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.     

Effect of preculture, pvs2 and vitamin C on survival of recalcitrant Nephelium ramboutan-ake shoot tips after cryopreservation by vitrification

This paper reports the cryopreservation of Nephelium ramboutan-ake shoot tips derived from in vitro shoot multiplication and in vitro seed germination using vitrification. Preculture with either 0.5 M sucrose for 2 days or a combination of 0.3 M sucrose and 0.5 M glycerol for 3 days enhanced dehydration tolerance and resulted in the highest survival of shoot tips; however, none of the shoot tips withstood liquid nitrogen (LN) exposure. The use of a lower temperature (0 degree C) during exposure to plant vitrification solution (PVS2) led to higher survival of shoot tips, compared to exposure at 25 degree C. The survival percentage of shoot tips exposed to PVS2 for up to 20 min at 0°C was 83.3 percent. It was only 53.3 percent when shoot tips were exposed to PVS2 at 25 degree C for 5 min. The importance of vitamin C for reducing oxidative stress in shoots tips was demonstrated. The addition of 0.28 mM vitamin C during critical steps of the vitrification process resulted in a high survival (96.7 percent) without LN exposure, compared to 73.3 percent for shoot tips not treated with vitamin C. Moreover, 3.3 percent shoot tips withstood LN exposure when vitamin C was added during the loading step. This result suggests that cryopreservation is possible for this tropical, recalcitrant seeded tree species.     

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.     

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.     

Palm cryobanking

We describe the development of an efficient cryopreservation protocol for proembryogenic masses (PEMs) of date palm variety 'Barhee'. Proembryos were induced by inoculating small pieces of juvenile leaves on MS medium supplemented with 0.3 mg per liter 2,4-D. Application of these in vitro conditions led to true-to-type plants as observed after plant fructification. When compared to the standard vitrification protocol, the ultra-rapid droplet-vitrification technique proved to be superior. Sucrose preculture considerably increased post-cryopreservation recovery. The highest regeneration after cryogenic exposure reached 63.3 percent when PEMs were treated with PVS2 for 30 min at 0 degree C and 56.7 percent when PVS2 treatment lasted for 15 min at 25 degree C. The first signs of regrowth of cryopreserved PEMs were observed after 2 to 3 weeks. Cryopreservation did not affect the morphogenetic capacities of the plant material. Moreover, highly proliferating suspension cultures could be established from the cryopreserved material. The overall production of somatic embryos from 500 mg cryopreserved PEMs reached 1030 +/- 50 units after 1 month. The morphological study of date palms regenerated from cryopreserved material confirmed the stability of clonal material following cryopreservation.     

Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification

Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation.     

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 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 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.     

Implementation of garlic cryopreservation techniques in the national plant germplasm system

The USDA-ARS National Plant Germplasm System (NPGS) maintains more than 200 Allium sativum (garlic) accessions at the Western Regional Plant Introduction Station in Pullman, WA. All accessions must be grown out in the field annually since garlic plants from these accessions do not reliably produce seeds and bulbs do not store well. Shoot tips excised from garlic cloves can be successfully cryopreserved using either Plant Vitrification Solution 2 (PVS2; 15 percent v/v DMSO, 15 percent v/v ethylene glycol, 30 percent v/w glycerol, 0.4 M sucrose) or Plant Vitrification Solution 3 (PVS3; 50 percent v/w sucrose, 50 percent v/w glycerol). We compared regrowth of shoot tips representing diverse garlic germplasm after exposure to either PVS2 or PVS3 during the cryopreservation procedure. At the USDA-ARS National Center for Genetic Resources Preservation, a component of the NPGS, we consider accessions successfully preserved if a minimum of 40 percent of explants exhibit regrowth after liquid nitrogen exposure and at least 60 viable shoot tips remain in long-term storage. Ten of twelve diverse garlic accessions were successfully cryopreserved using either PVS2 or PVS3 as cryoprotectants. Five genotypes had the best post liquid nitrogen regrowth after exposure to PVS2, four genotypes had the best regrowth after exposure to PVS3, and three genotypes performed equally well using either cryoprotectant solution. This project is part of an ongoing program to cryopreserve accessions of NPGS clonal crop collections.     

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 shoot tips of blackberry and raspberry by encapsulation-dehydration and vitrification

Encapsulation-dehydration and PVS2-vitrification cryopreservation protocols were evaluated for the long-term conservation of a diverse group of Rubus germplasm. Cold acclimation for a 4-week period prior to cryopreservation was necessary for regrowth of shoot apices from blackberry and raspberry genotypes. For the encapsulation-dehydration protocol, encapsulated apices were pretreated in 0.75 M sucrose for 20 h, desiccated 6-h under laminar flow to c. 20 percent moisture content, then plunged in liquid nitrogen (LN) and rapidly warmed. The PVS2-vitrification protocol included pretreating shoot tips on 5 percent dimethyl sulfoxide (DMSO) medium for 48 h, exposure to loading solution (LS) and PVS2 for 20 min each at 25 degree C , followed by immersion in LN and rapid warming. Shoot tips of 25 genotypes in 9 Rubus species and 9 Rubus hybrids were successfully cryopreserved with recovery of 60 to 100 percent using the encapsulation-dehydration protocol. Four genotypes of 3 species were tested using the vitrification protocol with 71 percent average regrowth. The present results indicate that both of these improved cryopreservation protocols can be applied to a diverse range of Rubus genetic resources.     

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.     

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 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 chayote (Sechium edule JACQ. SW.) zygotic embryos and shoot-tips from in vitro plantlets

This paper presents the development of cryopreservation protocols for zygotic embryos and apices of chayote (Sechium edule Jacq. Sw.), a tropical plant species with recalcitrant seeds. Zygotic embryos of two cultivars, Ccocro negro (CN) and Claudio (Cl) could withstand cryopreservation, with survival percentages of 10 and 30 %, after desiccation to 23 and 19 % moisture content (fresh weight basis), respectively. Apices sampled on in vitro plantlets of cultivars Cl, 13 and JM were successfully cryopreserved using a vitrification technique. Optimal conditions included the culture of mother-plants for 22 days on medium containing 0.3 M sucrose, culture of excised apices on the same medium for 1 day, loading of apices for 20 min with 2M glycerol + 0.4M glycerol, treatment with a series of diluted PVS2 solution (60 % PVS2 followed by 80 % PVS2 solution for 15 min (cultivar Cocoro Blanco [CB]) or 30 min (cultivars CN and Cl) at each concentration), rapid freezing and thawing, washing of shoot-tips with a 1.2 M sucrose solution, followed by recovery on media with progressively decreasing sucrose concentrations until the standard concentration of 0.1 M was reached. The highest survival percentages achieved ranged between 17 and 38 %, depending on the cultivar.     

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 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.     

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.     

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.