The transcription factor WRKY22 is required during cryo-stress acclimation in Arabidopsis shoot tips.

Storage of meristematic tissue at ultra-low temperatures offers a mean to maintain valuable genetic resources from vegetatively reproduced plants. To reveal the biology underlying cryo-stress, shoot tips of the model plant Arabidopsis thaliana were subjected to a standard preservation procedure. A transcriptomic approach was taken to describe the subsequent cellular events which occurred. The cryoprotectant treatment induced the changes in the transcript levels of genes associated with RNA processing and primary metabolism. Explants of a mutant lacking a functional copy of the transcription factor WRKY22 were compromised for recovery. A number of putative downstream targets of WRKY22 were identified, some related to phytohormone-mediated defense, to the osmotic stress response, and to development. There were also alterations in the abundance of transcript produced by genes encoding photosynthesis-related proteins. The wrky22 mutant plants developed an open stomata phenotype in response to their exposure to the cryoprotectant solution. WRKY22 probably regulates a transcriptional network during cryo-stress, linking the explant's defense and osmotic stress responses to changes in its primary metabolism. A model is proposed linking WRKY53 and WRKY70 downstream of the action of WRKY22.

Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.

ATP Content and Cell Viability as Indicators for Cryostress Across the Diversity of Life.

In many natural environments, organisms get exposed to low temperature and/or to strong temperature shifts. Also, standard preservation protocols for live cells or tissues involve ultradeep freezing in or above liquid nitrogen (-196°C or -150°C, respectively). To which extent these conditions cause cold- or cryostress has rarely been investigated systematically. Using ATP content as an indicator of the physiological state of cells, we found that representatives of bacteria, fungi, algae, plant tissue, as well as plant and human cell lines exhibited similar responses during freezing and thawing. Compared to optimum growth conditions, the cellular ATP content of most model organisms decreased significantly upon treatment with cryoprotectant and cooling to up to -196°C. After thawing and a longer period of regeneration, the initial ATP content was restored or even exceeded the initial ATP levels. To assess the implications of cellular ATP concentration for the physiology of cryostress, cell viability was determined in parallel using independent approaches. A significantly positive correlation of ATP content and viability was detected only in the cryosensitive algae Chlamydomonas reinhardtii SAG 11-32b and Chlorella variabilis NC64A, and in plant cell lines of Solanum tuberosum. When comparing mesophilic with psychrophilic bacteria of the same genera, and cryosensitive with cryotolerant algae, ATP levels of actively growing cells were generally higher in the psychrophilic and cryotolerant representatives. During exposure to ultralow temperatures, however, psychrophilic and cryotolerant species showed a decline in ATP content similar to their mesophilic or cryosensitive counterparts. Nevertheless, psychrophilic and cryotolerant species attained better culturability after freezing. Cellular ATP concentrations and viability measurements thus monitor different features of live cells during their exposure to ultralow temperatures and cryostress.

An Improved Cryopreservation Protocol for Mentha spp. Based on Pvs3 as the Cryoprotectant.

BACKGROUND: Vitrification approaches are widely used to cryopreserve Mentha spp. genetic resources. OBJECTIVE: Here, we compared the response of 20 different Mentha species and hybrids during cryopreservation and elucidated the efficacy of two cryoprotectants. MATERIALS AND METHODS: One hundred and fifty three Mentha spp. accessions were cryopreserved using in vitro plants maintained under slow-growth storage and PVS2 or PVS3 as cryoprotectants. RESULTS: The cryoprotectant PVS2 was effective for all species, except M. requienii and M. villosanervata. The use of PVS3 increased the proportion of explants able to regrow after rewarming. The outbreak of endophytes upon rewarming was both less frequent and less severe when PVS3 replaced PVS2. CONCLUSION: Both PVS2 and PVS3 can be used as cryoprotectant for all the species and accessions of Mentha spp. surveyed. Since higher regenerations were achieved using PVS3, and since the risk of an endophyte outbreak was reduced, this cryoprotectant should be preferred in future for cryopreserving Mentha spp.

The changes in the reproductive barrier between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.): different states lead to different fates.

