Maize pollen oil suspension

Abstract

Farmers need an ability to reliably collect and store viable maize pollen on one day or in one location and deliver that pollen to a field of females another day and / or at another location. To meet this need, methods of delivering maize pollen to female plants are provided. In one embodiment, one collects an amount of fresh maize pollen, optionally treats the collected pollen with a carrier and adds the pollen and optional carrier to an oil. The pollen suspension in oil may be delivered to female maize plants, resulting in successful fertilization. In one aspect, the carrier is microcrystalline cellulose, activated magnesium silicate, talc powder, or silica powder.

Description

[0001] MAIZE POLLEN OIL SUSPENSION FIELD OF THE INVENTION This invention relates to the field of maize breeding and human-induced pollination, and particularly the field of applying stored maize pollen in maize production fields and greenhouses. BACKGROUND Pollen storage has long been both a need and a goal for plant breeders. See generally W.M. King, Report of chief on seed divisions, In REPORT OF THE COMMISSIONER OF AGRICULTURE (YEARBOOK), Washington D.C., GPO, 47–61 (1885) (articulating the desire for stored pollen “so that we might use it when and where convenient to ourselves.”). In some plants, pollen is quite hardy and long-lived. For example, gingko tree pollen can be collected and stored for six months or more with no specific care required. In contrast, other plants have pollen that is fragile and susceptible to rapid decay within hours if left exposed to the elements. Maize (corn) is one such plant. In maize commercial hybrid production fields, current practice is to alternate four rows of female inbred plants with two rows of male inbred plants. The females are detasseled to prevent self-pollination, while the males are grown solely for their ability to pollinate the neighboring females. This arrangement works best where the female plants and the male plants are of similar maturity groups—that is, the males shed pollen at about the same time the females are receptive to the pollen. However, a farmer risks no successful pollination, and therefore the loss of a crop, if the males and the females are of different maturity groups or if the reproductive maturity of the males and females are not in sync. Without pollen storage, the farmer risks having the male plant shed pollen too early or too late and could lose an entire field due to failed pollinations. With pollen storage and efficient methods for delivery of the stored pollen, pollen can be delivered at precisely the right time regardless of flowering time challenges. Interbreeding different maturity groups could be more easily accomplished, thus expanding the genetic pool and improving maize plant breeding, for example, by making maize lines that are more drought and / or disease resistant. Such methods would also enable introgression of desirable traits from one maize line to another. 1 83189-US-L-ORG-NAT-1 SUMMARY Farmers need an ability to reliably collect and store viable maize pollen on one day or in one location and deliver that pollen to a field of females another day and / or at another location. To meet this need, methods of delivering maize pollen to female plants are provided. In one embodiment, one collects an amount of fresh maize pollen, optionally treats the collected pollen with a carrier, and adds the pollen and optional carrier to an oil. The pollen suspension in oil may be delivered to female maize plants, resulting in successful fertilization. In one aspect, the carrier is microcrystalline cellulose, activated magnesium silicate, talc powder, or silica powder. 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. 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. 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. “Oil” refers to non-polar liquids into which pollen is added to form suspensions. “Oil” may refer to triglyceride mixtures derived from plants, hydrocarbon mixtures derived from petroleum, or synthetic substances with properties similar to plant derived triglyceride mixtures and petroleum derived hydrocarbon mixtures. The specific type of oil is listed in each outlined experiment. Further use of the term “oil” in a given section refers to the initially stated type of oil. “Clarification” as used herein refers to the process by which an oil is treated to remove certain components from the oil. Treatment processes include mixing an oil with a powder with a high specific surface area or filtering the oil through the same powder. Powders with high specific surface areas may include an acid or base component to assist in clarification. A step where the oils is passed through a filter to exclude the powders may be included. Filtration through a filter or bed of powder may be accomplished through gravity or with the assistance of a vacuum pump. Powders used for clarification include, without limitation, activated charcoal, Magnesol®, and Florisil®. As used herein, a “container” or “vessel” refers to an object capable of holding pollen within it. For example, a container may refer to a Magenta GA7 box. Said container or vessel also comprises a breathable barrier. As used herein, “Breathable Barrier” refers to a component of a storage container or vessel for maize pollen. The breathable barrier component of the container or vessel allows for sufficient gas exchange with minimal water vapor transmission. Breathable barriers may include, but are not limited to, Parafilm, Tyvek, Micropore tape, perforations in an otherwise airtight storage container or vessel (e.g., mason jar and lid), or use of non-airtight containers or vessels with aperture for gas exchange manufactured to a specified total surface area (e.g., clamshell container, Magenta GA7 box, VWR® cell culture flasks 25-850ml capacity). For cell culture flasks, see generally us.vwr.com / store / product / 12585790 / vwr-cell-culture-flasks. A non-airtight container may also include, for example, a mason jar and with a perforated lid. “Carrier,” as used herein, means a compound, preferably in powdered form, which acts as an agent to accompany collected pollen. Suitable carrier compounds can be, but are not limited to, talc powder, silica powder, microcrystalline cellulose, and the like. “Clumping,” “Aggregating,” and similar terms, as used herein, refers to the tendency of pollen to bind together, whether due to excess moisture or other cause, in the absence of a carrier and / or suitable storage conditions. Pollen that has clumped is not flowable and cannot be blown by air onto a silk. Clumped pollen is unlikely to adhere to a silk sufficiently to cause pollination. As used herein, the term “comprising” or “comprise” is open-ended. When used in connection with a method comprising series of steps, that method is still practiced so long as the series of steps are performed, even additional steps are performed. “Crystalline silica,” as used herein, refers to a powdered form of silica derived from quartz or other natural rock formations. The terms “crystalline silica,” “SiO2,” and “polycrystalline silica” are used interchangeably throughout. Crystalline silica has different structural properties than talc or amorphous silicas, which include but are not limited to a higher Mohs mineral hardness, higher bulk density, and lower specific surface area. In one embodiment, the crystalline silica comprises an average particle size between 1 nanometer (1 nm) and 100 micrometers (100 μm). In another embodiment, the crystalline silica comprises an average particle size between 1 micrometer (1 μm) and 10 micrometers (10 μm). Unless otherwise specified, particle size values provided herein are the average size. “Activated magnesium silicate” as used herein, refers to a synthetic powdered magnesium silicate. The terms “activated magnesium silicate,” “synthetic amorphous activated magnesium silicate,” and “MgO3Si” are used interchangeably throughout. “FLORISIL®” is a commercially available source of activated magnesium silicate. See www.ussilica.com / products / florisil. Activated magnesium silicate is characterized by an amorphous structure and high specific surface area. In one embodiment, the activated magnesium silicate comprises an average particle size between 1 nanometer (1 nm) and 100 micrometers (100 μm). In another embodiment, the activated magnesium silicate comprises an average particle size between 1 micrometer (1 μm) and 10 micrometers (10 μm). “Microcrystalline cellulose” as used herein, refers to a synthetic polymer derived from alpha cellulose feedstock. “Avicel®” is a commercially available source of microcrystalline cellulose (see, for example, pharma.iff.com / industry-segments / pharma- solutions / products / avicel). Microcrystalline cellulose is characterized by a crystalline structure and low specific surface area. In one embodiment, the microcrystalline cellulose comprises an average particle size of 42 micrometers (42 μm). In another embodiment, the microcrystalline cellulose comprises an average particle size of 167 micrometers (167 μm). In another embodiment, the particle size is expressed a percentage of particles that pass through a 75-micrometer sieve (75 μm, US 200 MESH). Particle sizes may be described using standard mesh sizes, as defined by ASTM E11-22 (www.astm.org / e0011-22.html). “Flowable,” as used herein, means the ability of a powder-like substance to be easily moved by application of air, wind, sound, or to be poured with unbroken continuity and proceed steadily and easily. As used herein, the term “gas” refers to a gas mixture (e.g., the normal air composition or a gaseous combination of oxygen-enriched air) or a substantially pure gas (e.g., pure oxygen). In one embodiment, when referring to gas, one may be referring to oxygen and carbon dioxide. 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” 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. “Heterotic group,” as used herein, refers to a breeding categorization of inbred line. “Heterotic group” and “heterotic pool” are used interchangeably and refer to the relationship between breeding pools of maize populations. Broadly, the primary designations for heterotic pool are: Stiff Stalk (“SS,” also called Iowa Stiff Stalk Synthetic, or “BSSS”), Non Stiff Stalk (“NSS”), and Iodent (“IDT”). See J. v. Hweerwaarden, et al., Historical genomics of North American maize, PROC. NAT’L ACAD. SCI. U.S.A.109(31): 12420-25 (2012). These are not exclusive, however, and other designations are known, e.g., Lancaster Sure Crop ("LSC"). See, e.g., C. Livini, et al., Genetic diversity of maize inbred lines with and among heterotic groups revealed by RFLPs, THEOR. APPL. GENET.84: 17-25 (1992). See further Hallauer et al. (1998) COM BREEDING, p.463-564; G.F. Sprague and J.W. Dudley (ed.) CORN AND CORN IMPROVEMENT; Smith, et al. (1990) Theor. Appl. Gen.80:833-840; Mikel and Dudley (2006) Crop Set 46: 1193-1205. See also WO2020 / 205334 and WO2021 / 041077. 