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 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.     

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.     

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.     

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 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.     

Applications of differential scanning calorimetry in developing cryopreservation strategies for Parkia speciosa, a tropical tree producing recalcitrant seeds

Shoot-tips of Parkia speciosa, a recalcitrant seed producing tropical leguminous tree withstood cryopreservation using encapsulation-vitrification in combination with trehalose preculture. Differential scanning calorimetry (DSC) revealed that trehalose moderated the thermal characteristics of the shoot-tips. A 30 min PVS2 treatment had the lowest glass transition temperature (Tg) (-50.2 +/- 1.1 degree C) when applied in combination with 5% (w/v) trehalose. The Tg increased to -40.2 +/- 1.0 degree C as the sugar concentration was decreased to 2.5 percent (w/v). Tg heat capacity for shoot-tips treated with 2.5 percent and 5 percent (w/v) trehalose and exposed to PVS2 for 30 min increased from 0.17 +/ 0.05 to 0.23 +/- 0.01 J per gram, respectively. Enthalpies of the melt-endotherm varied in proportion to trehalose concentration, for the 30 min PVS2 treatment, whereas the melt enthalpy for control shoots was greater than 150 J per gram and decreased to ca. 60 J per gram with 2.5 percent (w/v) trehalose. For 5 percent and 10 percent (w/v) trehalose treatments, enthalpy declined to ca. 24 and 12 J per gram respectively and freezing points were depressed to -75 degree C and -85 degree C with 2.5 percent and 5 percent trehalose (w/v), respectively. DSC elucidated the critical points at which vitrification occurred in germplasm exposed to trehalose and PVS2. A 60 min PVS2 treatment supporting ca. 70 percent survival was found optimal for stable glass formation during cooling and on rewarming.     

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 in vitro-grown shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree

Cryopreservation of in vitro axillary shoot tips of Crateva nurvala Buch. Ham, an important medicinal tree, was investigated. Axillary buds (c. 1mm in length) excised from 4-week-old in vitro cultures, were pre-cultured on liquid MS medium supplemented with 0.4 M sucrose for 16 h. These were incubated in 2 M glycerol+0.4 M sucrose for 20 min at 25 degree C before being dehydrated with PVS2 solution for 40 min The dehydrated shoot tips were directly immersed in LN. Following cryopreservation and after rapid warming at 40 degree C, shoot tips were quickly washed with MS+1.2 M sucrose solution for 20 min and then plated on top of filter paper placed on MS medium supplemented with 0.1 mg l-1 BAP, kept in darkness for one day followed by placement of shoots directly on the medium and incubation in darkness for a day more, before transfer of cultures to light. Average survival in terms of normal shoot formation after 4 weeks of plating was 56.6 percent. The rescued shoot tips were bulked up by subsequent nodal cultures and when put onto 0.02 mg l-1 NAA showed a rhizogenic response. Thus, in vitro-grown shoot tips of Crateva nurvala were successfully cryopreserved following the optimization of the PVS2-vitrification protocol.     

Cryopreservation of in vitro-grown shoot tips of cassava by encapsulation-vitrification method

Shoot tips of cassava (Manihot esculenta Crantz) in vitro plantlets were successfully cryopreserved using the encapsulation-vitrification technique. Nodal cuttings of 5 mm length with one leaf were cultured on modified MS medium in Petri dishes (90 mm x 20 mm) for about 28 days. Excised shoot tips were precultured on sucrose enriched (0.3 M) medium for 16 h, encapsulated and osmoprotected with a mixture of 2 M glycerol and 0.6 M sucrose for 90 min at 25 degree c before dehydration with PVS2 at 0 degree C for 4 h, then plunged in liquid nitrogen. Successfully vitrified shoot tips resumed growth within 3 days, without intermediary callus formation, and developed shoots. Shoot tips sampled from 21 day-old plantlets produced the highest survival of 80 %. The percentage survival of vitrified shoot tips differed from 38 to 80 % depending on the day of excision. The protocol was successfully applied to four cultivars of cassava with about 80 % average percentage of survival.     

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.     

