Storage In LN Research Articles

High regrowth of potato crop wild relative genotypes after cryogenic storage

Potatoes are consumed by millions of people and are the survival food in several countries. Cultivated varieties of potato (Solanum tubersosum L.) are results of selection and crossing of many wild species. Only 8–13% of wild potato species used for food are preserved by either in situ or ex situ methods. The U.S. National Potato Germplasm Collection maintains over 5900 accessions, of which 75% are crop wild relatives (CWR). The objective of the study was to investigate regrowth of cryogenically stored clonal propagules (shoot tips) of selected CWR accessions maintained in the collection. Sixty-nine accessions from 30 Solanum species and six accessions that are not yet assigned to a species were cryopreserved by a droplet vitrification method at the NLGRP. The post cryopreservation regrowth varied from 40 to 100% (average 68%) but was not significantly different between the tested accessions. Regrowth of six accessions tested after 10 years of cryogenic storage was between 35 and 90% (average 66%) and was significantly different from their initial regrowth (average 87%); the largest viability loss was in S. condolleanum; but for the other five accessions the regrowth was between 45 and 90% (average 72%) and suggested at least 10 years of successful storage in LN was possible. Twelve potato wild species cryopreserved in this study were reported in literature as important for developing cultivated varieties for changed weather conditions.

Effect of different conditions of storage on seed viability and seedling growth of six European wild fruit woody plants

Key messageMalus sylvestris(L.) Mill.,Pyrus communis(L.),Sorbus aucuparia(L.),Prunus avium(L.),Prunus padus(L.), andCornus sanguinea(L.) are related, co-occurring species producingorthodoxseeds. However, we observed differences in their response to storage conditions, such as storage at different seed moisture contents (5%, 8%, and 11%) and/or temperatures (− 3 °C, − 18 °C, and − 196 °C). Severe desiccation to ca. 5% of MC negatively affected seeds ofM. sylvestris. Seeds ofP. aviumwere sensitive to storage in LN or at − 18 °C.S. aucupariaseeds are best stored at − 3 °C, whereasC. sanguineaseeds tolerate desiccation and storage in LN. In general, species with deeper physiological dormancy (S. aucuparia,P. padus, andC. sanguinea) tended to be more tolerant to desiccation and low temperatures. For all species, storage conditions did not affect seedling growth.ContextWild fruit woody species face many threats such as genetic loss, population fragmentation, and alien species; thus, their genetic variability should be preserved.AimsTo examine the effect of storage conditions on seed viability and the initial growth of seedlings of six European wild fruit species: Malus sylvestris (L.) Mill., Pyrus communis (L.), Sorbus aucuparia (L.), Prunus avium (L.), Prunus padus (L.), and Cornus sanguinea (L.).MethodsSeeds were desiccated to three different levels of moisture content (ca. 5, 8, or 11%) and stored at three different temperatures (− 3°, − 18°, or − 196 °C; liquid nitrogen, LN) for up to 3 years. Germination and seedling emergence tests were performed as well as measurement of seedling growth.ResultsDesiccation of M. sylvestris seeds from 10.7 to 4.9% significantly lowered germination from 91 to 77% and seedling emergence from 88 to 74%. In P. avium, LN storage significantly inhibited seedling emergence, both in the laboratory and the greenhouse, but did not affect total seed germination. In P. communis, P. padus, and C. sanguinea, neither germination nor seedling emergence was affected by seed storage conditions. There were small or no differences in stem height and root collar diameter in the first year of seedling growth of stored seeds.ConclusionSpecies with deeper physiological dormancy (S. aucuparia, P. padus, C. sanguinea) tended to be more tolerant of various storage conditions. Seeds of P. padus and C. sanguinea can be stored long term at − 18 °C or in LN at 5–8% MC without losing viability. M. sylvestris and P. avium seeds are sensitive to desiccation below 6% MC or low temperature of storage at − 18 °C or − 196 °C, respectively. We observed that storage conditions had significant influence on germination and seedling emergence but had no effect on seedling growth after the first growing season.

