Gayle M. Volk, Department of Horticulture and Landscape Architecture (affiliate), Colorado State University, 301 University Ave., Fort Collins, Colorado 80523. Gayle.Volk@colostate.edu

Bradford D. Hall, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.

Adam Henk, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521.

John Bamberg, USDA-ARS U.S. Potato Genebank, 4312 Hwy 42, Sturgeon Bay, Wisconsin 54235.

Katheryn Chen, Department of Soil and Crop Sciences, Colorado State University, 307 University Ave., Fort Collins, Colorado 80523.

Outline

1. Introduction
2. In vitro cultures and preconditioning
3. Shoot tip excision
4. Shoot tip processing and storage
5. Shoot tip recovery, regrowth, and assessment
6. References
7. Additional information
8. Acknowledgments

The potato shoot tip cryopreservation procedure is available to download here.

The potato shoot tip recovery procedure is available to download here.

1. Introduction

In 2021, the U.S. potato sector contributed $101 billion to the economy and generated about 714,000 jobs in the American workforce (Ellis, 2023). The U.S. potato industry depends on having high quality cultivars for production as well as sources of novel germplasm that provide resistance to environmental and biological threats and desirable quality traits so breeding programs can develop new cultivars that meet producer and consumer needs.

The U.S. National Plant Germplasm System (NPGS) potato collection, also known as the U.S. Potato Genebank (USPG) in Sturgeon Bay, Wisconsin, includes 6026 accessions representing 89 taxa of cultivated potato and its Solanum crop wild relatives. These materials are acquired from domestic and international sources, as well as potato germplasm that are publicly available after their Plant Variety Protection certificates have expired. The NPGS provides access to these unique collections, and in 2023, distributed more than 7500 potato samples for breeding, research, and education projects. These cultivars are usually not available from other sources, yet provide key genetic diversity needed to improve potato quality and agricultural productivity.

While most wild potato relatives at the USPG are maintained as seeds, cultivated potato accessions (as well as some wild species) are maintained clonally using in vitro technologies to ensure the desirable genotypes are conserved (Bamberg et al., 2016). These in vitro accessions are the most vulnerable part of the USPG, and their maintenance is resource-intensive; cryopreservation offers an opportunity to preserve these critical and vulnerable collections at a low annual cost (Bamberg et al., 2016; Jenderek et al., 2023).

Early potato cryopreservation methods used two-step cooling and ultra-rapid freezing methods (Kaczmarczyk et al., 2011), which were then followed by a DMSO droplet vitrification method, which was successfully implemented at the Leibniz Institute of Plant Genetics and Crop Plant Research (Schäfer-Menuhr et al. 1996; Kaczmarczyk et al., 2011). With the advent of newer cryopreservation techniques, such as droplet vitrification and V cryo-plate methods, whereby shoot tips are cryopreserved within droplets of vitrification solutions on foil strips or on cryo-plates, methods became more uniformly successful across diverse potato germplasm, making it possible to cryopreserve a wide range of species and cultivar germplasm using a single procedure (Kim et al., 2006; Yamamoto et al., 2015; Senula and Nagel, 2020; Vollmer et al., 2022; Zhang et al., 2023).

The NLGRP in Fort Collins, Colorado serves as the secure back-up location for NPGS crop collections. In addition to storing a back-up of Solanum seed collections for crop wild relatives, shoot tip cryopreservation methods are employed to secure the in vitro collections from the USPG (Jenderek and Reed, 2017; Jenderek et al., 2023). NLGRP also maintains Plant Variety Protection (PVP) potato voucher specimens as cryopreserved shoot tips.

As of 2025, there are a total of 618 accessions of Solanum tuberosum and S. tuberosum subspecies cryopreserved at NLGRP: 475 accessions are PVP accessions (or former PVP accessions for which the certificates have expired), and 143 accessions are S. tuberosum cultivars from USPG that have been cryopreserved at NLGRP since 2021. These USPG cultivars have an average viability of 68% and an average of 115 predicted viable propagules across the stored vials. These accessions complement the 69 cryopreserved accessions representing 30 Solanum wild species reported by Jenderek et al. (2023).

The methods reported herein follow those originally published by Kim et al. (2006), and reflect practices currently used at the NLGRP to cryopreserve meristematic tissues of Solanum tuberosum. This ongoing effort will continue to preserve the remaining USPG potato cultivars that are maintained in vitro, helping to protect these unique and valuable genetic resources.

2. In vitro cultures and preconditioning

In vitro potato cultures at the USPG (Bamberg et al., 2016) are maintained in a disease-free state that makes routine potato shoot tip cryopreservation possible. Healthy in vitro cultures are sent to the NLGRP, where they are grown at 25 °C, 18 hour daylength. Potato cultures are maintained on Potato Propagation Medium—Murashige & Skoog Basal Medium (MS) buffered with MES to pH 5.7, 3% sucrose, 0.7% agar—and subcultured as needed.

