Cost Action 871

General Overview

This COST Action will focus on the scientific understanding of the effects of cryoprotection, the development of efficient cryopreservation protocols and the dissemination of the findings for application in plant germplasm collections. The scientific programme has been developed with the input of researchers from 17 different COST countries. Taking into account their research priorities 2 main Working Groups (WGs) have been identified.

WG1 : Fundamental aspects of cryopreservation/cryoprotection and genetic stability

Within this WG, two major research topics can be distinguished : 1.1 fundamental aspect of cryopreservation and cryoprotection and 2.1 genetic stability and traceability.

1.1. Fundamental aspect of cryopreservation and cryoprotection

This part mainly aims at the elucidation of the physico-biochemical background of cryoprotection and cryopreservation. For this, physico-biochemical changes associated with cryoprotection will be studied and correlated with post-thaw viability rates.

Specific objectives of this part are:

1. To define changes of the “Key parameters” during the application of different cryopreservation protocols;
2. To determine the relation between these changes and cryopreservation ability
3. To find treatments which improve the cryopreservation ability of plant tissue through a manipulation of the “Key parameters”

1.2. Genetic stability and authenticity

This part mainly deals with the assessment of the genetic integrity of plants to determine if they are ‘true to type’ after cryopreservation.

Specific objectives of this part are;

1. To develop recommendations and techniques to assess genetic stability of plants and plant tissues after cryopreservation
2. To give a guidance to cryobiologists how to limit mutational events and genetic changes during the cryopreservation procedure as well as during storage in liquid nitrogen.

WG1 : Fundamental aspects of cryopreservation/cryoprotection and genetic stability

1.1. Fundamental aspect of cryopreservation and cryoprotection

Currently, for each species and tissue type, a cryopreservation protocol needs to be developed / adapted to the natural cold, freezing and desiccation resistance of the species, explant size and type.

Cryopreservation protocols are largely developed through empirical studies and care is thereby taken to avoid ice intracellular crystallisation during the freezing process causing physical damage to the tissue.

The only way to prevent ice crystal formation at ultra-low temperatures without an extreme reduction in moisture content is through vitrification i.e. non-crystalline solidification of water (Sakai et al., 1990). To obtain a vitrified solution, it needs to be sufficiently concentrated and/or cooling rates need to be high (Fahy et al., 1984).

The existing cryogenic strategies rely on freeze-dehydration, addition of cryoprotective substances including the recently developed plant vitrification solutions, desiccation and acclimatisation or combinations of these processes. It is now generally accepted that the critical step to achieve post-thaw survival lies in the dehydration step and not in the freezing step itself.

The key for successful cryopreservation thus lies in the induction of tolerance to dehydration / desiccation. In practice, this tolerance is induced by sugar treatment, osmotic treatment, cold acclimatisation, ABA treatment, etc., depending on the plant species, tissue type and research group.

The induction of freezing tolerance in nature is now more and more subject to intensive investigations. A wide range of studies indicates that the cell membrane systems are the primary site of freezing injury in plants. Freeze tolerance mechanisms depend on the membrane stabilisation (through changes in lipid composition, production of membrane protecting polypeptides), the water status in the plant cell, the change of the proteins and cytoskeletal proteins, the accumulation of sugars, polyamines and the induction of anti-oxidative mechanisms.

The most damaging physical event during cryopreservation is caused by developing intracellular ice crystals during the cooling/thawing process. This can be prevented through vitrification, i.e. solidification of water in an amorphous (non-crystalline) form. Vitrification of intracellular water can be achieved by reducing the amount of freezable water in cells and/or the use of very high cooling/thawing rates. Studies of all parameters and their relation to cryopreservation ability thus offers considerable potential for the improvement of conservation methods.

