Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) by vitrification

The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.Abbreviations DMSO dimethyl sulfoxide - PVS2 vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - BA 6-benzylaminopurine - MT Murashige-Tucker basal medium - INAA naphthaleneacetic acid     

Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification

Cultured cells and somatic embryos derived from the mesophyll tissue of asparagus (Asparagus officinalis L.) were cryopreserved by vitrification. The vitrification solution (PVS) contains (w/v) 22% glycerol, 15% ethylene glycol, 15% propylene glycol and 7% DMSO in Murashige-Skoog medium enriched with 0.5M sorbitol. After initial cryoprotection with sorbitol supplemented MS medium containing 12% ethylene glycol, cells or embryos were exposed stepwise to 85% PVS at 0°C. They were loaded into 0.5 ml transparent straws, and were then plunged directly into liquid nitrogen. After rapid warming, PVS was removed and diluted stepwise. The highest survivals of vitrified cells and embryos were about 65 and 50%, respectively. Surviving embryos developed into plantlets.Abbreviations DMSO dimetyl sulfoxide - PVS vitrification solution - LN liquid nitrogen - DSC differential scanning calorimeter - MS Murashige-Skoog salt medium - NAA naphthalene acetic acid - BA 6-benzyladenine     

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant <Emphasis Type="Italic">Emmenopterys henryi</Emphasis> Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS₂) for 30 min at 0°C and followed by 100% PVS₂ for 40 min at 0°C. After changing the solution with fresh 100% PVS₂, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l⁻¹, α-naphthaleneacetic acid 0.1 mg l⁻¹, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.     

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

Cryopreservation of grapevine (Vitis vinifera L.) in vitro shoot tips

In this work, we compared the efficiency of encapsulation-dehydration and droplet-vitrification techniques for cryopreserving grapevine (Vitis vinifera L.) cv. Portan shoot tips. Recovery of cryopreserved samples was achieved with both techniques; however, droplet-vitrification, which was used for the first time with grapevine shoot tips, produced higher regrowth. With encapsulationdehydration, encapsulated shoot tips were precultured in liquid medium with progressively increasing sucrose concentrations over a 2-day period (12 h in medium with 0.25, 0.5, 0.75 and 1.0 M sucrose), then dehydrated to 22.28% moisture content (fresh weight). After liquid nitrogen exposure 37.1% regrowth was achieved using 1 mm-long shoot tips and only 16.0% with 2 mm-long shoot tips. With droplet-vitrification, 50% regrowth was obtained following treatment of shoot tips with a loading solution containing 2 M glycerol + 0.4 M sucrose for 20 min, dehydration with half-strength PVS2 vitrification solution (30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% dimethylsulfoxide and 0.4 M sucrose in basal medium) at room temperature, then with full strength PVS2 solution at 0°C for 50 min before direct immersion in liquid nitrogen. No regrowth was achieved after cryopreservation when shoot tips were dehydrated with PVS3 vitrification solution (50% (w/v) glycerol and 50% (w/v) sucrose in basal medium).     

Cryopreservation of in vitro-grown apical meristems of lily by vitrification

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige & Skoog (1962) medium - PVS2 vitrification solution     

Cryopreservation of <Emphasis Type="Italic">Dendrobium candidum</Emphasis> Wall. ex Lindl. protocorm-like bodies by encapsulation-vitrification