Cryopreservation of somatic embryos for avocado germplasm conservation

Presently avocado germplasm is conserved ex situ in the form of field repositories across the globe including Australia. The maintenance of germplasm in the field is costly, labour and land intensive, exposed to natural disasters and always at the risk of abiotic and biotic stresses. The aim of this study was to overcome these problems using cryopreservation to store avocado (Persea americana Mill.) somatic embryos (SE). Two vitrification-based methods of cryopreservation were optimised (cryovial and droplet-vitrification) using four avocado cultivars (‘A10′, ‘Reed’, ‘Velvick’ and ‘Duke-7′). SE of the four cultivars were stored for short-term (one hour) in liquid nitrogen using the cryovial-vitrification method and showed a viability of 91%, 73%, 86% and 80% respectively. While when using the droplet vitrification method viabilities of 100%, 85% and 93% were recorded for ‘A10′, ‘Reed’ and ‘Velvick’. For long-term storage, SE of cultivars ‘A10′, ‘Reed’ and ‘Velvick’ were successfully recovered with viability of 65–100% after 3 months of LN storage. For cultivar ‘Reed’ and ‘Velvick’ SE were recovered after 12 months of LN storage with viability of 67% and 59%, respectively. The outcome of this work contributes towards the establishment of a cryopreservation protocol that is applicable across multiple avocado cultivars.

Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. Key message: Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

Ex situ conservation of the endangered species Androcalva perlaria (Malvaceae) by micropropagation and cryopreservation

Micropropagation and cryopreservation protocols were developed for the threatened Australian species Androcalva perlaria. Vegetative shoots were brought into culture using a simplified surface sterilisation process with between 26 and 100 % of shoots successfully initiated across all genotypes. Shoots were multiplied on ½ MS basal salts medium (BM) with 1.25 µM 6-furfurylaminopurine (K) + 0.125 µM 6-benzylaminopurine (BAP). Cryopreservation was then developed for a single genotype to facilitate long-term ex situ storage for conservation purposes. Highest survival (>80 %) of shoot tips was achieved by preculture on 1.2 M glycerol for 48 h, incubation in PVS2 solution at 0 °C for 30 min, followed by rapid LN immersion then recovery. Application of this cryogenic approach to shoot tips from a range of genotypes gave variable post-cryopreservation regeneration results; survival for one genotype was only 3 %, while for four other genotypes survival varied between 60 and 80 % which compared favourably with post-cryopreservation regeneration (85 %) of the genotype used to develop the protocol. Callus production was achieved by culturing stem segments on ½ MS BM with 2.5 µM α-naphthaleneacetic acid + 2.5 µM BAP. Adventitious shoots were best regenerated from callus through incubation on BM only. Small callus pieces were successfully cryopreserved from 16 genotypes (1–88 % regeneration). Using a callus tissue pathway plant material was placed into LN storage after 6–8 weeks from the time of collection (compared to ~6 months using shoot tips). Plants derived from cryogenically preserved callus tissues were re-established in soil 28 weeks after removal from LN. This study demonstrates how biotechnology can be effectively utilised for the rapid ex situ conservation of endangered flora while ensuring that a significant range of genetically diverse samples can be conserved for long-term biosecurity.

C-2001: Survival of short-lived desiccation tolerant seeds during long-term storage in liquid nitrogen: Implications for the management and conservation of plant germplasm collections

Ex situ seed conservation is a widespread and powerful tool for the preservation of plant genetic resources. However, long-term storage of short-lived seeds is challenging in standard seed bank temperatures (4 and −20 °C), and cryopreservation has been acknowledged as the only effective option for their long-term conservation. However, as seed aging has been demonstrated during cryostorage, the potential and limits of LN storage of short-lived seeds need to be evaluated. We have examined the viability and growth of seeds from species with short-lived seeds stored up to 20 years in CREW’s CryoBioBank®, including Plantago cordata, Populus deltoides, several Salix species, and two species in the genus Betula. We have found survival in short-lived seeds after >14 years of LN storage (−196 °C). P. cordata seeds did not show a significant decrease in germination over this time. However, Salix sp. and P. deltoides accessions tested showed a final germination ranging between 0% and 96% of initial germination. Seeds of P. deltoides stored at 4 and −20 °C did not show any survival after the same storage period. Seed viability in P. deltoides and Salix sp. was overestimated when measured as total germination, rather than normal germination. Furthermore, 38–43% of seedlings from normal seedlings of P. deltoides did not continue normal development during early growth of the young plants. In addition, initial seed quality was correlated with seed longevity. These results suggest that: (1) monitoring of short-lived seeds cryostored as in this study (at 8–10% moisture) should be done at 2–5 year intervals, similar to the recommendation by IPGRI for seeds stored at −18°C, and regeneration should not exceed 10–15 years; (2) in order to minimize loss of seed quality prior to cryostorage, processing of short-lived seeds after harvest must be fast and gentle; (3) based on losses during early seedling growth and development, it is very important not to exceed the time to reach 85% of initial normal germination when planning regeneration of Populus and Salix collections to avoid large genetic erosion.

