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Cryopreservation of adventitious shoot tips of Paraisometrum mileense by droplet vitrification

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Background: Gesneriaceae family contains numerous species endemic to China, and many of them are listed as endangered species. There is a need for a simple and efficient method for long-term conservation of these species. Objective: The study aimed to establish an efficient procedure for cryopreserving Paraisometrum mileense, a critically endangered species endemic to Yunnan, China. Methods: Effects of sucrose concentration of preculture solution, duration of sucrose preculture, duration of plant vitrification solution 2 (PVS2) treatment, and cold acclimation on regeneration of cryopreserved adventitious shoot tips (ASTs) were assessed. Results and conclusion: Among different sucrose preculture regimes tested, preculture with 0.3M sucrose for 24h resulted in best regeneration of cryopreserved ASTs. PVS2 treatment also affected regeneration considerably with the maximum survival of ASTs after incubation in PVS2 for 90 min at 0 degrees C. With the optimised parameters, the level of shoot regeneration from cryopreserved ASTs reached 86%. No morphological abnormalities were observed during one year's growth of the plantlets developing from cryopreserved ASTs. Procedure established in this research is a promising technique for the cryopreservation of ASTs of this species.
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... Cryostorage strategies applied for ex situ conservation of endemic plant diversity comprise multiple explant types, such as: shoot tips (Sharma et al., 2005); axillary nodes (Barraco et al., 2013); in vitro grown buds (Edesi et al., 2020); callus (Kaczmarczyk et al., 2013); protocorms (Cripps & McGregor, 2006); intact or scarified seeds (Khanna et al., 2014;Voronka & Kholina, 2010); and pollen (Ajeeshkumar & Decruse, 2013). In recent decades, among the most frequently used methods for the cryopreservation of various endemic and threatened plant species were droplet-vitrification (Halmagyi et al., 2020;Kaczmarczyk et al., 2013), vitrification (Lin et al., 2014;Whiteley et al., 2016), encapsulation-vitrification (Ciringer et al., 2018), and encapsulation-dehydration (Coelho et al., 2014). ...
... However, a few studies targeting seed cryopreservation protocols at the global scale for endemic and threatened species, including taxa from our list, exist (Illyés et al., 2005;Nikishina et al., 2006) (SM 3). While several endemic and threatened plant species worldwide were successfully cryopreserved with survival or regrowth percentages above 50% (Coelho et al., 2014;Lin et al., 2014), our cryopreservation protocols showed higher survival and regeneration rates in the targeted species (Coste et al., 2012;Halmagyi et al., 2020). This might be explained by the variables that can affect the success of cryopreservation such as: explant type, pretreatment conditions, cryopreservation procedures and post-thaw treatment (Nakkanong & Nualsri, 2018). ...
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Romania hosts a relatively high species diversity, including 3,829 vascular and 979 non-vascular spontaneous plant taxa. Multiple national Red Lists exist, with the number of taxa assessed as threatened varying greatly between them, from 548 to 1,438, and with number of taxa assigned to a given threat category also varying between the different sources. A composite list including all taxa mentioned in at least one of the threat categories for Romania is required in order to compensate for this lack of consensus and to assess their ex situ conservation status. In this study, we synthesized data from the national Red Lists and counted 1,220 spontaneous vascular plant species and 201 subspecies, of which 77 are endemic and 76 subendemic for Romania. In addition, 18 non-Red-Listed endemics and 14 subendemics have been added, bringing the total to 1,453 threatened and (sub)endemic plant taxa, representing almost 38% of the total native vascular flora of Romania. Despite the large network of protected areas in Romania, many taxa are still being threatened with extinction in the region mainly due to anthropogenic pressure. Several ex situ conservation measures have been employed to assure a more substantial buffer against plant extinction in the wild, supported by thorough and adequate conservation strategies and multiple means to reintroduce taxa back to their natural habitats. Consequently, our second aim was to evaluate the ex situ conservation status of these threatened and (sub)endemic plants from Romania, focusing on both conventional methods (cultivation in botanic gardens, seed banking) and biotechnological approaches (in vitro tissue culture, medium-term storage and cryostorage). Of the 1,453 taxa included in our list, 642 (44.2%) are conserved by ex situ approaches. Of these, 524 are harboured in the most important botanic gardens throughout Romania, while 156 are currently held in long-term seed banks locally or in the Millennium Seed Bank of the Royal Botanic Garden, Kew (UK). Conversely, only 64 taxa from the list are preserved at the national level through in vitro cultures, and cryopreservation protocols have been developed for only 8 taxa. Overall, more than half of the threatened and (sub)endemic vascular flora from Romania remains unprotected outside the classical in situ conservation measures. For Red Listed bryophytes, only 0.6% are preserved in national ex situ collections. Moreover, some aspects related to population genetic studies and the genetic stability of ex situ coneserved plants are also briefly discussed, as essential prerequisites for applied biodiversity conservation programs. Considering the distribution range of targeted taxa, we included a synthesis of biotechnological approaches at both national and international level. Our study presents not only a first assessment of the ex situ conservation status of national Red List flora, but also, to our knowledge, the most comprehensive and updated overview of the rare, threatened and (sub)endemic taxa from Romania. This evaluation will provide a supporting tool for national decision- and policy-making actions for biodiversity conservation, using both in situ and ex situ approaches. We also highlight the need for an updated Red List for the Romanian flora that accurately follows the IUCN assessment criteria and protocols.
... Therefore, after cryostorage and rewarming, these characteristics enable direct shoot formation from cryopreserved shoot tips and maintenance of the genetic integrity [14,77,78]. Several endemic plant species, such as Paraisometrum mileense W. T. Wang (endemic to Yunan, China) [79], Thymus moroderi Pau ex Martínez (endemic to South-eastern Spain) [80,81], and Tuberaria major (Willk.) P. Silva and Rozeira (endemic to the south of Portugal) [82], were successfully cryopreserved using shoot tips, with survival (swollen green shoots or callus formation) or regrowth (normal shoot development) percentages above 50%. ...
... These authors also proved that preculture had no effect on the regeneration of A. altaicus cryopreserved shoot tips, but the composition of the recovery medium was relevant to improve shoot tips regrowth. A regeneration of 86% was achieved for P. mileense when shoot tips were precultured with 0.3 M sucrose for 24 h and then exposed to PVS2 for 90 min at 0 • C, before immersion in LN [79]. Regeneration of P. mileense shoot tips was not improved by cold acclimation. ...
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Endemic plant species are usually more vulnerable to anthropogenic threats and natural changes and, therefore, hold a higher extinction risk. The preservation of these species is a major concern on a worldwide context and in situ protection alone will not guarantee their conservation. Ex situ conservation measures must be undertaken to support the conservation of these species, and seed banking is the more efficient and cost-effective method. However, when seed banking is not an option, alternative approaches should be considered. Biotechnological tools provide new and complementary options for plant conservation including short-, medium-, and long-term strategies, and their application for plant species conservation has increased considerably in the last years. This review provides information about the status of the use biotechnology-based techniques for the conservation of endemic plant species. Particular attention is given to cryopreservation, since is the only long-term ex situ conservation strategy that can complement and support the other conservation measures. The cryopreservation of plant genetic resources is, however, more focused on crop or economically important species and few studies are available for endemic plant species. The plant material used, the cryopreservation methods employed, and the assessment of cryogenic effects are reviewed. The reasons to explain the difficulties in cryopreserving these species are discussed and new strategies are proposed to facilitate and increase the interest on this matter. We expect that further studies on the conservation of endemic plant species will increase in a near future, thus contributing to maintain these valuable genetic resources.
... Diverse cryopreservation methods have been applied to shoot tips of endangered species, including vitrification (13,14), droplet-vitrification (3,11,15,20), encapsulation-dehydration (13,14,23,31,32) and encapsulation-vitrification (13,23,31). In a multi-stage procedure, such as in droplet-vitrification, every step, including pre-culture, osmoprotection, vitrification solution (VS) treatment and unloading, may become critical for survival. ...
