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The droplet vitrification method was improved for maneuverability by embedding shoot tips in gelled droplets before osmoprotection. This newly modified cryopreserving method gelled droplet vitrification - was compared with other P...
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The droplet vitrification method was improved for maneuverability by embedding shoot tips in gelled droplets before osmoprotection. This newly modified cryopreserving method gelled droplet vitrification - was compared with other PVS2-based cryopreservation methods using potato shoot tips. Survival rates of each cryogenic procedure held at 25°C were about 40% by cryotube-vitrification procedures (vitrification and encapsulation vitrification methods) and about 70% by PVS2-droplet procedures (droplet vitrification and gelled droplet vitrification methods). Much higher cooling rates of PVS2-droplet procedures than cryotube-vitrification procedures increased their survival rates. The gelled droplet vitrification method was applied to shoot tips of 26 potato cultivars and six wild potatoes. After a little modifications of the conditions for preculture, osmoprotection and dehydration, all cultivars and wild potatoes produced high enough survival rates to be of value to genebanks and all surviving shoot tips developed normal shoots within 3 weeks.
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In this paper, we compared three vitrification-based cryopreservation techniques, viz vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos Viability of somatic embryos w...
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In this paper, we compared three vitrification-based cryopreservation techniques, viz vitrification, encapsulation-vitrification and droplet-vitrification for cryopreserving sugarcane somatic embryos Viability of somatic embryos was evaluated by measuring electrolyte leakage and by regrowth on recovery medium Droplet-vitrification was the most efficient technique Optimal conditions included loading with a solution containing 1..5 M glycerol and 0 3 M sucrose for 30 min at 25°C, treatment with the PVS2 solution for 20-40 min at 0°C followed by rapid immersion in liquid nitrogen of clumps of somatic embryos placed in microdroplets of cryoprotectant solution Under such conditions, viability of cryopreserved somatic embryos reached 55 %.
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A cryopreservation protocol has been developed for embryogénie callus cultures of castor aralia (Kalopanax septemlobus), a deciduous tree which is widely used in oriental medicine and in landscape design. Three preculture treatme...
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A cryopreservation protocol has been developed for embryogénie callus cultures of castor aralia (Kalopanax septemlobus), a deciduous tree which is widely used in oriental medicine and in landscape design. Three preculture treatments, four loading and six vitrification solutions were tested in a vitrification procedure. Preculture of embryogenic callus (EC) with high sucrose concentrations (up to 0.7 M) showed no effect on regrowth after cryopreservation. Loading for 20 min at ambient temperature improved regrowth of cryopreserved EC by 70-75% compared with non-loaded samples, regardless of the composition of the loading solution. Among vitrification solutions, the highest regrowth of 95-100% after cryopreservation was obtained after incubation of EC in a vitrification solution A3-80% comprising (w/v) 33.3% glycerol + 13.3% DMSO + 13.3% EG + 20.1% sucrose for 40 min at 0°C. Profiling of crystallization and recrystallization events using differential scanning calorimetry (DSC) confirmed that freezing injury was minimized in samples after loading and cryoprotection with this vitrification solution. Unlike many other papers, the droplet-vitrification protocol did not produce higher post-cryopreservation regrowth of Kalopanax EC, compared with the vitrification procedure. When samples are sufficiently cryoprotected during VS treatment, vitrification using cryovials may be preferred, since droplet-vitrification is more complex and requires skilled personnel. Cryopreserved callus grew rapidly and produced numerous somatic embryos, which developed similarly to embryos obtained from non-cryopreserved samples.
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The efficiency of a new vitrification technique using embryos obtained after abnormal fertilization was assesed. A total of 100 embryos were vitrified, 48 with the automatic dehydrated carrier (ADC) technique and 52 with cryotop. ...
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The efficiency of a new vitrification technique using embryos obtained after abnormal fertilization was assesed. A total of 100 embryos were vitrified, 48 with the automatic dehydrated carrier (ADC) technique and 52 with cryotop. The survival rates in ADC and cryotop groups were similar (85.4% versus 92.3%). The loss rates of 10.4% for the ADC group and 1.9% for the cryotop group were not significantly different. The intact rate was significantly lower in the ADC group (26.8% versus 64.6%; P = 0.001). After 48 h of culture, blastocyst formation rates in the ADC and cryotop groups were 14.6% and 25.0%, respectively, which were not significantly different. In conclusion, the practicability of ADC vitrification was demonstrated. The study has some limitations, which necessitates further work. (C) 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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Previous studies on the vitrification of peach palm (Bactris gasipaes Kunth) embryogenic cluster highlighted PVS3 as essential for survival, but toxic nonetheless. The objective of this work was to identify methods to improve embr...
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Previous studies on the vitrification of peach palm (Bactris gasipaes Kunth) embryogenic cluster highlighted PVS3 as essential for survival, but toxic nonetheless. The objective of this work was to identify methods to improve embryogenic cluster survival through either modification to PVS3 or other changes to the established protocol. PVS3 reduced to 80% strength was less toxic to non-vitrified controls and showed no significant difference in vitrified embryogenic cluster at any incubation time (60, 120, 180, or 240 min) compared to full strength PVS3. The addition of 0.1 M KCl, MgCl2, MgSO4, or K2H(PO4) to 80% PVS3 increased vitrified embryogenic cluster regrowth. D2O had no significant effect on embryogenic cluster regrowth when used as a solvent for PVS3 compared to H2O. Larger embryogenic cluster showed greater regrowth than smaller ones. Regrowth increased with rising rewarming temperature up to 70 degrees C, the highest tested temperature. The duration of rewarming between 1, 2, or 3 min had no overall significant effect, suggesting that the most critical events in rewarming occur within the first minute. The most effective strips used for droplet vitrification were made from aluminum or silver, which were significantly more effective than polystyrene and expanded polystyrene. Droplet vitrification was initially more effective than conventional cryovial immersion vitrification at 60 min incubation in PVS3, but not significantly different at 120, 180 or 240 min incubations. Partial dehydration for 1 h in a laminar flow hood significantly increased regrowth, but longer dehydration times decreased regrowth when combines with longer PVS3 incubation times.
