Plant cryopreservation is important for genetic resources that cannot maintained as seeds. For the last decades, sophisticated protocols have been empirically elaborated and comprise shoot tip preparation, osmotic adjustment, cryoprotection, cooling and rewarming. These treatments are accompanied by multiple stresses, i.e., dehydration, cold and chemical toxicity, that induce reactive oxygen species (ROS). ROS can function as signaling molecules influencing gene, redox and antioxidant regulation and may determine the success of post cryogenic survival. To decipher the detailed molecular processes during cryopreservation, the potential of the model plant Arabidopsis is used and efficient protocols have been developed. In particular, plant material can be obtained from homogeneous seeds. Effects of preculture environment and endophytic contamination can be avoided and Arabidopsis shoot tip cryopreservation results in high regrowth percentage after cryo-treatment. Previous cryopreservation experiments have elucidated that members of the transcription factor families WRKY, MYB and AP2 EREBP, such as DREBs/CBFs are involved in the stress response mechanisms. Some are known to transduce ROS signals and are participate in complex networks. Therefore, we may speculate that higher resistance to cryo-stress is related to cross-tolerances with other stresses, such as cold and osmotic stress. Understanding these specific signaling cascades involved in cryopreservation of Arabidopsis could be useful to improve and develop successful cryopreservation protocols for other plant genetic resources.

The genus Allium has been one of the main research objects of the Gatersleben institute for many years. Due to the high proportion of accessions that had been collected from the original sites a comprehensive living collection was established. Maintenance of living material was performed with a spectrum of methods, namely field culture, seed and in vitro storage, and cryopreservation. Vegetatively propagated material is very vulnerable both in the field and also in vitro as recent data on field loss and endophytic threat show. Endophytes may influence plant development in vitro and regeneration of explants without their directly visible appearance. Resulting from this experience, permanent storage in liquid nitrogen turned out to be the safest conservation method in the long term. Suppression of dangerous outbreaks after rewarming is a possible strategy to store material with high bacterial load. Various antibiotics were applied to rescue these explants. A case study of an accession lost in the field and re-established from liquid nitrogen storage is illustrating the value of cryopreservation. The main method of cryopreservation is storage of explants from shoot tips out of the bulbs, cloves, bulbils, or in vitro plants. More recently other organ sources were introduced such as basal parts of young inflorescences. This was performed with garlic. To support the taxonomic research collection, which is difficult to maintain, this method was also adopted for cryopreservation of wild Allium species. Furthermore, the maintenance of the latter very complex collection is supported by pollen cryopreservation. Pollen is a very suitable object for investigations by cryomicroscopy, which is recently a main research topic in the cryopreservation unit. Storage of pollen of the botanical family Amaryllidaceae to which Allium belongs seems to be facilitated by its low water content, which was demonstrated by Differential Scanning Calorimetry. Resulting from the accumulated experience in cryopreservation, recommendations will be given how to apply the various techniques in the complex genebank collection.

Cryopreservation as a method to store plant organs in liquid nitrogen is a widely applied routine procedure. It is suitable for preserving germplasm endangered by genetic erosion, especially for vegetatively propagated material, species that cannot produce storable seeds, and pollen representing vulnerable populations. The IPK applies cryopreservation for large collections of potato (>1400 accessions), garlic, mint shoot tips and Allium pollen. Various methods are routinely applied, such as dimethyl sulfoxide (DMSO) droplet freezing, vitrification, and droplet vitrification. In contrast to well-responding genotypes, however, there are many types, which do not or only poorly respond. The reasons for such differences are unknown and more basic research is needed to elucidate underlying mechanisms. The effects of various cryoprotectants on the state of water in the tissues are measured by differential scanning calorimetry (DSC). In some methods like in DMSO droplet freezing, the presence of freezable water is proved by DSC and tolerated as survival and regrowth rates show. In addition, differences in the structure between meristem and surrounding cells cause differential survival of these tissues in cryopreservation. Likewise viruses are unequally distributed between the various tissue types and may be eliminated by liquid nitrogen treatment. Recently, at the IPK studies on components of cryo-stress were performed. Reactive oxygen species and changes in the proteome and metabolite patterns were investigated. Oxygen was found to play a crucial role in stress-mediated injury and regeneration after rewarming from cryopreservation. The elucidation of components involved in this process allows improvements in large-scale cryopreservation protocols for the valuable germplasm in genebanks.

