Maize pollen storage and carrier

Abstract

Corn pollen is known to be fragile and prone to decomposition unless properly stored. Unlike some woody pollen, which is very hardy and may be able to successfully fertilize for months or years after scattering, corn pollen remains viable for only a few hours after scattering before it begins to decompose. Described herein is an invention for storing corn pollen, where the pollen is collected and stored in a refrigerated, but not frozen, environment with a breathable barrier. Pollen stored as described herein can remain viable for up to 12 days or 2 weeks or more. The addition of a carrier compound can extend the viability of the pollen.

Description

[Technical field]

[0001] The present invention relates to the field of corn breeding and human induced pollination, in particular to the collection, storage and application of stored corn pollen in fields and greenhouses for corn production. [Background technology]

[0002] Pollen storage has long been a need 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) (clarifying the desire for stored pollen "so that it may be used at convenient times and places"). In some plants, pollen is very hardy and long-lived. For example, ginkgo tree pollen can be collected and stored for six months or more without special care. In contrast, pollen of other plants is fragile and prone to rapid breakdown within a few hours if left exposed to the elements. Maize (corn) is one such plant.

[0003] In commercial hybrid corn production, 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 adjacent females. This arrangement works best when the female and male plants are of similar maturity groups; that is, the males shed pollen at approximately the same time that the females are receptive to pollen.

[0004] However, a risk with current practices is that if the males and females are of different maturity groups, pollination may fail and thus the crop may be lost. Without pollen storage, growers run the risk of male plants shedding pollen too early or too late and losing the entire field due to pollination failure. With pollen storage, the pollen can be delivered at exactly the right time, regardless of flowering time challenges. Because crossing different maturity groups can be more easily achieved, the gene pool can be expanded and maize plant breeding can be improved, for example, by creating more drought-resistant and / or disease-resistant maize lines. Summary of the Invention [Means for solving the problem]

[0005] Growers need the ability to reliably collect and store corn pollen on 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 in a container is sealed with a breathable barrier, and the pollen is stored in a refrigerated environment. The pollen collected and stored in this manner remains viable for up to 20 days, at least up to 12 days. In one aspect, the carrier is talc powder or silica powder. In another aspect, the carrier is a metal powder or mica mineral. The carrier can be added 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 one embodiment, the breathable barrier can be Parafilm, Tyvek, 3M Micropore Tape, cellulose, nitrocellulose, and non-airtight container. Preferably, the breathable barrier is 3M Micropore Tape. In another embodiment, a carbon dioxide sequestering agent can be added to the sealable container. Additionally, provided herein is a pollen storage container with a total breathable barrier surface area. The pollen storage container can have multiple openings, and the openings can be covered with a breathable barrier. [Brief description of the drawings]

[0006] [Figure 1]Photographs of seed set produced using pollen stored in a breathable barrier surface area factorial using a breathable barrier of Tyvek and micropore covering the perforations of a normally airtight storage container with added soda lime (Example 4, Experiment 5). From left to right, ears were produced using pollen stored in a normally airtight container with a single 1 / 6 inch opening covered with Tyvek, a single 1 / 8 inch opening covered with Tyvek, a single 3 / 8 inch opening covered with Tyvek, a single 1 / 16 inch opening covered with micropore tape, a single 1 / 8 inch opening covered with micropore tape, a single 1 / 4 inch opening covered with micropore tape, and a single 3 / 8 inch opening covered with micropore tape treatment, respectively. [Diagram 2] Photographs of seed set produced using pollen stored in a breathable barrier surface area factorial using open perforations in a normally airtight storage container with added soda lime (Example 4, Experiment 6). From left to right, ears were produced using pollen stored in normally airtight containers with a single 3.175 mm (1 / 8 inch) open perforation, a single 1.588 mm (1 / 16 inch) open perforation, a single 3.175 mm (1 / 8 inch) open perforation, and a single 6.35 mm (1 / 4 inch) open perforation treatments covered with Tyvek, respectively, and the last two ears on the right are from a normally airtight container with a single 9.525 mm (3 / 8 inch) open perforation. [Diagram 3] Photographs of seed set produced using pollen stored in a breathable barrier surface area factorial using a breathable barrier of Tyvek and micropores covering large perforations in a normally airtight storage container with added soda lime (Example 4, Experiment 7). The three ears on the far left in the image are from a treatment where the large perforations were covered with micropore tape. Perforation size increases from left to right across the three ears. The three ears on the far right in the image are from a treatment where the large perforations were covered with Tyvek. Perforation size increases from left to right across the three ears. [Figure 4]Photograph showing seed set induced using pollen deposited 6.5 cm deep in a 50 ml conical tube with a 71.3 mm2 opening covered with micropore tape without the addition of soda lime (Example 4, Experiment 8). The leftmost spike is from the top 1.5 cm of the pollen-carrier mixture deposited in the tube. The middle spike is from the middle part of the pollen pile, approximately 4 cm below the surface. The rightmost spike is from the base of the pollen pile, 6.5 cm from the surface. [Diagram 5] Photographs of seed set produced using pollen stored on a breathable barrier surface area using a microporous breathable barrier to cover the perforations in a normally airtight storage container with and without the addition of soda lime (Example 4, Experiment 9). The four ears on the left side of the image are from the treatment with no soda lime added. The four ears on the right side of the image are from the treatment with soda lime added. For each set of four ears, the perforation size increases from left to right. [Figure 6] 14 shows pressure-normalized oxygen percentage during 9 days of pollen storage from Example 6. [Figure 7] Shown is the stock version of the culture flask. The stock vented cap has been removed to show a VWR® cell culture flask using 3M Micropore Tape (indicated using black arrow) as a breathable barrier. This view shows the larger surface area flat side that served as the upturned top of the flask during use. [Figure 8] The stock version of the culture flask is shown. The stock vented cap has been removed to show a VWR® cell culture flask using 3M Micropore Tape (indicated using black arrow) as a breathable barrier. This view shows the smaller surface area flat side of the flask, which served as the downturned bottom of the flask during use. This smaller surface area is due to the taper created in the 47 mm outer high side wall of the flask. [Figure 9]Shown is the stock version of the culture flask. The stock vented cap has been removed to show a VWR® cell culture flask using 3M Micropore Tape as a breathable barrier. This view shows the side of the flask when standing on the end opposite the end of the 3M Micropore Tape breathable barrier. [Figure 10] Shown is the stock version of the culture flask. The stock vented cap has been removed to show a VWR® cell culture flask using 3M Micropore Tape (indicated using black arrow) as a breathable barrier. This view shows the angled top and side when the flask is seated on a downward facing bottom with tapered sidewalls. [Figure 11] Shown is the stock version of the culture flask. The stock vented cap has been removed to show a VWR® cell culture flask using 3M Micropore Tape (indicated using black arrow) as a breathable barrier. The figure shows the flask from the end of the vessel opposite the 3M Micropore Tape breathable barrier, with the flask seated face down. [Figure 12] Shown is the stock version of the culture flask. The stock vented cap has been removed to show a VWR® cell culture flask using 3M Micropore Tape (indicated using black arrow) as a breathable barrier. This view shows the side of the flask when standing on the end opposite the 3M Micropore Tape breathable barrier. This is the opposite side to the view in Figure 9. [Figure 13] Depicting the 22-hole configuration of the pollen storage vessel. Shown is a VWR® cell culture flask with 11 holes drilled into the flat side of the larger surface area facing upwards. The total of 22 holes are indicated by black arrows (11 per figure). [Figure 14] Depicting the 22-hole configuration of the pollen storage vessel. Shown is a VWR® cell culture flask with 11 holes drilled into the side of the smaller surface area with tapered sidewalls. The total of 22 holes are indicated by black arrows (11 per figure). [Figure 15]1 depicts the 22-hole configuration of the pollen storage vessel. 2 shows a VWR® cell culture flask with 11 holes drilled into the flat side of the larger surface area facing upwards. 3 shows a side view. 4 shows the 11 holes drilled into the flat side of the larger surface area facing upwards, indicated by black arrows. [Figure 16] 1 depicts the 22-hole configuration of the pollen storage vessel. A VWR® cell culture flask is shown with 11 holes drilled into the flat side of the larger surface area facing up. View from the end opposite the vent cap. The 11 holes drilled into the flat side of the larger surface area facing up are indicated by black arrows. [Figure 17] Depicts the 22 hole configuration of the pollen storage vessel. Shows a VWR® cell culture flask with 11 holes drilled on the larger surface area flat side facing up and 11 holes drilled on the smaller surface area side with tapered sidewall. However, all 22 holes are covered with 3M Micropore Tape, which acts as a breathable barrier. View of the larger surface area flat side facing up. Shows the cap side closest to the camera. Indication of the 3M Micropore Tape is indicated by the black arrow. [Figure 18] Depicts the 22 hole configuration of the pollen storage vessel. Shows a VWR® cell culture flask with 11 holes drilled into the larger surface area flat side facing up and 11 holes drilled into the smaller surface area side with tapered sidewall. However, all 22 holes are covered with 3M Micropore Tape, which acts as a breathable barrier. View of the larger surface area flat side facing up. From the opposite end of the stock cap. Indication of the 3M Micropore Tape is indicated by the black arrow. [Figure 19]Depicts the 22 hole configuration of the pollen storage vessel. Shows a VWR® cell culture flask with 11 holes drilled into the larger surface area flat side facing up and 11 holes drilled into the smaller surface area side with tapered sidewall. However, all 22 holes are covered with 3M Micropore Tape, which acts as a breathable barrier. View of the larger surface area flat side facing up. Side view with stock cap on the left. Indication of 3M Micropore Tape indicated by black arrow. [Figure 20] Depicts the 22 hole configuration of the pollen storage container. Shows a VWR® cell culture flask with 11 holes drilled on the larger surface area flat side facing up and 11 holes drilled on the smaller surface area side with a tapered sidewall. However, all 22 holes are covered with 3M Micropore Tape, which acts as a breathable barrier. View of the smaller surface area side with tapered sidewall and facing down during use. Indication of 3M Micropore Tape is indicated by black arrow. [Figure 21] 1 depicts the 28-hole configuration of the pollen storage vessel. A VWR® cell culture flask is shown with 11 holes drilled into the flat side of the larger surface area facing upwards and 11 holes drilled into the smaller surface area side with a tapered sidewall. The stock cap is shown closest to the camera. [Figure 22] 1 depicts the 28-hole configuration of the pollen storage container. A VWR® cell culture flask is shown with 11 holes drilled into the flat side of the larger surface area facing upwards and 11 holes drilled into the side of the smaller surface area with a tapered sidewall. Viewed from the end opposite the stock cap. [Diagram 23] 1 depicts the 28-hole configuration of the pollen storage container; 2 shows a VWR® cell culture flask with 11 holes drilled into the flat side of the larger surface area facing upwards and 11 holes drilled into the side of the smaller surface area with a tapered side wall; 3 shows a side view with the stock cap facing left; [Figure 24]Depicts the 28-hole configuration of the pollen reservoir. A VWR® cell culture flask is shown from the side, standing upright (vertical) on the end opposite the stock cap. The view shows three holes drilled into the side of the flask. [Diagram 25] Depicts the 28 hole configuration of the pollen reservoir. Shows a VWR® cell culture flask with the tapered sidewall facing up, providing a smaller surface area. Eleven holes are drilled into the flat side. [Figure 26] 23 depicts the 28-hole configuration of the pollen reservoir. A VWR® cell culture flask is shown from the side, standing upright (vertical) on the end opposite the stock cap. This view shows three holes drilled into the side of the flask. This is the opposite side to that shown in FIG. 24. [Figure 27] Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. [Figure 28] Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. [Figure 29] Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. [Diagram 30] Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. [Diagram 31] Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. [Diagram 32]Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. [Diagram 33] Depicts the 28-hole configuration of the pollen reservoir. Shows a culture flask with a 28-hole configuration, but the holes are covered with 3M Micropore Tape as a breathable barrier. Indication of the Micropore Tape is indicated by the black arrow. 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 defined below. References to technology used herein are intended to refer to technology commonly understood in the art, including variations of those technologies and / or equivalent technology replacements that would be apparent to those skilled in the art. Although 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 of this disclosure.

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

[0009] The term "about" as used herein refers to a normal range of error for the respective value, readily known to one of ordinary skill in the art, e.g., ±20%, ±10% or ±5%, within the intended meaning of the recited value.

[0010] As used herein, a "container" or "receptacle" refers to an object capable of holding pollen therein. For example, a container may refer to a Magenta GA7 box. The container or receptacle also includes a breathable barrier. As used herein, a "breathable barrier" refers to a component of a storage container or receptacle for corn pollen. The breathable barrier component of the container or receptacle allows sufficient gas exchange with minimal water vapor transmission rate. Breathable barriers may include, but are not limited to, the use of parafilm, Tyvek, micropore tape, perforations in normally airtight storage containers or receptacles (e.g., mason jars and lids) or non-airtight containers or receptacles with openings for gas exchange made in a designated total surface area (e.g., clamshell containers, Magenta GA7 boxes, VWR® cell culture flasks 25-850 ml capacity). For cell culture flasks, see generally us.vwr.com / store / product / 12585790 / vwr-cell-culture-flasks. Non-airtight containers can also include, for example, mason jars, and can include perforated lids.

[0011] As used herein, "carbon dioxide sequestration" refers to the process of isolating carbon dioxide ("CO 2 ") 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, excess CO 2 Optionally, the sequestrant is a 2 10 mmol CO per liter of chamber headspace 2 Prevent the load from exceeding the limit.

[0012] As used herein, "carrier" refers to a compound, preferably in powder form, that acts as an agent that accompanies the collected pollen. Suitable carrier compounds can be, but are not limited to, talc powder, silica powder, etc.

[0013] As used herein, "agglomeration," "clumping," and similar terms refer to the tendency of pollen to stick together in the absence of a carrier and / or proper storage conditions, whether due to excess moisture or other causes. Agglomerated pollen is not flowable and cannot be blown onto the silk with air. Agglomerated pollen is unlikely to adhere sufficiently to the silk to cause pollination.

[0014] As used herein, the terms "comprising" or "comprises" are open ended. When used in the context of a method including a series of steps, the method remains performed as long as the series of steps are performed, even if additional steps are performed.

[0015] As used herein, "crystalline silica" refers to a powdered form of silica derived from quartz or other natural rock formations. The terms "crystalline silica", "SiO 2 " and "polycrystalline silica" are used interchangeably throughout. Crystalline silica has different structural properties than talc or amorphous silica, including, but not limited to, higher Mohs mineral 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 size values ​​provided herein are average sizes.