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 maize plant. As used herein, the term “plant line” refers to a single plant material or a genetically identical set of materials. “Refrigerated environment,” as used herein, means any condition where the temperature is less than ambient temperature (or room temperature), but does not fall below the temperature at which water freezes. Said another way, if ambient temperature is 25°C, then a refrigerated environment comprises temperatures greater than 0°C and less than 25°C. Likewise, a refrigerated environment comprises temperatures between 2°C and 10°C. “Refined” as used herein refers to the processing of oils in their production prior to distribution. This includes expeller pressing, cold pressing, naturally refining, roasting, heat processing, and winterizing. Oils tested have various degrees of refining, ranging from unrefined (the least processed) to lightly refined (medium degree of processing) to refined (the most processed). “Sealable container,” as used herein, means any container capable of forming an air-tight seal. Preferably, a sealable container is also capable of holding a vacuum. “Seed Set,” as used herein, means the number of kernels produced on a cob from a successful pollination event. Seed set may be expressed qualitatively (e.g., low, good, or high) or quantitatively. In a quantitative measurement, the measurement may be given as either a percentage or a number of seeds per ear. The term generally refers to the percentage or number of normal kernels (i.e. non-aborted, endosperm-viable kernels). Achieving a good seed set is a goal of a controlled pollination event. “Storage,” as used herein, refers to the act of storing pollen for a suitable period. “Sparging” as used herein refers to bubbling a gas through an oil, or through an oil and pollen mixture. The gas used in sparging may be, without limitation, atmospheric air, 100% oxygen or 100% nitrogen. Sparging may be performed at diverse gas pressures and flow rates. “Treatment,” as used herein, means intentional application of compounds or environmental constraints to pollen. In particular, a pollen treatment may include addition of a carrier compound to the pollen to preserve the pollen’s flowability and viability. “Viable” as used herein refers to a given subject's ability to perform as intended, to fully carry out its intended role and remain productive. Viable pollen is pollen that is alive and capable of germinating. Viable oils are oils that support the survival of pollen and facilitate germination of pollen grains. “Vigor,” as used herein, means the ability of pollen to adhere to silks, germinate pollen tubes, and successfully fertilize egg cells. “Viable,” “Viability,” and similar terms, are used interchangeably with “Vigor.” “Canola oil” as used herein refers to food-grade oils derived from rapeseed cultivars bred for low erucic acid content. Rapeseed cultivars producing food-grade oils are assumed to be derived from Brassica napus and Brassica rapa genetics with an oil erucic acid content of ≤2%. As used herein, the term transgenic “event” refers to a recombinant plant produced by transformation and regeneration of a single plant cell with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term “event” refers to the original transformant and / or progeny of the transformant that include the heterologous DNA. The term “event” also refers to progeny produced by a sexual outcross between the transformant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and the flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome of a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected. Thus, for example, “event 3272”, “3272” or “3272 event” as used herein, means the original 3272 transformant and / or progeny of the 3272 transformant and / or plants derived in any way from the original 3272 transformant. For 3272, See WO06 / 098952. Other examples of transgenic events include, but are not limited to, MIR162 (See WO07142840), Bt11 (See US6114608 (construct) and WO8705629 (gene)), GA21 (See WO9704103 (gene) WO9844140 (cassette)), MIR604 (See WO05103301), MZIR098 (See WO18231890), 5307 (See WO10077816), DAS40278 (See US8598413), TC1507 (See WO04099447), DAS-59122-7 (See WO06 / 039376), NK603 (See US6825400), MON810 (See US6713259), MON863 (See US7705216), MON89034 (See WO07140256), MON88017 (See WO05059103), DP-4114 (See WO11084621), and MON87411 (See WO13169923). “Pollen:Carrier Ratio,” as used herein, means the proportion of pollen present in a mixture with a carrier. For example, and not by way of limitation, a mixture of pollen and carrier with a pollen:carrier ratio of 2:1 comprises two parts pollen measured by weight or volume and one part carrier compound, e.g., talc, measured by weight or volume. DETAILED DESCRIPTION Producibility in maize seed production (i.e., a measure of whether the required quantities of inbred or hybrid seed can be produced through self-pollination or cross pollination at an economical cost that does not exceed the value of the seed being produced) is a critical factor for success in developing maize inbred parent lines, as large quantities of inbred parent line seed are required to produce the hybrid seed sold to customers. A maize inbred parent line with low producibility may be discontinued due to excessive costs in parent seed production, even if that inbred parent line can produce hybrids with characteristics that are desirable to customers (e.g., leading GM and genome edited traits, high yield, disease resistance). Pollen storage and delivery technologies can be used to enhance the producibility of inbred maize parent lines used in hybrid seed production. Challenges to producibility that may be addressed by pollen storage and delivery technologies include but are not limited to, low pollen production, low total pollen shed, short duration of pollen shed, short duration of silk receptivity, and GM or genome edited traits that may impact plant reproductive characteristics. An additional challenge with self- pollination may be a long self-split, which is defined by the number of days between when pollen starts shedding and when silks emerge and become available for pollination. In some iterations, self-split can be a negative value, where silks emerge for pollination before the start of pollen shed. The observed self-split may be a result of the inbred parent line genetics or a result of stress in the growing environment that reduces the rate of silk extension and increases the number of days between the start of pollen shed and silk availability for pollination. To address these producibility challenges, pollen storage and delivery technologies may be used to collect pollen during the optimal window for pollen shed, store that pollen while maintaining pollen viability, then apply the pollen during the optimal window for silk emergence and receptivity. In some iterations, pollen collection may be conducted multiple times per day. In other iterations, pollen may be collected on multiple days throughout the duration of pollen shed. Application of stored pollen may combine pollen collected over multiple days and multiple applications may take place on the same day or across multiple days. Pollen application may combine pollen collected from multiple field locations into a single application to one location. In some iterations, pollen may be collected in one geography and applied to silks in a different geography. The geographies may be different fields at the same production location, fields in different states or municipalities within country, or fields in different countries. In some iterations, pollen is collected from temperate maize inbred parent lines grown in a temperate location and applied to sub-tropical or tropical maize inbred parent lines grown in sub-tropical or tropical locations. In other iterations, pollen is collected from subtropical or tropical maize inbred parent lines grown in sub- tropical or tropical locations and applied to temperate maize inbred parent lines grown in a temperate location. Technologies for pollen delivery that enable seed production in the recipient maize lines are needed to allow for the collected and stored maize pollen to be used for seed production. By addressing these challenges to producibility, pollen storage and delivery technologies may enable seed increase for desirable maize inbred parent lines that will produce new hybrids with desirable characteristics for sale to customers. Pollen storage and delivery technologies may also enable economical hybrid seed production for combinations of temperate, sub-tropical, and tropical maize inbred parent lines that are not currently feasible. Accordingly, an embodiment provides a method of delivering stored maize pollen to a recipient maize plant and producing seeds thereby. In an embodiment, the pollen has an optional carrier added to it. The carrier may be talc, microcrystalline cellulose, activated magnesium silicate, crystalline silica, or another suitable molecule. In an embodiment, the carrier is present in a pollen: carrier ratio selected from the group consisting of 1:20, 1:30, 1:10, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, and any ratio between 1:20 and 50:1 The pollen: carrier ratio is preferably 2:1. In an embodiment, the carrier is microcrystalline cellulose with an average particle size between about 42 μm and about 167 μm, or microcrystalline cellulose with a particle size distribution that is expressed as a sieve pass through fractional using sieves ranging between 20 and 500 micrometers (between US MESH 635 and US MESH 35). In an embodiment, pollen may be delivered to recipient plants in an oil suspension. The oil may be a paraffin oil such as light paraffin oil, paraffin lamp oil, paraffin heavy oil, or may be a plant-derived oil. The plant-derived oil may be derived from a species selected from the group consisting of cotton (Gossypium hirsutum), Brassica napus, Brassica rapa, Brassica nigra, Brassica juncea, olive (Olea europaea), sunflower (Helianthus annuus), peanut (Arachis hypogaea), hemp (Cannabis sativa), safflower (Carthamus tinctorius), coconut (Cocos nucifera), pumpkin (Curcurbita pepo), soybean (Glycine max), walnut (Juglans regia), rice (Oryza sativa), avocado (Persea americana), mustard (Rhamphospermum nigrum), jojoba (Simmondsia chinensis), grape (Vitis vinifera), pine (Pinus pinea), amaranth (Amaranthus cruentus), milk thistle (Silybum marianum), and maize (Zea mays). In an embodiment, the plant-derived oil may be refined or clarified, for example by using activated charcoal, prior to the addition of the pollen. In an embodiment, the pollen suspended in oil may have a gas sparged through it. In a preferred embodiment, the gas may be an oxygen-containing gas such as, for example, atmospheric air or 100% oxygen. In an embodiment, the pollen suspended in oil may have an emulsifier such as egg yolk added to it. In an embodiment, pollen may be delivered to a recipient plant using a sprayer. In an embodiment, the sprayer produces a spray pattern selected from the group consisting of a stream, fan, and cone spray pattern, and may produce a spray angle of about 110°, about 120°, about 130°, or a stream. In an embodiment, the sprayer produces a stream, fine, coarse, ultra coarse, or extra coarse droplet size, or a droplet size between about 341-403 μm, between about 503-665 μm, greater than about 665 μm, or a stream. In an embodiment, the sprayer is a rotary atomizer. In an embodiment, the maize pollen is transgenic maize pollen. In another embodiment, the transgenic maize pollen comprises a transgenic event selected from the group consisting of MIR162, Bt11, GA21, MIR604, MZIR098, 5307, 3272, DAS40278, TC1507, DAS-59122-7, NK603, MON810, MON863, MON89034, MON88017, DP-4114, and MON87411. In one embodiment, the maize pollen comprises transgenic events Bt11, GA21, and MIR162. In another embodiment, the maize pollen comprises transgenic events Bt11 and MIR162. In an embodiment, the maize pollen comprises transgenic event MIR162. EXAMPLES Example 1. Collection Maize plants were grown in field and in greenhouse conditions. Once tassels emerged and began shedding pollen, bags were placed over the tassels to collect the pollen. Bags were typically placed during the late afternoon and removed the following morning. Collected pollen, after sifting away any anthers or other tassel material, was then placed in an appropriate container sealed with a breathable barrier. The pollen was stored according to the methods described in PCT / US2023 / 023203 and PCT / US2022 / 033575, both of which are incorporated by reference herein in their entirety. Alternatively, pollen is collected by harvesting the pre-shed tassels from the maize plants. The tassels can be placed in a beaker of water and allowed to shed pollen normally, or the tassels can be dried, macerated, and filtered to collect the pollen mechanically. See, e.g., U.S. Patent No.8,252,988 (filed June 27, 2007).

