Cryopreservation of Arabidopsis thaliana shoot tips

Development of a successful shoot tip cryopreservation method for Arabidopsis thaliana L. will enable researchers to use molecular tools to study processes important for successful cryopreservation in this model organism. We demonstrate that Arabidopsis can be successfully cryopreserved using either plant vitrification solution 2 (PVS2) or plant vitrification solution 3 (PVS3) as cryoprotectants prior to rapidly cooling shoot tips in liquid nitrogen (LN). Shoot tip regrowth after PVS2 cryoprotectant treatment was improved after cold acclimation treatments of 8 or 18 days. All of the shoots tips regrew after LN exposure when cryoprotected with PVS3 for 60 min at 22 degree C. In addition, shoot tips could be cryopreserved using a two-step cooling procedure with PGD (polyethylene glycol-glucose-dimethyl sulfoxide) as a cryoprotectant. The high levels of shoot formation after LN exposure of Arabidopsis shoot tips makes this a desirable system in which molecular tools can be used to examine how alterations in biochemical, metabolic and developmental processes affect regrowth after cryoprotective treatments.     

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 %.     

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 cultivated and wild potato varieties by droplet vitrification: effect of subculture of mother-plants and of preculture of shoot tips

In this paper, we studied the effect of subculture of mother-plants and of preculture of shoot tips of two potato varieties (Dejima, cultivated and STN13, wild) cryopreserved using the droplet-vitrification technique. The subculture conditions (light intensity, aeration and planting density) significantly affected survival of both non-cryopreserved and cryopreserved shoot-tips in both varieties. The subculture duration and the position of the shoot tips on the axis of the in vitro plantlets had a significant (P<0.0001) effect on survival of cryopreserved shoot tips. The optimal subculture duration was 7 and 5 weeks and the optimal size of shoot tips was 1.5-2.0 and 1.0-1.5 mm for var. Dejima and STN13, respectively. Survival of cryopreserved shoot tips was influenced by the sucrose concentration in the preculture medium and the preculture duration. The highest survival of cryopreserved shoot tips was observed after preculture with 0.3 M sucrose for 8 h followed by 0.7 M sucrose for 18 h. These results indicate that the parameters of the subculture of mother-plants and of preculture of shoot tips should be carefully optimized, especially in the case of wild species.     

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 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 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.     

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.     

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 Arachis species by vitrification of in vitro-grown shoot apices and genetic stability of recovered plants

A storage protocol at cryogenic temperature was established for shoot apices from in vitro plants of the cultivated groundnut (Arachis hypogaea) and wild Arachis species (A. retusa and A. burchellii) using a basic vitrification protocol with direct immersion in liquid nitrogen (LN). The effect of pre-treatments of donor-plants with ABA as well as of different supplements in the post-thaw culture medium was studied. After rapid warming at 40 C, the explants were cultured on MS medium devoid of growth regulators (MS0) or MS supplemented with 4.4(M benzylaminopurine (BAP) and 0.5(M naphthalene acetic acid (NAA) plus 5(M silver nitrate (AgNO3), 0.25% polyvinylpyrrolidone (PVP) or 0.2% activated charcoal. Non-frozen explants from the three species formed one shoot through meristematic amplification when cultured on MS0 medium. These explants also developed callus on MS supplemented with growth regulators (4.4(M BAP and 0.5(M NAA) alone or plus PVP or AgNO3. Callus formation was suppressed in the presence of activated charcoal. Post-thaw regeneration ocurred only through indirect organogenesis on media containing AgNO3 or PVP. Preculturing on medium supplemented with abscisic acid (ABA) improved regrowth rate in these media. Recovery failed to occur in the presence of activated charcoal. The genetic stability of shoots of A. burchellii originated from shoot apices was analyzed through Random Amplified Polymorphic DNA (RAPD) markers.     

Cryopreservation of Citrus madurensis embryonic axes encapsulation-dehydration

In this paper, we demonstrate that C. madurensis embryonic axes can withstand cryopreservation using the encapsulation-dehydration technique. Up to 57.5 % survival was achieved using a standard encapsulation-dehydration protocol, which included pregrowth of encapsulated axes for 16 h in medium containing 0.8 M sucrose + 1 M glycerol, desiccation of beads to around 30 % moisture content (fresh weight basis) followed by rapid freezing. A slightly higher survival percentage (65 %) was obtained using a modified encapsulation-dehydration protocol, which included pretreatment of axes with 2 M glycerol + 0.6 M sucrose for 1 h, concomitantly with their encapsulation in 3 % calcium alginate beads, followed by desiccation of the beads to around 30 % moisture content.     

