Plant Cryopreservation Importance, Approaches and Future Trends

Abstract

Plant cryopreservation is useful for long term storage of clonal germplasm and endangered species. Clonally propagated crops which produce recalcitrant seeds cannot be easily conserved using conventional methods. Preservation of plants in vitro is limited to two years and not ideal for germplasm storage for a very long time. The need to conserve plant genetic resources through cryopreservation techniques to mitigate the effects of climate change such as extinction of certain plant species cannot be underestimated. Different cryopreservation methods including dehydration, programmed freezing, vitrification and v cryo-plate are employed in the long-term storage of different plants. These methods are usually based on the principle of the removal of freezable water from tissues by physical or osmotic dehydration followed by ultra-rapid freezing. There have been several advancements in the identification and use of cryoprotective agents, nonetheless, its toxicity remains a challenge. To accelerate plant cryopreservation, there is the need for the development of global expertise. The current practice for the conservation of germplasm in the Biotechnology Laboratory in Ghana is through the use of slow growth media. Moving forward, there is the need to work on developing cryopreservation protocols for preservation of germplasm using liquid nitrogen and cryogenic refrigerators.

1. Introduction

As early as 2000 BC, archaeological findings has shown that icehouse were used throughout Mesopotamia to store foods [1]. Since time immemorial, the preservation of biological material has been known. The storage of biological material at ultra-low temperatures is referred to as cryopreservation. In broad terms, cryopreservation refers to the study of life at low temperatures [2]. Plant cryopreservation is a conservation method that permits long-term storage of tissue samples at very low temperatures of −135°C to -196°C with little risk of causing variation. Cells can successfully be cryopreserved in liquid nitrogen when extracellular water has been removed to the extent that any remaining water form the so-called biological glass (vitrification), thereby mitigating the adverse effect of ice crystal formation and growth [3]. Cryopreservation for storage of plant cells, tissues, and organs became operational in the 1960s till date. Long term storage of in vitro cultures of secondary metabolite cell cultures, embryogenic cultures, clonal germplasm, endangered species, and transgenic products remains a sine qua non for many scientists, organizations and companies [4]. In the case of clonally propagated crops which produce recalcitrant seeds and cannot be readily conserved by conventional methods through seed preservation, cryopreservation is important for long term conservation. Over the years, a lot of research on different crops to study the feasibility of the long-term storage of plant species has taken place. Prof. Akira Sakai, researched on mulberry twigs after exposing them to liquid nitrogen. This study is reported to be the pioneer in plant cryopreservation research [2, 5]. Research in cryopreservation in the twentieth century was devoted to basic studies of ice formation, vitrification of solutions and the beginnings of cryopreservation as a long-term storage technique [4]. In recent times, cryopreservation research has focused on practical procedures for gene bank storage, thereby enabling cells and meristems to be cryopreserved by direct transfer into liquid nitrogen. The development of simple and reliable methods for cryopreservation has led to cryo-banking [6].

2. Importance of cryopreservation of plants

A prerequisite for the short- and long-term survival of plant species in their natural habitat is genetic diversity [7]. Biological diversity conservation importance was recognized in 196 countries this led to the generation of a treaty that includes the sustainable use of its components, fair and equitable participation in the benefits derived from the use of plant resources [3]. The long-term conservation of tissues using cryopreservation has been increasingly used in recent years as it requires very little storage space, minimal upkeep, and eliminates the risk of contamination, makes the germplasm available for posterity, and its applicability to a wide range of plant tissues [6, 8].

2.1 Advantages of cryopreservation over other methods

Plant genetic resources are usually conserved in their natural habit (in situ) or other sites (ex situ). Preservation off site is partially used or for the entire population when preservation in situ is extremely challenging usually as a result of lack of complete control over many factors that influence the survival of plant materials and its genetic make-up [3]. Maintenance of plant genetic material in vitro is more efficient and secure than conservation in the field. In vitro conservation has been reported for the long-term conservation of germplasm for approximately two years without sub-culture such as in the case of potato. This notwithstanding, in vitro preservation is not ideal for long term germplasm conservation because it is labour consuming, costly, and carries risks of losing germplasm due to human error, such as contamination and mislabeling during sub-culturing [9]. Furthermore, erratic power supply, malfunctions in air-conditioning and lighting system could sometimes pose a challenge for in vitro conservation. Moreover, another setback of tissue culture for long term conservation is the induction of genetic variation or somaclonal variation during prolonged sub-culture [6]. Besides, mites, thrips, and other small arthropods can cause extensive fungal contaminations in tissue culture and are difficult to eliminate. In addition, tissue culture collections are constrained by the occurrence of cellular aging and senescence during prolonged cultivation. The effect of cellular aging may appear in parallel with slow growing endophytic microbes that can accumulate over time [10].