223 CRYOPRESERVATION OF IMMATURE BOVINE CUMULUS–OOCYTE COMPLEXES BY SLOW RATE FREEZING AND VITRIFICATION

Cryopreservation of mature oocytes can result in damage to the metaphase spindle due to the temperature sensitivity of microfilaments and microtubules. Cryopreservation of immature oocytes may circumvent this problem because these structures have not formed yet and the genetic material is enclosed within a nuclear envelope. Because intact oocyte cumulus-oocyte complexes (COC) are essential for normal maturation, we chose to cryopreserve immature intact COC. The aim of this study was to determine if immature COC cryopreserved by slow rate freezing or vitrification would resume meiosis upon thawing. In 2 separate experiments, immature COC (n = 102 and 79) were collected from cross-bred cattle by transvaginal ultrasound-guided aspiration and divided into 2 groups. For both experiments, the first group (n = 64 and 40) was placed directly into maturation medium (TCM 199 supplemented with 10% fetal bovine serum, 0.2 mM sodium pyruvate, 2 mM glutamine, and 5 μg mL–1 of FSH) and cultured for 22 h under 5% CO2 in air atmosphere at 39°C. In experiment 1, COC (n = 38) in the second group were cryopreserved by a slow rate freezing protocol. The COC were equilibrated for 5 min in 1.5 M ethylene glycol (EG), and 5 COC were loaded into 0.25-mL straws and placed into the cooling chamber of a Freeze Control unit at –6°C. After 5 min, straws were seeded, then cooled at 0.5°C per min to –35°C before plunging into LN. After storage in LN for 5 days, straws were removed from LN, thawed by placing straws in a 35°C water bath, and COC put into maturation as described above. In experiment 2, COC (n = 39) in the second group were cryopreserved by vitrification using a 3-step procedure. The COC were equilibrated in solutions consisting of 10% glycerol then 10% glycerol and 20% EG in PBS for 5 min each. The COC were then placed in a solution of 24% glycerol and 26% EG, and 1 to 3 COC were placed onto a cryotop device in minimal medium and plunged into LN within 45 s. After storage in LN for 2 days, COC were thawed by placing the cryotop device directly into a warmed dilution solution consisting of 0.5 M galactose in PBS. After 5 min in the dilution solution, COC were put into maturation as described above. For all groups, after 22 h of maturation cumulus cells were stripped from the oocytes by vortexing and oocytes were placed on slides for fixation in methanol acetic acid and stained with 1% orcein to determine the nuclear stage. In experiment 1, oocytes cryopreserved by slow rate freezing matured to MII at the same rate as control oocytes (45 v. 55%, P = 0.44). In experiment 2, oocytes cryopreserved by vitrification matured to MII at a lower rate than controls (49 v. 79%, P = 0.01). These results show that cryopreservation of immature intact COC is a viable alternative to cryopreservation of mature oocytes. Further studies are needed to optimize either slow rate freezing or vitrification of intact COC and determine the developmental competence of cryopreserved oocytes following fertilization.

74. Cryopreservation of in vitro shoot tips of potato varieties by V- Cryo-plate method

Cryopreservation procedure using aluminium cryo-plates (V-Cryo-plate method) was successfully applied to in vitro -grown potato ( Solanum tuberosum L.) shoot tips. The shoots were cultured at 25 °C on MS medium containing 3 % (w/v) sucrose and 2.9 mM calcium over a period of 14 d after subculture. The shoot tip (1–1.5 × 1 mm) was dissected from the shoot and precultured at 25 °C for 1 d on MS medium containing 0.3 M sucrose. The precultured shoot tips were placed on aluminium cryo-plates (7 mm × 37 mm × 0.5 mm) with ten wells (diameter 1.5 mm, depth 0.75 mm) and embedded with alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 °C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in another 25 ml pipetting reservoirs filled with PVS2 for 30 min at 25 °C. Then, the cryo-plate was transferred in an uncapped 2 ml cryotube and directly plunged into liquid nitrogen. After storage in LN 2 , shoot tips attached to the cryo-plate were directly rewarmed by immersion into a cryotube containing 2 ml 1 M sucrose solution. Using this procedure, the regrowth rate of cryopreserved shoot tips of cultivar ‘Sayaka’ reached more than 95%. This protocol was successfully applied to 12 different cultivars, which had quite high regrowth levels ranging from 93% to 100% and the average regrowth level of those cultivars reached 99%. This new method has many advantages and will facilitate the cryostorage of varieties of potato germplasms.