[0002] Example 2. Pollen Storage Carriers Maize pollen was collected and stored for five days prior to mixing with various carriers. Talc and crystalline silica were mixed with pollen at a ratio of two parts pollen, one part carrier by weight. Microcrystalline cellulose carriers were mixed with pollen at a ratio of two parts pollen, one part carrier by volume. These mixtures of pollen and carrier were used to pollinate maize stigmas, with 0.5mL of the pollen and carrier mix applied to each stigma. The resulting seed set was observed and is summarized in Table 1. Table 1 demonstrates that each of these mixtures of pollen and carrier was capable of successfully pollinating maize stigmas and producing seeds. Table 1: Seed set from pollen stored with six carriers. N = five replicate pollinations per pollen donor by carrier combination. Data are presented as average number of seeds per ear plus or minus one standard deviation. ND = No data Carrier Carrier Male Particle Example 3. Varying pollen:carrier ratios Increasing the amount of carrier relative to the amount of pollen is desirable, as this could allow for a given amount of collected pollen to be used for pollination of an increased number of plants. To test whether increased dilution with microcrystalline cellulose could support successful pollination, maize pollen was collected and mixed with Avicel® PH101 at a ratio of two parts pollen to one-part Avicel® PH101 by volume. This mixture was stored for four days prior to use in pollination. Following storage, the 2:1 mixture of pollen and Avicel® PH101 was further diluted using additional Avicel® PH101. Exact quantities of Avicel® PH101 were added to generate 1:1, 1:2, 1:5, and 1:10 pollen in Avicel® PH101 dilutions by volume. The original 2:1 stored pollen mixture and the four dilutions created using the 2:1 stored pollen mixture were used to pollinate maize stigmas, with 0.5mL of the pollen and carrier mix applied to each stigma. The resulting seed set was observed and is summarized in Table 2. A “Seed Set Efficiency” was calculated by dividing the number of seeds produced from each pollination by the volume of pollen applied. Table 2: Seed set from stored pollen across five dilutions using Avicel® PH101. N = 10-20 replicate pollinations per pollen donor by carrier combination. Data are presented as average number of seeds per ear plus or minus one standard deviation and as the calculated number of seed set per one milliliter of applied pollen. Pll V lAvicel® PH101 A S d Seed Set y The data in Table 2 demonstrates that pollen stored with microcrystalline cellulose can be further diluted with different volumes of microcrystalline cellulose after storage and result in successful pollinations. A 1:2 dilution may be preferred in instances where the number of available ears is limiting and maximizing the average seed set per ear is prioritized. A higher dilution (e.g., 1:5) may be preferred where the amount of available stored pollen is limiting and producing the maximum total number of seed per volume of pollen over many ears is prioritized. Example 4. Fluorescent Pigments to Track Pollen Tracking the location where applied stored pollen lands on silks and which silks have already pollinated is a critical component of efficient pollen application. To enable visual tracking following stored pollen application, stored pollen was mixed with Rolio® Daylight Fluorescent Pigments (≥97% CAS# 25035-75-7, <3% 68427-35-0, <3% 3068-39-1). These formaldehyde polymer fluorescent pigments were selected to maximize contrast color visibility under normal lighting conditions and to enable fluorescent detection under UV light. Base pigment color and fluorescence were visible by both human eye observation and camera visualization. Orange, blue, red, and green pigments were all tested and were non- toxic to stored pollen. To test whether this fluorescent pigment was compatible with maize pollination, maize pollen was collected and mixed with Avicel® PH101 at a ratio of two parts pollen to one-part Avicel® PH101 by volume and stored for five days prior to use in pollination. Following storage, a subsample of the 2:1 mixture of pollen and Avicel® PH101 was mixed with 10% Rolio® Daylight Fluorescent Red Pigment by volume. Application of pigment to silks prior to pollen application was tested by dusting 0.05 mL of Rolio® Daylight Fluorescent Red Pigment on the silks of selected maize ears.0.5mL of the pollen and carrier mix was applied to check ears and ears treated with pigment prior to pollination.0.5 mL of the pollen and carrier mix with added pigment was applied to untreated silks for comparison. The resulting seed set was observed and is summarized in Table 3. Table 3: Seed set from stored pollen mixed with Rolio® Daylight Fluorescent Red Pigment compared to silks treated with the same pigment prior to pollination. Each N = 14-20 replicate pollinations per pollen donor by carrier combination. Data are presented as average number of seeds per ear plus or minus one standard deviation. k) No Pigment No Pigment 388 ± 108 ere Example 5. Electrostatic Application Electrostatic powder coating technology was tested for applying maize pollen stored with the microcrystalline cellulose powder. Field testing was conducted using a Sames electrostatic powder coating system consisting of an eight-liter Inotank powder fluidization chamber with a CS130 powder pump connected to an Inogun A powder gun. The system was controlled using an Inobox H control unit supplied with compressed air at 7 bar pressure. Various Inocoat system settings were tested, as summarized in Tables 4 and 4B. Table 4: Inocoat powder coating system settings tested for stored pollen application. n s tested for stored pollen application. Air speed was collected using an Alnor AVM440 Velometer Thermal Anemometer. Airspeed Airspeed Airspeed Pollen was stored for three days as a mixture of two parts pollen to one part Avicel® PH101 Microcrystalline Cellulose by volume. Hand pollinated checks received 0.5mL of stored pollen mix each and Avicel® PH101 only checks received 0.5mL of microcrystalline cellulose each. While applied volumes may vary, it was estimated that each ear pollinated by the Inocoat system received approximately 0.5mL of stored pollen mix. This approximate applied volume per pollination was consistent across all four settings. All Inocoat system applications were conducted at a standoff distance of 10 cm between the application gun nozzle and the targeted maize silks. Table 5 summarizes the number of seeds produced from these pollinations using the Inocoat system. Table 5: Seed set outcome of stored maize pollen applied using the Inocoat powder coating system on four different settings. N = 14-20 replicate pollinations per treatment. Data are presented as average number of seeds per ear plus or minus one standard deviation. Avg Seed Set / Ear The data in Table 5 pported successful pollination of maize silks to produce seeds. To test whether electrostatic charge would affect pollination when pollen was applied from increasing distances from the target silks, Inocoat settings C and D (Table 4) were used at standoff distances of 10 cm, 50 cm, and 100 cm. Pollen was stored for four days as a mixture of two parts pollen to one part Avicel® PH101 microcrystalline cellulose by volume. While applied volumes may vary, it was estimated that each ear pollinated by the Inocoat system received approximately 0.5mL of stored pollen mix. This approximate applied volume per pollination was consistent across both settings. Inocoat system applications were conducted at set standoff distances using a meter ruler held between the application gun nozzle and the targeted maize silks. Table 6: Seed set outcome of stored maize pollen applied using the Inocoat powder coating system on two different settings at increasing application standoff distances. N = five replicate pollinations per treatment. Data are presented as average number of seeds per ear plus or minus one standard deviation. Standoff DistanceInocoat Setting C Inocoat Setting DThe data sh is beneficial, as Setting D re g p g ch of the three standoff distances tested. Example 6. Suspending Pollen in Oils Stored maize pollen mixed with a carrier compound can be applied directly to maize silks as a flowable powder and result in seed set. While direct application of flowable powders is an effective practice for hand pollination, efficient application of stored pollen in a liquid carrier would enable scale up to mechanized application using spray equipment. It is established in scientific literature that freshly collected pollen can be suspended in paraffin oil to expose the pollen to active ingredients (Neuffer and Coe 1978 Maydica 23:21-28). These pollen in paraffin oils suspensions can be applied to silks and result in seed set. From this basis, pollen in paraffin oil suspensions were tested to determine the optimal concentration of pollen in oil. Sunflower oil and canola oil were also tested in this experiment. Pollen was stored for three days as a mixture of two parts pollen, one part silica by weight. Suspensions were mixed for ten minutes with horizontal agitation prior