Vitrification Protocol Research Articles

Vitrification as a method for genome resource banking oocytes from the endangered Tasmanian devil (Sarcophilus harrisii)

Populations of Australia's largest terrestrial marsupial carnivore, the Tasmanian devil (Sarcophilus harrisii), are rapidly declining in the wild due to Tasmanian Devil Facial Tumour Disease (TDFTD). One tool which can reduce the loss of genetic diversity is genome resource banking. This study examines the application of an oocyte vitrification protocol, initially developed in a model marsupial carnivore, to the endangered Tasmanian devil. Ovarian tissue was transported to the laboratory on ice from Tasmania which took up to 48 h. Individual granulosa oocyte complexes (GOC) were isolated enzymatically and the viability of oocytes from primary GOC was assessed immediately following isolation or after exposure to cold shock, vitrification and thawing media without exposure to liquid nitrogen or the full vitrification and thawing process. There was no decline in oocyte viability following cold shock or exposure to the vitrification and thawing media. Following the full vitrification and thawing process there was a decline in oocyte viability (chi(2)=20.0, P<0.001) but approximately 70% of oocytes remained viable. This study provides further evidence that oocyte vitrification is a promising strategy for genome resource banking in carnivorous marsupials and suggests that it should be considered in conservation plans for the survival of the iconic Tasmanian devil.

Immature cat oocyte vitrification in open pulled straws (OPSs) using a cryoprotectant mixture

Cryopreservation of gametes is an important tool in assisted reproduction programs to optimise captive breeding programmes of selected felid species. In this study the vitrification was evaluated in order to cryopreserve the immature domestic cat oocytes by assessing the survival of cumulus-oocyte complexes (COC), and the development competence after IVM and IVF by fresh cat epididymal sperms. From a total of 892 COC obtained from queens after ovariectomy were divided into two groups: Experiment 1 for viability evaluation (150 vitrified and 100 control COC) and Experiment 2 for assessing the developmental competence (414 vitrified and 228 control COC). The viability was evaluated by double staining with carboxyfluorescein and Trypan blue, while the developmental competence was evaluated by in vitro maturation (IVM), in vitro fertilisation (IVF) by fresh epididymal spermatozoa and in vitro culture (IVC). The vitrification was performed in OPS into sucrose medium (1 M sucrose in HSOF + 6% BSA) containing dimethyl sulfoxide (DMSO) (16.5% final concentration) and ethylene glycol (EG) (16.5% final concentration) as cryoprotectants. Percentage of non-viable COC was significantly higher in Experimental 1 vs Control 1 (11% vs 54.5%; P < 0.01), while cleavage rate were significantly lower for vitrified oocytes (Experimental 2) than control 2 (18.6% vs 48.2%; P < 0.01). Blastocyst rate on day 8 was higher for control oocytes than vitrified counterparts (4.3% vs 20.6% P < 0.01). This vitrification protocol ensured a development to blastocyst stage and it is the first report of development of vitrified GV COC.

Effects of oocyte vitrification on histone modifications

Vitrification has been widely used as an assisted reproductive technology in animals and humans, yet the impact of oocyte vitrification and warming on survival and histone modifications has to be evaluated. In the present study, the survival of mouse MII oocytes was assessed after freezing, as were changes in histone 3 lysine 9 (H3K9) dimethylation, histone 4 lysine 5 (H4K5) acetylation and histone 3 lysine 14 (H3K14) acetylation. The results show that, in oocytes subjected to vitrification, H3K9 methylation and H4K5 acetylation were increased. H3K14 acetylation could not be detected in either non-vitrified or vitrified oocytes. Oocytes are very sensitive to changes in H3K9 and H4K5 following vitrification. Both these histone modifications could be useful markers to monitor epigenetic perturbations induced by various experimental vitrification protocols and eventually for optimising the cryopreservation of human oocytes.