To prepare for cryopreservation, approximately 200 nodal or apical sections are cut from vigorous 4- to 5-week-old cultures, using aseptic tissue culture techniques. These are plated onto Potato Propagation Medium prepared in sterile 100×25 mm petri dishes, 25-30 nodal sections per plate. Petri plates are then wrapped with PVC sealing film, and grown at 25 °C, 18 hour daylength , for six to eight days prior to shoot tip excision.

3. Shoot tip excision

Apical shoot tips are excised from nodal sections using forceps and a scalpel or thin razor blade. Shoot tips (including the apical meristem, 1-2 leaf primordia, and a small supportive base) may range in size from 0.5 to 3 mm, with a target of approximately 1 to 1.5 mm. Note that this and all other steps where plant tissues are handled must be performed using standard aseptic technique.

Shoot tips are immediately placed into Pre-loading Solution 1 (0.3 M sucrose in buffered MS, pH 5.7) as they are cut. Once 180 or more shoot tips have been excised and placed into Petri dishes containing Pre-loading Solution 1, the plates are sealed and incubated overnight at ambient room temperature (~24 °C) on a platform shaker (60-100 rotations per minute).

After about 18 hours, Pre-loading Solution 1 is removed and replaced with the higher-concentrated Pre-loading Solution 2 (0.7 M sucrose in buffered MS, pH 5.7) and returned to the shaker for 6-8 hours.

4. Shoot tip processing and storage

Shoot tips are then transferred to room temperature PVS2 (Sakai et al., 1990) for 20 to 30 minutes, at which time they are moved over to foil strips. 18 foil strips should be cut, sterilized, and folded (if desired) in advance. A small pool of PVS2 can be added to each strip using a sterile glass capillary pipette. The same pipette can be used to transfer individual shoot tips until there are 10 on each foil strip.

Just before shoot tips are ready to freeze (20-30 minutes after first PVS2 exposure), the pipette is used to remove excess liquid and ensure shoot tips are distributed evenly across the foil. Foils containing shoot tips are then plunged into liquid nitrogen (LN) and loaded into 1.2 mL cryovials (Sigma-Aldrich, St. Louis, MO, USA) that were previously labeled and chilled in a shallow dewar with LN. Excess LN can then be poured out of vials, and the vials capped.

Vials must remain in LN until they can be transferred onto chilled cryocanes. Four vials are placed onto each of four cryocanes, which are then covered with labeled and chilled sleeves. The remaining two vials are placed onto an additional cane, which will be used for viability testing. All canes are stored in a temporary holding tank in LN or the vapor phase of liquid nitrogen (LNV).

The cane with two vials is used for an initial viability assessment. Once the viability assays are complete, the remaining four cryocanes are transferred to the NLGRP vault. Two canes are placed in a cryotank in LN and two canes are placed into a cryotank in LNV. Duplicated storage locations protect against loss in the rare case that there is catastrophic failure in one tank. All data are recorded in GRIN-Global.

5. Shoot tip recovery, regrowth, and assessment

Foil strips containing cryopreserved shoot tips are removed from cryovials and immediately plunged into 10-15 mL of preheated (38-40 °C) Unloading Solution (0.8 M sucrose in MS, pH 5.7) in a 60 mm Petri dish (Kim et al., 2006). Cryovials should have remained completely frozen in LN or LNV until this point. Shoot tips should incubate in Unloading Solution at ambient room temperature for 30 minutes. Intermittent swirling helps to dilute the PVS2.

Thawed shoot tips are transferred to Kim Potato Recovery Medium (Murashige and Skoog (Phytotech Labs. M519) pH 5.7, 2.5% sucrose, 0.6% agar, and supplemented with 0.05 mg/L IAA, 0.05 mg/L GA3, and 0.3 mg/L zeatin riboside; Kim et al., 2006). Shoot tips should be evenly spaced with their bases in the medium and their tips pointed up. Plates are wrapped with PVC sealing film, then placed into a 25 °C growth room with 16-hour photoperiod and covered with a paper towel to lower the light intensity. After 7-14 days (when elongation occurs), shoot tips are transferred to Potato Propagation Medium, typically in Magenta cubes.

Viability levels are measured after 4 weeks, and shoot tips are considered viable when resulting plants are green and vigorous with three or more leaves and a length of at least 5 mm. At NLGRP, viability cryopreserved shoot tips must be 40% or greater to be considered successfully backed up. Material with viability lower than 40% may be stored until it can be preserved again. Once viability data are recorded, material is either discarded or transferred to Magenta cubes for further growth and evaluation.