... transsilvanicum and H. umbellatum, respectively (3), and after 20 min PVS2 treatment in Centaurea ultreiae (13). A longer PVS2 treatment of 90 min at 0°C was beneficial for cryopreservation of Paraisometrum mileense (11) and Dioscorea deltoidea (14) shoot tips. VS A3-90% treatment at 0 °C for 60 min was optimal for shoot tips of Betula lenta (25). ...
Article
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Background: Aster altaicus var. uchiyamae Kitam is an endemic and endangered species in urgent need of a comprehensive conservation strategy. Objective: To develop an efficient cryopreservation protocol using in vitro shoot tips to complement traditional conservation approaches in case seeds are not available or insufficient for conservation programs. Methods: Shoot tips of in vitro plants were cryopreserved using a droplet-vitrification method following improvement of pre-culture, osmoprotection, vitrification solution (VS), unloading and post-culture treatments. The starting protocol included step-wise pre-culture with 10% and 17.5% sucrose for 55 h and 17 h, respectively, followed by osmoprotection with C4-35% (17.5% glycerol + 17.5% sucrose) for 30 min, and cryoprotection with B5-80% (40% glycerol + 40% sucrose) for 60 min. Results: Shoot tips of A. altaicus were found to be moderately sensitive to the osmotic stress. Pre-culture and osmoprotection were not critical for the regeneration of cryopreserved explants when either of these treatments was applied. Osmoprotection with C4-35% on ice for 60 min followed by cryoprotection with A3-80%, a modified and diluted PVS2, on ice for 60 min resulted in the highest (65.3%) regeneration of cryopreserved shoot tips. Among alternative VSs tested, A3-80% and B5-80% were superior to PVS2 and PVS3 used under the same conditions. Step-wise recovery of shoot tips on ammonium-free medium followed by GA3-containing medium and medium without growth regulators were critical for the normal regeneration of both VS-treated and cryopreserved shoot tips. Conclusions: Cryopreservation of in vitro shoot tips using droplet-vitrification was developed as a complementary conservation approach for A. altaicus. Adjustment of the composition of regrowth media depending on recovery stage was important for the regeneration of healthy plants from cryopreserved shoot tips.
... Additional experiments indicated that germinating seedlings and plantlets derived from shoot organogenesis showed extraordinary drought tolerance (data not shown). An efficient procedure was also established for the cryopreservation of O. mileense (Lin et al. 2014). Oreocharis mileense was among the first groups of plants with extremely small population size used to establish small-scale protected areas in China (Ma et al. 2013). ...
... Micropropagation of O mileense is therefore the prerequisite for additional studies of this species. All in vitro grown adventitious shoots of O. mileense used for cryopreservation in the study of Lin et al. (2014) originated from in vitro culture. In vitro culture is an important and effective method for preserving plant germplasm. ...
Article
Oreocharis mileense (W.T. Wang) M. Möller & A. Weber is endemic to China and was considered to be extinct because it had not been seen in the wild since the first collection in 1906. In 2006, the species was rediscovered in Shilin County, Yunnan Province. Oreocharis mileense was considered critically endangered for its narrow geographic range and extremely small population. An efficient method to preserve plant germplasm by in vitro culturing of O. mileense has not been reported. In this study, an orthogonal array with three factors (6-benzyladenine, BA; α-naphthaleneacetic acid, NAA; and sucrose), at four levels was performed, and shoot induction as well as shoot proliferation were recorded. The results were analyzed to determine the most significant components and the optimum combination for micropropagation of O. mileense. The results showed that: (1) organogenesis was easily induced by different combinations of plant-growth regulators and sucrose; (2) NAA and sucrose had the most significant effect on shoot induction and shoot multiplication, and (3) the optimum induction and proliferation media were 0.5 mg L⁻¹ BA + 0.2 mg L⁻¹ NAA + 30 g L⁻¹ sucrose and 1 mg L⁻¹ BA + 0.1 mg L⁻¹ NAA + 30 g L⁻¹ sucrose, respectively.