Ethylene Glycol-supplemented Calcium-free Media Improve Zona Penetration of Vitrified Rat Oocytes by Sperm Cells

Cryopreservation of matured oocytes is a useful technique because the oocytes can be used for some assisted reproductive technologies after warming. Even though rats, like mice, have been used in various research fields including reproductive technology, information about cryopreservation of rat oocytes is limited. The objective of the present study was to improve the vitrification protocol for matured rat oocytes. To determine the optimal equilibration time, oocytes were equilibrated in 7.5% ethylene glycol (EG) + 7.5% dimethyl sulfoxide (DMSO) + 20% fetal calf serum (FCS) for 1, 4, 7 or 10 min at 24 C and then 15% EG + 15% DMSO + 0.5 M sucrose + 20% FCS for 1 min at 24 C before being plunged into liquid nitrogen on Cryotops. Oocytes exposed to equilibration medium for 4 min showed higher survival and cleavage rates after artificial activation than those of oocytes exposed for 1, 7 or 10 min. The survival and cleavage rates of vitrified oocytes after activation were 98.3 and 78.4%, respectively. However, the perivitelline spaces of most of the vitrified/warmed oocytes (6/168, 3.6%) could not be penetrated by sperm after in vitro fertilization, and cortical granule exocytosis (CGE) was observed in the oocytes. Therefore, the inhibitory effect of calcium and cryoprotectants in vitrification medium on CGE was examined. In most of the oocytes vitrified in calcium-free media, CGE was strongly suppressed independent of cryoprotectants. Oocytes vitrified in EG-supplemented calcium-free media showed high survival rates after warming (79.4%). After artificial activation, the cleavage and blastocyst formation rates of the oocytes were also high (72.8 and 23.1%, respectively). The zona penetration rate of vitrified/warmed oocytes was dramatically improved by using EG-supplemented calcium-free media after in vitro fertilization (111/155, 63.9%). Thus, our data suggest that EG-supplemented calcium-free media improve zona penetration of vitrified rat oocytes by sperm cells.

123 BOVINE OOCYTE VITRIFICATION USING THE CRYOTOP METHOD: EFFECT OF CUMULUS CELLS AND VITRIFICATION PROTOCOL ON SURVIVAL AND SUBSEQUENT DEVELOPMENT

The ability to successfully cryopreserve mammalian oocytes has numerous practical and economic ethical benefits that may positively affect animal breeding programs and assisted conception in humans. However, oocyte survival and development following cryopreservation remain poor. The aim of the present study was (1) to evaluate the effect of the presence of cumulus cells on the outcome of vitrification of immature (GV) or mature (MII) oocytes, (2) to compare empirical and theoretical vitrification protocol, and (3) to assess the effect of adding ice blockers to vitrification media on survival and development competence of bovine oocytes following vitrification using the Cryotop method. Bovine oocytes were collected from the ovaries of slaughtered cross-bred beef heifers. In Experiment 1, cumulus-enclosed and partially denuded GV and MII oocytes were vitrified in 15% EG + 15% DMSO + 0.5 M sucrose in 2 steps. In Experiment 2, GV oocytes were vitrified as above or using theoretical modelling based on permeability and osmotic tolerance characteristics (Wang et al. Reprod. Fertil. Dev. 21 141) in 30% EG +11.4% trehalose in 3 steps or 40% EG + 11.4% trehalose in 4 steps. In Experiment 3, GV oocytes were vitrified in media supplemented or not with 1 of 2 ice blockers (21st Century Medicine, Fontana, CA, USA) 1% X-1000, 1% Z-1000, or both in 3 steps. The survival, cleavage, and blastocyst rate of cumulus-enclosed oocytes was significantly higher (ANOVA) than those of partially denuded oocytes when vitrified at GV (93.8% v. 81.3%, 65.8% v. 47.3%, 11.3% v. 4.0%, respectively, P &lt; 0.05). However, no significant effect of cumulus cover was detected between the two groups when vitrified at MII (93.0% v. 91.8%, 35.2% v. 36.8%, 5.0% v. 4.4%, respectively). Furthermore, cumulus-enclosed oocytes vitrified at the GV stage exhibited a significantly higher development competence than those vitrified at MII (P &lt; 0.05). In Experiment 2, there were no significant differences in the survival, cleavage, and blastocyst rates among 3 protocols (86.0% v. 92.8% v. 91.2%, 44.8% v. 54.4% v. 45.6%, 5.0% v. 5.4% v. 4.0%, respectively). However, cleavage and blastocyst rate were significantly lower (P &lt; 0.05) than nonvitrified control oocytes. In Experiment 3, the presence of ice-blockers did not improve rate or blastocyst development (P &lt; 0.05). In conclusion, cumulus-enclosed GV bovine oocytes survived vitrification and subsequently developed at higher rates than MII oocytes. Theoretical analysis of permeability characteristics and tolerance limits alone may not be sufficient to improve vitrification protocols.

