Chilling Out for Conservation

23 March 2023

CGIAR genebanks are using cryopreservation for the long-term conservation of crops that can’t be stored as seeds.

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Molecules in living organisms are always racing around. That movement is what keeps us alive – and what eventually leads to aging and death. But molecules slow down when it gets super cold. In fact, at about −196°C – the temperature of liquid nitrogen – molecular motion is pretty much halted and all metabolic reactions in a living cell come to a stop. But, fortunately, that’s temporary and reversible.

Cryopreservation, storing materials in liquid nitrogen, uses this property to ensure the long-term conservation of crops that cannot be conserved as seeds because they are vegetatively propagated – planted using plant parts such as tubers (e.g. potatoes) or cuttings (e.g. cassava) – or have recalcitrant seeds – seeds that cannot be dried and stored at low temperatures like most seeds. Well-known examples include potato, sweetpotato, banana, yam, cassava, taro and garlic. Such crops are most often conserved as live plants in the field or on artificial growth media in flasks or test tubes in the lab (in vitro), but this makes them particularly vulnerable to contamination or infection with pests and diseases.

Putting the Freeze On

Scientists have succeeded in showing that tissues of these crops can be cryopreserved, and revived again to recover whole plants, but there are very few cases of the technique being applied on the scale necessary to secure thousands of different varieties. “It has only been during the last few decades that plant cryopreservation has really been used for storing large collections,” said Bart Panis, a cryopreservation specialist who works with bananas at the International Musa Germplasm Transit Center (ITC).

In 2017, as part of an expert study, the Crop Trust, together with Bioversity International and the International Potato Center (CIP), conducted a survey to assess the status of cryopreserved collections of Annex 1 crops and under Article 15 of the International Treaty on Plant Genetic Resources for Food and Agriculture worldwide. The study found that 100,000 unique samples of vegetatively propagated crops and crops with recalcitrant seed are held in field and in vitro genebanks, but that fewer than 10,000 samples held in 15 genebanks are cryopreserved and only eight crops are represented by more than 100 accessions in cryopreservation.

“The advantages of cryopreservation are many,” said Elena Popova, head of the All-Russian Plant Cell Culture Collection. “Low maintenance costs, optimized space usage, extended – or rather, theoretically indefinite – storage period, low risk of mixing up or losing samples, good genetic stability [i.e. few mutations or other genetic changes], no selection pressure and high probability to maintain pathogen-free material clean over time.”

“During a 20-year conservation period, a sweetpotato sample held in vitro is renewed every six months, or about 40 times,” said Rainer Vollmer, CIP’s lead cryo scientist. “Every time you renew in vitro material, you have a potential risk of human mistake or loss of genetic integrity [changes in the genetic make-up of the sample]. In cryo, you process a sample only once to put it into storage.”

Cooling It

Cryopreservation of plant materials isn’t easy though. Just imagine. You’re sticking a living organism in the VERY deep freeze, and you need to be super-confident that years later you’ll be able to bring it back to life. Scientists have had to overcome many challenges. Plants have a large amount of water inside their cells that can convert into damaging ice crystals during freezing. Plant tissues therefore need to be dehydrated before freezing, otherwise they will be damaged. That has to be done carefully. To avoid ice crystallization, scientists use chemicals called cryoprotectors, but these can be applied in a multitude of ways and there is no universal protocol that works for all species. The technique needs to be tailored for each crop and sometimes even for different varieties within a crop.

Once the right protocols are developed, it takes skillful personnel to apply them correctly and consistently. One tricky part is excising the shoot tips – the growing part of the plant that is used as the main material for conservation. “One skilled staff member can prepare and cryopreserve between 40 and 70 samples per year,” said Bart. “If you are dealing with collections of thousands of samples, you can imagine how much work is involved.”

There is a huge difference between successfully testing a protocol on a handful of samples as part of a research project and the cryopreservation of a collection of thousands of samples. The latter requires a totally reliable protocol and an assembly-line approach, with meticulously organized workflow involving trained staff at each step. It’s the difference between administering a few home remedies and undertaking successful heart surgery. What’s worse, it is not uncommon for staff who have been trained for several months to implement a particular protocol in one lab to be unable to repeat the same success in another lab.

And then there is the issue of funding. In the long run, maintaining cryopreserved collections is considerably less expensive than holding in vitro and field collections. But getting the sample into a cryotank filled with liquid nitrogen in the first place is expensive and few labs can afford to do so.

“At CIP, we estimated that the cost of introducing a new sample to the cryobank is USD 400 based on a throughput rate of 500 samples per year,” said Rainer. “The cost for maintaining that sample in the cryobank once it’s in there is about USD 2 per year, which is pretty much just the cost of refilling tanks with liquid nitrogen every once in a while.”

Getting Warm

Despite the challenges, CGIAR genebanks are making great progress in improving and scaling up cryo activities. A project at CIP supported by the former CGIAR Genebank Platform reached rates of cryopreservation never seen before. “We have trained a team of 16 technicians who can now prepare more than 500 potato samples and about 70 sweetpotato samples in a year,” said Rainer. “This scaling up has meant that we now have 4,100 potato samples in our cryotanks, which is about 84% of the whole in vitro collection. Five years ago we only had 35%.”

A lot of the success at CIP can be attributed to the experience of the staff. “We have invested a lot in developing our team,” said Rainer. The team continuously performs small experiments, which have enabled them to simplify their protocols. “This has helped us to reduce the workload and make time available for cryopreserving more samples,” said Rainer. “It’s crucial to have just-in-time production in place. If you run out of plants, your throughput will break down.”

Protocols are also improving and being standardized. “A lot of the current cryopreservation work is focusing on the use of the droplet vitrification or cryoplate techniques,” said Bart. In these protocols, dehydrated plant material is frozen inside a drop of a concentrated cryoprotector solution that is placed on an aluminum foil or plate. This helps to achieve rapid freezing and rewarming, which is critical for regrowth. The research focus is maximizing the regeneration of the frozen 1-mm-sized shoot tips and, of course, eventually a full healthy plant.

Backing It All Up

There is no equivalent of Svalbard Global Seed Vault for crops that are vegetatively propagated or have recalcitrant seeds. And even though most cryopreserved samples have backups in field and in vitro collections, most of them are not safety duplicated in other cryo facilities.

An exception is CIP’s cryobanked potato collection. A backup of the 4,100 cryobanked potato samples is stored in a separate cryotank and is ready for shipment to a remote partner institute as soon as the required agreements are signed. Additionally, CIP is building up a separate national safety-backup of its cryo collection that will be stored at its regional office in the Peruvian highlands (over 3,200 meters above sea level). 

The 2017 expert study report concluded that a major global initiative is urgently needed to accelerate the development and implementation of cryopreservation for the conservation of crop diversity. 

“Crops stored in the field and in vitro are indeed vulnerable, so we need a game changer to ensure they are cryopreserved and backed up safely as soon as possible,” said Elena. The vision is to set up a global initiative that would link CGIAR genebanks, national agricultural research system and cryo research labs to solve major problems in cryopreservation technology and facilitate large-scale implementation of cryopreservation and safety duplication of collections at risk.

Cryopreservation may slow down molecules, but it certainly doesn’t slow down scientists like Rainer, Bart and Elena who are racing to save our crops by chilling them out.

This article was originally published by the CGIAR Genebank Platform.

Category: Genebank Platform