... In vitro conservation is one of the plant tissue culture techniques that is widely used for the rapid and efficient propagation of several endangered and endemic medicinal species under sterile conditions (Debnath et al., 2006). In recent years, intensive efforts have been made to conserve and propagate several endangered medicinal plants such as Paraisometrum mileense and Tuberaria major by in vitro conservation techniques from a small quantity of the explant sources without any notable effects on wild-type populations (Coelho et al., 2014;Lin et al., 2014;Monemi et al., 2014). On the basis of these facts, the present investigation aimed to evaluate the effect of 6-benzylaminopurine (BAP), thidiazuron (TDZ), α-naphthalene acetic acid (NAA), and indole-3-butyric acid (IBA), either alone or in combination, on callus induction, shoot organogenesis, and multiplication of in vitro cultured S. araxensis leaf, petiole, and stem explants. ...
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The present study introduced an in vitro shoot organogenesis protocol for the medicinal plant Scutellaria araxensis (Lamiaceae). Stem, leaf, and petiole explants were cultured in half-strength Murashige and Skoog (MS) medium containing different concentrations of 6-benzylaminopurine (BAP) alone or in combination with thidiazuron (TDZ), indole-3-butyric acid (IBA), or α-naphthalene acetic acid. Callus formation occurred from stem and petiole explants in most cultures; however, in leaf explants, it was observed only in cultures containing 0.5 mg/l BAP supplemented with TDZ at all concentrations. The highest frequency of indirect shoot induction (100 and 90%) with an average of 20.33 and 12 shoots per explant was observed in stem-derived calli cultured on half-strength MS medium containing 2.0 mg/l BAP plus 0.5 and 1.5 mg/l TDZ, respectively. The best direct shoot organogenesis (40%) was observed in stem explants cultured on half-strength MS medium containing 0.5 mg/l BAP and 0.5 mg/l IBA with a mean of 18 shoots per stem explant. The regenerated micro-shoots were elongated on a medium fortified with 0.5 mg/l gibberellic acid and then successfully rooted in half-strength MS medium supplemented with 0.5 mg/l IBA. The obtained plantlets were acclimatized in a growth chamber with a survival rate of 100%. This study is the first report of a simple and efficient in vitro shoot organogenesis and regeneration protocol for S. araxensis by using stem explants, which could be useful for the conservation, genetic manipulation, and exploitation of biological molecules of this valuable genetic source.
... sobria and A. peacockii) tested until now, post-cryopreservation results have exceeded 80% in the first case (Tin and Folgado 2019), and 90% in our studies. Different protocols following droplet-vitrification approach have been successfully applied to cryopreserve organized structures of several endemic and endangered plant species such as adventitious shoot-tips of Paraisometrum mileense (Lin et al. 2014); shoot-tips of Castilleja levisecta Greenm (Salama et al. 2018); shoot apices and axillary buds of Dianthus taxa (Halmagyi et al. 2020); shoot-tips of Pogostemon yatabeanus (Lee et al. 2021). ...
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More than 50% out of 129 of Agave species are endemic to Mexico. Among them, Agave peacockii is among the list of threatened species that require special protection. In this work, we aimed at developing new supplementary strategies to achieve micropropagation and perform cryopreservation of in vitro grown shoot-tips of A. peacockii. For multiplication, the addition of two cytokinins, 6-benzylaminopurine (26.6 μM) and kinetin (27.84 μM) to MS semisolid medium significantly favoured the morphogenetic response and produced the highest (87.00 ± 17.18) shoot generation number after 60 days of culture. This interaction was more effective than using the same growth regulators separately. Propagated and rooted plantlets were acclimatized with 100% survival and normal morphological development. For cryopreservation, an optimized protocol following droplet-vitrification allowed obtaining 98% and 96% regrowth before and after cryopreservation, respectively. Shoot-tips (1 mm in length × 1 mm wide) were excised of in vitro-propagated plants, subjected to preculture on MS semisolid medium with 0.3 M sucrose for 1d, loaded in solution with 0.4 M sucrose and 1.6 M glycerol for 20 min, exposed to Plant Vitrification Solution 2 for 15 min, and then, immersed in liquid nitrogen in droplets of PVS2 placed on aluminium foil strips. The vegetative growth of cryo-derived plants and of the in vitro propagated plants was compared under greenhouse conditions. No significant differences were detected in most assessed characteristics after 120 days of culture. The results presented here constitute new viable biotechnological approaches for the in vitro propagation and long-term conservation of endangered Agave germplasm.