153. Changes in alginate bead water status and dry weight content during encapsulation–dehydration and encapsulation–vitrification protocols

153. Changes in alginate bead water status and dry weight content during encapsulation–dehydration and encapsulation–vitrification protocols

Vitrification and warming of in vivo–derived porcine embryos in a chemically defined medium

The aim of this study was to design a protocol for vitrification and warming of porcine embryos in a chemically defined medium. A total of 663 morulae and blastocysts were collected from weaned crossbred sows (Large White-Landrace) 5 to 6 d after estrus and vitrified with the Superfine Open Pulled Straw method. In Experiment 1, embryos were vitrified using as a basic medium TCM-199–HEPES supplemented with 20% newborn calf serum (NBCS) or with 0, 0.1%, 0.5%, or 1% polyvinyl alcohol (PVA). Nonvitrified embryos were used as a fresh control group. Survival and hatching rates were evaluated after 72 h of in vitro culture to assess embryo viability. In addition, some hatched blastocysts derived from morulae and blastocysts were processed to determine the total cell number and the cell proliferating index as measures of their quality. Within each stage of embryo development, the different vitrification groups and the fresh control group showed similar high embryo survival (range, 70.5 ± 7.1% to 84.9 ± 8.1% and 85.3 ± 8.1% to 98.4 ± 8.2% for morulae and blastocysts, respectively) and hatching rate (range, 46.3 ± 10.1% to 66.7 ± 11.2% and 73.7 ± 11.3% to 89.4 ± 11.2% for morulae and blastocysts, respectively) and quality after in vitro culture. In Experiment 2, embryos were vitrified using 0.1% PVA and warmed with TCM-199–HEPES–0.13 M sucrose supplemented with 20% NBCS or either 0 or 0.1% PVA. Nonvitrified embryos were used as a fresh control group. As in Experiment 1, no significant differences were detected in embryo survival (range, 67.9 ± 6.6% to 74.5 ± 6.6% and 91.9 ± 7.0% to 99.5 ± 6.3% for morulae and blastocysts, respectively) and hatching rate (range, 47.0 ± 7.2% to 64.8 ± 9.9% and 89.4 ± 7.4% to 98.2 ± 6.9% for morulae and blastocysts, respectively) and quality among the warming groups or among vitrified and fresh control embryos. In both experiments, the developmental embryo stage influenced the survival and hatching rates, as well as the number of cells (P < 0.01), with the blastocyst stage yielding the best results. In conclusion, PVA can be used as a substitute for serum in vitrification and warming solutions without detrimental effects on the in vitro development of in vivo–derived porcine morulae and blastocysts.