[0016] As used herein, "activated magnesium silicate" refers to synthetic powdered magnesium silicate. The terms "activated magnesium silicate," "synthetic amorphous activated magnesium silicate," and "MgO 3"Si" 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).

[0017] The term transgenic "event" as used herein 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 "event" refers to the original transformant and / or progeny of the transformant containing the heterologous DNA. The term "event" also refers to progeny produced by sexual outcrossing between the transformant and another corn line. Even after repeated backcrossing to the recurrent parent, the inserted and flanking DNA from the transformed parent are present in the progeny of the cross at the same chromosomal location. Typically, transformation of plant tissue produces multiple events, each of which represents the insertion of a DNA construct into a different location in the genome of the plant cell. A particular event is selected based on the expression of the transgene or other desirable characteristics. Thus, for example, "event 3272," "3272," or "3272 event" as used herein refers to the original 3272 transformant. and / or progeny of the 3272 transformant, and / or plants derived in some way from the original 3272 transformant. For 3272, see WO 06 / 098952.

[0018] Other examples of transgenic events include MIR162 (see WO 07142840), Bt11 (see U.S. Pat. No. 6,114,608 (constructs) and WO 8705629 (gene)), GA21 (see WO 9704103 (gene), WO 9844140 (cassette)), MIR604 (see WO 05103301), MZIR098 (see WO 18231890), 5307 (see WO 10077816), DAS40278 (see U.S. Pat. No. 8,598,413), TC1507 (WO 04 099447), 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).

[0019] As used herein, "flowable" refers to the ability of a powdered material to move easily with the application of air, wind, sound or be poured in an uninterrupted continuous stream to move steadily and easily.

[0020] As used herein, the term "gas" refers to a gas mixture (e.g., a gas combination of normal air composition or oxygen-enriched air) or a substantially pure gas (e.g., pure oxygen). In one embodiment, references to gas may refer to oxygen and carbon dioxide.

[0021] As used herein, "hybrid vigor group" refers to a breeding classification of inbred lines. "Hybrid vigor group" and "hybrid vigor pool" are used interchangeably and refer to the relationship between breeding pools of a corn population. In general, the main designations for hybrid vigor pools are Stiff Stalk ("SS" also called Iowa Stiff Stalk Synthetic 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) Corn breeding, p. 463-564; GFS Prague and JW 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 WO 2020 / 205334 and WO 2021 / 041077, which are incorporated by reference in their entireties.

[0022] 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 with proven genetic superiority. For example, for a given environment or geographic region, the phrases "unadapted germplasm," "untreated germplasm," and "exotic germplasm" refer to plant material of unknown or unproven genetic value, for example, for a given environment or geographic region. Thus, the phrase "unadapted germplasm" 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.

[0023] The term "mica" as used herein refers to the general chemical formula X2Y4-6Z8O20(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 can be silicon, aluminum or other transition metals.

[0024] As used herein, "starting oxygen content" refers to the amount of oxygen (measured as an absolute measurement, as a percentage, or by other methods) present in the atmosphere of the chamber containing the pollen as it is initially collected and once initially sealed. In one embodiment, the starting oxygen content is 0.12 mmol O 2 / g pollen / storage day ~0.57mmol O 2 / g pollen / day of storage (inclusive). In another embodiment, the starting oxygen content is 0.24 mmol O 2 / g pollen / storage day ~0.57mmol O 2 / g pollen / storage date (inclusive). "Starting oxygen content", "Starting O 2 mmol”, “start O 2 mmol / g pollen" and "Start O 2 "mmol / g pollen / day of storage" are used interchangeably herein.

[0025] A "plant" is any 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 set of genetically identical material.

[0026] As used herein, "platform" means a surface within the 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.

[0027] As used herein, "pollen:carrier ratio" refers to the proportion of pollen present in a mixture with a carrier. For example, and without limitation, a mixture of pollen and carrier with 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.

[0028] As used herein, a "refrigerated environment" refers to any condition in which the 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° C. and 10° C.

[0029] As used herein, "seed set" refers to the number of kernels produced on the cob from successful pollination. Seed set can be expressed qualitatively (e.g., low, good, or high) or quantitatively. In quantitative measurements, the measurement can be given as a percentage or number of seeds per ear. The term generally refers to the percentage or number of normal kernels (i.e., non-degenerate endosperm viable kernels). For normal corn lines (i.e., not haploid derivative lines), seed set of more than 80% (or more than 300 kernels / ear) is considered good seed set. Achieving good seed set is the goal of pollination management.

[0030] "Storage" as used herein refers to the act of storing pollen for a suitable period of time. A suitable storage period can be as short as 24 hours or as long as 12 days.

[0031] As used herein, "treatment" refers to the deliberate application of a compound or environmental constraint to pollen. In particular, pollen treatment may include the addition of a carrier compound to pollen to preserve the mobility and viability of the pollen.

[0032] "Container pressure" as used herein refers to any atmospheric pressure artificially applied within a container. Containment pressure values ​​are measured herein in units of standard atmosphere "atm", e.g., 0.5 atm, but may be measured in other units as needed to measure the container pressure (e.g., Torr or Pascals or "Pa", 1 Pa=9.8692×10 -6 atm) may be used. It is expressly contemplated that the vessel pressure may be 1 atm or greater. Under conditions where the vessel pressure is between 0 and 1 atm, "vessel pressure" and "vacuum" have the same meaning and are used interchangeably. However, "vessel pressure" is the preferred term as it contemplates both vacuum conditions and conditions where artificially imposed atmospheric pressure exceeds the ambient atmosphere (e.g., 1 atm).

[0033] As used herein, "pollen viability" refers to the ability of a pollen grain to germinate a pollen tube that grows through the stigma (silk) during a pollination event and delivers two sperm cells to the female gametophyte to fertilize the egg cell and the two polar nuclei, resulting in the formation of the embryo and endosperm, respectively.

[0034] As used herein, "pollen vigor" refers to the time interval between physical contact of a pollen grain with corn silk and subsequent fertilization. Pollen vigor is important for seed set because viable pollen grains must complete pollen tube growth through the stigma and subsequent fertilization before losing viability due to desiccation or other environmental stressors.

[0035] Because large quantities of inbred parent line seeds are required to produce hybrid seeds sold to customers, productivity in 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 a key factor for successful development of corn inbred parent lines. A corn inbred parent line with low productivity may be discontinued due to excessive costs in parent seed production, even if the inbred parent line is capable of producing hybrids with characteristics desirable to customers (e.g., major GM and genome editing traits, high yield, disease resistance). Pollen storage techniques can be used to increase the productivity of inbred corn parent lines used for hybrid seed production.

[0036] Challenges to productivity that may be addressed by pollen storage techniques include, but are not limited to, low pollen production, low total pollen shedding, short pollen shedding period, short silking period, and GM or genome editing traits that may affect plant reproductive characteristics. An additional challenge with self-pollination may be long self-division, defined by the number of days from when pollen starts shedding until silks appear and are available for pollination. In some repeat phases, self-division may be negative, with silks appearing for pollination before pollen shedding begins. The observed self-division may be the result of the genetics of the inbred parent line, or it may be the result of stresses in the growing environment that slow down the rate of silk elongation and increase the number of days from the start of pollen shedding to silk availability for pollination.

[0037] To address these productivity challenges, pollen storage techniques can be used to collect pollen at the optimal time for pollen dispersal, store the pollen while maintaining pollen viability, and then apply the pollen at the optimal time for silk emergence and receptivity. In some repetitive phases, pollen collection can be done multiple times per day. In other repetitive phases, pollen can be collected on multiple days throughout the period of pollen dispersal. The application of stored pollen can combine pollen collected over multiple days, and multiple applications can be made on the same day or over multiple days. Pollen application can combine pollen collected from multiple field locations into a single application and directed to one location. In some repetitive phases, pollen can be collected at one terrain and applied to silks in different terrains. The terrains 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 recurrent phases, pollen is collected from a temperate corn inbred parent line grown in a temperate location and spread onto a subtropical or tropical corn inbred parent line grown in a subtropical or tropical location. In other recurrent phases, pollen is collected from a subtropical or tropical corn inbred parent line grown in a subtropical or tropical location and spread onto a temperate corn inbred parent line grown in a temperate location. By addressing these challenges to productivity, pollen storage technology may enable seed expansion of desirable corn inbred parent lines to produce new hybrids with desirable characteristics for sale to customers. Pollen storage technology may also enable economical hybrid seed production for combinations of temperate, subtropical and tropical corn inbred parent lines that are not currently feasible.

[0038] Thus, one embodiment provides a method of storing viable corn pollen comprising collecting a quantity of fresh corn pollen, optionally adding a carrier to the collected corn pollen, obtaining a quantity of treated corn pollen, placing the quantity of fresh corn pollen or the quantity of treated corn pollen in a container, sealing the container with a breathable barrier, and storing the pollen in the container in a refrigerated environment. In one embodiment, the quantity of fresh corn pollen or the quantity of treated 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. In another embodiment, the amount of fresh or processed corn pollen is about 0.3 grams to 10 kilograms, and in another embodiment, the amount of fresh or processed corn pollen is about 2 grams to 1 kilograms. In one embodiment, the carrier is selected from the group consisting of crystalline silica, talc, metal powder, and mica mineral. In another embodiment, the carrier is crystalline silica and comprises an average particle size. The average particle size can be about 1 nanometer to about 100 micrometers. In another embodiment, the average particle size is 10 micrometers. In yet another embodiment, the metal powder is a metal oxide powder or a metal carbide powder. In another embodiment, the metal powder comprises an average particle size can be about 1 micrometer to about 100 micrometers. In one embodiment, the average particle size is about 10 micrometers spherical. In another embodiment, the metal powder is a stainless steel powder.

[0039] In another 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 one embodiment, the container comprises a volume between 0.1 milliliters and 10 liters. In another embodiment, the container comprises a volume between 10 milliliters and 1500 milliliters. In a further embodiment, the container comprises a volume between 100 milliliters and 1250 milliliters. The vessel contains a CO2 absorbent 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 It may further comprise a sequestrant. In one embodiment, the sequestrant is soda lime.

[0040] In one embodiment, the breathable barrier is selected from the group consisting of Parafilm, Tyvek, 3M Micropore Tape, cellulose, nitrocellulose, and a non-airtight container. In another embodiment, the breathable barrier is 3M Micropore Tape. In another embodiment, the non-airtight container comprises an opening for gas exchange. The opening may also comprise at least one perforation. In a further embodiment, the opening comprises at least one perforation having a diameter size between 0.10 millimeters and 30 millimeters. In one embodiment, the breathable barrier comprises a perforation having a diameter size between 0.49 mm per gram of fresh or processed pollen. 2 ~47.5mm per gram of fresh or processed pollen 2 In another embodiment, the breathable barrier comprises a surface area of ​​1.98 mm per gram of fresh or processed pollen. 2 ~26.72mm per gram of fresh or processed pollen 2 In another embodiment, the breathable barrier comprises a surface area of ​​4.45 mm per gram of fresh or processed pollen. 2 ~11.88mm per gram of fresh or processed pollen 2 Includes a surface area of

[0041] The stored corn pollen may remain 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 embodiment, 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 another embodiment, the refrigerated environment comprises a temperature of approximately 6° C. In a further embodiment, 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 embodiment, the pollen is stored for 12 days or less.

[0042] In one embodiment, the corn pollen is transgenic corn pollen. In another embodiment, the transgenic corn pollen comprises a transgenic event selected from the group consisting of MIR162, 3272, Bt11, GA21, MIR604, MZIR098, 5307, DAS40278, TC1507, DAS-59122-7, NK603, MON810, MON863, MON89034, MON88017, DP-4114, and MON87411. In one embodiment, the corn pollen comprises the transgenic events Bt11, GA21, and MIR162. In another embodiment, the corn pollen comprises the transgenic events Bt11 and MIR162. In one embodiment, the corn pollen comprises the transgenic event MIR162.

[0043] In yet another embodiment, a device for storing pollen is provided. The device includes a container having a total breathable barrier surface area. In one embodiment, the container includes a plurality of openings. In another embodiment, the container has a total breathable barrier surface area of ​​0.49 mm per gram of fresh or processed pollen. 2 ~47.5mm per gram of fresh or processed pollen2 The pollen storage container may include at least 1 opening, at least 2 openings, at least 3 openings, at least 4 openings, at least 5 openings, at least 6 openings, at least 7 openings, at least 8 openings, at least 9 openings, at least 10 openings, at least 11 openings, at least 12 openings, at least 13 openings, at least 14 openings, at least 15 openings, at least 16 openings, at least 17 openings, at least 18 openings, at least 19 openings, at least 20 openings, at least 21 openings, at least 22 openings, at least 23 openings, at least 24 openings, at least 25 openings, at least 26 openings, at least 27 openings, at least 28 openings, at least 29 openings, or at least 30 openings.

[0044] In one embodiment, the individual openings in the device are 15.2 mm 2 ~660.5mm 2 The individual openings are selected from the group consisting of circles, ovals, squares, rectangles, triangles, and any other two-dimensional shape. In one embodiment, the openings are circular. In another embodiment, the container includes 22 openings. The 22 openings may be circular. In another embodiment, the container includes 28 openings. The 28 openings may be circular. In one embodiment, the total of 28 circular openings is 2199.1 mm 2 has a total surface area of

[0045] In one embodiment, the plurality of apertures is 0.49 mm per gram of fresh or processed pollen. 2 ~47.5mm per gram of fresh or processed pollen 2 The plurality of apertures may be circular holes having a diameter of 5-15 mm. The plurality of apertures may also be circular holes having a diameter of 10 mm. In another embodiment, the circular holes have a diameter of 78.5 mm. 2 Each of the particles has an individual surface area of ​​100 mm.

[0046] In one embodiment, the plurality of openings are individually covered with a breathable barrier, the plurality of openings being distributed such that the pollen grains are no more than 47 mm from the nearest breathable barrier, or any other distribution that optimizes the furthest possible distance between the pollen grains and the nearest breathable barrier. In one embodiment, the breathable barrier is selected from the group consisting of Parafilm, Tyvek, 3M Micropore Tape, cellulose, nitrocellulose, and a non-airtight container. The breathable barrier may be 3M Micropore Tape.

