Successful cryopreservation of Quercus robur plumules

Successful cryopreservation of Q. robur germplasm as plumules (i.e. shoot apical meristems of embryos) is described in this paper. After excision from the recalcitrant seeds and preliminary storage in 0.5 M sucrose solution (18 h), the plumules were subjected to cryoprotection (in 0.75 M sucrose, followed by 1.0 M sucrose and 1.5 M glycerol solutions), and next to desiccation (over silica gel or in nitrogen gas) and cooling (in slush at –210°C or in vials filled with liquid nitrogen, LN, −196°C), and were then cryostored for 24 h. High percentage of survival was obtained after cryostorage (21–67%, depending on pretreatment, assessed in vitro by greening plumules that increased in size). Desiccation of plumules over silica gel resulted in significantly higher survival after cryopreservation (58%) in comparison with desiccation in nitrogen gas (29%), with regrowth (shoots with leaves) 5–18%. The extent of plumule desiccation was comparable in both methods, in which drying of plumules for 20 min decreased the water content to 0.5–0.6 g H₂O g⁻¹ dry weight before LN exposure. The type of LN exposure did not significantly influence plumule survival and regrowth after cryostorage. Plumules isolated from acorns of four provenances survived cryostorage after cryoprotection followed by desiccation over silica gel and direct cooling in vials with LN (survival 51–76%, regrowth 8–20%). Normal plants developed from the recovered shoots after rooting. The presented protocol for Q. robur plumule cryopreservation may offer a potential approach for establishing germplasm conservation in gene banks for Quercus species.     

Cryopreservation of white mulberry (<Emphasis Type="Italic">Morus alba</Emphasis> L.) by encapsulation-dehydration and vitrification

Shoot apices of in vitro-grown plantlets of white mulberry, Morus alba L. cv Florio, were cryopreserved using either encapsulation-dehydration or vitrification. For encapsulation-dehydration, alginate beads containing apices were dehydrated for 1, 3, 5 or 7 days in a liquid medium containing various sucrose concentrations (0.5, 0.75, 1.0 or 1.25 M). Bead desiccation was performed using silica gel for either 0, 4, 6, 8, 9 or 14 h. For vitrification, apices were directly immersed for either 5, 15, 30 or 60 min in a vitrification solution (PVS2). Following encapsulation-dehydration, treatment of alginate beads with 0.75 M sucrose was more effective in promoting re-growth of explants after immersion in liquid nitrogen than in the presence of 0.5 M sucrose for either 1 or 3 days. Re-growth of explants was also observed following vitrification and this reached 47% with increasing duration of the PVS2 treatment from 5 to 30 min. Overall, the highest frequency of explant re-growth was obtained when explants were subjected to encapsulation-dehydration in the presence of 0.75 M along with a 3 day sucrose dehydration pre-treatment and followed by desiccation for 9 h in silica gel.     

Cryopreservation of protocorm-like bodies (PLBs) of <Emphasis Type="Italic">Phalaenopsis bellina</Emphasis> (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.     

Cryopreservation of Quercus faginea embryonic axes

Embryonic axes, excised or included in cotyledonary tissue, of Quercus faginea were tested for viability after rapid freezing and storage in liquid nitrogen (−196 °C). Explants were previously pretreated by desiccation at different moisture contents or by soaking in cryoprotectant (15% dimethyl sulfoxide). Best germination response after freezing (60%) was observed when embryonic axes were desiccated from 53 to 21% moisture content (on a fresh weight basis). Desiccation below 39% moisture content or freezing produced damage resulting in loss of organized growth and the induction of callus formation.     

A quantitative evaluation of two methods for preserving hair samples

Hair samples are an increasingly important DNA source for wildlife studies, yet optimal storage methods and DNA degradation rates have not been rigorously evaluated. We tested amplification success rates over a one‐year storage period for DNA extracted from brown bear (Ursus arctos) hair samples preserved using silica desiccation and ?20 °C freezing. For three nuclear DNA microsatellites, success rates decreased significantly after a six‐month time point, regardless of storage method. For a 1000 bp mitochondrial fragment, a similar decrease occurred after a two‐week time point. Minimizing delays between collection and DNA extraction will maximize success rates for hair‐based noninvasive genetic sampling projects.     

