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 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 human ovarian tissue by direct plunging into liquid nitrogen: negative effect of disaccharides in vitrification solution

Given that it has been possible to successfully cryopreserve human ovarian tissue by direct plunging into liquid nitrogen, this study was designed to establish the future direction to be taken in this line of research. Bovine oviductal epithelial fragments (as a tissue model) and large biopsy fragments (approximately 2.0 cubic mm) of human ovarian tissue were used for cryopreservation. Two protocols were tested: with permeable cryoprotectants (dimethyl sulphoxide, propylene glycol, glycerol) + egg yolk + sucrose or trehalose + a synthetic blocker of ice nucleation, Supercool X-1000; and using only permeable cryoprotectants (glycerol and ethylene glycol) + egg yolk + Supercool X-1000. The cryopreserved tissue specimens were subsequently thawed and the cryoprotectants removed by dilution in graded sucrose solutions. Both the dynamic growth and hormonal activity of the ovarian tissue pieces, vitrified using only permeable cryoprotectants, were greater than after vitrification in a mixture of permeable cryoprotectants and sucrose. Unlike the case for other reproductive tissue (spermatozoa, oocytes, embryos), these findings suggest that the cryopreservation of ovarian tissue by direct plunging into liquid nitrogen must be achieved by vitrification using only permeable cryoprotectants and agents that prevention ice formation.     

Cryopreservation of shoot-tips of citrus using vitrification: effect of reduced form of glutathione

A commercial citrus scion cultivar, '439' tangor [C. Suavissima Hort. et Tanaka x C. sinensis (L.) Osbesk cv.Gailiangcheng] was used to investigate whether GSH (reduced form of glutathione) could improve survival of a vitrification procedure. The optimal pre-growth treatment was a 3-day pre-culture on basal pre-culture medium (BPM: MT basal medium containing 0.5 mol/L glycerol and 5 % sucrose at pH 5.8). GSH of 40 mg/L in the pre-culture medium improved shoot tip survival after cryopreservation. GSH in the recovery medium also improved survival, with 10 mg/L giving the best result. GSH of 40 mg/L in the loading and vitrification solutions also improved survival. The optimal cryopreservation protocol was successfully applied to 12 other citrus cultivars. This is the first report on the successful cryopreservation of shoot-tips from commercial citrus scion cultivars.     

Vitrification of caudal fin explants from zebrafish adult specimens

No data on vitrification of tissue samples are available in fishes. Three vitrification solutions were compared: V1: 20% ethylene glycol and 20% dimethyl sulphoxide; V2: 25% propylene glycol and 20% dimethyl sulphoxide, and; V3: 20% propylene glycol and 13% methanol, all three prepared in Hanks' buffered salt solution plus 20 percent FBS, following the same one step vitrification procedure developed in mammals. Caudal fin tissue pieces were vitrified into 0.25 ml plastic straws in 30s and stored in liquid nitrogen for 3 days minimum, warmed (10s in nitrogen vapour and 5s in a 25 degree C water bath) and cultured (L-15 plus 20% FBS at 28.5 degree C). At the third day of culture, both attachment and outgrowing rates were recorded. V3 led to the worst results (8% of attachment rate). V1 and V2 allow higher attachment rates (V1: 63% vs V2: 50%. P < 0.05) but not significantly different outgrowing rates (83% to 94%). Vitrification of caudal fin pieces is advantageous in fish biodiversity conservation, particularly in the wild, due to the simplicity of procedure and equipment.     

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.     

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 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.     

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.     

Studies on cryoprotectant toxicity to zebrafish (Danio rerio) ovarian tissue fragments