Figure : Key parameters involved in cryoprotection

Differential Scanning Calorimetry (DSC) is a well-established method to characterise and identify the water thermal behaviour (phase transitions) over a large temperature range. While the potential of this technique in the understanding of mechanisms involved in the tolerance of plant materials to ultra-low temperature exposure has been demonstrated, its use for plant cryopreservation studies is still rare and should be expanded

It has been observed that cold acclimatisation in nature often leads to the accumulation of proteins like Heat Shock Proteins, Cold Regulated Proteins and dehydrines. Many of these proteins would play an important role in the stabilisation of membranes against freeze-induced injury. A change of the protein pattern through cryoprotection using a sugar treatment was described for somatic embryos of Daucus carota. The most critical aspect of this approach is to find methods which are sensitive enough to detect such proteins and its applicability to the different plant tissues/species of this project. In many cases immunological methods have been used to detect those proteins among other cell proteins. However, immunological methods often identify only a specific protein and antisera do not always show the cross-reactivity between different plant species. Using a 2DE protein analytical method and cross species tandem mass spectrometry identification, it is possible to identify proteins that are significantly up-regulated and down-regulated due to a cryoprotective treatment for banana meristems. The identified proteins represent a broad range of pathways. Down regulated proteins deal with cell growth, cellulose and storage protein biosynthesis while up-regulated proteins are among others related to glycolysis, cell rescue and defence, secondary metabolisms and signal transduction.

Soluble sugars (di- and oligo-saccharides) have been proposed to play an important role in plant tolerance to desiccation and freezing because of their stabilisation of membranes and the vitrification of the intracellular aqueous phase. During the last 15 years, numerous studies, using liposomes, pollen and animal tissues, have shown that desiccation or freeze-induced desiccation can lead to at least two types of membrane damages which cause cell death: lateral phase separation of membrane components and formation of non-lamellar phases, such as the inverse hexagonal phase. The protective effect of soluble sugars on membrane stability during desiccation and freeze-induced-desiccation relies on their ability to form hydrogen-bonds with the polar headgroups of membrane phospholipids and thus to replace water molecules bound to phospholipids in the hydrated state. This would prevent the reduction of the spacing between phospholipids and thus maintaining membranes in a fluid phase, and avoiding lateral phase separation. The second mechanism relies on the fact that sugars are excellent glass formers. Since vitrification of the intracellular aqueous phase can be crucial for survival to ultra-low temperature exposure, this second mechanism could also be involved in the differences in sensitivity to liquid nitrogen exposure observed between different plant materials and after various pre-treatments.

Besides proteins, sterols and phospholipids are the major components of plant membranes. Plant sterols not only are able to regulate membrane fluidity and permeability, but also can modulate the activity of membrane-bound enzymes. It is now also recognised that phospholipids are not only just structure components of membranes, but they can also act as co-factors for membrane enzymes, signal precursors or signalling molecules themselves. Fatty acids of phospholipids influence membrane flexibility and permeability. The unsaturation degree of phospholipid fatty acids is closely associated with abiotic stress resistance in plants. The proportion of non-bilayer phospholipids and destabilising lipids, such as phosphatidylethanolamine (PE) and phosphatidic acid (PA), and destabilizing lipids, such as free fatty acids (FFA), is supposed to be important regarding the deleterious formation of non-bilayer structures, such as inverse hexagonal phase, which follows lateral phase separation of membrane PL according to their calorimetric properties during desiccation or freeze-induced dehydration. In recent years, there is increasing evidence that lipids also function as mediators in many plant processes including signal transduction, cytoskeleton rearrangements and membrane trafficking. For example, free fatty acids (and more specifically oleic acid) are considered as stimulators of the signalling enzyme PLDd, which has an anti-cell death function suggesting that free oleic acid is a signalling messenger and that PLDd is one of its potential targets. In addition, it was also proven that the saturated fatty acids, palmitic and stearic acid were involved in cell regulation.

Polyamines are polycations and thus bind readily to important cellular polyanions like DNA, RNA, phospholipids and acidic protein residues and cell wall components. Their involvement in various cell functions such as cell division, growth, differentiation and senescence has widely been widely recognised. There are more and more indications that polyamine metabolism is also strongly involved in the plant response to environmental challenges such as osmotic stress, salinity, hypoxia, cold stress and environmental pollutants. It was shown that cold acclimatisation as well as an osmotic shock induces the accumulation of polyamines. The mode of action, however, is still far from understood. Binding of polyamines to membranes renders them more stress resistant and reduces leakage. Also aromatic amines may also respond to several abiotic stresses. A correlation between the presence of specific polyamines and cryopreservation ability was described by Ramon and coworkers (2002).

