings confirmed the non-systemic effects of the chemical in sunflowers (See FIG.15). Example 2: HFE7500 prevents self-crossing in Nicotiana benthamiana flowers (1) Flower sterility was induced in Nicotiana benthamiana using HFE7500 To test the hypothesis that fluorocarbon chemicals may also be effective in preventing self- crossing in other species, HFE7500 was applied to the model plant Nicotiana benthamiana. In sunflowers, these chemicals shorten anther filaments to create a physical separation between anthers and stigmas, thus preventing self-pollination. However, in Nicotiana benthamiana, the plant naturally has short stamens located near the corolla tube's mouth. During flowering, the anthers’ positions are closer to the stigma regardless of their length, so the stamen length has less impact on self-pollination. In sunflower, it was observed that the anthers of flowers treated with HFE7500 were indehiscent. Evaluation in tobacco was done to see if the same occurred. The Nicotiana benthamiana plants were grown in 3.5-inch soil pots and placed in a growth chamber. HFE7500 was applied to the flowering buds at developmental stages 4-5 when the corolla had just emerged from the calyx (developmental stage 4; See FIG.16A) or extended about 1 cm or less outside of it (developmental stage 5; See FIG.16B). For each flower, a volume of 5-20 μL was dropped into the corolla using a pipette. The results shown in Table 2 demonstrated that all treatments were effective in inducing flower sterility. Optimal sterility was achieved with the application of 10-20 uL of HFE7500 onto each flower. Table 2: HFE7500 induced sterility in Nicotiana benthamiana flowers Experiment Exp. 1 Exp. 2 Exp. 3Exp.Exp. Exp. Docket no. PAT-109962-WO-SEC-1 Flower development stage4 4 5 5 5 5ul0 (2) The treatment causes defects in anther dehiscence A comparative analysis between treated flowers and non-treated flowers of Nicotiana benthamiana was done to evaluate the effect on anther dehiscence. The control flowers exhibited typical anther dehiscence, with white pollen grains adhering to the stigma and surrounding areas. In contrast, the flowers treated with HFE7500 did not demonstrate anther dehiscence at any stage of flowering, including post-flowering. Although the stigma of the treated flowers appeared healthy and green, they did not undergo self-pollination. Pollen viability was assessed by germinating the pollen grains on medium plates and found that pollen from treated flowers germinated with no significant differences observed compared to untreated pollen. Chemical treatment using HFE7500 altered anther dehiscence and prevented self-pollination. (3) HFE7500 treatment does not affect female organ fertility - enabling crosses without emasculation To assess whether the treatments solely affected the male reproductive organs while leaving female fertility unaffected, pollination testing was conducted. Two days after chemical treatment using HFE7500, the Nicotiana benthamiana flowers were manually pollinated using healthy pollen from other flowers on the same plant. In the absence of pollination, the treated flowers exhibited 100% sterility. However, when the treated flowers were manually pollinated, the flower was fertile and produced seeds as shown in Table 3. It was observed that flowers treated at developmental stage 5 exhibited a higher rate of fertility compared to those treated at developmental stage 4. This difference in fertility rate may be attributed to the readiness of the stigma for pollen reception. Docket no. PAT-109962-WO-SEC-1 Table 3: Identification of the optimized conditions for treatment and pollination Experiment Exp-1 Exp-2 Manually Treated and Treated, but not Treated and Treated, but not To validate the previously obtained results, a total of 40 Nicotiana benthamiana flowers at developmental stage-5 were treated. Among them, 30 flowers were pollinated within 2 days after treatment, while the remaining 10 flowers were pollinated 1 day after treatment. The flowers that were pollinated after 2 days displayed a fertility rate of 80%. However, the flowers that were pollinated after 1 day exhibited a lower fertility rate of only 30% (See Table 4). This discrepancy in fertility rates may suggest that the female reproductive structures were not fully prepared to receive pollination at the earlier time point. Table 4: The confirmation of female fertility in Nicotiana benthamiana under optimized treatment and pollination conditions. ExperimentFlowerNumber Amount of HFE7500 Pollination Number Flower y (4) The seeds obtained from "treated and manually pollinated" flowers exhibited normal germination Docket no. PAT-109962-WO-SEC-1 To evaluate the quality of the seeds derived from the "treated and pollinated" Nicotiana benthamiana flowers, the seeds were sown in soil. The seeds germinated normally, indicating that the HFE7500 treatment did not adversely impact seed germination. Example 3: Preliminary testing of HFE7500's effect on inducing sterility in pepper and tomato Experiments on sunflower and tobacco show that chemically induced flower sterility primarily results from impaired anther dehiscence and suppressed stamen growth. These effects create spatial or temporal separation between pollen and stigmas. To assess the chemical's efficacy on other plant species, preliminary tests on pepper and tomato plants were conducted. The experimental procedure involved applying HFE7500 to flower buds one day before anthesis, followed by an evaluation of the treatment's effectiveness. For tomato, a single