[0047] In one embodiment, the pollen storage container is a VWR® cell culture flask with a vented cap, used in its stock configuration with the nitrocellulose membrane in the vented cap acting as a breathable barrier. In another embodiment, the container is a VWR® cell culture flask used in its stock configuration with the cap removed and the mouth of the container covered with 3M Micropore Tape to act as a breathable barrier. In another embodiment, the container is a VWR® cell culture flask modified to include 28 additional openings distributed over the entire surface of the container, the additional openings covered with a breathable barrier, and the stock non-vented cap is left sealed over the mouth of the container. In another embodiment, the container is a VWR® cell culture flask modified to include any number of additional openings covered with a breathable barrier in any distribution over the entire surface of the container, with a solid or vented cap left in place over the mouth of the container, or the cap removed and replaced with a breathable barrier. In yet another embodiment, the vessel is any brand of cell culture flask in stock or modified configuration. The vessel can be any plastic, metal or ceramic vessel with one or more openings for a breathable barrier. EXAMPLES

[0048] 1. Collection Corn plants were grown under field and greenhouse conditions. Once the tassels emerged and began to shed pollen, the tassels were covered with bags to collect the pollen. The bags were typically placed in the evening and removed the following morning. The collected pollen was then placed in a suitable closed container, discarding any anthers or other tassel material, optionally mixed with a carrier.

[0049] Alternatively, pollen is collected by harvesting pre-shed tassels from corn plants. The tassels can be placed in a beaker of water and the pollen allowed to shed as normal, or the tassels can be dried, macerated and filtered to mechanically collect the pollen. See, e.g., U.S. Patent No. 8,252,988, filed June 27, 2007, which is incorporated herein by reference in its entirety.

[0050] 2. An experiment in which a mixture of pollen and talc was stored in Magenta GA7 boxes with added soda lime and sealed with parafilm as a breathable barrier. Experiments 1, 2 and 3: 1) breathable barrier (parafilm around GA7 boxes) with added soda lime (5 days); 2) breathable barrier (parafilm around GA7 boxes) with added soda lime and a higher starting pollen load than in experiment 1 (5 days); 3) breathable barrier (parafilm around GA7 boxes) with added soda lime and a higher starting pollen load than in experiment 2 (5 days).

[0051] For experiment 1, 2.522 g of pollen was mixed with 1.26 g of talc for a total of 3.783 g of pollen mixture, and the initial fresh pollen-talc mixture moisture content ("PMC") was 36.71%. Pollen was mixed in a weight ratio of 2 parts pollen, 1 part carrier. A total of 3.513 g of pollen mixture (6.5 ml) was added to a 400 mL Magenta GA7 box containing 0.44 g of soda lime. For experiment 2, a larger amount of pollen (5.998 g) was mixed with 2.999 g of talc (2:1 weight pollen:talc) for a total of 9.997 g of pollen mixture compared to experiment 1 above, and the initial fresh mixture PMC was 34.16%. A total of 8.1 g of pollen mixture (15 ml) was added to a 400 mL Magenta GA7 box containing 1.02 g of soda lime. For experiment 3, a larger amount of pollen (23.89 g) than used in experiments 1 and 2 was mixed with 11.944 g of talc (2:1 weight pollen:talc) for a total of 35.82 g of pollen mixture, with an initial fresh mixture PMC of 35.97%. A total of 29.7 g of pollen mixture (55 ml) was added to a 400 mL Magenta GA7 box containing 3.74 g of soda lime. For all three experiments, the soda lime was placed in the bottom of the GA7 box and contained within a 50 ml conical tube cap. The pollen mixture was contained inside the base of a polylactic acid plastic (PLA) cup (approximately 45 mm x 30 mm) and placed over the open soda lime container. A iButtonLink DS1923-F5# DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture container to monitor the internal relative humidity (rH) of the pollen storage container. The GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide Parafilm around the opening. The Parafilm was stretched as is common practice in plant tissue culture to provide a tight seal. The gas exchange gap surface area from the GA7 lid to the box was measured to be a total of 42 mm 2 The pollen storage container was stored at 6°C and 1 atm for 5 days. After 5 days, the pollen viability, final PMC relative to initial PMC, and O 2 and CO 2The end point gas analysis was evaluated.

[0052] In all three experiments, the iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that a saturated rH of over 98% was obtained throughout the entire storage period inside the storage container. For experiment 1, six replicate pollinations were performed on the receptive silks of the primary ear on the hybrid corn plants. After 5 days of storage, the pollen viability was 62% and the relative PMC was 96.3%. For experiment 2, six replicate pollinations were performed on the receptive silks of the primary ear on the hybrid corn plants. After 5 days of storage, the pollen viability was 66% and the relative PMC was 99.6%. For experiment 3, 12 replicate pollinations were performed on the receptive silks of the primary ear on the hybrid corn plants. The pollen viability was 87% after 5 days of storage, significantly increased compared to the fresh viability. The relative PMC was 100.9%. Inner vessel rH and final relative PMC data are for the exposed 42 mm 2 The results show that the use of Parafilm as a breathable barrier to seal the surface area of ​​the storage container and pollen mixture reduced moisture loss from the storage container and pollen mix. Internal gas samples taken from the storage container after wrapping air-impermeable tape in the gas exchange gap between the box and the lid showed 19.7% O in experiment 1. 2 Content and 0.1% CO 2 content, 19.5% O in experiment 2 2 Content and 0.1% CO 2 content, 16.2% O in experiment 3 2 Content and 0.3% CO 2 The use of Parafilm as a breathable barrier allowed for sufficient gas exchange while minimizing water vapor transmission. The presence of soda lime, in conjunction with the breathability of the barrier, allowed for CO 2The average seed set of 480 kernels indicates that pollen was viable after 5 days of storage in experiment 1. The average seed set of 347 kernels indicates that pollen was viable after 5 days of storage in experiment 2. Experiment 2 shows that the soda lime breathable barrier allows for the storage of larger amounts of pollen and talc while maintaining sufficient gas exchange and minimal water vapor transmission, compared to experiment 1. 2 Although the O content was lower than previously observed for experiments 1 and 2, which were performed with lower pollen mixture amounts, there was sufficient O to support pollen respiration. 2 was available. The average seed set of 598 kernels indicates that pollen was viable after 5 days of storage for experiment 3. The high seed set indicates that a large amount of pollen and talc mixture can be stored in a container using a breathable barrier.

[0053] [Table 1]

[0054] [Table 2]

[0055] Experiments 4 and 5: 4) Pollen and talc mixtures were stored in Magenta GA7 boxes sealed with parafilm as a breathable barrier, with and without the addition of soda lime. For experiment 4, 11.362 g of pollen was mixed with 5.681 g of talc (2:1 by weight pollen:talc) for a total pollen mix of 17.043 g, with an initial fresh mix PMC of 35.31%. Two treatments were prepared to compare protocol performance in the storage bin with and without added soda lime. This comparison was performed using CO2 throughout the storage period. 2A total of 5.4 g of pollen mixture (10 ml) was added to a 400 mL Magenta GA7 box per treatment. In treatment 1, 0.68 g of soda lime contained within a 50 ml conical tube cap was placed in the bottom of the GA7 box. In treatment 2, an empty 50 ml conical tube cap was placed in the bottom of a 400 mL Magenta GA7 box.

[0056] For experiment 5, 30.527 g of pollen was mixed with 15.264 g of talc (2:1 pollen:talc) for a total of 45.791 g of pollen mixture, with an initial fresh mixture PMC of 34.96%. A total of 8.1 g of pollen mixture (15 ml) was added to a 400 mL Magenta GA7 box and an empty 50 ml conical tube cap was placed on the bottom without adding soda lime. The pollen mixture was contained inside a PLA plastic cup (45 mm x 30 mm) and placed on top of an open 50 ml conical tube cap containing soda lime or on top of an empty 50 ml conical tube cap in all treatments. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture container to monitor the internal relative humidity of the pollen storage container. The GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and lid was wrapped with a single layer of 25.4 mm (1 inch) wide Parafilm. The Parafilm was stretched as is common practice in plant tissue culture to provide a tight seal. The gas exchange gap surface area from the GA7 lid to the box was measured to give a total of 42 mm 2 The pollen storage container was then stored at 6°C and 1 atm for 5 days. After 5 days, the pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0057] In experiment 4, nine replicate pollinations were performed on the receptive silks of the primary ear on hybrid corn plants for both treatments. Pollen viability was qualitatively scored on a scale of 4 (41-60% germination, see Table 29) after 5 days of storage, with both treatments scored similarly. Treatment 1, with soda lime added, had a relative PMC of 105.6% and treatment 2, without soda lime added, had a relative PMC of 101.4%. Final relative PMC data were collected from 42 mm 400 mL Magenta GA7 boxes. 2 The use of Parafilm as a breathable barrier to seal the gas exchange surface area of ​​the storage containers and pollen mixtures reduced water loss from the storage containers and pollen mixtures. For both treatments, the water content of the pollen mixtures increased over time due to additional new water produced from cellular respiration. After wrapping air-impermeable tape over the gas exchange gaps, internal gas samples were taken from each treatment. Treatment 1 was at endpoint O 2 Content 19.2%, CO 2 The content was 0.2%. Treatment 2 was at end point O 2 Content is 19.5%, CO 2 The CO content was 1.2%. 2 The content was higher than in treatment 1 (with soda lime added), but the 1.2% CO 2 Content is measured through a Parafilm breathable barrier 2 They demonstrated that gas exchange occurs. The use of Parafilm as a breathable barrier allowed sufficient gas exchange while minimizing water vapor transmission. The presence of soda lime, in conjunction with the breathability of the barrier, allowed for CO 2 The barrier is breathable, allowing toxic levels of CO 2 Enough CO to avoid accumulation 2 and O 2Gas exchange of 100% was achieved. These results suggest that soda lime may not be required for pollen storage if a breathable barrier is available for gas exchange. The average seed set of 381 kernels / ear for the treatment with soda lime and 426 kernels / ear for the treatment without soda lime indicates that pollen was viable after 5 days of storage in both treatments. The higher seed set for the treatment without soda lime further supports that soda lime may not be required for pollen storage if a breathable barrier is available for gas exchange.

[0058] In experiment 5, three replicate pollinations were performed on the receptive silks of the primary ear on hybrid corn plants. The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that greater than 98% saturation rH was achieved throughout the storage period inside the storage container. After 5 days of storage, pollen viability was annotated at 66% and the relative PMC was 102.1%. Inner container rH and final relative PMC data were obtained from the exposed 42 mm 2 These results indicate that the use of parafilm as a breathable barrier to seal the gas exchange surface area of ​​the storage container reduced water loss from the pollen mix. The water content of the pollen mix increased over time due to additional new water produced by cellular respiration. After wrapping air-impermeable tape over the gas exchange gap, internal gas samples taken from the storage container showed 19.6% O. 2 Content and 0.9% CO 2 The use of Parafilm as a breathable barrier allowed adequate gas exchange while minimizing water vapor transmission. 2 Levels remain below 1.0%, a toxic level of CO 2 Accumulation was avoided, suggesting that soda lime may not be required for this approach to pollen storage. Average seed set of 514 kernels / ear indicates that pollen was viable after 5 days of storage.

[0059] [Table 3]

[0060] [Table 4]

[0061] [Table 5]

[0062] [Table 6]

[0063] 3. An experiment in which a mixture of pollen and crystalline silica was stored in a Magenta GA7 box containing added soda lime. Experiment 1: Breathable barrier (parafilm around GA7 box) with crystalline silica and soda lime (7 days) A quantity of 5.955g of pollen was mixed with 2.9775g of 10μm crystalline silica (2:1 weight pollen:silica) for a total of 8.933g of pollen mixture, with an initial fresh mixture PMC of 33.57%. A total of 8.85g of pollen mixture (12.5ml) was added to a 400mL Magenta GA7 box containing 1.02g of soda lime. The soda lime was placed at the bottom of the GA7 box and contained within a 50ml conical tube cap. The pollen mixture was contained inside a PLA plastic cup (45mm x 30mm) and placed on top of the open soda lime container. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture container to monitor the internal relative humidity of the pollen storage container. The GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide Parafilm around the open gap. The Parafilm was stretched as is common practice in plant tissue culture to provide a tight seal. The GA7 gas exchange gap between the box and lid surface area was 42 mm in total.2 The pollen storage container was stored at 6°C and 1 atm for 7 days. After 7 days, the pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0064] Four replicate pollinations were performed on the receptive silks of the primary ear on hybrid corn plants. The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that a saturated rH of >98% was obtained throughout the entire storage period inside the storage container. Pollen viability was qualitatively scored with a ranking of 4 (41-60% germination, see Table 29) after 7 days, and the relative PMC was 104.9%. The inner container rH and final relative PMC data indicate that the use of parafilm as a breathable barrier reduced moisture loss from the storage container and pollen mix. The moisture content of the pollen mix increased over time due to additional water produced by cellular respiration. After wrapping a seal with air impermeable tape, an internal gas sample taken from the storage container showed 19.4% O. 2 Content and 0.1% CO 2 The use of Parafilm as a breathable barrier allowed for sufficient gas exchange while minimizing water vapor transmission. The presence of soda lime, in conjunction with the breathable barrier, reduced CO 2 The average seed set of 670 kernels / ear indicates that the pollen was viable after 7 days of storage.

[0065] [Table 7]

[0066] [Table 8]

[0067] Experiments 2 and 3: 2) crystalline silica, soda lime, and a breathable barrier (parafilm around the GA7 box) with reduced headspace (5 days); 3) crystalline silica, soda lime, and a breathable barrier (parafilm around the GA7 box) with reduced headspace and a higher starting amount of pollen than in experiment 2 (5 days). In experiment 2, 23.623 g of pollen was mixed with 11.812 g of 10 μm crystalline silica (2:1 weight ratio of pollen:silica) for a total of 35.4345 g of pollen mixture, with an initial fresh mixture PMC of 35.62%. A total of 8.5 g of pollen mixture (12.5 ml) was added to each of the four Magenta GA7 box treatments. Three Magenta GA7 boxes were modified by partially filling the boxes with epoxy resin to achieve final headspace volumes of 100 ml, 200 ml, and 300 ml, as required. The epoxy resin was cured in a pressure chamber set at 3 atmospheres absolute for a minimum of 24 hours prior to use for pollen storage. The fourth box (400 ml headspace) was used as a positive control. 1.05 g of soda lime was placed in a 50 ml conical tube cap and placed in the bottom of each box for the 200 ml, 300 ml, and 400 ml headspace treatments. For the 100 ml headspace treatment, the soda lime was placed directly on top of the hardened epoxy resin but was kept separate from the container of pollen:silica mixture.

