Cryopreservation of somatic embryos of paradise tree (Melia azedarach L.)

In paradise tree (Melia azedarach L.), immature zygotic embryos sampled from immature fruits are the starting material for the production of somatic embryos. These somatic embryos are employed for freezing experiments. Immature fruits could be stored at 25 degrees C for up to 80 days without impairing the embryogenic potential of zygotic embryos, which represents a four-fold increase in immature fruit storage duration, compared with previous studies. Among the three cryopreservation techniques tested for freezing paradise tree somatic embryos, namely desiccation, encapsulation-dehydration and pregrowth-dehydration, only encapsulation-dehydration and pregrowth-dehydration led to successful results. The optimal protocol was the following: i) somatic embryos (encapsulated or not) pretreated in liquid Murashige & Skoog medium with daily increasing sucrose concentration (0.5 M/0.75 M/1.0 M); ii) dehydrated with silica gel to 21 - 26% moisture content (fresh weight basis), for encapsulation-dehydration, or to 19% moisture content, for pregrowth-dehydration; iii) frozen at 1 degree C/min from 20 degrees C to -30 degrees C with a programmable freezing apparatus; iv) rapid immersion in liquid nitrogen. The highest recovery achieved was 36% with encapsulation-dehydration and 30% with pregrowth-dehydration. Regrowth of frozen embryos was direct in most cases, as secondary embryogenesis originating from the root pole was observed on only around 10% of cryopreserved somatic embryos. Plants recovered from cryopreserved embryos presented the same phenotypic traits as non-frozen control plants.     

Is sperm cryopreservation at -150 degree C a feasible alternative?

A series of experiments was carried out to validate a -150 degree C ultra-low temperature freezer for its possible use to properly freeze and store semen. In the first part, crude sample handling was simulated to see whether temperature of stored samples was maintained within a safe range; also, the freezing point and latent heat of fusion plateau of a semen extender were monitored. In the second part, buck semen was (i) frozen in liquid nitrogen and stored in the ultra-low freezer, (ii) frozen and stored in the ultra-low freezer, and (iii) frozen and stored in liquid nitrogen, to compare sperm cryosurvival between freezing methods. Both, frequent removal of samples and long opening of the freezer door did not negatively affect stored sample temperature; latent heat of fusion plateau was 5 minutes long. Semen stored either at -150 degree C or at -196 degree C cryosurvived similarly after 2 days and after 2 months of cryopreservation.     

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells without dimethyl sulfoxide

Cryopreservation of umbilical cord blood-derived mesenchymal stem cells (UCB-derived MSCs) is crucial step for its clinical applications in cell transplantation therapy. In the cryopreservation of MSCs, dimethyl sulfoxide has been widely used as a cryoprotectant (CPA). However, it has been proved that DMSO has toxic side effects to human body. In this study, DMSO-free CPA solutions which contained ethylene glycol (EG), 1, 2-propylene glycol (PG) and sucrose as basic CPAs, supplemented with polyvinyl alcohol (PVA) as an additive, were developed for the cryopreservation of UCB-derived MSCs. The cryopreservation of UCB-derived MSCs was achieved by vitrification via plunging into liquid nitrogen and by programmed freezing via an optical-DSC system respectively. The viability of thawed UCB-derived MSCs was tested by trypan blue exclusion assay. Results showed that the viability of thawed UCB-derived MSCs was enhanced from 71.2% to 95.4% in the presence of PVA for vitrification, but only < 10% to 45% of viability was found for programmed freezing. These results indicate that PVA exerts a beneficial effect on the cryopreservation of UCB-derived MSCs and suggest the vitrification in combination with the dimethyl sulfoxide free CPA solutions supplemented with PVA would be an efficient protocol for the cryopreservation of UCB-derived MSCs.     