... Por otro lado, después de una hora de almacenamiento en NL se observó que los valores de viabilidad se mantuvieron constantes y bajos, indicando que ninguno de los tiempos de contacto con PVS2 fue adecuado para brindar protección a las yemas y, por tanto, mantener su viabilidad celular. Estos resultados son similares a lo reportado por , donde observaron que la solución de PVS2 causó mayor daño en los ápices de papa, dando como resultado más tiempo de recuperación y baja regeneración de los tejidos después estar en NL; así como lo reportado en brotes adventicios de Paraisometrum mileense, donde el tratamiento con PVS2 afectó considerablemente la regeneración y se obtuvo un óptimo de sobrevivencia a los 90 min (Lin, Yuan, Wang, & Li, 2014). ...
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La crioconservación ha revolucionado el campo de la biotecnología. Congelar en nitrógeno líquido (NL) preserva células por largo tiempo. En ese sentido, en este trabajo se evaluaron tres condiciones de crioconservación basados en la vitrificación de yemas de vid. Las yemas fueron sometidas a PVS2, PVS3 y glicerol por 0-420 min, y colocadas en NL por una hora. De modo posterior a cada tiempo de incubación se cuantificó la pérdida de iones como medida de viabilidad y se evaluó el daño mediante observación en estereoscopio. Basados en el porcentaje de viabilidad el mejor método fue empleando PVS3 (30% viabilidad), seguido de glicerol (25%) y PVS2 (<10%). Las imágenes de las yemas expuestas a PVS3 no muestran daño en el tejido, a diferencia de PVS2 y glicerol, los cuales resultaron insuficientes para preservar el tejido.
... Por otro lado, después de una hora de almacenamiento en NL se observó que los valores de viabilidad se mantuvieron constantes y bajos, indicando que ninguno de los tiempos de contacto con PVS2 fue adecuado para brindar protección a las yemas y, por tanto, mantener su viabilidad celular. Estos resultados son similares a lo reportado por , donde observaron que la solución de PVS2 causó mayor daño en los ápices de papa, dando como resultado más tiempo de recuperación y baja regeneración de los tejidos después estar en NL; así como lo reportado en brotes adventicios de Paraisometrum mileense, donde el tratamiento con PVS2 afectó considerablemente la regeneración y se obtuvo un óptimo de sobrevivencia a los 90 min (Lin, Yuan, Wang, & Li, 2014). ...
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The cryopreservation has revolutionized the field of biotechnology. Frozen in liquid nitrogen (LN) preserves long living cells. In this sense, in this paper were evaluated three conditions of cryopreservation based on vitrification of buds of grapevine. The buds were subjected to the PVS2, PVS3 and glycerol for 0-420 min, and submerged in LN for one hour. After the incubation time electrolyte leakage was determined as a viability measurement, and tissue damage was evaluated through stereoscopic observation. Based on the viability percentage the best preservation method was using PVS3 solution (30%) followed by glycerol (25%) and PVS2 (<10%). The images of the buds exposed to PVS3 shows no tissue damage unlike to PVS2 and glycerol, were not suficient to preserve buds tissue. The results shown here suggest that using PVS3 as protocol can be considered for buds grapevine germplasm preservation.