Vitamins C and E improve regrowth and reduce lipid peroxidation of blackberry shoot tips following cryopreservation

Oxidative processes involved in cryopreservation protocols may be responsible for the reduced viability of tissues after liquid nitrogen exposure. Antioxidants that counteract these reactions should improve recovery. This study focused on oxidative lipid injury and the effects of exogenous vitamin E (tocopherol, Vit E) and vitamin C (ascorbic acid, Vit C) treatments on regrowth at four critical steps of the plant vitrification solution number 2 (PVS2) vitrification cryopreservation technique; pretreatment, loading, rinsing, and regrowth. Initial experiments showed that Vit E at 11-15 mM significantly increased regrowth (P < 0.001) when added at any of the four steps. There was significantly more malondialdehyde (MDA), a lipid peroxidation product, at each of the steps than in fresh untreated shoot tips. Vit E uptake was assayed at each step and showed significantly more alpha- and gamma-tocopherols in treated shoots than those without Vit E. Vit E added at each step significantly reduced MDA formation and improved shoot regrowth. Vit C (0.14-0.58 mM) also significantly improved regrowth of shoot tips at each step compared to the controls. Regrowth medium with high iron concentrations and Vit C decreased recovery. However, in iron-free medium, Vit C significantly improved recovery. Treatments with Vit E (11 mM) and Vit C (0.14 mM) combined were not significantly better than Vit C alone. We recommend adding Vit C (0.28 mM) to the pretreatment medium, the loading solution or the rinse solution in the PVS2 vitrification protocol. This is the first report of the application of vitamins for improving cryopreservation of plant tissues by minimizing oxidative damage.

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro–matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro–matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.

La vitrification : technique d’avenir pour la cryoconservation ovarienne ? Bases physiques de cryobiologie, avantages et limites

Ovarian cryopreservation is presently indicated in patients who undergo a gonadotoxic treatment, most commonly for anticancer procedures. These procedures can strongly alter fertility by damaging the follicular ovarian reserve. Although six human live births have been described in the world after ovarian tissue cryopreservation and autografting, the techniques of cryopreservation techniques are not consensual. Vitrification is a physical process that allows cryopreservation without formation of ice crystals, by transformation of a highly concentrated solution in a glassy or amorphous state. Vitrification is at present rapidly expanding in the biology of reproduction. With the classic methods of freezing, formation of ice crystals within the ovarian tissue is systematic and can entail cellular lesions. Which is why more and more teams question the theoretical advantage of the vitrification for ovarian cryopreservation. Our objective was to summarize the fundamental physical basis of cryobiology, necessary for an understanding of vitrification. From our experience, we also wanted to point out the practical difficulties of this technique, and we are proposing a model of evaluation and validation that uses differential scanning calorimetry, applicable to any protocol of vitrification.

Multiplication and cryopreservation of vanilla (Vanilla planifolia ‘Andrews’)

A simple and efficient method for multiplication of vanilla (Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters of plantlets, by cutting the remaining base of these plant clusters into segments of about 1 cm in length. After 4 months of culture on multiplication medium, 100% of IFEs produced up to 15 new shoots per explant, providing an efficient additional method for in vitro propagation of vanilla that maximizes the use of available material. Cryopreservation of apices from in vitro grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of in vitro plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to plant vitrification solution PVS3 for 30 min at room temperature. Even though the cryogenic protocol needs to be optimized to improve results, this work represents the first successful report of cryopreservation of vanilla apices.

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of rare and endangered plant Emmenopterys henryi Oliv.