The only ex situ conservation method that allows long term survival of organisms at very low temperature and using reagents such as liquid nitrogen is cryopreservation. Plant materials stored in liquid nitrogen have indefinite lifespan in spite of the fact that no biological specimen is immortal [11]. Again, it is the only ex situ conservation method used for long-term conservation of plant materials that cannot be stored in seed banks, for instance clonal crops or species with a low number of progenies or recalcitrant seeds. Furthermore, it requires only a minimum space and maintenance efforts (Figure 1a and b). It has become a very important tool for the long-term storage of plant genetic material [11, 12]. Moreover, in addition to its use for the conservation of genetic resources, cryopreservation can also be applied for the safe storage of plant tissues with specific characteristics. Plant cells of different types, gametic cells, tissues and organs can be cryopreserved [13]. Due to the totipotency, various plant cells can be manipulated to enhance regrowth after cryopreservation, paying attention to genetic integrity.

Figure 1.

Comparison of conservation of 150 plantain accessions on (a) RITA temporary immersion bioreactor system compared with the use of a (b) cryo-freezer in terms of space for storage at CSIR-CRI, Kumasi-Ghana.

2.2 Food security, biotechnology and breeding

The world’s most important food crops for food, nutrition, and livelihoods most especially for the poorest people are vegetatively propagated crops. Examples of some of these crops include banana (Musa sapientum), plantain (Musa paradisiaca), sweetpotato (Ipomoea batatas), cassava (Manihot esculenta), yam (Dioscorea spp.), citrus (Citrus spp.) and coconut (Cocos nucifera) [14]. Plant genetic resources constitute the store of genome information and are important for world food security, crop improvement and conservation of genetic diversity [15]. It is important in breeding programs to obtain new or more productive plants that are resistant to biotic and abiotic stresses, due to the changing weather patterns [2]. Globally, food, feed and fiber utilization are restricted to very few species, hence, advanced biotechnology techniques such as cryopreservation represents an efficient alternative method for ex situ conservation of germplasm of various crop species. It helps in overcoming several challenges of storage by conventional means. In recent times over 10,000 accessions through initial in vitro introduction and subsequent preservation using cryogenic methods have been used for several crops. Above 80% of these crops belong to crops that are widely consumed such as potato, cassava, bananas, mulberry and garlic [16].

In modern breeding programs, cryopreservation is important for providing long-term storage and international access to various genetic materials. The genetic materials accessible internationally include seeds, pollen and meristematic apices and buds. Plant breeders and horticulturists involved in fruit and forest tree improvement are very much particular about pollen storage. Techniques for pollen culture have been used for decades to obtain haploids or homozygous diploid plants from various plant species such as maize and rice. Regular supply of viable pollen provided by pollen banks takes away seasonal, geographical or physiological limitations of hybridization programs and supports hybrid development between genera and species. Large field areas are required by traditional pollen banks at different stages to synchronize flowering both of which are very labour intensive and needs a lot of funds to be operational. Other methods of pollen banking for the purposes of breeding for short-term storage such as freeze drying, freeze storage, vacuum drying and cold storage in organic solvents lead to frequently observed sharp reduction in pollen viability. The most efficient means of pollen storage is cryopreservation that does not require any expensive cryostats. This is so because pollen grains can be directly immersed in liquid nitrogen for long-term storage [16].

