Corn Pollen Storage and Carrier

Abstract

Corn pollen is notoriously fragile and susceptible to deterioration unless properly stored. Unlike some tree pollen, which is fairly hardy and can successfully fertilize for months or years after pollen dispersal, corn pollen remains viable for only a few hours after dispersal before it begins to deteriorate. An invention for storing corn pollen is described herein, where the pollen is collected and stored in a refrigerated environment rather than a frozen environment. Pollen stored as described herein can remain viable for 12 days, or 2 weeks, or longer. Pollen viability can be enhanced by adding a carrier compound.

Description

[Technical field]

[0001] The present invention relates to the field of maize breeding and artificial pollination, in particular to the field of collection, storage and application of stored maize pollen in maize producing fields and greenhouses. [Background technology]

[0002] Pollen storage has been a necessity and goal of plant breeders. See generally W. M. King, Report of chief on seed divisions, In REPORT OF THE COMMISSIONER OF AGRICULTURE (YEARBOOK), Washington DC, GPO, 47-61 (1885) (articulating a desire for stored pollen "so that we may use it at times and places convenient to us"). In some plants, pollen is fairly cold-hardy and long-lived. For example, ginkgo tree pollen can be collected and stored for six months or more without requiring any particular care. In contrast, other plants have pollen that is brittle and subject to rapid senescence within a few hours if left exposed to the elements. Maize (corn) is such a plant.

[0003] In commercial hybrid corn production, it is current practice to alternate four rows of female inbred plants with two rows of male inbred plants. The females are detasseled to prevent self-pollination, and males are grown solely for their ability to pollinate adjacent females. This arrangement works best when the female and male plants are of similar maturity group, i.e., when the males shed their pollen at approximately the same time that the females are receptive to pollen.

[0004] However, the risk with the current practice is that pollination may not be successful and therefore the crop may be lost if the male and female are of different maturity groups. Without storing pollen, the male plants have a greater risk of pollen shedding too early or too late, and the entire field may be lost due to failed pollination. With stored pollen, the pollen can be delivered at exactly the right time, regardless of flowering exposure. Cross-breeding of different maturity groups can be more easily achieved, thus expanding the gene pool and improving maize plant breeding, for example, by creating maize lines that are more drought and / or disease resistant. Summary of the Invention

[0005] Growers need the ability to reliably collect and store corn pollen in one day or at one location, and deliver the pollen to female fields on another day or at another location. To meet this need, a method for storing corn pollen is provided. In one embodiment, a quantity of fresh corn pollen is collected; optionally, the collected pollen is treated with a carrier; the pollen is sealed in a container, and optionally, a container pressure is set on the container; and the pollen is stored in a refrigerated environment. The pollen thus collected and stored is viable for up to 20 days, and at least up to 12 days. In one embodiment, the carrier is talc powder, or silica powder. In another embodiment, the carrier is metal powder or mica. The carrier may be applied at a pollen:carrier ratio of 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 1:2 and 50:1. Preferably, the pollen:carrier ratio is 2:1. In another embodiment, once placed in a sealable container, such as a bottling jar, the container is sealed and container pressure is applied to the container until the container pressure is between 1 atm and 0.01 atm, or between 0.6 atm and 0.3 atm, or between 0.4 atm and 0.35 atm. In another embodiment, a carbon dioxide sequestering agent is added to the sealable container. In another embodiment, the amount of pollen collected may be between about 1 mg and about 54 g. In another embodiment, the amount of pollen collected may be between about 1 mL and about 150 mL or more. In another embodiment, the amount of pollen collected is a desired amount. [Brief description of the drawings]

[0006] [Figure 1]Four seed sets are shown after pollination. The first ear (far left) was pollinated with fresh pollen. Pollen used to pollinate all other ears was stored in a similar manner: pollen was mixed with talc in a 2:1 ratio, then 0.5 mL of the pollen / talc mixture was stored on an aluminium dish in a 125 mL glass container. The container was sealed and then a vacuum was applied until the container pressure reached 0.4 atm before storing at 6 °C. The second ear from the right was pollinated with pollen stored for 3 days. The third ear from the left was pollinated with pollen stored for 5 days. The fourth ear (far right) was pollinated with pollen stored for 7 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] definition All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise specified below. References to technology used herein are intended to mean technology as commonly understood in the art, including variations on the technology and / or equivalent technology substitutes that would be apparent to one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are provided to facilitate the description of the subject matter disclosed herein.

[0008] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" optionally includes a combination of two or more such molecules, and the like.

[0009] As used herein, the term "about" refers to the normal range of error for each value readily known to one of ordinary skill in the art in the field of the present invention, for example, ±20%, ±10%, or ±5% within the intended meaning of the stated value.

[0010] As used herein, the term "carbon dioxide sequestration" means that carbon dioxide ("CO2") is captured by a carbon dioxide sequestrant, such as soda lime, activated charcoal, ethanolamine, zeolite 4A, lithium hydroxide (LiOH), or activated magnesium silicate (e.g., FLORISIL®). In this way, excessive CO2 buildup in the chamber is prevented. Optionally, the sequestrant prevents the chamber from exceeding 10 mmol of CO2 per liter of headspace.

[0011] As used herein, the term "carrier" refers to a compound, preferably in powder form, that acts as an agent to accompany the collected pollen. Suitable carrier compounds can be, but are not limited to, talc powder, silica powder.

[0012] As used herein, "clumping", "aggregating" and similar terms refer to the tendency of pollen to stick together, whether due to excess moisture or other causes, in the absence of a carrier and / or proper storage conditions. Clumped pollen is not mobile and cannot be blown onto the silk by air. Clumped pollen is unlikely to adhere well enough to the silk to be pollinated.

[0013] As used herein, the terms "comprising" or "comprises" are open-ended. When used in the context of a method that includes a series of steps, the method is still carried out as long as the series of steps are performed, even if additional steps are performed.

[0014] As used herein, "crystalline silica" refers to the powdered form of silica derived from quartz or other natural stone structures. "Crystalline silica", "SiO2", and "polycrystalline silica" are used interchangeably throughout. Crystalline silica has structural characteristics that differ from talc or amorphous silica, including, but not limited to, higher mineral Mohs hardness, higher bulk density, and lower specific surface area. In one embodiment, the crystalline silica comprises an average particle size of 1 nanometer (1 nm) to 100 micrometers (100 μm). In another embodiment, the crystalline silica comprises an average particle size of 1 micrometer (1 μm) to 10 micrometers (10 μm). Unless otherwise specified, particle sizes provided herein are average particle sizes.

[0015] As used herein, the term "activated magnesium silicate" refers to powdered synthetic 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 a high specific surface area. In one embodiment, the activated magnesium silicate comprises an average particle size of seventy-five micrometers (75 μm) to one hundred forty-nine micrometers (149 μm). In another embodiment, the activated magnesium silicate comprises an average particle size of less than seventy-five micrometers (<75 μm).

