Cryopreservation of in vitro grown shoots of ginger (Zingiber officinale Rosc.)

An efficient cryopreservation technique for in vitro grown shoots of ginger (Zingiber officinale Rosc) was developed based on encapsulation dehydration, encapsulation vitrification and vitrification procedures. Pregrowth and serial preculture were needed to obtain the best regrowth for all techniques. The vitrification procedure resulted in higher regrowth (80%) when compared to encapsulation vitrification (66%) and encapsulation dehydration (41%). In the vitrification procedure shoots were: precultured in liquid Murashige-Skoog medium containing 0.3 M sucrose for 3 days; cryoprotected with a mixture of 5% DMSO and 5% glycerol for 20 min at room temperature; osmoprotected with a mixture of 2 M glycerol and 0.4 m sucrose for 20 min at 25 degrees C; before being dehydrated with a highly concentrated vitrification solution (PVS2) for 40 min at 25 degrees C. The dehydrated shoots were transferred to 2 ml cryotubes, suspended in 1 ml PVS2 and plunged directly into liquid nitrogen. In all the three cryopreservation procedures tested, shoots grew from cryopreserved shoot tips without intermediary callus formation. The genetic stability of cryopreserved ginger shoot buds were confirmed using ISSR and RAPD profiling.     

Comparison of cryopreservation techniques for long-term storage of ash (Fraxinus excelsior L.)

The main purpose of this study was to develop a cryopreservation protocol for ash and to highlight the importance of testing different clones and plant material of different ontogenetic states. In vitro-grown ash (Fraxinus excelsior L.) shoot tips were successfully cryopreserved following optimization of the PVS2-vitrification protocol. Pretreatment conditions were optimized and three cryopreservation techniques (encapsulation/dehydration, PVS2-vitrification and encapsulation-vitrification) were tested one after another. PVS2-vitrification proved to be the most suitable technique. In vitro-grown shoot tips of ash were successfully cryopreserved with a mean regrowth of 73% for juvenile clones and 67% for selected mature trees. The optimum preculture conditions and the initial protocol were: 10 days cold hardening, preculture for 2 days on medium with 0.8 M glycerol, incubation in 2 M glycerol solution for 20 min at 22 degrees C followed by PVS2 for 25 min at 0 degrees C on ice and direct immersion in liquid nitrogen. Warming was carried out in 43 degree C water for 1 min followed by 22 degree C water for 10 sec. The encapsulation/dehydration method was not successful for shoot tips of F. excelsior because the shoots were sensitive to osmotic dehydration. The encapsulation/vitrification method resulted in a mean regrowth of only 16%. PVS2 vitrification can now be used to store important ash germplasm of either juvenile or mature trees.     

Cryopreservation of garlic bulbil primordia by the droplet-vitrification procedure

The droplet-vitrification protocol, a combination of droplet-freezing and solution-based vitrification was applied for cryopreserving garlic bulbil primordia. The highest survival and regeneration percentages of cryopreserved primordia (90.1 to 95.0 percent and 82.7 to 85.0 percent, respectively) were achieved after preculture for 2-4 days at 10 degree C on solid medium with 0.1 - 0.3 M sucrose, loading for 50 minutes in liquid medium with 2 M glycerol + 0.5 M sucrose, dehydration with PVS3 vitrification solution for 90-150 min, cooling primordia in 5 microl droplets of PVS3 vitrification solution placed on aluminum foil strips by dipping these strips in liquid nitrogen, warming them by plunging the foil strips into pre-heated (40 degree C) 0.8 M sucrose solution for 30 s and further incubation in the same solution for 30 minutes. The optimized droplet-vitrification protocol was successfully applied to bulbil primordia of five garlic varieties originating from various countries and to immature bulbils of two vegetatively propagated Allium species, with regeneration percentages ranging between 77.4 - 95.4 percent.     

Cryopreservation of Citrus madurensis embryonic axes by vitrification: importance of loading and treatment with vitrification solution

This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 degree C or for 60 min at 0 degree C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 degree C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.     

