Maize pollen storage and carrier

Abstract

As is well known, maize pollen is fragile and is easy to degrade if not stored properly. Unlike some tree pollen that can be very cold-resistant and can be successfully fertilized over several months or years after it is scattered, maize pollen remains viable over only several hours before it is degraded after it is scattered. Described herein are inventions for storing maize pollen, wherein the pollen is collected and stored in a refrigerated but non-frozen environment having a breathable strap. The stored pollen as described herein can remain viable for up to twelve days, or two weeks, or more. The vitality of the pollen can be prolonged by adding a carrier compound.

Description

Technical Field

[0001] The present invention relates to the fields of corn breeding and artificially induced pollination, and in particular to the fields of collecting, storing and applying stored corn pollen in corn production fields and greenhouses. Background Art

[0002] Pollen storage has long been a need and goal for plant breeders. See generally WM King, Report of chief on seed divisions, In Report of the Commissioner of Agriculture (Yearbook), Washington, D.C., GPO, 47-61 (1885) (stating the expectation for stored pollen "so that we can use it at our own convenient time and place."). In some plants, pollen is very hardy and long-lived. For example, Ginkgo pollen can be collected and stored for six months or more without special care. In contrast, the pollen of other plants is very fragile, and if exposed to these elements, they are prone to rapid decay within hours. Maize (corn) is such a plant.

[0003] In maize commercial hybrid production fields, current practice is to alternate four rows of female inbred plants with two rows of male inbred plants. Females are emasculated to prevent self-pollination, while males are planted only for their ability to pollinate neighboring females. This arrangement works best when female and male plants belong to similar maturity groups (i.e., males shed pollen at about the same time that females are available to pollinate).

[0004] However, if males and females belong to different maturity groups, the risk of current practice is the possibility of unsuccessful pollination, and therefore the loss of crops. In the absence of pollen storage, for growers, there is the risk of male plants shedding pollen too early or too late, and the entire field may be lost due to pollination failure. In the case of pollen storage, no matter how the challenge of flowering time, pollen can be accurately delivered at the right time. Interspecific hybridization of different maturity groups can be completed more easily, thereby expanding the genetic pool and improving maize plant breeding, for example, by preparing maize lines with more drought and / or disease resistance. Summary of the invention

[0005] Planters need to be able to reliably collect and store corn pollen in one day or in one position, and deliver the pollen to female fields on another day or in another position. In order to meet this need, a method for storing corn pollen is provided. In one embodiment, a certain amount of fresh corn pollen is collected; the collected pollen is optionally treated with a carrier; the pollen is sealed in a container with an air-permeable barrier and the pollen is stored in a refrigerated environment. The pollen collected and stored in this way remains viable for up to 20 days and at least up to 12 days. On the one hand, the carrier is talcum powder or silica powder. On the other hand, the carrier is a metal powder or a mica mineral. The carrier can be applied with a pollen 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: carrier ratio. Preferably, the pollen: carrier ratio is 2: 1. On the one hand, the breathable barrier can be parafilm, Tyvek, 3M microporous tape, cellulose, nitrocellulose, and a non-airtight container. Preferably, the breathable barrier is 3M microporous tape. On the other hand, a carbon dioxide sequestrant can be added to a sealable container. In addition, a pollen storage vessel with a total breathable barrier surface area is provided herein. The pollen storage vessel can have multiple openings, and the openings can be covered in the breathable barrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 Is a photograph of seed set produced using pollen stored at air barrier surface area factors using Tyvek and Micropore air barriers covering perforations in otherwise airtight storage containers with added soda lime (Example 4, Experiment 5). From left to right, these ears were produced using pollen stored in otherwise airtight containers having a single 1.588 mm (1 / 6 inch) opening covered in Tyvek, a single 3.175 mm (1 / 8 inch) opening covered in Tyvek, a single 9.525 mm (3 / 8 inch) opening covered in Tyvek, a single 1.588 mm (1 / 16 inch) opening covered in Micropore tape, a single 3.175 mm (1 / 8 inch) opening covered in Micropore tape, a single 6.35 mm (1 / 4 inch) opening covered in Micropore tape, and a single 9.525 mm (3 / 8 inch) opening covered in the Micropore tape treatment, respectively.

[0007] Figure 2is a photograph of seed set produced using pollen stored at an air permeable barrier surface area factor using open perforations in otherwise airtight storage containers with added soda lime (Example 4, Experiment 6). From left to right, these ears were produced using pollen stored in otherwise airtight containers treated 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, a single 6.35 mm (1 / 4 inch) open perforation covered in Tyvek, respectively, and the last two ears on the right were from otherwise airtight containers with a single 9.525 mm (3 / 8 inch) open perforation.

[0008] Figure 3 is a photograph of the fruit set rate produced by pollen stored with a breathable barrier surface area factor using Tyvek and microporous tape breathable barriers, which are covered with large perforations in an otherwise airtight storage container with the addition of soda lime (Example 4, Experiment 7). The three ears on the far left in the image are from the treatment where the large perforations are covered in microporous tape. The perforation size increases from left to right in these three ears. The three ears on the far right in the image are from the treatment where the large perforations are covered in Tyvek. The perforation size increases from left to right in these three ears.

[0009] Figure 4 is a photograph showing the fruit set rate using pollen deposited 6.5 cm deep in a 50 ml conical tube with a 71.3 mm diameter tube covered in microporous tape. 2 Open, no soda lime added. (Example 4, Experiment 8). The leftmost ear is from the top 1.5 cm portion of the pollen carrier mixture deposited in the tube. The middle ear is from the central portion of the pollen pile approximately 4 cm below the surface. The rightmost ear is from the base portion of the pollen pile 6.5 cm from the surface.

[0010] Figure 5 are photographs of seed set rates produced using pollen stored with air permeable barrier surface areas using microporous tape air permeable barriers covering perforations in otherwise airtight storage containers 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 where no soda lime was added. The four ears on the right side of the image are from the treatment where soda lime was added. For each series of four ears, the perforation size increases from left to right.

[0011] Figure 6 The pressure normalized oxygen percentage during 9 days of pollen storage from Example 6 is shown.

[0012] Figure 7-12 A stock version of a culture flask is shown.

[0013] Figure 7 Shows Cell culture flask with the stock vent cap removed and 3M Micropore Tape (shown using black arrow) used as a breathable barrier. The image shows the larger surface area flat side that serves as the top of the flask facing upward during use.

[0014] Figure 8 Shows Cell culture flask with the stock vent cap removed and 3M microporous tape (shown with black arrows) used as a breathable barrier. The figure shows the smaller surface area flat side of the flask, which serves as the bottom side of the flask facing downward during use. The surface area is low due to the taper made on the 47 mm outer height side wall of the flask.

[0015] Fig. 9 Shows Cell culture flask with stock vent cap removed and 3M Microporous Tape used as a gas permeable barrier. The figure shows a side view of the flask standing on its end opposite the 3M Microporous Tape gas permeable barrier end.

[0016] Fig.10 Shows Cell culture flask with the stock vent cap removed and 3M microporous tape (shown with black arrow) used as a breathable barrier. The figure shows the angled top and side views of the flask as it sits on a downward facing bottom with tapered side walls.

[0017] Fig.11 Shows Cell culture flask with the stock vent cap removed and 3M Micropore Tape (shown using black arrow) used as a gas permeable barrier. The figure shows the flask from the opposite end of the vessel to the 3M Micropore Tape gas permeable barrier, with the flask placed face down.

[0018] Fig.12 Shows Cell culture flask with the stock vent cap removed and 3M Microporous Tape (shown with black arrow) used as a breathable barrier. This figure shows a side view of the flask standing on its end opposite the 3M Microporous Tape breathable barrier. Fig. 9 on the opposite side of the view.

[0019] Figure 13-20 Depicted is a 22-well configuration of a pollen storage vessel.

[0020] Figure 13-14 Shows Cell culture flask with 11 holes drilled in the larger surface area flat side facing upward ( Fig.13) and 11 holes drilled in the smaller surface area side with tapered sidewalls ( Fig.14 ). A total of 22 holes are indicated by black arrows - each Fig.11 indivual.

[0021] Figure 15-16 Shows A cell culture flask with 11 wells drilled in the larger surface area flat side facing upward. Fig.15 is a view from the side, and Fig.16 This is a view from the end opposite the vent cap. The 11 holes drilled in the larger surface area flat side facing upward are indicated by the black arrows.

[0022] Figure 17-20 Shows A cell culture flask having 11 holes drilled in the larger surface area flat side facing upward and 11 holes drilled in the smaller surface area side with tapered sidewalls. However, a total of 22 holes were covered with 3M microporous tape used as a breathable barrier. Figure 17-19 It is a view with the flat side of the larger surface area facing upward; Fig.17 The side of the cap closest to the camera is shown, while Fig.18 from the end opposite to the reserve cap. Fig.19 is a side view with the reserve cap on the left side. Fig. 20 is a view of the smaller surface area side that has a tapered sidewall and faces downward during use. A representation of the 3M Microporous Tape is indicated by the black arrow.

[0023] Figure 21-33 Depicted is a 28-well configuration of a pollen storage vessel.

[0024] Figure 21-23 Shows A cell culture flask having 11 holes drilled in the larger surface area flat side facing upward and 11 holes drilled in the smaller surface area side with tapered side walls. Fig.21 The observation was made with the reserve cap closest to the camera. Fig. 22 is viewed from the end opposite to the reserve cap, and Fig.23 is a side view with the reserve cap on the left side.

[0025] Fig.24 Shown from the side Cell culture flask standing on the end opposite the stock cap (vertical). This view shows the 3 holes drilled in the side of the flask.

[0026] Fig.25 Shows Cell culture flask with the smaller surface area side with tapered sidewalls facing up. The flat side has 11 holes drilled into it.

[0027] Fig.26 Shown from the side Cell culture flask standing on the end opposite the stock cap (vertical). This view shows the 3 holes drilled in the side of the flask. Fig.26 yes Fig.24 Opposite side shown.

[0028] Figure 27-33 A 28-well configuration culture flask is shown, but the wells are covered with 3M microporous tape as a breathable barrier. Representation of the microporous tape is indicated by the black arrow.

[0029] definition

[0030] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. Reference to the technology employed herein is intended to refer to the technology commonly understood in the art, including variations of those technologies and / or alternatives to equivalent technologies that are clear to those of ordinary skill in the art. Although it is believed that the following terms will be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the interpretation of the subject matter disclosed by the present invention.

[0031] As used herein, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to an "antibody" optionally includes a combination of two or more such molecules, and the like.

[0032] As used herein, the term "about" means that a person skilled in the art would readily know that the typical error range for the corresponding value (eg, ±20%, ±10%, or ±5%) is within the intended meaning of the recited value.

[0033] As used herein, "container" or "vessel" refers to an object that can hold pollen therein. For example, a container may refer to a Magenta GA7 box. The container or vessel also includes a breathable barrier. As used herein, "breathable barrier" refers to a component of a storage container or vessel for corn pollen. The breathable barrier component of a container or vessel allows adequate gas exchange and minimal water vapor penetration. The breathable barrier may include, but is not limited to, parafilm, Tyvek, microporous tape, perforations in otherwise airtight storage containers or vessels (e.g., mason jars and lids), or the use of non-airtight containers or vessels (e.g., clamshell containers, Magenta GA7 boxes, 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 may also include, for example, mason jars and have perforated lids.

[0034] As used herein, "carbon dioxide sequestration" means the removal of carbon dioxide from the atmosphere by a carbon dioxide sequestering agent (e.g., soda lime, activated carbon, ethanolamine, zeolite 4A, lithium hydroxide (LiOH), or activated magnesium silicate (e.g., )) captures carbon dioxide ("CO2"). In this way, excess CO2 accumulation in the chamber is prevented. Optionally, the sequestering agent will prevent CO2 from exceeding 10 mmol CO2 / liter of chamber headspace.

[0035] As used herein, "carrier" means a compound that acts as an agent accompanying the collected pollen, preferably in powdered form. Suitable carrier compounds can be, but are not limited to, talc powder, silica powder, and the like.

[0036] As used herein, "clumping," "aggregation," and similar terms refer to the tendency of pollen to bind together in the absence of a carrier and / or suitable storage conditions, whether due to excess moisture or other reasons. Agglomerated pollen is non-flowable and cannot be air-blown onto the silk. Agglomerated pollen is less likely to be sufficiently attached to the silk to cause pollination.

[0037] As used herein, the terms "comprising" and "comprise" are open ended. When used in conjunction with a method comprising a series of steps, even if additional steps are performed, the method is still practiced as long as the series of steps are performed.

[0038] As used herein, "crystalline silica" refers to a powdered form of silica derived from quartz or other natural rock formations. The terms "crystalline silica", "SiO2" and "polycrystalline silica" are used interchangeably throughout the text. Compared with talc or amorphous silica, crystalline silica has different structural characteristics, including but not limited to higher Mohs mineral hardness, higher bulk density and lower specific surface area. In one embodiment, crystalline silica has an average particle size between 1 nanometer (1nm) and 100 microns (100 μm). In another embodiment, crystalline silica has an average particle size between 1 micron (1 μm) and 10 microns (10 μm). Unless otherwise specified, the particle size values ​​provided herein are average sizes.

[0039] As used herein, "active magnesium silicate" refers to synthetic powdered magnesium silicate. The terms "active magnesium silicate", "synthetic amorphous active magnesium silicate" and "MgO3Si" are used interchangeably throughout the text. is a commercially available source of active magnesium silicate. See www.ussilica.com / products / florisil. Active magnesium silicate is characterized by an amorphous structure and a high specific surface area. In one embodiment, the active magnesium silicate has an average particle size between 75 microns (75 μm) and 149 microns (149 μm). In another embodiment, the active magnesium silicate has an average particle size of less than 75 microns (<75 μm).

[0040] As used herein, the term transgenic "event" refers to a recombinant plant produced by transforming and regenerating a single plant cell with heterologous DNA (e.g., an expression cassette comprising a target gene). The term "event" refers to an original transformant comprising heterologous DNA and / or the progeny of the transformant. The term "event" also refers to the progeny produced by sexual outcrossing between a transformant and another maize strain. Even after repeated backcrossing with the recurrent parent, the insertion DNA and flanking DNA from the transformed parent are present in the same chromosome position of the hybrid progeny. Typically, the transformation of plant tissues produces multiple events, in which each event represents the different locations of the plant cell genome inserted by a DNA construct. Based on the expression of transgenic or other desirable features, a specific event is selected. Therefore, for example, "event 3272," "3272," or "3272 event" as used herein means the progeny of the original 3272 transformant and / or the 3272 transformant and / or the plant derived from the original 3272 transformant in any way. For 3272, see WO 06 / 098952.