Cryopreservation of ovarian tissue is a viable alternative to cryopreservation of oocytes and embryos in many species but it has not been studied in fish. Selection of cryoprotectant is an important step in designing cryopreservation protocols. In order to identify the optimum cryoprotectant (CPA) in a suitable concentration for zebrafish ovarian tissue cryopreservation, studies on toxicities of five commonly used cryoprotectants methanol, ethanol, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were carried out. Experiments were conducted on ovarian tissue fragments consisting of stage I and stage II ovarian follicles. Ovarian tissue fragments were incubated in 90% L-15 medium (pH 9) containing 1-4M cryoprotectants for 30min at 22°C. Three different tests were used to assess ovarian tissue fragment viability: trypan blue (TB) staining, fluorescein diacetate (FDA) combined with propidium iodide (PI) staining and adenosine 5′- triphosphate (ATP) assay. Results from these tests showed that ATP assay was more sensitive than FDA+PI or TB staining for assessing cryoprotectant toxicity to follicles in tissue fragments. Methanol and ethanol were the least toxic cryoprotectants tested. Cryoprotectant toxicity increased in the order of methanol/ethanol, DMSO, PG and EG. Ethanol was used for zebrafish ovarian tissue for the first time and the results showed that the effect of methanol and ethanol on ovarian tissue fragments were comparable. As methanol has been shown to be the most effective cryoprotectant for zebrafish ovarian follicles in our laboratory, the use of ethanol will also be considered in assisting future freezing protocol design. The present study also showed that stage II ovarian follicles are more sensitive to cryoprotectant treatment than stage I follicles in tissue fragments. The results obtained in this study provided useful information for ovarian tissue fragment cryopreservation protocol design in the future.     

Cryopreservation of Citrus madurensis embryonic axes by vitrification: importance of loading and treatment with vitrification solution

This paper investigates the importance of loading and treatment with a vitrification solution on the survival of Citrus madurensis embryonic axes cryopreserved using a vitrification protocol. Among the seven different loading solutions tested, the solution containing 2 M glycerol + 0.4 M sucrose was the most efficient. Of the six vitrification solutions tested, the PVS2 vitrification solution, applied for 20 min at 25 degree C or for 60 min at 0 degree C, ensured the highest survival. A three-step vitrification protocol, involving the treatment of embryonic axes at 0 degree C with half strength PVS2 solution for 20 min then with full strength PVS2 for an additional 40 min was more efficient than a two-step protocol that involved treatment of axes directly with full strength PVS2 solution for 60 min. After rapid immersion in liquid nitrogen, rapid rewarming, unloading in a 1.2 M sucrose solution for 20 min, culture on solid medium with 0.3 M sucrose for 1 day and growth recovery for 4 weeks on standard medium, survival of C. madurensis embryonic axes reached 85 % following the three-step process, compared with 70 % for the two-step process.     

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

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

Cryopreservation of lateral buds of in vitro-grown innala plants (Solemostemon rotundifolius) by vitrification

We succeeded in cryopreserving of innala (Solenostemon rotundifolius) in vitro-grown young lateral buds by vitrification. Nodal segments from in vitro-grown shoots (2-4 mm in length) were cultured on MS medium containing 0.1M sucrose in Petri dishes for 3 weeks under 16-h photoperiod at 25 degree C. This pre-growth induced a large number of uniform young lateral buds. Nodal segments (0.5 to 1.0 mm in length) with two lateral buds were dissected from the shoots and precultured with 0.3 M sucrose for 2 days at 25 degree C. They were then treated with loading solution containing 2 M glycerol and 0.4 M sucrose (LS solution) for 20 min at 25 degree C and dehydrated with the PVS2 vitrification solution for 18 min at 25(C prior to either rapid immersion in liquid nitrogen. Surviving lateral buds resumed growth within 3 days and developed shoots without intermediary callus formation. The average growth recovery after cryopreservation amounted to 85%.     

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

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

Ultrastructure of Gentiana tibetica proembryogenic cells before and after cooling treatments

The influence of increased concentrations of sucrose, 0.4 M sorbitol, DMSO and vitrification solution (PVS2) on the ultrastructure of non-frozen and frozen suspensions of Gentiana tibetica King ex Hook. F.tissue cells was investigated. Embryogenic aggregates were composed of three groups of cells of different size with various types of plastids. The ultrastructural changes resulting from increasing the sucrose concentration in the medium from 3 to 6 percent for 4 weeks and from treatment with 0.4 M sorbitol for 48 h were similar. Observations showed replacement of large vacuoles by numerous small ones, condensation of cytoplasm, accumulation of starch, and fragmentation of endoplasmic reticulum. Treatment with PVS2 led to degradation of starch, coalescence of amyloplasts and to shrinking of nucleoli from the third group of cells when originating from 6 percent sucrose medium. The mitochondria initially had various shapes, but after PVS2 treatment showed only spherical shapes with sparse cristae. After programmed freezing of tissue protected by sorbitol and DMSO, lethal damage was observed: membrane and nuclei degradation, and cell destruction. Reversible changes after freezing were observed in tissue pretreated with vitrification solution: dilation of cell membranes, mitochondria with electron-lucent vessels, aggregation of numerous vesicles, and degradation of starch in amyloplasts. In cells cooled by a vitrification method, cell organelles appeared normal as early as 5 h after thawing, and anomalies were not observed after 48 h of post-thawing culture.     

