Dead pollen contents have been proven to cause live pollen grains to die more quickly than they would if the live pollen grains were separated from the dead pollen contents (see, for example, U.S. Pat. No. 12,245,587). Evidently, cellular components leached from the dead pollen interact with viable pollen grains and causes their death or loss of viability. The present invention seeks to minimize and/or prevent such interaction. U.S. Pat. No. 12,245,587 uses solid particulates to separate pollen grains and separate live pollen grains from interactions with dead pollen contents. The present invention demonstrates that when a specific percentage of starch is mixed with a solid particulate diluent and then used in pollen preservation, there are surprising and unexpected benefits.

Collected pollen stored in the diluent of the present invention may be stored for any length of time, including but not limited to just long enough to transfer pollen to a plant in the same or a neighboring field, minutes, hours, days, weeks, months, and/or years. For example, in some cases, the pollen is stored in the diluent of the present invention for 30 minutes, for 1 hour, for 2 hours, for 3 hours, for 4 hours, for 5 hours, for 8 hours, for 10 hours, for 12 hours, for 15 hours, for 20 hours, for 24 hours, for 2 days, for 3 days, for 5 days, for a week, for several weeks, for a month, for several months, for a year, or for several years. In the embodiments of the invention, surface to surface contact between grains of pollen and/or the cellular content of grains of pollen is prevented or limited.

For the purposes of this invention, the pollen and diluent mixture can be stored in a vessel of any shape, size, and construction suitable for the containment of the pollen and diluent mixture, such as a container, a cartridge, an enclosure, a tube, or any other type of vessel. Furthermore, the vessel may optionally be aerated and may also optionally be housed within a controlled environment. For example, the vessel may optionally be stored within a refrigerated environment, a freezer, a cooler, or any other environment in which the temperature may be controlled.

The starch used in the diluent mixture can be derived from any plant type or plant organ that accumulates starch as temporary carbohydrate storage. For example, it can be extracted from various plant types, including but not limited to root vegetables, grains, legumes, and palms. In particular, root vegetable starches are useful in the practice of the invention, including, for example, starch extracted from potato, sweet potato, cassava, taro, and yam. Likewise, grain starches may be used in the practice of the invention, such as, for example, starch extracted from corn, barley, buckwheat, wheat, and rice. Another starch that is useful for the practice of the invention is sago starch. Sago starch is extracted from the pith of various tropical ‘sago palms’ such as Metroxylon sagu and other Metroxylon species, but also from cycad species, in particular, Cycas revoluta. In addition, arrowroot starch can be used in the practice of the invention. Arrowroot starch is obtained from the rhizomes of various tropical plants, including, but not limited to, Maranta arundinacea, Zamia integrifolia, Manihot esculenta, Tacca leontopetaloides, and Pueraria lobata. Starch extracted from legume seeds may also be used in the practice of the invention. For example, mature seeds of chickpea, cowpea, lentil, mung bean, pea, and pigeon pea may be particularly useful in the practice of this invention as they are comprised of 40 to 60% starch (Tayade, et al (2019) Frontiers Plant Science Vol 10. <doi: 10.3389/fpls.2019.01213>).

Furthermore, starches used in embodiments of the invention may be unique genetic variant starches or transgenically developed or modified starches. Additionally, the starch may be entirely artificially synthesized rather than being extracted or derived from a plant. Accordingly, starches used in the practice of the invention may be naturally, chemically, or transgenically derived or created.

It is beneficial that the diluent mixtures of the invention regulate pollen moisture content. Furthermore, the diluents may provide increased flowability when the pollen is applied to a female plant, such as during mechanical application. It is also desirable to use diluents that are cost effective for large scale operations, which is one benefit of solid substances or particulates generally. Another benefit of solid particulates is that they are more easily portable or transportable, which can be especially beneficial when the method is performed on pollen as it is collected, such as in a field. It is a benefit of the present invention that the diluents are not harmful to the plant and/or the environment. Furthermore, it is beneficial that the diluent mixture does not interfere with the normal function of the stigmas of the female parent plant onto which the pollen will be applied. Similarly, it is beneficial that the diluent and the pollen grains can separate during or immediately after application, such that the diluent doesn't bind to the pollen and/or female parent plant, including but not limited to pollen receptors, during application to female parent plants.