[0041] Other examples of transgenic events include, but are not limited to, MIR162 (see WO 07142840), Bt11 (see US 6114608 (construct) 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 US 8598413), TC1507 (see WO 04099447), DAS-59122-7 (see WO 06 / 039376), NK603 (see US 6825400), MON810 (see US 6713259), MON863 (see US 7705216), MON89034 (see WO 07140256), MON88017 (see WO 05059103), DP-4114 (see WO 11084621) and MON87411 (see WO 13169923).

[0042] As used herein, "flowable" means the ability of a powder-like substance to be easily moved by the application of air, wind, sound, or poured with uninterrupted continuity and to be stable and easy to carry out.

[0043] 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, when referring to a gas, it may refer to oxygen and carbon dioxide.

[0044] As used herein, "hybrid group" refers to the breeding classification of inbred lines. "Hybrid group" and "hybrid pool" are used interchangeably and refer to the relationship between the breeding pools of a maize population. In summary, the main names of hybrid pools are: Stiff Stalk ("SS", also known as Iowa Stiff Stalk Synthetic or "BSSS"), Non Stiff Stalk ("NSS") and Iodent ("IDT"). See JvHweerwaarden et al., Historical genomics of North American maize [Historical genomics of North American maize], Proc.Nat'l Acad.Sci.USA [Proceedings of the National Academy of Sciences of the United States] 109 (31): 12420-25 (2012). However, these are not exclusive, and other names are known, for example, Lancaster Sure Crop ("LSC") ("LSC"). See, e.g., C. Livini et al., Genetic diversity of maize inbred lines with and among heterotic groups revealed by RFLPs, Theor. Appl. Genet. 84: 17-25 (1992). See also Hallauer et al. (1998) Corn breeding, pp. 463-564; GF Sprague and JW Dudley (eds.) 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 herein by reference in their entirety.

[0045] The term "germplasm" refers to the totality of the genotypes of a population or other group of individuals (e.g., species or plant strains). The phrase "adapted germplasm" refers to plant material that has been proven to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original 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" refers in some embodiments to plant material that does not belong to an established breeding population and has no known relationship to a member of an established breeding population.

[0046] As used herein, the term "mica" refers to the general chemical formula X2Y4-6Z8O20(OH,F)4, where X is an alkali metal or alkaline earth metal, Y is a transition metal, a post-transition metal, or an alkaline earth metal, and Z is silicon, aluminum, or may include other transition metals.

[0047] As used herein, "initial oxygen content" refers to the amount of oxygen present in the atmosphere of the chamber that initially contains the collected pollen and is initially sealed (whether measured as an absolute measurement, a percentage, or otherwise). In one embodiment, the initial oxygen content is between 0.12 mmol O2 / g pollen / storage day and 0.57 mmol O2 / g pollen / storage day, inclusive. In another embodiment, the initial oxygen content is between 0.24 mmol O2 / g pollen / storage day and 0.57 mmol O2 / g pollen / storage day, inclusive. "Initial oxygen content", "initial mmol O2", "initial mmol O2 / g pollen" and "initial mmol O2 / g pollen / storage day" are used interchangeably herein.

[0048] "Plant" is any plant at any stage of development, particularly a seed plant. In particular, in the context of the present disclosure, a plant refers to a maize plant. As used herein, the term "plant line" refers to a single plant material or a genetically identical group of materials.

[0049] As used herein, "platform" means a surface within a container that is in direct contact with the pollen and carrier mixture and prevents direct contact with the container itself. For example, the platform can be filter paper or an aluminum tray.

[0050] As used herein, "pollen: carrier ratio" means the proportion of pollen present in a mixture with a carrier. For example, and not by way of limitation, a mixture of pollen and a carrier having a pollen: carrier ratio of 2:1 comprises two parts pollen measured by weight or volume and one part carrier compound, such as talc, measured by weight or volume.

[0051] As used herein, "refrigerated environment" means any condition where the temperature is lower than the ambient temperature (or room temperature) but not lower than the temperature at which water freezes. In other words, if the ambient temperature is 25°C, the refrigerated environment includes temperatures greater than 0°C and less than 25°C. Likewise, the refrigerated environment includes temperatures between 2°C and 10°C.

[0052] As used herein, "seed setting rate" means the number of grains produced on the cob due to successful pollination. Seed setting rate can be expressed qualitatively (e.g., low, good or high) or quantitatively. In quantitative measurements, the measured value can be in terms of the percentage or quantity of seeds per ear. The term generally refers to the percentage or quantity of normal grains (i.e., non-aborted grains with endosperm vigor). For normal maize strains (i.e., not haploid induced strains), a seed setting rate higher than 80% (or higher than 300 grains per ear) is considered to be a good seed setting rate. Achieving a good seed setting rate is the goal of controlled pollination.

[0053] As used herein, "storage" refers to the act of storing pollen for a suitable period of time. Suitable storage periods may be as little as 24 hours or as much as 12 days.

[0054] As used herein, "treatment" means the deliberate application of compounds or environmental constraints to pollen. In particular, pollen treatment may include the addition of carrier compounds to pollen to preserve the flowability and viability of the pollen.

[0055] As used herein, "vessel pressure" refers to any artificially applied atmospheric pressure within a vessel. The vessel pressure value is measured in standard atmospheric pressure "atm", such as 0.5 atm, however, other units (e.g., Torr or Pascal or "Pa"; 1Pa = 9.8692×10 -6 The vessel pressure is measured as 1 atm. It is explicitly contemplated that the vessel pressure may reach or exceed 1 atm. Under conditions where the vessel pressure is between 0 and 1 atm, "vessel pressure" and "vacuum" have the same meaning and are used interchangeably. However, "vessel pressure" is the preferred term because it contemplates both vacuum conditions and conditions where an artificially imposed atmospheric pressure exceeds the ambient atmospheric pressure (e.g., 1 atm).

[0056] As used herein, "pollen viability" refers to the ability of a pollen grain to germinate a pollen tube. During a pollination event, this pollen tube will grow through the stigma (silk), deliver two sperm cells to the female gametophyte, which will fertilize the egg cell and two polar nuclei, which will lead to the formation of an embryo and endosperm, respectively.

[0057] As used herein, "pollen viability" refers to the time interval between when a pollen grain physically contacts a maize silk and when subsequent fertilization occurs. Pollen viability is critical to seed set rate because viable pollen grains must complete pollen tube growth through the stigma and subsequently fertilize before losing viability due to desiccation or other environmental stressors. DETAILED DESCRIPTION

[0058] The productivity of maize seed production (i.e., a measure of whether the desired amount of inbred or hybrid seed can be produced by self-pollination or cross-pollination at an economic cost that does not exceed the value of the produced seed) is a key factor in the successful development of maize inbred parent lines, because a large amount of inbred parent line seeds are required to produce hybrid seeds sold to customers. Maize inbred parent lines with low productivity may be discontinued due to excessive parent seed production costs, even if the inbred parent line can produce hybrids with customer-desired characteristics (e.g., leading GM and genome-edited traits, high yields, disease resistance). Pollen storage technology can be used to enhance the productivity of inbred maize parent lines used in hybrid seed production.

[0059] The production capacity challenges that can be solved by pollen storage technology include but are not limited to low pollen yield, low total pollen scattering, short pollen scattering duration, short duration of tassel silk receptivity, and the GM or genome editing traits that may affect plant reproduction characteristics. Another challenge of self-pollination may be a long self-split period, which is defined by the number of days between when pollen begins to scatter and when tassel silk appears and becomes available for pollination. In some iterations, the self-split period may be a negative value, wherein before pollen scattering begins, tassel silk appears for pollination. The observed self-split period may be the result of inbred parent strain genetics or the result of stress in the growth environment, and the stress reduces the rate of tassel silk extension and increases the number of days between pollen scattering and tassel silk that can be used for pollination.

[0060] In order to solve the challenge of these production capacities, pollen can be collected in the best window of pollen scattering using pollen storage technology, store this pollen and maintain pollen viability simultaneously, then apply pollen in the best window of fringe appearance and receptivity.In some iterations, pollen collection can be carried out multiple times in one day.In other iterations, in the whole process of pollen scattering duration, pollen can be collected in multiple days.The application of the pollen stored can combine the pollen collected in multiple days, and can be applied multiple times on the same day or multiple days.The pollen application can combine the pollen collected from multiple field positions, and is applied to a position in a single time.In some iterations, pollen can be collected in a geographic region and applied to fringe in different geographic regions.Geographical region can be different fields in the same production position, fields in different states or cities in a country or fields in different countries.In some iterations, collect pollen from the temperate maize inbred parent strains grown in temperate locations, and be applied to the subtropical or tropical maize inbred parent strains grown in subtropical or tropical locations. In other iterations, pollen is collected from the subtropical or tropical maize inbreeding parent lines grown in subtropical or tropical locations, and is applied to the temperate maize inbreeding parent lines grown in temperate locations. By solving these challenges to production capacity, pollen storage technology can be used to increase the seed of desired maize inbreeding parent lines, and this will produce the new hybrid with the desired feature for sale to the client. Pollen storage technology can also realize the economic hybrid seed production of the combination of currently unfeasible temperate, subtropical and tropical maize inbreeding parent lines.

[0061] Therefore, the embodiment provides the method for storing vigorous corn pollen, the method comprises collecting a certain amount of fresh corn pollen, optionally a carrier is applied to the corn pollen collected, obtaining a certain amount of treated corn pollen, the fresh corn pollen of this amount or the treated corn pollen of this amount is placed in a container, the container is sealed with a breathable barrier, and pollen is stored in the container in a refrigerated environment. In one embodiment, the fresh corn pollen of this amount or the treated corn pollen of this amount is 0 day age, 1 day age, 2 day age, 3 day age, 4 day age, 5 day age, 6 day age, 7 day age, 8 day age, 9 day age, 10 day age, 11 day age, 12 day age, 13 day age, 14 day age, 15 day age, 16 day age, 17 day age, 18 day age, 19 day age, 20 day age or longer. In another embodiment, the amount of fresh corn pollen or treated corn pollen is about 0.3 grams to 10 kilograms, and in another embodiment, the amount of fresh corn pollen or treated corn pollen is about 2 grams to 1 kilogram. In one embodiment, the carrier is selected from the group consisting of the following: crystalline silica, talc, metal powder and mica mineral. In another embodiment, the carrier is crystalline silica and includes an average particle size. The average particle size can be between about 1 nanometer and about 100 microns. In another embodiment, the average particle size is 10 microns. In another embodiment, the metal powder is a metal oxide powder or a metal carbide powder. In another embodiment, the average particle size of the metal powder can be between about 1 micron and about 100 microns. In one embodiment, the average particle size is about 10 microns spherical. In another embodiment, the metal powder is a stainless steel powder.

[0062] 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 has a volume of 0.1 ml to 10 liters. In another embodiment, the container has a volume of 10 ml to 1500 ml. In another embodiment, the container has a volume of 100 ml to 1250 ml. The container may further include a CO2 sequestering agent 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., In one embodiment, the sealing agent is soda lime.

[0063] In one embodiment, the breathable barrier is selected from the group consisting of: parafilm, Tyvek, 3M microporous tape, cellulose, nitrocellulose, and a non-airtight container. In another embodiment, the breathable barrier is 3M microporous tape. In another embodiment, the non-airtight container includes an orifice for gas exchange. The orifice may also include at least one perforation. In another embodiment, the orifice includes at least one perforation with a diameter size of 0.10 mm to 30 mm. In an embodiment, the breathable barrier has a diameter of 0.49 mm. 2 / g fresh or processed pollen to 47.5mm 2 / gram of fresh or treated pollen surface area. In another embodiment, the breathable barrier has a surface area of ​​1.98 mm 2 / g fresh or processed pollen to 26.72mm 2 / gram of fresh or treated pollen surface area. In another embodiment, the breathable barrier has a surface area of ​​4.45 mm 2 / g fresh or processed pollen to 11.88mm 2 / g surface area of ​​fresh or treated pollen.

[0064] The stored maize pollen can maintain viability 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 an embodiment, the refrigerated environment comprises a temperature range selected from the group consisting of 1° C.-10° C., 4° C.-8° C., and 5.5° C.-6.5° C. In another embodiment, the refrigerated environment comprises a temperature of about 6° C. In another embodiment, the pollen is stored in a 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 than 1 day. In one embodiment, the pollen is stored for 12 days or less.

[0065] In an embodiment, the maize pollen is transgenic maize pollen. In another embodiment, the transgenic maize pollen comprises a transgenic event selected from the group consisting of: MIR162, 3272, Bt11, GA21, MIR604, MZIR098, 5307, DAS40278, TC1507, DAS-59122-7, NK603, MON810, MON863, MON89034, MON88017, DP-4114, and MON87411. In an embodiment, the maize pollen comprises transgenic events Bt11, GA21, and MIR162. In another embodiment, the maize pollen comprises transgenic events Bt11 and MIR162. In an embodiment, the maize pollen comprises transgenic event MIR162.

[0066] In yet another embodiment, a device for storing pollen is provided. The device includes a vessel having a total air permeable barrier surface area. In one embodiment, the vessel includes a plurality of openings. In another embodiment, the vessel has a total air permeable barrier surface area of ​​0.49 mm. 2 / g fresh or processed pollen with 47.5mm 2 / gram of fresh or treated pollen. The pollen storage vessel may include at least one opening, at least two openings, at least three openings, at least four openings, at least five openings, at least six openings, at least seven openings, at least eight openings, at least nine openings, at least ten openings, at least eleven openings, at least twelve openings, at least thirteen openings, at least fourteen openings, at least fifteen openings, at least sixteen openings, at least seventeen openings, at least eighteen openings, at least nineteen openings, at least twenty openings, at least twenty-one openings, at least twenty-two openings, at least twenty-three openings, at least twenty-four openings, at least twenty-five openings, at least twenty-six openings, at least twenty-seven openings, at least twenty-eight openings, at least twenty-nine openings, or at least thirty openings.

[0067] In an embodiment, the single opening of the device has a 15.2 mm 2 Up to 660.5mm 2 The single opening is selected from the group consisting of a circle, an oval, a square, a rectangle, a triangle, and any other two-dimensional shape. In one embodiment, the opening is a circle. In another embodiment, the vessel includes twenty-two openings. The twenty-two openings may be circular. In a separate embodiment, the vessel includes twenty-eight openings. The twenty-eight openings may be circular. In an embodiment, the total of twenty-eight circular openings have a surface area of ​​2199.1 mm. 2 total surface area.

[0068] In an embodiment, the plurality of openings have a range of 0.49 mm. 2 / g fresh or processed pollen with 47.5mm 2 / gram of fresh or processed pollen. The plurality of openings may be circular holes with a diameter of 5-15 mm. The plurality of openings may also be circular holes with a diameter of 10 mm. In another embodiment, the circular holes have a diameter of 78.5 mm. 2 of individual surface area.