An improved cryopreservation protocol for pineapple apices using encapsulation-vitrification

Several modifications to the cryogenic protocols previously described for pineapple apices were performed using vitrification and encapsulation-vitrification. Pregrowth of apices in sucrose-proline before loading significantly reduced the exposure duration to PVS2 and PVS3 required for successful cryopreservation. Encapsulation and treatments with PVS3 at 0 degree C gave the highest survival before and after cooling. Optimal conditions involved the encapsulation of pineapple apices in calcium alginate (3 percent) followed by a 2-d preculture in liquid medium with 0.16 M sucrose + 0.3 M proline for 24 h and then transfer to 0.3 M sucrose + 0.3 M proline for an additional 24 h. After preculture, samples were loaded in 0.75 M sucrose + 1 M glycerol solution at room temperature (25 min) and dehydrated with PVS3 at 0 degree C for 60 min before immersion into liquid nitrogen. Following this procedure 54 percent and 83 percent of apices from MD-2 and Puerto Rico varieties respectively survived.     

Thermal analysis of garlic shoot tips during a vitrification procedure

The thermal behavior of garlic shoot tips was analyzed during the course of a vitrification protocol using the PVS3 vitrification solution. The size of shoot tips did not significantly influence the thermal behavior of garlic shoot tips. Though there was no significance, endo-thermal enthalpy from melting of crystalline ice increased as preculture duration increased to 6 days. Preculture on medium with 0.5 M sucrose significantly lowered exo- and endothermal enthalpies of dehydration-control shoot tips. By contrast, after dehydration with PVS3 solution, the concentration of sucrose in preculture medium had no significant effect on the value of enthalpies. A big thermal event was observed in garlic shoot tips air-dried for 1-3 h before dehydration. Both vitrification solution and dehydration duration significantly (P < 0.0001) influenced exo- and endothermal enthalpies. After dehydration with PVS1, PVS2, Fahy or Steponkus solutions for 120 min, only a small peak was detected in some shoot tips, but recovery of cryopreserved shoot tips was low. Dehydration duration with PVS3 solution significantly (P < 0.0001) influenced exo- and endothermal enthalpies and onset temperatures during cooling and warming. After dehydration for 150 and 180 min with PVS3 vitrification solution, no crystallization was observed during cooling and warming in most replicates, and recovery of cryopreserved shoot tips was highest (> 80%). There was a significant (P < 0.001) negative correlation between moisture content of shoot tips and concentration of sucrose and glycerol, and regeneration of cryopreserved shoot tips. By contrast, there was a significant (P < 0.001) positive correlation between MC and enthalpy of ice melting, and onset temperature of crystallization. Overall, the results of the analysis of the thermal behavior of garlic shoot tips coincide very well with their recovery after cryopreservation and provide a very useful tool for the establishment and optimization of cryopreservation protocols.     

Cryopreservation of Centaurea ultreiae (Compositae) a critically endangered species from Galicia (Spain)

Ex situ conservation of endangered plants is an important aim in order to preserve biodiversity of flora in threatened ecosystems. Among the biotechnological techniques which can be used, cryopreservation is emerging as a preferred option in many instances. This study describes a cryopreservation technique developed for shoot tips of the endangered species Centaurea ultreiae (Compositae) using a vitrification procedure. Basal medium (BM) for preculture and loading phases consisted of 1/2 MS basal salts with modified vitamins (3 microM thiamine). For preculturing shoot tips, BM with five osmotic treatments were investigated: 0.3 M sucrose +/- 20 microM ABA, 0.6 M glycerol +/- 20 microM ABA and 0.25 M sucrose + 0.25 M glycerol + 10 microM ABA. A loading solution treatment (BM with 2 M glycerol and 0.4 M sucrose) was applied prior to exposure of shoot tips to PVS2 and found to be indispensable to obtaining successful post-LN recovery. Highest (95.5%) regrowth of LN immersed shoot tips was obtained following incubation on BM + 0.3 M sucrose + 20 microM ABA or 0.25 M sucrose + 0.25 M glycerol + 10 microM ABA, with loading treatment and PVS2 exposure for 20 minutes at 0 degree C. Keywords: cryopreservation, encapsulation, endangered species, ex situ conservation, vitrification.     

Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification

This paper investigates the effect of the origin and size of the explants employed and of the preconditioning (cold acclimation, preculture) and loading treatments on survival and regeneration of cryopreserved garlic shoot apices using vitrification with the PVS3 vitrification solution. Both the origin and size of explants had a significant effect on regeneration of cryopreserved apices. Higher regeneration was generally observed with apices excised from bulbs and bulbils, followed by cloves, and those originated from larger propagules regrew more rapidly. Smaller apices (1.5 or 3.0 mm in diameter) displayed higher regeneration than large ones (4.5 mm in diameter). Cold acclimation at 5 degree C of apices before freezing had no positive effect on regeneration after cryopreservation. Preculture of apices at 10 or 23 degree C for more than 3 days had a detrimental effect on regeneration. The optimal sucrose concentration in the preculture medium was 0.3-0.5 M. Loading apices for 30 or 60 min at 23 degree C in medium containing 2 M glycerol + 0.4 M sucrose or 1 M glycerol + 0.8 M sucrose had no effect on regeneration after cryopreservation, in comparison with apices cryopreserved without loading treatment. Under optimal conditions, regeneration of cryopreserved apices sampled from large cloves was above 90 percent.     

Cryopreservation of protocorm-like bodies of the hybrid orchid Bratonia (Miltonia flavescens × Brassia longissima)

In this study, cryopreservation of Bratonia (Miltonia flavescens (Lindl.) Lindl. × Brassia longissima (Reichb.) Nash), a hybrid tropical orchid, was achieved using protocorm-like bodies (PLBs) multiplied in vitro. Cryopreservation was performed using a vitrification protocol including pretreatment of PLBs with a loading solution (LS, 2.0 M glycerol + 0.4 M sucrose) for 15 min followed by treatment with modified PVS2 vitrification solution (containing PEG instead of ethylene glycol) for 1 h. Increasing benzyladenine (BA) concentration in the recovery medium to 5.0 or 10.0 mg l?1 during the initial 3 weeks after rewarming provided 20.4 % post-cryopreservation regrowth. By contrast, preliminary culture of PLBs with abscisic acid (ABA) and high sucrose concentrations (up to 0.3 M) as well as addition of reduced glutathione during the preculture, loading and post-culture steps were not beneficial. Forty to 45 plants were regenerated from each PLB which withstood cryopreservation. No morphological differences were observed between plants regenerated from cryopreserved and untreated PLBs. Investigations into the functional activity of photosystems I and II in PLBs suggest that electron transport was retained in the reaction centers of both photosystems shortly after cryopreservation.     

Cryopreservation of sugarcane somatic embryos

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 degree C, treatment with the PVS2 solution for 20-40 min at 0 degree 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 percent.     

Development of a shoot-tip vitrification protocol and comparison with encapsulation-based procedures for plum (Prunus domestica L.) cryopreservation

Cryopreservation of plum (Prunus domestica L.), cv Regina Claudia, was obtained by a vitrification/one-step cooling procedure of shoot tips from cold-hardened in vitro-grown plants. Best survival (57%) was obtained when the shoot tips were precultured at 4 degree C for 2 days on 0.09 M sucrose-containing Quoirin and Le Poivre medium, loaded for 30 min with a cryoprotectant (2 M glycerol and 0.4 M sucrose), incubated with the PVS2 solution at 0 C for 90 min, and directly plunged into liquid nitrogen. After re-warming in a waterbath at 40 degree C, the shoot tips were washed in a 1.2 M-sucrose MS solution for 20 min and finally plated on a regrowth medium. In comparison with the one-step cooling procedure, both the slow cooling (-0.5 degree C/min up to -45 degree C), and the two-step cooling (-160 degree C for 25 min, then -196 degree C) gave lower percentages of shoot-tip survival. Among the other cryogenic procedures tested, the performance of the encapsulation-vitrification method was similar to the vitrification protocol in terms of shoot-tip regrowth (47.5%), while encapsulation-dehydration was unsatisfactory.     