to being pipetted on to silks. One milliliter of suspension was pipetted onto each silk. Table 7: Seed set outcome of stored maize pollen suspended in sunflower oil, canola oil, and light paraffin oil at three different concentrations. N = three replicate pollinations per oil by pollen concentration combination. Data are presented as average number of seeds per ear plus or minus one standard deviation. Pollen Oil Concentration Avg Seed (m / ml) Set / Ear (k) Prior to pollinat centrations exceeding 100 mg / ml were prone to clumping. The seed set data summarized in Table 7 demonstrated that increasing the concentration of stored pollen suspended in oil increased seed set. Stored pollen mixed with silica at two parts pollen, one part silica by weight has a bulk density of approximately 0.9-1.0 mg / ml, therefore, 10% stored pollen in oil on a weight per volume basis and a volume per volume basis were considered interchangeable in future experiments. To further optimize maize pollen application in oil suspension, application temperature was tested. Oils were adjusted to either the 6°C optimal pollen storage temperature or held at 25°C to represent standard room temperature. In addition to light paraffin oil, paraffin lamp oil and heavy paraffin oil were included in this experiment, along with canola oil and cottonseed oil. Table 8: Seed set outcome of stored maize pollen suspended in five oils adjusted to 6°C or 25°C. Seed set for each condition is shown as kernels per ear ± standard deviation. N = nine replicate pollinations per oil by pollen concentration combination. 6°C Incubation 25°Incubation Oil T m r t r T m r t r There was no significant difference in seed set produced by stored maize suspended in oil when comparing 6°C and 25°C incubation temperatures for any of the five oils tested (Tukey’s HSD, p<0.05). Therefore, it was concluded that cooling oil to 6°C is not a requirement for efficacy. The resulting seed set from pollen suspended in canola and cottonseed oil led the team to search for other plant derived oils that could deliver seed set efficacy similar to that of paraffin oils. Plant derived oils offer advantages for use in maize pollen suspension application compared to oils derived from petroleum. Plant derived oils generally have lower flash points, lower environmental toxicity, and reduced risks to workers. Plant derived oils include those derived from the endosperm (canola, cottonseed, etc.), those derived from the embryo (corn, etc.), those derived from mesocarp (olive, avocado), and those derived from the aleurone and pericarp (rice bran oil). Plant derived oils may be refined into their constituent fatty acids (oleic acid, squalene, linoleic acid) or triglycerides (coconut medium chain triglycerides). A panel of these oils was assembled to test comparative efficacy in stored pollen application. Pollen was stored for between one and five days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 10% pollen to oil, mixing by hand, and incubated without agitation prior to application. One milliliter of suspension was pipetted onto each silk. Table 9: Seed set outcome of stored maize pollen suspended in a panel of oils. N = three replicate pollinations per oil. Data are presented as average number of seeds per ear plus or minus one standard deviation. Avg Seed Helianthus annuus High Linoleic Sunflower 210 ± 180 Juglans regia Walnut 100 ± 70 The ize pollen application, with many plant derived oils equaling or exceeding seed set performance of paraffin oils in previous experiments. Stored pollen suspended in two oils, extra virgin olive oil and black mustard oil, showed zero seed set. Interestingly, stored pollen suspended in refined olive oil showed average seed set comparable to cottonseed or canola oil. A similar trend was observed with avocado oils, where seed set produced by pollen suspended in refined and lightly refined avocado oil suspensions was higher than seed set produced by pollen in an extra virgin avocado oil suspension. An additional unexpected result was that a stored pollen suspension using black mustard oil derived from Rhamphospermum nigrum showed zero seed set, while a similar stored pollen suspension in canola oil derived from Brassica napus showed the highest average seed set in the experiment. The trend in stored maize pollen suspension seed set performance was less clear when comparing pure oleic acid to sunflower oil and safflower oil products selected for high oleic acid or high linoleic acid composition. Pure oleic acid resulted in zero seed set, while high oleic acid safflower oil resulted in higher average seed set than high linoleic safflower oil. Sunflower oils showed the opposite trend, with high linoleic sunflower oil suspension resulting in higher seed set than high oleic sunflower oils. These results suggest that the oleic to linoleic fatty acid composition of commercial plant derived oils is not a significant factor in performance of stored pollen suspensions. Pure fatty acid preparations produced from plant derived oils, namely pure oleic acid, may be detrimental to suspended pollen performance and are unlikely to be feasible for large scale application. In addition to testing pollen in oil suspensions using test pollinations, additional plant derived oils were tested using a pollen tube germination assay. Pollen tube formation has been shown previously to correlate with pollen viability. Pollen was stored for between one and five days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 20% pollen to oil, mixing by hand, and incubated without agitation prior to application.700 microliters of suspension were pipetted onto a pollen tube germination media plate. A section of the pollen tube germination media was removed by hand prior to suspension application and the plate was set at a fifteen-degree angle to allow oil to drain away from the pollen into the well. Plates were imaged 45 minutes after application. Stored maize pollen in canola oil and cottonseed oil suspensions were included in these assays as positive checks with known high pollen tube germination ratings, while stored pollen in black mustard oil suspension was included as a negative check with a known low pollen tube germination rating. The data in Table 10 shows that pollen suspended in amaranth or milk thistle oil can form pollen tubes well, while pollen suspended in pine nut oil performs poorly in a pollen tube germination assay. Germination ratings were scored as follows: germination rate >60%, scored as 5; germination rate 41-60%, scored as 4; germination rate 21-40%, scored as 3; germination rate 1-20%, scored as 2; germination rate 0%, scored as 1. Table 10: Pollen tube germination ratings of stored maize pollen suspended in seven oils. N = three replicate pollinations per oil. Pollen Tube Germination Example 7. Oil Clarification Improves Pollination Performance Without being bound by theory, certain oils may comprise components that inhibit pollen tube formation and ultimately inhibit seed set. We hypothesized that processing of the oils prior to formation of the pollen suspension could remove these possible inhibitory components in the oil. Plant derived oil processing technologies were investigated to test whether this processing could result in improved seed set. Typical oil clarification solutions involve filtering plant derived oils through powders with high specific surface areas. One example of a powder than can filter plant derived oils is activated charcoal. Other examples include magnesium silicate and silica gel. These magnesium silicate powders may include an acid or base component to improve clarification performance. The oil is generally passed through a cloth filter after exposure to these high specific surface area powders to remove any suspended powder particulate matter. Activated charcoal was tested as an oil clarification agent. Ultrafine grade activated charcoal was mixed with extra virgin olive oil at a rate of one part extra virgin olive oil, one part activated charcoal by volume. The resulting suspension was incubated overnight at room temperature and passed through a 0.22μm filter to remove any suspended activated charcoal. The same activated charcoal clarification treatment was applied to cottonseed oil as a positive check for seed set from stored pollen suspension application. Pollen was stored for three days as a mixture of two parts pollen, one part crystalline silica by weight. Suspensions were formed by adding 10% (v / v) pollen / crystalline silica mixture to oil, mixing by hand, and incubated without agitation prior to application. One milliliter of suspension was sprayed onto silks using an oil spritzer. Table 11: Seed set outcome of stored maize pollen suspended in two oils with or without activated charcoal clarification. Data is presented as kernels per ear ± one standard deviation. N = three replicate pollinations per oil. Oil Source Species Commercial Oil Name No ClarificationActivated CharcoalClarificationT rcoal resulted in seed set, while untreated extra virgin olive oil resulted in no seed set. Activated charcoal clarification of cottonseed oil did not result in a statistically different