Protocorm-like bodies (PLBs) of Dendrobium candidum Wall. ex Lindl., orchid, were successfully cryopreserved using an encapsulation vitrification method. PLBs were precultured in liquid Murashige and Skoog (MS) medium containing 0.2 mg l⁻¹ α-naphthalene acetic acid and 0.5 mg l⁻¹ 6-benzyladenine enriched with 0.75 M sucrose, and grown under continuous light (36 μmol m⁻² s⁻¹) at 25 ± 1°C for 5 days. PLBs were osmoprotected with a mixture of 2 M glycerol and 1 M sucrose for 80 min at 25°C and dripped in a 0.5 M CaCl₂ solution containing 0.5 M sucrose at 25 ± 1°C and left for 15 min to form Ca-alginate beads (about 4 mm in diameter). Then, these were dehydrated with a plant vitrification solution 2 (PVS₂) consisting of 30% (w/v) glycerol, 15% (w/v) ethylene glycol, and 15% (w/v) dimethyl sulfoxide in 0.5 M sucrose, pH 5.8, for 150 min at 0°C. Encapsulated and dehydrated PLBs were plunged directly into liquid nitrogen for 1 h. Cryopreserved PLBs were then rapidly re-warmed in a water bath at 40°C for 3 min and then washed with MS medium containing 1.2 M sucrose for three times at 10 min intervals. Within 60 days, plantlets with the cryopreserved PLBs developed normal shoots and roots, and without any observed morphological abnormalities, were obtained. The survival rate of encapsulated-vitrified PLBs was above 85%. Thus, this encapsulation-vitrification method was deemed promising for cryopreservation of PLBs of D. candidum.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Cryoprotectants and components induce plasmolytic responses in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> (L.) Lam.) suspension cells

Plant genebanks often use cryopreservation to securely conserve clonally propagated collections. Shoot tip cryopreservation procedures may employ vitrification techniques whereby highly concentrated solutions remove cellular water and prevent ice crystallization, ensuring survival after liquid nitrogen exposure. Vitrification solutions can be comprised of a combination of components that are either membrane permeable or membrane impermeable within the timeframe and conditions of cryoprotectant exposure. In this study, the osmotic responses of sweet potato [Ipomoea batatas (L.) Lam.] suspension cell cultures were observed after treatment with plant vitrification solution 2 [PVS2; 15% (v/v) dimethyl sulfoxide (DMSO), 15% (v/v) ethylene glycol, 30% (v/v) glycerol, 0.4 M sucrose], plant vitrification solution 3 (PVS3; 50% (v/v) glycerol, 50% (w/v) sucrose), and their components at 25 and 0°C, as well as cryoprotectant solution, PGD (10% (w/v) PEG 8000, 10% (w/v) glucose, 10% (v/v) DMSO) at 25°C. At either 25 or 0°C, sweet potato cells plasmolyzed after exposure to PVS2, PVS3, and PGD solutions as well as the PVS2 and PVS3 solution components. Cells deplasmolyzed when the plasma membrane was permeable to the solutes and when water re-entered to maintain the chemical potential. Sweet potato suspension cells deplasmolyzed in the presence of 15% (v/v) DMSO or 15% (v/v) ethylene glycol. Sweet potato plasma membranes were more permeable to DMSO and ethylene glycol at 25°C than at 0°C. Neither sucrose nor glycerol solutions showed evidence of deplasmolysis after 3 h, suggesting low to no membrane permeability of these components in the timeframes studied. Thus, vitrification solution PVS2 includes components that are more membrane permeable than PVS3, suggesting that the two vitrification solutions may have different cryoprotectant functions. PGD includes DMSO, a permeable component, and likely has a different mode of action due to its use in two-step cooling procedures.     

Development of vitrified bovine secondary and primordial follicles in xenografts

The objective was to evaluate the effect of various vitrification conditions on the morphology of bovine secondary and primordial follicles, and to use xenografting to confirm their developmental ability. Secondary follicles were placed in vitrification solution containing 15% (v:v) ethylene glycol (EG), 15% (v:v) dimethyl sulfoxide (DMSO), 20% (v:v) fetal calf serum (FCS), and 0, 0.25, or 0.5 M sucrose at room temperature for 1 or 30 min, or at 4 °C for 30 min before being plunged into liquid nitrogen (LN₂). Ovarian tissues with primordial follicles were equilibrated in a solution containing 7.5% EG, 7.5% DMSO, and 20% FCS for 5 or 15 min, and then treated with a vitrification solution (15% EG, 15% DMSO, and 20% FCS) containing 0 or 0.5 M sucrose at room temperature for 1 min, and then plunged into LN₂. One week later, follicles and tissues were warmed, and morphology assessed histologically. Secondary follicles vitrified in sucrose-free solution had more oocytes with shrinkage of the nucleus and abnormal cytoplasm relative to those vitrified in sucrose-containing solution. When primordial follicles were equilibrated for 5 min and vitrified in sucrose-free solution, the percentage of morphologically normal primordial follicles was higher than in the other groups (P < 0.05). After 4 wk and 6 mo of xenografting of vitrified-warmed secondary and primordial follicles, respectively, in SCID mice, follicles developed to the antral stage and oocytes grew. In conclusion, bovine secondary follicles were successfully cryopreserved in sucrose-containing vitrification solutions and maintained their ability to develop to the antral stage and grow oocytes, whereas primordial follicles vitrified in sucrose-free solution maintained their morphology and developed to the antral stage, with oocyte growth.     