Cryopreservation of embryogenic callus of Aesculus hippocastanum L. by vitrification or one-step freezing

An effective procedure for the cryopreservation of horse chestnut (Aesculus hippocastanum L.) embryogenic callus by vitrification/one-step freezing is described here. In particular, the study focused on the possibility of recovering the full proliferation potential of the embryogenic lines after storage in liquid nitrogen. The developmental stage of the embryogenic lines was shown to play an important role. Ninety-min incubation in PVS2 and preservation at -196 degrees C of callus samples, containing a prevalence of embryogenic masses at an advanced stage of somatic embryo maturation (i.e., the torpedo stage), gave optimum regrowth of healthy and proliferating embryogenic callus. Moreover, raising the thawing temperature to 45 degrees C yielded the maximum survival (94%) of torpedo-stage embryogenic samples, recovery of proliferation and, in more than 70% of cases, maturation to the cotyledonary stage. This study opens the way to the possibility of safe, long-term storage in liquid nitrogen of valuable embryogenic lines of horse chestnut, avoiding repeated subculturing.     

Cryopreservation of cryosensitive basidiomycete cultures by application and modification of perlite protocol

Homolka's perlite protocol (HPP) for cryopreservation of fungal cultures was evaluated in 12 strains (7 species) of cryosensitive basidiomycete cultures maintained in NBRC culture collection by investigating viability, time to recover, and basic morphological study after freezing and storing at -80 degree C for 6 months. The viability of the fungal strains was 60 percent in Phallus hadriani and 100 percent in remaining 11 strains, indicating the efficacy of HPP method for cryopreservation of some cryosensitive basidiomycetes. The HPP method was modified by changing the addition of cryoprotectant (glycerol) from prior precultivation to post precultivation, limiting the cryoprotectant exposure time to 48 hours, and increasing the glycerol concentration from 5 percent to 12 percent. The viability of P. hadriani strain increased from 60 percent to 100 percent with the modified perlite protocol after storage at -80 degree C for 6 months.     

Cryopreservation of Quercus suber and Quercus ilex embryonic axes: in vitro culture,desiccation and cooling factors

This study examines different factors included in the cryopreservation protocols for Quercus ilex and Q. suber embryonic axes. In vitro incubation temperature played an important role in the appropriate development of Q. ilex axes, as 15 degrees C was superior to 25 degrees C. Q. suber axes proved to be more sensitive to desiccation and cooling. Poor survival (35%) was observed when axes were included into cryovials and then in liquid nitrogen, and none when immersed in sub-cooled liquid nitrogen (-210 degrees C). Q. ilex axes showed poorly organised development in vitro (c. 50% of non-cooled axes showed shoot development). However, c. 80% survival was observed after cryopreservation (either in liquid nitrogen or sub-cooled liquid nitrogen at 0.34 g water / g dry weight), of which c. 15% showed shoot development.     

Vitrification of human spermatozoa without cryoprotectants

The aim of our study was to investigate the effects of vitrification (cooling rate approximately 10000(C/min) without cryoprotectants on swim-up prepared human spermatozoa in comparison to standard conventional freezing with cryoprotectants. Motility, morphology, rate of viability and acrosome reaction of spermatozoa were evaluated. The described method of cryopreservation of human spermatozoa by direct plunging into liquid nitrogen slush without cryoprotectants was effective and could be recommended for routine IVF.     

Seed cryopreservation and longevity of two Salix hybrids

The effects of desiccation and storage temperature on the viability and longevity of willow seeds was investigated using two hybrids, Salix rehderiana x (Salix x capreola) [cross 458] and Salix x sericans x Salix viminalis [cross 512]. Freshly harvested seed of both crosses survived silica gel drying down to c. 3 to 5% moisture content. Hybrid 458 seed stored in liquid nitrogen (-196C) for 3 d retained viability when equilibrated to < or = 45% RH (pre-storage), showed slightly reduced survival at 65% RH and exhibited no survival at > or =82% RH. The level of survival after 68 d for seeds pre-equilibrated to either c. 10 or 65% (5 or 10% moisture) and stored at four temperatures was -196C > -20C > 2C > 16C. At all temperatures, drier seed stored better than wetter seed. For hybrid 512, seed longevity at 20C > 40C > 60C, and a 10% fall in pre-storage seed RH resulted in a c. 2-fold increase in longevity at each storage temperature. The response of hybrid willow seeds to desiccation and cooling raises possibilities for the long-term seed conservation of Salix species by cryopreservation.     