[0069] In an embodiment, the plurality of openings are individually covered in a breathable barrier, wherein the plurality of openings are distributed such that no pollen particle is greater than 47 mm from the nearest breathable barrier, or any other distribution that optimizes the farthest possible distance between a pollen particle and the nearest breathable barrier. In an embodiment, the breathable barrier is selected from the group consisting of: parafilm, Tyvek, 3M microporous tape, cellulose, nitrocellulose, and a non-airtight container. The breathable barrier can be 3M microporous tape.

[0070] In an embodiment, the pollen storage container is A cell culture flask wherein the vent cap is used in its stock configuration and the nitrocellulose membrane in the vent cap acts as a gas permeable barrier. In another embodiment, the vessel is used in its stock configuration A cell culture flask with the cap removed and the mouth of the vessel covered in 3M microporous tape to act as a breathable barrier. In another embodiment, the vessel is A cell culture flask modified to include 28 additional openings distributed over the vessel surface, wherein the additional openings are covered in a gas permeable barrier and a reserve, non-vented cap remains sealed over the vessel mouth. In another embodiment, the vessel is Cell culture flasks, which are modified to include any number of additional openings, which are covered with gas permeable barriers arbitrarily distributed on the vessel surface, a solid or vented cap left on the vessel mouth in place, or the cap is removed and replaced with a gas permeable barrier. In another embodiment, the vessel is a cell culture flask of any brand in reserve or modified configuration. The vessel can be any plastic, metal, or ceramic container with one or more openings for a gas permeable barrier.

[0071] Examples

[0072] 1. Collection

[0073] Maize plants are planted under field and greenhouse conditions. Once the tassel appears and begins to shed pollen, a bag is placed on the tassel to collect the pollen. The bag is typically placed in the late afternoon and removed the next morning. The collected pollen is then placed in a suitable sealed container after sifting out any anther or other tassel material and optionally mixing with a carrier.

[0074] Alternatively, pollen can be collected by gathering the tassel before scattering from the maize plant. The tassel can be placed in a beaker with water and allowed to scatter pollen normally, or the tassel can be dried, soaked and filtered to mechanically collect pollen. See, for example, U.S. Patent No. 8,252,988 (filed on June 27, 2007), which is incorporated herein by reference in its entirety.

[0075] 2. Experiments in which the mixture of pollen and talc was stored in Magenta GA7 boxes supplemented with soda lime and sealed with parafilm as a breathable barrier.

[0076] Experiments 1, 2 and 3: 1) air permeable barrier (Parafilm around GA7 boxes) with added soda lime (5 days); 2) air permeable barrier (Parafilm around GA7 boxes) with added soda lime (5 days) and a larger starting pollen load than Experiment 1; 3) air permeable barrier (Parafilm around GA7 boxes) with added soda lime (5 days) and a larger starting pollen load than Experiment 2.

[0077] For Experiment 1, 2.522 g of pollen was mixed with 1.26 g of talc to give a total of 3.783 g of pollen mixture, wherein the initial fresh pollen-talc mixture moisture content ("PMC") was 36.71%. The pollen was mixed in a ratio of two parts pollen to one part carrier by weight. 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 pollen: talc by weight) compared to Experiment 1 above to give a total of 9.997 g of pollen mixture, wherein 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 pollen by weight: talc) to obtain a total of 35.82 g of pollen mixture, wherein the initial fresh mixture PMC was 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. In all 3 experiments, soda lime was placed at the bottom of the GA7 box and contained in a 50 ml conical tube cap. The pollen mixture was contained in the base of a polylactic acid plastic (PLA) cup (approximately 45 mm x 30 mm) and was located above an open soda lime container. An iButtonLink DS1923-F5#DS1923-F5#Hygrochron temperature and humidity data logger was placed above the pollen mixture container to monitor the internal relative humidity (rH) of the pollen storage vessel. 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 to create a tight seal, as is common practice in plant tissue culture. The GA7 lid to box gas exchange gap surface area was measured and calculated to be a total of 42 mm 2 The pollen storage vessels were stored at 6°C, 1 atm pressure for 5 days. After 5 days, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0078] In all three experiments, the iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger showed that a saturated rH above 98% was obtained during the entire storage period in the storage vessel. For Experiment 1, six repetitions of pollination were performed on the receptive silks of the primary ears of hybrid maize plants. After 5 days of storage, the pollen viability was 62% and the relative PMC was 96.3%. In Experiment 2, six repetitions of pollination were performed on the receptive silks of the primary ears of hybrid maize plants. After 5 days of storage, the pollen viability was 66% and the relative PMC was 99.6%. In Experiment 3, twelve repetitions of pollination were performed on the receptive silks of the primary ears of hybrid maize plants. After 5 days of storage, the pollen viability was 87%, and significantly increased compared to fresh viability. The relative PMC was 100.9%. Internal vessel rH and final relative PMC data indicate that the use of parafilm as a breathable barrier to seal the exposed 42 mm of a 400 mL Magenta GA7 box is 2 The surface area reduces the water loss from the storage vessel and the pollen mixture. The internal gas samples obtained from the storage vessel after wrapping the gas exchange gap between the box and the lid with an impermeable tape showed that the O2 content was 19.7% and the CO2 content was 0.1% in Experiment 1, the O2 content was 19.5% and the CO2 content was 0.1% in Experiment 2, and the O2 content was 16.2% and the CO2 content was 0.3% in Experiment 3. The use of paraffin film as a breathable barrier provides sufficient gas exchange while minimizing water vapor penetration. The presence of soda lime, together with the breathable properties of the barrier, provides sufficient CO2 sequestration. In Experiment 1, the average fruit set rate of 480 kernels showed that the pollen was viable after 5 days of storage. In Experiment 2, the average fruit set rate of 347 kernels showed that the pollen was viable after 5 days of storage. Compared with Experiment 1, Experiment 2 shows that the breathable barrier using soda lime allows the storage of larger amounts of pollen and talc while maintaining sufficient gas exchange and minimal water vapor penetration. The O2 content in Experiment 3 was lower than that observed in Experiments 1 and 2, which were performed with smaller pollen mixes, but sufficient O2 was available to support pollen respiration. For Experiment 3, the average seed set rate of 598 kernels indicated that the pollen was viable after 5 days of storage. The high seed set rate indicates that large amounts of pollen and talc mixes can be stored in containers using a breathable barrier.

[0079] Table 1. Pollen viability and gas composition analysis of Experiments 1, 2 and 3.

[0080]

[0081]

[0082] NA = Not applicable

[0083] Table 2. Average fruit set rate for Experiments 1, 2 and 3.

[0084]

[0085] Experiments 4 and 5: 4) Experiments in which a mixture of pollen and talc was stored in Magenta GA7 boxes sealed with parafilm as a gas permeable barrier, with and without the addition of soda lime.

[0086] For Experiment 4, 11.362g of pollen was mixed with 5.681g of talc (2:1 pollen:talc by weight) to give a total of 17.043g of pollen mixture with an initial fresh mixture PMC of 35.31%. Two treatments were prepared to compare the performance of the scheme in storage containers with and without the addition of soda lime. This comparison was performed to determine whether CO2 would escape through the parafilm air permeable barrier during the entire storage process. A total of 5.4g of pollen mixture (10ml) was added to a 400mL Magenta GA7 Box for each treatment. In treatment 1, 0.68g of soda lime contained in a 50ml conical tube cap was placed at the bottom of the GA7 box. In treatment 2, an empty 50ml conical tube cap was placed at the bottom of the 400mL Magenta GA7 box.

[0087] For experiment 5, 30.527g pollen was mixed with 15.264g talcum (2:1 pollen: talcum) to obtain a total of 45.791g pollen mixture, wherein the initial fresh mixture PMC was 34.96%. A total of 8.1g pollen mixture (15ml) was added to a 400mL Magenta GA7 box, and an empty 50ml conical tube cap was placed at the bottom and no soda lime was added. The pollen mixture was contained in a PLA plastic cup (45mm x 30mm) and was located above the open 50ml conical tube cap containing soda lime or above the empty 50ml conical tube cap in all treatments. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was placed above the pollen mixture container to monitor the internal relative humidity of the pollen storage vessel. The GA7 lid was placed on the top of the GA7 box, and the gas exchange gap between the box and the lid was wrapped in a single layer 25.4mm (1 inch) wide paraffin film. Following common practice in plant tissue culture, the parafilm was stretched to create a tight seal. The GA7 lid-to-box gas exchange gap surface area was measured and calculated to be a total of 42 mm 2 The pollen storage vessels were then stored at 6°C, 1 atm pressure for 5 days. After 5 days, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis for O2 and CO2 were assessed.

[0088] In Experiment 4, nine replicate pollinations were performed on the receptive silks of the primary ears of hybrid maize plants for both treatments. After 5 days of storage, pollen viability was qualitatively scored as 4 (41%-60% germination rate; see Table 29), and both treatments were scored similarly. Treatment 1, with the addition of soda lime, had a relative PMC of 105.6%, and treatment 2, without the addition of soda lime, had a relative PMC of 101.4%. The final relative PMC data showed that the use of parafilm as a breathable barrier to seal the 42 mm of a 400 mL Magenta GA7 box was 2 Gas exchange surface area reduces water loss from storage vessels and pollen mixture. For both treatments, the moisture content of the pollen mixture increases over time due to the additional new water produced by cellular respiration. After wrapping the gas exchange gap with impermeable tape, internal gas samples were obtained from each treatment. The endpoint O2 content of treatment 1 was 19.2% and the CO2 content was 0.2%. The endpoint O2 content of treatment 2 was 19.5% and the CO2 content was 1.2%. The CO2 content of treatment 2 (without adding soda lime) was higher than that of treatment 1 (adding soda lime), but the CO2 content of 1.2% showed that CO2 gas exchange occurred through the paraffin film breathable barrier. Using paraffin film as a breathable barrier provides sufficient gas exchange while minimizing water vapor penetration. The presence of soda lime provides sufficient CO2 sequestration together with the breathable properties of the barrier. The breathable properties of the barrier provide sufficient gas exchange of CO2 and O2 to avoid toxic levels of CO2 accumulation. These results indicate that if a breathable barrier can be used for gas exchange, soda lime may not be needed for pollen storage. The average seed set of 381 kernels / ear for the treatment with soda lime added and 426 kernels / ear for the treatment without soda lime added indicated that pollen in both treatments was viable after 5 days of storage. The higher seed set in the treatment without soda lime further supports that soda lime may not be necessary for pollen storage if a gas permeable barrier is available for gas exchange.

[0089] In Experiment 5, three replicate pollinations were performed on viable silks of primary ears of hybrid maize plants. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that greater than 98% of saturation rH was achieved throughout the storage period within the storage vessel. Pollen viability after 5 days of storage was annotated to be 66% and relative PMC to be 102.1%. Internal vessel rH and final relative PMC data indicated that the use of parafilm as a breathable barrier to seal the exposed 42 mm of a 400 mL Magenta GA7 box was sufficient to maintain the pollen viability. 2The gas exchange surface area reduced water loss from the storage vessel and the pollen mixture. The moisture content of the pollen mixture increased over time due to the additional new water produced by cellular respiration. After wrapping the gas exchange gap with impermeable tape, internal gas samples taken from the storage vessel indicated an O2 content of 19.6% and a CO2 content of 0.9%. The use of paraffin film as a breathable barrier provided adequate gas exchange while minimizing water vapor transmission. CO2 levels were kept below 1.0%, and toxic levels of CO2 accumulation were avoided, indicating that soda lime may not be required for this pollen storage method. The average fruit set rate of 514 kernels / ear indicated that the pollen was viable after 5 days of storage.

[0090] Table 3. Pollen viability assessment and gas composition analysis of treatments in Experiment 4.

[0091]

[0092] Table 4. Average fruiting rate (Experiment 4)

[0093]

[0094] Table 5. Pollen viability assessment and gas composition analysis (Experiment 5) NA = Not applicable

[0095] Table 6. Average fruiting rate (Experiment 5)

[0096] 3. Experiments in which a mixture of pollen and crystalline silica was stored in Magenta GA7 boxes with the addition of soda lime.

[0097] Experiment 1: Gas-permeable barrier with crystalline silica and soda lime (Parafilm around GA7 boxes) (7 days)

[0098] Pollen of 5.955g amount is mixed with 2.9775g 10μm crystalline silica (2:1 pollen by weight: silica) to obtain a total of 8.933g pollen mixture, wherein the initial fresh mixture PMC is 33.57%. A total of 8.85g pollen mixture (12.5ml) is added to a 400mL Magenta GA7 box containing 1.02g soda lime. Soda lime is placed at the bottom of the GA7 box and contained in a 50ml conical tube cap. The pollen mixture is contained in a PLA plastic cup (45mm x 30mm) and is located above an open soda lime container. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger is placed above the pollen mixture container to monitor the internal relative humidity of the pollen storage vessel. The GA7 lid is placed on the top of the GA7 box, and the gas exchange gap between the box and the lid is sealed by wrapping a single layer of 25.4mm (1 inch) wide paraffin film around the open gap. Following common practice in plant tissue culture, the parafilm was stretched to create a tight seal. The GA7 gas exchange gap between the box and the lid surface area was calculated to be a total of 42 mm 2 The pollen storage vessels were stored at 6°C, 1 atm pressure for 7 days. After 7 days, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0099] Four replicate pollinations were performed on the receptive silks of the primary ears of hybrid maize plants. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger showed that a saturated rH greater than 98% was obtained throughout the storage period in the storage vessel. After 7 days, pollen viability was qualitatively scored as 4 (41%-60% germination rate; see Table 29), and the relative PMC was 104.9%. The internal vessel rH and final relative PMC data showed that the use of paraffin film as a breathable barrier reduced the water loss from the storage vessel and the pollen mixture. Due to the additional water produced by cellular respiration, the moisture content of the pollen mixture increased over time. Internal gas samples obtained from the storage vessel after wrapping and sealing with an impermeable tape showed that the O2 content was 19.4% and the CO2 content was 0.1%. The use of paraffin film as a breathable barrier provides sufficient gas exchange while minimizing water vapor penetration. The presence of soda lime provides sufficient CO2 sequestration together with the breathable barrier. The average seed setting rate of 670 kernels / ear indicated that the pollen was viable after 7 days of storage.

[0100] Table 7. Final PMC evaluation and gas composition analysis for Experiment 1.

[0101]

[0102] Table 8. Average fruit set rate in Experiment 1.

[0103]

[0104] Experiments 2 and 3: 2) a breathable barrier (Parafilm surrounding GA7 boxes) with crystalline silica, soda lime, and reduced headspace (5 days); 3) a breathable barrier (Parafilm surrounding GA7 boxes) with crystalline silica, soda lime, reduced headspace, and a greater pollen initiation than Experiment 2 (5 days).