It is a benefit of the present invention that the diluent mixture that includes starch can impart an improvement or enhancement in pollen performance, stability, and viability compared to the same diluent that is lacking the starch component. When used without the starch present, some diluents maintain or enhance pollen performance or stability, but the use of starch as a blended component of the diluent shows a surprising and unexpected benefit. As further demonstrated in the working examples of this disclosure, this benefit is observed whether pollen viability is assessed in terms of pollen germination in vitro or in terms of kernel set measured in planta.

The concentration of starch in the diluent mixture may vary depending on a number of factors including, but not limited to, the type of pollen being preserved, the type of starch(es) being used in the diluent mixture, and the nature of the other solid particulates in the diluent mixture. The starch may be added as a percentage of the mixture by weight or by volume. One of skill in the art will understand and appreciate that the decision to measure the starch component by weight or by volume will depend on the nature and density of the other components of the diluent mixture. In the case of the blends used in the working examples of this disclosure, the starch was typically blended by weight.

As discussed above, the methods disclosed herein improve and/or extend the viability of pollen in storage. Viable pollen can successfully germinate and commonly possesses the vigor necessary to promote fertilization and initiation of seed development. Not all viable pollen is also fertile pollen. In some cases, even when a pollen grain germinates and commences with pollen tube growth, it may lack the vigor necessary to reach the ovule and promote fertilization. Non-viable pollen grains cannot successfully germinate. Thus pollen with good viability is desirable for use with the methods in the present disclosure. In maize, the level of viability and fertility required for successful pollinations is typically accepted or defined in the art to be an average of four grains of fresh pollen per maize silk, or four to ten grains of preserved pollen which has been preserved according to the methods of the present disclosure.

As discussed above, many embodiments of the present invention require collection of freshly shed pollen or acquiring pollen which has previously been collected. Pollen collection for the purposes of the invention may take place by any method and in any location where plants are grown, such as a growth chamber, a greenhouse, a glasshouse, a shade house, a hoop house, a vertical farming facility, a hydroponic facility, or any growing facility providing cultured tassels, as described below. Pollen may, optionally, be field-conditioned prior to storage or preservation, such as according to the methods set forth in U.S. Pat. No. 10,575,517. Field conditioning, if used, may also take place using other methods known in the art, now or in the future. In some embodiments, field conditioning may occur immediately upon collection or at any point between collection and storage. In addition, it is anticipated that field conditioning may include methods of the present invention.

The collection of fresh pollen may be conducted in ways commonly known in the art. For example, pollen may be collected from freshly shedding flowers or male flower structures produced in any variety of manners. In the case of maize, and many other species, the plant is monoecious and contains separate male and female inflorescences on a single plant. In the practice of breeding, pollination, cross-pollination, and hybridization, some plants act as the male parent plant from which pollen is collected for use in pollinations, and some plants act as the female parent plant being the recipient of the pollen. In the case of self-pollinations, a single plant is acting as both the male parent and the female parent because the female flowers are fertilized by pollen from its own male flowers. Similar to self-pollinations, sib-pollinations occur when plants of the same genetics (such as a neighboring plant from the same field) act as both the male parent and the female parent. For purposes of this disclosure, the term “self-pollination” includes sib-pollinations, as the same genetics are involved in both situations. Still referring to maize as an example, pollen is collected from freshly shedding male flowers borne on tassels, which may be attached or detached from the plant.

Pollen from any type of plant may be collected from plants grown in any environment suitable for plant growth. Such environments include, but are not limited to, a field, a growth chamber, a greenhouse, a glasshouse, a shade house, a hoop house, a vertical farming facility or a hydroponic facility. Alternatively, pollen may be collected directly from anthers by crushing or grinding the anthers or by letting them naturally continue to expel pollen as the anther dries, thereby releasing the pollen and allowing for its collection. In addition, pollen may be collected from a tassel culture facility (Pareddy et al. (1989) Theor Appl Genet 77:521-526). Mature tassels removed from plants grown in any type of controlled environment facility or the field are maintained under controlled conditions that promote pollen shed. Also, pollen can be collected from cultured tassels derived from tissue harvested from immature flower structures and developed into tassels that produce viable pollen.