MAIN CONCLUSION: The changes in the reproductive barrier between hexaploid wheat ( Triticum aestivum L.) and rye ( Secale cereale L.) can be induced using in situ embryo rescue of abnormal embryos, yielding stable fertile amphidiploid plants. In intergeneric crosses between hexaploid wheat (Triticum aestivum L.) and rye (Secale cereale L.), postzygotic barriers may occur at different stages of hybrid development. One such mechanism is embryo lethality, which is genetically determined by the interaction and expression of two incompatible genes in wheat (Eml-A1) and rye (Eml-R1). Using in vitro culture methods as stressors, we overcame this hybrid lethality. Normal and abnormal embryos were observed to build embryogenic calli and produce regenerated plantlets in a similar manner. The high regenerative capacity of the abnormal embryos led us to conclude that the reproductive barrier in these intergeneric hybrids may have an epigenetic origin that can be easily overcome by culturing immature embryos via callus induction. After colchicine treatment during callus culture, amphidiploid plants were obtained. However, most of these plants did not produce seeds, due mainly to sterility of the pollen but also of the embryo sacs. These findings demonstrate that hybrid sterility affects both male and female gametophytes in plants obtained from abnormal embryos. The key roles of double fertilization and stress factors in the implementation of the apical meristem formation program in embryos from incompatible intergeneric crosses between hexaploid wheat and rye during in vitro culture are discussed. We also propose a hypothetical model for a wheat-rye lethality system involving differential expression of incompatible wheat Eml-A1 and rye Eml-R1b alleles in an identical genetic background.

A simple method for cryopreservation of shoot tips of Arabidopsis genotypes.

　　BACKGROUND: The use of the model plant Arabidopsis could be a valuable tool to elucidate the basic mechanisms involved in plant cryopreservation. OBJECTIVE: A simple and powerful protocol, independent of Arabidopsis genotypes, was established using a PVS2 protocol. MATERIALS AND METHODS: Two PVS2 (a and b), one PVS3 droplet-vitrification and one DMSO droplet-freezing protocol were tested with alternating temperatures during the growing phase of donor plants. RESULTS: PVS2 protocols, including cold acclimation of donor plants, resulted in highest recovery. The PVS2a protocol was successfully applied to a collection of different Arabidopsis genotypes with an average recovery of 94%. In addition, Differential Scanning Calorimetry confirmed the occurrence of glass transitions in the PVS3 and PVS2 protocols. CONCLUSION: The PVS2a protocol is suitable to screen the large collection of Arabidopsis mutants and transgenic lines with the aim to identify cellular functions associated with cryopreservation tolerance.

Melatonin-loaded alginate beads improve cryopreservation of yam (Dioscorea alata and D. cayenensis).

BACKGROUND: The cryopreservation of yam is constrained with many challenges. OBJECTIVE: This study tested the effects of melatonin on shoot tips of D. alata and D. cayenensis accessions exposed to water and liquid nitrogen (LN) stresses. MATERIALS AND METHODS: Sucrose pretreatment (0.3 M) was applied for 48 h before cryopreservation. Shoot tips were encapsulated in beads loaded with 0.75 M sucrose, with and without melatonin and desiccated over sterile dry silica gel for 0 - 9 h. RESULTS: The beads moisture content declined from 100% to ~ 13% after 9 h. The 3 h desiccation period without melatonin produced a significantly higher regeneration compared to 6 h and 9 h. Shoot tips with melatonin had significantly higher regeneration after 3 - 6 h desiccation compared to 9 h and the regeneration of all accessions after 6 h was >80%. Regeneration following 6 h desiccation and LN was significantly greater for melatonin-treated shoot tips compared to non-treated ones. CONCLUSION: The results indicate that melatonin significantly increased regeneration from 15% to 35%.

Influence of oxygen deficiency and the role of specific amino acids in cryopreservation of garlic shoot tips.

BACKGROUND: Garlic has lost its ability to form seeds in the course of its domestication. Therefore, the germplasm storage via cryopreservation is increasingly applied. The progression of the various steps within the cryopreservation procedure is accompanied by declining survival rates of the explants. Much of the recent work on cryo-stress has been focussed on osmotic and cold stress components. However, two decades after invention of garlic cryopreservation, the function of metabolites and oxygen in and around the cryopreserved tissues is still largely obscure. METHODS: In this study, hypoxia was characterized in cryopreservation of garlic with oxygen sensors and amino acid metabolism. Furthermore, malondialdehyde, soluble sugars and ammonium were quantified to demonstrate the influence of cryo-stress in declining survival rates. RESULTS: To better understand the possible reasons for a reduction in the survival rate at the subsequent steps of cryopreservation, the concentration of amino acids, ammonium, γ-aminobutyric acid (GABA), soluble sugars, malondialdehyde (MDA), and oxygen were measured in garlic shoot tips undergoing cryopreservation. Using microsensors, a very low oxygen concentration (<0.1 µM) was detected within the central meristem region of the shoot apex. When apices were immersed in cryoprotectant solution, the well-oxygenated peripheral regions (foliage leaf bases) became likewise hypoxic within a few minutes, probably resulting from strongly restricted gaseous diffusion. CONCLUSIONS: Tissue level oxygen measurements supported the occurrence of hypoxia while biochemical analysis indicated adaptive responses, in particular the modulation in alanine and glutamate metabolism. The possible role of serine and glycine metabolism during cryopreservation is also discussed.