Published evidence indicated that a modified cytoskeleton can improve cell survival after cryopreservation and other studies indicate that both cytoskeletal stability and the activity of Ca2+ dependent protein cross-linking enzyme transglutaminase are both affected differently by the above cryo-preservation pre-treatments. Since such treatments involve physical stresses, they are also likely to affect stress related cell signaling pathways. Hence studies are ongoing towards the identification of key molecular events associated with exposure of cells to different cryo-preservation pre-treatments and post thaw recovery, with particular emphasis on stress related changes in the cytoskeleton and associated enzyme activities involved in its regulation.

Since free radical-mediated oxidative damage is a generic stress response in aerobic organisms, it is also a key factor in cryoinjury. Metabolic uncoupling occurs leading to the production of toxic free radicals (molecules with unpaired electrons), which cause lethal incipient cellular damage by extracting electrons from essential macromolecules. For example, hydroxyl radical adducts of DNA (e.g. 8-hydoxyguanosine) are correlated with mammalian cancers and mutagenesis. Similarly, free radicals can attack proteins and lipids forming highly toxic reaction products such as the unsaturated aldehydic compounds 4-hydroxyalkenals and malondialdehyde. These extremely reactive compounds cross-link with enzymes and proteins (via Michel additions and Schiff’s base reactions) and impair metabolic and regulatory processes. Medical cryobiologists have utilised a fundamental knowledge of free radical damage and antioxidant protection to assist the development of improved low temperature storage protocols. These are based on the avoidance of free radical reactions and the amelioration of oxidative stress through the application of antioxidants and their pharmacological analogs; these approaches have also been successfully applied to freeze-recalcitrant algae. It is now accepted that free radical mediated oxidative stress can potentially occur in all components of plant cryopreservation protocols, including in vitro manipulations, cryoprotection and desiccation. For example, it has been shown recently that desiccation of coffee seeds to optimal water status for cryopreservation (0.2 g H2O.g?1 dw) leads to extensive loss and oxidation of antioxidants, lipid hydrolysis and phospholipid loss.

WG1 : Fundamental aspects of cryopreservation/cryoprotection and genetic stability

1.2 Genetic stability and authenticity

This part mainly deals with the assessment of the genetic integrity of plants to determine if they are ‘true to type’ after cryopreservation.

There are many issues and concerns regarding investigations into the genetic stability of plant germplasm after recovery from cryopreservation. One central theme is the lack of international agreement and consensus in approaching generic solutions acceptable to a wide range of conservation activities. Although, it is recognised, that the effects of cryoinjury upon the genome are often unknown, and any accumulative DNA polymorphisms may not be induced by cryopreservation per se but as the result of the whole culture-cryoprotection-regeneration process, it is generally experimentally difficult to detect what specific stages generate genomic instability. Higher plant cells exhibit the phenomenon of somaclonal variation, which can be manifest as manifold plant variations. Consequently, successful cryopreservation is often judged by the survival of the tissue and ability to regenerate into complete plants, thereby it is desirable to assess the genetic integrity of plants to determine if they are ‘true to type’ after cryopreservation

Techniques to assess genetic stability
The genetic integrity of plants at the phenotypic, cytological, biochemical and molecular levels and its relevance to stability investigations have been reviewed (Harding, 2004; Harding et al., 1997). Briefly, these include:

· Assessments of phenotypic variation with defined morphological descriptors for a given species, whereby stem, leaf, root, flower, fruit and growth habit can be analysed collectively by a range of multi-variate statistical techniques.
· Cytological techniques to detect various types of chromosomal instability. Variant cells with gross chromosomal changes may include: polyploidy, aneuploidy and other mitotic abnormalities.
· Biochemical metabolite/protein (isozyme) profiles that are useful to compare changing patterns in gene expression in plants recovered from cryopreservation.
· Genomic DNA sequences that can be analysed using a range of hybridisation and Polymerase Chain Reaction (PCR) techniques for which several investigations report evidence of stability after cryopreservation.
· Epi-genetic variation in chromatin and DNA methylation of gene sequences which have been found in plants after cryopreservation, suggesting altered patterns of gene expression.