6. References

Bamberg JB, Martin MW, Abad J, Jenderek MM, Tanner J, Donnelly DJ, Nassar AMK, Veilleux RE, Novy RG. 2016. In vitro technology at the US Potato Genebank. In Vitro Cellular & Developmental Biology—Plant 52:213-225. https://doi.org/10.1007/s11627-016-9753-x

Ellis S. 2023. Report: potatoes have massive impact on U.S. economy. Idaho Farm Bureau Federation. Accessed 11 May 2025. https://www.idahofb.org/news-room/posts/report-potatoes-have-massive-impact-on-u-s-economy/

Jenderek MM, Ambruzs BD, Tanner JD, Bamberg JB. 2023. High regrowth of potato crop wild relative genotypes after cryogenic storage. Cryobiology 111:84-88. https://doi.org/10.1016/j.cryobiol.2023.03.006

Jenderek MM & Reed BM. 2017. Cryopreserved storage of clonal germplasm in the USDA National Plant Germplasm System. In Vitro Cellular & Developmental Biology—Plant 53:299-308. https://doi.org/10.1007/s11627-017-9828-3

Kaczmarczyk A, Rokka V-M, Keller ERJ. 2011. Potato shoot tip cryopreservation. A review. Potato Research 54:45-79. https://doi.org/10.1007/s11540-010-9169-7

Kim H-H, Yoon J-W, Park Y-E, Cho E-G, Sohn J-K, Kim T-S, Engelmann F. 2006. Cryopreservation of potato cultivated varieties and wild species: Critical factors in droplet vitrification. CryoLetters 27:223-234. https://www.ingentaconnect.com/content/cryo/cryo/2006/00000027/00000004/art00004#

Nagel M, Pence V, Ballesteros D, Lambardi M, Popova E, Panis B. 2024. Plant cryopreservation: Principles, applications, and challenges of banking plant diversity at ultralow temperatures. Annual Review of Plant Biology 75:797-824. https://doi.org/10.1146/annurev-arplant-070623-103551

Niino T & Valle Arizaga M. 2015. Cryopreservation for preservation of potato genetic resources. Breeding Science 65:41-52. https://doi.org/10.1270/jsbbs.65.41

Sakai A, Kobayashi S, Oiyama I. 1990. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. Var. Brasiliensis Tanaka) by vitrification. Plant Cell Reports 9:30-33. https://doi.org/10.1007/BF00232130

Schäfer-Menuhr A. 1996. Refinement of cryopreservation techniques for potato. Final report for the period September 1, 1991-August 31, 1996. International Plant Genetic Resources Institute, Rome, Italy. pp. 1-41.

Senula A & Nagel M. 2020. Cryopreservation of Plant Shoot Tips of Potato, Mint, Garlic, and Shallot Using Plant Vitrification Solution 3. In: Wolkers WF & Oldenhof H (editors). Cryopreservation and Freeze-Drying Protocols. Methods in Molecular Biology 2180. Humana, New York, NY. Pp. 647-661. https://doi.org/10.1007/978-1-0716-0783-1_35

Vollmer R, Villagaray R, Castro M, Cárdenas J, Pineda S, Espirilla J, Anglin N, Ellis D, Azevedo VCR. 2022. The world’s largest potato cryobank at the International Potato Center (CIP) – Status quo, protocol improvement through large-scale experiments and long-term viability monitoring. Frontiers Plant Science 13:1059817. https://doi.org/10.3389/fpls.2022.1059817

Yamamoto S-i, Wunna Rafique T, Valle Arizaga M, Fukui K, Cruz Gutierrez EJ, Castillo Martiez CR, Watanabe K, Niino T. 2015. The aluminum cryo-plate increases efficiency of cryopreservation protocols for potato shoot tips. American Journal of Potato Research 92:250-257. https://doi.org/10.1007/s12230-014-9425-5

Zhang A-L, Wang M-R, Li Z, Panis B, Bettoni JC, Vollmer R, Xu L, Wang Q-C. 2023. Overcoming challenges for shoot tip cryopreservation of root and tuber crops. Agronomy 13:219. https://doi.org/10.3390/agronomy13010219

7. Additional Information

The potato shoot tip cryopreservation procedure is available to download here.

The potato shoot tip recovery procedure is available to download here.

8. Acknowledgements

Citation: Volk GM, Hall BD, Henk A, Bamberg J, Chen KY. 2026. Potato Shoot Tip Cryopreservation (Droplet Vitrification). In: Volk GM (Eds.) Training in Plant Genetic Resources: Cryopreservation of Clonal Propagules. Fort Collins, Colorado: Colorado State University. Date accessed. Available from https://colostate.pressbooks.pub/clonalcryopreservation/chapter/potato-cryopreservation/

This training module was made possible by:

Editors: Katheryn Chen

Content providers: Gayle Volk, Bradford Hall, Adam Henk, John Bamberg, Katheryn Chen

Videographers: Gayle Volk

This project was funded by Colorado State University and the USDA-Agricultural Research Service. 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.