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More than 50% of Agave species are endemic to Mexico. Among them, Agave peacockii is listed within the list of threatened species that require special protection. In this work, we aimed at developing new supplementary strategies to achieve micropropagation and perform cryopreservation of in vitro -grown shoot-tips of A. peacockii . For multiplication, the addition of two cytokinins, 6-benzylaminopurine (26.6 μM) and kinetin (27.84 μM) to MS semisolid medium significantly favoured the morphogenetic response and produced the highest shoot generation (87.00±17.18) after 60 d of culture. This interaction was more effective than using the same growth regulators separately. Propagated and rooted plantlets were successfully acclimated with 100% survival and a normal morphological development during greenhouse performance. For cryopreservation, an optimized protocol following droplet-vitrification approach allowed obtaining 98% and 96% regrowth before and after cryopreservation, respectively. Shoot-tips were excised of in vitro -propagated plants, subjected to preculture on MS semisolid medium with 0.3 M sucrose for 1d, loaded in solution with 0.4 M sucrose and 1.6 M glycerol for 20 min, exposed to vitrification solution PVS2 for 15 min, and then, immersed in liquid nitrogen in droplets of PVS2 placed on aluminium foil strips. The vegetative growth of cryo-derived plants and of the in vitro propagated plants was compared under greenhouse culture conditions. No significant differences were detected in most assessed characteristics after 120 d of acclimatization. The results presented here constitute new viable biotechnological approaches for the in vitro propagation and long-term conservation of endangered Agave germplasm.
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The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.
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Teucrium polium L. with the common name of Felty Germander is one of the plants flora that is widely used in folk medicine in many Middle East countries, it is an endangered plant species and must be highly considered for preservation. Cryopreservation of T. polium by vitrification and encapsulation-dehydration was successfully achieved in this study. Shoot-tips were excised aseptically from in vitro grown plants and incubated for 3 days on solid hormone free-Murashige and Skoog (HF-MS) media supplemented with 0.3 M sucrose under complete darkness at 24 ± 1 °C. In vitrification, shoot-tips were loaded in 0.4 M sucrose and 2 M glycerol for 20 min followed by desiccation with different combinations and concentrations of plant vittrification solution 2 (PVS2), before immersion in Liquid Nitrogen (LN). Whereas for the encapsulation-dehydration; shoot-tips were encapsulated in calcium alginate and dehydrated under laminar air flow cabinet for 0, 3, 6, or 9 h. A total of 60 % of the cryopreserved vitrified shoot-tips survived when desiccated in concentrated PVS2 solution for 20 min, whereas, 28 % of the cryopreserved vitrified shoot-tips were regrown after 20 min of desiccation by two step increase in PVS2 concentration. Complete survival were obtained for the non-cryopreserved encapsulated shoot-tips treated for 3 days in 0.5 M sucrose with MS media without or with 3 h of dehydration, whereas, only 20 % of the cryopreserved encapsulated shoot-tips were regrown. The procedures developed in this study are easy to handle and produced a high levels of shoot formation.
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A simple and efficient method was developed for cryopreservation ofin vitro-grown shoot tips of `Troyer' citrange byencapsulation-vitrification. Excised shoot tips were precultured withincreasing sucrose concentrations of 0.3, 0.5, 0.75 and 1 M for 4 days.Precultured shoot tips were encapsulated and simultaneously osmoprotectedwith a loading solution of 2 M glycerol and 1 M sucrose.Osmoprotected and encapsulated shoot tips were dehydrated with a highlyconcentrated vitrification solution prior to direct immersion in LN for 1 h.Optimal survival of cryopreserved shoot tips was obtained when preculturedshoot tips were osmoprotected for 60 min during encapsulation.Dehydration by exposure to modified PVS2 solution for 90 min at24 C and for 180 to 210 min at 0 C was found optimalfor survival of cryopreserved shoot tips. Preculture duration largelyinfluenced survival of cryopreserved shoot tips, with the best result obtainedafter 2 to 5 days of preculture with stable 1 M sucrose. With theoptimized parameters, 100% survival of cryopreserved shoot tips wasachieved. Morphologies of plants regenerated from cryopreserved shoottips were similar to those of the seedlings.