Alternatively, anthers may be collected directly, instead of pollen. Anthers may be collected prior to the pollen maturing in the anther, during the maturation time, or when the pollen is mature and being released. Whole anthers may also be stored for later use by storing them in the compositions of the invention. Pollen that is either immature, partially mature, close to mature but not yet being released, or mature dehisced pollen, may be released from collected anthers by stripping, grinding, shaking, drying, or other similar methods. Grinding is particularly effective for immature pollen because the grinding of the entire anther along with the immature pollen can be conducted in the composition of the invention. This process will automatically release the pollen into the ideal environment where the surfaces of each pollen grain are limited in their ability to touch other grains. Moreover, anthers may be stored employing methods of the present invention, including but not limited to, with solid, liquid, gas, and/or physical separation of pollen grains to separate dead pollen contents from live pollen grains.

This disclosure outlines both the methods of minimizing surface-to-surface contact between pollen grains, and the diluents that are used to minimize pollen surface-to-surface contact to improve viability and fertility. The diluent may be refreshed or replaced over time, including with the same or a new diluent. Accordingly, this may include switching pollen from one diluent to another during storage.

Embodiments of the invention utilize solid particles to separate pollen grains from dead pollen contents and/or each other, thus preventing interaction between dead pollen contents and live pollen grains. In many embodiments, the solid particles minimize surface-to-surface contact between grains of pollen. In the case of solid particle substances, preferably the size of the particles should be determined in relation to the size of the pollen grains intended to be stored. If the solid particles are too large, they can sometimes be ineffective at preventing contact between pollen grains because the pollen can collect in pockets between the particles. If the solid particles are too small, they can also be ineffective at separating the pollen grains and dead pollen contents because they do not provide enough separation. Different species of plant pollen can vary in size, ranging from approximately 5 to more than 125 μm in diameter. In addition, some pollen grains have surfaces that are not perfectly round which result in a larger surface area. Accordingly, using solid particle compositions that contain a range of particle sizes and/or shapes will allow use with pollen from different species. Alternatively, routine experimentation will easily determine the ideal size of solid particles based upon the size and morphology of the pollen grains to be stored.

A further consideration is the ratio of solid particulate diluent to pollen grains. High ratios of diluent to pollen tend to increase separation between dead pollen contents and live pollen grains. Moreover, higher ratios result in more dilution of the pollen, which is sometimes beneficial for application of the pollen to the female plants. In some examples of embodiments, the ratio may be anywhere from 1:1 to 1:100 of pollen grains to diluent. In some examples, a ratio of 1:4 pollen grains to diluent is used. Moreover, the ideal ratio can depend on the amount of time for which the pollen will be stored. Over the short term, the ratio has been shown to be less important. On the other hand, as time progresses, the ratio becomes more important in maintaining viability of preserved pollen. The ratio can be optimized for benefits including, but not limited to, enhancing storage duration, diluting pollen, and/or optimizing flowability.

Preferably, the nature of any solid particle composition used to separate dead pollen contents from live pollen grains includes particles that do not cause negative interactions with the pollen. In some embodiments, the solid particles are of a non-reactive nature so that the pollen is essentially unaffected by the particles. For example, hydrophilic or hygroscopic solid particles must not remove too much moisture from the pollen; the loss of moisture impacts the viability of pollen, with greater moisture loss resulting in greater viability loss, and eventually the death of the pollen grain. A solid particle with a slight hydrophilic or hygroscopic nature, however, can be beneficial because it can separate dead pollen contents from live pollen grains. More specifically, and without being bound to a particular theory, experimentation has shown that substances that are slightly hydrophilic absorb dead pollen contents that escape or leak from dead and/or dying pollen. Because the hydrophilic substances absorb this material, it is separated from live pollen grains. Moreover, slightly hydrophilic materials help regulate pollen moisture content, which can oftentimes be important to pollen viability. Slightly hydrophilic substances often do not desiccate pollen in the same way that more hydrophilic substances may. Hydrophobic particles may be less desirable because they may have a natural tendency to avoid physical contact with the pollen grains which could result in a gradient between the pollen and the particle. Nonetheless, some hydrophobic substances can be beneficial. Namely, some hydrophobic substances may attach to the surface of pollen grains and at least partially coat the grains. Such coating can result in separation of dead pollen contents from live pollen grains and/or separation between living pollen grains.