Post-vitrification survival and in vitro maturation rate of buffalo (Bubalus bubalis) oocytes: effect of ethylene glycol concentration and exposure time

The present study was undertaken to investigate the effects of ethylene glycol concentration and time of exposure to equilibration solution on the post-thaw morphological appearance and the in vitro maturation rate of buffalo oocytes. Vitrification solution-I (VS-I) consisted of 4.5M ethylene glycol (EG), 3.4M dimethyl sulphoxide, 5. 56mM glucose, 0.33mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), whereas vitrification solution-II (VS-II) contained 3.5M EG, with other constituents at same concentrations as in VS-I. The equilibration solutions-I and II were prepared by 50% dilution (v/v) of VS-I and VS-II, respectively, in DPBS. Prior to vitrification, the cumulus-oocyte complexes (COCs) were exposed to equilibration solution-I or II for 1 or 3min at room temperature (25-30 degrees C). Groups of four to five oocytes were then placed in 15microl of respective vitrification solution, and immediately loaded into 0. 25ml French straws, each containing 150microl of 0.5M sucrose in DPBS. The straws were placed in liquid nitrogen (LN(2)) vapour for 2min, plunged and stored in LN(2) for at least 7 days. The straws were thawed by keeping in warm water at 28 degrees C for 20s, and the oocytes were equilibrated for 5min in 0.5M sucrose for one-step dilution. The percentage of oocytes found to be morphologically normal varied from 89 to 96% for the two equilibration solutions and the two exposure times. Among the damaged oocytes, cracking of zona pellucida was the abnormality observed most frequently. The nuclear maturation rate of oocytes equilibrated in equilibration solutions-I and II for 1 (28 and 24%, respectively) or 3min (32 and 33%, respectively) did not differ significantly. These results show that it is possible to cryopreserve buffalo oocytes by vitrification using a combination of 3.5M EG and 3.4M DMSO with an exposure time of 3min.     

Plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells cryopreserved by vitrification

Summary Rice cells were precultured for 10 d in medium containing 60 g/L sucrose and subsequently for 1 d in medium supplemented with 0. 4 M sorbitol. After loading with 25%PVS2 at 22°C for 10 min and dehydration in 100%PVS2 at 0°C for 7. 5 min,they were plunged into liquid nitrogen directly. Survival was 45. 0 ±5.1% (based on the reduction of triphenyl tetrazolium chloride)following warming and unloading. For regrowth, cells were plated on semi-solid medium replenished with 40%(w/v) starch for 2d prior to reculture. Cell suspensions were reestablished and plants were regenerated from recovered cells. Twenty eight plants set seeds in the greenhouse.Abbreviations PVS plant vitrification solution - P preculture - LN liquid nitrogen - TTC triphenyl tetrazolium chloride - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - EG ethylene glycol - BSA bovine serum albumin     

Development of a modified vitrification strategy suitable for subsequent scale-up for hepatocyte preservation