[0068] In experiment 3, 10.484 g of pollen was mixed with 5.242 g of 10 μm crystalline silica (2:1 pollen:silica ratio) for a total of 15.726 g of pollen mixture, with an initial fresh mixture PMC of 35.93%. A total of 15.72 g of pollen mixture (22 ml) was added to a modified Magenta GA7 box. The box was modified by partially filling it with epoxy resin to achieve a final headspace volume of 100 ml. 1.87 g of soda lime was placed directly on top of the hardened epoxy resin, but separate from the container of pollen:silica mixture. For both experiments, the pollen mixture was contained inside a PLA plastic cup (45 mm x 30 mm) and placed on an open soda lime container or beside the soda lime placed directly on top of the hardened epoxy resin. For each treatment, a GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide Parafilm around the opening. The Parafilm was stretched as is common practice in plant tissue culture to provide a tight seal. The GA7 gas exchange gap between the box and lid surface area was 42 mm in total. 2 The pollen storage container was stored at 6°C and 1 atm for 5 days. After 5 days of storage, the pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0069] For experiment 2, three replicate pollinations per treatment were performed on receptive silks of the primary ear on hybrid corn plants. Pollen viability was qualitatively scored with a ranking of 5 (>60% germination, see Table 29) after 5 days of storage for all treatments. Relative PMC ranged from 99.0% to 104.8% across treatments. Internal gas samples were taken for each treatment from the storage container after wrapping air-impermeable tape over the gas exchange gap. 2 The content ranged from 18.6 to 19.3% throughout the treatment, and CO 2 The content was 0.1%.

[0070] For experiment 3, six replicate pollinations were performed on the receptive silks of the primary ear on the hybrid maize plants. The relative PMC was 97.4%. Internal gas samples were taken from the storage container after wrapping the gas exchange gap with air-impermeable tape. 2 The content is 19.6%, and CO 2 The content was 0.1%. In both experiments, the use of Parafilm as a breathable barrier allowed sufficient gas exchange while minimizing water vapor transmission. The presence of soda lime, in conjunction with the breathable barrier, reduced CO 2 This achieved sufficient isolation.

[0071] The reduction in headspace from 400 ml to 100 ml in experiments 2 and 3 was due to the initial starting molar amount of O2 present in the vascular volume. 2 Does not restrict pollen breathing and is provided with 42mm 2 We demonstrated that a Parafilm breathable barrier of 100 μm surface area covers the gas exchange gap within each GA7 Magenta box. O 2 is constantly replenished into the pollen storage container. In experiment 2, the average seed set for the 400 ml, 300 ml, 200 ml and 100 ml headspace storage containers was 535, 510, 502 and 668 kernels / ear, respectively, indicating that the pollen was viable after 5 days of storage. In experiment 3, an average seed set of 564 kernels / ear indicated the same conclusion.

[0072] [Table 9]

[0073] [Table 10]

[0074] Experiment 4: Reduction of breathable barrier surface area in Magenta GA7 boxes containing pollen mixed with crystalline silica with the addition of soda lime To evaluate the reduction in breathability barrier surface area, 24.871 g of pollen was mixed with 12.436 g of 10 μm crystalline silica (2:1 pollen:silica) for a total of 37.307 g of pollen mixture with an initial fresh mixture PMC of 34.94%. A total of 9.2 g of the pollen mixture was added to each of four 400 ml Magenta GA7 boxes. Three of the four boxes were modified by sealing the gas exchange gap between the box and lid with modelling clay to seal gas exchange on one, two or three sides of the lid, respectively. The sealed edges reduced the surface area available for gas exchange by 10.5 mm 2 , 21mm 2 and 31.5mm 2 A fourth box with the same 400 ml headspace and no gas exchange gap sealed with modelling clay was used as a positive control (surface area 42 mm 2 ). A 50 ml conical tube lid containing 1.11 g soda lime was placed at the bottom of each box. The pollen mixture was contained inside a PLA plastic cup (45 mm x 30 mm) and placed over the open soda lime container. For each treatment, a GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide parafilm around the opening. The parafilm was stretched as is normal practice in plant tissue culture to provide a tight seal. The pollen storage containers were stored at 6°C and 1 atmosphere for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC as well as O were measured. 2 and CO 2 End point gas analysis was evaluated for each treatment.

[0075] Pollen viability ranged from 46% to 50% after 5 days of storage, and relative PMC ranged from 96.9 to 99.9% across treatments. Final PMC increased as breathable barrier surface area decreased. Internal gas samples were taken for each treatment from the storage container after wrapping air-impermeable tape in the gas exchange gap between the box and the lid. 2 The content ranged from 17.8 to 19.1% throughout the treatments, and CO 2The content was 0.1% in all cases. As the breathable barrier surface area decreased, the final O 2 The content was also reduced. By using a reduction in the surface area covered with a Parafilm breathable barrier, the water vapor transmission rate was reduced to 10.5 mm. 2 The presence of soda lime, in conjunction with the breathable barrier, allows for sufficient gas exchange while minimizing CO 2 This achieved sufficient isolation.

[0076] [Table 11]

[0077] [Table 12]

[0078] 4. Alternative Storage Containers – Plastic Clamshell Containers Experiments 1 and 2: 1) Plastic clamshell boxes as a breathable barrier (i.e., non-airtight containers with gaps between sections) with crystalline silica and soda lime added - no pollination (5 days); 2) Plastic clamshell boxes as a breathable barrier (i.e., non-airtight containers with gaps between sections) with crystalline silica and soda lime - with pollination (5 days). In experiment 1, 11.609 g of pollen was mixed with 5.805 g of 10 μm crystalline silica (2:1 pollen:silica by weight) for a total of 17.414 g of pollen mixture, with an initial fresh mix PMC of 33.82%. A total of 17.00 g of pollen mixture was added to a 630 ml polyethylene terephthalate glycol clear plastic clamshell Phytatray™ brand container. 2.07 g of soda lime contained within a 50 ml conical tube cap was placed in the bottom of the clamshell container. In experiment 2, 19.876 g of pollen was mixed with 9.938 g of 10 μm crystalline silica (2:1 pollen:silica) for a total of 29.814 g of pollen mixture, with an initial fresh mix PMC of 35.9%. A total of 22.3 g of the pollen mixture was added to a 630 ml clear deep plastic clamshell container and 3.15 g of soda lime was added directly to the bottom of the container. For both experiments, the pollen mixture was contained inside a PLA plastic cup (45 mm x 30 mm) and placed next to the open soda lime container or the free soda lime. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed next to the pollen mixture container to monitor the internal relative humidity of the pollen storage container. The clamshell lid was placed on top of the clamshell bottom and snapped into place. Any gaps present between the two clamshell container sections were not sealed with any form of barrier. The pollen storage container was then stored at 6°C and 1 atm for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, as well as O 2 and CO 2 The end point gas analysis was evaluated.

[0079] In experiment 1, the iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that a saturated rH of greater than 98% was achieved throughout the entire storage period inside the storage container. After 5 days of storage, pollen viability was 51% and the relative PMC was 95.8%. The inner container rH and final relative PMC data indicate that the clamshell container with sections sealed together using only the manufactured locking mechanism acted as a breathable barrier. An internal gas sample taken from the storage container showed 19.8% O. 2 Content and 0.1% CO 2 The clamshell container allows for sufficient gas exchange while minimizing water vapor transmission, while the presence of soda lime, in conjunction with the gaps between the clamshell sections that act as a breathable barrier, allows for the ingress of CO 2 The clamshell breathable barrier achieved comparable pollen tube germination (51%) to the fresh pollen mix control (54%).

[0080] For experiment 2, four replicate pollinations were performed on receptive silks of the primary ear on hybrid corn plants. Pollen viability was qualitatively scored with a ranking of 3 (>21-40% germination, see Table 29) after 5 days of storage, and the relative PMC was 99.3%. Internal gas samples taken from the storage containers were 19.9% ​​O. 2 Content and 0.1% CO 2 The clamshell container allows for sufficient gas exchange while minimizing water vapor transmission, while the presence of soda lime, in conjunction with the gaps between the clamshell sections that act as a breathable barrier, allows for the ingress of CO 2 The average seed set of 784 kernels / ear indicated that the pollen was viable after 5 days of storage.

[0081] [Table 13]

[0082] [Table 14]

[0083] 5: Tyvek, micropore tape and open perforated breathable barrier surface area typically used for airtight containers. In experiments 1, 2, 3, and 6 of Example 4, Tyvek was used to reduce water vapor transmission from the pollen storage container while allowing gas exchange. In experiments 4, 5, 6, and 7, micropore tape was evaluated, which, like Tyvek, allows gas exchange while reducing water vapor transmission from the pollen storage container. In experiments 1-7 and 9 of Example 4, 100 μl of H 2 O was added to the bottom of the storage container to account for the low humidity (30% rH) in the test environment. The addition of water encouraged the internal atmosphere of the storage container to reach saturated relative humidity.

[0084] Experiment 1: Tyvek as an alternative breathable barrier with added soda lime (5 days) To evaluate Tyvek as a breathable barrier, 9.7551 g of pollen was mixed with 4.8776 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 14.633 g of pollen mixture, with an initial fresh mixture PMC of 37.02%. A total of 14.0 g of pollen mixture was added to a 500 ml wide-mouth mason jar. A PLA plastic cup containing 1.73 g of soda lime was added to a 500 ml wide-mouth mason jar. The pollen mixture was contained inside an aluminum pan (45 mm x 35 mm). The pollen mixture pan was placed on top of the soda lime cup and separated by a metal 841 micrometer mesh screen. The wide-mouth lid was equipped with a septum for gas analysis, and a 3.175 mm perforation was formed in the lid by a handheld die press to act as a breathable barrier. A disk of Tyvek was cut from a standard FedEx envelope and taped over the 3.175 mm (1 / 8 inch) perforation. An impermeable plate sealing film was applied around the perimeter of the Tyvek to seal it to the container lid. The 3.175 mm (1 / 8 inch) perforation was sealed to the 7.9 mm 2The air permeability barrier surface area of ​​the pollen storage container represents the area of ​​air permeability barrier of the pollen storage container. A iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mix container to monitor the internal relative humidity of the pollen storage container. The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was separated from the pollen mix container using a metal 841 micrometer mesh screen. The lid of the pollen storage container was sealed with a mason jar lid band and stored at 6°C and 1 atmosphere for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and O were measured. 2 and CO 2 The end point gas analysis was evaluated.

[0085] Two replicate pollinations were performed on the receptive silks of the primary ear on the hybrid corn plants. Pollen viability was qualitatively scored with a ranking of 3 (>21-40% germination, see Table 29) after 5 days of storage, and the relative PMC was 102.5%. Internal gas samples taken from the storage containers showed 19.1% O 2 Content and 0.1% CO 2 The 3.175 mm (1 / 8 inch) perforations covered by Tyvek allowed adequate gas exchange while minimizing water vapor transmission. The presence of soda lime, in conjunction with the breathable barrier, allowed for good CO 2 The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that the rH was greater than 98% saturated throughout the storage period inside the storage container. The average seed set of 789 kernels / ear indicates that the pollen was viable after 5 days of storage.

[0086] [Table 15]

[0087] [Table 16]

[0088] Experiment 2: Tyvek breathable barrier surface area factorials with soda lime additive In this experiment, 22.608 g of pollen was mixed with 11.304 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 33.912 g of pollen mixture, with an initial fresh mixture PMC of 34.08%. A total of 4.0 g of pollen mixture (2.67 g of pollen) was added to each of seven 125 ml Regular Mouse mason jars. A 50 ml conical tube cap containing 0.56 g of soda lime was added to the bottom of each jar. The pollen mixture was contained inside an aluminum pan (45 mm x 15 mm) and deposited on top of the soda lime containers separated by a piece of metal 841 micrometer mesh screen. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture containers to monitor the internal relative humidity of the seven 125 ml pollen storage containers. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a piece of metallic 841 micrometer mesh screen. Regular Mouse mason jar lids were equipped with septa for gas analysis and a series of different sized perforations was created in each lid by a handheld die press (i.e., each lid received a different size perforation). Perforation sizes included diameters of 0.0, 1 / 16 inch, 3 / 32 inch, 1 / 8 inch, 3 / 16 inch, 1 / 4 inch, and 3 / 8 inch. Disks of Tyvek were cut from a standard FedEx envelope and taped over the perforations. This series of progressively larger perforations covered with a Tyvek breathable barrier was used to evaluate the effect of breathable barrier surface area on pollen storage. A series of different perforation sizes are available, from 2.0mm 2 ~71.3mm 2A range of surface areas of 125 ml were provided (Table 17). A non-perforated lid (0.0 mm in Table 18) was used as a negative control and the pollen mixture was allowed to respire in a sealed container until anaerobic conditions were achieved and the stored pollen was non-viable. Standard mouse mason jar lids were sealed to 125 ml mason jars with mason jar lid bands.

[0089] A positive control was included by adding 4.89 g of the pollen mix to a 400 ml GA7 Magenta box containing 0.68 g of added soda lime. The soda lime was contained within a 50 ml conical tube cap placed at the bottom of the GA7 box. 100 μl of water was added to the bottom of the GA7 container. The pollen mix was contained inside a PLA plastic cup (approximately 45 mm x 30 mm) and placed over the open soda lime container. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed over the pollen mix container to monitor the internal relative humidity of the GA7 Magenta box. The GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and the lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide parafilm around the open gap. The parafilm was stretched as per normal practice in plant tissue culture to provide a tight seal. The GA7 gas exchange gap between the box and the lid surface area is 42mm in total 2 All treatments were stored at 6°C and 1 atm for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0090] For all treatments, except for the negative control treatment without a breathable barrier, two replicate pollinations were performed on the receptive silks of the primary ear on the hybrid corn plants. The negative control was non-viable after 5 days of storage, with the vessel pressure decreasing to -22 kPa gauge due to pollen respiration and internal O 2 The content is 0.4%, and the internal CO 2The content was 0.1% and the final relative PMC was 103.2%. Pollen viability ranged from 64% to 78% after 5 days of storage for the positive control and treatments with Tyvek covered perforations ranging from 1.588 mm (1 / 16 inch) to 9.525 mm (3 / 8 inch). All treatments with Tyvek covered perforations ranging from 1.588 mm (1 / 16 inch) to 9.525 mm (3 / 8 inch) had higher pollen tube germination rates than the GA7 Magenta boxes and parafilm with soda lime positive control. The relative final PMC ranged from 97.3% to 108.2% after 5 days of storage for the positive control and treatments with Tyvek covered perforations ranging from 1.588 mm (1 / 16 inch) to 9.525 mm (3 / 8 inch). The positive control had the lowest final PMC. As the Tyvek breathable barrier surface area increased, the relative final PMC appeared to decrease. Internal gas samples were taken from each treatment and showed O ranging from 19.1% to 19.5%. 2 Content and CO in the range of 0.1% to 0.3% 2 The higher Tyvek breathable barrier surface area treatments and the positive control had lower O content than the lower Tyvek breathable barrier surface area treatments. 2 Low content and CO 2 The positive control and all Tyvek treatments provided adequate gas exchange while minimizing water vapor transmission across all surface areas tested. The presence of soda lime, along with both types of breathable barrier, provided adequate CO 2 The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that all treatments achieved greater than 98% saturation rH throughout the entire storage period inside the storage container. Seed set was observed to increase as the surface area of ​​the Tyvek breathable barrier increased compared to the positive control. The average seed set ranged from 686 kernels / ear to 788 kernels / ear, indicating that pollen was viable after 5 days of storage.