Cryopreservation of shoot apices of in-vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols

Using vitrification and encapsulation-vitrification protocols, we successfully cryopreserved shoot apices from in-vitro plants of different Gentiana cultivars (lines). Although both protocols gave high survival percentages after storage in liquid nitrogen, the encapsulation-vitrification protocol had several distinct advantages over the vitrification protocol: (i) survival was higher under optimal conditions, (ii) the range of optimal exposure periods to the plant vitrification solution 2 (PVS2) was broader, and (iii) regrowth of cryopreserved shoot apices was apparently more vigorous and faster. Shoot apices from ten cultivars/lines of three Gentiana species (G. scabra, G. triflora, and G. pneumonanthe) were successfully cryopreserved using the two protocols with average survival of 49.0 percent and 73.7 percent for vitrification and encapsulation-vitrification, respectively. These results indicate that the two protocols optimized in the present study are promising for cryopreservation of a wide range of Gentiana genetic resources.     

Cryopreservation of shoot tips and cotyledons of the north american ginseng (Panax quinquefolius l.)

North American ginseng (NAG) (Panax quinqueolius L.) is a medicinal plant in high demand due to its health benefits. Cryopreservation is a good alternative for long-term conservation of NAG germplasm. Pretreatments of shoot tips (0.8-1 mm) and cotyledons (1-2 mm) on sucrose and abscisic acid (ABA) enriched medium were tested to determine the effects on regrowth following cryopreservation in liquid nitrogen. The maximum regrowth (60 percent) following PVS2 vitrification occurred with shoot tips after three weeks of cold acclimation and pretreatment on sucrose (0.3 M) or a combination of ABA (0.1 M) and sucrose in the third week. Cotyledon recovery was best with the combination pretreatment. Shoot tips showed normal development and cotyledons produced embryogenic callus after the cryopreservation process. This is the first report on cryopreservation of shoot tips and cotyledons of Panax species. This cryopreservation protocol provides a safe long-term storage method for important NAG selections and makes it possible to use cryopreservation for improving the security of NAG germplasm.     

Cryopreservation of in vitro grown shoot tips of Diospyros kaki thunb. using different methods

The objective of this study was to compare the potential of different cryopreservation strategies for in vitro shoot tips of Diospyros kaki Thunb. The treatments consisted of three different cryopreservation methods: vitrification, droplet-vitrification and modified droplet-vitrification. The following variables were assessed: cold acclimation, sucrose concentration in the preculture medium and PVS2 treatment time. A higher average survival level was obtained using the modified droplet-vitrification method compared to the other two methods.     

Cryopreservation of black iris (Iris nigricans) somatic embryos by encapsulation-dehydration

Somatic embryos of black iris (Iris nigricans) were cryopreserved using encapsulation-dehydration. Embryos size of 2-4 mm gave the highest survival after cryopreservation. Preculturing embryos on medium containing 0.75 M sucrose for 3 d at 22 degree C, then at 30 degree C for 1 d ensured maximum survival (60%). Viable embryos were brown in color after cryopreservation and during early recovery while dead ones where light brown or creamy. The first sign of regrowth was noted after 15 d. The final regrowth percentage of living embryo was 90% after 35 d. Only 10% of the embryos in all treatments showed secondary embryogenesis. Direct sowing in vivo of cryopreserved embryos was not successful in achieving germination.     

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 'Nabali' olive (Olea europea l.) somatic embryos by encapsulation-dehydration and encapsulation-vitrification

Olive (Olea europea L.) somatic embryos were successfully cryopreserved using encapsulation-dehydration and encapsulation-vitrification. In the encapsulation-dehydration procedure, a maximum of 48% embryo survival was obtained when bead moisture content was decreased to 21.1% after 4 h dehydration. Preculture of embryos for 4 d in medium containing 0.75 to 1.25 M sucrose produced higher (40 to 34 %, respectively) regrowth after cryopreservation using encapsulation-dehydration procedure. Dehydration of beads for 3 h in PVS2 ensured higher survival (64%) of encapsulated-vitrified and cryopreserved (EVN) somatic embryos. Thermal treatment of embryogenic callus for 1 d at 30 degree C was very effective to increase survival of encapsulated-dehydrated and cryopreserved (EDN) (58%) and EVN (68%) embryos. Plantlets produced from control and cryopreserved embryos were phenotypically similar.     

Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification

Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.     

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 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.