An Overview of Pollen Cryopreservation
Gayle M. Volk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521. Gayle.Volk@usda.gov
Ashley N. Shepherd, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Katheryn Y. Chen, Department of Soil and Crop Sciences, Colorado State University, 307 University Ave., Fort Collins, Colorado 80523.
The purpose of this chapter is to provide an overview of the processes the National Plant Germplasm System has adopted to collect and prepare pollen for long-term preservation, as well as to evaluate pollen viability after recovery from cryogenic storage. This chapter is designed to be complementary to subsequent chapters, which focus on the unique qualities of individual crops and the specific methodologies for collecting and conserving their pollen.
Outline
- Introduction to pollen conservation
- Conserving pollen in the NPGS
- Pollen collection and initial processing
- Moisture equilibration and cryopreservation
- Preparing for and conducting viability assays
- References
- Acknowledgments
1. Introduction to pollen conservation
Plant genebanks across the world strive to conserve genetic diversity from cultivars, landraces, and wild relatives for research, education and plant breeding. With preservation of diversity as a core genebank function, it follows that pollen—a form of germplasm that captures diverse alleles in relatively small samples—serves well for long-term preservation in genebanks. These resources are particularly valuable for supplementing collections that are complicated or infeasible to manage as seeds or clonally propagated materials.
In cases where flexibility in plant breeding is a high priority, pollen is considered a practical means of conserving whole chromosomes and their allelic content. Because stored pollen is immediately useable upon rewarming, plant breeders can make crosses immediately, which might not be possible with other forms of germplasm. By maintaining cryopreserved pollen, breeding programs can be less subject to the variabilities of pollen production. Pollen can be kept from late-blooming plants and pollinate early-blooming germplasm the subsequent year. Distributing cryopreserved pollen could further alleviate some resource bottlenecks, especially in dioecious trees where breeders need access to pollen from plants that are not co-located with the female trees. It might even be possible to transport pollen across international borders because shipping pollen is also less likely to promote disease transfer as compared to shipping other forms of germplasm (Hoekstra, 1995).
2. Conserving pollen in the NPGS
The USDA-ARS National Plant Germplasm System (NPGS) safeguards our nation’s important crop genetic resources. Each crop is maintained at an NPGS genebank unit, typically in cooperation with a land grant university, located in a region suited to its maintenance. For crops that are vegetatively propagated, such as many fruits, nuts, and some vegetables and ornamentals, the primary collections are maintained as actively growing plants in the field, in greenhouses/screenhouses or in vitro. Collections maintained in the field or greenhouse/screenhouse are available for genotypic characterization and phenotypic evaluation, and they are readily accessible for plant breeding, research, and education.
Actively growing plants in NPGS collections are particularly susceptible to abiotic and biotic threats. To secure these resources, collections are ideally maintained in multiple locations and in different forms. The USDA-ARS National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado plays an essential role within the NPGS. NLGRP strives to completely back up NPGS collections in quiescent (slow-growing) forms that can be regenerated as needed. Most of the plant germplasm at NLGRP is maintained as seeds in cold storage or in the vapor phase of liquid nitrogen. Clonally propagated crops are largely represented by cryopreserved dormant buds or tiny shoot tips.
Recently, NLGRP scientists and curators of the active NPGS collections have cooperated to develop, improve, and apply standardized pollen preservation procedures to back up several crop germplasm collections as pollen in liquid nitrogen conditions. This eBook describes the pollen preservation methods that have been successfully implemented at NLGRP, as measured by in vitro pollen germination assays (Connor and Towill, 1993). Although these techniques can guide those starting or expanding their pollen preservation efforts, it is important to note that programs with differing scopes, resources, or conservation goals might require some modifications to accommodate their specific needs.
3. Pollen collection and initial processing
In the NPGS, pollen cryopreservation has been performed for crops in which seed, dormant bud, and shoot tip cryopreservation methods are either unavailable or technical support is not sufficient to cryopreserve these crops via alternative propagule types. Recognizing that cryopreserved pollen can’t be directly regenerated into plants that are identical to the source material, pollen preservation is viewed as a method to preserve the alleles as assembled into chromosomes of the accession and to make them available for crossing with plants that have maternal flowers.
