The survival of in vitro shoot tips of Garcinia mangostana L. after cryopreservation by vitrification

This report highlights the first successful cryopreservation protocol for shoot tips of Garcinia mangostana L. achieved by using vitrification technique. We investigated the effects of different temperatures and exposure periods to a plant vitrification solution 2 (PVS2), sucrose concentrations and preculture periods, and unloading treatments in steps of the vitrification protocol on the survival of G. mangostana shoot tips after cryopreservation. Exposure to PVS2 for 25 min gave beneficial effects with 10.4 ± 1.8 % survival at 0 °C with average water content of 1.1 ± 0.3 g g^?1 dry mass. Survival was 13.7 ± 5.5 % when using preculture medium with full-strength Murashige and Skoog (MS) medium supplemented with 0.6 M sucrose for 2 days. A significant difference was observed in survival of shoot tips when treated with various sucrose concentrations in preculture which strengthens their importance towards enhancing survival of shoot tips after cryopreservation. MS with 0.4 M sucrose and 2 M glycerol applied as an unloading solution increased the survival of shoot tips to 44.1 ± 6.5 %. Experiments on the effect of ascorbic acid were also conducted for each step of vitrification. Our results showed higher survival of 45.8 ± 3.8 % but there were no significant effects compared with the control (without ascorbic acid). Further study on the recovery dark/light period was conducted. Survival of shoot tips significantly increased to 50.0 ± 16.7 % when subjected to 7 days in the dark before transferring to 16 h/8 h light/dark photoperiod. These studies strengthen suggestions that cryopreservation through vitrification is possible for ex situ conservation of germplasm of this tropical recalcitrant species.     

Cryopreservation of the critically endangered golden paintbrush (<Emphasis Type="Italic">Castilleja levisecta</Emphasis> Greenm.): from nature to cryobank to nature

In this study conservation of Castilleja levisecta Greenm., a globally endangered species was addressed through in vitro cryopreservation of shoot tips. In vitro cultures were successfully established using seedlings received from British Columbia, Canada. Shoot tips excised from in vitro propagated plants were cryopreserved using a droplet-vitrification method following optimization of individual protocol steps such as pre-culture, treatment with vitrification solutions, and unloading. The highest plant regrowth after cryopreservation (66%) was achieved when shoot tips were pre-cultured in 0.3 M sucrose for 17 h followed by 0.5 M sucrose for 4 h, incubated in an osmo-protectant solution (17.5% [v/v] glycerol and 17.5% [w/v] sucrose) for 20 min, exposed to vitrification solution A3 (37.5% [v/v] glycerol plus 15% [v/v] dimethylsulfoxide (DMSO) plus 15% [v/v] ethylene glycol (EG) plus 22.5% [w/v] sucrose) on ice for 40 min, and unloaded in 0.8 M sucrose solution for 30 min. Healthy plants were developed from cryopreserved shoot tips and propagated in vitro using nodal segments. Plants derived from in vitro culture and from cryopreserved tissues were successfully rooted and acclimated in a greenhouse with 100% survival rate. Acclimatized plants were reintroduced in a naturalized propagation area at the Conservation Nursery at Fort Rodd Hill, Canada. Twenty of 94 reintroduced plants (21%) survived the transit from lab to field and some had started to flower. This is the first report for cryopreservation of C. levisecta, an important step in conserving and re-introducing this critically imperiled species in nature.     

Cryopreservation of shoot tips from the endangered endemic species Tuberaria major

Tuberaria major is an endangered endemic species from the Algarve, in the south of Portugal. We investigated two techniques for the cryopreservation of T. major shoot tips, namely vitrification and encapsulation-dehydration. Before the cryopreservation trials, shoot tips were precultured for 1 day on liquid Murashige and Skoog (MS) medium containing 0.3 M sucrose. For the vitrification method, shoots tips were exposed for 0, 30, 60, 90 and 120 min to plant vitrification solution 2 (PVS2). As for the encapsulation-dehydration method, shoot tips were dried inside a laminar air flow cabinet for 0, 1, 2, 3, 4, 5 and 6 h at room temperature. The highest regrowth percentages were approximately 60 and 67 % for vitrification and encapsulation-dehydration, respectively. The best times were 60 min exposure to PVS2 for vitrification and 3 h desiccation for encapsulation-dehydration. Though these are preliminary results, the use of the cryopreservation techniques tested here proved to be an important asset in the conservation of this endangered species and will complement the conservation strategies previously developed.     

