Concentrated Vitrification Solution Research Articles

Model biological systems demonstrate the inducibility of pathways that strongly reduce cryoprotectant toxicity

Cryoprotectant toxicity is a limiting factor for the cryopreservation of many living systems. We were moved to address this problem by the potential of organ vitrification to relieve the severe shortage of viable donor organs available for human transplantation. The M22 vitrification solution is presently the only solution that has enabled the vitrification and subsequent transplantation with survival of large mammalian organs, but its toxicity remains an obstacle to organ stockpiling for transplantation. We therefore undertook a series of exploratory studies to identify potential pretreatment interventions that might reduce the toxic effects of M22. Hormesis, in which a living system becomes more resistant to toxic stress after prior subtoxic exposure to a related stress, was investigated as a potential remedy for M22 toxicity in yeast, in the nematode worm C. elegans, and in mouse kidney slices. In yeast, heat shock pretreatment increased survival by 18-fold after exposure to formamide and by over 9-fold after exposure to M22 at 30 °C, and at 0 °C and with two-step addition, treatment with 90% M22 resulted in 100% yeast survival. In nematodes, surveying a panel of pretreatment interventions revealed 3 that conferred nearly total protection from acute whole-worm M22-induced damage. One of these protective pretreatments (exposure to hydrogen peroxide) was applied to mouse kidney slices in vitro and was found to strongly protect nuclear and plasma membrane integrity in both cortical and medullary renal cells exposed to 75–100% M22 at room temperature for 40 min. These studies demonstrate for the first time that endogenous cellular defenses, conserved from yeast to mammals, can be marshalled to substantially ameliorate the toxic effects of one of the most toxic single cryoprotectants and the toxicity of the most concentrated vitrification solution so far described for whole organs.

Ascorbate Peroxidase Activity of Aranda Broga Blue Orchid Protocorm-like Bodies (PLBs) In Response to PVS2 Cryopreservation Method.

Throughout the cryopreservation process, plants were exposed to a series of abiotic stresses such as desiccation and osmotic pressure due to highly concentrated vitrification solution. Abiotic stress stimulates the production of reactive oxygen species (ROS) which include hydrogen peroxide, superoxide radicals, and singlet oxygen. Higher production of ROS may lead to oxidative stress which contributes to the major injuries in cryopreserved explants. Antioxidant enzymes in plant such as ascorbate peroxidase (APX) can protect plants from cell damage by scavenging the free radicals. This study was determined based on APX enzyme activity of Aranda Broga Blue orchid's protocorm-like bodies (PLBs) in response to PVS2 (Plant Vitrification Solution 2) cryopreservation treatments at different stages. PLBs that were precultured at 0.25 M sucrose for 3 days were subjected to vitrification cryopreservation method. Results obtained showed that the highest APX activity was achieved at PVS2 cryoprotectant treatment prior liquid nitrogen (LN) storage. This phenomenon indicating that accumulation of osmotic and dehydrating stress throughout the cryopreservation treatment resulted in oxidative burst which in turn leads to higher APX activity in order to control the excess production of ROS. To conclude, PVS2 treatment was revealed as the most detrimental step throughout cryopreservation treatment. Thus, this research also suggested that exogenous antioxidant such as ascorbic acid can be added throughout cryopreservation procedure especially at PVS2 treatment in the future experiments to aid in regrowth of cryopreserved explants by reducing oxidative stress.

A simple and efficient protocol for cryopreservation of embryogenic calli of the medicinal plant Anemarrhena asphodeloides Bunge by vitrification

Embryogenic calli of Anemarrhena asphodeloides Bunge were successfully cryopreserved by vitrification. Excised embryogenic calli from in vitro grown tillers were precultured in liquid basal tissue culture medium from Murashige and Skoog (MS medium) containing 2 mg L−1 Kinetin (Kn), 0.1 mg L−1α-naphthalene acetic acid (NAA) and 0.5 M sucrose for 2 days under continuous light at 25 ± 1°C. Following preculture the calli were transferred to a 2 mL plastic cryotube. The calli was then loaded with a mixture of 2 M glycerol and 0.4 M sucrose for 30 min at 25 ± 1°C and dehydrated with a highly concentrated vitrification solution (PVS2) for 40 min at 0°C. After changing the solution with fresh PVS2, the calli were directly immersed in liquid nitrogen (LN). After rapid thawing in a water-bath at 35°C for 5 min, the calli were then transferred onto solid MS medium supplemented with Kn 2 mg L−1, NAA 0.1 mg L−1, 3% (w/v) sucrose and 0.75% (w/v) agar. The cultures were kept in the dark for 5 days prior to exposure to the light (14 h light/dark cycle). After 30 days, the embryoids developed from embryogenic calli were transferred to fresh solid basal MS media supplemented with Kn 2 mg L−1, NAA 0.1 mg L−1, 3% (w/v) sucrose and 0.75% (w/v) agar (regeneration medium) and developed normal shoots. The regrowth rate of vitrified embryogenic calli reached over 60%. Cryopreservation did not affect the plant regeneration potential of A. asphodeloides Bunge embryogenic calli through somatic embryogenesis. Morphologies of plants regenerated from cryopreserved calli were similar to those of the seedlings. This efficient cryopreservation protocol would be useful for the ex-situ conservation of A. asphodeloides Bunge germplasm in gene banks at very low temperatures.

