Plant material for cryopreservation
In general, cryopreservation of crops that are clonally propagated or have non-orthodox seeds is performed using winter buds (apple and other temperate fruit trees), shoot tips or axillary buds of in vitro grown plants, or using isolated embryonic axes. Some genebanks also cryopreserve seeds and pollen upon their sufficient dehydration.
Methods for plant cryopreservation
Until 15 years ago, cryopreservation protocols for plant tissues were mainly based on slow, or programmed, freezing of samples in the presence of cryoprotective mixtures containing dimethyl sulphoxide, sugars, glycerol and/or proline. Slow freezing results in a freeze-dehydration, leaving less water in the cells to form lethal ice crystals upon exposure to extreme low temperatures. While this method was very convenient for many plant tissues, especially undifferentiated callus or suspension cells, it was not very effective for crops of tropical origin that are usually sensitive to dehydration under low temperatures.
During the past two decades, several new cryopreservation procedures have been established. Among them, the protocols termed encapsulation/dehydration, encapsulation/vitrification, vitrification, droplet-vitrification and D- or V-cryoplate. All these techniques involve the extraction of freezable water from the tissue cells before rapid cooling. As a result, if the cell interior is sufficiently dehydrated, it undergoes the process called “vitrification”: direct transition from the liquid phase into an amorphous phase (or “glassy state”), whilst avoiding the formation of lethal crystalline ice. Normally, the vitrification technique involves a series of treatments: preculture with 0.3-0.7 M sucrose, treatment with cryoprotective solution (osmoprotection) and dehydration with highly concentrated vitrification solution followed by rapid immersion into liquid nitrogen. To withdraw the material from the cryotank, the process is repeated in reverse: after rapid rewarming samples are put in unloading solution(s) with deceasing sucrose concentrations (1.2-0.8 M) and then transferred to nutrient media for recovery and plant regrowth.
A modification of this technique, that further reduces the chance for lethal ice-crystal formation through the application of ultra-fast cooling and rewarming rates using aluminum foils and small amounts of cryoprotectant solutions called “droplet vitrification” has now been developed for different crops.
V- and D- cryoplate methods developed recently involve adhesion of samples to aluminum plates using Ca-alginate gel followed by air dehydration or treatment with cryoprotectant solutions. These methods has been successfully tested under laboratory conditions for range of crops including potato and cassava.
Despite the abundancy of methods available for plant cryopreservation, there is no universal protocol that could be applied to all species. Each species requires specific set of treatments that must be carefully followed to ensure maximum regrowth.
Prioritization of accessions for cryopreservation
Expenses for the accession to be introduced into a cryobank vary from $30 to $1800 depending on crop, protocol and genebank location. Therefore, it is crucial to have clear and well-thought policy in prioritizing accessions for cryopreservation. The following rules generally apply for cryobanking the majority of crops:
- Accessions should be true-to-type
- Accessions should be phytosanitary clean
There may be different approaches to prioritizing accessions that should be cryobanked:
- Accessions from the core collection
- Accessions that are not in high demand and/or have not been distributed within the past 3-5 years
- Accessions that are in danger of extinction caused by break down of pests, deceases or by other factors
As cryobanks allow conservation for the long-term, decision on which part or the whole collection should be cryopreserved is strategic and should be made based on genebank policies and long-term goals and manager’ experience.
Practical tips
- Liquid nitrogen (LN) constantly boils up and evaporates from the storage tanks. Ensure regular delivery of LN to your facility from a local supplier to top up storage tanks or consider purchasing a small-scale LN producing plant (available at different capacities from various manufacturers). Use an alarm system to indicate low liquid nitrogen levels. Regularly monitor temperature in the storage tanks.
- Transfer cryopreserved material quickly between vessels to avoid rewarming.
- Storage container vary with the size of the seeds or tissues. 1.8-2.0 ml cryogenic vials are the most common. Larger canisters can be used for scions/winter buds.
- For meristems and shoot tips, 10-20 samples can be placed in one 2.0 ml vial.
- The number of replications and samples put in storage depends on the survival, crop type, speed of propagation, stability in culture and material available. Probalistic tools have been developed to assist in the establishment and management of cryopreserved collections (Dussert et al. 2003; Volk et al. 2017)
- Currently, there is no standard requirement for minimum percentage of plant regrowth from cryopreserved material. At CIP, CIAT and IITA, 30% minimum plant regrowth is used as a threshold for potato, yam and cassava and 20% for sweetpotato. Regrowth is counted strictly as development of the whole plant.
- Cryogenic Dry Shippers can be used to transport cryopreserved materials without rewarming. A range of vessels with different volume/storage period is available from different manufactures.
- Some contemporary cryogenic tank models allow storage in both liquid nitrogen and in its vapor phase. There is no standard recommending one type of storage over another type. Most genebankers consider storage in liquid phase to be more secure in terms of temperature fluctuation and sample longevity, others prefer storage in vapor phase due to safer and easier sample handling.
Basic safety measures
(consult Occupational Health and Safety team at your institute before working with liquid nitrogen or putting even a small cryotank in the room)
- Ensure good air circulation in the room where cryogenic storage tanks are placed because nitrogen gas is constantly boiling from the tanks. Oxygen monitors should be placed around the room for detection of oxygen content of the air.
- Use emergency fans that are triggered by the oxygen monitors or emergency buttons to increase air exchange in case of build up of nitrogen gas
- Wear cryogenic-proof protection glasses, gloves and aprons and closed shoes when working with liquid nitrogen.
Literature
Dussert S, Engelmann F, Noirot M. 2003. Development of probabilistic tools to assist in the establishment and management of cryopreserved plant germplasm collections. CryoLetters 24: 149-160.
Volk GM. Henk AD. Jenderek MM, Richards CM. 2017. Probabilistic viability calculations for cryopreserving vegetatively propagated collections in genebanks. Genet Resour Crop Evol 64: 1613–1622.