[0105] In experiment 2, 23.623g pollen was mixed with 11.812g 10 μm crystalline silica (2: 1 pollen by weight: silica) to obtain a total of 35.4345g pollen mixture, wherein the initial fresh mixture PMC was 35.62%. A total of 8.5g pollen mixture (12.5ml) was added to each of four Magenta GA7 box treatments. Three Magenta GA7 boxes were modified by partially filling the box with epoxy resin to achieve a final headspace volume of 100ml, 200ml, and 300ml as required. Before use in pollen storage, epoxy resin was cured in a pressure chamber set to 3atm absolute pressure for a minimum of 24 hours. The fourth box (400ml headspace) was used as a positive control. 1.05g soda lime was included in a 50ml conical tube cap and placed at the bottom of each box for 200ml, 300ml, and 400ml headspace treatments. In the 100 ml headspace treatment, soda lime was placed directly on top of the cured epoxy, but kept separate from the container of the pollen:silica mixture.

[0106] In experiment 3, 10.484g pollen was mixed with 5.242g 10μm crystalline silica (2:1 pollen: silica ratio) to obtain a total of 15.726g pollen mixture, wherein the initial fresh mixture PMC was 35.93%. A total of 15.72g pollen mixture (22ml) was added to a modified Magenta GA7 box. The box was modified by partially filling the box with epoxy resin to achieve a final headspace volume of 100ml. 1.87g soda lime was placed directly on top of the cured epoxy resin, but separated from the pollen: silica mixture container. For these two experiments, the pollen mixture was contained in a PLA plastic cup (45mm x 30mm) and was located above an open soda lime container or directly next to the soda lime placed on top of the cured 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 the lid was sealed by wrapping a single layer of 25.4 mm (1 inch) wide parafilm around the opening. The parafilm was stretched to create a tight seal, as is common practice in plant tissue culture. The GA7 gas exchange gap between the box and lid surface area was calculated to be a total of 42 mm 2 The pollen storage vessels were stored at 6°C and 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0107] For Experiment 2, three replicate pollinations / treatments were performed on the receptive silks of the primary ears of hybrid maize plants. For all treatments, after 5 days of storage, pollen viability was qualitatively scored as 5 (>60% germination rate; see Table 29). Relative PMC ranged from 99.0% to 104.8% during treatment. After wrapping the gas exchange gap with impermeable tape, internal gas samples of each treatment were obtained from the storage vessel. O2 content ranged from 18.6% to 19.3% during treatment, and CO2 content was 0.1%.

[0108] For Experiment 3, six replicate pollinations were performed on the receptive silks of the primary ears of hybrid maize plants. The relative PMC was 97.4%. After wrapping the gas exchange gap with an impermeable tape, internal gas samples were obtained from the storage vessel. The O2 content was 19.6% and the CO2 content was 0.1%. In both experiments, the use of paraffin film as a breathable barrier provided sufficient gas exchange while minimizing water vapor penetration. The presence of soda lime provided sufficient CO2 sequestration together with the breathable barrier.

[0109] The reduction of the headspace from 400 ml to 100 ml in Experiments 2 and 3 demonstrated that the initial starting molar amount of O2 present in the vessel volume was not limiting for pollen respiration, with the 42 mm 2A parafilm gas-permeable barrier of surface area covered the gas exchange gap in each GA7 Magenta box. O2 was continuously replenished to the pollen storage vessels while water loss was limited. In Experiment 2, the average seed set of 400 ml, 300 ml, 200 ml, and 100 ml headspace storage vessels was 535, 510, 502, and 668 kernels / ear, respectively, indicating that pollen was viable after 5 days of storage. In Experiment 3, the average seed set of 564 kernels / ear indicated the same conclusion.

[0110] Table 9. Final PMC evaluation and gas composition analysis for Experiments 2 and 3.

[0111]

[0112] Table 10. Average fruit set rate for Experiments 2 and 3.

[0113] Experiment 4: Reduction of air barrier surface area in Magenta GA7 boxes containing pollen mixed with crystalline silica and with the addition of soda lime.

[0114] To evaluate the reduction in the surface area of ​​the gas permeable barrier, 24.871 g of pollen was mixed with 12.436 g of 10 μm crystalline silica (2:1 pollen:silicon dioxide) to give 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 the lid with modeling clay to seal gas exchange on one, two or three sides of the lid, respectively. The sealed edge effectively reduced the surface area available for gas exchange to 10.5 mm 2 , 21mm 2 , and 31.5mm 2 A fourth box with the same 400 ml headspace and without any gas exchange gaps sealed with modeling clay was used as a positive control (42 mm 2Surface area). A 50 ml conical tube lid containing 1.11 g of soda lime was placed at the bottom of each box. The pollen mixture was contained in a PLA plastic cup (45 mm x 30 mm) and located above the open soda lime container. For each treatment, a GA7 lid was placed on the 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 opening. Following common practice in plant tissue culture, the parafilm was stretched to produce a tight seal. The pollen storage vessels were stored at 6 ° C, 1 atm pressure for 5 days. After storage for 5 days, the pollen viability of each treatment, the final PMC relative to the initial PMC, and the endpoint gas analysis of O2 and CO2 were evaluated.

[0115] Pollen viability after 5 days of storage ranged from 46% to 50%, and relative PMC ranged from 96.9% to 99.9% across treatments. Final PMC increased as the gas permeable barrier surface area decreased. Internal gas samples were taken from the storage vessels for each treatment after wrapping the gas exchange gap between the box and the lid with an impermeable tape. O2 levels ranged from 17.8% to 19.1% across treatments, and CO2 levels were 0.1% across treatments. Final O2 levels also decreased as the gas permeable barrier surface area decreased. Using a reduced surface area covered by a parafilm gas permeable barrier provides adequate gas exchange while minimizing water vapor transmission to 10.5 mm 2 The presence of soda lime together with the breathable barrier provides adequate CO2 sequestration.

[0116] Table 11. Pollen viability assessment and gas composition analysis for Experiment 4.

[0117]

[0118]

[0119] NA = Not applicable

[0120] Table 12. Final vessel oxygen content for four gas permeable membrane surface areas.

[0121] <![CDATA[Gas permeable barrier surface area (mm 2 )]]> <![CDATA[O2% at 1 atm and 22 °C]]> 10.5 17.8 21 18.2 31.5 19.0 42 19.1

[0122] 4. Alternative Storage Containers – Plastic Clamshell Containers

[0123] Experiments 1 and 2: 1) Plastic clamshell boxes as air-permeable barrier (i.e., non-airtight container with gaps between sections), use of crystalline silica and addition of soda lime - no pollination (5 days); 2) Plastic clamshell boxes as air-permeable barrier (i.e., non-airtight container with gaps between sections), use of crystalline silica and soda lime - with pollination (5 days).

[0124] 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) to give a total of 17.414 g of pollen mixture with an initial fresh mixture PMC of 33.82%. A total of 17.00 g of pollen mixture was added to a 630 ml polyethylene terephthalate clear plastic clamshell Phytatray TM brand container. 2.07g soda lime contained in a 50ml conical tube cap was placed at the bottom of a clamshell container. In experiment 2, 19.876g pollen was mixed with 9.938g 10μm crystalline silica (2:1 pollen:silicon dioxide) to obtain a total of 29.814g pollen mixture, wherein the initial fresh mixture PMC was 35.9%. A total of 22.3g pollen mixture was added to a 630ml clear deep plastic clamshell container, and 3.15g soda lime was added directly to the bottom of the container. For both experiments, the pollen mixture was contained in a PLA plastic cup (45mm x 30mm) and located next to an open soda lime container or loose 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 vessel. The clamshell lid was placed on top of the clamshell bottom and snapped into place. Any gap that existed between the two clamshell container sections was not sealed by any form of barrier. The pollen storage vessels were then stored at 6°C, 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis for O2 and CO2 were assessed.

[0125] In Experiment 1, an iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that greater than 98% of saturation rH was achieved throughout the storage period within the storage vessel. Pollen viability was 51% and relative PMC was 95.8% after 5 days of storage. Internal vessel rH and final relative PMC data indicated that the clamshell container with only sections sealed together using the manufactured locking mechanism acted as a breathable barrier. Internal gas samples taken from the storage vessel indicated an O2 content of 19.8% and a CO2 content of 0.1%. The clamshell container provided adequate gas exchange while minimizing water vapor transmission, while the presence of soda lime provided adequate CO2 sequestration together with the gaps between the clamshell sections acting as a breathable barrier. The clamshell breathable barrier provided pollen tube germination (51%) comparable to the fresh pollen mixture control (54%).

[0126] For Experiment 2, four replicate pollinations were performed on the fertile silks of the primary ears of hybrid maize plants. After 5 days of storage, pollen viability was qualitatively scored as Grade 3 (>21% to 40% germination rate; see Table 29), and the relative PMC was 99.3%. Internal gas samples obtained from the storage vessels showed that the O2 content was 19.9% ​​and the CO2 content was 0.1%. The clamshell container provided sufficient gas exchange while minimizing water vapor penetration, and the presence of soda lime provided sufficient CO2 sequestration together with the gaps between the clamshell parts used as a breathable barrier. The average fruit set rate of 784 grains / ears showed that the pollen was viable after 5 days of storage.

[0127] Table 13. Pollen viability assessment and gas composition analysis for Experiments 1 and 2.

[0128]

[0129] ND = No Data

[0130] Table 14. Average fruit set rate in Experiment 2.

[0131] 5: Tyvek, microporous tape, and open perforated breathable barrier surfaces applied to otherwise airtight containers.

[0132] In Experiments 1, 2, 3, and 6 in Example 4, Tyvek was used to allow gas exchange while reducing water vapor transmission out of the pollen storage container. In Experiments 4, 5, 6, and 7, microporous tape was evaluated in the same manner as Tyvek to allow gas exchange while reducing water vapor transmission out of the pollen storage container. In Experiments 1-7 and Experiment 9 in Example 4, 100 ul of H2O was added to the bottom of the storage container to cope with the low humidity (30% rH) in the test environment. The addition of water helps the internal atmosphere of the storage container reach saturated relative humidity.

[0133] Experiment 1: Tyvek as an alternative breathable barrier with the addition of soda lime (5 days)

[0134] In order to evaluate Tyvek as a breathable barrier, 9.7551g pollen was mixed with 4.8776g 10μm crystalline silica (2:1 pollen by weight: silica) to obtain a total of 14.633g pollen mixture, wherein the initial fresh mixture PMC was 37.02%. A total of 14.0g pollen mixture was added to a 500ml wide-mouthed mason jar. A PLA plastic cup containing 1.73g soda lime was added to a 500ml wide-mouthed mason jar. The pollen mixture was contained in an aluminum pan (45mm x 35mm). The pollen mixture pan was stacked above the soda lime cup and separated by 841 micron metal mesh screens. The wide-mouthed lid was equipped with a diaphragm for gas analysis, and a 3.175mm perforation was made in the lid with a handheld molding press to serve as a breathable barrier. A Tyvek disk was cut from a standard FedEx envelope and taped over a 3.175 mm perforation (1 / 8 inch). An impermeable sheet seal was applied to the outer perimeter of the Tyvek to seal the Tyvek to the container lid. The 3.175 mm (1 / 8 inch) perforation represented a 7.9 mm 2 Gas permeable barrier surface area. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of the pollen storage vessel. An 841 micron metal mesh screen was used to separate the iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger from the pollen mixture container. The pollen storage vessel lid was sealed with a Mason jar lid strip and stored at 6°C and 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were evaluated.

[0135] Two replicate pollinations were performed on the fertile silks of the primary ears of hybrid maize plants. After 5 days of storage, pollen viability was qualitatively scored as Grade 3 (>21% to 40% germination rate; see Table 29), and the relative PMC was 102.5%. Internal gas samples obtained from the storage vessel showed that the O2 content was 19.1% and the CO2 content was 0.1%. The Tyvek cover with 3.175 mm (1 / 8 inch) perforations provided sufficient gas exchange while minimizing water vapor penetration. The presence of soda lime provided sufficient CO2 sequestration together with the breathable barrier. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger showed that a saturated rH greater than 98% was obtained throughout the storage period in the storage vessel. The average fruit set rate of 789 grains / ears showed that the pollen was viable after 5 days of storage.

[0136] Table 15. Pollen viability assessment and gas composition analysis

[0137]

[0138] Table 16. Average fruiting rate

[0139]

[0140] Experiment 2: Tyvek breathable barrier surface area factor with soda lime added

[0141] In this experiment, 22.608g of pollen was mixed with 11.304g of 10μm crystalline silica (2:1 pollen:silicon dioxide by weight) to obtain a total of 33.912g of pollen mixture, where the initial fresh mixture PMC was 34.08%. A total of 4.0g of pollen mixture (2.67g pollen) was added to each of seven 125ml regular mouth mason jars. A 50ml conical tube cap containing 0.56g of soda lime was added to the bottom of each jar. The pollen mixture was contained in an aluminum pan (45mm x 15mm) and stacked above the soda lime container separated by a piece of 841 micron metal mesh screen. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of the seven 125ml pollen storage vessels. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a piece of 841 micron metal mesh screen. Conventional Mason jar lids were equipped with septa for gas analysis, and a series of different sized perforations were made in each lid using a handheld molding machine (i.e., each lid received a different sized perforation). The perforation sizes included 0.0, 1.588 mm (1 / 16 inch), 2.381 mm (3 / 32 inch), 3.175 mm (1 / 8 inch), 4.763 mm (3 / 16 inch), 6.35 mm (1 / 4 inch), and 9.525 mm (3 / 8 inch) diameters. Tyvek disks were cut from standard FedEx envelopes and taped over the perforations. This series of increasingly larger perforations covered in the Tyvek breathable barrier was used to evaluate the effect of the breathable barrier surface area on pollen storage. The series of different perforation sizes provided a range of 2.0 mm to 4.5 mm diameters. 2 Up to 71.3mm 2 The surface area range of the jar was 1.30 mm (Table 17). A lid without perforations (0.0 mm in Table 18) was used as a negative control and the pollen mixture was allowed to breathe in the sealed container until anaerobic conditions were achieved and the stored pollen became nonviable. A standard mouth Mason jar lid was sealed to a 125 ml Mason jar using Mason jar lid tape.