IN VITRO PROPAGATION AND CRYOPRESERVATION OF STEVIA REBAUDIANA USING A VITRIFICATION METHOD

The present study aims at developing a reliable protocol for in vitro propagation and cryostorage shoot tips of Stevia rebaudiana using a vitrification method. The highest multiplication rate (8.9) was achieved on Murashige and Skoog (MS) medium supplemented with 1.5 mg/L benzyl amino purine (BAP) and 0.2 mg/L Indole-3-butryic acid (IBA). For cyropreservation of shoot tips in liquid nitrogen, several pretreatment trials prior to cryopreservation were conducted to enhance shoot tips recovery after storage in liquid nitrogen. Culturing plantlets in vitro for four weeks in MS medium containing 30 g/L sucrose, excision of shoot tips; preculture of shoot tips on media supplemented with 0.4 M sorbitol for 2 d, followed by loading shoot tips with 80% PVS2 for 20 mint, then dehydrated with a highly concentrated vitrification solution (100% PVS2) for 60 min at 0°C prior plunge into liquid nitrogen produced the highest re-growth (up to 68.8%) post thawing at 45°C for 2 min. After six months post cryopreservation none of the growth parameters had been significantly affected by the cryopreservation process. The procedure developed in this study is easy to handle and produced high levels of shoot formation. Thus, the protocol is applicable for long-term storage of S. rebaudiana germplasm in liquid nitrogen.

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.

Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification

A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after de- and rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0°C during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure.

Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l−1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.

Simplified cryopreservation of sweet potato [ Ipomoea batatas (L.) Lam.] by optimizing conditions for osmoprotection.

Shoot tips of sweet potato were successfully cryopreserved using an encapsulation vitrification method. Encapsulated shoot tips were pre-incubated in liquid Murashige-Skoog medium containing 30 g/l sucrose for 24 h, then precultured in sucrose-enriched medium (0.3 M sucrose) for 16 h. Shoot tips were osmoprotected with a mixture of 2 M glycerol and 1.6 M sucrose for 3 h before being dehydrated with a highly concentrated vitrification solution (PVS2) for 1 h at 25 degrees C. The encapsulated and dehydrated shoot tips were transferred to a 2 ml cryotube, suspended in 0.5 ml PVS2, and plunged directly into liquid nitrogen. Rapidly warmed shoot tips developed normal shoots and roots in 21 days without any morphological abnormalities after plating on a recovery medium. High levels (average of about 80%) of shoot formation were obtained for three cultivars of sweet potato. This encapsulation vitrification method appears promising for cryopreservation of sweet potato germplasm.

Cryopreservation of persimmon (Diospyros kaki Thunb.) by vitrification of dormant shoot tips

Shoot tips excised from dormant axillary buds of persimmon (Diospyros kaki Thunb.) were cryopreserved by vitrification. These excised shoot tips were dehydrated in a highly concentrated vitrification solution for 20min at 25°C and then plunged directly into liquid nitrogen. After rapid warming in water at 40°C, the shoot tips were rinsed in a 1.2M sucrose solution for 20min and then plated on a solidified culture medium. Successfully vitrified shoot tips resumed growth within 10days of plating and developed shoots within 3weeks without intermediary callus formation. This simple protocol was successfully applied to the 16 cultivars found in the temperate zone. The average rate of shoot formation was 89%. Even the subtropical species of Diospyros demonstrated a very high recovery growth when the shoot tips had been previously osmoprotected with a mixture of 2M glycerol plus 0.4M sucrose for 20min following sucrose preculture. Little or no contamination occurred in the cryopreserved shoot tips excised from sterilized winter axillary buds. Thus, this simple and reliable vitrification protocol using dormant shoot tips appears to be promising as a routine method for the long-term conservation of Diospyros germplasm of both temperate and subtropical origins.

Cryopreservation of in vitro-grown axillary shoot-tip meristems of mint (Mentha spicata L.) by encapsulation vitrification.