[0016] 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, e.g., an expression cassette containing a gene of interest. The term "line" refers to an initial transformant containing heterologous DNA and / or the progeny of the transformant. The term "line" also refers to the progeny produced by sexual outcrossing of a transformant with another corn line. Even after repeated backcrossing with the recurrent parent, the inserted DNA and the flanking DNA from the transformed parent are present at the same chromosomal location in the progeny of the cross. Usually, transformation of plant tissue produces multiple lines, each of which represents an insertion of the DNA construct at a different location in the genome of the plant cell. A particular line is selected based on the expression of the transgene or other desirable characteristics. Thus, as used herein, "line 3272," "3272," or "3272 line" refers to the original 3272 transformant and / or the progeny of the 3272 transformant and / or a plant derived from the original 3272 transformant in whatever form. For 3272, see WO 06 / 098952.

[0017] Other examples of transgenic lines include, but are not limited to, MIR162 (see WO 07142840), Bt11 (see U.S. Pat. No. 6,114,608 (constructs) and WO 8705629 (genes)), GA21 (see WO 9704103 (genes), WO 9844140 (cassettes)), MIR604 (see WO 05103301), MZIR098 (see WO 18231890), 5307 (see WO 10077816), DAS40278 (see U.S. Pat. No. 8,598,413), T C1507 (see WO 04099447), DAS-59122-7 (see WO 06 / 039376), NK603 (see U.S. Pat. No. 6,825,400), MON810 (see U.S. Pat. No. 6,713,259), MON863 (see U.S. Pat. No. 7,705,216), MON89034 (see WO 07140256), MON88017 (see WO 05059103), DP-4114 (see WO 11084621), and MON87411 (see WO 13169923).

[0018] "Flowable" as used herein means the ability of a powdered material to move easily with the application of air, wind, or sound, or to be poured in a continuous manner and progress steadily and easily.

[0019] As used herein, "hybrid vigor group" refers to a breeding classification of inbred lines. "Hybrid vigor group" and "hybrid vigor pool" are used interchangeably to refer to the relationship between breeding pools of maize populations. Broadly, the main names for the hybrid vigor pools are: Stiff Stalk (also called Iowa Stiff Stalk Synthetic, "SS", or "BSSS"), Non Stiff Stalk ("NSS"), and Iodent ("IDT"). See JvHweerwaarden, et al., Historical genomics of North American maize, PROC. NAT'L ACAD. SCI. USA 109(31):12420-25 (2012). However, these are not exclusive and other names are known, such as Lancaster Sure Crop ("LSC"). See, for example, 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 also Hallauer et al.(1998) COM BREEDING, p.463-564; GF Sprague and JW Dudley (ed.) CORN AND CORN IMPROVEMENT; Smith, et al.(1990) Theor.Appl.Gen.80:833-840; Mikel and Dudley(2006) Crop Set46:1193-1205. See also WO 2020 / 205334 and WO 2021 / 041077, which are incorporated herein by reference in their entireties.

[0020] The term "germplasm" refers to the totality of the genotype of a population or other group of individuals (e.g., a species or plant line). The phrase "adapted germplasm" refers to plant material of proven genetic superiority; e.g., with respect to a given environment or geographic region; the phrases "unadapted germplasm," "raw germplasm," and "exotic germplasm" refer to plant material of unknown or unproven genetic value; e.g., with respect to a given environment or geographic region; the phrase "unadapted germplasm" itself, in some embodiments, refers to plant material that is not part of an established breeding population and has no known relationship to members of an established breeding population.

[0021] As used herein, the term "mica" refers to a compound having the chemical formula X2Y4-6Z8O 20 It refers to a group of minerals generally having the formula (OH,F)4, where X is an alkali or alkaline earth metal, Y is a transition metal, post-transition metal, or alkaline earth metal, and Z is silicon, aluminum, or may include other transition metals.

[0022] As used herein, "starting oxygen content" refers to the amount of oxygen (whether measured in absolute terms, as a percentage, or otherwise) present in the atmosphere of the chamber containing the collected pollen at its initial time and since it was first sealed. In one embodiment, the starting oxygen content is between 0.12 mmol O2 / g pollen / day (days stored) and 0.57 mmol O2 / g pollen / day (days stored). In another embodiment, the starting oxygen content is between 0.24 mmol O2 / g pollen / day (days stored) and 0.57 mmol O2 / g pollen / day (days stored). "Starting oxygen content", "starting O2 mmol", "starting O2 mmol / g pollen", and "starting O2 mmol / g pollen / day (days stored)" are used interchangeably herein.

[0023] A "plant" is a plant at any developmental stage, particularly a seed plant. In particular, in the context of this disclosure, a plant refers to a corn plant. As used herein, the term "plant line" refers to a single plant material or a genetically identical set of material.

[0024] As used herein, "platform" means a surface within a container that is in direct contact with the pollen and carrier mixture and prevents direct contact with the container itself. For example, the platform can be a filter paper or an aluminum tray.

[0025] As used herein, "pollen:carrier ratio" refers to the ratio of pollen present in a mixture with a carrier. For example, and without limitation, a mixture of pollen and carrier having a pollen:carrier ratio of 2:1 contains 2 parts pollen (measured by weight or volume) and 1 part carrier compound, such as talc (measured by weight or volume).

[0026] As used herein, a "refrigerated environment" refers to any condition whose temperature is below ambient (or room) temperature, but not below the temperature at which water freezes. In other words, if the ambient temperature is 25°C, then a refrigerated environment includes temperatures above 0°C and below 25°C. Similarly, a refrigerated environment includes temperatures between 2 and 10°C.

[0027] As used herein, a "sealable container" refers to a container that is capable of forming an air-tight seal. Preferably, the sealable container is also capable of holding a vacuum.

[0028] As used herein, "seed set" refers to the number of kernels produced on a cob resulting from successful pollination. Seed set may be expressed qualitatively (e.g., low, good, or high) or quantitatively. In quantitative measurements, the measurement may be given as a percentage or number of seeds per corn plant. In general, the term refers to the percentage or number of normal kernels (i.e., non-sterile, endosperm viable kernels). For normal corn lines (i.e., not haploid derivatives), greater than 80% seed set (or greater than 300 kernels per plant) is considered good seed set. Achieving good seed set is the goal of controlled pollination.

[0029] "Storage" as used herein refers to the act of storing pollen for a suitable period of time, which may be as little as 24 hours or as long as 12 days.

[0030] As used herein, "treatment" refers to the deliberate application of a chemical compound or environmental constraint to pollen. In particular, pollen treatment may involve the addition of a carrier compound to pollen to preserve pollen fluidity and viability.

[0031] As used herein, "vessel pressure" refers to the artificially applied atmospheric pressure within a vessel. Vessel pressure values ​​are measured herein in units of standard atmospheric pressure "atm", e.g., 0.5 atm, although other units may be used as desired to measure vessel pressure (e.g., Torr or Pascal or "Pa"; 1 Pa=9.8692×10 -6atm). It is expressly contemplated that the vessel pressure can meet or exceed 1 atm. Under conditions where the vessel pressure is between 0 and 1 atm, "vessel pressure" and "vacuum" have the same meaning and are used interchangeably. Although "vessel pressure" is the preferred term, both vacuum conditions and conditions where the artificially applied air pressure exceeds the ambient atmosphere (e.g., 1 atm) are contemplated. In some embodiments, the vessel pressure is 1 atm, 2 atm, or 3 atm absolute. In some embodiments, the vessel can be pressurized with pure oxygen gas at 18 mmol, 24 mmol, or 27 mmol of O2 per liter of reservoir headspace.