[0142] Positive controls were included by adding 4.89g of pollen mixture to a 400ml GA7 Magenta box containing 0.68g of added soda lime. Soda lime was contained in a 50ml conical tube cap placed at the bottom of the GA7 box. 100ul of water was added to the bottom of the GA7 container. The pollen mixture was contained in a PLA plastic cup (approximately 45mm x 30mm) and was located above an open soda lime container. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of the GA7 Magenta box. The GA7 lid was placed on the top of the GA7 box, and the gas exchange gap between the box and the lid was sealed by wrapping a single layer of 25.4mm (1 inch) wide paraffin film around the open gap. According to common practice in plant tissue culture, the paraffin film was stretched to produce a tight seal. The GA7 gas exchange gap between the box and the lid surface area was calculated to be a total of 42mm 2 All treatments were stored at 6°C, 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0143] Except the negative control treatment without air permeable barrier, for all treatments, two repetitions of pollination were carried out on the receptive tassels of the primary ear of hybrid maize plants. The negative control was inactive after storage for 5 days, because the pollen respirator pressure had been reduced to -22 kPa gauge, internal O content was 0.4%, internal CO content was 0.1%, and final relative PMC was 103.2%. For the positive control and the treatment with the perforation covered by Tyvek with a scope between 1.588 mm (1 / 16 inch) and 9.525 mm (3 / 8 inch), pollen viability was 64% to 78% after storage for 5 days. The treatment with the perforation covered by Tyvek with a scope between 1.588 mm (1 / 16 inch) and 9.525 mm (3 / 8 inch) all had a higher pollen tube germination rate than the GA7 Magenta box and the paraffin film positive control that added soda lime. For the positive control and the treatments with Tyvek-covered perforations ranging between 1.588 mm (1 / 16 inch) and 9.525 mm (3 / 8 inch), the relative final PMC ranged from 97.3% to 108.2% after 5 days of storage. The positive control had the lowest final PMC. As the surface area of ​​the Tyvek gas permeable barrier increased, the relative final PMC appeared to decrease. Internal gas samples were obtained from each treatment and indicated that the O2 content ranged between 19.1% and 19.5% and the CO2 content ranged between 0.1% and 0.3%. The treatments with higher Tyvek gas permeable barrier surface areas and the positive control had lower O2 content and higher CO2 content compared to the treatments with lower Tyvek gas permeable barrier surface areas. The positive control and all Tyvek treatments provided adequate gas exchange while minimizing water vapor penetration at all tested surface areas. The presence of soda lime provided adequate CO2 sequestration together with the two types of gas permeable barriers at the tested surface areas. iButtonLink DS1923-F5#Hygrochron temperature and humidity data loggers indicated that for all treatments, greater than 98% of saturation rH was achieved throughout the storage period within the storage vessels. Increased seed set was observed with increased surface area of ​​the Tyvek breathable barrier compared to the positive control. Average seed set ranged from 686 to 788 kernels / ear, indicating that pollen was viable after 5 days of storage.

[0144] Table 17. Perforation diameter and surface area and Tyvek area per gram of pure pollen

[0145]

[0146] Table 18. Pollen viability assessment and gas composition analysis

[0147]

[0148] NA = Not applicable

[0149] Table 19. Average fruiting rate

[0150]

[0151] Experiment 3: Surface Area Factor of a Perforation in an Otherwise Airtight Container (i.e., Mason Jar and Lid) with Addition of Soda Lime

[0152] For the following experiment, 24.832g of pollen was mixed with 12.416g of 10μm crystalline silica (2:1 pollen:silicon dioxide by weight) to obtain a total of 37.248g of pollen mixture, wherein the initial fresh mixture PMC was 35.41%. A total of 4.0g of pollen mixture (2.67g pollen) was added to each of six 125ml regular mouth mason jars. A 50ml conical tube cap containing 0.56g of soda lime was added to the bottom of each mason jar. The pollen mixture was contained in an aluminum pan (45mm x15mm). The pan containing pollen was stacked above the soda lime container and separated by a piece of 841 micron metal mesh screen. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of the six 125ml pollen storage vessels. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a piece of 841 micron metal 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 and sealed with standard Mason jar lid tape.

[0153] Using a handheld molding machine, one mason jar lid is modified to include a 1.588mm perforation (1 / 16 inch), and a second mason jar lid is modified to include a 3.175mm (1 / 8 inch) perforation. A Tyvek disk is cut from a standard FedEx envelope and taped to each of the two perforations to serve as a breathable barrier. An impermeable plate sealing film is applied to the outer periphery of the Tyvek to seal the Tyvek to the container lid. Two Tyvek breathable barrier treatments are used as positive controls. Each of the remaining four 125ml pollen storage vessels receives perforations of different sizes on the lid. Perforations are produced using needles of various gauge sizes (18 gauge, 20 gauge, 23 gauge, and 30 gauge needles). These needles provide open microperforations from the inside of the pollen storage vessel to the external atmosphere and from 0.015mm 2 Up to 0.552mm 2The open surface area range was 125 ml Mason jar pollen storage vessels were sealed with Mason jar lid strips. All treatments were stored at 6°C, 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were evaluated.

[0154] Except 30 gauge micro-perforation treatment, for all treatments, two repetitions of pollination were carried out on the receptive tassel silk of the primary ear of hybrid maize plants.30 gauge treatment was inactive after storage for 5 days, anaerobic internal O2 content was 0.2%, CO2 content was 0.1%, and the final relative PMC was 98.5%.Pollen tube germination was 48% for 3.175mm perforation (1 / 8 inch), and 50% for 1.588mm (1 / 16 inch) perforation positive control Tyvek air permeable barrier treatment.For 18 gauge, 20 gauge and 23 gauge treatment, pollen tube germination ranged between 61% and 62% after storage for 5 days.Compared with Tyvek treatment, open micro-perforation treatment demonstrated higher pollen tube germination.For all treatments, the relative final PMC ranged from 94.4% to 103.0% after storage for 5 days. Internal gas samples were taken from each Tyvek treatment and indicated that the O2 content ranged between 18.5% and 18.8%, while the CO2 content ranged between 0.1% and 0.2%. Both Tyvek breathable barrier treatments provided adequate gas exchange while minimizing water vapor permeation at all tested surface areas. Open microperforations reduced and restricted gas exchange. Internal gas samples measured O2 content of 8.2%, 2.2%, and 6.1%, respectively, from 18 gauge, 20 gauge, and 23 gauge open microperforation treatments. For all three treatments, the measured CO2 content was 0.2%. The presence of soda lime provided adequate CO2 sequestration. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that for all treatments, saturated rH above 98% was obtained throughout the storage period within the storage vessel. As the surface area of ​​the Tyvek breathable barrier increased, an increase in fruit set was observed. As the open area of ​​the microperforations increased from 23 gauge to 18 gauge, an increase in seed set was observed. The Tyvek breathable barrier treatment had a greater seed set than any of the open microperforation treatments. The average seed set ranged between 629 kernels / ear and 770 kernels / ear indicating that the pollen was viable after 5 days of storage.

[0155] Table 20. Perforation diameter versus surface area and perforation area per gram of pure pollen. All treatments used 2.67 g of pure pollen per container.

[0156] Needle Gauge Perforation diameter(mm) <![CDATA[Surface area (mm 2 )]]> <![CDATA[Perforation area mm 2 / g pure pollen]]> 30 0.14 0.015 0.110 23 0.318 0.079 0.250 20 0.584 0.268 0.459 18 0.838 0.552 0.658

[0157] Table 21. Perforation diameter versus surface area and Tyvek area per gram of pure pollen. All treatments used 2.67 g of pure pollen per container.

[0158] Perforation diameter(mm) <![CDATA[Surface area (mm 2 )]]> <![CDATA[Tyvek surface area / gram pollen (mm 2 )]]> 1.588 2.0 0.74 3.175 7.9 2.97

[0159] Table 22. Pollen viability assessment and gas composition analysis

[0160]

[0161] NA = Not applicable

[0162] Table 23. Average fruiting rate

[0163]

[0164] Experiment 4: 3M Microporous Tape as an Alternative Breathable Barrier in the Presence of Soda Lime (5 Days)

[0165] For the microporous tape evaluation, 10.396g of pollen was mixed with 5.190g of 10μm crystalline silica (2:1 pollen:silicon dioxide by weight) to obtain a total of 15.5856g of pollen mixture, wherein the initial fresh mixture PMC was 34.90%. A total of 4.0g of pollen mixture (2.67g pollen) was added to three 125ml regular mouth mason jars, and a 50ml conical tube cap containing 0.56g of soda lime was added to the bottom of each mason jar. The pollen mixture was contained in an aluminum pan (about 45mm x 15mm). The pan containing the pollen mixture was stacked above the soda lime container and separated by a piece of 841 micron metal mesh screen. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of each of the three 125ml pollen storage vessels. Use a piece of 841 micron metal mesh screen to separate the iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger from the pollen mixture container. Conventional mason jar lids are equipped with diaphragms for gas analysis. Among the three mason jar lids used, a lid is modified to include a 1.588mm (1 / 16 inch) perforation using a handheld molding machine, and two lids are modified to include a 3.175mm (1 / 8 inch) perforation. Cut the Tyvek disk from the standard FedEx envelope and tape it to the 3.175mm (1 / 8 inch) perforation in one of the three lids. Impermeable plate sealing film is applied to the outer periphery of Tyvek to seal Tyvek to the container lid. Use 3.175mm (1 / 8 inch) perforation with Tyvek breathable barrier as a positive control. 3M brand microporous tape was placed over the 1.588 mm (1 / 16 inch) perforation in the second lid, and the same microporous tape was placed over the 3.175 mm (1 / 8 inch) perforation in the third lid. The lids were sealed to 125 ml Mason jar pollen storage vessels using Mason jar lid tape. All treatments were stored at 6°C, 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis for O2 and CO2 were evaluated.

[0166] For each treatment, single pollination was carried out on the receptive silk of the primary ear of hybrid maize plants. For all treatments, after storage for 5 days, pollen viability was qualitatively scored as 4 levels (>41% to 60% germination rate; see Table 29). Relative PMC ranged from 103.8% to 106.0% during the treatment. Internal gas samples were obtained from each jar, and the range of O2 content was 18.4% to 19.4%, while the range of CO2 content was 0.1% to 0.2%. 1.588mm (1 / 16 inch) microporous tape treatment showed that the lowest O2 content was 18.4%, while it also showed that CO2 content was 0.2%. Tyvek and microporous tape breathable barriers both provide enough gas exchange, while minimizing water vapor penetration at the surface area of ​​the test. The presence of soda lime provides enough CO2 sealing together with the breathable barrier at the test surface area. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that for all treatments, greater than 98% saturation rH was achieved throughout the storage period in the storage vessel. Both the 3.175 mm (1 / 8 inch) Tyvek breathable barrier treatment and the 1.588 mm (1 / 16 inch) Microporous Tape breathable barrier treatment reached 100% saturation rH, while the 3.175 mm (1 / 8 inch) Microporous Tape breathable barrier treatment only reached 98% rH. Seed set rates of 711 kernels / ear, 751 kernels / ear, and 696 kernels / ear were obtained for the 3.175 mm (1 / 8 inch) Tyvek breathable barrier, 1.588 mm (1 / 16 inch) Microporous Tape breathable barrier, and 3.175 mm (1 / 8 inch) Microporous Tape breathable barrier, respectively, indicating that the pollen was viable after 5 days of storage.

[0167] Table 24. Pollen viability assessment, gas composition analysis, and seed setting rate (k)

[0168]

[0169] Experiment 5: Surface Area Factor Using Tyvek Compared to Microporous Tape in the Presence of Soda Lime (5 Days)

[0170] For further evaluation, 10.89 g of pollen was mixed with 5.445 g of 10 μm crystalline silica (2: 1 pollen: silica by weight) to obtain a total of 16.335 g of pollen mixture, wherein the initial fresh mixture PMC was 33.77%. A total of 2.0 g of pollen mixture (1.335 g of pollen) was added to eight 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 in an aluminum pan (45 mm x 15 mm). The pan containing the pollen mixture was stacked above the soda lime container and separated by a piece of 841 micron metal mesh screen. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture in each container to monitor the internal relative humidity of the eight 125 ml pollen storage vessels. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was separated from the pollen mixture container using a piece of 841 micron metal screen. Conventional mason jar lids were equipped with diaphragms for gas analysis. Using a handheld molding press, a total of eight mason jar lids were modified to include a perforation for each lid. Two lids were modified to include 1.588mm (1 / 16 inch), 3.175mm (1 / 8 inch), 6.35mm (1 / 4 inch), and 9.525mm (3 / 8 inch) perforations. Tyvek disks were cut from standard FedEx envelopes and taped to a lid with each preformed size. Impermeable sheet sealing film was applied to the outer periphery of Tyvek to seal Tyvek to the lid. At each perforation size, a piece of 3M microporous tape was applied to the second lid. In addition to evaluating the microporous tape for Tyvek, the experiment also evaluated the impact of increasing or reducing the surface area of ​​the permeable barrier. The perforated lid was sealed onto each 125 ml Mason jar pollen storage vessel using Mason jar lid tape. All treatments were stored at 6°C, 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0171] For each treatment, a single replicate pollination was performed on the receptive silks of the primary ear of hybrid maize plants. After 5 days of storage, pollen tube germination ranged between 55% and 61% for the Tyvek treatment and between 47% and 62% for the microporous tape treatment. Internal gas samples were obtained from each treatment. O2 content ranged from 19.5% to 19.9% ​​during the Tyvek treatment, while CO2 content was measured to be 0.1%. O2 content ranged from 19.3% to 19.9% ​​during the microporous tape treatment, while CO2 content was measured to be 0.1%. O2 content increased with the increase of the surface area of ​​the two types of permeable barriers. The presence of soda lime provided sufficient CO2 sequestration together with the two types of permeable barriers at the surface area tested. The relative final PMC ranged from 102.2% to 84.3% during the Tyvek treatment and was 101.5% to 78.6% for the microporous tape treatment. A decrease in relative final PMC was observed with increasing surface area of ​​both types of breathable barriers. Both the Tyvek and microporous tape covered 9.525 mm (3 / 8 inch) perforation treatments had high pollen moisture losses. Overall, the Tyvek breathable barrier treatment retained higher pollen moisture content compared to the microporous tape treatment. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that greater than 98% of saturated rH was achieved throughout the storage period for both the Tyvek and microporous tape 1.588 mm (1 / 16 inch) and 3.175 mm (1 / 8 inch) perforation treatments. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that greater than 96% of rH was achieved throughout the storage period for the Tyvek covered 6.35 mm (1 / 4 inch) and 9.525 mm perforations and the 6.35 mm (1 / 4 inch) microporous tape covered perforation treatments. For the perforated treatment covered with 9.525 mm (3 / 8 in.) microporous tape, rH above 94% was observed throughout the storage period. For all eight treatments, seed set of more than 500 kernels / ear indicated that pollen was viable ( Figure 3 ).

[0172] Table 25. Pore diameter versus surface area and barrier area per gram of pure pollen. All treatments used 1.335 grams of pollen per container.