Pollen cryopreservation was initiated to support preservation of the complex outbreeding Allium species collection at IPK Gatersleben. Pollen germination was counted and results compared with staining by fluorescein diacetate in order to test viability. Germination tests gave more reliable results than staining. A screening of 176 accessions belonging to most of the subtaxa of genus Allium was performed in three subsequent summer seasons. In 159 direct comparisons within 78 Allium species, the mean germination rate of cryopreserved pollen was 78% of the value obtained with fresh pollen. A feasibility study was performed comparing pollen germination and fertilization of female test plants between fresh and cryopreserved pollen. Two species were used, Allium cepa and A. obliquum. In A. obliquum, seed set with cryopreserved pollen was 60% of that obtained with fresh pollen (calculated on a maximum of six seeds per flower), whereas in A. cepa, the results were only 25% of the result obtained with fresh pollen. The individual rates revealed high variability, but no dependence on species, accession, nor on the season. Although the general usability of pollen cryopreservation was clearly shown for genus Allium, further studies are needed to gain more insight into harvest and storage conditions to further improve the germination rates.

At IPK, routine application of cryopreservation started in the year 1998 on potato and garlic and, in 2006, on mint. Allium pollen is the newest target. Main methods are: DMSO droplet freezing in potato, vitrification in garlic, droplet vitrification in mint, and direct cooling of pollen in Allium. Across the years, some changes in detailed protocol steps were introduced improving the methods. Since more than 25 years, accessions of IPK's Potato Collections have been maintained via in vitro culture. For potato, a protocol including microtuber formation was applied since the beginning with only minimal modifications. Garlic, however, is not amenable to in vitro storage over such long periods and quickly declining quality requires using fresh material. Accumulating endophytic colonisation revealed to be the main obstacle for prolonged use of in vitro plantlets as source for cryopreservation. Further investigations were performed on morphogenesis, tissue water conditions and genetic stability. For potato, studies were conducted analyzing the methylation patterns of different potato cultivars under three different maintenance systems (field, in vitro, in cryo) in 2012. Although this study revealed differences in the methylation status of different single plants of the same genotype these differences could not be traced back to one maintenance system in particular. Hence, it can be stated that in cryo maintenance does not induce additional genetic variation. In mint, no differences between the in vitro source plants and regenerants from cryopreservation were found using RAPDs. Cost estimations were done in potato and garlic. With respect to cost factors, the large potato collection had been prioritized depending on the request frequency so far experienced for the various genetic resources.

Allium, including onion, garlic and leek, consists of about 850 species, many of which are recently used or potentially usable. A taxonomic project running at Gatersleben for already more than 30 years elucidated infrageneric relationships and led to the establishment of new systematic treatments. More than 40 collecting missions together with material exchange resulted in a large living collection of 3354 accessions. A considerable proportion of vegetatively propagated material from collecting missions makes this collection very valuable. Field and pot culture is performed for vegetatively maintained material and for seed production. To counteract the vulnerability of field and pot collections, laboratory methods were developed, beginning with slow growth culture on artificial media. Meristem culture was used to clean material from viruses. In vitro storage was found to be possible, but longer storage caused increasing weakness of plantlets and endophytic accumulation. Cryopreservation turned out to be the better alternative resulting in increasing numbers of stored accessions, at present amounting to 84 accessions. Explants from bulbs, bulbils, young inflorescence bases and in vitro cultures are cryopreserved. In garlic, average regeneration of 40% was attained. The method is extended to other taxa such as hybrids and wild species like Allium obliquum. Furthermore, pollen storage was initiated enabling preservation of broader populations of material. Based on the living collections and field studies a herbarium of about 8050 Allium specimens and about 10,000 voucher photographs were assembled. Intensive analyses of general morphology, seed testa structure, flower, scape, root and leaf anatomy, and phenological, biochemical and karyological studies were performed. Along with the development of molecular methods, isoenzyme, RFLP, RAPD, AFLP, and sequence-based analyses resulted in a revised taxonomic classification. The genus Allium proved to be monophyletic and consists of three major evolutionary lineages. Fifteen subgenera are accepted and 79 sections are circumscribed.