[0032] As used herein, "vigor" refers to the ability of pollen to attach to silks, grow a pollen tube, and successfully fertilize an egg cell. "Vigor," "viable," "vigor," and similar terms are used interchangeably with "vigor."

[0033] Detailed Description Because large quantities of inbred parental line seeds are required to produce hybrid seeds sold to consumers, the productivity of corn seed production (i.e., a measure of whether the required amount of inbred or hybrid seeds can be produced by self- or cross-pollination at an economic cost that does not exceed the value of the seeds produced) is an important factor in the successful development of corn inbred parental lines. Even if the inbred parental lines are capable of producing hybrids with characteristics that are desirable to customers (e.g., GM and genome editing traits leading to high yields, disease resistance), corn inbred parental lines with low productivity may be discontinued due to excessive costs in parental seed production. Pollen storage techniques can be used to increase the productivity of inbred corn parental lines used in hybrid seed production.

[0034] Challenges to production potential that may be addressed by pollen storage techniques include, but are not limited to, low pollen production, low total pollen shedding, short duration of pollen shedding, short window of silk pollination, and GM or genome edited traits that may affect plant reproductive characteristics. An additional challenge with self-pollination is long self-split, defined by the number of days between when pollen shedding begins and when silk appears and becomes available for pollination. In some replicates, self-split may be a negative value, where silk appears for pollination before the onset of pollen shedding. The self-split observed may be the result of inbred parent line genetics, or it may be the result of stresses in the growing environment that reduce the rate of silk elongation and increase the number of days between the onset of pollen shedding and the availability of silk for pollination.

[0035] To address the issue of producibility, pollen storage techniques can be used to collect pollen during the optimal window of pollen dispersal, store the pollen while maintaining pollen viability, and then apply the pollen during the optimal window for silk emergence and pollination. In some iterations, pollen collection can be performed at multiple times per day. In other iterations, pollen can be collected on multiple days throughout pollen dispersal. The application of stored pollen uses combined pollen collected over multiple days, and multiple applications can be made on the same day or over multiple days. In pollen application, combined pollen collected from multiple field locations can be used in one application to one location. In some iterations, pollen can be collected in one geography and applied to silk in different geographies. The geographies can be different fields in the same production location, fields in different states or municipalities within a country, or fields in different countries. In some iterations, pollen is collected from a temperate corn inbred parent line grown in a temperate region and applied to a subtropical or tropical corn inbred parent line grown in a subtropical or tropical region. In other iterations, pollen is collected from a subtropical or tropical corn inbred parent line grown in a subtropical or tropical region and applied to a temperate corn inbred parent line grown in a temperate region. By addressing these challenges to production potential, pollen storage techniques may enable increased seed production of desirable corn inbred parent lines that produce new hybrids with desired characteristics to be sold to customers. Pollen storage techniques may also enable economical hybrid seed production for combinations of temperate, subtropical, and tropical corn inbred parent lines that are not currently feasible.

[0036] Thus, one embodiment provides a composition comprising corn pollen and crystalline silica. In one aspect of the composition, the crystalline silica comprises an average particle size. In another aspect, the average particle size is from about 1 nanometer to about 100 micrometers. In another aspect, the average particle size is from about 1 micrometer to about 10 micrometers. In yet another aspect, the corn 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 older.

[0037] Another embodiment provides a method of storing viable corn pollen comprising the steps of: a) collecting a quantity of fresh corn pollen; b) optionally applying a carrier to the collected corn pollen of step a) to obtain a quantity of treated corn pollen; c) placing the quantity of fresh corn pollen or the quantity of treated corn pollen in a sealable container and optionally setting a container pressure; and d) storing the product of step c) in a refrigerated environment. In one aspect of the method, the stored corn pollen remains viable for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days. In one aspect, the container comprises a volume between 1 mL and 100 L, or the container comprises a volume between 10 mL and 20 L, or the container comprises a volume of about 12 L, about 1.8 L, about 1 L, 500 mL, or about 125 mL. In another embodiment of the method, the amount of fresh or processed corn pollen is at least about 54 g, or at least about 25 g, or at least about 11 g, or at least about 720 mg, or at least about 360 mg, or at least about 180 mg, or at least about 90 mg, or at least about 45 mg, or at least about 1 mg. In another embodiment of the method, the vessel pressure is about 0.6 atm to 0.3 atm, or the vessel pressure is about 0.4 atm to 0.35 atm. In another embodiment of the method, the carrier is selected from the group consisting of crystalline silica, activated magnesium silicate, talc, metal powder, and mica mineral. In one embodiment, the metal powder is a metal oxide powder or a metal carbide powder. In another embodiment, the metal powder is a powder of an average particle size. In one embodiment, the average particle size is spherical with a particle size of 10 μm. In another embodiment, the metal powder is a stainless steel powder.

[0038] In another aspect, 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. In one aspect, the pollen:carrier ratio is 2:1. In another embodiment of the method, the sealable container comprises a platform. In one aspect, the platform comprises a material that reduces pollen clumping due to clot formation. In another aspect, the platform comprises an aluminum tray, a copper tray, a nickel tray, or a stainless steel tray. In another embodiment, the sealable container comprises a material that reduces pollen clumping due to clot formation. In another aspect, the sealable container is fabricated from glass, aluminum, acrylic, or stainless steel. In another embodiment of the method, the refrigerated environment comprises a temperature range selected from the group consisting of 1° C. to 10° C., 4° C. to 8° C., and 5.5° C. to 6.5° C. In one aspect, the refrigerated environment comprises a temperature of about 6° C. In another embodiment of the method, the pollen is stored in the refrigerated environment for 20 days or less, 19 days or less, 18 days or less, 17 days or less, 16 days or less, 15 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. In one aspect, the pollen is stored for 12 days or less. In another embodiment of the method, the sealable container comprises an initial oxygen content. In one aspect, the starting oxygen content is between 0.12 mmol O2 / g pollen / day (days stored) and 0.57 mmol O2 / g pollen / day (days stored). In another aspect, the starting oxygen content is between about 0.24 mmol O2 / g pollen / day (days stored) and 0.57 mmol O2 / g pollen / day (days stored). In another embodiment of the method, the sealable container comprises a CO2 sequestering means.In one embodiment, the sequestering agent is selected from the group consisting of activated carbon, ethanolamine, zeolite 4A, lithium hydroxide (LiOH), soda lime, calcium silicate (Ca2O4Si), and activated magnesium silicate (e.g., FLORISIL®).