Cryopreservation of in vitro shoot apices of Oxalis tuberosa Mol

Oca (Oxalis tuberosa Mol.) is an under-utilized tuber crop from the Andean region. Cryopreservation would allow the safe and long-term preservation of the genetic resources of this crop. A protocol for the cryopreservation of in vitro grown shoots has been developed using the vitrification solution PVS2. Two genotypes were studied (G1 and G27). Nodal segments were cultured on MS medium and incubated at 10 degree C with 16 h photoperiod and 10 mol per square meter per second irradiance, for two weeks. Apices were then excised and cultured on MS+0.15 M sucrose for 3 days at 5 degree C in darkness. Subsequently, apices were immersed in a loading solution (liquid MS medium+2 M glycerol+0.4 M sucrose), and then treated with the vitrification solution PVS2 for 0 to 40 minutes. Cryovials were then immersed in liquid nitrogen. Four weeks after rewarming and culture on recovery medium, genotype G1 showed approximately 60 percent recovery (normal growth) with 20 min PVS2 treatment. Genotype G27 showed lower recovery (30 percent). Differential scanning calorimetry yielded a Tg midpoint for PSV2 solution of ca. -120 degree C. Calorimetric studies on apices at different stages of the cryopreservation protocol showed a change in calorimetric parameters consistent with a decrease in the amount of frozen water as the protocol advanced.     

Cryopreservation of hairy roots of Rubia akane (Nakai) using a droplet-vitrification procedure

An efficient protocol for the cryopreservation of madder (Rubia akane Nakai) hairy root cultures was developed using droplet-vitrification and alternative loading and vitrification solutions formulated previously in our laboratory. Among eight preculture treatments tested, the highest post-cryopreservation regeneration was obtained for explants incubated in liquid half-strength MS medium with progressively increased sucrose concentration (0.3 M for 54 h, then 0.5 M for 16 h). Loading of precultured explants improved their post-cryopreservation regeneration by 50-75% compared with non-loaded control. Combining loading solution C4 (35% PVS3) and vitrification solution B5 (80% PVS3) was the most effective, while applying six PVS2-based solutions at room temperature resulted in low post-cryo regeneration. Treatment duration was optimized to 30 min for loading and to 10-20 min for vitrification solution. Apices of primary and secondary hairy roots showed similar post-cryo regeneration (88 and 95%, respectively), which was significantly higher than regeneration of root sections without apices (65%). Droplet-vitrification produced higher post-cryo regeneration than 'classical' vitrification in cryovials. Our results suggest that droplet-vitrification using alternative loading and vitrification solutions is an efficient method for cryopreservation of R. akane hairy root cultures.     

V-cryo-plate procedure as an effective protocol for cryobanks: case study of mint cryopreservation

A vitrification procedure using aluminium cryo-plates (V-Cryo-plate procedure) was successfully developed and adjusted for in vitro-grown mint (Mentha spp.) shoot tips. Shoots were cultured at 25°C on MS medium containing 0.088 M sucrose for 7 to 14 days after the last subculture. Shoot tips with a basal part (1-1.5 mm × 1 mm) were dissected from the shoots and precultured at 25°C for 1 day on the same medium. Precultured shoot tips were placed on aluminium cryo-plates with 10 wells and embedded in alginate gel. Osmoprotection was performed by immersing the cryo-plates for 30 min at 25 degree C in 25 ml pipetting reservoirs filled with loading solution (2 M glycerol + 0.8 M sucrose). For dehydration, the cryo-plates were transferred and immersed in 25 ml pipetting reservoirs filled with PVS2 for 20 min at 25 degree C. Then the cryo-plates were transferred in uncapped 2 ml cryotubes and directly plunged into liquid nitrogen. For rewarming, shoot tips attached to the cryo-plates were immersed in cryotubes containing 2 ml 1 M sucrose solution at room temperature. Using this procedure, regrowth of cryopreserved shoot tips of line 'Fukuyamajisei' reached over 90 percent. This protocol was successfully applied to 16 additional Mentha lines, with regrowth ranging from 73 percent to 100 percent. This V-Cryo-plate method will facilitate the cryostorage of mint germplasm in our genebank.     