Alginate-coated meristems from in vitro-grown axillary buds of mint (Mentha spicata L.) were successfully cryopreserved by vitrification. Excised meristems from nodal segments cold hardened at 4  °C for 3 weeks were encapsulated and osmoprotected by a mixture of 2 M glycerol plus 0.4 M sucrose. These meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 h at 0  °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems developed shoots within a week after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 90%. This procedure was successfully applied to other Mentha species. It was also confirmed that encapsulated vitrified meristems produced a much higher rate of shoot formation than the encapsulated dried meristems. Thus, this revised encapsulation vitrification method appears promising for the cryopreservation of mint and other germplasm.

Cryopreservation of in vitro-grown meristems of potato (Solanum tuberosum L.) by encapsulation-vitrification

Alginate coated meristems from in vitro-grown axillary buds of potato (Solanum tuberosum L.) were successfully cryopreserved by vitrification. Excised meristems were precultured on sucrose-enriched MS medium and then encapsulated. To induce dehydration tolerance (osmotolerance), encapsulated meristems were treated with a mixture of 2 M glycerol plus 0.6 M sucrose for 90 min. These encapsulated meristems were dehydrated with a highly concentrated vitrification solution (PVS2 solution) for 3 hr at 0°C prior to a plunge into liquid nitrogen. Successfully vitrified meristems developed shoots within 3 weeks after plating without intermediary callus formation. The average rate of shoot formation amounted to nearly 70%. No difference was observed in RAPD analysis using 17 primers between cryopreserved and non-treated plantlets. The cryogenic protocol was successfully applied to 14 cultivars. It was also confirmed that the encapsulated vitrified meristems produced much greater shoot formation than the encapsulated dried meristems. Thus, the encapsulation vitrification protocol appears promising for cryopreservation of potato germplasm.

Cryopreservation of Shoot Tips of Tetraploid Potato (Solanum tuberosumL.) Clones by Vitrification

In vitro-grown shoot tips of five tetraploid potato (Solanum tuberosumL.) clones were cryopreserved by vitrification. Excised shoot tips (0.5–0.7 mm) were pre-cultured on filter paper discs over half strength liquid Murashige and Skoog (MS) medium supplemented with 8.7 μmGA3and different combinations of sucrose (0.3, 0.5 and 0.7m) plus mannitol (0, 0.2 and 0.4m) for 2 d under a 16 h photoperiod at 24 °C. The pre-cultured shoot tips were either successively loaded with 20 and 60% PVS 2 solutions or directly exposed to concentrated vitrification solution before physical vitrification during liquid nitrogen treatment. The vitrified shoot tips were warmed rapidly and treated with dilution mixture (MS+1.2msucrose) for 30 min before plating on regrowth medium. Addition of mannitol to the pre-culture medium improved survival of vitrified shoot tips. Direct dehydration of pre-cultured shoot tips with concentrated PVS 2 was detrimental to survival of vitrified shoot tips. Shoot tips pre-cultured on medium containing 0.3msucrose plus 0.2mmannitol, and loaded with 20% PVS 2 for 30 min followed by 15 min incubation in 60% PVS 2 and 5 min incubation in 100% PVS 2 at 0 °C resulted in up to 54% survival after vitrification. About 50% of vitrified and warmed shoot tips formed shoots directly. Post-thaw culturing of vitrified shoot tips on medium containing an elevated level of sucrose (0.2m) under diffuse light for the first week enhanced the survival rate. Continuous culturing of vitrified shoot tips on high-sucrose medium induced multiple shoot formation.

CRYOPRESERVATION OF WOODY PLANTS BY AXILLARY BUD VITRIFICATION: A FIRST APPROACH WITH POPLAR

Axillary buds from in vitro-grown stock plants of White Poplar (Populus alba L.) were utilized to test the efficiency of the vitrification technique, as cryoprotective during direct freezing in liquid nitrogen. Excised axillary buds were precultured for 2 days at 5°C on a gelled MS medium, loaded for 20 min at 25°C with a cryoprotectant (2 M glycerol and 0.4 M sucrose, 1: 1), and then exposed to a concentrated vitrification solution (PVS2: 30% glycerol, 15% ethylene glycol, 15% DMSO in MS medium). After vitrification, the buds were immersed directly into liquid nitrogen for at least one hour. Successfully vitrified buds remained green after plating, and resumed growth within 2 weeks. The highest percentage of bud survival (90%) was obtained when the buds were precultured on MS medium containing 0.09 M sucrose, and vitrified by exposure to PVS2 for 60 min at 0°C. The average rate of healthy shoot development from the surviving buds amounted to about 60%. Vitrification procedure can be proposed as an useful technique for germplasm cryopreservation of woody plants.