Starch is hygroscopic. It can absorb moisture up to 10-17% of its weight when equilibrated at normal atmospheric conditions (Leach, 1965, Starch: Chemistry and Technology. Vol. 1. New York: Academic Press; p. 20.). It is commonly used as an absorbent in drug formulations to keep powders dry and ensure the stability of drugs that are liable to deteriorate by hydrolysis and other chemical reactions. Starch is one of the most widely used pharmaceutical excipients because it is nontoxic, odorless, inexpensive, widely available, and biocompatible. It is widely used as a binder in the wet granulation process to ensure uniform dosages in tablets. Starch also is a convenient and effective disintegrant of tablets due to its natural swelling properties in the presence of water (Olobayo, 2020, Chemical Properties of Starch. IntechOpen. doi: 10.5772/intechopen.89811).

When a starch grain is exposed to water, the water molecules pass through the surface of the starch granules into the interior. As water molecules continue to penetrate, they eventually reach the amorphous region of the starch granules and hydrate the amylose and amylopectin chains. This hydration causes starch granules to expand (Zhang et al., 2024, Trends in Food Science & Technology 144: 104321). The capacity of native starch to absorb water depends on the physico-chemical properties of the starch grain and its components. These properties include the molecular composition of the starch, granule surface structure, long-range crystalline structures, short-range ordered structures, and hydrophilic groups. In particular, the amylopectin (branched) chain length and amylose content are positively correlated with starch water absorption capacity. Likewise, the presence of large numbers of concavities, cracks, and holes on the starch surface accelerates water penetration into the starch granules, resulting in a higher water absorption rate.

While not wishing to be bound to any one theory, it is postulated that the benefits of including starch in a diluent in the proper proportion may serve to stabilize the moisture content of the storage environment that contains recalcitrant pollen grains at 50% to 60% moisture. Too little starch allows the pollen to retain moisture and continue to respire rapidly; too much starch serves to accelerate pollen drying and rapid loss of viability.

Accordingly, pollen moisture content and regulation of same is beneficial in certain embodiments of the invention. In some examples of embodiments, the method may include pollen moisture content at 15-60%. More specifically, pollen moisture content may be 40-58%. In even further embodiments, pollen moisture content may be 45-55%. In some embodiments of the invention, it has been found that the pollen can be kept at higher moisture levels than has previously been considered optimal in the art. Accordingly, in some embodiments, it can be beneficial if substance used in the invention can enable maintenance of higher pollen moisture content without detriment.

Solid particles that can be mixed with starch to form diluents useful for the disclosed invention include inorganic or organic particles, including, but not limited to the following: dormancy regulators, osmotic regulators, cellular respiration inhibitors and disruptors, electron transport inhibitors, uncouplers of membrane electron transport, membrane stabilizers and conditioners, cell wall stabilizers and conditioners, growth regulators, plant hormones, cryoprotectants, monosaccharides, disaccharides, polysaccharides, anti-flocculants, dispersal agents, bentonite, cellulose, sand, desiccants, flow agents, dry sugars, and activated charcoal, or any combinations or mixtures thereof. It is to be understood that the selection of the solid particles to be combined with the starch may be influenced by the type of pollen to be stored. For example, the selection may be based on size and/or how tolerant specific pollen is to certain interactions and characteristics. An ideal composition may include a single solid particle type or a combination of particle types that are chosen to maximize pollen viability and longevity. It may also contain a range of particle sizes to ensure that both larger and smaller grains of pollen are being separated with equal efficiency.

In addition, it may be beneficial to add a substance that will increase flowability of the stored pollen such that the pollen can be easily applied to plants at a later date. Such a compound must not cause negative interactions between other solid particles in the storage medium, nor between the pollen grains. Examples of compounds that separate dead pollen contents from live pollen grains and also aid in flowability include, but are not limited to, various forms of silica, clays, talc, zeolite, magnesium chloride, and stearic acid.

Pollen can be subjected to additional preservation steps to increase its ability to retain viability and fertility over storage periods. The preparation of pollen for storage requires a careful process including an environment with known relative humidity levels, as well as specific temperature and atmospheric pressure conditions. For example, depending upon the preparation methods used, the temperature may be −80° C. to about 25° C. In some embodiments, relative humidity may be 50-100%. Moreover, air pressure may be 15 kPa-150 kPa. In addition, the addition or removal of certain gases will assist in maximizing storage success. Such gases may include but are not limited to nitrogen, carbon dioxide, nitrous oxide, oxygen, or a mixture of these gases.