STORAGE STABILITY USING CRYOPRESERVATION: A CASE STUDY IN PAPAYA

ISHS XXVIII International Horticultural Congress on Science and Horticulture for People (IHC2010): III International Symposium on Plant Genetic Resources STORAGE STABILITY USING CRYOPRESERVATION: A CASE STUDY IN PAPAYA

Dehydration status of ABA-treated and cold-acclimated Hypericum perforatum L. shoot tips subjected to cryopreservation

To improve the vitrification protocol for Hypericum perforatum shoot tips, calorimetric measurements of the intensity of thermal transitions in the samples during warming using differential scanning calorimetry were assessed. Comparison of ABA treatment and cold-acclimation revealed higher content of crystallizable water in the latter. Regeneration capacity of cryopreserved H. perforatum shoot tips increased progressively with the exposure time in PVS3 cryoprotection mixture reaching the maximum between 59.3 and 71.3% after 180min when the proportion of freezable water ranged between 0.0 and 0.4% of total water content respectively. Glass transitions were observed in all samples dehydrated with PVS3 when freezable water was present. However, the decreasing recovery of H. perforatum shoot tips after prolonged PVS3 dehydration (without LN storage) indicated some injuries of meristematic tissue induced by PVS3 dehydration rather than ice nucleation and resulted from genotype-dependent tolerance to abiotic stresses accompanying the entire process.

Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb.f.) christenson by encapsulation-dehydration

Protocorm-like bodies (PLBs) of Phalaenopsis bellina were successfully cryopreserved by the encapsulation-dehydration approach. Various stages in obtaining successful cryopreservation using this method were optimized. Encapsulated PLBs precultured in half-strength MS medium supplemented with 0.75 M sucrose for 3 days exhibited the highest viability in terms of 2,3,5-triphenyltetrazoliumchloride (TTC) reduction. The amount of sucrose in the PLBs after incubation in different concentrations of sucrose for different periods of time determined by HPLC. The highest sucrose concentration was 7 mg/g of PLBs for the PLBs treated with 0.75 M sucrose for 3 days as compared to the control which had only 1 mg/g sucrose. After sucrose preculture, the PLBs were subjected to desiccation using one of two methods. Desiccation using silica gel was more efficient in reducing PLBs moisture content. After 6 h of desiccation, PLBs desiccated using laminar air flow had 43.5% moisture content while for those desiccated using silica gel had 32% moisture content. PLBs desiccated to different moisture contents were plunged into LN. After storage in LN the encapsulated PLBs were re-warmed. Two weeks after re-warming PLBs viability was determined by TTC reduction and re-growth assessed. Encapsulated PLBs precultured with 0.75 M sucrose for 3 days followed by desiccated using silica gel for 5 h resulting in a moisture content of 39% lead to the highest post re-warming viability in terms of TTC reduction (46.6% of control PLBs) and 30% re-growth.

In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length

Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1–9 d; b) 10–30 d; c) 31–90 d; d) 1–3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN 2) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN 2 has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage.

La cryoconservation des semences dormantes orthodoxes des arbres forestiers : le merisier (Prunus avium L.)

• The sensitivity of dormant seeds of mazzard cherry (Prunus avium L.) of Polish provenances to extreme desiccation and/or the ultra-low temperature of liquid nitrogen, LN (−196 ◦ C), was investigated to identify the optimum seed water content (w.c.) at this temperature. • Germination and seedling emergence tests in this study showed that there is no critical seed w.c. for deeply desiccated seeds (stone w.c. 1.6%), although such desiccated seeds were sensitive to the temperature of LN in the seedling emergence test. For seeds frozen in LN, the highest germinability was observed at w.c. of 9.0–16.9%, but seedling emergence was then significantly lower than in nonfrozen seeds. The 2-year of storage in LN of seeds desiccated to 7.8% w.c. did not decrease germinability after thawing, in comparison with 2-year of storage at −3 ◦ C. Storage in LN showed that if seeds were stored after breaking of their dormancy, germinability after storage was lower because of the necessity of seed desiccation to the lower level (∼ 8%) of w.c. after stratification (before storage). Secondary dormancy was induced in seeds desiccated after stratification. • The results of this study demonstrate the potential for long-term cryopreservation of mazzard cherry seeds in forest gene banks.