Cryopreservation of in vitro-grown meristems of strawberry (Fragaria × ananassa Duch.) by encapsulation-vitrification

Alginate-coated meristems from in vitro-grown strawberry (Fragaria × ananassa Duch.) were successfully cryopreserved following dehydration by a vitrification solution. Excised meristems from cold-hardened plantlets at 4 °C for two weeks in the dark were encapsulated into alginate-gel beads containing a mixture of 2 M glycerol plus 0.4 M sucrose. These encapsulated and cryoprotected meristems were dehydrated with a highly concentrated vitrification solution (designated PVS2) for 2 hr at 0 °C prior to a plunge into liquid nitrogen. Successfully encapsulated vitrified meristems remained green and then developed shoots within one week after plating without intermediary callus formation. The average rate of shoot formation of encapsulated vitrified meristems amounted to nearly 90%. The cryogenic protocol was successfully applied to four cultivars of strawberry. It was also confirmed that encapsulated vitrified meristems cooled to - 196 °C produced higher shoot formation than encapsulated dried meristems. Besides, the recovery growth was much earlier than the latter. The encapsulation-vitrification method is easy to handle and produces high levels of shoot formation. Thus, the protocol promises for cryopreservation of strawberry germplasm.

Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) by vitrification.

The seeds of a Japanese terrestrial orchid (Bletilla striata Rchb.f.) were germinated and cultured on solidified new Dogashima (ND) medium for 10 days. These embryos were then precultured on ND medium supplemented with 0.3 M sucrose for 3 days at 25°C in continuous dark. The embryos were then overlaid with a mixture of 2 M glycerol and 0.4 M sucrose for 15 min at 25°C and finally dehydrated with highly concentrated vitrification solution (PVS2) for 3 h at 0°C prior to immersion into liquid nitrogen for 30 min. After rapid warming, the embryos were washed with liquid ND medium supplemented with 1.2 M sucrose for 20 min and then plated on ND medium. Successfully vitrified and warmed embryos developed into normal plantlets. The rate of plant regeneration amounted to about 60%. This vitrification method appears to be a promising technique for cryopreservation of orchids.

Cryopreservation of invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1. Investigation of basic conditions of the vitrification procedure.

Invitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott.) were successfully cryopreserved by vitrification. Excised shoot tips precultured on solidified MS supplemented with 0.3M sucrose and maintained under a 16 h phtoperiod at 25°C for 16 h were loaded with a mixture of 2M glycerol plus 0.4M sucrose for 20 min at 25°C. The shoot tips were then sufficiently dehydrated with a highly concentrated vitrification solution (PVS2) for 20 min at 25°C prior to immersion into liquid nitrogen. Successfully vitrified and warmed shoot tips resumed growth within 7 days and developed shoots directly without intermediate callus formation. The average rate of shoot recovery amounted to around 80%, and the vitrification protocol appeared to be very promising for the cryopreservation of taro germplasm.

Cryopreservation of encapsulated shoot primordia induced in horseradish (Armoracia rusticana) hairy root cultures.

Shoot primordia induced inArmoracia rusticana Gaertn. Mey. et Scherb. (horseradish) hairy root cultures were successfully cryopreserved by two cryogenic procedures. Encapsulated shoot primordia were precultured on solidified Murashige-Skoog medium supplemented with 0.5M sucrose for 1 day and then dehydrated with a highly concentrated vitrification solution (PVS2) for 4 h at 0°C prior to a plunge into liquid nitrogen. The survival rate of encapsulated vitrified primordia amounted to 69%. In a revised encapsulation-dehydration technique, the encapsulated shoot primordia were precultured with a mixture of 0.5M sucrose and 1M or 1.5M glycerol for 1 day to induce dehydration tolerance and then subjected to air-drying prior to a plunge into liquid nitrogen. The survival rate of encapsulated dried primordia was more than 90%, and the revived primordia produced shoots within 2 weeks after plating. A long-term preservation of shoot primordia was also achieved by the technique. Thus, this revised encapsulation-dehydration technique appears promising as a routine method for the cryopreservation of shoot primordia of hairy roots.

Vitrificational cryopreservation and subsequently fertile plant regeneration from rice (Oryza sativa L.) embryogenic suspension cells

CRYOPRESERVATION by vitrification is a new ultralow-temperature-storage technique developed in the 1980s. Operationally, procedures for vitrification of biological specimens consist of the following basic steps: treatment of the samples in a highly concentrated vitrification solution and then plunging the specimens into liquid nitrogen. The word vitrification is sometimes used to describe the high moisture of the in vitro cultures or regenerants. However, in this note, this word means a biophysical process in which the cells and the solution are transformed into a homogeneous and amorphous state. The water in cells does not change into ice, but in a...

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.

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

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.