Advanced biotechnology application such as cryopreservation is a very good efficient method for ex-situ conservation of plant germplasm. This method supersedes the challenges and limitations of conventional methods seed banks and conventional orchards [2]. Preservation of plant germplasm for plant breeding and biotechnology has long been recognized and it is very essential for enhancing breeding activities. It has been reported that easy access to diverse plant germplasm is a pre-requisite for breeding more productive cultivars. This in the long run ensures food security [16, 17]. With respect to biotechnological interventions, the consistently evolving area of phyto-chemical production via biotechnological methods is supported by cryo-banking of root cultures, embryogenic and non-embryogenic cell lines to ensure their genetic and biochemical stability [16, 18].

2.3 Agrobiodiversity

Plants are recognized as a vital component of biodiverse ecosystems (the carbon cycle, food production and bio-economy) [19]. An important issue concerning human population worldwide is the conservation of plant biodiversity. Plant biodiversity is a natural source of products to the food industries. Provision of basic raw materials is its hallmark. Maintenance of plant biodiversity in their natural habitat, as well as domesticated and cultivated species on the farm or in the surroundings where they have developed their distinctive characteristics, represent the in situ strategies. Due to heavy loss of species, populations and ecosystem composition leading to loss of biodiversity, ex situ conservation is a viable way for saving plants from extinction, and in some instances, it is the only possible strategy to conserve certain species [17]. Plant genetic resources are highly important for agro-biodiversity because they can be used to breed new or more productive crops that can withstand biological and environmental stresses [12, 13].

2.4 Cryotherapy for virus elimination

Systemic pathogens such as viruses, phytoplasmas and bacteria could be eliminated by treating shoot tips with liquid nitrogen using cryopreservation protocols. It is a novel approach for pathogen eradication in plants. The uneven distribution of viruses and other pathogens in shoot tips allows the elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from surviving pathogen-free meristematic cells. Cryopreservation methods have been useful in pathogen eradication by means of shoot tips cryotherapy [17]. The use of cryotherapy to remove viruses from vegetatively propagated crops has been reported [4]. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Consequently, it has the potential to replace more traditional methods like meristem culture, chemo- and thermo-therapies. This method has been utilized for eradication of severe pathogens in banana, citrus, grapevine, raspberry, potato and sweetpotato [17].

2.5 Importance of cryopreservation in the era of climate change

Greater risks of extreme weather and changes in climate variables such as prolonged drought and storms are events that biomes will have to adapt as one of the measures to prevent extinction [11, 20]. Effects of climate change on biodiversity, agricultural production and food security have been a matter of great concern [21]. The need to adopt strategies to conserve plant genetic resources to mitigate the effects of climate change that has a potential of causing the extinction of certain plant species cannot be underestimated. One strategy to address the issues of climate change in order not to lose endangered species is cryopreservation. For instance, critically endangered species growing in the wild in Finland has been successfully cryopreserved to enable its long-term conservation through the use of droplet vitrification protocol. Additionally, protocol development for cryogenic preservation of plant species is an additional tool to ex situ conservation toolbox for the maintenance of plants to avert the effects of climate change [11].

3. Stages in cryopreservation

Depending on the selected technique, cryopreservation is made up of different stages which includes preparation and explant excision, preculture, cryoprotection, vitrification/dehydration, fast cooling in liquid nitrogen, rewarming, cryoprotector elimination, regeneration and plant culture [8, 22].

In an effort to preserve biological materials for cryopreservation, the following steps are followed. The first step involves harvesting or selection of material, the growth stage has to be considered where applicable. Much attention should be paid to volume or size, density, pH and morphology. The second stage has to do with addition of cryo-protectant agents that include glycerol, salts, sugars, glycols that are added to samples. This stage is then followed by the application of different methods of freezing to protect cells from damage and cell death by their exposure to the warm solutions of cryoprotective agents (CPA). After all these have taken place, the cryopreserved samples are stored in −80°C in a freezer for at least 24 hours before transferring it to storage vessels. Finally, the process of thawing is initiated which involves warming the biological samples in order to control the rate of cooling and prevention of cell damage caused by crystallization [23].

4. Plant material used for cryopreservation and cryopreservation agents

The state of mother plant especially with regards to physiological state is a key factor for the success of cryopreservation. For cryopreservation techniques, any totipotent tissue may be used. Most commonly used tissues are shoot tips, and to a lesser extent, somatic embryos and embryonic axes. Shoot tips and somatic embryos for cryopreservation require tissue culture systems with established micropropagation regimes [24].