Cryopreservation of In Vitro. Grown Apical Shoot Tips of Strawberry by Vitrification.

After the strawberry shoots on culture medium were cold-hardened at 5°C for 20-30 days, shoot tips were dissected and precultured at 5°C for 1 day on MS medium containing 2 M glycerol and 0.3M sucrose. Precultured 10 shoot tips were placed in 2.0ml cryotubes each, and treated with a mixture of 2M glycerol and 0.3M sucrose for 20min at 25°C. They were then treated with PVS2 solution for 50min at 25°C, and immersed into LN. After rapid rewarming in water at 35°C, they were transferred onto the MS medium containing 1gl-1 PVP and 0.2mgl-1 BA and grown under standard conditions. By this protocol, survival rate of cryopreserved “Donner” shoot tips reached 93%. This protocol was successfully applied to 7 other races or lines of strawberry with high survival rates. These results prompt us to setting up large-scale strawberry LN storage system.

Modulation of the cryopreservation cap: elevated survival with reduced dimethyl sulfoxide concentration

The development of cryopreservation (CP) strategies has traditionally focused on the cellular chemo-osmometric characteristics attendant to the freeze-thaw process. This approach coupled with a limited understanding of cellular physiological and biochemical responses to the CP process often yields sub-optimal cell survival. Recently, we have reported on the benefits of the utilization of an intracellular-like preservation solution, HypoThermosol (HTS), as well as incorporating a molecular approach to improving CP outcome [In Vitro Cell. Dev. Biol. Anim. 36(4) (2000) 262]. We now report on the elucidation of a cryoprotective agent (CPA)-dependent survival limit (cap) associated with standard CP methodologies. We further demonstrate an elevation and shift in the CP cap through the utilization of HTS coupled with a reduction in CPA levels necessary to achieve “successful” cell preservation. Methods. Human fibroblasts, keratinocytes, hepatic, and renal cells were cryopreserved in a standard fashion ( ∼1 ° C min −1 cooling and storage in LN 2) in culture media (serum-free) or HTS with varying levels of dimethyl sulfoxide (Me 2SO). Samples were allowed to recover for 24-h prior to survival assessment. Survival was assessed using alamarBlue (metabolic activity indicator) and calcien-AM (membrane integrity stain) in comparison with non-frozen controls. Results. (1) A limit in cell survival was identified following CP in media-based CP solutions yielding a cell-type specific CPA-dependent survival limit, (2) peak cell survival resulted in the identification of “optimal” Me 2SO concentrations for CP of each cell type, (3) incorporation of HTS as the carrier medium at typical Me 2SO concentrations substantially elevated survival, and (4) utilization of HTS allowed for the successful preservation of all systems examined at significantly reduced Me 2SO levels. Conclusion. The data presented in this study illustrate that the utilization of HTS as the carrier medium during CP facilitated a significant improvement in efficacy at reduced Me 2SO levels. Further, the utilization of HTS offers the potential for successful Me 2SO-free CP. These findings may prove significant to the advancement in the development of cell-based clinical therapies by providing an improved biocompatible CP methodology.

Cryopreservation of Digitalis obscura selected genotypes by encapsulation-dehydration.

Shoot-tips from several genotypes of the cardenolide-producing perennial shrub Digitalis obscura L. were successfully cryopreserved using the encapsulation-dehydration technique. Precultivation on MS medium containing 0.5 M sucrose, followed by 2.5 h dehydration (final weight 30 %) induced shoot regrowth in 42 % of cryopreserved shoot-tips. Cold-hardening of the in vitro cultures before sucrose treatment dramatically increased shoot recovery up to 86 %. The optimized cryopreservation protocol was then employed using different shoot cultures from five D. obscura genotypes. Responses to cryopreservation depended mainly on the genotype, best results being obtained when shoot tips from HU3 and LL11 were used. Prolonged subcultures reduced proliferation rates in both control and cryopreserved HU3 shoot-tips, whereas long-term storage in LN did not affect the shoot recovery rate of the genotype. RAPD markers were employed to evaluate possible somaclonal variation occurring in shoots regenerated through successive subcultures and after cryopreservation. The band patterns revealed differences between the original parent plant and the shoots grown in vitro, especially after a prolonged subculture (84.9 % of matches for HU3 after 16 subcultures vs. 93.4 % for AY3 after 2 subcultures). Nevertheless, match percentages were higher (98.6 % to 99.5 %) when band patterns from subcultured shoots were compared to those obtained from their respective control or frozen progenies indicating that cryopreservation ensure genetic stability of selected Digitalis obscura genotypes.