[0091] [Table 17]

[0092] [Table 18]

[0093] [Table 19]

[0094] Experiment 3: Surface Area Factorial of Perforations in Normally Airtight Containers (i.e. Mason Jars and Lids) with Addition of Soda Lime For the following experiment, 24.832 g of pollen was mixed with 12.416 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 37.248 g of pollen mixture, with an initial fresh mixture PMC of 35.41%. A total of 4.0 g of pollen mixture (2.67 g of pollen) was added to each of six 125 ml Regular Mouse mason jars. A 50 ml conical tube cap containing 0.56 g of soda lime was added to the bottom of each mason jar. The pollen mixture was contained inside an aluminum pan (45 mm x 15 mm). The bread containing the pollen was deposited on top of the soda lime container and separated by a piece of metal 841 micrometer mesh screen. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture container to monitor the internal relative humidity of the six 125 ml pollen storage containers. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a piece of metallic 841 micrometer mesh screen. A Regular Mouth mason jar lid equipped with a septum for gas analysis was placed on top of each 125 ml mason jar lid and sealed with a standard mason jar lid band.

[0095] Using a handheld die press, one mason jar lid was modified to contain 1.588 mm (1 / 16 inch) perforations and a second mason jar lid was modified to contain 3.175 mm (1 / 8 inch) perforations. Disks of Tyvek were cut from a standard FedEx envelope and taped over each of the two perforations to act as breathable barriers. An impermeable plate sealing film was applied to the perimeter of the Tyvek to seal it to the container lid. Two Tyvek breathable barrier treatments were used as positive controls. The remaining four 125 ml pollen storage containers each received a different size perforation in the lid. Needles of various gauge sizes (18 gauge, 20 gauge, 23 gauge and 30 gauge needles) were used to create the perforations. The needles were used to create open microperforations from the inside of the pollen storage container to the outside atmosphere and 0.015 mm microperforations. 2 ~0.552mm 2 The 125 ml Mason lid pollen storage containers were sealed with a Mason jar lid band. All treatments were stored at 6° C. and 1 atm for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0096] All treatments were pollinated twice on receptive silks of the primary ear on hybrid corn plants, except for the 30-gauge micro-drilled treatment, which was non-viable after 5 days of storage and was pollinated in anaerobic internal O. 2 Content is 0.2%, CO 2 The content was 0.1% and the final relative PMC was 98.5%. Pollen tube germination was 48% for the 3.175 mm perforated (1 / 8 inch) and 50% for the 1.588 mm (1 / 16 inch) perforated positive control Tyvek breathable barrier treatments. Pollen tube germination ranged from 61% to 62% after 5 days of storage for the 18 gauge, 20 gauge, and 23 gauge treatments. The open microperforated treatments showed higher pollen tube germination compared to the Tyvek treatments. The relative final PMC ranged from 94.4% to 103.0% after 5 days of storage for all treatments. Internal gas samples were taken from each Tyvek treatment and O2 While the content ranges from 18.5% to 18.8%, CO 2 The results showed that the O content ranged from 0.1% to 0.2%. The two Tyvek breathable barrier treatments allowed adequate gas exchange while minimizing water vapor transmission across all surface areas tested. The open microperforations reduced and inhibited gas exchange. Internal gas samples showed 8.2%, 2.2%, and 6.1% O from the 18-gauge, 20-gauge, and 23-gauge open microperforation treatments, respectively. 2 The content was measured. 0.2% CO 2 The presence of soda lime reduced the CO 2 The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that all treatments achieved greater than 98% saturation rH throughout the entire storage period inside the storage container. Seed set was observed to increase as the surface area of ​​the Tyvek breathable barrier increased. Seed set was observed to increase as the open area of ​​the microperforations increased from 23 gauge to 18 gauge. The Tyvek breathable barrier treatments had greater seed set than any of the open microperforation treatments. Average seed set ranged from 629 kernels / ear to 770 kernels / ear indicating that pollen was viable after 5 days of storage.

[0097] [Table 20]

[0098] [Table 21]

[0099] [Table 22]

[0100] [Table 23]

[0101] Experiment 4: 3M Micropore Tape as an Alternative Breathable Barrier with Soda Lime (5 days) For the micropore tape evaluation, 10.396 g of pollen was mixed with 5.190 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 15.5856 g of pollen mixture, with an initial fresh mixture PMC of 34.90%. A total of 4.0 g of pollen mixture (2.67 g of pollen) was added to three 125 ml regular mouth mason jars and a 50 ml conical tube cap containing 0.56 g of soda lime was added to the bottom of each mason jar. The pollen mixture was contained inside an aluminum pan (approximately 45 mm x 15 mm). The pan containing the pollen mixture was placed on top of the soda lime container and separated by a piece of metal 841 micrometer mesh screen. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture container to monitor the internal relative humidity of each of the three 125 ml pollen storage containers. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a piece of metallic 841 micrometer mesh screen. Regular Mouse mason jar lids were equipped with septa for gas analysis. One lid of the three mason jar lids used was modified to contain a 1.588 mm (1 / 16 inch) perforation using a handheld die press, and two lids were modified to contain a 3.175 mm (1 / 8 inch) perforation. A disk of Tyvek was cut from a standard FedEx envelope and taped over the 3.175 mm (1 / 8 inch) perforation in one of the three lids. An impermeable plate sealing film was applied around the perimeter of the Tyvek to seal the Tyvek to the container lid. The 3.175 mm (1 / 8 inch) perforation with a Tyvek breathable barrier was used as a positive control. 3M brand micropore tape was placed over the 1 / 16 inch perforation in the second lid and the same micropore tape was placed over the 1 / 8 inch perforation in the third lid. The lids were sealed onto the 125 ml mason jar pollen storage containers using mason jar lid bands. All treatments were stored at 6°C and 1 atmosphere for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and overall PMC were measured. 2 and CO2 The end point gas analysis was evaluated.

[0102] A single pollination was performed on receptive silks of the primary ear on hybrid corn plants per treatment. Pollen viability was qualitatively scored on a ranking of 4 (>41-60% germination, see Table 29) after 5 days of storage for all treatments. Relative PMC ranged from 103.8% to 106.0% across treatments. Internal gas samples were taken from each jar and O 2 The content ranged from 18.4% to 19.4%, while CO 2 The O content ranged from 0.1% to 0.2%. The 1.588 mm (1 / 16 inch) micropore tape treatment had a minimum O content of 18.4%. 2 While the content was shown, 0.2% CO 2 The content of the gas was also shown. Both the Tyvek and Micropore Tape breathable barriers allowed sufficient gas exchange while minimizing water vapor transmission over the surface area tested. The presence of soda lime, in conjunction with the breathable barrier, allowed for CO 2 The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that all treatments achieved greater than 98% saturated rH throughout the entire storage period inside the storage container. The 3.175mm (1 / 8 inch) Tyvek breathable barrier treatment and the 1.588mm (1 / 16 inch) Micropore Tape breathable barrier treatment both reached 100% saturated rH, while only the 3.175mm (1 / 8 inch) Micropore Tape breathable barrier treatment reached 98% rH. Seed set of 711 kernels / ear, 751 kernels / ear, and 696 kernels / ear was achieved for the 3.175mm (1 / 8 inch) Tyvek breathable barrier, 1.588mm (1 / 16 inch) Micropore Tape breathable barrier, and 3.175mm (1 / 8 inch) Micropore Tape breathable barrier, respectively, indicating that the pollen is viable after 5 days of storage.

[0103] [Table 24]

[0104] Experiment 5: Surface Area Factorial with Tyvek vs. Micropore Tape with Soda Lime (5 Days) For further evaluation, 10.89 g of pollen was mixed with 5.445 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 16.335 g of pollen mixture, with an initial fresh mixture PMC of 33.77%. A total of 2.0 g of pollen mixture (1.335 g of pollen) was added to eight 125 ml Regular Mouse mason jars. A 50 ml conical tube cap containing 0.28 g of soda lime was added to the bottom of each mason jar. The pollen mixture was contained inside an aluminum pan (45 mm x 15 mm). The pan containing the pollen mixture was placed on top of the soda lime container and separated by a piece of metal 841 micrometer mesh screen. The internal relative humidity of the eight 125 ml pollen storage containers was monitored by placing an iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger on the pollen mixture in each container. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a metallic 841 micrometer screen strip. Regular Mouse mason jar lids were equipped with septa for gas analysis. A total of eight mason jar lids were modified to contain one perforation per lid using a handheld die press. Two lids were modified each to contain 1.588 mm (1 / 16 inch), 3.175 mm (1 / 8 inch), 6.35 mm (1 / 4 inch) and 9.525 mm (3 / 8 inch) perforations. Disks of Tyvek were cut from a standard FedEx envelope and taped over one lid at each preformed size. An impermeable plate sealing film was applied around the perimeter of the Tyvek to seal the Tyvek to the lid. One strip of 3M Micropore tape was applied over the second lid at each perforation size. In addition to evaluating Micropore Tape against Tyvek, this experiment also evaluated the effect of increasing or decreasing breathable barrier surface area. Perforated lids were sealed to each 125 ml Mason jar pollen storage container using Mason jar lid bands. All treatments were stored at 6°C and 1 atm for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0105] A single repeat pollination was performed on receptive silks of the primary ear on hybrid corn plants for each treatment. Pollen tube germination ranged from 55% to 61% for the Tyvek treatments and 47% to 62% for the Micropore Tape treatments after 5 days of storage. Internal gas samples were obtained from each treatment. 2 The content ranged from 19.5% to 19.9% ​​throughout the Tyvek treatments, but CO 2 The content was measured at 0.1%. 2 The content ranged from 19.3% to 19.9% ​​throughout the micropore tape treatment, but CO 2 The content was measured at 0.1%. 2 The content increased with increasing surface area of ​​both types of breathable barrier. The presence of soda lime reduced CO2 absorption in the tested surface area, in conjunction with both types of breathable barrier. 2The relative final PMC ranged from 102.2% to 84.3% throughout the Tyvek treatments and 101.5% to 78.6% for the Micropore Tape treatments. The relative final PMC was observed to decrease as the surface area of ​​both types of breathable barrier increased. Both the 3 / 8 inch perforated treatments covered with Tyvek and Micropore Tape had higher pollen moisture loss. In general, the Tyvek breathable barrier treatments retained higher pollen moisture content compared to the Micropore Tape treatments. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that greater than 98% saturation rH was achieved throughout the storage period for both the 1 / 16 inch and 1 / 8 inch perforated treatments of Tyvek and Micropore Tape. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that rH greater than 96% was obtained throughout the storage period for the 6.35mm (1 / 4 inch) and 9.525mm perforation treatments covered with Tyvek and the 6.35mm (1 / 4 inch) micropore tape-covered perforation treatment. An rH greater than 94% was observed throughout the storage period for the 9.525mm (3 / 8 inch) micropore tape-covered perforation treatment. Seed set of greater than 500 kernels / ear for all eight treatments indicated that pollen was viable (Figure 3).

[0106] [Table 25]

[0107] [Table 26]

[0108] [Table 27]

[0109] Experiment 6: Surface Area Factorials with Perforation and Soda Lime in Normally Airtight Containers (i.e. Mason Jars and Lids) (5 Days) For experiment 6, 6.48 g of pollen was mixed with 3.24 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 9.72 g of pollen mixture, with an initial fresh mixture PMC of 35.78%. A total of 1.95 g of pollen mixture (1.3007 g of pollen) was added to four 125 ml regular mouth mason jars, and 1.6 g of pollen mixture (1.0672 g of pollen) was added to the fifth 125 ml regular mouth mason jar. A 50 ml conical tube cap containing 0.28 g of soda lime was added to the bottom of each container. The pollen mixture was contained inside an aluminum pan (45 mm x 15 mm). The pan containing the pollen mixture was placed on top of the soda lime container and separated by a metal 841 micrometer mesh screen piece. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mixture container to monitor the internal relative humidity of each of the eight 125 ml pollen storage containers. The iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was separated from the pollen mixture pan using a piece of metallic 841 micrometer mesh screen. Five regular mouth mason jar lids were equipped with septa for gas analysis. One lid was modified to include a 3.175 mm (1 / 8 inch) perforation made by a handheld die press. A disk of Tyvek was cut from a standard FedEx envelope and taped over the 3.175 mm (1 / 8 inch) perforation. An impermeable plate sealing film was applied to the perimeter of the Tyvek to seal the Tyvek to the container lid. A 3.175 mm perforated (1 / 8 inch) Tyvek breathable barrier jar received 1.95 g of the pollen mixture and was used as a positive control in the experiment.