This work explores the design of a vitrification solution (VS) for scaled-up cryopreservation of hepatocytes, by adapting VS_basic (40% (v/v) ethylene glycol 0.6 M sucrose, i.e. 7.17 M ethylene glycol 0.6 M sucrose), previously proven effective in vitrifying bioengineered constructs and stem cells. The initial section of the scale-up study involved the selection of non-penetrating additives to supplement VS_basic and increase the solution’s total solute concentration. This involved a systematic approach with a step-by-step elimination of non-penetrating cryoprotectants, based on their effect on cells after long/short term exposures to high/low concentrations of the additives alone or in combinations, on the attachment ability of hepatocytes after exposure. At a second stage, hepatocyte suspension was vitrified and functions were assessed after continuous culture up to 5 days.Results indicated Ficoll as the least toxic additive. Within 60 min, the exposure of hepatocytes to a solution composed of 9% Ficoll + 0.6 M sucrose (10⁻³ M Ficoll + 0.6 M sucrose) sustained attachment efficiency of 95%, similar to control. Furthermore, this additive did not cause any detriment to the attachment of these cells when supplementing the base vitrification solution VS_basic. The addition of 9% Ficoll, raised the total solute concentration to 74.06% (w/v) with a negligible 10⁻³ M increase in molarity of the solution. This suggests main factor in inducing detriment to cells was the molar contribution of the additive.Vitrification protocol for scale-up condition sustained hepatocyte suspension attachment efficiency and albumin production. We conclude that although established approach will permit scaling-up of vitrification of hepatocyte suspension, vitrification of hepatocytes which are attached prior to vitrification is more effective by comparison.     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Cryopreservation: A tool to conserve date palm in Saudi Arabia

The cryostoring of embryogenic tissue of the date palm (Phoenix dactylifera L. cv. Sagai) was examined through dehydrated-encapsulation, vitrification, and vitrification-encapsulation. The most extreme regeneration rate (53.33%) of epitomized, cryostored liquid nitrogen (+LN) treated embryos was observed when pre-embryonic masses were hatched with 0.5 M sucrose for 48 h pursued by 6 h air drying out. The most noteworthy survival rate (80.0%) of epitomized, cryopreserved embryonic cluster came about when calli were hatched with 0.3 or 0.7 M sucrose for 48 h pursued by four hours of lack of hydration, or with 0.5 M sucrose for 48 h without air drying out or with 2 h of air drying out. Following cryopreservation utilizing the embodiment vitrification convention, the most astounding survival (86.7%) as well as the greatest growth (46.7%) was accomplished when the typified vitrified, cryopreserved calli were treated with Vitrification Solution 2 for plants (PVS2) for 60 min at 25 °C. Cryopreservation utilizing the vitrification convention brought about the most extreme recuperation of 53.3%, when vitrified-cryopreserved calli were subjected to PVS2 solution for 30 min at 25 °C. Most extreme (40%) regeneration of vitrified, cryopreserved embryonic calli was seen when these calli were treated with PVS2 solution for 60 min at 25 °C. The outcome got amid this investigation of regrowth after cryopreservation of the cv. Sagai was over the base suitable for a cryo-germplasm bank. Recovery and regrowth were above 30% for all the techniques developed for the cv. Sagai.     

Successful vitrification of pronuclear-stage rabbit zygotes by minimum volume cooling procedure

Rabbit zygotes at the pronuclear-stage were cryopreserved by vitrification using a gel-loading tip (GL-tip), Cryoloop or Cryotop. In GL-tip and Cryoloop methods, zygotes were first exposed to 10% ethylene glycol (EG)+10% DMSO in TCM199+20% fetal bovine serum (FBS) for 2 min, and then equilibrated for 30 s in a vitrification solution composed of 20% EG+20% DMSO+0.6 M sucrose in TCM199+20% FBS. In Cryotop method, zygotes were first exposed to 7.5% EG+7.5% DMSO+20% FBS in TCM199 for 3 min, and then equilibrated for 1 min in a vitrification solution composed of 15% EG+15% DMSO+0.5 M sucrose+20% FBS in TCM199. In vitro culture of vitrified-warmed zygotes using GL-tip and Cryoloop resulted in low cleavage rates (2 and 5%, respectively) and no development into blastocysts. In contrast, zygotes vitrified-warmed using Cryotop exhibited higher proportions of cleavage (58%) and development into blastocysts (24%). When compacted morulae or early blastocysts were vitrified by these three procedures, 80-93% of them exhibited blastocoele expansion or zona hatching during the subsequent 48 h of culture. Use of Cryotop instead of GL-tip or Cryoloop for zygote vitrification, without changing conditions of solutions and periods for exposure, equilibration and post-warm dilution, resulted in cleavage and blastocyst development rates of 88 and 45%, respectively. A longer exposure time (10 min) of zygotes to 7.5% EG+7.5% DMSO+20% FBS in TCM199 resulted in higher proportions of zygotes cleaving (94%) and developing into blastocysts (51%) after Cryotop vitrification. Proportions of post-warm zygotes (10-min exposure group) and fresh control zygotes developing into newborn offspring were 36 and 53%, respectively. Pronuclear-stage rabbit zygotes were successfully cryopreserved by vitrification using the Cryotop method.     