Cryobionomics
The concept of ‘Cryobionomics’ was first introduced and explored by Harding (2004). It addresses the issues and inherent weakness in current genetic stability investigations leading to concerted action for the basis of an acceptable international agreement in the characterisation of cryopreserved germplasm. The conceptual impact of ‘Cryobionomics’ on European germplasm repositories and culture collections is fundamental for this COST Action in order to fully explore its implication.

WG2: Technology, application and validation of plant cryopreservation

Within this WG, two major research topics can be distinguished; i.e. (i) the technology aspects of the different cryopreservation techniques and (ii) Impact and applications: links to and integration with conventional plant resources and genebanks, establishment of cryo-bank, dissemination of results.

2.1. Technology aspects of cryopreservation

This part mainly deals with the applications of cryopreservation protocols to different plant species and tissues.

The cryopreservation technologies that will be used are based on existing protocols that are mainly developed through empirical studies, knowledge of traditional controlled rate freezing and contemporary protocols based on the multidisciplinary fundamental approaches aimed at understanding cryoinjury and tolerance as described under WG1.

Specific objectives of this part are:

1. To develop efficient cryopreservation protocols partially based on the elucidation of the mode of action of cryoprotection /tolerance to cryopreservation.
2. To apply the cryopreservation protocols to a wide variety of plant species/tissues derived from species exhibiting different natural tolerances to cold, freezing and desiccation stress.

2.2. Impact and applications of cryopreservation in plants

This part mainly deals with making links between the more technical aspects of cryopreservation (see previous parts) and plant genetic resources and genebanks, establishment of cryo-bank and dissemination of results

Specific objectives of this part are:

1. To disseminate the improved cryopreservation protocols developed by the COST Action participants to relevant European industries, research institutes and genebanks through the development of a plant cryopreservation manual and the organisation and implementation of specialised training courses.

2. To provide policy makers with first hand information on the role of existing and the potential of the new cryopreservation protocols in the sustainable utilisation of Europe’s plant genetic resources in key application sectors and in meeting the needs of the international conservation agendas.

WG2: Technology, application and validation of plant cryopreservation

2.1. Technology aspects of cryopreservation

This part mainly deals with the applications of cryopreservation protocols to different plant species and tissues.

Two requirements must be met to prevent lethal ice crystallisation during cryopreservation: (i) rapid freezing rates, and (ii) a concentrated cellular solution.

The cell cytosol can be concentrated through air drying, freeze dehydration, application of penetrating or non-penetrating substances (cryoprotectants), or adaptive metabolism (hardening).

For a solution to be vitrified at high cooling rates, a reduction in water content to at least 20-30% is required. Most hydrated tissues, however, do not withstand dehydration to moisture contents needed for vitrification (20-30%) due to solution and mechanical effects.

Exceptions are pollen, seeds, dormant buds and somatic embryos of most temperate seed species.

The key for successful cryopreservation is thus shifted from freezing tolerance to dehydration tolerance.

This tolerance can be induced by chemical cryoprotection with substances like sugars, amino acids, DMSO, glycerol, etc. but can also be induced by adaptive metabolism like cold hardening in temperate species and/or for less cold tolerance species.

All this is subject of investigations executed under WG1. The following cryopreservation protocols can be distinguished.

Air drying (evaporative drying, flash drying)

Classical slow-cooling (or slow-freezing) protocol

Encapsulation/dehydration

Vitrification

Other protocols

Air drying (evaporative drying, flash drying)

This method is directly applicable to seeds, dormant buds, zygotic embryos and pollen of many common agricultural and horticultural species. Flash (or ultra-rapid) drying proved to be beneficial for recalcitrant zygotic embryos of some plant species (Berjak et al., 2000). <top>

Classical slow-cooling (or slow-freezing) protocol

This was the first ‘standard’ protocol that was developed for hydrated plant tissues (Withers and King, 1982). It is based on slow cooling of specimens (at a rate of 0.5-2°C/min) in the presence of a cryoprotectant solution, generally containing DMSO at a 5-15% concentration. When during the slow-cooling process a temperature of about –40°C is reached, the intra-cellular solution is considered to be concentrated enough to vitrify upon a subsequent liquid nitrogen plunging. Now, this method is mainly used for cryopreservation of dormant buds and non-organised tissues, like cell suspensions and calli. This “more traditional” freezing method still has an outermost important place in plant cryopreservation and more specifically in biotechnology, cereal breeding and forestry (e.g. conifers) were they are used extensively, particularly in the commercial sector (Cyr, 2000). <top>

