Establishing cryopreservation methods for conserving european plant germplasm collections

Assessment of the extent of modifications/loss of proteins associated with the cytoskeleton and signalling pathways is a powerful means of pinpointing critical injurious points of cryo-conservation protocols; application of which is used to improve cryo- methods. Result 6 is the development of transglutaminase assays and identification of markers of desiccation and freezing tolerance for profiling cryoinjury; the result can be implemented to improve and design new cryo-conservation protocols for diverse species ensuring wide application potential. Critical success factors: - Formulation of storage solutions based on informed knowledge of cryoinjury rather than empirical know-how. - Increased efficiency of storage development through identification of critical points using markers of protein modification/loss. Hence minimising germplasm sacrificed in cryostorage work up - Enhanced confidence in cryostorage by improved amelioration of cell injury. Innovative benefits: bespoke techniques for application by end users: germplasm repositories/coillections, , botanic gardens and biotechnology companies. Helping to meet their needs to hold germplasm in cryogenic storage as the only long-term means of securing non-orthodox germplasm ex situ. The major strength of the result is the integration of technological and fundamental approaches to provide markers of cell injury that assist the design less damaging cryostorage protocols, particularly for storage recalcitrant germplasm.

Sugar analysis methods have been optimised for very low amounts (down to 1 mg) of the different plant materials generally employed in plant germplasm cryobanks: meristems, seeds, somatic embryos. Using these improved analytical methods, it was clearly shown that tolerance to desiccation and cryopreservation is enhanced by pre-treatments designed to increase the sucrose content of the plant material. Therefore, sugar analysis can constitute a valuable tool to optimise rapidly the pre-treatment step (medium composition, duration), thus reducing the number and/or size of trial-and-error tests usually performed to set up a cryopreservation protocol for any new given accession. Since standard procedures for plant genetic resource cryopreservation usually include a pre-treatment step, by reducing the time required for its optimisation, Result 3 should allow to speed up the introduction of endangered genetic resources in European plant germplasm cryobanks. Important notes: - Increasing the tissue sucrose content is not always sufficient to confer tolerance to desiccation and storage at ultra-low temperature. - Sugar composition alone cannot explain the high variability observed between cultivars or species for their cryo-ability and cannot be employed as a predictor of post-thaw survival.

New / adapted cryopreservation protocols have been established. The protocols are not only applicable to the mandate plant species, which were initially foreseen in the project, but also are readily applicable other plant species. Key developed protocols: Vitrification droplet method for apical meristems and highly proliferating meristem clumps: With this method, cryopreservation of banana became a routine procedure since all banana accessions can now be cryopreserved (up to date, more than 400 banana accessions are stored in liquid nitrogen for long term at K.U.Leuven). Moreover, this method can be efficiently applied to a wide variety of plant species like potato, chicory, strawberry, taro, pelargonium and date palm. Other plant species are currently being tested. Simple freezing method: A very unique method only based on a preculture phase on high concentrations of sucrose. The application of specific sterols, polyamines or aromatic amines as well as some mono-unsaturated fatty acids into the sucrose preculture medium could enhance the post thaw shoot regeneration of proliferating meristems of different banana cultivars. Encapsulation / dehydration cryopreservation method: The encapsulation/dehydration method as applied to Ribes spp. utilized DSC thermal analysis studies to evaluate and optimise the loading of sucrose into the alginate matrix and the critical desiccation times required for silica gel treatment as compared to evaporative airflow. This method is also applicable to garlic, apple and almond, hawthorn, service tree and strawberry tree, etc. The encapsulation method was combined with vitrification for several woody plant species in vitro germplasm types demonstrating that this method is particularly useful in aiding post storage survival in desiccation sensitive systems. Controlled rate freezing method: This method has been applied to olive. Seed cryopreservation method: Five key factors of non-orthodox seed cryopreservation procedures have been investigated for the first time (seed-lot quality, effect of slow drying, rewarming procedure) or re-examined (optimal hydration status, cooling rate). For each of these five factors, important findings were obtained, allowing immediate modifications of standard cryopreservation procedure for coffee seeds and formulation of important new recommendations for the setting up of a cryopreservation protocol in any new given species producing non-orthodox oily seeds. Droplet freezing method: Compared with other methods, the ultra-rapid droplet freezing method is the simplest approach for the cryopreservation of potato germplasm. Innovative benefits: bespoke techniques for application by end user practitioners: germplasm repositories, seed banks, culture collections, botanic gardens and biotechnology companies. Helping to meet their needs to hold germplasm in cryogenic storage provides the only long-term means of securing non-orthodox germplasm ex situ. The major strength of the result is the integration of technological and fundamental approaches to assist end users to cryopreserve their germplasm collections.