seed set as compared with unclarified cottonseed oil. Florisil® (activated magnesium silicate trioxide) was previously described as an effective solid carrier for use in maize pollen storage technology (PCT / US2023 / 023203; PCT / US2022 / 033575). Oil clarification experiments included application of <200 mesh Florisil® to extra virgin olive oil and extra virgin avocado oil. Florisil® clarification was also applied to cottonseed oil as a positive check for seed set from stored pollen suspension application. To facilitate clarification, oils were mixed with <200 mesh Florisil® powder at a rate of approximately 10% Florisil® by volume. The resulting suspension was incubated at room temperature, during which the Florisil® settled out of suspension. After 12 to 24 hours incubation, the oil fraction was decanted off for use in stored pollen suspension application. For these experiments, pollen was stored for between one and five days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 10% pollen to oil, mixing by hand, and incubated without agitation prior to application. Table 12: Seed set outcome of stored maize pollen suspended in three oils before and after Florisil® clarification. One milliliter of suspension was sprayed onto silks using an oil spritzer. Data are presented as kernels per ear ± one standard deviation. N = three replicate pollinations per oil. Oil Source Species Commercial Oil Name No Clarification Florisil®ClarificationT for all three of the oils tested, and made seed set possible with extra virgin olive oil, which results in no seed set without a clarification treatment. Based on these results, stored pollen suspensions in off-the-shelf cottonseed oil and Florisil® cottonseed oil were tested at higher replication. Pollen was stored for three days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 20% pollen to oil, mixing by hand, and incubated without agitation prior to application. One milliliter of suspension was sprayed onto silks using an oil spritzer. This experiment confirmed that Florisil® clarification of cottonseed oil resulted in increased seed set relative to unclarified cottonseed oil. To further investigate the impact of oil clarification on diverse plant derived oils, a series of experiments were conducted to test additional oil clarification technologies. In addition to activated charcoal and Florisil®, the experiments included Magnesol®, a magnesium silicate product. Table 13: Seed set outcome of stored maize pollen suspended in cottonseed oil before and after Florisil® clarification. Data are presented as kernels per ear ± one standard deviation. N = seventeen replicate pollinations per oil. Commercial Clarification Avg Seed Oil S r S i Oil N m Tr tm nt S t E k In the first experiment, activated charcoal, <200 mesh Florisil®, and Magnesol® were separately suspended in extra virgin olive oil at a rate of approximately 10% clarification agent by volume. The suspensions were incubated at room temperature and the clarification agents settled out of suspension. After incubation and settling, the oil fractions were decanted for testing. In addition to standard extra virgin olive oil, delicate olive oil was included in this experiment. Delicate olive oil is extra virgin olive oil derived from olives harvested at later stages of ripeness, which may result in a different fatty acid and organic compound profile than standard extra virgin olive oil that bulks oil derived from olives over a range of olive ripeness. Refined olive oil was included in the experiment for comparison to previous seed set data. Refined olive oil has been processed to remove suspended chlorophyll and reduce the concentration of free oleic acid. <200 mesh Florisil® was suspended in delicate olive oil and refined olive oil at a rate of approximately 10% clarification agent by volume. The suspensions were allowed to incubate at room temperature and the clarification agents settled out of suspension. After incubation and settling, the oil fractions were decanted for testing. Pollen was stored for three days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 20% pollen to oil, mixing by hand, and incubating without agitation prior to application.700 microliters of each suspension were pipetted onto a pollen tube germination media plate. A section of the pollen tube germination media was removed by hand prior to suspension application and the plate was set at a fifteen-degree angle to allow oil to drain away from the pollen into the well. Plates were imaged 45 minutes after application. The data demonstrated that multiple clarification agents can be used to improve pollen tube germination for maize pollen stored in various olive oils. Germination ratings were scored as follows: germination rate >60%, scored as 5; germination rate 41-60%, scored as 4; germination rate 21-40%, scored as 3; germination rate 1-20%, scored as 2; germination rate 0%, scored as 1. Table 14: Maize pollen tube germination ratings of stored pollen suspended in preparations of olive oil. N = three replicate plates per oil. Commercial Oil Name NoActivated al Extra Virgin Olive 1 3 2 2 Refined Olive 2 4 not tested not tested d In ol® were separately suspended in cottonseed oil at a rate of approximately 10% clarification agent by volume. The suspensions were allowed to incubate at room temperature and the clarification agents settled out of suspension. After incubation and settling, the oil fractions were passed through cloth filters to remove any remaining clarification agent particles. A separate sample of unclarified cottonseed oil was passed through the cloth filter to act as a check. Pollen was stored for three days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 20% pollen to oil, mixing by hand, and incubating without agitation prior to application.700 microliters of each suspension were pipetted onto a pollen tube germination media plate. A section of the pollen tube germination media was removed by hand prior to suspension application and the plate was set at a fifteen-degree angle to allow oil to drain away from the pollen into the well. Germination ratings were scored as follows: germination rate >60%, scored as 5; germination rate 41-60%, scored as 4; germination rate 21-40%, scored as 3; germination rate 1-20%, scored as 2; germination rate 0%, scored as 1. Table 15: Maize pollen tube germination ratings of stored pollen suspended in preparations of cottonseed oil. Plates were imaged 45 minutes after application. N = three replicate plates per oil. CommerciPollen Tube Oil Source SpeciesalClarification MethodGermination These data demonstrated that oil clarification technology could increase the pollen tube germination performance of stored maize pollen suspended in cottonseed oil following clarification compared to the same oil in its off-the-shelf composition. Passing the oil through a cloth filter alone did not alter the pollen tube germination performance of stored pollen suspended in the filtered oil. In the third experiment, oils derived from the seed of three different mustard plant species were compared in their off-the-shelf composition and after clarification using <200 mesh Florisil®. <200 mesh Florisil® was suspended in each of the three oils at a rate of approximately 10% clarification agent by volume. The suspensions were allowed to incubate at room temperature and the clarification agents settled out of suspension. After incubation and settling, the oil fractions were decanted for further use. Pollen was stored for one day as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 20% pollen to oil, mixing by hand, and incubated without agitation prior to application.700 microliters of each suspension were pipetted onto a pollen tube germination media plate. A section of the pollen tube germination media was removed by hand prior to suspension application and the plate was set at a fifteen-degree angle to allow oil to drain away from the pollen into the well. Plates were imaged 45 minutes after application. Germination ratings were scored as follows: germination rate >60%, scored as 5; germination rate 41-60%, scored as 4; germination rate 21-40%, scored as 3; germination rate 1-20%, scored as 2; germination rate 0%, scored as 1. The data suggested that Florisil® clarification of B. juncea or R. nigrum oil had a minimal effect on maize pollen tube germination. Table 16: Pollen tube germination ratings of stored maize pollen suspended in various preparations of mustard oil. N = three replicate plates per oil. Oil source No Clarification Florisil®Clarification Example 8. Addition of An Emulsifier to Pollen in Oil Suspensions A challenge observed in using maize pollen in oil suspensions is that pollen rapidly settles out of suspension in a pure oil. The addition of an emulsifier to increase interaction between pollen grain membrane surfaces and oil could improve suspension stability. A pollen in oil suspension with stable dispersion of singulate pollen grains may improve the resulting seed by increasing the rate of direct contact between individual pollen grains and silk membranes following application. Egg yolk was selected as a representative emulsifier