[0173]

[0174] Table 26. Pollen viability assessment and gas composition analysis

[0175]

[0176] NA = Not applicable

[0177] Table 27. Average fruiting rate

[0178]

[0179] Experiment 6: Surface Area Factor with Perforations and Soda Lime in an Otherwise Airtight Container (i.e., Mason Jar and Lid) (5 Days)

[0180] For Experiment 6, 6.48 g of pollen was mixed with 3.24 g of 10 μm crystalline silica (2: 1 pollen: silica by weight) to give 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 in an aluminum pan (45 mm x 15 mm). The pan containing the pollen mixture was stacked above the soda lime container and separated by a piece of 841 micron metal mesh screen. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger is stacked above the pollen mixture container to monitor the internal relative humidity of each of the eight 125ml pollen storage vessels. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger is separated from the pollen mixture dish using a piece of 841 micron metal mesh screen. Five conventional mason jar lids are equipped with diaphragms for gas analysis. One lid is modified to include a 3.175mm perforation (1 / 8 inch) made with a handheld molding press. A Tyvek disk is cut from a standard FedEx envelope and taped to a 3.175mm (1 / 8 inch) perforation. Impermeable plate sealing film is applied to the outer periphery of the Tyvek to seal the Tyvek to the container lid. The 3.175mm perforation (1 / 8 inch) Tyvek breathable barrier jar receives 1.95g of pollen mixture and is used as a positive control in the experiment.

[0181] The remaining four lids were modified to include perforations using a handheld molding machine. One lid was modified to include 1.588mm (1 / 16 inch), 3.175mm (1 / 8 inch), 6.35mm (1 / 4 inch) or 9.525mm (3 / 8 inch) diameter perforations. During storage, the perforations in each of the four lids were opened to the atmosphere to determine whether perforations could serve as a breathable barrier without adding a covering. The impact open perforation surface area was also evaluated in the experiment. 1.588mm (1 / 16 inch), 3.175mm (1 / 8 inch) and 6.35mm (1 / 4 inch) open perforations were processed to receive 1.95g of pollen mixture each, while 9.525mm (3 / 8 inch) open perforations were processed to receive 1.6g of pollen mixture. Each lid was sealed to a 125ml mason jar using a mason jar lid tape. All processes were stored for 5 days at 6°C, 1atm pressure. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0182] For each treatment, single pollination was carried out on the receptive tassel of the primary ear of hybrid maize plants. After storage for 5 days, except 9.525mm (3 / 8 inch) open perforation treatment, the pollen tube germination of all treatments was between 50% and 55%. After 60 minutes of pollen tube germination, 9.525mm (3 / 8 inch) open perforation treatment was qualitatively graded as 2 levels (<10% germination rate; referring to Table 29). The hysteresis of pollen tube germination was observed, and the sample was incubated for five hours again under 100% rH. 9.525mm (3 / 8 inch) open perforation treatment confirmed 43% pollen tube germination after five hours. Each treatment all obtains internal gas samples, and the scope of O2 content is 19.9% ​​to 20.0%, and all CO2 content is measured as 0.1%. The presence of soda lime provides enough CO2 sealing together with Tyvek and open perforated air permeable barrier. The relative final PMC for the 3.175 mm (1 / 8 inch) Tyvek covered punch positive control was 91.7%. The internal rH in the 6°C storage chamber fluctuated between 30% and 50% during storage. The relative final PMC ranged from 102.2% to 84.3% during the Tyvek treatment and from 94.9% to 46.0% for the open punch treatments in this series. The relative final PMC decreased as the surface area of ​​the open punch increased. The 6.35 mm (1 / 4 inch) and 9.525 mm (3 / 8 inch) open punch treatments had large pollen moisture losses. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that greater than 98% of saturated rH was achieved throughout storage for the 3.175 mm (1 / 8") Tyvek covered perforation positive control treatment and for both the 1.588 mm (1 / 16") and 3.175 mm open perforation (1 / 8") treatments. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger data indicated that greater than 92% of rH was achieved throughout storage for the 6.35 mm (1 / 4") open perforation treatment. For the 9.525 mm (3 / 8") open perforation treatment, greater than 85% of rH was observed throughout storage. 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 perforation treatments, seed set of over 700 kernels / ear was obtained, indicating that the pollen was viable after 5 days of storage. The 9.525 mm (3 / 8 inch) open perforation treatment produced 322 kernels / ear, indicating that greater moisture loss from the pollen during storage negatively affected seed set.

[0183] Table 28. Pollen viability assessment and gas composition analysis

[0184]

[0185] NA = Not applicable

[0186] Table 29. Average fruiting rate

[0187]

[0188] Table 30. Grading scale for qualitative pollen tube germination used in the experiments. For all pollen tube germination assessments in the above examples, N=4.

[0189] Scale Germination rate 5 >60％ 4 41％-60％ 3 21％-40％ 2 1％-20％ 1 0％

[0190] Experiment 7: Larger Perforations Using Tyvek and Microporous Tape Breathable Barriers in the Presence of Soda Lime (5 Days)

[0191] To evaluate greater perforation, 11.8924 g of pollen was mixed with 5.946 g of 10 μm crystalline silica to give a total of 17.839 g of pollen mixture, with an initial fresh mixture PMC of 35.9%. The pollen was mixed in a ratio of two parts pollen to one part carrier by weight. 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 in an aluminum pan (45 mm x 15 mm). The pollen mixture containers were stacked above the soda lime containers and separated by a piece of 841 micron metal mesh screen. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of each of the six 125 ml pollen storage vessels and separated from the pollen mixture container by a piece of 841 micron metal mesh screen. A regular mouth Mason jar lid was equipped with a septum for gas analysis.

[0192] Six lids were modified to include perforations using a handheld molding press. Two lids were each modified to include 3.175mm (1 / 8 inch), 11.113mm (7 / 16 inch), and 12.7mm (1 / 2 inch) perforations. Tyvek disks were cut from standard FedEx envelopes and taped to one instance of each perforation size, for a total of three Tyvek treatments. Impermeable plate sealing film was applied to the outer periphery of Tyvek to seal Tyvek to the container lid. A piece of 3M microporous tape was applied to the second set of lids, one for each of the three perforation sizes. In addition to evaluating the microporous tape compared to the Tyvek breathable barrier, the experiment also evaluated the effects of increasing or decreasing surface area on the two breathable barriers. Mason jar lid tape was used to seal the lids to six 125ml Mason jar pollen storage vessels. All treatments were stored for 5 days at 6°C, 1atm pressure. After 5 days of storage, pollen viability and final PMC relative to the initial were evaluated.

[0193] For each treatment, a single pollination was performed on the receptive silk of the primary ear of hybrid maize plants. After storage for 5 days, the pollen tubes of the Tyvek treatment were between 53% and 55%, and the pollen tubes of the microporous tape treatment were 47% to 50%. The relative final PMC of the Tyvek treatment ranged from 101.5% to 98.0% and the microporous tape treatment was 94.2% to 83.3%. With the increase of the surface area of ​​the two types of air permeable barriers, it was observed that the relative final PMC decreased. The Tyvek treatment maintained a higher pollen moisture content than the equivalent microporous tape treatment. The iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger showed that for both the 3.175mm perforations (1 / 8 inch) covered by Tyvek and microporous tape, a saturated rH higher than 98% was obtained during the entire storage period. iButtonLink DS1923-F5#Hygrochron temperature and humidity data indicate that for the Tyvek covered 11.113 mm (7 / 16 inch) (7 / 16 inch) and 12.7 mm (1 / 2 inch) perforation treatments, rH above 96% was achieved throughout the storage period. iButtonLink DS1923-F5#Hygrochron temperature and humidity data indicate that for the microporous tape covered 11.113 mm (7 / 16 inch) and 12.7 mm (1 / 2 inch) perforation treatments, a hysteresis of approximately 36 hours was required to achieve rH above 98%. The microporous tape covered 11.113 mm (7 / 16 inch) and 12.7 mm (1 / 2 inch) perforation treatments had relative final PMCs of 83.3% and 90.1%, respectively, indicating that these surface areas per gram of pollen were too large for the microporous tape to maintain adequate pollen moisture content. The seed set rate ranged from 614 to 755 kernels / ear, indicating that the pollen was viable after 5 days of storage.

[0194] Table 31. Perforation diameter versus surface area and barrier area per gram of pure pollen. 1.355 grams of pure pollen were used for all treatments.

[0195]

[0196] Table 32. Pollen viability assessment and seed setting rate NA = Not Applicable; ND = No Data

[0197] Experiment 8: Higher pollen load, direct accumulation in tubes, and Microporous tape-alkali-free lime (5 days)

[0198] In this experiment, 20.19 g of pollen was mixed with 10.096 g of 10 μm crystalline silica to give a total of 30.286 g of pollen mixture, wherein the initial fresh mixture PMC was 36.10%. The pollen was mixed in a ratio of two parts pollen to one part carrier by weight. A total of 29.675 g of the pollen mixture (approximately 40 ml) was added directly to a 50 ml conical tube. The 50 ml conical tube cap was modified to include a 9.525 mm (3 / 8 inch) diameter perforation drilled into the center of the cap. The perforation was covered with 3M microporous tape to provide a 71.3 mm 2 The modified cap was placed on a 50 ml conical tube filled with pollen and tightened down to seal with the tube. The capped conical tube was stored at 6°C, 1 atm pressure in a vertical orientation (cap directly facing upward) for 5 days. After 5 days of storage, pollen viability and final PMC relative to the initial were assessed.

[0199] The pollen mixture was subsampled at a depth of 1.5 cm (top), 4.0 cm (middle), and 6.5 cm (bottom) below the surface of the pollen mixture. For each depth, a single pollination was performed on the receptive silk of the primary ear of the hybrid maize plant. Pollen tube germination was 57% at 1.5 cm, 57% at 4.0 cm, and 59% at 6.5 cm. The relative final PMC was 98.4% at 1.5 cm, 102.7% at 4.0 cm, and 103.0% at 6.5 cm. The relative final PMC increased at a greater depth from the surface of the pollen mixture. Pollen tube germination evaluation showed that the pollen mixture could be directly deposited in a storage container up to 7 cm deep with 10 ml of head space without problem. Relative final PMC and pollen viability evaluation showed that 71.3 mm 2 The microporous tape breathable barrier surface area provided sufficient gas exchange to support pollen respiration while minimizing water vapor transmission from the storage container. A seed set rate of 773 kernels / ear was obtained using pollen subsampled at a depth of 1.5 cm, 749 kernels / ear at 4.0 cm, and 689 kernels / ear at 6.5 cm, indicating that the pollen was viable after 5 days of storage.

[0200] Table 33. Pollen viability assessment and seed setting rate

[0201]

[0202] NA = Not Applicable; ND = No Data

[0203] Experiment 9: Microporous Tape Surface Area Factor - No Alkali Lime vs. Alkali Lime (5 Days)

[0204] For Experiment 9, 19.43 g of pollen was mixed with 9.72 g of 10 μm crystalline silica to obtain a total of 29.15 g of pollen mixture, wherein the initial fresh mixture PMC was 37.22%. The pollen was mixed in a ratio of two parts pollen to one part carrier by weight. 3.5 g of the pollen mixture was added to each of eight 125 ml regular mouth mason jars. The pollen mixture was contained in 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. The empty 50 ml conical tube cap was placed at the bottom of the remaining four 125 ml regular mouth mason jars. The pan containing the pollen mixture was stacked above the 50 ml conical tube cap soda lime container. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was stacked above the pollen mixture container to monitor the internal relative humidity of each of the eight 125ml pollen storage vessels and separated from the pollen mixture container using a piece of 841 micron metal mesh screen. Conventional mason jar lids were equipped with diaphragms for gas analysis. A total of eight lids were modified to include perforations using a handheld molding press. Two sets of lids were each modified to include 1.588mm (1 / 16 inch), 3.175mm (1 / 8 inch), 6.35mm (1 / 4 inch), or 9.525mm (3 / 8 inch) perforations. For all eight lids, a piece of 3M microporous tape was applied to the perforations. This produced two sets of repeated lids, each with a microporous tape breathable barrier surface area of ​​2.0mm 2 、7.9mm 2 、31.7mm 2 , and 71.3mm 2 The purpose of this experiment was to determine if a minimal surface area microporous tape gas permeable barrier was required per gram of pollen mixture in the absence of soda lime to avoid accumulation of CO2 from cellular respiration to toxic levels. The modified lids were sealed to 125 ml Mason jar pollen storage vessels using Mason jar lid tape. All treatments were stored at 6°C, 1 atm pressure for 5 days. After 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis for O2 and CO2 were assessed.

[0205] For each treatment, a single pollination was performed on the fertile silk of the primary ear of hybrid maize plants. For all treatments, pollen tube germination was between 43% and 56%. It was observed that the treatments with soda lime had a lower percentage of pollen tube germination than the treatments without soda lime. Internal gas samples were taken for each treatment, and the O2 content ranged from 19.7% to 19.2%. For the treatments with soda lime, the CO2 content was all 0.2%. The presence of soda lime, together with the microporous tape air permeable barrier at all tested surface areas, provided adequate CO2 sequestration. For the 2.0 mm 2 、7.9mm 2 、31.7mm 2 , and 71.3mm 2 The CO2 levels for the treatments without soda lime were 1.2%, 0.6%, 0.4%, and 0.4% for each surface area tested without soda lime and for the amount of pollen mixture stored, there was sufficient gas exchange to maintain CO2 accumulation below toxic levels that would affect pollen. The relative final PMC ranged from 100.5% to 87.3% across all 3M Microporous Tape treatments. No pattern of increase or decrease in relative final PMC was observed, regardless of the presence or absence of soda lime. iButtonLink DS1923-F5#Hygrochron temperature and humidity data loggers indicated that greater than 98% of saturated rH was achieved throughout the storage period for all treatments, regardless of the presence or absence of soda lime. Fruit set from a single test pollination for all eight treatments exceeded 500 grains / ear, indicating that pollen was viable after 5 days of storage (Table 35). Fruit set and pollen tube germination results indicate that the breathable barrier is able to provide sufficient gas exchange to maintain CO2 below toxic levels in the absence of added soda lime.

[0206] Table 34. Pollen viability assessment

[0207]

[0208] NA = Not applicable

[0209] Table 35. Average fruiting rate

[0210] Experiment 10: Comparison of single layer versus double layer use of microporous tape in the absence of soda lime (5 days)

[0211] This experiment examined the impact of doubling the layer of microporous tape breathable barrier. 27.84g of pollen was mixed with 13.92g of 10μm crystalline silica to obtain a total of 41.76g of pollen mixture, wherein the initial fresh mixture PMC was 36.06%. The pollen was mixed in a ratio of two parts pollen and one part carrier by weight. A total of 20.0g of pollen mixture was added directly to the bottom of two 125ml regular mouth mason jars. Regular mouth mason jar lids were equipped with diaphragms for gas analysis. The two lids were modified to include identical 9.525mm (3 / 8 inch) diameter perforations using a handheld molding press. A single layer of 3M microporous tape was applied to the perforations on one lid. The perforations on the second lid were covered with two layers of 3M microporous tape, one layer was applied to the top of the lid covering the perforations, and the second layer was applied to the bottom surface of the lid covering the perforations. The same 9.525 mm (3 / 8 inch) diameter perforation in each cover covered with 3M Microporous Tape provides a 71.3 mm 2 The experimental design tested whether the use of two layers of microporous tape breathable barriers could increase the relative final PMC of pollen during storage and determine whether the rate of gas exchange through the two layers compared to a single layer was sufficient to support pollen respiration. The treatments were stored at 6°C and 1 atm pressure for 5 days. At 5 days of storage, pollen viability, final PMC relative to initial PMC, and endpoint gas analysis of O2 and CO2 were assessed.