Cell membrane systems are the primary site of freezing injury in plant. Freeze tolerance mechanisms depend on the membrane stabilization: through changes in lipid composition, production of membrane protecting polypeptides etc. Sterols and phospholipids are the major components of plant membrane. In order to unravel possible mechanisms involved in cryo-protection on the membrane level, a detailed analysis of sterols, phospholipids and fatty acids bound to different lipid fractions as well as well as free fatty acids should be performed. Sucrose pretreatment and desiccation are both are essential to survive the cryopreservation for many plant species. Result 4 focused on the change in membrane components induced by sucrose pretreatment / desiccation. The knowledge was subsequently implemented to improve the existing cryopreservation protocols for diverse plant species ensuring wide application potential. Key results: - Sucrose pretreatment / desiccation resulted in change in sterols, phospholipids, bound and free fatty acids; - Such changes are closely associated with cryopreservation ability; - Application of specific sterols, fatty acids during the preculture process could improve post thaw regeneration after cryopreservation. This achievement is not only helpful for improve the conservation of plant germplasm but also provides valuable information with respect to possible mechanisms of abiotic stress (especially drought and frost) resistance in plants.

Two training workshops were organized by IPGRI in collaboration with other CRYMCEPT partners. The aim of the workshops was to disseminate the results from the various work packages, in particular the improved cryopreservation protocols and analytical techniques developed by the project partners to relevant scientific and technical staff of selected genebanks and research institutes in Europe and beyond. The workshops had the following two main objectives: - To create awareness among decision-makers on the importance of cryopreservation and how it can be useful effectively to conserve plant genetic resources; and - To train germplasm collection holders on how to develop and optimise cryopreservation techniques and protocols to more effectively conserve germplasm of importance to their regions, countries and institutions. The procedure for the organization of the courses was discussed at the 3rd CRYMCEPT Steering Committee held in Derby in September 2004. During this meeting the venue of the training courses, the contents, resources persons for the course, the selection process for trainees, workshops announcement and training materials and budget were discussed and agreed upon. A selection committee was set up at the third CRYMCEPT steering committee to follow on the workshop arrangements. Close contact was maintained between IPGRI and the workshop hosting institutions (KUL and IRD) and other the members of the selection committee by email and one teleconference was held.

Polyamines are polycations and thus bind readily to important cellular polyanions like DNA, RNA, phospholipids and acidic protein residues and cell wall components. There are more and more indications that polyamine metabolism is also strongly involved in the plant response towards environmental challenges such as osmotic stress, salinity, hypoxia, cold stress and environmental pollutants. Analysis of polyamines and aromatic amines is a best way to understanding possible mechanisms involved in the cryo-protecting process. Sugar (particularly sucrose) pretreatment is essential to survive cryopreservation for most of plant species. Result 5 focused on the change in polyamines and aromatic amines induced by sugar / sucrose pretreatment. The knowledge is implemented to improve the existing cryopreservation protocols for diverse plant species ensuring wide application potential. Key results: - Previously unknown aromatic amines were identified; - Sugar / sucrose pretreatment resulted in change in polyamines and aromatic amines; - Such change is closely associated with cryopreservation ability; - The polyamine level could be comparable to the control during the post thaw regrowth - Application of specific polyamines or aromatic amines during the preculture process could improve post thaw regeneration after cryopreservation. This achievement is not only helpful for improve the conservation of plant germplasm but also provides valuable information with respect to possible mechanisms of abiotic stress (especially drought and frost) resistance in plants.