Droplet-vitrification cryopreservation of <Emphasis Type="Italic">in vitro</Emphasis>-grown shoot tips of grapevine (<Emphasis Type="Italic">Vitis</Emphasis> spp.)

An efficient and broad-spectrum protocol for cryopreservation of Vitis spp. shoot tips by droplet-vitrification is reported. Shoot tips (1.0 mm) containing 5–6 leaf primordia (LPs) were precultured for 3 d with a preculture medium containing 0.3 M sucrose, 0.16 μM glutathione, and 0.14 μM ascorbic acid. Precultured shoot tips were treated for 20 min at 24°C with a loading solution composed of 2 M glycerol and 0.4 M sucrose, followed by exposure at 0°C to half-strength plant vitrification solution 2 (PVS2) for 30 min, and then full-strength PVS2 for 50 min. Dehydrated shoot tips were transferred into 2.5-μL PVS2 carried on aluminum foil, prior to a direct immersion in liquid nitrogen. With this method, an average shoot regrowth level of 50.5% was obtained from cryopreserved shoot tips in six V. vinifera genotypes (three wine cultivars, two table cultivars, and one rootstock) and two V. pseudoreticulata genotypes. Vegetative growth of the regenerants recovered from cryopreservation, significantly increased as the number of subculture cycles increased and was greater than the control after the third subculture following cryopreservation. Inter-simple sequence repeats (ISSR) and random amplification of polymorphic DNA (RAPD) analyses did not detect any polymorphic loci in the plants of V. vinifera L. cv. ‘Cabernet Sauvignon’ from cryopreserved shoot tips compared to the original cultures. This droplet-vitrification cryopreservation method provides a technical platform to set up cryobanks of Vitis spp.     

Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique

 Sugar beet shoot tips from cold-acclimated plants were successfully cryopreserved using a vitrification technique. Dissected shoot tips were precultured for 1 day at 5  °C on solidified DGJ0 medium with 0.3 M sucrose. After loading for 20 min with a mixture of 2 M glycerol and 0.4 M sucrose (20  °C), shoot tips were dehydrated with PVS2 (0  °C) for 20 min prior to immersion in liquid nitrogen. Both cold acclimation and loading enhanced the dehydration tolerance of shoot tips to PVS2. After thawing, shoot tips were deloaded for 15 min in liquid DGJ0 medium with 1.2 M sucrose (20  °C). The optimal exposure time to both loading solution and PVS2 depended on the in vitro morphology of the clone. With tetraploid clones a higher sucrose concentration during cold acclimation and preculture further enhanced survival after cryopreservation. Survival rates ranged between 60% and 100% depending on the clone. Since only 10–50% of the surviving shoot tips developed into non-hyperhydric shoots, regrowth was optimized. Received: 13 September 1999 / Revision received: 2 March 2000 / Accepted: 16 March 2000     

Heterologous expression of <Emphasis Type="Italic">PDH47</Emphasis> confers drought tolerance in <Emphasis Type="Italic">indica</Emphasis> rice