Optionally, during the period of pollen storage, the pollen and starch diluent mixture contained within the storage vessel may be agitated mechanically. Agitation may be occasional or continuous. In some embodiments, agitation is continuous. The agitation serves to expose the maximum surface area of the pollen to the diluent, thereby ensuring a homogenous mix of the pollen with the diluent and preventing surface-to-surface contact of pollen grains, clumping of the pollen, and the spread of pollen necrosis. The continued, regular, and/or occasional agitation of pollen grains in the composition, including dead or dying pollen grains, may be achieved in a number of ways, including the use of vibration, forced air, rotation, mixing, or other means.

The temperature of the storage vessel or the environment in which the storage vessel containing the pollen composition is stored may be an ambient temperature. Alternatively, the pollen composition may be refrigerated or frozen, or maintained in cryogenic storage. Pollen that is frozen or stored cryogenically may need to be appropriately prepared for such storage. The preparation may depend on the species of pollen to be stored. Many pollen species must be dried to a specific pollen moisture content before they can be successfully stored at temperatures below freezing. For example, the pollen composition of the invention may therefore be preserved and stabilized using techniques such as those disclosed in U.S. Patent Application Publication No. 2024/0237637. Alternatively, the pollen may be frozen or cryogenically stored and mixed with the diluent after being removed from storage and acclimated. Accordingly, the compositions of the invention may be maintained at temperatures ranging from about −196° C. to about 30° C. Temperatures at which the pollen in the diluent can be stored include −196° C., −150° C., −120° C., −80° C., −70° C., −60° C., −50° C., −40° C., −30° C., −20° C., −10° C., 0° C. 1° C. to about 10° C., including about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C. about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., and about 25° C. In some cases, the pollen in the diluent can be stored at higher temperatures, including temperatures ranging from about 10° C. to about 25° C. While lower temperatures are often preferable, this invention also has the ability to maintain and/or increase pollen viability at temperatures higher than those that have previously been disclosed in the art.

In examples of embodiments, pollen subjected to the disclosed method may be used in any application where pollen is a commercial or experimental unit. In one example, the preserved pollen may be used to produce seed (whether hybrid, parent, or otherwise) in any setting, including but not limited to a laboratory, greenhouse, and field. In another example, the pollen may be used to produce grain (whether hybrid or otherwise) in any setting, including but not limited to a laboratory, greenhouse, and field. The preservation techniques disclosed in this invention are intended to successfully preserve pollen such that the preserved pollen maintains its viability to the extent that a sufficient number of viable pollen grains are delivered to stigmas to ensure successful fertilization. For example, in corn, about 4 to about 10 grains of pollen per stigma (silk) are sufficient for successful fertilization.

Although various representative embodiments of this invention have been described above with a certain degree of particularity, those skilled in the art could make numerous alterations to the disclosed embodiments without departing from the spirit or scope of the inventive subject matter set forth in the specification and claims. In some instances, in methodologies directly or indirectly set forth herein, various steps and operations are described in one possible order of operation, but those skilled in the art will recognize that steps and operations may be rearranged, replaced, or eliminated without necessarily departing from the spirit and scope of the present invention. It is intended that all matter contained in the above description or shown in the accompanying drawings shall be interpreted as illustrative only and not limiting. Changes in detail or structure may be made without departing from the spirit of the invention as defined in the appended claims.

Although the present invention has been described with reference to the embodiments outlined above, various alternatives, modifications, variations, improvements and/or substantial equivalents, whether known or that are or may be presently foreseen, may become apparent to those having at least ordinary skill in the art. Listing the steps of a method in a certain order does not constitute any limitation on the order of the steps of the method. Accordingly, the embodiments of the invention set forth above are intended to be illustrative, not limiting. Persons skilled in the art will recognize that changes may be made in form and detail without departing from the spirit and scope of the invention. Therefore, the invention is intended to embrace all known or earlier developed alternatives, modifications, variations, improvements, and/or substantial equivalents.

The following examples illustrate the present invention in more detail and are illustrative of how the invention described herein could be implemented in pollen of various species.

Example 1: Seed set resulting from pollinations using maize pollen stored in diluent mixtures of lactose and corn starch.