Decisions concerning the choice of a plant material for cryopreservation are dependent on plant type and reason for storage. Based on knowledge of plant vulnerability, curators need to make decisions on which plants to store based on their knowledge of plant vulnerability. The decision to select a plant part for cryopreservation technique depends on growth conditions. Generally, practice shows that plants that are diseased or not thriving for any reason are generally poor candidates for cryopreservation. Plant materials should be in an optimal growth phase, dormant materials should fully break dormancy, and where appropriate fully cold acclimated [25]. The question thus remains about how amenable plants indigenous to the tropical regions can respond successfully to cryopreservation.

Meristems and embryos are explants normally conserved using encapsulation techniques. Alginate beads which contain mineral salts and organic substances are used for the encapsulation of meristems and embryos. Cryopreservation agents are used for the treatment of plant genetic material as in the case of vitrification methods. The most commonly applied vitrification solutions include vitrification solution number 2, which contains glycerol, ethylene glycol and sucrose. These reagents are used by synseeds during regrowth so that they quickly grow to prevent loss of viability. Vitrification solutions contain penetrating and non-penetrating cryoprotective substances to preserve both inside and outside of plant genetic material and prevent the formation of lethal ice crystal so that cells remain viable for a long period of time [26].

Pollens are cryopreserved for breeding purposes. Viability of pollen after cryopreservation depends on a number of factors. Pollen moisture content, freezing and thawing procedure, physiological stage of mother plant, flowering stage, plant vigor and genotypic differences are the factors that determine pollen viability [16, 24].

5. Methods of cryopreservation and application

Different techniques are employed in the long-term storage of different plants. These techniques include dehydration, controlled-rate cooling and vitrification [4]. Cryopreservation technique is based on the principle of the removal of freezable water from tissues by physical or osmotic dehydration followed by ultra-rapid freezing. In cryopreservation procedures, water plays a central role in preventing freezing injury and in maintaining post-thaw viability of cryopreserved cells stored in a small volume, requiring a very limited maintenance. Classical freezing procedures encapsulates the use of different cryoprotective solutions combined with pre-growth of material followed by slow cooling (0.5–2.0°C/min) to a determined pre-freezing temperature (usually around −40°C), rapid immersion of samples in liquid nitrogen, storage, rapid thawing and recovery [17]. Cells with low water content which includes pollen, seeds, and dormant tissues of stress-tolerant species, may be introduced to low temperatures such as in the case of using liquid nitrogen without lethal damage. On the other hand, plant cells with higher water content present considerable problem as a result of ice crystal growth causing cell bursting [3].

For any cryopreservation to be successful, it is important to avoid the lethal intracellular freezing that occurs during rapid cooling. Consequently, in any cryogenic procedure, the cells and shoot tips must be sufficiently dehydrated in order to preclude freezing and to allow vitrification in liquid nitrogen [12].

In the past 25 years, many cryopreservation techniques have been established based on the conventional slow freezing techniques. The different approaches used include vitrification, droplet vitrification, dehydration and pre-growth and pre-growth dehydration [19]. Cryopreservation methods are commonly used globally. It has been reported that new cryogenic methods using cryo-plates (the V cryo-plate and D cryo-plate) are advantageous over early developed methods. Advantages are manifested in ease of handling during the procedure and high regrowth rates after cryopreservation [12].

5.1 Dimethyl sulfoxide droplet

Since 1866, dimethyl sulfoxide (DMSO) has been commonly used for the cryopreservation of tissues because of its low cost and relatively low level of cytotoxicity [27]. DMSO acts by reducing the electrolyte concentration in the residual unfrozen solution in and around a cell at any given temperature. With this method, plant materials are treated with a 10% DMSO in liquid Murashige Skoog (MS) medium with 30 g sucrose, 0.5 mg/l zeatin riboside, 0.2 mg/l GA3 and 0.5 mg/l IAA. This method appears to be simple because only 10% DMSO in liquid medium is used as cryoprotectant solution. The explants (shoot tips of 2–3 mm) are then incubated in MSTo medium overnight at 22°C and treated with cryoprotectant solution (10% DMSO in MSTo medium) for 1–3 h at RT followed by transfer into droplets of 2.5 μl cryoprotectant solution one by one on aluminium foil. The aluminium foil is then immersed directly into cryotube filled with liquid nitrogen [6]. DMSO droplet has been routinely used for by the for safe and long-term conservation storage of shoot tips of sweetpotato by the International Potato Center (CIP) [9].