The genebank of the IPK contains 216 mint accessions, which are maintained in the field or in vitro. In 2005, routine cryopreservation of seed-sterile mint was started, using axillary shoot tips from cold-acclimated nodal segments and a simple droplet-vitrification method with PVS 2 (Senula et al., 2007). At present, 35 accessions from 12 species are cryopreserved by this method. The average regeneration rate was 65% however, there was high variation in regeneration % (between 13 to 100%), both between accessions and between two repetitions of the same accession. This was correlated to the genotype and endophytes in the in vitro donor plants that were previously found to be free of visible infections. After rewarming, bacterial infections often broke out of and reduced or even inhibited regeneration of plantlets. A screening of endophytes using single donor plants prior to multiplication and cryopreservation was executed. Since bacteria are not homogeneously distributed in the plants, the results of the screening test reflected only the given situation in the tested material proper, but not that in the whole plant. Therefore, the use of these pre-tests alone is not conclusive. Furthermore, some endophytes do not grow on culture media alone, which makes detection difficult. The preventive use of antibiotics in the regeneration medium after cryopreservation resulted in higher plant regeneration, when the donor plants were infected. The antibiotics so far tested (Cefotaxim, Ticarcillin, Vancomycin) delayed the regeneration process, but did not reduce regeneration rates.

Cryopreservation, storage of genetic material in or above liquid nitrogen, is the safest and most cost-effective method to maintain vegetatively propagated plant germplasm and such species, which possess recalcitrant seeds. Experience has been accumulated in many species. Along the main line consisting in explant trimming, dehydration, cryoprotection and temperature reduction several parameters can be variegated. Active reactions of the tissue prior to cryopreservation treatments may be used, such as cold adaptation. Dehydration conditions and cooling speed are other important factors. Finally repair of cryo-injury and regeneration must be investigated. Using these basic components several methods were developed, such as dormant bud method, two-step freezing, vitrification, encapsulation and various droplet methods, also in combination. Although being suitable for most targeted species, considerable differences were found in success rates depending on genotype and presence of endophytes. In vitro storage uses slow growth methods for medium-term preservation. These methodical surroundings to cryopreservation can also be used in parallel to it, especially when material is more often requested. Genebanks need to maintain highly diverse collections and cannot focus on well-investigated model organisms. Therefore, logistic questions are important and considerations concerning regeneration probability, storage safety and economy. Various scenarios can be followed depending on given situations like highly safe storage of important material or emergency rescue in case of acute danger of loss. It is also necessary to assess relations of storage and request frequency. Finally, cryopreservation and in vitro culture methods can also be potential contributions to store seed-producing populations in case of limitations in true-seed reproduction. Criterion for success is safe maintenance of healthy and genetically unchanged germplasm. At present, the genebank of IPK Gatersleben maintains 1307 accessions in cryopreservation, mainly of potato, garlic and mint. The characteristics of in vitro storage and cryopreservation are exemplified using the experience with these crops.

Differential scanning calorimetry (DSC) belongs to thermal analysis methods that can be used for measurement and determination of glass transition phases and crystallization in tissues used for cryopreservation. The aim of this study was to analyze the physical state of water in potato shoot tips during cooling and rewarming applying the DMSO droplet method. Results should give insight into the function of the method. Thermal characterization was done with freshly isolated and cryoprotected shoot tips as well as for the applied solutions. Two accessions were compared. These were the frost-resistant, wild potato Solanum demissum and the frost-sensitive, cultivated potato S. tuberosum 'Desiree'. Influence of cooling rate on thermal characteristics of free water was compared for slowly (10 degrees C/min) and ultra-rapid (ca. 12.000 degrees C/min) directly in liquid nitrogen quenched samples. Results showed glass transitions for the 10% DMSO solution and cryoprotected samples of S. tuberosum 'Desiree'. In contrast, S. demissum did not show glass transition. Freshly isolated and cryoprotected shoot tips as well as the cryoprotectant solution proper showed melting of ice during rewarming. Of the three investigated materials, the amount of freezable water was the smallest in the cryoprotected samples. It can be concluded that DMSO lowers the freezing point and the amount of freezable water in the tissue. The comparison of cooling rates in S. tuberosum 'Desiree' showed two glass transition phases during rewarming of the samples cooled directly in liquid nitrogen in comparison to one transition in samples cooled within the DSC device. Therefore, it can be concluded that the DMSO droplet method is based on ultra-rapid cooling with partial ice formation in the tissue.