Mushroom cryopreservation and its effect on survival, yield and genetic stability

Mycelial stock cultures of Agaricus bisporus, A. Bitorquis, Pleurotus flabellatus, P. Sajor-caju, P. Ostreatus, P. Sapidus, Auricularia polytricha, Lentinula edodes, Morchella esculenta and Volvariella volvacea were maintained by frequent subculturing at an interval of two months and separately as wheat grain spawn in liquid nitrogen with 15 percent glycerol. Preservation of mushroom stock cultures as wheat grain spawn under liquid nitrogen proved to be the better method of maintenance. The percent recoveries of stored samples were unchanged from the first recovery after six months to the last recovery after 42 months in nine out of 11 stock cultures preserved under liquid nitrogen. However, a marginal decline in survival of 10 % was recorded in Auricularia polytricha and Volvariella volvacea. Yields before preservation of mushroom stock cultures and after 30 months of preservation exhibited static biological efficiency and fruitbody weight. The comparison of Random Amplified Polymorphic DNA (RAPD) and Internal Transcribed Spacers (ITS) PCR amplified products did not exhibit DNA fragment variation in banding patterns at the intraspecific level during preservation of stock cultures by either method. The modified cryopreservation protocol and experimental demonstration of genetic stability of stock cultures reported here validate the use of mushroom cryopreservation techniques and supports studies on genetic stability of preserved biological materials.     

The incidence of mitotic abnormalities in cryopreserved eight-cell early and compacted mouse embryos

The effects of slow freezing-rapid thawing on viability and chromosomal complement of eight-cell early and eight-cell compacted mouse embryos were investigated. The abnormalities connected with damage to the mitotic apparatus, and with chromosome damage were investigated in cryopreserved embryos by cytological analysis. The embryos were preserved using 1M glycerol as cryoprotectant and a slow cooling regime to -30 degrees C before transfer to liquid nitrogen. The proportion of mitotic abnormalities in compacted embryos was significantly higher (13.1%) than in early embryos (5.9%) and unfrozen control embryos (3.9%) in these studies performed after thawing. This was reflected after cryopreservation by a reduced viability of the embryos as judged by culture to the hatching blastocyst stage--compacted 8-cell embryos (71.8+/-4.7%) versus controls (78.4+/-5.9%) and cryopreserved early-stage 8-cell embryos (86.1+/-4.0%)--p<0.05 in each case. However, aneuploidy rates were low in all groups in both fresh and cryopreserved embryos (around 3%).     

Optimization of cryopreservation of stem cells cultured as neurospheres: comparison between vitrification, slow-cooling and rapid cooling freezing protocols

We compared cryopreservation of mammalian neural stem cells (NSCs) cultured as neurospheres by slow-cooling (1 C/min) in 10% (v/v) DMSO and cryopreservation by immersion into liquid nitrogen in ethylene glycol (EG)-sucrose solutions that support vitrification (40% (v/v) EG, 0.6 M sucrose) or that do not (37% v/v) EG, 0.6 M sucrose and 30% (v/v) EG, 0.6 M sucrose); the concentration of penetrating cryoprotectant in the last two solutions was lowered with the intention to reduce their toxicity towards NSCs. To protect against contamination a straw-in-straw technique was employed. Vitrification offered the best combination of preservation of structural integrity of neurospheres, cell viability (>96%), multipotency and karyotype. Rapid cooling in 37% (v/v) EG, 0.6 M sucrose afforded good viability but did not preserve structural integrity. Rapid cooling in 30% (v/v) EG, 0.6 M sucrose additionally reduced cell viability to 77%. Slow-cooling reduced cell viability to 65% and damaged the neurospheres. This study suggests that, in contrast to freezing, vitrification has immense potential for the cryopreservation of stem cells cultured as neurospheres or in other structured cultures.     

Low temperature survival of slender naiad (Najas flexilis) seeds

The response to drying and storage at -20 degrees C or in liquid nitrogen was studied in seeds of the freshwater aquatic plant Najas flexilis. The seeds of this species show some desiccation sensitivity, although post-harvest storage in water at 16 degrees C resulted in improvements in desiccation tolerance. There was 63% germination of seeds dried to 9.5% moisture content (30% RH) following this maturation period. Optimum moisture contents for seeds stored at -20 degrees C for 3 months and in liquid nitrogen for 1 week were ~11% and ~15%, respectively.     