[0039] Another embodiment provides a method of applying stored corn pollen to the stigma comprising: a) obtaining stored corn pollen by the method described above; and b) applying stored pollen to silk, wherein the stored corn pollen is applied to the silk after collection. In another embodiment, the stored corn pollen is applied to the stigma at least one day after collection. In one aspect, the stigma is corn silk. In another aspect, the corn silk is of a different hybridization group than the corresponding hybridization group of the stored corn pollen. In yet another aspect, the corn silk is from a tropical or subtropical hybridization group and the stored corn pollen is from a temperate hybridization group; or the corn silk is from a temperate hybridization group and the stored corn pollen is from a tropical or subtropical hybridization group. In another aspect, the hybridization group is selected from the group consisting of Stiff Stalk, Non-Stiff Stalk, Iodent, and Lancaster. In another embodiment, the corn silk is of a different maturity group than the maturity group corresponding to the stored corn pollen. In yet another embodiment, the stigma is a wheat stigma.

[0040] In another embodiment, the stored corn pollen is transgenic corn pollen. In another aspect, the transgenic corn pollen comprises a transgenic line 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 aspect, the transgenic corn pollen comprises the transgenic lines Bt11, GA21, and MIR162. In another aspect, the transgenic corn pollen comprises the transgenic lines Bt11 and MIR162. In yet another aspect, the transgenic corn pollen comprises the transgenic line MIR162.

[0041] In another embodiment, the vessel pressure used during the storage method is pressurized with standard atmospheric oxygen and pressurized with pure oxygen gas. In one embodiment, the standard atmospheric oxygen is at 1 atm absolute pressure. In one embodiment, the standard atmospheric oxygen is at 2 atm absolute pressure. In another embodiment, the standard atmospheric oxygen is at 3 atm absolute pressure. In another embodiment, the pure oxygen gas is at 4 mmol O2 per liter of storage vessel headspace. In another embodiment, the pure oxygen gas is at 7 mmol O2 per liter of storage vessel headspace. EXAMPLES

[0042] 1. Collection Corn plants were grown under field and greenhouse conditions. When the tassels emerged and began to shed pollen, the tassels were covered with bags to collect the pollen. The bags were usually covered during the late afternoon and removed the following morning. After being sieved from the anthers or other tassel material and optionally mixed with a carrier, the collected pollen was then placed in a suitable sealed container. Alternatively, pollen is collected from the corn plants by harvesting the tassels from which pollen has previously been shed. The tassels can be placed in a beaker of water and the pollen can be normally removed, or the tassels can be dried, soaked to macerate, filtered, and the pollen collected mechanically. See, for example, U.S. Patent No. 8,252,988 (filed June 27, 2007), the entire contents of which are incorporated herein by reference.

[0043] 2. Microbial growth Microbial analysis was performed to understand whether bacteria or fungi were present in the collected pollen in sufficient quantities to contribute to the deterioration of fresh pollen. The effect of microbial pressure was first visually confirmed in pollen stored at 23°C. All pollen samples were mixed in a pollen:talc ratio of 2:1 before storage. After 4 days of sealed storage at 23°C, the stored pollen was covered with swathes of microbial colonies. To better understand the composition of these microorganisms and how they change depending on the environmental conditions, 3M Petrifilm was used on fresh pollen from the greenhouse and the field. In particular, the presence of molds, aerobic bacteria, lactic acid bacteria, and heterotrophic bacteria was assayed. Not surprisingly, pollen collected from field-grown plants probably served as a host to several orders of magnitude more microbial pressure (Table 1). Furthermore, heterotrophic bacteria, absent in greenhouse pollen, were found in numbers similar to aerobic bacteria in field pollen, and in both cases the presence of lactic acid bacteria was equivocal, whereas the presence of molds was significant (Table 1).

[0044] Table 1. Colony forming units (CFU) of various microorganisms in 50 uL of fresh greenhouse and field pollen. [Table 1]

[0045] The presence of these microorganisms could be controlled by antimicrobial treatment of the pollen or by changing the storage environment. Therefore, temperature was assayed as a means of effective control of microbial growth on stored pollen. 0.36 g of pollen as a 2:1 mixture of pollen:talc was stored in 125 mL sealed glass containers at 23°C, 6°C, and 2°C at 1 atm pressure for 7 days, and O2 consumption and CO2 production in the sealed storage containers were recorded on each day. At 4 days of storage, the 23°C samples no longer germinated pollen tubes in the medium, nor were they stained with the enzyme stain, MTT (Tables 2 and 3). As previously described, visible microbial colonies also grew on these samples. Conversely, the 6°C and 2°C samples did not show any visible signs of microbial growth, and pollen tubes successfully germinated and stained with MTT for more than 6 days of storage (Tables 2 and 3). At 7 days of storage, the 2°C samples no longer germinated but stained positively with MTT, indicating how capable the pollen tubes were to germinate before complete loss of enzyme activity on pollen desiccation.

[0046] Table 2. Observations of pollen tube germination in pollen samples stored at different temperatures. [Table 2] ND = no data

[0047] Table 3. Enzyme staining observations in pollen samples stored at different temperatures. [Table 3] ND = no data

[0048] Over the course of the first 6 days of storage, oxygen consumption and CO2 production in samples stored at 6°C and 2°C were less than 17% of those observed in samples stored at 23°C (Table 4). This difference is due to the forced reduction in metabolic rates of both pollen and microorganisms at the lower temperature. This is highlighted by the absence of visible microbial growth in the 6°C and 2°C samples seen in the 23°C samples after 4 days of storage. A further reduction in metabolic rate was observed between samples stored at 6°C compared to those stored at 2°C, with overall vitality being nearly the same up to 6 days (Tables 4, 2, and 3). Taken together, these results indicate that reduced temperature impedes microbial respiration and acts as an effective treatment against undesirable microbial growth on stored pollen.

[0049] Table 4. Final amounts of O2 and CO2 and rates of O2 use and CO2 production on the sixth day of storage at different temperatures. [Table 4]

[0050] 3.Storage temperature As shown above, storage temperature clearly plays an important role in determining pollen and microbial metabolic rates during storage. Pollination was also performed to test the effect of temperature on pollen vigor. A reduction in seed set over time occurred for all temperatures tested. However, the reduction in seed set over time was magnified at 2°C and 23°C relative to 6°C (Table 5). To further evaluate the effect of temperature on pollen vigor, additional experiments were conducted for 7 days using storage temperatures ranging from about 4.3 to 8.7°C (Table 6). For all treatments, 0.18 g of pollen was mixed with talc in a pollen:talc ratio of 2:1 prior to storage in 125 mL glass containers at 0.4 atm. Combining the data from Table 5, reduction in seed set occurred at temperatures below 4°C and above 6°C, with the most dramatic reductions identified at 2°C and 23°C. The temperatures tested for pollination were not completely prohibitive to storage success, but instead affected shelf life (Tables 5 and 6). In conclusion, these studies indicate that temperatures of at least 2°C to 23°C are acceptable for pollen storage, although longer storage periods may be achieved at lower temperatures of about 2-8°C.

