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

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

Maria Jenderek, USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, Colorado 80521. Maria.Jenderek@usda.gov

Outline

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

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

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

1. Introduction

The USDA-ARS National Plant Germplasm System (NPGS) collection of banana and plantains (Musa spp.) is housed at the USDA-ARS Tropical Agriculture Research Station (TARS) based in Mayagüez, Puerto Rico (Video 1). This collection is maintained as field plantings (Fig. 1) and in vitro cultures (Fig. 2), many of which are sent to the USDA-ARS National Laboratory for Genetic Resources Preservation (NLGRP) in Fort Collins, Colorado to provide a secondary backup of the germplasm. NLGRP maintains these cultures until they are able to cryopreserve the accession as shoot tips stored in liquid nitrogen.

As a tropical crop, successful cryopreservation of banana depends on ultra-fast freezing and thawing rates. For this reason, NLGRP favors cryopreserving apical banana meristems through droplet vitrification, as described in “Cryopreservation of Musa germplasm: 2nd edition” (Panis, 2009). This method, initially published by Panis et al. (2005), combined the concept of droplet vitrification (Schäfer-Menuhr et al., 1997) with an established banana vitrification protocol (Thinh et al., 1999). While Musa vitrification is typically more successful in genotypes with more B-genome—such as AB and ABB, which are more associated with drought resistance—this method can be used to successfully cryopreserve all Musaceae, including diploid, triploid, and tetraploid banana cultivars as well as wild Musa and even the related genus Ensete (Panis et al., 2005).

2. In vitro cultures

Healthy field plants at TARS are used to establish in vitro cultures (Fig. 2). These are then sent to NLGRP, where they are tested for culturable endophytes using bacterial growth medium described by Panis (2009), then placed into quarantine for 14 days prior to moving to CI medium (MS, 30 g L^-1 sucrose, 2 g L^-1 gelrite at pH 5.8).

All plants are grown individually in 25×150 mL test tubes, caps wrapped with PVC film, on 25 mL CI medium. They are cultured at 25±2 °C under continuous 50 μE m^-2 s^-1 illumination provided by 36W Osram cool-white fluorescent tubes. Plants are subcultured every 4-6 weeks until robust and well-rooted in vitro plants are obtained with a corm diameter of 5-8 mm that forms an appropriate source for the excision of shoot tips. To multiply cultures, shoots of 3-5 cm in length are separated and transferred to CII medium (MS, 30 g L^-1 sucrose, 10 μM BAP, 1 μM IAA, 2 g L^-1 gelrite at pH 5.8) in test tubes.

3. Shoot tip excision

Plants are 5-8 weeks post-subculture at time of shoot tip excision (Fig. 3). At this stage, plants should appear healthy and be free of visible contaminates. Shoots should be 3-5 cm in length and have a corm diameter of 5-8 mm.

Shoot tip excision, shown in Video 2, must take place in a laminar flow hood to maintain sterile conditions. Individual apical meristems are excised under a clean binocular microscope. Leaves are removed until the apical dome is visible but still partially covered by 1-2 young leaf primordia, and the leaf base (corm tissue) is trimmed to 1 mm in diameter (Fig. 4). The dissected shoot tips are immediately transferred to the loading solution (MS, 0.4 M sucrose, 2 M glycerol at pH 5.8) in the dark at room temperature, and remain in the solution until all excisions are complete.

4. Shoot tip processing and storage

Shoot tips should be exposed to loading solution for no less than 20 minutes and may remain in the solution for up to ~5 hours (Panis et al., 2005). After loading, the solution is replaced by ice-cooled PVS2 solution for 30-40 minutes. at 0 °C. Just prior to the end of PVS2 treatment, shoot tips are transferred to sterile foil strips. Each foil should contain one pool of PVS2 holding 10 shoot tips. All steps should take place on top of a frozen cooling element or ice. After the PVS2 treatment, the aluminum strip is plunged into a shallow dewar of liquid nitrogen.