The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs

The short-term storage (at 0 degrees C) and cryopreservation of spermatozoa may be useful for providing gametes for fertilisations performed in programmes for the conservation and management of endangered amphibians. The current study was undertaken to examine the applicability of amphibian spermatozoa storage protocols developed with the cane toad (Bufo marinus) to a wider range of amphibian species, with a view to ultimately using these protocols for endangered species. In Australia, at least 29 species of recently extinct or endangered frogs are from the families the Myobatrachidae and the Hylidae. This study investigated the applicability of short-term storage and cryopreservation protocols developed for cane toad (Bufo marinus) spermatozoa to those of hylid and myobatrachid species. Storage of spermatozoa in intact testes or in suspensions for six days at 0 degrees C showed spermatozoa maintained higher motility in suspensions than those in testes, and hylid spermatozoa maintained greater motility than myobatrachid spermatozoa. However, the protocols for optimal storage at 0 degrees C varied with testis size when spermatozoa were stored in whole testes. Spermatozoa from 13 frog species representing both families were cryopreserved using sucrose as diluent with Me(2)SO or glycerol as cryoprotectants. After cryopreservation hylid spermatozoa showed a greater recovery than myobatrachid spermatozoa and Me(2)SO provided higher recovery than glycerol. The freeze-thaw recovery of spermatozoa was independent of testes weight of the species studied. These results show spermatozoa from the Hylidae and Myobatrachidae may be stored both in the short-term (at 0 degrees C) and long-term by cryopreservation using protocols established for B. marinus.     

Recovery of potato apices after several years of storage in liquid nitrogen

A method for the systematic cryopreservation of potato apices was developed by the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and the Institute for Crop and Grassland Science of the Federal Agricultural Research Centre (FAL, Braunschweig). Designed specifically for routine use in genebanks, this method uses a very simple ultra-rapid freezing approach and was applied to a wide range of varieties within the Federal Centre for Breeding Research on Cultivated Plants (BAZ, Quedlinburg) Potato Collection. After several years of storage in liquid nitrogen, shoot tips from a random sample of 51 varieties were thawed and the survival and shoot regeneration percentages compared to those measured immediately after freezing. There were no major changes in either survival or recovery of frozen apices. Data presented are not the outcome of a systematic experiment but from that accumulated during our work from 1992 to 1999.     

High viability of dormant Malus buds after 10 years of storage in liquid nitrogen vapour

Three hundred and sixty two Malus accessions from the Canadian Clonal Genebank of Plant Gene Resources of Canada were cryopreserved as dormant buds at the USDA-ARS National Center for Genetic Resources Preservation in 1996. According to grafting data collected on 165 of these accessions in 1999, 80 percent of the accessions had at least 40 percent viability. A subsample of these accessions was processed for cryopreservation by either adjusting the moisture content of the budwood sections containing dormant buds to 32 or 37 percent moisture (fresh weight basis) or by not drying the budwood sections (46 percent moisture fresh weight basis) prior to cooling. Budwood sections were then slow-cooled at 1 degree C per hour to -3 degree C, held for 24 h at -30 degree C and then rapidly transferred to the vapour phase of liquid nitrogen. Cryopreserved buds from 13 accessions that were dried using the various techniques were warmed and grafted in both 1999 and 2006 to determine viability. Overall, bud viability was high at both storage times. At the 10 year time point, some accessions had higher bud growth when they were desiccated prior to slow-cooling when compared to those that were not.     

Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification

Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation.     

Cold acclimation improves recovery of cryopreserved grass (Zoysia and Lolium sp.)

Cold acclimation of Lolium L. and Zoysia Willd. Grass cultivars significantly increased regrowth of cryopreserved meristems. One wk of cold acclimation improved recovery following cryopreservation but extended acclimation (4-8 wk) resulted in the best regrowth. Cold acclimation also significantly increased the dehydration tolerance of both Zoysia and Lolium meristems. Lolium apices cold acclimated for 4 wk produced 60-100% regrowth following cryopreservation by slow freezing or encapsulation-dehydration. Cold-acclimated Zoysia had greater than 60% regrowth following encapsulation-dehydration when beads were dehydrated to less than 22% water content. Non-acclimated meristems of both genera had little or no regrowth. Thawed meristems grew quickly without callus formation and the plantlets produced were transplanted to pots in the greenhouse after 4 to 6 wk. Samples of each cultivar were stored in liquid nitrogen as part of the U.S. National Plant Germplasm System.     