Jerusalem artichoke (Helianthus tuberosus L.) cultivars are conserved in genebanks for use in breeding and horticultural research programs. Jerusalem artichoke collections are particularly vulnerable to environmental and biological threats because they are often maintained in the field. These field collections could be securely conserved in genebanks if improved cryopreservation methods were available. This work used four Jersualem artichoke cultivars (‘Shudi’, ‘M6’, ‘Stampede’, and ‘Relikt’) to improve upon an existing procedure. Four steps were optimized and the resulting procedure is as follows: preculture excised shoot tips (2–3 mm) in liquid MS medium supplemented with 0.4 M sucrose for 3 days, osmoprotect shoot tips in loading solution for 30 min, dehydrate with plant vitrification solution 2 for 15 min before rapid cooling in liquid nitrogen, store in liquid nitrogen, rapidly rewarm in MS liquid medium containing 1.2 M sucrose, and recover on MS medium supplemented with 0.1 mg L^?1 GA₃ for 3–5 days in the dark and then on the same medium for 4–6 weeks in the light (14 h light/10 h dark). After cryopreservation, Jerusalem artichoke cultivar ‘Shudi’ had the highest survival (93%) and regrowth (83%) percentages. Cultivars ‘M6’, ‘Stampede’, and ‘Relikt’ achieved survival and regrowth percentages ranging from 44 to 72%, and 37–53%, respectively. No genetic changes, as assessed by using simple sequence repeat markers, were detected in plants regenerated after LN exposure in Jerusalem artichoke cultivar ‘Shudi’. Differential scanning calorimetry analyses were used to investigate the thermal activities of the tissues during the cryopreservation process and it was determined that loading with 2.0 M sucrose and 0.4 M sucrose dehydrated the shoot tips prior to treatment with PVS2. Histological observations revealed that the optimized droplet vitrification protocol caused minimal cellular damage within the meristem cells of the shoot tips.     

Cryopreservation of in vitro sugar beet shoot tips using the encapsulation-dehydration technique: influence of abscisic acid and cold acclimation

It has been previously shown that shoot tips of in vitro plantlets of sugar beet (Beta vulgaris L. clone SES1) can be cryopreserved using the encapsulation-dehydration technique (survival rate of 37% after freezing). This article reports the influence of abscisic acid (ABA) and cold acclimation on survival after cryopreservation. When ABA was added to the multiplication medium of the plants, the survival rate of shoot tips after cryopreservation was not increased (45%). After cold acclimation of the plants, their growth pattern differed (plants became apically dominant) and the survival rate of the shoot tips after cryopreservation clearly increased (70% survival and 50% plant regeneration after freezing). This improved protocol was successfully applied to three other clones. Received: 28 October 1996 / Revision received: 28 January 1997 / Accepted: 15 March 1997     

Cryopreservation of ex-vitro-grown Rosa chinensis ‘Old Blush’ buds using droplet-vitrification and encapsulation-dehydration

Axillary buds from greenhouse-grown plants of Rosa chinensis ‘Old Blush’ were successfully used to establish cryopreservation protocols using both droplet-vitrification and encapsulation-dehydration methods. In droplet vitrification, regrowth occurred after exposure to liquid nitrogen even without pre-culture in the loading solution (LS) before immersion in the plant vitrification solution 2 (PVS2). However, a 20–80 min LS step followed by a short immersion in PVS2 for 3 or 15 min, at 0 °C gave the best regrowth rates (82–86 %). In encapsulation dehydration, the level of dehydration significantly influenced shoot regrowth. The best regrowth rate, 60 %, was obtained at a bead water content of 0.35 g water per g dry weight. These results demonstrate the possibility of using greenhouse plants of rose for cryopreservation by droplet vitrification and encapsulation dehydration.     

Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species

Cell membranes are the primary sites of cryopreservation injury and measuring changes to membrane composition arising from cold acclimation may assist with providing a rationale for optimising cryopreservation methods. Shoot tips from two south-west Western Australian species, Grevillea scapigera and Loxocarya cinerea, and Arabidopsis thaliana (reference species) were subjected to cryopreservation using the droplet vitrification protocol. Two pre-conditioning regimes involving a constant temperature (23 °C, CT with a 12 h light/dark cycle) or an alternating temperature (AT) regime (20/10 °C with a 12 h light/dark cycle) were compared. Soluble sugars, sterols and phospholipids present in the shoot tips were analysed. Use of AT pre-conditioning (acclimation) resulted in a modest decrease in cryotolerance in A. thaliana, increased cryotolerance in G. scapigera, and increased survival in the non-frozen control explants of L. cinerea in comparison to CT pre-conditioning. Increased cryotolerance was accompanied by a higher total sugar sterol and phospholipid content, as well as an increase in strong hydrating phospholipid classes such as phosphatidylcholine. The double bond index of bound fatty acyl chains of phospholipids was greater after AT pre-conditioning, mostly due to a higher amount of monoenes in A. thaliana and trienes in G. scapigera and L. cinerea. These findings suggest that AT pre-conditioning treatments for in vitro plants can have a positive influence on cryotolerance for some plant species and this may be related to observed changes in the overall composition of cell membranes. However, alternative factors (e.g. oxidative stress) may be equally important with other species (e.g. L. cinerea).     