A common garden experiment with Porphyra umbilicalis (Rhodophyta) evaluates methods to study spatial differences in the macroalgal microbiome

While macroalgal microbiomes are the focus of many recent studies, there is little information about microbial spatial diversity across the thallus. Reliance on field material makes it difficult to discern whether recovered microbiomes belong to the host or its epiphytes, and technical comparisons of macroalgal samples for microbial studies are needed. Here, we use a common garden approach that avoids the problem of epiphytes, particularly at holdfasts, to examine the microbiome of Porphyra umbilicalis (strain Pum1). We used the V6 hypervariable region of the 16S rDNA with Illumina HiSeq sequencing and developed PNA clamps to block recovery of organelle V6 sequences. The common garden approach allowed us to determine differences in the microbiome at the holdfast versus blade margin. We found a notable increase in the relative abundance of Planctomycetes and Alphaproteobacteria at the holdfast, particularly of the possible symbiont Sulfitobacter sp. Nonadjacent 1.5 cm² samples of blade margin had microbiomes that were not statistically different. The most abundant phylum in the overall microbiome was Proteobacteria, followed by Bacteroidetes. Because phycologists often work in remote sites, we compared three stabilization and preparation techniques and found silica gel desiccation/bead‐beating and flash‐freezing/lyophilization/bead‐beating to be interchangeable. Core taxa (≥0.1% of sequences) across treatments were similar and accounted for ≥95% of all sequences. Finally, statistical conclusions for all comparisons were the same, regardless of which microbial community analysis tool was used: mothur or minimum entropy decomposition.     

The cryopreservation of shoot tips of Rosa multiflora

Shoot tips of in vitro grown plantlets of Rosa multiflora were cryopreserved using an encapsulation/dehydration procedure. The influence of sucrose and silica gel pretreatments on pre- and post-freeze shoot growth were examined. Shoot tips recovered from liquid nitrogen only grew after 24h pretreatment in medium containing 0.5 M sucrose, followed by 2 h drying with silica gel and rapid freezing.Abbreviations RSC1 modified Murashige and Skoog medium for Rosa multiflora shoot culture     

Effect of desiccation level and storage temperature on green spore viability of Osmunda japonica

In order to effectively preserve green spores, which have relatively higher water content and lose viability more quickly than non-green spores, we studied the effect of desiccation level and storage temperature on Osmunda japonica spores. The water content of fresh spores was 11.20%. After 12 h desiccation by silica gel, the water content decreased to 6% but spore viability did not change significantly. As the desiccation continued, the decrease in water content slowed, but spore viability dropped. For almost all storage periods, the effects of storage temperature, desiccation level, and temperature × desiccation level were significantly different. After seven days of storage, spores at any desiccation level stored at 4 °C obtained high germination rates. After more than seven days storage, liquid nitrogen (LN) storage obtained the best results. Storage at −18 °C led to the lowest germination rates. Spores stored at room temperature and −18 °C all died within three months. For storage at 4 °C and in LN, spores desiccated 12 and 36 h obtained better results. Spores without desiccation had the highest germination rates after being stored at room temperature, but suffered the greatest loss after storage at −18 °C. These results suggest that LN storage is the best method of long-term storage of O. japonica spores. The critical water content of O. japonica spores is about 6% and reduction of the water content to this level improves outcome after LN storage greatly. The reason for various responses of O. japonica spores to desiccation and storage temperatures are discussed.     

Preservation of Thiobacillus ferroxidans and Thiobacillus thiooxidans with activity check

Cultures of Thiobacillus ferrooxidans and Thiobacillus thiooxidans, used in biohydrometallurgical processes of economic importance, are very difficult to preserve by conventional methods. Hence, to preserve the cultures with their activity intact, various techniques were tried, after determining their respective activity in terms of Iron Oxidation Rate (IOR) and Sulfur Oxidation Rate (SOR). Among the methods tested, along with the recommended method of serial transfer in a liquid medium, were methods such as lyophilization, storage in a liquid nitrogen and mixing with sterile, inert carriers like lignite or chalcopyrite ores. After a period check-up at 4 months and 8 months storage, it was found that out of these methods, mixing with sterile ore followed by storage at 8°C, kept both types of activities intact. The temperature of storage was observed to have a definite effect on activity, in that when the preserved cultures were stored at 8°C, the activity was retained, whereas at 28–30°C (RT) storage, the activity of all the cultures preserved by various techniques, dropped significantly.     