In vitro-grown shoot tips of Emmenopterys henryi Oliv. were successfully cryopreserved by vitrification. Shoot tips excised from 3-month old plantlets were precultured in a liquid hormone-free Murashige and Skoog (MS) medium supplemented with 0.5 M sucrose for 3 days at 25°C and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose (LS solution) for 40 min at 25°C. Osmo-protected shoot tips were first dehydrated with 60% vitrification solution (PVS2) for 30 min at 0°C and followed by 100% PVS2 for 40 min at 0°C. After changing the solution with fresh 100% PVS2, the shoot tips were directly plunged into liquid nitrogen. After rapid warming in a water-bath at 40°C for 2 min, the shoot tips were washed for 20 min at 25°C with liquid MS medium containing 1.2 M sucrose and then transferred onto solid MS medium supplemented with kinetin 2 mg l−1, α-naphthaleneacetic acid 0.1 mg l−1, 3% (w/v) sucrose and 0.75% (w/v) agar. The shoot tips were kept in the dark for 7 days prior to exposure to the light (12 h photoperiod cycle). Direct shoot elongation was observed in approximately 12 days. The regeneration rate was approximately 75–85%. This method appears to be a promising technique for cryopreserving shoot tips of Emmenopterys henryi Oliv. germplasm.

The global DNA methylation of mouse oocytes following vitrification

OBJECTIVE: Recently it has been reported that oocyte survival rate was improved tremendously by vitrification method following cryopreservation. However, there is paucity of information about oocyte methylation following vitrification. Therefore, the purpose of this study is to determine the global DNA methylation following vitrification in order to ensure the safe protocol for oocyte vitrification.

A modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes

In this study, we examined a modified cryoloop vitrification protocol in the cryopreservation of mature mouse oocytes. The mature mouse oocytes were first vitrified and then warmed up in a modified cryoloop vitrification medium [15% ethylene glycol (EG) + 15% dimethyl sulphoxide (ME2SO) + 5.8 mg/ml Ficoll 400 (F) + 0.58 mol/l sucrose (S)]. These oocytes were later studied along with fresh oocytes, which served as the control group.Based on the post-warm-up incubation time, the oocytes in the study group were divided into three subgroups: 0 h, 1 h and 2 h. We then examined the configurations of spindles and chromosomes, the fragmentation of DNA, and the oocyte's ability to be fertilized and developed into blastocysts. By evaluating the vitrified oocytes' morphology, we confirmed that 601 out of 612 (98.2%) oocytes survived this protocol. The percentage of oocytes with normal spindle and chromosome configurations in the study groups 0 h, 1 h and 2 h were all quite similar to each other and not statistically different from that of the control group. Similar results were also observed in the percentage of oocytes containing fragmented DNA. The fertilization rate and blastocyst formation rate of the thawed oocytes were not statistically different from that of the control group either. However, if not handled properly (too much remnant medium on oocytes in the process of freezing or too long a time of oocytes in the vitrification medium before freezing), the cryopreserved oocytes could show dramatic difference from the control group in terms of the morphologically survival rate, the configuration of the spindles and chromosomes, and the DNA fragmentation. In conclusion, when followed correctly, this modified cryoloop vitrification protocol had little effect on the survival rate and development potential of mature mouse oocytes.

Cryopreserved human blastocysts after vitrification result in excellent implantation and clinical pregnancy rates

We conducted a retrospective review of all frozen ETs from May 2002 to October 2007 using our new vitrification technique in order to determine its efficacy. We found high pregnancy (70.5%; 79/112) and implantation (40.6%; 110/271) rates with the use of this new method.

Oocyte cryopreservation: a technical and clinical update

The primary objective of this article is to review the most recent progress in oocyte cryopreservation using various slow-freezing and vitrification protocols. There is a significant improvement in the clinical outcomes with higher pregnancy rates following mature metaphase II oocyte cryopreservation. Moreover, some reassuring data were recently reported regarding the long-term safety of this technology. However, overall clinical pregnancy rates following oocyte cryopreservation remain less efficient than the more established practice of embryo cryopreservation. Oocyte cryopreservation is a viable alternative for fertility preservation for women at risk of premature ovarian failure due to diverse factors, such as gonadotoxic chemotherapy or radiotherapy. Currently, there are two distinct techniques for oocyte cryopreservation: slow freezing and vitrification. Oocyte vitrification is gaining popularity as the technique of choice; however, well-designed, randomized, controlled trials are still required to e...