[0051] Table 5. Decay of seed set over time at different storage temperatures. [Table 5]

[0052] Table 6. Seed set from pollen stored for 7 days at various storage temperatures. [Table 6]

[0053] 4. Oxygen and Carbon Dioxide As shown in Example 2, pollen remains metabolically active during storage - utilizing O2 and producing CO2 during aerobic respiration. As pollen continues to respire during storage, O2 is inevitably depleted at the same time as CO2 increases. To ascertain the effect that these changes in container atmosphere may have on pollen, several experiments were performed. First, several weights of pollen were stored as a 2:1 pollen:talc mixture at 6°C at 0.4 atm in 125 mL standard containers (e.g., 4 oz. Ballmason jars) with starting oxygen of 0.44 mmol (20.9%) and starting CO2 of 0.00084 mmol (0.04%). After 7 days of storage, the end point oxygen and CO2 readings were measured (Table 7). Samples of 0.54 g or more were depleted to less than 0.1 mmol of oxygen and showed no pollen tube germination on the medium. Samples over 0.54 g were nearly depleted of all available oxygen and failed to stain with MTT, indicating a complete lack of viability. In this same experiment, CO2 was confirmed to accumulate in a negative linear pattern with oxygen depletion. Thus, by the end of the 7-day storage period, samples that had exhausted most of the available oxygen had accumulated over 0.4 mmol of CO2.

[0054] Table 7. Oxygen utilization and CO2 production from various weights of pollen stored for 7 days. [Table 7]

[0055] A second experiment was conducted to test the effect of CO2 accumulation on pollen vigor. In this experiment, 125 mL containers containing 0.36 g pollen mixed with 0.18 g talc were spiked with increasing amounts of CO2 before storage (maintaining total container pressure at 1 atm) and then stored at 6°C. Pollen tube germination was then assayed on a 1 to 5 scale for each treatment once per day for 6 days (Tables 8 and 9). As expected, a baseline starting CO2 (approximately 0.002 mmol, the amount present in 125 mL of natural air at room temperature) resulted in good germination throughout the 6-day storage period. Pollen longevity responded negatively to increasing starting CO2, with all containers with more CO2 than atmospheric starting CO2 showing a reduction or loss of pollen tube germination earlier than the baseline (Table 9). These results demonstrate the toxicity of accumulated CO2 on pollen grains and the importance of its control and / or sequestration during storage.

[0056] Table 8. Germination rating scale. [Table 8]

[0057] Table 9. Pollen tube germination over time for pollen stored with increasing amounts of starting CO2. [Table 9]

[0058] To further investigate the metabolic rate within the storage vessel, we calculated the average O2 utilization and CO2 production from 245 individual samples. All samples were stored at 6°C for 5-7 days with a 2:1 pollen:talc ratio. At the end of storage, samples were included only if their germination score was 2 or higher. Samples were stored under pressures ranging from 0.13 to 1 atm and with pollen amounts ranging from 0.09 to 25 g. For pollen undergoing aerobic respiration, O2 and CO2 are exchanged 1:1 stoichiometrically. As a result, the amount of oxygen used and the amount of CO2 produced during aerobic respiration were approximately equal. Based on the 245 samples, we found that the average O2 utilization and CO2 production / g pollen / day was approximately 0.17 mmol (Table 10). The maximum metabolic rate was almost double at 0.30 mmol / g pollen / day, and the minimum metabolic rate was as low as 0.01 mmol / g pollen / day.

[0059] Table 10. Mean, maximum and minimum metabolic rates of pollen during storage. [Table 10]

[0060] After showing that CO2 accumulation during storage is harmful to the pollen, our next step was to identify a way to passively sequester CO2 safely during storage. There are many chemical compounds that actively bind CO2 and remove it from the atmosphere. In a new experiment, we tested the ability of soda lime to sequester CO2 in a storage vessel setup. Soda lime utilizes water vapor and binds CO2 in a reaction that produces CaCO3 and heat. Therefore, it is expected that water vapor in the storage environment will be consumed as CO2 is produced by the pollen and subsequently sequestered by the soda lime. To offset this loss of water vapor, we also tested the addition of liquid water to the storage vessel. In each 125 mL vessel, the starting oxygen was about 1 mmol, the starting CO2 was about 0.002 mmol, and the vessel pressure was 1 atm. Vessels were tested with and without 0.05 g soda lime and with and without 10 mL water. Pollen amounts also varied from 0.18 to 0.72 g, but all pollen was mixed with talc in a pollen:talc ratio of 2:1 5 days before storage at 6°C. Percent pollen moisture content (PMC) was measured as the difference between fresh and dry weights. The initial pollen moisture content in this experiment was 53%.

[0061] For all pollen amounts, the containers containing soda lime showed positive sequestration of CO2, reflected in lower residual CO2 mmol relative to the same weight of pollen in containers without soda lime (Table 11). Due to the detection limit of the equipment used, it was not possible to detect the complete absence of CO2, since samples with 0.01 mmol of residual CO2 were considered to have all CO2 sequestered. For pollen amounts above 0.36 g, 0.05 g of soda lime was insufficient to completely sequester all CO2 over the 5 days. Therefore, the amount of soda lime had to be scaled to the amount of pollen in the container (approximately 0.02 g soda lime / g pollen / day). In containers that did not contain additional water, the PMC was significantly reduced - likely resulting in moisture being pulled from the pollen into the atmosphere as water vapor is utilized by the soda lime, restabilizing humidity (Table 11). Interestingly, the containers containing water showed a slight increase in final PMC relative to the initial PMC. This may indicate a moderate uptake of water vapor by the pollen over time. Together the results of this experiment demonstrate the effectiveness of soda lime as a CO2 sequestrant for pollen storage and the importance of a secondary water vapor source to offset the drying effect of the soda lime reaction.

[0062] Table 11. CO2 sequestration in containers with and without soda lime and water after 5 days of storage with various amounts of pollen. [Table 11]

[0063] After confirming the effectiveness of soda lime in sequestering released CO2 in the storage vessel, we performed additional experiments on the second day of storage to assay the effectiveness of other sequestering compounds. We assayed activated carbon, ethanolamine, zeolite 4A, lithium hydroxide (LiOH) and activated magnesium silicate, FLORISIL®, at 100-200 mesh and below 200 mesh. When measuring particle size, "mesh" is defined by the number of holes in a linear inch, e.g., a 200 mesh screen has 200 holes in a linear inch and a 100 mesh screen has 200 holes in a linear inch. Thus, a 200 mesh powder has finer particles than a 100 mesh powder. See, e.g., ASTM E11-20, Standard Specification for Woven Wire Test Sieve Cloth and Test Sieves, ASTM International, West Conshohocken, PA, 2020, www.astm.org. Soda lime and no sequestering agent treatments were used as controls. To assay the effectiveness of each CO2 sequestering agent, 0.02-9.6 g of sequestering agent was added to 125 mL glass jars containing 1 mL of water (Table 12). To mimic the typical amount of CO2 released / g pollen / day, the jars were sealed, the container pressure was reduced to 0.9 atm, and then the jars were filled with compressed CO2 until the internal pressure reached 1 atm. This resulted in approximately 0.4 mmol of CO2 and approximately 0.95 mmol of oxygen per container. The containers were then placed at 6°C for 2 days. After this period, the containers were returned to room temperature (approximately 21°C) and the final CO2 and oxygen contents were measured (Table 12). In general, all sequestering agents were successful in sequestering some of the provided CO2, but the amount required and the efficiency of sequestration varied by compound.