[0110] The remaining four lids were modified to contain perforations using a handheld die press. One lid was modified to contain perforations measuring 1 / 16", 1 / 8", 1 / 4" or 3 / 8" in diameter. The perforations in each of the four lids were left open to the atmosphere during storage to determine if the perforations could act as a breathable barrier without additional covering. This experiment also evaluated the effect on open perforation surface area. The 1 / 16", 1 / 8", and 1 / 4" open perforation treatments each received 1.95g of pollen mixture, while the 3 / 8" open perforation treatment received 1.6g of pollen mixture. Each lid was sealed onto a 125ml mason jar using a mason jar lid band. All treatments were stored at 6°C and 1 atm for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0111] A single pollination was performed on receptive silks of the primary ear on hybrid corn plants for each treatment. Pollen tube germination was 50%-55% for all treatments except for the 9.525 mm (3 / 8 inch) open punch treatment after 5 days of storage. The 9.525 mm (3 / 8 inch) open punch treatment was originally qualitatively scored with a ranking of 2 (<10% germination, see Table 29) after 60 minutes of pollen tube germination. A delay in pollen tube germination was observed. Samples were incubated for another 5 hours in 100% rH2O. The 9.525 mm (3 / 8 inch) open punch treatment showed 43% pollen tube germination after 5 hours. Internal gas samples were taken for each treatment and O 2 The content ranged from 19.9% ​​to 20.0%, while CO 2 All content measurements were at 0.1%. The presence of soda lime, in conjunction with Tyvek and the open perforated breathable barrier, reduced CO 2The relative final PMC for the 3.175 mm (1 / 8 inch) Tyvek covered perforated positive control was 91.7%. The internal rH inside the 6°C storage chamber varied from 30% to 50% during the storage period. The relative final PMC ranged from 102.2% to 84.3% across the Tyvek treatments and 94.9% to 46.0% for the range of open perforation treatments. The relative final PMC decreased as the surface area of ​​the open perforation increased. The 6.35 mm (1 / 4 inch) and 9.525 mm (3 / 8 inch) open perforation treatments had greater pollen moisture loss. The iButtonLink DS1923-F5# Hygrochron Temperature and Humidity Data Logger showed that for both the 1 / 8 inch Tyvek covered perforated positive control treatment and the 1 / 16 inch and 1 / 8 inch open perforated treatments, greater than 98% of the saturated rH was achieved throughout the storage period. The iButtonLink DS1923-F5# Hygrochron Temperature and Humidity Data Logger data showed that for the 1 / 4 inch open perforated treatment, greater than 92% of the rH was achieved throughout the storage period. For the 3 / 8 inch open perforated treatment, greater than 85% of the rH was observed throughout the storage period. Seed set of over 700 kernels / ear was obtained for the 3.175 mm (1 / 8 inch) Tyvek, 1.588 mm (1 / 16 inch), 3.175 mm (1 / 8 inch), and 6.35 mm (1 / 4 inch) open-perforated treatments, indicating that the pollen was viable after 5 days of storage. The 9.525 mm (3 / 8 inch) open-perforated treatment yielded 322 kernels / ear, suggesting that greater moisture loss from the pollen during storage adversely affected seed set.

[0112] [Table 28]

[0113] [Table 29]

[0114] [Table 30]

[0115] Experiment 7: Larger Perforations with Tyvek and Micropore Tape Breathable Barrier with Soda Lime (5 Days) To evaluate larger perforations, 11.8924 g of pollen was mixed with 5.946 g of 10 μm crystalline silica for a total of 17.839 g of pollen mixture, with an initial fresh mixture PMC of 35.9%. Pollen was mixed in a weight ratio of 2 parts pollen, 1 part carrier. A total of 2.0 g of pollen mixture (1.335 g of pollen) was added to six 125 ml regular mouth mason jars. A 50 ml conical tube cap containing 0.28 g of soda lime was added to the bottom of each mason jar. The pollen mixture was contained inside an aluminum pan (45 mm x 15 mm). The pollen mixture container was placed on top of the soda lime container and separated by a metal 841 micrometer mesh screen piece. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mix container to monitor the internal relative humidity of each of the six 125 ml pollen storage containers, separated from the pollen mix containers using a piece of metallic 841 micrometer mesh screen. Regular mouth mason jar lids were equipped with septa for gas analysis.

[0116] Six lids were modified to contain perforations using a handheld die press. Two lids were modified to contain 1 / 8", 7 / 16", and 1 / 2" perforations, respectively. Disks of Tyvek were cut from standard FedEx envelopes and taped over one example of each perforation size for a total of three Tyvek treatments. An impermeable plate sealing film was applied to the perimeter of the Tyvek to seal it to the container lids. Strips of 3M Micropore Tape were applied over a second set of lids, one for each of the three perforation sizes. In addition to evaluating Micropore Tape compared to the Tyvek breathable barrier, this experiment also evaluated the effect of increasing or decreasing the surface area for both breathable barriers. Lids were sealed to six 125 ml Mason jar pollen storage containers using Mason jar lid bands. All treatments were stored at 6°C and 1 atm for five days. After 5 days of storage, pollen viability relative to the initial period and final PMC were assessed.

[0117] A single pollination was performed on receptive silks of the primary ear on hybrid corn plants for each treatment. Pollen tube germination was 53%-55% for the Tyvek treatments and 47%-50% for the Micropore Tape treatments after 5 days of storage. Relative final PMC ranged from 101.5%-98.0% throughout for the Tyvek treatments and 94.2%-83.3% for the Micropore Tape treatments. Relative final PMC was observed to decrease with increasing surface area for both breathable barrier types. The Tyvek treatments maintained higher pollen moisture content than equivalent Micropore Tape treatments. iButtonLink DS1923-F5# Hygrochron temperature and humidity data loggers showed that greater than 98% saturated rH was obtained throughout the storage period for both the Tyvek and Micropore Tape covered 3.175 mm perforated (1 / 8 inch) treatments. iButtonLink DS1923-F5# Hygrochron temperature and humidity data showed that the 7 / 16" and 1 / 2" perforation treatments covered with Tyvek achieved rH greater than 96% throughout the storage period. iButtonLink DS1923-F5# Hygrochron temperature and humidity data showed that the 7 / 16" and 1 / 2" perforation treatments covered with Micropore Tape achieved rH greater than 98% with a delay of approximately 36 hours. The 7 / 16" and 1 / 2" perforation treatments covered with Micropore Tape had relative final PMCs of 83.3% and 90.1%, respectively, indicating that the surface area per gram of pollen for Micropore Tape was too large to maintain sufficient pollen moisture content. Seed set ranged from 614 kernels / panicle to 755 kernels / panicle, indicating that pollen was viable after 5 days of storage.

[0118] [Table 31]

[0119] [Table 32]

[0120] Experiment 8: Higher pollen load, direct deposition in tube and micropore tape - no soda lime (5 days) In this experiment, 20.19 g of pollen was mixed with 10.096 g of 10 μm crystalline silica for a total of 30.286 g of pollen mixture, with an initial fresh mixture PMC of 36.10%. Pollen was mixed in a weight ratio of 2 parts pollen, 1 part carrier. A total of 29.675 g of pollen mixture (approximately 40 ml) was added directly into a 50 ml conical tube. A 50 ml conical tube cap was modified to include a 9.525 mm (3 / 8 inch) diameter perforation drilled in the center of the cap. The perforation was covered with 3M Micropore tape to remove 71.3 mm 2 The modified cap was placed over the 50 ml conical tube filled with maximized pollen and screwed down to seal with the tube. The capped conical tubes were stored in a vertical orientation (cap directly up) at 6° C. and 1 atm for 5 days. After 5 days of storage, pollen viability relative to initial and final PMC were assessed.

[0121] The pollen mixture was subsampled at depths of 1.5 cm (top), 4.0 cm (middle) and 6.5 cm (bottom) below the surface of the pollen mixture. A single pollination was performed on receptive silks of the primary ear on the hybrid maize plants at each depth. Pollen tube germination was 57% at 1.5 cm, 57% at 4.0 cm and 59% at 6.5 cm. Relative final PMC was 98.4% at 1.5 cm, 102.7% at 4.0 cm and 103.0% at 6.5 cm. Relative final PMC increased with increasing depth from the surface of the pollen mixture. Pollen tube germination evaluations showed that the pollen mixture could be directly deposited in storage containers up to 7 cm deep with 10 ml headspace without issue. Relative final PMC and pollen viability evaluations were 71.3 mm. 2We demonstrated that the breathable barrier surface area of ​​the micropore tape provides sufficient gas exchange to support pollen respiration while minimizing water vapor transmission from the storage container. Using pollen subsampled at a depth of 1.5 cm, seed set was obtained at 773 kernels / panicle, 749 kernels / panicle at 4.0 cm, and 689 kernels / panicle at 6.5 cm, indicating that pollen was viable after 5 days of storage.

[0122] [Table 33]

[0123] Experiment 9: Micropore Tape Surface Area Factorial-No Soda Lime vs. Soda Lime (5 days) For experiment 9, 19.43 g of pollen was mixed with 9.72 g of 10 μm crystalline silica for a total of 29.15 g of pollen mixture, with an initial fresh mixture PMC of 37.22%. Pollen was mixed in a weight ratio of 2 parts pollen, 1 part carrier. 3.5 g of pollen mixture was added to each of eight 125 ml regular mouth mason jars. The pollen mixture was contained inside an aluminum pan (45 mm x 15 mm). A 50 ml conical tube cap containing 0.49 g of soda lime was added to the bottom of four 125 ml regular mouth mason jars. An empty 50 ml conical tube cap was placed on the bottom of the remaining four 125 ml regular mouth mason jars. The pan containing the pollen mixture was placed on top of the 50 ml conical tube cap soda lime container. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mix container to monitor the internal relative humidity of each of the eight 125 ml pollen storage containers, separated from the pollen mix container using a piece of metallic 841 micrometer mesh screen. Regular Mouth Mason jar lids were equipped with septa for gas analysis. All eight lids were modified to contain perforations using a handheld die press. Two sets of lids were each modified to contain 1 / 16 inch, 1 / 8 inch, 1 / 4 inch, or 3 / 8 inch perforations. For all eight lids, a piece of 3M Micropore tape was applied over the perforations. This resulted in a Micropore Tape breathable barrier surface area of ​​2.0 mm2 each. 2 , 7.9mm 2 , 31.7mm 2 and 71.3 mm 2 The objective of this experiment was to determine whether a micropore tape breathable barrier with minimal surface area per gram of pollen mixture could effectively absorb cellular respiration-derived CO in the absence of soda lime. 2The objective of this study was to determine how much chlorine was required to prevent the accumulation of chlorine to toxic levels. The modified lids were sealed onto 125 ml Mason jar pollen storage containers using Mason jar lid bands. All treatments were stored at 6°C and 1 atm for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and O were assessed. 2 and CO 2 The end point gas analysis was evaluated.

[0124] A single pollination was performed on the receptive silks of the primary ear on the hybrid corn plants for each treatment. Pollen tube germination ranged from 43% to 56% for all treatments. Treatments with soda lime were observed to have a lower percentage of pollen tube germination compared to treatments without soda lime. Internal gas samples were taken for each treatment and O 2 The content ranged from 19.7% to 19.2%. 2 The presence of soda lime, in conjunction with the Micropore Tape breathable barrier, reduced CO2 absorption in all tested surface areas. 2 The results showed that sufficient sequestration of CO was achieved without soda lime. 2 The content is 2.0 mm 2 , 7.9mm 2 , 31.7mm 2 and 71.3 mm 2 For each surface area and amount of pollen mixture stored tested without soda lime, the CO 2There was sufficient gas exchange to keep accumulation below toxic levels that would affect pollen. Relative final PMC ranged from 100.5% to 87.3% across all 3M Micropore Tape treatments. No pattern of increase or decrease in relative final PMC was observed, regardless of the presence of soda lime. iButtonLink DS1923-F5# Hygrochron temperature and humidity data loggers showed that greater than 98% saturated rH was achieved throughout the storage period for all treatments, regardless of the presence of soda lime. Seed set for single test pollinations from all eight treatments exceeded 500 kernels / ear, indicating that pollen was viable after 5 days of storage (Table 35). Seed set and pollen tube germination results showed that the breathable barrier was able to withstand CO2 without the presence of added soda lime. 2 It has been shown that sufficient gas exchange can be achieved to keep the concentration of CO2 below toxic levels.

[0125] [Table 34]

[0126] [Table 35]

[0127] Experiment 10: Comparison of single layer vs. double layer of Micropore Tape without soda lime (5 days) The experiment investigated the effect of doubling the layers of Micropore Tape breathable barrier. 27.84 g of pollen was mixed with 13.92 g of 10 μm crystalline silica for a total of 41.76 g of pollen mixture, with an initial fresh mixture PMC of 36.06%. Pollen was mixed in a weight ratio of 2 parts pollen, 1 part carrier. A total of 20.0 g of the pollen mixture was added directly to the bottom of two 125 ml Regular Mouth Mason jars. The Regular Mouth Mason jar lids were equipped with septa for gas analysis. The two lids were modified to contain identical perforations measuring 9.525 mm (3 / 8 in) in diameter using a handheld die press. A single layer of 3M Micropore Tape was applied over the perforations in one lid. The perforations in the second lid were covered with two layers of 3M Micropore Tape. One layer was applied to the top of the lid covering the perforation and the second layer was applied to the bottom side of the lid covering the perforation. Identical 3 / 8 inch (9.525 mm) diameter perforations in each lid covered with 3M Micropore tape were tapered to 71.3 mm. 2 The experimental design tested whether two layers of the micropore tape breathable barrier could be used to increase the relative final PMC of pollen during storage and to determine if the rate of gas exchange through two layers was sufficient to support pollen respiration compared to a single layer. Treatments were stored at 6°C and 1 atm for 5 days. On the fifth day of storage, pollen viability, final PMC relative to initial PMC, and O 2 and CO 2 The end point gas analysis was evaluated.

[0128] Three replicate pollinations were performed on receptive silks of the primary ear on hybrid corn plants per treatment. Pollen tube germination was annotated at 55% for the single layer of micropore tape and 56% for the double layer of micropore tape. The relative final PMC was 102.7% for the single layer and 104.3% for the double layer, indicating that the relative final PMC increased when a second layer of micropore tape was added. Internal gas samples were taken for each of the two storage containers. O for the single layer was 100.0%. 2 The content is 19.3%, and CO 2 The content was 0.7%. 2The content is 18.2%, and CO 2 The single-layer treatment supported greater gas exchange compared to the double-layer treatment. The double-layered micropore tape allowed sufficient O2 to support pollen respiration. 2 The level was maintained. 2 The levels remained lower than those toxic to pollen during storage with two layers of micropore tape, but CO 2 The pollen tube germination assessment was 71.3 mm in single or double layers. 2 We demonstrated that the surface area of ​​the micropore tape barrier provided sufficient gas exchange to support pollen respiration while minimizing water vapor transmission from the storage container. Average seed set of 707 kernels / ear for a single layer of micropore tape coverage and 663 kernels / ear for a double layer coverage was observed, indicating that pollen was viable after 5 days of storage.