for testing based on the favorable interaction between egg yolk and plant derived oils that is commonly used in food processing. Pollen was stored for six days as a mixture of two parts pollen, one part silica by weight. Suspensions with emulsifier were formed by adding powdered egg yolk to oil by weight and mixed using a vortex mixer. Pollen in oil with emulsifier suspensions were formed by adding 25% pollen to oil, mixing by hand, and incubated without agitation prior to application. One milliliter of suspension was sprayed onto silks using an oil spritzer. Table 17: Seed set outcome of stored maize pollen suspended in cottonseed oil with and without added powdered egg yolk as an emulsifier. N = three replicate pollinations per treatment. Data are presented as average number of kernels per ear plus or minus one standard deviation. Commercial Egg Yolk Added Avg Seed Oil Source Species Oil Name (% w / w) Set / Ear (k) These in plant derived oil suspensions. The addition of less than three percent egg yolk by weight to the oils effectively doubled average seed set of suspended pollen when compared to the same batch of pollen suspended in the same cottonseed oil without an emulsifier. Example 9. Enabling Pollen Metabolic Gas Exchange in Oil Suspensions As demonstrated previously (PCT / US2023 / 023203; PCT / US2022 / 033575), maintaining normal metabolic gas exchange is critical to maize pollen grain survival and pollination performance. A lack of gas exchange can lead to a buildup of carbon dioxide in the pollen, which may cause a lag in pollen tube germination and could lead to pollen death. Similarly, a lack of oxygen may also induce pollen tube germination lag. Given that plant derived oils block metabolic gas exchange, there is a risk of excessive carbon dioxide accumulation or oxygen depletion in the pollen if the pollen is retained in an oil suspension for an excessive period of time. To enable scale-up of pollen in oil suspensions to field applications where the suspensions may be held in a sprayer tank for hours, gas sparging was investigated as a means to enable pollen metabolic gas exchange in an oil suspension. Gas sparging was accomplished by delivering pressurized gas to the base of a maize pollen in oil suspension. Gas was delivered through a bubbling stone to ensure dispersion into fine bubbles that would maximize interaction between suspended pollen grains and the sparging gas. Standard atmospheric air (≈78% nitrogen, ≈21% oxygen, <0.1% carbon dioxide) was tested as a baseline for normal gas exchange.100% oxygen was tested as a sparging gas to evaluate whether providing a higher oxygen concentration than atmospheric air resulted in higher or lower pollination performance.100% nitrogen was included as a sparging gas to separately test the action of gas sparging from providing supplemental oxygen. Sparging with 100% nitrogen was expected to decrease carbon dioxide accumulation in suspension without providing additional oxygen in suspension. Pollen was stored for three days as a mixture of two parts pollen, one part silica by weight. Pollen in oil suspensions were formed by adding 20% pollen to oil, mixing by hand, and incubating without agitation prior to application. One milliliter of suspension was sprayed onto silks using an oil spritzer. Table 18: Seed set outcome of stored maize pollen suspended in cottonseed oil with and without gas sparging for three hours prior to pollination. N = three replicate pollinations per treatment. Data are presented as average number of seeds per ear plus or minus one standard deviation. Commercial Avg Seed Thes s exchange in an oil suspension for at least three hours and obtain a higher seed set from the suspended pollen when an oxygen-containing gas is sparged through the oil. The presence of oxygen in the sparging gas as a pure gas or as a component of atmospheric air is necessary to maintain metabolic gas exchange. The act of gas sparging unto itself does not facilitate pollen metabolic gas exchange as evidenced by the lower seed set from pollen suspended in oil that was sparged with 100% nitrogen gas. Example 10. Pollen in Oil Suspension Application Systems To apply stored maize pollen in plant derived oil suspensions at field scale, a series of experiments was conducted to identify application equipment parameters that delivered optimal seed set. These parameters included testing both pneumatic and hydraulic pressurized application systems, as well as testing different nozzle tips with varying orifice diameters and spray patterns. The first experiment tested off-the-shelf products developed to spray plant derived oils in culinary applications. A panel of eight oil spritzers was purchased to test diverse spray patterns. Spray nozzle orifice diameter was observed to vary across the panel of spritzers. All eight spritzers were hydraulically powered in that the spritzer trigger depressed a plunger that directly pressurized and expelled the oil. Pollen was stored for two or three days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 25% pollen to cottonseed oil, mixing by hand, and incubated without continuous agitation prior to application. Suspensions were agitated by shaking spritzer bottles immediately prior to application. Between one and two milliliters of suspension was sprayed onto each test silk. Table 19: Seed set outcome of stored maize pollen suspended cottonseed oil and applied through eight different oil spritzers. N = three replicate pollinations per spritzer. Data are presented as average number of seeds per ear plus or minus one standard deviation. Average Avg Spray Seed r 6 5 0 1 0 0 The small proof of concept that pollen suspensions in oil could be sprayed onto maize flowers, resulting in successful seed set. Based on these learnings, testing was scaled up to commercial agricultural spray application systems. In the experiments with agricultural spray application systems, stored maize pollen suspended in canola oil or cottonseed oil was applied to silks using a panel of spray nozzles. The panel of spray nozzles included products from various manufacturers that delivered a variety of spray patterns (stream, fan, and half-dome; spray angles between 110°-130°) and a variety of droplet sizes ranging from a stream to extra coarse droplets. Droplet sizes were classified based on ASABE 572.1 standards (American Society of Agricultural and Biological Engineers; http: / / www.asabe.org / ). Based on nozzle compatibility with existing spray equipment, nozzles were tested using either a pneumatically pressurized CO2application system or a hydraulically pressurized diaphragm pump application system. Manufacturer recommended application pressures were followed based on the nozzle being tested. In general, application pressures ranged between 20 and 45 PSI. Pollen was stored for between four and six days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 10% pollen to cottonseed oil, mixing by hand, and incubated without continuous agitation prior to application. Suspensions were agitated by shaking spray equipment immediately prior to application. While applied volumes vary between the nozzles being tested, it was estimated that each ear received between one to two milliliters of suspension. The data confirmed that high seed sets could be obtained from stored maize pollen in oil suspensions applied through sprayer nozzles. Table 20: Seed set outcome of stored maize pollen suspended in canola oil or cottonseed oil and applied through a panel of sprayer nozzles. N = three or four replicate pollinations per spritzer. Data is presented as average number of seeds per ear plus or minus one standard deviation. Product Manufacturer Spray Pattern Droplet Size Spray Seeds / Ear (k) Name (μm) Angle (°) AIC Tip TeeJet® Fan 503-665 110 315 ± 125 (extremely p , p p applied to silks using a Herbi 4 spinning disk, controlled droplet application (CDA) sprayer (www.microngroup.com / the-herbi-4 / ). In this rotary atomizer system, the pollen in oil suspension was gravity fed onto the spinning disk that delivered large droplets of oil with suspended pollen onto maize silks. Pollen was stored for three days as a mixture of two parts pollen, one part silica by weight. Suspensions were formed by adding 10% pollen to canola or sunflower oil, mixing by hand, and incubated without continuous agitation prior to application. Suspensions were agitated by shaking spray equipment immediately prior to application. While applied volumes varied, it was estimated that each ear received between one to two milliliters of suspension. The data confirmed that high seed sets could be obtained from stored maize pollen in oil suspensions applied through a spinning disk rotary atomizer. This alternative application system could consistently apply a pollen in oil suspension without damaging pollen viability. Table 21: Seed set outcome of stored maize pollen suspended in canola oil or sunflower oil and applied through a spinning disk sprayer. N = three replicate pollinations per oil. Data are presented as average number of seeds per ear plus or minus one standard deviation. Oil Source Commercial Oil SpeciesNameAvg Seed Set / Ear (k)

