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 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 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.     

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.     

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.     

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 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 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.     

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 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.     

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-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 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-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 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 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 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 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.     

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.