Cryopreservation of cold-acclimated mint (Mentha spp.) shoot tips using a simple vitrification protocol

Accessions of Mentha x piperita, M. x villosa, and M. spicata were evaluated for regrowth after cooling in liquid nitrogen using shoot tips from in-vitro grown plantlets and a simple vitrification protocol with aluminium foil as a carrier. The influences of plant preculture, loading solution and loading time and of the effects of the cryoprotectant PVS 2 on plant re-growth after re-warming were investigated. Nodal segments were cultivated at constant temperatures of 20 or 25 degree C or in alternating temperature regimes (25/15C or 25/-1C). The illumination was always 16 h per day. The re-growth levels after re-warming were significantly higher in plants pre-cultured at 25/-1C regime than in plants cultivated at 20C or 25C or at 25/15C regime for all nine tested accessions. The mean re-growth levels increased from 36 percent at 20C to 69percent at alternating temperatures, respectively. The maximum of plant re-growth after re-warming was 89 percent. A pre-culture at alternating temperatures of 25/15C did not increase the recovery of plants. Loading in sucrose solutions with different dehydration capacities did not alter the plant re-growth. Differences in the loading time between 20 min and 2 h were not important for re-growth either. No significant differences were found between freezing without and with PVS 2 droplets on the aluminium foil. Re-grown shoots rooted easily on the re-growth medium and plantlets were successfully transferred to soil.     

Improved cryopreservation of chrysanthemum (Chrysanthemum morifolium) using droplet-vitrification

A droplet-vitrification protocol has been established for cryopreserving Chrysanthemum morifolium cv. Peak using axillary shoot tips and apical shoots of in vitro plants. In the optimized procedure, explants were submitted to a step-wise preculture in liquid sucrose-enriched medium (0.3, 0.5 and 0.7 M for 31,17 and 7 h, respectively). Precultured explants were treated for 40 min with C4 loading solution comprising (w/v) 17.5 percent glycerol + 17.5 percent sucrose, then dehydrated with PVS3 vitrification solution (w/v, 50 percent glycerol + 50 percent sucrose) for 60 min (axillary shoot tips) or 90 min (apical shoots). Explants were cryopreserved by direct immersion in liquid nitrogen in minute drops of PVS3 attached to aluminum foil strips. The optimal age of donor plants was 4-5.5 weeks for apical shoots and 7 weeks for axillary shoot tips, producing post-cryopreservation regeneration percentages of 81.9 percent and 84.9 percernt, respectively. Plants regenerated from cryopreserved samples showed no phenotypical abnormalities and similar profiles of relative DNA content were recorded for control and cryopreserved plants. Our results suggest that the modified droplet-vitrification protocol described in this paper is highly effective and may prove user-friendlier than the cryopreservation protocols already published for chrysanthemum.     