Comparison of different vitrification protocols on viability after transfer of ovine blastocysts in vitro produced and in vivo derived

We compare different vitrification protocols on the pregnancy and lambing rate of in vitro produced (IVP) and in vivo derived (IVD) ovine embryos. Ovine blastocysts were produced by in vitro maturation, fertilization and culture of oocytes collected from slaughtered ewes or superovulated and inseminated animals. Embryos were cryopreserved after exposure at room temperature either for 5 min in 10% glycerol (G), then for 5 min in 10% G + 20% ethylene glycol (EG), then for 30 s in 25% G + 25% EG (glycerol group), or for 3 min in 10% EG + 10% dimethyl sulphoxide (DMSO), then for 30s in 20% EG + 20% DMSO + 0.3 M sucrose (DMSO group). One group of in vitro produced embryos was cryopreserved similarly to the DMSO group, but with 0.75 M sucrose added to the vitrification solution (DMSO 0.75 group). Glycerol group embryos were then loaded into French straws or open pulled Straws (OPS) while the DMSO group embryos were all loaded into OPS and directly plunged into liquid nitrogen. Embryos were warmed with either a one step or three step process. In the one step process, embryos were placed in 0.5 M sucrose. The three-step process was a serial dilution in 0.5, 0.25 and 0.125 M sucrose. The embryos of DMSO 0.75 group were warmed directly by plunging them into tissue culture medium-199 (TCM-199) + 20% foetal bovine serum (FBS) in the absence of sucrose (direct dilution). Following these manipulations, the embryos were transferred in pairs into synchronised recipient ewes and allowed to go to term. The pregnancy and the lambing rate within each group of IVP and IVD embryos indicated that there was no statistical difference among the vitrification protocols.     

Investigating the cryopreservation of nodal explants of Lithodora rosmarinifolia (Ten.) Johnst., a rare, endemic Mediterranean species

In this study, we investigated the possibility of using the droplet-vitrification technique for cryopreserving nodal segments of in vitro plantlets of the endangered plant species Lithodora rosmarinifolia. Among the three vitrification solutions tested, only solutions B1, containing (w/v) 50 % glycerol and 50 % sucrose, and B3, containing 40 % glycerol and 40 % sucrose, were able to induce cryotolerance in nodal explants, resulting in intermediate survival and recovery after cryopreservation. A three-step vitrification protocol, including an additional dehydration treatment with half-strength vitrification solution for 30 min before the treatment with full-strength vitrification solution, did not lead to any improvement in survival and recovery compared with the two-step protocol. The optimal protocol was the following: preculture of nodal segments in liquid medium with 0.3 M sucrose for 16 h and 0.7 M sucrose for 5 h, treatment for 20 min in loading solution containing 1.9 M glycerol + 0.5 M sucrose, dehydration with vitrification solution B1 (glycerol 50.0 %, sucrose 50.0 %, w/v) for 60 min at room temperature, rapid cooling in minute droplets of vitrification solution, and rapid rewarming by immersion of nodal segments for 20 min in unloading solution containing 1.2 M sucrose. Under these conditions, 33 % recovery of cryopreserved nodal explants was achieved. Regrowth of cryopreserved samples was rapid and direct. These results indicate that long-term storage of L. rosmarinifolia by means of cryopreservation of nodal segments is possible, thereby contributing to securing the diversity of this rare and endangered plant species.