elite tomato line (SYN-T1) was cultivated in the greenhouse. Ten flowering buds were selected at stages 4-5 where the petal tip was just visible in the flowering bud (See FIG.17A), and each was treated with 50 μL of HFE7500 applied directly to the petal tip using a pipette. The results were as follows: 6 of the treated flower buds failed to develop fruits and eventually abscised; 3 buds produced very small fruits; and 1 bud developed a small fruit (See FIG.17B). These outcomes suggest that HFE7500 is effective in inducing flower sterility. To develop an efficient method for inducing sterility, additional research is necessary, including testing chemicals at various dosages and different plant developmental stages. A pepper line (SYN-P1) was cultivated in the greenhouse and flower buds at stages 4 and 5 were selected (See FIG.18A). For chemical treatment, 50μL of HFE7500 was applied directly to the tip of each flower bud using a pipette. The results were similar to those observed in tomato experiments: 4 flower buds did not develop into fruits and eventually dropped from the plants while the other 6 flower buds developed into very small peppers (See FIG.18B). The non-treated pepper flowers developed into normal size pepper fruit (See FIG. 18C). Dissection of the very small peppers shown in FIG.18B revealed only a few seeds within (See FIG.18D), which might be due to limited pollen availability for fertilization, resulting in reduced fruit size. It is evident that HFE7500 was effective in inducing flower sterility, although the effect was partial. Additional work is needed to optimize the treatment Docket no. PAT-109962-WO-SEC-1 dosage and to identify the most suitable flower developmental stages to achieve complete sterility. Example 4: Fluorocarbons are different from conventional gametocides. They do not damage mature pollen, therefore are also suitable as a medium for pollen storage. (1) HFE7500 does not have negative impacts on pollen viability after 24 hours of treatment compared to untreated pollen Pollen grain samples from greenhouse-grown maize plants were evaluated for viability through germination on an artificial medium. Initial assessment immediately after collection revealed a 51% germination rate. Two concurrent experiments were conducted to evaluate HFE7500's impact on pollen survival rate. In experiment-1, the collected pollen was divided into two 5 mL capped microtubes: One tube contained pollen suspended in HFE7500, while the other held dry pollen without any treatment. The experiment-2 followed an identical design but used uncapped tubes. After 24 hours of incubation at room temperature, pollen germination rates were reassessed for all groups, with results summarized in Table 5. Table 5: Pollen germination rates after 24 hours suspended HFE7500 Pollen Pollen germination rate after 24 hours germination rate y The data revealed a decrease in pollen viability for all treatments when stored at room temperature. However, the application of HFE7500 did not have any detrimental effects on pollen viability compared to the untreated control group. In the case of pollen stored in tubes with the caps open, the untreated pollen completely lost its viability, likely due to moisture loss. Interestingly, the viability of HFE7500-treated pollen was less affected by the presence of caps. (2) Pollen viability was not impacted after different time periods post treatment Based on the previous experiment, it was evident that moisture had a significant impact on pollen viability. To comprehensively assess the influence of HFE7500, further evaluations were done by storing the pollen exclusively in capped tubes. Pollen germination was assessed Docket no. PAT-109962-WO-SEC-1 at various time points after treatment with HFE7500. These experiments were carried out at room temperature, and the results of pollen germination rates are summarized in Table 6. The data represents the average of two independent experiments. Table 6: Pollen germination rates after storage at room temperature. Hours after pollen collected Pollen germination rate on plates and treated (room The data indicated that there was no significant decrease in pollen viability between the HFE7500-treated and untreated groups under room temperature storage conditions, suggesting that HFE7500 did not negatively impact pollen germination. However, pollen treated with HFE7500 did exhibit longer pollen tubes across most of the treatments. This observation suggests that HFE7500 may contribute to improved pollen survival. Figures 19A and 19B depict the differences between pollen tubes in HFE7500-treated pollen (FIG.19B) compared to its respective control (FIG.19A) after 8 hours of treatment. (3) HFE7500 enhanced pollen viability under 4°C storage Pollen viability is significantly affected by temperature. Lower temperatures favor pollen storage. To more comprehensively assess the influence of HFE7500 on pollen viability, pollen was stored at 4°C for different time periods and evaluated for germination. The results, which are averaged from two independent experiments, are summarized in Table 7. Table 7. Pollen germination rates after storage at 4oC Hours after pollen collected Pollen germination rate on plates Docket no. PAT-109962-WO-SEC-1 72 hours 20% 30% NA = not applicable (See Table 7). It was also observed that the pollen treated with HFE7500 exhibited longer pollen tubes across most of the treatments (See FIG.20A and 20B). Based on these results, HFE7500 has the potential to be used as a liquid pollen storage medium.