Three vitrification-based cryopreservation procedures cause different cryo-injuries to potato shoot tips while all maintain genetic integrity in regenerants.

We previously reported successful cryopreservation of shoot tips of potato 'Zihuabai' by three vitrification-based protocols. In the present study, cryo-injury to shoot tips and genetic stability in regenerants recovered from cryopreserved shoot tips by the three vitrification-based protocols were further investigated. The results showed that sucrose preculture caused no obviously different injuries, while dehydration with plant vitrification solution 2 (PVS2) was the step causing major damage to cells of shoot tips, regardless of the cryogenic procedures. Compared with droplet-vitrification and encapsulation-vitrification, vitrification caused the most severe injury to cells of the shoot tips, thus resulting in much longer time duration for shoot recovery and much lower shoot regrowth rate. Cells in apical dome and the youngest leaf primordia were able to survive and subsequently some of them regrew into shoots following all three vitrification-based cryopreservation procedures. Analyses using inter-simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) markers in shoots regrown from all three vitrification-based protocols did not find any polymorphic bands. The results reported here suggest that vitrification-based cryo-procedures can be considered promising methods for long-term preservation of potato genetic resources.

Micropropagation and cryopreservation of garlic (Allium sativum L.).

Garlic (Allium sativum L.) is a very important medicinal and spice plant. It is conventionally propagated by daughter bulbs ("cloves") and bulbils from the flower head. Micropropagation is used for speeding up the vegetative propagation mainly using the advantage to produce higher numbers of healthy plants free of viruses, which have higher yield than infected material. Using primary explants from bulbs and/or bulbils (shoot tips) or unripe inflorescence bases, in vitro cultures are initiated on MS-based media containing auxins, e.g., naphthalene acetic acid, and cytokinins, e.g., 6-γ-γ-(dimethylallylaminopurine) (2iP). Rooting is accompanying leaf formation. It does not need special culture phases. The main micropropagation methods rely on growth of already formed meristems. Long-term storage of micropropagated material, cryopreservation, is well-developed to maintain germplasm. The main method is vitrification using the cryoprotectant mixture PVS3.

Influence of cryopreservation on the cytosine methylation state of potato genomic NDA.

Shoot tips of Solanum tuberosum (Désirée) were successfully cryopreserved by the DMSO droplet method and stored for almost 7 years, while control material was maintained in vitro for the same period of time. To analyse potential epigenetic changes, the DNA methylation status was assayed by methylation-sensitive amplified polymorphism (MSAP) analysis using restriction endonucleases MspI and HpaII. An amount of 93.6% of the analysed MSAP signals were stable among all cryopreserved and in vitro maintained samples tested, indicating extensive stability of DNA methylation. Only 0.9 % of MSAP signals showed results that differed between the two treatments and at the same time matched for all three biological replications within each treatment. These can be seen as indicating directed effects of the two treatments on the DNA methylation. Cryopreserved samples displayed in comparison to in vitro stored samples consistent hypomethylation for 0.6 % (3 of 469) of MSAP signals (Table 4, pattern 4) and consistent hypermethylation for 0.2% (1 of 469), respectively. For 5.6% of all MSAP signals, inconsistent results were observed among the three biological replications at least for one of the two treatments. These were interpreted as resulting from stochastic DNA methylation changes in individual samples. As results for two biological replications were identical and different from the result for the third biological replication, the direction of methylation change could be determined in those cases. Cases of stochastic loss of CG methylation in cryopreserved samples were most frequent among them, adding up to 3.4% of MSAP signals. Stochastic loss of CG methylation was also found in material maintained in vitro, only for 0.6% of all MSAP signals. In conclusion, methylation changes occurred in long-term cryopreservation of potato, in a random rather than directed fashion. Hence, cryopreservation and long-term in vitro maintenance both induce limited changes of DNA methylation status. The order of magnitude of methylation changes observed was consistent with other studies, where similar rates of DNA methylation changes have been found.

The effect of olestra on the absorption, excretion and storage of 2,2',5,5' tetrachlorobiphenyl; 3,3',4,4' tetrachlorobiphenyl; and perfluorooctanoic acid.