Cryopreservation of the conchocelis of the marine alga Porphyra yezoensis Ueda (Rhodophyta) in liquid nitrogen

Cryopreservation og the conchocelis of Porphyra yezoensis by the two-step cooling method was attempted. A solution composed of 10% DMSO and 0.5 M sorbitol in 50% seawater had the most favorable cryoprotective effect on the conchocelis. The appropriate cryopreservation procedure was as follows: DMSO was added gradually over a period of 15 min followed by an equilibration period of several minutes. Conchocelis cells were then prefrozen to −40°C at 0.1–1°C/min prior to immersion in LN. After storage in LN, the conchocelis suspension was thawed quickly by agitation of the vial in a water bath at 40°C, and cryoprotectants were washed off by gradual dilution with seawater. The survival was independent of the period of storage at least for 300 days. The survival evaluated by staining with neutral red was approx. 60%. The conchocelis cells stored in LN were able to form colonies, and retained the ability to form conchospores which grew into gametophytic thalli.

Comment on the loss of immunogenicity of tumor cells caused by freezing

The freezing of cells to temperatures below about 1 3 0 ° C permits their extended storage in a state of "suspended animation" for decades if not longer. Such long-term storage of viable biological material offers potential benefits in a variety of research and clinical situations. The thesis advocated by Bartlett et al. (1977) is a good example. They suggest that the storage of human tumor tissue in the frozen state might permit its later use as an autologous vaccine in the immunotherapy of cancer. Unfortunately, the data of Bartlett et al., who used a model system of vaccines consisting of BCG and irradiated L10 hepatoma cells, seem to invalidate their own thesis. They reported that L10 cells that had been frozen and stored in liquid nitrogen (LN2) at --196 ° C possessed little or no capacity to prevent the progressive growth of challenge tumor cells, despite the fact that about 50% of the frozen-thawed cells excluded trypan blue. Bartlett et al. conclude that there was "clear evidence that tumor cells which were 'viable' after storage in LN 2 were significantly less immunogenic than cells used without prior freezing". A reasonable extension of that conclusion, if correct, would be that frozen-thawed cells are not suitable for immunotherapy. I believe there are reasons for suggesting that the conclusion of Bartlett et al., and its corollary, may be unduly pessimistic. Those authors have sought to use one of the major potential advantages of cryobiology, namely, the preservation of viable cells in the frozen state. I believe they have underestimated the present level of mechanistic understanding of low temperature biology, however. In this specific instance, this misreading of cryobiology might preclude its future application to a clinical situation in which freezing would be a requisite for the therapy.

On the storage space requirement of consecutive retrieval with redundancy

A very economic method of organizing records in an information retrieval system is due to Ghosh [1,2] (see also refs. [5,6]): the records relevant to a query are stored in consecutive storage locations, and one location may be significant for several queries. Thus, the record sets corresponding to queries form overlapping segments in storage. Unfortunately, this may in general not be done without repetitions, so redundancy has tobe taken into account (see refs. [3,7]). Putting it into more abstract terms, the problern is as follows: given a family cm_ == {M1,M2, ... ,M11 } of subsets of a finite set X, find an arrangement of all elements of X suchthat (i) each Mi forms a segrnent and (ii) the total number of repetitions is minimal. The problern (for linear arrangements) has been shown tobe NP-complete, see ref. [4]. In i:he present paper we give methods for producing suboptimal solutions, and we calculate the storage space required. Our basic assumptionisthat each component of the given family cm_ contains exactly one element. (A component of cm_ is a block of the product of all partitions {MbX\MJof X, see ref. [8]). This assumption seems tobe not too restrictive, if we consider the method e.g. developed by Marek and Pawlak [8]: each component is stored elsewhere and referred to by a pointer; thus, we have 2n pointers referred to by the 2n binary n-tuples as keys. Our problern now is to find arrangements of these n-tuples such that, for each position i, 1 < i < n, there is a segrnent all of whose items have ith position 1, while the segment contains all possible 2n-l items, each one exactly once. This Situation is somewhat related to the inverted file organization with n binary attributes in the case ofuniformly distributed keys. Here,Ln == n2n-l storage locations are required. The storage space required by our linear method (section 1) will be 2 shown tobe about 3Lw In section 2, the method is generalized to the arrangement of items in acyclic f-graphs as introduced by the second author [5] (see also ref. [6]). This organization will be shown to need approximately ~Ln storage locations.