5.2 Dehydration

It involves dehydration of samples by either air current, silica gels, or incubation with cryoprotectant followed by rapid freezing or two-step freezing. It usually results in 100% recovery rate after liquid nitrogen drying in a laminar flow hood until 5–15% moisture content. For this technique, shoot tips or embryo are precultured on 0.3–0.6 M sucrose medium for 1–3 days. This is followed by encapsulation into alginate beads and treated with highly concentrated sucrose solution (approx. 0.8 M) for 16 h. These treatments induce tolerance in the samples. Following the pretreatment, plant genetic materials are dehydrated on silica gels or in a laminar flow cabinet to reach their optimal hydration levels. The advantage of this method is that it eliminates the need for other cryoprotectants that have been implicated in inducing genetic changes after cryopreservation such as DMSO and ethylene glycol [12].

5.3 Programmed freezing

With this method, samples are pretreated in cryoprotectants. The cryoprotectant agents used include DMSO, ethylene glycol and sucrose alone or in low concentration mixtures. Pretreated samples are dehydrated while frozen slowly (0.3–1°C/min) between −40°C and −70°C, then plunged directly into liquid nitrogen. This method is based on the principle of free-induced dehydration. Programmed freezer that is expensive is required and it is a major disadvantage. Additionally, relatively long exposure of samples to subzero temperatures, which can be deleterious for cold-sensitive species is also a disadvantage [16].

5.4 Vitrification

The physical process by which a highly concentrated cryoprotective solution super cools to very low temperatures and finally solidifies into a metastable glass without undergoing crystallization at a practical rate. It was proposed as a method for the cryopreservation of biological materials because it avoid the potentially detrimental effects of extracellular and intracellular freezing [25]. This process involves pre-culturing of plant tissues on basal medium supplemented with cryoprotectants, pre-treatment with loading solution, dehydration with PVS, and rapid freezing rewarming. In general, vitrification protocols have been very useful for cryopreserving complex organs like shoot tips, and somatic embryos that could not be effectively frozen following classical protocols. The vitrification method uses a highly concentrated solution. This solution sufficiently dehydrates tissues and does not lead to injury. This leads to the formation of a stable glass along with the surrounding highly concentrated solution plunged in liquid nitrogen. Cells or shoot tips must be sufficiently dehydrated with highly concentrated vitrification solution at 0°C or 25°C and should not lead to injury. Recovery rate is 74.5% with 5-day pre-culture on 0.5 M sucrose followed by PVS2 treatment for 1 h at 0°C. This method has been applied to several plants that includes tropical and subtropical species. It has been applied in the cocoa industry through cocoa somatic embryos [12].

5.5 Droplet-vitrification

Droplet-vitrification is a protocol derived from combination of droplet procedure with droplet freezing technique. With regards to all the steps, droplet-vitrification is similar to vitrification method but the only difference is that materials are cryopreserved on foil strips in drops of vitrification solution. It has been successfully used for rubber shoot tips. It has a relatively lower recovery rate of 43% regrowth with pre-culture on basal + proline (0.193 M) for 24 h in the dark at 25°C and PVS2 15 minutes at 0°C.

5.6 V cryo-plate

This method involves the culturing of plant material such as nodal segments in the case of potato on solid MS medium containing 30 g/l sucrose and 0.3 g/l CaCl₂ at 20°C for 2 weeks. The shoot tips are then excised from the in vitro grown shoots and pre-cultured on MS medium containing sucrose at 25°C overnight. Pre-cultured shoot tips are then placed on aluminium cryo-plates with ten wells and embedded with calcium alginate gel. The next step is the performance of osmo-protection by immersing the cryo-plates for 30 minutes at 25°C in 25 ml pipetting reservoirs filled with MS medium with 2 M glycerol and 0.8 M sucrose. For dehydration step, the cryo-plates are transferred and immersed in another reservoirs filled with PVS2 for 30 minutes at 25°C. This is followed by the transfer of the cryo-plate in an uncapped 2 ml cryotube in liquid nitrogen and immersed in a 2 ml cryotube and directly plunged into liquid nitrogen. The cryo-plate is then retrieved for rewarming in the in liquid nitrogen and immersed in a 2 ml cryotube containing 2 ml MS basal medium with 1 M sucrose, in which it is incubated for 15 minutes at room temperature. Rewarmed shoot tips are placed on solid MS medium and cultured under standard conditions [6].