Long-term preservation of anammox bacteria

Deposit of useful microorganisms in culture collections requires long-term preservation and successful reactivation techniques. The goal of this study was to develop a simple preservation protocol for the long-term storage and reactivation of the anammox biomass. To achieve this, anammox biomass was frozen or lyophilized at two different freezing temperatures (−60°C and in liquid nitrogen (−200°C)) in skim milk media (with and without glycerol), and the reactivation of anammox activity was monitored after a 4-month storage period. Of the different preservation treatments tested, only anammox biomass preserved via freezing in liquid nitrogen followed by lyophilization in skim milk media without glycerol achieved stoichiometric ratios for the anammox reaction similar to the biomass in both the parent bioreactor and in the freshly harvested control treatment. A freezing temperature of −60°C alone, or in conjunction with lyophilization, resulted in the partial recovery of the anammox bacteria, with an equal mixture of anammox and nitrifying bacteria in the reactivated biomass. To our knowledge, this is the first report of the successful reactivation of anammox biomass preserved via sub-zero freezing and/or lyophilization. The simple preservation protocol developed from this study could be beneficial to accelerate the integration of anammox-based processes into current treatment systems through a highly efficient starting anammox biomass.     

Cryopreservation of seed of Western Australian native species

The ability of seed of native Western Australian species to be stored using cryopreservation methods was investigated by subjecting seed of 90 native species representing 84 genera and 33 families to storage in liquid nitrogen. Seed of 68 native Western Australian species were germinated after storage in liquid nitrogen for two weeks following treatments which involved direct plunging into liquid nitrogen or slow cooling at 0.4°C min^–1 in 15% dimethyl sulphoxide (DMSO) or slow cooling at 0.4°C min^–1 in 35% DMSO. The largest number of species (37) responded positively to direct plunging without pretreatments, with only 10 species responding to slow cooling in 15% DMSO. Thirty one species had enhanced germination and 10 species depressed germination after any of the liquid nitrogen treatments. There were no trends in a species ability to survive liquid nitrogen storage and freezing regime, moisture content, seed size or taxonomic relatedness. However, hard seeded species belonging to the families Caesalpinaceae and Papilionaceae showed a consistently high degree of tolerance to liquid nitrogen storage. Significant physical damage to seed and cotyledons only occurred in Templetonia retusa (Papilionaecae) and this was alleviated by nicking the seed coat. This study indicates that seed of a large proportion of native Western Australian species may be amenable to storage in liquid nitrogen and that at least 40% of the listed rare and endangered species of Western Australia could be maintained in this way.     

Cryopreservation of embryogenic tissues of <Emphasis Type="Italic">Picea omorika</Emphasis> (Serbian spruce)

Two-year-old embryogenic tissues (ET) of Picea omorika (Pančić) Purk. were successfully cryopreserved after preculture with sucrose, air-drying for 2 h, and freezing in liquid nitrogen (LN). The preculture protocol consisted of passaging the ET onto standard Litvay medium containing increasing concentrations of sucrose (0.25 M sucrose for 24 h, 0.5 M for 24 h, 0.75 M for 2 days, and 1.0 M for 3 days) for 7 days, at 25°C, and in the dark. The clumps were subsequently air-dried over silica gel, down to a 20% water content (based on fresh weight), placed in cryovials, and immersed in liquid nitrogen (LN) for 24 h. These were thawed at 42°C and progressively rehydrated in phytagel-solidified LM media containing decreasing concentrations of sucrose. After 3 weeks of in vitro culture, surviving clumps were friable and white in color, similar to their morphology prior to cryostorage. The frequency of bacterial and fungal contamination was higher if ET was frozen in LN-containing vials than in LN-free vials. This efficient cryopreservation protocol would be useful for the ex-situ conservation of P. omorika germplasm in gene banks at very low temperatures.     