Cryopreservation for eradication of Jujube witches' broom phytoplasma from Chinese jujube (Ziziphus jujuba)

Here, we report efficient eradication of Jujube witches' broom phytoplasma (Candidatus Phytoplasma ziziphi) from Chinese jujube (Ziziphus jujuba) by cryopreservation. Shoot tips (1.0 mm in size) with 5–6 leaf primordia (LPs) excised from diseased in vitro stock shoots were subject to droplet‐vitrification cryopreservation. Shoot tips following cryopreservation were post‐cultured on a recovery medium for survival. Plantlet regeneration was obtained by micrografting of surviving shoot tips upon in vitro rootstocks. With this protocol, 85% of shoot tips survived following cryopreservation, among which 75% regenerated into whole plantlets and all of them were free of phytoplasma, regardless of the sizes used for cryopreservation. Ultrastructural studies demonstrated that phytoplasma was absent in the apical dome, and leaf primordia (LPs) 1 and 2, while abundance of phytoplasma was present in the lower parts of shoot tips, leaf primordium 3 and older tissues. Histological observations showed that much more damage was found in cells located in the lower part of apical dome, leaf primordium 3 and older tissues than in those at the upper part of apical dome and in the LPs 1 and 2. These cells were most likely to survive and regenerate into phytoplasma‐free plantlets following cryopreservation and micrografting. Ploidy levels analyzed by flow cytometry (FCM) were maintained in plantlets regenerated from cryopreservation followed by micrografting. Results reported here would provide technical support for production of phytoplasma‐free plants and for long‐term storage of germplasm of Chinese jujube.     

Cryopreservation by encapsulation-dehydration of ‘Christmas bush’ (<Emphasis Type="Italic">Ceratopetalum gummiferum</Emphasis>) shoot tips

Summary Christmas bush (Ceratopetalum gummiferum Sm) is a shrubby tree species of the east coast of New South Wales in Australia. It is much prized as a cut flower crop because of its bright, pinky red floral calyces. New varieties are being developed, the storage of which is an important issue. In this study, it was shown that shoot tips sampled from in vitro plantlets withstood cryopreservation using the encapsulation-dehydration technique. The protocol leading to optimal regrowth was the following: excised shoot tips were pretreated for 1 d in the dark on hormone-free Murashige and Skoog (MS) medium with 0.3 M sucrose, then encapsulated in 3% calcium alginate and precultured in liquid MS medium with 0.5 M sucrose for 3 d. Precultured beads were dehydrated for 6 h in the air current of the laminar flow cabinet to 24.3% moisture content (fresh weight basis) before rapid immersion in liquid nitrogen. Under these conditions, regrowth of shoot tips after cryopreservation reached 61.4%. Regrowth of cryopreserved shoot tips was not affected by the period of cold acclimation of in vitro mother plants.     

香蕉离体茎尖超低温保存研究

以香蕉(Musaspp.)试管苗为试材,对其离体茎尖小滴玻璃化法超低温保存的影响因素进行了研究。小滴玻璃化法和玻璃化法超低温保存后再生率的差异表明,香蕉更适合用小滴玻璃化法进行超低温保存。香蕉小滴玻璃化法超低温保存的方案如下:试管苗在60g/L蔗糖的MS培养基上培养1~2个月,剥离带有1~2片叶原基的茎尖,室温下装载30m in(可延长至4h),0℃下PVS2处理40~50m in。6个基因型的14个品种的再生率平均为46.9%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。该结果为香蕉种质资源的长期保存提供了理论依据和技术支撑。     

Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification

In vitro-grown shoot tips of apples (Malus domestica Borkh. cv. Fuji) were successfully cryopreserved by vitrification. Three-week-old in vitro apple plantlets were cold-hardened at 5°C for 3 weeks. Excised shoot tips from hardened plantlets were precultured on a solidified Murashige & Skoog agar medium (MS) supplemented with 0.7 M sucrose for 1 day at 5°C. Following preculture shoot tips were transferred to a 2 ml plastic cryotube and a highly concentrated cryoprotective solution (designated PVS2) was then added at 25°C. The PVS2 contains (W/V) 30% glycerol, 15% ethylene glycol and 15% dimethylsulfoxide in medium containing 0.4 M sucrose. After dehydration at 25°C for 80 min, the shoot tips were directly plunged into liquid nitrogen. After rapid warming, the shoot tips were expelled into 2 ml of MS medium containing 1.2 M sucrose and then plated on agar MS medium. Direct shoot elongation was observed in approximately 3 weeks. The average rate of shoot formation was about 80%. This vitrification method was successfully applied to five apple species or cultivars and eight pear cultivars. This method appears to be a promising technique for cryopreserving shoot tips from in vitro-grown plantlets of fruit trees.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - PVS2 vitrification solution - LN liquid nitrogen - BA 6-benzylaminopurine - NAA -naphthaleneacetic acid - SE standard error - ABA abscisic acid     

Development of a common PVS2 vitrification method for cryopreservation of several fruit and vegetable crops

Many cryopreservation techniques are currently available, and it is common for new modifications to be developed for individual crops or specific genotypes. In this study, results of variations of the PVS2 cryopreservation protocol are compared to provide evidence for the suitability of a standard form of this technique for cryopreservation of a range of fruit, berry crops, and potato. Shoot cultures of Malus, Solanum, Lonicera, and Berberis were tested with variations of cold acclimation, pretreatment media, and PVS2 exposure times. A general protocol with some modifications was produced that was suitable for all four genera. The regenerative capacity of shoot tips after cryopreservation by this method exceeded a mean of 50% for Malus, Solanum, Lonicera, and Berberis, which is sufficient for setting storage in a cryobank. After liquid nitrogen storage, the shoot cultures that survived had a healthy appearance and developed rapidly. For each species tested, the only optimization required was the preparation of donor plants by cold acclimation and pretreatment. The choice of one common method simplifies the methodology for conducting experiments and storing a range of germplasm. The use of the PVS2 vitrification method with a 0.3-M sucrose pretreatment is multiuse and can be recommended as the most effective method for the cryopreservation of shoot tips from many plant species.     

Cryopreservation of wheat (Triticum aestivum L.) egg cells by vitrification

A procedure has been developed for the cryopreservation of wheat female gametes. The procedure involves loading the cells with 25% concentrated vitrification solution consisting of 30% glycerol, 10% sucrose, 120 mM ascorbic acid (AA) and 5% propylene glycol (PG), dehydration in 80% concentrated vitrification solution, droplet vitrification and storage in liquid nitrogen, unloading and rehydration of the cells by gradual addition of isolation solution. Supplementation with AA significantly increased the proportion of viable egg cells after de- and rehydration. During the early phase of rehydration AA reduced the probability of membrane damage caused by rapid water uptake. Maintaining the temperature of the cells at 0°C during the de- and rehydration processes increased cell survival. Microscopic examination of the semi-thin sections of untreated and viable cryopreserved cells revealed that the vitrification process might cause changes in cell structure.     

Anatomy and osmotic potential of the Vitis rootstock shoot tips recalcitrant to cryopreservation

This study was carried out on Kober 5BB (Vitis Berlandieri × V. riparia) grape rootstock shoot tips during the preparatory steps preceding the direct immersion in liquid nitrogen, in order to overcome until now unsuccessful cryopreservation with this species. The exposure of shoot tips to 0.3–0.4 M sucrose leads to a high cell solute concentration. The treatment with plant vitrification solution (PVS2) alone, i.e., not followed by storage in liquid nitrogen, markedly affected shoot tip survival. After a 30 min exposure, regrowth percentage of shoot tips decreased from 94 % (control) to 57 %, and dropped to 15 % when the treatment was prolonged up to 60 min. After a 90 min exposure, no regrowth occurred. In addition, plantlets regenerated from shoot tips which underwent 60 min or more exposure to PVS2 showed signs of malformation. Microscope observations of shoot tips treated with 0.3 or 0.4 M sucrose and 30 min PVS2 showed the presence of cells starting to plasmolyze, localized in the area surrounding the apical meristem. A limited presence of starch grains in meristem and bract cells was also noted. However, the most conspicuous consequence of prolonged PVS2 treatment was convex plasmolysis. The phenomenon was dependent on the time of PVS2 exposure. Indeed, after a 30 min treatment, plasmolysis was minimal or absent, but it increased with longer exposure to PVS2 at 4 °C.     