Claims

What is claimed is:

1. A composition comprising maize pollen and microcrystalline cellulose.

2. The composition of claim 1, wherein said microcrystalline cellulose: a. comprises an average particle size between about 42 and about 167 micrometers; or b. comprises a particle size distribution that is expressed as a sieve pass through fractional using sieves ranging between 20 and 500 micrometers (between US MESH 635 and US MESH 35).

3. The composition of claim 1 wherein said composition comprises a maize pollen:microcrystalline cellulose ratio selected from the group consisting of 1:20, 1:30, 1:10, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, and any ratio between 1:20 and 50:

1.

4. The composition of claim 3 wherein said maize pollen:microcrystalline cellulose ratio is 2:

1.

5. The composition of claim 1, wherein said composition is suspended in an oil.

6. The composition of claim 5, wherein said oil is selected from the group consisting of light paraffin oil, paraffin lamp oil, paraffin heavy oil, and a triglyceride derived from a plant.

7. The composition of claim 6, wherein said plant is a species or hybrid selected from the group consisting of cotton (Gossypium hirsutum), Brassica napus, Brassica rapa, Brassica nigra, Brassica juncea, Brassica carinata, Brassica oleracea, olive (Olea europaea), sunflower (Helianthus annuus), peanut (Arachis hypogaea), hemp (Cannabis sativa), safflower (Carthamus tinctorius), coconut (Cocos nucifera), pumpkin (Curcurbita pepo), soybean (Glycine max), walnut (Juglans regia), rice (Oryza sativa), avocado (Persea americana), mustard (Rhamphospermum nigrum), jojoba (Simmondsia chinensis), grape (Vitis vinifera), pine (Pinus pinea), amaranth (Amaranthus cruentus), milk thistle (Silybum marianum), and maize (Zea mays).

8. The composition of claim 6, wherein said triglyceride derived from a plant is clarified prior to suspending maize pollen in oil.

9. The composition of claim 8, wherein said triglyceride derived from a plant is clarified using activated charcoal.

10. The composition of claim 9, wherein said activated charcoal is suspended in the oil at a ratio of about one part oil, one part activated charcoal by volume.

11. The composition of claim 9 wherein said activated charcoal is removed from the oil by a method selected from the group consisting of filtration, centrifugation, and decanting prior to suspending said maize pollen in said oil.

12. The composition of claim 11, wherein said activated charcoal is removed from the oil by filtration.

13. The composition of claim 5, wherein said maize pollen is suspended in said oil at a concentration of between about 1 mg / mL and about 100 mg / mL.

14. The composition of claim 13, wherein said maize pollen is suspended in said oil at a concentration of about 20% pollen by weight.