Pollen processing methods must be identified and optimized for each crop and environmental growth condition. Pollen cryopreservation is currently performed for NPGS crops with easily-harvested and processed pollen that is desiccation tolerant. At this time, this includes mostly nut crops, such as pecan, walnut, and pistachio as well as some fruit crops such as date palm and Prunus. Candidate crops for pollen preservation are selected based on availability of materials, ease of processing, and available technical support. It is truly a collaborative effort, with site personnel collecting the male floral organs and then sending the dehisced pollen to NLGRP in Fort Collins, Colorado for moisture adjustment, viability assessment, and liquid nitrogen storage. Pollen processing must occur quickly; consequently, the harvest, shipment, and cryopreservation procedures must be carefully synchronized.
When a new crop is selected for pollen cryopreservation, the focus for the first season is typically identifying the correct stage of flower/catkin development, conditions for successful pollen collection from the flowers/catkins, and identifying the conditions for successful cryopreservation and viability assessment. It is generally helpful to discuss pollen collection methods with breeders of the selected crop because they likely collect and apply pollen. The subsequent chapters in this eBook provide examples of how pollen is harvested and then cryopreserved from a wide range of crops in the NPGS.
The optimal stage of flowering for pollen harvest might be short, so careful timing is essential for success. All staff, equipment, and facilities needed for the process should be on standby prior to flowering. Flowers, catkins, or other reproductive structures should be monitored daily as they near maturity. In many cases, precautions might be needed to prevent the loss of pollen. This could include tying closed spathes, as is the case in date palm. Collections of plant genetic resources often have a wide range of genetic diversity resulting in a span of flowering times across the collection. Therefore, the season when flowering occurs could extend for up to a month as different accessions in the collection produce flowers. Biennial bearing in some crops might limit the number of trees that are flowering in a given year. In addition, environmental conditions can affect the availability of flowers in a season. For example, a particularly rainy period during flower production might not produce high quality and quantities of pollen that can be cryopreserved. Weather events can also compress the flowering season so that many trees all produce flowers at the same time.
Whole flowers or catkins of a single tree will typically be collected just prior to anthesis (when the flower opens and pollen is released). In some cases, the same tree might need to be sampled multiple times to collect the desired amount. For some crops, air drying the harvested male floral organs might be sufficient to release pollen. For other species, the flower may need to be crushed and/or sifted to release the anthers and the pollen. Depending on the crop, pollen is cryopreserved either with or without anthers.
When handling material from multiple accessions, it essential to prevent cross contamination. Flowers must be kept in well-labeled bags/containers. Tables and racks used for air drying should also be labeled and kept separate, sometimes in different rooms. Any instruments or workspaces that come into contact with pollen should be cleaned with ethanol or isopropyl alcohol after use.
Longevity of pollen after collection varies based on species, genotype, and environmental factors. Because pollen viability might drop rapidly after harvest, all steps should be completed as quickly as possible. Pollen must be shipped the cryopreservation site if it is distant from the collection site, adding another time delay. When shipping, it is important to sift insects from the pollen (and anthers) to limit spread of pests, seal containers tightly to prevent the loss or mixing of pollen, label material clearly, and ship the package with overnight delivery.
4. Moisture equilibration and cryopreservation
Upon receipt at the cryoprocessing facility (such as NLGRP), all steps including weighing, moisture equilibration, packaging, and cryopreserving should be conducted as quickly as possible to ensure the longevity of the pollen.
Determining moisture content
Moisture content is a key factor for the success or failure of cryopreservation. To better understand viability of cryopreserved material and to help optimize protocols in the future, it can be extremely useful to determine the exact moisture content of the pollen. At NLGRP, moisture content is established for every sample of pollen both before and after the moisture equilibration step. This requires that a small amount of material (about 15 mg) to be sacrificed for this purpose.
To determine the moisture contents, a supply of small weigh boats is needed. At the NLGRP, these are crafted by wrapping a small triangle of aluminum foil around the end of an unsharpened pencil. The method for creating these weigh boats is demonstrated in the video below.