Claims

Docket no. PAT-109962-WO-SEC-1 What is claimed is:

1. A method to emasculate a plant; comprising applying a chemical emasculation agent to the flowers of a plant, wherein the chemical emasculation agent is not a gametocide, and wherein application of the emasculation to the flowers of the plant occurs at a desired developmental stage.

2. The method of claim 1, wherein the chemical emasculation agent comprises a compound selected from the group consisting of fluorocarbons, hydrofluoroethers, perfluorooctyl bromides, hydrofluoroalkyl esters, and fluorooctanoyl chlorides.

3. The method of claim 2, wherein the chemical emasculation agent comprises a hydrofluoroether compound.

4. The method of claim 3, wherein the chemical emasculation agent comprises a compound selected from the group consisting of HFE7500 and FC-40.

5. The method of claim 1, wherein the plant is a dicot selected from the group consisting of sunflower, soybean, tobacco, pepper, and tomato.

6. The method of claim 5, wherein the dicot is sunflower, and the desired developmental stage is selected from the group consisting of R2, R3, R4 and both R3 and R4.

7. The method of claim 5, wherein the dicot is sunflower, and the desired developmental stage is all stages R2, R3, and R4.

8. The method of claim 5 or 6, wherein the sunflower is treated with the emasculation agent at a volume of 0.4 milliliters to 1.5 milliliters.

9. The method of claim 5, wherein the dicot is tobacco, and the desired developmental stage is stage 4-5.

10. The method of claim 9, wherein the tobacco is treated with the emasculation agent at a volume of 5 to 20 microliters.

11. The method of claim 5, wherein the dicot is pepper, and the desired developmental stage is stage 4-5.

12. The method of claim 11, wherein the pepper is treated with the emasculation agent at a volume of 50 microliters.

13. The method of claim 5, wherein the dicot is tomato, and the desired developmental stage is stage 4-5.

14. The method of claim 13, wherein the tomato is treated with the emasculation agent at a volume of 50 microliters.Docket no. PAT-109962-WO-SEC-1 15. The method of claim 1, wherein the emasculation agent is applied in liquid form uniformly across the flower buds.

16. The method of claim 1, wherein the female organ remains fertile.

17. The method of claim 1, wherein the chemical emasculation agent is non-systemic.

18. The method of claim 1, wherein the emasculation agent application results in at least 90% sterility of the plant.

19. The method of claim 1, wherein the chemical emasculation agent is applied using a pipette.

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