The family Alliaceae contains several crop species. Some of them do not form seeds (garlic, A. hookeri, top onions, other primary hybrids) or are, for other reasons, multiplied as clones (shallot, ornamental alliums, Tulbaghia). Furthermore, the outbreeding character causes complications in management of large collections. As field maintenance is labour-intensive and risky and in vitro storage suffers from accumulation of endophytes, cryopreservation is the only safe method for long-term storage of Alliaceae germplasm. Several methods help to improve the cryopreservation success. Thus, preculture of in vitro plantlets at alternating temperatures (25/-1 degrees C) increased the regeneration percentages in garlic from such source organs up to 80%. Using such preculture now regularly, the number of cryopreserved accessions increased to 64 considering the statistical model of Dussert and coworkers for safety storage. Several clones of wild species, A. obliquum and A. hookeri, as well as hybrids were cryopreserved. Two methods were used: vitrification and droplet vitrification using PVS3. The latter method resulted in 24% regeneration in A. obliquum. Preculture of donor plantlets of the hybrid A. cepa x saxatile on higher sucrose (8 vs. 3%) resulted in better regeneration (11.8 vs. 6.7%). Covert endophytes are a problem in Allium cryopreservation. They lead sometimes to unexpected bacteria outbreaks. Although the safety conditions require larger samples of clonal material, storing of populations consisting of smaller clones might be sensible in case of emergency rescue actions.

In living plant collections, vegetatively propagated accessions are outstanding material with respect to vulnerability and labour amount. This is also true for the main vegetative material, held in the IPK, Gatersleben. A survey of the preservation of potato, garlic and other alliums, mint and yam is given. More than 630 accessions are in slow growth conditions. Amongst them, 99 clones of garlic and 35 of shallot have been tested to be virus-free. Cryopreservation is routinely applied for potato using the droplet method. The cryo-collection contains more than 1000 accessions, a part of which has been integrated from another collection, formerly established at Braunschweig. Cryopreservation of garlic has been used to store the accessions of the European core collection. Cryopreservation is successful in three Dioscorea species using a combination of the original vitrification with the droplet method. Investigations about morphogenesis and ultrastructural cell parameters before, during and after cryopreservation were included in these activities.

Allium and Mentha represent two genera with important species of medicinal and aromatic plants. Large proportions of Allium (1000 of 3500) and Mentha accessions (160 of 220) are traditionally maintained vegetatively in field plots in the genebank of IPK Gatersleben. The main species used for medicinal purposes (A. sativum and M. x piperita) are seed-sterile. The field cultivation is endangered by risks (diseases, bad weather conditions). Studies were done on the field infection for LYSV, OYDV, GCLV, SLV and allexi viruses in garlic and some other Allium species. Significant differences were found between species without infection (A. saxatile), species infested by one virus mostly (A. obliquum with LYSV, A. globosum with SLV) and multiple infection (A. sativum). Meristem culture was performed on 100 garlic accessions resulting in virus free clones in 95 of them. Both medicinal plants were maintained in slow-growth culture cycles including storage at reduced temperature (2 or 10 degrees C) on medium MS without hormones for 12 months (garlic) and 15 months (mint). Cryopreservation of garlic was successful with regrowth rates of 100% (clove explants) and 73-80% (bulbil explants). Lower success was achieved with material from in vitro culture. The vitrification technique and the droplet method were compared.

Crops, which cannot be maintained as seeds, need high input, thus requiring development of alternative methods. In vitro culture is used for maintenance and pathogen cleaning. Cryopreservation is increasingly effective in long-term conservation. Whereas most vegetative crops are tropical, potato, Allium, and mint are examples of temperate crops. Potato is maintained for long periods by shoot tip or microtuber storage. Combinations of thermotherapy with meristem culture are used for virus elimination. Several methods work for potato cryopreservation; droplet method is applied in Germany. The main vegetative Allium species, garlic, is virus-cleaned by meristem culture. Slow growth storage works well, whereas only some organs of in vivo plants can be used for cryopreservation. Mint, an important medicinal herb, is maintained by slow growth storage. Vitrification protocols exist for cryopreservation. Important are the physiological state of donor plants prior to explant excision and the cellular water content. Cold hardening may improve storability. For germplasm preservation, contamination and hyperhydration are of increasing influence. Genebanks are often challenged with respect to genetic stability of plant material. However, proof of genetic stability or demonstration of instability are difficult and cannot be established beyond doubt.