This study was conducted in July 2023 on field-grown plants in Ankeny, IA to evaluate the longevity of maize pollen stored in various diluent mixtures of lactose and starch for three days at 5° C. in sealed containers. Fresh pollen collected from Alexho hybrid was mixed with the diluents in a 1:4 ratio of pollen: diluent by weight. Pollen-diluent mixtures were applied by hand (scoop and tube method) to female plants in amounts that limited kernel set. Pure pollen was applied to control plants at 4 times the rate to assess potential kernel set of similarly aged plants (3 reps of 8 plants each per treatment). The resulting kernel number per ear was evaluated at least 14 days after pollination. Diluents tested include: 100% lactose, 100% corn starch, 97% lactose blended with 3% corn starch, 95% lactose blended with 5% corn starch, 90% lactose blended with 10% corn starch, and 80% lactose blended with 20% corn starch by weight.

FIG. 1 shows that when fresh pollen is stored for three days in 100% lactose diluent, pollinations conducted using that stored pollen result in seed set significantly above the negative (unpollinated) controls, as expected from numerous previous studies. Substituting a proportion of this diluent with corn starch had a variable impact on seed set, depending on the concentration of starch in the diluent. Diluents containing 10 to 20% starch increased seed set 3 to 4-fold over the 100% lactose diluent. Lower concentrations of starch were far less effective, and 100% corn starch completely failed to maintain pollen viability. The dramatic improvement in viability (as measured by the ability to affect fertilization) when pollen was stored in a diluent containing 10 to 20% corn starch was a completely novel and unexpected discovery, given the limited benefit of the pure diluent components on maintaining pollen viability in this experiment.

Example 2: Comparison of diluent mixtures containing corn, potato, or cassava starch.

This study was conducted in February 2024 in Ames, IA to evaluate the longevity of maize pollen stored in various diluents containing starch obtained from various plant sources. Fresh pollen collected from inbred PHM81 growing in a controlled environment was mixed with lactose and starch blended diluents in a 1:4 pollen: diluent ratio by weight. The mixture was then stored for three days at 5° C. in sealed containers. Diluents evaluated included: 97% lactose blended with 3% corn starch, 100% potato starch, 80% lactose blended with 20% potato starch, 100% cassava starch, and 80% lactose blended with 20% cassava starch by weight. Maintenance of pollen viability in storage was determined by in vitro germination. Pollen grains were counted as germinated if an intact pollen tube emerged and was longer than the diameter of the pollen grain after one hour incubation at 20° C. in germination media.

FIG. 2 shows the percent germination of maize pollen observed after storage for three days in lactose diluent containing starch from maize, cassava, or potato. As observed in Example 1, storing pollen in 97% lactose blended with 3% corn starch maintained pollen viability, as measured by emergence of a pollen tube in vitro. Storing pollen in potato starch or cassava starch at 20% of the lactose diluent mixture also maintained pollen viability, albeit to a greater extent than the diluent containing 3% corn starch. Similar to the results presented in Example 1, these data indicate the addition of plant starch to a lactose diluent at 20% by weight (w/w) had a dramatic positive impact on maintaining the viability of maize pollen in storage, regardless of the source of starch.

This result is highly unexpected because these seed-and tuber-sourced starches vary dramatically in starch granule size, granule crystalline and surface morphology, amylopectin-amylose content, and wetting characteristics (Remya, R, & Jyothi A N (2015) Journal of Root Crops 41:37-47, Tarique J, et al. (2022) Polymers (Base1) <doi: 10.3390/polym14030388>, Moorthy N (2002) Starch 54:559-592). These results also are quite surprising since cereal and tuber starches vary dramatically in phosphorous content, which directly impacts starch granule microstructure, hydration capacity, and their surface physico-chemical properties (Carciofi M, et al (2011) Journal of Cereal Science 54:339-346).

Example 3: Viability of maize pollen stored in diluent mixtures containing 20, 50% or 100% more starch.

This study was conducted in February 2024 in Ames, IA to evaluate whether concentrations of starch greater than 20% by weight in the diluent mixture could be beneficial for extending pollen viability in storage. As in Example 2, pollen viability was assessed by quantifying percent germination in liquid media. Fresh pollen collected from inbred PHM81 growing in a controlled environment was mixed with lactose and starch diluents in a 1:4 pollen:diluent ratio by weight and stored for three days at 5° C. in sealed containers. Starches were sourced from maize kernels, cassava tubers, potato tubers, and arrowroot stolons. Each source of starch was evaluated in lactose diluent mixtures at 0/100, 50/50 and 80/20 (lactose/starch) ratios by weight.