Encapsulation/dehydration

In this method, developed by Fabre and Dereuddre (1990), explants (usually meristems or embryos) are firstly encapsulated in alginate beads (which can contain also mineral salts and organics), thus forming “synthetic seeds” (“artificial seeds” or “synseeds”). Then, the synseeds are treated with a high sucrose concentration, dried down to a moisture content of 20-30% (under airflow or using silica gel) and subsequently rapidly frozen in liquid nitrogen. Although the procedure can be considered rather lengthy and labour-intensive, it is observed that the presence of a nutritive matrix (the bead) surrounding the explant can promote its regrowth after thawing. <top>

Vitrification

First reports on the use of a vitrification solution with plant tissues appeared in 1989 (Langis et al., 1989; Uragami et al., 1989). The technique relies on treatment of explants with a concentrated vitrification solution for variable periods of time (from 15 minutes up to 2 hours), followed by a direct plunge into liquid nitrogen (“vitrification/one-step freezing”). This results in both intra- and extra-cellular vitrification. Freezing rates of about 6°C/sec are normally obtained by plunging explants enclosed in a cryovial into liquid nitrogen. Higher cooling rates can be obtained by enclosing the meristems in semen straws, resulting in cooling rates of about 60°C/sec, or using a “droplet freezing protocol” where the material is placed on aluminium foil strips that are plunged directly into liquid nitrogen, giving rise to cooling rates of 130°C/sec (Panis et al., 2005; Schäfer-Menuhr et al., 1997). The vitrification solution consists of a concentrated mixture of penetrating and non-penetrating cryoprotectant substances. The most commonly applied solution, named “PVS2” (Plant Vitrification Solution n° 2), consists of 30% glycerol, 15% ethylene glycol, 15% DMSO (all v/v) and 0.4 M sucrose (Sakai et al., 1990). Fifteen years after its first report, vitrification is today a widely used cryopreservation protocol. The success of the procedure can be attributed to its easiness, high reproducibility and to the fact that it can successfully be applied to a wide range of tissues and plant species. <top>

Other protocols

Other available methods are the “droplet freezing” (Schäfer-Menuhr et al., 1997), the “preculture method” (Panis et al., 1996), the “encapsulation/ vitrification” (Sakai., 2000) and the“preculture/dehydration” (Dumet et al., 1993). These techniques have been up to now applied to only a limited number of plant species.<top>

WG2: Technology, application and validation of plant cryopreservation

2.2. Impact and applications of cryopreservation in plants

This part mainly deals with making links between the more technical aspects of cryopreservation (see previous parts) and plant genetic resources and genebanks, establishment of cryo-bank and dissemination of results

In Europe, there is a large number of crops, including roots and tubers but also fruit trees, as well as forest tree species for which cryopreservation represents the only option for safe and cost-effective long-term conservation of genetic resources. However, if in vitro slow growth storage is used in a number of genebanks for medium-term conservation of different species, cryopreservation is currently only used routinely in a few exceptional cases only (Frison and Serwinski, 1995; Turok et al., 1995). There is an urgent need to disseminate information on this technology and to train scientists and technicians in genebanks in the implementation of cryopreservation protocols. In this part, the new cryopreservation methods will be applied to the germplasm collections of the collaborating institutes. At the same time the results will be validated and disseminated through a plant cryopreservation manual and specialised training workshops which will be attended by end users (European industries and scientific and technical staff of relevant research institutes and genebanks) as well as by decision makers.

In this part the choice of IPGRI (International Plant Genetic Resources Institute) as a partner is obvious. It is the largest international agricultural research institute involved solely in research on conservation of plant genetic resources. Its role in this COST Action is to help disseminating the results and translating them into practical applications to improve cryopreservation procedures for other crops than those incorporated in this COST Action. In Europe, IPGRI has two main programmes; (i) the European Cooperative Programme for Crop Genetic Resources Networks (ECP/GR) and (ii) European Forest Genetic Resources Programme (EUFORGEN).