[0129] [Table 36]

[0130] 6: Water vapor permeability of Tyvek, micropore tape, open perforation and Parafilm Experiments 1 and 2: Water vapor transmission rate to atmosphere across a Tyvek barrier, a 3M Micropore Tape barrier, and an open perforation (uncovered) Two experiments were conducted to investigate the water vapor transmission rate through Tyvek and 3M Micropore Tape breathable barriers compared to perforations open to the atmosphere. All three breathable barriers had a transmission rate of 71.3 mm. 2The samples had identical surface areas of 1000 μm x 1000 μm. Water vapor transmission rates were measured by the weight loss of steady-state water vapor flow over unit time covering a unit area of ​​the breathable barrier under specific temperature and humidity conditions. Water vapor transmission rates were measured at 23° C. and 58%-61% relative humidity following a similar approach as shown in the ASTM Standard Test Methods for Water Vapor Transmission of Materials (E96 / E96M-16). See generally www.astm.org / standards / e96. An approximately 4 liter acrylic vacuum chamber (Terra Universal model 5235-01B) with interior dimensions of 28 cm (11 in) x 28 cm (11 in) x 12.7 cm (5 in) was placed in a Fisher Scientific benchtop incubator (model 6500). A glycerol solution was prepared at 80% pure glycerol by weight in deionized water. The specific gravity of the glycerol solution was 1.4374. 500 ml of this glycerol water solution was placed in an open container measuring 8.89 cm (3.5 in) by 15.24 cm (6.0 in). The open container of glycerol water solution was placed in an acrylic chamber to achieve the proper humidity within the chamber. Approximately 60 ml of deionized water was placed into each of twelve 125 ml Ball Mason jars. The deionized water was approximately 19 mm (3 / 4 in) below the lid of each jar. The twelve Mason jar lids were die pressed to a depth of 71.3 mm using a handheld die press. 2The lids were modified to include a 3.175 mm perforation. Disks of Tyvek were cut from a standard FedEx envelope and taped over the 3.175 mm perforation in three of the 12 lids. An impermeable plate sealing film was applied to the perimeter of the Tyvek to seal the Tyvek to the container lids. 3M Micropore tape was placed over the perforations in three of the 12 lids. The perforations in the remaining six of the 12 lids were left uncovered. The lids were placed over identical mason jars containing deionized water and sealed using mason jar lid bands. All twelve sealed jars were inserted into an acrylic chamber and the acrylic chamber was placed in a Fisher incubator to maintain a controlled atmosphere for up to eight days. Weight loss measurements were collected to calculate the water vapor transmission rate from all twelve jars encompassing the three breathable barrier treatments.

[0131] The Tyvek breathable barrier was observed to reduce water vapor loss by 48.3% at 60% rH and 23°C under static airflow compared to open perforations. The Micropore Tape breathable barrier was observed to reduce water vapor loss by 23.2% at 60% rH and 23°C under static airflow compared to open perforations. The Tyvek barrier was more effective than the Micropore Tape barrier in reducing the water vapor transmission rate of the storage container by 32.6% at 60% rH and 23°C under static airflow.

[0132] [Table 37]

[0133] Continuous gas analysis during storage at 7:6°C Experiment 1: Continuous gas application during pollen storage with a breathable barrier (9 days) In the continuous gas experiment, 9.699 g of pollen was mixed with 4.845 g of 10 μm crystalline silica (2:1 weight pollen:silica) for a total of 14.53 g of pollen mixture, with an initial fresh mixture PMC of 36.39%. A total of 14.4 g of pollen mixture was added to a 400 mL Magenta GA7 box. A 50 ml conical tube cap containing 2.03 g of soda lime was placed at the bottom of the GA7 box. The pollen mixture was contained inside a PLA plastic cup (45 mm x 30 mm) and the container was placed on top of an open soda lime container. Due to 30% rH in the lab environment during the experimental setup, 100 μl of water was added to the bottom of the GA7 box. The addition of water was intended to increase the rate at which the internal atmosphere of the storage container reached saturated relative humidity. An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger was placed on top of the pollen mix container to monitor the internal relative humidity of the pollen storage container. The GA7 lid was placed on top of the GA7 box and the gas exchange gap between the box and lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide parafilm around the opening. The parafilm was stretched as is normal practice in plant tissue culture to provide a tight seal. The GA7 gas exchange gap between the box and lid surface area was a total of 42 mm 2 The pollen storage container was stored at 6°C and 1 atm for 9 days. 2 and CO 2 A Quantek 902P gas analyzer was set up for continuous measurements of O. The input and output lines of the analyzer were attached to two 23 gauge precision glide needles and inserted into two septa placed in the side wall of the GA7. The input and output lines of the analyzer were passed through stoppers into the side wall of the freezer unit. The analyzer was kept outside of the freezer unit at room temperature. After 9 days, pollen tube germination, final PMC versus initial PMC, and O were measured. 2 and CO 2 Continuous gas analysis was evaluated.

[0134] An iButtonLink DS1923-F5# Hygrochron temperature and humidity data logger showed that greater than 98% saturation rH was obtained throughout the entire storage period inside the storage container. Pollen tube germination was qualitatively scored with a ranking of 2 (1% to 20% germination, see Figure 10) after 9 days. The relative PMC was 97.6%. The inner container rH and final relative PMC data showed that the use of parafilm as a breathable barrier to seal the 400mL Magenta GA7 box reduced moisture loss from the storage container and pollen mix. Continuous internal gas sampling was taken from the storage container with a 1 minute sampling interval. The continuous gas data was normalized to the atmospheric pressure change. O 2 The content decreased to a minimum of 19.4% within the first few hours of storage at 6°C and CO 2 The content remained below 0.1%. 2 The content remained at a steady state of 20.0% throughout the first 8 days of the storage experiment. 2 The content increased to 20.4%, likely due to a reduction in pollen viability and associated reduction in pollen respiration. The use of parafilm as a breathable barrier allowed sufficient gas exchange while minimizing water vapor transmission. The presence of soda lime, in conjunction with the breathable barrier, reduced CO 2 The breathable barrier provided high steady-state levels of oxygen to support pollen respiration. No reduction in oxygen content to anaerobic levels was measured inside the storage container.

[0135] 8. Pollen storage scale-up using non-airtight containers with manufactured openings without soda lime (i.e. 250 ml VWR® cell culture flasks) Experiments 1, 2, and 3: 1) Breathable barrier 250ml cell culture flasks with stock nitrocellulose barrier - no soda lime (days 5, 6, 7, 8, and 9); 2) Breathable barrier 250ml cell culture flasks stock cap vented covered with 3M Micropore Tape - no soda lime (days 5, 6, 7, 8, and 9); 3) Breathable barrier 250ml cell culture flasks with cap removed and entire opening covered with one layer of 3M Micropore Tape - no soda lime (days 5, 6, 7, 8, and 9). For experiments 1, 2, and 3, fresh pollen was collected and mixed with 10 μm crystalline silica (2:1 by weight pollen:silica) on three different days. The bulk pollen mixture was added directly to 250 ml VWR® cell culture flasks without the addition of soda lime. Each experiment consisted of a different culture flask cap breathable barrier treatment. Each treatment per experiment included one replicate.

[0136] For experiment 1, a total of 51.15 g of pollen mixture (34.42% initial fresh mixture PMC) was added to a 250 ml VWR® cell culture flask. A stock vented cap from a cell culture flask with stock embedded in nitrocellulose barrier was placed on the flask and tightened to create a seal. For experiment 2, a total of 48.4 g of pollen mixture (36.97% initial fresh mixture PMC) was added directly to a 250 ml VWR® cell culture flask. The stock embedded nitrocellulose barrier was removed from the vented cap and the vent was covered with one layer of 3M Micropore Tape. For experiment 3, a total of 18.06 g of pollen mixture (40.7% initial fresh mixture PMC) was added directly to a 250 ml VWR® cell culture flask. The stock vented cap was completely removed from the flask and the entire opening was covered with one layer of 3M Micropore Tape. For experiments 1 and 2, the stock cap vent was 36.4 mm in diameter to act as a breathable barrier. 2 For experiment 3, the lid was removed and the entire open neck of the flask was covered with micropore tape to provide a total surface area of ​​486.95 mm 2 This resulted in a breathable membrane surface area of ​​100 mm.

[0137] In each experiment, the pollen storage containers were laid flat and stored at 6°C and 1 atm for 9 days. Subsamples of the pollen mixture were taken from each pollen storage container for pollination onto one receptive ear of the hybrid maize plants on the 5th, 6th, 7th, 8th and 9th days of storage. On each day of sampling and pollination, the pollen storage containers were removed from storage, sampled, resealed with micropore tape and returned to storage at 6°C in experiments 2 and 3. Seed set of 518 kernels / ear, 380 kernels / ear, 443 kernels / ear, 403 kernels / ear and 467 kernels / ear was obtained on the 5th, 6th, 7th, 8th and 9th days, respectively, in experiment 1. The 250 ml cell culture flasks with stock caps and nitrocellulose barriers provided sufficient gas exchange while maintaining pollen viability. For experiment 2, seed set was achieved at 338 kernels / ear, 425 kernels / ear, 409 kernels / ear, and 2 kernels on days 6, 7, 8, and 9, respectively. Stock caps without embedded nitrocellulose barriers and with vents covered with 3M Micropore Tape provided sufficient gas exchange to maintain pollen viability for up to 8 days. For experiment 3, seed set was achieved at 457 kernels / ear, 517 kernels / ear, 461 kernels / ear, 30 kernels / ear, and 27 kernels / ear on days 5, 6, 7, 8, and 9, respectively. 3M Micropore Tape covering the open neck of a 250 ml cell culture flask provided sufficient gas exchange while maintaining pollen viability for up to 9 days. Seed set results for each experiment were based on CO 2 These results indicate that the presence of soda lime was not required to maintain HO below toxic levels and that the use of a breathable barrier provided sufficient gas exchange to support aerobic respiration. Cell culture flasks are suitable pollen storage vessels, with the open neck of the flask providing sufficient surface area for gas exchange through the breathable barrier while minimizing water vapor transmission from the vessel.

[0138] [Table 38]

[0139] 9. Breathable Barrier Pollen Storage with Crystalline Silica, Metal Powder and Mica Carrier Crystalline quartz silica is an effective carrier for corn pollen storage. It inhibits agglomeration interactions between pollen grains during storage, but does not overly coat the pollen grains due to carrier grain collapse immediately after addition or during handling. Crystalline silica also does not act as a desiccant during storage.

[0140] Metal powders are effective carriers for pollen storage. They prevent clumping interactions between adjacent pollen grain barriers during storage but do not overly coat the pollen barrier surface and inhibit attachment to the stigma of corn silks or other plant bodies. This lack of inhibition allows for effective pollen tube germination. 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 performance, including solid reduction, electrolysis, chemical reaction, high temperature combustion, gas atomization, ultra-high pressure 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, although other particle sizes and surface properties may be superior 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 pollen grain membranes, modify pollen and microbial respiration during storage, or inhibit microbial growth during storage. These active ingredients can include nucleic acids, proteins, pesticides, or biostimulants. Metal carriers include elements with a known biological role in plants that can enhance pollen performance, and elements with no known biological role that do not affect pollen performance. Ferromagnetic carriers may be preferred in applications where the carriers can be magnetically removed from the pollen-carrier mixture after storage to increase the concentration of pollen in the mixture.

[0141] Micas are a group of minerals defined by a general chemical formula and complete basal cleavage. Complete basal cleavage results in flat sheet-like particles that are effective in preventing interactions between adjacent pollen grain membranes. In addition to the physical properties that make mica minerals effective carriers in pollen storage, the high reflectance of mica minerals can serve as a visual indicator during pollen application. This reflectance property can be visualized by a protocol operator or machine camera to track the distribution of pollen during application or to verify where pollination has occurred.

[0142] Table 39 details the performance of crystalline silica, metal powder carriers, and mica as carriers in pollen storage. All carriers in this test perform similarly to crystalline silica, as detailed in U.S. Provisional Application No. 63 / 289,299, filed December 14, 2021, which is incorporated herein by reference.

[0143] [Table 39]

[0144] All carriers were mixed with pollen in a weight ratio of 2 parts pollen to 1 part carrier. 10 μm 316L stainless steel powder was produced by high-temperature combustion, and the individual particles have an amorphous structure. This was optimized for 3D printing with ultra-high pressure water and gas atomization by milling to produce uniform spherical particles.

[0145] 10. Pollen Storage of Plants Containing Transgenic Events The male rating is an overall assessment of performance as a pollen source that accounts for all data types collected during the development of the inbred parent lines. The ratings from best to worst male performance are desirable, acceptable, slight and no improvement.

[0146] Pollen from five inbred parental lines containing transgenic events was collected separately and mixed with crystalline silica carrier in a weight ratio of 2 parts pollen, 1 part weight carrier. Fresh self-pollination (0 day storage) was performed using a portion of the pollen mixed with crystalline silica. The remaining pollen + crystalline silica mixture was stored in a closed container with added soda lime at 6°C environment. After 5 days, the stored pollen was applied to silks of the same inbred line that provided the pollen (i.e. self-pollination). All ears received the same volumetric amounts of fresh pollen and carrier or stored pollen and carrier.

[0147] [Table 40]

[0148] 11.Breathable Barrier Pollen Storage-Container Design Experiment 1: 25 ml VWR® cell culture flasks with vented caps compared to 850 ml VWR® cell culture flasks with pollen stored from the same collection Pollen was collected from field trials and mixed with a silica carrier in a weight ratio of 2 parts pollen to 1 part silica. The total amount of pollen collected after mixing with silica was 1,009 grams. 1000 grams of treated pollen was placed in an 850 ml VWR® cell culture flask, the flask cap was removed, and the mouth of the container was covered with a single layer of 3M Micropore Tape to act as a breathable barrier (see, e.g., Figures 7-12). This storage container configuration had a 0.7 mm 2 The surface area of ​​660.5 mm 2 The total breathable barrier surface area of ​​1.7 mm was calculated for this configuration. In this configuration, the maximum distance between the pollen grain and the breathable barrier was calculated based on trigonometry to be 274 mm. 9 grams of treated pollen was placed in a 25 ml VWR® cell culture flask and a stock breathable cap with nitrocellulose membrane was used as the breathable barrier. This storage vessel configuration provided a total breathable barrier surface area of ​​1.7 mm per gram of treated pollen. 2 The surface area of ​​15.2 mm 2This provided a total breathable barrier surface area of ​​1000 nm. In this configuration, the maximum distance between the pollen grains and the breathable barrier was calculated to be 72 mm. The oxygen content of the air in the pore space between the pollen grains and the silica carrier particles in the 850 ml flask was measured during storage by inserting a needle through an airtight septum placed at the rear of the breathable barrier or storage container. The oxygen content of the air in the pore space was found to have dropped by 0.7% within the first 2 hours of storage and was observed to stabilize at 13.1% throughout the 5 day storage period at 6°C. After 5 days of storage, the pollen from each storage container was mixed separately to uniformly combine the stored pollen throughout each storage container. The mixed pollen was applied to pollen tube germination plates and tester ears. All ears received the same amount of stored pollen.