Each frozen foil strip with shoot tips is quickly transferred to an appropriately labelled 1.2 mL cryovial, pre-cooled with liquid nitrogen. Vials are then quickly capped and placed back into liquid nitrogen until they are transferred to cryocanes. Once cryovials are attached to pre-cooled cryocanes, canes are inserted into labelled cryosleeves, then transported in a liquid nitrogen-filled vessel to a temporary holding tank. When viability assays are complete, the samples are transferred to cryotanks in the NLGRP vault. This entire process is shown in Video 3.

5. Shoot tip recovery

To establish that the cryopreservation process was successful, 2 vials (a total of 20 shoot tips) must be recovered for a viability assay. This process is shown in Video 4. Viability assays are ideally conducted on shoot tips that have been stored in cryovials in liquid nitrogen for 20 minutes or longer. Viability assays my be conducted several days or even weeks after storage.

Cryovials are removed from liquid nitrogen, then the aluminum foil strips are quickly transferred to the unloading solution (MS, 1.2 M sucrose at pH 5.8) at room temperature. Once shoot tips are released from the foil, they remain for another 15 minutes in the unloading solution. Unloaded shoot tips are then placed onto two sterile filter papers on top of the semi-solid RI medium (MS, 0.3 M sucrose, 2 g L^-1 gelrite at pH 5.8) in small Petri plates. Plates are wrapped with PVC sealing film and then aluminum foil to prevent light exposure. These are stored in the dark in in a growth chamber set to 25 °C. After two days, the shoot tips are then transferred onto RII medium (MS, 30 g L^-1 sucrose, 10 μM BAP, 1 μM IAA, 2 g L^-1 gelrite at pH 5.8) in small Petri plates without filter papers. The first week of culture always takes place in the dark. After that, recovering shoot tips are placed in full light (50 μE m^-2 s^-1 ) with a 16 hour photoperiod. Shoot tips should be moved to fresh RII medium every 7-10 days.

6. Viability assessment

Shoot tips may be evaluated four to six weeks following cryopreservation (Fig. 5). At this time, four types of reactions can be distinguished:

1. White shoot-tips resulting from an immediate death of the tissue without blackening;
2. Completely or partially black shoot-tips, indicating that there was enzymatic reaction following cryopreservation (production and oxidation of polyphenols);
3. Unorganized callus growth, representing the outgrowth of small isolated areas of the apical dome and/or primordial tissues; and
4. Shoot-tip regeneration resulting from the survival of a substantial part of the apical dome.

Shoot tips are scored as “viable” if a green and elongated (at least 0.5 cm) shoot can be observed. Calluses never produced shoots. Accessions with at least 40% viability are considered successfully backed up and ready to receive a storage location in the NLGRP vault cryotanks.

7. References

Panis B. 2009. Cryopreservation of Musa germplasm: 2nd edition. Technical guidelines No. 9 (F. Engelmann and E. Benson, eds). Bioversity International, Montpellier, France. https://alliancebioversityciat.org/publications-data/cryopreservation-musa-germplasm

Panis B, Piette B, Swennen R. 2005. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Science 168:45-55. https://doi.org/10.1016/j.plantsci.2004.07.022

Schäfer-Menuhr A, Martin Schumacher H, Mix-Wagner G. 1997. Cryopreservation of potato cultivars – Design of a method for routine application in genebanks. Acta Horticulturae 447:477-482. https://doi.org/10.17660/ActaHortic.1997.447.97

Thinh NT, Takagi H, Yashima S. 1999. Cryopreservation of in vitro grown shoot tips of banana (Musa spp) by vitrification method. CryoLetters 20:163-174.

8. Additional information

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

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

9. Acknowledgments

Citation: Skogerboe D, Chen KY, Jenderek M. 2026. Banana 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/banana-cryopreservation/

This training module was made possible by:

Editors: Katheryn Chen

Content providers: Dianne Skogerboe, Katheryn Chen, Maria Jenderek

Videographers: Katheryn Chen

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.