[0064] Table 12. Sequestration capacity of various chemical CO2 sequestrants in vessels starting at approximately 0.4 mmol of CO2 and approximately 0.95 mmol of oxygen per container. [Table 12]

[0065] Additionally, the inventors tested the effectiveness of mixing one of the sequestration compounds, FLORISIL®, directly with pollen. In this experiment, 0.18 g of pollen was mixed with 0.09 g of FLORISIL® at <200 mesh. The pollen-FLORISIL® mixture was then placed in a 125 mL container, the container pressure was reduced to 0.4 atm, and the container was then placed at 6° C. for 5 days. After storage, the pollen-FLORISIL® mixture was used in pollinations, using 0.5 mL of the mixture for each pollination. The average seed set from these pollinations was 107 seeds, with a standard deviation of 76 seeds. Thus, it is possible to mix pollen directly with CO2 sequestration and achieve seed set even after storage.

[0066] A follow-up experiment was conducted to test the combined effect of increasing the initial oxygen content and sequestering CO2 produced during aerobic respiration. In this study, 0.9g of pollen mixed with 0.45g of talc was stored for 5 days in 125mL containers at 6°C and 1 atm with 0.208g of soda lime and various initial amounts of oxygen (Table 13). In contrast to the experiment from Table 7 above, where 0.9g of pollen used up 0.44mmol of available O2 before the end of storage, the presence of excess oxygen in this experiment allowed the pollen to maintain the ability to germinate and set seeds for the 5 days tested. Neither metabolic rate nor PMC was affected by the different oxygen treatments. However, germination rate appeared to decrease as the initial oxygen concentration increased. These results demonstrate the benefits of adjusting the initial oxygen content and passive sequestering of respired CO2 in our closed loop system.

[0067] Table 13. Effect of different starting oxygen concentrations on the vitality characteristics of stored pollen. [Table 13]

[0068] Further testing included various experiments with different inbreds, hybrids, and storage duration conditions in pressurized container storage systems. Across these experiments, we evaluated absolute pressures (atm) up to 3 atm, as well as starting O2 mmol per liter of storage container headspace. As can be seen from Table 14 below, we were able to recover seeds from stored pollen in conditions using sealed containers containing standard atmosphere or a mixture of standard atmosphere and pure oxygen gas, pressurized to 3 atm absolute pressure. Additionally, we were able to recover seeds when pollen was stored in containers with starting oxygen concentrations up to 27 mmol O2 per liter of container storage headspace.

[0069] Table 14. Average stored pollen performance, using pollen stored in containers pressurized with standard atmosphere or a mixture of standard atmosphere and pure oxygen gas. This table shows a comparison between the various conditions tested. [Table 14] N = number of spikelets

[0070] 5. Carrier Compounds Crystalline quartz silica is a better carrier for pollen storage than amorphous silica and silicates. Existing examples of pollen storage technology use talc (magnesium silicate hydrate) or amorphous silica (precipitated, pyrogenic, or silica gel) as a carrier to prevent pollen clumping. Synthetic amorphous activated magnesium silicate (e.g., FLORISIL®) can also be used to prevent pollen clumping. While these existing carriers are effective in preventing pollen clumping by inhibiting the interaction between the cell membranes of pollen grains, their structure can also inhibit the interaction between pollen grains and silk during pollination with stored pollen. Furthermore, the unique properties of amorphous silica and silicates, which are not found in crystalline silica, are detrimental to pollen viability in storage.

[0071] Talc exists as phyllosilicate sheets with the chemical formula Mg3Si4O 10Talc is a soft clay mineral of (OH)2. These soft, lamella-like sheets break apart when mixed with pollen, completely covering the pollen surface. This coating inhibits both the cohesive interactions between pollen particles and the pollen-silk interactions necessary for the initiation of pollen tube germination. The act of mixing pollen with talc can reduce the potential seed set that can be produced by the pollen. Common alternatives to talc in pollen storage applications include amorphous forms of silica (precipitated, pyrogenic, or silica gel). Synthetic amorphous activated magnesium silicate (e.g., FLORISIL®) is also an effective carrier in pollen storage, depending on the storage method used. All forms of silica share the chemical formula SiO2, and activated magnesium silicate is represented by the chemical formula MgO3Si. Precipitated silica, silica gel, and synthetic amorphous magnesium silicate offer similar benefits to talc by inhibiting the cohesive interactions between pollen particles. However, they are detrimental to pollen viability in storage. These compounds have a high specific surface area and have been shown to dehydrate pollen during storage. Pyrogenic silica consists of low density, polymer-like, silica agglomerates and is the most efficient at inhibiting cohesive interactions between pollen particles. Pyrogenic silica also acts as a desiccant in storage, which is detrimental to pollen viability. When pollen is mixed with pyrogenic silica, the pollen particles bind to the silks and the pollen tubes are inhibited from germinating, thus pyrogenic silica eliminates the possibility of fresh or stored pollen seed set to the same extent as if the pollen were not viable. Due to their desiccation, low mineral hardness, and high specific surface area properties, talc, amorphous silica, and amorphous silicates are not effective carriers in pollen storage techniques.

[0072] Silica is commonly found in nature as the crystalline mineral, quartz. Crystalline silica can exist in multiple polymorphic crystalline forms. A mixture of these polymorphs can be called polycrystalline silica. Crystalline silica has structural properties that differ from talc or synthetic amorphous silica, including but not limited to higher mineral Mohs hardness, higher bulk density, and lower specific surface area. Crystalline silica inhibits the cohesive interactions between pollen particles during storage, but does not overly coat the pollen particles immediately upon application or during handling, which would cause the carrier particles to break apart. Additionally, crystalline silica does not act as a desiccant.

[0073] Pollen samples mixed with crystalline silica show more uniform and higher seed set after storage than talc (Tables 17, 18, and 19). The preferred average crystalline silica particle size is believed to be 10 μm (Table 19), although particle sizes ranging from nanoparticles (e.g., 1 nm) to 100 μm may be used in various applications. Pollen samples mixed with activated magnesium silicate show similar performance to pollen mixed with talc (Tables 16 and 20).

[0074] Table 15. Particle size, specific surface area, and bulk density of pollen storage carriers. [Table 15]

[0075] Table 15 describes the important properties of the pollen storage carriers that differentiate crystalline silica from other silicates. Particle size and specific surface area values ​​are taken from the manufacturer's specifications. Bulk density was measured directly on the carrier preparations used during the pollen storage experiments.