[0212] For each treatment, three replicate pollinations were performed on the receptive silks of the primary ear of hybrid maize plants. Pollen tube germination was noted as 55% for a single layer of microporous tape and 56% for a double layer of microporous tape. The relative final PMC was 102.7% for a single layer and 104.3% for a double layer. This indicates that the relative final PMC increases when a second layer of microporous tape is added. Internal gas samples were obtained for each of the two storage containers. The O2 content was 19.3% and the CO2 content was 0.7% for a single layer. The O2 content was 18.2% and the CO2 content was 2.2% for a double layer. The single layer treatment supported greater gas exchange compared to the double layer treatment. Sufficient O2 levels were maintained with the double layer of microporous tape to support pollen respiration. CO2 levels remained below levels toxic to pollen during storage with the double layer of microporous tape, but CO2 was higher than the levels determined for the single layer. Pollen tube germination assessment showed that the 71.3 mm diameter of the single or double layer microporous tape barrier 2 The surface area provided sufficient gas exchange to support pollen respiration while minimizing water vapor transmission from the storage container. An average seed set rate of 707 kernels / ear for single-layer microporous tape coverage and 663 kernels / ear for double-layer microporous tape coverage was observed, indicating that the pollen was viable after 5 days of storage.

[0213] Table 36. Pollen viability assessment and seed setting rate

[0214]

[0215] NA = Not Applicable; ND = No Data

[0216] 6: Water Vapor Transmission of Tyvek, Microporous Tape, Open Perforations, and Parafilm Experiments 1 and 2: Water Vapor Transmission Rate through Tyvek Barrier, 3M Microporous Tape Barrier, and Open Perforations (No Cover) to Atmosphere

[0217] Two experiments were conducted to examine the water vapor transmission rate through Tyvek and 3M Microporous Tape breathable barriers compared to perforations open to the atmosphere. All three breathable barriers had 71.3 mm 2 The same surface area of ​​the film. Water vapor transmission rate is measured by the weight loss of steady-state water vapor flow per unit area per unit time on the breathable barrier under specific temperature and humidity conditions. A similar method as expressed in the ASTM standard test method for water vapor transmission of materials (E96 / E96M-16) is followed, and water vapor transmission rate is measured at 23°C and a relative humidity of 58% to 61%. See generally www.astm.org / standards / e96. An approximately 4-liter acrylic vacuum chamber (Terra Universal Model 5235-01B) with an internal dimension of 28cm (11 inches) x 28cm (11 inches) x 12.7cm (5 inches) is placed in a Fisher Scientific benchtop incubator (Model 6500). A glycerol aqueous solution is prepared with 80% by weight of pure glycerol to deionized water. The specific gravity of the glycerol aqueous solution is 1.4374. 500 ml of the glycerol aqueous solution was placed in an 8.89 cm (3.5 in) x 15.24 cm (6.0 in) open container. The open container of glycerol aqueous solution was placed in an acrylic chamber to provide appropriate humidity in the chamber. Approximately 60 ml of deionized water was placed in 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 modified to include 71.3 mm using a handheld molding press. 2Perforations. A Tyvek disk was cut from a standard FedEx envelope and taped to a 3.175 mm perforation on three of the twelve lids. An impermeable plate sealing film was applied to the outer perimeter of the Tyvek to seal the Tyvek to the container lid. 3M Microporous Tape was placed over the perforations on three of the twelve lids. The perforations on the remaining six of the twelve lids were left uncovered. The lids were placed on identical Mason jars containing deionized water and tightly sealed using Mason jar lid tape. 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 8 days. Weight loss measurements were collected to calculate the water vapor transmission rate from all twelve jars covering the three breathable barrier treatments.

[0218] It was observed that the Tyvek breathable barrier reduced water vapor loss by 48.3% at 60% rH and 23°C under static airflow compared to open perforations. It was observed that the microporous tape breathable barrier reduced water vapor loss by 23.2% at 60% rH and 23°C under static airflow compared to open perforations. Under static airflow, at 60% rH and 23°C, the Tyvek barrier was more effective than the microporous tape barrier, reducing water vapor transmission of the storage container by 32.6%.

[0219] Table 37. Through three different 71.3mm 2 Water vapor loss through a breathable barrier.

[0220]

[0221]

[0222] 7: Continuous gas analysis during storage at 6°C

[0223] Experiment 1: Continuous gas application during pollen storage using a gas-permeable barrier (9 days)

[0224] In the continuous gas experiment, 9.699g pollen was mixed with 4.845g 10μm crystalline silica (2:1 pollen: silica by weight) to obtain a total of 14.53g pollen mixture, wherein the initial fresh mixture PMC was 36.39%. A total of 14.4g pollen mixture was added to a 400mL Magenta GA7 box. A 50ml conical tube cap containing 2.03g soda lime was placed at the bottom of the GA7 box. The pollen mixture was contained in a PLA plastic cup (45mm x 30mm) and the container was located above the open soda lime container. Due to the 30% rH in the laboratory environment during the experimental setup, 100ul water was added to the bottom of the GA7 box. Adding water is intended to increase the rate at which the internal atmosphere of the storage container reaches saturated relative humidity. An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger was placed above the pollen mixture container to monitor the internal relative humidity of the pollen storage vessel. 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 opening. The parafilm was stretched to create a tight seal, as is common practice in plant tissue culture. The GA7 gas exchange gap between the box and lid surface area was calculated to be a total of 42 mm 2 . The pollen storage vessels were stored at 6°C and 1 atm pressure for 9 days. A Quantek 902P gas analyzer was set up for continuous measurement of O2 and CO2. The input and output lines of the analyzer were attached to two 23 gauge precision slide needles and inserted into two spacers installed in the side wall of the GA7. The input and output lines of the analyzer passed through the side wall of the refrigeration unit through plugs. The analyzer was maintained outside the refrigeration unit at room temperature. After 9 days, pollen tube germination, final PMC relative to initial PMC, and continuous gas analysis of O2 and CO2 were evaluated.

[0225] An iButtonLink DS1923-F5#Hygrochron temperature and humidity data logger indicated that greater than 98% of saturation rH was achieved throughout the storage period in the storage vessel. After 9 days, pollen tube germination was qualitatively scored as 2 levels (1%-20% germination rate; see Fig.10). The relative PMC was 97.6%. The internal vessel rH and final relative PMC data indicate that the use of paraffin film as a breathable barrier to seal the 400 mL Magenta GA7 box reduced water loss from the storage vessel and pollen mixture. Continuous internal gas sampling with 1 minute sampling intervals was obtained from the storage vessel during storage. The continuous gas data were normalized for atmospheric pressure changes. Within the first few hours of storage at 6°C, the O2 content decreased to a minimum of 19.4%, and the CO2 content remained below 0.1%. Over the duration of the first 8 days of the storage experiment, the internal O2 content remained at a steady state of 20.0%. On the 9th day, the O2 content increased to 20.4%. This may be due to a decrease in pollen viability and an associated decrease in pollen respiration. The use of paraffin film as a breathable barrier provides adequate gas exchange while minimizing water vapor transmission. The presence of soda lime, together with the breathable barrier, provides adequate CO2 sequestration. The breathable barrier provides high steady-state levels of oxygen to support pollen respiration. No reduction in oxygen levels to anaerobic levels was measured within the storage vessels.

[0226] 8. Use a non-airtight container with a manufactured opening that does not contain soda lime (i.e., 250 ml Scale-up of pollen storage in cell culture flasks

[0227] Experiments 1, 2, and 3: 1) Breathable Barrier 250 ml Cell Culture Flasks using stock nitrocellulose barrier - without soda lime (5, 6, 7, 8, and 9 days); 2) Breathable Barrier 250 ml Cell Culture Flasks stock cap vent covered with 3M Micropore Tape - without soda lime (5, 6, 7, 8, and 9 days); 3) Breathable Barrier 250 ml Cell Culture Flasks, cap removed and entire opening covered with 1 layer of 3M Micropore Tape - without soda lime (5, 6, 7, 8, and 9 days);

[0228] For Experiments 1, 2, and 3, fresh pollen was collected and mixed with 10 μm crystalline silica (2:1 pollen:silica by weight) on three different days. The bulk pollen mixture was added directly to 250 ml Cell culture flasks were prepared without the addition of soda lime. Each experiment consisted of a different treatment of the gas permeable barrier of the culture flask caps. Each treatment in each experiment included 1 replicate.

[0229] For Experiment 1, a total of 51.15 g of pollen mixture (34.42% of the initial fresh mixture PMC) was added to 250 ml The stock vent cap of the cell culture flask (with the stock embedded nitrocellulose barrier) was placed on the flask and tightened to form a seal. For Experiment 2, a total of 48.4 g of pollen mixture (36.97% of the initial fresh mixture PMC) was added directly to the 250 ml Cell culture flasks were placed in the flasks. The stock embedded nitrocellulose barrier was removed from the vent cap and the vent hole was covered with a layer of 3M Micropore Tape. For Experiment 3, a total of 18.06 g of pollen mixture (40.7% of the initial fresh mixture PMC) was added directly to the 250 ml Cell culture flasks. Remove the stock vent cap completely from the flask and cover the entire opening with a layer of 3M microporous tape. For Experiments 1 and 2, the stock cap vent hole provided 36.4 mm 2 For Experiment 3, the cap was removed and the entire open neck of the flask was covered with microporous tape to create a total surface area of ​​486.95 mm 2 The surface area of ​​the breathable membrane.

[0230] In each experiment, pollen storage vessel is laid flat and stored 9 days under 6 ℃, 1atm pressure.At the 5th, 6, 7, 8 and 9th day of storage, from each pollen storage vessel, obtain the subsample of pollen mixture, be used for pollination on a fertile ear of hybrid maize plant.Every day of sampling and pollination, pollen storage vessel is taken out from storage, sampling, reseals with microporous tape in experiment 2 and 3, and is put back in the storage under 6 ℃.In experiment 1, obtained the fruiting rate of 518 grains / ear, 380 grains / ear, 443 grains / ear, 403 grains / ear and 467 grains / ear at 5 days, 6 days, 7 days, 8 days and 9 days respectively.The 250ml cell culture flask with reserve cap and nitrocellulose barrier provides enough gas exchange, keeps pollen viability simultaneously. In experiment 2, obtained the fruiting rate of 338 grains / ear, 425 grains / ear, 409 grains / ear and 2 grains at 6 days, 7 days, 8 days and 9 days respectively.The reserve cap of the nitrocellulose barrier without embedding and the vent covered with 3M microporous tape provide enough gas exchange, to maintain pollen viability for up to 8 days.For experiment 3, obtained the fruiting rate of 457 grains / ear, 517 grains / ear, 461 grains / ear, 30 grains / ear and 27 grains / ear at 5 days, 6 days, 7 days, 8 days and 9 days respectively.The 3M microporous tape covering the open neck of 250ml cell culture flask provides enough gas exchange, keeps pollen viability for up to 9 days simultaneously.The fruiting rate result in each experiment shows that the existence of soda lime is not needed to maintain CO2 below toxicity level, and uses breathable barrier to provide enough gas exchange to support aerobic respiration. Cell culture flasks are suitable pollen storage vessels, and the open neck at the bottom of the flask provides sufficient surface area for gas exchange through a gas permeable barrier while minimizing water vapor transmission from the vessel.

[0231] Table 38. From using 250ml Scale-up results of pollen set in cell culture flasks

[0232]

[0233] ND = No Data

[0234] 9. Breathable barrier pollen storage using crystalline silica, metal powder, and mica carriers

[0235] Crystalline quartz silica is an effective carrier for maize pollen storage. Crystalline silica inhibits agglomeration interactions between pollen grains during storage, but does not over-coat pollen grains immediately after application or due to breakage of carrier particles during handling. In addition, crystalline silica does not act as a desiccant in storage.

[0236] Metal powder is an effective carrier for pollen storage. Metal powder prevents the agglomeration interaction between adjacent pollen grain barriers during storage, but does not over-coat the pollen barrier surface and does not inhibit the attachment to corn silk or other plant stigmas. This lack of inhibition enables effective pollen tube germination. Elemental metal powders, metal oxide powders, and metal carbide powders are all effective pollen storage carriers. These powders can be manufactured by various techniques to optimize function, including solid-state reduction, electrolysis, chemical reaction, high temperature combustion, gas atomization, ultra-high pressure water atomization, pressing and sintering, centrifugal atomization, grinding, and other polishing techniques that optimize particle size and particle surface properties. The best particle type of metal powder in corn pollen storage is considered to be 10 μm polished spherical particles, but other particle sizes and surface features may be superior to other pollen types. In some applications, metal powders can be coated in polymers to change the interaction between the particle surface and the pollen membrane. In other applications, metal particles can be coated in active ingredients to modify the interaction with the pollen grain membrane, alter the respiration of pollen and microorganisms in storage, or inhibit the reproduction of microorganisms during storage. These active ingredients can include nucleic acids, proteins, pesticides, or biostimulants. Metal carriers include elements with known biological effects in plants that can enhance pollen performance; and those without known biological effects that have no effect on pollen performance. Ferromagnetic carriers may be preferred in applications where the carrier can be magnetically removed from the pollen-carrier mixture after storage to enrich the pollen concentration in the mixture.

[0237] Micas are a group of minerals defined by a general chemical formula and perfect basal cleavage. Perfect basal cleavage produces flat, flaky particles that effectively prevent interaction between adjacent pollen grain membranes. In addition to the physical properties that make mica minerals effective carriers in pollen storage, the high reflectivity of mica minerals can serve as a visual indicator during pollen application. These reflective properties can be visualized by a program operator or machine camera to track the distribution of pollen during application or to verify where pollination has occurred.

[0238] Table 39 details the performance of crystalline silica, metal powder carriers, and mica as carriers in pollen storage. All carriers in this test showed similar performance to the crystalline silica detailed in U.S. Provisional Application No. 63 / 289,299, filed December 14, 2021, which is incorporated herein by reference.

[0239] Table 39: Fruit setting rate of pollen stored in ten carriers.

[0240]

[0241]

[0242] All carriers were mixed with pollen at a ratio of two parts pollen to one part carrier by weight. 10 μm 316L stainless steel powder was produced by high temperature combustion, and the individual particles had an amorphous structure. It was optimized for 3D printing by ultra-high pressure water and gas atomization and grinding to produce uniform spherical particles.

[0243] 10. Pollen storage of plants containing transgenic events

[0244] The male rating is an overall assessment of performance as a pollen source that accounts for all types of data collected during the development of the inbred parent lines. The male performance ratings from highest to lowest are ideal, acceptable, insignificant, and no improvement.