15. The composition of claim 5, wherein a gas is sparged through said oil.

16. The composition of claim 15, wherein said gas is atmospheric air.

17. The composition of claim 15, wherein said gas is 100% oxygen, 100% nitrogen, or any mixture of oxygen, nitrogen, carbon dioxide, or other gas.

18. The composition of claim 15, wherein said gas is sparged through said oil before said maize pollen is suspended in said oil.

19. The composition of claim 18, wherein said gas is sparged through said oil throughout the duration during which said maize pollen is suspended in said oil.

20. The composition of claim 5, wherein an emulsifier is added to said maize pollen suspended in said oil.

21. The composition of claim 20, wherein said emulsifier is egg yolk.

22. The composition of claim 1, wherein said maize pollen is 0 days old, 1 day old, 2 days old, 3 days old, 4 days old, 5 days old, 6 days old, 7 days old, 8 days old, 9 days old, 10 days old, 11 days old, 12 days old, 13 days old, 14 days old, 15 days old, 16 days old, 17 days old, 18 days old, 19 days old, 20 days old, or more.

23. A method of applying maize pollen to a stigma, comprising: a. collecting maize pollen; b. suspending said maize pollen in an oil; c. applying said maize pollen suspended in oil to a stigma; wherein the maize pollen is applied to the stigma at least one day after collection.

24. The method of claim 23 wherein said oil is selected from the group consisting of light paraffin oil, paraffin lamp oil, paraffin heavy oil, and a triglyceride derived from a plant.

25. The method of claim 24 wherein said plant is a species selected from the group consisting of cotton (Gossypium hirsutum), Brassica napus, Brassica rapa, Brassica nigra, Brassica juncea, Brassica carinata, Brassica oleracea, olive (Olea europaea), sunflower (Helianthus annuus), peanut (Arachis hypogaea), hemp (Cannabis sativa), safflower (Carthamus tinctorius), coconut (Cocos nucifera), pumpkin (Curcurbita pepo), soybean (Glycine max), walnut (Juglans regia), rice (Oryza sativa), avocado (Persea americana), mustard (Rhamphospermum nigrum), jojoba (Simmondsia chinensis), grape (Vitis vinifera), pine (Pinus pinea), amaranth (Amaranthus cruentus), milk thistle (Silybum marianum), and maize (Zea mays).

26. The method of claim 25 wherein said triglyceride derived from a plant is clarified prior to suspending maize pollen in oil.

27. The method of claim 26 wherein said triglyceride derived from a plant is clarified using activated charcoal.

28. The method of claim 27 wherein said activated charcoal is suspended in the oil at a ratio of about one part oil, one part activated charcoal by volume.

29. The method of claim 27 wherein said activated charcoal is removed from the oil by a method selected from the group consisting of filtration, centrifugation, and decanting prior to suspending said pollen in said oil.

30. The method of claim 29 wherein said activated charcoal is removed from the oil by filtration.

31. The method of claim 23 wherein said maize pollen is suspended in said oil at a concentration of 20% pollen by weight.

32. The method of claim 23 further comprising sparging a gas through said oil.

33. The method of claim 32 wherein said gas is atmospheric air.

34. The method of claim 32 wherein said gas is 100% oxygen, 100% nitrogen, or any mixture of oxygen, nitrogen, carbon dioxide, or other gas.

35. The method of claim 32 wherein said sparging occurs before said maize pollen is suspended in said oil.

36. The method of claim 35 wherein said sparging occurs throughout the duration during which said maize pollen is suspended in said oil.

37. The method of claim 23 wherein an emulsifier is added to said maize pollen suspended in said oil.

38. The method of claim 37 wherein said emulsifier is egg yolk.

39. The method of claim 23 wherein said stored maize pollen is applied to said stigma using a sprayer.

40. The method of claim 39 wherein said sprayer is hydraulically or pneumatically pressurized.

41. The method of claim 39 wherein said sprayer produces a spray pattern selected from the group consisting of a stream, fan, and cone spray pattern.

42. The method of claim 39 wherein said sprayer produces a spray pattern with a spray angle of about 110°, about 120°, about 130°, or a stream.

43. The method of claim 39 wherein said sprayer comprises a nozzle that produces a stream, fine, coarse, ultra coarse, or extra coarse droplet size.

44. The method of claim 39 wherein said sprayer comprises a nozzle that produces a droplet size between about 341-403 μm, between about 503-665 μm, greater than about 665 μm, or a stream.

45. The method of claim 39 wherein said sprayer comprises a rotary atomizer.

46. The method of claim 23, wherein said stigma is a maize silk.

47. The method of claim 46, wherein said maize silk is a different heterotic group than the heterotic group corresponding to the stored maize pollen.

48. The method of claim 46, wherein the maize silk is from a tropical or sub-tropical heterotic group and the stored maize pollen is from a temperate heterotic group; or the maize silk is from a temperate heterotic group and the stored maize pollen is from a tropical or subtropical heterotic group.

49. The method of claim 47, wherein the heterotic group is selected from the group consisting of Stiff Stalk, Non-Stiff Stalk, Iodent, and Lancaster.

50. The method of claim 46, wherein the maize silk is a different maturity group than the maturity group corresponding to the stored maize pollen.

51. The method of claim 23, wherein said stigma is a wheat stigma.

52. A method of accelerated trait introgression in the genome of a plant, the method comprising: a. providing a first plant being of a first maturity group; b. cross pollinating the first plant of (a) by the method of claim 23 with stored pollen from a second plant being of a second maturity group and further having a desired trait or phenotype; and, c. selecting a progeny plant from step (b) comprising the desired trait or phenotype; and d. optionally, backcrossing the progeny plant of (c) as the pollen donor onto a recurrent parent plant and selecting progeny plants comprising the desired trait or phenotype.

53. The method of claim 52, wherein the first plant is a maize plant and the second plant is a maize plant.

54. The method of claim 52, wherein the first maturity group is greater than one maturity group away from the second maturity group.

55. The method of claim 23 wherein a carrier is added to said maize pollen prior to adding said maize pollen to said oil.

56. The method of claim 55 wherein said carrier is selected from the group consisting of microcrystalline cellulose, crystalline silica, activated magnesium silicate, and talc.

57. The method of claim 56 wherein said carrier is microcrystalline cellulose.

58. The method of claim 57 wherein said microcrystalline cellulose a. comprises an average particle size between about 42 and about 167 micrometers, or b. comprises a particle size distribution that is expressed as a sieve pass through fractional using sieves ranging between 20 and 500 micrometers (between US MESH 635 and US MESH 35).

59. The method of claim 55, wherein said carrier is present in a pollen:carrier ratio selected from the group consisting of 1:20, 1:30, 1:10, 1:5, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, and any ratio between 1:20 and 50:

1.

60. The method of claim 59, wherein the pollen:carrier ratio is selected from the group consisting of 2:1, 1:1, 1:2, 1:5, and 1:10.

61. The composition of claim 1, wherein the maize pollen is transgenic maize pollen.

62. The composition of claim 61, wherein the transgenic maize pollen comprises a transgenic event selected from the group consisting of MIR162, Bt11, GA21, MIR604, MZIR098, 5307, 3272, DAS40278, TC1507, DAS-59122-7, NK603, MON810, MON863, MON89034, MON88017, DP-4114, and MON87411.

63. The method of claim 23, wherein the maize pollen is transgenic maize pollen.

64. The method of claim 63, wherein the transgenic maize pollen comprises a transgenic event selected from the group consisting of MIR162, Bt11, GA21, MIR604, MZIR098, 5307, 3272, DAS40278, TC1507, DAS-59122-7, NK603, MON810, MON863, MON89034, MON88017, DP-4114, and MON87411.

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