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 potato cultivated varieties and wild species: critical factors in droplet vitrification

The applicability of cryopreservation protocols to a broad range of genotypes is a key issue for genebanks. We tried to identify the critical factors causing differences in survival of cryopreserved shoot tips using potato varieties coming from cultivated and wild species. The droplet-vitrification method, a combination of droplet-freezing and solution-based vitrification, was selected from several protocols. High survival after freezing was observed after dehydration with PVS2 for 20 min, cooling shoot tips placed in a droplet of PVS2 solution on aluminum foil strips by immersing the foil strips in liquid nitrogen, warming them by plunging the foil strips into a 0.8 M sucrose solution (at 40 degrees C) for 30 s and unloading in 0.8 M sucrose for 30 min. This optimized protocol was successfully applied to 12 accessions with survival ranging between 64.0 and 94.4%.     

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.     

Vitrification, encapsulation-vitrification and droplet-vitrification: a review

This paper discusses the importance of the successive steps of the vitrification technique and reviews the current development and use of vitrification and of the two derived protocols, encapsulation-vitrification and droplet-vitrification. Vitrification refers to the physical process by which a highly concentrated cryoprotective solution supercools to very low temperatures and finally solidifies into a metastable glass, without undergoing crystallization at a practical cooling rate. Samples are thus cryopreserved without detrimental intracellular ice formation. In a standard vitrification protocol, excised explants are precultured on medium enriched with sucrose, treated (loaded) with a loading solution composed of 2 M glycerol + 0.4 M sucrose, dehydrated with a highly concentrated vitrification solution [e.g. the PVS2 vitrification solution, which contains 30 percent (w/v) glycerol, 15 percent (w/v) ethylene glycol and 15 percent (w/v) DMSO and 0.4 M sucrose], frozen and rewarmed rapidly, unloaded with basal culture medium supplemented with 1.2 M sucrose, and then transferred to standard culture conditions. In the encapsulation-vitrification technique, the explants are encapsulated in alginate beads, loaded and dehydrated with a vitrification solution before rapid immersion in liquid nitrogen. In the droplet-freezing technique, excised explants are loaded, treated with the vitrification solution and frozen in individual microdroplets of vitrification solution placed on aluminium foils, which are immersed rapidly in liquid nitrogen. These three techniques have been applied to different tissues of over 100 plant species from temperate and tropical origins and the number of cases where they are being tested on a large scale or applied routinely is increasing.     

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.     

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 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.     

Cryopreservation of garlic (allium sativum L.) using plant vitrification solution 2

Most cryopreservation procedures are optimized using a small number of germplasm accessions. We classified the garlic (Allium sativum L.) accessions that were selected for our studies based on genotype as identified using amplified fragment length polymorphism markers. Although recovery was variable, shoots regenerated from a broad range of the accessions after cryo-exposure. Garlic shoot tips were excised from cloves, surface sterilized, and placed on media at 5 degree C for 2 days prior to cryopreservation. Shoot tips were then treated with sucrose-glycerol for 20 minutes, plant vitrification solution 2 (PVS2; 15 percent w/v ethylene glycol, 15 percent w/v DMSO, 30 percent w/v glycerol, 13.7 percent w/v sucrose) at 0 degree C, and then plunged on foils into liquid nitrogen slush. Explants were recovered in 1.2 M sucrose for 20 minutes and then plated onto Gamborgs B5 medium containing alpha-naphthaleneacetic acid (NAA) and 6-(gammagamma-dimethylallylamino purine) (2-iP). Our results demonstrate that genotypically diverse accessions of garlic can be successfully cryopreserved.     

Cryopreservation of garlic shoot tips by vitrification: effects of dehydration, rewarming, unloading and regrowth conditions

This paper investigates the effect of dehydration, rewarming, unloading and regrowth conditions and of bulb post-harvest storage duration on survival and regeneration of cryopreserved garlic shoot tips. PVS3 was the most effective of the seven vitrification solutions compared. Treating shoot tips with PVS3 for 150-180 min ensured 92 % regeneration after freezing. An air-drying treatment, performed either before or after the PVS3 treatment, was detrimental to regeneration of cryopreserved shoot tips. Rapid rewarming in a water-bath at 37 degree C gave higher regeneration than the slower rewarming procedures employed. Regeneration was similar using either sucrose or sorbitol unloading solutions. The growth regulator content of the recovery medium did not influence percentage regeneration. However, the fresh weight of explants cultured on medium containing 0.3 mg/L zeatin and 0.3 mg/L gibberellic acid was significantly higher than on other media. Post-harvest storage duration of bulbs dramatically influenced survival and regeneration of non-cryopreserved and cryopreserved shoot tips, which were nil for samples cryopreserved immediately after harvest and highest after 3 and 6 months of storage. The optimized cryopreservation protocol was applied to ten different garlic varieties, with regeneration percentages ranging between 72 and 95 %.     

