Walnut 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
Jenny Smith, USDA-ARS National Clonal Germplasm Repository for Tree Fruit, Nut Crops, and Grapes, One Shields Ave, University of California, Davis, California 95616.
Ashley N. Shepherd, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.
Outline
- Walnut in the NPGS
- Collecting catkins
- Collecting pollen from catkins
- Moisture equilibration, packaging, and cryopreservation
- Retrieval, rehydration, and germination
- References
- Additional information
- Acknowledgments
1. Walnut in the National Plant Germplasm System (NPGS)
Juglans regia is the most commonly grown walnut species for nut production, and there are about 20 additional species of Juglans that are native to eastern Asia, southeastern Europe, and North America. The NPGS walnut collection maintained at the USDA National Clonal Repository for Tree Fruit, Nut Crops and Grapes in Davis, California has over 400 accessions representing 10 taxa that are maintained as trees in the field (Smith et al., 2021). These trees serve a wide range of research and breeding programs. At this time, the NPGS walnut collection is not backed up as seeds/nuts, dormant buds, or shoot tips at a secondary site such as the National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado; however, walnut pollen is plentiful and is being cryopreserved to help secure the NPGS walnut collection.
2. Collecting catkins
Walnut trees in the Wolfskill Experimental Orchard, where the collections of the USDA Clonal Germplasm Repository for Tree Fruit, Nut Crops, and Grapes in Davis, California are maintained, are monitored regularly for catkin development in mid-spring. When the catkins just begin to shed pollen, staff quickly collect catkins that have not completely dehisced. Harvested catkins are placed into labeled paper bags (lunch bag size, approximately 2500-3000 cubic centimeters in volume), then brought directly to the laboratory for processing. This will yield approximately 50 mL of pollen.
3. Collecting pollen from catkins
In the lab in Davis, CA, the catkins are spread out in a single layer inside labeled trays. These are allowed to dry at room temperature (~22 °C) for 24-48 hours. Released pollen becomes visible at the bottom of trays.
After the catkins have shed pollen, a clean paintbrush can help remove any extra pollen from the catkins. Catkins are placed into a coarse strainer to separate the pollen into a large container. The paintbrush can collect loose pollen clinging to the trays. After initial sifting, insects, plant tissues, or other large debris will be present. To extract just the pollen, carefully pour contents from the large container into a fine mesh sieve. Sift the pollen onto clean paper. This process of capturing pollen from walnut catkins is shown in the video below.
The sieved pollen is decanted from the paper into clean 50 mL tubes. These must be well-labeled and stored in refrigeration (ideally less than one day) until they can be packaged on ice and shipped to the National Laboratory for Generic Resources Preservation (NLGRP) in Fort Collins, Colorado.
All materials must be sanitized with alcohol between accessions to prevent any cross contamination.
4. Moisture equilibration, packaging, and cryopreservation
Once received, moisture contents are determined by weighing a small aliquot of pollen, which is then dried in an oven and reweighed after several days. The moisture content of all remaining walnut pollen is then adjusted to approximately 6-10% (fresh weigh basis). This is achieved by placing pollen into a Petri dish, which is left overnight in a sealed desiccation chamber with a calcium nitrate saturated salt solution (46% relative humidity). The following day, another small aliquot of pollen is weighed, then dried in an oven and reweighed after several days. Moisture content is calculated and recorded. More information on saturated salt solutions, moisture equilibration, and moisture calculations are described in an earlier chapter.
After moisture adjustment, pollen is placed in fifteen 2 mL cryovials, which are then loaded onto canes and stored in cryocanisters in the vapor phase of liquid nitrogen in a cryotank. This provides 15 distributions of pollen for crosses, 2 mL of pollen per distribution. The video below shows the process of moisture adjusting, packaging, and cryopreserving pollen.
5. Retrieval, rehydration, and germination
To test viability of stored material, one cryovial containing pollen must be removed from the vapor phase of liquid nitrogen, ensuring the remaining vials do not warm. The cryovial warms at room temperature (~22 °C) for 10 minutes.
After pollen comes to room temperature, the technician scoops a very small amount from the vial (<15 mg needed) and places it into a small Petri dish. To rehydrate the pollen, the small Petri dishes are placed in a high-humidity environment. At NLGRP, this is achieved by placing them on a water-saturated paper towel within a larger Petri dish with a lid, then letting them rehydrate for 2 hours at room temperature in the dark.
Walnut pollen viability is assessed through in vitro germination. Germination medium (15% sucrose + 0.01% boric acid + 0.03% calcium nitrate + 0.02% magnesium sulfate + 0.01% potassium nitrate + 2% agarose) is dispensed into 10×35 mm plastic Petri dishes in advance (Marquard, 1992). To germinate pollen, the technician uses a small, soft bristled paint brush to carefully pick up a dusting of pollen with just the tips of the bristles. A very gentle tap of the brush lightly dusts pollen across the surface of the medium. Pollen should be evenly spread. The lid is placed back onto the Petri dish, which is then placed into the dark at room temperature for about 18-24 hours.
The pollen grains on the medium are observed at 200x magnification with a compound microscope. Viability percentages are calculated by counting 100 pollen grains on each of three plates for each cryopreserved inventory. Pollen tubes longer than the diameter of the pollen grain are considered germinated. Inventories are kept in long-term preservation when the average viability of the three plates is greater than 5%.
6. References
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
Smith J, Volk GM, Preece JE. 2021. Walnut collection. In: Volk GM, Preece JE (Eds.) Field tour of the USDA National Clonal Germplasm Repository for Tree Fruit, Nut Crops, and Grapes in Davis, California. Fort Collins, Colorado: Colorado State University. Accessed January 2025. Available from: https://colostate.pressbooks.pub/davisrepositoryfieldtour/chapter/walnuts/
7. Additional information
Download the walnut pollen cryopreservation procedure used at the NLGRP.
Download the walnut pollen recovery procedure used at the NLGRP.
8. Acknowledgments
Chapter citation: Volk GM, Smith J, Shepherd AN. 2025. Walnut pollen cryopreservation. In: Volk GM, Chen KY (Eds.) Pollen Preservation. Fort Collins, Colorado: Colorado State University. Date accessed. Available from https://colostate.pressbooks.pub/pollenpreservation/chapter/walnut-pollen-cryopreservation/
This training module was made possible by:
Content providers: Gayle M. Volk, Jenny Smith, Ashley N. Shepherd
Videographers: Jenny Smith, Emma Balunek, Remi Bonnart, Katheryn Y. Chen
This project was funded 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.