APPLICATION OF CRYOPRESERVATION FOR PISTACHIO GERMPLASM CONSERVATION

Pistachio (Pistacia vera L.) which has an economical importance especially for Mediterranean countries, is today under the threat of genetic erosion. Therefore, it is very important to develop efficient biotechnological strategies for the safeguard conservation of pistachio biodiversity. Currently, cryopreservation is the only technique available that enables to maintain plant germplasm without alteration or modification for a theoratically unlimited period of time. Hence, in this study experiments were conducted to develop and optimize efficient cryogenic protocols for the preservation of seeds and axillary buds of pistachio. By using dehydration and one-step freezing technique, 90% germination was obtained with decreasing the moisture content of the seeds to 11.7% (FW basis) by 8 hours drying over silica gel prior to their direct immersion in liquid nitrogen (LN). As regards the long-term preservation of axillary buds of in vitro-grown pistachio shoots, vitrification and one-step freezing technique was also tested with incubation of buds in PVS2 solution for various periods of time (0, 30, 60, 90 min) and different temperatures (0°C and 25°C) before immersion in LN. Following the optimization of the vitrification protocols, trials are now in progress for the development of effective cryopreservation protocols for pistachio axillary buds both by using encapsulation-vitrification and encapsulation-dehydration techniques.

Cryopreservation of Panax ginseng Adventitious Roots

We tested desiccation and/or vitrification procedures to cryopreserve the adventitious roots of Panax ginseng, the source of commercially produced ginsenosides. When only desiccation was applied, the post-freeze survival of 3- to 4-mm root tips was <14% regardless of the composition of the preculture medium or the explant origin. Callus formation was frequently observed after cryopreservation. In contrast, 90% survival and 32.5% root formation efficiency were achieved after cryopreservation when a vitrification protocol was followed. Adventitious root cultures in flasks and bioreactors were reestablished from root tips cryopreserved by vitrification. A prolonged lag-phase and lower biomass production were recorded in post-freeze-regenerated cultures compared with control roots that were subcultured four times in flasks. However, biomass accumulations did not differ between control and regenerated roots at the end of the sixth subculturing period. After 40 days of culture in bioreactors, a mean value of 12.5 g dw L−1 was recorded for post-freeze-regenerated cultures versus 9.1 g dw L−1 for the control roots. Production of triol and diol ginsenosides in our bioreactor cultures also was enhanced after cryopreservation, by 41.0% and 89.8%, respectively. These results suggest that the vitrification method is successful for cryopreservation of P. ginseng adventitious roots.

Comparison of the offspring sex ratio between fresh and vitrification-thawed blastocyst transfer

There was a significant difference in the sex ratio between offspring from fresh and from vitrification-thawed blastocyst transfer. The live birth sex ratio influenced by the blastocyst-stage embryo transfer correlated with top-quality embryo morphology.

High-efficiency vitrification protocols for cryopreservation of in vitro grown shoot tips of transgenic papaya lines

In vitro grown shoot tips of transgenic papaya lines (Carica papaya L.) were successfully cryopreserved by vitrification. Shoot tips were excised from stock shoots that were preconditioned in vitro for 45–50-day-old and placed on hormone-free MS medium with 0.09 M sucrose. After loading for 60 min with a mixture of 2 M glycerol and 0.4 M sucrose at 25°C, shoot tips were dehydrated with a highly concentrated vitrification solution (PVS2) for 80 min at 0°C and plunged directly into liquid nitrogen. The regeneration rate was approximately 90% after 2 months post-thawing. Successfully vitrified and warmed shoot tips of three non-transgenic varieties and 13 transgenic lines resumed growth within 2 months and developed shoots in the absence of intermediate callus formation. Dehydration with PVS2 was important for the cryopreservation of transgenic papaya lines. This vitrification procedure for cryopreservation appears to be promising as a routine method for cryopreserving shoot tips of transgenic papaya line germplasm.