Optimization of droplet-vitrification protocol for carnation genotypes and ultrastructural studies on shoot tips during cryopreservation

This study was carried out to optimize a modified droplet-vitrification procedure for the cryopreservation of shoot tips from different carnation genotypes. The best procedure was developed by applying orthogonal tests to the experimental data and by further investigation of the effects on the regrowth percentage. It consisted in preculturing shoot tips in liquid Murashige and Skoog (MS) medium with 0.3 M sucrose for 2 days, pretreating them in liquid MS medium with 5 % Dimethyl sulfoxide +5 % glycerol + 0.3 M sucrose for 10 min, osmoprotecting in Loading solution for 20 min at 25 °C, cryoprotecting with Plant vitrification solution No.2 (PVS2) for 60 min at 0 °C, transferring in drops of fresh PVS2 over aluminum strips and finally storing them in Liquid nitrogen. With the application of the optimized protocol, four carnation cultivars (‘Master’, ‘Calibra’, ‘Lamour’ and ‘Ofcar’) achieved regrowth percentage after cryopreservation ranging from 41 to 73 %. Ultrastructural observations investigated by using transmission electron microscopy showed that the cells encountered the stress during cryopreservation and the main damages occurred during the dehydration step. For surviving cells, the most of the damaged cells could be repaired after recovery growth. This modified protocol will aid in the long-term conservation of carnation germplasm and the ultrastructural studies will benefit for understanding the damage and recovery of the cells during cryopreservation.     

Cryopreservation of red raspberry cultivars from the VIR <Emphasis Type="Italic">in vitro</Emphasis> collection using a modified droplet vitrification method

The goal of the present study was to analyze the post-cryogenic recovery of 12 red raspberry cultivars from the N.I. Vavilov All-Russian Institute of Plant Genetic Resources in vitro collection. The 1.1–1.8 mm shoot tips of microplants were subjected to cryopreservation using the modified droplet vitrification method. The current modifications to the droplet vitrification protocol included the elimination of the initial pretreatment stage of the microplants, and the use of modified media at the stages of initial micropropagation, explant isolation, and post-cryogenic regeneration. The optimized method reduced the duration of some cryopreservation stages compared to the initial protocol, and reduced the total procedure from 14 to 11 wk. This modified cryopreservation method also demonstrated a relatively high level of post-cryogenic regeneration. Depending on the genotype, the shoot recovery of explants after rewarming varied from 24.2–89.3% and averaged 58.8 ± 5.3%. There was a statistically significant influence of the genotype on the shoot recovery after rewarming. No differences in inter simple sequence repeats and in start codon targeted marker spectra were found between post-cryopreservation microplants and donor in vitro plants from two red raspberry cultivars.     

Survival and genetic stability of shoot tips of <Emphasis Type="Italic">Hedeoma todsenii</Emphasis> R.S.Irving after long-term cryostorage

Endangered and rare species for which seed banking is not possible require alternative methods of ex situ conservation for long-term preservation. These methods depend primarily on cryopreservation methods, such as shoot tip cryopreservation, but there are few datasets with information on the long-term survival of shoot tips stored in liquid nitrogen. In this study, survival and genetic stability of shoot tips of the endangered species, Hedeoma todsenii, banked over multiple years were examined. In vitro cultures cryopreserved with both the encapsulation dehydration and the encapsulation vitrification methods showed good average survival after up to 13 yr of storage in liquid nitrogen. The application of droplet vitrification to this species increased survival significantly, with an average of 72%, compared with 24–45% survival obtained with other methods. As measured with microsatellite and sequence-related amplified polymorphism (SRAP) markers, the genetic stability of the same genotypes stored over different periods of time typically did not change. However, there was an average of 10.4% band loss between replicate samples that did indicate a potential change in DNA composition. These results demonstrate the use of shoot tip cryopreservation as an effective ex situ conservation tool for this species, but genetic stability of the cryopreserved tissues should be closely monitored.     

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.