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 shoot tips of Picrorhiza kurroa Royle ex Benth,an indigenous endangered medicinal plant,through vitrification

The cryopreservation of shoot tips of Picrorhiza kurroa Royle ex Benth (IC 266698), an endangered medicinal plant of India was investigated. Shoot tips (about 1 mm in length) excised from four-week-old proliferating shoot cultures were precultured on MS medium supplemented with various osmotica before dehydrating with PVS2 solution at 0 degrees C. The dehydrated shoot tips were directly immersed in LN2. Following cryopreservation, and after rapid rewarming at 45 degrees C, shoot tips were quickly washed with 1.2 M sucrose solution and then plated on solidified shoot culture medium. Shoot tips were successfully cryopreserved by vitrification, when they were precultured on medium supplemented with 5% DMSO at 4 degrees C for two days before dehydrating in PVS2 for 10-20 minutes at 0 degrees C. Average survival in terms of normal shoot formation after 4 wks of plating was about 20% without callus formation. Cold hardening of shoot cultures for four weeks at 4 degrees C significantly improved the survival and shoot regeneration of cryopreserved shoot tips to 70% and 35%, respectively.     

Cryopreservation of in vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant: effect of cryogenic procedure and storage duration

In vitro shoot tips of Dioscorea deltoidea Wall., an endangered medicinal plant, were successfully cryopreserved using the vitrification and the encapsulation-dehydration techniques with subsequent high frequency plant regeneration. Using vitrification, post-liquid nitrogen (LN) shoot regeneration up to 83% was recorded when excised shoot tips were pretreated overnight on MS medium containing 0.3 M sucrose followed by loading with MS containing 2 M glycerol plus 0.4 M sucrose for 20 min at 25 degree C, dehydration with PVS2 for 90 min at 0 degree C and quenching in LN. After 1 h of storage in LN, the shoot tips were rewarmed in a water-bath at 40 degrees C, unloaded with 1.2 M sucrose solution for 20 min and cultured on recovery growth medium. While using encapsulation-dehydration, the highest regeneration frequency recorded was 76% when sucrose-pretreated shoot tips were encapsulated with 3% calcium alginate, precultured in 0.75 M sucrose for 3 days, dehydrated to 25% moisture content (FW basis) under the laminar air flow, stored in LN for 1h and rewarmed at 40 degree C. The cryopreserved shoot tips maintained their viability and an unaltered level of regeneration capability after up to one year of storage in LN.     

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.     

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.     

High viability of dormant Malus buds after 10 years of storage in liquid nitrogen vapour

Three hundred and sixty two Malus accessions from the Canadian Clonal Genebank of Plant Gene Resources of Canada were cryopreserved as dormant buds at the USDA-ARS National Center for Genetic Resources Preservation in 1996. According to grafting data collected on 165 of these accessions in 1999, 80 percent of the accessions had at least 40 percent viability. A subsample of these accessions was processed for cryopreservation by either adjusting the moisture content of the budwood sections containing dormant buds to 32 or 37 percent moisture (fresh weight basis) or by not drying the budwood sections (46 percent moisture fresh weight basis) prior to cooling. Budwood sections were then slow-cooled at 1 degree C per hour to -3 degree C, held for 24 h at -30 degree C and then rapidly transferred to the vapour phase of liquid nitrogen. Cryopreserved buds from 13 accessions that were dried using the various techniques were warmed and grafted in both 1999 and 2006 to determine viability. Overall, bud viability was high at both storage times. At the 10 year time point, some accessions had higher bud growth when they were desiccated prior to slow-cooling when compared to those that were not.     

Evolution of DMSO concentration in garlic shoot tips during a vitrification procedure

In this paper, the evolution of dimethylsulfoxide (DMSO) concentration and moisture content (MC) of garlic shoot tips was studied during the course of a vitrification protocol using the PVS2 vitrification solution. DMSO concentration of shoot tips increased rapidly, reaching 34.1 mg per g fresh weight after 20 min of PVS2 treatment and remained stable afterwards, while moisture content decreased from 82 to 60 percent, reaching 53 percent after 60 min. A reverse process was observed during unloading. There was a highly significant negative correlation between shoot tip moisture content and DMSO concentration during the dehydration and unloading treatments. Using unloading solutions with osmolarities between 0.42 and 2.29 Osm led to very different shoot tip MCs, between 63.55 and 81.24 percent, while DMSO concentration was between 14.83 and 19.97 mg per g fresh weight. After 24 h on recovery medium, DMSO concentration of shoot tips had decreased to 3.2 mg per g fresh weight.     

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 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 %.