A large collection of Allium crop and wild species is maintained at Gaters-leben. Additionally to the field maintenance, in vitro culture and cryopreservation are used to increase the maintenance safety and plant quality. One third of the 3500 Allium accessions are held vegetatively. Vegetative maintenance of germplasm is usually accompanied by virus accumulation. Onion yellow dwarf (OYDV), leek yellow stripe (LYSV), garlic common latent (GCLV), shallot latent (SLV) and allexiviruses have been detected by ELISA and tissue print. Virus susceptibility is species-specific. Together cultivated A. albidum, A. globosum, A. hymenorrhizum, A. lineare, and A. obliquum were more infected than A. rubens, saxatile, and senescens. Similar differences were found in their hybrids with A. cepa. Virus elimination is performed by meristem culture partly in combination with chemotherapy. Virus elimination is better in shallot. (82.2% of 191 tested plants) than in garlic (34.6% of 379 tested plantlets). In garlic, it was possible to establish a virus-free core collection of the most important accessions. This collection has been described by internationally standardised morphological descriptors. Cryopreservation methods are in development for garlic. The cryoprotectant mixture PVS3 (50% glycerol + 50% sucrose) is used. For cryopreservation, meristematic regions of the plants are used. Differences were found in plantlet regeneration with respect to the source organs - ripe cloves, bulbils of different age and size, in vitro cultures. Explants from cloves regenerate well, but they suffer from contamination. Therefore, bulbils are the most frequently used organs. Finally, in vitro cultures offer the chance to use virus-free material for storage, but cryopreservation of them is still difficult. Therefore, several pre-treatments will be necessary such as dehydration and cold hardening.

A part of the Gatersleben garlic (Allium sativum) collection, amounting to 50 accessions has been selected for morphological characterisation and establishment of virus-free in vitro clones. Several characters have been described which more or less closely correspond to previously found isozyme/Random amplified polymorphic DNA (RAPD) groups: the characters describing the inflorescence formation, bulb structures and pseudostem shape have been analysed. The clones have been screened for virus infection by means of 6 different ELISA tests. The infection rates varied from 8 mite borne filaments virus (MbFV) to 94 % garlic common latent virus (GCLV). Most accessions were infected with more than one virus, mostly latent viruses accompanying another form. Meristem cultures were performed to eliminate the viruses. Meristem explants reacted in 8-33 %. The regenerated plantlets have been retested by ELISA, a total of 38 % of the plantlets have been proved virus-free.

An attempt was made to establish a large number of virus- free garlic accessions in vitro. Virus- free in vitro plants of 87 accessions have been obtained via meristem culture. The virus- free status has been determined for the most frequent garlic viruses: OYDV, LYSV, GCLV, SLV and MbFV. In another 7 accessions one or more viruses were still present. The most persistent virus was GCLV. The occurrence of the other viruses was definitely lower. OYDV and LYSV have been eliminated to 85- 95 %. The detection of viruses in the plant was mainly performed by means of the ELISA technique using polyclonal and monoclonal antibodies. ISEM and decoration methods were used for the verification of the ELISA results. The results of ISEM confirmed the virus- free status of plants in all cases, as determined by ELISA. In some cases viruses detected by ELISA could not be found by ISEM or the decoration method. On the other hand, in tests on MbFV with doubtful ELISA results, decoration tests gave a clear indication of virus infection. Small meristems from bulbils with one leaf primordium (0.3- 0.8 mm) were used for the establishment of meristem- tip cultures. The rate of plant regeneration varied between 1 % and 80 % of cultivated meristems and was dependent on the genotype. Meristem culture failed to result in plantlet regeneration in only 6 accessions. The influence of the culture medium was low compared with that of the genotype. The addition of ribavirin to the induction medium reduced the regeneration of plantlets, but virus elimination was significantly increased (from 19.6 to 59.3 %). Hot air treatment of bulbils at 36 degreesC for 6 weeks did not reduce the survival of meristems. It was, however, favourable for virus elimination. Virus elimination was especially high, when thermotherapy and addition of ribavirin to the induction medium were combined.