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.     

Simplified technique of human ovarian tissue freezing: quick cooling from -36 degree C

Standard protocol of freezing of human ovarian tissue presupposes the very slow cooling (-0.3 C/min) from auto-seeding to -40 C, then slow cooling (-10 C/min) to -140 C and then direct plunging into liquid nitrogen. The aim of this investigation was to compare the -10 C/min cooling rate of human ovarian tissue from -40 C to -140 C with the -220 C/min cooling rate (direct plunging into liquid nitrogen) from -36 degree C. After post-thawing in vitro culture of tissue, hormonal activity as well as follicle viability was evaluated. After culture of fresh tissue pieces (Group 1), pieces after freezing and thawing with slow cooling (-10 C/min) from -40 C (Group 2) and pieces after freezing and thawing with direct plunging into liquid nitrogen (-220 C/min) from -36 C (Group 3), the supernatants showed estradiol 17-ss concentrations of 481, 441 and 459 pg per ml, respectively, and progesterone concentrations of 9.05, 5.06, 4.87 ng per ml, respectively. It is concluded that 94, 96, and 98 percent follicles for Groups 1, 2 and 3, respectively, were normal. Technique of human ovarian tissue cryopreservation with very slow cooling to -36 C and then direct plunging into liquid nitrogen with -220 C/min cooling rate is tolerated without apparent detriment.     

Vitrification of encapsulated hepatocytes with reduced cooling and warming rates

We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400 degrees C/min and above) and three warming rates (650 degrees C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.     

Ultra rapid freezing and vitrification of human embryos derived from abnormally fertilised zygotes

The present study was undertaken to compare the developmental capacity of human embryos derived from abnormally fertilised zygotes (1 PN, > 3 PN; 16-18 hours after ICSI) cryopreserved using two techniques: ultra rapid freezing and vitrification. At 2-4 cell stage, (48 hours after ICSI), these abnormally fertilised embryos were then distributed in three groups: a) embryos that were cryopreserved by ultra rapid freezing (URF Group), b) embryos cryopreserved by vitrification (V Group) and c) embryos that were not cryopreserved (Control group). Survival rates and embryo development after 24 hours of in vitro culture (72 hours after ICSI) were compared. 42 embryos were cryopreserved by ultra rapid freezing in 0.5 mL straws, using a mixture of dimethyl sulphoxide (3M) and sucrose (0.25M) in a base solution consisting of IVF medium plus 20 percent (v/v) of Human Serum Albumin (HSA), and 24 embryos were vitrified in 0.25 ml straws, using a two step protocol with an equilibration solution consisting of 10 percent ethylene glycol (1.79 M) and 10 percent dimethyl sulphoxide (1.41 M) in a base solution of modified phosphate buffered saline (PBS) with 20 percent of HSA and a vitrification solution consisting of 20 percent ethylene glycol (3.58 M), 20 percent dimethyl sulphoxide (2.82 M) and 0.5 M sucrose in base solution. The recovery rate after thawing/warming was lower for the vitrification group (75 percent V; 83 percent URF). The number of embryos with less than 50 percent of intact blastomeres after cryopreservation was significantly higher for the URF group (0 percent V; 34 percent URF). After in vitro culture, the rate of embryos not cryopreserved (Control group) that developed in vitro (72 hours after ICSI) was the highest (86 percent), followed by group V (50 percent), while group URF was the lowest (13 percent). These differences were statistically significant. This straw method of vitrification is successful and safe.     

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.