6. Future trends and challenges

There have been several advancements in the identification and use of CPA in cryopreservation procedures. However, CPA toxicity remains a challenge in cryopreservation techniques. Mechanisms of the toxicity of CPA has not been understood fully [1]. Researchers are still working to better understand how different protective chemicals work to protect cells from the rigid temperature of liquid nitrogen.

Cryopreservation protocols have been developed for several crops. However, the number of crops represented in cryo-banks is still limited. Also the ability to successfully repeat the protocol in another laboratory has been a challenge. There is still more room for improvement for the cryopreservation of vegetatively propagated crops and the system requires a lot of optimisation. There is also the need for the development of efficient protocols, challenges related to cryo-banking capacities such as insufficient funding, lack of equipment and infrastructure, inadequate skilled personnel with knowledge on plant genetic resources [10]. The need for the acceleration of plant cryopreservation procedures especially for vegetatively propagated crops requires the development of global expertise. There should be a community of practice initiative involving curators of gene banks, researchers, advocacy organizations, academic institutions and other stakeholders to address the unmet need for cryopreservation advances. Other challenges should be outlined, underinvestment and untapped opportunities should also be identified [14]. There is the need for the establishment of pollen cryo-banks to facilitate a regular supply of pollen to support breeding programs for anther culture activities. The Biotechnology Laboratory at the Council for Scientific and Industrial Research (CSIR)-Crops Research Institute (CRI) in Kumasi, Ghana in sub-Sahara Africa receives germplasm of vegetatively propagated crops such as sweetpotato and cassava from research scientists in Ghana, CIP, International Institute of Tropical Agriculture (IITA) and other centers of the Consultative Group for International Agricultural Research (CGIAR). The current practice for preservation of these plant materials is conservation using slow growth media. Moving forward, the Biotechnology Laboratory in Ghana, should work on developing cryopreservation protocols for preservation of germplasm using liquid nitrogen and cryogenic refrigerators. Also, there is the need for the development of cryotherapy protocols for virus elimination of vegetatively propagated crops sent to the laboratory for in vitro propagation and long-term conservation.

A new threat for conserving global biodiversity in addition to other human activities that could lead to global mass extinction of germplasm is climate change. Recent approaches to in situ conservation are not very reliable to address anticipated changes. Therefore, there is the urgent need for the creation of new cryogenic models, protocols and technologies to mitigate the threats of climate change. Since cryopreservation is the safest and most cost-effective strategy for long-term conservation of germplasm of economically important plant species as well as endangered species [16].

7. Conclusions

Cryopreservation has been in existence since 2000 BC as demonstrated in archaeological findings that icehouse were used throughout Mesopotamia to store foods. In simple terms cryopreservation refers to the study of life at low temperatures. Plant cryopreservation on the other hand refers to conservation method for long-term storage of tissue samples at very low temperatures of −135°C to −196°C. The risk of causing variation is usually less. Cryopreservation methods are suitable are very useful for long-term storage of in vitro cultures of secondary metabolite cell cultures, embryonic cultures, clonal germplasm, endangered species, and transgenic products. The advantages of cryopreservation of plant genetic materials are enormous with several advantages of cryopreservation over other methods. Cryotherapy for virus elimination hold a lot of potential for crop germplasm dissemination.

The development of cryopreservation protocols are enormous. However, the number of crops represented in cryo-banks are still limited. For the acceleration of cryopreservation, there is the need for the development of global expertise. It is recommended that a community of practice initiative involving gene banks, researchers, advocacy organizations, academic institutions and other stakeholders come together to address the gaps in cryopreservation advances

Acknowledgments

The authors wish to express their sincere thanks to scientists and technicians at the CSIR-Crops Research Biotechnology Laboratory who contributed in one way or the other towards the preparation of this book chapter.

Conflict of interest

The authors declare no conflict of interest.