Techniques have been established for the extraction and 2-dimensional separation of proteins isolated from banana, apple and potato. These techniques have been applied to investigate protein patterns of those tissues, which are used for the cryopreservation of these plants. In banana several defined metabolic changes could be identified which go along with sucrose pre-treatment providing increased cyrotolerance of the plant tissues investigated. In apple the success of any cryopreservation procedure is influenced by two major components: first the survival of plant cells during preculture treatment and cooling and secondly the regeneration process of an intact plant from the thawed tissue. Proteins found to be up- or down-regulated or de novo synthesized during a cryopreservation procedure may contribute either to the first or the second process or to both. Mass spectra analyses of the different proteins will help to better understand the metabolic pathway involved in the tolerance to treatments. In potato it was demonstrated that several protein spots appeared to be up- and down-regulated in an initial overnight incubation phase preceding cryopreservation. Nevertheless a strongly up regulated protein spot indicating a major induced stress mechanisms could not be found in any of the cultivars investigated before the freezing step. Innovative benefits: For banana a major success was the definition of several metabolic changes caused by sucrose pre-treatment. A major success for the work with apple, almond and also potato was the scale down of method for the use of excised apical meristems; in fact proteins were extracted from low amount of material. The techniques can in general be applied for the investigation of stress metabolism.

Monitoring oxidative damage is a powerful means of pinpointing components of storage protocols that cause injury, application of this fundamental knowledge is used to improve cryo-conservation methods. Result 7 is an analytical toolbox for profiling oxidative processes in cryopreserved germplasm the result is implemented to improve and design new cryo-conservation protocols for diverse species ensuring wide application potential. Critical success factors: - Formulation of storage solutions based an informed knowledge of cryoinjury rather than empirical now-how. - Increased efficiency of storage development through identification of critically injurious points using generic oxidative stress markers. - Implicit to minimisation of germplasm sacrificed in cryostorage work up. - Enhanced confidence in cryostorage due to enhanced amelioration of stress. Innovative benefits: bespoke techniques for application by end user practitioners: germplasm repositories, seed banks, culture collections, botanic gardens and biotechnology companies. Helping to meet their needs to hold germplasm in cryogenic storage provides the only long-term means of securing non-orthodox germplasm ex situ. The major strength of the result is the integration of technological and fundamental approaches to provide generic stress markers that assist end users to design less damaging cryostorage protocols, particularly for difficult to conserve, germplasm that hitherto is storage recalcitrant.

The study of the thermal behaviour of water using Differential Scanning Calorimetry (DSC) analysis is a powerful tool to optimise plant cryopreservation protocols based on osmotic or evaporative dehydration before cooling. DSC can be used to predict the optimal: - Time of exposure to the Plant Vitrification Solution 2 (PVS2) (vitrification technique) - Duration of desiccation over silica-gel (encapsulation-dehydration technique) - Period of drying in the laminar air flow of a sterile cabinet (encapsulation-dehydration technique) - Optimal water content for non-orthodox oily seed cryopreservation (equilibrium drying technique). In all cases, DSC is employed to ascertain how to avoid/minimize the formation of lethal intracellular ice crystals in the plant material to be cryopreserved. The study of the thermal behaviour of water could therefore be a valuable way to reduce the number and/or size of trial-and-error tests usually performed to set up a cryopreservation protocol for any new given accession. Since protocols based on the osmotic or evaporative dehydration of the plant material before cooling represent to date the most employed procedures for plant genetic resources cryopreservation, by reducing the time required for protocol optimisation, Result 1 should allow to speed up the introduction of endangered genetic resources in European plant germplasm cryobanks.

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