[0076] Table 16. Fresh pollen seed sets for three types of carriers. [Table 16]

[0077] The industry standard has been to use talc as a carrier when working with fresh pollen. See, for example, U.S. Patent No. 2,570,511, filed May 22, 1946. Both crystalline silica and activated magnesium silicate perform similarly to talc when working with fresh pollen. All carriers were mixed with pollen in a ratio of 2 parts pollen:1 part carrier by weight. Pollination occurred within 1 hour on receptive silks.

[0078] Table 17. Stored pollen seed sets for two types of carriers. [Table 17]

[0079] This experiment demonstrates that storing pollen with crystalline silica results in greater seed set than pollen stored with talc. Both carriers were mixed with pollen in a ratio of 2 parts pollen:1 part carrier by weight. Pollination was carried out after 4 days of storage on acceptable silk.

[0080] Table 18. Stored pollen seed sets for two types of carriers. [Table 18]

[0081] The standard deviations measured in this experiment further demonstrate that the use of crystalline silicate carriers improves pollination performance by reducing variability in seed set among ears. Both carriers were mixed with pollen at a ratio of 2 parts pollen:1 part carrier by weight. Pollination was performed after 7 days of storage on acceptable silks.

[0082] Table 19. Comparison of different average sizes of crystalline SiO2. [Table 19]

[0083] This experiment demonstrates that 1 μm or 10 μm average particle size preparations of crystalline silica are excellent choices for storing corn pollen. The larger 45 um average particle size preparation was successful in preserving pollen viability in storage and may be an excellent choice for other pollen types. All carriers were mixed with pollen at a ratio of 2 parts pollen:1 part carrier by weight. Pollination was performed after 7 days of storage on acceptable silk.

[0084] Metal powders are effective carriers for pollen storage that do not exhibit the same disadvantages as talc powder and amorphous silicates. Metal powders prevent cohesive interactions between adjacent pollen grain membranes during storage, but do not overly cover the pollen membrane surface and inhibit attachment to the stigma of corn silks or other plants. This lack of inhibition allows for effective pollen tube germination and makes these carriers superior to talc powder and amorphous silica. Elemental metal powders, metal oxide powders, and metal carbide powders are all effective pollen storage carriers. These powders can be produced by a variety of techniques to optimize functionality, such as solid-state reduction, electrolysis, chemical reaction, high-temperature combustion, gas atomization, water atomization, pressing and sintering, centrifugal atomization, grinding, and other polishing techniques to optimize particle size and particle surface properties. The optimal particle type for metal powders in corn pollen storage is believed to be 10 μm polished spherical particles, but other particle sizes and surface properties may be better for other pollen types. In some applications, metal powders can be coated with polymers to modify particle surface interactions with the pollen membrane. In other applications, metal particles can be coated with active ingredients to modify interactions with the pollen grain membrane, to modify pollen and microbial respiration in storage, or to inhibit microbial growth during storage. These active ingredients include nucleic acids, proteins, pesticides, or biostimulants. Metal carriers include weathering with a known biological role in plants that can enhance pollen performance, and carriers with no known biological role that do not affect pollen performance. Ferromagnetic carriers may be preferred in applications where the carrier can be magnetically removed from the pollen carrier after storage to increase the concentration of pollen in the mixture.

[0085] Mica is a group of minerals defined by a general chemical formula and perfect basal cleavage. Perfect basal cleavage results in platelet-shaped particles that are effective in preventing interactions between adjacent pollen grain membranes. In addition to the physical properties that make mica minerals effective carriers for pollen storage, the high reflectance of mica minerals can act as a visual indicator during pollen application. These reflective properties can be visualized by protocol operators or camera machines to follow the distribution of pollen during application or to confirm where pollination has occurred.

[0086] Table 20 details the performance of crystalline silica, metal powder supports, and mica as supports in pollen storage. All supports in this test perform similarly to crystalline silica.

[0087] Table 20: Seed set from pollen stored on 10 different carriers. [Table 20]

[0088] All carriers were mixed with pollen in a ratio of 2 parts pollen:1 part carrier by weight. 10μm 316L stainless steel powder was produced by high temperature combustion, with individual particles having amorphous properties. Ultra-high pressure water and gas atomization along with milling to form uniform spherical particles are optimized for 3D printing.

[0089] Table 21. Stored pollen seed sets for two types of carriers in two independent experiments. [Table 21]

[0090] These experiments demonstrate the conflicting results obtained from using talc or activated magnesium silicate carriers on pollen stored for 5 days. The mean and standard deviation for experiment 2 are the same whole numbers after rounding. For both experiments, the carrier was mixed with the pollen in a ratio of 2 parts pollen:1 part carrier by weight. After 5 days of storage, pollination was performed on the same population of acceptable silks.

[0091] 6. Pollen Storage of Plants Containing Transgenic Lines The male score is an overall assessment of performance as a pollen source that accounts for all data types collected during the development of the inbred parent line. The male performance scores from highest to lowest are Desirable, Acceptable, Marginal, and Do Not Advance. Pollen from the five inbred parent lines containing transgenic lines that received an overall male score of Desirable, Acceptable, Marginal, and Do Not Advance was collected separately and mixed with crystalline silica carrier in a ratio of 2 parts pollen:1 part carrier by weight. A portion of the pollen mixed with crystalline silica was used to perform fresh self-pollination (0 days of storage). The remaining pollen + crystalline silica mixture was stored in a sealed container with added soda lime in a 6°C environment. After 5 days, the stored pollen was applied to silks on the same inbred line providing the pollen (i.e., self-pollination). All ears received the same amount (by volume) of fresh pollen and carrier or stored pollen and carrier.

[0092] Table 22. Seed set from stored pollen for five different inbred parent lines. Inbred lines 1-4 include the transgenic lines Bt11, GA21, and MIR162. Inbred line 5 includes the transgenic lines Bt11 and MIR162. [Table 22]

[0093] Materials and Methods 1.MTT staining The use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as a colorimetric assay for cell viability is common. Positive staining of cells with MTT (visible purple coloration) indicates activity of NAD(P)H-dependent cellular oxidoreductases and is therefore an indicator of metabolic activity of live cells. In our study, 200 uL of 0.9% MTT, 5% sucrose solution was added to approximately 20 uL of pollen, covered and left at room temperature for 5 minutes, then stained and intensity recorded. Enzyme activity is one of the last processes to be stopped in the drying of pollen and is a reliable indicator of whether the tissue is truly dead (in which case a complete lack of staining is confirmed). Positive staining should be combined with other assays such as pollen tube germination and pollination to fully assay the vitality of the pollen and its ability to fertilize.

[0094] 2. Pollen Tube Germination Prior to fertilization of the embryo, maize pollen must germinate a pollen tube through the silk threads to deliver sperm cells to the ovary. This ability to germinate pollen tubes can be assayed on solid media. Pollen is sprinkled on the surface of the medium, allowed to rest on the bench for 60 minutes at room temperature, and then observed or described. The number of pollen grains with pollen tubes is counted or scored. Quantitative scoring of germinated pollen grains is possible, but very time consuming. As an alternative, a ranking on a categorical scale can be assigned to indicate the degree of pollen tube germination / germination potential of a given pollen sample. In our studies, a scale of 1-5 was used, where 1=no germination, 2=1-20% of pollen grains germinated, 3=21-40% of pollen grains germinated, 4=41-60% of pollen grains germinated, and 5=>60% of pollen grains germinated. We counted pollen grains with pollen tubes longer than or equal to the diameter of a single grain after 60 min of incubation as successful germination.