The tare weight is recorded on a precision balance. A small aliquot (<5 mg) of pollen is placed into the weigh boat, which is gently closed with forceps. The weight is recorded and the tare weight is subtracted to determine the fresh weight (FW) of the sample. Three replicates per accession are weighed in this manner to ensure moisture calculations are as accurate as possible.
After measuring the fresh weight, sealed weigh boats are placed into a labeled tray, which is put into an oven set at 90 °C. After 2-4 days, the tray is removed from the oven and the packet is reweighed. The tare weight is once again subtracted to establish the dry weight (DW) of the sample.
Moisture contents are calculated either on a fresh weight basis or dry weight basis with the formulae below. These may also be expressed as percentages.
Moisture content (fresh weight basis):
((FW – DW) ÷ FW)
Moisture content (dry weight basis):
((FW – DW) ÷ DW)
Moisture equilibration
Pollen preservation and regeneration methods rely on controlling moisture content. Low humidity is needed to desiccate pollen in preparation for cryopreservation, whereas high humidity is needed to rehydrate material after cryostorage. Exact moisture is generally achieved by allowing the pollen to reach equilibrium in a controlled humidity environment.
Many of the moisture equilibration procedures described in this eBook incorporate saturated salt solutions at room temperature to achieve the desired humidity; this approach is common practice in some seed research programs. Identifying the appropriate saturated salt solution (and resulting relative humidity) and duration of exposure are the result of protocol optimization.
The saturated salt solution is made by adding salts (such as calcium nitrate, which achieves a relative humidity of 46% at 22 °C) to a large Petri dish (140 x 20 mm), with enough water to form a slurry. The dish should be filled within a few millimeters of the top for easier handling. The Petri dish containing the saturated salt solution is left uncovered at the bottom of the desiccation chamber and monitored daily to ensure that undissolved salts are present in the slurry. Excess water might need to be decanted. Techniques for preparing salt solutions are shown in the video below.
Pollen should be poured into an appropriately sized clean, dry Petri dish, ensuring the pollen is evenly distributed with a depth of no more than a few millimeters. Uncovered Petri dishes containing pollen are carefully placed into the prepared desiccation chamber that contains the saturated salt solution to control the relative humidity of the chamber, which is then sealed shut and left at room temperature overnight. After approximately 18 hours, the pollen moisture content should be adjusted. To determine the extent to which moisture has been adequately adjusted, moisture content should be determined and recorded according to the methods described in the previous section.
Packaging and cryopreservation
After moisture equilibration, pollen is placed into cryovials. Only one Petri dish containing pollen should be removed from the desiccation chamber and the pollen from that dish is quickly placed into vials and sealed to limit uncontrolled moisture changes. The ideal vial size should be determined by the smallest volume needed to achieve breeding goals because one vial will be thawed at a time for making crosses (often ranging from 0.5 mL to 4 mL volume). Cryovials are labeled according to data management protocols at the cryostorage facility. These must be on labels rated for liquid nitrogen storage. Cryovials are either placed on aluminum canes and stored in cryocanisters, or are organized in boxes designed for cryovial storage.
After appropriate labeling and packaging, pollen is ready for cryopreservation. Materials can be brought to the liquid nitrogen tank where they will be stored. Containers with cryovials of pollen can be placed directly into the vapor phase of liquid nitrogen (approximately -140 °C). No intermediate cooling steps are needed for protocols included in this eBook. Pollen is always stored in the vapor phase (above the liquid line); if placed in liquid, buoyancy, loss of vials, loss of pollen, or cross contamination could occur.
5. Preparing for and conducting viability assays
Viability assays are essential for determining if the pollen was successfully cryopreserved. When possible, an aliquot for each accession is warmed, rehydrated, and germinated at three different stages: immediately after arriving at the cryo facility, after the moisture equilibration step, and most crucially after cryoexposure. These tests can be repeated as often as is appropriate to ensure continued viability over years of storage. Viability tests can be conducted within a day of cryoexposure, particularly when methods are being identified. For routine processing, it might be more practical to perform the post-cryopreservation viability assays after the pollen cryopreservation has been concluded for the season if staff availability is limited.