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The cryopreservation of ovarian tissue has been proved to be effective for fertility preservation. To find a better cryopreservation method, we tested the efficacy of cryovial monolayer vitrification method in comparison with that...
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The cryopreservation of ovarian tissue has been proved to be effective for fertility preservation. To find a better cryopreservation method, we tested the efficacy of cryovial monolayer vitrification method in comparison with that of needle immersed vitrification (NIV). Ovaries from 10 female kunming mice aged 6-8 weeks were cut into pieces and allocated into group A (cryovial monolayer vitrification method), group B (NIV method) and group C (fresh control). In group A, pieces of ovarian tissue were layered around the inner wall of cryovial as a monolayer; and in group B, pieces of ovarian tissue were pierced with a needle. Other than the difference in the carrier for ovarian tissue, the cryoprotectants and the protocols in group A and B were the same. The viability and in vitro growth potential of the follicles after warming in groups A and B were evaluated respectively and compared with those of fresh control. The results showed that the viabilities of the follicles were not statistically different among three groups. The average diameter of follicles did not show statistically significant difference among three groups before culture and between group A and group B after culture (p > 0.05), but demonstrated statistically significant difference between group A and group C (p < 0.01), group B and group C (p < 0.01), respectively. In the procedure of loading ovarian tissue onto carriers, group A took less time compared with group B. In addition, the small pieces and debris which were harder or impossible to be pierced with needle in group B could be easily layered onto the inner wall of the cryovial in group A. Hence the follicles existed within the small pieces and debris of the ovarian tissue could also be cryopreserved. It is concluded that, in cryopreserving both viability and growth potential of follicles, the cryovial monolayer vitrification method is comparable with NIV method but with higher efficiency.
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Surface pressure-area isotherms, surface potential-area isotherms and fluorescence microscopy were employed to study the behavior of phospholipid monolayers at the air/water interface when trehalose was added to the aqueous subpha...
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Surface pressure-area isotherms, surface potential-area isotherms and fluorescence microscopy were employed to study the behavior of phospholipid monolayers at the air/water interface when trehalose was added to the aqueous subphase. In the presence of this sugar, the critical area corresponding to the onset of surface potential increases, indicating that trehalose is participating in the network of hydrogen bonds between the phospholipid polar heads. In addition, it causes an expansion of the isotherm, hindering the formation of the liquid-condensed phase. The collapse area is significantly increased, indicating that trehalose takes part in the monolayer structure without being expelled even at high surface pressures. A quantitative comparison of the collapse areas and critical areas for surface potential in the presence and in the absence of the sugar shows that an almost fixed number of trehalose molecules interacts with the monolayer independently of the surface packing, thus indicating that the observed effects can be ascribed to a tight binding of trehalose to the polar heads in a defined ratio. No similar effects were observed in the presence of glucose; We rationalize the reported data in light of the water replacement hypothesis, developed to explain the preservation of biomembranes by trehalose; this hypothesis suggests that trehalose forms hydrogen bonds with the membrane polar headgroups, thus replacing the water of hydration at the membrane-fluid interface and maintaining the headgroups at their hydrated position. [References: 19]
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Cryopreservation of the threatened orchid Cymbidium eburneum L. was successfully achieved using encapsulation-vitrification and vitrification. Comparing the two methods tested, it was observed that regeneration of protocorms cryop...
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Cryopreservation of the threatened orchid Cymbidium eburneum L. was successfully achieved using encapsulation-vitrification and vitrification. Comparing the two methods tested, it was observed that regeneration of protocorms cryopreserved using encapsulationvitrification was higher than with vitrification. To achieve optimal regrowth after cryopreservation, protocorms were precultured for 24 h with 0.2 M sucrose for vitrification and with 0.7 M sucrose for encapsulation-vitrification, reaching 60% and 70% regeneration, respectively. With both techniques employed, a 20 min exposure duration to Plant Vitrification Solution 2 (PVS2) led to optimal regeneration after cryopreservation. A maximum of 66% regeneration was achieved after cryopreservation using encapsulationvitrification, whereas it was only 50% after cryopreservation using vitrification. The same regeneration pattern was observed with protocorms cryopreserved using both techniques employed. This is the first report of long-term conservation by cryopreservation of C. eburneum protocorms.
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A simple and efficient method for multiplication of vanilla (Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters o...
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A simple and efficient method for multiplication of vanilla (Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters of plantlets, by cutting the remaining base of these plant clusters into segments of about 1 cm in length. After 4 months of culture on multiplication medium, 100% of IFEs produced up to 15 new shoots per explant, providing an efficient additional method for in vitro propagation of vanilla that maximizes the use of available material. Cryopreservation of apices from in vitro grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of in vitro plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20-30 min; and exposure to plant vitrification solution PVS3 for 30 min at room temperature. Even though the cryogenic protocol needs to be optimized to improve results, this work represents the first successful report of cryopreservation of vanilla apices.
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