Geneticallly enginereed trehalose accumulation improves cryopreservation tolerance of chrysanthemum (Dendranthema grandiflorum kitam.) shoot-tips

Shoot-tips isolated from two transgenic lines of chrysanthemum (Dendranthema grandiflorum Kitam.) var. Indianapolis in vitro plantlets with induced capacity to biosynthesize trehalose, and from a non-transformed line, were subjected to cryopreservation using a vitrification procedure. After dissection, apices were precultured on semi-solid MS medium with 0.3 M sucrose for 4 days, loaded in a 0.4 M sucrose + 2 M glycerol solution for 20-30 min and exposed to PVS2 or PVS3 vitrification solutions for 0, 20, 40 or 60 min at room temperature prior to rapid immersion in liquid nitrogen. The highest shoot regeneration after cryopreservation was obtained with exposure to either PVS solution for 40 min. Plant regeneration from cryopreserved shoot-tips ranged between 48 percent and 67 percent for transgenic lines and between 33 percent and 36 percent for non-transgenic lines. No polymorphic loci were detected in plantlets regenerated from cryopreserved and non-cryopreserved shoot-tips with RAPD techniques using eight primers that amplified 101 monomorphic loci.     

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 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.     

Cryopreservation of coconut (Cocos nucifera L.) zygotic embryos by vitrification

The present study investigates the effect of preculture conditions, vitrification and unloading solutions on survival and regeneration of coconut zygotic embryos after cryopreservation. Among the seven plant vitrification solutions tested, PVS3 was found to be the most effective for regeneration of cryopreserved embryos. The optimal protocol involved preculture of embryos for 3 days on medium with 0.6 M sucrose, PVS3 treatment for 16 h, rapid cooling and rewarming and unloading in 1.2 M sucrose liquid medium for 1.5 h. Under these conditions, 70-80 survival (corresponding to size enlargement and weight gain) was observed with cryopreserved embryos and 20-25 percent of the plants regenerated (showing normal shoot and root growth) from cryopreserved embryos were established in pots.     

Development of a vitrification-based cryopreservation protocol for the storage of saltcedar (Tamarix boveana Bunge)

We cryopreserved in vitro shoot tips of saltcedar (Tamarix boveana Bunge) using the vitrification technique. The success of the cryopreservation protocol was strongly affected by preculture, loading duration, dehydration duration in plant vitrification solution 2 (PVS2), and medium composition during post-warming regrowth. The highest explant regrowth (50 percent) occurred when the following conditions were employed: preculture in 0.4 M glycerol; treatment with a loading solution (LS) consisting of 2 M glycerol + 0.4 M sucrose in culture medium for 40 min at room temperature; and dehydration in PVS2 at 0 degree C for 45 min before rapid immersion in liquid nitrogen (LN). Rewarming was performed in a water-bath at 40 degree C for 2 min. Explants were then immersed in unloading solution for 10 min before plating on recovery medium supplemented with 0.01 mg per liter thidiazuron (TDZ). TDZ was progressively eliminated from the medium over a period of 6 weeks. Plantlets were transferred to a double-layer medium to enhance rooting. This protocol was successfully applied to three individuals of T. boveana harvested from the wild.     

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 seeds of a Thai orchid (Doritis pulcherrima lindl. ) by vitrification

Seeds from selfing of a Thai orchid (Doritis pulcherrima Lindl.) were successfully cryopreserved in liquid nitrogen (LN) using the vitrification method. Seeds from 3-month-old pods were sufficiently dehydrated in 2 ml cryotubes filled with highly concentrated vitrification solution (PVS2) at 25 +/- 2 degree C for 50 min. The seeds were then rapidly plunged into LN. After rapid warming, the PVS2 solution was replaced with 0.5 ml of 1.2M sucrose in modified Vacin and Went (1949) (VW) solution and kept at 25 +/- 2 degree C for 20 min prior to transfer on VW agar medium. About 62% of cryopreserved seeds treated with PVS2 solution were able to develop into normal seedlings while without that treatment there was no survival. This vitrification protocol appears to be a promising technique for the cryopreservation of some Thai orchid germplasm     

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.     

Cryopreservation of basal stem buds of in vitro-grown mat rush (Juncus spp.) by vitrification

An optimal protocol for the cryopreservation of in vitro-grown mat rush (igusa) buds by vitrification has been successfully developed. Established multiple stemmed cultures, which were induced in liquid MS medium containing 8.9 microM BA by roller culture, were cut into small clumps, plated on solid MS medium and cultured for three weeks at 25 degree C. Clumps that grew many buds were cold-hardened at 5 degrees C, with an 8 h photoperiod, for more than 30 d. The basal stem bud (1 to 2 mm long) was dissected from the clumps and precultured at 5 degrees C for 2 d on solid MS medium containing 0.3 M sucrose. The precultured buds were placed in 2 ml plastic cryotubes and osmoprotected with 1 ml loading solution containing 2 M glycerol and 0.6 M sucrose for 30 min at 25 degree C. Then they were dehydrated in 1 ml PVS2 solution at 25 degree C for 30 min and immersed in liquid nitrogen. Using this protocol, the survival level of cryopreserved igusa 'NZ219' buds reached 87 percent. This protocol was successfully applied to 42 different lines from three Juncus species, which had relatively high survival levels ranging from 30 to 90 percent and an average of 63 percent.     

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.     

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.