Warming
Material for viability assays must be warmed, but any material remaining in storage must stay cold. Accidental warming and re-cooling could negatively impact viability. To warm pollen, an entire vial can be sacrificed; this is removed from liquid nitrogen vapor and placed directly in room temperature to warm. Alternatively, a technician with the appropriate personal protective equipment can hold the vial in the nitrogen vapor, then quickly open it and pour out a small amount of the pollen into a Petri dish. The vial must be closed immediately and replaced into its designated storage location while the Petri dish is left to warm at room temperature. If the latter method results in accidental rewarming of the vial, the vial should not be replaced into storage.
Rehydration
Once at room temperature, pollen must be rehydrated. To achieve this, pollen should be placed in an open Petri dish and placed in a high humidity environment. At the NLGRP, the high humidity environment is created in one of two ways: a saturated salt solution with a high average relative humidity can be placed into a desiccator, or water-saturated paper towels can be placed into a small, enclosed container. The rehydration process will be species dependent and might require optimization as the method is being developed.
Germination
At the NLGRP, in vitro germination serves to determine pollen viability. Marquard medium is typically used, although alternate media or variations to the Marquard recipe might be needed depending on the species (Marquard, 1992). Media preparation should occur in advance of germination tests. Information on media preparation can be found in “Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules”. Media is dispensed in 10 x 35 mm plastic Petri dishes.
Although the medium must be sterilized to prevent microbial cultures during storage, the pollen itself is not sterile. When pollen is ready to be tested, the germination medium no longer requires aseptic conditions. Pollen should be applied, left to germinate for a designated amount of time, then quickly counted. After viability is recorded, the plates are discarded.
Rehydrated pollen is applied to the media with a small paint brush, or by other means of lightly dispensing the pollen grains across the surface. Pollen must not be applied too heavily or in clumps, otherwise it will be impossible to differentiate individual grains or pollen tubes. It is ideal to apply pollen to several plates as multiple replicates will improve accurate reporting: 3 replicates are typically prepared for each pollen viability test at the NLGRP. Pollen should then be left to germinate overnight in specific conditions, typically in the dark, at room temperature.
Viability assessment
After the designated time, usually overnight, Petri plates should be examined under a microscope. At NLGRP this is typically a compound microscope at 100x magnification. All grains in the field of vision are methodically counted in a left-to-right, top-to-bottom fashion until 100 have been counted. Tally counters contribute to accurate counting. Pollen tubes longer than the diameter of the pollen grain are considered germinated.
6. References
Connor KF, Towill LE. 1993. Pollen-handling protocol and hydration/dehydration characteristics of pollen for application to long-term storage. Euphytica 68:77-84. DOI: 10.1007/BF00024157
Hoekstra FA. 1995. Collecting pollen for genetic resources conservation. In: Guarino L, Ramanatha Rao V, Reid R (Eds.) Collecting Plant Genetic Diversity: Technical Guidelines. CAB International, Wallingford, UK. pp. 527-550. Available from cropgenebank.sgrp.cgiar.org
Marquard RD. 1992. Pollen Tube Growth in Carya and Temporal Influence of Pollen Deposition on Fertilization Success in Pecan. Journal of American Society of Horticulture Science 117:328-331. DOI: 10.21273/JASHS.117.2.328
7. Acknowledgments
Chapter citation: Volk GM, Shepherd AN, Chen KY. 2025. An overview of pollen cryopreservation. In: Volk GM, Chen KY (Eds.) Pollen Preservation. Date accessed. Available from: https://colostate.pressbooks.pub/pollenpreservation/chapter/an-overview-of-pollen-cryopreservation/
This training module was made possible by:
Content providers: Gayle M. Volk, Ashley N. Shepherd, Katheryn Y. Chen
Videography by: Gayle M. Volk, Ashley N. Shepherd, Remi Bonnart, Katheryn Y. Chen, Emma Balunek
Funding by the USDA-ARS and by the USDA-NIFA Higher Education Challenge Program grant 2020-70003-30930. USDA is an equal opportunity provider, employer, and lender. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