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H₂O₂ up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H₂O₂. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

     

A Simple and Rapid Cryopreservation Technique for Ciliates: A Long‐Term Storage Procedure Used for Marine Scuticociliates

Pseudocohnilembus persalinus is a free‐living marine scuticociliate that, as a new model organism, has been used in a wide variety of studies. However, long‐term laboratory maintenance for this species is mainly achieved by subculture that requires rigorous culture environments and, too often, cultures of the organism die out for a variety of reasons. Successful transport of viable cultures also poses problems for researchers. This study describes a simple and rapid protocol for long‐term cryopreservation of P. persalinus. The effects of physiological states of individuals before freezing, the type and concentration of cryoprotectant, and optimal temperatures for freezing and thawing were assessed. A cryopreservation protocol, using a mixture of 30% glycerol and 70% concentrated P. persalinus cell culture, incorporating rate‐controlled freezing at ?80 °C before liquid nitrogen storage, maintained a high recovery efficiency after 8 wk of storage. These results suggest that broader application of this protocol to build a cryopreserved marine protozoa culture bank for biological studies may be possible.     

A cheap,reliable and rapid method of extracting high‐quality DNA from plants

Here we describe a rapid method for extracting DNA from plant material using a microtitre plate system. This extraction procedure has been tested using three species, Brassica napus, Brassica napus/rapa hybrids and Arum maculatum, stored frozen, in silica gel or used fresh. The length of storage was between 7 days and 2 years, and in all cases high molecular weight DNA was reliably purified. Polymerase chain reaction (PCR) testing, using multiplex and single product amplification, inter simple sequence repeats (ISSRs) and microsatellites, was always reliable. This method combines the speed of commercial 96‐well plate methods with the economies associated with readily available laboratory chemicals.     

The effect of two cryopreservation methods on human sperm DNA damage

Several methods are currently available for selection when conducting sperm cryopreservation, however, these methods might cause different degrees of damage on sperm DNA. The aim of the this study is to compare the effects of storage at −80 °C (in ultra-low temperature refrigerator) and at −196 °C (in liquid nitrogen) on sperm DNA damage, thus to provide a reference for choosing the right method according to different aims. We randomly collected 28 semen samples from college students of Chongqing city. The samples stored at −80 °C were neat semen samples and the samples stored at −196 °C were mixed with additional cryoprotectants. Each sample was subjected to two freezing-thawing cycles, and the sperm DNA damage levels of fresh and thawed samples were measured by single cell gel electrophoresis (SCGE) and sperm chromatin structure assay (SCSA). Both SCGE and SCSA assays showed cryopreservation induced significant damage to sperm DNA. However, storage at −196 °C lead to more severe damage to sperm DNA than storage at −80 °C measured by SCSA. Sperm DNA damage increased simultaneously with the higher frequency of freezing-thawing cycles. We concluded that storage of neat semen samples at −80 °C had milder damage to sperm DNA than storage at −196 °C mixed with cryoprotectants. To avoid additional sperm DNA damage, repeated freezing and thawing should be prevented.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

Sperm cryostorage in a dry tank: An accurate alternative

This prospective study aimed to determine the effects of dry nitrogen cryostorage on human sperm characteristics in comparison with liquid nitrogen cryostorage. For this purpose, 42 men undergoing routine semen analysis (21 normozoospermia and 21 with altered semen parameters) were analyzed. After slow freezing, half of the straws of each sample were randomly stored in liquid and dry tanks, at the top and bottom levels of the latter. After 6 months storage, thawed samples were treated by density gradient centrifugation and sperm characteristics were compared. There was no difference in sperm progressive motility (15.1% ± 14.2% vs. 15.1% ± 12.7%; p = 0.76), sperm vitality (25.5% ± 17.7% vs. 26.2% ± 19%; p = 0.71), percentages of acrosome-reacted spermatozoa (38% ± 8.5% vs. 38.5% ± 7.4%; p = 0.53) and DNA fragmentation spermatozoa (27.3% ± 12.4% vs. 28.5% ± 12.9%, p = 0.47) after cryostorage in the dry or the liquid nitrogen tank. Moreover, we did not observe differences between either cryostorage system for normal and altered sperm samples. This lack of difference was also observed whatever the floor level of cryostorage in the dry tank. The temperature measurement of the dry tank showed a stable temperature at −194 °C throughout storage whatever the storage floor level, guaranteeing the stability of the low temperatures suitable for human sperm storage. Because of its greater safety, dry storage without contact with the liquid phase should be preferred and can be a useful alternative for the cryostorage of human sperm samples.     

Double freezing of bovine semen

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3 h incubation at 37 °C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P < 0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P ≤ 0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P < 0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P = 0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.