[0095] 3. Measurement of pollination and seed set with stored pollen There are several tools that can be used to understand the approximate viability of stored pollen. These tools include MTT staining, pollen tube germination, and impedance flow cytometry devices such as Ampha Z32 (Amphasys; amphasys.com / ampha-z32-pollen-analyzer / ). Each of these tools has its uses, and when used in combination, the ability of pollen to fertilize an ovule can be adequately estimated. However, seed set measurements remain the strongest indicator of this trait.

[0096] Seed set measured in the experiments described herein was produced from controlled greenhouse or field pollinations. Each ear used for pollination was bagged before silking to prevent contamination from wind-borne pollen. Pollen used in storage is usually collected from multiple tassels of the same line and then combined into a single batch of pollen. Each batch was thoroughly mixed before being distributed into individual sample containers. After storage, the pollen can be applied directly to the ear from the storage container or it can be combined again and a subsample used for pollination. Pollination is done manually - the ear bag is removed long enough to perform hand pollination, and then the ear is bagged with a larger ear bag to prevent contamination from outside pollen sources. The kernels are then allowed to develop for 12-14 days, when the ears are harvested for kernel counting. The kernels are counted manually or with image analysis software. Kernels that did not fully develop are not included in the kernel count.

Claims

1. A composition comprising corn pollen and crystalline silica, wherein the crystalline silica has an average particle size.

2. The composition according to claim 1, wherein the average particle size is from about 1 nanometer to about 100 micrometers, or from about 1 micrometer to about 10 micrometers.

3. The composition according to claim 1, wherein the corn 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 older.

4. a) collecting a quantity of fresh corn pollen; b) applying a carrier to the collected corn pollen in step a) to obtain a quantity of treated corn pollen; c) placing the quantity of fresh corn pollen or the quantity of treated corn pollen in a sealable container and optionally setting the container pressure; and d) storing the product of step c) in a refrigerated environment, wherein the stored corn pollen maintains a viable state for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days A method for storing viable corn pollen, comprising.

5. The method according to claim 4, wherein the container has a volume of 1 mL to 100 L.

6. The method according to claim 4, wherein the quantity of fresh corn pollen or treated corn pollen is from about 1 mg to 54 g.

7. The method according to claim 4, wherein the container pressure is from about 0.6 atm to 0.3 atm.

8. The method according to claim 4, wherein the carrier is selected from the group consisting of crystalline silica, activated magnesium silicate, talc, metal powder, and mica mineral.

9. The method according to claim 8, wherein the metal powder is a metal oxide powder or a metal carbide powder.

10. The method according to claim 9, wherein the particle size of the metal powder is an average of 10 μm in spherical shape.

11. The method according to claim 10, wherein the metal powder is stainless steel powder.

12. The method according to claim 4, wherein the carrier is present at a pollen:carrier ratio selected from the group consisting of ratios 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 from 1:20 to 50:

1.

13. The method according to claim 4, wherein the sealable container includes an aluminum tray, a copper tray, a nickel tray, or a stainless steel tray platform.

14. The method according to claim 4, wherein the sealable container is made of glass, acrylic resin, aluminum, or stainless steel.

15. The method according to claim 4, wherein the refrigerated environment includes a temperature range selected from the group consisting of 1 to 10 °C, 4 to 8 °C, and 5.5 to 6.5 °C.

16. The method according to claim 4, wherein the pollen is stored in the refrigerated environment for a period of 20 days or less, 19 days or less, 18 days or less, 17 days or less, 16 days or less, 15 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less than 1 day.

17. The method according to claim 4, wherein the sealable container comprises an initial oxygen content of from O2 0.12 mmol / g pollen / day (number of days stored) to O2 0.57 mmol / g pollen / day (number of days stored). **Claim 18** The method according to claim 4, wherein the sealable container comprises a CO selected from the group consisting of activated carbon, ethanolamine, zeolite 4A, lithium hydroxide (LiOH), soda lime, calcium silicate (Ca2O4Si), and activated magnesium silicate (e.g., FLORISIL®). 2 blocking agent. **Claim 19** The method according to claim 8, wherein the crystalline silica comprises an average particle size of from about 1 nanometer to about 100 micrometers, or from about 1 micrometer to about 10 micrometers. **Claim 20** A method of applying stored corn pollen to a stigma, comprising: a) obtaining the stored corn pollen by the method according to claim 6; b) applying the stored pollen to the stigma. The method, wherein the stored corn pollen is applied to the stigma after collection. **Claim 21** The corn pollen according to claim 20, wherein the corn pollen is applied to the stigma at least 1 day after collection. **Claim 22** The method according to claim 20, wherein the stigma is a corn silk. **Claim 23** The method according to claim 22, wherein the corn silk is a heterosis group different from the heterosis group corresponding to the stored corn pollen. **Claim 24** The maize silk is silk from a tropical or subtropical heterotic group, and the stored maize pollen is pollen from a temperate heterotic group; or the maize silk is silk from a temperate heterotic group, and the stored maize pollen is pollen from a tropical or subtropical heterotic group, wherein the heterotic group is selected from the group consisting of Stiff Stalk, Non-Stiff Stalk, Iodent, and Lancaster, the method according to claim 23.

25. The method according to claim 22, wherein the maize silk is from a maturity group different from the maturity group corresponding to the stored maize pollen.

26. The method according to claim 21, wherein the stigma is a wheat stigma.

27. A method for enhanced introgression of a trait gene in a plant genome, comprising: a) providing a first plant that is a first maturity group; b) cross-pollinating the first plant of (a) with stored pollen from a second plant that is a plant of a second maturity group and further has a desired trait or phenotype; and c) selecting a progeny plant from step (b) that includes the desired trait or phenotype; and d) optionally, backcrossing the progeny plant of (c) as a pollen donor onto a recurrent parent plant and selecting a progeny plant that includes the desired trait or phenotype; comprising, The method, wherein the stored pollen is obtained by the method according to claim 6.

28. The method according to claim 27, wherein the plant is a maize plant.

29. The method according to claim 27, wherein the first maturity group is a maturity group more than one removed from the second maturity group.

30. The method according to claim 27, wherein the stored pollen from the second plant is applied to the first plant at least one day after collection.

31. The method according to claim 4, wherein the maize pollen is transgenic maize pollen comprising at least one transgenic line 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.

32. The method according to claim 4, wherein the container pressure is pressurized with standard atmosphere or a mixture of standard atmosphere and pure oxygen gas, the standard atmosphere is at an absolute pressure of 1 to 3 atm, and the pure oxygen gas is 18 to 27 mmol of O2 per liter of the storage container headspace.

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