Development of a cryopreservation protocol for the microcyclic rust-fungus Puccinia spegazzinii

The rust fungus Puccinia spegazzinii (Basidiomycotina: Uredinales) has been identified as a potential classical biological control agent for the invasive weed Mikania micrantha (Asteraceae). Long-term, live storage of this pathogen is required for reference. As biotrophs, almost all rusts species cannot be preserved by traditional cryopreservation protocols, which rely on in vitro culture techniques. In addition, the embedded teliospores and delicate basidiospores of this microcyclic rust are not amenable to direct plunge freezing. Continuous culture of the rust on living plants is both laborious and expensive, so a variety of approaches for cryopreservation and storage were tested. These methods included traditional approaches to fungal cryopreservation such as variation of cooling rate regime and alginate encapsulation techniques. However, an in situ cryopreservation technique was the only method identified as having any potential for the long-term cryopreservation of the 10 isolates tested. Material from either petiole or stem tissue remained viable after cryopreservation, determined by the ability of the material to produce basidiospores. However, despite great progress being made in developing an optimal cryopreservation method, infection of the host plant by basidiospores produced from previously cryopreserved teliospores, embedded in leaf petioles, was not achieved.     

Effect of temperature increase and freezing on intravascular elastography

Intravascular ultrasound (IVUS) elastography is a technique that assesses the local strain in the vessel wall and plaque. The strain is an important parameter for characterization of different plaque components. These regions are related to plaque vulnerability. IVUS elastography was validated in vitro using human coronary and femoral arteries. These experiments were performed on specimens that were stored frozen and measured at room temperature for practical issues. The aim of this study is to determine the influence of freezing and measuring the tissues at room temperature (23 degrees C instead of 37 degrees C) on the elastic properties. Four human coronary, one carotid and one femoral arteries were first measured at 23 degrees C and next at 37 degrees C. Additionally they were stored at -80 degrees C for up to 24 h and finally measured at 23 degrees C. Acquisitions at intraluminal pressures of 80 and 100 mmHg were performed using an EndoSonics 20 MHz Visions catheter. Elastograms were determined from the IVUS rf-data (sampled at 100 MHz in 12 bits) that were obtained from a digital interface. Qualitative and quantitative analysis of the elastograms obtained from fresh and frozen specimens measured at 23 degrees C reveals that storage of the specimen at -80 degrees C has no significant influence. In vitro experiments can be performed at room temperature after storage of the tissue at -80 degrees C without significant affection of the information with respect to measuring fresh ex vivo material at body temperature.     

Cryopreservation, encapsulation and promotion of shoot production of embryonic axes of a recalcitrant species Ekebergia capensis, Sparrm

A study on zygotic axes of the recalcitrant seeds of Ekebergia capensis compared two cryopreservation methods, partial desiccation, and encapsulation-dehydration, and also investigated a method to promote shoot production. High (80 percent) survival (assessed as root production) was obtained after direct immersion into liquid nitrogen of axes rapidly dehydrated by flash drying for 20 min to a water content about 0.4 g water per g dry mass. In contrast, no survival at all was obtained of axes that were first encapsulated, then desiccated for three hours to the same water concentration as those fast-dried, and then placed in a cryovial and immersed in liquid nitrogen. Axes encapsulated after cryopreservation germinated both in vitro and in soil, and could be stored at room temperatures for several weeks while maintaining germinability, thus producing synseeds capable of distribution. However, shoot production after cryopreservation was seldom observed. The inclusion of the plant growth regulator, N6-benzyl adenine (BA) in the MS-based recovery medium promoted vigorous multiple shoot formation. Microscopical examination of embryos of E. capensis revealed that the cotyledonary insertions were contiguous with the shoot apex, leading to the conclusion that injury to, and ultimate necrosis of, the apical meristem following severing of these connections was a primary cause of the observed lack of, or poor, shoot development in excised axes (whether cryopreserved or not). The study demonstrated that it may be possible to resolve two of the problems facing attempts at cryopreservation of axes of recalcitrant seeds; lack of shoot production and difficulty of distribution of cryopreserved material for re-introduction.