[0245] Pollen of five inbred parent lines containing transgenic events was collected separately and mixed with a crystalline silica carrier at a ratio of two parts pollen to one part carrier by weight. A portion of the pollen mixed with crystalline silica was used for fresh self-pollination (storage day 0). The remaining pollen and crystalline silica mixture was stored in a sealed container with soda lime added in a 6°C environment. After five days, the stored pollen was applied to the tassels of the same inbred line that provided the pollen (i.e., self-pollination). All tassels received the same amount of fresh pollen and carrier or stored pollen and carrier by volume.

[0246] Table 40: Seed setting of stored pollen of five different inbred parent lines. Inbred lines 1 to 4 contain transgenic events Bt11, GA21 and MIR162. Inbred line 5 contains transgenic events Bt11 and MIR162.

[0247]

[0248]

[0249] 11. Breathable barrier pollen storage-container design

[0250] Experiment 1: 25 ml with vent cap Cell culture flask with 850 ml of pollen stored from the same collection Cell culture flasks compared

[0251] Pollen was collected from the field trial and mixed with a silica carrier at a ratio of two parts pollen to one part silica by weight. The total amount of pollen collected after mixing with silica was 1,009 grams. One kilogram of treated pollen was placed in 850 ml In a cell culture flask, the flask cap was removed and the vessel opening was covered with a single layer of 3M Micropore Tape to act as a breathable barrier (see, e.g., Figure 7-12 ). This storage container is configured to have a 0.7 mm 2 The surface area ratio provides 660.5mm 2 Total permeable barrier surface area. In this configuration, the maximum distance between the pollen particles and the permeable barrier was calculated to be 274 mm based on trigonometric calculations. 9 g of treated pollen was placed in 25 ml Cell culture flasks were placed in the same container and a stock vent cap with a nitrocellulose membrane was used as a gas barrier. This storage vessel was configured to provide 1.7 mm per gram of treated pollen. 2 The surface area ratio provides 15.2mm 2 Total air permeable barrier surface area. In this configuration, the maximum distance between the pollen grains and the air permeable barrier was measured to be 72 mm. During storage, the oxygen content of the air within the pore space between the pollen grains and the silica carrier particles within the 850 ml flask was measured by inserting a needle through the air permeable barrier or through a gas-tight septum mounted on the rear of the storage vessel. The oxygen content of the air within the pore space was found to drop to as low as 0.7% within the first two hours of storage and was observed to stabilize at 13.1% over the duration of five days of storage at 6°C. After five days of storage, the pollen from each storage vessel was mixed separately to evenly merge the pollen stored in each storage vessel. The mixed pollen was applied to pollen tube germination plates and test spikes. All spikes received the same amount of stored pollen.

[0252] Table 41. Seed set obtained from pollen stored for five days, unamended 850 ml Cell culture flasks with unmodified 25 ml Cell culture flasks compared.

[0253]

[0254] Experiment 2. Modified 850ml Cell culture flasks

[0255] After observing the delayed fruiting rate performance of pollen stored in 850 ml flasks when compared to the same batch of pollen stored in 25 ml flasks, it was hypothesized that the scaled-up storage conditions may have affected pollen viability. It was hypothesized that anaerobic conditions early in the storage process and reduced oxygen availability throughout the storage period reduced pollen viability, so that pollen tested as viable on pollen tube germination medium could not complete pollen tube growth and fertilization when placed on tassels, and subsequently died from environmental stress. To test this hypothesis, the 850 ml flask was modified to reduce the maximum distance between the pollen grain and the nearest air permeable barrier, while still maintaining the ratio of total air permeable barrier surface area to grams of pollen within 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 larger surface area flat side of the flask in a three by three grid pattern, with a spacing of 72 mm between the holes. Where applicable, the center of the hole was 12 mm from the nearest edge. This larger surface area flat side was designed to be the top of the flask facing upward during use. The same three by three grid pattern of 10 mm holes is drilled through the smaller surface area flat side of the flask, with 64 mm spacing between the holes. Where applicable, the holes are centered 12 mm from the nearest edge. This smaller surface area flat side is designed to be the bottom of the flask facing downward during use, and has a lower surface area due to the taper made on the 47 mm outer height side wall of the flask. Two additional 10 mm holes are drilled in the two forward facing obtuse corners of the larger surface area top side of the flask. Each hole is centered 12 mm from the two nearest edges of the flask. Two additional 10 mm holes are drilled in the two forward facing obtuse corners of the smaller surface area bottom side of the flask. Each hole is centered 12 mm from the two nearest edges of the flask. Three 10 mm holes are drilled on each side of the flask on an axis extending parallel to the direction of the mouth of the vessel, resulting in a total of six additional holes. These holes are placed at 64 mm spacing, and each hole is centered 12 mm from the edge of the bottom surface of the flask. See, for example, Figure 21-26 .

[0256] For Experiment 2, pollen was collected from a field trial and mixed with a silica carrier at a ratio of two parts pollen to one part silica by weight. The total amount of pollen collected after mixing with silica was 860 grams. Eight hundred and fifty grams of the treated pollen was placed in an optimized 850 ml flask with 28 added 10 mm holes. A non-vented vessel cap was tightly sealed on the vessel mouth, and each of the 28 added 10 mm holes was covered in a single layer of 3M microporous tape (see, e.g., Figure 27-30 ). This storage container is configured to have a 2.6 mm 2 The surface area ratio provides 2199.1mm 2 Total permeable barrier surface area. In this configuration, the maximum distance between the pollen grain and the permeable barrier was calculated to be 47 mm based on trigonometric calculations. Ten grams of treated pollen were placed in a 25 ml flask, and a stock vent cap with a nitrocellulose membrane was used as a permeable barrier. This storage vessel configuration was 1.5 mm per gram of treated pollen. 2 The surface area ratio provides 15.2mm 2 Total breathable barrier surface area. In this configuration, the maximum distance between the pollen grains and the breathable barrier was measured to be 72 mm. During storage, the oxygen content of the air within the pore spaces between the pollen grains within the optimized 850 ml flask was measured by inserting a needle through the breathable barrier or through a gas-tight septum mounted on the rear of the storage vessel. The oxygen content of the air within the pore spaces did not fall below 18.7% during the six days of storage. After six days of storage, the pollen from each storage vessel was mixed separately to evenly merge the pollen stored in each storage vessel. The mixed pollen was applied to pollen tube germination plates and test spikes. All spikes received the same amount of stored pollen.

[0257] Table 42. Seed set obtained from pollen stored for six days, using the optimized 850 ml Cell culture flasks with unmodified 25 ml Cell culture flasks compared.

[0258]

[0259] Experiment 3. With optimized 850ml Cell culture flasks compared to unmodified 850ml Cell culture flasks.

[0260] After observing the increased fruit set obtained from pollen stored in optimized cell culture flasks, experiments were performed to directly compare the performance of equivalent pollen stored in unmodified 850 ml flasks and optimized 850 ml flasks with 28 added openings for a gas permeable barrier (see e.g., Figure 21-26). The same optimized 850 ml flask described in Experiment 2 with 28 added openings for a breathable barrier was used during Experiment 3. Pollen was collected from the field trial and mixed with a silica carrier at a ratio of two parts pollen to one part silica by weight. The total amount of pollen collected after mixing with silica was 2,000 grams. One kilogram of treated pollen was placed in an optimized 850 ml flask with 28 added 10 mm holes. A non-vented vessel cap was tightly sealed over the vessel mouth, and each of the 28 added 10 mm holes was covered in a single layer of 3M Microporous Tape (see, e.g., Figure 27-31 ). This storage container is configured to have 2.2 mm per gram of processed pollen. 2 The surface area ratio provides 2199.1mm 2 Total breathable barrier surface area. In this configuration, the maximum distance between the pollen grain and the breathable barrier was calculated to be 47 mm based on trigonometric calculations. One kilogram of treated pollen was placed in an unmodified 850 ml flask, the flask cap was removed, and the mouth of the vessel was covered with a single layer of 3M microporous tape to act as a breathable barrier. This storage vessel configuration was 0.7 mm per gram of treated pollen. 2 The surface area ratio provides 660.5mm 2 Total breathable barrier surface area. In this configuration, the maximum distance between the pollen grains and the breathable barrier was calculated to be 274 mm based on trigonometrics. After three days of storage, the pollen from each storage vessel was mixed separately to evenly merge the pollen stored in each storage vessel. The mixed pollen was applied to pollen tube germination plates and test spikes. All spikes received the same amount of stored pollen. The mixed pollen was also mechanically broadcast on receptive spikes to further test pollination efficiency. During broadcast application, the pollen dose to individual spikes was unequal.

[0261] Table 43. Seed set obtained from pollen stored for three days, using the optimized 850 ml Cell culture flask with unmodified 850ml Cell culture flasks compared.

[0262]

[0263]

Claims

1. A method for storing viable corn pollen, the method comprising: a) collecting a certain amount of fresh corn pollen; b) optionally applying a carrier to the collected corn pollen of step a) to obtain a certain amount of treated corn pollen; c) placing the amount of fresh corn pollen or the amount of treated corn pollen in a container; d) sealing the container with a gas permeable barrier; and e) storing the product of step d) in a refrigerated environment.

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

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

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

5. The method of claim 1, wherein the carrier 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 has an average particle size.

8. The method of claim 7, wherein the average particle size is between about 1 nanometer and about 100 microns.

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

10. The method of claim 5, wherein the metal powder is a metal oxide powder or a metal carbide powder. The method of claim 10 , wherein the metal powder has an average particle size.

12. The method of claim 11, wherein the average particle size is between about 1 micron and about 100 microns.

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

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

15. The method of claim 1, wherein the vector is present in a pollen:vector 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. The method of claim 15, wherein the vector is present in a 2:1 pollen:vector ratio.

17. The method of claim 1, wherein the container has a volume of 0.1 ml to 10 liters.

18. The method of claim 17, wherein the container has a volume of 10 ml to 1500 ml.

19. The method of claim 18, wherein the container has a volume of 100 ml to 1250 ml.

20. The method of claim 1, wherein the container further comprises a CO2 sequestering agent.

21. The method of claim 20, wherein the sealing agent is selected from the group consisting of activated carbon, ethanolamine, zeolite 4A, lithium hydroxide (LiOH), soda lime, calcium silicate (Ca2O4Si), and activated magnesium silicate (e.g., ).

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

23. The method of claim 1, wherein the air permeable barrier is selected from the group consisting of Parafilm, Tyvek, 3M Microporous Tape, cellulose, nitrocellulose, and a non-airtight container.

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

25. The method of claim 23, wherein the non-airtight container comprises ports for gas exchange.

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

27. The method of claim 26, wherein the orifice comprises at least one perforation having a diameter size of 0.10 mm to 30 mm.

28. The breathable barrier of claim 23, wherein the breathable barrier has a thickness of 0.49 mm 2 / g fresh or processed pollen to 47.5mm 2 / g surface area of ​​fresh or treated pollen.

29. The breathable barrier of claim 28, wherein the breathable barrier has a thickness of 1.98 mm. 2 / g fresh or processed pollen to 26.72mm 2 / g surface area of ​​fresh or treated pollen.

30. The breathable barrier of claim 29, wherein the breathable barrier has a thickness of 4.45 mm 2 / g fresh or processed pollen to 11.88mm 2 / g surface area of ​​fresh or treated pollen.

31. The method of claim 1, wherein the stored maize 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. The method of claim 1, wherein the refrigerated environment comprises a temperature range selected from the group consisting of 1°C-10°C, 4°C-8°C, and 5.5°C-6.5°C.

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

34. 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 than 1 day.

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

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

37. The method of claim 36, wherein the transgenic maize 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. The method of claim 37, wherein the maize pollen comprises transgenic events Bt11, GA21, and MIR162.

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

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

41. A device for storing pollen, the device comprising a vessel having a total gas permeable barrier surface area.

42. The apparatus of claim 41, wherein the vessel comprises a plurality of openings.

43. The device of claim 41, wherein the vessel has a diameter of 0.49 mm. 2 / g fresh or processed pollen with 47.5mm 2 / g of total air barrier surface area between fresh or treated pollen.

44. An apparatus as described in claim 42, wherein the vessel comprises at least one opening, at least two openings, at least three openings, at least four openings, at least five openings, at least six openings, at least seven openings, at least eight openings, at least nine openings, at least ten openings, at least eleven openings, at least twelve openings, at least thirteen openings, at least fourteen openings, at least fifteen openings, at least sixteen openings, at least seventeen openings, at least eighteen openings, at least nineteen openings, at least twenty openings, at least twenty-one openings, at least twenty-two openings, at least twenty-three openings, at least twenty-four openings, at least twenty-five openings, at least twenty-six openings, at least twenty-seven openings, at least twenty-eight openings, at least twenty-nine openings, or at least thirty openings.

45. The device of claim 44, wherein the at least one opening has a 15.2 mm 2 Up to 660.5mm 2 total surface area.

46. ​​The device of claim 45, wherein the at least one opening is selected from the group consisting of: circular, oval, square, rectangular, triangular, and any other two-dimensional shape.

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

48. The apparatus of claim 44, wherein the vessel comprises twenty-two openings.

49. The apparatus of claim 44, wherein the vessel comprises twenty-eight openings.

50. The device of claim 48, wherein the twenty-two openings are circular.

51. The device of claim 49, wherein the twenty-eight openings are circular.

52. The device of claim 42, wherein the plurality of openings have a range of 0.49 mm 2 / g fresh or processed pollen with 47.5mm 2 / g total combined surface area between fresh or treated pollen.

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

54. The device of claim 53, wherein the circular hole has a diameter of 10 mm.

55. The device of claim 54, wherein the circular hole has a diameter of 78.5 mm. 2 of individual surface area.

56. The device of claim 51, wherein the total of twenty-eight circular openings have a diameter of 2199.1 mm. 2 total surface area.

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

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

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

60. The device of claim 41, wherein the vessel is A cell culture flask wherein the vent cap is used in its stock configuration and the nitrocellulose membrane in the vent cap acts as a gas permeable barrier.

61. The apparatus of claim 41, wherein the vessel is used in its reserve configuration Cell culture flask with the cap removed and the vessel mouth covered in 3M micropore tape to act as a breathable barrier.

62. The device of claim 41, wherein the vessel is A cell culture flask modified to include 28 additional openings distributed over the surface of the vessel, wherein the additional openings are covered in a gas permeable barrier and a stock, non-vented cap remains sealed over the vessel port.

63. The device of claim 41, wherein the vessel is A cell culture flask modified to include any number of additional openings covered with gas permeable barriers randomly distributed over the surface of the vessel, with a solid or vented cap left in place over the vessel mouth, or with the cap removed and replaced with a gas permeable barrier.

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

65. The apparatus of claim 41, wherein the vessel is any plastic, metal, or ceramic container having one or more openings for a gas permeable barrier.

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