Mice were gavaged with either (14)C-labeled 2,2'5,5' tetrachlorobiphenyl; 3,3',4,4' tetrachlorobiphenyl; or perfluorooctanoic acid. Absorption of these compounds was determined by assay of feces collected for 48 h after the gavage. Part of the animals received test diets containing olestra during this 48-hour period to determine its effect on absorption of the compounds. Mice that received the diet without olestra during this period were divided into groups that either continued the diet without olestra or changed to a diet containing olestra. These diets were continued for 7 days, and a second 48-hour fecal collection was made to measure the effect of olestra on enterohepatic circulation of the compounds and their metabolites. The animals were sacrificed, and blood, fat, and liver concentrations of (14)C were measured. Olestra decreased the absorption of 2,2',5,5' tetrachlorobiphenyl. It also reduced tissue and blood concentrations of this compound. Olestra also decreased the absorption of 3,3',4,4' tetrachlorobiphenyl, but it did not alter enterohepatic circulation or tissue concentrations. Olestra significantly increased the excretion of perfluorooctanoic acid in the second 48-hour collection, suggesting an effect on enterohepatic circulation. It did not, however, alter tissue concentrations of perfluorooctanoic acid. These data are consistent with previously observed effects of olestra on the absorption and storage of lipophilic compounds.

Influence of alternating temperature preculture on cryopreservation results for potato shoot tips.

Cryopreservation is the most suitable long-term storage method for genetic resources of vegetatively maintained crops like potato. In the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) the DMSO droplet method is applied, and so far more than 1000 accessions are cryopreserved with an average regeneration rate of 58%. New experiments with four potato accessions using alternating temperatures (22/8 degrees C day/night temperature, 8 h photoperiod, 7 d) prior to cryopreservation showed improved regeneration. The influence of this preculture on the shoot tips was studied for two wild, frost resistant species Solanum acaule and S. demissum and for two cultivated, frost sensitive potatoes S. tuberosum 'Désirée' and 'King Edward'. Comparison of liquid and solid media after cryopreservation showed improved regeneration on solid media with higher regeneration percentages, less callus formation and better plantlet structure. In comparative analyses biochemical factors like soluble sugars, starch, and amino acid concentrations were measured. Shoot tips after constant and after alternating temperature preculture were analyzed. Total concentrations of soluble sugars (glucose, fructose, and sucrose) were higher for all accessions after the alternating temperature preculture, which could be the reason for improved cryopreservation results.

Ultrastructural changes associated with cryopreservation of potato (Solanum tuberosum l.) shoot tips.

The ultrastructure of cells within shoot tips of S. tuberosum 'Désirée' was studied after different steps of the DMSO droplet cryopreservation method. After 2 h of DMSO treatment, cells contained numerous small vesicles, while at the same time mitochondria and chloroplasts had increased in size and vacuoles had assumed an irregular shape. After rapid cooling in liquid nitrogen, subsequent rewarming, and 1 h incubation there were no apparent changes in the ultrastructural organization of the cells, suggesting that they might be still intact. However, two days after rewarming, the meristematic dome area and part of the epidermis showed signs of extensive damage. Rupture of plasmalemma, plasmolysis and destruction of cell organelles as well as strong heterochromatisation of nuclei were observed. Survival and regeneration of cells were found mainly in leaf primordial regions. Here cells were very active, containing many mitochondria and intact or regenerating chloroplasts. Alternating temperature preculture of donor plants before shoot tip isolation improved the cryopreservation results (plant regeneration 46.5 percent) as compared to constantly warm precultured shoot tips (plant regeneration 20.0 percent), which showed slightly stronger damage after rewarming from liquid nitrogen.

Cryopreservation for plant genebanks - a matter between high expectations and cautious reservation.

Cryopreservation is the best method of storing germplasm efficiently and safely, particularly for the maintenance of vegetatively propagated material. In IPK cryopreservation is used for potato, garlic, mint and yam. IPK collaborates with other cryobanks and research groups (ECPGR, COST, EURALLIVEG) and finds considerable differences in the adoption of cryopreservation between crops and their host institutes, depending on crop, local and historical circumstances. A better understanding of the long-term benefits of cryopreservation and its further integration into general genebank management is therefore needed. Recommended approaches include: comparative validation of methods between different laboratories, detailed comparisons of crop-based methods, economical analyses, efficient integration strategies of cryobanks by genebanks; including safe duplication of cryopreserved resources for the limitation of risk of loss Importantly, there has been recent progress in the development of quality management systems. Cryopreservation is, however, characterized by high expectations. Therefore, to ensure its sustainable and practicable use, basic knowledge of storage protocols must be combined with increased awareness of the rationales required to validate, implement and apply cryobanking technologies in working genebanks.

Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol.

Accessions of Mentha x piperita, M. x villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant re-growth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25 degree C or in alternating temperature regimes (25/15C or 25/-1C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25/-1C regime than in plants cultivated at 20C or 25C or at 25/15C regime for all nine tested accessions. The mean re-growth levels increased from 36 percent at 20C to 69percent at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89 percent. A pre-culture at alternating temperatures of 25/15C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.

Cryopreservation of yams using vitrification modified by including droplet method: effects of cold acclimation and sucrose.

Cryopreservation of yams (Dioscorea spp.) is important for the preservation of valuable genotypes for food, medicine and breeding purposes. This study on four species of yams was conducted to evaluate the influence of cold acclimation in an alternating 5 degree C and 28 degree C, 12 h thermo-photo-period and of two sucrose concentrations in the preculture medium using a modified droplet method. Acclimation of a 3-week period provided the best preconditioning treatment averaged over four genotypes. Effect of sucrose concentration in the preculture medium depended on the genotype (significant genotype x sucrose interaction; P = 0.036). High survival (67 to 70%) with 30% to 50% shoot recovery was obtained for D. bulbifera, D. polystachya and D. cayenensis, compared to 20 percent survival without shoot recovery for D. alata.

Improvement of cryopreservation results in garlic using low temperature preculture and high-quality in vitro plantlets.

The efficiency of garlic cryopreservation is, amongst other factors, depending on the origin of the donor explants. So far, in vitro grown material has always been the least responding one with respect of the regrowth rates. On the other side, the possibility to produce virus-free material via meristem culture and to keep these clones then under isolated conditions in a clean culture induced studies to increase the efficiency of cryopreservation using this kind of material. Experiments have been performed to use various materials and cultivation temperatures for a vitrification protocol. Best results (up to 70 percent regrowth) were obtained with cultures grown for only 10 months under in vitro conditions including a cold preculture of two months either at alternating or at permanently low temperatures. The conclusion was drawn that the quality of the explants and temperature conditions play a major role for the efficiency of cryopreservation using in vitro plantlets.

Chemical characterization of interspecific hybrids between Allium cepa L. and Allium kermesinum Rchb.

Interspecific hybridization between wild and cultivated species of the genus Allium has been performed to generate plant material possessing biochemical properties of both parental plants. These cross-breeding experiments should lead to Allium plants with higher amounts of valuable constituents. The chemical characterization of interspecific hybrids between A. cepa and A. kermesinum is described on the basis of their sulfur-containing constituents and secondary metabolites. In addition, the hybrid character has been proven by random amplified polymorphic DNA (RAPD) analysis of the progenies obtained from the crosses. It has been shown that the distribution of the cysteine sulfoxides as well as the volatile secondary metabolites in the hybrids is not uniform. The profiles are mainly determined by the paternal wild species A. kermesinum. It has been ascertained that the gas chromatography profiles of the hybrids show increasing amounts of unsymmetrical substituted oligosulfides, which are known to be physiologically active substances. On the basis of statistical calculations, three different types of hybrids can be separated. The chemical analysis of cysteine sulfoxides and volatile sulfur-containing substances is shown to be a useful tool for breeding purposes as it allows an effective selection with regard to optimal distribution and amount of valuable constituents.

A comparison of Guatemalan and USA nurses' attitudes towards nursing.

BACKGROUND: Student nurses from the United States of America (USA) spent 5 weeks working with Guatemalan nurses in an acute care setting in Guatemala. This experience led to a heightened awareness of the global scope of nurses' discontent and a desire to better understand the driving factors and drawbacks to practising nursing in both the USA and Guatemala. AIM: The purpose of this research was to identify those factors that discourage nurses and those that motivate nurses to continue in their practice despite the drawbacks. METHOD: Qualitative interviews using field notes were conducted with five Guatemalan and five USA nurses. Themes were derived through qualitative content analysis. FINDINGS: Nurses in both the USA and Guatemala had similar reasons for choosing and staying in nursing. The different health care systems presented different problems resulting in different discontents. CONCLUSION: The two groups of nurses had much in common, especially in their reasons for staying in nursing. The Guatemalan nurses were most discontent with the lack of resources to treat patients, while the USA nurses focused on work environment drawbacks. IMPLICATIONS FOR PRACTICE: Strategies to support nurses and nursing in developing countries need to be developed and implemented. As nurses reach out to their colleagues in other nations, understanding our commonalities and differences will help us to support each other in improving health throughout the world.