Cryopreservation of in vitro-grown shoot-tips of tropical taro (Colocasia esculenta var. esculenta) by vitrification

In vitro shoot-tips of three cultivars of tropical taro (Colocasia esculenta var. esculenta (L.) Schott) were successfully cryopreserved by vitrification. Different conditioning treatments were required for each of the cultivars, while the vitrification protocol was constant for all. For the cultivars E399 and CPUK, shoot-tips from three-month-old in vitro plants grown on solidified MS were preconditioned on MS with 0.3 M sucrose in the dark for 16 h at 25 degree C. For the cultivar TNS, donor plants were preconditioned on solid MS with 90 g per liter sucrose for seven weeks before cryopreservation. For vitrification, the shoot-tips were loaded with a solution of 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydrated with PVS2 for 12 min at 25 degree C and plunged in liquid nitrogen. Vials were warmed by rapid shaking in a water bath at 40 degree C for 1 min 30. Shoot-tips were rehydrated in liquid MS with 1.2 M sucrose for 15 min at 25 degree C then plated on recovery medium. Shoot-tips resumed growth within a week and developed into plantlets six to eight weeks later without any callus formation. The best mean recoveries for the three cultivars were 21, 29 and 30 percent for E399, CPUK and TNS, respectively. This protocol was evaluated with five other taro cultivars with no success. However, this study has shown that vitrification has potential for cryopreserving tropical taro.     

Cryopreservation of Vanda coerulea protocorms by encapsulation-dehydration

Protocorms of Vanda coerulea were successfully cryopreserved by encapsulation-dehydration in combination with a loading solution. Protocorms were selected 70 days after sowing seeds harvested from 7-month-old fruits. After encapsulation in an alginate matrix composed of 2 percent Na-alginate, 2 M glycerol plus 0.4 M sucrose (loading solution), the protocorms were precultured in modified Vacin and Went (1949) (VW) liquid medium supplemented with 0.7 M sucrose on a shaker (110 rpm) at 25 +/- 3 degree C for 20 h. Encapsulated protocorms were then dehydrated in a sterile air-flow in a laminar air-flow cabinet at 25 +/- 3 degree C for 0-10 h and then directly plunged into liquid nitrogen for 1 d. After thawing at 40 degree C for 2 min, cryopreserved beads were cultured on modified VW agar medium for regrowth. The highest regrowth of 40 percent was observed with cryopreserved beads with 35 percent water content after 8 h dehydration. No morphological variation was detected between non-cryopreserved and cryopreserved plantlets, and ploidy level was unchanged as a result of cryopreservation.     

Cryopreservation of lateral buds of in vitro-grown innala plants (Solemostemon rotundifolius) by vitrification

We succeeded in cryopreserving of innala (Solenostemon rotundifolius) in vitro-grown young lateral buds by vitrification. Nodal segments from in vitro-grown shoots (2-4 mm in length) were cultured on MS medium containing 0.1M sucrose in Petri dishes for 3 weeks under 16-h photoperiod at 25 degree C. This pre-growth induced a large number of uniform young lateral buds. Nodal segments (0.5 to 1.0 mm in length) with two lateral buds were dissected from the shoots and precultured with 0.3 M sucrose for 2 days at 25 degree C. They were then treated with loading solution containing 2 M glycerol and 0.4 M sucrose (LS solution) for 20 min at 25 degree C and dehydrated with the PVS2 vitrification solution for 18 min at 25(C prior to either rapid immersion in liquid nitrogen. Surviving lateral buds resumed growth within 3 days and developed shoots without intermediary callus formation. The average growth recovery after cryopreservation amounted to 85%.     

Cryopreservation of Pelargonium apices by droplet-vitrification

The droplet-vitrification method was adapted to Pelargonium apices by optimizing the duration of the loading solution (LS) as well as the plant vitrification solution 2 (PVS2). The excised apices were dehydrated in two steps (20 min in LS and 15 min in PVS2) and then immersed directly in liquid nitrogen (LN). After thawing and unloading in the recovery solution at room temperature for 15 min, apices were plated onto semi-solid Murashige and Skoog medium. This simple protocol without any pretreatment was successfully applied to eight cultivars with a survival level ranging between 55.6 - 96.2 percent and a regrowth level between 9.1 and 70.6 percent. These results prove the feasibility of the long-term storage of Pelargonium germplasm through cryopreservation.