[0149] [Table 41]

[0150] Experiment 2. Modified 850 ml VWR® Cell Culture Flask After observing the delayed seed set of pollen stored in 850 ml flasks compared to the same batch of pollen stored in 25 ml flasks, it was hypothesized that the scaled-up storage conditions may have affected the vigor of the pollen. It was hypothesized that the initial anaerobic conditions in the storage process and the reduced oxygen availability throughout the storage period reduced the vigor of the pollen such that pollen that tested viable on pollen tube germination medium would not be able to complete pollen tube growth and fertilization before dying from environmental stress when placed on silk. To test this hypothesis, the 850 ml flask was modified to reduce the maximum distance between the pollen grain and the nearest breathable barrier while still maintaining the total breathable barrier surface area to gram of pollen ratio within the established metrics. The flask was modified by drilling 28 holes of 10 mm diameter at selected points on the flask. Nine holes were drilled through the flat side of the larger surface area of ​​the flask in a 3×3 grid pattern with 72 mm spacing between holes. The centers of the holes were 12 mm from the nearest edge, where applicable. This larger surface area flat side was designed to be the upward facing top of the flask in use. The same 3×3 grid pattern of 10 mm holes was drilled through the smaller surface area flat side of the flask with 64 mm hole to hole spacing. The centers of the holes were 12 mm from the nearest edge, where applicable. This smaller surface area flat side was designed to be the downward facing bottom of the flask in use and the surface area is smaller due to the taper manufactured in the 47 mm exterior height side wall of the flask. Two additional 10 mm holes were drilled in the larger surface area top side of the flask at two forward facing obtuse angles. The centers of each hole were 12 mm from the two nearest edges of the flask. Two additional 10 mm holes were drilled in the smaller surface area bottom side of the flask at two forward facing obtuse angles. The centers of each hole were 12 mm from the two nearest edges of the flask. Three 10 mm holes were drilled into each side of the flask on an axis extending parallel to the neck of the vessel for a total of six additional holes. These holes were spaced 64 mm apart, with the center of each hole being 12 mm from the bottom facing the edge of the flask. See, for example, Figures 21-26.

[0151] For experiment 2, pollen was collected from the field trial and mixed with a silica carrier in a weight ratio of 2 parts pollen to 1 part silica. The total amount of pollen collected after mixing with silica was 860 grams. 850 grams of treated pollen was placed into an optimized 850 ml flask with 28 added 10 mm holes. A non-vented container cap was sealed onto the mouth of the container and each of the 28 added 10 mm holes was covered with a single layer of 3M Micropore Tape (see, e.g., Figures 27-30). This storage container configuration resulted in 2.6 mm per gram of treated pollen. 2 The surface area of ​​2199.1 mm 2 In this configuration, the maximum distance between the pollen grain and the breathable barrier was calculated to be 47 mm based on trigonometry. 10 grams of treated pollen was placed in a 25 ml flask and a stock breathable lid with nitrocellulose membrane was used as the breathable barrier. This storage vessel configuration provided a total breathable barrier surface area of ​​1.5 mm per gram of treated pollen. 2 The surface area of ​​15.2 mm 2 This provided a total breathable barrier surface area of ​​1000 nm. In this configuration, the maximum distance between the pollen grains and the breathable barrier was calculated to be 72 mm. The oxygen content of the air in the pore spaces between the pollen grains in the optimized 850 ml flasks was measured during storage by inserting a needle through the breathable barrier or an airtight septum placed at the rear of the storage container. The oxygen content of the air in the pore spaces did not fall below 18.7% over the 6 days of storage. After 6 days of storage, the pollen from each storage container was mixed separately to uniformly combine the stored pollen throughout each storage container. The mixed pollen was applied to pollen tube germination plates and tester ears. All ears received the same amount of stored pollen.

[0152] [Table 42]

[0153] Experiment 3. Unmodified 850ml VWR® Cell Culture Flasks compared to optimized 850ml VWR® Cell Culture Flasks. After observing the increased seed set obtained from pollen stored in the optimized cell culture flasks, an experiment was conducted to directly compare the performance of the equivalent pollen stored in an unmodified 850 ml flask and an optimized 850 ml flask with the addition of 28 openings for breathable barrier (see, e.g., Figures 21-26). The same optimized 850 ml flask with the addition of 28 openings for breathable barrier described in experiment 2 was used during experiment 3. Pollen was collected from the field trial and mixed with a silica carrier in a weight ratio of 2 parts pollen to 1 part silica. The total amount of pollen collected after mixing with silica was 2,000 grams. 1000 grams of treated pollen was placed in the optimized 850 ml flask with the addition of 28 10 mm holes. The unvented container cap was left sealed on the container mouth and each of the 28 added 10 mm holes was covered with a single layer of 3M Micropore tape (see, e.g., Figures 27-31). The composition of this reservoir is 2.2 mm per gram of processed pollen. 2 The surface area of ​​2199.1 mm 2 This provided a total breathable barrier surface area of ​​0.7 mm per gram of treated pollen. In this configuration, the maximum distance between the pollen grain and the breathable barrier was calculated based on trigonometry to be 47 mm. 1000 grams of treated pollen were placed in an unmodified 850 ml flask, the flask cap was removed, and the mouth of the container was covered with a single layer of 3M Micropore tape to act as a breathable barrier. This storage container configuration provided a total breathable barrier surface area of ​​0.7 mm per gram of treated pollen. 2 The surface area of ​​660.5 mm 2 This configuration provided a total breathable barrier surface area of ​​274 mm. In this configuration, the maximum distance between the pollen grains and the breathable barrier was calculated based on trigonometry to be 274 mm. After 3 days of storage, the pollen from each storage container was mixed separately to combine the stored pollen evenly throughout each storage container. The mixed pollen was applied to pollen tube germination plates and tester ears. All ears received the same amount of stored pollen. The mixed pollen was also mechanically dusted onto receptive ears to further test pollination efficiency. The pollen dose to each ear was not equal during dust application.

[0154]

Table 43

Claims

1. 1. A method for storing viable corn pollen comprising the steps of: a) collecting a quantity of fresh maize pollen; b) optionally adding a carrier to the collected corn pollen of step a) to obtain a quantity of treated corn pollen; c) placing said amount of fresh corn pollen or said amount of processed corn pollen into a container; d) sealing said container with a breathable barrier; e) storing the product of step d) in a refrigerated environment; The method includes:

2. 2. The method of claim 1, wherein the amount of fresh corn pollen or the amount of processed 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.

3. 3. The method of claim 2, wherein the amount of fresh or processed corn pollen is about 0.3 grams to 10 kilograms.

4. 4. The method of claim 3, wherein the amount of fresh or processed corn pollen is about 2 grams to 1 kilogram.

5. 2. The method of claim 1, wherein the support is selected from the group consisting of crystalline silica, talc, metal powders and mica minerals.

6. The method of claim 5 , wherein the support is crystalline silica.

7. The method of claim 6 , wherein the crystalline silica comprises an average particle size.

8. The method of claim 7, wherein the average particle size is from about 1 nanometer to about 100 micrometers.

9. The method of claim 8 , wherein the average particle size is 10 micrometers.

10. The method of claim 5 , wherein the metal powder is a metal oxide powder or a metal carbide powder.

11. The method of claim 10 , wherein the metal powder comprises an average particle size.

12. The method of claim 11 , wherein the average particle size is from about 1 micrometer to about 100 micrometers.

13. The method of claim 12, wherein the average particle size is spherical and is about 10 micrometers.

14. The method of claim 11 , wherein the metal powder is a stainless steel powder.

15. 2. The method of claim 1, wherein 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.

16. 16. The method of claim 15, wherein the carrier is present in a pollen:carrier ratio of 2:

1.

17. The method of claim 1 , wherein the container comprises a volume between 0.1 milliliters and 10 liters.

18. The method of claim 17, wherein the container comprises a volume between 10 milliliters and 1500 milliliters.

19. The method of claim 18, wherein the container comprises a volume between 100 milliliters and 1250 milliliters.

20. The container contains CO 2 The method of claim 1 further comprising a sequestration agent.

21. 21. The method of claim 20, wherein the sequestrant 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®).

22. 22. The method of claim 21, wherein the sequestrant is soda lime.

23. 10. The method of claim 1, wherein the breathable barrier is selected from the group consisting of Parafilm, Tyvek, 3M Micropore tape, cellulose, nitrocellulose, and a non-airtight container.

24. 24. The method of claim 23, wherein the breathable barrier is 3M Micropore Tape.

25. 24. The method of claim 23, wherein the non-airtight container includes an opening for gas exchange.

26. The method of claim 25 , wherein the opening comprises at least one perforation.

27. 27. The method of claim 26, wherein the opening comprises at least one perforation having a diameter size between 0.10 millimeters and 30 millimeters.

28. 0.49 mm per gram of fresh or processed pollen 2 ~47.5 mm per gram of fresh or processed pollen 2 24. The breathable barrier of claim 23 comprising a surface area of

29. 1.98 mm per gram of fresh or processed pollen 2 ~26.72 mm per gram of fresh or processed pollen 2 30. The breathable barrier of claim 28 comprising a surface area of

30. 4.45 mm per gram of fresh or processed pollen 2 ~11.88 mm per gram of fresh or processed pollen 2 30. The breathable barrier of claim 29 comprising a surface area of

31. 10. The method of claim 1, wherein 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.

32. 10. The method of claim 1, wherein 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.

33. 33. The method of claim 32, wherein the refrigerated environment comprises a temperature of approximately 6°C.

34. 2. The method of claim 1, wherein 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.

35. 35. The method of claim 34, wherein the pollen is stored for 12 days or less.

36. The method of claim 1 , wherein the corn pollen is transgenic corn pollen.

37. 37. The method of claim 36, wherein the transgenic corn pollen comprises a transgenic event selected from the group consisting of MIR162, 3272, Bt11, GA21, MIR604, MZIR098, 5307, DAS40278, TC1507, DAS-59122-7, NK603, MON810, MON863, MON89034, MON88017, DP-4114, and MON87411.

38. 38. The method of claim 37, wherein the corn pollen comprises the transgenic events Bt11, GA21 and MIR162.

39. 38. The method of claim 37, wherein the corn pollen comprises the transgenic events Bt11 and MIR162.

40. 38. The method of claim 37, wherein the corn pollen comprises transgenic event MIR162.

41. An apparatus for storing pollen comprising a container having a total breathable barrier surface area.

42. 42. The apparatus of claim 41, wherein the container includes a plurality of openings.

43. The container contains 0.49 mm per gram of fresh or processed pollen. 2 ~47.5 mm per gram of fresh or processed pollen 2 42. The device of claim 41, comprising a total breathable barrier surface area of

44. 43. The apparatus of claim 42, wherein the container comprises at least 1 aperture, at least 2 apertures, at least 3 apertures, at least 4 apertures, at least 5 apertures, at least 6 apertures, at least 7 apertures, at least 8 apertures, at least 9 apertures, at least 10 apertures, at least 11 apertures, at least 12 apertures, at least 13 apertures, at least 14 apertures, at least 15 apertures, at least 16 apertures, at least 17 apertures, at least 18 apertures, at least 19 apertures, at least 20 apertures, at least 21 apertures, at least 22 apertures, at least 23 apertures, at least 24 apertures, at least 25 apertures, at least 26 apertures, at least 27 apertures, at least 28 apertures, at least 29 apertures, or at least 30 apertures.

45. The at least one opening is 15.2 mm 2 ~660.5mm 2 45. The device of claim 44 having a total surface area of

46. 46. ​​The apparatus of claim 45, wherein the at least one opening is selected from the group consisting of a circle, an oval, a square, a rectangle, a triangle, and any other two-dimensional shape.

47. 47. The apparatus of claim 46, wherein the at least one opening is circular.

48. 45. The apparatus of claim 44, wherein the container includes 22 openings.

49. 45. The apparatus of claim 44, wherein the container includes 28 openings.

50. 49. The apparatus of claim 48, wherein the 22 openings are circular.

51. 50. The apparatus of claim 49, wherein the 28 openings are circular.

52. The plurality of openings may be arranged to accommodate 0.49 mm per gram of fresh or processed pollen. 2 ~47.5 mm per gram of fresh or processed pollen 2 43. The device of claim 42 comprising a total combined surface area in the range of

53. 53. The apparatus of claim 52, wherein the plurality of openings are circular holes having a diameter of 5 to 15 mm.

54. 54. The apparatus of claim 53, wherein the circular hole is 10 mm in diameter.

55. The circular hole is 78.5 mm 2 55. The device of claim 54, having an individual surface area of

56. The total of 28 circular openings is 2199.1 mm 2 52. The device of claim 51 having a total surface area of

57. 43. The apparatus of claim 42, wherein the plurality of openings are individually covered with a breathable barrier, and the plurality of openings are distributed such that no pollen grain is more than 47 mm from the nearest breathable barrier, or any other distribution that optimizes the furthest distance possible between a pollen grain and the nearest breathable barrier.

58. 58. The device of claim 57, wherein the breathable barrier is selected from the group consisting of Parafilm, Tyvek, 3M Micropore tape, cellulose, nitrocellulose, and a non-airtight container.

59. 59. The device of claim 58, wherein the breathable barrier is 3M Micropore Tape.

60. 42. The apparatus of claim 41, wherein the vessel is a VWR® cell culture flask having a vented cap, the VWR® cell culture flask being used in its stock configuration, with a nitrocellulose membrane in the vented cap acting as a breathable barrier.

61. 42. The apparatus of claim 41, wherein the vessel is a VWR® cell culture flask used in its stock configuration with the cap removed and the mouth of the vessel covered with 3M Micropore tape which acts as a breathable barrier.

62. 42. The apparatus of claim 41, wherein the vessel is a VWR® cell culture flask modified to include 28 additional openings distributed across a surface of the vessel, the additional openings being covered with a breathable barrier, and a stock non-vented cap is left sealed over the mouth of the vessel.

63. 42. The apparatus of claim 41, wherein the vessel is a VWR® cell culture flask modified to include any number of additional openings covered with a breathable barrier in any distribution across the surface of the vessel, and a solid or vented cap is left in place over the mouth of the vessel or the cap is removed and replaced with a breathable barrier.

64. 42. The apparatus of claim 41, wherein the vessel is any brand of cell culture flask in stock or modified configuration.

65. 42. The device of claim 41, wherein the container is any plastic, metal or ceramic container having one or more openings for a breathable barrier.

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